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New insights into HDV persistence: The role of interferon response and implications for upcoming novel therapies

  • Zhenfeng Zhang
    Affiliations
    Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
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  • Stephan Urban
    Correspondence
    Corresponding author. Address: Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany. Tel.: +49-6221-564902.
    Affiliations
    Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany

    German Center for Infection Research (DZIF) - Heidelberg Partner Site, Heidelberg, Germany
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Open AccessPublished:November 30, 2020DOI:https://doi.org/10.1016/j.jhep.2020.11.032

      Summary

      Chronic hepatitis D (CHD), a global health problem, manifests as the most severe form of viral hepatitis. The causative agent, HDV, is the smallest known human virus; it replicates its circular single-stranded RNA genome in the nucleus of hepatocytes. HDV requires HBV-encoded envelope proteins for dissemination and de novo cell entry. However, HDV can also spread through cell division. Following entry into hepatocytes, replicative intermediates of HDV RNA are sensed by the pattern recognition receptor MDA5 (melanoma differentiation antigen 5) resulting in interferon (IFN)-β/λ induction. This IFN response strongly suppresses cell division-mediated spread of HDV genomes, however, it only marginally affects HDV RNA replication in already infected, resting hepatocytes. Monotherapy with IFN-α/λ shows efficacy but rarely results in HDV clearance. Recent molecular insights into key determinants of HDV persistence and the accelerated development of specifically acting antivirals that interfere with the replication cycle have revealed promising new therapeutic perspectives. In this review, we briefly summarise our knowledge on replication/persistence of HDV, the newly discovered HDV-like agents, and the interplay of HDV with the IFN response and its consequences for persistence. Finally, we discuss the possible role of IFNs in combination with upcoming therapies aimed at HDV cure.

      Keywords

      Introduction

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      In this review, we briefly summarise current knowledge on HDV replication and newly described HDV-like agents, and expand on the novel discoveries relating to cell division-mediated HDV spread and the interplay between HDV and the IFN response. We also briefly review recent basic and clinical data on novel antiviral drugs and discuss the implications of these data for the development of potentially curative therapies.

      HDV replication, persistence, and pathogenesis

      HDV replication cycle

      The HDV virion comprises a ribonucleoprotein (RNP) core complex and an HBV-derived envelope. The genome is a circular, single-stranded, backfolded, negative sense GC-rich RNA. Due to its high degree (~74%) of intramolecular base pairing, the HDV genome folds into a viroid-like rod structure
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      which associates with 2 forms of the solitary HDV encoded protein, small HDAg (S-HDAg) and large HDAg (L-HDAg), to form the HDV RNP complex. The 3 membrane-embedded HBV envelope proteins small (S-), middle (M-) and large (L-), collectively termed HBsAg, are encoded by the covalently closed circular DNA (cccDNA) in HBV-replicating hepatocytes and the integrated HBV DNA in chromosomes.
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      on the basolateral (sinusoidal) membrane of hepatocytes. This step is mandatory to initiate the highly specific interaction of the preS1 domain of the HBV L-HBsAg with the hepatocyte-specific receptor sodium taurocholate co-transporting polypeptide (NTCP).
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      After membrane fusion by an unknown mechanism, the HDV RNP is transported to the nuclear pore complex and released into the nucleoplasm. Replication proceeds via a double rolling circle amplification mechanism, generating linear multimeric antigenomic and genomic RNAs which are cleaved to monomers by 2 intrinsic ribozymes. Monomers are self-ligated to (+) and (−) single-stranded RNA circles. Genomic RNA is the template for the mRNAs encoding the 2 forms of HDAg. During replication, antigenomic RNA is edited by host adenosine deaminase acting on RNA 1 (ADAR1), introducing an A→G mutation in the amber stop codon of the S-HDAg open reading frame (ORF). This introduces a Trp codon. Accordingly, a second mRNA is produced coding for the elongated L-HDAg with a C-terminal ORF extension of 19 or 20 (genotype dependent) amino acids. L-HDAg becomes prenylated by the cellular farnesyl transferase at a conserved C-terminal Cys residue within the extension. Progeny HDV RNAs assemble to RNPs containing S-HDAg, as well as prenylated and non-prenylated L-HDAg.
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      HDV depends on HBV envelope proteins for dissemination via the receptor NTCP but can also maintain its genome in the liver through cell division.
      Figure thumbnail gr1
      Fig. 1HDV replication cycle.
      HDV firstly binds HSPGs and then the viral receptor NTCP. The viral membrane fuses either with the plasma membrane or undergoes an endocytosis-mediated fusion process to release the RNP complex which is transported into the nucleus to initiate RNA replication. The genomic RNA with negative polarity serves as the template for the first RCA step. The resulting AG multimer is cleaved in cis by intrinsic ribozyme activity and ligated into the circular viral AG. After a second RCA using AG as the template. The G multimer is produced and further cleaved and ligated into the circular G. A portion of AG is edited by ADAR1 resulting an extended HDAg ORF. These G (with or without ADAR1 edition) are used as the template for mRNA transcription which are translated into S-HDAg and L-HDAg. Some L-HDAg molecules are prenylated by farnesyltransferase which is needed for envelope acquirement. S-HDAg and L-HDAg are transported into the nucleus to regulate viral replication or bind to circular G to form RNPs which are exported to the cytosol. L-HDAg is responsible for RNP's interaction with the HBV envelope proteins which are expressed by cccDNA or integrated HBV DNA. HDV particles are presumably released through the classic secretory pathway. ADAR1, adenosine deaminase acting on RNA 1; AG, antigenome; cccDNA, covalently closed circular DNA; G, genome; L/S-HDAg, large/small-hepatitis delta antigen; (L/S/M)-HBsAg, (large/small/middle)-HBV surface antigen; HSPGs, heparan sulphate proteoglycans; NTCP, human sodium taurocholate co-transporting polypeptide; ORF, open-reading frame; RCA, rolling circle amplification; RNP, ribonucleoprotein.

      HDV persistence, dissemination and spread

      Since HDV replicates via episomal RNAs, persistence probably requires continuous de novo RNA synthesis (although we presently cannot exclude the presence of inactive HDV RNAs serving as a reservoir for reactivation). Accordingly, 2 modes of HDV RNA maintenance in the liver can be envisaged (Fig. 2). Firstly, the extracellular mode of spreading involves HBV envelope protein-mediated secretion of viral progeny followed by continuous de novo infection of NTCP-expressing naïve or already infected hepatocytes (Fig. 2A). This extracellular spreading pathway can be blocked by entry inhibitors like Hepcludex®/bulevirtide (formerly Myrcludex B)
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      Figure thumbnail gr2
      Fig. 2HDV spreading pathways and the targets of antivirals.
      (A) De novo infection-mediated extracellular spreading pathway. HBV/HDV coinfection of naive hepatocytes produces progeny HD virions; HDV infection of hepatocytes carrying integrated HDV DNA also produces progeny HD virions. These progeny HD virions (middle) infect neighbouring intact hepatocytes (right) or super-infect HBV-positive hepatocytes (left). Bulevirtide and lonafarnib efficiently block the extracellular spreading pathway as an entry inhibitor and a secretion inhibitor, respectively. IFN response inhibits early steps of HDV de novo infection. (B) Cell division-mediated HDV spread. HDV survives cell division and efficiently establishes replication in both daughter cells. IFN response efficiently suppresses cell division-mediated HDV spread and RNA amplification. HSPGs, heparan sulphate proteoglycans; IFN, interferon; NTCP, human sodium taurocholate co-transporting polypeptide.

