Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 μM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART

Blockade of the PD-1 axis alone is not sufficient to activate HIV-1 virion production from CD4+ T cells of individuals on suppressive ART.

Blockade of the programmed cell death protein/ligand 1 (PD-1/PD-L1) pathway with monoclonal antibodies (mAb) is now commonly used for cancer immunotherapy and has therapeutic potential in chronic viral infections including HIV-1. PD-1/PD-L1 blockade could augment HIV-1-specific immune responses and reverse HIV-1 latency, but the latter effect has not been clearly shown. We tested the ability of the human anti-PD-L1 mAb BMS-936559 and the human anti-PD-1 mAb nivolumab to increase HIV-1 virion production ex vivo from different peripheral blood mononuclear cell populations obtained from donors on suppressive antiretroviral therapy (ART). Fresh peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, total CD4+ T cells, and resting CD4+ T cells were purified from whole blood of HIV-1-infected donors and cultured in varying concentrations of BMS-936559 (20, 5, or 1.25μg/mL) or nivolumab (5 or 1.25μg/mL), with or without anti-CD3/CD28 stimulatory antibodies. Culture supernatants were assayed for virion HIV-1 RNA by qRT-PCR. Ex vivo exposure to BMS-936559 or nivolumab, with or without anti-CD3/CD28 stimulation, did not consistently increase HIV-1 virion production from blood mononuclear cell populations. Modest (2-fold) increases in virus production were observed in a subset of donors and in some cell types but were not reproducible in longitudinal samples. Cell surface expression of PD-1 and PD-L1 were not associated with changes in virus production. Ex vivo blockade of the PD-1 axis alone has limited effects on HIV-1 latency

<i>Ex vivo</i> activation of CD4⁺ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells

<div><p>The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, <i>et al</i>. recently showed that CD4⁺ T-cells containing intact proviruses can clonally expand <i>in vivo</i> and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4⁺ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an <i>ex vivo</i> cell culture system involving stimulation of CD4⁺ T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1–7), followed by rest (day 7–21), and then repeat stimulation (day 21–28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4⁺ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.</p></div

Levels of HIV DNA during cell culture.

<p>The frequency of HIV-infected cells was quantified using qPCR.</p

Neighbor-joining distance tree of virion-associated HIV RNA and proviral-associated HIV DNA sequences illustrates proviral population dynamics after CD4⁺ T-cell activation (Donor 1).

<p>The tree was constructed using the neighbor-joining p-distance method. All sequences were rooted to a consensus sequence of HIV subtype B. Hypermutant sequences are boxed. The Viral Outgrowth Assay (VOA) was performed using day 7 and day 28 cells from Donor 1 (Experiment 2) total CD4⁺ T-cells. The day 7 cells were seeded into 6 wells at 1x10⁶ cells/well and the day 28 cells were seeded into 6 wells at 3x10⁵ cells/well.</p

Levels of HIV virion production during cell culture.

<p>HIV virion production was measured on cell-free supernatants using the Roche COBAS AmpliPrep/TaqMan HIV-1 test. The cumulative levels of virus produced spontaneously after 24 hours in separate experiments were < 20 copies/mL.</p

Proviral expression and dynamics in total CD4⁺ T-cells after sequential stimulation.

<p>Different proviral population outcomes are quantified for experiments with total CD4⁺ T-cells. Each outcome is calculated as either 1) the frequency of proviruses displaying a given outcome relative to the total number of unique proviral sequences observed over the entire duration of cell culture, or 2) the frequency of unique virion sequences displaying a given outcome relative to the total number of unique virion sequences observed over the entire duration of cell culture. IQR = interquartile range.</p

Experimental system schematic.

<p>PBMC or purified total CD4⁺ T-cells were cultured with 300 nM efavirenz (EFV), 300 nM raltegravir (RAL), and 20 IU/mL of recombinant human IL-2 (rhIL-2). Cells were sequentially stimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL). The stimulation duration was seven days with an inter-stimulation period of fourteen days. Cells were washed twice weekly. Aliquots of cells from blood and from <i>ex vivo</i> cultures on days -1, 7, 21, and 28 were saved for HIV DNA qPCR and/or single-genome sequencing (SGS). Aliquots of supernatant were removed once weekly for virion-associated HIV RNA qRT-PCR and SGS.</p

Characteristics of study subjects.

<p>Characteristics of study subjects.</p

Inhibitors of Histone Deacetylases: CORRELATION BETWEEN ISOFORM SPECIFICITY AND REACTIVATION OF HIV TYPE 1 (HIV-1) FROM LATENTLY INFECTED CELLS

Deacetylation of histone proteins at the HIV type 1 (HIV-1) long terminal repeat (LTR) by histone deactylases (HDACs) can promote transcriptional repression and virus latency. As such, HDAC inhibitors (HDACI) could be used to deplete reservoirs of persistent, quiescent HIV-1 proviral infection. However, the development of HDACI to purge latent HIV-1 requires knowledge of the HDAC isoforms contributing to viral latency and the development of inhibitors specific to these isoforms. In this study, we identify the HDACs responsible for HIV-1 latency in Jurkat J89GFP cells using a chemical approach that correlates HDACI isoform specificity with their ability to reactivate latent HIV-1 expression. We demonstrate that potent inhibition or knockdown of HDAC1, an HDAC isoform reported to drive HIV-1 into latency, was not sufficient to de-repress the viral LTR. Instead, we found that inhibition of HDAC3 was necessary to activate latent HIV-1. Consistent with this finding, we identified HDAC3 at the HIV-1 LTR by chromatin immunoprecipitation. Interestingly, we show that valproic acid is a weak inhibitor of HDAC3 (IC50 = 5.5 mm) relative to HDAC1 (IC50 = 170 μm). Because the total therapeutic concentration of valproic acid ranges from 275 to 700 μm in adults, these data may explain why this inhibitor has no effect on the decay of latent HIV reservoirs in patients. Taken together, our study suggests an important role for HDAC3 in HIV-1 latency and, importantly, describes a chemical approach that can readily be used to identify the HDAC isoforms that contribute to HIV-1 latency in other cell types