Effect of Histone Deacetylase Inhibitors on HIV Production in Latently Infected, Resting CD4+ T Cells From Infected Individuals Receiving Effective Antiretroviral Therapy (original) (raw)

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

Correspondence: Tae-Wook Chun, PhD, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bldg 10, Rm 6A32, 9000 Rockville Pike, Bethesda, Maryland 20892 (twchun@nih.gov).

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Bethesda, Maryland

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Department of Chemistry

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Stanford University

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Stanford, California

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Received:

05 December 2011

Accepted:

27 February 2012

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Jana Blazkova, Tae-Wook Chun, Bietel W. Belay, Danielle Murray, J. Shawn Justement, Emily K. Funk, Amy Nelson, Claire W. Hallahan, Susan Moir, Paul A. Wender, Anthony S. Fauci, Effect of Histone Deacetylase Inhibitors on HIV Production in Latently Infected, Resting CD4+ T Cells From Infected Individuals Receiving Effective Antiretroviral Therapy, The Journal of Infectious Diseases, Volume 206, Issue 5, 1 September 2012, Pages 765–769, https://doi.org/10.1093/infdis/jis412
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Abstract

Persistence of the latent viral reservoir has been recognized as a major obstacle to eradicating human immunodeficiency virus (HIV) in infected individuals receiving antiretroviral therapy. It has been suggested that histone deacetylase inhibitors (HDACis) may purge HIV in the latent viral reservoir. However, the effect of HDACis on the degree and extent of HIV expression in the latent viral reservoir has not been fully delineated. Here we demonstrate that HDACis do not induce HIV production in the latent viral reservoir of aviremic individuals. Therefore, alternative therapeutic strategies may be necessary to eliminate HIV in the latent viral reservoir.

The majority of individuals infected with human immunodeficiency virus (HIV) receiving antiretroviral therapy (ART) suppress HIV plasma viremia for significant periods of time [1]. Nonetheless, eradication of HIV by ART alone has not been achieved, likely in part due to the persistence of infected cells in peripheral blood and tissues [2]. In particular, it has been demonstrated that a pool of latently infected CD4+ T cells persists in virtually all infected individuals receiving ART for prolonged periods of time, and it has been suggested that the existence of this pool of cells is one of the major obstacles to achieving eradication of virus [2]. In order to eliminate the latent viral reservoir, a therapeutic strategy aimed at deliberately inducing HIV expression in infected cells has been proposed [2]. Such a strategy hypothesizes that “purging” agents could stimulate HIV expression in the latent viral reservoir and lead to elimination of infected cells via virus-induced cytopathic effects while preventing spread of infection by ART [2]. Indeed, nearly a decade ago, it was demonstrated that intermittent administration of interleukin 2 (IL-2) could reduce the infectious HIV burden in patients receiving ART [3]. However, administration of IL-2 [3] or anti-CD3 antibody, a potent T-cell stimulator [4], did not lead to eradication of HIV in infected individuals receiving ART; the treatment failed to prevent plasma viral rebound upon cessation of antiretroviral drugs [5]. Subsequently, a number of studies have addressed the molecular mechanisms of HIV persistence and have suggested that repression of chromatin structure may play a role in inhibition of viral transcription [2]. More recently, it has been shown in vitro that inhibitors of histone deacetylases (HDACis) promote acetylation and remodeling of the chromatin structure, which in turn allow expression of HIV RNA to occur [6]. Among HDACis, valproic acid (VPA), a drug clinically used to treat epilepsy and bipolar disorder, has been tested in humans as a potential virus “purging” agent and was shown in one study to diminish the size of the latent viral reservoir in infected individuals receiving ART [7]. However, subsequent studies have demonstrated no appreciable reduction of the latent viral reservoir following treatment with VPA [6, 8, 9]. Suberoylanilide hydroxamic acid (SAHA), another HDACi, has been shown to induce HIV expression in several in vitro and ex vivo systems [6, 10]. Given these conflicting results and considering the fact that a major thrust of HIV therapeutic research at the present time is the development of strategies for eradicating virus, it is of considerable interest and importance to carefully evaluate the capacity of HDACis to induce HIV expression in the latent viral reservoir of infected individuals receiving ART. We conducted the present study to address this issue.

METHODS

Clinical Samples

Twenty-seven HIV-infected individuals receiving ART for a median of 2 years were included in this study (Table 1). All individuals received various antiretroviral regimens and maintained undetectable levels of plasma viremia (<50 copies/mL) at the time of study (Table 1). Leukapheresis products were obtained from the study participants in accordance with protocols approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases.

Table 1.

