JIM-11846; No of Pages 8 Journal of Immunological Methods xxx (2014) xxx–xxx

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Review

Immunotherapeutic interventions in chronic hepatitis B virus infection: A review Li Wang a,⁎, Zhi Qiang Zou a, Cheng Xia Liu b, Xiang Zhong Liu a a b

Infectious Disease Hospital of Yantai, Huanshan Road 62, Zhifu District, 264001, Yantai, Shandong, China Digestive Department, Affiliated Hospital of Binzhou Medical College, Huanghe Second Road 661, 256603, Shandong, China

a r t i c l e

i n f o

Article history: Received 25 April 2013 Received in revised form 20 February 2014 Accepted 2 April 2014 Available online xxxx Keywords: Chronic hepatitis B Immunity Antiviral agents Immune-based therapy Immunotherapeutic intervention

a b s t r a c t Chronic hepatitis B virus (HBV) infection is a public health challenge worldwide. Antiviral agents (nucleos(t)ide analogues, NAs) and immune-based therapies (IFN-α or Pegylated-IFN-α) are two therapeutic approaches available currently against chronic hepatitis B (CHB). However, these approaches are associated with the development of acquired drug resistance or poor response rates and are accompanied by numerous side effects. Furthermore, due to defective innate and adaptive immune responses, HBV cannot be effectively controlled or completely eliminated, which may ultimately result in liver decompensation and hepatocelluar carcinoma. The imperative for development of new approaches targeting CHB cannot be overstated. Various immunotherapeutic interventions have been tried as adjuvants to inhibit HBV replication. In this paper, we will review immunotherapeutic interventions in the treatment of CHB. © 2014 Published by Elsevier B.V.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Currently available treatments for CHB . . . . . . . . . . . . . . . . . . . . 2.1. Nucleos(t)ide analogues . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Immune-based therapies . . . . . . . . . . . . . . . . . . . . . . . 3. Immunotherapeutic interventions for CHB . . . . . . . . . . . . . . . . . . . 3.1. DC and HBV-specific cytotoxic T lymphocytes (CTLs)-based immunization 3.2. NK cell-based therapy . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Cytokines and thymosin . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Blockage or depletion of immunoinhibitory cells . . . . . . . . . . . . 3.5. Blockage of immunoinhibitory signals . . . . . . . . . . . . . . . . . 3.6. Activation of signaling pathways leading to IFN-alpha production . . . . 3.7. Therapeutic vaccines . . . . . . . . . . . . . . . . . . . . . . . . . 4. Future challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Tel.: +86 05356232253. E-mail address: [email protected] (L. Wang).

http://dx.doi.org/10.1016/j.jim.2014.04.004 0022-1759/© 2014 Published by Elsevier B.V.

Please cite this article as: Wang, L., et al., Immunotherapeutic interventions in chronic hepatitis B virus infection: A review, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.04.004

