In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors

Abstract

The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects.

Importance: The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiency in vitro using primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

FIG 1

HDACis efficiently reactivate HIV-1 replication from latently infected resting memory CD4 T cells isolated from long-term-treated HIV-1-infected subjects. (A) Schematic representation of the modified VOA. (B) Proportion of responders to HDACis treatment based on the detection of HIV-1 RNA (n = 10; 5 replicates per condition). Individuals having at least one replicate with detectable HIV-1 RNA (≥200 HIV-1 RNA copies/ml) are indicated as “responders” for the condition tested. (C) Proportion of responders to HDACi treatment based on the detection of P24 (n = 10; 5 replicates per condition). Individuals having at least one replicate with detectable P24 (≥1 ECL unit/ml) are indicated as “responders” for the condition tested. (D) Proportion of HIV-1 RNA-positive wells induced following HDACis treatment (n = 10; 5 replicates per condition). Wells with detectable HIV-1 RNA (≥200 HIV-1 RNA copies/ml) are indicated to as HIV-1 RNA positive for the condition tested. (E) Proportion of P24-positive wells induced following HDACis treatment (n = 10; 5 replicates per condition). Wells with detectable P24 (≥1 ECL unit/ml) are indicated as P24 positive for the condition tested. (F) Levels of HIV-1 RNA copies per milliliter induced following HDACis treatment (n = 10; 5 replicates per condition). (G) Levels of P24 (ECL units per milliliter) induced following HDACis treatment (n = 10; 5 replicates per condition). (H and I) Frequencies of inducible replication-competent virus as measured by replication-competent (RNA) units per million (RUPM) (H) or as measured by infectious units per million (IUPM) (I). (J) Correlation between P24 and HIV-1 RNA levels (n = 350; 10 subjects, 7 conditions, 5 replicates per condition). Panels B to G and J were generated using the 5 replicates of the lowest dilution of cells (5 × 10⁵ cells) of all conditions by modified VOA. Subjects were color coded, and each color corresponds to a subject (F to J). Histograms correspond to the mean (B to I), and red error bars correspond to the standard error of the mean (SEM) (F to I). Blue lines correspond to the median (F to I). Red asterisks indicate statistical significance compared to unstimulated or unexposed (US) (P < 0.05). Green asterisks indicate statistical significance compared to vorinostat (P < 0.05). romi, romidepsin; pano, panobinostat; givi, givinostat; beli, belinostat. Statistical significance (P values) was obtained using two-tailed chi-square analysis for comparison of positive proportions (B to E), by one-way ANOVA (Kruskal-Wallis test) followed by Wilcoxon matched-pair two-tailed signed rank test (F to I), or by using Spearman's rank correlations (J). Bonferroni's correction was applied for multiple comparisons.

FIG 2

Assessment of functional activity and toxicity of the HDACis tested. (A and B) Mean fluorescence intensity (MFI) of acetyl histone H3 (A) or histone H4 (B). Blood mononuclear cells isolated from 4 HIV-seronegative individuals were exposed or not (unexposed control [US]) to vorinostat (400 nM), romidepsin (Romi [5 nM]), panobinostat (Pano [15 nM]), givinostat (Givi [400 nM]), or belinostat (Beli [400 nM]) for 24 h, and acetyl histone H3 or H4 MFI was analyzed on CD4 T cells by flow cytometry. (C) Percentage of CD4 T-cell proliferation upon HDACi treatment. CFSE-labeled blood mononuclear cells isolated from 3 HIV-seronegative individuals were exposed or not (US) to vorinostat (400 nM), romidepsin (5 nM), panobinostat (15 nM), givinostat (400 nM), or belinostat (400 nM) for 24 h and stimulated for 6 days with anti-CD3/CD28 MAbs, and the percentage of proliferating CD4 T-cell proliferation (CFSE low) was assessed by flow cytometry. The percentage of annexin V-positive (D) or Aqua-positive (E) CD4 T cells was assessed at 24, 48, 96, and 144 h.CD4 T cells isolated from 3 HIV-uninfected individuals were exposed or not to vorinostat (400 nM), romidepsin (5 nM), panobinostat (15 nM), givinostat (400 nM), and belinostat (400 nM) in the presence of allogeneic CD8-depleted PBMCs isolated from HIV-uninfected subjects. Red horizontal lines or histograms correspond to the mean, and red error bars correspond to the SEM. Red asterisks indicate statistical significance (P < 0.05) compared to unexposed conditions. Statistical significance (P values) was obtained using a one-way ANOVA (Kruskal-Wallis test) followed by a paired t test (A to C).

