present and future therapies of hepatitis b from discovery to cure
TRANSCRIPT
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
1/16
REVIEW
Present and Future Therapies of Hepatitis B: From
Discovery to Cure
T. Jake Liang,1
Timothy M. Block,2
Brian J. McMahon,3
Marc G. Ghany,1
Stephan Urban,4
Ju-Tao Guo,2
Stephen Locarnini,5 Fabien Zoulim,6 Kyong-Mi Chang,7 and Anna S. Lok 8
Hepatitis B virus (HBV) is a significant global pathogen, infecting more than 240 mil-lion people worldwide. While treatment for HBV has improved, HBV patients oftenrequire lifelong therapies and cure is still a challenging goal. Recent advances in tech-nologies and pharmaceutical sciences have heralded a new horizon of innovative thera-peutic approaches that are bringing us closer to the possibility of a functional cure of chronic HBV infection. In this article, we review the current state of science in HBV therapy and highlight new and exciting therapeutic strategies spurred by recent scientificadvances. Some of these therapies have already entered into clinical phase, and we will
likely see more of them moving along the development pipeline. Conclusion: With grow-ing interest in developing and efforts to develop more effective therapies for HBV, thechallenging goal of a cure may be well within reach in the near future. (HEPATOLOGY 2015;62:1893-1908)
Despite the availability of effective vaccines forthree decades and improvement of treatment,the prevalence of chronic hepatitis B viral
(HBV) infection worldwide has declined minimally from 4.2% in 1990 to 3.7% in 2005.1 Moreover, the
actual number of persons who are chronically infected isestimated to have increased slightly from 223 million to240 million during this same period. Treatment for thisinfection, while advancing to the stage that viral replica-tion can be effectively suppressed and disease success-fully controlled, is still handicapped by variouslimitations and cannot be considered as curative. Recog-nizing that HBV therapeutics is at the cusp of innova-tions and breakthroughs, this review summarizes new targets among the HBV viral and host immune systemsfor which drugs are now in late preclinical developmentand clinical testing. In addition, novel and potentially promising therapeutic strategies that would likely result
in more durable and complete responses are highlighted.To put these advances in the context of the current stateof the science, we summarize the current HBV therapiesand their limitations and spotlight the continued impactof fundamental scientific discoveries in advancing the
research and development of new HBV therapies.
Natural History of Chronic Hepatitis B
The course of chronic HBV infection has beengrouped into four phases: the immune tolerant phase,the immune active/hepatitis B e antigen (HBeAg)–posi-tive chronic hepatitis phase, the HBeAg-negative inac-tive phase, and the immune active/HBeAg-negativechronic hepatitis phase. However, these terms may notaccurately reflect the immunological status of patients ineach phase but are useful for prognosis and determining need for therapy.2,3 The duration of each phase varies
Abbreviations: anti-HBs, antibody to HBsAg; CAR, chimeric antigen receptor; cccDNA, covalently closed circular DNA; HBeAG, hepatitis B e antigen; HBsAg,
hepatitis B surface antigen; HBV, hepatitis B virus; IFN, interferon; IL, interleukin; ISG, interferon-stimulated gene; NRTI, nucleos(t)ide reverse transcriptase
inhibitor; NTCP, sodium/taurocholate cotransporter; PEG-IFN, pegylated interferon; RNAi, RNA interference; TLR, toll-like receptor; WHV, woodchuck hepatitis virus.
From the 1Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD; 2 Baruch S.
Blumberg Institute, Doylestown, PA; 3National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Anchorage, AK; 4 Department of Infectious Diseases, Molecular Virology and German Center for Infection Diseases, University Hospital Heidelberg, Heidelberg, Germany;5 Hepatology Department, Lyon University and Cancer Research Center of Lyon, INSERM U1052, Lyon, France; 6 Victorian Infectious Diseases Reference Labora-
tory, Doherty Institute, Melbourne, VIC, Australia; 7 Department of Medicine, Philadelphia Veterans Affairs Medical Center and the University of PennsylvaniaPerelman School of Medicine, Philadelphia, PA; 8 Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, MI.
Received March 29, 2015; accepted July 31, 2015.
1893
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
2/16
from months to decades. Transition can occur from anearlier to a later phase, but regression back to an earlierphase can also occur.4 It should be noted that not allpatients go through all four phases. Furthermore, whilethe cutoff levels of alanine aminotransferase used todefine different phases were traditionally based on upperlimits of normal determined by clinical diagnostic labo-ratories, recent studies suggest that the true normal val-ues are lower.5
HBV Replication: From Basic Science toDrug Development
Advances in understanding the molecular biology andreplication cycle of HBV have provided unprecedentedinsight into the mechanisms of action and treatmentresponse of currently available drugs against HBV as
well as potential future targets for therapeutic develop-ment (Fig. 1). HBV gains entry into hepatocytes initially through a low-affinity interaction between heparan sul-fate proteoglycans on the hepatocytes involving the anti-genic loop (“a” determinant or antibody neutralizationdomain) of the HBV envelope proteins6,7 and then a high-affinity interaction of the myristoylated pre-S1domain with the liver-specific receptor sodium/tauro-cholate cotransporter (NTCP).8 NTCP is exclusively expressed on the basolateral/sinusoidal membrane of hepatocytes. Its natural function is to transport conju-gated bile salts (e.g., taurocholate) into hepatocytes as
part of the enterohepatic pathway.9 Accordingly, NTCPplays a key role in the liver tropism of HBV.10,11 NTCPis also crucial for the host specificity of HBV. Two shortsequence motifs within NTCP are sufficient to renderthe respective proteins from cynomolgus monkey andmouse functioning as an HBV receptor.12,13 Additionalhost factors are probably required for efficient HBV entry. Fusion of HBV particles and release of nucleocap-sids into the cells involves receptor-mediatedendocytosis.14,15
The HBV genome–containing nucleocapsid is trans-
ported into the nucleus through a yet-undefined path-
way, probably involving microtubule and nuclearimportin machinery.16 In the nucleus, the relaxed circu-lar, partially double-stranded genome is then repaired toa full-length, circular DNA by covalently attached viralpolymerase (P) and other incompletely understood
mechanisms probably involving tyrosyl DNA phospho-diesterase of the topoisomerase and DNA repair path- way.17 The circularized protein-free genome thencomplexes with host histone and nonhistone proteinsincluding various histone-modifying enzymes into a minichromosome that functions as the template fortranscription.18 Its transcriptional activity is regulatedby epigenetic modifications and specific host transcrip-tional factors, such as hepatocyte nuclear factor 4.19
HBV core and X proteins are also present on the mini-chromosome and probably play an important role inHBV transcription.18,20,21 The covalently closed circular
DNA (cccDNA) is transcribed to three classes of HBV RNAs: genome-length RNAs (pregenomic and precoreRNAs coding for core gene products and P protein), SRNAs (S proteins), and X RNA (HBx protein). Thepregenomic RNA transcript is reverse-transcribed by theP protein to relaxed circular DNA in the core-containing nucleocapsid. The nucleocapsid can eitherassemble into an infectious virion with the envelopeproteins through the multivesicular body pathway 22 orrecycle back to the nucleus for cccDNA amplification ina process probably controlled by the pre-S1 envelope
protein and other host factors.
23
The steady-state popu-lation of cccDNA is about one to 10 molecules perinfected hepatocyte.24
Current Therapies of Hepatitis B andMechanisms of Action
There are currently two classes of drugs approved forthe treatment of hepatitis B: nucleos(t)ide reverse tran-scriptase inhibitors (NRTIs) and interferon-a (IFN-a).The first-line antiviral HBV medications include a nucleoside analogue, entecavir; a nucleotide analogue,
tenofovir; and pegylated IFN-a (PEG-IFN-a), used as
Address reprint requests to: T. Jake Liang, LDB/NIDDK/NIH, Bldg. 10-9B16, 10 Center Drive, Bethesda, MD 20892-1800. E-mail: [email protected]; tel:
11-301-496-1721. fax: 11-301-402-0491.
Copyright VC 2015 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the U.S.A.
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.28025 Potential conflict of interest: Dr. Guo received grants from Janssen. Dr. Block is on the Board of and owns stock in Contravir. He received grants and holds
intellectual property rights with Oncore-Tekmira. Dr. Lok consults and received grants from Gilead. She consults from GlaxoSmithKline, Merck, MYR, and
Tekmira. She received grants from Bristol-Myers Squibb. Dr. Chang advises Genentech, Arbutus, and Alnylam. Dr. Zoulim consults and received grants fromRoche, Gilead, and Novira. He consults for Janssen. Dr. Locarnini received royalties and holds intellectual property rights with Melbourne Health. He consults and
received fees from Arrowhead. He consults for Gilead.
1894 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
3/16
monotherapy.25-27 PEG-IFN is administered for 48-52 weeks. While it has a weaker antiviral activity thanNRTIs, it is associated with a higher rate of HBeAg andhepatitis B surface antigen (HBsAg) loss, possibly through a combination of direct antiviral and immuno-modulatory effects. By contrast, NRTIs target only thereverse-transcription of pregenomic RNA to HBV DNA and have no direct effect on cccDNA. Long-term treat-ment with more potent NRTIs can lead to progressive
loss of HBeAg and HBsAg with time.IFN-a, as a front-line host defense against viral infec-
tions, is known to induce IFN-stimulated genes (ISGs), which have promiscuous antiviral functions against a variety of viruses. Depending on the viruses, these ISGsplay a diverse and pleiotropic role in targeting variousviral functions at different steps of the viral replicationcycle and potently suppress viral infection and spread.IFN-a has a direct anti-HBV effect and acts on multiplesteps of the HBV replication cycle (Fig. 1).28,29 In addi-tion, it has an immunomodulatory effect that can indi-
rectly inhibit HBV replication by affecting cell-mediatedimmunity in vivo.30 Studies of the HBV kinetics inIFN-a-treated patients suggest a more relevant role of the latter mechanism in mediating IFN-a’s anti-HBV effects.31
Despite targeting multiple steps of HBV replication,the molecular mechanisms underlying IFN-a’s actionremain to be fully defined. IFN-a is thought to inducespecific ISGs that inhibit HBV transcription or prevent
the formation of nucleocapsid or target it for degrada-tion.28,29,32 The responsible ISGs have not been clearly defined. IFN-a’s effect on HBV transcription is partly mediated by epigenetic modifications of the cccDNA minichromosome.33 Recent development of infectiousHBV cell culture systems provided the much neededtools and models to study the effects of antivirals,including IFN, on HBV replication.33,34 A recent study demonstrated that IFN-a and another putative antiviralcytokine, lymphotoxin-b, induce the degradation of cccDNA in infectious cell culture systems.35 This effect
Fig. 1. HBV life cycle and targets of therapeutic development. The complete HBV life cycle including entry, trafficking, cccDNA formation, tran-
scription, encapsidation, replication, assembly, and secretion is shown. The functions of the HBV gene products are incorporated into the life
cycle. Drugs or biologics, in clinical use or development, targeting various steps of the HBV life cycle, are illustrated in red. See text for detailsof these drugs. Abbreviations: ER, endoplasmic reticulum; HSPG, heparan sulfate proteoglycan; siRNA, small interfering RNA.