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1. Introduction Chronic hepatitis B virus (HBV) infection is a global health problem. Despite the availability of a prophylactic vaccine, approximately 350–400 million people worldwide are infected with HBV. The noncytopathic virus triggers immune responses resulting in persistent hepatic inflammation and progressive fibrosis. These immune-mediated pathological processes may ultimately lead to cirrhosis and hepatocellular carcinoma (HCC), accounting for up to 1 million deaths per year (Lau et al., 2007). The goal of therapy is to improve quality of life and survival by preventing progression to cirrhosis, decompensated cirrhosis, HCC and death through sustained suppression of HBV replication (European Association For The Study Of The Liver, 2012). However, sustained clearance of HBV DNA and anti-HBs seroconversion are rarely achieved, probably due to defective reconstitution of an efficient HBV-specific adaptive immune response (Bertoletti et al., 2009). Acute viral hepatitis, which successfully clears the virus, involves a vigorous, polyclonal and multi-specific, HBV-specific cytotoxic T lymphocytes (CTLs) response to multiple epitopes in the viral nucleocapsid, envelope, and polymerase. The relatively weak CTL response leads to persistent infection in chronically infected patients. Clearance of HBV is T-cell dependent, although B cells are involved in the presentation to CD4+ T cells and production of neutralizing antibodies (Lau et al., 2007). Noncytolytic viral eradication by HBV-specific CTLs accounts for recovery from acute HBV infection in that most HBV is cleared from hepatocytes with only a fraction of the hepatocytes destroyed. HBV-specific CTLs are barely detected in the peripheral blood of patients with CHB, especially those with high levels of HBV DNA (Boni et al., 2007). The essential role of multi-specific immune responses in the control of HBV infection implies the need for multimodal therapeutic strategies against chronic HBV infection. Dendritic cells (DCs) are specialized antigen-presenting cells, with subtypes used as vaccines to stimulate both innate and adaptive immunity (Apostolopoulos et al., 2013). In patients with CHB, both HBV particles and purified hepatitis B surface antigen (HBsAg) may directly contribute to the dysfunction of myeloid (mDC) and plasmacytoid DCs (pDC) (Op den Brouw et al., 2009; Woltman et al., 2011). In addition, multiple inhibitory receptors, such as programmed cell death 1 (PD-1), T-cell immunoglobulin domain and mucin domain-containing molecule-3(TIM3) and CD244 (Raziorrouh et al., 2010) may play a role in the negative regulation of T cell functions in HBV infection. CD4+ CD25+ regulatory T cells (Tregs) were linked to an impaired immune response in patients with CHB. The enhanced suppressor function of Tregs induced by HBV infection inhibited tumor surveillance and suppressed the anti-tumor immune response to HCC tumor antigen (Zhang et al., 2010). The innate immune system represents the first line of defense against viral infection. The potential contribution of the innate system in controlling HBV infection is now an important area of controversy. HBV appears to be able to induce a long-lasting inhibition of innate immunity (Ait-Goughoulte et al., 2010). As a component of innate immune system, NK cells rapidly eliminate HBV-specific T cells in a contact-dependent manner and negatively regulate antiviral immunity in chronic HBV infection (Peppa et al., 2013). These mechanisms suggest that combination of enhanced host immune response by

specific immunotherapeutic interventions, effective viral load suppression and blockage of inhibitory receptors and cells are needed to promote sustained viral clearance and achieve desirable antiviral effects in CHB (Nebbia et al., 2012a). In this review, we focus on current immunotherapeutic interventions in chronic HBV infection in humans and animal models. 2. Currently available treatments for CHB The two therapeutic approaches available for the suppression of HBV replication include antiviral agents (nucleos(t)ide analogues, NAs) and immune-based therapies (IFN-α or Pegylated-IFN-α). However, most patients undergoing these therapies do not manifest long-term, durable control of infection after treatment withdrawal. In particular, rates of HBsAg loss and seroconversion to HBsAb are very low (Fletcher and Delaney, 2013). 2.1. Nucleos(t)ide analogues NAs, including lamivudine (LMV); adefovir (ADV); entecavir (ETV); telbivudine (LdT); and tenofovir (TFV) target the HBV polymerase and therefore, interfere with viral DNA replication. Antiviral therapies for CHB using NAs have become standard treatment modalities, based on several independent guidelines. Inhibition of viral replication with NAs leads to strong and long-term viral suppression, reversal or restoration of impaired function of monocyte-derived DCs and HBVspecific T cells (Boni et al., 2007; Zheng et al., 2007; Lu et al., 2008a). However, restoration of T-cell response might be transient (Boni et al., 2003). In addition to exorbitant costs, it entails prolonged therapy with a low rate of hepatitis B e antigen (HBeAg) seroconversion in HBeAg positive patients, which is often temporary (Reijnders et al., 2010). Drugresistant HBV mutants frequently arise, leading to treatment failure and disease progression. Furthermore, reactivation rates of HBV replication and HBeAg seroreversion are high after treatment cessation (Tseng et al., 2012). Immune therapies that increase the antiviral T-cell response might increase the likelihood of complete HBV control in patients undergoing long-term NAs treatment. 2.2. Immune-based therapies IFN-α or Pegylated IFN-α (Peg-IFN-α) stimulates the host antiviral immune response as well as direct but modest antiviral activity. Compared with NAs, IFN treatment is associated with a finite duration, absence of resistance, and a higher rate of anti-HBe and anti-HBs seroconversion. A finite duration of treatment with Peg-IFNα improves the probability of achieving sustained virological responses. IFN-α mediates divergent effects on the innate and adaptive arms of the immune system, in vivo. Peg-IFN-α therapy cannot significantly restore the depleted HBV-specific CTL response. However, it may enhance recovery of memory T cells in CHB patients by down-regulating inhibitory receptors (PD-1 and CD244) and up-regulating effector molecules, including CD127 and CXCR4 (Liu et al., 2012). However, IFN-α therapy is associated with high costs, the need for subcutaneous injections, moderate antiviral effect (effective in only 30–40% of CHB patients), the contraindication profiles

