Introdução: A vacina tríplice acelular de adultos (dTpa) foi introduzida no Programa Nacional de Imunizações (PNI) em novembro de 2014, sendo recomendada para gestantes e profissionais de saúde (PS) que têm contato com gestantes e recém-nascidos. De abril a dezembro de 2015, foram implementadas várias estratégias para aumentar a cobertura vacinal entre os profissionais do Instituto Central do Hospital das Clínicas da FMUSP. Objetivos: Avaliar a cobertura vacinal entre os PS após implementação de cada estratégia e ao término de um ano; avaliar as variáveis associadas à vacinação; e avaliar os principais motivos de não vacinação entre os PS com indicação para tal. Métodos: Estratégias implementadas: divulgação, no boletim do hospital, de texto relembrando da necessidade de vacinação de coqueluche; reforço da necessidade da vacinação, via correio eletrônico, para as chefias de enfermagem das Divisões de Clínica Obstétrica, Neonatologia e Anestesia; aulas sobre a vacina dTpa nas reuniões científicas das Divisões de Clínica Obstétrica e Neonatologia; e vacinação ativa dos profissionais na Divisão de Clínica Obstétrica, Neonatologia e Anestesia. A cobertura vacinal foi avaliada ao fim de cada mês até abril de 2016, por meio do sistema informatizado de vacinação usado no CRIE-HC. Foi usado o modelo de regressão de Poisson com variância robusta para avaliação das variáveis associadas com a vacinação com dTpa. As razões de prevalência foram calculadas e seus intervalos de confiança de 95% estimados. Para avaliar os motivos de não vacinação, foram realizadas ligações telefônicas para os profissionais que não receberam a vacina e aplicado questionário padronizado. Resultados: Entre os 515 PS elegíveis para vacinação, 59 não possuíam registro no sistema informatizado de vacinação e foram excluídos. Assim, este estudo incluiu 456 PS. Após as intervenções, a cobertura vacinal com dTpa aumentou de 2,9% para 41,2%. As coberturas vacinais após a implementação de cada estratégia foram: 3,7% após publicação no Boletim do hospital; 10,5% após mensagem de correio eletrônico para as chefias de enfermagem; 16,2% após aula sobre a vacina em reuniões científicas das Divisões de Clínica Obstétrica e Neonatologia; 27,9% após vacinação ativa na Divisão de Clínica Obstétrica; 40,6% após vacinação ativa na Divisão de Neonatologia e 41,2% após vacinação ativa na Divisão de Anestesia. Na análise multivariada, ser médico (a), trabalhar nas Divisões de Clínica Obstétrica ou Anestesia e ter recebido a vacina de influenza de 2015 foram associados à vacinação com dTpa. Foi feito contato telefônico com 39 profissionais que não receberam a vacina em nosso serviço; apenas 9 (23%) referiram ter recebido a vacina em outros serviços; e dos 30 não vacinados, 27 (90%) alegaram desconhecimento da recomendação. Conclusões: Conhecimento sobre a doença e a recomendação de vacinação são importantes para aumentar a cobertura vacinal entre PS. Porém, mesmo sabendo do efeito cumulativo na cobertura vacinal a cada estratégia realizada, a vacinação ativa dos PS em seus locais de trabalho parece ter sido a estratégia que mais contribuiu para o aumento da cobertura. A cobertura vacinal final de dTpa permanece baixa e maiores esforços são necessários para aumentá-la / Introduction: The acellular pertussis vaccine for adults (Tdap) was introduced in the Brazilian National Immunization Program (PNI) in November 2014, being recommended for pregnant women and healthcare workers (HCWs) who have contact with pregnant women and newborns. From April to December 2015, interventions to raise Tdap coverage among HCWs of the Instituto Central do Hospital das Clínicas were implemented. Objective: To evaluate the cumulative vaccine coverage after each intervention; identify factors associated to Tdap vaccination among HCWs; and evaluate the main reasons for HCWs not receiving Tdap. Methods: Interventions implemented: a note on the hospital\'s internal newsletter, reminding HCWs of the importance of pertussis vaccination; email to the nurse´s teams