As meningites representam um importante agravo no quadro sanitário nacional, por suas características epidemiológicas e por seu impacto sócio-econômico. O Haemophilus influenzae tipo b (HIB), causa infecções respiratórias e doenças invasivas como meningites, pneumonias, epiglotites, sinusites, bacteremias, otites e artrites. Entre essas enfermidades, a meningite tem sido a mais estudada, em virtude de sua alta morbi-mortalidade e por ser de notificação compulsória. A proposta deste trabalho foi a de avaliar a tendência, em série histórica, da morbimortalidade e da letalidade das meningites caudadas por HIB no Brasil, como uma alternativa para avaliação do impacto da vacinação específica. O estudo seguiu um delineamento observacional do tipo ecológico e relativo ao período de 1983 a 2002. Foram calculados os coeficientes de incidência, mortalidade e letalidade de meningites por HIB, a partir de base de dados do Ministério da Saúde e da Fundação Instituto Brasileiro de Geografia e Estatística. Para a análise de tendência destes indicadores foram estimadas retas de predição, com intervalos de confiança de 95%. Os softwares utilizados para a fase de gerenciamento e para a análise de dados foram: Excel, Epiinfo e Stata. No Brasil, entre 1983 e 2002, foram notificados 23.921 casos de meningites por HIB, dos quais 10.524 (43,99%) em menores de 1 ano e 9.269 (38,75%) na faixa de 1 a 4 anos. Os coeficientes de incidência, mortalidade e letalidade de maior magnitude foram observados nas faixas etárias de menores de 1 ano e de 1 a 4 anos. A partir de 1988, houve incremento na magnitude dos coeficientes de incidência e mortalidade. As regiões Norte e Nordeste apresentaram menor magnitude para esses indicadores, enquanto que para a letalidade, a região Nordeste apresentou indicadores de maior magnitude em menores de 1 ano. De modo geral, embora a letalidade tenha apresentado um padrão de declínio desde o inicio da série, sua magnitude permaneceu elevada até 2001. O impacto da vacinação sobre a letalidade, apenas a partir de 2002, sugere que esta pode levar até três anos, em média, para exercer efeito sobre o prognóstico das meningites por HIB. Com exceção da região Nordeste, a tendência para a incidência e a mortalidade no Brasil e regiões, foi semelhante, no período prévacinação (ascensão), detectando-se declínio após a introdução da vacina na rotina do Programa Nacional de Imunização. No Brasil e nas regiões Norte, Nordeste, Sul e Sudeste (nesta, em menores de 1 ano), a tendência da letalidade foi de declínio em toda a série. Nas regiões Centro-Oeste e Sudeste, o impacto da introdução da vacina sobre a incidência e a mortalidade foi mais precoce (1999-2000), em relação às outras regiões do estudo, talvez pelo fato dessas regiões terem apresentado cobertura vacinal mais elevada. O impacto da vacinação foi mais evidente sobre a incidência e a mortalidade do que sobre a letalidade. Os resultados revelaram um impacto positivo das estratégias de vacinação contra HIB, no Brasil e macrorregiões, inclusive em faixas etárias não vacinadas. Recomenda-se a monitorização contínua desses agravos, através de ações de Vigilância Epidemiológica, com aprimoramento do Sistema de Informação em Saúde. / Meningitis is an important disease within the national health system, due to its epidemiological characteristics and socioeconomic impact. The Haemophilus influenzae (HIB) type B causes respiratory infections as well as invasive diseases such as meningitis, pneumonias, epiglottitis, sinusitis, bacteremias, otitis and arthritis. Among these diseases, the meningitis has been the most studied due to its high morbi-mortality rate and also because requires compulsory notification. The purpose of this study was to evaluate the trend, considering the history series, regarding morbi-mortality and case fatality rates of meningitis caused by Hib in Brazil, as an alternative for the evaluation of the impact of a specific vaccination. This research was an ecologic observation from 1983 to 2002. The author calculated the incidence, mortality and case fatality rates of Hib meningitis, based on data from the National Health Department and the Geography and Statistics Brazilian Foundation Institute. In order to analyze these indicators\' trends, authors estimated prediction lines, with confidence intervals of 95%. The softwares used to manage and analyze data were: Excel, Epiinfo and Stata. In Brazil, from 1983 to 2002, 23.921 cases of Hib meningitis were notified. Among them, 10.524 (43.99%) in babies with less than one year and 9.269 (38.75%) in children with age varying from 1 to 4 years. The higher magnitude of incidence, mortality and case fatality rates were observed in the ages less than 1 year and varying from 1 to 4 years. Since 1988, the author observed an increment in the magnitude of the incidence and mortality rates. The North and Northeast regions presented a lower magnitude for these indicators, while for case fatality, the Northeast region presented indicators of higher magnitude in babies with age less than 1 year old. In general, although the case fatality rates presented a decreasing pattern since the beginning of the series, its magnitude was high until 2001. The impact of vaccination on case fatality rates was perceived only after 2002, suggesting that it can take three years, in average, to have some effect on the Hib meningitis prognosis. With exception to the Northeast region, the trends regarding the incidence and mortality in Brazil were similar, in the pre-vaccination period (increase), detecting a decrease after the introduction of vaccination in the routine of the do Programa Nacional de Imunização. In Brazil and in the North, Northeast, South and Southeast (age less than one year) regions, the case fatality trend was of decline during all series. In the Centerwest and Southwest regions, the impact of the vaccination introduction on the incidence and mortality rates was precocious (1999-2000), if compared to the other regions studied and maybe due to the fact that in these regions the vaccination achieved a greater amount of the population. The impact of vaccination was clearer on the incidence and mortality than on the case fatality rates. Results revealed a positive impact of the vaccination strategies against Hib in Brazil and in macro regions, including the age ranges that were not vaccinated. The author recommends the continuous monitoring of these diseases, through epidemiological surveillance actions and the improvement of the Health Information System.

