AB STRAC T B O O K
www.eswiconference.org
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
TAB LE OF
CO NTENTS
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
II
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
LECTURE ABSTRACTS
SPB103
SPA1: CLINICAL IMPACT AND DIAGNOSTIC
APPROACHES
Assembly of Influenza virus particles: Signals
for targeting of HA and M2 to the budding
site
L2
SPA101
Ability of the Luminex xTAG RVP to detect
zoonotic influenza viruses with pandemic
potential
Veit, Michael (1); Brett, Katharina (1); Kordyukova, Larisa
(2); Serebryakova, Marina (2); Siche, Stefanie (1); Thaa,
Bastian (1); deVries, Maren (1); Herrmann, Andreas (3)
SPB104
L9
Conserved and host-specific features of
influenza virion architecture
Jörn Winter, Christine Warnes and Stephen Lindstrom
SPA102
L8
L3
Hutchinson, Edward; Charles, Philip; Hester, Svenja;
Thomas, Benjamin; Trudgian, David; Martínez-Alonso,
Mónica; Fodor, Ervin
L4
SPA2: EPIDEMICS AND PANDEMIC THREATS
A review of the link between influenza and
non-communicable diseases
Palache, Abraham Mozes (1); Tainijoki-Seyer, Dr. Julia (2)
SPA103
Influenza B disease burden in general
population in France. Results of
the IBVD retrospective study from 2003 to
2014
SPA201
The Epidemiological Characteristics of
Influenza B Compared to Influenza A: Results
of the Global Influenza B Study
Cohen, Jean Marie (1,2); Daviaud, Isabelle (1,2); Debost,
Emmanuel (2); Valette, Martine (3); Enouf, Vincent (4);
Mosnier, Anne (1,2)
L5
SPA104
Severity related factors in patients with
severe acute respiratory infections (SARI):
results from the Belgium SARI surveillance
during the Influenza season 2012-2013
Caini, Saverio (1); Huang, Sue (2); Ciblak, Meral Akçay
(3); Schellevis, François (1); Plotkin, Stanley (4); Paget,
John (1)
SPA202
Gefenaite, Giedre (1); Caini, Saverio (2); Gross, Diane
(3); Meerhoff, Tamara (4); Pereyaslov, Dmitriy (3); Paget,
John (2); Brown, Caroline Sarah (3)
SPA203
L6
The novel functional site in the PB2 subunit
of RNA-dependent RNA polymerase essential
for acetyl-CoA interaction, RNA polymerase
activity and viral replication
Wu, P (1); Jiang, H (2); Wu, JT (1); Chen, E (3); He, J (4);
Zhou, H (2); Wei, L (1); Yang, J (2); Yang, B (1); Qin, Y (2);
Fang, VJ (1); Li, M (2); Tsang, TK (1); Zheng, J (2); Lau, EHY
(1); Cao, Y (2); Chai, C (3); Zhong, H (4); Li, Z (2); Leung,
GM (1); Feng, L (2); Gao, GF (5,6); Cowling, BJ (1); Yu, H (2)
SPA204
Hatakeyama, Dai (1); Shoji, Masaki (1); Yamayoshi, Seiya
(2); Hirota, Takenori (1); Nagae, Monami (1); Yanagisawa,
Shin (1); Kawaoka, Yoshihiro (2); Kuzuhara, Takashi (1)
L14
Molecular determinants of influenza A/H7N9
virus HA receptor binding and stability
L7
Role of segment-specific genome packaging
signals in genetic reassortment of influenza
A viruses
L13
Effectiveness of closure of live poultry
markets in reducing influenza A(H7N9)
transmission during the 2013-14 winter
wave
SPB1: VIRUS STRUCTURE AND REPLICATION
SPB102
L11
Seasonal influenza in the WHO European
Region: analysis of 2008-2013 influenza
surveillance data in 48 countries
Nathalie Bossuyt (1), Anneleen Hombrouck (2), Isabelle
Thomas (2), Françoise Wuillaume (1), Viviane Van
Casteren (1), Steven Van Gucht (2) , the Belgian Network
of sentinel general practitioners, the Belgian Network of
sentinel hospitals, Sophie Quoilin (1)
SPB101
L10
Schrauwen, Eefje J.A. (1); Richard, Mathilde (1); Herfst,
Sander (1); Bestebroer, Theo (1); Lexmond, Pascal (1);
Burke, David F. (2); Rimmelzwaan, Guus F. (1); Osterhaus,
Albert D.M.E. (1); Fouchier, Ron A.M. (1)
Isel, Catherine (1); Essere, Boris (2); Yver, Matthieu (2);
Gavazzi, Cyrille (1); Terrier, Olivier (2); Fournier, Emilie (1);
Giroux, Fabienne (2); Textoris, Julien (3); Julien, Thomas
(2,4); Socratous, Clio (2); Rosa-Calatrava, Manuel (2,4);
Lina, Bruno (2,5); Marquet, Roland (1); Moulès, Vincent
(2,4)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
III
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
SPA304
SPB2: VIRAL FACTORS IN PATHOGENESIS
SPB201
L15
Human cytotoxic T lymphocytes directed to
influenza B viruses cross-react with viruses of
two phylogenetic lineages
L16
SPB3: MATHEMATICAL MODELING
The Epidemiological Characteristics of
Influenza B Compared to Influenza A: Results
of the Global Influenza B Study
L23
Van De Sandt, Carolien E.; Dou, Yingying; Vogelzangvan Trierum, Stella E.; Westgeest, Kim B.; Osterhaus,
Albert D.M.E.; Fouchier, Ron A.M.; Hillaire, Marine L.B.;
Rimmelzwaan, Guus F.
Baumann, Jan; Monougu, Nancy; Klenk, Hans Dieter;
Matrosovich, Mikhail
SPB202
Single cell analysis reveals strain-specific
inhibition of ISG-expression by the viral NS1
protein in influenza A virus infected cells
SPB301
von Recum, Jessica; Weinheimer, Viola K; Wolff, Thorsten
L17
SPB203
Neuraminidase-negative avian influenza
viruses of subtype H5 and H7: Variation of
virulence in the chicken model
L19
SPB204
Reperant, Leslie (1); Kuiken, Thijs (1); Grenfell, Bryan (2);
Osterhaus, Albert (1)
SPB302
Hoffmann, Donata (1); Röhrs, Susanne (1); Bogs, Jessica
(1); Stech, Jürgen (2); Beer, Martin (1)
Assessment of transmission, pathogenesis
and adaptation of H2 subtype influenza
viruses using the ferret model
Markovic Delabre, Rosemary (1,2); Salez, Nicolas (3);
Lemaitre, Magali (4); Leruez-Ville, Marianne (5,6); de
Lamballerie, Xavier (3,7,8); Carrat, Fabrice (1,2,9)
SPB303
SPA3: IMMUNOLOGY
Wu, Joseph (1); Leung, Kathy (1); Perera, Ranawaka APM
(1); Chu, Daniel KW (1); Lee, Cheuk Kwong (2); Hung,
Ivan FN (3); Lin, Che Kit (2); Lo, Su-Vui (4); Lau, Yu-Lung
(5); Leung, Gabriel M (1); Cowling, Benjamin J (1); Peiris,
JS Malik (1)
CD8+ T cells correlate with protection against
symptomatic illness following natural
pandemic influenza infection in humans
SPA4: ANTIVIRALS AND RESISTANCE
L21
SPA401
L30
Influenza A(H7N9) virus gains neuraminidase
inhibitor resistance without loss of in vivo
virulence or transmissibility
Krammer, Florian
Polyclonal and Isotype-Specific Effects
of Hemagglutinin Stalk-Mediated
Neutralization
L29
Miller, Mark Andrew; Collaborating Team, MISMS
Exploring the humoral cross-reactome
against the influenza virus surface
glycoproteins proteins hemagglutinin and
neuraminidase
SPA303
SPB304
The MISMS program: highlights of an
international influenza research network
Sridhar, Saranya (1); Begom, Shaima (1); Bermingham,
Alison (2); Hoschler, Katja (2); Adamson, Walt (3);
Carman, William (3); Bean, Thomas (4); Barclay, Wendy
(1); Deeks, Jonathan (5); Lalvani, Ajit (1)
SPA302
L28
Inferring Influenza Infection Attack Rate
from Seroprevalence Data
L20
L26
Seroprotection threshold and persistence
of seroprotective titer among 2007H1N1
infected subjects: results from the Flurec
cohort study
Claudia Pappas, Hua Yang, Hui Zeng, Paul J. Carney,
James Stevens, Jacqueline M. Katz, and Terrence M.
Tumpey.
SPA301
L24
The immune response and within-host
emergence of pandemic influenza virus
L22
Hai, Rong (1); Schmolke, Mirco (1,2); Leyva-Grado, Victor
H. (1); Thangavel, Rajagowthamee R. (1); Margine, Irina
(1); Jaffe, Eric L. (1); Krammer, Florian (1); Solórzano,
Alicia (4); García-Sastre, Adolfo (1,2,3); Palese, Peter
(1,3); Bouvier, Nicole M. (1,3)
Miller, Matthew S.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
IV
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
L31
SPA402
L38
SPB404
Influenza antiviral susceptibility monitoring
in Europe – taking stock of 10 years’
experience, 2004-2014
Isolation of H15N7 influenza virus from wild
birds in Eastern Europe and its relationship to
viruses from other regions
Meijer, Adam (1,10); Lackenby, Angie (2,10); Pereyaslov,
Dmitriy (3); Enouf, Vincent (4,10); Kossyvakis, Athanasios
(5,10); Lina, Bruno (6,10); Rebelo-de-Andrade, Helena
(7,10); Pozo Sánchez, Francisco (8,10); Hay, Alan (9);
McCauley, John (9); Daniels, Rod (9); Zambon, Maria (2)
Muzyka, Denys (1);
Fereidouni, Sasan (3)
L33
SPA403
Evidence for clinical efficiency potential of
anti-H5N1 polyclonal immunoglobulins
Fabenflu®
L34
Takashita, Emi (1); Ejima, Miho (1); Fujisaki, Seiichiro (1);
Yokoyama, Masaru (2); Nakamura, Kazuya (1); Shirakura,
Masayuki (1); Sugawara, Hiromi (1); Sato, Aya (1); Sato,
Hironori (2); Tashiro, Masato (1); Odagiri, Takato (1)
SPA5: VACCINES: CURRENT AND NOVEL
APPROACHES
L35
de Vries, Rory Dylan (1); Kreijtz, Joost (1); Sutter, Gerd (2);
Osterhaus, Albert (1); Rimmelzwaan, Guus (1)
SPA503
SPA504
L36
European surveillance network for influenza
in pigs (ESNIP): Surveillance programs,
diagnostic tools and swine influenza viruses
identified in 14 European countries from
2010 to 2013
L42
Evaluation of heterosubtypic immune
responses in older people following prepandemic H5N1 influenza vaccination
M Levine (1)*, K Nicholson (2), S Batham (2), D Wang (1),
B Dighero-Kemp (1), ZN Li1, V Veguilla (1), F Liu1, XH Lu
(1), J Katz (1).
Simon, Gaëlle (1); Larsen, Lars E (2); Dürwald, Ralf (3);
Foni, Emanuela (4); Van Reeth, Kristien (5); Reid, Scott M
(6); Harder, Timm (7); Dan, Adam (8); Markowska-Daniel,
Iwona (9); Maldonado Garcia, Jaime (10); Huovilainen,
Anita (11); Billinis, Charalambos (12); Davidson, Irit (13);
Agüero Garcia, Montserrat (14); Bublot, Michel (15);
Brown, Ian H (6); Loeffen, Willie (16)
SPB5: GENETICS AND EVOLUTION OF VIRUS AND
HOST
L37
Pathobiology of novel genotype of H9N2
containing polymerase (PB2, PB1, PA) and
non-structural (NS) gene segments derived
from high pathogenicity H7N3 avian
influenza virus
L41
Specific induction of neutralizing antibodies
against HA, and T cell
responses against conserved influenza
antigens
Grødeland, Gunnveig (1); Mjaaland, Siri (2); Baranowska,
Marta (1); Fredriksen, Agnete (1); Bogen, Bjarne (1)
Gildea, Sarah; Cullinane, Ann
SPB403
L40
Mva-based influenza vaccines – bringing
these viral vectors from bench to bedside
Equine Influenza virus surveillance in Ireland
– findings of international
significance
SPB402
L39
Florek, Nicholas W. (1,2); Weinfurter, Jason T. (1,2);
Jegaskanda, Sinthujan (3); Brewoo, Joseph N. (4); Powell,
Tim D. (4); Young, Ginger R. (4); Das, Subash C. (4); Hatta,
Masato (1); Broman, Karl W. (1); Hungnes, Olav (5);
Dudman, Susanne G. (5); Kawaoka, Yoshihiro (1); Kent,
Stephen J. (3); Stinchcomb, Dan T. (4); Osorio, Jorge E.
(1,4); Friedrich, Thomas C. (1,2)
SPA502
SPB4: ANIMAL FLU-ECOLOGY AND EPIDEMIOLOGY
OF ANIMAL INFLUENZA
(2);
Modified vaccinia Ankara encoding influenza
hemagglutinin induces heterosubtypic
immunity in macaques
A community cluster of influenza A(H1N1)
pdm09 virus exhibiting cross-resistance
to oseltamivir and peramivir in Japan,
November 2013 to February 2014
SPB401
Mary
SPA501
Herbreteau, Cécile Hélène (1); Buchy, Philippe (2); Bal,
Céline (1); Durand, Caroline (1); Tambyah, Paul (3);
Lépine, Bertrand (1)
SPA404
Pantin-Jackwood,
SPB501
L43
Two independent evolutionary pathways of
HPAIV
Stech, Olga; Veits, Jutta; Abdelwhab, Sayed; Wessels,
Ute; Mettenleiter, Thomas C.; Stech, Juergen
Iqbal, Munir (1); Yaqub, Tahir (2); Mukhtar, Nadia (2);
Shabbir, Muhammad Z (2); McCauley, John W (3)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
V
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
L44
SPB502
Dynamics of influenza virus reassortment in
a co-infected host
Tao, Hui; Steel, John; Lowen, Anice
Mapping the antigenic drift of human
influenza A(H1N1) and B viruses
since the 1940s
Risk factors for severe outcomes and impact
of vaccination on pneumonia and influenza
within active US military populations 20002012
Van Kerkhove, Maria D (1); Cooper, Michael J (2); ErikCost, Angelia A (2); Sanchez, Jose L (2); Riley, Steven (1)
Koel, Björn
L46
High co-infection rates and multiplicity
reactivation facilitate incomplete gene
segment packaging in vivo
Huang, Q. Sue (1); Turner, N. (2); Baker, MG (3); McArthur,
C (4); Williamson, D (1,2,4); Grant, C (2); Roberts, S (4);
Aley, D (4); Trenholme, A (5); Conroy, C (5); Taylor, S
(5); Lawrence, S (5); Davey, K (5); Wood, T (1); Bissielo,
A (1); Radke, S (1); Bandaranayake, D (1); Seeds, R (1);
Mackereth, G (1); Todd, A (1); Pierse, N (3); Thomas, P (6);
Webby, R (6); Thompson, M (7); Duque, J (7); Gross, D (7);
Widdowson, MA (7)
SPA6: EVALUATION OF VACCINE SAFETY AND
EFFECTIVENESS
L47
SPA601
Live attenuated vaccines against potentially
pandemic influenza viruses: rationale for
genetic stability
Blak, Betina (1); Rajaram, Sankarasubramanian (1);
Steffey, Amy (2); Caspard, Herve (2)
L48
Mohn, Kristin G-I (1); Bredholt, Geir (1); Brokstad, Karl
(1); Aarstad, Hans Jørgen (2); Pathirana, Rishi (1); Tøndel,
Camilla (2); Cox, Rebecca Jane (1,2)
L49
Brown, Caroline
Michala
Sarah;
Hegermann-Lindencrone,
SPA701
L55
Flu vaccination of pregnant women in
Kazakhstan
Kuatbayeva, Ainagul Mukhanovna
Cowling, Benjamin; Chan, Kwok-Hung; Feng, Shuo;
Chan, Eunice; Peiris, Malik; Chiu, Susan
SPA702
L50
Influenza revaccination with MF59®adjuvanted or nonadjuvanted trivalent
inactivated seasonal influenza vaccines in
30–96 month-old children primed two years
previously
Pandemic preparedness in Europe – five years
on from the 2009 pandemic
SPA7: MATERNAL IMMUNIZATION
Effectiveness of influenza vaccination in
preventing hospitalizations in children in
Hong Kong, 2009-13
SPA604
L54
SPB604
Cellular immune responses elicited after
intranasal influenza vaccination in children,
a clinical trial
SPA603
L53
SPB603
Extension of the United Kingdom Influenza
Immunisation Programme 2013/2014 to
Children and the Implications for High-Risk
Group Uptake
Kiseleva, Irina (1); Larionova, Natalie (1); Dubrovina,
Irina (1); Bazhenova, Ekaterina (1); Fedorova, Ekaterina
(1); Isakova-Sivak, Irina (1); Kuznetsova, Victoria (1);
Stukova, Marina (2); Erofeeva, Marianna (2); Pisareva,
Maria (2); Desheva, Julia (1); de Jong, Jorgen (3); Ross,
Ted (4); Flores, Jorge (5); Tsvetnitsky, Vadim (5); Rudenko,
Larisa (1)
L52
SPB602
Southern hemisphere influenza and vaccine
effectiveness research and surveillance
(SHIVERS) in New Zealand
Brooke, Christopher Byron
SPA602
L51
SPB601
L45
SPB503
SPB504
SPB6: RISK MANAGEMENT AND MITIGATION
L56
The Norwegian Influenza cohort (NorFlu):
immune responses in pdmH1N1 exposed
versus non-exposed pregnant women
Savic, Miloje (1,4); Laake, Ida (2); Dembinski, Jennifer
(1,4); Tunheim, Gro (1,4); Oftung, Fredrik (1,4); Hungnes,
Olav (3); Næss, Lisbeth (1); Trogstad, Lill (2); Cox, Rebecca
(5); Mjaaland, Siri (1,4); Waalen, Kristian (3)
Vesikari, Timo (1); Forsten, Aino (1); Arora, Ashwani (2);
Tsai, Theodore (2); Clemens, Ralf (2)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
VI
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
SPB7: HOST FACTORS IN PATHOGENESIS
L57
SPB701
Quantitative proteomic analysis of protein
signatures in permissive vs. non-permissive
influenza A virus infections in human host
cells
L58
Gerlach, Thomas; Matrosovich, Tatyana; Klenk, HansDieter; Matrosovich, Mikhail
L59
The role of endothelial cells in influenza A
virus pathogenesis
PL0504
L64
Ghoneim, Hazem E. (1); Smith, Amber M. (1); McCullers,
Jonathan A. (1,2)
L60
L63
Comparison of aerosol and intra-nasal
challenge with 2009 pandemic influenza
virus (H1N1) in cynomolgus macaques
showed clear differences in the distribution
of virus
Secondary Bacterial Pneumonia Suppresses
Pulmonary CD8 and CD4 T Cell Responses
after Influenza Infection
Short, Kirsty Renfree (1); Kasper, Jennifer (2); Fouchier,
Ron (1); Kirkpatrick, Charles (2); Kuiken, Thijs (1)
1: Erasmus MC, the Netherlands; 2: Department of
Pathology, Medical University of Mainz, Mainz, Germany
SPB704
Nakatsu, Sumiho (1); Sagara, Hiroshi (2); Noda, Takeshi
(1,3); Kawaoka, Yoshihiro (1,4,5)
Marriott, Anthony Colin (1), Sally Sharpe (1), Mike Dennis
(1), Jennifer Kane (1), Claudia Prevosto (1), Nigel Silman
(1), Emma Rayner (1), Simon Bate (1), Saranya Sridhar
(2), Karl J. Staples (3), Ajit Lalvani (2), Tom Wilkinson (3),
Miles W. Carroll (1)
Inhibition of avian and human influenza
viruses by human airway mucins
SPB703
Understanding the genome packaging
mechanism of influenza viruses
PL0503
Sadewasser, Anne (1); Paki, Katharina (1); Eichelbaum,
Katrin (2); Selbach, Matthias (2); Wolff, Thorsten (1)
SPB702
L62
PL0502
Type I interferon receptor 2 (Ifnar2) plays an
important role in limiting inflammation and
disease following influenza virus infection
PL0505
L65
Pathogenicity of airborne-transmissible
H5N1 viruses.
Mathilde Richard, Sander Herfst, Judith van den Brand,
Pascal Lexmond, Theo Bestebroer, Ab Osterhaus, Thijs
Kuiken and Ron Fouchier
Tate, Michelle D; Dowling, Jennifer K; Piganis, Rebecca
A; Hertzog, Paul J
PL05: LATE BREAKERS
PL0501
L61
Post-pandemic Review of anti-Influenza Drug
Effectiveness (PRIDE Study): an investigation
of the impact of neuraminidase inhibitor
antiviral use on pneumonia and length
of hospital stay in hospitalized influenza
A(H1N1)pdm09 patients
Muthuri, Stella; Myles, Puja; Venkatesan, Sudhir;
Leonardi-Bee, Jo; Nguyen-Van-Tam, Jonathan
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
VII
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
POSTER ABSTRACTS
SPA1P06
SPA1: CLINICAL IMPACT AND DIAGNOSTIC
APPROACHES
COMPARATIVE ANALYSIS OF mariPOC SYSTEM
AND rRT-PCR IN DETECTION OF INFLUENZA A
VIRUS IN CLINICAL SAMPLES
P2
SPA1P01
Clinical usefulness of the BD Veritor System
for Rapid Detection of Flu A+B to detect
influenza viruses
Diagnosis of human influenza A virus
infection using single chain fragment variable
antibody-based competitive inhibition ELISA
Rajput, Roopali (1); Sharma, Gaurav (2); Saxena, Latika
(1); Khanna, Madhu (1); Pradhan, Hare Krishna (3);
Pattnaik, B. (2)
Hospitalizations with influenza in the
northern hemisphere 2013-2014 influenza
season. Results from the Global Influenza
Hospital Surveillance Network
SPA1P09
Influenza in pregnant women during 20122014, Moscow, Russia
P14
Epidemiology of patients infected by
Influenza B viruses in a northern Spanish
population during 2012-2013 Influenza
season
P8
Aushakhmetova, Zabira T.; Kiyanbekova, Lyazzat S.; Abeev,
Arman B.; Demesinova., Balzira M.; Kasenova, Zhulduz K.
Ferro Bricks, Lucia (1); Falleiros, Luiza Helena (2);
Mascareñas, Cesar (3); el Guerche-Séblain, Clotilde (4)
SPA1P10
Trushakova, Svetlana (1); Kisteneva, Lidiya (1,2);
Kruzhkova, Irina (2); Mukasheva, Evgeniya (1);
Krasnoslobodtsev, Kirill (1); Siluyanova, Elina (1); Garina,
Ekaterina (1); Kolobukhina, Ludmila (1,2); Burtseva, Elena
(1)
Laboratory monitoring of influenza like
illnesses (ILI) and severe acute respiratory
infections (SARI) in the flu sentinel
surveillance system in the Republic of
Kazakhstan during last three seasons – 20112014
P13
Influenza A and B burden in Latin American
countries
P6
SPA1P05
P11
Importance of Influenza infection in the
etiology of exacerbations of COPD during a
singular pandemic and post-pandemic period
Sanz, Iván (1,2); Tamames, Sonia (1,3); Rojo, Silvia (1,2);
Justel, Mar (2); Vega, Tomás (3); Ortiz de Lejarazu, Raúl
(1,2)
Puig-Barberà, Joan (1); Tormos, Anita Marie Catherine
(1); Sominina, Anna (2); Ciblak, Meral (3); Mira-Iglesias,
Ainara (1); Natividad-Sancho, Angels (1); Buigues-Vila,
Amparo (1); Burtseva, Elena (4)
SPA1P04
Grover, Sandeep (1); Weick, Anja (1); Obermeier, Patrick
(1); Chen, Xi (1); Seeber, Lea (1); Tief, Franziska (1);
Karsch, Katharina (1); Muehlhans, Susann (1); Hoppe,
Christian (1); Adamou, Eleni (1,2); Behrens, Stephanie
(1); Reiche, Janine (2); Schweiger, Brunhilde (2); Rath,
Barbara (1)
SPA1P08
P4
SPA1P03 P10
Seizure following respiratory viral infection
versus immunization in infants and children
– a syndromic surveillance study
P3
Petrova, Ekateria; Krivitskaya, Vera; Pisareva, Maria;
Buzitskaya, Zhanna; Sukhovetskaya, Vera; Danilenko,
Daria; Suddenkova, Polina; Sominina, Anna A.
SPA1P07
Noh, Ji Yun (1); Choi, Won Suk (1); Lee, Jacob (2); Seo, Yu
Bin (2); Lee, Jung Hwa (3); Song, Joon Young (1); Cheong,
Hee Jin (1); Kim, Woo Joo (1,4)
SPA1P02
P9
Ortiz de Lejarazu, Raúl (1,2); Rojo, Silvia (1,2); López,
Irene (2); López, Cristina (2); Tamames, Sonia (1,3); Sanz,
Iván (1,2)
SPA1P11
P15
Co-circulation of two lineages of influenza B
viruses in South Korea
Noh, Ji Yun (1); Lee, Han Sol (2); Song, Joon Young (1);
Choi, Won Suk (1); Lee, Jacob (3); Seo, Yu Bin (3); Lee, JinSoo (4); Wie, Seong-Heon (5); Jeong, Hye Won (6); Kim,
Young Keun (7); Park, Kyung Hwa (8); Kim, Shin Woo (9);
Lee, Sun Hee (10); Lee, Jung Hwa (11); Kim, Dong Hyun
(12); Woo, Sung Il (13); Lim, Chae Seung (14); Cho, Kyung
Soon (15); Cheong, Hee Jin (1); Kim, Woo Joo (1,16)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
VIII
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P16
SPA1P12
Factors that influence the clinical outcome in
patients admitted with influenza like illness
and the effect of glucocorticoid treatment
SPA2: EPIDEMICS AND PANDEMIC THREATS
Tanriover, Mine Durusu (1); Ozisik, Lale (1); Akcay Ciblak,
Meral (2); Yurtcu, Kubra (2); Unal, Serhat (3); Badur,
Selim (2)
P18
SPA1P13
Bo Shu, Kai-Hui Wu, William Davis, LaShondra Berman,
Christine Warnes, Shannon Emery, Nathelia Barnes,
Lakshmi Malapati, Julie Villanueva and Stephen
Lindstrom
P19
Shieh, Happy K. (1); Cheng, Ming-Chu (2); Chang, PoaChun (1)
P21
Replication of MDCK-adapted viruses with
different receptor specificity in vitro and in
vivo
Eropkin, Mikhail; Danilenko, Daria; Konovalova, Nadejda;
Komissarov, Andrey; Pisareva, Maria; Grudinin, Mikhail;
Lobova, Tamara; Schekanova, Svetlana; Kornilova,
Ekaterina; Eropkina, Elena; Suddenkova, Polina
P29
SPA2P05
Excess mortality risk attributable to
influenza infection in Hong Kong, 1998-2012
Kuznetsova, Victoria; Isakova-Sivak, Irina; Rudenko,
Larisa
Wu, P (1); Goldstein, E (2); Ho, LM (1); Wu, JT (1); Ip, DKM
(1); Leung, GM (1); Cowling, BJ (1)
P22
Structural insight into extracellular domain
of matrix protein 2 of influenza A virus
P30
SPA2P06
Community psychological and behavioral
responses to the threat of A(H7N9) in Hong
Kong
Kim, Kyung Hyun (1); Cho, Ki Joon (1,2,3); Schepens, Bert
(2,3); Seok, Jong Hyeon (1); Kim, Sella (1); Rose, Kenny
(2,3); Lee, Ji-Hye (1); Gallardo, Rodrigo (4); Schymkowitz,
Joost (4); Rousseau, Frederic (4); Fiers, Walter (2,3);
Saelens, Xavier (2,3); Chung, Mi Sook (5)
Wu, P; Fang, VJ; Liao, Q; Ng, DMW; Wu, JT; Leung, GM;
Fielding, R; Cowling, BJ
P24
da Silva, Diogo V; Nordholm, Johan; Dou, Dan; Daniels,
Robert
P28
SPA2P04
INFLUENZA EPIDEMIC SEASON 2013-2014
IN RUSSIA: CHARACTERISTICS OF ISOLATED
STRAINS
Application of next-generation sequencing
technologies to study the evolution of H5N2
avian influenza viruses in Taiwan
The transmembrane domain of influenza NA
has co-evolved with the distal head domain
P27
Van Kerkhove, Maria D (1); Laurie, Karen (2); Engelhardt,
Othmar (3); Wood, John (4); ., CONSISE (5)
P20
SPB1P04
Wong, Jessica; Tsang, Tim K.; Wu, Peng; Lau, Eric H. Y.; Ip,
Dennis K. M.; Cowling, Benjamin J.
Improving Standardisation and Timeliness
of Seroepidemiological Studies through the
Global Partnership CONSISE (the Consortium
for the Standardization of Influenza
Seroepidemiology)
SPB1: VIRUS STRUCTURE AND REPLICATION
SPB1P03
Real-time estimation of the hospitalization
fatality risk of influenza A(H1N1)pdm09 in
Hong Kong
SPA2P03
Kai-Hui Wu, C. McCord, B. Shu, C. Warnes, and S.
Lindstrom
SPB1P02
P26
SPA2P02
Performance validation of CDC real-time
RT-PCR influenza assays on the AB TaqMan
Array Card
SPB1P01
Application of next-generation sequencing
technologies to study the evolution of H5N2
avian influenza viruses in Taiwan
Wong, Jessica Y. (1); Kelly, Heath (2,3); Cheung, ChungMei M. (4); Shiu, Eunice Y. (1); Wu, Peng (1); Ni, Michael Y.
(1); Ip, Dennis K. M. (1); Cowling, Benjamin J. (1)
Differentiation of influenza A(H3N2) variant
viruses from human seasonal A(H3N2)
viruses by real-time RT-PCR
SPA1P14
P25
SPA2P01
SPA2P07
P31
The avian origin H9N2 polymerase, common
to multiple human lethal strains, possesses
robust activity in mammalian cells
Cox, Andrew George (1,2); Kim, Yoel (1); O’Dell, Colleen
(1); Schmierer, Jordana (1); Smith, Andrew (1,2);
Dewhurst, Stephen (1)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
IX
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P32
SPA2P08
The Global Influenza B Study (GIBS): a
Research Platform for the Study of Influenza
B
Transmission of the human influenza virus
A/NL/602/09, but not the triple reassortant
A/swine/Kansas/77778/07, occurs efficiently
from low infectious dose in guinea pigs
Paget, John (1); Caini, Saverio (1); Huang, Sue (2); Ciblak,
Meral (3); Schellevis, Francois (1); Plotkin, Stanley (4)
P33
SPA2P09
Possible explanations for why some countries
were harder hit by Influenza A(H1N1)pdm09
– a global mortality impact modeling study
Evolution of highly pathogenic avian
influenza (H5N1) virus of domestic poultry in
Vietnam between 2011 and 2013
P40
SPB2P01
P35
ANALYSIS OF INFLUENZA SENTINEL
SURVEILLANCE RESULTS IN KAZAKHSTAN,
2013-2014 EPIDEMIOLOGICAL SEASON
Relative role of individual complement
pathway during influenza infection
Rattan, Ajitanuj
P40
SPB2P02
Flt3 signaling is critical for host response
after influenza A infection
Ozhanova, Alfiya Ibiraimovna; Mirzabekova, Gulfairuz
Kuralbekovna
P36
SOME APPROACHES TO PREDICTION OF
SEASONAL INFLUENZA EPIDEMIC BURDEN
Kühn, Nora (1); Hatesuer, Bastian (1); Dengler, Leonie
(1); Wilk, Esther (1); Schäkel, Friederike (1); Pils, Marina
(2); Beutler, Bruce (3); Schughart, Klaus (1)
SPB2P03
Sominina, Anna A.; Karpova, Ludmila; Smorodintseva,
Elizaveta; Grudinin, Mikhail; Pisareva, Maria;
Komissarov, Andrei; Eropkin, Mikhail; Konovalova,
Nadejda; Danilenko, Daria
P37
SPA2P13
Hooiveld, Mariëtte (1); Donker, Gé (1); Meijer, Adam (2);
Zock, Jan-Paul (1); Schellevis, François (1)
SPB2: VIRAL FACTORS IN PATHOGENESIS
Lee, Eun-Kyoung (1); Kang, Hyun-Mi (1); Song, ByungMin (1); Jeung, Jipseol (1); Choi, Jun-Gu (1); Thanh, Long
To (2); Tho, Dang Nguyen (2); Choi, Kang-Seuk (1); Kye,
Soo-Jeong (1); Kim, Ji-Ye (1); Lee, Hee-Soo (1); Lee, YounJeong (1)
SPA2P12
P39
Influenza surveillance in the Netherlands: a
new system covering 5% of the population
that uses Electronic Medical Records
P34
SPA2P11
Campbell, Patricia J. (1); Danzy, Shamika (1); Ramos,
Irene (2); Fernandez-Sesma, Ana (2); Lowen, Anice C. (1);
Steel, John (1)
SPA2P15
Morales, Kathleen (1,2); Paget, John (1); Spreeuwenberg,
Peter (1)
SPA2P10
P38
SPA2P14
P41
H7N9 and H6N1 influenza A virus
hemagglutinins engineered to bind human
type receptors reveal a novel layer of
specificity beyond the α2-6 linkage of sialic
acid
Influenza surveillance systems in Europe – a
recent survey update
de Vries, Robert P; Peng, Wenjie; McBride, Ryan; Paulson,
James C
Meerhoff, Tamara (1); Vermaire, Jorien (2); Jorgensen,
Pernille (2); Gross, Diane (2); Pereyaslov, Dmitriy (2);
Brown, Caroline (2)
SPB2P04
P42
Activation of coagulation and tissue fibrin
deposition in experimental influenza in
ferrets
Goeijenbier, Marco (1); van Gorp, Eric C.M. (1); van den
Brand, Judith (1); Stittelaar, Koert (2); Bakthiari, Kamran
(2); Roelofs, Joris T.H. (2); van Amerongen, Geert (1);
Kuiken, Thijs (1); Martina, Byron (1); Meijers, Joost C.M.
(3); Osterhaus, Albert D.M.E. (1)
SPB2P05
P43
Analysis of the host response to influenza
A virus infection in the Collaborative Cross
founder strains
Leist, Sarah R. (1); Pilzner, Carolin (1); Kollmus, Heike (1);
Schughart, Klaus (1,2,3)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
X
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P44
SPB2P06
P51
SPB2P13
Type I interferon-mediated signaling
is inhibited upon influenza A virus and
Staphylococcus aureus co-infection
Ubiquitination of the influenza A virus
PB1-F2 protein is crucial for its biological
functions
Warnking, Kathrin; Klemm, Carolin; Löffler, Bettina;
Niemann, Silke; Peters, Georg; Ludwig, Stephan;
Ehrhardt, Christina
Kosik, Ivan (1); Praznovska, Margareta (1); Kosikova,
Martina (1); Vareckova, Eva (1); Kostolansky, Frantisek (1);
Polakova, Katarina (2); Russ, Gustav (1)
SPB2P07
P45
Saelens, Xavier; De Baets, Sarah; Verhelst, Judith;
Van den Hoecke, Silvie; Smet, Anouk; Roose, Kenny;
Schotsaert, Michael; Schepens, Bert; Fiers, Walter
Klemm, Carolin (1); Warnking, Kathrin (1); Löffler,
Bettina (2); Peters, Georg (2); Ludwig, Stephan (1);
Ehrhardt, Christina (1)
P46
SPB2P08
Shin, Dai-Lun (1,2); Hatesuer, Bastian (1); Schughart,
Klaus (1,2,3)
Mooij, Petra; Koopman, Gerrit; Mortier, Daniella; van
Heteren, Melanie; Fagrouch, Zahra; de Laat, Rudy;
Remarque, Edmond J.; Kondova, Ivanela; Verschoor,
Ernst J.; Bogers, Willy M.J.M.
Insights into the interaction of influenza
A virus RNA polymerase with cellular RNA
polymerase II
P48
High basal expression of interferonstimulated genes in transformed human
bronchial epithelial (BEAS-2B) cells
contributes to influenza A virus resistance
P49
van Riel, Debby (1); Leijten, Lonneke M. (1); Verdijk, Rob
M. (2); GeurtsvanKessel, Corine (1); van der Vries, Erhard
(1); van Rossum, Annemarie M.C. (3); Osterhaus, Albert.
D.M.E. (1); Kuiken, Thijs (1)
Pantin-Jackwood, Mary Josephine; Costa-Hurtado, Mar;
Afonso, Claudio; Miller, Patti; Shepherd, Eric; Smith,
Diane
Virus dose-dependent neutrophil and
lymphocyte proportions in peripheral blood
during influenza A infection of mice
Kostolansky, Frantisek (1); Dugovicova, Veronika (1);
Janulikova, Jana (1); Mucha, Vojtech (1); Mistrikova, Jela
(2); Vareckova, Eva (1)
P57
Heterogeneous pathological outcomes after
experimental pH1N1 influenza infection in
ferrets correlate with viral replication and
host immune responses in the lung.
P58
SPB2P19
P50
Experimental co-infection of chickens with
lentogenic, mesogenic and velogenic strains
of Newcastle disease viruses and highly
pathogenic avian influenza viruses
P56
Vidana, Beatriz (1,3); Martínez, Pamela (3); Martínez,
Jorge (1,3); Montoya, María (3,4); Martorell, Jaime (2); G.
Migura, Lourdes (3); Majó, Natàlia (1,3)
The olfactory nerve: A shortcut for influenza
viruses into the CNS in humans !?
SPB2P12
Janulikova, Jana; Bobisova, Zuzana; Mucha, Vojtech;
Kostolansky, Frantisek; Vareckova, Eva
SPB2P18
Seng, Lai-Giea; Daly, Janet; Chang, Kin-Chow; Kuchipudi,
Suresh
SPB2P11
Genetic background influences the antiviral
activity of Mx1 gene to influenza virus
infection in mice
SPB2P17
Martínez-Alonso, Mónica; Hengrung, Narin; Vreede,
Frank; Fodor, Ervin
SPB2P10
P55
SPB2P16
P47
P54
SPB2P15
Genetic background influences the antiviral
activity of Mx1 gene to influenza virus
infection in mice
Pandemic swine-origin H1N1 influenza virus
replicates to higher levels and induces more
fever and acute inflammatory cytokines in
Cynomolgus versus Rhesus monkeys and can
replicate in Common Marmosets
SPB2P09
P53
SPB2P14
In vivo Virus Tropism and Protection by
a Matrix Protein 2 Ectodomain-Specific
Monoclonal Antibody revealed by a Novel
GFP-expressing Influenza A Virus
Lipoteichoic acid of Staphylococcus aureus
as main contributor to the enhancement of
influenza A virus induced mitogen-activated
protein kinase signaling
Species-specific host factors for Influenza A
Virus replication
Martin-Sancho, Laura (1); Karlas, Alexander (1); Imai, Aki
(1); Barclay, Wendy (2); Meyer, Thomas F. (1)
SPB2P20
P59
The relevance of cell cycle manipulation of
influenza A virus to virus pathogenicity
Xu, Ke
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XI
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P60
SPB2P21
TMPRSS2 is a host cell factor that is essential
for pneumotropism and pathogenicity of
H7N9 and H1N1 influenza A virus in mice
The adaptation of avian influenza viruses to
the respiratory epithelium of pigs
Yang, Wei (1); Meng, Fandan (1); Punyadarsaniya,
Darsaniya (2); Hoffmann, Markus (1); Stech, Juergen
(3); Hoeper, Dirk (3); Beer, Martin (3); SchwegmannWessels, Christel (1); Ren, Xiaofeng (4); Herrler, Georg (1)
Tarnow, Carolin (1); Engels, Geraldine (2); Arendt, Annika
(1); Schwalm, Folker (1); Sediri, Hanna (1); Garten,
Wolfgang (1); Klenk, Hans-Dieter (1); Gabriel, Gülsah
(2); Böttcher-Friebertshäuser, Eva (1)
P61
SPB2P22
SPB2P29
The effect of Streptococcus suis co-infection
on the infection of well-differentiated
porcine respiratory epithelial cells by swine
influenza viruses
Streptococcus suis affects the replication of
swine influenza virus in porcine tracheal cells
Wu, Nai-Huei (1); Meng, Fandan (1); Seitz, Maren (2);
Valentin-Weigand, Peter (2); Herrler, Georg (1)
P63
Virus and host determinants of influenza A
virus restriction by human MxA
Patzina, Corinna; Riegger, David; Haller, Otto; Kochs,
Georg
Viral replication kinetics and pathogenesis
of pandemic H1N1 and H7N9 influenza virus
infection in isogenic guinea pigs
P69
PI3K-gamma activation during influenza
A infection induces type I and type III IFN
responses and resolution of inflammation via
p38 activation
Garcia, Cristiana Couto (1); Tavares, Luciana Pádua (1);
Dias, Ana Carolina Fialho (1); Queiroz-Junior, Celso (1);
Lima, Braulio Henrique Freire (1); Machado, Alexandre
Magalhães Vieira (2); Sousa, Lirlândia Pires (1); Russo,
Remo Castro (1); Teixeira, Mauro Martins (1)
P70
Replicative Fitness of Avian Influenza
A(H7N9) Virus Variants with Reduced
Susceptibility to Neuraminidase Inhibitors in
Mice and Ferrets
Henju Marjuki (1), Vasiliy P. Mishin (1), Anton P.
Chesnokov (1), (2), Joyce Jones (1), Juan A. De La Cruz
(1), (2), Katrina Sleeman (1), Ho-Sheng Wu3, Feng-Yee
Chang3, Ming-Tsan Liu3, Alicia M. Fry (1), Nancy J. Cox
(1), Julie M. Villanueva (1), Charles T. Davis (1), Larisa V.
Gubareva (1)
Wiersma, Lidewij (1); Kreijtz, Joost (1); van Trierum,
Stella (1); Nieuwkoop, Nella (1); van Run, Peter (1); van
Amerongen, Geert (1); Ladwig, Mechthild (2); Banneke,
Stefanie (2); Schaefer, Hubert (3); Osterhaus, Ab (1);
Rimmelzwaan, Guus (1)
SPB2P26
SPB2P30
SPB2P31
P64
SPB2P25
P68
Bertram, Stephanie (1); Schwalm, Folker (2); Preuß,
Annette (1); Klenk, Hans-Dieter (2); Gabriel, Gülsah (1)
P62
SPB2P23
H7N9 influenza A viruses exhibit importin-α
mediated replication in the mammalian
respiratory tract
Meng, Fandan (1); Wu, Nai-Huei (1); Seitz, Maren (2);
Valentin-Weigand, Peter (2); Ren, Xiaofeng (3); Herrler,
Georg (1)
SPB2P24
P67
SPB2P28
P65
The contribution of the PB1-F2 protein to
viral virulence and pathogenesis of avain
influenza virus
SPA3: IMMUNOLOGY
James, Joe (1,2); Moncorgé, Olivier (2); Barclay, Wendy
(2); Shelton, Holly (1)
SPA3P01
P66
SPB2P27
Hemagglutinin 222D/G polymorphism of
pandemic influenza A (H1N1) 2009 viruses
enables fast intra-host evolution and causes
severe biphasic influenza in mice
Seidel, Nora; Sauerbrei, Andreas; Wutzler, Peter;
Schmidtke, Michaela
P71
Back-boost of antibody landscapes after
influenza virus infection or vaccination
Fonville, Judith (1,2); Wilks, Sam (1,2); James, Sarah
(1); Aban, Malet (3); Xue, Lumin (3); Jones, Terry (1,2);
Ventresca, Mario (1); Fox, Annette (4); Nguyen Minh
Hoa, Le (4); Quang Thai, Pham (5); Nhu Duong, Tran (5);
Wong, Yan (1); Mosterin, Ana (1,2); Katzelnick, Leah (1,2);
van der Net, Guido (6); Skepner, Eugene (1,2); Russell,
Colin (2,7); Kaplan, Todd (8); Rimmelzwaan, Guus (6);
Masurel, Nic (6); de Jong, Jan (6); Palache, Abraham (9);
Beyer, Walter (6); Quynh Mai, Le (5); Tran Hien, Nguyen
(5); Wertheim, Heiman (4); Hurt, Aeron (3); Osterhaus,
Ab (6); Barr, Ian (3); Fouchier, Ron (6); Horby, Peter (4);
Smith, Derek (1,2)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XII
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P73
SPA3P02
SPA3P09
Exposure history or immunosenescence –
what determines the antibody response to
influenza vaccine in elderly persons?
Antibody response against unchanged
influenza vaccine components in elderly
people vaccinated in consecutive years
Mosterín Höpping, Ana (1,4); McElhaney, Janet (2);
Fonville, Judith (1,4); Beyer, Walter (3); Smith, Derek
(1,4,3)
Basileo, Michela (1); Iorio, Anna Maria (1); Bartolini,
Guido (2); Tozzi, Paolo (3); Camilloni, Barbara (1)
P75
SPA3P03
Serodiagnostic studies during the
development of new pandemic caused
A(H1N1)pdm09.
P82
Searching for a universal biomarker of
human T-cell responses to Influenza A virus
infections
Dembinski, Jennifer Lynn (1,3); Oftung, Fredrik (1,3);
Savic, Miloje (1,3); Kim, Yohan (2); Peters, Bjoern (2);
Mjaaland, Siri (1,3)
Mukasheva, Evgeniya (1); Kolobukhina, Ludmila
(1); Kisteneva, Lidiya (1); Zaplatnikov, Andrey (2);
Smolonogina, Tatyana (3); Desheva, Yuliya (3); Burtseva,
Elena (1)
SPA3P11
P76
SPA3P04
SPA3P10
P81
Avian myotubes are highly susceptible to
influenza virus infection and replication
Chang, Kin-Chow (1); Baquero-Pereza, Belinda (1);
Kuchipudi, Suresh V. (1); Ho, Jemima (1); Sebastian,
Sujith (1); Brookesb, Sharon, M. (2); Brownb,
Ian H. (2)
P83
LPS-modified outer membrane vesicle
(fmOMV) adjuvant provides protective
innate immunity against influenza infection
prior to the induction of vaccine-induced
adaptive immune response
Bae, Eun Hye; Lee, Tae Young; Lee, Sang Ho; Yeom, Min
Joo; Na, Woon Seong; Kim, Chang Ung; Song, Dae Sub;
Kim, Doo Jin; Kim, Sang Hyun
P77
SPA3P05
Investigating B and T cell repertoire
signatures in response to influenza infection
SPB3: MATHEMATICAL MODELING
Lu, I-Na; Farinelle, Sophie; Muller, Claude P.
P78
SPA3P06
Influenza virus infection in quail (Coturnix
coturnix): characterization of humoral
immune response
P79
Humoral and cellular immune responses
following the 2009 pandemic
Petukhova, Galina; Kuznetsova, Svetlana; Losev, Igor;
Isakova-Sivak, Irina; Kuznetsova, Victoria; Korenkov,
Daniil; Rudenko, Larisa; Naykhin, Anatoly
P86
The relationship between school holidays
and transmission of influenza in England and
Wales 1967-2008
Jackson, Charlotte (1,2,3); Vynnycky, Emilia (1,2);
Mangtani, Punam (1)
P80
Contribution of single mutations specific
for A/Leningrad/134/17/57 (H2N2) master
donor virus in influenza A virulence and
immunogenicity in a mouse model
Tempia, Stefano (1,2,3); Walaza, Sibongile (3); Cohen,
Adam (1,2); von Mollendorf, Claire (3); Moyes, Jocelyn
(3,4); Mhlanga, Sarona (3); McAnerney, Johanna (3);
Cohen, Cheryl (3,4)
SPB3P02
Oftung, Fredrik (1); Korsvold, Gro Ellen (1); Dembinski,
Jennifer (1); Tunheim, Gro (1); Savic, Miloje (1);
Kristoffersen, Anne Cathrine (1); Haneberg, Bjørn (1);
Germundsson Hauge, Anna (1); Kim, Yohan (2); Peters,
Bjoern (2); Berdal, Jan Erik (4); Monceyron Jonassen,
Chrstine (3,4); Mjaaland, Siri (1,5)
SPA3P08
P84
Mortality Associated with Seasonal and
Pandemic Influenza among Pregnant and
Non-Pregnant Women of Childbearing Age in
a High HIV-Prevalence Setting – South Africa,
1999-2009
Majo, Natalia (1,2); Busquets, Núria (1); Garcia, Beatriz
(1); Bensaid, Albert (1); Dolz, Roser (1); Oliver, Salvador
(1); Bertran, Kateri (1); Ramis, Antoni (1,2); Rivas, Raquel
(1)
SPA3P07
SPB3P01
SPB3P03
P87
A drug-disease model to investigate
the effect of combination therapy with
oseltamivir on influenza virus progression
Kamal, Mohamed A (1); Gieschke, Ronald (2); LemenuelDiot, Annabelle (2); Beauchemin, Catherine AA (3); Smith,
Patrick F (4,5); Rayner, Craig R (4,6)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XIII
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P89
SPB3P04
Dynamic modeling of the cost-effectiveness
of sick leave practices for influenza illness
SPA4P08
Comparative analysis and anti-viral activity
of medicinal plant extracts against Influenza
A virus: An in-vitro study.
Edwards, Christina Hansen (1); de Blasio, Birgitte
Freiesleben (1,2); Scalia Tomba, Gianpaolo (3)
Roy, Saugata; Saxena, Latika; Kumar, Binod; Khanna,
Madhu
SPA4P09
SPA4: ANTIVIRALS AND RESISTANCE
P90
SPA4P01
Evaluation of heterosubtypic crossprotection by active infection and TIVvaccination of human seasonal influenza
virus in ferret models
Oseltamivir inhibits influenza virus
replication and transmission following
ocular-only aerosol inoculation of ferrets
P92
Vidana, Beatriz (1,3); Martorell, Jaime (2); Martínez,
Jorge (1,3); Martínez, Pamela (3); G. Migura, Lourdes
(3); córdoba, Lorena (3); Casas, Inmaculada (5); Pozo,
Francisco (5); Majó, Natàlia (1,3); Montoya, María (3,4)
P93
Is it possible to fight influenza by targeting
intracellular redox state?
P101
Hemagglutination (HA)-based
neuraminidase (NA) inhibitory assays to
search for novel NA inhibitors (NAIs)
P94
Neuraminidase Inhibitors and the Cochrane
Reviews – a critical appraisal
Richter, Martina (1); Walther, Elisabeth (1); Bohn,
Kathrin (1); Xu, Zhongli (1); Grienke, Ulrike (2); Rollinger,
Judith M. (2); von Grafenstein, Susanne (3); Liedl, Klaus
R. (3); Kirchmair, Johannes (4); Sauerbrei, Andreas (1);
Schmidtke, Michaela (1)
SPA4P13
Lehnert, Regine Magdalene; Matz, Sibylle Renate
P95
SPA4P06
P100
Lina, Bruno (1); Boucher, Charles (2); Osterhaus, Albert
(2); Schutten, Martin (2); Monto, Arnold (3); Whitley,
Richard J (4); Nguyen-Van-Tam, Jonathan (5)
SPA4P12
Palamara, Anna Teresa (1,2); Sgarbanti, Rossella (3);
Amatore, Donatella (1,4); Celestino, Ignacio (1,4);
Fraternale, Alessandra (5); Magnani, Mauro (5); Garaci,
Enrico (3); Nencioni, Lucia (1)
SPA4P05
SPA4P11
Five years of monitoring for the emergence
of oseltamivir resistance in patients with
influenza A virus infections in the Influenza
Resistance Information Study (IRIS)
Oseltamivir resistant and wild type pH1N1
virus infection in ferrets
SPA4P04
P99
Novel inhibitors targeting influenza virus and
pneumococcal neuraminidase (NA)
Walther, Elisabeth (1); Richter, Martina (1); Xu,
Zhongli (1); Bohn, Kathrin (1); Rollinger, Judith (2); von
Grafenstein, Susannne (3); Liedl, Klaus (3); Kirchmair,
Johannes (4); Sauerbrei, Andreas (1); Pfister, Wolfgang
(5); Schmidtke, Michaela (1)
Belser, Jessica A; Maines, Taronna R; Creager, Hannah M;
Katz, Jacqueline M; Tumpey, Terrence M
SPA4P03
Viral shedding and susceptibility
to oseltamivir in hospitalised
immunocompromised patients with
influenza in the Influenza Resistance
Information Study (IRIS)
SPA4P10
P91
P98
Fraaij, Pieter (1); Schutten, Martin (1); Javouhey, Etienne
(2); Burleigh, Laura (3); Outlaw, Russell (4); Kumar,
Deepali (5); Boucher, Charles (1)
Choi, Young-Ki
SPA4P02
P97
Antiviral drugs for influenza -A
comprehensive review of the evidence with
emphasis on relevant clinical endpoints
P102
Identification of amino acids important for
binding of novel oseltamivir and zanamivir
derivatives by using recombinant influenza
viruses
Hoffmann, Anja (1); Schade, Dennis (2); Kirchmair,
Johannes (3); Sauerbrei, Andreas (1); Schmidtke,
Michaela (1)
Lehnert, Regine Magdalene (1); Pletz, Mathias W (2);
Schaberg, Tom (3); Reuss, Annicka (4); Haas, Walter (4)
P96
SPA4P07
Heterologous Polyclonal Antibodies, Past and
Present, with Future Applications to Avian
Influenza and Other Neglected Viruses.
Dixit, Rashmi (1); Herbreteau, Cécile Hélène (2); Herz,
Jenny (3); Booy, Robert (1); Lepine, Bertrand (2)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XIV
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P103
SPA4P14
P110
SPB4P05
Antiviral effect of new oseltamivir derivates
against influenza A virus
Serological monitoring of equine influenza
after vaccination in Korea in 2013
García-Machorro, Jazmín (1); Neri-Bazan, Rocio M. (1);
Correa-Basurto, José (1); Trujillo-Ferrara, José G. (1);
Tolentino-López, Luis (1); Martínez-Ramos, Federico (2);
Velazquez-Quiroz, Isaac (3); Jímenez-Estrada, Juan M.
(3); Soriano-Ursúa, Marvin A. (1); Aguilar-Faisal, José L.
(1)
Kim, Bo-Hye (1); Yang, Sunjoo (2); Kim, Eun-Ju (1); Shin,
Ye-Jin (1); Song, Jae-Young (1); Shin, Yeun-Kyung (1)
SPB4P06
P104
SPA4P15
Monitoring Avian Influenza Virus in a
small Spanish wetland using non-invasive
sampling methods.
Torrontegui, Olalla (1); Álvarez, Vega (1); Gerrikagoitia,
Xeider (1); Höfle, Ursula (2); Barral, Marta (1)
Indirect effects of oseltamivir treatment of
an influenza-infected index case-patient
on secondary household transmission: A
randomized placebo-controlled clinical trial
in a crowded urban area in Bangladesh
P112
SPB4P07
Influenza A(H1N1)pdm09 virus in pigs, Togo,
2013
Alicia M Fry (1), Doli Goswami (2), Kamrun Nahar (2),
Amina Tahia Sharmin (2), Mustafizur Rahmun (2),
Larisa Gubareva (1), Tasnim Azim (2), Joseph Bresee (1),
Stephen P Luby (2) (3), W Abdullah Brooks (2)
Ducatez, Mariette (1); Webby, Richard (2); Awoume,
Félix (3)
P113
SPB4P08
SPB4: ANIMAL FLU-ECOLOGY AND EPIDEMIOLOGY
OF ANIMAL INFLUENZA
P106
SPB4P01
Phylogenetic relationships of genes of
hemagglutinin and matrix protein of
influenza A/equine/Kazakhstan/236/12
(H3N8) virus
P107
Surveillance of influenza virus subtypes H1,
H2, H3 among wild birds in Ukraine in 20062012
P108
Barral, Marta (1); Alvarez, Vega (1); Busquets, Nuria
(2); Gerrikagoitia, Xeider (1); Minguijón, Esmeralda (1);
Costa, Tania (2); Ramis, Antonio (2); Majo, Natalia (2)
P109
Sero-prevalence of antibodies against
H5N1 and H9N2 avian influenza viruses
among Egyptians: Results of a prospective,
controlled, sero-epidemiological study.
Kayali, Ghazi (1); Webby, Richard J (1); Ali, Mohamed A
(2)
P114
Prior infection of pigs with a European H3N2
SIV confers partial protection against a North
American swine-origin H3N2v influenza virus
SPB4P10
Tissue distribution of low pathogenic avian
influenza virus in naturally infected wild
birds.
SPB4P04
Krauss, Scott L. (1); Webster, Robert G. (1); Danner,
Angela (1); Friedman, Kimberly (1); Knowles, James (1);
Ghazi, Kayali (1); Niles, Lawrence J. (2); Dey, Amanda D.
(3); Raven, Garnet (4); Pryor, Paul (4); Stucker, Karla (5);
Schobel, Seth (5); Stockwell, Timothy B. (5); Lin, Xudong
(5); Wentworth, David (5)
QIU, YU; Mancera Gracia, José Carlos; Li, Yewei; Trus,
Ivan; Nguyen Van, Ut; Van Reeth, Kristien
Muzyka, Denys (1); Pantin-Jackwood, Mary (2); Stegniy,
Borys (1)
SPB4P03
Long term surveillance of H7 influenza
viruses in American wild aquatic birds:
Concordance with emergence of variants
with pathogenic potential?
SPB4P09
Khan, Yelizaveta
SPB4P02
P111
P115
Biosecurity practices in small commercial
poultry farms, Bangladesh 2011-12
Rimi, Nadia Ali; Sultana, Rebeca; Muhsina, Mushtari;
Uddin, Baktiar; Haider, Najmul; Nahar, Nazmun; SturmRamirez, Katharine; Luby, Stephen P.
SPA5: VACCINES: CURRENT AND NOVEL
APPROACHES
SPA5P01
P116
Multi-component adjuvants for inactivated
influenza vaccines: comparative studies in a
preclinical setting
Vasiliev, Y.M.; Kashirina, O.S.; Chernikova, M.I.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XV
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
SPA5P02
P117
Characteristics of chitosan-based adjuvants
determine immunogenicity of inactivated
influenza vaccines in mice
P118
A comparative study of adjuvants with
different mechanisms of action for influenza
vaccines in mice
P119
Epidemiologic performance and influenza
vaccination prophylaxis in Russia
P120
Comparison of immune response induced by
candidate recombinant influenza vaccines
based on heterologous M2e linked to fulllength and truncated forms of flagellin
Stepanova, Liudmila (1); Kotlyarov, Roman (2); Kovaleva,
Anna (1); Potapchuk, Marina (1); Korotkov, Alexandr (1);
Sergeeva, Maria (1); Kasianenko, Marina (1); Kuprianov,
Victor (2); Ravin, Nicolai (2); Tsybalova, Liudmila (1)
A Novel Influenza Vaccination Strategy based
on DNA vaccines encoding M2 ectodomain
fused with adjuvant proteins against
Pandemic Influenza Viruses
Humoral immunity induced by seasonal
influenza vaccine in humans protects against
H5N1 challenge in mice
P129
Adjuvation broadens the protective efficacy
of a seasonal influenza vaccine in mice
SPA5P14
P130
Universal influenza vaccine development
based on the MVA vector platform
P124
Roos, Anna; Roozendaal, Ramon; Theeuwsen, Jessica;
Riahi, Sarra; Vaneman, Joost; Tolboom, Jeroen; Koldijk,
Martin; Dekking, Liesbeth; Koudstaal, Wouter;
Goudsmit, Jaap; Radošević, Katarina
P128
Cross-reactive antibodies and specific CD8
T-cells induced by conserved consensus HA2
of influenza viruses A/H2N2 delivered with
flagellin
Cox, Freek; Saeland, Eirikur; Baart, Matthijs; Koldijk,
Martin; Goudsmit, Jaap; Radosevic, Katarina
Roozendaal, Ramon; Tolboom, Jeroen; Roos, Anna;
Riahi, Sarra; Theeuwsen, Jessica; Bujny, Miriam V.;
Klaren, Vincent; Korse, Hans J.W.M.; Dekking, Liesbeth;
Grootenhuis, Arijan; Weverling, Gerrit Jan; Koudstaal,
Wouter; Goudsmit, Jaap; Radošević, Katarina
Humoral protection against H5N1 influenza
by multiple immunizations with seasonal
influenza vaccine in mice
Isakova-Sivak, Irina (1); de Jonge, Jorgen (2);
Smolonogina, Tatiana (1); Rekstin, Andrey (1); Kiseleva,
Irina (1); Kuznetsova, Victoria (1); Donina, Svetlana (1);
Petukhova, Galina (1); Naykhin, Anatoly (1); Pisareva,
Maria (3); Stukova, Marina (3); Erofeeva, Mariana (3);
Tsvetnitsky, Vadim (4); Flores, Jorge (4); Rudenko, Larisa
(1)
SPA5P13
P123
SPA5P08
P127
Stepanova, Liudmila; Potapchuk, Marina; Kuznetsov,
Vasiliy; Sergeeva, Maria; Kovaleva, Anna; Korotkov,
Alexandr; Tsybalova, Liudmila
Yi, Hwajung; Seo, Ki-Weon; Lee, Mi-Seon; Kang, Chun;
Kim, Kisoon
SPA5P07
Development, pre-clinical and clinical
evaluation of potentially pandemic H2N2
live attenuated influenza vaccine
SPA5P12
P122
SPA5P06
Vidyaeva, Inna; Potapchuk, Marina; Petrov, Sergei;
Sergeeva, Maria; Tsybalova, Liudmila
SPA5P11
Briko, Nikolay Ivanovich
SPA5P05
P126
SPA5P10
Evaluation of attenuate and reproductive
properties of influenza A vaccine strain
RA35 (H7N9) based on new donor strain A/
HongKong/1/68/162/35.
Chernikova, M.I. (1); Kashirina, O.S. (1); Khasanova, L.M.
(1,2); Vasiliev, Y.M. (1)
SPA5P04
Improved vaccine-induced immune
responses to influenza antigens.
Gilbert, Sarah Catherine (1); Antrobus, Richard D
(1); Berthoud, Tamara K (1); Mullarkey, Caitlin E (1);
Hoschler, Katja (2); Coughlan, Lynda (1); Lambe, Teresa
(1); Tully, Claire (1); Zambon, Maria (2); Hill, Adrian VS (1)
Khasanova, L.M. (1,2); Kashirina, O.S. (1); Chernikova, M.I.
(1); Vasiliev, Y.M. (1)
SPA5P03
P125
SPA5P09
Altenburg, Arwen F.; Kreijtz, Joost H.C.M.; de Vries, Rory
D.; De Gruyter, Heidi L.M.; Osterhaus, Albert D.M.E.;
Rimmelzwaan, Guus F.
SPA5P15
P131
Influence of different schedules of chicken
immunization with DNA vaccine against
avian influenza H5N1 virus on antibody
response
Góra-Sochacka, Anna; Stachyra, Anna; Sirko, Agnieszka
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XVI
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
SPA5P16
P132
A universal influenza virus vaccine strategy
based on the conserved stalk domain of the
hemagglutinin
P133
P134
Yeom, Minjoo (1,2); Na, Woonsung (1,2); Hong, Minki
(1,2); Kim, Doo-Jin (1); Jung, Dae-Gwin (1,2); Kim, JungKi (3); Kim, Sang-Hyun (1); Song, Daesub (1,2)
SPA5P26
Evaluation of the campaign’s impacts of
influenza vaccination in the productivity of
workers of brazilians industries
Adam Johnson (1), Emily Winne (2), David Cureton (1),
Callie Ridenour (1), Wanda Santana (2), Taejoong Kim
(1), John R. Barr (2), Ruben O. Donis (1), Julie Villanueva
(1), Tracie Williams (2), Li-Mei Chen (1)
P135
SPA5P27
Cross-protection by human and avian M2edisplaying virus-like particles
P136
Mice immunization against influenza with
three variants of DNA vaccine encoding
hemagglutinin.from H5N1.
Redkiewicz, Patrycja (1); Góra-Sochacka, Anna (1);
Kosson, Piotr (2); Sirko, Agnieszka (1)
P137
SPA5P21
Assessment of the public health benefit of
quadrivalent influenza vaccine in the United
Kingdom from 1996–97 to 2008–09 seasons
SPB5: GENETICS AND EVOLUTION OF VIRUS AND
HOST
P144
Adaptation of a human influenza virus to
two mouse strains with different genetic
backgrounds
P138
Evaluation of immunogenicity and crossprotective efficacy of MVAtor™-based
universal influenza vaccine candidates
in mice against H1N1, H5N1 and H7N9
influenza strains
Stricker, Ruth L.O. (1); Leist, Sarah R. (1); Schughart, Klaus
(1,2,3)
SPB5P02
P145
Host-specific distribution of (+)-RNA
secondary structures in segment 8 of
influenza A viruses
Auer, Sebastian; Wachs, Frank-Peter; Leyrer, Sonja
P139
SPA5P23
Callie Ridenour (1), Adam Johnson (1), Emily Winne
(2), Jaber Hossain (1), Guaniri Mateu-Petit (1), Amanda
Balish (1), Wanda Santana (2), Taejoong Kim (1), Todd
Davis (1), John R. Barr (2), Ruben O. Donis (1), Julie
Villanueva (1), Tracie Williams (2), Li-Mei Chen (1)
SPB5P01
Matias, Goncalo (1); Taylor, Robert J. (2); Haguinet,
Francois (1); Kim, WonGyu Lewis (2); Lustig, Roger (2);
Fleming, Douglas M. (3)
SPA5P22
P143
Development of influenza A(H7N9)
candidate vaccine viruses with improved
hemagglutinin antigen yield in eggs
Christopoulou, Ioanna (1,2); Schotsaert, Michael (1,2);
Roose, Kenny (1,2); Fiers, Walter (1,2); Saelens, Xavier
(1,2)
SPA5P20
P142
Increased Hemagglutinin Yield of Influenza
Vaccine Viruses by Incorporation of
Homologous PB1 Gene
Mattos, Bianca Ramos de Freitas (1); Araujo, Gabriela
Tannus Branco (2); Fonseca, Marcelo Cunio Machado
(2); Sansone, Dayan (2); Homsani, Sheila (3); Bricks,
Lucia Ferro (3); Liao, Paulo Sti Lin (3); Guarino, Hubert (3)
P141
Timerized Hemagglutinin Stalk Domain
Based Vaccine Protect against Canine
Influenza Virus
Poulsen Nautrup, Barbara (1); Meier, Geneviève (2);
Anastassopoulou, Anastassia (3); Welte, Robert (3);
Gregg, Meghann (2)
SPA5P19
P140
Magini, Diletta (1,2); Buccato, Scilla (2); Mangiavacchi,
Simona (2); Giovani, Cinzia (2); Tavarini, Simona (2);
Maione, Domenico (2); Brito, Luis (3); Nuti, Sandra (2);
Mason, Peter (3); Settembre, Ethan (3); Montomoli,
Emanuele (1); Geall, Andrew (3); Brazzoli, Michela (2);
Bertholet, Sylvie (2)
SPA5P25
Cost-effectiveness of quadrivalent versus
trivalent influenza vaccine in Germany: a
Markov multi-cohort model
SPA5P18
Characterization of T cell responses induced
by Flu SAM®(NP) and SAM®(M1) vaccines
Krammer, Florian (1); Margine, Irina (1,2); Nachbagauer,
Raffael (1); Pica, Natalie (1,2); Hai, Rong (1); Albrecht,
Randy A. (1,4); García-Sastre, Adolfo (1,3,4); Palese, Peter
(1,3)
SPA5P17
SPA5P24
Vasin, Andrey (1,2); Petrova, Alexandra (1,2); Plotnikova,
Marina (1); Klotchenko, Sergey (1); Egorov, Vladimir (1);
Kiselev, Oleg (1)
Prime-boost DNA vaccination protects
chickens against challenge with homologues
and heterologues H5N1 virus
Stachyra, Anna (1); Smietanka, Krzysztof (2); Minta,
Zenon (2); Gora-Sochacka, Anna (1); Sirko, Agnieszka (1)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XVII
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P146
SPB5P03
Changes in receptor binding properties
of influenza A(H3N2) and influenza B
viruses and their impact on antigenic
characterization
H1N1 IAVs exploited the co-translational
insertion process to decrease their NA
transmembrane domain hydrophobicity
Dou, Dan; da Silva, Diogo V; Nordholm, Johan; Daniels,
Robert
Lin, Yipu; Gu, Yan; Wharton, Stephen; Whittaker, Lynne;
Gregory, Victoria; Li, Xiaoyan; Metin, Simon; Cattle,
Nicholas; Daniels, Rodney; Hay, Alan; McCauley, John
SPB5P04
SPB5P11
P147
Evolution of highly pathogenic avian
influenza H7 virus from low pathogenicity
precursors in ovo
SPB5P12
P148
Molecular peculiarities of influenza A and
B viruses circulating in Russia during two
epidemic seasons in 2012-2014
Phylogenetic and principal component
analyses of influenza virus sequence data
from the first five years (2009–2013)
of the IRIS study show subtype distinct
geographical patterns in global epidemics
P157
Comparison of common genotypes of
Chinese H7 avian influenza viruses for
replication and transmission in chickens
Identifying Antigenic Regions of H9
Haemagglutinin of Avian Influenza
David L. Suarez and Marisela Rodriguez
SPB5P15
P158
Sequencing artifacts in the type A influenza
database and attempts to correct them
Peacock, Thomas Philip (1,2); Reddy, Kolli B (1); Shelton,
Holly (1); Barclay, Wendy S (2); Iqbal, Munir (1)
David L. Suarez (1), and Nikki Chester (2),
P151
SPB5P08
EPITOPE MAPPING OF THE HEMAGGLUTININ
MOLECULE OF INFLUENZA B/YAMAGATA
AND B/VICTORIA LINEAGE VIRUSES USING
MONOCLONAL ANTIBODIES
SPA6: EVALUATION OF VACCINE SAFETY AND
EFFECTIVENESS
Sorokin, Evgenii; Tsareva, Tatiana; Sominina, Anna A.;
Pisareva, Maria; Komissarov, Andrei; Kosheleva, Anna
SPA6P01
P152
SPB5P09
Analysis of mammalian adaptation in
Influenza from surveillance data
Burke, David Francis; Smith, Derek J
Strain specificity of serum hemagglutinationinhibition antibody responses in humans
infected with 2009 pandemic influenza
A(H1N1) virus
SPB5P14
P150
P156
Xiuhua Lu, Feng Liu, Vic Veguilla, Min Levine, and
Jacqueline M Katz
van der Vries, Erhard (1); Zhang, Jitao David (2); Boucher,
Charles (1); Schutten, Martin (1)
SPB5P07
P155
Use of deep-sequencing to evaluate the
intrinsic heterogeneity of human influenza
type A viruses directly in nasal swabs
SPB5P13
P149
P154
Barbezange, Cyril (1); Blanc, Hervé (2); Isakov, Ofer (3);
Enouf, Vincent (1); Shomron, Noam (3); van der Werf,
Sylvie (1); Vignuzzi, Marco (2)
Komissarov, Andrey; Kosheleva, Anna; Fadeev, Artem;
Elpaeva, Ekaterina; Mikhailova, Maria; Pisareva, Maria;
Buzitskaya, Janna; Stukova, Marina; Danilenko, Daria;
Konovalova, Nadejda; Eropkin, Mikhail; Grudinin,
Mikhail
SPB5P06
Influenza A and B intertypic reassortment is
restricted by incompatible viral packaging
signals
Baker, Steven F. (1); Nogales, Aitor (1); Finch, Courtney
(4); Tuffy, Kevin (1); Domm, William (1); Perez, Daniel R.
(4); Topham, David J. (1,2,3); Martínez-Sobrido, Luis (1)
Hanna, Amanda (1,2); Howard, Wendy A (1); Núñez,
Alejandro (1); Hicks, Daniel (1); Barclay, Wendy S (2);
Banks, Jill (1)
SPB5P05
P153
SPB5P10
P159
Effectiveness of influenza and pneumococcal
vaccines in preventing pneumonia
development and hospitalization: a
prospective cohort study
Song, Joon Young (1,10); Lee, Jin Soo (2); Wie, SeongHeon (3); Kim, Hyo Youl (4); Lee, Jacob (5); Seo, Yu Bin (5);
Jeong, Hye Won (6); Kim, Shin Woo (7); Lee, Sun Hee (8);
Park, Kyung Hwa (9); Noh, Ji Yun (1,10); Choi, Won Suk
(1); Cheong, Hee Jin (1); Kim, Woo Joo (1,10)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XVIII
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P160
SPA6P02
Influenza vaccine effectiveness to prevent
admissions with influenza in adults
belonging to target groups for vaccination
in the Valencia Hospital Network. A testnegative study, 2013-2014 influenza season
SPA6P07
P168
Immunogenicity and safety of an AS03adjuvanted H7N1 vaccine in an elderly
population
Madan, Anu (1); Ferguson, Murdo (2); Rheault, Paul (3);
Seiden, David (4); Friel, Damien (5); Soni, Jyoti (6); Li, Ping
(1); Innis, Bruce L. (1); Schuind, Anne (1)
Puig-Barberà, Joan (1,2); Carballido-Fernández, Mario
(3); Limón-Ramírez, Ramón (4); Tortajada-Girbés, Miguel
(5); Otero-Reigada, María Carmen (6); Mollar-Maseres,
Juan (6); Carratalá-Munuera, Concha (7); Gil-Guillén,
Vicente (7); Natividad-Sancho, Angels (1); Tormos, Anita
(1); Buigues-Vila, Amparo (1); Mira-Iglesias, Ainara (1);
López-Labrador, Francisco Xavier (1,8); Díez-Domingo,
Javier (1)
SPA6P08
P169
Influenza A/H3N2 mutations causing vaccine
mismatches and possibly associated with
more severe disease, Singapore, 2009-2013
Lee, Hong Kai
P162
SPA6P03
Influenza vaccine effectiveness and averted
medically-attended influenza cases, seasons
2011/12, 2012/13 and 2013/14
SPB6: RISK MANAGEMENT AND MITIGATION
Reuss, Annicka; Buda, Silke; An der Heiden, Matthias;
Schweiger, Brunhilde; Wedde, Marianne; Haas, Walter;
Buchholz, Udo
SPB6P01
P164
SPA6P04
Vaccine effectiveness estimates in preventing
laboratory-confirmed mild and moderate-tosevere influenza in the Belgian population
during the 2012-2013 season
Leclercq, India (1,2); Pariente, Kevin (1); Sawoo, Olivier
(1,2); Feher, Maxence (1); Vandenbogaert, Mathias (1);
Manuguerra, Jean-Claude (1)
SPB6P02
Hombrouck, Anneleen (1); Bossuyt, Nathalie (2); Van
Casteren, Viviane (2); Van Gucht, Steven (1); Quoilin,
Sophie (2); Wuillaume, Françoise (2); Thomas, Isabelle
(1)
Immunogenicity and safety of AS03adjuvanted H7 vaccines in adults ≤64 years
of age: Phase I/II, randomized, observerblind, placebo-controlled, multi-center trials
in the United States and Canada
SPB7P01
Prevato, Marua (1); Nandi, Avishek (3,4); Lilja, Anders (4);
Giusti, Fabiola (1); Ferlenghi, Ilaria (2); Buccato, Scilla (2);
Cozzi, Roberta (2); Giovani, Cinzia (2); Maione, Domenico
(2); Brito, Luis (4); Geall, Andrew (4); Montomoli,
Emanuele (1); Bertholet, Sylvie (2); Legay, Francois (2);
Bonci, Alessandra (2)
P172
Emerging anti-influenza agents
Kainov, Denis
P166
The «forgotten antigen» of Influenza virus:
a novel approach to study immunity to
neuraminidase
Edwards, Christina Hansen (1); de Blasio, Birgitte
Freiesleben (1,2); Tomba, Gianpaolo Scalia (3)
SPB7: HOST FACTORS IN PATHOGENESIS
Madan, Anu (1); Segall, Nathan (2); Ferguson, Murdo
(3); Sheldon, Eric (4); Frenette, Louise (5); Chu, Laurence
(6); Rheault, Paul (7); Kroll, Robin (8); Friel, Damien (9);
Soni, Jyoti (10); Li, Ping (1); Innis, Bruce L. (1); Schuind,
Anne (1)
SPA6P06
P171
Recommendations and policies for sick leave
during seasonal and pandemic influenza in
Europe
P165
SPA6P05
P170
Identification of molecular determinants
involved in Influenza A H5N1 virus survival in
the environment
SPB7P02
P173
The impact of school dismissal and
vaccination on influenza transmission: an
alternative to school closure
Voirin, Nicolas (1); Payet, Cécile (1); Régis, Corinne (1);
Barrat, Alain (2); Cattuto, Ciro (3); Pinton, Jean-François
(4); Lina, Bruno (5); Vanhems, Philippe (1)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XIX
TA B L E OF CON TE N TS POSTE R A BSTRAC TS
P174
SPB7P03
Understanding reasons behind HCW
participation and non-participation in
seasonal influenza vaccination in Podgorica,
Montenegro to tailor strategies that increase
uptake.
Meier, Geneviève; Gregg, Meghann
PL05P07
P175
Pre-existing neutralizing antibodies against
potential pandemic influenza viruses in
middle-aged adults vaccinated annually with
seasonal influenza vaccines
P176
PL05P09
P178
Ferret models to study clinical intervention
strategies against influenza.
Szurgot, Inga (1); Szolajska, Ewa (1); Laurin, David (2);
Lambrecht, Benedicte (3); Chroboczek, Jadwiga (1,2)
PL05P10
Stittelaar, Koert (1); Mallett, Corey (2); de Waal, Leon
(1); Veldhuis Kroeze, Edwin (1); van Amerongen, Geert
(1); van den Brand, Judith (3); van der Vries, Erhard (3);
Osterhaus, Ab (1,3)
P179
Results of phase II and phase III studies of
AS03-adjuvanted cell culture-based H5N1
influenza vaccine (KD-295)
P185
Exploratory Study for Risk Group of
Psychogenic Illness Following Vaccination:
Experience from Mass Immunization
Campaign Against Pandemic Influenza A
(H1N1) 2009 in Republic of Korea
Yang, Taeun; Park, Young-Joon; Kim, Hee Jung; Lee, Yeon
Kyeong; Park, Ok
PL05P11
Naruse, Takeshi; Tanabe, Tetsuro; Fukuda, Tadashi;
Ichikawa, Munetaka; Oda, Yoshiaki; Tochihara, Shinji;
Kino, Yoichiro
P186
Sensitivity, specificity and predictive values
of clinical definitions of influenza
P180
Investigating the potential for temporary
immunity between influenza viruses in the
ferret model
P184
Self-adjuvanting influenza candidate
vaccine carrying HA and M1 epitopes on a
proteinaceous multivalent nanoplatform
Jorge Tavel, Jacqueline McBride, Rong Deng, Michael
Derby, Tracy Burgess, Ning Chai,
PL05P05
P183
Effectiveness of inactivated influenza vaccine
in children less than 3 years of age over
multiple influenza seasons
Chang, Chia-Kun (1); Tsuei, Hsiang (2); Chih, Yi-Chien (1);
Lee, Chia-Lin (1); Chou, Shu-Mei (1); Yang, Chih-Hui (1)
Clinical Safety and Efficacy of MHAA4549A,
A Novel Monoclonal Antibody for the
Treatment of Severe Influenza A: Results
of a Randomized, Double-Blind, Placebo
Controlled Clinical Trial
PL05P04
Liu, Marcy Heng (1); Robinson, Stuart (2); Ben-Yedidia,
Tamar (3); Wanderley, Wilson Caparros (2); Pleguezuelos,
Olga (2); Gottlieb, Tanya (3); Babecoff, Ron (3); Schmidt,
Ed (4); Huckriede, Anke (5); Van Doorn, Eva (1); Hak,
Eelko (1)
PL05P08
Wei Wang, Esmeralda Alvarado, Qiong Chen, Dorothy
Scott, Russell Vassell, and Carol D. Weiss
PL05P03
P182
Design of randomized, double-blind,
controlled, multi-center phase IIb trials
as part of the EU-funded UNISEC project
to assess the safety, immunogenicity and
clinical efficacy of cross-seasonal universal
influenza vaccines with or without pandemic
influenza vaccine in healthy adults.
PL05: LATE BREAKERS
PL05P02
P181
Cost-effectiveness analysis of quadrivalent
influenza vaccination in the elderly: an
update of a united kingdom analysis
Likhite, Nathalie Kavita (1); Kavaric, Nebosja (2); Grbovic,
Mensud (3); Terzic, Natasa (4); Brajovic, Mina (5);
Jorgensen, Pernille (1); Caroline, Brown (6)
PL05P01
PL05P06
Cohen Jean Marie 1, Gaston De Serres 2, Isabelle
Daviaud1, Anne Mosnier 1, Tan Tai Bui 1, Vincent Enouf
3,
Martine Valette 4, Michel Lamure 5 with the help of
doctors and virologists in the GROG network.
Laurie, Karen (1), Teagan Guarnaccia(1),(2), Louise
Carolan(1), Ada Yan(3), Malet Aban(1), Patrick
Reading(1), Anne Kelso(1), Jennifer Mosse (2), James
McCaw(3),(4), Ian Barr(1)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XX
TA B L E OF CON TE N TS L E C TU R E A BSTRAC TS
P188
PL05P12
PL05P18
Efficacy of chicken immunization against
H5N1 influenza virus with bacterially
produced hemagglutinin protein.
The fraction of influenza virus infections that
are asymptomatic: a systematic review and
meta-analysis
Saczynska, Violetta (1); Romanik, Agnieszka (1); Florys,
Katarzyna (1); Cecuda-Adamczewska, Violetta (1);
Kęsik-Brodacka, Małgorzata (1); Łukasiewicz, Natalia
(1); Sokołowska, Iwona (1); Minta, Zenon (2); Smietanka,
Krzysztof (2); Szewczyk, Bogusław (3); Płucienniczak,
Grazyna (1); Płucienniczak, Andrzej (1)
Leung, Nancy; Xu, Cuiling; Ip, Dennis; Cowling, Benjamin
PL05P19
P189
PL05P13
Airway fluids from mice and ferret contain
different innate immune proteins that
mediate antiviral activity against influenza A
viruses
P194
P195
Expression, purification and immunological
properties of glycosylated influenza H5N1
hemagglutinin produced in Pichia pastoris
Kopera, Edyta; Zagórski-Ostoja, Włodzimierz; Grzelak,
Krystyna; Protas-Klukowska, Anna Maria; Zdanowski,
Konrad; Pietrzak, Maria; Macioła, Agnieszka
PL05P20
P196
RSV infection in neonates
Job, Emma (1,2); Short, Kirsty (2); Deng, Yi-Mo (3);
Laurie, Karen (3); Brooks, Andrew (2); Saelens, Xavier (1);
Reading, Patrick (2,3)
Hammoud, Majeda Sabri
P190
PL05P14
Diagnostic reassortant influenza strains for
anti-neuraminidase antibody detection in
clinical studies of live influenza vaccine
Smolonogina, Tatiana; Desheva, Yulia; Donina, Svetlana;
Rekstin, Andrey; Rudenko, Larisa
PL05P15
P191
MUC1 enhances influenza binding and
infection of epithelial cells
Allen, Robert John; Brown, Lorena Elizabeth; McAuley,
Julie Louise
P192
PL05P16
Use of government-funded antiviral drugs for
containment of an influenza A(H1N1)pdm09
outbreak in a long-term care psychiatric
facility, 2014
Lin, Mei-Ling (1); Teng, Ya-Wen (2); Chih, Yi-Chien (1);
Chang, Chia-Kun (1); Chou, Shu-Mei (1); Yang, Chih-Hui
(1)
PL05P17
P193
Circular DNAzyme against the BM2 gene
transcript of influenza B virus: a potent
inhibitor of virus replication in host cells
Kumar, Binod; Rajput, Roopali; Kumar, Prashant; Khanna,
Madhu
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
XXI
L E C TU R E A BSTRAC TS
LECTU RE
A B STR ACTS
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L1
L E C TU R E A BSTRAC TS
SPA1: CLINICAL IMPACT AND DIAGNOSTIC APPROACHES
SPA101
OMEGA
Ability of the Luminex xTAG RVP to detect zoonotic influenza viruses
with pandemic potential
Jörn Winter, Christine Warnes and Stephen Lindstrom
Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA 30333, U.S.A.
The Luminex xTAG Respiratory Virus Panel (RVP) is a PCR-based, qualitative assay used for the diagnosis and
surveillance of multiple human respiratory viruses, including human influenza virus. Due to country-specific
regulations, the number of viruses detected by the assay varies. While the xTAG RVP is intended to detect clinical
infections with human influenza viruses, it is important to understand the ability of the test to detect zoonotic
influenza type A viruses that have caused sporadic infections in humans such as swine-origin A(H1N1), A(H1N2) or
A(H3N2)v viruses as well as avian A(H7N3), A(H7N9), and highly pathogenic A(H5N1) influenza. Animal viruses that
have the potential to cause outbreaks among humans differ genetically from human seasonal viruses and may show
variable genetic diversity. In this study we evaluated the analytical performance of the xTAG RVP to demonstrate the
ability of the assay to detect zoonotic influenza viruses with pandemic potential.
Analytical performance of the xTAG RVP was evaluated by testing serial dilutions of RNA extracted from grown
influenza reference viruses of swine and avian origin belonging to Eurasian or North American lineages. All RNA
dilutions were tested in duplicate by xTAG RVP and analyzed by TDAS-RVP software. The limit of detection of an RVP
assay target was determined to be the lowest RNA concentration where two reactions were both positive.
Highly pathogenic avian A(H5N1) viruses of clades 1, 2.1, 2.2, 2.2.1, 2.3.2, 2.3.4, and 7 were identified as influenza
type A and subtype H5 at RNA concentrations equivalent to 102.2 to 104.1 infectious units. Viruses of clades 2.1.3,
2.1.3.1, 2.3.2.1, and 2.3.4 demonstrated variable reactivity with FluA and/or H5 targets and were often not identified
correctly even at RNA concentrations equivalent to 109.2 to 109.5 infectious units. Other avian influenza A subtypes
including H6 (Eurasian lineage), and H9 (clade B Y280, clade G1) and North American A(H7N3) were identified as
influenza type A at low RNA concentrations. However, avian A(H7N9) viruses recently identified in humans in China
were negative for influenza A at all concentrations tested. The xTAG RVP identified A(H1N2) human-origin influenza
virus as influenza A subtype H1 virus at low RNA concentrations. A(H3N2) (v) viruses were identified as seasonal
influenza A subtype H3 viruses at high concentrations of RNA only, while lower concentrations were identified as
influenza A only.
Our analytical performance evaluation showed that the xTAG RVP’s demonstrated variable ability to detect zoonotic
influenza viruses with pandemic potential. Most non-human influenza A viruses tested here were identified as
“unsubtypable” influenza type A. Importantly, A(H7N9) and some avian A(H5N1) viruses were not detected at any
concentration tested. These results emphasize the need to consider alternative testing methods for specimens
from suspect human cases of influenza viruses of pandemic potential, particularly A(H5N1) and A(H7N9) influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L2
L E C TU R E A BSTRAC TS
SPA102
OMEGA
A review of the link between influenza and non-communicable diseases
Palache, Abraham Mozes (1); Tainijoki-Seyer, Dr. Julia (2)
1: IFPMA, Switzerland; 2: The World Medical Association, France
Background
Approximately 5-10% of adults and 20-30% of children will contract seasonal influenza annually resulting in about 5
million severe cases and 500,000 deaths. Influenza in persons with non-communicable diseases (NCDs) can increase
the risk of long-term exacerbation of chronic conditions, hospitalization, and death. Diabetes increases the risk
of hospitalization from influenza three-fold. Heart attacks are 5 times more likely in persons with cardiovascular
disease, and stroke is 3 times more likely in the 3 days following systemic respiratory infection. Case fatality rates
from influenza A for persons with chronic obstructive pulmonary disease can be 30% or more, compared to 0.1%
or less in the healthy. Since NCDs account for 63% of the annual global mortality, and 84% of these deaths are
caused by cardiovascular disease (48%), cancer (21%), respiratory disease (12%), and diabetes (3%), the World
Health Organization recommends influenza vaccination for people living with NCDs to reduce the consequent
complications, hospitalizations and deaths. We reviewed the best available evidence of the impact of influenza
vaccination on complications, hospitalizations and deaths from NCDs.
Methods
A search of the literature was conducted for quantifiable impacts of influenza immunization on deaths,
hospitalizations, and complications from non-communicable diseases. The search was limited to the impacts of
influenza vaccination on diabetes, heart attack, stroke and respiratory disease, leading causes of mortality from
non-communicable diseases.
Results
Influenza increases the risk of deaths, hospitalization and complications in diabetes, heart disease, stroke, and
respiratory disease. In diabetics, Influenza vaccination was found to reduce the risk of death from all causes by 28%.
Furthermore, influenza vaccination in persons with diabetes reduced the risk of hospitalization during influenza
epidemics by 79%. In a meta-analysis of randomized control trials, the risk of heart attack was shown to be reduced
by over 50% in persons at high risk for cardiovascular disease who received influenza vaccination compared to
placebo or other control groups. The risk of stroke was reduced by 24% in the vaccinated compared to controls.
Influenza vaccination has also been shown to prevent exacerbations of chronic respiratory conditions.
Conclusions: Vaccination against influenza has the potential to significantly improve public health, particularly in
people living with NCDs. Our review indicates that influenza vaccination can prevent not only the infection caused
by the flu virus, but also the complications from cardio-vascular diseases, such as heart attack and stroke, and from
respiratory diseases and diabetes. Our findings support the WHO recommendation to provide vaccinations against
seasonal influenza to all persons with NCDs and encourage the full implementation of this recommendation.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L3
L E C TU R E A BSTRAC TS
SPA103
OMEGA
Influenza B disease burden in general population in France. Results of
the IBVD retrospective study from 2003 to 2014
Cohen, Jean Marie (1,2); Daviaud, Isabelle (1,2); Debost, Emmanuel (2); Valette, Martine (3); Enouf, Vincent (4); Mosnier, Anne (1,2)
1: Open Rome, France; 2: Réseau des Grog, France; 3: NRC influenza, Lyon; 4: NRC influenza, Paris
Background
If much attention has been paid to the flu over the past three decades, few studies have focused specifically on
influenza B. Although, two distinct lineages (Yamagata and Victoria) operate alternately or simultaneously since
1997, leading to some “misadequation” of B vaccine strain. In France, since 1984, part of the influenza surveillance
is performed by the Regional Groups for Influenza Observation network (Réseau des GROG), based on general
practitioners and paediatricians weekly describing the intensity of their activity and swabbing part of their patients
consulting for acute respiratory infection (ARI). Demographical, clinical and virological data are collected in a
centralized data base called Vircases. The IBVD (Influenza B in Vircases Database study IBVD - funded by GSK) was
launched with the purpose of describing the circulation and the burden of influenza B, from the 2003-2004 to
the 2012-2013 influenza season in France. Data of the declining 2013-2014 influenza season will be added to this
database.
Methods
The project is based on routine influenza surveillance data collected since 2003 at national level by the GROG
network in France. Every patient consulting a GROG practitioner for an ARI and whose rhino-pharyngeal swab has
confirmed the presence of any influenza virus, is included. We performed a retrospective descriptive analysis of the
2003-2013 influenza cases included in Vircases and will complete this analysis with 2013-2014 data.
Results and discussion
This powerful data base leads to get a comprehensive picture of influenza B disease burden for the period 20032014. Between 2003 and 2013, IBVD study is based on 16,335 virologically confirmed influenza cases, including
3,859 influenza B cases (23.6 %). Among the 2,266 B virus whose lineage is known, 1,422 (62.8%) belonged to
the Yamagata lineage and 844 (37.2 %) to the Victoria lineage. Significant influenza B circulation with vaccine
“misadequation” was observed in three seasons (2005-2006, 2007-2008 and 2008- 2009). Although considered as
not very problematic, notably because without pandemic potential, influenza B circulated with a significant impact
(estimated number of cases ≥ 1 million of the French population) during six of the ten seasons. The number of
influenza B detections was higher than the one of A (H3N2) or A(H1N1) in three seasons (2005-2006, 2010-2011
and 2012-2013). The impact of seasonal circulation of influenza B was significant and highly variable: between 2003
and 2013, it is estimated that 20.5 million people (nearly one in three French people) have consulted a primary care
practitioner for an ARI due to influenza B. Particularly common in 5-14 years old children, influenza B also concerns
other age groups, especially the frail elderly (≥ 65 years old). Depending on the season and vaccine matching, elderly
represent up to 11.7 % of influenza B cases.
Paper Keywords: Influenza B; influenza B lineage; disease burden; primary care; France Conflicts of Interest:
Presented study has been funded by GSK
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L4
L E C TU R E A BSTRAC TS
SPA104
OMEGA
Severity related factors in patients with severe acute respiratory
infections (SARI): results from the Belgium SARI surveillance during the
Influenza season 2012-2013
Nathalie Bossuyt (1), Anneleen Hombrouck (2), Isabelle Thomas (2), Françoise Wuillaume (1), Viviane Van Casteren (1), Steven Van Gucht (2) ,
the Belgian Network of sentinel general practitioners, the Belgian Network of sentinel hospitals, Sophie Quoilin (1)
1: Scientific Institute of Public Health, Operational Directorate of Public Health and Surveillance, Brussels, Belgium 2: Scientific Institute of
Public Health, Operational Directorate of Communicable and Infectious Diseases, Viral Diseases, Brussels, Belgium
Background
In order to provide an early estimation of the severity of the seasonal flu epidemic, sentinel SARI surveillance was
implemented in Belgium in 2011. The aim of the current analysis is to describe the SARI infections in season 20122013 in terms of severity and to provide insight in the association of characteristics of patient and pathogen with
severity and mortality.
Methods
Six general hospitals (including emergency departments, intensive care units (ICU) and pediatric, internal medicine
and geriatric wards) participated in the surveillance. Around the 2012-2013 influenza epidemic in Belgium (2012w51
- 2013w19), data were collected for all patients with an acute respiratory illness with onset within the last 7 days
of (history of or measured) fever of ≥ 38°C and cough or dyspnoea (shortness of breath or difficulty to breath) and
requiring hospitalisation (≥24). For each patient a respiratory swab was tested on influenza and other respiratory
viruses by the National Reference Centre Influenza. An infection was defined severe if the patient either stayed on
ICU, required invasive respiratory assistance or ECMO, developed ARDS or died. The association between severity and
mortality and patient or pathogen characteristics was estimated using multivariable logistic regression models with
severity/mortality as outcome and agegroup, sex, comorbidities, seasonal vaccination status, influenza test result,
other respiratory viruses test result and month as linear predictors.
Results
The surveillance included 990 SARI cases in season 2012-2013, among which 148 (15%) severe cases: 11% ICU, 7
% invasive respiratory assistance, 6 % ARDS, 0% ECMO and 4% mortality (N=40). Among all patients severity was
positively related to age (OR for respectively agegroups 15-44y 45-65y, 65-84y and ≥84y vs 0-4y: 3.73 (95%CI: 1.698.22), 3.86 (95%CI: 1.85-8.04), 3.51 (95%CI: 1.71-7.24) and 2.54 (95%CI: 1.05-6.13)), having a comorbidity (OR: 3.63
(95%CI: 1.93-6.81)), having cardiovascular comorbidity (OR: 1.75 (95%CI: 1.07-2.86)) and negatively to a confirmed
influenza diagnosis (OR: 0.66 (95%CI: 0.44-1.00)). Mortality was positively related to age (OR for respectively
agegroups 45-65y, 65-84y and ≥84y vs 0-4y: 4.86 (95%CI: 1.07,22.1), 6.55 (95%CI: 1.67-25.7) and 9.45 (95%CI:
2.08,43.0)), having at least 1 comorbidity (OR: 4.82 (95%CI: 1.02-22.7)) and negatively to respiratory comorbidity
(OR: 0.28 (95%CI: 0.09-0.87)). Among the subset of laboratory confirmed influenza cases, patients having renal (OR:
8.03 (95%CI: 1.36,47.4)) or cardiovascular comorbidity (OR: 6.77 (95%CI: 1.25-36.8)) were at higher risk of dying. No
relation between either severity or mortality and subtype (B, H1N1 or H3N2) could be found.
