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How to achieve functional cure of HBV: Stopping NUCs, adding interferon or new drug development?

  • Grace L.H. Wong
    Affiliations
    Medical Data Analytics Centre, Department of Medicine and Therapeutics, and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong
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  • Ed Gane
    Affiliations
    New Zealand Liver Transplant Unit, Auckland City Hospital, University of Auckland, New Zealand
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  • Anna S.F. Lok
    Correspondence
    Corresponding author. Address: Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Tel.: 734-936-4882, fax: 734-936-7392.
    Affiliations
    Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
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      Summary

      Functional cure of hepatitis B is defined as sustained undetectable circulating HBsAg and HBV DNA after a finite course of treatment. Barriers to HBV cure include the reservoirs for HBV replication and antigen production (covalently closed circular DNA [cccDNA] and integrated HBV DNA), the high viral burden (HBV DNA and HBsAg) and the impaired host innate and adaptive immune responses against HBV. Current HBV therapeutics, 1 year of pegylated-interferon-α (PEG-IFNα) and long-term nucleos(t)ide analogues (NUCs), rarely achieve HBV cure. Stopping NUC therapy may lead to functional cure in some Caucasian patients but rarely in Asian patients. Switching from a NUC to IFN after HBV DNA suppression increases the chance of HBsAg clearance mainly in those with low HBsAg levels. Novel antiviral strategies that inhibit viral entry, translation and secretion of HBsAg, modulate capsid assembly, or target cccDNA transcription/degradation have shown promise in clinical trials. Novel immunomodulatory approaches including checkpoint inhibitors, metabolic modulation of T cells, therapeutic vaccines, adoptive transfer of genetically engineered T cells, and stimulation of innate and B-cell immune responses are being explored. These novel approaches may be further combined with NUCs or PEG-IFNα in personalised strategies, according to virologic and disease characteristics, to maximise the chance of HBV cure. The development of curative HBV therapies should be coupled with the development of standardised and validated virologic and immunologic assays to confirm target engagement and to assess response. In addition to efficacy, curative therapies must be safe and affordable to meet the goal of global elimination of hepatitis B.

      Keywords

      Background

      Despite the availability of safe and effective vaccines for forty years, chronic HBV infection remains a serious threat to global public health, affecting approximately 257 million people worldwide.
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      In June 2016, the World Health Organization published the first global health sector strategy on viral hepatitis, with the goal of reducing the incidence of viral hepatitis by 90% and its associated mortality by 65%, by 2030.

      World Health Organization. Global health sector strategy on viral hepatitis 2016–2021. Website: https://www.who.int/hepatitis/strategy2016-2021/ghss-hep/en/. Accessed on 20 April 2021.

      In addition to ensuring universal vaccination of all newborns, and improving diagnosis and linkage to care, much effort has been devoted to developing a cure for chronic hepatitis B.

      Definitions and clinical implication of functional cure of HBV

      Functional cure of HBV

      Sterilising HBV cure with elimination of both covalently closed circular DNA (cccDNA) and integrated HBV DNA is unlikely to be feasible in the foreseeable future. The consensus of experts is to aim for functional HBV cure, defined as the sustained loss of detectable HBsAg and HBV DNA in serum, after a finite course of treatment.
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      Functional HBV cure is defined as sustained undetectable circulating HBsAg and HBV DNA after a finite course of treatment.
      HBsAg seroclearance is an indication of marked suppression of HBV replication and cccDNA transcription but HBV is still present in the liver as transcriptionally inactive cccDNA or integrated HBV DNA, and reactivation of HBV replication can occur upon immune suppression. Nonetheless, HBsAg seroclearance is associated with an additional clinical benefit – a further reduction in risk of hepatocellular carcinoma – beyond that of HBV DNA suppression without HBsAg seroclearance.
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      It also allows for the discontinuation of nucleos(t)ide analogues (NUCs) with very low likelihood of seroreversion and viral relapse.
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      From the patients’ perspective, HBsAg seroclearance is a major milestone, removing the stigma that can limit social life and occupational opportunities.

      Partial cure of HBV

      Partial HBV cure, defined as detectable HBsAg but persistently low or undetectable HBV DNA in serum after completing a course of antiviral therapy, has been proposed as an intermediate step towards HBV cure. Patients who achieve partial HBV cure have normal liver enzymes and inactive liver disease, and favourable clinical outcomes compared to their viraemic counterparts, but outcomes are inferior compared to those in patients who achieve functional cure. This inactive state represents disease remission rather than cure because reactivation can occur either spontaneously or following immunosuppression.
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      • Lam K.L.
      • Lui G.C.
      • et al.
      HBsAg seroclearance further reduces hepatocellular carcinoma risk after complete viral suppression with nucleos(t)ide analogues.

      Barriers to HBV cure

      The major barriers to HBV cure include the reservoirs for HBV replication and antigen production (cccDNA and integrated HBV DNA), the high viral burden (HBV DNA and HBsAg) and the impaired host innate and adaptive immune responses against HBV. Episomal cccDNA is a key intermediate in the HBV life cycle, serving as a transcriptional template for all HBV RNAs responsible for DNA replication and antigen production.
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      Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.
      This minichromosome is located within the nucleus of infected hepatocytes, protected from host immune responses and not targeted by NUCs. The transcriptional activity of cccDNA is responsible for ongoing antigen production during NUC therapy and for virologic relapse following NUC withdrawal. cccDNA persists following HBsAg clearance and is responsible for HBV reactivation in patients with resolved HBV infection following rituximab treatment or following transplantation of livers from anti-HBc-positive donors into HBV-naïve recipients.
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      Rituximab-associated hepatitis B virus (HBV) reactivation in lymphoproliferative diseases: meta-analysis and examination of FDA safety reports.
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      • et al.
      Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection.
      Barriers to HBV cure include high viral burden and impaired immune responses against HBV.
      Any effective strategy for functional cure will require therapies that can directly target cccDNA, through silencing of transcriptional activity, mutagenesis, or degradation. Gene editing with CRISPR Cas-9 could directly degrade cccDNA (by targeting cccDNA genes) or indirectly silence cccDNA transcriptional activity (by targeting the cccDNA-associated histones). Gene editing studies in preclinical models have demonstrated significant reductions in cccDNA activity.
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      • et al.
      Novel anti-HBV therapies using CRISPR/cas9 targeting HBV genome strongly suppress HBV.
      However, cure of chronic HBV infection would require 100% editing efficiency in infected hepatocytes, which is not yet achievable with current liver-targeting delivery systems. Earlier studies suggested that the half-life of cccDNA is long, years or decades, and elimination of cccDNA relies on turnover of infected hepatocytes.
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      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      However, a recent study showed that the half-life of cccDNA may be shorter, weeks or months, suggesting that elimination of cccDNA may be feasible.
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      • et al.
      Rapid turnover of hepatitis B virus covalently closed circular DNA indicated by monitoring emergence and reversion of signature-mutation in treated chronic hepatitis B patients.
      The shorter estimated half-life of cccDNA in this study is in part related to the realisation that NUCs do not suppress HBV DNA replication completely. Additional studies are needed to confirm this new finding.
      Another reservoir for HBV antigen production is integrated HBV DNA. Integration occurs early in the natural history of chronic HBV Infection and increases with duration of infection, although there is recent evidence that this trend may be partly reversed by long-term viral suppression.
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      • et al.
      Long term nucleos(t)ide analogue therapy reduced the extent of HBV DNA integration in chronic hepatitis B patients.
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      • Yang P.S.
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      • et al.
      Tenofovir disoproxil fumarate treatment reduces the number of transcriptionally active viral integrations in chronically infected HBV patients.
      During the early phase of HBV infection (HBeAg positive), >95% of HBsAg is derived from cccDNA transcripts, whilst in the late phase of infection (HBeAg negative), >50% of HBsAg is derived from integrated HBV DNA.
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      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      Thus, any HBV cure will need to target both cccDNA and integrated HBV DNA.
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      • Manns M.P.
      Hepatitis: No cure for hepatitis B and D without targeting integrated viral DNA?.
      Another major barrier to HBV functional cure is the presence of a dysfunctional innate and adaptive immune response to the virus. The high antigen burden, particularly HBsAg, in chronic HBV infection creates a tolerogenic intrahepatic and extrahepatic environment. HBV produces a huge excess of spherical and filamentous subviral particles (SVPs), which contain HBsAg but lack a nucleocapsid and are therefore non-infectious. The high concentration of circulating HBsAg is thought to promote HBV persistence via several mechanisms: acting as a decoy for neutralising hepatitis B surface antibody; downregulating HBsAg- and HBeAg-specific T-cell immunity
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      and suppressing innate immunity through dendritic cell and natural killer cell dysfunction.
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      • et al.
      Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.
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      • et al.
      Altered functions of plasmacytoid dendritic cells and reduced cytolytic activity of natural killer cells in patients with chronic HBV infection.
      Inhibition of HBV DNA replication and HBsAg production may suffice to restore immune responses to HBV in some patients but additional immunomodulatory therapies may be needed for others.

