INVITED REVIEW ARTICLE
Nagoya J. Med. Sci. 74. 217 ~ 232, 2012

IMMUNOREGULATION OF HEPATITIS B VIRUS
INFECTION–RATIONALE AND CLINICAL APPLICATION–
TETSUYA ISHIKAWA
Division of Pathophysiological Laboratory Sciences, Department of Radiological and Medical Laboratory
Sciences, Nagoya University Graduate School of Medicine

ABSTRACTHepatitis B virus (HBV) is susceptible to the cellular immune responses, especially to the signal ofinterferon (IFN)-g. The action of IFN-g is pleiotropic, and causes downregulation of HBV in protein,RNA, and possibly DNA levels. Therefore, therapeutic vaccination to induce cellular immune responses to
HBV is a promising approach for controlling chronic HBV infection. A number of clinical trials with thisapproach have been conducted to date, however, they have not been as successful as initially expected.
T-cell exhaustion induced by the excessive HBV antigens caused by persistent infection is thought tobe one of the main causes of poor responses to therapeutic vaccination. In this review, the mechanisms
behind immunoregulation of HBV replication and immunodysfunction during chronic HBV infection aresummarized, and novel approaches to improve the efficacy of therapeutic vaccination, from basic research
to clinical trials, are introduced.
Key Words: chronic hepatitis B, interferon-g, adaptive immune response, therapeutic vaccination,
T-cell exhaustion

INTRODUCTION
Hepatitis B virus (HBV) is a type of the hepadnavirus which is spread by contact with infectedblood and body fluids, and causes acute and chronic necroinflammatory liver diseases.1-3) Since
HBV is a noncytopathic virus, inflammation in the liver is mediated by host immune responses
to the HBV-infected hepatocytes. HBV infection in immunocompetent adults results in a selflimited, transient liver disease, and subsequent viral clearance is achieved in more than 95%
of adults, whereas more than 90% of neonates exposed to HBV at birth become persistently
infected.1-3) Even in persistently infected individuals, hepatitis B e antigen (HBeAg)/antibody (Ab)
seroconversion (SC) with marked reduction of HBV replication, associated with biochemical andhistologic regression of liver inflammation, occurs in the majority of patients in their natural
course. If a reduction of HBV replication does not occur, the infected individuals are under agreater risk of developing cirrhosis (LC), liver failure, and hepatocellular carcinoma (HCC).1-3)
Received: June 4, 2012; accepted: June 5, 2012
Corresponding author: Tetsuya Ishikawa MD, PhD
Division of Pathophysiological Laboratory Sciences, Department of Radiological and Medical Laboratory
Sciences, Nagoya University Graduate School of Medicine,
1-1-20 Daikominami, Higashi-ku, Nagoya 461-8673, Japan.
Phone/Fax: +81-52-719-1561, E-mail: ishikawa@met.nagoya-u.ac.jp
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Chronic liver diseases associated with chronic HBV infection are serious public healthproblems worldwide. It is estimated that 370 million people are chronically infected with HBV,
and that up to 1.2 million people die every year due to the complications of HBV-related
chronic liver diseases, such as LC and HCC.4-6) HBV infection has been the most significant
factor associated with the development of liver cancer, which is one of the most malignant
cancers; the second most frequent cause of cancer death in men, and the sixth leading causeof cancer death in women.4-6) HBV infection accounts for about 60% of all HCC occurrencesin developing countries and about 23% of cancer occurrences in developed countries; the corresponding percentages for hepatitis C virus (HCV) infection are 33% in developing countries
and 20% in developed countries.6) As of 2008, a total of 177 countries (91%) had introduced
the HBV vaccine for their national infant immunization schedules.7,8) This efficient vaccination
method decreases the incidence of HBV infection; however, chronic HBV infection remains aserious problem in countries with a higher prevalence of chronic HBV infection, such as thosein Asia and sub-Saharan Africa, where the prevalence exceeds 8% of their populations.6,9) Even
in Japan, where the HBV carrier rate is about 1% and is gradually decreasing, thanks to thesuccess of a national program for immunoprophylaxis of perinatal HBV transmission, the number
of HBV-related HCC is not decreasing10,11). It is obvious that there is an urgent need for theeffective treatment control and termination of HBV infection.
It is considered that permanent and profound suppression of viral replication is beneficial
for preventing complications of chronic hepatitis B (CHB), as persistent HBV viremia has been
proven to be the most important predictor of progression to LC, hepatic failure, and developmentof HCC.12) Therefore, the initial goal of the treatment is to suppress active viral replication andto subsequently restrain the activity of hepatitis.
Currently, CHB patients are treated mainly with antiviral reagents, i.e., nucleoside or nucleotide
analogues (NAs). NAs target the reverse transcriptase of HBV and are potent inhibitors of viral
replication. NA treatment usually results in a rapid decline of serum HBV DNA levels, andlong-term therapy results in reduction in hepatic ﬁbrosis, hepatic decompensation, and liver-related
mortality.13-15) Induction of NAs opened a new era in the treatment of CHB, providing a safe,
effective and well-tolerated therapeutic option. However, at the same time, there is a drawbackof NA treatment, which is not negligible. Since the complete eradication of HBV infection israrely achieved with this treatment, NA must be administered for an extremely long period oftime. And long-term treatment is occasionally associated with an increased risk for developmentof viral resistance to the drugs, which eventually results in recurrence and progression of thedisease.16-18)
Interferon (IFN)-a/b is another option for CHB treatment. It has a direct antiviral effect,
but is thought to act mainly by augmenting host immune response specific to the HBV-related
antigens.15,19,20) Because of its immunomodulatory mechanism of action, IFN-a/b requires only alimited treatment period, and brings out a durable response even after the discontinuation of thetreatment if the endpoint response is favorable.21) In addition, recently approved pegylated IFN(Peg-IFN)-a shows a slightly increased efficacy compared to standard IFN-a/b.15,22-25) However,
the response rate by IFN therapy is not necessarily high, despite the use of Peg-IFN-a (sustained
response rate: 10 to 30%),15,22-25) and IFN-based therapy is associated with a wide spectrum ofadverse events, including flu-like symptoms, decrease of white blood cells and platelets, anddepression. In addition, IFN is contraindicated in patients with decompensated LC.
As seen in the case of IFN treatment, the key factor for obtaining permanent control over HBV
is to induce effective virus-specific immune responses which are strong enough and effectively
regulate viral replication. In other words, therapeutic vaccination is a promising candidate forthe treatment of chronic HBV infection.

