HBV cure: why, how, when?

HBV cure: why, how, when?Massimo Levrero1,2,3,4,5, Barbara Testoni1,2 andFabien Zoulim1,2,3,6,7

Available online at www.sciencedirect.com

ScienceDirect

Current HBV treatments control replication and liver disease

progression in the vast majority of treated patients. However,

HBV patients often require lifelong therapies due to the

persistence of transcriptionally active viral cccDNA mini-

chromosome in the nucleus, which is not directly targeted by

current antiviral therapies. A true complete cure of HBV would

require clearance of intranuclear cccDNA from all infected

hepatocytes. An intermediate but still relevant step forward that

would allow treatment cessation would be reaching a functional

cure, equivalent to resolved acute infection, with a durable

HBsAg loss � anti-HBs seroconversion, undetectable serum

DNA and persistence of cccDNA in a transcriptionally inactive

status. Recent advances in technologies and pharmaceutical

sciences, including the cloning of the mayor HBV receptor (i.e.

the NTCP transporter) and the development in vitro HBV

infection models, have heralded a new horizon of innovative

antiviral and immune-therapeutic approaches.

Addresses1 Cancer Research Center of Lyon (CRCL), Lyon 69008, France2 INSERM, U1052, Lyon 69003, France3 Hospices Civils de Lyon (HCL), 69002 Lyon, France4 Department of Internal Medicine – DMISM, Sapienza University,

00161 Rome, Italy5 CLNS@SAPIENZA, Istituto Italiano di Tecnologia (IIT), 00161 Rome,

Italy6 University of Lyon, UMR_S1052, UCBL, 69008 Lyon, France7 Institut Universitaire de France (IUF), 75005 Paris, France

Corresponding author: Levrero, Massimo ([email protected])

Current Opinion in Virology 2016, 18:135–143

This review comes from a themed issue on Antiviral strategies

Edited by Raymond F Schinazi and Erik de Clercq

For a complete overview see the Issue and the Editorial

Available online 19th July 2016

http://dx.doi.org/10.1016/j.coviro.2016.06.003

1879-6257/Published by Elsevier B.V.

IntroductionCurrent treatments for chronic hepatitis B (CHB) (see the

companion paper by Zoulim et al. [1]) are able to control

viral replication and liver disease progression in the vast

majority of treated patients. Although these improve-

ments have radically changed the management and out-

come of individual CHB patients the global control HBV

infection is still a largely unattained goal. Due to the

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inability of all available treatments to eliminate HBV

infected hepatocytes, there is no cure for HBV infection,

lifelong therapies are often required and very few patients

maintain a sustained viral and clinical remission off-ther-

apy [2]. The persistence of the virus in most patients

under treatment also contributes to the substantial resid-

ual risk to develop an hepatocellular carcinoma (HCC)

[3]. The suboptimal to very low access to anti-HBV

therapies and to the HBV prophylactic vaccine in many

highly endemic regions, together with the partial effect of

treatments on HCC development, concur to explain why

still more than 240 million people worldwide are chroni-

cally infected with HBV and up to a million patients die

every year from HBV-related liver cancer and end stage

liver disease [4]. Despite all these limitations, the avail-

ability of HBV prophylactic vaccines and the potential of

anti-HBV therapies to control HBV transmission make

HBV eradication, in principle, a reachable objective. The

lack of a cure for chronic HBV is due in part to the

continued presence of transcriptionally active cccDNA

mini-chromosome in the nucleus, which is not directly

targeted by current antiviral therapies, and by the pro-

found depression of both intrahepatic innate immunity

and adaptive HBV specific T cell and B responses [2].

Recent advances in understanding the molecular biology

and replication cycle of HBV, including the cloning of the

mayor HBV receptor (i.e. the NTCP transporter) and the

development of in vitro HBV infection models [5], have

provided new insight on potential future targets for drug

development and boosted the efforts to develop innova-

tive antiviral and immune-therapeutic approaches.

Targeting the HBV replication cycleHBV entry into hepatocytes is initiated by the low-affini-

ty interaction between the antigenic loop (‘a’ determi-

nant) of the HBV envelope proteins with heparan sulfate

proteoglycans on the hepatocytes surface [6,7�], followed

by the high-affinity interaction of the myristoylated pre-

S1 domain of the HBV large envelope protein with the

liver-specific receptor sodium/taurocholate cotransporter

(NTCP) [8�,9], the fusion of HBV particles with the cell

membrane mediated by receptor-mediated endocytosis

[10,11] and the release of the HBV genome — containing

nucleocapsids into the cells. Nucleocapsids transport into

the nucleus probably involves microtubules and the nu-

clear import in machinery [12]. In the nucleus, the relaxed

circular, partially double-stranded HBV genome is

repaired to a full-length, circular DNA by the covalently

attached viral polymerase (P) and still poorly understood

cellular factors involving the topoisomerases and DNA


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136 Antiviral strategies

repair pathways. The tyrosyl DNA phosphodiesterase

TDP2 mediates the first step of cccDNA formation from

incoming rcDNA (i.e. removal of the phosphodiester

bond between the viral polymerase and viral minus strand

DNA) [13] but its role in the formation of active cccDNA

precursors has been challenged [14]. The circularized

protein-free genome then complexes with host histone

and non-histone proteins including various histone-mod-

ifying enzymes into a minichromosome that functions as

the template for the transcription of the three classes of

HBV RNAs [i.e. genome-length RNAs (pregenomic and

precore RNAs coding for c gene products and P protein),

S RNAs (S proteins), and X RNA (HBx protein)].

cccDNA transcriptional activity is regulated by epigenet-

ic modifications and specific host transcriptional factors

[15�]. The HBV core and X proteins are also present on

the minichromosome and HBx play an important role in

HBV transcription [16,17,18�]. The pregenomic RNA

transcript is reverse-transcribed by the P protein to re-

laxed circular DNA in the core-containing nucleocapsid.

The nucleocapsid can either assemble into an infectious

virion with the envelope proteins through the multivesi-

cular body pathway [19] or recycle back to the nucleus

[20] to amplify/replenish the cccDNA pool (1–10 mole-

cules for infected hepatocyte) [21].

