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Hepatitis B surface antigen seroclearance: Immune mechanisms, clinical impact, importance for drug development

  • Issam Tout
    Affiliations
    University Paris Diderot, Sorbonne Paris Cité, CRI, UMR 1149, Inserm, F-75018 Paris, France

    Department of Hepatology, AP-HP Hôpital Beaujon, Clichy 92110, France
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  • Dimitri Loureiro
    Affiliations
    University Paris Diderot, Sorbonne Paris Cité, CRI, UMR 1149, Inserm, F-75018 Paris, France

    Department of Hepatology, AP-HP Hôpital Beaujon, Clichy 92110, France
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  • Abdellah Mansouri
    Affiliations
    University Paris Diderot, Sorbonne Paris Cité, CRI, UMR 1149, Inserm, F-75018 Paris, France

    Department of Hepatology, AP-HP Hôpital Beaujon, Clichy 92110, France
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  • Vassili Soumelis
    Affiliations
    Hôpital Saint Louis, Saint Louis Research Institute, Paris, France

    INSERM U976, Human Immunology, Physiopathology and Immunotherapy, Paris, France
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  • Nathalie Boyer
    Affiliations
    University Paris Diderot, Sorbonne Paris Cité, CRI, UMR 1149, Inserm, F-75018 Paris, France

    Department of Hepatology, AP-HP Hôpital Beaujon, Clichy 92110, France
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  • Tarik Asselah
    Correspondence
    Corresponding author. Address: Viral Hepatitis, INSERM, UMR 1149, Hôpital Beaujon, 100 Boulevard du General Leclerc, Clichy 92110, France. Tel.: +33 (0) 140875579, fax: +33 (0) 147309440.
    Affiliations
    University Paris Diderot, Sorbonne Paris Cité, CRI, UMR 1149, Inserm, F-75018 Paris, France

    Department of Hepatology, AP-HP Hôpital Beaujon, Clichy 92110, France
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Published:April 22, 2020DOI:https://doi.org/10.1016/j.jhep.2020.04.013

      Summary

      HBsAg seroclearance occurs rarely in the natural history of chronic hepatitis B (CHB) infection and is associated with improved clinical outcomes. Many factors are associated with HBsAg seroconversion, including immune and viral factors. However, the immune mechanisms associated with HBsAg seroclearance are still difficult to elucidate. HBsAg seroclearance is the ideal aim of HBV treatment. Unfortunately, this goal is rarely achieved with current treatments. Understanding the mechanisms of HBsAg loss appears to be important for the development of curative HBV treatments. While studies from animal models give insights into the potential immune mechanisms and interactions occurring between the immune system and HBsAg, they do not recapitulate all features of CHB in humans and are subject to variability due to their complexity. In this article, we review recent studies on these immune factors, focusing on their influence on CHB progression and HBsAg seroconversion. These data provide new insights for the development of therapeutic approaches to partially restore the anti-HBV immune response. Targeting HBsAg will ideally relieve the immunosuppressive effects on the immune system and help to restore anti-HBV immune responses.

      Keywords

      Introduction

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      Figure thumbnail gr1
      Fig. 1Schematic model of Dane particle and HBsAg proteins.
      HBV genome is housed in a capsid structure formed by core (HBcAg) proteins surrounded by 3 different HBsAg, L-, M- and S-HBsAg. All HBsAg share the S domain which contains 4 putative transmembrane domains. M-HBsAg contains an additional PreS2 domain and L-HBsAg have both PreS1 and PreS2 domains. The NTCP binding domain is present in the PreS1 domain and is important for HBV infection. The “a” determinant is immunogenic in its S domain which is important for antibody neutralisation. NTCP, sodium taurocholate co-transporting polypeptide.
      Figure thumbnail gr2
      Fig. 2HBV replication cycle and main effects in infected hepatocytes.
      HBsAg is produced from both cccDNA transcripts and HBV integration in the host genome. M- and L-HBsAg can act as transcriptional activators for host genes. HBsAg proteins are accumulated in ER and can induce the activation of cellular stress pathways. HBsAg plays a crucial role in HBV encapsidation and virions secretion. Secreted HBsAg can subvert the antiviral immune response. cccDNA, covalently closed circular DNA; ER, endoplasmic reticulum; HSPG, heparan sulphate proteoglycan; L-HBsAg, large HBsAg; M-HBsAg, medium HBsAg; NTCP, sodium taurocholate co-transporting polypeptide; rcDNA, relaxed circular DNA; S-HBsAg, small HBsAg; SVP, sub-viral particle.

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      HBsAg seroclearance: prevalence and clinical significance

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      Interestingly, HBsAg seroclearance was associated with a lower baseline HBV DNA level (6.61 log10 IU/ml vs. 7.71 log10 IU/ml) and a lower baseline HBsAg level (2.74 log10 IU/ml vs. 3.90 log10 IU/ml). HBsAg seroclearance was not associated with gender, HBV genotype or treatment history. Heterogeneity was substantial across the studies. HBsAg seroclearance is associated with a reduced risk of HCC compared with patients who are HBsAg-persistent carriers.
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      Immune cells and HBsAg

      In CHB, liver necroinflammation which is the driver for fibrosis, is affected by a dynamic imbalance of HBV, liver cells, and the host's immune system. Although many useful immunological insights into HBV pathogenesis have been made by studying peripheral blood, a large proportion of relevant responses are enriched in the liver as tissue-resident immune subsets play vital roles in front-line immunosurveillance in the liver and other organs. Liver biopsies and fine needle aspiration have enabled the identification of tissue- and liver-resident immune cells, not seen in the peripheral blood, including HBV-specific programmed cell death protein 1 (PD-1)hi T cells and natural killer (NK) cells, together with PD-L1 (or CD274)-expressing hepatocytes. A preferential accumulation of PD-1hi Tbethi atypical memory B cells (atMBCs) in the liver was shown compared to the peripheral blood.
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      • Suveizdyte K.
      • Amin O.E.
      • Swadling L.
      • et al.
      Circulating and intrahepatic antiviral B cells are defective in hepatitis B.
      The liver is also enriched with several innate-like populations such as mucosal-associated invariant T cells and liver sinusoidal endothelial cells.
      HBsAg seroclearance is associated with a reduced risk of HCC.
      HBV particles can inhibit innate immune responses in hepatocytes, leading to decreased expression of antiviral cytokines.
      • Luangsay S.
      • Gruffaz M.
      • Isorce N.
      • Testoni B.
      • Michelet M.
      • Faure-Dupuy S.
      • et al.
      Early inhibition of hepatocyte innate responses by hepatitis B virus.
      HBsAg is involved in immune evasion processes which are presented in Table 1. Many immune cells contribute to the immunopathogenesis of HBV infection, such as NK cells, cytotoxic T lymphocytes (CTLs), dendritic cells (DCs), memory and plasma B cells, and myeloid-derived suppressor cells (MDSCs), among others.
      • Kondo Y.
      • Shimosegawa T.
      Significant roles of regulatory T cells and myeloid derived suppressor cells in hepatitis B virus persistent infection and hepatitis B virus-related HCCs.
      • Martinet J.
      • Dufeu-Duchesne T.
      • Bruder Costa J.
      • Larrat S.
      • Marlu A.
      • Leroy V.
      • et al.
      Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
      • Shi B.
      • Ren G.
      • Hu Y.
      • Wang S.
      • Zhang Z.
      • Yuan Z.
      HBsAg inhibits IFN-α production in plasmacytoid dendritic cells through TNF-α and IL-10 induction in monocytes.
      • Kalathil S.
      • Lugade A.A.
      • Miller A.
      • Iyer R.
      • Thanavala Y.
      Higher frequencies of GARP(+)CTLA-4(+)Foxp3(+) T regulatory cells and myeloid-derived suppressor cells in hepatocellular carcinoma patients are associated with impaired T-cell functionality.
      • Xu X.
      • Shang Q.
      • Chen X.
      • Nie W.
      • Zou Z.
      • Huang A.
      • et al.
      Reversal of B-cell hyperactivation and functional impairment is associated with HBsAg seroconversion in chronic hepatitis B patients.
      • Salimzadeh L.
      • Le Bert N.
      • Dutertre C.-A.
      • Gill U.S.
      • Newell E.W.
      • Frey C.
      • et al.
      PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.
      • Liu S.
      • Peng N.
      • Xie J.
      • Hao Q.
      • Zhang M.
      • Zhang Y.
      • et al.
      Human hepatitis B virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways.
      However, the immune mechanisms underlying HBsAg loss have not been studied in detail. Understanding the cellular basis of these immune interactions may help in the development of improved strategies for viral clearance. We will discuss the role of innate and adaptive immune cells in CHB, as well as reviewing their interactions and correlations with HBsAg (Table 1).
      Table 1HBsAg effects on immune cells.
      Immune cell typeHBsAg effectsReference(s)
      Kupffer cellDirectly interacts with KCs in vivo and in vitro

