Clinical Infectious Diseases SUPPLEMENT ARTICLE
Eradication Strategies for Chronic Hepatitis B Infection Eleanor M. P. Wilson, Lydia Tang, and Shyam Kottilil Division of Clinical Care and Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore
Chronic hepatitis B infection affects >300 million people worldwide and is a leading cause of liver failure and cancer. Current approaches to treatment for chronic hepatitis B involve suppression of hepatitis B virus (HBV) DNA with the use of nucleoside analogues. Chronic suppressive therapy rarely results in a “functional cure” or absence of detectable HBV DNA in plasma and loss of detectable hepatitis B surface antigen after cessation of therapy. The major obstacles to achieving a functional cure are the presence of covalently closed circular DNA and ineffective/exhaustive immune system. This review focuses on novel approaches to target viral life cycle and host immunity to achieve a functional cure. Keywords. HBV; eradication; exhaustion.
Current therapies for chronic hepatitis B virus (HBV) infection seek to suppress viral replication and reduce viremia in order to prevent the progression of liver disease. Whereas sustained suppression of circulating HBV viremia improves clinical outcomes, including slowing or even reversing fibrosis [1] and reducing rates of hepatocellular carcinoma (HCC) [2], it does not address intrahepatic viral persistence and thus does not constitute a cure. Some patients are able to achieve functional cure after prolonged therapy with interferon (IFN) or nucleos (t)ide (NUC)–based therapies, or a combination of these [3– 7]. However, rates of viral clearance are low: A 48-week course of pegylated IFN-α–based therapy leads to viral clearance and hepatitis B surface antigen (HBsAg) loss in just 3%–7% of patients [4, 6] and is associated with treatment-related side effects, resulting in low rates of viral control and high rates of discontinuation. Although oral NUC-based regimens are better tolerated, these function as suppressive therapy for most, with even fewer patients (about 1% per year) achieving durable suppression and HBsAg loss [3, 5]. Many patients will require lifelong therapy, and interruption of NUC-based therapy may result in a resurgence of viral replication and flare of hepatic disease [8]. The pitfalls of long-term suppressive therapy include adverse events from long-term use of NUC therapy and the potential for the development of viral resistance to widely used NUC analogues. Here, we will discuss ongoing efforts to control HBV replication, purge covalently closed circular DNA (cccDNA) reservoirs, and develop long-lasting protective immunity in patients with chronic HBV infection.
Correspondence: S. Kottilil, Institute of Human Virology, University of Maryland, 725 W Lombard St, Baltimore, MD 21201 ([email protected]). Clinical Infectious Diseases® 2016;62(S4):S318–25 Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: 10.1093/cid/ciw044
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DEFINING CURE: HEPATITIS B ERADICATION ENDPOINTS AND MECHANISMS OF PERSISTENCE
There are several markers used to evaluate the efficacy of current HBV therapies, including virologic and serologic endpoints. Suppression of hepatitis B viral replication without measurable serum HBV DNA when tested by a sensitive polymerase chain reaction assay during and after therapy is used as a virologic endpoint, but viral replication typically rebounds after treatment is stopped. Serological response for HBsAg is defined as HBsAg loss. This can occur with or without seroconversion, which is the development of protective anti–hepatitis B surface antibody [9]. HBsAg loss is by far the most valuable surrogate marker for treatment outcome and correlates well with prevention of complications such as cirrhosis and HCC [10, 11]. Functional cure is defined as the absence of plasma HBV DNA and HBsAg after stopping antiviral therapy, with or without HBsAg seroconversion. Understanding HBV persistence is critical for designing eradication strategies for chronic HBV infection. Impaired or ineffective host immune responses, resulting in uncontrolled HBV replication and cccDNA formation, characterize chronic infection. Both viral and host factors contribute to HBV persistence, and both must be addressed to eliminate the infection. To achieve an eradication of HBV that is synonymous with cure, novel therapeutics (Table 1) must target the reservoir that persists in hosts, cccDNA, and reconstitute the exhausted, ineffective T-cell responses that have failed to clear the infection. Viral Targets
HBV consists of a partially double-stranded relaxed circular DNA (rcDNA) genome (Figure 1); upon entry into human hepatocytes, this rcDNA is converted into cccDNA in the host cell nucleus, where it is sequestered, tightly coiled in association with histones and DNA chaperone proteins as a minichromosome, and thus able to evade detection by innate DNA sensing
Table 1.
Novel Therapeutics Against Hepatitis B Virus
Drug Name
Class
Phase
Company
Clinical Trials Registration
Viral targets Niuliva HBV
HBV immunoglobulin
3
Institute Ginfols, S.A.
Myrcludex-B
Entry inhibitor
Preclinical
Stephan Urban, DKFZ
NCT01131065
TAF
Nucleotide analogue
3
Gilead Sciences, Inc
NCT02296853 NCT01940341 NCT01940471 NCT02071082
ANA380
Nucleotide analogue
2
Anadys
NCT02300688
CMX157
Nucleotide analogue
1
ContraVir
NCT01080820
ARC-520
RNAi
2a
Arrowhead Research
NCT01872065
Bay 41-4109
Capsid formation inhibitor
Preclinical
AiCuris
NVR 3-778
HBV core inhibitor
1b
Novira Therapeutics
NCT02112799 NCT02401737
REP 2139-Ca
Blocks HBsAg release
1b
REPLICor Inc
NCT02233075
Interferon γ-1b (Actimmune)
IFN immune enhancer: immunomodulatory
. . .
Huntington Medical Research Institute
Approved in US NCT00753467
GS-9620
TLR-7 agonist
1
Gilead Sciences, Inc
NCT02166047 NCT01591668 NCT01590654 NCT01590641
Nivolumab
PD-1 antibody
1
Bristol-Myers Squibb
NCT01658878
DV-601
Therapeutic vaccine: viral antigen complex based
1b
Dynavax
NCT01023230
Hi-8 HBV
Therapeutic vaccine: viral antigen complex based
2
Oxxon
ePA-44
Therapeutic vaccine: viral antigen complex based
2
Chongqing Jiachen Biotechnology Ltd
Host targets
NCT00869778 NCT01326546
HB-110
Therapeutic vaccine: DNA based
1
Genexine
HBV-DNA plasmid pdpSC18 vaccine
Therapeutic vaccine: DNA based
1
PowderMed/Pfizer
GS-4774
Therapeutic vaccine: Tarmogen based
2
Gilead Sciences, Inc
NCT02174276
TG1050
Therapeutic vaccine: Adenovirus based
1/1a
Transgene
NCT02428400
Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IFN, interferon; RNAi, RNA interference; TAF, tenofovir alafenamide; TLR, Toll-like receptor.
Figure 1.
Viral targets. Abbreviations: cccDNA, covalently closed circular DNA; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.
