The broad purpose of this program-project is to show that advances AAV-based gene therapy enable stable, long-term suppression of HIV-1 replication. Our immediate goal is to stably suppress an ongoing SHIV-infection in rhesus macaques, using specific combinations of antiviral proteins delivered by self-complementary AAV vectors. Any effective therapy for HIV-1 infection has to solve the problem of viral escape. This project addresses the problem of viral escape from a cocktail of antibodies iteratively, by rapidly identifying viral escape pathways and adjusting these cocktails so that these pathways are blocked. We are aided in this effort by a variant of CD4-lg, fused to a 14-amino-acid peptide that closely mimics a high-affinity binding region of CCR5. This enhanced CD4-lg (eCD4-lg) neutralizes as efficiently as the best neutralizing antibodies and binds only necessarily conserved regions of gp120. Thus escape from eCD4-lg is more difficult, and may coincide with a decrease viral fitness. We therefore first determine which anfi-gp120 CD4- and CCR5-binding site antibodies best complement escape from eCD4-lg. To do so, we develop libraries of SHIV proviruses diversified in their env genes. Using several such libraries, we select in parallel a number of envelope glycoproteins resistant to eCD4-lg. We determine which antibodies best neutralize these resistant variants, and generate two antibody cocktails which we hypothesize will best suppress in vivo viral escape. We then evaluate these cocktails in vivo, using self-complementary AAV (scAAV) vectors to deliver scFv-Fc forms of these antibodies to SHIV infected macaques. Envelope glycoproteins from viruses that escape In vivo will be cloned, characterized, and used to select by phage display new variants of already broadly neutralizing antibodies. These antibody variants can either replace or complement the original antibody in a cocktail. We then repeat the process to develop new antibody cocktails and again characterize their efficacy in macaques. This project will: (1) determine whether and under what circumstances viral escape can be suppressed, (2) generate broader and more potent forms of already exceptional HIV-1 neutralizing antibodies, and (3) identify a set of scAAV-deliverable inhibitors that may suppress HIV-1 replication in humans.