The goals of this project are to define conserved epitopes in the V1/V2 domain of HIV-1 gp120 that mediate virus neutralization, and to use this information to develop an HIV vaccine based on modified V1/V2 miniproteins. Novel monoclonal antibodies (MAbs) directed against functional targets in the V1/V2 domains will be isolated from infected humans and from mice immunized with HIV antigens, including V1/V2 miniproteins, recombinant gp120/140 molecules, and mouse cells expressing native HIV-1 Env proteins. Human MAbs will be isolated by EBV transformation of B cells followed by fusion with myeloma cells to generate hybridomas. Mouse MAbs will be made from transgenic mouse strains (Xenomouse, developed by Abgenix) that produce human immunoglobulins. These MAbs will be characterized for their epitope specificities, crossreactivity and ability to mediate viral neutralization and enhancement. Further information regarding the V1/V2 epitopes that mediate either neutralization or enhancement will be obtained by fractionation of antibodies possessing these activities from polyclonal human or macaque sera. For these experiments we will use human sera from patients infected with viruses from clades A, B, and E that possess crossreactive anti-V1/V2 antibodies, and macaque sera that neutralize primary viruses obtained from macaques immunized with V1/V2 fusion proteins. Antibodies will be isolated by chromatography on V1/V2 affinity columns, and characterized for specificity for V1/V2 conformers, for crossreactivity against distantly related sequences, and for neutralizing (or enhancing) activities against various viruses. In order to further define neutralization epitopes additional mutant V1/V2 miniproteins, based on both the CaseA2 and SF162 sequences, will be generated. Mutations to be studied will include deletions, modifications of N-linked glycosylation sites, and alanine substitutions of key residues. The immunoreactivities 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 determined by using these proteins in depletion assays with human and macaque sera that possess such activities. Selected mutant proteins which preferentially express neutralization epitopes will be used to immunize rats and macaques, 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.
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