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. 2023 Apr 20;15(4):1009.
doi: 10.3390/v15041009.

Intact Proviral DNA Analysis of the Brain Viral Reservoir and Relationship to Neuroinflammation in People with HIV on Suppressive Antiretroviral Therapy

Dana Gabuzda  1   2 , Jun Yin  1 , Vikas Misra  1 , Sukrutha Chettimada  1 , Benjamin B Gelman  3
Affiliations

Intact Proviral DNA Analysis of the Brain Viral Reservoir and Relationship to Neuroinflammation in People with HIV on Suppressive Antiretroviral Therapy

Dana Gabuzda et al. Viruses. .

Abstract

HIV establishes a persistent viral reservoir in the brain despite viral suppression in blood to undetectable levels on antiretroviral therapy (ART). The brain viral reservoir in virally suppressed HIV+ individuals is not well-characterized. In this study, intact, defective, and total HIV proviral genomes were measured in frontal lobe white matter from 28 virally suppressed individuals on ART using the intact proviral DNA assay (IPDA). HIV gag DNA/RNA levels were measured using single-copy assays and expression of 78 genes related to inflammation and white matter integrity was measured using the NanoString platform. Intact proviral DNA was detected in brain tissues of 18 of 28 (64%) individuals on suppressive ART. The median proviral genome copy numbers in brain tissue as measured by the IPDA were: intact, 10 (IQR 1-92); 3' defective, 509 (225-858); 5' defective, 519 (273-906); and total proviruses, 1063 (501-2074) copies/106 cells. Intact proviral genomes accounted for less than 10% (median 8.3%) of total proviral genomes in the brain, while 3' and 5' defective genomes accounted for 44% and 49%, respectively. There was no significant difference in median copy number of intact, defective, or total proviruses between groups stratified by neurocognitive impairment (NCI) vs. no NCI. In contrast, there was an increasing trend in intact proviruses in brains with vs. without neuroinflammatory pathology (56 vs. 5 copies/106 cells, p = 0.1), but no significant differences in defective or total proviruses. Genes related to inflammation, stress responses, and white matter integrity were differentially expressed in brain tissues with >5 vs. +5 intact proviruses/106 cells. These findings suggest that intact HIV proviral genomes persist in the brain at levels comparable to those reported in blood and lymphoid tissues and increase CNS inflammation/immune activation despite suppressive ART, indicating the importance of targeting the CNS reservoir to achieve HIV cure.

Keywords: HIV reservoirs; HIV-associated neurocognitive disorders; intact proviral DNA assay; intact proviral genomes; neuroinflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
IPDA analysis of HIV proviruses in brain tissue from 28 HIV+ individuals on ART. Log10 copy number (standardized to 106 cells) of proviral genomes defined as intact (blue), 3′ defective (green), 5′ defective (yellow), or total (sum of intact, 3′ defective, and 5′ defective; red) was measured in autopsy brain tissue (frontal white matter) from HIV+ individuals on ART (n = 28) (top left). Log10 copy number of intact and total proviruses in groups by neurocognitive impairment (NCI, n = 15) vs. no neurocognitive impairment (No NCI, n = 13), last CD4 count <350 (n = 18) vs. last CD4 count ≥350 (n = 10), or HIVE score 0 (n = 19) vs. HIVE score 1 or 2 (n = 9) (top right and bottom panels). Horizontal bars represent medians, boxes span the interquartile range (IQR), and whiskers extend to extreme data points within 1.5 times the IQR.
Figure 2
Figure 2
Correlation analysis of intact versus total HIV provirus or gag DNA levels in brain tissue from HIV+ individuals on ART. (A) Correlation between log10 intact provirus copy number and log10 total provirus copy number or log10 gag DNA copy number (standardized to 106 cells) in brain tissue (frontal lobe white matter) among 28 HIV+ individuals. (B) Correlation analysis as described in sensitivity analysis (A) restricted to 18 HIV+ individuals with detectable intact proviruses (>1 intact copy per 106 cells). Spearman’s rho and p-values are shown.
Figure 3
Figure 3
Differential expression of genes related to neuroinflammation and white matter integrity in groups stratified by level of intact proviruses in brain tissue from 28 HIV+ individuals on ART. Differential gene expression in brain tissue (frontal white matter) from 28 HIV+ individuals stratified by frequency of intact proviruses per 106 cells in brain tissue >5 copies vs. ≤5 copies per 106 cells (n = 12 and n = 16, respectively). Horizontal bars represent medians, boxes span the interquartile range (IQR), and whiskers extend to extreme data points within 1.5 times the IQR. p-values calculated using Welch’s t-test.
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