Clin J Gastroenterol (2009) 2:71–79 DOI 10.1007/s12328-009-0074-z

CLINICAL REVIEW

Immunopathogenesis of hepatitis B persistent infection: implications for immunotherapeutic strategies Yasuteru Kondo Æ Yoshiyuki Ueno Æ Tooru Shimosegawa

Received: 4 March 2009 / Accepted: 9 March 2009 / Published online: 27 March 2009 Ó Springer 2009

Abstract It has been shown that cellular immunity, especially by cytotoxic T lymphocytes (CTLs), NK cells and NK-T cells, plays a central role in the control of virus infection. In addition, CD4? T cells facilitate both CTL and B-cell responses. Hyporesponsiveness of HBV-specific T cells in peripheral blood has been shown in patients with chronic HBV infection. Interferon and nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir and tenofovir, are the currently available treatments. Unfortunately, the efficacy of nucleos(t)ide analogs is limited by viral reactivation by the emergence of escaped mutants in cases of prolonged treatment. Therefore, immunotherapy is one of the significant options to eradicate or control HBV replication without drugs. The aim of immunotherapies is to decrease the levels of viral replication and to eradicate infected hepatocytes. For this reason, new strategies for immunotherapies by vaccination target not only the induction or stimulation of CD4? and CD8? T cell responses, but also the induction of proinflammatory cytokines capable of controlling viral replication. We will review the immunopathogenesis of persistent HBV infection, especially focusing on the mechanisms of immune suppression. Then we will review the immunotherapy for HBV persistent infection. Keywords HBV
Immunotherapy
Vaccine
CTL
Tregs
NK
NK-T
DC
TLR

Y. Kondo
Y. Ueno (&)
T. Shimosegawa Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai 980-8574, Japan e-mail: [email protected]

Introduction Hepatitis B virus (HBV) is a basically noncytopathic DNA virus which causes chronic hepatitis and hepatocellular carcinoma as well as acute hepatitis and fulminant hepatitis [1]. HBV now affects more than 400 million people worldwide [2] and in approximately 5% of adults and 95% of neonates who become infected with HBV, persistent infection develops. It has been shown that cellular immunity, especially by cytotoxic T lymphocytes (CTLs), NK cells and NK-T cells, plays a central role in the control of virus infection [3, 4]. In addition, CD4? T cells facilitate both CTL and B-cell responses [5]. Hyporesponsiveness of HBV-specific T cells in peripheral blood has been shown in patients with chronic HBV infection [6]. There may be several mechanisms in this CTL hyporesponsiveness, including immune suppression induced by high viral and antigen load that is typically observed in patients with chronic HBV infection [7]. The role of viral load on antiviral T cell responses has been well characterized in animal models and human studies of viral infection, most of which show that sustained presence of viral antigens leads to the functional decline of virus-specific CD8 responses [8, 9]. On the other hand, the induction of tolerance caused by CD25?CD4?FOXP3? regulatory T cells and/or liver environment have been also proposed to suppress efficient immune responses [10–16]. Interferon-a and nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir and tenofovir, are the currently available treatments [17–21]. Unfortunately, interferon-a is effective in around 30% of cases and even less so in younger cases [17]. Nucleos(t)ide analogs inhibit HBV replication very efficiently and improve the liver histology. However, their efficacy is limited by viral reactivation by the emergence of escaped mutants in cases of prolonged treatment [18–21].

