Transcriptomic Signatures of Human Immunodeficiency Virus Post-Treatment Control

Abstract

Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

Keywords: ART; HIV DNA; HIV posttreatment controllers; RNA splicing; analytic treatment interruption; cell-mediated immune response; human immunodeficiency virus; noncontrollers; splicing; transcription; transcriptional completion; transcriptional profile; treatment interruption; viremic suppression.

FIG 1

PTCs and NCs show different temporal changes in plasma and cell-associated HIV RNA after ATI. (A and B) Plasma viral loads were measured by clinical assays in posttreatment controllers (A) and in noncontrollers (B) before ATI (pre), at early and late times after ATI, and at the time of virologic failure (VF). (C and D) Initiated (TAR), 5′-elongated (Long LTR), mid-transcribed (Pol), completed (PolyA), and multiply-spliced (Tat-Rev) cell-associated HIV transcripts were measured at each time point in posttreatment controllers (C) and in noncontrollers (D) using RT-ddPCR. Each participant is represented by a different color or symbol. Open symbols denote PTCs who eventually experienced rebound. Maroon-colored lines indicate medians. Nondetectable transcripts were assigned a value of 1 cp/μg. Statistics were calculated using the Wilcoxon signed-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

PTCs have lower levels of measured HIV-1 transcripts at all time points studied. Viral loads (A) and cell-associated HIV RNA levels at pre-ATI (B), early ATI (C), and late ATI (D) time points were compared between posttreatment controllers (PTCs) and noncontrollers (NCs). PTCs are represented by unique white or black symbols. Open symbols denote PTCs who eventually experienced virologic rebound. NCs are represented by unique red symbols. Blue lines indicate median detectable transcripts. All viral loads of <50 copies/mL were assigned a value of 25 copies/mL, and undetected transcripts were assigned a value of 1 cp/μg. Statistics were calculated using the Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistically trending data are denoted using blue bars, with the associated P values being above the data sets.

FIG 3

PTCs and NCs show different temporal changes in average HIV RNA levels per provirus (HIV RNA/HIV DNA) after ATI. To account for differences in infection frequency, levels of each HIV transcript were normalized to the total HIV DNA (A and C) and the intact HIV DNA (B and D) levels, as measured using the intact proviral DNA assay (IPDA) in a subset of individuals. Each participant is represented by a different color or symbol. Open symbols denote PTCs who eventually experienced viral rebound. Maroon lines indicate ratio medians. Ratios with a value of 0 were assigned a value of 0.0001. Statistics were calculated using the Wilcoxon signed-rank test or the Mann-Whitney U-test. *, P < 0.05. Statistically trending data are denoted using blue bars, with the associated P values being above the data sets.

FIG 4

Average HIV RNA levels per provirus do not differ between NCs and PTCs before ATI, but differences start to appear by late ATI. The average levels of each HIV transcript per provirus (HIV RNA/total HIV DNA) were compared between PTCs and NCs at pre-ATI (A), early ATI (B), and late ATI (C). Blue lines indicate medians. Ratios with a value of 0 were assigned a value of 0.0001. Statistics were calculated via the Mann-Whitney U test. Statistically trending data are denoted using blue bars, with the associated P values being above the data sets.

FIG 5

PTCs have lower levels of HIV transcriptional completion and splicing after ATI. The ratios of one HIV RNA to another were used to express the extent of progression through various blocks to HIV transcription, including: (A) 5′ elongation (elongated/initiated HIV RNA, (B) completion (completed/elongated HIV RNA), (C) multiple splicing (multiply-spliced/completed HIV RNA), and (D) multiply-spliced as a fraction of elongated (multiply-spliced/elongated HIV RNA). Each participant is represented by a different color. Statistics were calculated via the Wilcoxon signed-rank test for within-group comparisons and the Mann-Whitney U test for between-group comparisons. *, P < 0.05; **, P < 0.01. Statistically trending data are denoted using blue bars, with the associated P values being above the data sets.

FIG 6

PTCs had distinct transcriptomic features from NCs during ATI. PTCs had lower levels of inflammation, cell death, and HIV-dependent factors during ATI. (A) Normalized Enrichment Scores (NES) for the curated pathways, highlighting the enrichment of inflammatory, cell death, and HIV-dependent pathways in NCs, compared to PTCs, during pre-ATI, early ATI, and late ATI. (B) Gene set enrichment in selected pathways at different time points. NES, normalized enrichment score. (C) Longitudinal NES for different pathways in PTCs and NCs. The NES were derived from a within-group comparison, namely, the transcriptome obtained during ATI versus that obtained in pre-ATI in PTCs and in NCs, respectively.