SUMMARY/ABSTRACT Herpes simplex type virus-1 (HSV-1) infects over 3.72 billion people worldwide, including 200 million individuals in the United States. Following primary infection of the cornea, HSV-1 establishes latency in sensory neurons of the trigeminal ganglia (TG). Reactivation of HSV-1 from latently infected TG leads to shedding of the virus in tears causing recurrent ocular herpetic disease, a major cause of infectious blindness in the Western world. Currenly, an FDA-approved herpes simplex vaccine is unavailable. Our long-term goal is to develop an immunotherapeutic ocular herpes vaccine. While a role for CD8+ T cells (but not CD4+ T cells) in reducing HSV- 1 reactivations from latently infected TG is gaining wider acceptance, the small numbers of functional tissue- resident memory CD8+ TRM cells that are present in latently infected TG are not enough to prevent virus reactivation. We have made several significant findings, during the last funding period: (1) HSV-specific CD8+ T cells from “naturally protected” HLA-A*0201-positive asymptomatic individuals (who never develop recurrent ocular herpetic disease despite being infected) mainly targeted five HSV-1 epitopes; (2) Phenotypic and transcriptomicprofiling indicates that frequent HSV-specific CD8+ TRM cells, which expressed high levels of tissue-homing and tissue-residency receptors (i.e. CXCR3, IL-2R/IL-15R, CD69, and CD103), found in the TG of HSV-1 infected HLA-A*0201 transgenic rabbits (HLA Tg rabbits) are associated with decreased virus shedding; (3) Topical ocular delivery to latently infected HLA Tg rabbits of prototype neurotropic adeno- associated virus (AAV8) constructs, which express either the T cell attracting CXCL11 chemokine (CXCR3 ligand) or IL-2/IL-15 cytokines (IL-2Rb/IL-15Rb ligands), increased the frequency of TG-resident CD8+ TRM cells specific to the five immunodominant epitopes; (4) Increased numbers of exhausted TG-resident CD8+ TRM cells were associated with increased virus shedding in HLA Tg rabbits; and (5) Ex vivo blockade of T cells exhaustion pathways PD-1, LAG-3 and TIGIT, ex vivo, in rabbit TG explants significantly reduced virus reactivation. Building on the above published and preliminary results, the central hypothesis of this revised competitive renewal proposal is that a TG-targeted vaccine that boosts the number, function and longevity of anti-viral TG-resident CD8+TRM cells will reduce virus reactivation and shedding. Specific Aims: Aim 1: Test the hypothesis that a tissue-targeted Prime/Pull/Keep therapeutic vaccine (designated as PPK vaccine) that incorporates the five immunodominant HSV-1 CD8+ TRM cell epitopes (prime), CXCL11 (pull) and IL-2/IL-15 (keep) will boost the number and longevity of TG-resident CD8+ TRM cells and significantly decrease HSV-1 reactivation in latently infected HLA Tg rabbits. Aim 2: Test the hypothesis that tissue-targeted PPK vaccine combined with blockade of PD-1, LAG-3 and/or TIGIT immune checkpoints will increase...