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Immunotherapy is one of the significant options to eradicate HBV. Understanding of the HBV immunopathogenesis is necessary for developing new strategies for HBV eradication. Here, we will review the immunopathogenesis of persistent HBV infection, especially focusing on the mechanisms of immune suppression. Then we will review the immunotherapy for HBV persistent infection. Early response of HBV infection (innate immune response) The initial phase of HBV replication is delayed by the consequence of HBV inhibition of the innate immune response. The innate immune response during the initial phases of viral infection is mainly characterized by the production of type 1 interferon (IFN)-a/b cytokines and the activation of natural killer (NK) cells. The expression of type 1 IFN can be induced by the presence of viral RNA or DNA [22, 23]. NK cells are activated by the recognition of stress-induced molecules and the modulation of major histocompatibility complex (MHC)-class 1 molecules on the surface of infected cells [24, 25]. In a model of acutely infected chimpanzees, there was clear evidence that NK/ NK-T cells could be responsible for the initial control of HBV replication [26]. Immediately after the exponential phase of HBV expansion, chimpanzees able to control the virus show a typical acute phase of disease with strong activation of INF-c, TNF-a [26] and many cellular genes related to a T helper type 1 (Th1) type cellular response (IFN-c, IP-10, etc.) [27, 28]. These initial immune responses to HBV are primarily sustained by NK/NK-T cells [27]. Recently, a group reported that the blockade of NKG2D on NKT cells prevents hepatitis and the acute immune response to hepatitis B virus in a mouse model [29]. These findings proved the importance of NKT cells in the pathogenesis of HBV liver injury. The risk of HBV infection becoming chronic after the acute infection depends on the age when contacting the HBV. For infants infected either at birth or within the first year of life, the risk is over 90% [30]. The detailed mechanism of persistent infection is unknown but may be related to the immunological immaturity of the infants. We could speculate that the immaturity of the innate immune response in addition to that of the adaptive immune response has an important role in the failure of HBV eradication since infants exposed to HBV usually do not have symptoms of liver damage. Recently, a group reported that the Toll-like receptor (TLR)-mediated immune response could be suppressed by hepatitis B protein [31]. The innate immune system detects so-called pathogenassociated molecular patterns via the TLR system, which is involved in the regulation of innate immune responses via

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pro-inflammatory signaling cascades. It was demonstrated that HBV-bearing supernatants, purified HBV virions, and recombinant HBsAg or HBeAg can suppress the innate immune response elicited by TLR stimulation of hepatocytes and nonparenchymal liver cells [31]. Adaptive immunity The adaptive immune response is comprised of various kinds of effector cells, all of which play important roles in the development of immunity to HBV. CD4? type-1 helper T cells (Th1) produce type 1 cytokines (IFN-c etc.) and are required for efficient development of cytotoxic CD8? T cells (CTLs) [5]. CD4? type-2 helper T cells produce type 2 cytokines (IL-4 etc.) and are required for B-cell antibody production [5]. CTLs can recognize viral antigens presented by HLA-class I molecules and clear HBV-infected hepatocytes through cytolytic and noncytolytic mechanisms [32, 33] (Fig. 1). There are many differences in the adaptive immunity of subjects with persistent or resolved HBV infection (Table 1). Cytotoxic T lymphocytes Patients who have spontaneously recovered from hepatitis B maintain virus specific CD8? T cell responses that are readily detectable in the peripheral blood for decades [34– 36]. In animal models, depletion of CD8? T cells prior to challenge with HBV leads to persistent infection. Therefore, these responses contribute to the control of HBV [37]. Patients with chronic hepatitis B display narrowly focused and weak HBV-specific T cell responses [6, 38]. The majority of these studies were carried out using peptides able to bind specifically to HLA-A2 molecules [39–41]. However, HBV-specific cytotoxic epitopes restricted by different HLA-class I molecules have also been identified [14, 42–44]. HBV specific CTLs isolated from peripheral blood of such patients have lost their ability to proliferate and to produce cytokines [14, 39]. CTL dysfunction has been attributed to high levels of persisting viral antigens [14, 39]. This loss of T cell function has been termed exhaustion. Frequent encounter with viral antigens has been proposed as one of the mechanisms of T cell exhaustion in HBV [7, 45]. Viral mutations also contribute to CTL impairment [39, 46]. However, viral mutations are more frequent in HCV than in HBV infection since HBV has more sequence constraints related to overlapping reading frames [47]. Signaling via the programmed death 1 (PD-1) molecules has been identified as a major inhibitory mechanism of virus-specific T cells. PD-1 is an inhibitory receptor of the CD28 costimulatory family and can be expressed by T cell, B cells, and myeloid cells [48–51]. Interaction of PD-L1 with PD1 on liver-infiltrating T cells

Clin J Gastroenterol (2009) 2:71–79 Fig. 1 Presumed scheme of immune responses during HBV infection

