Received: 27 November 2017 | Accepted: 27 November 2017

DOI: 10.1111/liv.13656

REVIEW ARTICLE

Towards HBV curative therapies

Raymond F. Schinazi1 | Maryam Ehteshami1 | Leda Bassit1 | Tarik Asselah2

1
Center for AIDS Research, Emory University
School of Medicine, Atlanta, GA, USA Abstract
2
Department of Hepatology, Centre de Tremendous progress has been made over the last 2 decades to discover and develop
Recherche sur l’Inflammation, Viral Hepatitis
approaches to control hepatitis B virus (HBV) infections and to prevent the develop-
INSERM UMR 1149, AP-HP Hôpital
Beaujon, Université Paris Diderot, Clichy, ment of hepatocellular carcinoma using various interferons and small molecules as
France
antiviral agents. However, none of these agents have significant impact on eliminating
Correspondence HBV from infected cells. Currently the emphasis is on silencing or eliminating cccDNA,
Raymond F. Schinazi, PhD, DSc, Center for
which could lead to a cure for HBV. Various approaches are being developed including
AIDS Research, Emory University School of
Medicine, Atlanta, GA, USA. the development of capsid effectors, CRISPR/Cas9, TALENS, siRNA, entry and secre-
Email: rschina@emory.edu
tion inhibitors, as well as immunological approaches. It is very likely that a combination
of these modalities will need to be employed to successfully eliminate HBV or prevent
Funding information
This work was supported by NIH grant 1R01-­ virus rebound on discontinuation of therapy. In the next 5 years clinical data will
AI-­132833 and in part by funding from the emerge which will provide insight on the safety and feasibility of these approaches and
NIH funded Emory Center for AIDS Research
grant P30-­AI-­050409 to RFS. if they can be applied to eradicate HBV infections globally. In this review, we summa-
rize current treatments and we highlight and examine recent therapeutic strategies
Handling Editor: Zobair Younossi
that are currently being evaluated at the preclinical and clinical stage.

KEYWORDS
antiviral agents, cccDNA, HBV cure, immunotherapy

1 | INTRODUCTION 2 | HBV REPLICATION CYCLE AND

Chronic HBV infection affects around 240 million people worldwide
(Figure 1A) and long-­term risks such as cirrhosis and hepatocellular
carcinoma (HCC) account for approximately 600 000 deaths annu-
1
ally. HCC is one of the most frequent cancers in Africa and Asia
and fibrosis is the most important prognostic predictor of survival.2
Despite the availability of several FDA-­approved drugs (Figure 1B),
continuous treatment is necessary for virus replication control.
Therefore, current HBV research and development programmes aim
to achieve a curative strategy that either eliminates or permanently
silences HBV infection. This review will discuss approved therapies
and will review novel therapeutic agents currently in development.
THERAPEUTIC TARGETS
HBV belongs to the hepadnaviridae family of viruses. Similar to
other related species such as duck hepatitis virus and woodchuck
hepatitis virus, the relatively small genome of HBV consists of a
3.2 kb partially double-­stranded DNA, referred to as relaxed cir-
cular DNA (rcDNA). HBV infects hepatocytes through a recently
identified human sodium taurocholate cotransporting polypep-
tide (hNTCP) receptor.3 Upon fusion with the host membrane, the
rcDNA-­containing nucleocapsid is released into the cytoplasm and
travels to the nucleus (Figure 2). Once inside, rcDNA is converted
to a highly stable episomal DNA known as covalently closed circular

Abbreviations: AAV, adeno-associated virus; AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis
B; eGFRCG, estimated glomerular filtration rate, Cockcroft-Gault method; ETV, entecavir; HAP, heteroarylpyrimidines; HBeAg, Hepatitis B e antigen; nucleocapsid. precore protein; HBeAg-ve,
antigen-negative; HBeAg+ve, HBe antigen-positive; HBsAg, Hepatitis B surface antigen; HBV, hepatitis B virus; HBx, viral protein X; HCC, hepatocellular carcinoma; hNTCP, human sodium
taurocholate cotransporting polypeptide; IFN-α, interferon alpha; LAM, lamivudine; LdT, telbivudine; mRNA, messenger RNA; NA, nucleoside analogue; NI, nucleoside inhibitors; NNI, non-­
nucleoside inhibitors; ORF, open reading frames; PEG-IFN, pegylated interferon; pgRNA, pregenomic RNA; PP, phenylpropenimides; QD, once daily; rcDNA, relaxed circular DNA; SBA, sulfam-
oylbenzamide; siRNA, small interfering RNA; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate; TFV, tenofovir; TLR, toll-like receptor.

102 | © 2018 John Wiley & Sons A/S. wileyonlinelibrary.com/journal/liv Liver International. 2018;38(Suppl. 1):102–114.
Published by John Wiley & Sons Ltd