      ↓ the AIM2-inflammasome and ↓ the production of IL-1β
      • Zannetti C.
      • Roblot G.
      • Charrier E.
      • Ainouze M.
      • Tout I.
      • Briat F.
      • et al.
      Characterization of the inflammasome in human Kupffer cells in response to synthetic agonists and pathogens.
      ,
      • Boltjes A.
      • van Montfoort N.
      • Biesta P.J.
      • Op den Brouw M.L.
      • Kwekkeboom J.
      • van der Laan L.J.W.
      • et al.
      Kupffer cells interact with hepatitis B surface antigen in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function.
      Monocyte/macrophage↓ TLR2 and c-Jun N-terminal protein kinase (JNK), thus ↓ production of IL-12
      • Wang S.
      • Chen Z.
      • Hu C.
      • Qian F.
      • Cheng Y.
      • Wu M.
      • et al.
      Hepatitis B virus surface antigen selectively inhibits TLR2 ligand-induced IL-12 production in monocytes/macrophages by interfering with JNK activation.
      mDC↓ frequency and function
      • van der Molen R.G.
      • Sprengers D.
      • Binda R.S.
      • de Jong E.C.
      • Niesters H.G.M.
      • Kusters J.G.
      • et al.
      Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.
      ,
      • Op den Brouw M.L.
      • Binda R.S.
      • van Roosmalen M.H.
      • Protzer U.
      • Janssen H.L.A.
      • van der Molen R.G.
      • et al.
      Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus.
      pDC↓ TLR9-mediated activation and IFN-α production
      • Martinet J.
      • Dufeu-Duchesne T.
      • Bruder Costa J.
      • Larrat S.
      • Marlu A.
      • Leroy V.
      • et al.
      Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
      ,
      • Shi B.
      • Ren G.
      • Hu Y.
      • Wang S.
      • Zhang Z.
      • Yuan Z.
      HBsAg inhibits IFN-α production in plasmacytoid dendritic cells through TNF-α and IL-10 induction in monocytes.
      ,
      • Woltman A.M.
      • Op den Brouw M.L.
      • Biesta P.J.
      • Shi C.C.
      • Janssen H.L.A.
      Hepatitis B virus lacks immune activating capacity, but actively inhibits plasmacytoid dendritic cell function.
      ,
      • Xu Y.
      • Hu Y.
      • Shi B.
      • Zhang X.
      • Wang J.
      • Zhang Z.
      • et al.
      HBsAg inhibits TLR9-mediated activation and IFN-alpha production in plasmacytoid dendritic cells.
      PBMC↓ the interaction of MVP with MyD88 in infected cells, thus ↓ type I IFN responses
      • Liu S.
      • Peng N.
      • Xie J.
      • Hao Q.
      • Zhang M.
      • Zhang Y.
      • et al.
      Human hepatitis B virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways.
      NK cell↓ cytolytic activity
      • Martinet J.
      • Dufeu-Duchesne T.
      • Bruder Costa J.
      • Larrat S.
      • Marlu A.
      • Leroy V.
      • et al.
      Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
      T cellExhaustion of CD4+ and CD8+ T cells by ↑ of PD-1 and Lag-3 expression
      • Ye B.
      • Liu X.
      • Li X.
      • Kong H.
      • Tian L.
      • Chen Y.
      T-cell exhaustion in chronic hepatitis B infection: current knowledge and clinical significance.
      ,
      • Dong Y.
      • Li X.
      • Zhang L.
      • Zhu Q.
      • Chen C.
      • Bao J.
      • et al.
      CD4+ T cell exhaustion revealed by high PD-1 and LAG-3 expression and the loss of helper T cell function in chronic hepatitis B.
      B cell↓ TLR9 expression and B cell related functions
      • Tout I.
      • Gomes M.
      • Ainouze M.
      • Marotel M.
      • Pecoul T.
      • Durantel D.
      • et al.
      Hepatitis B virus blocks the CRE/CREB complex and prevents TLR9 transcription and function in human B cells.
      Monocyte/MDSC↑ the differentiation and ↑ the expansion of monocytes into MDSCs
      • Fang Z.
      • Li J.
      • Yu X.
      • Zhang D.
      • Ren G.
      • Shi B.
      • et al.
      Polarization of monocytic myeloid-derived suppressor cells by hepatitis B surface antigen is mediated via ERK/IL-6/STAT3 signaling Feedback and restrains the activation of T cells in chronic hepatitis B virus infection.
      AIM2, absent in melanoma 2; IFN, interferon; IL-, interleukin; KC, Kupffer cell; LAG-3, lymphocyte-activation gene 3; mDC, myeloid dendritic cell; MDSC, myeloid-derived suppressor cell; MVP, major vault protein; Myd88, Myeloid differentiation primary response 88; NK cell, natural killer cell; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death protein 1; pDC, plasmacytoid dendritic cell; TLR, Toll-like receptor.

      Innate immune cells

      Kupffer cells

      Kupffer cells (KCs) are the resident macrophages of the liver,
      • Naito M.
      • Hasegawa G.
      • Takahashi K.
      Development, differentiation, and maturation of Kupffer cells.
      accounting for approximately 20% of liver parenchymal cells. Their most important function is the removal of toxins from the circulating blood, but KCs can also effectively remove viruses, bacteria, and other pathogens mostly via tumour necrosis factor-α (TNF-α), interleukin-1 (IL)-1, IL-6, oxygen free radicals and the inflammasome. HBV and HBsAg can abrogate absent in melanoma 2 (AIM2) inflammasome responses by deregulating IRF7 (interferon regulatory factor 7) expression and binding on the AIM2 promoter in human KCs.
      • Zannetti C.
      • Roblot G.
      • Charrier E.
      • Ainouze M.
      • Tout I.
      • Briat F.
      • et al.
      Characterization of the inflammasome in human Kupffer cells in response to synthetic agonists and pathogens.
      Furthermore, KCs directly interacted with HBsAg in vivo and in vitro
      • Boltjes A.
      • van Montfoort N.
      • Biesta P.J.
      • Op den Brouw M.L.
      • Kwekkeboom J.
      • van der Laan L.J.W.
      • et al.
      Kupffer cells interact with hepatitis B surface antigen in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function.
      which induced the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and CXCL8 (C-X-C motif chemokine ligand 8).
      HBsAg-induced cytokine production by KCs and monocyte-derived macrophages and subsequent NK cell activation may be an early event in viral containment, potentially supporting the induction of HBV-specific immunity upon HBV infection.

