Advertisement

Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: Perspectives and challenges

  • Marie-Louise Michel
    Correspondence
    Corresponding author at: Laboratoire de Pathogenèse des virus de l’hépatite B, Institut Pasteur and INSERM U845, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: +33 1 45 68 88 49; fax: +33 1 40 61 38 41.
    Affiliations
    Institut Pasteur, Laboratoire de Pathogénèse des virus de l’hépatite B, département de virologie, 75015 Paris, France

    INSERM U845, Centre de recherche «Croissance et signalisation», Faculté de Médecine Paris Descartes, 75015 Paris, France

    AP-HP, Paris, France
    Search for articles by this author
  • Qiang Deng
    Affiliations
    Institut Pasteur of Shanghai, Unit of Tumor Virology, 200025 Shanghai, China
    Search for articles by this author
  • Maryline Mancini-Bourgine
    Affiliations
    Institut Pasteur, Laboratoire de Pathogénèse des virus de l’hépatite B, département de virologie, 75015 Paris, France

    INSERM U845, Centre de recherche «Croissance et signalisation», Faculté de Médecine Paris Descartes, 75015 Paris, France
    Search for articles by this author
Open AccessPublished:January 14, 2011DOI:https://doi.org/10.1016/j.jhep.2010.12.031
      The treatment of chronic hepatitis B virus (HBV) infection has greatly improved over the last 10 years, but alternative treatments are still needed. Therapeutic vaccination is a promising new strategy for controlling chronic infection. However, this approach has not been as successful as initially anticipated for chronic hepatitis B. General impairment of the immune responses generated during persistent HBV infection, with exhausted T cells not responding correctly to therapeutic vaccination, is probably responsible for the poor clinical responses observed to date. Intensive research efforts are now focusing on increasing the efficacy of therapeutic vaccination without causing liver disease. Here we describe new approaches to use with therapeutic vaccination, in order to overcome the inhibitory mechanisms impairing immune responses. We also describe innovative strategies for generating functional immune responses and inducing sustained control of this persistent infection.

      Keywords

      Abbreviations:

      HBV (hepatitis B virus), cccDNA (covalently closed circular DNA), HBeAg (hepatitis B “e” antigen), HBsAg (hepatitis B surface antigen), NK (natural killer), IFN (interferon), IL (interleukin), TNF (tumor necrosis factor), DCs (dendritic cells), PD-1 (programmed death-1), PD-L1 (programmed death-ligand 1), CTLA-4 (cytotoxic T lymphocyte antigen 4), Bim (Bcl2-interacting mediator of cell death), Tregs (regulatory T cells), mDCs (myeloid DCs), pDCs (plasmacytoid DCs), IC (immune complexes), HBcAg (hepatitis B core antigen), Th1 (T helper 1), PBMCs (peripheral blood mononuclear cells), CIK (cytokine-induced killer), TCR (T-cell receptor), ALT (alanine amino-transferase)

      Linked Article

      • Progress in the development of preventive and therapeutic vaccines for hepatitis C virus
        Journal of HepatologyVol. 54Issue 6
        • Preview
          Hepatitis C virus (HCV) is a blood borne disease estimated to chronically infect 3% of the worlds’ population causing significant morbidity and mortality. Current medical therapy is curative in approximately 50% of patients. While recent treatment advances of genotype 1 infection using directly acting antiviral agents (DAAs) are encouraging, there is still a need to develop vaccine strategies capable of preventing infection. Moreover, vaccines may also be used in future in combination with DAAs enabling interferon-free treatment regimens.
        • Full-Text
        • PDF
        Open Access

      Introduction

      Worldwide, two billion people have been infected with the hepatitis B virus (HBV) at some time, more than 370 million currently have chronic HBV infection, and about one million die each year from HBV-related liver diseases. HBV is a non-cytopathic virus, and the host immune response determines whether the virus is cleared or whether immunopathy and liver damage are induced. Persistent inflammation during chronic HBV infection results in liver cirrhosis or hepatocellular carcinoma in 25% of patients. HBV is currently the second leading carcinogen after tobacco, with up to 80% of hepatocellular carcinoma cases worldwide attributable to HBV [
      • Ganem D.
      • Prince A.M.
      Hepatitis B virus infection – natural history and clinical consequences.
      ]. Individuals with chronic HBV infection also serve as the primary reservoir for viral spread. Preventive vaccination is the most effective way to reduce the global incidence of hepatitis. Efficient vaccines against hepatitis B have been available for over 20 years, and their use should decrease the incidence of HBV infection [
      • Chen D.S.
      Hepatitis B vaccination: the key towards elimination and eradication of hepatitis B.
      ]. By contrast, the treatment of chronic HBV infection is based on the use of antiviral agents. These agents have improved considerably over the last 10 years, with the development of molecules targeting the HBV polymerase and decreasing viral replication. The major goals of anti-HBV treatment are to prevent the development of progressive disease, specifically cirrhosis, liver failure, and hepatocellular carcinoma. However, although currently available antiviral drugs efficiently decrease serum viral load to undetectable levels, they fail to eradicate infection due to the persistence of HBV covalently closed circular DNA (cccDNA) in hepatocytes and the emergence of resistant viruses [
      • Dienstag J.L.
      Hepatitis B virus infection.
      ,
      • Zoulim F.
      • Locarnini S.
      Hepatitis B virus resistance to nucleos(t)ide analogues.
      ]. In addition, such drugs rarely result in the long-term immunological control of HBV infection through the elimination of residual infected hepatocytes, hepatitis B “e” antigen (HBeAg) seroconversion and/or hepatitis B surface antigen (HBsAg) clearance. Moreover, long-term treatment is expensive and may result in problems of toxicity and intolerance.
      Both the adaptive and innate immune responses are known to be involved in viral clearance during HBV infection [
      • Rehermann B.
      • Nascimbeni M.
      Immunology of hepatitis B virus and hepatitis C virus infection.
      ]. There is a clear dichotomy in the profile of the immune responses observed, depending on whether patients naturally resolve viral infection or develop chronic infection. Patients with self-limited acute hepatitis B display multi-specific CD4 and CD8 T-cell responses, with the secretion of antiviral cytokines and the production of anti-HBV antibodies. By contrast, patients suffering from chronic infection have very weak or functionally impaired immune responses. Therapeutic vaccination has been proposed as a potentially promising strategy for controlling viral infection, based on these observations. Therapeutic vaccination aims to eliminate persistent viral infection, by stimulating the patient’s immune responses.
      In this review, we first outline the characteristics of cell-mediated immune responses and the immunosuppressive environment generated during chronic HBV infection. We then describe current and future approaches for manipulation of the immune system to achieve sustained control of this persistent infection.

      How is host immunity altered during chronic HBV infection?

