Inducers of the NF-κB pathways impair hepatitis delta virus replication and strongly decrease progeny infectivity in vitro

Abstract

Background & aims: HDV superinfection of chronically HBV-infected patients is the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis/cirrhosis and increased risk of liver failure, hepatocellular carcinoma, and death. While HDV infection is not susceptible to available direct anti-HBV drugs, suboptimal responses are obtained with interferon-α-based therapies, and the number of investigational drugs remains limited. We therefore analyzed the effect of several innate immune stimulators on HDV replication in infected hepatocytes.

Methods: We used in vitro models of HDV and HBV infection based on primary human hepatocytes (PHHs) and the non-transformed HepaRG cell line that are relevant to explore new innate immune therapies.

Results: We describe here, for the first time, anti-HDV effects of Pam3CSK4 and BS1, agonists of Toll-like receptor (TLR)-1/2, and the lymphotoxin-β receptor (LTβR), respectively. Both types of agonists induced dose-dependent reductions of total intracellular HDV genome and antigenome RNA and of HDV protein levels, without toxicity in cells monoinfected with HDV or co/superinfected with HBV. Moreover, both molecules negatively affected HDV progeny release and strongly decreased their specific infectivity. The latter effect is particularly important since HDV is thought to persist in humans through constant propagation.

Conclusions: Immune-modulators inducing NF-κB pathways in hepatocytes can inhibit HDV replication and should be further evaluated as a possible therapeutic approach in chronically HBV/HDV-infected patients.

Lay summary: Hepatitis delta virus causes the most severe form of viral hepatitis. Despite positive recent developments, effective treatments remain a major clinical need. Herein, we show that immune-modulators that trigger the NF-κB pathways could be effective for the treatment of hepatitis delta infections.

Keywords: HDV-AG(s), HDV anti-genome(s); HDV-G(s), HDV genome(s); Hepatitis B virus; Hepatitis D virus; IFN, interferon; IL-, interleukin-; L-HDAg, large HDV antigen; LTβR, lymphotoxin-β receptor; NF-κB; NTCP, Na+-taurocholate cotransporting polypeptide; PHH, primary human hepatocyte; Peg-IFN-α, pegylated interferon-α; RNP, ribonucleoprotein; S-HDAg, small HDV antigen; TLR, Toll-like receptor; TNF, tumor necrosis factor; antiviral activity; dHepaRG, differentiated HepaRG cells; hepatocytes; lymphotoxin beta receptor; rh, recombinant human; toll-like receptor; vge, viral genome equivalent.

Graphical abstract

Fig. 1

Inducers of the NF-κB pathway such as Pam3CSK4 and BS1 reduced the levels of intracellular HDV RNA and protein. (A, B) dHepaRG cells were infected with HBV and then with HDV 6 days later with 100 vge/ml per cell for HBV and 10 vge/ml per cell for HDV. Cells were treated 3 days after HDV infection with BS1 (0.5 μg/ml) or Pam3CSK4 (0.5 μg/ml), IFN-α (1,000 U/ml), control IgG (0.5 μg/ml), tenofovir (10 μM), TNF-α (50 ng/ml) or recombinant human IL-6 (rhIL-6, 100 ng/ml) for 11 days. Cells were collected and the levels of total intracellular (A) HBV DNAs and (B) HDV RNAs were analyzed by qPCR and RT-qPCR respectively. Results are the mean ± SD of 2 (for HBV RNAs) or 3 (for HDV RNAs) independent experiments each performed with 3 biological replicates and statistical analyses were performed using Mann-Whitney tests. (C,D,E) dHepaRG cells were infected with HDV (HDV), HBV and with HDV 6 days later (HBV->HDV) or coinfected with HBV and HDV (HBV+HDV) with 100 vge per cell for HBV and 10 vge per cell for HDV. Cells were treated 3 days after HDV infection with (C, D) BS1 (0.1 μg/ml), Pam3CSK4 (0.1 μg/ml) or (E) IFN-α (1,000 U/ml) for 11 days. Cells were collected and the levels of total (C, E) intracellular HDV RNAs as well as the levels (C) HDV proteins were analyzed by (C) RT-qPCR, (E) Northern Blot or (C) western blot, respectively. (C) Results are the mean ± SD of 2 independent experiments each performed with 4 biological replicates and statistical analyses were performed using Mann-Whitney tests. (D) Cells were fixed and the number of living cells as well as % of HDV-positive cells was determined by immunofluorescent staining and quantification with ImageJ. Results are the mean ± SD of 3 independent experiments (each dot represent the quantification of one field) and statistical analyses were performed using Mann-Whitney tests. (F) dHepaRG cells were coinfected with HBV and HDV (HBV+HDV) with the indicated vge/cell. Cells were treated 6 days later (at the pic of HDV RNAs level) with BS1 (0.1μg/ml) or Pam3CSK4 (0.1 μg/ml) for 11 days. Cells were collected and the levels of total intracellular HDV RNAs as well as the levels of HDV proteins were analysed by RT-qPCR and western blot, respectively. Results are the mean ± SD of 2 independent experiments each performed with 3 biological replicates and statistical analyses were performed using Mann-Whitney tests. (G) dHepaRG or freshly isolated PHHs were coinfected with HBV (100 vge per cell) and with HDV (10 vge per cell). Cells were treated 3 days after with BS1 (0.5 μg/ml for dHepaRG or 2.5 μg/ml for PHHs) or Pam3CSK4 (0.5 μg/ml for dHepaRG or 2.5 μg/ml for PHHs) for 10 days. Supernatants were collected and release of LDH (assessing toxicity) were measured. Results are the mean ± SD of 2 independent experiments (2 per cell type) each performed with 3 to 4 biological replicates. (H) Freshly isolated PHHs from 4 distinct donors were coinfected with HBV (100 vge per cell) and with HDV (10 vge per cell). Cells were treated 3 days after with BS1 (1 μg/ml) or Pam3CSK4 (1 μg/ml) for 10 days. Cells were collected and the levels of total intracellular HDV RNAs were analysed by RT-qPCR. Results are the means of experiments performed with 4 donors with 3 or 4 biological replicates per experiment. dHepaRG, differentiated HepaRG; PHH, primary human hepatocyte; RT-qPCR, reverse-transcription quantitative PCR; vge, viral genome equivalent.