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1895
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
4/16
is mediated by induction of the APOBEC3 family of proteins, specifically APOBEC3A by IFN-a and APO-BEC3B by lymphotoxin-b; APOBEC3 functions torestrict foreign DNAs, such as those from invading microbial genomes, which activate the IFN response
including induction of APOBEC3s as ISGs.
36
The APOBEC3s are DNA editing enzymes and deaminateforeign double-stranded DNA cytidines to uridines.36
This conversion can lead to either C to T mutations ordegradation of foreign DNA. In contrast, cellulargenomic DNA is unaffected. APOBEC3s are known totarget human immunodeficiency virus, adeno-associatedvirus, and possibly other DNA viral genomes for degra-dation.36 For HBV, the role of APOBEC3 has been con-troversial. APOBEC3G was shown to inhibit HBV replication in cell culture, but the mechanism had beenattributed to either direct inhibition of HBV replication
or hypermutations from DNA editing.37-40 All of theearlier studies were performed in HBV DNA transfec-tion systems that could not be used to investigatecccDNA. In an HBV infectious culture system, theinduced APOBEC3 interacted with the core proteinand translocated to the nucleus to target cccDNA.35
The development of nucleoside analogues owes muchof its success to the comprehensive understanding of how HBV replicates. Based on the model of HBV repli-cation, the P protein has been the primary therapeutictarget in HBV drug development (Fig. 1). While this
advance represents a pivotal step in the chronicle of HBV treatment, knowledge of the mechanism of actionof this class of anti-HBV drugs also exposes its limita-tion, as discussed above.
While the second-generation NRTIs, such as enteca-vir and tenofovir, can potently suppress the DNA syn-thesis step of HBV replication, they have little effect onthe level and activity of cccDNA, which has a long half-life and can persist for decades in the infected liverdespite successful antiviral treatment.24 This limitationexplains the necessity for a prolonged, possibly indefi-nite, treatment with this class of anti-HBV drugs. The
turnover of cccDNA has been the subject of intenseresearch because of its fundamental importance in HBV replication and therapy. Several mechanisms appear toexplain the turnover of cccDNA in vivo. First, the directcytopathic effect of activated HBV-specific T lympho-cytes can cause death of infected cells. Second, gradualloss of the cccDNA pool by cell proliferation in injuredliver can account partly for gradual loss of cccDNA.Finally, a noncytopathic mechanism of eliminating cccDNA from infected cells contributes to the turnoverof cccDNA.41 IFNs and other cytokines have been
implicated in this “cell cure” mechanism, but the precisemechanism is unknown.41
Entecavir and tenofovir can decrease the level of HBV DNA by 6 logs within 1 year of treatment andhave low rates of antiviral drug resistance (0%-1% after
5 years of continued treatment).
42-44
However, rates of HBeAg seroconversion (20% after 1 year and 40%-50% after 5 years) and HBsAg loss (5%-10% after 5years) are low. Therefore, most patients require many years and often lifelong treatment with associated costsand risks of adverse reactions, drug resistance, and non-adherence.45 Despite these limitations, antiviral treat-ment can reverse liver fibrosis and even cirrhosis,prevent cirrhosis complications, and reduce, though noteliminate, the risk of hepatocellular carcinoma.44,46
Derivatives of tenofovir as prodrugs with improvedpharmacological properties are being developed and
may be of benefit in certain situation.47For patients who do not have cirrhosis or do not
require immunosuppressive therapy, professional society guidelines recommend treating those in the immuneactive phase,25-27 although treatment at an earlier stagehas been proposed to minimize unrecognized yet signifi-cant liver damage.48 However, treatment during theimmune tolerant phase is associated with a low rate of HBeAg seroconversion and failure to completely sup-press HBV DNA to nondetectable levels.49
The ultimate goal of antiviral therapy would be to
eliminate all forms of potentially replicating HBV, butthis may not be feasible because even in persons whorecover from acute HBV infection with HBsAg to anti-body to HBsAg (anti-HBs) seroconversion, HBV per-sists in the liver in the form of cccDNA and can bereactivated during immunosuppressive therapy. A morerealistic goal is a “functional cure” in which HBV DNA is not detectable after the completion of a finite courseof treatment with loss of HBsAg and minimization of hepatocellular carcinoma risk over time. To accomplishthis goal, a combination of antiviral drugs that targetdifferent steps in the HBV life cycle or immunomodula-
tory therapies to restore host immune response to HBV will be needed.
Combination Studies of Current Therapies
Given that only two classes of anti-HBV agents arecurrently available, combination therapy consist of twoNRTIs or an NRTI plus PEG-IFN. In the latter case, anNRTI and PEG-IFN may be combined simultaneously,sequentially, starting with either drug first, or as an add-on strategy with either drug first.
1896 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
5/16
Initially, the clinical need to increase the potency of first-generation antivirals and to prevent emergence of antiviral resistance was the primary reason to test combi-nation therapy with NRTIs. Unfortunately, thisapproach suffered from the fact that all NRTIs have thesame virological target, the HBV polymerase. Thus, thetreatment response observed in patients was similar tothat of the most potent agent in the combination. Theissue of antiviral resistance is now greatly diminished
with the development of second-generation NRTIs,such as entecavir and tenofovir. The efficacy and safety of entecavir and tenofovir combination therapy werecompared to entecavir monotherapy in previously untreated HBV patients.50 A greater proportion of sub-
jects receiving combination therapy achieved viral sup-pression compared to entecavir alone, but the difference
was not statistically significant.50 However, HBeAg-
positive subjects with baseline HBV DNA 108
IU/mLreceiving combination therapy had a significantly higherrate of virological response compared to those receiving monotherapy.50
Conceptually, combination of PEG-IFN with anNRTI would be more likely to result in synergy becausethe drugs have different mechanisms of action, the con-cept being that inhibition of viral replication with anNRTI may augment the immune effects of PEG-IFN.Unfortunately, while studies of PEG-IFN in combina-tion with first-generation NRTIs did show synergy inachieving viral suppression and reducing the incidence
of antiviral resistance, off-treatment responses were simi-lar to that of PEG-IFN alone.51,52 The availability of more potent, second-line NRTIs together with a renewed interest in achieving HBsAg clearance hasstimulated interest in combining these agents togetheror in combination with PEG-IFN.
Simultaneous PEG-IFN and tenofovir was evaluatedin treatment-naive patients with HBeAg-positive andHBeAg-negative chronic hepatitis B.43 Patients receiving PEG-IFN and tenofovir had a higher rate of HBsAg lossthan those receiving either drug along.43 Although theseresults are encouraging, they represent a small increase
(6%) in HBsAg loss over PEG-IFN monotherapy, and a benefit was mainly observed in those with genotype A infection.
Sequential therapy beginning either with an NRTIfollowed by PEG-IFN or vice versa for variable dura-tions has been conducted in both HBeAg-positive andHBeAg-negative subjects. In general, these studies havenot demonstrated a substantial benefit in terms of eitheron-treatment or sustained off-treatment HBV DNA suppression or HBeAg and HBsAg loss compared toPEG-IFN as a historical control.53-55
Starting with NRTI first and adding PEG-IFN later would seem to be the most logical approach to combi-nation therapy. The idea is that the NRTI would rapidly lower viral load and restore T-cell responsiveness, thenadding PEG-IFN might hasten the decline of circulating
and intrahepatic viral antigens leading to an improve-ment in the innate immune response.56 Several recentstudies seem to support such an approach.57-59 Among HBeAg-positive subjects, higher rates of HBeAg sero-conversion were achieved with add-on combinationtherapy of PEG-IFN and NRTI (27%) compared toNRTI only (0%).58 Among HBeAg-negative subjects,HBsAg loss was reported in 6.6% of subjects at the endof therapy in the combination arm versus 1% in theNRTI-only arm.59 None of these studies included anarm using PEG-IFN monotherapy, and when comparedto historical studies of PEG-IFN monotherapy, the
results obtained with combination therapy arecomparable.
A recent study compared PEG-IFN alone to PEG-IFN followed by add-on entecavir or entecavir followedby add-on PEG-IFN.60 Rates of HBeAg seroconversionposttreatment were similar across treatment groups.60
With an add-on strategy, a longer duration of NRTIbefore add-on PEG-IFN and a longer duration of PEG-IFN therapy was associated with higher rates of HBeAg and HBsAg loss.
In summary, there are insufficient data at present torecommend the use of combination therapy except in
very special circumstances, such as in subjects with very high baseline viral levels (>108 IU/mL) or for manage-ment of subjects who have failed a first-line agent due toa suboptimal response or the development of multidrug resistance. Further studies are needed to address the ben-efit of various formats of combination therapy withPEG-IFN and more potent NRTIs.
HBV Entry Inhibitors
Entry inhibitors have been used successfully in treat-ing viral infections. In particular, small molecules and
antibody-based treatments are quite effective in treating acute viral infections.61 For chronic viral infection likehuman immunodeficiency virus, entry inhibitors havealso been successfully developed.62 For HBV, entry inhibitors can be applied in two ways. The first is in a preventive setting: entry inhibition, such as using anti-HBs antibodies, blocks de novo HBV infection. Thisapplication has been successfully demonstrated in ani-mal models63 and is clinically a standard of care using HBsAg-specific immunoglobulins to prevent reinfectionafter liver transplantation, to avoid vertical transmission
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1897
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
6/16
of HBV from infected mothers to children, and forpostexposure prophylaxis.64 Regarding chronic hepatitisB patients, whether entry inhibition would be a viabletherapeutic option is debatable. It is conceivable thatpotent blockade of HBV reinfection in chronically infected patients can reduce viral load due to the turn-over of HBV-infected hepatocytes.65 Previous studiessuggested that hepatocyte turnover is indeed much fasterin HBV-infected liver than in healthy hepatocytesbecause of immune-mediated cytotoxicity.66 A sustainedinhibition of de novo formation of cccDNA in hepato-cytes may contribute to the eventual clearance of thevirus with prolonged therapy, especially if it is used incombination with other potent anti-HBV drugs.Because hepatitis D virus shares the same entry pathway,another potential application of entry inhibitors is inHBV/hepatitis D virus coinfection.
