hepatitis b cure: from discovery to regulatory … b cure: from discovery to regulatory approval...

Review

Hepatitis B cure: From discovery to regulatory approval

Anna S. Lok1,⇑, Fabien Zoulim2, Geoffrey Dusheiko3, Marc G. Ghany4

Summary
Keywords: Chronic hepatitis B;
Review

Antiviral therapy; Immune mod-ulatory therapy; Hepatitis B sur-face antigen; Covalently closedcircular DNA.

Received 11 May 2017; accepted 11May 2017

1Division of Gastroenterology andHepatology, University of Michigan,

The majority of persons currently treated for chronic hepatitis B require long-term or lifelongtherapy. New inhibitors of hepatitis B virus entry, replication, assembly, or secretion andimmune modulatory therapies are in development. The introduction of these novel com-pounds for chronic hepatitis B necessitates a standardised appraisal of the efficacy and safetyof these treatments and definitions of new or additional endpoints to inform clinical trials. Tomove the field forward and to expedite the pathway from discovery to regulatory approval, aworkshop with key stakeholders was held in September 2016 to develop a consensus on treat-ment endpoints to guide the design of clinical trials aimed at hepatitis B cure. The consensusreached was that a complete sterilising cure, i.e., viral eradication from the host, is unlikely tobe feasible. Instead, a functional cure characterised by sustained loss of hepatitis B surfaceantigen with or without hepatitis B surface antibody seroconversion, which is associated withimproved clinical outcomes, in a higher proportion of patients than is currently achieved withexisting treatments is a feasible goal. Development of standardised assays for novel biomark-ers toward better defining hepatitis B virus cure should occur in parallel with development ofnovel antiviral and immune modulatory therapies such that approval of new treatments canbe linked to the approval of new diagnostic assays used to measure efficacy or to predictresponse. Combination of antiviral and immune modulatory therapies will likely be neededto achieve functional hepatitis B virus cure. Limited proof-of-concept monotherapy studiesto evaluate safety and antiviral activity should be conducted prior to proceeding to combina-tion therapies. The safety of any new curative therapies will be paramount given the excellentsafety of currently approved nucleos(t)ide analogues.� 2017 by the American Association for the Study of Liver Diseases, published by WileyPeriodicals Inc and � 2017 European Association for the Study of the Liver, published byElsevier B.V. All rights reserved.

Introduction
Ann Arbor, MI, United States;2Cancer Research Center of Lyon–INSERM U1052, Hospices Civils deLyon, Lyon University, Lyon, France;3University College London MedicalSchool and Kings College Hospital,London, UK;4Liver Diseases Branch, NationalInstitute of Diabetes and Digestiveand Kidney Diseases, NationalInstitutes of Health, Bethesda, MD,United States
⇑ Corresponding author. Address:Division of Gastroenterology andHepatology, University of Michi-gan Health System, 1500 EastMedical Center Drive, 3912 Taub-man Center, SPC 5362, Ann Arbor,MI 48109, United States.E-mail address: [email protected] (A.S. Lok).

The advent of several novel antiviral and immunemodulatory therapies for chronic hepatitis B nownecessitates a standardised appraisal of the efficacyand safety of these therapies, and definitions ofnew or additional endpoints to inform clinical tri-als. To move the field forward, and to expeditethe pathway from discovery to regulatory approval,the American Association for the Study of Liver Dis-eases and the European Association for the Study ofthe Liver jointly organised the Hepatitis B Treat-ment Endpoints Workshop on September 8-9,2016, in Alexandria, Virginia. The primary goal ofthis workshop was to assemble key stakeholdersfrom regulatory agencies (US Food and DrugAdministration and European Medicines Agency),biopharmaceutical and biotechnology companiesengaged in development of diagnostic tests andtherapeutic agents for hepatitis B, and academia

Journal of Hepato

Please cite this article in press as: Lok AS et al. Hepatitis Bjhep.2017.05.008

in order to develop a consensus on treatment end-points to guide the design of clinical trials aimedat hepatitis B cure.

Sixty-six (33%) of 202 participants completed apremeeting survey, including four from regulatoryagencies, 31 from industry, 28 from academia, andthree from other health care sectors. During theworkshop, experts reviewed the natural history ofchronic hepatitis B virus (HBV) infection, efficacyof currently approved treatments, potential antiviraltargets and approaches to restore immune respon-siveness to HBV, and preclinical and early-phaseclinical trial data on novel antiviral and immunemodulatory therapies for chronic HBV. The work-shop concluded with a session on the definition ofHBV cure; efficacy endpoints, safety assessments,target populations, and design of clinical trials;and diagnostic assays needed to support develop-

logy 2017 vol. xxx j xxx–xxx

cure: From discovery to regulatory approval. J Hepatol (2017), http://dx.doi.org/10.1016/j.

http://dx.doi.org/10.1016/j.jhep.2017.05.008


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Review

Review

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ment of curative therapies. These topics were fur-ther discussed during a closed session involving23 experts (including the four authors) represent-ing all constituent groups. This report summarisesthe discussions and consensus opinions of the 2-day meeting.

Definition of HBV cure

The goal of developing new therapies is to achieveHBV cure, i.e., elimination of HBV, thereby allowingtreatment to be stopped with no risk of virologicalrelapse and no risk of liver disease progression.However, a true cure may not be feasible becauseHBV DNA is integrated into the host genome; evenamong persons who have recovered from acuteHBV, viral covalently closed circular DNA (cccDNA)can be detected in the liver, explaining the reactiva-tion of HBV replication when these ‘‘recovered” per-sons are profoundly immunosuppressed. However,the observation that hepatitis B surface antigen(HBsAg) may become undetectable in serum afterclinical recovery from acute hepatitis B, sponta-neously during the course of chronic HBV infection,and during or after nucleosid(t)e analogue (NA) orinterferon (IFN) therapy, despite the likelihood ofpersistent integrated HBV genomes, argues for thefeasibility of achieving undetectable levels of HBsAg.

A key objective of the meeting was to establish adefinition of cure. Three definitions of HBV curewere proposed at this meeting: (i) complete sterilis-ing cure with undetectable HBsAg in serum anderadication of HBV DNA including intrahepaticcccDNA and integrated HBV DNA; (ii) functionalcure with sustained, undetectable HBsAg and HBVDNA in serum with or without seroconversion tohepatitis B surface antibody (anti-HBs) after com-pletion of a finite course of treatment, resolutionof residual liver injury, and a decrease in risk ofhepatocellular carcinoma (HCC) over time (severallevels of functional cure including complete shut-down of cccDNA transcription, elimination ofcccDNA, complete resolution of liver damage, andelimination of risk of HCC were discussed); (3) par-tial cure with detectable HBsAg but persistentlyundetectable HBV DNA in serum after completionof a finite course of treatment.

The vast majority (87.9%) of survey respondentsselected functional cure (sustained HBsAg loss) asthe goal for new HBV therapies. This selectionwas endorsed by other participants and the expertpanel as a feasible goal. In addition, functional cureoffers several other advantages: it is easy to assessand tests are widely available, it is associated withimproved clinical outcomes and lower rates of dis-ease reactivation, and once achieved, there is nofurther requirement for therapy.

There was less consensus regarding the neces-sity of achieving anti-HBs seroconversion becauseit is unclear whether durability of HBsAg loss and

: Lok AS et al. Hepatitis B cure: From discovery to regulato

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clinical benefits of HBsAg loss are dependent onthe development of anti-HBs. Importantly, few ofthe participants believed elimination of cccDNAwas a mandatory criterion for functional cure, andless than half required that cccDNA be renderedtranscriptionally inactive, reflecting uncertaintiesover whether new therapies in development cansilence or clear cccDNA, as well as pragmatic diffi-culties in measuring cccDNA.

