AAC Accepted Manuscript Posted Online 20 April 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00574-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

Histone Deacetylase Inhibitor Romidepsin

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Inhibits de novo HIV-1 Infections

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Kasper L. Jønsson1,2§, Martin Tolstrup3,4§, Johan Vad-Nielsen3, Kathrine Kjær1, Anders Laustsen1,2,

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Morten N. Andersen1; Thomas A. Rasmussen3, Ole S. Søgaard3,4, Lars Østergaard3,4, Paul W.

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Denton3,4,5# and Martin R. Jakobsen1,2#.

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1

Institute of Biomedicine, Faculty of Health, Aarhus University, Denmark

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Aarhus Research Centre of Innate Immunology, Denmark

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Department of Infectious Diseases, Aarhus University Hospital Skejby, Denmark

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4

Institute of Clinical Medicine, Aarhus University, Denmark

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Aarhus Institute for Advanced Studies, Aarhus University, Denmark

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§

These authors contributed equally to this work.

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#

Equal contributors and co-corresponding authors:

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Paul W. Denton, PhD Department of Infectious Diseases (Q) Aarhus University Hospital, Skejby Palle Juul-Jensens Boulevard 99 8200 Aarhus N, Denmark [email protected]

Martin R. Jakobsen, PhD Institute of Biomedicine Faculty of Health, Aarhus University Wilhelm Meyers Alle 4 8000 Aarhus C, Denmark [email protected]

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Running title: Romidepsin Inhibits de novo HIV Infections

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Keywords: HIV, romidepsin, viral outgrowth, latency, HDACi

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ABSTRACT

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Adjunct therapy with the histone deacetylase inhibitor (HDACi) romidepsin increases plasma

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viremia in HIV patients on combination antiretroviral therapy (cART). However, a potential

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concern is that reversing HIV latency with an HDACi may reactivate the virus in anatomical

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compartments with sub-optimal cART concentrations leading to de novo infection of susceptible

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cells in these sites. We tested physiologically relevant romidepsin concentrations known to

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reactivate latent HIV in order to definitively address this concern. We found that romidepsin

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significantly inhibited HIV infection in PBMCs and CD4+ T cells, but not in monocyte-derived-

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macrophages. In addition, romidepsin impaired HIV spreading in CD4+ T cell cultures. When we

31

evaluated the impact of romidepsin on quantitative viral outgrowth assays with primary resting

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CD4+ T cells, we found that resting CD4+ T cells exposed to romidepsin exhibited reduced

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proliferation and viability. This significantly lowered assay sensitivity when measuring the efficacy

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of romidepsin as an HIV latency reversal agent. All together our data indicate that romidepsin-

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based HIV eradication strategies are unlikely to reseed a latent T cell reservoir, even under sub-

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optimal cART conditions, because romidepsin profoundly restricts de novo HIV infections.

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INTRODUCTION Combination antiretroviral therapy (cART) greatly reduces HIV disease-related mortality, but

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does not cure HIV infection. During cART, transcriptionally silent HIV persists in resting CD4+ T

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cells as a latent HIV reservoir (1). If cART is interrupted, viral replication is reinitiated from this

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reservoir. The resumption of viral replication typically manifests clinically as rebound in plasma

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viremia accompanied by a rapid decline in CD4+ T cells. Thus, lifelong cART is essential for

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continued virus suppression. The primary goal of HIV eradication therapies is to eliminate the latent

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HIV reservoir such that cART can be interrupted without viral rebound (2). Currently, activating

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HIV from latency such that viral cytopathic effects or the host immune system kills the infected

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cells is under investigation as a curative strategy. Key to the success of such strategies, collectively

47

referred to as “kick and kill” approaches, is the ability to effectively reverse HIV latency in vivo (3).

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The mechanisms by which HIV establishes latency are complex and include enzymatic

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processes that affect the chromatin organization of the HIV-promoter region, one of the key

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determinants of virus transcriptional activity (4-7). Histone deacetylation mediated by histone

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deacetylases (HDAC) leads to structural changes in chromatin that inhibit transcription (2, 6, 8).

