Expert Opinion on Investigational Drugs

ISSN: 1354-3784 (Print) 1744-7658 (Online) Journal homepage: http://www.tandfonline.com/loi/ieid20

New antivirals for the treatment of chronic

hepatitis B

Vincent Soriano, Pablo Barreiro, Laura Benitez, Jose M. Pea & Carmen de
Mendoza

To cite this article: Vincent Soriano, Pablo Barreiro, Laura Benitez, Jose M. Pea & Carmen
de Mendoza (2017) New antivirals for the treatment of chronic hepatitis B, Expert Opinion on
Investigational Drugs, 26:7, 843-851, DOI: 10.1080/13543784.2017.1333105

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EXPERT OPINION ON INVESTIGATIONAL DRUGS, 2017
VOL. 26, NO. 7, 843851
https://doi.org/10.1080/13543784.2017.1333105

REVIEW

New antivirals for the treatment of chronic hepatitis B

Vincent Sorianoa, Pablo Barreiroa, Laura Benitezb, Jose M. Peaa and Carmen de Mendozab
a
Infectious Diseases Unit, La Paz University Hospital & Autonomous University, Madrid, Spain; bDepartment of Internal Medicine, Puerta de Hierro
Research Institute, Majadahonda, Spain

ABSTRACT ARTICLE HISTORY

Introduction: Current treatment with oral nucleos(t)ides entecavir or tenofovir provide sustained Received 26 January 2017
suppression of HBV replication and clinical benefit in most chronic hepatitis B virus (HBV) infected Accepted 17 May 2017
persons. However, HBV rebound generally occurs upon drug discontinuation due to persistence of KEYWORDS
genomic HBV reservoirs as episomic cccDNA and chromosomic integrated HBV-DNA. There is renewed Hepatitis B; capsid inhibitors;
enthusiasm on HBV drug discovery following recent successes with antivirals for hepatitis C and cccDNA; entry inhibitors;
immunotherapies for some cancers. immunomodulators; RNAi;
Areas covered: New drugs that target distinct steps of the HBV life cycle are been developed, including TAF; novel therapy;
inhibitors of viral entry, new polymerase inhibitors, capsid and assembly inhibitors, virus release nucleos(t)ide analogues;
blockers, and disruptors of cccDNA formation and transcription. Alongside these antivirals, agents polymerase inhibitors;
that enhance anti-HBV specific immune responses are being tested, including TLR agonists, checkpoint therapeutic vaccines; TLR7
agonist
inhibitors and therapeutic vaccines.
Expert opinion: The achievement of a functional cure for chronic HBV infection, with sustained HBsAg
clearance and undetectable viremia once medications are stopped, represents the next step in the pace
towards HBV elimination. Hopefully, the combination of new drugs that eliminate or functionally inactivate
the genomic HBV reservoirs (cccDNA and integrated HBV-DNA) along with agents that enhance or activate
immune responses against HBV will lead to a definitive cure for chronic HBV infection.

