The goals of this project are to define conserved epitopes in the V1/V2 domain of HIV-1 gp120 that mediate virus neutralization and enhancement, and to use this information to develop an HIV vaccine based on modified V1/V2 miniproteins. The structures of the alternate V1/V2 conformers will be determined by MALDI analysis of proteolytic fragments of the purified isomers. A number of human sera have been identified that contain antibodies that react with conserved V1/V2 epitopes, including some that recognize alternate conformational forms of V1/V2. Also available are sera from V1/V2 immunized macaques that possess either neutralizing or enhancing activities for specific M-tropic HIV-1 isolates. These antibodies will be fractionated by affinity chromatography and characterized for specificity for V1/V2 conformers, cross reactivity against distantly related sequences, and for neutralizing (or enhancing) activity against various viruses. Novel monoclonal antibodies (Mabs) directed against functional targets in the V1/V2 domains will be isolated from hybridoma prepared from mice immunized with various antigens, including V1/V2 miniproteins, recombinant gp120/140 molecules, and mouse cells expressing native HIV-1 Env proteins. These studies will utilize transgenic XenoMouse strains developed at Abgenix that produce human immunoglobulins, in order to isolate human Mabs. Because of the human nature of these antibodies, any Mabs with potent neutralizing activities that are isolated in these experiments could have direct utility as passive immunotherapeutic agents in humans. Hybridomas will be generated by standard technologies and screened against the relevant immunogens. These Mabs will be used to map epitopes that mediate viral neutralization and enhancement. Finally, a panel of mutant V1/V2 miniproteins, based on both the CaseA2 and SF162 sequences, will be prepared. Mutations to be studied will include N-linked glycosylation sites, deletions and alanine substitutions. The immunoreactivies of the mutant proteins will be measured with available Mabs and fractionated human and macaque sera, and the distribution of neutralization and enhancement epitopes on the mutant proteins will be determine by absorption of human and macaque antibodies that possess such activities. Selected mutant proteins that preferentially express neutralization epitopes will be used to immunize rats, and the neutralizing/enhancing activities of the resulting sera compared to that of the parental antigen. Modified V1/V2 miniproteins that induce effective neutralizing responses against clinically relevant HIV isolates would provide the basis of a V1/V2-based vaccine against HIV-1.