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Conversely, histone deacetylase inhibitors (HDACi) turn on gene transcription by promoting

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acetylation of lysine residues on histones leading to chromatin relaxation (9). There are 4 classes of

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HDACs into which the 18 described enzymes have been categorized (10). Relevant to HIV

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eradication strategies, class I HDACs are particularly important for maintaining HIV latency (10,

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11). HDACi, including romidepsin and panobinostat, may serve as latency reactivating agents

58

(LRA) via direct interference with HDAC maintained latency (9, 12). Romidepsin exhibits

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inhibitory activity in the lower nanomolar range against class I HDACs and panobinostat exhibits

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activity against class I/II HDACs (13). Consistent with the close interactions between HDACs and

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the maintenance of HIV latency, HDACi have the ability to reactivate and induce HIV expression

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from latently infected cells in both ex vivo and in vivo studies (9, 14-16).

63 64

Recently, Lucera, et al. reported that supra-physiological concentrations (200 nM) of

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romidepsin increased the susceptibility of CD4+ T cells to HIV infection (17). These data, as well

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as those of others (18), raise the concern that inducing latent HIV in anatomical compartments with

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sub-optimal cART concentrations (19) could lead to the infection of new target cells and reseeding

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of the latent reservoir. To determine the likelihood of such paradoxical outcomes, we

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comprehensively analyzed the impact of HDACi on de novo virus infection employing a broad

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panel of ex vivo experimental systems. We found that romidepsin-based HIV eradication strategies

71

are unlikely to reseed the latent reservoir because this drug profoundly restricted de novo HIV

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infections. Further, resting CD4+ T cells exposed to romidepsin exhibited reduced proliferation and

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viability in the viral outgrowth assay which led to significantly lower assay sensitivity when

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measuring the efficacy of romidepsin as an HIV latency reversal agent.

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METHODS AND MATERIALS

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Source and Isolation of Primary Human Cells

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Healthy blood donor buffy coat fractions were obtained from the Department of Clinical

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Immunology Blood Bank at Aarhus University Hospital, Denmark. Peripheral blood mononuclear

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cells (PBMCs) were isolated using Ficoll density separation. CD4+ T cells were enriched by

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negative depletion from PBMCs using EasySep Human CD4+ T Cell Enrichment Kit (Cat #:

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19052; Stem Cell Technologies) or CD4+ T Cell Isolation Kit (Cat #: 130-096-533, Miltenyi

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Biotec) according to the respective manufacturer’s protocol. Monocyte-derived-macrophages

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(MDMs) were generated from monocytes purified from PBMCs by plastic adherence, with

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subsequent culture for 7 days in “RPMI growth medium” [RPMI 1640; 50,000 I.U. Penicillin;

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50,000 µg Streptomycin; 10% fetal calf serum; 1% glutamine; IL-2 (20U/ml)] supplemented

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with 10 ng/mL M-CSF and 1 ng/mL GM-CSF (PeproTech). Resting CD4+ T cells were enriched

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from cryopreserved PBMCs by negative depletion via a 2-step protocol as previously described

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(20). Briefly, the first step was to enrich CD4+ T cells from PBMCs using Miltenyi CD4+ T Cell

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Isolation Kit (Cat #: 130-096-533) according to the manufacturer’s protocol. The second step was to

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further enrich for resting CD4+ T cells via depletion of cells expressing CD69, CD25 or HLA-DR

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(Miltenyi Cat #: CD69 Microbeads Kit II– 130-092-355; CD25 Microbeads II – 130-092-983;

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HLA-DR Microbeads– 130-046-101). All cell incubations at 37°C unless otherwise noted. The

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purity of enriched cells populations was assessed by flow cytometry using either a LSR Fortessa

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(BD Biosciences) or a FACSVerse (BD Biosciences) flow cytometer and FlowJo software

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(Treestar).

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Viability Assay for TZM-bl Cells, PBMCs and Activated CD4+ T Cells

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The viability of TZM-bl cells (21-25) and primary human cells was assessed following exposure to

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romidepsin (Selleckchem) or panobinostat (Selleckchem) (26-28). We tested a range of drug doses

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that spanned the physiologically relevant doses of each drug [i.e. 40nM romidepsin (14) and 30nM

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panobinostat (15, 29)] and in select experiments, sub-physiological doses of romidepsin were

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included along with media controls to facilitate the observation of dose-dependent effects on HIV

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infection.

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TZM-bl cells in “cDMEM” [DMEM; 50,000 I.U. Penicillin; 50,000 µg Streptomycin; 10% fetal

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calf serum; 1% glutamine] were seeded (5x103 cells/well) in 96-well plates and incubated for 24

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hours- and then primed (8 or 18 hours) with romidepsin or panobinostat at the indicated range of

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concentrations (two-fold dilutions). Following priming, the cells were washed with cDMEM and

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incubated for 48 hours. TZM-bl cells were trypsinized, resuspended in 100uL cDMEM, transferred

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to 96-well ELISA plates and 20uL of MTS substrate (Promega - CellTiter 96® AQueous One

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Solution Cell Proliferation Assay) was added to each well. Plates were incubated for 4 hours and

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formazan production was then analyzed with an ELISA plate reader (Biotek - ELx808). Cell

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viability was calculated according to the Promega CellTiter-protocol.