Conclusions
The first analysis of factors contributing to severity in SARI patients revealed that patient characteristics (age and
(cardiovascular) comorbidity) rather than characteristics of the pathogen were associated with severity. Analysis of
further seasons is necessary to corroborate these results.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L5
L E C TU R E A BSTRAC TS
SPB1: VIRUS STRUCTURE AND REPLICATION
SPB101
ALFA
The novel functional site in the PB2 subunit of RNA-dependent RNA
polymerase essential for acetyl-CoA interaction, RNA polymerase
activity and viral replication
Hatakeyama, Dai (1); Shoji, Masaki (1); Yamayoshi, Seiya (2); Hirota, Takenori (1); Nagae, Monami (1); Yanagisawa, Shin (1); Kawaoka,
Yoshihiro (2); Kuzuhara, Takashi (1)
1: Tokushima Bunri University, Japan; 2: University of Tokyo, Japan
Background
Influenza A is still considered a serious global health issue. To combat the influenza A virus, neuraminidase inhibitors
are widely used as antiviral drugs. However, since the viral neuraminidase gene mutates easily, many strains were
found to be resistant to these drugs. Therefore, the identification of novel target molecules is required to develop
antiviral therapies. Viral
RNA-dependent RNA polymerase (RdRp), which is constructed by the PA, PB1 and PB2 subunits, is a promising target
in the development of antiviral drugs. This RdRp is essential for viral transcription and replication. PB2 subunit
binds to the host RNA cap (m7GTP) and supports the endonuclease activity of PA to “snatch” the cap from host
pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this
study, we found that the Val/Arg/Gly (VRG) site in PB2 required for acetyl-CoA interaction, RNA polymerase activity
of viral RdRp and viral growth.
Methods
(1) We performed series of biochemical experiments using wild-type and mutant recombinant proteins of PB2. The
mutant PB2 contained amino-acid substitutions in the VRG site. (2) RNA polymerase activities of wild-type and
mutant PB2 were measured by luciferase assays and strand-specific qRT-PCR.
(3) Effects of VRG site amino-acid mutations on viral growth were investigated using wild-type and mutant
recombinant viruses.
Results
In-vitro experiments using radioisotope-marked acetyl-CoA revealed that the recombinant PB2 cap-binding domain
that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked
by excess amount of CoA and various HAT inhibitors, epigallocatechin gallate, curcumin, anacardic acid and garcinol.
Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting
with acetyl-CoA and m7GTP in a number of ways. To elucidate the importance of the VRG site on PB2 function and
viral replication, we constructed a PB2 recombinant proteins and recombinant viruses including several patterns of
amino-acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the
VRG site to alanine did not affect binding abilities of m7GTP, but significantly reduced PB2’s binding ability to acetylCoA and its RNA polymerase activity. Strand-specific qRT-PCR showed that mRNA levels specifically decreased by
point mutations in the VRG site, suggesting that this site plays crucial roles in transcriptional activity. Recombinant
viruses containing the same mutations could not be replicated in cultured cells.
Conclusion
These results indicate that the VRG site in PB2 is a functional site that is essential for acetyl-CoA interaction, RNA
polymerase activity and viral replication. This is the first report to identify a new functional site that is required for
replication of the influenza virus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L6
L E C TU R E A BSTRAC TS
SPB102
ALFA
Role of segment-specific genome packaging signals in genetic
reassortment of influenza A viruses
Isel, Catherine (1); Essere, Boris (2); Yver, Matthieu (2); Gavazzi, Cyrille (1); Terrier, Olivier (2); Fournier, Emilie (1); Giroux, Fabienne (2); Textoris,
Julien (3); Julien, Thomas (2,4); Socratous, Clio (2); Rosa-Calatrava, Manuel (2,4); Lina, Bruno (2,5); Marquet, Roland (1); Moulès, Vincent (2,4)
1: Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg, France; 2: Virologie et
Pathologie Humaine, Université Lyon 1, EA4610, Faculté de Médecine RTH Laennec, 69008 Lyon, France; 3: Unité de Recherche sur les Maladies
Infectieuses et Emergentes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7278, Institut National de la Santé et de la
Recherche Médicale U1095, Faculté de Médecine Timone, Université Aix-Marsei; 4: VirNext, Université Lyon 1, EA4610, Faculté de Médecine RTH
Laennec, 69008 Lyon, France; 5: Laboratoire de Virologie Est, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon,69677 Bron Cedex,
France
The segmented nature of the genome of influenza A viruses (IAVs) complicates virion assembly as IAVs
must incorporate at least one copy of each vRNA to be infectious but allows the virus to rapidly evolve by
genetic reassortment. We recently showed, for a human H3N2 virus, that the 8 viral ribonucleoproteins
are interconnected, that each vRNA interacts with at least one other partner in vitro and that the regions
involved in several of these interactions match known packaging signals. We also showed that the interactions
formed by the vRNAs of an avian H5N2 IAV are different and involve sequences not previously recognized as
packaging signals. Importantly, we demonstrated that a vRNA/vRNA interaction identified in vitro is crucial for
optimal viral replication of the avian H5BN2 virus and for the selective co-packaging of the interacting vRNAs.
A selective incorporation model of IAV vRNAs has important implications for genetic reassortment and for
the design and production of vaccines. However, although genetic reassortment is clearly not random, little
is known about the rules underlying this process. Genetic reassortment is limited by incompatibilities at
the protein level but nothing was known about possible incompatibilities between vRNAs from different
IAVs. We postulated that vRNA/vRNA interactions not only govern packaging, but also genetic reassortment.
To investigate this hypothesis, we studied genetic reassortment between the human H3N2 and the avian H5N2
viruses, focusing our interest on reassortant viruses that bear the avian H5 HA segment. We found that only a
limited subset of all the possible genotypes containing the H5 HA gene were produced. Importantly, the wild type
H5 HA gene never entered the human genetic background alone but was always accompanied by the avian PA and
M segments. Furthermore, introduction of the 5’ and 3’ packaging sequences of the H3 HA gene into an otherwise
avian HA backbone allowed efficient incorporation of the chimerical HA vRNA into the human genetic background,
suggesting that packaging signals are important for genetic reassortment. Accordingly, we showed that forcing the
incorporation of the avian M segment into the human genetic background was sufficient to drive coincorporation
of the H5 HA segment. Finally, introducing five silent point mutations into the 3’ packaging region of the human
M segment was sufficient to drive incorporation of the H5 HA segment into the human genetic background
during in vitro co-infections, and to affect the genotype of reassortant viruses. Altogether, these results indicate
that suboptimal compatibility between the vRNA packaging signals limit genetic reassortment. Interestingly,
the 3’ region of the human M segment, which drives co-incorporation of the H5 HA segment, has been shown to
mediate interaction between the human HA and M vRNAs in vitro, suggesting a role for intermolecular vRNA/vRNA
interactions in genetic reassortment.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L7
L E C TU R E A BSTRAC TS
SPB103
ALFA
Assembly of Influenza virus particles: Signals for targeting of HA and M2
to the budding site
Veit, Michael (1); Brett, Katharina (1); Kordyukova, Larisa (2); Serebryakova, Marina (2); Siche, Stefanie (1); Thaa, Bastian (1); deVries, Maren (1);
Herrmann, Andreas (3)
1: Free University Berlin, Virology, Germany; 2: Lomonosov Moscow State University, Physico-Chemical Biology, Russia; 3: Humboldt University
Berlin, Molecular Biophysics, Germany
Background
Influenza virus assembly occurs in nanodomains of the plasma membrane enriched in spingolipids and cholesterol
(“rafts”). HA is supposed to target to rafts by acylation of three conserved cysteine residues located in its cytoplasmic
tail and by the hydrophobic amino acids VIL in the outer part of the transmembrane region (TMR). M2 causes
scission of virus particles by wedge-like insertion of an amphiphilic helix located in its cytoplasmic tail. The helix
is palmitoylated and binds cholesterol, features that have been proposed to target M2 to the edge of the viral
budozone.
Methods
We established Foerster Resonance Energy Transfer by Fluorescence Life-time Imaging Microscopy (FLIM-FRET), a
method that measures close proximity of two fluorophores, to investigate assembly of virus particles in transfected
cells. Targeting signals identified by FLIM-FRET were exchanged in the context of the viral genome to analyze their
significance for virus replication. Mass spectrometry was used to identify fatty acid species bound to HA.
Results
We demonstrate that deletion of the two raft-targeting features of HA reduced raft association. In addition, exchange
of VIL, but not of the acylation sites severely retards transport of HA through the Golgi. Recent data suggest that VIL
may be part of a sequence motif that binds cholesterol.
Using mass spectrometry of HA we demonstrate that a cysteine at the cytoplasmic border of the TMR is modified
with stearate, while two cysteines in the cytoplasmic tail carry exclusively palmitates. Recent results show that the
location of the cysteine rather than the sequence context determines whether palmitate or stearate are attached.
We (and others) have shown that acylation of HA is essential for virus replication. Recombinant viruses (WSN strain)
with single disrupted palmitoylation sites could not be rescued and deletion of the stearoylation site caused a
growth defect.
In contrast, both raft-targeting features in M2 affect association of the cytoplasmic tail with membranes, but are not
required for association of M2 with the viral budozone and do not affect virus replication. Thus, M2 must associate
with the viral assembly site by other means.
Conclusions
Enzymes for acylation of HA have not been identified, but DHHC-proteins are likely candidates. 23 different DHHCs
with distinct, partly overlapping substrate specificities exist in mammalian cells, but only a few might catalyze
acylation of HA. These DHHCs are promising drug candidates, since their inhibition might not compromise acylation
of cellular proteins.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L8
L E C TU R E A BSTRAC TS
SPB104
ALFA
Conserved and host-specific features of influenza virion architecture
Hutchinson, Edward; Charles, Philip; Hester, Svenja; Thomas, Benjamin; Trudgian, David; Martínez-Alonso, Mónica; Fodor, Ervin
Sir William Dunn School of Pathology, University of Oxford, United Kingdom
Background
Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral
transmissibility and immunogenicity. However, the virions of influenza and of many other viruses are complex and
pleomorphic, which has made them difficult to analyse in detail.
Methods
Making use of recent developments in mass spectrometry, we identified and quantified the hundreds of viral and
host-encoded proteins that make up the pleomorphic virions of influenza A and B viruses. By comparing different
combinations of virus and host, we produced the first complete and quantified model of influenza virions.
Results
We identified a conserved influenza virion architecture which is maintained across diverse combinations of virus
and host. This ‘core’ architecture includes substantial quantities of host proteins, most of which had not previously
been identified in virions. It also includes NS1, a viral suppressor of immunity which has previously been considered
non-structural but which we show to be stably associated with virions. We found that this conserved ‘core’ virion
architecture was elaborated with abundant host-dependent features. As a result, influenza virions produced by
mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an
underlying protein composition with the exosomes shed by uninfected cells.
Conclusions
Quantitative mass spectrometry allows detailed analysis even of complex, pleomorphic virions. In the case of
influenza we show that the host plays a substantial role in shaping virion architecture, and suggest that this is
because the virus has co-opted parts of the exosome assembly pathway for virion production. The host-dependent
features of the virion mean that egg-grown vaccines have a different composition to the virions shed from
mammalian cells, and suggest that virion architecture may change during pandemic emergence.15 SEPTEMBER
2014
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L9
L E C TU R E A BSTRAC TS
SPA2: EPIDEMICS AND PANDEMIC THREATS
SPA201
OMEGA
The Epidemiological Characteristics of Influenza B Compared to Influenza
A: Results of the Global Influenza B Study
Caini, Saverio (1); Huang, Sue (2); Ciblak, Meral Akçay (3); Schellevis, François (1); Plotkin, Stanley (4); Paget, John (1)
1: NIVEL, The Netherlands; 2: The WHO National Influenza Centre, Institute of Environmental Science and Research, New Zealand; 3: Istanbul
University, Istanbul, Turkey; 4: University of Pennsylvania, Philadelphia, USA
Background
The Global Influenza B Study (GIBS) has collected epidemiological and virological data from influenza surveillance
systems from twenty-six countries worldwide. The GIBS database includes 935,673 influenza cases collected
between 2000 and 2013. Here we analyse: (1) the epidemiological impact of influenza B, (2) the age distribution of
influenza B cases, (3) the proportion of influenza B over total influenza cases, and (4) the frequency of influenza B
vaccine mismatches.
Methods
To assess the epidemiological impact of influenza B, we tested the correlation between the proportion of influenza
cases due influenza B and the maximum value of the country-specific Z-score of weekly influenza-like illness (ILI) rate
during the same season. The correlation was defined as strong (0.4-0.69), moderate (0.3-0.39), weak (0.20-0.29) or
negligible (0.01-0.019). We tested whether the age distribution of influenza B cases differed compared to influenza
A. Finally, we calculated the proportion of influenza B cases, the proportion of Victoria and Yamagata lineages in each
country and season, and determined the frequency of influenza B vaccine mismatches. All analyses were performed
overall and separately for countries situated in the Southern or Northern hemisphere or in the intertropical belt.
Results
The correlation between the proportion of influenza B and the maximum ILI-rate (Z-score) during the same season
was -0.31 in Southern hemisphere (p=0.15), -0.09 in the inter-tropical belt (p=0.62), and -0.31 in the Northern
hemisphere (p=0.03). Patients infected with influenza B were younger than influenza A patients; in particular, there
was a consistently higher proportion of influenza B cases in the 5-17 age group and influenza A cases in the 18-64
years age group. Age differences were more evident in countries in Southern and Northern hemispheres and less
evident in countries in the intertropical belt. The overall median proportion of influenza B over all influenza cases was
22.6% (inter-quartile range [IQR]: 8.3%-37.7%); it was 17.8% in the Southern hemisphere (IQR 3.5%-30.4%), 24.3%
in the intertropical belt (IQR 10.2%-40.8%), and 21.4% in the Northern hemisphere (IQR 7.3%-38.0%). Victoria and
Yamagata lineages predominated during 64% and 36% of seasons respectively. A vaccine mismatch was observed in
38% of seasons; this occurred more frequently in the Northern (50.0% of seasons) and Southern (46.2% of seasons)
hemispheres, and less frequently in the intertropical belt (13.3% of seasons).
Conclusions
Influenza B accounted for roughly 20% of total influenza cases in all regions of the world during 2000-2013; it usually
co-circulated with influenza A and was moderately correlated with lower ILI rates in the Northern and Southern
Hemisphere. Influenza B was frequently associated with a vaccine mismatch and was more frequent in the young
(5-17 years), with both of these findings being more evident in temperate zones. These findings are important for
planning and implementing influenza control and prevention policies, especially vaccination campaigns, in the
different regions of the world.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L10
L E C TU R E A BSTRAC TS
SPA202
OMEGA
Seasonal influenza in the WHO European Region: analysis of 2008-2013
influenza surveillance data in 48 countries
Gefenaite, Giedre (1); Caini, Saverio (2); Gross, Diane (3); Meerhoff, Tamara (4); Pereyaslov, Dmitriy (3); Paget, John (2); Brown, Caroline Sarah
(3)
1: Department of Pharmacoepidemiology & Pharmacoeconomics, University of Groningen, Groningen, the Netherlands; 2: Netherlands
Institute for Health Services Research (NIVEL), Utrecht, the Netherlands; 3: WHO Regional Office for Europe, Denmark; 4: Department of
Primary and Community Care, Radboud University Medical Centre, Nijmegen, the Netherlands
Background
Understanding the diversity of seasonal influenza activity across regions and over time is vital to developing effective
preventive and control measures. In order to better understand seasonal influenza patterns in the WHO European
Region, we assessed five consecutive influenza seasons.
Methods
Data on influenza seasons from 2008-2013 was obtained from the WHO Regional Office for Europe platform for
Influenza Surveillance (EuroFlu), which contains surveillance data from 48 Member States. Countries were included
in the analysis if their weekly laboratory-confirmed influenza, influenza-like illness (ILI), or acute respiratory
infections (ARI) data were reported for 20 consecutive weeks between surveillance weeks 40 to 20, allowing one
week of missing data. The laboratory-confirmed influenza median peak week and its interquartile range (IQR), length
(weeks) and spread (Pearson’s correlation between peak week and latitude/longitude) were estimated for the
Region, by influenza seasons, across six geographical sub-regions, and for influenza virus type. The influenza season
length for each country was defined as the period between the first and last week with 5% of laboratory-confirmed
influenza cases that season, The influenza peak activity was correlated with the latitude and longitude of the middle
of each country. Statistical analysis was performed with one-way ANOVA, t-test and nonparametric median test
using SPSS 20.0.0.2. The 2009/2010 pandemic season was excluded from the pooled seasonal influenza analysis. A
p-value <0.05 indicated statistical significance.
Results
Excluding the 2009/2010 pandemic season, the length of the influenza season was similar in countries throughout
the Region and averaged approximately 8.9 weeks (SD 3.6), with no significant variation between sub-regions,
influenza seasons or influenza virus type. Seasonal influenza activity based on laboratory-confirmed influenza
usually peaked in countries between weeks 4 and 9, but peaks were different between sub-regions(p-value < 0.05),
first peaking in week 5 in northern and western Europe and central Asia, and last in eastern Europe in week 8. There
was west to east spread in 2008/2009, 2010/2011 and 2012/2013 influenza seasons, and south to north spread in
2011/2012 season (Table 1). During the pandemic, influenza peaked in week 47 and it lasted about approximately
7.5 weeks (SD=4.1). The pandemic season’s length was not significantly different from the other years or between
sub-regions, but there was evidence for moderate west to east spread (R2=0.33, p-value >0.5).
Discussion
From 2008-2013, the length of the influenza season was similar across the Region typically lasted for about nine
weeks independent of the sub-region, but it was rather diverse in its timing. There was also evidence of west to
east spread of influenza. These results might be used to strengthen public health action plans related to influenza
preparedness, and, particularly in severe seasons, to share experience between first-affected countries and those
not yet experiencing peak influenza activity.
Keyword
Influenza, WHO European Region, Peak activity, Duration
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L11
L E C TU R E A BSTRAC TS
Fig. 1. Sub-regions of the WHO European Region.
Fig. 1. Sub-regions of the WHO European Region
Subregions
Central Asia
Eastern Europe
Northern Europe
Southern Europe
West Asia
Western Europe
Table 1. Median influenza peak activity, mean season length, and spread by season based on the laboratoryconfirmed influenza between 2008/2009 and 2012/2013. W-E: west to east spread; S-N: south to north spread;
*p-value <0.05; **p-value <0.01; IQR: interquartile range
Season
N
Median peak activity
Mean season length
week (IQR)
weeks (SD)
W-E (R2)
S-N (R2)
2008/2009
38
4 (3-9)
8.42 (4.42)
0.69**
-0.16
2009/2010
50
47 (46-48)
7.56 (4.13)
0.33*
0.02
4
2010/2011
47
4 (3-7)
8.96 (3.43)
0.37*
-0.14
2011/2012
52
9 (6-11)
8.75 (4.12)
0.10
0.33*
2012/2013
54
6.5 (5-9)
9.59 (2.40)
0.31*
-0.15
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L12
L E C TU R E A BSTRAC TS
SPA203
OMEGA
Effectiveness of closure of live poultry markets in reducing influenza
A(H7N9) transmission during the 2013-14 winter wave
Wu, P (1); Jiang, H (2); Wu, JT (1); Chen, E (3); He, J (4); Zhou, H (2); Wei, L (1); Yang, J (2); Yang, B (1); Qin, Y (2); Fang, VJ (1); Li, M (2); Tsang, TK
(1); Zheng, J (2); Lau, EHY (1); Cao, Y (2); Chai, C (3); Zhong, H (4); Li, Z (2); Leung, GM (1); Feng, L (2); Gao, GF (5,6); Cowling, BJ (1); Yu, H (2)
1: School of Public Health, The University of Hong Kong, Hong Kong S.A.R. (China); 2: Division of Infectious Disease, Key Laboratory of
Surveillance and Early-warning on Infectious Disease, Chinese Center for Disease Control and Prevention, Beijing, China; 3: Zhejiang Provincial
Centre for Disease Control and Prevention, Hangzhou, Zhejiang Province, China.; 4: Guangdong Provincial Centre for Disease Control and
Prevention, Guangzhou, Guangdong Province, China; 5: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing, China; 6: Office of Director-General, Chinese Center for Disease Control and Prevention,
Beijing, China
Background
The novel avian influenza A(H7N9) virus has caused two epidemic waves in mainland China during 2013-2014
since it was first identified in March 2013, with 417 laboratory-confirmed human cases reported by 28 April 2014.
Temporary closure of live poultry markets was suggested to be highly effective in reducing in the incidence of H7N9
infections in a few major cities involved in the 2013 spring wave. This study aims to estimate the impact of market
closure in reducing risk of human infections during the 2013-14 winter wave.
Methods
Nine urban areas in the two most affected provinces in the 2013-14 winter wave were selected for the study. The
ratio of the estimated incidence rates of H7N9 virus infection during versus prior to LPM closure from the model
indicated the effectiveness of intervention by assuming a constant incidence rate of infection before and during
the market closure and a lognormal distribution of the incubation period of the H7N9 infection. An overall model
combining data from all selected areas was also fitted assuming the same incidence rate ratio across all areas.
Results
Point estimates of the effectiveness of LPM closure varied from 61-89% in the nine areas with generally wide
credibility intervals while the effectiveness was estimated to be 97% (95%CI: 89%, 100%) in the overall model. The
incubation period distribution has a mean 3.4 days (95% CI: 2.2-5.0).
Conclusions
Our analysis found that live poultry market closure was highly effective in reducing the risk of human infections
with H7N9 virus during the 2013-14 winter wave, similar to the estimates from the study on the 2013 spring wave.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L13
L E C TU R E A BSTRAC TS
SPA204
OMEGA
Molecular determinants of influenza A/H7N9 virus HA receptor binding
and stability
Schrauwen, Eefje J.A. (1); Richard, Mathilde (1); Herfst, Sander (1); Bestebroer, Theo (1); Lexmond, Pascal (1); Burke, David F. (2); Rimmelzwaan,
Guus F. (1); Osterhaus, Albert D.M.E. (1); Fouchier, Ron A.M. (1)
1: ErasmusMC, Rotterdam, Netherlands The; 2: University of Cambridge, Cambridge, United Kingdom
Background
In the spring of 2013, an avian origin A/H7N9 influenza virus emerged in Eastern China and was transmitted to
humans. These human infections are believed to have resulted from exposure to infected poultry. Although A/H7N9
viruses harbor genetic traits associated with adaptation of avian viruses to humans and increased transmission
between mammals, no sustained human-to-human transmission has been reported. Several studies have shown
that transmission of A/H7N9 viruses via the airborne route (respiratory droplets and aerosols) between ferrets was
limited, suggesting that A/H7N9 viruses still lack crucial determinants for airborne transmission (e.g. Richard et
al., Nature 501:560-3, 2013). Receptor specificity and stability of the hemagglutinin (HA) are thought to contribute
substantially to airborne transmission of influenza viruses.
Methods
Amino acid substitutions that are known to affect receptor binding and stability were investigated in the context
of the A/Anhui/1/13 HA in vitro. Using a model of the trimeric structure of the H7 HA, additional amino acid
substitutions that might affect stability were predicted and investigated. To address the question whether this virus
could acquire other determinants for adaptation to mammals, A/Anhui/1/13 wild type virus and A/Anhui/1/13
virus containing a G219S substitution in the receptor binding site of HA (G228S in H3 numbering), were passaged in
the upper respiratory tract (URT) of ferrets.
Results
The HA of A/Anhui/1/13 with the G219S substitution in the receptor binding site displayed a change in receptor
affinity towards increased human receptor preference. However, this H7 with G219S displayed a decrease in
temperature and acid stability. A substitution known to stabilize the H5 HA (K58I in HA2), increased the temperature
and acid stability of H7 with G219S. Another amino acid substitution, which was found during the earlier ferret
transmission experiments with the A/Anhui/1/13 isolate (A210E) was also found to increase the acid and
temperature stability of HA. Surprisingly, A/Anhui/1/13 and A/Anhui/1/13 with G219S acquired only a few amino
acid substitutions in HA after passage in the URT of ferrets.
Conclusions
Here, we showed that a switch towards increased human receptor specificity coincided with a decrease in stability
of HA of A/H7N9 virus. These results are in line with what has been recently shown for airborne transmissible H5N1
virus, for which increased human receptor specificity also coincided with decreased stability of the HA. For the H5N1
virus, an increase in stability was required for the virus to be transmitted via the airborne route. Here, several amino
acid substitutions in the hemagglutin had the ability to restore the stability of HA of A/Anhui/1/13 with a G219S
mutation. These results could help identifying amino acid substitutions which alter the hemagglutinin stability and
binding preference, properties that have been shown to influence airborne transmission. Therefore these results
provide essential information for the surveillance and assessment of the pandemic potential of currently circulating
H7N9 viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L14
L E C TU R E A BSTRAC TS
SPB2: VIRAL FACTORS IN PATHOGENESIS
SPB201
ALFA
The Epidemiological Characteristics of Influenza B Compared to Influenza
A: Results of the Global Influenza B Study
Baumann, Jan; Monougu, Nancy; Klenk, Hans Dieter; Matrosovich, Mikhail
Philipps University Marburg, Germany, Germany
Background
The repeated introduction of influenza A viruses of wild aquatic birds into land-based birds and mammals emphasizes
the need to determine viral properties which contribute to interspecies transmission. Alteration of the receptorbinding specificity of the viral hemagglutinin (HA) is known to be essential for the avian-to-mammalian adaptation.
Less is known about potential host-specific differences in the membrane fusion activity of the HA and their role in
interspecies transmission. We addressed these questions by comparing fusogenic properties of phylogenetically
related H1N1 influenza viruses isolated from wild aquatic birds, pigs, and humans.
Materials and methods
To analyze pH dependency of viral cell entry we studied neutralization of viral infectivity in MDCK cells by lysosomotropic
agent ammonium chloride, which interferes with endosomal acidification. Hemolysis assay was used to determine
HA-mediated membrane fusion activity of the viruses. Viral stability at low pH was assessed by incubating viruses in
buffers with pH values from 5.0 to 7.0 followed by titration of remaining infectivity in MDCK cells. The effects of amino
acid substitutions on the HA fusion activity was studied using the plasmid encoded HA gene of A/dk/Bavaria/1/77
(H1N1) virus modified by site directed mutagenesis. The modified HAs were expressed in HeLa cells, and polykaryon
formation was determined after incubation at low pH.
Results
Swine viruses were less sensitive to neutralization by ammonium chloride than avian and human viruses (IC50% of
NH4Cl: 1.5- 2.5 mM for swine viruses and 0.6-1.0 mM for avian and human viruses). This finding suggests that swine
viruses can fuse with endosomal membranes and enter the cells at a higher pH. Indeed, the pH optimum of the viral
hemolytic activity was found to be 0.2 to 0.5 units higher in the case of swine viruses than in the cases of avian and
human viruses. Accordingly, swine viruses were more sensitive than avian and human viruses to inactivation under
low pH conditions. To identify amino acids responsible for the observed differences between Eurasian avian-like swine
viruses and their putative avian precursors, we compared viral HA sequences and found eight host-specific amino acid
differences. We introduced the swine-virus-like amino acids into an avian HA, assayed membrane fusion activity of the
HA variants and found that substitutions T393S and S457F raised the pH of HA-mediated fusion of the avian HA by
0.2 pH units.
Conclusion
Because avian and human H1N1 viruses have a lower pH optimum of membrane fusion activity and are more stable
than closely related porcine viruses, we assume that fusion-related characteristics of the HA represent a host-range
factor restricting avian-to-swine and swine-to-human transmission of influenza viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L15
L E C TU R E A BSTRAC TS
SPB202
ALFA
Single cell analysis reveals strain-specific inhibition of ISG-expression by
the viral NS1 protein in influenza A virus infected cells
von Recum, Jessica; Weinheimer, Viola K; Wolff, Thorsten
Div of Influenza Viruses and Other Respiratory Viruses, Robert Koch Institut,Berlin, Germany
Background
Influenza A virus (IAV) infection provokes an antiviral immune response including activation of interferon (IFN) and
IFN-stimulated genes like ISG15 but the spatio- temporal spread of this alarm response throughout a tissue or
culture is not very well understood. The aim of this study was to elucidate the expression patterns of ISGs and viral
antigen in infected vs. non-infected cells within the same culture and to determine viral and cellular factors involved
in this process.
Methods
In order to assess the induction and distribution of the antiviral IFN response in single cells within an IAV infected cell
culture we devised a FACS-based assay enabling simultaneous viral antigen and ISG detection. Further approaches
involved the use of small molecule inhibitors targeting relevant signaling pathways, transfection-based reporter
assays to identify viral regulatory factor(s), as well as GST pulldown assays and reverse genetic analysis.
Results
Analysis of human cell cultures infected with seasonal H3N2 and H1N1 IAV revealed an unexpected distinction of
cell populations expressing either viral antigen or ISG15, but not both. This bisection was robustly observed across a
number of experimental variations including doses and time-points of infection indicating ISG upregulation in noninfected, but not in infected cells. Interestingly, the proportion of infected cells that were also ISG15-positive was
significantly increased upon infections with low- pathogenic avian IAV or the emerging avian-origin H7N9 IAV in
comparison to seasonal IAV, indicating that these non-human-adapted viruses are debilitated in ISG15 suppression.
A seasonal mutant virus lacking the NS1 gene increased the percentage of infected ISG15-positive cells up to 6 times
indicating a role for NS1 in inhibiting ISG15-expression in infected cells. In contrast, NS1-deficiency barely affected
the proportions of non-infected ISG expressing cells despite of strongly increased type I and III IFN induction.
This finding illustrates that NS1 reduces ISG induction primarily in infected cells, but hardly affects ISG levels in
neighbouring cells. Transfection-based experiments confirmed that NS1 of seasonal IAV strongly reduced IFNstimulated ISG15-induction whereas this was not observed for NS1 of avian IAV or H7N9. Sequence comparisons
revealed that NS1 proteins unable to suppress ISG15 had amino acid changes related to the loss of binding to the
polyadenylation factor CPSF30. In fact, reverse genetic analysis of viral NS1 proteins showed a direct correlation
between CPSF30 binding and suppression of ISG15, MxA and ISG56 expression in infected cells.
Conclusions
Single cell analyses revealed an unexpected restriction of ISG suppression by NS1 proteins of seasonal IAV to infected
cells that correlates with their ability to bind CPSF30. The data not only provide insights into the ways IAV counteract
the IFN response in infected cells, but also implicate that even NS1-expressing IAV strains face substantial ISG
induction in neighbouring cells during multicycle replication.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L16
L E C TU R E A BSTRAC TS
SPB203
ALFA
Neuraminidase-negative avian influenza viruses of subtype H5 and H7:
Variation of virulence in the chicken model
Hoffmann, Donata (1); Röhrs, Susanne (1); Bogs, Jessica (1); Stech, Jürgen (2); Beer, Martin (1)
1: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany; 2: Institute of Molecular Virology and Cell
Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany
Background
A neuraminidase (NA)-negative virus originating from highly pathogenic avian influenza virus of subtype H5
(EscEgg50A; H5NAneg) was recently characterized as replication competent in vitro and highly attenuated in adult
chicken (Kalthoff et al., 2013. J Virol. 87(24):13556-68). The suitability of the H5NAneg virus variant as a model for
the induction of a very early onset of protection was demonstrated within the adult chicken, mouse and ferret
model (Röhrs et al., 2014. Vaccine. 32(22):2631-6).
Material and Methods
The H5 of the EscEgg50 strain was exchanged by reverse genetics with H7 including a polybasic cleavage site resulting
in strain H7NAneg. Both viruses were further evaluated in the chicken model using day-old chicks (H5NAneg) or 6
week-old adult chickens (H7NAneg).
Results
In contrast to the outcome with older chickens, day-old-chicks exhibited typical disease symptoms of highly
pathogenic avian influenza infection after intramuscular as well as oronasal infection with H5NAneg (Fig 1A). The
animals succumbed to the disease or had to be euthanized within 4 days (i.m. application) or 12 days (oronasal
application). In-contact animals were also infected in both groups. Viral RNA loads from different tissues (e.g.
myocard, lung, brain, skeletal muscle) confirmed a systemic infection. However, in one-week-old chickens inoculation
with H5NAneg again did not result in clinical signs.
Furthermore, adult chickens, intramuscularly inoculated with H7NAneg showed the typical clinical picture (Fig. 1B)
of highly pathogenic avian influenza with 100% mortality until 4 days post inoculation. Transmission to sentinel
chicken could be demonstrated in 2 out of 5 animals. Again, different organ samples scored positive for viral RNA,
demonstrating severe systemic infection.
Conclusions
We evaluated the virulence of replication-competent NA-negative viruses in chickens of different ages. Interestingly,
the virulence of H5NAneg is very much age-dependent. Day-old-chicks without maternally derived antibodies
succumb to a systemic infection, while chickens one week of age do not show any clinical signs at all. Therefore, the
day-old-chick organism is most likely not able to restrict replication of the virus as older chickens do. In addition to this
host-derived component of virulence, our results from the H7NAneg inoculation experiment clearly demonstrated
a hemagglutinin subtype-dependent variation in virulence despite using an identical genetic backbone. Inoculation
with H7NAneg exhibited a highly pathogenic phenotype even in adult chickens, providing evidence, that the
virulence of the H7 subtype might be less affected by the neuraminidase protein than that of the H5 virus.
Our work with NA-negative avian influenza viruses provides a unique tool to study the role of the neuraminidase
protein in replication cycle and pathogenesis of highly pathogenic avian influenza viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L17
L E C TU R E A BSTRAC TS
Fig 1: Typical clinical signs (hemorrhages on the feet; lethargy) from (A) H5NAneg infected day-old-chicks, and (B)
Fig 1: Typical clinical signs (hemorrhages on the feet; lethargy) from (A) H5NAneg
H7NAneg-inoculated
infected
day-old-chicks, and (B)chicken
H7NAneg-inoculated chicken
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L18
L E C TU R E A BSTRAC TS
SPB204
ALFA
Assessment of transmission, pathogenesis and adaptation of H2 subtype
influenza viruses using the ferret model
Claudia Pappas, Hua Yang, Hui Zeng, Paul J. Carney, James Stevens, Jacqueline M. Katz, and Terrence M. Tumpey.
Influenza Division, National Center for Immunization and Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, GA 30333,
USA
Background
After their disappearance from the human population in 1968, influenza viruses of the H2 subtype have continued
to circulate in the natural avian reservoir. In 2006, the sudden emergence of H2N3 subtype viruses in the swine
population increased the awareness that H2 viruses still pose a threat to public health. These viruses contained a
Q226L substitution in the hemagglutinin (HA) receptor binding site which is a key amino acid for specific binding
to α2,6 sialic acid receptors found in mammalian cells. The present work evaluated the transmissibility and
pathogenicity of H2 viruses isolated from avian, swine and human hosts using the ferret model.
Methods
For each experiment, ferrets were inoculated with either avian, swine or human viruses of the H2N2 or H2N3
subtypes. Clinical signs were monitored and replication of H2 viruses in ferret tissues was determined. Because
the HA1 is important for recognition of sialic acid receptors and efficient transmission in mammals, we performed
sequence analysis of the HA receptor binding region from infected ferret tissues to look for the presence of
genetically distinct viral subpopulations. A detailed glycan array approach was also used to analyze the repertoire of
sialylated glycans recognized by swine H2 virus.
Results
Results revealed that despite a low pathogenic phenotype, avian H2 viruses were able to replicate in the respiratory
tracts of ferrets at different levels and could be transmitted between ferrets in the presence of direct contact, but not
by respiratory droplets. Virus quasispecies were present in the nasal washes and tissues of ferrets, some containing
amino acids mutations in the HA receptor binding region that included a Q226L substitution. Ferrets inoculated
with swine H2N3 virus displayed greater clinical symptoms and higher infectious virus titers throughout the ferret
respiratory tract when compared to human H2N2 virus-infected ferrets. Moreover, the swine H2N3 virus spread
efficiently between cohoused ferrets, similar to that observed for human H2N2 viruses. Furthermore swine H2N3
virus possessed the capacity to be transmitted via respiratory droplets to 2 out of 3 ferrets, while a human H2N2
virus transmitted to 3 out of 3 ferrets. Glycan microarray analysis of the swine recombinant HA revealed a strong
binding preference for the α2-6-linked sialosides and some binding to mixed α2-3/α2-6 branched sialosides.
Conclusions
The presence of leucine at position 226 in the HA suggests that following replication, H2 viruses have ability to
mutate and adapt to mammals by altering their receptor binding specificity. Further studies are underway to
characterize additional genetic mutations that confer host adaption of potentially pandemic H2 viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L19
L E C TU R E A BSTRAC TS
SPA3: IMMUNOLOGY
SPA301
OMEGA
CD8+ T cells correlate with protection against symptomatic illness
following natural pandemic influenza infection in humans
Sridhar, Saranya (1); Begom, Shaima (1); Bermingham, Alison (2); Hoschler, Katja (2); Adamson, Walt (3); Carman, William (3); Bean, Thomas
(4); Barclay, Wendy (1); Deeks, Jonathan (5); Lalvani, Ajit (1)
1: Imperial College London, United Kingdom; 2: Public Health England, Colindale, UK; 3: Glasgow University, UK; 4: Public Health
England,Porton Down, UK; 5: University of Birmingham, UK
Background
Experimental challenge studies in humans lacking pre-existing antibodies demonstrated an inverse association for
influenza-specific T-cells and symptomatic illness providing proof-of-concept for the protective capacity of T-cells
against influenza-illness. Whether cross-reactive T-cells impact clinical outcomes of natural influenza infection
remains unanswered. The 2009 pandemic provided a unique natural experiment to determine whether crossreactive cellular immunity is associated with limiting symptomatic illness following influenza infection in antibodynaive individuals.
Methods
We followed 342 healthy adults through the pandemic from 2009-11. We correlated pre-existing cellular immune
responses to live pH1N1 virus and conserved CD8 T- cell epitopes from virus core proteins with clinical outcomes
following incident pH1N1 infection.
Results
Individuals developing mild illness without fever (or cough or sore throat) had higher frequencies (p=0.04) of preexisting influenza-specific cross-reactive T-cells than those with symptoms or fever. Total symptom score inversely
correlated with frequencies of pre-existing cross-reactive T-cells to pH1N1 virus (r=-0.39, p=0.05) and conserved CD8
epitopes (r=-0.5, p=0.01). Of three functional T-cell subsets, only IFN-γ+IL-2- T-cells were associated with limiting
illness severity and viral shedding. Within this influenza-specific functional T-cell subset, CD8+ cells of late-effector
CD45RA+CCR7- phenotype inversely correlated with symptom score (r=-0.49, p=0.02) and expressed CCR5 tissuehoming and CD107 degranulation markers on exposure to live virus.
Conclusions
In the absence of protective antibodies, cross-reactive CD8+ T-cells correlate with protection against symptomatic
pandemic influenza. This immune correlate of protection could guide development and evaluation of T-cell-inducing
influenza vaccines and inform vaccination policy.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L20
L E C TU R E A BSTRAC TS
SPA302
OMEGA
Exploring the humoral cross-reactome against the influenza virus surface
glycoproteins proteins hemagglutinin and neuraminidase
Krammer, Florian
Icahn School of Medicine at Mount Sinai, United States of America
Background
Influenza viruses continue to be a major public health burden worldwide. In addition to seasonal influenza, zoonotic
viruses pose a constant pandemic threat. Recently, it has been shown that natural infection with influenza viruses
can induce antibodies against the major surface glycoprotein, the hemagglutinin, that are able to broadly neutralize
different subtypes. However, it is unknown to which extend these cross-reactive antibodies are induced by natural
infection with H1N1 or H3N2 in animal models and in humans, how prevalent they are in the population and how
broad their reactivity is in terms of binding and neutralization. Furthermore, little is known about cross-reactivity for
the second viral surface glycoprotein, the neuraminidase.
Methods
To recapitulate the human exposure to influenza viruses we sequentially infected mice with seasonal and pandemic
H1N1 strains or with H3N2 viruses that are separated by 29 years of antigenic drift. Sera from these animals were
than analyzed for binding to more than 30 different recombinant hemagglutinins including all 18 subtypes and
many strains within subtypes by quantitative endpoint titer ELISA. Similarly, reactivity to all neuraminidase subtypes
was tested. Importantly, all hemagglutinin and neuraminidase proteins used as substrate were expressed as trimers
and tetramers respectively, folded correctly and fully functional.
Results
We found that animals sequentially infected with H1N1 strains developed broad reactivity against group 1
hemagglutinins and low reactivity to group 2 hemagglutinins suggesting the presence of group 1 specific stalkreactive antibodies. Interestingly, sera from H1N1 infected animals did not react with recombinant H3 proteins
at all. Animals sequentially infected with H3N2 viruses developed broad reactivity to group 2 hemagglutinins but
also reacted strongly to H2, H5 and H6 group 1 hemagglutinins in the absence of reactivity to H1. This suggests the
presence of cross-reactive epitopes in the globular head domain of H2, H3, H5 and H6 hemagglutinins. The complete
lack of H1-H3/H3-H1 cross-reactivity might also suggest that these viruses - which co-circulate in humans - have
been under evolutionary pressure to exclude cross-reactive epitopes from their surface proteins. Cross-reactivity
within neuraminidase subtypes was good but no reactivity across subtypes was detected.
Conclusions
We are now expanding our studies to guinea pigs, ferrets and humans and we are also exploring the functionality
of these antibodies. The data generated will allow novel insights in circulation dynamics of seasonal influenza virus
strains in the human population and will also help us to understand to which extend these crossreactive antibodies
contribute to the species barrier that protects the human population from emerging zoonotic influenza viruses like
H5N1 and H7N9. Finally, we believe that insights into the cross- reactome will further advance the development of
universal influenza virus vaccines and antibody therapies.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L21
L E C TU R E A BSTRAC TS
SPA303
OMEGA
Polyclonal and Isotype-Specific Effects of Hemagglutinin Stalk-Mediated
Neutralization
Miller, Matthew S.
Icahn School of Medicine at Mount Sinai, United States of America
Background
Broadly-neutralizing antibodies that bind to the hemagglutinin (HA) stalk domain have received substantial
attention for their ability to provide potentially ‘universal’ protection against influenza A virus (IAV) infection.
While considerable progress has been made in understanding the structure and function of monoclonal HA stalkbinding IgGs, little is known about how these antibodies behave in the context of a polyclonal response, or as other
isotypes. This information will be essential in informing universal influenza virus vaccine design, and will advance
comprehension of the complex interplay between the diverse antibody clonotypes elicited during natural humoral
immune responses.
Methods
Polyclonal donor-matched IgG and IgA was purified from the serum of healthy adults. The functional activities of
these antibodies were compared to well-characterized monoclonal antibodies with specificity for the HA head or
stalk domain. VH and VL regions of monoclonal HA stalk binding antibodies were then cloned into chimeric human
IgG or IgA backbones in order to assess how isotype affected antibody function.
Results
In a polyclonal context, the neutralizing potency of HA-stalk binding antibodies was markedly increased relative to
that of antibodies that bind to the HA head domain. While serum-derived IgG and IgA were capable of neutralizing
virus via the HA stalk domain to a similar extent, IgAs were comparably weaker in mediating HA head domainmediated neutralization. This was due to an isotype-specific bias, whereby IAV-specific IgA were most likely to be
HA stalk-specific, whereas the majority of IgG was specific for the HA head domain. Furthermore, when expressing
identical VH and VL regions, IgA was capable of neutralizing virus with superior potency relative to IgG. Taken
together, these observations highlight an important role for IgA in contributing to HA stalk-mediated neutralization
that should inform universal IAV vaccine design.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L22
L E C TU R E A BSTRAC TS
SPA304
OMEGA
Human cytotoxic T lymphocytes directed to influenza B viruses crossreact with viruses of two phylogenetic lineages
Van De Sandt, Carolien E.; Dou, Yingying; Vogelzang-van Trierum, Stella E.; Westgeest, Kim B.; Osterhaus, Albert D.M.E.; Fouchier, Ron A.M.;
Hillaire, Marine L.B.; Rimmelzwaan, Guus F.
ErasmusMC, Netherlands, The
Background
Influenza virus infections are an important cause of respiratory tract infections worldwide. Seasonal influenza
epidemics are caused by influenza type A and B viruses. The yearly return of these viruses can be attributed to
their ability to evade the virus neutralizing antibody response by accumulating mutations in the hemagglutinin and
neuraminidase. It has been shown that cytotoxic T lymphocytes (CTLs) directed against conserved internal proteins
of influenza A viruses contribute to cross-protection against infection with antigenically distinct intrasubtypic
and heterosubtipic variants. Influenza B viruses fall into two antigenically distinct lineages, namely the B/
Victoria/02/1987 and B/Yamagata/16/1986 lineage. It is unknown to what extent T cells directed to viruses of one
lineage cross-react with viruses of the other lineage. Here, we assessed the cross- reactivity of CTLs with influenza
B viruses of both lineages.
Materials and Methods
Influenza viruses B/Yamagata/16/1988, B/Victoria/02/1987 and two recent epidemic strains B/
Netherlands/712/2011 and B/Netherlands/455/2011 belonging to either lineage were propagated in MDCK
cells and used in the present study. Amino acid sequence homology of the internal proteins was determined by
BLAST analysis. Immune-epitope database and SYFPEITHI were used to predict putative epitopes shared by these
viruses. Polyclonal CTL populations were obtained from HLA-typed donors after in vitro stimulation with influenza
viruses B/Yamagata/16/1988 or B/Victoria/02/1987. The (cross-)reactivity of these CTLs with the respective viruses
was assessed by determining IFN-γ production using intracellular cytokine staining and flowcytometry and their
capacity to lyse MHC class I-matched cells infected with the respective viruses.
Results
Although the viral HA of these lineages is variable, the internal proteins were relatively conserved and several
predicted epitopes were present in viruses of both lineages. This indicates the possibility of CTLs to cross-react
between viruses of different lineages. Polyclonal CTL populations obtained after in vitro stimulation with influenza
viruses B/Victoria/02/1987 or B/Yamagata/16/1988 displayed a high degree of cross-reactivity with drifted variants
of the corresponding lineage or viruses of the heterologous lineage.
Conclusions
Thus, influenza B virus specific CTLs display a high degree of cross-reactivity and it is anticipated that these cells
afford cross-protection against infection with drift variants of the homologous lineage or viruses of the alternate
lineage. These findings may have implications for the development of vaccines that aim at the induction of broadly
protective immunity based on cross-reactive T cell responses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L23
L E C TU R E A BSTRAC TS
SPB3: MATHEMATICAL MODELING
SPB301
ALFA
The immune response and within-host emergence of pandemic influenza
virus
Reperant, Leslie (1); Kuiken, Thijs (1); Grenfell, Bryan (2); Osterhaus, Albert (1)
1: Erasmus Medical Centre, Netherlands, The; 2: Princeton University, USA
Background
Pandemic influenza viruses have emerged four times in the past hundred years. Their origins are zoonotic influenza
viruses transmitted from animal reservoirs to humans, in which they have evolved to acquire efficient human-tohuman transmissibility. The conditions favoring these rather rare events are poorly understood. Variants with the
efficient human-to-human transmissibility trait must randomly evolve during zoonotic infection and then replicate
to titers allowing transmission to a new individual. We propose that the latter step occurs rarely because the immune
response triggered by zoonotic influenza virus also controls transmissible mutants that emerge during infection.
Methods
Here we use mathematical models of within-host infection dynamics to evaluate the conditions for emergence of
a transmissible mutant during zoonotic influenza virus infection (emergence being defined as the mutant’s ability
to replicate and increase titers). We used deterministic systems of coupled differential equations to follow in time
the number of cells infected with the zoonotic and transmissible virus variants, as the latter evolves at different
time points after the start of the zoonotic infection. The dynamics of innate and adaptive immune responses were
modeled and varying their strengths accounted for immunocompetent and immunocompromised conditions.
Results
We found that within-host emergence of a transmissible variant could occur in an immunocompetent individual,
provided it evolved early during infection, specifically before the replication peak of the zoonotic virus. The later
the transmissible variant evolved, the less it replicated; variants evolving after the replication peak of the zoonotic
virus did not emerge due to immune-mediated exclusion (Fig.). In contrast, in an immunocompromised individual,
transmissible variants could emerge irrespective of the time of their evolution during zoonotic infection. Furthermore,
because of different receptor binding affinity, the transmissible variant had a fitness advantage and predominated
in the upper respiratory tract (Fig.).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L24
L E C TU R E A BSTRAC TS
Figure: Replication of zoonotic (wild-type) and transmissible (mutant) viruses after mutant evolution (from Reperant
et al. The Lancet, 2014).
Figure: Replication of zoonotic (wild-type) and transmissible (mutant) viruses
after mutant evolution (from Reperant et al. The Lancet, 2014).
• Conclusions:
Conclusions
The evolution of a novel pandemic influenza virus may rarely occur in humans,
when
deficient immune
fail to controlinfluenza
the replicationvirus
of a transmissible
The
evolution
of a responses
novel pandemic
may rarely occur in humans, when deficient immune responses
variant during zoonotic infection. Immunocompromised individuals thus may
represent
the
elusive
index
cases
for
the
generation
of
a
transmissible
variant
of
fail to control the replication of a transmissible variant
during
zoonotic infection. Immunocompromised individuals
zoonotic influenza virus, to titers capable of initiating chains of human-to-human
thus
may represent the elusive index cases for the generation of a transmissible variant of zoonotic influenza virus,
infection.
to titers capable of initiating chains of human-to-human infection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L25
L E C TU R E A BSTRAC TS
SPB302
ALFA
Seroprotection threshold and persistence of seroprotective titer among
2007H1N1 infected subjects: results from the Flurec cohort study
Markovic Delabre, Rosemary (1,2); Salez, Nicolas (3); Lemaitre, Magali (4); Leruez-Ville, Marianne (5,6); de Lamballerie, Xavier (3,7,8); Carrat,
Fabrice (1,2,9)
1: Institut National de la Santé et de la Recherche Médicale, UMR-S 707, Paris, France; 2: Université Pierre et Marie Curie-Paris 6, UMR-S 707,
Paris, France; 3: Unité des Virus Emergents, UMR-D 190, Aix-Marseille université and Institut de Recherche pour le Développement, Marseille,
France; 4: National Agency for the Safety of Medicine and Health Products, St Denis; 5: Université Paris Descartes, Sorbonne Paris Cité, EA 3620, Paris, France; 6: Laboratoire de Virologie, Hôpital Necker, AP-HP, Paris, France; 7: Laboratoire de Virologie, Pôle hospitalier de Microbiologie
et Maladies Infectieuses, Assistance Publique, Hôpitaux de Marseille, France; 8: Ecole des Hautes Etudes en Sante Publique, Rennes, France; 9:
Assistance Publique-Hôpitaux de Paris,Hôpital Saint Antoine, Unité de Santé Publique, F-75012 Paris, France
Background
Haemagglutination-inhibiting (HI) titers, following natural infection and/or vaccination, and their persistence
through time, are important to determining individual susceptibility to infection. However, few studies have
reported long-term data regarding the magnitude and duration of HI titers following infection. Using data from a
French cohort study, designed to identify risk factors of recurrent influenza infection, we studied antibody dynamics
among subjects with and without confirmed seasonal 2007H1N1 infection over time.
Methods
This study focuses on the 2007-2008 H1N1 influenza epidemic and concerns cohort subjects recruited in 2008.
Subjects were recruited during general practitioner visits and stratified by age (10-year age groups) and medical
visit reason (influenza-like illness (ILI) or an illness unrelated to ILI). Serological samples and information regarding
vaccinal status were collected at all three annual study visits. Infection was defined as laboratory-confirmed
influenza A/2007H1N1 on a nasal swab by RT-PCR collected for all ILI subjects at inclusion. This analysis depended
on a total of 621 samples for 224 unvaccinated subjects. Subjects were analyzed according to ILI and infection
status at inclusion (Infected ILI, Non-Infected ILI, and Non-ILI). 2007H1N1 titers ≥ 40, according to conventional
standards, were considered protective. General additive mixed models were fit to log-transformed HI data to study
the evolution of HI titer during study follow-up. Seroprotection threshold titer, defined as the titer associated with
50% protection against infection, was determined using generalized linear model with a logistic regression fit.
Results
Among the 103 ILI subjects, 2007H1N1 infection was identified in 34. A total of 55 (infected ILI subjects n=22;
uninfected ILI subjects n=33) had titers ≥40 by the 2008 annual study visit. Infected ILI subjects reached peak GMT
(73.1, 95CI: 55.4, 96.5) up to 8 months following infection (Figure 1); up to 22 months following infection, 50%
of subjects had titers ≥40. However, titers rise again late during the 2009-2010 influenza season. Titers among
uninfected ILI subjects remained stable throughout study duration. A 2007H1N1 titer of 20.2 (95CI: 7.1; 28.7) was
associated with 50% protection against 2007H1N1 infection.
Conclusions
Subjects reaching HI titer ≥40 after infection were able to maintain this titer level for almost 2 years post-infection.
Rise in 2007H1N1 titers during the 2009-2010 influenza season suggest a cross-reactive antibody response with
H1N1pdm09. We found that the conventional protective titer (HI titer ≥ 40) was conservative compared
to our seroprotection threshold estimate. These results have important implications in the understanding of longterm immunogenicity.
Figure 1: 2007H1N1 antibody dynamics among subjects with 2007H1N1 titer ≥40 at first annual visit (2008):
Infected ILI subjects vs Non-Infected ILI subjects
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L26
to our seroprotection threshold estimate. These results have important implications in
the understanding of long-term immunogenicity.
L E C TU R E A BSTRAC TS
Word count: 399 + 50 (figure)
Figure 1: 2007H1N1 antibody dynamics among subjects with 2007H1N1 titer ≥40 at first annual visit
(2008): Infected ILI subjects vs Non-Infected ILI subjects
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L27
L E C TU R E A BSTRAC TS
SPB303
ALFA
Inferring Influenza Infection Attack Rate from Seroprevalence Data
Wu, Joseph (1); Leung, Kathy (1); Perera, Ranawaka APM (1); Chu, Daniel KW (1); Lee, Cheuk Kwong (2); Hung, Ivan FN (3); Lin, Che Kit (2); Lo,
Su-Vui (4); Lau, Yu-Lung (5); Leung, Gabriel M (1); Cowling, Benjamin J (1); Peiris, JS Malik (1)
1: School of Public Health, The University of Hong Kong, Hong Kong S.A.R. (China); 2: Hong Kong Red Cross Transfusion Services, Hong Kong
S.A.R. (China); 3: Department of Medicine, The University of Hong Kong, Hong Kong S.A.R. (China); 4: Hospital Authority, Hong Kong S.A.R.
(China); 5: Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong S.A.R. (China)
Background
Seroprevalence studies are crucial for estimating infection attack rate and disease severity in an epidemic such as
influenza. These studies typically entail selecting a titer threshold for seropositivity (e.g. microneutralization [MN]
1:40) and assuming the probability of seropositivity per infection (infection-seropositivity probability, ISP) among
community infections is 100% or similar to that among clinical cases. Different thresholds applied to the same
seroprevalence data may give different IAR estimates if serologic responses of clinical cases is not representative
(e.g. clinical cases have higher ISP).
Methods and Results
To illustrate this conjecture, we fitted a transmission model to the 2009 pandemic influenza A/H1N1 (pdmH1N1)
hospitalization and seroprevalence data in Hong Kong. We estimated that while almost all pdmH1N1 infections
became MN1:20 seropositive, only 72%, 62%, 58% and 34% of infections among age 3-12, 13-19, 20-29, 30-59
became MN1:40 seropositive, much lower than the 90%-100% observed among clinical cases. The fitted model was
consistent with prevailing consensus on pdmH1N1 transmission characteristics (e.g. initial reproductive number
of 1.28 and mean generation time of 2.4 days), hence our ISP estimates were consistent with the transmission
dynamics and temporal buildup of population-level immunity.
Conclusions
Incidence of pdmH1N1 in Hong Kong and other countries might have been previously underestimated, especially
among adults, due to overestimations of ISP. Because antibody kinetics among asymptomatic or mild community
infections will likely to be lacking during a pandemic, real-time seroprevalence studies should evaluate the
consistency of IAR estimates across multiple thresholds to provide timely and robust estimates of disease burden
and severity.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L28
L E C TU R E A BSTRAC TS
SPB304
ALFA
The MISMS program: highlights of an international influenza research
network
Miller, Mark Andrew; Collaborating Team, MISMS
FIC/NIH/DHHS, United States of America
Introduction
The Multinational Influenza Seasonal Mortality Study (MISMS) network was initiated in 2001 by the Fogarty
International Center, to research the epidemiology and evolutionary dynamics of influenza and build analytical
capacity. We highlight the main findings of the MISMS program from the past year with a focus on influenza
transmission dynamics.
Methods
Multinational and bilateral collaborations through MISMS researchers facilitate the collection and analysis
of international influenza related data and disseminate findings through scientific publications, conference
presentations, and interactive workshops for investigators and decision‐makers. To date, influenza epidemiological
and genetic sequence data have been shared between more than 30 countries, representing ~3.2 billion people,
resulting in over 220 peer‐reviewed publications and more than 9400 citations.
Findings
The MISMS network has a focus on assessing the transmission dynamics and dissemination of emerging influenza
threats, particularly as it related to trade and connectivity. Leveraging increased influenza sampling from Africa in
the context of the 2009 A/H1N1 pandemic, MISMS researchers demonstrated the multiyear persistence of two
pandemic virus lineages in West Africa, likely due to asynchronous timing of influenza activity and low global
connectivity of this region. These findings highlight the potential for new virus variants to evolve within Africa and
the need for intensified surveillance in this and other undersampled regions.
In the United States, MISMS researchers have shown that the fall wave of the 2009 pandemic travelled over
a 3‐month period in a highly diffusive pattern across the US, unlike seasonal epidemics which spread over a few
weeks through large population centers. High attack rates among children, who may not travel as much as adults,
combined with the relatively low transmissibility, may explain the slow diffusion of the pandemic virus in the US.
From an historical perspective, review of data from the 1918 pandemic has revealed great variation in the overall
burden, timing, and age patterns of excess mortality between countries, potentially due to regional differences in
connectivity in the 19th and early 20th century correlated prior immunity from differential past viral exposures.
Finally, phylogeographic analyses have highlighted the importance of swine trade in the dissemination of swine
viruses both within the US and globally, echoing the diffusion of influenza in humans. In contrast to human viruses
however, diffusion of a new swine influenza variants may take from months to years.
Conclusions
MISMS research findings demonstrate the value of contrasting the ecology and evolutionary dynamics of influenza
across hosts and countries, and the benefits of grounding and calibrating models against empirical data. Further
work is needed to fully understand the global migration of pandemic and seasonal influenza viruses in human,
swine and other hosts, and how this relates to host movements, demographic factors, and prior immunity.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L29
L E C TU R E A BSTRAC TS
1 6 SEPTEM B ER 20 14
SPA4: ANTIVIRALS AND RESISTANCE
SPA401
OMEGA
Influenza A(H7N9) virus gains neuraminidase inhibitor resistance
without loss of in vivo virulence or transmissibility
Hai, Rong (1); Schmolke, Mirco (1,2); Leyva-Grado, Victor H. (1); Thangavel, Rajagowthamee R. (1); Margine, Irina (1); Jaffe, Eric L. (1); Krammer,
Florian (1); Solórzano, Alicia (4); García-Sastre, Adolfo (1,2,3); Palese, Peter (1,3); Bouvier, Nicole M. (1,3)
1: Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L Levy Place, Box 1124, New York, New York 10029, USA;
2: Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, One Gustave L Levy Place, Box 1124, New York,
New York 10029, USA; 3: Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, One Gustave L Levy
Place, Box 1124, New York, New York 10029, USA; 4: Public Health Research Institute and Regional Biocontainment Laboratory, New Jersey
Medical School, RUTGERS, The State University of New Jersey, 225 Warren Street, Newark, New Jersey 07103, USA
Background
Since there is no baseline human immunity to the novel avian origin influenza A (H7N9) virus emerged in eastern
China in the spring of 2013, the neuraminidase (NA) inhibitors oseltamivir and zanamivir are vital for controlling viral
replication in severe infections. An amino acid change associated with drug resistance, NA-R292K (N2 numbering),
has been found in patients infected with H7N9 influenza A viruses and treated with NA inhibitors. However,
resistance mutations usually result in a loss in viral fitness, which may be restored by additional compensatory
changes in the viral genome.
Methods
Here, we investigate whether the R292K-encoding NA of a recent H7N9 clinical isolate confers NA inhibitor resistance
and whether it affects the replication or pathogenicity of this strain in primary human tracheobronchial epithelial
(hTBE) cell culture and in a mouse virulence model. We also explore the replicative ability of oseltamivir-sensitive
and -resistant H7N9 viruses in the lungs of mice treated with oseltamivir or zanamivir, as well as the respiratory
droplet transmissibility of these viruses in the guinea pig model.
Results
Our results demonstrate that the acquisition of high-level oseltamivir resistance, via the NA-R292K mutation, can
significantly impair the sialidase activity of the H7N9 NA without compromising viral replication, virulence, or
transmissibility in these experimental models.
Conclusions
Compared to seasonal A(H3N2) viruses bearing the R292K resistance mutation, H7N9 IAV replication and
pathogenicity in these models are not substantially altered by the acquisition of high-level oseltamivir resistance.
These findings underscore the multigenic and sometimes unpredictable nature of the fitness of influenza A viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L30
L E C TU R E A BSTRAC TS
SPA402
OMEGA
Influenza antiviral susceptibility monitoring in Europe – taking stock of
10 years’ experience, 2004-2014
Meijer, Adam (1,10); Lackenby, Angie (2,10); Pereyaslov, Dmitriy (3); Enouf, Vincent (4,10); Kossyvakis, Athanasios (5,10); Lina, Bruno (6,10);
Rebelo-de-Andrade, Helena (7,10); Pozo Sánchez, Francisco (8,10); Hay, Alan (9); McCauley, John (9); Daniels, Rod (9); Zambon, Maria (2)
1: National Institute for Public Health and the Environment, Bilthoven, The Netherlands; 2: Public Health England Colindale, London, United
Kingdom; 3: Division of Communicable Diseases, Health Security, & Environment, World Health Organization Regional Office for Europe,
Copenhagen, Denmark; 4: Centre National de Référence du Virus Influenza Région North, Institut Pasteur, Paris, France; 5: National Influenza
Reference Laboratory of Southern Greece, Institut Pasteur Hellénique, Athens, Greece; 6: Centre National de Référence du Virus Influenza
Région Sud, Laboratoire de Virologie Est, Institut de Microbiologie Bat A3, Centre de biologie et de pathologie Est, HCL, Lyon, France; 7:
Instituto Nacional de Saúde, and Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal; 8: National Influenza Centre, Influenza
and Respiratory Viruses Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; 9: World Health
Organization Collaborating Centre for Reference and Research on Influenza, MRC-National Institute for Medical Research, The Ridgeway, Mill
Hill, London, United Kingdom; 10: ECDC antiviral susceptibility task group (AVTG) members
Background
Influenza antiviral drug susceptibility monitoring has become an important activity of WHO National Influenza
Centres (NICs) and ERLI-Net in Europe. With the availability, stockpiling, and wider use of neuraminidase inhibitors
Background
(NAI) and the emergence of natural NAI resistant variants, identification of variant viruses with reduced inhibition
Influenza antiviral drug susceptibility monitoring has become an important activity of WHO
by NAI
andCentres
M2 (NICs)
ion-channel
blockers
is With
necessary
to inform
the clinical management of influenza patients and
National
Influenza
and ERLI-Net
in Europe.
the availability,
stockpiling,
and wider
use of neuraminidase
inhibitors (NAI) and the emergence of natural NAI resistant
outbreak
management.
variants, identification of variant viruses with reduced inhibition by NAI and M2 ion-channel
blockers is necessary to inform the clinical management of influenza patients and outbreak
management.
Methods
Antiviral susceptibility (AS) testing coordinated with surveillance and reporting via a web-based system was initiated
Methods
through VIRGIL and EISS EU funded projects in 2004 (Figure 1). A database with online data analysis facilities was
Antiviral susceptibility (AS) testing coordinated with surveillance and reporting via a web-based
developed,
to collate
AS EISS
dataEUgenerated
nationally
and1).centrally
within Europe, together with epidemiological
system
was initiated 1)
through
VIRGIL and
funded projects
in 2004 (Figure
A
database
online data,
data analysis
was developed,
1) toincollate
AS data generated
andwith
clinical
and facilities
2) to assist
countries
the analysis
and interpretation of AS data. Data collection and further
nationally and centrally within Europe, together with epidemiological and clinical data, and 2) to
of ASandtesting
capabilities
have
continued
through the ERLI-Net and EuroFlu networks. The AVTG
assistdevelopment
countries in the analysis
interpretation
of AS data. Data
collection
and further
development
of AS testing
capabilities development
have continued through
ERLI-Net protocols,
and EuroFlu guidance and tools for testing and interpretation of
contributed
to database
andthe
provided
networks. The AVTG contributed to database development and provided protocols, guidance and
AS data.
tools for testing and interpretation of AS data.
Results
The usefulness of centralized data collection and analysis, with efforts to increase the capability for national AS
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L31
L E C TU R E A BSTRAC TS
monitoring, were fully validated by the response to the events of the 2007/2008 winter season when emergence
of high levels of natural neuraminidase (NA)-H275Y oseltamivir resistant A(H1N1) viruses was first demonstrated
in Europe. The database was the key tool used at European and global level for accurate dissemination of A(H1N1)
NA-H275Y prevalence and geographical spread data. Also the emergence of natural adamantane resistant A(H1N1)
and A(H3N2) viruses with M2-S31N was closely monitored, which gained increased importance, as the options for
antiviral treatment and prophylaxis became limited due to the simultaneous emergence of A(H1N1) NA-H275Y.
Again, during the 2009 A(H1N1)pdm09 pandemic its usefulness was emphasized by allowing the identification
and tracking of (highly) reduced inhibition by neuraminidase inhibitors, mainly due to NA-H275Y, among A(H1N1)
pdm09 influenza viruses in Europe. Furthermore, collection of clinical data allowed the pooled analysis of the
potential effect of A(H1N1) NA-H275Y on the severity of infection; which appeared to be absent.
Conclusions
Joined efforts increased the capacity for AS monitoring in the WHO European Region, reflected in effective responses
to emergencies and continuous vigilance by weekly analysis and reporting of AS surveillance data. Ongoing
challenges are to introduce AS monitoring in a wider number of NICs and further standardization of analyses
performed on pooled phenotypic and genotypic AS monitoring data.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L32
L E C TU R E A BSTRAC TS
SPA403
OMEGA
Evidence for clinical efficiency potential of anti-H5N1 polyclonal
immunoglobulins Fabenflu®
Herbreteau, Cécile Hélène (1); Buchy, Philippe (2); Bal, Céline (1); Durand, Caroline (1); Tambyah, Paul (3); Lépine, Bertrand (1)
1: Fab’entech, France; 2: Institut Pasteur, Cambodia; 3: National University Hospital, Singapore
Background
Regarding the insufficiency of therapeutic solutions for the management of H5N1 infected patient, specific antiH5N1 polyclonal immunoglobulins were developed. A strong neutralizing (and cross-neutralizing) activity was
previously demonstrated by the pre- clinical characterization of anti-H5N1 immunoglobulin F(ab’)2 fragments
(Fabenflu®) (Herbreteau et al., 2014). Following these studies, a product for human use was produced and a phase
I clinical trial was designed and conducted in Singapore to assess the safety of the proposed therapeutic protocol,
and determine the pharmacokinetic profile of intravenous injections. The excellent safety profile of the product was
confirmed by this study.
In addition to the phase I clinical trial, complementary studies were performed to (1) document the efficacy of
Fabenflu® in human plasma or serum at concentrations equivalent to the ones measured during the phase I clinical
trial and (2) evaluate the residual neutralizing activity of the human plasmas collected in healthy human volunteers
during the phase I clinical trial.
Methods
Human plasmas and sera spiked with Fabenflu®, or human plasmas collected in healthy human volunteers during
the phase I clinical trial, were tested by classical in vitro serum neutralization and hemagglutination inhibition
assays on H5N1 A/Vietnam/1194/04 strain.
Results
A neutralizing activity was evidenced in human serum and plasma spiked with Fabenflu® even at low concentrations,
from 5 μg/mL. Neutralization titer rates up to 1:12 were found in the plasma of human healthy volunteers sampled
during the phase I clinical trial. The serum neutralization activity increase with the augmentation of plasmatic
concentration of Fabenflu® measured by ELISA.
Conclusions
The neutralizing efficacy of Fabenflu® was confirmed in a human matrix, and at therapeutic concentration.
Fabenflu® neutralizing activity on the H5N1 avian influenza virus was confirmed by both the spiking experiment
and the analysis of the human plasmas collected during the phase I clinical trial.
These data, associated with safety and pharmacokinetic data from the phase I clinical trial and cross-reactivity data
on 21 H5N1 strains, demonstrate the excellent potential of Fabenflu® as a new therapeutic approach in the clinical
management of H5N1 infected patient.
Special accesses are explored in order to answer to public health need and ensure the availability of this agent in
exposed countries, pending the granting of a marketing authorization.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L33
L E C TU R E A BSTRAC TS
SPA404
OMEGA
A community cluster of influenza A(H1N1)pdm09 virus exhibiting crossresistance to oseltamivir and peramivir in Japan, November 2013 to
February 2014
Takashita, Emi (1); Ejima, Miho (1); Fujisaki, Seiichiro (1); Yokoyama, Masaru (2); Nakamura, Kazuya (1); Shirakura, Masayuki (1); Sugawara,
Hiromi (1); Sato, Aya (1); Sato, Hironori (2); Tashiro, Masato (1); Odagiri, Takato (1)
1: National Institute of Infectious Diseases, Influenza Virus Research Center, Japan; 2: National Institute of Infectious Diseases, Pathogen
Genomics Center, Japan
Background
In Japan, four neuraminidase (NA) inhibitors, oseltamivir, peramivir, zanamivir and laninamivir, are approved for
chemotherapy against influenza and are prescribed with the highest frequency in the world. Therefore, Japan
could be at high risk of the emergence and spread of NA inhibitor-resistant viruses. We have conducted nationwide
monitoring for NA inhibitor-resistant viruses in cooperation with 74 local public health institutes. The nationwide
monitoring is important for public health planning and clinical recommendations for antiviral use.
Methods
Clinical specimens were collected in 500 sentinel hospitals consisting of pediatrics and internal medicine as part
of the National Epidemiological Surveillance of Infectious Diseases in Japan. Most specimens were collected
at the first visit to clinics before drug use. Influenza viruses were isolated from the specimens in 74 local public
health institutes. Between September 2013 and May 2014, 2,221 A(H1N1)pdm09 viruses were detected and then
screened by allelic discrimination assay and/or NA gene sequencing to detect an H275Y substitution, which confers
resistance to oseltamivir and peramivir. The susceptibility of the viruses to four NA inhibitors approved in Japan was
determined by fluorescent enzyme inhibition assay. Results were expressed as
the drug concentrations required to inhibit NA activity by 50% (IC50).
Results
We found that 97 (4.4%) of the 2,221 A(H1N1)pdm09 viruses possessed the H275Y substitution. Thirty-four of
the 97 H275Y mutant viruses were detected in Sapporo, the capital city of Hokkaido, the second-largest island
in Japan, between November 2013 and February 2014. In Sapporo, 108 A(H1N1)pdm09 viruses were detected
between September 2013 and May 2014, and 34 (31.5%) of the 108 viruses possessed the H275Y substitution.
Elsewhere in Hokkaido, 5 (19.2%) of the 26 A(H1N1)pdm09 viruses possessed the H275Y substitution. Outside
Hokkaido, 58 (2.8%) of the 2,087 A(H1N1)pdm09 viruses were the H275Y mutants. All H275Y mutant viruses
exhibited highly reduced inhibition by oseltamivir and peramivir, but remained fully susceptible to zanamivir and
laninamivir. No epidemiological link among the H275Y cases in Hokkaido could be identified and none of them had
received NA inhibitors before specimen collection. The haemagglutinin and NA genes of the H275Y mutants in
Hokkaido were closely related to one another, suggesting clonal spread of a single mutant virus. The H275Y mutant
viruses in Hokkaido possessed three additional substitutions in NA and single substitution in PB1, nucleoprotein,
haemagglutinin and matrix protein (M1), respectively. Studies are underway to elucidate the effect of these
substitutions on the stability and transmissibility of the mutant virus.
Conclusions
A community cluster of A(H1N1)pdm09 virus exhibiting cross-resistance to oseltamivir and peramivir occurred in
Sapporo, Japan, between November 2013 and February 2014. The nationwide monitoring should be continued,
particularly for the choice of antivirals.
This study was conducted by collaboration with the influenza virus surveillance group of Japan.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L34
L E C TU R E A BSTRAC TS
SPB4: ANIMAL FLU-ECOLOGY AND EPIDEMIOLOGY OF ANIMAL INFLUENZA
SPB401
ALFA
Equine Influenza virus surveillance in Ireland – findings of international
significance
Gildea, Sarah; Cullinane, Ann
Irish Equine Centre, Ireland
Background
Equine influenza (EI) is considered the most important respiratory pathogen of the horse and outbreaks in endemic
populations continue to cause economic loss despite widespread mandatory vaccination. Ireland is the 4th largest
producer of Thoroughbred horses in the world and the majority of these are exported worldwide. The gross value
of the equine industry to our economy is in excess of €1 billion euros per annum. The aim of this study was to carry
out a comprehensive analysis of EI outbreaks in Ireland, to identify key management and environmental factors that
determine the risk of horses contracting EI, to genetically characterise EI viruses responsible for outbreaks and to
highlight any evidence of vaccine breakdown.
Methods
Between 2007 and 2014 epidemiological investigations involving 587 horses on 28 premises where EI outbreaks
occurred were undertaken.
Results
Outbreaks occurred among Thoroughbred horses, non-Thoroughbred horses, ponies and donkeys. With one exception,
all outbreaks were associated with movement of horses but no epidemiological link between affected premises was
established. Veterinary advice was sought on average six days after first clinical signs were observed. The majority
of diagnosis was made by RT-PCR. Of the 587 horses, 47% were clinically affected, 41% were identified as confirmed
cases and 10% were subclinically affected. Vaccination status, age, housing type, teaser stallions and fomites were all
identified as risk factors involved in virus spread. Analysis of the HA1 sequence data generated for 44 viruses identified
indicated that with the exception of those which circulated from November 2009 to January 2010 all viruses belong
to clade 2 of the Florida sublineage of the American lineage. Viruses identified from November 2009 to January 2010
belonged to clade 1 of the Florida sublineage. This was the first time clade 1 viruses were identified in Ireland. Following
several outbreaks of EI in the UK and the first outbreak of EI in Australia where the index cases were horses which had
travelled from Ireland, sequence data generated during this study highlighted that the index cases during an outbreak
may not necessarily be the original source of the virus. Viruses identified during this study were submitted annually
for review by the OIE Expert Surveillance Panel which makes recommendations for updating vaccine strains. None of
the EI vaccines available in Ireland have been updated in line with OIE recommendations of 2010 to include both clade
1 and clade 2 viruses. Vaccine failure was observed in this study among horses vaccinated with all four commercially
available vaccines.
Conclusions
The identification of key management and environmental factors which assist in EI virus spread along with appropriate
preventative and controls measures should reduce the economic losses associated with EI. The findings of this study
endorse the OIE recommendations.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L35
L E C TU R E A BSTRAC TS
SPB402
ALFA
European surveillance network for influenza in pigs (ESNIP): Surveillance
programs, diagnostic tools and swine influenza viruses identified in 14
European countries from 2010 to 2013
Simon, Gaëlle (1); Larsen, Lars E (2); Dürwald, Ralf (3); Foni, Emanuela (4); Van Reeth, Kristien (5); Reid, Scott M (6); Harder, Timm (7); Dan,
Adam (8); Markowska-Daniel, Iwona (9); Maldonado Garcia, Jaime (10); Huovilainen, Anita (11); Billinis, Charalambos (12); Davidson, Irit (13);
Agüero Garcia, Montserrat (14); Bublot, Michel (15); Brown, Ian H (6); Loeffen, Willie (16)
1: Anses, Ploufragan, France; 2: DTU, Copenhagen, Denmark; 3: IDT-Biologika GmbH, Dessau-Rosslau, Germany; 4: IZSLER, Parma, Italy; 5:
Ghent University, Ghent, Belgium; 6: AHVLA, Weybridge, United Kingdom; 7: FLI, Reims, Germany; 8: Nébih, Budapest, Hungary; 9: NVRI,
Pulawy, Poland; 10: HIPRA, Gerona, Spain; 11: EVIRA, Helsinki, Finland; 12: Thessaly University, Karditsa, Greece; 13: KVI, Rishon L’Tzion, Israel;
14: LCV, Algete, Spain; 15: Merial, Lyon, France; 16: CVI, Lelystad, The Netherlands
Swine influenza causes concern for global veterinary and public health. Thus, ongoing knowledge of viral strains and
lineages circulating in pigs is indispensable not only for the prevention and control of the disease in pigs, but also
to detect reassortant viruses that may pose a threat to the human population, as was the case for the novel H1N1
virus responsible for the last pandemic in 2009 (H1N1pdm).
In continuation to two previous coordination actions that initiated the surveillance for swine influenza viruses
(SIVs) circulating in European pigs between 2001 and 2008, a third phase of the European Surveillance Network
for Influenza in Pigs (ESNIP3), aimed to expand widely the knowledge of the epidemiology of European SIVs from
2010 to 2013. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the
coordination of appropriate diagnostic tools and subtyping methods. Thus, in an extensive virological monitoring,
mainly conducted through passive surveillance programs, more than 9 000 herds in 17 countries were examined
over a period of three years.
Influenza A viruses were detected in 31% of these herds, from which 1887 viruses were subtyped preliminary. The
dominating lineages were identified to represent the three previously known European enzootic SIVs, i.e. the avianlike swine H1N1 (53.6%), the human-like reassortant swine H1N2 (13%) and the human-like reassortant swine
H3N2 (9.1%), but also viruses from the H1N1pdm lineage (10.3%). Viruses from these four lineages co-circulated in
several countries, but with very different relative levels of incidence. The H3N2 subtype, for instance, was missing
from some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, areas free of H3N2
were those that exhibited the highest frequencies of H1N2 viruses in circulation. H1N1pdm viruses were detected
at an increasing rate in some countries throughout the three year period, indicating that this subtype has become
established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these
four lineages. Among them, reassortants between older enzootic SIVs and H1N1pdm emerged at the same time in
several countries. They were detected at an increasing frequency in 2012-2013, indicating that some of them might
become established in pig herds and potentially provide implications for zoonotic infections.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L36
L E C TU R E A BSTRAC TS
SPB403
ALFA
Pathobiology of novel genotype of H9N2 containing polymerase (PB2,
PB1, PA) and non-structural (NS) gene segments derived from high
pathogenicity H7N3 avian influenza virus
Iqbal, Munir (1); Yaqub, Tahir (2); Mukhtar, Nadia (2); Shabbir, Muhammad Z (2); McCauley, John W (3)
1: Avian Viral Diseases Programme,The Pirbright Institute, United Kingdom; 2: University of Veterinary and Animal Sciences, Lahore, Pakistan;
3: Division of Virology, MRC National Institute for Medical Research, Mill Hill, London
Background
Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we
identified a novel genotype of H9N2 viruses in widespread circulation in domestic poultry on the Indian subcontinent
and in the Middle East containing the polymerase (PB2, PB1 and PA) and non-structural (NS) gene segments which
were identical to highly pathogenic H7N3 viruses. The other genes shared homology to the G1-like H9N2 virus
lineage. Infections with H9N2 have been reported in a diverse variety of wild avian species including quail, crows and
sparrows. Limited data are available that describe the relative infectivity and transmissibility of these novel H9N2
viruses in different poultry breeds and terrestrial wild bird species. Here, we investigated the potential of this novel
genotype of H9N2 virus to cause disease in poultry and wild terrestrial avian species and assess the transmission
capability between these species.
Materials and methods
Groups of chicken broilers, chicken layers, jungle fowl, quail, sparrows or crows were inoculated with a representative
strain (A/chicken/UDL-01/08) of this novel H9N2 virus and then co-housed with naïve birds of the same breed or
species. Transmission between different species was examined by co-housing infected broilers with naïve sparrows
and vice versa. Pathogenesis in birds was monitored by recording clinical disease signs. Buccal and
cloacal swab samples were taken and analysed for virus shedding and blood samples allowed the detection of H9N2
virus-specific antibodies using haemagglutination inhibition tests.
Results
With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with
quail showing the most pronounced clinical signs. Viral shedding was detected in all infected birds, with quail
showing the greatest levels of virus secretion; however, only very low levels of virus were found in directly infected
crow samples. Efficient virus intra-species transmission was observed within each group with the exception of
crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens
and sparrow and vice versa and efficient transmission was seen in both directions.
Conclusions
Our results indicated that reassortant H9N2 viruses currently circulating on the Indian sub- continent and in the
Middle East can cause clinical disease in experimentally infected commercial and backyard chicken breeds as well
as in quail and house sparrows. Our observations of efficient transmission between different chicken breeds, from
quail to quail, and from chickens to sparrows or vice versa is a worrying scenario in which the quail and sparrows
could be potential intermediate hosts for maintenance of viruses and the transmission of these viruses to poultry.
Crows seem less of a problem because although they became infected they did not transmit the virus to contacts
and had only low virus titres in swabs. These data, along with the recent identification in China of a human zoonotic
H7N9 virus that contains the polymerase genes from a H9N2 virus, warrant an increased surveillance of circulating
H9N2 viruses. Further experimental infection studies of new reassortant H9N2 viruses will be needed to assess their
pathogenicity and transmissibility in different poultry hosts and terrestrial wild bird species, especially those that
might act as carriers and as a reservoir of infection and potentially shuttle the viruses between poultry and, possibly,
hence to humans.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L37
L E C TU R E A BSTRAC TS
SPB404
ALFA
Isolation of H15N7 influenza virus from wild birds in Eastern Europe and
its relationship to viruses from other regions
Muzyka, Denys (1); Pantin-Jackwood, Mary (2); Fereidouni, Sasan (3)
1: National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, Ukraine; 2: Southeast Poultry Research Laboratory,
Agricultural Research Service, USDA, Athens, Georgia; 3: WESCA Wildlife Network; Friedrich Loeffler Institute, Riems, Germany
Background
Continuous monitoring of influenza viruses circulating in the natural reservoir in wild birds is important for early
warning and forecasting of epizootic and epidemiological situation with influenza. These studies allow getting
much valuable information on the environmental features of influenza viruses’ circulation, new ecological niches of
viruses, to detect new variants of the virus and establish their introduction into new geographic regions.
Methods
Monitoring of AIVs in wild birds of 27 families and 11 genera was conducted in 2006-2012 in the Azov-Black Sea
region of Ukraine.
Results
As part of the monitoring of wild bird influenza in 2010 an influenza virus subtype H15N7 was isolated (А/mallard/
Novomychalivka/2-23-12/10) from a clinically healthy wild mallard (Anas platyrhynchos) in the Azov-Black Sea
region of Ukraine. This virus grew well in chicken embryos without causing their death. The HA titer was 1:512, and
the infectious titer was 6,5 lg EID50/0.2 ml. This subtype of virus circulated only in wild birds of Australia, but in 2008
the virus A/teal/Chany/7119/2008 (H15N4) was isolated from wild birds in Western Siberia (Russia).
Phylogenetic analysis of HA15 gene Ukrainian virus (А/mallard/Novomychalivka/2-23- 12/10 H15N7) showed that
the virus was closely related to the virus A/teal/Chany/7119/2008 (H15N4) from Russia, and the N7 gene it was
related to avian influenza viruses of H7N7 and H10N7 subtypes circulating in wild birds in Western Europe. M gene
sequencing revealed a close relationship with AIV of different subtypes from Russia and Mongolia, and analysis of
the NP, NS, and PB1 genes revealed that the Ukrainian virus is related to influenza viruses of different subtypes from
Norway and Sweden, and the PB2 gene sequence was similar to viruses of Asian origin.
Conclusions
The H15N7 avian influenza subtype was isolated for the first time from wild birds in Eastern Europe. This virus is a
reasortant between influenza viruses of different subtypes from Western Europe, Western Siberia and Asia. These
results support the need for regular monitoring of influenza viruses circulating in the Azov-Black Sea region in order
to study the characteristics of their circulation and the possible introduction of new genetic variants of the virus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L38
L E C TU R E A BSTRAC TS
SPA5: VACCINES: CURRENT AND NOVEL APPROACHES
SPA501
OMEGA
Modified vaccinia Ankara encoding influenza hemagglutinin induces
heterosubtypic immunity in macaques
Florek, Nicholas W. (1,2); Weinfurter, Jason T. (1,2); Jegaskanda, Sinthujan (3); Brewoo, Joseph N. (4); Powell, Tim D. (4); Young, Ginger R. (4);
Das, Subash C. (4); Hatta, Masato (1); Broman, Karl W. (1); Hungnes, Olav (5); Dudman, Susanne G. (5); Kawaoka, Yoshihiro (1); Kent, Stephen J.
(3); Stinchcomb, Dan T. (4); Osorio, Jorge E. (1,4); Friedrich, Thomas C. (1,2)
1: University of Wisconsin, USA; 2: Wisconsin National Primate Research Center, USA; 3: University of Melbourne, Australia; 4: Takeda Vaccines,
USA; 5: Norwegian Institute of Public Health, Norway
Background
Current influenza vaccines primarily aim to induce neutralizing antibodies (NAbs). These vaccines are highly effective
against well-matched strains, but do not offer broad protection against emerging strains. A vaccine that elicits both
T cell and B cell responses might induce more broadly protective immunity. Modified vaccinia Ankara (MVA) is a safe
and well-characterized vector for inducing both antibody and cellular immunity; in this study, we evaluated the
immunogenicity and protective efficacy of MVA vaccines expressing hemagglutinin (HA) and/or nucleoprotein (NP)
in a translational macaque model.
Methods
We tested 4 MVA constructs encoding either HA from an H1N1 virus; the H1 HA with NP from an H5N1 virus; HA
and NP from an H5N1 virus; or H5N1 NP alone. Vaccines were given to cynomolgus macaques (Macaca fascicularis)
intradermally in 2 doses of 108 pfu 4 weeks apart. 8 weeks later, animals were challenged with 107 pfu of the
pandemic isolate A/California/04/2009 (H1N1pdm). During the vaccination and challenge phases, we monitored
neutralizing and HA- binding antibody responses; virus-specific CD4+ and CD8+ T cells; and antibody-dependent
natural killer (NK) cell activation.
Results
MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs,
but did not stimulate strong T cell responses prior to challenge. However, animals vaccinated with MVA encoding
influenza antigens made strong virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm
infection, while animals vaccinated with MVA encoding irrelevant antigens did not. There was little or no H1N1pdm
replication in animals that received vaccines encoding H1 (homologous) HA, while in contrast, the vaccine encoding
only NP from an H5N1 virus provided no protection. Surprisingly, shedding of H1N1pdm was reduced in animals
vaccinated with MVA encoding HA and NP from an H5N1 isolate. This partial protection was associated with crossreactive antibodies capable of binding H1 HA protein and eliciting antibody-dependent cellular cytotoxicity (ADCC)
effector functions from NK cells.
Conclusions
Our results suggest that ADCC may play a previously unrecognized role in heterosubtypic immunity to influenza.
Vaccines optimized to induce cross-reactive “ADCC antibodies” may provide an important measure of protection
against emerging antigenic variants, including emerging pandemic viruses, when NAbs are ineffective.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L39
L E C TU R E A BSTRAC TS
SPA502
OMEGA
Mva-based influenza vaccines – bringing these viral vectors from bench
to bedside
de Vries, Rory Dylan (1); Kreijtz, Joost (1); Sutter, Gerd (2); Osterhaus, Albert (1); Rimmelzwaan, Guus (1)
1: Viroscience department, Erasmus MC, Rotterdam, the Netherlands; 2: LMU, Munich, Germany
Conventional influenza vaccines contain inactivated viruses or components thereof and have been used for over 50
years. They form the cornerstone of the seasonal influenza vaccination campaigns organized to effectively protect
high-risk patients against influenza annually. In contrast, executing an effective vaccination campaign against a
pandemic outbreak of influenza is a complex challenge. Pandemic influenza vaccine development suffers from many
hurdles that complicate the production, distribution and timely availability of sufficient amounts of vaccines with
MVA-BASED INFLUENZA VACCINES – BRINGING THESE VIRAL VECTORS FROM BENCH TO BEDSIDE
good immunogenicity. Several
of the these hurdles have been overcome by the use of reverse genetics for vaccine
Rory D. de Vries
[email protected]
strain construction, the
availability of cell-culture based production platforms and the development and registration
Joost
Kreijtz PhD, Viroscience
lab, the
Rotterdam,
The Netherlands
of several
adjuvants.
To address
hurdles
in pandemic influenza vaccine development new vaccine platforms are
Guus Rimmelzwaan PhD, Viroscience lab, Rotterdam, The Netherlands
Gerd
Sutter
PhD,
LMU,
Munich,
Germany
explored and under development. A promising platform is that of viral vector vaccines such as adenoviral vectors
Albert Osterhaus PhD, Viroscience lab, Rotterdam, The Netherlands
and poxviral vectors. In the last category, Modified Vaccinia virus Ankara (MVA) is a promising candidate.
Conventional influenza vaccines contain inactivated viruses or components thereof and have been used for over
50 years. They form the cornerstone of the seasonal influenza vaccination campaigns organized to effectively
MVA
is a replication
originally
tested
as safe
alternative for the traditional poxvirus vaccine. It is
protect high-risk
patients
against influenzadeficient
annually. Inpoxvirus,
contrast, executing
an effective
vaccination
campaign
against a pandemic outbreak of influenza is a complex challenge. Pandemic influenza vaccine development
being evaluated as a vaccine platform for different infectious diseases and as a therapeutic vaccine. These studies
suffers from many hurdles that complicate the production, distribution and timely availability of sufficient
amounts of vaccines
good immunogenicity.
Several of the these
haveevaluation.
been overcome The
by theavailable
use of
are in with
various
phases of pre-clinical
andhurdles
clinical
data demonstrate that the vector is highly
reverse genetics for vaccine strain construction, the availability of cell-culture based production platforms and
the development
and
registration
of
several
adjuvants.
To
address
the
hurdles
in
pandemic
influenza
vaccine
immunogenic, even in the presence of pre-existing immunity to the vector.
development new vaccine platforms are explored and under development. A promising platform is that of viral
vector vaccines
such as adenoviral
vectors and poxviral
vectors. Invaccines,
the last category,
Modified Vaccinia virus
To construct
recombinant
MVA-vector
the hemagglutinin
(HA) gene can be cloned into the MVA- genome
Ankara (MVA) is a promising candidate.
through homologous recombination. This was done for the HA gene of influenza virus A/Vietnam/1194/04 (H5N1),
MVA is a replication deficient poxvirus, originally tested as safe alternative for the traditional poxvirus vaccine. It
a highly
pathogenic
influenza
mice
and cynomolgus
macaques the MVA-H5 vaccine proved to be safe
is being evaluated
as a vaccine
platform avian
for different
infectious virus.
diseasesIn
and
as a therapeutic
vaccine. These
studies are in various phases of pre-clinical and clinical evaluation. The available data demonstrate that the
and
it induced
immunity
against
homologous
vector is highly
immunogenic,
evensterile
in the presence
of pre-existing
immunity
to the vector. and heterologous challenge viruses. Because of its favourable
and excellent
safety
profile(HA)
MVA
a bepromising
candidate
as a future pandemic influenza vaccine
To constructproperties
recombinant MVA-vector
vaccines, the
hemagglutinin
geneiscan
cloned into the
MVAgenome through homologous recombination. This was done for the HA gene of influenza virus
production platform.
A/Vietnam/1194/04 (H5N1), a highly pathogenic avian influenza virus. In mice and cynomolgus macaques the
MVA-H5 vaccine proved to be safe and it induced sterile immunity against homologous and heterologous
challenge viruses. Because of its favourable properties and excellent safety profile MVA is a promising
candidate asFor
a future
influenza
vaccine production
thepandemic
further
development
the platform.
MVA-H5 was adjusted to make it suitable for use in humans and in 2013/2014
For the further
development
the MVA-H5
was adjusted
to make ittrial
suitable
for use
in humans andResults
in 2013/2014
the
first-in-man
phase
I/II clinical
was
conducted.
obtained indicate
the first-in-man phase I/II clinical trial was conducted. Results obtained indicate that the vaccine is safe and
immunogenicimmunogenic
in humans and holdsin
promise
as
an
influenza
vaccine
candidate.
humans and holds promise as an influenza vaccine candidate.
that the vaccine is safe and
Figure: Clinical trial layout
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L40
L E C TU R E A BSTRAC TS
SPA503
OMEGA
Specific induction of neutralizing antibodies against HA, and T cell
responses against conserved influenza antigens
Grødeland, Gunnveig (1); Mjaaland, Siri (2); Baranowska, Marta (1); Fredriksen, Agnete (1); Bogen, Bjarne (1)
1: University of Oslo, Norway; 2: Institute of Public Health, Norway
Background
Current influenza vaccines are mostly aiming at the induction of specific neutralizing antibodies. While these are
important for protection against a particular strain, T cells can recognize epitopes that will offer a broader protection
against influenza. We have previously developed a DNA format in which antigens can be targeted specifically to
receptors on antigen presenting cells (APCs). Here, we have investigated whether targeting to different receptors can
polarize the immune system to different arms of immunity. The aim is thus to construct vaccines that specifically
can induce the best suited type of immune responses against a particular influenza antigen.
Methods
We have constructed DNA vaccines where influenza antigens are bivalently targeted to Major Histocompatibility
Complex (MHC) class II molecules or various chemokine receptors (CCR1/3/5) on antigen presenting cells (APCs) in
mice. The DNA encoded vaccine molecules are homodimers, each chain consisting of a targeting unit, a dimerization
unit, and an antigen.
Results
A single DNA immunization in mice with MHC class II-targeted hemagglutinin (HA) can induce long-lasting
neutralizing and protective antibodies against influenza. By contrast, targeting of HA to CCR1/3/5 will predominantly
induce protective CD8+/Th1 responses. In order to fully take advantage of the targeting dependent polarization,
we constructed targeted vaccines encoding internal influenza antigens. Again, we found that the polarization
of protective immune responses was dependent on the selection of receptor on APCs. Substitution of influenza
antigens with ovalbumin (OVA) further indicated that the polarization of immune responses holds true for a variety
of antigens.
Conclusions
Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of
adaptive immune responses by specifically targeting different surface molecules on APCs. This could be used to
develop vaccines that specifically induce high levels of neutralizing antibodies against HA, while simultaneously
inducing high levels of CD8+ T cells against conserved internal influenza antigens.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L41
L E C TU R E A BSTRAC TS
SPA504
OMEGA
Evaluation of heterosubtypic immune responses in older people
following pre-pandemic H5N1 influenza vaccination
M Levine (1)*, K Nicholson (2), S Batham (2), D Wang (1), B Dighero-Kemp (1), ZN Li1, V Veguilla (1), F Liu1, XH Lu (1), J Katz (1).
1: Centers for Disease Control and Prevention, Atlanta, Georgia, United States; 2: Infectious Disease Unit, University Hospitals Leicester, United
Kingdom
Background
Vaccination is the primary public health measure to control seasonal and pandemic influenza. However,
vaccine responses may be influenced by host factors including age and levels of baseline antibodies. Previously, it was demonstrated that a proportion of older adults exhibited low baseline serum antibody titers to
H5N1 virus. We evaluated the impact of baseline H5 antibody on the outcome of H5N1 vaccine responses in
older adults and compared antibody responses with younger adults that received H5N1 vaccine; the possible virus priming history of vaccinees was investigated by examining the cross-reactive antibody responses
to a panel of historic H1N1 viruses.
Materials and Methods
Older adults (64-81 yrs, n=27) and young adults (19-39 yrs n=20) received two doses of subvirion H5N1
vaccine (A/Vietnam/1194/2004 NIBRG14) with MF-59 adjuvant. Sera were tested by HI and MN assays
against H5N1 vaccine virus, and by HI against a panel of H1N1 viruses (BPL inactivated A/SC/1/18 RG virus,
A/MT/43, A/FM/1/47, A/USSR/90/77, A/TW/1/86, A/NC/20/99, and A/MX/4108/09). Antibody cross adsorption studies were conducted with magnetic beads and Luminex® multiplex assays using rHAs from the
above subtypes.
Results
To compare the effect of priming, older adults were divided into 2 subgroups based on their baseline HI
titers to H5N1: primed (HI≥8, n=11) and unprimed (HI<8, n=16). After one dose of H5N1 vaccine, primed
older adults mounted significantly higher HI and MN responses (91% achieved titer ≥40 by HI, 73% ≥40 by
MN), than unprimed older adults (6% ≥40 by HI, 13% ≥40 by MN) and young adults (30% ≥40 by HI, 15% ≥40
by MN), and similar to those in young adults after 2 doses of vaccine (80% ≥40 by HI, 70% ≥40 by MN).
