Recently discovered broadly HIV-neutralizing antibodies (bnAbs) are being actively investigated for HIV/AIDS treatment, functional cure and/or prevention. A variety of such bnAbs are known and classified according to their epitopes clusters on the HIV envelope (Env). Rigorous preclinical studies evince potential advantages of engineered bnAbs over currently used antiretroviral drugs (ARVs); including infrequent administration, lower risk of side effects and genotoxicity, capacity to target latent HIV reservoirs, promotion of host antiviral immune responses, and insensitivity to conventional ARV resistance. Currently, the major obstacle for realizing the clinical potential of single bnAbs is that all of them exhibit limits in covering epitope variability, which allows virus escape. One logical mitigation strategy is to utilize combinations of three bnAbs, each targeting a distinct epitope cluster. Combinations that promote extensive concurrent binding of bnAbs to the same targets (virions or trimers) in plasma virus populations are expected to be especially escape resistant. Yet whether and how this goal can be obtained for HIV prevention or therapy remains unclear. Conventional neutralization assays do not directly measure bnAb-virion binding but have shown that combining bnAb classes improves breadth and potency. Models predict that bnAb class combinations can act collectively on a single virus strain. However, clinical trials suggest a more complex picture. Several trials have tested combined bnAbs, but sustained suppression of viremia has not yet been demonstrated. Collectively, these findings introduce several important questions regarding concurrent bnAb binding in vivo: Are certain bnAb class combinations distinguishable as “superior” in establishing concurrent virion/trimer binding within major fractions of plasma virus swarms? How consistently does such desirable coverage occur across individuals and subtypes? Does HIV+ human plasma contain immunoreactive particles (exosomes, immature virions); circulating anti-Env antibodies or other factors that perturb desirable concurrent binding patterns? Goal of the project is to answer these questions by direct analyses of bnAb-virion interactions in native plasma. Our hypothesis is that this unique endeavor can be accomplished by novel quantitative single molecule and fluorescence correlation spectroscopy (FCS) detection methods applied to plasma virions in situ. Two Specific Aims are: Aim 1. Establish the immunoreactivity patterns of bnAb combinations against single virions or envelope trimers; Aim 2. Characterize interactions of bnAbs and bnAb combinations with single virions in HIV+ plasma. Project output will be an unprecedented tier of data informing the nature of combined bnAb action in vivo and prospects for triple bnAb combinations to counter HIV escape. Our data will uniquely define principles, limits, and opportunities in using bnAb combinations to target circulating virions in the HIV+ ...