      The immune response to viral infection is orchestrated in several stages, which function together to eliminate the pathogen and leave the host with memory cells for defense against subsequent infections. During the early phases of acute HBV infection, natural killer (NK) cells are the first line of host defense, and the activation of these cells helps to reduce viral load, through the secretion of interferon (IFN)-γ [
      • Mondelli M.U.
      • Varchetta S.
      • Oliviero B.
      Natural killer cells in viral hepatitis: facts and controversies.
      ]. However, the activation of these cells is rapidly checked and this is temporally associated with a surge in interleukin (IL)-10 at the time of peak viremia [
      • Dunn C.
      • Peppa D.
      • Khanna P.
      • Nebbia G.
      • Jones M.
      • Brendish N.
      • et al.
      Temporal analysis of early immune responses in patients with acute hepatitis B virus infection.
      ]. Adaptive immunity is then induced by 5–6 weeks post-infection, the CD8 and CD4 T-cell populations expand and become major contributors to viral control. HBV-specific T-cell responses participate in the elimination of infected hepatocytes, initially through antiviral cytokine secretion (IFN-γ and tumor necrosis factor (TNF)-α) and then through the production of cytotoxic molecules, once the level of MHC-class I peptide complexes on infected cells has decreased [
      • Bertoletti A.
      • Maini M.K.
      Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection?.
      ,
      • Isogawa M.
      • Furuichi Y.
      • Chisari F.V.
      Oscillating CD8(+) T cell effector functions after antigen recognition in the liver.
      ,
      • Fisicaro P.
      • Valdatta C.
      • Boni C.
      • Massari M.
      • Mori C.
      • Zerbini A.
      • et al.
      Early kinetics of innate and adaptive immune responses during hepatitis B virus infection.
      ]. The complete control and eradication of infection are achieved by humoral responses, in which circulating viral particles are neutralized by protective anti-HBs antibodies.
      The functional impairment of immune responses is a key feature of chronic HBV infection (Fig. 1) [
      • Rehermann B.
      Chronic infections with hepatotropic viruses: mechanisms of impairment of cellular immune responses.
      ]. When T cells encounter HBV antigens presented by intrahepatic antigen-presenting cells, such as liver-resident dendritic cells (DCs), Kupffer cells, or liver sinusoidal endothelial cells, the co-stimulation signals received by T cells are too weak, driving the immune response toward tolerance rather than functional activation. The IFN-α and IL-8 cytokines produced by hepatocytes and inflammatory cells in liver promote NK cell-mediated hepatocyte death and liver damage [
      • Maini M.K.
      • Schurich A.
      The molecular basis of the failed immune response in chronic HBV: therapeutic implications.
      ]. T-cell defects are directly related to the sustained exposure of these cells to viral antigens, such as HBsAg and HBeAg, which are produced in large amounts over a period of decades during chronic infection. This chronic exposure to antigens leads to the progressive exhaustion of T cells, which lose their effector functions, such as cytokine production (TNF-α and IL-2), cytotoxicity, and proliferation. Two major inhibitory receptors at the cell surface, programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), have been identified as regulators of CD8 and CD4 T-cell effector function [
      • Barber D.L.
      • Wherry E.J.
      • Masopust D.
      • Zhu B.
      • Allison J.P.
      • Sharpe A.H.
      • et al.
      Restoring function in exhausted CD8 T cells during chronic viral infection.
      ,
      • Wherry E.J.
      • Ha S.J.
      • Kaech S.M.
      • Haining W.N.
      • Sarkar S.
      • Kalia V.
      • et al.
      Molecular signature of CD8+ T cell exhaustion during chronic viral infection.
      ]. PD-1 is over expressed on HBV-specific T cells, and its level of expression is correlated with viral load [
      • Fisicaro P.
      • Valdatta C.
      • Massari M.
      • Loggi E.
      • Biasini E.
      • Sacchelli L.
      • et al.
      Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B.
      ]. PD-1-over expressing T cells produce only small amounts of IFN-γ and cannot differentiate into memory cells [
      • Wherry E.J.
      • Barber D.L.
      • Kaech S.M.
      • Blattman J.N.
      • Ahmed R.
      Antigen-independent memory CD8 T cells do not develop during chronic viral infection.
      ]. Ultimately, the HBV-specific T-cell population may be entirely deleted following prolonged exposure to high doses of HBV antigens. For example, CD8 T cells specific for dominant epitopes have been shown to be undetectable in the liver and peripheral blood of patients with HBV viral loads exceeding 107 copies/ml [
      • Webster G.J.
      • Reignat S.
      • Brown D.
      • Ogg G.S.
      • Jones L.
      • Seneviratne S.L.
      • et al.
      Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy.
      ]. It has also recently been shown that pro-apoptotic genes are more strongly expressed in HBV-specific CD8 T cells from patients with chronic infection than in those from patients who resolve infection. The molecule most strongly upregulated in this context is the Bcl2-interacting mediator of death (Bim) protein, a member of the Bcl2 family that plays a crucial role in the initiation of lymphocyte apoptosis [
      • Lopes A.R.
      • Kellam P.
      • Das A.
      • Dunn C.
      • Kwan A.
      • Turner J.
      • et al.
      Bim-mediated deletion of antigen-specific CD8 T cells in patients unable to control HBV infection.
      ].
      Figure thumbnail gr1
      Fig. 1Impairment of T-cell responses in chronic hepatitis B. T cells circulate in the liver through the sinusoidal network, in which they come into contact with hepatocytes and antigen-presenting cells (Kupffer cells and liver sinusoidal endothelial cells). During chronic hepatitis B virus infection, CD8 T cells in the liver encounter virus particles, viral antigens, and infected hepatocytes. They interact with infected hepatocytes and Kupffer cells through the T-cell receptor (TCR)-MHC class I-peptide complex. Hepatocytes over-expressing PD-L1 during chronic infection provide CD8 T cells with an inhibitory signal via the PD-1 pathway. This negative signal results in the impairment of T-cell functions: decreases in proliferation, cytotoxic function and in the production of anti-viral cytokines (IFN-γ and TNF-α). The presence of a large number of regulatory T cells (Tregs) and high levels of IL-10 secretion also contribute to T-cell exhaustion.
      Other major actors potentially involved in the global collapse of immune responses during chronic hepatitis B infection include regulatory T cells (Tregs) and DCs. Patients with a high HBV load have a high proportion of Tregs in the liver, but not in blood [
      • Xu D.
      • Fu J.
      • Jin L.
      • Zhang H.
      • Zhou C.
      • Zou Z.
      • et al.
      Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B.
      ,
      • Stoop J.N.
      • Claassen M.A.
      • Woltman A.M.
      • Binda R.S.
      • Kuipers E.J.
      • Janssen H.L.
      • et al.
      Intrahepatic regulatory T cells are phenotypically distinct from their peripheral counterparts in chronic HBV patients.
      ]. However, the role of Tregs in the modulation of CD8 T-cell effector functions and the contribution of these cells to T-cell dysfunction in vivo remain a matter of debate [
      • Stoop J.N.
      • van der Molen R.G.
      • Baan C.C.
      • van der Laan L.J.
      • Kuipers E.J.
      • Kusters J.G.
      • et al.
      Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection.
      ,
      • Franzese O.
      • Kennedy P.T.
      • Gehring A.J.
      • Gotto J.
      • Williams R.
      • Maini M.K.
      • et al.
      Modulation of the CD8+-T-cell response by CD4+ CD25+ regulatory T cells in patients with hepatitis B virus infection.
      ]. The high frequency of Tregs in the liver may also be a direct consequence of liver inflammation, because these cells are induced to protect organs against strong, potentially damaging immune responses. Tregs have, therefore, never been considered as possible targets for the treatment of chronic hepatitis B. As professional antigen-presenting cells, DCs play an important role in the induction of functional T cells. Patients with chronic HBV infection display not only a decrease in the number of circulating DCs, but also functional defects in these cells that may contribute to the dysfunction of HBV-specific T cells [
      • Woltman A.M.
      • Boonstra A.
      • Janssen H.L.
      Dendritic cells in chronic viral hepatitis B and C: victims or guardian angels?.
      ]. Functional impairment of both myeloid (mDCs) and plasmacytoid DCs (pDCs) from patients with HBV-related chronic hepatitis has been described [
      • van der Molen R.G.
      • Sprengers D.
      • Binda R.S.
      • de Jong E.C.
      • Niesters H.G.
      • Kusters J.G.
      • et al.
      Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.
      ]. However, it has been shown that DCs from such patients cannot support HBV replication, despite being able to take up viral antigens and viral DNA, ruling out a direct role of viral replication [
      • Untergasser A.
      • Zedler U.
      • Langenkamp A.
      • Hosel M.
      • Quasdorff M.
      • Esser K.
      • et al.
      Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection.
      ]. Recent data have shown that B7-H1 (PD-L1), the known ligand of PD-1, is upregulated on mDCs from patients with chronic liver inflammation, including patients with chronic hepatitis B [
      • Chen L.
      • Zhang Z.
      • Chen W.
      • Li Y.
      • Shi M.
      • Zhang J.
      • et al.
      B7–H1 up-regulation on myeloid dendritic cells significantly suppresses T cell immune function in patients with chronic hepatitis B.
      ]. It remains to be determined whether the T-cell defects in these patients are due to inhibitory signals transmitted to the T cells by DCs over-expressing PD-L1 or to liver inflammation.
      The functional changes to the immune system, which have only recently been discovered, may account for the poor results obtained during initial attempts to develop therapeutic vaccines [
      • Pol S.
      • Michel M.L.
      Therapeutic vaccination in chronic hepatitis B virus carriers.
      ,
      • Bertoletti A.
      • Gehring A.
      Therapeutic vaccination and novel strategies to treat chronic HBV infection.
      ]. Improvements in our understanding of the role of the immune response in the control of viral infection and liver damage, will facilitate the development of new strategies based on immunotherapy that, when combined with current antiviral treatments, could accelerate viral clearance, making life-long treatment unnecessary (Table 1).
      Table 1Characteristic of chronic HBV infection and current or future strategies to improve the immune-mediated control of viral infection.

      Are classical preventive vaccines good candidates for use in therapeutic vaccination?

