New Perspectives on Development of Curative Strategies for Chronic Hepatitis B

A functional cure of chronic hepatitis B defined as sustained hepatitis B surface antigen loss after finite course of therapy is rarely achieved with current therapy but is the goal of novel treatments. Understanding the virological and immunological mechanisms of hepatitis B virus persistence has enabled the identification of novel treatment targets, drug discovery, and the evaluation of novel agents in clinical trials. Lessons were learned from early phase 1 and phase 2 trials regarding the antiviral activity and safety profile of these agents. There is a strong rationale to combine agents to reduce viral replication, reduce viral antigen load, invigorate immune responses, and induce specific adaptive immune responses. Nucleos(t)ide analogs will likely remain an essential backbone of future combinations to control viral replication and prevent resistance to antiviral drugs. In this review, we discuss perspectives on approaches to achieving functional cure, with a review of virological and immunological strategies, highlighting challenges and unresolved questions with the various attempts to achieve cure, as well as exploring alternative endpoints such as partial cure and new noninvasive viral and immunological biomarkers to stratify patients and predict/monitor antiviral response. A functional cure of chronic hepatitis B defined as sustained hepatitis B surface antigen loss after finite course of therapy is rarely achieved with current therapy but is the goal of novel treatments. Understanding the virological and immunological mechanisms of hepatitis B virus persistence has enabled the identification of novel treatment targets, drug discovery, and the evaluation of novel agents in clinical trials. Lessons were learned from early phase 1 and phase 2 trials regarding the antiviral activity and safety profile of these agents. There is a strong rationale to combine agents to reduce viral replication, reduce viral antigen load, invigorate immune responses, and induce specific adaptive immune responses. Nucleos(t)ide analogs will likely remain an essential backbone of future combinations to control viral replication and prevent resistance to antiviral drugs. In this review, we discuss perspectives on approaches to achieving functional cure, with a review of virological and immunological strategies, highlighting challenges and unresolved questions with the various attempts to achieve cure, as well as exploring alternative endpoints such as partial cure and new noninvasive viral and immunological biomarkers to stratify patients and predict/monitor antiviral response. Despite the availability of safe and effective vaccines for 40 years, hepatitis B virus (HBV) infection remains a global health problem, with an estimated 1.5 million new infections, 296 million chronically infected persons, and 820,000 HBV-related deaths in 2019.1World Health Organization Hepatitis B Fact Sheet. WHO Press, Geneva, Switzerland2021Google Scholar Current treatment of chronic hepatitis B (CHB) using pegylated interferon alfa (pegIFNα) or nucleos(t)ide analogs (NAs) can suppress HBV DNA replication, decrease liver inflammation and fibrosis, and decrease risk of cirrhosis, hepatocellular carcinoma (HCC), and liver-related deaths. However, hepatitis B surface antigen (HBsAg) loss rarely occurs, 2%–3% after 1 year of pegIFNα increasing to 8%–14% after 3–5 years post-treatment follow-up overall, with lower rates in patients with non-A genotype, and only 2%–5% after 10 years of NA therapy irrespective of genotype.2Jeng W.J. Lok A.S. Should treatment indications for chronic hepatitis B be expanded?.Clin Gastroenterol Hepatol. 2021; 19: 2006-2014Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Virological relapse is almost universal when treatment is stopped prior to HBsAg loss, necessitating retreatment in the case of pegIFNα or long-term therapy in the case of NA. While some studies found that cessation of NA after at least 2–4 years of HBV DNA suppression had higher rates of HBsAg loss than continuing NA, this finding pertains mainly to Caucasian patients, with Asian patients having 6.8- to 8.3-fold lower odds of HBsAg loss.3Hirode G. Choi H.S.J. Chen C.H. et al.Off-therapy response after nucleos(t)ide analogue withdrawal in patients with chronic hepatitis B: an international, multicenter, multiethnic cohort (RETRACT-B study).Gastroenterology. 