InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Medicine » Infectious Diseases » "Practical Management of Chronic Viral Hepatitis", book edited by Gaetano Serviddio, ISBN 978-953-51-1109-2, Published: May 29, 2013 under CC BY 3.0 license. © The Author(s).

Chapter 3

Immunotherapy and Vaccine Development in Viral Hepatitis

By Kazuto Tajiri and Yukihiro Shimizu
DOI: 10.5772/55525

Article top

Immunotherapy and Vaccine Development in Viral Hepatitis

Kazuto Tajiri1 and Yukihiro Shimizu2

1. 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 HBeAg patients after cessation of the treatment, although drug-free viral controls would be better than long-term administration of the drugs in terms of control of medical costs and avoidance of adverse effects of these agents. Therefore, it would be beneficial to achieve long-term viral eradication even after cessation of nucleos(t)ide analogs in combination with efficient immunotherapy.

On the other hand, antiviral oral drugs, such as protease inhibitor or polymerase inhibitor showing potent antiviral ability [2], have been developed for chronic HCV infection. However, not all patients treated with these drugs could achieve sustained virological response and high medical cost for each patient is a global serious problem. Effective immunotherapy combined with these drugs may improve their antiviral effects and control medical costs also in hepatitis C.

2. Problems and recent advances in HBV vaccination

2.1. Non-responder for HBV vaccine (Table 1)

In HBV infection, HBV is transmitted at a high incidence rate by parenteral, percutaneous or sexual contact. Therefore, primary protection is very important and universal vaccination regardless of maternal HBsAg status is recommended. Most of countries have introduced HBV vaccination into their national immunization programs and more than 80% of infants have received HBV vaccine three times. In Taiwan, universal vaccination program of all newborns was introduced in 1986. After twenty years of the program, the rate of chronic HBV infection decreased from 9.8% to 1.2% and the risk of childhood HCC has been decreased by 70% [3, 4].

Host factors
Age > 40 years
Genetics, certain HLA types
Other medical conditions
Renal failure
Conditions requiring immunosuppressive therapy
Unrecognized chronic HBV infection
Subcutaneous administration
Freezing of vaccine

Table 1.

Causes of non-responder to HBV vaccine

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 three-dose 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 long-term 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 [10-12]. 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].

Materials Mechanism Effectiveness ref
GM-CSFdendritic cell activationMeta-analysis: OR 4.63[7]
Levamisoleinterferon inducerMeta-analysis: OR 2.43[8]
upregulation of defective immune functionMeta-analysis: OR 2.77[9]
Interleukin-2enhanced cellular immunityRCT: no significant effect[13, 14]
HB-AS04upregulation of CD86, increased cytokineRCT: Significant at month 3[10]
(aluminium salt, monophosphoryl lipid (MPL))RCT: Significant[11]
HB-AS02TLR4 agonist, improve antigen presentationRCT: Significant[12]
(MPL, QS21: extract from Quillaja saponaria)
Sci-B-Vac/BioHepBblock HBV-entry to hepatocyte? preS1 antibody 50~60% positive[15, 16]

Table 2.

Human trials to improve the effectiveness of HBV vaccination

[i] - GM-CSF;granulocyte macrophage-colony stimulating factor, OR; Odds ratio, RCT; randomized controlled trial, TLR; toll-like receptor

2.2. 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.

3. 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 [29-31]. 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 challenge [20,32-37]. Because many chimpanzees spontaneously resolve acute hepatitis C in unvaccinated control groups, definite conclusions for the efficacy of HCV vaccination should be analyzed in human studies. Most vaccine candidates were successful in inducing immune response and reducing viral load. However, protection of infection following challenge with heterologous strains was limited. Although these limited protection shows the difficulty in developing a vaccine against different isolates, these preclinical trials certainly provides information on potential and design of vaccine candidates. As phase I human clinical trial, E1/E2 vaccine adjuvanted with MF59 (an oil-in-water emulsion) was administered to healthy adults, and neutralizing antibodies could be induced without adverse events [38]. Furthermore, these antibodies showed the neutralizing capacities against heterologous virus strains [39]. Further trials should be made for the development of effective HCV vaccines.

Materials Outocome ref
E1/E2 protein21 vaccinated/24 controls[32]
2/12 chronic infection after homologous challenge
1/9 chronic infection after heterologous challenge
Strong humoral immune response
15/24 chronic infection in controls
DNA plasmid: E22 vaccinated/1 controls[33]
2/2: viral clearance
High anti-E2 antibodies in one vaccine
E2-specific CD4 T-cell response in the second
DNA plasmid: Core-E1-E2, NS3-56 vaccinated/2 controls[34]
2/6: viral clearance
HCV-specific antibody and T-cell response
Reduced peak viral load in all animals
1 sterilizing immunity: high anti-E2 and strong cellular response
1/2 controls: viral clearance
Adenovirus NS3-NS5B5 vaccinated/5 controls[20]
4/5: viral clearance after 18 months
Peripheral and intrahepatic CD8 T-cell response
3/5 controls: viral clearance
HCV like particle: Core E1-E24 vaccinated/4 controls[35]
4/4: viral clearance after 12 months
HCV specific CD4 and CD8 T-cell response
1/4 controls: viral clearance
DNA plasmid: Core E1-E2 NS34 vaccinated/2 controls[36]
1/4: viral clearance
Reduction of HCV load in serum and liver
Strong HCV CD4 response
Anti-E1 and anti-E2 specific antibodies
Vaccinia virus Core E1-E2-P7 -NS3-NS34 vaccinated/2 controls[37]
1/4: viral clearance after homologous
T-cell response: vigorous IFNγ production and moderate proliferation

Table 3.

Preclinical trials of HCV vaccines in chimpanzees

4. 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.

4.1. 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 HBsAg-specific 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].

4.2. 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].

4.3. 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 liver-infiltrating 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.

4.4. Immunosuppressive mechanisms responsible for persistent hepatitis virus infection

4.4.1. 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].

4.4.2. 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.

Intrahepatic HBV-specific CD8+ T cells express higher levels of PD-1, and upregulation of intrahepatic PD-1/PD-L1 is associated with liver inflammation and ALT elevation [64]. PD-1/PD-L1 blockade increased CD8+ T cell proliferation and enhanced IFN-γ and IL-2 production by intrahepatic lymphocytes [61].

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].

4.4.3. 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 HBcAg-specific 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].