To better understand the priming effect, sera from older and young adults were tested by HI with 8 H1N1
viruses circulated from 1918 to 2009; the age range of older adults was consistent with exposure to H1N1
viruses early in life. For older adults, titers to A/SC/1/18 and A/MT/43 viruses were significantly higher in
primed compared with unprimed group, suggesting that these priming viruses contributed to the baseline
H5 antibody titer. Younger adults exhibited priming by more modern era viruses including A/TW/1/86 and
A/NC/20/99. Primed elderly were also more likely to seroconvert to H1N1 viruses following H5N1 vaccination than either unprimed older or younger adults. Analysis of antibody adsorption studies is ongoing to
determine the effect of priming on the quality of the H5N1 vaccine response.
Conclusions
Compared with unprimed young and older adults, older adults with a baseline H5N1 titer mounted robust
antibody responses to adjuvanted H5N1 vaccine after a single dose. This study provides further understanding of effects of priming on vaccine responses that will inform better vaccination strategies.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L42
L E C TU R E A BSTRAC TS
SPB5: GENETICS AND EVOLUTION OF VIRUS AND HOST
SPB501
ALFA
Two independent evolutionary pathways of HPAIV
Stech, Olga; Veits, Jutta; Abdelwhab, Sayed; Wessels, Ute; Mettenleiter, Thomas C.; Stech, Juergen
Friedrich-Loeffler-Institut, Germany
Background
HPAIV develop from low-pathogenic precursors of hemagglutinin (HA) serotypes H5 or H7, requiring the acquisition
of a polybasic HA cleavage site (HACS). However, introduction of a polybasic HACS into LPAIV does not necessarily
result in high virulence for chicken indicating that, beside the essential polybasic HACS, the existence of additional
virulence determinants. Those additional adaptive changes might accumulate in the low-pathogenic precursors
during their circulation in gallinaceous poultry prior to emergence of an HPAIV. Since this evolutionary process is not
well understood, we aimed to unravel the genetic determinants which, beside the polybasic HACS, are required for
transformation of LPAIV into HPAIV.
Results
To select a minimal gene constellation sufficient for a highly pathogenic virus, we co-transfected plasmids coding for
all eight genes from an H5N1 HPAIV and seven, except HA, from an H5N1 LPAIV, and used the supernatant to infect
chickens. Shed reassortants carried the HPAIV PB2, NP, HA, NA, and M genes; a virus reconstituted by reverse genetics,
was highly pathogenic and transmissible like the wild-type.
Furthermore, by stepwise exchange of the HPAIV genes, we eventually found that an LPAIV reassortant carrying
only the HPAIV HA and NA contains the minimum set of HPAIV genes enabling high virulence, albeit at a prolonged
mean death time. Furthermore, we abolished the NA stalk deletion leading to considerably reduced lethality and no
transmission in chicken. Conversely, an LPAIV reassortant carrying only the HPAIV HA but the LPAIV NA with engineered
stalk deletion displayed 100% lethality both after primary or contact infection. Therefore, the NA stalk deletion is an
essential virulence determinant beside the polybasic HACS. Remarkably, the LPAIV NA which contains no stalk deletion,
introduced into the H5N1 HPAIV exclusively, did not reduce virulence and transmission compared to the HPAIV parent
virus.
Taken together, the HPAIV HA and NA are sufficient for high virulence in an LPAIV background. Moreover, the NA
stalk deletion along with the polybasic HACS form a minimum set of virulence determinants. However, our selection
experiment in chickens, infected with transfection supernatants, indicates that beside the HPAIV HA and NA, the PB2,
NP, and M enable maximal virulence.
For that reason, we surveyed all public PB2, NP, and M1 protein sequences from Genbank and GISAID. Data mining
revealed that there are natural HPAIV strains corresponding to the here described HA/NA reassortant, confirming the
HA with polybasic HACS and the NA with stalk deletion as a minimum set of virulence determinants. However, natural
HPAI strains without stalk deletion but with several NP mutations from the HPAIV studied, corresponding to the NA/
HPAIV reassortant, indicate an alternative set of virulence determinants.
Conclusions
Therefore, those two sets of virulence determinants indicate two independent pathways of evolution of LPAIV into
HPAIV.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L43
L E C TU R E A BSTRAC TS
SPB502
ALFA
Dynamics of influenza virus reassortment in a co-infected host
Tao, Hui; Steel, John; Lowen, Anice
Emory University, United States of America
Background
The segmented nature of the influenza virus genome allows reassortment between co- infecting viruses. This
process of genetic exchange combines with genetic drift to generate the extensive diversity of circulating influenza
viruses. The importance of reassortment to public health is clear from its role in the emergence of a number of
epidemiologically important viruses, including novel pandemic and epidemic strains. To gauge its impact on withinhost genomic variation, we tracked reassortment in co- infected guinea pigs over time and given matched or
discordant doses of co-infecting viruses.
Methods
To ensure unbiased detection of reassortants, we used parental viruses of equivalent fitness that differ only by noncoding nucleotide changes. Silent mutations were introduced into influenza A/Panama/2007/1999 (H3N2) virus to
generate a variant referred to as Pan/99var virus. These mutations allow differentiation of each genome segment
from those of the Pan/99 wild type (wt) virus using high resolution melt analysis. The mutations introduced did
not attenuate Pan/99var virus relative to the Pan/99wt virus; thus, all parental and reassortant progeny arising
from mixed infection between Pan/99wt and Pan/99var viruses are of equivalent fitness. Co-infections with these
two well-matched viruses were performed in a guinea pig model and virus was sampled through the collection of
nasal washes. To evaluate the intra-host diversity achieved through reassortment, the full genotypes of clonal virus
isolates derived from nasal washes were determined.
Results
At a relatively low dose of 102 PFU of each virus, one parental virus was absent from each guinea pig throughout
the time course, indicating the presence of a bottleneck. With an intermediate dose of 103 PFU, genomic diversity
present in nasal wash samples increased from day 1-3 post-infection (p.i.) and then declined by day 6 p.i. as the
virus was cleared. With a high dose of 106 PFU, however, reassortment levels were high (avg. 59%) on day 1 p.i. and
remained stable. Even late in the course of infection, parental viruses were not eclipsed by reassortants, suggesting
that a uniformly high multiplicity of infection was not achieved in vivo. When the relative doses of parental viruses
were altered from 1:1 to approximately 1:10, the frequency of reassortant progeny was not reduced. The spectrum
of genotypes produced changed significantly, however, with gene segments from the more abundant parental
strain predominating within reassortant genomes.
Conclusions
Our data indicate the importance to reassortment efficiency of total dose, relative dose and time after infection and
reveal the potential for reassortment to contribute to intra- host diversity in mixed influenza virus infection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L44
L E C TU R E A BSTRAC TS
SPB503
ALFA
Mapping the antigenic drift of human influenza A(H1N1) and B viruses
since the 1940s
Koel, Björn
Erasmus MC, Netherlands, The
Background
Influenza viruses are able to evade humoral immunity built up in the human population during the annual influenza
epidemics by accumulating mutations in the surface proteins haemagglutinin and neuraminidase. This process
is called antigenic drift and is thought to facilitate the annual return of these viruses. For the annual update of
the influenza vaccine composition, the antigenic reactivities of recent epidemiological strains are compared with
candidate vaccine strains using haemagglutination inhibition (HI) assays. For influenza A(H3N2) viruses, the
antigenic drift since their introduction in 1968 was visualised using the method of antigenic cartography (Smith DJ,
Lapedes AS, Jong JC de, et al. Mapping the antigenic and genetic evolution of influenza virus. Science 2004;305:3716). The goal of the present study was to map the antigenic evolution of human influenza A(H1N1) and B viruses
since the 1940s.
Methods
Thirty-nine influenza A(H1N1) viruses from the periods 1947 to 1957 and 1977 to 2009, and 24 influenza B virus
strains from the period 1949 to 2011, isolated in eggs or cell culture, were selected and in part used to prepare postinfection antisera in ferrets. The antisera were titrated in HI assays against the mentioned 39 influenza A(H1N1) and
24 influenza B viruses, using turkey erythrocytes in a WHO standard protocol.
Results
Like influenza A(H3N2) virus, the antigenic evolution of influenza A(H1N1) and B virus proceeded in a punctuated
fashion rather than gradually. Among both the influenza A(H1N1) and the influenza B viruses, a number of clusters
could be distinguished, the strains of the phylogenetic B/Yamagata16/88 line forming a single cluster. The antigenic
profiles moved away from the strains circulating in the 1940s, though not as straightforward as with influenza
A(H3N2) viruses. The changes occurred at a lower rate compared with influenza A(H3N2) viruses, that seems in
line with the lower incidence of influenza A(H1N1) and B virus infections in the human population compared with
influenza A(H3N2) viruses.
Conclusions
Influenza A(H1N1) and B viruses evolved antigenically in a punctuated way. Their antigenic profiles were found to be
more stable than those of influenza A(H3N2) viruses, a feature that may be related to the lower levels of circulation
of the influenza A(H1N1) and B viruses compared with influenza A(H3N2) viruses. The results provide background
information for the selection of influenza virus strains for vaccine production.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L45
L E C TU R E A BSTRAC TS
SPB504
ALFA
High co-infection rates and multiplicity reactivation facilitate
incomplete gene segment packaging in vivo
Brooke, Christopher Byron
National Institute of Allergy and Infectious Diseases, United States of America
Background
The influenza A virus (IAV) genome is divided into eight distinct RNA segments, all of which are required to encode
a productive infection. IAV maintains a highly specific packaging mechanism that ensures that a single copy of each
gene segment is selectively packaged into budding virions, however the efficiency of this process is not known. We
recently reported that the vast majority of infectious virions fail to express gene products from one or more segments,
suggesting that most virions carry incomplete genome sets. These findings raised the possibility that incomplete
genome packaging might be beneficial to the virus under some conditions. Consistent with this hypothesis, we
identified a single substitution (F346S) in the NP of PR8 that enhanced replication and transmissibility in guinea pigs
while selectively reducing packaging of the NA gene segment.
Methods
We assessed gene packaging rates both by quantitative RT-PCR on both highly purified virus preparations and guinea
pig nasal wash, and by measuring the fraction of infectious virions capable of expressing different gene products
by flow cytometry. To measure the in vivo co-infection frequency in guinea pigs, we exploited the observation that
only a minor fraction of virions in nasal wash co-expressed HA, NA, and NP when used to infect MDCK cells ex vivo
under low MOI conditions, but that these co-expression frequencies increased predictably as the MOI increased due
to complementation. We compared these ex vivo co-expression frequencies to the co-expression frequencies that
we observed in cells isolated from guinea pig nasal turbinates at different times post- infection. We also examined
the effect of the NP:F346S mutation on burst size and multiplicity reactivation in vitro.
Results
We observed high frequency co-infection within the upper airways of infected guinea pigs within the first 48 hours of
infection, despite using a relatively modest inoculum dose (500 TCID50). The fraction of NP:F346S virions harboring
incomplete genome sets complemented each other under high MOI conditions, resulting in productive infection.
Conclusions
IAV gene packaging is more variable than previously thought, and reduced gene packaging rates can be associated
with increased in vivo fitness and transmissibility. The high frequency of co-infection in vivo allows particles
carrying incomplete sets of gene segments to propagate through complementation. Under these conditions, the
costs of incomplete packaging are minimized while the benefits, such as increased sexual recombination through
reassortment, are maximized.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L46
L E C TU R E A BSTRAC TS
SPA6: EVALUATION OF VACCINE SAFETY AND EFFECTIVENESS
SPA601
OMEGA
Live attenuated vaccines against potentially pandemic influenza viruses:
rationale for genetic stability
Kiseleva, Irina (1); Larionova, Natalie (1); Dubrovina, Irina (1); Bazhenova, Ekaterina (1); Fedorova, Ekaterina (1); Isakova-Sivak, Irina (1);
Kuznetsova, Victoria (1); Stukova, Marina (2); Erofeeva, Marianna (2); Pisareva, Maria (2); Desheva, Julia (1); de Jong, Jorgen (3); Ross, Ted (4);
Flores, Jorge (5); Tsvetnitsky, Vadim (5); Rudenko, Larisa (1)
1: Institute of Experimental Medicine, St Petersburg, Russian Federation; 2: Institute of Influenza, St Petersburg, Russian Federation; 3: Centre
for Infectious Disease Control, RIVM, Bilthoven, the Netherlands; 4: Vaccine & Gene Therapy Institute of Florida, FL, USA; 5: Program for
Appropriate Technologies in Health, Seattle, WA, USA
Background
Genome stability is one of the main properties of any live attenuated influenza vaccine (LAIV). This study describes
the results of virus shedding and clinical isolates’ testing of a double-blinded randomized placebo-controlled phase
I clinical trials of LAIVs against potentially pandemic influenza viruses in healthy adults.
Methods
LAIV candidates were generated in eggs by classical reassortment of potentially pandemic influenza viruses H2N2,
H5N1 and H7N3 with A/Leningrad/134/17/57 (H2N2) master donor virus (MDV) (H2-LAIV, H5-LAIV and H7-LAIV,
respectively). All LAIV candidates retained temperature sensitive (ts) and cold-adapted (ca) phenotypes of the MDV
and were attenuated for ferrets. For each vaccine tested 40 subjects were randomly distributed into two groups to
receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. The subjects were followed in an isolation
unit for one week. Nasal swabs were examined for vaccine shedding by culturing in eggs or by PCR. Determination
of ts/ca phenotypes of LAIV isolates was performed by titration in eggs at optimum, low and elevated temperatures.
Nucleotide sequencing was performed using a Genetic Analyzer (Applied Biosystems).
Results
Vaccine virus detection. The majority of PCR- and eggs-positive specimens were detected for the first day following
vaccination. LAIVs tested shed vaccine virus after the first vaccination at different rate from 13.3% (H7-LAIV) to 33.3%
(H5-LAIV) and 39.3% (H2-LAIV), respectively. After revaccination LAIV virus was isolated in 0% (H7-LAIV), 20.7% (H5LAIV) and 29.6% (H2-LAIV) of vaccinated subjects, respectively. PCR method demonstrated higher sensitivity then
routine virus isolation in eggs. The percentage of positive subject varied from 90.0% (dose 1 of H5-LAIV) to 74.1%
(dose 2 of H2-LAIV). In general, no significant differences between the first and the second vaccinations regarding
detection of LAIV virus were revealed. None of the placebo recipients had detectable shed vaccine virus.
Phenotype of recovered viruses. The attenuated phenotype of LAIV isolates has been proven in experiments in eggs.
All viruses isolated from study subjects retained the ts/ca phenotypic characteristics of the MDV.
Confirmation of the presence of attenuating mutations in recovered viruses. All clinical isolates tested were shown
to preserve all attenuating mutations described for MDV. These data suggest high level of vaccine virus genetic
stability after replication in humans. During manufacture process no additional mutations occurred in the genome
of H2- LAIV. In contrast, the acquisition of additional mutations in the HA of manufacture vaccine lots of H5-LAIV
(two) and H7-LAIV (one) was detected, but they didn’t affect their attenuated phenotype.
Conclusions
Our clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from those volunteers who
shed vaccine virus. In addition, vaccine virus was not detected in placebo group supporting a notion for the lack of
person-to-person transmission of the live vaccine virus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L47
L E C TU R E A BSTRAC TS
SPA602
OMEGA
Cellular immune responses elicited after intranasal influenza vaccination
in children, a clinical trial
Mohn, Kristin G-I (1); Bredholt, Geir (1); Brokstad, Karl (1); Aarstad, Hans Jørgen (2); Pathirana, Rishi (1); Tøndel, Camilla (2); Cox, Rebecca Jane
(1,2)
1: University of Bergen, Norway, Norway; 2: Haukeland University hospital, Bergen, Norway
Background
Children represent the main source of spread of influenza infection in the community. The palatine tonsils have a
central role in protecting the upper respiratory tract and are a site for activation of protective immune responses
towards respiratory pathogens such as the influenza virus. Despite 10 years on the global market, there exists no
good correlate of protection after LAIV immunization. The aim of this study was to investigate local and systemic
immune responses with focus on cellular immunity elicited toward the vaccine in young children, and to identify
better efficacy/protective markers.
Methods
Fifty-five children aged 2-17 (median 4) years old, scheduled for elective tonsillectomy, were recruited from the EarNose-Throat clinic. Thirty-nine children were immunized with a seasonal live attenuated influenza vaccine (LAIV),
of whom 30 children received 2 doses of vaccine, and 16 children were controls. Two children withdrew after the
first dose. Influenza specific immune responses were evaluated using a direct antibody secreting cell, memory B cell
(MBC) and IFN-γ enzyme- linked immunosorbent spot assay (ELISPOT). CD4+ Th1 cells were evaluated by intracellular
cytokine staining for INF-γ, IL-2 and TNFα using multi parametric flow cytometry. The serological response was
measured by HAI assay.
Results
The LAIV vaccine was easily administered and well tolerated, and we saw no significant changes in cytokine or
chemokine profile in the plasma. We observed a great degree of strain variation in the immune response, with
a difference between the influenza A and B groups. Overall the B strain elicited the most significant increases in
immune responses. Influenza specific INF-γ secretion increased after vaccination and was maintained one-year
post vaccination. The increase in MBC response was dependent on the priming status of the child. Primed children
had high levels of MBC, without further increase after vaccination, suggesting a biological threshold in the MBC
response. The unprimed children showed a significant increase in MBC for H3N2 strain after the first vaccine dose
with an additional boosting after the second dose. Flow cytometry analysis showed a significant increase in the
influenza specific Th1 T-cell cytokine production (INF-γ, IL-2 and TNF-a) after the second dose.
Conclusions
This study is unique since it is studying a relative large number of very young children and many samples have
been collected constituently during the one-year prospective study. The LAIV vaccine elicited long-lasting cellular
immune responses in young children. This vaccine may represent a good alternative for immunization of high-risk
children against seasonal influenza in Europe, and may possibly provide some protection against the threat of a new
pandemic before a vaccine is available.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L48
L E C TU R E A BSTRAC TS
SPA603
OMEGA
Effectiveness of influenza vaccination in preventing hospitalizations in
children in Hong Kong, 2009-13
Cowling, Benjamin; Chan, Kwok-Hung; Feng, Shuo; Chan, Eunice; Peiris, Malik; Chiu, Susan
The University of Hong Kong, Hong Kong S.A.R. (China)
Background
Influenza vaccination is widely recommended every year to protect individuals against influenza virus infection and
illness. There are few published estimates of influenza vaccine effectiveness against hospitalization in children or
from subtropical regions.
Methods
We conducted a test-negative year-round study between October 2009 and September 2013, recruiting children 6
months to 17 years of age admitted to two hospitals in Hong Kong with a febrile acute respiratory infection. Cases
were tested for influenza A and B and conditional logistic regression was used to estimate vaccine effectiveness
comparing influenza vaccination history of the trivalent influenza vaccine (TIV) among cases testing positive versus
negative for influenza, adjusting for age and sex and matching by calendar week of recruitment.
Results
Overall vaccine effectiveness against hospitalization with laboratory- confirmed influenza A and B was estimated
to be 61.7% (95% CI: 43.0%, 74.2%). The estimated vaccine effectiveness against hospitalization with confirmed
influenza A(H3N2) was 36.6% (95% CI: -25.5%, 67.9%) compared to 71.5% (95% CI: 39.4%, 86.6%) for A(H1N1)pdm09
and 68.8% (95% CI: 41.6%, 83.3%) for B. Vaccine effectiveness was lower for children 6m to 2y of age (34.1%; 95% CI:
-27.3%, 65.9%) compared to children 3-5y (70.4%; 95% CI: 36.3%, 86.3%) and 6-17y (66.1%; 95% CI: 25.8%, 84.5%).
Conclusions
Vaccine effectiveness against hospitalization in children varied from year to year, but was moderate to high overall
even in an area with influenza activity throughout the year.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L49
L E C TU R E A BSTRAC TS
SPA604
OMEGA
Influenza revaccination with MF59®-adjuvanted or nonadjuvanted
trivalent inactivated seasonal influenza vaccines in 30–96 month-old
children primed two years previously
Vesikari, Timo (1); Forsten, Aino (1); Arora, Ashwani (2); Tsai, Theodore (2); Clemens, Ralf (2)
1: University of Tampere Medical School, Tampere, Finland; 2: Novartis Vaccines and Diagnostics, Cambridge, MA, USA.
Background
After showing that MF59®-adjuvanted influenza vaccine is significantly more efficacious than non-adjuvanted
vaccine against influenza in children (Vesikari et al. NEJM 2011;365:1406–16), we followed up participants after
interruption by the 2009 A/H1N1 pandemic.
Methods
In this phase IIIb, observer-blind, extension study at 15 sites across Finland, 197 children (29 aged 30–36 months and
168 aged 36–96 months) vaccinated two years previously with placebo, or two half or full doses of trivalent MF59®adjuvanted influenza vaccine (aTIV, Fluad®) or non-adjuvanted vaccine (TIV, Agrippal®) were revaccinated with 20102011 season aTIV or TIV. Immunogenicity was assessed by hemagglutination inhibition (HI) against 2010–2011
season antigens in sera obtained at Day 1, Day 22 and Day 181. Parents reported reactogenicity on diary cards.
Results
Two years after priming, aTIV- and TIV-primed groups (N=166, excluding subjects previously treated with placebo) had
similar HI titers for A/H1N1 and B strains, but aTIV-primed had higher A/H3N2 titers than TIV-primed. Revaccinating
aTIV- or TIV-primed with aTIV elicited robust responses to all three strains, higher for A/H3N2 and B strains in aTIVaTIV than TIV-aTIV. TIV vaccination in aTIV-primed subjects produced higher titers than in TIV-primed against the A/
H3N2 and B strains, but similar responses to A/H1N1. Patterns of responses were similar when analyzed in sub-sets
of children not vaccinated with pandemic or seasonal vaccine since priming. High titers persisted to Day 181 in most
cases, except against B strain in TIV-primed subjects.
Reactogenicity was slightly higher for aTIV than TIV, but generally acceptable, consisting of mainly mild or moderate
reactions expected of childhood vaccinations, with no vaccine-related SAEs.
Conclusions
Revaccinating aTIV-primed children with either aTIV or TIV produced higher responses than after TIV-priming, with
acceptable tolerability.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L50
L E C TU R E A BSTRAC TS
SPB6: RISK MANAGEMENT AND MITIGATION
SPB601
ALFA
Risk factors for severe outcomes and impact of vaccination on pneumonia
and influenza within active US military populations 2000-2012
Van Kerkhove, Maria D (1); Cooper, Michael J (2); Erik-Cost, Angelia A (2); Sanchez, Jose L (2); Riley, Steven (1)
1: Imperial College London, Department of Infectious Disease Epidemiology, United Kingdom; 2: Armed Forces Health Surveillance Center,
Global Emerging Infections Surveillance and Response System Division, Silver Spring, Maryland, USA
Background
Respiratory infections are responsible for up to 350,000 of medical encounters each year among US military
personnel.
Methods
We obtained data on 15,210 hospitalizations for pneumonia and influenza (P&I) between 01/01/00 and
31/12/12. From these, we identified 335 SARI episodes using standard case definitions. We evaluated the effect
of demographic and occupational characteristics, comorbid conditions, and history of influenza vaccination on the
risk of a hospitalized P&I case becoming a SARI case. We also evaluated the risk of SARI and the length of time since
vaccination.
Results
The median age of subjects was 22 years (range, 17-64) and subjects were predominantly male (89.5%). Risk factors
for developing SARI included age (≥45 years, RR=1.8, 95% CI 1.2-2.7), American Indian or Alaskan Native ethnicity
(RR=3.0, 95% CI 1.0-8.7), and service in the Air Force (RR=1.7, 95% CI 1.3-2.3), Coast Guard (RR=2.7, 95% CI 1.3-5.3) or
Navy (RR=1.4, 95% CI 1.0-2.0). Being male, born in mainland US (vs outside of mainland US) and recent vaccination
(within 180 days of episode) were protective against developing SARI. Among comorbid conditions, risk factors for
SARI included chronic or liver disease (RR=5.7, 95% CI 4.5-7.1), diabetes (RR=2.8, 95% CI 1.7-4.6), immune disorders
(RR=2.6, 95% CI 1.1-6.1), obesity (RR=1.76, 95% CI 1.26-2.46), cancer (RR=1.7, 95% CI 1.3-2.2), and chronic obstructive
pulmonary disease (RR=1.5, 95% CI 1.16-1.96).
Male gender (RR=0.7, 95% CI 0.5-0.9) and influenza vaccination (RR=0.7, 95% CI 0.5-0.97) remained significantly
protective and, while serving in the Coast Guard (RR=2.1, 95% CI 1.1-4.1) or Air Force (RR=1.6, 95%CI 1.2-2.1) and
renal or liver disease (RR=5.0, 95% CI 3.8-6.5) were significantly associated with developing SARI in the multivariate
model. Our results also suggest that the closer the vaccination was to hospitalization, the less likely the individual
was to develop SARI, with substantially reduced odds of SARI for delays of 1 vs. 6 months).
Conclusions
These data suggest that timing of influenza vaccination is critical in reducing severity of P&I hospitalizations;
additional analysis of delays in vaccination may impact future military immunization policy.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L51
L E C TU R E A BSTRAC TS
SPB602
ALFA
Southern hemisphere influenza and vaccine effectiveness research and
surveillance (SHIVERS) in New Zealand
Huang, Q. Sue (1); Turner, N. (2); Baker, MG (3); McArthur, C (4); Williamson, D (1,2,4); Grant, C (2); Roberts, S (4); Aley, D (4); Trenholme, A (5);
Conroy, C (5); Taylor, S (5); Lawrence, S (5); Davey, K (5); Wood, T (1); Bissielo, A (1); Radke, S (1); Bandaranayake, D (1); Seeds, R (1); Mackereth, G
(1); Todd, A (1); Pierse, N (3); Thomas, P (6); Webby, R (6); Thompson, M (7); Duque, J (7); Gross, D (7); Widdowson, MA (7)
1: Institute of Environmental Science and Research, New Zealand; 2: University of Auckland, Auckland, New Zealand; 3: University of Otago,
Wellington, New Zealand; 4: Auckland District Health Board, Auckland, New Zealand; 5: Counties Manakau District Health Board, Auckland,
New Zealand; 6: WHO Collaborating Centre, St Jude Children’s Research Hospital, Memphis, USA; 7: Centers for Disease Control and Prevention
(CDC), Atlanta, USA
Background
The “Southern Hemisphere Influenza and Vaccine Effectiveness Research and Surveillance” (SHIVERS) project was
established in 2012 for a five year period. It is a multi-centre and multi-disciplinary collaboration. The SHIVERS project
has 9 objectives: 1∗) understand severe acute respiratory infections; 2∗) assess influenza vaccine effectiveness; 3∗)
investigate interaction between influenza and other respiratory pathogens; 4∗) ascertain the causes of respiratory
mortality; 5∗) understand non-severe respiratory illness; 6) estimate influenza infection through a serosurvey; 7∗)
determine influenza risk factors; 8) study the immune response to influenza; and 9) determine the healthcare and
societal economic burden and vaccine cost-effectiveness of influenza.
Materials and methods
To address these objectives, we established two surveillance systems for the 838,000 residents living in central,
east and south of Auckland: 1) hospital- based: active, prospective, continuous, population-based surveillance for
all hospitalized severe acute respiratory infection (SARI) cases and a proportion of non-SARI respiratory cases, all ICU
admissions and deaths caused by influenza and other respiratory pathogens; and 2) sentinel (18 providers) general
practice-based: active, prospective, population-based surveillance (covering 12% of the catchment population) for
influenza-like illness (ILI) cases caused by influenza and other respiratory pathogens. A case test-negative design
was used to estimate influenza vaccine effectiveness in preventing SARI and ILI.
Results
During the 2013 influenza season (29-April to 29-September 2013), influenza- associated SARI rates were highest
at the extremes of age (122/100,000; 49/100,000; 72/100,000 and 69/100,000 population in persons aged <1; 1-4;
65-79 and ≥80 years respectively), in Pacific (51/100,000) and Maori (27/100,000) ethnic groups and those from the
lowest socio-economic (SES) group (51/100,000). In contrast, influenza-associated general practice ILI consultation
rates were highest in children aged 5-19 years (640/100,000) and 1-4 years (488/100,000), in Asian ethnic group
(573/100,000) and those from the highest SES group (498/100,000). Influenza vaccination provided 52% (95%CI 3266) effectiveness against laboratory-confirmed influenza hospitalization and 56% (95%CI 34- 70) against laboratory
confirmed influenza presenting to general practice.
Conclusions
Results from SARI and ILI surveillance of the 2013 influenza season showed contrasting socio-demographic patterns
with inverse ethnic and deprivation gradients seen for influenza-associated ILI in sentinel practices compared with
influenza-associated SARI in hospitals. Influenza vaccine appeared equally effective at preventing SARI and ILI,
though the populations presenting with influenza are markedly different in general practices to those admitted
to hospital. The hospital-based SARI and sentinel practice-based ILI surveillance systems will provide a research
platform over the next three years to comprehensively investigate influenza epidemiology, virology, immunology
and vaccine effectiveness. These results will enable SHIVERS to develop methods to improve clinical detection of
influenza, measure the impact of vaccination, guide targeted vaccination delivery, inform vaccine design, optimize
clinical management and laboratory diagnosis, identify risk factors, understand host immune responses and identify
better immune diagnostic markers.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L52
L E C TU R E A BSTRAC TS
SPB603
ALFA
Extension of the United Kingdom Influenza Immunisation Programme
2013/2014 to Children and the Implications for High-Risk Group Uptake
Blak, Betina (1); Rajaram, Sankarasubramanian (1); Steffey, Amy (2); Caspard, Herve (2)
1: AstraZeneca, London, United Kingdom; 2: AstraZeneca, Gaithersburg, MD, USA
Background
The Departments of Health of the United Kingdom (UK) extended the 2013/2014 influenza immunisation
programme to children with a routine offer of vaccination to all 2- and 3-year-olds as well as geographical pilots
for 4- to 11-year-olds. Children with high-risk conditions are part of the targeted groups in the schedule historically,
but rates of vaccination in this population have been well below the elderly uptake rate until now. The aim of this
study was to evaluate the impact of the influenza immunisation programme on vaccination rates in children with
high-risk conditions.
Methods
All children aged 2 to 17 years on September 1, 2012 (for season 2012/2013) or September 1, 2013 (for season
2013/2014), with at least 12 months of prior medical history documented in the observational UK Clinical Practice
Research Datalink (CPRD), were retained in this analysis. Administration of influenza vaccine was retrieved from
immunisation, clinical, and therapy records between September 1 and December 31. High-risk conditions were
defined prior to September 1 using operational definitions adapted from the specifications published by PRIMIS at
the University of Nottingham. Vaccination rates were analyzed for each season using time to event methods.
Results
A total of 807,551 and 747,847 children aged 2 to 17 years were retained in the analysis for seasons 2012/2013
and 2013/2014, respectively. During season 2013/2014, 6.4% of all children presented with at least one high risk
condition. The most frequent high risk conditions were asthma/chronic respiratory diseases (4.8%), followed by
chronic heart diseases (0.9%), chronic neurological diseases (0.5%), and diabetes (<0.3%). These figures were similar
for the 2012/2013 season, with 6.6% of all children presented with at least one high risk condition.
Vaccination rates by age category as of December 31, 2012 and December 31, 2013, in children with and without
high-risk conditions are presented in the table below:
High-Risk Condition
Age (years)
No High-Risk Condition
Season
Season
Season
Season
2012/2013, %
2013/2014, %
2012/2013, %
2013/2014, %
2 to 3
39.2
59.2
1.0
42.1
4 to 8
40.4
43.5
1.1
3.0
9 to 17
39.7
40.8
1.3
2.5
Conclusions
Extension of the 2013/2014 influenza immunisation programme to children sharply increased (40 percentage
points) the vaccination rate for 2- to 3-year-olds without high-risk conditions. Vaccination rates also increased for
children with high-risk conditions in all age categories, with the most profound increase (20 percentage points) in
2- to 3-year-olds. These results are consistent with those supported by ImmForm surveys and published by the UK
public health agencies. This study was sponsored by MedImmune, biologicals arm of AstraZeneca.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L53
L E C TU R E A BSTRAC TS
SPB604
ALFA
Pandemic preparedness in Europe – five years on from the 2009
pandemic
Brown, Caroline Sarah; Hegermann-Lindencrone, Michala
WHO Regional Office for Europe, Denmark
Background
On 6 June 2014, it is the five year anniversary for the declaration of the H1N1 pandemic in 2009. It was the first
pandemic for which countries had pandemic preparedness plans in place and for which sophisticated surveillance
and monitoring systems existed. Substantial information was collected, new evidence became available and a broad
range of national and international experiences were gained related to pandemic preparedness and response.
Five years down the line, less than 10 of the 53 WHO European Member States have incorporate the new evidence
and experiences gained by revising and publishing their national pandemic preparedness plans. Only one country
has revised its plan after the publication of the new global guidelines for pandemic preparedness, i.e. the Pandemic
Influenza Risk Management WHO interim guidance.
Methods
A review of those pandemic plans that have been revised and published subsequent to the pandemic in 2009 was
performed to discover what has changed in pandemic plans in post- pandemic versions. Five plans out of seven
published plans were included. Two were excluded because of translation issues.
Results
The main changes to the plans published after the pandemic in 2009 compared to earlier plans are that they are
placing greater emphasis on flexibility and on a response that is proportionate with the risk. Plans are largely moving
away from basing their response on WHO pandemic phases and several plans state that they are intended for use
for pandemic response as well as response to other outbreaks. There is in general more emphasis on multisectoral
work and on business continuity planning.
Conclusions
The experience from pandemic planning and the response to the 2009 pandemic is so substantial that other areas of
preparedness are now benefitting from the work done and knowledge gained during the 2009 pandemic. Not only
preparedness for outbreaks of other disease but also environmental preparedness etc is drawing from the lessons
learned. It is important that all countries incorporate new evidence and national experience from the pandemic and
publish their updated plans. By publishing plans, countries improve interoperability and support countries that may
learn from these plans – and the plans are used as basis for generic preparedness to other health hazards. Sharing
the new knowledge is fundamental to improve preparedness in Europe.
Keywords Pandemic preparedness, Europe, pandemic plans16 SEPTEMBER 2014
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L54
L E C TU R E A BSTRAC TS
SPA7: MATERNAL IMMUNIZATION
SPA701
OMEGA
Flu vaccination of pregnant women in Kazakhstan
Kuatbayeva, Ainagul Mukhanovna
Research & Practice Center of Sanitary Epidemiological Expertise and Monitoring of the Agency of Consumers Rights Protection of Kazakhstan
Republic, Kazakhstan
Introduction
In Kazakhstan, annually, 750,000 – 1,300,000 people are vaccinated against influenza (approximately 10% of the
population), with free vaccination provided for: “frequently sick” children; persons >65 years-of-age; children in
children’s homes and orphanages; and healthcare workers. The global morbidity of influenza among pregnant
women during the H1N1-09 pandemic influenced the Kazakhstan Ministry of Health (MOH) to offer free vaccination
to pregnant women in 2012. We studied the effectiveness of implementation of this policy.
Methods
Government vaccination records determined the numbers of women vaccinated and side effects. To determine the
effectiveness of vaccination, we used the weekly monitoring data of the routine epidemiological surveillance system
for acute respiratory virus infections (ARVI) and pneumonia morbidity and mortality among pregnant women.
Sentinel surveillance and public health laboratory records were reviewed to determine influenza virus recovery from
pregnant women.
Results
Prior to onset of the epidemic season of 2012/2013, >75,000 women were vaccinated, and prior to the 2013/2014
season, > 150,000 pregnant women were vaccinated. Comparing ARVI case rates in the 2013/2014 season with the
2012/2013 influenza season, the number of ARVI cases per 1,000 pregnant women decreased 1.8 times, i.e., 63.7
cases compared to 111.7 cases; pneumonia case-rates were reduced 2.4 times; and, the number of hospitalized
pregnant women was reduced 2.2 times. No woman was reported to have had side effects from the vaccine.
Influenza virus was not recovered from any vaccinated pregnant women
In 2014, a scientific study was started in Almaty to assess the impact of ARVI and flu on the pregnancy course and
health condition of children under one year in pregnant women who were vaccinated and pregnant women who
were not vaccinated during pregnancy (data pending).
Conclusions
Vaccination of the future mothers against flu is an important and likely cost-effective way of reducing rates of ARVI
due to influenza protecting two high-risk groups simultaneously – pregnant women and infants.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L55
L E C TU R E A BSTRAC TS
SPA702
OMEGA
The Norwegian Influenza cohort (NorFlu): immune responses in
pdmH1N1 exposed versus non-exposed pregnant women
Savic, Miloje (1,4); Laake, Ida (2); Dembinski, Jennifer (1,4); Tunheim, Gro (1,4); Oftung, Fredrik (1,4); Hungnes, Olav (3); Næss, Lisbeth (1);
Trogstad, Lill (2); Cox, Rebecca (5); Mjaaland, Siri (1,4); Waalen, Kristian (3)
1: Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Oslo, Norway; 2: Department of Vaccines, Norwegian
Institute of Public Health, Oslo, Norway; 3: Department of Virology, Norwegian Institute of Public Health, Oslo, Norway; 4: KG Jebsen Centre for
Influenza Vaccine Research, Oslo, Norway; 5: Influenza Centre at the University of Bergen, Bergen, Norway
Background
Short and long-term consequences of in utero exposure to influenza need detailed clarification. Furthermore, the
impact of the specific immunological challenges in pregnancy on women’s responses to influenza infection is
largely unknown. Two major aims that the Norwegian Influenza cohort (NorFlu) cohort will address are: 1) perinatal
outcomes and children’s mental and cognitive development depending on mothers’ H1N1pdm09 exposure status,
and 2) the correlation of risk factors for severe influenza in pregnancy and the effect on children’s immune response.
The specific aim of this study was to analyze the correlation between humoral and cellular responses to influenza
exposure and the risk of influenza illness symptoms.
Methods
In Norway, a cohort of pregnant women and their children, the Norwegian Influenza Pregnancy Cohort (NorFlu),
was established during the influenza A H1N1 pandemic in 2009 (pdmH1N1). The cohort comprises about 3200
mother and child pairs of which 2600 paired biological samples were collected. Information on immunization,
health and diseases is available from questionnaire data and national health registries. A nested case-control study
among women who were not vaccinated against pdmH1N1 was initiated. Selection criteria for the cases were: last
menstrual period between 1st May and 1st December (corresponding to the pregnancy in the period between 1st
October and 31st December 2009, which was the main pandemic peak in Norway), and hemagglutination inhibition
assay (HAI) titer values equal or above 20 HAI units. Controls were randomly selected among the women who were
pregnant in the same time period, had a HAI titer of 0, and reported not feeling influenza illness symptoms.
Results
Based on the inclusion criteria 82 cases and control pairs were selected. The median value of the HAI titers for the
cases was 40 (range from 20 to 320). The median age was 30 (age range: 19–41) for cases and 32 (age range: 17–40)
for controls. Among cases 51% reported experiencing influenza illness symptoms: fever and cough or fever and
sneezing. Detailed immunological analyses of serum and plasma have been conducted. Initial association study
between antibody level and risk of experiencing the symptoms upon exposure suggests there may not be significant
correlation. Characterization of cellular responses by analyzing the expression of cytokines from peripheral blood
mononuclear cells using Elispot, Multiplex and multiparametric flow cytometry assays is in progress.
Conclusions
Preliminary epidemiological analyses indicate no significant correlation between antibody levels and the risk of
experiencing influenza related symptoms. The NorFlu cohort provides a unique opportunity to assess perinatal
risks of maternal exposure to influenza virus, as well as to study the interplay of humoral and cellular immunity in
pregnancy during the first pandemic of the 21st century.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L56
L E C TU R E A BSTRAC TS
SPB7: HOST FACTORS IN PATHOGENESIS
SPB701
ALFA
Quantitative proteomic analysis of protein signatures in permissive vs.
non-permissive influenza A virus infections in human host cells
Sadewasser, Anne (1); Paki, Katharina (1); Eichelbaum, Katrin (2); Selbach, Matthias (2); Wolff, Thorsten (1)
1: Div. of Influenza viruses and other Respiratory Viruses; Robert Koch-Institut, Seestr. 10, 13353 Berlin, Germany; 2: Max-Delbrück-Center for
Molecular Medicine; Robert-Roessle-Str. 10, 13125 Berlin, Germany
Background
Influenza virus infections are the major cause for respiratory disease in humans, which affect all age groups and
result in extensive global mortality and morbidity, as well as substantial economic costs. Human and most avian
influenza A virus (IAV) strains differ largely in their replication efficiency in and activation of human cells despite
successful cell entry. We hypothesize that the distinct outcome of an IAV infection with a given virus strain is
determined by the differential interplay between specific host and viral factors, which remain to be defined in their
entity.
Methods
By using mass spectrometry-based quantitative proteomics we aim to characterize the sets of cellular and viral
factors whose abundance is specifically up- or down- regulated in permissive vs. non-permissive IAV infection,
respectively. Our analysis involves a “Spike-in SILAC (stable isotopic labeling by amino acids in cell culture)” approach
in human A549 cells that are highly permissive for a seasonal H3N2 IAV strain, but restrict replication of an avian
H3N2 virus. This approach allows for the definition and quantitative comparisons of changes in the host cell
proteomes in response to infections with these two viruses.
Results
The analysis resulted in the identification of distinct sets of cellular factors influenced by infections with the human
or the avian strain, respectively. Many identified cellular and viral proteins were similarly regulated by both virus
strains, but also candidates with distinct changes in permissive vs. non-permissive infection were found. Ongoing
studies involve the validation of the detected proteins and bioinformatic and functional analyses to elucidate their
roles in IAV infection and their potential contributions to IAV adaptation to human cells.
Conclusions
In conclusion, we will present initial results of a comprehensive analysis of complex host pathogen interaction
networks by using systems biology tools. We expect to identify key parameters related to efficient IAV replication
and host specificity and thereby to further our understanding of IAV biology.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L57
L E C TU R E A BSTRAC TS
SPB702
ALFA
Inhibition of avian and human influenza viruses by human airway mucins
Gerlach, Thomas; Matrosovich, Tatyana; Klenk, Hans-Dieter; Matrosovich, Mikhail
Philipps University Marburg, Germany
Background
In humans, influenza viruses replicate in the respiratory epithelium of conducting airways which is covered by the
so-called mucous blanket. Mucins, the major components of the blanket, are large multimeric heavily glycosylated
sialoglycoproteins. One of their functions is to serve as decoy receptors for carbohydrate-binding bacteria, viruses
and toxins. It is generally believed that human airway mucins (HAM) mainly express 3-linked sialic acids and that
avian viruses are more sensitive than human viruses to neutralization by HAM (Couceiro et al, 1993). This longstanding concept has been recently challenged by several reports, which revealed high sensitivity of human viruses
and low sensitivity of avian viruses to HAM (Roberts and Shelton et al., 2011; Limsuwat et al., 2013). In this study,
we wished to address this apparent discrepancy and to clarify the role of HAM in protection of the human airways
from influenza infection.
Material and Methods
Human airway mucins were collected from the apical surfaces of differentiated cultures of human tracheo-bronchial
epithelial cells (HTBE) and were used without fractionation. We tested neutralization of representative avian and
human influenza viruses by HAM in three different cell systems. MDCK cells were used to reproduce experiments
performed in previous studies. MDCK-SIAT1 cells differed from MDCK cells by increased expression of 6-linked sialic
acids and decreased expression of 3-linked sialic acids. This pair of cell lines was employed to determine the effects
of receptor density on target cells on virus neutralization by mucins. HTBE cultures were used to mimic conditions of
influenza virus infection in human airway epithelium in vivo. Neutralization experiments were performed by mixing
viruses with serial dilutions of HAM, inoculation of cell monolayers and immunostaining of infected cells for the
viral nucleoprotein after one cycle of virus replication.
Results
In agreement with the recent reports, neutralization experiments in MDCK cells showed that human viruses were
more sensitive to HAM than avian viruses. However, human viruses were less sensitive and avian viruses were more
sensitive to HAM when MDCK-SIAT cells were used for the assay. In HTBE cultures, avian and human IAVs were
neutralized by HAM with comparable efficiency.
Conclusions
Our results indicate that neutralization of influenza viruses by competitive sialic acid-containing inhibitors strongly
depends on the concentration of specific viral receptors on target cells. As a result, neutralization experiments
performed in standard laboratory cells, such as MDCK cells, which do not mimic the spectrum of sialic acids in
human airway epithelium may lead to erroneous conclusions. Our data on neutralization in differentiated HTBE
cultures suggest that during the infection in the human respiratory tract both avian and human influenza viruses
can be inhibited by airway mucus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L58
L E C TU R E A BSTRAC TS
SPB703
ALFA
The role of endothelial cells in influenza A virus pathogenesis
Short, Kirsty Renfree (1); Kasper, Jennifer (2); Fouchier, Ron (1); Kirkpatrick, Charles (2); Kuiken, Thijs (1)
1: Erasmus MC, the Netherlands; 2: Department of Pathology, Medical University of Mainz, Mainz, Germany
Background
Influenza A virus (IAV) is an important cause of respiratory disease worldwide. The primary cellular target of IAV is
epithelial cells within the respiratory tract.Within the lower respiratory tract, alveolar epithelial cells are in close
proximity to the underlying endothelium, with only 0.5 μm separating the airspace from the capillary. During IAV
infection the endothelium is therefore exposed to free virus particles produced by infected epithelial cells. The role
of pulmonary endothelial cells in IAV pathogenesis remains controversial. Numerous in vitro studies have suggested
that IAV can infect endothelial cells, trigger apoptosis and facilitate pulmonary oedema. However, there is little if
any evidence to suggest that IAV can infect pulmonary endothelial cells in vivo. Instead, studies in mice suggest that
endothelial cells produce a large amount of pro-inflammatory cytokines during IAV infection, although these results
are yet to be confirmed using human cells.
Methods
Here, we use an in vitro co-culture model to assess the role of endothelial cells in IAV pathogenesis.
are seeded on the upper half of a transwell membrane whilst human endothelial cells are seeded
on the lower half. These cells are then grown in co-culture and influenza virus is added to the
Briefly, human epithelial cells
upper chamber. Unlike conventional in vitro monocultures of endothelial cells, this system
allows us to model the dynamic interplay that occurs in vivo between pulmonary epithelial and
endothelial cells.
Results
We show that IAV infection of the epithelium (with viruses of the H1N1, H3N2, H7N9 and H5N1 subtypes) does
not result in the infection or death of the underlying endothelial cells, with the epithelium serving to ‘protect’ the
endothelium from infection. We further demonstrate that whilst IAV damages pulmonary epithelial cells, this
damage occurs independently of the presence of endothelial cells. Instead, we show that endothelial cells drive
epithelial cell cytokine production. Specifically, epithelial cells grown in co-culture with endothelial cells produced
significantly more pro-inflammatory cytokines than epithelial cells grown in a conventional monoculture. This
increased cytokine production was associated with increased expression of RAGE (receptor for advanced glycation
endproducts) and its ligand HMGB1 (high mobility group protein B1), suggesting a potential role for RAGE activation
in this epithelial-endothelial crosstalk.
Conclusions
These data provide the first evidence that endothelial cells are not a key target of IAV infection and instead play
an essential role in driving cytokine production by epithelial cells during IAV infection. This epithelial-endothelial
crosstalk may then have important consequences for the pathogenesis of IAV in vivo.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L59
L E C TU R E A BSTRAC TS
SPB704
ALFA
Type I interferon receptor 2 (Ifnar2) plays an important role in limiting
inflammation and disease following influenza virus infection
Tate, Michelle D; Dowling, Jennifer K; Piganis, Rebecca A; Hertzog, Paul J
MIMR-PHI Institute of Medical Research, Australia
Background
The innate immune system provides a critical first line of defence following influenza virus infection, however,
excessive inflammation is associated with severe infections in both mice and humans. Type I interferons (IFNs) are
an important component of the innate anti-viral response. The type I IFN receptor (Ifnar) is comprised of two chains,
Ifnar1 and Ifnar2. Binding of IFNα/β to Ifnar initiates signaling cascades that lead to the upregulation of hundreds
of interferon-stimulated genes (ISGs) with immunomodulatory and anti-viral properties.
Studies have illustrated that Ifnar1 knockout mice are more susceptible to influenza virus infection than wild-type
mice. The role of Ifnar2 during influenza virus infections is currently not known. We hypothesized that Ifnar1 and
Ifnar2 may play differing roles in controlling influenza virus infections.
Methods
To examine the susceptibility of mice lacking components of the type I IFN receptor to influenza virus, wild-type,
Ifnar1-/- and Ifnar2-/- mice on a C57BL/6 background were intranasally inoculated with 102 PFU of HKx31 (H3N2).
Mice were weighed daily and examined for signs of disease. Viral loads in lung and nasal tissue homogenates were
determined by standard plaque assay. Flow cytometry was utilized to examine cellular infiltrate in the airways and
cytokines in the bronchoalveolar lavage (BAL) fluid and sera were examined by cytokine bead array and ELISA. The
protein concentrations of BAL fluid and lung wet to dry ratios were utilised to examine vascular leakage and edema,
respectively.
Results
Mice lacking Ifnar2 were more susceptible to influenza virus infection than Ifnar1 knockout and wild-type mice. By
day 7 post-infection, ifnar2-/- mice displayed evidence of severe disease and had to be euthanised. In comparison to
wild-type mice, viral replication was elevated in both the lungs and nasal tissues of Ifnar1 and Ifnar2 knockout mice
at days 1, 3 and 7 post- infection, however there were no striking differences between Ifnar1-/- and Ifnar2-/- mice.
Severe disease displayed by Ifnar2-/- mice was associated with increased neutrophil infiltration as well as elevated
levels of IL-1β in the airways and blood. Furthermore, mice lacking Ifnar2 displayed evidence of edema and vascular
leakage.
Conclusions
Overall, our data suggests the Infar1 and Infar2 chains of the type I IFN receptor play distinct roles in host defence
during influenza virus infections. In particular, Ifnar2 appears to play an important role in controlling the production
of IL-1β and the induction of severe disease. Understanding further this role of Ifnar2 and how type I IFNs control
inappropriate or damaging immune responses will be of great significance to reduce mortality and morbidity
associated with influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L60
L E C TU R E A BSTRAC TS
PL05: LATE BREAKERS
PL0501
OMEGA
Post-pandemic Review of anti-Influenza Drug Effectiveness (PRIDE
Study): an investigation of the impact of neuraminidase inhibitor
antiviral use on pneumonia and length of hospital stay in hospitalized
influenza A(H1N1)pdm09 patients
Muthuri, Stella; Myles, Puja; Venkatesan, Sudhir; Leonardi-Bee, Jo; Nguyen-Van-Tam, Jonathan
Division of Epidemiology and Public Health, University of Nottingham, United Kingdom
Background
The aim of this study was to investigate the impact of neuraminidase inhibitor (NAI) antiviral treatment on
influenza-related pneumonia and length of hospital stay during the 2009-10 A(H1N1)pdm09 influenza pandemic.
Methods
We conducted an individual patient data meta-analysis of observational data on hospitalised A(H1N1)pdm09
pandemic influenza cases (n=34,910) contributed by 78 research groups from 38 countries within 6 WHO regions.
Multilevel logistic regression modelling was conducted separately for the outcomes: influenza–related pneumonia
(yes vs no) and length of stay (LOS) in hospital (> 5 days vs ≤ 5days; median LOS= 5 days). We adjusted for propensity
scores (based on age, sex, comorbidity and disease severity at admission), treatment with antibiotics and steroids.
Results are presented for laboratory confirmed A(H1N1)pdm09 cases of all ages as adjusted odds ratios (aOR) with
95% confidence intervals (CI).
Results
Analyses for influenza-related pneumonia showed treatment with NAI (at any time) versus no NAI treatment was
associated with a significantly increased likelihood of pneumonia (aOR, 1.26 [95% CI, 1.10 – 1.43]). However, we
found reduced likelihood of pneumonia when early treatment (≤48h after symptom onset) was compared with later
treatment (aOR, 0.51 [95% CI, 0.46 – 0.57]) or no NAI treatment (aOR, 0.83 [95% CI, 0.70 – 0.99]). Compared with
no NAI treatment, treatment with NAI (at any time) was significantly associated with longer stay in hospital (aOR,
1.55 [95% CI, 1.40 – 1.71]; early treatment when compared with no treatment was associated with a non-significant
increase in hospital stay (aOR, 1.11 [95% CI, 0.95 to 1.29]); whilst early compared with later treatment (aOR, 0.68
[95% CI, 0.62 - 0.75]) showed significant reduction in the duration of hospital stay.
Conclusions
Early treatment with NAI was associated with a significant reduction in influenza A(H1N1)pdm09 pneumonia
compared with either no or later NAI treatment. A significant reduction in the duration of hospital stay was only
seen when early NAI treatment was compared with later NAI treatment.
Prospero Registration. CRD42011001273.
Funding. Unrestricted educational grant from F. Hoffmann-La Roche
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L61
L E C TU R E A BSTRAC TS
PL0502
OMEGA
Understanding the genome packaging mechanism of influenza viruses
Nakatsu, Sumiho (1); Sagara, Hiroshi (2); Noda, Takeshi (1,3); Kawaoka, Yoshihiro (1,4,5)
1: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Japan; 2: Medical
Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Japan; 3: PRESTO, Japan Science and Technology Agency, Japan; 4:
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, USA; 5: International Research Center
for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Japan
Introduction
Influenza A and B viruses infect humans and cause epidemics. Therefore, both types of influenza virus are considered
important pathogens. Although many studies have attempted to elucidate the nature of influenza viruses, their
replication mechanisms in host cells remain a mystery.
The genomes of influenza A and B viruses possess eight-segmented RNAs. Each viral RNA segment associates with
the nucleoproteins and a polymerase, forming a rod-like structure called the ribonucloeprotein (RNP) complex. The
lengths of the RNPs vary from 30 to 120 nm depending on the RNA segments. When progeny virions bud from
infected cells, they need to package at least one copy of each of the eight different RNPs to be infectious; however, the
detailed mechanisms of RNP packaging are not yet fully understood. Previously, our group showed that a laboratory
strain of influenza A virus selectively packages eight kinds of RNPs. However, it remains unknown whether influenza
B viruses and other influenza A viruses, including clinically isolated strains, employ a similar mechanism. Here, to
determine whether the selective packaging mechanism is common to influenza A and B viruses, laboratory and
clinical strains of influenza A and B viruses were examined by use of electron microscopy.
Material and Methods
A/WSN/33 (H1N1) and B/Lee/40 strains were used as laboratory strains; A/Yokosuka/UT-Y291/11 (H1N1) and B/
Yokosuka/UT-Y23/11 were used as clinical strains. The number of RNPs within transversely sectioned virions on
ultrathin section was counted manually and compared between each type of virus. The configuration of the RNPs
within the virions was examined by using electron tomography.
Results & Discussion
In transmission electron microscopy (TEM), 8 RNPs were observed within the virions for all of the strains examined,
although the percentages of those virions differed for each strain. Electron tomography of the laboratory and clinical
strains of influenza A and B virus revealed that most of the virions packaged 8 RNPs, implying that the influenza A
and B viruses generally package 8 RNPs. Interestingly, some virions packaged less than 8 RNPs and, therefore, lacked
a complete set. These results suggest that a small number of noninfectious particles are also produced, which is
consistent with previous reports that some virus-infected cells fail to express all viral proteins (Martin and Helenius,
Cell 1991; Brook et al., J Virol 2013).
As shown in this study, most virions package 8 RNPs, and strict adherence to this genome packaging mechanism
ensures their efficient replication. However, some virions packaged less than 8 RNPs, which are non-infectious.
Producing non-infectious viruses would seem to be a disadvantage for the virus; however, that non-robustness in
genome incorporation may contribute to the evolution of viruses by allowing easier genome reassortment events
when multiple kinds of viruses infect a host in nature.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L62
L E C TU R E A BSTRAC TS
PL0503
OMEGA
Comparison of aerosol and intra-nasal challenge with 2009 pandemic
influenza virus (H1N1) in cynomolgus macaques showed clear
differences in the distribution of virus
Marriott, Anthony Colin (1), Sally Sharpe (1), Mike Dennis (1), Jennifer Kane (1), Claudia Prevosto (1), Nigel Silman (1), Emma Rayner (1), Simon
Bate (1), Saranya Sridhar (2), Karl J. Staples (3), Ajit Lalvani (2), Tom Wilkinson (3), Miles W. Carroll (1)
1: Public Health England, Porton Down, Salisbury SP4 0JG, UK.; 2: Imperial College London, London W2 1PG, UK.; 3: University of Southampton,
Southampton SO16 6YD, UK.
Background
The aim is to develop non-human primate models for upper and lower respiratory tract (URT and LRT) infection
with clinically relevant seasonal influenza viruses. Previous studies using H1N1pdm09 virus challenge in macaques
have used high doses of virus delivered by the intra-tracheal (IT) route in order to target virus to the LRT, whereas
human challenge studies are restricted to URT infection. NHP models have several advantages over human studies
including, ability to sample a range of tissues at necropsy (e.g. for pathology), use of more clinically relevant strains,
use of aerosol challenge and LRT infection routes, in addition to much reduced set-up time and cost.
Methods
We have compared the outcomes of challenge by the IT route, the intra-nasal (IN) route, and by small-particle nasally
inhaled aerosol. Cynomolgus macaques (male, aged 3-4 years) were challenged with 106 (IT or IN), 105 (aerosol) or
103 (IN) pfu of A/Cal/04/09 virus.
Results
None of these challenge routes resulted in overt clinical disease (fever, weight loss, respiratory symptoms). However
all the animals were infected, as determined by evidence of virus replication, virus shedding and/or seroconversion.
IN challenge led to URT infection, with virus replication confined to the nasal cavity. The high IN dose (106 pfu)
led to virus shedding on days 3-6 post-infection, but when the dose was reduced to 103 pfu, the infection was
nonproductive. Thus the sensitivity of the NHPs to H1N1 virus resembles that seen in human challenge studies,
rather than the much more sensitive ferret model. IT and aerosol challenges led to LRT infections. The IT challenge
resulted in localized lung infection, whereas the aerosol challenge led to more widespread virus replication in the
LRT and URT. Some animals in each group also shed virus in nasal washes. Immune responses in BAL and PBMCs are
being analysed for T-cell memory and lung homing markers. The cellular innate immune response in lung mirrored
the kinetics of viral replication.
Conclusions
For the first time, we have demonstrated and directly compared URT and LRT models for H1N1pdm in the macaque,
with virus tropism being determined by route of challenge. Furthermore, we showed that a low IN challenge dose
(103 pfu) fails to result in productive infection, in contrast to the ferret, which can be productively infected with ≤
10 pfu influenza virus. These models will be of great value in defining immune correlates of protection and memory
responses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L63
L E C TU R E A BSTRAC TS
PL0504
OMEGA
Secondary Bacterial Pneumonia Suppresses Pulmonary CD8 and CD4 T
Cell Responses after Influenza Infection
Ghoneim, Hazem E. (1); Smith, Amber M. (1); McCullers, Jonathan A. (1,2)
1: Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN, United States of America.; 2: Department of
Pediatrics, University of Tennessee Health Science Center, Memphis, TN, United States of America.
Background
Secondary bacterial pneumonia (SBP) is a major complication during influenza virus infections
that leads to increased morbidity and mortality during influenza pandemics epidemics, and
seasonal periods. Although several mechanisms underlying this viral-bacterial synergism
have been studied, viral-induced alterations in lung microenvironment and antibacterial
immunity that enhances the susceptibility to SBP are most studied. However, the effect of
the subsequent bacterial infections on the host adaptive immune responses against primary
influenza infections is unclear.
Methods
To initiate SBP, we intranasally infected BALB/c mice with a sublethal dose of influenza virus PR8 followed by a
sublethal dose of Streptococcus pneumoniae (pneumococcus) using serotypes 3 and 2 strains A66.1 and D39,
respectively. We analyzed the number and function of total CD8 and CD4 T cells in the alveolar airspaces, lung tissues
and mediastinal lymph nodes at different timepoints after single influenza infection or secondary pneumococcal
challenge. We also analyzed pulmonary PR8-specific CD8 T cell numbers and viral titers in single influenza infected
and co-infected mice.
Results
The absolute number of CD8 T cells, CD4 T cells and influenza-specific CD8 T cells are significantly decreased in
the lungs and alveolar airspaces of co-infected mice compared to those with single influenza infection, which is
correlated with successful development of SBP in co-infected mice. Furthermore, the ability of these cells to produce
IFN γ intracellularly is significantly decreased after ex vivo stimulation. Progression into SBP is associated with a
significant increase in influenza viral titers and delayed viral clearance.
Conclusions
We identified a novel immunosuppression mechanism during co-infection such that SBP suppresses CD8 and
CD4 T cell responses against the ongoing influenza virus replication. This immunosuppression is associated with
significant increase in influenza viral titers and delayed viral clearance, which may complicate the severity of SBP.
Our current and previous findings
suggest a bidirectional interaction between primary influenza and secondary bacterial infections. That is,
primary influenza infection can suppress host protective antibacterial immunity in the lungs leading to SBP
while secondary bacterial infections may also suppress the virus-specific adaptive immunity. Our findings may
highlight the importance of combining anti-viral with anti-bacterial therapeutic approaches to treat co-infected
hosts. Furthermore, this defect in effector T cell immunity might have negative outcomes on the development of
memory T cell responses as well. Therefore, it is crucial to determine the impact of this early immunosuppression
on the development of effector and central memory T cells against influenza infections. Further exploration of
this immunosuppression mechanism and its outcomes might result in novel therapeutic approaches for lung coinfections.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L64
L E C TU R E A BSTRAC TS
PL0505
OMEGA
Pathogenicity of airborne-transmissible H5N1 viruses.
Mathilde Richard, Sander Herfst, Judith van den Brand, Pascal Lexmond, Theo Bestebroer, Ab Osterhaus, Thijs Kuiken and Ron Fouchier
Department of Viroscience. ErasmusMC. Rotterdam. Netherlands.
Background
Since 2003 H5N1 viruses have been continuously circulating in poultry in Eurasia, occasionally spilling over to wild
birds and mammals, including humans. This circulation has led to an ongoing evolution of the H5N1 viruses in poultry.
As a consequence, it is feared that H5N1 viruses with pandemic potential, i.e. with the ability to transmit via the
air between humans, might emerge. Herfst et al. demonstrated that H5N1 virus could indeed acquire substitutions
conferring airborne transmissibility between ferrets. Five to nine mutations of these substitutions were found to
be of particular importance for airborne transmissibility: PB2 E627K, PB1 H99Y, PB1 I368V, NP R99K, NP S345N,
HA Q222L, HA G224S, HA T156A and HA H103Y. Now that light has been shed on the determinants necessary for
avian influenza viruses to become transmissible via the air, new studies are crucial in order to understand, in depth,
the impact of such findings for public health. There are no studies to date characterizing the pathogenicity of fully
avian-origin airborne-transmissible influenza viruses in mammals nor in poultry, such as chickens. Such studies are
of importance in order to address the chances of airborne-transmissible H5N1 emerging in chickens, and the impact
that this may have on human health.
Methods
Reverse genetics techniques were used to design H5N1 viruses with all or subsets of airborne mutations. We
inoculated ferrets and chickens with airborne-transmissible H5N1 viruses and wild-type H5N1 virus. Respiratory
samples were collected daily to assess viral shedding. Detailed necropsies followed by pathological examination
were performed at fixed time points to analyze the pathogenicity, replicative capacity and tissue tropism of airbornetransmissible H5N1 viruses in comparison with wild-type H5N1 virus.
Results
Airborne-transmissible H5N1 viruses were attenuated, both in terms of mortality and morbidity, in ferrets and
in chickens. The contribution of different substitutions or combinations of substitutions to the attenuation and
phenotype of the airborne-transmissible H5N1 viruses is currently under investigation.
Conclusion
Here, we provide the first in depth characterization of the pathogenicity of airborne-transmissible H5N1 viruses in
both ferrets and chickens. The findings of these studies emphasize the need to study airborne-transmissible H5N1
viruses as they help to assess both the likelihood of these viruses emerging in poultry and the consequences that
these viruses may have for human morbidity and mortality.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
L65
POSTE R A BSTRAC TS
P OSTER
A B STR ACTS
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P1
POSTE R A BSTRAC TS
SPA1: CLINICAL IMPACT AND DIAGNOSTIC APPROACHES
SPA1P01
Clinical usefulness of the BD Veritor System for Rapid Detection of Flu
A+B to detect influenza viruses
Noh, Ji Yun (1); Choi, Won Suk (1); Lee, Jacob (2); Seo, Yu Bin (2); Lee, Jung Hwa (3); Song, Joon Young (1); Cheong, Hee Jin (1); Kim, Woo Joo (1,4)
1: Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea, Republic of (South
Korea); 2: Division of Infectious Diseases, Department of Internal Medicine, Kangnam Sacred Heart Hospital, Hallym University School of
Medicine, Seoul, Korea, Republic of (South Korea); 3: Department of Pediatrics, Korea University College of Medicine, Seoul, Korea, Republic of
(South Korea); 4: Transgovernmental Enterprise for Pandemic Influenza in Korea, Seoul, Korea, Republic of (South Korea)
Background
Rapid antigen test has been used widely for prompt diagnosis of influenza. The aim of this study is to evaluate the
clinical usefulness of the BD VeritorTM System for Rapid Detection of Flu A+B in patients with influenza-like illness
(ILI).
Methods
Respiratory specimen was obtained from 434 patients with ILI in three teaching hospitals, South Korea during 20132014 influenza season. The performance of the BD VeritorTM System for Rapid Detection of Flu A+B was compared
with the results of SD Bioline Influenza Ag and the Humasis Influenza A/B antigen Test.
Results
The number of patients who participated in the study was 434. Median age of the adult patients (≥15 years) was 37
years (IQR 30-53) and that of children was 4 years (IQR 2-7). The median time to sample collection from symptom
onset was 2 days (IQR 1-3) in adults. Among 434 specimens, 210 (48.4%) were positive for influenza A virus and 56
(11.6%) were influenza B viruses.
To detect overall influenza viruses, the BD VeritorTM System for Rapid Detection of Flu A+B showed 80.8% sensitivity,
88.5% specificity, 91.7% positive predictive value, and 74.6% negative predictive value in adult patients, and 93.3%
sensitivity, 83.9% specificity, 89.4% positive predictive value, and 89.7% negative predictive value in children. For
influenza A virus, BD VeritorTM System for Rapid Detection of Flu A+B yielded higher sensitivity than the SD Bioline
Influenza Ag and the Humasis Influenza A/B antigen Test in adults: 81.5%, 60.1%, and 43.8%, respectively. For
influenza B virus, BD VeritorTM System for Rapid Detection of Flu A+B showed 71.4% sensitivity, while sensitivities
of the SD Bioline Influenza Ag and the Humasis Influenza A/B antigen Test were 83.3% and 42.9% in adults.
In children, the BD VeritorTM System for Rapid Detection of Flu A+B showed 93.8% sensitivity in detection of
influenza A viruses, and the sensitivities of the SD Bioline Influenza Ag and the Humasis Influenza A/B antigen Test
were 81.3% and 53.1% for influenza A viruses.
Conclusions
The BD VeritorTM System for Rapid Detection of Flu A+B showed a good sensitivity to detect influenza viruses. It
would be useful for early diagnosis of influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P2
POSTE R A BSTRAC TS
SPA1P02 Diagnosis of human influenza A virus infection using single chain
fragment variable antibody-based competitive inhibition ELISA
Rajput, Roopali (1); Sharma, Gaurav (2); Saxena, Latika (1); Khanna, Madhu (1); Pradhan, Hare Krishna (3); Pattnaik, B. (2)
1: Dept. of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi-110007, India; 2: Project Directorate on Foot and
Mouth Disease, Mukteswar, Nainital-263138, India; 3: Former National Consultant (Avian Influenza), WHO-India office, Delhi-110001, India
Background
Influenza A viruses pose a major concern for public health and are responsible for significant morbidity and
mortality not only in humans but also in the other host species. Precise diagnosis of influenza A virus infection is
critical for the treatment of the viral infection and prevent frequent occurrence of the viral outbreaks. Thus, efficient
sero-diagnostic strategies are required for assessment of pre-existing and/ or cross-protective immunity among
human population to devise better preparedness plans. The relative ease of large-scale production and high specific
reactivity led us to develop recombinant single chain variable fragment (scFv) antibodies against the NP and NS1
proteins of pandemic influenza H1N1 (2009) virus for development of an ELISA-based sero-diagnostic test, which
efficiently differentiated among the vaccinated and influenza A virus infected individuals.
Methods
The scFv antibodies were developed from spleen cells of the mice, hyper- immunized with pandemic influenza H1N1
(2009) virus. The antibody genes were amplified from the isolated mRNA and cloned in pSEX81 phagemid vector
for generation of phage display antibody library against the viral proteins. The NS1- or NP- specific scFv-displaying
phages were selected, from the library, by bio-panning against the respective antigens. The recombinant phage
clones, showing high yield in antigen-binding phage ELISA, were used for production and purification of functional
scFv antibodies in bacterial cells, by transfer of the scFv cassette from the positive phagemid clones into the plasmid
vector. The antibodies were purified and analyzed for their antigen binding efficacy. The phage displayed antibodies
were used for development of enzyme linked immunosorbent assay in a competitive inhibition format for its higher
sensitivity and specificity over the indirect ELISA (Bhatia et al., 2008).
Results
The reactivity of soluble scFv antibodies was higher in antigen capture ELISA than the antigen tracing format. Of
the total 50 samples, 28 samples were from patients, who acquired natural infection of influenza A (H1N1) virus,
12 from individuals vaccinated against influenza virus and 10 served healthy controls. Anti- NS1 antibodies were
detected only in the influenza virus infected patients, except 6 H3N2 samples, whereas anti- NP antibodies were
found in both virus infected and vaccinated samples.
Conclusions
Our findings suggest that the recombinant anti-NS1 and anti-NP scFv antibodies developed in this study prove to
be significant for sero-diagnosis of human influenza A virus infection and epitope mapping of the antigens. The test
may be used for evaluation of pre-existing immunity against various strains/ sub-types of influenza A virus. It may
also help in assessment of vaccine efficacy, as the dual-parameter test can differentiate between the infected and
non-infected individuals. The test may also be exploited as a cheaper alternative to the presently used real time RTPCR diagnostic assay, in developing nations like India.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P3
POSTE R A BSTRAC TS
SPA1P03 Hospitalizations with influenza in the northern hemisphere 20132014 influenza season. Results from the Global Influenza Hospital
Surveillance Network
Puig-Barberà, Joan (1); Tormos, Anita Marie Catherine (1); Sominina, Anna (2); Ciblak, Meral (3); Mira-Iglesias, Ainara (1); Natividad-Sancho,
Angels (1); Buigues-Vila, Amparo (1); Burtseva, Elena (4)
1: FISABIO-Salud Pública, Spain; 2: Research Institute of Influenza, St. Petersburg, Russian Federation; 3: National Influenza Reference
Laboratory Capa-Istanbul, Istanbul, Turkey; 4: D.I. Ivanovsky Institute of Virology, Moscow, Russian Federation
The GIHSN (Global Influenza Hospital Surveillance Network) Group members: F Aktaş, Faculty of Medicine, Gazi University, Ankara, Turkey;
S Badur, Istanbul Faculty of Medicine, Istanbul, Turkey; A Buigues Vila, FISABIO-Salud Pública, Valencia, Spain; S Borekci, Cerrahpaşa Faculty
of Medicine, Istanbul University, Istanbul, Turkey; M Carballido, Hospital General, Castellón, Spain; C Carratalá Munuera, Universidad
Miguel Hernández, San Juan de Alicante, Spain; M Ciblak, National Influenza Reference Laboratory Capa-Istanbul, Istanbul, Turkey; S Çelebi,
Uludağ University Faculty of Medicine, Bursa, Turkey; Denef B. Deniz, Dr. Siyami Ersek Göğüs Kalp ve Damar Cerrahisi Eğitim ve Araştırma
Hastanesi Istanbul, Turkey; M Durusu, Hacettepe Üniversitesi, Ankara, Turkey; C El Guerche-Seblain, Sanofi-Pasteur, Lyon, France; S Gencer,
Dr. Lütfi Kırdar Kartal Training andResearch Hospital, Istanbul, Turkey ; V Gil Guillén, Universidad Miguel Hernández, San Juan de Alicante,
Spain; M Hacımustafaoğlu, Uludağ University, Bursa, Turkey; Selda Hancerli, Istanbul Faculty of Medicine, Istanbul University, Istanbul,
Turkey; L Kolobukhina, D.I Ivanovsky Institute of Virology, Moscow, Russian Federation; R Limón Ramírez, Hospital La Plana, Vila-real, Spain; C
Mahé, Sanofi-Pasteur, Lyon, France; A Mira Iglesias, FISABIO-Salud Pública, Valencia, Spain; J Mollar Maseres, Hospital La Fe, Valencia, Spain;
A Natividad Sancho, FISABIO-Salud Pública, Valencia, Spain; L Ozisik, Hacettepe University, Ankara, Turkey; L Osidak, Research Institute of
Influenza, North-West Department of Russian Academy of Medical Sciences, St. Petersburg, Russian Federation; MC Otero Reigada, Hospital La
Fe, Valencia, Spain; S Özer, Dr. Lütfi Kırdar Kartal Training and Research Hospital, Istanbul, Turkey; M Pisareva, Research Institute of Influenza;
St.Petersburg Russian Federation; J Puig-Barberà, FISABIO-Salud Pública, Valencia, Spain; A Eren Şensoy, Dr. Siyami Ersek Göğüs Kalp ve
Damar Cerrahisi Eğitim ve Araştırma Hastanesi, Istanbul, Turkey; S Şimşek Yavuz, Dr. Siyami Ersek Göğüs Kalp ve Damar Cerrahisi Eğitim ve
Araştırma Hastanesi, Istanbul, Turkey; Solmaz Çelebi, Uludağ University Faculty of Medicine, Bursa, Turkey; A Sominina, Research Institute of
Influenza; St. Petersburg, Russian Federation; K Stolyarov, Research Institute of Influenza, St. Petersburg, Russian Federation; A Tormos, FISABIOSalud Pública, Valencia, Spain; M Tortajada Girbés, Hospital Dr Peset,Valencia, Spain; S Trushakova, D.I Ivanovsky Institute of Virology, Moscow,
Russian Federation.
Background
The Global Influenza Hospital Surveillance Network (GIHSN) was launched in 2012 to address growing awareness
that influenza-related hospitalization is a significant burden that remains insufficiently characterised. The GIHSN
is a partnership between industry and public health institutions that use active surveillance and a common core
protocol to collect data on the epidemiology of severe influenza. We here present the preliminary results for the
2013-2014 northern hemisphere influenza season.
Methods
During the main influenza season of 2013-2014, the GIHSN included 17 hospitals from the northern hemisphere, 6
in Spain, 7 in Turkey and 4 in the Russian Federation. Hospitalized patients of all ages presenting influenza like-illness
(ILI) within 7 days between the onset of symptoms and admission were swabbed. Positives for influenza were real
time reverse transcription polymerase chain reaction (RT-PCR) positive for influenza A(H3N2), A(H1N1)pdm09, or B.
Results
Of the 6,887 patients screened, 4,217 were eligible and tested, 784 (19%) were positive for influenza and 3,272 (81%)
negative. A(H3N2) was dominant (n=390, 50%), followed by A(H1N1)pdm09 (n=334, 43%). Few cases of influenza B
were observed (n=32, 4%). A(H3N2) was most common in Turkey, St. Petersburg and Moscow in ages between 18 and
50 years old, and caused a mean length of stay at the hospital of 6.08 days [95% CI: 5.74 – 6.42]. For the GIHSN sites
of the northern hemisphere, the influenza season started beginning of December with a peak of A(H1N1)pdm09
from week 3 to week 5, and a second peak of A(H3N2) from week 8 to week 10. Socio-demographic characteristics
such as age, having a chronic conditions (OR 1.34; 95%CI: 1.14– 1.57), smoking habits (OR 1.23; 95%CI: 1.03–1.47)
and being pregnant (OR 3.17; 95%CI: 2.12–4.74) were significantly different between positives and negatives with
p-values of <0.05.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P4
POSTE R A BSTRAC TS
Conclusions
A(H3N2) was highly circulating in the younger in east and south-eastern Europe, and A(H1N1)pdm09 was present
mostly in the elderly in south-western Europe. There was a peak at a later appearance for A(H3N2) compared
to A(H1N1)pdm09. The data from this network provides information for influenza epidemiology giving a global
overview of the Influenza season 2013-2014 in the northern hemisphere.
Funding: This network activity is partly funded by Sanofi Pasteur.
Number of positives for influenza by epidemiological week
GIHSN
Spain
60
50
40
30
20
10
0
40
30
20
N=68
10
20
13
20 -50
13
20 -51
13
20 -52
14
20 -01
14
20 -02
14
20 -03
14
20 -04
14
20 -05
14
20 -06
14
20 -07
14
20 -08
14
20 -09
14
20 -10
14
20 -11
14
20 -12
14
20 -13
14
20 -14
14
-1
5
13
-5
0
13
-5
1
13
20 52
14
-0
20 1
14
20 02
14
-0
20 3
14
20 04
14
-0
20 5
14
20 06
14
-0
20 7
14
-0
20 8
14
-0
20 9
14
-1
20 0
14
-1
20 1
14
-1
20 2
14
20 13
14
-1
20 4
14
-1
5
20
Epidemiologcial Week at Admisison
>=50 - <65 yrs
>=65 - <75 yrs
Epidemiological Week at Admission
Moscow, Russia
Number of positives for influenza
50
40
30
20
10
60
N=248
N=108
50
>=75 - <85 yrs
40
N=77
>=85 yrs
30
20
10
Turkey
70
60
20
20
13
-5
0
13
-5
1
13
-5
20 2
14
-0
20 1
14
-0
20 2
14
20 03
14
20 04
14
-0
20 5
14
20 06
14
20 07
14
-0
20 8
14
20 09
14
20 10
14
-1
20 1
14
-1
20 2
14
-1
20 3
14
20 14
14
-1
5
20
20
Epidemiological Week at Admission
13
-5
0
13
-5
1
13
-5
20 2
14
-0
20 1
14
-0
20 2
14
-0
20 3
14
-0
20 4
14
-0
20 5
14
-0
20 6
14
-0
20 7
14
-0
20 8
14
-0
20 9
14
-1
20 0
14
-1
20 1
14
-1
20 2
14
-1
20 3
14
-1
20 4
14
-1
5
0
0
20
Number of positives for influenza
N=63
N=103
>=18 - <50 yrs
70
60
20
>=5 - <18 yrs
50
St. Petersburg, Russia
70
N=88
N=28
Epidemiological Week at Admission
H1N1
H3N2
A-Not subtyped
Yamagata
Victoria
B-Not subtyped
50
Virus distribution for hospitalized patients by age group in years
40
30
20
10
20
20
13
-5
0
13
-5
1
13
20 52
14
-0
20 1
14
-0
20 2
14
20 03
14
-0
20 4
14
20 05
14
-0
20 6
14
20 07
14
-0
20 8
14
-0
20 9
14
20 10
14
-1
20 1
14
-1
20 2
14
-1
20 3
14
-1
20 4
14
-1
5
0
20
Number of positives for influenza
>=1 - <5 yrs
60
0
20
20
>=0 - <1 yrs
70
Num ber of positives for influenza
Number of positives for influenza
70
Epidemiological Week at Admission
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P5
POSTE R A BSTRAC TS
SPA1P04
Influenza in pregnant women during 2012-2014, Moscow, Russia
Trushakova, Svetlana (1); Kisteneva, Lidiya (1,2); Kruzhkova, Irina (2); Mukasheva, Evgeniya (1); Krasnoslobodtsev, Kirill (1); Siluyanova, Elina
(1); Garina, Ekaterina (1); Kolobukhina, Ludmila (1,2); Burtseva, Elena (1)
1: D.I.Ivanovsky Research Institute of Virology, Russian Federation; 2: Hospital #1 for Infectious Deseases, Moscow, Russian Federation
Background
Pregnant women are one of the main groups at risk for influenza infection and subsequent complications. Results
of influenza monitoring in hospitalized pregnant women in 2012-2014 are presented.
Methods
Pregnant women with influenza like-illness (ILI) were admitted to Hospital #1 for infectious diseases in Moscow.
Patients were interviewed based on the Global Influenza Hospital Surveillance Network (GIHSN) protocol. Reliable
patients having ILI within 7 days between the onset of symptoms and admission were swabbed. RT-PCR was applied
to detect influenza A(H3N2), A(H1N1)pdm09 and B viruses.
Results
Since December 2012 to March 2014 2050 hospitalized patients were tested for influenza infection. Among them
there were 738 pregnant women aged 15-40: in the season 2012-2013 – 523 women, in 2013-March 2014 – 215
women. The number of influenza positive cases in pregnants was 48% in the season 2012-2013 (A(H1N1)pdm09
– 29%; A(H3N2) – 9%; B – 10%) and 44% in the season 2013-2014 (A(H1N1)pdm09 – 5%; A(H3N2) – 36%; B – 3%).
The onset of the disease was acute with such symptoms as high fever, malaise, headache and sore throat. The
most common underlying medical conditions among pregnants were renal impairment, cardiovascular and
chronic obstructive pulmonary disease. According to the season 2012-2013 most of admitted women were in
the second (36%) and third (37%) trimesters of pregnancy. Threatening miscarriages were registered in 41% of
pregnants, miscarriages - in 5% of them. In obstetric ward 18 childbirths happened in women with confirmed
influenza. Complications like as urinary tract infections, bronchitis and sinusitis were registered in 27% of pregnants,
pneumonia – in 3,6% of them. Smoking while pregnant was reported in 5,5% of women. No one of pregnants was
vaccinated in the studied period, but 18-20% of them were vaccinated in the previous seasons.
To detect overall influenza viruses, the BD VeritorTM System for Rapid Detection of Flu A+B showed 80.8% sensitivity,
88.5% specificity, 91.7% positive predictive value, and 74.6% negative predictive value in adult patients, and 93.3%
sensitivity, 83.9% specificity, 89.4% positive predictive value, and 89.7% negative predictive value in children.
For influenza A virus, BD VeritorTM System for Rapid Detection of Flu A+B yielded higher sensitivity than the SD
Bioline Influenza Ag and the Humasis Influenza A/B antigen Test in adults: 81.5%, 60.1%, and 43.8%, respectively. For
influenza B virus, BD VeritorTM System for Rapid Detection of Flu A+B showed 71.4% sensitivity, while
sensitivities of the SD Bioline Influenza Ag and the Humasis Influenza A/B antigen Test were 83.3% and 42.9% in
adults.
In children, the BD VeritorTM System for Rapid Detection of Flu A+B showed 93.8% sensitivity in detection of
influenza A viruses, and the sensitivities of the SD Bioline Influenza Ag and the Humasis Influenza A/B antigen Test
were 81.3% and 53.1% for influenza A viruses.
Conclusions
The results showed the involvement of pregnant women in epidemic process was high independently of etiology
of influenza epidemics. At the same time the morbidity among pregnants corresponded to the etiology of influenza
epidemics in 2012-2014: the predominant types of influenza were A(H1N1)pdm09 in the 2012-2013 season and
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P6
POSTE R A BSTRAC TS
A(H3N2) – in the 2013- 2014 season. Influenza is able to induce different complication including threatening
miscarriage. Therefore vaccination, rapid hospitalization and early antiviral therapy are the main way to prevent and
protect against influenza among high risk population groups.
This work is partly funded by Sanofi Pasteur.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P7
POSTE R A BSTRAC TS
SPA1P05
Laboratory monitoring of influenza like illnesses (ILI) and severe acute
respiratory infections (SARI) in the flu sentinel surveillance system in the
Republic of Kazakhstan during last three seasons – 2011-2014
Aushakhmetova, Zabira T.; Kiyanbekova, Lyazzat S.; Abeev, Arman B.; Demesinova., Balzira M.; Kasenova, Zhulduz K.
Center of Sanitary Epidemiological Expertise of the Agency of Consumers’ Rights Protection of Kazakhstan Republic, Kazakhstan
Background
The Republic of Kazakhstan (RK) implemented influenza sentinel surveillance in 2008, with currently seven sites
conducting among influenza-like illness (ILI) and severe acute respiratory illness (SARI) surveillance in seven
different geographical areas – mostly on the bordering countries and in the capital of RK with active migration of
the population. Improvement of laboratory diagnosis is necessary to understand the epidemiology of cases, manage
cases, and understand the economic consequences of annual flu epidemics.
Methods
There were 1739 ILI patients and 2747 SARI patients investigated during the last three influenza seasons – 20112014. Nasal and pharyngeal specimens were collected from patients during the first three days from onset of
clinical symptoms meeting the standard definitions of ILI and SARI. The samples were tested using real time RT-PCR
according to the CDC protocol. Zonal virology laboratories (ZVL) used the lyophilized panel prepared by “Vector Best”
company (Russia, Novosibirsk) with different type and subtypes of the viruses in different concentrations, including
H5N1, for an external control quality program (ECQP) for the seven sentinel laboratories. One ZVL is regularly doing
retesting of the all positives and 10% of negatives from the six other sentinel sites using CDC/Atlanta primers for
ECQP.
Results
Influenza-positive results among patients with ILI increased from 16% (n=553) in the season of 2011-2012 to 27%
(n=517) in the last season of 2013-2014. Influenza-positive results among SARI patients increased from 22% (n=814
SARI patients in the 2011-2012 season) to 30% (n=879) during the season 2013/2014. During all three seasons,
flu virus of A type was identified in both of ILI and SARI patients in 83% cases, and flu virus of B type in 17% cases.
Subtypes of flu virus of A type varied during all three seasons and in different areas. There was active circulation of
both subtypes of Influenza A – H3N2 and H1N1-09-- in west/south of RK, in the east and in the capital of RK during
2013-2014 season. Predominant subtype in the north/west was H1N1-09 – 88% (42/48); predominant virus H3N2
was in the far north 92% (35/38) and in the south - 75% (15/20). We tried to analyze from where the flu virus comes
to the RK. Among the seven sites, two cities with active migration first identified flu virus during last three seasons
as did one city in the south which has a border with Kyrgyzstan, Uzbekistan and China. Due to the ECQP program,
the retesting results from 6 laboratories improved from 90% to 98% during last three seasons.
Conclusions
Laboratory monitoring of circulating types and subtypes of flu viruses is permitting us to understand the importance
of influenza and its epidemiology in Kazakhstan. The ECQP helped to improve laboratory capacity in all sentinel
surveillance laboratories.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P8
POSTE R A BSTRAC TS
SPA1P06
COMPARATIVE ANALYSIS OF mariPOC SYSTEM AND rRT-PCR IN DETECTION
OF INFLUENZA A VIRUS IN CLINICAL SAMPLES
Petrova, Ekateria; Krivitskaya, Vera; Pisareva, Maria; Buzitskaya, Zhanna; Sukhovetskaya, Vera; Danilenko, Daria; Suddenkova, Polina; Sominina,
Anna A.
Research Institute of Influenza, Russian Federation
Background
This study aimed to compare PCR with new automated rapid diagnostic mariPOC system (ArcDia International Oy
Ltd, Finland).
Methods
The mariPOC system is based on the principle of two-photon excitation of fluorescence and applying of drychemistry reagents. Assay scheme includes: polymer microspheres coated by antibodies and fluorescently labeled
antibody conjugate. Fluorescent immunocomplexes are formed on the microsphere’s surface (Janne O. Koskinen et
al., J. Clin. Microbiol. – 2007. – Vol.45, No11. – p. 3581–3588). Sample buffer was added to nasopharyngeal swabs
and specimens were vortexed to disrupt the mucus before the analysis.
Results
Nasal swabs from 116 children (aged 9 days to 14 years) were investigated for presence of influenza A antigens using
both PCR and mariPOC in parallel. mariPOC system was the most efficient in detection of influenza A virus (IAV) in
samples with RNA content up to 25 cycles of amplification in PCR. Sixteen of investigated samples appeared to be
positive in PCR and 15 of them were positive in mariPOC. The sensitivity and the specificity in IAV detection using
mariPOC in comparison with PCR was 93,8% and 99,0% respectively. Total results coincidence was registered in
98,3% of cases. Negative and positive predictive values for detecting IAV were 93,8% and 99% respectively. Fourteen
specimens were analyzed for presence of IAV by isolation in MDCK cell culture. Eleven of them were positive in PCR.
Six samples were positive for IAV by culture. All of the samples positive by culture were positive in mariPOC as well.
Also 2 clinical samples which were negative by virus isolation but positive in PCR, IAV was detected by mariPOC.
Thus, detection of influenza A virus by the mariPOC system showed a higher sensitivity than the virus isolation
method.
Conclusions
Comparative investigation showed mariPOC as highly effective system in detection of influenza A virus. Simplicity
of analysis and rapid results are the great advantages of mariPOC in diagnosis of influenza A in clinical conditions.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P9
POSTE R A BSTRAC TS
SPA1P07
Seizure following respiratory viral infection versus immunization in
infants and children – a syndromic surveillance study
Grover, Sandeep (1); Weick, Anja (1); Obermeier, Patrick (1); Chen, Xi (1); Seeber, Lea (1); Tief, Franziska (1); Karsch, Katharina (1); Muehlhans,
Susann (1); Hoppe, Christian (1); Adamou, Eleni (1,2); Behrens, Stephanie (1); Reiche, Janine (2); Schweiger, Brunhilde (2); Rath, Barbara (1)
1: Department of Pediatrics, Division of Pneumonology-Immunology, Charité University Medical Center Berlin, Germany; 2: National Reference
Centre for Influenza, Robert Koch Institute, Berlin, Germany
Background
Pediatric seizures cause significant disease burden and distress to patients, parents and caregivers. Febrile seizures
in children are commonly triggered by acute respiratory infections. Complicated febrile seizures and afebrile seizures
may be early indicators of future motor manifestations. In rare instances, seizures may also represent adverse events
following immunization (AEFI). Syndromic hospital-based surveillance data of seizures following recent infection
versus immunization in infants and children are currently lacking.
Methods
This inception cohort study was conducted in the context of a quality management (QM) program for children with
influenza-like illness (ILI) at the Charité Department of Pediatrics in collaboration with the National Reference Centre
for Influenza at the Robert Koch Institute (Charité Influenza-Like Disease = ChILD Cohort). All pediatric patients (aged
0-18) fulfilling pre- defined ILI case criteria and presenting to the emergency room and inpatient units were enrolled
consecutively from 10/2009 through 11/2013. A specifically trained QM team performed highly standardized
clinical assessments in real-time. Seizure cases were ascertained in compliance with the Brighton Collaboration
AEFI definition. Nasopharyngeal swabs were collected for blinded real-time PCR testing for influenza A&B virus, RSV,
human metapneumovirus, human rhinovirus and adenovirus at the Robert Koch Institute. Multivariate analysis
correlating seizures to multiple clinical, laboratory and demographic variables was conducted using binary logistic
regression analysis.
Results
A total of 4158 ILI cases were included, with 414 patients fulfilling seizure criteria. More than 80% of seizure cases
were identified in children below three years of age. Among the respiratory viruses tested, multivariate analysis
identified a significant association of seizure with influenza A (H3N2) and adenovirus infections (OR=1.80, 95%
CI=1.06-3.11, p=0.031; OR=1.46, 95% CI=1.1-2.17, p=0.046). With respect to clinical risk factors, underlying hepatorenal conditions were strongly associated with a likelihood to develop seizures in the context of ILI (OR=2.41,
95% CI=1.64-3.54, p<0.0001). Multivariate analysis did not identify a significant association between seizure and
immunization per se. However, when analyzing the likelihood to develop seizure depending on the time elapsed
since the most recent routine childhood immunization, immunization against mumps, measles, rubella and/or
varicella were all significantly associated with seizure in the context of ILI (p<0.0001).
Conclusions
Influenza A (H3N2) and adenoviral infections, as well as underlying hepato-renal conditions may all increase the
likelihood for children to develop seizures in the context of ILI. Higher influenza vaccination may therefore decrease
the risk of seizures. Interestingly, recent immunization with mumps, measles, rubella and/or varicella vaccines
further increases the likelihood of seizure in the context of ILI. This study underlines the importance of timely
diagnostics and accurate immunization histories in the pediatric acute care and inpatient settings.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P10
POSTE R A BSTRAC TS
SPA1P08
Importance of Influenza infection in the etiology of exacerbations of
COPD during a singular pandemic and post-pandemic period
Sanz, Iván (1,2); Tamames, Sonia (1,3); Rojo, Silvia (1,2); Justel, Mar (2); Vega, Tomás (3); Ortiz de Lejarazu, Raúl (1,2)
1: Valladolid National Influenza Center, Spain; 2: Microbiology & Immunology Service - Hospital Clinico Universitario de Valladolid, Spain; 3:
Conserjería de Sanidad - Junta de Castilla y León, Spain
Background
Influenza and other respiratory virus infections are a known cause of acute exacerbations of COPD (AE-COPD).
Emergence of a new pandemic influenza virus in 2009 A/H1N1pdm09 has generated the opportunity to evaluate
the burden of this clinical event. The aim of this work was to study viral etiology of AE-COPD during pandemic
emergence of new influenza virus and following post-pandemic periods.
Methods
An observational retrospective study was conducted selecting 239 respiratory samples from AE-COPD patients
between 2009 pandemic (May 2009 – May 2010) and Post-pandemic (June 2010 – January 2013). Patients were
recruited from Hospitals and Influenza Surveillance Sentinel Network of Castilla y León (ISSN) (Spain) for viral
diagnostic following previously established clinical criteria. Clinical information was extracted from clinical petitions.
Microbiology diagnostic was done by means of a set of RT-PCR multiplex molecular techniques for the detection of
21 viruses in Valladolid National Influenza Centre and Microbiology and Immunology Service of Hospital Clinico
Universitario de Valladolid.
Results
During pandemic period (May 2009 – May 2010) Influenza A/H1N1pdm09 virus was the main virus affecting AECOPD patients (34.4%). However, in the following post-pandemic periods viral etiology of AE-COPD changed. In the
first inter epidemic period (June 2010 - September 2010) RSV was the most prevalent virus (16.7%). In the first
epidemic season after pandemic (October 2010 – May 2011), RSV and Influenza A/H1N1pdm09 were the viruses
most prevalent (16.7% each one), and in second inter epidemic season (June 2011 – September 2011) any virus was
the main responsible of AE-COPD. In the last Influenza epidemic season analyzed (October 2011 – May 2012) the
main virus affecting COPD patients was A/H3 Influenza virus, and any case A/H1N1pdm09 virus wasn ́t detected
in this period. During the last inter epidemic period analyzed (June 2012 – January 2013) it wasn ́t detected any
Influenza virus affecting COPD patients (Table1).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P11
POSTE R A BSTRAC TS
Table 1. Evolution of mean age, percentage of males and main respiratory viruses affecting AE- COPD patients during
pandemic and post-pandemic periods.
Table 1. Evolution of mean age, percentage of males and main respiratory viruses affecting AECOPD patients during pandemic and post-pandemic periods.
Conclusions
Viral
etiology in patients with AE-COPD has changed from pandemic to subsequent post-pandemic flu seasons.
Conclusions.
Influenza A/H1N1pdm09 virus was the most prevalent during pandemic, but patterns changed in post-pandemic,
Viral etiology in patients with AE-COPD has changed from pandemic to subsequent
post-pandemic
flu seasons.
Influenza
A/H1N1pdm09
the most
prevalent during
being
RSV and
A/H3
Influenza
virusvirus
thewasmain
etiological
agents causing AE-COPD. Mean age of patients raised
pandemic, but patterns changed in post-pandemic, being RSV and A/H3 Influenza virus the
during
all period
Proportion
malesraised
hadduring
beenallincreasing
since the beginning of the -study, being men the
main etiological
agents studied.
causing AE-COPD.
Mean age of
of patients
period
studied. Proportion of males had been increasing since the beginning of the -study, being men
majority
of viral etiology cases at the end of studied period.
the majority of viral etiology cases at the end of studied period.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P12
POSTE R A BSTRAC TS
SPA1P09
Influenza A and B burden in Latin American countries
Ferro Bricks, Lucia (1); Falleiros, Luiza Helena (2); Mascareñas, Cesar (3); el Guerche-Séblain, Clotilde (4)
1: Sanofi Pasteur, Brazil; 2: Marilia Medical School and Medical School of the Metropolitan University of Santos, Brazil; 3: Sanofi Pasteur,
Mexico; 4: Sanofi Pasteur, France
Background and aims
The majority of influenza infections are caused by influenza A. However, some seasons are dominated by influenza
B with two lineages co-circulating. Much of the scientific literature has focused on influenza A. It seems important
that further information on the global burden of influenza type B disease is obtained within the context of the recent
launch of new tetravalent influenza vaccines. The aim of this study is to present a review of influenza surveillance
data in the Latin American region.