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PBMCs CD4+ T cells were stimulated in “RPMI growth medium” supplemented with PHA

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(5µg/mL) for 48 hours. PHA containing media was replaced with fresh RPMI growth medium and

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cells were incubated for 24 hours. PBMCs or CD4+ T cells were then seeded (2x105 cells/well) in

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96-well plates and primed (8 or 18 hours) with romidepsin or panobinostat at the indicated range of

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concentrations (two-fold dilutions). Following priming, the cells were washed, resuspended in

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RPMI growth medium and incubated for 48 hours. Cells were resuspended in 100µL RPMI growth

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media and assayed for formazan production as described above.

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122 123

Single Round, VSV-g Pseudotyped HIV Infection Assay

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VSVg-pseudotyped HIV virions were produced by transfecting HEK293T cells with a packaging

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system consisting of 4 plasmids: pCCL-PGK-eGFP, pMD.2G, pRSV-REV, and pMDlg/p-RRE.

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Viral supernatants were harvested after 48 and 72 hours and concentrated through a 20% sucrose

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cushion (25,000 x g for 2 hours). Viral pellets were resuspended in PBS, pooled and aliquots were

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stored at -80oC.

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PBMCs or enriched CD4+ T cells were stimulated in RPMI growth medium supplemented with

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PHA (5ug/mL) for 48 hours. PHA containing media was replaced with fresh RPMI growth medium

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and PBMCs or enriched CD4+ T cells were incubated for 24 hours. MDMs were grown as

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described above. Cells were seeded (2x105 cells/well) in 48-well plates with RPMI growth medium

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and pulsed for 2 hours with romidepsin (10nM; 40nM) or panobinostat (30nM). VSVg-pseudotyped

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HIV virions were then inoculated into the cell cultures along with polybrene (6 µg/ml) followed by

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incubation for an additional 6 hours. Cells were then washed to remove unbound virions and

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resuspended in fresh RPMI growth medium and incubated for 48 hours. Cells were harvested by

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aspiration for PBMCs and T cells or following EDTA treatment for MDMs. Cells were incubated

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on ice with LIVE/DEAD Fixable Violet Dead Cell Stain Kit-405 (Life Technologies) according to

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manufacturer’s instructions. Data were collected using a LSR Fortessa flow cytometer. Viable cells

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exhibiting eGFP expression were characterized as infected.

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HIV Spreading Assay

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Enriched CD4+ T cells were stimulated in RPMI growth medium supplemented with PHA

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(5µg/mL) for 48 hours. PHA containing media was replaced with fresh RPMI growth medium and

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cells were incubated for 24 hours. CD4+ T cells were pulsed for 2 hours with romidepsin (10nM;

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40nM) or panobinostat (30nM). Cells were then infected with HIV-1HXB2 (30). After an additional 4

148

hours of incubation, the HDACi and virus inoculum were removed and cells were resuspended in

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fresh RPMI growth medium. At the indicated time points, culture supernatants were harvested and

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evaluated for p24 antigen by ELISA to determine the replication kinetics of HIV as previously

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described (9).

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Romidepsin and Interferon-Stimulated Genes in PBMCs

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PHA-activated PBMCs were seeded (1x105 cells/well) into a conical 96-well plate and primed for 2

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or 8 hours with romidepsin (10nM; 40nM) or IFNβ (1000 IU/mL). Following priming, RNA was

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collected from the cells using a High Pure RNA Isolation kit (Cat #: 11828665001, Roche)

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according to manufacturer’s instructions. The RNA expression levels of 39 different genes were

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subsequently analyzed using the Fluidigm 48-48 multiplex Gene array Biomark system, including

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TaqMan probes (Fluidigm, USA) according to manufacturer’s instructions.