4.4.4. T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3)

Recently, an inhibitory molecule, Tim-3, has been reported. A high frequency of Tim3-expressing 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 HCV-specific antigens [70].

4.4.5. 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.

5. 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.

Animal model Immunotherapy Results Ref
Peptide vaccination
HBV transgenic mice A synthesized fusion peptide, consisting HBcAg18-27 and HIV Tat49-57 adjuvanted with CpG ODN Decreased in serum HBV DNA levels and the expression levels of HBsAg and HBcAg in the liver [79]
Protein vaccination
HBV transgenic mice HBsAg vaccine Most of the mice showed reduction of HBV DNA levels and disappearance of HBeAg and HBsAg [83]
Woodchuck hepatitis Virus infection Combination of vaccine of HBV large surface protein and clevudine Restored T-cell response to Pre-S and S region.[85]
MiceChimeric HBsAg-preS1 proteinPrimed both HBcAg-specific T cells and antibodies to preS1.[86]
Balb/c and HBV transgenic miceChimeric HBsAg-preS1 proteinInduced strong anti-HBc and moderate anti-preS1 immune response, and reduced HBsAg and HBV DNA in HBV-Tg mice. [96]
Balb/c miceChimeric protein with HBcAg and carboxy terminus of the Hsp65Induced moderate anti-HBc immune response and strong HBcAg-specific T cells response.[97]
Balb/c and HBV transgenic miceHBsAg, HBcAg and heat shock protein gp96Decreased serum HBsAg and HBcAg expression in hepatocytes by 45% and 90%, respectively. Decreased serum HBV DNA to below or close to the detection limit.[98]
Balb/c and HBV transgenic miceFusion protein with protein transduction domains from HIV-1-Tat and HBcAgInduced HBcAg-specific CTLs and enhanced production of IFN-γ, IL-2, IL-4 and IL-10. Reduced HBV DNA and HBsAg in the serum and HBsAg expression in liver tissue of HBV transgenic mice.[99]
DNA immunization
WoodchuckDNA vaccine expressing WHsAg was administered by electroporationInduced dose-dependent antibody and T cell responses to WHsAg more efficiently than conventional hypodermic needle injection.[104]
Acute DHBV infection DNA vaccine expressing DHBc and Pre-S/S and entecavir
Boosted with fowl poxvirus vectors expressing DHBc and Pre-S/S
Cleared DHBV infection at a rate of 100%. [105]
Chronic DHBV
DNA vaccine encoding the HBV large envelope and/or core protein with or without lamivudine Reduced viremia and liver DHBV cccDNA in 33% of ducks.
Seroconversion to anti-pre S in 67% of ducks showing cccDNA clearance.
HBV transgenic miceDNA vaccine expressing HBcAg fused to extracellular domain of CTLA-4. Mice were challenged by pAAVNBV1.2Reduced serum HBV DNA and HBcAg in the liver. Clearance of serum HBsAg was also observed. [107]
HBV transgenic miceDNA vaccine expressing HBsAg fused to extracellular domain of CTLA-4. Serum levels of HBsAg and HBV DNA were decreased by induction of anti-HBs Ab and HBsAg-specific CD8+ T cell response. [108]
DC immunization
HBV transgenic mice Activated bone marrow-derived DCs Broke CTL tolerance to HBsAg. [121]
HBV transgenic mice HBV-specific peptide-pulsed DCs Reduced in the serum HBsAg and HBV DNA. [122]
HBV transgenic mice Anti-CD40 agonistic monoclonal Ab Induced noncytopathic inhibition of HBV replication mediated by antiviral cytokines (IL-12 and TNF-α) produced by activated intrahepatic APCs.[123]
Cytokines and adjuvants
HBV transgenic mice Recombinant IL-12 Markedly inhibited HBV replication in the liver. [132]
HBV transgenic mice α-galactosylceramide that can activate NKT cellsInduced complete inhibition of HBV replication. [133]
HBV transgenic mice Recombinant IL-18Inhibited HBV replication noncytopathically, mediated by activation of resident intrahepatic NK cells and NKT cells. [134]
Gene therapy
HBsAg transgenic mice Lentivectors expressing HBsAg and IgFc fusion Ag Induced seroconversion to anti-HBs. [100]
HBV transgenic miceCationic lipid DNA complexes and HBsAgSuppressed HBV DNA in hepatocytes non-cytopathically.[101]
WoodchuckCationic liposomes and non-coding DNA was administered with WHsAg intramuscularlyInduced rapid and high Ab and T cell response to WHsAg.[102]

Table 4.

Immunotherapeutic approaches for animal models of HBV infection

[i] - CpG ODN; CpG oligodeoxynucleotide, WHV; woodchuck hepatitis virus, DHBV; duck hepatitis B virus, cccDNA; covalently closed circular DNA, DC; dendritic cells, CTL; cytotoxic T lymphocytes, APC; Antigen-presenting cells, NKT; natural killer T, Ig; immunoglobulin