1. Introduction across and within regions [2,16]. The risk of developing

chronic HBV infection is related to the age at infection.
Although an effective preventive HBV vaccine exists for over
Particularly those infected in early childhood, either perinatally
30 years [1], chronic hepatitis B virus (HBV) infection currently
or horizontally, depict the highest risk of developing a chronic
affects 240 million people worldwide [2]. This figure is by far
HBV carrier status [5]. The strong age-dependent nature of
greater than for other viruses that cause pandemic chronic
becoming chronic HBV carrier implies that areas with younger
infections, such as HIV or hepatitis C virus (HCV) that globally
ages at HBV exposure like sub-Saharan Africa and East Asia
affect 39 and 80 million people, respectively. Indeed, hepatitis
have disproportionate higher endemicity of chronic HBV infec-
B is the major cause of cirrhosis and liver cancer globally,
tion [2,16].
accounting for more than 750,000 deaths annually [3].
Most countries have experienced a decline in chronic
The introduction of lamivudine in 1997, the first oral antiviral
hepatitis B rates over the last decades, largely as result of
drug to treat HBV infection, represented a huge advance in the
improved socio-sanitary conditions and HBV vaccination
management of chronic hepatitis B. Long-term lamivudine ther-
programs [17]. HBV vaccines were introduced in the early
apy was associated with viral suppression, liver enzyme normal-
1980s; however, safer and more effective recombinant HBV
ization, and prevention of cirrhosis [4]. However, emergence of
vaccines only became available after 1986 [1]. Nowadays
resistance over time is associated with loss of sustained benefits
only 5% of HBV vaccinated people fail to response. While
of lamivudine monotherapy [5]. The arrival of newer anti-HBV
some countries started vaccination in the early 1990s, in
agents more potent and with higher resistance barrier, such as
many countries HBV vaccines were introduced into univer-
entecavir and tenofovir, has made feasible lifelong suppression
sal childhood HBV vaccination schedules only in the 2000s.
of HBV replication with oral therapy [68]. However, HBV
Moreover, implementation began very often with low cov-
rebound occurs in more than 90% of patients following drug
erage rates. This is why the expected huge benefit of HBV
discontinuation [9], as HBV is not eradicated with current antiviral
vaccines is still far away [18,19].
therapies [10]. The recent development of curative therapies for
hepatitis C and the strong commitment for HIV eradication
initiatives have led to international calls seeking also for a cure 2. HBV life cycle and therapeutic targets
for hepatitis B [1115].
HBV is a member of the Hepadnaviridae family and can only
Although roughly 3.6% of the global population is chroni-
infect humans and primates. The virion is constituted by a
cally infected with HBV, a large heterogeneity exists both
small 3.2 kb partially double-stranded circular DNA

CONTACT Vincent Soriano vicente.soriano@inv.uam.es; vsoriano@dragonet.es Autonomous University, Madrid 28035, Spain
2017 Informa UK Limited, trading as Taylor & Francis Group
844 V. SORIANO ET AL.
Article highlights
Current treatment of HBV infection with nucleos(t)ide analogs pro-
vides long-term serum HBV-DNA suppression, significantly ameliorat-
ing liver damage and extrahepatic complications; however, the risk of
liver cancer remains increased.
New antivirals that target distinct steps of the HBV life cycle including
entry, assembly and/or release inhibitors, along with drugs acting on
HBV reservoirs (cccDNA disruptors or transcription blockers) hopefully
would lead to serum HBsAg clearance, the next step in HBV ther-
apeutics or functional cure.
Adding immune-modulators that enhance specific anti-HBV immune
responses hold promise as adjunctive therapy with antivirals for
eliminating the HBV reservoir, the definite HBV cure (eradication).
This box summarizes key points contained in the article.
surrounded by the envelope that interact with hepatocytes.
HBV first binds with low-affinity to heparin sulfate proteogly-
cans on hepatocytes. Subsequently, the pre-S1 lipopeptide of
the large envelope protein binds to its higher affinity receptor
on the hepatocyte, the bile acid transporter NCTP (sodium
taurocholate cotransporting polypeptide). Then, the virus
enters the cytoplasm by endocytosis, as shown in Figure 1
[20,21].
After uncoating within the cytoplasm, the nucleocapsid
containing the viral genome goes to the nucleus wherein
the relaxed DNA is converted to covalently closed circular
DNA (cccDNA). To a lesser extent, HBV-DNA is integrated
into host chromosomes. The HBV cccDNA is stable within
hepatocytes and acts as template for producing mRNAs
HBV
HSPG
NTCP
pdsDNA
cccDNA
POL-II
Integration
Figure 1. HBV life cycle.
HSPG: heparin sulfate proteoglycan; NTCP: sodium/taurocholate cotransporting polypeptide; cccDNA: covalently closed circular DNA; pdsDNA: partially double
stranded DNA; POL-II: cellular polymerase II; RT: viral reverse transcriptase; pgRNA: pregenomic RNA; preS/SRNA: Surface messanger RNA; XRNA: X messanger RNA;
HBV: hepatitis B virus; HBc: capsid; HBs: surface protein.
mediated by host polymerase II. On average, 15 cccDNA
copies per infected hepatocyte are found [22]. These
include the pregenomic RNA that serves as template for
full genome replication and encodes the viral polymerase,
which is a reverse transcriptase, as well as the viral core
protein (HBcAg).
When sufficient HBV envelope molecules are present,
nucleocapsids are directed to their secretory pathway and
cccDNA amplification ceases. Mature virions might exit
cells through exosomes and go to infect other hepatocytes
or be redirected to the nucleus for replenishing the
cccDNA pool [20,21]. Interestingly, the hepatitis B surface
antigen (HBsAg) is synthetized much more in excess than
required for viral enveloping purposes, and most likely
overwhelms any benefit of anti-HBs responses, causing
immune tolerance.
3. Limitations of current HBV antiviral therapy
Liver enzymes are a poor predictor of liver damage and
disease progression in chronic hepatitis B. Serum HBV-DNA
is currently the stronger predictor of cirrhosis and hepato-
cellular carcinoma (HCC) [23]. Serum HBsAg level serves as
a complementary marker to viral load for the prediction of
HBV-related clinical outcomes in patients with low viremia.
In other words, those with low viral load but high serum
HBsAg levels show a higher risk of cirrhosis and HCC [24].
Following the administration of current approved HBV
nucleos(t)ide reverse transcriptase inhibitors (entecavir or
HBV
HBe
HBs X
HBc
DNA
RT
pgRNA
SRNA
XRNA

