Introdução: A coqueluche é caracterizada por tosse paroxística, pode levar menores de um ano de idade ao óbito, deixar seqüelas e exacerbar quadros respiratórios crônicos. A imunidade após a doença ou vacina não é para toda a vida. Nos países desenvolvidos, apesar de altas coberturas vacinais e do controle da doença entre as décadas de 50 e 80, desde o final dos anos 80 é observado o aumento dos casos em adolescentes, adultos e lactentes, sendo indicado o reforço vacinal para adolescentes e adultos. No Brasil a doença aparenta estar sob controle, mas há um estudo teórico que demonstra a possibilidade de aumento dos casos. Objetivo: Avaliar novas estratégias de reforço vacinal contra a coqueluche no município de São Paulo. Metodologia: Desenvolvimento de um modelo matemático determinístico, dinâmico e dependente da idade dos indivíduos. Simulações com o esquema vacinal atual e: (i) novo reforço aos 12 anos com coberturas vacinais de 10%, 35%, 45% e 70%; (ii) reforços aos 12 anos e aos 20 anos de idade, com 35% e 70% de cobertura, respectivamente. Introdução de contato heterogêneo da população com o uso de uma matriz de contato. Fontes dos dados: Banco de dados do Sistema Único de Saúde (DATASUS), Centro de Vigilância Epidemiológica da Secretaria de Estado da Saúde de São Paulo e a literatura nacional e internacional. Uso dos programas Berkeley Madonna® para resolução das equações diferenciais e Microsoft Excel® para o cálculo da matriz de contato e das forças de infecção. Realização de teste de sensibilidade do modelo. Resultados: A vacinação com cobertura de 10% aos 12 anos de idade reduziu os casos entre os próprios adolescentes (10 a 19 anos); com cobertura de 35%, 45% e 70% reduziu os casos em 59%, 65% e 73%, respectivamente; a vacinação em conjunto aos 12 anos com cobertura de 35% e aos 20 anos com cobertura de 70% reduziu 62% dos casos. Conclusões: Há benefício ao vacinar os adolescentes, inclusive com baixa cobertura vacinal, portanto tal estratégia demonstra-se promissora para o controle da coqueluche. Não houve ganho ao acrescentar apenas um reforço para os adultos (20 anos). Os resultados são concordantes com o que há na literatura e permitiram um primeiro panorama para auxiliar na abordagem do problema. Estudos com diferentes estratégias de vacinação de adultos e estudos de custo-benefício são recomendados. / Background: Whooping cough is a respiratory tract infection caused by Bordetella pertussis and characterized by paroxysmal cough that usually causes complications for infants, including death, and for people with chronic respiratory diseases. Immunity against pertussis after infection or vaccination is not everlasting. Despite of high childhood immunization coverage and the disease control from the 50's to 80's, since late 80's developed countries notified high levels of pertussis in adolescents and adults. This reappearance has not being detected in Brazil yet, but at least one formal study has demonstrated the possibility of this change in the next years. Objective: Evaluating new pertussis vaccine's booster for adolescents and adults in São Paulo city. Methods: Development of a deterministic, compartmental and age-dependent model accounting for immunity waning. The data was retrieved from literature, Surveillance Center of the State of São Paulo (CVE), and the Brazilian national health data system (DATASUS). Data manipulation used Berkeley Madonna® and Microsoft Excel®. Vaccination strategies included the current vaccination scheme, plus (i) 10%, 35%, 45% or 70% vaccine coverage for those at the age of 12 and (ii) both 35% and 70% vaccine coverage at the ages 12 and 20, respectively. The Who Acquire Infection from Whom (WAIFW) matrices' method was used to assume age related transmission rates. Sensitivity analysis was performed. Results: Booster vaccination for 12 years youths, at 10% coverage, yields disease reduction only among adolescents (10 to 19 years); coverage up to 35% yields disease reduction for all ages; at 35%, 45% and 70% coverage, the reduction achieves 59%, 65% and 73%. Booster vaccination at 12 and 20 years, with coverage at 35% and 70% respectively, yields 62% cases reduction. Discussion: Results suggest that adolescent's vaccine booster could reduce pertussis occurrence for all ages, including infants, as also demonstrated by other authors. In contrast, only one vaccine booster for adults (20 years) achieves insignificant results. In conclusion, we have been able to demonstrate that, in São Paulo, the adolescent vaccine booster strategy is a promising police to further reduce whooping cough occurrence. However, cost effective analysis and other adults' vaccination strategies are recommended.