FIG 3

HIV-1 RNA quantification in culture supernatants using Roche TaqMan assay versus the Abbott RealTime HIV-1 assay. Culture supernatants containing a range of HIV-1 RNAs, induced following HDACi treatments, anti-CD3/CD28 MAb treatment, givinostat plus anti-CD3/CD28 MAb treatment, or unexposed cells (n = 38) were assessed for HIV-1 RNA using the Roche TaqMan assay (Roche) and Abbott RealTime HIV-1 assay (Abbott). HIV-1 RNA quantification was performed using Roche TaqMan assay versus the Abbott RealTime HIV-1 assay in VOA culture supernatants obtained following anti-CD3/CD28 MAb treatment (A) or treatment with vorinostat (B), romidepsin (C), panobinostat (D), givinostat (E), belinostat (F), or givinostat plus anti-CD3/CD28 MAbs (G). Panel H shows the total results. (I) Correlation between HIV-1 RNA quantification using the Roche TaqMan assay and Abbott RealTime HIV-1. Histograms correspond to the mean, and red error bars correspond to the SEM. NS, not significant. Statistical significance (P values) was obtained using Wilcoxon's matched-pair two-tailed signed rank test (A to H) or Spearman's rank correlations (I).

FIG 4

Correlation between P24 and HIV-1 RNA levels detected in culture supernatants following the viral outgrowth assay. Correlation between P24 and HIV-1 RNA levels (n = 50; 10 subjects, 5 replicates per condition) was obtained following anti-CD3/CD28 MAb treatment (A) or treatment with vorinostat (B), romidepsin (C), panobinostat (D), givinostat (E), or belinostat (F). Each circle corresponds to one replicate, and each color corresponds to one HIV-1-infected subject. Dotted lines correspond to the limit of detections. Statistical significance (P values) was obtained using Spearman's rank correlations.

FIG 5

Estimation of the proportion of provirus induced following treatment with anti-CD3/CD28 MAbs and HDACis. (A) Proportion of provirus induced following treatments with anti-CD3/CD28 MAbs and HDACis as assessed by the ratio of RUPM and integrated (INT) DNA (n = 10). (B) Proportion of provirus induced following treatment with anti-CD3/CD28 MAbs and HDACis as assessed by the ratio of IUPM and integrated DNA (n = 10). (C) Proportion of provirus induced following HDACi treatment compared to treatment with anti-CD3/CD28 MAbs as assessed by RUPM. (D) Proportion of provirus induced following treatment with HDACis compared to anti-CD3/CD28 MAbs as assessed by IUPM (n = 6). Red asterisks indicate statistical significance (P < 0.05). Statistical significance (P values) was obtained using one-way ANOVA (Kruskal-Wallis test) followed by Wilcoxon's matched-pair two-tailed signed rank test.

FIG 6

Infectivity of HIV-1 reactivated by HDACi treatment. (A) Proportion of P24-positive wells at days 3 and 10 after in vitro HIV-1 inoculation of activated allogeneic CD8-depleted mononuclear cells from HIV-1-negative subjects in the modified VOA culture supernatants (obtained from P24-positive supernatants at day 14) (n = 27). (B) P24 values at days 3 and 10 following in vitro HIV-1 infection (n = 27). (C) Correlation between P24 levels detected in the cell culture supernatants from the in vitro infection assay and the P24 levels of the modified VOA supernatants (n = 27). Subjects were color coded, and each color corresponds to a subject (B and C). Each condition is depicted with a unique symbol. Red asterisks indicate statistical significance (P < 0.05). Statistical significance (P values) was obtained using two-tailed chi-square analysis for comparison of positive proportions (A), Wilcoxon's matched-pair two-tailed signed rank test (B), or Spearman's rank correlations (C).