Methods
An extensive search of published information in PUBMED, SCIELO and WHO, PAHO and Latin American Ministry
of Health sites, from January/2000 until March/2014 was performed. After a review of the abstracts, articles were
selected based on their relevance. Additional references were included when considered relevant for the review.
Results
Before 2009, the majority of information about A and B strains circulation was based on passive surveillance of
influenza-like illness (ILI), mostly using Immunofluorescence tests. It was observed large variation on influenza
strains circulation in the region. On average, FLU A strains were responsible for about 2/3 of ILI cases and B strains,
for 1/3. The strain A(H3N2) caused the majority of serious cases, but all A and B FLU strains were identified in ILI and
hospitalized cases. Since 2002, there is evidence that both B lineages (Victoria and Yamagata) co-circulated with
A(H1N1) and A(H3N2), and in some years, the B strains were the predominant ones (>50%). After 2009, the majority
of countries implemented the surveillance for serious acute respiratory syndrome (SARS), and introduced RT-PCR
tests. The strain A(H1N1)pdm09 was the predominant one in both, ILI and SARS cases, but the strains A(H3N2)
and both B lineages co-circulated in the region, with variable impact in different countries and seasons. Limited
information about mismatch pointed that in about 50% of the seasons there was B strain mismatch with the
linages included in trivalent vaccines used in Brazil and Mexico, and disproportional impact of B strains in children,
adolescents and young adults.
Conclusions
Influenza A and B caused substantial morbidity and mortality in the region. In about 50% of the last 10 years, there
was B strain mismatch with the vaccine recommended for Northern and Southern Hemisphere, and the introduction
of a tetravalent vaccine can reduce substantially influenza cases and complications.
Note: this study was supported by Sanofi Pasteur
2010 a 2014
A(H1N1)pdm09
%
A(H3N2)
%
A?
%
B
%
Total
Argentina
3,781
27
2,180
16
5,355
39
2,525
18
13,841
Brasil
4,418
53
1,961
23
135
2
1,860
22
8,374
Chile
2,818
32
3,915
44
411
5
1,675
19
8,819
Colômbia
1,818
60
742
24
322
11
154
5
3,036
Costa Rica
1,491
53
917
32
47
2
363
13
2,818
Ecuador
1,397
53
755
29
181
7
294
11
2,627
México
10,784
54
5,582
28
1,670
8
1,920
10
19,956
Panamá
137
18
373
48
12
2
248
32
770
Paraguay
665
21
1,738
55
12
1
720
23
3,135
2,089
51
945
23
402
10
669
16
4,105
320
35
335
37
8
2
237
26
900
Country
Peru
Uruguay
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P13
POSTE R A BSTRAC TS
SPA1P10
Epidemiology of patients infected by Influenza B viruses in a northern
Spanish population during 2012-2013 Influenza season
Ortiz de Lejarazu, Raúl (1,2); Rojo, Silvia (1,2); López, Irene (2); López, Cristina (2); Tamames, Sonia (1,3); Sanz, Iván (1,2)
1: Valladolid National Influenza Center, Spain; 2: Microbiology & Immunology Service - Hospital Clinico Universitario de Valladolid, Spain; 3:
Conserjería de Sanidad - Junta de Castilla y León, Spain
Background
Influenza B lineages Victoria and Yamagata have been co-circulating globally since 25 years ago. Before the
emergence of A/H1N1 pandemic virus in 2009, Influenza B type viruses appeared as predominant virus every 2-4
years. After 2009 pandemic, 2012-2013 influenza season was the first epidemic with predominant B virus. The aim
of this study is to evaluate the characteristics of patients infected by Influenza B viruses Yamagata and Victoria
during 2012-2013 influenza season.
Methods
A retrospective observational and descriptive study was designed selecting 109 patients positives for Influenza B
virus from Hospital and Influenza Sentinel Surveillance Network of Castilla y León (ISSN) (Spain). These samples
were diagnosed at Valladolid National Influenza Centre and Microbiology and Immunology Service of Hospital
Clinico Universitario de Valladolid (Spain), by means of cellular culture, commercial and in-house RT-PCR multiplex
molecular techniques. Clinical information as sex, age and other clinical base conditions was extracted from clinical
petitions of each patient.
Results
Globally during 2012-2013 flu season, 30.3% of patients diagnosed for Influenza B infection (33) were hospitalized
and 69.7% (76) were non-hospitalized patients. Among hospitalized patients, 66.7% were infected by Influenza B
Yamagata lineage and in 27.3% by Victoria lineage (6.1% can ́t be subtyped by molecular assays). Among nonhospitalized patients, 64.5% were diagnosed as Yamagata lineage and 31.6% as Victoria lineage (3.9% can ́t be
subtyped by molecular assays). The most affected age group by Influenza B infection was 1-14 years (46.8%) in both
lineages, followed by 31-60 age group (26.6%). Globally, prevalence of Influenza B in both lineages was higher in man
than woman (62% for Yamagata lineage and 75.6% for Victoria lineage). In the same way, in all age groups described
also men was the gender group with higher prevalence of Influenza B infection, however, prevalence was higher for
women of 15- 30 age group.
Conclusions
Although it has been proved the epidemic joint circulation of both lineages of Influenza B, a higher prevalence of
circulation of Yamagata lineage in general population and also in hospitalized patients was observed in 2012-2013
influenza season. Most affected age group was 1-14 years, but patients with ages between 31-60 years had also
high rates of infection. Yamagata lineage infection had similar sex ratio, but Victoria lineage had higher proportion
of infected men. Influenza B is a cause of hospitalization in an important proportion of cases.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P14
POSTE R A BSTRAC TS
SPA1P11
Co-circulation of two lineages of influenza B viruses in South Korea
Noh, Ji Yun (1); Lee, Han Sol (2); Song, Joon Young (1); Choi, Won Suk (1); Lee, Jacob (3); Seo, Yu Bin (3); Lee, Jin-Soo (4); Wie, Seong-Heon (5);
Jeong, Hye Won (6); Kim, Young Keun (7); Park, Kyung Hwa (8); Kim, Shin Woo (9); Lee, Sun Hee (10); Lee, Jung Hwa (11); Kim, Dong Hyun (12);
Woo, Sung Il (13); Lim, Chae Seung (14); Cho, Kyung Soon (15); Cheong, Hee Jin (1); Kim, Woo Joo (1,16)
1: Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea; 2: Department of
Biomedical Sciences, Korea University College of Medicine, Seoul, Korea; 3: Division of Infectious Diseases, Department of Internal Medicine,
Kangnam Sacred Heart Hospital, Hallym University School of Medicine, Seoul, Korea; 4: Division of Infectious Diseases, Department of Internal
Medicine, Inha University College of Medicine, Incheon, Korea; 5: Division of Infectious Diseases, Department of Internal Medicine, The
Catholic University of Korea, School of Medicine, St. Vincent’s Hospital, Suwon, Korea; 6: Division of Infectious Diseases, Department of Internal
Medicine, College of Medicine, Chungbuk National University, Cheongju, Korea; 7: Department of Infectious Diseases, Yonsei University Wonju
College of Medicine, Wonju, Korea; 8: Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea; 9:
Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, Korea; 10: Department of Internal Medicine,
Pusan National University School of Medicine, Busan, Korea; 11: Department of Pediatrics, Korea University College of Medicine, Seoul,
Korea; 12: Department of Pediatrics, Inha University College of Medicine, Incheon, Korea; 13: Department of Pediatrics, College of Medicine,
Chungbuk National University, Cheongju, Korea; 14: Department of Laboratory Medicine, Korea University College of Medicine, Seoul, Korea;
15: Division of Virology, Busan Metropolitan City Institute of Health and Environment, Busan, Korea; 16: Transgovernmental Enterprise for
Pandemic Influenza in Korea, Seoul, Korea
Background
Two antigenically distinct lineages of influenza B viruses have circulated globally: Yamagata and Victoria lineages.
Current trivalent influenza vaccines include only one lineage of influenza B viruses, and it leads to the reduced
influenza vaccine effectiveness especially when the predominant circulating influenza B virus does not match
vaccine strain. We aimed to characterize the lineages of influenza B viruses obtained from clinical specimens.
Methods
Respiratory specimens from the patients with laboratory-confirmed influenza B from 2007-2008 influenza season
to 2013-2014 season were used. RT- PCR for the partial hemagglutinin gene of influenza B virus was performed to
differentiate Yamagata and Victoria lineages and phylogenetic tree was generated. B/Brisbane/60/2008 virus and B/
Wisconsin/01/2010 virus were used as representative strains of Victoria lineage and Yamagata lineage, respectively.
Results
In South Korea, influenza B viruses generally circulated every two years recently. Therefore, samples of 2007-2008,
2009-2010, 2011-2012, and 2013-2014 influenza seasons were available for the analysis. Victoria lineage influenza
B viruses were predominant in 2007-2008 influenza season (42/49, 85.7%) and in 2009-2010 season (90/99, 90.9%).
In 2007-2008 and 2009-2010 seasons, the influenza B vaccine strain was well matched to circulating strains. During
2011-2012 season, among 152 samples which lineage was specified, 90 (59.2%) belonged to Yamagata lineage group.
Also, in 2013-2014 season, two lineages of influenza B viruses have co-circulated: Yamagata lineage 53.9%(83/154)
and Victoria lineage 46.1%(71/154). Among 121 adult patients with influenza B in 2013-2014 season, there was
no significant difference in seasonal influenza vaccination rate (27.1% vs. 24.5%, P=0.83) and hospitalization rate
(6.9% vs. 10.2%, P=0.52) between patients infected with matched strain (Yamagata lineage) and those infected with
mismatched strain (Victoria lineage).
Conclusions
Co-circulation of two influenza B lineages was found in South Korea. Quadrivalent influenza vaccine would improve
the effectiveness of influenza vaccine.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P15
POSTE R A BSTRAC TS
SPA1P12
Factors that influence the clinical outcome in patients admitted with
influenza like illness and the effect of glucocorticoid treatment
Tanriover, Mine Durusu (1); Ozisik, Lale (1); Akcay Ciblak, Meral (2); Yurtcu, Kubra (2); Unal, Serhat (3); Badur, Selim (2)
1: Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey; 2: Istanbul University, Faculty of Medicine,
Department of Microbiology and Clinical Microbiology, Istanbul, Turkey; 3: Hacettepe University Faculty of Medicine, Department of Infectious
Diseases and Clinical Microbiology, Ankara, Turkey
Background
Viral respiratory infections, mainly influenza can be devastating in adults, especially in those with chronic diseases.
Severe cases may require hospitalization. Glucocorticoids are questionable in the treatment of viral infections,
however patients with chronic obstructive lung disease or severe bronchospasm may require systemic glucocorticoid
treatment.
Methods
Patients admitted from the Adult Emergency Department of a University Hospital were screened for influenza
like illness between January 16 and March 16, 2013. Those who required hospitalization for at least 24 hours
and who consented were sampled with nasal/nasopharyngeal swab. Samples were collected in Virocult (Medical
Vire&Equipment, England) and transferred to the lab for testing. All samples were stored at -80C if not tested
upon arrival. High-Pure Viral Nucleic Acid Kit (Roche, Germany) was used for total RNA extraction. CDC rRT-PCR
protocol was used for detection of influenza viruses. (http://www.who.int/csr/resources/publications/swineflu/
CDCRealtimeRTPCR_Swine H1Assay-2009_20090430.pdf).In-house multiplex rRT-PCR with TaqMan probes were
used for detection of respiratory viruses other than influenza viruses.
Results
A total of 75 patients were enrolled during the study period. The viral panel yielded at least one viral etiology in 25
of the patients (Table 1). Seven patients died. Those who had a lower body mass index, who smoked and who had
a malignancy had a higher mortality rate (Table 2). The mortality rate of the patients diagnosed to have influenza
A H1N1pdm09 infection was 23.5%. The only significant difference in terms of laboratory data was the lymphocyte
number; those who survived had a higher lymphocyte number than those who died (mean 1289 /mm3 vs 614/
mm3, respectively). Glucocorticoid treatment (either systemic or inhaled) did not have an effect on mortality or
admission to intensive care unit. Most of the patients with pneumonia received glucocorticoid therapy than those
without pneumonia (82.4% vs 25% respectively). None of the patients who died were vaccinated and only half of
them received oseltamivir treatment (p=0.053). Multi-organ failure was the cause of death in most of the fatalities.
Conclusions
This small, pilot surveillance study has demonstrated that respiratory viral infections may be fatal in adult patients;
one fourth of patients who had influenza A infection diagnosed died. Systemic or inhaled glucocorticoid treatment
does not seem to have a negative impact on the course of viral respiratory diseases. Smoking, having a malignancy
and not receiving oseltamivir therapy and not being vaccinated seemed to be risk factors for death in those patients
who were admitted with influenza like illness and who had a viral etiology diagnosed; the statistical significance can
probably be demonstrated in future larger studies.
Table 1. Viruses that were identified as the etiology of the respiratory infection
Table 2. The characteristics of patients with a viral etiology (n=25)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P16
POSTE R A BSTRAC TS
Table 1. Viruses that were identified as the etiology of the respiratory infection
Table 2. The characteristics of patients with a viral etiology (n=25)
Virus
Number (%)
Influenza A H1N1pdm09
17 (68)
İnfluenza A
1 (4)
Influenza A H3N2
1 (4)
Rhinovirus
Age
Survived
discharge
67 (26-90)
Female/male
to Died
p
72 (18-88)
NS
9/9
1/6
NS
Body mass index
25.2 (19.3-45.1)
21.5 (19.5-35.2)
NS
3 (12)
Smoking
9 (50)
5 (71.4)
NS
Parainfluenza virus type 2
1 (4)
Pregnancy
1 (5.6)
0
NS
Parainfluenza virus type 2 +
metapneumovirus
CoronavirusOC43
1 (4)
Chronic heart disease
10 (55.6)
3 (42.9)
NS
COPD
10 (55.6)
3 (42.9)
NS
Diabetes
5 (27.8)
2 (28.6)
NS
Renal disease
6 (33.3)
1 (14.3)
NS
Malignancy
3 (16.7)
3 (42.9)
NS
İmmune suppression
3 (16.7)
1 (14.3)
NS
Rheumatological disease
2 (11.1)
0
NS
Neuromuscular disease
0
1 (14.3)
NS
Chronic liver disease
1 (5.6)
0
NS
Influenza A infection
15 (83.3)
3 (50)
NS
Oseltamivir treatment
17 (94.4)
4 (57.1)
NS
Antimicrobial treatment
16 (88.9)
7 (100)
NS
Glucocorticoid treatment
12 (66.7)
4 (57.1)
NS
Influenza vaccination in 4 (22.2)
2012-13 season
Pneumonia
10 (55.6)
0
NS
7 (100)
0.057
Respiratory failure
9 (50)
7 (100)
0.027
Acute respiratory distress
0
2 (28.6)
0.070
Acute renal failure
2 (11.1)
6 (85.7)
0.001
Acute liver dysfunction
0
5 (71.4)
<0.001
Heart failure
2 (11.1)
1 (14.3)
NS
Disseminated
intravascular coagulation
Sepsis
0
1 (14.3)
NS
0
6 (85.7)
<0.001
Encephalitis
1 (5.6)
0
NS
Intensive care admission
3 (16.7)
1 (14.3)
NS
1 (4)
NS, statistically nonsignificant (p >0.05); COPD, chronic obstructive pulmonary
disease
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P17
POSTE R A BSTRAC TS
SPA1P13
Differentiation of influenza A(H3N2) variant viruses from human
seasonal A(H3N2) viruses by real-time RT-PCR
Bo Shu, Kai-Hui Wu, William Davis, LaShondra Berman, Christine Warnes, Shannon Emery, Nathelia Barnes, Lakshmi Malapati, Julie Villanueva
and Stephen Lindstrom
Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA
Background
Influenza A(H3N2) variant (A(H3N2)v) viruses have been detected in humans in the United States (US) every year since
2009, and from 2011-2013 greater than 300 cases have been identified. A(H3N2)v virus is a swine-origin influenza
virus that circulates enzootically in US swine populations that posses the hemagglutinin (HA) and neuraminidase
(NA) genes originating from human A(H3N2) viruses that were introduced into swine population in late 1990s and
have since evolved independently from human seasonal A(H3N2) viruses. Subsequent evolutionary divergence of
the respective HA genes of human A(H3N2) viruses and swine A(H3N2) viruses allows for differentiation by genetic
analysis. We have developed a real-time RT-PCR (rRT-PCR) assay to rapidly differentiate A(H3N2)v viruses found in the
US from human seasonal A(H3N2) viruses.
Materials and Methods
Analytical performance of rRT-PCR assays designed to differentiate the HA gene of A(H3N2)v viruses (H3v) from
human seasonal A(H3N2) viruses (H3s) were evaluated by testing ten-fold serial dilutions of RNA extracted from
recent A(H3N2) vaccine seed viruses A/Perth/16/2009 and A/Victoria/361/2011, as well as A(H3N2)v candidate
vaccine viruses A/West Virginia/06/2011 and A/Minnesota/11/2010. Reactivity of the H3v and H3s assays was
further evaluated by testing other seasonal human A(H3N2), A(H3N2)v viruses as well as other human and animal
influenza virues.
Performance of H3v/H3s rRT-PCR assays with clinical specimens was demonstrated by testing 329 A(H3N2)v suspect
respiratory specimens and 20 human seasonal A(H3N2) specimens received by CDC from the US state public health
laboratories in 2012 and 2013. Using the CDC Human Influenza Virus Real-Time rRT-PCR Diagnostic Panel (CDC Flu
rRT-PCR Dx Panel), all 329 A(H3N2)v suspect specimens were positive for universal influenza A (InfA; M gene target),
pdm influenza A (pdmInfA; NP gene target) and H3 (HA gene target) assays, and were negative for H1 and pandemic
H1 (pdmH1; HA gene target ) assays.
Results
The analytical performance studies demonstrated the H3v and H3s assays are comparable to CDC InfA rRT-PCR assay.
Cross reactivity was not observed when human seasonal influenza A(H3N2) viruses were tested against H3v assay,
or when A(H3N2)v viruses were tested against H3s assay. Likewise, H3v and H3s assays did not react with influenza
A viruses of other subtypes. The H3v and H3s assays demonstrated 100% agreement when testing 329 human
specimens that were confirmed positive for A(H3N2)v and 20 specimens confirmed positive for human A(H3N2).
Conclusion
The H3v and H3s rRT-PCR assays specifically detect A(H3N2)v viruses and human seasonal A(H3N2) viruses,
respectively. Use of both assays allow for rapid differentiation of A(H3N2)v viruses found in the US from human
seasonal A(H3N2) viruses when used in conjunction with CDC Flu rRT-PCR Dx Panel.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P18
POSTE R A BSTRAC TS
SPA1P14
Performance validation of CDC real-time RT-PCR influenza assays on the
AB TaqMan Array Card
Kai-Hui Wu, C. McCord, B. Shu, C. Warnes, and S. Lindstrom
Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA
Background
The TaqMan® Array Card (TAC) is a 384 microfluidic card that enables simultaneous testing of up to 48 real-time
PCR assays on the AB ViiA7 platform. The CDC real-time RT-PCR (rRT-PCR) influenza assays are designed for universal detection of influenza type A and B, and determination of influenza A subtypes and discrimination of influenza
A and B genotypes on 96-well format thermocycler systems such as the Applied BiosystemsTM (AB) 7500 Fast Dx.
In this study we evaluated the performance of the CDC rRT-PCR influenza assays on TAC on the AB ViiA7 Real-Time
PCR.
Materials and Methods
Performance of twelve rRT-PCR assays for detection and characterization of influenza including A(H3), A(H3)v,
A(H1pdm09), avian A(H5), A(EuH7), B(Victoria lineage) (VIC), and B(Yamagata-lineage) (YAM) were compared by
performing side-by-side limit of detection (LoD) analysis with TaqMan® array cards on the ViiA7 system and 96well plates on the AB 7500 Fast Dx system. Serial dilutions of viral RNA were tested with both systems using the
same reagent preparation. The LoD for each primer and probe set was calculated to indicate the range of lowest
detectable concentration of influenza virus at which ≥95% of all replicates tested positive. Assay reactivity with
clinical specimens was evaluated by testing specimens positive for influenza A(H3N2), A(H3N2)v, A(H1N1), A(H1N1pdm09), B(YAM), B(VIC), as well as specimens negative for influenza A and B.
Results & Conclusion
All assays tested showed comparable sensitivity on ViiA7 when compared with AB 7500 Fast Dx. Sensitivity of the
assays was approximately 5 fold lower on TAC when 46uL RNA per 100uL reaction was tested on the TAC compared
with 5 uL RNA per 25 uL reaction on AB 7500 Fast Dx system. This is expected due to the substantially lower reaction volume of the microfluidic array (approximately 1.4uL per reaction). Performance when testing 96 positive
and 30 negative clinical specimens for influenza showed 100% agreement between both formats. Results from
this evaluation demonstrated that CDC Flu rRT-PCR assays can be used in the TAC microfluidic format on the ViiA7
instrument with limited loss of sensitivity and that the influenza rRT-PCR assays performed comparably on TAC
and 96-well formats.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P19
POSTE R A BSTRAC TS
SPB1: VIRUS STRUCTURE AND REPLICATION
SPB1P01
Application of next-generation sequencing technologies to study the
evolution of H5N2 avian influenza viruses in Taiwan
Shieh, Happy K. (1); Cheng, Ming-Chu (2); Chang, Poa-Chun (1)
1: National Chung Hsing University, Taiwan, Republic of China; 2: Animal Health Research Institute, Taiwan, Republic of China
Background
Sporadic outbreaks of low pathogenic H5N2 influenza viruses have occurred in Taiwan since 2003. The aim of this
study was to investigate the change in the virulence and HA cleavage site sequence of the H5N2 viruses after
consecutive passages in chicken embryonic eggs.
Methods
The low pathogenic H5N2 virus isolated in Taiwan in 2003 was serially passaged in 14‐day‐old chicken embryonic
eggs. The viruses obtained at 5th, 10th, 20th, 30th, 40th and 50th passages were collected. The intravenous
pathogenicity index (IVPI) of these viruses were determined, and the sequence of the HA, NA and PB2 genes of the
viruses were determined by RT‐PCR combined with next‐generation sequencing analysis.
Results
Before the passage, the H5N2 virus isolated in 2003 had an IVPI of 0 and contained only three basic amino acids
(REKR) at the HA cleavage site. At the 10th passage, the IVPI of this virus increased to 0.45 and heterogeneity in the
HA cleavage sequence was observed. Namely, about 53% of the virus contained three basic amino acids (REKR) at
the HA cleavage site, 11% contained four basic amino acids (RKKR), 35% contained five basic amino acids (RRKKR
or RKKKR) and 0.4% contained six basic amino acids (RRRKKR). At 20th and 30th passages, the IVPI of the virus
increased to 1.45 and the percentages of viruses containing four or more basic amino acids increased. All the viruses
examined in this study did not contain E627K mutation at their PB2 protein, and the number of amino acid deletion
at the stalk region of the NA protein is stable.
Conclusions
The low pathogenic H5N2 viruses isolated in Taiwan could mutate into high pathogenic viruses. This result highlights
the importance of eradication of these viruses even when they are low pathogenic.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P20
POSTE R A BSTRAC TS
SPB1P02
Replication of MDCK-adapted viruses with different receptor specificity
in vitro and in vivo
Kuznetsova, Victoria; Isakova-Sivak, Irina; Rudenko, Larisa
Institute of Experimental Medicine RAMS, Russian Federation
Background
Influenza pandemics are caused by influenza viruses that switch receptor binding preference from avian-like α2,3–
sialic acid (SA) to the human-like α2,6–SA receptors. To model the natural process of virus adaptation to a new
host eggs can be used as avian-type cells which predominantly express α2,3–SA and MDCK cells may be used as
mammalian host. During adaptation to the new host viruses usually gain mutations in their surface proteins –
hemagglutinin (HA) and neuraminidase (NA). It is not fully understood how these mutations may influence the
main viral properties such as virulence, replication, transmissibility, immunogenicity and antigenicity.
Materials and Methods
A pair of highly related influenza viruses derived from A/Singapore/1/57 (H2N2) heterogeneous population was
used in this study. These viruses differed by three amino acids in their HA: E156K, Q226L and G228S (H3 numbering),
where EQG variant displayed α2,3–SA receptor binding specificity, whereas KLS had α2,6–SA receptor specificity.
Adaptation of H2N2 viruses to MDCK cells was performed by their serial passaging at MOI=0.001. Cloning of the virus
population was performed by plaque purification on MDCK cells. HAs of MDCK-adapted viruses were sequenced to
find adaptive changes. Replication of the viruses in vitro was assessed by EID50/ml and TCID50/ml. Five viruses
differing by their HAs were used to infect groups of 4 female CBA mice by i.n. route at a dose of 105.0EID50.
Results
Adaptation of KLS variant to MDCK cells resulted in acquisition of two mutations in HA1: G158E and L321P (KLS–EP).
Residue 158 is located in receptor binding site, and receptor specificity of the MDCK-adapted variant significantly
changed. Adaptation of the EQG variant to MDCK cells resulted in two clones with substitution P221S in HA1
(EQG–S) and A96V HA2 (EQG–V). All EQG variants showed high titers in eggs (8.5-8.9 log10EID50/ml) whereas their
titers in MDCK cells significantly varied: the titer of EQG variant didn’t exceed 7.4 log10TCID50/ml while EQG–S
and EQG–V variants grew to the titers over 8.6 log10TCID50/ml. KLS virus replication was low efficient in both
substrates (6.7 log10EID50/ml vs 7.1 log10TCID50/ml) whereas KLS–EP variant demonstrated increased replication
in MDCK cells (7.3 log10EID50/ml vs 8.3 log10TCID50/ml). Replication of all EQG variants in nasal turbinates of mice
was undetectable whereas their titers in lungs significantly varied: 4.4 log10TCID50/ml for EQG variant and 6.5
log10TCID50/ml for EQG–S and EQG–V variants. In contrast, KLS variants were able to replicate in nasal turbinates
(2.2-3.1 log10TCID50/ml) and titers in lungs were comparable: 5.8 log10TCID50/ml for KLS and 6.3 log10TCID50/ml
for KLS–EP variant.
Conclusions
MDCK-adapted mutations of influenza viruses with avian-type receptor specificities affect viral replication in
mammalian cells which can have an impact on virus virulence, transmissibility, immunogenicity and antigenicity.
This work was supported by RFBR Grant No No14-04-32088.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P21
POSTE R A BSTRAC TS
SPB1P03
Structural insight into extracellular domain of matrix protein 2 of
influenza A virus
Kim, Kyung Hyun (1); Cho, Ki Joon (1,2,3); Schepens, Bert (2,3); Seok, Jong Hyeon (1); Kim, Sella (1); Rose, Kenny (2,3); Lee, Ji-Hye (1); Gallardo,
Rodrigo (4); Schymkowitz, Joost (4); Rousseau, Frederic (4); Fiers, Walter (2,3); Saelens, Xavier (2,3); Chung, Mi Sook (5)
1: Korea University, Korea, Republic of South Korea; 2: VIB Inflammation Research Center, 9052 Ghent, Belgium; 3: Department of Biomedical
Molecular Biology, Ghent University, 9052 Ghent, Belgium; 4: VIB Switch Laboratory, Department for Cellular and Molecular Medicine,
Katholieke Universiteit Leuven, 3000 Leuven, Belgium; 5: Department of Food and Nutrition, Duksung Women’s University, Seoul 132-714, Kore
Background
The extracellular domain of matrix protein 2 (M2e) of influenza A is extensively explored as a universal influenza
A vaccine candidate because of its strong sequence conservation. Although crystal structures of the extracellular
domain of HA and NA have been known, either as a free form or as a complex form with an Fab of monoclonal
antibodies, the ectodomain structure of M2 has remained elusive.
Methods
M2e-tGCN4 and M2e-Fab65 complex were characterized by biochemical methods and their structures were solved
by X–ray crystallography. Mutation studies and reverse genetics system were also used.
Results
FTIR and crystallographic studies of M2e-tGCN4 revealed that the leucine zipper domain of M2e-tGCN4 is
superimposed well with the transmembrane domain of M2 determined by high resolution NMR, but M2e was
invisible possibly due to high flexibility (Figure 1). In contrast, the M2e-Fab65 complex crystal structure showed that
the complementary determining regions interact with M2e that adopts a compact U-shaped conformation (Figure
2). Several highly conserved M2e residues are critical for interaction with Fab65: Trp15 is essential for stabilizing the
conformation of M2e and Glu6 and Glu8 are critical for antibody binding.
Conclusions
We determined the structures of M2e and its complex with Fab fragment of an M2e-specific monoclonal antibody
65 (mAb65). M2e appears to have an extended conformation and is highly flexible, whereas it adopts a U-shaped
conformation that is stabilized by the engagement of crucial residues that are highly conserved among human and
nonhuman influenza A viruses.
This work was supported by grants from Mid-career Researcher Program (2010-0029242) through NRF funded by
the MEST and from TEPIK (A103001) funded by the Ministry for Health, Welfare & Family Affairs, Korea.
Figure 1. Structure of M2e-tGCN4. The leucine zipper domain of M2e-tGCN4 is superimposed well with that of
M2 determined by NMR (green and marine colors, respectively), whereas M2e was invisible possibly due to high
respectively), whereas M2e was invisible possibly due to high flexibility (orange and red)
flexibility (orange and red)
Figure 1. Structure of M2e-tGCN4. The leucine zipper domain of M2e-tGCN4 is
superimposed well with that of M2 determined by NMR (green and marine colors,
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P22
POSTE R A BSTRAC TS
Leu3
cys19
Figure
2. Structures
ofwith
M2e
within Fab65. The Fab (marine and green) in complex with M2e (magenta)
Figure 2. Structures
of M2e complexed
Fab65.complexed
The Fab (marine and green)
complex with M2e (magenta) (upper panel) and the compact U-shaped conformation of
(upper panel) and the
compact U-shaped conformation of M2e (lower panel).
M2e (lower panel).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P23
POSTE R A BSTRAC TS
SPB1P04
The transmembrane domain of influenza NA has co-evolved with the
distal head domain
da Silva, Diogo V; Nordholm, Johan; Dou, Dan; Daniels, Robert
Stockholm University, Sweden
Abstract
Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as hydrophobic
membrane anchors for functional domains. Influenza neuraminidase (NA) exemplifies this concept as it retains
enzymatic function upon proteolytic release from the membrane. However, we recently showed the NA TMDs have
become increasingly less hydrophobic in human H1N1 viruses, which suggests the TMD is changing to maintain
compatibility with the distal enzymatic head domain. Here, we investigated this relationship by analyzing an ‘old’
1933 H1N1 virus (WSN33) where the amphipathic NA TMD was exchanged for a ‘contemporary’ 2009 subtype
1 NA TMD, and an engineered hydrophobic TMD. Each exchange decreased the NA folding efficiency resulting in
significant fitness loss in viral replication at 37°C, but very little loss at 33°C where NA folds more efficiently. The
chimera viruses restored the viral infectivity and NA folding efficiency at 37°C by selecting for mutations in the NA
TMD, which corresponded with their assembly properties (polar/hydrophobic). These results likely explain why the
amphipathic NA TMD in H1N1 viruses became more polar with subsequent changes in the head domain, and show
that the folding equilibrium of NA can significantly impact viral replication likely by altering the enigmatic ‘HA-NA’
equilibrium.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P24
POSTE R A BSTRAC TS
SPA2: EPIDEMICS AND PANDEMIC THREATS
SPA2P01
Application of next-generation sequencing technologies to study the
evolution of H5N2 avian influenza viruses in Taiwan
Wong, Jessica Y. (1); Kelly, Heath (2,3); Cheung, Chung-Mei M. (4); Shiu, Eunice Y. (1); Wu, Peng (1); Ni, Michael Y. (1); Ip, Dennis K. M. (1);
Cowling, Benjamin J. (1)
1: School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China; 2:
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia; 3: National Centre for Epidemiology and Population
Health, Australian National University, Canberra, Australian Capital Territory, Australia; 4: Medical School, University College London, London,
United Kingdom
Background
During the 2009 influenza pandemic, uncertainty surrounding the seriousness of human infections with the
H1N1pdm09 virus hindered the calibration of the public health response. One measure of seriousness is the
hospitalization fatality risk (HFR), defined as the probability of mortality among cases of H1N1pdm09 who
required hospitalization for medical reasons. The objective of our study was to review published data on the HFR of
H1N1pdm09.
Methods
We searched for relevant studies in PubMed, MEDLINE and EMBASE. Studies that reported population-based
estimates of the HFR for H1N1pdm09 were included. We excluded studies that reported estimates of the HFR in
population subgroups such as pregnant women or those at higher risk of severe outcome if infected (e.g. individuals
with underlying chronic diseases).
Results
We included 187 estimates of the HFR from 184 published studies, reporting a total of 151,754 hospitalized cases
and 7,010 deaths. In total, our analysis was based on reports from 49 countries or regions in every continent except
Antarctica. We identified heterogeneity in published HFR estimates, with crude estimates of the risk of death
ranging from 0% to 48% but in wealthy countries the estimate ranged from 1% to 4%. The point estimates of the HFR
increased substantially with age, with lower GDP and in second and subsequent years of H1N1pdm09 circulation. In
children, the HFR of H1N1pdm09 and inter-pandemic influenza did not have substantial difference.
Conclusions
There was some variability in published estimates of the HFR, but much less variability than we found in our review
of published estimates of the case fatality risk (Wong J et al., 2013 Epidemiol). Lower HFR in children could be due to
reduced seriousness of infection, or a lower threshold for admitting children. Early in the next pandemic, estimation
of the HFR may provide a reasonable picture of seriousness of infection and thereby inform risk assessment of the
severity of the pandemic strain, particularly if presented in comparison with HFR for inter-pandemic influenza in
similar setting.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P25
POSTE R A BSTRAC TS
SPA2P02
Real-time estimation of the hospitalization fatality risk of influenza
A(H1N1)pdm09 in Hong Kong
Wong, Jessica; Tsang, Tim K.; Wu, Peng; Lau, Eric H. Y.; Ip, Dennis K. M.; Cowling, Benjamin J.
School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
Background
During the early stage of an epidemic, timely and reliable estimation of the seriousness are important for predicting
the impact that the influenza viruses will have in the population. The hospitalization fatality risk (HFR), which is a
measure of seriousness of infection, is defined as the probability of death among cases who required hospitalization
for medical reasons. The objective of our study was to determine optimal methods for real-time estimation of the
HFR of H1N1pdm09 in 2009 in Hong Kong.
Methods
We obtained age-specific deaths and hospitalizations among patients with laboratory-confirmed H1N1pdm09
infections from May 2009 through July 2010 from the Hong Kong Hospital Authority. We estimated HFR in real-time,
using crude estimation or allowing for right-censoring for final status in some patients (Jewell et al., 2007 Stat Med;
Garske et al., 2009 BMJ) using Bayesian inference.
Results
We applied the four different estimators to the H1N1pdm09 data and found that the estimate was relatively stable.
Models allowing for censoring preformed better than models without adjustments. We were able to obtained
reliable estimates of HFR in mid-September, early-September and mid- October for 20-44y, 45-64y and ≥65y
respectively. The risk of deaths among hospitalized patients with confirmed H1N1pdm09 increased with age, with
point estimates ranging from approximately 1% in younger adults to 5% in the elderly.
Conclusions
We obtained reliable estimate of the HFR before the peak of the 2009 pandemic in adults but after the peak in
the elderly using models allowing for censoring. Unable to obtain timely HFR estimate in the elderly until could
be due to very few deaths at the early stage of the pandemic. During an ongoing epidemic, we would be able to
obtain reasonable real-time estimates of theseriousness of human infection, if appropriate models are prepared in
advance.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P26
POSTE R A BSTRAC TS
SPA2P03
Improving Standardisation and Timeliness of Seroepidemiological
Studies through the Global Partnership CONSISE (the Consortium for the
Standardization of Influenza Seroepidemiology)
Van Kerkhove, Maria D (1); Laurie, Karen (2); Engelhardt, Othmar (3); Wood, John (4); ., CONSISE (5)
1: Imperial College London, United Kingdom; 2: WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia;
3: Division of Virology, National Institute for Biological Standards and Control, United Kingdom; 4: Formerly Division of Virology, National
Institute for Biological Standards and Control, United Kingdom; 5: Consortium for the Standardization of Influenza Seroepidemiology
Background
Serological studies can detect infection with influenza viruses in the absence of symptoms or virus detection,
providing additional information on infection beyond estimates from epidemiological, clinical and virological data.
Identification of both asymptomatic and symptomatic infections enables estimates of severity and transmissibility
for a novel influenza virus to be calculated. Furthermore, possible susceptible populations can be identified and
the potential impact of a novel influenza virus estimated. A large number of serological studies were conducted
in 2009 and 2010 describing the impact of the pandemic influenza virus around the world. However the timing
of many publications limited their usefulness to inform public policy and differences in methodology made direct
comparison of studies difficult.
Methods
In 2011, CONSISE, the Consortium for the Standardization of Influenza Seroepidemiology, a global partnership,
was formed to develop influenza investigation protocols and standardise seroepidemiology to inform public health
policy. The activities of CONSISE are performed by two inter-linked working groups, laboratory and epidemiology,
and a steering committee.
Results
The Epidemiology Working Group is developing several generic yet comprehensive epidemiological, virological and
serological protocols for influenza and other respiratory viruses and question banks to facilitate rapid and informative
studies. The Laboratory Working group focuses on improving serological assay comparability and standardisation
through consensus assay development, comparative laboratory testing and quality assurance. Two comparative
laboratory studies assessing microneutralisation (MN) assay protocols have been performed. A third comparative
laboratory study assessing the Enzyme-linked lectin assay (ELLA), that detects antibodies to neuraminidase, is
underway. A further comparison of both MN and haemagglutination inhibition assay protocols will begin in late
2014.
Conclusions
CONSISE is playing a role in public health evaluation and management of newly emerging respiratory agents,
including MERS-CoV and A(H7N9). Our materials are open access and shared on the Global Health Network website
(http://CONSISE.tghn.org/). We seek additional members from public health agencies, academic institutions and
other interested parties.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P27
POSTE R A BSTRAC TS
SPA2P04
INFLUENZA EPIDEMIC SEASON 2013-2014 IN RUSSIA: CHARACTERISTICS
OF ISOLATED STRAINS
Eropkin, Mikhail; Danilenko, Daria; Konovalova, Nadejda; Komissarov, Andrey; Pisareva, Maria; Grudinin, Mikhail; Lobova, Tamara; Schekanova,
Svetlana; Kornilova, Ekaterina; Eropkina, Elena; Suddenkova, Polina
Research Institute of Influenza, Saint-Petersburg, Russian Federation
Background
influenza surveillance in Russian Federation during 2013-2014 epidemic season.
Methods
virus isolation, identification, antigenic analysis in HI-test, sequencing, phylogenetic analysis, MUNANA-test of
sensitivity to neuraminidase inhibitors.
Results
Epidemic season 2013-2014 in Russia was characterized by a relatively low intensity. By the end of April only 739
strains were isolated, among them 366 strains (49,5%) – influenza А/H1N1рdm09, 333 strains (45 %) – influenza
A/H3N2 and only 35 strains (4,7%) – influenza B. The essential exceeding of the epidemic morbidity thresholds was
observed only in the Far East where more than 130 strains were isolated. Nevertheless in this season we succeeded
to isolate, antigenically characterize and sequence three pre-epidemic strains – A/St.Petersburg/428/13(H3N2), A/
Moscow/18/13 (H1N1)pdm09 and B/Khabarovsk/43/13 which were isolated in October-December 2013. In the
Asian part of Russia the prevailing strains were А/H1N1pdm09 (89,5 % of all isolates) while in the European part – А/
H3N2 (78,5 %). The impact of influenza B into the epidemic process was minor (3,7 % of all isolated strains). Antigenic
analysis in HI-test has shown in general the conformity with the strain composition of seasonal flu vaccines proposed
by the WHO. Influenza virus А/H1N1pdm09 were similar to the reference strain A/California/07/09, while a slight
gradual drift was observed (HI-titer of a part of strains was 4-8-fold of homological titer). Sequencing of those strains
has demonstrated their belonging to sub-clade 6B (similar to the etalon А/Stockholm/15/13). Viruses А/H3N2 were
similar to the etalon A/Texas/50/12 with aminoacid substitutions specific for the sub-clade 3C.3 A/Victoria/361/11like strains (a typical representative - A/Samara/73/13). We did not reveal any mutations of resistance to oseltamivir
and zanamivir in the sequences of neuraminidase gene of all analyzed strains but all of them were genetically
resistant to rimantadine. All analyzed strains proved to be sensitive to the neuraminidase inhibitors also according
the results of the fluorescent MUNANA-test. Between the analyzed influenza B strains 3 strains isolated in St.Petersburg, Yekaterinburg and Vladivostok belonged to Yamagata lineage (B/Massachusetts/2/12-like) while 2
strains from Khabarovsk were of Victorian lineage (B/Brisbane/60/08-like). Its HA gene belonged to the clade 1A.
Conclusions
The conformity of strains circulated on the territory of Russia with the WHO-proposed strain composition of
influenza vaccines for the epidemic season 2014-2015 was endorsed on the session of the National Commission
on vaccine and diagnostic influenza strains which took place in the Research Institute of Influenza on Feb. 27 2014.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P28
POSTE R A BSTRAC TS
SPA2P05
Excess mortality risk attributable to influenza infection in Hong Kong,
1998-2012
Wu, P (1); Goldstein, E (2); Ho, LM (1); Wu, JT (1); Ip, DKM (1); Leung, GM (1); Cowling, BJ (1)
1: School of Public Health, The University of Hong Kong, Hong Kong S.A.R. (China); 2: 2Center for Communicable Disease Dynamics,
Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA
Background
Seasonal influenza viruses have caused considerable morbidity and mortality in humans. Estimates from previous
studies indicated that the disease burden attributable to influenza virus infection was much more substantial than
the observed influenza cases confirmed by laboratory testing. We aimed to estimate the excess mortality from
major underlying causes associated with different influenza types and subtypes in Hong Kong, a subtropical city
with influenza circulating throughout the year, from 1998 through 2012.
Methods
We used multi-linear regression models to fit age and sex-specific all-cause and cause- specific mortality rates
in Hong Kong from 1998 to 2012. The model allows activities of different types/subtypes of influenza virus and
co-circulating respiratory syncytial virus to be adjusted as the product of influenza-like-illness consultation rates
from sentinel surveillance and laboratory detection rates of each specific virus (Wu P et al., J Infect Dis 2012).
Environmental factors and periodic temporal trends in mortality rates during the study period were also adjusted.
The influenza-associated excess mortality was measured as the differences between estimated mortality rates in
the presence or absence of influenza activity in the model. Sex-specific attributable fractions were estimated for
each cause of death.
Results
The annual influenza-associated all-cause excess death rates varied between 7 and 14 per 100,000 person-years
in 1998-2012 during which influenza A(H3N2) contributed more than half of the deaths attributed to influenza.
On average, influenza caused higher mortality rate in men than in women. The attributable fractions of influenzaassociated excess mortality were generally higher in women than in men in different causes of death except for
cardio-respiratory death. Most influenza-associated excess deaths occurred in the elderly both for men and women.
Cardio-respiratory diseases accounted for more influenza-associated excess deaths in men in women.
Conclusions
Influenza was associated with a substantial number of deaths in Hong Kong between 1998 and 2012 in both men
and women particularly in the elderly. Cardio-respiratory diseases accounted for the majority of the influenza
associated excess deaths in both sexes especially among men although the proportion of excess cardiovascular
deaths was relatively lower in Hong Kong than western countries.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P29
POSTE R A BSTRAC TS
SPA2P06
Community psychological and behavioral responses to the threat of
A(H7N9) in Hong Kong
Wu, P; Fang, VJ; Liao, Q; Ng, DMW; Wu, JT; Leung, GM; Fielding, R; Cowling, BJ
School of Public Health, The University of Hong Kong, Hong Kong S.A.R. (China)
Background
The novel influenza A(H7N9) virus has causes epidemics of infections in poultry and humans since its first detection
although evidence suggested that the risk of human-to-human transmission is low. Laboratory-confirmed human
cases dramatically decreased following the closure of live poultry markets in affected cities in China. While risk of
H7N9 appears to be low in Hong Kong, nine H7N9 cases among Hong Kong residents have been reported since
December 2013. We therefore conducted a study aiming to measure levels of exposure to live poultry, risk perception,
psychological and behavioral responses towards H7N9, and attitudes towards specific control measures at different
stages of the epidemic in the population.
Methods
A series of cross-sectional population surveys were conducted in 2013. The first survey was carried out on in April
shortly after the first H7N9 case was announced in mainland China. A second survey was conducted in December
once incidence of human cases of H7N9 began to rise in the winter and the first local case was notified in Hong Kong.
Subjects who were Cantonese/Mandarin- speaking Hong Kong Chinese adults (≥18 y) were selected for interview
through randomly dialed landline numbers generated by computer. The questionnaire included demographics and
items investigating the state of anxiety, H7N9- related risk perception, attitudes towards closure of live poultry
markets.
Results
There were 1,556 and 1,000 interviews completed during the April and December surveys with response rates of
68.9% and 68.0%, respectively. The level of general anxiety in the population remained low. Respondents reported
low perceived susceptibility of infection, lower perceived severity of influenza A(H7N9) than SARS, higher perceived
severity of influenza A(H7N9) than influenza A(H5N1) and seasonal influenza, and low levels of influenza-like
symptoms induced worry. Across the population, the average numbers of annual visits to live poultry markets in
Hong Kong and mainland China to be 17.6 and 0.53 visits per person, respectively. A total of 17.5% respondents
reported avoidance of visiting live poultry markets because of influenza A(H7N9), whereas 35.9% reported that they
would support permanent closure of markets. Younger age, lower educational attainment, and visit to live poultry
markets >1 time in the preceding year were independently associated with a lower probability of support for market
closures.
Conclusions
Our study identified generally low anxiety levels among the population in Hong Kong related to the threat of
influenza A(H7N9). Higher symptom-induced worry and higher perceived severity of influenza A(H7N9) compared
with seasonal influenza were associated with avoidance of live poultry markets. Our results suggested that
obtaining support from the public for permanent closure of live poultry markets might be difficult, particularly
among younger adults and adults with lower educational attainment.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P30
POSTE R A BSTRAC TS
SPA2P07
The avian origin H9N2 polymerase, common to multiple human lethal
strains, possesses robust activity in mammalian cells
Cox, Andrew George (1,2); Kim, Yoel (1); O’Dell, Colleen (1); Schmierer, Jordana (1); Smith, Andrew (1,2); Dewhurst, Stephen (1)
1: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, United States of America; 2:
University of Medical Scientist Training Program, University of Rochester School of Medicine and Dentistry, United States of America
Background
Influenza A Viruses (IAV) adapted to their natural avian host do not normally replicate in mammalian cells due to
multiple restrictions. In order to replicate effectively in humans, IAV require both adaptive mutations within the
polymerase as well as within hemagglutinin to allow for efficient mammalian infection and replication. Contrary
to this dogma, the H9N2 virus (A/quail/Hong Kong/G1/97) that donated the internal segments to the lethal H5N1
virus replicates efficiently in a human lung epithelial cell line at 37oC. Interestingly, this viral polymerase does not
possess any of the previously described mutations required for mammalian adaptation including PB2 627K and
PB1 99Y. Of further interest, the novel H7N9 and H10N8 viruses also contain a H9N2 polymerase. We therefore
hypothesize that the avian H9N2 polymerase may be uniquely primed for function in mammalian cells, and that it
has acquired this functionality through a novel mechanism.
Methods
We used luciferase based mini genome reporter assays to assess viral polymerase activity, beginning with either
a vRNA or cRNA template of varying lengths in mammalian A549 cells and avian DF-1 cells at physiologically
relevant temperatures. We compared this avian origin H9N2 virus to a well-studied avian origin virus A/chicken/
Nanchang/03-120/2001 H3N2, known to have restricted polymerase activity in mammalian cells. We also have
compared activity to the avian polymerase of A/wild bird/Korea/A14/2011 H7N9 that donated the N9 segment to
the human lethal H7N9 viruses. As positive controls we have utilized the well characterized mammalian adapted A/
WSN/1933 H1N1 and A/Puerto Rico/8/1934 H1N1 polymerases.
Results
We observed that the H9N2 polymerase has robust activity in mammalian cells at 37 and 39oC with all templates,
but low levels of activity at 33oC. Surprisingly; this H9N2 polymerase has greater activity than the prototypical
mammalian adapted strains of PR8 and WSN at 37 and 39oC, but lower at 33oC. The residues of the H9N2
polymerase that enable this increased activity within mammalian cells are located with the PB2 and PA segments.
Conclusions
The polymerase of A/quail/Hong Kong/G1/1997 H9N2 displays robust activity in mammalian cells despite not
possessing mammalian amino acid identity at the previously described amino acids. Through further characterization
we have identified a cohort of novel residues within the N termini of PA and middle of PB2 that permit avian origin
polymerases to function within mammalian cells. These results suggest that the H9N2 avian IAV polymerase
has unique properties that may account for the serial incorporation of this polymerase into avian influenza virus
reassortants capable of infecting humans. Future studies will identify specific amino acid residues with the viral PA
and PB2 segments that contribute to this phenotype, and thereby elucidate the mechanism underlying the unique
functional characteristics of the H9N2 avian IAV polymerase.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P31
POSTE R A BSTRAC TS
SPA2P08
The Global Influenza B Study (GIBS): a Research Platform for the Study
of Influenza B
Paget, John (1); Caini, Saverio (1); Huang, Sue (2); Ciblak, Meral (3); Schellevis, Francois (1); Plotkin, Stanley (4)
1: NIVEL, The Netherlands; 2: The WHO National Influenza Centre, Institute of Environmental Science and Research, New Zealand; 3: Istanbul
University, Istanbul, Turkey; 4: Univesity of Pennsylvania, Philadelphia, USA
Background
Background: Much of the scientific literature to date has focused on influenza A in temperate countries, and we
still have a relatively poor understanding of the epidemiology of influenza B, especially in the inter-tropical belt.
Launched in 2012, the Global Influenza B Study (GIBS) collects surveillance data on influenza B worldwide in order
to support influenza prevention and control policies around the world.
Methods
We contacted National Influenza Centres in 43 countries and asked for surveillance data during 2000-2013. Each
country was asked to provide: the weekly number of reported influenza cases broken down by age group, virus
type/subtype and lineage (for influenza B); the weekly influenza-like illness (ILI) or acute respiratory illness (ARI)
rates per 100,0000 population or 100 consultations. Large countries that covered multiple climatic regions we asked
to provide regional data. Participating countries were also asked to complete a brief questionnaire regarding the
characteristics of their national influenza surveillance system.
Results: Twenty-six countries (accounting for over one third of world’s population) joined GIBS
Results
(Table): seven
in the Northern
hemisphere,
five in the Southern
fourteen
the inter- population) joined GIBS (Table): seven
Results:
Twenty-six
countries
(accounting
for hemisphere,
over oneandthird
of inworld’s
intropical
the Northern
hemisphere,
five
in the
Southern
hemisphere,
and
in the inter- tropical belt. The GIBS
belt. The GIBS
database includes
935,673
influenza
cases collected
between 2000
andfourteen
2013,
database
includes
935,673
influenza
between
andwere
2013, of which 178,206 (19.0%) were cases
of which 178,206
(19.0%)
were cases
of influenzacases
B. Thecollected
proportion of
influenza A2000
cases that
of influenza B. The proportion of influenza A cases that were subtyped was 65.1% and information on strains was
subtyped was 65.1% and information on strains was available for 17.2% of influenza B cases.
available for 17.2% of influenza B cases. Information on age was available for 59.1% of influenza B cases and 47.6%
on age
was available for 59.1% of influenza B cases and 47.6% of influenza A cases.
ofInformation
influenza
A cases.
Table.
Countries
participating
in the
Global
Influenza
B Study
Table. Countries
participating
in the Global
Influenza
B Study
(GIBS), May
2014 (GIBS), May 2014
Region
Countries
No. influenza
cases
Influenza A cases Influenza B cases
(No., %)
(No., %)
Southern
hemisphere
Argentina (Santa Fe), Australia, Chile, New
Zealand, South Africa
210,301
176,742
84.0%
33,559
16.0%
Intertropical
belt
Brazil, Cameroon, Costa Rica, El Salvador,
Guatemala, Honduras, Indonesia, Ivory
Coast, Kenya, Madagascar, Nicaragua,
Panama, Singapore, Viet Nam
62,794
48,847
77.8%
13,947
22.2%
Northern
hemisphere
Bhutan, China, England, Italy, Turkey,
Ukraine, USA
662,578
531,878
80.3%
130,700
19.7%
Total
26 countries
935,673
757,467
81.0%
178,206
19.0%
Conclusions
Conclusions: Thanks to the participation of a very large number of countries from most regions of the
Thanks to the participation of a very large number of countries from most regions of the world, we have built a
world, we have built a unique global influenza database as it is case-based, includes age specific data
unique global influenza database as it is case-based, includes age specific data (the FluNet database collects
(the FluNet database collects aggregated data that is not age specific), and contains nearly 1 million
aggregated data that is not age specific), and contains nearly 1 million cases of influenza. The wealth of info that
casesbeen
of influenza.
The wealth
of GIBS
info that
has been collected
means GIBS
represents
valuable
has
collected
means
represents
a valuable
platform
thata can
be exploited to address influenza research
projects
that
help support
prevention
policies
around
the world.
platform that
canwill
be exploited
to addressinfluenza
influenza research
projects that
will help
support influenza
prevention policies around the world.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P32
POSTE R A BSTRAC TS
SPA2P09
Possible explanations for why some countries were harder hit by
Influenza A(H1N1)pdm09 – a global mortality impact modeling study
Morales, Kathleen (1,2); Paget, John (1); Spreeuwenberg, Peter (1)
1: NIVEL, The Netherlands; 2: Sage Analytica, Rockville, MD. USA
Background
Since the first confirmed death of Influenza A(H1N1)pdm09 in Mexico in April 2009, researchers and public health
officials have continued to search for clarity on the global morbidity and mortality impact of Influenza A(H1N1)
pdm09 and its associated risk factors in order to improve future pandemic response. The GLaMOR study (Plos
Med 2013), found prominent regional mortality variations in 2009 for Influenza A(H1N1)pdm09, with the highest
burden in the Americas and the lowest burden in Europe. Our study attempts to identify factors that explain why
the pandemic mortality burden was high in some countries and low in others.
Methods
As a starting point, we identified possible risk factors worth investigating for Influenza A(H1N1)pdm09 mortality
through a targeted literature search. We then used Rubin’s Causal Inference Model to identify factors that could
explain differences in respiratory mortality due to Influenza A(H1N1)pdm09 and to determine their impact. We ran
Netherlands Institute for Health Services research (NIVEL), Utrecht, The Netherlands Sage Analytica, Rockville, MD.
USA sixteen different Causal Inference Models to produce robust results and draw conclusions. In order to assess
the role of each factor in explaining differences in excess pandemic mortality, we calculated the mean proportion of
pandemic mortality country variance that can be explained by each factor
Results
The literature search identified 124 publications and 47 possible risk factors. Of the 47 possible risk factors, we
identified appropriate global datasets for 27 factors based on four selection criteria (e.g. we could identify enough
data from one global source for at least 50 countries). The Causal Inference Models indicated that age structure
(explaining 40% of the mean proportion of pandemic mortality country variance), latitude (8%), influenza A and B
viruses circulating during the pandemic (3-8%), influenza A and B viruses circulating during the preceding influenza
season (2-6%), air pollution (pm10; 4%) and the prevalence of other infections (HIV and TB) (4-6%) were factors that
explained differences in mortality around the world. Healthcare expenditure, levels of obesity and air travel did not
explain global pandemic mortality differences
Conclusions
Our study found that countries with a large proportion of young persons and those located in the inter-tropical
belt had higher pandemic mortality rates in 2009. These results are difficult to interpret as age is associated with a
number of factors (e.g. living in the tropics and HIV) and latitude could be driven by the low mortality rates in the
WHO Euro and Western Pacific regions. Interestingly, the co-circulation of influenza viruses during the pandemic
and the circulation of influenza viruses during the preceding season were also associated with pandemic mortality
rates. Our findings suggest that public health officials confronted with a future pandemic should probably target
pandemic preparedness activities in the young and in countries with high rates of HIV/TB.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P33
POSTE R A BSTRAC TS
SPA2P10
Evolution of highly pathogenic avian influenza (H5N1) virus of domestic
poultry in Vietnam between 2011 and 2013
Lee, Eun-Kyoung (1); Kang, Hyun-Mi (1); Song, Byung-Min (1); Jeung, Jipseol (1); Choi, Jun-Gu (1); Thanh, Long To (2); Tho, Dang Nguyen (2);
Choi, Kang-Seuk (1); Kye, Soo-Jeong (1); Kim, Ji-Ye (1); Lee, Hee-Soo (1); Lee, Youn-Jeong (1)
1: Animal and Plant Quarantine Agency, Korea, Republic of (South Korea); 2: National Center for Veterinary Diagnosis, Department of Animal
Health, Vietnam
Background
Since the H5N1 highly pathogenic avian influenza (HPAI) virus was first detected in 1996 in southern china, the virus
has spread to more than 60 countries across Asia, the Middle East, Europe and Africa. With a threat to public health,
H5N1 has caused major losses in poultry flocks in affected areas through either direct infection or preventive culling.
In spite of vaccination campaigns, H5N1 viruses continue to circulate in Vietnam for domestic poultry. To estimate
the prevalence of AIV in Vietnam, surveillance was conducted between November 2011 and Feburary 2013.
Methods
A total of 450 samples, collected in 54 provinces of Vietnam, were obtained from the National Center of Veterinary
Diagnostics of Vietnam (NCVD). After initial screening for Matrix gene of AIV using real-time RT-PCR, AIV positive
samples were inoculated into embryonated eggs for virus isolation. For subtyping of AIV, Hemagglutination (HA)
and Neuraminidase (NA) gene of all AIV isolates were amplified with gene-specific primers (Hoffmann, et al., 2001)
using the One-Step RT- PCR kit (Qiagen, USA) and confirmed by sequencing analysis, which were identified by
BLAST searches of the National Center for Biotechnology Information (NCBI) database. To analyze the phylogenetic
analysise of the Vietnamese H5 subtype AIVs in this study, of all isolates, 31 AIVs for Surface genes and 17 AIVs for
internal genes were selected according to the HA clades, region and prevalence.
Results
We report on the genetic analysis of 311 HPAI H5 viruses isolated from poultry in Vietnam and investigated possible
genetic relationships with neighboring countries. Two H5N1 HPAI clades were circulating in the Southern provinces
(clade 1.1) and Northern provinces (clade 2.3.2.1) of Vietnam. Phylogenetic analysis of the H5 AIVs revealed clade
1.1 is geographical circumscribed group related with Cambodia and subclade 2.3.2.1C were emerged in June 2012
and was predominat in 2013 in Northern and Central province. In the NA gene tree, most of H5N1 isolates can be
divided into major two groups and HA clade groups shared also NA lineages. In gene constellation, Clade 2.3.2.1C
viruses were found to be reassortants between Clade 2.3.2.1A(PB2, PA) and new subtype of Clade 2.3.2.1B(PB1, NP,
NA, M, NS). Interestingly, Enzootic vietnamese clade 2.3.2.1C H5 virus subsequently reassorted with N2 originated
from wild bird to generated H5N2 HPAI, which isolated from duck in live bird market(LBM).
Conclusions
The results of the present study revealed that HPAI H5N1 viruses are endemic in Vietnam. Vietnamese viruses
have genetic diversity depend on regions and the genetic subclade have been changed. Recently Evolution and
genetic reassortment events of Clade 2.3.2.1 were identified from phylogenetic analysis. Further investigation of
inter-country surveillance programs are required if we are to understand the epidemiology of these viruses and to
manage the emergence of novel types.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P34
POSTE R A BSTRAC TS
SPA2P11
ANALYSIS OF INFLUENZA SENTINEL SURVEILLANCE RESULTS IN
KAZAKHSTAN, 2013-2014 EPIDEMIOLOGICAL SEASON
Ozhanova, Alfiya Ibiraimovna; Mirzabekova, Gulfairuz Kuralbekovna
Zhambyl oblast Consumers Rights Protection Department of of Consumers Rights Protection Agency of Kazakhstan Republic, Kazakhstan
Introduction
Since 2008, the practice of year-round sentinel surveillance (SS) for influenza- like illnesses (ILI) and severe acute
respiratory infections (SARI) was adopted in the Republic of Kazakhstan to complement the routine surveillance for
acute respiratory infections (ARI) and influenza. During the last epidemiological season of 2013-2014, SS included
22 hospitals and 24 clinics in 7 cities. Following WHO recommendations, in 2011 the epidemiology of ILI and SARI
sentinel surveillance was strengthened and today all persons with ILI and persons admitted to hospital with SARI are
included into the overall statistics. We determined the characteristics of the 2013-2014 influenza season.
Methods
Data in the online database, as well weekly data collected by the routine surveillance for ARI and influenza
epidemiological season, were reviewed.
Results
In the 2013-2014 epidemiological season, the ILI incidence rate was 400 cases per 100,000 population. The highest
ILI incidence was observed during the 7th through the 12th weeks of 2014, being highest among children under 5,
ranging between 81.3 to 108.9 cases per 100,000. The lowest ILI incidence rate occurred among people above 65
years of age, ranging between 5.2 to 7.6 cases per 100,000. The percentage of PCR-positive influenza tests among ILI
patients was 25.6% (140/545). Influenza virus circulated among ILI patients during the 45th through the 16th week,
with the largest number of PCR+ influenza cases occurring during weeks 7-9 (55%, 59% and 58%, respectively). Of
the 140 confirmed influenza cases, the proportion of influenza virus type A comprised 96.4%, type B – 3.6%. Both
influenza virus A subtypes, A/H1N1-09 – 45.9% and A/H3N2 – 54.1%, circulated concomitantly. SARI incidence in
the 2013-2014 season was as high as 247.5 per 1,000 admissions to sentinel hospitals. The highest SARI incidence
was from week 44 through the week 50 (ranging 294-327 per 1,000 admissions) due to other respiratory pathogens,
because SARI patients began to test positive for influenza from week 50. Influenza circulation among SARI patients
was observed from week 49 to week 16. High SARI rates were registered mainly among children under 5 throughout
the season, ranging from 100 to 319 per 1,000 admissions. The proportion of PCR-positive tests among SARI patients
was 30.4% (271/891), mainly driven by influenza type A – 99.6% (270/271) and only one case of influenza type B
(0.4 %). The A/H1N1-09 influenza subtype was found in 47% (127/270) and A/H3N2 in 53% of all cases (143/270).
Conclusions
The 2013-2014 epidemiological season saw an active circulation of influenza type A, and only a few isolated cases of
influenza type B in ILI and SARI patients. There was concomitant circulation of influenza A subtypes A/H1N1-09 and
A/H3N2. A high incidence of ILI and SARI was recoded in children under 5 years of age.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P35
POSTE R A BSTRAC TS
SPA2P12
SOME APPROACHES TO PREDICTION OF SEASONAL INFLUENZA EPIDEMIC
BURDEN
Sominina, Anna A.; Karpova, Ludmila; Smorodintseva, Elizaveta; Grudinin, Mikhail; Pisareva, Maria; Komissarov, Andrei; Eropkin, Mikhail;
Konovalova, Nadejda; Danilenko, Daria
Research Institute of Influenza, Russian Federation
Background
New knowledge on annual influenza activity and correlations with peculiarities of influenza virus type/sub-type
circulation in Russia are helpful for prediction of epidemic impact on country level and for understanding of the
spread of epidemics and future pandemic on a global level. Goal of this investigation was complex analysis of
routine and sentinel epidemiological data, antigenic and genetic peculiarities of viruses circulated in Russia for the
5 years period since the emerging of new pandemic influenza virus in 2009.
Methods
Results of routine epidemiological surveillance based on investigation of WHO NIC of Russia in collaboration with
regional base laboratories. The methods used were described earlier (A.A.Sominina at al., AJID, 9(3):77-93, 2013)
Results
Enhanced epidemiology capacity and infrastructure for disease surveillance with weekly analysis of ILI and ARI
morbidity by age groups both for early recognition of epidemic start and for evaluation of epidemic intensity,
geography spread and hospitalization rates achieved last years in Russia. Three influenza season 2009-2010, 20102011 and 2012-2013 associated with domination of influenza A(H1N1)pdm09 virus characterized with the highest
morbidity, hospitalization and lethality index in the European part of Russia. At that influenza A(H3N2) circulated
simultaneously in Far East and Siberian regions having close relations with Japan and China, correspondingly.
Majority of influenza associated death cases in Russia was caused by virus subtype A(H1N1)pdm09. During 20112012 and 2013-2014 seasons influenza activity caused mainly by subtype A(H3N2) was moderate or low. Data of
sentinel surveillance indicated that SARI cases were registered mainly among not vaccinated patients. Main risk
factors for SARI was pregnancy, chronic cardiovascular and lung diseases and diabetes.
No pronounced changes were observed in antigenic properties both of circulated influenza A(H1N1)pdm09
viruses closely related to influenza A/California/07/09 virus and influenza A(H3N2) viruses antigenically related to
reference viruses A/Victoria/361/11 and A/Texas/50/12. Influenza A(H1N1)pdm09 viruses genetically belonged to
phylogenetic group 6 (A/St.Petersburg/27/2011-like strains) and A(H3N2) viruses belonged to A/Victoria/361/2011like clade (group 3C.3). Decrease of intensity of influenza A(H1N1)pdm09 activity from year to year was associated
apparently with increase of population immunity level to this virus registered during last years. Influenza B activity
during analyzed period was less significant. Both viruses of Victorian (B/Brisbane/60/08-like) and Yamagata (B/
Massachusetts/02/12 or B/Wisconsin/01/2010-like) lineage were isolated in different proportions during these five
seasons.
Conclusions
Enhanced integration of laboratory and epidemiologic surveillance for influenza make it possible to recognize
increase of influenza activity and epidemic start, determine etiology and anticipated severity of upcoming epidemic.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P36
POSTE R A BSTRAC TS
SPA2P13
Influenza surveillance systems in Europe – a recent survey update
Meerhoff, Tamara (1); Vermaire, Jorien (2); Jorgensen, Pernille (2); Gross, Diane (2); Pereyaslov, Dmitriy (2); Brown, Caroline (2)
1: Radboud university medical center, Department of Primary and Community Care, Nijmegen, the Netherlands; 2: WHO Regional Office for
Europe, Denmark
Background
Influenza surveillance plays an important role in determining appropriate control and intervention measures
and informing case management recommendations by providing timely data on virological and epidemiological
indicators for influenza. In Europe, a regional influenza surveillance network is coordinated by the World Health
Organization (WHO) Regional Office for Europe and the European Center for Disease Prevention and Control (ECDC).
Combined clinical and virological data are collected by primary care facilities from patients with influenza-like illness
(ILI) and/or acute respiratory infections (ARI). Since the emergence of influenza A(H1N1)pdm09 in 2009, countries
have established hospital-based surveillance for influenza. Little is known about the current case definitions and
surveillance methods used, this information is essential to interpret and compare data across Europe and to further
improve surveillance. In 2001, a survey on the surveillance systems in 21 countries in Europe was carried out. At
present, influenza surveillance data are available for 50 Member states. The purpose of this study was to provide an
updated review on current surveillance systems in the WHO European Region.
Methods
WHO Regional Office for Europe and ECDC conducted an email survey in March 2014. The survey included questions
on the presence of primary care, hospital and other types of surveillance, case definitions, laboratory testing and
epidemiological data collected in each of these systems.
Results
47 out of 50 Member States completed the survey. All countries perform primary care surveillance and 31 countries
perform hospital-based surveillance. Practically all countries report data from primary care sources while for severe
disease surveillance only 48% of the countries report data to the European network. Sentinel systems reporting ILI/
ARI are primarily used and sentinel doctors generally cover 1-15% of the population. The majority of countries use
the WHO or the European Union (EU) case definition for ILI (73%) or ARI (64%). For severe acute respiratory infection
surveillance 51% use the WHO case definition. Generally a standard protocol is used when taking respiratory
specimens and patient details are collected routinely. However, data on onset of illness, underlying conditions and/
or outcome of the disease are collected by less than 60% of the countries.
Conclusions
This survey provides insight in current influenza surveillance systems in the WHO European Region and has identified
strengths and weaknesses in the data collection. Practically all countries that collect primary care surveillance data
report to the European network, while half of countries that have hospital-based surveillance are reporting these
data. The data collection on onset of illness, underling diseases and outcome can be improved. The results will be
used to further strengthen the quality of data collected, to help interpret the surveillance data and will lead to
appropriate control and intervention measures that need to be taken in an epidemic.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P37
POSTE R A BSTRAC TS
SPA2P14
Transmission of the human influenza virus A/NL/602/09, but not the
triple reassortant A/swine/Kansas/77778/07, occurs efficiently from
low infectious dose in guinea pigs
Campbell, Patricia J. (1); Danzy, Shamika (1); Ramos, Irene (2); Fernandez-Sesma, Ana (2); Lowen, Anice C. (1); Steel, John (1)
1: Emory University, United States of America; 2: Mount Sinai School of Medicine, United States of America
Background
Swine influenza viruses of the triple reassortant (TRIG) lineage do not transmit efficiently among humans.
Nonetheless, this virus lineage donated 6 of the 8 gene segments present in the highly transmissible 2009 pandemic
influenza virus. Neither the genetic determinants nor the mechanistic bases responsible for the difference in
transmissibility are fully delineated. We have previously shown that the guinea pig model broadly recapitulates the
differences in transmissibility between swine and human strains of influenza virus, using inoculation doses of ~10
000 PFU per animal. In the present study, we reasoned that an influenza virus with optimal fitness at a population
level should exhibit both a low median infectious dose and the ability to transmit rapidly to a new host following
infection at the minimal infectious dose. Toward understanding their differing potentials for spread in the human
population, we applied this concept to influenza A/swine/Kansas/77778/07 (H1N1) [KAN07] and A/NL/602/09
(H1N1) [NL602] viruses.
Methods
Utilizing plasmid-based reverse genetics, we generated recombinant NL602 and KAN07 viruses. Specific reassortant
viruses containing segments from two backgrounds were also generated. Growth phenotypes were assayed in vitro
in MDCK and HTBE cells, and infectivity, replication and transmission were assessed using a guinea pig contact
transmission model. Receptor binding preferences of each virus were assessed using a solid phase binding assay as
well as a flow cytometry based assay utilizing labelled sialic acid analogues.
Results
We show that NL602 virus possesses a lower 50% infectious dose than KAN07 in the guinea pig model (GPID50
= 3.16 PFU for NL602 versus 213.8 PFU for KAN07). Moreover, when donor animals were inoculated with a
minimal infectious dose, NL602 virus transmitted from each infected animal to a naïve contact, whereas KAN07
virus transmitted only from animals receiving higher initial doses. In groups of guinea pigs inoculated with
approximately 100, 10 or 1 GPID50 of NL602 virus, 100% transmission from infected animals to cagemates was
observed. In animals inoculated with ≤100 GPID50 of KAN07 virus, no greater than 50% transmission efficiency was
seen. Using recombinant viruses possessing reassortant genotypes of NL602 and KAN07, we mapped the observed
phenotypes to the HA and NA segments of NL602 virus. Preliminary receptor binding studies indicate that NL602
virus possesses alpha 2,6- linked sialic acid receptor specificity only, whereas KAN07 possesses both alpha 2,3- and
alpha 2,6-linked sialic acid binding. Studies are underway to test the importance of this difference to infectious dose
and transmissibility.
Conclusions
Unlike KAN07, the human influenza virus strain NL602 infects guinea pigs from low dose inoculation, and transmits
onwards efficiently to contact animals. We speculate that low infectious dose and the ability to transmit rapidly
from low dose are key characteristics distinguishing human viruses from viruses capable of zoonotic infection of
humans.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P38
POSTE R A BSTRAC TS
SPA2P15
Influenza surveillance in the Netherlands: a new system covering 5% of
the population that uses Electronic Medical Records
Hooiveld, Mariëtte (1); Donker, Gé (1); Meijer, Adam (2); Zock, Jan-Paul (1); Schellevis, François (1)
1: NIVEL, Netherlands institute for health services research, Utrecht, the Netherlands; 2: National Institute for Public Health and the
Environment (RIVM), Bilthoven, the Netherlands
Background
The surveillance of influenza is important to monitor the spread and impact of the disease and to detect the
emergence of new viruses. Traditional surveillance systems have been based on reporting by sentinel General
Practitioners (GPs) and laboratory testing. With electronic medical records, automatic data collection of routinely
recorded information from health care providers is possible. In the Netherlands, a new syndromic surveillance
system has recently been established to complement the existing sentinel influenza-like illness (ILI) surveillance
system.
Methods
Daily information on morbidity is automatically collected once a week from 250 general practices, participating in
NIVEL Primary Care Database. Every Dutch citizen is obligatory enlisted in a general practice and the GP acts as a
gatekeeper for specialized, secondary health care. The electronic medical records kept by the GP, therefore, provide
the most complete picture of the population’s health. In addition, some 40 practices within NIVEL Primary Care
Database report patients with clinically diagnosed ILI (Sentinel General Practices, since 1970) and take nose- and
throat swabs for virological analyses (since 1992).
Results
In total, the syndromic surveillance system covers a population of 1 million people, about 5% of the Dutch
population. The larger population allows for stratification by smaller groups or for instance in areas with a high
density of poultry. Weekly numbers on patients consulting for an Acute Respiratory Infection (ARI, including ILI) or
pneumonia are published on the NIVEL website. Although ARI is less specific for an influenza virus infection than
ILI, seasonal data are highly correlated. The weekly number of patients consulting the GP for ARI or pneumonia were
lower during season 2013/2014 compared to previous seasons. A mild ILI-epidemic was observed during weeks 2 to
16, with ILI incidences around the baseline threshold and influenza virus detection in 0 to 50% of the ILI specimens
collected per week. Rhinovirus and respiratory syncytial virus explained a relatively large part of the ILI incidence.
Conclusions
The new syndromic surveillance system complements the traditional sentinel surveillance of influenza-like illness in
the Netherlands. Integration of the two systems provides more detailed information on patients with ARI and likely
influenza, and the power of a large-scale network. There are plans to make the ARI data from this new surveillance
system available to ECDC/WHO Euro and the global surveillance system WHO FluID.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P39
POSTE R A BSTRAC TS
SPB2: VIRAL FACTORS IN PATHOGENESIS
SPB2P01
Relative role of individual complement pathway during influenza
infection
Rattan, Ajitanuj
National Center For Cell Sciences (NCCS), India
SPB2P02
Flt3 signaling is critical for host response after influenza A infection
Kühn, Nora (1); Hatesuer, Bastian (1); Dengler, Leonie (1); Wilk, Esther (1); Schäkel, Friederike (1); Pils, Marina (2); Beutler, Bruce (3); Schughart,
Klaus (1)
1: Department of Infection Genetics, Helmholtz Centre for Infection Research, University of Veterinary Medicine Hannover, and University
of Tennessee Health Science Center, 38124 Braunschweig, Germany; 2: Mousepathology, Helmholtz Centre for Infection Research, 38124
Braunschweig, Germany; 3: Center for Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
FMS-like tyrosine kinase 3 (Flt3) is a hematopoietic receptor tyrosine kinase important for the development of many
immune cells including dendritic cells (DC) and natural killer (NK) cells. Binding of its ligand Flt3l results in stimulation
of the growth of progenitor cells in the bone marrow and blood. The Flt3wmfl/wmfl (warmflash – wmfl) strain
was identified in an ENU (N-ethyl-N- nitrosourea) mutagenesis screen for susceptibility to mouse cytomegalovirus
(MCMV). The induced point mutation (G to A) causes a shift of the splice donor site and an in-frame deletion of two
amino acids in Flt3. Here, we studied the host response to influenza A infections in Flt3wmfl/wmfl mice.
Flt3wmfl/wmfl mice are smaller than wild type of same age and sex. Furthermore they exhibit an abnormal loss
of hair and show increased urination. Flow cytometry analysis of lung cell populations indicates a decreased
CD11chigh MHCIIhigh subpopulation representing alveolar DCs in uninfected Flt3wmfl/wmfl lungs. Additionally a
reduced NKp46+ cell population (NK cells) was found compared to wild type mice. Also, mutant mice exhibit lower
numbers of CD11c+ MHCII+ cells in the draining lymph nodes (dLN).
Flt3wmfl/wmfl mutants showed an increased susceptibility to influenza A virus infection compared to wild type
mice. After infection with a low virulent H1N1 PR8M virus, Flt3wmfl/wmfl mice lost weight rapidly and died 6 to 8
days post infection. In contrast, the wild type controls regained body weight after day 7 and survived. At days 1 to 5
after infection, Flt3wmfl/wmfl mice exhibit a similar viral load in the lungs as wild type mice. Flt3wmfl/wmfl mice
respond to influenza A infection with a higher granulocyte count in peripheral blood compared to wild type. The
immune cell infiltration in the lung is currently under investigation.
In summary, the absence of Flt3 signaling in mice results in high susceptibility to influenza A infections. We
hypothesize that a defect in the link between innate and adaptive immune response due to reduced numbers of
DCs and NK cells in Flt3wmfl/wmfl mice might be responsible for this increased susceptibility to influenza virus
infection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P40
POSTE R A BSTRAC TS
SPB2P03
H7N9 and H6N1 influenza A virus hemagglutinins engineered to bind
human type receptors reveal a novel layer of specificity beyond the α2-6
linkage of sialic acid
de Vries, Robert P; Peng, Wenjie; McBride, Ryan; Paulson, James C
The Scripps Research Institute, United States of America
Influenza A virus (IAV) is a serious pathogen in livestock and humans. The virus resides in the wild waterfowl
population but is able to cross the species barrier to various mammals. One part of this barrier is the recognition
of sialic acid receptors. Avian viruses prefer binding to sialic acid with an α2-3 linkage to the penultimate galactose
(avian type), whereas human viruses bind to sialic acid with an α2-6 linkage (human type). All previous human
pandemics were of avian or swine origin and were accompanied by amino acid mutations in HA that shifted binding
from avian to human type. With the continuous threat of avian viruses, H5N1, H6N1, H7N9 and H10N8, infecting
humans that if adapted to bind human type receptors, could spark another pandemic, it is vital to know which amino
acid changes are required in these subtypes for this specificity shift. Several assays have been developed to assess
receptor-binding specificity of IAVs, all have some drawbacks such as being only qualitative, using only terminal
parts of more complex glycans, but most importantly lacking the representation of biological relevant glycans found
in the human lung. Recently it has been reported that the glycome of human respiratory tissues contain highly
complex N-linked glycans containing multiple Galβ1-4GlcNAc (LacNAc) repeats on their antennae. To represent
these glycans on our glycan-arrays we created a series of bi- and tri-antennary N- linked glycans with 2 to 5 LacNAc
repeats by chemo-enzymatic synthesis. These structures were sialylated with either α2-3 or α2-6 linked sialic acid
and printed on a glycan-array and then probed with human and avian recombinant hemagglutinins (HA). Although
the paradigm of avian viruses binding to α2-3 and human viruses binding to α2-6 linked sialic acids was maintained.
Exemplified by human H7N9 and H6N1 viruses, not yet adapted to human type receptors, that bound to avian type
receptors. However when selective mutations were introduced in the conserved binding pocket, we were able to
create H7 and H6 HAs that specifically bound to human type receptors. Inspection of the glycans bound revealed
another layer of specificity towards complex N-linked glycans bearing 3 to 5 LacNAc repeats. These glycans were very
similar to those bound by the human 2009 pandemic Cal/04/09 HA. Cal/04/09 did not bound to any glycan arrays
before, as previous arrays did not contain these highly complex structures, but perhaps more importantly, Cal/04/09
transmits highly efficient by respiratory droplets between ferrets and humans. We hypothesize that the arms of
these N-linked glycan can bind to two monomers within the HA trimer, furthermore we are currently exploring the
biological consequences of binding these highly complex structures by engineered H7N9 and H6N1 viruses.
(Supported by NIH Grant AI099275 and a Rubicon Grant of the Netherlands organization for scientific research (to
RPdV).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P41
POSTE R A BSTRAC TS
SPB2P04
Activation of coagulation and tissue fibrin deposition in experimental
influenza in ferrets
Goeijenbier, Marco (1); van Gorp, Eric C.M. (1); van den Brand, Judith (1); Stittelaar, Koert (2); Bakthiari, Kamran (2); Roelofs, Joris T.H. (2); van
Amerongen, Geert (1); Kuiken, Thijs (1); Martina, Byron (1); Meijers, Joost C.M. (3); Osterhaus, Albert D.M.E. (1)
1: ErasmusMC, Netherlands, The; 2: Viroclinics, Netherlands, The; 3: Academic Medical Center, Netherlands, The
Background
Epidemiological studies relate influenza infection with vascular diseases including myocardial infarction. In several
animal model studies the procoagulant effects of influenza virus infection have been investigated. Since these
studies made use of animals only susceptible to laboratory animal adapted influenza viruses, their results are hard
to translate to human influenza. Therefore we decided to study the influence of different human influenza virus
infections on coagulation in the ferret influenza model.
Methods
Ferrets were infected with either a seasonal-, pandemic- or highly pathogenic avian influenza (HPAI-H5N1) virus
strains, or mock,infected. In a 14 day interval (only 4 days for HPAI-H5N1) with in total 7 time points, 4 animals were
euthanized per timepoint and citrated plasma was tested for prothrombin time (PT), activated partial thromboplastin
time (aPTT), Von Willebrand factor activity (VWF), thrombin-antithrombin complex levels and D-dimer. Lung tissue
was used for fibrin staining.
Results
All influenza virus infected animals showed alterations in hemostasis. Specifically on day 4 post infection, a four
second rise in both PT and aPTT was observed. D-dimer concentrations increased in all 3 influenza groups with
the highest concentrations in the pandemic influenza group. Von Willebrand factor activity levels increased early
in infection suggesting endothelial cell activation. Mean thrombin-antithrombin complex levels increased in both
pandemic and HPAI-H5N1 virus infected ferrets. At tissue level, fibrin staining showed intracapillary fibrin deposition
especially in HPAI-H5N1 virus infected ferrets.
Discussion
This study showed hemostatic alterations both at the circulatory and at the tissue level upon infection with
different influenza viruses in an animal model closely mimicking human influenza virus infection. Alterations largely
correlated with the severity of the respective influenza virus infections.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P42
POSTE R A BSTRAC TS
SPB2P05
Analysis of the host response to influenza A virus infection in the
Collaborative Cross founder strains
Leist, Sarah R. (1); Pilzner, Carolin (1); Kollmus, Heike (1); Schughart, Klaus (1,2,3)
1: Helmholtz Centre for Infection Research, Germany; 2: University of Veterinary Medicine Hannover, Germany; 3: University of Tennessee
Health Science Center, Memphis, USA
Background
Influenza A virus poses a major health threat and caused multiple severe pandemics in the last century with
millions of death. The course and outcome of an influenza A infection is influenced by viral as well as host factors.
We use mouse genetic reference populations (GRPs) to identify genetic host factors that contribute to resistance or
susceptibility to influenza A infections. The recently established Collaborative Cross (CC) is a GRP derived from eight
genetically different founder strains. Amongst them are classical lab strains (A/J, C57BL/6J, 129S1/SvlmJ) as well as
mouse models for human diseases like diabetes (NOD/ShiLtJ) and obesity (NZO/HILtJ). The addition of three wildderived strains (PWK/PhJ, CAST/EiJ and WSB/EiJ) strongly enhances the genetic diversity. These founder strains were
bred in a specific breeding funnel to generate 500 – 700 recombinant inbred CC lines. Each of the CC lines represents
a unique mosaic of the genetic information of the eight founder strains.
Methods
Here, we present our analysis of the eight CC founder strains and four CC lines (OR13140, OR13067, IL16188,
IL16211) after infection with the mouse-adapted influenza virus strain A/HK/01/68 (H3N2). Female as well as male
mice were infected intra-nasally with up to three different concentrations of H3N2. Body weight and survival was
monitored for 14 days after infection. In addition, we characterized the host response in more detail by analyzing
the hemogram of peripheral blood and determining viral loads in infected lungs.
Results
We found large differences in the host response between the different CC founder and CC lines. We observed
‘expected’ phenotypes e.g. high viral loads accompanied by a strong increase in immune cell counts in the blood in
case of lethal outcomes of infection, but also unexpected phenotype combinations were seen. In general, the lethal
or non-lethal outcome of the infection was mainly influenced by the different Mx1 alleles segregating in the CC
founder strains. However, we could also show that the function of Mx1 is modulated by the genetic background in
one of the four CC lines.
Conclusions
The genetic diversity present in the CC founder strains is reflected by the different phenotypic outcomes after
influenza A infection. Moreover, CC lines exhibit additional phenotypic combinations which are not observed in the
eight founder strains. Thus, CC lines represent a valuable resource for genetic mapping studies to unravel novel host
factors that influence susceptibility and resistance to influenza A infections.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P43
POSTE R A BSTRAC TS
SPB2P06
Type I interferon-mediated signaling is inhibited upon influenza A virus
and Staphylococcus aureus co-infection
Warnking, Kathrin; Klemm, Carolin; Löffler, Bettina; Niemann, Silke; Peters, Georg; Ludwig, Stephan; Ehrhardt, Christina
Westfälische Wilhelms-University Münster, Germany
Background
Influenza A viruses (IAV) are the causative agents of severe respiratory diseases. The majority of disease fatalities
are linked to secondary bacterial pneumonia, caused by pathogens such as Staphylococcus aureus (S. aureus). One
major problem of the increased pathogenicity is the dysregulation of the cellular immune response. While this is a
well-known complication, there is only scarce knowledge about the interplay of IAV with S. aureus during infection
on a molecular level. Thus we assessed the regulation of type I interferon (IFN) response in an IAV/S. aureus coinfection model in vitro.
Methods
To investigate cellular signal transduction processes in the human lung epithelial cell- line (A549) upon IAV/S. aureus
co-infection we established a complex infection procedure. For co-infection various IAV subtypes, the S. aureus strain
6850 and heat killed S. aureus (HKSA) were employed. In control experiments viral RNA, different bacterial factors
and IFN beta were used as stimuli. Viral titers were analysed by standard plaque assays. Regulation of pathogeninduced type I IFN-mediated signaling was investigated on mRNA and protein levels via qRT-PCR, Western Blot
analysis and co-immunoprecipitation assays.
Result
By using an in vitro co-infection model of A549 cells, we were able to show that IAV replicate more efficiently in the
presence of S. aureus. Investigation of pathogen- induced signaling mechanisms during co-infection surprisingly
revealed a stronger induction of the innate immune response, most importantly increased levels of type I IFN mRNA.
In disagreement with that, mRNA levels of strictly IFN-stimulated genes, such as MxA or OAS, were rather decreased,
correlating with reduced IFN-induced protein expression. Based on these results, we hypothesize that there seems
to be a block of type I IFN signaling provoked by the bacteria. In fact, we were able to show that independent of the
initial stimulus that drives IFN beta up-regulation, metabolically active intracellular S. aureus inhibits type I IFNmediated STAT1 phosphorylation and subsequently STAT1-STAT2 dimerization.
Conclusions
In the presence of S. aureus the first line of defence against influenza viruses is interrupted, resulting in a boost of
viral replication, which may lead to enhanced pathogenicity.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P44
POSTE R A BSTRAC TS
SPB2P07
Lipoteichoic acid of Staphylococcus aureus as main contributor to the
enhancement of influenza A virus induced mitogen-activated protein
kinase signaling
Klemm, Carolin (1); Warnking, Kathrin (1); Löffler, Bettina (2); Peters, Georg (2); Ludwig, Stephan (1); Ehrhardt, Christina (1)
1: Westfaelische Wilhelms-University Muenster, Germany; 2: University Hospital of Muenster, Germany
Background
Bacterial co-infections are a major complication of influenza A virus (IAV) infections leading to severe illness and
fatal outcomes. Recent findings suggest that beside the pathogen load, a dysregulated immune response of the
host also contributes to increased morbidity and mortality. Toll-like receptors (TLRs) play an important role in the
innate immune response by pathogen sensing and activating signaling cascades leading to the induction of proinflammatory cytokines and chemokines. Although several in vivo studies demonstrate elevated levels of cytokines
and chemokines upon IAV and bacterial co-infections resulting in a massive influx of immune cells into the lung and
severe tissue damage, the underlying molecular signaling mechanisms still remain to be elucidated. This knowledge
is crucial for development of new therapeutic approaches.
Methods
In this study, we investigated the signaling events upon co-infection of IAV and Staphylococcus aureus (S. aureus)
in a human epithelial cell line (A549) in vitro. We established a co-infection protocol including serial pathogen
incubation combined with an antibiotic wash. Furthermore we used viral and bacterial components to mimic
infection by applying viral RNA or several bacterial factors. Manipulations of different signaling pathways were
achieved by siRNA approaches. Phosphorylation and activation of mitogen-activated protein kinases (MAPKs) were
investigated by immunoblotting and quantitative Real-Time-PCR was performed to analyse mRNA levels of several
cytokines and chemokines.
Results
Upon co-infection with IAV and S. aureus we observed elevated levels of cytokines and chemokines as described in
in vivo models. Analyses of cellular signaling mechanisms regulating these immune responses revealed significantly
increased activation of the MAPKs JNK and p38 in presence of both pathogens compared to IAV-infected cells.
Similar results were obtained, when IAV infection was replaced with viral RNA or S. aureus infection was restored by
lipoteichoic acid (LTA) stimulation, but not with other bacterial components.
Conclusions
Our data indicate a correlation of hyper-activation of MAPKs and hyper-transcription of pro-inflammatory cytokines
and chemokines to the activation of TLRs. We will provide deeper insights in the regulation of pathogenicity during
IAV and S. aureus co-infections on a molecular level, which contributes to the lethal synergism of these pathogens.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P45
POSTE R A BSTRAC TS
SPB2P08
Pandemic swine-origin H1N1 influenza virus replicates to higher
levels and induces more fever and acute inflammatory cytokines in
Cynomolgus versus Rhesus monkeys and can replicate in Common
Marmosets
Mooij, Petra; Koopman, Gerrit; Mortier, Daniella; van Heteren, Melanie; Fagrouch, Zahra; de Laat, Rudy; Remarque, Edmond J.; Kondova,
Ivanela; Verschoor, Ernst J.; Bogers, Willy M.J.M.
Biomedical Primate Research Centre, Netherlands, The
Background
Because of the close immunologic and physiologic resemblance with humans, non-human primates have provided
valuable models in studying influenza virus pathogenesis, immunity and vaccine efficacy against infection. Although
both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of
susceptibility to infection and disease had not yet been performed.
Methods and Results
In the current study a recently described swine-origin pandemic H1N1 strain, derived from a single stock and given at
the same dose and route of infection, showed in cynomolgus macaques significantly higher levels of virus replication,
both in peak level and duration of virus production in the upper airways and in the lungs, as well as higher increases
in body temperature relative to rhesus macaques. In contrast, clinical symptoms including respiratory distress were
easier observable in rhesus macaques. The study also shows that common marmosets, a new world non-human
primate species, are susceptible to infection with pandemic H1N1.
Conclusions
The study results favour the cynomolgus macaque as model for influenza virus research because of the more
uniform and high levels of virus replication as well as temperature increases.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P46
POSTE R A BSTRAC TS
SPB2P09
Insights into the interaction of influenza A virus RNA polymerase with
cellular RNA polymerase II
Martínez-Alonso, Mónica; Hengrung, Narin; Vreede, Frank; Fodor, Ervin
Sir William Dunn School of Pathology, University of Oxford, United Kingdom
Background
The influenza virus RNA polymerase is known to interact with the carboxy-terminal domain (CTD) of cellular RNA
polymerase II (Pol II) but the molecular details of this interaction and its biological role remain uncharacterised.
Whether influenza virus polymerase binds directly to the CTD of Pol II or if this interaction is mediated by host
factors remains controversial. In addition, it is unclear if the binding occurs between Pol II and free polymerase or
polymerase that is part of vRNPs. This study addresses these questions under the hypothesis that direct binding to
Pol II would facilitate cap-snatching for viral transcription.
Methods
HEK 293T cells were infected with influenza A/WSN/33 virus and subjected to RNA immunoprecipitation (RIP) to
analyse the presence of viral RNAs in Pol II complexes. Infected cell lysates were also subjected to peptide pulldowns
using synthetic peptides consisting of four repeats of the consensus sequence of Pol II CTD (YSPTSPS) with
modifications to represent different phosphorylation states. The set of peptides was also used to analyse in vitro
binding of purified viral polymerase. For these experiments, the viral polymerase was either purified from HEK 293T
cells coexpressing a short vRNA template, or expressed in insect cells and bound to synthetic RNA oligonucleotides
mimicking the vRNA promoter. The presence of RNA in the peptide-bound complexes was analysed by radiolabelling
and the activity of the peptide-bound polymerase was assessed by an in vitro transcription assay.
Results
Viral RNAs coimmunoprecipitated with Pol II complexes, suggesting that the viral polymerase that interacts with
Pol II is assembled into vRNPs. In addition, vRNPs were also pulled down from infected cell lysates with a Pol II
CTD mimic peptide phosphorylated on serine 5. This phosphorylation state mimics the form of Pol II engaged in
transcription initiation. Recombinant viral polymerase also bound specifically to the serine 5 phosphorylated CTD
mimic peptide, and RNA was also present in the bound complexes. Peptide-bound viral polymerase was shown to
be able to perform transcription in vitro.
Conclusions
Our data suggest a model in which vRNPs bind directly via the viral polymerase to Pol II in a transcriptional context,
a scenario that could facilitate cap-snatching for viral transcription.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P47
POSTE R A BSTRAC TS
SPB2P10
High basal expression of interferon-stimulated genes in transformed
human bronchial epithelial (BEAS-2B) cells contributes to influenza A
virus resistance
Seng, Lai-Giea; Daly, Janet; Chang, Kin-Chow; Kuchipudi, Suresh
University of Nottingham, United Kingdom
Background
Influenza A viruses (IAV) continue to threaten human and animal health globally. Respiratory epithelial cells play
a key role in IAV pathogenesis and host innate response. Transformed human respiratory cell lines are widely used
in the study of virus-host interactions due to their relative convenience, and the inherent technical limitations of
working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper
assessments of different respiratory cell types in their responses to IAV infection are therefore needed to ensure that
the cell line chosen will provide results that are of relevance in vivo.
Methods
Replication kinetics of avian H2N3 (A/mallard duck/England/7277/06) and human H1N1 (A/USSR/77) IAVs were
compared between normal primary human bronchial epithelial (NHBE) cells and two commonly used transformed
human respiratory epithelial (BEAS-2B and A549) cells. Influenza virus receptor distribution was analysed using sialic
acid linkage- specific lectins. IAV replication was assessed by Western blotting of viral matrix and PB1 proteins, and
by infectious progeny virus production assays. Host antiviral gene expression was quantified by reverse transcription
real time-PCR (qRT-PCR).
Results
IAV replication was distinctly poor in BEAS-2B cells in comparison with Madin-Darby canine kidney (MDCK),
NHBE and A549 cells. BEAS-2B cells co-expressed both human and avian influenza virus receptors indicating that
resistance to IAV replication was not due to the absence of influenza receptors. IAV resistance in BEAS-2B cells was
accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7),
stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Pre-treatment of BEAS-2B cells with a pan-Janusactivated-kinase (JAK) inhibitor (pyridine 6) led to increased IAV replication and marked reduction of ISGs.
Conclusions
BEAS-2B cells are not typical human respiratory epithelial cells in that this cell line is highly resistant to influenza A
virus infection, in part due to high constitutive expression of anti-viral ISGs.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P48
POSTE R A BSTRAC TS
SPB2P11
The olfactory nerve: A shortcut for influenza viruses into the CNS in
humans !?
van Riel, Debby (1); Leijten, Lonneke M. (1); Verdijk, Rob M. (2); GeurtsvanKessel, Corine (1); van der Vries, Erhard (1); van Rossum, Annemarie
M.C. (3); Osterhaus, Albert. D.M.E. (1); Kuiken, Thijs (1)
1: Erasmus MC, Department of Viroscience, The Netherlands; 2: Erasmus MC, Department Pathology, The Netherlands; 3: Erasmus MCSophia,Department of Paediatric Infectious Diseases and Immunology, The Netherlands
Background
Influenza virus associated CNS disease is the most common extra-respiratory tract complication of influenza in
humans. The pathogenesis of influenza virus-associated CNS disease, including the route of entry is largely
unknown. Recently we have shown that in ferrets highly pathogenic H5N1 virus is able to enter the CNS via the
olfactory nerve, causing a severe meningo- encephalitis. The olfactory nerve directly connects the nasal cavity with
the CNS and could therefore function as a shortcut into the CNS. Here, we report on a human case in which we
found strong evidence for CNS invasion via the olfactory nerve.