      The first therapeutic vaccine trials were based on prophylactic recombinant vaccines containing HBV envelope proteins carrying HBsAg (Table 2). This was a logical initial choice, because the final goals of any anti-HBV treatment are the clearance of HBsAg and the induction of protective anti-HBs antibodies. Pilot clinical studies demonstrated that standard vaccination induced a significant increase in HBeAg seroconversion [
      • Pol S.
      • Michel M.L.
      Therapeutic vaccination in chronic hepatitis B virus carriers.
      ]. Although the percentage of serum samples in which HBV DNA levels decreased or became undetectable was higher in some vaccinated groups than in control groups, the effects were not sustained. HBV vaccine-specific T-cell responses, mostly involving CD4 T cells, were also induced. However, few patients displayed HBsAg clearance and anti-HBs seroconversion, highlighting the need for vaccines with a greater therapeutic potential.
      Table 2Preclinical studies, past and ongoing clinical trials, and future approaches.
      • Mancini M.
      • Hadchouel M.
      • Tiollais P.
      • Pourcel C.
      • Michel M.L.
      Induction of anti-hepatitis B surface antigen (HBsAg) antibodies in HBsAg producing transgenic mice. a possible way of circumventing “nonresponse” to HBsAg.
      ,
      • Akbar S.M.
      • Kajino K.
      • Tanimoto K.
      • Kurose K.
      • Masumoto T.
      • Michitaka K.
      • et al.
      Placebo-controlled trial of vaccination with hepatitis B virus surface antigen in hepatitis B virus transgenic mice.
      ,
      • Malanchere-Bres E.
      • Payette P.J.
      • Mancini M.
      • Tiollais P.
      • Davis H.L.
      • Michel M.L.
      CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice.
      ,
      • Kakimi K.
      • Isogawa M.
      • Chung J.
      • Sette A.
      • Chisari F.V.
      Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections.
      ,
      • Loirat D.
      • Mancini-Bourgine M.
      • Abastado J.P.
      • Michel M.L.
      HBsAg/HLA-A2 transgenic mice. a model for T cell tolerance to hepatitis B surface antigen in chronic hepatitis B virus infection.
      ,
      • Schirmbeck R.
      • Dikopoulos N.
      • Kwissa M.
      • Leithauser F.
      • Lamberth K.
      • Buus S.
      • et al.
      Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross-reactive, natural HBsAg variants.
      ,
      • Lu M.
      • Hilken G.
      • Kruppenbacher J.
      • Kemper T.
      • Schirmbeck R.
      • Reimann J.
      • et al.
      Immunization of woodchucks with plasmids expressing Woodchuck Hepatitis Virus (WHV) core antigen and surface antigen suppresses WHV infection.
      ,
      • Rollier C.
      • Sunyach C.
      • Barraud L.
      • Madani N.
      • Jamard C.
      • Trepo C.
      • et al.
      Protective and therapeutic effect of DNA-based immunization against hepadnavirus large envelope protein.
      ,
      • Pol S.
      • Nalpas B.
      • Driss F.
      • Michel M.L.
      • Tiollais P.
      • Denis J.
      • et al.
      Efficacy and limitations of a specific immunotherapy in chronic hepatitis B.
      ,
      • Jung M.C.
      • Gruner N.
      • Zachoval R.
      • Schraut W.
      • Gerlach T.
      • Diepolder H.
      • et al.
      Immunological monitoring during therapeutic vaccination as a prerequisite for the design of new effective therapies: induction of a vaccine-specific CD4+ T-cell proliferative response in chronic hepatitis B carriers.
      ,
      • Wang X.Y.
      • Yao X.
      • Guo L.M.
      • Xu L.F.
      • Shen X.L.
      • Xu D.Z.
      • et al.
      Advances on antigen-antibody immunogenic complex therapeutic vaccine for viral hepatitis B.
      ,
      • Depla E.
      • Van der Aa A.
      • Livingston B.D.
      • Crimi C.
      • Allosery K.
      • De Brabandere V.
      • et al.
      Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections.
      ,
      • Hill A.V.
      • Reyes-Sandoval A.
      • O’Hara G.
      • Ewer K.
      • Lawrie A.
      • Goodman A.
      • et al.
      Prime-boost vectored malaria vaccines: progress and prospects.
      .
      #Recombinant HBsAg, preS2/HBsAg or preS1/preS2/HBsAg with Alum as adjuvant.
      DNAs encoding HBV proteins (DNA encoding small and middle envelope proteins or mixed DNAs encoding envelope, capsid and polymerase proteins and human IL-12).
      HBsAg complexed to human anti-HBs immunoglobulins with Alum as adjuvant.
      °Cytokine-induced killer cells.
      §Dendritic cells loaded with HBsAg or HBV-derived peptides.
      NCT, clinicaltrials.gov identifier (http://clinicaltrials.gov).
      Several ways of increasing vaccine efficacy have been proposed, including changes in vaccine composition and vaccine delivery route. A double-blind placebo-controlled Phase II B clinical trial has been carried out on 242 patients with chronic hepatitis B infection. These patients received injections of antigen–antibody-complexes (HBsAg-anti-HBs immune complexes (IC)) with alum as the adjuvant, with the aim of targeting DCs. DCs incubated with IC secrete large amounts of IL-12. They upregulate functional markers in vitro are thought to prime CD8 T cells in vivo. A significant virological effect was observed 24 weeks after the last of six IC injections. The HBeAg seroconversion rate was 21.8% in the group immunized with 60 μg IC, and only 9% in the control group immunized with alum alone [
      • Xu D.Z.
      • Zhao K.
      • Guo L.M.
      • Li L.J.
      • Xie Q.
      • Ren H.
      • et al.
      A randomized controlled phase IIb trial of antigen-antibody immunogenic complex therapeutic vaccine in chronic hepatitis B patients.
      ]. However, the immune responses accounting for this therapeutic effect were not analyzed in this study.
      Most of the therapeutic HBV vaccines designed to date have used envelope proteins as the target antigen, but core antigen (HBcAg) and the polymerase are also specific targets of the immune response during self-limiting hepatitis B. A novel vaccination approach, based on the use of a combination of recombinant HBsAg and HBcAg and known as “NASVAC”, is currently being developed for both the prevention and cure of hepatitis B. Both these antigens form virus-like particles. When they are mixed together, they form highly immunogenic superstructures, with the two antigens working together in the development of T- and B-cell responses [
      • Aguilar J.C.
      • Lobaina Y.
      • Muzio V.
      • Garcia D.
      • Penton E.
      • Iglesias E.
      • et al.
      Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.
      ]. This novel vaccine formulation has been administered intranasally to individuals taking part in a Phase I trial [
      • Betancourt A.A.
      • Delgado C.A.
      • Estevez Z.C.
      • Martinez J.C.
      • Rios G.V.
      • Aureoles-Rosello S.R.
      • et al.
      Phase I clinical trial in healthy adults of a nasal vaccine candidate containing recombinant hepatitis B surface and core antigens.
      ]. The vaccine was found to be safe and immunogenic, eliciting anti-HBc and anti-HBs seroconversion. Preliminary results of the ongoing Phase I/II trial in patients with chronic hepatitis B infection in Bangladesh were presented at “The 20th conference of the Asian Pacific association for the study of the liver” [
      • Akbar S.M.
      • Al-Mahtab M.
      • Rahman S.
      • Aguilar J.C.
      • Onji M.
      • Mishiro S.
      A therapeutic nasal vaccine combining both HBsAg and HBcAg wassafe, has antiviral potential and induced antigen-specific immunity in patients with chronic hepatitis B.
      ].

      Is decreasing viral load before vaccination a rational choice?

      A decrease in HBV load seems to precede the detection of HBV-specific T-cell responses, both in patients resolving natural infections and in those displaying flare-ups of hepatitis associated with HBeAg seroconversion during chronic infection. Reducing HBV load by antiviral chemotherapy may, therefore, increase the responsiveness of HBV-specific T cells, which are hyporesponsive in cases of persistent HBV or viral antigen stimulation. Indeed, HBV-specific T cells are detectable during the first few months of lamivudine treatment [
      • Boni C.
      • Bertoletti A.
      • Penna A.
      • Cavalli A.
      • Pilli M.
      • Urbani S.
      • et al.
      Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B.
      ]. However, this restoration of T-cell activity is partial and transient and does not lead to an increase in HBeAg seroconversion [
      • Boni C.
      • Penna A.
      • Bertoletti A.
      • Lamonaca V.
      • Rapti I.
      • Missale G.
      • et al.
      Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.
      ]. Classical preventive recombinant vaccines do not themselves have a strong antiviral potential, but pilot studies have shown that they are most effective in patients with a low HBV load at the start of treatment [
      • Pol S.
      • Michel M.L.
      Therapeutic vaccination in chronic hepatitis B virus carriers.
      ]. Rational therapeutic approaches based on these findings have been developed. They combine the vaccine-induced exogenous stimulation of protective T- and B-cell responses with the concomitant suppression of viral replication by antiviral drug treatment. Proof-of-concept has been demonstrated in animal models, notably duck and woodchuck, in which a combination of antiviral drugs and vaccination had sustained therapeutic effects [
      • Le Guerhier F.
      • Thermet A.
      • Guerret S.
      • Chevallier M.
      • Jamard C.
      • Gibbs C.S.
      • et al.
      Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model.
      ,
      • Menne S.
      • Roneker C.A.
      • Korba B.E.
      • Gerin J.L.
      • Tennant B.C.
      • Cote P.J.
      Immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-l-arabinofuranosyl)-uracil (L-FMAU) breaks humoral and cell-mediated immune tolerance in chronic woodchuck hepatitis virus infection.
      ,
      • Roggendorf M.
      • Yang D.
      • Lu M.
      The woodchuck: a model for therapeutic vaccination against hepadnaviral infection.
      ] (Table 2).
      Lamivudine is the nucleoside analog most widely combined with multiple administrations of vaccine. In most published clinical trials, efforts to prevent the emergence of escape mutants during the prolonged use of lamivudine have been based on the use of this analog for a short period only (6–12 months). The combination of vaccine and lamivudine is well tolerated, with no serious adverse effects. Responses to combination therapy have generally been evaluated on the basis of virological and serological parameters, such as the decrease in HBV DNA levels, the loss of HBeAg and HBsAg and seroconversion. Many of the patients enrolled in clinical trials have never been treated before and have high serum ALT levels. Serum ALT levels are an important predictor of the response to lamivudine treatment [
      • Leung N.
      Treatment of chronic hepatitis B: case selection and duration of therapy.
      ]. In two recent studies, following 6 months of combination therapy, regardless of the composition of the vaccine, HBeAg/anti-HBe seroconversion was described in about 25% of treated patients [
      • Al-Mahtab M.
      • Rahman S.
      • Akbar S.M.
      • Khan S.I.
      • Uddin H.
      • Karim F.
      • et al.
      Combination therapy with antiviral drugs and hepatitis B vaccine in incidentally-detected and asymptomatic chronic hepatitis virus B carriers at Bangladesh.
      ,
      • Senturk H.
      • Tabak F.
      • Ozaras R.
      • Erdem L.
      • Canbakan B.
      • Mert A.
      • et al.
      Efficacy of pre-S-containing HBV vaccine combined with lamivudine in the treatment of chronic HBV infection.
      ]. These results should be compared with the current rate of HBeAg seroconversion in lamivudine-treated patients of about 15%. In one study, patients were followed for more than 6 years following cessation of treatment. Twenty-four percent of them were classified as sustained responders with HBV DNA below detection limit, persistence of anti-HBe and no relapse in hepatitis [
      • Senturk H.
      • Tabak F.
      • Ozaras R.
      • Erdem L.
      • Canbakan B.
      • Mert A.
      • et al.
      Efficacy of pre-S-containing HBV vaccine combined with lamivudine in the treatment of chronic HBV infection.
      ]. However, this study had two major drawbacks. First, it has no control group and second, the technique used for HBV-DNA quantification was less sensitive than that used in other trials. This raised the question of whether the virus is really cleared or just decreased to below detection limit.
      In a randomized controlled study of 180 patients assigned to three groups, Hoa et al. evaluated the relative contributions of a preventive vaccine and antiviral drugs to the treatment of chronic HBV patients during active disease. Patients received vaccine monotherapy, lamivudine monotherapy, or a combination treatment. The combination treatment was found to give significantly higher and earlier rates of viral suppression than lamivudine or vaccine monotherapy. HBeAg seroconversion rates did not differ significantly between the three groups of patients. However, a continual increase in the rate of seroconversion was observed in patients from the vaccinated groups, suggesting that the vaccine enhances the antiviral immune response [
      • Hoa P.T.
      • Huy N.T.
      • Thule T.
      • Nga C.N.
      • Nakao K.
      • Eguchi K.
      • et al.
      Randomized controlled study investigating viral suppression and serological response following pre-S1/pre-S2/S vaccine therapy combined with lamivudine treatment in HBeAg-positive patients with chronic hepatitis B.
      ]. Results of these trials have to be compared with those of a randomized controlled study in which lamivudine was given with a hepatitis B vaccine mixed with a strong Th-1 adjuvant [
      • Vandepapeliere P.
      • Lau G.K.
      • Leroux-Roels G.
      • Horsmans Y.
      • Gane E.
      • Tawandee T.
      • et al.
      Therapeutic 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.
      ]. In this study, despite 12 injections of vaccine over a 1 year period, neither improvement in HBe-seroconversion rate nor decrease in HBV-DNA levels was observed in the vaccine group compared to treatment with lamivudine alone.
      In combination treatment, the time between the administration of the antiviral drug and that of the therapeutic vaccine also appears to be crucial for the immune responses induced. T-cell restoration upon lamivudine treatment accompanies the decrease in viral replication, but this effect disappears after 6 months, even in cases of continuous antiviral treatment [
      • Boni C.
      • Penna A.
      • Bertoletti A.
      • Lamonaca V.
      • Rapti I.
      • Missale G.
      • et al.
      Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.
      ]. The CD8 T-cell response, to HBcAg in particular, is also closely correlated with viral load [
      • Webster G.J.
      • Reignat S.
      • Brown D.
      • Ogg G.S.
      • Jones L.
      • Seneviratne S.L.
      • et al.
      Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy.
      ]. It would, therefore, seem logical to begin vaccination when serum HBV DNA levels have just become undetectable. The results obtained for patients with chronic hepatitis B undergoing an intradermal vaccination program with an HBsAg-based preventive vaccine beginning 3 months after the initiation of antiviral treatment provide support for this strategy. Indeed, the rate of seroconversion from HBeAg to anti-HBe was 56% for the combination regimen, whereas it was only 16% for lamivudine monotherapy [
      • Horiike N.
      • Fazle Akbar S.M.
      • Michitaka K.
      • Joukou K.
      • Yamamoto K.
      • Kojima N.
      • et al.
      In vivo immunization by vaccine therapy following virus suppression by lamivudine: a novel approach for treating patients with chronic hepatitis B.
      ]. It should be noted that this high rate of seroconversion was significantly associated with an initial low viral load. Interestingly, none of the patients receiving the combination therapy displayed HBV DNA or hepatitis breakthrough during the 3-month post-treatment follow-up period of the study, whereas such problems were observed in the monotherapy group and have been reported in other studies.
      HBV DNA loss or decrease was generally used as an end point to assess viral control in the various studies. However, it is difficult to reconcile the results as none of these studies used the same criteria to define virological responders. In addition, the patients enrolled in the clinical trials may have a completely different disease history depending on the mode of virus transmission, the age at which contamination occurred, and duration of chronic infection.
      Only a few reports have documented humoral or cellular immune responses. The humoral response has been assessed through the detection of anti-HBs antibodies, whereas proliferation and cytokine production have been used as indicators of cellular responses. Vaccination clearly stimulates the humoral response, because 50–85% of vaccinated patients with chronic HBV infection develop antibodies against HBsAg. However, HBsAg clearance has only rarely been observed [
      • Hoa P.T.
      • Huy N.T.
      • Thule T.
      • Nga C.N.
      • Nakao K.
      • Eguchi K.
      • et al.
      Randomized controlled study investigating viral suppression and serological response following pre-S1/pre-S2/S vaccine therapy combined with lamivudine treatment in HBeAg-positive patients with chronic hepatitis B.
      ,
      • Vandepapeliere P.
      • Lau G.K.
      • Leroux-Roels G.
      • Horsmans Y.
      • Gane E.
      • Tawandee T.
      • et al.
      Therapeutic 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.
      ]. Furthermore, although the HBsAg-specific lymphoproliferative responses in patients with chronic hepatitis B were found to be similar to those in healthy vaccine-recipients, the T cells of these patients secreted less IFN-γ. This suggests that the anticipated Th1 stimulation in response to vaccination may not be achieved in this setting [
      • Vandepapeliere P.
      • Lau G.K.
      • Leroux-Roels G.
      • Horsmans Y.
      • Gane E.
      • Tawandee T.
      • et al.
      Therapeutic 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.
      ]. In addition, no association was found between HBsAg-specific T-cell proliferation and any clinical or virological improvement in the few patients for which immune response was analyzed.