HBV entry depends in part on the pre-S1 sequence,more specifically the myristoylated N terminus of thelarge envelope protein. Both the myristoylation and theN-terminal 75 amino acids are required for infectivity of HBV.67,68 It was shown that synthetic lipopeptidesrepresenting this subdomain potently inhibit HBV infection. The mode of action of such peptidic inhibi-tors (Myrcludex B, for example) can be attributed tospecific receptor binding.11 Myrcludex B successfully passed phase 1 clinical trials.69 Moreover, because thenatural role of NTCP as a bile salt transporter has beenstudied in some detail, molecules already known to bind
or inhibit the function of NTCP have been tested.Cyclosporin A and its derivatives (e.g., alisporivir) orapproved drugs like ezetimibe are among those thathave been demonstrated to inhibit HBV entry.70-72
Myrcludex B, cyclosporin A, and other substrate ana-logues inhibit bile salt transport by NTCP. Accordingly,these molecules may elevate bile salts and other trans-ported substrates in the serum of patients. This concernmay be a clinically manageable problem. First, people withpolymorphisms in NTCP resulting in a functional knock-down show very moderate clinical symptoms and do notdevelop any specific pathology.73 Second, NTCP knock-
out mice are viable and show elevated conjugated bile saltlevels without symptoms but have a slight retardation ingrowth during development.74 Most importantly, the anti-viral effect of Myrcludex B and cyclosporin A is already apparent at a much lower concentration than that requiredfor inhibiting bile acid transport (>100-fold differ-ence).12,71 Thus, entry inhibition should be clinically achievable without significant interference with the trans-porter function of the receptor.
Myrcludex B is currently being tested in two ongoing clinical trials.75 Preliminary results suggested that Myr-
cludex B is safe and well tolerated in HBsAg-positivepatients with or without HDV coinfection. A decline inthe HBV DNA level (>1 log 10) was reported in 87% of patients at 12 weeks of treatment (10 mg/day), and thedecline continued with extended treatment beyond 12
weeks. Myrcludex B treatment at high doses was associ-ated with some bile acid elevation.
HBV Capsid Inhibitors
Several classes of inhibitors of pregenomic RNA pack-aging and HBV capsid assembly have been identified.They function to dysregulate or selectively inhibit eitherpregenomic RNA encapsidation or nucleocapsid assem-bly or both. The first of these were the phenylpropena-mide derivatives AT-61 and AT-130.76 Thesecompounds selectively inhibit viral pregenomic RNA
packaging
77
and are active against both wild-type andlamivudine-resistant HBV.78,79 As a class and at themolecular level, these agents have been shown to inducetertiary and quaternary structural changes in HBV cap-sids. AT-130 binds to a promiscuous pocket at the coredimer–dimer interface.80 This binding decreases viralproduction by initiating virion assembly prematurely inthe replication cycle, resulting in morphologically nor-mal capsids that are empty and noninfectious.77
The second group of inhibitors is the heteroaryldihy-dropyrimidines, which inhibit HBV virion productionin vitro and in vivo by preventing capsid formation.81
The best studied of the heteroaryldihydropyrimidines,Bay 41-4109, has a dual mechanism of action by inhibi-ting encapsidation directly and causing a concomitantreduction in the half-life of the core protein. Structuralstudies of this class of inhibitors revealed that they induce inappropriate capsid assembly at low concentra-tions and, when in excess, promote a misdirected assem-bly reaction and decreased capsid stability.82,83 Like thephenylpropenamides, the heteroaryldihydropyrimidinesare active against NRTI-resistant strains of HBV.79
Other inhibitors targeting the nucleocapsid are being
developed by several biotech companies (Tables 1).
84
Invitro studies have demonstrated strong synergy whenthese inhibitors are used in combination with currently approved NRTIs.78,79
Inhibition of HBV Gene Expression
Persistence of HBV results from an ineffective antivi-ral immune response against the virus, and one of the
ways HBV orchestrates this is through excess productionof subviral particles containing HBsAg. These noninfec-tious subviral particles may act as a decoy for the
1898 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
7/16
immune system,85 especially for mopping up potentially neutralizing anti-HBs. High levels of HBsAg, in therange of 400 lg/mL (0.4% of total serum protein), arecommonly found in the blood of patients with chronichepatitis B86 and may interfere with HBV-specificimmune responses.87,88
A molecular approach to inhibit HBV gene expres-sion has been successfully achieved in vitro using molecule-based therapies targeting the viral messengerRNA. Viral messenger RNA can be directly targetedusing antisense oligonucleotides, ribozymes, or RNA interference (RNAi).89 Of these, RNAi appears most
Table 1. Experimental HBV Therapeutics in Late Preclinical or Clinical Stage*
Compound Mechanism/ Target†
Stage of Development Sponsor Reference
Direct-acting antivirals:
GS-7340 (tenofovir
alafenamide fumarate
Polymerase (prodrug of
tenofovir)
Phase 2/3 Gilead Sciences 47; NCT0194047; NCT01940341‡
CMX157 Polymerase (prodrug of
tenofovir)
Phase 1/2§
Contravir (Chime rix) 14 6; NCT01080820‡
NVR1221/3778 Capsid Phase 1/2 Novira 84; NCT02112799‡
Sulfamoylbenzamides Capsid Animal Oncore 147
GLS4 Capsid Phase 1 HEC Pharm Group, China 148
Bay41-4109 Capsid Phase 1 AiCuris 83
REP 2139-Ca Assembly/HBsAg Phase 1/2 Replicor NCT02233075‡
ARC-520 RNAi Phase 1/2 Arrowhead 94; sponsor’s website;
NCT02065336‡
TKM-HBV RNAi Phase 1 Tekmira Sponsor’s website; NCT02041715‡
ALN-HBV RNAi Animal Alnylam Sponsor’s website
DNA-directed RNAi RNAi Animal Benitec Sponsor’s website
ISIS HBV Antisense Phase 1 Isis Sponsor’s website
Host targeting agents:
Myrcludex B Entry/NTCP Phase 1/2 Myr-GmbH/Hepatera 75
Birinapant Apoptosis/second
mitochondrial activator of caspases
Phase 1 Tetralogic Sponsor’s website; NCT02288208‡
Flavonoids STING agonist (pattern
recognition receptor)
Animal Oncore 149
NVP018 Cyclophilins, IRF-9 Animal Oncore (NeuroVive) Sponsor’s website
Epitope HBV Glucosidase/therapeutic
vaccine
Animal Blumberg Institute 150
Immune modulatory agents:
GS-9620 TLR-7 agonist Phase 2 Gilead Sciences 122; NCT02166047‡
Nivolumab PD-1 blockade Phase 1||
BMS 151; Sponsor’s website,
NCT01658878‡
SB 9200HBV RIG-I and NOD2 activation Phase 1/2 INC/Springbank 152; NCT01803308‡
GS-4774 Therapeutic vaccine Phase 2/3 Gilead Sciences/GlobeImmune 144; NCT02174276‡
ANRS HB02 Therapeutic vaccine Phase 1/2 French National Agency for
Research on AIDS and Viral
Hepatitis
141; NCT02166047‡
Heplisav B Dynavax 601 Therapeutic vaccine Phase 1 Dynavax 153; NCT01023230‡
Nasvac Therapeutic vaccine Phase 2/3 CGEB, Cuba 154
TG1050 Therapeutic vaccine Phase 1/1b Transgene NCT02428400
HBIG1GM-CSF 1HBV vaccine Therapeutic vaccine Phase 1/2 Beijing 302 Hospital NCT01878565
HBV vaccine1 IFN-a2b1 IL-2 Therapeutic vaccine Phase 2/3 Tongji Hospital NCT02360592 (labeled as Phase 4)
HBV vaccine–activated dendritic
cells
Therapeutic vaccine Phase 1/2 Third Affiliated Hospital, Sun
Yat-Sen University
NCT01935635
Euvax 1 PEG-IFN-a Therapeutic vaccine P hase 2/3 Seoul National University N CT0 2097004 ( labeled as Phas e 4)
PD-1 monoclonal antibody PD1 blockade Animal AcadSin 155
Altravax HBV Therapeutic vaccine Animal Altravax Sponsor’s website
INO-1800 Therapeutic vaccine Animal Innovio Sponsor’s website
*Compounds are organized by names and targets with developmental phase based on authors’ estimates derived from the literature where available or the spon-
sor’s website and presentation information.†
Mechanisms are characterized as either direct acting antiviral, indicating action against a virus-specified gene product; immune modulatory agent, activating
host immune response; or host targeting agent, which targets a host function required for the HBV replication cycle.‡
Identifier for Clinicaltrials.gov.§
In phase 2 for human immunodeficiency virus.k Trial i ndication is for treatment of HBV-associated hepatocellular carcinoma.
Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; HBIG, hepatitis B immune globulin.
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1899
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
8/16
promising because efficient in vivo delivery systems havebeen developed by a number of biotech companies(Table 1).
RNAi is a process by which small interfering RNA molecules of 21-25 nucleotides induce gene silencing at
the posttranscriptional level to effectively knock downthe expression of the gene(s) of interest. Such shortinterfering RNA can lead to transcriptional silencing ortranslational repression.90 These processes are critical incell growth regulation and tissue differentiation andinvolve the Drosha and Dicer enzyme complexes, theRNA-induced silencing complex, and the nuclease
Argo.90 The extensive use of overlapping RNAs andopen reading frames within the HBV genome makes foran attractive target for inhibition by RNAi.91 Both cellculture and mouse model studies have shown thatRNAi, delivered as an expression plasmid, is able to
inhibit all steps of HBV replication.92
In transgenicmice, RNAi expression has been shown to significantly reduce the secretion of HBsAg in serum, reduce bothHBV messenger RNAs and genomic DNA in the liver,and eliminate hepatocytes stained positive for core anti-gen.93 These mouse studies have been extended to a chronically infected chimpanzee.94 Currently, a phase 2placebo-controlled dose-escalation study with the DPC-NAG-ARC-520 formulation has been initiated inHBeAg-negative chronic hepatitis B whose viremia wascontrolled by entecavir and showed a 50% drop inHBsAg levels in treated compared to placebo patients.95
Another RNAi platform has demonstrated similarsuccess in its preclinical evaluation with a 2.3-log 10reduction in HBsAg in chronically HBV-infected chim-panzees (Alnylam’s company press release).96 OtherRNAi-based regimens are currently being developed andtested (Table 1).
