Identification of initial sites of SIV rebound after treatment cessation

Antiretroviral therapy (ART) suspends HIV replication, but virus persists and rebounds after ART discontinuation. Although much is known about the persistent viral population, the tissue origin(s) and the earliest viral dynamics of post-ART viral rebound remain obscure. Here, using barcoded SIVmac239 in rhesus macaques (RMs) and extensive necropsy tissue sampling on ART and early post ART, we defined the spectrum of low-level barcode-specific viral RNA expression in tissues during ART and then assessed initial clonal rebound by identifying barcodes in individual tissues that exceeded this distribution limit ("outliers"). Eight such outlier barcodes were identified in 4 of 11 aviremic (<1 copy/mL) post-ART RM, with 16 additional outliers in 5 of 6 post-ART RM with low viremia (5-30 copies/ml). Nine of these 16 barcodes were also identified in rebound viremia, confirming specific tissues as rebound origin sites. An RM with post-ART viremia of 4700 copies/mL showed 8 outlier barcodes that ranged from a single site to anatomically discontinuous, multi-site spread. Among all identified outlier barcodes, 27 were determined to reflect rebound origins, of which 96% were in the gastrointestinal (GI) tract (26%) or GI-tract-draining lymphoid tissues (70%). These results indicate that distinct tissue sites differentially restrict/promote post-ART viral rebound, with potential therapeutic implications for interventions designed to prevent or control these events.