Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb;6(2):281-296.
doi: 10.1002/hep4.1804. Epub 2021 Aug 25.

Capsid Allosteric Modulators Enhance the Innate Immune Response in Hepatitis B Virus-Infected Hepatocytes During Interferon Administration

Keisuke Fukutomi  1 , Hayato Hikita  1 , Kazuhiro Murai  1 , Tasuku Nakabori  1 , Akiyoshi Shimoda  1 , Makoto Fukuoka  1 , Takuo Yamai  1 , Yuichiro Higuchi  2 , Kei Miyakawa  3 , Hiroshi Suemizu  2 , Akihide Ryo  3 , Ryoko Yamada  1 , Takahiro Kodama  1 , Ryotaro Sakamori  1 , Tomohide Tatsumi  1 , Tetsuo Takehara  1
Affiliations

Capsid Allosteric Modulators Enhance the Innate Immune Response in Hepatitis B Virus-Infected Hepatocytes During Interferon Administration

Keisuke Fukutomi et al. Hepatol Commun. 2022 Feb.

Abstract

Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen-associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV-infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41-4109 (BAY41), to HBV-infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)-stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN-α in HBV-infected PHHs but did not change ISG induction by IFN-α in uninfected PHHs. Compared with BAY41 or IFN-α alone, coadministration of BAY41 and IFN-α significantly suppressed extracellular HBV-DNA levels. HBV-infected human liver-chimeric mice were treated with vehicle, BAY41, pegylated IFN-α (pegIFN-α), or BAY41 and pegIFN-α together. Compared with the vehicle control, pegIFN-α highly up-regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN-α further enhanced intrahepatic antiviral ISG expression, which was up-regulated by pegIFNα. The serum HBV-DNA levels in mice treated with the combination of BAY41 and pegIFN-α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN-α, likely due to increased cytoplasmic extracapsid pgRNA.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Intracellular ISG expression is not altered by HBV infection. (A‐E) PHHs isolated from human liver–chimeric mice were inoculated with HBV (100 GEq/cell or 1,000 GEq/cell), and the HBc‐positive cell ratio, intracellular pgRNA levels, and antiviral ISG expression levels were evaluated at indicated times. Each samples were analyzed after washing. (A) Schematic of the experimental procedure. (B) Representative image of fluorescence immunostaining (red, HBc; blue, nucleus). The HBc‐positive cell ratio was calculated based on the average of four fields of view. (C) Western blot of intracellular HBc protein and its quantifications (n = 3‐4). (D) Intracellular total pgRNA levels (n = 4). (E) The mRNA expression levels of antiviral ISGs (n = 4). Dot plot shows individual values. (F) Uninfected PHHs or HBV‐infected PHHs at 20 dpi were transfected with 0.01 μg/mL Poly (I:C). Two days later, the mRNA expression levels of antiviral ISGs were measured (n = 4). For reference, uninfected HepG2‐derived NTCP‐expressing cells (HepG2‐hNTCP C4 cells) or HBV‐infected (5,000 GEq/cell) HepG2‐hNTCP C4 cells at 8 dpi were transfected with 0.1 μg/ml Poly (I:C). Two days later, the mRNA expression levels of antiviral ISGs were measured (n = 4). Dot plot shows individual values. The data are shown as the mean ± SEM. *P < 0.05.
FIG. 2
FIG. 2
BAY41 reduces intracellular HBc and pgRNA levels. Cells were incubated with HBV inoculum (1,000 GEq/cell) for 24 hours and then treated with BAY41 or TFV beginning on 20 dpi. (A) Schematic of the experimental procedure. (B) WST assay at indicated days after treatment (n = 6). (C) Representative image of fluorescence immunostaining (red, HBc; blue, nucleus). (D) Western blot of intracellular HBc protein after indicated days after treatment, and its quantifications (n = 3‐4). (E) Intracellular total pgRNA, HBV DNA, HBsAg, and HBeAg levels in the culture supernatants at the end of the 12‐day treatment (n = 4). Each value was compared with that in the untreated control. The data are shown as the mean ± SEM. *P < 0.05.
FIG. 3
FIG. 3
