The aim of this proposal is to identify and isolate the immunodominant antigen Treponema pallidum (Nichols) which are responsible for eliciting protective immunity in rabbits and man. Lymphocyte hybridomas producing monoclonal antibodies directed specifically against T. pallidum antigens have been produced through the fusion of T. pallidum-sensitized mouse spleen cells with mouse plasmacytoma cells. Additional hybridomas producing monoclonal antibodies against treponemal antigens will be produced and identified by employing radioimmunoassay (RIA) using T. pallidum cells as solid-phase antigen and protective IgG isolated from immune rabbit serum as a positive control. Hybridomas producing cross-reactive monoclonal antibodies of questionable utility and directed against treponemal group antigens are identified using T. phagedenis biotype Reiter cells as antigen in the RIA screen. Monoclonal antibodies directed specifically against relevant immunodominant antigens of T. pallidum are tentatively identified by employing radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems utilizing syphilitic human serum or protective immune rabbit serum as positive controls. Monoclonal antibodies against immunodominant T. pallidum antigens will be isolated and purified in large quantities from respective hybridoma cell cultures, and used in affinity chromatography to bind the protective antigens from fractionated T. pallidum cells. Isolated antigens will be collected, concentrated, and assayed for physical and chemical composition by routine analytical methods. Antigen preparations will also be tested for immunogenicity in rabbits. Antibodies produced by rabbits in response to these antigens will be investigated with regard to their ability to inactivate virulent T. pallidum cells, as demonstrated through the in vitro-in vivo rabbit neutralization test of Bishop and Miller. Purified T. pallidum antigen preparations will also be tested for efficacy as vaccinogens against syphilitic infection in rabbits.
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