Certain broadly HIV-neutralizing antibodies (bnAbs) are being actively investigated as agents for HIV/AIDS treatment, functional cure and/or prevention. These bnAbs are classified according to cognate epitopes clusters on the HIV envelope (Env). Rigorous preclinical studies evince several potential advantages of bnAbs over currently used antiretroviral drugs (ARVs) in various settings. Currently, optimization of breadth, potency, and virus escape resistance are primary goals for clinical development. A major issue is that all single bnAbs exhibit limited coverage of epitope variability/mutability among HIV strains, allowing virus escape. A lead mitigation strategy proposes triple combinations of bnAb classes for clinical intervention under the premise that polyspecific reactivity will boost potency and breadth. However, the identification of promising combinations is challenging. Screening through all possible triple combinations; bnAb class members; and engineered class members via clinical trials or animal models is an expensive and lengthy process. Therefore, the advancement of bnAb combinations requires a rational, preclinical selection process based on in vitro analytical systems that inform prospects for efficacy. Standardized neutralization assays will continue to play an important role; however, the data can overestimate in vivo potency and escape resistance, fail to capture important determinants of combination bnAb action, and appear incongruous with clinical outcomes in bnAb prevention or therapy trials. Past trials of mono or dual bnAb therapy did not achieve sustained virus suppression of viremia; in some cases, viral rebound occurred via resistance to only one bnAb. This experience reinforces the need for new analytical techniques that capture additional determinants of potency and escape resistance more thoroughly. Such efforts should focus on bnAb interactions with wild type virus swarms in plasma, as these are targets for infused bnAbs and mediate transmission and disease. The development of combination ARV therapies points to instantaneous inhibition and concurrent bnAb class action as highly relevant measures of potency, breadth, and escape resistance. The goal of this project is to introduce new capacities to take these measures for various bnAb combination and test settings, including wild type virus populations in plasma. In ongoing studies, we developed novel quantitative single molecule and fluorescence correlation spectroscopy (FCS) detection methods that can directly measure qualitative and quantitative aspects of triple bnAb class binding to virus populations in plasma. We hypothesize that our goals will be met by innovative applications of these techniques toward analyses of bnAb combinations and plasma viruses. Two Specific Aims are: 1) Establish and interpret the interactions of bnAb combinations with single virions or Env trimers measured by FCS techniques. 2) Characterize interactions of bnAbs and bnAb combinations...