      Approaches to achieve functional cure of HBV

      Current treatment approaches (Table 1)

      Currently available treatment for chronic HBV infection includes 1 year of pegylated-interferon α (PEG-IFNα) and long-term NUCs. Both approaches result in very low rates of HBsAg seroclearance. A 1-year course of PEG-IFNα results in an overall HBsAg seroclearance rate of 2-3% at the end of treatment, increasing to 3-8% after 3 years of post-treatment follow-up; however, these rates are much lower in patients infected with non-A HBV genotypes.
      • Ren H.
      • Huang Y.
      Effects of pegylated interferon-alpha based therapies on functional cure and the risk of hepatocellular carcinoma development in patients with chronic hepatitis B.
      Continuous treatment with second generation NUCs, entecavir or tenofovir disoproxil fumarate (TDF) for up to 10 years, results in overall HBsAg seroclearance rates of 0-5%, with higher rates in HBeAg-positive patients.
      • Jeng W.J.
      • Lok A.S.
      Should treatment indications for chronic hepatitis B Be expanded?.
      ,
      • Yip T.C.
      • Wong G.L.
      • Wong V.W.
      • Tse Y.K.
      • Lui G.C.
      • Lam K.L.
      • et al.
      Durability of hepatitis B surface antigen seroclearance in untreated and nucleos(t)ide analogue-treated patients.
      HBV genotype is the strongest predictor of HBsAg seroclearance in patients who received PEG-IFNα treatment while low baseline HBsAg level is the best predictor of HBsAg seroclearance in patients receiving NUC treatment.
      • Cornberg M.
      • Wong V.W.
      • Locarnini S.
      • Brunetto M.
      • Janssen H.L.A.
      • Chan H.L.
      The role of quantitative hepatitis B surface antigen revisited.
      Table 1HBsAg seroclearance rates with pegylated-interferon-α or nucleos(t)ide analogue therapy.
      • Marcellin P.
      • Wong D.K.
      • Sievert W.
      • Buggisch P.
      • Petersen J.
      • Flisiak R.
      • et al.
      Ten-year efficacy and safety of tenofovir disoproxil fumarate treatment for chronic hepatitis B virus infection.
      ,
      • Hou J.
      • Ning Q.
      • Duan Z.
      • Chen Y.
      • Xie Q.
      • Wang F.S.
      • et al.
      3-year treatment of tenofovir alafenamide vs. Tenofovir disoproxil fumarate for chronic HBV infection in China.
      Antiviral agents (dose)HBsAg seroclearance (%)
      HBeAg-positiveHBeAg-negative
      PEG-IFNα-2a (180 ug)3 (EOT; 5 in genotype A, 3 non-genotype A)

      7 (3 years)
      7 (EOT; 11 genotype A; 3 non-genotype A)

      9 (3 years)
      ETV (0.5 mg)2 (1 year)

      3 (5 years)
      0 (1 year)

      1 (5 years)
      TDF (300 mg)3 (1 year)

      5 (10 years)
      0 (1 year)

      3 (10 years)
      TAF (25 mg)1 (1 year)

      4 (3 years)
      0 (1 year)

      3 (3 years)
      EOT, end of treatment; ETV, entecavir; PEG-IFNα, pegylated-interferon α; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate.
      Table 2Summary of studies reporting HBsAg loss in patients with chronic hepatitis B who had stopped nucleos(t)ide analogues (modified from Kao et al.).
      • Kao J.H.
      • Jeng W.J.
      • Ning Q.
      • Su T.H.
      • Tseng T.C.
      • Ueno Y.
      • et al.
      APASL guidance on stopping nucleos(t)ide analogues in chronic hepatitis B patients.
      Author/study designNNUCCirrhosis (%)Tx (years)Consolidation Tx (months)FU (months)HBsAg loss
      HBeAg-positive
      Liem/RCT18ETV/TDF07.941180% (vs. 0% in control)
      Cao/Pros60Mixed04252410%
      mixed with HBeAg-positive and HBeAg-negative patients.
      Liu/Pros138Mixed02.4211200%
      Su/Pros28ETV/TDF03.126480%
      Chi/Pros71Mixed03.926314%
      He/Retro97Mixedn.a.2.848120%
      Chen/Retro148LAM/ETV02.7-9619.6%
      Kuo/Retro-Pros154ETV/TDF03.1>12244.7% (ETV)

      0% (TDF)
      Song/Retro262Mixed20.62.917.573.39.5% (median FU 6.1 yr)
      HBeAg-negative
      Hadziyannis/Pros33ADV04–5456939.4%
      Berg /RCT21TDF0>4>423619% (vs. 0% in control)
      Liem/RCT27ETV/TDF07.485184% (vs. 5% in control)
      Van Bommel/RCT79Mixed0>4n.a.2410.3% at 96 weeks (vs. 0% in control)
      Cao/Pros22Mixed04354810%
      mixed with HBeAg-positive and HBeAg-negative patients.
      Papatheodoridis/Pros57ETV/TDF0>4641825%
      Liu/Pros85Mixed02.42112014%
      Su/Pros72ETV/TDF03.126480%
      Chi/Pros29Mixed03.91063110%
      Jeng/Retro-Pros691ETV/TDF452.9253613% by 6 years
      Chen/Retro263LAM/ETV02.99633.1%
      Kuo/Retro-Pros353ETV/TDF03.1>123610% (ETV)

      15.4% (TDF)
      Chen/Retro-Pros250ETV03.2>125820.8%
      Song/Retro226Mixed36.42.72573.314.6% (median FU 6.1 yr)
      Hirode/Retro1,183Mixed531715% by 4 years
      Studies selected if RCT or prospective design with > 20 patients or retrospective design with >30 patients. If several studies from the same sites, the largest number of patients and the latest was chosen.
      ADV, adefovir; CR, clinical relapse; ETV, entecavir; FU, follow-up; LAM, lamivudine; LdT, telbivudine; n.a., not available; NUC, nucleos(t)ide analogue; Pros, prospective study; Retro, retrospective study; RCT, randomised controlled trial; TDF, tenofovir disoproxil fumarate; Tx, treatment; VR, virological relapse.
      # mixed with HBeAg-positive and HBeAg-negative patients.

      Stopping NUCs (Table 2)

