Understanding the distribution of genetically intact, and therefore potentially replication-competent, proviruses in different CD4+ T cell subsets and within different anatomical sites is important for identifying targets for future eradication strategies.
Naïve (NV), central (CM), transitional (TM) and effector memory (EM) cells were isolated from both the peripheral blood (PB, 13 participants) and lymph nodes (LN, paired, 5 participants) of HIV positive individuals on long-term ART (3-17 years). HIV proviral sequences were obtained using the Full-Length Individual Proviral Sequencing assay (FLIPS) which amplifies single, near full-length (92% of genome) HIV proviruses followed by Next-Generation Sequencing. Genetically intact HIV proviruses were identified as those lacking inversions, stop codons/hypermutation, insertions, deletions or frameshifts.
We sequenced 1893 genomes and identified genetically intact proviruses in all cell subsets except LN EM (3 participants). The frequency of infection with any HIV genome (intact and defective) differed across the cell subsets in LN and PB (P<0.001). We observed the highest levels in TM and EM cells, followed by CM and then NV cells, with the trend significant in blood (P<0.05). The infection frequency of genetically intact proviruses also differed across the subsets in blood (P<0.001) and LN (P=0.02). In blood, intact genomes showed a trend of EM>NV>TM>CM with evidence for EM>CM (P=0.01). In LN, the intact genomes showed a different trend: NV>CM>TM>EM. Overall, the infection frequencies of intact proviruses within the LN and PB anatomic sites were similar (P=0.67), but this result was sensitive to 2 participants with high levels of intact proviruses in LN NV cells (10% and 26% were intact of all LN NV sequences). The intact LN sequences were genetically unique. We did not identify any identical intact sequences between PB and LN.
The distribution of genetically intact proviruses within T cell subsets in blood do not necessarily reflect their distribution in lymph nodes. In lymphoid tissues, the frequency of intact proviruses is highest in less differentiated cells such as NV cells, while in blood the frequency is highest in more differentiated EM cells. Tissue-based NV T cells may act as progenitors of the total reservoir during ART, whereas in the periphery this reservoir is maintained within the EM T cell population, perhaps by clonal proliferation.