Please cite this article as: Wang, L., et al., Immunotherapeutic interventions in chronic hepatitis B virus infection: A review, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.04.004

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and significant side effects limiting its use (Walsh and Locarnini, 2012). The effects of these antiviral therapies are far from satisfactory as they are unable to eradicate HBV from the liver. Some studies have shown that the efficacy of IFN-α plus NAs combination therapy was superior to IFN-α monotherapy (Huang et al., 2013) and NA monotherapy, such as lamivudine (Vassiliadis et al., 2007). In other studies, the combination therapy failed to show significant association between virological and biochemical endpoints and histological improvement compared with Peg-IFN-α monotherapy (van Zonneveld et al., 2006; Enomoto et al., 2013). Current AASLD guidelines recommend monotherapy with tenofovir or entecavir in IFN-α non-responders (Lok and McMahon, 2009). Whether combination therapy of two NAs or NAs and IFN-α conferred an additional benefit compared with monotherapy remains controversial. Combination of NAs or IFN-α with other immunotherapies may be beneficial. A recent study showed that cytokines from peripheral blood mononuclear cells (PBMC) from HBV-negative individuals stimulated with CpG ODN strongly inhibited HBV viral replication as well as HBsAg and HBeAg secretion from HepaRG and HepG2 cells following transduction or HBV inoculation (Vincent et al., 2009). Immunizations with antigen-antibody immune complexes or DNA (IC/DNA) vaccines lead to an anti-woodchuck hepatitis virus surface antibody response and significant reductions of viral load and antigenemia (Lu et al., 2008b). Such immunotherapeutic strategies should be evaluated in vivo to assess restoration and duration of anti-HBV-specific immune responses. Thus, it is imperative to develop more effective and safe but affordable anti-HBV strategies to improve therapeutic outcomes in CHB (Liaw, 2013). An ideal therapy combines activation of antiviral immunity with induction of efficient and long-lasting control of viral replication. The inherent capacity of the immune system to efficiently contain HBV in acutely infected adults who resolve infection provides a strong rationale for the development of treatments based on immune boosting therapeutic strategies. 3. Immunotherapeutic interventions for CHB 3.1. DC and HBV-specific cytotoxic T lymphocytes (CTLs)-based immunization HBV-specific CD4+ and CD8+ T cell frequency and function are elevated in patients who resolve HBV infection compared with subjects afflicted with chronic infection. Impaired DC function in patients with CHB may lead to insufficient T cell response to HBV, which may be associated with persistent viral infection. The mechanism of impaired DC function in patients with CHB is unclear. HBV particles and purified HBsAg may both contribute to the dysfunction of myeloid DCs (mDCs) (Apostolopoulos et al., 2013; Op den Brouw et al., 2009), and directly inhibit the production of IFN-α (Shi et al., 2012a). Recent research suggested that HBsAg/ hepatitis B core antigen (HBcAg)-pulsed human blood DC of CHB patients and DC from HBV transgenic mice (HBV TM) induced HBsAg-specific and HBcAg-specific CTLs (Akbar et al., 2010). Immunization of HBV TM with HBcAg-pulsed DC resulted in HBsAg disappearance, production of anti-HBs, and