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Currently, the only available vaccine for HBV is the hepatitis B surface antigen (HBsAg)
vaccine, which is usually used for the prophylaxis of HBV infection, as mentioned above. The
HBsAg vaccine elicits a level of anti-hepatitis B surface antigen antibody (HBsAb) high enoughto protect against the infection of newly intruding HBV in more than 90% of vaccinees, if properly administered.26) However, therapeutic use of this HBsAg vaccine has not brought satisfactory
results for controlling viral replication at present.27.28) Therefore, a new vaccine or novel strategy
is definitely needed to achieve sustained control of chronic HBV infection.
In this review, the mechanisms of immune responses that control HBV replication, theirapplication to therapy, and current and future approaches for developing a vaccination strategy
to achieve sustained control or eradication of HBV, are described.
Immune responses that control HBV replication
In general, the immune responses that terminate viral infection work in several steps. During
the early phases of acute viral infection, natural killer (NK) cells and natural killer T-(NKT)
cells are the first lines of defense, and the activation of these cells helps to reduce the viral load
through the secretion cytokines, such as IFN-g.29-32) In the liver, the frequencies of NK and NKT
cells are higher than in other organs,33) and these cells mediating innate immunity are thought
to play certain roles in the pathogenesis of HBV infection. Indeed, during the early phases ofacute HBV infection, the activation of NK cells helps to reduce the viral load through the actionof IFN-g.34) However, this activation is rapidly suppressed by interleukin (IL)-10 at the peak ofviremia, indicating that the roles of NK cells on HBV regulation are rather limited.35) In fact,
in chimpanzees infected by HBV, the innate immune cells, such as NK and NKT cells, do notsignificantly contribute to the pathogenesis of viral hepatitis during the symptomatic phase of
acute or chronic HBV infection.36) Therefore, it is assumed that a crucial role in the pathogenesis
of HBV infection and the control of HBV replication is played by the adaptive immune response
primed after the initial response by the innate immunity.
HBV transgenic mouse hepatitis model for analysis of liver pathogenesis
The roles of specific immune responses on the pathogenesis of HBV infection and viralclearance have been well examined in the hepatitis model with HBV transgenic mouse (Fig.
1). The adoptive transfer of HBV-specific cytotoxic T-lymphocyte (CTL) lines and clones into
immunologically tolerant HBV transgenic mice produced liver disease with hepatocyte necrosis
and inflammation that is histologically very similar to acute viral hepatitis in human.1,3, 37-43)
The initial step in the disease process after adoptive transfer is apoptosis caused by the
direct attack of CTLs against viral antigen-positive hepatocytes. Apoptosis occurs immediately
after the transfer of CTLs to mice and is necessary for further steps in the disease process.
The direct CTL-target cell interaction results in the appearance of widely scattered apoptotic
hepatocytes.37-39,42) After the initial step of direct CTL killing, many host-derived inflammatory
cells, such as polymorphocnuclear neutrophils (PMNs), antigen-nonspecific T-cells, NK cells,
and macrophages, are recruited around apoptotic hepatocytes and virus-specific CTLs, and form
necroinflammatory foci, which resembles the histology observed in acute hepatitis in men.37-39,42)
This process, from hepatocyte apoptosis to necroinflammatory foci, is shown to be associatedwith the action of activated platelets,43) cytokines such as IFN-g and the tumor necrosis factor(TNF)-a,37,40,41) chemokines such as CXCL9 and CXCL10, 44) and matrix metalloproteinases(MMPs) produced by PMNs (Fig. 2).45)
Mechanisms of HBV elimination by adaptive immune response
In the same HBV transgenic mouse model, the mechanisms of viral elimination by adaptive