      HDV-induced liver damage

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      • Accapezzato D.
      • Bonino F.
      • Rosina F.
      • Santantonio T.
      • et al.
      Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles.
      others have demonstrated only weak or undetectable HDV-specific T cell responses.
      • Schirdewahn T.
      • Grabowski J.
      • Owusu Sekyere S.
      • Bremer B.
      • Wranke A.
      • Lunemann S.
      • et al.
      The third signal cytokine interleukin 12 rather than immune checkpoint inhibitors contributes to the functional restoration of hepatitis D virus-specific T cells.
      ,
      • Landahl J.
      • Bockmann J.H.
      • Scheurich C.
      • Ackermann C.
      • Matzat V.
      • Heide J.
      • et al.
      Detection of a broad range of low-level major histocompatibility complex class II-restricted, hepatitis delta virus (HDV)-specific T-cell responses regardless of clinical status.
      HDV but not HBV replication activates innate immune responses in hepatocytes, which may lead to direct killing, e.g. by natural killer (NK) cells, or indirectly by activating inflammation and T cell responses. Concise overviews of the pathogenesis of CHD are provided in.
      • Koh C.
      • Heller T.
      • Glenn J.S.
      Pathogenesis of and new therapies for hepatitis D.
      ,
      • Sureau C.
      • Negro F.
      The hepatitis delta virus: replication and pathogenesis.
      ,
      • Botelho-Souza L.F.
      • Vasconcelos M.P.A.
      • Dos Santos A.O.
      • Salcedo J.M.V.
      • Vieira D.S.
      Hepatitis delta: virological and clinical aspects.

      Novel HDV-like agents

      HDV was assumed to be a fully helper-dependent virus because it uses the HBV envelope proteins for dissemination. This assumption has been shaken by the recent discoveries of HDV-like agents in rodents,
      • Paraskevopoulou S.
      • Pirzer F.
      • Goldmann N.
      • Schmid J.
      • Corman V.M.
      • Gottula L.T.
      • et al.
      Mammalian deltavirus without hepadnavirus coinfection in the neotropical rodent Proechimys semispinosus.
      snakes,
      • Hetzel U.
      • Szirovicza L.
      • Smura T.
      • Prahauser B.
      • Vapalahti O.
      • Kipar A.
      • et al.
      Identification of a novel deltavirus in Boa Constrictors.
      birds,
      • Wille M.
      • Netter H.J.
      • Littlejohn M.
      • Yuen L.
      • Shi M.
      • Eden J.S.
      • et al.
      A divergent hepatitis D-like agent in birds.
      fish, amphibians, and even invertebrates,
      • Chang W.S.
      • Pettersson J.H.
      • Le Lay C.
      • Shi M.
      • Lo N.
      • Wille M.
      • et al.
      Novel hepatitis D-like agents in vertebrates and invertebrates.
      especially since these agents lack the association with a respective animal hepadnavirus. Among HDV-like agents identified to date, only the ones from rodents and snakes may encode a large delta antigen (L-DAg) according to bioinformatics analysis. However, within the putative L-DAg a prenylation site is lacking, indicating that adaptation to a hepadnavirus-derived envelope association is not mandatory for dissemination. This also implies that adaptation of HDV to HBV envelope proteins was rather an occasional and recent evolutionary event that accelerated the spread of HDV in humans. Vice versa the discovery of this whole family of viroid-like agents in the animal kingdom supports the hypothesis that spreading through mitosis, as described before for HDV, is a general mechanism and helper virus adaptation is an alternative.
      Novel HD-like agents have been found in several other species and are not associated with respective hepadnaviruses.
      An intriguing novel finding relates to the observation that HDV RNPs can be artificially packaged in vitro into the envelopes of vesiculo-, flavi- and hepaciviruses, allowing egress of HDV RNPs from accordingly infected cells and subsequent entry into cell lines expressing their respective receptors.
      • Perez-Vargas J.
      • Amirache F.
      • Boson B.
      • Mialon C.
      • Freitas N.
      • Sureau C.
      • et al.
      Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo.
      This raises the possibility that HDV could utilise alternative envelopes. In a recent study, HDV RNA was reported to be positive in 1 of 160 HCV patients without detectable HBV markers.
      • Chemin I.
      • Pujol F.H.
      • Scholtes C.
      • Loureiro C.L.
      • Amirache F.
      • Levrero M.
      • et al.
      Preliminary evidence for hdv exposure in apparently non-HBV-infected patients.
      However, 2 other studies did not observe HBV-independent HCV/HDV coinfection in more than 2,000 individuals with HCV.
      • Cappy P.
      • Lucas Q.
      • Kankarafou N.
      • Sureau C.
      • Laperche S.
      No evidence of HCV-assisted HDV propagation in a large cohort of hepatitis C positive blood donors.
      ,
      • Pfluger L.S.
      • Schulze Zur Wiesch J.
      • Polywka S.
      • Lutgehetmann M.
      Hepatitis delta virus propagation enabled by hepatitis C virus-scientifically intriguing, but is it relevant to clinical practice?.
      Larger studies on this subject are needed in regions with a high prevalence of chronic HBV/HDV and HCV, and regions where parental infection routes have been studied and are known. Similarly, more investigations are needed to clarify whether HDV-like agents spread with the help of viruses other than hepadnaviruses in respective infected hosts. Lastly, it cannot be excluded that RNPs may even disseminate without a helper virus, like viroids in plants, once physical transmission into host cells has occurred.

      Novel insights into the interplay between HDV and the IFN response

      IFN signalling during RNA virus infection

      The cellular innate immune system plays a crucial role in the suppression of invading viruses through initiation of direct antiviral responses
      • Medzhitov R.
      • Janeway Jr., C.
      Innate immunity.
      and induction of the adaptive immune system.
      • Jain A.
      • Pasare C.
      Innate control of adaptive immunity: beyond the three-signal paradigm.
      It thereby determines if clearance or persistence occurs, with the latter often associated with chronic inflammation.
      • Wang L.
      • Wang K.
      • Zou Z.Q.
      Crosstalk between innate and adaptive immunity in hepatitis B virus infection.
      Innate immune responses are initiated following the recognition of invading pathogens by pathogen-associated molecular patterns (PAMPs). For RNA viruses, the incoming viral genomes (e.g. Influenza virus
      • Weber M.
      • Gawanbacht A.
      • Habjan M.
      • Rang A.
      • Borner C.
      • Schmidt A.M.
      • et al.
      Incoming RNA virus nucleocapsids containing a 5'-triphosphorylated genome activate RIG-I and antiviral signaling.
      ) or replication intermediates thereof (e.g. double-stranded RNA) are sensed by specific PRRs, i.e. toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). RLRs include RIG-I, melanoma differentiation antigen 5 (MDA5) and laboratory of physiology and genetics 2 (LGP2), which share similar domains but recognise different RNA ligands. Sensing of PAMPs triggers a cascade of signalling events that lead to the production of type I (IFN-α/β) and type III IFNs (IFN-λ). After secretion, these IFNs amplify the signal by binding to their cognate receptors on the membrane of infected and non-infected neighbouring cells, thus activating Janus kinases 1/2 (JAK1/2), tyrosine kinase 2 (TYK2), signal transducer and activator of transcription 1/2 (STAT1/2), interferon regulatory factor 9 (IRF9) and consequently inducing hundreds of ISGs which either directly interfere with viral replication or indirectly affect the spread of viruses by lowering permissiveness of host cells and activating adaptive immune responses. More details on innate immune responses during viral replication are provided in.
      • Rehwinkel J.
      • Gack M.U.
      RIG-I-like receptors: their regulation and roles in RNA sensing.
      ,
      • Schneider W.M.
      • Chevillotte M.D.
      • Rice C.M.
      Interferon-stimulated genes: a complex web of host defenses.