      Long-term treatment with NUCs is associated with very low rates of HBsAg seroclearance. Paradoxically, recent studies suggest that stopping NUCs after >2-3 years treatment and persistently undetectable serum HBV DNA may result in higher rates of HBsAg seroclearance. The first study found that after a median follow-up of 5.5 years, 14 of 33 (42.4%) HBeAg-negative patients who stopped adefovir after 4-5 years of treatment cleared HBsAg.
      • Hadziyannis S.J.
      • Sevastianos V.
      • Rapti I.
      • Vassilopoulos D.
      • Hadziyannis E.
      Sustained responses and loss of HBsAg in HBeAg-negative patients with chronic hepatitis B who stop long-term treatment with adefovir.
      Most studies evaluating stop NUC approaches only included HBeAg-negative patients. One study in Taiwan included 411 non-cirrhotic patients, of whom 148 were HBeAg positive at baseline – including 96 who remained HBeAg positive when NUCs were stopped after a mean treatment duration of 2.5 years. The 3- and 8-year cumulative rates of HBsAg loss were 5.2% and 19.6%, respectively, in patients who were HBeAg positive before treatment and 10.5% and 33.1% in patients who were HBeAg negative before treatment.
      • Chen C.H.
      • Hung C.H.
      • Wang J.H.
      • Lu S.N.
      • Hu T.H.
      • Lee C.M.
      Long-term incidence and predictors of hepatitis B surface antigen loss after discontinuing nucleoside analogues in noncirrhotic chronic hepatitis B patients.
      During follow-up, nearly half the patients required re-treatment and 5 patients experienced hepatic decompensation of whom 1 died.
      Another study in Taiwan included 1,075 HBeAg-negative patients, of whom 691 stopped NUCs after 3 (1.2-8.3) years of treatment. The cumulative incidence of HBsAg seroclearance after stopping NUCs was 13% at 6 years, with an estimated annual incidence of 1.8% compared to 0.9% in patients who continued NUCs.
      • Jeng W.J.
      • Chen Y.C.
      • Chien R.N.
      • Sheen I.S.
      • Liaw Y.F.
      Incidence and predictors of hepatitis B surface antigen seroclearance after cessation of nucleos(t)ide analogue therapy in hepatitis B e antigen-negative chronic hepatitis B.
      Predictors of HBsAg seroclearance included achieving undetectable HBV DNA within 12 weeks of starting a NUC, HBsAg reduction during therapy by >1 log10 during treatment, end-of-treatment HBsAg <100 IU/ml, sustained clinical response after stopping NUC treatment, and no retreatment among clinical relapsers. Clinical relapse (HBV DNA >2,000 IU/ml and alanine aminotransferase [ALT] >2 times the upper limit of normal) occurred in 61% patients, and hepatic decompensation in 2.3% patients after stopping NUCs. Results of a study of 1,076 HBeAg-negative patients in Hong Kong who stopped NUCs after 82±35 months of treatment were less encouraging with only 11 (1%) patients achieving HBsAg seroclearance after stopping NUCs and a cumulative 3-year rate of HBsAg seroclearance of 4.9%.
      • Wong G.L.
      • Chan H.L.
      • Yuen B.W.
      • Tse Y.K.
      • Luk H.W.
      • Yip T.C.
      • et al.
      The safety of stopping nucleos(t)ide analogue treatment in patients with HBeAg-negative chronic hepatitis B.
      Such large differences in HBsAg seroclearance rates after cessation of NUCs in the studies above may in part be related to differences in timing of resumption of NUCs. One study from Taiwan suggested that early resumption of NUCs in patients with clinical relapse may be associated with a lower likelihood of HBsAg seroclearance.
      • Jeng W.J.
      • Chen Y.C.
      • Chien R.N.
      • Sheen I.S.
      • Liaw Y.F.
      Incidence and predictors of hepatitis B surface antigen seroclearance after cessation of nucleos(t)ide analogue therapy in hepatitis B e antigen-negative chronic hepatitis B.
      Three prospective randomised controlled trials have been conducted to evaluate the benefits and harms of stopping NUCs. The first trial, the FINITE study, included 42 non-cirrhotic HBeAg-negative German patients who had received TDF for at least 4 years, randomly assigned to either stop (n = 21) or continue (n = 21) TDF.
      • Berg T.
      • Simon K.G.
      • Mauss S.
      • Schott E.
      • Heyne R.
      • Klass D.M.
      • et al.
      Long-term response after stopping tenofovir disoproxil fumarate in non-cirrhotic HBeAg-negative patients - FINITE study.
      HBsAg seroclearance at 2 years was achieved in 19% of those who stopped TDF compared to 0% in patients who continued. Only 2 patients met stringent criteria for restarting treatment and none experienced hepatic decompensation. The second trial, the Toronto STOP study, included 67 patients of whom 27 were HBeAg positive at the start of treatment and had undergone HBeAg seroconversion 4 years prior to the trial.
      • Liem K.S.
      • Fung S.
      • Wong D.K.
      • Yim C.
      • Noureldin S.
      • Chen J.
      • et al.
      Limited sustained response after stopping nucleos(t)ide analogues in patients with chronic hepatitis B: results from a randomised controlled trial (Toronto STOP study).
      Most were receiving TDF and mean duration of treatment prior to the trial was 7.3 years. The patients were randomised 2:1 to stop or to continue NUCs. During follow up of 72 weeks, only 2 patients had HBsAg seroclearance, one in each group, and 38% of those who stopped NUCs had to resume treatment. In the third trial, the Stop-NUC trial, 158 German patients who had received NUCs for at least 4 years were randomised to continue or to stop NUCs. At 96 weeks, 8 of 79 (10%) who had stopped NUCs vs. 0 of 79 who continued NUCs achieved HBsAg seroclearance, and only 14% meeting stringent criteria resumed treatment.
      • van Bommel F.
      • Stein K.
      • Heyne R.
      • Möller H.
      • Petersen J.
      • Buggisch P.
      • et al.
      Response to discontinuation of long-term nucleos(t)ide analogue treatment in HBeAg negative patients: results of the Stop-NUC trial (LBO06).
      The exact reasons for the differences in outcomes of these 3 trials with similar designs are not clear. One possible reason might be differences in race and duration of infection. All but 3 patients in the Toronto trial were Asians while the 2 German trials enrolled mostly (>85%) Caucasians. A retrospective study of 1,541 patients at 12 centres in North America, Europe and Asia who stopped NUCs found that the cumulative rate of HBsAg seroclearance at year 4 was 14%, and that Caucasian race was the only independent predictor of HBsAg seroclearance, with a hazard ratio of 5.8 (95% CI 3.6-9.5).
      • Hirode G.
      • Choi H.S.
      • Su T.H.
      • Wong G.L.
      • Seto W.K.
      • Wong R.J.
      HBsAg loss is higher among Caucasians compared to Asians after stopping nucleos(t)ide analogue therapy: results from a large, global, multiethnic cohort of patients with chronic hepatitis b (RETRECT-B study).
      Another difference is that 40% of patients in the Toronto trial were HBeAg positive before treatment while all patients in the German trials were HBeAg negative before treatment.
      Collectively, these studies suggest that stopping NUCs after >4 years of treatment in HBeAg-negative patients might be associated with higher rates of HBsAg seroclearance compared to continuing NUCs, but the rates varied from 2.7-16.7%/year in Caucasian patients and are lower, 0-3.8%/year, among Asian patients.
      • Kao J.H.
      • Jeng W.J.
      • Ning Q.
      • Su T.H.
      • Tseng T.C.
      • Ueno Y.
      • et al.
      APASL guidance on stopping nucleos(t)ide analogues in chronic hepatitis B patients.
      While the stop NUC approach is intriguing, it may only be appropriate for highly selected patients who agree to close monitoring after treatment is stopped, and it appears to be less applicable to Asian patients. Several factors have been identified that predict clinical remission and/or HBsAg seroclearance after stopping NUCs.
      • Hadziyannis E.
      • Hadziyannis S.
      Current practice and contrasting views on discontinuation of nucleos(t)ide analog therapy in chronic hepatitis B.
      The most consistent predictor of functional cure is low HBsAg level at the time of NUC withdrawal. By contrast, hepatitis flares are not predictive of HBsAg seroclearance or clinical remission after stopping NUCs and may lead to hepatic decompensation and death.
      Stopping NUCs after viral suppression might increase HBsAg seroclearance in selected patients.

      Antiviral and immunomodulatory therapy

      Interferon has been used to treat hepatitis B for almost 50 years; however, its exact mechanisms of action are still unclear. While IFN has a less potent inhibitory effect on HBV DNA replication compared to NUCs, it results in higher rates of HBeAg and HBsAg seroclearance, particularly among patients with genotype A infection. Recent studies suggest that IFN may have direct effects on the stability or transcription of cccDNA, and in turn translation of HBV proteins. In vitro studies suggest that IFN-α may increase degradation of nuclear cccDNA via lymphotoxin-β-receptor activation.
      • Lucifora J.
      • Xia Y.
      • Reisinger F.
      • Zhang K.
      • Stadler D.
      • Cheng X.
      • et al.
      Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.
      IFN also has immunomodulatory effects, boosting innate and adaptive immunity.
      • Nishio A.
      • Bolte F.J.
      • Takeda K.
      • Park N.
      • Yu Z.X.
      • Park H.
      • et al.
      Clearance of pegylated interferon by Kupffer cells limits NK cell activation and therapy response of patients with HBV infection.
      Clinical trials of PEG-IFNα monotherapy have shown HBsAg seroclearance rates of 3-7% after 1 year of treatment increasing to 8-14% after 3-5 years of post-treatment follow-up; with marked differences between genotype A vs. non-A.
      • Jeng W.J.
      • Lok A.S.
      Should treatment indications for chronic hepatitis B Be expanded?.
      Various strategies combining NUCs and PEG-IFNα have been evaluated with the aim of increasing the rate of HBsAg loss. Early studies of the de novo combination of PEG-IFNα and NUCs, predominantly lamivudine, found that HBsAg seroclearance was rare. One study found that the de novo combination of TDF and PEG-IFNα for 48 weeks resulted in a higher HBsAg seroclearance rate at 24 weeks post-treatment compared to monotherapy with PEG-IFNα or TDF, 8.1% vs. 2.9% and 0%, respectively.
      • Marcellin P.
      • Ahn S.H.
      • Ma X.
      • Caruntu F.A.
      • Tak W.Y.
      • Elkashab M.
      • et al.
      Combination of tenofovir disoproxil fumarate and peginterferon alpha-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B.
      A recent meta-analysis of 60 studies found that the de novo combination of a NUC and IFNα improved HBsAg seroclearance rates compared to NUC monotherapy but not IFNα monotherapy.
      • Liu J.
      • Wang T.
      • Zhang W.
      • Cheng Y.
      • He Q.
      • Wang F.S.
      Effect of combination treatment based on interferon and nucleos(t)ide analogues on functional cure of chronic hepatitis B: a systematic review and meta-analysis.
      Among patients with virus suppression on NUCs, switching to IFNα appeared to have a more marked effect on increasing HBsAg seroclearance (relative risk [RR] 12.15, 95% CI 3.99– 37.01) compared to adding IFNα, with both strategies being superior to continuing NUCs alone.
      • Liu J.
      • Wang T.
      • Zhang W.
      • Cheng Y.
      • He Q.
      • Wang F.S.
      Effect of combination treatment based on interferon and nucleos(t)ide analogues on functional cure of chronic hepatitis B: a systematic review and meta-analysis.
      Although criteria for selecting patients to switch to or to receive add-on IFNα was variable, HBsAg seroclearance was strongly associated with HBsAg level at the start of IFNα therapy. These studies suggest that in highly selected patients who can tolerate IFN and who have low HBsAg levels and undetectable HBV DNA after a few years of NUC therapy, switching treatment to IFNα may enhance the rate of HBsAg seroclearance.