tenofovir), serum HBV-DNA declines to undetectable levels
and liver inflammation improves in most treated patients [5
8]. This occurs with 30% of patients treated with subcutaneous
peginterferon- [58]. However, cccDNA and integrated HBV
DNA within host chromosomes persist as does high levels of
secreted HBsAg. As previously highlighted, persistence of
HBsAg production is associated with an increased risk of liver
cancer, even in the absence of productive HBV replication
[24,25]. Thus, new drugs and treatment strategies are needed
to confront properly HBV infection.
4. Prospects for HBV cure
Persons who recover from acute HBV infection typically
achieve HBsAg loss, with antibody to HBsAg (anti-HBs) sero-
conversion. Even these patients, however, harbor cccDNA and
integrated HBV DNA for life and are potentially at risk for HBV
reactivation if they undergo chemotherapy for cancer [26],
immunosuppression for transplantation [27], or antiviral ther-
apy for hepatitis C [28].
The advent of curative therapies for hepatitis C has renewed
the enthusiasm for curing HBV infection, moving beyond the
achievement of viral suppression. Like in HIV infection, unde-
tectable viremia with antivirals is not enough in chronic hepatitis
B patients to completely avert clinical complications. In other
words, chronic inflammation and persistent immune activation
in uncontrolled HBV infection might be associated with an
accelerating aging, mimicking what already has been reported
for HIV or HCV infections [29]. Therefore, expression of HBV
proteins and inflammatory phenomena should need to be com-
pletely halted if virus cannot be eradicated [30].
HBV cure can be defined as either complete or sterilizing
cure (elimination of all viral replicative intermediates, includ-
ing HBsAg and cccDNA of infected cells) or functional cure
(undetectable serum HBV-DNA and loss of HBsAg, despite
persistence of cccDNA and integrated viral sequences within
host chromosomes) [11,13,14]. Functional HBV cure is scienti-
fically plausible given that it occurs spontaneously in >90% of
adults exposed to the virus as well as in a small proportion of
chronically infected patients that clear circulating HBsAg and
HBV-DNA spontaneously or following treatment with pegin-
terferon- or nucleos(t)ide analogs [5]. Complete HBV cure
represents a greater challenge, as neither current therapies
nor spontaneous resolution of infection eliminate the
cccDNA and/or integrated HBV-DNA that may account for
late reactivations, with reappearance of circulating HBV-DNA
and/or HBsAg [2628]. Figure 2 summarizes the path toward
HBV cure, with interventions required and biomarkers moni-
toring for distinct goals.
5. New HBV antivirals
Following the discovery of NCTP as the major HBV cell
receptor, more reliable cell culture systems have been
developed, allowing a better understanding of the HBV
life cycle [3134]. New drugs that target distinct steps of
the HBV cell infection process are been tested, including
inhibitors of viral entry, new polymerase inhibitors, capsid
and assembly inhibitors, virus release blockers, and
EXPERT OPINION ON INVESTIGATIONAL DRUGS 845
Prevention Functional cure Definite cure
Goal Protection Suppression Eradication
Interferon-,
Intervention Vaccine Nucleos(t)ide New drugs
analogues
HBsAg & cccDNA
Biomarker Anti-HBs
HBV-DNA
1980 2000 2020
Figure 2. The global path towards hepatitis B eradication.
disruptors of cccDNA formation and transcription
(Figure 3) [1214,3540]. On the other hand, a second
group of agents that potentiate host immune responses
are being developed to fill the gap between just viral
suppression and virus extinction (Table 1)[41].
5.1. HBV entry inhibitors
The identification of NCTP, a key bile acid transporter
expressed on hepatocytes, as a critical mediator for HBV cell
entry [20], has triggered a major interest for HBV entry block-
ers. The pre-S1 region of the HBsAg appears to bind the
extracellular loops of NCTP, promoting endocytosis of the
receptor-virus complex.
Myrcludex-B is a lipopeptide that mimics the pre-S1
domain and can be administered subcutaneously, blocking
the new infection of hepatocytes by HBV throughout inhibi-
tory competition [42]. Given that NCTP acts as viral receptor
for both HBV and the hepatitis delta virus, myrcludex-B has
been tested in patients with either chronic hepatitis B or delta
[43,44]. However, myrcludex has to be administered subcuta-
neously and recent results have been poorer than expected
[22,44]. The drug is currently been tested as combination
therapy in phase II trials.
5.2. HBV polymerase inhibitors
Entecavir and tenofovir are the current recommended
nucleos(t)ide analogs to treat HBV infection [68]. Both
are specific inhibitors of the HBV retrotranscriptase,
although tenofovir is also active against the HIV reverse
transcriptase, being one of the most widely used antire-
troviral agents [45]. Both entecavir and tenofovir are given
orally once a day. While the efficacy of entecavir may be
hampered in chronic hepatitis B patients with lamivudine-
resistant strains [68], individuals treated with tenofovir do
not develop drug resistance. Occasional reports [46,47] do
not preclude that even in monotherapy the drug has been
shown to be effective for managing multidrug-resistant
HBV [48].
The use of tenofovir disoproxil fumarate (TDF) has been
hampered by the development of renal tubulopathy and
occasionally kidney insufficiency [49]. Furthermore, chronic
tubular damage due to long-term TDF exposure has been
846 V. SORIANO ET AL.

