REVIEW

New Viral and Immunological Targets for Hepatitis BTreatment and Cure: A Review

Joop E. Arends . Faydra I. Lieveld . Shazaad Ahmad . Andrew Ustianowski

Received: August 22, 2017 / Published online: October 25, 2017� The Author(s) 2017. This article is an open access publication

ABSTRACT

Although current therapies can be successful atsuppressing hepatitis B viral load, long-termviral cure is not within reach. Subsequentstrategies combining pegylated interferon alfawith nucleoside/nucleotide analogues have notresulted in any major paradigm shift. Animproved understanding of the hepatitis B virus(HBV) lifec ycle and virus-induced immunedysregulation has, however, revealed manypotential therapeutic targets, and there arehopes that treatment of hepatitis B could soonbe revolutionized. This review summarizes thecurrent developments in HBV therapeutics—both virus directed and host directed.

Keywords: Antiviral therapy; Hepatitis B virus;Immunomodulators; Nonnucleoside antivirals;Nucleoside analogues

INTRODUCTION

Chronic infection with hepatitis B virus (HBV)remains a major healthcare problem, with anestimated 240 million persons infected world-wide [1]. In patients with untreated HBV infec-tion the 5-year incidence of evolution tocirrhosis is 8–20%, and among those with cir-rhosis the annual risk of developing hepatocel-lular carcinoma is 2–5% [2–4]. Although theimplementation of hepatitis B vaccines havebeen effective in reducing the incidence of HBVin vaccine recipients, significant declines inend-stage liver disease rates have not yet beenseen.

Sustained virological response [i.e., hepati-tis B surface antigen (HBsAg) seroconversion] isonly seldom observed with currently approvedHBV antiviral drugs [pegylated interferon(PEG-IFN) and nucleoside/nucleotide analogues(NUCs)] because of the persistence of covalentlycircular closed DNA (cccDNA) in the nucleus ofhepatocytes [5, 6]. Moreover, the widely usedNUCs most likely have to be taken lifelong toprevent rebound [7], and while treatment withPEG-IFN does have a finite duration, theresponses are suboptimal at best [7]. Noveltherapeutic strategies are therefore needed.

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J. E. Arends (&) � F. I. LieveldDepartment of Internal Medicine, Section InfectiousDiseases, University Medical Center Utrecht,Utrecht, The Netherlandse-mail: j.e.arends@umcutrecht.nl

J. E. Arends � F. I. LieveldUtrecht University, Utrecht, The Netherlands

S. Ahmad � A. UstianowskiRegional Infectious Diseases Unit, NorthManchester General Hospital, Manchester, UK

Infect Dis Ther (2017) 6:461–476

DOI 10.1007/s40121-017-0173-y

With the recent revolutionary advances inthe treatment of viral diseases such as hepatitisC virus (HCV) infection (with direct-actingantivirals) and the development of multipleclasses of anti-human immunodeficiency virus(HIV) agents, the way may now be paved forsimilar advances in HBV therapeutics—withdirect-acting antivirals and host-directed thera-peutic strategies [8–10]. Improved understand-ing of the HBV life cycle has illustrated multiplepotential antiviral targets, and there are manyagents in preclinical and early clinical investi-gation, and other promising strategies for HBVclearance include immune modulators, whosepotential is strongly supported by the fact thatnatural immune responses are capable of effec-tively preventing chronic HBV infection in 90%of infected adults.

This review aims to give a comprehensiveoverview of the major new drug developments inthe treatment of HBV infection, specificallyfocusing on novel direct-acting antivirals andhost-directed anti-HBV therapeutics. This articleis based on previously conducted studies anddoes not involve any new studies of human oranimal subjects performed by any of the authors.