Immunotherapy Results Ref
Peptide vaccination
A vaccine with HBc18-27 peptide comprised of a T-helper cell epitope and two palmitic acid residuesLow levels of CTL activity were induced but no significant changes in liver biochemistry or viral serology were observed. [80]
Protein vaccination
PreS2/S (GenHevac B) or S (Recombivax). HBe/anti-HBe seroconversion was observed in 13% and HBV DNA negativity was in 16% of the treated patients. [84]
Oral administration of HBV envelope proteins (HBsAg+preS1+preS2)Induced histological improvement in 30%, HBeAg negativity in 26.3% and HBsAg-specific T cell proliferation in 78% of the treated patients.[87]
Combination of lamivudine and HBsAg vaccine Induced sustained negativity of HBV DNA in 1/4 of the patients. [88]
The combination with lamivudune and HBsAg vaccine in HBeAg+ casesNo improvement of HBe seroconversion rate was observed in comparison with lamivudine therapy alone.[89]
Combination of lamivudine and HBsAg vaccine HBV DNA became undetectable in 64% of the patients, and was decreased in the remaining patients. [90]
Intradermal HBsAg vaccine and laimvudine in combination with IL-2 Induced significant HBV DNA loss in the serum in two of five of the treated patients. [91]
IFN-α-2b monotherapy (9 months) or IFN-α-2b plus pre-S2/S vaccineInduced greater reduction in HBV DNA in patients with combination HBV therapy than those who received IFN-α-2b monotherapy. [92]
Complexes composed of yeast-derived hepatitis B surface antigen (HBsAg) and antibodies HBeAg seroconversion rate was 21,6% and was correlated with decrease of HBsAg and HBV DNA. [93]
HB preS/S vaccine (GenHevac B)Caused no effect on HBV DNA and seroconversion of HBeAg to HBeAg in the immunotolerant phase of children with chronic HBV infection.[94,95]
DNA immunization
DNA vaccine encoding HBV envelope protein Induced an increase in HBV-specific IFN-γ-secreting T cells in nonresponders to conventional therapies, and HBV DNA levels were transiently decreased in 50% of vaccinated patients. [109]
DNA vaccine encoding PreS and S in patients with lamivudine breakthrough Induced IFN-γ-producing T cells specific for preS or S antigen. Two of 10 patients showed seroconversion to anti-HBe. [110]
DNA vaccine encoding HBsAg followed by recombinant modified vaccinia virus Ankara expressing HBsAgFailed in decrease in AST or ALT and did not reduce HBV viremias. [111]
DC immunization
Activated DCs from PBL pulsed with HBsAgInduced anti-HBs and HBsAg-specific cellular immnunity in some patients. [124]
PBL-derived DCs from chronic hepatitis B incubated with a cocktail of cytokines: IL1-β, PGE2, IL-6 and TNF-α, and pulsed with HBsAg or HBcAgInduced autologous T cell proliferation and Ag-specific IFN-γ production.[125]
Peripheral blood-derived DCs, activated with GM-CSF and IL-4 pulsed with HBsAg. Both patients with normal and elevated ALT responded equally to DC vaccine and 53% of the patients showed induction of HBeAg negativity. [126]
Activated DCs from PBL with GM-CSF and IL-4, pulsed with two peptides, HBc18-27 and PreS2 44-53. Undetectable HBV DNA was achieved in 46.3% and 3.13% of HBeAg- and HBeAg+ patients, respectively. ALT normalization was observed in 69% and 30.5% of HBeAg- and HBeAg+ patients, respectively. [127]
GM-CSFSafe and tolerable up to 1.0mg/kg body weight, and induced HBV DNA negativity in 4/8 patients.[135]
Combination therapy with GM-CSF and HBsAg vaccine in HBV carrier childrenSignificantly 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]
Thymosin-α 1(Talpha1)
Combination of Talpha1 and IFN-α No significant differences was observed as compared with IFN-α monotherapy with respect to HBeAg seroconversion, changes in histology, normalization of ALT or loss of HBV DNA. [140]
Talpha1 aloneAt 12 months after cessation of therapy, 36.4% of patients treated with 1.6mg of Talpha1 achieved ALT normalization, 15% achieved HBV DNA clearance by transcription-mediated amplification, and 22.8% achieved clearance of HBeAg. [141]
Comparative effect of Talpha1 and IFN-α Talpha1 treatment was more effective in achieving ALT normalization and HBV DNA negativity at the end of the follow-up period than IFN-α. [142]
Combination of Talpha1 and lamivudine No any additional antiviral effect compared with lamivudine monotherapy as assessed by HBe seroconversion and the emergence of viral breakthrough. [143]
Combination therapy with lamivudine and Talpha1 Induced significantly higher rates of ALT normalization, virological response, and HBeAg seroconversion than lamivudine monotherapy. [145]

Table 5.

Immunotherapeutic trials for chronic HBV infection in humans

[i] - CTL; cytotoxic T lymphocytes, GM-CSF; granulocyte macrophage-colony stimulating factor

Animal model Vaccine Results Ref.
Protein vaccination
ChimpanzeeRecombinant HCV-like particles containing core, E1 and E2 proteinsIncreased in peripheral and intrahepatic T cell proliferative responses against the HCV proteins. [35]
DNA immunization
HCV transgenic mouse modelThe combination of DNA vaccination encoding HCV core and mouse IL-2Broke tolerance against HCV and activates previously tolerant T cells.[112]
Mice expressing HCV antigens in the liverHCV NS3/NS4 DNA vaccineInduced HCV-specific CD8+ T cells expressing IFN-γ and CCR5 and cleared HCV NS3 expressing hepatocytes.[113]
MiceMurine DCs with CFm40L transfected with adenovirus encoding HCV NS3Induced CD4+ and CD8+ T cell response against HCV NS3.[114]
Mice DCs transfected with adenovirus encoding HCV NS3 Induced multiepitopic CD4+ (Th1) and D8+ T cell response and down-regulated the expression of HCV RNA in the liver. [115]
Balb/c and HLA-A2.1 trangenic miceDCs transfected with adenovirus encoding HCV NS3 Induced NS3-specific cell mediated and humoral immune response.[116]
Chimpanzee with HCV-challenge Recombinant adenoviral vectors encoding the HCV NS3-5B (genotype 1b) and with NS3-5B–encoding plasmid DNA in a combined modality regimenHCV NS3-NS5, HCV-specific T cells appeared earlier, maintained better functionality, and persisted at higher frequencies. The T cells controlled HCV-challenge.[117]
DC immunization
HCV transgenic mouse modelDCs treated with peptide inhibitors of IL-10Induced strong anti-HCV T cell responses[128]
Gene therapy
C57BL/6 and BALB/c miceAdenovirus-besed HCV vaccine by fusing HCV NS3 to MHC class II chaperone protein invariant chainInduced CD8+ T cells expressing IFN-γ, TNF-α, IL-2, CD27 and CD127. The CD8+ T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains [129]

Table 6.

