Maraviroc Is Associated with Latent HIV-1 Reactivation through NF-κB Activation in Resting CD4+ T Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy

Abstract

Maraviroc is a CCR5 antagonist used in the treatment of HIV-1 infection. We and others have suggested that maraviroc could reactivate latent HIV-1. To test the latency-reversing potential of maraviroc and the mechanisms involved, we performed a phase II, single-center, open-label study in which maraviroc was administered for 10 days to 20 HIV-1-infected individuals on suppressive antiretroviral therapy (EudraCT registration no. 2012-003215-66). All patients completed full maraviroc dosing and follow-up. The primary endpoint was to study whether maraviroc may reactivate HIV-1 latency, eliciting signaling pathways involved in the viral reactivation. An increase in HIV-1 transcription in resting CD4⁺ T cells, estimated by levels of HIV-1 unspliced RNA, was observed. Moreover, activation of the NF-κB transcription factor was observed in these cells. To elucidate the mechanism of NF-κB activation by maraviroc, we have evaluated in HeLa P4 C5 cells, which stably express CCR5, whether maraviroc could be acting as a partial CCR5 agonist, with no other mechanisms or pathways involved. Our results show that maraviroc can induce NF-κB activity and that NF-κB targets gene expression by CCR5 binding, since the use of TAK779, a CCR5 inhibitor, blocked NF-κB activation and functionality. Taking the results together, we show that maraviroc may have a role in the activation of latent virus transcription through the activation of NF-κB as a result of binding CCR5. Our results strongly support a novel use of maraviroc as a potential latency reversal agent in HIV-1-infected patients.IMPORTANCE HIV-1 persistence in a small pool of long-lived latently infected resting CD4⁺ T cells is a major barrier to viral eradication in HIV-1-infected patients on antiretroviral therapy. A potential strategy to cure HIV-1-infection is the use of latency-reversing agents to eliminate the reservoirs established in resting CD4⁺ T cells. As no drug has been shown to be completely effective so far, the search for new drugs and combinations remains a priority for HIV cure. We examined the ability of maraviroc, a CCR5 antagonist used as an antiretroviral drug, to activate latent HIV-1 in infected individuals on antiretroviral therapy. The study showed that maraviroc can activate NF-κB and, subsequently, induce latent HIV-1-transcription in resting CD4⁺ T cells from HIV-1-infected individuals on suppressive antiretroviral therapy. Additional interventions will be needed to eliminate latent HIV-1 infection. Our results suggest that maraviroc may be a new latency-reversing agent to interfere with HIV-1 persistence during antiretroviral therapy.

Keywords: HIV infected; HIV-1; LRA; NF-κB; latency; maraviroc; persistence; reservoir.

FIG 1

Maraviroc promotes HIV-1 latency reversal in resting CD4⁺ T cells. HIV-1 latency reversal was estimated by the number of copies per million of HIV-1 usRNA in resting CD4⁺ T cells from all patients as determined by nested qRT-PCR. (A) Individual changes in usRNA levels are shown for each study participant. (B) Expression of the number of copies of usRNA per million resting CD4⁺ T cells per day of the study. Significant changes between time points were determined using the Wilcoxon signed-rank test. Central lines indicate the median. (C) Expression of usRNA before, during, and after 10-day intensification of maraviroc in resting CD4⁺ T cells of the patients. Samples were stratified into the following groups: baseline (Pre-MVC); day 1, day 3, day 10 (On MVC); and day 28 (Post-MVC). Comparisons of expression of usRNA among the premaraviroc, on-maraviroc, and off-maraviroc time periods were also performed using the Wilcoxon signed-rank test. (D) Fold change in usRNA following maraviroc treatment in resting CD4⁺ T cells compared to baseline. The maximum fold change in usRNA during the study (dark gray bar) and the change at day 28 (light gray bar) are shown for resting CD4⁺ T cells, and the median (IQR) change is shown for all participants. The solid line indicates 1-fold change. MVC, maraviroc.

FIG 2

Maraviroc induces NF-κB, but not AP-1 or NFAT, in resting CD4⁺ T cells. AP-1, NFAT, and NF-κB activities in resting CD4⁺ T cells were estimated by a DNA binding assay coupled with ELISA (Active Motif). (A) Fold change in transcription factor activity in resting CD4⁺ T cells following maraviroc treatment compared to baseline. Results are shown as geometric means, with error bars denoting 95% confidence intervals. (B to D) Fold changes in AP-1 (B), NFAT (C), and NF-κB (D) are shown for each participant (*, P < 0.05; **, P < 0.01). Blue lines represent patients infected with R5-tropic virus, red lines those infected with non-R5-tropic virus, and green lines those with indeterminate tropism; the solid black line indicates the median value. MVC, maraviroc.

FIG 3

Maraviroc induces expression of the NF-κB target genes for IFN-γ (A), IL-6 (B), IL-10 (C), and TNF-α (D) in resting CD4⁺ T cells. Significant changes between time points were determined using the Wilcoxon signed-rank test. Central lines indicate the median at each time point, and results are expressed as the fold change in mRNA expression compared to the baseline (without maraviroc). MVC, maraviroc.

FIG 4

Maraviroc induces NF-κB activity in CCR5-overexpressing HeLa P4 C5 cells. The cells were treated with 5 µM, 10 µM, and 20 µM maraviroc for 2, 6, 12, and 24 h. NF-κB p65 activity was significantly higher in HeLa P4 C5 cells at 2 h with 5 µM maraviroc. PMA was used as a positive control. NF-κB activity was not observed in HeLa wild-type cells at any of the concentrations and times or in HeLa P4 C5 cells treated with TAK-779, a CCR5 inhibitor, for 2 h with maraviroc (5 µM). Mean values were calculated from triplicates of a representative experiment. MVC, maraviroc; PMA, phorbol 12-myristate 13-acetate.

FIG 5

Maraviroc induces NF-κB target genes for IFN-γ, IL-6 and TNF-α in HeLa P4 C5 cells. Representative results of qRT-PCR experiments for relative expression of NF-κB target genes in HeLa P4 C5 cells upon maraviroc treatment are shown. MVC, maraviroc.