CURRENT TREATMENT OPTIONSFOR HBV INFECTION

The presently available antiviral treatments forHBV infection include two classes of therapeu-tic agents: PEF-IFN-a and NUCs. NUCs, whichtarget HBV through inhibition of viral poly-merase, are the most commonly used and havean excellent safety profile and tolerability. Mostpatients, however, require indefinite NUCtherapy because of frequent relapse or reactiva-tion of HBV infection after cessation of treat-ment, which is a hindrance for patienttreatment adherence [9]. In contrast, PEG-IFN-ahas predominant immunoregulatory effectsalong with limited direct antiviral properties[11, 12], and has a finite duration of therapyand a slightly higher occurrence of attaininganti-hepatitis B e antigen (HBeAg) and anti--HBsAg seroconversion than with NUCs [13, 14].However, interferon (IFN) sensitivity differsamong the different HBV genotypes, and the

main drawback of PEG-IFN-a is poor tolerabilitywith frequent severe side effects, which con-siderably limits its use. Neither NUCs norPEG-IFN-a is therefore optimal or results inlong-term control or cure except in a minorityof HBeAg-negative noncirrhotic individualswho experience durable HBsAg seroconversion,in which case therapy can be stopped underclose monitoring.

There has therefore been interest in whetherincreased efficacy could be obtained by combi-nation of these classes. Conceptually, combin-ing PEG-IFN-a and NUC therapy could result inimproved HBV control due to potential syner-gistic effects of their different mechanisms ofactions, and there has been interest in simulta-neous use (i.e., commencing both a NUC andPEG-IFN-a together) or sequentially or add-onadministration [15, 16]. Earlier studies intosimultaneous use of lamivudine with PEG-IFN-ashowed more pronounced on-treatment viro-logical response although without clear long--term benefit [14, 17]. Some newer studiesinvestigating PEG-IFN-a combined with teno-fovir disoproxil fumarate (TDF) describe a sig-nificantly larger proportion of participantsattaining HBsAg loss (9.1% vs 0% and 2.8%,respectively) and conversion to anti-HBsAgamong patients receiving TDF plus PEG-IFN-athan in those receiving either TDF or PEG-IFN-aalone [18]. Two recent studies analyzingPEG-IFN-a therapy as an add-on to long-termentecavir (ETV) therapy demonstrated higherHBeAg seroconversion rates in the combinationarm than in the ETV monotherapy arm [15, 16].Nonetheless, a third trial examining PEG-IFN-aadd-on therapy in patients receiving ETV didnot establish superiority in comparison with thePEG-IFN group or patients allocated to ETVadd-on treatment when receiving PEG-IFN-a[19]. A summary of recent published articles onthis subject is depicted in Table 1. Overall,recent evidence suggests that in patientsreceiving long-term NUC therapy, both anadd-on approach and sequential administrationof PEG-IFN-a may have some minor advantages[15, 16, 20]. However, such combination ther-apy appears unlikely to lead to a paradigm shiftor significant improvement for most HBV-in-fected individuals.

462 Infect Dis Ther (2017) 6:461–476

Table1

Summaryof

recent

mainrand

omized

controlledtrialsdemonstrating

hepatitisBvirus(H

BV)virologicalresponsesin

pegylatedinterferon

(PEG-IFN

)and

nucleoside/nucleotideanalogue

treatm

entstrategies

Stud

ySample

size

(n)

Patient

status

HBV

geno

type

Treatmentregimen

Follo

w-up

(weeks)

Virological

respon

se

HBeA

gconversion

(%)

HBsA

gconversion

(%)

HBV

DNA

supp

ression

(%)

Add-onstrategy

Brouw

eret

al.

[15]

(ARES

study)

175

HBeA

gpositive

A7%

B20%

C42%

D31%

1.ETVfor48

weeks

?PE

G-IFN

-a

inweeks

24–4

8

2.ETVfor48

weeks

9626

a

13

1 0

77(\

202IU

/

ml)

72

Xieet

al.[19]

218

HBeA

gpositive

Treatmentnaıve

HBVDNAC10

5 copies/ml

–1.

PEG-IFN

-afor

48weeks

2PE

G-IFN

-afor48

weeks

?ETVin

weeks

13–3

6

3.ETVfor24

weeks

?PE

G-IFN

-a

inweeks

21–6

8

72–9

231 25 26

1.4

4.1

1.4

40.3

(\10

3

copies/m

l)

31.5

37.0

Sequentialstrategy

Ninget

al.[16]

200

HBeA

gpositive

ETVfor9—

36months

HBVDNA\

104copies/m

l

–1.

