Changes in gene expression in DC-induced latently infected CD4⁺ T cells.

<p>(<b>A</b>) Fold change scatterplot comparing gene expression between HIV T (+DC) (CD4⁺ T cells cultured with DC and HIV), and Mock T (+DC) (CD4⁺ T cells cultured with only DC) relative to their controls, HIV T (CD4⁺ T cells cultured with HIV) and Mock T (CD4⁺ T cells cultured in media alone) respectively. The solid line indicates absolute 2-fold change. (<b>B</b> and <b>C</b>) Top two gene interaction networks as ranked by Ingenuity Pathway Analysis. The networks were built from the list of differentially expressed genes induced by HIV T (+DC), relative to Mock T (+DC) after subtracting HIV T and Mock T from each group respectively. Genes highlighted in red were up-regulated and those in blue were down-regulated. The different node shapes indicate genes in different functional categories according to the legend. The interactions between the different nodes are shown as solid (direct interaction) or dashed (indirect interaction) lines (edges). (<b>D</b>) Fold change in gene expression values for selected genes from the Illumina BeadArrays plotted against Real-Time PCR (qPCR) deltaCt values for each target gene. PCR targets were mapped to BeadArray probes by matching the official gene symbols.</p

Soluble factors in DC-induced HIV latency.

<p>(<b>A</b>) Resting CD4⁺ T cells were cultured alone (light grey) or with sorted pDC (grey) or mDC (dark grey). At day 5 post-infection, cytokines and chemokines were quantified in culture supernatants using cytometric bead arrays, n = 3. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01 (paired t-test). (<b>B</b>) Latent infection was quantified in eFluor670^hiEGFP⁻ resting CD4⁺ T cells that were cultured either alone or with sorted pDC in the presence of media alone (light grey), anti-IgG (grey) or anti-IFN-alpha (dark grey) following stimulation with anti-CD3/CD28 in the presence of L8, n = 5. (<b>C</b>) Resting CD4⁺ T cells were co-cultured with mDC with (grey) or without (light grey) the addition of equal numbers of pDC. Productive infection was determined at day 5 post-infection. Latent infection was determined in sorted eFluor670^hiEGFP⁻ CD4⁺ T cells following stimulation with anti-CD3/CD28 in the presence of L8, n = 5. (<b>D</b>) Latent infection was quantified in eFluor670^hiEGFP⁻ resting memory CD4⁺ T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or neutralising antibodies (dark grey) to IL-6, IL-10-receptor, CXCR3 or CCL19, n = 5. (<b>E</b>) eFluor670-labelled resting CD4⁺ T cells were cultured either alone (light grey) or with blood mDC (dark grey). Virus was added to (<b>i</b>) CD4⁺ T cells cultured alone; (<b>ii</b>) CD4⁺ T cells co-cultured with mDC; (<b>iii</b>) CD4⁺ T cells cultured with mDC in the presence of a 0.4 µm membrane transwell and latency determined at day 5 post-infection, n = 5. (<b>F</b>) eFluor670-labelled resting CD4⁺ T cells were cultured either (<b>i</b>) alone (light grey) or (<b>ii</b>) with blood mDC (dark grey) and infected. (<b>iii</b>) Following 24 hours, supernatant from infected mDC-T cell co-cultures was added to uninfected resting CD4⁺ T cells and these cells were then infected, n = 3. Columns represent the median of 3–5 donors and error bars indicate the interquartile range. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01 (Wilcoxon signed-rank test).</p

Myeloid DC induce post-integration latency in non-proliferating memory CD4⁺ T cells.

