H epatitis B

Current Status of Therapy and Future Therapies

Elias Spyrou, MD, PhDa,b, Coleman I. Smith, MD

a
,
Marc G. Ghany, MD, MHScc,*

KEYWORDS

Vaccines
Infection
Cirrhosis
Hepatocellular carcinoma

Antiviral/immunomodulatory therapy

KEY POINTS

The number of persons with chronic hepatitis B virus (HBV) infection is high.

These persons are at risk for complications of cirrhosis and hepatocellular carcinoma.

There is a need for novel, finite therapy that can cure chronic HBV infection.

This article highlights key developments in antiviral/immunomodulatory therapy, the ratio-
nale for choosing these approaches, and possible therapeutic regimens.

INTRODUCTION

Chronic hepatitis B virus (HBV) infection is a substantial global public health problem.
Despite the availability of a protective vaccine for more than 3 decades, the preva-
lence of infection remains high. Worldwide it is estimated there are 257 million persons
with chronic HBV infection, which results in 887,000 deaths annually primarily from
complications of cirrhosis and hepatocellular carcinoma (HCC).1 Although currently
approved treatments, peginterferon and nucleos(t)ide analogues, are effective at
inhibiting viral replication and reducing complications of chronic HBV infection, they
are not curative and, in the case of nucleos(t)ide analogues, must be administered
long term, if not indefinitely, because of persistence of the covalently closed circular
DNA (cccDNA) in the hepatocyte nucleus.2 Therefore, there is a need for new treat-
ments that could lead to sustained, off-treatment inhibition of viral replication and

Funding: This work was in part supported by the Intramural Research Program of NIDDK, NIH.
M.G. Ghany is an employee of the NIH.
a
MedStar Georgetown Transplant Institute, MedStar Georgetown University Hospital, Wash-
ington, DC, USA; b Nazih Zuhdi Transplant Institute, INTEGRIS Baptist Medical Center, Okla-
homa City, OK, USA; c Liver Diseases Branch, National Institute of Diabetes, Digestive and
Kidney Diseases, National Institutes of Health, Building 10, Room 9B-16, 10 Center Drive MSC
1800, Bethesda, MD 20892-1800, USA
* Corresponding author.
E-mail address: marcg@[Link]

Gastroenterol Clin N Am 49 (2020) 215–238

[Link] [Link]
0889-8553/20/ª 2020 Elsevier Inc. All rights reserved.
216 Spyrou et al

loss of hepatitis B surface antigen (HBsAg). The search for new antiviral agents with
activity against HBV has been hampered by the small and compact nature of the
HBV genome with few druggable viral targets and a lack of experimental systems or
animal models that faithfully recapitulate the viral life cycle in the presence of an intact
immune system. Nevertheless, recent improvements in model systems have facili-
tated the identification of several novel therapeutic approaches against HBV, as
shown in Fig. 1. There are now more than 30 agents under investigation that either
directly or indirectly target the HBV.3 This article highlights key developments in anti-
viral/immunomodulatory therapy, the rationale for choosing these approaches, and
possible therapeutic regimens.

GOALS OF NOVEL THERAPY AND DEFINITIONS OF CURE

The goals of novel therapies for chronic hepatitis B are to cure the chronic infection
and thereby prevent complications of chronic liver disease, cirrhosis, HCC, and
liver-related death. It is important to appreciate that any definition of a cure should
encompass eradication of the chronic viral infection as well as resolution of the under-
lying liver disease. It is expected that complete viral eradication should result in reso-
lution of liver disease, but this may not be true for patients with cirrhosis. The optimal
treatment end point would be eradication of intrahepatic cccDNA and integrated HBV
DNA, loss of HBsAg, and undetectable HBV DNA, referred to as a complete sterilizing
cure. This end point may not be a feasible one, because currently it is not possible to
eradicate cccDNA and integrated HBV DNA. In addition, complete sterilizing cure may
not be necessary to reduce complications of chronic HBV infection. Current efforts are
focused on achieving loss of HBsAg and undetectable HBV DNA in serum with or
without seroconversion to hepatitis B surface antibody (anti-HBs) after completion
of a finite course of treatment, resolution of residual liver injury, and a reduced risk
of cirrhosis and HCC, referred to as functional cure. This end point may still be chal-
lenging given the recent observation that HBsAg may originate from integrated HBV

Fig. 1. Hepatitis B cell life cycle and main classes of medications under development. AAA,
poly adenosine monophosphate; CD, cluster of differentiation; HBc, hepatitis B core/capsid
protein; HBe, hepatitis B e; HBeAg, hepatitis B e antigen; HBs, hepatitis B surface protein;
HBXs, HBV X proteins; HSP, heparan sulfate proteoglycans; NK, natural killer; NTCP, sodium
taurocholate cotransporting polypeptide; pgDNA, pregenomic DNA; rcDNA, relaxed circular
DNA; siRNA, small interfering RNA.
 Hepatitis B Therapy 217

DNA.4 An alternate and perhaps more attainable goal may be complete viral suppres-
sion but persistence of HBsAg in serum after completion of a finite course of treat-
ment, referred to as a partial cure. This end point is observed in a proportion of
patients, but, in the absence of HBsAg loss, is unlikely to be durable because there
is a 15% to 40% lifetime risk of disease reactivation, and risk of HCC may be higher
compared with patients who achieve HBsAg loss.5

NEW THERAPEUTIC APPROACHES FOR CHRONIC HEPATITIS B

A greater understanding of the HBV life cycle and the host immune response has led to
the development of many new therapeutic approaches to treat chronic HBV infection.
Broadly they can be viewed as agents that target the virus directly (direct-acting anti-
viral) or indirectly through modulation of a host factor (indirect-acting antiviral) or the
host immune response (immunotherapy). These various strategies are discussed in
more detail later.

