Introdução: A vacina pneumocócica é recomendada para crianças com IRC, pelo maior risco de adquirir a forma invasiva da doença. O Streptococcus pneumoniae é o principal agente etiológico causador de pneumonia e otite média aguda, e a segunda causa de meningite na criança. A vacina conjugada 7 - valente tem se mostrado imunogênica e com efeito duradouro em crianças normais, porém em pacientes com IRC, devido às alterações imunológicas associadas à uremia, pode resultar uma resposta sub - ótima e de vida curta. Objetivo: Determinar as concentrações de anticorpos da classe IgG contra os sorotipos 4,6B, 9V, 14, 18C, 19F e 23 F do Streptococcus pneumoniae, antes e depois da administração da vacina antipneumocócica conjugada 7 - valente, em crianças portadoras de insuficiência renal crônica em tratamento conservador e diálise, e comparar a resposta entre eles. Métodos: Selecionamos 48 crianças, com idade de 1 ano a 9 anos, portadoras de IRC, com depuração de creatinina menor ou igual a 70 ml/min/1,73m 2 , calculada pela fórmula de Schwartz, divididos em: grupo 1 (G1, tratamento conservador), e grupo 2 (G2,tratamento dialítico). Os pacientes receberam duas doses da vacina conjugada 7 - valente, com intervalo de 60 dias. A sorologia foi colhida antes da primeira dose, e 30 a 60 dias após o reforço. Os títulos de anticorpos para os sorotipos presentes na vacina (4, 6B, 9V, 14, 18C, 19F, 23F) foram determinados pelo método imunoenzimático (ELISA). Resultados: A análise da concentração pré - vacinal de IgG mostrou maior percentual dos pacientes do grupo 1 e 2 com IgG <0,6 para os sorotipos 4, 9 e 18, e maior percentual com IgG maior ou igual a 1,3 ug/ml para os sorotipos 14 e 19. Para avaliar a concentração de IgG pós - vacinal foi utilizado o cálculo de freqüências com intervalo de confiança 95%(IC95), para os 7 sorotipos nos 2 grupos, utilizando-se 3 critérios : Critério A: IgG pós vacinal maior ou igual a 1,3 ug/ml, por este critério verificou-se freqüência de resposta do G1 de 0,650 (IC95: 0,407-0,864) a 1,0 (IC95: 0,663 - 1,0) e do G2 de 0,777 (IC95: 0,399 -0,971) a 1,0 (IC95: 0,663 - 1,0), critério B: ou seja, delta (pré/pós) maior ou igual a 4X, freqüência de resposta G1 de 0,458 (IC95: 0,255 - 0,671) a 0,708 (IC95%: 0,488 - 0,873) e do G2 de 0,458 (IC95%: 0,255 - 0,671) a 0,708 (IC95%: 0,488 - 0,973), e critério C:isto é, delta (pré/pós) maior ou igual a 4X e IgG maior ou igual a 1,3 ug/ml, freqüência de resposta do G1 de 0,416 (IC95%: 0,221 - 0,633) a 0,625 (IC95%: 0,406 - 0,811) e do G2 de 0,416 (IC95%: 0,221 - 0,633) a 0,666 (IC95%: 0,447 - 0,843). G1 e G2 mostraram comportamento semelhante na análise das freqüências de resposta, segundo os 3 critérios. Conclusão: Este estudo é pioneiro na avaliação da vacina antipneumocócica conjugada em crianças com IRC, e demonstrou uma boa resposta para cada critério analisado separadamente, com comportamento semelhante nos 2 grupos. A dificuldade inerente à definição de critérios de soroconversão, demonstra a necessidade de estudos multicêntricos com acompanhamento clínico e laboratorial em longo prazo para avaliar a sustentabilidade da soroconversão e a imunogenicidade da vacina. / Introduction: The pneumococcal vaccine is recommended for children with CRF due to their increased risk in acquiring the invasive form of the disease. Streptococcus pneumoniae is the main etiological agent that causes pneumonia and acute middle otitis and the second cause of meningitis in children. The 7-valent conjugated pneumococcal vaccine has been shown to be immunogenic and to have a lasting effect in normal children; however, in children with CRF, the vaccine can produce a suboptimal and short-lasting response due to the immunological alterations associated to uremia. Objective: to determine pneumococcal IgG antibodies to serotypes 4, 6B, 9V, 14, 18C, 19F e 23 F before and after 7 - valent conjugated pneumococcal vaccine, in children with chronic renal failure in conservative treatment and dialysis. Methods: 48 children with CRF, aged 1 to 9 years, with a creatinine clearance of menor ou igual a 70 ml/min/1.73 calculated by the Schwartz formula, were selected for the present study and divided in two groups: Group 1: conservative treatment and Group 2: dialytic treatment. The patients received two doses of the 7- valent conjugated vaccine, with a 60-day interval between them. Serological samples were collected before the first dose and 30 to 60 days after the second one. Antibody titers for the serotypes present in the vaccine (4, 6B, 9V, 14, 18C, 19F, 23F) were determined by the immunoenzymatic method (ELISA). Results: The analysis of the pre-vaccinal IgG concentration showed a higher percentage of patients from Groups 1 and 2 with IgG < 0.6 for the serotypes 4, 9 and 18, and a higher percentage with pre-vaccinal IgG maior ou igual a 1.3 ug/ml for the serotypes 14 and 19. To assess the post-vaccinal IgG concentration, the calculation of frequencies with 95% confidence interval (95CI) was employed for the 7 serotypes in both groups, using three criteria: Criterion A: post-vaccinal IgG maior ou igual a 1.3 ug/ml, this criterion verified the frequency of response from G1 0,650 (IC95: 0,407-0,864) to 1,0 (IC95: 0,663 - 1,0) and G2 0,777 (IC95: 0,399 - 0,971) to 1,0 (IC95: 0,663 - 1,0), criterion B: i.e., delta (pre/post) maior ou igual a 4X, frequency of response from G1 0,458 (95CI: 0.255 ? 