Immunotherapeutic approaches for animal models of HCV infection

Immunotherapy Results Ref.
Peptide vaccination
A vaccine, IC41, containing 7 relevant HCV T cell epitopes and the Th1 adjuvant poly-L-arginineInduced HCV-specific Th1/Tc1 responses in a subset of HCV patients not responding to or relapsing from standard therapy. However, only a minimal decrease in HCV viremia was induced by the vaccination.[81]
Vaccination with a peptide derived from HCV core proteinInduced both cellular and humoral responses in nearly all HCV patients with different HLA class I-A alleles, and reduced serum ALT and AFP levels in 29% and 50% of patients, respectively.[82]
DNA vaccination
A new vaccine, CIGB0230, consisting of a mixture of plasmid expressing HCV structural antigens and HCV recombinant core proteinInduced specific T cell proliferation and IFN-γ production in 73%. More than 40% of the vaccines showed improvement of liver histology, despite persistent detection of HCV RNA.[99]
DC vaccination
Human DCs from HCV-infected patienst with CFh40L transfected with adenovirus encoding HCV NS3Induced CD4+ and CD8+ T cell response against HCV NS3 in HCV-infected patients[114]
Monocyte-derived DCs loaded with lipopeptides consisting of HCV-specific HLA-A2.1-restricted CTL epitopesInduced HCV-specific CD8+ T cell responses with IFN-γ production in PBL in HCV patients in whom conventional IFN-based therapy has failed. However, ALT levels were not elevated and viral load was not decreased. [118]
Human DCs infected with adenoviral vectors harboring HCV core and NS3Induced CD4+ and CD8+ T cell response against HCV core and NS3 in healthy subjects[130]
Human DCs infected with adenoviral vectors harboring HCV NS genesDCs transfected with adenovirus NS3/NS4 efficiently induced HCV-specific immunity in healthy subjects[131]
Talpha1Patients with chronic HCV infection who had been nonresponders to prior IFN-α and ribavirin were treated with Talpha1, PEG-IFN α-2a, and ribavirin for 48 weeks. Twenty-four percent of the treated patients with genotype 1 achieved a sustained virological response.[144]
Blockade of inhibitory signals
Blocking PD-1, CTLA-4 and IL-10 combined with therapeutic vaccinationSynergistically enhanced functional CD8+ T cell response and improve viral control in chronically infected mice. Moreover, addition of stimulatory signals, such as IL-2, could further increase the efficacy of the therapy in chronic viral infection[146]
Combined blockade of CTLA-4 and PD-1Combined blockade of CTLA-4 and PD-1, but not blocking of either molecule, can reverse CD8+ T cell exhaustion in HCV infected patients[147]
Blocking Tim-3/Tim-3 ligandBlocking Tim-3/Tim-3 ligand induced intrahepatic T cell proliferation and IFN-γ production in response to HCV antigens in HCV-infected patients[70]
Blockade of Tim-3 on human HCV-specific CTLsBlockade of Tim-3 on human HCV-specific CTLs fron HCV-infected patients increased cytotoxicity against an HCVAg-expressing hepatocyte cell line that expresses HCV epitopes[149]
Gene transfection
Human T cells transduced with HCV TCR specific for HCV NS3 1071-1081 (HLA A2-restricted epitope)T cells recognized the peptide and produced IFN-γ, IL-2 and TNF-α in healthy subjects[119]
Two adenoviral vectors expressing NS3, 4 and 5 proteins from HCV genotype 1B Induced HCV-specific CD4+ and CD8+ T cells subsets secreting IL-2, IFN-γ, and TNF-α and could be sustained for at least a year after boosting. [120]
Recombinant virus
Recombinant poxvirus vaccine, TG4040, that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. It was safe and well-tolerated. It induced HCV-specific immune response and a transient decrease in HCV viremia (>1log) in 33% of HCV-infected patients.[103]

Table 7.

Immunotherapeutic approach for chronic HCV infection in humans

[i] - CTLA-4; cytotoxic T lymphocyte antigen-4, PD-1; programmed death-1, Tim-3; T cell Immunoglobulin and Mucin domain containing-3

5.1. 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.

5.2. Induction of immune response to hepatitis virus

5.2.1. 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].

5.2.2. 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.

Recently, HB vaccine containing not only S protein but also preS has been used with increased immunogenicity [84-88], or has been combined with lamivudine [89,90], IL-2 [91] or IFN-α [92] leading to potential improvement of clinical efficacy [93-95]. Moreover, vaccines containing HBcAg have been developed, and some showed significant anti-viral effect in HBV transgenic mice [96-99]. Because T cell response to HBcAg is important for viral control, these vaccines may have a promising immunothepapeutic potential also in humans. Recently, some trials to enhance the immunogenicity of HBV vaccine in combination with adjuvant or by using viral vectors have been made [100-102].

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.

5.2.3. 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 [109-111].

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 [113-116]. 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].

5.2.4. 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.

5.2.5. 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 [137-139] 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 [140-143] 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%).


Figure 1.

5.2.6. 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 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.

6. 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.