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Resting CD4+ T Cell Proliferation and Viability Assay

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Resting CD4+ T cells were incubated with eFluor-670 (Cat. #: 65-0840-85; eBioscience) according

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to manufacturer’s instructions. Dyed resting CD4+ T cells (1x106 cells per well) were placed into a

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24-well plate in “cRPMI+IL2 medium” [RPMI with L-glutamine; 1% streptomycin and penicillin;

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10% fetal calf serum; IL-2 (10,000U/ml); conditioned media from a mix lymphocyte reaction

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culture as described in (31)] plus PHA (0.5 µg/ml) and/or romidepsin (4 nM; 40 nM). Following an

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18-hour incubation, the cultures were washed twice (2 hour interval) with cRPMI+IL2 medium and

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then maintained in 2ml cRPMI+IL2 medium for 4 additional days. Cells were incubated on ice with

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LIVE/DEAD Fixable Green Dead Cell Stain Kit-488 (Cat. #: L-23101; Life Technologies)

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according to manufacturer’s instructions. Data were collected using a FACSVerse flow cytometer.

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Viable cells exhibiting a dilution of eFluor-670 were characterized as having proliferated during the

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culture period. Fold proliferation was determined relative to the respective human donor “media

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only” culture. The percentage of viable cells represents the proportion of resting CD4+ T cells

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which excluded the fixable green dead cell stain.

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Activation of Latent HIV in ACH2 Cells

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ACH2 cells (12, 32, 33) were incubated for 18 hours +/- PHA (0.5 µg/ml) or romidepsin (4 nM; 40

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nM), washed twice (2 hour interval) with cRPMI+IL2 medium and then incubated in fresh

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cRPMI+IL2 medium for 48 hours. Viral p24 antigen was measured in culture supernatants by

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ELISA as an indicator of the ability of either romidepsin or PHA to induce virus production from

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this latent HIV cell model (9).

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Modified Viral Outgrowth Assay

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The viral outgrowth assay was performed essentially as described (34). Briefly, resting CD4+ T

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cells were isolated from healthy blood donor buffy coat fractions. On Day 0, 1x106 resting CD4+ T

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cells per well from each donor were plated into a 24-well plate together with the indicated number

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of ACH2 cells in cRPMI+IL2 medium with PHA (0.5 µg/ml) or romidepsin (4 nM; 40 nM). After

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18 hours, the cultures were washed twice (2 hour interval) with cRPMI+IL2 medium, cultures were

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moved into 6-well plates and fed with 5x105 MOLT-4/CCR5 cells in a final volume of 8ml

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cRPMI+IL2 medium. On Day 7, 6ml of the culture volume was replaced with fresh cRPMI+IL2

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medium. On Day 14, 200ul of each culture supernatant was transferred to TZM-bl cells in duplicate

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and luciferase activity from these reporter cells was measured on Day 16 as an indicator of HIV

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production within a given culture (15).

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Statistics

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Comparisons of infectivity within the single round infection assay were performed using t-tests of

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each drug condition versus DMSO controls. Differences in replication (area-under-the-curve; AUC)

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in the HIV spreading assay were evaluated by a one-way repeated measures ANOVA. Comparisons

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between modified viral outgrowth conditions were made using a two-way ANOVA where the

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sources of variation were either the latency reactivation treatment or the number of ACH2 cells per

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well.

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RESULTS

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Limited HDACi-induced Cell Death in Primary Human Cells

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Our research focus was to determine the extent of romidepsin’s impact on de novo HIV

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infection. As a control for the generalizability of our observations with romidepsin to other HDACi,

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we also evaluated the impact of panobinostat on de novo HIV infection in selected assays. To

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design the best strategies for evaluating the impact of HDACi on de novo viral infection, we first

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determined the toxicity of each drug in: (i) HIV reporter TZM-bl cells; (ii) primary human PBMCs;

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and (iii) primary CD4+ T cells. The dose range examined included physiologically relevant doses

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of each drug [i.e. 40nM romidepsin (14) and 30nM panobinostat (15, 29)]. Furthermore, we

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designed these analyses to detect potentially delayed toxic effects of drug exposure by measuring

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viability at both 24 and 48 hours following removal of the drug (Figure 1A-left and middle).

