Thesis (D.P.H.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 22, 2008). Includes bibliographical references.

Area-wide vaccination against pertussis, also known as whooping cough, has been going on since the 40th - 50th of the last century. In our country this vaccination started in 1958. The World Health Organization estimates that worldwide 20 to 30 million people per year contract pertussis. Patients from developing countries account for 90% of cases. Pertussis is the cause of the death of around 200 000 - 300 000 people per year, up to 85% of whom are children under 2 years of age. As the only source of the bacteria Bordetella pertussis are humans, they are also suitable candidate for the elimination. Therefore, the WHO established an ambitious plan to reduce the incidence of pertussis below 0.1 per 100 000 inhabitants by the year 2000. However, no country succeeded in meeting this plan´s objectives on a long-term basis and since the 80th of the 20th century there has been an increasing trend of the disease incidence across all age groups worldwide. As the proof of severity of this issue pertussis was categorized among the so-called "emerging infectious diseases", that is re-emerging diseases. Diseases whose incidence has increased in recent decades and which could become a major health problem in the near future are classified into this category. In the case of pertussis, the most effective prevention is high vaccination rates in the population, especially children. The theoretical part of the thesis summarizes the current knowledge about pertussis. In the practical part I focused on the incidence and development of the disease in the South Bohemian Region in the years 2005 - 2010, with regard to individual age groups. The results show that in the South Bohemian Region the incidence was rising. The highest incidence was in the age group of 10 to 14 years. Another purpose of the research was to compare the incidence of pertussis in the South Bohemian Region with other regions of the CR. The South Bohemian region in the surveyed 6-year-period ranked among the regions with the highest incidence.

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Made available in DSpace on 2014-09-09T12:27:46Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 174.pdf: 1434437 bytes, checksum: d0f5b3f583c5599b623f3257623452b8 (MD5) Previous issue date: 2003 / De acordo com reunião entre produtores e comitê de expertos da OMS, realizada em novembro de 1998, ficou estabelecida a necessidade do desenvolvimento de novas metodologias alternativas que atestem a atividade imunogênica de novas formulações de vacinas pertussis acelulares e de outras preparações sem a participação de estudos clínicos complementares. O aumento do número de notificações ocorrido nos últimos vinte anos, reforça a busca de modelos experimentais que assegurem a qualidade das vacinas disponíveis no mercado. No presente trabalho lotes de vacina pertussis celular e acelular foram testados pelos métodos de desafio intranasal (DIN) e ELISA. Os resultados alcançados foram comparados aos resultados obtidos pelo ensaio de desafio intracerebral (DIC). Em nossos estudos percebemos a existência variações intra e interlaboratoriais quanto aos resultados obtidos pelo método clássico de DIC. Análises estatísticas demonstraram que os métodos DIN e ELISA apresentaram repetibilidade e capacidade em discriminar os produtos testados. Somado a isto, foi possível uma acentuada redução no número de animais utilizados bem como no nível de estresse proporcionado aos mesmos. A reprodutibilidade dos ensaios sugere que os métodos podem ser utilizados como alternativa, também sob nossas condições, na verificação de consistência de produção lote a lote, análise prévia, além de auxiliar nos estudos visando melhor entendimento dos componentes envolvidos nos mecanismos de infecção e de resposta imune à infecção por Bordetella pertussis. / De acordo com reunião entre produtores e comitê de expertos da OMS, realizada em novembro de 1998, ficou estabelecida a necessidade do desenvolvimento de novas metodologias alternativas que atestem a atividade imunogênica de novas formulações de vacinas pertussis acelulares e de outras preparações sem a participação de estudos clínicos complementares. O aumento do número de notificações ocorrido nos últimos vinte anos, reforça a busca de modelos experimentais que assegurem a qualidade das vacinas disponíveis no mercado. No presente trabalho lotes de vacina pertussis celular e acelular foram testados pelos métodos de desafio intranasal (DIN) e ELISA. Os resultados alcançados foram comparados aos resultados obtidos pelo ensaio de desafio intracerebral (DIC). Em nossos estudos percebemos a existência variações intra e interlaboratoriais quanto aos resultados obtidos pelo método clássico de DIC. Análises estatísticas demonstraram que os métodos DIN e ELISA apresentaram repetibilidade e capacidade em discriminar os produtos testados. Somado a isto, foi possível uma acentuada redução no número de animais utilizados bem como no nível de estresse proporcionado aos mesmos. A reprodutibilidade dos ensaios sugere que os métodos podem ser utilizados como alternativa, também sob nossas condições, na verificação de consistência de produção lote a lote, análise prévia, além de auxiliar nos estudos visando melhor entendimento dos componentes envolvidos nos mecanismos de infecção e de resposta imune à infecção por Bordetella pertussis.