      Dendritic cells

      DCs are crucial immune sentinels which orchestrate antiviral immunity. They can detect viruses and their components through multiple pattern recognition receptors (PRRs). DCs then produce large quantities of antiviral cytokines, especially type I and type III IFNs, and cooperate with other immune effectors, as well as performing cross-presentation and priming virus-specific cytotoxic T cells.
      • Patente T.A.
      • Pinho M.P.
      • Oliveira A.A.
      • Evangelista G.C.M.
      • Bergami-Santos P.C.
      • Barbuto J.A.M.
      Human dendritic cells: their heterogeneity and clinical application potential in cancer immunotherapy.
      Myeloid or conventional dendritic cells (cDCs) that express Toll-like receptor (TLR)3, TLR4 and TLR8 can be distinguished from plasmacytoid dendritic cells (pDCs) which express mainly TLR7 and TLR9.
      • Hémont C.
      • Neel A.
      • Heslan M.
      • Braudeau C.
      • Josien R.
      Human blood mDC subsets exhibit distinct TLR repertoire and responsiveness.
      • Sittig S.P.
      • Bakdash G.
      • Weiden J.
      • Sköld A.E.
      • Tel J.
      • Figdor C.G.
      • et al.
      A comparative study of the T cell stimulatory and polarizing capacity of human primary blood dendritic cell subsets.
      • Collin M.
      • Bigley V.
      Human dendritic cell subsets: an update.
      In CHB infection, functional perturbations in DCs have been described in numerous studies.
      • Martinet J.
      • Dufeu-Duchesne T.
      • Bruder Costa J.
      • Larrat S.
      • Marlu A.
      • Leroy V.
      • et al.
      Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
      ,
      • van der Molen R.G.
      • Sprengers D.
      • Binda R.S.
      • de Jong E.C.
      • Niesters H.G.M.
      • Kusters J.G.
      • et al.
      Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.
      • Op den Brouw M.L.
      • Binda R.S.
      • van Roosmalen M.H.
      • Protzer U.
      • Janssen H.L.A.
      • van der Molen R.G.
      • et al.
      Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus.
      • Woltman A.M.
      • Op den Brouw M.L.
      • Biesta P.J.
      • Shi C.C.
      • Janssen H.L.A.
      Hepatitis B virus lacks immune activating capacity, but actively inhibits plasmacytoid dendritic cell function.
      • Xu Y.
      • Hu Y.
      • Shi B.
      • Zhang X.
      • Wang J.
      • Zhang Z.
      • et al.
      HBsAg inhibits TLR9-mediated activation and IFN-alpha production in plasmacytoid dendritic cells.
      ,
      • van Montfoort N.
      • van der Aa E.
      • van den Bosch A.
      • Brouwers H.
      • Vanwolleghem T.
      • Janssen H.L.A.
      • et al.
      Hepatitis B virus surface antigen activates myeloid dendritic cells via a soluble CD14-dependent mechanism.
      ,
      • Vincent I.E.
      • Zannetti C.
      • Lucifora J.
      • Norder H.
      • Protzer U.
      • Hainaut P.
      • et al.
      Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells.
      Moreover, two recent studies showed that HBsAg can affect the maturation of DCs
      • Li X.
      • Zhou L.
      • Gu Y.
      • Chen L.
      • Gu L.
      • Huang Y.
      Quantative HBsAg level correlates dendritic cells maturation in chronic hepatitis B patients.
      and that HBV subverts DCs in both the blood and liver.
      • Ouaguia L.
      • Leroy V.
      • Dufeu-Duchesne T.
      • Durantel D.
      • Decaens T.
      • Hubert M.
      • et al.
      Circulating and hepatic BDCA1+, BDCA2+, and BDCA3+ dendritic cells are differentially subverted in patients with chronic HBV infection.
      A study on the peripheral blood mononuclear cells of patients undergoing HBsAg seroclearance showed increased DC frequencies with enhanced expression of TLRs, as well as increased CD8+ T cell and plasma B cell frequencies, suggesting that DCs may play a crucial role in HBsAg seroclearance.
      • Vyas A.K.
      • Sharma B.C.
      • Sarin S.K.
      • Trehanpati N.
      Immune correlates of hepatitis B surface antigen spontaneous seroconversion in hepatitis B e antigen negative chronic hepatitis B patients.

      NK cells

      NK cells are important immune lymphocytes in the liver, accounting for approximately one-third of intrahepatic lymphocytes.
      • Norris S.
      • Collins C.
      • Doherty D.G.
      • Smith F.
      • McEntee G.
      • Traynor O.
      • et al.
      Resident human hepatic lymphocytes are phenotypically different from circulating lymphocytes.
      NK cell receptors have activating or inhibitory properties upon engagement by molecules on the surface of target cells. The balance between these signals controls immediate effector functions: cytotoxicity and IFNγ secretion. They have an important role in the defence against intracellular pathogens and tumours
      • Vivier E.
      • Tomasello E.
      • Baratin M.
      • Walzer T.
      • Ugolini S.
      Functions of natural killer cells.
      and they play a crucial antiviral role during HBV infection.
      • Zhang J.
      • Chen Q.
      • Feng H.
      Relationship between chronic hepatitis B virus infection and nature killer cells.
      • Guidotti L.G.
      • Borrow P.
      • Hobbs M.V.
      • Matzke B.
      • Gresser I.
      • Oldstone M.B.
      • et al.
      Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver.
      • Xia Y.
      • Stadler D.
      • Lucifora J.
      • Reisinger F.
      • Webb D.
      • Hösel M.
      • et al.
      Interferon-γ and tumor necrosis factor-α produced by T cells reduce the HBV persistence form, cccDNA, without cytolysis.
      During CHB, NK cells have been shown to express an inhibitory phenotype with blunt functional responses
      • Lunemann S.
      • Malone D.F.G.
      • Hengst J.
      • Port K.
      • Grabowski J.
      • Deterding K.
      • et al.
      Compromised function of natural killer cells in acute and chronic viral hepatitis.
      and to mediate virus-specific CD8+ T cell depletion through a death receptor pathway.
      • Peppa D.
      • Gill U.S.
      • Reynolds G.
      • Easom N.J.W.
      • Pallett L.J.
      • Schurich A.
      • et al.
      Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell-mediated deletion.
      Furthermore, HBV can generate suppressive monocytes, which initiate regulatory NK cell differentiation resulting in T cell inhibition.
      • Li H.
      • Zhai N.
      • Wang Z.
      • Song H.
      • Yang Y.
      • Cui A.
      • et al.
      Regulatory NK cells mediated between immunosuppressive monocytes and dysfunctional T cells in chronic HBV infection.
      Changes in the NK cell phenotype may predict efficient immune reconstitution before anti-HBsAg seroconversion. Furthermore, induction of the proliferation and expansion of CD56 bright NK cell numbers in peripheral blood and increased cytotoxicity and IFNγ expression were associated with decreased serum HBsAg levels.
      • Micco L.
      • Peppa D.
      • Loggi E.
      • Schurich A.
      • Jefferson L.
      • Cursaro C.
      • et al.
      Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B.
      • Tan A.T.
      • Hoang L.T.
      • Chin D.
      • Rasmussen E.
      • Lopatin U.
      • Hart S.
      • et al.
      Reduction of HBV replication prolongs the early immunological response to IFNα therapy.
      • Gill U.S.
      • Peppa D.
      • Micco L.
      • Singh H.D.
      • Carey I.
      • Foster G.R.
      • et al.
      Interferon alpha induces sustained changes in NK cell responsiveness to hepatitis B viral load suppression in vivo.
      Increased NK cell function is associated with active hepatitis and HBsAg seroclearance following structured NA cessation.
      • Zimmer C.L.
      • Rinker F.
      • Höner Zu Siederdissen C.
      • Manns M.P.
      • Wedemeyer H.
      • Cornberg M.
      • et al.
      Increased NK cell function after cessation of long-term nucleos(t)ide analogue treatment in chronic hepatitis B is associated with liver damage and HBsAg loss.
      However, the role of NK cells in the process of HBsAg loss is still unknown.

      Adaptive immune cells

      Cytotoxic CD8+ T cells

      HBV-specific CD8+ T cell responses have been suggested to play an important role in viral clearance. In contrast to CD4+ T cells, CD8+ T cell responses have been studied more widely. The presence of strong and multiple CTL responses in the peripheral blood of patients with acute hepatitis B reinforced this concept,
      • Maini M.K.
      • Boni C.
      • Ogg G.S.
      • King A.S.
      • Reignat S.
      • Lee C.K.
      • et al.
      Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection.
      ,
      • Penna A.
      • Artini M.
      • Cavalli A.
      • Levrero M.
      • Bertoletti A.
      • Pilli M.
      • et al.
      Long-lasting memory T cell responses following self-limited acute hepatitis B.
      while such responses were barely detectable in the peripheral blood of patients with CHB.
      • Kennedy P.T.F.
      • Sandalova E.
      • Jo J.
      • Gill U.
      • Ushiro-Lumb I.
      • Tan A.T.
      • et al.
      Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B.
      T cell exhaustion has been documented in the literature: CTLs are unable to mediate complete eradication of the virus, and they subsequently recruit HBV-non-specific inflammatory cells, including bystander T cells, NK cells, and neutrophils, that inevitably cause the immunopathology of CHB.
      • Rehermann B.
      Pathogenesis of chronic viral hepatitis: differential roles of T cells and NK cells.
      The low clearance rate of HBsAg is possibly related to a weak CTL response to HBsAg. CD8+ T cell exhaustion in chronic HBV infection mirrors that described in other chronic viral infections in mice and humans, with the sustained expression of inhibitory receptors, such as PD-1, TIM-3 (T cell immunoglobulin and mucin-domain containing-3) and CD244 ( 2B4), reduced proliferative capacity and poor effector functions (reduced IFNγ and IL-2 secretion).
      • Ye B.
      • Liu X.
      • Li X.
      • Kong H.
      • Tian L.
      • Chen Y.
      T-cell exhaustion in chronic hepatitis B infection: current knowledge and clinical significance.
      ,
      • Reignat S.
      • Webster G.J.M.
      • Brown D.
      • Ogg G.S.
      • King A.
      • Seneviratne S.L.
      • et al.
      Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection.
      Inhibition of indoleamine 2,3-dioxygenase activity enhances the HBsAg-specific Tc1 immune response and induction of CTLs after immunisation with HBsAg, α-GalCer, IL-2, and IL-12β, which are vital cytokines for inducing the antigen-specific Tc1 response.
      • Ito H.
      • Ando T.
      • Ando K.
      • Ishikawa T.
      • Saito K.
      • Moriwaki H.
      • et al.
      Induction of hepatitis B virus surface antigen-specific cytotoxic T lymphocytes can be up-regulated by the inhibition of indoleamine 2, 3-dioxygenase activity.
      ,
      • Ito H.
      • Ando K.
      • Ishikawa T.
      • Nakayama T.
      • Taniguchi M.
      • Saito K.
      • et al.
      Role of Valpha14+ NKT cells in the development of Hepatitis B virus-specific CTL: activation of Valpha14+ NKT cells promotes the breakage of CTL tolerance.
      A genome-wide expression profiling of exhausted HBV-specific CD8+ T cells from patients with CHB revealed an extensively downregulated gene-expression programme when compared to functionally competent CD8+ T cells from patients who spontaneously resolved infection.
      • Fisicaro P.
      • Barili V.
      • Montanini B.
      • Acerbi G.
      • Ferracin M.
      • Guerrieri F.
      • et al.
      Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B.
      Among the various dysregulated processes, mitochondrial function seemed to be extensively defective, and its restoration by mitochondria-targeted antioxidants elicited functional T cell reconstitution. These results may lead to novel strategies for improving HBsAg clearance.
      HBsAg contributes to the deregulation of both innate and adaptive immune cells.