      HDV-induced innate immune responses

      In contrast to HBV, which is considered a “stealth” virus,
      • 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.
      ,
      • Mutz P.
      • Metz P.
      • Lempp F.A.
      • Bender S.
      • Qu B.
      • Schoneweis K.
      • et al.
      HBV bypasses the innate immune response and does not protect HCV from antiviral activity of interferon.
      • Wieland S.
      • Thimme R.
      • Purcell R.H.
      • Chisari F.V.
      Genomic analysis of the host response to hepatitis B virus infection.
      • Suslov A.
      • Boldanova T.
      • Wang X.
      • Wieland S.
      • Heim M.H.
      Hepatitis B virus does not interfere with innate immune responses in the human liver.
      HDV replication induces innate immune responses in in vitro infection models (PHH, NTCP-expressing HepG2 and HepaRG cells, etc.),
      • Alfaiate D.
      • Lucifora J.
      • Abeywickrama-Samarakoon N.
      • Michelet M.
      • Testoni B.
      • Cortay J.C.
      • et al.
      HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes.
      ,
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      as well as in uPA/SCID/beige mice repopulated with human hepatocytes,
      • 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.
      NTCP transgenic mice,
      • He W.
      • Ren B.
      • Mao F.
      • Jing Z.
      • Li Y.
      • Liu Y.
      • et al.
      Hepatitis D virus infection of mice expressing human sodium taurocholate co-transporting polypeptide.
      ,
      • Winer B.Y.
      • Shirvani-Dastgerdi E.
      • Bram Y.
      • Sellau J.
      • Low B.E.
      • Johnson H.
      • et al.
      Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection.
      and a AAV vector-mediated HDV replication mouse model.
      • Suarez-Amaran L.
      • Usai C.
      • Di Scala M.
      • Godoy C.
      • Ni Y.
      • Hommel M.
      • et al.
      A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-beta induction.
      HDV infection induces IFN-β/λ but not IFN-α.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      Notably HDV is not a strong IFN stimulator when compared to other RNA viruses like, Sendai- or Mengo-Zn virus
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      or synthetic ligands like poly (I:C).
      • Winer B.Y.
      • Gaska J.M.
      • Lipkowitz G.
      • Bram Y.
      • Parekh A.
      • Parsons L.
      • et al.
      Analysis of host responses to hepatitis B and delta viral infections in a micro-scalable hepatic co-culture system.
      In addition, the cellular responses investigated so far were based on acute HDV infection applying relatively high multiplicities of genome equivalents. Whether this represents the situation in livers of chronically infected patients requires further investigation. Moreover, their IFN responses may vary significantly due to the profound difference in viral loads. Accordingly, individual treatment responses under IFN therapy may depend on the level of the HDV-induced endogenous IFN response.
      HDV RNA replication is sensed by the pattern recognition receptor MDA5, which activates IFN-β/λ. The IFN response suppresses cell division-mediated intrahepatic HDV spread with only a marginal effect on HDV replication in resting cells.

      Innate immune sensing of HDV replication

      An overview of the HDV-induced IFN response is depicted in Fig. 3, with a focus on key players in RNA recognition and their associated signalling pathways. Active HDV RNA replication – leading to the production of genomic, antigenomic and mRNA – is required to induce innate immune responses in hepatocytes.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      By knocking down RIG-I, MDA5 and TLR3 in HepG2-NTCP and HepaRG-NTCP cells, it was demonstrated that the MDA5 pathway, but not TLR3 or RIG-I, is essential for initiating IFN-β/λ-mediated signalling.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      This finding is in line with the observation that mitochondrial antiviral signalling protein (MAVS), a key downstream adaptor of MDA5, plays an essential role in innate immune activation in AAV-HDV transduced mice.
      • Suarez-Amaran L.
      • Usai C.
      • Di Scala M.
      • Godoy C.
      • Ni Y.
      • Hommel M.
      • et al.
      A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-beta induction.
      Figure thumbnail gr3
      Fig. 3HDV-induced IFN production and signalling.
      HDV RNA is recognised by the PRR MDA5 probably in the cytoplasm. The recognition triggers the activation of MAVS on mitochondria and downstream signalling molecules and transcription factors like IRF3/7 and NF-κB. Activated transcription factors are translocated into the nucleus and initiate the transcription of IFN-β/λ. IFN-β/λ are secreted and bind to their receptors (IFNAR1/IFNAR2 for IFN-β and IFNLR1/IL10R2 for IFN-λ) on the infected cell or neighbouring cells, which further activates JAK1/TYK2 and transcription factors STAT1/2 and IRF9. STAT1/2 and IRF9 are translocated into the nucleus and activate ISGs which inhibit intracellular RNA replication (weak effect) or prevent new infections (strong effect). AG, antigenome; G, genome; HSPG, heparan sulphate proteoglycans; IFN, interferon; IFNAR, interferon alpha receptor; IFNLR, interferon lambda receptor; IRF9, interferon regulatory factor 9; ISG, IFN-stimulated genes; JAK, Janus kinase; LGP2, laboratory of physiology and genetics 2; MAVS, mitochondrial antiviral signalling protein; MDA5, melanoma differentiation antigen 5; NTCP, human sodium taurocholate co-transporting polypeptide; PRR, pattern recognition receptor; RCA, rolling circle amplification; RIG-I, retinoic acid-inducible gene I; RNP, ribonucleoprotein; TLR, Toll-like receptor; TYK2, tyrosine kinase 2.
      The discovery of MDA5 as a key sensor of HDV replication raised a couple of questions regarding how HDV replication is recognised. For instance, which type of HDV RNA is sensed by MDA5 and where within the cell does recognition occur? MDA5 preferentially (if not exclusively) locates in the cytoplasm, while HDV RNA replicates in the nucleus, and the RNA intermediates are confined to this compartment. The progeny HDV genomic RNP complexes are delivered to the cytoplasm where they might be sensed by MDA5. A recent study demonstrated that a minor fraction of RIG-I locates in the nucleus and captures Influenza A virus RNP complexes.
      • Liu G.
      • Lu Y.
      • Thulasi Raman S.N.
      • Xu F.
      • Wu Q.
      • Li Z.
      • et al.
      Nuclear-resident RIG-I senses viral replication inducing antiviral immunity.
      So, the possibility that nuclear localised HDV RNAs are captured by nuclear MDA5 cannot be completely ruled out.
      Besides the infected hepatocytes, dendritic cells and macrophages may also be sources of IFNs during HDV infection.
      • Ali S.
      • Mann-Nuttel R.
      • Schulze A.
      • Richter L.
      • Alferink J.
      • Scheu S.
      Sources of type I interferons in infectious immunity: plasmacytoid dendritic cells not always in the driver's seat.
      Lacking NTCP, these cells are not susceptible to HDV infection. However, they might capture replication intermediates via unspecific uptake pathways. Extracellular vesicles (EVs) are important for the transfer of viral RNA to these cells.
      • Kouwaki T.
      • Okamoto M.
      • Tsukamoto H.
      • Fukushima Y.
      • Oshiumi H.
      Extracellular vesicles deliver host and virus RNA and regulate innate immune response.
      • Assil S.
      • Coleon S.
      • Dong C.
      • Decembre E.
      • Sherry L.
      • Allatif O.
      • et al.
      Plasmacytoid dendritic cells and infected cells form an interferogenic synapse required for antiviral responses.
      • Dreux M.
      • Garaigorta U.
      • Boyd B.
      • Decembre E.
      • Chung J.
      • Whitten-Bauer C.
      • et al.
      Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity.
      • Webster B.
      • Assil S.
      • Dreux M.
      Cell-cell sensing of viral infection by plasmacytoid dendritic cells.
      Recently, Jung et al. observed that HDV-infected hepatoma cell lines and PHHs secrete EVs. Incubation of peripheral blood mononuclear cells and macrophages with the enriched EVs activated pro-inflammatory cytokines, however type I IFN induction was not reported.
      • Jung S.
      • Altstetter M.S.
      • Wilsch F.
      • Shein M.
      • Schütz A.K.
      • Protzer U.
      Extracellular vesicles derived from hepatitis-D virus infected cells induce a proinflammatory cytokine response in human peripheral blood mononuclear cells and macrophages.
      More investigations are needed to make clear the role of these immune cells in HDV RNA sensing and IFN production.

