Chronic hepatitis B (CH-B) is characterized by inflammatory

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1 PERSPECTIVES IN CLINICAL HEPATOLOGY New Targets and Possible New Therapeutic Approaches in the Chemotherapy of Chronic Hepatitis B Jordan Feld, 1 Jia-yee Lee, 2 and Stephen Locarnini 2 Chronic hepatitis B (CH-B) is characterized by inflammatory liver disease of variable severity driven by persistent replication of the hepatitis B virus (HBV). 1 The development of a safe and effective hepatitis B surface antigen recombinant vaccine was an important milestone towards achieving control of CH-B, and its widespread implementation has dramatically reduced the incidence of infection. 2 However, for those individuals chronically infected with HBV, antiviral chemotherapy represents the best prospect of controlling active replication and thereby preventing life-threatening hepatic disease. 1 In this article, potential virus and host targets for possible future therapeutic intervention will be highlighted and reviewed, because existing therapies, either approved or in clinical trial, still have the disadvantage of low response rates and selection of resistance. 3-5 Antiviral Therapy: Goals of Therapy Abbreviations: CH-B, chronic hepatitis B; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; IFN-, interferon alfa; LMV, lamivudine; ADV, adefovir dipivoxil; IL-2, interleukin 2; cccdna, covalently closed circular DNA; pgrna, pregenomic RNA; APC, antigen-presenting cell; Th, helper T lymphocyte; CTL, cytotoxic T lymphocyte; TNF-, tumor necrosis factor ; sirna, small interfering RNA; FCV, famciclovir. From the 1 Department of Medicine, The Toronto Hospital, University of Toronto, Toronto, Ontario, Canada; and 2 Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia. Received March 27, 2003; accepted June 27, Address reprint requests to: Stephen Locarnini, M.D., Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn St., North Melbourne, Victoria 3051, Australia. stephenlocarnini@compuserve.com; fax: (61) Copyright 2003 by the American Association for the Study of Liver Diseases /03/ $30.00/0 doi: /jhep The complex interplay between the HBV-infected hepatocyte and the host immune response greatly influences the clinical course of disease and, consequently, strategies for clinical management. CH-B infection generally consists of an initial replicative phase with active liver disease developing during the elimination stage of hepatitis B e antigen (HBeAg)-positive CH-B, which involves HBeAg seroconversion and remission of liver disease. 1 After HBeAg-seroconversion, a number of patients continue to experience active hepatitis due to the selection and persistence of HBV mutants that are unable to express HBeAg, a hallmark of HBeAg-negative CH-B. 1 As morbidity and mortality in CH-B are linked to the development of cirrhosis and hepatocellular carcinoma, the goals of antiviral therapy are (1) to induce disease remission, (2) to arrest disease progression to cirrhosis, and (3) to block the sequelae of liver failure and/or hepatocellular carcinoma. Interferon alfa (IFN- ), lamivudine (LMV) and adefovir dipivoxil (ADV) are the only approved treatments for CH-B. While these treatments have similar short-term benefits in HBeAg-positive and HBeAg-negative CH-B patients, they have limited long-term efficacy. There is, therefore, a need for new treatment strategies for CH-B that can overcome some of these limitations. Several anti- HBV compounds are currently under evaluation (Table 1). Some of these including entecavir, 6 emtricitabine 7 (the 5-fluorinated derivative of LMV), clevudine (L- FMAU), 3,4 and other L-nucleosides such as L-deoxythymidine (L-dT), 8 are at various stages of clinical trials. The efficacy of immunomodulatory approaches such as interleukin 2 (IL-2), IFN-, thymosin or vaccinebased therapies have either been disappointing or have produced conflicting results. 3,4 Therapeutic and DNA vaccine approaches, although theoretically attractive, have not progressed beyond clinical trials. 4,5 Two pegylated interferons, PEG-IFN -2b (Peg-Intron) and PEG- IFN -2a (Pegasys), which have shown promise for the treatment of chronic hepatitis C, 9 are currently being evaluated for CH-B. The future development of these approaches should provide clinicians with more therapeutic options. While new therapeutic agents for CH-B are on the horizon, issues such as long-term efficacy and drug resistance will continue to pose challenges in patient management. 3 Furthermore, it is not known whether combinations of antivirals and immunomodulatory agents will improve the clinical end points for CH-B. 10 The Viral Life Cycle: Where Are the New Targets? The mature HBV virion is composed of a nucleocapsid core surrounded by a lipid bilayer incorporating the viral 545