PEG-IFN

-afor48

weeks

2.ETVfor48

weeks

4814.8a

6.1

4.3

0

72.0

(\10

3

copies/m

l)

97.8a

Piccoloet

al.[81]

30HBeA

gnegative

HBVDNAC

203copies/m

l

D87%

1.PE

G-IFN

-afor24

weeks

?LbT

in

weeks

25–4

8

2.LbT

for24

weeks

?PE

G-IFN

-a

inweeks

25–4

8

72–

0 0

13.3(\

203IU

/

ml)

46.7a

Infect Dis Ther (2017) 6:461–476 463

Table1

continued

Stud

ySample

size

(n)

Patient

status

HBV

geno

type

Treatmentregimen

Follo

w-up

(weeks)

Virological

respon

se

HBeA

gconversion

(%)

HBsA

gconversion

(%)

HBV

DNA

supp

ression

(%)

Concomitantstrategy

Liu

etal.[82]

61HBeA

gpositive

HBVDNAC

204copies/m

l

–1PE

G-IFN

-a?

ADF

for52

weeks

2PE

G-IFN

-afor52

weeks

5236.7

25.8

3.3

0

76.7a

(und

etectable)

29.0

Tangkijvanich

etal.[83]

125

HBeA

gnegative

Treatmentnaıve

HBVDNAC

203copies/m

l

B19.8%

C78.6%

Other

1.6%

1.PE

G-IFN

-a?

ETV

for48

weeks

2.PE

G-IFN

-afor48

weeks

96–

3.2

7.9

38.1(\

203IU

/

ml)

41.3

Marcellinet

al.

[84]

(stopped

becauseof

adverseevents)

159

HBeA

gpositive

Treatmentnaıve

A17%

B24%

C19%

D28%

Other

4%

Unknown

9%

1.PE

G-IFN

-a?

LbT

for24

weeks

2.LbT

for24

weeks

3.PE

G-IFN

-afor24

weeks

248 4 12

0 0 0

71c(\

302

copies/m

l)

35 7

464 Infect Dis Ther (2017) 6:461–476

Table1

continued

Stud

ySample

size

(n)

Patient

status

HBV

geno

type

Treatmentregimen

Follo

w-up

(weeks)

Virological

respon

se

HBeA

gconversion

(%)

HBsA

gconversion

(%)

HBV

DNA

supp

ression

(%)

Marcellinet

al.

[18]

740

HBeA

gpositive

ornegative

Treatmentnaıve

HBVDNAC

204copies/m

l

(HBeA

gpositive)or

C20

3

copies/m

l(H

BeA

g

negative)

A8%

B27%

C42%

D21%

E–H

1%

1.PE

G-IFN

-a?

TDF

forweeks

2.PE

G-IFN

-afor16

weeks

?TDFfor48

weeks

3.TDFfor120weeks

4.PE

G-IFN

-afor48

weeks

7225.0b

23.8

12.8

24.5

8.1

0.6

0 2.9

9.1(\

15IU

/

ml)

6.5

71.9d

9.2

PEG-IFN

-awas

administeredat

180mg/weekor

1.5lg/kg/w

eek.

ADFadefovir10

mg/day,ETVentecavir0.5mg/day,HBeAghepatitisBeantigen,HBsAghepatitisBsurfaceantigen,LdT

telbivudine600mg/day,TDFtenofovir

disoproxilfumarate300mg/day

aP\

0.05.

bP\

0.05

versus

arm

3.cP\

0.05

versus

arms2and3.

dP\

0.05

versus

arms1and2.

Infect Dis Ther (2017) 6:461–476 465

FOR WHAT SHOULD WE BEAIMING?

The presently available agents can suppressplasma viremia and may occasionally changethe plasma antigen profiles (HBeAg or HBsAgloss/seroconversion). They, however, have no(in the case of NUCs) or little (in the case ofPEG-IFN) impact on hepatocyte HBV DNAlevels, let alone in any way influence cccDNAlevels intrahepatically. Therefore, these agentscan control active disease, but do not lead to aradical virological cure [where all viral nucleicacid (i.e., cccDNA) is removed from the patient]or long-term control when the patient is notreceiving treatment in most individuals. Eventhose few individuals who currently naturally orpharmacologically clear circulating HBV infec-tion remain prone to reactivation/relapse ofHBV infection if they become significantlyimmunosuppressed.

