Lack of cytokine gene upregulation in PHH treated with REP 2055.

<p>The expression levels of <i>TNF</i> (a), <i>IL6</i> (b), <i>IL10</i> (c), <i>IFNA4</i> (d) and <i>IFNB1</i> (e) genes were assessed by quantitative RT-PCR 6 hr after treatment with the NAP REP 2055 (0.01–10 μM) or without treatment (w/o), ODN 2216 (a CpG oligonucleotide TLR-9 agonist; 2 μM), poly I:C (a double stranded RNA TLR-3 agonist; 25 μg/ml) or Pam3CK4 (a TLR-1/2 agonist; 1 μM). Values represent mean ± SEM (normalized to 100,000 copies of beta actin mRNA). Statistically significant changes compared to untreated controls are reported for p< 0.05 (*) and p< 0.01 (**).</p

<i>In vivo</i> Experiment 2: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.

<p>^aNS or REP 2055 was administered via IP injection.</p><p>^b--- = no liver tissue harvested (ducks autopsied at 103 dpi, 9 weeks of follow-up)</p><p>^cLower limit of detection of DHBsAg positive hepatocytes is 0.001%.</p><p>^dLower limit of detection of DHBcAg positive hepatocytes is 0.006%.</p><p><i>In vivo</i> Experiment 2: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.</p

<i>In vivo</i> Experiment 1.

<p><b>Response to various REP 2055 treatment regimens in Groups 1–4</b>. Individual duck data for serum DHBsAg (top row) and serum DHBV DNA (bottom row). Days of REP 2055 treatment are indicated at the top of each graph by inverted triangles (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140909#pone.0140909.g001" target="_blank">Fig 1A</a>). LLOQ for DHBsAg (0.88 μg/ml) and DHBV DNA (24000 copies/ml) are shown as dashed lines.</p

Experimental design of REP 2055 and NS treatment in <i>in vivo</i> Experiment 1 and 2.

<p>Fourteen-day-old ducks were infected with 5x10⁸ DHBV genome equivalents via the jugular vein. In Experiment 1 (a) all ducks were treated by IP injection with REP 2055. Group 1 received 10 mg/kg/day from 1 day prior to DHBV infection to 14 dpi; Group 2 received 10 mg/kg/day from 12–19 dpi and 10 mg/kg once weekly for 49 days; Group 3 received 10 mg/kg/day from 4–18 dpi and; Group 4 received 2 mg/kg/day from 4–18 dpi. After treatment, ducks in Groups 1, 3 and 4 were followed for an additional 49 days, from 19–68 dpi. Liver biopsies were performed prior to treatment in Group 2 and at the end of treatment in Groups 1, 3 and 4. Autopsies were performed at 68 dpi at the end of treatment in Group 2 and the end of follow-up in Groups 1, 3 and 4. In Experiment 2 (b), 14 DHBV-infected ducks were treated by IP injection with 10 mg/kg/day of REP 2055 from 12–40 dpi. A control group of 14 DHBV-infected ducks received daily IP injections of NS. Blood samples were collected during treatment and from 41–103 dpi during the first 9 weeks of follow-up. Based on interim analysis of serum DHBV DNA, 7 REP 2055-treated ducks that maintained control of their infection and 7 randomly selected NS-treated ducks, were followed from 103–155 dpi (total 16 weeks of follow-up). Liver biopsies were performed in all animals at 12 dpi prior to treatment. Biopsies or autopsies were performed at 103 dpi (9 weeks follow-up) and additional autopsies were performed in 7 animals per group at 155 dpi (16 weeks follow-up).</p

<i>In vivo</i> Experiment 1: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.

<p>^a--- = no liver tissue harvested</p><p>^bLower limit of detection of DHBsAg positive hepatocytes is 0.001%.</p><p>^cLower limit of detection of DHBcAg positive hepatocytes is 0.006%.</p><p>^dThe liver of duck 296 contained amyloid deposits</p><p>^eND = not done due to sample exhaustion</p><p><i>In vivo</i> Experiment 1: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.</p