SB 9200 treatment induces dose-dependent and sometimes long-lasting expression increases of type I IFNs, cytokine, and ISGs in peripheral blood.

<p>Changes in mean blood transcript levels of IFN-α, IFN-β, and IL-6 (<b>top panels</b>) and of CXCL10, OAS1 and ISG15 (<b>bottom panels</b>) in response to SB 9200 treatment at a low (<b>A, C</b>) or high dose (<b>B, D</b>). Changes in mean serum WHV DNA relative to T₀ is plotted on the left y-axis. Error bars represent the standard error of the mean.</p

SB 9200 treatment induces dose-dependent, transient reduction in hepatic WHV nucleic acids.

<p>Changes in mean hepatic levels of WHV cccDNA (<b>left panels</b>), WHV RI DNA (<b>middle panels</b>) and WHV RNA (<b>right panels</b>) relative to week -1 (pretreatment baseline) in response to SB 9200 treatment at a low (<b>A, D, G</b>) or high dose (<b>B, E, H</b>) and mean of each group (<b>C, F, G</b>). At week -1, WHV DNA RI levels were 1830.9 and 1965.7 pg/μg total DNA and WHV RNA levels were 42.9 and 44.3 pg/μg total RNA in the low and high dose groups. Error bars represent the standard error of the mean.</p

SB 9200 treatment results in dose-dependent increases in total plasma exposure of SB 9000.

<p>Changes in mean plasma levels of SB 9000 during daily, oral treatment with SB 9200 for 12 weeks in woodchucks administered a low (<b>A</b>) or high dose (<b>B</b>). Changes in mean serum WHV DNA relative to T₀ is plotted on the left y-axis. Error bars represent the standard error of the mean. Because plasma values represent single time-point values at 2 hours post-dosing, it is likely that the peak individual plasma levels do not represent the actual C<i>max</i> since the T<i>max</i> in individual woodchucks could vary. Peak levels in individual woodchucks were between 25–150 and 75–170 ng/mL in the low and high dose groups, respectively.</p

SB 9200 treatment results in transient reduction of WHcAg expression and inflammation in liver.

<p>Changes in mean immunohistochemistry (IHC) score for cytoplasmic WHcAg expression in liver (<b>top panels</b>) and liver histology score for portal and lobular sinusoidal hepatitis (<b>bottom panels</b>) in response to SB 9200 treatment at a low (<b>A, C</b>) or high dose (<b>B, D</b>). Changes in mean serum WHV DNA relative to T₀ is plotted on the left y-axis. Error bars represent the standard error of the mean.</p

SB 9200 treatment is associated with dose-dependent, transient increases of SDH but not of other liver enzymes.

<p>Changes in mean serum levels of SDH, AST and ALT in response to SB 9200 treatment at a low (<b>A</b>) or high dose (<b>B</b>). Changes in mean serum WHV DNA relative to T₀ is plotted on the left y-axis. Error bars represent the standard error of the mean.</p

Oligonucleotides used for analyses of RIG-I/NOD2 pathway and host innate immune responses in blood and liver.

<p>Oligonucleotides used for analyses of RIG-I/NOD2 pathway and host innate immune responses in blood and liver.</p

SB 9200 treatment induces dose-dependent, transient suppression of serum viremia and antigenemia.

<p>Changes in WHV DNA (<b>left panels</b>) and WHsAg levels (<b>right panels</b>) relative to T₀ (pretreatment baseline) during daily, oral treatment with SB 9200 for 12 weeks in individual woodchucks administered a low (<b>A, D</b>) or high dose (<b>B, E</b>) and mean of each group (<b>C, F</b>). At T₀, mean WHV DNA levels were 4.78x10¹⁰ and 5.27x10¹⁰ genomic equivalents/mL serum and mean WHsAg levels were 3.30x10⁵ and 4.32x10⁵ ng/mL serum in the low or high dose groups, respectively. Error bars represent the standard error of the mean.</p

Intrahepatic expression of genes induced by both low and high dose wIFN-α or induced by neither.

<p>RNA-Seq data for select genes significantly induced by both low dose (20 μg) and high dose (100 μg) wIFN-α (top 9 rows), or induced by neither (bottom 5 rows). The full list of genes induced by both low dose and high dose wIFN-α (n = 775 genes) is displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.s019" target="_blank">S8B Table</a>.</p><p>^aFold-change ratio values relative to time-matched placebo animals (n = 5–12) are displayed, with underlining denoting statistical significance (FDR<0.05). Fold-change ratio was calculated as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.t002" target="_blank">Table 2</a>.</p><p>^bHugo symbols are used for gene names. Alternative gene names; <i>CD274</i>: <i>PD-L1</i>, <i>TNFSF10</i>: <i>TRAIL</i>, <i>KLRC1</i>: <i>NKG2A</i>, <i>FCGR3A</i>: <i>CD16</i>, <i>NCAM1</i>: <i>CD56</i>.</p><p>^cFold-change ratios for <i>FASLG</i> at week 3 and <i>PRF1</i> at week 11 were close to statistical significance (FDR = 0.097 and FDR = 0.062, respectively).</p><p>W: week.</p><p>Intrahepatic expression of genes induced by both low and high dose wIFN-α or induced by neither.</p

Modular analysis of intrahepatic transcriptional signatures in the wIFN-α treatment group.

<p>Data from all available wIFN-treated animals (n = 5–11) were included at each time-point. Spot intensity (red: over-expressed; blue: under-expressed) denotes the percentage of transcripts significantly changed in each module (M) and is defined by the scale bar. The functional interpretation of each module [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.ref029" target="_blank">29</a>] is displayed on the right. Only modules with enrichment greater than 10% at one or more time-point are displayed. At each time-point, all genes selected for modular analysis had an absolute fold-change > 1.5 with a Benjamini-Hochberg corrected <i>FDR</i><0.05 relative to the time-matched placebo group. The horizontal bars together with the week numerators indicate the study stage, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.g001" target="_blank">Fig 1</a>.</p

wIFN-α treatment of chronic WHV carriers induces suppression of serum antigenemia and viremia.

<p>Change in serum (a) WHsAg and (b) WHV DNA relative to week -3 (pre-treatment baseline). Circles indicate the mean of each group (open: placebo, closed: wIFN-α), and the error bars represent the standard error of the mean. The WHsAg level for two wIFN-treated animals was ≤ lower limit of detection (LLOD; 20 ng/mL) at various times during the study (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.s002" target="_blank">S2 Fig</a>); the LLOD was used to estimate the WHsAg decline at these timepoints. Note that seven animals (three in the placebo group, four in the wIFN-α group) died during the study, and one animal in the wIFN-α group (M1004) was excluded from the analysis since it was likely naturally clearing WHV as the study initiated (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.t001" target="_blank">Table 1</a>).</p