2 546 FELD, LEE, AND LOCARNINI HEPATOLOGY, September 2003 Table 1. Therapeutic Agents Available for Treating HBV Infections Registered Company Under Development Company Interferon-alfa Roche/Schering Plough Entecavir Bristol Myer Squibb Lamivudine GlaxoSmithKline Emtricitabine [( )FTC] Gilead Sciences Adefovir Dipivoxil Gilead Sciences Clevudine (L-FMAU) Gilead Sciences Tenofovir (HIV Indication) Gilead Sciences Telbivudine (L-dT) Novartis-Idenix Thymosin-alpha SciClone Valtorcitabine (Valyl-L-dC) Novartis-Idenix LY Eli Lilly and Mitsubishi Pharma Corporation Racevir ( FTC) Pharmasset MIV-210 (FLG-prodrug*) Medivir/GlaxoSmithKline PEG-interferon Roche/Schering Plough *FLG, 2,3 -dideoxy-3 -fluoroguanosine. Some Asian countries. surface proteins. Residing within the nucleocapsid is the compact 3.2-kb partially double-stranded DNA genome that exists in a relaxed circular configuration with the genetic information organized into overlapping but frame-shifted open reading frames coding for the surface (L, M, S), core, precore (HBeAg), polymerase (pol), and the X proteins. An understanding of the HBV life cycle is crucial for the identification of potential antiviral targets. 11,12 HBV replication begins when the virion attaches to an as yet identified receptor on the hepatocyte surface (Fig. 1). Following viral entry, the virus uncoats and is transported to the nucleus where the relaxed circular genome is converted by host cellular machinery to the covalently closed circular DNA (cccdna); the cccdna is, in turn, organized into viral minichromosomes. This key replicative intermediate is the transcriptional template for the production of the various HBV RNAs, including the pregenomic RNA (pgrna), that are necessary for viral replication and represents one of the major obstacles in the development of effective treatments for the control of CH-B. Fig. 1. Diagrammatic representation of HBV replication in the context of its life cycle. The early events in the life cycle of receptor-mediated endocytosis and nuclear transport are not fully elucidated. The intracellular conversion pathway (ICP) where newly replicated HBV genomes are shuttled back to the nucleus for conversion to cccdna to replenish the viral minichromosome pool is marked. The key virus-specific events of packaging and reverse transcription of the pregenomic RNA (pgrna) are shown and expanded in Figs. 2 and 3, respectively. Modified from Delaney et al. 3,4

3 HEPATOLOGY, Vol. 38, No. 3, 2003 FELD, LEE, AND LOCARNINI 547 occurs. This is facilitated by a short redundancy (r) in the negative-strand DNA template, which anneals to the r region on the 5 end of the positive-strand DNA, thereby circularizing the genome. 11,12 Premature termination of the positive-strand DNA synthesis by the pol protein results in the characteristic partially double-stranded genome. The HBV nucleocapsid containing the partial dsdna is either recycled back to the nucleus to increase the supply of cccdna, or undergoes further processing in the endoplasmic reticulum and Golgi for virion assembly (Fig. 1). Mature virions are subsequently exported from the cell via the constitutive secretory pathway. Fig. 2. The model of packaging of HBV pregenomic RNA into nucleocapsids of replicating genomic DNA based on Hu et al. 13 The viral polymerase/reverse transcriptase [RT1] associates with the cellular Hsp-90 chaperone complex, which causes the viral polymerase to undergo a conformation change [RT2-Hsp-90]. This then binds to the on pgrna and the viral polymerase undergoes a further conformational change [RT3- ]. This then provides the signal for nucleocapsid assembly and initiation of viral DNA synthesis [RT4 dgaa]. The Hsp90 complex is depicted together with the chaperone partner p23 and other factors marked as X as described in Hu et al. 13 Modified from Hu et al. 13 with permission from EMBO Journal. Transcription of the 3.5-kb pgrna serves three important roles. First, its translation leads to production of the core and pol proteins. Second, it participates in the nucleocapsid packaging reaction, the specificity of which is provided by a unique stem-bulge-stem structure known as epsilon ( ) on the 5 and 3 ends of the pgrna (Fig. 2). 13 Following translation of the pgrna, the pol protein binds to the 5 -end. Host proteins such as heat shock protein 90 (Hsp90) stabilize this pol- interaction (Fig. 2). 13 The cis-translated core proteins 14 dimerize around the pgrna-pol complex and self-assemble to form viral nucleocapsids (Fig. 2). Once packaged into a nucleocapsid, the pgrna serves its third and most important role as the template for reverse transcription and DNA synthesis (Fig. 3). The pol protein is bound to the 5 -structure and acts as its own primer for initiation and synthesis of the first 3 nucleotides of the negative-strand DNA. 11,12 This nascent DNA is then translocated to the 3 end of the pgrna where it binds to the complementary sequence within a 12-nt region known as direct repeat 1 (DR1*; Fig. 3). From here, negative-strand DNA synthesis proceeds and the RNase H activity of the pol protein degrades all but the last few nucleotides of the template pgrna. This RNA oligomer is translocated to the 3 copy of the 12-nt repeat known as DR2 from which point positive-strand DNA synthesis begins (Fig. 3). Elongation of the positive-strand DNA proceeds to the 5 end of the negative-strand DNA where a third strand transfer Fig. 3. The HBV reverse transcription of pgrna into genomic DNA. The terminally redundant pgrna is indicated by a wavy line and the 3 direct repeats are shown as numbered boxes. The 2 epsilon stem-loop structures are located at the 5 and 3 ends. The newly replicated DNA is indicated by a solid line. The polymerase/reverse transcriptase (Pol) is represented by the shaded oval. Following translation of the pgrna, the pol binds to the 5 copy of, protein primes, and then copies part of the bulge (GAA), after which the complex is translocated to DR1* and the primer is extended from there. Modified from Tavis, 11 and Nassal and Schaller. 12