As meningites bacterianas constituem um sério problema de Saúde Pública em todo mundo, por sua incidência, sua letalidade e pela frequência das sequelas que os sobreviventes apresentam. Os agentes etiológicos Haemophilus influenzae, Neisseria meningitidis e Streptococcus pneumoniae são responsáveis por cerca de 60 a 80 por cento dos casos. O presente estudo tem como objetivo conhecer o comportamento epidemiológico das meningites por H.influenzae, S. pneumoniae e por bacilos Gram-negativo, especialmente as enterobactérias 1 no Município de São Paulo no pertodo 1960- 77. O levantamento foi realizado por uma equipe formada por professores do Departamento de Medicina Social da Faculdade de Ciências Médicas da Santa Casa de São Paulo, por médicos sanitaristas e por acadêmicos de medicina. Os dados, colhidos diretamente do prontuário dos pacientes, foram anotados em uma ficha pré-codificada. A meningite por H.influenzae somente foi confirmada quando se identificava o agente na cultura. A confirmação da meningite por S.pneumoniae se dava pela bacterioscopia e/ou pela cultura do líquor. As meningites por bacilo Gram-negativo foram subdivididas em 3 grupos. No primeiro, incluíram-se os casos em que a bacterioscopia e/ou cultura revelaram a presença de um bacilo Gram-negativo sem, contudo, haver a especificação do agente. No segundo, classificaram-se os casos em que, na cultura, foi isolada uma bactéria do gênero Salmonella. O último grupo correspondeu áquele em que se identificou a presença de uma outra enterobactéria. Os subdistritos ou distritos do Municipio de São Paulo foram agrupados de 3 maneiras. As duas primeiras corresponderam às 3 ou 6 áreas homogêneas especificadas pela Fundação SEADE. A última se baseou na distibuição da população economicamente ativa segundo sua participação nos diferentes setores da economia. A população dos subdistritos e distritos do Município de São Paulo segundo faixa etária para os anos compreendidos no estudo foi estimada pelo método geométrico modificado. No período estudado foram confirmados 900 casos de meningite por H.influenzae com um coeficiente médio de 0,89 por 100000 habitantes. Os menores de 5 anos contribuíram com 91,2 por cento dos casos, dos quais 63 por cento eram em menores de um ano. O coeficiente médio para menores de um ano foi de 23,3 por 100000 habitantes. As zonas central, intermediária e periférica não apresentaram incidências significantemente diferentes. Os coeficientes de morbidade padronizados segundo idade foram 0,8, 0,8 e 0,9 para as zonas central, intermediária e periférica, respectivamente. A letalidade média no período de 1960-77 foi de 31 por cento . As crianças menores de um ano apresentaram a maior taxa de letalidade, 40 por cento . No período 1960-77 foram confirmados 1951 casos de meningite por S.pneumoniae com um coeficiente médio de 1,9 por 100000 habitantes. As crianças menores de 5 anos contribuíram com 52 por cento dos casos dos quais 38.5 por cento eram menores de um ano. Os coeficientes médio por 100000 habitantes, para os menores de um ano, foram 37,1 e 29,7 para 1960-69 e 1970-77, respectivamente. A incidência por 100000 habitantes na zona periférica (2,2) na primeira década foi, praticamente, o dobro da zona central, (1,2). Os coeficientes padronizados segundo idade foram 1,6, 1,5 e 2,0 para as zonas central, intermediária e periférica, respectivamente. No período seguinte estes valores foram 1,4, 1,5 e 2,0. A letalidade média no período foi de 44 por cento . Ela foi inversamente proporcional ao número de leucócitos no llquor de entrada. A letalidade na faixa etária menores de um ano foi de 60 por cento no período estudado. No período estudado foram identificados 290 casos de meningite por Salmonella dos quais 10 por cento o foram na primeira década. O coeficiente médio por 100000 habitantes foi de 0,3. A S.typhimurium foi a espécie mais frequente com 112 casos. Os menores de um ano contribuíram com 91 por cento dos casos, dos quais 52 por cento ocorreram no primeiro trimestre de vida. A incidência média por zona não mostrou diferencas estatisticamente significantes. A letalidade média foi de 87 por cento . Os menores de um ano apresentaram um valor ainda maior, 89 por cento . No período estudado foram identificados 211 casos de meningite por outras enterobactérias com um coeficiente médio de 0,2. A primeira década contribuiu com 32 por cento dos casos. Os gêneros Escherichia e Enterobacter foram os mais frequentes sendo responsáveis por 71 por cento dos casos. Os menores de um ano contribuiram com 57 por cento dos casos, com coeficiente de 4.0 e 4.5 para os períodos 1960-69 e 1970-77 respectivamente. A letalidade média foi de 65 por cento sendo o grupo etário maior de 60 anos o de maior letalidade. A incidência por zona não diferiu significantemente. A meningite por bacilo Gram-negativo apresentou um comportamento epidemiológico distinto da meningite por H.influenzae e das enterobactérias, revelando ser composto por uma miscelânea de agentes. No período de estudo foram identificados 25455 casos de meningite bacteriana, com coeficiente médio de 25 por 100000 habitantes. O coeficiente passaria a ser de 36 por 100000 habitantes se acrecentássemos as meningites bacterianas de etiologia indeterminada. Este índice representou 1 caso para 2782 habitantes. A meningite por N.meningitidis ocupou o primeiro lugar, com 84 por cento dos casos e um coeficiente médio de 21 por 100000. Na primeira década ocorreram 2657 casos, com um coeficiente médio de 5,4 por 100000 habitantes. As três principais etiologias foram responsáveis por 89 por cento dos casos. No octênio seguinte a meningite por meningococo foi responsável por 90 por cento dos casos. No ano de 1974, acme da epidemia de meningite meningocócica, foram identificados 18069 casos de meningite representando um coeficiente de 264 por 100000. Este valor representaria que 1 em cada 379 habitantes foi acometido pela doença naquele ano. / Bacterial meningitis is an intectious disease of major public health throughout the world because of its high incidence and case fatality rates and the permanent sequelae that are seen in the survivors. Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae are the etiologic agents responsible for 60 to 80 per cent of cases. The purpose of this study is to better understand the epidemiology of meningitis caused by H. influenzae, S. pneumoniae and gram-negative bacilli, especially, the Enterobacteriaceae, in the city of São Paulo during the period 1960-77. The survey was performed by a group of professors from the Department of Social Medicine of the \"Faculdade de Ciências Médicas da Santa Casa de São Paulo\", public health physicians and medical students. Data were obtained directly from the patient\'s records and registered on a pre-coded form. Cases of H.influenzae meningitis were confirmed by culture while S.pneumoniae cases were confirmed by gram stain and/or culture of the cerebrospinal fluid (CSP). The cases of gram negative bacillary meningitis were divided into three groups. The first included the cases that were diagnosed by gram stain and culture; the second, the cases where salmonella species were isolated in the culture: and the third, the cases where the presence of other Enterobacteriaceae were identified. The districts of the city of São Paulo were grouped in three ways: two corresponding to the homogenous areas specified by the \"Fundação SEADE\", and the third one based on the distribution of the economically active population according to its participation in the different branches of economic activity. The population of São Paulo by districts included in the study was estimated by the modified geometric method. During the study, 900 cases ot H. influenzae meningitis were confirmed, giving an average rate ot 0.89 cases per 100,000 population. Children <5 years old represented 91 per cent ot the cases, 63 per cent of them being less than one year old . The average rate for children <1 year old was 23.3 cases per 10O,OOO population. The average case fatality rate for the period 1960-77 was 31 per cent . The hightest case fatality rate ocurred in children <1 year old and was 40 per cent . The central, intermediate and peripheria zones didn\'t show significant different rates of incidence. The age standartized morbidity rates for these zones were, respectively, 0.8, 0.8 and 0.9. During 1960-77, 1,951 cases of S.pneumoniae meningitis were confirmed, giving an average rate of 1.9 per 100,000 population. Children <5 years old accounted for 52.4 per cent ot cases and 38.5 per cent were <l year old. The average rates for children <1 year of age were 37.1 and 29.7 per 100,000 population, for the periods of 1960-69 and 1970- 77, respectively. The incidence rate for the peripheria zone -2.2 per 100000 population- pratically doubled the rate for the central area- 1.2 per 100000 population- in the 1960\'s. The age standardized rates were 1.6, 1.5 and 2.0 tor central, intermediate, and peripheric zones, respectively. In the 1970\'s these rates were 1.4, 1.5 and 2.0. The average case fatality rate for the period was 46.9 per cent , which inversely proportional to the number ot CSF leucocytes at first examination. For children <1 year old, the case tatality rate was 60 per cent during the same period. Two hundred ninety cases of Salmonella meningitis were indentitied during the study, 10 per cent of them during the first decade. The average rate was 0.3 cases per 100,000 population. S. typhimurium was the most frequently isolated species, with 112 cases. Children <1 year old represented 91 per cent of the cases and 52 per cent ot these ocurred in children <3 months of age. The averages rates of incidence in the different zones didn\'t show statistically significant differences. The average case fatality rate was 87 per cent children <1 year old had a rate of 89 per cent . During the study period, 211 cases of meningites by other Enterobacteriaceae were indentified , giving an average rate of 0.2 per 100,000. Almost one-third of these cases ocurred in the first decade of the study period. The genus Escherichia and the genus Enterobacter were the most frequent, being responsible for 32 per cent of the cases. For children under one year 1 the rates were 4.0 and 4.5 for the periods of 1960-69 and 1970-77, respectively, representing 57 per cent of the total of cases. The average case fatality rate was 65 per cent , the hightest being among persons >60 years old. The rates of incidence by zone didn\'t show significant differences. The epidemiology of gram-negative bacillary meningitis was distinct from that of H.influenzae meningitis and meningitis due to the Enterobacteriaceae, giving evidence of being composed by a mixture of agents. In the same period, 25,455 cases of bacterial meningitis were identified, giving an average rate of 25 cases per 100,000 population. This rate would be 36.0 per 100,000 if we added the cases ot bacterial meningitis of unknown etiology. This represents one case per 2,782 inhabitants. N. meningitidis meningitis was the most frequent etiologic agent representing 84 per cent of the total, giving an average rate of 21 per 100,000. From 1960-69, 2,657 cases ocurred, giving an average rate of 5,4. The three principal etiologies were responsible for 89 per cent of cases. During the next eight years, 90 per cent of cases of meningitis were meningococcal. In year of 19/4, during the peak of the meningoccocal meningitis epidemic, 18,069 cases of meningitis were identified, representing a rate of 264 per 100,000. Put another way, 1 in 379 inhabitants developed menngitis.