      Th CD4+ cells

      Virus-specific CD4+ T cells are key regulators of both efficient B cell/antibody and CD8+ T cell responses.
      • Milich D.R.
      • McLachlan A.
      • Thornton G.B.
      • Hughes J.L.
      Antibody production to the nucleocapsid and envelope of the hepatitis B virus primed by a single synthetic T cell site.
      ,
      • Böcher W.O.
      • Herzog-Hauff S.
      • Herr W.
      • Heermann K.
      • Gerken G.
      • Meyer Zum Büschenfelde K.H.
      • et al.
      Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection.
      However, in CHB infection, T cell responses are described as hardly detectable and display a functionally exhausted phenotype.
      • Dong Y.
      • Li X.
      • Zhang L.
      • Zhu Q.
      • Chen C.
      • Bao J.
      • et al.
      CD4+ T cell exhaustion revealed by high PD-1 and LAG-3 expression and the loss of helper T cell function in chronic hepatitis B.
      ,
      • Boni C.
      • Fisicaro P.
      • Valdatta C.
      • Amadei B.
      • Vincenzo P.D.
      • Giuberti T.
      • et al.
      Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection.
      T cells of patients with subsequent HBsAg loss, following cessation of NA treatment, had a less exhausted and more activated phenotype, compared to patients with retained HBsAg.
      • Rinker F.
      • Zimmer C.L.
      • Höner Zu Siederdissen C.
      • Manns M.P.
      • Kraft A.R.M.
      • Wedemeyer H.
      • et al.
      Hepatitis B virus-specific T cell responses after stopping nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B.
      T cells expressed low levels of PD-1 and KLRG1 (killer cell lectin like receptor G1). Furthermore, PD-1+ CD8+ T cells positively correlated with HBsAg levels at baseline. In HBV-infected mice and blood from patients with CHB, a T follicular helper (TFH)-cell response to HBsAg is required for HBV clearance, and blocking regulatory T (Treg) cell activity restored the ability of TFH cells to clear HBV infection.
      • Wang X.
      • Dong Q.
      • Li Q.
      • Li Y.
      • Zhao D.
      • Sun J.
      • et al.
      Dysregulated response of follicular helper T cells to hepatitis B surface antigen promotes HBV persistence in mice and associates with outcomes of patients.
      Circulating PD-1hi CXCR5+ CD4+ T cells were associated with decreased HBsAg levels in patients with CHB receiving PEG-IFNα therapy.
      • Zhang L.
      • Li H.
      • Ren H.
      • Hu P.
      Circulating PD-1hiCXCR5+CD4+ T cells are associated with a decrease in hepatitis B surface antigen levels in patients with chronic hepatitis B who are receiving peginterferon-α therapy.
      Indeed, HBV-specific IFNγ producing CD4+ T cells are associated with viral clearance in patients with CHB and their frequency was positively correlated with the decrease of HBsAg.
      • Wang H.
      • Luo H.
      • Wan X.
      • Fu X.
      • Mao Q.
      • Xiang X.
      • et al.
      TNF-α/IFN-γ profile of HBV-specific CD4 T cells is associated with liver damage and viral clearance in chronic HBV infection.
      Finally, in a cohort of 209 patients with CHB, there was a strong negative correlation between IL-4-secreting CD4+ T cells and quantitative HBsAg levels.
      • Gu Y.
      • Lian Y.
      • Gu L.
      • Chen L.
      • Li X.
      • Zhou L.
      • et al.
      Correlations between cytokines produced by T cells and clinical-virological characteristics in untreated chronic hepatitis B patients.

      B cells

      B cells are major contributors in the humoral immune response and have dual roles: being professional antigen-presenting cells, they recognise antigens and prime T cells. They also differentiate into memory cells and antibody-producing plasma cells which are responsible for the production of antibodies. B cells play a vital role during HBV infection by secreting anti-HBsAg, a sign of the resolution of the infection. Conversely, HBsAg could disrupt the mechanisms of innate and adaptive immunity and result in the suppression of immune responses against HBV.
      • Tout I.
      • Gomes M.
      • Ainouze M.
      • Marotel M.
      • Pecoul T.
      • Durantel D.
      • et al.
      Hepatitis B virus blocks the CRE/CREB complex and prevents TLR9 transcription and function in human B cells.
      However, while the quantity and function of HBV-specific T cells have been clearly defined in patients with CHB,
      • Fisicaro P.
      • Barili V.
      • Montanini B.
      • Acerbi G.
      • Ferracin M.
      • Guerrieri F.
      • et al.
      Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B.
      ,
      • Bengsch B.
      • Martin B.
      • Thimme R.
      Restoration of HBV-specific CD8+ T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation.
      less attention has been paid to the role of neutralising anti-HBsAg and detailed characterisation of the anti-HBV-specific B cell response is lacking. In addition, B cell-depleting drugs, such as the anti-CD20 antibody rituximab, induce HBV reactivation in HBV carriers and even in patients with “resolved” HBV in whom nuclear HBV cccDNA persists as a viral reservoir for decades.
      • Loomba R.
      • Liang T.J.
      Hepatitis B reactivation associated with immune suppressive and biological modifier therapies: current concepts, management strategies, and future directions.
      B cells from patients with CHB had a reduced proliferative capacity and were incapable of producing anti-HBsAg upon stimulation. This functional defect reverted after HBsAg loss.
      • Xu X.
      • Shang Q.
      • Chen X.
      • Nie W.
      • Zou Z.
      • Huang A.
      • et al.
      Reversal of B-cell hyperactivation and functional impairment is associated with HBsAg seroconversion in chronic hepatitis B patients.
      ,
      • Oliviero B.
      • Cerino A.
      • Varchetta S.
      • Paudice E.
      • Pai S.
      • Ludovisi S.
      • et al.
      Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections.
      Recently, two studies were able to characterise HBsAg-specific B cells. While B cells were present in similar frequencies in chronic and resolved infections, they were unable to mature into anti-HBsAg-secreting cells in CHB. Instead, they presented functional alterations that resemble atMBCs, with low expression of CD21 and CD27 and high expression of PD-1 and T-bet.
      • Burton A.R.
      • Pallett L.J.
      • McCoy L.E.
      • Suveizdyte K.
      • Amin O.E.
      • Swadling L.
      • et al.
      Circulating and intrahepatic antiviral B cells are defective in hepatitis B.
      ,
      • Salimzadeh L.
      • Le Bert N.
      • Dutertre C.-A.
      • Gill U.S.
      • Newell E.W.
      • Frey C.
      • et al.
      PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.
      The function of these HBsAg-specific B cells can be partially restored in vitro by PD-1 blockade.
      • Salimzadeh L.
      • Le Bert N.
      • Dutertre C.-A.
      • Gill U.S.
      • Newell E.W.
      • Frey C.
      • et al.
      PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.
      These results suggest that B cell dysfunction rather than antibody depletion is the main reason for the lack of anti-HBsAg; thus, B cells are a possible target for novel antiviral strategies.