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development of HBsAg- and HBcAg-specific CTL in the spleen and the liver. Significantly higher levels of activated endogenous DC were detected in HBV TM immunized with HBcAgpulsed DC compared with HBsAg-pulsed DC (Akbar et al., 2012). DCs loaded with HBV subviral particles (HBVsvp) and HBV-derived peptides overcome tolerance against HBV and elicit functional virus-specific T cells and reactivate B cell responses in HBV TM (Farag et al., 2012) and CHB patients (Martinet et al., 2012), but were not sufficient to lead to virus control in these mice (Chen et al., 2008). Activated pDCs acted synergistically with HBcAg-pulsed monocyte-derived DCs in the induction of HBV-specific CTL response (Boni et al., 2012). Therefore, a DC-based immunotherapeutic approach may disrupt tolerance against HBV and restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV infection. HBV-specific T cells play a key role in clearance of the virus. Although HBV-specific T cells from NAs-treated patients with complete control of infection were dysfunctional ex vivo, they showed efficient responses after expansion in vitro. Similar responses were seen in patients who spontaneously resolved acute HBV infection. Though NAs-treated patients with negative HBV DNA, and positive HBsAg showed lower levels of T-cell responses, responses were greater than those of untreated patients (Boni et al., 2012). A low frequency of T cell immune responses to HBsAg was found in Chinese subjects with HBsAg seroclearance following antiviral therapy, which indicated that HBsAg-specific immune responses were not responsible for HBsAg seroclearance (Liang et al., 2011). Robust anti-core T cell responses were found in patients with reduced HBsAg serum levels, suggesting that core-specific T cell responses mediated a protective effect in HBV control (Loggi et al., 2013). These results suggested that expansion of HBV-specific T cells in vitro through HBcAg stimulation may be one of immunotherapeutic interventions. Experimental evidence in HBV TM also suggests that HBcAg-specific CTLs are essential for the control of HBV replication and prevention of liver damage in patients with CHB. The capacity of HBcAg to induce HBcAg and HBsAg-specific CTLs, and activate endogenous DC in HBV TM without inducing liver damage suggests that HBcAg was an integral component of the therapeutic vaccine against CHB (Akbar et al., 2012). Genetically modified T cells were also used to reconstitute virus-specific T cell immunity in CHB patients (Gehring et al., 2011). 3.2. NK cell-based therapy One of the significant differences between the immune composition of the liver and the peripheral blood is the proportion of NK cells. The inherent enrichment of NK cells in the human liver underscores their potential importance in the control of hepatotropic viral infections. NK cells manifest potential antiviral activity in HBV infection either directly or indirectly, by modulating T cell responses. Direct NK cell effects involve lysis of infected hepatocytes through granzyme/perforin or death receptor pathways at the expense of infected hepatocytes. Thus, NK cells are capable of exerting antiviral and immunoregulatory functions while also contributing to the pathogenesis of liver injury (Maini and Peppa, 2013). In CHB patients, the cytotoxic capacity was retained, but NK cell activation and subsequent IFNγ and

Please cite this article as: Wang, L., et al., Immunotherapeutic interventions in chronic hepatitis B virus infection: A review, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.04.004

