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12 Review

4 Chronic hepatitis B: Virology, natural history, current management5 and a glimpse at future opportunities

6

7

8 Robert G. Gish a,, Bruce D. Given b, Ching-Lung Lai c, Stephen A. Locarnini d, Johnson Y.N. Lau e,9 David L. Lewis b, Thomas Schluep b

10 aDivision of Gastroenterology and Hepatology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA11 bArrowhead Research Corporation, Pasadena, CA, USA12 c The University of Hong Kong, Hong Kong, China13 dVictorian Infectious Diseases Reference Laboratory, Victoria, Australia14 eHong Kong Polytechnic University, Hong Kong, China

1516

1 8a r t i c l e i n f o

19 Article history:20 Received 7 May 201521 Accepted 16 June 201522 Available online xxxx

23 Keywords:24 Hepatitis B virus25 Trained immunity26 Functional cure27 Virological cure28 Antiviral therapy29 Phases of infection30

3 1a b s t r a c t

32The host immune system plays an important role in chronic hepatitis B (CHB), both in viral clearance and33hepatocellular damage. Advances in our understanding of the natural history of the disease have led to34redening the major phases of infection, with the high replicative, low inammatory phase now replac-35ing what was formerly termed the immune tolerant phase, and the nonreplicative phase replacing36what was formerly termed the inactive carrier phase. As opposed to the earlier view that HBV estab-37lishes chronic infection by exploiting the immaturity of the neonates immune system, new ndings on38trained immunity show that the host is already somewhat matured following birth, and is actually very39capable of responding immunologically, potentially altering future hepatitis B treatment strategies. While40existing therapies are effective in reducing viral load and necroinammation, often restoring the patient to41near-normal health, they do not lead to a cure except in very rare cases and, in many patients, viremia42rebounds after cessation of treatment. Researchers are now challenged to devise therapies that will elim-43inate infection, with a particular focus on eliminating the persistence of viral cccDNA in the nuclei of hep-44atocytes. In the context of chronic hepatitis B, new denitions of cure are emerging, such as functional45and virological cure, dened by stable off-therapy suppression of viremia and antigenemia, and the nor-46malization of serumALT and other liver-related laboratory tests. Continued advances in the understanding47of the complex biology of chronic hepatitis B have resulted in the development of new, experimental ther-48apies targeting viral and host factors and pathways previously not accessible to therapy, approacheswhich49may lead to virological cures in the near term and functional cures upon long term follow-up. This article50forms part of a symposium in Antiviral Research on An unnished story: from the discovery of the51Australia antigen to the development of new curative therapies for hepatitis B.52 2015 Published by Elsevier B.V.53

54

5556 Contents

57 1. The global importance of chronic hepatitis B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0058 2. HBV structure, molecular biology and replication cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0059 3. Natural history of chronic hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0060 4. Host immune response and phases of CHB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0061 5. Immune escape in CHB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0062 6. Goals of therapy and definitions of cure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0063 7. Existing therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0064 7.1. Reverse transcriptase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0065 7.2. Interferon-a and PEG-interferon-a. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

http://dx.doi.org/10.1016/j.antiviral.2015.06.0080166-3542/ 2015 Published by Elsevier B.V.

Corresponding author at: Liver Transplant Program, Stanford University Medical Center, 6022 La Jolla Mesa Drive, San Diego, CA 92037, USA.E-mail address: rgish@robertgish.com (R.G. Gish).

Antiviral Research xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Antiviral Research

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66 8. Current treatment recommendations for chronic hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0067 8.1. High-replicative, low-inflammatory and nonreplicative phases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0068 8.2. HBeAg(+) chronic hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0069 8.3. HBeAg() chronic hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0070 9. New therapeutic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0071 10. Future directions and opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0072 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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74

75 This year marks the 50th anniversary of the discovery of the76 Australia antigen, a serendipitous nding which led to the identi-77 cation of the hepatitis B virus (HBV), the virtual elimination of HBV78 from the blood supply in industrialized countries, the widespread79 introduction of a protective vaccine and the development of a80 range of antiviral therapies. Current treatments are effective in81 restoring patients with chronic hepatitis B infection (CHB) to82 near-normal health, but are unable to eliminate all virus from all83 hepatocytes. Researchers now face the major challenge of devising84 new therapies that will provide a denitive cure for CHB, as is now85 being achieved for another major human disease, hepatitis C.86 Dening more accurately the phases of infection and the interac-87 tion of the immune system with the virus will aid in the develop-88 ment of newmedications that can target cccDNA and possibly even89 eliminate cells with integrated HBV DNA.90 This article reviews the virology, natural history and current91 management of CHB, and offers a glimpse at future opportunities92 for curative therapy. It forms part of a symposium in Antiviral93 Research on An unnished story: from the discovery of the94 Australia antigen to the development of novel curative therapies95 for hepatitis B.

96 1. The global importance of chronic hepatitis B

97 The World Health Organization estimates that 240 million peo-98 ple are chronically infected with HBV (Ott et al., 2012).99 Approximately 45% of the world population lives in areas of high

100 endemicity, including many African and Asian countries (The101 EASL Jury, 2003), with about 75% of infected persons in Asia and102 12% in Africa (Gust, 1996). Markers of HBV infection are present103 in 3562% of the population of China (Yao, 1996), 5698% of the104 population of sub-Saharan Africa (Kew, 1996; Kiire, 1996), 82% of105 the Melanesian population of Fiji (Zhuang et al., 1982) and 69%106 of the population of the Pacic island of Nauru (Speed et al.,107 1989). Up to 2.2 million persons with CHB live in the United108 States, with a particularly high prevalence (3.45%) among the109 foreign-born (Kowdley et al., 2012). Although the majority of110 patients do not develop hepatic complications, it is estimated that111 during their lifetimes 1540% may develop serious sequelae of112 infection (Lok and McMahon, 2009).