      Regulatory cells

      Treg cells are a specialised subpopulation of T cells that act to suppress T cell proliferation and cytokine production, thereby maintaining homeostasis. HBsAg can enhance Treg cell activity and mDC costimulatory molecules and can suppress monocyte activation and pDC function.
      • Kondo Y.
      • Ninomiya M.
      • Kakazu E.
      • Kimura O.
      • Shimosegawa T.
      Hepatitis B surface antigen could contribute to the immunopathogenesis of hepatitis B virus infection.
      Monocytic MDSCs are strong inhibitors of the T cell response.
      • Kong X.
      • Sun R.
      • Chen Y.
      • Wei H.
      • Tian Z.
      γδT cells drive myeloid-derived suppressor cell-mediated CD8+ T cell exhaustion in hepatitis B virus-induced immunotolerance.
      A recent study found that HBsAg levels were positively correlated with monocytic MDSC frequency in patients with CHB and that HBsAg can maintain HBV persistence by increasing the differentiation of monocytes into monocytic MDSCs.
      • Fang Z.
      • Li J.
      • Yu X.
      • Zhang D.
      • Ren G.
      • Shi B.
      • et al.
      Polarization of monocytic myeloid-derived suppressor cells by hepatitis B surface antigen is mediated via ERK/IL-6/STAT3 signaling Feedback and restrains the activation of T cells in chronic hepatitis B virus infection.
      The polarisation of MDSCs by HBsAg can restrain the activation of T cells in CHB infection.
      • Fang Z.
      • Li J.
      • Yu X.
      • Zhang D.
      • Ren G.
      • Shi B.
      • et al.
      Polarization of monocytic myeloid-derived suppressor cells by hepatitis B surface antigen is mediated via ERK/IL-6/STAT3 signaling Feedback and restrains the activation of T cells in chronic hepatitis B virus infection.
      Despite these data, several key questions remain unanswered. The ideal would be to determine immune parameters associated with persistence, clearance and recurrence of HBV and to study the function and phenotype of both peripheral and intrahepatic lymphocyte populations, as well as hepatocytes, which may aid in the rational design of immunotherapeutic strategies.
      The decrease of HBsAg can potentially restore anti-HBV immune responses.

      Cytokines and HBsAg

      Cytokines act as key coordinators of the inflammatory responses involved in HBV pathogenesis. IL-6 is produced by a variety of cells and is involved in many biological processes, including induction of cell differentiation, generation of B immunoglobulin, promotion of T cell proliferation. A recent study showed that IL-6 polymorphisms 572G/C and -597G/A are significantly associated with CHB risk.
      • Riazalhosseini B.
      • Mohamed Z.
      • Apalasamy Y.D.
      • Shafie N.S.
      • Mohamed R.
      Interleukin-6 gene variants are associated with reduced risk of chronicity in hepatitis B virus infection in a Malaysian population.
      Furthermore, Bouezzedine et al. show that IL-6 can strongly inhibit HBsAg secretion which was confirmed by observation of a severe reduction in cccDNA after IL-6 treatment.
      • Bouezzedine F.
      • Fardel O.
      • Gripon P.
      Interleukin 6 inhibits HBV entry through NTCP down regulation.
      IL-12 also has numerous roles: promoting the differentiation of Th1 cells, enhancing NK cell cytotoxicity and activation of IFNγ pathways, and rescuing the antiviral function of exhausted HBV-specific T cells.
      • Schurich A.
      • Pallett L.J.
      • Lubowiecki M.
      • Singh H.D.
      • Gill U.S.
      • Kennedy P.T.
      • et al.
      The third signal cytokine IL-12 rescues the anti-viral function of exhausted HBV-specific CD8 T cells.
      Direct interaction of HBsAg with human monocytes and macrophages regulates the production of IL-12.
      • Fang Z.
      • Li J.
      • Yu X.
      • Zhang D.
      • Ren G.
      • Shi B.
      • et al.
      Polarization of monocytic myeloid-derived suppressor cells by hepatitis B surface antigen is mediated via ERK/IL-6/STAT3 signaling Feedback and restrains the activation of T cells in chronic hepatitis B virus infection.

      TLRs and HBsAg

      The early and non-specific detection of pathogens generally occurs at subcellular/molecular levels, via the recognition of pathogen-associated molecular patterns by innate immunity sensors, also called PRRs expressed in various types of epithelial/endothelial cells, as well as professional and non-professional immune cells.
      • Crump K.E.
      • Sahingur S.E.
      Microbial nucleic acid sensing in oral and systemic diseases.
      Among PRRs, TLRs belong to a conserved family of transmembrane glycoprotein receptors capable of sensing a wide variety of pathogen- and damage-associated molecular patterns. Viral proteins have been suggested to interfere with innate signalling pathways in hepatocytes and immune cells. In fact, HBsAg is capable of impairing the activation of all TLR pathways.
      • Jiang M.
      • Broering R.
      • Trippler M.
      • Poggenpohl L.
      • Fiedler M.
      • Gerken G.
      • et al.
      Toll-like receptor-mediated immune responses are attenuated in the presence of high levels of hepatitis B virus surface antigen.
      HBsAg downregulates TLR9 in pDCs leading to the inhibition of IFNα production.
      • Vincent I.E.
      • Zannetti C.
      • Lucifora J.
      • Norder H.
      • Protzer U.
      • Hainaut P.
      • et al.
      Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells.
      ,
      • Xu N.
      • Yao H.-P.
      • Lv G.-C.
      • Chen Z.
      Downregulation of TLR7/9 leads to deficient production of IFN-α from plasmacytoid dendritic cells in chronic hepatitis B.
      In B cells, HBsAg has been shown to downregulate TLR9 expression and function, leading to deficient TLR9-mediated B cell responses.
      • Tout I.
      • Gomes M.
      • Ainouze M.
      • Marotel M.
      • Pecoul T.
      • Durantel D.
      • et al.
      Hepatitis B virus blocks the CRE/CREB complex and prevents TLR9 transcription and function in human B cells.
      Moreover, HBsAg can selectively inhibit TLR2 ligand–induced IL-12 production in monocytes/macrophages by interfering with JNK (c-Jun N-terminal protein kinase) activation.
      • Wang S.
      • Chen Z.
      • Hu C.
      • Qian F.
      • Cheng Y.
      • Wu M.
      • et al.
      Hepatitis B virus surface antigen selectively inhibits TLR2 ligand-induced IL-12 production in monocytes/macrophages by interfering with JNK activation.
      These data may suggest that strategies targeting HBsAg production or secretion may lead to the restoration of an efficient immune response in patients with CHB and therefore combination therapy should be considered. Fig. 3 represents the proposed immune mechanisms of HBsAg seroclearance, from decreased HBsAg levels to the potential restoration of anti-HBV immune responses.
      Figure thumbnail gr3
      Fig. 3Proposed immune mechanisms for HBsAg seroclearance.
      Decreased HBsAg levels could facilitate the recovery of the host's immune system. mDCs regain their antigen-presenting capacities to activate T cells, as well as TNFα secretion. pDCs restore TNFα and IFNγ secretion and activate NK cells. Restoration of NK cell effector functions: cytotoxicity and IFNγ secretion, activation of T cells. Recovery from T cell exhaustion: restored proliferation, increase in HBsAg-specific CTLs, direct cytolytic activity on infected hepatocytes and IFNs secretion. Suppression of excessive functions of Treg cells and MDSCs. KCs induce cytokine production, have restored inflammasome functions and activate NK cells. Restoration of functional HBsAg-specific plasmocytes secreting neutralising anti-HBsAg. Upward green arrows signify a restoration of function and/or frequency while downward red arrows signify a decrease in function and/or frequency. Anti-HBsAg, hepatitis B surface antigen antibodies; CTLA4, cytotoxic T lymphocyte-associated protein 4; IFN, interferon; IL-, interleukin; KC, Kupffer cell; mDC, myeloid dendritic cell; MDSC, myeloid-derived suppressor cell; NK cell, natural killer cell; PD-1, programmed cell death protein 1; pDC, plasmacytoid dendritic cell; TCR, T cell receptor; TGF-β, transforming growth factor-β; Th, T helper; TNF-α, tumour necrosis factor alpha; Treg, regulatory T.