SCHINAZI et al.
DNA (cccDNA) via the host DNA repair machinery molecule.4,5 cc-
cDNA is the transcriptional template for subsequent virus gene ex-
pression and generation of pregenomic RNA (pgRNA).6 HBV DNA
is also found integrated into the host chromosome. Integration of
viral DNA does not appear to play a direct role in virus replication.
Instead, it is thought that HBV DNA integration may render the
cellular environment more permissive to virus replication through
modulating gene expression. It also likely plays an important role in
hepatocellular carcinogenesis (reviewed in Ref. 7).
Gene organization of cccDNA is uniquely intricate whereby sev-
eral overlapping open reading frames (ORFs) code for 7 viral pro-
teins: pgRNA serves as the template for viral polymerase-­mediated
reverse transcription and subsequent synthesis of rcDNA. Another
gene product of HBV is the core protein, also known as hepatitis B
core antigen (HBcAg), which forms the viral nucleocapsid. Precore
protein (hepatitis B e antigen, HBeAg), is a proteolytically processed
viral protein that is often secreted from the infected cells and can
8
serve as a marker for disease stage. Viral surface proteins, S, M and
L (named based on their small, medium and large sizes respectively)
are coded from the S gene whereby S is translated from S mRNA,
M is translated from preS2 + S mRNA and L is translated from
preS1 + PreS2 + S mRNA. All surface proteins of different length
are collectively referred to as HBsAg. Finally, cccDNA also codes
for viral protein X (HBx), a nonstructural protein that likely acts as a
transcriptional transactivator and plays a role in regulating viral gene
expression.
Once all viral proteins are synthesized and the rcDNA-­
containing nucleocapsid is formed, it can travel through the cel-
lular secretion pathway and be released as an enveloped and
infectious virion. Alternatively, the nucleocapsid can cycle back
to the nucleus intracellularly, whereby the recently synthesized
rcDNA serves to replenish the cccDNA pool. In this way, cccDNA
can be maintained even in the absence of observable viremia.9
Furthermore, cccDNA can remain dormant for a long time and
only become transcriptionally activated years or decades after
the initial infection. Although several therapeutic strategies have
been developed against HBV replication, targeting or eliminat-
ing cccDNA, as an inert, yet highly stable mini-­chromosome has
proven challenging. In this way, cccDNA elimination, or at the very
least, transcriptional control, lies at the heart of the quest for a
cure for chronic hepatitis B.
In theory, any step of the virus replication cycle can be a target for
10
antiviral therapeutics (Figure 2). The most successful target so far
has been the reverse transcription activity of pol. Interferon (IFN) treat-
ment has also shown modest success in inhibiting virus replication.
Other areas of therapeutic research include targeting of the core/cap-
sid protein, mRNA transcription and cccDNA stability and formation.
In addition to gene expression targeting technologies such as CRISPR/
Cas9 and small interfering RNA (siRNA), several immune-­modulatory
strategies that aim to enhance both the innate and adaptive response
to CHB infection are also under investigation. The following sections
will give a more detailed account of these novel strategies in develop-
ment and their mechanism of action.
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| 103
Key points
• Chronic hepatitis B affects an estimated 240 million peo-
ple worldwide.
• Current treatment includes the use of nucleoside ana-
logue inhibitors and interferons which can control virus
replication but are not curative.
• Several novel therapeutic strategies are in development.
These include immunotherapy, gene-editing technology
and small molecule inhibitors that target the viral capsid
and cccDNA.
• These novel therapies aim to provide a functional cure for
chronic hepatitis B.
3 | APPROVED THERAPIES FOR
TREATMENT OF HBV INFECTION
The goal of CHB therapy is to improve survival by preventing risk
of cirrhosis and end-­stage liver disease, and to improve quality of
life.11,12 Several therapies have been approved for CHB infection
(Figure 1B). Historically, under long-­term lamivudine (LAM) treatment,
HBV DNA suppression leads to reduction in cirrhosis decompensa-
tion and HCC (Figure 1C).13,14 Thus, achieving HBV DNA clearance
allows for reduction in necro-­inflammation and reduction in the risk
of fibrosis progression. There are currently 2 principal therapeutic
strategies approved for both HBe antigen-­positive (HBeAg+ve) and
antigen-­negative (HBeAg-­ve) patients (Table 1). This includes a finite
treatment course of interferon alpha (IFN-­α)/pegylated interferon
(PEG-­IFN) or, a long-­term maintenance treatment with nucleoside
analogues (NAs).
A 1-­year treatment with PEG-­IFN offers the potential for immune
control of HBV infection, with higher rates of HBeAg seroconversion
and the possibility of viral suppression after stopping treatment, with
loss of hepatitis B surface antigen (HBsAg) in a significant propor-
tion of patients who maintain undetectable HBV DNA (Figure 3).15-19
However, PEG-­IFN is administered by subcutaneous injection and is
associated with poor tolerability and a risk of depression. Furthermore,
PEG-­IFN is contraindicated in subjects with decompensated cirrhosis,
in persons with autoimmune disease and during pregnancy.
NAs suppress HBV replication via direct antiviral activity. Usually,
tolerability is good and compliance to treatment appears adequate.
Entecavir (ETV) and tenofovir disoproximal fumarate (TDF) are potent
HBV inhibitors with a high barrier to resistance and should be used
as first-­line monotherapies. The chemical structures of various HBV
reverse transcriptase inhibitors are depicted in Figure 1B.20,21 ETV
is a nucleoside analogue that disrupts DNA elongation as a delayed
chain-­terminator, whereas tenofovir (TFV) is a nucleotide (adenosine)
analogue that causes immediate chain termination when incorporated
into HBV DNA during reverse transcription. ETV is unusual in that it is
a D-­enantiomer of guanosine, and this in part accounts for its tighter
binding in the active site of the enzyme as part of the mechanism of
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104 | SCHINAZI et al.

interference. Normal nucleotides can be incorporated following ETV indirect (or delayed) chain terminator, in contrast to the other agents
at the 3′ hydroxyl; nevertheless, chain termination occurs shortly that are used to treat HBV. TFV, in contrast, lacks a 2-­deoxyribose
thereafter because of structural disruptions to the enzyme, so it is an (cyclic) moiety including the 3′ hydroxyl group, and therefore once

(A)

≥ 8% = High
2-7% = Intermediate
<2% = Low

(B)

ADV TDF

ETV

TAF
ADV ETV TDF Approved Jan, 2017

1998 2002 2005 2006 2008 2016

LAM Peg-IFN LdT

TAF
LdT
LAM

(C)

Placebo (n = 215) P = 0.047

Patient with Diagnosis (%)

