Detection Assays for Virion Susceptibility to HIV Broadly Neutralizing Antibodies in Plasma and Culture Fluids - Broadly HIV-neutralizing antibodies (bnAbs) with extreme breadth and potency represent promising options for HIV/AIDS treatment, functional cure and/or prevention. However, all bnAb epitope classes exhibit distinct gaps in covering viral Env diversity in populations of people living with HIV. Consequently, prevention trials experience unpredictably restricted efficacy. Further, treatment trials inadvertently enroll infected participants harboring bnAb-resistant variants in plasma and/or tissue compartments. As noted in RFA-AI-22-022, such subjects have little chance of clinical benefit, and therefore negatively skew estimates of efficacy and clinical utility. There is an unmet need for innovative clinical assays that rapidly and prospectively detect bnAb- resistant HIV variants in potential therapy/cure trial participants; and characterize resistance in breakthrough infections in prevention trials. New tests must also meet the additional complexities of testing triple bnAb class combinations, which seems imminent. Current screening approaches do not meet these needs as they are time consuming, labor intensive, technically complex, involve pseudovirus production and testing, and require RNA or DNA sequencing which may be unreliable, depending on the subject and sample. A promising approach toward expediting and expanding neutralization resistance testing is based on direct, quantitative detection of antibody-target binding within a subject’s virus population. The underlying rationale is that neutralization stems from virion binding; resistant variants are by nature poorly (or not) immunoreactive. Thus, a certain fraction of bnAb-unbound virus in a population will presage viral outgrowth/escape during treatment by the test bnAb. Also, bnAb-virion binding can be rapidly and readily quantified. However, this approach demands technology with the sensitivity to quantify bnAb-bound (sensitive) and unbound (resistant) virus fractions in plasma samples or culture fluids, with minimal target perturbations. To date, such technology has not come forward. Our ongoing studies now show that these demands may be met by confocal fluorescence correlation spectroscopy (FCS) techniques for detecting antibody binding at the single virion level. Using fluorescent labeled bnAbs in FCS, we can simultaneously count, in one multiplex assay, the numbers of virions in a sample population that are bound by one or more bnAb types or avoid recognition altogether. Further, virions concurrently bound to multiple bnAb types can be counted by cross- correlated signals. Our goal for this R61/R33 project is to translate this technology into a rapid, economical, multiplex clinical test for blood or culture samples that: 1) determines the presence and proportions of virus sub- populations in a human sample that are covered (or ignored) by one or more bnAbs in a triple class combination and thus reliably and sensitively 2) identifies people living with HIV who harbor resistant viruses risking rebound under bnAb treatment or 3) characterizes the nature of breakthrough infections in prevention trials. The impact of this project will be to advance bnAb resistance detection capacities to support numerous bnAb clinical trial activities, from screening volunteers to tailoring subject-specific bnAb combinations for treatment or cure.
