Cell-Associated HIV-1 Unspliced-to-Multiply-Spliced RNA Ratio at 12 Weeks of ART Predicts Immune Reconstitution on Therapy

Abstract

Incomplete restoration of CD4⁺ T-cell counts on antiretroviral therapy (ART) is a major predictor of HIV-related morbidity and mortality. To understand the possible mechanisms behind this poor immunological response despite viral suppression, we longitudinally measured more than 50 virological and immunological biomarkers in a cohort of HIV-infected individuals at several time points during the first 96 weeks of virologically suppressive ART. No baseline virological or immunological marker was predictive of the degree of immune reconstitution. However, the cell-associated HIV-1 unspliced-to-multiply-spliced (US/MS) RNA ratio at 12 weeks of ART positively correlated with markers of CD4⁺ T-cell activation and apoptosis and negatively predicted both the absolute and relative CD4⁺ T-cell counts at 48 and 96 weeks. A higher US/MS RNA ratio may reflect the higher frequency of productively infected cells that could exert pressure on the immune system, contributing to persistent immune activation and apoptosis and subsequently to a poor immunological response to ART.IMPORTANCE Human immunodeficiency virus (HIV) infection is currently managed by antiretroviral drugs, which block virus replication and promote immune restoration. However, the latter effect is not universal, with a proportion of infected individuals failing to sufficiently reconstitute their immune function despite a successful virological response to antiretroviral therapy (ART). No reliable predictive markers of immunological failure have been identified, and there is still no efficient therapeutic strategy, apart from ART itself, to facilitate immune reconstitution. Here, we measured more than 50 viral and host biomarkers at five time points during the first 2 years of ART and identified the cell-associated HIV-1 unspliced-to-multiply-spliced RNA ratio at 12 weeks of ART as a predictive factor for the immunological response to therapy. Moreover, the same marker positively correlated with markers of CD4⁺ T-cell activation and apoptosis. The fact that a virological biomarker performed better than any immunological biomarker in predicting an immunological outcome highlights the importance of considering the residual HIV activity on ART as a correlate and a possible cause of the residual immune dysfunction that frequently occurs despite virologically suppressive ART.

Keywords: AIDS; cell-associated RNA; human immunodeficiency virus; immune reconstitution; viral persistence; viral reservoirs.

FIG 1

Longitudinal dynamics of CD4⁺ counts, CD4/CD8 ratios, and virological biomarkers during the first 96 weeks of ART. Participants are color-coded. Open circles depict undetectable values, censored to the detection limits. For the US RNA/total DNA ratios, open circles correspond to the samples where US RNA was undetectable and censored to the detection limits; because total DNA in these samples was detectable, these circles depict the upper limits of the US RNA/total DNA ratios. For the US/MS RNA ratios, open crossed circles correspond to the samples where MS RNA was undetectable and censored to the detection limits; because US RNA in these samples was detectable, these circles depict the lower limits of the US/MS RNA ratios. Two samples where both US and MS RNA were undetectable were excluded from the US/MS ratio calculations. For plasma viral load (VL), limits of detection of the commercial assays are shown with dashed lines. Repeated-measures mixed-effects P values (representing the significance values of the change of the parameter between the time points) as well as P values of pairwise comparisons between the biomarker values at different time points (adjusted to account for multiple comparisons) are depicted to the right of the corresponding graphs. Significant effects are shown in red. BL, baseline.

FIG 2

Longitudinal dynamics of CD4⁺ and CD8⁺ T-cell subsets during the first 96 weeks of ART. Participants are color-coded. Repeated-measures mixed-effects P values (representing the significance values of the change of the parameter between the time points) as well as P values of pairwise comparisons between the biomarker values at different time points (adjusted to account for multiple comparisons) and of comparisons between 96-week values of HIV-infected participants and those of healthy donors (HD) are depicted to the right of the corresponding graphs. Significant effects are shown in red.

FIG 3

Longitudinal dynamics of markers of CD4⁺ and CD8⁺ activation and senescence during the first 96 weeks of ART. Participants are color-coded. Repeated-measures mixed-effects P values (representing the significance values of the change of the parameter between the time points) as well as P values of pairwise comparisons between the biomarker values at different time points (adjusted to account for multiple comparisons) and of comparisons between 96-week values of HIV-infected participants and those of healthy donors (HD) are depicted to the right of the corresponding graphs. Significant effects are shown in red.

FIG 4

Longitudinal dynamics of markers of CD4⁺ and CD8⁺ apoptosis during the first 96 weeks of ART. Participants are color-coded. Repeated-measures mixed-effects P values (representing the significance values of the change of the parameter between the time points) as well as P values of pairwise comparisons between the biomarker values at different time points (adjusted to account for multiple comparisons) and of comparisons between 96-week values of HIV-infected participants and those of healthy donors (HD) are depicted to the right of the corresponding graphs. Significant effects are shown in red.

FIG 5

Spearman correlogram of CD4⁺ counts, CD4/CD8 ratios, and virological and immunological biomarkers at baseline. A heat map is used to indicate the strengths of associations between biomarkers. Red indicates a negative correlation, and blue indicates a positive correlation.

FIG 6

Spearman correlogram of CD4⁺ counts, CD4/CD8 ratios, and virological and immunological biomarkers at 12 weeks of ART. A heat map is used to indicate the strengths of associations between biomarkers. Red indicates a negative correlation, and blue indicates a positive correlation.

FIG 7

The CA HIV-1 US/MS RNA ratio predicts the immunological response to ART. (A) Spearman correlations between the US/MS RNA ratio at 12 weeks of ART and absolute and relative CD4⁺ counts at 48 and 96 weeks of ART. (B) Comparisons of immunological responses to ART between participants with high and those with low values of US/MS RNA ratios at 12 weeks of ART. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01.