Despite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.

Bovine respiratory disease (BRD) is the principal cause of morbidity and mortality among feedlot cattle. Mannheimia haemolytica is consistently implicated in this condition, but treatment options are diminishing with the rise of antimicrobial resistance and intensifying consumer pressure to reduce reliance on conventional therapies. Thus, sustainable alternatives like vaccination are required. In this study, the phenotypic and genotypic diversity of BRD pathogens were examined with the objective to identify vaccine targets using reverse vaccinology, an innovative approach to identify antigens via genomic sequence. Preliminary surveillance confirmed M. haemolytica serotype 2 isolates were predominant in healthy animals (75.5%) while serotypes 1 (70.7%) and 6 (19.5%) were common in diseased animals. Pathogens of BRD, including M. haemolytica, Pasteurella multocida and Histophilus somni were also isolated from North American BRD mortalities, and compared using pulsed-field gel electrophoresis and antimicrobial susceptibility. Concurrently, polymerase chain reaction detection of bacterial and viral agents confirmed that M. haemolytica with bovine viral diarrhea virus were the most prevalent. Whereas isolates from live cattle were found to have a relatively low level of resistance, several pathogens from the mortalities were found to contain integrative conjugative elements (ICE) conferring resistance to seven antimicrobial classes. These ICEs were transferred via conjugation to other bacterial species, emphasizing the need for alternative antimicrobial therapies. Collectively, data from these investigations informed the selection of 11 diverse M. haemolytica strains for whole genome sequencing and comparative analyses. Several bacteriophage associated genes and CRISPR-Cas regulated gene expression systems were identified and are likely contributing to virulence in M. haemolytica. Coding sequences across all genomes were screened using pan-genome analysis, identifying 291 candidates with cell-surface associated signatures. Using a cell-free translation system and enzyme-linked immunosorbent assay the candidates were screened against serum from cattle challenged with serovar 1, 2 or 6 of M. haemolytica, and ranked according to immunogenicity. The top five vaccine candidates included Ssa1, ComE, a solute binding protein, an outer membrane protein, and the periplasmic component of an ABC transporter. With further characterization, these unique antigenic candidates could be developed into a vaccine to effectively reduce the dependence on antimicrobial therapies.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2018-04-25T20:07:07Z No. of bitstreams: 2 Dissertação - Moisés Morais Inácio - 2018.pdf: 2052518 bytes, checksum: 343f0c40d2a797544b4af9800b9e5522 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-26T11:15:33Z (GMT) No. of bitstreams: 2 Dissertação - Moisés Morais Inácio - 2018.pdf: 2052518 bytes, checksum: 343f0c40d2a797544b4af9800b9e5522 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-26T11:15:33Z (GMT). No. of bitstreams: 2 Dissertação - Moisés Morais Inácio - 2018.pdf: 2052518 bytes, checksum: 343f0c40d2a797544b4af9800b9e5522 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Toxoplasma gondii is the causative agent of congenital toxoplasmosis, which manifests as mild chorioretinitis, miscarriage, mental retardation, microcephaly, hydrocephalus, and seizures. Treatment of this disease is limited and a new vaccine represents the best strategy for prevention of the infection. In the present study, the reverse vaccinology combined with immunomics was applied for the development of a vaccine against T. gondii. Using an in silico approach, we identified T. gondii’s proteins that contain signal peptide and transmembrane domain using the ToxoDB® database. We evaluated the homology of these proteins with the human proteome and predicted their epitopes using Blastp, NetMHCpan 3.0 and NetMHCIIpan 3.1 tools. Class I and II HLA alleles with frequency greater than 1% in the population of South America, North America and Europe were obtained using the dbMHC database. Processing of the MHC class I epitopes were evaluated by MHC I Processing on the