The overall objective of this proposal is to utilize recombinant DNA technology to isolate the major immunogens of Treponema pallidum for examination of their roles in the immunopathogenesis of syphilis. We recently described the identification of a major immunodominant 47,000 (47K) mol wt surface immunogen of T. pallidum. E. coli recombinant DNA clones expressing the major 47K immunogen of T. pallidum also have been isolated. A 1.75 kb DNA fragment encoding the 47K immunogen will be sequenced to derive the first amino acid sequence for a major immunogen of T. pallidum. Simultaneously, "native" and "recombinant DNA-derived" 47K immunogens will be characterized using radioimmunoprecipitation, Western blotting, peptide mapping, N-terminal amino acid sequencing, and hydrophilicity analysis. Genes encoding a 44,000 mol wt immunogen and other immunogens of T. pallidum also will be analyzed by these techniques. Respective "recombinant" immunogens will be analyzed by E. coli cell-free in vitro coupled transcription/translation, or by expression in minicells or maxicells. High level expression (production) of cloned immunogens will be achieved by subcloning genes into the expression vector pKC30, followed by isolation of the product(s) using polyclonal or monoclonal antibody immunoaffinity chromatography. Pre-established (pBR322) or newly-generated (pKC30; controlled expression) T. pallidum genomic clone banks in E. coli will continue to be screened by solid-phase radioimmuno-colony blot (RICB) assay employing immune rabbit serum, human syphilitic serum, and selected anti-T. pallidum monoclonal antibodies, to detect additional E. coli clones expressing relevant T. pallidum immunogens that may not have been recovered by previous strategies. The biological relevance of recombinant immunogens will be tested through immunogenicity studies in animals to determine 1) if antibodies raised against recombinant immunogens are reactive in the T. pallidum immobilization test; 2) if they can block the attachment of T. pallidum to host cells; and 3) their activity in the in vitro-in vivo neutralization test. Long term studies include vaccine trials in animals. Selected genetic studies will determine the genomic complexity of T. pallidum by DNA-DNA hybridization (Cot) analysis using "total clones insert DNA." The activity of T. pallidum gene promoters in E. coli will be examined through the expression of various cloned immunogen genes.