Novartis Vaccines Academy Immune

Novartis Vaccines Academy
Learning around the Globe
PhD Scholarship Project – Università degli Studi di Bologna
Immune-competent human skin equivalents for
predicting Staphylococcus aureus vaccine
efficacy
Supervisor
Group
composition
Fabio Bagnoli [email protected]
Dr. Clarissa Pozzi, Dr. Silvia Maccari, Dr. Paolo Ruggiero, Prof. Jos Van Strijp
Research interest of the Group
Staphylococcus aureus is a major human pathogen and current antibiotics are not efficacious
against emerging multidrug resistant strains. Therefore, there is an urgent need to develop
vaccines to target this pathogen. However, S. aureus vaccine development is hindered by the
lack of known correlates of protection. staphylococcal cutaneous infections are among the
leading cause of emergency department visits and represent an important target for clinical
trials.
We believe that three dimensional organotypic human skin models are informative and tractable
experimental systems and they may represent a valid alternative to animal infection models.
Indeed, S. aureus expresses several human-specific toxins, immune evasion and complement
binding factors. Therefore, no reliable correlates of protection are expected to be identified on
the basis of animal models. One of the aims of this proposal is to integrate key immune
components in the 3D epithelial matrix. Role of staphylococcal vaccine antibodies in neutralizing
skin pathogenesis and preventing immune evasion mechanisms within the humanized matrix
will be evaluated. We believe that this research will put the basis for developing the first human
skin vaccine model.
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PhD Scholarship Project – Università degli Studi di Bologna
PhD project
Human organotypic models are emerging as promising predictive preclinical methods. In
addition, research on alternative methods to animal models is an ethical priority of the scientific
community. However, at the present such models have been rarely used in the S. auerus
research field and, to our knowledge, never with immune-competent models. The major aim of
this proposal is to put the basis for the development of the first human skin vaccine model and
use it to understand mechanisms of protection against S. aureus skin infection. To do that, we
will integrate key immune components such as antibodies against S. aureus vaccine antigens,
complement factors, cytokines and professional phagocytes in the epithelial matrix. To
accomplish this goal the PhD student will have to be familiar with basics of Immunology.
Although some of the immune components will be already available, the student will need to
purify phagocytes from fresh blood. In collaboration with the University of Utrecht the student
will take advantage of cutting-edge technologies (e.g., induced pluripotent stem cell technology),
which will be used to generate macrophages and neutrophils.
Mechanisms of protection triggered by vaccine antibodies, cytokines, and professional
phagocytes against staphylococcal invasive processes of the skin layers will be studied by
histology as well as confocal microscopy. Bacterial viability will be determined by standard
microbiology techniques. These studies will benefit of the availability both at Novartis and at the
University of Utrecht of mutant strains for vaccine antigens and most of the virulence factors of
S. aureus. Furthermore, GFP, mRFP and YFP expressing plasmids will also be part of the toolbox
available for this project, which will greatly facilitate confocal microscopy studies.
Other aspects that the student will characterize are the immune response elicited in the skin
cells by the pathogen as well as the influence of the host-pathogen interaction on bacterial gene
and protein expression. This will be key to understand how skin response could be modulated
by cytokines/adjuvants to improve vaccine efficacy and if target antigens are expressed during
skin infection. Therefore, the student will handle RNA preparations that will be retro-transcribed
and analysed by RT-PCR and/or DNA microarrays. On the other hand, cytokine expression from
the skin cells will be confirmed by Luminex Technology. S. aureus protein expression will be
evaluated by quantitative mass spectrometry taking advantage of the expertise available at
Novartis on this innovative technology. Last but not least, our team is setting up collaboration
with the Plastic Surgery Department at the University of Siena. Through this collaboration we will
have access to human skin explants from non-infected patients as well as with S. aureus
infections. Confirming data obtained with the skin equivalent model studying natural human skin
infections will certainly represent an important added value of the project
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PhD Scholarship Project – Università degli Studi di Bologna
Technologies employed
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Human skin equivalents
Handling of professional phagocytes
Basics of the induced pluripotent stem cell technology
RNA preparation/RT-PCR and/or DNA microarray
Cytokine measurement by Luminex
Microbial mutagenesis
Confocal microscopy
Relevant publications on the topic
1. The staphylococcal Esx proteins modulate apoptosis and release of intracellular
Staphylococcus aureus during infection in epithelial cells" by Charalampia Korea, Giuliana
Balsamo, Alfredo Pezzicoli, Christina Merakou, Simona Tavarini, Fabio Bagnoli, Davide
Serruto, and Meera Unnikrishnan. (paper submitted to IAI)
2. Three-dimensional human skin models to understand Staphylococcus aureus skin
colonization and infection. Lauren Popov, Joanna Kovalski, Guido Grandi, Fabio Bagnoli and
Manuel R. Amieva. Front Immunol. 2014 Feb 5.
3. Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.
Kuhn ML, Prachi P, Minasov G, Shuvalova L, Ruan J, Dubrovska I, Winsor J, Giraldi M, Biagini
M, Liberatori S, Savino S, Bagnoli F, Anderson WF, Grandi G. FASEB J. 2014 Jan 13.
4. An auto-assembling Staphylococcus aureus alpha-hemolysin as a vaccine mimicking natural
immune response against the toxin. Luigi Fiaschi, Benedetta Di Palo, Maria Scarselli, Bruno
Galletti, Vincenzo Nardi Dei, Letizia Arcidiacono, Ravi Mishra, Elena Mori, Michele Pallaoro,
Fabiana Falugi, Maria Rita Fontana, Marco Soriani, Guido Grandi, Ilaria Ferlenghi & Fabio
Bagnoli. Journal of Clinical Investigation (paper under revision).
5. Host immune responses against Staphylococcus aureus infections and mechanisms of
protection: closing the knowledge gap to develop efficacious vaccines. Research topic,
Frontiers in Immunology (Edited by Fabio Bagnoli and Sylvie Bertholet)
6. Protective Activity of the CnaBE3 Domain Conserved among Staphylococcus aureus Sdr
Proteins. Becherelli M, Prachi P, Viciani E, Biagini M, Fiaschi L, Chiarot E, Nosari S, Brettoni
C, Marchi S, Biancucci M, Fontana MR, Montagnani F, Bagnoli F, Barocchi MA, Manetti AG.
PLoS One. 2013 Sep 17;8(9).
7. Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FLIPr) and its homologue
FLIPr-like are potent FcγR antagonists that inhibit IgG-mediated effector functions.
Stemerding AM, Köhl J, Pandey MK, Kuipers A, Leusen JH, Boross P, Nederend M, Vidarsson
G, Weersink AY, van de Winkel JG, van Kessel KP, van Strijp JA. J Immunol. 2013 Jul
1;191(1):353-62.
8. Staphylococcal Ecb protein and host complement regulator factor H enhance functions of
each other in bacterial immune evasion. Amdahl H, Jongerius I, Meri T, Pasanen T,
Hyvärinen S, Haapasalo K, van Strijp JA, Rooijakkers SH, Jokiranta TS. J Immunol. 2013 Aug
15;191(4):1775-84.
9. The staphylococcal toxin Panton-Valentine Leukocidin targets human C5a receptors. Spaan
AN, Henry T, van Rooijen WJ, Perret M, Badiou C, Aerts PC, Kemmink J, de Haas CJ, van
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PhD Scholarship Project – Università degli Studi di Bologna
Kessel KP, Vandenesch F, Lina G, van Strijp JA. Cell Host Microbe. 2013 May 15;13(5):58494.
10. Mining the bacterial unknown proteome: identification and characterization of a novel
family of highly conserved protective antigens in Staphylococcus aureus. Schluepen C, Malito
E, Marongiu A, Schirle M, McWhinnie E, Lo Surdo P, Biancucci M, Falugi F, Nardi-Dei V,
Marchi S, Fontana MR, Lombardi B, De Falco MG, Rinaudo CD, Spraggon G, Nissum M,
Bagnoli F, Grandi G, Bottomley MJ, Liberatori S. Biochem J. 2013 Jul 29.
11. Structural and functional characterization of the Staphylococcus aureus virulence factor and
vaccine candidate FhuD2. Mariotti P, Malito E, Biancucci M, Lo Surdo P, Mishra RP, NardiDei V, Savino S, Nissum M, Spraggon G, Grandi G, Bagnoli F, Bottomley MJ. Biochem J.
2013 Feb 1;449(3):683-93.
12. The staphylococcal toxin Panton-Valentine Leukocidin targets human C5a receptors. Spaan
AN, Henry T, van Rooijen WJ, Perret M, Badiou C, Aerts PC, Kemmink J, de Haas CJ, van
Kessel KP, Vandenesch F, Lina G, van Strijp JA. Cell Host Microbe. 2013 May 15;13(5):58494
13. Opsonic and protective properties of antibodies raised to conjugate vaccines targeting six
Staphylococcus aureus antigens. Pozzi C, Wilk K, Lee JC, Gening M, Nifantiev N, Pier GB.
PLoS One. 2012;7(10):e46648
14. Rudkin JK, Bowden MG, Edwards A, Brown EL, Pozzi C, Waters EM, Chan WC, Williams P,
O’Gara JP and. Massey RC. Methicillin Resistance Reduces the Virulence of HealthcareAssociated Methicillin-Resistant Staphylococcus aureus by Interfering With the agr Quorum
Sensing System. J Infect Dis. 2012 Mar; 205 (5):798-806.
15. Pozzi C, Waters EM, Rudkin JK, Schaeffer C, Pier GB, Fey PD, Massey RC and O’Gara
JP.Methicillin resistance alters the biofilm phenotype and attenuates virulence in
Staphylococcus aureus device-associated infection. PloS Pathog 2012 Apr;8(4):e1002626.
16. Houston P, Rowe SE, Pozzi C, Waters EM, O’Gara JP. An essential role for the major
autolysin in the fibronectin-binding protein- mediated Staphylococcus aureus biofilm
phenotype. Infect.Immun. 2011 Mar; 79 (3):1153-65
17. Collins J, Rudkin J, Recker M, Pozzi C, O'Gara JP, Massey RC. Offsetting virulence and
antibiotic resistance costs by MRSA. ISME J. 2010 Apr; 4(4): 577-84.
18. Vaccines and antibiotic resistance. Mishra RP, Oviedo-Orta E, Prachi P, Rappuoli R, Bagnoli
F. Curr Opin Microbiol. 2012 Oct;15(5):596-602.
19. Functional basis for complement evasion by staphylococcal superantigen-like 7. Bestebroer
J, Aerts PC, Rooijakkers SH, Pandey MK, Köhl J, van Strijp JA, de Haas CJ. Cell Microbiol.
2010 Oct;12(10):1506-16. doi:
20. A novel computational method identifies intra- and inter-species recombination events in
Staphylococcus aureus and Streptococcus pneumoniae. Sanguinetti L, Toti S, Reguzzi V,
Bagnoli F, Donati C. PLoS Comput Biol. 2012;8(9)
21. Inferring reasons for the failure of Staphylococcus aureus vaccines in clinical trials. Bagnoli
F, Bertholet S, Grandi G. Front Cell Infect Microbiol. 2012;2:16.
22. Staphylococcus aureus FhuD2 is involved in the early phase of staphylococcal dissemination
and generates protective immunity in mice. Mishra RP, Mariotti P, Fiaschi L, Nosari S,
Maccari S, Liberatori S, Fontana MR, Pezzicoli A, De Falco MG, Falugi F, Altindis E, Serruto D,
Grandi G, Bagnoli F. J Infect Dis. 2012 Oct 1;206(7):1041-9.
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PhD Scholarship Project – Università degli Studi di Bologna
23. Fabio Bagnoli and Rino Rappuoli. Vaccine Design: Innovative Approaches and Novel
Strategies. Horizon Scientific Press. 2011 Sept (Book Editor)
24. Hwan Keun Kim, Andrea DeDent, Alice G. Cheng, Molly McAdow, Fabio Bagnoli, Dominique
M. Missiakas and Olaf Schneewind. IsdA and IsdB antibodies protect mice against
Staphylococcus aureus abscess formation and lethal challenge. Vaccine. 2010 Aug
31;28(38):6382-92
25. Pozzi C, O’Neill E, Houston P, Loughman A, Humphreys H, Foster TJ, O’Gara JP. A novel
Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins,
FnBPA and FnBPB. J Bacteriol. 2008 Jun; 190 (11): 3835-50.
26. Fabio Bagnoli, Monica Moschioni, Claudio Donati, Valentina Dimitrovska, Ilaria Ferlenghi,
Claudia Facciotti, Alessandro Muzzi, Fabiola Giusti, Carla Emolo, Antonella Sinisi, Markus
Hilleringmann, Werner Pansegrau, Stefano Censini, Rino Rappuoli, Antonello Covacci, Vega
Masignani & Michele A. Barocchi. J Bacteriol. 2008 Aug;190(15):5480-92.
27. O’Neill E, Pozzi C, Houston P, Smyth D, Humphreys H, Robinson DA, O’Gara JP. Association
between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates
from device-related infections. J Clin Microbiol. 2007 May; 45(5): 1379-88.
28. Staphylococcal complement evasion by various convertase-blocking molecules. Jongerius I,
Köhl J, Pandey MK, Ruyken M, van Kessel KP, van Strijp JA, Rooijakkers SH. J Exp Med.
2007 Oct 1;204(10):2461-71
29. Bagnoli F., Buti L., Tompkins L., Covacci A., Amieva M. Helicobacter pylori CagA induces a
transition from polarized to invasive phenotypes in MDCK cells. Proc Natl Acad Sci U S A.
2005 102(45):16339-44*.
30. Rooijakkers SH, Ruyken M, Roos A, Daha MR, Presanis JS, Sim RB, van Wamel WJ, van
Kessel KP, van Strijp JA. Immune evasion by a staphylococcal complement inhibitor that
acts on C3 convertases. Nat. Immunol. 2005 Sep;6(9):920-7. Epub 2005 Aug 7.
31. de Haas CJ, Veldkamp KE, Peschel A, Weerkamp F, Van Wamel WJ, Heezius EC, Poppelier
MJ, Van Kessel KP, van Strijp JA. Chemotaxis inhibitory protein of Staphylococcus aureus, a
bacterial antiinflammatory agent. J Exp Med. 2004 Mar 1;199(5):687-95. PubMed PMID:
14993252;
PhD technical development
The student will acquire the following major technical skills:
1)
2)
3)
4)
5)
6)
7)
Development of immune-competent human skin equivalents
Handling of professional phagocytes
Basics of the induced pluripotent stem cell technology
RNA preparation/RT-PCR and/or DNA microarray
Cytokine measurement by Luminex
Microbial mutagenesis
Confocal microscopy
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