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cellular machinery. Hepatocytes with integrated HBV DNA, in either form, have a long half-life, which allows the maintenance of chronicity and acts as a reservoir for reactivation of viral genome replication [12, 13]. Although these factors, including the production of cccDNA from rcDNA, are crucial to the establishment of chronic HBV infection, they remain poorly understood. Preventing, inactivating, or eliminating cccDNA remains an important goal of chronic HBV eradication efforts. Blocking Viral Entry
HBV is an enveloped virus with tropism to infect hepatocytes; one effective way to address the persistence of cccDNA is to prevent the entry of HBV into cells. Viral entry is mediated through specific interactions of viral membrane proteins with cellular receptors, in particular the sodium taurocholate co-transporting polypeptide (NTCP), a functional receptor identified by Yan et al in the treeshrew Tupaia [14]. NTCP is a sodium-dependent transporter for taurocholic acid expressed at the basolateral membrane of hepatocytes and responsible for most Na+dependent bile acid uptake in hepatocytes. Antagonizing the interaction of NTCP with synthetic viral protein mimics is a novel method to treat chronic HBV, and therapeutic strategies that block viral entry are under active investigation. Myrcludex-B, a synthetic lipopeptide derived from the pre-S1 domain of the HBV envelope protein, which specifically targets the NTCP, has been shown to efficiently block HBV infection in vitro [15] and in a humanized mouse model (a uPA+/+-Severe Combine Immune Deficient [SCID] mouse reconstituted with human hepatocytes and infected with HBV). Mice treated with Myrcludex-B had lower HBV viremia, intrahepatic cccDNA, and antigen levels than untreated mice, showing that the synthetic lipopeptide can prevent the spread of HBV from infected human hepatocytes in vivo and hinder the amplification of the cccDNA pool in previously infected hepatocytes [16]. Inhibiting Transcription
Current NUC-based therapy blocks the production and accumulation of new cccDNA, but blocking replication alone will not impact existing cccDNA reservoirs in infected hepatocytes. With a long half-life of approximately 33–50 days [12], the intrahepatic viral cccDNA functions as a library of potential viral escape variants generated by the error-prone HBV viral polymerase, a potential source for the emergence of drug resistance or viral rebound upon cessation of effective antiviral therapy. Although NUC-based therapies alone will not address the viral reservoir, HBV eradication efforts will likely employ innovative strategies in conjunction with suppressive NUC-based therapies. An investigational NUC, tenofovir alafenamide, a prodrug of the currently approved tenofovir disoproxil fumarate, has been shown to concentrate in human hepatocytes and undergo conversion to the active form of the drug [17]. Phase 3 clinical trials are currently under way to study the S320
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drug in both human immunodeficiency virus (HIV) and HBV infection, and results are eagerly anticipated. Silencing or disrupting cccDNA, if possible, may deplete this viral reservoir. One approach to target cccDNA is to repress its transcription, making it epigenetically silent while allowing it to remain intact in the nucleus of infected hepatocytes. Treatment with IFN-α has been shown to inhibit HBV replication by reducing transcription of pregenomic RNA from the HBV cccDNA minichromosome, both in vitro and in the uPA+/+-SCID humanized HBV-infected mouse model [18]. While IFN-α therapies have high rates of treatment-limiting side effects, their ability to mediate epigenetic repression of HBV cccDNA transcriptional activity is an effect that may prove useful as part of a strategy to achieve a functional cure for chronic HBV infection. Another strategy to target cccDNA is to inactivate it. Gene therapy has undergone an evolution and expansion in recent years, and there are now a variety of sequence-specific DNA modification technologies available. Multiple different gene therapy approaches, including zinc finger nuclease (ZFN), transcription activator–like effector nuclease (TALEN), and clustered regularly interspaced palindromic repeat applications have provided options for the study and treatment of liver diseases [19, 20]. ZFN, also known as zinc finger proteins, are a versatile and effective class of gene-targeting reagents [21] that can be used to block the transcription and replication of cccDNA and production of viral proteins from infected cells in vitro [22], but have been recently also used to cleave and degrade HBV cccDNA in hepatoma cells in vivo. A ZFN pair designed by Cradick et al cleaves the HBV core gene open reading frame, reducing the pregenomic RNA by 29% [23]. More recently, Weber et al designed 3 ZFNs to target HBV polymerase, core, and X genes, creating double-stranded breaks in the cccDNA, which, when imprecisely repaired by nonhomologous end joining, led to inactivating mutations with durable effects after a single treatment in vitro [24]. Another class of nucleases, TALENs can cleave sequencespecific DNA targets. TALENs designed to target conserved regions of the HBV viral genomic DNA across genotypes and expressed in transfected cell culture can inactivate and reduce cccDNA, interfering with the production of viral antigens without apparent cytotoxic effects [25]. Interestingly, these effects were maintained in the humanized mouse model and were observed to work synergistically with IFN-α to restore HBV-suppressed IFN-stimulated response element–directed transcription [25]. RNA interference (RNAi) is another methodology that has been employed in targeting HBV transcription. Viral messenger RNA (mRNA) can be directly targeted using small interfering RNA molecules that silence genes posttranscriptionally, effectively knocking down the expression of a specific gene or genes. RNAi can be used to target mRNA for degradation
and, due to the extensive use of open reading frames within HBV’s DNA genome, multiple HBV RNAs could serve as RNAi targets [26, 27]. McCaffrey et al have demonstrated that RNAi could be applied to inhibit production of HBV replicative intermediates in mice transfected with a HBV plasmid at multiple steps: HBV RNA and DNA were substantially reduced in mouse liver, RNAi expression reduced HBsAg secreted into serum, and the number of cells with HBV core antigen staining was substantially decreased [28]. Another RNAi-based drug currently in clinical trials is a dynamic polyconjugate of an RNAi trigger and cholesterol, which, when coinjected with a hepatocyte-targeted, membrane-active peptide, is well tolerated and reduces HBsAg in a dose-dependent manner for up to 2 months [27]. Capsid Inhibitors
HBV persistence and transmission require ongoing cycles of HBV replication and assembly of the virus particle, composed of the capsid protein, polymerase, and pregenomic DNA. Interference with the assembly step is an attractive target for HBV therapeutics. Multiple classes of compounds may be able to dysregulate or inhibit virion assembly and encapsidation. Members of the family of heteroaryldihydropyrimidines have shown the ability to reduce HBV viremia in vitro and in the humanized mouse model [29]. Treatment with one heteroaryldihydropyrimidine, Bay 41–4109 [30], resulted in incorrectly assembled or disassembled core capsids into monomers or dimers, from which they could be further degraded into peptides [31]. Preclinical studies with Bay 41–4109 and the related compound GLS4 have shown potent antiviral activity, including sustained suppression of HBV DNA after cessation of treatment in most humanized mice, without toxicity to hepatocytes [32]. Additionally, another chemical group, the phenylpropenamides, has also been found to inhibit viral encapsidation and is active against lamivudine-resistant strains of HBV [33, 34]. Phenylpropenamides induce tertiary and quaternary structural changes in HBV capsids. One candidate, AT-130 (a phenylpropenamide derivative), has been shown to bind a promiscuous pocket at the dimer–dimer interface that favors a unique quasiequivalent binding site in the capsid and can serve as an effective antiviral agent. Viral production is disrupted after virion assembly is initiated prematurely, resulting in morphologically normal capsids that are empty and noninfectious [35]. The clinical efficacy of compounds from this family has not yet been reported. A novel compound, NVR 3-778, prevents production of infectious virus by causing core proteins to misassemble into capsid-like particles in vitro [36]. Preclinical studies have shown that treatment of HBV-producing cell lines with NVR 3-778 leads to inhibition of RNA encapsidation and downstream disruption of the production and secretion of infectious HBV virions. NVR 3-778 was active against both wild-type and several tested HBV NUC-resistant variants [36]. The compound
