Project Summary Broadly neutralizing antibodies (bNAb) against HIV Env represent a novel class of antivirals that have the potential to neutralize viral entry, while simultaneously activating non-neutralizing immune effector functions. However, a major obstacle for their clinical use is identifying the virologically suppressed HIV+ patients that are best suited for a given bNAb or bNAb combination. Indeed, early-stage therapeutic studies with bNAb have not found uniform concordance between viral outgrowth assay sequences (in vitro) and the Env resistance mutations that arise in treated patients in vivo. Our laboratory has developed robust and quantitative flow cytometry-based assays for neutralization of both cell-free and cell-to-cell infection. We have observed decreases in the potency and efficacy of Abs using cell-associated versus cell-free HIV-1 neutralization assays. The detection of these cell-associated phenotypes may be particularly important for cure-based approaches where functional reactivity of bNAbs against infected cells may be important for in vivo efficacy. To overcome the limitations of PCR-first based approaches that often identify non-functional Env, we propose the development of a single cell Viral Outgrowth Neutralization Assay (scVONA) that will be performed in the presence and absence of bNAb. This scVONA will simultaneously quantify viral latent reservoir sequences and determine their sensitivity to bNAb. To accomplish this, scVONAs will leverage and enhance a highly sensitive single cell reporter cell line to detect infection and identify cells to test for neutralization of cell-to-cell spread. The ultimate readout are single cell flow cytometry measures that enable capture of resistant env genes by sorting and sequencing insensitive versus sensitive clones. Long read Env sequencing will be employed for bioinformatic analyses to determine linked sequences that correlate with resistance. Titration of bNAb will also provide estimated IC50 and sequences may allow a measure of the fraction of sensitive clones. We believe that scVONA will provide a rapid <2 week readout of neutralization sensitivity and simultaneously provide a measure of functionally intact env genes for confirmatory analyses, significantly reducing the time, cost and labor associated with identifying bNAb resistant sequences from HIV+ patient samples.