      Can combination therapy be improved further?

      The use of strong Th1-inducing adjuvants or cytokines might increase the efficacy of therapeutic vaccination. Several trials have evaluated the use of a therapeutic vaccine combined with IL-2 to treat human immunodeficiency virus or HBV infection [
      • Goujard C.
      • Marcellin F.
      • Hendel-Chavez H.
      • Burgard M.
      • Meiffredy V.
      • Venet A.
      • et al.
      Interruption of antiretroviral therapy initiated during primary HIV-1 infection: impact of a therapeutic vaccination strategy combined with interleukin (IL)-2 compared with IL-2 alone in the ANRS 095 Randomized Study.
      ,
      • Dahmen A.
      • Herzog-Hauff S.
      • Bocher W.O.
      • Galle P.R.
      • Lohr H.F.
      Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis B.
      ]. The withdrawal of antiviral treatment at the end of vaccination had almost no effect on the risk of viremia relapse. However, one potential drawback of IL-2 treatment is that it induces Tregs, which constitutively express IL-2Rα. Other cytokines, such as IL-7 and IL-15, are essential for the homeostatic proliferation of T cells in vivo, the survival of effector cells and the maintenance of memory T cells during viral infection. These cytokines may increase the pool of naïve T cells available for vaccine-mediated stimulation and the survival of activated T cells. Studies on animals have shown that IL-7 improves anti-tumor responses and survival after a vaccine-induced immune response [
      • Pellegrini M.
      • Calzascia T.
      • Elford A.R.
      • Shahinian A.
      • Lin A.E.
      • Dissanayake D.
      • et al.
      Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies.
      ]. A clinical trial combining antiviral treatment with a classical preventive vaccine and multiple IL-7 injections is currently underway in HBe-negative chronic hepatitis B patients with no detectable HBV DNA during at least 3 months of anti-viral treatment (www.ClinicalTrials.gov, Identifier: NCT01027065).
      Experiments in mice with persistent lymphocytic choriomeningitis virus infection have shown that anti-PD-L1 antibodies and therapeutic vaccination synergistically improve viral control [
      • Ha S.J.
      • Mueller S.N.
      • Wherry E.J.
      • Barber D.L.
      • Aubert R.D.
      • Sharpe A.H.
      • et al.
      Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection.
      ], by blocking the PD-1/PD-L1 inhibitory signals on exhausted T cells. In vitro studies have shown that it may be possible to at least partly restore intrahepatic HBV-specific T-cell responses in patients with chronic HBV infection, by blocking the PD-1 pathway [
      • Fisicaro P.
      • Valdatta C.
      • Massari M.
      • Loggi E.
      • Biasini E.
      • Sacchelli L.
      • et al.
      Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B.
      ]. Targeting of the PD-1 pathway, in combination with vaccination and antiviral treatments, therefore, constitutes another possible approach to restoring T-cell function and generating long-term memory T cells [
      • Watanabe T.
      • Bertoletti A.
      • Tanoto T.A.
      PD-1/PD-L1 pathway and T-cell exhaustion in chronic hepatitis virus infection.
      ].

      Are DNA-based vaccines more powerful?