is now in phase 1b clinical trials, with results expected as early as summer 2016. Preventing HBV Antigen Secretion
The exact mechanisms by which HBV escapes immunity remain poorly understood. Recent reports have suggested that the immunosuppression in the early stages of HBV infection is a direct result of high circulating levels of HBsAg, acting directly on dendritic cells to limit cytokine production [37, 38], leading to T-cell exhaustion [39]. One innovative therapeutic strategy targets the secretion of HBsAg with a synthetic small molecule, HBF-0259, an aromatically substituted tetrahydrotetrazolo-(1, 5-a)-pyrimidine. HBF-0259 selectively inhibits the secretion of subviral HBsAg and DNA-containing viral particles, without inhibiting HBeAg secretion, which is not a constituent of HBV viral particles, or HBV viral replication [40]. A derivative of this compound has been shown to be more potent and has been shown to have favorable kinetics in the humanized mouse model [41]. It remains unclear whether these compounds will effectively block secretion of HBsAg in humans, and whether blockade, in acute or chronic HBV infection, will reverse the observed T-cell anergy. Host Factors
In chronic HBV, as opposed to other chronic viral infections, there are several factors that favor the search for eradication strategies. Widely available and effective vaccines against hepatitis B have established correlates of protection. The fact that the majority of individuals acutely infected with hepatitis B will recover has established correlates of viral clearance. And finally, despite low tolerability and efficacy, the ability of IFN-based therapies to eradicate chronic HBV infection in select patients has established correlates of treatment response. Liver damage due to chronic HBV is primarily mediated by the host inflammatory response rather than a direct viral cytopathic effect [42]. An exaggerated host immune response to HBV is not only ineffective at viral eradication, but also triggers hepatic inflammation. Recovery from acute HBV infection is associated with robust and effective innate and adaptive immune responses. Strategies that address host factors seek to tip the scales toward an effective response that results in viral clearance, rather than an excessive inflammatory response that may potentiate hepatic damage. The innate immune response is the first line of defense against viral infections, resulting in production of type 1 IFNs when Toll-like receptors (TLRs) on the surface of innate immune cells are engaged (Figure 2). The type 1 IFNs, in turn, suppress viral replication, recruit natural killer cells to kill infected hepatocytes, and support the efficient maturation and recruitment of adaptive immunity through stimulating antigen-presenting cells to prime a strong and effective adaptive immune response [43]. The principal producers of type 1 IFNs are plasmacytoid dendritic cells, which also stimulate tumor necrosis factor alpha (TNF-α), interleukin 6, and cell
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Figure 2. Innate immunity and mechanisms of hepatitis B virus (HBV) persistence. Abbreviations: CCL3, C-C ligand 3; DC, dendritic cell; IFN, interferon; IL, interleukin; NK cells, natural killer cells; NKT cells, natural killer T cells; PAMP, pathogen-associated molecular patterns; PRR, pattern recognition receptor; TLR, Toll-like receptor.
surface costimulatory molecules when TLRs are stimulated. Strategies that enhance HBV-specific immune responses may risk increasing liver damage, and safety is an important consideration for any approach that seeks to modify host immune responses to HBV. Triggering Innate Immunity
In individuals with chronic HBV infection, TLR signaling is downregulated [44]; targeting the recruitment and activation of innate immune cells by agonizing the remaining TLRs, particularly TLR-7, has the potential to accelerate HBV-specific immune reconstitution and assist with clearance of HBV in chronically infected individuals. Although HBV circumvents endogenous type 1 IFNs by decreasing expression of TLRs in hepatocytes, it is plausible that the use of exogenous IFN induction using the TLR-7 pathway may reinstate the IFN-α response. There are commercially available TLR agonists that may be orally administered, allowing for rapid uptake by the liver. An investigational orally administered selective TLR-7 agonist, GS-9620, has been shown to stimulate production of IFNα and various other cytokines and chemokines and produce prolonged suppression of circulating and hepatic HBV DNA in chimpanzees when administered over 4 weeks [45]. In a randomized, double-blind phase 1b study of both treatment-naive and virologically suppressed patients with chronic HBV, GS9620 induces peripheral interferon-stimulated genes following a single or double dose in this short-term study [46]. Longerterm studies are needed to evaluate whether stimulation of TLR-7 is capable of suppressing HBV replication in humans. S322
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Reversing Immune Exhaustion
Correlates of an adaptive immune response against HBV have been shown to be protective against HBV infection in both recovered and vaccinated subjects [47]. T cells are needed to simultaneously stimulate B cells to manufacture antibodies targeted to E and S antigens while inducing cellular immune responses to kill infected hepatocytes (Figure 3). Chronic HBV infection is associated with functionally impaired immune responses, particularly within HBV-specific lymphocytes. The persistent exposure to viral antigens leads to virus-specific CD4 and CD8 cell dysfunction or exhaustion. Recent data suggest that these exhausted virus-specific T cells overexpress the PD-1 cell surface molecule [48]. In vitro data suggest that inhibition of the interaction of PD-1 with its ligands (PD-L1/2) improves the antiviral function of these exhausted T cells. Fisicaro et al examined the role of T-cell exhaustion in the pathogenesis of chronic HBV by comparing the phenotype and function of intrahepatic and circulating HBV-specific T cells and the effect of PD-1/PD-L1 blockade, and they reported that intrahepatic HBV-specific CD8 T cells express higher PD-1 and lower levels of the interleukin 7 receptor CD127 [49]. These alterations were reversed by PD-1/PD-L1 blockade, with improvement in T-cell function in peripheral and intrahepatic lymphocyte compartments. Sherman et al report that PD-1 blockade improves cytokine secretion and survival of HBV-specific CD8 T cells, from HBV-monoinfected and HIV/HBV-coinfected patients treated with adefovir [50]. Collectively, these data evidence that PD-1 blockade, in combination with nucleotide inhibitors, might provide a strategy to address the immune defects seen in chronic
Figure 3. Adaptive immunity in chronic hepatitis B infection. Abbreviations: APC, antigen-presenting cell; HBeAb, hepatitis B e antibody; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; TCR, T cell receptor; Th1, type 1 helper T cell; Th2, type 2 helper T cell; TNF, tumor necrosis factor.
