This proposal aims to use a genome wide screen of the entire Y. pestis complement of proteins to identify novel protective antigens for inclusion in a multivalent vaccine designed to protect military personnel and the civilian community against possible terrorist attacks with weaponized plague bacteria. Of the more than 4000 predicted protein coding genes identified by complete genome sequencing, approximately 476 are predicted to be secreted and/or associated with bacterial membrane and are, as such, potentially protective antigens. We propose to clone the genes for each of these proteins, to express each as a recombinant protein in Escherichia coli and, using antibodies raised in mice against these antigens, definitively determine their cellular location by ELISA and flow cytometry on intact bacteria. Complete genome hybridizations will be used to identify, among these potential candidates, genes common to the majority of clinical isolates of Y. pestis. In addition, the candidate list will be further refined by analysis of gene expression in vitro and in vivo using DNA chip technology. Finally, the capacity of the candidate antigens to induce immunologic protection will be assessed by in vivo challenge experiments in mice and by assays of serum bactericidal activity in vitro. We expect to identify several novel antigens capable of conferring protection to mice against aerosol challenge with virulent Y. pestis bacteria. The approach of using modern genomic technologies in vaccine design has been pioneered in our laboratories and has been termed "reverse vaccinology". The approach has been remarkably successful in the identification of novel antigens that induce protection against Neisseria meningitidis, some of which are included in a vaccine currently undergoing clinical evaluation. [unreadable] [unreadable]