4 548 FELD, LEE, AND LOCARNINI HEPATOLOGY, September 2003 Pathogenesis of CH-B: What Is the Host Doing? Because the liver disease caused by acute or chronic HBV infection is largely immune mediated, understanding the complex and diverse immune responses to HBV has potential immunotherapeutic implications. To this end, animal models such as HBV-infected chimpanzees and HBV transgenic mice have led to significant milestones in understanding the role of the immune system in causing liver disease and in controlling and/or eliminating the virus. 15,16 Although useful, there are limitations to the use of animal models, particularly the HBV-transgenic mouse model, which lacks the key HBV replication event, namely, the generation and processing of cccdna. The course of HBV infection is influenced by the rigor of the immune response. Activation of both the innate and adaptive immune responses during the early stages of infection is critical to HBV clearance. Antigen-presenting cells (APC), particularly dendritic cells, NK, and NKT cells, play an important role in stimulating CD4 helper T lymphocytes (Th) and CD8 cytotoxic T lymphocytes (CTL) for the control of HBV replication in the liver. 17,18 Th cells are divided into Th-type 1 (Th1) and Th-type 2 cells (Th2), based on their cytokine-secreting profile. Th1 cells predominantly produce IL-2, which induces the primarily cell-mediated immunity, whereas Th2 cells produce IL-4, IL-5, and IL-10, which trigger the humoral immune response for the production of antibodies. HBV clearance can be achieved when both the Th1 cells and CTL are activated to generate a multispecific response to the viral envelope, pol, and core proteins. 16 An immune response that predominantly activates Th2 cells for antibody production most likely leads to HBV persistence. 19 Thus, HBV persistence is likely caused by a discrepancy in the T-cell repertoire resulting in tolerance to HBV proteins during acute infection of neonates or adults. The simplistic dogma of HBV clearance via cytokinemediated destruction of HBV-infected cells through CTL-activated perforin- or Fas-dependent pathways has now been supplanted by a new paradigm placing emphasis on a cytokine-mediated noncytolytic HBV clearance. 16 This new paradigm emerged through studies in acutely HBV-infected chimpanzees 15 and in HBV transgenic mice. 16 Cytokines, predominantly tumor necrosis factor (TNF- ), IFN-, and IFN /, were found to inhibit HBV replication without necessarily killing infected hepatocytes. Induction of these cytokines in the transgenic mouse model has been shown to interfere with at least two pathways of the HBV life cycle: post-transcriptional degradation of HBV RNA 20 and post-translational loss of HBV core protein via the elimination of immature nucleocapsids containing pgrna. 21 Key cellular processes are likely to be involved. IFN- / appears to activate two cellular pathways involving dsrna-dependent protein kinase and dsrna-dependent 2 5 oligoadenylate synthetase. It is likely that induction of PKR leads to inhibition of HBV protein synthesis, while induction of 2 5 oligoadenylate synthetase activates RNase L to degrade the HBV RNA. Involvement of cellular processes was also shown in studies reporting IFN- / and IFN- induced changes to the transcription profile of cellular genes. 22 However, the molecular antiviral significance of these cytokine-induced effects on HBV replication remains unclear. The underlying molecular basis for the post-transcriptional degradation of HBV RNA is emerging through HBV transgenic mice studies. 16 These studies have revealed a role for the cellular protein, La, in viral RNA degradation following induction of TNF- and IFN. In HBV, this protein binds specifically to a predicted stemloop structure located in the 5 end of the post-transcriptional regulatory element within the viral RNA and appears to mediate the nuclear export of the HBV RNA. Recent studies have suggested that specific HBV RNA binding sites on the La protein could serve as potential antiviral targets. 