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Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment

Open AccessPublished:January 23, 2023DOI:https://doi.org/10.1016/j.jhepr.2023.100673

      Highlights

      • Responsiveness of three HDV strains to interferon alpha (pegIFNα) was tested in vivo
      • A newly cloned HDV-1 and an HDV-3 isolate strongly responded to pegIFNα
      • In contrast, a commonly used HDV-1 strain was IFN-resistant in vivo
      • PegIFNα reduced HBV in all co-infected mice and induced similar innate responses
      • Study points out existence of virus-specific determinants of IFNα responsiveness

      Abstract

      Background & Aims

      Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of hepatitis D virus (HDV)-infected patients. However, its mode of action (MoA) in HDV-infected cells remains elusive and only a minority of patients responds to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to IFNα in vitro (Zhang, J.Hepatol. 2018), a new HDV strain (HDV-1p) we isolated from a patient achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3) (Casey, PNAS 1993).

      Methods

      PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analyzed by qPCR, sequencing, immunoblotting, RNA in situ hybridization (ISH) and immunofluorescence staining.

      Results

      PegIFNα treatment efficiently reduced HDV RNA viremia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viremia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-HDAg posttranslational modification sites among all three isolates.

      Conclusions

      Our comparative study points out the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness.

      Lay summary

      Understanding factors counteracting hepatitis D virus (HDV) infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

      Graphical abstract

      Keywords

      Conflict of interest

      The authors declare no competing interests.

      Financial support

      The study was supported by the German Research Foundation (DFG) by a grant to MD and ML (SFB 841, A8), and to DG (GL595/9-1 and SFB1021, B08). MD and DG also received funding from the German Center for Infection Research (DZIF-BMBF; TTU-hepatitis 05.820; 05.822; 05.714). The National Reference Center for Hepatitis B Viruses and Hepatitis D Viruses is supported by the German Ministry of Health via the Robert Koch Institute. All funding sources supporting the work are acknowledged and authors have nothing to disclose.

      Author contributions

      ML and MD initiated and supervised the study. ML, MD and KG designed experiments. AV, LA and TV generated chimeric mice. KG, LHen, PPG, JK and LHer performed analyses and generated data. KG performed HDV full genome sequencing. NG and DG cloned the HDV-1p and tested their infectivity in HepG2hNTCP cells. JHB and JP provided clinical data. KG and MD wrote the manuscript. AV, JP and ML discussed the data. NG, DG, AV, LA and PPG corrected the manuscript.

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      . In this study, we assessed and compared the antiviral and intrinsic host response to pegIFNα treatment in vivo using human liver chimeric mice stably infected with HBV and either with HDV-1a or two different patient-derived cloned HDV strains: a novel genotype 1 (HDV-1p) and an HDV-3 (table 1).
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      Table 1Characteristics of the source patients and cloned viruses. Summary of information available regarding the three HDV strains used in this study, including known patient characteristics, cloning strategies used, referring accession numbers and references. *Sequence analyses revealed that this HDV-1 clone shows 99.7% identity with M21012 and 99.5% with AJ00058.
      HDV-1aHDV-1pHDV-3peru
      Gendermalemalemale
      Patient ageunknown52y18y
      OriginunclearTurkeyPeru
      Clinical characteristicsunclearchronic HBV and HDV infectionsevere acute HBV and HDV infection
      IFN response in patientuntreatedResponsiveuntreated
      Specificsserially passaged in chimpanzees, in a woodchuck and then clonedNAs treatment when serum was collected; serially passaged in HBV infected humanized mice and then clonednot passaged; initially HBV envelope of genotype F
      Accession numberM21012
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      AJ00058
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      *
      OL825606L22063
      Year virus cloning198820191993
      HDV plasmid for cloningHDV trimer in pSVLHDV tandem dimer in pcDNA3.1(+)pCMV3-Peru-1.2
      HBV envelope-expressing plasmid for cloningpT7HB2.7 (genotype D)HBV subgenome in pcDNA3.1(+)

      (genotype D)
      pT7HB2.7 (genotype D)
      References
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      • Mason W.
      • Gerin J.
      • Taylor J.
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      ,
      • Langon T.
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      • Kay A.
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      • et al.
      Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.
      ,
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      High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.
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      Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen.