Lamivudine (n = 436)
Excluding 5 cases in yr1: HR = 0.47; P = 0.052
F I G U R E 1 Global distribution of HBV
10% and current therapies. (A) Worldwide
distribution of chronic HBV infection.
Placebo (B) Approved therapies for chronic HBV
infection: ETV (entecavir), PEG-­IFN
(pegylated-­interferon), TDF (tenofovir),
Lamivudine 5% LAM (lamivudine), LdT (telbivudine), TAF
(tenofovir alafenamide). (C) Long-­term
lamivudine therapy leads to reduction in
Time to Diagnosis (months) cirrhosis decompensation and HCC13,14
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SCHINAZI et al. | 105

incorporated, it terminates polymerization. For TFV, the innovation is
the use of the acyclic phosphonate group, which achieves 2 functions:
(a) this group is resistant to cellular esterases that otherwise might
F I G U R E 2 Schematic representation of mechanisms of HBV
replication and inhibition. The virus replication begins with the
attachment of the virion to hepatocyte cell surface receptor hNTCP.
This step can be blocked with entry inhibitors such as myrcludex
B. Upon entry, the virion is released in the cytoplasm and the
nucleocapsid travels to the nucleus where rcDNA enters the nucleus
and cccDNA is formed. Novel cccDNA formation, or maintenance
of already formed cccDNA can theoretically be disrupted with small
molecule inhibitors or gene-­editing technology such as CRISPR/Cas9.
Viral mRNA and pregenomic RNA are transcribed from cccDNA.
Inhibition of transcription suppresses viral gene expression. HBV DNA
also gets integrated into the host chromosome. Secretion inhibitors
can prevent the release of HBsAg, which can be independent of
the virus replication cycle. Capsid effectors/inhibitors can mislead
proper nucleocapsid formation while nucleoside analogues inhibit
reverse transcription inside the nucleocapsid. Secretion inhibitors
can subsequently inhibit the release of enveloped virions. Finally,
immunomodulators such as therapeutic vaccines and TLR agonists
can enhance the immune response to chronic hepatitis B
serve to remove the phosphate group to form a nucleoside derivative,
and (b) in contrast to other nucleoside analogues, this stable alteration
circumvents the need for the cellular addition of the alpha phosphate,
which is the rate-­limiting step in the synthesis of 2′-­deoxynucleoside
triphosphates, the active substrates for reverse transcriptase.
The phosphonate group of TFV differs from a normal phosphate
group in that a carbon is directly linked to the phosphorus. For a phos-
phate group, the phosphorus would be directly linked to an oxygen
atom in place of the carbon atom in the TFV diagram above. The phos-
phonate is referred to as acyclic because in naturally occurring nucle-
otides, the phosphate group is linked to a sugar moiety (cyclic, ribose
or deoxyribose), whereas this is absent in this drug. The nucleoside
analogues used for HBV treatment are cyclic nucleosides (ETV, lami-
vudine [LAM] and telbivudine). Adefovir (ADV), in spite of its different
characteristics, differs from TFV only by the lack of the methyl group
(Figure 1B).
More than 95% of persons treated with the highly potent TDF
and ETV achieve virological clearance. NAs are administered orally,
and tolerance is good. The safety of these drugs over lifelong ther-
apy remains to be established. Regarding the risk of drug resistance,
although common in the past with earlier, less potent NAs such as
LAM and ADV, resistance has become extremely rare with TDF and
ETV. Long-­term clinical data up to 6 years and beyond are emerging
for the newer NAs that are providing reassuring data on their efficacy
and safety. There are large data that long-­term blockage of HBV repli-
cation by the most potent drugs (ETV and TDF) results in an improved
long-­term survival with a decreased risk of progression to cirrhosis,
end-­stage liver disease and HCC. Furthermore, a study analysing liver
histology in persons treated with TDF for 5 years demonstrated fibro-
sis regression in the majority of patients and also cirrhosis reversal
(Figure 4).22,23 In addition, the cirrhosis reversal was observed during
treatment in 75% of patients with cirrhosis, probably associated with
a decreased risk of HCC and improved survival. Real-­world data with
12 11
Proportion of patients

10
AgHBe-positive (%)

10
8 6
6 5
T A B L E 1 Advantages and disadvantages of PEG-IFN and 4 3
2
nucleoside analogues 2
0
Nucleoside pegIFN LAM ADV ETV LdT TDF
Peg-­IFN analogues 3-4 years 2 years 4-5 years 2 years 3 years 5 years
12 11
Proportion of patients
AgHBe-negative (%)

Advantages Finite duration High efficacy 10

Higher rates of HBs loss Favourable 8
6 5
and/or seroconversion tolerability
4
No resistance Oral 2 0 0 0,5 0*
administration 0
pegIFN LAM ADV ETV LdT TDF
Disadvantages Poor tolerability Long life 3-4 years 2 years 4-5 years 2 years
duration
Moderate efficacy Unknown F I G U R E 3 HBsAg loss after therapy. Loss of HBsAg is the most
long-­term reliable indicator to measure a functional cure of hepatitis B. In
toxicity HBeAg(+) patients, HBsAg loss was around 11% with Peg-­IFN after
Risk of adverse events Costs 4 y, and around 10% after 5 y of TDF. In HBeAg(−) patients, HBsAg
loss was still around 11% with Peg-­IFN. However, no HBsAg loss was
Subcutaneous injection
observed after 2 y of TDF or ETV treatment15-17,22,23