IEDB® database and the B lymphocyte epitopes were obtained through the Bcpred and BCTOPE servers. Finally, the antigenicity of the potential targets was analyzed by the VAxiJen server. A total of 1228 proteins were obtained, from which 349 showed no homology with human proteins. For the South American population, among the proteins identified with promiscuous epitopes, we observed proteins that are part of the virulence arsenal of the pathogen such as ROP8, ROP7, ROM4, Cathepsin C / B, rhoptry neck protein and LMBR1 family region protein. In the North American and European populations, we identified common proteins to both populations, such as MIC15, ROP7, HECT-domain (ubiquitin-transferase) domain-containing protein and rhoptry neck protein. ROP31 and subtilisin SUB2 are exclusive to the North American population. These proteins are involved in the invasion process and were shown to be positive in all the parameters adopted in this study. Regarding B lymphocyte epitopes, proteins such as ROP7, ROP8, ROM4, MIC15, HECT were identified. These proteins also presented promiscuous epitopes to class I and II HLAs from the analyzed populations. In addition, MIC2, ROM5, ROP9, MIC8, and MIC9 also showed B lymphocyte epitopes, but MIC9 was noteworthy with the highest score, high expression in the bradyzoite stage, and lack of vaccine test. ROP7, ROP8, ROM4, MIC8 and MIC9 were selected for in vivo and in vitro testing. Thus, our results demonstrate that immunochemical reverse vaccination has been shown not only to identify potential vaccine candidates against pathogens with complex life cycles. / Toxoplasma gondii é o agente etiológico da toxoplasmose congênita, que pode se manifestar como coriorretinite leve, aborto espontâneo, retardo mental, microcefalia, hidrocefalia e convulsões. O tratamento dessa doença é limitado e uma nova vacina representaria a melhor estratégia para a prevenção da infecção. No presente estudo, adotamos a vacinologia reversa associada a imunômica foi aplicada na construção de uma vacina contra T. gondii. Utilizando uma abordagem in silico, selecionamos as proteínas do patógeno que possuem peptídeo sinal e domínio transmembranar utilizando o banco de dados no ToxoDB®. Avaliamos a homologia dessas proteínas com o proteoma humano e predizemos os epítopos utilizando as ferramentas Blastp. NetMHCpan 3.0 e NetMHCIIpan 3.1. Os alelos de HLAs de classe I e II com frequência ≥ 1% na população da América do Sul, América do Norte e Europa foram obtido através do banco de dados dbMHC. O processamento dos epítopos de MHC de classe I foram avaliados pelo MHC I Processing no banco de dados IEDB® e os epítopos de linfócitos B foram obtidos através dos servidores Bcpred e BCTOPE. Por fim, a antigenicidade dos potenciais alvos foi analisada pelo servidor VAxiJen. Um total de 1228 proteínas foi obtido, das quais 349 não apresentaram homologias em humanos. Para a população sul-americana, entre as proteínas com epítopos promíscuos identificadas, observamos proteínas que fazem parte do arsenal de virulência do patógeno tais como ROP8, ROP7, ROM4, cathepsin C/B, rhoptry neck protein e LMBR1 family region protein. Em relação às populações norte-americana e europeia, a identificação de epítopos promíscuos revelou proteínas comums às duas populações tais quais MIC15, ROP7, HECT-domain (ubiquitin-transferase) domain-containing protein e rhoptry neck protein e exclusivas à população norte americana, como ROP31 e subtilisina SUB2. Essas proteínas estão envolvidas no processo de invasão e/ou foram positivas em todos os parâmetros adotados neste estudo. Com relação aos epítopos de linfócitos B, obteve-se 93 proteínas, dentre elas, ROP7, ROP8, ROM4, MIC15, HECT que também apresentaram epítopos promíscuos aos HLAs de classe I e II das populações analisadas. Além delas, MIC2, ROM5, ROP9, MIC8 e MIC9 também apresentaram epítopos de linfócitos B, mas MIC9 destacou-se com o maior score; pela elevada expressão no estágio de bradizoíto e pela inexistência de testes vacinais. ROP7, ROP8, ROM4, MIC8 e MIC9 foram selecionadas para teste in vivo e in vitro. Dessa forma, nossos resultados demonstram que vacinologia reversa associada a imunômica mostrou-se capaz de identificar fortes candidatos vacinais contra patógenos de ciclo vida complexo.

