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

Serum WHsAg and liver enzymes for individual wIFN-treated animals.

<p>Serum WHsAg (black open circles) is plotted on the left y-axis. Serum ALT (red circles), AST (blue circles) and SDH (green circles) are all plotted on the right y-axis. The treatment response group classifications (a-d) are described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.t001" target="_blank">Table 1</a>. *Animals died prior to end-of-study. Note all data was from pre-dose (or equivalent).</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

Comparable induction of intrahepatic expression of most ISGs with low dose and high dose wIFN-α.

<p>(a) Unsupervised hierarchical clustering of differentially expressed intrahepatic ISGs of animals that were responders (R, n = 3), partial responders (PR, n = 2) or non-responders (NR, n = 2) to wIFN-α treatment. Note that there was no week 19 sample for the responder group animal M1002 and the week 25 sample (end-of-study for this responder animal) was included at week 23 (end-of-study for most animals) for ease of data comparison. The sources of the ISGs are described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.s014" target="_blank">S3 Table</a>. Heatmap columns represent samples from individual animals collected at the indicated times, and rows represent different genes (n = 209). Red and blue coloring of cells represents high and low expression levels (normalized count data), respectively, as indicated by the scale bar for log₂ normalized values. (b) qRT-PCR data expressed as fold-change relative to week -3 (pre-treatment baseline). The bar height indicates the mean of each group, and the errors bars represent the standard error of the mean. Placebo: maximum induction at 6 hours post-dose at weeks 0 and 7. 20 μg: sample collected 6 hours post-first dose of 20 μg wIFN-α (week 0). 100 μg: sample collected 6 hours post-first dose of 100 μg wIFN-α (week 7). Animals from the wIFN-α treatment group were only included if both 20 μg wIFN-α (week 0) and 100 μg wIFN-α (week 7) data was available (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.s015" target="_blank">S4 Table</a>). Statistical significance was calculated with log-transformed values by one-way ANOVA with Tukey's multiple comparison correction.</p

Serum and liver WHV measurements in the wIFN-α and placebo groups.

<p>N/A: not applicable.</p><p>^aTreatment response groups were defined as follows: R, responder ≥1 log₁₀ reduction in WHsAg at week 15 (end-of-treatment) and week 23 (end-of study); PR, partial responder ≥1 log₁₀ reduction in WHsAg at week 15 but not week 23; NR, non-responder <1 log₁₀ reduction in WHsAg at week 15 and week 23.</p><p>^bSerum WHV DNA and WHsAg levels at week -3 (pre-treatment baseline).</p><p>^cThe serum and intrahepatic viral endpoint differentials at weeks 0–23/25 (end-of-study) were calculated relative to the week -3 (pre-treatment) timepoint.</p><p>^dWHsAg levels for animals M1002, M1004 and F1022 were ≤ lower limit of detection (LLOD; 20 ng/mL) at one or more timepoints; the LLOD was used to calculate the maximum WHsAg decline for these animals.</p><p>^eM1004 had substantially lower week-3 baseline WHsAg and WHV DNA relative to the other study animals; WHsAg levels in this animal dropped by 1.5 log₁₀ between week -3 and the start of treatment (week 0), and then fell to ≤ LLOD after only 2 weeks of low dose wIFN-α. This animal subsequently developed very high titer anti-WHs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.s012" target="_blank">S1 Table</a>).</p><p>†Animal died during the study: M1006 (wIFN-α group); euthanized week 3 due to deteriorating health conditions which were likely related to underlying metabolism abnormalities, M1007 (wIFN-α group); died week 11, likely due to biopsy-related hemorrhage, F1020 (wIFN-α group); died week 16, cause of death unknown, F1023 (wIFN-α group); died week 2, likely related to severe pneumonia, M1010 (placebo group); euthanized week 17 due to symptoms associated with terminal HCC, F1017: (placebo group); euthanized week 17 due to symptoms associated with terminal HCC, F1024 (placebo group); died week 11, likely due to biopsy-related hemorrhage.</p><p>Serum and liver WHV measurements in the wIFN-α and placebo groups.</p

Differential induction of whole blood gene expression by low dose and high dose wIFN-α.