113 2. HBV structure, molecular biology and replication cycle

114 HBV virions are double-shelled particles, 4042 nm in diameter,115 with an outer lipoprotein envelope that contains three related116 envelope glycoproteins (surface antigens) (Dane et al., 1970;117 Ganem, 1991). Within the envelope is the viral nucleocapsid, or118 core (Robinson and Lutwick, 1976). The viral capsid contains a viral119 genome, a relaxed-circular, partially duplex DNA of 3.2 kb, and a120 polymerase (also serving as a reverse transcriptase) that is121 responsible for the synthesis of viral DNA in infected cells and122 mRNA transcripts (Summers et al., 1975). Multiple viral genotypes123 (10) and serotypes have been identied, each with a characteristic124 geographic distribution (Kao, 2002). In addition to virions,125 HBV-infected cells produce two distinct subviral lipoprotein

126particles: 20-nm spheres and lamentous forms of similar dia-127meter (Robinson and Lutwick, 1976). These hepatitis B surface128antigen (HBsAg) particles contain only envelope glycoproteins129and host-derived lipids and are produced in 34 log excess over130virions (Robinson and Lutwick, 1976).131HBV contains a small, partially double-stranded genome that132consists of a full-length negative strand and an incomplete positive133strand (Fig. 1) (Warner and Locarnini, 2012). The genome contains134four promoters, two enhancer regions (Enh1, Enh2), and two direct135repeats (DR1, DR2). During virus replication, the partially136double-stranded DNA, also named relaxed circular (RC) DNA, is137repaired into covalently closed circular (ccc) DNA. This process138requires removal of polymerase and involves cellular proteins,139although the exact steps are not well understood (Nassal, 2008).140cccDNA is a minichromosome that serves as the template for viral141transcription, generating mRNAs that are 3.5-, 2.4-, 2.1-, and 0.7-kb142in size (Warner and Locarnini, 2012).

Fig. 1. HBV genome organization. The genome consists of a circular, partiallydouble-stranded DNA molecule 3.2 kb in length. During viral replication, thepartially double-stranded genome, also named relaxed circular (RC) DNA, isrepaired into covalently closed circular (ccc) DNA. cccDNA is a mini-chromosomethat serves as the template for viral transcription, generating ve major mRNAs thatare 3.5-, 2.4-, 2.1-, and 0.7-kb in size. One 3.5 kb mRNA called pre-C mRNA serves asthe template for translation of the HBeAg (pre-core protein), and one slightlyshorter mRNA called pgRNA serves as the template for core and polymeraseproteins, as well as the pre-genomic RNA used as a template during viralreplication. The 2.4 kb transcript (pre-S1 mRNA) is used in the translation of thelarge surface antigen (L); the 2.1 kb transcripts (pre-S2 mRNAs) are used to producethe M and S surface antigens; and the 0.7 kb transcript is used in the production ofthe X protein.

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143 The HBV genome has four long open reading frames (Fig. 1)144 (Ganem, 1991; Robinson and Lutwick, 1976; Warner and145 Locarnini, 2012). The presurface/surface (preS1/preS2/S) region of146 the genome encodes the three viral surface antigens, the 24-kD S147 protein (HBsAg), the M (or preS2) protein, and the L (or preS1) pro-148 tein. The S protein constitutes the bulk of the envelope proteins,149 while the L proteins are essential in the binding of the virus to150 host-cell receptors (see below) and in the assembly of the virion151 and its release from the cell.152 The PC/CP regions encode hepatitis B core antigen (HBcAg) and153 hepatitis B e antigen (HBeAg) (Ganem, 1991; Warner and Locarnini,154 2012). The PC region encodes a signal sequence that directs it to be155 processed in the Golgi and secreted as HBeAg. HBeAg plays no role156 in viral assembly, but has been reported to play a role in modulating157 the host immune response (Walsh and Locarnini, 2012). While it is158 not required for viral replication, mutants bearing chain-159 terminating codons within the PC region replicate well in culture160 and, in fact, arise frequently during natural infection.161 HBcAg itself is essential for capsid assembly and regulation of162 replication, including the encapsidation of RNA, DNA synthesis,163 and transport of partially double-stranded DNA into the nucleus164 to help replenish cccDNA, as well as for virus maturation and165 release (Bock et al., 1996; Lewellyn and Loeb, 2011; Scaglioni et166 al., 1997). Additionally, it has been suggested that HBcAg binds167 to CpG (cytosinephosphateguanine) islands of HBV168 cccDNA and thereby promotes an epigenetic permissive state169 (Guo et al., 2011).

170The polymerase coding region is specic for the viral polymerase,171a multifunctional enzyme involved in DNA synthesis and RNA172encapsidation (Jones and Hu, 2013). The X open reading frame173encodes the viral X protein (HBx) which modulates host-cell signal174transduction and can directly and indirectly affect host and viral175gene expression (Tang et al., 2006). X-protein activity is required176for efcient in vivo replication and spread of the virus. It has also177been found to promote chronic infection by preventing178immune-mediated apoptosis of infected hepatocytes, by promoting179the establishment and persistence of brosis and cirrhosis preced-180ing the development of HCC, and by promoting the remodeling of181extracellular matrix (ECM) during tumor progression (Feitelson et182al., 2009). The integration of partial or possibly full strands of HBV183DNA into the hepatocyte genome is not completely understood.184The ability of the integrated virus to cause liver cancer is well185known; the ability to produce viral sequences, proteins or full length186genomes is controversial (Saitta et al., 2015; Tarocchi et al., 2014).187HBV enters the hepatocyte through the sodium-taurocholate188cotransporting polypeptide (NTCP) receptor (Yan et al., 2014, in189press) and is uncoated in the cytoplasm (Fig. 2). Core particles190are transported to the nucleus where their DNA genomes are con-191verted to a covalently closed circular (ccc) form which serves as the192transcriptional template for host RNA polymerase II. This enzyme193generates ve major RNA transcripts, including two genomic and194three subgenomic RNA transcripts. In the cytoplasm, the prege-195nomic RNA (pgRNA) is translated into the core protein and the viral196polymerase; the subgenomic RNA is translated into the 3 envelope

Fig. 2. Schematic of the HBV life cycle. HBV enters the hepatocyte through the sodium-taurocholate cotransporting polypeptide (NTCP) receptor and is uncoated in thecytoplasm. Core particles are transported to the nucleus, where their genomes are converted to a covalently closed circular (cccDNA) form which serves as the transcriptionaltemplate for host RNA polymerase II. This enzyme generates six genomic and subgenomic RNA transcripts. In the cytoplasm, the pregenomic RNA (pgRNA) is translated intothe core protein and the viral polymerase; the subgenomic RNA is translated into the 3 envelope proteins and the X protein. Next, nucleocapsids are assembled in the cytosol,and during this process a single molecule of genomic RNA and as well as viral polymerase are incorporated into the assembling viral core. Once the viral RNA is encapsidated,reverse transcription begins. Synthesis of the two viral DNA strands is sequential. The rst DNA strand is made from the encapsidated RNA template which is degraded by theRNAse function of the viral polymerase; the second DNA strand is then synthesized, using the newly made rst strand as a template. The core particle that contains thegenome can either be reimported into the nucleus to form additional cccDNA or be enveloped with L, M, and S surface antigens and secreted from the cell.