0.671) to 0.708 (95CI: 0.488 - 0.873) and G2 0,458 (IC95: 0,255 - 0,671) to 0,708 (IC95: 0,488 - 0,873); criterion C: i.e., delta (pre/post) ?4X and IgG maior ou igual a 1.3 ug/ml, frequency of response from G1 0.416 (95CI: 0.221 - 0.633) to 0,625(95CI: 0,406 - 0,811) and G2 0,416 (9CI: 0,221 - 0,633) to 0.666 (95CI: 0.447 - 0.843). Groups 1 and 2 showed similar behavior at the analysis of response frequencies according to the three criteria. Conclusion: this is the first study to assess the conjugated pneumococcal vaccine in children with CRF, which showed good response, with a similar behavior in both groups. The inherent difficulty in defining criteria for seroconversion demonstrates the need for multicentric studies with long-term clinical and laboratory follow up, in order to assess the seroconversion duration and the vaccine immunogenicity.

INTRODUÇÃO: A vacina pneumocócica conjugada 10-valente (VPC10) foi introduzida no calendário de imunização infantil do Programa Nacional de Imunizações em 2010. Este estudo analisou as taxas de incidência de Meningite Pneumocócica (MP) no Brasil, por faixa etária e unidade da federação (UF); a distribuição espacial das taxas de incidência de MP em menores de cinco anos no Brasil, por UF, no período pré (2005-2009) e pós-vacinação (2011-2013); e associações com variáveis socioeconômicas e cobertura vacinal. Foram analisadas a distribuição espacial das taxas de incidência de MP em menores de cinco anos, por município do estado de São Paulo (SP), no período pré e pós-vacinação, e a existência de aglomerados espaciais e espaço-temporais. Através de estatística espacial, foram analisadas associações das taxas de incidência de MP, por microrregiões do estado, com variáveis socioeconômicas e cobertura vacinal. MÉTODOS: Estudo ecológico de base populacional, que utilizou dados do Sistema de Informação de Agravos de Notificação. Cobertura vacinal e o Índice de Desenvolvimento Humano (IDH) foram utilizados na análise do Brasil. Na análise de SP, as unidades ecológicas foram municípios e microrregiões, e a variável socioeconômica foi o Índice Paulista de Responsabilidade Social (IPRS) da Fundação Sistema Estadual de Análise de Dados. Foram construídos mapas temáticos para as taxas de incidência de MP em menores de cinco anos, nos períodos pré e pós vacinação, cobertura vacinal e IDH, por UF. Também foram construídos mapas temáticos das taxas de incidência de MP em menores de cinco anos, por microrregião de SP, nos períodos pré e pós-vacinação, cobertura vacinal e IPRS, utilizando o software QGis 2.6.1. Para SP, foi utilizada a técnica de varredura (software SatScan 9.2) para analisar aglomerados. O modelo Gaussiano latente Bayesiano com modelos inflados de zeros de Poisson, através da aproximação de Laplace aninhada e integrada (INLA), foi utilizado na análise espacial para avaliar associações entre taxa de incidência de MP, cobertura vacinal e variáveis socioeconômicas. RESULTADOS: De 2005 a 2013, foram notificados 10.769 casos de MP. Crianças menores de cinco anos foram as mais acometidas. No período pós-vacinação (2011-2013), a taxa de incidência de MP diminuiu nos menores de cinco anos, especialmente nos menores de um ano (de 10,42/100.000, em 2005, para 4,13/100.000, em 2013). No Brasil, maiores taxas de incidência de MP ocorreram nos estados com maior IDH. Em SP ocorreu o mesmo, sendo encontrados, no período pré-vacinação, dois aglomerados de municípios - um de baixo risco para MP, no noroeste do estado (RR, 0,45, p=0,00025), e outro de alto risco no sudeste, englobando a capital do estado, (RR, 1,62, p=0,0000051). No período pós-vacinação, apenas um aglomerado de maior risco se manteve na mesma região (RR, 1,97, p=0,057). Na análise Bayesiana, riqueza foi identificada como fator de risco para MP (RR, 1,026, IC: 1,002-1,052) no período pré-vacinação. Cobertura vacinal, longevidade e escolaridade não foram significativas. CONCLUSÕES: Maior IDH e maior riqueza foram fatores de risco para MP, sugerindo necessidade de maior investimento na capacidade diagnóstica de MP nas áreas estudadas, avanços na qualificação da vigilância e notificação da doença / INTRODUCTION: The 10-Valent pneumococcal conjugate