Non-nucleoside reverse transcriptase inhibitor-based combination antiretroviral therapy is associated with lower cell-associated HIV RNA and DNA levels compared to protease inhibitor-based therapy

Abstract

Background: It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI).

Methods: CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts (n = 100, n = 124) of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4⁺ count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART.

Results: In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals (p_adj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (p_adj = 0.048 and p_adj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals.

Conclusions: All current classes of antiretroviral drugs only prevent infection of new cells but do not inhibit HIV RNA transcription in long-lived reservoir cells. Therefore, these differences in CA HIV RNA and DNA levels by treatment regimen suggest that NNRTIs are more potent in suppressing HIV residual replication than PIs, which may result in a smaller viral reservoir size.

Funding: This work was supported by ZonMw (09120011910035) and FP7 Health (305522).

Keywords: HIV; antiviral drugs; infectious disease; medicine; microbiology; virus; virus infection.

Figure 1.. Associations of clinical and virological variables, time of virological suppression, and ART regimens (NNRTI-based vs PI-based) with the levels of cell-associated human immunodeficiency virus (HIV) unspliced RNA (US RNA) and total HIV DNA in (A) COBRA cohort (n = 100) and (B) AIMS cohort (n = 124).

Units of measurement are US RNA: log₁₀ copies/μg total RNA, total DNA: log₁₀ copies/10⁶ peripheral blood mononuclear cells (PBMC), CD4 count and CD4 nadir: cells/mm³, plasma HIV RNA zenith: log₁₀ copies/ml. Levels of significance were calculated by Spearman correlation analyses or Mann-Whitney tests, as appropriate. In all correlation graphs, non-nucleoside reverse transcriptase inhibitor (NNRTI)- and protease inhibitor (PI)-treated participants are color-coded (NNRTI - blue, PI - red).

Figure 1—figure supplement 1.. Effects of duration of cumulative virological suppression and duration of the current regimen on the levels of cell-associated HIV US RNA and total HIV DNA in the AIMS cohort (n = 124).

Units of measurement are US RNA: log₁₀ copies/μg total RNA, total DNA: log₁₀ copies/10⁶ PBMC. Levels of significance were calculated by Spearman correlation analyses.

Figure 1—figure supplement 2.. Differences in duration of continuous and cumulative virological suppression, duration of current regimen, and in adherence to ART between participants with undetectable vs low-level detectable pVL in the AIMS cohort.

Levels of significance were calculated by Mann-Whitney tests.

Figure 2.. Regression analyses to identify variables associated with cell-associated human immunodeficiency virus (HIV) unspliced (US) RNA and total HIV DNA levels in (A) COBRA and (B) AIMS cohorts.

Effect sizes and 95% confidence intervals for US RNA are plotted as log₁₀ copies per microgram of total cellular RNA and for total DNA as log₁₀ copies per million peripheral blood mononuclear cells (PBMC). Effect sizes were obtained by fitting generalized linear models. Variables associated with HIV RNA or DNA with p-values <0.1 in the univariable analyses were included in the multivariable analyses.

Figure 2—figure supplement 1.. Regression analyses to identify variables associated with cell-associated HIV US RNA and total HIV DNA levels in the AIMS cohort, taking into account either (A) duration of cumulative virological suppression on ART or (B) duration of current ART regimen, prior to the measurements.

Effect sizes and 95% confidence intervals for US RNA are plotted as log₁₀ copies per microgram of total cellular RNA and for total DNA as log₁₀ copies per million PBMC. Effect sizes were obtained by fitting generalized linear models. Variables associated with HIV RNA or DNA with p-values <0.1 in the univariable analyses were included in the multivariable analyses.

Figure 2—figure supplement 2.. Regression analyses to identify variables associated with cell-associated HIV US RNA and total HIV DNA levels in the AIMS cohort, taking into account both duration of continuous virological suppression on ART and duration of current ART regimen, prior to the measurements.

Effect sizes and 95% confidence intervals for US RNA are plotted as log₁₀ copies per microgram of total cellular RNA and for total DNA as log₁₀ copies per million PBMC. Effect sizes were obtained by fitting generalized linear models. Variables associated with HIV RNA or DNA with p-values <0.1 in the univariable analyses were included in the multivariable analyses.

Figure 3.. Sensitivity analysis and associations of individual antiretroviral drugs with cell-associated human immunodeficiency virus (HIV) RNA and DNA in the pooled cohort.

Associations of antiretroviral therapy (ART) regimens (non-nucleoside reverse transcriptase inhibitor (NNRTI)-based vs protease inhibitor (PI)-based) with the levels of cell-associated HIV unspliced RNA (US RNA) and total HIV DNA in either (A) the total pooled cohort (n = 213) or (B) limiting the analysis to participants with undetectable plasma viral loads (pVLs) and >6 months of virological suppression on ART (n = 178). (C) Differences in the levels of US RNA and total HIV DNA between participants treated with ART regimens based on efavirenz (EFV), nevirapine (NVP), or PIs in the total pooled cohort. (D) Differences in the levels of US RNA and total HIV DNA between participants treated with ART regimens based on different ritonavir-boosted PIs: atazanavir (ATZ/r), darunavir (DRV/r), or lopinavir (LPV/r) in the total pooled cohort. Units of measurement are US RNA: log₁₀ copies/μg total RNA, total DNA: log₁₀ copies/10⁶ peripheral blood mononuclear cells (PBMC). Levels of significance were calculated by Mann-Whitney tests or Kruskal-Wallis tests with Dunn’s post-tests, as appropriate. For three-group comparisons, Kruskal-Wallis p-values are shown on top of the graphs and Dunn’s significance levels of pairwise comparisons are shown by asterisks only where significant; **0.001 < p < 0.01; *0.01 < p < 0.05. Participant numbers per regimen are indicated below the graphs.

Figure 3—figure supplement 1.. Association of ART regimen (NNRTI-based vs PI-based) with the cell-associated HIV US RNA/total HIV DNA ratio in the total pooled cohort (n = 213).

Level of significance was calculated by Mann-Whitney test.

Figure 3—figure supplement 2.. Associations of ART regimen (NNRTI-based vs PI-based) with the levels of cell-associated HIV US RNA and total HIV DNA in the total pooled cohort.

Participants were grouped according to the time of continuous virological suppression: 0–1 years, 2–5 years, 6–9 years, and 10 years or more. Units of measurement are US RNA: log₁₀ copies/μg total RNA, total DNA: log₁₀ copies/10⁶ PBMC.

Figure 3—figure supplement 3.. Levels of US RNA and total HIV DNA in participants treated with ART regimens based on efavirenz (EFV), nevirapine (NVP), ritonavir-boosted atazanavir (ATZ/r), ritonavir-boosted darunavir (DRV/r), or ritonavir-boosted lopinavir (LPV/r) in the total pooled cohort.

Units of measurement are US RNA: log₁₀ copies/μg total RNA, total DNA: log₁₀ copies/10⁶ PBMC. Levels of significance were calculated by Kruskal-Wallis tests. Participant numbers per regimen are indicated below the graphs.