1 - B Rehermann, Immunopathogenesis of viral hepatitis. Bailliere’s clinical gastroenterology 199610483500
2 - E. J Gane, S. K Roberts, C. A Stedman, et alOral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial. Lancet 201037614671475
3 - Y. H Ni, L. M Huang, M. H Chang, et alTwo decades of universal hepatitis B vaccination in taiwan: impact and implication for future strategies. Gastroenterology 200713212871293
4 - M. H Chang, S. L You, C. J Chen, et alDecreased incidence of hepatocellular carcinoma in hepatitis B vaccinees: a 20-year follow-up study. Journal of the National Cancer Institute 200910113481355
5 - M. C Rangel, V. G Coronado, G. L Euler, R. A Strikas, Vaccine recommendations for patients on chronic dialysis. The Advisory Committee on Immunization Practices and the American Academy of Pediatrics. Seminars in dialysis 200013101107
6 - F Fabrizi, S Andrulli, G Bacchini, M Corti, F Locatelli, Intradermal versus intramuscular hepatitis b re-vaccination in non-responsive chronic dialysis patients: a prospective randomized study with cost-effectiveness evaluation. Nephrol Dial Transplant 19971212041211
7 - F Fabrizi, S. V Ganeshan, V Dixit, P Martin, Meta-analysis: the adjuvant role of granulocyte macrophage-colony stimulating factor on immunological response to hepatitis B virus vaccine in end-stage renal disease. Alimentary pharmacology & therapeutics 200624789796
8 - F Fabrizi, V Dixit, P Messa, P Martin, Meta-analysis: levamisole improves the immune response to hepatitis B vaccine in dialysis patients. Alimentary pharmacology & therapeutics 201132756762
9 - S. M Alavian, S. V Tabatabaei, Effects of oral levamisole as an adjuvant to hepatitis B vaccine in adults with end-stage renal disease: a meta-analysis of controlled clinical trials. Clinical therapeutics 201032110
10 - N. K Tong, J Beran, S. A Kee, et alImmunogenicity and safety of an adjuvanted hepatitis B vaccine in pre-hemodialysis and hemodialysis patients. Kidney international 20056822982303
11 - N. C Kong, J Beran, S. A Kee, et alA new adjuvant improves the immune response to hepatitis B vaccine in hemodialysis patients. Kidney international 200873856862
12 - C. L Tielemans, J Vlasak, D Kosa, et alImmunogenicity and safety of an investigational AS02(v)-adjuvanted hepatitis B vaccine in patients with renal insufficiency who failed to respond or to maintain antibody levels after prior vaccination: results of two open, randomized, comparative trials. Vaccine 20112911591166
13 - P Jungers, P Devillier, H Salomon, J. E Cerisier, A. M Courouce, Randomised placebo-controlled trial of recombinant interleukin-2 in chronic uraemic patients who are non-responders to hepatitis B vaccine. Lancet 1994344856857
14 - J. M Mauri, M Valles, Effects of recombinant interleukin-2 and revaccination for hepatitis B in previously vaccinated, non-responder, chronic uraemic patients. Collaborative Group of Girona. Nephrol Dial Transplant 199712729732
15 - UB Hellstrom, , K Madalinski, , SP Sylvan, . Pre, 1 epitope recognition in newborns after vaccination with the third-generation Sci-B-Vac vaccine and their relation to the antibody response to hepatitis B surface antigen. Virology journal 2009;6:7
16 - S. P Sylvan, K Madalinski, U. B Hellstrom, Anti-preS responses influence the anti-HBs response in newborns after vaccination with the third generation Sci-B-Vac vaccine. Vaccine 200928446451
17 - W. F Carman, A. R Zanetti, P Karayiannis, et alVaccine-induced escape mutant of hepatitis B virus. Lancet 1990336325329
18 - S. L Stramer, U Wend, D Candotti, et alNucleic acid testing to detect HBV infection in blood donors. The New England journal of medicine 2011364236247
19 - W. O Osburn, B. E Fisher, K. A Dowd, et alSpontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection. Gastroenterology 2010138315324
20 - A Folgori, S Capone, L Ruggeri, et alA T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nature medicine 200612190197
21 - J. M Pestka, M. B Zeisel, E Blaser, et alRapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C. Proceedings of the National Academy of Sciences of the United States of America 200710460256030
22 - T Von Hahn, J. C Yoon, H Alter, et alHepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo. Gastroenterology 2007132667678
23 - B Bartosch, J Dubuisson, F. L Cosset, Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. The Journal of experimental medicine 2003197633642
24 - E Scarselli, H Ansuini, R Cerino, et alThe human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. The EMBO journal 20022150175025
25 - B Bartosch, A Vitelli, C Granier, et alCell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. The Journal of biological chemistry 20032784162441630
26 - A. M Owsianka, J. M Timms, A. W Tarr, et alIdentification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. Journal of virology 20068086958704
27 - M Perotti, N Mancini, R. A Diotti, et alIdentification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. Journal of virology 20088210471052
28 - G Leroux-roels, A. H Batens, I Desombere, et alImmunogenicity and tolerability of intradermal administration of an HCV E1-based vaccine candidate in healthy volunteers and patients with resolved or ongoing chronic HCV infection. Human vaccines 200516165
29 - S Cooper, A. L Erickson, E. J Adams, et alAnalysis of a successful immune response against hepatitis C virus. Immunity 199910439449
30 - F Lechner, D. K Wong, P. R Dunbar, et alAnalysis of successful immune responses in persons infected with hepatitis C virus. The Journal of experimental medicine 200019114991512
31 - A Grakoui, N. H Shoukry, D. J Woollard, et alHCV persistence and immune evasion in the absence of memory T cell help. Science (New York, NY 2003302659662
32 - Q. L Choo, G Kuo, R Ralston, et alVaccination of chimpanzees against infection by the hepatitis C virus. Proceedings of the National Academy of Sciences of the United States of America 19949112941298
33 - X Forns, P. J Payette, X Ma, et alVaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV. Hepatology (Baltimore, Md 200032618625
34 - J. W Youn, S. H Park, D Lavillette, et alSustained E2 antibody response correlates with reduced peak viremia after hepatitis C virus infection in the chimpanzee. Hepatology (Baltimore, Md 20054214291436
35 - G. A Elmowalid, M Qiao, S. H Jeong, et alImmunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees. Proceedings of the National Academy of Sciences of the United States of America 200710484278432
36 - C. S Rollier, G Paranhos-baccala, E. J Verschoor, et alVaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity. Hepatology (Baltimore, Md 200745602613
37 - J. W Youn, Y. W Hu, N Tricoche, et alEvidence for protection against chronic hepatitis C virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus. Journal of virology 2008821089610905