2022; 162: 757-771.e4Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar,4Sonneveld M.J. Chiu S.M. Park J.Y. et al.Probability of HBsAg loss after nucleo(s)tide analogue withdrawal depends on HBV genotype and viral antigen levels.J Hepatol. 2022; 76: 1042-1050Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The success of hepatitis C cure has spurred renewed efforts to develop curative therapy for HBV, although the unique pathobiology of chronic HBV infections raises different challenges. The major barriers to HBV cure include the reservoirs for HBV replication and antigen production and the impaired innate and adaptive immune responses against HBV.5Testoni B. Zoulim F. Liver capsule: Validated and potential novel targets to treat hepatitis B virus.Hepatology. 2015; 62: 1619Crossref PubMed Scopus (2) Google Scholar,6Fanning G.C. Zoulim F. Hou J. et al.Therapeutic strategies for hepatitis B virus infection: towards a cure.Nat Rev Drug Discov. 2019; 18: 827-844Crossref PubMed Scopus (299) Google Scholar Episomal covalently closed circular DNA (cccDNA) located in the hepatocyte nucleus serves as a transcriptional template for all HBV RNAs including pregenomic RNA (pgRNA), which is reverse-transcribed into HBV DNA, and for messenger RNAs, which are translated into viral proteins.7Martinez M.G. Boyd A. Combe E. et al.Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar cccDNA is derived not only from incoming virions, but also from intracellular recycling of nucleocapsids. The dual source of cccDNA and its long half-life, estimated to be years, explain why cccDNA concentrations are minimally decreased even after many years of NA therapy.8Werle-Lapostolle B. Bowden S. Locarnini S. et al.Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.Gastroenterology. 2004; 126: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar, 9Boyd A. Lacombe K. Lavocat F. et al.Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.J Hepatol. 2016; 65: 683-691Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Wong D.K. Seto W.K. Fung J. et al.Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos(t)ide analogues of different potency.Clin Gastroenterol Hepatol. 2013; 11: 1004-1010.e1Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar HBV DNA can also be integrated into host genomic DNA, with recent data suggesting that NA therapy may reduce integration events.11Hsu Y.C. Suri V. Nguyen M.H. et al.Inhibition of viral replication reduces transcriptionally active distinct hepatitis B virus integrations with implications on host gene dysregulation.Gastroenterology. 2022; 162: 1160-1170.e1Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,12Chow N. Wong D. Lai C.L. et al.Effect of antiviral treatment on hepatitis B virus integration and hepatocyte clonal expansion.Clin Infect Dis. 2023; 76: e801-e809Crossref PubMed Scopus (8) Google Scholar Integrated HBV DNA is not replication competent, but full-length S gene and transcripts are generally preserved, and integrated HBV DNA can be a source of HBsAg expression.13Wooddell C.I. Yuen M.F. Chan H.L. et al.RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.Sci Transl Med. 2017; 9eaan0241Crossref PubMed Scopus (305) Google Scholar, 14Freitas N. Lukash T. Gunewardena S. et al.Relative abundance of integrant-derived viral RNAs in infected tissues harvested from chronic hepatitis B virus carriers.J Virol. 2018; 92e02221-17Crossref PubMed Google Scholar, 15Zhao L.H. Liu X. Yan H.X. et al.Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma.Nat Commun. 2016; 712992Google Scholar Deletions have been observed in the 5′-UTR/pre-S1 domain, the pre-S2 domain, and the 3′ UTR, suggesting that the small S is generally more preserved than the other S species.14Freitas N. Lukash T. Gunewardena S. et al.Relative abundance of integrant-derived viral RNAs in infected tissues harvested from chronic hepatitis B virus carriers.J Virol. 