      IFN-mediated inhibition of HDV replication

      Although not highly curative, IFN-α/λ treatment significantly suppresses HDV replication in HBV/HDV-coinfected patients (see below) and humanised 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.
      Knocking out the IFN-α/β receptor
      • He W.
      • Ren B.
      • Mao F.
      • Jing Z.
      • Li Y.
      • Liu Y.
      • et al.
      Hepatitis D virus infection of mice expressing human sodium taurocholate co-transporting polypeptide.
      or MAVS
      • Suarez-Amaran L.
      • Usai C.
      • Di Scala M.
      • Godoy C.
      • Ni Y.
      • Hommel M.
      • et al.
      A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-beta induction.
      promoted HDV replication in mouse models. Regarding the mode of action, an early study showed that IFN-α treatment of HDV-infected PHHs preferentially affected early stages of infection (establishment of replicative intermediates) with high dosing of IFN-α (600 units/ml).
      • Han Z.
      • Nogusa S.
      • Nicolas E.
      • Balachandran S.
      • Taylor J.
      Interferon impedes an early step of hepatitis delta virus infection.
      In a recent study, poly (I:C) was used to artificially activate IFN either 12 h before or 12 days after HBV/HDV infection, resulting in a significant inhibition of HDV replication in the pre-treatment group but only marginal effects in the late-treatment group.
      • Winer B.Y.
      • Gaska J.M.
      • Lipkowitz G.
      • Bram Y.
      • Parekh A.
      • Parsons L.
      • et al.
      Analysis of host responses to hepatitis B and delta viral infections in a micro-scalable hepatic co-culture system.
      Using NTCP-expressing resting HepG2 and HepaRG cells, our group could show that early treatment (day 1–7) with IFN-α (100 IU/ml) and IFN-λ1 (10 ng/ml) caused an ~50% reduction in the establishment of HDV-positive cells, while later treatment (day 5–11) barely interfered with HDV replication.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      These insignificant short-term effects contrast with the long-term effect of IFN-α treatment in patients and require additional explanations.
      In patients, monotherapy with IFN-α/λ is partially efficient but rarely clears HDV in the presence of HBV envelope proteins (HBsAg).
      As described earlier, cell division-mediated HDV amplification might contribute to HDV persistence. We recently demonstrated that this type of HDV spread is highly sensitive to both the HDV-induced IFN response and exogenous IFN-α/λ treatment.
      • Zhang Z.
      • Ni Y.
      • Urban S.
      Endogenous and exogenous IFN responses suppress HDV persistence during proliferation of hepatocytes in vitro.
      The severe loss of HDV replicative intermediates during cell division may be explained by the resolution of nuclei and exposure of viral RNA to induced ISGs. Such ISGs may either cause direct degradation of HDV RNA or inhibit the re-establishment of replication in the nuclei of daughter cells. This novel finding has profound implications for future combination treatments with IFNs and drugs blocking de novo infection, indicating possible additive/synergistic effects against HDV (see below).
      Besides the effect on itself, the HDV-induced IFN response probably also contributes to the suppression of HBV replication (in the same cell but also in neighbouring cells). An in vitro study demonstrated that the repression of HBV replication was associated with RNA replication of super-infected HDV, and the kinetics of HBV marker reduction correlated with those of IFN activation.
      • Alfaiate D.
      • Lucifora J.
      • Abeywickrama-Samarakoon N.
      • Michelet M.
      • Testoni B.
      • Cortay J.C.
      • et al.
      HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes.
      This is along the line of another study showing that HBV reactivation after HCV clearance is mainly due to a reduction in the HCV-induced IFN response.
      • Cheng X.
      • Uchida T.
      • Xia Y.
      • Umarova R.
      • Liu C.J.
      • Chen P.J.
      • et al.
      Diminished hepatic IFN response following HCV clearance triggers HBV reactivation in coinfection.

      Viral countermeasures to HDV-induced IFN responses

      The strategies employed by viruses to counteract innate immune responses can be roughly classified into 2 categories: i) blocking IFN signalling and the function of ISGs; and ii) avoiding recognition by PRRs and antiviral effectors. Following transfection of hepatoma cells with an HDV genome, an early study showed that HDV downregulated the transcription of ISGs in response to IFN-α treatment by inhibiting phosphorylation and nuclear translocation of STAT1/2.
      • Pugnale P.
      • Pazienza V.
      • Guilloux K.
      • Negro F.
      Hepatitis delta virus inhibits alpha interferon signaling.
      In another study using stable HDV-replicating 293 cell lines, suppression of poly (I:C) activated IFN production by HDV.
      • Han Z.
      • Nogusa S.
      • Nicolas E.
      • Balachandran S.
      • Taylor J.
      Interferon impedes an early step of hepatitis delta virus infection.
      Using HBV/HDV-coinfected humanised mice, it was demonstrated that hepatocytes with high levels of HDV replication exhibited lower levels of STAT1 activation.
      • 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.
      However, recent work from our group demonstrated that ISGs (e.g. Mx1) are induced in HDV-replicating HepG2-NTCP and HepaRG-NTCP cells, arguing against a profound blockade of IFN signalling by HDV.
      • Zhang Z.
      • Filzmayer C.
      • Ni Y.
      • Sultmann H.
      • Mutz P.
      • Hiet M.S.
      • et al.
      Hepatitis D virus replication is sensed by MDA5 and induces IFN-beta/lambda responses in hepatocytes.
      Further investigations using authentic infection systems are needed to clarify these discrepancies.
      Besides direct blockade of IFN signalling, HDV probably also applies strategies to escape from recognition and clearance by the IFN system. Firstly, HDV replicates in the nucleus. Since PRRs and most IFN-induced effectors principally localise in the cytoplasm, HDV replication intermediates might escape recognition through compartmentalisation. This assumption is indirectly supported by the observation that only HDV mRNA but not genomic and antigenomic RNA is targetable by siRNA.
      • Chang J.
      • Taylor J.M.
      Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action.
      Secondly, the HDAg but also RNP-associated HBV envelope proteins may protect HDV RNA from recognition by PRRs. Electron microscopy studies revealed that the HDV RNP complex is a highly ordered structure.
      • Ryu W.S.
      • Netter H.J.
      • Bayer M.
      • Taylor J.
      Ribonucleoprotein complexes of hepatitis delta virus.
      ,
      • Griffin B.L.
      • Chasovskikh S.
      • Dritschilo A.
      • Casey J.L.
      Hepatitis delta antigen requires a flexible quasi-double-stranded RNA structure to bind and condense hepatitis delta virus RNA in a ribonucleoprotein complex.
      Binding of HDAg can protect HDV RNA from degradation by cellular RNases.
      • Lazinski D.W.
      • Taylor J.M.
      Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging.
      So, HDAg binding probably prevents the recognition of HDV RNA by PPRs as well. Application of newly developed cell culture models, e.g. cell lines expressing both the HDV receptor and the HBV envelope that thereby support the full HDV life cycle,
      • Lempp F.A.
      • Schlund F.
      • Rieble L.
      • Nussbaum L.
      • Link C.
      • Zhang Z.
      • et al.
      Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation.
      ,
      • Ni Y.
      • Zhang Z.
      • Engelskircher L.
      • Verch G.
      • Tu T.
      • Lempp F.A.
      • et al.
      Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus.
      will be helpful to better understand to what extent these HDV-specific countermeasures contribute to the evasion of immune recognition.
      Monotherapy with upcoming direct-acting antivirals (bulevirtide, lonafarnib and REP-2139) leads to more profound therapeutic effects but requires long-term treatment.

