Immunotherapy and Vaccine Development in Viral Hepatitis

More than 300 million people and 170 million people are chronically infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), respectively. To eradicate those viral infections, both prophylactic and therapeutic approaches are required. In HBV infection, there are global programs for prophylactic vaccination. However, some subjects, especially under immuno‐ compromized state, are unable to acquire ant-HBs antibody (Ab) with conventional vaccina‐ tion, and several attempts to improve the immunogenicity of HB vaccine have been made. On the other hand, development of effective prophylactic HCV vaccine has not been achieved mainly because of high rates of escape mutations within HCV envelope genes. We first focus on the recent development of prophylactic vaccine for HBV and HCV infections.


Introduction
More than 300 million people and 170 million people are chronically infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), respectively. To eradicate those viral infections, both prophylactic and therapeutic approaches are required. In HBV infection, there are global programs for prophylactic vaccination. However, some subjects, especially under immunocompromized state, are unable to acquire ant-HBs antibody (Ab) with conventional vaccination, and several attempts to improve the immunogenicity of HB vaccine have been made. On the other hand, development of effective prophylactic HCV vaccine has not been achieved mainly because of high rates of escape mutations within HCV envelope genes. We first focus on the recent development of prophylactic vaccine for HBV and HCV infections.
In the second half of the review, we summarized immunotherapeutic approach for both viral infections. Neither HBV nor HCV is cytopathic, and hepatitis is caused by the host immune response against virus-related peptides expressed on hepatocytes in conjunction with human leukocyte antigens (HLA). In acute self-limiting hepatitis, a broad immune response occurs that is strong enough to eradicate the virus or suppress viral replication [1], indicating that efficient induction of anti-viral immune response could have a potential to control viral infections. However, in chronic hepatitis, there are many mechanisms that hamper the antiviral immune response leading to persistent viral infection.
In chronic HBV infection, strong long-term viral suppression can now be achieved with various nucleoside and nucleotide analogs. However, there are some problems that must be solved in the near future. One of the problems in the treatment of nucleos(t)ide analogs is a low rate of HBe seroconversion even after long-term administration in HBeAg + patients. Moreover, reactivation rate of HBV replication is high in both HBeAg + and HBeAgpatients after cessation of the treatment, although drug-free viral controls would be better than long-term adminis-Non-responder for HBV vaccination and vaccine escape mutants are important problems to be focused in the future. The general recommendation for non-responders is to repeat a threedose schedule; 50-75% is expected to respond to the second dose. Non-responders to the second dose should be evaluated for underlying chronic HBV infection. For hemodialysis patients, response may be improved by using double-dose vaccine [5]. Intradermal administration have been tried to improve the effectiveness of vaccination but is technically difficult [6] and longterm efficacy has not been demonstrated. Several trials to improve the effectiveness of HBV vaccination particularly in hemodialysis patients have been investigated (Table 2). Combined use of granulocyte macrophage-colony stimulating factor or levamisole as an adjuvant of HBV vaccination is a promising strategy [7-9]. New chemical adjuvant has also shown an excellent potential [10][11][12]. On the other hand, Interleukin (IL)-2 is not shown to be effective when used as an adjuvant [13,14]. Recently, a new HBV vaccine including preS lesion, which is known as an essential site for HBV-entry to hepatocyte, has been tried [15,16]. (preS1/PreS2/preS) GM-CSF;granulocyte macrophage-colony stimulating factor, OR; Odds ratio, RCT; randomized controlled trial, TLR; tolllike receptor Table 2. Human trials to improve the effectiveness of HBV vaccination

Escape mutant
Mutations in the small-S protein, commonly glycine to arginine substitution at codon 145 (G145R), have been found in some children born from mothers infected with hepatitis B [17]. Although these mutants have been found in many parts of the world, the prevalence appears to be low and decline in the efficacy of HBV vaccine has not been reported. However, a recent report showed that the mutant HBV was transmitted by sexual contact with a subject who had received universal HBV vaccination [18]. Application of HBV vaccine including preS protein to block infection of HBs mutant could be an effective strategy and should be investigated in the future.