HEPATITIS B VIRUS LIFE CYCLE

To better understand the mechanism of action of direct-acting antiviral agents under

development, review of the viral life cycle is informative (see Fig. 1). HBV initiates
infection by first loosely attaching to heparan sulfate proteoglycans on the hepatocyte
membrane, then it binds to its entry receptor, the hepatocyte-specific bile acid trans-
porter, sodium taurocholate, cotransporting polypeptide (NTCP) through an interac-
tion with the pre-S1 lipopeptide of the large (L) envelope protein. This stage is
followed by fusion of the HBV envelope with the endosomal membrane and endocy-
tosis of the virus.6–8 Next, there is uncoating with release of the double-stranded
relaxed circular DNA genome (rcDNA) and its transport into the hepatocyte nucleus.
In the nucleus, host cellular enzymes repair the rcDNA to form cccDNA.2,9 The
cccDNA serves as the transcriptional template for all messenger RNAs (mRNAs) as
well as the pregenomic RNA (pgRNA), which is the template for viral replication.
Four viral transcripts (polymerase, core, surface, and X) are exported to the cyto-
plasm where they are translated into 7 viral proteins. In the cytoplasm, core proteins
self-assemble into a viral nucleocapsid and the pgRNA and viral polymerase are pack-
aged into the newly formed nucleocapsid through an encapsidation reaction. Viral
replication is achieved using an RNA template, pgRNA, within the nucleocapsid.
The mature viral capsids containing rcDNA are then enveloped with the small, me-
dium, and large (S, M, L) surface proteins in the endoplasmic reticulum and secreted
from the infected cell as intact virions or transported back to the nucleus to replenish
the cccDNA pool.

ENTRY INHIBITORS

HBV entry into hepatocytes requires the coordinated binding of the virus to heparin
sulfate proteoglycans, a low-affinity receptor that mediates hepatocyte attachment,
followed by a high-affinity interaction with the NTCP receptor for viral internaliza-
tion.8,10 The NTCP receptor confers the species specificity to HBV.11,12 Knowledge
of this process has led to the development of several classes of specific and nonspe-
cific inhibitors of viral entry (Table 1).13–16 Entry inhibitors may lead to clearance of
cccDNA by preventing de novo infection of uninfected hepatocytes.
Agents such as heparin, suramin, and synthetic antilipopolysaccharide peptides
can bind to the virus or cellular heparan sulfate proteoglycans (poly-L-lysine) and
 218
Spyrou et al
Table 1
Agents in the development pipeline for chronic hepatitis B

Target Mechanism of Action Class Compounds in Development

Viral entry Antibodies targeting pre-S1 or small surface Monoclonal antibodies GC1102
protein
Attachment inhibitors that block viral Heparin —
interaction with entry receptors Poly-L-lysine —
Reversibly/irreversibly block the NTCP receptor Conjugated bile salts —
Synthetic N-acylated pre-S1 Myrcludex B (Bulevirtide)
Cyclosporine —
cccDNA Inactivate cccDNA Zinc finger nucleases —
Transcription activator–like —
effector nuclease
CRISPR/cas9 system EBT106
HBV CRISPR-CAS-9 lipid nanoparticle
Degrade cccDNA Interferon a, g —
TNF-a —
Lymphotoxin-b receptor agonists —
Functionally silence cccDNA Epigenomic modifiers —
Viral Degrade mRNA siRNA AB-729
transcripts ARB-1467
ARB-1740
DCR-HBVS
Hepbarna (BB-HB-331)
JNJ-3989 (ARO-HBV)
Lunar-HBV
Vir-2218 (ALN-HBV)
Bind viral mRNA to prevent viral protein Antisense oligonucleotides IONIS-HBVRx (GSK3228836)
production IONIS-HBVLRx (GSK33389404)
RG6004
RO7062931
Cause degradation of HBV RNA in the nucleus DHQ AB452
RG7834
Downregulate viral mRNA FXRa agonist EYP001
Core Inhibit encapsidation of pregenomic RNA or Heteroaryldihydropyrimidines Morphothiadin (GLS4)
protein nucleocapsid assembly Phenylpropenamide AT-61; AT-130
assembly Pyridazinone derivatives 3711
modulators Sulfamoylbenzamide AB-423, AB 506
JNJ-6379, JNJ-0440, NVR 3-778
Isothiafludine NZ-4
2-amino-n-(2,6-dichloropyridin-3-yl) acetamide BCM-599
derivatives
5,50 -bis[8-(phenylamino)-1- Bis-ANS
naphthalenesulfonate]
— ABI-H2158, ABI-H0731, ABI-H3733
— RG7907
— QL-007
— EDP-514
— CB-HBV-001
— —
HBsAg Synthetic oligonucleotides that bind HBsAg Nucleic acid polymers Rep 2139
release Rep 2165
inhibitors
Targeting Apoptosis Apoptosis inducer APG-1387
host Cyclophilin Cyclophilin inhibitor CRV 431 (CPI 431–32)
pathways
Boost innate Agonists of sensing arm of innate immune Toll-like receptor-7 agonist AL-034
immunity system ANA773
RO6864018 (RG7795)
RG7854
Toll-like receptor-8 agonist GS-9688

Hepatitis B Therapy
RIG-I and NOD2 agonist Inarigivir (SB9200)
STING agonists —
TCR-like antibodies —

(continued on next page)

219
 220
Table 1
(continued )

Spyrou et al
Target Mechanism of Action Class Compounds in Development
Boost — Anti-HBs HBIg
humoral — Anti-HBs monoclonal antibody GC1102
immunity
Boost Checkpoint inhibitors Anti–CTLA-4 —
adaptive Anti–PD-1 Nivolumab
immunity Engineering new HBV-specific T cells TCR gene transfer LTCR-H2-1
CAR-T cells —
Therapeutic Induction of HBV-specific B and T cells T-cell vaccines HepTcell
vaccines DNA vaccines HB-110
INO-1800
JNJ 64300535
Viral vectors expressing HBV proteins TG1050
TomegaVax HBV
Virus-like particle vaccine VBI-2601
Inactivated parapoxvirus nonspecific vaccine AIC 649
Antigen-antibody fusion vaccine Chimigen HBV