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Romidepsin and panobinostat treatment of TZM-bl cells resulted in only a minor impact on cell

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viability at 24 hours, with the exception of the very high doses of romidepsin. In contrast, cell

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viability at 48 hours was dramatically affected by romidepsin treatment, where cell death exceeded

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90% at 100nM and above (Figure 1A-middle). Cell death was less pronounced for panobinostat

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even at high doses (Figure 1A-left and middle). In these experiments, TZM-bl cells were exposed to

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the HDACi for 8 hours. Given that key ex vivo analyses have incorporated drug exposures with

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these HDACi of 18 hours (34); we also evaluated 18 hours of drug exposure on TZM-bl cells and

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observed a notably greater reduction in cell viability (Figure 1A-right). As little as 7nM romidepsin

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resulted in more than 80% of TZM-bl cells dying within 24 hours. We observed ~50% cell death at

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30nM panobinostat and 80% cell death at 125nM. Next, to explore whether HDACi toxicity also

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occurred in primary human cells, we determined the viability of both PBMCs and CD4+ T cells

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exposed to romidepsin or panobinostat for a total of 8 hours. We found that romidepsin-induced cell

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death in primary cells was less pronounced versus TZM-bl cells. Specifically, approximately 70-

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80% of PBMCs and CD4+ T cells remained viable for 48 hours following exposure to the drug

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concentrations used in our subsequent virus infection assays (Figure 1B and C).

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Romidepsin Inhibits VSV-g Pseudotyped HIV Infection of CD4+ T Cells To understand the impact of HDACi on de novo viral infection, we performed a series of

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primary human cell experiments to measure de novo infection following HDACi treatment. In the

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first line of experiments, IL-2/PHA activated PBMCs were primed with increasing amounts of

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either romidepsin or panobinostat for 2 hours prior to incubation with VSVg-pseudotyped single-

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round HIV virions expressing eGFP (Figure 2A). The live cells in each culture were examined

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using flow cytometry to quantitate the proportion of eGFP positive cells as a measure of infectivity

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(Figure 2B). We found that priming cells with panobinostat did not alter the proportion of infected

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PBMCs (Figure 2B&C); however, romidepsin significantly reduced infectivity of VSVg-

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pseudotyped HIV at a relatively low concentration (10nM, P ≤ 0.01; 95% CI of difference 1.874 to

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8.240) as well as at the more physiologically relevant concentration (40nM, P ≤ 0.001; 95% CI of

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difference 2.845 to 9.211) (Figure 2B&C). Collectively, our experiments with activated PBMCs

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showed a substantial reduction in infectivity following exposure to HDACi although this

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experimental approach did not permit cell lineage specific data interpretation.

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To examine whether the observed inhibition of infectivity by romidepsin specifically affected

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CD4+ T cells and/or macrophages, we sorted CD4+ T cell populations from activated PBMCs prior

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to HDACi priming and generated MDMs. Similar to the results described above, significantly fewer

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primary CD4+ T cells were infected following priming with 10nM or 40nM romidepsin (P ≤ 0.01;

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95% CI of difference 3.017 to 13.32 and P ≤0.0001; 95% CI of difference 5.910 to 16.22;

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respectively) (Figure 2C). In contrast, MDMs were generally permissive for infection by the VSVg-

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pseudotyped single-round HIV virions regardless of HDACi treatment (Supplemental Figure 1).

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Specifically, we observed more than 20% infection in DMSO, romidepsin and panobinostat treated

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MDMs derived from one of the donor whose CD4+ T cells exhibited profoundly reduced infection

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following exposure to romidepsin. Together, these data indicate that de novo infection of activated

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CD4+ T cells by VSVg-pseudotyped HIV was inhibited by romidepsin.

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Romidepsin Inhibits Wild-type HIV Infection of CD4+ T Cells Next we examined whether the mechanism of inhibition was specific to the pH-dependent

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fusion that characterizes VSVg-pseudotyped virions or if wild-type HIV envelope fusion was

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similarly affected by treatment. To do this, HDACi-primed activated CD4+ T cells were challenged

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with replication competent HIV-1HXB2, a highly pathogenic, CXCR4-tropic, laboratory-adapted

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viral isolate (30) (Figure 3A). CD4+ T cell cultures primed with 10nM romidepsin exhibited

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significantly reduced infection after 2 days in culture as measured by Gagp24 in supernatants (P ≤

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0.05) (Figure 3B&C). Similar effects were observed for romidepsin 40nM (P ≤0.05), but not for

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30nM panobinostat. By Day 5, reduced HIV replication was only observed in the cultures primed

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with 40nM romidepsin (P ≤0.001). This pronounced effect was maintained through 7 days of the

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culture, despite the fact that the drug pulse was relatively short in duration (Figure 3B&C). Thus

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exposure of activated CD4+ T cells to physiologically relevant doses of romidepsin significantly

268

decreased viral spreading and thus the effect of romidepsin on de novo HIV infection is not

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dependent on the viral route of entry.

