Baseline characteristics, telomere length and telomerase activity by treatment arm.

<p>T/S ratio: TL was expressed as a ratio to a single (S) copy housekeeping gene 36B4 (T/S ratio). All parameters are shown as mean (SD), unless otherwise stated.</p><p>Baseline characteristics, telomere length and telomerase activity by treatment arm.</p

MONET trial- Telomere sub-study patient disposition.

<p>MONET trial- Telomere sub-study patient disposition.</p

Latency is established with higher efficiency following co-culture of resting CD4+ T-cells with mDC.

<p>Resting CD4+ T-cells from 2 donors were labelled with eFluor670 cytoplasmic dye and cultured for 24 hours either untreated (<i>red</i>), with 100 nM CCL19 (<i>purple</i>) or with autologous mDCs at 1 mDC:10 T-cell ratio (<i>blue</i>). Cells were incubated with increasing TCID₅₀ per cell of X4-tropic HIV^NL4.3-EGFP. After 2 hours, cells were washed, cultured for 5 days in a low concentration of IL-2 (2 U/ml) and analysed for EGFP expression by flow cytometry to quantify productive infection (A). Additionally, non-proliferating eFluor670^hiEGFP- cells were sorted, cultured for 3 days with anti-CD3/anti-CD28 plus IL-7, in the presence of the integrase inhibitor L-870812 (L8), to induce EGFP expression from post-integration latent infection (B). As a comparative control, aliquots of sorted cells were also cultured for 3 days with L-870812 (L8) but no reactivation stimuli in order to measure background, spontaneous EGFP expression during 3 further days of culture (C). The true level of post-integrated latency in cultures was calculated by subtracting the percentage of EGFP+ cells in the spontaneous cultures from the percentage of EGFP+ cells in reactivated cultures (D). The percentage of EGFP+ cells per 10⁴ cultured cells is shown. Columns represent the median of donor pairs with each donor shown as a different symbol.</p

High titres of R5-tropic HIV.EGFP reporter virus are needed to infect resting CD4+ T-cells.

<p>Resting CD4+ T-cells from 4 donors were labelled with eFluor670 proliferation dye and cultured without (<i>red</i>) or with autologous mDCs at 1 mDC:10 T-cell ratio (<i>blue</i>) for 24 hours. Cells were incubated with increasing TCID₅₀ per cell of HIV^{NL4.3(AD8)-EGFP} for 2 hours and cultured for 5 days in a low concentration of IL-2 (2 U/ml). 5 days post-infection, cultures were analysed for EGFP expression by flow cytometry to quantify productive infection (A). Non-proliferating eFluor670^hiEGFP- cells were also sorted and cultured for 3 days with anti-CD3/anti-CD28 plus IL-7 to induce EGFP expression from latent infection (B). The percentage of EGFP+ cells per 10⁴ cultured cells is shown. Columns represent the median of donors tested with each donor shown as a different symbol.</p

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

<div><p></p><p>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.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT01365065" target="_blank">NCT01365065</a></p></div

Induction of changes in acetylation of histone 3, histone 4 and lysine by vorinostat.

<p>Changes in histone (H) acetylation were quantified using flow cytometry which is shown (for representative participant) as (A) fold change in mean fluorescence intensity (MFI) of antibody to acetylated (Ac) H3, Ac lysine (K) and Ac H4 in lymphocytes by size prior to, during and following vorinostat and (B) histograms of the change in MFI with antibody to Ac H3 following vorinostat. (C) PBMC were analysed by western blot using an antibody to Ac H3 and actin (as a control for total protein). A positive control (pos) of splenocytes from a mouse with acute myeloid leukemia treated with the HDACi panobinostat is also shown. (D) Fold change in acetylated (A) Histone 3 (red), (B) Lysine (orange) and (C) Histone 4 (blue) in total lymphocytes is shown for each study participant (solid circle) and the median (IQR) fold change above baseline is shown. *p<0.01, **p<0.001. Grey shaded box represents the time on vorinostat.</p

Changes in host genes were associated with an increase in CA-US HIV RNA after vorinostat.