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Liver

Cytokine secretion

NK

DC

HBV antigen uptake

Maturation

Lymph node

CD8

Th1 cytokines

CD4

MHC-class I resticted TCR stimulation

MHC-class II resticted TCR stimulation

DC

Th2 cytokines

Inhibition by Tregs

B cell CD4+ CD25+ Treg

Liver

CD8+ CTL

Cytokine secretion, Fasmediated cell killing

results in inhibition of the antiviral effector function and apoptosis of T cells [52–54]. Functional modulation of virus-specific T cells in association with PD-1 expression was shown in a mouse model of HBV expression [52–54]. CD4? helper T lymphocyte A key factor that could influence the functional potential of CTL is the amount of functional CD4? helper T lymphocyte. CD4? helper T lymphocytes may help CTLs either by directly activating dendritic cells and CTLs via CD40-dependent co-stimulation or by indirectly supporting the CTL response by secreting cytokines such as IL-4, IL2 and IFN-c [55]. CTL exhaustion is amplified when CD4? helper T lymphocytes were unavailable in a mouse model of LCMV-induced hepatitis [56, 57]. High viral load and

Poly-and monoclonal B cell expansion

signaling via PD-1 could affect not only CTL exhaustion but also CD4? helper T lymphocyte exhaustion [58, 59]. The fact that lamivudine treatment can restore the HBV specific CD4? helper T cell response and that HIV patients with low CD4? cell counts have suppressed HBV-specific cellular immune responses might indirectly prove the effects of high viral load [60–62]. CD4?CD25? regulatory T cells Numerous experimental models have provided evidence that a population of specialized T cells is able to regulate the immune response. CD4?CD25? regulatory T cells (Tregs) constitutively express the forkhead transcription factor 3 (FOXP3), the IL-2 alpha chain (CD25), and the glucocorticoid-induced tumor necrosis factor receptor

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Table 1 Differences in the adaptive immunity of subjects with persistent or resolved HBV infection Target

Function

Mechanism of suppression

Toll-like receptor

Detection of so-called pathogen-associated molecular patterns

Direct effect of HBV protein (HBV virion, HBsAg, HBeAg)

NK/NK-T cells

Independent of epitope, cell killing, IFN-c secretion

Unknown

CD8? T cells (CTL)

HLA-class-I related cell killing and IFN-c secretion

High viral load, exhaustion, PD-1 related signaling, viral mutation

CD4? T cells (Th)

HLA-class-II related cytokine secretion

High viral load, exhaustion, PD-1 related signaling

Tregs

Suppression of immune response by cell to cell contact,

Increasing of the frequency of Tregs and function

Innate immune response

Adaptive immune response

Il-10, TGF-b secretion Dendritic cells

Antigen-presentation

The different steps of binding, uptake and subsequent replication of HBV could all compromise DC function

Activate naı¨ve T cells and stimulate B cells

Controversial

Induction of immunological tolerance

The immunological features of the liver might contribute to the induction of immunological tolerance

Other factors Liver environment

family-related gene (GITR) [63–65]. These cells have been shown to suppress immunological responses against self and foreign antigens. The detailed mechanisms that mediate the regulatory effect of CD4?CD25? cells are still controversial, with evidence supporting regulation through either suppressive cytokines or direct cell-to-cell contact [66–68]. CD4?CD25? Tregs might be derived from activated effector cells or from natural or induced Treg populations [63–65, 68]. The circulating CD4?CD25? Tregs involved in the HBV pathogenesis have been analyzed. Increased frequencies of circulating Tregs in subjects with persistent infection have been reported in some studies. Subjects with HBV persistent infection showed higher frequencies of CD25?CTLA4?CD4? T cells [11] and FOXP3 messenger RNA levels [12] in the blood than healthy subjects and subjects with resolved hepatitis B. The relevance of Tregs in persistent HBV infection was confirmed by a Treg depletion study [14]. The depletion of CD25? cells from peripheral blood samples of HBV persistent infection resulted in enhanced in vitro proliferation of and IFN-c production by lymphocytes [15]. On the other hand, reconstitution of the CD25? depleted cell population with Tregs results in a dose-dependent reduction of HBV specific IFN-g secreting cells [15]. It is possible that Tregs are excessively activated in vivo to suppress the expansion of the HBV-specific CTL and other favorable immune responses that might be able to control HBV.