Inhibitors of HBV cccDNA Formation andStability
Because the cytoplasmic nucleocapsid DNA is theprecursor for cccDNA biosynthesis, complete inhibition
of viral DNA replication in the nucleocapsids with poly-merase inhibitors should preclude de novo cccDNA for-mation. However, clinical studies demonstrated thatalthough NRTI monotherapy for 48-52 weeks reducedcirculating viremia by 5 log 10 and cytoplasmic HBV DNA levels in hepatocytes by approximately 2 log 10,reduction of cccDNA was much less pronounced, only by 0.11 to 1.0 log 10.
24,97 Moreover, sequential analysesof viral DNA replicative intermediates and core antigen-positive hepatocytes in the livers of woodchuck hepatitisvirus (WHV)-infected woodchucks before and during
clevudine (an NRTI) therapy revealed that after morethan 6 weeks of therapy, all WHV DNA replicativeintermediates were markedly reduced, with the excep-tion of cccDNA, which remained as the predominantviral DNA species in the liver.98
Concerning the failure of prolonged NRTI therapy toeradicate cccDNA, one possibility is that the currently available NRTIs do not completely inhibit viral DNA synthesis in every infected hepatocyte in vivo, allowing for continuous replenishment of the cccDNA poolthrough the intracellular amplification pathway. NRTIsare prodrugs requiring activation by host cellularnucleoside kinases, the expression and function of whichmay be heterogeneous in the liver. Therefore, hepato-cytes may have varying abilities to activate the NRTIs,resulting in incomplete inhibition of HBV DNA repli-cation. Emergence of drug-resistance mutations during
apparently effective NRTI therapy suggests that residualHBV replication and de novo cccDNA synthesis stilloccur at a low level.99
Alternatively, failure to eradicate cccDNA by pro-longed NRTI therapy may also be due to the extraordi-nary stability of cccDNA.100 cccDNA may persist in a “latent” state amid the host chromosomes and remain asa reservoir for later HBV replication. Healthy hepato-cytes in the absence of immune response or inflamma-tory reaction have a half-life of over 6 months.101,102
What we have learned from NRTI therapy is thateradication of cccDNA is essential for the cure of
chronic hepatitis B. Combination therapies with NRTIsand one or multiple novel antiviral drugs targeting dif-ferent steps of HBV replication may completely inhibitHBV DNA replication and thus accelerate the reductionof cccDNA. The other approach would be to directly purge the preexisting cccDNA or permanently silencecccDNA transcription.
A recent strategy to cleave cccDNA molecules orinhibit their transcription by generating cccDNA sequence-specific endonucleases with zinc-finger nucle-ase, transcription activator-like effector nuclease, orCRISPR/cas9 technology has been tested in cell cultures
and a mouse model103,104; but efficient and targeteddelivery of these antiviral genes to all HBV-infected cellsin vivo is a major challenge for clinical application.
Another approach is to target the other enzymatic func-tion of HBV polymerase, RNaseH, which is requiredfor HBV replication and cccDNA formation. Recentstudies have identified potential inhibitors of HBV RNaseH.105
Further understanding the molecular mechanism of cccDNA metabolism and functional regulation is essen-tial for identifying and validating molecular targets for
1900 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
9/16
rational development of antiviral drugs to eradicate ortranscriptionally silence cccDNA. As discussed above,recent studies on the molecular mechanism of immunecontrol of HBV infection by IFN-a demonstrated thatcccDNA can be specifically targeted for degradation by
a cytidine-deamination mechanism
35
and its transcrip-tion can be silenced by epigenetic modification.33,106
These findings raise a potentially exciting possibility of targeting cccDNA through pharmacological activationor augmentation of the host intrinsic antiviral pathways.Moreover, investigation into the role and mechanism of HBx and core protein in cccDNA metabolism and func-tion may reveal virus–host interactions for selectiveelimination or silencing of cccDNA.20
Additional efforts have been made to discovercccDNA-targeting compounds through high-throughputcell-based phenotypic screening. This unbiased approach,
while attractive, is currently hampered by a lack of effi-cient HBV infection cell culture systems and convenientassays for high-throughput quantification of HBV replica-tion and cccDNA quantification. Disubstituted sulfona-mides were identified as cccDNA formation inhibitors ina screen of 85,000 small molecular compounds.107 Therecent rapid progress in the establishment of an efficientHBV infection cell culture system may ultimately allow the development of cell-based assays for high-throughputscreening of cccDNA-targeting antivirals.
Immune Mechanisms of HBV Control and
Implications for Therapy
The pathogenesis of chronic HBV infection involvesnot only viral mechanisms by which HBV establishes a persistent infection but also the host responses to infec-tion. The latter includes the response of hepatocytes toHBV infection as well as the interplay of the virus andinfected cells with the other parenchymal and nonpar-enchymal cells in the liver, i.e., Kupffer cells, endothe-lial cells, fibroblasts, and nonresident immune cellsthat are recruited to the site of infection. HBV hasevolved mechanisms to counteract and escape these dif-
ferent host responses to establish a chronic infection.Recent studies point out a critical role of the livermicroenvironment in the elimination or control of HBV (Fig. 2). 108,109 While much has been learnedabout the HBV-specific adaptive immunity, the early and innate immune response during acute HBV infec-tion remains largely unknown. In addition, few studieshave examined intrahepatic immune responses inpatients with chronic HBV infection. Available data suggest impaired responses, but the mechanism of thisimpairment is unclear.109
In chronic hepatitis B, the antiviral B- and T-cellresponses are quantitatively and/or qualitatively defec-tive. For example, anti-HBs is generally undetectable inthe setting of excess circulating HBsAg. Furthermore,antiviral T cells show impaired antiviral effector func-tion in vitro. However, this host immune response,despite being dysfunctional, exerts at least partial viralcontrol in vivo because immune suppression withimmunosuppressive therapies results in increased vire-mia.58,110 HBV persistence with antiviral immune dys-function is also associated with the induction of immune inhibitory pathways including PD-1, CTLA-4,Bim, arginase, and FoxP31 regulatory T cells.111-116
These pathways, likely induced in response to continuedinflammation, viral replication, and antigen expression,can dampen both cytopathic inflammatory responses as
well as noncytopathic antiviral effector functions. Thus,
the antiviral effector T-cell function may be enhancedby blocking one or more of these inhibitory path- ways,112,117 raising the possibility for potential thera-peutic application in chronic viral infections such aschronic hepatitis B.
Based on our knowledge of the immune mechanismsof chronic HBV infection, several approaches to restoreinnate or adaptive immunity or both to control HBV infection in combination with other direct antiviralstrategies have been applied.108,109 These approachescan be broadly divided into virus-nonspecific and virus-specific modalities. The first involves general immuno-
modulatory agents, and the latter aims to activate theHBV-specific immune response by applying the tech-nologies of therapeutic vaccination. As discussed above,the efficacy of IFN-a therapy can be partly attributed toits immunostimulatory effect. A promising approachemerges from the field of toll-like receptors (TLRs). Var-ious TLR agonists with potent immunostimulatory effects have been developed.118 Their administration toHBV patients leads to both intrahepatic and extrahe-patic induction of type 1 interferons and other cytokinesthat may contribute directly to antiviral activity or indi-rectly result in activation of innate and adaptive immune
responses. The second approach involves the blockadeof negative immunoregulatory pathways (i.e., coinhibi-tory signals, inhibitory cytokines, regulatory T cells),
which may induce a partial restoration of HBV-specificT cells. Third, engineering of redirected T cells may result in a de novo reconstitution of functionally activeHBV-specific T cells and activation of heterologous Tcells. Whether inhibition of a suppressive effect(s) of HBV can lead to restoration of HBV-specific innate andadaptive immune responses remains a challenging ques-tion. Several lines of evidence suggest that HBV
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1901
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
10/16
interferes negatively with these host immune responses. A more detailed understanding of the specific mecha-nisms is mandatory before new ways of restoring immune responses by targeting virus-specific factors canbe explored. HBV-specific strategies may prove moreeffective and safer than virus-nonspecific approaches.
A concern of the various immunotherapies is thepotential risk of autoimmunity and/or exacerbation of liver damage by immune-mediated death of hepatocytesin vivo. Careful consideration of benefit versus risk and
close clinical monitoring would be needed in theseapproaches.
HBV-Nonspecific Immunomodulatory Agents
TLR Agonists. The antiviral effect of TLR agonists,particularly TLR-7, through activation of innate immu-nity has been evaluated in HBV chronically infectedchimpanzees and woodchucks. Upon stimulation of TLR-7, plasmacytoid dendritic cells produce IFN-a and
Fig. 2. Innate and adaptive HBV-specific immune responses and immune-based therapeutic development. Immune cells involved in innate
and adaptive immune responses activated by HBV infection and their mechanisms of antiviral actions are shown. They are virus-specific CD81 T
cells that inhibit viral replication by both direct killing of infected hepatocytes and cytokine-mediated antiviral mechanisms; virus-specific CD41
T
cells, which provide essential help for CD81
T-cell priming and effector functions as well as antiviral cytokines; regulatory T cells, which suppress
virus-specific T-cell functions; B cells, which mature to plasma cells, producing neutralizing antibodies and potentially participating in antigen pre-
sentation; natural killer cells, which display antiviral but also regulatory activity by eliminating activated virus-specific CD81
T cells; natural killer
T cells that sense virus-infected hepatocytes, produce antiviral cytokines, and activate adaptive immune responses; other immune cells in the
liver that play important roles in the activation and coordination of the innate and adaptive responses such as Kupffer, myeloid, and plasmacy-
toid dendritic cells. Therapeutic approaches designed to activate various pathways of the innate and adaptive immunities are illustrated in red.
See text for details of these approaches. Abbreviations: CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFNAR, IFN-a receptor; IFNGR, IFN-creceptor; IFNLR, IFN-k receptor; JAK/STAT, Janus kinase/signal transducer and activator of transcription; Mu, macrophage; NK, natural killer; NKT,NK T cell; TNF-L, tumor necrosis factor–like molecule (e.g., lymphotoxin-b); TNF-LR, TNF-L receptor; Treg, regulatory T cell.
