CORE C PROJECT SUMMARY A comprehensive understanding of the dynamic interplay between immune responses and the virus within the spatial context of infected tissues after SIV challenge will be essential to dissect the mechanisms responsible for initiating and maintaining 68-1 RhCMV/SIV vaccine-mediated SIV replication arrest efficacy and to define the basis of the ability of MHC-E-restricted CD8+ T cells, but not MHC-Ia or MHC-II-restricted CD8+ T cells, to mediate this type of protection. In order to capture the dynamic breadth and nature of the complex immunologic processes within tissues in early primary SIV infection and to unravel the mechanisms of RhCMV/SIV vaccine efficacy, the Advanced Spatial Analysis Core (Core C) will utilize a battery of novel high-dimensional spatial approaches to elucidate 1) the state of the virus, including identity of infected cells, viral gene and protein expression, and infected cell transcriptional patterns (S.A.1), and 2) essential immune cell profiles, including phenotypes, subsets, activation/immunoregulatory states, cytokine signaling, effector functions and transcriptomic landscapes (S.A.2) that will define the virology and immunology of established replication arrest (Project 1) and will distinguish the in vivo immune interception of primary SIV infection by protective MHC-E-restricted CD8+ T cells from non-protective MHC-E-, MHC-Ia- or MHC-II-restricted CD8+ T cells elicited by differentially programmed RhCMV/SIV vectors, or MHC-Ia-restricted CD8+ T cell responses elicited by a conventional prime- boost vaccine (Project 2). The Core will interrogate spatial virologic, phenotypic, and transcriptomic data on single-cells, populations, and cell neighborhoods in tissues associated with SIV transmission using high- dimensional RNAscope/DNAscope, PANINI-CODEX spatial phenotyping, and GeoMx DSP and CosMx SMI spatial transcriptomics platforms, and will link spatial data sets with bulk and single-cell transcriptomic data sets on matched tissues (and blood), to comprehensively define gene expression signatures, networks and immunological responses (but with additional spatial context) associated with RhCMV/SIV vaccine-mediated SIV replication arrest. Core D will apply cutting edge bioinformatics approaches to model the functional outputs and inform the mechanisms of action by which RhCMV/SIV vaccination programs the immune response for protection against SIV infection. Core C is directed by Dr. Jacob Estes, who will work closely with Core D, Drs. Michael Gale, Jr. and Ben Bimber, and with Core A, statistician Dr. Paul Edlefsen, to provide consistent, high-quality functional multi-omics data generation, integrated data analysis, biostatistical analyses and computational modeling in support of the advanced spatial analysis being performed for this scientific program. 1