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197 proteins and the X protein. Next, nucleocapsids are assembled in198 the cytosol, and during this process a single molecule of genomic199 RNA and as well as viral polymerase are incorporated into the200 assembling viral core.201 Following viral RNA is encapsidated, reverse transcription202 begins. Synthesis of the two viral DNA strands is sequential. The203 rst DNA strand (called, the minus strand) is made from the204 encapsidated pgRNA template which is degraded by the ribonu-205 clease H (RNase H) function of the viral polymerase; the second206 DNA strand is then synthesized, using the newly made rst DNA207 strand as a template. The core particle that contains the HBV208 DNA genome can either be reimported into the nucleus to form209 additional cccDNA or be enveloped with L, M, and S surface anti-210 gens and secreted from the cell. Each of these steps is a target or211 potential target for antiviral therapy as well as interactions of the212 virus with host cellular pathways.

213 3. Natural history of chronic hepatitis B

214 CHB is usually acquired perinatally or in childhood in Asia and215 Africa (Lavanchy, 2004). Infection is usually the result of close216 person-to-person contact during childhood in Alaska, sub-Saharan217 Africa, and the Mediterranean region (Bortolotti et al., 2006;218 Dusheiko et al., 1989; Lok and McMahon, 2009; McMahon et al.,219 1985). In persons infected perinatally, there is commonly a pro-220 longed period with normal serum alanine aminotransferase (ALT)221 levels, positive sera for hepatitis B e antigen (HBeAg), high HBV222 DNA levels, and minimal or no liver inammation (McMahon,223 2005). Infected persons may remain in this high replicative, low224 inammatory phase (previously termed immune tolerant) for225 many years without disease progression (Hong et al., 2015).226 However, it is during this period that DNA integration into the liver227 cell genome probably occurs, with its subsequent effect on onco-228 genicity, although such integration is not required for productive229 HBV replication (Shafritz et al., 1981). The integrated templates230 can produce viral proteins such as HBsAg. In HBeAg(+) children, ele-231 vated ALT and high HBV DNA levels are common, with HBeAg ser-232 oconversion usually occurring around the onset of puberty (Lok and233 McMahon, 2009). In Western countries where prevalence is low,234 HBV infection in adulthood usually occurs through sexual exposure235 or injection drug use, with immediate entry into the immune236 clearance phase (McQuillan et al., 1999; Stroffolini et al., 2000).

237 4. Host immune response and phases of CHB

238 The age of infection largely dictates the type and level of the239 immune response to acute HBV infection and the subsequent nat-240 ural history of CHB (Fig. 3). CHB can be divided into at least 5 major241 phases (Fig. 4):

242 An initial high replicative, low inammatory phase (pre-243 viously known as immune tolerant), which is characterized244 by high serum viral DNA, HBeAg(+) status, normal or low serum245 ALT, mild or no liver necroinammation, lower production of246 IL-10 and pro-inammatory cytokines (IL-6, IL-8 and TNF-a)247 and no or slow progression of brosis. In this phase the immune248 system is trained and can be further activated by changes or249 maturation of subsets of the immune response (Hong et al.,250 2015).251 A phase of immune clearance, characterized initially by252 widely oscillating serum HBV DNA and ALT levels, ultimately253 decreasing from high to low or undetectable DNA and from high254 to normal ALT (

297 mutations (Baqai et al., 2015; Chu et al., 2003; Takahashi et al.,298 1995) which transitions later to a dominant, but not pure,299 HBeAg() disease. If infection is transmitted from a person with300 mixed infection, the person who becomes infected (for example,301 the child in mother-to-child transmission) develops HBeAg(+)302 disease.303 The rate of spontaneous HBeAg seroconversion in CHB patients304 with elevated ALT (>20 for women and >30 IU/mL for men) ranges305 from 8% to 12% per year (Fattovich et al., 1986; Hoofnagle et al.,306 1981; Lok et al., 1987; McMahon et al., 2001). Most of those who307 achieve HBeAg seroconversion (up to 80%) transition to the308 non-replicative phase characterized by normal ALT and low or309 undetectable HBV DNA or transition directly to HBeAg() CHB.310 (Bortolotti et al., 2006; Fattovich et al., 1986; Hadziyannis and311 Vassilopoulos, 2001; Hoofnagle et al., 1981; Hsu et al., 2002; Lok312 et al., 1987).

313 5. Immune escape in CHB

314 A high percentage of infected neonates develop CHB, but the315 vast majority of acutely infected adults are able to spontaneously316 clear the virus from the blood, (Ganem and Prince, 2004).317 However, in most cases, those individuals who clear HBsAg main-318 tain a low level of infection throughout their lives. In order for HBV319 to cause chronic infection, it must use strategies that enable it to320 evade or modify the host immune response to prevent clearance.321 Innate and adaptive immune responses in chronic HBV infection322 are also thought to cause the related liver damage, leading to pro-323 gression of brosis, hepatic decompensation and hepatocellular324 carcinoma (Iannacone et al., 2006). Some of these mechanisms325 are discussed below.326 Immune responses to viral infection can be divided into innate327 responses, mediated by complement, phagocytes and natural killer328 (NK) cells), and adaptive responses, mediated by B lymphocytes,329 CD4+ T lymphocytes, CD8+ T lymphocytes, and antigen-330 presenting cells such as dendritic cells and macrophages. Hong331 and colleagues have recently reported that HBV exposure in utero332 appears to trigger a state of trained immunity, in which there is333 enhanced innate immune cell maturation and increased Th1