      Material and Methods

      Virus generation

      The patient derived HDV-1 isolate (HDV-1p) was isolated from a CHD male outpatient from the university clinic of Hamburg,
      • Bockmann J.-H.
      • Grube M.
      • Hamed V.
      • von Felden J.
      • Landahl J.
      • Wehmeyer M.
      • et al.
      High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.
      passaged in human liver chimeric uPA/SCID/beige/IL2RG-/- (USG) mice, sequenced, cloned as genome-sense tandem dimer in pcDNA3.1(+) and infectious particles were produced in cell culture (table1). The HDV-3 isolate (Peru-1) was obtained from a young man from Peru, who developed severe acute hepatitis, and was cloned by Casey et al..
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      • Colan E.J.
      • Wignall F.S.
      • Gerin J.L.
      A genotype of hepatitis D virus that occurs in northern South America.
      The origin of the first HDV clone available in the research community is less clear: it is a genotype 1 strain, patient(s) sera were passaged through various chimpanzees at NIH (J. Taylor personal communication), inoculated in a woodchuck and then cloned (table1). For the production of the HDV-1a and HDV-3 strains, the HDV recombinant plasmid pSVL(D3) (kindly provided by John Taylor, Philadelphia, PA USA)
      • Kuo M.Y.
      • Chao M.
      • Taylor J.
      Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen.
      and pCMV3-Peru-1.2 (kindly provided by John Casey, Washington D.C, USA)
      • Casey J.L.
      • Gerin J.L.
      Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen.
      were used. Infectious HDV-1a, HDV-1p and HDV-3 particles were generated in HuH7 cells as previously described.
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      (HDV-1a, HDV-3) encoding the surface proteins of HBV genotype D using Fugene HD Transfection Reagent (Promega, Madison, USA) (table1).

      Treatment

      In vivo, pegIFNα treatment was started when HBV/HDV-1a-, HBV/HDV-1p- or HBV/HDV-3-infected mice reached stable HBV and HDV viremia levels. Mice received pegIFNα (Pegasys; Roche, Basel, Switzerland) twice a week subcutaneously (25 ng/g body weight)
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      • et al.
      Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.
      ,
      • Allweiss L.
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      • Pirosu A.
      • Volz T.
      • Muench R.C.
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      • et al.
      Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.
      for 4 or 9 weeks. Mice were sacrificed 24 h after the last pegIFNα injection. Cultured PHHs (isolated from an HBV/HDV-infected mouse) received IFNα (1000 international units (IU)/ml; Roferon-A; Roche, Basel, Switzerland) starting 1 day after plating. The culture medium was changed twice a week.
      More methods (e.g. generation and infection of mice, virological measurements, RNA in situ hybridization) can be found in the Supplement.

      Results

      Production and infectivity of HDV-1p in vitro and in vivo. As summarized in table 1, the HDV-1p strain was obtained from a CHD patient receiving NUCs treatment (lamivudine plus adefovir), which resulted in undetectable serum HBV DNA levels (figure 1A). HDV viremia was detectable and immunohistology confirmed abundance of HDAg-positive hepatocytes (figure 1A, C). Eventually, after discontinuation of NUC treatment, the patient received pegIFNα for 48 weeks, which resulted in a sustained HDV response and temporary increase of HBV viremia,
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      • Hamed V.
      • von Felden J.
      • Landahl J.
      • Wehmeyer M.
      • et al.
      High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.
      but led to HBV and HBsAg loss and seroconversion to anti-HBs years later (figure 1B).
      Figure thumbnail gr1
      Figure 1Patient characteristics (HDV-1p). A) HDV and HBV viremia (above) and HBsAg and anti-HBs levels (below) during and after NUC (green box) and pegIFNα treatment (yellow box) of the infected patient. The black arrow indicates the time-point when serum was collected to infect human liver chimeric mice for passaging the virus. B) Immunohistology of HDAg (brown nuclei) in a liver biopsy of the patient before treatment with pegIFNα (overview, 10x and close up, 20x).
      The HDV-positive isolate obtained before IFN treatment was first shown to be infectious in HBV-infected human liver chimeric USG mice
      • Giersch K.
      • Homs M.
      • Volz T.
      • Helbig M.
      • Allweiss L.
      • Lohse A.W.
      • et al.
      Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.
      and now sequenced and cloned as described in Material and Methods (Table 1). The full genome sequence of the HDV-1p strain is available at NCBI (accession number: OL825606). HDV-1p virus stocks were produced in HuH7 cells and their infectivity was tested first in vitro using HepG2hNTCP cells. Seven days after HDV-1p inoculation (multiplicity of infection; MOI=1), HDAg staining (figure 2A) confirmed in vitro infectivity of the HDV-1p particles produced after cloning the virus. The in vitro infectivity of HDV-1p appeared similar to the cloned strains HDV-3 (MOI=1) and HDV-1a (MOI=2) (figure 2A).
      Figure thumbnail gr2
      Figure 2In vitro and in vivo infectivity of the cloned strains HDV-1p, HDV-3 and HDV-1a. A) HDAg (red, IF staining) in HepG2hNTCP cells 7 days after mono-infection with HDV-1p, HDV-3 or HDV-1a. B) Development of HDV viremia in humanized mice (HDV-1p left, HDV-3 middle, HDV-1a right). IF staining of HDAg (red), HBcAg (green) and the human marker CK18 (turquoise) (C) and Western blot analysis of S- and L-HDAg (D) in stable HBV/HDV infected mice and in the liver biopsy obtained from the HDV-1p infected patient.
      To assess the infectivity of the cloned HDV-1p strain in vivo, HBV-infected humanized USG mice were super-infected with HDV-1p from the supernatant of HuH7 cells (4x106 GE/mouse). In these HBV infected mice, where HDV infection is first established in a low number of PHHs, HDV disseminated among PHHs and viremia increased up to week 6 after super-infection and remained stable until the end of the observation time (9 weeks post super-infection) (figure 2B). In line with previous studies,
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      we observed a decrease of HBV viremia (5-fold) during HDV-1p super-infection (data not shown), while HDAg was clearly detected in human hepatocytes (figure 2C). Moreover, amounts and distribution of S- and L-HDAg resembled those detected in the patient liver biopsy (figure 2D). Likewise, HDV-3 and HDV-1a reached stable HDV titers after around 5 weeks of HDV super-infection and showed similar ratios of S- and L-HDAg (figure 2B, D). The amount of HDV-infected human hepatocytes determined by IF tended to be higher in mice infected with HDV-3 (63%) compared to those infected with HDV-1a (32%) or HDV-1p (42%), suggesting that HDV-3 may display superior spreading capacities in vivo than the HDV-1 strains. Overall, these comparative analyses show that human liver chimeric mice are well suited to study patient-derived viral strains.