      Clinical aspect: current therapies for CHD

      IFN monotherapy

      Until the recent conditional marketing authorisation (CMA) of bulevirtide in Europe, off-label IFN-α (subtype 2a and 2b) was the only drug for the treatment of patients with CHD. Several pilot investigations were conducted to evaluate the efficiency of IFN and pegylated-IFN (Peg-IFN) in HDV-infected patients. In 1994, Farci et al. reported suppression of HDV serum RNA after 48 weeks of IFN-α treatment; 71% of patients treated with the high dose (9 million units) had undetectable RNA at the end of treatment. However, only 43% of patients remained RNA negative 6 months post therapy and virologic relapse occurred in all patients after 39 months of follow-up.
      • Farci P.
      • Mandas A.
      • Coiana A.
      • Lai M.E.
      • Desmet V.
      • Van Eyken P.
      • et al.
      Treatment of chronic hepatitis D with interferon alfa-2a.
      Of note, the sensitivity of quantification assays for HDV RNA was low at that time
      • Bremer B.
      • Anastasiou O.E.
      • Ciesek S.
      • Wedemeyer H.
      Automated nucleic acid isolation methods for HDV viral load quantification can lead to viral load underestimation.
      and therefore rates of serum RNA negativation were probably overestimated. Peg-IFN-α has higher efficacy than standard IFN-α
      • Alavian S.M.
      • Tabatabaei S.V.
      • Behnava B.
      • Rizzetto M.
      Standard and pegylated interferon therapy of HDV infection: a systematic review and meta- analysis.
      but still could not eliminate HDV in most patients.
      • Castelnau C.
      • Le Gal F.
      • Ripault M.P.
      • Gordien E.
      • Martinot-Peignoux M.
      • Boyer N.
      • et al.
      Efficacy of peginterferon alpha-2b in chronic hepatitis delta: relevance of quantitative RT-PCR for follow-up.
      Some studies indicated that treatment extension, e.g. for 2 years with standard IFN-α
      • Gunsar F.
      • Akarca U.S.
      • Ersoz G.
      • Kobak A.C.
      • Karasu Z.
      • Yuce G.
      • et al.
      Two-year interferon therapy with or without ribavirin in chronic delta hepatitis.
      or even 5 years with Peg-IFN-α
      • Heller T.
      • Rotman Y.
      • Koh C.
      • Clark S.
      • Haynes-Williams V.
      • Chang R.
      • et al.
      Long-term therapy of chronic delta hepatitis with peginterferon alfa.
      did not significantly improve the response rates, while 1 retrospective study showed that prolongation of IFN treatment increased efficacy.
      • Yurdaydin C.
      • Keskin O.
      • Kalkan C.
      • Karakaya F.
      • Caliskan A.
      • Kabacam G.
      • et al.
      Interferon treatment duration in patients with chronic delta hepatitis and its effect on the natural course of the disease.
      The effect of IFN-α was more comprehensively evaluated in the HIDIT-I and -II studies. In the HIDIT-I study, 48 weeks treatment with 180 μg of Peg-IFN-α weekly resulted in HDV RNA negativation in about 24% of patients with CHD.
      • Wedemeyer H.
      • Yurdaydin C.
      • Dalekos G.N.
      • Erhardt A.
      • Cakaloglu Y.
      • Degertekin H.
      • et al.
      Peginterferon plus adefovir versus either drug alone for hepatitis delta.
      However long-term follow-up demonstrated high relapse rates.
      • Heidrich B.
      • Yurdaydin C.
      • Kabacam G.
      • Ratsch B.A.
      • Zachou K.
      • Bremer B.
      • et al.
      Late HDV RNA relapse after peginterferon alpha-based therapy of chronic hepatitis delta.
      Neither application of HBV suppressing nucleot(s)ide analogues nor prolongation of the treatment to 96 weeks in the HIDIT-II study improved responses
      • Wedemeyer H.
      • Yurdaydin C.
      • Hardtke S.
      • Caruntu F.A.
      • Curescu M.G.
      • Yalcin K.
      • et al.
      Peginterferon alfa-2a plus tenofovir disoproxil fumarate for hepatitis D (HIDIT-II): a randomised, placebo controlled, phase 2 trial.
      (for a detailed review on IFN treatments and meta-analyses see references
      • Alavian S.M.
      • Tabatabaei S.V.
      • Behnava B.
      • Rizzetto M.
      Standard and pegylated interferon therapy of HDV infection: a systematic review and meta- analysis.
      ,
      • Mentha N.
      • Clement S.
      • Negro F.
      • Alfaiate D.
      A review on hepatitis D: from virology to new therapies.
      • Abbas Z.
      • Khan M.A.
      • Salih M.
      • Jafri W.
      Interferon alpha for chronic hepatitis D.
      • Triantos C.
      • Kalafateli M.
      • Nikolopoulou V.
      • Burroughs A.
      Meta-analysis: antiviral treatment for hepatitis D.
      ). Again, the sensitivity and specificity of the applied reverse transcription PCR method to determine serum HDV levels were limited in these trials. A WHO HDV standard was introduced in 2013
      • Pyne M.T.
      • Mallory M.A.
      • Xie H.B.
      • Mei Y.
      • Schlaberg R.
      • Hillyard D.R.
      Sequencing of the hepatitis D virus RNA WHO international standard.
      ,
      • Chudy M.
      • Hanschmann K.M.
      • Bozdayi M.
      • Kreß J.
      • Nübling C.M.
      the-Collaborative-Study-Group
      Collaborative Study to Establish a World Health Organization International Standard for Hepatitis D Virus RNA for Nucleic Acid Amplification Technique (NAT)-Based Assays.
      and more sensitive pan-genotypic RNA assays were developed in 2016,
      • Le Gal F.
      • Brichler S.
      • Sahli R.
      • Chevret S.
      • Gordien E.
      First international external quality assessment for hepatitis delta virus RNA quantification in plasma.
      so direct comparisons of these early responses with those of recent trials should be undertaken with caution.
      These clinical observations on IFN responses are consistent with the in vitro and in vivo findings (see above) demonstrating that HDV replication in hepatocytes is only marginally affected by “self-induced” or “therapeutically applied” IFNs. The direct effect of IFNs preferentially occurs during cell division or by inducing cellular immune responses. Since induction of eliminating T cell responses is rare and escape from T cell recognition has been described,
      • Kefalakes H.
      • Koh C.
      • Sidney J.
      • Amanakis G.
      • Sette A.
      • Heller T.
      • et al.
      Hepatitis D virus-specific CD8(+) T cells have a memory-like phenotype associated with viral immune escape in patients with chronic hepatitis D virus infection.
      ,
      • Karimzadeh H.
      • Kiraithe M.M.
      • Oberhardt V.
      • Salimi Alizei E.
      • Bockmann J.
      • Schulze Zur Wiesch J.
      • et al.
      Mutations in hepatitis D virus allow it to escape detection by CD8(+) T cells and evolve at the population level.
      ,
      • Karimzadeh H.
      • Kiraithe M.M.
      • Kosinska A.D.
      • Glaser M.
      • Fiedler M.
      • Oberhardt V.
      • et al.
      Amino acid substitutions within HLA-B∗27-restricted T cell epitopes prevent recognition by hepatitis delta virus-specific CD8(+) T cells.
      monotherapy with IFN-α is only partially effective and not curative for most patients. Nevertheless, overall, long-term follow-up of IFN-α-treated patients has demonstrated better clinical outcomes, especially in those who experienced a >2 log reduction in HDV RNA levels.
      • Farci P.
      • Roskams T.
      • Chessa L.
      • Peddis G.
      • Mazzoleni A.P.
      • Scioscia R.
      • et al.
      Long-term benefit of interferon alpha therapy of chronic hepatitis D: regression of advanced hepatic fibrosis.
      • Wranke A.
      • Serrano B.C.
      • Heidrich B.
      • Kirschner J.
      • Bremer B.
      • Lehmann P.
      • et al.
      Antiviral treatment and liver-related complications in hepatitis delta.
      • Wranke A.
      • Hardtke S.
      • Heidrich B.
      • Dalekos G.
      • Yalcin K.
      • Tabak F.
      • et al.
      Ten-year follow-up of a randomized controlled clinical trial in chronic hepatitis delta.