      DNA-based vaccines are increasingly being tested, for both preventive and therapeutic vaccination purposes, and encouraging results have been obtained in mice and chimpanzees [
      • Mancini M.
      • Hadchouel M.
      • Davis H.L.
      • Whalen R.G.
      • Tiollais P.
      • Michel M.L.
      DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.
      ,
      • Prince A.M.
      • Whalen R.
      • Brotman B.
      Successful nucleic acid based immunization of newborn chimpanzees against hepatitis B virus.
      ]. The functional profile of the immune responses generated by immunization with plasmid DNA is similar to that observed in patients with resolved HBV infection and self-limited disease [
      • Loirat D.
      • Lemonnier F.A.
      • Michel M.L.
      Multiepitopic HLA-A∗0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization.
      ]. This type of vaccination has the advantage of inducing both humoral and cellular immune responses, including cytotoxic and Th1 responses [
      • Donnelly J.J.
      • Wahren B.
      • Liu M.A.
      DNA vaccines: progress and challenges.
      ].
      The first Phase I trial of therapeutic DNA immunization for chronic hepatitis B was carried out with a DNA vaccine encoding HBV envelope proteins [
      • Mancini-Bourgine M.
      • Fontaine H.
      • Scott-Algara D.
      • Pol S.
      • Brechot C.
      • Michel M.L.
      Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers.
      ] (Table 2). At the time of inclusion, the treated HBV patients presented viral breakthrough on lamivudine and active HBV replication. Despite this unfavorable environment, a decrease in viral load and HBeAg seroconversion was observed in six and two of 10 patients, respectively. Seroconversion occurred in the two patients with the lowest viral load at the beginning of the trial, highlighting the importance of HBV DNA titers for successful immunotherapy. DNA vaccination led to the induction of IFN-γ-secreting T cells specific to several envelope-derived epitopes and cytotoxic CD8+ T cells, demonstrating the ability of this approach to induce or to increase the number of HBV-specific T cells. This HBV-specific IFN-γ T-cell response was also correlated with an increase in the percentage of a specific NK-cell subset known to produce cytokines in abundance, the CD56bright NK-cell population [
      • Scott-Algara D.
      • Mancini-Bourgine M.
      • Fontaine H.
      • Pol S.
      • Michel M.L.
      Changes to the natural killer cell repertoire after therapeutic hepatitis B DNA vaccination.
      ]. Nevertheless, despite the stimulation of innate and adaptive immune responses, no severe adverse events were reported with the exception of a transient increase in ALT levels in two patients, probably due to the restoration of T-cell responses. However, T-cell reactivity gradually declined at the end of treatment, reflecting the profound defect in immune responses observed during chronic HBV infection.
      A Phase I/II trial is currently underway to determine whether the DNA vaccination of patients with chronic HBV infection treated with nucleos/tide analogs can lead to a T-cell restoration and delayed viral reactivation after treatment discontinuation (www.ClinicalTrials.gov, Identifier: NCT00536627). Seventy patients with effective antiviral treatments have been randomized to two treatment groups: one group receiving two series of three and two DNA injections, with 6 months between the two series, and a control group. Antiviral treatment will be stopped in both groups at a time point corresponding to 2 weeks after the last DNA injection. The primary endpoint of this trial is vaccine protection against the virological breakthrough that may occur during treatment with analogs and against reactivation after the end of analog treatment. The secondary endpoints include the immune responses in both groups (see Fig. 2).
      Figure thumbnail gr2
      Fig. 2General scheme of combined vaccination and antiviral drug treatment. In trials of combination treatment, patients suffering from chronic hepatitis B are enrolled and assigned to one of two groups (Groups A and B). Patients from both groups receive antiviral drugs. At the beginning of antiviral treatment or once HBV-DNA viral load has decreased to undetectable levels, multiple shots of the vaccine are administered to patients from group A. HBV DNA viral load, % HBeAg and HBsAg seroconversion are assessed. During antiviral treatment, viral and antigenic loads decrease and dysfunctional HBV-specific T cells are expected to recover. At the end of the clinical trial (after the last vaccine injection) treatment is stopped in both groups. ALT levels are continually monitored to ensure the rapid detection of liver damage. Viral rebound is expected in the patients of group B, whereas viremia control is expected in the patients of group A. In the patients of group A, HBV-specific and vaccine-activated T cells are expected to exert their antiviral functions, including the secretion of cytokines (IFN-γ, IL-2 and TNF-α) and proliferation.
      Similarly, DNA vaccines encoding multiple HBV proteins and a modified human IL-12 molecule were administered once monthly, for 12 months, in combination with lamivudine treatment, to improve the efficacy of DNA vaccination and to broaden immune responses [
      • Yang S.H.
      • Lee C.G.
      • Park S.H.
      • Im S.J.
      • Kim Y.M.
      • Son J.M.
      • et al.
      Correlation of antiviral T-cell responses with suppression of viral rebound in chronic hepatitis B carriers: a proof-of-concept study.
      ]. IL-12 promotes Th1 cell development, cell-mediated cytotoxicity, and IFN-γ production and is involved in the control of viremia in HBeAg-positive chronic hepatitis B carriers [
      • Rigopoulou E.I.
      • Suri D.
      • Chokshi S.
      • Mullerova I.
      • Rice S.
      • Tedder R.S.
      • et al.
      Lamivudine plus interleukin-12 combination therapy in chronic hepatitis B: antiviral and immunological activity.
      ]. A virological response was observed in half the treated patients at the end of combination therapy and this was significantly higher compared to lamivudine monotherapy in historical control groups. Memory T cells were detected in virological responders, persisted for 40 weeks after vaccination and were closely associated with the suppression of viral rebound after treatment was stopped. Two of the virological responders still had undetectable viral load 3 years after cessation of combined therapy [
      • Im S.J.
      • Yang S.H.
      • Yoon S.K.
      • Sung Y.C.
      Increase of plasma IL-12/p40 ratio induced by the combined therapy of DNA vaccine and lamivudine correlates with sustained viremia control in CHB carriers.
      ]. The presence of modified IL-12 as a genetic adjuvant may have contributed as well to the induction of long-lasting memory T cells. Whether the HBV-specific T cells were present in patients at the beginning of the trial and were reactivated by the combined treatment or if they were de novo activated by the vaccine is not known as pretreatment samples were not analyzed.
      Finally, DNA vaccines may be used to prime immunity before boosting with viral vectors, such as poxviruses or adenoviruses encoding the same antigenic proteins. Such combinations result in very large numbers of multifunctional T cells, as demonstrated in trials of vaccines for malaria and tuberculosis [
      • Gilbert S.C.
      • Moorthy V.S.
      • Andrews L.
      • Pathan A.A.
      • McConkey S.J.
      • Vuola J.M.
      • et al.
      Synergistic DNA-MVA prime-boost vaccination regimes for malaria and tuberculosis.
      ].

      Are cell-based therapies an attractive alternative to vaccination?

      As an alternative to vaccination, DCs loaded ex vivo with HBV antigens could also be used to stimulate T cells (Table 2). The use of autologous DCs pulsed with tumor-related antigens as immunotherapy for cancer has been largely documented [
      • Ueno H.
      • Schmitt N.
      • Klechevsky E.
      • Pedroza-Gonzalez A.
      • Matsui T.
      • Zurawski G.
      • et al.
      Harnessing human dendritic cell subsets for medicine.
      ]. Few studies have reported the use of DCs for diseases related to viral infections [
      • Lu W.
      • Arraes L.C.
      • Ferreira W.T.
      • Andrieu J.M.
      Therapeutic dendritic-cell vaccine for chronic HIV-1 infection.
      ,
      • Leen A.M.
      • Myers G.D.
      • Sili U.
      • Huls M.H.
      • Weiss H.
      • Leung K.S.
      • et al.
      Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.
      ]. A pilot study was carried out in five patients with chronic hepatitis B, to evaluate the safety of HBsAg-pulsed autologous DCs [

      Akbar SM, Furukawa S, Horiike N, Abe M, Hiasa Y, Onji M. Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B. J Viral Hepat 2010. May 17 [Epub ahead of print].

      ]. Monocyte-derived DCs were isolated from peripheral blood from a given patient by a classical method using granulocyte macrophage colony-stimulating factor and IL-4. They were then pulsed with purified HBsAg and re-injected into the patient. This approach had previously been shown to induce anti-HBs antibodies when administered to vaccine-non-responders [
      • Fazle Akbar S.M.
      • Furukawa S.
      • Onji M.
      • Murata Y.
      • Niya T.
      • Kanno S.
      • et al.
      Safety and efficacy of hepatitis B surface antigen-pulsed dendritic cells in human volunteers.
      ]. Administration was safe and no exacerbation of liver damage was observed in patients with chronic hepatitis B. In another recent trial, autologous DCs pulsed with HLA-A2-restricted peptides derived from HBcAg (core 18–27 epitope) and from the N-terminal part of the middle envelope protein were re-injected on 9 occasions into 380 chronic hepatitis B patients in an autologous manner [
      • Luo J.
      • Li J.
      • Chen R.L.
      • Nie L.
      • Huang J.
      • Liu Z.W.
      • et al.
      Autologus dendritic cell vaccine for chronic hepatitis B carriers: a pilot, open label, clinical trial in human volunteers.
      ]. Interestingly, a significant response, as demonstrated by a decrease in HBV DNA levels to below 103 cp/ml and transaminase normalization, was observed in half the HBeAg-negative/anti-HBe-positive patients, whereas only 13% of the HBeAg-positive patients displayed complete responses. This treatment was most successful in HBeAg-negative patients and patients with a viral load <105 cp/ml, some of whom had cleared HBsAg. A transient increase in ALT levels was observed in some patients during the first few weeks of treatment, possibly reflecting an effect of the treatment on infected hepatocytes, but ALT levels returned to normal by the end of treatment. However, in that study, the immune response-mediated mechanisms underlying the observed serologic and virological effects were not documented. An increase in the number of CD8+ T cells was reported, but it was not determined whether these T cells are specific for the HBV peptides loaded onto DCs. Furthermore, it has been reported that the core 18–27 peptide used to load DCs frequently induces T cell responses in HLA-A0201 Caucasian patients, but is rarely able to induce CD8+ T cells in HLA-A0206 or HLA-A0203 Chinese patients [
      • Tan A.T.
      • Loggi E.
      • Boni C.
      • Chia A.
      • Gehring A.J.
      • Sastry K.S.
      • et al.
      Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire.
      ]. Moreover, the sequences of the two peptides used in the former study vary according to HBV genotype, raising questions about the specificity of this DC-mediated therapy.
      Animal and clinical studies have demonstrated that the transfer of HBV-specific memory cells from an immune donor, through bone marrow transplantation or the transfer of peripheral blood mononuclear cells (PBMCs), can induce the serological clearance of HBsAg and lead to seroconversion in patients with chronic hepatitis B [
      • Ilan Y.
      • Nagler A.
      • Adler R.
      • Tur-Kaspa R.
      • Slavin S.
      • Shouval D.
      Ablation of persistent hepatitis B by bone marrow transplantation from a hepatitis B-immune donor.
      ,
      • Hui C.K.
      • Lie A.
      • Au W.Y.
      • Leung Y.H.
      • Ma S.Y.
      • Cheung W.W.
      • et al.
      A long-term follow-up study on hepatitis B surface antigen-positive patients undergoing allogeneic hematopoietic stem cell transplantation.
      ]. These observations have led to the development of the autologous transfusion of cytokine-induced killer (CIK) cells into patients with chronic hepatitis. CIK cells consist of heterologous cell populations, including cells with both the T-cell and NK cell markers (CD3+ CD8+ and CD3+ CD56+), with cytotoxic and IFN-γ secretion functions. These cells are obtained by the ex vivo stimulation of PBMCs with IFN-γ, anti-CD3 antibody and IL-2. After 2 weeks of ex vivo stimulation, the CIK cells are transferred back into patients, in an autologous manner. In a recent study, CIK cells were generated from 21 chronic hepatitis B virus carriers with high HBV DNA loads and high ALT levels. They were then transferred back into the patients, through three intravenous transfusions. Patients were followed up for over 48 weeks and a significant effect on viral load and HBeAg clearance was observed, mostly in patients with baseline serum ALT levels above normal values. This improvement was achieved despite the absence of HBV-specific CD8 T cells among the re-infused CIK cells. The biochemical and virological effects in responders were attributed to the higher proportion of CD3+ CD56+ cells than of other cells among CIK cells and to the effector capacity of CIK cells [
      • Shi M.
      • Fu J.
      • Shi F.
      • Zhang B.
      • Tang Z.
      • Jin L.
      • et al.
      Transfusion of autologous cytokine-induced killer cells inhibits viral replication in patients with chronic hepatitis B virus infection.
      ]. These results although encouraging are tempered by the difficulties in developing individualized treatment on a large scale.

