HIV envelope (env) proteins are among the most highly glycosylated eucaryotic proteins, and recent studies have emphasized the importance of glycosylation in the formation of conformational structures necessary for proper function and immunogenicity of HIV env. In the present proposal, we will use a novel recombinant retroviral system for expressing correctly folded and glycosylated domains of HIV env proteins in native forms. These domains can be produced as fusion proteins to the amino-terminal domain of the murine leukemia virus (MuLV) env protein- gp70. We will express several native regions of HIV-1 gp120, including the V1/V2 domain, the V3 loop, the V4/CD4 domain, and dtermine their immunoreactivities. The expressed fragments will be purified by immunoaffinity methods and the immunogenicity of each domains will be evaluated. We will use this system to precisely map the epitopes in V1/V2 recognized by strongly neutralizing monoclonal antibodies. We will also characterize the immunoreativities and immunogenicities of different glycopeptide subregions of the V1/V2 domains and analyze the functional activities of natural antibodies against these domains that are present in sera of infected individuals and that are induced in animals upon immunization with purified fusion proteins. The goal of this work is to define native epitopes important for HIV neutralization and to identify additional domains and key epitopes capable of producing conserved neutralizing responses, that may have been missed by previous studies because of their conformationally active and glycan-dependent nature. Once such sites have been identified, novel features of the MuLV-based expression system can be used to produce these regions in highly immunogenetic forms, suitable as effective HIV vaccines.
