Neisseria meningitidis antigens expressed in infection

描述（由申请人提供）：脑膜炎奈瑟菌是脑膜炎和败血症的重要病因。包膜B群菌株占所有病例的30-80%，开发疫苗以预防这种疾病的传统方法在很大程度上是不成功的，因此需要确定新的候选疫苗。迄今为止，所有候选抗原都是用人工培养基或上皮细胞或内皮细胞单层培养的生物来鉴定的。然而，人们对脑膜炎奈瑟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，在菌血症的婴儿大鼠模型中生长的脑膜炎奈瑟菌或在人全血中生长的脑膜炎奈瑟菌与在人工培养中表达的基因相比，会显示出特定的基因被上调或激活。此外，在侵袭性感染期间被上调的基因编码脑膜炎奈瑟菌中保守的新蛋白，并且预计可表面接近，将成为有希望的候选疫苗。我们拟采用实时荧光定量PCR技术，研究从感染的幼鼠和人血液感染模型中分离的脑膜炎奈索菌在不同时间点的时间基因表达谱，并与肉汁中培养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，以及作为大肠杆菌外膜囊泡中的“天然”蛋白的免疫原性。免疫小鼠的抗血清将通过ELISA， FACS，血清杀菌活性和婴儿大鼠模型的被动保护进行分析。拟议的研究可能鉴定出能够引发广泛保护性抗体的新抗原，以预防脑膜炎奈瑟菌病，包括目前没有疫苗的B组菌株。此外，在体内模型中表征基因表达将有助于更好地了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.