      Novel HBV therapeutics (Table 3)

      Current antiviral therapy for chronic hepatitis B is associated with low rates of HBsAg clearance; thus, long-term treatment is required to maintain virologic response. Therefore, there is great interest in developing finite treatment strategies that will achieve durable off-treatment clearance of circulating HBsAg and HBV DNA. This will require therapeutic strategies that target the high viral and antigen burden as well as inadequate host immune responses.
      Table 3Mechanisms of novel HBV therapeutics.
      ClassMechanismExamplesDevelopment stage
      Antiviral approaches
      Nucleos(t)ide analoguesInhibit reverse transcriptase, and HBV DNA replicationEntecavir, tenofovir disoproxil fumarate, tenofovir alafenamideRecommended
      Lamivudine, adefovir, telbivudine, clevudineNot recommended
      Liver-targeting prodrugATI-2173 is a phosphoramidate nucleotide converted to clevudine-5’-triphosphate in hepatocytesPhase II
      Capsid assembly modulatorsInterfere with capsid assembly and disassemblyJNJ-6379, JNJ-440, RO7049389, ABI-H0731, ABI-3733, ALG001075, AB-836, AT-130, BAY41-4109, HAP-12, HAP-R01, GLS94JHS, EP-027367, EDP-514Phase I/II
      NVR 3-778, AB-506, AB-423, ABI-H2158, RG-7907, JNJ-440Discontinued
      Entry inhibitorBlocks HBV entry receptor NTCPBulevirtidePhase III for hepatitis D
      Translational inhibitor
       Small-interfering RNAmRNA degradation via RISCJNJ-3989, AB-729, RG-6346, VIR-2218,Phase II
       Antisense oligonucleotidemRNA degradation via RNaseHGSK-3228836, ALG-020572Phase I/II
      RO70629311Discontinued
       mRNA destabilisersPPAD5/7 inhibitionEDP-721Phase I
      AB-492, RO7239958Discontinued
      HBsAg secretion inhibitorDecrease S antigen production and secretion and increase proteasomal and lysosomal degradationNucleic acid polymers

      REP-2055/ REP-2139
      Phase II/III
      S-antigen traffic-inhibiting oligonucleotide polymers

      ALG-010133
      Phase 1
      Immunomodulatory approaches
      Innate immune activator
       TLR-7 agonistsActivation of TLR-7 pathway plasmacytoid dendritic cells, Induction of IFN-α, ISGs, NK cellsRO7020531Phase I and II
      Vesatolimod, JNJ64794964Discontinued
       TLR-8 agonistsActivation of TLR-8 pathway intrahepatic innate and adaptive immune responsesSelgantolimodPhase II
       RIG-I agonistsImmunomodulatory effect and direct antiviral effectInarigivirDiscontinued
      Adaptive immune activator
       Therapeutic vaccinesExpand HBV-specific T-cell responsesBRII-179 (VBI-2601), VRON-0200, TG-1050Phase I
      GS-4774, HB-100, ED-DNA.PS2.S, pCMV.PS2.S, CY-1899, YIC, NASVAC, Hepavax-Gene TF, Sci-B-Vac, ENGERIX-B, Gen Hevac BDiscontinued
       Checkpoint inhibitorsRemove T-cell exhaustion by blocking the PD-1/PD-L1 pathwayNivolumab Anti-PD1 mAbPhase II
      RG-6084 PD1 LNA,Phase II
      GS-4224 PD1 small moleculeDiscontinued
      Soluble bispecific TCRsTCR-redirected CD8 cellsIMC-I109VPhase I
      Fc-engineered antibodyInhibition of viral entry, clearance of circulating HBsAg and increase Ag presentation to dendritic cells, thereby stimulating T-cells (vaccinal effect)VIR-3434Phase I/II
      Direct cccDNA approaches
      cccDNA silencing through transcriptional inhibitorsEpigenetic modificationHBV X inhibitorsPreclinical/phase I
      FXR agonist
      GS-5801 demethylase inhibitorDiscontinued
      cccDNA eliminationCleavage of cccDNA by endonucleasesCRISPR/Cas9; TALENS, ZFNsPreclinical
      cccDNA, covalently closed circular DNA; FXR, farnesoid X receptor; NTCP, sodium taurocholate co-transporting polypeptide; RIG-I, retinoic acid-inducible gene 1; RISC, RNA-induced silencing complex; TCR, T cell receptor; TLR, toll-like receptor; ZFN, zinc finger nuclease.