334development, along with higher production of IL-12p40 and lower335production of IL-10 and pro-inammatory cytokines (IL-6, IL-8336and TNF-a) (Hong et al., 2015).337As opposed to the earlier view that HBV establishes chronic338infection by exploiting the immaturity of the neonates immune339system, the researchers note that their ndings do not support340the hypothesis that HBV induces a state of immune tolerance.341Instead, they propose that there may be defective priming of342HBV-specic T cells which predisposes to chronicity. Noting that343the way in which in utero exposure to HBV induces trained immu-344nity is not known, they hypothesize that these complex immune345changes may result from cytokine alterations, including the346increased production of IL-12p40 and, at least in some patients,347increased levels of IFN-a2 (in their study, seen only in the cord348blood of Asian CHB mothers), both of which cytokines are known349to skew T-cell development towards Th1 maturation (Brassard et350al., 2002b; Cooper and Khader, 2007; Nguyen et al., 2002; Pien et351al., 2002). The recent ndings by Vanwolleghem et al. that innate352IFN and B-cell responses are highly active during this phase, with353peripheral blood genomic data suggesting that during this phase354the antiviral sensing and effector machinery helps to control high355levels of viral replication, provide further support that the pre-356sumed immune tolerant phase of chronic infection needs to be357reconsidered (Vanwolleghem et al., 2015).358The innate immune response does not signicantly contribute359to the pathogenesis of liver injury or to viral clearance, indicating360that HBV can spread and remain undetected until an adaptive361immune response is mounted (Iannacone et al., 2006). During the362initial phase of infection, the virus appears to suppress toll-like363receptor (TLR)-mediated innate immune responses (Riordan et364al., 2006; Wu et al., 2009). Studies have shown that HBeAg (precore365protein) is the effector protein that modulates the innate immune366response through interaction with TLR domains to suppress signal-367ing cascades and down-regulate downstream inammatory factors368such as nuclear factor kappa B (NF-jB) (Walsh and Locarnini,3692012). In another study, pretreatment of hepatocytes with370HBsAg, HBeAg, or HBV virions almost completely abrogated371TLR-induced antiviral activity (Wu et al., 2009). During acute372HBV infection, NK cell activation and capacity for interferon

High Replicave,Low Inammatory

High HBV DNANormal or low ALTHBeAg(+)High serum levels of

HBeAg & HBsAgMild or no

necroinammaonNo or slow brosis

progressionDecreased IL-10, IL-6,

IL-8 & TNF- No HBV DNA mutaons

Immune Clearance High changing to low

or undetectable HBVDNA

High decreasing tonormal ALT

Acute or intermient hepas

Declining HBeAg & HBsAg Eventual loss of HBeAg High changing to minimal

necroinammaon Emergence of core and

precore mutaons

HBeAg(-) Chronic

Moderate to high HBV DNA

High but uctuangALT

Low HBsAg levels Persistent hepas Necroinammaon Progressive liver

disease Immune clearance

aemptsineecve

Non-Replicave Low or

undetectable HBV DNA

HBeAg(-) Very low HBsAg

levels Normal ALT

HBsAg Loss/Occult Hepas B

Serum HBV DNAphases, alternangundetectable and very low but detectable

Detectable HBV DNAin the liver

Intrahepac replicaon-competentHBV genomes such as HBV cccDNA

Integrated HBV DNA

Serum HBV DNA

ALT

Normal ALT / Undetectable HBV DNA

High Replicave,Low Inammatory

High HBV DNANormal or low ALTHBeAg(+)High serum levels of

HBeAg & HBsAgMild or no

necroinammaonNo or slow brosis

progressionDecreased IL-10, IL-6,

IL-8 & TNF- No HBV DNA mutaons

Immune Clearance High changing to low

or undetectable HBVDNA

High decreasing tonormal ALT

Acute or intermient hepas

Declining HBeAg & HBsAg Eventual loss of HBeAg High changing to minimal

necroinammaon Emergence of core and

precore mutaons

HBeAg(-) Chronic

Moderate to high HBV DNA

High but uctuangALT

Low HBsAg levels Persistent hepas Necroinammaon Progressive liver

disease Immune clearance

aemptsineecve

Non-Replicave Low or

undetectable HBV DNA

HBeAg(-) Very low HBsAg

levels Normal ALT

HBsAg Loss/Occult Hepas B

Serum HBV DNAphases, alternangundetectable and very low but detectable

Detectable HBV DNAin the liver

Intrahepac replicaon-competentHBV genomes such as HBV cccDNA

Integrated HBV DNA

Serum HBV DNA

ALT

Normal ALT / Undetectable HBV DNA

Fig. 4. Major phases of chronic hepatitis B virus infection. The natural history of CHB can be divided into 5 major phases: high replicative, low inammatory; immuneclearance; HBeAg() chronic hepatitis; non-replicative; and HBsAg loss/occult hepatitis. These phases do not occur in all patients, and transitions between them are dynamicand can be non-consecutive.

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373 (IFN)-gamma production is reduced (Dunn et al., 2009). Chronic374 infection was also found to impair NK cell cytolytic function and375 cytokine production (Lunemann et al., 2014). In addition to sup-376 pressing innate immune responses, the use of a transcriptional377 template (cccDNA) sequestered in the nucleus of infected cells378 may allow the virus to evade detection by the innate immune sys-379 tem (Ferrari, 2015).380 HBV also interferes with the adaptive immune response through381 multiple mechanisms. Early functional virus-specic CD4+ and382 CD8+ T-cell responses are attenuated, coinciding with a surge in383 the immunosuppressive cytokine IL-10, while type I IFN, IL-15,384 and IFN-k1 are not induced (Dunn et al., 2009). Patientswho resolve385 HBV infection have superior HBV specic CD4+ and CD8+ T cell fre-386 quency and function when compared to patients who become387 chronically infected (Bertoletti et al., 2009). CHB has been asso-388 ciated with an impaired ability of CD8+ T cells to lyse, proliferate,389 and produce certain cytokines (TNF-a and IL-2), as well as with a390 reduced ability to produce IFN-c (Bertoletti et al., 2009). It has been391 postulated that this T cell exhaustion is caused by prolonged expo-392 sure to high levels of soluble HBV antigens (HBeAg and, particularly,393 HBsAg). High amounts of HBsAg have been shown to exhaust CD8+394 and CD4+ T cells (Kondo et al., 2013).395 Reduction of serum HBV antigen levels may therefore be one396 strategy to restore T-cell responsiveness. Indeed, in HBeAg()397 chronic HBV patients on NUC therapy, a gradual reduction in398 HBsAg levels was associated with gradual and persistent recovery399 of HBV-specic T-cell function which got progressively more com-400 plete as a function of the HBsAg decline (Boni et al., 2012). Novel401 therapies are being developed that may result in a more complete402 and/or more rapid reduction in viral antigens with the hope that403 this will lead to an even higher degree of T-cell restoration.404 Additional improvement in T-cell function may be achieved by405 combinations with other strategies that synergize with antigen406 reduction. Among these are TLR9 agonists (Jiang et al., 2010),407 TLR7 agonists (Lanford et al., 2013) and programmed death 1408 (PD-1) inhibitors (Tzeng et al., 2012).

