Objective: To establish stable transgenic non-toxic plants as a platform for plant-based vaccine production as well as potential oral delivery system of vaccine antigens for sexually transmitted infections (STIs). The concept is to immunize the mucosal immune system present in the gut-associated lymphoid tissues (GALT). HIV-1 p24 subtype C protein has been used as the main antigen model, in parallel with an engineered unique chimeric MOMP antigen from Chlamydia trachomatis serovar E. Methods: Chimeric MOMP and p24 vaccine antigens were successfully inserted into the nuclear genomes of Arabidopsis thaliana and Daucus carota via Agrobacterium-mediated gene transfer. The characteristics of the genetic inserts and corresponding mRNAs and recombinant proteins in planta were described using several methods, including northern, Southern, and western blotting, ELISA, and a commercial HIV Ag/Ab combination assay. Immunogenicity of the antigens was studied in mice models. Results: Transgenes of both plant species expressing p24 or chimeric MOMP were successfully generated. Additional HIV-1 vaccine antigen candidates were introduced and the genetic inserts have been confirmed in Arabidopsis thaliana. The Arabidopsis thaliana expressing p24 and chimeric MOMP were demonstrated to be stable over generations and antigenicity analyses showed that plant-derived HIV-1 p24 and chimeric MOMP retained immunological epitopes when they were expressed in planta. Oral administration of transgenic plant material generated a priming effect of the immune competent cells present in the GALT, shown by the presence of antigen-specific-IgG in mice sera after boosting. Mice immunized with plant-derived HIV-1 p24 antigen were also analyzed for antigen-specific faecal IgA as well as cellular immune responses. However, detectable levels of the two latter immune responses were not observed. The Chlamydia trachomatis chimeric MOMP antigen was further evaluated for its potential as a vaccine antigen candidate, with positive results indicating a more rapid clearance of the Chlamydia trachomatis infection post immunization. Conclusion: Stable non-toxic transgenic plants expressing either HIV-1 p24 or a novel  Chlamydia trachomatis chimeric MOMP antigens have successfully been developed. The two plant-produced STI vaccine antigens have in initial mice feeding studies provided important proof-of-concept for the oral vaccination approach. Now, immunization studies to expand, en-hance, and improve knowledge of the immune responses generated by the orally delivered transgenic plants are of high priority. / Kemi/biokemi

[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cell–mediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.

La voie de signalisation impliquant l’interleukine (IL)-22 a un rôle majeur dans le maintien des fonctions de barrière des surfaces exposées du corps humain. Elle est indispensable pour la promotion de l'immunité antimicrobienne épithéliale, de l'inflammation et de la réparation tissulaire. Des situations pathologiques impliquant une altération de cette voie de signalisation ont déjà été décrites, mais rarement au niveau pulmonaire. Ce travail étudie cette voie de signalisation dans trois pathologies respiratoires. Lors de la broncho-pneumopathie chronique obstructive, un excès de sécrétion de protéases à sérine secondaire au recrutement de neutrophiles est responsable d’une protéolyse du récepteur à l’IL-22. Cette protéolyse inhibe les mécanismes de défense épithéliale dépendante de l’IL-22. Lors d’infection pulmonaire à P. aeruginosa, un facteur de virulence sécrété par cette bactérie dégrade l’IL-22 et inhibe la sécrétion épithéliale de peptides antimicrobiens. Enfin, une surexpression du récepteur à l’IL-22 a été observée dans le cancer du poumon non à petites cellules. Cette surexpression est associée à une surmortalité. / The (interleukin) IL-22/IL-22 receptor (R) pathway is critical in the maintenance of barrier function at exposed surface of the body. This pathway is also essential to promote innate mucosal immunity, inflammation and tissue homeostasis. Dysregulation of IL-22/IL-22R pathway has been described in human diseases, but has been barely studied in respiratory pathologies. This work reveals three lung diseases with altered IL-22/IL-22R pathway. During chronic obstructive pulmonary disease, the proteolytic action of neutrophil-derived enzymes cleave the IL-22R and inhibit IL-22-mediated epithelial cell response. Next, we demonstrated that P. aeruginosa used its own proteolytic system to escape from host defenses through the proteolysis of IL-22 leading to negative regulation of antimicrobial peptides. Finally, we observed that higher IL-22R expression is correlated with squamous cell lung carcinoma and is associated with increase mortality.