      HBsAg seroclearance in animal models

      Animal models are important tools to explore mechanisms of HBV immunopathogenesis and to evaluate new therapies. For example, chimpanzees chronically infected with HBV were used to test the efficacy of GS-9620, a TLR7 agonist.
      • Lanford R.E.
      • Guerra B.
      • Chavez D.
      • Giavedoni L.
      • Hodara V.L.
      • Brasky K.M.
      • et al.
      GS-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees.
      A 100-fold decline in viral load and a dramatic drop in the number of HBsAg-positive cells was observed during therapy. A phase II study with GS-9620, in virally suppressed patients on NAs showed evidence of immune activation but no significant decline in HBsAg.
      • Janssen H.L.A.
      • Brunetto M.R.
      • Kim Y.J.
      • Ferrari C.
      • Massetto B.
      • Nguyen A.-H.
      • et al.
      Safety, efficacy and pharmacodynamics of vesatolimod (GS-9620) in virally suppressed patients with chronic hepatitis B.
      Another study assessed the efficacy of ARC-520, an RNAi antiviral targeting HBV transcripts including HBsAg, in combination with NAs, and reported a profound reduction in HBsAg, with a maximum reduction of more than 2 logs.
      • Wooddell C.I.
      • Yuen M.-F.
      • Chan H.L.-Y.
      • Gish R.G.
      • Locarnini S.A.
      • Chavez D.
      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      Additionally, a novel long-acting modified IFNα (PASylated-IFNα) induced anti-HBsAg seroconversion in HBV-transgenic mice after 3 weeks.
      • Xia Y.
      • Schlapschy M.
      • Morath V.
      • Roeder N.
      • Vogt E.I.
      • Stadler D.
      • et al.
      PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.
      Recently, GLP-26, a capsid assembly modulator, was tested in a humanised mouse model in combination with entecavir (ETV) and induced a decrease in viral load and viral antigens that was sustained for up to 12 weeks after treatment cessation.
      • Amblard F.
      • Boucle S.
      • Bassit L.
      • Cox B.
      • Sari O.
      • Tao S.
      • et al.
      Novel hepatitis B virus capsid assembly modulator induces potent antiviral responses in vitro and in Humanized mice.

      HBsAg seroclearance and current therapies

      The goal of treatment is to improve survival, by preventing the risk of end-stage liver disease and HCC, and to improve quality of life. However, it is difficult to demonstrate improvements in survival and surrogate markers are needed. HBsAg seroclearance is a surrogate marker of survival and is therefore the ideal endpoint for treatment. Current treatments include PEG-IFN, which have been shown to exert dual actions, including an immunomodulatory effect and minimal direct antiviral activity against HBV or NAs such as lamivudine, telbivudine, adefovir dipivoxil, ETV, tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF), which directly target the reverse transcription functions of the viral polymerase, and thus suppress HBV replication effectively, when used as monotherapy. Both approaches offer limited efficacy in achieving HBsAg loss.
      • Schinazi R.F.
      • Ehteshami M.
      • Bassit L.
      • Asselah T.
      Towards HBV curative therapies.
      PEG-IFN has the advantage of inducing sustained response after a defined course of treatment (usually 48 weeks), although response rates and tolerability are poor. NAs require life-long administration because they do not eliminate the persistent cccDNA within the infected hepatocytes. Among NAs, ETV, TDF and TAF are preferred as first choice therapy because of their high antiviral potency and the low risk of resistance. A significantly greater proportion of patients receiving TDF plus PEG-IFN for 48 weeks had HBsAg loss than those receiving TDF or PEG-IFN alone, and this combination is suitable in a subset of patients.
      • Marcellin P.
      • Ahn S.H.
      • Ma X.
      • Caruntu F.A.
      • Tak W.Y.
      • Elkashab M.
      • et al.
      Combination of tenofovir disoproxil fumarate and peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B.
      However, the observed rate of HBsAg loss in the study was lower than that assumed in the study design, reducing the power for comparison between groups. Finally, prolonged follow-up of patients who had not restarted TDF treatment would be required to determine the long-term benefits of response and durability of outcome. Further studies are required to identify the optimal combination regimen that would allow more patients to achieve and sustain HBsAg loss.

      HBsAg seroclearance during IFN therapy

      A 48-week treatment with PEG-IFN has the potential to elicit immune control of HBV infection, leading to higher rates of HBeAg seroconversion (than achieved with NAs) and the possibility of viral suppression after stopping treatment, with HBsAg loss in a proportion of patients who maintain undetectable HBV DNA. After PEG-IFN, sustained off-therapy virological response (SVR) is defined as serum HBV DNA levels <2,000 IU/ml after the end of therapy. In HBeAg-negative patients with CHB, a phase III trial evaluating PEG-IFN monotherapy reported SVR rates of 44% at 6 months and 28% at 3 years after the end of therapy.
      • Marcellin P.
      • Lau G.K.K.
      • Bonino F.
      • Farci P.
      • Hadziyannis S.
      • Jin R.
      • et al.
      Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B.
      In HBeAg-negative patients with genotype D or E, PEG-IFN was less effective with an SVR of around 20%. The rate of HBsAg loss progressively increased after PEG-IFNα discontinuation, from 3% at month 6, to 9% at year 3, and to 12% at year 5 in the registrational trial. Overall, among sustained responders, approximately 30% clear HBsAg in the long-term.
      Combination of antivirals and immune therapy is crucial for drug development.
      HBsAg loss rates increase after the end of PEG-IFNα therapy in initially HBeAg-positive patients with SVR.
      • Lau G.K.K.
      • Piratvisuth T.
      • Luo K.X.
      • Marcellin P.
      • Thongsawat S.
      • Cooksley G.
      • et al.
      Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B.
      Of the patients with an initial HBeAg seroclearance, 30% experienced HBsAg seroclearance after 3 years of follow-up. The sustainability of HBsAg loss and seroconversion after PEG-IFNα have been documented.
      • Marcellin P.
      • Ahn S.H.
      • Ma X.
      • Caruntu F.A.
      • Tak W.Y.
      • Elkashab M.
      • et al.
      Combination of tenofovir disoproxil fumarate and peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B.
      HBsAg loss was evaluated in patients receiving the combination of TDF and PEG-IFNα-2a for a finite duration in a randomised trial, and compared to TDF monotherapy and PEG-IFN monotherapy.
      • Marcellin P.
      • Ahn S.H.
      • Ma X.
      • Caruntu F.A.
      • Tak W.Y.
      • Elkashab M.
      • et al.
      Combination of tenofovir disoproxil fumarate and peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B.
      A total of 740 patients with CHB were randomly assigned to receive TDF plus PEG-IFN for 48 weeks, TDF plus PEG-IFN for 16 weeks followed by TDF for 32 weeks, TDF for 120 weeks, or PEG-IFN for 48 weeks. At week 72, 9% of patients in the group receiving TDF plus PEG-IFN for 48 weeks had HBsAg loss compared with less than 3% in the other groups.

