This proposal seeks to identify novel neutralization epitopes formed on the HIV-1 envelope protein following its interaction with cellular receptors. The approach is to reconstitute in vitro the molecules required for HIV-1 fusion using purified recombinant HIV-1 envelope proteins, the chemokine receptors CCR5 and CXCR4, and soluble CD4. Because CD4 and a fusogenic chemokine receptor appear to be necessary and sufficient cellular mediators of HIV-1 fusion, it is hypothesized that their binding to the envelope protein may induce it to adopt a fusogenic conformation in vitro. Envelope proteins imparting different viral tropisms will be examined both as monomeric gpl20 proteins and as cleaved ectodomain gpl60 proteins since the two forms may interact with cellular receptors in substantially different manners. These interactions will be measured using quantitative surface plasmon resonance methods that will provide a detailed understanding of the interactions leading from virus attachment to virus fusion. The most promising bi- and trimolecular soluble CD4-envelope-chemokine receptor complexes will be used as immunogens in mice and as antigens for panning phage display libraries of antibody genes prepared from HIV+ donors. The antibodies thus generated will be screened for binding specificity and for neutralizing activity in HIV-1 infection and fusion assays.