leaders strengthening vaccine recommendations; lectures on pertussis and Tdap for physicians at the clinical meetings of the Obstetrics and Neonatology Clinics; on-site vaccination by mobile teams at the Obstetrics, Neonatology, and Anesthesiology Clinics. The vaccine coverage was evaluated at the end of each month until April-2016. A multivariate Poisson regression model with robust error variance was used to evaluate variables associated with Tdap vaccination. Prevalence ratios (PR) and their 95%CI were estimated. To evaluate the reasons for HCWs not to be vaccinated, those who have not received Tdap were called by phone and a standard questionnaire was applied. Results: Among 515 HCWs eligible for immunization, 59 professionals were not registered in the vaccination data system and were excluded because information about Tdap vaccine could not be achieved. The study included 456 HCWs. After the interventions, Tdap coverage raised from 2.9% to 41.2%. The vaccine coverage after each intervention was: 3.7% after a note on the hospital\'s internal newsletter; 10.5% after email to the nurse´s teams leaders strengthening vaccine recommendations; 16.2% after lectures on pertussis and Tdap for physicians at the clinical meetings of the Obstetrics and Neonatology Clinics; 27.9% after on-site vaccination by mobile teams at the Obstetrics Clinic; 40.6% after on-site vaccination at the Neonatology Clinic and 41.2% after on-site vaccination at the Anesthesiology Clinic. In the multiple analysis, occupation, working place and having received influenza vaccination in 2015 were independently associated to Tdap vaccination. Thirty-nine HCWs that have not received Tdap were contacted by phone: 90% of them claimed they did not know the vaccine recommendation. Conclusions: Knowledge about pertussis and the recommendation of vaccination are important to raise vaccine coverage between HCWs. Even knowing the cumulative effect of each strategy on vaccine coverage, HCWs vaccination in their workplaces seems to be the most effective strategy in raising coverage. The final Tdap coverage remains low and greater efforts are needed to increase it

Atualmente, a coqueluche representa um crescente problema de saúde pública em países desenvolvidos. Embora ainda não existam evidências de um aumento do número de casos de coqueluche no nosso país, não se pode descartar a hipótese de uma futura re-emergência da doença, pois dados epidemiológicos de algumas regiões revelam um aumento da incidência, indicando que provavelmente ocorra uma baixa notificação de novos casos ao Ministério da Saúde. A maioria dos casos ainda ocorre em lactentes menores de seis meses de idade, ou seja, crianças ainda não completamente imunizadas. Diversos trabalhos demonstraram a aquisição de anticorpos IgG reativos com Bordetella pertussis pelo recém-nascido através da passagem transplacentária, mas a partir dos dois meses de vida, observa-se um declínio substancial do título destes anticorpos. Neste caso, outro modo de conferir proteção ao neonato é através da transmissão de anticorpos IgA específicos pelo aleitamento materno, que poderia suprir a falta de anticorpos IgG até que o esquema de vacinação esteja completo. Os objetivos deste trabalho foram analisar a transferência passiva de anticorpos IgG e IgA anti-B. pertussis para o recémnascido a termo e investigar a habilidade destes anticorpos em neutralizar a patogenicidade bacteriana em um modelo experimental in vivo utilizando camundongos desafiados por via intracerebral com B. pertussis viável. Foram coletadas 40 amostras pareadas de sangue materno, de cordão umbilical e de colostro. Foram demonstrados títulos equivalentes de anticorpos IgG anti-B. pertussis entre as amostras de soro materno e de cordão (medianas de 1:225 e 1:265, respectivamente) com taxa de transferência de 118%. Foram observados títulos variáveis de anticorpos IgA específicos nas amostras de colostro materno com mediana de 1:74. O Immunoblotting realizado com extrato bruto de B. pertussis e Pools de soro