<p>(A) Heatmap showing the fold change in gene expression using linear regression analysis between CA-US HIV RNA and DEG at two hours following the initial dose of vorinostat (n = 9). CA-US HIV RNA is plotted as a continuous variable (ranging from low to high – light green to dark green) and correlated with distinct gene expression profiles. The copy number of CA-US HIV RNA per million cells two hours following vorinostat for each participant is listed next to the patient identification code at the bottom of each column. The top 50 regression features (of a total of ∼2000 at nominal p-value<0.05) are shown. (B) Pathway analysis was performed on the regression features and a checkerboard map showing the top common enriched pathways on the y-axis and leading edge analysis (gene members contributing most to enrichment) plotted along the x-axis. Up- and down-regulated genes at two hours versus baseline are plotted as log₂ fold change (FC); red squares correspond to upregulated gene expression and blue downregulated gene expression. Genes associated with MAPK signal transduction pathways are annotated with black arrows and cell cycle regulators annotated with green arrows. Genes associated with the ER stress response and apoptosis are annotated with red arrows.</p

Changes in gene expression over the duration of study with most early changes occurring in T-cells.

<p>(A) ANOVA (F-test) heatmap of top 50 differentially expressed genes (DEGs) from matched donor supervised analysis (n = 9) comparing gene expression one, 14 and 84 days following the initial dose of vorinostat. Gene Expression was adjusted for baseline expression and represented as a gene-wise standardized expression (Z-score), with p-values<0.05. (B) Checkerboard map of differentially expressed genes 84 days after the initial dose of vorinostat (70 days post cessation of drug) compared to baseline showing the top 10 enriched pathways using gene subset enrichment analysis (GSEA) on the y-axis and leading edge analysis (gene members contributing most to enrichment) plotted along the x-axis. Scale represents log₂ fold change where red corresponds to up- and blue down-regulated genes respectively. (C) Pathway heatmap illustrates enrichment of gene expression in PBMC subsets at different timepoints compared to baseline. Red and blue represent up and down regulated expression of gene subsets respectively. (D) Checkerboard map of DEG at each timepoint compared to baseline. The cell subsets (modules) are plotted on the y-axis and gene members contributing to enrichment plotted on the x-axis. Scale represents log₂ fold change. Red and blue boxes represent up and down gene regulation respectively. mDC = myeloid cells; pDC = plasmacytoid dendritic cells; NK = natural killer cells.</p

Vorinostat induced a transcriptional burst and chromatin perturbations that are recurrent with subsequent dosing.

<p>(A) ANOVA (F-test) heatmap of top 50 differentially expressed genes (DEGs) from matched donor supervised analysis (n = 9) comparing gene expression two hours (2 h), eight hours (8 h) and one day following the initial dose of vorinostat. Gene expression was adjusted for baseline expression and represented as a gene-wise standardized expression (Z-score), with p-values<0.05. DEGs are annotated with colored arrowheads: red, transcription coactivators and adaptors; blue, DNA damage and ER stress response; purple, apoptosis resistance; black, chromatin remodeling factors; orange, mSIN3a histone-deacetylase complex subunits. (B) Checkerboard map of DEG two hours after the initial dose of vorinostat compared to baseline showing the top 10 enriched pathways on the x-axis and leading edge analysis (gene members contributing most to enrichment) plotted along the y-axis. Scale represents log₂ fold change where red corresponds to up- and blue down-regulated genes at two hours versus baseline. Genes associated with viral transcriptional activity are annotated with colored arrowheads: red, BAF component SMARCB1 (SNF5) and CDK9; black, splicesome and nuclear export proteins; orange, mSIN3A HDAC subunits.</p

Baseline characteristics of study participants.

a<p>Values represent n (% with characteristic) or median (interquartile range).</p>b<p>Patient had a single HIV RNA viral load to <200 copies/mL during period of suppression.</p>c<p>Antiretroviral agents separated by “/” were fixed dose combination.</p><p>“+r” denotes administration of ritonavir.</p><p>NOTE: TDF, Tenofovir; 3TC, Lamivudine; FTC, Emtricitabine; EFV, Efavirenz; NVP, Nevirapine; ABC, Abacavir; 3TC, Lamivudine; DRV, Darunavir; RTV, Ritonavir; AZT, Zidovudine; LPV, Lopinavir; RAL, Raltegravir; ATV, Atazanavir; NVP SR Nevirapine (slow release).</p><p>Baseline characteristics of study participants.</p