Typhoid fever remains an important public health problem globally. Cluster randomized effectiveness trials with typhoid Vi polysaccharide vaccine were conducted in Kolkata, India and Karachi, Pakistan, to provide evidence for vaccine introduction. While efficacy trials are limited to estimate vaccine's performance on the vaccine recipients, effectiveness trials consider the public health impact, notably the herd protection, or indirect effect, which can only be seen when vaccines are administered to groups rather than to individuals. The observed total protection by the Vi polysaccharide vaccine in school-aged children was consistent in Kolkata and Karachi (61% and 56%, respectively), and was associated with minimal side-effects. The total protection in young children, however, was different (80% in Kolkata and no protection in Karachi). The Kolkata trial demonstrated significant herd protective effects, as demonstrated by indirect protection of non-vaccinees (45%), which was not shown in the Karachi trial. The difference in the effectiveness estimates between the trials may be due to the difference in study design and the population characteristics. Immunogenicity studies were undertaken for randomly selected persons from both sites at pre-vaccination, 6 weeks, and 2 years post-vaccination. Serum Vi antibody titres (IgG) were measured through ELISA. At baseline, the GMTs were below the protective level for both sites. At six weeks after vaccination, though there is a significant increase in the GMTs in children from both site, the level of GMTs were significantly lower from those in Karachi (2,307.0 ELU vs. 1,189.1 ELU). GMT declined from 6 week to 2 year testing points for both sites but maintained the protective level. These effectiveness trials gave a conclusive evidence of the protection conferred by the Vi polysaccharide vaccine in children older than 5 years of age. Targeted vaccination programme in high endemic areas, as stipulated in the WHO Position Paper, suggest the potential for effective control of typhoid fever in places like India and Pakistan with the school-based Vi vaccination.

Long-term non-progressors (LTPNs) in HIV infection target the structural protein Gag more frequently than individuals who progress to disease. However, the targeting of Gag per se does not always distinguish these two groups. Various factors have been put forth as likely explanations for this discrepancy including differences in the breadth and magnitude of observed responses, the HLA type of the host, the nature of the individual epitopes targeted and the ability of the virus to mutate these antigenic regions. The purpose of this thesis was to examine, using PBMCs isolated from HIV-2 infected LTNPs and CTL clones established in vitro, the clonotypic architecture and quality of an immunodominant HIV-2 Gag-specific response directed towards the HLA-B*3501-restricted epitope NPVPVGNIY (NY9: Gag245-253). The data presented in this thesis show that in spite of the expression of multiple inhibitory receptors on the surface of NY9-specific CD8+ T-cells, the NY9-response, which is a clonotypically 'private' response, bears a signature characterised by an increased cytotoxic sensitivity and the production of an array of cytokines, most notably IFN-γ and MIP-1β. Moreover, the results of this thesis indicate that the NY9-specific CD8+ T-cells are able to cross-recognise and lyse target B-cells pulsed with the corresponding HIV epitope PY9 and its variants at functional avidities (EC50) that are close to those exhibited by PY9-specific T-cells. However, not all mobilised TCR clonotypes are equally sensitive or equally cross-reactive. When individual CTL clones were studied it emerged that dominant clonotypes within the NY9-specific CD8+ T-cell memory pool possessed a higher avidity for tetramer and sensitivity for antigen than subdominant ones and demonstrated a better cross-reactive potential towards variants of the HIV-2 epitope. Hence, future HIV vaccine strategies may benefit from the inclusion of epitopes like NY9, the presentation of which appears to mobilise CD8+ T-cells with superior functional profiles.