      PegIFNα treatment in mice infected with different HDV strains

      Human liver chimeric mice stably infected with HBV and one of the three HDV clones received pegIFNα twice a week (figure 3A). In HBV/HDV-1p and HBV/HDV-3 infected mice, 4-week pegIFNα treatment reduced HDV viremia by more than 2.0-log and by 1.7-log, HBV viremia by 2.0-log and 1.7-log, respectively, as well as levels of circulating HBsAg (figure 3B-D, and figure S1A-D). Intrahepatic analyses revealed a clear reduction of HDV RNA (HDV-1p: 0.9-log, HDV-3: 1.5-log) (figure 3E) and HBV pgRNA levels relative to human hepatocyte contents (figure 3F) in mice infected with HBV/HDV-1p (0.5-log) or HBV/HDV-3 (0.6-log) in comparison to untreated control mice. In strikingly contrast, 4-week treatment of HBV/HDV-1a infected mice clearly reduced HBV DNA (2-log) and HBsAg in serum (figure 3C, D and figure S1B); intrahepatic pgRNA (1.5-log) (figure 3E), but had no effect on HDV in serum (figure 3B and figure S1E) and liver (figure 3E). Even after extending pegIFNα treatment to 9 weeks, serum HDV RNA levels remained comparable in treated and untreated mice harboring the HDV-1a strain (figure 3B, figure S1E), while HBV viremia (3-log) and HBsAg levels decreased even further (figure 3C and D, figure S1F).
      Figure thumbnail gr3
      Figure 3PegIFNα treatment in HBV/HDV-1a, HBV/HDV-1p and HBV/HDV-3 infected mice. A) Experimental design: Stable HBV/HDV-1a, HBV/HDV-1p and HBV/HDV-3 infected mice received pegIFNα for 4 weeks or remained untreated. Some mice infected with HBV/HDV-1a were treated for 9 weeks. Serum HDV (B), HBV (C) and HBsAg (D) are depicted as fold change from baseline in HBV/HDV-1a infected untreated (grey line, square symbols; n=4) or treated mice (blue line, square symbols; n=3), in HBV/HDV-1p infected untreated (grey line, round symbols; n=4) or treated mice (pink line, round symbols; n=5) and in HBV/HDV-3 infected untreated (grey line, triangular symbols; n=4) or treated mice (light red line, triangular symbols; n=5). Curves show median levels with range. PCR measurements of liver HDV RNA (E) and HBV pgRNA (F) in untreated and treated HBV/HDV-1a, HBV/HDV-1p and HBV/HDV-3 infected mice. Bars show median levels black dots represent individual mice. HBV/HDV-1a infected mice are shown as clear square or black dots when they were treated for 4 (n=1) or 9 weeks (n=2), respectively.
      In line with liver HDV RNA levels, IF staining of HDV-1p- and HDV-3-infected mice 4 weeks after pegIFNα treatment demonstrated lower numbers of HDAg-positive PHHs (HDV-1p: 4%, HDV-3: 30%) compared to untreated controls (HDV-1p: 42%, HDV-3: 63%) (figure 4A). This confirms that both HDV-1p and HDV-3 isolates are highly responsive to IFN treatment in vivo in mice stably infected with both HBV and HDV. In mice infected with the IFN-resistant HDV-1a strain the amount of HDAg-positive PHHs appeared comparable between untreated controls (32%) and mice that received pegIFNα for 4 (33%) or 9 weeks (31%) (figure 4A).
      Figure thumbnail gr4
      Figure 4IF staining, genomic/antigenomic HDV RNA and S-/L-HDAg. A) IF staining of HDAg (red) and CK18 (human hepatocytes, aqua) in mouse livers of all groups. Nuclei are stained with Hoechst 33258 (blue). B) Genomic (left) and antigenomic HDV RNA (right) (qPCR assay using biotinylated magnetic beads) in untreated and treated mice infected with HBV/HDV-1a, HBV/HDV-1p and HBV/HDV-3. HBV/HDV-1a infected mice are shown as clear square or black dots when they were treated for 4 (n=1) or 9 weeks (n=2), respectively. C) Western blot analysis of S-HDAg (23 kDa) and L-HDAg (27 kDa) in livers of two untreated and two treated mice (four weeks) infected with either HBV/HDV-1a, HBV/HDV-1p or HBV/HDV-3. The amount of human hepatocytes per liver specimen used to extract proteins was estimated by detecting human albumin (67 kDa).
      RNA in situ hybridization showed a clear reduction of genomic HDV RNA in treated HDV-1p and HDV-3 infected mice, while the amount of genomic HDV RNA positive PHHs remained similar in untreated and treated HDV-1a infected mouse livers (figure S2). Moreover, genomic and antigenomic HDV RNA levels determined by a strain-specific qPCR assay using biotinylated magnetic beads
      • Giersch K.
      • Homs M.
      • Volz T.
      • Helbig M.
      • Allweiss L.
      • Lohse A.W.
      • et al.
      Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.
      revealed that pegIFNα reduced both HDV RNA forms in HDV-1p and HDV-3 infected mice but mice harboring HDV-1a remained unaffected (figure 4B). Accordingly, levels of S- and L-HDAg decreased in HDV-1p and HDV-3 infected mouse livers receiving pegIFNα administration but remained comparable in untreated and treated HDV-1a infected mice (figure 4C).