materno, de soro de cordão e de colostro com alto e baixo título de anticorpos específicos revelou um perfil de reconhecimento idêntico entre os Pools de soro materno e dos respectivos neonatos. Os Pools de colostro apresentaram, em seu perfil de reconhecimento, diferentes intensidades que variaram de acordo com os títulos de anticorpos IgA específicos. No desafio intracerebral com B. pertussis, embora todos os Pools de soro materno, de cordão e de colostro tenham apresentado capacidade significativa de neutralizar a patogenia bacteriana quando comparados ao controle positivo, os Pools com alto título de anticorpos revelaram maior capacidade neutralizante. Os Pools de soro e colostro absorvidos com B. pertussis e, portanto, sem anticorpos IgG e IgA específicos, protegeram 30% dos animais testados e anticorpos IgG purificados, apresentando alto título de anticorpos anti-B. pertussis (1:2.560), protegeram 65% dos camundongos. Nossos dados confirmaram a transferência de anticorpos reativos com B. pertussis para o neonato via placenta e aleitamento materno e sua eficácia na neutralização da patogênese bacteriana, o que pode proteger a criança contra infecções respiratórias causadas por Bordetella pertussis. / Pertussis is currently considered an important public health problem in developed countries. Although there is no evidence of an increase in the number of pertussis cases in our country, can not rule out the hypothesis of a future re-emergence of disease, as epidemiological data from some regions show an increase in incidence, indicating that probably there is a low report of new cases to the public health authorities. Most cases still occurs in infants under six months of age, i.e. children not fully immunized. Several works have demonstrated the acquisition of IgG antibodies reactive with Bordetella pertussis by the newborn through placental transfer, but by age of two months it was observed a substantial decay of titers these antibodies. In this case, another way to confer protection the neonate is through the transmission of IgA antibodies via breast-feeding, which could supply the lack of IgG antibodies until the vaccination schedule will be completed. The aims of this work were to analyze the passive transfer of IgG and IgA anti-B. pertussis antibodies to term newborns and to investigate the ability of these antibodies to neutralize the bacterial pathogenicity in an experimental model in vivo using mice intracerebrally challenged with viable B. pertussis. It was collected 40 paired samples of maternal blood, cord umbilical blood and colostrum. Equivalent titers of anti-pertussis IgG antibodies were demonstrated between maternal and cord serum samples (medians of 1:225 and 1:265, respectively) with transfer rate of 118%. It was observed variable specific IgA titers in maternal colostra with a median of 1:74. Immunoblotting performed with B. pertussis crude extract and Pools of maternal serum, cord serum and colostrum with high and low specific antibody titers revealed an identical recognition profile between paired maternal and newborn serum Pools. Colostrum Pools presented, in their recognition profile, different intensities that varied according to specific IgA antibody titers. In the intracerebral challenge with B. pertussis, although all maternal and cord serum and colostrum Pools presented a significant bacterial neutralizing ability when compared with positive control group, Pools with high antibody titers revealed higher neutralizing capacity. Serum and colostrum Pools absorbed with B. pertussis and, thus, without specific IgG and IgA antibodies, protected 30% of the animals tested and purified IgG antibodies, presenting a high anti-pertussis antibody titer (1:2,560), protected 65% of the mice. Our data confirmed the transfer of antibodies reactive with B. pertussis to the neonate via placenta and breast-feeding and their effectiveness in bacterial pathogenesis neutralization, which could protect infants against respiratory infections caused by Bordetella pertussis.

