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.
The goal of this proposal is to investigate how HIV-1 can escape an exceptionally broad and hard-to-escape HIV-1-entry inhibitor, eCD4-Ig. To do so, we will apply a novel and powerful approach developed in our laboratories for identifying inhibitor-resistant HIV-1 isolates. These studies will provide insights necessary for the forthcoming clinical trials of this inhibitor.