      HBsAg seroclearance during NA therapy

      ETV and TDF are potent HBV inhibitors with a high barrier to resistance and should be used as first-line monotherapies.
      • Papatheodoridis G.V.
      • Sypsa V.
      • Dalekos G.
      • Yurdaydin C.
      • van Boemmel F.
      • Buti M.
      • et al.
      Eight-year survival in chronic hepatitis B patients under long-term entecavir or tenofovir therapy is similar to the general population.
      More than 95% of patients treated with the highly potent TDF and ETV achieve virological undetectability. NAs are administered orally, tolerability is favourable and efficacy is good.
      • Marcellin P.
      • Asselah T.
      Long-term therapy for chronic hepatitis B: hepatitis B virus DNA suppression leading to cirrhosis reversal.
      In a mainly Caucasian population of HBeAg-positive patients, HBsAg loss was around 10% after 5 years of TDF and was more likely to occur in Caucasian patients.
      • Marcellin P.
      • Buti M.
      • Krastev Z.
      • de Man R.A.
      • Zeuzem S.
      • Lou L.
      • et al.
      Kinetics of hepatitis B surface antigen loss in patients with HBeAg-positive chronic hepatitis B treated with tenofovir disoproxil fumarate.
      No HBsAg loss was observed after 2 years of TDF or ETV. Table 2 represents the spontaneous and after treatment HBsAg seroclearance rates reported to date (Table 2).
      Table 2Spontaneous and after treatment HBsAg seroclearance.
      ModeTreatmentTherapy duration (weeks)Follow-up duration (weeks)Number of participants (n)HBsAg seroclearance (%)Reference(s)
      Spontaneousn.a.n.a.260–5201,965–42,5884.03–8.1
      • Chu C.-M.
      • Liaw Y.-F.
      HBsAg seroclearance in asymptomatic carriers of high endemic areas: appreciably high rates during a long-term follow-up.
      ,
      • Zhou K.
      • Contag C.
      • Whitaker E.
      • Terrault N.
      Spontaneous loss of surface antigen among adults living with chronic hepatitis B virus infection: a systematic review and pooled meta-analyses.
      Under current treatment (monotherapy)ETV52–26052–260146–7091.4–5.1
      • Chang T.-T.
      • Lai C.-L.
      • Kew Yoon S.
      • Lee S.S.
      • Coelho H.S.M.
      • Carrilho F.J.
      • et al.
      Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B.
      • Gish R.G.
      • Chang T.-T.
      • Lai C.-L.
      • de Man R.
      • Gadano A.
      • Poordad F.
      • et al.
      Loss of HBsAg antigen during treatment with entecavir or lamivudine in nucleoside-naïve HBeAg-positive patients with chronic hepatitis B.
      • Chang T.-T.
      • Gish R.G.
      • de Man R.
      • Gadano A.
      • Sollano J.
      • Chao Y.-C.
      • et al.
      A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B.
      TDF48–26048–260266–5853–11
      • Marcellin P.
      • Buti M.
      • Krastev Z.
      • de Man R.A.
      • Zeuzem S.
      • Lou L.
      • et al.
      Kinetics of hepatitis B surface antigen loss in patients with HBeAg-positive chronic hepatitis B treated with tenofovir disoproxil fumarate.
      ,
      • Marcellin P.
      • Gane E.
      • Buti M.
      • Afdhal N.
      • Sievert W.
      • Jacobson I.M.
      • et al.
      Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label follow-up study.
      ,
      • Marcellin P.
      • Heathcote E.J.
      • Buti M.
      • Gane E.
      • de Man R.A.
      • Krastev Z.
      • et al.
      Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B.
      TAF96965761
      • Agarwal K.
      • Brunetto M.
      • Seto W.K.
      • Lim Y.-S.
      • Fung S.
      • Marcellin P.
      • et al.
      96 weeks treatment of tenofovir alafenamide vs. tenofovir disoproxil fumarate for hepatitis B virus infection.
      PEG-IFN48–5248–52136–1774–7
      • Marcellin P.
      • Lau G.K.K.
      • Bonino F.
      • Farci P.
      • Hadziyannis S.
      • Jin R.
      • et al.
      Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B.
      ,
      • Janssen H.L.A.
      • van Zonneveld M.
      • Senturk H.
      • Zeuzem S.
      • Akarca U.S.
      • Cakaloglu Y.
      • et al.
      Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial.
      Combination therapyETV+TDF92–969657–1971.7–3.6
      • Petersen J.
      • Ratziu V.
      • Buti M.
      • Janssen H.L.A.
      • Brown A.
      • Lampertico P.
      • et al.
      Entecavir plus tenofovir combination as rescue therapy in pre-treated chronic hepatitis B patients: an international multicenter cohort study.
      ,
      • Lok A.S.
      • Trinh H.
      • Carosi G.
      • Akarca U.S.
      • Gadano A.
      • Habersetzer F.
      • et al.
      Efficacy of entecavir with or without tenofovir disoproxil fumarate for nucleos(t)ide-naïve patients with chronic hepatitis B.
      PEG-IFN+ETV4848851.17
      • Brouwer W.P.
      • Xie Q.
      • Sonneveld M.J.
      • Zhang N.
      • Zhang Q.
      • Tabak F.
      • et al.
      Adding pegylated interferon to entecavir for hepatitis B e antigen-positive chronic hepatitis B: a multicenter randomized trial (ARES study).
      PEG-IFN+TDF48721869.1
      • Marcellin P.
      • Ahn S.H.
      • Ma X.
      • Caruntu F.A.
      • Tak W.Y.
      • Elkashab M.
      • et al.
      Combination of tenofovir disoproxil fumarate and peginterferon α-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B.
      ETV, entecavir; n.a., not applicable; PEG-IFN, pegylated interferon; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate.

      HBsAg decrease as a predictor of treatment response

      To identify responders in the early phase of PEG-IFNα-2a therapy, decreased serum quantitative HBsAg is a validated on-treatment marker predicting sustained off-treatment response.
      • Martinot-Peignoux M.
      • Lapalus M.
      • Asselah T.
      • Marcellin P.
      HBsAg quantification: useful for monitoring natural history and treatment outcome.
      In a proof of concept study of 48 HBeAg-negative patients receiving PEG-IFNα-2a, a decrease of 0.5 and 1 log10 IU/ml of serum HBsAg levels at weeks 12 and 24 of therapy had a 90% negative predictive value and a 89% positive predictive value for week 12 and 97% negative predictive value and a 92% positive predictive value for sustained response at week 24, respectively.
      • Moucari R.
      • Mackiewicz V.
      • Lada O.
      • Ripault M.-P.
      • Castelnau C.
      • Martinot-Peignoux M.
      • et al.
      Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.
      This was the first study to suggest that the early kinetics (week 12) of HBsAg might differentiate sustained responders from non-responders to PEG-IFN.

      The role of HBsAg in NA cessation

      Interestingly, in a retrospective study, Chen et al. evaluated the role of HBsAg quantification in predicting HBsAg loss and HBV relapse.
      • Chen C.-H.
      • Lu S.-N.
      • Hung C.-H.
      • Wang J.-H.
      • Hu T.-H.
      • Changchien C.-S.
      • et al.
      The role of hepatitis B surface antigen quantification in predicting HBsAg loss and HBV relapse after discontinuation of lamivudine treatment.
      End of treatment HBsAg levels of <300 IU/ml, 300–1,000 IU/ml and >1000 UI/ml in HBeAg-positive patients were associated with sustained HBeAg loss in 55.6%, 7.7% and 3.3%, respectively. End of treatment HBsAg cut-offs <120 IU/ml, 120–1,000 IU/ml and >1,000 IU/ml in HBeAg-negative patients were associated with HBsAg loss in 79.2%, 14.3% and 0%, respectively, and HBsAg cut-offs <200 IU/ml, 200–1,000 IU/ml and >1,000 IU/ml were associated with an SVR in 93%, 11.1% and 15.4%, respectively. Similar results were reported in patients whose treatment was discontinued; an end of treatment HBsAg level <100 IU/ml was associated with high SVR, while >1,000 IU/ml was associated with a 1-year post-treatment relapse in 70%.
      • Liang Y.
      • Jiang J.
      • Su M.
      • Liu Z.
      • Guo W.
      • Huang X.
      • et al.
      Predictors of relapse in chronic hepatitis B after discontinuation of anti-viral therapy.
      A systemic review to summarise the role of HBsAg in NA cessation among Asian patients with CHB showed that HBsAg loss ranged between 21.1–58-8% in patients with HBsAg <100 IU/ml, compared to 3.3–7.4% in patients with HBsAg >100 IU/ml.
      • Liu J.
      • Li T.
      • Zhang L.
      • Xu A.
      The role of hepatitis B surface antigen in nucleos(t)ide analogues cessation among Asian patients with chronic hepatitis B: a systematic review.