4. Release
HBV HBV
1. Entry HSPG

NTCP 3. Encapsidation
& Assembly

HBe
HBs X
HBc
pdsDNA
2. Reverse DNA
transcription
RT

pgRNA
POL-II
cccDNA SRNA
Integration
XRNA

5. cccDNA 6. Transcription

Figure 3. Major HBV therapeutic targets.

HSPG: heparin sulfate proteoglycan; NTCP: sodium/taurocholate cotransporting polypeptide; cccDNA: covalently closed circular DNA; pdsDNA: partially double
stranded DNA; POL-II: cellular polymerase II; RT: viral reverse transcriptase; pgRNA: pregenomic RNA; preS/SRNA: Surface messanger RNA; XRNA: X messanger RNA;
HBV: hepatitis B virus; HBc: capsid; HBs: surface protein.

Table 1. New HBV armamentarium.

Mechanisms of action Drugs
Virus life cycle (antivirals) Entry inhibitors Myrcludex
Polymerase inhibitors TAF, CMX-157, AGX-1009, Besifovir, Lagociclovir
Capsid blockers GLS-4, NVR 3778
Release inhibitors Rep-2139, Rep-2165
cccDNA cleavage (gene editing) CRISPR/Cas9, TALENS, ZFNs
Transcription inhibitors (RNA interference) ARC-520, ARC-521
Host immune response (immunomodulators) Innate immunity GS-9620, Birinapant
Adaptive immunity Therapeutic vaccines (GS-4774),
Engineered T cells

associated with hypophosphatemia, bone demineralization,
and fractures [50]. The advent of a new prodrug of tenofovir,
named tenofovir alafenamide (TAF) that provides greater
intra-hepatocyte concentrations with less plasma drug levels,
has become an important advantage for patients treated with
tenofovir, since kidney abnormalities and osteopenia are less
frequent with TAF than with TDF [51,52]. In the HIV field, TAF
has already replaced TDF, and the same shift is expected soon
in hepatitis B therapeutics.
Several next-generation nucleos(t)ide analog pro-drugs are
being tested looking for increased antiviral activity and safety
against HBV, including CMX-157, AGX-1009, besifovir, and
lagociclovir [22,40]. However, it is worth to mention that
long-term administration of potent nucleos(t)ide analogs
may produce a reduction in cccDNA in the liver of treated
patients [53], but this finding mostly reflects hepatocyte pro-
liferation [54].
5.3. HBV nucleocapsid inhibitors
The HBV core protein that assembles into the viral capsid
is involved in many different steps of the HBV life cycle.
Accordingly, targeting it may lead to simultaneous inhibi-
tion of multiple steps of HBV replication within infected
hepatocytes [22,40]. Small molecules that disrupt the
encapsidation of HBV pgRNA have been known for some
time. The pioneering discovery of BAY-4109 by Bayer scien-
tists was a milestone. However, recent advances in the
understanding of nucleocapsid formation, how compounds
interact with core protein, and drug development have
only recently progressed the study of capsid assembly
modulators to the clinic [55].
The first compounds acting as allosteric inhibitors of
core assembly that have entered clinical trials are NVR
3778 and GLS-4. The HBV capsid assembly modulator
NVR 3778 suppresses serum HBV-DNA in the humanized
mouse model of HBV infection with continuous declines
and without evidence of selecting nucleotide changes at
the core coding HBV genome that could be associated to
resistance [56].
GLS-4 is a pyrimidine analog that can interfere with the
assembly of HBV core particles. Given its short half-life, it has
to be boosted pharmacokinetically with ritonavir. In a recent
multiple dose-escalating study, 24 chronic hepatitis B patients
 EXPERT OPINION ON INVESTIGATIONAL DRUGS 847