Therefore, paradigm shifts are required tolead to either long-term off-treatment suppres-sion in most individuals or to complete radicalvirological cure. Can the newer treatmentsbeing investigated potentially lead to these endpoints as monotherapies or in combination?

NOVEL DIRECT-ACTINGANTIVIRALS FOR HBV

Very significant progress has been made inelucidating the life cycle of HBV and therebyidentifying potential targets and agents fortherapy. We will examine the major steps in thelife cycle, illustrating the main potential drugclasses and some of the leading contenders fordirect-acting antivirals (Fig. 1).

Binding and Attachment of HBVto the Hepatocyte

HBV attaches initially to heparan sulfate pro-teoglycans on the hepatocyte cell surface, andthen with high affinity binds its specific recep-tor—sodium–taurocholate cotransportingpolypeptide (NTCP) [21]. This latter step can betargeted with a specific synthetic lipopeptide,

myrcludex B, which has been shown to signifi-cantly inhibit viral NTCP binding and therebyreduce HBV viremia and serum surface antigenlevels in humans [22, 23]. To date little toxicityhas been demonstrated, although as the NTCPreceptor’s natural activity is bile acid trans-portation, it will be of interest to determine ifthere will be some pharmacokinetic interac-tions with drugs metabolized via this route.Although seemingly potent at inhibiting theinfection of uninfected hepatocytes, as amonotherapy it would not be expected to sig-nificantly influence hepatocyte HBV DNA levels(as these are replenished via pathways withinthe cell not requiring entry via NTCP binding;see later). It may well have an interesting role incombination, however, with other novel HBVagents and also in protecting the graft frominfection in the setting of liver transplant inHBV infection.

Entry into the Cytoplasm, NucleocapsidRelease, and Entry of DNAinto the Nucleus and Conversionto cccDNA

After fusion with the NTCP receptor, the virusenters the cytoplasm of the hepatocyte, isuncoated, and the HBV nucleic acid, in the formof relaxed circular DNA (rcDNA), leaves thenucleocapsid to subsequently enter the nucleus.Within the nucleus the rcDNA is converted intocccDNA. This is a vital step for the chronicnature of HBV infection as this cccDNA ishighly stable and acts as a long-lived templatefor subsequent transcription of HBV RNA andproteins. It is the cccDNA (which effectivelypersists as an HBV minichromosome not inte-grated into the host DNA) that allows reactiva-tion of apparently cleared HBV on significantimmunosuppression, and is the major obstacleto radical cure of HBV infection.

Multiple agents are in preclinical develop-ment to target this cccDNA moiety: from zincfinger nucleases to disubstituted sulfonamidecompounds, CRISPR–Cas9 technologies, andlymphotoxin beta receptor agonists [24–29].However, to date, it is RNA interference meth-ods that have entered human studies [30].

466 Infect Dis Ther (2017) 6:461–476

Mixtures of small interfering RNA moleculeshave shown activity in humans in decreasingserum surface antigen levels for prolongedperiods after single dosing [31]. They are beingdeveloped to be pangenotypic, and some com-pounds are being targeted specifically at hepa-tocytes by combination with with ligands suchas N-acetylgalactosamine, which promotesuptake via asialoglycoprotein receptor [32, 33].However, issues remain with the developmentof stable delivery systems and, to date, studieshave been small.

Transcription by cccDNA, Productionof Viral Proteins, and Capsid Assembly

The cccDNA serves as a template for the tran-scription of viral RNA of two types. The first isthe pregenomic RNA (pgRNA) that will producethe core and capsid proteins and also thenucleic acid template that ultimately enters thenew nucleocapsid and is converted into DNA,creating a new infectious virion. The second ismessenger RNA that is translated into the viralproteins—predominately the surface proteins(HBsAg and HBeAg) and X protein.