HBV infection. Antibodies blocking the interaction of PD-1/ PD-L1 have been tested in patients with advanced malignancies, and have induced durable tumor regression and prolonged stabilization of disease [51], while unfortunately causing a relatively high frequency (approximately 14%) of high-grade adverse events, including severe autoimmune complications [52]. Blockade of PD-1 offers an opportunity to revive exhausted T cells in chronic HBV infection, allowing restoration of adaptive immunity against HBV. With the first PD-1 antibody, nivolumab, approved by the US Food and Drug Administration in 2014, the safety profile and risks of PD-1 blockade are being actively studied in patients with HBV-associated HCC. If it can be administered safely, further investigation into PD-1/PD-L1 blockade may inform approaches to eradication strategies for chronic HBV infection. Transfer of Adaptive Immunity
Reversal of immune exhaustion will not work if there are no HBV-specific T cells present. Studies have pointed to the clonal deletion of HBV-specific CD8+ T cells as a mechanism of HBV persistence [43]. Chimeric antigen receptors (CARs) are synthetic, engineered receptors that can engage cell-surface molecules in their native conformation, independent of antigen processing by the target cell and major histocompatibility complex [53]. T-cell responses may be “trained” to recognize infected cells by expressing HBV-specific CARs. In 2013, Krebs et al were able to show that murine CD8 T cells infected with retroviral vectors induced CARs that bind HBV envelope proteins and activate T cells [54]. They demonstrated that CD8 T
cells expressing HBV-specific CARs recognized HBV and were able to engraft and expand in an immunocompetent transgenic mouse model, where they efficiently relocated to hepatic tissues and controlled HBV replication, causing only transient liver damage [54]. Therapeutic Vaccination
Therapeutic vaccination presents a promising strategy to address HBV-specific T-cell exhaustion. A therapeutic vaccine that could induce a potent CD4 T-cell response could counteract immune tolerance to activate humoral and cytolytic immune responses against one or more HBV antigens. Several categories of therapeutic vaccines are being developed for chronic HBV infection. A yeast-based immunotherapy platform using targeted molecular antigens, or tarmogen, engineered to express HBV X, S, and C antigens were able to stimulate HBV-specific CD4 and CD8 Tcell responses, including lymphocyte proliferation, interferon-γ release, and intracellular cytokine production. When used to stimulate peripheral blood mononuclear cells isolated from patients with chronic HBV in combination with autologous dendritic cells, the candidate vaccine produced pronounced expansions of HBV antigen–specific T cells possessing a cytolytic phenotype [55]. This yeast-based immunotherapeutic vaccine platform may be capable of eliciting a functional adaptive immune response in patients with chronic HBV. A second investigational vaccine platform, an engineered vesicular stomatitis virus expressing HBV middle surface envelope glycoprotein and generating virus-like vesicles (VLVs), has been
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shown to induce functional CD8 T-cell responses in the transgenic mouse model of chronic HBV infection [56]. The ability of VLVs to stimulate T-cell responses in a tolerogenic model of chronic infection indicates that highly immunogenic viral vectors may offer hope in human chronic HBV. Adenoviral vector-based vaccines have been shown to be safe and immunogenic, but are often limited by preexisting adenoviral immunity. One nonreplicative adenovirus serotype 5 (Ad5) encoding candidate vaccine, TG1050, has been shown to induce robust, multispecific, and long-lasting HBV-specific T cells targeting 3 encoded HBV immunogens and the capacity to produce both IFN-γ and TNF-α as well as stimulating cytolytic functions. In the transgenic mouse model of chronic HBV, the candidate vaccine was shown to reduce circulating levels of HBV DNA and HBsAg, whereas alanine aminotransferase levels remained within normal limits [57]. It remains to be seen whether this vaccine strategy will be able to elicit these bifunctional responses in patients with preexisting Ad5 immunity. CONCLUSIONS
Current antiviral therapy does not induce sustained virologic remission in the majority of patients with chronic HBV infection. Novel therapeutic strategies are urgently needed. A rational therapeutic design will be required to circumvent the various factors by which HBV induces immune tolerance and adaptive immune system exhaustion and establishes a longterm viral reservoir. Targeting a combination of viral and host factors is likely to offer the best chance for accomplishing this goal. If the benefits of these approaches can be shown to outweigh the risks, these approaches may offer hope in the ongoing efforts to control HBV replication, purge cccDNA reservoirs, and develop long-lasting protective immunity. Notes Supplement sponsorship. This article appears as part of the supplement “Hepatitis B,” sponsored by the CDC Foundation and Gilead. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Marcellin P, Gane E, Buti M, et al. Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label followup study. Lancet 2013; 381:468–75. 2. Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006; 295:65–73. 3. Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med 2006; 354:1001–10. 4. Janssen HL, van Zonneveld M, Senturk H, et al. Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 2005; 365:123–9. 5. Lai CL, Gane E, Liaw YF, et al. Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med 2007; 357:2576–88. 6. Lau GK, Piratvisuth T, Luo KX, et al. Peginterferon alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med 2005; 352:2682–95.
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35. Katen SP, Tan Z, Chirapu SR, Finn MG, Zlotnick A. Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure. Structure 2013; 21:1406–16. 36. Lam A, Ren S, Vogel R, et al. Inhibition of hepatitis B virus replication by the HBV core inhibitor NVR 3-778. Hepatology 2015; 62(suppl 1):222–5A. 37. Op den Brouw ML, Binda RS, Geijtenbeek TB, Janssen HL, Woltman AM. The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells. Virology 2009; 393:84–90. 38. Xu Y, Hu Y, Shi B, et al. HBsAg inhibits TLR9-mediated activation and IFN-alpha production in plasmacytoid dendritic cells. Mol Immunol 2009; 46:2640–6. 39. Wieland SF, Chisari FV. Stealth and cunning: hepatitis B and hepatitis C viruses. J Virol 2005; 79:9369–80. 40. Dougherty AM, Guo H, Westby G, et al. A substituted tetrahydro-tetrazolopyrimidine is a specific and novel inhibitor of hepatitis B virus surface antigen secretion. Antimicrob Agents Chemother 2007; 51:4427–37. 41. Yu W, Goddard C, Clearfield E, et al. Design, synthesis, and biological evaluation of triazolo-pyrimidine derivatives as novel inhibitors of hepatitis B virus surface antigen (HBsAg) secretion. J Med Chem 2011; 54:5660–70. 42. Guidotti LG, Isogawa M, Chisari FV. Host-virus interactions in hepatitis B virus infection. Curr Opin Immunol 2015; 36:61–6. 43. Bertoletti A, Ferrari C. Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection. Postgrad Med J 2013; 89:294–304. 44. Momeni M, Zainodini N, Bidaki R, et al. Decreased expression of Toll like receptor signaling molecules in chronic HBV infected patients. Hum Immunol 2014; 75:15–9. 45. Lanford RE, Guerra B, Chavez D, et al. GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees. Gastroenterology 2013; 144:1508–17, 1517.e1–10. 46. Gane EJ, Lim YS, Gordon SC, et al. The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection. J Hepatol 2015; 63:320–8.