Recent advances in HCV vaccine
In the analysis of secondary HCV infection after spontaneously clear primary infection, increased rate of spontaneous viral clearance and broader T-cell responses was found [19]. Actually, an animal study using chimpanzee showed that T-cell vaccine elicits effective immunity against HCV challenge thorough early CD4 + and CD8 + T-cell response [20]. These indicate the induction of protective memory responses against HCV during natural infection and also suggest a possibility for the development of effective prophylactic HCV vaccines.
B-cell response against HCV through the production of neutralizing antibodies has been analyzed for the development of HCV vaccine. In acute HCV infection, the detection of neutralizing antibodies and consequent rapid clearance of HCV had been reported [24]. On the other hand, delayed induction of these neutralizing antibodies has been shown in patients developing chronic HCV infection, [21]. These observations represent that the neutralizing antibodies could not control HCV infection and there are escape mechanisms of HCV from those antibodies. Actually, rapid evolution in the envelope glycoprotein sequences has been demonstrated as the mechanism of HCV evasion [22]. The HCV envelope glycoproteins, E1 and E2, have proven to be the essential region not only for viral attachment but also for viral endocytosis into hepatocytes [23]. Hypervariable region (HVR) is known to be located at the N-terminus of E2 region and is highly immunogenic and the region is necessary for binding to scavenger receptor class B type I (SR-BI), a lipoprotein receptor molecule involved in HCV entry [24,25]. Furthermore, conformational epitope within E2 is known to be conserved among various genotypes of HCV and is necessary for binding to CD81 [26,27]. While, E1 displays a relatively high degree of conservation within subtypes, also suggesting a high degree of intergenotypic cross-neutralization potential [28].
CD4 + and CD8 + T-cell are also important in viral clearance [29][30][31]. CD4 + T-cells against conserved protein epitopes, such as HCV core, nonstructural (NS)3, NS4 and NS5, have been associated with self-limited infection of HCV. Trials for HCV prophylactic vaccines can be divided into two strategies to induce T-cell response or neutralizing antibodies, but both should be addressed together for an effective prophylaxis strategy. Since acute HCV infection is usually asymptomatic and is not associated with liver failure, prevention from acute to chronic HCV infection is another key goal of vaccine development.
Preclinical evaluation of prophylactic vaccine has been performed in chimpanzee, the only established model for the study of HCV infection in an immunocompetent host. These preclinical trials and the results are summarized in Table 3. Not only humoral responses but also cellular responses were elicited by vaccination, leading to viral clearance after HCV

Immnunotherapy for viral hepatitis and vaccine development
To develop efficient immunotherapy, understanding of immune response for eradication or suppression of hepatitis virus during acute hepatitis is important. Moreover, the immunosuppressive mechanisms leading to persistent viral infection need to be analyzed.

Immune response in acute viral hepatitis
Immunological analysis has been extensively performed in transgenic and chimpanzee models of acute HBV infection. In one model, transgenic mice, in which infectious HBV virions replicate in the liver with expression of all HBV-related antigens, were injected with HBsAgspecific cytotoxic T lymphocytes (CTLs) that had been induced in nontransgenic mice. The transgenic mice produced interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which purged viral RNA and DNA without destroying infected hepatocytes [40,41]. Importantly, this noncytolytic clearance of intracellular HBV is more efficient at controlling HBV replication than the killing of infected hepatocytes. This was confirmed in a chimpanzee infection model [42] and incubation phase of acute hepatitis B in humans [43].
The same is essentially true in acute HCV infection. Multispecific and vigorous CTL responses against HCV antigens are important for successful eradication of the virus. Moreover, a CD4 + T cell response at an early stage of acute infection and persistence of the response are apparent in acute infection [44]. In contrast to acute HBV infection, the majority of patients with acute HCV infection progress to persistent infection, and the mechanisms underlying failure to eradicate the virus have been analyzed. The failure of CD4 + T cell function is a key factor in HCV persistence and CD4 + T cells from persistent infection do not produce Th1 cytokines, such as IFN-γ and IL-2, but produce IL-4 and IL-10, clearly distinct from those seen in patients with recovery [45]. Moreover, an early and strong Th1 response has been shown to play an important role in disease resolution.
The contributions of CD4 + and CD8 + T cells to the control of viral infection were analyzed in a chimpanzee model of acute hepatitis B and C by depleting either T cell population with monoclonal antibodies (Abs). The data showed that both CD4 + and CD8 + T cells are required for virus elimination [46,47].

