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Novartis Vaccines Academy
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PhD Scholarship Project – Università degli Studi di Bologna
Understanding and exploiting lipoprotein
translocation in Gram negative pathogens for
improved vaccine antigen delivery platforms
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
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
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.
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):
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.
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.