A eficácia da associação quádrupla (DPT + BCG) foi estudada através da proteção e comparação da soro conversão das vacinas, usando como adjuvante o hidróxido de alumínio e ou BCG. A potência da vacina pertussis foi avaliada pelo teste de proteção em camundongos e o BCG pelo teste de consumo de oxigênio e de vitalidade, sendo considerada satisfatória. Sendo os toxóides diftérico e tetânico considerados proteínas inertes e estáveis, suas potências não foram determinadas depois de associadas. Os níveis de anticorpos foram determinados para difteria e tétano pela reação imunoenzimática, para vacina pertussis pela reação de imunofluorescência e para o BCG pela conversão tuberculínica. Os níveis de conversão foram satisfatórios, revelando ser possível a associação sem prejuízo para nenhum dos antígenos. A associação DPT + BCG não causou reação local ou geral significante, possibilitando uma simplificação operacional. / The efficacy of the quadruple association (DPT + BCG) was studied though protection and comparison ot the conversion serum in vaccines using aluminium hydroxide or BCG as adjuvant. Both the protection power of the Pertussis vaccine evaluated by the protection test in mice and BCG by the oxygen uptake and counts of viable particles were considered satisfactory. Being difteria and tetanus toxoids considered inert and stable proteins, their protection wasn\'t determined after their combination. The antibody levels produced by the antigens were determined by the enzyme-linked immunosorbent assay to the difteria and tetanus toxoids, by the fluorescent antibody technique to the Pertussis vaccine and by the tuberculin conversion to the BCG. The conversion levels were satisfatory and there was no damage in their association. There was no meaningful local or general reaction in this association making an operational simplification possible.

La toxine de pertussis (PTX) est une A-B protéine considérée comme l’un des principaux facteurs de virulence de Bordetella pertussis, l’agent bactérien responsable de la coqueluche. Aujourd’hui, cette maladie représente encore un réel danger pour les nouveaux-nés et les nourrissons non ou partiellement immunisés. Actuellement, la coqueluche provoque encore la mort d’environ 350.000 individus par an. La toxicité de la PTX est liée à l’activité enzymatique de sa sous-unité A capable d’inhiber les voies de signalisation associées aux protéines Gi. La partie B, quant à elle, permet l’entrée de cette sous-unité A dans le cytoplasme des cellules cibles en se liant spécifiquement à son ou ses récepteurs membranaires toujours inconnus de nos jours. Des études réalisées chez la souris et chez l’homme ont montré que les vaccins anticoquelucheux combinés à différents antigènes vaccinaux étaient capables de moduler leurs réponses humorales spécifiques. Par ailleurs, la PTX est couramment qualifiée d’agent immunostimulant. En effet, des modèles murins de vaccination permirent d’identifier des propriétés adjuvantes de la PTX coadministrée avec des antigènes non relevants. Le travail développé dans ce manuscrit étudie les effets de la PTX sur 2 types cellulaires primordiaux sollicités lors d’une vaccination : la cellule dendritique (DC) et le lymphocyte T CD4+ naïf. Les DC sont les seules cellules présentatrices d’antigènes aptes à initier une réponse immune primaire. Dans un premier temps, nous avons montré que la PTX était capable d’activer des DC générées in vitro à partir de monocytes. En effet, elles acquièrent un phénotype mature caractérisé par une augmentation de l’expression membranaire des molécules costimulatrices et du CMH de classe II, démontrant un effet direct et spécifique de la PTX sur les DC myéloïdes. Parallèlement, ces DC produisent du TNF-a, de l’IL-12p40 et de l’IL-12p70 et activent NF-kappaB, un facteur de transcription essentiel au processus de maturation. Nous avons obtenu des résultats similaires avec une toxine génétiquement modifiée qui est enzymatiquement inactive. A partir de sang total incubé avec la PTX, nous avons par ailleurs observé que les DC circulantes du nouveau-né étaient déficientes dans leur maturation et leur sécrétion d’IL-12p70 comparées aux DC de l’adulte. D’autre part, il a été décrit précédemment que la PTX exerçait des effets mitogènes sur les lymphocytes T humains et murins. Cependant, le rôle qu’elle joue sur la population des lymphocytes T CD4 naïfs reste peu connu. A l’issue de notre second travail, nous pouvons dès lors affirmer que la PTX est également capable d’activer des lymphocytes T CD4+CD45RA+ naïfs isolés à partir des cellules mononuclées du sang périphérique, et ce indépendamment de son activité enzymatique. En effet, ces lymphocytes T CD4+ naïfs stimulés par la PTX prolifèrent, synthétisent des quantités non négligeables d'ARN messagers codant pour l’IL-2 et le TNF-a, augmentent l’expression membranaire des molécules CD40L, CD69 et CD25 et expriment la protéine Foxp3. Cette activation s’accompagne de la translocation nucléaire de NF-kappaB et NFAT. Parallèlement à l’adulte, la PTX active les lymphocytes T CD4 néonataux. Néanmoins, ceux-ci prolifèrent moins bien et expriment plus faiblement le CD40L à leur surface. Enfin, la PTX induit la sécrétion de taux importants d’IFN-g par des T CD4+CD45RA+ naïfs adultes mis en présence de DC autologues. Nous terminerons en proposant l’hypothèse suivante : La PTX pourrait exercer ses propriétés adjuvantes par l’intermédiaire de différents mécanismes comprenant notamment la maturation des DC d’origine myéloïde et l’activation des lymphocytes T CD4+CD45RA+ naïfs. Ces 2 populations cellulaires sont en effet les principaux protagonistes impliqués dans la réponse immune primaire.

