PROJECT SUMMARY : Cytomegalovirus (CMV) has a major negative impact in solid organ transplant recipients (SOTxR) due to limitations in current preventive and therapeutic strategies, especially in CMV seronegative recipients (R-) of organs from seropositive donors (D+) [D+R-]. The D+R- subset comprises ~25% of all SOTxR but >80% of CMV disease and is independently associated with worse long-term survival after SOTx. The disproportionate impact in D+R- SOTxR results from an impaired ability to develop a primary immune response to donor-transmitted CMV infection in the context of immunosuppression. Strategies that elicit or enhance CMV-specific immunity prior to SOTx could lead to more effective prevention/control of CMV after SOTx, minimizing the need for toxic CMV antiviral therapy (AVT). We have developed a modified vaccinia Ankara virus vaccine, Triplex, that expresses immunodominant CMV antigens pp65, IE-1, and IE-2 that are targets of protective T cell immunity. Triplex elicits robust, long-lasting, and functional CMV-specific CD4 and CD8 T cells. Triplex was safe, accelerated reconstitution of CMV protective immunity, and reduced significant CMV infection by ~50% in a phase 2 study of allogeneic hematopoietic cell TxR. Our long-term goals are to harness vaccine-induced CMV- specific cellular immunity to reduce the impact of CMV in D+R- SOTxR and to define immune correlates of risk (CoR) and for protection (CoP) (i.e. immune correlates of Triplex vaccine efficacy [VE]). The central hypothesis is that pre-Tx Triplex vaccination of CMV seronegative LTx candidates elicits functional CMV-specific CD4 and CD8 T cells, leading to improved immune control of CMV and significantly decreases the need for CMV AVT post- Tx in D+R- LTxR who receive preemptive therapy (PET) for CMV prevention. We further hypothesize that there are specific immune CoR for CMV outcomes and CoP of Triplex vaccine. We will leverage our established consortium and preliminary studies in a target population of D+R- LTxR with high unmet need (no FDA-approved available antiviral prophylaxis options, highly susceptible to valganciclovir toxicity, and significant CMV- associated morbidity, mortality, and cost). The objectives of this proposal are to assess the efficacy, safety and immunogenicity of Triplex in D+R- LTxR in a phase 2 study and to define the immune CoR and CoP using state- of-the-art polyfunctional T cell assays and novel analytic approaches (COMbinatorial Polyfunctionality analysis of Antigen-Specific T cell Subsets [COMPASS]). An effective pre-Tx CMV vaccination approach would transform CMV prevention strategies in SOTx. The proposed studies will define immune CoR for clinical outcomes, which will facilitate efficient evaluation of future immune-based strategies, and lead to broader implementation of the more effective PET CMV prevention strategy in D+R- LTxR.