Some members of the expert panel consideredpartial cure (sustained suppression of HBV replica-tion off treatment but persistent presence of HBsAg)an acceptable intermediary step toward functionalcure because partial cure is more achievable in theshort term, has been shown to lead to a reductionin clinical outcomes,1 and could expedite drugdevelopment.

Natural history of chronic HBV infection

The natural history of chronic HBV infection is vari-able and dependent on a complex interplay betweenthe host immune response and the virus. ChronicHBV infection comprises four phases definedby threeclinical parameters: serum alanine aminotransferase(ALT) concentrations, serum HBV DNA levels, andhepatitis B e antigen (HBeAg) status (Fig. 1). The firstphase is characterised by the presence of HBeAg andhigh serum HBV DNA but normal ALT levels. It hasbeen called the ‘‘immune-tolerant” phase, thoughrecent studies have challenged the concept ofimmune tolerance. HBV-specific T-cell responseshave been observed in patients in the immune-tolerant phase with similar frequency as in patientsin the immune-active phase. This finding has ledsome to propose that the immune response is bettercharacterised as low inflammatory during theimmune-tolerant phase (as opposed to inflammatoryduring the immune-active phase).2–4 The immune-tolerant phase is followed by the ‘‘HBeAg-positiveimmune-active” phase, when ALT levels become ele-vated. After varying intervals, seroconversion fromHBeAg to hepatitis B e antibody occurs, and a major-ity of patients transition to the ‘‘inactive carrier”phase, during which ALT levels return to normaland serum HBV DNA levels are low or undetectable.In some patients, serum HBV DNA and ALT levelsbecome elevated again, after years or decades. Thesepatients are considered to be in the ‘‘HBeAg-negativeimmune-active” phase, which is characterised byfluctuating HBV DNA and ALT levels. The annual inci-dence of HBeAg-negative immune-active hepatitisamong inactive carriers is estimated to be 0.37%.5

Some patients do not fit into any of these conven-tional phases. SerumHBsAg levels are highest duringthe immune-tolerant phase and lowest during theinactive carrier phase. Consequently, quantificationof serum HBsAg levels may help in determining thephase of infection, particularly for HBeAg-negativepersons,6 and to predict the risk of disease progres-

ry approval. J Hepatol (2017), http://dx.doi.org/10.1016/j.

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HBeAg

Anti-HBe

0 20 40 60Years

HBV DNA

HBsAg

ALT

Immunetolerant

Immune clearanceHBeAg-positivechronic hepatitis

Inactivecarrier state

ReactivationHBeAg-negativechronic hepatitis

Fig. 1. Phases of chronic HBV infection. Immune tolerant: HBeAg-positive, high serum HBV DNA butnormal ALT levels; immune clearance/HBeAg-positive chronic hepatitis: HBeAg-positive, high serum HBVDNA and elevated ALT levels, HBeAg seroconversion to hepatitis B e antibody occurs after varyingduration; inactive carrier: HBeAg-negative, serum HBV DNA low (generally \2000 IU/ml) or unde-tectable; reactivation/HBeAg-negative chronic hepatitis: HBeAg-negative, elevated levels of HBV DNA andALT in serum, HBV precore and/or basal core promoter variant often present. Traditionally phases ofchronic HBV infection are defined by HBeAg status, serum HBV DNA, and ALT levels. Quantitative HBsAglevels are different in each phase and are generally highest in the immune-tolerant phase and lowest inthe inactive carrier phase. While most patients progress from one phase to the next, not all patients gothrough each phase, and reversion to an earlier phase can occur. ALT, alanine aminotransferase; HBeAg,hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.

JOURNAL OF HEPATOLOGY
sion and HCC in HBeAg-negative patients with lowviraemia (HBV DNA\2,000 IU/ml).7 Because phasesof chronic HBV infection are defined based on clini-cal and not immunologicmeasures, the recent Euro-pean Association for the Study of Liver Diseasesproposed to describe these four phases as phases 1,2, 3, and 4.8 Patients in phases 1 and 2 would beHBeAg-positive, those in phases 3 and 4 would beHBeAg-negative, thosewith inactive disease (phases1 and 3) would be considered to have chronic infec-tion,while thosewith active disease (phases 2 and4)would be considered to have chronic hepatitis.
Some patients spontaneously clear HBsAg, butthis event is rare, occurring at a rate of 0.5%-1%per year. These patients remain positive for hepati-tis B core antibody, and some may develop anti-HBs. The majority of patients who clear HBsAg haveundetectable HBV DNA in serum, but HBV DNA per-sists in the serum in some and in the liver in allpatients. These patients are considered to haveoccult HBV infection. While the risk of cirrhosisand end-stage liver disease is greatly diminished,the risk of HCC after HBsAg loss remains substan-tial, particularly if HBsAg loss occurred after theage of 50 or after development of cirrhosis.9–11

Importantly, HBV can be reactivated uponimmunosuppression, suggesting that eradicationof HBV from the host is rarely achieved.12

Identifying individuals at greatest risk for devel-opment of cirrhosis and HCC is an important goal inthe management of chronic HBV infection. Recentstudies have highlighted the importance of viralload in predicting risk of cirrhosis and HCC.13,14

However, many other host (sex, age, family historyof HCC, obesity, diabetes), viral (HBV genotype andvariants, coinfection with other viruses: hepatitis C,hepatitis D, human immunodeficiency virus), andenvironmental (alcohol, smoking, carcinogens) fac-tors contribute to liver disease progression.

Several risk models have been developed to pre-dict risk of HCC.15–19 Most of these models werederived from data on Asians. The applicability ofthese models to all racial/ethnic groups and HBVgenotypes, patients with or without cirrhosis, anduntreated patients as well as those receiving antivi-ral therapy has not been confirmed. Cirrhosis is themajor risk factor for HCC. In the past decade, nonin-vasive assessment—serum marker panels and liverstiffness measurements—have largely replaced liverbiopsies in staging of liver fibrosis. These noninva-sive tests have also been shown to predict survivaland HCC in patients with chronic HBV infectionbecause they have high accuracy in diagnosing cir-rhosis.20–24

Current status of HBV treatment

Goals of treatment and efficacy assessment

The goals of HBV therapy are to prevent the devel-opment of cirrhosis, hepatic decompensation, HCC,


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and death from HBV-related liver disease. The even-tual clinical outcomes of chronic hepatitis B requireyears, if not decades, of surveillance; therefore, bio-chemical (normalisation of ALT), virological (sup-pression of serum HBV DNA), serological (HBeAgand/or HBsAg loss with or without seroconversionto hepatitis B e antibody and anti-HBs), and histo-logical (decrease in hepatic necroinflammation withor without improvement in fibrosis) markers havebeen used as surrogates for clinical outcomes—cir-rhosis, hepatic decompensation, HCC, and HBV-related mortality—and to assess indications fortreatment, response, and prognosis. The durabilityof responses after treatment discontinuation isvariable.

