Interval dosing with the HDAC inhibitor vorinostat effectively reverses HIV latency

Abstract

Background: The histone deacetylase (HDAC) inhibitor vorinostat (VOR) can increase HIV RNA expression in vivo within resting CD4+ T cells of aviremic HIV+ individuals. However, while studies of VOR or other HDAC inhibitors have reported reversal of latency, none has demonstrated clearance of latent infection. We sought to identify the optimal dosing of VOR for effective serial reversal of HIV latency.

Methods: In a study of 16 HIV-infected, aviremic individuals, we measured resting CD4+ T cell-associated HIV RNA ex vivo and in vivo following a single exposure to VOR, and then in vivo after a pair of doses separated by 48 or 72 hours, and finally following a series of 10 doses given at 72-hour intervals.

Results: Serial VOR exposures separated by 72 hours most often resulted in an increase in cell-associated HIV RNA within circulating resting CD4+ T cells. VOR was well tolerated by all participants. However, despite serial reversal of latency over 1 month of VOR dosing, we did not observe a measurable decrease (>0.3 log10) in the frequency of latent infection within resting CD4+ T cells.

Conclusions: These findings outline parameters for the experimental use of VOR to clear latent infection. Latency reversal can be achieved by VOR safely and repeatedly, but effective depletion of persistent HIV infection will require additional advances. In addition to improvements in latency reversal, these advances may include the sustained induction of potent antiviral immune responses capable of recognizing and clearing the rare cells in which HIV latency has been reversed.

Trial registration: Clinicaltrials.gov NCT01319383.

Funding: NIH grants U01 AI095052, AI50410, and P30 CA016086 and National Center for Advancing Translational Sciences grant KL2 TR001109.

Figure 1. Study participant allocation and outcome.

Figure 2. Ex vivo modeling of serial VOR exposures.

(A) Schematic of ex vivo dosing experiment. Resting CD4⁺ T cells from aviremic participants were exposed to media or 335 nM VOR (or PHA as a comparative control) for 6 hours, rca-HIV RNA was measured in an aliquot (B), and the remaining cells were washed and placed in culture. Cells then received no VOR or a second 6-hour pulse of VOR at 18 hours, 42 hours, or 66 hours following the first pulse to model daily, every-2-day, or every-3-day dosing regimens. rca-HIV RNA was measured in cells that had never been exposed to VOR (circles), were pulsed once (squares), or pulsed twice (triangles) at 24 hours (C), 48 hours (D), and 72 hours (E). Each data point represents 1 million resting CD4⁺ T cells. n = 3 independent experiments. Data represent the mean ± SD. *P < 0.05,**P < 0.005, and ***P < 0.0001, by Mann-Whitney U test.

Figure 3. A 6-hour exposure to VOR ex vivo increased HIV gag RNA in resting CD4⁺ T cells isolated from aviremic donors.

Pools of 24 million resting CD4⁺ T cells isolated from aviremic donors were exposed to 335 nM VOR or mitogen (PHA and 60 U/ml IL-2) for 6 hours, and rca-HIV RNA levels were measured. Data represent the mean ± SD. P < 0.05 to P < 0.0001 (Mann-Whitney U test) for all comparisons between untreated cells and ex vivo VOR–treated cells.

Figure 4. In vivo administration of a single 400-mg dose of VOR led to a significant increase in rca-HIV RNA in the resting CD4⁺ T cells of aviremic participants.

Data represent the mean ± SD. P < 0.0001 (Mann-Whitney U test) for all comparisons between baseline and VOR treatment.

Figure 5. Two sequential doses of VOR given every 72 hours, but not 48 hours, resulted in a sustained increase in rca-HIV RNA in participants.

(A) A 48-hour interval between sequential doses for 1 participant resulted in an increase in rca-HIV RNA levels that only became statistically significant when the interval between doses was increased to 72 hours. (B) A significant increase in rca-HIV RNA levels was observed in 5 of 6 additional participants at the 72-hour interval. Data represent the mean ± SD. **P < 0.01 and ***P < 0.0001, by Mann-Whitney U test. Baseline rca-HIV RNA levels are from samples collected at screening, either before the first VOR dose (V-3, V-4, V-5, and V-10), or from a second baseline sample collected prior to the paired dosing for participants with more than 1 year of elapsed time from administration of the first dose (V-1, V-2, and V-11).

Figure 6. Multiple (10 doses) of in vivo VOR administered every 72 hours resulted in a sustained increase in HIV RNA in 2 of 3 participants.

Data represent the mean ± SD. ***P < 0.0001, by Mann-Whitney U test.

Figure 7. Repeated exposure to VOR (10 doses) had a limited effect on ex vivo T cell function.

(A) Following in vivo exposure to VOR, CD8⁺ T cell proliferation in response to Flu peptide (FEC), HIV Gag/Nef peptide (CTLA), and other HIV peptides (CTLB) was evaluated by CFSE staining. Study clinic visits: 4–6 hours after a single VOR dose (visit 2); 4–6 hours after the second of paired VOR doses (visit 3); 4 hours and 7 months after the last of 10 VOR doses (visits 4 and 5, respectively). (B) In participants V-3, V-4, and V-5, in vivo exposure to VOR over a 1-month period did not reduce the antiviral capacity of CD8⁺ T cells when challenged with autologous targets infected with AR virus in a virus inhibition assay.