(Project 2 ? Limiting anti-drug antibodies against AAV-delivered bNAbs) Given the remarkable collection of human monoclonal antibodies with potent neutralizing activity against a broad range of HIV-1 isolates (bNAbs), it has become possible to envision long-term delivery of a combination of such antibodies as a means for achieving stringent, long-term virological control in the absence of continuing antiviral drug therapy. In preliminary data and recently published paper in Immunity, we shown that sustained AAV-mediated expression of two bNAbs, 10-1074 and 3BNC117, introduced 86 weeks after an untreated SHIV-AD8 infection resulted in robust long-term suppression of viral replication for over three years. This monkey has been referred to as ?the Miami monkey?. We also nonetheless show that consistent delivery of bNAbs using AAV has been severely hampered by anti-drug antibody (ADA) responses to the bNAb in the majority of macaques. Although the broad antibody-like immunoadhesin eCD4-Ig is less immunogenic, it also raises ADA that could potentially limit its efficacy or its safety in humans. This project is therefore committed to finding approaches that can effectively and practically suppress ADA responses to an antibody, using the immunogenic bNAb 3BCN117 as our primary test case. In this project we will evaluate three approaches for doing so: (1) elimination of CpG motifs in the transgene, (2) oral tolerization, and (3) tolerization with a bispecific antibody that coordinates bNAb binding to erythrocytes. In addition, we will assist in evaluating the ADA responses from different capsids and promoters (Project 1), and those from an AAV transgene whose expression has been delayed by four months (Project 4). We will then use the best combination of these approaches and AAV- expressed bNAbs to establish and characterize functional cures in ART-treated and untreated rhesus macaques. This project will therefore address a central challenge to the use AAV-expressed bNAbs for HIV-1 prophylaxis and therapy.