Streptococcus mutans é o principal agente etiológico da cárie dental humana, uma doença infecciosa para a qual não há vacina disponível. A influência dos sistemas de transporte ABC na virulência bacteriana suporta seu uso como alvos vacinais. Em S. mutans a proteína ligadora de fosfato (PstS) influencia a expressão de fatores de virulência. A proposta deste trabalho foi caracterizar uma estratégia vacinal de mucosa anti-cárie baseada na proteína PstS como antígeno alvo. Inicialmente, a forma recombinante da proteína foi obtida em E. coli. A caracterização biofísica revelou uma estrutura secundária estável, semelhante a outras proteínas ligadoras e capaz de interagir com seu ligante. Epítopos antigênicos conservados foram identificados na proteína recombinante pela reatividade com soro anti-S. mutans. A proteína rPstS foi imunogênica por via sublingual, combinada ou não à LTK4R e anticorpos rPstS-específicos interferiram na colonização oral in vivo por S. mutans. Os resultados indicam que a proteína rPstS pode ser explorada em estratégias vacinais contra a cárie. / Streptococcus mutans is the main etiological agent of human dental caries, an infectious disease for which there is no vaccine available. The influence of ABC transport systems in bacterial virulence supports its use as vaccine targets. In S. mutans the phosphate binding protein (PstS) influences the expression of virulence traits. The purpose of this work was characterizing an anti-caries mucosal vaccine based on the PstS protein as target antigen. First, a recombinant form of the protein was obtained in E. coli. The biophysical characterization showed a stable secondary structure, similar to other binding proteins and able to interact with its ligand. Conserved antigenic epitopes were identified in the recombinant protein by reactivity with anti- S. mutans serum. The rPstS protein was immunogenic by the sublingual route in combination or not with LTK4R and rPstS-specific antibodies interfered with S. mutans oral colonization in vivo. The results indicated that the recombinant PstS protein can be exploited in vaccine strategies against dental caries.

O objetivo deste trabalho foi a construção de linhagens geneticamente modificadas de B. subtilis capazes de expressar porções de intimina, principal componente envolvido na capacidade de colonização de linhagens enteropatogênicas de Escherichia coli (EPEC), como estratégia vacinal de administração oral contra diarréias infecciosas. As vacinas desenvolvidas empregaram cinco regiões da intimina de EPEC e linhagens de B. subtilis capazes de expressar e acumular proteínas recombinantes no citoplasma. Além disso, avaliamos o uso de esporos e células vegetativas como veículos vacinais para a entrega de antígenos recombinantes a partir de sistema de expressão epissomal. A eficácia do modelo vacinal foi demonstrada pela: (i) produção de anticorpos sistêmicos (IgG) e secretados (sIgA) contra intimina, (ii) capacidade de neutralização das intiminas expressas por diferentes linhagens de EPEC pelos anticorpos específicos gerados nos animais imunizados; e (iii) proteção a desafio com linhagens de EPEC a partir de modelo experimental que emprega camundongos recém-nascidos. Os resultados representam uma etapa importante na validação de uma nova estratégia vacinal para o controle de patógenos entéricos. Além disto, propomos a utilização de um modelo animal como uma nova ferramenta para se avaliar o potencial protetor de vacinas contra EPEC. / The objective of this work was the construction of genetically modified strains of B. subtilis able to express portions of intimin, the main component involved in colonization by enteropathogenic Escherichia coli strains (EPEC) as a strategy of oral vaccination against infectious diarrhea. The vaccines employed five regions of EPEC intimin and B. subtilis strains expressing recombinant proteins in the cytoplasm. Furthermore, we evaluated the use of spores and vegetative cells as vaccine vehicles for the delivery of recombinant antigens based on an epissomal expression system. The efficacy of the vaccines was demonstrated by: (i) production of systemic (IgG) and mucosal (sIgA) antibody responses to intimin, (ii) neutralizing of intimin expressed by different strains of EPEC by the antibodies generated in immunized animals, and (iii) protection to lethal challenges carried out with EPEC strains using an experimental model based in newborn mice. The results represent an important step in the validation of a new vaccine strategy for the control of enteric pathogens. Moreover, we propose the use of an animal model as a new tool to evaluate the protective potential of vaccines against EPEC.