409 6. Goals of therapy and denitions of cure

410 Historically, the goals of therapy for CHB have been the411 reduction of viremia and ameliorization of hepatic dysfunction,412 with the expectation that these would lead to the prevention or413 slowdown of progression to cirrhosis, liver decompensation, and414 development of HCC. To that end, various treatment endpoints415 have been chosen, including virologic (HBV DNA levels), serologic416 (HBeAg/HBsAg loss/seroconversion), biochemical (normalization417 of ALT), and histologic (reduced necroinammation or reversion418 of brosis) responses (Kuo and Gish, 2012).419 With the advent of new, curative therapies for patients with420 hepatitis C, a discussion has developed to dene what it means421 to be cured of chronic HBV. Recently, Block et al. proposed422 denitions of a cure, based on a patients residual risk of death423 from liver disease (Table 1) (Block et al., 2013). They identied424 three possible types of cure:

425 absolute, in which the patient is virus-free and returned to his426 state of health prior to illness, including his age- and427 gender-matched likelihood of developing cirrhosis or HCC;428 functional, in which the patient has returned to a state of429 health equivalent to that of a person who has recovered430 spontaneously from HBV infection, and has similar likelihood431 of developing cirrhosis or HCC; and432 apparent virologic, dened as a sustained off-drug433 suppression of virologic markers and the normalization of liver434 function.

435

436The latter denition encompasses a sustained virological437response (SVR), a continuous suppression of viral load following438the cessation of therapy, but adds the loss of all circulating viral439markers (seroclearance), possibly with suppression of cccDNA.440One complicating factor in the case of HBV in contrast to HCV is441the observation that patients who have achieved a serologic reso-442lution of infection (loss of HBsAg, undetectable serum HBV DNA,443appearance of anti-HBs) can experience reactivation of their dis-444ease as a consequence of immunosuppression or the use of445anti-inammatory medications (Hoofnagle, 2009; Perrillo et al.,4462015; Reddy et al., 2015; Seetharam et al., 2014). In addition, it447is important to note that in occult infection, despite the complete448loss of HBsAg and nondetectable or very low HBV DNA in serum,449an increased risk may remain for progression to cirrhosis and the450development of HCC (Pollicino and Raimondo, 2014; Pollicino451and Saitta, 2014; Raimondo et al., 2013).452A cure that involves the complete elimination of all viral DNA453from the body (a sterilizing cure) with removal of all cccDNA454and integrated virus may therefore be difcult or impossible to455prove and may turn out to be unachievable. However, this should456be the ultimate goal of future therapies. In any event, given the457improved outcome of patients who seroclear after acute infection,458as inmost adults, there is still a large public health benet in achiev-459ing functional cure or apparent virological cure in chronically460infected patients, even if a sterilizing cure proves to be unattainable.461Apparent virologic or functional cure as a desirable end-462point for therapy is supported by a recent study looking at the risk463of HCC in patients with or without spontaneous seroclearance of464HBV seromarkers (Liu et al., 2014). In that study, the hazard ratio465of developing HCC after seroclearance of HBeAg, HBV DNA and466HBsAg during follow-up was 0.63, 0.24, and 0.18, respectively.467The authors concluded that HBeAg seroclearance itself may not468be adequate to reduce HCC risk, but that loss of HBV DNA alone469and loss of HBV DNA together with HBsAg seroclearance were470important predictors of reduced HCC risk. Among HBeAg()471patients with detectable serum DNA at study entry, the lifetime472cumulative incidence of HCC in those with seroclearance in both473HBV DNA and HBsAg, seroclearance of HBV DNA alone, or sero-474clearance of neither was 4.0%, 6.6% and 14.2%, respectively.

4757. Existing therapies

476Seven therapeutics are now approved by the US Food and Drug477Administration for the treatment of CHB: standard or pegylated478IFN-alpha and 5 oral nucleos(t)ide analogs. Current guidelines call

Table 1Proposed denitions of absolute, functional and apparent virological cure, fromBlock et al. (2013).

Parametermeasured

Absolute cure Functional cure Apparentvirologicalcure

Risk of deathfrom liverdisease

Same as a personwho was neverinfected

Same as a person withnaturally resolvedinfection

To bedetermined

Viral load Undetectable Undetectable UndetectableHBsAg Undetectable Undetectable UndetectablecccDNA Undetectable Undeteca Undetectable

or repressedHBsAb Present Variable Present or

absentTreatment

statusOff-drug Off-drug Off-drug

a Functional cure is not universally associated with loss of viral DNA as indicatedby the reactivation events observed in patients receiving immunosuppressivedrugs, even after decades of off treatment viral control (Perrillo et al., 2015;Seetharam et al., 2014).

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479 for the treatment of chronic HBV patients with elevated circulating480 levels of viral DNA, ALT and AST, as they are at the greatest risk of481 developing progressive liver disease and HCC (EASL, 2009; Liaw et482 al., 2008; Lok and McMahon, 2009).

483 7.1. Reverse transcriptase inhibitors

484 Nucleos(t)ide analogs (NUCs) target the reverse transcriptase485 (RT) function of the HBV polymerase and therefore interfere with486 the synthesis of viral DNA from pregenomic RNA (Fig. 2). NUCs487 approved for the treatment of HBV fall into three groups:

488 L-nucleosides, including lamivudine and telbivudine; acyclic phos-489 phonates, including tenofovir and adefovir; and deoxyguanosine490 analogs, including entecavir. Table 2 shows the rates of response491 for current NUCs. Entecavir and tenofovir have more potent anti-492 viral activity, leading to high rates of patients with loss of circulat-493 ing HBV DNA, ALT normalization and/or histologic improvement.494 This has been shown to lead to long-term benets for patients. A495 meta-analysis pooling 5 studies showed that the incidence of496 HCC was reduced by 78% in the NUC treatment arm, especially in497 HBeAg(+) patients (Sung et al., 2008). In HBeAg(+) patients, the loss498 of serum HBeAg and seroconversion to an anti-HBe status have tra-499 ditionally been considered major therapeutic milestones, as clear-500 ance of HBeAg, whether spontaneous or after antiviral therapy, was501 found to reduce the risk of hepatic decompensation and improve502 survival (Lok and McMahon, 2009).503 Therapy with NUCs leads to HBeAg seroconversion in approxi-504 mately 20% of patients at 4852 weeks; however, when treatment505 is stopped, up to 50% of patients revert back to HBeAg positivity506 within 6 months (Chien and Liaw, 2008). Additionally, as the507 increasing prevalence of long-term NUC therapy has resulted in508 an increased number of HBeAg() patients, it has become clear509 that these patients still have a high risk of fatal outcomes related510 to liver cancer or cirrhosis, such that HBeAg status can no longer511 be considered the most useful marker to begin or end therapy512 (Frenette and Gish, 2009; Liu et al., 2014).513 NUCs are administered orally and are very well tolerated (Jafri514 and Lok, 2010). A major concern with long-term NUC treatment515 is the selection of antiviral resistance mutations. The rate at which516 resistant mutants are selected is related to pretreatment serum517 HBV DNA level, rapidity of viral suppression, duration of treatment,518 and prior exposure to NUC therapies. Among the approved NUC519 therapies for hepatitis B, lamivudine is associated with the highest520 and entecavir and tenofovir with the lowest rates of drug resis-521 tance in NUC-nave patients (Jafri and Lok, 2010).522 NUCs are administered until the desired endpoint is achieved.523 Ideally, treatment should continue until HBsAg loss is conrmed.524 However, in contrast to the viral DNA level, which may drop multi-525 ple logs during the rst year of NUC therapy, the level of HBsAg526 reduction is generally less than 1 log. Rates of HBsAg loss after