      IFNα responsiveness of HDV-1a and HDV-1p in vitro using PHHs isolated from infected mice

      To further assess the different responsiveness of HDV-1a and HDV-1p to IFNα in a different experimental setting, PHHs were isolated from humanized mice that were either stable HBV/HDV-1a infected (1.8x108 HBV DNA copies/ml, 1.1x107 HDV RNA copies/ml) or HBV/HDV-1p infected (2.5x109 HBV DNA copies/ml, 1.5x107 HDV RNA copies/ml). Plated cells received IFNα for 2 weeks (figure 5A). In HDV-1p infected PHHs, HDV RNA levels remained stable over time, whereas 7- and 14-day IFNα treatment decreased intracellular HDV RNA levels by 1.0-log (89%) and 1.6-log (98%), respectively, compared to untreated controls (figure 5B). IFNα also lowered HDV RNA levels in cell culture supernatant by 0.7-log (figure 5C), intracellular HBV pgRNA levels by 0.3-log (80%) (figure 5D). In contrast, but in line with the in vivo results, 2-week IFN treatment did not reduce HDV RNA levels in HDV-1a infected PHHs (figure 5B), although intracellular pgRNA was reduced by 0.5-log (65%) (figure 5D). The total amount of cells, determined as ng RNA per well, was not substantially altered over time and by treatment (figure 5E).
      Figure thumbnail gr5
      Figure 5IFNα treatment in HBV/HDV-1a and HBV/HDV-1p infected PHHs isolated from humanized mice. A) Experimental design: PHHs were isolated from a stable HBV/HDV-1a or HBV/HDV-1p infected mouse and treated with IFNα. HDV RNA levels in PHHs (percent change from median controls at day 7) (B) and in cell culture supernatant (IU/ml; BL, day 4 and 7: n= 6; day 11 and 14: n=3 for both groups) (C) of infected PHHs 7 or 14 days post IFNα treatment. Percent change of HBV pgRNA levels (D) and the amount of total RNA per well (E) in treated and untreated HBV/HDV-1a or HBV/HDV-1p infected PHHs. Bars show median levels, black dots represent replicates. Blue bars: treated HDV-1a-infected PHHs, pink bars: treated HDV-1p-infected PHHs, grey bars: untreated PHHs. Curves display median levels and range.

      PegIFNα-mediated human ISG induction in HBV/HDV-1a, HBV/HDV-1p and HBV/HDV-3 infected mice

