Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes

Abstract

Hepatitis B virus (HBV) infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs) are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV) and to exert an antiviral activity against established DHBV infection in vitro and in vivo. In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2'O-methyl ribose, 5-methylcytidine) were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2'O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.

Fig 1. Effect of multiple nucleic acid polymers treatments starting at the time of viral inoculation on HBV replication in HepaRG cells and primary human hepatocytes.

(A) Treatments procedure: HBV infected HepaRG cells and primary human hepatocytes were treated every two days starting at the time of inoculation. Supernatants and cell lysates were harvested for extracellular HBsAg, HBeAg and intracellular total HBV RNA analysis at day 4 (data not shown) and day 8 post-inoculation. The antiviral effect of (B) REP 2055 at 0.1 μM, 1 μM and 5 μM final concentrations and (C) REP 2006, REP 2139, REP 2165, REP 2031 and REP 2138 at 5 μM final concentration was assessed on differentiated HepaRG cells at day 8 post-inoculation by measuring secreted HBsAg, HBeAg and total HBV RNA. The antiviral effect of (D) REP 2055 and (E) REP 2139 at 0.1 μM, 1 μM and 5 μM final concentrations was assessed on primary human hepatocytes at day 8 post-inoculation by measuring secreted HBsAg, HBeAg and total HBV RNA. The solvent of NAP compounds was used as a non-treated condition. Heparin (HEP) was used as a positive control for entry inhibition at a concentration of 300 μg/ml. All data, expressed as means ± standard deviation, were independently reproduced three to four times, except for the effect of REP 2055 and REP 2139 on HBV RNA in HepaRG which was reproduced two times. Statistical analysis was conducted with R software using an ordinary one-way ANOVA with random effect for comparison to non-treated sample; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 2. Effect of post-viral inoculation treatment with nucleic acid polymers on HBV replication in HepaRG and primary human hepatocytes.

(A) Treatments procedure: HBV infected cells were treated every two days starting two days post-inoculation with (B) REP 2055 at 0.1 μM, 1 μM and 5 μM final concentrations,(C) REP 2006, REP 2139, REP 2165, REP 2031 and REP 2138 at 5 μM final concentration in differentiated HepaRG cells and (D) REP 2055 and (E) REP 2139 at 0.1 μM, 1 μM and 5 μM final concentrations in primary human hepatocytes at day 8 post-inoculation by measuring secreted HBsAg, HBeAg and total HBV RNA (See legend to Fig 1. for experimental details). The solvent of NAP compounds was used as a non-treated condition. Heparin (HEP) was used as a positive control for entry inhibition at a concentration of 300 μg/ml. All data, expressed as means ± standard deviation, were independently reproduced two to four times, except for the effect of REP 2139 on HBV RNA which was reproduced one time in PHH. Statistical analysis was conducted with R software using an ordinary one-way ANOVA with random effect for comparison to non-treated sample; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 3. Effect of a single nucleic acid polymers treatment at the time of viral inoculation on HBV replication in HepaRG cells and primary human hepatocytes.

(A) Treatments procedure: HBV infected cells were treated at the time of inoculation for the duration of inoculation with REP 2055 at 0.1 μM, 1 μM and 5 μM final concentrations in (B) differentiated HepaRG cells and (C) primary human hepatocytes at day 8 post-inoculation by measuring secreted HBsAg, HBeAg and total HBV RNA. (D) In primary human hepatocytes, in a side by side experiment with 5 μM REP 2055, heparin (HEP) was used as a positive control for entry inhibition at a concentration of 300 μg/ml (See legend to Fig 1. for experimental details). All data originate from three independent experiments except in (D) where two independent experiments have been performed. Results are expressed as means ± standard deviation. Statistical analysis was conducted with R software using an (B, C) ordinary one-way ANOVA with random effect for comparison to non-treated sample and (D) unpaired, 2-tailed t-tests for comparison of specific samples using GraphPadPrism6 software; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 4. Relationship between NAPs sequence, amphipathicity, chemical modification and anti-HBV antiviral activity.

(A) Treatments procedure: HBV infected HepaRG cells were treated at the time of inoculation for the duration of inoculation. Supernatants and cell lysates were harvested for extracellular HBsAg, HBeAg and intracellular total HBV RNA analysis at day 4 (data not shown) and day 8 post-inoculation. (B) To determine the effect of different NAPs chemical modifications on antiviral activity, HepaRG cells were treated once at the time of viral inoculation with REP 2172, REP 2147, REP 2006, REP 2107, REP 2055 and REP 2139 NAP compounds containing or not PS (phosphorothioation), 2’-OMe (2’O-methyl ribose) and 5-MeC (5-methylcytidine) at a 5 μM concentration and secreted HBsAg, HBeAg and total HBV RNA were measured at day 8 post-inoculation. The solvent of NAP compounds was used as a non-treated condition. All data originating from two independent experiments are expressed as means ± standard deviation. Statistical analysis was conducted with unpaired, 2-tailed t-tests, for comparison of specific samples; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, ns; non-significant.

Fig 5. Relationship between NAPs size and anti-HBV antiviral activity.

(A) Treatments procedure: HBV infected HepaRG cells were treated at the time of inoculation for the duration of inoculation. Supernatants and cell lysates were harvested for extracellular HBsAg, HBeAg and intracellular total HBV RNA analysis at day 4 (data not shown) and day 8 post-inoculation. (B) The effect of NAPs size on antiviral activity was assessed by measuring the effect of REP 2055 analogs of different length from 60 to 10 nucleotides length on secreted HBsAg, HBeAg and total HBV RNA in HepaRG cells treated once at the time of viral inoculation at a 5 μM NAPs concentration. The solvent of NAP compounds was used as a non-treated condition. All data originating from two independent experiments are expressed as means ± standard deviation. Statistical analysis was conducted with R software using an ordinary one-way ANOVA with random effect for comparison to non-treated sample; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.