Defective HBV specific immune responsesAcute HBV infections are cleared by a coordinated acti-

vation of innate and adaptive immune responses in about

95% of immune-competent adults but only in 5–10% of

children. In acute self-limited infections, HBV replication

is suppressed by non-cytopathic mechanisms involving

cytokines [22] well before the onset, usually delayed by

4–6 weeks post infection, of the strong polyclonal CD8+

T and CD4+ T cell responses that are thought to be

instrumental for the elimination of infected hepatocytes,

antibody production and clinical recovery [23]. During

chronic HBV infection, the unrestricted viral replication

and the ongoing liver damage are accompanied by the

accumulation of non HBV-specific T cells in the liver,

whereas HBV-specific CD8+ T cells are found at very low

frequency and have an exhausted phenotype character-

ized by over-expression of inhibitory checkpoint mole-

cules such as PD-1, CTLA-4, CD244, and Tim3. CD4+ T

cell responses are also affected with an impaired produc-

tion of IL-2 and low proliferation potential and Treg and

IL-10 secreting T cells accumulate in the liver [24]. In

keeping with the notion that the HBV viral structures

(double strand viral double strand DNA and RNA–DNA

hybrids) that are potentially capable to stimulate the

innate immunity sensors, are sequestered into the cyto-

plasmic capsid particles [25] and the lack of intrahepatic

gene modulation following the onset of viremia in HBV-

infected chimpanzees [26] HBV has been considered as a

‘stealth’ virus. More recent data have challenged this

notion suggesting that the virus is detected by the innate

immunity sensors but it rapidly blocks innate immunity at


several levels in hepatocytes as well as in other liver cells

(for review, see [27,28]).

Definition of HBV cureAlthough the term cure is being increasingly used in HBV

infected patients, several concepts around the definition

of a cure of HBV infection need to be clarified.

True complete cure of HBV would require the clearance of

intra-nuclear cccDNA from all infected hepatocytes,

thereby allowing treatment cessation and preventing

the risk of reactivation in case of a loss of immune control

[2]. The requirement to eliminate an intranuclear reser-

voir is a clear distinction from HCV infection, where the

entire replication cycle occurs within the cytoplasm. As an

intermediate but still relevant step toward HBV cure there

is consensus on aiming at reaching a functional cure,

equivalent to a resolved acute infection: durable HBsAg

loss (with or without anti-HBs seroconversion), undetect-

able serum DNA, persistence of cccDNA in a transcrip-

tionally inactive status and the absence of spontaneous

relapse after treatment cessation [2]. According to this

definition functional cure equals the natural condition

known as occult HBV infection. Due to the lack of solid

data regarding the off therapy stability of viral suppression,

patients treated with current anti-viral treatments who

reach and maintain low serum HBsAg levels, equivalent to

those observed in HBV inactive carriers, are not included in

the definition of functional cure. These definitions do not

take into account the possible presence and persistence of

integrated HBV DNA in the host genome. The integra-

tion of viral DNA into the host genome, that occurs

randomly in regenerating infected hepatocytes [29], does

not contribute to HBV replication but is an important

factor in viral pathogenesis both by cis-acting mechanisms

(insertional mutagenesis) and by the continuous expres-

sion of trans-acting wild type and truncated HBx or

truncated pre-S/S polypeptides bearing enhanced trans-

forming properties [3]. Clonal expansions of hepatocytes

containing unique virus-cell DNA junctions formed by

the integration of HBV DNA can be detected in patients at

various stages of chronic infection [30]. Whether these

integrants must be eliminated to prevent all HBV-related

pathogenicity in the absence of other liver co-morbidities

is still debated. Terms like HBV eradicating or sterilizingtherapies, although evocative, should be avoided.

Current antiviral therapies for chronic HBV disease is

limited to direct acting antivirals (DAAs) that target the

viral DNA polymerase, the most efficacious of which are

the nucles(t)ides analogs (NUCs) entecavir and tenofovir,

and interferon [namely the long acting pegilated interfer-

on (PEG-IFN)] [31]. DAAs act directly on viral replication

by inhibiting the reverse transcription of pre-genomic

RNA to DNA and the subsequent replication of the (+)

strand of HBV DNA [29]. Interferon up-regulates a range

of antiviral ‘interferon stimulated genes’ (ISGs) to modify


HBV cure Levrero, Testoni and Zoulim 137

the cccDNA epigenome, control viral replication and

stimulate NK cell activity with little or no effect on

adaptive HBV specific T cell responses [32,33,34�]. The

efficacy of interferon therapy is HBV genotype depen-

dent, working best for genotypes A and B and less well in

genotypes C and D, and is far from satisfactory, leading to

HBsAg clearance only in a minority of patients. Altogether

these treatments are not curative, since they do not

directly target cccDNA, although it has been recently

shown, by us and by others, that interferon treatment

diminish cccDNA transcriptional activity [34�,35,36], a

property that may explain its ability to lower serum HBsAg

levels and to induce HBsAg loss in a proportion of patients.

IFNa also induces, at least in some cell culture systems, a

partial loss of cccDNA [37�,38].

Strategies for HBV cureSeveral strategies targeting all steps of the HBV replica-

tion cycle (HBV entry, HBV cccDNA production and

Figure 1

Polymerase inhibitors

• Nucleoside analogues• Non-nucleosides

Targeting cccDNA

Entry inhibitors• Lipopeptides, e.g.

Myrcludex-B• Cyclosporin derivatives

Inhibitors of HBsAg releaseReplicor

“exaustion”“high antigen load”

PD-1

CD8+ T cell

DysfunctionalT-cell response

nuclear cccDNA

Integrated

HBV DNA

immatureRNA

nucleocapsid

matureRC-DNAnucleo-capsid

envelopedRC-DNA

virion

The landscape of HBV cure efforts. All steps of the HBV replication cycle (v

mRNAs, envelope protein secretion, core proteins and the capsid, Pol enzy

deficient adaptive HBV specific B and T cell responses are currently being


activity, viral replication and viral proteins expression)

and/or aiming at stimulating the intrahepatic innate im-

mune responses or restoring the deficient adaptive HBV

specific B and T cell responses are currently being ex-

plored (Figure 1). Mechanistically all these different

approaches can be classified in 4 functional categories

according to their mode of action and their objective or

targets (Figure 2):

(a) complete inhibition of HBV replication;

(b) restoration of host innate immunity and adaptive anti-

HBV T and B cell responses;

(c) selective sensitization of HBV infected hepatocytes

to immune elimination;

(d) direct targeting of cccDNA.