      Novel therapeutics and responses in patients

      The availability of HBV/HDV susceptible/permissive cell culture systems has enabled specific and more effective drugs to be identified and successfully translated into clinical development (Table 1). The unmet medical need for patients with CHD means that 2 of these drugs (lonafarnib and bulevirtide) are being developed under special programmes from the FDA and EMA (orphan drug status, break through therapy designation, prime eligibility).
      Table 1Approved and investigational HDV drugs.
      DrugSubstanceMode of actionAvailability
      Peg-IFN-αProteinCytokine, activating innate immune systemApproved for HBV, off-label use for HDV
      Peg-IFN-λ1ProteinCytokine, activating innate immune systemPhase II
      BulevirtidePreS1 peptideNTCP binding, blocking HBV/HDV entryPhase III, CMA by EMA in July 2020
      LonafarnibSmall moleculeInhibiting L-HDAg prenylation and HDV secretionPhase III
      REP 2139Nucleic acid polymerInhibiting HBsAg/HBV /HDV secretion, possibly also HBV/HDV entryPhase II
      CMA, conditional marketing authorisation; L-HDAg, large hepatitis delta antigen; Peg-IFN, pegylated interferon; NTCP, human sodium taurocholate co-transporting polypeptide.
      Bulevirtide is a myristoylated 47-aa long peptide derived from an optimised consensus sequence of the preS1-domain of the HBV L-protein, which efficiently blocks NTCP, the receptor of HDV/HBV, thereby inhibiting virus entry.
      • Lempp F.A.
      • Urban S.
      Hepatitis delta virus: replication strategy and upcoming therapeutic options for a neglected human pathogen.
      ,
      • Tu T.
      • Urban S.
      Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections.
      Its ability to completely prevent virus entry at very low concentrations in vitro
      • Gripon P.
      • Cannie I.
      • Urban S.
      Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein.
      ,
      • Glebe D.
      • Urban S.
      • Knoop E.V.
      • Cag N.
      • Krass P.
      • Grun S.
      • et al.
      Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes.
      and in mice transplanted with PHHs
      • Lutgehetmann M.
      • Mancke L.V.
      • Volz T.
      • Helbig M.
      • Allweiss L.
      • Bornscheuer T.
      • et al.
      Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation.
      ,
      • Volz T.
      • Allweiss L.
      • Ben M.M.
      • Warlich M.
      • Lohse A.W.
      • Pollok J.M.
      • et al.
      The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus.
      enables low dosing in patients.
      • Bogomolov P.
      • Alexandrov A.
      • Voronkova N.
      • Macievich M.
      • Kokina K.
      • Petrachenkova M.
      • et al.
      Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: first results of a phase Ib/IIa study.
      ,
      • Wedemeyer H.
      • Bogomolov P.
      • Blank A.
      • Allweiss L.
      • Dandri M.
      • Bremer B.
      • et al.
      Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV/HDV co-infection.
      A dose of 2 mg (s.c.) daily, which does not block bile salt transport of NTCP but already exhibits strong effects on virus entry, induced a 1.67 log10 and 2.84 log10 median decline of serum HDV RNA after 24 weeks
      • Bogomolov P.
      • Alexandrov A.
      • Voronkova N.
      • Macievich M.
      • Kokina K.
      • Petrachenkova M.
      • et al.
      Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: first results of a phase Ib/IIa study.
      and 48 weeks (Myr-202 trial) of treatment, respectively.
      • Wedemeyer H.
      • Bogomolov P.
      • Blank A.
      • Allweiss L.
      • Dandri M.
      • Bremer B.
      • et al.
      Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with Tenofovir in patients with chronic HBV/HDV co-infection.
      A dose increase to 10 mg/day enhanced the antiviral response to 4.58 log10 median reduction at week 48 of treatment (Myr-203 extension study). As expected for an entry inhibitor, the kinetics of HDV RNA decline followed zero-order kinetics with a continuous reduction of serum RNA, arguing for prolonged treatment. Liver biopsy revealed a loss of infected hepatocytes during therapy. Remarkably, the recently presented final results of the Myr-203 extension study showed that 10 mg bulevirtide led to a higher response rate (33.3% HDV RNA negativation and 47% RNA negativation or >2 log10 reduction 24 weeks post (!) treatment cessation) compared to the combination with IFN, although a pronounced synergistic effect was observed during treatment.
      • Wedemeyer H.
      • Schöneweis K.
      • Bogomolov P.
      • 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.
      Based on its good safety, continuous antiviral efficacy and the fast normalisation of ALT levels during therapy, the EMA granted CMA for bulevirtide (2 mg) in July 2020 within its “prime eligibility” and “orphan drug” programme. A multicentre phase III trial (Myr-301) investigating the curative potential of long-term treatment with 10 mg bulevirtide is ongoing.
      Lonafarnib is a farnesyl transferase inhibitor preventing prenylation of the L-HDAg. Developed as an anti-cancer drug that interferes with essential signalling pathways involved in cell cycle regulation (e.g. the RAS pathway),
      • Berndt N.
      • Hamilton A.D.
      • Sebti S.M.
      Targeting protein prenylation for cancer therapy.
      it also interferes with secretion of HDV virions.
      • Koh C.
      • Canini L.
      • Dahari H.
      • Zhao X.
      • Uprichard S.L.
      • Haynes-Williams V.
      • et al.
      Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial.
      ,
      • Yurdaydin C.
      • Keskin O.
      • Kalkan C.
      • Karakaya F.
      • Caliskan A.
      • Karatayli E.
      • et al.
      Optimizing lonafarnib treatment for the management of chronic delta hepatitis: the LOWR HDV-1 study.
      According to this direct antiviral effect, lonafarnib exhibits biphasic kinetics, lowering serum RNA levels to steady state levels. A phase IIa pilot clinical trial showed that 28 days of lonafarnib treatment led to a substantial reduction in serum HDV RNA, although adverse events (mostly gastrointestinal) were frequent, especially when higher doses were applied.
      • Koh C.
      • Canini L.
      • Dahari H.
      • Zhao X.
      • Uprichard S.L.
      • Haynes-Williams V.
      • et al.
      Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial.
      Adding the cytochrome P450 3A4 inhibitor ritonavir, which elevates local drug concentrations in the liver, allows for a reduction of lonafarnib dosing and consequently better control of side effects (LOWR-HDV-1 trial).
      • Yurdaydin C.
      • Keskin O.
      • Kalkan C.
      • Karakaya F.
      • Caliskan A.
      • Karatayli E.
      • et al.
      Optimizing lonafarnib treatment for the management of chronic delta hepatitis: the LOWR HDV-1 study.
      Subsequent studies (LOWR-HDV-2/3/4 studies) demonstrated >2 log10 decline or undetectable serum HDV RNA levels in most patients following 24 weeks of lonafarnib plus ritonavir treatment.
      • Koh C.
      • Surana P.
      • Han T.
      • Fryzek N.
      • Kapuria D.
      • Etzion O.
      • et al.