106 | SCHINAZI et al.
ETV and TDF in routine clinical practice are confirming the favourable
safety and excellent efficacy of these therapies.
4 | TENOFOV IR ALAFENAMIDE (TAF)
TAF, as a single-­agent treatment, has been developed for CHB. It is a
novel prodrug of TFV with potent antiviral activity against HBV. The
drug is more stable in plasma with more than 90% reduction in cir-
24
culating TFV level compared to TDF. The dose is once-­daily, oral,
25 mg tablet. Chemical structures of TAF is provided in Figure 1B. In
the phase III clinical trials,25-27 treatment with TAF through 72 weeks
demonstrated: viral suppression (HBV DNA < 29 IU/mL) similar to
TDF; higher rates of alanine aminotransferase (ALT) normalization;
no resistance development in either treatment group at Week 48;
rates of HBeAg loss and seroconversion similar to TDF in Study 110;
good tolerance in HBeAg-­negative and -­positive subjects; treatment-­
emergent AEs similar to TDF; significantly less declines in hip and
spine bone mineral density (BMD) compared to TDF with improved
bone biomarkers; and significantly smaller decreases in estimated
­
glomerular filtration rate by Cockcroft-­Gault (eGFRCG) compared to
TDF, with improved markers of renal tubular function (Figure 5).
5 | RECENT DEVELOPMENTS REGARDING
APPROVED THERAPIES
5.1 | Combination therapy (TDF plus PEG-­IFN)
In a recent study, HBsAg loss was evaluated in patients receiving
the combination of TDF and PEG-­IFN for a finite duration.
p<0.001
p<0.001
100
90
80
70
60
Pa ents (%)
50
40
30
20
10
0 tect uninfected cells from HBV entry and subsequent establishment
Baseline Year 1 Year 5
F I G U R E 4 Fibrosis regression under TDF. Advanced liver fibrosis
and particularly cirrhosis were previously considered to be largely
irreversible. Data from small studies suggest this may be possible.
The histological benefits of 5 y of TDF therapy in the largest study
of paired liver biopsies in HBV over 5 y of therapy are shown.
The histogram shows that the proportion of patients with Ishak
stages 0-­2 increased over the study period, whereas, in contrast,
the proportion with Ishak stages 4-­6 decreased, thus dramatically
illustrating a marked overall decrease in liver scarring for this
cohort22,23
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open-­label, active-­controlled study, 740 patients with CHB were ran-
domly assigned to receive TDF plus PEG-­IFN for 48 weeks (group A),
TDF plus PEG-­IFN for 16 weeks followed by TDF for 32 weeks (group
B), TDF for 120 weeks (group C) or PEG-­IFN for 48 weeks (group D).
At week 72, 9.1% of subjects in group A had HBsAg loss compared
with 2.8% of subjects in group B, none of the subjects in group C and
2.8% of subjects in group D. A significantly higher proportion of sub-
jects in group A had HBsAg loss than in group C (P < .001) or group
D (P = .003; Figure 6). However, the proportions of subjects with
HBsAg loss did not differ significantly between group B and group
C (P = .47) or group D (P = .88). HBsAg loss in group A occurred in
HBeAg-­positive and HBeAg-­negative patients with all major viral gen-
otypes. Finally, a significantly greater proportion of subjects receiving
TDF plus PEG-­IFN for 48 weeks had HBsAg loss than those receiving
TDF or PEG-­IFN alone.
6 | NOVEL THERAPEUTIC STRATEGIES
IN DEVELOPMENT
6.1 | Entry inhibitors
The discovery of human sodium taurocholate cotransporting poly-
peptide (hNTCP) as the cellular receptor that mediates HBV fusion
has facilitated drug development efforts with regard to inhibition of
HBV entry. Myrcludex B represents the most advanced therapeutic
agent that targets hNTCP-­mediated HBV entry into the cell (Table 2).
It is a synthetic 47 amino acid, N-­myristoylated lipopeptide that is de-
rived from the amino acid sequence of L surface protein. By binding
to this receptor in a competitive manner, Myrcludex B excludes viral
28
In an HBsAg binding, and thus inhibiting virus entry.29 Although Mycludex
B showed positive effects on virus control and intracellular cccDNA
accumulation,29 some serious adverse events were observed in phase
I clinical trials with healthy volunteers.30 Myrcludex B has also shown
Ishak score promise for the treatment of hepatitis D virus, which uses the same
6
hNTCP receptor as HBV.31 Another molecule of interest for inhibition
5
4 of HBV entry is irbesartan.32 Originally, this molecule was developed
3 against hypertension and received FDA approval in 1997. Because
2
irbesartan interferes with hNTCP receptors, it was also shown to be
1
0 able to inhibit HBV virus entry in HepG2 cell culture.33 It remains to
be determined whether the addition of entry inhibitors to the current
arsenal of HBV therapeutics would provide any additional benefits,
since at least in theory, this class of inhibitors should be able to pro-
of HBV cccDNA pool.34
6.2 | RNA interference
RNA interference is a relatively novel strategy that targets virus rep-
lication at the level of gene expression (Table 2). Briefly, short RNA
molecules are designed to target specific viral mRNA sequences.
Upon entry into the cell, they hybridize to viral mRNA and the re-
sulting double-­stranded RNA is targeted for degradation.35 ARC-­520
represents a leading siRNA molecule that when conjugated with
 14783231, 2018, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.13656 by Cochrane Israel, Wiley Online Library on [02/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SCHINAZI et al. | 107

cholesterol, can be delivered to the liver upon intravenous adminis-
tration. Experiments on chimpanzees showed that ARC-­520 is highly
effective at reducing circulating HBV DNA levels as well as serum
35,36
HBeAg and HBsAg. ARC-­520 has shown to be relatively well
tolerated in a phase I clinical trial, although some hypersensitivity
reactions were observed. Therefore, the authors recommend future
37
co-­administration with antihistamines. Based on these promising
results, ARC-­520 has advanced to phase II trials. However, these
trials were recently terminated because of formulation toxicities.38
Interestingly, a recent study has identified a novel small molecule
inhibitor that can reduce viral messenger RNA (mRNA) and pgRNA
39
expression. Despite strong evidence that RG7834 does not func-
tion in as RNAi, it is not yet clear how this therapeutic agent can
suppress HBV transcription in a virus-­specific manner. As well, it has
been shown that RG7834 can indeed reduce levels of viral antigens
in the humanized mouse model, although it is still not clear whether
this small molecule would also reduce HBsAg levels expressed from
integrated HBV DNA as seen in individuals with chronic hepatitis B.
6.3 | Gene-­editing strategies
Recent advancement in novel sequence-­specific nuclease technology
has led to the development of several novel therapeutic strategies
against HBV (Table 2). These include zinc finger nucleases, TALENs
(transcription activator-­like effector nucleases) and, CRISPR/Cas9 sys-
tems, which through viral gene editing, have shown some success in
reducing viral DNA and cccDNA levels in both cell culture and animal
models.40-42 Most antiviral gene-­editing therapeutics act as mutagenic
agents that, upon cell entry, cause DNA damage at sequence-­specific
sites within cccDNA. DNA repair via non-­homologous recombination
at these sites is error-­prone and this in turn, results in generation of
insertion/deletions within cccDNA.43
Zinc finger nucleases have successfully been used in tissue
culture to reduce HBV levels.44-47 For example Weber et al devel-
oped a zinc finger nuclease that targeted the core and pol regions
of cccDNA. Using an adeno-­associated virus (AAV) delivery system,
they showed that HBV production from HepAD38 cells could be
significantly controlled upon zinc finger nuclease delivery. However,
they were unable to show specific mutations within cccDNA in a
reproducible manner.41 The efficacy of TALENs in reducing HBV
levels in cell culture was first demonstrated by Bloom et al in 2013
whereby targeting the S and C regions in cccDNA resulted in disrup-
tion of 35% of cccDNA molecules in the HepG2 cell line.48 In agree-
ment with these findings, it was demonstrated that TALENs could
also reduce the circulation of viral particle equivalents in the murine
hydrodynamic injection model48 and cause reductions in secreted
HBeAg levels.49 Use of CRISPR/Cas9 for treatment of HBV was first
evaluated in 2014.42 Through the simultaneous administration of
multiple small guiding RNAs specific for various regions of the HBV
genome, several studies have demonstrated successful inhibition of
virus replication and production of viral markers such as HBsAg.50-53 One
study also showed a marked decrease in cccDNA levels in HepAD38.
This reduction was associated with rampant presence of mutations
within viral DNA.40 Overall, preclinical studies with gene-­editing
technologies have been promising. However, several challenges still
remain. For one, in vivo delivery methods that would maximize ef-
ficacy and minimize immune response to therapeutic gene editors
are still being developed. There is also a concern for viral escape
mutants as a result of treatment with sequence-­specific zinc finger
nucleases, TALENs or CRISPR/Cas9 systems. Finally, adverse events
need to be carefully investigated with these agents, both in terms
of non-­specific cleavage of host chromosomes, and chromosome
destabilization as a result of cleavage of viral DNA integrants.44
Despite these concerns, some gene-­editing agents such as EBT106
(Excision Biotherapeutics) are cautiously advancing towards clinical
development (https://www.excisionbio.comBiotherapeutics 2017,
posting date. [Online]).
6.4 | Secretion inhibitors
In addition to infectious virion secretion, a large number of incomplete
viral particles are released from infected hepatocytes. These include
spherical and filamentous particles made up of HBsAg, also known as
Australia antigen. It is hypothesized that these particles are released

