PROJECT SUMMARY/ABSTRACT Despite the remarkable success of the combination antiretroviral therapy (cART) to control HIV-1 infection, viral reservoirs persist indefinitely under treatment. These remaining viral populations constitute the principal burden for an effective HIV-1 cure, as they lead to a rapid rebound in viremia when treatment fails or after analytic treatment interruption (ATI). Unfortunately, our understanding of the mechanisms of HIV-1 persistence during cART is limited because the viral reservoir population size is very small and it is established within a wide variety of susceptible tissues, inaccessible to evaluation. Hence these difficulties, the majority of the reservoir studies do not include tissue samples or study a very limited sampling of the tissue reservoirs. Therefore, we are not able to properly study these viral populations and their main characteristics remain unknown. Understanding the nature and properties of these tissue reservoirs during treatment is key to design a successful HIV-1 cure strategy. The team comprised by the Hope lab and Dr. Villinger has optimized and refined their 89Zr-labelled FAB2 probe that allows for the in vivo detection of cells expressing SIV env in an infected macaque. Using this approach, they have generated a new PET-CT-based workflow for the identification and downstream characterization of foci of SIV infection in tissues. This updated “beacon-guided system” is exquisitely sensitive and it is extremely powerful to find and characterize small foci of infected cells during cART or early after ATI, prior to any detectable viremia. These foci represent extremely rare events that are key to the understanding of underlying SIV biology and yet, to date, were only found through random and/or biased survey of tissue. In the current proposal, we intend to use the Hope lab ability to localize SIV active viremia sites in infected macaques and complementary genomic analysis workflows optimized by our team, to perform in-depth spatio-temporal studies of the reservoir viral population dynamics. We will use phylogenetic, evolutionary, and phyloanatomy analysis to study the characteristics tissue reservoirs. We will also assess the contribution of biological processes such as clonal expansion, residual viral replication, and viral production bursts in the process of reservoir persistence during cART. Additionally, we will analyze the characteristics and transcriptional profiles of the different cell types that harbor the reservoir in tissues using our multiple complementary genomic techniques. We will use PET-CT-guided RNAseq and PET-CT-guided spatial transcriptomics from SIV positive tissues, and isolate specific types of infected cells to analyze their transcriptional profiles using single-cell analysis. Due to Hope lab’s findings indicating that mast cells could be key in the reservoir persistence, we plan to specifically study the role of these mast cells in the viral population dynamics of the t...