La toxine pertussique (PTX) est une exotoxine produite uniquement par Bordetella pertussis, un pathogène de la coqueluche. Les effets de la toxine au cours d'une infection bactérienne sont bien connus, et sont pour la plupart liés à son activité ADP-ribosyltransférase qui cible les GPCRs. Or, la PTX est un antigène majeur permettant d’établir une réponse immunitaire contre B. pertussis ce qui en fait donc un composant principal de tous les vaccins anti-coqueluche actuels. De nombreux travaux sur la PTX concernent ses mécanismes moléculaires et son rôle durant la phase d'infection. Mais, il y a un manque d'information sur le rôle immunogène de la PTX.En utilisant un modèle d'infection intranasale par B. pertussis, nous avons constaté que la génération de lymphocytes T CD4 mémoires résidant (Trm) dans les poumons dépendait de l'exposition à la PTX. La toxine pertussique est couramment utilisée pour inhiber la réponse aux chimiokines, dans l'étude de la migration des cellules T. Etant donné que la plupart des récepteurs aux chimiokines sont des GPCRs, la mobilité de nombreuses cellules immunitaires, y compris les cellules T, est facilement affectée par la PTX. La migration des cellules T est un phénomène sophistiqué régulé spatio-temporellement. Nos résultats démontrent que la PTX n’affecte pas les étapes de la migration dépendantes des intégrines lorsque les cellules T sont activées.Ce travail s’intéresse à l'impact de la PTX sur la biologie des cellules T en étudiant son rôle dans la réponse immunitaire adaptative in vivo, dans un modèle animal d'infection et son impact sur la migration des lymphocytes T in vitro. / Pertussis toxin (PTX) is an exotoxin uniquely produced from Bordetella pertussis, a human respiratory tract pathogen causing pertussis disease, also known as whooping cough. The toxin is well described its virulence effects during bacterial infection. Most of these effects are due to ADP-ribosyltransferase activity of the molecule that targets G-protein coupled receptors (GPCR). On the other hand, PTX is an important antigen that provides protection against pertussis disease and a major component of all current pertussis vaccines. There are numerous literatures on PTX about its molecular mechanisms and its role during infection phase. Instead, lack of information on how PTX contributes host’s adaptive immunity has incurred confusion in understanding the immunogenic role of PTX. With intranasal infection model of B. pertussis, we detected the generation of CD4 lung-resident memory T cells (Trm) were depending on PTX exposure. For T cell migration study, PTX is being used to inhibit chemokine response. Because most of chemokine receptors are GPCR, the motility of many immune cells including T cells is easily affected by PTX. T cell migration is a sophisticate phenomenon regulated space-temporally. The results demonstrated, once T cells become activated and effector, are less influenced than inactivated T cells.This thesis reports the impact of PTX on T cells in two parts; 1) Role of PTX in adaptive immune response by in vivo infection system and 2) Influence of PTX on T cell motility by in vitro assays.

A coqueluche vem reemergindo enquanto importante problema de saúde pública em vários países do mundo, apesar das altas coberturas vacinais na infância. O objetivo geral deste estudo foi avaliar a morbimortalidade da coqueluche no Brasil e os objetivos específicos foram: estimar as taxas de mortalidade, incidência e letalidade anuais, geral e por faixa etária, por unidade da federação e regiões do país; caracterizar a sazonalidade da doença; estimar as taxas de hospitalização anuais por faixa etária e verificar as características clínicas, histórico de contato e vacinação prévia dos casos notificados da doença. Métodos: estudo descritivo, baseado nos casos de coqueluche notificados ao Sistema de Informação de Agravos de Notificação (SINAN), de 2006 a 2013. Os resultados mostraram aumento nas taxas de incidência de coqueluche no Brasil, a partir de 2011. Em 2013, foram confirmados 6.523 casos de coqueluche no país, três vezes o número de casos confirmados em 2011, com incidência geral de 3,24 /100.000 habitantes e incidência em menores de um ano de 125,82/100.000 habitantes, as maiores durante o período estudado. As crianças menores de um ano foram as mais acometidas pela doença em todas as macrorregiões. Em 2013, todas as regiões, exceto a região sul, apresentaram suas maiores taxas de incidência geral, com destaque para as regiões sudeste e centro-oeste com 4,0 e 3,1 por 100.000 habitantes, respectivamente. As maiores taxas de letalidade foram observadas na faixa etária menor de dois meses