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. ? ?