The capsid is created by multiple copies ofthe core protein combining. This step is one ofthe main targets of new HBV therapies, and

Table 2 Overview of ongoing clinical trials for new hep-atitis B virus therapeutics

Compound Phase

Entry inhibitors

Myrcludex B Phase I

RNA interference

ALN-HBV Phase I–II

ARC-520 Phase II

ARB-1467 Phase II

Lunar-HBV Preclinical

BB-HB-331 Preclinical

Ionis HBVRx (GSK3228836) Phase I

IONIS-HBVLRx (GSK33389404) Phase I

Capsid assembly modulators/core inhibitors

GLS-4 (morphothiadine mesilate) Phase II

NVR 3-778 Phase Ia

BAY41-4109 Phase I

JNJ56136379 Phase I

Core protein allosteric modifier Phase I

Nucleoside/nucleotide analogues

AGX-1009 (prodrug of tenofovir) Phase III

LB80380 (besifovir) Phase III

CMX-157 (prodrug of tenofovir) Phase IIa

Surface antigen/release inhibitors

REP 2139 and REP 2165 Phase II

RO7020322 (RG7834) Phase I

GC 1102 Phase II

Therapeutic vaccines

GS-4774 (recombinant antigen containing

X, Env, core epitopes)

Phase II

ABX-203 (recombinant antigen containing

HBsAg and HBcAg)

Phase II

TG-1050 (nonreplicative adenovirus

encoding a large fusion protein (truncated

core, modified Pol, and 2 Env domains)

Phase I

Table 2 continued

Compound Phase

INO-1800 (DNA plasmids encoding HBsAg

and HBcAg)

Phase I

FP-02.2 (HepTcell) (peptide encoding

CD4? and CD8? epitopes)

Phase I

Innate immune defense pathway

GS 9620 Phase II

RO6864018 (RG7795, ANA773) Phase II

SB9200 Phase II

This table provides a current overview of compounds inclinical development to the best of our knowledge.HBcAg hepatitis B core antigen, HBsAg hepatitis B surfaceantigen

Infect Dis Ther (2017) 6:461–476 467

current capsid assembly modulators/core inhi-bitors generally form one of two main classes.The first class contains compounds that pro-mote capsid assembly but inhibit the entry ofpgRNA into the immature nucleocapsid. Thisresults in nucleocapsids that appear to be nor-mal in geometry and size but are empty ofnucleic acid and therefore noninfectious. Suchcompounds include phenylpropenamides andsulfamoylbenzamide derivatives [34, 35]. Theother class contains compounds that directlyinhibit the correct formation of the nucleocap-sid itself, resulting in virus particles that aredeformed with abnormal structure and size andappear noninfectious. Examples include theheteroaryldihydropyrimidines (e.g.,BAY41-4109) [36] and NVR 3-778 [37, 38].

The activities of a further viral protein, theX protein, remain poorly defined. It appears tohave a role in inhibiting the SMC5–6 complexand thereby promoting productive HBV geneexpression [39]. Some preclinical work is ongo-ing at targeting this protein.

Nucleocapsid Coating and Conversionof pgRNA to rcDNA

HBV surface proteins (which have been pro-cessed within the host Golgi apparatus) nowcoat the nucleocapsid, and within this structurethe pgRNA is converted by HBV polymerase torcDNA. This latter step is the point of action ofthe currently available NUCs. There are newNUCs in development (e.g., besifovir, CMX 157,AGX-1009, and MIV-210) but it is currentlyunclear what advantages they will have com-pared with the existing agents [31, 40, 41].

Surface Protein Secretionfrom the Hepatocyte

It has long been recognized that large quantitiesof viral proteins, especially HBsAg, are secretedfrom the hepatocyte unassociated with virions.It is thought that this extra protein acts toabsorb potentially neutralizing antibodies inthe host plasma and also to induce a state of

Fig. 1 Life cycle of hepatitis B virus within the hepatocyte.cccDNA covalently closed circular DNA, ER endoplasmicreticulum, NTCP sodium–taurocholate cotransporting

polypeptide, pgRNA pregenomic RNA, rcDNA relaxedcircular DNA

468 Infect Dis Ther (2017) 6:461–476

immune exhaustion and tolerance to the virus.HBsAg release inhibitors have been developed(e.g., REP 2139 and REP 2165) that appearpotent in preventing the release of HBsAg inhumans and thereby decreasing serum HBsAglevels and also potentially promoting surfaceantibody seroconversion [42, 43]. Whetherthese compounds may cause detrimental intra-hepatocyte accumulation of HBsAg is still to bedetermined.