      What future approaches could be used to engineer functional T cells?

      In addition to treatments designed to stimulate HBV-specific T cells in vivo by vaccination strategies or ex vivo with cytokines or DCs, a novel approach is based on the genetic modification of T cells by T-cell receptor (TCR) gene transfer [
      • Engels B.
      • Uckert W.
      Redirecting T lymphocyte specificity by T cell receptor gene transfer–a new era for immunotherapy.
      ]. This involves (1) the isolation of the HBV-specific TCR from T cells obtained from individuals with self-resolved hepatitis, (2) the insertion of this TCR into a DNA or viral vector and (3) the transfer of this TCR specificity to T cells from patients with chronic HBV infection. Retroviral vectors have been used to introduce HLA-A2-restricted HBV-specific TCR into T cells from chronic patients. The TCR-transduced T cells produced IFN-γ, TNF-α, and IL-2 following target cell recognition and lysed hepatocyte-like cell lines expressing cognate HBV antigens. In vivo functionality was also assessed by the adoptive transfer of TCR-redirected T cells in immunodeficient mice, leading to the rejection of grafted tumor cells expressing HBV antigens. This strategy should make it possible to reconstitute functional HBV-specific T cells in patients with chronic infection [
      • Bertoletti A.
      • Gehring A.
      Therapeutic vaccination and novel strategies to treat chronic HBV infection.
      ,
      • Gehring A.
      • Xue S.-A.
      • Ho Z.Z.
      • Teoh D.Y.L.
      • Ruedl C.
      • Chia A.
      • et al.
      Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.
      ]. Alternatively, artificial chimeric TCRs composed of a single-chain antibody fragment recognizing HBV envelope proteins, and cytoplasmic regions of the co-stimulatory CD28 molecule followed by the CD3-ζ signaling domain have been constructed. These chimeric receptors, when delivered by retroviruses and expressed on the cell surface, enable primary human T cells to recognize HBsAg-positive hepatocytes, release IFN-γ and IL-2, and, most importantly, lyse HBV-replicating cells. [
      • Bohne F.
      • Chmielewski M.
      • Ebert G.
      • Wiegmann K.
      • Kurschner T.
      • Schulze A.
      • et al.
      T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.
      ]. However, it remains unclear whether the re-injected TCR-redirected T cells will proliferate in vivo and reach the target organ, and whether the newly engineered T cells will be subject to a tolerizing environment.
      If anti-HBV immunotherapy is to be efficient, the activated T-cell response must specifically reach the liver. Deng et al. [
      • Deng Q.
      • Mancini-Bourgine M.
      • Zhang X.
      • Cumont M.C.
      • Zhu R.
      • Lone Y.C.
      • et al.
      Hepatitis B virus as a gene delivery vector activating foreign antigenic T cell response that abrogates viral expression in mouse models.
      ], therefore, designed a recombinant HBV (rHBV) containing a modified viral core gene for the specific delivery of foreign (i.e. non-HBV) epitopes to the liver. This recombinant virus was engineered to self-maintain only in hepatocytes already infected with HBV through capsid complementation. The activated non-HBV specific CD8+ T-cell response is expected to be functional and to compensate for the deficiencies of HBV-specific anti-viral immunity. It should target infected liver cells and would not be subject to functional exhaustion during chronic hepatitis B. Proof-of-concept for this approach was demonstrated through a protocol for rHBV-based active immunization in HLA-A2/DR1-transgenic mice (see Fig. 3). A strong polyepitope-specific T-cell response was first primed in the periphery by DNA immunization. In absence of a mouse model susceptible to HBV infection, hydrodynamic injection was used to mimic rHBV replication and gene expression in the mouse liver. This method allows direct in vivo transfection of hepatocytes and to bypass the strict host-range of the virus for human hepatocytes [
      • Yang P.L.
      • Althage A.
      • Chung J.
      • Chisari F.V.
      Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection.
      ]. The expression of foreign antigenic epitopes in hepatocytes recruited a vigorous T-cell response in situ. Most liver-infiltrating polyepitope-specific CD8+ T cells proved to be functional effectors. Following DNA priming and hydrodynamic injection, the rHBV-based expression of HBsAg in mouse liver was completely inhibited without major liver injury. Studies in HLA-A2/DR1/HBsAg-transgenic mice as a surrogate model for HBV chronic infection further validated this approach. In these mice the polyepitope-specific functional T cell response not only controlled the rHBV expression but also controlled HBV transgene expression in the liver. Thus, rHBV and foreign antigen-based active immunotherapy constitute a promising strategy for the treatment of persistent HBV infection. This strategy could potentially be generalized and extended to other chronic viral diseases.
      Figure thumbnail gr3
      Fig. 3Gene therapy for chronic HBV infection. An immunotherapy protocol is established, combining vaccination and liver gene therapy. A recombinant HBV virus (rHBV) is designed, both as a gene delivery vector and as an antigen carrier, for intrahepatic expression of a foreign antigenic polyepitope. In this strategy, functional poly-epitope-specific T-cell responses will be generated in patients by vaccination before applying rHBV gene therapy. An rHBV is created in which the viral core gene is disrupted by the insertion of the foreign antigenic sequence. This core-deficient virus is not competent for replication unless the host hepatocyte provides the wild-type viral capsid protein in trans. The rHBV specifically infects human hepatocytes, as does the wild-type virus (wtHBV). However, rHBV cannot replicate in healthy recipients, and is maintained in chronically infected patients only in hepatocytes persistently infected with wtHBV. In the presence of HBV liver-specific promoters, rHBV proteins (polymerase, L, M, S envelopes, HBx) and the foreign antigenic polyepitope are produced, processed and presented as peptides on liver cells. The presentation of the foreign epitopes recruits vigorous functional T-cell response to the liver that should compensate for the dysfunctional HBV-specific T-cell response observed during persistent viral infection.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      References

