Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection

Abstract

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.

Conflict of interest statement

Competing Interests: This study was funded by Replicor Inc. Dr. Andrew Vaillant is employed by Replicor Inc. NAPs are protected by patents wholly owned by Replicor Inc and are currently in development for the treatment of hepatitis B / hepatitis D infection. Relevant patents are: US 8,067,385 Antiviral oligonucleotides targeting HBV US 8,008,270 Antiviral oligonucleotides targeting viral families US 7,358,068 Antiviral oligonucleotides, US 8,008,269 Antiviral oligonucleotides. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1. Experimental design of REP 2055 and NS treatment in in vivo Experiment 1 and 2.

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).

Fig 2. Lack of cytokine gene upregulation in PHH treated with REP 2055.

The expression levels of TNF (a), IL6 (b), IL10 (c), IFNA4 (d) and IFNB1 (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 (**).

Fig 3. In vivo Experiment 1.

Response to various REP 2055 treatment regimens in Groups 1–4. 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 Fig 1A). LLOQ for DHBsAg (0.88 μg/ml) and DHBV DNA (24000 copies/ml) are shown as dashed lines.

Fig 4. In vivo Experiment 2.

Response to treatment with NS or REP 2055. Levels of DHBsAg (a), anti-DHBs antibodies (b), DHBV DNA (c and e) and anti-DHBc antibodies (d) in the serum and DHBV total and cccDNA in the liver (f) during and after treatment are shown. For NS-treated ducks in all panels, n = 13 except for the last 7 measurements, where n = 7 (see *). Horizontal lines in (a-e) represent the lower limit of quantification. For REP 2055duck n = 6 but SVR duck 188 is plotted separately in (a) and (e). For REP 2055 NO SVR ducks, n = 5 but NO SVR duck 194 is plotted separately in (a) and (e). Duck 194 and duck 188 are plotted separately in (a) and (e) as the response of these ducks was different from the other SVR or NO SVR ducks. Treatment interval in (a-e) is indicated on the x-axis by the shaded bar. Differences in mean responses of REP 2055 SVR and REP 2055 NO SVR were significantly different from NS in a, b and c (p