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TNFα production, especially of the CD56(dim) subset, were strongly hampered. This selective defect in NK cell function may be attributed to the influence of interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) in the liver, since it was restored following blockade of these immunosuppressive cytokines, in vitro (Peppa et al., 2010). Restoration of the NK cell cytokine-producing capacity by viral load reduction, therefore, contributed to clearance of the virus (Tjwa et al., 2011). Attempts to manipulate NK cell immunity in the setting of chronic HBV infection need to be carefully timed and tailored to prevent pathogenic effects while harnessing vital protective functions. 3.3. Cytokines and thymosin IL-12 is an immunomodulatory cytokine that promotes cellular immunity. In vitro, IL-12 has been reported to restore the hyporesponsiveness of T cells induced by autologous DCs to viral antigens from patients with CHB (Löhr et al., 2002). Combined therapy of DNA vaccine and lamivudine induced plasma IL-12 level increased and the IL-12/p40 ratio is associated with long-term sustained virological response (SVR) in HBV carriers (Im et al., 2009). Combination treatment with lamivudine plus recombinant human IL-12 enhanced T-cell reactivity to HBV and IFN-γ production (Rigopoulou et al., 2005). High viremia may inhibit IFN-γ production upon IL-12 stimulation in lymphocytes (Crettaz et al., 2009). IL-12 boosts functional HBV-specific CD8+ T cell responses within the liver and promotes HBV-specific central memory CD8+ T cell and HBeAg seroconversion (Schurich et al., 2013; Xiong et al., 2007). IL-12 stimulation can also lead to down-regulation of the co-inhibitory molecule PD-1 (He et al., 2012). Though IL-12 has a pleiotropic effect in restoring functional HBV-specific CTLs, IL-12 does not maintain inhibition of HBV replication in HBeAg-positive patients after lamivudine withdrawal. Therefore, IL-12 could be used as an adjuvant agent to break immunological tolerance in CHB treatment. Tumor necrosis factor (TNF)-α and IFN-γ are two important cytokines involved in the immune responses to HBV infection. Compared with lamivudine alone, cytokine (IFN-γ + TNF-α) treatment and sequential therapy with cytokine and lamivudine showed a stronger inhibition of HBV covalently close circular DNA (cccDNA). Lamivudine pretreatment significantly reduced IFN-γ + TNF-α-mediated toxicity of HepG2.2.15 cells (Shi et al., 2012b). Phillips et al. (2010) demonstrated that virus-specific CD8+ T cells inhibited HBV replication in HBV-producing hepatocytes, with minimal cell lysis. Blockade of IFN-γ and TNF-α abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines secreted from virus-specific CD8+ T cells mediated the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-γ and rTNF-α resulted in an efficient suppression of viral replication without cytotoxicity. These results provide direct evidence that virus-specific CD8+ T cells efficiently control HBV replication by noncytolytic mechanisms mediated by IFN-γ and TNF-α. Thymosin alpha-1 (Tα1) is a 28-amino acid polypeptide with immunomodulatory activities produced synthetically. In vitro studies have shown that Tα1 affected T-cell production and maturation, stimulated production of Th1 cytokines, such as IFN-γ and IL-2, and activated NK cell-mediated cytotoxicity