HBeAg- HBeAg+

TAF: 92.6%
TDF: 92.1%
Proporon of Paents, % (95% CI)

TAF: 71.6%
TDF: 71.9%

Treatment difference: −0.9 (−7.0, 5.2); p=0.78

Treatment difference +0.6 (-5.3, +6.4); p=0.84

Weeks Weeks
F I G U R E 5 Efficacy of TAF in phase III
clinical trials. In the phase III clinical trials, ALT Normalizaon (AASLD Criteria)¤
in both HBeAg-­positive and HBeAg-­
negative chronic hepatitis B, treatment
Paents with ALT
Normalizaon, %

‡ ‡ †
‡ ‡
‡ ‡ ‡ TAF: 49.1%
with TAF through 72 wk demonstrated ‡ ‡
‡ ‡
TDF: 39.0%
† *p<0.05
comparable viral suppression (HBV †
†p<0.005
*
DNA < 29 IU/mL) to TDF and improved ‡p<0.001
rates of ALT normalization25,26
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108 | SCHINAZI et al.

in copious amounts to induce immune tolerance and exhaustion.54
Based on this, several inhibitors such as REP 2139 and REP 2165 have
been developed that inhibit the secretion of HBsAg containing sub-
particles (Table 2). Phase I clinical trials have shown positive results
with regard to safety, tolerability and control of HBV viraemia. Based
on these findings, Phase II clinical trials are underway.
6.5 | Polymerase inhibitors
Currently, nucleoside analogue inhibitors are the backbone of chronic
HBV treatment. As discussed elsewhere, when administered as mono-
therapy, nucleoside analogues are quite successful at suppressing viral
replication, but do not eliminate the virus from the human body. There
are currently several nucleoside analogue inhibitors in development
that aim to provide therapeutic, pharmacological and tolerability im-
provements on the existing drug regimens. These include CMX-­157,
a novel prodrug of TAF and MIV-­210, a nucleoside analogue with
57
antiviral activity in woodchucks. Another novel acyclic nucleoside
analogue is besifovir (LB80380), which in comparison to entecavir,
was shown to achieve similar therapeutic results in treatment-­naïve
chronic hepatitis B patients.58 It remains to be seen whether these
novel nucleoside analogues, as mono or combination therapy, provide
substantial advantages over current FDA-­approved therapies.
6.6 | Immunomodulators
An important characteristic of chronic hepatitis B is the development
of immune exhaustion as a result of continuous exposure of T cells
to high levels of viral antigens. Overexpression of several inhibitory
receptors such as PD1 and CTLA-­4 has been shown to be associated
with T-­cell exhaustion. As such, it has been hypothesized that small
molecules that inhibit PD-­1 expression, may result in T-­cell reactiva-
tion, and subsequent mounting of an efficient immune response that
54
may be able to eliminate chronic hepatitis B.
most immune checkpoint inhibitors have the potential to be used in
any number of diseases where immune reactivation is desired. These
include important human diseases such as melanoma, lymphoma and
54
other cancers. A list of current PD-­1 and other immune checkpoint
inhibitors is provided in Table 2.
Recent developments with regard to toll-­like receptor (TLR) ago-
nists have also been shown to be effective in modulating the innate
immune response against HBV. Activation of virus-­specific TLRs
can lead to production of IFN-­α and -­β, both of which play import-
ant roles with regard to suppression of virus replication (See review
55
59). Specifically, TLR-­7 plays a critical role in activating the immune
response within the liver environment without causing systemic in-
flammation.60-62 Stimulation of TLR-­8 as a result of viral infection was
shown to result in IFN-­γ production and subsequent virus control.63
At the same time, it was found that TLR-­8 function was impaired in
patients with chronic HBV infection.64 Based on this, several thera-
peutic strategies have been developed to harness the innate immune
response in response to chronic hepatitis B. GS-­9620 is a TLR-­7 ag-
onist that showed promising results in animal studies with chimpan-
zee65 and woodchuck models.66 Treatment with GS-­9620 resulted in
56
reduction in circulating HBV DNA and woodchuck surface antigen.66
Conversely, when GS-­9620 was administered to patients with chronic
hepatitis B in a phase II trial, it was found that the T cell response was
improved in a transient manner, but no significant reduction in HBsAg
levels was detected. It was hypothesized that variations in dosing may
account for this discrepancy.67,68
Development of therapeutic vaccines represents another recent
strategy against chronic hepatitis B. It is hypothesized that the stimu-
lation of T cells with HBV antigens may result in the generation of an
effective and HBV-­specific adaptive immune response that may elimi-
nate the chronic infection. Based on this, several therapeutic vaccines
are currently being evaluated in the clinic. Examples include GS-­4774,
which consists of highly immunogenic recombinant HBcAg, HBsAg
and HBx epitopes, ABX-­203 consisting of HBcAg and HBsAg epi-
topes,69 and INO-­1800 consisting of DNA plasmids coding for HBcAg
and HBsAg. Other therapeutic vaccines currently in clinical trials are
listed in Table 2. Despite promising outcomes of recent immune ther-
apies, several potential challenges still remain. For one, manipulation
It is worth noting that of the innate or adaptive immune response may be accompanied by
related adverse events in either a local, or systemic manner. This was
most acutely observed in recent clinical trials involving PD-­1 agonists
and other immune-­modulatory therapies whereby the liver and the en-
docrine system were negatively affected.70,71

