Background:
An HIV cure is elusive due to the viral persistence in tissue reservoirs. Upon antiretroviral therapy (ART) interruption (ATI), the virus can reemerge from tissue reservoirs and spread through the body. In order to develop better HIV therapies, a deeper understanding of the tissue and cellular origin of early rebound is critical.
Methods:
In this study, the barcoded virus SIVmac239M was used to infect 7 Chinese rhesus macaques. The animals were treated with ART for 42 weeks beginning at 12 weeks post-infection, and followed by a 3-week ATI (ATI1). ART resumed for 9-16 weeks, and a second ATI (ATI2) was started one week before each individual necropsy. To evaluate potential sources of viral rebound, and possible cellular factors contributing to viral reactivation, we used deep sequencing, single genome amplification, quantitative real-time PCR, intact proviral DNA assay, immunofluorescence analysis, combined CODEX/RNAscope/in situ hybridization (Comb-CODEX-RNAscope) analysis, and single-cell RNA sequencing (scRNA-seq).
Results:
All animals rebounded within 20 days after the start of ATI1. In addition, despite PVL levels were < 15 copies/ml during ART and at necropsy after ATI2, a total of 11 viral barcodes were detected across the 4 animals, with mesenteric lymph node (mesLN), inguinal lymph nodes (ingLN), and spleen exhibiting viral barcode clonotype overlap with plasma virus. These tissue sites also had higher viral barcode clonotypic diversity, larger intact proviral reservoir size, and consistent production of vRNA detected by Comb-CODEX-RNAscope. Moreover, scRNA-seq analysis of these sites revealed expression of genes related to immune activation, mitochondria damage, and epigenetic modulation in these 4 animals. While the remaining 3 animals had no measurable plasma rebound viremia at ATI2, similar viral reservoirs were identified in lymphoid tissues comparable to those found in rebounding animals.
Conclusions:
Our results suggest that tissues with higher levels of virus, particularly mesLN, ingLN, and spleen, appear to be potential sources of early viral rebound during the first week after ATI, despite PVL levels below 15 copies/ml, with transcriptomic profiles providing hints of potential cellular correlates of viral reactivation. Further validation of these findings may help guide efforts to develop targeted strategies to reduce or eliminate rebound-competent viral reservoirs.