      A phase 2 study exploring once daily dosing of ritonavir boosted lonafarnib for the treatment of chronic delta hepatitis – end of study results from the LOWR HDV-3 study.
      • Wedemeyer H.
      • Port K.
      • Deterding K.
      • Wranke A.
      • Kirschner J.
      • Bruno B.
      • et al.
      A phase 2 dose-escalation study of lonafarnib plus ritonavir in patients with chronic hepatitis D: final results from the Lonafarnib with ritonavir in HDV-4 (LOWR HDV-4) study.
      • Yurdaydin C.
      • Kalkan C.
      • Karakaya F.
      • Caliskan A.
      • Karatayli S.
      • Keskin O.
      • et al.
      Subanalysis of the LOWR HDV-2 study reveals high response rates to Lonafarnib in patients with low viral loads.
      Based on these studies, longer treatment durations with lonafarnib are being tested in phase III registration trials.
      Combinations of direct-acting antivirals with IFNs accelerate the reduction of viral loads by synergistically addressing both pathways of persistence.
      Nucleic acid polymers (NAP) are amphipathic molecules with broad antiviral activities and multiple but only partially understood modes of action. In patients with CHB, monotherapy with REP-2055/REP-2139 led to >2 log10 reductions in serum HBsAg and >3 log10 reductions in serum HBV DNA in 9 of 12 patients.
      • Al-Mahtab M.
      • Bazinet M.
      • Vaillant A.
      Safety and efficacy of nucleic acid polymers in monotherapy and combined with immunotherapy in treatment-naive Bangladeshi patients with HBeAg+ chronic hepatitis B infection.
      REP-2139 was also reported to be associated with profound reductions in HBsAg and HDV RNA in a subset of patients in a pilot study, although there have been no other reports on REP-2139 in patients with HDV. In vitro studies indicated that REP-2139 inhibits HBsAg release from hepatocytes.
      • Blanchet M.
      • Sinnathamby V.
      • Vaillant A.
      • Labonte P.
      Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.
      • Vaillant A.
      Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.
      • Boulon R.
      • Blanchet M.
      • Lemasson M.
      • Vaillant A.
      • Labonte P.
      Characterization of the antiviral effects of REP 2139 on the HBV lifecycle in vitro.
      In addition some NAPs also interfere with HBV
      • Guillot C.
      • Martel N.
      • Berby F.
      • Bordes I.
      • Hantz O.
      • Blanchet M.
      • et al.
      Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes.
      and HDV
      • Beilstein F.
      • Blanchet M.
      • Vaillant A.
      • Sureau C.
      Nucleic acid polymers are active against hepatitis delta virus infection in vitro.
      entry into hepatocytes. However, the observed in vitro effects are moderate and may not entirely explain the strong virological effects in patients.
      IFN-λ, a type III IFN, showed antiviral activity against HDV in a humanised mouse model
      • 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 in a reported phase II clinical trial (LIMT-HDV study) in patients with CHD.
      • Etzion O.
      • Hamid S.
      • Lurie Y.
      • Gane E.J.
      • Yardeni D.
      • Bader N.
      • et al.
      End of study results from LIMT HDV study: 36% durable virologic response at 24 weeks post-treatment with pegylated interferon lambda monotherapy in patients with chronic HDV infection.
      The rationale behind the use of IFN-λ in patients with HDV is its reported improved liver targeting and its higher tolerability. Unlike IFN-α whose receptor (IFNAR1/2) is ubiquitously expressed on nucleated cells, the IFN-λ receptor (IFNLR1) is preferentially expressed in epithelial tissues of the lung, liver, and gut.
      • Doyle S.E.
      • Schreckhise H.
      • Khuu-Duong K.
      • Henderson K.
      • Rosler R.
      • Storey H.
      • et al.
      Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes.
      • Meager A.
      • Visvalingam K.
      • Dilger P.
      • Bryan D.
      • Wadhwa M.
      Biological activity of interleukins-28 and -29: comparison with type I interferons.
      • Sommereyns C.
      • Paul S.
      • Staeheli P.
      • Michiels T.
      IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo.
      • Baldridge M.T.
      • Lee S.
      • Brown J.J.
      • McAllister N.
      • Urbanek K.
      • Dermody T.S.
      • et al.
      Expression of Ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus.
      Accordingly, IFN-λ causes fewer systemic side effects, with comparable antiviral efficacy, which has been shown in chronic HCV-infected patients.
      • Muir A.J.
      • Arora S.
      • Everson G.
      • Flisiak R.
      • George J.
      • Ghalib R.
      • et al.
      A randomized phase 2b study of peginterferon lambda-1a for the treatment of chronic HCV infection.
      Results of the LIMT-HDV trial revealed that Peg-IFN-λ (180 μg weekly) bears antiviral activity, leading to a mean reduction in serum HDV RNA levels of 2.3 log10 at week 48, with 36% of patients demonstrating HDV RNA below the limit of quantification (BLQ) 24 weeks after stopping therapy.
      • Etzion O.
      • Hamid S.
      • Lurie Y.
      • Gane E.J.
      • Yardeni D.
      • Bader N.
      • et al.
      End of study results from LIMT HDV study: 36% durable virologic response at 24 weeks post-treatment with pegylated interferon lambda monotherapy in patients with chronic HDV infection.
      As in multiple prior studies,
      • Muir A.J.
      • Arora S.
      • Everson G.
      • Flisiak R.
      • George J.
      • Ghalib R.
      • et al.
      A randomized phase 2b study of peginterferon lambda-1a for the treatment of chronic HCV infection.
      ,
      • Chan H.L.Y.
      • Ahn S.H.
      • Chang T.T.
      • Peng C.Y.
      • Wong D.
      • Coffin C.S.
      • et al.
      Peginterferon lambda for the treatment of HBeAg-positive chronic hepatitis B: a randomized phase 2b study (LIRA-B).
      IFN-λ exhibited superior patient tolerability compared to IFN-α, with reversible liver-specific adverse events in some patients, and may become an alternative to Peg-IFN-α in upcoming combination therapies.
      • Hamid S.
      • Etzion O.
      • Lurie Y.
      • Bader N.
      • Yardeni D.
      • Channa S.M.
      • et al.
      A phase 2 randomized clinical trial to evaluate the safety and efficacy of pegylated interferon lambda monotherapy in patients with chronic hepatitis delta virus infection. Interim results from the LIMT HDV study.
      It is currently being evaluated in combination with lonafarnib and ritonavir in a phase II trial (LIFT-study), where 25 of 26 (96%) patients had a >2 log decline and 58% of individuals achieved BLQ or undetectable HDV RNA after just 24 weeks of treatment.
      • Koh C.
      • Hercun J.
      • Rahman F.
      • Huang A.
      • DA B.
      • Surana P.
      • et al.
      A phase 2 study of peginterferon lambda, lonafarnib and ritonavir for 24 weeks: end-of-treatment results from the LIFT HDV study.
      Nevertheless, more results on the effectiveness of Peg-IFN-λ, especially in patients with high viral loads, are needed to determine its possible superiority to Peg-IFN-α.
      Conditional marketing authorisation of bulevirtide by the EMA in July 2020 provides the first specific treatment option for HDV-infected patients.