Haemophilus influenzae (Hi) é uma bactéria Gram-negativa comensal da nasofaringe e um patógeno oportunista cujo único hospedeiro natural conhecido é o ser humano. As cepas de Hi que possuem cápsula de polissacarídeo estão associadas a doenças invasivas mais graves, sendo as de sorotipo b (Hib) as principais causadoras da meningite bacteriana em populações não vacinadas. Para produzir a vacina contra Hib, o polissacarídeo purificado desta bactéria é conjugado quimicamente ao toxóide tetânico. Industrialmente, a produção do polissacarídeo é realizada cultivando esse micro-organismo em biorreatores, entretanto o rendimento em polissacarídeo é baixo, mesmo com fornecimento de nutrientes, controle de pH e outros ajustes das condições no decorrer do cultivo. O estudo dos diferentes perfis fisiológicos da população bacteriana de Hib no decorrer do cultivo através da transcritômica traz a possibilidade de aprofundar o conhecimento sobre o metabolismo desse micro- organismo. As taxas de transcrição dos genes expressos em diferentes momentos considerados como pontos metabolicamente significativos do cultivo de Hib linhagem GB 3291 em batelada alimentada conduzido em Biorreator de 10 L, com aeração submersa e controles de pH (7,0) e temperatura (30° C) foram obtidas através de sequenciamento de RNA paralelo massivo (RNA-seq). A análise de co-expressão dos genes foi realizada com WGCNA, em que oito módulos de genes co-expressos foram identificados, quatro dos quais apresentaram correlação alta com dados fenotípicos dos cultivos, inclusive produtividade de acetato e de polissacarídeo. Análise de enriquecimento funcional identificou vias metabólicas associadas a ribossomo, síntese de parede celular, transportadores e consumo de carbono. A análise de expressão diferencial permitiu observar o comportamento desta bactéria durante o cultivo. Através da análise das taxas de transcrição dos genes foi possível identificar as principais vias de síntese de acetato e de polissacarídeo capsular, sendo esta última feita principalmente através da via de pentose fosfato, em detrimento da via de interconversão pentose-glucuronato. Nossos dados mostram que as diferentes etapas do cultivo de Hib leva à ação conjunta de vários grupos de genes, com destaque àqueles ligados às funções celulares básicas, como a síntese de proteínas e de parede celular, o transporte e a síntese de aminoácidos. Esses resultados contribuem para o entendimento dos processos bioquímicos e celulares que ocorrem durante o processo de cultivo de Hib, possibilitando que sejam feitas sugestões de modificações genéticas em Hib e alteração no processo de cultivo com propósito de diminuir produção de acetato e aumentar produção do polissacarídeo. / Haemophilus influenzae (Hi) is a nasopharynx commensal Gram-negative bacterium and an opportunistic pathogen whose only known natural host is human being. Hi strains with polysaccharide capsule are related to more severe invasive diseases, wherein type b capsule (Hib) strains are the main cause of bacterial meningitis in unvaccinated population. To produce Hib vaccine, purified polysaccharide of this bacterium is chemically conjugated to tetanus toxoid protein. Industrially, polysaccharide production is performed by cultivating this micro-organism in bioreactors; however, the yield of polysaccharide is low, even with supply of nutrients, pH control and further adjustments of the fermentation conditions during cultivation. The study of different physiological profiles of Hib bacterial population during cultivation by transcriptomics brings the possibility to deepen the knowledge about the metabolism of this micro-organism. Transcription of genes expressed at different times considered metabolically significant points of Hib strain GB 3291 grown in fed-batch conducted in a 10 L bioreactor with submerged aeration and pH (7.0) and temperature (30 ° C) control rates were obtained through massive parallel RNA sequencing (RNA-seq). Gene co-expression analysis was performed with WGCNA, in which eight modules of co-expressed genes were identified, four of which showed high correlation with cultivation data traits, including acetate and polysaccharide productivity. Enrichment analysis identified pathways related to ribosome, cell wall synthesis, transports and carbon consumption. Differential expression analysis allowed to observe this bacteria behaviour during cultivation. Through transcription rate analysis, it was possible to identify the main pathways for acetate and polysaccharide synthesis, which is through pentose phosphate pathway instead of glucoronate-pentose pathway. Our data show that different stages in Hib cultivation leads to joint action of several gene groups, highlighting genes related to basic cellular roles, like protein and cell wall synthesis, transport and aminoacid synthesis. These results contribute to the understanding of biochemical and cellular processes that ocurr during Hib cultivation process, allowing suggestions to be made to modify Hib gene circuitry and to change cultivation process in order to decrease acetate production and to decrease acetate production and increase polysaccharide production.