Abbreviations: CAR-T, chimeric antigen receptor T; CRISPR/cas9, clustered regularly interspaced short palindromic repeats–associated nuclease 9; CTLA-4, cyto-
toxic T-lymphocyte–associated antigen 4; DHQ, dihydroquinolizinone; FXR, farnesoid X receptor alpha; PD-1; programmed cell death protein 1; STING, stimu-
lator of IFN genes; TCR, T-cell receptor; TNF-a, tumor necrosis factor-a.
 Hepatitis B Therapy 221

nonspecifically interrupt viral attachment to proteoglycans but none of them have

been evaluated in the clinic.10,17–19
More specific agents are ones that either target the antigenic loop of the HBV S
domain or N-terminal epitopes in the pre-S1 domain, such as neutralizing antibodies
(Ma18/7, KR127, 17.1.41/19.79.5, hepatitis B immunoglobulin [HBIg]), or ones that
reversibly (taurocholate and ezetimibe)6,15 or irreversibly bind to NTCP (Myrcludex
B, cyclosporin A, and its derivatives such as SCYX1454139).10,20,21 HBIg, a mixture
of antibodies that target HBsAg purified from the plasma of vaccinated individuals,
is the only approved medication but requires parenteral administration and large
doses to be effective. It has not been tested as a therapeutic agent in clinical
studies.22–25 Similarly, taurocholate and ezetimibe require high concentrations to be
effective but their short half-lives at the receptor may limit their clinical application.
In contrast, Myrcludex B (Bulevirtide), an HBV large surface protein–derived syn-
thetic lipopeptide, has a long half-life at the receptor and irreversibly blocks the
NTCP receptor in nonsaturating concentrations.26 Myrcludex was evaluated in a
phase IIa proof-of-concept study in 40 patients with hepatitis B e antigen (HBeAg)–
negative chronic hepatitis B without cirrhosis. Patients were randomized to receive
Myrcludex B at one of 5 dosages: 0.5 mg, 1 mg, 2 mg, and 5 mg for 12 weeks, and
10 mg once daily for 24 weeks. HBV DNA declined greater than 1 log in 6 out of 8 pa-
tients in the 10-mg dosing group and in 7 out of 32 patients in the lower dosing groups;
however, no patient experienced HBsAg loss.27 Studies of Myrcludex either alone or in
combination with pegylated interferon in patients with chronic delta hepatitis yielded
more promising results. Among 60 patients with HBV/hepatitis D virus (HDV) coinfec-
tion who were randomized to receive pegylated interferon alone, pegylated interferon
plus Myrcludex B 2 or 5 mg, or Myrcludex B 2 mg for 48 weeks, 3 out of 15 (20%) pa-
tients in the pegylated interferon plus 2 mg of Myrcludex B arm achieved HBsAg loss
24 weeks off therapy and this number increased to 4 out of 15 (27%) 48 weeks off ther-
apy. No HBsAg loss was observed in any of the other treatment arms.28 Thus, Myrclu-
dex B in combination with other antiviral agents may be a promising treatment of
chronic delta hepatitis.

TARGETING COVALENTLY CLOSED CIRCULAR DNA

cccDNA plays a key role in the viral life cycle, where it serves as the template for viral
transcription and the pgRNA, the template for viral replication. Elimination of cccDNA
is considered the Holy Grail of HBV treatment because its persistence in the nucleus of
infected hepatocytes is the major reason why cure of HBV is currently not possible.
Strategies to block cccDNA formation, enhance its destruction, silence its transcrip-
tion, and stimulate cell division to promote its elimination are currently under
investigation.
DNA cleavage enzymes, such as homing endonucleases or meganucleases, zinc
finger nucleases, transcription activator–like effector nucleases, and clustered regu-
larly interspaced short palindromic repeats–associated system 9 (CRISP/Cas-9)
proteins, act by inducing targeted breaks in double-stranded DNA. The breaks in
double-stranded DNA are repaired by homologous repair in the case of homologous
template such as double-stranded DNA and single-stranded DNA or nonhomologous
end repair in the absence of template and the mutated cccDNA is eventually lost.
In vitro studies showing inhibition of HBV replication have provided evidence that
such an approach can work; however, specificity for viral targets and efficient and
safe delivery of the gene editing agent to eliminate cccDNA from all infected hepato-
cytes are major challenges that need to be overcome.29–36
222 Spyrou et al

Another approach being pursued is to use agents that upregulate apolipoprotein B

mRNA editing enzyme catalytic subunit 3A and 3B (APOBEC3A/B) deaminases. Upre-
gulation of APOBEC3A/B by interferon-a (IFN-a) and lymphotoxin-b has been shown
to lead to noncytolytic degradation of cccDNA in vitro, but the degradation of the
cccDNA pool is incomplete.37 Other cytokines including IFN-g, tumor necrosis fac-
tor-a (TNF-a), and interleukin (IL) 1b can also lead to cccDNA degradation via a similar
mechanism.38,39
In the nucleus, the viral cccDNA is organized into a chromatinlike structure that
makes it amenable to epigenetic manipulation.40 Several compounds have been
shown in vitro to silence cccDNA transcription. IFN-a inhibits transcription of
genomic and subgenomic RNAs derived from cccDNA, both in HBV-replicating cells
in culture and in HBV-infected chimeric urokinase-type plasminogen activator/se-
vere combined immunodeficiency (uPA/SCID) mice repopulated with primary human
hepatocytes.41 The HBV X protein (HBX), which is essential for viral transcription, has
been shown to act through degradation of the host structural maintenance of chro-
mosomes (Smc) complex Smc5/6, which can selectively block extrachromosomal
DNA transcription. HBX destroys the Smc5/6 complex, thereby removing the inhibi-
tion on transcription and permits HBV gene expression.42,43 Thus, targeting the HBX
might be a viable approach to silencing cccDNA. Pevonedistat, a neural-precursor-
cell-expressed developmentally down-regulated 8 (NEDD-8)-activating enzyme in-
hibitor, restored Smc5/6 protein levels and suppressed viral transcription in cultured
hepatocytes.44 However, the observation that there is reactivation of cccDNA as
soon as HBX becomes available again may be a major limitation to this
approach.45,46 At present, no cccDNA targeting strategies have been evaluated in
clinical trials.