38 - S. E Frey, M Houghton, S Coates, et alSafety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults. Vaccine 20102863676373
39 - Z Stamataki, S Coates, S Abrignani, M Houghton, J. A Mckeating, Immunization of human volunteers with hepatitis C virus envelope glycoproteins elicits antibodies that cross-neutralize heterologous virus strains. The Journal of infectious diseases 2011204811813
40 - L. G Guidotti, T Ishikawa, M. V Hobbs, et alIntracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 199642536
41 - L. G Guidotti, F. V Chisari, Noncytolytic control of viral infections by the innate and adaptive immune response. Annual review of immunology 2001196591
42 - L. G Guidotti, R Rochford, J Chung, et alViral clearance without destruction of infected cells during acute HBV infection. Science (New York, NY 1999284825829
43 - G. J Webster, S Reignat, M. K Maini, et alIncubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology (Baltimore, Md 20003211171124
44 - N Semmo, P Klenerman, CD4+ T cell responses in hepatitis C virus infection. World J Gastroenterol 20071348314838
45 - S. L Tsai, Y. F Liaw, M. H Chen, C. Y Huang, G. C Kuo, Detection of type 2-like T-helper cells in hepatitis C virus infection: implications for hepatitis C virus chronicity. Hepatology (Baltimore, Md 199725449458
46 - R Thimme, S Wieland, C Steiger, et alCD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. Journal of virology 2003776876
47 - S. E Bassett, B Guerra, K Brasky, et alProtective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology (Baltimore, Md 20013314791487
48 - G Webster, A Bertoletti, Quantity and quality of virus-specific CD8 cell response: relevance to the design of a therapeutic vaccine for chronic HBV infection. Molecular immunology 200138467473
49 - M Cucchiarini, A. R Kammer, B Grabscheid, et alVigorous peripheral blood cytotoxic T cell response during the acute phase of hepatitis C virus infection. Cellular immunology 2000203111123
50 - N. H Shoukry, J Sidney, A Sette, C. M Walker, Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections. J Immunol 2004172483492
51 - F. V Chisari, C Ferrari, Hepatitis B virus immunopathology. Springer seminars in immunopathology 199517261281
52 - C Ferrari, A Penna, A Bertoletti, et alCellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol 199014534423449
53 - M. J Koziel, D Dudley, N Afdhal, et alHLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. The Journal of clinical investigation 19959623112321
54 - A. J Freeman, Y Pan, C. E Harvey, et alThe presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection. Journal of hepatology 200338349356
55 - J. N Stoop, M. A Claassen, A. M Woltman, et alIntrahepatic regulatory T cells are phenotypically distinct from their peripheral counterparts in chronic HBV patients. Clinical immunology (Orlando, Fla 2008129419427
56 - R Cabrera, Z Tu, Y Xu, et alAn immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection. Hepatology (Baltimore, Md 20044010621071
57 - T Boettler, H. C Spangenberg, C Neumann-haefelin, et alT cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. Journal of virology 20057978607867
58 - N Sturm, M. A Thelu, X Camous, et alCharacterization and role of intra-hepatic regulatory T cells in chronic hepatitis C pathogenesis. Journal of hepatology 2010532535
59 - L. M Francisco, P. T Sage, A. H Sharpe, P. D The, pathway in tolerance and autoimmunity. Immunological reviews 2010236219242
60 - B. T Fife, K. E Pauken, The role of the PD-1 pathway in autoimmunity and peripheral tolerance. Annals of the New York Academy of Sciences 201112174559
61 - P Fisicaro, C Valdatta, M Massari, et alAntiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B. Gastroenterology 2010138682693e681-684
62 - L Golden-mason, B Palmer, J Klarquist, et alUpregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. Journal of virology 20078192499258
63 - A Penna, M Pilli, A Zerbini, et alDysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection. Hepatology (Baltimore, Md 200745588601
64 - Z. Q Yao, E King, D Prayther, D Yin, J Moorman, T cell dysfunction by hepatitis C virus core protein involves PD-1/PDL-1 signaling. Viral immunology 200720276287
65 - L Golden-mason, J Klarquist, A. S Wahed, H. R Rosen, Cutting edge: programmed death-1 expression is increased on immunocytes in chronic hepatitis C virus and predicts failure of response to antiviral therapy: race-dependent differences. J Immunol 200818036373641
66 - R Sabat, G Grutz, K Warszawska, et alBiology of interleukin-10. Cytokine & growth factor reviews 201021331344
67 - J Li, W Wu, G Peng, et alHBcAg induces interleukin-10 production, inhibiting HBcAg-specific Th17 responses in chronic hepatitis B patients. Immunology and cell biology 201088834841
68 - L Barrett, M Gallant, C Howley, et alEnhanced IL-10 production in response to hepatitis C virus proteins by peripheral blood mononuclear cells from human immunodeficiency virus-monoinfected individuals. BMC immunology 2008
69 - Y Ju, N Hou, X. N Zhang, et alBlockade of Tim-3 pathway ameliorates interferon-gamma production from hepatic CD8+ T cells in a mouse model of hepatitis B virus infection. Cellular & molecular immunology 200963543
70 - L Golden-mason, B. E Palmer, N Kassam, et alNegative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells. Journal of virology 20098391229130
71 - F. S Wang, L. H Xing, M. X Liu, et alDysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection. World J Gastroenterol 20017537541
72 - S Beckebaum, V. R Cicinnati, X Zhang, et alHepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape. Immunology 2003109487495
73 - X. Z Duan, M Wang, H. W Li, et alDecreased frequency and function of circulating plasmocytoid dendritic cells (pDC) in hepatitis B virus infected humans. Journal of clinical immunology 200424637646
74 - A. M Woltman, Ter Borg MJ, Binda RS, et al. Alpha-galactosylceramide in chronic hepatitis B infection: results from a randomized placebo-controlled Phase I/II trial. Antiviral therapy 200914809818
75 - E. A Eksioglu, J. R Bess, H Zhu, et alHepatitis C virus modulates human monocyte-derived dendritic cells. Journal of viral hepatitis (2010).,17757769
76 - A Takaki, M Tatsukawa, Y Iwasaki, et alHepatitis C virus NS4 protein impairs the Th1 polarization of immature dendritic cells. Journal of viral hepatitis 201017555562
77 - A Dolganiuc, E Paek, K Kodys, J Thomas, G Szabo, Myeloid dendritic cells of patients with chronic HCV infection induce proliferation of regulatory T lymphocytes. Gastroenterology 200813521192127
78 - C Boni, A Penna, G. S Ogg, et alLamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy. Hepatology (Baltimore, Md 200133963971
79 - S Wang, Q Han, N Zhang, et alHBcAg18-27 epitope fused to HIV-Tat 49-57 adjuvanted with CpG ODN induces immunotherapeutic effects in transgenic mice. Immunology letters 2010127143149