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Dendritic cells Dendritic cells (DC) represent a specialized antigen presenting cell population necessary for the induction of the adaptive immune response. DC play an important role in antiviral immunity and have the unique capacity to activate naı¨ve T cells and stimulate B and natural killer cells [69]. Depending on the maturation status, represented by the expression level of costimulatory and human leukocyte antigen (HLA) molecules and the capacity to produce proinflammatory cytokines, DC can induce either immunity or tolerance. Some groups reported that the myeloid DC (mDC) of patients with chronic HBV were impaired in their capacity to mature [70]. Whether HBV directly interferes with the function of DC is still not clear [69]. The different steps of binding, uptake and subsequent replication of HBV could all compromise the function of DC. Contradictory results have been reported about the possible presence and active replication of HBV in in vitro generated mDC of patients with chronic HBV. Recently, a group reported that myeloid DC actively internalize HBsAg and that the presence of either purified HBsAg or intact viral particles during mDC maturation gives rise to mDC with a significantly reduced immunogenic phenotype and function as demonstrated by the reduced expression of costimulatory molecules and decreased T cell stimulatory capacity [71]. In contrast, no evidence was found of reduced allogeneic or HBc antigen-specific T cell stimulation by mDC

Clin J Gastroenterol (2009) 2:71–79 Table 2 New strategies for immunotherapies by vaccination target not only the induction or stimulation of CD4? and CD8? T cell responses

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Category

Explanation

Protein vaccine

Recombinant HBsAg, Recombinant HBsAg and anti-HBs immunoglobulins, Recombinant HBsAg with IL2, etc Most of these protein vaccines were administered intramuscularly and could induce Th1 cytokines

DNA vaccine

IM administration of plasmid DNA encoding HBV surface proteins DNA vaccination could induce both humoral and cellular responses DNA vaccine encoding multiple HBV proteins together with a genetically modified interleukin-12 gene, etc

T cell epitope vaccine

HLA-class-I restricted CTL epitope derived from the hepatitis B core and surface protein The efficacy is limited (The restoration of exhausted T cells is difficult)

Immunomodulating agents

Th1 related cytokines (IL2, IL12, etc.), TLR agonist is not currently available for HBV Clinical trials of TLR7 and nine agonist were already carried out in HCV patients

from subjects with HBV persistent infection [69]. We need to wait for more convincing results concerning the function of DC in HBV persistent infection [69, 70, 72–75]. Other factors The immunological features of the liver might contribute to the induction of immunological tolerance present in HBV persistent infection. Data produced in mice models have shown that CD8 T cell induction, expansion and survival are changed following activation by antigens present in liver [71, 76]. However, this hypothesis is becoming controversial as recent work in mice has shown rapid activation of naı¨ve or effector CD8 T cells in liver [76]. Immunotherapies Interferon and nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir and tenofovir are the currently available treatments. Unfortunately, interferon-a is effective around 30% of cases and even less so in younger cases [77]. The efficacy of nucleos(t)ide analogs is limited by viral reactivation by the emergence of escaped mutants in cases of prolonged treatment [78, 79]. Therefore, immunotherapy is one of the significant options to eradicate or control HBV replication. The aim of immunotherapies is to decrease the levels of viral replication and to eradicate infected hepatocytes [80, 81]. For this reason, new strategies for immunotherapies by vaccination target not only the induction or stimulation of CD4? and CD8? T cell responses, but also the induction of proinflammatory cytokines capable of controlling viral replication (Table 2).

Protein vaccines Many groups have described that various kinds of recombinant anti-hepatitis B vaccines could have a specific but transient effect on viral replication in hepatitis B surface antigen (HBsAg) positive chronic hepatitis B patients [82]. Most of these protein vaccines were administered intramuscularly and could induce Th1 cytokines [82–84]. However, little is known about the CTL response and histological changes [82–84]. In a remarkable study, a vaccine was administered orally, in saline, three times per week for five to six months. During treatment, the virus titers decreased significantly in 36% of the patients, with 26% displaying anti-HBe-Ab and most of them showing histological improvement in the liver. Moreover, this vaccine could induce CTL responses [85]. Another remarkable vaccine consisting of recombinant HBsAg and anti-HBs immunoglobulins could induce HBs-specific T cells efficiently since the formation of Ag–Ab immune complexes could be easily captured and taken up by antigen-presenting cells [86]. Treatments combining protein vaccine and immunomodulatory drugs such as IL2 have been also tested. However, the administration of low dose IL2 did not seem to increase the efficacy of the vaccine [87]. Recently, a combination therapy of lamivudine and protein vaccine was tried in patients with hepatitis B persistent infection. Three months after administration of the last vaccine the rate of HBe seroconversion was significantly higher in patients receiving combination therapy than in controls with lamivudine therapy alone [88]. Therefore, modifications such as by combinations with DC, Th1 cytokines, interferon and nucleos(t)ide analogue are required to increase the efficacy of protein vaccine [89].