      IFN-based combination therapies

      Although the direct-acting antivirals bulevirtide and lonafarnib exhibit clear therapeutic efficacy as monotherapy, HDV rebound was frequently observed within 1 year of treatment withdrawal. So, it is still questionable whether either drug can eliminate HDV when administered as monotherapy. Long-term treatment and full blockade of particular steps during replication will be needed to achieve this goal. Strikingly, recent clinical studies have demonstrated that combinations of lonafarnib, bulevirtide and REP-2139 with Peg-IFN-α (or -λ) exhibited additive or, in the case of bulevirtide and lonafarnib, even strong synergistic antiviral effects in terms of faster and more profound reductions in serum HDV RNA, higher off treatment responses and lower relapse rates.
      • Bazinet M.
      • Pantea V.
      • Cebotarescu V.
      • Cojuhari L.
      • Jimbei P.
      • Albrecht J.
      • et al.
      Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial.
      • Bazinet M.
      • Pântea V.
      • Cebotarescu V.
      • Cojuhari L.
      • Jimbei P.
      • Krawczyk A.
      • et al.
      Ongoing analysis of virologic control/functional cure of HBV and HDV infection following REP 2139-Ca and pegylated interferon alpha-2a therapy in patients with chronic HBV/HDV co-infection: 3.5-year follow-up results from the REP 301-LTF study.
      • Koh C.
      • Da B.L.
      • Surana P.
      • Huang A.
      • Kapuria D.
      • Rotman Y.
      • et al.
      A phase 2 study of lonafarnib, ritonavir and peginterferon lambda for 24 weeks: interim end-of-treatment results from the LIFT HDV study.
      • Wedemeyer H.
      • Schöneweis K.
      • Bogomolov P.
      • Voronkova N.
      • Chulanov V.
      • Stepanova T.
      • et al.
      Final results of a multicenter, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of myrcludex B in cwith PEG-interferon Alpha 2a in patients with chronic HBV/HDV co-infection.
      For example bulevirtide (2 mg) plus Peg-IFN-α (180 mg) (Myr 203 trial)
      • Wedemeyer H.
      • Schöneweis K.
      • Bogomolov P.
      • Voronkova N.
      • Chulanov V.
      • Stepanova T.
      • et al.
      Final results of a multicenter, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of myrcludex B in cwith PEG-interferon Alpha 2a in patients with chronic HBV/HDV co-infection.
      resulted in median serum HDV declines of 4.81 log10 at week 48, compared to a reduction of 2.84 log10 and 1.30 log10 after bulevirtide or Peg-IFN-α monotherapy, respectively.
      • Wedemeyer H.
      • Schöneweis K.
      • Bogomolov P.
      • Voronkova N.
      • Chulanov V.
      • Stepanova T.
      • et al.
      Final results of a multicenter, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of myrcludex B in cwith PEG-interferon Alpha 2a in patients with chronic HBV/HDV co-infection.
      Raising the dose of bulevirtide to 5 mg or 10 mg in the combination with Peg-IFN-α even increased the virological response rates to a decline of 5.59 log10 and 6.09 log10, respectively (Myr 203 and Myr-203 extension).
      It is presently not completely clear how a combination of IFNs and direct-acting antivirals suppresses HDV in an additive or even synergistic manner. This is mostly due to our limited knowledge concerning how IFNs act on HDV persistence. Besides activating the innate and adaptive immune systems (by complex and yet unclear mechanisms), the additive/synergistic effect may be a consequence of blocking both HDV spreading pathways (de novo infection and cell division-mediated) discussed earlier. Using an in vitro infection model supporting both pathways, we confirmed the suppression of cell division-mediated HDV spread by IFN-α/λ and blockade of de novo infection by bulevirtide and lonafarnib. Of interest, co-treatment with inhibitors targeting different spreading pathways, e.g. bulevirtide plus IFN-α, showed a strong synergistic effect compared to monotherapy.
      • Zhang Z.
      • Walther T.
      • Lempp F.A.
      • Ni Y.
      • Urban S.
      Synergistic suppression of HDV persistence in vitro by co-treatment with investigational drugs targeting both extracellular and cell division mediated spreading pathways.
      Clinical observations that Peg-IFN-α can further cause profound serum RNA decline even under conditions (e.g. 10 mg bulevirtide) that completely block the extracellular route of HDV spread, also support the assumption that IFNs preferentially inhibit de novo independent maintenance pathways of HDV. If this hypothesis holds true, fast curative treatment approaches should aim at blocking both pathways (Fig. 4). In the future it will be interesting to learn whether other modulators of innate immune signalling pathways (e.g. orally available PRR agonists with minor side effects compared to IFNs) act in the same way.
      Figure thumbnail gr4
      Fig. 4HDV therapies and the speculated outcomes.
      HBV envelope-dependent de novo infection (upper row), HBV independent cell division-mediated spread (middle row) and long-lasting replication in resting cells (bottom row) all contribute to HDV persistence. The extracellular spread inhibitors e.g. bulevirtide and lonafarnib efficiently block de novo infection-mediated spread, but have marginal effect on cell division-mediated spread and HDV replication in resting cells (3rd column). IFN-α/λ inhibits extracellular spread (less effective than extracellular spread inhibitors) and efficiently suppresses cell division-mediated spread (4th column). Combinations of extracellular spread inhibitors and IFNs can block both de novo infection and cell division-mediated spread (last column), nevertheless, their effect on HDV replication in resting cells is still questionable. #, HDV markers in resting cells might be cleared by long-term direct antiviral effects of the IFN response, or cell death caused by apoptosis, T cells, etc. IFNs, interferons.

      Perspectives

      HDV is the smallest known human virus with a unique RNA structure and replication strategy. Beside HBV-dependent de novo infection, HDV also spreads and persists though cell division. HDV replication activates MDA5-mediated IFN-β/λ responses in hepatocytes. IFNs inhibits HDV replication predominantly by destroying HDV RNA during cell division. The efficacy of IFN-α as a monotherapy is not satisfactory. However, combining IFNs with bulevirtide and drugs under development (lonafarnib, REP-2139) has shown promising clinical results. Regrowing interest in HDV and the application of novel cell culture systems in the future will lead to a deeper understanding of (i) the mechanism of HDV persistence, (ii) the interplay between HDV and the IFN response and (iii) how to design combination therapies to cure HDV infection. These studies will also help to elucidate whether HDV cure can be achieved without the elimination of HBsAg-expressing cells (hepatocytes carrying replicating HBV or those encoding HBsAg from integrates) from the liver. Since the latter goal is still some way off, drugs that control HDV and normalise liver function are of urgent need for patients.

      Abbreviations

      AAV, adeno-associated virus; ADAR1, adenosine deaminase acting on RNA 1; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; CHD, chronic hepatitis D; CMA, conditional marketing authorisation; DAg, delta antigen of HDV-like agents; EV, extracellular vesicle; HSPG, heparan sulphate proteoglycans; IFN, interferon; IFNAR, interferon alpha receptor; IFNLR, interferon lambda receptor; IL-6, interleukin 6; ISG, IFN-stimulated gene; IRF9, interferon regulatory factor 9; LGP2, laboratory of physiology and genetics 2; (L/S/M)-HBsAg, (large/small/middle) HBV surface antigen; (L/S)-HDAg, (large/small) hepatitis delta antigen; MAVS, mitochondrial antiviral signalling protein; MDA5, melanoma differentiation antigen 5; NAP, nucleic acid polymer; NK, natural killer; NTCP, human sodium taurocholate co-transporting polypeptide; ORF, open reading frame; PAMPs, pathogen-associated molecular patterns; Peg-IFN, pegylated interferon; PHH, primary human hepatocyte; PRR, pattern recognition receptor; RIG-I, retinoic acid-inducible gene I; RLR, RIG-I like receptor; RNP, ribonucleoprotein; STAT, signal transducer and activator of transcription; TLR, toll-like receptor.

      Financial support

      The authors received funding by from the “ Deutsche Forschungsgemeinschaft ” (DFG, German Research Foundation ) - Project No. 272983813 - TRR 179 (TP 15); DFG - Project No. 240245660 - SFB 1129 (TP 16), and the “ Deutsches Zentrum für Infektionsforschung ” (DZIF, German Center for Infection Research ) - TTU 05.904, TTU 05.807, TTU 05.804, TTU 05.704.

      Authors' contributions

      Both authors wrote the article, revised and approved the final text.

      Conflict of interest

      SU is inventor and co-applicant of intellectual property protecting Hepcludex® (bulevirtide/Myrcludex B).
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We thank Dr. Camille Sureau for critical reading of the manuscript. We thank Drs. Katrin Schöneweis, Christine Bekker and Stefan Seitz for their help in preparing the figures, Mattis Hilleke and Eva Gnimah Gnouamozi for polishing the manuscript. We apologize to numerous researchers whose original contributions could not or only partly be referenced for space limitations.

      Supplementary data

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