Les campylobactérioses sont les infections intestinales bactériennes d’origine alimentaire les plus fréquemment rapportées au sein de l’Union Européenne et sont principalement associées à la consommation de viande de volailles. Une diminution de la colonisation intestinale des volailles par Campylobacter de 2 à 3 log10 UCF/g permettrait de réduire l’incidence des cas humains de 76 à 100 %. La vaccination aviaire constitue un moyen de lutte potentiel mais, malgré de nombreuses études, aucun vaccin commercial n’est actuellement disponible. L’objectif de ce projet a été d’identifier de nouveaux antigènes vaccinaux contre Campylobacter en appliquant la stratégie de la vaccinologie inverse et d’évaluer leurs pouvoirs immunogène et protecteur contre la colonisation intestinale des volailles. Sur la base de leur localisation subcellulaire, leur antigénicité, leur densité en épitopes B et leur homologie de séquence avec l’ensemble des souches de C. jejuni et C. coli, quatorze antigènes ont été sélectionnés. Six d’entre eux ont été produits et testés in vivo en appliquant un protocole vaccinal optimisé. Quatre antigènes ont montré des diminutions significatives de la charge intestinale des oiseaux de 2 à 4,2 log10 UFC/g associées à l’induction de réponses humorales spécifiques. L’immunogénicité de ces candidats vaccins et l’efficacité protectrice de deux antigènes ont été observées à nouveau. Ces premiers résultats montrent l’intérêt et la fiabilité de la vaccinologie inverse. L’évaluation du potentiel vaccinal de ces nouveaux antigènes doit être poursuivie et approfondie lors de futures expérimentations. / Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis reported in the European Union and is mainly associated to consumption of poultry meat. Reducing the intestinal colonization of broilers by Campylobacter from 2 to 3 log10 CFU/g could decrease human cases incidence by 76 to 100%. Vaccination of poultry could be a potential strategy but despite many studies, no efficient vaccine is available yet. The aim of this project was to identify new vaccine antigens against Campylobacter using the reverse vaccinology strategy and to assess their immune and protective powers against the avian intestinal colonization. Based on their sub-cellular localization, immunogenicity, B-epitopes density and their sequence conservation among C. jejuni and C. coli strains, fourteen antigens were selected. Six out of them were produced and in vivo tested according to an optimized avian vaccine protocol. Four antigens showed intestinal load decreases from 2 to 4.2 log10 CFU/g correlated with the induction of specific humoral responses. Vaccine candidates’ immunogenicity and the protective efficiency of two antigens were observed again. These first results highlight the interest and reliability of the reverse vaccinology. The assessment of these new antigens vaccine potential needs to be continued and deepened in next experiments.

Cette thèse de doctorat s'est déroulée dans le cadre d'un projet européen FP7 (7th Framework Program) MSCA (Marie Sklodowska-Curie action) ITN (Initiale Training Network) EID (European Industrial Doctorates) appelé DISCo (a multidisciplinary Doctoral Industrial School on novel preventive strategies against Escherichia coli infections) coordonné par Mariagrazia Pizza et co-coordonné par Mickaël Desvaux. Ainsi, ce doctorat s'est déroulé pour moitié en Italie au centre de recherche GSK (GlaxoSmithKline) sur le site de Sienne sous la supervision de Roberto Rosini et la direction de Fabio Polticelli de Universita degli Studi Roma Tre. L'autre moitié de la thèse s'est déroulée en France à l'INRA, centre Auvergne-Rhône-Alpes sur le site de Theix sous la direction de Mickaël Desvaux et Grégory Jubelin comme co-encadrant. Cette thèse de doctorat participe au développement de nouvelles stratégies préventives aux infections aux E. coli pathogènes intestinaux (InPEC), en particulier E. coli entérohémorragiques (EHEC), par une stratégie vaccinale. Dans ce contexte, une approche de vaccinologie inverse a été mise en œuvre pour identifier de nouveaux antigènes candidats qui ont ensuite été délivrés par la technologie GMMA (Generalized Modules for Membrane Antigens). Par ailleurs, un domaine épitope potentiel chez les autotransporteurs, i.e. l'autochaperon, a été caractérisé par analyse des séquences protéiques et modélisation structurale. / Enterohemorrhagic E. coli (EHEC) are a major cause of large outbreaks mainly affecting developed countries. From 1982 to 2002, a total of 350 E. coli O157 outbreaks were reported in the United States. EHEC infection causes diarrheal disease often associated with clinical complications like hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although efforts focused on hygiene have been implemented in the food supply chain to reduce the risk of the foodborne E. coli O157 infection, outbreaks caused by this pathogen are still common. In addition, antibiotic-based therapy is discouraged for their potential undesirable effect in releasing shiga-toxin from the bacteria. Among non-antibiotic preventing strategies, vaccine development is warranted, still nowadays a licensed vaccine specific for human use against EHEC is not available. In this study, we used the Reverse Vaccinology approach applied on the EHEC O157:H7 genome to select new potential vaccine candidates. We identified a panel of 24 of potential protein antigens and we successfully expressed three of them in Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing these vaccine candidates resulted to be immunogenic, raising a specific antibody response for two of the selected antigens. In particular, immunization with MC001 candidate was able to reduce intestinal EHEC O157:H7 colonization lowering the bacterial count in feces, colon and ceacum tissues in mice. This candidate was found to be homologue to the Salmonella Typhimurium Lipid A deacylase enzyme (LpxR) and to our knowledge this study was the first report describing it as vaccine candidate. Also, gene distribution and sequence variability analysis showed that MC001 was mainly present and conserved in EHEC O157:H7 and in some EPEC. Given the high genetic variability among and within these pathotypes, the identification and inclusion of this conserved candidate in a vaccine might cover against major intestinal pathogenic strains. Furthermore, because it has been showed that during the infection process some autotransporters, as MC021 can be reactive, we also analysed molecular determinant with an important role for their proper secretion and folding, namely the autochaperon (AC) domain. It appeared the AC is a common feature of autotransporters but strictly associated with passenger domains exhibiting a –helix fold. Their exposition at the bacterial cell the surface further positions the AC as a potential antigenic target and/or development of new treatments. These findings further provide new research directions for the development of non-antibiotic preventive strategy against InPEC in human but also animal.