1902 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
11/16
other cytokines/chemokines and induce the activation of natural killer cells and activation of cytotoxic lympho-cytes, thereby orchestrating both innate and adaptiveimmune responses.119 The altered responsiveness of plasmacytoid dendritic cells may contribute to the
reduced innate and adaptive immune responses during chronic viral infections. Agonist-induced activation of TLR-7 therefore represents a novel approach for thetreatment of chronic viral infections.120 GS-9620, anorally administered agonist of TLR-7, was tested inHBV-infected chimpanzees.121 Short-term administra-tion of the TLR-7 agonist provided long-term suppres-sion of serum and liver HBV DNA. Serum levels of HBsAg and HBeAg and numbers of HBV antigen-positive hepatocytes were reduced. In parallel, GS-9620administration induced the production of IFN-a andother cytokines and chemokines, up-regulated ISG
expression, and activated natural killer cells and lympho-cyte subsets, confirming the activation of TLR-7 signal-ing. Similar effects were also observed in chronically infected woodchucks. Phase 1 clinical evaluation hasbeen performed, and patients are now being enrolled ina phase 2 trial combining tenofovir and GS-9620 incomparison to tenofovir monotherapy.122
PD1 and Other Coinhibitory Blockers. In chronicHBV infection, loss of viral control has been explainedby exhausted T cells. One approach would be to recoverexisting T cells by correcting the balance between coin-hibitory (PD1, CTLA-4, Tim-3, Lag-3) and costimulat-
ing (41BB, interleukin-12 [IL-12]) signals.123 Recentstudies in the field of cancer therapy have highlightedthe clinical relevance of PD1 blockade to restore antitu-mor immunity to improve survival.124 As chronic HBV infection and tumor immunology share similar charac-teristics in terms of immune subversion and the role of PD1, PD1 blockade may be an attractive concept forHBV therapy. A recent study in chronically infected
woodchucks tested the combination therapy of entecavirand an anti-PD1 ligand monoclonal antibody together
with a WHV DNA vaccine. PD1 blockade was shown
to synergize with entecavir and therapeutic vaccinationto control viral replication and restore WHV-specific T-cell responses.125
HBV-Specific Modified T Cells
As discussed above, HBV-specific T cells are eitherexhausted or nonresponsive in chronic HBV infection.This therapeutic approach is designed to provide geneti-cally engineered T cells to target and eliminate HBV-infected hepatocytes. The strategy to genetically modify patient’s T cells to express HBV-specific T-cell receptors
and then infuse them into the same patients with HBV-associated hepatocellular carcinoma showed some prom-ise.126,127 But the variable and major histocompatibility complex–restricted nature of the interaction between T-cell receptor and its ligand and the skepticism that
whether one or two such modified T cells would be suf-ficient to mount an effective T cell–based immuneresponse may limit the clinical application of thisapproach. The recent emerging technology of chimericantigen receptor (CAR) in the field of cancer therapeu-tics has been extended to treatment of persistent viralinfections.128 The CAR approach is to generate a chi-meric receptor expressing an extracellular target-binding domain, a hinge and membrane-anchoring region, andone (or more) intracellular signaling domain.128 Thetarget-binding domain is derived from the light andheavy chain sequences of a single-chain variable frag-
ment of the immunoglobulin. In the case of HBV, thetarget could be the cell-surface form of HBsAg and thesingle-chain variable fragment derived from a construct
with high-affinity anti-HBs activity.129 The binding of the CAR-modified T cells to HBV-infected hepatocytescan trigger proliferating or activating signals to initiatean effective anti-HBV T-cell response. This strategy hasbeen applied to HBV animal models with some prom-ise.130 It remains to be seen whether CAR-modified Tcells can achieve a broadly acting and potent anti-HBV response that is sufficient for viral clearance in chronicHBV-infected patients.
Therapeutic Vaccines
The goal for therapeutic vaccination in chronic hepa-titis B is to induce sufficient anti-HBV immuneresponses to eliminate and/or cure infected hepatocytes
without undue host cell damage, prevent viral spread tonew hepatocytes, and promote long-term viral control.These approaches leverage our accumulating knowledgeof the adaptive immune responses of HBV infectionand focus on restoring or activating endogenous HBV-specific immune responses that initially targeted HBsAg
and later expanded to other HBV antigens using recombinant proteins, cytotoxic T-lymphocyte epitopevaccine, viral vectors, and DNA vaccination. These vac-cines are being combined with antiviral drugs andimmune modulators to maximize their effects. Anintriguing strategy to personalize antigen presentation toinduce anti-HBV immune response involving mono-cytes has been recently proposed.131
HBsAg-Based Vaccine. Because HBsAg-based pro-phylactic vaccine can induce protective virus-neutralizing antibodies, the initial studies involved the
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1903
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
12/16
use of HBsAg in small trials, with virological and sero-logical responses in some patients.132,133 Combinationof an HBsAg vaccine with lamivudine showed promiseinitially in smaller studies.134 However, no difference inclinical efficacy was shown between vaccinated and con-trol groups despite the induction of vigorous HBsAg-specific cellular and humoral immune responses in a large open-labeled randomized controlled trial of HBV patients receiving 12 doses of recombinant HBsAg and
ASO2 adjuvant with 52 weeks of lamivudine.135 Simi-larly, the use of yeast-derived recombinant HBsAg andhepatitis B immune globulin immune complex showedpromise in a phase 1/2 study,136 but this was not repro-duced in a larger phase 3 study.137
Cytotoxic T-Lymphocyte Epitope Vaccine. Immu-nization with recombinant proteins (e.g., HBsAg) canpromote antibody and CD4 helper T-cell responses, but
generally not those of CD8 T cells, which require endo-genously processed viral peptides. Given the relevance of antiviral CD8 T cells in HBV clearance, direct augmen-tation of HBV-specific CD8 T cells was attempted in a pilot study using a lipopeptide encoding a single immu-nogenic human leukocyte antigen A2–restricted HBV core 18-27 CTL epitope.138 Despite their immunoge-nicity in healthy adults, this epitope vaccine was notimmunogenic in patients with chronic hepatitis B anddid not significantly change the HBV DNA titers orHBeAg status. Inclusion of other epitopes in thisapproach may be necessary.
DNA Vaccination With or Without Immunomo-dulators. DNA vaccination can promote antiviralCD8 T-cell as well as CD4 T-cell and antibody responses.139 In this regard, intramuscular injection of DNA encoding only pre-S2/S was safe, well-tolerated,and at least transiently immunogenic but only margin-ally effective in reducing HBV DNA levels in a phase 1study of chronic hepatitis B patients who did notrespond to IFN-a and/or lamivudine.140,141 It also didnot prevent viremic relapse in the phase 1/2 ANRSHB02 VAC-AND trial.141 Another phase 1 study using plasmid DNA encoding all HBV open reading frames
and human IL-12 in addition to daily lamivudineshowed a 50% HBV DNA suppression at 1 year post–treatment cessation.142 However, in a subsequent largerstudy, a related HBV plasmid DNA (all HBV open read-ing frames except HBx) and human IL-12 with daily adefovir showed only a tendency for greater HBeAg lossand HBV DNA suppression compared to adefoviralone.143
Other Therapeutic Vaccine Trials. Currently,open-label therapeutic HBV vaccine trials on clinical-trials.gov (as of May 2015) include (1) GS4774, a heat-
killed recombinant yeast expressing HBV S, core, andHBx fusion protein144; (2) ABX203 with recombinantHBsAg and hepatitis B core antigen in the setting of PEG IFN-a and oral antivirals; (3) INO-1800, a multi-antigen DNA vaccine encoding HBsAg and hepatitis Bcore antigen electroporated alone or combined withINO-9112 encoding IL-12 in patients on either enteca-vir or tenofovir; (4) TG1050, a nonreplicative E1/E3-deleted human adenovirus encoding a fusion proteincombining modified HBV core, polymerase and enve-lope145; (5) HBV vaccine with hepatitis B immuneglobulin and granulocyte-macrophage colony-stimulat-ing factor; (6) HBV vaccine with IFN-a2b and IL-2; (7)HBV vaccine activated dendritic cells combined withPEG IFN-a or nucleos(t)ide analogues; (8) intensifiedEuvax (HBV S) vaccination with PEG-IFN-a.
Therapeutic Pipeline and ConclusionBased on the literature, expert input, publicly dis-
closed information of various pharmaceutical compa-nies, the clinicaltrial.gov website, we generated a tablesummarizing the current status of various anti-HBV drugs or biologics in the development pipeline (Table1). While many of them are still in preclinical develop-ment, several have advanced to clinical trials. As dis-cussed above, some of them showed early promise and
will likely advance to the late clinical trial phase formore definitive proof of the preliminary success. In this
review, we have summarized the major therapeuticapproaches and novel molecular targets for anti-HBV drug development and provided a knowledge-basedrationale behind these various strategies. It is possiblethat new and additional technologies may emerge as thefield advances. To achieve a more sustained and effectivecontrol of HBV infection, a combination of the existing HBV therapies and one or more of the above modalities,either small-molecule drugs or biologics, will be neces-sary. With the concerted efforts of private and publicsectors, the next milestone in the therapy of HBV infec-tion, a functional “cure” that has remained elusive, is
likely within our grasp within the next decade.
References
1. Ott JJ, Stevens GA, Groeger J, Wiersma ST. Global epidemiology of
hepatitis B virus infection: new estimates of age-specific HBsAg sero-
prevalence and endemicity. Vaccine 2012;30:2212-2219.
2. Kennedy PT, Sandalova E, Jo J, Gill U, Ushiro-Lumb I, Tan AT,
et al. Preserved T-cell function in children and young adults with
immune-tolerant chronic hepatitis B. Gastroenterology 2012;143:637-
645.
3. Vanwolleghem T, Hou J, van Oord G, Andeweg AC, Osterhaus AD,
Pas SD, et al. Re-evaluation of hepatitis B virus clinical phases by
1904 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
13/16
systems biology identifies unappreciated roles for the innate immune
response and B cells. HEPATOLOGY 2015;62:87-100.
4. McMahon BJ. The natural history of chronic hepatitis B virus infec-
tion. HEPATOLOGY 2009;49:S45-S55.
5. Prati D, Taioli E, Zanella A, Della Torre E, Butelli S, Del Vecchio E,
et al. Updated definitions of healthy ranges for serum alanine amino-
transferase levels. Ann Intern Med 2002;137:1-10.
6. Schulze A, Gripon P, Urban S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate pro-
teoglycans. HEPATOLOGY 2007;46:1759-1768.