were randomized to receive a 28-day course of ritonavir- Viral clearance during acute hepatitis B is mediated by
boosted GLS-4 or entecavir. The mean decline from baseline CD8-positive cytotoxic T lymphocytes (CTL) that kill infected
in HBV-DNA was 2.4 log IU/mL and the mean decline in HBsAg cells and secrete antiviral cytokines that non-cytolytically
was close to 104 IU/mL [57]. inhibit HBV gene expression and replication. In contrast, the
CTL-induced mechanisms that mediate clearance of the
nuclear pool of viral cccDNA during chronic hepatitis B
5.4. HBsAg release inhibitors involve interferon gamma [63], most likely by upregulating
the APOBEC deaminases, which induce non-hepatotoxic
Clearance of HBsAg is a critical step in the functional control of
degradation of cccDNA [64].
HBV infection. It will unveil the unmaking of the existing anti-
DNA cleavage enzymes, including homing endonucleases
HBs response, which would favor clearance of virions, both
or meganucleases, zinc-finger nucleases (ZFNs), TAL effector
infectious and noninfectious. In other words, it would remove
endonucleases (TALENs), and clustered regularly interspaced
the immunosuppression mediated by the HBsAg protein and
short palindromic repeats (CRISPR)-associated system 9 (Cas9)
permit anti-HBs seroconversion. In other words, the ability to
proteins, that specifically target the cccDNA have been engi-
clear HBsAg is the next frontier in hepatitis B therapeutics
neered and tested in chronic hepatitis B [65].
[21,58].
Disabling cccDNA is key to curing HBV infection and appli-
Synthetic oligonucleotides that incorporate a sulfur atom
cation of gene editing technology, such as harnessing the
depict high stability and may interact with glycoproteins. Used
CRISPR/Cas9 system, is currently the one in most advanced
as antivirals, these amphipatic (hydrophobic) nucleic acid
steps for curative potential [66]. Several studies have reported
polymers (NAPs) function as broad spectrum viral attach-
good efficacy when employing CRISPR/Cas9 technologies to
ment/entry inhibitors. Interestingly, a few of these compounds
disable HBV replication in cultured cells and modified mice
work against HBV with a unique post-entry activity blocking
[6769]. Although preclinical work shows that CRISPR/Cas9
HBsAg release from infected hepatocytes [59].
technology has potential to overcome infection with HBV,
Different NAPs (REP-2055, REP-2139 and REP-2165) block
significant challenges need to be met [66]. Ensuring specificity
the release of HBsAg [60]. A trial in 40 patients was
for viral targets and efficient delivery of the gene editing
recently conducted in Moldova. All received tenofovir for
sequences to virus-infected cells are particularly important.
26 weeks as a lead-in. They were then randomized to one
Another intriguing question refers to the role played in HBV
of two treatment groups for an additional 48 weeks.
rebounds by integrated HBV-DNA in host chromosomes. The
Patients in the NAP group received tenofovir and pegin-
field is at an interesting stage and the future of curative
terferon- plus one of the two NAPs, either REP-2139 or its
antiviral drug regimens for chronic HBV infection may well
derivative, REP-2165. Those in the control group received
entail use of combinations that include derivatives of
the two antiviral agents alone. In the polymer groups,
CRISPR/Cas9 [69].
most patients cleared HBsAg and nearly half developed
anti-HBs [61].
In the REP 401 study, 30 chronic hepatitis B patients 5.6. HBV transcription inhibitors
received at least 12 weeks of NAP. Serum HBsAg response
Once HBV enters the infected hepatocytes, partially double-
over time continued to improve with reductions from baseline
stranded DNA (pdsDNA) goes to the nucleus and is converted
>1 log in 29, >2 log in 25, and >4 log in 19 patients. Moreover,
to cccDNA. Wrapped by histones, the cccDNA forms a mini-
HBsAg loss occurred in 14 patients [62]. Thus, NAPs may allow
chromosome. Subject to epigenetic control by acetylation/
functional control of HBV infection. An intriguing finding of
methylation, the cccDNA is the unique template for producing