Haemophilus influenzae type b (Hib) é uma importante bactéria Gram-negativa causadora de pneumonia, meningite e septicemia em crianças abaixo dos 5 anos de idade. A prevenção contra a doença pode ser alcançada pela imunização da população com vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficiente. As vacinas conjugadas disponíveis comercialmente custam para o governo brasileiro cerca de US2,7 a dose, sendo necessárias no mínimo 3 doses para imunização completa. O presente estudo desenvolveu um novo método de conjugação de polissacarídeo capsular de Hib (PRP) com toxóide tetânico (TT). O método hidrazona baseia-se em 3 etapas simples: oxidação e derivatização de PRP com espaçador molecular e conjugação com TT na presença de uma carbodiimida e de um éster amino reativo. Após um estudo detalhado de cada etapa do método hidrazona, o novo processo mostrou excelentes resultados de rendimento mesmo após escalonamento. A imunogenicidade e o índice de avidez do conjugado hidrazona foram avaliados e os resultados encontrados foram comparáveis a vacina comercial Hiberix®. A técnica de HPSEC mostrou-se eficaz e o perfil cromatográfico do conjugado hidrazona foi muito similar ao da vacina Hiberix. Finalmente, o novo processo de conjugação de vacina permitiu o desenvolvimento de uma poderosa tecnologia constituindo uma excelente opção para o governo brasileiro. / Haemophilus influenzae type b (Hib) is an important encapsulated bacteria, which causes pneumonia, meningitis and septicaemia in infants. Prevention against infection is achieved by the currently available polysaccharide protein conjugate vaccine. However, due to its high production costs (around U$ 2,7 per dose) this formulation cannot be used in mass immunization programs in Brazil. In the present study, we developed a new method for the conjugation of Hib polysaccharide (PRP) and tetanus toxoid (TT). The hydrazone method is based on 3 singles steps: PRP oxidation, PRP derivatization with linker spacer and conjugation with TT in the presence of carbodiimide and an amino reactive ester. After detailed study of each step of method, the new process showed very good yield of conjugation even when it was scaled-up. The immunogenicity and the avidity index of hydrazone conjugate were evaluated and the results were comparable with those obtained with the commercial vaccine Hiberix®. The HPLC hydrazone profile was very similar to HPLC Hiberix profile. Finally, the new conjugation process allows the development of a powerful vaccine technology, constituting an excellent choice for the brazilian government.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-23T15:50:27Z No. of bitstreams: 1 elo-de-oliveira-rodrigues.pdf: 697936 bytes, checksum: fb3899c2c6d659498d8e8ab0a1a0b33a (MD5) / Made available in DSpace on 2012-11-23T15:50:27Z (GMT). No. of bitstreams: 1 elo-de-oliveira-rodrigues.pdf: 697936 bytes, checksum: fb3899c2c6d659498d8e8ab0a1a0b33a (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O Instituto de Tecnologia em Imunobiológicos, Biomanguinhos, é uma unidade da FIOCRUZ produtora de vacinas, biofármacos e reativos. O Departamento de Controle de Qualidade, pertencente a unidade de Biomanguinhos, é responsável pelos diversos ensaios físico-químicos, microbiológicos e biológicos para liberação dos produtos finais, produtos intermediários e matérias-primas. Devido à necessidade de normalizar seus ensaios referentes a produtos finais e intermediários, várias medidas têm sido tomadas como: calibração e qualificação de equipamentos, validação de métodos analíticos, aquisição de padrões, entre outras atividades de melhoria. Uma das dificuldades encontradas pelos laboratórios de controle de qualidade é a aquisição de padrões que tenham características semelhantes aos produtos produzidos em Biomanguinhos. A disponibilidade de materiais de referência/padrões que atendam às necessidades do laboratório e a dificuldade em obtê-los, além dos custos elevados, os tornam impeditivos para uso nos ensaios rotineiros. Esta dissertação tem como objetivo estabelecer a prática da produção de material de referência in housepara os métodos que são utilizados para o controle de qualidade de produtos obtidos em Biomanguinhos. O planejamento eelaboração do material de referência de trabalho serão realizados considerando-se todas as condições necessárias para que a substância candidata atenda às normas vigentes relacionadas à normalização de métodos de controle de qualidade. A implantação da metodologiae dos requisitos necessários para obtenção do material serão descritos neste trabalho. O material “candidato” a material de referência é opolirribosil-ribitol fosfato, o PRRP, que após conjugação com a proteína monomérica tetânica, torna-se o princípio ativo da vacina contra Haemophilus influenzae, a vacina Hib. A avaliação do material de referência candidato é baseada nos estudos de caracterização, homogeneidade e estabilidade, utilizando-se ferramentas estatísticas adequadas, visando à atribuição do seu valor com uma incerteza de medição associada, atendendo aos propósitos desejados e agregando maior confiabilidade aos produtos analisados pelo laboratório. Além do uso interno, há a intenção de produzir este material de referência emitindo certificado de acordo com as normas vigentes, e assim fornecê-lo também para o INCQS, órgão da FIOCRUZ responsável pelo controle de qualidade nacional de vacinas e medicamentos. / The Institute of Technology in Immunobiologicals, Biomanguinhos, is a vaccine, biopharmaceuticals, and diagnostic kits production unit that belongs to Fiocruz. The Quality Control Department is responsible for the many physical-chemical, microbiological, and biological assays performed to release the final and intermediate products and the raw materials. Due to the need of standardization of the assays, some measures have been being taken, such as equipments’ calibration and qualification, validation of analytical methods, and standards purchase. One of the challenges faced by the quality control laboratories is the acquisition of standards that have the same characteristics as the Biomanguinhos products. The low availability of standards and reference materials that attend the laboratories’ needs and the difficulties in obtaining these products, besides the high costs, make their use in the laboratories routine almost impossible. This thesis intends to establish the production practice for the in-house reference materials used in Biomanguinhos’ quality control assays. The planning and elaboration of the reference materials will be made according to the current legislation that concerns the standardization of quality control methods. The deployment of the methodology and of the requirements for the material obtainment will be discussed in this work. The ‘candidate’ to be a reference material is the polyrribosil ribitol phosphate (PRRP) that, after conjugation with the tetanical monomeric protein, becomes the active substance of the Haemophilus influenzaevaccine (Hib). The evaluation of the candidate material is based in characterization, homogeneity and stability studies, using suitable statistical tools, in order to assign its value with an associated measurement uncertainty. It aggregates reliability to the products analyzed in the laboratories. Besides the internal use, the purpose of this work is to certify the reference material in accordance with the current regulations, so that itcan be more trustable and therefore be used by INCQS, Fiocruz unit responsible for the nationalquality control of vaccines and other pharmaceutical products.

