Fiona Wightman

Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. Trial Registration ClinicalTrials.gov NCT01365065

Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4+ T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4+ T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4+ T cells during normal chemokine-directed recirculation of CD4+ T cells between blood and tissue.

Comparison of HDAC inhibitors in clinical development: effect on HIV production in latently infected cells and T-cell activation.

Objective: We aimed to compare the potential for inducing HIV production and the effect on T-cell activation of potent HDAC inhibitors undergoing clinical investigation. Design: In vitro study Methods: The latently infected cell lines ACH2 and U1 were treated with the HDAC inhibitors panobinostat, givinostat, belinostat, vorinostat and valproic acid. Viral induction was estimated by p24 production. Peripheral blood mononuclear cells from uninfected donors were treated with the HDAC inhibitors and the expression of activation markers on T-cell phenotypes was measured using flow cytometry. Finally, the ability of givinostat, belinostat and panobinostat to reactivate latent HIV-1 expression in primary T-cells was investigated employing a CCL19-induced latent primary CD4+ T cell infection model. Results: The various HDAC inhibitors displayed significant potency differences in stimulating HIV-1 expression from the latently infected cell lines with panobinostat > givinostat ≈belinostat > vorinostat > valproic acid. Panobinostat was significantly more potent than all other HDAC inhibitors and induced virus production even in the very low concentration range 8–31 nM. The proportion of primary T-cells expressing the early activation marker CD69 increased moderately in all HDAC inhibitor-treated cells compared with untreated cells. Finally, proof was obtained that panobinostat, givinostat and belinostat induce virus production in latently infected primary cells at therapeutic concentrations with panobinostat being the most potent stimulator. Conclusion: At therapeutic concentrations panobinostat stimulate HIV-1 expression in latently infected cells with greater potency than other HDAC inhibitors undergoing clinical investigation. These findings warrant further investigation and panobinostat is now being advanced into clinical testing against latent HIV infection.

HDAC inhibitors in HIV

Combination antiretroviral therapy (cART) has led to a very substantial reduction in morbidity and mortality in HIV‐infected patients; however, cART alone is unable to cure HIV and therapy is lifelong. Therefore, a new strategy to cure HIV is urgently needed. There is now a concerted effort from scientists, clinicians and funding agencies to identify ways to achieve either a functional cure (long‐term control of HIV in the absence of cART) or a sterilizing cure (elimination of all HIV‐infected cells). Multiple strategies aiming at achieving a cure for HIV are currently being investigated, including both pharmacotherapy and gene therapy. In this review, we will review the rationale as well as in vitro and clinical trial data that support the role of histone deacetylase inhibitors as one approach to cure HIV.

Both CD31+ and CD31- Naive CD4+ T Cells Are Persistent HIV Type 1-Infected Reservoirs in Individuals Receiving Antiretroviral Therapy

BACKGROUND Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31⁻ naive T cells to immune reconstitution and viral persistence is unknown. METHODS In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31⁻ naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31⁻ naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS After ART, both CD31(+) and CD31⁻ naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.

Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells

BackgroundWe recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency.ResultsHigh levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the nef open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat.ConclusionsIn this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with ex vivo findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent in vitro model of HIV latency.

Reduced Hepatitis B Virus (HBV)-Specific CD4+ T-Cell Responses in Human Immunodeficiency Virus Type 1-HBV-Coinfected Individuals Receiving HBV-Active Antiretroviral Therapy

ABSTRACT Functional hepatitis B virus (HBV)-specific T cells are significantly diminished in individuals chronically infected with HBV compared to individuals with self-limiting HBV infection or those on anti-HBV therapy. In individuals infected with human immunodeficiency virus type 1 (HIV-1), coinfection with HBV is associated with an increased risk of worsening liver function following antiviral therapy and of more rapid HBV disease progression. Total HBV-specific T-cell responses in subjects with diverse genetic backgrounds were characterized by using a library of 15-mer peptides overlapping by 11 amino acids and spanning all HBV proteins. The magnitude and breadth of CD4+ and CD8+ T-cell responses to HBV in peripheral blood were examined by flow cytometry to detect gamma interferon production following stimulation with HBV peptide pools. Chronic HBV carriers (n = 34) were studied, including individuals never treated for HBV infection (n = 7), HBV-infected individuals receiving anti-HBV therapy (n = 13), and HIV-1-HBV-coinfected individuals receiving anti-HBV therapy (n = 14). CD4+ and CD8+ HBV-specific T-cell responses were more frequently detected and the CD8+ T-cell responses were of greater magnitude and breadth in subjects on anti-HBV treatment than in untreated chronic HBV carriers. There was a significant inverse correlation between detection of a HBV-specific T-cell response and HBV viral load. HBV-specific CD4+ and CD8+ T-cell responses were significantly (fivefold) reduced compared with HIV-specific responses. Although, the frequency and breadth of HBV-specific CD8+ T-cell responses were comparable in the monoinfected and HIV-1-HBV-coinfected groups, HBV-specific CD4+ T-cell responses were significantly reduced in HIV-1-HBV-coinfected individuals. Therefore, HIV-1 infection has a significant and specific effect on HBV-specific T-cell immunity.

The phenotype of hepatitis B virus–specific T cells differ in the liver and blood in chronic hepatitis B virus infection

Hepatitis B virus (HBV)‐specific T cells play a key role in clearance of the virus and in the pathogenesis of liver disease. Peripheral blood (n = 25) and liver biopsies (n = 19) were collected from individuals with chronic untreated HBV infection. Whole blood, cultured peripheral blood mononuclear cells (PBMCs), and cultured liver‐infiltrating lymphocytes (LILs) were each stimulated with an overlapping peptide library to the whole HBV genome. The expression of T helper 1 (Th1) cytokines [interferon gamma (IFN‐γ), tumor necrosis factor alpha (TNF‐α), and interleukin 2 (IL‐2)] and interleukin 10 (IL‐10) was analyzed by intracellular cytokine staining and flow cytometry. In ex vivo whole blood, more lymphocytes produced Th1 cytokines than IL‐10. When comparing cultured LILs with cultured PBMCs, we found a significantly higher magnitude of CD8+ T cells from the liver producing IL‐10 (P = 0.044), primarily in hepatitis B e antigen positive (HBeAg+) individuals. A positive correlation resulted between the magnitude of HBV‐specific TNF‐α+ CD4+ T cells in the liver and the degree of liver inflammation and fibrosis (P = 0.002 and P = 0.006, respectively). Conclusion: The differences in cytokine production from HBV‐specific T cells in blood and liver may explain the capacity for HBV to persist in the absence of significant hepatic destruction and highlights the balance between cytokine‐mediated viral control and liver damage. (HEPATOLOGY 2007.)

Effect of ipilimumab on the HIV reservoir in an HIV-infected individual with metastatic melanoma.