Release of Virions and IntrahepatocytecccDNA Replenishment

Once coated in surface protein, the virions arereleased from the hepatocyte to infect new cells.But not all nucleocapsids are released, withsome diverted to replenish the nuclear cccDNAcompartment. Some agents that affect the for-mation of nucleocapsids, such as the capsidassembly inhibitor JNJ-379, have been shown toultimately decrease cccDNA levels. Berke et al.[35] suggested that this is presumably by pre-venting the cccDNA replenishment cyclethrough blocking of pgRNA synthesis.

Therefore, multiple potential therapeutictargets have become apparent as a result ofimproved understanding of the cellular lifecycle of HBV (Table 2). It is quite probable,however, that none of these agents asmonotherapies will result in radical cure of HBVinfection, and combination direct-actingantivirals may be required. Even with suchcombinations it may well be that host-directedtherapies will also be required to reverseimmune exhaustion and tolerance, and there-fore promote viral clearance and cure.

NOVEL IMMUNOLOGICALTARGETS FOR HBV THERAPY

Cellular Immune Response

The human cellular immune response plays akey role in attaining immune control and viralclearance after HBV infection [44, 45]. Cyto-toxic T lymphocytes (CTLs) have been identi-fied to be the major contributing factor for HBV

clearance [46]. Two signal types are mandatoryto activate T cells: interaction between T-cellreceptors and antigens presented by major his-tocompatibility complex molecules on anti-gen-presenting cells (APCs) on the one hand,and interaction between co-stimulatory orco-inhibitory receptors on T cells with theirligands on APCs on the other [47, 48]. The lattermolecules can amplify or inhibit active immuneresponses and are called ‘‘immune checkpoints’’[49]. Immune checkpoints are physiologicallynecessary for maintaining self-tolerance andminimizing collateral host tissue destruction[50].

As a consequence of persistently high anti-gen levels in chronic viral infections, CTLs andCD4? cells have been observed to becomefunctionally exhausted [51–53]. This was firstdiscovered in mice infected with lymphocyticchoriomeningitis virus infected that exhibitedCTLs with reduced capability to kill infectedcells and reduced cytokine secretion despitepersisting indefinitely [54, 55]. To date, T-cellexhaustion, classified by the overexpression ofinhibitory receptors such as programmeddeath 1 (PD1), CTL-associated antigen 4, andlymphocyte activation gene 3 protein, is ahallmark of chronic viral infection and has beenobserved in infections with HIV, HCV, humanT-cell lymphotropic virus, and HBV [56, 57].

In HBV, enhanced PD1 expression by CTLsand CD4? cells has been demonstrated inmouse models of persistent HBV infection [54]and on human HBV-specific exhausted CTLs inchronic HBV infection [58].

Therefore, targeting these inhibitory recep-tors and thereby reversing CTL responses (i.e.,rescuing exhausted T cells) is one of the thera-peutic strategies currently being explored(Table 3). For example, in chronic infectionwith woodchuck hepatitis virus (WHV; a virusclosely related to HBV), infected woodchuckswere treated with a combination of ETV, ther-apeutic DNA vaccination, and PD1 ligand 1(PD-L1) antibodies, which led to sustainedimmune control of the infection and even viralclearance in some woodchucks [57]. In addi-tion, an ex vivo study using intrahepatic andperipheral T cells from patients with chronicHBV or HCV infection observed increased

Infect Dis Ther (2017) 6:461–476 469

cytokine secretion of HBV-specific T cells in thepresence of APCs treated with anti-PD-L1 incombination with stimulation of the co-stimu-latory receptor CD137 [59].

However, some important points should betaken into account. First, sufficient presence ofHBV-specific exhausted T cells may be crucial tothe effectiveness of these drugs [38]. As such itmay prove to be difficult to determine whichpatients should be treated with immunecheckpoint inhibitors. Second, longer infectionduration with excessive antigen exposure maylead to the irreversible exhaustion of T cells[52, 60]. Combined treatment with otherantiviral drugs may thus be necessary to bringdown antigen levels before initiation ofimmunotherapy [39]. Lastly, because of themechanisms of action of immune checkpointinhibitors, important immune-related adverseevents have been observed in trials withCTL-associated antigen 4, PD1, and PD-L1

antibodies, affecting several organ systems,including the liver [51, 61–63].

