Neisseria meningitidis antigens expressed in infection

描述（由申请人提供）：N。脑膜炎是脑膜炎和败血症的重要原因。开发用于预防由荚膜组B菌株（占所有病例的30-80%）引起的疾病的疫苗的常规方法在很大程度上是不成功的，因此需要鉴定新的候选疫苗。迄今为止，所有候选抗原都是使用在人工培养基中或在上皮或内皮细胞单层上生长的生物体鉴定的。然而，关于N.体内感染期间的脑膜炎。我们的假设是N.在菌血症的幼鼠模型或人全血中生长的脑膜炎球菌将显示与人工培养物中表达的那些相比被上调或激活的特异性基因。此外，在侵入性感染过程中上调的基因编码在N.脑膜炎，预计是表面接触，将是有希望的候选疫苗。我们拟采用实时荧光定量PCR技术研究N.在不同时间点从感染的幼大鼠的血流和从人血液感染模型分离的脑膜炎球菌的表达谱，并且还将各自的表达谱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标记或GST融合蛋白以及作为E.大肠杆菌外膜囊泡。将通过ELISA、FACS、血清杀菌活性和幼年大鼠模型中的被动保护来分析来自免疫小鼠的抗血清。这些研究可能会发现新的抗原，能够激发广泛的保护性抗体，用于预防N。脑膜炎病，包括目前没有可用疫苗的B组毒株。此外，在体内模型中的基因表达的表征将导致更好地理解脑膜炎球菌的发病机制。

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.