vaccine (PCV10) was introduced into the childhood immunization schedule of the Brazilian National Immunization Program in 2010. This study analyzed Pneumococcal Meningitis (PM) incidence rates in Brazil, by age group and federation unit (FU), the spatial distribution of PM incidence rates in under-5 children in Brazil, by FU, in the pre (2005-2009) and post-vaccination (2011-2013) periods, and associations with socioeconomic variables and vaccination coverage. We conducted spatial analysis of PM incidence rates in under-5 children, by municipality in SP, in pre and post-vaccination periods, and evaluated the existence of spatial and spatial-temporal clusters. Spatial statistics was used to test associations of PM incidence rates with socioeconomic variables and vaccine coverage, by state micro regions. METHODS: This is a population-based ecological study using data from the Sistema de Informação de Agravos de Notificação. Vaccine coverage and the Human Development Index (HDI) were used in the Brazilian analysis. In SP analysis, the ecological units were municipalities and micro regions, and the socio-economic variable was the Índice Paulista de Responsabilidade Social (IPRS) of the Fundação Sistema Estadual de Análise de Dados. Thematic maps were built for PM incidence rates in under-5 children, in the pre- and post-vaccination periods, vaccine coverage and HDI, by FU. Thematic maps were also built for PM incidence rates in under-5 children by SP micro regions, in the pre- and post-vaccination periods, vaccination coverage and IPRS using QGis 2.6.1 software. The scanning technique (SatScan 9.2 software) was used to analyze spatial and spatiotemporal clusters in SP. A Bayesian latent Gaussian model with zero-inflated Poisson model through the integrated nested Laplace approximation (INLA) was used in the spatial analysis to evaluate associations of PM incidence rates with vaccine coverage and socioeconomic variables. RESULTS: From 2005 to 2013, 10,769 PM cases were reported. Under-5 children were the most affected in the whole period. In the post-vaccination period (2011-2013), PM incidence rates decreased among under-5 children, especially among infants (from 10.42/100,000, in 2005, to 4.13/100,000, in 2013). Higher PM incidence rates occurred in states with higher HDI. The same occurred in SP, where two municipalities clusters were found in the pre-vaccination period - one of low risk for PM in the northwest of the state (OR, 0.45, p=0.00025), and another of high risk in the southeast, including the state capital (OR, 1.62, p=0.0000051). In the post-vaccination period, only one cluster of higher risk remained in the same area (RR, 1.97, p=0.057). In Bayesian analysis, wealth was identified as a risk factor for PM (RR, 1.026, CI: 1.002-1.052). Vaccination coverage, longevity and education were not important. CONCLUSIONS: A higher HDI as well as greater wealth were risk factors for PM. This result highlights the need to improve the diagnostic capacity of PM in studied areas, advancing in the surveillance quality and disease notification

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La BPCO se traduit rapidement par l'apparition d'une susceptibilité aux infections liées aux atteintes des mécanismes de défense du poumon. Les travaux antérieurs de l’équipe montrent qu'une altération de la réponse IL-17/IL-22 et de la fonction des cellules dendritiques (DC) participe au développement de l’exacerbation de la BPCO par les bactéries. Les mécanismes responsables de ce défaut de réponse ne sont pas élucidés. Au cours de cette thèse, nous nous sommes intéressés aux points suivants :1-Mécanismes cellulaires responsables du défaut de production d'IL-17 et d'IL-22 au cours de l'infection.Les cellules présentatrices d'antigène (APC) et en particulier, les DC jouent un rôle essentiel dans la réponse antimicrobienne, par leur fonction de phagocytes et par l’activation et la polarisation de cellules immunitaires innées et adaptatives. Sur des modèles murins d’exacerbation de la BPCO par Streptococcus pneumoniae ou Haemophilus influenzae non typable (NTHi), nous avons réalisé des tris de macrophages, DC et monocytes inflammatoires du poumon par cytométrie en flux. Ces analyses montrent que les cellules APC pulmonaires présentent des altérations fonctionnelles aboutissant à une limitation de leur capacité à polariser la réponse Th17 de Lymphocytes T CD4+. Une analyse transcriptomique est également effectuée sur les ARN des APC triées afin de préciser les altérations fonctionnelles de ces cellules par rapport aux souris contrôles.2-Rôle des cytokines IL-20 dans la susceptibilité à l'infection et l'exacerbation de la BPCO Myles et al ont montré en 2013 que les cytokines IL-20 (IL-19, IL-20 et IL-24) jouent un rôle délétère dans la réponse