In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a "Block-and-Lock" Strategy for HIV-1 Treatment

Abstract

HIV-1 Tat activates viral transcription and limited Tat transactivation correlates with latency establishment. We postulated a "block-and-lock" functional cure approach based on properties of the Tat inhibitor didehydro-Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4+ T cells isolated from aviremic individuals. Combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increasing nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Adding dCA to ART-suppressed mice systemically reduces viral mRNA in tissues. Moreover, dCA significantly delays and reduces viral rebound levels upon treatment interruption. Altogether, this work demonstrates the potential of block-and-lock cure strategies.

Keywords: HIV latency; HIV-1; HIV-1 transcription; Tat inhibitor; block-and-lock; didehydro-Cortistatin A; epigenetics; humanized mouse model; infected CD4+T cells; latent reservoir.

Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Addition of dCA to ART promotes rapid HIV-1 suppression from primary human CD4+ T cells isolated from infected individuals and inhibits viral rebound during reactivation and treatment interruption

A. PBMCs are extracted from successfully treated aviremic HIV-infected individuals (at least 3 years on ART). CD4+ T cells are isolated and expanded in PHA, IL-2, feeder cells (irradiated PBMCs from 3 healthy donors). B. Expanded primary human CD4+ T cells from 5 HIV-infected individuals were kept on IL-2 alone and treated with a cocktail of ART with or without 50 nM dCA. Viral RNA levels in the supernatant of ART and ART+dCA treated cells measured every 7 days. After 35 days in culture viral RNA production in the supernatant was below the detection limit (*p<0.05). C. Total HIV DNA was determined up to 60 days in cells treated with ART or ART+dCA. Limit of detection for the qPCR is 3 viral copies per million cells and error bars represent standard error. D. On day 35 cells were stimulated overnight with 1 µM prostratin without ART or dCA. Viral production in supernatant quantified by RT-qPCR. E. Aggregate plot of data from D. Significant decrease (**p<0.01) in viral rebound after stimulation in dCA treated compared to ART alone. F. Treatment interruption does not result in immediate viral rebound even during strong cellular activation. On day 35, all drugs (ART and dCA) were removed and viral output in the supernatant quantified for the next 25 days by RT-qPCR. Cells were re-expanded with PHA, IL-2 and feeder cells at day 42 to maintain cell cultures and provide a stimulation. Results show viral RNA levels in the supernatant for each individual sample. *p<0.05, **p<0.01, ***p<0.001. ND, not detected; sup, supernatant.

Figure 2. Characterization of chromatin structure and RNAPII recruitment to the HIV genome after long term treatment with dCA using the OM10.1 latency model

A. dCA inhibits viral production in the OM-10.1 cell line to almost-undetectable levels. OM-10.1 cells were split and treated on average every 3 days in the presence of ARVs with or without dCA (100 nM). Capsid production was quantified via p24 ELISA. Data are representative of four independent experiments. B. On days 263, 276 and 286 cells treated with ARVs and ARVs+dCA (100 nM) were stimulated with SAHA (2.5 µM) for 8 hours (highlighted with ⦿/☒ in panel A). cDNAs prepared from total RNA quantified by RT-qPCR with Nef region primers. Results were normalized as number of viral mRNA copies per GAPDH mRNA. Viral mRNA generated in the ARVs control was set to 100%, error bars represent the standard deviations of 3 independent experiments. C. The chromatin structure of the HIV LTR assessed by Micrococcal nuclease (MNase) protection assay. The chromatin profile of cell samples from B was determined by normalizing the amount of the MNase digested PCR product to that of the undigested product using the ΔC(t) method (y-axis), which is plotted against the midpoint of the corresponding PCR amplicon (x-axis). The X-axis represents base pairs units with 0 as the start of HIV LTR. Error bars represent the standard deviations of 3 independent experiments. D. H3 ChIP of cell samples from B. The promoter of GAPDH was used as a reference. The results are presented as percent immunoprecipitated DNA over input. Data are average of 3 independent experiments, and error bars represent the standard deviations of 3 experiments for each primer set. Statistical significance was determined using paired t-test, *p<0.05. E. Distribution of RNAPII on the HIV genome. ChIP to RNAPII was performed using cells samples from panel B. Data are average of 3 independent experiments, and error bars represent the standard deviations of 3 experiments for each primer set.

Figure 3. Impact of dCA on ART mediated suppression of viremia and residual viral RNA expression in tissues of humanized mice

A. Diagram outlining the experimental design and highlighting the two different treatments (red and blue). B. Aggregate plot of all HIV-infected BLT humanized mice receiving dCA (blue, n=7) or vehicle (red, n=7) in addition to ART shows similar kinetics of viral suppression and no increases in plasma viral load as a result of dCA treatment. C. dCA administration does not affect plasmas viral load levels or the levels of CD4+ or CD8+ T cells in peripheral blood. D. (Left) Residual HIV-1 RNA levels in spleens, bone marrow, lymph nodes, and peripheral blood mononuclear cells from control (red) and dCA treated mice (blue). (Right) Reductions in viral RNA for all individual tissues from all animals are graphed together. Each mouse was assigned a different shape, and tissues are coded by color: spleen-red, bone marrow-blue, lymph node-green, PBMC-black. E. Residual HIV-1 RNA levels in brain tissue from control and dCA treated animals. HIV RNA levels were normalized to the levels of TATA box binding protein RNA. Error bars in D and E indicate standard error of the mean. Statistical significance was determined using the Mann-Whitney U test.

Figure 4. dCA treatment results in a delay in rebound viremia in HIV-1 infected, suppressed BLT humanized mice following therapy interruption

A. Diagram outlining the experimental design and experimental groups receiving ART+dCA (blue) or ART alone (red). B. Rebound viremia following therapy interruption (day 0) in individual HIV infected ART treated mice administered vehicle, or C. Rebound viremia following therapy interruption (day 0) in individual HIV-infected, ART-treated mice that were administered dCA. D. Comparison of the median viral load of mice treated with vehicle and with dCA following therapy interruption. In these plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data. Blue boxes indicate dCA treated mice and red boxes indicate control mice. * statistical significant differences between control and dCA treated mice as determined by the Mann-Whitney U-test. E. Time to event (Kaplan-Meier) plot demonstrating the significant delay in viral rebound observed in animals treated with dCA (p=0.0040, exact rank test).