Indications for treatment

The decision to treat is based upon clinical assess-ment of the risk of disease progression, which isrelated to phase of disease. This risk assessment isbased primarily on HBV DNA and ALT levels andthe stage of disease, as assessed by liver biopsy ornoninvasive staging of hepatic fibrosis. Currentguidelines recommend treatment for patients withcirrhosis or decompensated liver disease and, forpatients without cirrhosis, evidence of modest tohigh viraemia and biochemical or histological evi-dence of hepatic necroinflammation.25–27 Continuedhigh levels of HBV replication together with hepaticinflammation increases the risk of cirrhosis andHCC; however, currently available therapies havelower efficacy in patients in the immune-tolerant


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phase, and treatment is not recommended for thesepatients.28,29

Approved therapies

Two classes of antiviral therapies have beenapproved for treatment of hepatitis B: IFNs andNAs. An advantage of IFN is that it results in higherrates of HBeAg and HBsAg loss (particularly inpatients with genotype A infection) compared toNAs. Pegylated IFN administered for 48–52 weeksresults in HBeAg seroconversion in 24%-27% andHBsAg loss in 3%-7% of patients compared to 12%-22% HBeAg loss and 0%-3% HBsAg loss after thesame duration of NA therapy.30 Response to IFN isalso more durable, and HBeAg and HBsAg lossmay occur after cessation of treatment, while viro-logical relapse, even after HBV DNA has becomeundetectable, is frequent after cessation of NA.31

However, IFN is less effective at suppressing viralreplication compared to NAs, requires parenteraladministration, is associated with numerousadverse effects, and is contraindicated in patientswith decompensated cirrhosis or severe exacerba-tions of hepatitis and those with autoimmune orpsychiatric illnesses. NAs are administered orallyand have negligible adverse effects. The recom-mended first-line NAs, entecavir and tenofovir,have low risk of drug resistance; but the require-ment for indefinite therapy in the majorityincreases the cost and the risk of nonadherenceand adverse effects.

Various combinations of IFN and NA have beenevaluated, but most studies have not shown anadded benefit compared to monotherapy. A recentstudy showed that combination of pegylated IFNand tenofovir increased the rate of HBsAg loss to9% at week 72, but the benefit was mainly observedin patients with genotype A.32 Therefore, there is anurgent need to develop new therapies for HBV thatcan result in durable suppression of HBV replica-tion and an ensuing decrease in hepatic inflamma-tion and fibrosis after a finite course of therapy. Inaddition, more research is needed to identify whichpatients can safely stop therapy. ImportantlyHBsAg loss at an older age and after the develop-ment of cirrhosis does not eliminate the risk ofHCC; however, the benefit of further treatment forpreventing HCC has not been proven.33 Treatmentthat can result in a functional cure at an earlierstage of disease might have a greater impact in pre-venting HCC.

Lack of impact on NAs on cccDNA

A major hurdle to HBV ‘‘cure” is the presence ofcccDNA in the hepatocyte nucleus in a noninte-grated form or episome. cccDNA serves as the tem-plate for transcription of all viral RNAs including



pregenomic RNA (pgRNA) and thus plays a key rolein the viral life cycle. There are two sources ofcccDNA: incoming virions and recycling of encapsi-dated DNA from the hepatocyte cytoplasm. Thehalf-life of cccDNA is long, thus explaining why itis difficult to cure HBV infection and why HBV canreactivate either spontaneously or followingimmune suppression, many years after clearanceof HBsAg. Chain terminating NAs block the reversetranscription of pgRNA to HBV DNA, but they havea marginal effect on cccDNA production, stability,or transcription. Continued transcription fromcccDNA and integrated viral genomes may explainthe relatively minor decrease in serum HBsAg levelsduring NA therapy despite undetectable serum HBVDNA levels.34 Unfortunately, current assays for cir-culating HBsAg cannot distinguish the transcriptionof HBsAg from cccDNA vs. integrated HBV DNA.

Impact of antiviral treatment on clinical outcomes

Most, but not all, long-term follow-up studies andmeta-analyses indicate that IFN and NA treatmentdecrease the risk of HCC and liver-related mortal-ity.35 A landmark randomised controlled trialshowed that the first-generation NA lamivudinedecreased the risk of disease progression and HCCin patients with advanced fibrosis or cirrhosis andhigh viraemia.1 Several studies have shown thatmaintained viral suppression during NA therapy isassociated with regression of fibrosis, reversal of cir-rhosis, and reduction in rates of hepatic decompen-sation.36,37 The risk of HCC is also diminished,although not eliminated, making it the only majorcomplication during NA treatment. The observedreduction in the incidence of HCC appears to bemore evident in persons with cirrhosis and afterseveral years of continued treatment.35

Novel antiviral therapies

Recent major scientific discoveries have allowed abetter understanding of the HBV life cycle, includingidentification of the cellular receptor for HBV entry,information on the key nuclear enzymes involved incccDNA formation and on its epigenetic control, theobservation of partial cccDNA degradation inducedby IFN or nuclear factor-jB signaling pathways,and identification of the role of the hepatitis B x pro-tein (HBx) in HBV transcription. Improved cellularand animal models have also enhanced the in vitroand in vivo assessment of the antiviral activity ofnovel compounds and their potential toxicity. Thesemajor advances in hepatitis B basic research havepaved the way for the identification of multiplenew therapeutic targets (Fig. 2), essential progresstoward a ‘‘cure” for hepatitis B. A list of novel antivi-ral therapies tested in clinical trials is provided inTable S1.


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HBV entry inhibitors
The pathway by which HBV enters hepatocytesinvolves virus attachment to heparan sulfate pro-teoglycans, allowing the binding of HBV pre-S1 tohuman sodium taurocholate cotransportingpolypeptide (NTCP), followed by membrane fusionand nucleocapsid release in the cytoplasm ofinfected cells.

Entry inhibitors can be classified according totheir modes of action. (i) Neutralising antibodiestarget the antigenic loop of HBV S-domain or N-terminal epitopes in the pre-S1 domain. These anti-bodies are highly specific but require parenteraladministration, and large quantities of antibodiesare necessary to neutralise circulating subviralenvelope particles. (ii) Attachment inhibitors arenegatively or positively charged drugs that bindthe virus (e.g., heparin) or cellular heparan sulfateproteoglycans (e.g., poly-l-lysine). They are efficientbut not specific. (iii) Substrates of NTCP includeconjugated bile salts (e.g., taurocholate, ezetimibe)and other small molecules that are transported byNTCP; the requirement for high concentrationsand short half-time at the receptor limit their clin-ical application. (iv) Irreversible NTCP inhibitors:myrcludex B (myristoylated pre-S1 peptide),cyclosporin A, and derivatives are allosteric inhibi-tors of NTCP. They irreversibly block receptor func-tion at nonsaturating concentrations and have longhalf-time at the receptor, but they can block trans-port of bile salts and other NTCP substrates athigher concentrations.

Entry inhibitors may prevent de novo cccDNAformation in noninfected hepatocytes and mightbe more effective at preventing mother-to-childtransmission or reinfection after liver transplanta-tion than at eliminating HBV in chronically infectedpersons.

Clinical trials of myrcludex B with or withoutpegylated IFN in chronic HBV and chronic hepatitisD virus infection are under way.38,39

Targeting cccDNA

Damage and destructionA number of studies demonstrate that it might bepossible to target cccDNA despite its location inthe protected sanctuary of the hepatocyte nucleus.Studies in cultured hepatocytes showed that sev-eral cytokines (IFN-a, lymphotoxin-b receptor ago-nists, IFN-c, and tumour necrosis factor-a) canmodulate pathways leading to the up-regulationof APOBEC3A/B deaminases, which in turn inducednonhepatotoxic degradation of cccDNA. However,only partial degradation was achieved in these tis-sue culture studies.40 DNA cleavage enzymes,including homing endonucleases or meganucleases,zinc-finger nucleases, TAL effector nucleases, andCRISPR-associated (cas) nucleases, specifically tar-geting cccDNA are being explored in experimental


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models.41 Some studies suggest that editing ofHBV DNA by CRISPR-associated (cas) nuclease cleav-age is more efficient than APOBEC-mediated cyto-sine deamination following treatment of infectedcells with IFN.42 Efficient delivery of these gene edit-ing approaches to HBV-infected hepatocytes with-out unintended off-target effects will need to beaddressed before they can be tested in clinical trials.