52735 years of NUC therapy are roughly in the 35% range (Chang528et al., 2010). It was therefore estimated that the predicted median529time to HBsAg loss would be 36 years for HBeAg(+) and 39 years530for HBeAg() patients (Zoutendijk et al., 2011). This is consistent531with their mechanism of action as RT inhibitors do not directly532suppress viral transcription, translation or cccDNA.533Complete suppression of viral replication would, in theory, after5341020 hepatocyte half-lives, lead to the death of all infected cells.535In practice, however, viral DNA persists even after signicantly536longer treatment periods (Block et al., 2013). This may be due to537incomplete suppression of replication even in patients with unde-538tectable serum HBV DNA or the existence of a drug-refractory viral539reservoir. This is consistent with the recent observation that con-540tinued NUC therapy (median 10.5 years) reduced serum viral loads541by more than 6 logs, while serum HBsAg, total intrahepatic HBV542DNA and cccDNA were reduced by 71.5%, 99.9% (3 logs), and54399.9% (3 logs), respectively (Lai et al., 2014). In the same study,54445% of patients had undetectable cccDNA, although a small amount545of HBV DNA was still detectable in all patients. In practice, this546means that NUC therapy of chronic HBV is highly effective at sup-547pressing viral replication, and over a prolonged period of time548results in a reduction in cccDNA, but rarely leads to the loss of549HBsAg and a functional or apparent virologic cure. In consequence,550long-term or life-long therapy is usually required.

5517.2. Interferon-a and PEG-interferon-a

552Alpha interferons (IFN-a) are pleiotropic cytokines with anti-553viral, antiproliferative, and immunomodulatory effects. Upon bind-554ing of IFN-a to its receptor, multiple interactions between Janus555kinases (JAKs) and signal transducers and activators of transcrip-556tion (STATs) cause a variety of signaling responses, including557induction of a diverse set of IFN-stimulated genes (ISGs) and many558antiviral proteins (Der et al., 1998; Rawlings et al., 2004). The abil-559ity of IFN-a to induce an antiviral state is important to host560immune defense against viral diseases (Goodbourn et al., 2000).561IFN-a has been shown to inhibit HBV transcription (Belloni et al.,5622012), nucleocapsid assembly and stability (Xu et al., 2010), HBV563protein processing (Robek et al., 2002), and the nuclear export564and stability of HBV RNAs and pgRNA (Pei et al., 2014). In addition565to direct antiviral effects, IFN-a affects nearly all phases of innate566and adaptive immune responses to viral infection (Goodbourn et567al., 2000). IFN-a enhances MHC class I protein expression and568thereby promotes CD8+ T cell responses. It affects other cell types569including the induction of macrophage activity, NK cell cytotoxi-570city, CTL activity and decrease in neutrophil activation (Brassard571et al., 2002a). Additionally, IFN-a participates in the transition of572the initial host innate response into an effective adaptive immune573response by regulating the activity of other cytokines and chemo-574kines (Brassard et al., 2002a).

Table 2Response rates of current therapies for HBeAg(+) and HBeAg() chronic HBV. Response rates are at 48 or 52 weeks of treatment. Histologic improvement is based on post-treatment biopsies. Data are from Lok and McMahon (2009) and Jafri and Lok (2010).

Lamivudine Entecavir Tenofovir PegIFNa PegIFNa + lamivudine Placebo

HBeAg(+)HBeAg seroconversion 1621 21 21 27 24 46Undetectable HBV DNA 4044 67 76 25 69 016HBsAg loss

575 IFN-a was licensed for the treatment of CHB in most countries576 in the early 1990s. Its advantages are a nite treatment period577 (6 months to 1 year) and absence of resistance mutations.578 Pegylated IFN-a (pegIFN-a) has the advantage of more convenient579 administration (weekly subcutaneous/sc injection for 48 weeks vs.580 thrice weekly sc injection for 612 months) when compared to581 standard IFN-a. In HBeAg(+) patients, treatment with pegIFN-a582 leads to loss of detectable HBV DNA in 25% of patients (Table 2).583 Viral suppression at the end of therapy was more pronounced in584 the group that received combination therapy with pegIFN-a and585 lamivudine. HBeAg seroconversion was observed in 27% of586 patients, a result comparable to combination therapy with587 pegIFN-a with lamivudine. In HBeAg() patients (Table 2), viral588 suppression was most marked for the combination therapy of589 pegIFN-a plus lamivudine. HBsAg loss is observed in approxi-590 mately 38% of peg-IFN-a treated patients at 4852 weeks in both591 HBeAg(+) and HBeAg() patients (Buster and Janssen, 2006;592 Lampertico, 2015; Lok and McMahon, 2009). HBsAg clearance593 has been shown to continue to occur in the years after treatment594 completion. In a retrospective evaluation of HBeAg(+) patients595 treated with IFN, HBsAg loss developed in 23% of patients during596 a median follow-up of 8.8 years (van Zonneveld et al., 2004). In597 another study of HBeAg() patients, 4 years after completion of a598 year of treatment with pegIFN-a, 11% of patients achieved HBsAg599 clearance, compared with only 2% of those treated with lamivudine600 (Marcellin et al., 2008).601 Several predictors for response to IFN-a therapy have been602 identied. In HBeAg(+) patients, an elevated pretreatment ALT603 level is the strongest predictor of HBeAg seroconversion. Other fac-604 tors include low HBV DNA level, high histologic activity index, and605 genotypes A and B (Heathcote, 2003; Lok and McMahon, 2009).606 The main disadvantages of IFN-a are the need for parenteral607 administration and the frequent adverse effects. IFN-a is contrain-608 dicated in patients with decompensated cirrhosis because of the609 risk of severe sepsis and worsening liver failure (Jafri and Lok,610 2010). It is also not advisable in patients with severe exacerbations611 of chronic HBV or acute liver failure, and in those undergoing612 immunosuppressive or cancer chemotherapy. The most common613 adverse effects of IFN-a are u-like symptoms of headaches, fevers,614 chills, myalgia, and malaise. Whereas these symptoms tend to615 diminish with continued therapy, the effect on mood does not dis-616 appear until treatment cessation; irritability and depression may617 even increase with duration of therapy (Heathcote, 2003). IFN-a618 treatment is associated with a are in ALT in 2540% of patients619 (Flink et al., 2005; Lok and McMahon, 2009). Host-induced hepati-620 tis ares, characterized by an ALT elevation with a concomitant or621 subsequent decrease in HBV DNA levels, are considered to be an622 indicator of a favorable response (Flink et al., 2005), but they can623 also lead to hepatic decompensation, especially in patients with624 underlying cirrhosis (Lok and McMahon, 2009). Combination ther-625 apy with NUCs and pegIFN-a is rapidly evolving with the advent of626 trials with tenofovir (TDF) and entecavir (ETV), given either in627 combination with pegIFN-a or sequentially.

