Cellular levels of HIV unspliced RNA from patients on combination antiretroviral therapy with undetectable plasma viremia predict the therapy outcome

Abstract

Background: Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia.

Methodology/principal findings: Here, we report the results of a retrospective longitudinal study of twenty-six HIV-infected individuals who initially responded to cART by having plasma viremia suppressed to <50 copies/ml. Eleven of these patients remained virologically suppressed, whereas fifteen experienced subsequent cART failure. Using sensitive methods based on seminested real-time PCR, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the peripheral blood mononuclear cells (PBMC) of these patients at multiple time points during the period of undetectable plasma viremia on cART. Median under-therapy level of usRNA was significantly higher (0.43 log(10) difference, P = 0.0015) in patients who experienced subsequent cART failure than in successfully treated patients. In multivariate analysis, adjusted for baseline CD4(+) counts, prior ART experience, and particular cART regimens, the maximal usRNA level under therapy was the best independent predictor of subsequent therapy failure (adjusted odds ratio [95% CI], 24.4 [1.5-389.5], P = 0.024). The only other factor significantly associated with cART failure was prior ART experience (adjusted odds ratio [95% CI], 12.3 [1.1-138.4], P = 0.042). Levels of usRNA under cART inversely correlated with baseline CD4(+) counts (P = 0.0003), but did not correlate with either baseline usRNA levels or levels of prDNA under therapy.

Conclusion: Our data demonstrate that the level of HIV-1 usRNA in PBMC, measured in cART-treated patients with undetectable plasma viremia, is a strong predictive marker for the outcome of therapy.

Figure 1. Levels of virological parameters and CD4⁺ counts.

Time points on the X axes correspond to the median times from cART initiation of PBMC samples for every of the sampling time groups (see Methods). Numbers of patients in every sampling time group are indicated on the graphs. For the upper panels, total numbers of patients are indicated. Median values and interquartile ranges of the studied parameters are indicated. Thin dotted lines on the upper panels indicate the limit of detection of the modern commercial assays (50 copies/ml). Levels of statistical significance are depicted on the graphs and signify the following P values: ***, P<0.001; **, 0.001<P<0.01; *, 0.01<P<0.05; ns (not significant), P>0.05. (A) All patients combined. Parameters were compared between baseline and time on cART-grouped PBMC samples. (B) Patient groups (red, failures; blue, successes). Parameters were compared between failures and successes. Numbers of failures and successes are indicated by n_F and n_S, respectively. To prevent overlap, data sets are nudged between the patient groups.

Figure 2. Longitudinal trends of the virological parameters and CD4⁺ count during the eclipse phase.

The dots represent all the PBMC samples used in this study. Best-fit lines and their 95% CI are shown by solid and dashed lines, respectively. (A) All patients combined. Levels of statistical significance of the longitudinal trends are shown on the graphs. (B) Patient groups (red, failures; blue, successes). Levels of statistical significance of the comparison of the parameters between the patient groups are shown on the graphs.

Figure 3. The risk of VR according to the maximal usRNA value during the eclipse phase.

Kaplan-Meier plot shows the risk of VR as a function of maximum usRNA level measured during the eclipse phase. Small vertical bars show successfully treated patients.

Figure 4. Correlations of virological parameters and CD4⁺ count.

Best-fit lines and 95% CI are shown by solid and dashed lines, respectively. Spearman correlation coefficients (r_s) and corresponding P values are indicated within the graphs. Mean values of the quantified under-therapy parameters per patient (geometric means of log₁₀-transformed values of usRNA and prDNA load and arithmetic means of CD4⁺ count) were used. The units of measurement are log₁₀ copies/10⁶ PBMC for prDNA, log₁₀ copies/µg total RNA for usRNA, log₁₀ copies/ml for plasma RNA, and cells/mm³ blood for CD4⁺ count. (A) Correlations between the under-therapy levels of the studied parameters. (B) Correlations between the baseline and under-therapy levels. (C) Schematic representation of the correlations shown in panels A and B. Arrows indicate significant correlations, and thickness of arrows indicates levels of statistical significance: thick arrows, P<0.001; intermediate arrows, 0.001<P<0.01; thin arrows, 0.01<P<0.05.