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Mes travaux de thèse ont pour but d’évaluer l’approche « épitope display » pour sa capacité d'induire les réponses cellulaires. Ad à capside modifiée par insertion de différents épitopes T issus de la ovalbumine ont étés produit. Après administration à des souris C57BL/6, j'ai mis en évidence l'induction de la réponse cellulaire dirigée contre les épitope insérés (grâce aux techniques ELISPOT, tétramères, ou bien en quantifiant la production d’IFNg par les splénocytes restimulés in vitro). J’ai démontré que ces réponses sont limités chez des souris préalablement immunisées avec Ad. Des manipulations en course ont pour but de confirmer ces résultats et d'évaluer la cinétique de ces réponses. Mon 2ème objectif est de comprendre les paramètres qui contrôlent l’immunogénicité des Ad présentant des épitopes. Ainsi, j’ai montré que l’ablation des interactions des Ad porteurs d’un épitope issu de l’ovalbumine avec les récepteurs/facteurs (intégrines, facteur X de la coagulation) impliqués dans l’entrée de l’Ad dans les cellules ne modifiait pas leur capacité à induire une réponse humorale contre l’ovalbumine. Ces résultats suggèrent que le processus d’infection virale n’est pas requis pour l’induction d’une réponse humorale par les Ad porteurs d’épitopes. / Recombinant adenoviruses (Ad) have recently been employed for a wide range of vaccination strategies. Unfortunately, highly prevalent pre-existing neutralizing antibodies (Abs), reduce their ability to trigger transgene expression. To avoid the step of gene transfer a new vaccination strategy has been proposed based on the use of Ad displaying epitopes inserted into their capsid proteins. Using an ovalbumin-derived B cell epitope, our group demonstrated that vaccination efficiency depends on both the site of peptide insertion and the host immune status towards Ad (Lanzi et al; 2011). The present work aims at (1) evaluating the potency of Ad displaying T-cell epitopes from ovalbumin to elicit cellular responses and (2) understanding the molecular bases controlling the efficacy of this vaccination strategy. 1) Ad displaying T-cell epitopes from ovalbumin were constructed, produced and characterized in vitro. First in vivo experiments in naive mice showed induction of cellular responses, assessed with techniques like ELISPOT, tetramer staining and in vitro splenocyte restimulation. Subsequent experiments showed that pre-exisitng anti-vector immunity is hampering the potent induction of anti-epitope cellular responses. Current work is aiming at confirming the obtained results as well as at evaluating the kinetics of cellular responses induced upon "epitope display" vaccination. 2)First, the influence of interactions of Ad (displaying OVA peptide) with their natural receptors was investigated. Different detargeted Ads were produced and characterized in vitro. Upon mice immunization these vectors led to unmodified anti-epitope humoral responses, suggesting that their efficacy does not depend on the ability to transduce cells. In parallel we sought to evaluate the impact of innate immunity on the outcome of anti-epitope adaptive immune responses. Upon immunization of WT and MyD88-/- mice with Ad displaying OVA epitope we observed that cellular responses induced in MyD88-/- mice are significantly diminished while humoral responses were not altered. These results remain to be confirmed but question the role of other innate immunity sensors in the immunogenicity of Ad-based vaccines. Altogether, our work is expected to provide the foundations for the development of Ad-based vaccines with minimized side effects and unaltered adjuvant properties.

Proteins govern practically every process in living organisms through inhibiting, activating, or acting on other proteins in different ways. With the large and growing number of known interactions through high throughput screening technologies, experimental determination of atomic-level details of these interactions is nigh impossible. Computational methods such as docking can speed up efforts of understanding these interactions. However, several issues ought to be addressed before docking can replace experimental methods. This thesis describes work on assessment of the state of the art in docking methods, implementation of a machine learning algorithm to improve model ranking and integration of docking with template-based modeling to expand its usage with a special focus on antibody-antigen interactions. Firstly, the performance of docking methods was rigorously assessed by using a diverse set of protein complexes with a special focus on ClusPro, one of the leading rigid-body docking servers. Different strengths and potential areas of improvement for ClusPro and rigid-body docking methods in general were highlighted. Secondly, one of the major short-comings of docking noted in the first project, poor ranking of good models, was addressed. A regression-based machine learning algorithm was introduced to improve the ranking. Finally, a server was developed to tackle the challenge of epitope mapping by integrating template-based modeling with docking. An intuitive ensemble approach to scoring residue likelihood using docking poses and different homologues is shown to yield great success. In addition to shifting docking’s purpose of conformational search to interface identification, this server also allows users to start with protein sequence inputs. / 2022-09-28T00:00:00Z

Vaccines play a vital role in public health by preventing infectious disease across the globe. Vaccine formulations represent a weakened form of a microbe or toxin that is injected into the human body to elicit an immune response, generating antibodies to protect against a future infection. To this day, it is a challenge to identify and engineer important antigens and epitopes to focus this immune response in a safe and effective manner. The example of Bordetella pertussis is used to highlight the problems and lessons learned in designing a vaccine for this global epidemic. In particular, this review will focus on the advantages and disadvantages of chemical versus genetic detoxification and whole cell versus acellular vaccines in the context of pertussis. The latter part of this review will provide a summary of general strategies, such as epitope mapping and manipulation, synthesis of truncated variants, reverse vaccinology, and structural vaccinology, that have been successful in addressing increasingly complex diseases. Collectively, these techniques provide an invaluable set of tools to focus the immune response by finding and engineering specific antigens and epitopes. / text

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Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

Monoclonal antibodies (mAbs) recognizing different regions of PrP are potential tools in the study of prion diseases and immunotherapy. We used shuffled recombinant prion protein containing elk and mouse PrP as antigen to produce monoclonal antibodies in mice. We found that mAb 5C6 mapped to a discontinuous epitope comprised of amino acid 132 and 158 (mouse numbering). Monoclonal anibody 9E9 which maps to a unique N-terminal epitope at amino acid preferentially recognized cervid PrP. In contrast, the epitope of mAb 9H9 is located in the C-terminus and only reacted with mouse and hamster. The epitope for mAb 7H11 appears to be affected by the glycosylation of PrP and by the presence or absence of the disulfide bond. To confirm the epitopes of these mAbs, we constructed elk and mouse mutants both with and without reactivity to 5C6 and 9E9. We then used these mutants to investigate the effect of each epitope on the conversion of PrPC to PrPsc. In one approach to map the epitopes of newly-generated monoclonal antibodies (mAbs), we generated a series of contiguous ten amino acids deletion constructs spanning amino acids 107 to 230 and expressed these recombinant proteins in mammalian cells (RK13) or bacteria. Using Western blotting, all deletion constructs could be recognized with antibodies to the extreme C-terminus of PrP, or the N-terminal region upstream of the structured globular domain of PrP. However, mAb 5C6 failed to react with all internally deleted PrP constructs expressed in mammalian cells, and to a lesser extent bacterially produced mutant recombinant proteins. We confirmed the surprising result using the well-defined antibodies 6H4 and D18, which recognize epitopes in the same internal region as 5C6. Our results suggest the formation of an ultra-stable, SDS-resistant conformation in PrP harboring deletions mutations in the globular domain of PrP. We hypothesize that epitope burying within this stable conformation(s) precludes mAb recognition by 5C6, 6H4 and D18. It will be of extreme interest to determine the relationship of this previously undefined PrP conformation to the pathogenic process of PrP conformational change.