47. Said ZN, Abdelwahab KS. Induced immunity against hepatitis B virus. World J Hepatol 2015; 7:1660–70. 48. Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol 2007; 81:4215–25. 49. Fisicaro P, Valdatta C, Massari M, et al. Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B. Gastroenterology 2010; 138:682–93, 693.e1–4. 50. Sherman AC, Trehanpati N, Daucher M, et al. Augmentation of hepatitis B virusspecific cellular immunity with programmed death receptor-1/programmed death receptor-L1 blockade in hepatitis B virus and HIV/hepatitis B virus coinfected patients treated with adefovir. AIDS Res Hum Retroviruses 2013; 29:665–72. 51. Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 2012; 366:2455–65. 52. Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012; 366:2443–54. 53. Maus MV, Grupp SA, Porter DL, June CH. Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 2014; 123:2625–35. 54. Krebs K, Bottinger N, Huang LR, et al. T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice. Gastroenterology 2013; 145:456–65. 55. King TH, Kemmler CB, Guo Z, et al. A whole recombinant yeast-based therapeutic vaccine elicits HBV X, S and core specific T cells in mice and activates human T cells recognizing epitopes linked to viral clearance. PLoS One 2014; 9:e101904. 56. Reynolds TD, Buonocore L, Rose NF, Rose JK, Robek MD. Virus-like vesicle-based therapeutic vaccine vectors for chronic hepatitis B virus infection. J Virol 2015; 89:10407–15. 57. Martin P, Dubois C, Jacquier E, et al. TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBVpersistent mice. Gut 2015; 64:1961–71.
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Eradication Strategies for Chronic Hepatitis B Infection Eleanor M. P. Wilson, Lydia Tang, and Shyam Kottilil Division of Clinical Care and Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore
Chronic hepatitis B infection affects >300 million people worldwide and is a leading cause of liver failure and cancer. Current approaches to treatment for chronic hepatitis B involve suppression of hepatitis B virus (HBV) DNA with the use of nucleoside analogues. Chronic suppressive therapy rarely results in a “functional cure” or absence of detectable HBV DNA in plasma and loss of detectable hepatitis B surface antigen after cessation of therapy. The major obstacles to achieving a functional cure are the presence of covalently closed circular DNA and ineffective/exhaustive immune system. This review focuses on novel approaches to target viral life cycle and host immunity to achieve a functional cure. Keywords. HBV; eradication; exhaustion.
Current therapies for chronic hepatitis B virus (HBV) infection seek to suppress viral replication and reduce viremia in order to prevent the progression of liver disease. Whereas sustained suppression of circulating HBV viremia improves clinical outcomes, including slowing or even reversing fibrosis [1] and reducing rates of hepatocellular carcinoma (HCC) [2], it does not address intrahepatic viral persistence and thus does not constitute a cure. Some patients are able to achieve functional cure after prolonged therapy with interferon (IFN) or nucleos (t)ide (NUC)–based therapies, or a combination of these [3– 7]. However, rates of viral clearance are low: A 48-week course of pegylated IFN-α–based therapy leads to viral clearance and hepatitis B surface antigen (HBsAg) loss in just 3%–7% of patients [4, 6] and is associated with treatment-related side effects, resulting in low rates of viral control and high rates of discontinuation. Although oral NUC-based regimens are better tolerated, these function as suppressive therapy for most, with even fewer patients (about 1% per year) achieving durable suppression and HBsAg loss [3, 5]. Many patients will require lifelong therapy, and interruption of NUC-based therapy may result in a resurgence of viral replication and flare of hepatic disease [8]. The pitfalls of long-term suppressive therapy include adverse events from long-term use of NUC therapy and the potential for the development of viral resistance to widely used NUC analogues. Here, we will discuss ongoing efforts to control HBV replication, purge covalently closed circular DNA (cccDNA) reservoirs, and develop long-lasting protective immunity in patients with chronic HBV infection.
Correspondence: S. Kottilil, Institute of Human Virology, University of Maryland, 725 W Lombard St, Baltimore, MD 21201 ([email protected]). Clinical Infectious Diseases® 2016;62(S4):S318–25 Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: 10.1093/cid/ciw044
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DEFINING CURE: HEPATITIS B ERADICATION ENDPOINTS AND MECHANISMS OF PERSISTENCE
There are several markers used to evaluate the efficacy of current HBV therapies, including virologic and serologic endpoints. Suppression of hepatitis B viral replication without measurable serum HBV DNA when tested by a sensitive polymerase chain reaction assay during and after therapy is used as a virologic endpoint, but viral replication typically rebounds after treatment is stopped. Serological response for HBsAg is defined as HBsAg loss. This can occur with or without seroconversion, which is the development of protective anti–hepatitis B surface antibody [9]. HBsAg loss is by far the most valuable surrogate marker for treatment outcome and correlates well with prevention of complications such as cirrhosis and HCC [10, 11]. Functional cure is defined as the absence of plasma HBV DNA and HBsAg after stopping antiviral therapy, with or without HBsAg seroconversion. Understanding HBV persistence is critical for designing eradication strategies for chronic HBV infection. Impaired or ineffective host immune responses, resulting in uncontrolled HBV replication and cccDNA formation, characterize chronic infection. Both viral and host factors contribute to HBV persistence, and both must be addressed to eliminate the infection. To achieve an eradication of HBV that is synonymous with cure, novel therapeutics (Table 1) must target the reservoir that persists in hosts, cccDNA, and reconstitute the exhausted, ineffective T-cell responses that have failed to clear the infection. Viral Targets
HBV consists of a partially double-stranded relaxed circular DNA (rcDNA) genome (Figure 1); upon entry into human hepatocytes, this rcDNA is converted into cccDNA in the host cell nucleus, where it is sequestered, tightly coiled in association with histones and DNA chaperone proteins as a minichromosome, and thus able to evade detection by innate DNA sensing
Table 1.
Novel Therapeutics Against Hepatitis B Virus
Drug Name
Class
Phase
Company
Clinical Trials Registration
Viral targets Niuliva HBV
HBV immunoglobulin
3
Institute Ginfols, S.A.
Myrcludex-B
Entry inhibitor
Preclinical
Stephan Urban, DKFZ
NCT01131065
TAF
Nucleotide analogue
3
Gilead Sciences, Inc
NCT02296853 NCT01940341 NCT01940471 NCT02071082
ANA380
Nucleotide analogue
2
Anadys
NCT02300688
CMX157
Nucleotide analogue
1
ContraVir
NCT01080820
ARC-520
RNAi
2a
Arrowhead Research
NCT01872065
Bay 41-4109
Capsid formation inhibitor
Preclinical
AiCuris
NVR 3-778
HBV core inhibitor
1b
Novira Therapeutics
NCT02112799 NCT02401737
REP 2139-Ca
Blocks HBsAg release
1b
REPLICor Inc
NCT02233075
Interferon γ-1b (Actimmune)
IFN immune enhancer: immunomodulatory
. . .
