The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gpl2O is the major target for antibodies which neutralize cell-free virus and inhibit cell-cell viral transmission. HIV-1 gpl2O also mediates the specific attachment of the virus to the target cell through a high-affinity interaction with the human cell surface glycoprotein, CD4. The development of effective AIDS vaccines and therapeutics would be greatly facilitated by the availability of large quantities of purified gpl2O for detailed immunogenicity and structure determination studies. During the Phase I period, we successfully generated molecular constructs encoding native HIV-1 gpl2O optimized for high-level expression and established stable mammalian cell lines in a system amenable to scale-up production. The major advantage of our system over already existing systems is that it encodes authentic, glycosylated, conformationally- intact gpl2O and that expression is stable and virus-free. During the Phase II period, we will generate molecular constructs, establish high-level expressing stable mammalian cell lines, and purify large quantities of 4 recombinant gpl2O glycoproteins derived from a laboratory-adapted strain and a primary isolate of HIV-1. The major aim is to test these glycoproteins as potential subunit vaccines that induce a broadly neutralizing humoral immune response with high-specificity, high- affinity, and high-titer antibodies against epitopes in gpl2O. In addition, we will produce crystals of gpl2O and gpl2O-CD4 complexes suitable for X-ray crystallographic analysis and structure determination. Finally, we will donate the purified gpl2O molecules to the AIDS Research and Reference Reagent Program of NIAID for worldwide distribution to AIDS researchers.