Methods
From a severely immunocompromised child, who died 3 days after being tested positive for influenza, samples were
collected during an extensive autopsy. These samples as well as previous samples collected for diagnostic purposes
were tested for the presence of influenza virus RNA and antigen. Subsequently, we determined for human (H3N2 &
H1N1) and avian influenza viruses (H5N1) (1) the attachment pattern to normal human and ferret olfactory mucosa
and (2) the infection efficiency in differentiated human neuronal cells.
Results
Influenza virus antigen could only be detected in neurons and glial cells of the olfactory bulb, olfactory tract, and
gyrus rectus, which is adjacent to the olfactory bulb. Virus RNA isolated from the olfactory bulb and broncho-alveolar
lavage fluid revealed the presence of an H3N2 virus. Virus antigen or RNA could not be detected in any other organs
or a serum sample. Virus attachment studies showed that both human and avian viruses attached to the apical
side of the human olfactory mucosa. Infection studies revealed that human differentiated neuronal cells could be
infected with both human and avian viruses.
Conclusions
The detection of virus antigen and RNA in the olfactory bulb, together with the absence of virus antigen or RNA in
any other organ or serum, suggests that virus entered the CNS via the olfactory nerve in this case. This finding is
supported by the ability of H3N2 viruses to attach to olfactory mucosa and to infect human neuronal cells. Virus
entry via the olfactory nerve might be an important route of entry into the CNS, even during mild upper respiratory
tract disease, which might lead to subclinical to severe disease. To obtain more insight into the frequency and
clinical impact of CNS invasion via the olfactory nerve, we emphasize that in any autopsy case involving influenza,
the olfactory tract should be examined for evidence of local virus replication.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P49
POSTE R A BSTRAC TS
SPB2P12
Experimental co-infection of chickens with lentogenic, mesogenic and
velogenic strains of Newcastle disease viruses and highly pathogenic
avian influenza viruses
Pantin-Jackwood, Mary Josephine; Costa-Hurtado, Mar; Afonso, Claudio; Miller, Patti; Shepherd, Eric; Smith, Diane
Southeast Poultry Research Laboratory. USDA, United States of America
Background
Avian influenza virus (AIV) and Newcastle disease virus (NDV) are two of the most economically important viruses
affecting poultry worldwide. Co-infections of poultry with AIV and NDV are a problem from the clinical point of view
and diagnosis of these viruses, but little is known on the interactions between these two viruses when infecting
birds. AIV and NDV can produce from mild to moderate upper respiratory diseases in their low pathogenic forms
[lentogenic or mesogenic NDV and low pathogenicity (LP) AIV], to severe systemic diseases with high mortality in
their more virulent forms [velogenic NDV and highly pathogenic (HP) AIV]. Exposure to NDV, either live vaccines or
field strains, is nearly unavoidable for commercial and non-commercial poultry worldwide and co-infections with
AIV are known to occur. The objective of this study was to determine if co-infection of chickens with different strains
of NDV could affect the outcome of infection with HPAI viruses.
Methods
We conducted three experiments in which we infected chickens with lentogenic, mesogenic or velogenic strains of
NDV, and high pathogenicity (HP) AIV’s, by giving the viruses simultaneously or sequentially. Pathogenesis (clinical
signs, lesions), presence of the viruses in tissues, duration and titer of virus shedding, and seroconversion to both
viruses were evaluated.
Results
We found that previous infection of chickens with mesogenic and velogenic strains of NDV (Pigeon 84 and CA2002
strains), but not a lentogenic NDV strain (LaSota) interfered with replication of a HPAIV (A/Ck/Queretaro/14588-19/95
H5N2) when given at a high titer dose [106.3 50% egg infectious dose (EID50)] 2 days after NDV inoculation;
however high mortality was still observed. Interestingly, chickens were refractory to infection, as measured by lack
of disease, virus shedding and seroconversion, when given lower titers (105.3 EID50) of the same HPAIV 3 days after
the mesogenic NDV (Pigeon 84), indicating that virus titer and timing of the second infection might play a role in
virus interference. This was corroborated by the results of a third study in which 10% mortality was observed among
chickens that received 105EID50 of a different HPAIV [A/chicken/Jalisco/CPA-12283-12/2012 (H7N3)] 3 days after
the same mesogenic NDV was given, when compared to 80% mortality observed in chickens challenged only with
the HPAIV.
Conclusions
Previous infection of chickens with NDV can affect HPAIV replication in tissues and consequently prevent disease
and mortality. This virus interference will depend on the virulence and titer of the co-infecting viruses, and the
timing of the infections. The information obtained from these studies helps understand the possible interactions
and outcomes of infection (disease and virus shedding) when AIV and NDV co-infect birds in the field.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P50
POSTE R A BSTRAC TS
SPB2P13
Ubiquitination of the influenza A virus PB1-F2 protein is crucial for its
biological functions
Kosik, Ivan (1); Praznovska, Margareta (1); Kosikova, Martina (1); Vareckova, Eva (1); Kostolansky, Frantisek (1); Polakova, Katarina (2); Russ, Gustav
(1)
1: Institute of virology Slovak Academy of Scince, Slovak Republic; 2: Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak
Republic
Background
PB1-F2 protein was shown to affect vRdRp activity, influences pathogenesis in mouse model and increase secondary
bacterial infection. It is not yet clear what the pathogenicity determinants in the PB1-F2 sequence are. Since PB1-F2
is rapidly degraded, we had supposed its biological function could be covered. Our goal was to identify determinants
of the PB1-F2 stability and consequences of higher expression level in vitro and in vivo.
Methods
It was shown, presence of the proteasome inhibitor increased expression level of the PB1-F2. We had concluded
PB1-F2 might be ubiquitinated, and we had identified C terminal lysine cluster (K73, 78, 85) as potential ubiquitination
site. Proximity ligation assay (PLA) was employed to identify ubiqitination status and site of the PB1-F2. Westernblot
and immunofluorescence were used to determine change in expression level of the PB1-F2. The firefly luciferase
minigenome assay was used to determine effect of the PB1-F2delUBQ (K73, 78, 85R) on vRdRp activity. Secreted
IFN-beta level was measured to characterize relation of the PB1-F2delUBQ on IFN-beta expression. Modulation of
the humoral immune response and protection induced by DNA vaccination with PB1-F2 wt or PB1-F2delUBQ was
also studied after sub lethal virus challenge. Plasmid based rescue system was used for determination of IAV PB1F2delUBQ viability and time laps microscopy was employed for observation of the cytopathic effect differences on
the MDCK cells.
Results
PLA assay clearly show that PB1-F2 is highly ubiqitinated and that change of the K73, 78, 85R suppresses it (Fig. 1).
As ubiquitination is required for proteasome targeting, its suppression led to increased expression of the PB1-F2. In
comparison to wt PB1-F2 enhanced expression of the PB1-F2delUBQ increased activity of the vRdRp about 40%. As
follows from body weight change and survival rate protection potential of the PB1-F2 is also enhanced in the case
of the PB1-F2delUBQ DNA vaccination. Time laps microscopy revealed accelerated cytopathic effect on MDCK cells
(Fig.2).
Conclusions
We had focused on posttranslational ubiquitination and its meaning for PB1-F2 functions. We had found out that
stability of the PB1-F2 is probably most important factor determining biological importance of this influenza A virus
protein.
Aknowledgement
This work was supported by grants VEGA 2/0085/10, 2/0176/12, 2/0100/13, 02/152/14, 02/153/14 and 2/0117/11
from the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and Slovak Academy of
Sciences. The topic was also supported by Grant no. APVV-0250-10, from the Slovak Research and Development
Agency. Appreciably topic was further supported by Eva Varečková (DO7RP-0025-10 from the Slovak Research and
Development Agency)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P51
POSTE R A BSTRAC TS
Figure. 1
Figure. 2
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P52
POSTE R A BSTRAC TS
SPB2P14
In vivo Virus Tropism and Protection by a Matrix Protein 2 EctodomainSpecific Monoclonal Antibody revealed by a Novel GFP-expressing
Influenza A Virus
Saelens, Xavier; De Baets, Sarah; Verhelst, Judith; Van den Hoecke, Silvie; Smet, Anouk; Roose, Kenny; Schotsaert, Michael; Schepens, Bert; Fiers,
Walter
Ghent University and VIB, Belgium
Background
Background: The severity of influenza-related illness is mediated by many factors, including the in vivo cell tropism,
the timing and magnitude of the immune response and the presence of pre-existing immunity. An easy way to
study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene such as GFP. However,
GFP-expressing influenza viruses are often attenuated and can be genetically unstable.
Methods
Methods: We generated PR8-NS1(1-73)GFP virus by reverse genetics. For this, we changed the wild type NSfragment into a tri-cistronic element consisting of the coding information for the first 73 amino acid residues of
NS1 fused to a heterologous dimerization domain. This truncated NS1 was followed by GFP and NEP. The virus was
rescued, serially passaged in vitro and analyzed by Illumina-based deep sequencing. Pathogenicity and tropism of
the PR8-NS1(1-73)GFP virus was analyzed in BALB/c mice that had been passively immunized with an M2e-specific
monoclonal antibody or treated daily with oseltamivir. Stromal as well as immune cells from the lungs of infected
mice were analyzed by flow cytometry.
Results
The recombinant GFP-expressing PR8-derived virus was successfully rescued. Serial in vitro passage and Illuminabased deep sequence analysis revealed that this virus was genetically stable. Compared to wild type PR8 the GFP
virus was approximately 10-fold attenuated in BALB/c mice. Based on the GFP expression levels, the main target
cells of the virus were found to be respiratory epithelial cells and myeloid cells, including dendritic cells (CD11c+)
and inflammatory monocytes (CD11b+ GR1+). Prophylactic treatment with anti- M2e monoclonal antibody or daily
administration of oseltamivir reduced GFP expression in all cell types studied. More than 95% of plaques derived
from lung homogenates prepared on day 5 after infection were GFP-positive, suggesting that the virus was also very
stable in vivo.
Conclusions
The in vivo cell tropism is an important virulence factors of influenza viruses. To study infection dynamics and cell
tropism, GFP-reporter viruses are very useful. However, most of these viruses are unstable and attenuated in vivo.
Here we describe a pathogenic PR8 virus, of which GFP expressing is stable in vitro and in vivo. Furthermore, we show
that this virus is very useful to determine the protective potential of antiviral treatments in vivo, like oseltamivir and
anti-M2e monoclonal antibody treatment.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P53
POSTE R A BSTRAC TS
SPB2P15
Genetic background influences the antiviral activity of Mx1 gene to
influenza virus infection in mice
Shin, Dai-Lun (1,2); Hatesuer, Bastian (1); Schughart, Klaus (1,2,3)
1: Helmholtz Centre for Infection Research, Germany; 2: University of Veterinary Medicine Hannover, Germany; 3: University of Tennessee,
Health Science Center, United-State
Background
In humans, there is circumstantial evidence that genetic factors may play an important role for susceptibility or
resistance to influenza A viruses (IAV) infection. In mice, It was shown that mice carrying a functional Myxovirus
resistance genes 1 (Mx1) allele are highly resistant against IAV. It acts as a restriction factor for virus importation
and block viral replication. Thus, the discovery of Mx1 as resistance factors dates back over 50 years, a detailed
phenotypic comparison between Mx1 mutant and wild type mice have not yet been performed. Furthermore, the
influence with genetic background and relationship with other genes were remaining unclear.
Methods
Our group demonstrated that genetic background could greatly influence the severity of disease after IAV infections
by comparing between the laboratory inbred strains DBA/2J (D2) and C57BL/6J (B6). In this study, we use B6.A2GMx1+/+ mice and A/Puerto Rico/8/34 H1N1 Freiburg variant virus (PR8F) as our standard setup, compared the
phenotype with B6 mice which contain nonfunctional Mx1 allele. Additionally, we changed the genetic background
for B6.A2G-Mx1+/+ mice from B6 into D2 background via backcross the mouse with D2 for 10 generations.
Phenotypes were measured by body weight loss, survival rate and virus titer from these mouse strains.
Results
D2.A2G-Mx1+/+ mice were highly susceptible to IAV infection. Mice with functional Mx1 allele in D2 background
reach 100% mortality after days 7 post infection. Performing the offspring F1 mice from D2.A2G-Mx1+/+ with
B6, which share half B6 and half D2 genetic background with one functional Mx1 allele, the mice could regain its
antiviral activity against IAV infection.
Conclusions
Our results suggest that B6 contain certain genes could initiate Mx1 antiviral function by restricting viral replication,
which were absence in D2 mouse strain.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P54
POSTE R A BSTRAC TS
SPB2P16
Genetic background influences the antiviral activity of Mx1 gene to
influenza virus infection in mice
Janulikova, Jana; Bobisova, Zuzana; Mucha, Vojtech; Kostolansky, Frantisek; Vareckova, Eva
Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic
Background
Several cases of human infection caused by avian influenza A viruses (IAV) were reported recently. Because they
were described as severe infections with high lethality, we studied the interaction of avian viruses with the immune
system of mammalian host. We focused on the specific antibody response induced after infection of mice with
avian or/and human influenza A viruses. The aim of this work was to simulate the situation, which could arise in
nature in humans, i.e. the infection with avian IAV after previous infection with human viruses and to analyze the
level of biologically active virus-specific antibodies induced after such subsequent mixed infection on mouse model.
Methods
Two avian IAV (A/Duck/56(H4N6), marked as “H4” and A/Vietnam/1203-04 (H5N1), marked as “H5”) and two human
IAV isolates (virus A/Miss/1/85(H3N2)-“H3” of medium virulence and A/PR8/34(H1N1) “H1” of high virulence) were
adapted to mice. The micro-neutralization and ELISA binding tests were used for analysis of virus-neutralizing (VN)
and for antigen-binding antibodies specific to HA2 gp. For the infection of mice various doses of human IAV were
used and specific antibody response of mice was compared with that obtained after a single or repeated infection
with two identical doses of avian IAV. In parallel, an antibody response to a mixed infection with one human and
subsequently with one avian influenza A virus was examined.
Results
We showed that two doses of identical IAV of H4 or H5 virus elicited in mice virus- specific neutralizing antibodies.
In mice previously infected with human IAV (of H3 or H1 subtype), infection with H4 subtype elicited antibodies
neutralizing H4 virus, but also titer increase of antibodies which neutralized the corresponding human virus used for
previous infection was recorded. After successive infection with H3 and H4 viruses a subpopulation of HA2-specific
antibodies cross-reactive with both subtypes was present. In mice infected with H5 virus after previous infection
with human IAV of H1 or H3 subtype, no measurable levels of antibodies neutralizing H5 virus were induced.
Notably, after infection with H5 virus, a significant titer increase of antibodies neutralizing IAV of H1or H3 subtype
(in dependence of the virus used for the first infective dose) was detected in sera of these mice.
Conclusions
Different antibody response to avian IAV after previous infection with human viruses can play an important role in
the epidemiology of newly emerged IAV of avian origin in human population.
Supported by grants: VEGA 2/0176/12, VEGA 2/0100/13, VEGA-2/0153/14, grants APVV- 0250-10 and
DO7RP-0025-2010 from the Slovak Research and Development Agency.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P55
POSTE R A BSTRAC TS
SPB2P17
Virus dose-dependent neutrophil and lymphocyte proportions in
peripheral blood during influenza A infection of mice
Kostolansky, Frantisek (1); Dugovicova, Veronika (1); Janulikova, Jana (1); Mucha, Vojtech (1); Mistrikova, Jela (2); Vareckova, Eva (1)
1: Institute of Virology, Bratislava, Slovak Republic; 2: Dept. Microbiology and Virology, Comenius University, Slovak Republic
Background
In this study, we focused on monitoring the changes in the proportion of cell types in the peripheral blood of mice
infected with mouse-adapted human influenza A viruses (IAVs). Our aim was to determine how the changes in the
differential leukocyte count correlate with the virulence and the dose of IAV used for the infection.
Methods
BALB/c mice were infected with sublethal dose (0.4 LD50 or 0.1 LD50) of moderately virulent IAV strain A/
Mississippi/1/85 (H3N2) – “Miss”, or of highly virulent A/PR/8/34 (H1N1) – “PR8”. Control (non-infected) mice were
given PBS. Differential leukocyte counts were done by microscopic examination of blood smears stained by the
May- Grunwald solution, followed by the Giemsa-Romanowski solution. Differential white blood cell counts were
calculated as a percentage of each kind of white blood cells.
Results
The infection with 0.4 LD50 of the virus Miss led to a 3-fold increase in the neutrophil count on day 2 p.i. with
subsequent decrease and returned to the normal level on day 10 p.i. In contrast, the proportion of lymphocytes
dropped by 18% on day 2 p.i., then gradually rose nearing the normal level. Other leukocyte subpopulations did not
show any marked changes during the IAV infection. The infection with a lower dose (0.1 LD50) of the Miss virus
revealed similar changes in monitored blood cell parameters: increase of neutrophil count by 8% and decrease of
lymphocyte count by 8% on day 2 p.i. The PR8 infection with 0.4 LD50 have shown the same picture: a significant
increase of neutrophil counts (more than two-fold compared with uninfected animals) on day 2 p.i., followed by a
decrease to the normal level. At the same time, the number of lymphocytes dropped by 15.6%. Again, no other group
of white blood cells displayed any significant changes. Similar effects occurred after infection with lower (0.1 LD50)
dose, with the return to the normal values on day 7 p.i.
Conclusions
In conclusion, the changes in differential leukocyte count caused by the infection with the human IAV were dosedependent, i.e. the changes were milder upon the infection with a lower virus dose. However, they did not depend
on the virulence of the virus used for infection.
Acknowledgements. This work was supported by Grants VEGA: 2/0117/11, 2/0176/12, 2/0100/13 and 1/0185/11
from the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and Slovak Academy of Sciences,
and by grant from the Slovak Research and Development Agency under the Contract No. APVV-0250-10.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P56
POSTE R A BSTRAC TS
SPB2P18
Heterogeneous pathological outcomes after experimental pH1N1
influenza infection in ferrets correlate with viral replication and host
immune responses in the lung.
Vidana, Beatriz (1,3); Martínez, Pamela (3); Martínez, Jorge (1,3); Montoya, María (3,4); Martorell, Jaime (2); G. Migura, Lourdes (3); Majó, Natàlia
(1,3)
Centre de Recerca en Sanitat Animal (CReSA), Spain; 2: Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona,
Spain; 3: Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Spain; 4: Institut de Recerca i Tecnologia
Agroalimentaria (IRTA), Barcelona, Spain
Background
The pH1N1 infection caused a variety of illness among the human population. The reason behind these varying
susceptibilities is unknown, but it has been suggested that may be due to host genetic variations associated with
inappropriate immune responses against viral characteristics. This study intends to evaluate whether the pathologic
patterns of influenza strains that produce different disease outcomes in healthy young humans could be reproduced
in a ferret model, and which immune-pathologic features were related to the disease outcome.
Methods
Ferrets were infected with two pH1N1 viral isolates (R11 and R61) from healthy young adults which developed
different disease outcomes (mild symptoms and death). Lung histopathological examination, viral detection and
quantification by immunohistochemistry (IHC) and RT-qPCR were carried out at 4 and 7 dpi. Phenotyping and
quantification of the inflammatory cell populations in the lungs were performed by IHC and correlated with the
lung expression of different proinflammatory molecules by RT-qPCR. The gene expression of pro- apoptotic markers
was quantified by RT-qPCR and related with the apoptotic cells observed in the lungs by IHC.
Results
Severity of infection did not correlate with the clinical phenotype observed in humans and did not correspond to any
viral isolate in particular (Figure 1). Severe lung lesion was associated with higher viral replication in alveolar areas and
with an exacerbated innate cellular immune response. This response was characterised by substantial phagocytic
and cytotoxic cell migration into the lungs (Figure 2), accompanied by elevated expressions of proinflammatory
cytokines and the down-regulation of IFNα in the lungs. Severe lesions were associated with greater up-regulations
of pro-apoptotic markers and higher accounts of apoptotic phagocytic cells.
Conclusions
Severe outcomes observed in infected ferrets were consequence of viral replication in alveolar areas which depended
on host capacities to mount an early efficient immune response. These results support viral replication and the
correct orchestration of the early inflammatory response as key factors in the outcome of the disease.
This work is currently submitted to the Veterinary Research Journal for revision.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P57
POSTE R A BSTRAC TS
SPB2P19
Species-specific host factors for Influenza A Virus replication
Martin-Sancho, Laura (1); Karlas, Alexander (1); Imai, Aki (1); Barclay, Wendy (2); Meyer, Thomas F. (1)
1: Max Planck Institute for Infection Biology, Germany; 2: Imperial College London, UK
Background
Typical avian influenza A virus (IAV) strains show limited replication in species other than aquatic birds, their natural
host. To become adapted to other hosts, the virus has to overcome host species barriers. It is hypothesized that
these barriers could be partially due to differences in host cell factor requirements. Five RNAi-based screens have
identified multiple host factors essential for IAV replication in human cells, but requirements in other host species
are unknown. The aim of this study is to identify similarities and differences in terms of host factor requirements in
the human, the avian and the porcine host.
Methods
Using our existing high-throughput-siRNA screening platform, chicken and porcine cells were transfected with
custom-made siRNAs. The screen assessed viral replication, cell viability and interferon induction. Hits were
validated in primary lung epithelial cells established in-house. Positive hits are subjected to polymerase assays,
confocal microscopy and immunoprecipitation assays for further investigation.
Results
Using data from the published screens, we identified 84 genes that have avian and porcine orthologs and confirmed
expression in our cell lines using microarrays. After excluding toxic and immunostimulatory siRNAs, bioinformatics
analyses revealed 30 genes required for influenza virus replication in all three species. Many of these were associated
with mRNA splicing, nuclear transport and cellular trafficking. In addition, we identified 13 genes required by the
virus in either human or porcine but not in avian cells. After validating these hits in lung epithelial cells, different
assays are currently ongoing to help us uncover the role of those factors in the viral cycle.
Conclusions
This siRNA cross-species screen will elucidate the role of specific host factors in IAV tropism, helping us to understand
the requirements for efficient replication and transmission of avian viruses in mammalian cells, and providing novel
target candidates for host-directed therapy.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P58
POSTE R A BSTRAC TS
SPB2P20
The relevance of cell cycle manipulation of influenza A virus to virus
pathogenicity
Xu, Ke
Institut Pasteur of Shanghai, Chinese Academy of Sciences, China, People’s Republic of
Background
Cell cycle is a fundamental activity for the host cells, especially when reproduction of cells is demanded under the
evasion of pathogens. By contrast, viruses develop different strategies to manipulate the host cell cycle for their own
benefits. We previously demonstrated that influenza A virus promotes its propagation by arresting the host cell
cycle at G0/G1 phase. However, whether cell cycle manipulation of influenza A virus is biological relevant to disease
outcomes and pathogenicity is still unknown.
Methods
The cell cycle profiles of cells infected with different strains and subtypes of influenza A viruses were analyzed by
FACS assay. A genomic-wide screening was applied to identify the responsible viral components for arresting the cell
cycle. A crucial host molecule was found to be targeted by influenza A virus for cell cycle arrest, and the interaction
motif was discovered by mutagenesis. Recombinant virus deficient in arresting the cell cycle was rescued by reverse
genetics to infect cell cultures and mice. The proliferation of T cells from the infected animals was further analyzed.
Results
We found that arresting the host cell cycle at G0/G1 phase is a universal strategy exploited by influenza A viruses, but
the strength of cell cycle arrest differs among subtypes. In general, it is more conserved for human viruses than for
avian viruses to induce a cell cycle arrest in human epithelium and T cells. The human cleavage and polyadenylation
specificity factor 30 (CPSF30), whose expression is essential for cell cycle progression, is targeted by viral proteins. By
binding to CPSF30 and subsequently inhibiting CPSF30 activity, influenza A virus heavily arrested the host cell cycle
at G0/G1 phase. The recombinant virus defective in cell cycle arrest was highly attenuated in CPSF30-competent
cells but not in CPSF30-deficient cells. In WT virus infected mice, the proliferation of T cells was severely inhibited
and the mice were totally dead under the challenge of high virus load. However, in mice infected with the same
amount of cell cycle arrest-deficient virus, we observe a normal T cell proliferation and 100% survival. The data
indicate that the ability to induce cell cycle arrest contributes to influenza A virus pathogenicity probably though
inhibiting the proliferation of immune cells.
Conclusions
With the fact that CPSF30 is curial for a normal cell cycle, influenza A virus uses two of its viral proteins to hijack
CPSF30 for manipulating host cell cycle. By arresting the cell cycle at G0/G1 phase, influenza A virus creates a
favorable in vivo environments for itself that will finally lead to a high pathogenicity.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P59
POSTE R A BSTRAC TS
SPB2P21
The adaptation of avian influenza viruses to the respiratory epithelium
of pigs
Yang, Wei (1); Meng, Fandan (1); Punyadarsaniya, Darsaniya (2); Hoffmann, Markus (1); Stech, Juergen (3); Hoeper, Dirk (3); Beer, Martin (3);
Schwegmann-Wessels, Christel (1); Ren, Xiaofeng (4); Herrler, Georg (1)
1: Institute of Virology, University of Veterinary Medicine, Hannover, Germany;; 2: Institute of Virology , Mahanakorn University of Technology,
Bangkok, Thailand;; 3: Friedrich Loeffler Institut, Bundesforschungsinstitut für Tiergesundheit, Greifswald, Germany; 4: College of Veterinary
Medicine, Northeast Agricultural University, Harbin, China
Background
Pigs are an important host for influenza A viruses and may play a crucial role in the interspecies transmission. To
analyze the infection by influenza viruses, we have established precision-cut lung slices from the porcine lung as
a culture system for differentiated respiratory epithelial cells. As differentiated repiratory epithelial cells are the
primary target cells for influenza virus infections, precision-cut lung slices provide an interesting system to analyze
the adaptation of avian influenza viruses to the respiratory epithelium of pigs. Avian influenza viruses H9N2 subtype
have been circulating worldwide in multiple avian species and have repeatedly infected mammalian to cause typical
disease. The continued avian-to-mammalian interspecies transmission of H9N2 viruses raises concerns about the
possibility of viral adaption with increased virulence for humans and poses a potential health risk to the public.
Methods
Avian influenza viruses H9N2 subtype were subjected to several passages in precision-cut lung slices. Then the
changes in the viral properties that are associated with the adaptation process were characterized by analyzing:
(1) duration of the growth cycle; (2) amount of infectious virus released into the supernatant; (3) extent of the
ciliostatic effect. Sequence analysis will reveal which amino acid changes occur during the different virus passages
Results
Adaptation of the avian viruses to growth in porcine cells was evident in a shortening of the growth cycle. Sequence
analysis revealed that few amino acid changes occurred during the different virus passages. The importance of the
individual mutations is currently analyzed by generating recombinant viruses that contain the respective mutated
proteins.
Conclusions
Our study will help to understand the processes involved in the adaptation of H9N2 influenza viruses to new hosts.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P60
POSTE R A BSTRAC TS
SPB2P22
The effect of Streptococcus suis co-infection on the infection of welldifferentiated porcine respiratory epithelial cells by swine influenza
viruses
Meng, Fandan (1); Wu, Nai-Huei (1); Seitz, Maren (2); Valentin-Weigand, Peter (2); Ren, Xiaofeng (3); Herrler, Georg (1)
1: Institute of Virology, University of Veterinary Medicine, Hannover, Germany; 2: Institute of Microbiology, University of Veterinary Medicine,
Hannover, Germany; 3: College of Veterinary Medicine, Northeast Agricultural University, Harbin, China
Background
Disease often occurs due to a combination of various factors including viral and bacterial pathogens as well as
environmental factors. A major factor responsible for severe virus infections may be bacterial co-infections.
As known, pigs are important hosts for influenza A viruses and may play an important role in the interspecies
transmission of influenza viruses. Primary target cells for Influenza viruses are the epithelial cells in the respiratory
tract. Differentiated airway epithelial cells contain special cell types such as ciliated cells or mucus-producing cells
that can’t be maintained as immortalized cell cultures.
Methods
We have recently reported a culture system for differentiated respiratory epithelial cells to analyze the infection of
porcine influenza viruses in their natural target cells. Therefore, the aims of this study are to analyze the effect of
Streptococcus suis (S.suis) co-infection on the infection of well-differentiated porcine respiratory epithelial cells by
porcine influenza virus types H1N1 and H3N2.The comparison of mono- and co-infection reveals to what extent the
bacterial infection enhances the severity of infection by porcine influenza virus.
Results
We compared five porcine viruses of the three subtypes currently prevalent in the swine populations (H3N2, H1N1,
H1N2) with respect to the following parameters: (1) duration of the growth cycle; (2) amount of infectious virus
released into the supernatant; (3) extent of the ciliostatic effect. These viruses showed differences in their growth
behavior and ciliostatic effect on PCLS and thus reflected the virulence properties of these viruses.
Conclusions
Our co-infection studies will reveal whether S.suis differentially affects influenza viruses differing in their virulence.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P61
POSTE R A BSTRAC TS
SPB2P23
Streptococcus suis affects the replication of swine influenza virus in
porcine tracheal cells
Wu, Nai-Huei (1); Meng, Fandan (1); Seitz, Maren (2); Valentin-Weigand, Peter (2); Herrler, Georg (1)
1: Institute for Virology, University of Veterinary Medicine Hannover, Germany; 2: Institute for Microbiology, University of Veterinary Medicine
Hannover, Germany
Background
Swine influenza viruses (SIV) are important pathogens affecting pigs of all ages. Surveillance data show that 31%
of European pig farms are exposed to SIV. Streptococcus suis is one of the most important bacterial respiratory
pathogens in the swine population. Secondary infection by S. suis may enhance the severity of disease in piglets
infected by SIV resulting in huge economic losses. To date, the relationship between SIV and S. suis still remains
unclear. In order to understand the interaction between SIV and S. suis, we established an in vitro co-infection model
based on newborn pig trachea cells (NPTr).
Methods
Two SIV variants A/sw/Bad Griesbach/IDT5604/2006 H1N1 and A/sw/Herford/IDT5932/2007 H3N2 were used to
compare subtype differences. Our previous studies showed that this H3N2 strain had a higher replication rate and
induced a strong ciliostatic effect in pig precision-cut lung slices, while the H1N1 strain only had a mild effect on
the ciliary activity. Wild type S. suis (WT) and an noncapsulated mutant strain (Δcps) were selected as secondary
bacterial pathogens in this study. NPTr cells were infected with different combinations, first inoculated with SIV,
followed by bacterial inoculation. The course of infection was monitored by immunofluorescence microscopy and
by determining the virus titers at different time points.
Results
After SIV infection, the adhesion rate of WT bacteria was enhanced. Different from Δcps, most of the WT bacteria
adhered to SIV infected cells. Furthermore, the viral replication rates of both H1N1 and H3N2 SIV were reduced
when cells were co-infected by S. suis WT strain. Interestingly, the virus titers in WT co-infected groups were ten-fold
lower than in SIV mono-infection or Δcps co-infected groups at 24 h.p.i.
Conclusions
These results indicate that S. suis and SIV affect each other in the infectious behavior in our NPTr cell model.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P62
POSTE R A BSTRAC TS
SPB2P24
Virus and host determinants of influenza A virus restriction by human
MxA
Patzina, Corinna; Riegger, David; Haller, Otto; Kochs, Georg
Institute for Virology, University Medical Center Freiburg, Germany
Background
Human myxovirus resistance protein MxA is an interferon-induced effector with a broad spectrum of antiviral
activity against divergent viruses, e.g. orthomyxo- or bunyaviruses. MxA-mediated inhibition of influenza A virus
(FLUAV) replication is dependent on the virus strain, as viruses of avian origin are more sensitive to MxA than human
strains. An accessible patch of amino acids in the viral nucleoprotein (NP) determines MxA sensitivity, implying that
MxA specifically recognizes FLUAV NP. However, it is still unclear how MxA can inhibit the replication of such a broad
range of viruses.
Methods
To validate the relationship between Mx resistance and adaptive mutations in NP, virus evolution assays were
performed in vivo. Therein, a recombinant human A/PR/8/34 carrying two avian NP mutations that render the virus
sensitive to Mx action, PR8(mut), was passaged in Mx1-positive mice and screened for emerging adaptive mutations.
To identify sites in human MxA that determine its specificity to FLUAV and to related orthomyxoviruses, a comparative
analysis of primate MxA proteins was performed. Sites with strong signatures of positive selection, a marker of hostpathogen interfaces, were characterized for their impact on orthomyxovirus restriction and vRNP recognition.
Results
PR8(mut) was highly attenuated in Mx1-positive mice when compared to the parental strain, but regained its
replicative potential upon consecutive lung passages in these mice. Thereby, two non-synonymous mutations
emerged in NP that are in close proximity to the previously characterized amino acid patch determining Mx resistance
of human FLUAV strains. In vitro experiments supported the increase of Mx resistance of PR8(mut) mediated by a
single adaptive NP mutation. In addition, an evolution-guided analysis of MxA revealed its overall high conservation
with a limited set of amino acids evolving under strong positive selection. These residues mainly cluster in loop L4,
a surface-exposed, flexible domain that protrudes from the highly ordered stalk of MxA. The minimal motif in loop
L4 of MxA required for orthomyxovirus specificity and vRNP recognition consists of the positively selected Phe- 561
and neighbouring residues.
Conclusions
We were able to confirm the ability of FLUAV to overcome restriction by Mx through single mutations in an accessible
region of NP. Interestingly, the virus did not adapt by back- mutating the initially altered residues, implying that
a potential Mx-NP interface in this region can be affected by divergent changes. The host protein MxA shows
signatures of continuous host-virus arms races, especially in the surface-exposed loop L4, wherein single residues
determined orthomyxovirus specificity.
We hypothesize that MxA restricts FLUAV by a direct interaction of loop L4 with NP, most likely by multiple contacts
of an MxA oligomer with FLUAV nucleocapsids, and we hope that further understanding the host’s defense system
might result in novel therapeutic strategies against highly pathogenic FLUAV strains.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P63
POSTE R A BSTRAC TS
SPB2P25
Viral replication kinetics and pathogenesis of pandemic H1N1 and H7N9
influenza virus infection in isogenic guinea pigs
Wiersma, Lidewij (1); Kreijtz, Joost (1); van Trierum, Stella (1); Nieuwkoop, Nella (1); van Run, Peter (1); van Amerongen, Geert (1); Ladwig,
Mechthild (2); Banneke, Stefanie (2); Schaefer, Hubert (3); Osterhaus, Ab (1); Rimmelzwaan, Guus (1)
1: Erasmus Medical Centre, Netherlands, The; 2: Federal Institute for Risk Assessment, Berlin, Germany; 3: Robert Koch Institute, Berlin,
Germany
Abstract
Isogenic guinea pigs uniquely combine the virtues of the mouse and ferret models for influenza A virus infection.
Like mice, they are inbred and therefore suitable for studies on cellular immunity and, like ferrets, they effectively
transmit the virus. In order to pave the way for new lines of research into viral transmission and cellular immunity, a
baseline study was performed to assess replication kinetics and the pathogenesis of pandemic H1N1 (pH1N1) and
H7N9 virus infection in this particular strain of guinea pigs.
Forty female isogenic guinea pigs 12-16 weeks were kept under biosafety level 3 conditions and inoculated with
pH1N1 virus intranasally (IN n=12) or intratracheally (IT n=10) and sacrificed on day 1, 2, 3, 4 or 7. Remaining animals
were inoculated IT with either H7N9 virus (n=12) or PBS (n=6) and sacrificed on day 2 and 7. Swabs were taken
daily and full necropsies were performed. Infectious virus titres in tissue samples and swabs were determined
using standard methods and immunohistochemical (IHC) and histopathological changes were evaluated by light
microscopy.
Virus inoculated animals showed peak average viral titres in nasal secretions at day 2 post-inoculation (pi) and by
day 7 pi infectious virus was no longer detectable for pH1N1 and low for H7N9 virus inoculated animals. Intranasal
inoculation with pH1N1 resulted in higher peak viral excretion via the nose than IT inoculation. pH1N1 IN inoculated
guinea pigs showed viral titres in only nasal epithelium at day 4. For pH1N1 IT inoculated animals, infectious virus
was recovered up to day 4 from nasal epithelium, trachea, lung and cerebral tissue. For H7N9 IT inoculated animals,
virus was recovered from nasal epithelium, lung, cerebrum and trachea on day 2 as well as nasal epithelium on
day 7. Histological evaluation revealed more widespread and severe lesions after IT than IN inoculation for pH1N1.
Overall, H7N9 inoculated animals showed the most severe lesions. Immunohistochemically, viral antigen positive
cells were restricted to the nasal cavity for pH1N1 IN inoculation. For IT inoculation of both pH1N1 and H7N9, viral
antigen was more diffusely dispersed in epithelial cells of the respiratory tract and alveolar macrophages around day
2 and still present in the lung and nasal epithelium respectively by day 7.
For future research, isogenic guinea pigs show promise as a model for cellular immunity and transmission of
influenza viruses. Additionally, the data highlight the importance of early time points and the advantages of the
intratracheal route of inoculation.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P64
POSTE R A BSTRAC TS
SPB2P26
The contribution of the PB1-F2 protein to viral virulence and
pathogenesis of avain influenza virus
James, Joe (1,2); Moncorgé, Olivier (2); Barclay, Wendy (2); Shelton, Holly (1)
1: Avian Infectious Disease Programme, The Pirbright Institute, Compton Laboratory, Compton, Berkshire, UK; 2: Faculty of Medicine, Imperial
College London, UK
Background
The PB1-F2 protein is translated from the +1 frame of the genetic segment that encodes for the influenza virus RNA
polymerase, PB1. Work in mammalian systems has shown PB1-F2 to have a range of effects dependent upon cell type
and viral strain. These activities include pro-apoptotic and pro-inflammatory functions, modulation of the innate
immune response, and regulation of viral polymerase activity. The PB1-F2 protein is often truncated or missing in
influenza viruses isolated from mammals. However 96% of all avian influenza isolates possess a full length PB1-F2
gene, suggesting a unique function in the avian host. To date, only limited studies have been undertaken in avian
systems and the role of PB1-F2 in the avian host remains to be fully elucidated. Here, using prototypical H9N2 and
H5N1 influenza strains of utmost importance to the poultry industry, we begin to examine the role of the PB1-F2
protein in influenza infection of poultry.
Methods
Using reverse genetics we generated isogenic viruses based on two different avian influenza virus backgrounds
that differed only in the length of the PB1-F2 protein which they produce but with no alteration to the other
viral proteins produced from the PB1 segment. We assessed the contribution to viral virulence by PB1-F2 using a
chicken embryonic lethality assay, where groups of chicken embryos were infected with decreasing concentrations
of influenza viruses and the percentage of eggs reaching the experimental endpoint determined. To assess viral
polymerase modulation, mutations to truncate the PB1-F2 protein were made in the context of a PB1 expression
plasmid and used a viral mini-replicon assay in DF-1 cells. Immune modulation was assayed via qRT-PCR for innate
immune related transcripts and with a chicken specific interferon-β promoter reporter.
Results
Using the in ovo lethality assays, we show that truncation of PB1-F2 from full length decreases the minimum lethal
dose. We show that full length PB1-F2 significantly up- regulated viral polymerase activity in chicken cells using
the in vitro minireplicon assay system. Although in the context of whole virus, PB1-F2 had little effect upon the
replication kinetics of a multi-cycle growth curve in chicken cells or embryos. However, expression of avian PB1-F2
was able to modulate the innate immune response via antagonism of the chicken type I interferon system.
Conclusions
We speculate that PB1-F2 modulates the innate immune response during virus infection in chickens and loss of
the C-terminus results in dysregulation and consequently a more severe outcome of infection. This knowledge will
facilitate enhanced predictions of pathogenicity in poultry based upon available sequence data, allowing for more
informed decisions about the course of action during influenza outbreaks in poultry.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P65
POSTE R A BSTRAC TS
SPB2P27
Hemagglutinin 222D/G polymorphism of pandemic influenza A (H1N1)
2009 viruses enables fast intra-host evolution and causes severe
biphasic influenza in mice
Seidel, Nora; Sauerbrei, Andreas; Wutzler, Peter; Schmidtke, Michaela
Jena University Hospital, Department of Virology and Antiviral Therapy, Jena, Germany
Background
The HA-222D/G polymorphism of pandemic (H1N1) 2009 influenza viruses (A(H1N1)pdm09) was frequently
reported in severe and fatal influenza in humans and mice. Its impact on viral pathogenicity and the course of
disease has been discussed controversially and the underlying mechanism remained unclear.
Methods
Using BALB/c mice infected with the once mouse lung- and cell-passaged A(H1N1)pdm09 isolate A/Jena/5258/09
(mpJena/5258; biphasic disease: Manchanda et al. 2014 Biosystems) and A/swine/Potsdam/15/81 (mpPotsdam/15;
monophasic disease), we followed the evolution of HA-222, which is within the host receptor binding and antigenic
sites, in different stages of infection and characterized its polymorphism in lung and trachea. Symptoms were
monitored daily based on body weight loss and clinical score.
Results
As expected, infection of mice resulted in one (mpPotsdam/15) or two symptom peaks (mpJena/5258) on day 7 or 4
and 7 p.i., respectively. In mpJena/5258-infected mice, HA-222D/G polymorphism was detected in virus isolates from
lung as well as trachea. Two virus titer maxima in lung and trachea preceded the symptom peaks. The first peak was
45-fold higher in the lung and the second was 13-fold higher in trachea virus titers. These results indicated a change
in organ tropism during virus infection. The change in organ tropism was accompanied by increased detection of
HA-G222 quasispecies in both organs. In the trachea, but not in the lung, HA-G222 became the major quasispecies
after day 4 p.i. Because HA-222 is part of the antigenic site Ca and the receptor binding site, both mpJena/5258
quasispecies were plaque-purified and the antibody binding as well as the receptor recognition was examined.
The results demonstrate an impaired neutralization and a broader receptor recognition including terminal Nacetylneuraminic acid α-2,3-linked to galactose (abundantly expressed in trachea) of HA-G222 quasispecies. The
latter was also confirmed for mpPotsdam/15 that exhibit only HA-G222. In additional mouse studies we showed
that these specific HA-G222 characteristics enabled replication of the plaque-purified HA-G222-mpJena/5258 and
mpPotsdam/15 virus with high viral titers in lung and trachea. A continuous worsening of symptoms in BALB/c mice
was observed. Only HA-G222 quasispecies were detected by sequencing of isolates from tissue samples.
Conclusions
These results demonstrate that quasispecies with a HA-222D/G polymorphism facilitate fast viral intra-host
evolution based on partial antibody escape and minor changes in receptor binding, resulting in an altered virus
tropism and severe influenza in mice. Detection of virus polymorphism in the early stage of infection might guide
to a better treatment of the disease.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P66
POSTE R A BSTRAC TS
SPB2P28
TMPRSS2 is a host cell factor that is essential for pneumotropism and
pathogenicity of H7N9 and H1N1 influenza A virus in mice
Tarnow, Carolin (1); Engels, Geraldine (2); Arendt, Annika (1); Schwalm, Folker (1); Sediri, Hanna (1); Garten, Wolfgang (1); Klenk, Hans-Dieter (1);
Gabriel, Gülsah (2); Böttcher-Friebertshäuser, Eva (1)
1: Institute of Virology, Philipps-University Marburg, Marburg, Germany; 2: Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology,
Hamburg, Germany
Background
Cleavage of the hemagglutinin (HA) by host proteases is a prerequisite for fusion activity and, thus, for influenza virus
infectivity. HA cleavage has been shown to be a prime determinant of avian influenza virus pathogenicity in poultry,
but little is known about its relevance for pathogenesis in mammals. We identified the serine protease TMPRSS2
(transmembrane protease serine S1 member 2) as a protease present in the human airways that activates influenza
virus HA with monobasic cleavage site. Here, we have analysed the role of TMPRSS2 on influenza virus pathogenesis
in a mouse model. We show that knockout mice that do not express TMPRSS2 are resistant to pulmonary disease
with lethal outcome when infected with H7N9 and H1N1 influenza A viruses, whereas they are not protected from
lethal H3N2 virus infection.
Methods and Results
Replication of the human H7N9 isolate A/Anhui/1/13 and of human H1N1 and H3N2 viruses was compared in
TMPRSS2-knockout (TMPRSS2-/-) and wildtype (WT) mice. Knockout of TMPRSS2 expression inhibited H7N9 influenza
virus replication in explants of murine trachea, bronchi and lung. Similarly, H1N1 virus replication was strongly
suppressed in airway explants of TMPRSS2-/- mice, while H3N2 virus replication was only marginally affected.
H7N9 and H1N1 viruses were apathogenic in TMPRSS2-/- mice, whereas WT mice developed severe disease with
mortality rates of 100% and 20%, respectively. Immunohistochemical analysis of viral antigen expression revealed
that knockout of TMPRSS2 prevented virus spread into the lung. In contrast, all H3N2 infected TMPRSS2-/- and WT
mice succumbed to lethal infection. Cleavage analysis in vitro showed that H7 and H1 are efficiently activated by
TMPRSS2, whereas H3 is less susceptible to the protease. It is currently under investigation what contributes to the
observed differences in protease specificity of H1 and H7, on one hand, and of H3, on the other hand.
Conclusions
Our data demonstrate that the host protease TMPRSS2 is essential for pneumotropism and pathogenicity of
H7N9 and H1N1 influenza virus in mice. In contrast, replication of H3N2 virus appears to depend on another,
not yet identified protease. These findings support the concept that human influenza viruses differ in protease
specificity, and demonstrate that expression of the appropriate protease along the respiratory tract is essential
for pneumotropism and pathogenicity. Furthermore, the data show that HA-activating proteases and in particular
TMPRSS2 are promising drug targets that should be considered for influenza therapy.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P67
POSTE R A BSTRAC TS
SPB2P29
H7N9 influenza A viruses exhibit importin-α mediated replication in the
mammalian respiratory tract
Bertram, Stephanie (1); Schwalm, Folker (2); Preuß, Annette (1); Klenk, Hans-Dieter (2); Gabriel, Gülsah (1)
1: Heinrich-Pette-Institute, Leibniz Institute for Experimental Virolo
Pneumonia complicated by respiratory distress is the leading cause of death in influenza A virus infected patients.
Previously, we have shown that the importin-α7 gene plays a major role in the development of pneumonia and
respiratory distress by promoting influenza virus replication in the lower respiratory tract of mammalian animal
models.
Here, we have analyzed whether a recently emerged avian H7N9 influenza virus that has crossed species barriers
and infected humans leading to high case fatality rates shows adaptive features towards human host factors that
promote virus replication in the lung. Using a cell-based polymerase activity assay in combination with siRNAmediated silencing for importin-α isoforms, we could detect a decreased H7N9 polymerase activity in human cells.
Consistently, virus replication was impaired in human importin-α7 silenced lung cells. Moreover, H7N9 infected
mice lacking the importin-α7 gene showed impaired pulmonary virus titres associated with reduced lung injuries
and enhanced survival compared to wildtype mice.
In summary, our results show that H7N9 influenza viruses show distinct features of adaptation to human host
factors. In particular, adaptation of H7N9 influenza viruses to importin-α7 might have contributed to elevated virus
replication in the lower respiratory tract leading to pneumonia and high case fatality rates among humans.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P68
POSTE R A BSTRAC TS
SPB2P30
PI3K-gamma activation during influenza A infection induces type I and
type III IFN responses and resolution of inflammation via p38 activation
Garcia, Cristiana Couto (1); Tavares, Luciana Pádua (1); Dias, Ana Carolina Fialho (1); Queiroz-Junior, Celso (1); Lima, Braulio Henrique Freire (1);
Machado, Alexandre Magalhães Vieira (2); Sousa, Lirlândia Pires (1); Russo, Remo Castro (1); Teixeira, Mauro Martins (1)
1: UFMG, Brazil; 2: CPqRR, Fiocruz, Brazil
Background
PI3Kγ is mainly expressed by leukocytes, is activated by GPCRs coupling and is involved in cell migration under
inflammatory conditions. Influenza A virus causes a common and severe pulmonary disease that affects millions of
people worldwide every year. Influenza NS1 binds and activates class IA PI3K and induces viral entry into host cell,
but also immune responses. Specific involvement of PI3Kγ was never assessed during influenza infection.
Methods
C57/BL6 WT and PI3Kγ KO mice from Center of Bioterism of UFMG were used for in vivo and ex vivo experiments.
Mice were infected with 104 PFU of influenza A/WSN/33 H1N1 or A/PR/8 H1N1 and were monitored for weight
loss and lethality for 21 days (WSN and PR8) or euthanized (WSN) at 3, 5 and 7 days after infection to analyze viral
loads (plaque assay), leukocyte infiltration (cell counts and FACS analysis) and activation (chemoluminescence), lung
damage (histology) and production of cytokines and ISGs (ELISA, qRT-PCR) and activation of signaling pathways
(Western Blot). Ex vivo infection with WSN (MOI 3.0) were performed in bone marrow derived macrophages (BMDM)
from WT and KO mice for assessment of signaling molecules and type I IFN and ISG RNA.
Results
PI3Kγ KO mice were more susceptible to WSN and PR8 infection with enhanced lethality and weight loss rates.
Neutrophil infiltration and ROS production in lungs and lung damage were higher in KO mice; however there is a
reduction in infiltration of macrophage, especially the pro-resolving population (Gr1- F4/80med CD11blow), and
NK and CD8+ T cells in KO mice after seven days. In all evaluated time points after infection, type I IFN (IFN-α4 and
IFN-β1) and type III (IFN-λ) RNA levels are strikingly reduced in lungs of KO mice compared to WT. Whereas RNA
from ISG15, Mx2 and ddx58 is highly enhanced in KO mice, protein levels of ISG15 and RIG-I were similar in WT and
KO. P-p38 is reduced in KO mice. Viral loads are increased in early and late time points in KO mice. To investigate the
mechanisms of ISG induction we performed ex vivo experiments. In BMDM, type I IFN RNA is enhanced in WT cells
after 24 hours but not in KO; ISG15 and Mx2 RNA is enhanced after 2 and 6 hours, before type I IFN induction and
P-p38 is reduced in KO. The blockade of P-p38 during infection reduces ISG15 protein in WT cells is same levels as
KO untreated cells.
Conclusions
PI3Kγ is necessary for effective type I IFN antiviral responses and resolution of inflammation induced by influenza
infection. PI3Kγ KO induces an IFN- independent induction of ISGs RNA that are not translated into protein. Therefore,
PI3Kγ via p38 might be involved in postracriptional regulation of ISGs.
Financial support: CNPq/CAPES/FAPEMIG
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P69
POSTE R A BSTRAC TS
SPB2P31
Replicative Fitness of Avian Influenza A(H7N9) Virus Variants with
Reduced Susceptibility to Neuraminidase Inhibitors in Mice and Ferrets
Henju Marjuki (1), Vasiliy P. Mishin (1), Anton P. Chesnokov (1), (2), Joyce Jones (1), Juan A. De La Cruz (1), (2), Katrina Sleeman (1), Ho-Sheng Wu3,
Feng-Yee Chang3, Ming-Tsan Liu3, Alicia M. Fry (1), Nancy J. Cox (1), Julie M. Villanueva (1), Charles T. Davis (1), Larisa V. Gubareva (1)
1: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA,
2: Battelle Memorial Institute, Atlanta, GA, USA, 3: Taiwan Centers for Disease Control, Taipei City, Taiwan
Background
Neuraminidase (NA) inhibitors (NAIs) have been used for treatment of patients with influenza A(H7N9) virus
infections. The efficacy of NAIs, however, can be compromised by the emergence of drug-resistant viruses. Four
NA variants with mixed bases encoding amino acid substitutions at positions E119V, I222K, I222R or R292K were
detected in a single virus isolate, A/Taiwan/1/2013 (H7N9), recovered from an oseltamivir-treated patient.
Methods
Virus variants with a single amino acid substitution in the NA were detected using the CDC pyrosequencing assay
during the initial screening of the isolate. Each NA variant was plaque-purified and sequenced for full genome
analysis. The fluorescent NA inhibition assay was used to assess susceptibility to NAIs zanamivir, oseltamivir,
peramivir, and laninamivir. Replicative fitness of each variant was assessed in cell cultures and in mice and ferrets.
Results
The NA substitutions identified in this study have the potential to reduce clinical susceptibility to one or more NAIs
based on the results of the NA inhibition assay. In MDCK cells, all NA variants replicated to comparably high titers,
while in MDCK-SIAT1 cells, the NA-R292K variant exhibited delayed growth. Depending on the inoculation dose
given, mice infected with any variant developed signs of severe disease with a high mortality rate. Notably, both
wild type and NA-R292K viruses were the least virulent in mice, while NA-I222K was the most virulent. An additional
internal protein gene substitution at position PB2-S714N appeared to cause increased virulence of the NA-I222K
variant in mice, while NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects. In ferrets,
all viruses replicated to high titers in the upper respiratory tract, but despite a high inoculation dose, none developed
severe infection. The NA-R292K virus showed reduced replicative fitness in ferrets, and infection with this virus did
not cause weight loss.
Conclusions
Collectively, the results suggested a host-dependent effect on replicative fitness of H7N9 NA variants analyzed in this
study. Three substitutions detected in PB2 and NS1 appeared to alter virus pathogenesis in mice, but had marginal
impact when tested in ferrets. Furthermore, our data highlighted the necessity of monitoring the emergence of
drug-resistant viruses during treatment of patients with NAIs.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P70
POSTE R A BSTRAC TS
SPA3: IMMUNOLOGY
SPA3P01
Back-boost of antibody landscapes after influenza virus infection or
vaccination
Fonville, Judith (1,2); Wilks, Sam (1,2); James, Sarah (1); Aban, Malet (3); Xue, Lumin (3); Jones, Terry (1,2); Ventresca, Mario (1); Fox, Annette
(4); Nguyen Minh Hoa, Le (4); Quang Thai, Pham (5); Nhu Duong, Tran (5); Wong, Yan (1); Mosterin, Ana (1,2); Katzelnick, Leah (1,2); van der
Net, Guido (6); Skepner, Eugene (1,2); Russell, Colin (2,7); Kaplan, Todd (8); Rimmelzwaan, Guus (6); Masurel, Nic (6); de Jong, Jan (6); Palache,
Abraham (9); Beyer, Walter (6); Quynh Mai, Le (5); Tran Hien, Nguyen (5); Wertheim, Heiman (4); Hurt, Aeron (3); Osterhaus, Ab (6); Barr, Ian
(3); Fouchier, Ron (6); Horby, Peter (4); Smith, Derek (1,2)
1: University of Cambridge, United Kingdom; 2: WHO Collaborating Center for Modeling, Evolution, and Control of Emerging Infectious
Diseases, Cambridge, United Kingdom; 3: WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia; 4: Oxford
University Clinical Research Unit and Wellcome Trust Major Overseas Programme, Hanoi, Vietnam; 5: National Institute of Hygiene and
Epidemiology, Hanoi, Vietnam; 6: Department of Virology, Erasmus Medical Center, Rotterdam, the Netherlands; 7: Department of Veterinary
Medicine, University of Cambridge, Cambridge; 8: bobblewire.com; 9: Abbott Laboratories, Weesp, the Netherlands
Background
We present a methodology that enables in-depth analyses of human serological data, which were previously
difficult to interpret because of complex, and usually unknown, exposure histories, and cross-reactivity due to
antigenic
relatedness
strains.
This technique,
Back-boost
of antibody
landscapes among
after influenza
virus infection
or vaccinationwhich we call the antibody landscape, achieves a quantitative
visualization and analysis of antibody-mediated immunity by accounting for antigenic variation among pathogen
• Background:
strains. Using this approach, we aim to improve our understanding of the typical characteristics of the human
We present a methodology that enables in-depth analyses of human serological data, which
were previously difficult to interpret because of complex, and usually unknown, exposure
serological response to infection and vaccination.
histories, and cross-reactivity due to antigenic relatedness among strains. This technique,
which we call the antibody landscape, achieves a quantitative visualization and analysis of
antibody-mediated immunity by accounting for antigenic variation among pathogen strains.
Using this approach, we aim to improve our understanding of the typical characteristics of the
human serological response to infection and vaccination.
Methods
The antibody landscape extends an antigenic map in an additional dimension, to show the serological titers, and
• Methods:
The antibody
extends an antigenic
map surface
in an additional
dimension,
to show the
we fitlandscape
a representative
smooth
showing
antibody
levels for viruses from 43 years of influenza A/H3N2 virus
serological titers, and we fit a representative smooth surface showing antibody levels for
figure).
Wevirus
created
landscapes
for samples taken annually from 2007 to 2012 from 69
virusesevolution
from 43 years(see
of influenza
A/H3N2
evolution antibody
(see figure). We
created antibody
landscapes for samples taken annually from 2007 to 2012 from 69 unvaccinated individuals
unvaccinated
(born between 1917-2005) in Vietnam. Additionally, we created pre- and post-vaccination
(born between
1917-2005) individuals
in Vietnam.
Additionally, we created pre- and post-vaccination antibody landscapes for 102 individuals
antibody
landscapes for
102 and
individuals
vaccinated
A/Nanchang/933/95 in 1997, and 123 individuals
vaccinated
with A/Nanchang/933/95
in 1997,
123 individuals
vaccinated with
with
A/Sydney/5/97 in 1998.
vaccinated with A/Sydney/5/97 in 1998.
Creating an antibody landscape. (A) Antigenic map of A/H3N2 showing virus strains color-­‐coded by antigenic cluster. (B) An additional dimension indicates antibody titers as impulses, and a smooth surface is fitted using locally weighted multiple linear regression to create the antibody landscape. TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P71
POSTE R A BSTRAC TS
Creating an antibody landscape. (A) Antigenic map of A/H3N2 showing virus strains color-coded by antigenic
cluster. (B) An additional dimension indicates antibody titers as impulses, and a smooth surface is fitted using locally
weighted multiple linear regression to create the antibody landscape.
Results
We assessed the serological changes in the Vietnam cohort, and found antibody landscapes to be stable from one
year to the next. Upon infection with A/H3N2, a back-boost was observed – an increase of antibodies across an
individual’s prior subtype-specific immunity. We retrospectively compared two vaccination studies with similar prevaccination landscapes. As expected following a vaccine update, average vaccination responses were significantly
greater against later antigenic clusters following vaccination with the antigenically advanced A/Sydney/5/97 virus.
The back-boost following infection was also observed for the vaccination studies: the antigenically advanced vaccine
virus showed similar or increased responses for across the complete antibody landscape.
Conclusions
Our results highlight underscore the importance of accounting for antigenic variation to better understand multiexposure sera, and provide a methodology for the direct visualization of otherwise complex serological patterns,
allowing basic insights into the breadth of the adaptive humoral immune response. We found that a pre-emptive
antigenic update of the vaccine has the dual advantage of being effective against the antigenically novel viruses to
which they were targeted while remaining effective for contemporary viruses if they continued to circulate, which
may improve influenza vaccine effectiveness in previously-exposed individuals.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P72
POSTE R A BSTRAC TS
SPA3P02
Exposure history or immunosenescence –what determines the antibody
response to influenza vaccine in elderly persons?
Mosterín Höpping, Ana (1,4); McElhaney, Janet (2); Fonville, Judith (1,4); Beyer, Walter (3); Smith, Derek (1,4,3)
1: Centre for Pathogen Evolution, University of Cambridge, Department of Zoology, Downing Street, Cambridge, CB2 3EJ, UK; 2: Advanced
Medical Research Institute of Canada, Sudbury, ON, P3E 5J1, Canada; 3: Erasmus MC, Department of Viroscience, Rotterdam, PB 2040, NL3000 CA Rotterdam, The Netherlands; 4: World Health Organization Collaborating Center for Modeling, Evolution, and Control of Emerging
Infectious Diseases, Cambridge CB2 3EJ, UK
Background
Numerous studies have explored whether the antibody response to influenza vaccination in elderly adults is as
strong as it is in young adults. Results vary, but tend to indicate lower post-vaccination titers (antibody levels) in the
elderly supporting the concept of immunosenescence.
Methods
We conducted a four-year study of serial annual immunisations with inactivated trivalent vaccines in 135 young
adults (16 to 39 years) and 105 elderly adults (62 to 92 years). None of the young had been vaccinated before
entering the study, but just as in other studies the elderly adults had all been vaccinated against influenza before
entering the study. This detailed panel dataset allowed us not only to investigate the effect of age, but to explain the
apparent contradiction in results reported so far based on investigations at an aggregate level.
Results
In response to the first vaccination, young adults produced higher postvaccination titers than elderly adults for
all three vaccine components. However, upon subsequent vaccinations the titer difference due to age declined
rapidly, for example, from a nearly two-fold ratio at first vaccination to one-fold after three vaccinations for A/H3N2.
Although age is an important factor when modeling the outcome of the first vaccination, this term lost relevance
with successive vaccinations. In fact, when we examined the data with the assumption that the elderly group had
received (on average) as few as two vaccinations prior to our study, the difference due to age disappeared.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P73
POSTE R A BSTRAC TS
In our interpretation, the initial age group difference in post-vaccination titers is not due to immunosenescence,
but is more parsimoniously explained by differing pre-study vaccination and infection histories. Since datasets
underlying other published studies are also affected by a difference in vaccination and infection history between
age groups, this interpretation may also explain the discrepancy between results reported.
Figure 1
Post-vaccination antibody levels for different age classes – notice there is a difference in post-vaccination titres
between, but not within the two age groups. Table 1
Independent
variables
Age
Estimates of regression model
A (y-intercept)
BAge
2
R
Old adults
Young adults
All
3.330*
0.005 (P=0.489)
0.001
4.319*
0.002 (P=0.747)
0.000
4.579*
-0.021 (P=0.000)
0.076
Summarized results of the regression post-titre = A+ BAge* Age for each of the two age groups, and for the
combined dataset. Notice that age only has a predictive effect in the combined dataset and not within either
age group.
Table 2
Independent
variables
Estimates of
regression model
Pre-titre
Age group
A (y intercept)
B pre
B agegroup
2
R
** p<0.01
Number of vaccinations (NV)
Total
2.43***
0.60**
1
3.54***
0.65***
-1.46***
0.29
*** p<0.001
2
2.36***
0.60***
-0.43**
0.42
3
1.79***
0.68***
-0.16
0.46
4
1.04***
0.84***
0.075
0.73
Regression analysis predicting post-titre, by pre-titre and excluding or including age group, by number of
vaccinations. Notice that the significance for age-group declines with number of vaccinations, until
agegeoup is small, insignificant and changes sign.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P74
POSTE R A BSTRAC TS
SPA3P03
Serodiagnostic studies during the development of new pandemic caused
A(H1N1)pdm09.
Mukasheva, Evgeniya (1); Kolobukhina, Ludmila (1); Kisteneva, Lidiya (1); Zaplatnikov, Andrey (2); Smolonogina, Tatyana (3); Desheva, Yuliya (3);
Burtseva, Elena (1)
1: D.I.Ivanovsky Research Institute of Virology, Russian Federation; 2: Russian Medical Academy of Postdegree Education, Russian Federation; 3:
Institute of Experimental Medicine of the NorthWest Branch of the Russian Academy of Medical Sciences, Russian Federation
Background
The aim of this study was to identify the features of the production factors of humoral immunity in response
to infection by new pandemic influenza A(H1N1)pdm09 virus among persons with different epidemiological
anamnesis.
Materials and methods.
Paired sera from 203 patients hospitalized with ILI symptoms (2009-2011) including pregnant women, single
sera from 287 schoolchildren living in boarding school in Moscow (2009), single sera from 83 pregnants and 20
newborns aged 1 day (2010), collected by staff Medical University named after V.I. Razumovsky, Saratov (March-April
2010) were included in study. To detect of specific influenza virus A(H1N1)pdm09 RNA in nasopharyngeal swabs
from hospitalized patients RT-PCR in real-time using commercial test kits was used. The level of specific antibodies
to hemagglutinin (anti-HA) and neuraminidase (anti-NA) and their dynamics were studied in HI and solid state
reaction inhibiting neuraminidase activity accordingly.
Results
The 4-fold and higher dynamics of the anti-HA antibodies was detected among A(H1N1)pdm09 positive by RT-PCR
hospitalized patients (28,6% among all patients and 14,0% – in pregnants); meantime in patients with A(H1N1)
pdm09 negative by RT-PCR these indexes were much lower (16,7% and 11,0% accordingly), but suggested A(H1N1)
pdm09 infection among them as additional diagnostic measures. Also 4-fold and higher dynamics of the anti-HA
antibodies to seasonal influenza A(H1N1) virus was found in these patients as well, meantime the active circulation
of it was not detected during studied period: among RT-PCR-positive – 22,4% and 14,0% accordingly; among RT-PCRnegative – 16,7 and 11,0% accordingly. Positive dynamics against strains of influenza virus A(H3N2) and B in the
sera was not detected. The results of screening sera from schoolchildren living in boarding school showed that by
March 2010 the most children (78%) have acquired specific antibodies to A(H1N1)pdm09. The transfer of maternal
antibodies to babies, including antibodies to the pandemic strain of influenza A(H1N1)pdm09 virus was found as
well. The results of this study showed that the immune response to hemagglutinin and neuraminidase (anti-HA and
anti-NA), as a result of natural infection by influenza A(H1N1)pdm09 virus, is formed simultaneously in more than
half of hospitalized patients: 57,7% – among all patients and 55,6% – pregnants.
Conclusions
Our results showed that during the first (2009-2010) and the second (2010-2011) wives of new pandemic caused
by influenza A(H1N1)pdm09 virus, population had different risk and time to have “contact” with this virus. It was
important to use serological tests to confirm influenza infection in RT-PCR-negative patients and confirm the
transfer the maternal antibodies to A(H1N1)pdm09 to newborn babies. Also the simultaneously forming of anti-HA
and anti-NA to A(H1N1)pdm09 in more then 50,0% hospitalized patients was found.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P75
POSTE R A BSTRAC TS
SPA3P04
Avian myotubes are highly susceptible to influenza virus infection and
replication
Chang, Kin-Chow (1); Baquero-Pereza, Belinda (1); Kuchipudi, Suresh V. (1); Ho, Jemima (1); Sebastian, Sujith (1); Brookesb, Sharon, M. (2); Brownb,
Ian H. (2)
1: School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Loughborough,
Leicestershire LE12 5RD, UK.; 2: Virology Department, Animal Health Veterinary Laboratories Agency Weybridge, Addlestone, Surrey KT15 3NB,
UK.
Background
Skeletal muscle can potentially play an important role in the transmission of highly pathogenic avian influenza
(HPAI) H5N1 viruses as demonstrated by their isolation from infected poultry. Furthermore, infection with HPAI
H5N1 viruses in ducks is often asymptomatic and meat from such birds could be a source of infection to humans,
either through handling or consumption of infected raw or undercooked meat. Little is known about the replication
of avian influenza viruses in avian skeletal muscle cells. To this end we used primary chicken and duck multinucleated
myotubes to examine the cyto-pathogenicity and corresponding host innate immune response to influenza virus
infection.
Methods.
Cytopathological effects of a low pathogenicity avian influenza (LPAI) H2N3 [A/mallard duck/England/7277/06]
and two HPAI H5N1 viruses [A/turkey/England/50-92/91 and A/turkey/Turkey/1/05, referred to as HPAI H5N1 5092 and HPAI H5N1 tyTy05 respectively], in primary chicken and duck muscle cells [myotubes and myoblasts] and
in relation to Madin-Darby canine kidney (MDCK) cells were examined, which included the determination virus
replication, relative cell viability, apoptotic changes and host innate immune response.
Results
We found that chicken and duck cultured myotubes were highly susceptible to infection with both LPAI and HPAI
viruses. Chicken and duck myotubes expressed both avian and human sialic acid receptors, and rapidly accumulated
viral nucleoprotein and M-gene RNA. Subsequent progeny virus release was accompanied by extensive cytopathic
damage of myotubes with hallmarks of apoptosis (widespread microscopic blebs, caspase 3/7 activation
and annexin-V binding at plasma membrane). Infected chicken myotubes produced significantly higher proinflammatory cytokines than corresponding duck cells. Additionally, in H5N1 virus infected chicken myotubes, IFN-β
and interferon-inducible genes, including MDA5, were down-regulated in contrast to corresponding H2N3 virus
infection.
Conclusions
Our findings indicate that avian skeletal muscle is a major amplification site for HPAI H5N1 virus replication,
highlighting the importance of skeletal muscle in the pathogenicity and transmission of virulent influenza virus
infection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P76
POSTE R A BSTRAC TS
SPA3P05
Investigating B and T cell repertoire signatures in response to influenza
infection
Lu, I-Na; Farinelle, Sophie; Muller, Claude P.
Laboratory of Immunology, Centre de Recherche Public de la Santé (CRP-Santé), Luxembourg
Background
The adaptive immune system provides protection against an enormous variety of pathogens. This protection is
mediated by receptors on the surface of B and T cells that bind to pathogen derived antigens.
Methods.
Using a model of murine influenza A virus infection, humoral and cellular immune responses are evaluated by
enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot) assay, and flow cytometry. B
and T cells from different organs (spleens, lymph nodes, bone marrows) are isolated applying magnetic-activated cell
sorting (MACS) technology. Next generation sequencing, such as the semiconductor-based Ion Torrent sequencing
platform, is used to investigate the B and T cell repertoire signatures.
Results
Influenza virus infection induces robust B and T cell responses. These responses are directed against immunodominant
surface structures that continuously undergo genetic changes such as the hemagglutinin protein, a major target
of neutralizing antibodies. The pathogenesis of influenza virus and the nature of the antiviral B and T cell responses
drive changes in repertoire diversity as the main mechanism of the influenza specific immunity. Here, we study
the B and T cell repertoire in response to influenza A virus in multiple organs and time points in order to clarify the
developmental evolution of the protective repertoire signatures.
Conclusions
This analysis of B and T cell repertoire signatures in response to influenza infection can provide important clues for
the design of successful vaccines and vaccination approaches.
Keywords: influenza; B cell repertoire; T cell repertoire
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P77
POSTE R A BSTRAC TS
SPA3P06
Influenza virus infection in quail (Coturnix coturnix): characterization of
humoral immune response
Majo, Natalia (1,2); Busquets, Núria (1); Garcia, Beatriz (1); Bensaid, Albert (1); Dolz, Roser (1); Oliver, Salvador (1); Bertran, Kateri (1); Ramis,
Antoni (1,2); Rivas, Raquel (1)
1: Centre de Recerca en Sanitat Animal (CReSA), Spain; 2: Facultat de Veterinaria, Universitat Autònoma de Barcelona
Since quail have emerged as a potential intermediate host in the spread of avian influenza viruses (AIV), new efforts
have been made to clarify the epidemiologic role of this species in avian influenza infection. In this study, we sought
to better understand the immune response generated by different influenza virus in quail. An experimental infection
with a low pathogenic avian influenza virus (LPAIV) H5N2 and a human H1N1 (pH1N1) virus were conducted in quail
to evaluate the humoral immune response, at both systemic and local levels.
Forty-eight quails were divided into three groups housed in independent isolation units at the biosafety level 3
(BSL-3) facilities of CReSA. Birds on group 1 (G1; n=20) were inoculated with H5N2 LPAIV (106EID50/50μL), birds on
group 2 (G2; n=20) were inoculated with pH1N1 (106EID50/50μL) and birds on group 3 (G3; n=8) were inoculated
with saline solution; thus served as a negative control. Five animals of each infected group and two animals of the
control group were euthanized at 3, 6, 10 and 12 days post-inoculation (dpi). Blood, choanal, tracheal and cloacal
swab samples were collected. Blood was used to evaluate seroconversion and antibodies isotype dynamics by
competition ELISA (cELISA). Swabs were used to study viral shedding by quantitative RT-PCR and total antibodies at
local level by cELISA. Direct ELISA tests were performed in order to determine if quail antibodies could be recognized
by commercial antibodies against chicken antibodies.
Quail supported the replication of H5N2 subtype, therefore, seroconversion was present in H5N2 infected animals.
On the contrary, only one bird belonging to G2 group (pH1N1) showed viral shedding on both choanal and tracheal
swabs at 3 dpi and they did not show seroconversion. Anti-chicken goat IgG polyclonal antibodies recognized IgY
and IgM quail antibodies in serum. Quail IgM isotype antibodies against influenza nucleoprotein (NP) were found
in serum samples from H5N2 infected quails with a strong humoral immune response. Moreover, total antibodies
against NP were detected in swab samples from one animal belonging to H5N2 infected group at 12 dpi.
Most of the animals inoculated with H5N2 LPAIV were capable to develop a humoral immune response in front of
the infection. These data correlate well with the replication capability of the viruses tested. We demonstrated that
anti-IgY and IgM chicken antibodies cross-reacted with quail antibodies and they can be used for serological studies
in quails. In H5N2 infected animals, IgM isotype antibodies could be detected as early as 6 dpi and a poor response
by IgY isotype antibodies was detected, indicating that the days sampled in this experimental infection were too
early to detect them effectively.
Work funded by Spanish National Institute for Agricultural and Food Research and Technology (INIA) (RTA201100111-C03).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P78
POSTE R A BSTRAC TS
SPA3P07
Humoral and cellular immune responses following the 2009 pandemic
Oftung, Fredrik (1); Korsvold, Gro Ellen (1); Dembinski, Jennifer (1); Tunheim, Gro (1); Savic, Miloje (1); Kristoffersen, Anne Cathrine (1); Haneberg,
Bjørn (1); Germundsson Hauge, Anna (1); Kim, Yohan (2); Peters, Bjoern (2); Berdal, Jan Erik (4); Monceyron Jonassen, Chrstine (3,4); Mjaaland, Siri
(1,5)
1: Norwegian Institute of Public Health, Oslo, Norway; 2: La Jolla Institute for Allergy and Immunology, California, USA; 3: Østfold Hospital
Trust, Norway; 4: Akershus University Hospital, Norway; 5: KG Jebsen Centre for Research on Influenza Vaccines
Background
Whereas humoral immunity confers protection against infection with influenza viruses through strain-specific
antibodies, recent findings suggest a major role of cross-reactive CD4 and CD8 T cells for protection against
heterotypic influenza infections by various effector mechanisms. In this study, we have characterized immune
responses following infection with the pH1N1 virus during the first wave of the 2009 pandemic.
Methods.
Serum, nasal secretions, and PBMC were collected from outpatients with laboratory confirmed pH1N1 virus 3 weeks
and 7 months after infection. Clinical parameters were recorded for all patients. Hemagglutination inhibition (HI)
assay, micro-neutralization (MN) assay, and ELISA against pH1N1 and two seasonal strains (NewCal/99/H1N1 and
Brisbane/H3N2) were performed on serum samples, whereas nasal secretions were only analyzed by ELISA. Cellular
immune responses were measured by IFN-γ ELISPOT assays using the same viral strains and selected peptide pools
representing known CD4 and CD8 epitopes from both surface and internal antigens.
Results
The HI and MN antibody titers correlated well. The titers against pH1N1 were significantly higher than for the
seasonal viruses, and all antibody levels declined over time. In contrast, the IgG antibody levels in serum and nasal
secretions measured by ELISA did not differentiate between pandemic and seasonal virus strains. IFN-γ ELISPOT
analysis of T cell responses showed cross-reactivity for pH1N1 virus and the two seasonal strains tested at both
time points. When analyzing the responses by using pools of common CD4 and CD8 peptide epitopes, we observed
a significant response against epitopes from conserved antigens, but not from surface antigens (HA and NA).
Moreover, the donors also responded to sets of peptides representing CD4 and CD8 epitopes unique to pH1N1
antigens.
Conclusions
In contrast to antibody responses against surface antigens, T cell responses involving both CD4 and CD8 subsets are
directed against sets of conserved epitopes common for both seasonal and pandemic influenza strains. The results
highlight that cross-reactive cellular immunity involving both subsets of T cells may be important for protection
against pandemic disease in the absence of strain-specific antibodies.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P79
POSTE R A BSTRAC TS
SPA3P08
Contribution of single mutations specific for A/Leningrad/134/17/57
(H2N2) master donor virus in influenza A virulence and immunogenicity
in a mouse model
Petukhova, Galina; Kuznetsova, Svetlana; Losev, Igor; Isakova-Sivak, Irina; Kuznetsova, Victoria; Korenkov, Daniil; Rudenko, Larisa; Naykhin, Anatoly
Institute Of Experimental Medicine RAMS, Russian Federation
Background
Live attenuated influenza vaccines (LAIVs) are able to provide protection by inducing significant mucosal and
cell-mediated immune responses. Vaccine strains for LAIV have temperature-sensitive (ts), cold-adapted (ca)
and attenuated phenotypes which are guaranteed by the presence of specific mutations in internal genes of A/
Leningrad/134/17/57 (Len/17) master donor virus (MDV). However it is known that the process of attenuation can
affect immunogenicity of live influenza viruses. Thus, study of the impact of single mutations in MDV on the virulence
of attenuated strains and immune response is necessary for better understanding of the LAIV immunogenicity
mechanisms.
Methods.
We used 11 reverse genetics–derived mutant viruses based on wild-type A/Leningrad/134/57 (H2N2) virus
(Len/134): nine single mutant reassortants (SMRs) carrying one MDV-specific mutation in the Len/134 background
(PB1-K265N, PB1-V591I, PB2-V478L, PA-L28P, PA-V341L, NP-N492S, M1-I15V, M1-F144L, NS2-M100I); and Len/134
and Len/17 as control viruses. Ts phenotype of the viruses was determined by plaque assay in MDCK cells at 33°C,
37°C and 38°C. All mutants were intranasally inoculated into CBA female mice at a dose of 5,5 log10TCID50. Lungs
and nasal turbinates (NT) were collected at 3 dpi and infectious virus titers were determined by titration of organ
homogenates in eggs. Antibodies against whole Len/134 wt virus in mouse sera were analyzed at 28 dpi by HAI
assay and ELISA.
Results
None of single mutations affected virus replication in MDCK cells at 37°C, whereas PB2-V478L and PB1-K265N
mutations had significant impact on virus ts phenotype at 38°C. Nevertheless, these two viruses replicated in
mouse lungs to the same level as Len/134 wt virus. Surprisingly, mutations in M1 gene had significant impact
on virus reproduction profile in mice: I15V mutation reduced virus titer in lungs whereas F144L change enhanced
virus replication in NT. Therefore no correlation was observed between ts phenotype and virus titers in lungs and/
or NT of these SMRs. Mutations PB1-K265N, PB1-V591I and NP-N492S slightly reduced HAI antibody titers at 28 dpi,
whereas PB2-V478L, PA-L28P, M1-I15V and M1-F144L substitutions enhanced virus immunogenicity if compared
to the control Len/134 virus, and the strongest effect was noted for PB2-V478L SMR. Combination of all tested
mutations resulted in the most attenuated and least immunogenic virus if measured by HAI and ELISA. HAI titers
correlated well with ELISA study of serum virus-specific IgG levels.
Conclusions
Most of single MDV-specific mutations do not affect virus replication in mouse respiratory organs with the exception
of mutations in M gene. Some mutations are able to increase or decrease virus-specific antibody levels in mouse
sera. Further studies of local humoral and cellular immune responses of these viruses in the mouse model will
provide additional data on the mechanisms involved in the immunogenicity of LAIV.
This work was supported by RFBR Grant No 14-04-32250.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P80
POSTE R A BSTRAC TS
SPA3P09
Antibody response against unchanged influenza vaccine components in
elderly people vaccinated in consecutive years
Basileo, Michela (1); Iorio, Anna Maria (1); Bartolini, Guido (2); Tozzi, Paolo (3); Camilloni, Barbara (1)
1: Dept. Exp. Med. University of Perugia, Italy; 2: Opera Pia B. Castori, Foligno, Italy; 3: Azienda USL2, Foligno, Italy
Background
Annual influenza vaccination is recommended for high risk groups because of the frequent mutations of influenza
viruses and the rapid decline of vaccine induced antibody titers. Field studies demonstrated that annually repeated
vaccination is efficacious in reducing morbidity and mortality. Serologic studies, however, showed divergent results
on the induction of protective antibodies after annually repeated vaccination and a decline of immune antibody
response was observed especially in the years when vaccine strains did not change. To further assess the effect of
annual vaccination we studied, over 16 consecutive seasons, from 1998/99 to 2013/14, the haemagglutination
inhibiting (HI) antibody response of elderly institutionalized yearly vaccinated volunteers against vaccine antigens
which for sequential years were not changed.
Methods.
The volunteers enrolled were elderly institutionalized people aged >60 years, annually vaccinated for influenza.
HI titers were determined in serum samples taken before and 1 month after vaccination by a standard microtiter
method. Differences in HI titers were analyzed by Student’s t-test for continuous statistics, and by chi-square test
for qualitative statistics.
Results
HI antibody responses were studied in 6 cohorts of volunteers against vaccine antigens not changed in consecutive
years. Responses against A/H1N1 antigens were examined in 16 subjects against A/New Caledonia/20/99 for 7
consecutive years (from 1998/99 to 2006/07), and in 36 subjects against A/California/7/09 for 4 years (from 2010/11
to 2013/14). Against A/H3N2 antigens 38 subjects were examined against A/Moscow/10/99 for 4 years (from
2000/01 to 2003/04). Responses for B antigens were studied in 51 subjects against B/Beijing/184/93 for 3 years
(from 1998/99 to 2000/01), and in 41 subjects against B/Brisbane/60/08 for 3 years (from 2009/10 to 2011/12).
The pre-vaccination HI antibody titers found after the first year of administration tended to be higher for A/H3N2
and B/Brisbane antigens and similar or slightly lower for the two A/H1N1 and for B/Beijing antigens. The postvaccination values observed in the sequential years were in general similar for B/Beijing, slightly lower for the two
A/H1N1 antigens and higher for B/Brisbane and A/H3N2 strains. The vaccine-induced HI antibody response against
the different antigens studied was substantially positive, comparing pre- and post-vaccination sera, the increases in
HI antibody titers resulted in most instances statistically significant. The requirements of the European Commission
were satisfied more frequently in the early years of administration with the exception for the B antigens.
Conclusions
The results obtained confirm the complexity of antibody induction in sequential annual influenza vaccination.
Although we cannot exclude other mechanisms, our data seem to support the possibility of an impairment of
antibody response against unaltered influenza A/H1N1 antigens that have circulated for prolonged periods whereas
the responses against the A/H3N2 and B vaccine strains, more frequently changed, tended to be similar or higher.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P81
POSTE R A BSTRAC TS
SPA3P10
Searching for a universal biomarker of human T-cell responses to
Influenza A virus infections
Dembinski, Jennifer Lynn (1,3); Oftung, Fredrik (1,3); Savic, Miloje (1,3); Kim, Yohan (2); Peters, Bjoern (2); Mjaaland, Siri (1,3)
1: Department of Bacteriology and Infection Immunology, Norwegian Institute of Public Health, Oslo, Norway; 2: La Jolla Institute for Allergy
and Immunology, La Jolla, CA, USA; 3: KG Jebsen Centre for Influenza Vaccine Research, Oslo, Norway
Background
The currently used split and subunit influenza vaccines induce antibody mediated immunity effective in protecting
against homologous influenza strains, but do not protect well against heterologous strains. The lack of robust cell
mediated responses is due to the lack of conserved epitopes in the vaccine. We aim to optimize influenza vaccination
and pandemic preparedness strategies by identifying conserved T-cell epitopes which have broad cross reactivity
against a range of seasonal and pandemic strains. The goal of the study was to develop a set of peptide defined
epitopes to be used as an efficient tool for monitoring Influenza A T-cell responses in the context of universal vaccine
development.
Methods.
Both MHC class I and class II restricted T-cell responses are based on the recognition of small peptide fragments
after antigen processing. Using this as a basis for prediction, we have defined a set of synthetic peptides using the
Immune Epitope Database (www.iedb.org). Scoring of the peptide candidates was calculated using parameters such
as epitope conservancy, prevalence, and HLA supertype coverage, and optimal sets of epitopes were defined by using
a greedy algorithm. A total of 33, 31, 14 or 5 peptides were selected which represent the general influenza CD4 or
CD8 epitope library or the 2009 pandemic specific CD4 or CD8 epitope library respectively. These libraries consist
of peptides which represent CD4 and CD8 epitopes from conserved (internal viral proteins) and variable (external
viral proteins) antigens. PBMCs were stimulated in vitro by the various peptide libraries to assess cellular immune
responses (cytokine expression) as measured by Elispot, multiplex and multiparameter flow cytometry.
Results
Utilizing available biobanks at the Norwegian Institute of Public Health, we evaluated cellular immune responses
from donors based on vaccination or infection history, and found that the peptides function well in detecting
both CD4 and CD8 responses in people with varying medical histories. When evaluating healthy donors, the CD4/
CD8 epitopes from the conserved antigens were more frequently recognized than the CD4/CD8 epitopes from
the variable antigens after stimulation by either the general or 2009 pandemic specific peptide libraries. We are
currently evaluating the peptides using PBMCs from donors based on previous vaccination and/or illness history in
those with confirmed seasonal or 2009 pandemic influenza infections.
Conclusions
By creating pooled peptide libraries, we are able to define more precise immune responses than from using whole
virus as antigenic stimulation. Peptide libraries may be a useful tool to monitor the efficacy of national vaccination
programs and clinical trials for the development of a universal influenza vaccine, as well as the immune status of
populations to predict pandemic preparedness. Taken together, we are optimistic that a universal influenza vaccine
is an achievable goal.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P82
POSTE R A BSTRAC TS
SPA3P11
LPS-modified outer membrane vesicle (fmOMV) adjuvant provides
protective innate immunity against influenza infection prior to the
induction of vaccine-induced adaptive immune response
Bae, Eun Hye; Lee, Tae Young; Lee, Sang Ho; Yeom, Min Joo; Na, Woon Seong; Kim, Chang Ung; Song, Dae Sub; Kim, Doo Jin; Kim, Sang Hyun
Korea research institute of bioscience and biotechnology, Korea, Republic of (South Korea)
Background
Outer membrane (OM) vesicles, naturally produced by Gram-negative bacteria, have important roles in bacterial
pathogenesis. It is also well known that OM vesicles can stimulate the host innate immune system through the
activation of toll like receptor (TLR) signaling pathways. Previous studies have revealed that TLR agonists can trigger
the innate immune response in the lung and protect against influenza infection. Therefore, it is shown to be a good
strategy to limiting the replication of influenza infection by innate immune response.
Methods.
C57BL/6 mice were intranasally administrated fmOMV(10ug) at various times (3, 7 or 14 days). Pre- treated mice
were then infected with 10MLD50 influenza A virus strain A/California/04/2009 (CA/04, H1N1 subtype) virus or
A/Puerto Rico/8/34 (PR8, H1N1 subtype) virus by aerosol and monitored for survival. Lung tissues were collected
to determine virus titers and to analyses immune cellular responses using multiparameter flow cytometry. The
cytokine and chemokine analysis were measured by the multiplex cytokine assay.
Results
In this study, we investigated the immunostimulatory effect of LPS-modified OM vesicle (fmOMV) in mice, as
well as the efficacy of fmOMV as an antiviral agent against influenza virus infection. Intranasal administration of
fmOMV dramatically enhanced the survival rate against lethal influenza A virus challenge. The protective immunity
maintained for a week. More importantly, pre-treatment of fmOMV conferred broad protection against different
influenza virus strains, while the vaccination provided only strain-specific immunity. fmOMV also significantly
increased the level of proinflammatory cytokines such as IL-6, RANTES, IL-1β, and TNF-α in the airway lumen, and
the neutrophil recruitment into the lungs.
Conclusions
These data suggest that fmOMV induces robust activation of innate immunity in the lungs and can be used as an
effective antiviral agent against influenza viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P83
POSTE R A BSTRAC TS
SPB3: MATHEMATICAL MODELING
SPB3P01
Mortality Associated with Seasonal and Pandemic Influenza among
Pregnant and Non-Pregnant Women of Childbearing Age in a High HIVPrevalence Setting – South Africa, 1999-2009
Tempia, Stefano (1,2,3); Walaza, Sibongile (3); Cohen, Adam (1,2); von Mollendorf, Claire (3); Moyes, Jocelyn (3,4); Mhlanga, Sarona (3);
McAnerney, Johanna (3); Cohen, Cheryl (3,4)
1: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America; 2: Influenza Division, Centers for
Disease Control and Prevention, Pretoria, South Africa; 3: Center for Respiratory Diseases and Meningitis, National Institute for Communicable
Diseases of the National Health Laboratory Service, Johannesburg, South Africa; 4: Faculty of Health Sciences, University of the Witwatersrand,
School of Public Health, Johannesburg, South Africa
Background
Information on the mortality burden associated with seasonal and pandemic influenza virus infection among
pregnant women is scanty in most settings, particularly in sub-Saharan Africa where pregnancy rates and HIV
prevalence are elevated.
Methods
We estimated the seasonal and A(H1N1)pdm09 influenza-associated mortality among pregnant and non-pregnant
women of childbearing age (15-49 years of age) from 1999 through 2009 in South Africa, using national vital
statistics data coded according to the International Classification of Diseases, Tenth Revision, adjusting for maternal
deaths underreporting. We modeled the excess mortality attributable to influenza infection by applying Poisson
regression models to monthly all-cause deaths among pregnant and non-pregnant women, using national influenza
laboratory surveillance data as covariates. Since the diagnosis of AIDS is rarely coded on the death certificate, we
indirectly estimated the annual influenza-associated deaths among HIV- infected and -uninfected pregnant and
non-pregnant women using Poisson regression models that incorporated year- and pregnancy status-specific HIV
prevalence and highly active antiretroviral treatment coverage as covariates. Mortality rates were expressed per
100,000 person-years. Because of the important differences in the age distribution of pregnant and non-pregnant
women of childbearing age, we used indirect standardization (using non-pregnant women as the standard
population) to compare the influenza- associated mortality rates between pregnant and non-pregnant women.
Results
During 1999-2009 the mean annual seasonal influenza-associated mortality rates were 12.6 (123 deaths) and 7.3
(914 deaths) among pregnant and non-pregnant women, respectively. Among pregnant women the mean annual
seasonal influenza-associated mortality rates were 74.9 (109 deaths) and 1.5 (14 deaths) among HIV-infected and –
uninfected individuals, respectively. Among non-pregnant women the mean annual seasonal influenza-associated
mortality rate was 41.2 (824 deaths) among HIV-infected and 0.9 (90 deaths) among HIV–uninfected individuals.
Pregnant women experienced an increased risk of seasonal influenza-associated mortality compared to nonpregnant women (HIV-adjusted and age-standardized relative risk [RR]: 2.8; 95% confidence intervals [CI]: 2.1-3.7). In
2009 the influenza A(H1N1)pdm09-associated mortality rates were 19.3 (181 deaths) and 9.4 (1189 deaths) among
pregnant and non-pregnant women, respectively (age-standardized RR for influenza A(H1N1)pdm09-associated
mortality due to pregnancy: 3.2; 95% CI: 2.3-4.1).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P84
POSTE R A BSTRAC TS
Conclusions
Among women of childbearing age, the majority of seasonal influenza-associated deaths occurred among HIVinfected individuals. Pregnant women experienced an increased risk of death associated with seasonal and
pandemic influenza infection compared to non-pregnant women. While the number of influenza A(H1N1)pdm09associated deaths was higher compared to seasonal influenza the relative risk of influenza-associated mortality
because of pregnancy was similar for seasonal and pandemic influenza. This suggests that the elevated number of
influenza A(H1N1)pdm09-associated deaths observed among pregnant women in 2009 may be due to the observed
heavy burden of the pandemic virus among adolescents and young adults, the age group where the majority of
pregnancies occur.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P85
POSTE R A BSTRAC TS
SPB3P02
The relationship between school holidays and transmission of influenza
in England and Wales 1967-2008
Jackson, Charlotte (1,2,3); Vynnycky, Emilia (1,2); Mangtani, Punam (1)
1: Public Health England, United Kingdom; 2: LSHTM; 3: UCL
Background
School closures is often considered as a possible intervention during influenza outbreaks, but its effects on
transmission are poorly understood, partly because data on how school closures affect contact patterns are limited.
Previous modelling studies have provided insight into the relationship between the school calendar and transmission
of influenza in settings such as France and The Netherlands, but it is unclear whether similar relationships exist
elsewhere.
Methods
An age-structured susceptible-infectious-recovered (SIR) model was fitted to GP consultation data for influenzalike illness, amongst 0-14 year-olds and ≥15 year-olds, in England and Wales, 1967-2008. Each influenza season,
defined to run from week 40 of one year to week 39 of the next, was analysed separately. Estimated parameters
for each season included the contact parameters (the per capita rates of effective contact within and between age
groups) and the relative change in the contact parameter representing contact between 0-14 year-olds comparing
school holidays to termtime. 95% confidence intervals were calculated as the central 95% of estimates obtained
by fitting the model to 1000 bootstrap datasets generated with weekly case numbers sampled from a Poisson
distribution with mean equal to the reported number for that week. The infectious period was assumed to be 3.5
days. In sensitivity analyses, we explored the effect of assuming that the infectious period was 2 or 4 days. Estimates
of the relative difference in the contact parameter during holidays compared to termtime were compared, using
the correlation coefficient, to those obtained from a simple mass action model which estimated weekly contact
parameters directly from the data for 5-14 year-olds. The agreement between the two estimates in the sign of the
change in the contact parameter was assessed using the kappa statistic.
Results
The estimated percentage difference in the contact parameter amongst 0-14 year-olds during holidays compared
to termtime ranged from a reduction of 100% (95% CI 88-100%) to an increase of 41% (95% CI 17% decrease to
50% increase). In >50% of the years considered, the contact parameter was estimated to be lower during holidays
than during termtime, with a 95% CI excluding zero. These estimates were insensitive to the assumed duration
of infectiousness. The correlation coefficient between the estimated difference in the contact parameter during
holidays compared to termtime from the age-structured model and that from the simple mass action model was
0.47 (95% CI 0.16-0.70). The kappa statistic for the agreement between the signs of the estimates was 0.44 (p=0.005).
Conclusions
School holidays can be associated with reductions in transmission of influenza amongst school-aged children in
England and Wales, although this was not consistent. School closure may reduce transmission during an influenza
pandemic. Reasons for the variability in the yearly estimates should be investigated.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P86
POSTE R A BSTRAC TS
SPB3P03
A drug-disease model to investigate the effect of combination therapy
with oseltamivir on influenza virus progression
Kamal, Mohamed A (1); Gieschke, Ronald (2); Lemenuel-Diot, Annabelle (2); Beauchemin, Catherine AA (3); Smith, Patrick F (4,5); Rayner, Craig R
(4,6)
1: Department of Clinical Pharmacology, Roche Translational Clinical Research Center Inc., New York, NY, USA; 2: F.Hoffmann-La Roche Ltd,
Basel, Switzerland; 3: Department of Physics, Ryerson University, Toronto, Canada; 4: d3 Medicine LLC, Parsippany, NJ, USA; 5: School of
Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY, USA; 6: Monash Institute of Pharmaceutical Sciences, Melbourne,
Australia
Background
A population drug-disease model was developed to describe the time course of influenza virus with and without
oseltamivir treatment.
Methods
Data included viral titres from 219 subjects across four studies who received placebo and oseltamivir 20–200 mg
twice daily for 5 days. A three compartment mathematical model consisting of target cells infected at rate β, free
virus produced at rate p and cleared at rate c, and infected cells cleared at rate δ was implemented in NONMEM with
an inhibitory Hill function on virus production (p) accounting for the effect of oseltamivir (Figure 1).
Results
In congruence with clinical data, the model predicts that the standard 75 mg regimen initiated 2 days after infection
decreased the duration of viral shedding by 1.5 days compared with placebo; the 150 mg regimen decreased
shedding by an additional 0.25 days. The model also predicts that initiating oseltamivir therapy more rapidly postinfection, specifically at Day 0.5 or 1, results in proportionally greater decreases in duration of viral shedding – 5
and 3.5 days, respectively (Figure 2). Furthermore, the model suggests that combining oseltamivir (acting to subdue
virus production rate) with an antiviral whose activity decreases viral infectivity (β) results in a moderate additive
effect dependent on therapy initiation time (Figure 2). In contrast, the combination of oseltamivir with an antiviral
whose activity increases viral clearance (c) shows significant additive effects independent of therapy initiation time.
Conclusions
The utility of the model for investigating pharmacodynamic effects of novel antivirals alone or in combination on
emergent influenza strains warrants further investigation.
Figure 1. Simple mechanistic model describing influenza virus progression.