80 - J Heathcote, J Mchutchison, S Lee, et alA pilot study of the CY-1899 T-cell vaccine in subjects chronically infected with hepatitis B virus. The CY1899 T Cell Vaccine Study Group. Hepatology (Baltimore, Md 199930531536
81 - C. S Klade, H Wedemeyer, T Berg, et alTherapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41. Gastroenterology 200813413851395
82 - S Yutani, N Komatsu, S Shichijo, et alPhase I clinical study of a peptide vaccination for hepatitis C virus-infected patients with different human leukocyte antigen-class I-A alleles. Cancer science 200910019351942
83 - S. M Akbar, K Kajino, K Tanimoto, et alPlacebo-controlled trial of vaccination with hepatitis B virus surface antigen in hepatitis B virus transgenic mice. Journal of hepatology 199726131137
84 - S Pol, B Nalpas, F Driss, et alEfficacy and limitations of a specific immunotherapy in chronic hepatitis B. Journal of hepatology 200134917921
85 - S Menne, B. C Tennant, J. L Gerin, P. J Cote, Chemoimmunotherapy of chronic hepatitis B virus infection in the woodchuck model overcomes immunologic tolerance and restores T-cell responses to pre-S and S regions of the viral envelope protein. Journal of virology 2007811061410624
86 - I. R Malik, A Chen, A Brass, et alA bi-functional hepatitis B virus core antigen (HBcAg) chimera activates HBcAg-specific T cells and preS1-specific antibodies. Scandinavian journal of infectious diseases 2012445559
87 - R Safadi, E Israeli, O Papo, et alTreatment of chronic hepatitis B virus infection via oral immune regulation toward hepatitis B virus proteins. The American journal of gastroenterology 20039825052515
88 - H Senturk, F Tabak, R Ozaras, et alEfficacy of pre-S-containing HBV vaccine combined with lamivudine in the treatment of chronic HBV infection. Digestive diseases and sciences 20095420262030
89 - P Vandepapeliere, G. K Lau, G Leroux-roels, et alTherapeutic vaccination of chronic hepatitis B patients with virus suppression by antiviral therapy: a randomized, controlled study of co-administration of HBsAg/AS02 candidate vaccine and lamivudine. Vaccine 20072585858597
90 - M Al-mahtab, S Rahman, S. M Akbar, et alCombination therapy with antiviral drugs and hepatitis B vaccine in incidentally-detected and asymptomatic chronic hepatitis virus B carriers at Bangladesh. Viral immunology 201023335338
91 - A Dahmen, S Herzog-hauff, W. O Bocher, P. R Galle, H. F Lohr, Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis B. Journal of medical virology 200266452460
92 - M Helvaci, A Kizilgunesler, E Kasirga, et alEfficacy of hepatitis B vaccination and interferon-alpha-2b combination therapy versus interferon-alpha-2b monotherapy in children with chronic hepatitis B. Journal of gastroenterology and hepatology 200419785791
93 - X. Y Wang, X. X Zhang, X Yao, et alSerum HBeAg sero-conversion correlated with decrease of HBsAg and HBV DNA in chronic hepatitis B patients treated with a therapeutic vaccine. Vaccine 20102881698174
94 - B Dikici, M Bosnak, H Ucmak, et alFailure of therapeutic vaccination using hepatitis B surface antigen vaccine in the immunotolerant phase of children with chronic hepatitis B infection. Journal of gastroenterology and hepatology 200318218222
95 - K Yalcin, R Danis, H Degertekin, et alThe lack of effect of therapeutic vaccination with a pre-S2/S HBV vaccine in the immune tolerant phase of chronic HBV infection. Journal of clinical gastroenterology 200337330335
96 - X Chen, M Li, X Le, W Ma, B Zhou, Recombinant hepatitis B core antigen carrying preS1 epitopes induce immune response against chronic HBV infection. Vaccine 200422439446
97 - B. F Yang, H. L Zhao, C Xue, et alRecombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response. Vaccine 20072544784486
98 - S Wang, L Qiu, G Liu, et alHeat shock protein gp96 enhances humoral and T cell responses, decreases Treg frequency and potentiates the anti-HBV activity in BALB/c and transgenic mice. Vaccine 20112963426351
99 - X Chen, J Lai, Q Pan, et alThe delivery of HBcAg via Tat-PTD enhances specific immune response and inhibits Hepatitis B virus replication in transgenic mice. Vaccine 20102839133919
100 - Y Hong, Y Peng, M Mi, et alLentivector expressing HBsAg and immunoglobulin Fc fusion antigen induces potent immune responses and results in seroconversion in HBsAg transgenic mice. Vaccine 20112939093916
101 - J. D Morrey, N. E Motter, S Chang, J Fairman, B Breaking, and T cell tolerance using cationic lipid--DNA complexes (CLDC) as a vaccine adjuvant with hepatitis B virus (HBV) surface antigen in transgenic mice expressing HBV. Antiviral research 201190227230
102 - P. J Cote, S. D Butler, A. L George, et alRapid immunity to vaccination with woodchuck hepatitis virus surface antigen using cationic liposome-DNA complexes as adjuvant. Journal of medical virology 20098117601772
103 - F Habersetzer, G Honnet, C Bain, et alA poxvirus vaccine is safe, induces T-cell responses, and decreases viral load in patients with chronic hepatitis C. Gastroenterology 2011141890899e891-894
104 - K. H Liu, M. A Ascenzi, C. A Bellezza, et alElectroporation enhances immunogenicity of a DNA vaccine expressing woodchuck hepatitis virus surface antigen in woodchucks. Journal of virology 20118548534862
105 - D. S Miller, D Boyle, F Feng, et alAntiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection. Virology 2008373329341
106 - A Thermet, T Buronfosse, B Werle-lapostolle, et alDNA vaccination in combination or not with lamivudine treatment breaks humoral immune tolerance and enhances cccDNA clearance in the duck model of chronic hepatitis B virus infection. The Journal of general virology 20088911921201
107 - Y Yin, C Wu, J Song, et alDNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic. PloS one 2011e22524
108 - C Zhou, G Peng, X Jin, J Tang, Z Chen, Vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4 enhances HBV-specific immune responses in mice: implication of its potential use as a therapeutic vaccine. Clinical immunology (Orlando, Fla 2010137190198
109 - M Mancini-bourgine, H Fontaine, D Scott-algara, et alInduction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers. Hepatology (Baltimore, Md 200440874882
110 - M Mancini-bourgine, H Fontaine, C Brechot, S Pol, M. L Michel, Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers. Vaccine 20062444824489
111 - J. S Cavenaugh, D Awi, M Mendy, et alPartially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection. PloS one 2011e14626
112 - J Encke, M Geissler, W Stremmel, J. R Wands, DNA-based immunization breaks tolerance in a hepatitis C virus transgenic mouse model. Human vaccines 200627883
113 - Lang Kuhs KAToporovski R, Ginsberg AA, et al. Peripheral immunization induces functional intrahepatic hepatitis C specific immunity following selective retention of vaccine-specific CD8 T cells by the liver. Human vaccines 2011713261335
