Project Summary Preclinical studies and early stage human trials evaluating passively transferred broadly neutralizing antibodies (bnAbs) suggest that a vaccine capable of eliciting bnAbs would provide effective protection from HIV infection. However, the difficulty of inducing bnAbs through vaccination has led to a focus in the field on vaccine strategies based on sequential immunizations meant to guide the developing B cell response. These sequential immunization strategies range from germline targeting to lineage-guided design to immunofocusing, and combinations thereof. While logical from a structural biology perspective, important immunological questions remain unanswered for such vaccines: In such a strategy, at what interval should sequential immunogens be administered? How does competition from antigen-specific but non-neutralizing B cell precursors impact vaccine “shepherding”? Is this sequential immunization process hindered by limited T cell help? In addition, vaccines comprised of 4 or more injections will be a challenge to implement globally. How do we make such a vaccine practical? In this phase R61/R33 application, we propose systematic studies in small animal models to evaluate fundamental vaccine immunology issues facing such strategies. We proposed several novel approaches to examine, and potentially solve, these issues: In aim 1 we will characterize the immunology of staggered sequential immunizations, in aim 2 we develop an approach to delete competitor B cells during vaccination, in aim 3 we develop vaccines employing augmented T cell help, and in aim 4, we propose technologies to enable sequential immunogen exposure following a single injection. We will test these concepts in the context of vaccines aiming to elicit bnAbs against the CD4 binding site (VRC01-class responses) and the Env fusion peptide, using physiologically relevant conditions. The most impactful of these immunization strategies will be downselected for testing in non-human primates (NHP) during the R33 phase, the animal model for HIV immunization closest to humans. Our work is guided by recent advances by the Irvine, Crotty, and Silvestri labs in understand GC kinetics, bnAb B cell competition,vaccine delivery systems, novel adjuvants, and roles of T cell help in rare B cell recruitment.