2018; 92e02221-17Crossref PubMed Google Scholar In fact, there is a body of evidence that integrated HBV DNA is the predominant source of HBsAg in hepatitis B e antigen (HBeAg)–negative patients, while cccDNA is the predominant source in HBeAg-positive patients.13Wooddell C.I. Yuen M.F. Chan H.L. et al.RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.Sci Transl Med. 2017; 9eaan0241Crossref PubMed Scopus (305) Google Scholar,16Meier M.A. Calabrese D. Suslov A. et al.Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.J Hepatol. 2021; 75: 840-847Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Permanent cccDNA elimination or silencing is the key objective to prevent any risk of viral reactivation, and thus cccDNA has long been held as the Achilles heel of HBV cure.7Martinez M.G. Boyd A. Combe E. et al.Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar However, if HBsAg loss is the only acceptable definition of successful therapy, this may require targeting of not only cccDNA, but also integrated HBV DNA. Currently, without a mechanism to reliably distinguish the source of circulating HBsAg, it is possible that a person may have cleared/silenced all cccDNA but remain HBsAg-positive from integrated DNA alone. Whether such a scenario is equivalent to clearance of HBsAg is currently unknown but highlights that tools to identify the source of HBsAg would be a useful advance to better define the natural history and the goals of therapy. Elimination of hepatocytes that harbor integrated HBV DNA may occur with restoration of HBV-specific immunity and hepatocyte turnover. Gene editing has been proposed as means to eliminate or permanently silence cccDNA and integrated HBV DNA, but there are major challenges and concerns with off target effects and delivery issues.17Martinez M.G. Smekalova E. Combe E. et al.Gene editing technologies to target HBV cccDNA.Viruses. 2022; 14: 2654Crossref PubMed Scopus (6) Google Scholar Epigenetic silencing has also been proposed but would require specific targeting to avoid major safety issues associated with general epigenome modifiers.7Martinez M.G. Boyd A. Combe E. et al.Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar However, patients with chronic HBV infection have impaired innate and adaptive immune responses to HBV.18Maini M.K. Burton A.R. Restoring, releasing or replacing adaptive immunity in chronic hepatitis B.Nat Rev Gastroenterol Hepatol. 2019; 16: 662-675Crossref PubMed Scopus (78) Google Scholar Innate immune responses are not triggered by acute HBV infection, and persistently high viral antigen levels had been incriminated to result in adaptive immune exhaustion. Recovery of HBV-specific immune responses has been demonstrated in patients with spontaneous, pegIFNα-induced, or NA-induced HBeAg or HBsAg loss, leading to the hope that reducing HBsAg levels may restore HBV-specific immune responses. Although this has become a major principle of most HBV curative strategies, it is only recently that there is suggestion of possible immune recovery in patients who experienced marked decreases in HBsAg levels during small interfering RNA (siRNA) therapy.19Ganchua S.C. Paratala B. Iott C. et al.Reduction of hepatitis B surface antigen mediated by RNA interference therapeutic AB-729 in chronic hepatitis B patients is associated with T cell activation and a decline in exhausted CD8 T cells.J Hepatol. 2022; 77: S851Abstract Full Text PDF Google Scholar These data hold promise that in some patients, sustained suppression of HBV DNA replication and HBsAg production may modify the immune environment and the level of HBV-specific T cell exhaustion. Ideally, cure would involve elimination of all traces of HBV with no risk of reactivation.20Lok A.S. Zoulim F. Dusheiko G. et al.Hepatitis B cure: from discovery to regulatory approval.J Hepatol. 