immunitaire cutanée contre Staphylococcus aureus par un mécanisme impliquant une inhibition indirecte d’IL-17 produite par les cellules T. La fonction des DC peut être affecté par l'environnement cytokinique. Comme les cytokines IL-20 sont surexprimées chez les souris BPCO, notre objectif a été de définir leur rôle au cours de l'exacerbation de la BPCO et l'impact de ces cytokines sur les DC dans ce contexte.Dans notre modèle d’exacerbation de la BPCO, nous avons bloqué cette voie en neutralisant l'IL-20RB qui est commune aux 2 récepteurs de ces cytokines afin d’étudier leur impact sur l'exacerbation et sur la réponse immune associée, notamment la réponse IL-17/IL-22. En parallèle, nous avons analysé la modulation de la fonction des DC humaines par ces cytokines dans un contexte d'infection bactérienne. Nos résultats montrent que le traitement avec l'anticorps bloquant anti-IL-20RB permet de bloquer le développement de l'exacerbation de la BPCO en diminuant la charge bactérienne et l'inflammation associée. Cet effet est associé à une diminution importante de la mobilisation des DC dans le poumon mais sans affecter sur la réponse IL-17/IL-22. In vitro, les cytokines IL-20 sont produites par les DC dans un contexte infectieux. De plus, ces cytokines sont capables de diminuer l'activation des ces cellules par les bactéries et de réduire leur capacité à activer les Lymphocytes T dans ce contexte.3-Capacité d'un immunostimulant à restaurer la réponse IL-17/IL-22, et à limiter le développement de l'exacerbation.L’utilisation d’immunostimulant dont la flagelline (agoniste du TLR-5, principale composante du flagelle bactérien) est souvent proposé comment pouvant promouvoir la réponse immunitaire des muqueuses et en particulier la réponse IL-17/IL-22. Nous avons analysé la capacité de cet agoniste du TLR-5 à améliorer la réponse à S. pneumoniae et NTHi dans notre modèle d’exacerbation de la BPCO.Le traitement par la flagelline permet de limiter les conséquences de l'infection bactérienne chez les souris BPCO en diminuant l'inflammation et les lésions pulmonaires associées. L'effet de ce ligand de TLR est au moins en partie dépendant de la production d'IL-22._A terme, ces données permettent d'envisager de nouvelles options thérapeutiques pour le traitement des exacerbations de la BPCO. / Patients with COPD often presented a susceptibility to respiratory infections. Previous works in our lab have showed that a defect of IL-17/IL-22 response and an altered dendritic cell (DC) function is involved in COPD exacerbation with bacteria. However, the mechanism responsible for this defect is not elucidated yet. In order to better define these mechanisms and to develop new therapeutic approaches against COPD exacerbation, we focused on the following points during this PhD project:1-Cellular mechanisms responsible of the defect of IL-17 and IL-22 production during infection in COPD.Antigen presenting cells (APC) particularly, DC are essential in antimicrobial immune response since they are professional phagocytes able to engulf and kill bacteria, but also by their essential role in the polarization of innate and adaptive immune responses. We worked on COPD exacerbation mice model infected with Streptococcus pneumoniae or Nontypeable Haemophilus influenzae (NTHi). APC including macrophages, DC and inflammatory monocytes were sorted by flow cytometry for phenotype and functional analysis. We found an altered function of these lung APC showing limited capacity of Th17 polarization. Transcriptional analysis on total RNA from sorted APC is performed to decipher the mechanisms involved in this functional alteration regarding to control mice.2-The role of IL-20 cytokines in the susceptibility to infection during COPD exacerbation.Myles et al showed in 2013 that IL-20 cytokines (IL-19, IL-20 and IL-24) are deleterious in skin immune response against Staphylococcus aureus. Indeed, these IL-20 cytokines indirectly inhibited IL-17 produced by T cells. The function of DC is also controlled by the presence of cytokines in the microenvironment. Because IL-20 cytokines are overexpressed in COPD, we aimed to determine their role during COPD exacerbation and the impact on DC.We used IL-20RB (common subunits of the 2 receptors) neutralizing antibodies to blocked IL-20 cytokines in COPD exacerbation mice model. We analyzed the impact of this treatment on the immune response, more particularly IL-17/IL-22 response. In addition, we analyzed the modulation of human monocyte derived DC (MDDC) function by IL-20 cytokines in the context of bacterial infection.Our results shows that treatment with IL-20 RB neutralizing antibodies limited COPD exacerbation by reducing the bacterial burden and the associated inflammatory response. This process was associated to reduced