Hierarchy of T cell response in viral hepatitis
The antigen-specificity of the T cell response to HBV in acute hepatitis has been analyzed, and it is clear that acute viral hepatitis involves a vigorous CTL response to multiple epitopes in the viral nucleocapsid, envelope, and polymerase proteins, while these are not seen in patients with chronic hepatitis [1]. Although multi-specificity of the CTL response is characteristic in acute hepatitis, there is known to be a hierarchy of epitope-specific CD8 + T cell responses determined by cytokine production after peptide stimulation. In acute hepatitis B, CD8 + T cell response to HBc18-27 (HLA-A2 restricted epitope) is dominant followed by the response to polymerase epitope (455 -463), whereas envelope epitopes are always subdominant [48]. The hierarchy is clearly distinct from that observed in chronic hepatitis, in which the CD8 + T cell response to envelope epitope (183 -191) is always dominant. Interestingly, chronic hepatitis patients with lower HBV DNA levels in the serum show greater responses to HBc18-27 than those with high HBV DNA. These findings imply that the T cell response to HBcAg is important for viral control, which is important for designing peptide vaccines for the treatment of chronic HBV infection.
In acute HCV infection, the CTL responses were directed against multiple viral epitopes, in particular within the structural (core) and nonstructural (NS) regions of the virus (NS3, NS4, and NS5), and the CTL frequencies were higher in patients with acute infection [30,49]than in those who develop persistent infection. The hierarchy of HCV epitopes has not been analyzed extensively, but resolution of primary infection in the chimpanzee was shown to be associated with a dominant CD4 + T cells response against epitopes including NS3 (GYKVLVLNPSV) [50].

Immune response in chronic viral hepatitis
In contrast to acute hepatitis, the T cell response to HBV is weak and is narrowly focused in chronically infected patients [51], suggesting that it may be a cause of persistent infection.
HBV-specific helper and CTLs are barely detectable in peripheral blood of patients with chronic hepatitis B (CHB) [52], possibly due to exhaustion by high viral load or tolerance to HBV.
In contrast to chronic HBV infection, CTL response against various HCV epitopes including core and envelope and NS regions can be detected in chronic HCV infection, especially in liverinfiltrating lymphocytes [53]. Although intrahepatic CTL response was shown associated with low viral load [54], the CTL response is not enough to terminate HCV infection possibly due to the presence of immunosuppressive mechanisms similar to chronic HBV infection.

Regulatory T cells (Tregs)
In HBV infection, significant accumulation of CD4 + CD25 + FoxP3 + Treg cells in the liver was found in patients with chronic HBV infection. Moreover, patients with high viral load have a higher proportion of Tregs in the liver [55], suggesting that intrahepatic Tregs suppress antiviral immune responses in the liver in chronic hepatitis B virus infection. In HCV infection, several groups have also shown a higher frequency of CD4 + CD25 + regulatory T cells in the blood of chronically HCV-infected patients versus recovered or healthy individuals [56,57] and the presence of CD4 + FoxP3 + T cells in the liver of chronically HCV-infected patients [58].

Programmed Death-1 (PD-1)
PD-1 is a surface receptor critical for the regulation of T cell function [59,60]. Binding to PD-1 by its ligands PD-L1 and PD-L2 results in the antigen-specific inhibition of T cell proliferation, cytokine production, and cytolytic function, leading to exhaustion of T cells.
In chronic HCV infection, circulating and intrahepatic HCV-specific CD8 + T cells were found to express high levels of PD-1 [62], and PD-1 expression level in the liver is higher than that in peripheral blood. Increased expression of PD-1 is associated with CD8 + T cell dysfunction, and functional restoration is achieved by blocking the signal from PD-1 [63]. Interestingly, HCV core protein induces PD-1 and PD-L1 on T cells from healthy donors [64], indicating that immunosuppressive ability of HCV core protein is mediated by the upregulation of inhibitory molecules on T cells. Increased PD-1 expression on HCV-specific CTLs was reported to be significantly associated with poor response to antiviral therapy [65].

IL-10
IL-10 is an important cytokine with anti-inflammatory properties, and is produced by activated monocytes/macrophages and T cell subsets, including Treg and Th1 cells [66]. In chronic HBV infection, HBcAg stimulates the production of IL-10, which negatively regulates HBcAgspecific Th17 cell responses in CHB patients [67].
In HCV infection, HCV proteins have been shown to induce IL-10 from monocytes in patients with chronic HCV infection, leading to suppression of antiviral immune response [68].

T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3)
Recently, an inhibitory molecule, Tim-3, has been reported. A high frequency of Tim3expressing CD4 + and CD8 + T cells are found in chronic HBV infection, and the frequency of Tim-3 + T cells was positively correlated with the severity of liver inflammation, and negatively correlated with plasma IFN-γ levels [69]. Tim-3 was also highly expressed on CD4 + and CD8 + T cells in HCV infection, with the highest levels seen on HCV-specific CTLs. Tim-3 expression is associated with reduced Th1/Tc1 cytokine production, and blocking the Tim-3 -Tim-3 ligand interaction could enhance CD4 + and CD8 + T cell proliferation in response to HCVspecific antigens [70].

