Infection courses, virological features and IFN-α responses of HBV genotypes in cell culture and animal models

Abstract

Background & aims: HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment.

Methods: Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model.

Results: HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock.

Conclusions: Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models.

Lay summary: Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both in vitro and in vivo models, which are valuable tools for antiviral development.

Keywords: Virus genotypes; infection model; liver disease; stable cell lines; treatment.

Fig. 1.. HBVcc genotypes infect PXB cells with different infectivity.

PXB cells were infected with HBVcc of different genotypes at an MOI of 50. (A) HBV DNA was performed with real-time qPCR and HBeAg /HBsAg were tested with ELISA at day 3, 7, 10 and 14 post-infection. The data from the ELISA is calculated as signal to noise ratio (S/N), with N defined as the average OD reading of multiple un-infected culture medium control. The ELISA is generally linear up to a S/N of 50. (B) Comparison of HBV DNA, HBeAg and HBsAg levels among different genotypes at day 14 post-infection. For samples with S/N greater than 50, they were diluted for further analysis. (C) PXB cells were infected with HBVcc genotypes at MOI of 30, 10, 3, and 1. HBeAg was tested with ELISA at day 7, 10 and 14 post-HBVcc infection. Dotted line shows the negative cutoff. Data are means ± SEM of triplicates. The results are representative of three independent experiments.

Fig. 2.. Antiviral activities of hIFN-α2a and entecavir in HBVcc genotypes-infected PXB cells.

PXB cells were infected with HBV genotypes A2, B2, C1, D3, E and F1b at 50 MOI and further treated with hIFN-α2a (500 U/mL) for 14 days from day 1 post-viral infection. (A, B and C) The anti-HBV effect of hIFN-α2a and entecavir on production of HBV DNA, HBeAg and HBsAg at day 3, 7, 10 and 14 were evaluated. Data are shown as means ± SEM of triplicates. *** P<0.001; ** P<0.01; * P<0.05. The results are representative of three independent experiments.

Fig. 3.. HBVcc genotypes infect humanized chimeric mice and produce variable levels of HBV DNA, HBeAg and HBsAg.

Humanized chimeric mice were infected intravenously through tail vein injection with 1×10⁶ copies of HBVcc genotypes A2, B2, D3, E, F1b or H and 5×10⁶ copies of HBVcc genotype C1 (3 mice per group). Serum was harvested weekly post-infection up to 23 weeks post-infection. (A) Serum was analyzed for HBV DNA, HBeAg, HBsAg and hAlb levels through real time qPCR and ELISA kits. The data of each mouse are denoted by a different color of symbols & lines. (B) Comparison of HBV DNA, HBeAg and HBsAg levels at week 10 post-infection. Data are shown as means ± SEM of triplicates.

Fig. 4.. Inhibition of HBV DNA, HBeAg and HBsAg by pegIFN-α2a in infected humanized chimeric mice.

HBVcc-infected humanized chimeric mice were treated with pegIFN-α2a (30 μg/kg) at week 10 post-infection and continued for 4 weeks. Mice serum was harvested weekly at indicated time points during the treatment period and continued for 6 weeks after treatment cessation. HBV DNA and hAlb in mice serum were analyzed using real time PCR and ELISA kit accordingly. (A) The changes of HBV DNA, HBeAg and HBsAg levels from the pre-treatment levels (week 10 of infection) were calculated and shown. The data of each mouse are denoted by a different color of symbols & lines. (B) the cumulative changes of HBV DNA during 4 weeks of pegIFN-α2a treatment were calculated as area under curve (AUC) from week 1–4 of treatment. The data are shown as means ± SEM of triplicates.

Fig. 5.. Infectivity and density gradient analysis of HBVmp and HBVcc.

(A) PXB cells were infected with HBVcc and the corresponding HBVmp harvested at week 10 of each infected mouse; an MOI of 1 based on HBV DNA copy number was used. Culture media on day 10 were harvested and assayed for HBV DNA and HBeAg. The data for HBVmp are shown as means ± SEM of individual mouse data, and HBVcc shown as means ± SEM in triplicates. The results are representative of three independent experiments. (B) Concentrated HBVcc genotype A2 (500 μL of 4.2×10⁹ HBV DNA copies/mL) and D3 (500 μL of 6×10⁹ HBV DNA copies/mL) stocks, HBVmp Gt D3 (100 μL of 5×10⁹ HBV DNA copies/mL) and HBVpt (500 μL of 9.4×10⁸ HBV DNA copies/mL) were layered on top of a preformed iodixanol density gradient and subjected the centrifugation density gradient analysis as described in the Methods. After centrifugation, 20 fractions (500 μL per fraction) were collected from each gradient. HBsAg, HBcAg and HBV DNA were measured in each fraction with ELISA kits or qPCR. The boxed number above fraction 10 where Dane particles are represents the relative infectivity, which was calculated by dividing HBV DNA level of PXB cells at day 10 post-infection over the infecting HBV DNA titer of fraction 10.