The overall aim of our studies is to design HIV envelope immunogens capable of eliciting broadly reactive neutralizing antibodies (NAbs) and to evaluate the protective potential of these antibodies in nonhuman primates in the context of homologous and heterologous viral challenge. We believe that in order to improve the potential of HIV envelope-based immunogens to elicit cross-reactive NAbs, a comprehensive, iterative, approach is required. We propose the following: First, identify modifications that result in the maximal increase in exposure of conserved neutralization epitopes on trimeric gp140 envelope proteins. Second, evaluate and compare in rabbits the immunogenic properties of such constructs, emphasizing on the generation and characterization of NAbs; and, third, utilize the information gathered from the immunogenicity studies to further ameliorate the design of our immunogens in an attempt to improve their ability to elicit cross-reactive NAbs. The most promising immunogens will be tested in non-human primates, where the protective potential of the antibodies elicited by such immunogens, during homologous and heterologous viral challenge, will be determined. Although we will continue to identify combination of modifications, such as hypervariable loop deletions or deglycosylation, that enhance the exposure of conserved neutralization epitopes, we also propose to engineer a new type of gp140 trimers, termed heterotrimers, as potential immunogens. These heterotrimers are formed by the assembly of gp140 monomers that differ in their amino acid composition and glycosylation pattern. We expect that neutralization epitopes will be differentially presented on gp140 heterotrimers than on the commonly used gp140 homotrimers. The following are the three specific aims of our proposal: (1) Engineer soluble gp140 immunogens that readily display neutralization epitopes; (2) Determine how the antigenic structure of HIV gp140 Envelope proteins relates to their immunogenic properties; and (3) Determine whether the anti-viral immune responses elicited by certain modified HIV Envelope gp140 immunogens allow for the less or more frequent emergence of escape mutants in macaques challenged with homologous and heterologous SHIVs.