Project Summary/Abstract An effective HIV vaccine is likely to be dependent on sufficient quantity and quality of HIV Envelope (Env)-specific antibody at the rectal and vaginal mucosa. Priming of the oral mucosa is viewed as a promising approach to generate mucosal antibody at HIV entry sites, however the understanding of the mechanisms that mediate this process are poorly understood. Our project seeks to precisely define the dynamics of inducing intestinal-mucosal plasma cells through direct intra-tonsillar (i.t.) immunization, and empirically test strategies to tune this process for obtaining an optimal mucosal antibody profile (localization, breadth, function, and durability) to achieve a protective HIV vaccine strategy. A main emphasis for inducing HIV-specific B cell responses has been to evaluate adjuvants/stimuli that target conventional B cell responses; leaving induction of non-conventional B cell responses, including those with direct relevance to mucosal antibody production largely under-explored. IgM memory, one such unconventional B cell population, is IgM memory, a major first line of defense against mucosal pathogens, and includes heterologous subsets such as marginal zone and B-1 B cells, which are major precursors of mucosal IgA plasma cells. Our research has demonstrated that IgM memory B cells are highly responsive to acute HIV infection and their maintenance is highly correlated with Env-specific antibody development. Mechanisms to induce robust IgM memory vaccine responses remain poorly defined, although our recent work has demonstrated the ability of IL- 9 and IL-33, known regulators of mucosal immunity, to promote the robust development of HIV Env-specific IgM, in addition to increasing the breadth, magnitude, and durability of the Env-specific IgG and IgA response when combined with the promising DNA/protein HIV Env immunogen platform VC10014 which elicits Tier 2 neutralizing antibody in rhesus macaques. Our central hypothesis is that conditioning the tonsil microenvironment with IL-9 or IL-33 will enable the rapid induction of durable and effective mucosal humoral immunity by the VC10014 HIV vaccine platform. This hypothesis will be tested by the following specific aims: 1) Define the effect of the tonsil microenvironment on the induction of mucosal humoral immunity, 2) Evaluate the protective ability of IL-9 or IL-33 adjuvanted VC10014 HIV vaccine platform, and 3) Identify strategies for enhancing human tonsil primary B cell responses to immunization. This project will significantly advance our insight into preventing HIV transmission and the mechanisms that control the development of protective humoral mucosal responses to HIV.