HBsAg loss
0.15
48 weeks 72 weeks
0.14
0.13
0.12 F I G U R E 6 Combination therapy with
Patients with HBsAg loss

0.11
0.10
TDF and PEG-­IFN. At week 72, 9.1%
Kaplan-Meier (%)

TDF + PEG 48 weeks. 9.0%

0.09 of subjects in group A had HBsAg loss
0.08 compared with 2.8% of subjects in group
0.07 p = 0.003
0.06
B, none of the subjects in group C and
0.05 PEG 48 weeks. 2.8% p < 0.001 2.8% of subjects in group D. A significantly
0.04 TDF + PEG 16 weeks. →TDF 32 weeks 2.8%
p = NS higher proportion of subjects in group A
0.03
0.02 p = NS
had HBsAg loss than in group C (P < .001)
0.01 TDF 120 weeks. 0%
or group D (P = .003). However, the
0.00 proportions of subjects with HBsAg loss
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 did not differ significantly between group B
Weeks and group C (P = .47) or group D (P = .88)28
 14783231, 2018, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.13656 by Cochrane Israel, Wiley Online Library on [02/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SCHINAZI et al. | 109

virions or amplified from encapsidated rcDNA from the hepatocyte

6.7 | Inhibitors of cccDNA formation
The major strategy to “cure” HBV is to silence or eliminate cccDNA
from the hepatocyte nucleus. cccDNA is the main element of viral
replication cycle as it serves as the template for transcription of all
viral RNAs including pgRNA, and subsequently the progeny HBV DNA
genomes are formed. cccDNA is either originated from new incoming
cytoplasm. Because hepatocytes have a long half-­life (>6-­months or
even years), the half-­life of cccDNA is also relatively long; therefore,
elimination of cccDNA by hepatocyte turnover is not a major means
of clearance.72 Reverse transcriptase nucleoside analogue (NA) inhibi-
tors have a negligible effect on cccDNA formation, stability or ampli-
fication, and therefore HBV commonly rebounds after cessation of

TABLE 2 Summary of recent anti-­HBV antiviral agents in clinical and preclinical development

Comments and
Drug class Antiviral agent Status Developer references

Entry inhibitors Myrcludex B Phase II MYR GmbH/Hepatera Bogomolov et al (2016)87

Irbesartan (Avapro) FDA-­approved Sanofi Research Approved for hyperten-
sion. Wang et al (2015)
33

Inhibitors of viral mRNA ALN-­HBV Phase I-­II Alnylam siRNA

ARC-­520 Terminated Arrowhead pharmaceuticals siRNA
ARB-­1467 Phase II Arbutus Biopharma siRNA88,89
ARB-­1740 Preclinial Arbutus Biopharma siRNA90
RO7020322 (RG7834) Phase I Roche Small molecule mRNA
inhibitor. Mueller et al
(2017)39
Ionis HBVRx Phase I Ionis Pharma/GSK Antisense molecule
(GSK3228836)
IONIS-­HBVLRx Phase I Ionis Pharma/GSK Antisense molecule91
(GSK33389404)
AB-­452 Preclinical Arbutus Biopharma RNA destabilizer92
Gene editing EBT106 Preclinical Excision Biotherapeutics CRISPR/Cas9 system93
Anti-­HBV sgRNA Preclinical CRISPR/Cas9 system.
Kennedy et al (2015)40
TALEN Preclinical Dryer et al (2016)94
Zinc finger nuclease Preclinical Weber et al (2014)41
HBsAg secretion inhibitor REP2139 & REP2165 Phase II Replicor Bazinet et al (2017)55,95
BM601 Preclinical Xu et al (2014)96
Nucleoside analogue LB80380 (besifovir, Phase III/Approved in S. Illong Pharmaceutical Yuen et al (2015)58
inhibitors besivo) Korea
CMX-­157 Phase IIa ContraVir Prodrug of tenofovir.
Painter et al (2007)56
Capsid assembly inhibitors/ GLS-­4 Phase II HEC Pharm, Sunshine China-­CFDA
modulators NVR 3-­778 Phase Ia Novira Pharmaceuticals/ Klumpp et al (2017)78
Janssen
BAY41-­4109 Phase I Berke et al (2017)79
JNJ56136379 Phase I JnJ Janssen NCT02662712. Zoulim
et al (2017)97
Core protein allosteric Phase I (ABI-­H0731) Assembly Biosciences ABI-­H0731 -­
modulators (CpAMs) IND enabling (ABI-­ NCT02908191
H2158) Huang et al AASLD
Clinical candidate (2017)98
(ABI-­Nx)
AB-­423 Phase I Arbutus Biopharma Eley et al AASLD (2017)99
AB-­506 IND enabling Arbutus Biopharma Mani et al AASLD
(2017)80

(Continues)
 14783231, 2018, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.13656 by Cochrane Israel, Wiley Online Library on [02/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
110 | SCHINAZI et al.