Functional silencingViral cccDNA is organised into a chromatin-likestructure and is subject to epigenetic regulation.43

Epigenome modifiers can potentially block cccDNAtranscriptional activity and shut down viral proteinexpression; however, if not specific, they mayinduce harmful effects on host cells. Some cytokines,including IFN-a, have been shown to decreasecccDNA transcription through epigenetic modifica-tions in preclinical models.44 HBx has been shownto be required for cccDNA transcription and viralreplication through degradation of the Smc5/6restriction factor; thus, HBx may be an attractiveviral target to silence not only cccDNA transcrip-tion45 but also several other HBx-dependent, virus-related cellular interactions. Current assays to detectcccDNA lack specificity, and a standardised methodto measure cccDNA or cccDNA transcription isneeded.

Targeting viral transcriptsThe use of RNA interference to inhibit replication ofHBV has been extensively evaluated in vitro and val-idated in animal models. Small interfering RNA(siRNA) can be designed to target any viral tran-script and induce its degradation by the RISC/Ago2complex, resulting in gene silencing. The potentiallimitations are the need for intravenous administra-tion, the risk of off-target binding, the potential tox-icity of the vehicle, and the risk of immuneactivation by pattern recognition receptors. ThreesiRNA formulations with different modes of deliveryare under preclinical evaluation and/or early-phaseclinical trial. Preliminary results of a phase II trialshowed that a single dose of ARC-520 in combina-tion with entecavir resulted in a profound and dur-able decrease in serum HBV DNA in both HBeAg-positive and HBeAg-negative patients and adecrease in HBsAg level in HBeAg-positive, but notin HBeAg-negative, patients.46,47 The siRNAs weredesigned to target the cotermini of all transcriptsfrom cccDNA, and the lack of effect on HBsAg levelin HBeAg-negative patients was postulated to bedue to altered viral transcript sequences derivedfrom integrated HBV DNA. Further studies areneeded to determine whether the decline in HBsAgproduction alone might be sufficient to restoreHBV-specific T-cell response or if addition of otherantiviral or immune modulatory therapies isneeded.

Antisense oligonucleotides targeting viral tran-scripts can block viral protein expression through





NTCP

Entry inhibitors

1. Entry

Inhibitors of rcDNAto cccDNA conversion

Targeting HBx

3. cccDNAformation

HBx

Targeting cccDNA-Degradation

-Epigenetic regulation

HBe

HBs

RNA interference

Interferons

MVB 8b. Morphogenesisand secretion

Assembly andsecretion inhibitors

CyclophilininhibitorsDNA+

DNA-pgRNA

Polymeraseinhibitors

Nucleocapsid assemblymodulators

8a. Trafficking

Hepatocyte

ccDNA

Host factors

mRNAs

Genomic DNA

Integration

9. Regulation of hostgenes by viral proteins

Pol

CoreRNA+

7. DNA synthesis0.7 kb2.4 kb2.1 kb3.5 kbRC

DNA

dsIDNA

4. cccDNAtranscription

5. Translation

2. Trafficking6. Nucleocapsid

formationpgRNA packaging

Fig. 2. HBV life cycle and antiviral targets. HBV entry: Lipopeptides mimicking pre-S1 domain competing with Dane particle for binding to NTCP (e.g., Myrcludex B). Othersmall molecule inhibitors are in development. Targeting cccDNA: Prevention of cccDNA formation. Damage and destruction through cytokines or cccDNA sequence-specificnucleases. Functional silencing through modulation of host cellular epigenetic-modifying enzymes by cytokines or inhibition of viral protein function. HBV mRNAs: siRNAapproaches or antisense oligonucleotides to block viral replication and viral protein expression. HBV polymerase: Reverse transcriptase inhibitors include approved NAs.Ribonuclease H inhibitors are in preclinical evaluation. Nucleocapsid assembly and pgRNA packaging: Capsid assembly modulators can affect nucleocapsid assembly andpgRNA encapsidation and may affect the nuclear functions of hepatitis B core protein (cccDNA regulation and IFN-stimulated gene expression). Targeting HBsAg:Phosphorothioate oligonucleotides inhibiting HBsAg release and monoclonal antibodies to decrease circulating HBsAg load are under evaluation. cccDNA, covalently closedcircular DNA; dslDNA, double-strand linear DNA; IFN, interferon; HBV, hepatitis B virus; MVB, multivesicular body; NTCP, sodium taurocholate cotransporting; pgRNA,pregenomic RNA; rcDNA, relaxed circular DNA.

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steric blockade of protein translation and/or RNAdegradation by ribonuclease H cleavage. In vitroand in vivo preclinical evaluations have shown theirpotential to inhibit viral replication and decreaseviral antigen load, but optimal delivery remains achallenge.

Nucleocapsid assembly and pgRNA packaging

Nucleocapsid formation and packaging of thepgRNA (the template for viral replication) are criti-cal steps of the viral life cycle. Therefore, develop-ing inhibitors or modulators of this process is anattractive therapeutic approach. The HBV core pro-tein is involved in many aspects of the viral lifecycle including transport of viral genome to thenucleus, uncoating to release relaxed circular DNA



in the nucleus, packaging of polymerase proteinand pgRNA, capsid assembly, modulation of reversetranscription, and interaction with envelope pro-teins for virus assembly. It may have additionalfunctions including modulation of cccDNA chro-matin and stability, nuclear export of viral RNAs,and modulation of innate immunity.

The HBV precore/core protein has recentlyemerged as a promising direct antiviral target. Withthe knowledge of the three-dimensional structure ofthe core protein, several classes of non-nucleosidesmall molecules called core protein assembly modula-tors have been developed, including phenylprope-namide and heteroaryldihydropyrimidinederivatives. These molecules can strengthen pro-tein–protein interaction, inhibit pgRNA encapsida-tion, and block plus strand DNA synthesis.48,49


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Results of the first dose-ranging phase Ib study ofNVR3-778 showed a decline in serum HBV DNA,HBV RNA, and HBsAg, with more pronounced effectwhen combined with pegylated IFN.50
Targeting HBsAg

Strategies inhibiting viral gene expression eitherthrough cccDNA transcription or viral mRNA trans-lation can decrease serum HBsAg levels. Owing tothe large amount of circulating HBsAg in personswith chronic HBV infection, monoclonal antibodiesaimed at neutralising and/or depleting HBsAg fromthe bloodstream are unlikely to be effective unlessused in combination with other approaches.

Nucleic acid polymers have been shown todecrease secreted HBsAg, through as yet unknownmechanisms. Clinical trials of REP 2055 and REP2139 used in monotherapy followed by combina-tion with pegylated IFN or thymosin alpha-1induced a marked decline in circulating HBsAglevels and viraemia and anti-HBs seroconversionin some patients.51 Similar results were observedin pilot studies of tenofovir and pegylated IFN incombination with REP 2139 or REP 2165.52 Theseresults need to be replicated in larger studies, andthe potential for cytotoxicity resulting from intra-cellular retention of HBsAg must be resolved.