Sialic acid (SA) is a known major virulence factor of Haemophilus influenza (Hi). This study aims to analyse the functions of some candidate sialometabolism genes, and to further our current understanding on the Hi sialometabolism gene regulation. Two candidate sialometabolism genes (HI0227 and HI0148) and their adjacent ORFs (HI0228, HI0148.1 and HI0149) were studied. HI0148.1 and HI0149 are transcribed as a single gene in screened NTHi strains, and we refer to the combined ORF as NTHI0236 (the designation in strain 86-028NP). Across Hi strains screened, the sequences of HI0227, HI0148 and NTHI0236 are conserved. However, the sequence of HI0228 is heterogeneous. Mutants that lack the functions of HI0227, HI0228, HI0148 and NTHI0236 were compared to their respective wild type parent strains for ability to grow on SA (in aerobic and microaerophilic conditions), their ability to sialylate LPS and their ability to resist complement mediated killing. The mutants did not exhibit major differences in the tested aspects of sialometabolism compared to their respective wild type strains. Changes observed in some of the mutants in serum bactericidal assays and LPS profiles were due to the effect of phase variable genes. The sialometabolism functions of HI0227, HI0148, and NTHI0236 remain obscure, and we postulate that HI0228 is a pseudogene. Investigation of Hi sialometabolism gene regulation was conducted using mutants that lack different steps of the Neu5Ac catabolism pathway and the Neu5Ac activation pathway. The expression of nanE and siaP, respectively representing the Neu5Ac catabolism and transport operons, were assessed using RT-PCR and qPCR. We investigated a temporal/concentration effect of Neu5Ac on the expression of sialometabolism operons, which highlights the importance of studying the Hi sialometabolism gene regulation as a dynamic process. We further demonstrated that GlcN-6P, a Neu5Ac intermediate from the catabolism pathway, is likely the SIS sugar that interacts with SiaR, the repressor protein of the Hi sialometabolism operons. We postulate that upon binding of GlcN-6P to SiaR, the SiaR-mediated repression on the Hi sialometabolism operons is relieved, resulting in the induction of the expression of Neu5Ac catabolism and transport genes.

Réalisé au sein du LISBP (Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, INSA Toulouse), ce travail porte sur l’étude du germe pathogène Haemophilus influenzae de type b (Hib). L’objectif du travail est l’amélioration de la compréhension du métabolisme cellulaire et de la synthèse du polyoside capsulaire chez Hib, utilisé pour la fabrication du vaccin. Ainsi, une étude bibliographique associée à une analyse in silico et à une démarche expérimentale rationnelle a permis de développer un milieu chimiquement défini répondant aux critères de robustesse du procédé et de qualité du produit. Par ce travail, il a pu être défini les facteurs nutritionnels et environnementaux influents sur la production de biomasse et de PRP. Plus généralement, ce travail a permis une meilleure compréhension de la physiologie cellulaire. Une étude Bioinformatique et Transcriptomique a permis l’établissement de cartes métaboliques spécifiques de la souche Hib de notre étude, et de mieux comprendre l’influence du milieu de culture sur la production de polyoside capsulaire. En complément, une étude Fluxomique a permis de développer les connaissances sur le métabolisme général de la souche et plus particulièrement sur la voie de synthèse du polyoside.Sur un plan plus appliqué, une corrélation directe entre la consommation de glucose et la production de polyoside a pu être établie, et ce, dans un environnement maîtrisé de culture en fermenteur. La combinaison du travail développé tant sur le procédé que sur le milieu de culture chimiquement défini ou la sélection de souche, a permis, à l’échelle laboratoire, une multiplication par 6,2 de la production de polyoside capsulaire (7,8 en spécifique) par rapport à la condition initiale de l’étude en milieu complexe. Ce résultat a alors pu être validé chez Sanofi-pasteur à l’échelle pré-industrielle (1000L), tout en conservant une qualité du produit conforme aux critères de production pharmaceutique / This work, undertaken at LISBP (Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, INSA Toulouse), concerns the study of the pathogen Haemophilus influenzae type b (Hib). The objective was to improve the understanding of cellular metabolism and the synthesis of the capsular polysaccharide in Hib, used for vaccine production. A literature review coupled with a rational experimental approach has enabled a chemically defined medium that meets the criteria of process robustness and product quality to be developed. This work has defined the key nutritional and environmental factors affecting biomass and PRP production. Bioinformatics and Transcriptomic studies have allowed the specific metabolic characteristics of the Hib strain to be mapped and enabled a better understanding of the influence of culture medium on capsular polysaccharide production to be obtained. A Fluxomics study points to specific organization of the central pathways and more specifically the interaction between the pentose phosphate pathway and the pathway of polysaccharide biosynthesis. On a more applied aspect, direct correlation between glucose consumption and production of polysaccharide was established in a batch culture. The combined knowledge obtained in this study enabled a 6.2-fold increase in production of capsular polysaccharide (7.8 in specific production) to be obtained in laboratory scale installations as compared to the initial fermentation process using complex media. This result was then validated at Sanofi-pasteur at the pilot-plant level (1000L), and shown to maintain product quality as defined by pharmaceutical production criteria