number of DC in the lung without impacting IL-17 and IL-22 production. In vitro, MDDC produced IL-20 cytokines upon bacterial infection. Additionally, these cytokines impaired MDDC activation following bacterial infection, which was associated to a reduced capacity of MDDC to activate T lymphocytes.3-The possibility to restore IL-17 and IL-22 response with immuno-stimulants in order to limit the development of COPD exacerbation.Flagellin (a TLR-5 agonist, the main component of bacterial flagellum) is an immuno-stimulant often used to promote mucosal immune response. This activity is related to its ability to promote IL-17 and IL-22 production. In this PhD work, we analyzed the capacity of this TLR-5 agonist to improve the immune response during S. pneumoniae and NTHi infection in COPD exacerbation mice model.Flagellin treatment reduced the bacterial burden and limited the consequences of bacterial infection in COPD mice, by lowering the inflammation and the associated lung remodeling. We also found that the immunomodulatory effect of flagellin was at least partially IL-22 dependent.Finally, these data allow to identify new therapeutic tools potentially useful for the treatment of COPD exacerbations.

Introdução: A Imunodeficiência Comum Variável (ICV) faz parte de um grupo de imunodeficiências primárias na qual os pacientes apresentam defeitos na maturação e diferenciação dos linfócitos B (LB), resultando em distúrbios funcionais além de alterações na distribuição de seus subtipos. Consequentemente, estes pacientes apresentam hipogamaglobulinemia, susceptibilidade a infecções e ausência de produção de anticorpos a antígenos específicos. Na tentativa de reduzir os episódios de infecções recorrentes, alguns trabalhos têm recomendado a vacinação com patógenos mortos ou subunidades e em trabalho anterior demonstramos a eficácia clínica da vacinação de pacientes com ICV, porém, a experiência com a administração de vacinas em imunocomprometidos é limitada. Objetivos: Avaliar a cinética da distribuição das subpopulações de linfócitos B antes e após a vacinação com antígenos proteicos e polissacarídicos em pacientes com ICV acompanhados no Ambulatório de Imunodeficiências Primárias do Hospital das Clínicas, FMUSP, além da produção de anticorpos específicos aos antígenos vacinais. Pacientes e Métodos: Um grupo de 35 pacientes com ICV e 16 controles foram vacinados contra Influenza, H1N1 e S. pneumoniae. Após as coletas nos tempos pré e pós 1, 3 e 6 meses foram realizados a separação de PBMC e cultura de linfócitos com lisado viral e hemaglutinina de Influenza, além da citometria de fluxo para identificação das subpopulações de LB naive, zona marginal (MZB), memória com troca de isotipo (SMB) e plasmoblastos (PBL). Foram dosados os anticorpos específicos e no grupo dos pacientes foi aplicado um score de sintomas antes e após a imunização. Resultados: Apesar da redução significativa na pontuação do score de sintomas, a maioria dos pacientes não produziu anticorpos específicos para Influenza, H1N1 e S. pneumoniae. A análise da cinética das subpopulações de LB revelou que em indivíduos saudáveis, a resposta contra Influenza apresentou duração de 6 meses, observada por meio da redução da subpopulação naive e aumento gradual da frequência de SMB a partir do primeiro mês. Observamos também redução da população de memória por volta do 3º mês, com aumento da população de PBL que permaneceu elevada até o 6º mês. Por outro lado, a despeito de os pacientes apresentarem aumento de SMB no primeiro mês após a vacinação, sua frequência foi inferior ao observado nos controles, decaindo ao terceiro mês. A população de PBL apresentou aumento precoce no primeiro mês após a vacinação, também muito menor do que observado nos controles, não sendo mantido no terceiro mês. Ainda, observamos uma correlação entre o aumento da expressão destas duas subpopulações no primeiro mês. Apenas a população de MZB apresentou aumento significativo no terceiro mês nos pacientes quando comparados aos controles. Ao dividirmos os pacientes de acordo com a expressão de SMB e PBL após 1 mês da administração das vacinas, observamos que os pacientes que apresentaram aumento na expressão de células B de memória foram os que exibiram uma melhora clínica mais expressiva, soroconverteram e desenvolveram soroproteção para H1N1.Conclusões: Apesar de não apresentarem eficaz diferenciação em células de memória e efetoras, resultando na resposta precoce e de curta duração, observamos que os pacientes foram capazes de reconhecer e responder às vacinas. Além disso, a elevada expressão de MZB no terceiro mês após a vacinação pode sugerir a atuação desta subpopulação na apresentação para os LT. Estes achados reforçam a necessidade de uma melhor compreensão da ativação do sistema imune em