Long-lived latently infected resting CD4+ T cells are the main reason why current antiretroviral therapy (ART) is unable to cure HIV infection [1]. Recent work has suggested that the expression of immune checkpoint markers, such as programmed death-1 (PD1), may play a role in viral persistence on ART via either suppression of virus transcription and/or reduced HIV-specific T cell activity [2,3], but the role of cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) in HIV persistence on ART is not clear. Ipilimumab (Yervoy, Bristol-Myers Squibb, New York, New York) is a human immunoglobulin G1 antibody to CTLA-4 that inhibits binding of CTLA-4, expressed on activated T cells and regulatory T cells (Tregs), to its ligands CD80 and CD86. The drug is used to treat metastatic melanoma and has been associated with multiple changes in immune function thought to enhance antitumor T cell function [4]. In HIV-infected individuals, CTLA-4 expression on CD4+ T cells correlates with HIV disease progression [5], and loss of HIV-specific CD4+ T cell function can be reversed in vitro by CTLA-4 blockade [5–7]. In a simian immunodeficiency virus (SIV) macaque model, CTLA-4 blockade led to an increase in T-cell activation and viral replication [8]. Here, we describe changes in the HIV reservoir in an HIV-infected patient on ART who received ipilimumab for the treatment of metastatic melanoma. At initiation of ipilimumab treatment in October 2013 for disseminated melanoma, the patient was a 51-year-old man diagnosed with HIV in 1986 and with a CD4+ nadir of 159 cells/μl in 1995. He was on ART since 1996 and plasma HIV RNA was less than 400 copies/ml from 2004 and less than 20 copies/ml from July 2012 (Fig. 1a). He received four doses of ipilimumab 3 mg/kg given at three-weekly intervals. Fig. 1 Clinical details and changes and impact of ipilimumab on virological and immunological parameters Whilst receiving ipilimumab, there was no overall change in plasma HIV RNA as measured by the Roche viral load assay [lower limit of detection (LLOD) = 20 copies/ml; Fig. 1c]. Using a sensitive single-copy HIV RNA assay (SCA) (LLOD = 0.3 copies/ml) [9], there was a cyclical decrease in plasma HIV RNA following each infusion and an overall decline from 60 to 5 copies/ml (Fig. 1c). Given more frequent sampling was performed with the SCA, we believe that longitudinal changes over time were best assessed with this assay. There was an increase in CD4+ T cells after each infusion (overall change from 610 to 900 cells/μl) (Fig. 1b). This increase was predominantly in total memory (Fig. 1d) and effector memory CD4+ T cells (Fig. 1e). Postinfusion increases in CD4+ T-cell activation were seen as measured by human leukocyte antigen-DR and CD38 and CCR5 expression (Fig. 1f). There were transient increases in CD8+ T cells following the second and third infusions, but no overall change in CD8+ T cell activation (Fig. 1g). Cell-associated unspliced HIV RNA in sorted CD4+ T cells was quantified with increases observed following the first and second infusions, with a maximum change from baseline of 19.6-fold (Fig. 1h). The changes in cell-associated unspliced HIV RNA was greater than those recently reported, following the administration of the histone deacetylase inhibitors vorinostat [10,11] or panobinostat [12], or following disulfiram [13]. There was no change in cell-associated HIV DNA (Fig. 1i), but any change in the small proportion of cells with HIV DNA containing inducible proviruses [14] may not have been detectable with the assays used here. Acknowledging the limitations deriving from this being a single case, we speculate the increase in cell-associated unspliced RNA could have been due to mechanisms, including an increase in HIV RNA transcription secondary to blocking the inhibitory effects of CTLA-4 on T cell transcription, similar to that described following ex-vivo anti-PD1 treatment of CD4+ T cells from HIV-infected patients on ART [15]; redistribution or expansion of effector memory CD4+ T cells that may have a higher ratio of cell-associated HIV RNA to HIV DNA [16] (Satish Pillai, San Francisco, UCSF, San Francisco, California, personal communication); or redistribution or expansion of activated T cells including Tregs. The increase in cell-associated unspliced HIV RNA and decline in SCA was intriguing, perhaps mediated by elimination of latently infected CD4+ T cells that were induced to express viral antigens. But the rapidity of the decline in SCA makes this somewhat unlikely. Blockade of CTLA-4 with ipilimumab in an HIV-infected patient on ART had significant effects on the total number and phenotype of CD4+ T cells and induced a profound increase in cell-associated unspliced HIV RNA with onset after the first dose and was associated with subsequent decline in plasma HIV RNA. Further studies are warranted to determine if ipilimumab could play a role in eliminating latently infected cells in HIV-infected patients on ART.

HIV Reservoirs and Strategies for Eradication

Combination antiretroviral therapy (cART) has led to a reduction in morbidity and mortality in HIV-infected patients but therapy is lifelong and there is no cure for HIV. The major barriers to cure include HIV latency, which has been identified in different T-cell subsets, as well as persistence of HIV in anatomical reservoirs. We review recent developments in our understanding of the major reservoirs of HIV in patients on cART as well as how latency is established and maintained in T cells. Finally, we review the scientific rationale of and clinical experience with pharmacotherapeutic strategies aimed at eliminating latently infected cells.