this study was that residual circulating HBsAg appeared to be
all viral RNA transcripts, including the pre-genomic RNA
almost completely derived from integrated HBV-DNA instead
[20,40].
of cccDNA.
RNA interference (RNAi) is an evolutionary conserved
A longer administration of NAPs would be needed to unveil
mechanism in which a double-stranded RNA (dsRNA) inhibits
their sustained virological effects and potential risk for side
gene expression by degrading mRNA or by blocking the
effects and selection of resistance when treating either HBV or
translation pathway of a specific gene [70]. During RNAi, exo-
HDV infections. The transition from intravenous to subcuta-
genous (e.g. viral RNA) or endogenous (e.g. microRNA) dsRNA
neous administration of NAPs has favored their further clinical
is processed and cut to 21- to 23-nucleotide RNA fragments by
development.
a ribonuclease called Dicer and assembled into a RNA-induced
silencing complex that destroy the passenger strand and
recognize and cleave the target mRNA [70]. New molecular
5.5. Anti-cccDNA agents
delivery systems such as nanoparticles and glycoconjugates
Even complete suppression of viral replication with potent are enabling RNAi oligonucleotides to become more efficient
HBV polymerase inhibitors would not stop episomic cccDNA in cell penetration.
or chromosomic integrated HBV DNA from being transcribed To date, several RNA oligonucleotides that can specifically
into RNA and/or sustain HBsAg secretion. Thus, any attempt to block transcription of HBV proteins have been designed [70].
eradicate HBV should silence or eliminate the episomic and Targeting HBV RNAs is particularly attractive as all HBV open-
integrated forms of the HBV genome within the infected reading frames use the same polyadenylation 3 terminus,
hepatocytes [40]. making possible to suppress all viral transcripts [22]. A few
848 V. SORIANO ET AL.
anti-HBV RNAi molecules have been tested against HBV in
human trials, including ARC-250, ARB-1467, and ALN-HBV. In
a pioneer phase II trial, ARC-520 produced deep and durable
knockdown of HBV-DNA and viral antigens [71,72].
Interestingly, ARC-520 only required administration once
monthly. Unfortunately, further clinical development of ARC-
520 has been halted due to concerns on toxicities of its
delivery agent [22,40].
It must be highlighted that a drawback of the RNAi tech-
nology in hepatitis B therapeutics is that blockage of HBV
transcripts does not affect cccDNA persistence within infected
hepatocytes. Thus, repeated courses of therapy should be
required. Hopefully, antiviral immunity might be restored in
some extent, enhancing spontaneous clearance rates. In this
regard, combination of NAPs with immune stimulatory agents
might act synergistically enhancing elimination of genomic
HBV reservoirs.
6. HBV immunotherapies
The therapeutic armamentarium against HBV infection can be
divided into two categories: those that target the virus, such
as nucleos(t)ide analogs, and those that target the host, such
as interferons. Patients with chronic hepatitis B exhibit very
weak HBV T-cell responses and lack anti-HBs. Based on this
observation, it makes sense to believe that achievement of
sustained virological response off medication for chronic
hepatitis B will require both blocking virus replication and
restoring the immune response [41,73].
Recent remarkable success of immunotherapy in cancer
(e.g. using anti-PD1 in melanoma and hypernephroma) offers
new and promising strategies for developing immune modu-
lators that may become important components of a wider
therapeutic approach to hepatitis B, some of which are now
in clinical development. As shown in Table 1, among the
immunomodulatory agents currently being investigated to
combat chronic HBV infection are boosters of innate immune
responses (e.g. toll-like receptor (TLR) agonists or immune
checkpoint inhibitors) [74,75] and agents that restore adaptive
immunity (e.g. therapeutic vaccines and engineered T
cells) [35].
Several TLR agonists are being tested in clinical trials,
including GS-9620. Advantages of GS-9620 include oral admin-
istration and its ability to induce interferon intrahepatically