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Romidepsin Selectively Activates Interferon-Stimulated Genes in PBMCs

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To gain further insights into a potential mechanism for the HIV inhibition exhibited by

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romidepsin, we investigated the gene profile in PBMCs challenged with either romidepsin or

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panobinostat. We found that nearly all tested innate immune sensors, as well as other genes

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examined, were affected by 2 or 8 hours of panobinostat exposure (e.g. downregulation of MX1,

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Viperin, and APOBEC3G, NFkB and IRF7) (Figure 4). In contrast, whereas the expression of most

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genes was unchanged following 2 hours of exposure to romidepsin, 8 hours of 40nM romidepsin

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exposure led to major alterations in the gene profile (Figure 4-right). Expression was downregulated

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for most of the examined genes; however, there were three notable exceptions: IFIT1, ISG15 and

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STAT1. Expression of each of these genes was increased following 8 hours of exposure to 40nM

281

romidepsin (Figure 4-right), indicative of differential regulation of innate restriction factors

282

important for viral confinement.

283 284

Romidepsin Inhibits Proliferation of Resting CD4+ T Cells and Reduces the Sensitivity of

285

Viral Outgrowth Assays

286

Given our observations that romidepsin reduced de novo HIV infection and virus spread in

287

culture, we investigated the impact of this drug specifically on resting CD4+ T cells. We chose

288

these cells because they are the primary cell type included in the viral outgrowth assays used to

289

quantitate the size of the latent reservoir in HIV patients (20, 31, 34-39). The impact of romidepsin

290

on the proliferation of magnetically-enriched, HIV-negative blood donor derived resting CD4+ T

291

cells was profound. We first confirmed a previously reported observation (40) that exposure to

292

romidepsin interferes with T cell proliferation when combined with mitogenic stimulation

293

(Supplemental Figure 2). However, it was essential that we also evaluate the impact of romidepsin

294

(18 hour drug exposure) in the absence of mitogenic stimulation to more closely approximate the

295

conditions utilized in viral outgrowth assays (34) (Figure 5A). We found that both proliferation

296

(Figure 5B-C) and cell viability (Figure 5D) of resting CD4+ T cells were dramatically reduced by

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18 hours of exposure to romidepsin.

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298 299

Latent HIV infection in resting CD4+ resting T cells is infrequent (~1 infectious unit per

300

million resting CD4+ T cells tested [IUPM]). Viral outgrowth assays are extended duration (~14

301

days), stimulatory cultures of resting CD4+ T cells that are optimized for the quantitation of the

302

latent HIV reservoir (20, 31, 34-39). To test our hypothesis that romidepsin’s broad impact on cell

303

viability, cell proliferation and de novo HIV infection could alter the sensitivity of viral outgrowth

304

assays and to experimentally overcome the limitation posed by the fact that latency events in cART-

305

suppressed HIV patients are rare, we established a modified viral outgrowth assay with 1x106

306

resting CD4+ T cells per culture from HIV-negative blood donors spiked with a defined number of

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ACH2 cells. ACH2 cells are a model for HIV latency (12, 32, 33), where cells can be stimulated to

308

produce HIV progeny virions by romidepsin exposure [as with latently infected resting CD4+ T

309

cells (14)], but not the mitogenic stimulator phytohemagglutinin (PHA) [unlike latently infected

310

resting CD4+ T cells (Figure 5E)]. Three different treatment conditions were evaluated in this

311

modified viral outgrowth system: media only, 0.5 µg PHA and 40nM romidepsin (Figure 5F).

312

When we tested the typical patient condition of 1 IUPM, we observed viral outgrowth in only 17%

313

(1 of 6) of PHA-treated wells tested and 0% in the media only (0 of 6) or romidepsin wells (0 of 8).

314

When a 10-fold higher IUPM was stimulated, we observed 0% viral outgrowth in the romidepsin

315

wells (0 of 8) whereas 33% of media only wells (2 of 6) and 50% of PHA-stimulated cultures (3 of

316

6) exhibited viral outgrowth. When 100 IUPM was evaluated only 50% of romidepsin cultures (4 of

317

8) versus 83% of media only wells (5 of 6) and 100% percent of PHA wells (6 of 6) were positive

318

for viral outgrowth. Even at 1000 IUPM, not all wells treated with romidepsin (5 of 6) were positive

319

for viral outgrowth as was the case for both the media control (6 of 6) and the PHA (6 of 6) treated

320

wells. We conclude from these data that romidepsin reduced the sensitivity of the viral outgrowth

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321

assay and that romidepsin’s induction effects are obscured within this assay at the typical patient

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condition of 1 IUPM.