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.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2018-06-08T14:07:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_tanise_fortes.pdf: 1277949 bytes, checksum: 4aec80165f53d0b2f587adbcc608f856 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-11T16:56:11Z (GMT) No. of bitstreams: 2 tese_tanise_fortes.pdf: 1277949 bytes, checksum: 4aec80165f53d0b2f587adbcc608f856 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-11T16:56:11Z (GMT). No. of bitstreams: 2 tese_tanise_fortes.pdf: 1277949 bytes, checksum: 4aec80165f53d0b2f587adbcc608f856 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A leptospirose é uma zoonose de ocorrência mundial causada por membros patogênicos do gênero Leptospira. Em países em desenvolvimento, como o Brasil, a doença representa um grave problema de saúde pública. Os hospedeiros suscetíveis adquirem a doença através do contato com urina, água ou solo contaminados com a bactéria. Além dos roedores, os bovinos e os caninos se destacam como importantes reservatórios de Leptospira spp, tornando a vacinação dos animais suscetíveis uma das principais formas de prevenção da doença. As vacinas disponíveis comercialmente são bacterinas que induzem resposta imune humoral predominantemente contra o lipopolissacarídeo (LPS) bacteriano e que não são capazes de fornecer proteção de longo prazo contra a infecção. As proteínas da membrana externa de Leptospira spp tem sido avaliadas como candidatos vacinais em modelo animal e são uma alternativa promissora às formulações comerciais em uso. Dessa maneira, o objetivo deste estudo foi realizar a triagem de preparações vacinais contendo proteínas recombinantes de Leptospira spp, visando o desenvolvimento de uma vacina contra a leptospirose. Cinco genes, LIC11889, LIC10666, LIC10498, LIC12666 e LIC10463, foram identificados e amplificados a partir do genoma de Leptospira interrogans sorovar Copenhageni cepa Fiocruz L1-130. Desses genes, quatro (LIC11889, LIC10666, LIC10498 e LIC10463) foram eficientemente expressos em sistema de expressão heteróloga em Escherichia coli e purificados através de cromatografia de afinidade. Duas proteínas recombinantes, rLIC11889 e rLIC10666 foram empregados na formulação de preparações vacinais, utilizadas para imunizar hamsters posteriormente desafiados com dose letal de L. interrogans sorovar Copenhageni cepa Fiocruz L1-130. Todos os animais vacinados com as preparações contendo as proteínas recombinantes sobreviveram à leptospirose aguda, tornando promissora sua utilização para a criação de vacinas contra a doença. / Leptospirosis is a worldwide zoonosis caused by pathogenic members of the genus Leptospira. In countries under development, like Brazil, the disease represents a serious problem of public health. The susceptible hosts acquire the disease through contact with urine, water or soil contaminated with the bacteria. As well as rodents, cattle and dogs are important reservoirs of Leptospira spp, making the vaccination of susceptible animals one of the main. ways of preventing the disease. The commercially available vaccines are bacterins that induce humoral immune response predominantly against the bacterial lipopolysaccharide (LPS) and are unable to provide long term protection against the infection. The outer membrane proteins of Leptospira spp have been evaluated as vaccinal candidates in animal models and are a promising alternative to usual commercial formulae. In this way, the objective of this study was to perform a trial of vaccinal preparations containing recombinant proteins of Leptospira spp, to develop a vaccine against leptospirosis. Five genes, LIC11889, LIC10666, LIC10498, LIC12666 and LIC10463 were identified and amplified from the genome of Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. From these genes, four (LIC11889, LIC10666, LIC10498 and LIC10463) were efficiently expressed in an heterologous expression system in Escherichia coli and purified through affinity chromatography. Two recombinant proteins, rLIC11889 and rLIC10666 were employed in the formulation of vaccinal preparations, used to immunize hamsters that were later challenged with the lethal dose of L. interrogans serovar Copenhageni strain Fiocruz L1-130. All the animals vaccinated with the preparations containing the recombinant proteins survived acute leptospirosis, making their use for the development of vaccines against the disease promising.