TARGETING VIRAL TRANSCRIPTS

It has been proposed that high levels of viral antigens may inhibit the host immune
response and contribute to persistence of HBV, based mostly on evidence from
host evasion strategies of other viruses. Thus, targeting the viral mRNA, through the
use of molecular approaches such as RNA interference, antisense oligonucleotides,
and ribozymes, may be an effective way to control HBV infection.9
Four viral genes are transcribed from the cccDNA template by host RNA polymer-
ase II in the hepatocyte nucleus. All mRNA transcripts share a common 30 terminus
and encode the 7 viral proteins: core, e, polymerase, small surface, middle surface,
large surface, and X. Therefore, targeting this region with a small interfering RNA
(siRNA) that can induce the cell’s RNA-induced silencing complex/argonaute 2 could
lead to degradation of all viral transcripts and would allow simultaneous suppression
of all viral proteins. Several siRNAs have been designed for this purpose, including
ARB-1467, ARB-1740, ALN-HBV, Hepbarna (BB-HB-331), Lunar-HBV, ARC-520,
and ARC-521.47–50 In clinical trials, ARC-520 and ARC-521, in combination with ente-
cavir, was well tolerated and showed a decrease of HBsAg, total HBV DNA, and
cccDNA in both HBeAg-negative and HBeAg-positive patients.51 An interesting
observation from these studies was that the decline in HBsAg levels was lower among
HBeAg-negative compared with HBeAg-positive patients. This reduced efficacy
among HBeAg-negative patients was subsequently shown in a chimpanzee model
to be caused by production of HBsAg from integrated HBV DNA and absence of
binding sites for the siRNA on the 30 end of the transcripts.4 Use of a second siRNA
targeting another region of preS/S was able to result in reduction of HBsAg levels in
HBeAg-negative chimpanzees that was similar to that achieved in HBeAg-positive
 Hepatitis B Therapy 223

animals. However, studies with this compound were discontinued because of the
possible fatal toxicity of the delivery vehicle in nonhuman primates.
An alternate approach to block viral protein expression is to use liver-directed
antisense oligonucleotides that act through steric hindrance and/or RNA degrada-
tion by ribonuclease H cleavage. Two antisense molecules, IONIS-HBVRx
(GSK3228836) and IONIS-HBVLRx (GSK33389404), linked to a trimer of N-acetyl-
galactosamine (GalNAc) moieties, allows delivery of the antisense molecule to the
liver via the hepatocyte-expressed asialoglycoprotein. Such an approach may
reduce off-target toxicities associated with antisense oligonucleotides.52 These
agents are currently in phase I trials. Note that both siRNA and antisense oligonu-
cleotides do not eliminate cccDNA, and rebound of HBsAg to pretreatment levels
after treatment is stopped has been observed, leading to concerns about the dura-
bility of response. In addition, viral mutations and quasispecies may contribute to
rebound and limit the applicability of this approach. Therefore, it is likely that
repeated courses of therapy or use in combination with other approaches would
most likely be required.53
RG7834 is a novel, small-molecule compound belonging to the dihydroquinolizi-
nones chemical class that leads to selective degradation of HBV transcripts in infected
hepatocytes and in the human liver chimeric uPA/SCID mouse model that acts similar
to siRNA but through a different mechanism.54

TARGETING VIRAL NUCLEOCAPSID ASSEMBLY

The HBV core protein is not only a structural component of the viral nucleocapsid but
is involved in nearly every stage of the HBV life cycle, including subcellular trafficking
and release of the HBV genome, RNA metabolism, capsid assembly and transport,
and reverse transcription,55 and modulation of the host innate immune response, mak-
ing the HBV core protein a promising target for HBV treatment.
Several compounds referred to as core protein assembly modulators (CpAMs) are
under investigation. All the compounds bind to a hydrophobic pocket on the capsid
formed at the interface between core protein dimers. These molecules inhibit nucle-
ocapsid assembly, encapsidation of pgRNA, or both, leading to inhibition of rcDNA
synthesis from pgRNA, as reverse transcription takes place only in the viral nucleo-
capsid.56,57 Two classes of CpAMs have been identified based on their mechanism
of action. Class I typified by heteroaryl dihyropyridines, increase the kinetics of
capsid formation, and lead to the formation of misassembled capsids. Class 2 is typi-
fied by phenylpropenamides, which accelerate capsid assembly and form morpho-
logically normal capsids that are empty and lack viral pgRNA and HBV polymerase.
Some CpAMs may have additional effects, such as affecting the conversion of
rcDNA to cccDNA. Importantly, CpAMs seem to be active against most HBV geno-
types and some have shown activity against nucleos(t)ide analogue-resistant
strains.58–61 There are now several CpAMs in differing stages of clinical development
(see Table 1).
NVR 3-778, a sulfamoyl benzamide derivative, was the first-in-class CpAM evalu-
ated either alone or with peginterferon in a proof-of-concept phase 1b trial among
HBeAg-positive patients without cirrhosis. Reductions in both HBV DNA and HBV
RNA were observed and were greatest in the group that received NVR 3-778 plus
peginterferon alfa (w2 log10 copies/mL for both respectively), compared with the
groups given NVR 3-778 or peginterferon alfa alone (reduction in HBV DNA of 1.43
log10 copies/mL and 1.06 log10 copies/mL respectively and reduction in HBV RNA
of 1.42 log10 copies/mL and 0.89 log10 copies/mL, respectively). NVR 3-778 dosed
224 Spyrou et al