Huntington Medical Research Institute
Approved in US NCT00753467
GS-9620
TLR-7 agonist
1
Gilead Sciences, Inc
NCT02166047 NCT01591668 NCT01590654 NCT01590641
Nivolumab
PD-1 antibody
1
Bristol-Myers Squibb
NCT01658878
DV-601
Therapeutic vaccine: viral antigen complex based
1b
Dynavax
NCT01023230
Hi-8 HBV
Therapeutic vaccine: viral antigen complex based
2
Oxxon
ePA-44
Therapeutic vaccine: viral antigen complex based
2
Chongqing Jiachen Biotechnology Ltd
Host targets
NCT00869778 NCT01326546
HB-110
Therapeutic vaccine: DNA based
1
Genexine
HBV-DNA plasmid pdpSC18 vaccine
Therapeutic vaccine: DNA based
1
PowderMed/Pfizer
GS-4774
Therapeutic vaccine: Tarmogen based
2
Gilead Sciences, Inc
NCT02174276
TG1050
Therapeutic vaccine: Adenovirus based
1/1a
Transgene
NCT02428400
Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IFN, interferon; RNAi, RNA interference; TAF, tenofovir alafenamide; TLR, Toll-like receptor.
Figure 1.
Viral targets. Abbreviations: cccDNA, covalently closed circular DNA; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.
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cellular machinery. Hepatocytes with integrated HBV DNA, in either form, have a long half-life, which allows the maintenance of chronicity and acts as a reservoir for reactivation of viral genome replication [12, 13]. Although these factors, including the production of cccDNA from rcDNA, are crucial to the establishment of chronic HBV infection, they remain poorly understood. Preventing, inactivating, or eliminating cccDNA remains an important goal of chronic HBV eradication efforts. Blocking Viral Entry
HBV is an enveloped virus with tropism to infect hepatocytes; one effective way to address the persistence of cccDNA is to prevent the entry of HBV into cells. Viral entry is mediated through specific interactions of viral membrane proteins with cellular receptors, in particular the sodium taurocholate co-transporting polypeptide (NTCP), a functional receptor identified by Yan et al in the treeshrew Tupaia [14]. NTCP is a sodium-dependent transporter for taurocholic acid expressed at the basolateral membrane of hepatocytes and responsible for most Na+dependent bile acid uptake in hepatocytes. Antagonizing the interaction of NTCP with synthetic viral protein mimics is a novel method to treat chronic HBV, and therapeutic strategies that block viral entry are under active investigation. Myrcludex-B, a synthetic lipopeptide derived from the pre-S1 domain of the HBV envelope protein, which specifically targets the NTCP, has been shown to efficiently block HBV infection in vitro [15] and in a humanized mouse model (a uPA+/+-Severe Combine Immune Deficient [SCID] mouse reconstituted with human hepatocytes and infected with HBV). Mice treated with Myrcludex-B had lower HBV viremia, intrahepatic cccDNA, and antigen levels than untreated mice, showing that the synthetic lipopeptide can prevent the spread of HBV from infected human hepatocytes in vivo and hinder the amplification of the cccDNA pool in previously infected hepatocytes [16]. Inhibiting Transcription
Current NUC-based therapy blocks the production and accumulation of new cccDNA, but blocking replication alone will not impact existing cccDNA reservoirs in infected hepatocytes. With a long half-life of approximately 33–50 days [12], the intrahepatic viral cccDNA functions as a library of potential viral escape variants generated by the error-prone HBV viral polymerase, a potential source for the emergence of drug resistance or viral rebound upon cessation of effective antiviral therapy. Although NUC-based therapies alone will not address the viral reservoir, HBV eradication efforts will likely employ innovative strategies in conjunction with suppressive NUC-based therapies. An investigational NUC, tenofovir alafenamide, a prodrug of the currently approved tenofovir disoproxil fumarate, has been shown to concentrate in human hepatocytes and undergo conversion to the active form of the drug [17]. Phase 3 clinical trials are currently under way to study the S320
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drug in both human immunodeficiency virus (HIV) and HBV infection, and results are eagerly anticipated. Silencing or disrupting cccDNA, if possible, may deplete this viral reservoir. One approach to target cccDNA is to repress its transcription, making it epigenetically silent while allowing it to remain intact in the nucleus of infected hepatocytes. Treatment with IFN-α has been shown to inhibit HBV replication by reducing transcription of pregenomic RNA from the HBV cccDNA minichromosome, both in vitro and in the uPA+/+-SCID humanized HBV-infected mouse model [18]. While IFN-α therapies have high rates of treatment-limiting side effects, their ability to mediate epigenetic repression of HBV cccDNA transcriptional activity is an effect that may prove useful as part of a strategy to achieve a functional cure for chronic HBV infection. Another strategy to target cccDNA is to inactivate it. Gene therapy has undergone an evolution and expansion in recent years, and there are now a variety of sequence-specific DNA modification technologies available. Multiple different gene therapy approaches, including zinc finger nuclease (ZFN), transcription activator–like effector nuclease (TALEN), and clustered regularly interspaced palindromic repeat applications have provided options for the study and treatment of liver diseases [19, 20]. ZFN, also known as zinc finger proteins, are a versatile and effective class of gene-targeting reagents [21] that can be used to block the transcription and replication of cccDNA and production of viral proteins from infected cells in vitro [22], but have been recently also used to cleave and degrade HBV cccDNA in hepatoma cells in vivo. A ZFN pair designed by Cradick et al cleaves the HBV core gene open reading frame, reducing the pregenomic RNA by 29% [23]. More recently, Weber et al designed 3 ZFNs to target HBV polymerase, core, and X genes, creating double-stranded breaks in the cccDNA, which, when imprecisely repaired by nonhomologous end joining, led to inactivating mutations with durable effects after a single treatment in vitro [24]. Another class of nucleases, TALENs can cleave sequencespecific DNA targets. TALENs designed to target conserved regions of the HBV viral genomic DNA across genotypes and expressed in transfected cell culture can inactivate and reduce cccDNA, interfering with the production of viral antigens without apparent cytotoxic effects [25]. Interestingly, these effects were maintained in the humanized mouse model and were observed to work synergistically with IFN-α to restore HBV-suppressed IFN-stimulated response element–directed transcription [25]. RNA interference (RNAi) is another methodology that has been employed in targeting HBV transcription. Viral messenger RNA (mRNA) can be directly targeted using small interfering RNA molecules that silence genes posttranscriptionally, effectively knocking down the expression of a specific gene or genes. RNAi can be used to target mRNA for degradation
