Direct detection and quantification of HIV particles and their envelope trimers bound by combinations of broadly neutralizing antibodies.

Broadly HIV-neutralizing antibodies (bnAbs) targeting viral envelope (Env) antigens are promising for HIV/AIDS treatment, functional cure, and prevention. However, individual bnAb epitope classes show distinct gaps in coverage of Env diversity, supporting the use of antibody combinations to improve breadth and potency. We examined how frequently multiple bnAb classes bind simultaneously to individual virions using quantitative single-particle fluorescence correlation spectroscopy (FCS) and FRET approaches. These methods quantified unmodified virion subpopulations from multiple strains bound by one or more fluorescently labeled bnAbs. Analysis of N49P7, PGT121, and PGDM1400, targeting the CD4-binding site, V3-glycan region, and trimer apex, respectively, revealed subpopulations bound by one, two, or three bnAbs. Across strains, the proportion of virions bound by all three antibodies correlated significantly with neutralization activity. These findings define the distribution of combinatorial bnAb binding across heterogeneous virion populations and support quantitative single-particle fluorescence approaches for evaluating antibody combinations against HIV.