      Antiviral strategies

      Currently the only approved direct-acting antivirals are NUCs. The HBV lifecycle provides many other targets for new molecular entities, including HBV entry, core protein assembly, viral protein synthesis and virion/subviral particle assembly and release (Fig. 1).
      Figure thumbnail gr1
      Fig. 1HBV lifecycle and target sites of novel direct-acting antiviral drugs.
      (1) Entry receptor inhibitors prevent virions entering hepatocytes by blocking binding to the NTCP receptor; (2) CAMs act by producing aberrant or empty core particles, which prevent pre-genomic RNA packaging, HBV DNA replication and virion production; (3) RNA interference (siRNA or ASOs), RNA destabilisers, and LNAs interfere with the transcription of viral RNA and in turn HBV DNA replication and production of HBV virions and proteins; (4) cccDNA epigenetic modifiers, destabilisers, and endonucleases act by decreasing cccDNA concentrations, stability or transcription; (5) HBx inhibitors act by interfering with HBx protein, which regulates cccDNA expression; (6) HBsAg release inhibitors: NAPs prevent subviral particle release from hepatocytes; (7) NUCs or HBV ribonuclease H(Rnase H) inhibitors inhibit reverse transcriptase or interfere with the RNaseH activity required for RNA cleavage causing accumulation of long RNA and blocking HBV DNA synthesis, respectively. ASO, antisense oligonucleotide; CAM, capsid assembly modulator; cccDNA, covalently closed circular DNA; NAP, nucleic acid polymer; NUC, nucleos(t)ide analogue; rcDNA, relaxed circular DNA; siRNA, short-interfering RNA; Reproduced from Jeng WJ and Lok ASF, Chapter on Is cure of hepatitis B infection a mission possible? In Hepatitis B Virus and Liver Disease, edited by JH Kao, publisher Springer Nature, with permission.
      Entry inhibitors: bulevirtide, a small myristoylated synthetic lipopeptide corresponding to the HBV preS1 sequence, blocks the binding of HBsAg to NTCP (sodium taurocholate co-transporting polypeptide), the entry receptor for both HBV and HDV. Bulevirtide’s antiviral effect is mediated via prevention of infection of uninfected hepatocytes, but it may also block entry of new virions to infected hepatocytes. Most studies of bulevirtide have focused on chronic HDV infection where 24 weeks of bulevirtide monotherapy has been shown to result in ≥2 log reductions in HDV RNA levels in 46-77% patients at the end of treatment, though viral relapse was observed in most patients during post-treatment follow-up.
      • Lok A.S.
      • Negro F.
      • Asselah T.
      • Farci P.
      • Rizzetto M.
      Endpoints and new options for treatment of chronic hepatitis D.
      Decline in HBsAg level by ≥1 log was rarely observed during bulevirtide monotherapy, but was more common with a combination of bulevirtide and PEG-IFNα though HBsAg clearance remained a rare event.
      • Lok A.S.
      • Negro F.
      • Asselah T.
      • Farci P.
      • Rizzetto M.
      Endpoints and new options for treatment of chronic hepatitis D.
      Inhibitors of core synthesis/capsid assembly modulators: the HBV core or capsid protein plays multiple essential roles in the HBV lifecycle and is an attractive target for small molecule inhibitors. The primary mechanism of action of core inhibitors or capsid assembly modulators (CAMs) is inhibition of HBV replication by interfering with HBV capsid assembly and encapsidation of pregenomic RNA (pgRNA). Secondary mechanisms of action of CAMs are inhibition of cccDNA establishment by interfering with capsid disassembly and inhibition of replenishment of cccDNA by interfering with intracellular recycling of HBV nucleocapsids.
      The first CAM to enter clinical trials, NVR3-778, had a modest antiviral effect.
      • Yuen M.F.
      • Gane E.J.
      • Kim D.J.
      • Weilert F.
      • Yuen Chan H.L.
      • Lalezari J.
      • et al.
      Antiviral activity, safety, and pharmacokinetics of capsid assembly modulator NVR 3-778 in patients with chronic HBV infection.
      Other CAMs (JNJ-379, RO7049389, vebicorvir and ABI-2158) are more potent and reduce HBV DNA by 3 logs and HBV RNA by 2 logs after 28 days.
      • Janssen H.L.A.
      • Hou J.
      • Asselah T.
      • Lim Y.S.
      • Kim H.J.
      • Tseng C.H.
      • et al.
      Efficacy and safety results of the phase 2 JNJ-56136379 JADE study in patients with chronic hepatitis B: interim week 24 data.
      • Zoulim F.
      • Lenz O.
      • Vandenbossche J.J.
      • Talloen W.
      • Verbinnen T.
      • Moscalu I.
      • et al.
      JNJ-56136379, an HBV capsid assembly modulator, is well-tolerated and has antiviral activity in a phase 1 study of patients with chronic infection.
      • Yuen M.F.
      • Agarwal K.
      • Gane E.J.
      • Schwabe C.
      • Ahn S.H.
      • Kim D.J.
      • et al.
      Safety, pharmacokinetics, and antiviral effects of ABI-H0731, a hepatitis B virus core inhibitor: a randomised, placebo-controlled phase 1 trial.
      • Yuen M.F.
      • Zhou X.
      • Gane E.
      • Schwabe C.
      • Tanwandee T.
      • Feng S.
      • et al.
      Safety, pharmacokinetics, and antiviral activity of RO7049389, a core protein allosteric modulator, in patients with chronic hepatitis B virus infection: a multicentre, randomised, placebo-controlled, phase 1 trial.
      Although modest (<0.5 log) reductions in HBsAg, HBeAg and hepatitis B core-related antigen (HBcrAg) levels were observed after 24 weeks of NUC+CAM in treatment-naïve HBeAg-positive patients, these were not different from those who received NUC monotherapy.
      • Janssen H.L.A.
      • Hou J.
      • Asselah T.
      • Lim Y.S.
      • Kim H.J.
      • Tseng C.H.
      • et al.
      Efficacy and safety results of the phase 2 JNJ-56136379 JADE study in patients with chronic hepatitis B: interim week 24 data.
      ,
      • Yuen M.F.
      • Agarwal K.
      • Ma X.
      • Zhou X.
      • Gane E.
      • Schwabe C.
      • et al.
      Antiviral activity and safety of the hepatitis B core inhibitor ABI-H0731 administered with a nucleos(t)ide reverse transcriptase inhibitor in patients with HBeAg-positive chronic hepatitis B infection in a long-term extension study.
      The combination of a CAM and a NUC may have synergistic antiviral activities. A pilot study of patients on vebicorvir plus entecavir showed that combination therapy resulted in more rapid and more marked decline in HBV DNA and HBV RNA levels compared to entecavir monotherapy, but despite having undetectable HBV DNA and HBV RNA levels, all patients rapidly relapsed when treatment was stopped at week 48.
      • Gane E.
      • Sulkowski M.
      • Ma X.
      • Verdon D.J.
      • Brooks A.E.
      • Gaggar A.
      • et al.
      Virologic response and safety following discontinuation of treatment with the core inhibitor vebicorvir and a nucleos(t)ide reverse transcriptase inhibitor in patients with HBeAg positive or negative chronic hepatitis B virus infection.
      For CAMs to become an integral part of finite HBV functional cure strategies, they will need to contribute to HBsAg loss through their secondary mechanism of action. The next-generation CAMs entering clinical development (ABI-H3733, AB-836, ALG-000184, VNRX-9945) are more potent and pharmacokinetic studies suggest that the hepatocyte concentration necessary for their secondary actions can be achieved.
      • Mani N.
      • Cole A.
      • Kultgen S.
      • Kim H.J.
      • Tseng C.H.
      • Coffin C.S.
      • et al.
      Preclinical antiviral profile of AB-836, a potent, highly selective hepatitis B virus capsid inhibitor.
      ,
      • Dawei Cai D.
      • Evanhcik M.
      • Yan R.
      • Mani N.
      • Cole A.
      • Kultgen S.
      • et al.
      Second generation Hepatitis B Virus core inhibitors ABI-H2158 and ABI-H3733 have enhanced potency and target coverage for both antiviral inhibition and covalently closed circular DNA establishment activities.
      There is emerging evidence that CAMs may be associated with liver-related toxicity. Several CAMs have been associated with treatment-emergent Grade 2-4 ALT elevations.
      • Yuen M.F.
      • Gane E.J.
      • Kim D.J.
      • Weilert F.
      • Yuen Chan H.L.
      • Lalezari J.
      • et al.
      Antiviral activity, safety, and pharmacokinetics of capsid assembly modulator NVR 3-778 in patients with chronic HBV infection.
      ,
      Assembly Bio
      Assembly Bio Announces Decision to Discontinue Clinical Development of ABI-H2158, September 01.
      Although RO7049389-induced ALT elevations are thought to be immune-mediated “good flares”, ALT elevations induced by other CAMs reflect drug-induced liver injury and their development has been halted. Naturally occurring HBV variants resistant to CAMs have been identified and monotherapy with CAMs has been reported to lead to the selection of resistant HBV variants and virologic breakthrough.
      • Verbinnen T.
      • Talloen W.
      • Shukla U.
      • Vandenbossche J.J.
      • Biermer M.
      • Beumont-Mauviel M.
      • et al.
      Viral sequence analysis of chronic hepatitis B (CHB) patients treated with the capsid assembly modulator (CAM-N) JNJ-56136379 (JNJ-6379) as monotherapy in the JADE phase 2a study.
      Thus, CAMs must be used as part of a combination therapy regimen and pre-existing CAM-resistant HBV variants may reduce the efficacy of some CAMs.
      Inhibitors of HBV protein synthesis/translation inhibitors: Translation inhibitors that silence HBV RNA could contribute to HBV cure by inhibiting virion and subviral particle production, thereby boosting host innate and HBV-specific immune responses. Strategies to inhibit translation include small-interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), which have very different pharmacodynamic properties, reflecting differences in their chemistry, cellular delivery and intracellular handling. siRNAs are duplex (double-stranded) RNAs with guide (23 nucleotide) and passenger (21 nucleotide) RNA strands, whilst ASOs are single-stranded DNAs (8-10 nucleotides). “Naked” ASOs can readily enter all cells, but siRNAs must be GalNAc conjugated (conjugation with N-acetylgalactosamine to bind to asialoglycoprotein receptors on hepatocytes) to ensure delivery to the hepatocyte. siRNAs accumulate within endosomes and form a stable guide RNA-argonaut (AGO) complex, that can cleave multiple target HBV mRNAs, thereby amplifying gene silencing and permitting infrequent dosing (monthly or less). In contrast, ASO binds to target HBV mRNA sequences, forming a DNA-RNA hybrid that is rapidly degraded by cytoplasmic RNase-H, necessitating frequent dosing (weekly or more).