and, due to the extensive use of open reading frames within HBV’s DNA genome, multiple HBV RNAs could serve as RNAi targets [26, 27]. McCaffrey et al have demonstrated that RNAi could be applied to inhibit production of HBV replicative intermediates in mice transfected with a HBV plasmid at multiple steps: HBV RNA and DNA were substantially reduced in mouse liver, RNAi expression reduced HBsAg secreted into serum, and the number of cells with HBV core antigen staining was substantially decreased [28]. Another RNAi-based drug currently in clinical trials is a dynamic polyconjugate of an RNAi trigger and cholesterol, which, when coinjected with a hepatocyte-targeted, membrane-active peptide, is well tolerated and reduces HBsAg in a dose-dependent manner for up to 2 months [27]. Capsid Inhibitors
HBV persistence and transmission require ongoing cycles of HBV replication and assembly of the virus particle, composed of the capsid protein, polymerase, and pregenomic DNA. Interference with the assembly step is an attractive target for HBV therapeutics. Multiple classes of compounds may be able to dysregulate or inhibit virion assembly and encapsidation. Members of the family of heteroaryldihydropyrimidines have shown the ability to reduce HBV viremia in vitro and in the humanized mouse model [29]. Treatment with one heteroaryldihydropyrimidine, Bay 41–4109 [30], resulted in incorrectly assembled or disassembled core capsids into monomers or dimers, from which they could be further degraded into peptides [31]. Preclinical studies with Bay 41–4109 and the related compound GLS4 have shown potent antiviral activity, including sustained suppression of HBV DNA after cessation of treatment in most humanized mice, without toxicity to hepatocytes [32]. Additionally, another chemical group, the phenylpropenamides, has also been found to inhibit viral encapsidation and is active against lamivudine-resistant strains of HBV [33, 34]. Phenylpropenamides induce tertiary and quaternary structural changes in HBV capsids. One candidate, AT-130 (a phenylpropenamide derivative), has been shown to bind a promiscuous pocket at the dimer–dimer interface that favors a unique quasiequivalent binding site in the capsid and can serve as an effective antiviral agent. Viral production is disrupted after virion assembly is initiated prematurely, resulting in morphologically normal capsids that are empty and noninfectious [35]. The clinical efficacy of compounds from this family has not yet been reported. A novel compound, NVR 3-778, prevents production of infectious virus by causing core proteins to misassemble into capsid-like particles in vitro [36]. Preclinical studies have shown that treatment of HBV-producing cell lines with NVR 3-778 leads to inhibition of RNA encapsidation and downstream disruption of the production and secretion of infectious HBV virions. NVR 3-778 was active against both wild-type and several tested HBV NUC-resistant variants [36]. The compound
is now in phase 1b clinical trials, with results expected as early as summer 2016. Preventing HBV Antigen Secretion
The exact mechanisms by which HBV escapes immunity remain poorly understood. Recent reports have suggested that the immunosuppression in the early stages of HBV infection is a direct result of high circulating levels of HBsAg, acting directly on dendritic cells to limit cytokine production [37, 38], leading to T-cell exhaustion [39]. One innovative therapeutic strategy targets the secretion of HBsAg with a synthetic small molecule, HBF-0259, an aromatically substituted tetrahydrotetrazolo-(1, 5-a)-pyrimidine. HBF-0259 selectively inhibits the secretion of subviral HBsAg and DNA-containing viral particles, without inhibiting HBeAg secretion, which is not a constituent of HBV viral particles, or HBV viral replication [40]. A derivative of this compound has been shown to be more potent and has been shown to have favorable kinetics in the humanized mouse model [41]. It remains unclear whether these compounds will effectively block secretion of HBsAg in humans, and whether blockade, in acute or chronic HBV infection, will reverse the observed T-cell anergy. Host Factors
In chronic HBV, as opposed to other chronic viral infections, there are several factors that favor the search for eradication strategies. Widely available and effective vaccines against hepatitis B have established correlates of protection. The fact that the majority of individuals acutely infected with hepatitis B will recover has established correlates of viral clearance. And finally, despite low tolerability and efficacy, the ability of IFN-based therapies to eradicate chronic HBV infection in select patients has established correlates of treatment response. Liver damage due to chronic HBV is primarily mediated by the host inflammatory response rather than a direct viral cytopathic effect [42]. An exaggerated host immune response to HBV is not only ineffective at viral eradication, but also triggers hepatic inflammation. Recovery from acute HBV infection is associated with robust and effective innate and adaptive immune responses. Strategies that address host factors seek to tip the scales toward an effective response that results in viral clearance, rather than an excessive inflammatory response that may potentiate hepatic damage. The innate immune response is the first line of defense against viral infections, resulting in production of type 1 IFNs when Toll-like receptors (TLRs) on the surface of innate immune cells are engaged (Figure 2). The type 1 IFNs, in turn, suppress viral replication, recruit natural killer cells to kill infected hepatocytes, and support the efficient maturation and recruitment of adaptive immunity through stimulating antigen-presenting cells to prime a strong and effective adaptive immune response [43]. The principal producers of type 1 IFNs are plasmacytoid dendritic cells, which also stimulate tumor necrosis factor alpha (TNF-α), interleukin 6, and cell
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Figure 2. Innate immunity and mechanisms of hepatitis B virus (HBV) persistence. Abbreviations: CCL3, C-C ligand 3; DC, dendritic cell; IFN, interferon; IL, interleukin; NK cells, natural killer cells; NKT cells, natural killer T cells; PAMP, pathogen-associated molecular patterns; PRR, pattern recognition receptor; TLR, Toll-like receptor.