without systemic induction of this cytokine, which is asso-
ciated to well-known side effects [76]. Ongoing studies are
combining GS-9620 with tenofovir [77]. Preliminary results,
however, have not been as good as expected [78].
A range of chronic viral infections, such as HIV, HCV, or HBV,
as well as various cancers, produces immune exhaustion [77].
A hallmark of this phenomenon of tired immune response
following an overwhelming antigen production is an increased
expression of programmed death-1 (PD-1) receptors [79]. The
interaction of PD-1 with its ligands inhibits the function of T
cells and tumor-infiltrating lymphocytes, leading cancer cells
to evade immune responses. In cancer immunotherapy, the
use of checkpoint PD-1 inhibitors has improved the prognosis
of patients with melanoma, renal cell carcinoma, and non-
small cell lung carcinoma [80]. Since T-cell exhaustion is a
major driver of progression of these cancers as well as of
persistence of chronic viral infections like HBV, testing of
checkpoint inhibitors as HBV therapy is being investigated.
Preliminary data are been obtained using some monoclonal
antibodies that block PD-1 and restores the activity of effector
T cells [81,82].
7. Expert opinion
It has been over 50 years since HBV discovery, yet 240 million
people worldwide suffer from chronic hepatitis B, resulting in
over 750,000 deaths per year. A cure is yet to be achieved, largely
due to a viral nuclear reservoir of transcriptionally active cccDNA
and/or chromosomic integrated HBV DNA. While current antiviral
therapies are effective at reducing viral replication, they have no
impact on the existing hepatocyte HBV reservoir. Identifying
mechanisms to either eliminate (complete cure) or inactivate
(functional cure) HBV cccDNA are a major focus of HBV research.
The ability of some viruses to establish chronic cellular reser-
voirs that escape to immune control becomes a major roadblock
that impedes the cure of these infections. HIV and HBV are major
paradigms of this challenge. In contrast, HCV does not establish
persistent cellular reservoirs and accordingly, complete viral sup-
pression for a short timeframe with antivirals drives to cure.
Therefore, new alternatives are needed to pursuit the eradication
of a persistent infection such as HBV. Gene silencing technologies
are in constant evolution from initially RNAi to the recently devel-
oped CRISPR/Cas9. They provide an outstanding sequence speci-
ficity that allows targeting any coding sequence of interest.
Therefore, they have become the most promising approach for
HBV eradication.
More than 30 drugs are currently in the pipeline been
tested as HBV therapy. Within the next months, TAF will
become the preferred nucleos(t)ide choice as HBV therapy,
given its convenient dosing, good safety profile, and high
resistance barrier [51,52].
While clinical trials are needed to evaluate HBV cure strate-
gies, it will not be possible to assess all of the outcomes
relevant to cure (e.g. potential for later toxicities, relapses,
and risk of liver cancer). In other words, the overall risks and
benefits, as well as costs, associated with any HBV cure strat-
egy will be difficult to capture in short-term clinical trials. On
the other hand, an effective HBV cure will probably require a
combination of approaches, which will be challenging to eval-
uate in any single trial. The potential implications of combin-
ing several investigational interventions also need to be
considered [83]. To facilitate this work, simulation modeling
approaches might be used, in conjunction with early data
from clinical trials, to assess the impact of different strategies
on long-term outcomes with strategies that vary based on
efficacy, early and late toxicity, relapse, liver cancer develop-
ment, and cost [19]. Besides, real-world studies once drugs are
approved should be conducted, as it is being done with
direct-acting antivirals already approved for hepatitis C, occa-
sionally unveiling unexpected findings [27,84].
The efficacy of some immune modulatory therapies is com-
promised by high viral antigen levels. Cutting edge technolo-
gies, including RNAi and CRISPR/Cas9, are now being