      The first siRNA, AR520, was designed to trigger at the common 3’ termini of HBV RNAs (pregenomic and messenger RNAs). While it led to marked decreases in HBsAg levels in HBeAg-positive patients, the effect was diminished in HBeAg-negative patients. Subsequent studies revealed that the trigger site is often missing in HBeAg-negative patients in whom the source of HBsAg is predominantly from integrated HBV DNA.
      • Wooddell C.I.
      • Yuen M.F.
      • Chan H.L.
      • 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.
      New siRNAs and ASOs target overlapping regions of the HBV genome or have multiple triggers to improve efficacy.
      The GalNAc-conjugated siRNAs in development (JNJ-3989; VIR-2218; RG-6346; AB-729) are safe and associated with predictable on-treatment HBsAg declines, durable for many months post-treatment.
      • Yuen M.F.
      • Lim T.H.
      • Kim W.
      • Verbinnen T.
      • Talloen W.
      • Shukla U.
      • et al.
      HBV RNAI inhibitor RG6346 in phase 1b-2a trial was safe, well tolerated, and resulted in substantial and durable reductions in serum HBsAg levels.
      • Gane E.
      • Lim Y.S.
      • Tangkijvanich P.
      • Hou J.
      • Asselah T.
      • Lim Y.S.
      • et al.
      Preliminary safety and antiviral activity of VIR-2218, an X-targeting RNAi therapeutic, in chronic hepatitis B patients.
      • Yuen M.F.
      • Berliba E.
      • Kim Y.J.
      • Verbinnen T.
      • Talloen W.
      • Shukla U.
      • et al.
      Safety and pharmacodynamics of the gal-Nac siRNA AB-729 in subjects with chronic hepatitis B infection.
      • Gane E.J.
      • Locarnini S.
      • Lim T.
      • Berliba E.
      • Sukeepaisarnjaroen W.
      • Kim D.J.
      • et al.
      Dose response with the RNA interference therapy JNJ-3989 combined with nucleos(t)ide analogue treatment in expanded cohorts of patients with chronic hepatitis B.
      HBsAg kinetics from phase I studies predicted that monthly siRNA dosing would lead to HBsAg loss after 12 months. However, phase II studies demonstrated plateauing of HBsAg after 16-20 weeks without HBsAg loss, despite continued administration for up to 48 weeks.
      • Yuen M.
      • Berliba E.
      • Sukeepaisarnjaroen W.
      • Strasser S.
      • Tangkijvanich P.
      • Holmes J.
      • et al.
      Repeat dosing of the GalNAc-siRNA AB-729 in subjects with chronic hepatitis B results in robust and sustained HBsAg suppression.
      Nonetheless, even in patients who received only 2-3 doses, decreased HBsAg levels were maintained for up to 9 months. The absence of ALT flares in the siRNA studies cast doubt on whether profound HBsAg reduction would restore host immune responses. However, a recent study of multiple doses of AB-729 observed that rapid HBsAg reduction was associated with moderate ALT elevations and upregulation of HBV-specific T-cell activation markers in some patients.
      • Paratala B.
      • Park J.
      • Ganchua S.
      • Gane E.
      • Yuen M.
      • Jucov A.
      • et al.
      Inhibition of hepatitis B surface antigen in chronic hepatitis B subjects by RNA interference therapeutic AB-729 is accompanied by upregulation of HBV-specific T cell activation markers.
      This suggests that efficacy of siRNA therapy may be enhanced by combining with immunomodulatory agents, a hypothesis that is supported by profound HBsAg reductions associated with ALT elevations in a study combining an siRNA with PEG-IFNα.
      • Yuen M.
      • Lim Y.
      • Cloutier D.
      • Paratala B.
      • Park J.
      • Ganchua S.
      • et al.
      Preliminary on-treatment data from a Phase 2 study evaluating VIR-2218 in combination with pegIFNα2a in patients with CHB.
      The only naked ASO in clinical development (GSK836) led to rapid and profound HBsAg reductions (3-4 log reduction within 28 days) followed by ALT elevations, thought to represent HBV-specific immune reconstitution.
      • Yuen M.F.
      • Heo J.
      • Jang J.
      • Agarwal K.
      • Ma X.
      • Zhou X.
      • et al.
      Hepatitis B virus (HBV) surface antigen (HBsAg) inhibition with ISIS 505358 in chronic hepatitis B (CHB) patients on stable nucleos(t)ide analogue (NA) regimen and in NA -naive CHB patients: phase 2a, randomized, double-blind, placebo-controlled study.
      Four patients in that study lost HBsAg by Day 29 and 2 remained HBsAg negative for more than 3 months. Additional studies of GSK836 and GalNAc-conjugated ASOs with enhanced antiviral efficacy are ongoing.
      • Hong J.
      • Tan H.
      • Lin T.
      • Berliba E.
      • Kim Y.J.
      • Verbinnen T.
      • et al.
      Combination of antisense oligonucleotides (ASOs) ALG-020572 and ALG-020576 against hepatitis B virus (HBV) improves activity and can be combined with other anti-HBV agents.
      cccDNA targeting: Complete or sterilising cure will require approaches that degrade or silence cccDNA. Direct cccDNA silencing: In in vitro experiments, combining Cas9 with guide RNAs which target conserved HBV sequences leads to profound reductions in cccDNA and all HBV proteins.
      • Murai K.
      • Kodama T.
      • Hikita H.
      • Saiki T.
      • Takizawa Y.
      • Okuno R.
      • et al.
      Novel anti-HBV therapies using CRISPR/cas9 targeting HBV genome strongly suppress HBV.
      The main challenges of translating gene editing to the clinic will be the need to achieve 100% efficiency of delivery to infected hepatocytes and to mitigate the long-term risks of possible chromosomal translocation following editing of integrated HBV sequences. Indirect cccDNA epigenetic silencing: small molecules that target host histone deacetylases, acetyltransferases and demethylases can also interrupt the normal epigenetic regulation of HBV gene expression thereby silencing cccDNA transcription. The oral (H3K4me3:H3) KDM5 demethylase inhibitor demonstrated potent reductions of HBV antigen production in vitro but clinical development was stopped because of safety concerns.
      • Gilmore S.
      • Tam D.
      • Dick R.
      • Mauss S.
      • Schott E.
      • Heyne R.
      • et al.
      Antiviral activity of GS-5801, a liver-targeted prodrug of a lysine demethylase 5 inhibitor, in a hepatitis B virus primary human hepatocyte infection model.
      EYP001, a farnesoid X receptor agonist, is a potent inhibitor of cccDNA transcription in vitro and is in clinical development.
      • Erken R.
      • Stelma F.
      • Roy E.
      • Sampson D.
      • Radreau P.
      • André P.
      • et al.
      First clinical evaluation in chronic hepatitis B patients of the synthetic farnesoid X receptor agonist EYP001.
      The HBx protein modifies the epigenetic regulation of cccDNA function. Small molecules, siRNAs and gene editing approaches to knockdown HBx are all being evaluated in preclinical studies.
      Inhibitors of HBsAg secretion (NAPs and STOPs): Nucleic acid polymers (NAPs) have been reported to rapidly reduce circulating HBsAg with minimal effect on other viral markers, when given as either monotherapy or in combination with IFNα.
      • 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.
      • Pantea 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 naive to nucleos(t)ide therapy.
      In a phase II trial of 40 patients, TDF for 24 weeks followed by the addition of PEG-IFNα and REP 2139 or REP 2165 for 48 weeks, resulted in 44% HBsAg loss at the end of treatment, and 39% of patients met criteria for functional cure >24 weeks after the end of treatment.
      • Bazinet M.
      • Pantea 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 naive to nucleos(t)ide therapy.
      The frequent ALT flares suggested hepatotoxicity from intracytoplasmic accumulation of HBsAg. However, in vitro studies have demonstrated that NAPs enhance intracellular degradation of HBsAg via proteasomal and lysosomal degradation.
      • Boulon R.
      • Blanchet M.
      • Lemasson M.
      • Vaillant A.
      • Labonte P.
      Characterization of the antiviral effects of REP 2139 on the HBV lifecycle in vitro.
      These encouraging results need to be confirmed by larger multicentre studies.
      S-antigen traffic-inhibiting oligonucleotide polymers (STOPs) are a new class of oligonucleotides that share structural similarity with NAPs but have novel chemical features which enhance antiviral activity and allow for subcutaneous administration, in contrast to NAPs which require intravenous administration.
      • Nie Y.
      • Tan H.
      • Kao C.
      • Hong J.
      • Tan H.
      • Lin T.
      • et al.
      ALG-010133, a representative S-antigen transport-inhibiting oligonucleotide polymer (STOPS) effectively inhibits hepatitis B surface antigen (HBsAg) secretion in multiple hepatitis B virus (HBV) cell models.
      ALG-010133 is the first STOP in clinical development.
      • Gane E.
      • Yuen M.
      • Jucov A.
      • Kultgen S.
      • Kim H.J.
      • Tseng C.H.
      • et al.
      Safety, tolerability and pharmacokinetics (PK) of single and multiple doses of ALG-010133, an S-antigen Transport Inhibiting Oligonucleotide Polymer (STOP) for the treatment of chronic hepatitis B.
      Novel antivirals and immunomodulatory therapies to increase functional cure rates are being tested in clinical trials.
      Host-targeting agents: Inarigivir activates the cellular retinoic acid-inducible gene I pathway, thereby inhibiting HBV polymerase and inducing endogenous IFN induction. Although early studies reported a dose-related reduction in HBV DNA levels, higher doses resulted in severe hepatotoxicity, with multiple cases of acute liver failure and one death, leading to its discontinuation.
      • Agarwal K.
      • Afdhal N.
      • Coffin C.
      • Tan H.
      • Kao C.
      • Hong J.
      • et al.
      Liver toxicity in the Phase 2 Catalyst 206 trial of inarigivir 400mg daily added to a nucleos(t)ide in HBeAg negative patients.
      ,
      • Yuen M.
      • Chen C.
      • Lui C.
      • Jeng R.
      • Elkhashab M.
      • Coffin C.
      • et al.
      Ascending dose cohort study of inarigivir - a novel RIG I agonist in chronic HBV patients: final results of the ACHIEVE trial.
      Other classes of antivirals that target host pathways include transcription inhibitors (KDM5 demethylase inhibitors, FXR agonists) and mRNA destabilisers (dihydroquinolizinone compounds, PPAD-5/7 inhibitors). Development of these classes of drugs has been hampered by off-target toxicities.