pacientes com ICV, para uma adequada subdivisão de acordo com o perfil de resposta após a vacinação / Introduction: Common Variable Immunodeficiency (CVID) is a primary antibody deficiency characterized by defects in B lymphocyte maturation, resulting in disturbed differentiation, distribution and functional variations on its subtypes. As a result , CVID patients have hypogammaglobulinemia and poor antibody response to specific antigens with increased susceptibility to infections. In an effort to minimize the recurrent episodes of infections, some studies have recommended immunization with inactivated pathogens or subunits and in a former study we have shown the clinical improvement determined by immunization in CVID patients, but the experience with vaccines\' administration to immunodeficient patients is limited. Objectives: To evaluate the changes in distribution of B cell subtypes before and after vaccination of CVID patients followed at the Division of Clinical Immunology and Allergy of University of São Paulo Medical School with protein and polysaccharide antigens, as well as specific antibody production . Methods: A group of 35 CVID patients and 16 controls were vaccinated against Influenza, H1N1 and S. pneumoniae vaccines. Blood samples were collected before and 1, 3 and 6 months post vaccination. PBMCs were stimulated with Influenza viral lysate and hemagglutinin peptide. Flow cytometry was performed to identify naïve B cells, marginal zone (MZB), switched memory B cells (SMB) and plasmablasts (PBL). Specific antibody production was measured and a symptoms score was applied for clinical evaluation before and after immunization. Results: In spite of the significant reduction in symptoms score after vaccination, most patients didn\'t produce specific antibodies to Influenza, H1N1 and S. pneumoniae. The analyzes of B cell subtypes changes in healthy individuals upon in vitro Influenza stimulation showed that the response endured up to 6 months post immunization. We observed a reduction in naïve B cell frequency while gradual increase in SMB frequency occurred already at 1 month after vaccination. Moreover, as the memory cell population declined, PBL population increased at the third month post vaccination until the sixth month. Although patients had an increase of SMB on the first month after vaccination, it was lower than that observed in controls, decreasing by the third month post vaccination. Plasmablast frequency had an early increase on the first month, also much lower than the observed in controls decreasing by the third month. In addition, we observed a correlation between the increased expression of SMB and PBL on the first month post vaccination. In patients, only MZB subtype presented a significant increase on the third month when compared to controls. We divided the patients according SMB and PBL expression after 1 month post vaccination and we observed that patients who were able to produce memory B cells showed a better clinical improvement, developed H1N1 seroconversion and seroprotection. Conclusion: Despite the defect on differentiation into memory and effector B cells resulting in early response with lowduration, we observed that patients were able to recognize and respond to vaccines. In addition, the over expression of MZB on the third month after vaccination may suggest the role of this subpopulation as an antigen presenting cell for T cells. These findings reinforce the need of a better understanding of immune system activation and response in CVID patients to propose a division according to vaccine (antigen) responders and non responders

Streptococcus pneumoniae (ou pneumocoque) est un agent pathogène humain responsable de maladies invasives et potentiellement mortelles. Les mécanismes impliqués dans le processus d'invasion restent largement inconnus, mais plusieurs observations suggèrent que les processus de signalisation impliquant la phosphorylation des protéines participeraient au caractère invasif du pneumocoque. Le génome de S. pneumoniae code pour une seule tyrosine-kinase (CpsD) et une seule sérine/thréonine-kinase (StkP). Cette dernière serait notamment impliquée dans la virulence, la compétence et la division cellulaire. Elle représente donc une cible thérapeutique potentielle intéressante pour lutter contre les infections liées au pneumocoque. L'objectif de cette thèse a donc été de caractériser le rôle de cette sérine/thréonine-kinase StkP dans la division cellulaire du pneumocoque. StkP est une protéine transmembranaire qui se caractérise par la présence de motifs structuraux conservés dans son domaine catalytique appelés motifs de Hanks. De plus, StkP possède un domaine extracellulaire composé de la répétition de quatre domaines PASTA (Penicillin-binding protein And Serine/Threonine