Toll-like Receptor 7 and Toll-like Receptor8 Agonists

Another HBV strategy, targeting the innateimmune system, is to activate Toll-like receptors(TLRs) as activation of virus-specific TLRs leadsto production of type I IFNs (mainly IFN-a andIFN-b) [54]. IFN-b in particular is capable ofinhibiting HBV replication through destabiliza-tion of pgRNA capsids and interfering with theirassembly. TLR7—present mainly in theendolysosomal compartment of plasmacytoiddendritic cells and B cells—can induce intra-hepatic type I IFN responses without causingsystemic harmful symptoms. As an activator ofthe innate immune response in the liver, itbecame a major focus in TLR agonist trialsfocusing on viral clearance of HBV [64].

Table 3 Summary of immune checkpoint inhibitors for targets of potential interest for hepatitis B virus immunotherapy

Target Target function Binding partner Drug Indication Phase

CTLA4 Inhibitory receptor CD80, CD86 Ipilimumab Melanoma

Multiple malignancies

FDA approved

Phase II/phase III

Tremelimumab Malignant mesothelioma FDA approved

PD1 Inhibitory receptor PD-L1, PD-L2 Pembrolizumab

Nivolumab

Pidilizumab

AMP-224

MDX-1106

Melanoma

Melanoma

Diffuse large B-cell lymphoma

Colorectal cancer

Hepatitis C virus

Multiple malignancies

FDA approved

FDA approved

Phase II

Phase I

Phase I

PD-L1 PD1 ligand PD-1 Avelumab

BMS-936559

MPDL33280A

MEDI4736

Multiple malignancies

HIV-1, multiple malignancies

Multiple malignancies

Multiple malignancies

Phase II

Phase I

Phase I

Phase I

CD137 Stimulatory receptor CD137L BMS-663513

PF-05082566

Solid tumors

Lymphoma

Phase I/II

Phase I

CD137L CD137 ligand, CTLA4 cytotoxic T lymphocyte associated antigen 4, HIV-1 human immunodeficiencyvirus 1PD1 programmed death 1, PD-L1 programmed death 1 ligand 1, PD-L2 programmed death 1 ligand 2

470 Infect Dis Ther (2017) 6:461–476

GS-9620, an oral TLR7 agonist, was demon-strated in animal studies to activate expressionof IFN-stimulating genes, while early humanstudies showed transient increases in the levelsof IFN-c-induced protein 10 (also known asIP-10) 8 h after GS-9620 administration, sug-gesting an IFN-y response [65, 66]. In a recentphase II trial, three different lengths of GS-9620dosing (4, 8, and 12 weeks) were investigated in156 chronic virally suppressed HBV infectedpatients [67]. Within each cohort, patients wererandomized to receive three different doses (1,2, or 4 mg). Although GS-9620 administrationwas safe and well tolerated, there was no sig-nificant decline in HBsAg levels. Additionalin-depth analysis of HBV-specific T-cell andnatural killer cell responses showed improvedinterplay between the two cell types, togetherwith a transient improvement of T-cell response[68]. This was most notable for the highest doseof GS-9620 (4 mg).

These somewhat disappointing results are incontrast to the results of earlier animal studiesin both chimpanzees and woodchucks in whichGS-9620 was demonstrated to induce rapidsustained reduction in serum and liver HBVDNA levels together with loss of woodchuckhepatitis surface antigen (WHsAg) in wood-chucks with chronic WHV infection [69, 70].One possible explanation could be the differ-ence in dosing—from 1 to 2 mg/kg in animaltrials to 1–4 mg per patient in human studies.However, further research into this TLR7 ago-nist was subsequently discontinued.

TLR8-mediated recognition has been associ-ated with viral infections. A first demonstrationof its possible role in HBV infection came froman in vitro study by Jo et al. [71] demonstratingdetectable intrahepatic IFN-c production onstimulation of mononuclear cells by a TLR8agonist [71]. Moreover, TLR8 expression andfunction was impaired in peripheral bloodmononuclear cells from patients with chronicHBV infection compared with healthy controls[72]. When these HBeAg-negative patients weretreated with a 48-week course of PEG-IFN-a,TLR8 messenger RNA level could discriminatebetween those who achieved a completeresponse and those who did not. This has led to

a number of drugs in development targetingTLR8.