at 400 mg or lower was ineffective at suppressing HBV DNA and HBV RNA. Viral
rebound was observed after treatment was stopped. Overall treatment was well
tolerated.62
A novel CpAM, JNJ 56136379 (JNJ-6379), showed promise as an antiviral in
treatment-naive patients with chronic hepatitis B without cirrhosis. Efficacy and safety
of JNJ-6379 at 3 doses of 25 mg, 75 mg, and 150 mg administered for 28 days was
evaluated in a randomized, double-blind, placebo-controlled phase 1b study among
36 HBeAg-positive and HBeAg-negative patients. Substantial dose-dependent reduc-
tions in HBV DNA and HBV RNA from baseline were observed, with up to 2.9 log IU/mL
reduction in HBV DNA and 1.7 log reduction in HBV RNA observed with the 150-mg
and 75-mg doses, respectively. The drug showed no dose-limiting toxicities, with
exposure increasing in a dose-dependent manner.63
RO7049389 is a small-molecule, class I HBV CpAM that induces formation of
abnormal HBV core aggregates, resulting in defective capsid assembly and thereby
suppressing HBV replication. RO7049389 administered in single and multiple
ascending doses among healthy volunteers was shown to be safe and well tolerated
across all tested doses. RO7049389 was administered at 200 mg twice a day for
28 days to 6 patients with chronic HBV infection, and showed a median decline in
HBV DNA of 2.7 log10 IU/mL from baseline and was less than the limit of detection
in 3 out of 6 patients. These preliminary but promising results suggest that
RO7049389 has excellent anti-HBV activity.64
Preliminary results from a phase 1b trial of AB-506, a potent, oral class II capsid
inhibitor, evaluating multiple doses of AB-506, with or without a nucleos(t)ide
analogue, once daily for 28 days among HBeAg-positive or HBeAg-negative sub-
jects with chronic hepatitis B reported a mean decline in HBV DNA of 2.0 log
(160-mg dose) and 2.8 log (400-mg dose) from baseline at day 28 and a
mean decline in HBV RNA of 2.4 log from baseline at day 28 for both doses.65 How-
ever, further development of AB-506 was discontinued because of the observation
of 2 cases of acute hepatitis in the phase 1a 28-day clinical trial in healthy
volunteers.
ABI-H0731 is a potent and selective oral HBV core protein inhibitor. Interim results
are available from 2 randomized, double-blind, placebo-controlled phase 2a studies.
In the first study (ABI-H0731-201), ABI-H0731, 300 mg once daily or placebo was
administered to HBeAg-positive or HBeAg-negative individuals with chronic HBV
infection already suppressed on standard-of-care nucleos(t)ide analogues
(n 5 73). In the second study (ABI-H0731-202), ABI-H0731, 300 mg plus entecavir
or entecavir plus placebo daily was given to treatment-naive HBeAg-positive individ-
uals (n 5 25). In the nucleos(t)ide-suppressed HBeAg1 cohort (ABI-H0731-201), sig-
nificant reductions in HBV RNA levels were observed in the group receiving
nucleos(t)ide analogues plus ABI-H0731compared with the those receiving nucle-
os(t)ide analogues plus placebo at week 12 (2.34 log10 IU/mL vs 0.05 log10 IU/mL,
P<.001). In the treatment-naive HBeAg1 cohort (ABI-H0731-202), significantly
greater declines in HBV DNA levels were noted with the combination of ABI-H0731
and entecavir compared with entecavir plus placebo at week 12 (4.54 vs 3.29
log10 IU/mL, P<.011) and HBV RNA levels at week 12 (2.27 vs 0.44 U/mL, P<.005).
No patient lost HBeAg or HBsAg, although decreases in HBeAg and HBsAg levels
were noted in some individuals in both studies.66 These encouraging early data sug-
gest that capsid inhibitors can result in substantial reduction in HBV DNA and HBV
RNA levels. Longer-term studies alone and in combination with other antiviral agents
will be needed to determine whether CpAMs will result in loss of serum HBsAg,
HBeAg, and cccDNA.
 Hepatitis B Therapy 225

HEPATITIS B VIRUS POLYMERASE INHIBITORS

All currently approved nucleos(t)ide analogues target the DNA polymerase activity
of the viral polymerase. The purpose of developing new HBV polymerase inhibitors
is to augment their antiviral efficacy and improve oral bioavailability and
safety compared with the current nucleos(t)ide analogues.9 Besifovir, CMX-157,
AGX-1009, and lagociclovir are newer HBV polymerase inhibitors in
development.53,67,68
Besifovir has entered phase III trials and in doses of 90 mg or 150 mg has shown the
same antiviral efficacy as 96 weeks of treatment with 0.5 mg of entecavir.69 Besifovir
had good antiviral activity against wild-type and drug-resistant mutant strains.70 The
drug was well tolerated, but dose-dependent carnitine depletion was observed in
some patients and, as a result, these patients had to receive carnitine
supplementation.71
Additional approaches are focused on targeting the ribonuclease H (RNase H) ac-
tivity of the HBV polymerase, which is required for production of new virions. Abolish-
ing RNase H activity results in the formation of defective HBV DNA. At present there
are no compounds that inhibit RNase H activity in clinical trials.

TARGETING HEPATITIS B SURFACE ANTIGEN

Clearance of HBsAg is the primary focus of drug development. Therefore, com-

pounds that can directly target HBsAg resulting in its clearance would be highly
desirable. Nucleic acid polymers (NAPs) are synthetic oligonucleotides that bind
HBsAg and block its release through a poorly understood mechanism.72 It has
been proposed that NAPs possibly interfere with the assembly/release of HBV sub-
viral particles.73,74 REP-2139 and REP-2165 are candidate NAPs in clinical devel-
opment and, in combination with pegylated interferon 2a or thymosin alpha-1,
showed significant decline of HBV DNA and HBsAg levels with anti-HBs serocon-
version in some HBeAg-positive Asian patients.75 REP 2139 and REP 2165 have
been studied in combination with tenofovir and pegylated interferon 2a. All patients
received lead-in tenofovir for 24 weeks, after which they were continued on tenofo-
vir and randomized to receive pegylated interferon 2a plus REP 2139 or pegylated
interferon 2a plus placebo for 24 weeks. Significant declines in HBsAg levels were
noted in patients receiving REP 2139, with 9 out of 10 patients achieving greater
than 4 log decline in HBsAg levels and 8 out of 10 experiencing HBsAg loss
compared with only 1 out of 10 patients not receiving REP 2139 24 to 48 weeks
off therapy.76 Further follow-up is required to determine whether these interesting
results are sustained.