Notably, some compounds or classes of compounds may

fit in more that one category (Figure 2).

Immune modulation

• Toll-like receptors agonists ,Gilead, Roche

• Anti-PD-1 mAbBMS, Merck

• Vaccine therapy: Transgene, Gilead, Roche Innovio, Medimmune, ITS

RNA interference,Arrowhead, Tekmira, Alnylam, GSK

Capsid inhibitorsNovira, Janssen, Abbvie

AssemblyPHARMA, Gilead,

plasma

membrane

Insufficient B-cell response

B cell

subgenomic RNAs

pgRNA

capcap

cap

cap

pApA

pA

pA

core protein+ P protein

Current Opinion in Virology

iral entry, cccDNA formation, chromatinization and transcription, viral

matic activities), the intrahepatic innate immune responses and the

explored as potential new therapeutic targets.



Figure 2

• Complete inhibition of HBV replication [1] [including entry inhibitors, capsid inbibitors, NAPs, si/shRNA approaches]

- avoid new hepatocytes infection and low level core particles recycling

• Restoration of host innate/adaptive antiviral immunity against HBV [2] - reduce HBs load [si/shRNA approaches; NAPs] - checkpoint inhibitors [anti-PD1/PDL1 …. others] - TCR engineering - Therapeutic vaccines [GS-4774, TG1050 …. ] - TLRs agonists [TLR7 …. others]

• Sensibilization to iimmune elimination of HBV infected hepatocytes [3] - cIAP inhibitor / Smac agonist Birinapant

• Direct targeting of cccDNA [4] - inhibit cccDNA formation - target cccDNA with endonucleases - transcriptional silencing of cccDNA - cccDNA bound viral proteins [HBc and HBx]

HBV cureStrategies

Immunesystem

CD8+ (CD4+)T cell

B cell

[2][3]

[4]

[1]Viral

targets

Integrated

HBV DNA

matureRC-DNAnucleo-capsid immature

RNAnucleocapsid

core protein+ P protein

cap pA

pAcapcap

capsubgenomic RNAsnuclear cccDNA

pApA

pgRNA

Current Opinion in Virology

HBV cure strategies classification according to their mode of action. The different approaches aiming at ‘HBV cure’ can be classified in

4 functional categories according to their mode of action and intended objective. Some compounds or classes of compounds may fit in more that

one category. (1) A complete inhibition of HBV replication can be achieved by entry inhibitors, capsid inhibitors, nucleic acids polymers (NAPs), si/

shRNAs, small molecules inhibiting the DNA polymerase/reverse transcriptase RNAse H activities of Pol). The aim is to avoid the de-novo infection

of uninfected hepatocytes and a complete blockade of the replenishment of the cccDNA pool through the recycling of mature core particles into

the nucleus, (2) the restoration of the defective host innate immunity and adaptive anti-HBV T and B cell responses can potentially be achieved

by: (a) si/shRNA approaches or NAPs to reduction HBsAg; (b) the anti-PD1/PDL1 or other checkpoint inhibitors; (c) therapeutic vaccines; (d)

transfer of engineered TCR bearing CD8 cells; (e) TLRs agonists; (3) a selective sensitization of HBV infected hepatocytes to immune elimination

using cIAP inhibitors/Smac agonists, (4) the direct targeting of cccDNA is pursued by multiple approaches aiming to: (a) inhibit cccDNA formation;

(b) destroy cccDNA molecules by targeted endonucleases; (c) silence the viral mini-chromosome by targeting the epigenetic control of cccDNA

transcription; (d) interfere with the activity of cccDNA bound viral proteins [HBc and HBx].

Complete inhibition of HBV replicationComplete suppression of the HBV polymerase should, in

theory, reduce viremia and intrahepatic levels of replica-

tive forms of HBV DNA to zero, and even cccDNA

should be eliminated, as the infected cells are eventually

replaced. According to this model, people with CHB

should be cured with DAAs alone if treated long enough

unless, as suggested by some recent observations [39],

despite the persistent negativity of serum HBV DNA, a

complete inhibition of the HBV polymerase is not

achieved. If the degree of suppression of viral replication


in infected hepatocytes is not complete new hepatocytes

infection and a low level of core particles recycling to the

nucleus will occur. In addition to new Pol and RNAseH

inhibitors, entry inhibitors, capsid inhibitors, NAPs and

si/shRNA-based approaches have all the potential to

cooperate with NUCs to achieve a complete inhibition

of HBV replication.

(a) Entry inhibitors that target the NTCP receptor prevent

the de novo infection of hepatocytes by HBV and HDV as

well as viral spreading from infected human hepatocytes



[5,8�,9,40]. The most prominent representative of this

category is the HBV ‘entry inhibitor’ Myrcludex B, which

is a myristlyated PreS1 peptide currently under clinical

trial [5].

(b) Anti-capsid inhibitors. The HBV capsid is essential for

HBV genome packaging, reverse transcription, intracel-

lular trafficking and the re-import of encapsidated HBV

genomes into the nucleus [41]. Several non-nucleoside

analog (NNA) inhibitors of pgRNA packaging and HBV

capsid assembly have been identified [41]. HBV capsid

proteins (HBV core/HBc) can also traffic to the nucleus of

infected cells where they bind the cccDNA mini-chro-

mosome [17] and the regulatory regions of a subset of

cellular genes [42]. Compounds that would also target the

nuclear functions of HBV core proteins, that is, regulation

of cccDNA transcriptional activity [43] and/or regulation

of ISGs expression, would have the potential for an

enhanced antiviral activity. At least two chemical classes

of anti-capsid drugs are in phase I and phase II clinical

assays [44].

Restoration of host innate immunity andadaptive anti-HBV T and B cell responsesThe key challenge for all immunomodulatory strategies

will be to stimulate antiviral immuno-mediated pathways

without triggering detrimental anti-HBV flares.

