Project Summary Herpes simplex virus-2 (HSV-2) is one of the most common sexually transmitted infections (STIs) with a high prevalence of 45 million in the USA. HSV-2 is primarily transmitted via exposure at the genital mucosal surfaces, which leads to the establishment of latency in the sacral ganglia. Although pharmacological interventions exist for HSV-2-related symptoms, there are no preventative vaccines or curative measures available for this disease. Towards developing vaccines to prevent HSV-2 transmission, a clear understanding of the mechanism by which immune responses are mediated within the relevant mucosal sites is necessary. Currently, the immune mechanisms of protection within the female genital mucosa are poorly understood. Our previous studies led to the understanding that memory T cell circulation to the vagina is restricted at steady state (Nakanishi et al, Nature, 2009). Based on this insight, we developed a new vaccine strategy we call, “Prime and Pull”, in which topical application of chemokines following parenteral vaccination establishes local memory CD8 T cells to dramatically improve protection afforded by parenteral vaccines (Shin and Iwasaki, Nature, 2012). We have also shown that intravaginal (ivag) immunization with TK- HSV-2 provides local protection based on establishment CD4 tissue resident memory T (TRM) cells (Iijima and Iwasaki, Science, 2014). Our Preliminary Studies reveal that memory B cells must migrate into the female reproductive tract (FRT) in order to restimulate the CD4 TRM. Strikingly, unlike T cells, B cells do not become tissue-resident, but rather, enter the FRT constitutively and in response to HSV-2 infection as “guest”, or B guest memory (BGM) cells. Memory B cell engagement of TRM results in their secretion of antiviral cytokine IFN-γ as well as B helper cytokine, IL-21. Consequently, BGM are induced to secrete virus-specific IgG in the presence of TRM within the FRT. The goal of this grant application is to understand the mechanism by which protective immunity against HSV-2 is orchestrated by BGM in the female genital tract, and to apply this understanding to design a robust vaccine against recurrent genital herpes through the following Specific Aims. We propose to dissect the mechanism of B cell recruitment to the FRT in locally immunized host following viral challenge (Aim 1), to determine the identity of memory B cell subset that stimulates CD4 TRM (Aim 2), and to determine the nature of CD4 help provided to memory B cells that converts them into antibody secreting plasma cells (Aim 3). By providing basic understanding of how migrating memory B cells and CD4 TRM mediate protection against HSV- 2 genital infection, and applying such knowledge to design a better vaccine approach, these studies will help to establish important foundation with which to design immunological interventions and preventative measures against genital herpes and other deleterious STI diseases in humans.