20 The noncytolytic processes of the immune response for the control of HBV replication in patients with CH-B are poorly understood. From a clinical management perspective, the challenge will be to develop an immunotherapy strategy that can overcome T-cell hyporesponsiveness with minimal infiltration of inflammatory cells or liver cell necrosis, 23,24 and to exploit noncytolytic clearance of HBV from hepatocytes. Possible New Targets, Therapies, and Directions The review of the HBV life-cycle (Fig. 1) reveals that, apart from reverse transcription (Table 1), most viral processes are dependent on host-cell machinery. The most important of these is the generation and persistence of cccdna. Conventional antiviral inhibitors of viral DNA synthesis such as nucleoside/nucleotide analogues can prevent or reduce the development of new molecules of cccdna (Figs. 1 and 3). However, successful elimination of the existing pool of hepadnaviral cccdna has only been achieved by either a noncytolytic Th1 immune response 15,19 or immune-mediated cell killing followed by hepatocyte cell division. 25,26 In this context, it is important to note that treatment of CH-B with nucleoside analogues can result in the (partial) restoration of (specific) immunoresponsiveness, which appears necessary for durable host-mediated control of infection. 27 Collectively,

5 HEPATOLOGY, Vol. 38, No. 3, 2003 FELD, LEE, AND LOCARNINI 549 the concept of successful therapy for CH-B is converging on the use of both antiviral and immunomodulating approaches. Small Molecule Inhibitors Polymerase Inhibitors: Nucleoside and Nucleotide Analogues. Nucleoside/nucleotide analogues are metabolized by cellular kinases into the triphosphate form, which can then selectively and competitively inhibit the catalytic properties of the viral polymerase. Incorporation of analogues that lack a functional equivalent of the 3 hydroxyl group on the sugar moiety (such as LMV or ADV) results in chain termination of nascent viral genomes. Furthermore, purine nucleoside analogues can potentially inhibit the priming activity of the polymerase since the nucleotides present in the primed sequence of HBV minus-strand DNA are 5 -GAA ,28 As shown in Table 1, a number of nucleoside/nucleotide analogues are being tested for use in CH-B. Except for entecavir, LY582563, and MIV-210, these compounds are in the unnatural L conformation. Thus, a major concern is that HBV-bearing LMV-resistance mutations such as rtl180m plus rtm204v/i may have significant cross-resistance to these L-nucleosides. 3-5 Viral Packaging Inhibitors. Several compounds have recently been developed that have a mechanism of HBV inhibition that is unrelated to the viral polymerase. The first of these are the phenylpropenamide derivatives, AT-61 and AT-130. King et al. 29 showed that AT-61 did not affect total HBV RNA production or HBV DNA polymerase activity but did significantly reduce the production of encapsidated RNA. Importantly, both AT-61 and AT-130 have identical antiviral activity against wildtype as well as a number of different LMV-resistant strains of HBV. 30 The mechanism of action of AT-130 has been examined by Feld and colleagues 31 using the recombinant baculovirus system that packages HBV RNA in cis, and an HBV core protein expression vector system, which packages in trans. In vitro studies using AT-130 showed significant inhibition of the production of encapsidated HBV RNA, but had no effect on total HBV RNA and did not affect core protein or nucleocapsid production, indicating an interference with the encapsidation process itself (Fig. 2). Steric inhibition or interaction with the host cell chaperone proteins such as Hsp90, may be possible mechanisms. Phenylpropenamides are not water-soluble and have extremely low bioavailability. Their future successful development as antiviral agents will depend on overcoming potential toxicity and medicinal chemistry issues. However, these observations represent an important proof of principle that both wild-type and drug-resistant HBV can be significantly and selectively inhibited at a level of replication that is independent of the viral polymerase. A second class of compounds, the heteroaryldihydropyrimidines, are also potent non-nucleoside inhibitors of HBV replication both in vitro and in vivo. 32 The heteroaryldihydropyrimidine compounds were developed by the Bayer Corporation and include the candidate molecule, Bay , and congeners, Bay and Bay Exposure of HBV-infected cells to Bay resulted in increased degradation of core protein through improper formation of viral nucleocapsids. These HAP compounds were shown to have efficacy against HBV in the HBV-transgenic mouse model and to possess suitable preclinical pharmacokinetic and toxicology profile. 32 Its novel mechanism of action and highly specific antiviral activity indicates that future clinical studies are warranted. A third compound, LY582563, is a 2-amino-6-arylthio- 9-phosphonomethoxyethylpurine bis (2,2,2-trifluoro-ethyl) ester, a novel nucleotide analogue derivative of phosphonomethoxyethyl purine, is in a structural class that is similar to ADV. This compound has excellent antiviral activity against HBV with a good preclinical toxicity profile. 33 It is also effective against LMV-resistant HBV 34 and its mechanism of action and early clinical development are under investigation by the Eli Lilly and Mitsubishi Pharma Corporation. Gene Therapy Approaches: The Promise of Gene Silencing Gene therapy is defined as the introduction of new genetic material into a target cell with a therapeutic benefit to the individual. 