TABLE 2 (Continued)

Comments and
Drug class Antiviral agent Status Developer references

cccDNA inhibitor Disubstituted Preclinical Cai et al (2012)100

sulphonamides
Therapeutic vaccine GS-­4774 Phase II GlobeImmune Recombinant X, S & C
epitopesa,101
ABX-­203 Phase II/III Abivax Recombinant S & C
epitopes
TG-­1050 Phase I Transgene Non-­replicative
adenovirus. C, pol & S
epitopes. Levin (2017)102
INO-­1800 Phase I Inovio DNA plasmids encoding S
&C
TLR agonists GS-­9620 Phase II Gilead Sciences TLR-­7 agonist. Boni et al
(2016)68
RO6864018 (RG7795) Phase II Roche TLR-­7 agonist
GS-­9688 Phase I Gilead Sciences TLR-­8 agonist
Immuno-­modulators PD-­1/PDL-­2 mAb Preclinical Merck Sharp and Dohme;
Bristol-­Myers Squibb
CTLA-­4 mAb Preclinical
SB9200 (Iranigivir) Phase II Spring Bank Pharmaceuticals RIG-­I and NOD-­2
agonist103
a
X = HBx, S = HBsAg, C = HBcAg.

treatment with NA.73 In addition, the presence of HBsAg in serum of
chronic hepatitis B (CHB) individuals with undetectable levels of HBV
DNA after or during NA therapy strongly suggest that this protein is
transcribed either from cccDNA or integrated HBV DNA in the nu-
cleus of hepatocytes. Enormous advances in understanding the HBV
replication cycle have facilitated the development of novel antiviral
therapies targeting multiple steps of viral replication with numerous
mechanistic actions. Both in vitro and in vivo models for testing these
novel antiviral therapies have also significantly improved and mutually
open a new era for extinction of hepatitis B chronic infection.74
Several in vitro studies demonstrate that it is possible to degrade
cccDNA in the nucleus of hepatocytes with minimal hepatotoxicity
effect. As discussed above, programmable RNA-­guided DNA endonu-
cleases, including CRISPR/Cas9 system have shown in both cell and
mouse models the potential to serve as effective tool for the deple-
tion of the cccDNA pool in HBV-­infected hepatocytes (Table 2).40,44,52
Moreover, up-­regulation of APOBEC3A/B deaminase by several cyto-
kines including IFN-­α, IFN-­γ, tumour necrosis factor-­α and lymphotox-
in-­β receptor agonist can cause partial degradation of cccDNA without
hepatotoxicity.75 These targets aim to prevent cccDNA formation by
damaging or destroying it. However, more research needs to be done
regarding off-­target effects of gene-­editing applications or the in vivo
delivery efficiency before they advance to humans.
6.8 | Silencing cccDNA with epigenetic drugs
Viral cccDNA is a mini-­chromosome that is inherently stable and si-
lencing its activity by epigenetic modifications with epigenetic drugs
(epidrugs) may represent a novel class of antivirals for treatment of
chronic hepatitis B. HBx binds to cccDNA and plays an important role
on activation of viral gene transcription, leading to high level of virus
replication. In contrast, inhibition of HBx binding to cccDNA leads to
suppression of its transcription and virus replication by modifying the
epigenetic regulation of cccDNA function with host restriction fac-
tors, including Smc5/6 complex and protein arginine methyltrans-
ferase PRMT1. HBV core inhibitors targeting cccDNA-­bound HBc
may potentially alter the epigenetics of cccDNA and in combination
with HBx inhibitors could in theory lead to a stronger synergistic ef-
fect on cccDNA transcriptional silencing and HBV replication.76
6.9 | Nucleocapsid assembly inhibitors or modulators
Novel direct acting core assembly inhibitors or modulators have an
important impact in HBV replication cycle. They can interfere with
several steps of the HBV replication cycle including capsid assem-
bly, reverse transcription, as well as pgRNA and polymerase protein
packaging. These molecules may also affect intracellular trafficking
of relaxed circular DNA in the nucleus, virus assembly by disrupt-
ing its interaction with envelope proteins, and core binding to cc-
cDNA.77 Two main classes of core assembly modulators have been
developed, including class I heteroarylpyrimidines (HAP), and class II
phenylpropenimides (PP) or sulfamoylbenzamide (SBA) and deriva-
tives (Table 2). NVR 3-­778 is a SBA compound developed by Novira
(later acquired by Johnson & Johnson, New Brunswick, NJ, USA) that
is in phase Ia clinical trial (NCT02112799 & NCT02401737), and
has shown synergistic effect when in combination with PEG-IFN.