MODULATING THE HOST IMMUNE RESPONSE

The immunologic response in patients with chronic HBV infection is characterized by a

weak innate and HBV-specific cellular immune response. The focus of current immu-
nologic approaches is to restore the host immune response to HBV that may lead to
viral clearance.77 Therapeutic strategies being pursued to induce innate immunity
include the use of cytokines and pattern-recognition receptor agonists to stimulate
production of interferon. Strategies to reconstitute functional HBV-specific immunity
include nonspecific approaches, such as checkpoint inhibitors to reawaken the
T-cell response, and more specific approaches using genetically engineered/modified
T cells and therapeutic vaccines.
226 Spyrou et al

BOOSTING INNATE IMMUNITY

HBV is considered a stealth virus and is not associated with induction of a strong
innate immune response.78 However, several lines of evidence suggest that an
adequate innate immune response can suppress HBV replication.79 As an example,
IFN-a, IFN-g, TNF-a, and IL-1a, produced by nonparenchymal liver cells, can sup-
press or even eradicate HBV from infected hepatocytes through a noncytolytic mech-
anism.41,80 In addition, lymphotoxin-b–mediated activation of APOBEC
(apolipoprotein B mRNA editing enzyme, catalytic polypeptide–like) or activation of
retinoic acid-inducible gene-I (RIG-I) has been shown to suppress HBV replica-
tion.37,81 These data suggest that strategies to boost the innate immunity are a rational
therapeutic approach.
Vesatolimod (GS-9620), a toll-like receptor (TLR) 7 agonist, activates intrahepatic
dendritic cells, triggering the production of type I and II interferons and activating intra-
hepatic natural killer (NK) and mucosal-associated invariant T (MAIT) cells. In proof-of-
principle studies, vesatolimod was shown to reduce HBV DNA and HBsAg levels in
chimpanzees and woodchucks but not in humans.82–84 Vesatolimod was tested in a
phase II, double-blind, randomized, placebo-controlled study in 162 patients stratified
by HBsAg levels and serum HBeAg status. Patients received once-weekly oral vesa-
tolimod (1-mg, 2-mg, or 4-mg doses) or placebo for 4, 8, or 12 weeks. No significant
decline in HBsAg level was observed at the primary (week 24) or secondary (weeks 4,
8,12, and 48) end points.85 Differences in response observed in animal and human
studies may relate to activity of TLR-7 and doses used in animal and human studies.
GS-9688, a TLR-8 agonist, activates intrahepatic dendritic cells and other myeloid
cells leading to production of IL-12/IL-18, activation of NK and MAIT cells, and even-
tually decline in serum woodchuck hepatitis virus (WHV) DNA and clearance of wood-
chuck hepatitis surface antigen. GS-9688 is currently under investigation.86
Inarigivir (SB9200), an oral dinucleotide RIG-I agonist, has shown antiviral activity
against HBV via a combination of activation of innate immunity and as a direct-
acting antiviral effect as a nonnucleotide reverse transcriptase inhibitor.87 The
ACHIEVE trial randomized 80 HBeAg-positive and HBeAg-negative patients to receive
one of 4 doses of inarigivir (25, 50, 100, and 200 mg) daily or placebo in a 4:1 ratio for
12 weeks, after which all patients were switched to tenofovir for 12 weeks. HBV DNA
reduction was achieved in a dose-dependent fashion for both HBeAg-positive and
HBeAg-negative patients, with a maximal reduction of 3.26 log10 in the 200-mg
dose. HBV RNA levels paralleled reductions in HBV DNA levels but HBsAg decline
was not dose dependent, indicating the potential importance of the host response
to inarigivir. The medication was well tolerated. Alanine aminotransferase (ALT) flares
were observed in 6 treated patients (10%) and 4 placebo patients (25%), and 1 patient
required dose discontinuation for ALT level greater than 400 IU. Further development
of this compound has been discontinued due to serious adverse events including the
death of one patient.88
An agonist of the mouse stimulator of IFN genes (STING), 5,6-dimethylxanthenone-
4-acetic acid (DMXAA), was found to induce a robust cytokine response in macro-
phages that efficiently suppressed HBV replication in mouse hepatocytes by reducing
the amount of cytoplasmic viral nucleocapsids.89
An alternate strategy to augment the intrahepatic innate immune response is to
selectively deliver cytokines to HBV-infected hepatocytes using T-cell receptor
(TCR)–like antibodies conjugated with cytokines such as IFN-a.90 These TCR-like an-
tibodies bind to the human leukocyte antigens that present HBV peptides at the sur-
face of the infected hepatocytes and they deliver the cytokines into the
 Hepatitis B Therapy 227

microenvironment of the infected cells. Studies in animal models or humans with TCR-
like antibodies have not yet been conducted.
In addition, IFN-g and TNF-a are known to control HBV in a noncytolytic fashion. In
Lymphotoxin-b–mediated activation of APOBEC or activation of RIG-I has been
shown to suppress HBV replication. Lymphotoxin-a (LTa), lymphotoxin-b (LTb), and
cluster of differentiation (CD) 258 are the natural ligands of lymphotoxin-b receptor
(LTbR). However, the potential for severe side effects with these cytokines limits their
therapeutic use. Superagonistic tetravalent bispecific antibody (BS1) and a bivalent
anti-LTbR monoclonal antibody (CBE11) are LTbR agonists. BS1 and CBE11 have
been tested in in vitro experiments using HBV-infected differentiated HepaRG (dHe-
paRG) cells and have been shown to decrease levels of cccDNA, intracellular HBV
DNA, pgRNA, and secreted HBeAg by w90% without toxicity.37

ADAPTIVE IMMUNITY CENTRAL TO CONTROL OF HEPATITIS B VIRUS

Control and resolution of acute HBV infection depend on the generation of a complex
repertoire of viral-specific B and T cells. HBV-specific CD8 T cells secrete cytokines
such as IFN-g that induce noncytolytic HBV clearance as well as recruitment of other
inflammatory immune cells that are critical for clearance of HBV. This process is regu-
lated by HBV-specific CD4 T cells.91 In chronic HBV infection, there are quantitative
and functional defects of the HBV-specific T-cell response that contribute to viral
persistence. T-cell exhaustion is a well-characterized phenomenon in chronic HBV
infection, and the underlying mechanisms are thought to be the persistent exposure
of T cells to HBV antigens92 and the increased expression of multiple coinhibitory mol-
ecules on HBV-specific T cells, programmed cell death protein 1 (PD-1), cytotoxic T
lymphocyte–associated antigen 4 (CLTA4), lymphocyte activation gene-3 (LAG-3),
CD160, T-cell immunoglobulin domain and mucin 3 (TIM-3), and 2B4.93,94 The
TRAIL-death receptor, TRAIL-2, is upregulated by the exhausted T cells, making
them susceptible to TRAIL-dependent NK cell lysis.95 Several other mechanisms
within the liver contribute to diminished T-cell function. The release of enzymes
from damaged hepatocytes depletes essential amino acids for T-cell function, such
as arginine and tryptophan.96 Myeloid suppressor cells can also produce arginase,
which also depletes arginine.97 In addition, regulatory T-cells, B cells, and stellate cells
can secrete cytokines, such as IL-10 and transforming growth factor-b, that suppress
T-cell function.98,99 Restoration of an adaptive immune responses is crucial for the
successful control of HBV.