kinase Associated). Le modèle actuel suggère que ces domaines PASTA seraient capables de fixer des fragments de la paroi cellulaire afin de permettre l'activation de StkP qui se comporterait donc comme un récepteur membranaire permettant de réguler la division cellulaire du pneumocoque. Mes travaux de thèse ont permis de revisiter ce modèle en démontrant que les domaines PASTA ne servent pas uniquement à contrôler l'activité protéine-kinase de StkP mais également à contrôler l'épaisseur de la paroi cellulaire et ainsi permettre la constriction de la cellule. Plus précisément, j'ai démontré que le domaine PASTA distal est spécialisé dans l'interaction avec une hydrolase de la paroi cellulaire alors que les trois autres domaines PASTA sont nécessaires à l'activité kinase de StkP mais également au positionnement du domaine PASTA distal. Ainsi, le domaine extracellulaire de StkP se comporterait comme une règle permettant de définir l'épaisseur de la paroi cellulaire de la bactérie. Ces travaux permettent donc de proposer un nouveau modèle d'activation et de régulation de la division cellulaire par la sérine/thréonine-kinase StkP / Streptococcus pneumoniae (the pneumococcus) is one of the most important human pathogens that causes potentially fatal invasive diseases. Mechanisms required for the pneumococcal invasion process remain largely unknown, but several observations suggest that phosphorylation-based signaling processes will be at play in the invasiveness of the pneumococcus. S. pneumoniae encodes only one tyrosine-kinase (CpsD) and one serine/threonine-kinase (StkP). The latter would be involved in virulence, competence, and cell division. StkP represent therefore a promising target to combat pneumococcal infections. My aims were to better understand the role of StkP in pneumococcal cell division. StkP is a transmembrane protein characterized by the presence of a series of conserved structural motifs called Hanks motifs in its catalytic domain. In addition, StkP possesses an extracellular domain composed of the repetition of four PASTA domains (Penicillin-binding protein And Serine/Threonine kinase Associated). The current model proposes that PASTA domains are able to bind cell wall fragments resulting in StkP kinase activation. StkP would thus behave as an authentic kinase receptor regulating cell division. My thesis works has allowed to revisit this model by showing that PASTA domains do not only serve StkP kinase activation. Rather, they contribute to determine the cell wall thickness and govern cell constriction. More precisely, I demonstrated that the distal PASTA domain possesses unique features for the binding of a cell wall hydrolase whereas the other three contributes to StkP kinase activation and the positioning of the distal PASTA domain. Thus, the extracellular domain of StkP acts as a ruler determining the cell wall thickness. This work allows to propose an alternative model of activation and regulation of cell division by the serine/threonine-kinase StkP

Malgré les modèles établis pour certaines bactéries, la morphogenèse de bactéries de formes atypiques est peu comprise. C'est le cas de la bactérie pathogène pour l'homme Streptococcus pneumoniae, ou pneumocoque, qui possède une forme ovo-diplococcale. Cependant, à mon arrivé au laboratoire, il avait été démontré qu'une sérine/thréonine protéine-kinase membranaire appelée StkP était indispensable à la division cellulaire et à la morphogenèse du pneumocoque. L'objectif de ma thèse a ainsi été de caractériser certains substrats de StkP et d'étudier leur rôle, ainsi que l'impact de leur phosphorylation, au cours du processus de division cellulaire. Dans ce contexte, j'ai montré que le substrat DivIVA et son paralogue GpsB coordonnent l'élongation et la division cellulaire du pneumocoque. Ces travaux permettent de proposer un nouveau modèle de morphogenèse du pneumocoque dans lequel la triade StkP/DivIVA/GpsB organise la synthèse de la paroi cellulaire nécessaire à l'élongation et à la division de la cellule. J'ai également mis en évidence que la protéine MapZ interagit avec la paroi cellulaire lors de l'élongation cellulaire afin de marquer de manière permanente le site de division, où elle recrute la protéine FtsZ. Ces travaux ont ainsi permis d'identifier un système inédit de régulation positive du positionnement du site de division chez les bactéries. Enfin, j'ai caractérisé les déterminants moléculaires du positionnement de MapZ au centre de la cellule. S. pneumoniae étant un pathogène humain important, nous pouvons anticiper que nos données pourraient servir de base fondamentale à des projets plus appliqués de lutte contre les infections bactériennes / Despite the established models for some bacteria, the morphogenesis of bacteria with atypical shapes is