      Since mice infected with HBV/HDV-1p and HBV/HDV-3 responded to IFNα treatment, whereas the HDV-1a strain resulted resistant, we investigated whether differences in IFN-responsiveness could be explained by the differential ability of these viruses to induce intrinsic innate responses in infected hepatocytes. In line with previous study,
      • Giersch K.
      • Allweiss L.
      • Volz T.
      • Helbig M.
      • Bierwolf J.
      • Lohse A.W.
      • et al.
      Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection.
      human ISGs (e.g. hISG15, hMxA, hOAS1, hSTAT1), pattern recognition receptors (hMDA5), and chemokines (e.g. hCXCL10) were similarly and strongly up-regulated (between 3- and 88-fold) upon HDV infection, regardless of whether the mice were infected with HBV/HDV-1a, HBV/HDV-1p, or HBV/HDV-3 (figure 6A+B, table 2). PegIFNα treatment induced a further enhancement (between 2- and 29-fold) of human ISGs compared to untreated, infected mice (figure 6A+B, table 2). Interestingly, despite the different HDV treatment outcomes, expression levels of analyzed genes appeared comparable among treated animals, suggesting that virological differences, rather than the different enhancement of innate host responses might be responsible for the antiviral effect of IFN in vivo. Furthermore, pegIFNα had no further effect on the expression of human cytokines (hIL28AB, hTGF-ß) and on the apoptosis marker human caspase 8, but led to a substantial decrease (3- to 4-fold) of hNTCP expression levels (figure 6B, table 2). Of note, hIL28AB and hTGF-ß baseline expression levels are close to the detection limit in PHHs, therefore slight expression changes among groups must be interpreted cautiously.
      Figure thumbnail gr6
      Figure 6PegIFNα-mediated hISG induction in HDV-1a, HBV/HDV-1p and HBV/HDV-3 infected mice. A) qPCR measurements of intrahepatic human hISG15, hMxA, hCXCL10 and hMDA5 mRNA levels in pegIFNα-treated or untreated infected mice compared to uninfected mice (n=6). Bars show median level, black dots represent individual mice. HBV/HDV-1a infected mice are shown as clear square or black dots when they were treated for 4 (n=1) or 9 weeks (n=2), respectively. B) The heat map shows mRNA expression levels of different genes in untreated and treated mice of all groups as log induction compared to uninfected controls. Baseline levels are grey, inductions red and reductions blue. C) RNA in situ hybridization (RNAScope) staining of antigenomic HDV RNA (red) and hMxA (aqua) in HBV/HDV-1a infected mice. Nuclei are stained with Dapi (blue).
      Table 2mRNA expression of human genes. Columns show relative expression levels in uninfected, HBV/HDV-1a-, HBV/HDV-1p- or HBV/HDV-3-infected as well as in infected pegIFNα treated mice. Fold induction (fold ind) of infected vs. uninfected mice and treated vs. untreated mice are shown as indicated in the table.
      geneuninfectedHDV-1aHDV-1pHDV-3
      median rel exprmedian rel exprfold ind to uninfmedian rel exprfold ind to uninfmedian rel exprfold ind to uninf
      hISG158,70E-032,00E-0123,02,55E-0129,44,03E-024,6
      hISG205,72E-032,86E-025,03,55E-026,22,39E-024,2
      hOAS17,15E-031,38E-0119,38,48E-0211,95,08E-027,1
      hMxA6,12E-033,04E-0149,71,75E-0128,61,06E-0117,3
      hHLA-E2,02E-023,18E-0115,81,61E-018,01,33E-016,6
      hADAR2,51E-021,48E-015,91,41E-015,61,59E-016,3
      hCXCL102,54E-032,23E-0188,01,14E-0145,02,10E-0182,9
      hTGF-β6,34E-047,64E-041,23,91E-036,28,81E-041,4
      hIL28AB2,55E-051,72E-0367,53,93E-03154,31,17E-044,6
      hSTAT11,53E-021,38E-019,01,46E-019,51,40E-019,2
      hMDA52,35E-031,14E-024,81,98E-028,49,71E-034,1
      hCasp81,18E-031,72E-031,54,87E-034,12,26E-031,9
      hNTCP3,12E-013,61E-011,21,54E-010,51,46E-010,5
      geneHDV-1a + pegIFNαHDV-1p + pegIFNαHDV-3 + pegIFNα
      median rel exprfold ind to HDV1ccmedian rel exprfold ind to HDV1pmedian rel exprfold ind to HDV3
      hISG151,50E+007,51,11E+004,31,15E+0028,5
      hISG201,44E-015,01,28E-013,61,52E-016,4
      hOAS15,58E-014,02,87E-013,45,04E-019,9
      hMxA1,20E+003,95,20E-013,07,80E-017,4
      hHLA-E1,41E+004,43,30E-012,01,41E+0010,6
      hADAR3,06E-012,11,62E-011,22,83E-011,8
      hCXCL101,25E+005,66,77E-015,96,50E-013,1
      hTGF-β2,98E-040,43,81E-031,01,55E-031,8
      hIL28AB3,41E-040,21,38E-030,47,54E-046,5
      hSTAT15,86E-014,26,04E-014,15,57E-014,0
      hMDA52,60E-022,33,58E-021,82,15E-022,2
      hCasp83,77E-032,24,01E-030,82,77E-031,2
      hNTCP9,89E-020,38,11E-020,55,46E-020,4
      Remarkably, simultaneous visualization of HDV RNA and human ISGs by RNA ISH revealed that MxA-positive human hepatocytes are still expressing high levels of antigenomic HDV-1a RNA. These results demonstrate that the IFN-resistant HDV-1a isolate does not hamper the IFN-mediated induction of classical human ISGs at the single cell level (figure 6C).

