Development of In Vitro assay for viral escape from broadly neutralizing antibodies

In vitro models to study HIV-1 escape from broadly neutralizing antibodies (bNAbs) are highly important for designing in vivo bNAb combination therapy. Frequently, short-term viral escape is studied in cell lines, which do not express physiological levels of receptors or with antigenic libraries that do not allow for the observation of concurrent escape or compensatory mutations. We designed an in vitro viral escape assay to measure the ability of HIV-1 to escape from single bNAbs in a high-throughput manner. We tested the multiplicity of infection (MOI) of virus, cloned and un-cloned virus stocks, and different concentrations of antibody. From these results, we developed a 56-day assay to measure escape from bNAbs by adding multiple concentrations of antibody that is gradually increased over time. In this assay, we observed both common escape mutations previously published, but also novel mutations that could be either escape or compensatory mutations. This in vitro bNAb escape assay will lead to a deeper understanding of viral escape, to better inform the design of highly effective bNAb cocktails targeting multiple conserved sites.