Dysfunction of Dendritic Cells (DCs)
In patients with CHB, maturation of DCs from peripheral blood of patients after incubation with cytokines is lower than that of normal subjects with lower expression of HLA-DR and costimulatory molecules in the former population [71], leading to low allostimulatory function of DCs from CHB patients. Interestingly, impaired function of monocyte-derived DCs from patients with CHB could be reversed by inhibiting viral replication with nucleoside analogs such as lamivudine [72]. Type 2 precursor plasmacytoid dendritic cells (pDCs), which are the most important cells in antiviral innate immunity, were also reported to have quantitative and qualitative impairment in patients with chronic HBV infection [73]. Recently, HBV itself was shown to inhibit the functions of pDCs [74].
In chronic hepatitis C, DCs from patients also show impaired immunostimulatory function, which could be induced by HCV [75] or NS4 protein [76]. Monocyte-derived DCs from HCV patients were shown to induce proliferation of CD4 + CD25 + FoxP3 + regulatory T cells, which limit proliferation of HCV-specific T lymphocytes [77]. DCs in HCV patients thus inhibit T cell responses via a variety of mechanisms.

Immunotherapeutic trials for viral hepatitis
Previous basic analyses and human trials in HBV infection are listed in Tables 4 and 5, respectively, and those in HCV infection are summarized in Tables 6 and 7. Combination therapy with GM-CSF and HBsAg vaccine in HBV carrier children Significantly reduced serum HBV DNA. [136] High dose of IL-12 (0.5μg/kg) HBV DNA clearance was observed in 25% of the patients. [137] Combination of IL-12 and lamivudine Stimulated T cell response to HBV with IFN-γ production.
However, IL-12 was unable to suppress re-elevation of HBV DNA after cessation of lamivudine. [138] Combination of IL-12 and IL-18 Stimulated IFN-γ production by CD4+ T cells isolated from peripheral blood in response to HBcAg, and the effect was greater than those observed with either cytokine alone. [139]

Suppression of viral replication
High viral load has been shown to suppress CD4 + and CD8 + T cells in addition to induction of Tregs, which could be reversed by antiviral therapy [78]. Therefore, immunotherapy followed by restoration of virus-specific T cell response with antiviral therapy could be more efficient.

Peptide immunization
A fusion peptide consisting of HBc18-27 and HIV Tat49-57 was synthesized, and the vaccination induced significant anti-viral effect in HBV transgenic mice [79]. However, in humans, peptide vaccine containing highly immunogenic HBc18-27 administered to CHB patients, showed disappointing results [80], because there was no induction of a significant antiviral T cell response.
In HCV infection, a vaccine, IC41, containing 7 HCV T cell epitopes and the Th1 adjuvant induced HCV-specific Th1/Tc1 responses in chronic HCV patients, but anti-viral effects were minimal [81]. Another HCV vaccine with a peptide derived from HCV core protein induced both cellular and humoral responses in HCV patients and reduced serum alanine aminotransferase (ALT) and alpha-fetoprotein (AFP) in some patients [82].

Protein immunization
In a model of HBV in transgenic mice, HBsAg vaccine in complete Freund's adjuvant once a month for 12 months induced reduction in HBV DNA, and the disappearance of HBeAg and HBsAg in most mice treated [83]. Interestingly, some mice developed anti-HBs in the sera. However, several human trials with HBsAg vaccine showed limited efficacy if used as monotherapy.
In HCV infection, a recombinant poxvirus vaccine expressing HCV NS3, NS4 and NS5B, TG4040, has been recently developed [103] and administered to HCV patients. The vaccine was safe and induced HCV-specific cellular immune response and reduction in viremia. These data are encouraging, and further large scale clinical trials need to be done.

DNA immunization
Injection of plasmid DNA has been shown to strongly elicit both cellular and humoral immune responses. DNA vaccine is now shown to be safe and well-tolerated, and has been tried in humans with some encouraging anti-viral effects both in mice and humans [104].
In a model of duck hepatitis B virus infection, DNA vaccine encoding HBV large envelope and/or core protein was shown to induce reduction in not only viremia [105] but also cccDNA in the liver in one third of ducks receiving DNA monotherapy or combination treatment along with lamivudine [106]. This finding is encouraging because clearance of cccDNA from the liver is the goal of treatment for HBV infection, but is difficult to achieve using IFN-α or nucleoside analogs. More recently, DNA vaccine expressing HBcAg or HBsAg in combination with extracellular domain of CTLA-4 have been developed and showed significant anti-viral effects in HBV transgenic mice [107,108]. In humans, safety and therapeutic potential of DNA vaccines have been already explored in chronic HBV carriers [109][110][111].
In HCV infection, DNA vaccine encoding HCV core and IL-2 breaks tolerance and activates previously tolerant T cells in HCV transgenic mice [112]. NS3-specific T cells were induced by DNA immunization in mice models [113][114][115][116]. In chimpanzee models, NS3-specific T cells were also induced and HCV-challenge could be controlled [117]. In HCV patients, a new DNA vaccine, CIGB0230, consisting of a mixture of plasmid expressing HCV structural antigens induced HCV-specific T cell response and improved liver histology [118]. Furthermore, trials to elicit HCV-specific T cells response by transduction of HCV-specific T cell receptor or by new type vaccines have been made [119,120].