A maioria dos estudos da interação parasito-hospedeiro tem focado na interação de um único patógeno. Porém, o hospedeiro em um ambiente natural é comumente exposto a múltiplos patógenos sequencialmente ou mesmo simultaneamente. Diversos estudos têm utilizado o modelo de Ligadura e perfuração do Ceco (CLP) para estudar a sepse, mas nenhum deles apresentou modelo de coinfecção ou estudo avaliando o papel de infecções prévias no desfecho da sepse polimicrobiana experimental. Neste contexto, nossa hipótese é de que a infecção crônica por parasitos poderia alterar o curso da resposta durante a sepse polimicrobiana. Para testar essa hipótese, animais C57BL/6 ou BALB/c foram infectados com 5 ou 20 cistos da cepa ME 49 de Toxoplasma gondii e 40 dias após a infecção os animais foram induzidos à sepse polimicrobiana. Em nosso estudo, 100% dos animais cronicamente infectados por T. gondii morreram num período de 24 horas após CLP. O mesmo não foi observado quando animais foram infectados cronicamente com os parasitos Leishmania major e Trypanosoma cruzi ou com o fungo Paracoccidioides brasiliensis. Um dado interessante em nosso estudo foi que, nos animais previamente infectados com T. gondii, constatamos melhora na eliminação de bactérias liberadas pela CLP e aumento do recrutamento celular para o sítio da infecção. Apesar de esses animais apresentarem melhora na resposta contra as bactérias, verificamos a presença de lesão intestinal e maior infiltrado inflamatório neste órgão, associado a um aumento da produção de citocinas pró-inflamatórias (IFN-, TNF-, IL-6 e IL-1) e consequente aumento de óxido nítrico (NO), num período de 24 horas depois da CLP. Verificamos que as células TCD4+ e TCD8+ são responsáveis pela produção de IFN- e TNF- nesse modelo de coinfecção, e em modelo in vitro, que macrófagos podem ser responsáveis pela produção de IL-1 dependente de ativação do inflamassoma NLRP3/ASC/Caspase 1. Neste estudo, observamos que a rápida resposta contra a CLP acontece em função da presença de células de memória de padrão Th1, induzidas na infecção por T. gondii. Dessa forma, esse trabalho mostra que a infecção crônica por T. gondii agrava a sepse polimicrobiana subletal, por aumentar a produção de citocinas pró-inflamatórias IL-6, TNF- e IL-1, com a indução de hipotensão, predispondo ao choque séptico. / Most studies of parasite-host interaction have focused on the interaction of a single pathogen with cells or organism of the host. However, in a natural enviroment, the host is commonly exposed to multiple pathogens sequentially or even simultaneously. Several studies have used the model of cecal ligation and puncture (CLP) to study sepsis, but none of them evaluated the effect of the presence of previous infections to the outcome of polymicrobial sepsis. In this context, we hypothesized that chronic infection with Toxoplasma gondii could alter the course of host response against polymicrobial sepsis. To test this hypothesis, C57BL/6 or BALB/c mice were orally infected with 5 or 20 cysts of ME-49 strain of T. gondii and 40 days post infection, they were subjected to CLP. When mice were chronically infected with T. gondii, 100% of the animals died within 24 hours after CLP. The same phenomenons were not observed in animals previously infected with other parasites, such as Leishmania major and Trypanosoma cruzi or the fungus Paracoccidioides brasiliensis. Interestingly, when we evaluated the response against the CLP in animals that were infected with T. gondii, we found an improvement in the killing of bacteria released by CLP and an increase in recruitment of inflammatory cells to the site of infection. However, despite the fact that these animals have improved response against the bacterial infection, they presented intestinal damage and increased inflammatory infiltrate in this organ. The animals also had increased pro-inflammatory cytokines (IFN-, TNF-, IL-6 and IL-1), and nitric oxide (NO) detected within 24 hours after CLP. We also found that the TCD4+ and TCD8+ cells were responsible to produce IFN- and TNF-, and, using an in vitro model, we verified that macrophages are primarily responsible for the production of IL-1 in a pathway dependent on the activation of NLRP3/ASC/Caspase 1 inflamassoma. In this study, we found that early response against CLP happens due to the presence of mainly Th1 memory cells, induced by T. gondii infection. Finally, we found that chronic infection with T. gondii aggravates sublethal polymicrobial sepsis by increasing the cytokines IL-6, TNF- and IL-1, with induction of hypotension that predispose to septic shock.