HIV-1 has claimed the lives of millions of people globally and continues to spread despite development of highly active antiretroviral therapy. In 2013, 2.1 million new infections occurred and over 35 million people were living with HIV-1 infection. A prophylactic HIV-1 vaccine that can prevent infection or reduce viremia and subsequent transmission will always be an important part of the solution to bring this epidemic under control. In this thesis, the first HIV-1 vaccine candidate to focus on conserved regions of the virus (HIVconsv) was assessed in a phase I clinical trial conducted in healthy HIV-1/2 negative volunteers in Oxford. The HIVconsv T-cell immunogen was delivered using three leading vaccine modalities (DNA (D), modified vaccinia virus Ankara (M) and chimpanzee adenovirus serotype 63 (C)), in several novel heterologous prime-boost regimens. The frequency of T cells elicited through HIVconsv vaccination in the CM and DDDCM regimens surpassed that of previous HIV-1 cell-mediated vaccines. A large proportion of these T cells produced multiple cytokines and proliferated in response to recall peptides. The breadth of T-cell responses were also greater than the non-efficacious STEP study vaccine, with an average of 10 T-cell epitopes per vaccine recipient recognised across CM and DDDCM regimens. In vitro HIV-1 control mediated by CD8⁺ T cells was demonstrated for all vaccinees receiving the CM regimen, mainly against clade A (U455) and clade B (IIIB) isolates. Two vaccinees, demonstrated superior control of 6/8 and 7/8 viruses from the panel. The CM regimen induced significantly higher magnitudes of viral inhibition compared to the DDDCM or DDDMC regimens, with this regimen showing potential to overcome the disadvantage for subjects of carrying non-protective HLA alleles. Investigation of T-cell specificities revealed that the frequencies of T cells specific for conserved Gag but more so Pol regions significantly correlated with in vitro virus control. Direct examination of peptide expanded T-cell lines showed that all Pol pool- and limited Gag pool-specific cell lines reduced HIV-1 replication in vitro. In most individuals, targeting multiple HIV-1 epitopes concomitantly resulted in higher levels of virus inhibition than targeting a single viral epitope and two T-cell specificities showed enhanced control of HIV-1; the first within Pol (TAFTIPSI) and second from Gag (TERQANFL). These data support further development of the conserved region strategy for T-cell vaccines against HIV-1.

Hepatitis C virus (HCV) is a major global pathogen estimated to infect over 170 million people worldwide. A recent study has shown that vaccination with adenoviral vectors, based on rare human and simian serotypes encoding the non-structural (NS) proteins of HCV, induces highly potent, multi-specific and durable T cell responses in healthy human volunteers. In this thesis I assess the safety and immunogenicity of these vaccines (ChAd3–NSmut and Ad6-NSmut), for the first time in HCV infected patients. This work also explores whether vaccine-induced T cell responses target in vivo circulating HCV antigens and common naturally occurring epitope variants. Patients with treatment naive chronic genotype 1 HCV infection were vaccinated (i.m.) with ChAd3-NSmut and Ad6-NSmut in a heterologous prime boost schedule, either with or without current IFN and ribavirin (IFN/RBV). Epitope-specific T cell responses were defined by fine mapping using HCV peptides. Circulating viral genomic sequence was determined in vaccinated patients at baseline and at any point of viral relapse. Cross-reactivity of vaccine-induced T cell responses was determined in T cell assays, using peptides corresponding to both circulating host virus and common population HCV epitope variants. An in vitro dendritic cell /T cell priming model was used to identify possible candidates for a cross-reactive vaccine immunogen at the most immunodominant epitope, NS3₁₄₀₆. 33 patients were vaccinated. Vaccination was well tolerated. At the highest vaccine dose (2.5 x 10¹⁰vp) vaccine-induced T cell responses were detectable in 11/20 patients receiving concurrent IFN/RBV and 2/4 patients receiving vaccination alone. In total 14 antigenic targets were identified, 2 of which have not previously been described. However, T cell responses were of lower magnitude and more narrowly focused than those observed in healthy volunteers vaccinated with the same regimen. Analysis of viral sequence showed that in many cases vaccine-induced T cells did not target the circulating virus. At the most immunodominant epitope (NS3₁₄₀₆), T cells induced by vaccination failed to target common circulating genotype 1 HCV variants. An in vitro model suggested that in order to target all genotype 1 sequences at this epitope, it would be necessary to insert both a genotype 1a and 1b version of this epitope into a vaccine immunogen. Vaccination with adenoviral vectors induces T cell responses in patients with chronic HCV infection, however immune responses are attenuated compared with healthy volunteers. Ultimately a successful therapeutic or prophylactic vaccine strategy will rely on inducing responses that target conserved or cross-reactive epitopes.

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.