Campylobacteriosis is a leading foodborne illness worldwide, primarily caused by Campylobacter jejuni (C. jejuni) which is associated with poultry consumption. The emergence of antibiotic resistance has emphasized the need for alternative strategies to control C. jejuni colonization in poultry. To assess the prevalence of C. jejuni in poultry, 270 cloacal swab samples were collected from broilers raised under No-Antibiotics Ever system. Among these samples, 16.3% were identified as C. jejuni positive. Notably, these isolates exhibited a diverse range of virulence factors and antimicrobial resistance genes, with 61.36% of isolates showing hyper-motile and 20.45% demonstrating multidrug resistance. Following isolation, whole genome sequencing was conducted on four selected strains using a hybrid sequencing approach. Subsequently, the complete genomes of these C. jejuni strains were analyzed to identify vaccine candidates using reverse vaccinology. Three conserved potential vaccine candidates were identified as suitable targets for vaccine development, namely phospholipase A (PldA), TonB dependent transporter (ChuA), and cytolethal distending toxin (CdtB). Furthermore, the gene expression of these candidates was examined in four C. jejuni strains during host-pathogen interactions using avian macrophage cell line HD11. Significant upregulation of all three candidate genes were observed in the four tested C. jejuni strains during interaction with host cells, indicating their crucial role in C. jejuni infection. Additionally, the expression of immune genes was evaluated in avian macrophage cells to understand the immune responses during C. jejuni infection. The infection resulted in the upregulation of toll-like receptor genes (TLR-4), pro-inflammatory genes (IL-1β, IFN-γ, IL-6, IL-8L1), anti-inflammatory gene (IL-10), and iNOS2 gene expression. The observed immune response demonstrates the potential of C. jejuni to induce host immunity for protection. In conclusion, our study identifies three conserved potential vaccine candidates and provides insights into the immune responses induced by C. jejuni infection in avian macrophage cells. These findings are crucial for the development of an effective vaccine against C. jejuni, aiming to reduce C. jejuni transmission through poultry consumption and the risk of human infection.

L’entérite nécrotique aviaire causée par Clostridium perfringens est une maladie économiquement dévastatrice et celle-ci est en émergence dans les troupeaux de poulets de chair éliminant l’usage des antibiotiques. À ce jour, aucune alternative en élevage ne permet de prévenir efficacement la maladie et un contrôle par une stratégie vaccinale serait des plus prisé. Une approche par génomique comparative jumelée à la vaccinologie réverse soustractive et comparative identifiant des protéines bactériennes de surface immunogènes figure parmi les approches méthodologiques des plus prometteuses pour le développement rapide d’un vaccin efficace. Une étude génomique comparative réalisée sur 48 souches de C. perfringens provenant de poulets de chair en santé ou affectés par l’entérite nécrotique a permis d’établir que les génomes analysés étaient composés de 155 700 protéines distinctes, où 13% étaient extracellulaires, 65% cytoplasmiques et 22% membranaires. L’évaluation du pouvoir immunogène de ces protéines à l’aide de l’outil de prédiction VaxiJen v.2.0 a permis d’identifier 4 catégories de scores pour les protéines identifiées, allant de 0,5 (seuil minimal recommandé) à 1,5. Les protéines présentant les scores les plus élevés ont été majoritairement associées à des localisations extracellulaires. La combinaison du score d’immunogénicité et de la localisation cellulaire des protéines analysées a mené à la sélection de 12 protéines candidates vaccinales, la plupart d’entre elles étant de fonction hypothétique. Une description plus approfondie de ces protéines permettra de mieux définir leur fonction, d’évaluer leur potentiel antigénique réel en caractérisant leur interaction avec le système immunitaire de la volaille et ultimement, d’évaluer leur rôle probable dans la pathogénie de l’entérite nécrotique. / Avian necrotic enteritis caused by Clostridium perfringens is a disease with a major economical impact, generating losses up to 6 billion dollars for the poultry industry worldwide. This disease appears in broiler chicken flocks that no longer employ the use of antibiotics. To date, no alternative method allows for the efficient prevention of necrotic enteritis (NE) and a control by a vaccinal strategy would be mostly prized. A comparative genomics approach as well as comparative and subtractive reverse vaccinology identifying immunogenic bacterial surface proteins is one of the most promising methodologies for the rapid development of an efficient vaccine. A comparative genomic study was performed on 48 C. perfringens strains isolated from healthy broiler chickens and from broilers affected by necrotic enteritis. From this study, it was established that the genomes analyzed were composed of 155 700 distinct proteins where 13% were predicted to have an extracellular expression, 65% at the cytoplasma level and 22% within the plasma membrane. The evaluation of the immunogenic potential of these proteins was established with the prediction software VaxiJen v2.0 for which a 0.5 threshold score allowed for the identification of four score categories among the identified proteins, from 0.5 to 1.5. For the most part, proteins with the highest scores were associated with an extracellular localisation. The combination of the immunogenicity score and localisation of the analysed proteins led to the selection of 12 vaccinal candidate proteins that were mostly identified as hypothetical. A more in-depth description of these proteins would allow the assessment of their function, the evaluation of their true immunogenic potential by characterizing their interaction with the avian immune system and ultimately, evaluate their probable role in the pathogenesis of necrotic enteritis.