628 8. Current treatment recommendations for chronic hepatitis B

629 8.1. High-replicative, low-inammatory and nonreplicative phases

630 Treatment is currently not generally recommended for patients631 in the high-replicative, low-inammatory or nonreplicative phases632 of infection due to the low efcacy of IFN-a or NUCs in patients633 with a normal ALT level when efcacy is dened as HBeAg con-634 version, HBsAg clearance or normalization of ALT. However, in this635 phase patients on nucleos(t)ide therapy will often achieve unde-636 tectable HBV DNA. Lamivudine treatment in HBeAg(+) patients

637has led to HBeAg seroconversion in only 2% of those with normal638ALT, compared to 21% of patients with ALT 25 times normal,639and 47% of patients with ALT >5 times normal (Perrillo et al., 2002).640A recent study compared tenofovir monotherapy to combina-641tion therapy with tenofovir and emtricitabine in HBeAg(+)642nucleos(t)ide-nave patients with high HBV DNA levels (mean643baseline level of 8.41 log10 IU/mL) and normal ALT (Chan et al.,6442014). At 192 weeks, viral suppression was better in the combina-645tion therapy group (76% with undetectable HBV DNA) compared to646the monotherapy group (55%) but the HBeAg seroconversion rate647was low in the tenofovir monotherapy group (3/64 patients; 5%)648and did not occur in the combination therapy group; HBsAg loss649did not occur in either group.650Prior to the initiation of antiviral therapy, the long-term risk of651developing resistance to NUCs must be considered, especially if the652likelihood of achieving a serologic endpoint is low (Chien et al.6531999; Perrillo et al., 2002; Marcellin et al., 2004; Lau et al.,6542005). However, development of resistance is a non-issue with655tenofovir and a very rare event with entecavir in treatment-nave656patients. Patients in the high-replicative, low-inammatory or657nonreplicative phases should be closely monitored with serial658ALT and HBV DNA measurements every 36 months, and imaging659such as elastography to conrm that they have not progressed or660reverted to chronic hepatitis (Lok and McMahon, 2009).

6618.2. HBeAg(+) chronic hepatitis B

662In HBeAg(+) chronic hepatitis, the rst treatment objectives are663viral suppression, normalization of ALT, and achievement of the664serologic endpoints of HBeAg loss, HBeAg seroconversion, and ulti-665mately HBsAg loss, with or without HBsAg seroconversion to666anti-HBs(+). IFN treatment can be considered in patients with pre-667dictors of IFN response (elevated ALT, moderate to low HBV DNA668level, and HBV genotypes A and B vs C or D). It offers the advantage669of a nite 12-month course of therapy (Erhardt et al., 2005; Janssen670et al., 2005). With NUCs, treatment duration is indenite. However,671once blood HBV DNA levels have become undetectable and HBeAg672in the blood is lost (with possible seroconversion), treatment dis-673continuation can be considered (but only with HBsAg loss). This674rationale for HBsAg control as a standard for discontinuation of675treatment is the lack of the durability of the HBeAg loss and sero-676conversion off treatment in many patients, although the rate of677HBeAg seroconversion may be increased with consolidation ther-678apy (continuation of oral therapy for at least 612 months beyond679when HBeAg seroconversion is achieved) (Frenette and Gish, 2009)680HBsAg loss is rare with short or intermediate-term therapy.

6818.3. HBeAg() chronic hepatitis B

682Because HBeAg seroconversion is not possible in HBeAg()683patients, and HBsAg seroconversion is rare, the objectives of ther-684apy are ALT normalization and achievement of undetectable HBV685DNA. Unlike in HBeAg(+) patients, there are no clear686pre-treatment IFN responsiveness predictors. NUC treatment687yields undetectable HBV DNA in a larger percentage of HBeAg()688patients, compared to those who are HBeAg(+), in large part689because of their 12 log10 lower average baseline HBV DNA levels.690Given the high risk of relapse with therapy discontinuation691(9097%), even in patients who have achieved sustained ALT nor-692malization and undetectable HBV DNA, lifelong therapy is gener-693ally recommended (Santantonio et al., 2000; Hadziyannis et al.,6942003; Lai et al., 2006; Lai et al., 2007; Marcellin et al., 2008;695Shouval et al., 2009). Unfortunately, HBsAg clearance is quite rare696in HBeAg() patients during NUC therapy and is less than 17% with697IFN. When quantitative HBsAg changes are used as predictors of698response or nonresponse on therapy, providers can guide

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699 treatment continuation or discontinuation with moderate to high700 level of quality.