Profile of 27 HIV-Infected Study Participants Receiving Effective Antiretroviral Therapy (ART)

aNucleoside reverse transcriptase inhibitors: Abacavir (ABC), Emtricitabine (FTC), Lamivudine (3TC), Tenofovir (TDF); nonnucleoside reverse transcriptase inhibitors: Efavirenz (EFV); protease inhibitors: Atazanavir (ATV), Atazanavir/Ritonavir (ATV/r), Darunavir (DRV), Darunavir/Ritonavir (DRV/r), Fosamprenavir/Ritonavir (FPV/r); integrase inhibitor: Raltegravir (RAL); CCR5 inhibitor: Maraviroc (MVC).

Table 1.

Profile of 27 HIV-Infected Study Participants Receiving Effective Antiretroviral Therapy (ART)

aNucleoside reverse transcriptase inhibitors: Abacavir (ABC), Emtricitabine (FTC), Lamivudine (3TC), Tenofovir (TDF); nonnucleoside reverse transcriptase inhibitors: Efavirenz (EFV); protease inhibitors: Atazanavir (ATV), Atazanavir/Ritonavir (ATV/r), Darunavir (DRV), Darunavir/Ritonavir (DRV/r), Fosamprenavir/Ritonavir (FPV/r); integrase inhibitor: Raltegravir (RAL); CCR5 inhibitor: Maraviroc (MVC).

Preparation and Cultivation of Resting CD4+ T Cells

Peripheral blood mononuclear cells (PBMCs) were obtained by leukapheresis and ficoll-hypaque centrifugation. CD4+ T cells were isolated using a cell separation system (StemCell Technologies). Subsequently, resting CD4+ T cells were isolated by depleting CD25+, HLA-DR+, and CD69+ CD4+ T cells using PE-conjugated antibodies (BD Biosciences) and anti-PE microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640-based medium containing antiretroviral drugs consisting of T-20 (100 µg/mL), tenofovir (1 µM), and emtricitabine (1 µM) in the absence or presence of the following compounds: VPA (40 µM), SAHA (0.5 µM), oxamflatin (0.5 µM), prostratin (50 nM), and plate coated anti-CD3 and soluble anti-CD28 antibody.

Quantitation of Cell-Free HIV

The copy number of virion-associated HIV RNA in the cell culture supernatants was determined using the Cobas Ampliprep/Cobas Taqman HIV-1 Test, version 2.0 (Roche Diagnostics) following 48 hours of incubation of cells with the above compounds. The limit of detection for this system is 20 copies/mL.

Quantitation of HIV Proviral DNA

To determine the frequency of resting CD4+ T cells carrying HIV DNA, real-time polymerase chain reaction (PCR) was performed on genomic DNA isolated from resting CD4+ T cells (Qiagen), as described elsewhere [11].

Flow Cytometric Analysis

In order to assess the level of cellular activation, resting CD4+ T cells in each culture condition were stained with anti-CD25, CD69, HLA-DR, and Ki67 antibody (BD Biosciences) and analyzed using a FACS Canto (BD Biosciences) and FlowJo software.

Heteroduplex Mobility Assay

To examine the degree of heterogeneity of virion-associated HIV RNA, heteroduplex mobility assay (HMA) was performed as described elsewhere [12] using cell-free culture supernatants. Briefly, HIV env (C2-V5) was amplified by nested RT-PCR using primers specific for HIV-1 envelope (ED5/ED12 and DR7/DR8). PCR products were then denaturated at 94°C for 2 minutes, reannealed by cooling on ice in annealing buffer, subjected to 5% polyacrylamide gel, and visualized by ethidium bromide.

Statistical Analysis

The Wilcoxon signed rank test was used to compare paired data. Correlations were determined by the Spearman rank method. The Bonferroni method was used to adjust P values for multiple testing.

RESULTS

To determine the degree and extent to which HDACis induce HIV expression in the latent viral reservoir, we isolated resting CD4+ T cells from 27 aviremic HIV-infected individuals receiving ART (Table 1) and incubated the cells with various HDACis and T-cell mitogens in the presence of antiretroviral drugs. We used 3 different HDACis, 2 of which are clinically approved for use in humans (VPA and SAHA), and oxamflatin, a hydroxamic acid selective for class I HDACs [13]. In addition, a protein kinase C agonist, prostratin [14], or anti-CD3 antibody were used as positive controls. Cell culture media was used as a negative control.

First, we investigated whether HDACis increase the level of cellular activation in resting CD4+ T cells. As shown in Figure 1A, incubation of resting CD4+ T cells with each of 3 HDACis did not result in upregulation of the cellular activation markers CD25, CD69, HLA-DR, and Ki-67 compared with cells cultured in media alone. In contrast, stimulation with prostratin and anti-CD3 dramatically increased the expression of these activation markers.

Figure 1.

