This project will use the transgenic """"""""XenoMouse"""""""" system, developed by Abgenix, to isolate novel human monoclonal antibodies against HIV-1 which possess potential clinical utility. These mice contain -2 megabases of the human Ig heavy and kappa light chain loci that functionally recapitulate the human humoral immune system. In preliminary studies, we have immunized Xenomice with a number of HIV envelope antigens, including recombinant fusion proteins expressing several of the variable loop domains of the viral surface protein, and a recombinant gpl20 derived from the primary virus isolate, SF162. This latter antigen was uccessful in eliciting Mabs that recognized native viral envelope proteins, including antibodies with potent neutralizing activities for the SF162 strain. Multiple types of epitopes were recognized, including some that had not been previously described. Domains recognized by neutralizing Mabs included the CD4-binding domain and other conserved conformational epitopes in gpl20, as well as sites in the V3 and V1/V2 hypervariable regions. The most potently neutralizing antibodies were directed against type-specific V1/V2 epitopes. In further proposed studies, the epitopes recognized by current neutralizing antibodies will be fully characterized, and the complete range of their neutralizing activities determined. Additional immunizations will be performed with related gpl20 immunogens derived from additional strains, including non-clade B strains. In order to elicit antibodies to native epitopes important for neutralization that may not survive gpl20 purification, immunizations will also be performed with native envelope complexes expressed on virion particles and on mouse B cells expressing the complete e n v gene. Immunizations will also be performed with viral particles expressing the HIV receptors, CD4, CCR5 and CXCR4, on their surfaces, as well as with mixtures of particles expressing both Env and HIV receptors, in order to generate human antibodies against 'activated gpl20' epitopes, or against the receptors themselves. To facilitate the identification of antibodies with functional activities, hybridomas will be screened directly by a sensitive neutralization assay, in addition to traditional binding assays. Epitopes recognized by Mabs with neutralizing activities will be fully mapped, and the full range of antiviral activities of interesting Mabs will be characterized. It is anticipated that these studies should help define the diversity of neutralization epitopes present both on the surface of HIV-1 and its cellular receptors. Ultimately, these studies may lead to the generation of a cocktail of novel human Mabs with antiviral activities against multiple clinical strains, that could serve as an effective immunotherapeutic reagent to control and prevent HIV infection.