SCHINAZI et al.
TABLE 3 Who to treat?
Phase Immune tolerant HBeAg-­positive CHB
HBeAg status Positive Positive
ALT Normal Elevated
HBV DNA Very high > 200 000 IU/mL >2000 IU/mL
Histology Normal or mild inflammation & Inflammation & fibrosis:
mild fibrosis moderate to severe
Disease progression Low Moderate to high
Treatment Not indicated Indicated
Evaluation of NVR 3-­788 with PEG-­IFN in mice with humanized liver
confirmed inhibition of both HBV DNA replication and HBV RNA pro-
78
duction. Recent in vitro studies with JNJ-­632 (SBA) or Bay41-­409
(HAP) compounds in human primary hepatocytes showed that both
compounds inhibited early and late steps of viral replication by (a)
preventing formation of cccDNA, and (b) reducing intracellular HBV
RNA levels and HBe and HBs antigens production when these com-
pounds were added at the time of infection.79 Clinical studies are
being conducted with 3 other potent core assembly modulators,
including morphothiadine mesilate GLS4 (HAP) in phase II (HEC
Pharm/Sunshie, Guangdong,­ China-­CFDA), JNJ56136379 in phase
I (JnJ Janssen, New Brunswick, NJ, USA -­ NCT02662712), and the
first-­generation Core protein Allosteric Modifier (CpAM) in phase I
Assembly Biosciences (NCT02662712). New improved second gen-
eration of CpAMs that induce formation of aberrant capsids were re-
cently developed that show favourable preclinical profile in inhibiting
4 HBV markers, including secretion of HBeAg and HBsAg, pgRNA
and cccDNA (Table 2). A new generation of capsid assembly inhibi-
tor AB-­506 (Arbutus Biopharma, Burnaby, BC, Canada) was devel-
oped that increases thermal stability to core protein compared to
first-­generation inhibitors, and it also inhibits pgRNA encapsidation
in HepAD38 cells and demonstrates potent in vivo activity in mouse
80
model. AB-­423 (Arbutus Biopharma) is another inhibitor of capsid
assembly that has demonstrated potent inhibition of HBV replication
by blocking pgRNA encapsidation. This compound is in phase I clinical
study (Table 2).81
7 | WHO TO TREAT: THE IMPORTANCE OF
FIBROSIS AS PROGNOSIS FACTOR
Fibrosis is the most important prognosis predictor of survival.
Therefore, subjects at risk of fibrosis progression or with advanced
fibrosis may be prioritized for treatment (Table 3). Several new
markers have been developed to assess fibrosis. For instance, our
group in France developed a simple scoring system to determine
the severity of fibrosis in persons with genotypes B or C HBV infec-
tion who are HBeAg-­positive.28 We developed 2 prediction scoring
systems (PSs). PS1 analysed data on HBV genotype (B vs C), patient
age (<30 vs ≥30 years), level of hepatitis B surface antigen (≤17 500
vs >17 500 IU/mL) and level of alanine aminotransferase (≤3-­fold
14783231, 2018, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/liv.13656 by Cochrane Israel, Wiley Online Library on [02/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
| 111
Inactive carrier HBeAg-­negative CHB
Negative Negative
Normal Elevated (fluctuating)
<2000 IU/mL >2000 IU/mL (fluctuating)
Normal or mild Inflammation and fibrosis:
inflammation moderate to severe
No, very low Moderate to high
Not indicated Indicated
vs >3-­fold the upper limit of normal). PS2 analysed data on only age
and level of hepatitis B surface antigen. Our system differentiated
persons with no or mild fibrosis (F0-­F1) from those with marked or
severe (F2-­F4) fibrosis with a high positive predictive value (PPV).
The high level of specificity for the identification of non-­severe
fibrosis (F0-­F2) limits the risk of overlooking patients with severe
fibrosis (F3-­F4).
In another study, the expression of 13 fibrosis-­related microRNAs
(miRNAs; miR-­20a, miR-­21, miR-­27a, miR-­27b, miR-­29a, miR-­29c,
miR-­92a, miR-­122, miR-­146a, miR-­155, miR-­221, miR-­222 and miR-­
224) was analysed in 194 serums and 177 liver biopsies of patients
with either CHB or chronic hepatitis C (CHC) to develop models to
diagnose advanced fibrosis and cirrhosis (Metavir F3-­F4).82 In CHB
subjects, the model (serum miR-­122, serum miR-­222, platelet count
and alkaline phosphatase) was more accurate than APRI and FIB-­4 to
discriminate in between mild and moderate fibrosis (F1-­F2) and F3-­
F4 (AUC of CHB model: 0.85 vs APRI: 0.70 and FIB-­4: 0.81). In CHC
persons, the model (hepatic miR-­122, hepatic miR-­224, platelet count,
albumin and alanine aminotransferase) was more accurate than both
APRI and FIB-­4 to discriminate in between patients with F3-­F4 and
F1-­F2 (AUC of the CHC model = 0.93 vs APRI: 0.86 and FIB-­4: 0.79).
Most of the miRNAs tested were differentially expressed in patients
with CHB and CHC. In particular, serum miR-­122 was 28-­fold higher
in patients with CHB than in those with CHC. Both CHB and CHC
models may help for the diagnosis of advanced fibrosis and cirrhosis
(F3-­F4).
8 | HBSAG QUANTIFICATION: A NEW
TOOL FOR HBV MONITORING
Quantifying HBsAg is certainly an important new tool for predict-
ing the severity of disease and distinguishing inactive carriers from
persons with HBeAg-­negative chronic active hepatitis. In this way,
HBsAg measurement helps tailor follow-­up and treatment manage-
ment.83,84 In addition, a decline in quantitative HBsAg during therapy
is a strong predictor of SVR after PEG-­IFN therapy and of the prob-
ability of HBsAg loss, which is the ultimate goal of therapy. In patients
treated with nucleoside analogues, a decline in HBsAg levels is also a
predictor of HBsAg loss, allowing therapy to be discontinued. A low
HBsAg level is predictive of favourable response. Baseline HBsAg

112 | SCHINAZI et al.
levels are reliable predictors of sustained response and several data
confirm this result. Baseline HBsAg levels are significantly lower in
patients who achieve an SVR than in non-­responders, both in HBeAg-­
positive and HBeAg-­negative patients.85,86
9 | CONCLUSIONS
The recent success in eliminating hepatitis C virus infection from the
liver using safe and potent combinations of direct antiviral agents
has raised hopes that this will also be possible for HBV infections.
Lifetime use of current anti-­HBV agents can decrease and suppress
the HBV replication to undetectable levels, but it does not produce
a cure. To have a sustained clinical cure, the discovery and develop-
ment of novel combined modalities that prevent virus rebound on
discontinuation of therapy is needed. There is also a need to silence
or eliminate cccDNA, and decrease or eliminate HBsAg, HBeAg,
pgRNA and possibly other HBV markers to undetectable levels. In
addition, restoring or increasing the host immune response should
lead to a functional and perhaps an absolute cure. Once these per-
sons are cured, theoretically they can be vaccinated against HBV to
prevent reinfection. Only then, we will have all the tools necessary
to eradicate HBV globally.
CO NFLI CTS OF I NTE RE ST
Raymond Schinazi is the founder and major shareholder of Cocrystal
Pharma, Inc. Tarik Asselah is a speaker and investigator for BMS,
Janssen, Gilead, Roche and Merck. Maryam Ehteshami and Leda
Bassit have no conflicts of interest to declare.
O RCI D
Tarik Asselah http://orcid.org/0000-0002-0024-0595
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How to cite this article: Schinazi RF, Ehteshami M, Bassit L,
et al. Towards HBV curative therapies. Liver Int. 2018;
38(Suppl. 1):102–114. https://doi.org/10.1111/liv.13656