de idade, variando de 4,0% (2008) a 9,5% (2010). As taxas de letalidade foram maiores em crianças menores de seis meses em todas as regiões, sendo as regiões nordeste e sudeste as que apresentaram maiores taxas ao longo dos anos, exceto em 2013, quando o centro-oeste superou o nordeste. Houve predomínio dos casos nos meses mais quentes, entre novembro e março. A maioria das hospitalizações ocorreu na faixa etária de menores de um ano, principalmente em menores de quatro meses, cuja frequência de hospitalização ficou em torno de 75%. A tosse e o paroxismo foram os sintomas mais frequentes, independente da faixa etária, e a cianose foi importante sintoma nos menores de dois meses, com uma frequência de 80% nos casos confirmados desta faixa etária. A complicação mais comum foi pneumonia (13,93%), principalmente na faixa etária menor de dois meses, com frequência de 27,5%. O critério mais utilizado para diagnóstico de coqueluche foi o clínico, seguido pelo laboratorial que aumentou a partir de 2011, ano em que foi responsável por 49,9% dos diagnósticos. A maioria dos casos confirmados (51%) não relatou contato prévio com casos suspeitos ou confirmados de coqueluche, no entanto quando presente, a maioria dos contatos ocorreu no domicílio (70,6%). Os resultados mostraram aumento dos casos de coqueluche no Brasil, a partir de 2011, com as maiores taxas de incidência, hospitalizações, complicações e letalidade na faixa etária de menores de um ano / Pertussis has reemerged as important public health problem in many countries, despite the high childhood vaccination coverage. The general aim of this study was to evaluate the morbimortality of pertussis in Brazil, and the specific objectives were: estimate the annual mortality, incidence and case-fatality rates, general and by age group, by federative units and country\'s regions; evaluate the disease seasonality; estimate the annual hospitalization rates by age group and verify the clinical characteristics, contact history and the previous vaccination status of the reported pertussis cases. Methods: Descriptive study, based on the pertussis cases reported to the Notifiable Diseases Information System (SINAN), from 2006 to 2013. In 2013, there were 6.523 confirmed pertussis cases in the country, three times the number of confirmed cases in 2011, with general incidence of 3.24/100,000 inhabitants, and incidence in children under one year of age of 125.82/100,000 inhabitants, the highest during the study period. Pertussis incidence rates were higher in children under one year old in all macroregions during the study. In 2013, higher general incidence rates were observed in all regions, except the south, particularly the southwest and Midwest with 4.0 and 3.1 per 100,000 inhabitants, respectively. The highest case-fatality rates were observed in infants under two months of age, varying from 4.0% (2008) to 9.5% (2010). Case-fatality rates were higher in children under six months in all regions; the northeast and southeast had the highest rates throughout the studied years, except in 2013, when the Midwest surpassed the northeast. More cases were reported in the warmer months, between November and March. Most hospitalizations occurred in the age group of children under one year old, mainly those under four months, for whom hospitalization rates were close to 75%. Cough and paroxysm were the most frequently symptoms, regardless of age, and cyanosis was important in children under two months, occurring in 80% of confirmed cases in this age group. The most common complication was pneumonia (13.93%), mainly in children under two months of age (27.5%). Clinical criteria were most frequent used for diagnosis, followed by laboratory, which increased since 2011, when 49.9% of cases had laboratory-confirmed diagnosis. Most confirmed cases (51%) had no recognized previous contact with pertussis cases. Among those with recognized previous contact, it mostly occurred at residence (70.6%).The results showed an increase in pertussis cases in Brazil, since 2011, with the highest incidence and lethality rates in children under one year of age