Effect of histone deacetylase inhibitors (HDACis; valproic acid [VPA], suberoylanilide hydroxamic acid [SAHA], or oxamflatin) and T-cell mitogens (prostratin or anti-CD3 antibody) on cellular activation and viral production in resting CD4+ T cells from aviremic individuals infected with human immunodeficiency virus (HIV). A, Level of cellular activation and proliferation of resting CD4+ T cells following treatment with study agents. B, Quantitation of cell-free HIV RNA in the culture supernatants of resting CD4+ T cells incubated with the corresponding agents. The median value and interquartile range is shown as dark gray bars and light gray lines, respectively. Undetectable levels of HIV RNA were scored as 19 copies/mL. The Wilcoxon signed rank test was used to compare paired data. C, Relationship between HIV DNA burden and virion production in the latent viral reservoir of aviremic individuals. The correlation between the frequency of cells carrying HIV DNA and the level of virion production by resting CD4+ T cells following treatment with study agents is shown. Correlations were determined by the Spearman rank method. D, Examination of viral quasi-species by heteroduplex mobility assay (HMA). Virion-associated HIV RNA from resting CD4+ T-cell cultures was subjected to amplification of HIV env by polymerase chain reaction and HMA.

Next, we measured the copy number of virion-associated HIV RNA in the culture supernatants of cells following incubation with media alone, HDACis, or T-cell activators (Figure 1B). In the majority of HIV-aviremic individuals receiving ART, HIV RNA were not detected when cells were incubated with media alone (median, 19 copies per mL; interquartile range, 19–27), VPA (median, 19; interquartile range, 19–37), SAHA (median, 19; interquartile range, 19–33), or oxamflatin (median, 25; interquartile range, 19–81). However, substantially higher levels of HIV RNA were detected when resting CD4+ T cells were incubated with prostratin (median, 2491; interquartile range, 889–10 356) or anti-CD3 antibody (median, 1966; interquartile range, 69–7121). The level of HIV RNA in the culture supernatant when cells were incubated with media alone was not significantly different when compared to that of cells incubated with VPA, SAHA, or oxamflatin (P = .38, .5, and .23, respectively). In contrast, prostratin and anti-CD3 antibody induced substantially higher levels of virions relative to that of cells incubated with media alone (P < .001 and .001, respectively). Of note, longer periods of incubation of resting CD4+ T cells with the HDACis did not increase the level of virion production relative to the copy numbers detected in the culture with media alone (data not shown).

We then determined the frequency of resting CD4+ T cells carrying HIV DNA in the study subjects prior to culture (median, 970 copies per 106 resting CD4+ T cells; range, 3–4285) (Figure 1C). There was a correlation between the level of HIV DNA in resting CD4+ T cells and the copy number of virion-associated HIV RNA in the cultures stimulated with either prostratin or anti-CD3 antibody (P = .02 and .02, respectively). However, no correlation was found between levels of HIV DNA in resting CD4+ T cells and the copy number of virion-associated HIV RNA in the cultures containing media alone or in those containing HDACis (Figure 1C).

Finally, we investigated the degree of heterogeneity of virion-associated HIV RNA in the culture supernatants upon incubation of resting CD4+ T cells with HDACis versus T-cell activating reagents. We isolated virion-associated HIV RNA from the HDACi-containing cultures (>100 copies) followed by amplification of C2-V5 region of the HIV envelope gene. The PCR products were then denatured and reannealed, resulting in the formation of homo- and/or heteroduplexes based on the degree of sequence heterogeneity. As shown in Figure 1D, the HIV env obtained after incubating the cells with prostratin and anti-CD3 antibody showed high levels of heterogeneity, likely a result of maximal stimulation of HIV expression in cells carrying inducible virus. In contrast, the HIV env originating from the resting CD4+ T-cell culture stimulated with HDACis showed relatively low degrees of heterogeneity, suggesting that HDACis may only stimulate HIV expression in a small fraction of latently infected, resting CD4+ T cells.

