PROJECT SUMMARY The goal of this proposal is to assess how HIV might escape eCD4-Ig, an exceptionally broad and difficult- to-escape entry inhibitor, using an approach we have recently developed for generating a highly diverse proviral library. eCD4-Ig is an engineered antibody-like molecule that potently neutralizes all 270 HIV-1, HIV-2 and SIV isolates that it has been tested against. When expressed by adeno-associated virus (AAV), eCD4-Ig can completely protect rhesus macaques from multiple high-dose challenges with both SHIV-AD8 and SIVmac239 (Gardner et al., Nature, 2015). Moreover, as we show in preliminary data, AAV-eCD4-Ig can suppress an established infection in macaques after cessation of antiretroviral therapy. Consistent with eCD4-Ig's exceptional breadth, our preliminary data also show that escape from eCD4-Ig is considerably more challenging than from broadly neutralizing antibodies. In fact, cell culture and in vivo efforts to select HIV-1 and SIV variants fully resistant to eCD4-Ig have so far been unsuccessful, even under the same conditions in which escape from bNAbs can be readily observed. We have recently developed a novel, highly parallel method for identifying pathways by which HIV-1 escapes an entry inhibitor. This technique allows the introduction of a controlled number of mutations into key targeted regions. We have shown that the library built using this method can be used to rapidly identify viruses resistant to every well- characterized VRC01-class CD4-binding site antibody (Otsuka et al., PLOS Pathogens, 2018). We propose to use the same approach to select HIV-1 variants that are fully resistant to eCD4-Ig, and characterize selected variants for their escape pathways, and their sensitivities to neutralization and ADCC. These studies will provide insights into whether escape from eCD4-Ig can be anticipated in forthcoming human trials, clarify the pathways by which such escape might occur, and perhaps suggest antibodies or other agents that could make such escape less likely.