Malgré l’existence de différents vaccins protecteurs à l’égard des infections par B. pertussis, la coqueluche reste une maladie infectieuse fréquente et est encore actuellement responsable de 300.000 décès par an dans le monde. Une meilleure compréhension des réponses immunitaires induites par les principaux facteurs de virulence de B. pertussis est donc importante afin d’optimaliser la vaccination. Alors que le rôle des anticorps dans la protection contre la coqueluche est reconnu depuis de nombreuses années, celui des réponses d’immunité cellulaire a été identifié mais est moins bien caractérisé. <p>Dans ce travail, nous avons analysé différents aspects des réponses d’immunité cellulaire induites par deux antigènes majeurs de B. pertussis, l’hémagglutinine filamenteuse (FHA) et la toxine pertussique (PT) chez des nourrissons. Nous avons étudié la persistance des réponses spécifiques des antigènes de B. pertussis après la primo vaccination des nourrissons et caractérisé différents facteurs qui modulent ces réponses, nous permettant ainsi de comprendre l’hétérogénéité des réponses d’immunité cellulaire observées tant qualitativement que quantitativement. <p>Nous avons montré que les réponses immunitaires spécifiques de la FHA et de la PT pouvaient persister jusqu’à neuf mois après la dernière administration du vaccin contre la coqueluche. Ces réponses immunitaires sont caractérisées par une production d’interféron-gamma (IFN-&61543;) par les cellules sanguines circulantes (PBMC), par une prolifération lymphocytaire ainsi que par une production d’anticorps. Bien que les lymphocytes T CD8+ participent à la sécrétion spécifique d’IFN-&61543; leur participation semble quantitativement moins importante que celle des lymphocytes T CD4+ et dépendante de ces derniers. Cependant, les PBMC de certains enfants vaccinés ne produisent pas ou peu d’IFN-&61472;&61543; après stimulation in vitro par les antigènes de B. pertussis. Cette inhibition, voire absence, de réponse immunitaire de type Th1 spécifique est associée à une sécrétion constitutive d’IL-10 par les monocytes ou à la présence de lymphocytes T CD8+. Enfin, étant donné que la FHA est inductrice d’IL-10 dans un modèle in vitro murin, nous avons analysé son effet sur la production in vitro de cette cytokine par les cellules dendritiques humaines (DCs). Nous avons montré que la FHA induit bien une production d’IL-10 par les DCs mais également les productions d’IL-12p70, d’IL-6 et d’IL-23, cytokines impliquées dans la différenciation des lymphocytes T naïfs en lymphocytes T effecteurs Th1 ou Th17. Parallèlement, nous avons testé une forme tronquée de FHA, la FHA 44 qui est également protectrice dans un modèle murin d’infection par B. pertussis. Nous avons montré que cette FHA tronquée n’induit pas de production d’IL-10 tout en induisant une production d’IL-12p70 et d’IL-23 par les DCs. La forme moléculaire de FHA présente dans les vaccins joue donc un rôle déterminant sur le type de réponses immunitaires induites et le remplacement de la FHA native dans les vaccins acellulaires par la FHA 44 devrait potentiellement permettre une meilleure réponse IFN-& / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

O habitat natural da Neisseria meningitis é a nasofaringe humana e a transmissão da bactéria é por contato direto ou por inalação de partículas durante a fase de transmissão N. meningitis é uma bactéria Gram-negativa responsável por uma significante mortalidade em todo o mundo. Embora existam vacinas polissacárides contras os sorogrupos A, C, W135 e Y , não há uma vacina adequada para crianças menores de 4 anos para o sorogrupo B. Estudos estão sendo direcionadas para pesquisa de antígenos vacinais que são derivados da proteínas de membrana externa(NOMV). Entretanto vacinas baseadas em NOMV são consideradas pouco imunogênicas , fazendo com que o uso de adjuvantes seja necessário. Este estudo investiga a imunogenicidade da NOMV de N. meningitidis administrada pela via intranasal/intramuscular em camundongos neonatos BALC/c, usando proteína de membrana externa (NOMC) obtido de uma cepa epidêmica de N. meningitidis B:4:P1:15. As cepas usadas para imunização dos camundongos foram selecionadas por colony-blot, usando anticorpo monoclonal anti L3,7,9 para maior expressão do LPS contra o imunotipo L3,7,9 presente na cepa (B:4:P1:15 3,7,9). Como adjuvantes de mucosa foram utilizados Bordetella pertussis (células íntegras) ou sobrenadante de cultura com 48 horas ou hidróxido de alumínio [Al(OH)3]. O soro dos camundongos imunizados foram analisados pelo método de ELISA à fim de se comparar os diferentes adjuvantes utilisados. O índice de avidez também foi determinado. IgG e IgM foram detectados nos soros dos camundongos após imunização, com índices de intermediária e alta avidez. Todos os adjuvantes foram capazes de aumentar a resposta imune contra NOMV de N.meningitidis. A via intranasal foi adequada para sensibilizar as células do sistema imune que foram rapidamente estimuladas pela via intramuscular usando os adjuvantes utilizados na presente investigação. Dados sugerem que o estudo da NOMV é importante na indução da imunidade de mucosa para N. meningitidis B, e que a qualidade e magnitude