PROJECT 1 - PROJECT SUMMARY A pre-clinical HIV vaccine approach based on strain 68-1 of rhesus cytomegalovirus expressing SIV antigens (RhCMV/SIV) elicits cellular immune responses that stringently arrest replication and spread of primary SIV infection in 59% of vaccinated rhesus macaques (RMs). The vast majority of these vaccine-protected RMs will maintain this SIV replication arrest (a state of aviremia with replication-competent SIV in tissues) until virus decays or is cleared to extinction over the subsequent 1-3 years. This pattern of immune-mediated viral suppression has no prior precedent and although the precise virologic and immunological mechanisms underpinning this phenomenon remain unknown, we have established that (a) replication arrest efficacy depends on RhCMV/SIV elicitation of SIV-specific CD8+ T cells that target SIV peptides in the context of major histocompatibility complex (MHC)-E rather than MHC-II or conventional MHC-Ia, and (b) that among RMs with MHC-E-restricted CD8+ T cell responses, efficacy is predicted by a sustained whole blood transcriptomic signature featuring IL-15 signaling. These data suggest that an innate immune/IL-15 signaling-facilitated, MHC- E-restricted, SIV-specific CD8+ T cell response mediates/coordinate replication arrest efficacy and that the in vivo functional outputs underlying this pattern efficacy are unique to this response and thus different from functional outputs of other types of SIV-specific CD8+ T cell responses. This, in turn, suggests that detailed functional comparison of the effective responses with other vaccine-elicited CD8+ T cell response types that fail to mediate replication arrest efficacy (including MHC-Ia- and MHC-II-restricted responses by programmed RhCMV/SIV and MHC-Ia-restricted responses by conventional prime-boost vaccination) will elucidate differences that underlie the replication arrest outcome. Thus, in S.A.1 we will utilize T cell receptor (TCR)- indexed single cell transcriptomics to identify cell-intrinsic transcriptional patterns that distinguish these SIV- specific CD8+ T cell responses in late vaccine phase and, importantly. correlate with subsequent SIV challenge outcome (replication arrest vs. progressive infection). In S.A.2, we will virologically characterize early replication arrest, determining the tissue extent, cellular distribution and state of residual SIV (transcription, translation, virion production) among 68-1 RhCMV/SIV vector-protected RM at necropsy. Our goal is to define the sites and cells hosting replication arrest and whether it involves suppression of SIV gene expression and virion production (analogous to deep latency) and/or an effect on target cells, rendering them resistant to viral infection. Finally, in S.A.3, we will characterize the cellular response (both CD8+ T cells and accessory cell types) to arrested SIV replication in RhCMV/SIV-protected RMs via bulk and single cell transcriptomics and spatial profiling in necropsy tissues. ...