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323

DISCUSSION

324

The rationale for incorporation of HDACi into HIV eradication strategies is that epigenetic

325

modulation of molecular mechanisms blocking transcription of integrated HIV DNA can reactivate

326

HIV expression in resting infected memory CD4+ T cells and disrupt latency (29). Numerous in

327

vitro, ex vivo and clinical studies have shown that HDACi reactivate latent HIV infection (9, 12-16,

328

37, 38, 40-48). However, a theoretical concern is that HDACi-induced viral production in cART

329

sanctuary sites could result in de novo HIV infections and reseeding of the latent reservoir (17, 18).

330

In the current study, we addressed this concern by comprehensively exploring the impact of

331

romidepsin on HIV infection. We found that this effective LRA profoundly restricts de novo HIV

332

infections of primary human T cells.

333 334

After demonstrating that the physiologically relevant dosages of the drugs that we used were not

335

overtly cytotoxic (Figure 1), we challenged primary human cells with romidepsin and then infected

336

the cells with either VSV-g pseudotyped HIV or highly pathogenic wild-type HIV, which in both

337

cases were virus models with robust infectivity rates (Figures 2&3). To our surprise, we observed

338

that de novo infection of activated PBMCs and activated CD4+ T cells by VSVg-pseudotyped HIV

339

was inhibited by physiological drug levels of romidepsin. In contrast, when we performed a similar

340

experiment with donor-matched MDM, we observed no appreciable HDACi effect on infection by

341

these VSVg-pseudotyped HIV virions which agrees with recent findings presented by Campbell, et

342

al. (18). When activated CD4+ T cells were treated with romidepsin and then infected with wild-

343

type HIV, we observed a significant reduction in viral spread within the cell culture. Taken

344

together, these two observations reveal that the mechanism of inhibiting de novo HIV was not

345

envelope-dependent. However, further investigations are needed to dissect the differences in

346

infectivity observed between romidepsin-treated CD4+T cells and MDMs.

17

347 348

Next we used Fluidigm gene expression analyses to begin to elucidate whether the mechanism of

349

inhibiting de novo HIV is dependent upon an HDACi-induced alteration in the innate antiviral

350

activities of the target cells. To do this we utilized a multiplex gene array (49) designed to

351

investigate mRNA levels for: (i) molecules involved in innate immune sensing pathways; (ii)

352

interferon stimulated genes (ISG); (iii) innate immunity signal transduction mediators; and (iv)

353

cytokines related to innate immune responses. We noted that after 8 hours of exposure to 40nM

354

romidepsin expression of IFIT1, ISG15 and STAT1 in PBMCs were significantly upregulated

355

(Figure 4-right). This is important, in short, because of known antiviral effects for each of these

356

proteins. Specifically, the members of the family of IFITs, including IFIT1, are known to be

357

upregulated after IFN treatment, viral infections and pathogen-associated molecular pattern

358

recognition (50-52). ISG15 is an ubiquitin-like protein shown to protect cells in numerous viral

359

infection models, but for HIV it is believed mainly to impair viral release (53, 54). STAT1

360

activation can often be correlated with cellular proinflammatory, anti-proliferative, and apoptotic

361

activities (55). While this analysis was focused on a subset of genes involved in innate immunity

362

and did not address other potential mechanisms of virus inhibition, the observation that romidepsin-

363

induced increases in expression for these genes suggests that HDACi inhibition of de novo HIV

364

infections could be mediated through HDACi-altered innate immunity versus direct interference

365

with virus replication.

366 367

In selected assays, we included panobinostat as a control to assess the generalizability of our

368

romidepsin-related findings to other HDACi. Romidepsin and panobinostat target class I and class

369

I/II HDAC, respectively (13). These two HDACi had similar cell viability profiles in activated,

370

primary PBMCs and CD4+ T cell cultures (Figure 1). When we examined the impact of these 2

18

371

HDACi on infection with VSV-g pseudotyped HIV and wild-type HIV, we observed a dose

372

dependent effect with romidepsin where the most profound inhibition of de novo infection was

373

observed at the highest tested dose (40nM). The dose of panobinostat (30nM) evaluated herein was

374

matched to the concentration of panobinostat that was attained in vivo with the dosing strategy

375

employed in a recent clinical trial (9, 15). At this dose (and also at the supra-physiological dose of

376

>100nM; data not shown), panobinostat did not exhibit the same level of inhibition of de novo HIV

377

infection that we observed for romidepsin (Figures 2&3).

