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Structural Vaccinology in the design of bionanoparticles with multi-copy antigen display for vaccines with enhanced efficacy

Periodic Reporting for period 1 - SVNanoVax (Structural Vaccinology in the design of bionanoparticles with multi-copy antigen display for vaccines with enhanced efficacy)

Reporting period: 2015-06-15 to 2017-06-14

Over the last century, vaccination has been the most effective medical intervention to reduce death and morbidity caused by infectious diseases. However, despite these advances, many diseases are not yet preventable by vaccination and some groups (e.g. the elderly, populations of developing countries, pregnant women and neonates) remain vulnerable. Infectious diseases also demand further efforts. The highly mutagenic potential of many pathogens and the increasing prevalence of drug-resistance germs stresses the need of broadly protective vaccines capable of preventing disease.
Compared to early vaccines containing killed or live-attenuated pathogens, modern sub-unit vaccines are safer but tend to be less immunogenic. Therefore, a key issue in vaccine research is how to improve their immunogenicity without reducing safety or tolerability.
SVNanoVax aimed to combine the approach of Structural Vaccinology with vaccine antigen display on protein bionanoparticles (BNPs).

Neisseria meningitidis serogroup B (MenB) causes severe sepsis and meningococcal meningitis, resulting in death or devastating long-term sequelae. Bexsero, an innovative research-based vaccine, is a multi-component vaccine composed of three meningococcal proteins plus outer membrane vesicles, and is conservatively estimated to provide 66-91% coverage against MenB strains worldwide. Thanks to the large amount of structural and immunological information of its components Bexsero represents an excellent tool to validate a BNPs-based antigen engineering strategy.

The first objective was to engineer stable antigens eliciting broadly protective antibody responses. Secondly, we sought to determine the most significant epitopes from an immunomodulatory perspective. The following objectives were conceived to design recombinant self-assembling protein BNPs for multicopy and surface display of the selected epitopes, thus recreating the antigen vast exposure format of virus like particles. Lastly, we set out to determine if presentation of multiple copies of these antigens on self-assembling BNPs will induce stronger immune responses.
The overall objectives of SVNanoVax focussed on the development of multi-copy antigen-BNPs for a second-generation vaccine against MenB that protects against all strains. Moreover, in addition to driving the development of an improved second-generation meningococcal vaccine with enhanced coverage, tolerability and efficacy, this research aimed at potentiating antigen-BNP technology possibly applicable to vaccines targeting other pathogens representing currently unmet medical needs.

During the SVNanoVax period we have determined the 3D structure of a broadly reactive human bactericidal antibody Fab targeting an important MenB antigen. To our knowledge, this work represents the first crystal structure of a vaccine-elicited human antibody bound to a bacterial antigen. We have also developed a system for the recombinant expression of BNPs with surface exposed, multi-copy and ordered arrays of a potent MenB antigen epitope. These BNPs will be tested in parallel with the current vaccines in order to determine the scope of protection against MenB.
We focused our initial efforts on factor H binding protein (fHbp), a MenB vaccine and virulent antigen factor. We have been able to determine the 3-dimensional structure of a human antibody (Ab) antigen binding fragment (Fab) bound to this important antigen. In the figure, one can see with high precision the binding site (epitope) of this human Ab on fHbp. Importantly, this human antibody was elicited upon vaccination of a human subject with a formulation containing fHbp. In addition, this antibody is of special interest as it targets representatives of the three different fHbp main variant groups in which the over one thousand fHbp naturally occurring mutants can be distributed. Furthermore, recognition of this novel epitope by this antibody triggers bactericidal activity also in strains bearing fHbp antigens of all three variant groups. Therefore, we wanted to proceed with an immunofocusing approach, and selected this important epitope to be displayed on the surface of two different self-assembling protein nanoparticle scaffolds, encapsulin and lumazine synthase. This approach takes advantage of the virus like particle distribution format, which provides a high-density and surface exposed presentation platform for immunogenic epitopes. We designed recombinant genes to fuse the fHbp immunodominant epitope with either of the two scaffolds. Next, we expressed these BNPs in non-pathogenic laboratory bacterial cells and purified them to homogeneity with chromatographic procedures. Purified BNPs were then characterized by transmission electron microscopy in order to confirm the correct assembly. Our analyses confirm that we have succeeded in the preparation of a system for the expression of chimeric encapsulin BNPs.
These results have been compiled into a manuscript for publication in a peer-review scientific journal in an open access format. We have also deposited the structural data of the Fab-fHbp complex in the Protein Data Bank (PDB) for its public release.
SVNanoVax has successfully delivered the first crystal structure of a bacterial antigen in complex with a human antibody raised by vaccination. This highly detailed data helps us to understand how the human immune system responds effectively to immunization with current meningococcal vaccines and helps to explain some of the factors that likely contribute to the broad protection conferred by such vaccines. SVNanoVax has also enabled design of new BNPs displaying a selected sub-fragment of a meningococcal antigen, which may allow immunization with greater responses. Moreover, this study demonstrates the feasibility of this approach in the design of novel vaccine antigens, either specifically for next-generation meningococcal vaccines, or more generally for other vaccines. As such, we expect that SVNanoVax will have an impact in the design of future vaccines, potentially applicable to combat several different pathogens that are currently unmet medical needs of global significance.
In addition to saving lives and suffering by relieving the medical burden of meningococcal and other diseases, the successful development of new vaccines would further establish the position of GSK Vaccines in Siena as a ‘Centre of Excellence’ for vaccines research and development in the heart of Europe. At a time when a number of other pharmaceutical companies have withdrawn from Italy, GSK and SVNanoVax together represented a fine example of continued European development in a field where we are globally competitive. Also, the movement of the Fellow from Chicago (USA) to GSK Siena has enriched the pool of highly-trained European researchers with multi-sector experience, an important objective for the structuring of the European research area. During SVNanoVax, the Fellow also developed BNPs by working briefly at a Spanish research centre. Collectively, these interactions may lead to future job opportunities for the Fellow. SVNanoVax has contributed to European Excellence and competitiveness by allowing the preparation of high impact scientific publications (one published, one under submission) and presentations at top European and international conferences, thus endorsing continued investment in the research sector.
The Fab 1A12-fHbp complex crystal structure