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DNA vaccines DNA vaccination for hepatitis B was carried out by the IM administration of plasmid DNA encoding HBV surface proteins. DNA vaccination could induce both humoral and cellular responses. Several studies described that DNA vaccines activated HBV-specific Th1 responses in healthy or infected individuals [90–93]. In a remarkable study, a vaccination with naked DNA encoding the small and middle envelope protein was evaluated in 10 patients chronically infected with HBV who did not respond to existing treatment. This DNA vaccine could activate not only HBV-specific T cells but also NK cells [92]. However, the responses nonetheless remained transient in most cases. A DNA vaccine encoding multiple HBV proteins together with a genetically modified interleukins-12 gene as an adjuvant and lamivudine therapy was also evaluated in 12 non-treated chronic HBV carriers. Virological responses were observed in half of the patients [94]. Thus, the efficacy of DNA vaccines has been limited so far. Therefore, a combination with antiviral treatment might be important to restore the adaptive immunity, especially in T cells. T cell epitope vaccines Over ten years ago, a T cell epitope-based vaccine was developed using a lipopeptide consisting of covalently linked components: an HLA-A2 restricted CTL epitope (amino acid 18–27) derived from the hepatitis B core protein, a T helper epitope derived from tetanus toxin, and tow molecules of palmitic acid. This vaccine was shown to be safe and able to induce an HBV-core-specific CTL response. However, a limitation of this vaccine is that it is only available for HLA-A2-restricted patients [95]. Therefore, a vaccine was developed consisting of a DNA vector coding for a string of 30 HBV-derived CTL epitopes linked to 16 Th epitopes that were expected to be presented to T cells by various kinds of HLA molecules [96]. This vaccine was shown to be safe and well tolerated in all healthy volunteers. So far, the efficacy of these vaccines has been very limited, since the restoration of exhausted T cells is difficult. Immunomodulating agents When developing specific vaccines, a combination of immunomodulating agents will be needed. Some of the protein and DNA vaccines are already combined with Th1 cytokines (IL2, IL12, etc.) [87, 94]. However, little is known about the appropriate timing of the administration and the amount of cytokines. In addition to conventional cytokines, the agonists of toll like receptor might be a powerful tool to induce the immune response. Clinical trials of TLR9 agonist

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and TLR7 agonist have already been carried out in HCV patients [97, 98]. The agonists of some kinds of TLR might be useful for persistent HBV infected patients.

Concluding remarks In this review, we focused on the mechanism of immune suppression and the immune therapies for chronic hepatitis B patients. Understanding the immunopathogenesis of HBV is necessary for developing immunotherapies. The main factors for the failure of HBV-specific T cells (CTL and Th1) are exhaustion, PD-1 signaling and the excessive function of regulatory T cells. In recent studies, vaccines were used with lamivudine and could rapidly reduce the viral antigens. The reduction of HBV antigens could restore the exhausted T cells function partially. In addition, we and other groups have shown that the reduction of viral antigens could reduce the levels of regulatory T cells. The combination therapies [nucleos(t)ide analogs and HBV vaccines] are reasonable approaches to induce an efficient HBV immune response, but the restoration from T cell exhaustion might be insufficient in some HBV patients. In addition to these combination therapies, we have to consider additional immune-modulating agents including several kinds of cytokines, antibodies for the suppression of Tregs, and agonists of TLR, etc. Moreover, we need to consider the function of NK/NK-T cells and TLR that are important in the pathogenesis of HBV. Acknowledgment This work was supported in part from Health and Labour Sciences Research Grants for the Research on Measures for Intractable Diseases (from the Ministry of Health, Labour and Welfare of Japan).

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