DISCUSSION

It has been firmly established that the persistence of latent viral reservoir is one of the major obstacles to eradicating HIV in infected individuals receiving ART [2]. To accelerate the decay of the latent viral reservoir, a strategy aimed at deliberately stimulating cells harboring replication-competent virus in the presence of antiretroviral drugs has been considered as a promising approach [2]. Despite the earlier observations that T-cell–activating agents, such as IL-2 and anti-CD3 antibody, did not succeed in completely clearing HIV in patients receiving ART [3, 4], a sustained effort has been directed in recent years toward testing newer agents that can induce HIV replication from the latent viral reservoir [2]. In particular, it has been suggested that VPA may lower the frequency of resting CD4+ T cells carrying infectious HIV in aviremic individuals by purging virus while avoiding toxicities associated with a polyclonal activation approach [7]. However, follow-up studies were inconsistent with the earlier finding and demonstrated no measurable efficacy when VPA was given to aviremic individuals [6, 8, 9]. Subsequently, SAHA was proposed as a virus purging agent on the basis of results obtained using cell lines and cells from infected individuals using a combination of HDACis and T-cell–stimulating agents [10, 15]. However, there have been only limited data directly addressing the degree and extent of HIV expression in HDACi-treated, highly purified resting CD4+ T cells from a large number of aviremic individuals. In the present study, we demonstrated that the overall levels of virion production by HDACi-stimulated resting CD4+ T cells are not statistically different from those of cells incubated with media alone. We chose to quantify the copy number of virion-associated HIV RNA instead of cell-associated HIV RNA because intracellular expression of HIV transcripts may not necessarily equate to production of viral particles; furthermore, virus-induced cytopathic effects would only occur following robust production of virions in latently infected cells. In addition, there was a lack of correlation between the frequency of cells carrying HIV DNA and the level of HDACi-induced virion production by the latent viral reservoir. These data suggest that HDACis would likely be ineffective at eliminating the latent viral reservoir in infected individuals receiving ART. Finally, we demonstrated that the quasi-species of HDACi-induced virions carried relatively homogeneous HIV env compared with that of virions collected from maximally stimulated cells. This finding indicates that HDACis, when they do induce virus production, may do so from only a restricted fraction of infected cells. It is also important to point out that ongoing HIV replication may occur in various tissue compartments in infected individuals receiving effective ART (<50 copies/mL) [11]. Under such circumstances, the administration of HDACis may not be able to eliminate persistently infected CD4+ T cells in aviremic individuals as these cells are already producing HIV.

The quest for eradication of HIV in infected individuals receiving ART remains an ongoing challenge for the AIDS research community. Despite extraordinary effort and interest in using HDACis to eliminate the latent viral reservoir in HIV-infected individuals, it is likely that alternative therapeutic strategies that incorporate HIV-specific targeting and/or more targeted immune activation will be necessary to clear cellular reservoirs of virus.

Notes

Acknowledgments. We thank the study volunteers for their participation in this study.

Financial support. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Author contributions. J. B. and T.-W. C. designed research; J. B., B. D., D. M., J. S. J., and T.-W. C. performed research; J. B., C. W. H., S. M., T.-W. C., and A. S. F. analyzed data; P. A. W. provided a reagent; E. K. F. and A. N. recruited and monitored study subjects; and J. B., T.-W. C., and A. S. F. wrote the article.

Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

1

et al.

The survival benefits of AIDS treatment in the United States

,

J Infect Dis

,

2006

, vol.

194

(pg.

11

-

9

)

2

The challenge of finding a cure for HIV infection

,

Science

,

2009

, vol.

323

(pg.

1304

-

7

)

3

et al.

Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1–infected patients receiving highly active antiretroviral therapy

,

Nat Med

,

1999

, vol.

5

(pg.

651

-

5

)

4

et al.

Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy

,

J Infect Dis

,

2002

, vol.

186

(pg.

1403

-

11

)

5

Reemergence of HIV after stopping therapy

,

Nature

,

1999

, vol.

401

(pg.

874

-

5

)

6

Histone deacetylase inhibitors and HIV latency

,

Curr Opin HIV AIDS

,

2011

, vol.

6

(pg.

25

-

9

)

7

et al.

Depletion of latent HIV-1 infection in vivo: a proof-of-concept study

,

Lancet

,

2005

, vol.

366

(pg.

549

-

55

)

8

et al.

Stability of the latent reservoir for HIV-1 in patients receiving valproic acid

,

J Infect Dis

,

2007

, vol.

195

(pg.

833

-

6

)

9

et al.

Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir

,

AIDS

,

2008

, vol.

22

(pg.

1125

-

9

)

10

Expression of latent HIV induced by the potent HDAC inhibitor suberoylanilide hydroxamic acid

,

AIDS Res Hum Retroviruses

,

2009

, vol.

25

(pg.

207

-

12

)

11

et al.

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir

,

J Clin Invest

,

2005

, vol.

115

(pg.

3250

-

5

)

12

et al.

Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes

,

Science

,

1993

, vol.

262

(pg.

1257

-

61

)

13

Inhibitors of histone deacetylases: correlation between isoform specificity and reactivation of HIV type 1 (HIV-1) from latently infected cells

,

J Biol Chem

,

2011

, vol.

286

(pg.

22211

-

8

)

14

Practical synthesis of prostratin, DPP, and their analogs, adjuvant leads against latent HIV

,

Science

,

2008

, vol.

320

(pg.

649

-

52

)

15

et al.

Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection

,

PLoS One

,

2009

, vol.

4

pg.

e6093

Author notes

a

J. B. and T.-W. C. contributed equally.

Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2012

Topic:

• hiv
• t-lymphocytes
• anti-retroviral agents
• histone deacetylase inhibitors

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