2017; 67: 847-861Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar However, a sterilizing cure, with elimination of cccDNA as well as integrated HBV DNA, is unlikely to be achievable in the foreseeable future. Furthermore, it may be difficult to verify sterilizing cure even if it were achievable. Thus, a functional cure, defined as sustained (>6 months post-treatment) HBsAg loss with or without seroconversion to hepatitis B surface antibody and undetectable HBeAg and serum HBV DNA after a finite course of therapy, was determined to be the goal at the 2016 and 2019 American Association for the Study of Liver Diseases and European Association for the Study of the Liver Joint HBV Treatment Endpoint Conference.20Lok A.S. Zoulim F. Dusheiko G. et al.Hepatitis B cure: from discovery to regulatory approval.J Hepatol. 2017; 67: 847-861Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,21Cornberg M. Lok A.S. Terrault N.A. et al.Guidance for design and endpoints of clinical trials in chronic hepatitis B - report from the 2019 EASL-AASLD HBV Treatment Endpoints Conference.J Hepatol. 2020; 72: 539-557Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar HBsAg loss was chosen because it is >90% durable if confirmed on a repeat test >6 months apart, the treatment can be stopped with a very low risk of virologic relapse, and it further reduces the risk of HCC compared with undetectable HBV DNA without HBsAg loss.22Yip T.C. Wong G.L. Chan H.L. et al.HBsAg seroclearance further reduces hepatocellular carcinoma risk after complete viral suppression with nucleos(t)ide analogues.J Hepatol. 2019; 70: 361-370Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Importantly, for patients, clearance of HBsAg is generally considered to mean resolution of infection, which has clinical benefit but also removes the stigma of HBV infection for many. However, cccDNA and integrated HBV DNA would still be present, and risk of HBV reactivation may occur during potent immunosuppression such as with anti-CD20 therapy.23Hwang J.P. Lok A.S. Management of patients with hepatitis B who require immunosuppressive therapy.Nat Rev Gastroenterol Hepatol. 2014; 11: 209-219Crossref PubMed Scopus (192) Google Scholar In 2017, it was thought that a functional cure as defined could be achieved in ∼30% of patients after 1–2 years treatment with combination of new antivirals targeting different steps in the HBV lifecycle and immunomodulatory therapies that activate innate/adaptive immune responses to HBV or remove immune blockades. Nonetheless, partial HBV cure, defined as HBsAg-positive (likely below a defined threshold), HBeAg-negative with undetectable serum HBV DNA after discontinuation of a finite course of treatment, has been proposed as an alternative intermediate endpoint that may be useful, particularly during the development of HBV cure, and a step forward compared with indefinite NA therapy (Figure 1). The HBV drug discovery and development field has made major progress in the last few years and has entered an exciting phase with many drug candidates that are direct-acting antiviral agents or host immune response modulators in clinical trial evaluation.6Fanning G.C. Zoulim F. Hou J. et al.Therapeutic strategies for hepatitis B virus infection: towards a cure.Nat Rev Drug Discov. 2019; 18: 827-844Crossref PubMed Scopus (299) Google Scholar,24Revill P.A. Chisari F.V. Block J.M. et al.A global scientific strategy to cure hepatitis B.Lancet Gastroenterol Hepatol. 2019; 4: 545-558Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar In this review, we discuss novel strategies that are active in clinical development, offering perspectives on the benefits and potential challenges of each approach (Table 1).Table 1HBV Antiviral Compounds Currently Under Clinical EvaluationCompoundMechanism of ActionClinical StageReference/Clinical TrialEntry inhibitorsBulevirtide (Hepcludex)Blocks NTCPIII (HBV/HDV coinfection)NCT04166266Capsid inhibitorsGLS4Core bindingIINCT04147208Canocapavir (ZM-H1505R)Core bindingIINCT05484466EDP-514Core bindingIINCT04470388NCT04008004PMID: 36382358ABI-H3733Core bindingINCT05414981ALG-000184Core bindingINCT04536337ABI-4334Core bindingINCT05569941HBsAg secretion inhibitorsREP 2139HBsAg