Immune response to HBV and Implications forimmune modulatory therapies

An orchestrated innate and adaptive immuneresponse is necessary to sense and control HBVinfection. The induction of innate proinflammatoryresponses and cytokine activation are weak inchronic HBV infection.53 IFN-c production fromnatural killer cells is detectable in acute hepatitisbut deficient in chronic hepatitis. HBV-specific Tcells necessary for HBV control (which may alsoplay a role in hepatic inflammation) are dysfunc-tional and may be depleted or deleted in chronicHBV.54,55 Their exhausted phenotype has beenattributed to persistent antigen exposure andincreased expression of T-cell inhibitoryresponses,3,53,56 providing the basis for the focuson reducing HBsAg production. B-cell dysfunctionin chronic hepatitis B is less well characterised,though the high rate of HBV reactivation in patientsreceiving anti–cluster of differentiation 20 suggeststhat B cells play a role in immune control of chronicHBV. Accordingly, several potential target mecha-nisms for immune modulation to engender orrestore HBV-specific immune responses in conjunc-tion with profound inhibition of HBV replicationand HBsAg production to attain immunologicalcontrol have been suggested (Fig. 3). The main con-cerns of immune modulatory therapy are theinduction of uncontrolled hepatitis flares andautoimmunity.


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Interferons

IFN-a has been used to treat chronic HBV andchronic hepatitis D virus. IFN-a induces expressionof IFN-sensitive genes encoding intracellular orsecreted effector proteins with antiviral properties.Recent studies have shown that IFN-a also inhibitspgRNA encapsidation, enhances cccDNA degrada-tion, and exerts epigenetic modification of cccDNAtranscription.40,57 Response to IFN-a (HBeAg sero-conversion) is durable in [70% of patients.58,59

Understanding the mechanisms for IFN nonrespon-siveness and the higher rates of response in geno-type A infection could lead to new targets forantiviral and/or immune modulatory therapies.

Pathogen recognition receptors

Pathogen recognition receptors are an importantgateway to sensing by the innate immune system.Pharmacological activation of the intrahepaticinnate immune response with toll-like receptor 7,8, or 9 has been studied. GS-9620, an oral agonistof toll-like receptor 7, induced a decline in serumHBV DNA and HBsAg levels as well as hepatic HBVDNA in HBV-infected chimpanzees.60 Similar effectswere also observed in woodchucks61 but not inhumans.62,63 The discrepancies between results inanimal models and humans highlight the impor-tance of testing new therapies in humans at an earlystage in drug development.

Stimulator of IFN gene agonists

Stimulator of IFN gene is the adapter protein of mul-tiple cytoplasmic DNA receptors and a pathogenrecognition receptor recognising bacterial secondmessenger and may be a potential target of pharma-cologic activation of the innate immune response.64

Stimulator of interferon gene agonists can also beused as adjuvants to therapeutic vaccination. Retinoicacid–inducible protein 1 has been shown not only toinduce IFNand cytokine productionbut also to inhibitHBV replication through sensing of the epsilon struc-ture of pgRNA.65 SB 9200, an oral prodrug of the din-ucleotide SB9000, is thought to activate retinoic acid–inducible protein 1 and nucleotide-bindingoligomerisation domain–containing protein 2, result-ing in IFN-mediated antiviral immune responses invirus-infected cells and decreased serum woodchuckhepatitis virus DNA and surface antigen levels.66 Clin-ical trials are ongoing.

Checkpoint modulation

The lack of a T cell–mediated response in chronicHBV is partly due to overexpression of coinhibitoryreceptors including programmed cell death, cyto-toxic T lymphocyte–associated antigen-4 lympho-cyte activation gene, and mucin domain to impair





Therapeutic vaccination

Blockade of immunosuppresive pathwaysCheckpoint inibitors

Stimulation of innate immunityTLR agonists

Small moleculescIAP inhibitors

IFNs and othersantiviral cytokines

Recovery of exhausted adaptive immunity

Engineering of T cells (HBV-specific TCR or CARS)

Naïve T cells priming to HBV antigens

Bispecific Abconstructs

TCR

CD4+

B cell

CD8+ MDSC NK

TGF-β

IL-10

Treg

NaiveT CD8+

IFNsIL-12IL-6

IL-18

TLR7TCR

CD1d

KC

CD40L/CD40CD40 agonist

CD8+

CD8+

NKT

PD-1

x x x

xx

Mature pDC

CCR7

CD80

CD86

MHC class II

x

Fig. 3. The immune liver microenvironment and immunotherapeutic targets. Innate immune responses: IFN-a exhibits antiviral activity in infected cells but alsocontributes to cell-mediated immunity in vivo. Toll-like receptor (TLR-7 and others) agonists to boost antiviral cytokine production and activation of natural killer cells, Bcells, and T cells are in clinical evaluation. Drugs antagonising cellular inhibitor of apoptosis can sensitise HBV-infected cells to tumour necrosis factor–mediated apoptosis.HBV-specific T-cell exhaustion: Approaches to block inhibitory pathways (checkpoint inhibitors, programmed cell death protein 1 blockade, and others) andimmunosuppressive cytokines (interleukin-10 and transforming growth factor beta) to achieve recovery of HBV-specific T cells and natural killer cells from chronichepatitis B patients are currently in evaluation. Engineering of redirected T cells through (i) transfer of HBV-specific T-cell receptors or HBV-specific chimeric antigenreceptors ex vivo in patient’s T cells or (ii) retargeting of immune effector cells toward HBV-infected cells using bispecific antibody constructs. Therapeutic vaccines:Antigenic stimulation by diverse approaches are currently being evaluated in phase I/II clinical trials in association with NAs to promote clusters of differentiation 4+ and 8+

T-cell antiviral activity and antibody responses. Ab, antibody; CARS, chimeric antigen receptors; CD, cluster of differentiation; cIAP, cellular inhibitor of apoptosis; HBV,hepatitis B virus; IFN, interferon; IL, interleukin; KC, Kupffer cell; MAIT, mucosa-associated invariant T; MDSC, myeloid-derived suppressor cell; MHC, majorhistocompatibility complex; NA, nucleos(t)ide analogue; NK, natural killer; NKT, natural killer T; PD-1, programmed cell death protein 1; pDC, plasmacytoid dendritic cell;TCR, T-cell receptor; TGF-b, transforming growth factor beta; TLR, Toll-like receptor; Treg, T regulatory.

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T-cell effector function. T-cell function may also beimpaired by immunosuppressive cytokines includ-ing interleukin 10. Recent cancer therapies haveindicated the potential of blockade of these coin-hibitory receptors with antibodies. Such inhibitionmay reverse immune dysfunction in hepatitis B asdemonstrated in studies in woodchucks andex vivo studies in humans.4,67–69 The main concernsof this approach are induction of uncontrolled hep-atitis flares and autoimmunity, which can lead tofatal organ damage.70

Therapeutic vaccine

The goal of therapeutic vaccination is to stimulateor boost the host immune response to restoreimmune control, resulting in sustained suppressionof HBV replication and ultimately HBsAg loss. Ther-apeutic vaccination strategies including conven-tional HBsAg vaccine with or without potentadjuvants, T-cell vaccine, immune complexes ofHBsAg and human anti-HBs, apoptotic cells thatexpress HBV antigens, DNA vaccines, and viral vec-tors expressing HBV proteins have been evaluatedwith limited success. Patients with high viral loadsmay be less responsive, and patients with cirrhosis



may be at higher risk for immune-mediated hepati-tis flares. Pretreatment with NAs to suppress HBVreplication may enhance HBV-specific T-cellresponse and prevent flares. GS-4774, a heat-inactivated yeast-based vaccine expressing HBV S/C/X fusion protein, induced HBV-specific T-cellresponses in healthy volunteers; but HBsAg declineand HBsAg loss were not observed in chronic hepati-tis B patients virally suppressed on NAs as well asthose who were NA-naive.71–73 A list of therapeuticvaccines tested in clinical trials is provided inTable S1.

