Genetically intact, and potentially replication competent, proviruses are a likely source for viremia during antiretroviral therapy (ART). Identifying the CD4+ T cell subsets that harbour these proviruses within different anatomic sites is important for future eradication strategies.
Near full-length proviral sequences were obtained from naïve (NV), central (CM), transitional (TM) and effector memory (EM) CD4+ T cells (sorted based on their expression of CD45RA, CD27 and CCR7), which were isolated from both the peripheral blood (PB, 13 participants) and lymph nodes (paired LN, 5 participants), using the Full-Length Individual Proviral Sequencing Assay (FLIPS). Proviral sequences were identified as genetically intact if they lacked inversions, stop codons/hypermutation, insertions, deletions or frameshifts. Genetically intact proviruses from 10 participants were compared to on-therapy plasma RNA (p6-RT region obtained by single-genome sequencing (SGS)).
We sequenced 1913 proviruses, and genetically intact proviruses were found in all cell subsets except for LNEM (n=3). We found that the infection frequency of genetically intact proviruses differed across the subsets in both PB and LN (P<0.001). In PB, the order of intact genomes was found to be EM>TM/NV>CM (all P<0.02), while in the LN the trend was NV>TM>CM>EM, with evidence for NV>CM (P=0.01). All 22 intact LN sequences were genetically unique. For the subsets that had more than 10 genetically intact DNA sequences (PBEM, PBNV and LNNV), we compared the genetically intact proviruses obtained by FLIPS to the on-therapy plasma RNA p6-RT sequences obtained by SGS. PBEM had the highest frequency of genetically intact DNA sequences matching 100% to the on-therapy RNA sequences (13/23, 57%). This was followed by PBNV, with 6/19 (32%) DNA sequences matching RNA, and LNNV, with 3/16 (19%) DNA sequences matching RNA.
The distribution of genetically intact proviruses differs between PB and LN. For the five participants with paired PB and LN cells available, NV cells had the highest frequency of intact proviruses in LN. In PB, however, the highest levels of intact genomes were found in EM cells. PBEM, PBNV and LNNV also had a high frequency of genetically intact proviruses matching to on-therapy plasma RNA p6-RT sequences, suggesting that the intact proviruses within these T cell subsets from different anatomic sites may contribute to ongoing viremia during ART.