BAY41 reduces intracapsid pgRNA levels per cell without inducing the host IFN response. (A‐D) Cells were incubated with HBV inoculum (1,000 GEq/cell) for 24 hours and treated with BAY41 for 2, 6, or 12 days beginning at 20 dpi. (A) Schematic of the experimental procedure. (B) Intracellular total pgRNA levels in untreated PHHs or PHHs treated with BAY41 over time (n = 4‐6). (C) Cytoplasmic total pgRNA and intracapsid pgRNA levels per cell after 2 days of treatment with BAY41 (n = 4). The cytoplasmic total pgRNA levels and intracapsid pgRNA levels were normalized to the cytoplasmic β‐actin mRNA level. (D) The mRNA expression levels of IFNs and antiviral ISGs in untreated PHHs and PHHs treated with BAY41 monotherapy (n = 4‐6). The results are shown as relative levels with the levels of each data of untreated PHHs set to 1. Dot plot shows individual values. The data are shown as the mean ± SEM. *P < 0.05. Abbreviation: N.D., not detected.
FIG. 4
FIG. 4
BAY41 enhances the antiviral ISG expression induced by IFN‐α in HBV‐infected PHHs. (A‐D) The cells were incubated with HBV inoculum (1,000 GEq/cell) for 24 hours and treated with BAY41 alone, IFN‐α alone, or in combination with BAY41 for indicated days beginning on 20 dpi. (A) Schematic of the experimental procedure. (B) Intracellular total pgRNA levels (n = 4‐6). (C) Cytoplasmic total pgRNA and intracapsid pgRNA levels (n = 4‐5). Total cytoplasmic pgRNA and intracapsid pgRNA levels in each sample were normalized to the cytoplasmic β‐actin mRNA level. (D) The mRNA expression levels of IFNs and ISGs in untreated PHHs or PHHs treated with BAY41 (10 μM) alone, IFN‐α (50 IU/ml) alone, or in combination with BAY41 (n = 4‐6). The results are shown as relative levels with the levels of each data of PHHs treated with IFN‐α alone set to 1. Dot plot shows individual values. (E,F) Uninfected PHHs were treated with BAY41 (10 μM) alone, IFN‐α (50 IU/ml) alone, or in combination with BAY41 and IFN‐α for 2 or 6 days beginning at 20 days after seeding. Same lot of PHHs as those in Fig. 4D were used. (E) Schematic of the experimental procedure. (F) The mRNA expression levels of the ISGs in uninfected PHHs after the treatment (n = 4‐6). The results are presented as relative levels, and the levels of each datapoint of PHHs treated with IFNα alone is set to 1. Dot plot shows individual values. The data are shown as the mean ± SEM. *P < 0.05.
FIG. 5
FIG. 5
The combination of BAY41 and IFN‐α reduces HBV‐DNA levels more effectively than BAY41 or IFN‐α monotherapy. (A‐C) HBV‐infected PHHs were treated with BAY41 alone, IFN‐α alone, or their combination for indicated days. (A) Schematic of the experimental procedure. (B) WST assay at indicated days after treatment (n = 6). Each value was compared to that in the untreated control. (C) cccDNA levels and HBV‐DNA, HBsAg, and HBeAg levels in the culture supernatants after 2 days or 12 days of treatment (n = 4). The data are shown as the mean ± SEM. *P < 0.05.
FIG. 6
FIG. 6
BAY41 enhances the intrahepatic ISG expression induced by pegIFN‐α in HBV‐infected human liver–chimeric mice, and BAY41 and pegIFN‐α combination therapy reduces serum HBV‐DNA levels more effectively than BAY41 or pegIFN‐α alone. HBV‐infected mice were treated with vehicle (n = 4), BAY41 (40 mg/kg, twice a day, p.o.) (n = 5), pegIFN‐α (25 mg/kg, twice per week, s.c.) (n = 4), or the combination of BAY41 and pegIFNα (n = 5) for 14 days. (A) Schematic of the experimental procedure. (B) Serum HBV‐DNA levels from inoculation to the end of the treatment. (C) Change in the serum HBV‐DNA levels over the treatment period. (D) Analysis of the intrahepatic mRNA expression levels of human‐specific IFN and ISGs. The results are presented as relative levels with the levels of each datapoint of mice treated with pegIFN‐α alone set to 1. Dot plot shows individual values. (E) Intrahepatic pgRNA and cccDNA levels at the end of the treatment. Dot plot shows individual values. (F) Representative image of HBc immunohistochemistry. The data are shown as the mean ± SEM. *P < 0.05. Abbreviations: p.o., per os; s.c., subcutaneous administration.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. World Health Organization . Fact sheet, Hepatitis B 2019, July 18. https://www.who.int/news‐room/fact‐sheets/detail/hepatitis‐b. Accessed January 14, 2021.
    1. European Association for the Study of the Liver . EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67:370‐398. - PubMed
    1. Terrault NA, Lok ASF, McMahon BJ, Chang K‐M, Hwang JP, Jonas MM, et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 2018;67:1560‐1599. - PMC - PubMed
    1. Suk‐Fong LA. Hepatitis B treatment: what we know now and what remains to be researched. Hepatol Commun 2019;3:8‐19. - PMC - PubMed
    1. Fanning GC, Zoulim F, Hou J, Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discov 2019;18:827‐844. - PubMed

Publication types