HBV Therapeutic Vaccines

The concept behind therapeutic vaccines is thegeneration of new HBV-specific T cells capableof controlling chronic HBV infection. HBVtherapeutic vaccines have been shown torestore both T-cell responses and IFN-c produc-tion [73]. In patients with high levels of virus inthe blood, HBV-specific CD8 T cells have beenshown to be dysfunctional although they arenecessary for future viral control [58, 74, 75].Therefore, in the setting of effective oralanti-HBV therapy with NUCs, therapeutic vac-cines may have a role in the restoration of T-cellcontrol.

The first HBV therapeutic vaccine to bestudied in HBV-infected humans was GS-4774, ayeast-based T-cell vaccine containing HBV core,surface, and X proteins that has been shown tobe immunogenic in mouse models and healthyvolunteers. In a small phase II study the effectsof GS-4774 were investigated on HBV-specificT-cell responses in 12 naıve HBeAg-negativegenotype D patients with chronic HBV infec-tion [76]. They received six consecutivemonthly doses of the vaccine in combinationwith TDF. Although T-cell function improved,mostly an effect on CD8 cells, it was insufficientto induce a substantial decline of HBsAg levels.Multiple other vaccines are currently in devel-opment (early phase). These vaccines includevaccines targeting the preS1 domain [77] andthe immune-stimulant vaccine ABX203 [78].

It might be that a combination of theaforementioned strategies (directed at reversalof T-cell exhaustion and generation of new T--cell responses) in patients with HBV infectionstably suppressed with NUCs might be a wayforward. A recent study in WHV-infectedwoodchucks investigated such a triple therapycombination, consisting of therapeutic vaccine(i.e., DNA plasmids expressing woodchuckhepatitis core antigen and WHsAg), theimmune checkpoint inhibitor anti-PD-L1, andETV. Sustained immunological control with thedevelopment of antibodies against WHsAg and

Infect Dis Ther (2017) 6:461–476 471

even viral clearance in some woodchucks wasachieved [79]. Recently, a phase I trial evaluat-ing the efficacy of anti-PD1 treatment with orwithout GS-4774 in HBeAg-negative chronichepatitis B patients was published [80]. A singledose of anti-PD1 resulted in a significant declinein HBsAg levels, but with no apparent addedbenefit of GS-4774.

CONCLUSIONS

Although treatment with the current nucle-oside/nucleotide inhibitors is very successful insuppressing HBV DNA to undetectable levels,functional let alone sterilizing cures are notwithin reach for the vast majority of HBV-in-fected patients. New exciting advances have ledto new compounds targeting multiple steps inthe viral life cycle as well as approaches toattenuate virus-induced immune dysregulation.Further research into these agents remainsproblematic however, with key issues needingto be resolved. First, firm therapy-related endpoints of cure need to be establish. Second, it islikely that combination therapies will berequired, and it is currently hard to envisagehow the most promising combination of drugtargets can be determined. Thereafter, however,the future looks bright for patients with chronicHBV infection.

ACKNOWLEDGEMENTS

No funding or sponsorship was received for thisstudy or publication of this article. All namedauthors meet the International Committee ofMedical Journal Editors criteria for authorshipfor the manuscript, take responsibility for theintegrity of the work as a whole, and have givenfinal approval for the version to be published.

Disclosures. Joop Arends provides advisoryservices for MSD, Abbvie, BMS, Janssen, Gileadand Viiv. Joop Arends has also received researchgrants from MSD, ViiV, BMS and Abbvie.Andrew Ustianowski provides advisory servicesfor Abbvie, Gilead, Janssen, MSD, ViiV. Faydra

Lieveld and Shazaad Ahmad have nothing todisclose.

Compliance with Ethics Guidelines. Thisarticle is based on previously conducted studies,and does not involve any new studies of humanor animal subjects performed by any of theauthors.

Open Access. This article is distributedunder the terms of the Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommer-cial use, distribution, and reproduction in anymedium, provided you give appropriate creditto the original author(s) and the source, providea link to the Creative Commons license, andindicate if changes were made.

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