      HBsAg seroclearance and novel therapies

      Several strategies, including antivirals targeting various stages of the HBV replication cycle (HBV entry, viral replication, cccDNA production, and viral protein expression), as well as immunotherapeutic agents, are being explored in experimental models or have reached clinical testing, which may have the potential to complement PEG-IFN- and NA-based therapy.
      • Asselah T.
      • Loureiro D.
      • Boyer N.
      • Mansouri A.
      Targets and future direct-acting antiviral approaches to achieve hepatitis B virus cure.
      ,
      • Bertoletti A.
      • Le Bert N.
      Immunotherapy for chronic hepatitis B virus infection.
      • Fanning G.C.
      • Zoulim F.
      • Hou J.
      • Bertoletti A.
      Therapeutic strategies for hepatitis B virus infection: towards a cure.
      • Boni C.
      • Janssen H.L.A.
      • Rossi M.
      • Yoon S.K.
      • Vecchi A.
      • Barili V.
      • et al.
      Combined GS-4774 and tenofovir therapy can improve HBV-specific T-cell responses in patients with chronic hepatitis.
      Strategies inhibiting viral gene expression either through cccDNA transcription or viral mRNA translation can decrease serum HBsAg levels. Nucleic acid-based polymers (NAPs) are a new class of broad-spectrum antiviral compounds which act against HBV infection by blocking the release of HBsAg from infected hepatocytes.
      • Vaillant A.
      Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.
      This pharmacological activity blocks replenishment of HBsAg in the circulation, enabling host-mediated clearance and has important clinical significance as it may potentiate the ability of immunotherapies (resumed in Fig. 4) to restore functional control of HBV infection. Removal of HBsAg would enhance the effect of PEG-IFNα-2a and could lead to favourable immunological activation, the appearance of free anti-HBsAg and the clearance of HBV virions in the blood. Among HBsAg-targeting antiviral strategies (Fig. 5), NAPs are being investigated in preclinical evaluations and in several clinical trials that have evaluated the activity of REP 2139, REP 2055 and REP 2165 in monotherapy and in combination with immunotherapy.
      • Vaillant A.
      Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.
      • Bazinet M.
      • Pântea 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.
      • 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.
      • Bazinet M.
      • Pântea V.
      • Placinta G.
      • Moscalu I.
      • Cebotarescu V.
      • Cojuhari L.
      • et al.
      Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naïve to nucleos(t)ide therapy.
      Out of 12 patients, a substantial reduction of HBsAg and seroconversion to anti-HBsAg was observed in response to REP 2139-Ca in 9 patients (NCT02646189).
      • Bazinet M.
      • Pântea 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.
      Recently, an open label, randomised, controlled, phase II study reported on the use of 2 different NAPs, REP 2139-Mg or REP 2165-Mg in association with PEG-IFN and TDF in 40 patients with CHB. No severe adverse events were reported. Efficacy was impressive with around half of the patients achieving HBsAg seroconversion.
      • Bazinet M.
      • Pântea V.
      • Placinta G.
      • Moscalu I.
      • Cebotarescu V.
      • Cojuhari L.
      • et al.
      Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naïve to nucleos(t)ide therapy.
      These interesting results need to be confirmed in larger studies. Furthermore, the potential for cytotoxicity resulting from intracellular retention of HBsAg should be investigated and the problem of NAPs accumulating in the liver should be addressed.
      Figure thumbnail gr4
      Fig. 4Developed immune-based approaches to clear HBV.
      (1) Therapeutic vaccination could restore dysfunctional T and B cell responses during CHB. (2) Stimulation of innate immunity by (i) TLRs and RIG-I agonists leading to the activation of hepatocytes, intrahepatic dendritic, NK and mucosal-associated invariant T cells. (ii) TCR-like antibodies allow direct recognition of HBV-infected hepatocytes. (iii) Cytokines inhibit HBV replication. (3) Antibody-mediated neutralisation could prevent HBV infection of hepatocytes and reduce HBsAg circulating levels. (4) Anti-HBV T cell boosting by (i) T cell engineering. (ii) and (iii) Checkpoint blockade, modulation of regulatory cells and metabolic modulation. (5) Functional maturation of dysfunctional HBsAg-specific B cells by boosting Th cells or anti-PD-1 therapy. CTLA4, cytotoxic T lymphocyte-associated protein 4; DC, dendritic cell; IFN, interferon; IL-, interleukin; KC, Kupffer cell; LAG-3, lymphocyte-activation gene 3; NKT cell, natural killer T cell; PD-1, programmed cell death protein 1; RIG-1, retinoic acid-inducible gene 1; TCR, T cell receptor; TGF-β, transforming growth factor-β; Th, T helper; Tim-3, T cell immunoglobulin and mucin-domain containing-3; TNF-α, tumour necrosis factor alpha; Treg, regulatory T.
      Figure thumbnail gr5
      Fig. 5HBsAg-targeting antiviral approaches.
      Schematic representation of HBsAg-targeting strategies. HBV replication cycle is shown, and different approaches are developed to reduce high levels of HBsAg in patients with CHB. (1) Inhibition of HBsAg release by NAPs. (2) Silencing and eliminating cccDNA. Targets against cccDNA include antiviral cytokines (PEG-IFN), blockade of rcDNA and epigenetic regulation to undergo its degradation. Technologies such as CRISPR/Cas9 are being utilised to eliminate cccDNA along with the use of histone deacetylase inhibitors. (3) Suppression of HBsAg expression by HBV-targeting siRNA. cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; ER, endoplasmic reticulum; NAPs, nucleic acid polymers; NTCP, sodium taurocholate co-transporting polypeptide; PEG-IFN, pegylated interferon; rcDNA, relaxed circular DNA; siRNA, small interfering RNA.

      Conclusion

      HBsAg seroclearance is a rare event in the natural history of HBV and it is associated with reduced risk of HCC. Current antiviral therapies using PEG-IFN and NAs can suppress HBV replication and improve the prognosis of CHB, but they fail to clear HBsAg. Several guidelines proposed HBsAg seroclearance as a crucial surrogate marker of thorough HBV clearance. HBsAg may contribute to the impairment of innate and adaptive immunity and the exhaustion of T cell and B cell responses. Therefore, a reduced serum HBsAg level could facilitate the recovery of the host's immune system. Numerous immune cells have been shown to interact with HBsAg, contributing to the immunopathogenesis of CHB, but the immune mechanisms underlying HBsAg seroclearance are still unclear. A better understanding of the interaction between HBsAg and these immune factors could contribute to the development of effective immunotherapies.
      The presence of cccDNA and integrated DNA on one hand, and the particular liver microenvironment and immune deregulations induced by chronic infection on another hand, are key challenges for antiviral approaches.
      • Fanning G.C.
      • Zoulim F.
      • Hou J.
      • Bertoletti A.
      Therapeutic strategies for hepatitis B virus infection: towards a cure.
      Overall, the safety and efficacy of the newly developed strategies, whether antivirals or immune based, need to be tested, and their ability to remove HBsAg needs to be investigated. Strategies focusing on reducing HBsAg by siRNA, NAPs or antibody-mediated neutralisation could be crucial in the restoration of effective immune responses. Developing effective combination therapies that target HBsAg may further induce the appearance of an anti-HBV-specific immune response and lead to a functional cure for CHB.

      Abbreviations

      ALT, alanine aminotransferase; anti-HBsAg, hepatitis B surface antigen antibodies; AST, aspartate aminotransferase; AIM2, absent in melanoma 2; atMBC, atypical memory B cells; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; CTL, cytotoxic T cell; CTLA4, cytotoxic T lymphocyte-associated protein 4; DCs, dendritic cells; ETV, entecavir; HCC, hepatocellular carcinoma; hNTCP, human sodium taurocholate co-transporting polypeptide; HSPG, heparan sulphate proteoglycan; IFN, interferon-; IL-, interleukin-; KC, Kupffer cells; LAG-3, lymphocyte-activation gene 3; L-HBsAg, large HBsAg; mDC, myeloid dendritic cell; MDSCs, myeloid-derived suppressor cells; M-HBsAg, medium HBsAg; MVP, major vault protein; MyD88, myeloid differentiation primary response 88; NA, nucleoside analogue; NAP, nucleic acid polymer; NK cells, natural killer cells; ORF, open reading frames; PBMCs, peripheral blood mononuclear cells; PD-1, programmed cell death protein 1; pDC, plasmacytoid dendritic cell; PEG-IFN, pegylated-interferon; pgRNA, pregenomic RNA; PRRs, pattern recognition receptors; rcDNA, relaxed circular DNA; RIG-1, retinoic acid-inducible gene 1; S-HBsAg, small HBsAg; siRNA, small interfering RNA; SVPs, sub-viral particles; TAF, tenofovir alafenamide; T-bet, T-box protein expressed in T cells; TCR, T cell receptor; TDF, tenofovir disoproxil fumarate; TFH, T follicular helper cell; TFV, tenofovir; TGF-β, transforming growth factor-β; Th, T helper; Tim-3, T cell immunoglobulin and mucin-domain containing-3; TLR, toll-like receptor; TNF-α, tumour necrosis factor alpha; Treg, regulatory T.

      Financial support

      The authors received no financial support to produce this manuscript.

      Authors' contributions

      TA designed and supervised the manuscript. IT and TA prepared the manuscript. All the authors contributed to the drafting of the review, the critical revision of the manuscript and the final approval of the version.

      Conflict of interest

      Tarik Asselah has acted as a speaker and investigator for Janssen, Gilead, Roche, and Merck. Nathalie Boyer has acted as a speaker and investigator for Janssen, Gilead, Roche and Merck. Issam Tout, Dimitri Loureiro, Abdellah Mansouri and Vassili Soumelis declare no competing interests.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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