bindingIINCT02565719PMID: 32147484Nucleos(t)ide analogsHS-10234Polymerase inhibitorIIINCT03903796Viral expression inhibitorsAntisense oligonucleotidesBepirovirsenASO targeting HBV transcriptsIIIIINCT05630820PMID: 36346079Small interfering RNAsJNJ-3989 (ARO-HBV)siRNA targeting HBV transcriptsIIIIIII/IINCT05275023NCT04439539NCT04535544NCT03365947PMID: 35870702VIR-2218siRNA targeting HBV transcriptsIIIIIII/III/IINCT04507269NCT04412863NCT04856085NCT03672188NCT05612581PMID: 34741731RG6346 (RO7445482)siRNA targeting HBV transcriptsIINCT04225715AB-729siRNA targeting HBV transcriptsIIIINCT04820686NCT04980482ALG-125755siRNA targeting HBV transcriptsINCT05561530Engineered antibodies against HBVVIR-3434HBsAg clearanceCross-presentation to and stimulation of T cellsInhibition of HBV entryIII/IINCT04856085NCT05612581Immunomodulatory compoundsImmune invigorationSelgantolimod (GS-9688)TLR8 agonistIIIINCT03615066NCT03491553RG7854TLR7 agonistIINCT04225715Envafolimab (ASC22)PDL1 inhibitorIINCT04465890Immune stimulationHepTcell (FP-02.2)Therapeutic vaccineIINCT04684914 NCT04465890TG-1050/T101Therapeutic vaccineIINCT04189276GSK3528869ATherapeutic vaccineIII/IINCT05276297NCT03866187VTP300Therapeutic vaccineIII/IINCT05343481NCT04778904ASO, antisense oligonucleotide; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; NTCP, sodium taurocholate cotransporting polypeptide; PDL1, programmed death ligand 1; siRNA, small interfering RNA; TLR, toll-like receptor. Open table in a new tab ASO, antisense oligonucleotide; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; NTCP, sodium taurocholate cotransporting polypeptide; PDL1, programmed death ligand 1; siRNA, small interfering RNA; TLR, toll-like receptor. Despite the fact that HBV codes for a single protein with enzymatic function (HBV-Pol), numerous steps in the viral lifecycle, from viral entry through virion secretion, have been targeted to enhance the inhibition of viral replication achieved with NA alone. The concept was to develop novel ways of decreasing the pool of cccDNA by more efficiently blocking both new rounds of infection and the intracellular recycling of the virus. While multiple strategies have been developed with early promising results, achieving functional cure with antiviral strategies alone has proven difficult to date. Approaches that have moved to human trials include entry inhibition, targeting of HBV RNA transcripts, blocking of encapsidation, more potent polymerase inhibition, and interference with viral and/or subviral particle secretion. While the discussion is limited to therapies that have entered human clinical trials, other approaches including powerful gene-editing strategies hold promise and have been reviewed elsewhere.17Martinez M.G. Smekalova E. Combe E. et al.Gene editing technologies to target HBV cccDNA.Viruses. 2022; 14: 2654Crossref PubMed Scopus (6) Google Scholar,25Dusheiko G. Agarwal K. Maini M.K. New approaches to chronic hepatitis B.N Engl J Med. 2023; 388: 55-69Crossref PubMed Scopus (15) Google Scholar Results of early-phase trials with antiviral agents have been nicely summarized in other review articles.26Lim S.G. Baumert T.F. Boni C. et al.The scientific basis of combination therapy for chronic hepatitis B functional cure.Nat Rev Gastroenterol Hepatol. 2023; 20: 238-253Crossref PubMed Scopus (12) Google Scholar,27Wong G.L.H. Gane E. Lok A.S.F. How to achieve functional cure of HBV: Stopping NUCs, adding interferon or new drug development?.J Hepatol. 2022; 76: 1249-1262Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar In the following sections we offer perspectives on each antiviral target based on existing data and considerations related to the mechanisms of action (Table 1 and Figure 1). Bulevirtide competes with HBV for binding to the sodium taurocholate cotransporting polypeptide, the primary HBV entry factor, and potently inhibits entry of HBV into hepatocytes.28Urban S. Bartenschlager R. Kubitz R. et al.Strategies to inhibit entry of HBV and HDV into hepatocytes.Gastroenterology. 