PROJECT SUMMARY We have developed an antibody-like HIV-1 entry inhibitor, eCD4-Ig, composed of the first two domains of CD4 fused to an antibody Fc domain and a short tyrosine-sulfated CCR5-mimetic peptide. eCD4-Ig has properties that make it an exceptionally promising tool in the fight against the HIV-1 pandemic. Specifically, it is broader than any broadly neutralizing antibody (bNAb), at least as potent at neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC), more difficult to escape, less immunogenic, and uniquely capable of amplifying the ADCC activity of non-neutralizing antibodies in patient sera. When expressed by an adeno-associated virus (AAV) vector, it can protect rhesus macaques from SHIV and SIV challenges more effectively than any conventional vaccine strategy, and, as we show here, it can suppress viral rebound after cessation of combined antiretroviral therapies (cART). In short, the case for optimizing eCD4-Ig is strong. Here we describe a series of cell-culture and animal studies that will further extend eCD4-Ig’s half-life, improve its potency, and reduce its immunogenicity. These improvements will increase the safety and efficacy of eCD4-Ig as an infused protein and as an AAV-expressed transgene, and bring us closer to our goals of sustained drug-free HIV- 1 remission and effective long-term prophylaxis against HIV-1 infection.