Novartis Vaccines Academy Learning around the Globe PhD Scholarship Project – Università degli Studi di Bologna Understanding and exploiting lipoprotein translocation in Gram negative pathogens for improved vaccine antigen delivery platforms Supervisor Group composition Isabel Delany [email protected] Andreas Haag, Marco Spinsanti Sara Borghi Research interest of the Group One of the major aims of our research is in the understanding of the regulatory circuits which allow pathogenic bacteria, to respond to environmental stimuli that they may meet in the host. In particular we investigate responses in gene expression to molecular signals correlated to the human host environment and undertake molecular characterization of the mechanisms of transcriptional or posttranscriptional control of bacterial antigens and genes often important for virulence. We use molecular genetic studies to characterize the role of virulence factors and candidate antigens in the bacterial systems in ex vivo and in vivo models of infection. We also use genetics to characterize the immunogenicity of bacterial antigens with respect to the identification of functional bactericidal epitopes. Furthermore, we apply bacterial genetics methodology for the design and construction of innovative vaccine strains, that generate generalized modules of membrane antigens (GMMA) enriched in important vaccine antigens that can be exploited as next generation acellular vaccine candidates. 1 http://www.novartisvaccines.it/ricerca/nva-eng.shtml PhD Scholarship Project – Università degli Studi di Bologna PhD project Bacteria can be genetically engineered to produce generalized modules of membrane antigens (GMMA) made up of nanoparticle vesicles of outer membrane, which represents an innovative vaccine platform. The Gram-negative outer membrane (OM) is an asymmetric lipid bilayer interspersed with integral OM proteins and peripheral lipoproteins which often are immunogenic and can be exploited as vaccine antigens. Two of the main components of a novel multicomponent vaccine against meningococcus B (4CMenB or Bexsero™) are lipoproteins, namely NHBA and fHBP. Lipoproteins destined for the OM are recognised by the Lol system which transports them across the periplasm and secures the proteins to the OM by incorporating the diacylglycerol moiety into the inner leaflet of the OM. Most lipoproteins are associated with a periplasmic facing OM orientation. In meningococcus, there are a large number of surface exposed lipoproteins, however the actual mechanism of translocation across the OM to the surface of the bacterium is completely unknown. In this project the PhD candidate will undertake multi-faceted approaches to identify, characterize and exploit molecular systems involved in translocation of lipoproteins for surface exposition. An increased understanding of these systems will allow the generation of next generation vaccine strains, which can express GMMA enriched in immunogenic lipoproteins. The full repertoire of lipoproteins of key Gram negative bacterial pathogens will be bioinformatically investigated and the possible regulatory sequences involved in their sorting and localization will be analysed. In addition, genetic approaches to identify the genes and loci in meningococcus that are responsible for flipping the surface exposed lipoproteins to the outside of the bacterium will be performed. Transposon-mutant libraries of meningococcus will be screened for those mutants devoid of surface exposure of model lipoproteins (NHBA or fHBP). In parallel, while E. coli does not express meningococcus lipoproteins on the surface, an expression library of the meningococcal genome in E.coli will be screened for those loci that can complement for surface exposure of the NHBA and fHBP model lipoproteins. Finally, the fruits of this project will be exploited to genetically enhance model antigenic lipoprotein expression on the surface of GMMAs for next generation vaccine design for gram negative pathogens. 2 http://www.novartisvaccines.it/ricerca/nva-eng.shtml PhD Scholarship Project – Università degli Studi di Bologna Technologies employed Expression libraries. Expression analyses: Use of state of the art reporter systems for in vitro protein expression, FLow cytometry analysis and sorting, confocal microscopy, SDS-PAGE & Western blot analysis. RNA analysis: Global gene expression analyses by microarray, RNA-seq, RT-PCR, Northern blot. Protein analysis: recombinant expression and purification of proteins, preparation of samples for proteomics. In vitro Assays: Relevant biochemical analyses of purified proteins: Basic bioinformatics: use of publically-available data bases and basic sequence analyses. Basic serology and immunology : preparation of vaccine candidates for mice immunization and serological analyses. Relevant publications on the topic Delany I, Rappuoli R, Seib KL. Vaccines, reverse vaccinology, and bacterial pathogenesis. Cold Spring Harb Perspect Med. 2013 May 1;3(5):a012476. doi: 10.1101/cshperspect.a012476. Fagnocchi L, Biolchi A, Ferlicca F, Boccadifuoco G, Brunelli B, Brier S, Norais N, Chiarot E, Bensi G, Kroll JS, Pizza M, Donnelly J, Giuliani MM, Delany I. (2013) Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine. Infect Immun. 81(2):560-9. Koeberling O, Delany I, Granoff DM. (2011) A critical threshold of meningococcal factor H binding protein expression is required for increased breadth of protective antibodies elicited by native outer membrane vesicle vaccines. Clin Vaccine Immunol 18(5):736-42. Seib KL, Pigozzi E, Muzzi A, Gawthorne JA, Delany I, Jennings MP, Rappuoli R. (2011) A novel epigenetic regulator associated with the hypervirulent Neisseria meningitidis clonal complex 41/44. FASEB J. 25(10):3622-33. Oriente, F, Scarlato V, Delany I. (2010). "Expression of factor H binding protein of meningococcus responds to oxygen limitation through a dedicated FNR-regulated promoter." J Bacteriol 192(3): 691-701. Seib, KL, Oriente F, Adu-Bobie J, Montanari P, Ferlicca F, Giuliani MM, Rappuoli R, Pizza M, Delany I. (2010). "Influence of serogroup B meningococcal vaccine antigens on growth and survival of the meningococcus in vitro and in ex vivo and in vivo models of infection." Vaccine 28(12): 2416-2427. Metruccio MM, Pigozzi E, Roncarati D, Berlanda Scorza F, Norais N, Hill SA, Scarlato V, Delany I. (2009). A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements. PLoS Pathog. Dec;5(12):e1000710. 3 http://www.novartisvaccines.it/ricerca/nva-eng.shtml PhD Scholarship Project – Università degli Studi di Bologna PhD technical development The PhD student will become skilled in all recombinant DNA technologies, and basic microbiological techniques that may be involved in bacterial genetic studies, manipulation of pathogens, and in vivo and in vitro characterization assays for the analysis of mutant phenotypes. Furthermore, they will be required to perform genetic screenings and molecular characterization of the mutants or expression clones identified. As well as using state of the expression and localization analysis of antigen based on Flow cytometry and microscopy. Furthermore indepth investigation of the molecular mechanisms involved will require a solid understanding and training in all classical molecular biology approaches and in vitro assays (see technologies employed). The design and generation of GMMA based vaccines will also require mouse immunizations and serological testing to be developed in this project. 4 http://www.novartisvaccines.it/ricerca/nva-eng.shtml