Combination of antiviral and immunemodulatory therapies

Combinations of antiviral therapy targeting multiplesteps in the HBV life cycle to suppress viral replica-tion and viral antigen production and immune mod-ulatory therapy to restore immune response to HBVwill likely be needed to achieve the goal of HBV‘‘cure,” but which specific combination of agentswill be needed is not certain because most antiviralor immune modulatory drugs in clinical evaluationare entering either phase Ib or phase II trials at this


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time. The possibility that HBV-specific T-cell func-tion can be partially restored has been demon-strated in patients with chronic HBV who hadspontaneous or antivirus (IFN or NA)–mediatedHBeAg or HBsAg loss.74,75 With the availability ofnovel approaches such as siRNA or combination ofantiviral agents targeting different steps in theHBV life cycle, marked reduction and even shut-down of HBsAg production may be achieved in ahigher proportion of patients. The reduction inantigen load might facilitate the restoration ofHBV-specific T-cell responses. Historically, immunemodulatory therapies have focused on patients inthe immune-active phase, but recent studies sug-gest that patients in the ‘‘immune-tolerant orhigh-replication noninflammatory” phase shouldalso be considered as HBV-specific T cells are pre-sent in this phase of disease.3,76 The ability to‘‘cure” hepatitis B at earlier stages of liver diseasewould theoretically have a greater impact onreducing risk of HCC.
Clinical trials aimed at HBV cure

Efficacy endpoints for clinical trials

Initial trials of IFN and NA used biochemical, viro-logical, serological, and histological endpoints toassess efficacy, while more recent trials havefocused on virological and serological endpointsbecause these endpoints have been shown to corre-late with improved clinical outcomes.36,77–79 Theuse of a biochemical endpoint is problematicbecause of the lack of a standardised definition ofnormal ALT. Furthermore, with increasing preva-lence of obesity and non-alcoholic fatty liver dis-ease, failure to normalise ALT may not necessarilyindicate ongoing liver inflammation induced byHBV. Indeed, phase III clinical trials of NAs haveconsistently found a lower percent of patientsachieving a biochemical vs. a virological response.Noninvasive assessments of liver fibrosis havereplaced liver biopsies in assessing liver disease inclinical practice, and a histological endpoint wouldno longer be practical or necessary for assessingfunctional cure. However, liver biopsies may berequired in proof-of-concept studies to confirm anovel mode of action and/or to validate noninva-sive surrogate markers of antiviral activity.

For both antiviral and immune modulatorytherapy trials, survey respondents ranked suppres-sion of serum HBV DNA to undetectable and loss ofHBsAg as the most important primary efficacy end-point for phase II and phase III trials, respectively;and the expert panel agreed. The difference in rank-ing of primary efficacy endpoint for phase II vs.phase III trials reflects a desire to have an earlierreadout in phase II trials and the potential difficultyin achieving HBsAg loss after shorter treatment.HBV DNA suppression will not be an appropriate


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endpoint for trials enrolling patients who are virallysuppressed on NAs. Decline in serum HBsAg levelhas been used as an endpoint in some trials, butthere is no consensus on whether the kinetics ofdecline in HBsAg level can predict ultimate HBsAgloss.

There was general consensus (�65%) that themost appropriate time to assess the primary efficacyendpoint for phase III trials of novel antiviral orimmune modulatory therapies should be month 6posttreatment. The choice reflected the intent toachieve a durable response (‘‘cure”) after a finitecourse of treatment. There was less consensus onthe optimal time for assessing efficacy in phase IItrials, with the responses divided between month6 on treatment vs. month 6 posttreatment. Theexpert panel indicated that the most appropriatetime to assess efficacy endpoints may depend onthe mechanism of action and half-life of the drug.For example, drugs that inhibit reverse transcriptionof HBV pgRNA to HBV DNA are expected to result inrapid decline in serum HBV DNA levels, while drugsaimed at restoring immune response may takelonger to have any measurable effects on viral load.The expert panel also emphasised the need for long-term follow-up to confirm durability of responsesand impact on clinical outcomes.

Diagnostic markers for new assays to determinetherapeutic efficacy

There was consensus on the need for standardisedassays to provide mechanistic insights into theeffects of novel antiviral or immune modulatoryagents and to have new surrogate markers to assessHBV ‘‘cure” (Table 1).

Serum HBsAg assaysHBsAg loss was ranked as the most important effi-cacy endpoint for phase III trials of both novelantiviral and immune modulatory therapies. Someexperts suggested the need for more sensitiveHBsAg assays. Others indicated that the persistenceof low levels of HBsAg in serum may stem not frompersistent cccDNA transcription but from integratedHBV DNA genomes. Assays that can differentiateHBsAg originating from integrated HBV DNA vs.cccDNA are required. Failure to detect HBsAg inserum may not represent shutdown of HBsAg pro-duction as circulating HBsAg may be complexed toanti-HBs. HBV produces a surplus of empty envelopeparticles, which can outnumber virions by [1,000-fold. Assays that can differentiate surface proteinsin virions vs. subviral particles (large, middle vs.small HBsAg proteins) would be informative.

Serum HBsAg decline may be used as a surrogateto explore efficacy in early-phase trials, although itsaccuracy in predicting HBsAg loss has not beenestablished. Quantitative HBsAg assays can also beused to categorise disease stage and to prognosti-cate. Several standardised quantitative HBsAg





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assays have been available in Europe and in Asia inthe last decade, and the consensus is that they willbe essential tools for new drug development.

cccDNA quantification and transcriptional activityMany new antiviral agents in development aim toenhance elimination or degradation of cccDNA orto epigenetically modify cccDNA transcription.Assays that reliably measure concentrations ofcccDNA and/or its transcriptional activity woulddirectly assess efficacy of these agents and wouldbe invaluable in proof-of-concept studies. Cur-rently, there are no standardised assays for intra-hepatic cccDNA. However, there was consensusthat an adequate sample of liver tissue would benecessary and that appropriate measures to ensurespecificity are needed, particularly in the presenceof abundant relaxed circular DNA.

Assays for serum markers that are reliable sur-rogates of intrahepatic cccDNA would be desirablefor clinical trials. Several studies have shown thatserum HBsAg level correlates better with hepaticcccDNA than serum HBV DNA level, suggesting thatit is an indirect marker of cccDNA.80,81 However,while the correlation is satisfactory in HBeAg-positive patients, it is suboptimal in HBeAg-negative patients.80,82 HBsAg can be translatedfrom subviral RNAs transcribed from cccDNA orfrom integrated HBV DNA. It has been suggestedthat HBsAg is predominantly derived from cccDNAin HBeAg-positive patients, while an increasingproportion is derived from integrated HBV DNA inHBeAg-negative persons. Recent studies suggestthat serum HBV RNA and hepatitis B core-relatedantigen (HBcrAg) levels may be more reliable sur-rogates of hepatic cccDNA than HBsAg levels.

Serum HBV RNA assaysThe vast majority of circulating HBV virions containpartially double-stranded relaxed circular DNA;however, circulating ‘‘virions” containing HBVRNA had been reported.83 These RNA-containing‘‘virions” are more abundant in patients receivingNA therapy, probably because inhibition of reversetranscription of pgRNA by NA leads to accumula-tion of encapsidated pgRNA, some of which maybe enveloped and secreted. Thus, the detection ofHBV RNA in serum in the absence of detectableserum HBV DNA in patients receiving NA therapycould infer ongoing cccDNA transcription and hasbeen shown to be a predictor of viral relapse afterdiscontinuation of NA therapy.84 Assays for serumHBV RNA levels, particularly if they are specificfor pgRNA, may provide a useful surrogate for tran-scriptionally active cccDNA.