NONSPECIFIC APPROACHES TO STIMULATE ADAPTIVE IMMUNITY

Nonspecific inhibition of PD-1, programmed death-ligand 1 (PD-L1), T-cell inhibitory

receptor Tim-3, and CTLA-4 could restore vigorous immune responses, and this
approach has been used in treatment of malignancies. However, a concern with
this approach is that nonspecific activation of the immune system may lead to auto-
immunity and/or flares of hepatitis.
Given the upregulation of PD-1 in patients with chronic hepatitis B, anti–PD-1 was
evaluated to restore exhausted HBV-specific T-cell function.100,101 The administration
of a combination of entecavir with an anti–PD-1 ligand monoclonal antibody together
with a WHV DNA vaccine to woodchucks with chronic WHV infection was associated
with restoration of WHV-specific T-cell responses and clearance of woodchuck hep-
atitis surface antigen (WHsAg). No significant increase in serum markers of hepatic
injury was observed in treated compared with untreated control animals.102 Data on
safety of nivolumab, a PD-1 inhibitor, in patients with chronic hepatitis B is available
228 Spyrou et al

from patients with HBV-related HCC. In 1 study, among 51 patients with HBV-related
HCC treated with nivolumab, all of whom were receiving nucleos(t)ide analogues with
HBV DNA level less than 100 IU/mL, none had reactivation of HBV and no patient
experienced anti-HBs seroconversion.103 A single dose of nivolumab with or without
GS-4774 (a therapeutic T-cell vaccine) was evaluated in HBeAg-negative, noncirrhotic
patients without HCC in a phase 1 study. The regimen showed a good safety profile
and modest decreases of HBsAg levels were observed.104 The safety of checkpoint
inhibitors with prolonged use and whether these medications can be safely adminis-
tered to patients with cirrhosis, those with underlying autoimmune disease, or who
are listed for liver transplant are important issues that need to be addressed.

SPECIFIC APPROACHES TO STIMULATE ADAPTIVE IMMUNITY

Restoring an adequate HBV-specific T-cell response in patients with chronic HBV

infection is likely to be challenging because of their low frequency and exhausted
phenotype. A possible solution may be the adoptive transfer of newly engineered
HBV-specific T cells.77 Evidence from patients with leukemia and chronic HBV infec-
tion who received bone marrow transplants from donors who developed HBV-specific
T-cell responses, either from vaccination or prior exposure to HBV with spontaneous
recovery, showing clearance of HBsAg and development of anti-HBs, suggests that
the approach of adoptive transfer of HBV-specific T cells may be feasible.105,106 Ge-
netic reprogramming to create functional T cells to eliminate HBV-infected hepato-
cytes could be achieved through TCR gene transfer or use of chimeric antigen
receptor (CAR) T cells. The main difference between the two approaches is the type
of antigen receptors used for reprogramming. In the case of TCR gene transfer, it is
the native alpha beta chains of the TCR, whereas, in the case of CAR, it is the extra-
cellular domain of virus-specific antibody. HBV-specific T cells with CAR or classic
TCRs tested in vitro and in HBV transgenic mice showed selective elimination of
HBV-infected cell lines and control of HBV replication with only transient liver damage,
respectively.107,108
Proof of principle of using adoptive transfer of reprogrammed HBV-specific T cells
was shown in a patient with metastatic HCC targeting the malignant cells expressing
HBV antigens. Tumor cells were recognized in vivo by lymphocytes engineered to ex-
press an HBV-specific TCR. The genetically modified T cells survived, expanded, and
mediated a reduction in HBsAg levels without exacerbation of liver inflammation or
other toxicity, but clinical efficacy was not observed in this patient.109

THERAPEUTIC VACCINATION

Although HBsAg-based vaccines have shown good efficacy at inducing anti-HBs pro-
duction and protective immunity in vaccinees, the use of vaccines as a therapeutic
modality has been generally disappointing. Vaccination has been used therapeutically
to break T-cell tolerance to HBV proteins and stimulate HBV-specific T-cell responses
with the goal of achieving sustained suppression of HBV replication and ultimately
HBsAg loss in patients with chronic HBV infection. However, studies of current pro-
tein, DNA, and T-cell vaccines in patients with chronic hepatitis B have been unsuc-
cessful, perhaps because they only targeted HBsAg.110
The development of newer DNA vaccines, heterologous prime-boost approaches,
vaccines against multiple HBV proteins, and novel adjuvants has renewed interest
in vaccines as a therapeutic modality for chronic hepatitis B.111 DNA vaccines, such
as INO-1800 or JNJ-64300535, are in phase 1 trials. INO-1800 is a mixture of recom-
binant DNA vaccines that encode the HBsAg and the consensus sequence of the
 Hepatitis B Therapy 229

hepatitis B core/capsid protein antigen (HBcAg). However, DNA vaccination alone is