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-02-15T16:51:53Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tamara Nascimento Ferreira.pdf: 2375661 bytes, checksum: 1a03fafd92daf583c5befdc68482ada8 (MD5) / Made available in DSpace on 2018-02-15T16:51:53Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Tamara Nascimento Ferreira.pdf: 2375661 bytes, checksum: 1a03fafd92daf583c5befdc68482ada8 (MD5) Previous issue date: 2015-02-25 / A tolerância oral é definida como uma supressão a respostas imunológicas específicas após imunização com um antígeno que foi previamente administrado por via oral. Esse método tem sido estudado, nas últimas décadas, como uma ferramenta na redução de respostas imunes humorais e celulares envolvidas em doenças autoimunes e alergias e parece ser uma alternativa para reduzir ou retardar a rejeição de transplantes. Neste estudo investigamos o potencial das toxinas dermonecróticas recombinantes do veneno da aranha marrom Loxosceles intermedia em induzir estado de tolerância de mucosas em modelo animal, avaliando os mecanismos celulares envolvidos e a resposta imune do animal tolerante frente a estímulos imunológicos específicos e inespecíficos. As toxinas recombinantes utilizadas para indução da tolerância oral foram a LiRecDT1 (com atividade dermonecrótica) e sua forma mutada, LiRecDT1H12A, (com atividade dermonecrótica residual). Para indução de tolerância oral foi utilizado protocolo com baixas doses de antígeno, onde foram tolerizados camundongos adultos Swiss, tratados oralmente com as toxinas recombinantes durante 21 dias, num total de 90g por animal. Para investigar possíveis mecanismos envolvidos na tolerância oral, foi avaliada a expressão fator de transcrição Foxp3 e da citocina TGF-β. Após o protocolo de tolerização os linfonodos mesentéricos foram processados para ensaio de western blotting e a expressão do fator de transcrição Foxp3 e da citocina TGF-β foram avaliados. Os grupos tolerizados com ambas as toxinas mostraram um aumento na expressão dos dois componentes, demonstrando que um dos mecanismos de tolerância oral no nosso modelo pode envolver aumento na população de linfócitos T regulatórios. Esse resultado pode ser reforçado pela dimimuição de edema de pata em camundongos naive que receberam, via intravenosa, esplenócitos de animais tolerizados com a toxina LiRecDT1 H12A (transferência adotiva). Além disso, pudemos observar que a tolerância induzida pela toxina LiRecDT1 H12A se estendeu a antígenos não relacionados (tolerância cruzada) com diminuição na produção de anticorpos IgG específicos e edema de pata em animais desafiados com veneno da vespa Polybia paulista. A tolerância oral induzida pela toxina LiRecDT1 H12A também permitiu aparente redução da resposta imunológica do animal tolerante frente ao enxerto alogênico de camundongos C57BL/6. Considerando os resultados obtidos é possível concluir que a tolerância oral induzida pelas toxinas LiRecDT1 e LiRecDT1 H12A pode envolver aumento na população de linfóticos T regulatórios cuja produção de citocinas anti-inflamatórias, pelo menos na tolerância induzida pela toxina LiRecDT1 H12A, pode reduzir a resposta imune a antígenos específicos e inespecíficos na tolerância cruzada e na redução de rejeição a transplantes. / Oral tolerance refers to physiologic induction of immunosuppression that occurs in mucosal induced by oral administration of an antigen. This method has been used to induce reduction of humoral and cellular responses involved in autoimmune diseases, allergies and may be an alternative to reduce transplant rejection. We investigated potential of dermonecrotic recombinant toxins of brown spider Loxosceles intermedia specie to induce a state of mucosal tolerance in a animal model. We did this by assessing the cellular mechanisms involved and the immune response of tolerant animals against specific and nonspecific immune stimulation. Dermonecrotic toxin LiRecDT1 and its mutated form, LiRecDT1 H12A (with residual dermonecrotic activity), has been used. Mice were treated with recombinant toxins, orally, for 21 days, with a total of 90g per animal. After oral tolerance protocol, mesenteric lymph nodes were processed for western blotting and the expression of nuclear transcription factor Foxp3 and cytokine Tgf-β were analyzed. LiRecDT1 and LiRecDT1 H12A tolerant groups showed an increase in expression of both proteins, which could be related to a increase in regulatory T-cells population. Adoptive transfer of splenocytes to naive mice from LiRecDT1 H12A tolerated group induced paw edema reduction, with supports this theory (adoptive transfer). Our results also demonstrated a bystander tolerance when LiRecDT1 H12A tolerated mice showed a decrease in IgG production and paw edema induction by Polybia paulista wasp venom. LiRecDT1 H12A tolerance also showed an apparent reduction in skin allograft rejection using C57BL/6 mice as donor and tolerated Swiss mice as receptor. Considering the results we concluded that oral tolerance induced by LiRecDT1 and LiRecDT1 H12A toxins could be involve increase in Tregs and non-inflammatory cytokines, and LiRecDT1 H12A tolerance can reduce specific and non specific immunology responses and allografts transplants rejection.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal. / Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen.

A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal. / Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen.

