Neisseria meningitidis antigens expressed in infection

描述(申请人提供)：脑膜炎奈瑟氏菌是脑膜炎和败血症的重要原因。占所有病例的30%-80%的B组衣壳病毒引起的疾病预防疫苗的传统方法大多不成功，因此需要确定新的候选疫苗。到目前为止，所有候选抗原都是使用在人工培养基中或在上皮细胞或内皮细胞单层上生长的生物体来鉴定的。然而，关于脑膜炎奈瑟氏菌在体内感染过程中的基因表达谱知之甚少。我们的假设是，在菌血症的幼鼠模型中或在人类全血中生长的脑膜炎奈瑟氏菌，与人工培养中表达的基因相比，将显示上调或激活的特定基因。此外，在侵袭性感染过程中上调的基因编码新的蛋白，这些蛋白在脑膜炎奈瑟氏菌中保守，预计可以在表面获得，将是有希望的候选疫苗。我们建议使用实时定量聚合酶链式反应来研究从感染幼鼠的血液和从人类血液感染模型中分离的不同时间点的脑膜炎奈瑟菌的时间基因表达谱，并将其与在肉汤中生长的细菌的表达谱进行比较。我们将评估重组蛋白作为纯化的His标记蛋白或GST融合蛋白，以及作为“天然”蛋白在大肠杆菌外膜囊泡中的免疫原性。免疫小鼠的抗血清将通过EL ISA、FACS、血清杀菌活性和被动保护幼鼠模型进行分析。拟议的研究可能会识别能够产生广泛保护性抗体的新抗原，以预防脑膜炎奈瑟氏菌病，包括目前尚无疫苗可用的B组菌株。此外，活体模型中基因表达的特征将有助于更好地理解脑膜炎双球菌的发病机制。

DESCRIPTION (provided by applicant): N. meningitidis is an important cause of meningitis and sepsis. Conventional approaches to develop a vaccine for prevention of disease caused by capsular group B strains, which account for 30-80% of all cases, have been largely unsuccessful, and therefore there is a need for the identification of new vaccine candidates. To date, all candidate antigens have been identified using organisms grown in artificial media or on epithelial or endothelial cell monolayers. Little is known, however, about the gene expression profile of N. meningitidis during infection in vivo. Our hypothesis is that N. meningitidis grown in the infant rat model of bacteremia or in human whole blood will show specific genes that are up-regulated or activated compared to those expressed in artificial culture. Furthermore, genes that are up-regulated during invasive infection that encode novel proteins that are conserved in N. meningitidis, and that are predicted to be surface-accessible, will be promising vaccine candidates. We propose to use quantitative real-time PCR to study the temporal gene expression profile of N. meningitidis isolated from the bloodstream of infected infant rats and from the human blood infection model at different time-points, and also to compare the respective expression profiles with those of bacteria grown in broth. We will evaluate the immunogenicity of the recombinant proteins as purified His-tagged or GST-fusion proteins, and as "native" proteins in E. coli outer membrane vesicles. Antisera from immunized mice will be analyzed by ELISA, FACS, serum bactericidal activity, and passive protection in the infant rat model. The proposed studies may identify new antigens capable of eliciting broadly protective antibody for prevention of N. meningitidis disease, including group B strains for which there is currently no vaccine available. Also, characterization of gene expression in an in vivo model will lead to a better understanding of meningococcal pathogenesis.