Background
More than 95% of HIV-1 proviruses detected are defective but often associated with RNA transcripts during antiretroviral therapy (ART). These transcriptionally active defective proviruses may contribute to persistent immune activation seen in people with HIV (PWH) receiving ART. The distribution and persistence of these transcriptionally active defective proviruses has not been described.
Methods
11 PWH originally enrolled in the Strategic Timing of Antiretroviral Therapy (START) trial were studied. Levels of HIV-DNA and total HIV-RNA in frozen whole blood were determined at the baseline and 6-11 years later by real-time PCR. Also, at the 2nd visit, CD4 cell subsets were isolated by flow cytometry sorting with CD45RO and CD27 antibody staining to identify naïve (NA), central memory (CM) and effector memory (EM) subsets. Sequences of HIV-DNA and cell-associated (CA) HIV-RNA in CD4 subsets were characterized by 5’LTR-to-3’LTR PCR single-genome amplification and direct amplicon sequencing. Levels of HIV-DNA and CA HIV-RNA in the CD4 subsets were determined per-cell as well as per 1mL of blood.
Results
All 11 participants (pts) were men, 55% white, 18% black, 9% Latino, median age 41 yrs, median baseline CD4 645 cells/µL. At the 2nd visit, after 4-11 yrs of suppressive ART, all 11 pts had ~2-log reduction in total HIV-RNA levels. While HIV-DNA levels also decreased, reductions were modest, at a median 0.5-log decrease. When analyzed on a per-cell basis, CA HIV-RNA levels were highest in EM, followed by CM and NA cells. In contrast, when analyzed based on total contribution per 1 mL of blood, CM cells had the highest median CA HIV-RNA levels, followed by EM and NA cells (Fig A). Of note, HIV-1 proviruses in the CD4 cell subsets were mostly defective, with median 9% of proviruses in a full-length intact form in NA cells, 4% in CM cells, and 2% in EM cells (Fig B). There was no evidence of transcription from these intact proviruses. Among 186 expanded provirus clones identified in 11 pts (2,473 seqs), 4% were shared among all CD4 cell subsets, with 43% of these being transcriptionally active.
Conclusions
Transcriptionally active “defective” HIV-1 proviruses were present in all CD4 subsets in a cohort of 11 pts after 4-11 yrs of ART. The majority resided in CM cells, identifying the CM cell pool as an important source for long-term HIV-1 transcriptional persistence on ART. This has implications for the mechanism of persistent immune activation among ART-treated pts.