Two main approaches have been developed to inhibitHBV gene expression at the post-transcriptional level in

order to reduce the excess production of sub-viral parti-

cles and, by relieving HBsAg-mediated immunosuppres-

sion, restore antiviral immunity:

a) RNA interference. RNA interference (RNAi) are in-

creasingly considered as a realistic new therapy for chron-

ic HBV, with recent clinical studies showing liver-specific

knockdown of HBV replication and protein expression

[45–48]. Their potential effect on immune restoration by

decreasing viral antigen load is actively investigated.

b) Nucleic acid polymers (NAPs) are sequence-independent

phosphoro-thioated oligonucleotides that display a broad

spectrum of antiviral activity against several enveloped

viruses, including HBV. REP9-AC (REP 2055), a 40-

nucleotide poly-cytidine amphipathic DNA polymer,

inhibits both HBV entry and HBsAg release from infected

hepatocytes. REP 2139 in combination therapy with

PEG-IFN alpha-2a is in clinical trial for both in chronic

HBV and HDV co-infection [49,50].

Check-point inhibitors. The exhausted HBV-specific CD8+

T cells in CHB patients express high levels of inhibitory

molecules including programmed cell death protein 1

(PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4),

lymphocyte activation gene 3 (LAG-3), T cell membrane

3 (Tim-3) and CD244 (2B4) [24]. Inhibitors of these


checkpoint molecules have a potent immunostimulatory

effect by rescuing virus specific CD8+ T cells [51–53].

Nivolumab (BMS-936558) and pembrolizumab (MK-

3475) are both anti-PD-1 monoclonal antibodies that have

progressed through phase 3 development in oncology.

Phase 1 studies of anti-PD-1 therapies for the treatment

of CHB patients with HCC have just started [54].

T-cell therapies. Engineering HBV-specific T cells

through transfer of HBV-specific T cell receptor or

HBV-specific chimeric antigen receptors represent prom-

ising alternative strategies to construct an HBV-specific

T-cell immunity in CHB patients [55]. Their potential is

supported by preclinical data and one patient case report

[56,57�].

Therapeutic vaccines. Attempt to break T cell tolerance to

HBV proteins (HBsAg, HBcAg) and stimulate HBV-spe-

cific T cell immunity in patients chronically infected with

HBV. Historical studies of therapeutic vaccination for

HBV have been disappointing but this approach is being

pursued with new vaccine candidates [58,59].

Innate immunity ligands. Toll-like Receptors (TLRs) are

pattern recognition receptors that play a key role in the

recognition of bacteria and viruses as part of the innate

immune response and are involved in the pathogenesis of

HBV. The TLR7 agonist GS-9620 is in clinical develop-

ment. In pre-clinical in vivo models it has been shown to

stimulate production of interferon-alpha as well as other

cytokines and to activate interferon-stimulated genes,

natural killer cells, and lymphocyte subsets, to suppress

serum and liver HBV DNA and to reduce HBsAg serum

levels [60,61�,62]. RIG-I, a member of the Retinoic acid-

inducible gene (RIG-I)-like RNA helicases (RLHs) class

of innate pattern recognition receptors, has dual antiviral

effect against HBV by inducing a type III interferon

response and by blocking the interaction of the HBV

polymerase with pgRNA to directly suppress viral repli-

cation [63�]. The SB-9200 an oral dinucleotide prodrug

has shown activity against both HCV and HBV in pre-

clinical models [64] and clinical trials in HBV populations

are planned to start in 2016.

Selective sensitization of HBV infectedhepatocytes to immune eliminationCellular inhibitor of apoptosis proteins (cIAPs) have

recently been shown to attenuate TNF signaling during

HBV infection and restrict the death of infected hepa-

tocytes, promoting viral persistence [65]. Birinapant, a

Smac mimetic that antagonizes cIAP, has shown anti-HBV

activity in preclinical models [66�] and a phase 1 study

was initiated in 2015.

Direct targeting of cccDNAStrategies targeting the cccDNA for HBV cure aim at

preventing cccDNA formation, eliminating existing



cccDNA or silencing cccDNA transcription. These aims

can be achieved by direct-acting antivirals (DAAs), host-

targeting agents (HTAs) [that inhibit key host factors

required for the viral replication cycle] and immune-

modulatory agents that elicit signals converging on the

cccDNA mini-chromosome. Mechanistically, control of

cccDNA can be obtained by inhibition of rcDNA entry

into nucleus, inhibition of conversion of rcDNA to

cccDNA, physical elimination of cccDNA, inhibition of

cccDNA transcription (epigenetic control), or inhibition

of viral/cellular factors contributing to cccDNA stability/

formation. All these approaches are currently at the

preclinical stage.

cccDNA formation. Di-substituted sulfonamide (DSS)

compounds have been identified as inhibitors of de novo

cccDNA formation in unbiased screenings [67] but their

molecular target is still unknown.cccDNA destruction approaches include two different

approaches. The first, direct, approach makes use of

transcription activator-like effector nucleases (TALENs)

[68] and the clustered regularly interspaced short palin-

dromic repeats (CRISPR) strategies [69–73]. The possi-

bility of targeting viral sequences integrated in the host

genome is obviously a plus but the open issue of potential

off-target effects induces some caution. The second is

based upon the ability, apparently shared by members of

the IFN and TNF family of cytokines, to decrease

cccDNA stability via the induction of deamination and

apurinic/apyrimidinic site formation in the cccDNA and

the up-regulation of nuclear APOBEC3 deaminases

[37�,38]. These results await confirmation and the safety

and therapeutic index of lymphotoxin-b receptor (LTbR)

agonists in patients with chronic HBV infection and liver

disease remains to be explored.

Functional silencing of cccDNA. These strategies are

intended to target histones modifying enzymes that have

been shown to bind to the cccDNA (including the inhi-

bition of histone acetyl-transferases [HATs] and the

activation of histone de-acetylases [HDACs] and histone

methyl-trasnferases [HMTs]) to induce a permanent/sta-

ble epigenetic inactivation of the cccDNA. Modulation of

these enzymatic activities with small compounds has

been shown to induce the expected PMTs on cccDNA

bound histones, to reduce cccDNA transcription and to

inhibition viral replication in cell systems, providing the

proof of concept for an epigenetic silencing of cccDNA

[74,75�]. However, efficacy must be confirmed invivo. The functional redundancy between the members

of the different classes of chromatin modifiers and the

possible off-target effects on the host genome should be

carefully considered.cccDNA bound viral proteins. The role of the viral proteins

HBx and HBc in cccDNA transcription and/or stability

[16,17,18�] has led to the development of drug discovery

programs targeting the cellular proteins [76] that are


bound and mediate the activities of HBx and HBc on

the cccDNA.