114 - I Echeverria, A Pereboev, L Silva, et alEnhanced T cell responses against hepatitis C virus by ex vivo targeting of adenoviral particles to dendritic cells. Hepatology (Baltimore, Md 2011542837
115 - A Zabaleta, D Llopiz, L Arribillaga, et alVaccination against hepatitis C virus with dendritic cells transduced with an adenovirus encoding NS3 protein. Mol Ther 200816210217
116 - M Xiang, C Eisenbach, C. M Lupu, et alInduction of antigen-specific immune responses in vivo after vaccination with dendritic cells transduced with adenoviral vectors encoding hepatitis C virus NS3. Viral immunology 200619210219
117 - I Zubkova, Y. H Choi, E Chang, et alT-cell vaccines that elicit effective immune responses against HCV in chimpanzees may create greater immune pressure for viral mutation. Vaccine 20092725942602
118 - E. J Gowans, S Roberts, K Jones, et alA phase I clinical trial of dendritic cell immunotherapy in HCV-infected individuals. Journal of hepatology 201053599607
119 - Y Zhang, Y Liu, K. M Moxley, et alTransduction of human T cells with a novel T-cell receptor confers anti-HCV reactivity. PLoS pathogens 2010e1001018
120 - E Barnes, A Folgori, S Capone, et alNovel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man. Science translational medicine 2012ra111
121 - Y Shimizu, L. G Guidotti, P Fowler, F. V Chisari, Dendritic cell immunization breaks cytotoxic T lymphocyte tolerance in hepatitis B virus transgenic mice. J Immunol 199816145204529
122 - W. Z Jiang, Y Fan, X Liu, et alTherapeutic potential of dendritic cell-based immunization against HBV in transgenic mice. Antiviral research 2008775055
123 - K Kimura, K Kakimi, S Wieland, L. G Guidotti, F. V Chisari, Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice. J Immunol 200216951885195
124 - S. M Akbar, S Furukawa, N Horiike, et alSafety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B. Journal of viral hepatitis 201118408414
125 - X. Z Duan, H. X He, H Zhuang, Restoration in vitro of impaired T-cell responses in patients with chronic hepatitis B by autologous dendritic cells loaded with hepatitis B virus proteins (R2). Journal of gastroenterology and hepatology 200621970976
126 - M Chen, Y. G Li, D. Z Zhang, et alTherapeutic effect of autologous dendritic cell vaccine on patients with chronic hepatitis B: a clinical study. World J Gastroenterol 20051118061808
127 - J Luo, J Li, R. L Chen, et alAutologus dendritic cell vaccine for chronic hepatitis B carriers: a pilot, open label, clinical trial in human volunteers. Vaccine 20102824972504
128 - N Diaz-valdes, L Manterola, V Belsue, et alImproved dendritic cell-based immunization against hepatitis C virus using peptide inhibitors of interleukin 10. Hepatology (Baltimore, Md 2011532331
129 - M Mikkelsen, P. J Holst, J Bukh, A. R Thomsen, J. P Christensen, Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain. J Immunol 201118623552364
130 - W Li, D. K Krishnadas, J Li, D. L Tyrrell, B Agrawal, Induction of primary human T cell responses against hepatitis C virus-derived antigens NS3 or core by autologous dendritic cells expressing hepatitis C virus antigens: potential for vaccine and immunotherapy. J Immunol 200617660656075
131 - Y Tian, H. H Zhang, L Wei, et alThe functional evaluation of dendritic cell vaccines based on different hepatitis C virus nonstructural genes. Viral immunology 200720553561
132 - V. J Cavanaugh, L. G Guidotti, F. V Chisari, Interleukin-12 inhibits hepatitis B virus replication in transgenic mice. Journal of virology 19977132363243
133 - K Kimura, K Kakimi, S Wieland, L. G Guidotti, F. V Chisari, Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. Journal of virology 2002761070210707
134 - K Kakimi, L. G Guidotti, Y Koezuka, F. V Chisari, Natural killer T cell activation inhibits hepatitis B virus replication in vivo. The Journal of experimental medicine 2000192921930
135 - J Martin, O Bosch, G Moraleda, et alPilot study of recombinant human granulocyte-macrophage colony-stimulating factor in the treatment of chronic hepatitis B. Hepatology (Baltimore, Md 199318775780
136 - J Wang, Q Zhu, T Zhang, H Yu, A pilot study on the combined therapy of granulocyte-macrophage colony-stimulating factor and hepatitis B vaccine on chronic hepatitis B virus carrier children. Chinese medical journal 200211518241828
137 - S Zeuzem, V Carreno, Interleukin-12 in the treatment of chronic hepatitis B and C. Antiviral research 200152181188
138 - E. I Rigopoulou, D Suri, S Chokshi, et alLamivudine plus interleukin-12 combination therapy in chronic hepatitis B: antiviral and immunological activity. Hepatology (Baltimore, Md 20054210281036
139 - A Szkaradkiewicz, A Jopek, J Wysocki, Effects of IL-12 and IL-18 on HBcAg-specific cytokine production by CD4 T lymphocytes of children with chronic hepatitis B infection. Antiviral research 2005662327
140 - Y Arase, A Tsubota, Y Suzuki, et alA pilot study of thymosin alpha1 therapy for chronic hepatitis B patients. Internal medicine (Tokyo, Japan) 200342941946
141 - S Iino, J Toyota, H Kumada, et alThe efficacy and safety of thymosin alpha-1 in Japanese patients with chronic hepatitis B; results from a randomized clinical trial. Journal of viral hepatitis 200512300306
142 - J You, L Zhuang, H. Y Cheng, et alEfficacy of thymosin alpha-1 and interferon alpha in treatment of chronic viral hepatitis B: a randomized controlled study. World J Gastroenterol 20061267156721
143 - H. W Lee, J. I Lee, S. H Um, et alCombination therapy of thymosin alpha-1 and lamivudine for HBeAg positive chronic hepatitis B: A prospective randomized, comparative pilot study. Journal of gastroenterology and hepatology 200823729735
144 - J. L Poo, Sanchez Avila F, Kershenobich D, et al. Efficacy of triple therapy with thymalfasin, peginterferon alpha-2a, and ribavirin for the treatment of hispanic chronic HCV nonresponders. Annals of hepatology 20087369375
145 - Y. Y Zhang, E. Q Chen, J Yang, Y. R Duan, H Tang, Treatment with lamivudine versus lamivudine and thymosin alpha-1 for e antigen-positive chronic hepatitis B patients: a meta-analysis. Virology journal 2009
146 - S. J Ha, E. E West, K Araki, K. A Smith, R Ahmed, Manipulating both the inhibitory and stimulatory immune system towards the success of therapeutic vaccination against chronic viral infections. Immunological reviews 2008223317333
147 - N Nakamoto, H Cho, A Shaked, et alSynergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade. PLoS pathogens 2009e1000313
148 - B Raziorrouh, W Schraut, T Gerlach, et alThe immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function. Hepatology (Baltimore, Md 20105219341947
149 - R. H Mcmahan, L Golden-mason, M. I Nishimura, et alTim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. The Journal of clinical investigation 201012045464557