        • Ganem D.
        • Prince A.M.
        Hepatitis B virus infection – natural history and clinical consequences.
        N Engl J Med. 2004; 350: 1118-1129
        • Chen D.S.
        Hepatitis B vaccination: the key towards elimination and eradication of hepatitis B.
        J Hepatol. 2009; 50: 805-816
        • Dienstag J.L.
        Hepatitis B virus infection.
        N Engl J Med. 2008; 359: 1486-1500
        • Zoulim F.
        • Locarnini S.
        Hepatitis B virus resistance to nucleos(t)ide analogues.
        Gastroenterology. 2009; 137 (e1591–e1592): 1593-1608
        • Rehermann B.
        • Nascimbeni M.
        Immunology of hepatitis B virus and hepatitis C virus infection.
        Nat Rev Immunol. 2005; 5: 215-229
        • Mondelli M.U.
        • Varchetta S.
        • Oliviero B.
        Natural killer cells in viral hepatitis: facts and controversies.
        Eur J Clin Invest. 2010; 40: 851-863
        • Dunn C.
        • Peppa D.
        • Khanna P.
        • Nebbia G.
        • Jones M.
        • Brendish N.
        • et al.
        Temporal analysis of early immune responses in patients with acute hepatitis B virus infection.
        Gastroenterology. 2009; 137: 1289-1300
        • Bertoletti A.
        • Maini M.K.
        Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection?.
        Curr Opin Microbiol. 2000; 3: 387-392
        • Isogawa M.
        • Furuichi Y.
        • Chisari F.V.
        Oscillating CD8(+) T cell effector functions after antigen recognition in the liver.
        Immunity. 2005; 23: 53-63
        • Fisicaro P.
        • Valdatta C.
        • Boni C.
        • Massari M.
        • Mori C.
        • Zerbini A.
        • et al.
        Early kinetics of innate and adaptive immune responses during hepatitis B virus infection.
        Gut. 2009; 58: 974-982
        • Rehermann B.
        Chronic infections with hepatotropic viruses: mechanisms of impairment of cellular immune responses.
        Semin Liver Dis. 2007; 27: 152-160
        • Maini M.K.
        • Schurich A.
        The molecular basis of the failed immune response in chronic HBV: therapeutic implications.
        J Hepatol. 2010; 52: 616-619
        • Barber D.L.
        • Wherry E.J.
        • Masopust D.
        • Zhu B.
        • Allison J.P.
        • Sharpe A.H.
        • et al.
        Restoring function in exhausted CD8 T cells during chronic viral infection.
        Nature. 2006; 439: 682-687
        • Wherry E.J.
        • Ha S.J.
        • Kaech S.M.
        • Haining W.N.
        • Sarkar S.
        • Kalia V.
        • et al.
        Molecular signature of CD8+ T cell exhaustion during chronic viral infection.
        Immunity. 2007; 27: 670-684
        • Fisicaro P.
        • Valdatta C.
        • Massari M.
        • Loggi E.
        • Biasini E.
        • Sacchelli L.
        • et al.
        Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B.
        Gastroenterology. 2010; 138 (693,e681–e684): 682-693
        • Wherry E.J.
        • Barber D.L.
        • Kaech S.M.
        • Blattman J.N.
        • Ahmed R.
        Antigen-independent memory CD8 T cells do not develop during chronic viral infection.
        Proc Natl Acad Sci USA. 2004; 101: 16004-16009
        • Webster G.J.
        • Reignat S.
        • Brown D.
        • Ogg G.S.
        • Jones L.
        • Seneviratne S.L.
        • et al.
        Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy.
        J Virol. 2004; 78: 5707-5719
        • Lopes A.R.
        • Kellam P.
        • Das A.
        • Dunn C.
        • Kwan A.
        • Turner J.
        • et al.
        Bim-mediated deletion of antigen-specific CD8 T cells in patients unable to control HBV infection.
        J Clin Invest. 2008; 118: 1835-1845
        • Xu D.
        • Fu J.
        • Jin L.
        • Zhang H.
        • Zhou C.
        • Zou Z.
        • et al.
        Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B.
        J Immunol. 2006; 177: 739-747
        • Stoop J.N.
        • Claassen M.A.
        • Woltman A.M.
        • Binda R.S.
        • Kuipers E.J.
        • Janssen H.L.
        • et al.
        Intrahepatic regulatory T cells are phenotypically distinct from their peripheral counterparts in chronic HBV patients.
        Clin Immunol. 2008; 129: 419-427
        • Stoop J.N.
        • van der Molen R.G.
        • Baan C.C.
        • van der Laan L.J.
        • Kuipers E.J.
        • Kusters J.G.
        • et al.
        Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection.
        Hepatology. 2005; 41: 771-778
        • Franzese O.
        • Kennedy P.T.
        • Gehring A.J.
        • Gotto J.
        • Williams R.
        • Maini M.K.
        • et al.
        Modulation of the CD8+-T-cell response by CD4+ CD25+ regulatory T cells in patients with hepatitis B virus infection.
        J Virol. 2005; 79: 3322-3328
        • Woltman A.M.
        • Boonstra A.
        • Janssen H.L.
        Dendritic cells in chronic viral hepatitis B and C: victims or guardian angels?.
        Gut. 2010; 59: 115-125
        • van der Molen R.G.
        • Sprengers D.
        • Binda R.S.
        • de Jong E.C.
        • Niesters H.G.
        • Kusters J.G.
        • et al.
        Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.
        Hepatology. 2004; 40: 738-746
        • Untergasser A.
        • Zedler U.
        • Langenkamp A.
        • Hosel M.
        • Quasdorff M.
        • Esser K.
        • et al.
        Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection.
        Hepatology. 2006; 43: 539-547
        • Chen L.
        • Zhang Z.
        • Chen W.
        • Li Y.
        • Shi M.
        • Zhang J.
        • et al.
        B7–H1 up-regulation on myeloid dendritic cells significantly suppresses T cell immune function in patients with chronic hepatitis B.
        J Immunol. 2007; 178: 6634-6641
        • Pol S.
        • Michel M.L.
        Therapeutic vaccination in chronic hepatitis B virus carriers.
        Expert Rev Vaccines. 2006; 5: 707-716
        • Bertoletti A.
        • Gehring A.
        Therapeutic vaccination and novel strategies to treat chronic HBV infection.
        Expert Rev Gastroenterol Hepatol. 2009; 3: 561-569
        • Xu D.Z.
        • Zhao K.
        • Guo L.M.
        • Li L.J.
        • Xie Q.
        • Ren H.
        • et al.
        A randomized controlled phase IIb trial of antigen-antibody immunogenic complex therapeutic vaccine in chronic hepatitis B patients.
        PLoS One. 2008; 3: e2565
        • Aguilar J.C.
        • Lobaina Y.
        • Muzio V.
        • Garcia D.
        • Penton E.
        • Iglesias E.
        • et al.
        Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.
        Immunol Cell Biol. 2004; 82: 539-546
        • Betancourt A.A.
        • Delgado C.A.
        • Estevez Z.C.
        • Martinez J.C.
        • Rios G.V.
        • Aureoles-Rosello S.R.
        • et al.
        Phase I clinical trial in healthy adults of a nasal vaccine candidate containing recombinant hepatitis B surface and core antigens.
        Int J Infect Dis. 2007; 11: 394-401
        • Akbar S.M.
        • Al-Mahtab M.
        • Rahman S.
        • Aguilar J.C.
        • Onji M.
        • Mishiro S.
        A therapeutic nasal vaccine combining both HBsAg and HBcAg wassafe, has antiviral potential and induced antigen-specific immunity in patients with chronic hepatitis B.
        Hepatol Int. 2010; 4: 159
        • Boni C.
        • Bertoletti A.
        • Penna A.
        • Cavalli A.
        • Pilli M.
        • Urbani S.
        • et al.
        Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B.
        J Clin Invest. 1998; 102: 968-975
        • Boni C.
        • Penna A.
        • Bertoletti A.
        • Lamonaca V.
        • Rapti I.
        • Missale G.
        • et al.
        Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.
        J Hepatol. 2003; 39: 595-605
        • Le Guerhier F.
        • Thermet A.
        • Guerret S.
        • Chevallier M.
        • Jamard C.
        • Gibbs C.S.
        • et al.
        Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model.
        J Hepatol. 2003; 38: 328-334
        • Menne S.
        • Roneker C.A.
        • Korba B.E.
        • Gerin J.L.
        • Tennant B.C.
        • Cote P.J.
        Immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-l-arabinofuranosyl)-uracil (L-FMAU) breaks humoral and cell-mediated immune tolerance in chronic woodchuck hepatitis virus infection.
        J Virol. 2002; 76: 5305-5314
        • Roggendorf M.
        • Yang D.
        • Lu M.
        The woodchuck: a model for therapeutic vaccination against hepadnaviral infection.
        Pathol Biol (Paris). 2010; 58: 308-314
        • Leung N.
        Treatment of chronic hepatitis B: case selection and duration of therapy.
        J Gastroenterol Hepatol. 2002; 17: 409-414
        • Al-Mahtab M.
        • Rahman S.
        • Akbar S.M.
        • Khan S.I.
        • Uddin H.
        • Karim F.
        • et al.
        Combination therapy with antiviral drugs and hepatitis B vaccine in incidentally-detected and asymptomatic chronic hepatitis virus B carriers at Bangladesh.
        Viral Immunol. 2010; 23: 335-338
        • Senturk H.
        • Tabak F.
        • Ozaras R.
        • Erdem L.
        • Canbakan B.
        • Mert A.
        • et al.
        Efficacy of pre-S-containing HBV vaccine combined with lamivudine in the treatment of chronic HBV infection.
        Dig Dis Sci. 2009; 54: 2026-2030
        • Hoa P.T.
        • Huy N.T.
        • Thule T.
        • Nga C.N.
        • Nakao K.
        • Eguchi K.
        • et al.
        Randomized controlled study investigating viral suppression and serological response following pre-S1/pre-S2/S vaccine therapy combined with lamivudine treatment in HBeAg-positive patients with chronic hepatitis B.
        Antimicrob Agents Chemother. 2009; 53: 5134-5140
        • Vandepapeliere P.
        • Lau G.K.
        • Leroux-Roels G.
        • Horsmans Y.
        • Gane E.
        • Tawandee T.
        • et al.
        Therapeutic 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. 2007; 25: 8585-8597
        • Horiike N.
        • Fazle Akbar S.M.
        • Michitaka K.
        • Joukou K.
        • Yamamoto K.
        • Kojima N.
        • et al.
        In vivo immunization by vaccine therapy following virus suppression by lamivudine: a novel approach for treating patients with chronic hepatitis B.
        J Clin Virol. 2005; 32: 156-161
        • Goujard C.
        • Marcellin F.
        • Hendel-Chavez H.
        • Burgard M.
        • Meiffredy V.
        • Venet A.
        • et al.
        Interruption of antiretroviral therapy initiated during primary HIV-1 infection: impact of a therapeutic vaccination strategy combined with interleukin (IL)-2 compared with IL-2 alone in the ANRS 095 Randomized Study.
        AIDS Res Hum Retroviruses. 2007; 23: 1105-1113
        • Dahmen A.
        • Herzog-Hauff S.
        • Bocher W.O.
        • Galle P.R.
        • Lohr H.F.
        Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis B.
        J Med Virol. 2002; 66: 452-460
        • Pellegrini M.
        • Calzascia T.
        • Elford A.R.
        • Shahinian A.
        • Lin A.E.
        • Dissanayake D.
        • et al.
        Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies.
        Nat Med. 2009; 15: 528-536
        • Ha S.J.
        • Mueller S.N.
        • Wherry E.J.
        • Barber D.L.
        • Aubert R.D.
        • Sharpe A.H.
        • et al.
        Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection.
        J Exp Med. 2008; 205: 543-555
        • Watanabe T.
        • Bertoletti A.
        • Tanoto T.A.
        PD-1/PD-L1 pathway and T-cell exhaustion in chronic hepatitis virus infection.
        J Viral Hepat. 2010; 17: 453-458
        • Mancini M.
        • Hadchouel M.
        • Davis H.L.
        • Whalen R.G.
        • Tiollais P.
        • Michel M.L.
        DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.
        Proc Natl Acad Sci USA. 1996; 93: 12496-12501
        • Prince A.M.
        • Whalen R.
        • Brotman B.
        Successful nucleic acid based immunization of newborn chimpanzees against hepatitis B virus.
        Vaccine. 1997; 15: 916-919
        • Loirat D.
        • Lemonnier F.A.
        • Michel M.L.
        Multiepitopic HLA-A∗0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization.
        J Immunol. 2000; 165: 4748-4755
        • Donnelly J.J.
        • Wahren B.
        • Liu M.A.
        DNA vaccines: progress and challenges.
        J Immunol. 2005; 175: 633-639
        • Mancini-Bourgine M.
        • Fontaine H.
        • Scott-Algara D.
        • Pol S.
        • Brechot C.
        • Michel M.L.
        Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers.
        Hepatology. 2004; 40: 874-882
        • Scott-Algara D.
        • Mancini-Bourgine M.
        • Fontaine H.
        • Pol S.
        • Michel M.L.
        Changes to the natural killer cell repertoire after therapeutic hepatitis B DNA vaccination.
        PLoS One. 2010; 5: e8761
        • Yang S.H.
        • Lee C.G.
        • Park S.H.
        • Im S.J.
        • Kim Y.M.
        • Son J.M.
        • et al.
        Correlation of antiviral T-cell responses with suppression of viral rebound in chronic hepatitis B carriers: a proof-of-concept study.
        Gene Ther. 2006; 13: 1110-1117
        • Rigopoulou E.I.
        • Suri D.
        • Chokshi S.
        • Mullerova I.
        • Rice S.
        • Tedder R.S.
        • et al.
        Lamivudine plus interleukin-12 combination therapy in chronic hepatitis B: antiviral and immunological activity.
        Hepatology. 2005; 42: 1028-1036
        • Im S.J.
        • Yang S.H.
        • Yoon S.K.
        • Sung Y.C.
        Increase of plasma IL-12/p40 ratio induced by the combined therapy of DNA vaccine and lamivudine correlates with sustained viremia control in CHB carriers.
        Immune Netw. 2009; 9: 20-26
        • Gilbert S.C.
        • Moorthy V.S.
        • Andrews L.
        • Pathan A.A.
        • McConkey S.J.
        • Vuola J.M.
        • et al.
        Synergistic DNA-MVA prime-boost vaccination regimes for malaria and tuberculosis.
        Vaccine. 2006; 24: 4554-4561
        • Ueno H.
        • Schmitt N.
        • Klechevsky E.
        • Pedroza-Gonzalez A.
        • Matsui T.
        • Zurawski G.
        • et al.
        Harnessing human dendritic cell subsets for medicine.
        Immunol Rev. 2010; 234: 199-212
        • Lu W.
        • Arraes L.C.
        • Ferreira W.T.
        • Andrieu J.M.
        Therapeutic dendritic-cell vaccine for chronic HIV-1 infection.
        Nat Med. 2004; 10: 1359-1365
        • Leen A.M.
        • Myers G.D.
        • Sili U.
        • Huls M.H.
        • Weiss H.
        • Leung K.S.
        • et al.
        Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.
        Nat Med. 2006; 12: 1160-1166
      1. Akbar SM, Furukawa S, Horiike N, Abe M, Hiasa Y, Onji M. Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B. J Viral Hepat 2010. May 17 [Epub ahead of print].

