Abstract B cell antigen receptor (BCR)-antigen interactions that govern the activation of B cells with a pre-vaccinated B- cell repertoire are not clearly defined for HIV-1 broadly neutralizing antibody (bnAb) lineages. Our recent studies show that B cell activation is dependent on antigen-binding association rate and not on the overall affinity (KD value), indicating that antigen sensing has a kinetic component. In this renewal application we will expand our studies to define the biophysical properties of antigen binding and cell surface interactions that lead to activation of B cells expressing germline precursor or early lineage intermediate BCRs of different HIV-1 bnAb specificities (CD4-binding site, V3-glycan). We will build upon our hypothesis that the exposure of the BCR to antigens that bind with enhanced association kinetics leads to re-organization and localization of receptors with co-stimulatory molecules on the B cell membrane, and together these parameters contribute to B cell activation and selection of functional mutations that lead to the development of neutralization breadth. In Aim 1, we will use Ramos B cell lines and purified BCR-complexes to perform biophysical interactions and structural analyses to define the properties of BCR-antigen interactions that govern signaling and activation of bnAb precursors and selection of functional mutations following immunizations in relevant knock-in mouse models. In Aim 2, we will define the role of the CH1 domain in class-specific antigen sensing, and the role of the immunoreceptor coupling and organization motifs (ICOM) interactome in the regulation and activation of HIV-specific BCRs on naïve and memory-type B cells. In Aim 3, we will define priming and boosting immunogen activation ranges for bnAb lineages to multiple Envelope targets and develop lineage-specific prime/boost combinations for testing in physiologically relevant mouse models. The overall objective of this grant is to bridge quantitative measurements of antigen-activated biophysical/biochemical events and B cell surface organization/interactions to optimal B cell priming and boosting in relevant ex-vivo and in-vivo B cell activation models, and to use this information for facilitating design and selection of immunogens for HIV-1 vaccine development.