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.

Thèse de doctorat : Sciences de la vie et de la santé : Lille 1 : 2001. / Résumé en français et en anglais. Textes en français et en anglais. Bibliogr. f. 232-270. Notes bibliogr.

JESSICA MASTRODOMENICO An Examination of the Socio-Demographic Characteristics Associated with Adult Vaccination Prevalence for Preventable Diseases in the United States Background: An estimated 50,000 adults in the United States (U.S.) die each year from one of 10 vaccine preventable diseases. For those who survive vaccine preventable infections, health care costs and loss of income become more significant. While children in the U.S. aged 0-2 exhibit vaccine prevalence rates of almost 90%, some adult vaccine prevalence rates in the U.S. population are reported to be nearly 30-40% less than the goals set forth by Healthy People 2010. The purpose of this study was to examine the associations between socio-demographic characteristics of U.S. adults and adult vaccination prevalence for pneumococcal, hepatitis A, hepatitis B, tetanus, and pertussis. Methods: Data from the 2008 National Health Interview Survey were assessed examining various health indicators and characteristics of non-institutionalized adults and children. The sample was restricted to adults ≥18 years of age. Odds ratios were calculated and multivariate logistic regression was also conducted. P-values of <0.05 and 95% confidence intervals were used to determine statistical significance. Results: There were 21781 total observations; 19.3% received the pneumococcal vaccine, 9.4% received the hepatitis A vaccine, 27.2% received the hepatitis B vaccine, 55.1% received the tetanus vaccine, and 15.2% received the pertussis vaccine. Of the socio-demographic characteristics examined, age, health insurance, marital status, and education were significant for either all five or at least four of the vaccines included in this study. As one might expect those who reported health insurance and those who had a higher level of education usually had a higher likelihood of vaccine receipt as compared to those without health insurance and those with less than a high school education. Age associations varied due to age-related recommendations for certain vaccines such as pneumococcal (recommended for adults ≥65). Compared to the married population (referent), marital status results varied, but for reasons unclear. Whites, the referent group, were the most likely to be vaccinated as compared to Blacks, Hispanics/Latinos, and Asians. Hispanics/Latinos typically had the lowest likelihood of vaccination in this examination. Conclusions: This study further explores the impact of socio-demographic disparities on vaccination status and adds new information to the literature regarding adult vaccination rates for preventable diseases. While research exists related to strengthening interventions such as patient reminder systems, those who do not see the same health care providers on a regular basis remain at risk for lower vaccination prevalence. It is important to better understand the role of social determinants of health, specifically in terms of vaccinations. Future research is needed to further characterize the association of socio-demographic factors with receipt of optional vaccines in adults.

El aumento creciente de la incidencia de Bordetella pertussis en poblaciones con elevada cobertura de vacunación ha puesto de manifiesto que las vacunas en uso presentan falencias que han cobrado importancia con el tiempo. Con el fin diseñar vacunas con mayor capacidad protectora nuestra investigación se ha orientado al estudio del fenotipo infectante. Parte de los esfuerzos en este sentido se han centrado en la identificación de los cambios fenotípicos inducidos en B. pertussis en respuesta a la falta de hierro libre, por ser éste el stress nutricional mas importante durante infección. Para ello se ha elegido una estrategia de trabajo que es una combinación de proteómica bidimensional comparativa y análisis serológico del proteoma. La ventaja de emplear esta estrategia es que permitió identificar potenciales factores de virulencia y, a la vez, seleccionar inmunógenos que se expresan en condiciones fisiológicas y que, sin embargo, no están incluidos en ninguna de las formulaciones vacunales en uso. En este trabajo de tesis, por un lado se amplió la caracterización del proteoma de B. pertussis cultivada bajo limitación de hierro mediante una proteómica shotgun, una técnica que presenta alta sensibilidad. Además se describen dos nuevos inmunógenos capaces de mejorar la protección conferida por la vacuna acelular actualmente en uso.

El objetivo general de esta tesis es generar conocimiento sobre los aspectos claves de la respuesta inicial frente a una infección respiratoria para aplicarlo en la formulación de estrategias de inmunoprofilaxis de la enfermedad. Este trabajo puede servir de prueba de concepto para contrastar las hipótesis enunciadas. Con este fin, emplearemos el modelo murino de infección por B. pertussis, analizando la contribución de distintos receptores TLR en la inducción de la defensa mucosal frente a la infección para luego evaluar cómo la activación de dichas vías en protocolos de inmunización intranasal son capaces de dar origen a respuestas protectivas frente al desafío intranasal con B. pertussis.