Tuberculosis (TB) is a global health problem, which is proving extremely difficult to control in the absence of an effective vaccine. Bacille Calmette-Guérin (BCG), the only vaccine currently licensed against TB, demonstrates variable efficacy in humans and cattle. A greater understanding of what constitutes a protective host immune response is required in order to aid the development of improved vaccines. Tissue-resident memory T cells (T_RM) are a recently-identified subset of T cells, which may represent an important aspect of protective immunity to TB. This thesis aims to characterise the role of lung T_RM in BCG-induced protection against TB. In a mouse model, intravascular staining allowed discrimination between lung-vascular and lung-parenchymal T cells. Experiments demonstrated that BCG vaccination induced a population of antigen-specific lung-parenchymal CD4⁺ T cells, a putative tissue-resident population. This lung-parenchymal population was significantly increased in frequency following mucosal BCG vaccination, compared to systemic BCG vaccination. This correlated with enhanced protection against Mycobacterium tuberculosis (M.tb) infection in the lungs of mice receiving mucosal BCG, compared to those receiving systemic BCG. Mucosal BCG induced lung-parenchymal CD4⁺ T cells with enhanced proliferative capacity and a PD1⁺KLRG1^- cell-surface phenotype, a memory-like phenotype associated with improved protection against M.tb infection. These cells may represent a BCG-induced lung T_RM population responsible for the enhanced protection observed following mucosal BCG. Overall, this thesis highlights the potential of mucosal vaccination to elicit lung T_RM and identifies this as a possible immunological mechanism underlying enhanced protection against M.tb infection. These cells may constitute an important target for future vaccination strategies.

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.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2018-12-04T17:08:35Z No. of bitstreams: 2 Tese - Lázaro Moreira Marques Neto - 2018.pdf: 4983908 bytes, checksum: 78504fbead82e1981e7577763889e31e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-12-05T10:32:28Z (GMT) No. of bitstreams: 2 Tese - Lázaro Moreira Marques Neto - 2018.pdf: 4983908 bytes, checksum: 78504fbead82e1981e7577763889e31e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-12-05T10:32:28Z (GMT). No. of bitstreams: 2 Tese - Lázaro Moreira Marques Neto - 2018.pdf: 4983908 bytes, checksum: 78504fbead82e1981e7577763889e31e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-10-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The search for new adjuvants is the main goal in vaccinology. Along with this, understanding the impact of using nanoparticles as a delivery system and immunomodulator in vaccine systems directly impacts the development of new vaccines. In this work, we seek to study and elucidate the adjuvanticity of magnetic nanoparticles, as well as its immunogenicity and protection of the vaccine systems. Initially, a literature review was made seeking scientific bases that demonstrated the possibility of using metallic nanoparticles (MeNPs) as innate immune system stimulators. It was also sought to find elements in which metallic nanoparticles could aid in the generation Th1, Th17 and T CD8 type cellular response. From this review, it was verified that the magnetic nanoparticles, or with metallic ions, were able to stimulate the activation of costimulatory molecules (CD80, CD40 and CD86), to induce secretion of cytokines (IL-1, IL-6, IFN-γ and TNF-α) as well as the humoral immune response, but no work demonstrated whether these nanoparticles were able to induce cellular response. Consequently, in the second part of the study, tuberculosis was used as model to verify if a vaccine formulation with a magnetic nanoparticle of manganese ferrite combined with recombinant fusion protein would have the ability to induce a protective cellular immune response, without adding other adjuvants. The nanoparticle was coated with recombinant CMX fusion protein and BALB/c mice were vaccinated with this formulation, in protocol with three vaccinations with 21-day intervals. Subsequently, the vaccinated animals were infected with Mycobacterium tuberculosis (H37Rv) to evaluate the protection conferred by the vaccine. The results showed that the nanoparticle was able to generate cellular immune responses of Th1, Th17 and T CD8 types, depending on the route of inoculation (subcutaneous, intranasal and mixed). The most preeminent response was Tc1 which was recalled after infection was able to protect against the challenge with Mtb. In addition, there was no appearance of side effects or damage to organs of infected animals, demonstrating that the formulation is safe. Finally, the vaccine formulations with MeNPs, more specifically with manganese ferrite, demonstrate potential application in vaccinology, and may be applied in vaccine formulations to generate cellular immune response, but the route must be considered and in case of use other adjuvants it should consider the possible interaction of NP with the molecule and their ligand. / A busca por novos adjuvantes é um dos objetivos principais dentro da vacinologia. Juntamente com isso, entender o impacto do uso de nanopartículas como sistema de entrega e imunomodulador em sistemas vacinais impacta diretamente no desenvolvimento de novas vacinas. Nesse trabalho, buscamos estudar e elucidar a adjuvanticidade de nanopartículas magnéticas, bem como a imunogenicidade e proteção de sistemas vacinais utilizando essas nanopartículas. Inicialmente foi feito uma revisão da literatura buscando bases científicas que demonstrassem a possiblidade do uso de nanopartículas metálicas (MeNPs) como estimuladores do sistema imune inato. Buscou-se também encontrar elementos em que as nanopartículas metálicas pudessem auxiliar na geração de uma resposta celular do tipo Th1, Th17 e T CD8. A partir dessa revisão, verificou-se que as nanopartículas magnéticas, ou com íons metálicos, eram capazes de estimular a ativação de moléculas coestimuladoras (CD80, CD40 e CD86), induzir secreção de citocinas (IL-1, IL-6, IFN-γ e TNF-α) bem como a resposta imune humoral, mas nenhum trabalho demonstrou se essas nanopartículas eram capazes de induzir resposta celular. Consequentemente, na segunda parte do trabalho utilizou-se a tuberculose como modelo de estudo para verificar se uma formulação vacinal com uma nanopartícula magnética de ferrita de manganês combinada com proteína de fusão recombinante, teria capacidade indutora de resposta imune celular protetora, sem adição de outros adjuvantes. A nanopartícula foi recoberta com a proteína de fusão recombinante CMX e os camundongos BALB/c foram vacinados com essa formulação, em protocolo com três vacinações com intervalos de 21 dias. Posteriormente, os animais vacinados foram infectados com Mycobacterium tuberculosis (H37Rv) para se avaliar a proteção conferida pela vacina. Os resultados mostraram que a nanopartícula teve capacidade de gerar resposta imune celular dos tipos Th1, Th17 e T CD8, dependendo da via de inoculação (subcutânea, intranasal ou mista). Essa resposta foi principalmente do tipo Tc1 e foi capaz de proteger contra o desafio com Mtb. Adicionalmente, não houve qualquer aparecimento de efeito colateral ou danos em órgãos dos animais infectados, demonstrando que a formulação é segura. Por fim, as formulações vacinais com MeNPs, mais especificamente com ferrita de manganês, então demonstram potencial aplicação em vacinologia, podendo ser aplicada em formulações vacinais para gerar resposta imune celular, mas deve-se levar em conta a rota e, caso for utilizar outros adjuvantes complementares, deve-se pensar na possível interação da NP com o adjuvantes e seus ligantes.