2014; 147: 48-64Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar The lack of effect of bulevirtide monotherapy on HBsAg levels, in HBV monoinfected patients as well as in patients with HDV coinfection,29Wedemeyer H. Schoneweis K. Bogomolov P. et al.Safety and efficacy of bulevirtide in combination with tenofovir disoproxil fumarate in patients with hepatitis B virus and hepatitis D virus coinfection (MYR202): a multicentre, randomised, parallel-group, open-label, phase 2 trial.Lancet Infect Dis. 2023; 23: 117-129Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar has dampened enthusiasm for entry inhibition as an approach to HBV cure. While clearly inadequate as monotherapy, entry inhibition may be a very useful addition to potent inhibition of viral replication or other antiviral strategies. It may be difficult to inhibit all HBV replication in the liver, but if the addition of an entry inhibitor blocks infection of new hepatocytes, thus preventing new integration events or formation of new cccDNA molecules, this may be adequate to effectively mop up the leak on viral inhibition and lead to clearance of HBV-infected hepatocytes through cell turnover. Such a strategy may require relatively prolonged therapy, but if safe and well tolerated, this may be acceptable to patients and providers. Potent monoclonal antibodies to HBsAg may also neutralize viral particles and prevent viral entry.30Hehle V. Beretta M. Bourgine M. et al.Potent human broadly neutralizing antibodies to hepatitis B virus from natural controllers.J Exp Med. 2020; 217e20200840Crossref PubMed Google Scholar This approach, unlike bulevirtide, also lowers circulating HBsAg levels, with the potential to reinvigorate immune responses to enhance viral clearance, which could also potentially be enhanced by modification of the Fc-gamma receptor to create a vaccinal effect by promoting stimulation of naïve T cells to enhance immune clearance of HBV.31Bournazos S. Corti D. Virgin H.W. et al.Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection.Nature. 2020; 588: 485-490Crossref PubMed Scopus (75) Google Scholar The major challenges with entry inhibition as a strategy will be the need for prolonged therapy, the potential for safety concerns due to inhibition of bile acid uptake by blocking sodium taurocholate cotransporting polypeptide, and for monoclonal antibodies or other methods for delivering hepatitis B surface antibody, the potential for immune complex-related complications. The compact genome of HBV with overlapping open reading frames allows for targeting of all HBV transcripts, including those from integrated HBV DNA, with individual siRNA or antisense oligonucleotides (ASOs).32Mak L.Y. Seto W.K. Yuen M.F. Novel antivirals in clinical development for chronic hepatitis B infection.Viruses. 2021; 13: 1169Crossref PubMed Scopus (17) Google Scholar Targeting HBV transcripts has the potential to inhibit HBV replication directly (pgRNA targeting), as well as reduce antigen production (messenger RNA targeting), which may in turn reinvigorate HBV-specific immune responses. Improvements of nucleic acid delivery systems, including GalNAc conjugation to increase uptake in hepatocytes and abandonment of lipid nanoparticles due to the frequency of infusion reactions, have made siRNA/ASO approaches feasible and attractive.32Mak L.Y. Seto W.K. Yuen M.F. Novel antivirals in clinical development for chronic hepatitis B infection.Viruses. 2021; 13: 1169Crossref PubMed Scopus (17) Google Scholar, 33Agarwal K. Buti M. Van Bommel F. et al.Efficacy and safety of finite 48-week treatment with the siRNA JNJ-3989 and the capsid assembly modulator (CAM-N) JNJ-6379 in HBeAg negative virologically suppressed (VS) chronic hepatitis B (CHB) patients: results from REEF-2 study.J Hepatol. 2022; 77: S8Abstract Full Text PDF Google Scholar, 34Gane E. Jucov A. Dobryanska M. et al.Safety, tolerability, and antiviral activity of the siRNA VIR-2218 in combination with the investigational neutralizing monoclonal antibody VIR-3434 for the treatment of chronic hepatitis B virus infection: preliminary results from the phase 2 MARCH trial.Hepatology. 