(Grimm et al., 2011). Higher-dose Tα1 exhibited better efficacy against HBV (Jiang et al., 2010). Among HBeAg positive patients, Tα1 and lamivudine combination therapy may be more effective than lamivudine monotherapy, providing superior rates of biochemical and virological responses, and HBeAg seroconversion (Zhang et al., 2009). Whereas, another study indicated that a short-term combination of Tα1 was not superior to PEG-IFN α-2a alone in HBeAg positive CHB patients on the basis of antiviral efficacy (Kim et al., 2012). And cessation of Tα1 treatment did not prevent the occurrence of viral and biochemical breakthroughs (Lee et al., 2008). Whether treatment with thymosin α-1 was superior to currently available antiviral therapies needs additional large-scale clinical studies. Granulocyte-macrophage colony-stimulating factor (GMCSF) is known to be a good adjuvant in improving immune response. Qing et al. (2010) demonstrated that when the HBV-S gene was fused to the GM-CSF gene, the immune effects of the HBV DNA vaccine both in normal and HBV-transgenic mice could be strengthened. HBV DNA vaccine containing HBV DNA plasmids fused with GM-CSF may be used in prophylactic and therapeutic intervention. However, Lin et al. (2010) found that changing the vaccine dose was associated with a better seropositive response than injecting the vaccine in combination with GM-CSF. GM-CSF as an adjuvant did not improve the Ab titer or the development of protective immunity to HBV vaccination in healthy adult non-responders receiving an accelerated vaccine schedule (Qing et al., 2010). GM-CSF also failed to improve responses to the booster HBV vaccination among HIV-infected persons (Overton et al., 2011). Thus, combination with GM-CSF was not the optimal vaccination strategy. IL-10 is a pleiotropic cytokine acting in a variety of immune cells through IL-10 receptor (IL-10R). Antiviral immunity may be enhanced by interfering with IL-10/IL-10R pathway. Blockade of IL-10 in vitro rescued polyfunctional virus-specific CD8+ T cell responses, which was regulated via IL-10-producing B cells (Das et al., 2012). TGF-β is an important cytokine with anti-inflammatory properties. TGF-β1 down-regulated the expression of NKG2D and 2B4 on NK cells leading to impaired effector function (Sun et al., 2012). Blockade of IL-10 +/− with TGF-β restored the capacity of peripheral and liver NK cells in CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity (Tjwa et al., 2011). TGF-β may also be able to effectively suppress HBV replication primarily through modulation of the expression of hepatocyte nuclear factor 4alpha (HNF-4α) gene (Hong et al., 2012). The levels of viral transcripts, core protein, and nucleocapsid were significantly diminished by TGF-β treatment (Chou et al., 2007). However, neutralization of IL-10 and TGF-β had no effect on Treg-mediated suppression of the anti-HBcAg response (Zhang et al., 2009). These data indicated that TGF-β might play a dual role in HBV infection, suppression of immune responses against viral infection and inhibition of viral replication (Chou et al., 2007). Therefore, TGF-β-based immunotherapy needs to be evaluated. 3.4. Blockage or depletion of immunoinhibitory cells Treg cells showed immunosuppressive effect against HBV-specific T helper cells. The presence of HBV-specific

Please cite this article as: Wang, L., et al., Immunotherapeutic interventions in chronic hepatitis B virus infection: A review, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.04.004

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Tregs contributed to inadequate immune response against the virus, leading to chronic infection (Stoop et al., 2005). Immune tolerance to HBV in CHB patients without hepatitis is under the control of the host forkhead box p3-expressing Tregs. The CD8+ T cell activity increased significantly after depletion of circulating Tregs (Koay et al., 2011). Tregs also reduced the response to treatment and non-responders to INF-α had higher level of Tregs. Furthermore, Tregs increased risk of HCC (Aalaei-Andabili and Alavian, 2012). Stross et al. (2012) initiated HBV infection via an adenoviral vector transferring a 1.3-fold oversized HBV genome (AdHBV) into transgenic DEREG mice, where Tregs were transiently but selectively depleted by injection of diphtheria toxin. Tregs mitigated immunomediated liver damage by downregulating the antiviral activity of effector T cells and by limiting cytokine production and cytotoxicity. However, Tregs did not influence development of HBV-specific CD8+ T cells or development of memory T cells. Exogenous TNF-α, partially abrogated the Treg-mediated suppression of antiHBcAg response (Stoop et al., 2007). The function of HBcAgspecific Treg cells was enhanced by soluble heat shock protein 60(sHSP60) produced from HBV-infected hepatocytes. Entecavir treatment suppressed the frequency and function of Treg cells (Kondo et al., 2010). Tregs determine the disease prognosis by reflecting infection progression and impaired immune response. Tregs are therapeutic targets for immunotherapy of HBV infection. 3.5. Blockage of immunoinhibitory signals Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic HBV infection. Limited evidence is available, supporting the nature of these receptors or their complex regulation, an excess of coinhibitory signals has been proposed to drive the T-cell exhaustion typical of persistent viral infections. PD-1/PD-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in HBV infection. Intrahepatic HBV-specific CD8+ T cells expressed higher levels of PD-1 and lower levels of CD127 than their peripheral counterparts (Fisicaro et al., 2010). PD-1 expression in CTLs may be related to the degree of liver damage in CHB patients. The HBV genotype C is associated with more severe damage (Xibing et al., 2013). Blockade of PD-1/PD-L1 interaction increased CD8+ T cell proliferation and IFN-γ and IL-2 production by circulating intrahepatic lymphocytes, even though anti-PD-L1 showed a stronger effect on intrahepatic compared with peripheral T cells (Stross et al., 2012). The reversed immune dysfunction and viral persistence of HBV infection suggested that the anti-PD-1 mAb might be a good therapeutic candidate in chronic HBV infection (Tzeng et al., 2012). CD244 and PD-1 are highly coexpressed in virus-specific CD8+ T cells in chronic HBV infection. Blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in CHB patients (Raziorrouh et al., 2010). T-cell immunoglobulin domain and mucin domaincontaining molecule-3 (Tim-3), a newly identified protein, negatively regulates antiviral responses of CD8+ T cells (Wu et al., 2012). T cells expressing Tim-3 showed an impaired ability to produce IFN-γ and TNF-α upon recognition of

