PROJECT SUMMARY/ABSTRACT Infection with Herpes simplex virus 2 (HSV-2) for which there is no vaccine is a significant cause of human morbidity both in the United States and worldwide. Current understanding of humoral immunity to HSV-2 is based on bulk metrics of antibodies circulating in bloodstream, however everyday battle between virus and host occurs in microscopic tissue microenvironment which represent a unique immunological niche. Our premise is that study of local humoral responses will reveal critical, currently unidentified mediators and mechanisms for immune control of HSV-2. After primary infection, HSV-2 establishes latency in sensory neurons. Clearance and subsequent control of constantly reactivating HSV-2 in tissue are believed to depend on T cells, but clinical observations and mathematical models present evidence consistent with essential role for local humoral responses required to achieve efficient virus control, Our preliminary data has shown that antibody secreting cells (ASCs) are actively present in genital skin during HSV-2 reactivation and long after virus outbreak is contained. Using the power of having sequential samples in individual tissue biopsies over time combined with recent advancements in single-cell RNA sequencing we demonstrated that these clonally expanded cells commit to the long-lived plasma cell lineage implying possible tissue residency, Isolation of antibodies from these unique cells has shown they recognize HSV-2 suggesting they may be directed to as yet unidentified HSV-2 antigens and/or provide functions important for virus control in the tissue. Therefore, the goal of this project is to thoroughly define the immunoprotective role of skin antibody-secreting cells (ASCs) against reactivating HSV-2. Underlying our proposed experiments is the idea that tissue resident ASCs have the ability to contain the virus over long timeframes via secreting HSV-2 specific antibodies that are essential for virus control due to their specificity and/or effector functions. Therefore, we focus on critical features of ASCs such as antigenic specificity, gene expression profile, clonal structure and functional activity of cognate antibodies during symptomatic HSV-2 reactivation and at multiple time points following clearance of a genital skin virus within microscopic sites of infection. We will also evaluate frequency of essential clones of skin ASCs among circulating B cells that have direct implication for rational vaccine design allowing targeted vaccination strategies. Ultimately, our goal is to use the gained insights to develop strategies that will allow for novel approaches to design successful herpes vaccine and development of immunotherapeutic approaches to treat chronic HSV-2 infection.