poorly understood. This is the case of the human pathogen Streptococcus pneumoniae, or pneumococcus, that displays an ovo-diplococcal shape. However, when I joined the lab, it had just been shown that a membrane serine/threonine kinase named StkP was crucial for the cell division and the morphogenesis of the pneumococcus. The goal of my thesis was to characterize the substrates of StkP and to study their function as well as the impact of their phosphorylation in the cell division process. First, I have shown that the substrate DivIVA together with its paralog GpsB coordinate cell elongation and division of the pneumococcus. Based on these observations, we propose a new model of pneumococcal morphogenesis in which the triad StkP/DivIVA/GpsB organizes cell wall synthesis involved in cell elongation and division. In a second part of my work, I have studied another substrate of StkP that was of unknown function and that we named MapZ. I have shown that MapZ interacts with the cell wall during the cell elongation to position at midcell. Then MapZ recruits the cell division protein FtsZ and controls the closure of the Z-ring. This work has uncovered a new mechanism of positive regulation for the positioning of the division site in bacteria. Finally, I characterized the molecular determinants of MapZ positioning at the division site. S. pneumoniae is an important human pathogen, we can thus anticipate that our work will represent a fundamental base for applied projects in order to develop new strategies against bacterial infections

Les bactéries possèdent une famille particulière de tyrosine-autokinases, les BY-kinases. Ces enzymes sont impliquées dans la régulation de plusieurs processus cellulaires dont la synthèse et l'export des polysaccharides extracellulaires qui jouent un rôle crucial dans la virulence de certaines bactéries pathogènes. Cependant, les mécanismes de régulation sous-jacents sont inconnus. L'objectif de ma thèse a été de caractériser d'un point de vue structural et fonctionnel le rôle des BY-kinases. Pour cela, j'ai réalisé trois études indépendantes dans trois modèles bactériens différents. Chez Escherichia coli, j'ai identifié et étudié la surface d'interaction entre la BY-kinase Wzc et sa phosphatase associée Wzb afin de comprendre comment Wzb déphosphoryle Wzc. Chez Staphylococcus aureus, j'ai participé à la caractérisation structurale de la pseudo-BY-kinase CapB1. Par comparaison avec la structure de son homologue actif CapB2, mes études ont permis de suggérer l'existence d'un mécanisme de régulation de la synthèse des polysaccharides extracellulaires impliquant CapB2 et CapB1. Enfin, chez Streptococcus pneumoniae, j'ai mis en évidence que si l'autophosphorylation de la BY-kinase CpsD était indispensable à la synthèse de la capsule polysaccharidique, elle était également indispensable à la division de la cellule. Ainsi, mes travaux ont permis de proposer l'existence d'un mécanisme de coordination de la production de la capsule et du cycle cellulaire du pneumocoque. D'un point de vue appliqué, l'ensemble de mes travaux représente une étape préalable et prometteuse au développement de nouvelles molécules, ciblant les BY-kinases de manière spécifique, afin de lutter contre la virulence de certaines bactéries pathogènes / Bacteria possess a particular family of tyrosine-autokinases named BY-kinases. These enzymes are involved in the regulation of numerous cellular functions including the synthesis and export of extracellular polysaccharides that play a critical role in the virulence of different bacterial pathogens. However, the mechanisms of these processes remain unknown. The aim of my thesis was to characterize the role of these BY-kinases by structural and functional approaches. For that, I have realized three independent studies in three bacterial models. In Escherichia coli, I have characterized the interaction surface between the BY-kinase Wzc and its cognate phosphatase Wzb to understand how Wzb dephosphorylates Wzc. In Staphylococcus aureus, I have studied structurally the pseudo-BY-kinase CapB1. By comparison with the structure of its active homologue CapB2, my studies have allowed to suggest the existence of a mechanism controlling capsular polysaccharides synthesis involving both CapB1 and CapB2. Last, in Streptococcus pneumoniae, I have showed that while the autophosphorylation of the BY-kinase CpsD is necessary for proper synthesis of capsular polysaccharides, it is also involved in cell division. Thus, my work shows that a mechanism coordinating capsule production and cellular cycle exists in the pneumococcus. These works constitute a preliminary and promising step toward the development of new molecules, targeting specifically BY-kinases and aim to combat the virulence of bacterial pathogens