35 Several genetic antiviral strategies including ribozymes, antisense oligonucleotides, 36 interfering peptides or proteins, and therapeutic DNA vaccination have been explored for the molecular therapy of CH-B. Furthermore, molecular strategies to treat or prevent the long-term sequelae of CH-B related cirrhosis and HCC are being investigated (see reviews 5,35,36 ). A novel molecular strategy that holds promise is the use of small interfering RNAs (sirna). RNA interference is a cellular process of sequence-specific gene silencing in which small duplexes of RNA target a homologous sequence for cleavage by cellular ribonucleases. 37 The introduction of approximately 22-nt sirnas into mammalian cells can lead to specific silencing of cellular mrnas without induction of the nonspecific interferon responses that are activated by longer RNA duplexes. Post-transcriptional gene silencing mediates resistance to both endogenous, parasitic, and exogenous pathogenic nucleic acids and can regulate the expression of protein-coding genes. 37 This approach has been successfully applied to HBV 38 and hepatitis C virus replicon RNAs in cell culture. 39 The

6 550 FELD, LEE, AND LOCARNINI HEPATOLOGY, September 2003 sirna molecules dramatically reduced virus-specific protein expression and RNA synthesis, and these antiviral effects were independent of IFN. While the approach of sirna, along with conventional gene therapy approaches, holds great promise, issues such as gene delivery, stability, toxicity, resistance, and safety need to be resolved. 5,35,36 Immunomodulatory Approaches to Controlling HBV Infection The goal of immunomodulatory therapy is to stimulate the host s immune response to HBV-infected cells, and thereby terminate/eliminate viral infection. As IFN- and thymosin- have been reviewed elsewhere, 4,5 this review will highlight other potential immunomodulatory agents for the treatment of CH-B. IL-12 and IL-18. IL-12 and IL-18 are secreted by activated macrophages and dendritic cells. 40 IL-12 is a cytokine that plays a critical role in the regulation of the immune system by promoting a Th1 response. 40 IL-18 is an IFN- inducing factor that can act on Th1 cells, NK cells, and dendritic cells to produce IFN- in the presence of IL ,41 The significance of the Th1 versus Th2 response with respect to HBV infection has been discussed in the section on pathogenesis of chronic hepatitis B. Experiments conducted in HBV-transgenic mice have indicated that IL-12 injection results in a marked downregulation of viral replication in the liver and extrahepatic sites. 42 In this model, IL-12 appears to exert antiviral effects indirectly by inducing lymphocytes locally to secrete IFN-, IFN- /, and TNF-. Examination of patients undergoing IFN- therapy has revealed that levels of IL- 12, as well as IL-2 and IFN- (also Th1 cytokines), were significantly higher in patients that responded to IFN- therapy in comparison to nonresponders. 43 A recent phase I/II study of IL-12 in CH-B patients showed that doses of 0.25 and 0.5 g/kg produced a significant reduction in serum HBV DNA after 12 weeks of treatment. However, the overall antiviral efficacy was lower than that reported for IFN- or LMV. 44 Importantly, this study showed that a short course of IL-12 therapy was safe, and with similar side effects to those observed with IFN- therapy. In transgenic mice studies, Kimura et al. 41 showed that IL-18 inhibits HBV replication in the liver, via a noncytopathic process that is mediated by IFN- and IFN- /. These investigators also showed that IL-18 can synergize with IL-12 to inhibit HBV replication, indicating that coadministration of IL-18 and IL-12 may have therapeutic potential for the treatment of CH-B. Dendritic Cell Vaccination. Dendritic cells play a vital role as APCs in stimulating adaptive immunity during viral infection as they prime naïve CD4 and CD8 T cells to produce cytokines that affect the Th1/Th2 immune balance. 45 To serve as APCs, dendritic cells internalize endogenous or exogenous antigens, which are processed in the MHC class I or II pathways for presentation to T cells. In addition to their APC function, activated mature dendritic cells secrete a variety of Th1 and Th2 type cytokines including IFN- /,TNF-, IL-1, IL-8, and IL Information on the role of dendritic cells in influencing Th1 or Th2 response during HBV infection is limited. It is likely that impairment of dendritic cell function may contribute to HBV chronicity. 46,47 Interestingly, while dendritic cells act as potent stimulators of immunity, these cell can also induce immune tolerance through T-cell anergy or depletion. 48 Dendritic cell mediated tolerance is not well understood, and it remains to be determined whether these cells contribute to neonatal tolerance leading to HBV persistence. The effectiveness of dendritic cells in manipulating the host immune response is currently being exploited clinically. The ex vivo culture and priming of human dendritic cells with antigens such as recombinant tumor and viral antigens, followed by autologous transfusion has been explored as a form of immunotherapy in several clinical trials. 