Figure 1. Simple mechanistic model describing influenza virus progression.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P87
Figure 2. Simulations predicting the combined in vivo pharmacologic effect on influenza
POSTE R A BSTRAC TS
viral dynamics with standard 75 mg twice daily oseltamivir therapy of decreasing viral
production, p, decreasing infection rate, β, increasing viral clearance rate, c, and
Figure 2. Simulations predicting the combined in vivo pharmacologic effect on influenza viral dynamics with standard
rate δ of infected
cells.
Treatment started
at 0.5, 1, 2 and
days
75increasing
mg twiceclearance
daily oseltamivir
therapy
of decreasing
viral production,
p, 3decreasing
infection rate, β, increasing viral
clearance
rate, Solid
c, and
increasing
rate δ of effect
infected
cells. Treatment
after infection.
lines
depict theclearance
single pharmacologic
of oseltamivir
75 mg started at 0.5, 1, 2 and 3 days after
infection. Solid lines depict the single pharmacologic effect of oseltamivir 75 mg twice daily for 5 days. Dashed lines
twice daily for 5 days. Dashed lines show combined pharmacologic effects. All
show combined pharmacologic effects. All secondary effects were changed 10-fold from baseline.
secondary effects were changed 10-fold from baseline.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P88
POSTE R A BSTRAC TS
SPB3P04
Dynamic modeling of the cost-effectiveness of sick leave practices for
influenza illness
Edwards, Christina Hansen (1); de Blasio, Birgitte Freiesleben (1,2); Scalia Tomba, Gianpaolo (3)
1: Norwegian Institute of Public Health, Norway; 2: Dept. of Biostatistics University of Oslo, Norway; 3: Dept. of Mathematics, University of
Rome, “Tor Vergata”, Italy
Background
Sick leave from work can contribute to reduce the number of cases of influenza in workplaces and in society. During
the 2009 influenza pandemic, recommendations on sickness absence were issued in several European countries,
but the net economic benefit of different recommendations has to our knowledge not been studied. We tested
the effectiveness of interventions promoting early withdrawal from work after symptom onset, and/or higher
proportion of work absence during illness (compliance) on reducing the health and economic burden of influenza in
a realistic simulation model of influenza spread based on real interview data.
Methods
We developed an age-structured SEIR compartmental model representing both seasonal (R_eff = 1.1-1.3) and
pandemic (R_0=1.2-1.6) influenza transmission. We calibrated the model to Norwegian data on work absence
during ILI illness and country- specific estimates of mixing in work places, schools, general population and at home.
Different scenarios were considered by varying i) level of compliance (65-90%), ii) delay in compliance following
symptoms (0.5-2 days), iii) proportion of symptomatic adults (25%-55%) and iv) transmissibility. The analysis was
performed from a societal perspective and the most effective was computed using a net benefit approach.
Results
Under base case assumptions (65% compliance, fully effective from day 4 after symptoms), the clinical attack rates
(AR) ranged from 0.7-13.2% in seasonal epidemics to 4.3-29.5% in pandemics. The number of avoided cases was
found to increase with the transmissibility. In a pandemic, changing sick leave policies could reduce the relative
AR by 1-42%; the relative reductions were found to decrease with higher R_0. Similar findings were obtained with
seasonal epidemics. The most effective strategy was 90% withdrawal to the home 0.5 days following symptom
onset. The least effective strategy was withdrawal after 2 days of symptom onset. With regard to the economic
consequences of the various interventions, the most central cost driver was productivity losses. The relative cost per
case avoided tended to increase with R_0 or R_eff. While final results on the net benefit of the various interventions
are in preparation, preliminary analyses indicate an economic benefit ranging from €180 to €390 per avoided case.
The economic benefit to society of the strategy yielding the greatest number of avoided cases was estimated to be
€22 million (in Norway).
Conclusions
Sick leave practices can have a substantial impact on the health and economic consequences of influenza. Improving
current recommendations can reduce the health and economic burden of influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P89
POSTE R A BSTRAC TS
SPA4: ANTIVIRALS AND RESISTANCE
SPA4P01
Evaluation of heterosubtypic cross-protection by active infection and
TIV-vaccination of human seasonal influenza virus in ferret models
Choi, Young-Ki
Chungbuk National University, Korea, Republic of (South Korea)
Background
Since 1997, highly pathogenic avian influenza (HPAI) H5N1 viruses circulated among domestic poultry and wild birds
in Asia, Africa and Europe and believed to have undergone genetic evolution. Moreover, World Health Organization
(WHO) reported a mortality rate of 59% out of the 630 human cases of HPAI H5N1 infections worldwide from 2003
to June 2013. There is, therefore, a growing concern that HPAI H5N1 viruses might cause the next pandemic. Here,
we investigated the long term heterosubtypic cross-protection by natural infection, or immunization with trivalent
human seasonal inactivated vaccine (TIV) followed by fatal HPAI A/Vietnam /1203/04 (H5N1) virus challenge in
ferret models.
Methods
Groups (n=6) of ferrets were inoculated intranasally with human seasonal A/H1N1 (A/California/07/2009, group A)
or A/H3N2 (A/Perth/16/2009, group B) virus for imitating natural infection, and additional ferrets were immunized
with seasonal TIV (group C) three times with two-week interval. Three months after infection or last immunization,
antibody responses were measured by hemagglutination inhibition (HI) and virus neutralization (VN) assays. After
serological assays, group A, B, C and non-treated ferrets (group D) were challenged with lethal dose of HPAI A/
Vietnam/1203/04(H5N1) virus, and evaluated the viral shedding from nasal washes, tissue samples and their
mortality.
Results
Serological assays revealed that the sera from groups A, B and C could elicit moderate titers against homologous
viruses at 4 weeks after natural infection or TIV immunization; cross reactivity against the HPAI H5N1 virus was not
detected. However, these serologic titers appeared to have receded after 12 weeks post- treatments (infection or
immunization). Following viral challenge with HPAI H5N1 virus, the ferrets from group A and B survived but those
from group C and D all died. Group A and B exhibited significantly attenuated viral loads in nasal washes, lung, brain
and spleen and showed recovery within 9 days post-infection. Notably, group A showed the most reduction of HPAI
H5N1 virus replication and attenuated clinical signs. In contrast, the ferrets in groups C and D demonstrated high
fever with substantial weight loss accompanied by high replication of the challenge virus at the upper respiratory
tract and tissues samples. Therefore immunization with the human seasonal TIV could not protect ferrets from fatal
HPAI H5N1 infection.
Conclusions
Taken together, our findings revealed that natural infection with human seasonal strains are more effective than
TIV immunization against fatal HPAI H5N1 infection. Data also suggest that natural virus exposure might provide a
long-term protective impact against the A/Vietnam/1203/04(H5N1) challenge virus. Thus, the inactivated human
seasonal influenza vaccine might not protect the vaccinated host, while healthy individuals who were previously
infected with seasonal influenza A viruses might be more protected from the lethal HPAI H5N1 virus infection.
Keywords: influenza vaccine, natural infection, seasonal influenza virus, HPAI H5N1
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P90
POSTE R A BSTRAC TS
SPA4P02
Oseltamivir inhibits influenza virus replication and transmission
following ocular-only aerosol inoculation of ferrets
Belser, Jessica A; Maines, Taronna R; Creager, Hannah M; Katz, Jacqueline M; Tumpey, Terrence M
Centers for Disease Control and Prevention, United States of America
Background
Neuraminidase inhibitors are frequently employed to limit influenza virus infection and spread following respiratory
exposure. However, ocular exposure to influenza virus can also lead to the establishment of a respiratory infection
in mammals. Despite a paucity of experimental evidence examining the efficacy of antiviral treatments following
non-respiratory exposure routes, oseltamivir prophylaxis has nonetheless been prescribed during outbreaks in
humans where conjunctivitis and/or ocular exposure has been reported. There is a need to understand the capacity
of currently available antiviral treatments to inhibit virus spread from ocular sites to the respiratory tract. Here,
we used a novel method of ocular-only aerosol (OA) inoculation of ferrets to investigate the mechanism of action
of the neuraminidase inhibitor oseltamivir in preventing influenza virus infectivity, disease progression, and
transmissibility following ocular exposure to the virus.
Methods
Influenza A viruses of the H5N1, H1N1, H7N3, and H7N9 subtypes were used in this study. OA inoculations were
performed by passing aerosolized virus through aerosol-delivery goggles fitted on sedated ferrets, which form a close
seal around each eye to prevent respiratory exposure to virus. Ferrets received placebo or 25 mg/kg of body weight/
day oseltamivir given orally twice daily from 2 hrs post-inoculation (p.i.) through day 5 p.i. Ferrets were observed
for clinical signs and symptoms, and nasal washes were collected to measure viral replication. Transmission was
assessed by placing naïve ferrets in direct contact with inoculated animals.
Results
All influenza viruses tested were capable of mounting a productive respiratory infection following OA exposure.
Oseltamivir administration following OA inoculation with H5N1 virus protected ferrets from a fatal and systemic
infection, with all antiviral- treated ferrets surviving the virus exposure. Ferrets inoculated by the OA route with a
2009 H1N1 pandemic virus, a HPAI H7N3 virus, or a LPAI H7N9 virus and treated with oseltamivir shed reduced levels
of virus in nasal washes with delayed kinetics compared with ferrets receiving placebo. Transmission of these viruses
in a ferret direct contact model was similarly reduced and delayed compared with the efficient transmission of these
viruses observed in the absence of antiviral treatment. Comparable results were observed when ferrets received a
joint ocular-respiratory exposure of aerosolized virus.
Conclusions
We demonstrate that oseltamivir treatment effectively limits the establishment of a respiratory infection in
ferrets exposed to aerosolized influenza virus by the ocular-only route, and reduces the transmission of viruses of
multiple subtypes to naïve contacts when a respiratory infection is ultimately mounted. These findings provide
critical experimental evidence in support of the use of neuraminidase inhibitors during outbreaks of influenza virus
resulting in ocular disease or ocular exposure.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P91
POSTE R A BSTRAC TS
SPA4P03
Oseltamivir resistant and wild type pH1N1 virus infection in ferrets
Vidana, Beatriz (1,3); Martorell, Jaime (2); Martínez, Jorge (1,3); Martínez, Pamela (3); G. Migura, Lourdes (3); córdoba, Lorena (3); Casas,
Inmaculada (5); Pozo, Francisco (5); Majó, Natàlia (1,3); Montoya, María (3,4)
Centre de Recerca en Sanitat Animal (CReSA), Spain; 2: Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona,
Spain; 3: Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Spain; 4: Institut de Recerca i Tecnologia
Agroalimentaria (IRTA), Barcelona, Spain; 5: Instituto de Salud Carlos III (ISCIII), Madrid, Spain
Background
Oseltamivir is a common therapy against IAV infections. It is believed that acquisition of oseltamivir resistance
(OsR) would decrease viral virulence. However, OsR pH1N1 viruses have demonstrated a potent ability to spread
throughout different populations. The objective of this work was comparing virulence of an OsR and a wild type (Wt)
pH1N1 virus, both causing fatal outcome in human patients.
Methods
Ferrets were divided in two groups; infected with OsR pH1N1 virus A/Baleares/RR6121/2009 (R6), isolated from
a young man with leukaemia, and Wt pH1N1 virus A/CastillaLaMancha/RR5911/2009 (F) isolated from a healthy
young woman. Clinical signs were daily recorded. Serum acute phase proteins were measured by ELISA at 1, 3, 6 and
10 dpi. Lung histopathology, viral quantification in the lungs and lung immunogenetic responses were measured
by immunohistochemistry (IHC) and RT-qPCR at 1, 3 and 10 dpi. Viral loads in nasal swabs were also determined in
PFUs at 1, 3 and 10 dpi and serum antibody response against both viral strains was performed by hemagglutination
inhibition assay (HI).
Results
Ferrets infected with F virus showed higher percentage of weight loss comparing with R6 infected animals still
4 dpi. R6 infected animals also showed higher clinical score still 7 dpi. However no differences were observed in
temperature variation between viral groups. F infected animals showed more severe histopathologic lesions at 1
and 3 dpi. Viral quantification by RT-qPCR was higher in the lungs of F virus infected animals at 1 but not at 3
dpi. Viral load in nasal swabs was also higher in F infected animals at 3 dpi. R6 infected animals showed higher
expression of IFNβ, TLR3, IL-6, CXCL10 and RIG-I at 1 dpi. However, F infected animals showed higher expression
of proinflamamtory cytokines such as CXCL10, IL-6, TNFα, IFNγ, and IL-8 at 3 dpi. Both infected animals showed
antibody cross- reactivity against both viral strains, further confirming similarities in the Hemagglutinin protein.
Conclusions
Ferrets infected with Wt pH1N1 virus (F) exhibited a stronger virulence than the OsR virus (R6). However, OsR R6
virus showed similar viral RNA expression in lungs at 3 dpi and a similar increment in temperature. In summary, even
when Wt pH1N1 virus exhibited increased fitness and pathogenicity, OsR R6 virus was also able to produce illness
with viral shedding and an inflammatory response.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P92
POSTE R A BSTRAC TS
SPA4P04
Is it possible to fight influenza by targeting intracellular redox state?
Palamara, Anna Teresa (1,2); Sgarbanti, Rossella (3); Amatore, Donatella (1,4); Celestino, Ignacio (1,4); Fraternale, Alessandra (5); Magnani,
Mauro (5); Garaci, Enrico (3); Nencioni, Lucia (1)
1: Dept. of Public Health and Infectious Diseases, Institute Pasteur, Cenci-Bolognetti Foundation, ‘‘Sapienza’’ University of Rome, Rome, Italy;
2: IRCCS San Raffaele Pisana Rome, Italy; 3: Telematic University San Raffaele Rome, Italy; 4: CEINGE Advanced Biotechnology, Naples, Italy; 5:
Dept. of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
Background
We have recently shown that influenza A virus replication is inhibited by treatment of infected cells or mice with a
derivative of GSH, GSH-C4. In particular, GSH-C4 is able to impair hemagglutinin (HA) maturation in the endoplasmic
reticulum and, as a consequence, to block the progression of virus replicative cycle. Interestingly, pro-GSH molecules,
including GSH-C4, are able to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the
immune response towards Th1 which plays a pivotal role in antiviral immunity.
On the basis of this background, our study was aimed at evaluating the effect of different redox modulating
compounds other than GSH-derivative, on influenza virus replication in lung epithelial cells and/or on virus-induced
cytokine production.
Materials and Methods
For in vitro study, human mucoepidermoid pulmonary carcinoma cells were infected with human and avian
influenza A viruses (A/Puerto Rico/8/34 H1N1; A/ULSTER/H7N1) and treated after viral adsorption with redox
modulating compounds such as GSH-C4, N-acetyl cysteine (NAC) and Trolox. After different hours of infection, viral
proteins, the intracellular redox state (ROS production) and mRNA of cytokines were evaluated through Western
blot, FACS analysis and real-time PCR, respectively. Viral titer was measured through hemagglutination and plaque
assays. For in vivo studies, Balb/C aged (13 months) mice were infected with mouse-adapted influenza virus PR8 and
treated daily with GSH-C4 for 7 days. Bronchoalveolar lavage fluid (BALF) was used for cytokine measure by means
of Bioplex multiplex system.
Results
Among different redox modulating compounds tested, differently from GSH-C4, N-acetyl cysteine (a GSH precursor)
and Trolox (a derivative of Vitamin E with ROS scavenging activity) exhibited short-term anti-influenza activity in
infected lung epithelial cells, without interfering with viral protein expression or HA folding. In addition, while
GSH-C4 was able to decrease the production of TNF-α induced by influenza virus, NAC and Trolox exacerbated the
release of this cytokine, suggesting that different redox-regulated pathways (GSH-mediated or ROS-mediated)
may be specifically involved in the regulation of virus life cycle and/or inflammatory responses. Moreover, in in vivo
studies GSH-C4 administration was able to induce a shift towards a Th1 response in infected aged mice.
Conclusions
Overall these results indicate that GSH-C4 is effective in both inhibiting influenza virus replication and blocking the
cytokine production responsible for the lethality of some viral strains. Moreover, the fact that GSH-C4 modulates
the immune response may be considered as an antiviral protective compound in aged people. Our data also
demonstrate that not all the so-called “antioxidant drugs” are able to control viral infection. Thus, the efficacy of
a specific compound depends on its real ability to affect redox-regulated pathways in different cellular contexts.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P93
POSTE R A BSTRAC TS
SPA4P05
Neuraminidase Inhibitors and the Cochrane Reviews – a critical appraisal
Lehnert, Regine Magdalene; Matz, Sibylle Renate
Federal Institute for Drugs and Medical Devices, Germany
Background
Shortly after the marketing authorisation of the two neuraminidase inhibitors (NI) zanamivir and oseltamivir,
the Cochrane Collaboration published their first meta‐analysis on NI use in prevention and therapy of influenza
infection in otherwise healthy adults. Subsequently this meta‐analysis underwent several revisions with the latest
review published in April 2014. Each revision of the review generated enormous media and public interest. This is
to describe the evolution of the Cochrane reviews from a methodological perspective and also in terms of contents.
The results are compared with the past and current licensing status of the neuraminidase inhibitors in Europe and
the USA.
Materials and Methods
All Cochrane Reviews on NI use in otherwise healthy adults were screened; those containing major revisions were
analyzed further. Altogether, five reviews were selected, the initial meta‐analysis from 1999 and the revisions
dated 2006 (Issue III), 2010, 2012 and 2014. The evolution of the main methodological features as well as the most
relevant results of the reviews are described and compared with the product information for the NIs, as approved by
the European Medicines Agency (EMA) and US Food and Drug Administration (FDA).
Results
Over time the results and conclusions of the reviews have changed, particularly with regard to the potential effects
of neuraminidase inhibitors on the reduction of influenza related complications. After the 2006 review no new
evidence has become available that could have led to the differing results. Instead, this evolution appears to be
primarily based on the authors’ decision to include or exclude certain data sources and to run increasing numbers
of different analyses on a variety of endpoints. At no time has there been a contradiction between the conclusions
of the Cochrane authors and the assessment and licensing status of the neuraminidase inhibitors in Europe or the
USA.
Conclusions
From the perspective of drug regulation these results are reassuring. However, the reasons for the great media
interest in each revision of this Cochrane review are not fully understood and could benefit from further exploration.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P94
POSTE R A BSTRAC TS
SPA4P06
Antiviral drugs for influenza -A comprehensive review of the evidence
with emphasis on relevant clinical endpoints
Lehnert, Regine Magdalene (1); Pletz, Mathias W (2); Schaberg, Tom (3); Reuss, Annicka (4); Haas, Walter (4)
1: Federal Institute for Drugs and Medical Devices, Germany; 2: Universitätsklinikum Jena; 3: Agaplesion Diakoniekrankenhaus, Rotenburg/
Wümme; 4: Robert Koch-Institut, Berlin
Background
In the past years and especially subsequent to the last pandemic in 2009/2010, ample clinical studies and meta‐
analyses on antivirals for influenza have become available. The objective of this review is to provide an overview
on the evidence on efficacy and safety of antiviral agents indicated for prevention and treatment of influenza in
Germany.
Methods
A comprehensive literature search of Cochrane Library, PubMed and Scopus was undertaken. We included all
available literature published before May 01 2014. As a starting point, we defined clinically relevant questions on the
use of antivirals. Data were regarded as appropriate evidence if derived from randomized controlled trials (RCTs) or
meta‐analyses thereof. Data were extracted primarily from meta‐analyses. If no meta‐analysis for certain research
questions was available, data from RCTs or observational studies were used. Data from observational studies were
considered as supportive, particularly in those areas where evidence from RCTs was missing. Titles, abstracts, and
full articles were screened by two experts. In case of disagreement a third reviewer was included as arbitrator.
AMSTAR criteria for the measurement of the methodological quality were applied where appropriate. Data from
these selected publications were extracted, summarized and assessed. In addition, information from regulatory
bodies, e.g. from public assessment reports/reviews was included for the safety evaluation.
Results
Ten meta‐analysis of high to very high quality according to AMSTAR were included. Overall, antivirals lead to a
shortening of symptoms of influenza of 0.5 to 1.0 days. For specific agents/in selected populations this effect was
more pronounced (e.g. for oseltamivir in children) or has not been proven (e.g. for elderly patients). Prophylactic use
reduced the frequency of symptomatic influenza by 60% to 80%. Effects on hospitalisations, serious complications,
mortality or other markers of disease severity have not been unequivocally demonstrated in RTCs. However, there
are observational studies that have shown statistically significant beneficial effects on severe disease and mortality.
Reduction in transmission by use of antivirals has not been proven. The side effects of neuraminidase inhibitors
are generally less pronounced than those of amantadine. Specifically, side effects predominantly occur in the
neuropsychiatric system (amantadine and possibly oseltamivir), respiratory system (zanamivir), gastrointestinal
system and skin (oseltamivir).
Conclusions
Despite the lack of evidence to answer some of the clinically relevant questions, the demonstrated effects and the
findings from observational studies do allow the conclusion of an overall positive benefit/risk ratio for the use of
antivirals against seasonal and pandemic influenza. In a pandemic situation these effects may become relevant
for the individual as well as for society, when antivirals may be used for prophylaxis and for therapy, particularly in
consideration of the lack of other options.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P95
POSTE R A BSTRAC TS
SPA4P07
Heterologous Polyclonal Antibodies, Past and Present, with Future
Applications to Avian Influenza and Other Neglected Viruses.
Dixit, Rashmi (1); Herbreteau, Cécile Hélène (2); Herz, Jenny (3); Booy, Robert (1); Lepine, Bertrand (2)
1: The Children’s Hospital, Westmead, Australia; 2: Fab’entech; 3: Bio-intellect
Background
Increasing neuraminidase inhibitor resistance, the emergence of pathogenic avian strains with pandemic potential,
and the lack of vaccines against novel strains each challenge influenza management. Polyclonal antibody therapy
has been applied to prophylaxis of rabies, and treatment of intoxications and envenomations. However, human
derived polyclonal antibodies production is limited. This review examines the historic of polyclonals use in human,
their future applications, and their potential application in both prophylaxis and treatment of influenza.
Methods
A literature review, published and grey, of the Medline database search was conducted, seeking original studies
and reviews on animal-derived polyclonal antibodies, their effectiveness and their safety, and current research into
potential applications.
Results
Serum therapy was first used in the 1890s against diphtheria. Its use expanded in the pre- antibiotic area to treat a
range of bacteria and viruses, including the 1918 Spanish Influenza pandemic. Crude preparation and purification
techniques yielded products contaminated with large amounts of animal proteins, associated with high rates of serum
sickness. With the development of antibiotics and high vaccine uptake, serum use contracted. However, enzymatic
digestion, and increased purification techniques such as ammonium sulphate precipitation, thermocaogulation
and chromatographic purification hugely raised their safety profile and allow the use of highly purified F(ab’)2
fragments for the management of intoxication or disease such as rabies, until today. The use of neutralising F(ab’)2
fragments, removed of the Fc fragment of the antibody (harbouring species specificity and responsible of mainly
of the adverse events) , allow a drastic reduction in hypersensitivity reaction. Modern animal derived polyclonal
antibody products are increasingly purified, very safe and effective. For seasonal and avian influenza, heterologous
polyclonal antibodies have been demonstrated to be effective in mouse models, and a recent phase I clinical trial
on Fabenflu® (anti-H5N1 F(ab’)2) gave reassuring safety data in human. Merely substituting one vaccine in horses
for a previous can enable rapid development of immunotherapy to emergent influenza strains. In the near future,
application of polyclonal serum therapy may be applied to a range of neglected viral diseases such as various viral
haemorrhagic fevers and bat-transmitted viruses.
Conclusions
Heterologous polyclonal antibody therapy is economical, safe, effective with large cross- reactivity activity and can be
rapidly produced, with application to many pathogens for which there are no efficient prophylactic and therapeutic
options. This mode of therapy may provide a viable strategy against pandemics of emergent influenza strains.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P96
POSTE R A BSTRAC TS
SPA4P08
Comparative analysis and anti-viral activity of medicinal plant extracts
against Influenza A virus: An in-vitro study.
Roy, Saugata; Saxena, Latika; Kumar, Binod; Khanna, Madhu
Vallabhbhai Patel Chest Institute, India
Background
Influenza virus is a respiratory pathogen of global importance which causes a high degree of morbidity and
mortality. Due to frequent antigenic and genetic changes, vaccines need to be formulated yearly thus requiring lot
of financial investment. Naturally derived products from medicinal plants have shown great potential in preventing
and or ameliorating diseases. This study aims at evaluating the antiviral efficacy of Trachyspermum ammi, Curcuma
longa and Solanum nigrum medicinal plant extracts, having expected antiviral activity for the development of an
alternative and effective therapy against Influenza A viruses.
Methods
Certified Plant materials were procured and extracts were prepared in ethanol. The extracts were dissolved in DMSO,
aliquoted and stored in -20°C. Cytotoxicity was checked by MTT assay after 24h and 48h in A549 cells. The Antiviral activity of the extract was assessed against Influenza A virus (A/PR/8/34-H1N1) infection by real time PCR.
Plaque reduction assay was performed in MDCK cells from cell supernatant of infected A549 cells treated with plant
extracts.
Results
A marked reduction in cytopathic effect caused by Influenza A Virus was observed post treatment Trachyspermum
ammi in A549 cells. A similar inhibition in viral RNA (56% approx) was observed by real time PCR when treated with
Trachyspermum ammi extracts whereas other plant extracts (Curcuma longa and Solanum nigrum) showed lesser
inhibition. Relative reduction of plaques was observed in Trachyspermum ammi treated cell supernatant of A549
cells.
Conclusions
Extract from Trachyspermum ammi showed potential inhibition of replication Influenza A virus in A549 cells.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P97
POSTE R A BSTRAC TS
SPA4P09
Viral shedding and susceptibility to oseltamivir in hospitalised
immunocompromised patients with influenza in the Influenza
Resistance Information Study (IRIS)
Fraaij, Pieter (1); Schutten, Martin (1); Javouhey, Etienne (2); Burleigh, Laura (3); Outlaw, Russell (4); Kumar, Deepali (5); Boucher, Charles (1)
1: Erasmus MC, Rotterdam, The Netherlands; 2: Hôpital Femme Mère Enfant, Lyon, France; 3: Roche Products Ltd, Welwyn, UK; 4: Micron
Research Ltd, Ely, UK; 5: Toronto General Hospital, Toronto, Canada
Background
Oseltamivir is widely used to treat influenza, especially in patients at risk of severe infection such as
immunocompromised individuals. Recent publications have raised concerns that influenza viruses may develop
resistance to oseltamivir in immunocompromised patients. As few prospective data are currently available on
this subject, the IRIS (NCT00884117) sub-study was initiated to assist the detection of resistance to antivirals in a
population of immunocompromised patients infected with influenza and describe their clinical outcomes.
Methods
This sub-study is a prospective, multicenter, information-gathering study. Between February 2012 and March 2013,
immunocompromised patients aged ≥1 year with a rapid diagnostic or PCR test positive for influenza up to 96 hours
after diagnosis, and displaying symptoms suggestive of influenza-like illness, were eligible for inclusion. The level of
immunosuppression was retrospectively scored on the likelihood of decreased viral clearance, ranging from 1 (very
unlikely) to 4 (highly likely). Clearance of virus was assessed by RT-PCR assay of viral RNA in nasal and throat swabs
taken on Day 1, then every 3 days until patients were virus-free. Patients were treated in accordance with local
routine clinical practice.
Results
A total of 42 patients were included in the study: 29 adults (age range, 22–83 years) and 13 children (aged 1–12 years).
The level of immunosuppression was mostly mild, with >80% of patients being scored as level 1 or 2. Twenty-nine
(69%) patients (mean age, 36 years) were influenza-positive by RT-PCR on Day 1, with H1N1pdm09 being the most
common infecting virus subtype (15/29 patients). No resistant viruses were detected at baseline. On Days 3 and 6,
the majority of patients tested remained influenza-positive (18/24 and 9/15, respectively). The H275Y mutation was
found in post-Day 1 samples from four adult patients (age range, 52–61 years); three patients cleared the virus, but
one severely immunocompromised patient was virus-positive until Day 15 when he died. Post-Day 9 samples were
available for five patients, of whom two continued to shed virus, including the aforementioned patient. Follow-up
beyond Day 9 was limited because symptoms resolved in most patients and they could then be discharged from
hospital, with no further follow-up required. Indeed, most patients only had mild symptoms; however, 10 patients
(seven influenza-positive) were admitted to the intensive care unit and three (two influenza-positive) died.
Conclusions
In this group of mostly mildly immunocompromised patients, emergence of oseltamivir-resistant viruses during
treatment was detected in four patients, but there was no clear association between resistance and prolonged
viral shedding. The clinical course of disease was benign in most patients; however, more serious disease was
encountered, including one fatal case in a patient with resistant virus and prolonged shedding.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P98
POSTE R A BSTRAC TS
SPA4P10
Novel inhibitors targeting influenza virus and pneumococcal
neuraminidase (NA)
Walther, Elisabeth (1); Richter, Martina (1); Xu, Zhongli (1); Bohn, Kathrin (1); Rollinger, Judith (2); von Grafenstein, Susannne (3); Liedl, Klaus
(3); Kirchmair, Johannes (4); Sauerbrei, Andreas (1); Pfister, Wolfgang (5); Schmidtke, Michaela (1)
1: Jena University Hospital, Department of Virology and Antiviral Therapy, Jena, Germany; 2: University of Innsbruck, Institute of
Pharmacognosy and Center for Molecular Biosciences, Innsbruck, Austria; 3: University of Innsbruck, Institute of Theoretical Chemistry and
Center for Molecular Biosciences, Innsbruck, Austria; 4: Swiss Federal Institute of Technology Zurich, Zurich, Switzerland; 5: Jena University
Hospital, Department of Medical Microbiology, Jena, Germany
Background
Secondary bacterial infections, in particular with Streptococcus pneumoniae, represent a major cause of excess
morbidity and mortality during influenza. Because of structural and functional similarities, pneumococcal NA
(NanA) can promote the release and spread of virus in a similar fashion than viral NA. Hence, bacterial NA needs to
be considered as valid therapeutic target for the treatment of flu infections. We identified a series of compounds
identified by virtual screening applying an ensemble-based molecular docking. The series shares a common
chemical scaffold with at least one acidic function and an aromatic core with diazenyl linker (here in referred to
as azo acid compounds). In the present study, we investigate these novel promising compounds with significant
inhibitory efficacy on influenza virus as well as pneumococci.
Methods
The susceptibility of a pneumococcal strain serotype 2 and a pandemic influenza A virus (A(H1N1)pdm09) and/
or their NA was studied against 37 azo acid compounds in human erythrocyte-based NA inhibition assays in
comparison to oseltamivir and zanamivir. Pneumococcal NA was obtained either as a component of bacterial
total surface protein via precipitation, or individually synthesized via in vitro expression system. By incubating
virus with in vitro-expressed NanA in A549 cells the dual-effect of NA inhibition was investigated in an in vitro
co-infection model. Here, the plaque reduction assay was used to analyze the compounds’ effect on virus yield and
immunohistochemical staining of viral nucleoprotein to evaluate the effect on virus spread.
Results
The susceptibility of NanA to oseltamivir as well as its resistance to zanamivir (Gut et al., J. Mol. Biol., 2011) was
confirmed. In comparison to viral NA, NanA was about 103-fold less sensitive to oseltamivir (IC50 of 0.002 μM and
2 μM, respectively). Eight and ten out of 37 tested compounds showed a mean inhibitory concentration equal or
lower than 10 μM against viral and bacterial NA, respectively. Four compounds active on both NAs were selected
for studying their inhibitory effect in an in vitro co-infection model. Here, NanA significantly increased viral yield
and spread. Zanamivir efficiently blocked viral replication in A549 cells. However, no effect was observed when
virus-infected A549 cells were co-incubated with in vitro-expressed NanA. In contrast, oseltamivir and the novel
compounds showed dose-dependent inhibition of the viral replication in the presence and absence of NanA.
Conclusions
Our results demonstrate that dual-active inhibitors targeting viral and pneumococcal NA prevent viral replication
in the presence and absence of pneumococcal NA. Therefore, this novel antimicrobial inhibition profile has the
potential to be optimized for the development of lead structures combating co-infected influenza.
This work is supported by the Austrian Science Fund (FWF: P24587 & P23051) and the Thuringian Ministry of
Economy, Labour and Technology funded by the European Social Fund (2011FGR0137).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P99
POSTE R A BSTRAC TS
SPA4P11
Five years of monitoring for the emergence of oseltamivir resistance in
patients with influenza A virus infections in the Influenza Resistance
Information Study (IRIS)
Lina, Bruno (1); Boucher, Charles (2); Osterhaus, Albert (2); Schutten, Martin (2); Monto, Arnold (3); Whitley, Richard J (4); Nguyen-Van-Tam,
Jonathan (5)
1: University Claude Bernard Lyon1, Lyon, France; 2: Erasmus MC, Rotterdam, The Netherlands; 3: University of Michigan School of Public
Health, Ann Arbor, MI, USA; 4: University of Alabama at Birmingham, Birmingham, AL, USA; 5: University of Nottingham, Nottingham, UK
Aim
Following naturally-occurring oseltamivir resistance in 2008, a global observational trial, the Influenza Resistance
Information Study (IRIS; NCT00884117) was initiated to study the emergence of neuraminidase inhibitor (NAI)
resistance and the clinical course of influenza in non-immunocompromised treated and untreated patients.
Methods
Patients with influenza-like illness and/or a positive rapid test result for influenza had throat/nose swabs collected
on Days 1, 3, 6 and 10 for real-time RT-PCR analyses of influenza type, subtype and NAI resistance. Positive samples
were cultured, sequenced (HA, NA and M2) and phenotypically tested for NAI resistance. Scores for influenza
symptoms (0 [absent], 1 [mild], 2 [moderate], 3 [severe]) were recorded on diary cards by the patient (Days 1–12).
Results
Of 3230 RT-PCR-positive patients with a single influenza strain infection in years 1 to 5, 2316 had influenza A of
whom 1216 received oseltamivir monotherapy within 2 days of symptom onset (9 seasonal H1N1; 662 H3N2; 545
have been detected.
the
followup,
11with
of the
43naturally
patients resistant
(25.6%) were
still RTPCR
H1N1pdm2009).
ExceptInfor
the
9 patients
the
seasonal
H1N1 positive
in 2008, no resistance has
been detected in the Day1 samples during the 5-year study. However, emergence of resistance (post-Day 1) was
at Day 10, but their symptoms resolved by Day 6. In 60% of the sample in which resistance first
detected by mutation-specific RT-PCR in 43 oseltamivir-treated influenza A patients (see Table), mostly with a mixed
genotype.
a limited
trend
of resistant viruses over the years (see Table). This was not correlated
appearedThere
viral was
loads
were too
lowof
forincrease
phenotyping.
to any sustained substitution in the NA or HA of the viruses. All resistant viruses had either the H275Y substitution
: In years 1–5 of IRIS, emergent resistance to oseltamivir in influenza A viruses was
for N1 viruses (27), or the R292K substitution for N2 viruses (16). No other substitutions associated to resistance
1detected
have beenonly
detected.
the
follow-up,
11 of the
43 patients
(25.6%)
were still RT-PCR
positive
during In
the
course
of antiviral
treatment,
mostly
in 1–5yearold
and with
mixedat Day 10, but their
symptoms resolved by Day 6. In 60% of the sample in which resistance first appeared viral loads were too low for
virus population. This post treatment emergence of resistance appears to be more frequent
phenotyping.
in the last 3 years. In the clinical management, this resistance had modest effect on viral
Conclusions
clearance
no effect
on symptom
resolution.
In
years 1–5and
of IRIS,
emergent
resistance
to oseltamivir in influenza A viruses was detected only during the course
of antiviral treatment, mostly in 1–5-year-old and with mixed virus population. This post treatment emergence of
resistance appears to be more frequent in the last 3 years. In the clinical management, this resistance had modest
effect
viral clearance
and no(mutationspecific
effect on symptomRTPCR)
resolution.
onEmergent
resistance
by age group and year in treated
patients with at least one postDay1 sample
Table. Emergent resistance (mutation-specific RT-PCR) by age group and year in treated patients with at least one
post-Day-1 sample
Resistance mutation
Age group
H275Y in H1N1 (2009)
2008-10
1–5 years
2010-11
1/48 (2,1%) 12/53 (22,6%)
Virus
Resistance
negativ e by rate (treated
R292K in H3N2
RTPCR at
influenza A
2011-12
2012-13
2008-10
2010-11
2011-12
2012-13
Day 10
patients)
1/5 (20%)
6/18 (33%)
0/1 (0%)
1/36 (2,8%)
2/16 (12,5%)
7/76 (9,2%)
24/30
30/253 (11,8%)
6–12 years
0/75 (0%)
1/30 ( 3,3%)
0/6 (0%)
1/4 (25%)
0/1 (0%)
0/42 (0%)
2/15 (13,3%)
1/76 (1,3%)
2/5
5/249 (2%)
≥13 years
1/95 (1,1%)
2/143 (1,4%)
0/15 (0%)
2/35 (5,7%)
0/20 (0%)
1/112 (0%)
0/66 (0%)
2/174 (1,2%)
6/8
8/660 (1.2%)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P100
POSTE R A BSTRAC TS
SPA4P12
Hemagglutination (HA)-based neuraminidase (NA) inhibitory assays to
search for novel NA inhibitors (NAIs)
Richter, Martina (1); Walther, Elisabeth (1); Bohn, Kathrin (1); Xu, Zhongli (1); Grienke, Ulrike (2); Rollinger, Judith M. (2); von Grafenstein,
Susanne (3); Liedl, Klaus R. (3); Kirchmair, Johannes (4); Sauerbrei, Andreas (1); Schmidtke, Michaela (1)
1: Jena University Hospital, Department of Virology and Antiviral Therapy, Jena, Germany; 2: Institute of Pharmacy/Pharmacognosy, Center
for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria; 3: Institute of General, Inorganic and Theoretical Chemistry,
Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria; 4: Department of Chemistry and Applied Biosciences,
Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
Background
Emergence and circulation of NAI-resistant influenza viruses underline the need to search for novel resistancebreaking NAIs. Flu-associated secondary bacterial pneumonia, most commonly caused by Streptococcus
pneumoniae, renders bacterial NA an additional druggable target. Chemiluminescence (CL)- and fluorescence (FL)based NA inhibitory assays can be applied to screen for NAIs. The reliability of results from both assays is under
discussion (Kongkamnerd et al., J Biomol Screen. 2011; Grienke et al., J Nat Prod. 2014). Due to quenching effects and
self- fluorescence, these assays can lead to false-positive or -negative results. The aim of this study was to establish
NA inhibition assays overcoming these drawbacks.
Methods
Inhibition of influenza A virus NA and bacterial NA (from S. pneumoniae, C. perfringens, V. cholerae) by well-known
(oseltamivir, zanamivir, DANA) and novel NAIs (isolated from medicinal plants or selected by structure similaritybased screening) was compared in (i) commercially available CL- and FL-based NA inhibitory assays and in (ii) HAbased NA inhibitory assays with human erythrocytes.
Results
When testing the approved NAIs, the CL-based NA inhibition assay reported higher sensitivity than its FL-based
counterpart. The results from both assays correlated well for viral as well as pneumococcal NA. In contrast to that,
the results markedly differed in these assays for some natural compounds, e.g. katsumadain A, because of strong
quenching effects or an increase in the FL signal (due to self-fluorescence). For several compounds showing activity
in the CL assay with viral NA, no or only poor inhibition was detected in the FL assay. However, these compounds
showed activity in both assay formats for pneumococcal NA. This corroborated our concerns regarding the reliability
of these assays and prompted us to search for alternative assays that overcome the interference of the test
compound with light signals as well as substrate differences that might be the reason for the observed artifacts.
As a result, viral and bacterial NA inhibitory assays based on human erythrocyte binding and elution were established
that work under physiological conditions. We found that these HA-based assays work most reliably with novel NAIs
acting against viral and/or bacterial NA.
Conclusions
The HA-based NA inhibitory assays enable the screening for novel NAIs under physiological conditions. FL- and/or
CL-based NA inhibition assays can be used to confirm the activity after exclusion of quenching and self-fluorescence.
This work is supported by the Austrian Science Fund (FWF: P24587 & P23051) and the Thuringian Ministry of
Economy, Labour and Technology funded by the European Social Fund (2011FGR0137).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P101
POSTE R A BSTRAC TS
SPA4P13
Identification of amino acids important for binding of novel oseltamivir
and zanamivir derivatives by using recombinant influenza viruses
Hoffmann, Anja (1); Schade, Dennis (2); Kirchmair, Johannes (3); Sauerbrei, Andreas (1); Schmidtke, Michaela (1)
1: Jena University Hospital, Department of Virology and Antiviral Therapy, Jena, Germany; 2: Department of Chemistry and Chemical Biology,
TU Dortmund University, Dortmund, Germany; 3: Swiss Federal Institute of Technology Zurich, Zurich, Switzerland
Background
Neuraminidase inhibitors (NAI; e.g. oseltamivir and zanamivir) are the only medicines approved for the prevention
and treatment of influenza. Emerging resistances urge for the development of novel resistance-breaking drugs
like the orally bioavailable amidine derivative of oseltamivir which was shown to be active against an oseltamivirresistant influenza A virus (Schade et al., J Med Chem. 2014). The aim of the present study is the identification of
amino acids (aa) that may influence the binding and thereby the activity of novel oseltamivir and zanamivir analogs,
which have been synthesized to overcome resistance mechanisms.
Methods
Several aa substitutions of neuraminidase (NA), known or presumed to cause drug resistance, were generated by sitedirected mutagenesis. Recombinant viruses were rescued with the help of an eight- plasmid transfection system.
The susceptibility of the variants and wild type WSN/1933 against amidine and guanidine analogs of oseltamivir
as well as a zanamivir analog was compared by fluorescence- and chemiluminescence-based NA inhibition assays.
Possible binding poses of the NAI into the NA active site have been modeled based on experimental data.
Results
By using a shared genetic background it was possible to attribute observed differences exclusively to the provided
mutations. The aa substitutions influenced the drug efficacy and resulted in different levels of resistance.
In chemiluminescence-based NA-activity assays (CL-assays) a strong increase in oseltamivir 50% inhibitory
concentration (IC50) was observed (250-fold for variant NA H275Y and 60- fold for variant NA N294S). Both, the
guanidine and the amidine analog of oseltamivir reduced this resistance considerably. In context of wild type A/
WSN/1933 virus these oseltamivir derivatives showed a higher inhibitory activity than their parent compound in
contrast to the zanamivir derivative that presented increased IC50-values. The substitution of tyrosine to histidine
at position 155 had little influence on the inhibitory effect of oseltamivir and its derivatives, whereas for zanamivir
a change in IC50 of up to 10-fold was observed when compared to the wild type. The zanamivir analog was unable
to break this resistance as determined by CL-assay. Variant NA Q136L had no influence on the inhibitory activity of
any of the investigated compounds. Results for oseltamivir derivatives were confirmed by fluorescence-based assay
(FL-assay). As shown previously (Nguyen et al., AAC 2010), the change in resistance measured with the FL-assay was
more pronounced than with the CL-assay. In case of the Y155H variant FL- and CL-assay data did not correlate well.
Conclusions
Using genetically modified influenza viruses, the underlying molecular mechanisms of action of novel oseltamivir
and zanamivir derivatives was shown. In particular the NA aa H275, Y155, and N294 are crucial for this mechanism
in A/WSN/1933. With this knowledge further strategies for developing resistance-breaking agents can be derived.
This work is supported by the European Social Fund (2011-FGR0137).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P102
POSTE R A BSTRAC TS
SPA4P14
Antiviral effect of new oseltamivir derivates against influenza A virus
García-Machorro, Jazmín (1); Neri-Bazan, Rocio M. (1); Correa-Basurto, José (1); Trujillo-Ferrara, José G. (1); Tolentino-López, Luis (1); MartínezRamos, Federico (2); Velazquez-Quiroz, Isaac (3); Jímenez-Estrada, Juan M. (3); Soriano-Ursúa, Marvin A. (1); Aguilar-Faisal, José L. (1)
1: Escuela Superior de Medicina. Instituto Politécnico Nacional, Mexico; 2: Escuela Nacional de Ciencias Biológicas. Instituto Politécnico
Nacional, México; 3: Instituto de Salud del Estado de México
Background
Recently, the emergence of drug resistant influenza A virus, such as H274Y mutation in neuraminidase that confers
high resistance to neuraminidase inhibitors, led to the design of new compounds derivates of oseltamivir which
increase the afinity on the mutant variants and on many subtypes of influenza. These new arylic-oseltamivir
derivates have as target a cluster of arginines residues highly conserved (Arg118, Arg292, and Arg371) between
different subtypes of the neuraminadase active site. According to bioinformatic tools, there are serveral interactions
including pi-cation and pi-pi interactions between the arylic compounds and aromatic or positively charged residues
present in the pocket site which improve properties as stability and recognition of oseltamivir. To confirm this, we
evaluate the antiviral activity in vitro of these compounds on a influenza virus strain AH1N1.
Methods
Based on virtual screening (molecular dynamics and docking studies) were selected six compounds with the best
scoring on Kd and ΔG, the best physicochemical (Lipinski rule) and toxicological properties, then these compounds
were synthesized. Then, we decided evaluate if the new compounds have any inhibitory activity in vitro against
influenza A virus H1N1 by Enzychrom neuraminidase assay kit. After, we determined the cytotoxicity of each
compound at different concentrations (250 nM to 5000 nM) on Vero, HeLa and MDCK cells by trypan blue at 24
and 48h. Furthermore antiviral activity was determinated at 250 nM (minimal cytotoxic concentration assayed) by
supernatant titration from MDCK cells infected with AH1N1 virus at 5 MOI. Finally, we analyzed the roughness on
infected, non infected and compound treated MDCK cell surface by Atomic Force Microscopy.
Results
In neuraminidase inhibition activity we determinated the IC50 of the arilic compounds and was lower (75 nM) than
the oseltamivir carboxyilate (200 nM), moreover only two compounds were cytotoxic at 250 nM concentration. We
found that the compound whit the group p-hidroxylaniline had the best antiviral activity (47% plaque reduction),
compared with the activity of oseltamivir caroxylate (35% plaque reduction) (ANOVA one way, α 0.05).
Conclusions
We found that as a virtual screening has predicted, the new oseltamivir derivates have antiviral activity better than
the oseltamivir carboxylate, in the case of the p-hidroxylaniline substitution compound. The compounds showed no
citotoxicity at high concentrations and the best compound has inhibitory effect better than the oseltamivir at lower
concentrations. On the other hand is necessary to verify the activity of the compounds in other influenza strains
including H274Y mutant.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P103
POSTE R A BSTRAC TS
SPA4P15
Indirect effects of oseltamivir treatment of an influenza-infected index
case-patient on secondary household transmission: A randomized
placebo-controlled clinical trial in a crowded urban area in Bangladesh
Alicia M Fry (1), Doli Goswami (2), Kamrun Nahar (2), Amina Tahia Sharmin (2), Mustafizur Rahmun (2), Larisa Gubareva (1), Tasnim Azim (2),
Joseph Bresee (1), Stephen P Luby (2) (3), W Abdullah Brooks (2)
1: Influenza Division, 3: Centers for Disease Control and Prevention, Atlanta, GA USA; 2: International Centre for Diarrhoeal Disease Research,
Bangladesh, Dhaka, Bangladesh
Background
Prophylactic use of oseltamivir can prevent influenza infection and illness when given prior to exposure or
immediately after exposure to an infectious person. However, during a pandemic, a large drug supply would be
needed if prophylactic use was recommended. Oseltamivir treatment reduces virus shedding and may reduce
infectiousness and subsequent virus transmission to susceptible individuals. However, studies supporting a
reduction in transmission are limited. Prior to the 2009 pandemic, we initiated a double blind, randomized, placebocontrolled trial in a crowded area in Dhaka, Bangladesh to determine the indirect effects of treatment of an index
case-patient on secondary household transmission.
Methods
From May 11, 2008 through December 31, 2010, we enrolled ill index patients with a positive rapid influenza test
and randomized 1:1 placebo to oseltamivir. The index patient and household members were monitored daily for
illness. Household members with one major symptom (e.g., fever, tachypnea, breathing difficulty) or two minor
symptoms (e.g., cough, sore throat, chills, myalgia, rhinorrhea,) were sent to the clinic for a nasal wash specimen.
All specimens were tested for influenza with reverse transcriptase-polymerase chain reaction (PCR). We compared
secondary attack rate (SAR) due to illness and PCR-confirmed influenza between households with an index patient
randomized to oseltamivir treatment versus placebo. Efficacy =1-SARo/SARp estimated from a log binomial model
adjusting for household clustering. Index patients and their households were pre-specified to be stratified by onset
of the index case-patient’s major symptoms relative to enrollment, <48 hours or 48-120 hours (“>48 hours”). All
analyses were intent to treat, except where noted.
Results
Among 6,544 households under surveillance, 1190 index case-patients and their households were enrolled into the
study; 794 (67%) index case-patients were enrolled <48 hours (median 1 day) and 396 (33%) were enrolled >48 hours
(median 3 days) since symptom onset. Overall, 2292 and 2402 household members were monitored from placebo
and oseltamivir treated index case-patient households. Median age of index case-patients was 5 years, interquartile
range (IQR) 2,9) and infecting viruses included influenza A(H3N2) (35%), seasonal influenza A (H1N1) (11%), A(H1N1)
pdm09 (18%), and influenza B virus (33%). The median household size, excluding index-patients, was 4 (IQR 3,5)
and the median age of household members was 24 years (IQR 10,34). The median interval from index enrollment
to household illness was 1 day; 32% of household illness occurred before or on same day as index enrollment and
27% occurred 1 day after enrollment, there were no differences between groups. The SAR for illness was less in
the oseltamivir group (196/2402 [8.1%]) compared to the placebo group (233/2292 [10%], p=0.02; efficacy= 0.23,
95% confidence interval [CI]: 0.02, 0.39). The SAR for PCR-confirmed illness was not statistically different between
placebo (4.5%) and oseltamivir groups (3.8%, p=0.20); however, only 57% of ill household members had a respiratory
specimen collected for PCR. The efficacy was similar after households with an illness before or on the same day
as the index case-patient enrollment, or with incomplete household illness onset dates, were excluded (n=208
households): illness efficacy=0.29 (CI: -0.005, 5) and PCR influenza efficacy= 0.24 (CI: -0.75, 67). Among these later
households with index case-patients enrolled > 48 hours from illness onset, the efficacy against illness = 0.46 (CI:
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P104
POSTE R A BSTRAC TS
0.06, 0.72) and the efficacy against PCR-confirmed illness = 0.53 (-1.9, 92). Among households with index casepatients enrolled< 48 hours from illness onset, the efficacy against illness = 0.22 (CI:-0.17, 0.49) and the efficacy
against PCR-confirmed illness = 0.12 (-1.2, 65).
Conclusions
In a crowded low income setting, oseltamivir treatment of an index case-patient resulted in a 20% reduction in
secondary illness among household members when treatment was started within120 hours after illness onset. In
our study, efficacy was higher among households with index case-patients enrolled > 48 hours from illness onset.
Future studies will explore these results and their relevance more completely.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P105
POSTE R A BSTRAC TS
SPB4: ANIMAL FLU-ECOLOGY AND EPIDEMIOLOGY OF ANIMAL INFLUENZA
SPB4P01
Phylogenetic relationships of genes of hemagglutinin and matrix
protein of influenza A/equine/Kazakhstan/236/12 (H3N8) virus
Khan, Yelizaveta
RSE “Institute of microbiology and virology” CS MES RK, Kazakhstan
The presently known horse influenza viruses belong to two subtypes: A (H7N7) and A (H3N8). The first strain of
influenza A virus (H7N7) was isolated in Czechoslovakia in 1956 (Bryant, 2009; Mumford, 2010). The latest outbreak
caused by H7N7 virus was registered in 1979, no more cases of its isolation have been reported.
In 1963, during a severe epizootic in Florida related to importation of horses from Argentina, influenza A virus
(N3N8) appeared, which by now widely is circulating in many countries (Australia, China, Mongolia, Japan, India,
etc. ) (Daly, 1996).
This paper presents data on isolation of influenza A (H3N8) virus from sick horses in southern Kazakhstan and study
of their biological properties.
In 2012, during a local outbreak of acute respiratory infection in South Kazakhstan region from sick horse
haemagglutinating agent was isolated identified by reverse transcription-polymerase chain reaction,
hemagglutination inhibition and inhibition of neuraminidase activity assays, as influenza A genus subtype H3N8
virus.
Its main biological properties: hemagglutinin (HA) thermal sensitivity, resistance to blood serum inhibitors,
spectrum of hemagglutinating activity, degree of adsorption and elution from chicken erythrocytes were studied,
and the phylogenetic relationships between HA and matrix protein (M) genes of A/equine/Kazakhstan/236/12
(H3N8) influenza virus and viruses of this subtype from GenBank were established.
Molecular genetic analysis revealed the highest changes in HA gene where five unique nucleotide substitutions
were found specific to cluster of Central Asian viruses.
It is shown that according to HA and M genes A/equine/Kazakhstan/236/12 isolate refers to Florida 2 subline cluster
of viruses within the American line, which includes viruses isolated in Europe and in the countries bordering with
Kazakhstan: China, Mongolia and North India. All viruses in this cluster phylogenetically ascend to the virus A/
equine/Newmarket/5/2003.
Isolation of influenza A (H3N8) virus from horses in South Kazakhstan and the epizootological situation in the
republic formed over the past 10 years, require constant monitoring of influenza dissemination and studying
antigenic and molecular biological properties of newly isolates.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P106
POSTE R A BSTRAC TS
SPB4P02
Surveillance of influenza virus subtypes H1, H2, H3 among wild birds in
Ukraine in 2006-2012
Muzyka, Denys (1); Pantin-Jackwood, Mary (2); Stegniy, Borys (1)
1: National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, Ukraine; 2: Southeast Poultry Research Laboratory,
Agricultural Research Service, USDA, Athens, Georgia, US
Background
Influenza is one of the most dangerous and unpredictable diseases of humans, other mammals and birds. There is
some danger of AIV of H5, H7 subtypes to human health. Influenza virus of H1, H2, H3 subtypes, circulate in humans
an cause seasonal influenza. Similar subtypes are also circulating in the natural reservoir, wild aquatic birds. Under
certain conditions, they can be donors of genes for new epidemic viruses. In this study, we examined the features of
circulating H1, H2, H3 influenza viruses in wild birds of various environmental groups in Ukraine.
Methods
Monitoring of circulating influenza virus of H1, H2, H3 subtypes was conducted in 2006-2012 in different regions
of Ukraine. Serology for the detection of influenza antibodies was conducted with samples collected from 946 wild
birds of 44 species and samples from 6281 wild birds of 84 species and 27 families were examined for the presence
of virus. All studies were performed as recommended by the OIE.
Results
6.06% of Dunlin, 5.88% of Grey Plover, 14.28 % of Curlew Sandpiper, 40% of Broad-billed Sandpiper in 2006, 15.38 %
of Mute Swan in 2008 and 6.97% of mallards in 2011 had antibodies to H1 influenza virus in serum. Also, antibodies
were found in egg yolks of Song Thrush, Blackbird, Jay in 2006, 3.22 - 4.16 % of mallard yolks in 2009. Antibodies
to H2 AIV in serum were found in 2006, 2009, 2011 at 13.04%, 1.61%, 1.55% -3.04 of mallards respectively, while
at egg yolks in 2006 at Song Thrush, Great Tit, Slender-billed Gull and egg yolks of mallards (12.5%). Antibodies
to H3 AIV were found in 2006 at egg yolks of Yellow-legged Gull (23.07%), in 2007 in yolks of Coot (33.3%) and
Grey Plover (14.28%), in 2008 at yolks of Common Tern, in 2009 at blood serum of mallards (12.90%). Ninety eight
hemagglutinating viruses were isolated, including 23 isolates of avian paramyxovirus and 69 AIV.. Of these AIV, 8
were H1 subtype (H1N1 - 7 isolates , H1N2 - 1), 5 subtype H3N8, and only 3 viruses H2 subtype (H2N3 - 2 isolates,
H2N? - 1). These viruses were isolated from wild waterfowl of two types - Mallard and Shelduck. H1 and H2 subtypes
of Influenza viruses were identified during autumn migration and wintering only, H3 influenza virus - during autumn
migration only. No virus was isolated during spring migration, nesting and after nesting movements.
Conclusions
Our results demonstrate that H1, H2 and H3 subtypes of AIV circulate widely in wild waterfowl. Particular attention
should be paid to the circulation in natural settings of H2 influenza viruses which have not been reported in humans
for many years.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P107
POSTE R A BSTRAC TS
SPB4P03
Tissue distribution of low pathogenic avian influenza virus in naturally
infected wild birds.
Barral, Marta (1); Alvarez, Vega (1); Busquets, Nuria (2); Gerrikagoitia, Xeider (1); Minguijón, Esmeralda (1); Costa, Tania (2); Ramis, Antonio (2);
Majo, Natalia (2)
1: NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario. Derio-Bizkaia, Spain; 2: Centre de Recerca en Sanitat Animal (CReSA). BellaterraBarcelona, Spain
Recent experimental infections with low pathogenic avian influenza virus (LPAIV) in birds have revealed interesting
data about clinical outcome, pathogenesis and virus shedding, but to date, little is known about natural LPAIV
infection in wild birds. The objective of this work was to gain knowledge about the pathogenesis of LPAIV infection
in naturally infected wild birds. During the course of surveillance studies, we had access to sixty-one carcasses
of wild birds that first resulted positive for avian influenza virus detection by real time RT-PCR when cloacal or
oropharyngeal swab were analysed. The majority of them were ducks (88%), being mallard (Anas platyrhynchos)
the most frequently studied species (N=42). A complete necropsy was performed and trachea, lung, thymus, liver,
kidney, heart, brain and intestine tissues were systematically collected. Histopathology, immunohistochemistry
and molecular detection of avian influenza virus by RT-PCR were carried out in collected tissue samples. In addition,
RT-PCR positive tissues and cloacal or oropharyngeal swabs were inoculated in SPF embrionated chicken eggs for
virus isolation and subtyping following specific RTPCR or sequencing. At least one tissue was RT-PCR positive in
20% of the animals. All analysed tissues from a yellow-legged gull were positive by RT-PCR. Attending to the type of
surveillance, 35.7% of birds obtained by passive surveillance showed almost one positive tissue by RT-PCR, whereas
only 6.1% were positive from those obtained by active surveillance (p<0.005). Histopathological findings included
tracheitis or laryngitis in 12% of individuals and lesions due to coinfection with other avian pathogens. Regarding
immunohistochemistry results, no positive reactions were observed in the studied animals. Virus isolation was
obtained from 45.9% of the birds and 16 different virus subtypes were identified, being H4N6, H1N1, H8N4, H3N6
and H6N8 the most frequently found, but low pathogenic H5 and H7 subtypes were also detected. Virus detection
in tissues was more frequent in birds found sick or dead suggesting a relation with other concomitant factors or
pathogens. Further studies should be made in order to better understand the dynamics of LPAIV infection in their
natural wild reservoirs.
Work funded by Spanish National Institute for Agricultural and Food Research and Technology (INIA) (FAU200600019-C03 and RTA2011-00111-C03), Department of Agriculture and Fisheries (Basque Government) and
Departament d’Agricultura, Ramadaria, Pesca, Alimentació i Medi Natural (Catalonia)
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P108
POSTE R A BSTRAC TS
SPB4P04
Sero-prevalence of antibodies against H5N1 and H9N2 avian influenza
viruses among Egyptians: Results of a prospective, controlled, seroepidemiological study.
Kayali, Ghazi (1); Webby, Richard J (1); Ali, Mohamed A (2)
1: St. Jude Children’s Research Hospital, United States of America; 2: National Research Centre, Egypt
Background
The extent of human infection with avian influenza H5N1 viruses remains debatable. A meta-analysis of published
sero-prevalence studies estimated that 1-2% of humans exposed to H5N1 are sero-positive. It concluded that a
large number of mild and/or subclinical cases have been missed thus over-estimating the case fatality rate. Experts
in the field responded that those findings have been over-interpreted since the analysis was based on limited and
uncertain data. Egypt is an H5N1-endemic hotspot where most of the world’s recent human cases were reported.
H9N2 viruses were detected in Egypt in 2011 and were found to co-circulate with H5N1, and co- infect the same avian
host. In this country, we set out to determine the extent of human infections with avian influenza by determining
the sero-prevalence of antibodies against these viruses and measuring the associated risk factors.
Methods
We conducted a prospective, controlled, sero-prevalence cohort study among 750 Egyptians exposed to poultry and
250 unexposed controls aged 2 and older. At baseline, we obtained a blood sample and completed a questionnaire
assessing demographic, health, and exposure variables. Subjects were followed for 2 years and a blood sample was
obtained annually. Sera were tested for antibodies against H5N1 and H9N2 viruses using a microneutralization
assay. Titer of >1:80 was considered positive. Logistic regression was used to determine risk factors.
Results
At baseline, 2.1% of the exposed group tested positive for anti-H5N1 antibodies compared to 0% in the control
group. In the follow-up years, the prevalence was 0.4% and 0.6% respectively in the exposed group and 0% in
the control group but the difference was not statistically significant. Having chronic respiratory problems was
significantly associated with infection (OR=12.6; 95% CI=3.8-41.7). At baseline, no significant difference was
detected in antibodies against H9N2 viruses between the 2 groups (1.1% vs 0%). During the follow-up years, when
H9N2 viruses were detected in Egypt, the sero-prevalence in the exposed group was 7.5% and 5.9% respectively,
significantly different than that in the control group (0.7% and 1.0% respectively). Older age, exposure to ducks, and
vaccinating poultry were associated with having antibodies against H9N2. Antibody titers against human influenza
viruses was not correlated to testing positive to H5N1 and H9N2.
Conclusions
Our data indicate that in an endemic setting where exposure is high and continuous, serology can be useful
in determining the extent of infection. In Egypt, 2% of the exposed had elevated antibody titers against H5N1,
supporting the notion that many non-severe infections go undetected. We also show that H9N2 viruses are infecting
humans in Egypt, and that infection with H9N2 is higher than that with H5N1. Avian influenza viruses continue to
be a serious threat to public health in Egypt.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P109
POSTE R A BSTRAC TS
SPB4P05
Serological monitoring of equine influenza after vaccination in Korea in
2013
Kim, Bo-Hye (1); Yang, Sunjoo (2); Kim, Eun-Ju (1); Shin, Ye-Jin (1); Song, Jae-Young (1); Shin, Yeun-Kyung (1)
1: Animal and Plant Quarantine Agency, Republic of Korea; 2: Korea Racing Authority, Gwacheon, Republic of Korea
Background
Equine influenza (EI) is an acute respiratory infection of horses, donkeys and mules caused by two distinct subtypes
(H7N7 and H3N8) of influenza A viruses (1). Viruses of the H7N7 subtypes have not been isolated since the late
1970s (1). As a result of antigenic drift the H3N8 subtype evolved into several distinct lineages (Eurasian, American,
Clade I, and Clade II). EI outbreaks have been reported all over the world with the exception of a few countries.
EI is endemic in Europe and North America and is considered to be of potentially major economic significance to
the equine industry (2). Rapid diagnosis, movement restrictions and vaccinations are the key control measures for
EI. EI vaccines are widely available and are routinely used in competition horses in worldwide (1). Because of the
antigenic differences among EI viruses, all equine influenza vaccines should contain epidemiologically relevant
strains of EI viruses. Expert Surveillance Panel (ESP) reviews the EI outbreak and makes recommendation (1).
Regular vaccination with proper strain is highly recommended since the disease can be spread quickly in the naïve
susceptible populations. Serological monitoring of EI has been performed to evaluate the seroconversion rate after
EI vaccination.
Methods
Equine serum samples were collected from horses maintained by Korea Racing Authority. Serum samples were
collected two times in 2013; 348 horse sera were collected in April and May and 474 serum samples were collected
between September and November. These horses have been regularly vaccinated against equine influenza. The virus
strain, H3N8 Florida Clade II (A/EQ/WILDESHAUSEN/08) was used as antigens for Hemagglutination Inhibition (HI)
test. The virus was kindly provided by Dr. A. Damiani from Institute of Virology, Veterinary Medicine, Free University
of Berlin, Germany. The HI test was performed according to OIE Terrestrial Manual, Chapter 2.5.7.
Results
At baseline, 2.1% of the exposed group tested positive for anti-H5N1 antibodies compared to 0% in the control
group. In the follow-up years, the prevalence was 0.4% and 0.6% respectively in the exposed group and 0% in
the control group but the difference was not statistically significant. Having chronic respiratory problems was
significantly associated with infection (OR=12.6; 95% CI=3.8-41.7). At baseline, no significant difference was
detected in antibodies against H9N2 viruses between the 2 groups (1.1% vs 0%). During the follow-up years, when
H9N2 viruses were detected in Egypt, the sero-prevalence in the exposed group was 7.5% and 5.9% respectively,
significantly different than that in the control group (0.7% and 1.0% respectively). Older age, exposure to ducks, and
vaccinating poultry were associated with having antibodies against H9N2. Antibody titers against human influenza
viruses was not correlated to testing positive to H5N1 and H9N2.
Results and Conclusion
In HI test using Clade II H3N8 virus as antigen of HI test, seropositive percentage was 81.3 among 348 serum
samples from the first half year; the horse sera collected in the second half showed 98.3. It could be presumed that
horses tested in this study were vaccinated with epidemiologically relevant EIV strains.
References
1. Equine influenza, Chapter 2.5.7. OIE Terrestrial Manual 2012.
2. Equine Influenza – A global perspective. Veterinary Microbiology. 167 (2013) 205~214
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P110
POSTE R A BSTRAC TS
SPB4P06
Monitoring Avian Influenza Virus in a small Spanish wetland using noninvasive sampling methods.
Torrontegui, Olalla (1); Álvarez, Vega (1); Gerrikagoitia, Xeider (1); Höfle, Ursula (2); Barral, Marta (1)
1: NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Derio-Bizkaia, Spain; 2: Instituto de Investigación en Recursos Cinegéticos IREC
(CSIC, UCLM, JCCM) Ciudad Real, Spain
Anseriformes and Charadriiformes are known to be the main reservoirs for Avian Influenza Virus (AIV) in which the
transmission within these occurs via faecal/oral contact in contaminated water. Understanding the Avian Influenza
Virus (AIV) persistence and transmission dynamics is crucial for predicting disease outbreaks and improving
surveillance strategies. The aim of this work was to monitor the presence and persistence of AIV in its natural
environment in relation to ecology of its reservoir hosts. During a 23 month period (March 2012-January 2014) the
relationship between AIV presence and wildfowl population movements was studied in a small wetland in the
north of Spain where the presence of different AIV had previously been recorded. This water ecosystem is considered
of special interest as it is an important site for wintering and breeding of waterfowl and as a stopover for waterfowl
migrating further south, being considerably small (209 Ha) and close to an urbanised area. Fresh faeces were
collected monthly from islets scattered along the wetland, used as roosting sites by the birds. Bird censuses and
meteorological data were gathered in parallel in order to relate AIV detection to waterfowl ecology. For AIV detection
RNA was extracted from the faecal matter and analysed by influenza A specific rRT-PCR. Positive samples were then
tested for H5 and H7 subtypes and inoculated into embrionated SPF chicken eggs for virus isolation. During the 23
months, a total of 2096 samples were analysed and AIV was detected in 0.29%. In regard to the wild bird population
movements, the highest AIV prevalence occurred along the breeding season (0.67%) and autumn migration (0.35%).
The highest prevalence 3.23% was recorded in June 2012. All positive samples contained low pathogenic AIV. The
concentration of positive results during the two above mentioned periods may be related to two scenarios in
waterfowl ecology; on one hand, high water bird aggregation densities and therefore a greater virus titre input to
the environment during migration and fledging of chicks and on the other hand, the presence of a naïve juvenile
population with a still less efficient immune system that will make them more susceptible towards an AIV infection.
More research is deemed necessary clarify the complex AIV ecology. In this regard, further studies are presently
being undertaken.
Work funded by Spanish National Institute for Agricultural and Food Research and Technology (INIA) (RTA201100111-C03-03) and Department of Agriculture and Fisheries (Basque Government).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P111
POSTE R A BSTRAC TS
SPB4P07
Influenza A(H1N1)pdm09 virus in pigs, Togo, 2013
Ducatez, Mariette (1); Webby, Richard (2); Awoume, Félix (3)
1: INRA, France; 2: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, TN, USA; 3: Laboratoire National
Vétérinaire de Lomé, Lomé, Togo
After Togo underwent a couple of outbreaks of highly pathogenic avian influenza H5N1 in 2007 and in 2008, a
large scale active surveillance study of animal influenza has been carried out from 2009 through 2014 with close to
10,000 avian and swine swabs tested for the presence of influenza virus genome. As observed in the neighboring
Benin and Côte d’Ivoire, none of the animal swabs collected in Togo through 2012 had been positive for influenza
A virus (irrespective of the subtype), nor had any animal serum tested positive for influenza antibodies (CouacyHymann et al, EID, 2012). We have previously hypothesized that the combination of climate and animal density
factors might be responsible for what appeared to be the absence of influenza virus in the backyard sector of Côte
d’Ivoire, Benin, and Togo.
From February through November 2013, influenza virus has been detected in swine from a slaughterhouse near
Lomé in Togo. 270 nasal swabs were collected from freshly slaughtered healthy pigs throughout the year. The
samples were kept at 4°C in virus transport medium for a few hours and stored at -80°C until further analyzed. They
were then pooled (5 samples per pool), RNA was extracted and influenza A specific RT-PCR was carried out. Eight
pools (15% of the pools) were positive for influenza A genome, which corresponds to a virus prevalence of 3 to 15%.
Subtyping PCRs were carried out and allowed the identification of the A(H1N1)pdm09 virus in all the influenza
A positive pools. We successfully isolated virus on MDCK cells from a representative specimen and confirmed by
Sanger sequencing that it harbored 8 genes very similar to A(H1N1)pdm09 viruses circulating in human in 2013.
While very little was known on swine influenza viruses in Africa until 2009, A(H1N1)pdm09 became pandemic
in human and emerged in swine in La Réunion Island in 2009 (Cardinale et al, EID, 2012), in Cameroon in 2010
(Njabo et al, Vet Microbiol, 2010), and in Nigeria in 2010 (Meseko et al, Vet Microbiol, 2014). We observe a delay in
A(H1N1)pdm09 emergence in pigs in Togo, emergence likely due to human to swine transmission. Further studies
are ongoing to assess the burden of the disease in swine in Togo.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P112
POSTE R A BSTRAC TS
SPB4P08
Long term surveillance of H7 influenza viruses in American wild aquatic
birds: Concordance with emergence of variants with pathogenic
potential?
Krauss, Scott L. (1); Webster, Robert G. (1); Danner, Angela (1); Friedman, Kimberly (1); Knowles, James (1); Ghazi, Kayali (1); Niles, Lawrence
J. (2); Dey, Amanda D. (3); Raven, Garnet (4); Pryor, Paul (4); Stucker, Karla (5); Schobel, Seth (5); Stockwell, Timothy B. (5); Lin, Xudong (5);
Wentworth, David (5)
1: St. Jude Children’s Research Hospital, Department of Infectious Diseases, Memphis, Tennessee, United States of America; 2: Conserve Wildlife
Foundation of New Jersey, Bordentown, New Jersey, USA; 3: Endangered and Nongame Species, New Jersey Division of Fish and Wildlife,
Trenton, New Jersey, USA; 4: Environment Canada, Canadian Wildlife Service, Edmonton, Alberta, Canada; 5: J. Craig Venter Institute, Rockville,
Maryland, USA
The emergence of influenza viruses in domestic avian species and associated transmissions to mammals, including
swine and humans, is currently unpredictable. Of current concern are the H7 influenza viruses where the H7N3
subtype has become highly pathogenic for poultry with occasional human infections in 4 separate outbreaks in
North and South America from 2002 to 2012. In addition low pathogenic H7N9 viruses in poultry have emerged in
China and are highly pathogenic in humans. Here we use long term surveillance for influenza viruses from North
America in shorebirds at Delaware Bay, USA (1985‐2012) and from ducks in Alberta, Canada (1976‐2012) to determine
whether shorebirds or ducks have potential for interspecies transmissions, and whether surveillance in wild birds
has any predictive value. For H7 influenza viruses shorebirds carried the majority of possible HA NA combinations ‐ 8
of the 9 neuraminidases subtypes found in aquatic birds in the Americas were detected in shorebirds while 6 of 9
neuraminidases were found in ducks. The most frequent combination was H7N3 which was found in both shorebird
and duck species but more frequently in shorebirds. Overall there was concordance between the peaks of activity of
low pathogenic H7N3 in wild birds and with the 4 outbreaks of highly pathogenic H7N3 in the Americas. Phylogenetic
analysis of the virus genomes shows that each of the 4 HPAI outbreaks of H7N3 in the Americas represents a separate
introduction of low pathogenic avian influenza to poultry from the wild aquatic bird reservoir ‐ most likely from
dabbling ducks. The outbreak of HPAI H7N3 in Chile in 2002 was generated by precursor viruses distinct from low
pathogenic avian influenza strains found in North America. Thus, enhanced biosecurity on poultry farms, coupled
with the potential forewarning of veterinary and public health officials by detection of increased activity of H7N3
influenza viruses in wild aquatic birds, could prevent emergence of highly pathogenic avian influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P113
POSTE R A BSTRAC TS
SPB4P09
Prior infection of pigs with a European H3N2 SIV confers partial
protection against a North American swine-origin H3N2v influenza virus
QIU, YU; Mancera Gracia, José Carlos; Li, Yewei; Trus, Ivan; Nguyen Van, Ut; Van Reeth, Kristien
Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Belgium
Background
H3N2 swine influenza virus (SIV) lineages in Europe and North America have distinct HA and NA genes that are
derived from human H3N2 viruses from the 1970s and 1990s, respectively. Of the multiple H3N2 genotypes
that circulate among US swine, one particular genotype - a reassortant with only the matrix gene from the 2009
pandemic H1N1 virus - has caused 340 human infections in 2011-2013. This H3N2 “variant” (H3N2v) virus and other
North American H3N2 SIVs have so far never been reported in Europe. This raises the question as to whether pigs
immune to the antigenically and genetically distinct European H3N2 SIVs would be protected against infection with
H3N2v if H3N2v were introduced in Europe.
Methods
Conventional 8-week-old influenza naïve pigs were inoculated intranasally with the H3N2v virus A/Indiana/08/11
(In/11-In/11 group), with the European H3N2 SIV sw/Gent/172/08 (Gent/08-In/11 group), or with phosphate
buffered saline. Eight weeks later, all pigs were challenged intranasally with A/Indiana/08/11. Both H3N2 viruses
have only 80% homology in their HA1 and all but the matrix gene segments have a phylogenetically distinct
origin. The extent of protection against challenge was determined by virus titration of nasal swabs (0-7 days post
challenge), lung, trachea and nasal mucosa (3 days post challenge). Serum antibody responses were measured
by hemagglutination-inhibition and virus-neutralization assays. Mononuclear cells from the nasal mucosa,
tracheobronchial lymph nodes and peripheral blood were examined for virus-specific IgG and IgA antibody secreting
cells (ASCs) and IFN-γ secreting cells (SCs) by ELISPOT assays.
Results
After the primary inoculation with A/Indiana/08/11 or sw/Gent/172/08, all pigs had high virus titers in nasal swabs
for 5-6 days. There was no or minimal serological cross-reactivity between both H3N2 viruses. After challenge with
A/Indiana/08/11, all challenge controls shed high amounts of virus for 6 days. Virus excretion was undetectable in
the In/11-In/11 group and significantly reduced in both magnitude and duration (3-4 days) in the Gent/08-In/11
group. Virus titrations of the respiratory tract tissues are pending. At challenge, the numbers of A/Indiana/08/11specific IgA and IgG ASC, but not IFN-γ SCs, were significantly higher in A/Indiana/08/11 pre-infected pigs than in
sw/Gent/172/08 pre-infected pigs. After challenge, the Gent/08-In/11 group showed vigorous early IgG and IgA ASC
recall responses to A/Indiana/08/11, which were by far most pronounced in the nasal mucosa and associated with
the shorter duration of nasal virus shedding. IFN-γ SC responses were boosted in the In/11-In/11 group but not in
the Gent/08-In/11 group.
Conclusions
There is partial cross-protection between the European H3N2 SIV lineage and the H3N2v virus, which may be due to
cross-reactive B cell responses in the nasal mucosa. The pig is a valuable model to study mucosal immune responses
and to improve our understanding of broad-spectrum immunity to influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P114
POSTE R A BSTRAC TS
SPB4P10
Biosecurity practices in small commercial poultry farms, Bangladesh 2011-12
Rimi, Nadia Ali; Sultana, Rebeca; Muhsina, Mushtari; Uddin, Baktiar; Haider, Najmul; Nahar, Nazmun; Sturm-Ramirez, Katharine; Luby,
Stephen P.
icddr,b, Bangladesh
Background
In Bangladesh, highly pathogenic avian influenza is endemic. Small scale poultry farms account for 262/543
confirmed outbreaks among commercial farms. Biosecurity measures may reduce the spread of avian influenza.
This study aimed to understand the biosecurity practices of small commercial poultry farmers.
Methods
During 2011-12, the research team conducted 57 hours of observations, 16 in-depth interviews and two group
discussions with poultry farmers in 16 small commercial farms from two districts to understand the practices for
three principle elements of biosecurity— segregation, cleaning and disinfection. We reviewed and summarized
emerging themes.
Results
None of the farms were segregated from the environment, people and animals. None maintained recommended
distances from the nearest households (median 6.7m, [IQR 1.65m-25.9m]; recommended: 500m), other poultry
farms (median 30.5m, [IQR 7m- 91.95m]; recommended: 200m), large trees (median 0m; recommended: 100m) or
roads (median 14.5m, [IQR 9m-41.35m]; recommended: 500m). Only one farm had intact fencing around all sides
of the poultry shed. People (other than the farmers), backyard poultry, and other birds were observed frequently
entering sheds. Farmers separated sick from well-appearing poultry inside sheds or bedrooms. Farmers, however,
did not allow the buyer’s to bring egg trays inside their sheds. Five farmers reported sleeping inside the shed at
night to protect chickens from rodents and foxes. Farmers used several locally available disinfectants to protect
chickens from disease. All the farmers reported cleaning and disinfecting sheds by spreading lime on the floor before
buying chicks. Six farmers reported spraying disinfectant inside and/or outside the shed daily. Four farmers reported
sweeping away feces daily but none was observed using any disinfectant on the floor after feces removal. All farmers
sold or used the feces as fish-feed without first treating it. Nine farmers used designated footwear for the shed. Two
farms had footbaths which were not used during our observation. Of the 94 times farmers entered sheds, the team
observed handwashing with soap by one farmer before entering and by two farmers after touching feces, and three
farmers disinfecting their feet before entering. Farmers were never observed using masks or gloves and changing
clothes before entering the shed. The farmers mentioned financial constraints and inconvenience as reasons for not
complying with certain biosecurity measures.
Conclusions
While we seldom observed flock segregation or farmers’ handwashing and using personal protective equipment,
some farmers used several measures that involved additional cost or effort, such as using disinfectants or separate
footwear for the shed and restricting buyer’s egg trays from entering the shed to protect their flock. This suggests
that farmers could be motivated by interventions that protect their investment. Future interventions could explore
the feasibility, desirability and effectiveness of low- cost alternatives such as locally available fencing materials and
disinfectant agents.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P115
POSTE R A BSTRAC TS
SPA5: VACCINES: CURRENT AND NOVEL APPROACHES
SPA5P01
Multi-component adjuvants for inactivated influenza vaccines:
comparative studies in a preclinical setting
Vasiliev, Y.M.; Kashirina, O.S.; Chernikova, M.I.
Mechnikov Research Institute of Vaccines and Sera, Russian Federation
Background
Influenza remains a global healthcare issue. Vaccines are the cornerstone of influenza prevention and control.
Adjuvants can boost immunogenicity of prepandemic and seasonal influenza vaccines. Novel adjuvants for
influenza vaccines could be based on depot-forming and immune stimulating components with an optimal safety
and immunogenicity profile (for individual components). Some of these components include chitosan-based
formulations and well-known adjuvants (aluminium hydroxide, CpG).
Methods
Groups of mice were immunized intramuscularly once or twice with adjuvanted inactivated whole-virion monovalent
influenza vaccines based on A/California/07/2009 X-179A (H1N1) strain. Immunogenicity was evaluated by HAI and
MN (in MDCK cells) sera antibodies against the vaccine and drift A/Brisbane/59/2007 IVR-148 (H1N1) strain. In the
first series of experiments a panel of commercially obtained chitosan- based adjuvants (different molecular weight
(Mw, Mn and/or Mv), viscosity and/or deacetylation degree) was studied (14 groups in total). In the second series
of experiments a panel of multi-component adjuvants as well as individual components (including aluminium
hydroxide, CpG, oil-in-water emulsions) were studied (19 groups in total).
Results
In the first series of experiments different chitosan-based adjuvants increased immunogenicity of the influenza
vaccine in varying patterns. The most immunogenic and a non-immunogenic (negative control) chitosan-based
adjuvants was selected for further studies. In the second series of experiments different adjuvants increased
immunogenicity of the influenza vaccine in varying patterns. A chitosan-based adjuvant was the most immunogenic
(antibody titers in some cases increased by 50 times compared with the unadjuvanted vaccine), highly immunogenic
2- and 4-component adjuvants as well as a 5-component oil-in-water emulsion were identified. Interestingly, a
chitosan-based adjuvant that was not immunogenic (negative control) potentiated the immunogenicity of a multicomponent adjuvant. Immunogenicity of the vaccine against the drift strain was very low regardless of the adjuvant
used.
Conclusions
Some of the multi-component adjuvants (depot-forming and immune stimulating components) significantly
increase immunogenicity of the intramuscularly administered inactivated influenza vaccine, further preclinical
studies elucidating mechanisms of action, optimal composition and safety are necessary.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P116
POSTE R A BSTRAC TS
SPA5P02
Characteristics of chitosan-based adjuvants determine immunogenicity
of inactivated influenza vaccines in mice
Khasanova, L.M. (1,2); Kashirina, O.S. (1); Chernikova, M.I. (1); Vasiliev, Y.M. (1)
1: Mechnikov Research Institute of Vaccines and Sera, Moscow, Russian Federation; 2: Centre “Bioengineering”, Moscow, Russian Federation
Background
Vaccination is the principal approach for influenza control. Immunogenicity of inactivated influenza vaccines
is suboptimal and addition of adjuvants has been suggested. However adjuvants with an optimal safety and
effectiveness as well as cost-effectiveness profile are currently lacking. Chitosan has been studied for various
biotechnology and biomedicine applications. Principle chitosan characteristics include deacetylation degree (DD),
molecular mass (Mm), polydispersion and source (e.g. shrimp, crab, fungi). Lack of chitosan characteristics in
published preclinical and clinical studies prevents conclusions regarding adjuvant properties for influenza vaccines.
Methods
A panel of characterized chitosan-based adjuvants was used. Chitosan substances with Mm of 700, 200, 10 kDa,
DD 85%; 200 kDa, DD 68% and 10 kDa, DD 98% and a succinylated chitosan were laboratory-produced, commercial
substances were obtained via Sigma-Aldrich, USA. Average viscous (Mv) molecular weight was determined by capillary
viscometry, weight average (Mw) and number average (Mn) molecular weight - by GPC, DD - by conductometric
titration and 1H NMR spectroscopy for all the chitosan substances. Groups of mice (15 in total) were immunized
intramuscularly once and twice with adjuvanted monovalent inactivated whole-virion influenza vaccines against A/
California/07/2009 X-179A (H1N1) strain. Immunogenicity was evaluated by sera hemagglutination inhibiting (HAI)
and neutralizing (MN in MDCK) antibodies against the vaccine and an antigenically distinct (A/Brisbane/59/2007
IVR-148 (H1N1)) strain.
Results
Adjuvants based on chitosan substances with different characteristics increased immunogenicity of the inactivated
influenza vaccine in varying patterns. Both DD and Mm had a significant effect on adjuvant properties of chitosanbased formulations. 700 kDa, 85% DD chitosan-based adjuvant was one of the most immunogenic, whereas
immunogenicity of adjuvants based on a low molecular weight chitosan (10 kDa, 85% DD) compared with high
molecular weight chitosans (200 and 700 kDa, 85% DD) was significantly lower (up to 3 and 7 times, respectively).
Conclusions
Further studies regarding the role of characteristics of chitosan-based adjuvants in immunogenicity of various
inactivated influenza and other vaccines are necessary.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P117
POSTE R A BSTRAC TS
SPA5P03
A comparative study of adjuvants with different mechanisms of action
for influenza vaccines in mice
Chernikova, M.I. (1); Kashirina, O.S. (1); Khasanova, L.M. (1,2); Vasiliev, Y.M. (1)
1: Mechnikov Research Institute of Vaccines and Sera, Moscow, Russian Federation; 2: Centre “Bioengineering”, Moscow, Russian Federation
Background
Influenza is one of the most common respiratory infectious diseases and is associated with high morbidity,
mortality and pandemic potential. Vaccination is one of the most effective approaches to prevent and control
influenza. Inactivated influenza vaccines are the most widely administered worldwide, however immunogenicity
in risk groups and against antigenically distinct strains is suboptimal. Adjuvants can increase immunogenicity
of inactivated influenza vaccines, however the search for effective, safe and affordable adjuvants is an ongoing
challenge complicated by a lack of data from direct comparative studies of adjuvants with different mechanisms of
action.
Methods
A panel of adjuvants was used: aluminium hydroxide, calcium phosphate, polyoxidonium, complete Freund’s adjuvant
(CFA), oligodeoxynucleotide CpG, cholera toxin subunit B (CTB), monophosphoryl lipid A (MPL), peptidoglycan,
poly-(lactide co- glycolide) [PLG], saponin, arabinogalactan as well as adjuvants based on characterized chitosan
substances (chitosan lactate, chitosan glutamate and oligochitosan glutamate solutions), an oil-in-water emulsion
and a multi-component formulation. Unimmunized mice, PBS, chicken embryo allantoic fluid (non-purified antigen)
and a live virus (model infection) were used as controls. Groups of mice (21 in total) were immunized intramuscularly
once or twice with adjuvanted inactivated whole-virion influenza vaccines based on A/California/07/2009 X-179A
(H1N1) strain. Immunogenicity was evaluated by sera hemagglutinin-inhibiting and neutralizing antibodies against
the vaccine and an antigenically distinct (A/Brisbane/59/2007 IVR- 148 (H1N1)) strain.
Results
Adjuvants with different mechanisms of action increased immunogenicity of the vaccine in varying patterns.
Immunogenicity against the antigenically distinct strain was very low regardless of the adjuvant used. Chitosan
lactate, oil-in-water emulsion and the multi-component formulation were the most immunogenic. Chitosan
glutamate and CTB were very highly immunogenic, and PLG, CFA and oligochitosan were highly immunogenic.
Conclusions
Chitosan-based adjuvants are promising candidates for influenza vaccines, however role of chitosan characteristics
and/or form (e.g. solution) in mechanisms of action should be further elucidated. Direct comparative studies provide
the basis for evaluation of novel as well as selection of optimal adjuvants for influenza vaccines.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P118
POSTE R A BSTRAC TS
SPA5P04
Epidemiologic performance and influenza vaccination prophylaxis in
Russia
Briko, Nikolay Ivanovich
I.M. Sechenov First Moscow Government Medical University, Russian Federation
Background
Analysis of influenza epidemiologic performance (morbidity rate, age pattern, seasonal fluctuation) and vaccination
prophylaxis status in the country.
Methods
Infection morbidity official data (F.2, approved by Rosstat) together with vaccination coverage statistical data (F.5,
approved by Rosstat), and scientific Russian literature publications were studied.
Results
In Russia, 20-25 mln. cases of morbidity with influenza and ARVIs are registered annually. Morbidity dynamics
involves case rates elevation and decline. In 2011, 308 829 cases of influenza were registered (217,6 per 100 000 of
population); in 2013 this value was to be 100 642 (70,4 per 100 000 of population). From them, pediatric population
comprised 38416 (145,6 per 100 000 of population). Economic loss from influenza and ARVIs provides about 77%
from total loss resulting from general infection diseases morbidity rate. In total temporary incapacity cases cohort,
influenza and ARVIs take 12–14%. Generally, in the period of 2005-2013 morbidity increase begins in December
achieving the maximum in February, and completed in April. Influenza morbidity high risk groups include pediatric
collectives of 1-2 years old, children of 3-6 years old, and elderly. In the season 2012-2013, simultaneous circulation
for influenza virus strains А (H3N2), A (H1N1), А(Н1N1)2009 and В was registered. Influenza vaccination is a part of
National Immunization Schedule, both for children from 6 months old, schoolchildren, and students, and for adults
as well. Since 2014, influenza vaccination is recommended for pregnant. Last 5 years, influenza vaccination coverage
in Russia grew up in 1.6 times. In 2013, 27.8% from all population was vaccinated (39 713 587). Vaccination coverage
in organized pediatric cohorts was 64 % (WHO recommends 75%).
Conclusions
Influenza and ARVIs bring significant social-economic loss due to high spread ability. To increase the influenza
vaccination coverage, more intensive information and public awareness activity is necessary, both in the form of
community outreach, and for medical professionals. Besides, it is necessary to engage extra-budgetary and nonstate and resources widely (insurance payments, charity funds, institutions and enterprises resources, citizens’
personal finances).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P119
POSTE R A BSTRAC TS
SPA5P05
Comparison of immune response induced by candidate recombinant
influenza vaccines based on heterologous M2e linked to full-length and
truncated forms of flagellin
Stepanova, Liudmila (1); Kotlyarov, Roman (2); Kovaleva, Anna (1); Potapchuk, Marina (1); Korotkov, Alexandr (1); Sergeeva, Maria (1);
Kasianenko, Marina (1); Kuprianov, Victor (2); Ravin, Nicolai (2); Tsybalova, Liudmila (1)
1: Research Institute of Influenza, Ministry of Health of the Russian Federation, St. Petersburg, Russia, Russian Federation; 2: Centre
“Bioengineering”, Russian Academy of Sciences, Moscow, Russia
Background
Flagellin protein presents the appropriate platform for development of recombinant vaccines against various
pathogens of viral and bacterial origin. Being the major structural protein of gram-negative bacteria flagellin
exhibits strong adjuvant properties when administered together with foreign antigens by parenteral, mucosal or
subcutaneous route. The ability of flagellin to be both a platform and an adjuvant for vaccine development was
demonstrated for various model infections including influenza. In this study we compared immune response and
protection of recombinant proteins where heterologous M2e were linked with full-length or truncated forms of
flagellin.
Methods
Recombinant proteins were generated by fusion of two tandem copies of consensus M2e sequence from human
influenza A viruses (M2eh) and two copies of M2e from A/H5N1 (M2ek) to C-terminus of full-length flagellin (Flg2M2eh2M2ek) or truncated form of flagellin with deletion of hypervariable (154-431aa) part (Flg(sh)-2M2eh2M2ek).
Balb/c mice were immunized intranasal 3 times at 2-weeks intervals with 30 μg of recombinant proteins. AntiM2e IgG, IgG1, IgG2a in serum and sIgA, IgG in bronchoalveolar lavage (BAL) were determined by ELISA. Sera of
immunized mice were tested for reactivity with MDCK cells infected by influenza A viruses. Immunized mice were
challenged with 1 LD50 A/PR/8/34 (H1N1) and monitored daily for 2 weeks for survival and weight loss.
Results
We demonstrated that intranasal immunization of Balb/c mice with both recombinant proteins Flg-2M2eh2M2ek
and Flg(sh)-2M2eh2M2ek resulted in similar antibody response to M2eh and M2ek (p>0.05) in serum and
BAL. Meanwhile it was not significant differences in titers of anti-M2e antibodies post immunization with Flg2M2eh2M2ek and Flg(sh)-2M2eh2M2ek (p>0.05). Intranasal immunization led to generation of specific IgG1 and
IgG2a, with predominant IgG1 (p<0.05), that suggested Th2-type immune response. Induced anti-M2e antibodies
Th2-type immune
response.
Induced
anti-M2e
antibodies
recognized
nativecells
M2einfected with A/PR/8/34 (H1N1) and A/
recognized
native M2e
epitopes
exposed
on the
surface
of the MDCK
epitopes
exposed
on
the
surface
of
the
MDCK
cells
infected
with
A/PR/8/34
(H1N1)
andof mice with recombinant proteins (FlgKurgan/05/05 RG (H5N1). Our results demonstrated that immunization
A/Kurgan/05/05
RG
(H5N1).
Our
results
demonstrated
that
immunization
of
mice
with
2M2eh2M2ek and Flg(sh)-2M2eh2M2ek) resulted in equal protection (90-100% survival), significantly less weight
recombinant
proteinsmice
(Flg-2M2eh2M2ek
Flg(sh)-2M2eh2M2ek)
resulted
in equal
loss
) than control
after lethal and
challenge
with A/PR/8/34
(H1N1).
Immunized mice experienced significant
protection
(90-100%
survival),
significantly
less
weight
loss
)
than
control
mice
after (table 1).
decrease lung viral titers than control mice (p<0.01) at 6th day post challenge
lethal challenge with A/PR/8/34 (H1N1). Immunized mice experienced significant
th
day post challenge (table 1).
decrease
lungtiters
viral titers
than control
(p<0.01)
at 6 challenge
Table
1. Viral
in mouse
lungmice
at 6th
day post
th
Table 1. Viral titers in mouse lung at 6 day post challenge
immunized mice
Flg-2M2eh2M2ek
Flg(sh)-2M2eh2M2ek
PBS (control)
influenza virus
А/PR/8/34 1 LD50
А/PR/8/34 1 LD50
А/PR/8/34 1 LD50
log10 TCID50
1,95±1,2 (р<0,01)
1,4±0,9 (р<0,01)
4,45±0,8
Conclusion. The results show the prospects for development of candidate
influenza vaccine based on truncated form of flagellin and heterologous M2e peptides
with broad spectrum recognition of influenza A viruses of distinct origin.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P120
POSTE R A BSTRAC TS
Conclusions
The results show the prospects for development of candidate influenza vaccine based on truncated form of flagellin
and heterologous M2e peptides with broad spectrum recognition of influenza A viruses of distinct origin.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P121
POSTE R A BSTRAC TS
SPA5P06
A Novel Influenza Vaccination Strategy based on DNA vaccines encoding
M2 ectodomain fused with adjuvant proteins against Pandemic
Influenza Viruses
Yi, Hwajung; Seo, Ki-Weon; Lee, Mi-Seon; Kang, Chun; Kim, Kisoon
Korea National Institute of Health, Korea Centers for Disease Control and Prevention
Background
There has been a growing concern about continuous risk of emerging novel influenza viruses, such as the 2009
pandemic and human infection of avian influenza viruses. Against the pandemic, it is essential to develop and
provide the influenza vaccines rapidly to the public. However, current influenza vaccines are only effective against a
narrow range of influenza strains and need at least 6 months for development and public-distribution for timeliness
of pandemic preparedness. Thus, novel vaccine techniques of rapid response to newly emerged influenza viruses are
required.
Methods
With advantages of DNA vaccine strategy, series of triple heterologous M2e (M2 ectodomain is highly conserved
region between influenza strains) derived from human, avian and swine influenza viruses into pCAG-EN vector
conjugated with various adjuvants (Flagellins, tetanus toxin C fragment, RSV-G proteins, E. coli endotoxin LTB,
Cholera toxin A and B subunits) were prepared and evaluated their immunogenicity.
Results
After confirming the expressions of the M2e series encoded in pCAG-EN in 293T cells by transfection, it was observed
that the thrice immunization of the M2e DNA vaccines into mice could induce the M2e specific antibodies. The
immunization of the M2e series into mice also elicited protective immunity against lethal infection of A/PR8/1934.
Moreover, it was shown that the M2e DNA vaccination conjugated with flagellin and LTB impose mice to more
efficient protection against the PR8 infection.
Conclusions
Only 7 weeks from the design and cloning of M2e series by gene-synthesis into DNA vaccine vector to the
confirmation of vaccine effectiveness to the influenza viruses were taken. These data suggests that DNA vaccine
strategy would be alternative to overcome limitation of the current vaccines based on influenza virus growing in
eggs or animal cells. This work was supported by intramural fund (#2013-NG43003-00) of the NIH, Korea.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P122
POSTE R A BSTRAC TS
SPA5P07
Humoral immunity induced by seasonal influenza vaccine in humans
protects against H5N1 challenge in mice
Roozendaal, Ramon; Tolboom, Jeroen; Roos, Anna; Riahi, Sarra; Theeuwsen, Jessica; Bujny, Miriam V.; Klaren, Vincent; Korse, Hans J.W.M.;
Dekking, Liesbeth; Grootenhuis, Arijan; Weverling, Gerrit Jan; Koudstaal, Wouter; Goudsmit, Jaap; Radošević, Katarina
Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis
Background
The search for vaccines that provide ‘universal’ protection against influenza has been fueled tremendously by the
identification of broadly influenza neutralizing antibodies (bnAbs). The isolation of bnAbs from humans recently
vaccinated with conventional influenza vaccines suggests that such vaccines could, in principle, elicit broadly
protective immunity. To assess the potential of seasonal influenza vaccine to induce broad protection against
influenza we established a novel human-to-mouse serum transfer and challenge model.
Methods
Human subjects in a vaccine safety and immunogenicity trial (INF-V-A017) were immunized three times in total
with a seasonal influenza vaccine. Serum samples were collected immediately prior to and one month after each
vaccination. Passive transfer of human serum into naïve mice was subsequently followed by a lethal challenge of an
either homologous or avian influenza strain. In parallel, we performed serological assays on human serum samples
to determine the titer of functional virus-reactive antibodies and examined to what extent these were correlated to
protection induced by the corresponding serum.
Results
We showed that passive protection of mice with serum from vaccinated humans recapitulates relevant features of
human protection against vaccine homologous influenza strains. Notably, protection against homologous H1N1
influenza was strongly correlated to challenge strain specific HAI and virus neutralization. Moreover, vaccination
of healthy adults induced humoral protection against highly pathogenic avian influenza of the H5N1 subtype. This
protection could not be detected nor accurately predicted by any of the in vitro immunogenicity assays performed.
Conclusions
This study demonstrates the ability of the humoral response induced by seasonal vaccine in humans to confer
protection against H5N1 influenza challenge in mice. The human-to-mouse serum transfer and challenge model
described can be used to assess the protective ability of vaccines in the absence of known in vitro correlates of
protection, and aid the development of ‘universal influenza vaccines’.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P123
POSTE R A BSTRAC TS
SPA5P08
Humoral protection against H5N1 influenza by multiple immunizations
with seasonal influenza vaccine in mice
Roos, Anna; Roozendaal, Ramon; Theeuwsen, Jessica; Riahi, Sarra; Vaneman, Joost; Tolboom, Jeroen; Koldijk, Martin; Dekking, Liesbeth;
Koudstaal, Wouter; Goudsmit, Jaap; Radošević, Katarina
Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis
Background
The development of an influenza vaccine which confers protection against a wide range of influenza viruses is of
great importance. Current seasonal influenza vaccines are believed to confer protection against a narrow range
of virus strains. However, isolation of broadly neutralizing antibodies from humans recently vaccinated with
conventional influenza vaccines suggests that the current vaccines may elicit a broader range of immune reactivity
than anticipated. In the current study we evaluate the ability of a seasonal trivalent influenza vaccine to elicit broad
protection against influenza challenge in mice and further investigate potential in vitro correlates of such protection.
Methods
BALB/c mice were immunized using different vaccination regimens composed of single or multiple immunizations
with seasonal influenza vaccine, or passively immunized by transfer of immune serum. Final immunization or
transfer was followed by a lethal challenge of homologous and avian influenza strains. Challenged mice were
monitored for survival, weight loss and clinical signs for 21 days. Antibody titers were determined in immune serum
and pre- challenge serum of mice after passive transfer and correlated to protection.
Results
Mice immunized with multiple shots of vaccine showed significant survival against heterosubtypic H5N1 influenza.
Immune serum obtained after three shots of vaccine induced a level of survival comparable to active immunization.
Cross-reactive neutralizing antibodies were not detectable for any of the vaccination regimens. Protection against
H5N1 challenge correlated with the level of vaccine-induced rH5 cross-reactive binding antibodies.
Conclusions
Our results demonstrate that repeated vaccination with a seasonal influenza vaccine broadens its range of
protection. Using passive transfer we show that humoral immunity is correlated to protection while virus
neutralizing antibodies are not detectable.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P124
POSTE R A BSTRAC TS
SPA5P09
Improved vaccine-induced immune responses to influenza antigens.
Gilbert, Sarah Catherine (1); Antrobus, Richard D (1); Berthoud, Tamara K (1); Mullarkey, Caitlin E (1); Hoschler, Katja (2); Coughlan, Lynda (1);
Lambe, Teresa (1); Tully, Claire (1); Zambon, Maria (2); Hill, Adrian VS (1)
1: The Jenner Institute, University of Oxford, UK; 2: Respiratory Virus Unit, Public Health England, London, UK
Background
The efficacy of influenza vaccines in adults aged over 65 years is much debated, but is significantly lower than
efficacy in younger adults, and may also decline before the end of the influenza season leaving vaccinated adults
vulnerable to a late surge in infections. Increasing vaccine efficacy in this age group could have a major public health
benefit. Attempts to improve efficacy have concentrated on increasing humoral responses to haemagglutinin (HA)
and have so far had little impact on efficacy. T cell responses to influenza antigens are known to be important in
protection against influenza in this age group, and we tested a method of boosting T cell responses to conserved
influenza antigens at the same time as inducing improved humoral responses to Trivalent Inactivated Vaccine (TIV).
Methods
MVA-NP+M1 is a replication-deficient viral vector expressing influenza nucleoprotein and matrix protein 1. We
vaccinated nine healthy volunteers (mean age 63.8 years) who had not received TIV that season with two vaccines,
TIV and MVA-NP+M1, and a further eight (mean age 59.6 years) with TIV plus saline placebo. We then measured
humoral responses to the three components of TIV by HI titre, and T cell responses to NP and M1 by interferongamma ELISpot assay.
Results
Co-administration of the two vaccines was well tolerated. The group receiving MVA- NP+M1 had significant
increases in T cell responses to NP and M1 whereas only very minor and transient responses were detected after TIV
with saline. HI titres to both influenza A components of the TIV were increased more from pre-vaccination levels in
volunteers receiving MVA-NP+M1 with TIV.
Conclusions
Increasing T cell responses to conserved influenza antigens may have greater impact on influenza vaccine efficacy
than increasing humoral responses to HA alone. Co- administration of MVA-NP+M1 with TIV results in increased T
and B cell responses compared to TIV alone and should now be tested for improved efficacy in older adults who are
in great need of an improved influenza vaccine. Since NP and M1 are highly conserved antigens, strain mismatch
is not expected to reduce vaccine efficacy. The assessment of the breadth of the humoral responses to HA in each
of the study groups is now underway, as the use of adjuvants or viral vectored delivery of HA has previously been
shown to increase the breadth of antibody recognition of HA.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P125
POSTE R A BSTRAC TS
SPA5P10
Evaluation of attenuate and reproductive properties of influenza
A vaccine strain RA35 (H7N9) based on new donor strain A/
HongKong/1/68/162/35.
Vidyaeva, Inna; Potapchuk, Marina; Petrov, Sergei; Sergeeva, Maria; Tsybalova, Liudmila
Research Institute of Influenza, Ministry of Health of the Russian Federation, St. Petersburg, Russia, Russian Federation
Background
Obtained in NII Influenza virus strain A/HongKong/1/68/162/35 (H3N2) (A/HK/ca), combines both properties
attenuate temperature – sensitive (ts) and cold-adapted (ca) and high growth in embryonated eggs (EE). Reassortants
that contain surface protein genes from various wild influenza viruses (H1N1, H3N2, H5N1, H3N8) and internal
protein genes from new vaccine donor strain A/HongKong/1/68/162/35 (H3N2) inherited main donor properties:
high growth in chicken embryos, ts- and ca- phenotype, safety in laboratory animals.
Methods
To obtain a reassortant strain of influenza virus A/Shanghai/HK/6:2 (H7N9) (RA35) we used the A/HK/ca strain as a
donor of internal protein genes. Surface antigens were derived from strain А/Shanghai/2/2013(H7N9) - PR8-IDCDCRG32A, that was kindly presented by CDC, Atlanta, USA. It was used method of classical reassortation in 10- days EE.
Gene constellation was determined by RT-PCR-RFLP analysis. Sequences of HA and NA were studied by RT-PCR with
subtype-specific primers. The degree of attenuation RA35 reassortant investigated by reproduction in EE at different
temperatures.
Results
According to data, of restriction analysis of amplified in RT-PCR viral RNA fragments, reassortant virus
strain A/Shanghai/HK/6:2 (H7N9) (RA35) had 6 genomic segments (PB2, PB1,PA, NP, M, NS) from donor A/
HongKong/1/68/162/35 (H3N2) and 2 (HA, NA) from virus PR8-IDCDC-RG32A strain of virus А/Shanghai/2/2013
(H7N9). It was shown that RA35 strain actively replicates in EE at 32°C, weakly at 26°C and does not replicate at 39°C.
RCT26 was 3.66 lg, RCT32 – 9.5 lg, RCT39 – <0.5 lg. For parent strain A/Shanghai/2/2013(H7N9)-PR8-IDCDC-RG32A
(H7N9) optimal reproduction temperature is 34-35°C (RCT34 – 6.5 lg). So replacement of internal protein genes of
the donor strain A/PR8 genes donor A/HongKong/1/68/162/35 led to the emergence of signs of attenuation and ts
ca. Moreover this reassortant had a higher reproductive activity. In hemagglutination inhibition test it was shown
that RA35 had bound with specific rat immune sera of types A(H7N3) and A(H7N9) only. Аfter a series of 5 passages
in chicken embryos the strain saves its infectious, heagglutinating and antigenic properties either.
Conclusions
Using new vaccine donor strain A/HongKong/1/68/162/35 (H3N2) we have obtained a reassortant strain with
surface antigens from actual high pathogenic influenza virus A(H7N9). The reassortant RA35 (A/Shanghai/
HongKong/6:2 (H7N9) had ca, ts phenotype and high-growth in embryonated eggs. The obtained RA35 strain has
all properties reguired for development as live as inactivated influenza vaccines.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P126
POSTE R A BSTRAC TS
SPA5P11
Development, pre-clinical and clinical evaluation of potentially
pandemic H2N2 live attenuated influenza vaccine
Isakova-Sivak, Irina (1); de Jonge, Jorgen (2); Smolonogina, Tatiana (1); Rekstin, Andrey (1); Kiseleva, Irina (1); Kuznetsova, Victoria (1); Donina,
Svetlana (1); Petukhova, Galina (1); Naykhin, Anatoly (1); Pisareva, Maria (3); Stukova, Marina (3); Erofeeva, Mariana (3); Tsvetnitsky, Vadim (4);
Flores, Jorge (4); Rudenko, Larisa (1)
1: Institute of Experimental Medicine RAMS, Saint Petersburg Russian Federation; 2: Centre for Infectious Disease Control, RIVM, Bilthoven, the
Netherlands; 3: Institute of Influenza, Saint Petersburg Russian Federation; 4: PATH Vaccine Development Global Program, Seattle, Washington,
USA
Introduction
H2N2 influenza viruses disappeared from circulation among humans in 1968 but continue to circulate in wild birds
causing potential pandemic threat. Live attenuated influenza vaccines (LAIV) prepared from avian influenza viruses
are usually poorly immunogenic in humans. Therefore we used human H2N2 wild-type viruses to prepare two LAIV
candidates using a well characterized A/Leningrad/134/17/57 (Len/17) master donor virus (MDV) as the backbone.
Methods
Two H2N2 LAIV candidates were prepared by classical reassortment of wild-type viruses A/Tokyo/3/67 and A/
California/1/66 with an intermediate reassortant virus carrying internal genes of Len/17 MDV and surface genes of
A/New Caledonia/10/99 (H1N1). These candidates were tested in mice and ferrets for attenuation, immunogenicity
and protective efficacy against homologous and heterologous H2N2 challenge. Pre-clinical evaluation also included
assessment of ts/ca phenotypes, genetic stability, antigenicity and toxicity. A/17/California/66/395 LAIV candidate
was studied in phase I clinical trial. 38 healthy adults aged 18 to 49 were randomized at 3:1 ratio to receive two
doses of H2N2 vaccine or placebo with 28 days interval. Nasal and throat swabs were collected prior vaccination
and at days 1 to 6 after first and second immunizations and tested for virus replication and genetic stability. Nasal
swabs and saliva were collected at days 0, 28 and 56 for the assessment of local immune responses. Blood samples
were collected at days 0, 6, 28, 56 and 112 after first vaccination. Antibody immune responses were assessed by HAI
and microneutralization assays (serum) and by ELISA (IgG, IgA in serum, nasal swabs and saliva). Cellular immune
responses were evaluated by the presence of virus-specific CD4+IFNγ+ and CD8+IFNγ+ PBMCs using flow cytometry
cytokine assay.
Results
Both H2N2 LAIV candidates had phenotypical characteristics comparable with Len/17 MDV. Pre-clinical studies
demonstrated safety, genetic stability, immunogenicity, cross-reactivity and protective efficacy of both H2N2
strains. A/California/1/66-based reassortant was more immunogenic and better protective against homologous
and heterologous challenge in ferrets than A/Tokyo/3/67- based candidate. Therefore A/17/California/66/395 was
selected for further clinical trials. A/17/California/66/395 was safe and well-tolerated for healthy adults. 11 of 28
and 19 of 28 volunteers shed the virus after first dose (and 8 of 27 and 20 of 27 after second dose) when tested
by culturing in eggs or qRT-PCR, respectively. Vaccine virus isolates were genetically stable and no mutations were
detected in HA and NA genes. 85.2% volunteers had systemic and/or local antibody immune responses if measured
by any assay. Nearly 30% of the volunteers had significant increases in levels of CD4+ and/or CD8+ virus-specific
IFNγ-secreting cells. Total 88.9% of volunteers had any immune response after one or two doses of H2N2 LAIV.
Conclusions
A/17/California/66/395 H2N2 LAIV candidate was attenuated, immunogenic and effective in pre-clinical trials and
proved to be safe and immunogenic in phase I clinical trial.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P127
POSTE R A BSTRAC TS
SPA5P12
Cross-reactive antibodies and specific CD8 T-cells induced by conserved
consensus HA2 of influenza viruses A/H2N2 delivered with flagellin
Stepanova, Liudmila; Potapchuk, Marina; Kuznetsov, Vasiliy; Sergeeva, Maria; Kovaleva, Anna; Korotkov, Alexandr; Tsybalova, Liudmila
Research Institute of Influenza, St. Petersburg, Russia, Russian Federation
Background
Isolation of cross-reactive monoclonal antibodies specific to conservative epitopes of the second subunit of influenza
virus hemagglutinin (HA2) suggest, that HA2 stem region may be a promising candidate for development of broadspectrum influenza vaccine. Anti-HA2 antibodies can prevent viral fusion step, inhibit viral replication and provide
protection of immunized mice from lethal influenza challenge. Considering that T-cell immunity also plays an
important role in clearance of the virus and decreases the severity of infection. We designed HA2-epitope vaccine on
the basis of flagellin, known to enhance T- and B-cell immune response. The aim of this investigation was to study
the capacity of recombinant protein comprising consensus fragment HA2(35-107) of influenza A/H2N2 viruses
linked to flagellin to induce cross-reactive antibodies and specific CD8+ T-cells.
Methods
We constructed a recombinant fusion protein FLG-HA2 containing consensus sequence of HA2(35-107) of
influenza A/H2N2 viruses linked to the C-terminus of full-length flagellin. The corresponding sequence was
synthesized and cloned in pQE30 vector. FLG-HA2 protein was expressed in E.coli, purified by chromatography
and used to immunize Balb/c(H-2b) and C57BL/6(H-2d) mice. Three intranasal immunizations with 50 μg of
FLG-HA2 were done in 2-week intervals, control mice received PBS. To estimate the cross-reactivity of induced
anti-HA2 IgG antibodies we performed ELISA with purified influenza A viruses: A/Singapore/1/57(H2N2),
A/PR/8/34(H1N1), A/Aichi/1/68(H3N2) and A/Kurgan/5/05 RG(H5N1) and with synthetic linear peptides
corresponding to HA2 regions 35-58(AADKESTQKAFDGITNKVNSVIEK), 59- 73(MNTQFEAVGKEFSNL) and 74107(ERRLENLNKKMEDGFLDVWTYNAELLVLMENERT). Intracellular cytokine staining of IFN-γ was used to study
antigen stimulated CD8+ T-cell activation in splenocytes of mice 10-30 days post immunization. Immunized mice
were challenged with 1LD50 of A/Singapore/1/57(H2N2) and monitored daily for survival and weight loss.
Results
Anti-HA2 antibodies (IgG in serum and IgG, sIgA in BAL) generated after immunization with FLG-HA2 reacted with
mentioned above synthetic peptides and most strongly with 74-107 peptide. Induced antibodies recognized HA2 of
whole influenza A/H2N2 virus as well as of other influenza A viruses from phylogenetic group 1 (H1N1 and H5N1).
We detected significant production of intracellular IFN-γ (p<0.05 as compared with control group) by CD8+ T-cells in
spleen of immunized Balb/c and C57BL/6 mice after stimulation by each of mentioned HA2 peptides and by whole
virus. We also observed significant reduction in weight loss and increase in survival rate for immunized mice upon
influenza A/H2N2 lethal challenge.
Conclusions
The data obtained shows that recombinant protein FLG-HA2 induce cross-reactive antibodies which can bind to
phylogenetic group 1 influenza A viruses and most strongly to HA2 peptide 74-107, that contains YNAELLVLMEN
sequence, shared by majority of experimental CD4+ and B-cell epitopes found in HA2. Furthermore FLG-HA2 induce
specific CD8+ T-cells. Thus immunization with FLG-HA2 can provide complete and cross-reactive immune response
against influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P128
POSTE R A BSTRAC TS
SPA5P13
Adjuvation broadens the protective efficacy of a seasonal influenza
vaccine in mice
Cox, Freek; Saeland, Eirikur; Baart, Matthijs; Koldijk, Martin; Goudsmit, Jaap; Radosevic, Katarina
Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, The Netherlands
Background
It remains a high priority to establish universal immunity to influenza by vaccination. Licensed seasonal influenza
vaccines induce strain-specific immunity and must be updated annually based on predicted strains that will
circulate in the upcoming season. Viral Hemagglutinin (HA) and Neuraminidase (NA) contribute to strain variability,
but these antigens also contain conserved epitopes that may be targeted by vaccination. In the current study, we
aim to broaden the protective efficacy of a seasonal influenza vaccine, in particular a Trivalent Virosomal Vaccine
(TVV, containing H1N1, H3N2 and B strains of the 2010/2011 season), by adjuvation with a saponin-based adjuvant
(Matrix-M).
Methods
Balb/c mice were immunized with TVV with or without Matrix-M three weeks apart. Four weeks after the final
immunization, mice were challenged with heterologous and avian influenza strains. Challenged mice were
monitored for survival, weight loss and clinical signs for 21 days. A separate experiment was performed where HAspecific antibody titers and Hemagglutination inhibition (HAI) titers were measured in serum samples of immunized
mice and IFN- producing T cells were measured by ELISPOT.
Results
Mice immunized with Matrix-M adjuvated TVV showed significant survival after challenge with heterologous
and avian influenza strains compared to the vehicle control whereas mice immunized with TVV alone did not.
Furthermore, TVV+Matrix-M immunized mice showed reduced body weight loss and clinical scores compared to the
vehicle control. Matrix-M enhanced both antibody and T cell responses induced by TVV against the influenza strains
used for challenge.
Conclusions
Our results demonstrate that adjuvation may effectively broaden the protective efficacy of a virosomal seasonal
influenza vaccine.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P129
POSTE R A BSTRAC TS
SPA5P14
Universal influenza vaccine development based on the MVA vector
platform
Altenburg, Arwen F.; Kreijtz, Joost H.C.M.; de Vries, Rory D.; De Gruyter, Heidi L.M.; Osterhaus, Albert D.M.E.; Rimmelzwaan, Guus F.
Erasmus MC, The Netherlands
Due to the antigenic drift of influenza viruses, current influenza vaccines need to be updated annually. These seasonal
influenza vaccines are based on the prediction of which strains will most likely circulate next season. However, if
the circulating strains do not match the strains used in the vaccine, the efficacy of the vaccine is greatly reduced.
Furthermore, in the case of a pandemic outbreak vaccines prepared with the matching virus strain often becomes
available too late. This illustrates the need for the development of a universal influenza vaccine that induces long
lasting and broadly protective immunity. Ideally, universal influenza vaccines induce both cross- reactive antibody
and T cell responses. In order to achieve this goal, we have made various modifications to conserved influenza virus
proteins to improve antigen processing and presentation to virus specific T cells. To improve cross-reactive antibody
responses to the hemagglutinin also this protein was modified to redirect the antibody response from the globular
head region to the more conserved stalk region. The modified antigens are expressed and presented to the host’s
immune system using the replication deficient Modified Vaccinia virus Ankara (MVA). MVA has proven to be a safe
method for antigen delivery and has been shown to be able to induce both T-cell as well as B-cell responses. The
various vaccine candidates are characterized in vitro to assess if the modifications have the desired effect. Next,
the vaccine candidates are tested for their potential to induce protective immunity against a lethal challenge with
homologous/heterologous influenza viruses in mice.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P130
POSTE R A BSTRAC TS
SPA5P15
Influence of different schedules of chicken immunization with DNA
vaccine against avian influenza H5N1 virus on antibody response
Góra-Sochacka, Anna; Stachyra, Anna; Sirko, Agnieszka
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland
Background
H5N1 virus is the causative agent of highly pathogenic avian influenza which leads to severe economic losses in
poultry industry. For the prevention and control of influenza infections vaccination is preferred strategy. Our study
is focused on DNA vaccine which is consider as safety, new generation vaccine consistent with DIVA strategy. We
engineered DNA vaccine for chickens against avian H5N1 influenza virus and analyzed humoral response after
its application. Different immunization schedules enabled us to compare dynamics of chicken humoral response
depending on the number of doses (one or two) and on the booster time (two or three weeks after the priming).
Methods
Mixure of plasmid carrying cDNA encoding hemagglutinin (HA) from H5N1 A/swan/Poland/305-135V08/2006
(EpiFluDatabase [http://platform.gisaid.org]; Accession No. EPI156789) with Lipofectin was used as a vaccine.
Broiler (Ross 308) chickens were immunized with DNA vaccine intramuscularly according to the following schemes:
(Scheme 1) immunization on day 7, (Scheme 2) immunization on days 7 and 21, (Scheme 3) immunization on days
7 and 35. The control group was immunized with mixture of the empty vector with Lipofectin. Regardless of the
scheme, the blood samples were collected four times: (i) two weeks after the first vaccination, (ii) on the day of a
booster, (iii) one week and (iv) two weeks after the booster. Indirect ELISA was performed for detection of anti-HA
IgY antibodies in blood samples.
Results
The level of anti-HA antibodies in serum samples of chickens immunized only once with DNA vaccine was marginally
higher than in control chickens (immunized with empty vector), and was shown as statistically insignificant. The
meaningful rise of specific antibodies was observed one week after the second (booster) immunization regardless of
the schedules. The significant level of antibodies was observed till the end of experiment (49th day of chickens life).
Conclusions
Comparison of three different immunization schedules indicated the need of booster dose of vaccine to achieve
efficient humoral response protecting chickens against influenza virus.
Acknowledgments: This work was supported by Innovative Economy Program, Grant No. WND POIG.01.01.02-00007/08.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P131
POSTE R A BSTRAC TS
SPA5P16
A universal influenza virus vaccine strategy based on the conserved stalk
domain of the hemagglutinin
Krammer, Florian (1); Margine, Irina (1,2); Nachbagauer, Raffael (1); Pica, Natalie (1,2); Hai, Rong (1); Albrecht, Randy A. (1,4); García-Sastre,
Adolfo (1,3,4); Palese, Peter (1,3)
1: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA; 2: Graduate School of Biological Sciences,
Icahn School of Medicine at Mount Sinai, New York, New York, USA; 3: Department of Medicine, Icahn School of Medicine at Mount Sinai, New
York, New York, USA; 4: Global Health & Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
Background
Influenza virus infections remain a significant cause of morbidity and mortality worldwide. Current vaccines show
good efficacy against antigenically matched viruses by inducing strain specific antibodies against the membranedistal globular head domain of the viral hemagglutinin, but fail to protect against drifted and pandemic strains.
Due to the rapid antigenic drift of influenza viruses - especially in the globular head domain - these vaccines have
to be re- formulated, generated and administered through a cumbersome and expensive process every year. The
membrane-proximal stalk domain of the viral hemagglutinin exhibits a high degree of both sequence and structural
conservation across influenza virus subtypes and monoclonal antibodies directed against this region typically show
broad neutralizing activity. However, these antibodies are rare and usually not induced/boosted by regular seasonal
vaccines. We hypothesize that a vaccine strategy that stimulates a robust immune response towards this region of
the hemagglutinin could provide universal influenza virus protection.
Methods
We developed a universal influenza virus vaccine based on the conserved stalk domain of group 1 and group 2
hemagglutinins. By sequential vaccination of mice with chimeric hemagglutinin constructs that share the same
stalk domain but have divergent head domains we were able to specifically boost broadly neutralizing antibody
titers against conserved epitopes in the hemagglutinin stalk.
Results
Mice vaccinated with our constructs were protected from morbidity and mortality induced by infection with a panel
of heterologous and heterosubtypic influenza A viruses. In the light of emerging viruses in Asia it is of note that our
vaccination regimen also protected animals from H6N1 and H7N9 virus challenges and reduced lung titers upon
H10 virus infection. In addition the chimeric HA based vaccination regimen also showed efficacy in ferrets, induced
high titers of broadly reactive antibodies against divergent hemagglutinins from different subtypes and significantly
reduced transmission in this model. Finally, we showed that stalk-reactive antibodies were boosted in individuals
that received an H5N1 vaccine in clinical trials. This supports the hypothesis that exposure to hemagglutinins with
divergent heads but conserved stalk induces such antibodies in humans.
Conclusions
The present data suggest that this vaccine strategy could be successfully developed in humans to provide broad
influenza virus protection and enhance our pandemic preparedness. A universal influenza virus vaccine, which similar to the ones developed for polio and measles viruses - requires a single or only a few immunizations, would
represent a major advance towards the control of influenza worldwide.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P132
POSTE R A BSTRAC TS
SPA5P17
Cost-effectiveness of quadrivalent versus trivalent influenza vaccine in
Germany: a Markov multi-cohort model
Poulsen Nautrup, Barbara (1); Meier, Geneviève (2); Anastassopoulou, Anastassia (3); Welte, Robert (3); Gregg, Meghann (2)
1: EAH-Consulting, Aachen, Germany; 2: GlaxoSmithKline Vaccines, Wavre, Belgium; 3: GlaxoSmithKline GmbH & Co. KG, München, Germany
Background
Co-circulation of influenza B lineages and mismatch in influenza vaccines can cause increased burden of disease
in a population. To minimize this burden a quadrivalent inactivated influenza vaccine (QIV) has been developed
including virus strains of two A sub-types and of both B lineages. This analysis estimates the cost-effectiveness of
adopting a quadrivalent vaccine versus the current trivalent vaccine (TIV) in Germany.
Methods
A Markov model was used to estimate cost-effectiveness. Cycle length was one year with a life- time horizon (100
years). A payer and societal perspective were taken and the vaccinated population is the entire German population
(considering reported vaccination coverage rates). Cost of QIV was €12.32, TIV €10.21 (average of TIV vaccines
in Germany). A meta-analysis was used to inform vaccine efficacy. Other input data was sourced via a targeted
literature review from peer-reviewed and government publications. Costs and effects were discounted at 3%.
Sensitivity analysis was conducted (univariate, scenario, and probabilistic). Published suggestions for incorporation
of indirect effects into static models were used for the approximation of the impact of a herd effect associated with
the vaccination of children in scenarios.
Results
The Incremental Costs Effectiveness Ratio (ICER) was €25,921 (payer perspective) and €17,628 (societal perspective).
It is estimated that in an average influenza season vaccinating with QIV instead of TIV averts 52,105 cases, 15,462
first line medical advice visits, 6,063 influenza complications, 1,408 hospitalisations, and 119 deaths. The most
sensitive model parameters were circulation of influenza A and matching of B lineage to trivalent vaccine. At a
€50,000 threshold, probabilistic sensitivity analysis estimates 85% of simulations to be cost- effective (payer),
and 90% (societal). Scenarios considering universal mass vaccination (at a coverage rate of 75%) of all individuals
<18 years yield an ICER of €25,571 (payer) and €13,044 (societal) without consideration of a herd effect. When
considering a generic or influenza-based herd effect, ICERs decrease to €23,563 or €17,704 (payer) and €10,651 or
€3,186 (societal), respectively.
Conclusions
Given a €50,000/quality adjusted life year (QALY) gained threshold the quadrivalent vaccine could be considered
cost-effective. Vaccine mismatch due to influenza B and co-circulation of B lineages has occurred in Germany in six
out of ten seasons (2000-2010). It is estimated that a quadrivalent vaccine would reduce the burden of influenza
in mismatch and co-circulation years. The estimated reduction being more prominent considering a herd effect
associated with vaccinating children.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P133
POSTE R A BSTRAC TS
SPA5P18
Evaluation of the campaign’s impacts of influenza vaccination in the
productivity of workers of brazilians industries
Mattos, Bianca Ramos de Freitas (1); Araujo, Gabriela Tannus Branco (2); Fonseca, Marcelo Cunio Machado (2); Sansone, Dayan (2); Homsani,
Sheila (3); Bricks, Lucia Ferro (3); Liao, Paulo Sti Lin (3); Guarino, Hubert (3)
1: SESI - Social Service of Industry, Brazil; 2: Axia.Bio Consulting; 3: Sanofi Pasteur
Background
Flu is still a big cause of absenteeism and presenteeism in Brazil. SESI (Social Service of Industry) is a private
Nationwide organization, supported by brazilian industries, acting since 1946, with the objective of promoting
better education and workplace safety and health. In 2013, SESI had a National Campaign against Flu, on all of 27
brazilian States with the objective of evaluate the economic and social impacts of flu vaccination and the return of
investment for brazilian Industries.
Methods
SESI provided flu vaccine to more than 900.000 workers distributed in 5.100 industries Nationwide, however, only
662.763 workers provide complete data to traceability. The cost-benefit analysis was made and validated through
mathematic model (Vaxincorp®), allowing the evaluation of the cost’s investiment on the vaccination campaign
and consequent absenteeism and presenteeism reduction. Some industries also provided economics internal data
for analysis. The sample used was 85 industries, 13 provided complete data, and 72 large industries used data from
research published in the Brazilian Magazine Exame – bigest and best of 2012, such as, operating profit, salary,
missing work among others.
Results
Data from Vaxincorp®, added with data from participating companies demosntrated that influenza vaccination
avoided 63.000 flu cases and 136.080 days of lost productivity, wich 22.680 days off per medical license and 113.400
presenteeism days. The total return of investiment was EUR 35,31 for vaccinated worker, which allows a prospective
analysis of EUR 23.426.932,80 for bazilian industries that participated on SESI’s campaign.
Conclusions
Based on theses results, Influenza Vaccination in companies is an action of health that generates a positive costbenefit analysis and should be considered annually, regardless of company size.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P134
POSTE R A BSTRAC TS
SPA5P19
Cross-protection by human and avian M2e-displaying virus-like particles
Christopoulou, Ioanna (1,2); Schotsaert, Michael (1,2); Roose, Kenny (1,2); Fiers, Walter (1,2); Saelens, Xavier (1,2)
1: VIB Inflammation Research Center, Technologiepark 927, Ghent, Belgium; 2: Department for Biomedical Molecular Biology, Ghent
University, Technologiepark 927, Ghent, Belgium
Background
Influenza is a global burden. The vaccines currently in use protect through the induction of neutralizing antibodies
against haemagglutinin (HA) and neuraminidase (NA) but need almost yearly update due to significant antigenic
variation (antigenic drift). In our lab we have developed a universal influenza A vaccine based on the extracellular
domain of matrix protein 2 (M2e) which is highly conserved. Here we assessed the potential cross-protection, using
a mouse model, of immunization with human(h) or avian(a) derived M2e against heterologous challenge.
Methods
Female BALB/c mice (SFP, 6-8 weeks old) were immunized twice intraperitoneally with either hM2e-VLP, aM2e-VLP,
or VLP alone. After the second immunization, 0,5LD50 of mouse-adapted X47 (H3N2) or A/Mallard/Alberta/676/79
(H3N6) viruses was administered intranasally to these mice. Mice were monitored for 14 days post challenge for
mortality/morbidity. Virus titers were measured in the lungs, T cell responses were analyzed by flow cytometry and
IFNγ producing cells were measured by ELISPOT.
Results
In a BALB /c mouse model, vaccination with hM2e-VLP or aM2e-VLP resulted in the induction of M2e specific serum
IgG antibody titers, strongly reduced morbidity and conveyed full protection after a A/Mallard/Alberta/676/79
(H3N6) or X47 (H3N2) challenge. We show that immunization reduced virus replication in the lungs, with the
hM2e-VLP being more effective in controlling virus replication than aM2e-VLP. IFNγ producing cells were also
highly induced with a 20-fold increase in the hM2e-VLP immunized group compared to control. Interestingly mice
immunized with hM2e-VLP had better IFNγ response when challenged with the avian like virus H3N6 compared to
aM2e-VLP immunized mice that had been challenged with the same virus. Finally, T cell response analysis showed
that TNFα and IFNγ production is augmented in both vaccinated groups.
Conclusions
We showed that vaccination with human or avian derived M2e-VLP universal vaccine protects against challenge
with viruses of different origin, as mice immunized with hM2e were equally protected against human and avian
origin influenza A viruses, whereas mice immunized with aM2e were also protected against avian influenza virus
and to some extend against human influenza A virus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P135
POSTE R A BSTRAC TS
SPA5P20
Mice immunization against influenza with three variants of DNA vaccine
encoding hemagglutinin.from H5N1.
Redkiewicz, Patrycja (1); Góra-Sochacka, Anna (1); Kosson, Piotr (2); Sirko, Agnieszka (1)
1: Institute of Biochemistry and Biophysics, PAS, Poland; 2: Mossakowski Medical Research Center PAS, Poland
Background
Our study was focused on the improvement of hemagglutinin (HA from H5N1) antigen expression in mammalian
cells in order to enhance its immunogenic potential as a DNA vaccine against influenza virus. For these purposes
three variants of the plasmids containing modified nucleotide sequence of HA were prepared and their immunogenic
potential was tested in mice.
Methods
4-6 weeks old BALB/c mice were immunized two times intramuscularly in the quadriceps muscle at two weeks
intervals with either 50 μg or 10 μg of plasmid DNA complexed with the lipid carrier (lipofectin). Two weeks after the
prime dose, one week after the boost dose and two weeks after the boost dose the blood samples were collected and
sera were prepared. Mice were scarified and spleens were isolated four weeks after the first dose. HA-specific IgG
titers in sera samples were determined by indirect ELISA. The proliferation ability of murine splenocytes stimulated
with commercial hemagglutinin H5 from A/Bar-headed Goose/Qinghai/12/05 H5N1 strain was determined using
CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS).
Results/ Conclusion
Both vaccine doses (50 μg and 10 μg) of all three tested plasmids elicited HA-specific IgG antibody response.
However, the level of the response depended on the variant and the dose. Additionally, the preliminary study of
splenocytes proliferation confirmed that the tested vaccines elicited the T-cell proliferative responses.
Acknowledgments: This work was supported by Innovative Economy Program, Grant No. WND- POIG.01.01.02-00007/08.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P136
POSTE R A BSTRAC TS
SPA5P21
Assessment of the public health benefit of quadrivalent influenza
vaccine in the United Kingdom from 1996–97 to 2008–09 seasons
Matias, Goncalo (1); Taylor, Robert J. (2); Haguinet, Francois (1); Kim, WonGyu Lewis (2); Lustig, Roger (2); Fleming, Douglas M. (3)
1: GlaxoSmithKline Vaccines, Wavre, Belgium; 2: Sage Analytica, Bethesda, MD, USA; 3: Independent Consultant, Birmingham, UK
Background
Influenza B burden might be reduced by the use of quadrivalent influenza vaccines (QIV, containing both circulating
B lineages, Yamagata and Victoria) instead of the current trivalent influenza vaccines (TIV, containing only one B
lineage). This study (ClinicalTrials.gov: NCT01520935) assessed the potential of QIV to reduce influenza B-attributable
events by preventing influenza B lineage mismatch in the United Kingdom (UK).
Methods
The hypothetical decrease in influenza B burden was estimated through a simple multiplier model based on Reed’s
probability model (Reed et al, Vaccine 2012). That model acted on seasonal burden estimates obtained by applying
Background: Influenza B burden might be reduced by the use of quadrivalent influenza
virus-guided regression models to time
series
ofcontaining
general both
practice
(GP)B visits,
hospitalizations
and instead
deathsoffor
vaccines
(QIV,
circulating
lineages,
Yamagata and Victoria)
the a
current trivalent
influenza
(TIV, Practice
containing Research
only one Database
B lineage). (GPRD),
This study
respiratory disease broadly defined outcome
extracted
fromvaccines
the General
the
(ClinicalTrials.gov: NCT01520935) assessed the potential of QIV to reduce influenza BHospital Episode Statistics (HES) database
andevents
the Office
of National
(ONS) in
mortality
For GP
attributable
by preventing
influenza Statistics
B lineage mismatch
the United database.
Kingdom (UK).
visits, the outcome definition included all respiratory diagnoses, respiratory symptoms, viral infections not otherwise
Methods: The hypothetical decrease in influenza B burden was estimated through a simple
specified (NOS) and otitis media; for hospitalizations
andondeaths,
the definition
respiratory
diagnoses,
multiplier model based
Reed’s probability
model included
(Reed et al,all
Vaccine
2012). That
model
acted on seasonal burden estimates obtained by applying virus-guided regression models to
viral infections NOS and septicemia (any
mention).
Summer
weeks
were excluded
model.
time
series of general
practice
(GP) 23–35
visits, hospitalizations
and from
deathsthe
for aregression
respiratory disease
broadly
defined Boutcome
extracted
from the
General Practice
Research
Database
the
The study used seasonal proportions of
influenza
from Public
Health
England,
vaccine
coverage
data (GPRD),
from GPRD
Hospital Episode Statistics (HES) database and the Office of National Statistics (ONS) mortality
and the vaccine effectiveness values used
by Reed
et al
(47%
matched
andincluded
0% forall
mismatched
seasons).
database.
For GP
visits,
thefor
outcome
definition
respiratory diagnoses,
respiratory
Results
symptoms, viral infections not otherwise specified (NOS) and otitis media; for hospitalizations
and deaths, the definition included all respiratory diagnoses, viral infections NOS and
septicemia (any mention). Summer weeks 23–35 were excluded from the regression model.
The study used seasonal proportions of influenza B from Public Health England, vaccine
studied seasons were B-mismatched,
B and
proportions
of 1%–67%;
there
was
one etB-dominant
coverage data fromwith
GPRD
the vaccine effectiveness
values
used
by Reed
al (47% for
matched
and
0%
for
mismatched
seasons).
season (2005–06). In an average season, 8,333 GP visits, 139 hospitalizations and 35 deaths were
6 of the 13
mismatched
estimated as preventable by QIV versus
TIV (table).
The
percentages
of preventable
GP B
visits,
hospitalizations
and
Results:
6 of the 13
studied
seasons were
B-mismatched, with
proportions
of 1%–67%; there
was one B-dominant mismatched season (2005–06). In an average season, 8,333 GP visits,
deaths by QIV versus TIV among all influenza
B-attributable corresponding events were highest in ≥65-year-olds,
139 hospitalizations and 35 deaths were estimated as preventable by QIV versus TIV (table).
The percentages of preventable GP visits, hospitalizations and deaths by QIV versus TIV
during 2005–06 (36% each).
Conclusions
among all influenza B-attributable corresponding events were highest in ≥65-year-olds, during
2005–06 (36% each).
Conclusions: The potential benefit of QIV depends on influenza B circulation, degree of
The potential benefit of QIV dependsvaccine
on influenza
circulation,
degree
of on
vaccine
and vaccine
coverage.
match andBvaccine
coverage.
However,
average,match
under reasonable
assumptions
of
vaccine effectiveness, substantial numbers of influenza-related GP visits, hospitalizations and
However, on average, under reasonable
assumptions of vaccine effectiveness, substantial numbers of influenzadeaths were estimated to be possibly QIV-preventable during the study period in the UK.
related GP visits, hospitalizations and deaths were estimated to be possibly QIV-preventable during the study period
Funding: GlaxoSmithKline Biologicals SA
in the UK.
Funding
GlaxoSmithKline Biologicals SA
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P137
POSTE R A BSTRAC TS
SPA5P22
Evaluation of immunogenicity and cross-protective efficacy of MVAtor™based universal influenza vaccine candidates in mice against H1N1,
H5N1 and H7N9 influenza strains
Auer, Sebastian; Wachs, Frank-Peter; Leyrer, Sonja
Emergent Biosolutions - Emergent Product Development Germany GmbH
Background
The current vaccination strategy against influenza virus consists of a live- attenuated or killed virus vaccine regimen
containing two influenza A subtypes and one or two B subtypes which are predicted to be most prevalent in the
upcoming influenza season. This approach requires adaptation of the vaccine every year and protective efficacy can
vary due to mismatches caused by strain mutation and evolution. Thus, there is a high demand for the development
of a universal vaccine that provides protection against all types of influenza viruses, including emergent pandemic
strains. This requires the identification and utilization of conserved regions in influenza proteins which show no or
only low antigenic variability by strain or over time, and directing the immune system to develop antibodies against
these conserved portions.
Emergent BioSolution has developed several MVAtorTM (modified vaccinia virus Ankara vector) based vaccine
candidates expressing conserved influenza A antigens. The tested antigens include nucleoprotein (NP), a
combination of six different external domains of matrix protein 2 (M2) designated multi-epitope tandem repeat
(METR) and variants of hemagglutinin (HA).
Methods
Recombinant MVAtor vaccine candidates were generated by insertion of influenza virus conserved antigens NP, METR
and HA in the MVA DelIII insertion site by homologous recombination. Vaccine candidates were plaque purified and
stocks were generated on chicken embryo fibroblast (CEF) cells or on the duck cell line AGE1.CR.pIX. Expression of
influenza antigens was confirmed by Western Blot, Direct ELISA or RT-PCR, and genomic stability of recombinant
MVAtor constructs was verified by sequential passaging on the production cell line and insert sequencing.
For a murine challenge study, BALB/c mice were immunized intramuscularly twice with 1x108 TCID50/animal on
days 1 and 22 with different combinations of vaccine candidates in homologous and heterologous prime/boost
regimens or controls. Vaccinated mice were challenged on day 43 with H1N1 A/Puerto Rico/8/34 (5/50 LD50), H7N9
A/Anhui/1/2013 (5/50 LD50), and H5N1 A/Vietnam/1203/2004 (10/100 LD50). Immune response, survival, weight
loss, and viral load in lungs were assessed.
Results
All generated recombinant MVAtor universal flu constructs were grown to high titer stocks and demonstrated good
growth rates comparable to the parental MVAtor vector. In vitro analysis of the vaccine candidates by Western Blot,
Direct ELISA or RT-PCR showed high expression levels of the antigens and the expected molecular weights for all
constructs. In a genomic stability analysis after six sequential passages on the production cell line AGE1.CR.pIX all
constructs showed full insert integrity and displayed no deletions or single nucleotide polymorphisms, indicating
genomic stability for at least six passages. Preliminary murine immunogenicity and protective efficacy studies
showed partial to full protection against various influenza strains. Confirmative murine in vivo efficacy studies are
in progress.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P138
POSTE R A BSTRAC TS
SPA5P23
Prime-boost DNA vaccination protects chickens against challenge with
homologues and heterologues H5N1 virus
Stachyra, Anna (1); Smietanka, Krzysztof (2); Minta, Zenon (2); Gora-Sochacka, Anna (1); Sirko, Agnieszka (1)
1: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Poland; 2: National Veterinary Research Institute, Poland
Background
DNA vaccination is a promising strategy to control and prevent the influenza virus circulation among domestic
birds. It fulfills a DIVA strategy and eliminates need for handling of highly pathogenic, zoonotic virus, it is relatively
easy and fast to produce. Despite the fact that many efforts were put in development and improvement of the DNA
vaccines immunogenicity only a few veterinary products have been registered to date and no human DNA vaccine
is available. In our study we optimized the nucleotide sequence of the main influenza antigen (HA) to improve
efficiency of translation and RNA stability. The DNA in our experimental vaccine was complexed with liposomal
carrier to improve the uptake of the plasmid by the host cells.
Methods
DNA vaccine encoding hemagglutinin from the H5N1 strain A/swan/Poland/305-135V08/2006 of influenza virus
was tested in near to field conditions on broiler chickens in prime-boost strategy in several combinations. The gene
had a deletion of the region encoding amino acids located at the proteolitic cleavage site and the codons were
optimized to chicken. The immunological potential of the vaccine was proved by ELISA and HI tests and the most
promising vaccination schedule (two doses, two weeks apart) was selected. Subsequently, the protective activity of
the HA-DNA vaccine was confirmed. Experimental infection of SPF chickens was conducted 3 or 8 weeks after the
second dose of the vaccine, using homologues and heterologues highly pathogenic virus strains to test the cross
protection. Besides serological tests, the level of viral RNA was determined by RT-PCR in swab samples.
Results
Challenge experiment turned up that 100% of vaccinated chickens were protected against H5N1 from the same
clade, without any clinical symptoms, whereas 70% of vaccinated chickens were protected against H5N1 from
distinct clade. Additionally, shedding of the virus was not detectable in the case of chickens challenge 3 weeks after
the booster or it was significantly reduced in the case of the chickens challenged 8 weeks after the boost.
Conclusions
Our experimental DNA vaccine has high efficacy, ensure partial cross protection and also limits transmission of the
HPAI virus to the control contact chickens.
Acknowledgments: This work was supported by Innovative Economy Program, Grant No. WND- POIG.01.01.02-00007/08.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P139
POSTE R A BSTRAC TS
SPA5P24
Characterization of T cell responses induced by Flu SAM®(NP) and
SAM®(M1) vaccines
Magini, Diletta (1,2); Buccato, Scilla (2); Mangiavacchi, Simona (2); Giovani, Cinzia (2); Tavarini, Simona (2); Maione, Domenico (2); Brito, Luis
(3); Nuti, Sandra (2); Mason, Peter (3); Settembre, Ethan (3); Montomoli, Emanuele (1); Geall, Andrew (3); Brazzoli, Michela (2); Bertholet, Sylvie
(2)
1: University of Siena, Italy; 2: Novartis Vaccines Srl, Siena; 3: Novartis Vaccines, Cambridge
Background
Vaccination remains the most cost-effective way to prevent and control Influenza outbreaks. However current flu
vaccines, including the two main surface antigens (HA and NA), do not provide efficacious heterosubtypic immunity.
Therefore, the development of a successful universal vaccination strategy is urgently needed. To achieve this goal,
several strategies are being tested. One approach is based on the induction of a T cell based response versus internal,
more conserved flu antigens, such as the Nucleoprotein (NP) and the Matrix protein 1 (M1).
The aim of this study is to improve the current HANA-based vaccines by including non-viral delivery of self-amplifying
mRNA encoding the NP and M1 antigens.
Methods
Self-amplifying RNA replicons expressing the NP and M1 antigens of the Influenza virus A/PR/8/34 H1N1 were
produced and tested for efficiency of self-amplification and antigen expression by flow cytometry. Immunogenicity
of SAM®(NP) and SAM®(M1) vaccines were further evaluated by characterizing T cell responses to NP and M1
antigens by flow cytometry.
Results
We demonstrate that both SAM(NP) and SAM(M1) vaccines are immunogenic in Balb/c mice. SAM(NP) vaccine
induced IFN-γ+ and LAMP-1+ NP-specific CD8+ T cells as well as multifunctional NP-specific CD4+ T cells. Furthermore,
we observed the development of NP-specific IFNγ+/TNFα+ Effector and Central Memory CD8+ T cells, in addition to
IL2+/TNFα+ memory CD4+ T cells. On the other hand, SAM(M1) vaccine induced a robust CD4 T cell-based response,
including Ag- specific multifunctional Effector and Central memory CD4+ T cells, but no CD8 T cells in Balb/c mice.
Experiments are currently ongoing to characterize how SAM(NP) and SAM(M1) vaccines may modify the phenotype
and intensity of the immune response induced by a HA protein-based vaccine. Furthermore, we are assessing the
possible contribution to protection of the individual and combined replicons to a HANA based vaccine.
Conclusions
SAM(NP) and SAM(M1) vaccines are immunogenic and induce T-cell immune responses that represent potent
mediator of heterosubtypic immunity, therefore they are promising antigens in the designing a cross-protective
flu vaccine. Since the SAM® vaccine technology offers several advantages compared to DNA or viral vector-based
vaccines, it may be useful in the development of universal flu vaccine candidates.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P140
POSTE R A BSTRAC TS
SPA5P25
Timerized Hemagglutinin Stalk Domain Based Vaccine Protect against
Canine Influenza Virus
Yeom, Minjoo (1,2); Na, Woonsung (1,2); Hong, Minki (1,2); Kim, Doo-Jin (1); Jung, Dae-Gwin (1,2); Kim, Jung-Ki (3); Kim, Sang-Hyun (1); Song,
Daesub (1,2)
1: Korea Research Institute of Bioscience and Biotechnology, Korea, Republic of (South Korea); 2: University of Science and Technology; 3: Korea
University Sejong campus
Background
The avian origin canine influenza virus H3N2 was reported in South Korea in 2007. Canine influenza H3N2 virus
has continually circulated in dogs’ population. During recent canine influenza surveillance, a novel H3N1 canine
influenza virus (CIV) that is a putative reassortant between pandemic H1N1 2009 and H3N2 CIVs was isolated.
Highly Effective whole virus killed vaccine is being commercialized. However Killed vaccine has limited protection
against heterologous viruses. To broaden the protective range against newly emerged reassortant viruses, highly
conserved region of influenza antigenic protein might be important for developing broad-spectrum influenza
vaccines. Recently, hemagglutinin stalk protein (HA2), stem region of influenza HA protein has gained increasing
attention, because of the conserved stalk based antibodies are able to broadly protect against wide spectrum of
influenza virus subtypes. Here, we successfully expressed partial hemagglutinin stalk proteins from PR8 H1N1
influenza virus. To increase immunogenicity, trimerized stalk domain protein was used for vaccine antigen. In this
study, the immunogenicity and efficacy of trimerized stalk domain (3xHA95) based vaccine were evaluated. Also the
synergistic effect after adding of 3xHA95 with canine influenza H3N2 vaccine was evaluated after virulent challenge
with canine H3N2 and pandemic H1N1 viruses.
The aim of this study is to improve the current HANA-based vaccines by including non-viral delivery of self-amplifying
mRNA encoding the NP and M1 antigens.
Methods
To evaluate vaccine efficacy, guinea pigs were randomly assigned to each groups. Each groups immunize twice
with vaccines contain inactivated whole virus, monomeric hemagglutinin stalk protein (mHA95), trimerized
hemagglutinin stalk protein(3xHA95) and trimerized hemagglutinin stalk protein added inactivated whole virus.
2 weeks after last immunization, challenge with canine H3N2 and pandemic H1N1 viruses. Blood samples were
collected at 0, 14, and 28 days after first immunization and nasal swab samples for the detection of viral shedding
were also collected daily for 8 days post-inoculation (DPI)
Results
The guinea-pigs with the purified stalk domain based vaccine injections intramuscularly 2 weeks apart successfully
produced stalk-specific antibodies. Serological investigation by an ELISA assay indicated that a significant increase in
antibody titer was displayed 2 weeks after the second vaccination. Especially 3xHA95 antibody titer was significantly
higher than that of HA95 monomer. Also we found that vaccinated guinea-pigs showed low viral shedding titers
and shorter shedding duration with lower loads than non-vaccinated controls’.
Conclusions
We successfully purified partial antigenic HA stalk domain protein from PR8 and conducted its vaccine efficacy study
for influenza virus challenges in guinea-pigs. In vaccinated animals, protective vaccine efficacy was observed against
canine H3N2 and pandemic H1N1 viruses. Our results are meaningful for further understanding of an applicable
range of the purified HA stalk domain protein and development of broad-spectrum vaccines, and this evokes further
studies in dogs which is co-infected with H3N2 CIV and H1N1 influenza virus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P141
POSTE R A BSTRAC TS
SPA5P26
Increased Hemagglutinin Yield of Influenza Vaccine Viruses by
Incorporation of Homologous PB1 Gene
Adam Johnson (1), Emily Winne (2), David Cureton (1), Callie Ridenour (1), Wanda Santana (2), Taejoong Kim (1), John R. Barr (2), Ruben O.
Donis (1), Julie Villanueva (1), Tracie Williams (2), Li-Mei Chen (1)
1: Influenza Division, National Center for Immunization and Respiratory Diseases; 2: Division of Laboratory Sciences, National Center for
Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
Background
Candidate viruses for production of inactivated influenza vaccines are traditionally generated by reassortment
between an influenza virus providing the genes encoding the HA and NA surface proteins and A/Puerto Rico/8/1934
(PR8) virus as donor of the internal genes (6:2 genotype) imparting high growth in eggs. Recent studies noted that
the HA yields of certain reassortant viruses with PB1 originating from the same virus as the HA and NA genes (5:3
genotype) were greater than those of classical 6:2 counterparts. In this study, we systematically evaluated the
impact of a homologous PB1 on hemagglutinin (HA) yields from diverse subtypes of viruses purified in sucrose
gradients.
Methods
A panel of eight virus pairs with PB1 genes from PR8 or the surface gene donor viruses was generated by reverse
genetics and purified by sucrose density gradient ultracentrifugation. Virus HA yields were quantified by isotope
dilution mass spectrometry (IDMS) and SDS-PAGE/densitometry analysis.
Results
Infectious titers from virus pairs were similar regardless of their PB1 source. In contrast, the total virus protein and
HA antigen yields from 4 of the 8 virus pairs increased by ~2-fold.
Conclusions
Our results suggest that increased HA antigen yields from eggs may be achieved for a subset of viruses by inclusion
of a homologous PB1 during reassortment. This finding may be important for improvement of seasonal vaccine
production and, most importantly, for rapid response to an emerging pandemic.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P142
POSTE R A BSTRAC TS
SPA5P27
Development of influenza A(H7N9) candidate vaccine viruses with
improved hemagglutinin antigen yield in eggs
Callie Ridenour (1), Adam Johnson (1), Emily Winne (2), Jaber Hossain (1), Guaniri Mateu-Petit (1), Amanda Balish (1), Wanda Santana (2),
Taejoong Kim (1), Todd Davis (1), John R. Barr (2), Ruben O. Donis (1), Julie Villanueva (1), Tracie Williams (2), Li-Mei Chen (1)
1: Influenza Division, National Center for Immunization and Respiratory Diseases; 2: Division of Laboratory Sciences, National Center for
Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
Background
The emergence of avian influenza A (H7N9) virus in poultry causing zoonotic human infections was reported on
April 1, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was
initiated as soon as multiple additional human cases were reported by Chinese public health authorities. Although
candidate vaccine viruses were quickly derived by reverse genetics with the internal genes of A/Puerto Rico/8/34
(PR8), the resulting A(H7N9) CVVs did not grow to sufficient titer in eggs. This would be problematic for rapid
production of pandemic vaccine if the H7N9 viruses acquired the ability to transmit from person to person.
Methods
Several strategies were developed to improve the HA antigen yield of A(H7N9) CVVs from eggs. The approaches
included evaluation of: (1) 3 alternative PR8 internal gene lineages; (2) modified non-coding region sequences
(NCR); (3) chimerization of the H7 hemagglutinin protein with termini of H1 from PR8 virus and, (4) serial passage
of the CVVs in embryonated eggs. The HA was quantified by isotope dilution mass spectrometry (IDMS) analysis of
sucrose gradient-purified virions.
Results
The internal gene set from one PR8 virus lineage was identified to support higher hemagglutinin antigen yield
from purified virus and was selected for subsequent studies. Further improvement of total viral protein yield was
observed from several egg-passaged viruses; HA quantification by IDMS indicated a greater than two-fold increase
in yield as compared to that of parental virus. Selected viruses were tested by hemagglutinin inhibition (HI) assays
with ferret antisera to confirm that the antigenic characteristics remain unchanged. Investigation of further
approaches exhibited little or no improvement in total viral protein yield.
Conclusions
If A(H7N9) viruses were to acquire sustained human to human transmissibility, the improved HA yield of the eggpassaged CVVs generated in this study could expedite vaccine manufacturing for pandemic mitigation.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P143
POSTE R A BSTRAC TS
SPB5: GENETICS AND EVOLUTION OF VIRUS AND HOST
SPB5P01
Adaptation of a human influenza virus to two mouse strains with
different genetic backgrounds
Stricker, Ruth L.O. (1); Leist, Sarah R. (1); Schughart, Klaus (1,2,3)
1: Helmholtz Centre for Infection Research, Department of Infection Genetics, Germany; 2: University of Veterinary Medicine, Hannover,
Germany; 3: University of Tennessee Health Service Center, USA
Background
So far, adaptation of influenza viruses from one species to another has been studied intensively. However, it is not
clear, if during the adaptation process to a new species, different virus variants become selected that depend on
variant host factors within this species. We aimed to investigate, if upon adaptation of a human influenza isolate to
mice a) the host genetic background influences selection of certain genetic variants, and b) different virus lineages
adapted within individuals of the same mouse strain are identical. We chose the two mouse strains C57BL/6J and
DBA/2J for this study. C57BL/6J mice are known to be more resistant to an influenza virus infection than DBA/2J
mice. A human isolate was chosen for adaptation [A/Panama/2007/99 (H3N2)], which is not pathogenic in either
mouse strain.
Methods
The Influenza A virus A/Panama/2007/99 (H3N2) was adapted as separate lineages in two mouse strains, C57BL/6J
and DBA/2J mice by lung-to-lung passages. For this, mice were infected i.n. with 50μl virus, body weight was
monitored every day and lungs were prepared on day three post infection. The lung homogenates were used for
infection of the next passage. A total of ten passages were performed. The tenth passage of each lineage was grown
on MDCK cells, titrated and sequenced by Next Generation Sequencing. C57BL/6J and DBA/2J mice were infected
with the mouse-adapted virus lineages and body weight and survival were monitored for 14 days post infection.
Results
We established two lineages of adapted virus by lung-to-lung passage in C57BL/6J and five lineages in DBA/2J mice.
The difference between body weight before infection and on day three post infection increased from passage to
passage in DBA/2J mice, but almost no difference in body weight loss upon subsequent passaging was observed in
C57BL/6J. The tenth passage of mouse-adapted virus lineages showed a broad range of virulence, from very mild
to severely pathogenic in C57BL/6J and DBA/2J, whereas DBA/2J mice exhibited more severe phenotypes. Sequence
analysis revealed that most mutations appeared in the globular head of HA. Mutations acquired in C57BL/6J
lineages were different from the ones in DBA/2J lineages.
Conclusions
The host genetic background influences the selection of certain genetic variants. Some mutations that were
acquired in the viral genome by repeated adaptation in one mouse strain were identical. In general it was observed
that in the more susceptible mouse strain DBA/2J viruses adapted faster than in a more resistant mouse strain
C57BL/6J. This can be explained by fact that viruses can replicate to higher titers in DBA/2J mice and with increased
virus replication cycles, viruses with adaptation mutations can establish themselves faster. Thus, DBA/2J mice may
represent a well suitable strain for influenza virus adaptation.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P144
POSTE R A BSTRAC TS
SPB5P02
Host-specific distribution of (+)-RNA secondary structures in segment 8
of influenza A viruses
Vasin, Andrey (1,2); Petrova, Alexandra (1,2); Plotnikova, Marina (1); Klotchenko, Sergey (1); Egorov, Vladimir (1); Kiselev, Oleg (1)
1: Research Institute of Influenza, Russian Federation; 2: St-Petersburg State Polytechnic University
Background
RNA secondary structures play an important role in the life cycle of influenza A viruses (IAVs). There have been found
at least two stable hairpin structures in the 82-148 and 497-564 nucleotide regions of the NS gene. Given that
evolutionary changes in the NS gene are strongly associated with host adaptation, pathogenicity, and transmissibility,
we analyzed the prevalence of stable hairpin structures in these regions among human as well as non-human IAVs.
Methods
The secondary structures of segment 8 (+)-sense RNAs from 10 097 human and 12 621 non-human IAV strains
were predicted using RNAfold software. Sequences were aligned using MAFFT; phylogenetic analysis was carried
out using the RAxML algorithm.
Results
Stable hairpins were found in the majority of avian, canine, and other host species IAVs. In equine IAVs, the presence
of a hairpin was predicted only in the 82-148 region, while in swine IAVs a hairpin was predicted only in the 497-564
region for one third of the strains. As for human IAVs, hairpin structures were found only in the minority of isolates
(such as H5N1, H7N7, and H1N11918). In order to perform a more detailed analysis of the RNA secondary structures
in human and swine IAVs, we clustered the NS gene sequences according to the results of the phylogenetic analysis.
In human IAVs, a hairpin in the 82-148 nucleotide region was predominantly predicted for H1N1 and H2N2, but not
for H3N2 or H1N1pdm2009 viruses. Stable hairpins in both regions were found only in 17 H1N1 strains, including A/
Brevig Mission/1/1918, which were isolated before 1946. Hairpins were also predicted in both regions for viruses of
avian origin, such as H5N1 or H7N7. In H1N1pdm2009 viruses with the NS gene of North-American classical swine
origin, hairpins were not predicted. However, in Eurasian swine IAVs, hairpins were predicted in the 497-564 region.
Finally, while hairpins were not found in the 82-148 region in classical swine and triple reassortant IAVs, they were
predicted in the 497- 564 region for 52% of the strains (predominantly in classical swine IAVs. We also evaluated
the NS hairpin distribution in human and swine IAVs that originated from the avian-origin ancestor NS gene of A/
Brevig Mission/1/1918 virus from different time periods. It would seem that the hairpin in the 497-564 region was
eliminated in the process of adaptation from an avian to human host, while a not highly stable hairpin structure in
the 82-148 region still remains in some IAV lineages (H1N1 and H2N2).
Conclusions
The predicted RNA secondary structures in the 82-148 and 497-564 nucleotide regions of the NS gene of IAVs show
host-dependent distribution. Hairpins are common among avian IAVs, but seem to be eliminated or destabilized in
human IAVs.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P145
POSTE R A BSTRAC TS
SPB5P03
Changes in receptor binding properties of influenza A(H3N2) and
influenza B viruses and their impact on antigenic characterization
Lin, Yipu; Gu, Yan; Wharton, Stephen; Whittaker, Lynne; Gregory, Victoria; Li, Xiaoyan; Metin, Simon; Cattle, Nicholas; Daniels, Rodney; Hay,
Alan; McCauley, John
MRC, National Institute for Medical Research, United Kingdom
Background
Recent evolution of A(H3N2) viruses has been accompanied by marked changes in receptor binding properties as
well as antigenicity. This evolution is reflected in the loss of agglutination of avian red blood cells (RBCs) and low
isolation rates in eggs and MDCK cells. Poor receptor binding by the haemagglutinin (HA) resulted in the selection of
mutations in the neuraminidase (NA) which contributed to agglutination of RBCs and so complicate interpretation
of antigenic comparisons by haemagglutination inhibition (HI). We have analysed the changes in HA and NA
genes occurring over recent evolution and during propagation and applied a microneutralization (MN) assay to
complement the HI assay in distinguishing between differences conferred by alterations in receptor binding and
true antigenic differences between viruses.
Methods
Viruses were passaged in MDCK cells or in hens’ eggs. Recombinant viruses were generated by reverse genetics.
Receptor binding was quantified by measuring virus binding to receptor analogues using surface biolayer
interferometry. An MN assay used high resolution imaging methods to estimate the infected cell population. The HI
assay was carried out using guinea pig RBCs.
Results
Quantitative receptor binding assays showed that influenza A(H3N2) viruses isolated after 2001 recognise α2,3 sialic
acid avian receptors at undetectable levels and binding to the human receptor analogue, α2,6-sialyl lactosamine,
decreased by 200-fold over the period of 2001–2004. By 2010, associated with the substitution D225N in HA1,
binding to the human receptor analogue had also become barely detectable under standard assay conditions.
Associated with the drop in receptor avidity, many H3N2 viruses propagated in MDCK cells acquired substitutions
in the NA at residues 148 or 151 which conferred NA-dependent haemagglutination that could be inhibited by
oseltamivir or anti-NA antiserum. The inhibition of the agglutination of RBCs by anti-NA antibody impacts on the
interpretation of the results of the HI assay. The optimized MN assay, in contrast to the HI assay, was insensitive
to the amino acid substitutions in NA of A(H3N2) viruses and so can complement the HI assay in examining virus
antigenicity.
Influenza B viruses also show adaptive changes during cultivation that affect receptor avidity. We have examined
the antigenic properties of influenza B viruses propagated in hens’ eggs or in cell culture in the MN assay. Changes
acquired on culture were shown to have less impact on the antigenic characterisation of viruses using MN than
using HI.
Conclusions
Changes in the receptor binding properties of the HA, through natural evolution or adaptation during cultivation, have
resulted in difficulties with antigenic analysis of circulating strains using the HI assay. An improved neutralization
assay has proved useful in complementing the HI assay to clarify the true antigenic relationships between viruses
for both influenza A(H3N2) and influenza B viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P146
POSTE R A BSTRAC TS
SPB5P04
Evolution of highly pathogenic avian influenza H7 virus from low
pathogenicity precursors in ovo
Hanna, Amanda (1,2); Howard, Wendy A (1); Núñez, Alejandro (1); Hicks, Daniel (1); Barclay, Wendy S (2); Banks, Jill (1)
1: AHVLA, United Kingdom; 2: Imperial College London, United Kingdom
Background
Outbreaks of highly pathogenic avian influenza virus (HPAIV) may result in the infection of millions of poultry, causing
devastating disease and up to 100% mortality. Observations from natural outbreaks and laboratory experiments
support the dogma that HPAIV can emerge from low pathogenicity avian influenza virus (LPAIV) precursors. HPAI
can result from infection with some, but not all, H5 and H7 subtypes of AIV with the multi-basic cleavage site
(MBCS) in the haemagglutinin (HA) gene being the main pathogenic determinant of HPAIV infection in chickens.
We aimed to further understand viral mechanisms of HPAIV evolution through development of an in ovo model of
LPAIV to HPAIV evolution.
Methods
Reverse genetics (RG) and wild type viruses containing different cleavage site (CS) motifs were inoculated and
passaged in 14-day-old embryonated chicken eggs. RG viruses were based on A/chicken/England/11406/08 (H7N7)
HPAIV. This is an example of a virus that has naturally evolved to high pathogenicity in the field from a LPAIV precursor
containing a rare di-basic CS (DBCS). A wild type avian influenza A(H7N9) Chinese virus isolate (A/Anhui/1/13) was
also passaged as an example of a globally significant circulating LPAIV. Embryos were harvested and screened for
evolution to a HPAIV genotype and phenotype by DNA sequencing and immunohistochemistry (IHC) respectively.
Results
Isogenic RG viruses based on the H7N7 England isolate were rescued containing either a DBCS (H7N7DBCS) as seen
in the putative LPAIV precursor or a more typical LPAIV single- basic CS (H7N7SBCS). After one passage, four out of
nineteen embryos (21%) inoculated with the recombinant H7N7DBCS LPAIV evolved to a HPAIV. DNA sequencing
confirmed the acquisition of a MBCS and influenza virus-specific IHC staining was present in embryo vascular
endothelial cells. In contrast, DNA sequencing from embryos inoculated with the H7N7SBCS LPAIV showed the
retention of the SBCS motif, although after two passages, two out of fourteen embryos (14%) exhibited IHC staining
indicative of a HPAIV phenotype.
Conclusions
We present an in ovo model that can be used to understand the mechanisms behind the molecular evolution and
emergence of HPAIV. In this system a H7N7 virus containing a SBCS is less able to mutate to HPAIV compared to those
containing a DBCS. We now intend to use this model to define the minimum genetic requirements for acquisition
of basic amino acids at the cleavage site, and assess the likelihood of a novel or emergent LPAIV to evolve into HPAIV.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P147
POSTE R A BSTRAC TS
SPB5P05
Molecular peculiarities of influenza A and B viruses circulating in Russia
during two epidemic seasons in 2012-2014
Komissarov, Andrey; Kosheleva, Anna; Fadeev, Artem; Elpaeva, Ekaterina; Mikhailova, Maria; Pisareva, Maria; Buzitskaya, Janna; Stukova,
Marina; Danilenko, Daria; Konovalova, Nadejda; Eropkin, Mikhail; Grudinin, Mikhail
Research Institute of Influenza, Russian Federation
Background
Continuous monitoring of variability of circulating influenza strains allows studying their relationship with vaccine
strains as well as revealing mutations coding for drug resistance. Influenza viruses A of two subtypes (H1N1pdm09,
H3N2) and B of Victoria and Yamagata lineages have circulated during 2012-2014 in Russia.
Methods
Virus isolation was performed in MDCK cell culture and 10-day-old chicken embryos (CE). More than 400 influenza A
and B viruses were isolated from clinical specimens (nasopharyngeal swabs) during two epidemic seasons.
Sequencing was carried out on ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, USA) with «BigDye
Terminator Cycle Sequencing Kit v3.1». Processing and analysis of sequences was performed using Vector NTI v10.1.1
(Invitrogen) and MEGA 5 (PSU, USA) software. Method of maximum likelihood was used to build phylogenetic trees.
Results
HA gene phylogenetic analysis of influenza A(H1N1pdm09) viruses showed that all 2012-2014 strains were genetically
homogenous and belonged to clade 6 (А/Saint- Petersburg/27/2011-like). Two strains contained substitution A141T
in HA (Ca2). 2014 strains were distinguished in a separate subgroup 6B (A/South Africa/3626/2013) with substitutions
K163Q (Sa) and A256T. Some analyzed strains had substitutions P189Q, L191P near HA receptor-binding site. All
2012-2014 A(H3N2) strains were A/Victoria/361/2011-like (subgroup 3C of A/Victoria/208/2009 clade) defined by
S45N (leading to emergence of a new previously unknown potential glycosylation site), T48I, S145N, A198S, V223I и
N312S in HA. Majority of strains bore substitution T128A in HA1 leading to the loss of glycosylation site. Strains with
T128A substitution also had R142G in the HA antigenic site A. Some 2014 strains of subgroup 3C by HA belonged
to subgroup 3B by their NA. All Russian influenza A strains were oseltamivir susceptible and adamantane resistant
by their genetic structure. Neuraminidase sequences of all strains isolated in 2012-2014 contained N369K in the
antigenic site 365-369 (N2 numbering). Some strains had D151N/G substitution in NA catalytic site. All strains of
B/Victoria lineage circulating in Russia in 2012-2014 fell into clade 1A and had common substitution K209T in HA1
unlike B/Victoria strains isolated in the previous period that fell into clades 1A and 1B and had N197K and T199A
substitutions in receptor-binding site. No intra-clade reassortant HA-1A/NA-4 was identified in these seasons.
B/Yamagata lineage strains belonged to clade 2 (B/Brisbane/03/2007-like) and clade 3 (B/Wisconsin/01/2010-like)
and bore R48K (in antigenic site BC), I150S (in BA), Y165N (in BB2), T181A (in BD). No changes conferring resistance
to oseltamivir or zanamivir were found in influenza B strains isolated till 2014.
Conclusions
Influenza A and B viruses circulating in Russia in 2012-2014 were characterized by relative genetic homogeneity
in groups. Early phylogenetic changes that could cause antigenic drift with further mutations were revealed. The
obtained data demonstrated that different lineages co-circulated and interacted through intra-subclade HA and NA
reassortment.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P148
POSTE R A BSTRAC TS
SPB5P06
Phylogenetic and principal component analyses of influenza virus
sequence data from the first five years (2009–2013) of the IRIS study
show subtype distinct geographical patterns in global epidemics
van der Vries, Erhard (1); Zhang, Jitao David (2); Boucher, Charles (1); Schutten, Martin (1)
1: Erasmus MC, Rotterdam, The Netherlands; 2: F. Hoffmann-La Roche Ltd, Basel, Switzerland
Background
Extensive virological and clinical data have been collected from patients participating in a global observational
trial, the Influenza Resistance Information Study (IRIS; NCT00884117). We investigated the phylogenetics, IC50 to
neuraminidase inhibitors (NAIs) and viral load of infecting viruses from baseline samples in Years 1–5 of IRIS.
Methods
Patients in Europe, North America, South-East Asia and Australia with influenza-like illness and/or a positive
rapid test result for influenza had throat/nose swabs collected on Day 1 (day of study entry) for real-time RT-PCR
analyses of influenza type, subtype and NAI resistance. Symptoms including fever were noted on diary cards. Positive
samples were cultured, sequenced and tested genotypically and phenotypically for NAI resistance. Full-length
haemagglutinin (HA), neuraminidase (NA) and matrix protein 2 (M2) sequences obtained from Day 1 samples from
individuals infected with A/2009H1N1, A/H3N2 and influenza B viruses were analysed by principal component
analysis (PCA) using concatenated HA, NA and M2 gene sequences. Temporal-spatial maps were constructed for
A/H3N2 and A/2009H1N1 to study changes in IC50 and baseline viral load over time (between 2009 and 2013).
Phylogenetic analysis was done by construction of maximum- likelihood trees.
Results
Sequences were obtained from 2983 patients: 1079 A/2009H1N1, 1035 A/H3N2, 581 B/Victoria and 288 B/
Yamagata. PCA plots from viruses split by continent confirmed the seeding phenomenon for A/H3N2 epidemics
from Asia into North America and Europe. Seeding from Asia was also observed for B/Yamagata; however, this was
not observed for A/2009H1N1 or B/Victoria. IC50 and viral load values both fluctuated over the course of the study.
Conclusions
Evolutionary history of influenza sequences collected in Years 1–5 of IRIS showed temporal and geographical
patterns in virus distribution and temporal change in baseline viral load. Mutations correlated with change in
NAI susceptibility were also identified. These are currently under analysis, together with additional phylogenetic
analyses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P149
POSTE R A BSTRAC TS
SPB5P07
Identifying Antigenic Regions of H9 Haemagglutinin of Avian Influenza
Peacock, Thomas Philip (1,2); Reddy, Kolli B (1); Shelton, Holly (1); Barclay, Wendy S (2); Iqbal, Munir (1)
1: The Pirbright Institute, United Kingdom; 2: Imperial College London, United Kingdom
Background
The H9N2 subtype of avian influenza virus is endemic in poultry throughout much of Asia and North Africa. It
poses an immediate threat to the poultry industry due to high morbidity and occasionally high mortality, as well
as a potential threat to human health both in itself as well as through its reassortant progeny, the outbreaks of
H7N9 and H10N8 in China being two recent examples. Antigenic drift in the haemagglutinin (HA) glycoprotein
of H9N2 (the receptor binding protein and major antigen) leads to the loss of in-field vaccine efficacy. Our aim is
to identify key amino acid motifs responsible for antigenic differences in H9N2 viruses to better understand the
mechanism of antigenic drift. As a result we aim to improve vaccine seed strain selection and the development of
broadly protective recombinant vaccines.
Methods
The coding regions of two genetically closely related but antigenically distinct H9N2 viruses were manipulated
using reverse genetics techniques. Utilising site-directed mutagenesis and Gibson Assembly, a library of mutant HA
genes were produced with single or multiple amino acids swapped between the antigenically distinct HAs. Using
panels of monoclonal antibodies and chicken post-infection antisera raised against one of the selected H9N2 strains
we determined critical residues for the antigenic properties of H9 HA.
Results
We observed that a short region constituting only 7 amino acid differences and located in the vicinity of the receptor
binding site (near the 220 loop) was responsible for the majority of the antigenic differences between these viruses.
Additionally we measured the individual contributions these amino acids have on the antigenic cross reactivity
against both viruses.
Conclusions
Here we show that a small region with only a few amino acid changes is responsible for the bulk antigenic diversity
observed between two H9N2 viruses. This information may help guide future vaccine seed-strain selection and
could provide a starting point for producing broadly cross-reactive recombinant vaccines.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P150
POSTE R A BSTRAC TS
SPB5P08
EPITOPE MAPPING OF THE HEMAGGLUTININ MOLECULE OF INFLUENZA
B/YAMAGATA AND B/VICTORIA LINEAGE VIRUSES USING MONOCLONAL
ANTIBODIES
Sorokin, Evgenii; Tsareva, Tatiana; Sominina, Anna A.; Pisareva, Maria; Komissarov, Andrei; Kosheleva, Anna
Research Institute of Influenza, Russian Federation
Background
Influenza is one of the most important infectious diseases in both industrial and developing countries. Influenza B
virus strains cause epidemics in humans as H1 and H3 subtype strains of influenza A virus. Compared with influenza
A influenza B virus is characterized by a low rate of antigenic change. Genetic reassortment, insertion and deletion
are responsible for its evolution.
Methods
Selection of escape mutants (EM) was conducted as described previously (Kaverin N. et al., J. Virol. 2007; 81: 1291112917), antigenic analysis of viruses and EM was performed in HI test, sequencing of wild viruses and EM was
conducted using BigDye Terninator v3.1 Cycle Sequencing Kit («Applied Biosystems», USA) and genetic analyzer
GA3130 («Applied Biosystems», USA). EM were named as the corresponding monoclonal antibodies (MAb) used for
their selection.
Results
For the identification of virus-neutralizing epitopes of influenza B virus (Yamagata lineage) the panel of six MAbs
to HA1 molecule was used: two MAbs (4H7 and D9) directed to B/Panama/45/90 and four MAbs (10F4, 8Н11, 9А3
and 8Н3) to B/Florida/04/06 strain. They were used to select EM of the viruses. These MAbs were highly specific and
reacted in HI and neutralization tests with viruses of Yamagata lineage only without any reactivity with B/Victorialike strains. Analysis of the deduced amino acid sequences of EM 4H7 identified the double amino acid substitution
at residue V75I (site BE) and D150T (site BA, loop 150). The single amino acid substitution Y40H, H85Y, R149I (site
BA, loop 150), N202K (site BB1, helix 190) and S242R (helix 240) were identified in EM 10F4, EM 8Н11, EM D9, EM
8Н3 and EM 9А3, respectively.
Additionally, the panel of four MAbs to HA1 molecule of influenza B virus of Victorian lineage with high virusneutralizing activity was developed: two MAbs (5B7 and B/4H1) to B/Shandong/07/97 virus and two MAbs (9F1 and
11F8) to B/Malaysia/2506/04 strain. For identification of virus neutralizing epitopes in HA1 EM were selected using
above indicated antibodies. EM 5B7, EM B/4H1(4) and EMB/4H1(6) had a single amino acid substitution H122N (site
BD, loop 120), K203T and K203I (site BB1, helix 190), respectively. EM 11F8 and EM 9F1 (2) had a single amino acid
substitution H122N and K203N, respectively; EM 9F1(1) had a double mutation A202E and A317V.
Conclusions
Identification of virus-neutralizing epitopes in changeable sites of viruses of both contemporary lineages is
important for prediction of possible future evolution ways of influenza B virus.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P151
POSTE R A BSTRAC TS
SPB5P09
Analysis of mammalian adaptation in Influenza from surveillance data
Burke, David Francis; Smith, Derek J
university of cambridge, United Kingdom
Background
The adaptation of avian influenza viruses with the ability to infect humans is an ongoing cause for concern and
infections of humans with A/H5N1 continue. The recent outbreak of human infections of A/H7N9 influenza viruses
in China in 2013 and 2014 and reports of human infections with H10 and H6 in Taiwan highlights the potential for
emergence of influenza viruses previously not detected in humans. A major worry is that an increase of incidences
of human infection with avian influenza could cause further mammalian adaptation which would eventually lead
to sustained human-to-human transmission.
Methods
Mutations within IAV genomes which confers an increased stability, replication rate or affinity of binding within
mammalian hosts would result in key mutations occurring more frequently in human infections. We have tested the
null hypothesis that the observed frequency of amino acid substitutions within avian and human-infection A/H5N1
sequences are the same at all amino acid positions in all gene segments. Any selection pressure for adaptation in
mammalian hosts would give rise to a low, statistically significant, p-value.
Results
This method is able to identify mutations (PB2: E627K, D701N) which are known to increase replication in
mammalian cells or cause amantadine/rimantidine resistance (MP Ser31Asn, Val27Ala). Many other mutations,
currently untested, have been identified, many of which are close to known sites of biological relevance.
Conclusions
Mutations which occur at significantly higher frequencies within sequences from human infections than within
sequences from an avian source and thus should be identifiable using such a statistical analysis. This method can
be applied to other subtypes of influienza such as H7 or H9. These data are useful in tracking evolutionary changes
of influenza viruses and identifying strains which may possess an increased risk of human infection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P152
POSTE R A BSTRAC TS
SPB5P10
H1N1 IAVs exploited the co-translational insertion process to decrease
their NA transmembrane domain hydrophobicity
Dou, Dan; da Silva, Diogo V; Nordholm, Johan; Daniels, Robert
Stockholm University, Sweden
Abstract
Bitopic (single-spanning) membrane proteins such as NA, HA and M2 are expected to have more stringent
hydrophobicity requirements on their transmembrane domains (TMDs) than multi-spanning membrane proteins.
However, we found that the strict hydrophobicity requirement applies to the HA and M2 TMDs, but not to the type
II NA TMDs, which co-translationally insert and invert. To identify the factors responsible for this difference, natural
NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, were examined using mammalian
cell localization and topology assays. Our results demonstrate that co-translational insertion is essential for the
marginally hydrophobic NA TMDs to properly invert and localize to the plasma membrane. However, for NA with
hydrophobic TMDs, that are similar to subtype 2, insertion and inversion can occur post-translationaly. In addition,
we found the highly conserve positively charged cytoplasmic tail residue is required for the TMD inversion process
likely explaining the viral morphology change that has been observed upon deleting the cytoplasmic tail. Thus, IAVs
exploited the co- translational insertion process to enable the human N1 TMDs to become less and less hydrophobic
overtime.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P153
POSTE R A BSTRAC TS
SPB5P11
Influenza A and B intertypic reassortment is restricted by incompatible
viral packaging signals
Baker, Steven F. (1); Nogales, Aitor (1); Finch, Courtney (4); Tuffy, Kevin (1); Domm, William (1); Perez, Daniel R. (4); Topham, David J. (1,2,3);
Martínez-Sobrido, Luis (1)
1: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, USA; 2: New York Influenza Center
of Excellence; 3: David H. Smith Center for Vaccine Biology and Immuology; 4: Department of Veterinary Medicine, University of Maryland
College Park and Virginia-Maryland Regional College of Veterinary Medicine, USA
Background
Co-circulation of influenza A and B viruses among humans seems to provide an evolutionarily advantageous
mixing-vessel for these two distinct, yet similar, viruses. However, intertypic reassortment, or swapping viral RNA
segments between influenza A and B viruses, has never been observed through either epidemiological observations
or forced attempts in laboratory settings. Recent discoveries suggest that viral ribonucleoprotein competition may
contribute to this lack of reassortment. However, this hypothesis does not explain the inability of influenza A virus
to incorporate influenza B virus hemagglutinin (HA) or neuraminidase (NA) in the absence of intertypic competition.
Methods
We first observed compatibility of full-length influenza B HA (BHA) protein with influenza A virus using a single-cycle
infectious Influenza A Virus (sciIAV). This virus contains modified HA viral RNA, in which GFP replaces the majority of
the viral glycoprotein open reading frame, except for the non-coding regions (NCR) and packaging signals (Ψ) at both
viral RNA segment termini. Growth of sciIAV was achieved in vitro using trans-complementing HA- expressing cell
lines. To further explore the ability of BHA to be incorporated into replication-competent viruses, we appended the
NCR and Ψ of influenza A HA and NA onto BHA and BNA to rescue recombinant viruses. In vitro and in vivo analysis
of recombinant viruses tested relative fitness as well as incorporation into other influenza A viruses in co-infection
tissue culture experiments.
Results
We observed that full-length BHA from multiple lineages could complement two influenza A (H1N1) viruses. Also,
influenza A virus HA or NA packaging signals were sufficient to incorporate BHA or BNA into replication- competent
influenza A viruses. We were able to rescue sciIBV using influenza B viral RNA regions that may comprise the BHA
Ψ. Consequently, both sciIAV and sciIBV restricted incorporation of a reporter GFP gene flanked with the NCR and
Ψ of the opposite virus. Lastly, influenza A viruses containing BHA and BNA were efficiently rescued and proved to be
immunogenic in mice, and retained the ability to reassort in vitro with another strain of influenza A (H1N1) virus.
Conclusions
When influenza A and B viral segments encode proteins compatible with both viruses, packaging signal
incompatibility inhibits intertypic reassortment. Although these packaging signal disparities likely arose gradually
during evolutionary divergence, it provides an additional barrier for influenza type speciation. Our findings suggest
that influenza A and B virus use similar mechanisms to specifically incorporate segments into budding virions.
To determine the mechanism of specific genome packaging, it may be beneficial to study both types of influenza
viruses. Additionally, these findings can be applied to the design of more efficient vaccine platforms, where one virus
backbone is used to generate all HA and NA (sub)types included in quadirivalent vaccine formulations.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P154
POSTE R A BSTRAC TS
SPB5P12
Use of deep-sequencing to evaluate the intrinsic heterogeneity of
human influenza type A viruses directly in nasal swabs
Barbezange, Cyril (1); Blanc, Hervé (2); Isakov, Ofer (3); Enouf, Vincent (1); Shomron, Noam (3); van der Werf, Sylvie (1); Vignuzzi, Marco (2)
1: Unit of Genetics of RNA Viruses, Institut Pasteur, UMR3569 CNRS, Univ Paris Diderot Sorbonne Paris Cité, France; 2: Unit of Viral
Populations and Pathogenesis, Institut Pasteur, UMR3569 CNRS; 3: University of Tel-Aviv, Sackler Faculty of Medicine, Department of Cell and
Developmental Biology, Tel-Aviv, Israel
Background
In 2009, a new influenza type A virus of the H1N1 subtype (H1N1pdm09) caused the first pandemic of the 21st
century. It replaced the previously circulating H1N1 virus and, along with the H3N2 subtype, is now responsible for
the seasonal influenza epidemics. The genome of influenza type A viruses is composed of eight single-stranded RNA
segments of negative polarity. RNA virus polymerases are considered to have low fidelity and a virus thus exists as
a cloud of closely-related sequences (referred to as a quasispecies). So far, the evolutionary potential of influenza
viruses has been mainly documented by consensus sequencing data and the quasispecies nature of influenza
viruses gained interest only recently with the development of Next Generation Sequencing (NGS) technologies.
Materials and Methods
The whole-genome genetic heterogeneity of the three subtypes of influenza type A viruses that circulated in humans
between 2007 and 2012 in France was analysed using specimens, collected from mild and severe human cases of
influenza corresponding to each subtype, The eight RNA segments of the viral genome was amplified by PCR after
RNA extraction and specific reverse transcription. Library preparations and Illumina GAII runs were performed by
multiplexing 6 samples per lane (2 tags per sample). Using bioinformatics tools developed for this purpose, short
sequences generated by Illumina technology were aligned to the respective reference sequences and underwent
different quality controls. The outputs giving the proportions of each base at each nucleotide position were then
used for analysis and comparison.
Results
Overall mutation frequencies and gene-specific mutation frequencies were first analysed to identify subtype
/ severity signatures for each virus subtype (pandemic H1N1, seasonal H3N2, seasonal H1N1) or each severity
group (mild versus severe within each subtype) and nucleotide polymorphisms were determined. Subtype-specific
positions in apparently similar proportions exist for all the samples of the given subtype we tested. Finally, common
or divergent positions between viruses of the two severity groups we searched for to try to identify sub-populations
that could be landmarks of the severity.
Conclusions
Using a significant number of samples for three influenza viruses that have been circulating in the human population
for the past 6 years this study provides information on the intrinsic heterogeneity of influenza viruses within the
human host. It will lead to further studies aiming at understanding more precisely how the viral quasispecies nature
is involved in evolution, pathogenicity and adaptability of influenza viruses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P155
POSTE R A BSTRAC TS
SPB5P13
Strain specificity of serum hemagglutination-inhibition antibody
responses in humans infected with 2009 pandemic influenza A(H1N1)
virus
Xiuhua Lu, Feng Liu, Vic Veguilla, Min Levine, and Jacqueline M Katz
Influenza Division, NCIRD, Centers for Disease Control and Prevention, USA.
Background
Exposure to influenza viruses in childhood influence future antibody responses to antigenically distinct viruses within
a subtype. The emergence of 2009 pandemic influenza A(H1N1) [A(H1N1)pdm09] virus provided an opportunity to
understand the complex interplay between influenza virus exposure and subsequent antibody responses. Here, we
characterized the serum hemagglutinin-inhibition (HI) antibody responses among A(H1N1)pdm09 virus-infected
patients of different ages and priming histories with former seasonal influenza A(H1N1) viruses circulating between
1977 and 2009.
Methods
Sera collected from US residents prior to the 2009 pandemic (n=343) and sera from A(H1N1)pdm09 virus-infected
persons were tested by HI assays using 0.5% turkey erythrocytes and 4 HA units (HAU) of virus against nine former
seasonal H1N1 (sH1N1) viruses circulating from 1977 to 2009 and an A(H1N1)pdm09 virus. To evaluate the relative
proportions of strain-specific versus cross-reactive antibodies, sera were adsorbed with 105 HAU of selected purified
viruses and retested by HI assay.
Results
The unique HI reactivity patterns of sera collected prior to the 2009 pandemic were identified; three distinct sH1N1
priming patterns were observed. Most children (median age 9 yrs; n=80) showed the highest antibody titers against
A/New Caledonia/20/99 (NC/99)-group viruses (NC/99-group-primed). Most young adults and a few older children
(median age 23 yrs; n=144) showed the highest antibodies to A/Taiwan/1/86 (TW/86)-group viruses (TW/86group-primed). Most adults (median age 39 yrs; n=119) exhibiting broad HI activity and were likely primed with A/
USSR/90/77 (USSR/77)-group viruses. Serologic evidence of sH1N1 exposure(s) could be used for determining the
priming sH1N1 virus in individuals with various ages.
Sera from 113 A(H1N1)pdm09-infected individuals (6-49 years old) were similarly grouped into one of the above
three priming patterns and then sera were adsorbed with A(H1N1)pdm09, the relevant priming former sH1N1,
and PBS as control. Infection with A(H1N1)pdm09 virus in NC/99-group-primed patients produced predominantly
strain-specific HI antibodies. In contrast, the majority of patients who were primed with TW/86-group viruses or
USSR/77-group viruses produced predominant cross-reacting antibodies.
Paired sera from 114 A(H1N1)pdm09-infected individuals (4-49 years old) were tested by cross-HI assays against
sH1N1 viruses and an A(H1N1)pdm09 virus. The NC/99-group-primed patients (median age 14 yrs; n=15) showed
significant rise of HI titers to A(H1N1)pdm09 virus but not to any sH1N1 viruses. However, some TW/86-groupprimed patients (median age 24 yrs; n=55) or USSR/77-group-primed patients (median age 39 yrs; n=44) showed
significant rise to one or more or all sH1N1 viruses tested.
Conclusion
Age and priming history with different former sH1N1 viruses impacted HI antibody formation following A(H1N1)
pdm09 virus-infection. Dampening of strain-specific antibody and enhancement of cross-reacting HI antibodies
occurred in the majority of TW/86-group-primed and USSR/77-group-primed individuals. These data highlight
potential differences in the quality of HI antibody responses to A(H1N1)pdm09 virus-infection that may have
implications for protective immunity.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P156
POSTE R A BSTRAC TS
SPB5P14
Comparison of common genotypes of Chinese H7 avian influenza viruses
for replication and transmission in chickens
David L. Suarez and Marisela Rodriguez
Southeast Poultry Research Laboratory, USDA, Athens, GA 30605
Background
In 2013 an outbreak of H7N9 was detected in humans and in poultry in China. Additionally H7N7 virus was also
found in poultry in China. Both groups of viruses appeared to be reassortant viruses having picked 6 internal gene
segments from the poultry adapted H9N2 that is endemic in China and having likely acquired the hemagglutinin
and neuraminidase genes from wild birds. With both groups of viruses, numerous polymorphisms were present
including 3 changes in the hemagglutinin protein. One of the changes at position 217 is in the receptor binding site
that typically impacts whether the virus attaches to alpha 2,3 (avian-like) or alpha 2,6 (human-like receptors) that
are predicted to affect virus replication. Two other changes were found in the human isolate that is rarely found in
poultry isolates.
Methods
Studies were performed using reverse genetics or natural mutations of both the H7N9 and H7N7 Chinese viruses
to examine virus replication and transmission in chickens. In addition variant viruses with these combinations of
three amino acid were also tested to see if they would affect virus replication and direct virus transmission. Studies
were performed in 4 week old chickens by directly inoculating some chickens and then adding naïve chickens to the
same cage 2 days later. Samples from both directly inoculated and transmission birds were also directly sequenced
to determine if viruses mutated to any particular genotype.
Results
Four different H7N9 viruses isolated from humans were tested, and all the viruses infected chickens, but did not
transmit to other chickens efficiently at the standard challenge dose of 10^6. All the human viruses had leucine
at position 217 (H7 numbering). Sequence from challenged birds showed a change in amino acid sequence from
leucine to glutamine, which is the amino acid typically found in avian H7 viruses. Transmission was seen at the
higher doses, with some evidence of increased replication and transmission in birds given virus variants with
glutamine at position 217.
Conclusions
There appears to be strong selection pressure for human viruses to have leucine at position 217, which is associated
with receptor binding to alpha 2,6 sialic acid, which is the predominant form found in humans. Chickens, which
primarily have alpha 2,3 sialic acid, appear to select for viruses with glutamine at that position, which is the type
found in almost all H7 avian influenza viruses. Although some differences were seen in transmission that seems to
correlate to position 217, the data is not clear enough to make firm conclusions.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P157
POSTE R A BSTRAC TS
SPB5P15
Sequencing artifacts in the type A influenza database and attempts to correct them
David L. Suarez (1), and Nikki Chester (2),
1: Exotic and Emerging Avian Disease Research Unit, Southeast Poultry Research Laboratory, Agricultural Research Service, USDA, 934 College
Station Road, Athens,GA 30605, 2: Athens Academy, 1281 Spartan Lane, Athens, GA 30606
Background
Type A influenza virus causes a wide range of disease in both man and animals, and considerable efforts have
been made to study and sequence large numbers of isolates. Currently there are over 300,000 gene sequences
representing over 50,000 strains in publicly available databases. However, the quality of the sequences submitted
to these public databases are determined by the contributor and many sequence errors are present in the databases,
which can affect sequence analysis and require significant curation of data by individual researchers to get usable
data. As part of a high school class project, bioinformatics analysis was performed on all eight gene segments of
influenza A virus.
Material and Methods
Sequences were selected in the Influenza Research Database website that were longer than the generally accepted
lengths of the individual segments, with the hypothesis that these sequences would have an error in the sequence.
Multiple sequence alignments were performed for each segment, and viral sequences. The virus sequences were
evaluated for common types of error associated with each sequence.
Results
A total of 1081 sequences were identified that met this criteria when the database was queried in March 2012,
which represents a 0.82% rate of potential errors.Three types of errors were commonly observed: non-influenza
primer sequence was not removed from the sequence; Taq polymerase added an adenine at the end of the PCR
product; and the PCR product was cloned and plasmid sequence was included in the sequence. Internal insertions
of nucleotides was also commonly observed, but in many cases it was unclear if the sequence was correct or actually
contained an error and therefore these sequences were not evaluated further. Students contacted some of the
sequence submitters alerting them that a sequence had been identified as likely having an error and if known what
type of error it probably was and asked to correct it. A total of 215
sequences, or 22.8% of the suspect sequences, were corrected in the public databases in the past year in part because
of the student project. Examination of the sequence database in 2013-14 showed an additional 409 sequences with
possible errors were added to GenBank in the last year.
Conclusions
The integrity of the public sequence databases is largely dependent on the scientists who submit the sequence
information. The identification of 409 new problematic sequences in the last two year highlights that errors are
still commonly reported. Additional awareness of the need for data integrity of sequences submitted to public
databases is needed to fully reap the benefits of the huge amount of data available for analysis.