      Immunomodulatory therapies

      Patients with chronic HBV infection have impaired immune responses to HBV.
      • Bertoletti A.
      • Ferrari C.
      Adaptive immunity in HBV infection.
      HBV-specific T cells are decreased in number and functionally defective with features of exhaustion as exemplified by upregulation of multiple co-inhibitory receptors, and transcriptional, metabolic, and epigenetic defects. Restoration of immune response is important in achieving functional cure to eliminate infected hepatocytes and/or block infection of new hepatocytes. Studies of patients who achieved HBeAg or HBsAg seroclearance after NUC or PEG-IFNα treatment showed that HBV-specific immune responses can be restored, suggesting that HBV-specific T cells are exhausted but not deleted.
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      Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues.
      ,
      • Rehermann B.
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      The hepatitis B virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response.
      Multiple approaches to stimulate or to remove blockade of HBV-specific immune responses have been studied, but results have been poor (Fig. 2). Several factors might have contributed to failure of these early attempts: persistent high levels of circulating HBsAg, use of HBV surface antigens/epitopes that patients are tolerant to, and reliance on single (in contrast to multi-prong) or subdued (for fear of severe flare if rigorous immune response is achieved) approaches.
      Figure thumbnail gr2
      Fig. 2Strategies to enhance adaptive and innate immune response to HBV.
      Cytokines, TLR7, TLR8 or RIG-1 and pegylated IFNα target innate immunity. IFN-a and IFN-b trigger the expression of ISGs downstream of IFN-stimulating response elements and the JAK-STAT pathway. RIG-1 triggers the secretion of IFN-a, IFN-b, IFN-g and activates NF-kB to produce inflammatory cytokines. TLR-7 and TLR-8 agonists stimulate antiviral cytokine production and activation of NK cells. Checkpoint inhibitors against PD-1 and PD-L1 reverse HBV-specific T cell exhaustion and restore immunity. Therapeutic vaccines stimulate host immune responses to restore HBV-specific adaptive immune control. cccDNA, covalently closed circular DNA; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; IL-, interleukin-; ISGs, IFN-stimulated genes; NK, natural killer; NOD-2, nucleotide-binding oligomerisation domain 2; PD-1, programmed death-1; PD-L1, programmed death ligand-1; rcDNA, relaxed circular DNA; RIG-I, retinoic acid-inducible gene I; TGF-β, transforming growth factor-β; TLR, Toll-like receptor; TNF, tumour necrosis factor; Treg, regulatory T cell. Reproduced from Jeng WJ and Lok ASF, Chapter on Is cure of hepatitis B infection a mission possible? In Hepatitis B Virus and Liver Disease, edited by JH Kao, publisher Springer Nature, with permission.
      Checkpoint inhibitors: In vitro studies showed that programmed death receptor 1 (PD-1) blockade alone is not sufficient to completely reverse the immune function impairment that is characteristic of chronic HBV infection. Studies in woodchucks showed that the combination of PD-L1 blockade with a NUC and therapeutic DNA vaccination may be more effective.
      • Kosinska A.D.
      • Liu J.
      • Lu M.
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      Therapeutic vaccination and immunomodulation in the treatment of chronic hepatitis B: preclinical studies in the woodchuck.
      A phase Ib study of 12 weeks treatment with nivolumab, a PD-1 inhibitor, with or without GS-4774, a yeast-based therapeutic T-cell vaccine that expresses HBV core, envelope and X antigens found that 3 of 22 patients in the high-dose group had ≥0.5 log decrease in HBsAg at week 24 with 1 patient having undetectable HBsAg that persisted at 12 months post-treatment.
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      Improvement in HBV-specific T cell responses was observed in some but not all patients, with the most marked improvement in the patient who achieved HBsAg seroclearance.
      Metabolic modulation: T-cell activation and function require dynamic metabolic adaptations. T cells in patients with chronic HBV infection have been shown to be deficient in arginine, and partial restoration of CD8+ T cell function may be restored by in vitro arginine replenishment.
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      HBV-specific T cells in patients with chronic HBV infection also exhibit mitochondrial dysfunction. A recent study found that mitochondria-targeted antioxidant therapy can improve HBV-specific T cell function in vitro.
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      Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B.
      Whether these approaches will restore HBV-specific T cell function in patients with chronic HBV infection remains to be determined.
      Therapeutic vaccines: Vaccines to boost HBV-specific T or B cell immune response might be needed to achieve functional cure. Multiple approaches, including with protein-, peptide-, DNA- and viral vector-based vaccines have been evaluated with very little success to date. Two recent studies using GS-4774, a yeast-based vaccine, in NUC-naïve and NUC-experienced virally suppressed patients, showed minimal reduction in serum HBsAg levels
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      Randomized phase II study of GS-4774 as a therapeutic vaccine in virally suppressed patients with chronic hepatitis B.
      ,
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      • Vecchi A.
      • Barili V.
      • et al.
      Combined GS-4774 and tenofovir therapy can improve HBV-specific T-cell responses in patients with chronic hepatitis.
      despite increased production of cytokines, such as IFNγ, by HBV-specific CD8 T cells.
      Recent approaches to therapeutic vaccines have focused on using viral vectors, inclusion of antigens from other regions of HBV (such as core and polymerase instead of surface) or modifications of the HBs epitope that appear to be better recognised by patients who spontaneously cleared HBsAg, as well as the addition of check-point inhibitors or metabolic modulators to overcome HBV-specific immune exhaustion. Interest in the viral vector approach has been spurred by COVID-19, for which several vaccines were developed using adenoviral vectors. The ChAdOx-HBV vaccine in combination with a PD-1 blocking monoclonal antibody will be tested in a phase I/IIa trial.
      • Chinnakannan S.K.
      • Cargill T.N.
      • Donnison T.A.
      • Ansari M.A.
      • Sebastian S.
      • Lee L.N.
      • et al.
      The design and development of a multi-HBV antigen encoded in chimpanzee adenoviral and modified Vaccinia Ankara viral vectors; A novel therapeutic vaccine strategy against HBV.
      Data on many of these new approaches are preliminary and some have only been tested in animal models or healthy volunteers; thus, the efficacy of these new vaccines in patients with chronic HBV infection remains to be seen.
      • Boni C.
      • Barili V.
      • Acerbi G.
      • Rossi M.
      • Vecchi A.
      • Laccabue D.
      • et al.
      HBV immune-therapy: from molecular mechanisms to clinical applications.
      Chronic exposure to high circulating levels of HBsAg has been identified to be a major contributor to HBV-specific immune exhaustion. Thus, most experts have agreed that in addition to suppression of HBV DNA replication, a decrease in HBsAg production and/or release would be important to increase the efficacy of therapeutic vaccines.
      Adoptive transfer of genetically engineered T cells: Transplantation of bone marrow from persons who have spontaneously recovered from HBV infection have been shown to result in HBsAg clearance in patients with chronic HBV infection demonstrating that transfer of HBV-specific T cells can achieve functional HBV cure.
      • Lau G.K.
      • Lok A.S.
      • Liang R.H.
      • Lai C.L.
      • Chiu E.K.
      • Lau Y.L.
      • et al.
      Clearance of hepatitis B surface antigen after bone marrow transplantation: role of adoptive immunity transfer.
      Several strategies to transfer genetically engineered T cells to restore HBV-specific immune response have been explored. Re-direction of patient T cells by transfer of HBV T-cell receptor genes has been tried in humans with HBV-related HCC and appeared to be safe and effective.
      • Qasim W.
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      • et al.
      Immunotherapy of HCC metastases with autologous T cell receptor redirected T cells, targeting HBsAg in a liver transplant patient.
      CAR (chimeric antigen receptor) T-cell therapies, which are increasingly being used for haematological malignancies, have been proposed, but genetically engineering T cells is technically complex and unlikely to be widely adopted as a treatment for chronic HBV infection.
      Stimulation of innate immune response: Innate immune response mediators may enhance antiviral immunity, but they lack precision. An oral toll-like receptor 7 (TLR7) agonist, GS9620, resulted in a sustained decrease in serum HBV DNA and HBsAg in chimpanzees with chronic HBV infection.
      • 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.
      However, a phase IIb trial in humans virally suppressed on NUC showed minimal decrease in HBsAg levels despite increased cytokine production by T cells and natural killer cell activation.
      • 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.
      ,
      • Boni C.
      • Vecchi A.
      • Rossi M.
      • Laccabue D.
      • Giuberti T.
      • Alfieri A.
      • et al.
      TLR7 agonist increases responses of hepatitis B virus-specific T cells and natural killer cells in patients with chronic hepatitis B treated with nucleos(T)ide analogues.
      Similarly, a recent trial of the TLR8 agonist selgantolimod, in combination with a NUC, resulted in minimal reduction in HBsAg levels.
      • Janssen H.L.A.
      • Lim Y.S.
      • Kim H.J.
      • Tseng C.H.
      • Coffin C.S.
      • Elkashab M.
      • et al.
      Safety and efficacy of oral TLR8 agonist, selgantolimod, in viremic adult patients with chronic hepatitis B.
      Inarigivir, which induces the intracellular IFN signalling pathways showed promise in early clinical studies, but development was terminated following the death of a trial participant.
      Stimulation of B-cell immune response: The importance of B-cell immune responses is evident from the high rate of HBV reactivation in patients receiving B cell depleting (anti-CD20) therapies. HBsAg-specific B cells persist in many patients with chronic HBV infection, albeit at low frequencies and functionally impaired.
      • 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.
      Boosting endogenous B cell responses has several potential advantages beyond increasing antibody production; however, an effective strategy remains elusive and will succeed only if HBsAg production is markedly suppressed. Infusion of antibodies to HBsAg may neutralise circulating HBsAg and prevent new infection of hepatocytes but the effects would be short-lived.
      New antiviral and immunomodulatory therapies may be combined with current treatment to increase HBV cure rates.
      Leverage NUCs or PEG-IFNα: Rapid suppression of HBV DNA replication during the first few weeks of NUC therapy had been shown to be associated with improvement in HBV-specific immune responses but this improvement was short-lived.
      • Boni C.
      • Penna A.
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      • Lamonaca V.
      • Rapti I.
      • Missale G.
      • et al.
      Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.
      Recent studies in patients who stopped NUCs found that HBV-specific T cell responses are partially restored in some patients who remained in clinical remission after stopping treatment. One study showed that patients who maintained virologic remission after NUC withdrawal had increased frequency of functional HBV-specific CD8+ T cells prior to stopping NUCs compared to those who required re-treatment, but there was no enhancement of HBV-specific T cell response after stopping NUC.
      • Garcia-Lopez M.
      • Lens S.
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      • Testoni B.
      • Rodriguez-Tajes S.
      • Marino Z.
      • et al.
      Viral and immune factors associated with successful treatment withdrawal in HBeAg-negative chronic hepatitis B patients.
      Another study found that T cells of patients who cleared HBsAg after stopping NUCs had a less exhausted and more activated phenotype compared to those who did not, with a progressive increase in HBV core but not surface or polymerase-specific CD4+ and CD8+ T cell responses for up to 48 weeks following NUC withdrawal.
      • Rinker F.
      • Zimmer C.L.
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      • 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.
      A third study showed a progressive increase in frequencies and functions of HBV-specific core and polymerase-specific T cells in patients who remained in clinical remission after stopping NUCs.
      • Rivino L.
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      • Gill U.S.
      • Kunasegaran K.
      • Cheng Y.
      • Tan D.Z.
      • et al.
      Hepatitis B virus-specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation.
      These data suggest that prolonged suppression of HBV DNA replication can restore HBV-specific T cell responses in some patients, enabling them to remain in clinical remission with or without HBsAg seroclearance, after NUCs are stopped. Further studies are needed to determine how often this occurs, how these patients can be identified, and why restoration of HBV-specific immune responses occurs in some but not all patients.
      The notion that IFN may provide an extra boost in HBsAg clearance, particularly in patients with low HBsAg levels, has led to a revival of interest in incorporating PEG-IFNα into new HBV therapies aimed at functional cure. Clinical trials suggest that combining PEG-IFNα and bulevirtide or REP 2139 or REP 2165 leads to a more marked decrease in HBsAg levels. Among the new direct-acting antiviral drugs in clinical trials, CAMs with or without NUCs have minimal effects on HBsAg decrease after 24 weeks of treatment. siRNAs and ASOs lead to a more marked decrease in HBsAg levels but HBsAg clearance remains rare. Studies are currently exploring whether switching to or adding on PEG-IFNα while HBsAg levels remain low and HBV DNA is suppressed might increase the rate of HBsAg seroclearance.