701 9. New therapeutic approaches

702 In order to achieve signicant rates of sustained virologic703 responses or functional cures, antivirals with new mechanisms of704 action are needed, with many currently under development705 (Table 3). Direct-acting antivirals (DAAs) targeting various steps706 of the HBV life cycle are under investigation. These new707 approaches are the subjects of many of the invited articles in the708 present symposium (see forthcoming review by Block and collea-709 gues and other papers cited below).710 Given the important role that core/capsid formation plays in the711 viral life cycle, multiple capsid inhibitors are being developed (see712 forthcoming review by Zlotnick et al. and (Delaney et al., 2002;713 Deres et al., 2003; Wang et al., 2012)). Capsid inhibitors interfere714 with HBV RNA packaging and capsid assembly, resulting in715 reduced intracellular capsids and ultimately undetectable HBV716 DNA. Because capsids are also recycled into the nucleus to replen-717 ish cccDNA, as well as exert additional nuclear functions that718 maintain viral infection, capsid inhibitors also indirectly affect719 cccDNA maintenance. Some capsid inhibitors have also been720 reported to directly interfere with cccDNA transcription and stabi-721 lity (Belloni et al., 2014).722 Novel therapies directly targeting cccDNA are also under devel-723 opment. Disubstituted sulfonamide (DSS) compounds target the724 conversion of rcDNA to cccDNA (Cai et al., 2012)while DNA cleavage725 enzymes are engineered to degrade cccDNA directly (see forthcom-726 ing paper by Guo and Guo in this symposium, and (Bloom et al.,727 2013; Cradick et al., 2010)). Viral expression vectors encoding these728 enzymes can be utilized for delivery to hepatocytes (Weber et al.,729 2014). Another direct antiviral strategy is based on RNA interfer-730 ence, which has resulted in signicant reductions in viral RNAs, pro-731 teins, and viral DNA (Wooddell et al., 2013). This approach is732 discussed in a forthcoming symposium paper by Gish et al.733 In addition to DAAs, indirect antiviral treatments are in devel-734 opment, which aim to interfere with host-cell pathways that sup-735 port viral persistence and/or to restore anti-HBV immune736 responses. Among these are entry inhibitors targeting the HBV737 receptor NTCP (see (Yan et al., in press) in this symposium and738 (Lucifora et al., 2013; Nkongolo et al., 2014; Petersen et al.,739 2008)). The HBV secretory pathway is another target for indirect740 antivirals. To date, multiple compounds that inhibit HBV secretion741 have been described (Mahtab et al., 2011; Xu et al., 2014; Yu et al.,742 2011), most of which interfere with intracellular processing and743 release of HBsAg (see forthcoming symposium articles by744 Cuconati and colleagues and by Vaillant et al.). Potential

745disadvantages of these therapies could be that intracellular746HBsAg accumulation could lead to storage diseases, and blockage747of virion synthesis could increase intracellular recirculation of748rcDNA, leading to increased cccDNA copy numbers (Zeisel et al.,7492015).750The emerging concept of trained immunity discussed above751potentially has substantial therapeutic implications. The earlier752model, in which HBV established chronic infection by exploiting753the immaturity of the neonates immune system, resulting in754immune exhaustion (immune tolerance), called for approaches755which would need to overcome anergy to HBV in the host. Doing756this would likely require a very broad-based immune or metabolic757activation, or even immune reconstitution, to overcome immune758exhaustion. Such reconstitution of innate and adaptive immune759responses against HBV is currently an area of active research and760development. Agents being studied include toll-like receptor 7761(TLR 7) (Lanford et al., 2013; Menne et al., 2015) and TLR 9 agonists762(Goldstein and Goldstein, 2009), lymphotoxin-b receptor agonists763(Lucifora et al., 2014), PD-1 and programmed death ligand 1764(PD-L1) inhibitors (Tzeng et al., 2012), and therapeutic vaccines765(Gaggar et al., 2014; Kosinska et al., 2013; Martin et al., 2014)766(see forthcoming symposium articles by Chang and Guo,767Durantel and colleagues and Roggendorf et al. on these topics).768With the trained immunity model, on the other hand, we must769consider that, rather than being tolerized, the host (the neonate and770probably the adolescent) is already matured somewhat following771birth, and is actually very capable of responding immunologically772with broad cross-protective responses to viral protein antigen773expression. We propose that it might be possible to use interfering774RNA and anti-sense tools to modulate viral protein expression, in a775way that would allow this competent immune system to become776fully active and achieve long-term suppression of HBV.

77710. Future directions and opportunities

778If the goal is to reduce the risk of progression of chronic hepa-779titis B to cirrhosis and HCC, currently available therapies, especially780lifelong NUC treatments, can be seen as an important advance.781However, as discussed above, to achieve maximum benet, espe-782cially regarding reduction of HCC risk, durable HBsAg seroclear-783ance must be obtained. The currently available therapies fall well784short in this regard. There is a clear need for treatments that not785only induce sustained virologic, biochemical and histologic786responses but also clear HBsAg (a functional cure). Patient787well-being would be importantly enhanced if this could be accom-788plished durably off-therapy. An increased understanding of trained789immunity and of how HBV establishes a permissive state in the790host that bears no characteristics of immunologic tolerance may

Table 3Categories of emerging therapies against chronic hepatitis B, from Zeisel et al. (2015). Symposium articles that review certain of these emerging therapies are identied in thetext.

Direct-acting antivirals Indirect-acting antivirals

Target Approach Target Approach

HBV polymerase Small molecule RT inhibitors Sodium taurocholate co-transporting peptide

Entry inhibitors

HBV capsid Capsid inhibitors Host factors involved in HBVsecretion and budding

HBV virion secretion inhibitors,HBsAg secretion inhibitors

rcDNA-ccc DNA conversion Disubstituted sulfonamide Innate immune response Lymphotoxin-b agonists, TLRagonists, cytokines, STING1

cccDNA DNA cleavage enzymes Adaptive immune response PD-1 and PD-L1 inhibitors,therapeutic vaccines

Modify histonesHBV RNA RNA interference, Antisense

1 Stimulator of interferon genes.

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791 allow for the development of therapies targeted towards patients792 not currently indicated for treatment. This could result in expand-793 ing therapeutic options to many more patients, including treat-794 ment at a younger age and a much earlier stage of disease.795 Optimally, treatment would provide complete loss of cccDNA as796 well as loss of viral DNA integrated into the host genome. This goal797 is complicated by the fact that HBV manipulates host responses in798 complex ways in order to sustain chronicity. New technologies are799 being developed that target viral and host factors and pathways800 previously not accessible to therapy. Given the complexity of this801 disease and precedents with other viruses such as human immu-802 nodeciency virus (HIV) and hepatitis C virus (HCV), it stands to803 reason that a combination of therapeutic approaches may be804 required to achieve the highest rates of response, including both805 a functional and virologic cure. Ultimately, we will test these indi-806 vidual and combined therapeutic approaches in the clinic, and will807 be able to determine which medication(s) can provide the best808 immunologic model of viral control and how we may be able to809 use immune modulation in combination with direct-acting anti-810 viral agents to achieve optimal therapeutic outcomes.

811 References

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