49 The role of dendritic cell immunotherapy for CH-B is still in its infancy, and strategies for the priming of dendritic cells with appropriate HBV proteins that will overcome immunologic tolerance and induce a multispecific immune response to HBV will need to be investigated. 50,51 Dynamics/Kinetic Studies and HBV: What Can Be Learned? Studies of viral dynamics in acute or chronic HBV have been performed and have provided important insights into the pathogenesis of host-virus responses Furthermore, improvements in HBV DNA testing technology and mathematical modelling have resulted in several important advances in the field. 52,56 The pathogenesis of CH-B is characterized by a dynamic equilibrium between viral production and clearance. 56,57 The introduction of antiviral therapy can upset this equilibrium by inhibiting virus production and causing a decline in the viral load. Indeed, the rate of decline in the viral load is a measure of the rate of viral clearance and by inference, must be equivalent to the rate of virus production before therapy. 56,57 The various models proposed to describe the fall in serum HBV differ principally in their underlying assumptions regarding at least three factors: the nature and efficacy of the inhibition of viral replication that is imposed on the virus-host system by

7 HEPATOLOGY, Vol. 38, No. 3, 2003 FELD, LEE, AND LOCARNINI 551 Table 2. Comparative Dynamics Among the Three Viral Infections Caused by HBV, HIV, and HCV HBV ADV* HBV LMV HIV Ritonavir HCV IFN- HBV LMV or LMV FCV Plasma virus Half-life (h) Mean lifespan (h) Mean viral generation time (d) Daily turnover (%) Daily production (plasma) ( ) Total load ( ) Infected cells Half-life (d) Mean lifespan (d) Daily turnover (%) *The HBV ADV data from Tsiang et al. 54 The HBV LMV data from Nowak et al. 53 The HIV and HCV data reviewed in Lewin et al. 57 The HBV LMV/LMV FCV data from Lewin et al. 57 Modified from Tsiang et al. 54 nucleoside analogue therapy, the behavior of the infected cell population after the commencement of therapy, and ultimately the residual effect of this population on the level of viremia. 57 Tsiang et al. 54 followed the decline in serum virus caused by ADV monotherapy in patients with CH-B, and quantified the level of serum HBV using the Roche MONITOR PCR-based assay (Roche Diagnostics, Branchburg, NJ). These investigators reported a consistent biphasic decay in treated patients. Regression analysis allowed the calculation of a mean viral half-life of days, a mean infected cell half-life of 18 7 days, and a mean antiviral efficacy of (Table 2). Lau et al., 55 using the Digene Hybrid Capture II assay (Digene Diagnostics, Beltsville, MD) to monitor viral load, compared the effect of LMV monotherapy and LMV plus famciclovir (FCV) combination therapy on the treatment dynamic equilibrium. A mean virus half-life of 2 days and a mean infected cell half-life of approximately 40 days were calculated. The antiviral efficacy of the combination therapy was found to be significantly greater than that of LMV monotherapy. 55 More recently, Lewin et al. 56 showed that the viremia of CH-B during potent combination antiviral therapy could not be entirely described by biphasic curves. Once therapy was initiated, there was a mean delay of 1.6 (from 1 to 5) days, before an antiviral effect could be detected, and in some patients, an increase in viral load was seen, after which a biphasic or typically a multiphasic decay of viremia occurred (see Fig. 4A vs. B). In most patients, two patterns for the first phase elimination (decay of viremia) was observed with either a rapid (t 1/2 of 1.0 hour) or a slow (t 1/2 of 92 hours) slope being observed. This was not related to pretreatment viral load, serum alanine aminotransferase level or the type of antiviral therapy (LMV monotherapy vs. LMV plus FCV combination therapy). For these patients, the second phase elimination (elimination of infected cells) was then either flat or slow with a t 1/ days. In some patients, a complex or stair-case pattern was seen with further phases of viral decline or phases with little change in viral load (Fig. 4A and B). These complex decay profiles could possibly represent impaired cytolytic and noncytolytic mechanisms of infected cell loss and highlight the complexity of the CH-B carrier, reflected in the outcome of the virus-cell-host immune response interaction. However, these profiles do need to be better understood in terms of developing alternative therapeutic approaches to improve the management of HBV-infected individuals. Conclusions: Is Combination Chemotherapy the Way Forward? The emergence of drug-resistant HBV as a consequence of long-term antiviral therapy and the low sustained/durable responses of existing therapies signal the need for new approaches. Viral dynamic studies have pointed to a problem with the second phase of HBV decline that follows on from the initial rapid response to chemotherapy. This will be a major challenge to overcome, but new insights into HBV pathogenesis and the development of novel immunotherapies may provide useful ways forward. Whether this is by using additional nucleoside/nucleotide analogues or adding existing immunemodulators to nucleoside/nucleotide analogue combinations is, at this time, unknown but warrants further clinical investigation. The next logical step may be to