7. Salisse J, Sureau C. A function essential to viral entry underlies the
hepatitis B virus “a” determinant. J Virol 2009;83:9321-9328.
8. Yan H, Zhong G , Xu G, He W, Jing Z, Gao Z, et al. Sodium tauro-
cholate cotransporting polypeptide is a functional receptor for human
hepatitis B and D virus. Elife 2012;1:e00049.
9. Doring B, Lutteke T, Geyer J, Petzinger E. The SLC10 carrier family:
transport functions and molecular structure. Curr Top Membr 2012;
70:105-168.
10. Schieck A, Schulze A, Gahler C, Muller T, Haberkorn U, Alexandrov
A, et al. Hepatitis B virus hepatotropism is mediated by specific
receptor recognition in the liver and not restricted to susceptible
hosts. HEPATOLOGY 2013;58:43-53.
11. Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to inhibitentry of HBV and HDV into hepatocytes. Gastroenterology 2014;
147:48-64.
12. Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M, et al.
Hepatitis B and D viruses exploit sodium taurocholate co-transporting
polypeptide for species-specific entry into hepatocytes. Gastroenterol-
ogy 2014;146:1070-1083.
13. Yan H, Peng B, He W, Zhong G, Qi Y, Ren B, et al. Molecular
determinants of hepatitis B and D virus entry restriction in mouse
sodium taurocholate cotransporting polypeptide. J Virol 2013;87:
7977-7991.14. Huang HC, Chen CC, Chang WC, Tao MH, Huang C. Entry of
hepatitis B virus into immortalized human primary hepatocytes by
clathrin-dependent endocytosis. J Virol 2012;86:9443-9453.
15. Macovei A, Radulescu C, Lazar C, Petrescu S, Durantel D, Dwek
RA, et al. Hepatitis B virus requires intact caveolin-1 function for
productive infection in HepaRG cells. J Virol 2010;84:243-253.
16. Schmitz A , Schwarz A , Foss M, Zhou L, Rabe B, Hoellenriegel J,
et al. Nucleoporin 153 arrests the nuclear import of hepatitis B virus
capsids in the nuclear basket. PLoS Pathog 2010;6:e1000741.
17. Koniger C, Wingert I, Marsmann M, Rosler C, Beck J, Nassal M.
Involvement of the host DNA-repair enzyme TDP2 in formation of
the covalently closed circular DNA persistence reservoir of hepatitis B
viruses. Proc Natl Acad Sci USA 2014;111:E4244-E4253.
18. Bock CT, Schwinn S, Locarnini S, Fyfe J, Manns MP, Trautwein C,
et al. Structural organization of the hepatitis B virus minichromo-
some. J Mol Biol 2001;307:183-196.19. Pollicino T, Belloni L, Raffa G, Pediconi N, Squadrito G, Raimondo
G, et al. Hepatitis B virus replication is regulated by the acetylation
status of hepatitis B virus cccDNA-bound H3 and H4 histones. Gas-
troenterology 2006;130:823-837.
20. Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, FanciulliM, et al. Nuclear HBx binds the HBV minichromosome and modifies
the epigenetic regulation of cccDNA function. Proc Natl Acad Sci
USA 2009;106:19975-19979.
21. Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero
M, et al. Hepatitis B virus X protein is essential to initiate and main-
tain virus replication after infection. J Hepatol 2011;55:996-1003.
22. Watanabe T, Sorensen EM, Naito A, Schott M, Kim S, Ahlquist P.
Involvement of host cellular multivesicular body functions in hepatitis
B virus budding. Proc Natl Acad Sci USA 2007;104:10205-10210.23. Lentz TB, Loeb DD. Roles of the envelope proteins in the amplifica-
tion of covalently closed circular DNA and completion of synthesis of
the plus-strand DNA in hepatitis B virus. J Virol 2011;85:11916-
11927.
24. Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J,
Lau G, et al. Persistence of cccDNA during the natural history of
chronic hepatitis B and decline during adefovir dipivoxil therapy. Gas-
troenterology 2004;126:1750-1758.
25. Lok AS, McMahon BJ. Chronic hepatitis B: update 2009. HEPATO-
LOGY 2009;50:661-662.
26. European Association for the Study of the Liver. EASL clinical prac-
tice guidelines: management of hepatitis C virus infection. J Hepatol2011;55:245-264.
27. Liaw YF, Leung N, Kao JH, Piratvisuth T, Gane E, Han KH, et al.
Asian-Pacific consensus statement on the management of chronic hep-
atitis B: a 2008 update. Hepatol Int 2008;2:263-283.
28. Xu C, Guo H, Pan XB, Mao R, Yu W, Xu X, et al. Interferons accel-
erate decay of replication-competent nucleocapsids of hepatitis B
virus. J Virol 2010;84:9332-9340.
29. Hao J, Jin W, Li X, Wang S, Zhang X, Fan H, et al. Inhibition of
alpha interferon (IFN-alpha)-induced microRNA-122 negatively
affects the anti-hepatitis B virus efficiency of IFN-alpha. J Virol 2013;
87:137-147.
30. Micco L, Peppa D, Loggi E, Schurich A, Jefferson L, Cursaro C,
et al. Differential boosting of innate and adaptive antiviral responses
during pegylated-interferon-alpha therapy of chronic hepatitis B.
J Hepatol 2013;58:225-233.31. ter Borg MJ, Hansen BE, Herrmann E, Zeuzem S, Cakaloglu Y,
Karayalcin S, et al. Modelling of early viral kinetics and pegylated
interferon-alpha2b pharmacokinetics in patients with HBeAg-positive
chronic hepatitis B. Antivir Ther 2007;12:1285-1294.
32. Sadler AJ, Williams BR. Interferon-inducible antiviral effectors. Nat
Rev Immunol 2008;8:559-568.
33. Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T,
et al. IFN-alpha inhibits HBV transcription and replication in cell
culture and in humanized mice by targeting the epigenetic regulation
of the nuclear cccDNA minichromosome. J Clin Invest 2012;122:
529-537.
34. Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, et al.
Infection of a human hepatoma cell line by hepatitis B virus. Proc
Natl Acad Sci USA 2002;99:15655-15660.
35. Lucifora J, Xia Y , Reisinger F, Zhang K, Stadler D, Cheng X, et al.Specific and nonhepatotoxic degradation of nuclear hepatitis B virus
cccDNA. Science 2014;343:1221-1228.
36. Stenglein MD, Burns MB, Li M, Lengyel J, Harris RS. APOBEC3
proteins mediate the clearance of foreign DNA from human cells.
Nat Struct Mol Biol 2010;17:222-229.
37. Liang G, Kitamura K, Wang Z, Liu G, Chowdhury S, Fu W, et al.
RNA editing of hepatitis B virus transcripts by activation-induced cyt-
idine deaminase. Proc Natl Acad Sci USA 2013;110:2246-2251.
38. Noguchi C, Hiraga N, Mori N, Tsuge M, Imamura M, Takahashi S,
et al. Dual effect of APOBEC3G on Hepatitis B virus. J Gen Virol
2007;88:432-440.
39. Renard M, Henry M, Guetard D, Vartanian JP, Wain-Hobson S.
APOBEC1 and APOBEC3 cytidine deaminases as restriction factors
for hepadnaviral genomes in non-humans in vivo. J Mol Biol 2010;
400:323-334.40. Turelli P, Mangeat B, Jost S, Vianin S, Trono D. Inhibition of hepa-
titis B virus replication by APOBEC3G. Science 2004;303:1829.
41. Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral
hepatitis. Annu Rev Pathol 2006;1:23-61.
42. Yapali S, Talaat N, Lok AS. Management of hepatitis B: our practice
and how it relates to the guidelines. Clin Gastroenterol Hepatol
2014;12:16-26.
43. Marcellin P, Ahn SA, Ma X, Caruntu FA, Tak WY, Elkashab M,
et al. HBsAg loss with tenofovir disoproxil fumarate plus peginter-
feron alfa-2a in chronic hepatitis B: results of a global randomized
controlled trial. HEPATOLOGY 2014;60:294A.
44. Marcellin P, Gane E, Buti M, Afdhal N, Sievert W, Jacobson IM,
et al. Regression of cirrhosis during treatment with tenofovir
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1905
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
14/16
disoproxil fumarate for chronic hepatitis B: a 5-year open-label
follow-up study. Lancet 2013;381:468-475.
45. Chotiyaputta W, Peterson C, Ditah FA, Goodwin D, Lok AS. Persist-
ence and adherence to nucleos(t)ide analogue treatment for chronic
hepatitis B. J Hepatol 2011;54:12-18.
46. Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, et al. Lam-
ivudine for patients with chronic hepatitis B and advanced liver dis-
ease. N Engl J Med 2004;351:1521-1531.47. Menendez-Arias L, Alvarez M, Pacheco B. Nucleoside/nucleotide ana-
log inhibitors of hepatitis B virus polymerase: mechanism of action
and resistance. Curr Opin Virol 2014;8:1-9.
48. Zoulim F, Mason WS. Reasons to consider earlier treatment of
chronic HBV infections. Gut 2012;61:333-336.
49. Chan HL, Chan CK, Hui AJ, Chan S, Poordad F, Chang TT, et al.
Effects of tenofovir disoproxil fumarate in hepatitis B e antigen-
positive patients with normal levels of alanine aminotransferase and
high levels of hepatitis B virus DNA. Gastroenterology 2014;146:
1240-1248.
50. Lok AS, Trinh H, Carosi G, Akarca US, Gadano A, Habersetzer F,
et al. Efficacy of entecavir with or without tenofovir disoproxil fuma-
rate for nucleos(t)ide-naive patients with chronic hepatitis B. Gastro-
enterology 2012;143:619-628.
51. Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S,Cooksley G, et al. Peginterferon alfa-2a, lamivudine, and the combi-
nation for HBeAg-positive chronic hepatitis B. N Engl J Med 2005;
352:2682-2695.
52. Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, et al.
Peginterferon alfa-2a alone, lamivudine alone, and the two in combi-
nation in patients with HBeAg-negative chronic hepatitis B. N Engl J
Med 2004;351:1206-1217.
53. Su W-W, Hsu C-W, Lee C-M, Peng C-Y, Chuang W-L, Kao J-H,
et al. Combination therapy with peginterferon alfa-2a and a nucleos(-
t)ide analogue for HBeAg-positive chronic hepatitis B patients: results
of a large, randomised, multicentre, double-blind, placebo-controlled
study. J Hepatol 2014;60:S47.