Conclusive remarksThe complexity of HBV replication cycle and virus–host

interactions that ensure HBV persistence represent a

formidable obstacle to a cure. Nevertheless the hunt

for new molecules and therapeutic strategies acting on

novel targets with the aim to set true combination thera-

pies is clearly on. Strategies targeting directly or indirect-

ly the cccDNA as well as the restoration of the immune

response against HBV infected cells will likely need to be

combined to reach at least a virological and immune

functional cure status similar to that observed in HBsAg

sero-converters, if not a complete cure. A major effort

must be dedicated to define the clinical, immunological

and virological endpoint that should be used to measure

the impact of the new treatments in future clinical trials.

Similarly, regulatory agencies (FDA, EMEA) must be

made aware of the strong need for early combination

trials. Clinical trial design will need to take into account

the clinical complexity of CHB and the need for selection

of homogeneous and well characterized CHB patients to

be included in the different trials. Whereas is not possible

to predict when a cure for HBV will be available, the

magnitude of the academic and industry-based research

and R&D efforts makes a breakthrough possible within a

time frame much shorter than initially expected. Working

to find a cure for CHB does not negate the need to further

implement mass HBV vaccination and increase the

access to existing DAAs in highly endemic areas in the

South of the world.

Conflict of interestFZ received research grants through INSERM from

Roche, Gilead Sciences, Novira, Janssen and Assembly

Bioscience, and consulting honoraria from Roche, Gilead

Sciences, Novira, Arbutus and Janssen.

ML participates to advisory boards of BMS, Assembly

Biosciences Gilead, Arbutus, Janssen, Galapagos, Med-

immune.

AcknowledgementsThis work was supported by grants from: Institut Hospitalo-Universitaire(IHU) OPeRa; LabEx DevWeCan of University of Lyon; InstitutUniversitaire de France; unrestricted research grant from Roche (to FZ);CARIPLO Foundation; University of Lyon-St Etienne (PALSEPROGRAM); the Agence National de la Recherche (ANR@TRACTION);ANRS AO 2.2015; Italian Ministry of Health (RF 2010-2317822) (to ML).

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8.�

Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B,Wang H et al.: Sodium taurocholate cotransportingpolypeptide is a functional receptor for human hepatitis B andD virus. Elife 2012, 1:e00049.

The AA shows, using near zero distance photo-cross-linking and tandemaffinity purification, that the receptor-binding region of pre-S1 specificallyinteracts with the sodium taurocholate co-transporting polypeptide(NTCP), a transmembrane transporter predominantly expressed in theliver. This is the first demonstration that NTCP is a functional receptor forboth HBV and HDV.

9. Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M,Stindt J, Koniger C, Nassal M, Kubitz R et al.: Hepatitis B and Dviruses exploit sodium taurocholate co-transportingpolypeptide for species-specific entry into hepatocytes.Gastroenterology 2014, 146:1070-1083.

10. Huang HC, Chen CC, Chang WC, Tao MH, Huang C: Entry ofhepatitis B virus into immortalized human primaryhepatocytes by clathrin-dependent endocytosis. J Virol 2012,86:9443-9453.

11. Macovei A, Radulescu C, Lazar C, Petrescu S, Durantel D,Dwek RA, Zitzmann N, Nichita NB, Hepatitis B: virus requiresintact caveolin-1 function for productive infection in HepaRGcells. J Virol 2010, 84:243-253.

12. Schmitz A, Schwarz A, Foss M, Zhou L, Rabe B, Hoellenriegel J,Stoeber M, Pante N, Kann M: Nucleoporin 153 arrests thenuclear import of hepatitis B virus capsids in the nuclearbasket. PLoS Pathog 2010, 6:e1000741.

13. Koniger C, Wingert I, Marsmann M, Rosler C, Beck J, Nassal M:Involvement of the host DNA-repair enzyme TDP2 in formation ofthe covalently closed circular DNA persistence reservoir ofhepatitisBviruses.ProcNatlAcadSciUSA2014,111:E4244-E4253.

14. Cui X, McAllister R, Boregowda R, Sohn JA, Cortes Ledesma F,Caldecott KW, Seeger C, Hu J: Does tyrosyl DNAphosphodiesterase-2 play a role in hepatitis B virus genomerepair. PLOS ONE 2015, 10:e0128401.

15.�

Pollicino T, Belloni L, Raffa G, Pediconi N, Squadrito G,Raimondo G et al.: Hepatitis B virus replication is regulated bythe acetylation status of hepatitis B virus cccDNA-bound H3and H4 histones. Gastroenterology 2006, 130:823-837.

The AA introduce the cccDNA-ChIP, a chromatin-immunoprecipitation(ChIP)-based assay, to analyze, in vitro and ex vivo, the transcriptionalregulation of HBV cccDNA minichromosome. First demonstration thatcccDNA transcription and HBV replication are regulated by epigeneticmodifications of cccDNA-bound histones.

16. Bock CT, Schwinn S, Locarnini S, Fyfe J, Manns MP, Trautwein C,Zentgraf H: Structural organization of the hepatitis B virusminichromosome. J Mol Biol 2001, 307:183-196.


17. Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, Fanciulli M,Raimondo G, Levrero M: Nuclear HBx binds the HBVminichromosome and modifies the epigenetic regulation ofcccDNA function. Proc Natl Acad Sci USA 2009, 106:19975-19979.

18.�

Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M,Levrero M, Zoulim F, Hantz O, Protzer U: Hepatitis B virus Xprotein is essential to initiate and maintain virus replicationafter infection. J Hepatol 2011, 55:996-1003.

Using primary human hepatocytes and differentiated HepaRG cellsallowing conditional trans complementation of HBx, the AA show thatHBx is required to initiate and maintain cccDNA transcription and HBVreplication and highlight HBx as the key regulator during the naturalinfection process.

19. Watanabe T, Sorensen EM, Naito A, Schott M, Kim S, Ahlquist P:Involvement of host cellular multivesicular body functions inhepatitis B virus budding. Proc Natl Acad Sci USA 2007,104:10205-10210.

20. Lentz TB, Loeb DD: Roles of the envelope proteins in theamplification of covalently closed circular DNA andcompletion of synthesis of the plus-strand DNA in hepatitis Bvirus. J Virol 2011, 85:11916-11927.

21. Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K,Petersen J, Lau G, Trepo C, Marcellin P, Goodman Z, Delaney WEet al.: Persistence of cccDNA during the natural history ofchronic hepatitis B and decline during adefovir dipivoxiltherapy. Gastroenterology 2004, 126:1750-1758.

22. Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R,Chisari FV: Viral clearance without destruction of infected cellsduring acute HBV infection. Science 1999, 284:825-829.

23. Rehermann B, Bertoletti A: Immunological aspects of antiviraltherapy of chronic hepatitis B virus and hepatitis C virusinfections. Hepatology 2015, 61:712-721.

24. Ferrari C: HBV and the immune response. Liver Int. 2015, 35:121-128.

25. Guidotti LG, Chisari FV: Immunobiology and pathogenesis ofviral hepatitis. Annu Rev Pathol 2006, 1:23-61.

26. Wieland S, Thimme R, Purcell RH, Chisari FV: Genomic analysisof the host response to hepatitis B virus infection. Proc NatlAcad Sci USA 2004, 101:6669-6674.

27. Lucifora J, Durantel D, Testoni B, Hantz O, Levrero M, Zoulim F:Control of hepatitis B virus replication by innate response ofHepaRG cells. Hepatology 2010, 51:63-72.

28. Isorce N, Lucifora J, Zoulim F, Durantel D: Immune-modulatorsto combat hepatitis B virus infection: From IFN-a to novelinvestigational immunotherapeutic strategies. Antivir Res2015, 122:69-81.

29. Seeger C, Mason WS: Molecular biology of hepatitis B virusinfection. Virology 2015, 479–480:672-686.

30. Tu T, Mason WS, Clouston AD, Shackel NA, McCaughan GW,Yeh MM, Schiff ER, Ruszkiewicz AR, Chen JW, Harley HA et al.:Clonal expansion of hepatocytes with a selective advantageoccurs during all stages of chronic hepatitis B virus infection. JViral Hepat 2015, 22:737-753.

31. EASL: Easl clinical practice guidelines: management ofchronic hepatitis B virus infection. J Hepatol 2012, 57:167-185.

32. Thimme R, Dandri M: Dissecting the divergent effects ofinterferon-alpha on immune cells: time to rethink combinationtherapy in chronic hepatitis B? J Hepatol 2013, 58:205-209.

33. Micco L, Peppa D, Loggi E, Schurich A, Jefferson L, Cursaro C,Panno AM, Bernardi M, Brander C, Bihl F et al.: Differentialboosting of innate and adaptive antiviral responses duringpegylated-interferon-alpha therapy of chronic hepatitis B. JHepatol 2013, 58:225-233.

34.�

Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T,Petersen J, Raimondo G, Dandri M, Levrero M: IFN-alpha inhibitsHBV transcription and replication in cell culture and inhumanized mice by targeting the epigenetic regulation of the


























































































































nuclear cccDNA minichromosome. J Clin Invest 2012, 122:529-537.

The AA show that IFN-a inhibits HBV replication by decreasing thetranscription of pregenomic RNA (pgRNA) and subgenomic RNA fromthe HBV covalently closed circular DNA (cccDNA) minichromosome, bothin cultured cells replicating HBV and in human liver chimeric-miceinfected with HBV. Mechanistically, IFN-a induces the epigenetic repres-sion of HBV cccDNA transcriptional activity by the active recruitment tothe cccDNA of transcriptional corepressors.

35. Liu F, Campagna M, Qi Y, Zhao X, Guo F, Xu C, Li S, Li W,Block TM, Chang J, Guo JT: Alpha-interferon suppresseshepadnavirus transcription by altering epigeneticmodification of cccDNA minichromosomes. PLoS Pathog2013, 9:e1003613.

36. Klumpp K, Shimada T, Allweiss L, Volz T, Luetgehetman M,Flores O, Hartman G, Lam A, Dandri M: High antiviral activity ofthe HBV core inhibitor NVR 3-778 in the humanized uPA/SCIDmouse model. J Hepatol 2015, 62:S214.

37.�

Lucifora J, Xia Y, Reisinger F, Zhang K, Stadler D, Cheng X,Sprinzl MF, Koppensteiner H, Makowska Z, Volz T et al.: Specificand nonhepatotoxic degradation of nuclear hepatitis B viruscccDNA. Science 2014, 343:1221-1228.

The APOBEC3A and APOBEC3B cytidine deaminases are induced byinterferon-a and lymphotoxin-b receptor activation and are recruitedby the HBV core protein on nuclear cccDNA, resulting in cytidine dea-mination, apurinic/apyrimidinic site formation, and finally cccDNA degra-dation.

38. Xia Y, Stadler D, Lucifora J, Reisinger F, Webb D, Hosel M,Michler T, Wisskirchen K, Cheng X, Zhang K et al.: Interferon-gand tumor necrosis factor-a produced by T cells reduce theHBV persistence form, cccDNA, without cytolysis.Gastroenterology 2015, 150:194-205.

39. Marcellin P, Gane EJ, Flisiak R, Manns MP, Kaita D, Gaggar A,Lin L, Kitrinos KL, Flaherty JF, Subramanian M et al.: Evidence forongoing low level viremia in patients with CHB receiving longterm NUC therapy. Hepatology 2014. S1:A1861.

40. Lutgehetmann M, Mancke LV, Volz T, Helbig M, Allweiss L,Bornscheuer T, Pollok JM, Lohse AW, Petersen J, Urban S,Dandri M: Humanized chimeric uPA mouse model for the studyof hepatitis B and D virus interactions and preclinical drugevaluation. Hepatology 2012, 55:685-694.

41. Zlotnick A, Venkatakrishnan B, Tan Z, Lewellyn E, Turner W,Francis S: Core prote pleiotropic keystone in the A HBVlifecycle. Antiviral Res 2015, 121:82-93.

42. Belloni, Palumbo L, Lupacchini GA, Li L, Reddy L, Chirapu S,Calvo S, Finn L, Lopatin MG, Zlotnick U, Levrero AM: Capsiddrugs HAP12 and AT130 target HBV core protein nuclearfunctions. J Hepatol 2015, 62:S513-S514.

43. Gruffaz M, Testoni B, Luangsay S, Fusil F, Ait-Goughoulte M,Mancip J, Petit AM, Javanbakth H, Cosset FL, Zoulim F,Durantel D: The nuclear function of Hepatitis B capsid (HBc)protein is to inhibit IFN response very early after infection ofhepatocytes. Hepatology 2013, 58:276A.