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HBV-peptides. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8+ T cells and cytokine production in response to HBV-specific antigens (Nebbia et al., 2012b). Similarly, enhanced cytotoxicity was also observed in PBMCs or NK cells in CHB patients treated with the Tim-3 blockade ex vivo (Ju et al., 2010). CTLA-4 is expressed by HBV-specific CD8+ T cells in the presence of high levels of the proapoptotic protein Bcl2interacting mediator (Bim) enhancing this proapoptotic phenotype (Schurich et al., 2011). Blocking CTLA-4 increases the expansion of IFN-γ-producing HBV-specific CD8+ T cells in both the peripheral and intrahepatic compartments. Suppression of expression of human CTLA4 mRNA in human lymphocytes using RNA interference (RNAi) in vitro induced Th1/ Th2 response. Thus, blockage of CTLA-4 and Tim is a new therapeutic strategy for chronic HBV infection (Yu et al., 2009). 3.6. Activation of signaling pathways leading to IFN-alpha production TLRs (Toll-like receptors) are evolutionarily conserved pattern recognition receptors. They play a crucial role in early host defense in a range of microbes, including viruses, by recognizing so-called PAMPs (pathogen-associated molecular patterns) leading to innate immune activation and orchestration of the adaptive immune response. They are expressed in many kinds of cells including hepatocytes and effector cells of the innate and adaptive immune systems. However, the relationship between HBV and TLRs in hepatocyte has been relatively unclear in comparison with HCV and TLRs (Kondo et al., 2011). A growing body of evidence suggests that HBV inhibited innate responses in CHB, in particular by regulating the expression of TLRs (Barton, 2007). HBV and purified HBV virions, and HBeAg, or HBsAg suppress TLRs-induced antiviral signaling in hepatocytes as well as nonparenchymal liver cells (Wu et al., 2009) and TNF-α production, which may in turn contribute to viral persistence and chronic HBV infection (Lang et al., 2011). Results indicated that TLR2 and TLR4 failed to increase the CTL responses, whereas stimulation by TLR3, 5, and 7 exhibited a moderate adjuvant function. In contrast, stimulation of TLR9 dramatically increased the CTL responses (Schwarz et al., 2003). Activation of pDC using synthetic TLR7 and TLR9 ligands or agonists is capable of producing large amounts of type I and III IFN in response to many viruses, including HBV, which contributes to the suppression of HBV replication (Guiducci et al., 2009). These data suggested that the combination of TLRs agonists, especially TLR9 agonist and appropriately timed immune therapy may be a promising approach in the treatment of HBV infection (Kondo et al., 2011). 3.7. Therapeutic vaccines Therapeutic vaccines, as an alternative to long-term antiviral treatment or to support only partially effective therapy, are currently being developed for chronic HBV infection. Several clinical trials with peptide vaccine containing highly immunogenic HBc18-27 and HBV surface based protein vaccine showed limited efficacy if used as monotherapy (Senturk et al., 2009). Buchmann et al. (2013) evaluated a novel vaccine formulation comprising particulate HBsAg and