      Sequencing of the three distinct HDV genome strains from infected mice

      Genome sequencing of intrahepatic HDV RNA in mice infected with HDV-1a, HDV-1p, or HDV-3 revealed that no mutations emerged after 4 or 9 weeks of pegIFNα treatment in vivo (data not shown). In addition, the occurrence of genomes encoding for the small (ACC) or large HDAg (ATC) (RNA editing at the amber/W site) remained similar in treated and untreated mice and was comparable between the different HDV isolates (figure S3A). The ribozyme site showed 100% identity between the two HDV-1 isolates and 89.6% identity between HDV-1 and HDV-3 (figure S3B). The open reading frame for the large HDAg (214 amino acids) showed 89.5% identity between HDV-1p and HDV-1a and 71.2% identity between HDV-1p and HDV-3, which translated into several amino acids changes (figure S3B, C). However, the amber/W site (aa196), the prenylation site (aa212) and other known posttranslational modification sites remained fully conserved between the three different isolates (figure S3C). In line with Le Gal et al., the two HDV-1 strains showed a proline residue at the nuclear export site at position 205, whereas HDV-3 harbors a glycine, suggesting different virion secretion efficiencies across genotypes.
      • Le Gal F.
      • Brichler S.
      • Drugan T.
      • Alloui C.
      • Roulot D.
      • Pawlotsky J.-M.
      • et al.
      Genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains.
      Two unique differences that exclusively occurred in the IFN-resistant HDV-1a-isolate were detected in the coiled coil domain at position aa41 (leucine instead of isoleucine) and aa44 (isoleucine instead of leucine) (figure S3C). Although these are conservative mutations, we cannot exclude their impact on SUMOylation rates at position 42.