8 552 FELD, LEE, AND LOCARNINI HEPATOLOGY, September 2003 Fig. 4. (A) Hypothetical schematic diagram showing the changes in serum HBV DNA levels (y axis; in copies/ml) following the initiation of potent antiviral therapy plotted against time (x axis). The first phase represents clearance of free virions, whereas the second phase represents clearance of infected hepatocytes. This pattern is typically seen in patients with chronic hepatitis C who have a sustained response to antiviral therapy. 56,57 LOD, level of detection of assay. (B) Composite diagram showing the complex and variable changes in serum HBV DNA viral load (y axis; in copies/ml) following initiation of combination nucleoside analogue (LMV plus FCV) therapy seen in two patients. 56,57 The slope of the first phase was found to vary considerably, whereas the slope of the second phase was often flat (broken line), or prolonged or with a staircase pattern of decay (unbroken line). This complex pattern includes further phases of viral DNA decline and phases with little change in viral load (unbroken line) or even an increase (broken line). Adapted from Lewin et al. 56,57 initiate trials of triple therapy with two nucleoside/nucleotide analogues in combination with PEG-IFN. In the meantime, the development of the future generation of anti-hbv antiviral agents including non-nucleoside analogues such as packaging inhibitors and immunotherapies such as dendritic cell vaccines, may well be needed to finally achieve a cure for CH-B. References 1. Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003;23: Kane M. Global status of hepatitis B immunization. Lancet 1996;348: Delaney WE IV, Locarnini S, Shaw T. Resistance of hepatitis B virus to antiviral drugs: current aspects and directions for future investigation. Antivir Chem Chemother 2001;12: Delaney WE IV, Bartholomeusz A, Locarnini S. Evolving therapies for the treatment of chronic hepatitis B virus infection. Expert Opin Investig Drugs 2002;11: Karayiannis P. Hepatitis B virus: old, new and future approaches to antiviral treatment. J Antimicrob Chemother 2003;51: Lai CL, Rosmawati M, Lao J, Van Vlierberghe H, Anderson FH, Thomas N, Dehertogh D. Entecavir is superior to lamivudine in reducing hepatitis B virus DNA in patients with chronic hepatitis B infection. Gastroenterology 2002;123: Gish R, Leung N, Wang C, Sacks S, Fried M, Wright T, Huy T, et al. Antiviral activity, safety and incidence of resistance in chronically infected hepatitis B patients (CHB) given once daily emtricitabine for 2 years [Abstract]. HEPATOLOGY 2002;36(Part 2):372A. 8. Lai C-L, Leung N, Teo E, Tong M, Wong F, Hann HW, Han S, et al. International multicenter trial of LDT (Telbivudine), alone and in combination with lamivudine, for chronic hepatitis B: an interim analysis [Abstract}. HEPATOLOGY 2002;36(Part 2):301A. 9. Reddy KR, Wright TL, Pockros PJ, Shiffman ML, Everson G, Reindollar R, Fried MW, et al. Efficacy and safety of pegylated (40-kd) interferon a-2a compared with interferon a-2a in noncirrhotic patients with chronic hepatitis C. HEPATOLOGY 2001;33: Shaw T, Bowden S, Locarnini S. Chemotherapy for hepatitis B: new treatment options necessitate reappraisal of traditional endpoints. Gastroenterology 2002;123: Tavis J. The Replication Strategy of the Hepadanaviruses. Viral Hepatitis Rev 1996;2: Nassal M, Schaller H. Hepatitis B virus replication an update. J Viral Hepatitis 1996;3: Hu J, Toft D, Seeger C. Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. EMBO 1997;16: von Weizsacker F, Kock J, Weiland S, Beck J, Nassal M, Blum H. Cis- Preferential recruitment of duck hepatitis B virus core protein to the RNA/ polymerase preassembly complex. HEPATOLOGY 2002;35: Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV. Viral clearance without destruction of infected cells during acute HBV infection. Science 1999;284: Guidotti LG, Chisari FV. Noncytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 2001;19: Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 2000; 192: Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice. J Immunol 2002;169: Thimme R, Wieland S, Steiger C, Ghrayeb J, Reimann KA, Purcell RH, Chisari FV. CD8( ) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol 2003;77: Horke S, Reumann K, Rang A, Heise T. Molecular characterization of the human La protein hepatitis B virus RNA.B interaction in vitro. J Biol Chem 2002;277: Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of alpha/ beta interferon eliminates viral RNA- containing capsids in hepatitis B virus transgenic mice. J Virol 2000;74: Wieland SF, Vega RG, Muller R, Evans CF, Hilbush B, Guidotti LG, Sutcliffe JG, et al. Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J Virol 2003; 77: Diepolder HM, Ries G, Jung MC, Schlicht HJ, Gerlach JT, Gr ner N, Caselmann WH, et al. Differential antigen-processing pathways of the hepatitis B virus e and core proteins. Gastroenterology 1999;116:

9 HEPATOLOGY, Vol. 38, No. 3, 2003 FELD, LEE, AND LOCARNINI Milich D. Do T cells see the hepatitis B core and E antigens differently? Gastroenterology 1999;116: Guo JT, Zhou H, Liu C, Aldrich C, Saputelli J, Whitaker T, Barrasa MI, et al. Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections. J Virol 2000;74: Kajino K, Jilbert AR, Saputelli J, Aldrich CE, Cullen J, Mason WS. Woodchuck hepatitis virus infections: very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte. J Virol 1994;68: Boni C, Bertoletti A, Penna A, Cavalli A, Pilli M, Urbani S, Scognamiglio P, et al. Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B. J Clin Invest 1998;102: Lanford RE, Notvall L, Beames B. Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells. J Virol 1995;69: King RW, Ladner SK, Miller TJ, Zaifert K, Perni RB, Conway SC, Otto MJ. Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-l- 2,3 dideoxy-3 -thiacytidine. Antimicrob Agents Chemother 1998;42: Delaney WE IV, Edwards R, Colledge D, Shaw T, Miller TG, Isom H, Furman P, et al. The phenylpropenamide derivatives AT-61 and AT-130 inhibit replication of both wild-type and lamiduvine resistant strains of hepatitis B virus in vitro. Antimic Agents Chemother 2002;46: Feld J, Colledge D, Sozzi T, Edwards R, Painter G, Locarnini S. The phenylpropenamide derivative AT-130 inhibits HBV replication at viral encapisdation and packaging [Abstract]. HEPATOLOGY 2002;36(Part 2): 300A. 32. Deres K, Schroder CH, Paessens A, Goldmann S, Hacker HJ, Weber O, Kramer T, et al. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids. Science 2003;299: Kamiya N, Kubota A, Iwase Y, Sekiya K, Ubasawa M, Yuasa S. Antiviral activities of MCC-478, a novel and specific inhibitor of hepatitis B virus. Antimicrob Agents Chemother 2002;46: Ono-Nita SK, Kato N, Shiratori Y, Carrilho FJ, Omata M. Novel nucleoside analogue MCC-478 (LY582563) is effective against wild-type or lamivudine-resistant hepatitis B virus. Antimicrob Agents Chemother 2002;46: Blum HE. Gene therapy of viral hepatitis. In: Tsuji T, Higashi T, Zeniya M, Meyer zum Buschenfelde KH, eds. Molecular Biology and Immunology in Hepatology. Chapter 8. Freiburg, Germany: Elsevier Science B.V., 2002: Branch A. A hitchhiker s guide to antisense and nonantisense biochemical pathways. HEPATOLOGY 1996;24: Hannon GJ. RNA interference. Nature 2002;418: Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference. HEPATOLOGY 2003;37: Randall G, Grakoui A, Rice C. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. PNAS 2003;100: Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol 2001;19: Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. J Virol 2002;76: Cavanaugh VJ, Guidotti LG, Chisari FV. Interleukin-12 inhibits hepatitis B virus replication in transgenic mice. J Virol 1997;71: Rossol S, Marinos G, Carucci P, Inger M, Williams R, Naoumov N. Interleukin-12 induction of Th1 cytokines is important for viral clearance in chronic hepatitis B. J Clin Invest 1997;99: Carreno V, Quiroga JA. Biological properties of interleukin-12 and its therapeutic use in persistent hepatitis B virus and hepatitis C virus infection. J Viral Hepat 1997;4(Suppl 2): Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392: Akbar SM, Horiike N, Onji M, Hino O. Dendritic cells and chronic hepatitis virus carriers. Intervirology 2001;44: Beckebaum S, Cicinnati VR, Dworacki G, Muller-Berghaus J, Stolz D, Harnaha J, Whiteside TL, et al. Reduction in the circulating pdc1/pdc2 ratio and impaired function of ex vivo-generated DC1 in chronic hepatitis B infection. Clin Immunol 2002;104: Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 2001;19: Nestle FO, Banchereau J, Hart D. Dendritic cells: On the move from bench to bedside. Nat Med 2001;7: Shimizu Y, Guidotti LG, Fowler P, Chisari FV. Dendritic cell immunization breaks cytotoxic T lymphocyte tolerance in hepatitis B virus transgenic mice. J Immunol 1998;161: You Z, Huang XF, Hester J, Rollins L, Rooney C, Chen SY. Induction of vigorous helper and cytotoxic T cell as well as B cell responses by dendritic cells expressing a modified antigen targeting receptor-mediated internalization pathway. J Immunol 2000;165: Whalley SA, Murray JM, Brown D, Webster GJ, Emery VC, Dusheiko GM, Perelson AS. Kinetics of acute hepatitis B virus infection in humans. J Exp Med 2001;193: Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H. Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci U S A 1996;93: Tsiang M, Rooney JF, Toole JJ, Gibbs CS. Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy. HEPA- TOLOGY 1999;29: Lau GK, Tsiang M, Hou J, Yuen S, Carman WF, Zhang L, Gibbs CS, et al. Combination therapy with lamivudine and famciclovir for chronic hepatitis B-infected Chinese patients: a viral dynamics study. HEPATOLOGY 2000;32: Lewin S, Ribeiro RM, Walter T, Lau GKK, Bowden DS, Locarnini S, Perelson AS. Analysis of hepatitis B viral load decline under potent therapy: complex decay profiles observed. HEPATOLOGY 2001;34: Lewin S, Walters T, Locarnini S. Hepatitis B treatment: rational combination chemotherapy based on viral kinetic and animal model studies. Antiviral Res 2002;55: