Abstract – Project 2 Globally, 600,000 young adults become infected with HIV-1 each year. Therefore, there is an urgent need to develop an early life vaccine strategy that protects young adults from HIV infection. This early life vaccine should elicit protective broadly neutralizing antibodies (bNAbs), which have been shown to be effective against infection in nonhuman primates and humans. Newer, rationally designed native-like HIV envelope trimer-based vaccines that engage germline B cell receptors and drive evolution of bNAbs have been developed. These B cell lineage- based vaccines induce bNAb precursors in 50–60% of infant and adult nonhuman primates, and early clinical trial data suggests some humans also develop bNAbs. While these findings offer significant promise that the long-standing goal of an effective HIV vaccine is on the horizon, there is a gap in our understanding of the determinants of bNAb induction that results in only half of vaccinees developing a favorable response. It is now clear that the microbiome is intricately intertwined with the development and modulation of the immune system, and that these microbiome–immune interactions are critical for vaccine responses. Our preliminary data confirm that the microbiome is a necessary and modifiable factor that influences vaccine responses, with specific bacterial taxa positively associated with antibody titers resulting from immunization with HIV Env-based vaccines. Thus, Project 2 proposes to define specific microbial features that can be harnessed as an endogenous adjuvant to help—along with optimal exogenous adjuvants identified by Project 1—drive induction of HIV bNAb precursors after vaccination with the BG505 GT1.1 SOSIP trimer immunogen. Specifically, Aim 1 of this project will couple shotgun metagenomic data and microbe–phenotype triangulation, a novel microbiome discovery platform we developed, to define microbial features that associate with the induction of HIV bNAb precursors. Aim 2 will test the hypothesis that some commensal bacteria predispose towards beneficial vaccine responses by helping induce vaccine-elicited antibodies that are cross-reactive with the microbiome. To enhance the translational potential of our work, we will examine microbiome cross-reactive antibodies in both infant NHPs and humans. Finally, Aim 3 will develop a gnotobiotic human B cell lineage knock-in mouse model to experimentally define the role of the microbiome in bNAb precursor induction, including functionally validating the bioinformatically identified microbial features. Taken together, the results of this Project will elucidate specific microbes that modulate the immune landscape such that a greater percentage of B cell lineage-based vaccine recipients develop bNAb precursors. Ultimately, these adjuvant-like microbes can be incorporated into future vaccine strategies.