Serum HBcrAg assaysThe HBV precore/core gene is translated into thecore protein, precore/core precursor, and HBeAg;these proteins can be collectively measured.HBcrAg can assemble into defective, enveloped par-



ticles that that do not contain HBV RNA or HBV DNA.A core-related antigen immunoassay measuring allthese forms of precore/core gene expression hasbeen developed. Serum HBcrAg levels have beenshown to correlate with hepatic HBV DNA and topredict viral relapse after discontinuation of NAtherapy.85 Assays for serum HBcrAg levels may pro-vide indirect evidence for transcriptionally activecccDNA.86

Immune response to HBVRestoration of immune response to HBV is a keystep toward HBV ‘‘cure.” Most immune modulatorytherapies have focused on restoring T-cell response.While markers of response to antiviral therapies canbe used to evaluate immune modulatory therapies,standardised assays to measure improvement inspecific immune responses to predict viral clearancewould be informative in early-phase drug develop-ment. The development of standardised assays toassess restoration of immune responses to HBV willrequire consensus on the requisite assays; theirmethodology, sensitivity, and specificity; their cor-rect interpretation and reproducibility in differentlaboratories; and cross-validation of the assays.

Approval of HBV diagnostic assaysDevelopment of standardised assays for surrogateendpoints for HBV cure should occur in parallel withdevelopment of novel antiviral and immune modu-latory therapies to expedite research. During theclosed session, the US Food and Drug Administrationand European Medicines Agency representativesproposed consideration of simultaneous testing ofmultiple intermediate secondary endpoints in allphase II and III trials. The research assays must bestandardised and data and assay platforms sharedto facilitate identification of the appropriate surro-gate markers of efficacy. It is important that theapproval of new treatments is linked to the develop-ment and approval of new diagnostic assays used tomeasure efficacy or to predict response.

Assessment of safety and stop rules

The remarkable safety profile of current NAsimposes a stringent requirement for the safety ofnew HBV therapies at all stages of the disease. Aunique concern in the development of hepatitis Btherapies is the risk of severe hepatitis flares, whichcan result in hepatic decompensation and death. TheUS Food and Drug Administration has explicit rec-ommendations on managing drug-induced liverinjury; however, these recommendations do notapply to patients with underlying liver disease. Fur-thermore, transient hepatitis flares are not alwaysharmful and may portend immune clearance ofinfected hepatocytes. Hepatitis flares may be dueto direct drug-induced liver injury, drug-inducedimmune-mediated hepatitis (e.g., checkpoint inhibi-tors), the underlying disease (incomplete viral sup-


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Table 1. Potential Diagnostic Tools/Surrogate Markers for HBV.

HBV Marker Purpose Rationale/Comments

HBsAg, ultrasensitivequalitative orquantitative assay

To detect minimal residual HBsAg, lower limit ofdetection of Lumipulse assay 0.004 IU/ml compared tocurrent assays 0.05 IU/ml

HBsAg loss is considered the most reliable indicator for functionalcure.

HBsAg fragments,epitope mapping

To determine whether residual HBsAg is translated fromcccDNA transcripts or integrated HBV DNA transcriptsTo detect antivirus-resistant/immune escape HBsAgvariants

Persistent detection of HBsAg may be from integrated HBV DNA andnot cccDNA. Integrated HBV DNA is often fragmented with deletionsand rearrangements, while cccDNA translates into full-lengthHBsAg. HBsAg variants may give rise to false-negative results orinaccuracy in quantification in current assays.

Large (L) vs. middle (M)vs. small (S) surfaceprotein

To differentiate complete virions from empty envelopeparticles

Complete virions are coated with L, M, & S surface proteins; butempty envelope particles comprise mostly S surface proteins.

HBsAg–anti-HBsimmune complex

To detect residual HBsAg masked by anti-HBs in immunecomplex

HBsAg loss is considered the most reliable indicator for functionalcure.

Quantitative HBsAglevel

To facilitate differentiation of inactive carriers fromHBeAg-negative chronic hepatitisTo predict outcome in HBeAg-negative patients with lowserum HBV DNA

Serves as an intermediary measure of HBsAg loss. HBsAg leveldeclines before it becomes undetectable. but accuracy in predictingHBsAg loss is low.

HBV RNA level To predict viral relapse when treatment is stopped Serves as a surrogate for transcriptionally active cccDNA,particularly if assay is specific for pgRNA. Encapsidated pgRNA canbe enveloped and secreted, levels higher in patients on NA becausereverse transcription of pgRNA to HBV DNA is blocked. Shown insome studies to predict viral relapse after discontinuation of NA.Specificity of current assays for pgRNA vs. subgenomic RNAs isunknown.

HBcrAg To correlate levels with intrahepatic HBV DNATo predict viral relapse when treatment is stopped

Translated from HBV precore/core gene, can assemble into defectiveparticles that are secreted. Shown in some studies to correlate withintrahepatic HBV DNA and cccDNA transcriptional activity and topredict viral relapse after discontinuation of NA.Limitation: lack of sensitivity, composite biomarker

cccDNA quantificationand transcriptionalactivity

To quantify cccDNA from treated and untreated patientsTo assess transcriptional activity: pgRNA/cccDNA ratio

cccDNA serves as a template for transcription of HBV RNA andtranslation of HBV antigens. Most direct measure of HBV cure.Adequate sample of liver tissue and stringent protocol to ensurespecificity are required.

Immune response toHBV antigens

To assess role as a biomarker for treatment response Restoration of immune response is considered a necessaryprerequisite for HBV cure. Unclear what types of immune responseare critical, what methods to use, and criteria for improvement orrestoration of response that will translate to cure.

cccDNA, covalently closed circular DNA; HBcrAg, hepatitis B core related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; pgRNA, pregenomic RNA.

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pression), or immune clearance of infected hepato-cytes (successful viral suppression and accompany-ing restitution of the host immune response). Thetiming and course of the flare, as well as the asso-ciated chronological changes in serum HBV DNAlevels, can help in differentiating the cause of most,but not all, hepatitis flares; for example, flaresrelated to drug-induced liver injury may beidiosyncratic and occur at any time. There was noconsensus on definition of hepatitis flares (magni-tude of ALT increase, absolute value, or fold-change compared to baseline); however, a majorityof survey respondents and the expert panel agreedthat flares accompanied by increase in bilirubin orprothrombin time and flares in patients with cir-rhosis should be considered severe. Other adverseevents can also occur, e.g., off-target effects ofsiRNA or epigenetic modification, autoimmunityassociated with checkpoint inhibitors, and hepato-toxicity from retention of dysfunctional viral parti-cles. There was no consensus on when a trial ordevelopment of a new agent should be stoppeddue to safety concerns; however, survey respon-


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dents and the expert panel indicated that any deathor liver transplantation, hepatic decompensation,irreversible autoimmunity, or incidence of severehepatitis flare in [5% of patients could prompt ahalt. Two-thirds of respondents to the survey andmost of the expert panel recommended that safetyand efficacy should be assessed for at least 6 monthsafter stopping therapy.

Design of clinical trials for HBV cure

A combination of antiviral and immune modulatorytherapies will most likely be needed to increase thelikelihood of HBV cures. There was consensus thatthe antiviral activity and safety of individual newagents used as monotherapy and infrequent orinsignificant drug interactions should be first estab-lished before progressing to clinical trials of combi-nation therapy. However, demonstration of efficacyas a monotherapy need not be required.