Fig. 2

Pam3CSK4 and BS1 on HDV triggered the activation of NF-κB pathways in HBV/HDV-coinfected dHepaRG cells. (A) dHepaRG cells were coinfected with HBV (100 vge per cell) and with HDV (10 vge per cell). Cells were treated 3 days later with BS1 (0.1 μg/ml) or Pam3CSK4 (0.1 μg/ml) for 10 days. Cells were collected and the levels of indicated proteins from the NF-kB pathways were analysed by western blot. (B) dHepaRG cells were coinfected with HBV (100 vge per cell) and with HDV (10 vge per cell). Cells were treated 3 days later with BS1 (0.1 μg/ml) or Pam3CSK4 (0.1 μg/ml) for 3 days. Cells were fixed and RelA and RelB were stained using an alkaline phosphatase reaction. Positive signals are pink. Bars represent the mean ± SD from 2 independent experiments each performed using 4 fields for quantification. (C) dHepaRG cells were infected with HBV (100 vge per cell) and with HDV (10 vge per cell) 6 days later. Cells were treated 3 days after HDV infection with BS1 (0.1 μg/ml) or Pam3CSK4 (0.1 μg/ml) for 48 h. Cells were collected, RNA extracted and sequenced. Fold changes were calculated by comparison to non-treated cells and genes with a p value-adj <0.05 were selected and submitted to the GSEA software using the MSigDB data base. Molecular signatures are represented as bar-plots (FDR <0.25). (D) dHepaRG cells were infected with HDV with 50 vge/cell and treated 3 days with BS1 (0.1 μg/ml) or Pam3CSK4 (0.1 μg/ml) in the presence or not of TPCA-1 (10 uM) for 11 days. Cells were collected and the levels of total intracellular HDV RNAs were analysed by RT-qPCR. Results are the mean ± SD of 3 independent experiments each performed with 3 biological replicates and statistical analyses were performed using Mann-Whitney tests. dHepaRG, differentiated HepaRG; FDR, false discovery rate; GSEA, gene set-enrichment analysis; RT-qPCR, reverse-transcription quantitative PCR; vge, viral genome equivalent.

Fig. 3

Pam3CSK4 and BS1 reduced the infectivity of HDV particles. dHepaRG cells were coinfected with HBV (500 vge per cell) and with HDV (50 vge per cell). Cells were treated or not 3 days later with (A, B, C, D) Pam3CSK4 (0.1 μg/ml) or (A, B, C, D) BS1 (0.1 μg/ml) or (C) Lamivudine (3TC; 10 μM) for 10 days. Supernatants were collected, concentrated by PEG precipitation and the levels of extracellular HDV RNAs (called HDV-2P for second passage) were assessed by qRT-PCR analyses before (A, B) being used for infection of naïve HuH7.5-NTCP cells with the indicated vge per cell. Six days later, (A) levels of intracellular HDV RNA were assessed by RT-qPCR analyses or (B) cells were stained with DAPI and with anti-HDAg antibodies. Results are the mean ± SD of two independent experiment each performed with 3 biological replicates and statistical analyses were performed using Mann-Whitney tests. (C) Concentrated supernatant were submitted to iodixanol gradients overnight. Fractions (FR) were collected and the levels of HDV RNA, HBV DNA, HBsAg, HDV protein were analysed by qRT-PCR, qPCR, ELISA and western blot respectively. (D) HDV RNAs were extracted from concentrated supernatants and sequences of HDV-G RNAs from HDV-2P were analysed after RT-PCR and cloning. Bars represent non-edited HDV-G and edited HDV-G at the amber termination codon (usually edited by ADAR1 to produce L-HDAg). See Fig. S9 for details of the sequencing. HDV-G, HDV genome; L-HDAg, large HDV antigen; MW, molecular weight; RT-qPCR, reverse-transcription quantitative PCR; vge, viral genome equivalent.