Antibiotic resistance in bacteria is a growing threat to global health. Many of the genes responsible for resistance are carried on mobile genetic elements which can be transferred laterally between strains and species. The most important of these are conjugative and mobilisable elements including plasmids and integrating and conjugating elements, ICEs. Haemophi/us influenzae is an important human pathogen, which was first identified as carrying antibiotic resistance genes in the 1970s. Much of this resistance is encoded by ICEHin1056, which is present in H. influenzae strains worldwide. The aims of this study were to describe features of the biology of ICEHin1056, with particular reference to the genetic site and control mechanisms responsible for instigating conjugative transfer. The origin of transfer has been localised to a sequence on ICEHin1056 and an environmental stressor initiating conjugative transfer, oxidative stress, has been identified. In addition, detailed phylogenetic analysis has demonstrated ICEHin1056 to be part of a much larger family of mobile genetic elements, widely distributed in proteobacteria and carrying accessory genes responsible for survival in adverse environments, virulence and antibiotic resistance. The ICEs in the family have conserved homology of gene content and synteny of gene arrangement over deep evolutionary time, challenging the accepted paradigm of modular mosaicism of mobile genetic elements. A key event in increasing dissemination of the ICE, acquisition of a phage type integrase gene has also been identified. The findings presented provide significant insight into the behaviour of ICEs and may in future allow predictions about the spread of virulence factors and antibiotic resistance genes, with important implications for human and animal health.

Heterogeneity in immunodominant outer membrane proteins has been proposed as a significant factor in the failure of an NTHi infection to induce immune protection against subsequent infections. This study has examined the vaccine potential of three outer membrane proteins in an attempt to identify conserved regions that could be targeted by an immune response after vaccination. The three proteins investigated were: TbpB, P5 and P48 (HI0164). The optimal route of immunisation in clearing a bolus inoculum of NTHi to the lung in the rat has been shown to be a combination of gut sensitisation with a respiratory boost and this regime was used in the present study. A panel of NTHi isolates was assessed to determine the frequency with which strains were able to bind transferrin and thus be targeted by a TbpBspecific immune response. A high proportion of strains was able to bind transferrin with similar frequencies in isolates associated with infection and those from normal throat swabs. A protocol was developed to purify nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase (GST)-rTbpB fusion protein and Glutathione-Sepharose affinity chromatography. Mucosal-directed immunisation with rTbpB significantly enhanced clearance of an NTHi challenge to the lung, however, whilst rTbpB-specific antibodies were cross-reactive on Western immunoblots, the cross-reactivity was variable in both transferrin binding inhibition assays and bactericidal activity. This suggested that the rTbpB-specific humoral response would be variable in the recognition of heterologous NTHi isolates. The secondary structure of P5 has been controversial with several reports suggesting that P5 was a fimbrin protein composed of coiled coils. In this present study the interstrain variation in P5 amongst isolates from diverse anatomical sites, as well as computer prediction methods and spectrophotometric analysis, generated a model of P5 based on the homologous E. coli protein, OmpA. This model suggested a B-barrel conformation with no evidence of coiled coils. Synthetic peptides corresponding to conserved regions of P5 that were thought to be surface exposed, as well as a region (H3) with some homology to a protective epitope in the P. aeruginosa protein, OprF, were then combined with a "promiscuous" T cell epitope from the measles virus F protein (MVF) and used for immunisation studies. Whilst variable protection was seen with the peptides, the MVF/H3 peptide was the most efficacious of the antigens assessed in this study in enhancing clearance of NTHi. This occurred in the absence of detectable peptide- or PS-specific antibody leading to the suggestion that cell mediated responses may have played an important role in enhancing clearance in this model. The highly conserved nature of the region in P5 represented by the H3 peptide suggests that further study should be focused on this peptide as a potential NTHi vaccine candidate. The last antigen, P48, is homologous to a A. pleuropneumoniae antigen, AopA, which has been proposed to have potential as a vaccine component against pleuropneumonia in pigs. Sequence analysis of the gene encoding P48 from several isolates showed that this protein was well conserved. Recombinant P48 was purified from a GST-rP48 fusion protein and used for immunisation, which also conferred significant protection. However, immunisation with rP48 was not as efficacious as immunisation with the MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody titres, no bactericidal activity could be detected indicating that bactericidal antibody had not contributed to the observed clearance. In addition, the rP48- specific serum IgG was predominantly of the IgG2a isotype suggesting that Thl cell mediated responses had been induced by immunisation with rP48.