      Discussion

      In CHD, PegIFNα is commonly used as an off-label treatment, although its mode of action in HDV-infected hepatocytes is still unclear and responsiveness to IFN in patients remains limited.
      • Urban S.
      • Neumann-Haefelin C.
      • Lampertico P.
      Hepatitis D virus in 2021: virology, immunology and new treatment approaches for a difficult-to-treat disease.
      Understanding HDV diversity and the mechanisms determining different antiviral responsiveness to treatment is paramount for improving therapeutic options. Experimental studies with the aim to unravel IFN MoA in HDV infection were hampered not only by the paucity of infection models available, but also by the limited availability of well-characterized HDV isolates. By chance, the first HDV-1 clone that was generated in 1988 (
      • Kuo M.Y.
      • Goldberg J.
      • Coates L.
      • Mason W.
      • Gerin J.
      • Taylor J.
      Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications.
      , herein HDV-1a), appeared IFN-resistant
      • Kuo M.Y.
      • Chao M.
      • Taylor J.
      Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen.
      ,
      • Kuo M.Y.
      • Goldberg J.
      • Coates L.
      • Mason W.
      • Gerin J.
      • Taylor J.
      Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications.
      both in transfected hepatoma cells and infected PHHs.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sültmann H.
      • Mutz P.
      • Hiet M.-S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes.
      ,
      • Han Z.
      • Nogusa S.
      • Nicolas E.
      • Balachandran S.
      • Taylor J.
      Interferon impedes an early step of hepatitis delta virus infection.
      ,
      • Ilan Y.
      • Klein A.
      • Taylor J.
      • Tur-Kaspa R.
      Resistance of hepatitis delta virus replication to interferon-alpha treatment in transfected human cells.
      HDV was reduced only when IFNα was administered around HDV infection time,
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sültmann H.
      • Mutz P.
      • Hiet M.-S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes.
      ,
      • Han Z.
      • Nogusa S.
      • Nicolas E.
      • Balachandran S.
      • Taylor J.
      Interferon impedes an early step of hepatitis delta virus infection.
      indicating that IFNα mainly limited new infection events. These in vitro studies led to the general assumption that HDV is resistant to IFN treatment when infection is already established. However, we observed that human liver chimeric mice infected with an HDV-positive patient-derived serum responded to pegIFNα.
      • Giersch K.
      • Homs M.
      • Volz T.
      • Helbig M.
      • Allweiss L.
      • Lohse A.W.
      • et al.
      Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.
      Interestingly, this HDV isolate (HDV-1p) was obtained from a patient who later achieved sustained HDV response upon pegIFNα treatment (figure 1 and
      • Bockmann J.-H.
      • Grube M.
      • Hamed V.
      • von Felden J.
      • Landahl J.
      • Wehmeyer M.
      • et al.
      High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.
      ). To assess the IFN responsiveness of the most commonly used HDV-1a clone in vivo, in HBV/HDV infected human hepatocytes, and to compare these data with distinct HDV strains, we cloned this new HDV-1p isolate and also employed an additional, genetically distant clone from an HDV-3 strain.
      • Casey J.L.
      • Brown T.L.
      • Colan E.J.
      • Wignall F.S.
      • Gerin J.L.
      A genotype of hepatitis D virus that occurs in northern South America.
      In stable HBV/HDV-1p- and HBV/HDV-3-infected humanized mice, pegIFNα treatment clearly reduced HDV markers in serum and liver, including the levels of genomic and antigenomic HDV RNA, as well as S- and L-HDAg. In striking contrast, we did not detect any antiviral effect on HDV parameters when stable HBV/HDV-1a infected mice received pegIFNα, although all HBV markers, including HBsAg, were clearly reduced. Of note, the IFN dose commonly applied for studies in humanized mice is rather high (adapted to mouse metabolism) and, most importantly, it is given twice per week. Nevertheless, even extending treatment to 9 weeks did not alter HDV RNA levels or the number of HDV-positive human hepatocytes (as determined by IF and RNA ISH) when the HDV-1a strain was used. Notably, HBsAg is needed for the release of HDV particles and the IFN-mediated reduction of HBV serological markers, particularly those linked to cccDNA activity, was shown to be associated with virological responses in CHD patients.
      • Sandmann L.
      • Yurdaydin C.
      • Deterding K.
      • Heidrich B.
      • Hardtke S.
      • Lehmann P.
      • et al.
      HBcrAg levels are associated with virological response to treatment with interferon in patients with hepatitis delta.
      Accordingly, IFN-mediated suppression of HBsAg production should also lower HDV RNA in serum. However, HBV infection and HBsAg levels are generally high in humanized mice and despite the decrease induced by IFN, HBsAg changes were not sufficient to induce substantial decrease of HDV-1a in IFN-treated mice. Our results indicate that also HDV viremia changes need to be monitored to predict virological responses during IFN treatment.
      Consistent with previous in vitro studies,
      • Kuo M.Y.
      • Chao M.
      • Taylor J.
      Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen.
      ,
      • Kuo M.Y.
      • Goldberg J.
      • Coates L.
      • Mason W.
      • Gerin J.
      • Taylor J.
      Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications.
      IFNα treatment of PHHs obtained from a stable HBV/HDV-1a-infected mouse had no effect on HDV RNA amounts. However, IFNα led to the reduction of HDV RNA levels in PHHs isolated from a stable HBV/HDV-1p-infected mouse. Being the virus strain the only variable in these experimental settings (we used the same human hepatocyte donor and HBV inoculum), the sharp differences determined by using different HDV isolates demonstrate for the first time the existence of virus-derived differences. The key IFN-mediated antiviral mechanism acting in stably infected PHHs needs however further investigations and the new genotype 1 HDV isolate could also serve for studies aiming at investigating HDV biology, as well as the antiviral activity of new compounds used alone and in combination with IFN.
      Since we did not measure induction of caspase 8 or detected substantial PHH loss in vitro and in vivo during treatment, the death of HDV-positive cells unlikely provides the main mechanism of intrahepatic HDV decline. Although we cannot rule out an increase of cellular stress induced both by HDV infection and interferon treatment, the strong HDV decrease determined in mice infected with HDV-1p or HDV-3 rather suggests that IFN inhibited HDV RNA synthesis and/or promoted its destabilization.
      The intrinsic innate responses of the hepatocytes are thought to be central to counteract HDV infection.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sültmann H.
      • Mutz P.
      • Hiet M.-S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes.
      ,
      • Dandri M.
      • Bertoletti A.
      • Lütgehetmann M.
      Innate immunity in hepatitis B and D virus infection: consequences for viral persistence, inflammation, and T cell recognition.
      To investigate whether these three HDV strains may alter the ability of the human hepatocytes to sense the infection and to respond to IFN administration, we comparatively analyzed classical human ISGs in infected and treated humanized mice.
      • Giersch K.
      • Allweiss L.
      • Volz T.
      • Helbig M.
      • Bierwolf J.
      • Lohse A.W.
      • et al.
      Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection.
      Both the two HDV-1 isolates and the HDV-3 strain similarly enhanced human ISGs, including chemokines and pattern recognition receptors, compared to uninfected control mice. PegIFNα treatment increased the levels of human IFN signaling genes even further and - interestingly - in a comparable manner between all three HDV isolates used, irrespective of their responsiveness to treatment. Even at the single cell level, HDV-1a-RNA-positive PHHs strongly co-expressed hMxA mRNA, suggesting that this IFN-resistant HDV-1a isolate does not hamper the broad IFN-mediated up-regulation of intrinsic innate responses of human hepatocytes in vivo. Nevertheless, since we analyzed a limited number of genes, we cannot exclude the possibility that other genes may be differentially expressed. Based on these results, it appears that the hepatocyte innate responses cannot be solely responsible for the strikingly different IFN responsiveness of distinct HDV isolates determined in stable HDV-infected cells, again hinting at the existence of virus-specific factors affecting the strength of IFN responsiveness. We did not identify clear mutations in the ribozyme and HDAg-coding region on the HDV genome or at the RNA editing site upon IFN treatment. Interestingly, all known posttranslational modification sites in the HDAg open reading frame, including the prenylation site at position 212 and the nuclear localization signal (aa-66-75) remained fully conserved among these three isolates. The two only differences that exclusively occurred in the IFN-resistant HDV-1a-isolate were detected in the coiled coil domain (leucine instead of isoleucine and vice versa) of the HDAg close to a SUMOylation site. However, leucine and isoleucine are both aliphatic, branched hydrophobic amino acids and these mutations may not influence SUMOylation rates at position 42. It remains to be investigated whether more complex sequence and conformational changes residing outside of the more conserved coding regions of HDV account for the intrinsic primary resistance to IFN determined with HDV-1a.
      Recently, Zhang et al. treated proliferating HDV-1a-infected hepatoma cells with IFNα and observed a strong block of cell division-mediated HDV spread, suggesting that HDV RNA molecules can be targeted by intrinsic innate responses preferentially during cell division, a mechanism permitting NTCP-independent HDV spreading among daughter cells.
      • Giersch K.
      • Bhadra O.D.
      • Volz T.
      • Allweiss L.
      • Riecken K.
      • Fehse B.
      • et al.
      Hepatitis delta virus persists during liver regeneration and is amplified through cell division both in vitro and in vivo.
      Experiments in human liver chimeric mice are usually started when human hepatocyte engraftment is completed and cell turnover is low.
      • Allweiss L.
      • Volz T.
      • Giersch K.
      • Kah J.
      • Raffa G.
      • Petersen J.
      • et al.
      Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.
      ,
      • Giersch K.
      • Bhadra O.D.
      • Volz T.
      • Allweiss L.
      • Riecken K.
      • Fehse B.
      • et al.
      Hepatitis delta virus persists during liver regeneration and is amplified through cell division both in vitro and in vivo.
      Herein, we treated HBV/HDV infected mice with IFN several weeks after PHH repopulation was accomplished and rates of PHH proliferation did not differ among mice infected with the distinct HDV isolates (Ki67-staining, data not shown). Although additional studies are required to explore the impact of IFN on distinct HDV strains during hepatocyte proliferation, cell turnover appears unlikely to explain the different IFN-responses determined in the experimental setting used here. The ability of IFN to limit not only HDV infection events but also to lower intrahepatic viral loads even in resting hepatocytes, would provide a rationale for the stronger anti-HDV effects determined in CHD patients receiving pegIFNα and the entry inhibitor Bulevirtide in combination (MYR-203 clinical trial, NCT02637999).
      • Wedemeyer H.
      • Schöneweis K.
      • Bogomolov P.O.
      • Chulanov V.
      • Stepanova T.
      • Viacheslav M.
      • et al.
      48 weeks of high dose (10 mg) bulevirtide as monotherapy or with peginterferon alfa-2a in patients with chronic HBV/HDV co-infection.
      In conclusion, our study showed that two new patient-derived HDV isolates of genotype 1 and 3 respond to IFNα treatment in immune deficient human liver chimeric mice. We also provide evidence that the commonly used HDV-1a isolate bears intrinsic capacities to resist to IFNα treatment in vivo, which were not determined in two other patient-derived isolates. The existence of virus-specific determinants of IFNα responsiveness raises awareness of the need to employ different HDV strains to evaluate virological and host-mediated mechanisms of IFN-responsiveness in HDV infection. The availability of new IFN-sensitive HDV strains could also contribute to the development of new therapies aiming at HDV cure.

