Overall Program Summary More than a decade ago our group initiated development of vaccine vectors based on the persistent β- herpesvirus Cytomegalovirus (CMV) because of the ability of CMV to elicit and indefinitely maintain high frequency effector-differentiated T cell responses in diverse tissues. Using the rhesus macaque (RM) model, we tested the concept that the early pathogen intercept afforded by such “in place” effector-memory T cells would result in superior protection against immune evasive pathogens like HIV/SIV, relative to the typical memory responses elicited by conventional vaccine approaches. We found that not only was Rhesus (Rh) CMV amenable to “vectorization” (e.g., we demonstrated that, even when spread-deficient, RhCMV vectors generate potent, durable, exogenous insert-specific effector-memory T cell responses in naturally RhCMV+ RM), but also that the SIV-specific responses elicited by these vectors were reproducibly able to stringently control and then clear mucosally-administered, highly pathogenic SIV from ~54% of vaccinated RM. Although this unprecedented “control and clear” protection was consistent with an early infection intercept, the nature of the protective immune response remained elusive until we discovered that the Rh157.5/Rh157.4 gene-deleted RhCMV vector used as the backbone of our vaccine had another very unusual immunologic property: all CD8+ T cell elicited by this vector were found to recognize epitopes that were restricted by either MHC-II or MHC-E, not MHC-Ia. We further demonstrated that this unconventional epitope targeting was reverted to conventional MHC-Ia restriction by repair of Rh157.5/Rh157.4 expression, which did not otherwise affect the functional or phenotypic characteristics of vector-elicited CD8+ T cells. Remarkably, the repaired RhCMV/SIV vectors failed to protect against SIV challenge, strongly suggesting that unconventional CD8+ T cell epitope recognition is required for RCMV/SIV vector efficacy. Recent work has demonstrated that conventional vs. unconventional CD8+ T cell priming is regulated by multiple RhCMV genes, the modification of which effectively programs RhCMV vectors to elicit CD8+ T cell responses with distinct epitope recognition patterns. In this program, we first seek to determine the mechanisms responsible for unconventional CD8+ T cell response generation, and develop RhCMV vectors that predominantly or exclusively elicit MHC-II vs. MHC-E-restricted CD8+ T cell responses (Projects 2-4). We will then use these new vectors to 1) identify the response type(s) needed for efficacy, 2) determine whether such response focusing improves efficacy, and 3) define quantitative and/or qualitative correlates of this protection (Project 1). Finally, we will use the insight gained in these RM studies to design (Project 4), manufacture and clinically test (Project 5) the safety and immunogenicity of a spread- deficient, “response-programmed” HCMV/HIV vector designed to elicit the protect...