Please cite this article as: Wang, L., et al., Immunotherapeutic interventions in chronic hepatitis B virus infection: A review, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.04.004

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HBcAg, and the saponin-based ISCOMATRIX™ adjuvant for its ability to stimulate T and B cell responses in C57BL/6 mice. The results suggested that the vaccine induced multifunctional HBsAg- and HBcAg-specific CD8+ T cells as well as high antibody titers against both antigens. Other studies demonstrated that immunization with a combined formulation of HBsAg and HBcAg antigens along with heat shock protein gp96 or ubiquitin (Ub) induced robust antiviral T-cell response with higher levels of IL-2 and IFN-γ as well as a greater percentage of HBcAg-specific CD8+ T cells and antibody against HBsAg and HBcAg, with significant decrease of serum HBsAg and HBV DNA levels in mice (Wang et al., 2011; Chen et al., 2011). Thus, only peptide- and protein-based vaccines combined with adjuvants effectively activate anti-viral immunity. An effective vaccine should both induce a strong antigenspecific immune response and the subsequent deployment of immune response to HBV in the liver. DNA vaccine encoding HBV large envelope and/or core protein was shown to induce reduction in viremia and cccDNA in the liver in a duck model (Thermet et al., 2008). The latter was achieved by boosting the hepatic immune response (Obeng-Adjei et al., 2012). Another study reported that HBsAg+ cationic lipid DNA complexes (CLDC) in C57BL/6 mice suppressed HBV DNA non-cytolytically via induced T cell and B cell response (Morrey et al., 2011). Although immunizing the TM with DNA encoding homologous HBsAg was sufficient to induce CD8+ T-cell responses, HBsAg from a heterologous subtype was required to induce a CD4+ T-cell response. Importantly, injection with plasmid DNA followed by protein induced the production of antibodies against the HBsAg expressed by the TM (Bourgine et al., 2012). These data indicate that DNA vaccines combined with viral antigens were potential HBV therapeutic vaccines. 4. Future challenges Although antiviral therapy of chronic HBV infection has improved dramatically during the last decades and several new approaches have achieved preclinical validation, a completely effective treatment is still not available. Thus, functional cure of chronic HBV infection remains an important therapeutic challenge. Affordable therapeutic strategies including combination immunotherapies that eradicate or control HBV, without major side effects, are strongly needed. Future immunotherapeutic strategies should aim to restore virus-specific T-cell defects, while blocking potential pathways of liver damage (Nebbia et al., 2012a). NK cell and DC-based therapy hold great promise. The development of new therapies that target viral proteins, such as HBeAg, which regulate the immune system, may offer newer strategies to circumvent progression to CHB (Liu et al., 2012). The involvement of TLRs signaling suppression during HBV persistent infection should be considered carefully. For example, the relationship between HBV genotypes and the level of TLRs suppression needs to be elucidated. The combination of TLR agonists and appropriately timed immune therapy should be considered in future studies. IFN-α/-β and/or IFN-γ play critical roles in the intracellular antiviral responses induced by HBV infection. The function of IFN-λ in the host immune response to HBV is not clear. IFN-λ may provide valuable insights into novel strategies for the elimination of HBV infection (Pagliaccetti and Robek, 2010).

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Please cite this article as: Wang, L., et al., Immunotherapeutic interventions in chronic hepatitis B virus infection: A review, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.04.004

Immunotherapeutic interventions in chronic hepatitis B virus infection: a review.

Chronic hepatitis B virus (HBV) infection is a public health challenge worldwide. Antiviral agents (nucleos(t)ide analogues, NAs) and immune-based the...
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