da resposta imune gerada pelas vacinas de mucosa são influenciadas tanto pelo adjuvante como pelo antígeno. Concluímos que NOMV juntamente com adjuvantes de mucosa tem considerável potencial no desenvolvimento de vacinas contra o meningococo do sorogrupo B. / The natural habitat of Neisseria meningitidis is the human nasopharynx, and the bacterium is transmitted by direct mouth-to-mouth contact or by the inhalation of released mucous particles during close contact. N meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharide-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Current studies are searching for vaccinal antigens that are derived from the native outer membrane (NOMV). However, vaccines based on NOMV are considered weak, making the use of adjuvants necessary. This study investigated the immunogenicity of NOMV of N. meningitidis administered intranasal/intramuscular in neonate BALB/c mice, using the native outer membrane complex (NOMC) obtained from an epidemic strain of N. meningitidis B:4:P1.15. The strains used for immunization of mice were selected by colony-blot, using anti L3,7,9 monoclonal antibodies, for the highest expression of LPS among the immunotypes (B:4:P1:15 L9á). As mucosal adjuvants, we used Bordetella pertussis (whole cells) or the supernatant of 48 h culture of this bacterium, followed by an intramuscular dose of the same protein adsorbed onto , B. pertussis (whole cells) or 48-h B. pertussis culture supernatant or aluminum hydroxide [Al(OH)3]. Sera of immunized mice were evaluated by ELISA in order to compare the different adjuvants used. We also determined their avidity index. IgG and IgM were detected in the serum of mice after immunization, with avidity indices that ranged from intermediate to high. All adjuvants were capable of increasing the immune response against NOMV of N. meningitidis in the homologous prime/boost schedule used. The intranasal route was suitable for sensitizing the cells of the immune system which were quickly stimulated by the intramuscular route using the adjuvants analysed in the present invertigation. Data suggest that the NOMV studied is important in the induction of mucosal immunity to N. meningitidis B, and that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the adjuvant as well as the antigen. In conclusion, nasal delivery of NoMV with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

Bordetella pertussis is the causative agent of whooping cough which is a worldwide acute respiratory disease that predominantly involves infants. Whooping cough is one of the ten most common causes of death from infectious diseases worldwide. The increased coverage of the primary pertussis vaccination (DaBT-IPA-Hib) decreased the incidence of disease in Turkey dramatically. However, in spite of the incidence decline, the circulation of B. pertussis has not yet been eliminated, and a change in the clinical spectrum and age-related incidence of the disease has been observed. On the other hand, in view of the moderate changes that have been observed in the genomic sequences of certain virulance factors over time, there are concerns about the gradual loss of the efficacy of the current pertussis vaccines as a result of antigenic drift and continuous selection of the least vaccine-sensitive clones. Proteomics deals with whole protein content (proteome) of cells as a function of space and time. Gel-based approach in proteomics involves two dimensional gel electrophoresis (2-DE) followed by peptide mass fingerprinting (PMF) employing matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). Immunoproteomics which is a combination of gel based proteomics and Western blot analysis determines tumor-specific antigens as well as immunoreactive proteins of pathogens by combining proteomics with Western blot technique. Although immunoproteomics is a rather new research tool, it has been quite effective to determine the virulence factors of various pathogenic microorganisms. The present study aims at comparing immunoproteome of the standard B. pertussis strain &ldquo / Tahoma I&rdquo / with those of two other strains, namely &ldquo / Saadet&rdquo / and &ldquo / Nursel&rdquo / , which are the local isolates that have been preferred as the vaccine strains for many years in our country for their ability to provide a better protection. Of a total of 38 immunogenic proteins identified, 14 were shown to be the novel antigens for B. pertussis. Among 14 proteins, one was detected as immunogenic in only Tohama I strain where two proteins were specific for Nursel strain. Among the strains compared, Saadet strain had the highest antigenic variety, than the others.

