HIV transcription persists in the brain of virally suppressed people with HIV

Abstract

HIV persistence in the brain is a barrier to cure, and potentially contributes to HIV-associated neurocognitive disorders. Whether HIV transcription persists in the brain despite viral suppression with antiretroviral therapy (ART) and is subject to the same blocks to transcription seen in other tissues and blood, is unclear. Here, we quantified the level of HIV transcripts in frontal cortex tissue from virally suppressed or non-virally suppressed people with HIV (PWH). HIV transcriptional profiling of frontal cortex brain tissue (and PBMCs where available) from virally suppressed (n = 11) and non-virally suppressed PWH (n = 13) was performed using digital polymerase chain reaction assays (dPCR). CD68+ myeloid cells or CD3+ T cells expressing HIV p24 protein present in frontal cortex tissue was detected using multiplex immunofluorescence imaging. Frontal cortex brain tissue from PWH had HIV TAR (n = 23/24) and Long-LTR (n = 20/24) transcripts. Completion of HIV transcription was evident in brain tissue from 12/13 non-virally suppressed PWH and from 5/11 virally suppressed PWH, with HIV p24+CD68+ cells detected in these individuals. While a block to proximal elongation was present in frontal cortex tissue from both PWH groups, this block was more extensive in virally suppressed PWH. These findings suggest that the brain is a transcriptionally active HIV reservoir in a subset of virally suppressed PWH.

Fig 1. HIV RNA transcripts are present in brain tissue from non-virally suppressed and virally suppressed PWH.

HIV RNA transcripts for HIV TAR, Long-LTR, Pol, PolyA and Tat/Rev quantified by QIAcuity digital PCR from frontal cortex brain tissue from (A) non-virally suppressed (n = 13) or (B) virally suppressed PWH (n = 10). HIV transcripts standardised to 10⁶ cell equivalents as measured by RRP30 DNA. Comparisons made using non-parametric paired Wilcoxon-tests where colored symbols represent individual PWH. (C) Comparative analysis of TAR, Long-LTR, Pol, PolyA and multiply spliced Tat/Rev HIV transcripts between virally suppressed (blue) and non-virally suppressed PWH (red). (D) Comparative analysis of HIV TAR RNA/Long-LTR RNA ratio in frontal cortex tissue from virally suppressed (VS; n = 6) or non-virally suppressed PWH (nVS; n = 11). Comparisons made using non-parametric unpaired Mann-Whitney tests. Median and interquartile ranges shown. *P<0.05, **P<0.01, ***P<0.001.

Fig 2. Levels of HIV RNA transcripts in brain tissue are directly associated with HIV total DNA levels in PWH.

Linear regression analysis of HIV RNA transcription (A) initiation (TAR), (B) elongation (Long-LTR), (C) Pol, (D) completion (PolyA) and (E) multiply spliced Tat/Rev transcripts vs total HIV DNA levels in frontal cortex brain tissue as measured by digital PCR for virally suppressed (n = 2–14; blue symbols) and non-virally suppressed PWH (n = 7–13; red symbols). All parameters log transformed; R², P values and 95% confidence intervals shown.

Fig 3. Expression of HIV p24 protein in human brain tissue from non-virally suppressed or virally suppressed PWH.

Representative images of cell nuclei (blue), CD68+ myeloid cells (green), CD3+ T cells (white) and HIV p24 protein (red) labelled cells by immunofluorescence imaging from human frontal cortex brain tissue from virally suppressed PWH (VS PWH; left panels) and non-virally suppressed (nVS PWH; right panels; n = 2 per group shown). Donor ID shown in white text. Larger images (40× magnification; 50 μm scale bar) and insets (40× magnification + 3× digital zoom; 10 μm scale bar).

Fig 4. Comparative analysis of HIV RNA transcripts in PBMCs relative to frontal cortex brain tissue from virally suppressed PWH.

(A) HIV RNA transcripts for HIV TAR, Long-LTR, Pol, PolyA and Tat/Rev quantified by QIAcuity digital PCR in PBMCs from virally suppressed PWH (n = 5). HIV transcripts standardised to 10⁶ cell equivalents as measured by RRP30 DNA. Colored symbols represent individual PWH. (B) Comparative analysis of levels of HIV RNA TAR, Long-LTR, Pol, PolyA and multiply spliced Tat/Rev transcripts between frontal cortex brain tissue (green) and PBMCs (orange) from virally suppressed PWH. (C) Comparison of ratio of TAR/LongLTR transcripts in frontal cortex and PBMCs from virally suppressed PWH (n = 5–6). Comparisons made using Mann-Whitney U tests. Median and interquartile ranges shown. *P<0.05, **P<0.01. P<0.05 considered statistically significant.