        • Fazle Akbar S.M.
        • Furukawa S.
        • Onji M.
        • Murata Y.
        • Niya T.
        • Kanno S.
        • et al.
        Safety and efficacy of hepatitis B surface antigen-pulsed dendritic cells in human volunteers.
        Hepatol Res. 2004; 29: 136-141
        • Luo J.
        • Li J.
        • Chen R.L.
        • Nie L.
        • Huang J.
        • Liu Z.W.
        • et al.
        Autologus dendritic cell vaccine for chronic hepatitis B carriers: a pilot, open label, clinical trial in human volunteers.
        Vaccine. 2010; 28: 2497-2504
        • Tan A.T.
        • Loggi E.
        • Boni C.
        • Chia A.
        • Gehring A.J.
        • Sastry K.S.
        • et al.
        Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire.
        J Virol. 2008; 82: 10986-10997
        • Ilan Y.
        • Nagler A.
        • Adler R.
        • Tur-Kaspa R.
        • Slavin S.
        • Shouval D.
        Ablation of persistent hepatitis B by bone marrow transplantation from a hepatitis B-immune donor.
        Gastroenterology. 1993; 104: 1818-1821
        • Hui C.K.
        • Lie A.
        • Au W.Y.
        • Leung Y.H.
        • Ma S.Y.
        • Cheung W.W.
        • et al.
        A long-term follow-up study on hepatitis B surface antigen-positive patients undergoing allogeneic hematopoietic stem cell transplantation.
        Blood. 2005; 106: 464-469
        • Shi M.
        • Fu J.
        • Shi F.
        • Zhang B.
        • Tang Z.
        • Jin L.
        • et al.
        Transfusion of autologous cytokine-induced killer cells inhibits viral replication in patients with chronic hepatitis B virus infection.
        Clin Immunol. 2009; 132: 43-54
        • Engels B.
        • Uckert W.
        Redirecting T lymphocyte specificity by T cell receptor gene transfer–a new era for immunotherapy.
        Mol Aspects Med. 2007; 28: 115-142
        • Gehring A.
        • Xue S.-A.
        • Ho Z.Z.
        • Teoh D.Y.L.
        • Ruedl C.
        • Chia A.
        • et al.
        Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.
        J Hepatol. 2010; (Nov 23 [Epub ahead of print])
        • Bohne F.
        • Chmielewski M.
        • Ebert G.
        • Wiegmann K.
        • Kurschner T.
        • Schulze A.
        • et al.
        T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.
        Gastroenterology. 2008; 134: 239-247
        • Deng Q.
        • Mancini-Bourgine M.
        • Zhang X.
        • Cumont M.C.
        • Zhu R.
        • Lone Y.C.
        • et al.
        Hepatitis B virus as a gene delivery vector activating foreign antigenic T cell response that abrogates viral expression in mouse models.
        Hepatology. 2009; 50: 1380-1391
        • Yang P.L.
        • Althage A.
        • Chung J.
        • Chisari F.V.
        Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection.
        Proc Natl Acad Sci USA. 2002; 99: 13825-13830
        • Mancini M.
        • Hadchouel M.
        • Tiollais P.
        • Pourcel C.
        • Michel M.L.
        Induction of anti-hepatitis B surface antigen (HBsAg) antibodies in HBsAg producing transgenic mice. a possible way of circumventing “nonresponse” to HBsAg.
        J Med Virol. 1993; 39: 67-74
        • Akbar S.M.
        • Kajino K.
        • Tanimoto K.
        • Kurose K.
        • Masumoto T.
        • Michitaka K.
        • et al.
        Placebo-controlled trial of vaccination with hepatitis B virus surface antigen in hepatitis B virus transgenic mice.
        J Hepatol. 1997; 26: 131-137
        • Malanchere-Bres E.
        • Payette P.J.
        • Mancini M.
        • Tiollais P.
        • Davis H.L.
        • Michel M.L.
        CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice.
        J Virol. 2001; 75: 6482-6491
        • Kakimi K.
        • Isogawa M.
        • Chung J.
        • Sette A.
        • Chisari F.V.
        Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections.
        J Virol. 2002; 76: 8609-8620
        • Loirat D.
        • Mancini-Bourgine M.
        • Abastado J.P.
        • Michel M.L.
        HBsAg/HLA-A2 transgenic mice. a model for T cell tolerance to hepatitis B surface antigen in chronic hepatitis B virus infection.
        Int Immunol. 2003; 15: 1125-1136
        • Schirmbeck R.
        • Dikopoulos N.
        • Kwissa M.
        • Leithauser F.
        • Lamberth K.
        • Buus S.
        • et al.
        Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross-reactive, natural HBsAg variants.
        Eur J Immunol. 2003; 33: 3342-3352
        • Lu M.
        • Hilken G.
        • Kruppenbacher J.
        • Kemper T.
        • Schirmbeck R.
        • Reimann J.
        • et al.
        Immunization of woodchucks with plasmids expressing Woodchuck Hepatitis Virus (WHV) core antigen and surface antigen suppresses WHV infection.
        J Virol. 1999; 73: 281-289
        • Rollier C.
        • Sunyach C.
        • Barraud L.
        • Madani N.
        • Jamard C.
        • Trepo C.
        • et al.
        Protective and therapeutic effect of DNA-based immunization against hepadnavirus large envelope protein.
        Gastroenterology. 1999; 116: 658-665
        • Pol S.
        • Nalpas B.
        • Driss F.
        • Michel M.L.
        • Tiollais P.
        • Denis J.
        • et al.
        Efficacy and limitations of a specific immunotherapy in chronic hepatitis B.
        J Hepatol. 2001; 34: 917-921
        • Jung M.C.
        • Gruner N.
        • Zachoval R.
        • Schraut W.
        • Gerlach T.
        • Diepolder H.
        • et al.
        Immunological monitoring during therapeutic vaccination as a prerequisite for the design of new effective therapies: induction of a vaccine-specific CD4+ T-cell proliferative response in chronic hepatitis B carriers.
        Vaccine. 2002; 20: 3598-3612
        • Wang X.Y.
        • Yao X.
        • Guo L.M.
        • Xu L.F.
        • Shen X.L.
        • Xu D.Z.
        • et al.
        Advances on antigen-antibody immunogenic complex therapeutic vaccine for viral hepatitis B.
        Zhonghua Gan Zang Bing Za Zhi. 2009; 17: 718-720
        • Depla E.
        • Van der Aa A.
        • Livingston B.D.
        • Crimi C.
        • Allosery K.
        • De Brabandere V.
        • et al.
        Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections.
        J Virol. 2008; 82: 435-450
        • Hill A.V.
        • Reyes-Sandoval A.
        • O’Hara G.
        • Ewer K.
        • Lawrie A.
        • Goodman A.
        • et al.
        Prime-boost vectored malaria vaccines: progress and prospects.
        Hum Vaccin. 2010; 6: 78-83