AIC649 reduces the HBV titer in HBV tg mice as effective as Tenofovir.

<p>HBV tg mice were treated i.p. with AIC649 (1 x 10⁸ viral particles / dose) twice weekly for 9 times in total. Tenofovir was administered twice daily by oral gavage at a total concentration of 100 mg/day. Negative control: Tenofovir vehicle, administration analogous to Tenofovir. Mice were sacrificed at the indicated time points and plasma samples were subjected to HBV DNA determination. HBV titers are expressed in % of the respective negative vehicle group +/- SD. * p < 0.05, ** p < 0.01 (vs respective treatment on day 0).</p

AIC649 induces a bi-phasic treatment effect on viremia levels in chronic WHV carrier woodchucks.

<p>AIC649 (3.9 x 10⁶ Units (ELISA-based titer)) or vehicle were administered i.m. twice weekly for 8 weeks (marked “Treatment”). At the indicated time points woodchucks were bled and serum was subjected to determination of WHV viral load. Values were normalized to individual baseline at day 0 and are given in %. n = 5 / group. Dotted lines: 100%, 50%, or 10% level. (A) Serum WHV DNA concentrations of individual woodchucks (identified by numbers) in the vehicle and AIC649 groups. (B) Group means of WHV DNA concentration +/- standard error of the mean (SEM). *p < 0.05.</p

AIC649 treatment induces cell-mediated immune responses in chronic WHV carrier woodchucks.

<p>AIC649 (3.9 x 10⁶ Units (ELISA-based titer)) or vehicle were administered i.m. twice weekly for 8 weeks (marked “Treatment”). At the indicated time points woodchucks were bled and PBMCs were subjected to determination of T cell proliferation after stimulation, whereas whole blood was used to analyze transcript levels of cytokines. Values were normalized to individual baseline at day 0 and are given in %. n = 5 / group. Dotted lines: 100% level. (A) Percent-change in mean T cell proliferation level from baseline at day 0 for woodchucks in the vehicle and AIC649 groups, respectively, following stimulation of PBMCs with WHsAg (WHsAg stim) or WHs peptide S226-245 (WHs peptide stim). (B) Percent-change in mean IFN-γ and TNF-α transcript level from baseline at day 0 in blood of woodchucks from both groups.</p

AIC649 treatment is associated with changes in serum activities of liver enzymes in chronic WHV carrier woodchucks.

<p>AIC649 (3.9 x 10⁶ Units (ELISA-based titer)) or vehicle were administered i.m. twice weekly for 8 weeks (marked “Treatment”). At the indicated time points woodchucks were bled and serum was used to analyze levels of liver enzymes. Values were normalized to individual baseline at day 0 and are given in %. n = 5 / group. Dotted lines: 100% or 50% level. (A). Group means of AST concentration +/- SEM. (B) Group means of GGT concentration +/- SEM.</p

AIC649 induces similar cytokine release kinetics in mice and woodchucks.

<p>Three healthy BALB/c mice / time point were treated once with 1 x 10⁵ U AIC649 i.p. (A) or 2.5 x 10⁴ U AIC649 i.m. (B). Mice were sacrificed at the indicated time points and plasma samples were pooled. The pooled plasma samples were subjected to duplicate cytokine determination. Plasma samples from non-injected mice were used as controls. Given are the means +/- SD. (C): Three healthy woodchucks were treated once with 3.9 x 10⁶ U AIC649 /ml or vehicle. Blood was drawn at the indicated time points and subjected to determination of cytokine transcript levels. Results are given as mean fold change relative to pre-treatment (t = 0) levels +/- SD.</p

iORFV (D1701) reduces antigen cross-presentation by liver sinus endothelial cells (LSEC) but enhances activation of T-cells.

<p>Activation of CD8+ T cells (B3Z) was measured by IL-2 secretion.</p

iORFV NZ2 is a more potent inhibitor of HBV than strain D1701 in HBV-transgenic mice.

<p>iORFV D1701, iORFV NZ2 or placebo was administered i.p. every third day, three times in total, to HBV-transgenic mice expressing 10⁷–10⁸ HBV genome equivalents per ml plasma (n = 7 per group). HBV-specific DNA from plasma was analyzed by quantitative PCR and from livers by dot-blot hybridization as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074605#pone.0074605-Weber1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074605#pone.0074605-Chromczynski1" target="_blank">[18]</a>. The figure shows means ± standard error of means (SEM) relative to placebo-treated animals where mean HBV-DNA content was set 100%. Treatment with iORFV reduced non-chromosomal HBV-DNA in the livers as compared to placebo animals <b>(a)</b> and in the plasma with the exception of the lowest dose of iORFV D1701 <b>(b)</b>. iORFV NZ-2 was more potent in terms of its potential to inhibit HBV replication compared to strain D1701 with the two lower dosages resulting in significantly higher reduction of HBV-DNA (one-way ANOVA and <i>post hoc</i> analysis [Newman-Keuls Multiple Comparison Test] *p<0.05, **p<0.01). <b>c)</b> Immunohistological analysis of HBcAg expression in the livers of placebo-treated animals. Diffuse cytoplasmic staining in periportal areas [arrows, central veins (CV)] indicates viral capsids and ongoing HBV replication in placebo-treated mice. Cytoplasmic HBcAg as well as nuclear HBcAg-specific stain (for empty capsids) was strongly reduced in both iORFV (NZ2)-treated <b>d)</b> and iORFV (D1701)-treated mice <b>e)</b>. Figures provide typical examples of the respective group of animals treated with a dose of 1.5×10⁶ TCID₅₀ iORFV NZ 2 or D1701, respectively.</p

Both ORFV strains induce cytokines suppressing HCV replication in an <i>in vitro</i> replicon system.

<p>Figures shows relative fluorescence units (RFU) as a measure for HCV replication. <b>a)</b> 20 µl (dose 1.6×10⁵ TCID₅₀) iORFV strain D1701 or NZ2 was added to whole blood and incubated for 3 days (see methods). Supernatants of cultured blood cells were added to replicon cells and HCV replication was determined three days later. Statistical analyses were performed using the one-way ANOVA and as <i>post hoc</i> analysis the Bonferroni Test. *p<0.05 vs. placebo. RFU-value from placebo (PBS) - treated supernatants was set as 100%. ConA served as a positive control. <b>b)</b> Cell viability of replicon-bearing cells was not affected after addition of supernatants from iORFV-incubated human blood cells. <b>c)</b> iORFV inhibits HCV replication in a dose-dependent manner. D 1701 or NZ2 were used for incubation with whole blood at doses ranging from 1.6×10⁵ TCID₅₀ to 1.6×10³ TCID₅₀. Values (n = 3) show means ± standard error of means (SEM).</p

Treatment with inactivated ORFV reduces HBV core antigen (HBcAg) in HBV-transgenic mice (n = 7/group).

*<p>Scores of anti-HbcAg stain: Nuclei: grade 3: the majority of nuclei in the liver specimens revealed intense immunostaining; grade 2: the number of positive nuclei was slightly reduced predominantly at the periphery of the liver lobule; grade 1: further reduction of positive nuclei was observed. Cytoplasm: grade 3: strong staining of numerous hepatocytes was observed around the central veins; grade 2: the cytoplasmic staining was reduced; grade 1: a further reduction or even absence of cytoplasmic immunostaining was observed. The histological slides were cross-checked by an independent pathologist and the results confirmed.</p><p>The severity of HBcAG-specific stain was quantified according to a scoring from 1 to 3*.</p