SPA6: EVALUATION OF VACCINE SAFETY AND EFFECTIVENESS
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P158
POSTE R A BSTRAC TS
SPA6P01
Effectiveness of influenza and pneumococcal vaccines in preventing
pneumonia development and hospitalization: a prospective cohort
study
Song, Joon Young (1,10); Lee, Jin Soo (2); Wie, Seong-Heon (3); Kim, Hyo Youl (4); Lee, Jacob (5); Seo, Yu Bin (5); Jeong, Hye Won (6); Kim, Shin
Woo (7); Lee, Sun Hee (8); Park, Kyung Hwa (9); Noh, Ji Yun (1,10); Choi, Won Suk (1); Cheong, Hee Jin (1); Kim, Woo Joo (1,10)
1: Korea University College of Medicine, Korea, Republic of (South Korea); 2: Inha University School of Medicine; 3: Catholic University Medical
College, St. Vincent’s Hospital; 4: Yonsei University, Wonju College of Medicine; 5: Hallym University College of Medicine; 6: Chungbuk National
University College of Medicine; 7: Kyungpook National University School of Medicine; 8: Pusan National University School of Medicine; 9:
Chonnam National University Medical School; 10: Transgovernmental Enterprise for Pandemic Influenza in Korea (TEPIK)
Background
Pneumonia and acute exacerbation of chronic illness are leading causes of influenza-related hospitalization,
particularly among patients with comorbidities. Therefore, influenza and pneumococcal vaccinations are strongly
recommended for the adults with comorbidities.
Methods
Using hospital-based influenza surveillance system (Hospital-based Influenza Morbidity and Mortality, HIMM), we
performed a multicenter, prospective cohort study of patients visiting emergency room with influenza-like illness
during the influenza epidemic period in 2013-2014. Patients aged ≥18 years were included, and clinical data were
collected regarding vaccination status (influenza and pneumococcus), comorbidities, complications, hospitalization
and mortality. Multivariate analyses were performed to estimate the effectiveness of influenza and pneumococcal
vaccination in preventing pneumonia development and hospitalization.
Results
Between 1 November 2013 and 30 April 2014, 2,217 patients with ILI were registered, and 44.6% (988 patients)
were laboratory-confirmed as influenza. Among 2,217 patients, 31.9% (707 patients) and 9.7% (216 patients) were
immunized by influenza and pneumococcal vaccine, respectively. The adjusted influenza vaccine effectiveness for
preventing pneumonia development and hospitalization were 58.0% (odds ratio 0.420, 95% CI 0.211-0.834) and
39.8% (odds ratio 0.602, 95% CI 0.430-0.842), respectively. Pneumococcal vaccination did not reduce the development
of pneumonia (odds ratio 1.450, 95% CI 0.672-3.126) and hospitalization (odds ratio 1.077, 95% CI 0.707-1.643).
Conclusions
Influenza vaccination was associated with a reduction in the risk of pneumonia development and hospitalization
during influenza season.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P159
POSTE R A BSTRAC TS
SPA6P02
Influenza vaccine effectiveness to prevent admissions with influenza
in adults belonging to target groups for vaccination in the Valencia
Hospital Network. A test-negative study, 2013-2014 influenza season
Puig-Barberà, Joan (1,2); Carballido-Fernández, Mario (3); Limón-Ramírez, Ramón (4); Tortajada-Girbés, Miguel (5); Otero-Reigada, María
Carmen (6); Mollar-Maseres, Juan (6); Carratalá-Munuera, Concha (7); Gil-Guillén, Vicente (7); Natividad-Sancho, Angels (1); Tormos, Anita (1);
Buigues-Vila, Amparo (1); Mira-Iglesias, Ainara (1); López-Labrador, Francisco Xavier (1,8); Díez-Domingo, Javier (1)
1: FISABIO-Salud Pública, Valencia, Spain; 2: Centro de Salud Pública de Castellón, Castellón, Spain; 3: Hospital General, Castellón, Spain; 4:
Hospital La Plana, Vila-real, Spain; 5: Hospital Dr Peset, Valencia, Spain; 6: Hospital La Fe, Valencia, Spain; 7: Universidad Miguel Hernández,
San Juan de Alicante, Spain; 8: CIBERESP, Instituto de Salud Carlos III, Madrid, Spain
Background
Influenza is associated with excess morbidity preventable by vaccination. Vaccinating risk groups is considered the
Influenza is associated with excess morbidity preventable by vaccination. Vaccinating risk groups is considered the
best preventive strategy. Because of antigenic drift of the virus and acquired immunity, vaccine effectiveness (VE)
is variable and questioned. Our aim was to estimate influenza VE to prevent admissions related to influenza in
immunized adults belonging to risk groups, coinciding with the 2013-2014 influenza season, in Valencia (Spain).
Methods
This was a hospital-based, test-negative, case-control study. We enrolled consenting, non-institutionalized subjects,
aged 18 or over, belonging to target groups for vaccination, consecutively admitted for acute processes related to
influenza in six participating hospitals, with influenza-like-illness (ILI) symptoms within seven days of admission.
We obtained pharyngeal and nasopharyngeal swabs within seven days of symptom onset in 97% of enrolled
subjects. We considered as cases those RT–PCR positive for influenza and as controls those with a negative result.
We ascertained vaccination by recall and by consulting the Valencia population based Vaccine Information System.
We considered a subject as immunized when vaccinated 15 or more days before ILI onset. We estimated influenza
VE unadjusted and adjusted by age, sex, previous admissions, days between onset of symptoms and swabbing,
hospital and epidemiological week when hospitalized.
Results
From week 52-2013 to week 11, 2014, we enrolled 1,172 subjects. In four, the sample was inadequate, leaving 1,168
subjects for analysis. Overall, 260 (22%) were influenza positive and considered cases, 201 (77%), A(H1N1)pdm; 54
(21%), A(H3N2 ); and 5 (2%), A not subtyped; 908 (78%) were negative controls.
The risk (odds ratio [OR]; 95% confidence interval [95CI]) of hospitalization with influenza
was higher in subjects younger than 65, OR, 2.2 (95%CI, 1.6 to 2.9); pregnant women, OR,
13.3 (2.1 to 85.0); with morbid obesity, OR, 5.3 (1.1 to 25.5); or in current smokers, OR 1.8
(1.3-2.5).
Vaccination was positively associated with age 65 and over, cardiovascular disease,
bronchitis, two or more visits to GP, previous admissions, not being a current smoker and
previous influenza vaccination, and negatively associated with being a current smoker or
pregnant.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P160
POSTE R A BSTRAC TS
Compared to 44% cases, 59% controls were immunized (p < 0.0001).
The unadjusted influenza VE was 46% (95%CI; 28% to 59%).
The adjusted VE was 32% (95% CI, 7% to 50%).
Conclusions
The vaccine conferred moderate protection against the risk of admission with influenza in
adults belonging to target groups. The adjusted estimate indicates that among immunized it prevented one in three
admissions with influenza.
Funding
Sanofi Pasteur funded the study.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P161
POSTE R A BSTRAC TS
SPA6P03
Influenza vaccine effectiveness and averted medically-attended
influenza cases, seasons 2011/12, 2012/13 and 2013/14
Reuss, Annicka; Buda, Silke; An der Heiden, Matthias; Schweiger, Brunhilde; Wedde, Marianne; Haas, Walter; Buchholz, Udo
Robert Koch Institute, Germany
Background
Influenza viruses evolve constantly and therefore the effectiveness of influenza vaccines (IVE) needs to be assessed
through observational studies each season. Our study objective was to estimate IVE against laboratory confirmed
influenza A(H1N1)pdm09, A(H3N2) and B in the German population and the number of potentially averted
medically-attended influenza (MAI) cases during the seasons 2011/12, 2012/13 and 2013/14.
Methods
We conducted a test-negative case-control study among outpatients with influenza-like-illness (ILI) whose nasal
swabs were tested by the national reference laboratory during the seasons 2011/12- 2013/14. Cases had laboratoryconfirmed influenza, controls were influenza-negative. We calculated IVE adjusted for age group and week of
symptom onset using multivariable logistic regression. Combining season-specific point estimates of IVE with the
estimated number of MAI cases allowed us to roughly calculate the subtype-specific number of potentially averted
MAI cases. We also took into account vaccination coverage, influenza attack rates and population size. Effects of herd
immunity were not considered.
Results
Depending on (sub)-type and season, point estimates of IVE against laboratory confirmed influenza ranged between
0% and 59% (figure). IVE against A(H1N1)pdm09 was 52% and 59%, against A(H3N2) 0% and 49% and against B
24% and 27%. The estimated total number of MAI cases were 1.1 million (2013/14), 2.1 million (2011/12) and 7.7
(2012/13), corresponding to 1-9% of the Germany population. In 2012/13, a season with high influenza activity,
seasonal influenza vaccination potentially averted almost a million MA influenza cases (table).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P162
80
70
60
50
40
30
20
10
0
IVE estimate in 2013/14 was assumed 0% for calculation
90
*
*
*
no IVE estimate in 2011/12 due to few cases
100
no IVE estimate in 2011/12 due to few cases
Influenza Vaccine Effectiveness (%)
POSTE R A BSTRAC TS
1 2011/12
2 2012/13
2 2013/14
* 95%‐CI includes negative values
*
0 A(H1N1)pdm09 A(H3N2) B
1
1
2
2
3
*
3
Study characteristics and estimates 2011/12
2012/13
2013/14
No. of ILI‐patients (influenza cases/controls) 2,002 (501/1,501) 2,129 (1,190/939) 1,033 (165/868) Vaccination rate among influenza cases/controls
5%/8% 7%/8% 11%/8% Proportion of A(H1N1)pdm09/A(H3N2)/B 1%/76%/23% 34%/30%/36% 28%/63%/9% Estimated MAI cases 2,100,000 7,700,000 1,100,000* Potentially averted MAI cases 334,000 917,000 0 * preliminary data Conclusions
Conclusions
estimated thatthat
seasonal
influenza vaccination
low to moderate
protection
WeWeestimated
seasonal
influenzaprovided
vaccination
provided
lowdepending
to moderate protection depending on (sub)-type
on (sub)-type and season. Low vaccination rate in our study population prevented analysis for
and season. Low vaccination rate in our study population prevented analysis for vaccination target groups. During
vaccination target groups. During influenza epidemics, physicians should consider influenza as
influenza
epidemics, physicians should consider influenza as diagnosis among patients with ILI, irrespective of
diagnosis among patients with ILI, irrespective of vaccination status. Despite a low to moderate
vaccination
status.
Despite
a lowmay
to have
moderate
influenza vaccination may have prevented a
protection, seasonal
influenza
vaccination
preventedprotection,
a substantial seasonal
number of MA
influenza cases.
substantial
number of MA influenza cases.
2
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P163
POSTE R A BSTRAC TS
SPA6P04
Vaccine effectiveness estimates in preventing laboratory-confirmed mild
and moderate-to-severe influenza in the Belgian population during the
2012-2013 season
Hombrouck, Anneleen (1); Bossuyt, Nathalie (2); Van Casteren, Viviane (2); Van Gucht, Steven (1); Quoilin, Sophie (2); Wuillaume, Françoise (2);
Thomas, Isabelle (1)
1: Scientific Institute of Public Health, Operational Directorate of Communicable and Infectious Diseases, Viral Diseases, Brussels, Belgium; 2:
Scientific Institute of Public Health, Operational Directorate of Public Health and Surveillance, Brussels, Belgium
Background
The influenza season 2012-2013 in Belgium was characterized by a very high intensity and duration as compared
to previous seasons. Three viruses co-circulated: A(H1N1), A(H3N2) and B with a predominance of A(H1N1) and B of
Yamagata lineage.
Methods
Mild cases of influenza were monitored through a sentinel network of 112 general practitioners (GPs), which
reported and sampled patients of any age who met the influenza-like-illness (ILI) case definition: sudden onset of
symptoms, fever (≥38°C), respiratory and systemic symptoms. Moderate-to-severe influenza cases were monitored
through a sentinel network of 6 hospitals, which reported and sampled patients with severe acute respiratory
infections (SARI), which were defined as patients of any age with an acute respiratory illness with onset within the
last seven days and fever of ≥ 38°C (or history of fever) and cough or dyspnoea, and requiring hospitalisation (24h
or more). Patients with swabs taken >7 days of symptoms onset and patients with no record of vaccination (date)
or vaccination <14 days before symptoms onset were excluded. We used a test-negative design and compared labconfirmed influenza-positive to influenza-negative patients with mild or moderate-to-severe illness. Using logistic
regression, we calculated adjusted vaccine effectiveness (AVE) by type/subtype and results were adjusted for
surveillance scheme (if applicable), age group, sex and month of sample collection.
Results
During the 2012-2013 season, 1425 samples from ILI and 1001 samples from SARI patients were collected. After
applying exclusion criteria, we included 1261 ILI patients and 665 SARI patients in the vaccine effectiveness (VE)
analysis. Overall trivalent influenza vaccine (TIV) adjusted VE against laboratory-confirmed influenza (mild and
moderate-to-severe cases) was estimated 39% (95%CI 17%-54%). TIV adjusted VE against influenza A(H1N1) was
48% (95%CI 20%-67%) and influenza B was 44% (95%CI 19%-61%). Data power was not sufficient to calculate
TIV adjusted VE for influenza A(H3N2). When comparing laboratory-confirmed mild influenza to moderate-tosevere influenza, overall TIV adjusted VE against mild influenza was estimated 42% (95%CI 12%-63%) and against
moderate-to- severe influenza was 35% (95%CI 0.7%-57%). TIV adjusted VE against mild influenza A(H1N1) alone
was 51% (95%CI 0.6%-65%) and against moderate-to-severe influenza A(H1N1) was 47% (95%CI 0.3%-71%). TIV
adjusted VE against mild influenza B alone was 56% (95%CI 27%-75%). The adjusted VE estimate against moderateto-severe influenza B was lower but the effect was not significant.
Conclusions
Estimation of VE was feasible in Belgium for the 2012-2013 season, because the number of ILI and SARI samples
collected in our routine surveillance systems were high due to the intensity of the epidemic. The data suggest a
moderate protection by TIV against mild and moderate-to-severe influenza (subtype A(H1N1) and B) during the
2012-2013 season.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P164
POSTE R A BSTRAC TS
SPA6P05
Immunogenicity and safety of AS03-adjuvanted H7 vaccines in adults
≤64 years of age: Phase I/II, randomized, observer-blind, placebocontrolled, multi-center trials in the United States and Canada
Madan, Anu (1); Segall, Nathan (2); Ferguson, Murdo (3); Sheldon, Eric (4); Frenette, Louise (5); Chu, Laurence (6); Rheault, Paul (7); Kroll, Robin
(8); Friel, Damien (9); Soni, Jyoti (10); Li, Ping (1); Innis, Bruce L. (1); Schuind, Anne (1)
1: GlaxoSmithKline Vaccines, King of Prussia, PA, USA; 2: Clinical Research Atlanta, Stockbridge, GA, USA; 3: Colchester Research Group, Truro,
NS, Canada; 4: Miami Research Associates, Miami, FL, USA; 5: QT Research, Sherbrooke, QC, Canada; 6: Benchmark Research, Austin, TX, USA; 7:
Medicor Research Inc., Sudbury, ON, Canada; 8: Seattle Women’s: Health, Research, Gynecology, University of Washington, Seattle, WA, USA; 9:
GlaxoSmithKline Vaccines, Wavre, Belgium; 10: GlaxoSmithKline Pharmaceuticals Ltd., Bangalore, India
Background
Following reports of H7N9 human infections in China, GlaxoSmithKline Vaccines first evaluated an influenza A/
H7N1 (A/mallard/Netherlands/12/2000) vaccine as a model H7 influenza vaccine, and, following the availability of
an H7N9 strain, has evaluated an A/H7N9 (A/Shanghai/2/2013) vaccine. Here we report immunogenicity and safety
results after two-dose immunization in healthy adults with different formulations of these two vaccines.
Methods
The first co-primary objective of these two trials (H7N1/NCT01934127; H7N9/NCT01999842) was to evaluate
whether two-dose vaccination, 21 days apart, with these monovalent vaccines containing nominal 3.75μg or 7.5μg
of hemagglutinin (HA) adjuvanted with AS03 (Adjuvant System containing α-tocopherol [11.86mg or 5.93mg] and
squalene in an o/w emulsion) results in an immune response (by hemagglutination inhibition [HI]) to the vaccinehomologous virus that meets CBER and CHMP guidance targets, 21 days post-dose 2 (Day 42). In each trial, ~420
subjects were randomized (1:1:1:1:1:2) to receive one of these four adjuvanted formulations, or a non-adjuvanted
formulation containing 15μg HA, or a placebo. For each of the adjuvanted formulations that met the CBER and CHMP
criteria, adjuvant effect was evaluated by comparing HI responses with the non-adjuvanted 15μg formulation. The
second co-primary objective was to assess safety/reactogenicity of the different vaccine formulations through Day
42. Solicited and unsolicited adverse events (AEs) were recorded for 7 and 21 days after each dose, respectively.
Medically attended AEs (MAEs), potential immune-mediated diseases (pIMDs) and serious AEs (SAEs) are presented
up to Day 42. Immunogenicity and safety follow-up up to 12 months post-vaccination is ongoing.
Results
In both trials, 21 days after the second dose, immune responses to all adjuvanted formulations, regardless of the
antigen dose or adjuvant, met CBER and CHMP criteria. The adjuvant effect was demonstrated for both vaccines. In
the H7N1 trial, a robust cross- reactive immune response to H7N9 was also observed. In both trials, pain was the most
common solicited injection site symptom and muscle ache, headache and fatigue were the most common solicited
general symptoms. A higher reactogenicity was seen with the adjuvanted formulations. Also, a trend for higher
reactogenicity was seen with the H7N9 compared to the H7N1 vaccine formulations. Occurrence of unsolicited
AEs and MAEs up to Day 42 was similar across all groups (adjuvanted, non-adjuvanted, placebo). Five days after
the administration of the second dose in the H7N1 trial, 1 SAE was reported (acute pancreatitis), assessed by the
investigator as not causally related to vaccination and resolved before the Day 42 visit. No pIMDs were reported.
Conclusions
Primary objectives for both studies were met: CBER and CHMP criteria were met in all adjuvanted groups, 21 days
after the second vaccine dose of either H7N1 or H7N9 vaccine. In both trials, the study vaccines were generally well
tolerated.
Funding
BARDA+GlaxoSmithKline Biologicals SA
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P165
POSTE R A BSTRAC TS
SPA6P06
The «forgotten antigen» of Influenza virus: a novel approach to study
immunity to neuraminidase
Prevato, Marua (1); Nandi, Avishek (3,4); Lilja, Anders (4); Giusti, Fabiola (1); Ferlenghi, Ilaria (2); Buccato, Scilla (2); Cozzi, Roberta (2); Giovani,
Cinzia (2); Maione, Domenico (2); Brito, Luis (4); Geall, Andrew (4); Montomoli, Emanuele (1); Bertholet, Sylvie (2); Legay, Francois (2); Bonci,
Alessandra (2)
1: University of Siena, Italy; 2: Novartis Vaccines, Siena, Italy; 3: Vaccine Research, Holly Springs, USA; 4: Vaccine Research, Cambridge, USA
Background
A detailed understanding of anti-neuraminidase (NA) immunity is not currently available due to a lack of suitable
tools to measure functional anti-NA antibodies. Currently available assays rely on hemagglutinin (HA) mismatched
reassortants (1) or detergent treated viruses (2) as sources of NA to overcome interference issues associated with
steric hindrance of anti-HA antibodies present in sera. The goal of this work is to setup an innovative EnzymeLinked Lectin Assay (ELLA) introducing NA pseudotyping as a simpler and easier method to generate sources of NA.
Furthermore, we explored the immunogenicity of a SAM®(NA) vaccine (3), which is based on the non- viral delivery
of a self-amplifying mRNA encoding the NA antigen from Influenza.
Methods
PseudoParticles (PPs) - Influenza PPs were generated by transfecting 293T cells with plasmids encoding for
the structural proteins and the NA of A/California/7/2009 H1N1 or A/turkey/Turkey/01/2005 H5N1 viruses;
PPs expressing both A/CA NA and the A/Vietnam/1194/04 mismatched H5 were also generated. Cell culture
supernatants containing PPs were harvested and filtered before storage, and further analyzed by SDS-PAGE and
western blotting for the presence of NA. NA activity and its inhibition were measured against a set of representative
sera by ELLA assay. NA inhibiting (NI) titers were also measured using the whole live and the Triton X-100 treated
viruses as sources of NA. SAM(NA) vaccine - The A/turkey/Turkey/01/2005 NA gene was cloned into the SAM vector,
linearized, in vitro transcribed and capped. NA protein expression was confirmed by western blotting and functional
activity by an ELLA test before being formulated with lipid nanoparticles and injected to mice intramuscularly.
Functional anti-NA antibodies were assayed in sera from immunized mice by ELLA assay.
Results
All preparations of PPs containing NA showed sialidase activity and were used as virus surrogate in the ELLA assay
to specifically measure functional anti-NA antibodies in a set of representative sera. Preparations expressing NA
alone or combined with the mismatched HA gave comparable NI titers. In contrast, titers measured using the whole
live virus were subjected to anti-HA antibody interference and NI titers obtained using Triton X-100 treated virus
were lower possibly because of the action of the detergent on the structure of NA. Immunization of mice with the
SAM(NA) vaccine generated an NA-specific antibody response, which was shown to have functional activity in a
novel and innovative ELLA assay. A dose response effect was observed when administering increasing amount of
RNA.
Conclusions
Our results highlight that a PPs-based NI assay has great potential to be developed as a sensitive, flexible, and easy
to handle and scale up serological tool to study immunity to NA. Moreover, the SAM technology is suitable to set up
new in vivo studies to better understand the role of NA immunity after infection or vaccination.
Bibliografie
(1) Sandbulte MR, et al. (2009) A miniaturized assay for influenza neuraminidase-inhibiting antibodies utilizing
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P166
POSTE R A BSTRAC TS
reverse genetics-derived antigens. Influenza Other Respir Viruses, 3, 233-240. (2) Jonges N, et al. (2010) Influenza
virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J Clin
Microbiol. 48(3), 928-940. (3) Deering RP, et al. (2014) Nucleic acid vaccines: prospects for non-viral delivery of mRNA
vaccines. Expert Opin. Drug Deliv. 11(6).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P167
POSTE R A BSTRAC TS
SPA6P07
Immunogenicity and safety of an AS03-adjuvanted H7N1 vaccine in an
elderly population
Madan, Anu (1); Ferguson, Murdo (2); Rheault, Paul (3); Seiden, David (4); Friel, Damien (5); Soni, Jyoti (6); Li, Ping (1); Innis, Bruce L. (1);
Schuind, Anne (1)
1: GlaxoSmithKline Vaccines, King of Prussia, PA, USA; 2: Colchester Research Group, Truro, NS, Canada; 3: Medicor Reasearch Inc., Sudbury, ON,
Canada; 4: Broward Research Group, Hollywood, FL, USA; 5: GlaxoSmithKline Vaccines, Wavre, Belgium; 6: GlaxoSmithKline Pharmaceuticals
Limited, Bangalore, India
Background
Development of influenza virus vaccines for potentially pandemic A subtypes is part of the World Health Organization’s
global strategy for pandemic preparedness. GlaxoSmithKline Vaccines developed an H7N1 influenza vaccine,
considered as a model vaccine in the context of emerging H7N9. This phase II, observer-blind, randomised, placebocontrolled study in the United States and Canada (ClinicalTrials.gov: NCT01949090) assessed the immunogenicity
and safety of different investigational monovalent A/mallard/Netherlands/12/2000 (H7N1) vaccine formulations
in a population of elderly adults, which represents the age segment most impacted by H7N9 infections.Methods
Adults ≥65 years of age in stable health were randomised (1:1:1:1:2) to receive 1 of 4 vaccine formulations (3.75 or
7.50μg hemagglutinin antigen (HA), each adjuvanted with AS03 (Adjuvant System containing α-Tocopherol (11.86
or 5.93 mg) and squalene in an o/w emulsion)) or saline placebo, administered as a 2-dose series (21 days apart).
Immune responses to the vaccine-homologous (A/mallard/Netherlands/12/2000 [H7N1]) and vaccine- heterologous
(A/Shanghai/2/2013 [H7N9], subset of subjects) strains were evaluated by hemagglutination inhibition (HI) assay
at Days 0, 21 and 42. Solicited and unsolicited adverse events (AEs) were recorded for 7 and 21 days after each dose,
respectively. Medically attended AEs (MAEs), potential immune-mediated diseases (pIMDs) and serious AEs (SAEs)
are presented up to Day 42. Immunogenicity and safety evaluations up to 12 months post- vaccination are ongoing.
Results
Of the 360 vaccinated subjects, 334 were included in the per-protocol cohort for immunogenicity. Robust HI
antibody responses were induced by all vaccine formulations, 21 days after the second dose. Strong cross-reactive
immune responses were also observed to the heterologous strain (H7N9). While no particular effect of the antigen
dose or adjuvant type was observed for the homologous responses, heterologous responses tended to be higher
for the formulations with the highest adjuvant content, with no observed effect of the antigen dose. Pain was the
most frequently reported injection site symptom (41.7%–65.0% of the H7N1 recipients). The most frequent general
symptoms were fatigue, headache and muscle ache (reported by 16.7%–35.0% of the H7N1 recipients). Within 21
days post-vaccination, unsolicited AEs were reported by 26.7%–35.0% of the H7N1 recipients (none grade 3 vaccinerelated). Up to Day 42, MAEs were reported by 3.3%–15.0% of the H7N1-vaccinated subjects; 1 and 7 of all vaccinated
subjects reported pIMDs and SAEs (none vaccine-related), respectively.
Conclusions
The AS03-adjuvanted H7N1 vaccines induced good immune responses to the vaccine-homologous and -heterologous
(H7N9) strains, 21 days following the second dose in a population aged 65 years or older and was well-tolerated.
Funding
Biomedical Advanced Research and Development Authority (BARDA) and GlaxoSmithKline Biologicals SA
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P168
POSTE R A BSTRAC TS
SPA6P08
Influenza A/H3N2 mutations causing vaccine mismatches and possibly
associated with more severe disease, Singapore, 2009-2013
Lee, Hong Kai
National University of Singapore, Singapore
Introduction
Recent studies have shown that amino acid (aa) substitutions, specifically those within influenza A/H3N2 HA1
epitopes A-E, particularly in A and B, result in antigenic drift. Such drift mutants may have reduced vaccine efficacy,
resulting in more severe influenza-related illness in Europe, Canada and the USA during the Northern hemisphere
2012/13 influenza season.
Methods
Influenza A/H3N2 clinical samples were collected from the National University Hospital and Singapore General
Hospital during April 2009- November 2013 (n=214). Patients were clinically stratified into groups with mild,
moderate and severe influenza-like illness (ILI), in whom chronic pulmonary (including asthma), cardiovascular, renal,
hepatic, neurological, hematologic, and metabolic disorders (including diabetes mellitus), immunosuppression
or pregnancy, were considered relevant comorbidities. Full-genome A/H3N2 sequences, obtained directly from
clinical samples without culturing, were compared against contemporaneous seasonal influenza A/H3N2 vaccine
sequences. Patients were stratified into 3 age groups for analysis (≤18, 19-64, ≥65 years). Dated, clinically-tagged
(‘mild’, ‘moderate’ or ‘severe’) viral sequence data (n=206) were analysed phylogenetically to examine the pattern of
clustering of each gene.
Results
Dated, aa-annotated, and clinically-tagged phylogenetic trees showed that A/H3N2 viruses mainly from subclade 3
have been causing more clinically severe disease since 2011, as compared to earlier years (2009 and 2010). During
the study period, 20/214 (9%) severe, 33/214 (15%) moderate, and 161/214 (75%) mild ILI cases were identified.
From multinomial logistic regression (adjusted for gender, age groups, and comorbidity), aa substitutions N145S,
A198S, and V223I were found with significantly higher prevalence in the HA1 genes (n=206) of the subclade 3
viruses obtained from patients with moderate/severe ILIs (all with p<0.05). Significant mutations detected in other
subclade 3 viral genes included (all with p<0.05): T613A (PB2, n=208); E4G, P8L, and H32R (PB1-F2, n=201); D396E,
V668I, and N675K (PA, n=201); L81P and N402D (NA, n=211); E26K (NS1, n=205). On the other hand, truncated
PB1-F2 in non-subclade 3 viruses was also found to be significantly associated with higher disease severity. The
aa mismatch frequencies in the epitopes A+B (epAB, Spearman correlation coefficient=0.149, p=0.033, n=206)
but not in the epitopes A-E (Spearman correlation coefficient=0.033, p=0.639, n=206) were positively correlated
with moderate/severe ILIs. Multinomial logistic regression analysis on the combined effect of factors (i.e. gender,
age groups, comorbidity, with total aa mismatches in epAB) on moderate/severe ILIs showed that comorbidity
(p<0.0001) was a more significant contributing factor than age (p<0.01) and total aa mismatches at the epAB loci
(p<0.05, OR 1.4) for moderate/severe ILIs.
Conclusions
Although pre-existing comorbidities contributed mostly to the moderate/severe ILIs caused by influenza A/H3N2
subclade 3 viruses, there was a small, possible contribution from aa mismatches. Further functional investigation of
these viral mutations is required to confirm these clinical disease associations.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P169
POSTE R A BSTRAC TS
SPB6: RISK MANAGEMENT AND MITIGATION
SPB6P01
Identification of molecular determinants involved in Influenza A H5N1
virus survival in the environment
Leclercq, India (1,2); Pariente, Kevin (1); Sawoo, Olivier (1,2); Feher, Maxence (1); Vandenbogaert, Mathias (1); Manuguerra, Jean-Claude (1)
1: Institut Pasteur, France; 2: Université Paris Diderot, France
Background
Influenza A viruses (IAVs) are a major cause of human infectious respiratory diseases and their transmission is
dependent upon the environment. However, the role of environmental factors on IAV survival outside the host
still raises many questions. Previous results from the laboratory showed that external viral structures, such as
glycoproteins (Hemaglutinin HA and Neuraminidase NA) and the lipid bilayer, in direct contact with the environment
are mostly involved in the loss of virus infectivity. Our study contributed to the understanding of the mechanism of
influenza virus survival outside the host by identifying molecular determinants involved in the loss or maintenance
of Influenza A viruses viability.Methods
The evolution of influenza A quasi-species during virus survival in water was assessed by using two A(H5N1) strains
submitted to a temperature of 35°C. Viral infectivity in water at 35°C was assessed over time by the TCID50 method.
Analysis by RT-PCR targeting the HA, NA and M genes was performed during the course of kinetic, at the beginning
of the kinetic (day 0) and at the end of the kinetic (day 45). RT-PCR products were analyzed by HTS (High-throughput
sequencing) method. Eighteen different amplicons from day 0 and day 45 corresponding to 5 HA and 4 NA regions
of 350 bp were sequenced using the 454 GsFLX technology from Roche Diagnostics.
Results
More than 100,000 sequences (5485 sequences in average per sample) were obtained, filtered then analyzed
by a workflow based on the Galaxy framework that uses the BWA sequence alignment program. Quantification
and detection of variants were assessed through an in-house SNP-caller program and visualized with IGV viewer
(Integrative Genomics Viewer). Minor variants for HA and NA genes were detected over time and their relative
abundance varied between day 0 and day 45. HA variants with a frequency over 0.1% were considered. Some of
these variants showed significant difference frequency between D0 and D45. Relevant mutations were chosen
and lentiviral pseudoparticles bearing mutated HA on their surface were generated. Survival kinetics with these
mutated pseudoparticles in water at different temperatures and salinities are in progress.
Conclusions
Expected data could highlight specific residus thus giving clues to the identification of molecular determinants
involved in Influenza A viruses viability outside the host.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P170
POSTE R A BSTRAC TS
SPB6P02
Recommendations and policies for sick leave during seasonal and
pandemic influenza in Europe
Edwards, Christina Hansen (1); de Blasio, Birgitte Freiesleben (1,2); Tomba, Gianpaolo Scalia (3)
1: Norwegian Institute of Public Health, Norway; 2: Dept. of Biostatistics University of Oslo, Norway; 3: Dept. of Mathematics, University of
Rome, “Tor Vergata”, Italy
Background
The European Centre for Disease Prevention and Control (ECDC) and other researchers have pointed out that
there is insufficient research on the transmission dynamics of influenza to establish the effect of workplace
interventions. During the 2009 H1N1p influenza pandemic, many European countries issued recommendations for
sickness absence. In this study, we review and compare the recommendations and policies issued across Europe in
conjunction with seasonal and pandemic influenza.
Methods
A web-based survey consisting of 21 questions was issued to health authorities in 31 European countries to assess
policies and recommendations on sick leave during seasonal and pandemic influenza epidemics. The questions
concerned whether recommendations were issued, what the recommendations were, the basis of knowledge upon
which the recommendations were based and the purpose of the recommendations. Identical questions were asked
for pandemic and seasonal influenza. In addition, the health authorities were questioned about general national
sick leave policies and perceived attitudes to sick leave.
Results
Health authorities in 18 of the 31 countries replied to the survey. Only one country introduced recommendation or
policies for sick leave during seasonal influenza. During the H1N1 2009 pandemic 9 (50%) of the 18 countries had
issued national policies or recommendations for sick leave. The most common recommendations included: staying
at home until fever cessation (67%) and staying at home for a specific number of days (5-10 days) following symptom
onset (22%). In countries where policy changes were made in response to the pandemic, more liberal sick leave
policies were introduced. Moreover, the general national sick leave policies also differed with respect to the number
of days of sick leave permitted prior to seeking medical advice (0-14 days), and the amount of payment received
during a short sick leave period of up to 10 days (0%-100%). The main purposes of issuing the recommendations
and policies was to reduce transmission in the population, to raise awareness, and to protect sick individuals. The
recommendations were based either on expert advice or on recommendations issued by other countries or by the
WHO.
Conclusions
Among the countries involved in the study recommendations or policies for sick leave during influenza were only
issued by one country during seasonal influenza, and by half the countries for pandemic influenza. Further research
is needed to evaluate various sick leave recommendations and policies to effectively achieve the aim of reducing
influenza transmission.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P171
POSTE R A BSTRAC TS
SPB7: HOST FACTORS IN PATHOGENESIS
SPB7P01
Emerging anti-influenza agents
Kainov, Denis
University of Helsinki, Finland
Influenza A viruses cause flu epidemics and pandemics with serious consequences for public health and global
economy. Different treatment and prevention options have been developed and applied with limited success. We
found that host-directed small-molecules, saliphenylhalamid, obatoclax, gemcitabine, MK2206 and SNS-032 inhibit
influenza A virus infection. Moreover, influenza A virus was unable to develop resistance to these host-directed
agents, in contrast to commercialy available virus-directed drugs. Saliphenylhalamid and SNS-032 have high
specificity and good pharmacological properties, which warrant further development of these agents as antiviral
drugs.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P172
POSTE R A BSTRAC TS
SPB7P02
The impact of school dismissal and vaccination on influenza
transmission: an alternative to school closure
Voirin, Nicolas (1); Payet, Cécile (1); Régis, Corinne (1); Barrat, Alain (2); Cattuto, Ciro (3); Pinton, Jean-François (4); Lina, Bruno (5); Vanhems,
Philippe (1)
1: Service d’Hygiène, Epidémiologie et Prévention, Hôpital Edouard Herriot, HCL & Université de Lyon, Lyon, France; 2: CNRS, CPT, UMR 7332,
Université Aix Marseille, Marseille & Université de Toulon, La Garde, France; 3: Data Science Lab, ISI Foundation, Torino, Italy; 4: Laboratoire
de Physique de l’Ecole Normale Supérieure de Lyon, CNRS UMR 5672, Lyon, France; 5: Laboratoire de Virologie Est, CNR des virus influenza &
VIRPATH EA4610 UCBL, France
Context
Given the important role of children and young adolescents in the community spread of pandemic influenza,
mitigation strategies including school closures are always debated. These closures have considerable social and
economic costs and alternatives need to be proposed, including vaccination.Aims
Using detailed contact data within a school and mathematical modelling, we wanted to test the impact of school
cases dismissal in combination or not with vaccination as alternative interventions to school closure.
Methods
We used a stochastic, individual-based model in discrete time with time-steps of 20 seconds. In this model, at each
time-step, each children and teacher can be S (Susceptible), V (Vaccinated), E (Exposed), I (Infected and symptomatic),
A (infected and Asymptomatic), D (Detected) or R (Recovered). Construction of the model was based on the French
academic year and typical school days and weeks. To model transmission, we made explicit use of detailed contact
data collected in a French primary school of 242 individuals (232 children and 10 teachers). The dynamic contacts
network was used for calculations. We modelled the transmission probability according to attack rates similar to
those observed in school outbreaks. Parents dismiss their children (i.e. do not allow them to go to school) when they
had symptoms for more than 12 hours before going to school with a 60% efficacy. Seven scenarios for influenza
vaccination were explored as well as 21 scenarios combining vaccination strategies and detection/isolation. For
each scenario 200 simulations were performed
Results
In the reference scenario the number of cases was 134 corresponding to an attack rate (AR) of 55% (134/242). In
this reference scenario, 21%, 60% and 19% of cases occurred during the pre-epidemic, epidemic and post- epidemic
periods respectively, and 90% of cases resulted from within school transmission. When no detection is practiced in
the school, the number of cases range from 134 (AR, 55%) to 39 (AR, 16%) and the reduction range from -21% (28
cases prevented) to -71% (95 cases prevented) when vaccination is introduced. When detection took place three
time a day, reduction range from -55% (73 cases prevented), in the absence of vaccination compared to -88% (118
cases prevented) in the best vaccine scenario.
Conclusions
This study confirms that alternative strategies to school closure can be used to mitigate dissemination of an
influenza virus in primary schools. Results indicate that vaccination can be an interesting prevention measure when
cases detection and isolation is insufficient. In absence of detection, 25% vaccination coverage leads to a 34% of
attack rate reduction and 50% coverage to a 71% reduction. Combining with intermediate cases detection and
isolation, these figures are 64% and 84% reduction respectively.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P173
POSTE R A BSTRAC TS
SPB7P03
Understanding reasons behind HCW participation and non-participation
in seasonal influenza vaccination in Podgorica, Montenegro to tailor
strategies that increase uptake.
Likhite, Nathalie Kavita (1); Kavaric, Nebosja (2); Grbovic, Mensud (3); Terzic, Natasa (4); Brajovic, Mina (5); Jorgensen, Pernille (1); Caroline, Brown
(6)
1: World Health Organization Regional Office for Europe, France; 2: Primary Health Care Centre Podgorica; 3: Ministry of Health Montenegro;
4: Institute of Public Health Montenegro; 5: WHO Montenegro Country Office; 6: World Health Organization Regional Office for Europe,
Copenhagen
Background
While seasonal influenza vaccination of health care workers (HCWs) is recommended in most European countries,
vaccination uptake remains low. The WHO Regional Office for Europe tested an innovative approach – the Guide to
Tailoring Immunization Programmes – with the Ministry of Health and Primary Health Care Centre (PHC) - Podgorica
in Montenegro to design evidence-based solutions to increase uptake of seasonal influenza vaccination among
HCWs. Formative research was conducted to explore and describe Montenegrin HCWs’ perceptions, attitudes,
beliefs and practices regarding seasonal influenza vaccination.
Methods
A self-administered questionnaire on risk perceptions of vaccine preventable diseases (VPD) and vaccination practices
was distributed to 400 frontline HCWs – physicians, nurses, laboratory technicians and ambulance personnel - in
the PHC Podgorica in December 2012. In addition, 23 semi-structured interviews exploring VPD risk perception,
motivations and barriers to influenza vaccination and strategies to increase uptake were conducted with HCWs and
their managers in January 2013. Multivariate statistical analysis was used to identify factors associated with being
vaccinated using data from the questionnaires. Content was analyzed from interviews to understand behavioural
patterns.
Results
A total of 291 (73%) HCWs completed the survey. PHC Podgorica HCWs generally share a positive attitude towards
immunization. Nevertheless, although almost 90%of HCWs believe seasonal influenza is the disease they are most
at risk of contracting at work and transmitting to their patients and family, only 19% of HCWs were vaccinated in
the 2012/2013 season. Main reasons for not vaccinating are low perceived susceptibility and disease severity (53%).
Twenty eight percent of non-vaccinating HCWs believe that they acquire immunity against seasonal influenza through
work. Having good health is another reason for non- vaccination. The only factor significantly associated with influenza
vaccination was increasing age. Profession, education, years of experience or general attitude towards vaccination were
not associated with vaccination status. Self protection is the most important reason for vaccination among vaccinating
HCWs (40%), followed by patient and family protection, fear of flu and have had flu in the past. Mandatory vaccination,
a supportive environment and improved communications emphasizing facts and negative effects of seasonal flu are
cited as effective strategies to increase uptake.
Conclusions
Seasonal influenza vaccination among HCWs is a matter of personal choice. Most PHC Podgorica HCWs perceive
seasonal influenza as a threat for risk groups, such as elderly, chronic disease patients or pregnant women. Though
HCWs are a priority target group for seasonal influenza vaccination, and despite evidence of nosocomial infection in
health care institutions and higher absenteeism, more tailored efforts are needed to emphasize vaccination benefits
and counter misconceptions for HCWs to adopt seasonal influenza vaccination as a positive professional practice, with
the objective of preventing nosocomial transmission of influenza and ensuring higher quality patient safety and care.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P174
POSTE R A BSTRAC TS
PL05: LATE BREAKERS
PL05P01
Pre-existing neutralizing antibodies against potential pandemic
influenza viruses in middle-aged adults vaccinated annually with
seasonal influenza vaccines
Wei Wang, Esmeralda Alvarado, Qiong Chen, Dorothy Scott, Russell Vassell, and Carol D. Weiss
Center for Biologics Evaluation and Research, US Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892
Background
Emergence of potential pandemic influenza A viruses raised questions about whether immunization with prior
influenza vaccines could confer any protection. We investigated whether persons immunized with recent seasonal
influenza vaccines harbor neutralizing antibodies to potential pandemic influenza viruses.
Methods
Sera from 50 volunteers aged 48-73 years, who received all annual seasonal influenza vaccinations from 20002001 to 2008-2009 seasons, were collected in September-December of 2009. Sera were assessed for neutralizing
antibody titers > 1:80 against H9, H7, H6, H5 and H3N2v subtype influenza viruses using an HA-pseudovirus
neutralization assay.
Results
We found that 77% have titers against HK/1073/99 (H9N2), 30% have titers against HK/G9/97 (H9N2), 17% have
titers against SE/81/02 (H6N1), 60% have titers against HK/SF4/01 (H6N1), 72% have titers against VN/1203/04
(H5N1), 44% have titers against EG/2321/07 (H5N1), 4% have titers against IND/5/05 (H5N1), 2% have titers
against VN/NCVD-016/08 (H5N1), 75% have titers against IN/08/11 (H3N2v ), while none had titers against
GY/337/06 (H5N1), SH/2/13 (H7N9) and NL/219/03 (H7N7). The neutralization titers to H9, H6 and H5 correlated
well with each other, but not with H3N2v. However, within subtypes, the sensitivities of influenza virus strains to
sera cross-neutralization varied.
Conclusions
These findings suggest that annual vaccination with seasonal influenza vaccines, especially multiple year
vaccinations, can elicit cross-neutralizing antibodies to potential pandemic influenza viruses that may potentially
contribute to protection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P175
POSTE R A BSTRAC TS
PL05P02
Clinical Safety and Efficacy of MHAA4549A, A Novel Monoclonal
Antibody for the Treatment of Severe Influenza A: Results of a
Randomized, Double-Blind, Placebo Controlled Clinical Trial
Jorge Tavel, Jacqueline McBride, Rong Deng, Michael Derby, Tracy Burgess, Ning Chai,
Summer Park, Min Xu and Lee Swem
Genentech, South San Francisco, CA 94080
Background
Novel therapeutics to treat severe influenza are a high unmet medical need.
MHAA4549A is a human monoclonal IgG1 antibody against influenza A virus that binds to a
highly conserved epitope on the influenza A hemagglutinin (HA) stalk region and allows
broad neutralization of the influenza A virus. Preclinical data demonstrate a significant
Clinical Safety and Efficacy of MHAA4549A, A Novel Monoclonal Antibody for the
response
lethalInfluenza
challenge
model
mice and ferrets
in both
group 1 (H1N1, H5N1) and
Treatmentto
of aSevere
A: Results
of in
a Randomized,
Double-Blind,
Placebo
Controlled Clinical Trial
group 2 (H3N2) strains of influenza A, including strong efficacy when dosed as late as 72
Jorge post-viral
Tavel, Jacqueline
McBride, Rong
Deng,
Michael
Derby,
Tracy Burgess,
Ning Chai,
hours
challenge.
Based
upon
these
findings,
a clinical
trial was executed to
Summer Park, Min Xu and Lee Swem
study
the safety
and
efficacy
MHAA4549A in an influenza challenge model.
Genentech,
South San
Francisco,
CAof
94080
Background: Novel therapeutics to treat severe influenza are a high unmet medical need.
Methods
MHAA4549A is a human monoclonal IgG1 antibody against influenza A virus that binds to a
epitope on the influenza A hemagglutinin (HA) stalk region and allows
Inhighly
this conserved
randomized,
double-blind, placebo controlled clinical trial, one hundred
broad neutralization of the influenza A virus. Preclinical data demonstrate a significant
healthy
weremodel
nasally
inoculated
H3N2
Wisconsin
Influenza A strain.
responsevolunteers
to a lethal challenge
in mice
and ferrets inwith
both group
1 (H1N1,
H5N1) and
group 2 (H3N2) strains of influenza A, including strong efficacy when dosed as late as 72
Nasopharyngeal
swabs were collected three times daily for viral load assessment. At 24
hours post-viral challenge. Based upon these findings, a clinical trial was executed to
the safety
and efficacy
of MHAA4549A
in an received
influenza challenge
model.
tostudy
36 hours
after
inoculation,
subjects
a single
intravenous infusion of
MHAA4549A
atrandomized,
three doses
– 400mg,
1200mg
and
3600mg
or placebo. Endpoints of
Methods: In this
double-blind,
placebo
controlled
clinical
trial, one –
hundred
healthy
volunteers
were reduction
nasally inoculated
with H3N2
Influenza
A strain.
the
study
included
in qPCR
viralWisconsin
load area
under
the curve (AUC) and clinical
Nasopharyngeal swabs were collected three times daily for viral load assessment. At 24
symptoms.
Comparison
of active
anda single
placebo
armsinfusion
was performed
using non-parametric
to 36 hours after
inoculation, subjects
received
intravenous
of
MHAA4549A at three doses – 400mg, 1200mg and 3600mg – or placebo. Endpoints of
Wilcoxon
rank-sum test.
the study included reduction in qPCR viral load area under the curve (AUC) and clinical
symptoms. Comparison of active and placebo arms was performed using non-parametric
Wilcoxon rank-sum test.
Results
Results:Nasopharyngeal
Median Nasopharyngeal
Viral AUC
Median
Viral AUC
There were no infusion reactions, no discontinuations, and no SAEs related to the study drug. The most common
drug related adverse event was elevated ALT and AST that did not occur at a rate greater than that observed in the
placebo group.
Conclusion
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P176
POSTE R A BSTRAC TS
MHAA4549A is a potent anti-influenza monoclonal antibody, producing up to a 98% decrease (p=0.0051) in the AUC
of nasopharyngeal viral shedding compared to controls, as well as a trend in decreased influenza symptoms. Clinical
data also suggest that MHAA4549A is safe and well tolerated. Currently, a study is being planned to evaluate the
safety and efficacy of MHAA4549A in hospitalized patients with severe influenza.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P177
POSTE R A BSTRAC TS
PL05P03
Ferret models to study clinical intervention strategies against influenza.
Stittelaar, Koert (1); Mallett, Corey (2); de Waal, Leon (1); Veldhuis Kroeze, Edwin (1); van Amerongen, Geert (1); van den Brand, Judith (3); van
der Vries, Erhard (3); Osterhaus, Ab (1,3)
1: Viroclinics Biosciences, Rotterdam, the Netherlands; 2: GlaxoSmithKline Biologicals, Laval, Québec, Canada; 3: Department of Viroscience,
Erasmus MC, Rotterdam, the Netherlands
Influenza is a moving target. Different influenza virus subtypes circulate across different avian and mammalian
populations, drift, reassort, causing disease and spreading rapidly. Ferrets have proven to be indispensable for
influenza virus research. Ferrets can be infected with primary and cultured human and avian influenza virus isolates
and develop a disease pattern which is very similar to that in humans. Ferrets have shown to be instrumental over
a broad spectrum of applications from the production of influenza-specific antiserum to the novel highly advanced
immunocompromised model. The appropriateness of the different ferret models and their read out parameters
for the assessment of clinical intervention strategies for influenza virus infection in humans, such as preventive
vaccination and the use of antivirals are discussed in the context of high pathogenic avian influenza A/H5N1,
pandemic influenza A/H1N1 and low pathogenic avian influenza A/H7N9 viruses
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P178
POSTE R A BSTRAC TS
PL05P04
Results of phase II and phase III studies of AS03-adjuvanted cell culturebased H5N1 influenza vaccine (KD-295)
Naruse, Takeshi; Tanabe, Tetsuro; Fukuda, Tadashi; Ichikawa, Munetaka; Oda, Yoshiaki; Tochihara, Shinji; Kino, Yoichiro
The Chemo-Sero-Therapeutic Research Institute (Kaketsuken), Kumamoto, Japan
Background
KD-295, derived from A/Indonesia/5/2005 (H5N1) / PR8-IBCDC-RG2 strain cultured in duck embryonic stem
cell-derived cell lines (EB66 ®), is a formulation mainly composed of HA protein obtained from inactivation and
purification and to be mixed at the time of use with the AS03 adjuvant.
Methods
In order to determine the recommended clinical dose of KD-295, immunogenicity and safety were investigated in a
double-blind, randomized, parallel-group study with 248 healthy adults (between 20 and 65 years of age). Subjects
were distributed into four groups (MA, MB, HA and HB) with different volumes of adjuvant and antigen. Subjects
received two 0.5 mL intramuscular administrations, with 21 day (± 7 day) intervals between administrations. To
examine the safety and immunogenicity of KD-295 (MA agent), we conducted a multicenter, non-controlled,
open-label study in 369 healthy adult subjects (between 20 and 65 years of age). Subjects received two 0.5 mL
intramuscular administrations with 21 ± 7-day intervals between administrations.
Results
For immunogenicity, the MA agent group, HA agent group, MB agent group, and HB agent group all met all three
evaluation criteria in the prototype vaccine guidelines after two administrations. However, higher antibody titer
trends were observed in the HA agent group and the MA agent group, which were administered the standard
volume of AS03 adjuvant, than in the HB agent group and the MB agent group, which were administered half the
standard volume of AS03 adjuvant. In regards to safety, no adverse events causing death, serious adverse events,
or adverse events requiring discontinuation of the study occurred. The HA agent group showed higher incidence
of fever than the MA agent group. Based on these results, we selected the MA agent (3.8 .g HA + AS03 adjuvant
standard amount) as the recommended clinical dose. Two administrations of the MA agent met all three evaluation
criteria in the prototype vaccine guidelines. In addition, we confirmed antibody titers in some subjects three and six
months after the second administration. Antibody titer decreased gradually, but antibody titers were retained even
six months after the second administration. In regards to safety, no adverse events causing death, or
adverse events requiring discontinuation of the study occurred. One case of potential immune-mediated disease
and two cases of serious adverse events occurred, but a causal relationship was denied.
Conclusion
Based on the above, the formulation appeared to have an acceptable safety profile and the sufficient immunogenicity
of this drug has been confirmed. Development of this agent was implemented as a pandemic influenza vaccine
development and production preparation project, and production and marketing approval for indication as
“prophylaxis for pandemic influenza (H5N1)” was granted in March 2014.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P179
POSTE R A BSTRAC TS
PL05P05
Investigating the potential for temporary immunity between influenza
viruses in the ferret model
Laurie, Karen (1), Teagan Guarnaccia(1),(2), Louise Carolan(1), Ada Yan(3), Malet Aban(1), Patrick Reading(1), Anne Kelso(1), Jennifer Mosse (2),
James McCaw(3),(4), Ian Barr(1)
1: WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, Peter Doherty Institute for Infection and Immunity, Melbourne,
Victoria, Australia; 2: Federation University Australia, School of Applied Sciences, Churchill, Victoria, Australia; 3: The University of Melbourne,
Melbourne School of Population Health, Melbourne, Victoria, Australia; 4: Royal Children’s Hospital, Murdoch Childrens Research Institute,
Vaccine and Immunisation Research Group, Melbourne, Victoria, Australia
Background
Epidemiological and modelling data suggest that following infection with influenza virus, there is a short period of
time during which the host experiences lower susceptibility to infection with any strain of influenza or perhaps any
respiratory virus. This phenomenon is independent of antigenic distance, and has been termed temporary immunity.
In this study, we used the ferret model of human influenza to investigate the hypothesis of temporary immunity and
the role it plays in determining the outcome of consecutive infections.
Methods
Ferrets were intranasally infected with a primary influenza virus then challenged 1 to 14 days later with a different
influenza virus. Nasal washings were collected daily as representative samples of upper respiratory tract infection
and the levels of virus shedding and immune markers measured. Primary infections were staggered to allow the
challenge to be performed on all animals on the same day. Combinations of influenza A(H1N1)pdm09, A(H3N2) and
influenza B viruses circulating in 2009 were tested to assess the potential for non-specific, as well as virus-specific,
temporary immunity.
Results
Infection with A(H1N1)pdm09 virus could temporarily protect against subsequent challenge with influenza B
virus, for short intervals. However a primary infection with influenza B virus did not protect against challenge with
A(H1N1)pdm09 virus, rather co-infection or delayed secondary infections, were observed. Interestingly, primary
infection with influenza B virus could block or delay subsequent challenge with A(H3N2) virus, yet A(H3N2) virus
had no effect on subsequent infection with influenza B virus. Primary infection with A(H1N1)pdm09 virus blocked
infection with A(H3N2); A(H3N2) virus and blocked A(H1N1)pdm09 virus challenge in some animals. Our results
indicate that temporary immunity may be mediated by both innate and adaptive immune mechanisms (depending
upon the virus pair and time interval), and studies to determine these mechanisms are ongoing.
Conclusions
These data support the hypothesis of temporary immunity. Infection with influenza A virus can prevent infection
with an unrelated influenza B virus and vice versa, yet this occurrence is entirely dependent on the combination of
viruses and the timing between primary infection and subsequent challenge. A hierarchy of the ability of infection
with one influenza virus to block or delay infection with another is suggested. Co-infection with influenza A viruses
and B viruses can occur in ferrets but only within a narrow period between challenges. Overall, these data indicate
prior infection with an influenza virus can not only protect against antigenically similar viruses, but also against
unrelated viruses. The impact of temporary immunity at a population level requires further investigation.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P180
POSTE R A BSTRAC TS
PL05P06
Cost-effectiveness analysis of quadrivalent influenza vaccination in the
elderly: an update of a united kingdom analysis
Meier, Geneviève; Gregg, Meghann
Health Economics, GlaxoSmithKline Vaccines, Wavre, Belgium
Background
The quadrivalent influenza vaccine (QIV), Fluarix Tetra®, has recently been made available in the United Kingdom (UK).
This analysis describes an update of a previously published cost-effectiveness evaluation of Fluarix Tetra®. However,
the present analysis considers only persons of 65 years old and older, as all persons in this age category are strongly
recommended to be vaccinated. This evaluation also applies the updated guidelines for cost-effectiveness analyses
from the JCVI (Joint Committee on Vaccination and Immunisation) and uses data from two recently completed
burden of disease and costing studies in the UK that were not available at the time of the original publication.
Methods: A recently published lifetime, multi-cohort, static Markov model that compares QIV to TIV (trivalent
influenza vaccine) was used and updated with the most recent circulation data. In addition, the pandemic season
2009/2010 was excluded (as a seasonal influenza vaccine should not have been included during this period),current
guidelines were applied, and the reference year for costs in the model was updated to 2013/2014. Results from two
database analysis which included recent information not available at the time of original publication were also
included in this update; the first one used the CPRD (Clinical Practice Research Datalink) with respect to mortality
and cases by influenza type A and B viruses. The second study included used the CPRD HES-linked database (Hospital
Episode Statistics) and examined the cost of influenza-related illness in the UK. The model perspective is that of the
NHS (National Health Service) and the robustness of the model outcomes were asses using a probabilistic sensitivity
analysis (PSA).
Results
The model projects that using quadrivalent influenza vaccination instead of trivalent vaccines in persons of 65 years
old and older, averted on average per year 13,908 cases, 2,760 physician visits, 1,702 influenza-related complications,
462 hospitalisations and 238 deaths. Over the period of the model (life-time) using the current price of Fluarix
Tetra® £9.94 versus the weighted average price of trivalent influenza vaccines (£6.43, Prescription Cost Analysis
England 2013), the incremental cost-effectiveness ratio (ICER) was £13,523/QALY gained (Quality Adjusted Life Year).
An analysis of the season 2005/2006 (highest level of mismatch for influenza B with highest circulation of influenza
B) predicted a substantial increase in the number of avert clinical outcomes with 70,577 cases, 14,008 physician
visits, 8,636 influenza-related complications, 2,343 hospitalisations and 1,208 deaths being averted had QIV been
available at the time. Sensitivity analyses confirmed the robustness of the results.
Conclusions
In the UK context, the use of quadrivalent influenza vaccination with Fluarix Tetra® instead of trivalent influenza
vaccines in persons 65 years and older is likely to be a cost-effective intervention.
Fluarix Tetra® is a registered trademark of the GlaxoSmithKline group of companies.
Conflict of interest
G Meier and M Gregg are employees of GlaxoSmithKline group of companies. G Meier holds stock or stock options
or restricted shares in the GlaxoSmithKline group of companies.
Source of funding
GlaxoSmithKline Biologicals SA Wavre, Belgium, funded this study and all costs related to the development of the
abstract and poster presentation.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P181
POSTE R A BSTRAC TS
PL05P07
Design of randomized, double-blind, controlled, multi-center phase
IIb trials as part of the EU-funded UNISEC project to assess the safety,
immunogenicity and clinical efficacy of cross-seasonal universal
influenza vaccines with or without pandemic influenza vaccine in
healthy adults.
Liu, Marcy Heng (1); Robinson, Stuart (2); Ben-Yedidia, Tamar (3); Wanderley, Wilson Caparros (2); Pleguezuelos, Olga (2); Gottlieb, Tanya (3);
Babecoff, Ron (3); Schmidt, Ed (4); Huckriede, Anke (5); Van Doorn, Eva (1); Hak, Eelko (1)
1: Department of PharmacoEpidemiology and PharmacoEconomics, University Center of Pharmacy, University of Groningen, Groningen, The
Netherlands; 2: SEEK, United Kingdom; 3: BiondVax Pharmaceuticals, Israel; 4: Department of Pharmaceutical Technology and Biopharmacy,
University Center of Pharmacy, University of Groningen, Groningen, The Netherlands; 5: Department of Medical Microbiology, Molecular
Virology Section, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Background
Current seasonal influenza vaccines mainly induce immune responses against viral membrane glycoproteins. These
proteins, however, undergo continuous mutations by antigenic drift. To prevent immune escape, annual vaccination
with the latest predicted viral strains is adopted. Such vaccination strategy not only poses inconvenience and costinefficiency, but also results in poor protective effectiveness when the vaccine strains are mismatched with the
actual circulating strains. The latter point is especially of concern during a pandemic outbreak caused by antigenic
shift, when a large geographical area is affected and the general population is naïve to the newly re-assorted viral
strain.
Aim
As part of the EU-funded Universal vaccines Secured (UNISEC) project (http:// www.unisec consortium.eu), we
aim to design phase IIb studies to evaluate the safety, immunogenicity and cross-seasonal clinical efficacy of two
universal influenza vaccines targeting different conserved immunogenic regions of influenza A and B viruses.
FLU-v and M-001 are both peptide based vaccines containing epitopes identified from the viral internal (structural)
proteins. M-001 contains also epitopes from the viral surface glycoproteins. While FLU-v is composed of synthetic
polypeptides, M-001 is composed of a single recombinant protein.
Methods
In two separate trials, a total of 1500 healthy adult study participants will be recruited from multiple centers in
Europe and randomized to receive placebo or the tested influenza vaccines at low or high antigen doses through a
double-blind procedure. Two parenteral administrations will be given with a 21 day interval. In one trial, additional
administrations of pandemic influenza vaccine will also be given 21 and 42 days after the second administration,
aiming to show enhanced response to pandemic influenza strains. Clinical symptom scores and adverse events
(AEs) will be collected throughout the whole study period. Immune correlates of protection will be assessed 21 days
after the second administration and after each pandemic vaccine administration. The (severity of) incident RT-PCRconfirmed influenza A and/or B infection will be recorded over two subsequent influenza seasons. This, together
with clinical symptom score will decide clinical efficacy of the tested vaccines.
Conclusion
Universal influenza vaccines are urgently needed to increase protection among vulnerable groups. Vaccine trial
design needs to incorporate safety, correlates of protection and clinical efficacy
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P182
POSTE R A BSTRAC TS
PL05P08
Effectiveness of inactivated influenza vaccine in children less than 3 years of age over
multiple influenza seasons
Chang, Chia-Kun (1); Tsuei, Hsiang (2); Chih, Yi-Chien (1); Lee, Chia-Lin (1); Chou, Shu-Mei (1); Yang, Chih-Hui (1)
1: Division of Preparedness and Emerging Infectious Diseases, Centers for Disease Control, Taipei, Taiwan, Republic of China; 2: Center for Drug
Evaluation, Taipei, Taiwan, Republic of China
Objectives
Preschool children are at increased risk for severe complications from influenza, but studies about influenza
vaccine effectiveness (VE) are rare. Taiwan launched a free influenza vaccination program for children aged 6 to 24
months in 2004, and expanded to children aged less than 36 months in 2008 according to Advisory Committee on
Immunization Practices. This study aims to assess trivalent inactivated influenza vaccine (TIV) effectiveness among
children aged 6 to 36 months over 7 consecutive influenza seasons in 2004-2010.
Methods
We conducted a nationwide retrospective-cohort study by using National Health Insurance Research Database
(NHIRD) and National Immunization Information System database (NIISD) in Taiwan. Study population was children
aged 6 to 36 months. The information of influenza vaccination status was obtained from NIISD, all enrollee were
divided into three groups: non-vaccinated, partially-vaccinated (one dose only) and full-vaccinated (two doses).
Study period was October to December per year (prior 3 months of flu season). The outcome of interest was medical
utilization and cost because of influenza-like illness (ILI) and the information was retrieved from NHIRD by ICD-9
during 2004-2010 seasons. Student t-test and chi-square test were used to analyze group differences and estimate
Risk Ratio (RR). Logistic regression was used to model the association between influenza vaccination and medical
utilization. Vaccine Effectiveness was calculated as (1-odds ration)*100.
Results
A total of 1,258,795 children were included during 2004-2010 in 7 seasons, full-vaccinated 644,063 (51.16%),
partial vaccinated 143,234 (11.38%) and non-vaccinated 471,498 (37.46%). Compared to other two groups, fullvaccinated group had lowest frequency of hospitalization and emergency department (ED) visits (mean: 0.0070.010, p<.001). RR also showed decreased hospitalization and ED visits in full-vaccinated group. After adjusted age,
gender and region, VE of full-vaccination were 4%-35% for hospitalization, 15%-45% for ED visits as compared to
those not vaccinated. The average medical point for hospitalization in full-vaccination group was 97.0-131.8, which
significantly saved 19%-45% and 43%-139% compared to non-vaccinated group and partially-vaccinated. For ED
visits in full-vaccination group was 29.8-53.3 with saving 14%-82% and -12%-45%. For Outpatient in full-vaccination
group was 288.1-536 with saving -26%-27% and -46%-6%.
Conclusion
This long-term retrospective cohort study found influenza vaccination can reduce the frequency and medical cost
of hospitalization and ED visits , which demonstrates moderate protective effectiveness among preschool children.
Keywords Influenza, vaccine effectiveness, cost-effectiveness, children, Taiwan
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P183
POSTE R A BSTRAC TS
PL05P09
Self-adjuvanting influenza candidate vaccine carrying HA and M1 epitopes on a
proteinaceous multivalent nanoplatform
Szurgot, Inga (1); Szolajska, Ewa (1); Laurin, David (2); Lambrecht, Benedicte (3); Chroboczek, Jadwiga (1,2)
1: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Poland; 2: TheREx, TIMC/IMAG, UMR CNRS/UJF 5525, Domaine de la
Merci, 38700 La Tronche, France; 3: CERVA Veterinary and Agrochemical Research Centre, 1180 Brussels, Belgium
Background
The traditional influenza vaccines, based on the induction of neutralizing antibodies against the influenza surface
glycoproteins have to be produced new every season to match the circulating viruses. It is thought that the vaccine
that could elicit cross-reactive T cell-mediated immunity targeting conserved regions of viral proteins could confer
broader protection across heterologous influenza strains. To meet these expectations we exploit the features of a
virus-like particle, the adenoviral dodecahedron (Dd), for engineering a vaccine carrying both, hemagglutinin (HA)
and the epitopes derived from highly conserved M1 matrix protein, for induction of neutralizing antibodies and, at
the same time, of cross-reactive CTLs.
Dd is a proteinaceous, biodegradable VLP, endowed with remarkable endocytosis activity. Two different M1
immunodominant epitopes were inserted in Dd external positions, whereas HA was attached to the vector surface
by a fragment of adenovirus fiber protein which enables stable interaction with Dd. The vaccine is spontaneously
formed upon expression in the highly flexible and safe baculovirus system, which allows for fast and cost-effective
production.
Methods
In the first step the vaccine in a form of Dd bearing 60 M1 epitopes was constructed, expressed in the baculovirus
system and purified. Its safety and immunogenicity was evaluated in vitro on human peripheral blood mononuclear
cells (PBMC), and in vivo on specific pathogen-free (SPF) chickens. In the second step the complete vaccine containing
both antigens was obtained and analyzed.
Results
The initial candidate vaccine containing immunodominant M1 epitopes strongly induced cell-mediated immunity
both in vitro and in the animal model. In vitro tests showed that Dd carrying M1 epitopes is a potent activator of
human myeloid dendritic cells (MDC). M1 peptides were efficiently presented in the context of HLA class II and crosspresented by the HLA class I molecules, activating both CD8+ memory T cells and CD4+ T cells that are known to
support the generation and maintenance of CD8+ memory T cells. Importantly, upon chicken vaccination with DdM1 both cellular and humoral immune responses were elicited in the absence of an adjuvant. Our data show that
the proposed vaccine is able to induce strong and possibly long-lasting cell-mediated immunity. The final vaccine
containing cellular immunity eliciting M1 immunodominant epitopes as well as humoral immunity eliciting HA
protein is at present prepared. Current work focuses on evaluation of immunogenicity of the complete vaccine (DdM1-HA) in the animal model. The molecular analysis of the final vaccine will be presented.
Conclusions
Development of a stable complex containing two different influenza antigenes on a multivalent presentation
platform - dodecahedron, that serves as a vector and an adjuvant is an important step toward a novel, long-lasting
influenza vaccine that could stimulate both humoral and cellular immunoresponses.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P184
POSTE R A BSTRAC TS
PL05P10
Exploratory Study for Risk Group of Psychogenic Illness Following Vaccination:
Experience from Mass Immunization Campaign Against Pandemic Influenza A (H1N1)
2009 in Republic of Korea
Yang, Taeun; Park, Young-Joon; Kim, Hee Jung; Lee, Yeon Kyeong; Park, Ok
Korea Centers for Disease Control and Prevention, Korea, Republic of (South Korea)
Backgrounds
Increased numbers of psychogenic illness cases following mass vaccination were reported during 2009-2010
influenza season in Korea.
Objectives
We aimed to investigate the characteristics of reported psychogenic illness cases following pandemic influenza mass
vaccination that needed further interventions of National Immunization Program and to estimate the economic
burden by identifying risk group, clinical course and medical cost.
Methods
We reviewed epidemiological features, clinical manifestations, management and medical cost from the reports of
vaccine injury compensation among the cases who were finally concluded as psychogenic illness following 2009
pandemic influenza A (H1N1) vaccination by Korean Advisory Committee on Vaccine Injury Compensation.
Results: During 2009-2010 influenza season, 13 million people were immunized against pandemic influenza. Of
28 psychogenic illness cases following immunization which claimed for compensation, 25 were vaccinated under
school-located mass immunization. Significant numbers of the psychogenic illness following mass vaccination
against pandemic influenza cases were female adolescents (68.0%) and had underlying emotional life-stressors
(36.0%). Their utilization of healthcare produced long duration of hospitalization (mean: 8.1 days) and high cost
(mean: USD $ 2,246 per case).
Conclusions
The female adolescents were high risk group of psychogenic illness following school-located mass immunization and
the medical expenses from their illness were remarkable. Health authorities, immunization managers, organizers
of future mass vaccination should be aware of and prepare psychogenic illness especially for those who are at risk
to achieve immunization goal.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P185
POSTE R A BSTRAC TS
PL05P11
Sensitivity, specificity and predictive values of clinical definitions of influenza
Cohen Jean Marie 1, Gaston De Serres 2, Isabelle Daviaud1, Anne Mosnier 1, Tan Tai Bui 1, Vincent Enouf 3,
Martine Valette 4, Michel Lamure 5 with the help of doctors and virologists in the GROG network.
1: Open Rome, Paris, France and GROG network, France; 2: University of Laval, Québec, Canada; 3: GMVR, Institut Pasteur, Paris, France; 4:
Virology laboratory, University Claude Bernard Lyon 1, France; 5: Health, Individual, Society Laboratory, University Lyon 1, France
Context.
The choice of a very reliable definition may facilitate the development of flu surveillance devices directly involving
the population, as is being experimented in the Netherlands and England, for example.
Flu surveillance systems call on multiple clinical definitions of the disease. Since 1984, most flu surveillance networks
have been based on a double surveillance, clinical and virological. Before an epidemic, their ability to alert is
maximized by a clinical definition that is both sensitive (so as not to “miss the start of the epidemic”) and that has a
strong negative predictive value (to avoid attributing an epidemic to the flu when in fact it is caused by another
virus).
The GROG network is a flu surveillance network in outpatient medicine, which since 2003 has collected
nasopharyngeal specimens in patients with an acute respiratory clinical picture who are tested by culture and/or by
a nucleic acid amplification (RT-PCR) technique, which identifies cases of flu with sensitivity and specificity. Access to
these virological data makes it possible to evaluate the performance of clinical definitions of flu currently used.
Objective.
To estimate the sensitivity (SE), specificity (SP) and positive predictive values (PPV) and negative predictive values
(NPV) of the most commonly used clinical definitions of flu.
Methods
Inclusion period: France, for 9 consecutive seasons of flu surveillance, from September 29th, 2003, to May 23rd, 2012.
Study population: 46,879 patients attending general practice or pediatrics for acute respiratory infection, having
undergone a nasopharyngeal swab along with a clinical file describing the clinical symptoms presented by the
patient.The virological result was positive for the flu in 13,819 of them. Definitions tested: ILI (influenza-like infection,
ECDC, WHO, US CDC), ARI (acute respiratory infection, ECDC/WHO Europe, GROG), FS39 (febrile ILI at 39° C
and higher, INSERM). Reference method: positive virological response to flu virus A or B after analysis of the sample
by culture on MDCK cells and/or by RT-PCR of the nasopharyngeal sample. Epidemic periods: predictive values
depending on the prevalence of infection, 3 periods, pre-, peri- and post-epidemic were considered. The epidemic
period is defined as all the weeks around the epidemic peak and covering 85% of positive samples for the flu.
Results
The most sensitive definitions in each period are those of the US CDC (ILI), the ECDC (ARI) and the GROG network
(ARI). Compared to these definitions, the ILI-WHO underestimates the frequency of flu by about 25% while the
definition limited to very febrile forms (39° C and above) has a higher specificity than other definitions, but its
sensitivity is so low that it detects 3-4 times fewer cases than the others.
In the pre-epidemic period, all definitions have a very low PPV (<13%) and high VPN (93.8% -96%).
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P186
POSTE R A BSTRAC TS
Pre-epidemic
ARI_GROG
ILI_ECDC
ARI _ECDC
ILI_WHO
ILI_US CDC
FS39_Inserm
SE
85.9
82.4
86.2
65.7
90.5
21.9
SP
26.3
32.6
25.2
55.9
20.4
88.3
VPP
8.0
8.4
7.9
10.0
7.9
12.3
VPN
96.1
96.1
96.0
95.6
96.6
93.8
In the epidemic period, all clinical definitions have a similar PPV varying between 46.8% and 56%. Similarly, the
VPN are similar, ranging from 58% to 71%.
Peri-epidemic
ARI_GROG
ILI_ECDC
ARI _ECDC
ILI_WHO
ILI_US CDC
FS39_Inserm
SE
84.3
80.3
84.4
61.5
89.9
19.3
SP
24.9
30.2
24.1
53.6
19.4
88.0
VPP
47.1
47.7
46.8
51.2
46.9
56.0
VPN
66.8
66.0
66.2
63.7
70.9
58.0
In the post-epidemic period, the VPPs are low but higher than in the pre-epidemic period while the VPNs remain at
high levels but lower than the VPNs in the pre-epidemic period.
Post-epidemic
ARI_GROG
ILI_ECDC
ARI _ECDC
ILI_WHO
ILI_US CDC
FS39_Inserm
SE
82.1
78.6
82.0
60.6
90.6
21.1
SP
26.5
31.9
25.7
55.2
20.6
89.2
VPP
17.2
17.7
17.0
20.1
17.5
26.6
VPN
88.9
88.9
88.5
88.3
92.2
85.9
Conclusion
The clinical definitions US CDC (ILI), ECDC (IRA and ILI) and GROG (ARI) have similar and reliable
performances. The low sensitivity of the definition FS39 (febrile flu syndrome at 39° C and higher), due to a desire to
gain specificity, underestimates the burden of influenza by 3 to 4 times compared with other definitions.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P187
POSTE R A BSTRAC TS
PL05P12
Efficacy of chicken immunization against H5N1 influenza virus with bacterially
produced hemagglutinin protein.
Saczynska, Violetta (1); Romanik, Agnieszka (1); Florys, Katarzyna (1); Cecuda-Adamczewska, Violetta (1); Kęsik-Brodacka, Małgorzata (1);
Łukasiewicz, Natalia (1); Sokołowska, Iwona (1); Minta, Zenon (2); Smietanka, Krzysztof (2); Szewczyk, Bogusław (3); Płucienniczak, Grazyna
(1); Płucienniczak, Andrzej (1)
1: Institute of Biotechnology and Antibiotics, Poland; 2: National Veterinary Research Institute, Poland; 3: Intercollegiate Faculty of
Biotechnology of University of Gdansk, Poland
The highly pathogenic avian influenza virus (HPAIV) H5N1 causes multi-organ disease and death in birds as well
as poses a permanent pandemic threat. Therefore, the development of efficacious vaccines against H5N1 HPAIV
is of both veterinary and public health significance. Moreover, the demand for technology that would allow the
efficient production of such a vaccine in a relatively short time, contrary to the traditional egg- or cell culture-based
manufacturing, is widely recognized.
Our research was performed with the use of relative ease, low-cost and efficient bacterial expression system for
the subunit vaccine production. Hemagglutinin (HA) - an immunodominant antigen of influenza viruses, has
been chosen as the target antigen since it is capable of elicit neutralizing antibodies, both during infection and
vaccination. The H5 HA protein, based on the sequence of A/swan/Poland/305-135V08/2006(H5N1) virus was
expressed in Escherichia coli in the form of inclusion bodies. Refolded and purified antigen was examined for
vaccination efficacy in the layer-type chicken model. The immunization was performed twice at six-week interval
by subcutaneous injection of 5, 10, 15 or 25 mcg of rH5 HA per dose with alhydrogel as an adjuvant. To evaluate
humoral immune response, serum samples were collected at different time points of experiment and analyzed for
the presence of anti-H5 HA antibodies by indirect ELISA, competitive ELISA (FLUAcH5, IDVet) and hemagglutination
inhibition (HI) test with heterologous H5N2 low pathogenic AIV.
It was shown that two doses of rH5 HA are necessary but sufficient to induce robust immune response in all
immunized chickens. The endpoint titers of anti-H5 IgY antibodies were as high as ~ 330 000, on the average.
Moreover, 60 - 90% birds immunized at applied dosage regimens were positive in FLUAc H5 test. Importantly,
protective HI antibody titers (≥ 1:16) were found in 100% and 80% of chickens immunized at 10 - 25 mcg and 5
mcg per dose, respectively, and HI-antibody titers reached high values up to 1:512. Altogether, these results indicate
potential of our H5 HA-based, bacterially produced, vaccine candidate to confer protection against H5N1 AIV.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P188
POSTE R A BSTRAC TS
PL05P13
Airway fluids from mice and ferret contain different innate immune proteins that
mediate antiviral activity against influenza A viruses
Job, Emma (1,2); Short, Kirsty (2); Deng, Yi-Mo (3); Laurie, Karen (3); Brooks, Andrew (2); Saelens, Xavier (1); Reading, Patrick (2,3)
1: Inflammation Research Centre, VIB and UGent, Belgium; 2: Department of Microbiology and Immunology, Peter Doherty Institute,
University of Melbourne; 3: World Health Organisation Collaborating Centre for Influenza, Peter Doherty Institute, Melbourne
Soluble neutralizing inhibitors in airway fluids of humans and animals provide an effective first-line barrier to
influenza A virus (IAV) infection. Ferrets and mice represent the most widely used animal models to study human
IAV and immune responses to IAV infection have been particularly well characterized in the mouse model.
Previous studies demonstrated that surfactant protein (SP)-D, a member of the collectin superfamily, is the major
neutralizing inhibitor of IAV in the upper and lower respiratory tract of mice. However, little is known regarding
the nature of soluble anti-IAV proteins in the ferret airways, nor whether these are similar to those in mice or in
humans. Our recent work has demonstrated that soluble neutralizing inhibitors in the airways of ferrets differ
markedly in their properties compared to those present in mice. We demonstrate that a SP-D-resistant mutant of
A/Philippines/2/1982 was highly virulent in mice, but showed no difference in its ability to infect and replicate in
ferret airways compared to wild-type virus. Although SP-D was detected in bronchoalveolar lavage fluids (BALF) from
naïve ferrets, it was not the major neutralizing activity against H3N2 subtype viruses. Instead, one or more sialic
acid α(2,6)-rich glycoproteins in the ferret airways were shown to act as potent inhibitors of H3N2 IAV. Mutants of
H3 subtype resistant to the sialylated glycoprotein inhibitor in ferret BALF was characterized by a single amino acid
substitution, L226Q, in the receptor-binding pocket of the HA. This mutation is typically associated with resistance
to α(2,6) sialylated glycoproteins and results in a change in receptor preference from dual recognition of SAα(2,3)
and SAα(2,6) to a sole recognition of SAα(2,3). Affinity chromatography and mass spectrometry techniques have
identified 5 candidate glycoproteins which are currently being explored further. Together, these data indicate clear
differences in the major neutralizing inhibitors in mouse and ferret BAL which are active against IAV.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P189
POSTE R A BSTRAC TS
PL05P14
Diagnostic reassortant influenza strains for anti-neuraminidase antibody detection in
clinical studies of live influenza vaccine
Smolonogina, Tatiana; Desheva, Yulia; Donina, Svetlana; Rekstin, Andrey; Rudenko, Larisa
Institute of Experimental Medicine, Russian Academy of Medical Sciences, Saint Petersburg, Russian Federation
Background
Both hemagglutinin (HA) and neuraminidase (NA) antigens contribute to protection against influenza illness.
WHO-organized meetings repeatedly underlined the importance of improvement of methodology for NA-specific
assays in order to evaluate different aspects of the immune response after influenza infection and vaccination.
One of the problems is the specificity of neuraminidase inhibition (NI) assays, since antibodies that bind to HA can
interfere with the interpretation of neuraminidase inhibition responses. The use of recombinant NAs can solve the
problem, but it is difficult to obtain NA in active multimeric form. Alternatively, antigenic hybrid influenza strains
with mismatched HA can be used as antigen. The aim of this study was to prepare a collection of well-characterized
diagnostic reassortant influenza A strains comprising NA of epidemic, pandemic and potentially pandemic viruses
for specific evaluation of quality of the anti-NA antibody response after influenza vaccination.
Materials and methods
Several diagnostic reassortant strains of A(H7N1) and A(H7N2) subtypes containing the NA of A/
Vietnam/1203/04(H5N1), A/California/07/09(H1N1), A/Leningrad/134/17/57(H2N2), A/California/1/66(H2N2)
and A/Victoria/361/11(H3N2) influenza viruses were generated by classic genetic reassortment in embryonated
chicken eggs. All reassortants comprised the HA of parent strain A/equine/Prague/1/56(H7N7). The peroxidaselinked lectin micro-procedure of NI assay previously reported by C.R. Lambre et al. was adjusted using these
reassortants. NI antibodies as well as hemagglutination-inhibiting (HI) or neutralizing antibodies were measured in
sera (390 samples) of volunteers who participated in clinical studies of live attenuated influenza vaccine candidates
of potentially pandemic HA-subtypes: A/17/duck/Potsdam/86/92(H5N2), A/17/turkey/Turkey/05/133(H5N2) and
A/17/California/66/395(H2N2).
Results
A panel of high-yield reassortant strains with NA of a variety of influenza A viruses and A/Leningrad/134/17/57(H2N2)
or A/Puerto Rico/8/34(H1N1) backbone was generated. Equine HA inherited by reassortant strains had low (40-47%)
amino acid sequence identity with HA of current epidemic and potentially pandemic strains (H5 or H2 subtypes) and
didn’t cross-react in HI assay with rat or human anti-sera to these influenza A viruses. Strong correlation (Rs=0.75
(95%CI: 0.66-0.79); n=44; p<0.0001) between anti-NA antibody titers obtained in NI assay using either whole
reassortant virus or recombinant multimeric NA suggests that the data obtained using diagnostic H7 strains were
reliable. NI assay revealed ≥2-fold increase of anti-NA antibody in sera of 13.8-48.1% of volunteers after two vaccine
doses. Coincidence of antibody titer increase in NI assay and microneutralization/HI test in the same pairs of sera
of vaccinated subjects ranged between 55.6% and 73.2% depending on the vaccine strain. 3.4-29.6% of vaccinated
volunteers developed anti-NA antibody response in the absence of increase in HI or neutralizing antibody titers.
Conclusions
The NI assay with diagnostic reassortant H7 strain as antigen specifically revealed serum anti-NA antibodies. Our
data suggest that the NI test may serve as an informative addition to the traditional serologic assays providing more
comprehensive evaluation of influenza vaccine immunogenicity.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P190
POSTE R A BSTRAC TS
PL05P15
MUC1 enhances influenza binding and infection of epithelial cells
Allen, Robert John; Brown, Lorena Elizabeth; McAuley, Julie Louise
The University of Melbourne, VIC, Australia
Background
Influenza A virus (IAV) haemagglutinin (HA) is known to interact with sialic acid (SA)- containing receptors to
facilitate viral entry to a host cell. However, the identity of the receptor molecule(s) that trigger endocytosis upon
virus binding remains unknown. Large sialylated glycoproteins on epithelial cells may potentially function either to
protect against or facilitate influenza infection. Herein, the cell-surface mucin MUC1 has been investigated for its
role in IAV infection.
Methods
Chinese hamster ovary K-1 (CHO) cells engineered to express human MUC1 (CHOMUC1+) were used alongside
unmodified CHO cells (CHO-Parent) in influenza infection experiments. These cells were either untreated or pretreated with bacterial neuraminidase to remove terminal SA structures in order to confirm SA-dependence of virion
attachment and entry. Cells were infected with A/Udorn/307/72 (H3N2) virus at a multiplicity of infection of 10
and harvested 1 or 8 hours later. Cells were analysed by flow cytometry and confocal microscopy to evaluate the
percentage of cells with virions attached by detection of HA (1hpi) and the amount of viral nucleoprotein (NP)
produced within the first round of replication (8hpi).
Results
Flow cytometric data on cells at 1hpi showed that a significantly greater percentage of CHO-MUC1+ cells are HA+
compared to CHO-Parent cells (87 vs 58%, p < 0.001; unpaired t test). Additionally, analysis of infected cell monolayers
by confocal microscopy demonstrated that significantly greater numbers of fluorescent pixels representing HA
staining were present on the surface of CHO-MUC1+ cells compared to CHO-Parent cells (HA pixels/nucleus 0.76
vs 0.45, p < 0.001; unpaired t test). In addition, HA staining co-localised with MUC1 staining on 89% of occasions,
indicating a possible close association. At 8hpi, a significantly higher proportion of CHO-MUC1+ cells were viral
NP+, compared to CHO-Parent cells (76 vs 48%, p < 0.001; unpaired t test). These interactions are dependent on the
presence of SA at the cell surface as all neuraminidase-treated cells had minimal levels of viral HA bound to the cell,
regardless of MUC1 expression.
Conclusions
The presence of MUC1 at the cell-surface of epithelial cells appears to enhance viral attachment and infection
of cells, at least in vitro. The interaction of virus with MUC1 was supported by co-localisation studies and was
dependent upon the presence of sialic acid. These data implicate MUC1 as a binding and entry receptor molecule
for influenza on epithelial cells. Knowledge of cell-surface receptors for influenza A virus may enhance research into
receptor-mediated viral endocytosis and provide a potential therapeutic target.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P191
POSTE R A BSTRAC TS
PL05P16
Use of government-funded antiviral drugs for containment of an influenza A(H1N1)
pdm09 outbreak in a long-term care psychiatric facility, 2014
Lin, Mei-Ling (1); Teng, Ya-Wen (2); Chih, Yi-Chien (1); Chang, Chia-Kun (1); Chou, Shu-Mei (1); Yang, Chih-Hui (1)
1: Division of Preparedness and Emerging Infectious Diseases, Centers for Disease Control, Taipei, Taiwan, Republic of China; 2: Eastern Regional
Center, Centers for Disease Control, Taipei, Taiwan, Republic of China
Objectives
During seasonal influenza epidemics, outbreaks of influenza in closed institutions are common and such
outbreaks might be associated with high morbidity, even with serious mortality. To effectively control seasonal
influenza outbreaks in long-term care facilities, the Taiwan Centers for Disease Control (Taiwan CDC) has released
and distributed stockpiled antiviral drugs (oseltamivir and zanamivir) for residents and health care providers as
treatment and chemoprophylaxis during flu season since 2011. From April 13 through May 4, 2014, eight clusters of
influenza-like illness (ILI) with influenza A(H1N1)pdm09 occurred in different psychiatric wards of the same facility.
We report the efficacy of chemoprophylaxis with antiviral drugs to control outbreaks in semiclosed environments.
Materials and Methods
There were 2,595 residents and 667 staff members in the facility with 10 wards, the seasonal influenza vaccination
coverage rate were 94.7% and 94.9% respectively. All personnel with suspected infection were tested, treated
with antiviral drugs and clinically isolated. In addition, we administered postexposure chemoprophylaxis with
antiviral drugs to all residents and staff members in six of the eight affected wards, including one acute ward.
The intervention dates varied across the six wards. Other preventative measures, including case isolation, personal
hygiene, wearing proper personal protective equipment, and intensive daily health surveillance, were implemented
during the outbreak period.
Results
A total of 829 personnel were at risk across the six affected wards, with 641 receiving chemoprophylaxis. There
were 174 ILI cases (174/228=76%, 228: total ILI cases) detected in the six wards during the investigation period
with a mean attack rate of 21% (174/829). The mean attack rate for the other two wards without chemoprophylaxis
intervention was 11.1% (54/488) A total of 21 nasopharyngeal swabs specimen were tested with 12 positive for
influenza A(H1N1)pdm09. A total of 151 personnel (86.8%) were infected before the intervention, and 23 (13.2%)
after the intervention. There was a significant reduction in the overall attack rate, from 18.2% (151/829) before the
intervention to 2.8% (23/829) after the intervention. Furthermore, the attack rates dropped obviously in five chronic/
mild wards with declining about 74-100% after chemoprophylaxis intervention, and the attack rate in the acute
ward declined 57% after the intervention.
Conclusion
Although the clusters were not reported in real-time which caused the delay of chemoprophylaxis and
underestimation, the study still found antiviral chemoprophylaxis together with early identification and isolation of
infected personnel, was effective in reducing the impact of outbreaks of influenza in closed facility.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P192
POSTE R A BSTRAC TS
PL05P17
Circular DNAzyme against the BM2 gene transcript of influenza B virus: a potent
inhibitor of virus replication in host cells
Kumar, Binod; Rajput, Roopali; Kumar, Prashant; Khanna, Madhu
Department of Respiratory Virology, Vallabhbhai Patel Chest Institute, University of Delhi, India
Background
The influenza B virus BM2 ion channel protein is highly conserved and essentially required during the trafficking,
assembly and budding processes of virus, thus an attractive target for designing antiviral drugs. Although single
stranded DNAzymes (Dz’s) may represent the most effective nucleic acid drug to date, they are nevertheless sensitive
to nuclease degradation and require modifications for in vivo application. The previously used PTO modifications
have been shown to reduce the catalytic efficiency and are toxic when applied in vivo.
Methods
We designed several 10-23 DNAzymes (Dz’s) targeting different regions of the matrix gene (BM2) of influenza B virus
and analyzed their ability to specifically cleave the target RNA in both cell free systems as well as in cell culture using
transient transfections. We further aimed to circularize the Dz to provide extended stability keeping the catalytic
efficiency intact.
Results
RT-PCR and real-time RT-PCR assays showed significant 45% inhibition of BM2 gene of influenza B viruses upon
specific Dz treatment. The transfection of MDCK cells with Dz showed reduced cytopathic effect caused by influenza
B virus (B/Yamagata/K270/2001) and considerably reduced the BM2 protein expression for a comparatively longer
duration of time. The catalytic efficiency of both linear and circular DNAzyme was found comparable. As expected,
the mutant-Dz did not hinder in virus replication showing high level of specificity of designed Dz towards the target
RNA.
Conclusions
Our results demonstrate substantial reduction in whole virus replication thereby paving new dimensions in antiviral
therapy. Our study, for the first time, has documented antiviral potential of circular-Dz against BM2 transcript of
influenza B virus. Thus, we propose that the 10-23 DNAzymes may be used as selective and effective inhibitor of
viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza
infection.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P193
POSTE R A BSTRAC TS
PL05P18
The fraction of influenza virus infections that are asymptomatic: a systematic review
and meta-analysis
Leung, Nancy; Xu, Cuiling; Ip, Dennis; Cowling, Benjamin
School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China.)
Background
Influenza virus infections lead to a wide range of clinical manifestations, from severe pneumonia through to
subclinical or even asymptomatic disease. There has been substantial controversy over thproportion oinfluenza virus
infections that are asymptomatic, referred to as the asymptomatic fraction (AF). Knowledge on the AF, variation in
the AF in certain groups, and the potential infectiousness of asymptomatic cases is extremely important in designing
public health control strategies sucas contact tracing and quarantine, and in estimating the burden of disease.
Methods
We conducted a systematic review to assess and summarize the AF of influenza virus infections. Influenza virus
infections were confirmed by RT-­PC oviral culture in respiratory specimens, while serologic evidence of recent
influenza virus infection was indicated by a 4-­fold or greater rise in antibody titer in paired sera or a high titer
ia single post­‐epidemic serum specimen. The AF is defined as the proportion of individuals with influenza virus
infection that experienced no symptomatic illness.
Results
We found that estimates of the AF were reported from two different types of studies: first, outbreak investigations
with short-­term followup ­of potentially exposed persons and virologic confirmation of infections; second, studies
conducted across epidemics typically evaluating rates of acute respiratory illness among persons with serologic
evidence of infection, in some cases adjusting for background rates of illness from other causes. Most point
estimates from studies of the first type fell ithe range 8%-­28%. Estimates from the second type of study without
adjustment were very heterogeneous (point estimates 0%-­93%; I2=97%), while estimates from studies that adjusted
for the rate of background illnesses were more consistent with point estimates in the range 65%-­85%.
Conclusions
Variation in estimates could be partially explained by differences in study design and analysis, and inclusion of mild
symptomatic illnesses as asymptomatic in some studies. Considerable differences between the AF of infections
confirmed by virologic versus serologic testing may indicate fundamental differences in the interpretation of these
two indicators.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P194
POSTE R A BSTRAC TS
PL05P19
Expression, purification and immunological properties of glycosylated influenza H5N1
hemagglutinin produced in Pichia pastoris
Kopera, Edyta; Zagórski-Ostoja, Włodzimierz; Grzelak, Krystyna; Protas-Klukowska, Anna Maria; Zdanowski, Konrad; Pietrzak, Maria; Macioła,
Agnieszka
Institute of Biochemistry and Biophysics Polish Academy of Sciences, Poland
Background
Indicate the purpose and objective of the research, the hypothesis that was tested or a description of the problem
being analyzed or evaluated;
It is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. The HA
gene mutates however fast making the production of a new vaccine very challenging since the vaccine production
lasts several months. In the attempt towards developing influenza vaccine production that would be faster and
safer we used surface antigen alone. In this report, we describe the expression of HA gene of H5N1 avian influenza
virus in simple Pichia pastoris system. rHA was produced in a soluble form with high yield and was shown to be
highly immunogenic in animals.
Methods
Describe the setting/location for the study, study design, study population, data collection and methods of analysis
used;
The A/swan/Poland/305-135V08/2006 (H5N1- subtype) hemagglutinin (HA) gene was cloned and expressed in
yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was
fused to α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1
(AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. rHA fusion protein
with His-6 affinity tag was secreted into the culture medium and was purified to homogeneity in one step using
Ni-NTA agarose. Glycosylation sites of rHA were determined using LC-MS-MS/MS (liquid chromatography coupled
to tandem mass spectrometry). The immunological properties of rHA antigen were tested in vivo. Mice were
immunized intradermally with three doses of rHA (1, 5 and 25 μg) given at 3-weekly intervals. The control group
received at the same intervals adjuvant only. Chicken were immunized subcutaneously twice with 25 μg of rHA
given at 4 weeks interval. After chicken and mice immunization sera samples were tested by ELISA.
Results
Present as clearly and detailed as possible the findings/outcome of the study, with specific results in summarized
form.
Recombinant HA antigen was secreted into the culture medium reaching the approximately 15 mg/ L (KM 71 strain).
rHA was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated
that the protein is cleaved into HA1 and HA2 domains linked with disulphide bond. Analysis of the N-linked glycans
revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. The
immunological activity of hemagglutinin protein was tested in chicken and mice, where rHA elicited high immune
response.
Conclusions
Briefly discuss the data and main outcome of the study. Emphasize the significance for influenza prevention,
treatment, care and/or support, and implications of the results.
The data presented here demonstrate that influenza antigen produced in P. pastoris with good efficiency is highly
immunogenic and might be consider as a candidate for subunit vaccine.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P195
POSTE R A BSTRAC TS
PL05P20
RSV infection in neonates
Hammoud, Majeda Sabri
Kuwait university, Kuwait
Background
Respiratory syncytial virus (RSV) causes frequent nosocomial outbreaks in general pediatric wards but is less
commonly reported in neonatal intensive care units. We investigated an outbreak of RSV infection in the neonatal
unit at Maternity Hospital and call for a review of the guidelines of palivizumab prophylaxis is suggested.
Methods
Chart review was performed after an RSV outbreak which occurred in our unit in February 2012. A case of RSV was
defined as an infant with a nasopharyngeal aspirate positive for RSV by PCR technique. Management of the infected
infants and their outcome were also recorded.
Results
During the outbreak, 12 preterm infants turned RSV positive (mean age at infection, 38days; mean birth weight,
1457 g; and mean gestational age, 31 weeks). Six infected infants became very sick and required intubation with
mechanical ventilation. There was no significant difference in birth weight or gestational age between those who
were intubated and the non-­‐intubated infants. The RSV-­positive patients were isolated, and infection control
measures were implemented. Palivizumab was administered to all patients and their contacts and no new cases
were subsequently identified. All infants survived Conclusion The current guidelines which recommend palivizumab
prophylaxis to preterm infants after discharge during winter season seem to be insufficient to prevent outbreaks in
NCUs. Palivizumab combined with infection control measures appears to prevent the spread of RSV in the neonatal
setting. More studies are needed to investigate whether the use of palivizumab prophylaxis should start at NCUs in
order to reduce RSV outbreaks.
TH E FI FTH ESWI I N FLU ENZA CON FER ENC E | 14-17 SEPTEMBER 2014 | R IGA
P196

UPDATE: Influenza Outbreak Lifted at RHC Rainycrest

Vaccine for Prevention of Influenza in Children

Approval of a Cell Culture Pandemic Influenza Vaccine (H5N1)

ILI - the Tennessee Department of Health

Number 4 - April 2014 - Northern Health Physicians

Strafanzeige - Partei Mensch Umwelt Tierschutz

Seasonal Influenza Vaccination

Read Full - Public Health Laboratory

Handout - College of American Pathologists

Update on U.S. Pandemic Influenza Vaccine Development