Nucleic acid polymers prevent the establishment of duck hepatitis B virus infection in vivo

Abstract

Nucleic acid polymers (NAPs) are novel, broad-spectrum antiviral compounds that use the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) as amphipathic polymers to block amphipathic interactions involved in viral entry. Using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection, NAPs have been shown to have both entry and postentry antiviral activity against DHBV infection in vitro in primary duck hepatocytes (PDH). In the current study, various NAPs were assessed for their prophylactic activity in vivo against DHBV infection in ducks. The degenerate NAP REP 2006 prevented the development of widespread and persistent DHBV infection in 14-day-old ducks, while the acidic-pH-sensitive NAP REP 2031 had little or no prophylactic effect. REP 2006 displayed significant toxicity in ducks, which was attributed to CpG-mediated proinflammation, while REP 2031 (which has no CpG motifs) displayed no toxicity. A third NAP, REP 2055, which was designed to retain amphipathic activity at acidic pH and contained no CpG motifs, was well tolerated and displayed prophylactic activity against DHBV infection at doses as low as 1 mg/kg of body weight/day. These studies suggest that NAPs can be easily and predictably tailored to retain anti-DHBV activity and to have minimal toxic effects in vivo. Future studies are planned to establish the therapeutic efficacy of NAPs against persistent DHBV infection.

Figures

Fig 1

Structures of NAPs used in this study. REP 2006 is degenerate, while REP 2031 [poly(C)] and REP 2055 [poly(AC)] have defined nucleic acid sequences. All NAPs are 40 nt in length.

Fig 2

Detection of DHBsAg by ELISA in the sera of ducks treated with REP 2006 (A), REP 2031 (B), and NS (C). Fourteen-day-old ducks were inoculated (i.v.) with 5 × 108 DHBV DNA genomes and treated with REP 2006 or REP 2031 or normal saline (NS) from day −1 to day 14 p.i., as follows: 10 mg/kg of REP 2006 i.p. once daily (QD) (A); 10 mg/kg of REP 2031 i.p. QD (B); NS i.p. QD (C). Serum samples were collected on days 1, 5, 10, and 14 p.i. and were diluted 1/100 for analysis. The normal duck serum (NDS) OD value was used as the cutoff point for the assay. Individual duck identifiers are indicated.

Fig 3

DHBV DNA levels in the liver of ducks on days 4 (A) and 14 (B) p.i. Fourteen-day-old ducks were inoculated (i.v.) with 5 × 108 DHBV genomes and treated with REP 2006 (10 mg/kg i.p. once daily [QD]) or REP 2031 (10 mg/kg i.p. QD) or normal saline (NS) (i.p. QD). Cellular and viral DNA extracts were tested for DHBV DNA by Southern blot hybridization as described in Materials and Methods. Expected positions of relaxed circular (RC), double-stranded linear (DSL), and single-stranded (SS) DHBV DNA are shown on the right. Individual duck identifiers are indicated above each lane. Note that the RC and DSL forms of DHBV DNA are occluded in some lanes in panel B due to a blotting artifact.

Fig 4

Detection of DHBsAg levels by ELISA in the sera of ducks treated with REP 2055 and normal saline (NS). Fourteen-day-old ducks were inoculated (i.v.) with 5 × 108 DHBV DNA genomes and treated with REP 2055 with 5 different dose regimens i.p. or with NS from 3 days prior to DHBV infection for 17 days: 0.5 mg/kg i.p. twice daily (BID)(A); 2 mg/kg i.p. BID (B); 3 mg/kg i.p. BID (C); 5 mg/kg i.p. BID (D); 10 mg/kg i.p. once daily (QD) (E); NS i.p. BID (F). Serum samples were collected on days 0, 5, 10, and 14 p.i. and were diluted 1/100 for analysis. The normal duck serum (NDS) OD value was used as the cutoff point for the DHBsAg assay. Individual duck identifiers are indicated. Ducks 502, 514, and 519 were excluded from analysis (see Results).

Fig 5

DHBV DNA levels in the liver of ducks on days 4 (A) and 14 (B) p.i. Fourteen-day-old ducks were inoculated i.v. with 5 × 108 DHBV genomes and treated twice daily (BID) with 0.5 to 5 mg/kg of REP 2055, once daily (QD) with 10 mg/kg of REP 2055, or with normal saline (NS). Cellular and viral DNA extracts were tested for DHBV DNA by Southern blot hybridization as described in Materials and Methods (radiographic exposure time, 24 h). The expected positions of relaxed circular (RC), double-stranded linear (DSL), and single-stranded (SS) DHBV DNA are shown on the right. Individual duck identifiers are indicated above each lane. Ducks 502, 514, and 519 were excluded from analysis (see Results).