Challenged by a huge and diverse antigenic stimulus, the intestinal mucosa has developed a unique immune system that mainly functions to maintain tolerance to innocuous antigens while retaining the ability to respond swiftly to pathogenic threats. Central to this specialised immune system are the Intraepithelial Lymphocytes (IELs). These cells are uniquely located between Intestinal Epithelial Cells (IECs) ready to respond to exogenous antigens in the intestinal lumen. The intestinal immune system is constantly influenced, not only by the commensal microbiota, but also by the nutritional status of the host and the availability of certain essential micronutrients that are derived from a healthy-balanced diet. Additionally, age has a significant impact on the efficiency of gut immunity in responding to infectious pathogens, as reflected by the increased burden of gastrointestinal infections at the extremes of age. In this thesis, using the Murine Norovirus (MNV) oral infection model, I aimed to characterize intestinal mucosal antiviral-responses with specific focus on the role of IELs, the impact of aging and the influence of certain micronutrients whose effects are mediated through the Aryl Hydrocarbon Receptor (AhR). Employing different knock-out and adoptive transfer experiments, I concluded that, at least in our experimental conditions and in a viral strain-specific manner, the activated IELs are not essential and may play a minor role in the protective response against MNV infection. This work also demonstrated that various MNV virus strains activate IELs differentially and for the first time (to our knowledge) revealed distinct abilities of these different Norovirus variants to infect IECs. Recognising an impaired response in old (2-year) mice, we were also able to identify a specific defect in the IFN-Lambda response of aged IECs. Furthermore, using the model of MNV infection to investigate the role of AhR signalling, the data I generated suggested a direct link between constitutive AhR signalling and innate interferon-mediated responses. These findings have uncovered a potential preventive/therapeutic targets for enhancing anti-viral responses.

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.

Orientador: Wirla Maria da Silva Cunha Tamashiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:43:40Z (GMT). No. of bitstreams: 1 Ruberti_Maristela_D.pdf: 10774061 bytes, checksum: 7afe7ee8aa8c7f97c1f80e66f0cd8bfa (MD5) Previous issue date: 2012 / Resumo: Trabalhos anteriores de nosso laboratório mostraram que camundongos transgênicos DO11.10, cuja maioria dos linfócitos T expressam TCR específico para ovalbumina (OVA) no contexto de...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Previous work from our laboratory showed that DO11.10 transgenic mice, in which the most of T lymphocytes express TCR specific for ovalbumin (OVA) in the context of...Note: The complete abstract is available with the full electronic document / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

Made available in DSpace on 2014-08-20T14:37:48Z (GMT). No. of bitstreams: 1 dissertacao_bianca_siedler.pdf: 968152 bytes, checksum: 4210f5ec4c0f10354b8e411cff54f4e5 (MD5) Previous issue date: 2012-02-14 / Mucosal immune system represents the initial barrier against many pathogens, including bovine herpesviruses (BoHV). After infection of mucous membranes, mainly nasal and genital, these viruses disseminate locally followed by viremia and neural spread. Innate defense mechanisms along with adaptive immunity confer protection to mucosal surfaces. In this context, secretory IgA (sIgA) plays an essential role in the mucosal humoral immunity, conferring protection to these body surfaces through different mechanisms, including viral neutralization. This class of antibody predominates in the vaginal mucosa of cattle playing an important role in the local defense against infections. Considering the importance of this infection route in herpesviruses pathogenesis, there is a growing interest in the development of vaccines that provide mucosal immunity against these viruses. In the present study, eight cows were divided in two groups (G1 and G2) and inoculated intravaginally with inactivated BoHV-5 associated with recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and xanthan (G1) and inactivated BoHV-5 associated with xanthan (G2). Local and systemic humoral immune response (IgA and IgG) induced after inoculation was measured by indirect ELISA. An increment in the levels of IgA and IgG was detected in sera, nasal and genital mucosa of all the immunized animals. Furthermore, the relative expression of interleukins 2 and 13 (IL-2 and IL-13) was investigated by real-time PCR indicating an increased mRNA expression of these cytokines in leukocytes collected from animals immunized with the experimental vaccine. These results demonstrate that the experimental intravaginal BoHV-5 vaccine induced a local and systemic immune response in cattle. The results also corroborated the immunostimulant activity of the rLTB in mucosal membranes and confirmed the use of xanthan as a delivery system for intravaginal vaccines. Our data also reinforce the importance of this route for administration of vaccines focused on providing local protection against pathogens. / O sistema imune de mucosa representa a barreira inicial frente a diversos patógenos que utilizam estas superfícies como porta de entrada no organismo, como é o caso dos herpesvírus bovinos (BoHV). Estes vírus utilizam as mucosas, principalmente nasal e genital, como ponto inicial de replicação, seguida de disseminação local, viremia sistêmica e disseminação neuronal. Mecanismos inatos de defesa em cooperação direta com mecanismos adaptativos conferem proteção a estas mucosas. A IgA secretora (sIgA) representa um papel fundamental na imunidade humoral destes locais, conferindo proteção a estas superfícies através de distintos mecanismos, incluindo a neutralização viral. Este anticorpo predomina na mucosa vaginal de bovinos, sendo essencial na defesa local frente a patógenos de transmissão genital. Devido a grande importância das vias mucosas na transmissão dos BoHV, torna-se evidenciado o interesse no desenvolvimento de vacinas que propiciem imunidade de mucosa contra estes agentes etiológicos. No presente estudo, oito fêmeas bovinas foram divididas em dois grupos (G1 e G2) e inoculadas por via intravaginal com BoHV-5 inativado associado à subunidade B recombinante da enterotoxina termolábil de Escherichia coli (rLTB) e xantana (G1) e BoHV-5 inativado associado a xantana (G2). A resposta humoral (IgA e IgG) local e sistêmica induzida nos animais inoculados foi mensurada através do teste de ELISA indireto. A vacina avaliada demonstrou-se capaz de incrementar os níveis de IgA e IgG no soro e nas mucosas nasal e vaginal dos bovinos imunizados. A expressão relativa das interleucinas 2 e 13 (IL-2 e IL-13) foi avaliada através da técnica de real time RT-PCR, a partir dos leucócitos dos animais vacinados, resultando em aumento na expressão de mRNA destas citocinas nos animais inoculados com a vacina experimental. Estes dados comprovam a capacidade da vacina de aplicação intravaginal em bovinos de estimular uma resposta imune local e sistêmica, além de corroborar a atividade imunoestimulante em mucosas da rLTB e também validar a utilização da xantana como sistema de entrega de vacinas de aplicação intravaginal. Portanto, esta via de administração de vacinas torna-se uma alternativa interessante, principalmente quando se objetiva gerar proteção local contra patógenos que utilizam as superfícies mucosas como porta de entrada no organismo.