<p>qRT-PCR data for (a) ISGs and (b) T_H1-type cytokine genes expressed as fold-change relative to week 0 pre-dose (pre-treatment baseline). The bar height indicates the mean maximal fold-change for each group, and the errors bars represent the standard error of the mean. Placebo: maximal induction at 6 hours post-dose at weeks 0, 1, 3, 7, 11 and 15. 20 μg: maximal induction at 6 hours post-dose at weeks 0, 1 and 3. 100 μg: maximal induction at 6 hours post-dose at weeks 7, 11 and 15. Animals from the wIFN-α treatment group were only included if data from all relevant time-points was available. Statistical significance was calculated with log-transformed values by one-way ANOVA with Tukey's multiple comparison correction.</p

High dose wIFN-α significantly induced intrahepatic expression of T cell, NK cell and IFN-γ response genes.

<p>RNA-Seq data for genes selectively induced by high dose (100 μg) but not by low dose (20 μg) wIFN-α. The full list of genes induced by high dose wIFN-α only (n = 468 genes) is displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.s019" target="_blank">S8C 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). For genes where wIFN-treated > placebo, the fold change ratio was calculated by 1 x (treated/placebo), i.e. 1.50 equals a 50% increase in treated vs. placebo. For genes where wIFN-treated < placebo, the fold change ratio was calculated by -1 x (placebo/ treated), i.e. -1.50 equals a 50% decrease in treated vs. placebo.</p><p>^bHugo symbols are used for gene names. Alternative gene names; <i>CXCL9</i>: <i>MIG</i>, <i>PDCD1</i>: <i>PD-1</i>, <i>TBX21</i>: <i>T-bet</i>, <i>KLRK1</i>: <i>NKG2D</i>.</p><p>^cFold-change ratio for <i>PLA2G2A</i> at week 11 was close to statistical significance (FDR = 0.063).</p><p>W: week.</p><p>High dose wIFN-α significantly induced intrahepatic expression of T cell, NK cell and IFN-γ response genes.</p

High dose wIFN-α significantly inhibits WHV.

<p>Maximum reductions in (a) serum and (b) intrahepatic viral parameters, and (c) maximum serum ALT and AST levels in response to placebo, low dose (20 μg) wIFN-α and high dose (100 μg) wIFN-α treatment. Changes in viral parameters were calculated relative to week -3 (pre-treatment baseline), with the exception of F1018 (wIFN-α group), for which week 0 was used as the baseline for the intrahepatic cccDNA and RNA analyses. The bar height indicates the mean of each group, and the errors bars represent the standard error of the mean. The lowest WHsAg level for two wIFN-treated animals (M1002 and F1022) was ≤ LLOD (20 ng/mL), and so the LLOD was used to estimate the maximum WHsAg decline. Per the sampling scheme outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#ppat.1005103.g001" target="_blank">Fig 1</a>, the following data was included in the analyses: maximum reduction in serum WHV DNA and WHsAg at weeks 0–16 (placebo), 0–7 (20 μg dose) and weeks 8–16 (100 μg dose); maximum reduction in intrahepatic cccDNA and RNA at weeks 0–15 (placebo), weeks 0 and 3 (20 μg dose) and weeks 7, 11 and 15 (100 μg dose); maximum serum ALT and AST levels at weeks 0–16 (placebo), 0–7 (20 μg dose) and weeks 11–16 (100 μg dose). Animals in the wIFN-α group were only included if data from all relevant time-points was available, with the exception of F1020, for which no week 16 sample was available for serum WHV DNA, WHsAg, ALT and AST analysis. Statistical significance was calculated by one-way ANOVA with Tukey's multiple comparison correction.</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

Design of the wIFN-α treatment study in woodchucks chronically infected with WHV.

<p>Chronic WHV carrier woodchucks were dosed three times a week (TIW) for 15 weeks with placebo, or for 7 weeks with 20 μg wIFN-α followed by another 8 weeks with 100 μg wIFN-α (15 weeks total). Animals were typically followed for additional 8 weeks after the treatment period (follow-up), although this was extended by two weeks for two wIFN-treated woodchucks (animals M1002 and M1004) that had no evidence of viral recrudescence at the end-of-study (week 23). *PK analysis of serum wIFN-α levels was not performed due to the lack of a sufficiently sensitive quantitative method (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005103#sec015" target="_blank">Methods</a>).</p