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.

Untreatable gonorrhea, caused by fully antimicrobial resistant Neisseria gonorrhoeae (GC), is a major global health threat. While a vaccine would greatly help address this crisis, development of a GC vaccine is complicated by the lack of lab models of symptomatic gonorrhea. We hypothesized that overt disease in animal models of gonorrhea is limited by the human-restriction of gonococcal virulence factors, and the impact of the reproductive hormone cycle (estrus and diestrus phases). We tested these hypotheses by examining the host response to infection in transgenic mice expressing targets of bacterial adhesion, human carcinoembryonic antigen-related cell adhesion molecules (hCEACAMs), in uterine versus vaginal infections, and in different phases of the reproductive cycle (estrus and diestrus phases). hCEACAM expression most impacted estrus phase infections, prolonging colonization in vaginal infection and inducing greater inflammation in uterine. Reproductive phase greatly influenced host response to uterine infection as diestrus infection was more inflammatory than estrus. Phase differences in uterine infection were driven by greater activation of a chemokine-centric common anti gonococcal response and unique induction of type 1 interferons in diestrus. These findings suggest that symptomatic uterine and vaginal GC infection can be modeled by transcervically infected wild-type diestrus mice and transgenic, vaginally-infected estrus mice, respectively. A novel approach to GC vaccine development is also needed. Mono-antigenic vaccines have failed to produce immunity suggesting a poly-antigenic antigen, like natural outer membrane vesicles (nOMVs) may be necessary. It has been shown that any GC vaccine must lack the bacterioprotective antigen, reduction modifiable protein (RMP), and no such nOMV has been previously described. Here we report successful isolation of RMP-deficient nOMVs through sequential size and weight restrictive filtration. Vesicle morphology, proteomics, and bioactivity was characterized via various methods. nOMVs were found to be consistent in size, shape and antigenic load. As antigens, nOMVs induced high serum titers and measurable vaginal levels of antigen and GC specific IgG that recognized several nOMV immunogens supporting the vaccine potential of GC nOMVs. These findings lay the groundwork for protective studies of nOMV vaccines in novel models of active gonorrhea moving the field closer to discovering the mechanism of protective anti-gonococcal immunity.