      Data availability statement

      All data, code, and materials used in the analysis are available upon reasonable request for collaborative studies regulated by materials/data transfer agreements (MTA/DTAs) to the corresponding author.

      Acknowledgements

      We are grateful to Roswitha Reusch and Sabrina Noster for excellent assistance with the mouse colony and Corinna Eggers and Martina Fahl for their great technical help. We thank John Taylor (Philadelphia, PA, USA) and John Casey (Washington, D.C., USA) for fruitful discussions and for providing the HDV recombinant plasmid pSVL(D3) for HDV-1a, and pCMV3-Peru-1.2 for HDV-3, respectively. We also thank Camille Sureau (INTS, Paris, France) for providing infectious HDV-1a and HDV-3 particles and Jiabin Huang for his help in uploading the HDV-1p sequence to NCBI.

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

      Abbreviations

      Actb
      actin beta
      AG
      antigenomic
      AJ
      AJ000558
      BSA
      bovine serum albumin
      CHD
      chronic hepatitis D
      CK18
      cytokeratin 18
      CXCL10
      C-X-C motif chemokine ligand 10
      DMSO
      dimethyl sulfoxide
      Eef2
      Eukaryotic elongation factor
      FCS
      fetal calf serum
      G
      genomic
      GAPDH
      glyceraldehyde-3 phosphate dehydrogenase
      hAAT
      Human alpha antitrypsin
      HBsAg
      hepatitis B virus surface antigen
      HBV
      hepatitis B virus
      HDAg
      hepatitis delta antigen (S, small, L, large)
      HCV
      hepatitis C virus
      HDV
      hepatitis delta virus
      HLA
      human leukocyte antigen
      HSA
      human serum albumin
      IL
      interleukine
      ISGs
      interferon stimulated genes
      IU
      international units
      LLoD
      lower limit of detection
      Mavs
      mitochondrial antiviral-signaling protein
      MDA5
      melanoma differentiation-associated protein 5
      MOI
      multiplicity of infection
      MxA
      myxovirus resistance gene A
      NK
      natural killer
      NTCP
      sodium (Na+) taurocholate cotransporting polypeptide
      NUCs
      nucleos(t)ide analogues
      OAS1
      2′-5′-oligoadenylatsynthetase 1
      p
      patient-derived
      PEG
      polyethylene glycol
      pegIFNα
      pegylated interferon alpha
      PFA
      paraformaldehyde
      pgRNA
      pregenomic RNA
      Rig-I
      retinoic acid-inducible gene I
      RNP
      ribonucleoprotein
      qPCR
      quantitative real time polymerase chain reaction
      SCID
      severe combined immunodeficiency
      STAT1
      signal transducers and activators of transcription 1
      uPA
      urokinase plasminogen activator
      USG
      uPA/SCID/ beige/ IL2RG-/-

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