2022; 76: S18PubMed Google Scholar, 35Yuen M.F. Locarnini S. Lim T.H. et al.Combination treatments including the small-interfering RNA JNJ-3989 induce rapid and sometimes prolonged viral responses in patients with CHB.J Hepatol. 2022; 77: 1287-1298Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 36Yuen M.F. Lim Y.S. Cloutier D. et al.Preliminary 48-week safety and efficacy data of VIR-2218 alone and in combination with pegylated interferon alfa in participants with chronic HBV infection.Hepatology. 2022; 76: S19Google Scholar, 37Gane E.J. Janczewska E. Takehara T. et al.Efficacy and safety of siRNA JNJ-73763989, capsid assembly modulator JNJ-56136379, nucleos(t)ide analog (NA), and pegylated interferon alpha-2a (PEGIFNα2a) for treatment of chronic hepatitis B (CHB): week 24 results from the phase 2 PENGUIN study.Hepatology. 2022; 76: LB5035Google Scholar, 38George J. S-PD Antonov K. et al.Evaluation of the Vebicorvir, NRTI and AB-729 combination in virologically suppressed patients with HBeAg negative chronic hepatitis B virus infection: interim analysis from an open label phase 2 study.Hepatology. 2022; 76: LB5064Google Scholar Early data with multiple siRNA compounds looked very promising, with steady declines in HBsAg and other HBV antigens seen.39Yuen M.F. Schiefke I. Yoon J.H. et al.RNA interference therapy with ARC-520 results in prolonged hepatitis B surface antigen response in patients with chronic hepatitis B infection.Hepatology. 2020; 72: 19-31Crossref PubMed Scopus (67) Google Scholar However, longer-term treatment has documented a consistent plateauing of HBsAg decline at 16–20 weeks with very slow reductions in HBsAg beyond that point. HBsAg loss has rarely been achieved with siRNA monotherapy (or with siRNA on an NA backbone).40Yuen M.F. Wong D.K. Schluep T. et al.Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.Gut. 2022; 71: 789-797Crossref PubMed Scopus (19) Google Scholar,41Yuen M.F.H.J. Strasser S.I. Leerapun A. Sukeepaisarnjaroen W. Tangkijvanich P. et al.Hepatitis B viral control maintained during extended follow up of HBeAg- chronic hepatitis B (CHB) subjects who discontinued nucleos(t)ide analogue (NA) therapy after completion of AB-729 treatment, and in HBeAg+ subjects still on NA therapy.Hepatology. 2022; 76: LB5047Google Scholar The reasons for the plateauing and the inability to drive HBsAg levels below the limit of detection remain poorly understood; however, the observation that this occurs with other siRNA strategies that target host proteins suggest that this may be a more generalized phenomenon, possibly due to saturation of the RISC complex or other intrinsic mechanisms related to microRNA processing. Strategies combining siRNA with hepatitis B surface monoclonal antibodies or pegIFNα suggest that there may be an additive effect on HBsAg decline, but durability and impact on HBsAg loss remain to be determined.34Gane E. Jucov A. Dobryanska M. et al.Safety, tolerability, and antiviral activity of the siRNA VIR-2218 in combination with the investigational neutralizing monoclonal antibody VIR-3434 for the treatment of chronic hepatitis B virus infection: preliminary results from the phase 2 MARCH trial.Hepatology. 2022; 76: S18PubMed Google Scholar,36Yuen M.F. Lim Y.S. Cloutier D. et al.Preliminary 48-week safety and efficacy data of VIR-2218 alone and in combination with pegylated interferon alfa in participants with chronic HBV infection.Hepatology. 2022; 76: S19Google Scholar siRNAs offer an opportunity to explore whether bringing down HBsAg levels alone restores HBV-specific T cell function. Although some preliminary data are suggestive of enhanced immune responsiveness after HBsAg decline with siRNA, comprehensive data on the impact of HBsAg reduction on immunological recovery are an important omission in the field to date. Understanding if and when HBV-specific immune responses recover with HBsAg decline will be critical to guide combination therapy, not only which agents to use in combination, but also the sequencing and timing of specific immunomodulatory approaches as well as the optimal patient characteristics for this approach.42Gehring A.J