DC immunization
DCs are specialized antigen-presenting cells that can induce strong immune responses in T and B cell. We have previously shown that activated bone marrow-derived DCs can break CTL tolerance to HBsAg in HBV transgenic mice [121]. Thereafter, several immunotherapies with activated DCs have been applied in both animals and humans. In a recent study performed in HBV transgenic mice, peptide-pulsed DCs were shown to significantly reduce the concentrations of serum HBsAg and HBV DNA [122], indicating therapeutic potential in chronic HBV infection. Moreover, when intrahepatic antigen-presenting cells, including DCs, were activated by injection of anti-CD40 agonistic Ab, HBV replication was inhibited by a noncytopathic mechanism possibly through production of antiviral cytokines such as TNF-α and IL-12 [123]. Although no CTL response against HBV antigens was reported in this study, the in vivo activation of DCs could be an alternative way for inducing antiviral immune responses including possible activation of CTLs against HBV. In humans, injection of activated DCs loaded with HBV peptide or protein achieved the induction of HBV-specific immunity [124,125] and a reduction in HBV DNA level in some patients [126,127]. HBeAg negativity was achieved in more than half of the treated patients in one study [126].
In HCV infection, DCs treated with peptide inhibitors of IL-10 were shown to induce strong anti-HCV T cells response in HCV transgenic mice [128], suggesting a strategy to augment the immunogenic function of DCs. Recently, murine DCs infected with adenovirus encoding HCV NS3 were used as vaccines, and showed induction of NS3-specific T cell response and antiviral effect [129]. In humans, DCs infected with adenovirus vectors harboring HCV core or NS genes, especially NS3, were administered in healthy subjects [130,131] and HCV patients [114,118], and those DCs induced CD4 + and CD8 + T cell response in both populations. Although preparation of activated and mature DCs incurs financial costs and requires experienced researchers, immunotherapy with DCs is a promising method.

Cytokines and Thymosin-α1 (Talpha1)
Cytokines, such as IL-12 [132] and IL-18 [133], and the activation of NKT cells [134] were shown to inhibit HBV replication noncytopathically in HBV transgenic mice. In humans, GM-CSF [135,136] and IL-12 [137][138][139] have been used for treatment with some antiviral effects. They have been used as monotherapy or in combination with HBsAg vaccine or lamivudine.
Talpha1, a synthetic 28-amino acid peptide, is able to enhance the Thl immune response and also exerts a direct antiviral mechanism of action. It has been used for the treatment of chronic HBV [140][141][142][143] and HCV [144] infection in humans, and showed antiviral effect with some efficacy. Although antiviral effect by the addition of Talpha1 to lamivudine or IFN-α therapy was controversial, a meta analysis demonstrated that the combination therapy with lamivudine and Talpha1 showed significantly higher rates of ALT normalization, virological response, and HBeAg seroconversion as compared with lamivudine monotherapy [145]. It is of note that HBeAg seroconversion rate was 45% in the combination group, which was significantly higher than that with lamivudine monotherapy (15%).

Blockade of inhibitory signals
There have been several basic attempts to improve the efficacy of immunotherapy. Among these reports, augmentation or restoration of T cell response by blocking the inhibitory signals have been extensively analyzed in vitro. It has been demonstrated that exhausted T cells

Activation of anti-viral immune response by vaccines and cytokines
• peptide • protein • DNA

Restoration of immune response
Effective induction of anti-viral immune response  express not only PD-1, but also CTLA-4 [146,147], CD244 [148] or Tim-3 [70,149], and blocking of these molecules in combination could be better than blocking any single molecule to achieve full activation of the exhausted T cells.

Conclusion
There have been several advances in immunotherapy and vaccine development both for prophylactic and therapeutic purposes in HBV and HCV infections and some of the data are promising. For therapeutic purposes, viral suppression, stimulation of antiviral immune response with vaccines with peptides, proteins, plasmid or DC, blockade of immunoinhibitory signals must be combined to achieve desirable antiviral effects (Fig.1). Further studies are required to explore the best protocols and their most efficient combinations to become a promising and practical treatment.