not very immunogenic. INO-1800, alone or in combination with IL-12 as an immune
activator, is currently being tested in adults with chronic hepatitis B. Preliminary results
have shown it is safe, well tolerated, and generated virus-specific T cells, including
CD81 killer T cells.112 JNJ-64300535 is being studied in combination with nucle-
os(t)ide analogues and results are awaited.111,113
A DNA prime-adenovirus boost approach was tested in WHV-transgenic mice and
was shown to elicit a potent and functional woodchuck hepatitis core antigen –specific
CD81 T-cell response that resulted in the reduction of the WHV load below the detec-
tion limit in more than 70% of animals. In addition, the combination of entecavir and
DNA prime-adenovirus boost immunization in chronic WHV carriers resulted in
WHsAg-specific and woodchuck hepatitis core antigen–specific CD41 and CD81
T-cell responses, which were not detectable in controls animals that received enteca-
vir alone.114 Woodchucks receiving the combination therapy showed a prolonged
suppression of WHV replication and lower WHsAg levels compared with entecavir-
treated controls.114 Moreover, 2 of 4 immunized carriers remained WHV negative after
the end of entecavir treatment and developed anti–woodchuck hepatitis surface pro-
tein antibodies. A clinical trial using a DNA prime-adenovirus boost approach is
planned.
Vaccines using multiple HBV proteins produced by different vectors in combination
with adjuvants have shown some efficacy in vitro and in animal models.77,114,115 GS-
4774 (a yeast-based, heat-inactivated, T-cell vaccine containing HBV core, surface,
and X protein) and TG-1050 (a nonreplicative adenovirus serotype 5 encoding a
unique large fusion protein composed of a truncated HBV core, a modified HBV poly-
merase, and 2 HBV envelope domains) have been shown to induce immunogenicity in
mice and healthy individuals.116,117 However, results of clinical trials were disap-
pointing.118–120 GS-4774 at 3 doses (2, 10, and 40 yeast units every 4 weeks for
24 weeks) was compared with tenofovir among HBeAg-positive and HBeAg-
negative viremic patients without cirrhosis. The primary end point was HBsAg loss.
No HBsAg loss was observed in any of the treatment arms and no significant differ-
ences in decline of HBsAg levels were observed between the GS-4774 dosing groups
and the tenofovir group. HB-110 (a vaccine composed of 3 plasmids that encode for
the HBV envelope proteins S and L, core protein, polymerase, and human IL-12) was
evaluated in combination with adefovir and compared with adefovir alone.121 HBV-
specific T-cell responses were induced in a portion of patients and a single patient
who received a high dose of HB-110 experienced HBeAg seroconversion.121 HepTcell
(FP-02.2), another candidate vaccine, composed of 9 synthetic peptides derived from
the most conserved domains of HBV, is currently in phase I trials.111 NASVAC
(ABX203), a vaccine based on recombinant HBsAg and HBV core proteins, did not
prevent viral relapse after discontinuing nucleos(t)ide analogues in HBeAg-negative
patients.122 Further results of therapeutic vaccine trials are awaited but, based on pre-
liminary results, it is unlikely that therapeutic vaccine alone would be effective and a
combination approach will be required.

COMBINATION THERAPY

Based on current management of other chronic viral infections, it is likely that a cock-
tail of antivirals targeting multiple steps in the viral life cycle or a combined antiviral/
immunomodulatory approach will be needed to achieve functional cure. Which spe-
cific agents will be required and in what sequence in a therapeutic regimen are
currently unknown because most of the drugs in the pipeline are in early-phase
230 Spyrou et al

Fig. 2. Possible future combination regimens to achieve functional cure. CAR-T, chimeric an-
tigen receptor T cells; NUC, nucleos(t)ide analogue; RNAi, RNA inhibitor.

development. Safety with monotherapy and then in combination therapy will need to
be shown before longer-duration studies can be conducted. One therapeutic
approach would be to use multiple direct-acting antiviral agents with different mech-
anisms of action to achieve complete inhibition of intrahepatic HBV replication. A
possible regimen might consist of a nucleos(t)ide analogue as a backbone plus 1 or
2 other agents, such as a CpAM, siRNA, entry inhibitor, or cccDNA inhibitor (Fig. 2).
A second approach might be to combine agents that inhibit viral replication with
ones that specifically reduce viral antigen load (see Fig. 2). Such a strategy is based
on the rationale that a high viral antigen burden may be a contributor to the exhausted
immune phenotype characteristic of chronic hepatitis B. Potential regimens might
include an siRNA, an NAP, or a cccDNA inhibitor in combination with a direct-acting
antiviral. The sequence of administration of agents in such a regimen is unknown.
Whether both agents would have to be used in combination initially or as an add-on
approach will require future study. Another approach might be to use a combination
antiviral/inhibitor of viral antigen burden with an immunomodulator to either boost
innate immune immunity (eg, TLR-RIG-I agonist) or to restore the HBV-specific
T-cell response, such as a TCR gene transfer or chimeric antigen receptor T (CAR-
T) cells or therapeutic vaccines (see Fig. 2).
It is also likely that treatment may need to be tailored for different patient popula-
tions, such as those who are HBeAg positive or negative, treatment naive versus treat-
ment experienced, those with high or low/suppressed viral replication, those with or
without cirrhosis, high versus low viral antigen burden, and perhaps also based on ge-
notype. These are issues that will need to be addressed in future trial designs.

SUMMARY

Although this is an exciting time for drug development in chronic hepatitis B, it is

important to remember that, if to have an impact on the global burden of HBV disease,
any future treatment regimen will have to be not only efficacious and safe but also
 Hepatitis B Therapy 231

tolerable, easy to administer, scalable, and above all affordable. Current antiviral ther-
apy satisfies most of these criteria but must be administered long term and is associ-
ated with very low rates of functional cure. There are safety concerns related to some
of the therapeutic approaches in development, such as the risk of hepatitis flares, he-
patic decompensation, autoimmunity, and drug toxicity, which will need to be
addressed. These issues are particularly relevant for resource-limited areas of the
world, where most chronic carriers reside and where the infrastructure and cost of
additional monitoring may be too burdensome. Although newer therapies that can
lead to functional cure are awaited, it is important to not forget some essential steps
in the efforts to eliminate HBV: better vaccination coverage, reduce vertical transmis-
sion, and improve population-wide testing and linkage to care of patients who require
treatment.

AUTHORS’ CONTRIBUTIONS

Drafting: E. Spyrou, C.I. Smith, M.G. Ghany. Critical revision: E. Spyrou, C.I. Smith,
M.G. Ghany. Final draft: M.G. Ghany.

DISCLOSURE

The authors have no financial discloses. This article has not been submitted to another
journal and has not been published in whole novel therapy is or in part elsewhere
previously.

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