New therapies are most needed for patients athigh risk of HBV-associated mortality (cirrhosis)and those in whom current therapies are less effec-





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tive (immune-tolerant, hepatitis D virus infection).However, in designing early-phase clinical trials,the target patient populations will necessarily bethose who are most likely to respond and able totolerate possible exacerbations of hepatitis. Theconsensus was to initially conduct trials intreatment-naïve, HBeAg-positive patients withactive disease or in HBeAg-positive or HBeAg-negative patients virally suppressed on NAs.Proof-of-principle and safety data would first beestablished in patients without cirrhosis.

A particular challenge with designing phase IIstudies is to identify a target population with suffi-cient heterogeneity to be representative of the pop-ulation with chronic hepatitis B but not so diversethat it hinders analysis because of multiple sub-groups. Among the survey respondents, 59% feltthat patient subpopulations should be studied sep-arately. If multiple patient populations wereincluded in the same trial, patients could be poten-tially stratified for cirrhosis, HBeAg status, HBsAglevel, HBV DNA level, treatment history, and HBVgenotype. Standardised criteria for ascertaining cir-rhosis by noninvasive serum markers and/or liverelastography are required.

Given the heterogeneity of the natural course ofchronic hepatitis B, there was consensus that ran-domised controlled trials are needed to establishefficacy and that comparison with placebo is feasi-ble and ethical in trials for patients in the immune-tolerant or inactive phases because current guideli-nes do not recommend treatment for thesepatients. For patients with active disease or cirrho-sis, investigational new agents can be compared toNA or IFN. Alternatively, the new drug can be testedagainst placebo as an additional therapy to NA.There was consensus that the trials should aim todemonstrate superiority of the investigationaltherapy.

Summary and recommendations

A summary of the recommendations on endpointsand design of clinical trials for HBV cure is pre-sented in Table 2. Improved understanding of theHBV life cycle and host immune response to HBVhas facilitated discovery and design of antiviraltherapies directed against multiple steps of theHBV replication cycle, as well as potentialimmunomodulatory therapies to restore immuneresponse to HBV infection. Development of novelHBV therapies will be further aided by the avail-ability of improved cellular and animal infectionmodels. While this progress has raised the possibil-ity of a cure for hepatitis B, a complete sterilisingcure, i.e., viral eradication from the host, may beunrealistic due to the presence of integrated HBVDNA. Although HBsAg clearance is infrequent, itdoes occur with NA and IFN therapy and is associ-ated with improved clinical outcomes. Therefore,



a functional cure (characterised by sustained lossof HBsAg with or without anti-HBs seroconversion)after a finite course of novel antiviral and immunemodulatory therapies in a higher proportion ofpatients than is currently achieved with existingtreatments is an attainable goal.

Cooperation between academia, industry, andregulatory agencies to standardise and validate sur-rogate markers for cure, in order to facilitate thedevelopment of curative therapies and to facilitatethe path from discovery to regulatory approval, isrequired. Limited proof-of-concept monotherapystudies to evaluate safety and antiviral activityshould be conducted prior to proceeding to combi-nation therapy. The safety of any new curative ther-apies will be paramount given the excellent safety ofcurrently approved NAs. Continued collaborationamong the stakeholders will make HBV ‘‘cure” areality for patients with chronic HBV.

Financial support

The HBV Treatment Endpoints Workshop wascosponsored by the American Association for theStudy of Liver Diseases and the European Associa-tion for the Study of the Liver. The contents of thispaper represent the opinions of the authors andnot necessarily the views of the US Food and DrugAdministration, Swedish Medical Products Agency,or European Medicines Agency.

Conflict of interest

Potential conflict of interest: Dr. Lok received grantsfrom Bristol-Myers Squibb and Gilead. Dr. Dusheikoconsults, advises, is on the speakers’ bureau, andreceived grants from Gilead. He consults, advises,and is on the speakers’ bureau for Bristol-MyersSquibb and Janssen. Dr. Zoulim consults andreceived grants from Arbutus, Gilead, Janssen,Roche, and Sanofi. He consults for Transgene. Hereceived grants from Assembly.

Authors’ contributions

All four authors contributed to concept and design,data collection and literature review, drafting, edit-ing and final approval of manuscript.

Acknowledgements

We are deeply grateful to the governing boards ofthe American Association for the Study of Liver Dis-eases and the European Association for the Study ofthe Liver for sponsoring the HBV Treatment End-point Workshop and all of the speakers, moderators,and participants for their contribution to the discus-sions. We also thank the members of the advisory


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Table 2. Summary of recommendations on endpoints and design of clinical trials for HBV cure.

Definition of functional HBV cure

� Sustained HBsAg loss with or without anti-HBs seroconversion and undetectable HBV DNA in serum after completion of a finite course of treatmentMain objective

� To demonstrate superiority of the investigational therapyControl arm

� Placebo or no treatment for patients in the immune-tolerant or inactive phase� NA or IFN as comparator therapy or add-on therapy to investigational therapy for patients with active disease or cirrhosis (IFN as comparator only

if no cirrhosis or compensated cirrhosis with no portal hypertension)Patient profile for initial trials

� HBeAg-positive patients with active disease, not currently on treatment� HBeAg-positive or HBeAg-negative patients with baseline active liver disease, who are virally suppressed on NAs� Factors to consider for stratificationo HBeAg statuso HBsAg levelo Cirrhosiso HBV DNA levelo Treatment historyo HBV genotype

Primary efficacy endpoint� Phase 2 trials: Suppression of HBV DNA in serum to undetectable month 6 on-treatment or month 6 posttreatment, decline in serum HBsAg level

may be used as exploratory endpoint in trials enrolling patients virally suppressed on NAs and in trials of compounds targeting HBsAg� Phase 3 trials: HBsAg and HBV DNA not detectable in serum month 6 posttreatment, must include long-term follow-up to confirm durability of

response after completion of treatmentSafety issues

� Evidence of safety of new drugs as monotherapy and evaluation of infrequent or insignificant drug interactions needed before new drugs arecombined

� Hepatitis flares: need to differentiate flares due to immune clearance vs. other causes� Severe flares: hepatitis flares accompanied by increase in bilirubin or prothrombin time or occurring in patients with cirrhosis� Any adverse event leading to death or liver transplantation, hepatic decompensation, irreversible autoimmunity, or incidence of severe hepatitis

flare in[5% of patients may prompt consideration for stopping trial

HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IFN, interferon; NA, nucleos(t)ide analogue.

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panel for their input into the program and surveydesign and their critical review of this manuscript.The advisory panel members include Debra Birnkr-ant, M.D., Eric F. Donaldson, Ph.D., Poonam Mishra,M.D., M.P.H., Center for Drug Evaluation andResearch, and Kathleen Whitaker, Ph.D., Center forDevices and Radiological Health, US Food and DrugAdministration; Timothy Block, Ph.D., Hepatitis BFoundation and Baruch S. Blumberg Institute; Fab-rice Carrat, M.D., Ph.D., France REcherche Nord&SudSida-HIV Hepatites (ANRS); Judith Falloon, Ph.D.,MedImmune; Lalo Flores, Ph.D., Novira Therapeu-tics; Bruce D. Given, M.D., Arrowhead Pharmaceuti-cals; Filip Josephson, M.D., Ph.D., Swedish MedicalProducts Agency; Rajen Koshy, Ph.D., NationalInstitute for Allergy and Infectious Diseases,National Institutes of Health; Stephen Locarnini,M.D., Ph.D., Victorian Infectious Diseases Reference

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Review

Please cite this article in press as: Lok AS et al. Hepatitis B cure: From discovery to regulatory approval. J Hepatol (2017), http://dx.doi.org/10.1016/j.jhep.2017.05.008

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