Made available in DSpace on 2014-07-28T18:10:50Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 98.pdf: 3463846 bytes, checksum: 8603b620b9560561e10f307f624ab539 (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:35Z (GMT). No. of bitstreams: 2 98.pdf: 3463846 bytes, checksum: 8603b620b9560561e10f307f624ab539 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Dentre os efeitos induzidos pela toxina pertussis (TP) em mamíferos, ocupa um lugar de destaque o fenômeno conhecido por sensibilização aos efeitos biológicos e letais da histamina, cuja intensidade e constância proporcionaram o estabelecimento de um ensaio in vivo de controle de qualidade para avaliação da segurança de vacinas contra a pertussis (coqueluche) e tríplice bacteriana contra a difteria, tétano e coqueluche (DTP). O ensaio de sensibilização à histamina (ESH) em camundongos NIH fêmeas mostrou-se altamente sensível à toxina pertussis de referência NIBSC 90/518 (TPR) detectando níveis tão baixos quanto 20 ng de TP/dose. Todas as 5 vacinas DTP de um produtor nacional noESH apresentaram níveis de TP ativa que variaram entre 84 a 147 ng/mL, valores inferiores ao valor limite de 1789 ng/mL obtido para a vacina pertussis de referência NIBSC 66/303 (VPR), logo, todas as vacinas foram aprovadas para uso humano. Embora o ESH tenha sido conclusivo quanto à alta especificidade à TP, o elevado número de animais, no mínimo, 40 por ensaio, acarretando alto custo e o sofrimento dos animais são fatores limitantes que dificultam o uso rotineiro como ensaio de controle de qualidade da vacina DTP. O objetivo do nosso estudo foi desenvolver uma metodologia in vitro em preparações de íleo isolado de cobaias Short Hair fêmeas (250 a 300 g) fornecidas pelo CECAL/FIOCRUZ /Rio de Janeiro para avaliação do fenômeno de sensibilização à histamina pela TPR. Todos os experimentos foram aprovados de acordo com as diretrizes estabelecidas pela CEUA/FIOCRUZ. Curvas concentração-efeito à histamina em íleos isolados de cobaias foram analisadas e os parâmetros de concentração efetiva média(CE50), concentração efetiva máxima (CEmax) e de constante de dissociação no equilíbrio do complexo droga-receptor (Kd) para histamina foram determinados [...]. / Among the effects induced by pertussis toxin (PT) in mammalian species, a prominence place is occupied by the phenomenon known as sensitization to the biological and lethal effects of the histamine, whose intensity and constancy promoted the establishment of an in vivo quality control assay to evaluate the safety of the pertussis vaccine (PV) against whooping cough and the triple bacterial vaccine, (DPT) against diphtheria, whooping cough and tetanus. The histamine sensitization assay (HSA) performed with NIH female mice was highly sensitive to reference pertussis toxin NIBSC 90/518 (RPT), detecting levels as low as 20 ng of administered RPT/dose, which caused 50% lethality. All five samples of DPT vaccines from one Brazilian producer presented active PT levels in the range of 84 and 147 ng/ml by the HSA, inferior to the limit value of 1789 ng/mL obtained for reference pertussis vaccine NIBSC 66/303 (RPV), thus all the vaccines were approved for use. Although the HSA has been conclusive in relation to its high specificity for RPT, the large number of mice used (at least 40 per assay) results in high costs and the suffering of the mice are limiting factors that make its routine use as a DPT vaccine quality control assay difficult. The aim of our study was to develop an in vitro methodology in ileum segments from female Short Hair guinea pigs (250-300 g) maintained in the animal facilities of the Oswaldo Cruz Foundation in Rio de Janeiro (FIOCRUZ), Brazil, to evaluate the histamine sensitization phenomenon by RPT. All experiments were approved in accordance with the guidelines of the Committee for Ethics in Animal Use of the FIOCRUZ. Concentration effects curves for histamine in guinea pig ileum were studied and the parameters of mean effective concentration (EC50), maximum effective concentration (ECmax) and dissociation constant of drug-receptor complex (Kd) were determined. No increase in ileum contractile response to histamine was detected in relation to control PBS 4 days after intraperitoneal treatment of guinea pigs with doses and dilutions corresponding to mean histamine sensitization dose (HSD50) obtained in NIH female mice of RPT (40 ng), RPV and of 5 DPT vaccines (0.26 IU). In all the ten assays performed on the experimental group, the data followed normal distribution, the variances were homogeneous and no significant differences occurred between assays. With doses 10 times higher than the HSD50 of RPT (400 ng) and of RPV (2.6 IU), analysis of the data showed the same behavior above. Contrary to the anticipated results, histamine EC50 and Kd values in ileum of guinea pigs treated in vivo with RPT were significantly higher than the control and RPV (p< 0.05) with no alteration in ECmax (p= 0.3672). In vitro 15 min treatment of guinea pig ileum with 30 ng/ml of RPT reduced the ECmax to about half in relation to control (p= 0.0028), with no significant reduction in the mean values of histamine EC50 and Kd (p= 0.09). In contrast, in vitro 15 min treatment of ileum with 40 ng/ml of RPT significantly reduced histamine ECmax (p< 0.0069), EC50 (p= 0.0261) and Kd (p= 0.0479) in relation to control ileum. In vitro 15 min treatment with PBS (390 and 520 µL in 13 mL of Tyrode) did not significantly alter the mean values of histamine EC50 (p=0.4043 and p= 0.1035), ECmax (p= 0.2366 and p= 0.2708) or KD (p= 0.4564 and p= 0.1158) in relation to control without treatment, demonstrating no effect of the control solvent (PBS) on ileum contractile response by histamine. In conclusion, increased histamine sensitization in female guinea pig ileum after in vitro treatment of 30 and 40 ng/ml of RPT was demonstrated.