FIG 7

Assessment of potential synergistic effect between givinostat treatment and TCR stimulation or PKC agonist on the reactivation of HIV-1 replication. (A) Proportion of HIV-1 RNA-positive wells following treatment with givinostat and/or anti-CD3/CD28 MAbs (n = 4). Wells with detectable HIV-1 RNA (≥200 HIV-1 RNA copies/ml) are indicated as HIV-1 RNA-positive wells for the condition tested. (B) Proportion of P24-positive wells following treatment with givinostat and/or anti-CD3/CD28 MAbs (n = 4). Wells with detectable P24 (≥1 ECL unit/ml) are indicated as P24 positive for the condition tested. (C) HIV-1 RNA (copies per milliliter) induced following treatment with givinostat and/or anti-CD3/CD28 MAbs (n = 4). (D) Levels of P24 (ECL units per milliliter) induced following treatment with givinostat and/or anti-CD3/CD28 MAbs (n = 4). (E) Proportion of HIV-1 RNA-positive wells following treatment with anti-CD3/CD28 MAbs or givinostat (Givi) and/or bryostatin (Bryo) (n = 3). Wells with detectable HIV-1 RNA (≥200 HIV-1 RNA copies/ml) are indicated as HIV-1 RNA-positive wells for the condition tested. US, unstimulated. (F) Proportion of P24-positive wells following treatment with anti-CD3/CD28 MAbs or givinostat and/or bryostatin (n = 3). Wells with detectable P24 (≥1 ECL unit/ml) are indicated as P24-positive wells for the condition tested. (G) Levels of HIV-1 RNA copies per milliliter induced following treatment with anti-CD3/CD28 MAbs or givinostat and/or bryostatin (n = 3). (H) Levels of HIV-1 P24 (ECL units per milliliter) induced following treatment with anti-CD3/CD28 MAbs or givinostat and/or bryostatin (n = 3). Subjects were color coded, and each color corresponds to a subject (C and D and G and H). Red error bars correspond to means ± SEM. Red asterisks indicate statistical significance (P < 0.05). Green asterisks indicate statistical significance compared to the givinostat (18-h) condition (P < 0.05). Statistical significance (P values) was obtained using two-tailed chi-square analysis for comparison of positive proportions (A, B, E, and F) or by one-way ANOVA (Kruskal-Wallis test) followed by Wilcoxon matched-pair two-tailed signed rank test (C, D, G, and H).

FIG 8

Prolonged/repeated exposure of resting memory CD4 T cells to HDACis is the primary mechanism responsible for efficient induction of HIV-1 replication by HDACi. (A) Levels of HIV-1 RNA (copies per milliliter) induced following givinostat treatment (n = 4; 5 replicates). (B) Levels of P24 (ECL units per milliliter) induced following givinostat treatment (n = 4; 5 replicates). Subjects were color coded, and each color corresponds to a subject. US, unstimulated or unexposed. Histograms correspond to the mean, and red error bars correspond to the SEM. Red asterisks indicate statistical significance (P < 0.05). Statistical significance (P values) was obtained using one-way ANOVA (Kruskal-Wallis test) followed by Wilcoxon's matched-pair two-tailed signed rank test.

FIG 9

Level of expression of activation markers and proliferation capacity of purified resting memory CD4 T cells exposed to allogeneic CD8-depleted mononuclear cells. (A to C) Percentages of CD69 (early) (A), CD25 (intermediate) (B), and HLA-DR (late) (C) activation marker expression on purified resting memory CD4 T cells (n = 3) following 0, 1, 2, 3, and 6 days of exposure to allogeneic CD8-depleted blood mononuclear cells or to anti-CD3/CD28 MAbs (positive control). Unexposed resting memory CD4 T cells were used as a negative control. (D) Percentage of proliferating resting memory CD4 T cells (CFSE_low [n = 3]) following 0, 1, 2, 3, and 6 days of exposure to allogeneic CD8-depleted mononuclear cells or to anti-CD3/CD28 MAbs (positive control). Unexposed resting memory CD4 T cells were used as a negative control. Red error bars correspond to the mean ± SEM. Red asterisks indicate statistical significance compared to unstimulated (US) (P < 0.05). Green asterisks indicate statistical significance compared to allogeneic CD8-depleted PBMCs (MLR) condition (P < 0.05). Statistical significance (P values) was obtained using paired Student's t tests.