surface costimulatory molecules when TLRs are stimulated. Strategies that enhance HBV-specific immune responses may risk increasing liver damage, and safety is an important consideration for any approach that seeks to modify host immune responses to HBV. Triggering Innate Immunity
In individuals with chronic HBV infection, TLR signaling is downregulated [44]; targeting the recruitment and activation of innate immune cells by agonizing the remaining TLRs, particularly TLR-7, has the potential to accelerate HBV-specific immune reconstitution and assist with clearance of HBV in chronically infected individuals. Although HBV circumvents endogenous type 1 IFNs by decreasing expression of TLRs in hepatocytes, it is plausible that the use of exogenous IFN induction using the TLR-7 pathway may reinstate the IFN-α response. There are commercially available TLR agonists that may be orally administered, allowing for rapid uptake by the liver. An investigational orally administered selective TLR-7 agonist, GS-9620, has been shown to stimulate production of IFNα and various other cytokines and chemokines and produce prolonged suppression of circulating and hepatic HBV DNA in chimpanzees when administered over 4 weeks [45]. In a randomized, double-blind phase 1b study of both treatment-naive and virologically suppressed patients with chronic HBV, GS9620 induces peripheral interferon-stimulated genes following a single or double dose in this short-term study [46]. Longerterm studies are needed to evaluate whether stimulation of TLR-7 is capable of suppressing HBV replication in humans. S322
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Reversing Immune Exhaustion
Correlates of an adaptive immune response against HBV have been shown to be protective against HBV infection in both recovered and vaccinated subjects [47]. T cells are needed to simultaneously stimulate B cells to manufacture antibodies targeted to E and S antigens while inducing cellular immune responses to kill infected hepatocytes (Figure 3). Chronic HBV infection is associated with functionally impaired immune responses, particularly within HBV-specific lymphocytes. The persistent exposure to viral antigens leads to virus-specific CD4 and CD8 cell dysfunction or exhaustion. Recent data suggest that these exhausted virus-specific T cells overexpress the PD-1 cell surface molecule [48]. In vitro data suggest that inhibition of the interaction of PD-1 with its ligands (PD-L1/2) improves the antiviral function of these exhausted T cells. Fisicaro et al examined the role of T-cell exhaustion in the pathogenesis of chronic HBV by comparing the phenotype and function of intrahepatic and circulating HBV-specific T cells and the effect of PD-1/PD-L1 blockade, and they reported that intrahepatic HBV-specific CD8 T cells express higher PD-1 and lower levels of the interleukin 7 receptor CD127 [49]. These alterations were reversed by PD-1/PD-L1 blockade, with improvement in T-cell function in peripheral and intrahepatic lymphocyte compartments. Sherman et al report that PD-1 blockade improves cytokine secretion and survival of HBV-specific CD8 T cells, from HBV-monoinfected and HIV/HBV-coinfected patients treated with adefovir [50]. Collectively, these data evidence that PD-1 blockade, in combination with nucleotide inhibitors, might provide a strategy to address the immune defects seen in chronic
Figure 3. Adaptive immunity in chronic hepatitis B infection. Abbreviations: APC, antigen-presenting cell; HBeAb, hepatitis B e antibody; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; TCR, T cell receptor; Th1, type 1 helper T cell; Th2, type 2 helper T cell; TNF, tumor necrosis factor.
HBV infection. Antibodies blocking the interaction of PD-1/ PD-L1 have been tested in patients with advanced malignancies, and have induced durable tumor regression and prolonged stabilization of disease [51], while unfortunately causing a relatively high frequency (approximately 14%) of high-grade adverse events, including severe autoimmune complications [52]. Blockade of PD-1 offers an opportunity to revive exhausted T cells in chronic HBV infection, allowing restoration of adaptive immunity against HBV. With the first PD-1 antibody, nivolumab, approved by the US Food and Drug Administration in 2014, the safety profile and risks of PD-1 blockade are being actively studied in patients with HBV-associated HCC. If it can be administered safely, further investigation into PD-1/PD-L1 blockade may inform approaches to eradication strategies for chronic HBV infection. Transfer of Adaptive Immunity
Reversal of immune exhaustion will not work if there are no HBV-specific T cells present. Studies have pointed to the clonal deletion of HBV-specific CD8+ T cells as a mechanism of HBV persistence [43]. Chimeric antigen receptors (CARs) are synthetic, engineered receptors that can engage cell-surface molecules in their native conformation, independent of antigen processing by the target cell and major histocompatibility complex [53]. T-cell responses may be “trained” to recognize infected cells by expressing HBV-specific CARs. In 2013, Krebs et al were able to show that murine CD8 T cells infected with retroviral vectors induced CARs that bind HBV envelope proteins and activate T cells [54]. They demonstrated that CD8 T
cells expressing HBV-specific CARs recognized HBV and were able to engraft and expand in an immunocompetent transgenic mouse model, where they efficiently relocated to hepatic tissues and controlled HBV replication, causing only transient liver damage [54]. Therapeutic Vaccination
Therapeutic vaccination presents a promising strategy to address HBV-specific T-cell exhaustion. A therapeutic vaccine that could induce a potent CD4 T-cell response could counteract immune tolerance to activate humoral and cytolytic immune responses against one or more HBV antigens. Several categories of therapeutic vaccines are being developed for chronic HBV infection. A yeast-based immunotherapy platform using targeted molecular antigens, or tarmogen, engineered to express HBV X, S, and C antigens were able to stimulate HBV-specific CD4 and CD8 Tcell responses, including lymphocyte proliferation, interferon-γ release, and intracellular cytokine production. When used to stimulate peripheral blood mononuclear cells isolated from patients with chronic HBV in combination with autologous dendritic cells, the candidate vaccine produced pronounced expansions of HBV antigen–specific T cells possessing a cytolytic phenotype [55]. This yeast-based immunotherapeutic vaccine platform may be capable of eliciting a functional adaptive immune response in patients with chronic HBV. A second investigational vaccine platform, an engineered vesicular stomatitis virus expressing HBV middle surface envelope glycoprotein and generating virus-like vesicles (VLVs), has been
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shown to induce functional CD8 T-cell responses in the transgenic mouse model of chronic HBV infection [56]. The ability of VLVs to stimulate T-cell responses in a tolerogenic model of chronic infection indicates that highly immunogenic viral vectors may offer hope in human chronic HBV. Adenoviral vector-based vaccines have been shown to be safe and immunogenic, but are often limited by preexisting adenoviral immunity. One nonreplicative adenovirus serotype 5 (Ad5) encoding candidate vaccine, TG1050, has been shown to induce robust, multispecific, and long-lasting HBV-specific T cells targeting 3 encoded HBV immunogens and the capacity to produce both IFN-γ and TNF-α as well as stimulating cytolytic functions. In the transgenic mouse model of chronic HBV, the candidate vaccine was shown to reduce circulating levels of HBV DNA and HBsAg, whereas alanine aminotransferase levels remained within normal limits [57]. It remains to be seen whether this vaccine strategy will be able to elicit these bifunctional responses in patients with preexisting Ad5 immunity. CONCLUSIONS
Current antiviral therapy does not induce sustained virologic remission in the majority of patients with chronic HBV infection. Novel therapeutic strategies are urgently needed. A rational therapeutic design will be required to circumvent the various factors by which HBV induces immune tolerance and adaptive immune system exhaustion and establishes a longterm viral reservoir. Targeting a combination of viral and host factors is likely to offer the best chance for accomplishing this goal. If the benefits of these approaches can be shown to outweigh the risks, these approaches may offer hope in the ongoing efforts to control HBV replication, purge cccDNA reservoirs, and develop long-lasting protective immunity. Notes Supplement sponsorship. This article appears as part of the supplement “Hepatitis B,” sponsored by the CDC Foundation and Gilead. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Marcellin P, Gane E, Buti M, et al. Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label followup study. Lancet 2013; 381:468–75. 2. Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006; 295:65–73. 3. Chang TT, Gish RG, de Man R, et al. A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B. N Engl J Med 2006; 354:1001–10. 4. Janssen HL, van Zonneveld M, Senturk H, et al. Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 2005; 365:123–9. 5. Lai CL, Gane E, Liaw YF, et al. Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med 2007; 357:2576–88. 6. Lau GK, Piratvisuth T, Luo KX, et al. Peginterferon alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med 2005; 352:2682–95.
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