<b>Supplemental Material - EDP-514 in healthy subjects and nucleos(t)ide reverse transcriptase inhibitor-suppressed patients with chronic hepatitis B</b>

Supplementary Material for EDP-514 in healthy subjects and nucleos(t)ide reverse transcriptase inhibitor-suppressed patients with chronic hepatitis B by Jordan J Feld, Eric Lawitz, Tuan Nguyen, Jacob Lalezari, Tarek Hassanein, Paul Martin, Steven-Huy Han, Douglas Dieterich, Jeanne-Marie Giard, Guy De La Rosa, Alaa Ahmad, Ed Luo, Annie L Conery, and Nathalie Adda in Antiviral Therapy.</p

Phylogenetic analysis of HIV sequences expressed ex vivo following the latency reversal.

<p>Resting CD4 T cells isolated from a cART-suppressed HIV-infected patient (high-responding patient from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#ppat-1004071-g007" target="_blank">Fig. 7</a>) were treated continuously with RMD or activation control (anti-CD3/CD28 antibodies). Single-genome sequencing was used to analyze patient HIV proviral DNA and plasma RNA at the initiation of treatment (day 0), together with the ex vivo induced HIV RNA in cell culture supernatants at the end of treatment (day 7). Total of 43 sequences were recovered for proviral DNA. Eighty-eight, 11, and 4 sequences of HIV RNA were collected in culture supernatants following the treatment with activation control, 7.5 nM RMD, and 2.5 nM RMD, respectively. Grey arrows indicate examples of full concordance between the sequence of proviral DNA and viral RNA induced either by RMD or activation control. No HIV RNA sequences were recovered in supernatants from cultures treated with control vehicle or 2 µM SAHA. Identified HIV DNA and RNA sequences were aligned and the phylogenetic tree was constructed using Clustal W and MEAG5.</p

Induction of extracellular viral RNA release from CD4 T cells treated with RMD and VOR.

<p>Memory or resting CD4 T cells isolated from HIV-infected patients on suppressive cART were treated with RMD or VOR, and viral RNA was quantified in cell culture supernatants 6 days after the addition of drugs. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#s2" target="_blank">Results</a> are depicted as fold increase in viral RNA relative to control cultures. Each symbol represents one HIV subject. Solid circles, p<0.05; open circles, p>0.05 compared to vehicle-treated controls from the same donors; solid squares, p value not calculated. Red lines represent the mean fold HIV induction across all analyzed donors. Symbols # and * denote a statistically significant difference (p<0.05) for RMD-mediated HIV induction vs. 0.5 and 1 µM VOR, respectively, across all tested donors. Data for each individual donor and condition including results of the statistical analysis are summarized in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#ppat.1004071.s008" target="_blank">Table S4</a>. (<b>A</b>) Memory CD4 T cells were treated with RMD and VOR for 4 and 24 hours, respectively. (<b>B</b>) Memory CD4 T cells were treated continuously for 6 days. (<b>C</b>) Resting CD4 T cells were treated continuously for 6 days.</p

RMD does not induce global activation of immune cell subsets.

<p>PBMCs isolated from four HIV-infected patients on suppressive cART were treated with a 4-hour pulse of RMD or continuously with vehicle control (DMSO), VOR, or PMA+ionomycin and stained for surface markers 48 hours after the treatment initiation. Fractions of CD69−, CD25−, and HLA-DR-positive cells in subsets of CD4+ T cells, CD8+ T cells, and CD19+ B cells were analyzed by flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#s4" target="_blank">Materials and Methods</a>. Each symbol represents one donor.</p

Ex vivo response to RMD in multiple longitudinal samples from the same donors.

<p>Resting CD4 T cells isolated from 2 cART-suppressed HIV-infected patients were treated continuously with RMD or with anti-CD3/CD28 antibodies (AC, activation control) for 7 days. HIV RNA in cell culture supernatants was quantified by COBAS on day 7. Data for a high-responding (<b>A</b>) and a low-responding donor (<b>B</b>) to RMD is shown; each donor was tested at 3 different time points separated by at least 2 weeks (Exp 1–3). VC/NDC, vehicle control/no-drug control. Asterisk (*) indicates no value due to COBAS analysis failure. Dashed lines indicate the limit of HIV quantification by COBAS (20 copies/ml). Data for each individual donor and condition including results of the statistical analysis are summarized in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#ppat.1004071.s008" target="_blank">Table S4</a>.</p

Ex vivo activation of HIV expression by RMD and VOR.

<p>CD4 T cells were isolated from virally suppressed HIV-infected patients and pulse-treated with RMD and VOR for 6 and 24 hours, respectively. Cell-associated total RNA was extracted, HIV RNA levels were quantified at the indicated time points, and fold increase in the cell-associated HIV RNA was determined relative to corresponding vehicle-treated control for each individual time point. The fold change for each donor and condition is based on mean number of HIV copies from 4 to 5 independent measurements. Red dashed line represents the mean fold HIV induction across all tested donors. Symbols # (p<0.01) and * (p<0.05) denote a statistically significant difference between fold HIV induction by RMD and VOR across all donors tested. Data for each individual donor and condition including results of statistical analysis are provided in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#ppat.1004071.s008" target="_blank">Table S4</a>. (<b>A</b>) Memory CD4 T cells were purified as the CD4(+)CD45RA(−) subset. (<b>B</b>) Resting CD4+ T cells were purified as the CD4(+)HLA-DR(−)CD69(−)CD25(−) subset.</p

RMD activates intracellular HIV expression at concentrations below the levels achieved by clinical dosing.

<p>Resting CD4 T cells were isolated from 3 cART-suppressed HIV-infected patients and pulse-treated with RMD for 4 hours with the indicated concentrations. Cell-associated HIV RNA levels were analyzed at each time point following the treatment initiation (t = 0 hour), and fold induction was determined relative to a background signal in vehicle-treated controls. Predicted i.v. dose and percentage of clinical exposure were calculated for each RMD concentration tested relative to the clinically approved dose of 14 mg/m²; calculations were performed based on the free fraction of drug in human plasma and cell culture media. Data represent mean ± SD from at least 3 HIV-infected donors. Data for each individual donor and condition including results of the statistical analysis are summarized in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004071#ppat.1004071.s008" target="_blank">Table S4</a>.</p

Activity and cytotoxicity of HDAC inhibitors in the in vitro HIV latency model.

<p>Latently infected primary CD4 T cells prepared from three independent healthy donors were treated with the indicated compounds continuously for 48 hours. EC₅₀ and CC₅₀ values were determined from each dose-dependent response using a multi-parameter algorithm. Data are mean ± SD from three independent experiments performed in quadruplicate.</p