<p>SNARF-labelled resting CD4⁺ T cells were cultured alone (light grey) or with syngeneic plasmacytoid (pDC; grey) or myeloid DC (mDC; dark grey). (<b>A</b>) Productive infection (EGFP⁺ cells) was determined by flow cytometry on day 5 post-infection. (<b>B</b>) Latent infection was quantified in SNARF^hiEGFP⁻ cells following either addition of PHA-activated PBMC, n = 5; or (<b>C</b>) direct activation with anti-CD3/CD28 in the presence or absence of the integrase inhibitor L8. (<b>D</b>) Integrated HIV DNA was quantified in the sorted SNARF^hiEGFP⁻ T cells by Alu-LTR real-time PCR, n = 3. (<b>E</b>) Productive and latent infection was determined in SNARF^hiEGFP⁻ CD4⁺ T cells from mDC-T cell co-cultures following infection with nef-deficient (-nef) or nef-competent EGFP HIV. (<b>F</b>) Latent infection was determined in sorted SNARF^hiEGFP^- CD4⁺ T cells, cultured alone or with mDC, following activation with PHA-PBMC. (<b>G</b>) Productive and latent infection was determined in SNARF^hiEGFP⁻ CD4⁺ T cells from mDC-T cell co-cultures with and without Staphylococcus Enterotoxin B (SEB), n = 4. (<b>H</b>) SNARF-labelled resting CD4⁺ T cells were cultured alone (light grey) or with syngeneic mDC (grey) at decreasing DC∶T cell ratios and latent infection quantified in sorted SNARF^hiEGFP⁻ T cells following addition of PHA-activated PBMC, n = 5. (<b>I</b>) Resting CD4⁺ T cells were cultured either alone or in the presence of mDC. At day 5 post-infection, SNARF^hiEGFP⁻ cells were sorted into naïve (light grey) or memory (grey) CD4⁺ T cells and latent infection quantified, n = 5. The lower limit of detection is represented by a dotted line. Columns represent the median of 3–7 donors and error bars indicate the interquartile range. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01; ns, not significant (Wilcoxon signed-rank test).</p

DC-induced latency in resting CD4⁺ T cells.

<p>(<b>A</b>) Resting CD4⁺ T cells were isolated from the blood of healthy donors and labelled with the proliferation dye SNARF, which decreases in intensity following each round of cell division allowing identification of non-proliferating cells. SNARF-labelled resting CD4⁺ T cells were cultured either alone or with syngeneic blood DC. Following 24 hours of culture, cells were infected with NL(AD8)-nef/EGFP at an MOI of 0.5. All culture media was supplemented with IL-2 (2 U/mL). (<b>B</b>) At day 5 post-infection, the number of productively infected (EGFP⁺) cells was determined and the non-proliferating (SNARF^hi) cells that were not productively infected (EGFP⁻) were sorted. The sorted SNARF^hiEGFP⁻ cells were stimulated with PHA/IL-2 in the presence of PBMC and cultured for 5 days to amplify any replication competent latent infection. (<b>C</b>) Productive infection and (<b>D</b>) latent infection following infection of T cells cultured alone (light grey) or in the presence of DC (grey) is shown. (<b>E</b>) Latent infection in the presence of DC cultured with (grey) or without (light grey) 0.1 µM Indinavir. (<b>F</b>) Expression of the early (CD69; black) and late (HLA-DR; grey) surface activation markers and the intracellular proliferation marker Ki67 (light grey) was quantified by flow cytometry on sorted SNARF^hiEGFP⁺ CD4⁺ T cells following HIV infection of T cells cultured alone or in the presence of DC. The lower limit of detection of each assay is represented by a dotted line. Columns represent the median of 5 independent experiments and error bars indicate the interquartile range. *<i>P</i>&lt;0.05 (Wilcoxon signed-rank test).</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

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

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

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

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 virological and immunological parameters from one patient with viral rebound on study.

<p>Changes in (A) CA-US HIV RNA (red), (B) plasma HIV RNA (green) and (C) CA-HIV DNA (blue) are shown as the mean± SD for replicates of CA-US HIV RNA and HIV DNA. Programmed death-1 (PD1) expression on CD4+ and CD8+ CD45RA- T-cells is shown. Grey shaded box represents the time on vorinostat. (D) Dot plot analysis of flow cytometry for co-expression of PD-1 and CD45RA on CD4+ (top panel) and CD8+ (lower panel) T-cells at baseline, after 7 days of vorinostat and at day 84 of follow up.</p