Despite the success of vaccines in routine use worldwide, there are substantial challenges hampering our ability to develop vaccines against extant diseases including malaria and tuberculosis. Novel approaches are urgently required to help us understand immunological correlates of protection against disease and facilitate our understanding of the impact of human genetic variation on the success of diverse vaccines. To identify host genetic factors responsible for variation in antibody responses against vaccine antigens delivered routinely to infants worldwide I performed a genome-wide association study (GWAS) involving 2,499 infants recruited from three diverse sites across Africa. I identified strong genetic associations between variants in the class II major histocompatibility complex (MHC) locus and responses against five antigens: pertussis toxin (PT), filamentous haemagglutinin (FHA) and pertactin; diphtheria toxin (DT); and hepatitis B surface antigen. To characterise these associations at the gene and allelic level I developed a large, high-resolution (6-digit 'G') population-specific human leukocyte antigen (HLA) imputation reference panel including 697 individuals from the vaccine GWAS typed at 11 genes, highlighting the diversity of HLA across the African continent. Using this panel I imputed HLA into the remaining GWAS dataset to fine-map the associations to specific HLA alleles, amino acid and single nucleotide polymorphism sites; some of which were found to be African specific. I then used these HLA association findings observed with PT response to correlate, through genetics, this trait with susceptibility to whooping cough in an independently recruited and analysed set of cohorts from the UK. I further used these genetic correlations to demonstrate the relevance of levels of PT-specific circulating follicular helper T-cells and TRBV29-1 T-cell receptor gene expression levels in the development of this protective immune response against PT. By using HLA-peptide binding studies I also demonstrate the diversity of mechanisms that are involved in HLA-disease association, showing that the breadth and affinity of DT-peptide binding are increased with HLA-DRB1 alleles associated with increased DT antibody responses. Taken together, these data represent the first comprehensive genetic association study of multiple vaccine responses undertaken in African infants. These results highlight the importance of human genetics in modulating protective responses against vaccine antigens and demonstrate how such associations can be harnessed to understand biological mechanisms of protective efficacy in greater detail that may in turn facilitate future vaccine development.

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Bordetella pertussis produces many virulent factors including adenylate cyclase toxin (CyaA) This toxin preferentially invades cells of immune system with integrin receptor CD11b/CD18 and weakens the immune system of the host. CyaA affects invaded cells in two ways. First, CyaA creates a cation-selective pores in the membrane of invaded cell and causes colloidal osmotic lysis. Second, CyaA converts cytosolic ATP into signal molecule cAMP, which causes a loss of physiological function of invaded cell and also leads to cellular death. The aim of my thesis was to test a suitability of a new model system composed from synthetic lipids - diphytanoyls, for a characterization of pore-forming properties of adenylate cyclase toxin. In the past, asolectin model system comprising many different lipid was used for characterization but it was found to be too complex for defining the role of individual lipids in CyaA activity. Further the effect of cholesterol for activity of CyaA was studied in a new model system because it was found recently that translocation of adenylate cyclase domain takes place at lipids rafts with high concentration of cholesterol. The last aim of my thesis was to characterize a newly discovered type of channel with the two conductance levels. Key words: Bordetella pertussis, adenylate...

The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...