378 379

The cells that compose the latent HIV reservoir are relatively infrequent (20, 31, 34-39). Because

380

positive signals detected by viral outgrowth assays are rare, we hypothesized that romidepsin’s

381

broad impacts on cell viability, cell proliferation and de novo HIV infection could alter the

382

sensitivity of this assay. Such an outcome could lead to under-estimation of efficacy when this

383

assay is utilized ex vivo to evaluate the function of potential LRA like romidepsin. Our modified

384

viral outgrowth assay was designed with defined IUPM values to allow us to test this hypothesis.

385

Two relevant comparisons were incorporated into the experiment. The “PHA versus media only”

386

comparison was included to evaluate whether resting CD4+ T cells that were activated by PHA

387

would in turn activate ACH2 cell HIV production while the “media only versus romidepsin”

388

comparison permitted us to discern whether romidepsin reduced the ability of this assay to detect

389

viral outgrowth from latently infected cells or not. Our results showed that ex vivo romidepsin

390

exposure profoundly impacted the ability of the viral outgrowth assay to quantitate the latent HIV

391

reservoir at the typical patient condition of 1 IUPM. Thus, these data indicate that caution should be

392

exercised when determining whether a given molecule is capable of reactivating latent HIV

393

infection from HIV patient-derived resting CD4+ T cells following ex vivo stimulation of with

394

potential LRA. We further demonstrate that the inhibition of HIV spreading in cell culture that

19

395

results from romidepsin treatment creates a technical barrier for the use of virus spreading assays,

396

such as the viral outgrowth assay, for assessing the potential of romidepsin to reverse HIV latency.

397 398

In conclusion, we show that romidepsin inhibits HIV infection in an envelope-independent manner.

399

Furthermore, we provide evidence that this inhibition could result from a romidepsin-mediated

400

enhancement in antiviral innate immunity. Altogether our data indicate that reseeding of the latent

401

reservoir is an unlikely outcome of a romidepsin-based HIV eradication strategy. Translation of our

402

ex vivo findings to the clinic, may best be facilitated through the use of an animal model of HIV

403

persistence where ART sanctuaries may exist (1). The in vivo testing of adjunct therapy with

404

romidepsin as an HIV eradication strategy in such a model is warranted to fully characterize the

405

likelihood of adjunct romidepsin treatment to enhance the risk of HIV infection and reservoir

406

reseeding during a kick and kill HIV eradication therapy.

20

407

ACKNOWLEDGEMENTS

408

This work was supported in part by: The Lundbeck foundation (KJ and MRJ), Aarhus University

409

Research Foundation (MRJ), The Danish Strategic Research Council Grant 0603-00521B (LØ) and

410

Danish Research Council Grant 12-133887 (OSS). The funders had no role in study design, data

411

collection, and analysis, decision to publish, or writing of the manuscript. The following reagents

412

were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl

413

cells (from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.); ACH-2 cells (from Dr.

414

Thomas Folks); MOLT-4/CCR5 cells (from Dr. Masanori Baba, Dr. Hiroshi Miyake, Dr. Yuji

415

Iizawa).

416 417

We thank the following for their technical assistance: Lene Svinth Jøhnke; Ane Kjeldsen; and the

418

Flow Cytometry core facilities at the Aarhus University Institute of Biomedicine.

21

419

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FIGURE LEGENDS

592

Figure 1. Differential impact of HDACi in immortalized versus primary human cells. (A-C)

593

The viability of TZM-bl cells (A) and primary human cells (B&C) was assessed by flow cytometry

594

after the cells were pulsed with either romidepsin or panobinostat for 8 or 18 hours and then

595

incubated for an additional 24 or 48 hours, as indicated in each sub-panel. Panel (A) presents

596

compiled results from (two) experiments performed in triplicate. Panels (B) and (C) present

597

compiled data for PBMCs or CD4+ T cells, triplicate measures respectively from 2 human donors.

598

Error bars: SD. Gray squares: romidepsin. Black circles: panobinostat.

599 600

Figure 2. Single round HIV infection of primary human cells with VSV-g pseudotyped virus

601

was inhibited by romidepsin. (A) Schematic representation of the experimental approach. (B)

602

Representative flow cytometry data highlight the reduced infection that results from romidepsin

603

treatment. Percentages of infected, eGFP positive cells are presented with each dot plot. (C)

604

Compiled data for PBMCs and for CD4+ T cells from 4 human donors are shown (mean +/- SEM).

605

**p