      Combination therapy – rational approach to combining treatments

      Why is combination needed?

      Multiple steps need to be satisfied to achieve the goal of functional HBV cure: i) complete suppression of HBV DNA replication, ii) inhibition of HBsAg production from both cccDNA and integrated HBV DNA, and iii) restoration of host innate and HBV-specific immune responses (Fig. 3). While one or two classes of drugs might suffice in achieving functional HBV cure in a small percentage of patients, the combination of several classes of drugs will be necessary to achieve this goal in a high percentage of patients.
      Figure thumbnail gr3
      Fig. 3Therapeutic approaches towards HBV cure.
      HBV functional cure requires combination therapy of different therapeutic approaches, that include (i) inhibition of HBV DNA replication that may be achieved with NUCs, CAMs, or entry inhibitors; and (ii) reduction in HBsAg production/secretion that may be achieved with siRNA, ASOs, NAPs, or STOP, with or without (iii) immune stimulation with TLR 7 or 8 agonists, anti-PD-1 or anti-PD-L1 drugs, B or T cell therapeutic vaccines, or IFN. ASOs, antisense oligonucleotides; CAMs, capsid assembly modulators; IFN, interferon; NAPs, nucleic acid polymers; NUCs, nucleos(t)ide analogues; PD-1, programmed death-1; PD-L1, programmed death ligand-1; siRNA, short-interfering RNAs; STOP, S-antigen traffic-inhibiting oligonucleotide polymers; TLR, Toll-like receptor.
      Recent studies showed that blood from patients with undetectable serum HBV DNA on NUC therapy contain residual virus and could transmit HBV.
      • Kosinska A.D.
      • Liu J.
      • Lu M.
      • Roggendorf M.
      Therapeutic vaccination and immunomodulation in the treatment of chronic hepatitis B: preclinical studies in the woodchuck.
      One study showed that the combination of a CAM and a NUC can lead to more rapid and marked suppression of HBV DNA as well as HBV RNA, but the effect on HBsAg level was small. The addition of entry inhibitors may contribute to suppression of HBV DNA replication. siRNAs and ASOs can result in a rapid decrease in HBsAg production but HBsAg clearance is rare. The addition of PEG-IFNα after HBsAg and HBV DNA have been suppressed to low levels might increase the likelihood of HBsAg seroclearance. Sustained functional cure will require restoration of HBV-specific immune responses. For some patients, this might be accomplished after complete suppression of HBV DNA replication and inhibition of HBsAg production but, for many, immunomodulatory therapies will be necessary.

      Personalised approach?

      Chronic HBV infection is a heterogenous condition with highly variable manifestations within a given patient and across patients. In addition to differences in host immune response, viral (HBV genotype, HBeAg status, HBV DNA level, HBsAg level) and disease characteristics (ALT level, fibrosis stage) that may determine which treatment approach has the greatest chance of success, patient preference (risk tolerance [benefit vs. risk], duration of treatment, route of administration, pill burden, out of pocket cost) should also be considered in recommending the best approach for functional HBV cure. It is hoped that multiple, equally efficacious, combination therapies with comparable safety will become available, so that treatment can be personalised according to virologic and disease characteristics, patient preference, and affordability – to maximise effectiveness in the real world.

      Conclusions and future perspectives

      The development of multiple classes of antivirals that target different steps in the HBV lifecycle, alongside immunomodulatory therapies that can enhance innate and adaptive T- and B-cell immune responses to HBV, provide hope that functional HBV cure can be achieved in a higher percentage of patients after a finite course of therapy than is currently possible. This will require collaborations not only between regulatory agencies, the pharmaceutical industry, scientists, and clinicians but also between companies to facilitate testing of the best combination of drugs. In addition, development of standardised and validated virologic and immunologic assays to assess response and to confirm target engagement should occur in parallel. Finally, chronic HBV infection is disproportionately prevalent in low-income countries, immigrants, and patients with limited resources and access to healthcare; thus, curative HBV therapies must be accessible and affordable for everyone with chronic HBV infection if we are to meet the goal of HBV elimination.

      Abbreviations

      ALT, alanine aminotransferase; ASO, antisense oligonucleotide; CAM, capsid assembly modulator; cccDNA, covalently closed circular DNA; NAP, nucleic acid polymer; NUC, nucleos(t)ide analogue; PD-1, programmed death receptor 1; PEG-IFNα, pegylated-interferon-α; siRNA, short interfering RNA; STOP, S-antigen trafficking-inhibitory oligonucleotide polymer; TDF, tenofovir disoproxil fumarate; TLR, toll-like receptor.

      Financial support

      This work was partly supported by the Health and Medical Research Fund (HMRF) of the Food and Health Bureau Commissioned Research on Hepatitis (CID-CUHK-D) awarded to Grace Wong.

      Authors’ contributions

      All authors were responsible for the interpretation of data, drafting, and critical revision of the manuscript for important intellectual content.

      Conflict of interest

      Grace Wong has served as an advisory committee member for Gilead Sciences and Janssen, as a speaker for Abbott, Abbvie, Bristol-Myers Squibb, Echosens, Furui, Gilead Sciences, Janssen and Roche, and received research grant from Gilead Sciences. Ed Gane has served as an advisory committee member and/or speaker for AbbVie, Abbott Diagnostics, Aligos, Arbutus, Arrowhead, Assembly, Avalia, Clear B Therapeutics, Dicerna, Enanta, Gilead Sciences, GlaxoSmithKline, Janssen, Merck, Roche and Vir Bio. Anna Lok has served as an advisory committee member / consultant for Arbutus, ClearB, Enanta, Enochian, GNI, Janssen, TARGET, and Viravaxx, and received research grants from Gilead and TARGET.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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