HBV
status
Hepatitis
Infection
(carrier)
Liver enzymes & fibrosis
Serum HBV-DNA
Serum HBsAg
Liver HBV-DNA (cccDNA &
integrated)
Figure 4. Stepwise therapy for HBV cure.
extensively applied and may ultimately prove to lower viral
antigen levels sufficiently to substantially increase the efficacy
of other HBV antivirals [22,40]. Figure 4 briefly summarizes the
multistepwise roadmap that would hypothetically lead to clear
HBV infection.
Following the recent recognition of trained immunity in
newborns from HBV-infected mothers, the classical immune
tolerance and immune clearance phases have been
renamed as the noninflammatory and inflammatory phases,
respectively [85]. New diagnostic and monitoring tools are
now available, including rapid diagnostic tests for HBsAg
and HBV-DNA detection, and quantification assays for
HBsAg, anti-HBc, HBV core-related antigen (HBcrAg), HBV-
DNA, and HBV-RNA [21].
With the development of new therapeutic agents target-
ing distinct steps of the HBV life cycle and improving the
host antiviral immune responses, a cure for chronic hepatitis
B should be envisioned within the next decade. Together
with full implementation of HBV vaccine programs world-
wide [86], it would pave the way to a HBV-free generation
by 2030.
Funding
This paper was not funded
Declaration of interest
The authors have no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial con-
flict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pending, or
royalties.
EXPERT OPINION ON INVESTIGATIONAL DRUGS 849
Nucleos(t)ide
monotherapy
Combination
antiviral
therapies
Combination
antiviral &
immune
therapies
Functional Definite
Cure Cure
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