44. Yuen MF, Kim DL, Weilert F, Chan HL, Jacob P, Lalezari JP,Hwang SG, Nguyen TT, Liaw S, Brown N et al.: Phase 1b efficacyand safety of NVR 3-778, a first-in- class HBV core inhibitor, inHBeAg-positive patients with chronic HBV infection.Hepatology 2015, 62:1385A.

45. Wooddell CI, Rozema DB, Hossbach M, John M, Hamilton HL,Chu Q, Hegge JO, Klein JJ, Wakefield DH, Oropeza CE:Hepatocyte-targeted RNAi therapeutics for the treatment ofchronic hepatitis B virus infection. Mol Ther 2013, 21:973-985.

46. Yuen MF, Chan HLY, Liu SHK, Given B, Schluep T, Hamilton J,Lai CL, Locarnini S, Lau JY, Ferrari C, Gish R: ARC-520 producesdeep and durable knockdown of viral antigens and DNA in aphase II study in patients with chronic hepatitis B. Hepatology2015, 62:LB9.

47. Wooddell CI, Chavez D, Goetzmann JE, Guerra B, Peterson RM,Lee H, Hegge JO, Gish R, Locarnini S, Anzalone CR et al.:Reductions in cccDNA under NUC and ARC-520 therapy inchimpanzees with chronic hepatitis B virus infection implicate


integrated DNA in maintaining circulating HBsAg. Hepatology2015, 62:32A.

48. Update on the preclinical development of an LNP-based HBVtherapeutic. 10th annual meeting of the oligonucleotidetherapeutics society. Edited by McLachlan I. CA: San Diego; 2014.

49. Bazinet M, Pantea V, Cebotarescu V, Cojuhari L et al.: Update onthe safety and efficacy of REP 2139 monotherapy andsubsequent combination therapy with pegylated interferonalpha-2a in chronic HBV/HDV co-infection in Caucasianpatients. Hepatology 2015, 62:222A.

50. Quinet P, Jamard C, Vaillant A, Cova L: Combination therapy withthe nucleic acid polymer REP 2139-Ca and nucleos(t)ideanalogues improves antiviral responses against hepatitis B in vivo.HepDart 2015. Grand Wailea; 2015:. December 6–10.

51. Raziorrouh B, Schraut W, Gerlach T, Nowack D, Gruner NH,Ulsenheimer A, Zachoval R, Wachtler M, Spannagl M, Haas J et al.:The immunoregulatory role of CD244 in chronic hepatitis Binfection and its inhibitory potential on virus-specific CD8+ T-cell function. Hepatology 2010, 52:1934-1947.

52. Fisicaro P, Valdatta C, Massari M, Loggi E, Ravanetti L, Urbani S,Giuberti T, Cavalli A, Vandelli C, Andreone P et al.: Antiviralintrahepatic T-cell responses can be restored by blockingprogrammed death-1 pathway in chronic hepatitis B.Gastroenterology 2010, 138:682-693.

53. Peppa D, Micco L, Javaid A, Kennedy PT, Schurich A, Dunn C,Pallant C, Ellis G, Khanna P, Dusheiko G et al.: Blockade ofimmunosuppressive cytokines restores NK cell antiviralfunction in chronic hepatitis B virus infection. PLoS Pathog2010, 6:e1001227.

54. Sangro S, Crocenzi TS, Welling TH, Inarrairaegui M, Prieto J,Fuertes C, Delanty L, Feely W, Anderson J, Grasela DM et al.:Phase I dose escalation study of nivolumab (Anti-PD-1; BMS-936558; ONO-4538) in patients (pts) with advancedhepatocellular carcinoma (HCC) with or without chronic viralhepatitis. J Clin Oncol 2013, 31:TPS3111.

55. Koh S, Tham CYL, Tanoto AT, Pavesi A, Kamm RD, Bertoletti A:Engineered HBV-specific T cells: disentangling antiviral fromkilling capacity. J Hepatol 2015, 62:S188.

56. Krebs K, Bottinger N, Huang LR, Chmielewski M, Arzberger S,Gasteiger G, Jager C, Schmitt E, Bohne F, Aichler M et al.: T cellsexpressing a chimeric antigen receptor that binds hepatitis Bvirus envelope proteins control virus replication in mice.Gastroenterology 2013, 145:456-465.

57.�

Qasim W, Brunetto M, Gehring AJ, Xue SA, Schurich A,Khakpoor A, Zhan H, Ciccorossi P, Gilmour K, Cavallone D et al.:Immunotherapy of HCC metastases with autologous T cellreceptor redirected T cells, targeting HBsAg in a livertransplant patient. J Hepatol 2015, 62:486-491.

First demonstration that autologous T cells genetically modified toexpress an HBsAg specific T cell receptor can survive, expand, recognizetumor cells in vivo and mediate a reduction in HBsAg levels withoutexacerbation of liver inflammation or other toxicity.

58. Lok AS, Pan CQ, Han SB, Trinh HN, Fessel WJ, Rodell T,Massetto B, Lin L: Randomized phase II study of GS-4774 as atherapeutic vaccine in virally suppressed patients with chronichepatitis B. J Hepatol 2016, 64:302 pii: S0168-8278(16)02-1.

59. Martin P, Dubois C, Jacquier E, Dion S, Mancini-Bourgine M,Godon O, Kratzer R, Lelu-Santolaria K, Evlachev A, Meritet JFet al.: TG1050, an immunotherapeutic to treat chronic hepatitisB, induces robust T cells and exerts an antiviral effect in HBV-persistent mice. Gut 2015, 64:1961-1971.

60. Lanford RE, Guerra B, Chavez D, Giavedoni L, Hodara VL,Brasky KM, Fosdick A, Frey CR, Zheng J, Wolfgang G et al.: GS-9620, an oral agonist of Toll-like receptor-7, inducesprolonged suppression of hepatitis B virus in chronicallyinfected chimpanzees. Gastroenterology 2013, 144:1508-1517.

61.�

Gane EJ, Lim YS, Gordon SC, Visvanathan K, Sicard E,Fedorak RN, Roberts S, Massetto B, Ye Z, Pflanz S et al.: The oraltoll-like receptor-7 agonist GS-9620 in patients with chronichepatitis B virus infection. J Hepatol 2015, 63:320-328.