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. It becomes widespread among adolescent and adults as well as infants. Although availability of effective pertussis vaccines seems to decrease the incidence of the disease, B. pertussis circulation in population has not been eliminated. It is thought that the antigenic drifts in major protective antigens and continued circulation of B. pertussis strains will result in gradual loss of the efficacy of the current pertussis vaccines. Therefore, development of more effective acellular pertussis vaccines with conserved protective proteins is a convenient strategy to provide a better protection against whooping cough. In this study, immune responses against putative peptidyl-prolyl cis-trans isomerase (PPIase) which was shown to be immunogenic in B. pertussis for the first time by our immunoproteome group and FimX whose expression was found higher in our local Saadet strain were determined in mice. The genes encoding FimX and putative PPIase were amplified by PCR, cloned into pGEM&reg / -T Easy vector and sequenced. The genes were then introduced into pET-28a (+) vector and they were expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins were purified by His-tag affinity chromatography and dialyzed. After Western blot analyses, 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase were used to immunize BALB/c mice (16-18 g) at day 0 and 21. The mice were challenged intranasally with 2.5 x 109 live B. pertussis Saadet cells. Before second immunization and challenge, the sera were collected to carry out ELISA for measurement of serum-specific IgG levels. According to ELISA results, IgG levels in the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX were found significantly higher than in control groups at both first and second vaccinations (p&lt / 0.01). On the other hand, immunization with 160 &micro / g recombinant putative PPIase provided a significant increase in IgG level (p&lt / 0.05) only at second vaccination. The lungs of the mice were removed at day 2, 5, 8 after challenge and bacterial colonization was determined. No significant decrease in bacterial colonization was observed in the lungs of the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase with respect to control groups. After respiratory challenge and second immunization (at day 30) with 20 &micro / g and 80 &micro / g recombinant FimX, the spleens of the mice were removed and a spleen cell culture was obtained. Supernatants were collected after induction of the cells with the recombinant protein and cytokine ELISA was carried out to measure IFN-&gamma / level. No significant difference was observed between control and vaccinated mice in terms of IFN-&gamma / production.

Bordetella pertussis is a gram-negative, human pathogen and etiologic agent of whooping cough (pertussis), a highly contagious, acute respiratory illness. In this study, the analysis of whole immunproteome and subproteomes of this microorganism was performed. The soluble cytoplasmic proteomes of B. pertussis Tohama I strain and a local isolate Saadet were separated by 2DE. By Western blot analysis, we identified 25 immunogenic proteins of three categories. In the first group, there were well-known proteins of the pathogen The second group comprised proteins which were already shown antigenic in certain pathogenic bacteria, but not in B. pertussis before. The third group of proteins were those which have not been shown to be immunogenic in any pathogen till the present study such as putative chromosome partition protein, preprotein translocase SecA subunit, carbamoyl-phosphate synthase large chain, PRP synthase, putative substrate-CoA ligase, lysyl-tRNA synthetase, fumaryl acetoacetase, putative peptidyl-prolyl cis-trans isomerase, aspartate-semialdehyde dehydrogenase, putative DNA-binding protein and a putative outer membrane protein. In our surfaceome study, surface proteins of two strains were identified by 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these techniques 45 proteins were identified by 2DE and 226 proteins by geLC-MS/MS. The immunogenicity of surface proteins on 2DE gels were analyzed by Western blotting and among 11 identified immunogenic proteins glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase were found to be immunogenic for the first time in Bordetella. It was also found that 16 proteins were differentially expressed in B. pertussis Saadet and Tohama I. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I. In the secretome study, we identified 40 proteins by 2DE and 357 proteins by geLC-MS/MS. It was found that 12 proteins were immunogenic by Western blot analysis and the immunogenicity of putative secreted protein (BP1047) was shown for the first time in this study. In our study, PT subunit 2 and putative outer protein D (BopD) were more abundant in Saadet while one protein, glutamate synthase subunit beta was expressed at a higher level in Tohama I. Four proteins were expressed only in Saadet (two capsular polysaccharide biosynthesis protein, protein FimX and putative outer membrane permeability protein). The present study comprehensively covered almost the entire proteome of a crucial pathogen, demonstrated many novel antigens and identified hundreds of membrane-bound proteins, cell surface-associated and extracellular proteins. Thus, it is anticipated to greatly aid in a better understanding of pathogen-host relations, rational design of novel drugs and developing new generation vaccines against B. pertussis.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-118).