Le virus de la grippe infecte les muqueuses du tractus respiratoire. Un vaccin intranasal induit une réponse immunitaire proche de celle faisant suite à une infection naturelle en bloquant le virus directement sur le site de l'infection et permet une vaccination sans aiguille. Par ailleurs, les vaccins à base de pseudo-particules virales ou Virus-like Particles (VLP) produites sur cellules représentent une alternative intéressante au vaccin classique produit sur oeufs. Les VLP sont des particules non-réplicatives qui ressemblent au virus et qui peuvent être immunogènes même sans adjuvant, en particulier par voie intranasale. Au cours de ma thèse, une plateforme de production de VLP grippales composées d'hémagglutinine, de neuraminidase et de protéine de matrice M1 a été développée par transfection transitoire des cellules de mammifères. Des immunisations de souris BALB/c ont montré que les VLP de type A et B, purifiées et caractérisées, étaient immunogènes à de faibles doses par voie intramusculaire. L'administration par voie intranasale de VLP avec la sous-unité B de la toxine cholérique, comme adjuvant muqueux, a permis d'obtenir des taux d'anticorps sériques comparables à ceux obtenus par immunisation en intramusculaire mais également une forte réponse IgA au niveau des muqueuses. Par ailleurs, le rendement des VLP s'est révélé souche-dépendant et lié aux protéines HA et NA à la surface de la particule. Pour contourner ce problème, un vaccin quadrivalent composé de deux VLP bivalentes exprimant chacune deux HA et NA différentes à la surface a été produit montrant ainsi la flexibilité de cette plateforme / The influenza virus infects the mucous membranes of the respiratory tract. An intranasal vaccine induces an immune response close to the one induced by the natural infection by blocking the virus directly at the site of infection and allows needle-free vaccination. In addition, vaccines based on Virus-like Particles (VLP) produced in cells represent an interesting alternative to the traditional egg-based vaccine. VLPs are non-replicative particles that mimic the virus. Studies on influenza VLPs have shown protection by the intranasal route without adding an adjuvant. During my thesis, a platform for the production of influenza VLPs composed of the hemagglutinin, the neuraminidase and the M1 matrix proteins was developed by transient transfection of mammalian cells. Immunizations of BALB/c mice showed that the purified and characterized type A and B VLPs were immunogenic at low doses by the intramuscular route. The intranasal administration of VLPs with the B subunit of cholera toxin as a mucosal adjuvant resulted in serum antibody levels comparable to those obtained by intramuscular immunization but also a strong IgA response in the mucosal secretions. In addition, VLP yield was found to be strain-dependent and linked to the HA and NA proteins on the surface of the particle. To overcome this problem, a quadrivalent vaccine based on two bivalent VLPs each expressing two different HAs and NAs at the surface was produced, demonstrating the flexibility of this platform

<p>The gastric mucosa is frequently exposed to endogenously secreted hydrochloric acid of high acidity. Gastric mucosal defense mechanisms are arranged at different levels of the gastric mucosa and must work in unison to maintain its integrity. </p><p>In this thesis, several mechanisms underlying gastric mucosal resistance to strong acid were investigated in anesthetized rats and mice. The main findings were as follows:</p><p>Only when acid secretion occurred did the pH gradient in the mucus gel withstand back-diffusion of luminal acid (100 mM or 155 mM HCl), and keep the juxtamucosal pH (pH_jm) neutral. Thus, when no acid secretion occurred and the luminal pH was 0.8-1, the pH gradient was destroyed. </p><p>Bicarbonate ions, produced concomitant with hydrogen ions in the parietal cells during acid secretion and blood-borne to the surface epithelium, were carried transepithelially through a DIDS-sensitive transport. </p><p>Prostaglandin-dependent bicarbonate secretion seemed to be less important in maintaining a neutral pH_jm. </p><p>Removal of the loosely adherent mucus layer did not influence the maintenance of the pH_jm. Hence, only the firmly adherent mucus gel layer, approximately 80µm thick, seemed to be important for the pH_jm. </p><p>Staining of the mucus gel with a pH-sensitive dye revealed that secreted acid penetrated the mucus gel from the crypt openings toward the gastric lumen only in restricted paths (channels). One crypt opening was attached to one channel, and the channel was irreversibly formed during acid secretion. </p><p>Gastric mucosal blood flow increased on application of strong luminal acid (155 mM HCl). This acid-induced hyperemia involved the inducible but not the neural isoform of nitric oxide synthase. These results suggest a novel role for iNOS in gastric mucosal protection and indicate that iNOS is constitutively expressed in the gastric mucosa. </p><p>It is concluded that a pH gradient in the gastric mucus gel can be maintained during ongoing acid secretion, since the acid penetrates the mucus only in restricted channels and bicarbonate is carried from the blood to the lumen via a DIDS-sensitive transporter.</p>