The results of two double-blind, phase 1b trials with the GS-9620 oralagonist of toll-like receptor 7 show that GS-9620 is safe, well tolerated,

































































































































and associated with induction of peripheral ISG15 production in theabsence of significant systemic IFN-alpha levels or related symptoms.

62. Steuer HM, Daffis S, Lehar SM, Palazzo A, Tharinger H, Frey C,Pflanz S, Niu C, Chang CY, Jin MQ et al.: Functional activation ofNK and CD8+ T cells in vitro by the toll-like receptor 7 agonistGS-9620. Hepatology 2015, 62:1187A.

63.�

Sato S, Li K, Kameyama T, Hayashi T, Ishida Y, Murakami S,Watanabe T, Iijima S, Sakurai Y, Watashi K et al.: The RNA sensorRIG-I dually functions as an innate sensor and direct antiviralfactor for hepatitis B virus. Immunity 2015, 42:123-132.

The AA show that the retinoic acid-inducible gene-I (RIG-I) senses the 50-eregion of the HBV pre-genomic RNA and prevents the interaction of theHBV polymerase with the 50-e region to suppress viral replication. Lipo-some-mediated delivery and vector-based expression of the e region-derived RNA in the liver abolished the HBV replication in human hepa-tocyte-chimeric mice.

64. Korolowicz K, Czerwinski S, Iyer R, Skell J, Yang J, Tucker R,Menne S: Antiviral efficacy and induction of host immuneresponses with SB 9200, an oral prodrug of the dinucleotideSB 9000, in the woodchuck model of chronic Hepatitis B Virus(HBV) infection. J Hepatol 2015, 62:557A.

65. Ebert G, Preston S, Allison C, Cooney J, Toe JG, Stutz MD,Ojaimi S, Scott HW, Baschuk N, Nachbur U et al.: Cellularinhibitor of apoptosis proteins prevent clearance of hepatitis Bvirus. Proc Natl Acad Sci U S A 2015, 112:5797-5802.

66.�

Ebert G, Allison C, Preston S, Cooney J, Toe JG, Stutz MD,Ojaimi S, Baschuk N, Nachbur U, Torresi J et al.: Eliminatinghepatitis B by antagonizing cellular inhibitors of apoptosis.Proc Natl Acad Sci U S A 2015, 112:5803-5808.

Cellular inhibitor of apoptosis proteins (cIAPs) impair the clearance ofHBV by preventing TNF-mediated killing/death of infected cells. Using animmuno-competent mouse model of chronic HBV infection the AA showthat birinapant and other Smac mimetics are able to rapidly reduce serumHBV DNA and serum HBV surface antigen, and they promote theelimination of hepatocytes containing HBV core antigen Smac mimetics.

67. Cai D, Mills C, Yu W, Yan R, Aldrich CE, Saputelli JR, Mason WS,Xu X, Guo JT, Block TM et al.: Identification of disubstitutedsulfonamide compounds as specific inhibitors of hepatitis Bvirus covalently closed circular DNA formation. AntimicrobAgents Chemother 2012, 56:4277-4288.

68. Bloom K, Ely A, Mussolino C, Cathomen T, Arbuthnot P:Inactivation of hepatitis B virus replication in cultured cellsand in vivo with engineered transcription activator-likeeffector nucleases. Mol Ther 2013, 21:1889-1897.


69. Kennedy EM, Kornepati AV, Cullen BR: Targeting hepatitis Bvirus cccDNA using CRISPR/Cas9. Antiviral Res 2015, 123:188-192.

70. Kennedy EM, Bassit LC, Mueller H, Kornepati AVR,Bogerd HP, Nie T, Chatterjee P, Javanbakht H, Schinazi RF,Cullen BR: Suppression of hepatitis B virus DNAaccumulation in chronically infected cells using a bacterialCRISPR/Cas RNA-guided DNA endonuclease. Virology2015, 476:196-205.

71. Ramanan V, Shlomai A, Cox DB, Schwartz RE, Michailidis E,Bhatta A, Scott DA, Zhang F, Rice CM, Bhatia SN: CRISPR/Cas9cleavage of viral DNA efficiently suppresses hepatitis B virus.Sci Rep 2015, 5:10833.

72. Seeger C, Sohn JA: Targeting Hepatitis B Virus with CRISPR/Cas9. Mol Ther Nucleic acids 2014, 3:e216.

73. Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, Sung KC, Lin YY,Wang HY, Wang CC, Shen YC et al.: The CRISPR/Cas9 systemfacilitates clearance of the intrahepatic HBV templates in vivo.Mol Ther Nucleic Acids 2014, 3:e186.

74. Palumbo GA, Belloni L, Valente S, Rotili D, Pediconi P, Mai A,Levrero M: Silencing HBV transcription by targeting cccDNA-bound chromatin modifiers. Hepatology 2014, 60:309A.

75.�

Tropberger P, Mercier A, Robinson M, Zhong W, Ganem DE,Holdorf M: Mapping of histone modifications in episomal HBVcccDNA uncovers an unusual chromatin organizationamenable to epigenetic manipulation. Proc Natl Acad Sci U S A2015, 112:E5715-E5724.

Using a new cccDNA ChIP-Seq approach the AA generate the firstgenome-wide maps of cccDNA-bound histone post-translational mod-ifications (PTMs) in de novo infected HepG2 cells, primary human hepa-tocytes and HBV-infected liver tissue. Transcription and active PTMs inHBV chromatin are reduced by the activation of the IFNa innate immunitypathway, and this effect can be recapitulated with a small moleculeepigenetic modifying agent.

76. Decorsiere A, Mueller H, van Breugel PC, Abdul F, Gerossier L,Beran RK, Livingston CM, Niu C, Fletcher SP, Hantz O, Strubin M:Hepatitis B virus X protein identifies the Smc5/6 complex as ahost restriction factor. Nature 2016, 531:386-389.

In this landmark paper, the AA show that the structural maintenance ofchromosomes (Smc) complex Smc5/6 act as a host restriction factor forHBV and that HBx relieves the inhibition to allow productive hepatitis Bvirus gene expression by hijacking the cellular DDB1-containing E3ubiquitin ligase.































































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HBV cure: why, how, when?

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