One of the major challenges of vaccine development is the conservation of immunogenicity and protective efficacy through the stages of design, production, formulation and delivery. The critical issue is that how and in what form an antigen is taken up by antigen presenting cells for proteolytic processing and presentation to the immune system bound to MHC can have dramatic effects on the activation of Th cells to drive clonal responses and induction of immunological memory. Nontypeable Haemophilus influenzae (NTHi) is a pathogenic commensal of the human respiratory tract that causes diseases with enormous socioeoconomic burdens. There is no licensed vaccine, although the potential for vaccination with outer membrane components to reduce the incidence of disease caused by NTHi has recently been demonstrated in clinical trials. The issue of immunomodulation was explored in this thesis in the context of the further evaluation of a leading NTHi vaccine candidate, the outer membrane protein OMP26. The efficacy of recombinant OMP26 (rOMP26) against NTHi challenge has been previously demonstrated in mice, rats and chinchillas. In rats, efficacy was shown to be restricted to the precursor form (containing the signal peptide) and not the mature form of rOMP26. The immunodulatory effects of changes to the rOMP26 structure were further investigated in this thesis. A range of structural variants of rOMP26 were constructed in view of reducing extraneous plasmid-derived sequence from the antigen and to introduce a unique cysteine residue as a potential conjugate site for multivalent vaccine development (Chapter 2). It was demonstrated that minor structural changes to rOMP26 such as the addition, deletion, modification or relative positioning of a single amino acid or bulky group, designed to increase the efficiency of production or introduce (cysteine) conjugation sites, altered the expression of the protein in E. coli and the immunogenicity in Balb/C mice. Furthermore, in contradiction to the published report (El-Adhami et al. 1999) and a new study in rats (Chapter 3), there was no positive effect of the signal peptide in mice, with precursor and mature forms of rOMP26 equally immunogenic (Chapter 2). Following confirmation of the need to retain the signal peptide for the immunogenicity of rOMP26 in rats, a precursor form (rOMP26VTAL) in which the conserved n-region of the signal peptide was deleted, and shown to reduce the efficiency of the cleavage of the signal peptide by signal peptidase during protein overexpression in E. coli (Chapter 3). Not only did this deletion result in an increase the yield and stability of the purified precursor protein, but rOMP26VTAL was highly immunogenic and enhanced the clearance of NTHi from the lungs of challenged rats. The potential for signal peptides to be exploited as an immune-enhancing moiety in a proteinaceous vaccine is discussed. Following the development of rOMP26VTAL as a production optimised variant of rOMP26, the next step was to test the feasibility of rOMP26VTAL as a component of a multivalent vaccine (Chapter 4). Two chimeras were constructed with LB1(f)2,1,3, a trivalent synthetic B-cell epitope from the extracellular loop 3 region of the P5 fimbrin protein of NTHi, positioned at the N- or C-terminus of rOMP26VTAL. The solubility of rOMP26VTAL was affected by the fusion, with both chimera constructs expressed only in the insoluble fraction, thus requiring a denaturing protocol for purification. Although rLB1(f)2,1,3-OMP26VTAL was expressed and purified as a more stable protein and in greater yield than rOMP26VTAL-LB1(f)2,1,3, the relative positioning of the fusion was important and rOMP26VTAL-LB1(f)2,1,3 was significantly more immunogenic in rats than rLB1(f)2,1,3-OMP26VTAL. In addition, rOMP26VTALLB1( f)2,1,3, but not rLB1(f)2,1,3-OMP26VTAL induced a significant degree of bacterial clearance following pulmonary challenge with NTHi, in levels comparable to the highly efficacious rOMP26VTAL construct. In the third part of the thesis, bacterial ghosts were evaluated as a novel mucosal delivery technology for rOMP26VTAL and rOMP26VTAL-LB1(f)2,1,3, (Chapter 5). To mimic the natural presentation of OMP26 and P5 fimbrin antigens on the cell surface of NTHi, an OmpA� sandwich fusion surface display system was developed for the outer membrane expression of the OMP26 constructs in E. coli ghosts. Following gut immunisation, but not intranasal immunisation even when co-administered with the cholera toxin�derived adjuvant CTA1-DD, bacterial ghosts were successful at presenting OMP26VTAL and rOMP26VTAL-LB1(f)2,1,3 to the immune system for the induction of enhanced clearance of NTHi in the rat pulmonary challenge model. Although this study was the first to demonstrate enhanced bacterial clearance induced by heterologous antigens expressed in the outer membrane of bacterial ghosts, future studies with ghosts will require optimisation of the expression levels of the OmpA� fusion proteins possibly to avoid cross-reactive responses related to high doses of ghosts in the inoculum. This thesis presents data that both supports the further evaluation of rOMP26 constructs for clinical trials, and has demonstrated the significant effects of structural changes, method of production and delivery system can have on the immunogenicity of a candidate vaccine. Such knowledge will contribute to and provide some new approaches for enhancing the efficiency of vaccine development against a range of diseases including those caused by NTHi. Major Outcomes: 1. Demonstration that the immunogenicity of rOMP26 antigen constructs is affected by structural modifications and their positioning within the construct, and by the delivery system. 2. Development of rOMP26VTAL, an rOMP26 construct with the KNIAK sequence deletion of the signal peptide n-region. This protein retains the immunogenicity and protective efficacy of rOMP26, but is produced with reduced cleavage of the signal peptide, resulting in higher yields and a stable protein. Lacks extraneous plasmidderived multiple cloning site sequence, and is produced in high yield as a stable protein. 3. Construction of a NTHi rOMP26VTAL-LB1(f)2,1,3 chimera antigen that induced enhanced clearance of NTHi in an acute pulmonary challenge model in rats. 4. Development of an OmpA� surface display system for the expression of rOMP26 antigen constructs in the outer membrane of E. coli/bacterial ghosts 5. Bacterial ghosts were successful as delivery vehicles for rOMP26 candidate vaccine constructs when delivered in the gut.