54. Ning Q, Han M, Sun Y, Jiang J, Tan D, Hou J, et al. Switching
from entecavir to PegIFN alfa-2a in patients with HBeAg-positive
chronic hepatitis B: a randomised open-label trial (OSST trial). J Hepatol 2014;61:777-784.
55. Chan HL, Leung NW, Hui AY, Wong VW, Liew CT, Chim AM,
et al. A randomized, controlled trial of combination therapy for
chronic hepatitis B: comparing pegylated interferon-alpha2b and lami-
vudine with lamivudine alone. Ann Intern Med 2005;142:240-250.
56. Thimme R, Dandri M. Dissecting the divergent effects of interferon-
alpha on immune cells: time to rethink combination therapy in
chronic hepatitis B? J Hepatol 2013;58:205-209.
57. Brouwer WP, Xie Q, Sonneveld MJ, Zhang N, Zhang Q, Tabak F,
et al. Adding peginterferon to entecavir for HBeAg-positive chronic
hepatitis B: a multicentre randomized trial (ARES study). HEPATOLOGY
2015;61:1512-1522.
58. Seto WK, Chan TS, Hwang YY, Wong DK, Fung J, Liu KS, et al.
Hepatitis B reactivation in patients with previous hepatitis B virus
exposure undergoing rituximab-containing chemotherapy for lym-phoma: a prospective study. J Clin Oncol 2014;32:3736-3743.
59. Bourliere M, Rabiega P, Ganne-Carrie N, Serfaty L, Marcellin P,
Pouget N, et al. HBsAg clearance after addition of 48 weeks of
PEGIFN in HBeAg negative CHB patients on nucleos(t)ide with
undetactable HBV DNA for at leat one year: a multicenter random-
ized controlled phase III trial ANRS-HB06 PEGAN study: prelimi-
nary findings. HEPATOLOGY 2014;60:1094A.
60. Xie Q, Zhou H, Bai X, Wu S, Chen JJ, Sheng J, et al. A randomized,
open-label clinical study of combined pegylated interferon alfa-2a
(40KD) and entecavir treatment for hepatitis B “e” antigen-positive
chronic hepatitis B. Clin Infect Dis 2014;59:1714-1723.
61. Vanderlinden E, Naesens L. Emerging antiviral strategies to interfere
with influenza virus entry. Med Res Rev 2014;34:301-339.
62. Haqqani AA, Tilton JC. Entry inhibitors and their use in the treat-
ment of HIV-1 infection. Antiviral Res 2013;98:158-170.
63. Petersen J, Dandri M, Mier W, Lutgehetmann M, Volz T, von
Weizsacker F, et al. Prevention of hepatitis B virus infection in vivo
by entry inhibitors derived from the large envelope protein. Nat Bio-
technol 2008;26:335-341.
64. Aspinall EJ, Hawkins G, Fraser A, Hutchinson SJ, Goldberg D. Hep-
atitis B prevention, diagnosis, treatment and care: a review. OccupMed (Lond) 2011;61:531-540.
65. Galun E, Eren R, Safadi R, Ashour Y, Terrault N, Keeffe EB, et al.
Clinical evaluation (phase I) of a combination of two human mono-
clonal antibodies to HBV: safety and antiviral properties. HEPATOLOGY
2002;35:673-679.
66. Mason WS, Xu C, Low HC, Saputelli J, Aldrich CE, Scougall C,
et al. The amount of hepatocyte turnover that occurred during resolu-
tion of transient hepadnavirus infections was lower when virus replica-
tion was inhibited with entecavir. J Virol 2009;83:1778-1789.67. Blanchet M, Sureau C. Infectivity determinants of the hepatitis B
virus pre-S domain are confined to the N-terminal 75 amino acid res-
idues. J Virol 2007;81:5841-5849.
68. Gripon P, Le Seyec J, Rumin S, Guguen-Guillouzo C. Myristylation
of the hepatitis B virus large surface protein is essential for viral infec-
tivity. Virology 1995;213:292-299.69. Blank A, Markert C, Hohmann N, Carls A, Mikus G, Lehr T, et al.
Myrcludex B: successful first-in-human administration of a first in
class hepatitis B and hepatitis D virus entry inhibitor. J Hepatol. Sub-
mitted for publication.
70. Lucifora J, Esser K, Protzer U. Ezetimibe blocks hepatitis B virus
infection after virus uptake into hepatocytes. Antiviral Res 2013;97:
195-197.
71. Nkongolo S, Ni Y, Lempp FA, Kaufman C, Lindner T, Esser-Nobis
K, et al. Cyclosporin A inhibits hepatitis B and hepatitis D virus entry
by cyclophilin-independent interference with the NTCP receptor.
J Hepatol 2014;60:723-731.
72. Watashi K, Sluder A, Daito T, Matsunaga S, Ryo A, Nagamori S,
et al. Cyclosporin A and its analogs inhibit hepatitis B virus entry
into cultured hepatocytes through targeting a membrane transporter,
sodium taurocholate cotransporting polypeptide (NTCP). HEPATOLOGY
2014;59:1726-1737.
73. Vaz FM, Paulusma CC, Huidekoper H, de Ru M, Lim C, Koster J,
et al. Sodium taurocholate cotransporting polypeptide (SLC10A1)
deficiency: conjugated hypercholanemia without a clear clinical pheno-
type. HEPATOLOGY 2015;61:260-267.74. Slijepcevic D, Kaufman C, Wichers CG, Gilglioni EH, Lempp FA,
Duijst S, et al. Impaired uptake of conjugated bile acids and hepatitis
B virus preS1-binding in Na-taurocholate cotransporting polypeptide
knockout mice. HEPATOLOGY 2015;62:207-219.75. Bogomolov P, Voronkova N, Allweiss L, Dandri M, Schwab M,
Lempp FA, et al. A proof-of-concept phase 2a clinical trial with
HBV/HDV entry inhibitor Myrcludex B. HEPATOLOGY 2014;60:
1279A-1280A.76. King RW, Ladner SK, Miller TJ, Zaifert K, Perni RB, Conway SC,
et al. Inhibition of human hepatitis B virus replication by AT-61, a
phenylpropenamide derivative, alone and in combination with (–)beta-L-2’,3’-dideoxy-3’-thiacytidine. Antimicrob Agents Chemother 1998;
42:3179-3186.
77. Feld JJ, Colledge D, Sozzi V, Edwards R, Littlejohn M, Locarnini SA.
The phenylpropenamide derivative AT-130 blocks HBV replication at
the level of viral RNA packaging. Antiviral Res 2007;76:168-177.
78. Delaney WE, Edwards R, Colledge D, Shaw T, Furman P, Painter G,
et al. Phenylpropenamide derivatives AT-61 and AT-130 inhibit repli-
cation of wild-type and lamivudine-resistant strains of hepatitis B
virus in vitro. Antimicrob Agents Chemother 2002;46:3057-3060.
79. Billioud G, Pichoud C, Puerstinger G, Neyts J, Zoulim F. The main
hepatitis B virus (HBV) mutants resistant to nucleoside analogs are
susceptible in vitro to non-nucleoside inhibitors of HBV replication.
Antiviral Res 2011;92:271-276.
1906 LIANG ET AL. HEPATOLOGY, December 2015
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
15/16
80. Katen SP, Tan Z, Chirapu SR, Finn MG, Zlotnick A. Assembly-
directed antivirals differentially bind quasiequivalent pockets to mod-
ify hepatitis B virus capsid tertiary and quaternary structure. Structure
2013;21:1406-1416.
81. Deres K, Schroder CH, Paessens A, Goldmann S, Hacker HJ, Weber
O, et al. Inhibition of hepatitis B virus replication by drug-induced
depletion of nucleocapsids. Science 2003;299:893-896.
82. Stray SJ, Bourne CR, Punna S, Lewis WG, Finn MG, Zlotnick A. A heteroaryldihydropyrimidine activates and can misdirect hepatitis B
virus capsid assembly. Proc Natl Acad Sci USA 2005;102:8138-8143.
83. Stray SJ, Zlotnick A. BAY 41-4109 has multiple effects on hepatitis B
virus capsid assembly. J Mol Recognit 2006;19:542-548.
84. Gane E, Schwabe C, Walker K, Flores L, Hartman G, Klumpp K,
et al. Phase 1a safety and pharmacokinetics of NVR 3-778, a poten-
tial first-in-class HBV core inhibitor. HEPATOLOGY 2014;60:1267A-
1290A.
85. Prange R. Host factors involved in hepatitis B virus maturation,
assembly, and egress. Med Microbiol Immunol 2012;201:449-461.
86. Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten
H, Gerlich WH. Large surface proteins of hepatitis B virus containing
the pre-S sequence. J Virol 1984;52:396-402.
87. Xu Y, Hu Y, Shi B, Zhang X, Wang J, Zhang Z, et al. HBsAg inhib-
its TLR9-mediated activation and IFN-alpha production in plasmacy-toid dendritic cells. Mol Immunol 2009;46:2640-2646.
88. Gehring AJ, Ann D’Angelo J. Dissecting the dendritic cell controversy
in chronic hepatitis B virus infection. Cell Mol Immunol 2015;12:
283-291.
89. Kapoor R, Kottilil S. Strategies to eliminate HBV infection. Future
Virol 2014;9:565-585.
90. Blazquez LC, Fortes P. Harnessing RNAi for the treatment of viral
infections. In: Arbuthnot P, Weinberg MS, eds. Applied RNAi. Cais-
ter Academic Press, Norfolk, UK; 2014:151-180.
91. Chen Y, Cheng G, Mahato RI. RNAi for treating hepatitis B viral
infection. Pharm Res 2008;25:72-86.
92. Klein C, Bock CT, Wedemeyer H, Wustefeld T, Locarnini S, Dienes
HP, et al. Inhibition of hepatitis B virus replication in vivo by nucleo-
side analogues and siRNA. Gastroenterology 2003;125:9-18.
93. McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H,et al. Inhibition of hepatitis B virus in mice by RNA interference.
Nat Biotechnol 2003;21:639-644.
94. Lanford R, Wooddell CI, Chavez D, Oropeza CE, Chu Q, Hamilton
HL, et al. ARC-520 RNAi therapeutic reduces hepatitis B virus
DNA, S antigen and e antigen in a chimpanzee with a very high viral
titer. HEPATOLOGY 2013;58(Suppl. 1):705A-730A.
95. Yuen M-F, Chan HL-Y, Given B, Hamilton J, Schluep T, Lewis DL,
et al. Phase II, dose ranging study of ARC-520, a siRNA-based thera-
peutic, in patients with chronic hepatitis B virus infection. H EPATO-
LOGY 2014;60:1267A-1290A.