Les plaques de Peyer (PP) sont les principaux sites inducteurs de la réponse immunitaire mucosale.L’épithélium associé aux follicules comprend des cellules épithéliales spécifiques, appelées cellules M et spécialisées dans le transport du matériel présent dans la lumière intestinale vers le dôme sous épithélial (SED) où il sera pris en charge par les cellules du système phagocytaire mononuclée (MPS) qui orchestreront ensuite les réponses immunitaires mucosales.Nous avons effectué une analyse complète du phénotype, de la distribution, de l’ontogénie, de la fonction et des profils transcriptomiques du MPS des PP. Nous avons montré que les monocytes donnent naissance à deux populations: les lysoDC et les lysoMac. La première exprime de fort niveau de CMH-II et de molécules de costimulation, a une courte durée de vie et est capable d’activer les lymphocytes T naïfs pour sécréter de l’IFNγ tandis que la deuxième exprime faiblement le CMH-II, à une longue durée de vie et n’est pas capable d’activer les LT naïfs. Ces deux populations ont toutefois des propriétés communes de phagocytose et de défense innée contre les virus et les bactéries entéropathogènes. Nous avons identifié deux populations distinctes de lysoMac selon l’expression de Tim4: les lysoMac Tim4+ situés dans l’IFR et la partie inférieure du follicule ; les lysoMac Tim4- situés dans le SED et la partie supérieure du follicule. Nous avons aussi déterminé 4 états de maturation pour les lysoDC suivant l’expression d’Emb, Jam-A et CD24. Nous avons également redéfini la localisation de chaque population du MPS des PP fournissant ainsi une base solide pour étudier le rôle de chacun de ses membres dans l’immunité mucosale. / Peyer’s patches (PPs) are primary inductive sites of mucosal immunity. The follicle-associated epithelium contains specialized epithelial cells, called M cells, that bind and rapidly transport microorganisms from the lumen to the subepithelial dome (SED) where they are internalized by cells of the mononuclear phagocyte system (MPS) which are involved in the initiation of the mucosal immune responses. MPS comprise monocytes, macrophages (Mφ) and dendritic cells (DC). Here, we provide a comprehensive analysis of the phenotype, distribution, ontogeny, function, and transcriptional profile of PP MPS. We show that monocyte give rise to two different cell populations named lysoDC and lysoMac. The former express high levels of MHCII and costimulatory molecules, have a short half life and are able to prime naïve T cells for IFNγ production while the latter display low levels of MHCII, have a long half life and are unable to prime naïve T cells efficiently. However, these two cell populations share common features such as phagocytosis and antimicrobial defense mechanisms. LysoMac can be separated in two subpopulations according to Tim4 expression: Tim4+ lysoMac located in the IFR and the lower part of the follicle; Tim4- lysoMac located in the SED and upper part of the follicle. LysoDC can be separated in four different maturation stages according to Emb, Jam-A and CD24 expression. Finally, we redefined the location of each PP MPS population. In summary, we provide a comprehensive map of the PP MPS which will allow to study its role in mucosal immune response initiation and regulation.

Infants are born with naive immune systems, making them susceptible to a variety of infections. In order to protect the newborn infant it is important that mothers be able to pass protective IgA antibodies to their infants through breast milk. B cells that produce IgA enter the mammary tissue during lactation and secrete IgA into the milk. During pregnancy, the mammary tissue expresses high levels of chemokines, molecules that allow lymphocytes to selectively home to specific tissues. The chemokine CCL28 has been shown to be upregulated during both pregnancy and lactation, and is vital for the ability of IgA-producing B cells to home to the mammary tissue during lactation. The aim of this study was to determine whether CCL28 expression is regulated by prolactin signaling.