Project Summary/Abstract Many potent and effective broadly neutralizing antibodies (bNAbs) directed to the HIV-1 envelope glycoprotein (Env) trimer have been studied mechanistically and structurally. Combined, their epitopes now cover most of the external surface of Env. Passive immunization with bNAbs, optimally mixed to minimize viral escape, holds promise for controlling HIV-1 infection, prevent it, or even helping to cure it. Eliciting bNAbs by active immunization, however, remains problematic. Our overall long-term goal is to contribute to solving that major problem. We will develop techniques for quantifying concentrations, affinities, kinetics, and stoichiometry of bNAb binding in polyclonal sera from infected or immunized animals and humans. We will determine how these quantities change as germline (GL) responses mature into bNAbs. So far, we have used soluble, native-like SOSIP trimers to dissect or induce Ab responses. The recent emergence of mRNA vaccination, however, allows the design of full-length Env constructs, opening the possibility of presenting external bNAb epitopes located near the membrane while shielding C-terminal neo-epitopes at the base of soluble trimers. We will therefore make virus-like particles that mimic HIV-1 virions in size, membrane composition, Env density, and Env interactions with interior viral proteins. The kinetics and stoichiometry of bNAb binding to these full-length membrane-anchored trimers, with and without changes in the cytoplasmic tail, will be compared with binding to SOSIP trimers. We will also extend in-depth binding analyses to SOSIP trimers derived from a global panel of neutralization-resistant HIV-1 isolates. We will dissect binding kinetics, stoichiometry, induced conformational changes, and the heterogeneity of the interactions, thereby identifying binding characteristics that correlate with neutralization breadth. In silico analyses of how the midpoints and Hill slopes (h) of the neutralization curves relate to neutralization breadth, for both single bNAbs and combinations, will guide further experimental dissections of how binding properties mold cross-reactivity. We will develop assays for measuring entry-fusion fitness to define the relationship between viral resistance to bNAbs and the efficiency of receptor-facilitated entry. To achieve these goals, we propose three Specific Aims: Aim 1. Analyze bNAb binding and neutralization properties relevant to passive and active immunization. Aim 2. Determine how bNAb breadth and binding properties are interrelated. Aim 3. Dissect the relationship between entry fitness and escape from bNAbs. In summary, we seek to define how dynamic properties of Ab binding are linked to neutralization breadth. Such fundamental information may help improve both active and passive immunization strategies, which are highly relevant to public health.