96. Alnylam announces new RNAi therapeutic program for the treatment
of HBV infection and reports an up to 2.3 log10 reduction of HBsAg
in chronically infected chimpanzees [press release]. Cambridge, MA:
Alnylam; February 12, 2014.
97. Sung JJ, Wong ML, Bowden S, Liew CT, Hui AY, Wong VW, et al.Intrahepatic hepatitis B virus covalently closed circular DNA can be a
predictor of sustained response to therapy. Gastroenterology 2005;
128:1890-1897.
98. Zhu Y, Yamamoto T, Cullen J, Saputelli J, Aldrich CE, Miller DS,
et al. Kinetics of hepadnavirus loss from the liver during inhibition of
viral DNA synthesis. J Virol 2001;75:311-322.
99. Zoulim F, Durantel D, Deny P. Management and prevention of drug
resistance in chronic hepatitis B. Liver Int 2009;29(Suppl. 1):108-
115.
100. Moraleda G, Saputelli J, Aldrich CE, Averett D, Condreay L, Mason
WS. Lack of effect of antiviral therapy in nondividing hepatocyte cul-
tures on the closed circular DNA of woodchuck hepatitis virus.
J Virol 1997;71:9392-9399.
101. MacDonald RA. “Lifespan” of liver cells. Autoradiographic study
using tritiated thymidine in normal, cirrhotic, and partially hepatec-
tomized rats. Arch Intern Med 1961;107:335-343.102. Magami Y, Azuma T, Inokuchi H, Kokuno S, Moriyasu F, Kawai K,
et al. Cell proliferation and renewal of normal hepatocytes and bile
duct cells in adult mouse liver. Liver 2002;22:419-425.
103. Chen J, Zhang W, Lin J, Wang F, Wu M, Chen C, et al. An efficient
antiviral strategy for targeting hepatitis B virus genome using tran-scription activator-like effector nucleases. Mol Ther 2014;22:303-311.
104. Weber ND, Stone D, Sedlak RH, De Silva Feelixge HS,
Roychoudhury P, Schiffer JT, et al. AAV-mediated delivery of zinc fin-
ger nucleases targeting hepatitis B virus inhibits active replication.
PLoS One 2014;9:e97579.
105. Tavis JE, Cheng X, Hu Y, Totten M, Cao F, Michailidis E, et al. The
hepatitis B virus ribonuclease H is sensitive to inhibitors of the
human immunodeficiency virus ribonuclease H and integrase
enzymes. PLoS Pathog 2013;9:e1003125.
106. Liu F, Campagna M, Qi Y, Zhao X, Guo F, Xu C, et al. Alpha-inter-
feron suppresses hepadnavirus transcription by altering epigenetic
modification of cccDNA minichromosomes. PLoS Pathog 2013;9:
e1003613.
107. Cai D, Mills C, Yu W, Yan R, Aldrich CE, Saputelli JR, et al. Identi-
fication of disubstituted sulfonamide compounds as specific inhibitorsof hepatitis B virus covalently closed circular DNA formation. Anti-
microb Agents Chemother 2012;56:4277-4288.108. Protzer U, Maini MK, Knolle PA. Living in the liver: hepatic infec-
tions. Nat Rev Immunol 2012;12:201-213.
109. Bertoletti A, Ferrari C. Innate and adaptive immune responses in
chronic hepatitis B virus infections: towards restoration of immune
control of viral infection. Gut 2012;61:1754-1764.110. Lau JY, Bird GL, Alexander GJ, Williams R. Effects of immunosup-
pressive therapy on hepatic expression of hepatitis B viral genome and
gene products. Clin Invest Med 1993;16:226-236.111. Das A, Hoare M, Davies N, Lopes AR, Dunn C, Kennedy PT, et al.
Functional skewing of the global CD8 T cell population in chronic
hepatitis B virus infection. J Exp Med 2008;205:2111-2124.
112. Lopes AR, Kellam P, Das A, Dunn C, Kwan A, Turner J, et al. Bim-
mediated deletion of antigen-specific CD8 T cells in patients unable
to control HBV infection. J Clin Invest 2008;118:1835-1845.
113. Chang KM. Hepatitis B immunology for clinicians. Clin Liver Dis
2010;14:409-424.
114. Bertoletti A, Maini MK, Ferrari C. The host–pathogen interaction
during HBV infection: immunological controversies. Antivir Ther
2010;15(Suppl. 3):15-24.115. Xu D, Fu J, Jin L, Zhang H, Zhou C, Zou Z, et al. Circulating and
liver resident CD41CD251 regulatory T cells actively influence the
antiviral immune response and disease progression in patients with
hepatitis B. J Immunol 2006;177:739-747.
116. Boni C, Fisicaro P, Valdatta C, Amadei B, Di Vincenzo P, Giuberti T,
et al. Characterization of hepatitis B virus (HBV)-specific T-cell dys-
function in chronic HBV infection. J Virol 2007;81:4215-4225.
117. Nakamoto N, Cho H, Shaked A, Olthoff K, Valiga ME, Kaminski
M, et al. Synergistic reversal of intrahepatic HCV-specific CD8 T cell
exhaustion by combined PD-1/CTLA-4 blockade. PLoS Pathog 2009;5:e1000313.
118. Baxevanis CN, Voutsas IF, Tsitsilonis OE. Toll-like receptor agonists:
current status and future perspective on their utility as adjuvants in
improving anticancer vaccination strategies. Immunotherapy 2013;5:
497-511.119. Zoulim F, Luangsay S, Durantel D. Targeting innate immunity: a
new step in the development of combination therapy for chronic hep-
atitis B. Gastroenterology 2013;144:1342-1344.
120. Hirsch I, Caux C, Hasan U, Bendriss-Vermare N, Olive D. Impaired
Toll-like receptor 7 and 9 signaling: from chronic viral infections to
cancer. Trends Immunol 2010;31:391-397.121. Lanford RE, Guerra B, Chavez D, Giavedoni L, Hodara VL, Brasky
KM, et al. GS-9620, an oral agonist of Toll-like receptor-7, induces
HEPATOLOGY, Vol. 62, No. 6, 2015 LIANG ET AL. 1907
-
8/18/2019 Present and Future Therapies of Hepatitis B From Discovery to Cure
16/16
prolonged suppression of hepatitis B virus in chronically infected
chimpanzees. Gastroenterology 2013;144:1508-1517.
122. Gane EJ, Lim YS, Gordon SC, Visvanathan K, Sicard E, Fedorak RN, et al. The oral toll-like receptor-7 agonist GS-9620 in patients
with chronic hepatitis B virus infection. J Hepatol 2015;63:320-328.
123. Bertoletti A, Gehring AJ. Immune therapeutic strategies in chronichepatitis B virus infection: virus or inflammation control? PLoS
Pathog 2013;9:e1003784.124. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC,
McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012;366:2443-2454.
125. Kosinska AD, Zhang E, Johrden L, Liu J, Seiz PL, Zhang X, et al.
Combination of DNA prime–adenovirus boost immunization withentecavir elicits sustained control of chronic hepatitis B in the wood-
chuck model. PLoS Pathog 2013;9:e1003391.
126. Koh S, Shimasaki N, Suwanarusk R, Ho ZZ, Chia A, Banu N, et al. A practical approach to immunotherapy of hepatocellular carcinoma
using T cells redirected against hepatitis B virus. Mol Ther Nucleic
Acids 2013;2:e114.
127. Qasim W, Brunetto M, Gehring AJ, Xue SA, Schurich A, Khakpoor A, et al. Immunotherapy of HCC metastases with autologous T cell
receptor redirected T cells, targeting HBsAg in a liver transplant
patient. J Hepatol 2015;62:486-491.128. Gill S, June CH. Going viral: chimeric antigen receptor T-cell therapy
for hematological malignancies. Immunol Rev 2015;263:68-89.
129. Bohne F, Chmielewski M, Ebert G, Wiegmann K, Kurschner T,Schulze A, et al. T cells redirected against hepatitis B virus surface
proteins eliminate infected hepatocytes. Gastroenterology 2008;134:
239-247.130. Krebs K, Bottinger N, Huang LR, Chmielewski M, Arzberger S,
Gasteiger G, et al. T cells expressing a chimeric antigen receptor that
binds hepatitis B virus envelope proteins control virus replication in
mice. Gastroenterology 2013;145:456-465.131. Gehring AJ, Haniffa M, Kennedy PT, Ho ZZ, Boni C, Shin A, et al.
Mobilizing monocytes to cross-present circulating viral antigen in
chronic infection. J Clin Invest 2013;123:3766-3776.132. Dienstag JL, Stevens CE, Bhan AK, Szmuness W. Hepatitis B vaccine
administered to chronic carriers of hepatitis b surface antigen. Ann
Intern Med 1982;96:575-579.133. Pol S, Driss F, Michel ML, Nalpas B, Berthelot P, Brechot C. Specific
vaccine therapy in chronic hepatitis B infection. Lancet 1994;344:
342.
134. Horiike N, Fazle Akbar SM, Michitaka K, Joukou K, Yamamoto K,Kojima N, et al. In vivo immunization by vaccine therapy following
virus suppression by lamivudine: a novel approach for treating
patients with chronic hepatitis B. J Clin Virol 2005;32:156-161.135. Vandepapeliere P, Lau GK, Leroux-Roels G, Horsmans Y, Gane E,
Tawandee T, et al. Therapeutic vaccination of chronic hepatitis B
patients with virus suppression by antiviral therapy: a randomized,controlled study of co-administration of HBsAg/AS02 candidate vac-
cine and lamivudine. Vaccine 2007;25:8585-8597.
136. Xu DZ, Zhao K, Guo LM, Li LJ, Xie Q, Ren H, et al. A randomizedcontrolled phase IIb trial of antigen-antibody immunogenic complex
therapeutic vaccine in chronic hepatitis B patients. PLoS One 2008;3:e2565.
137. Xu DZ, Wang XY, Shen XL, Gong GZ, Ren H, Guo LM, et al.Results of a phase III clinical trial with an HBsAg-HBIG immuno-
genic complex therapeutic vaccine for chronic hepatitis B patients:
experiences and findings. J Hepatol 2013;59:450-456.138. Heathcote J, McHutchison J, Lee S, Tong M, Benner K, Minuk G,
et al. A pilot study of the CY-1899 T-cell vaccine in subjects chroni-
cally infected with hepatitis B