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immune response is examined in detail. As mentioned above, HBV-specific CTLs directly kill
HBV-expressing hepatocytes, and the recruited host-derived inflammatory cells also cause damage

Fig. 1 HBV transgenic mice hepatitis model. Nontransgenic (B10.D2; H-2d) mice were immunized with either
recombinant vaccinia virus or HBsAg protein. Spleens were harvested and stimulated in vitro with
irradiated HBsAg-expressing P815 cells. Primed spleen cells were restimulated and expanded in vitro,and cloned by limiting dilution method. HBsAg-specific CTL clones or cell lines were intravenously
injected to HBV transgenic mice (1.3.32; inbred B10.D2, H-2d). Liver inflammation and viral clearance
were observed in HBV transgenic mice.

Fig. 2 Mechanisms of CTL-induced liver disease and viral clearance. HBV-specific CTLs kill antigen-expressing
hepatocytes via Fas lingad- and perforin-mediated pathways, and produce antiviral cytokines, such as
IFN-g, that inhibit HBV replication noncytopathically in a greater number of adjacent cells. IFN-g activates
hepatpcytes to produce chemokines that recruit antigen-nonspecific polymorphonuclear cells (PMNs) andantigen-nonspecific lymphomononuclear cells (e.g., NK cells, T-cells, and macrophages) into the liver.
Production of matrix metalloproteinases (MMPs) by PMNs also promotes migration of antigen-nonspecificlymphomononuclear cells. These antigen-nonspecific inflammatory cells amplify liver disease initiated by
CTLs. The figure is modified from that of ref #3.

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to infected hepatocytes, thus reducing the viral load to some extent (Fig. 2). However, more
importantly, IFN-g produced by activated CTLs following antigen recognition is responsible formost of the antiviral potential of the CTL-mediated immunity.40,41,47) The action of IFN-g on
HBV elimination is thought to be pleiotropic. It is indicated that IFN-g hinders the assembly of
pregenomic HBV RNA-containing nucleocapsid protein in a proteasome- and kinase-dependent
manner.48-50) Also, IFN-g causes destabilization of HBV RNAs in the nucleus by a La-dependent
mechanism. Briefly, HBV RNAs are stabilized in the nucleus by the binding of the full lengthof La, which is known as an autoantigen of Sjögren’s syndrome. However, IFN-g produced by
activated CTLs induces proteolytic cleavage of La, and the subsequent binding of cleaved La to
HBV RNAs results in the destabilization of HBV RNAs, i.e., facilitating the destruction of HBVRNAs by nuclear RNases.51-55) IFN-g also effectively downregulates replication intermediates of
HBV DNA in a nitric oxide (NO)-dependent manner, while the effect of IFN-g on covalently
closed circular DNA (cccDNA) in the nucleus is not yet clarified (Fig. 3).36,56,57) Thus, with all

Fig. 3 HBV life cycle and CTL-mediated viral clearance. The process of HBV entry into hepatocyte is poorly
understood, however, it is thought to be a receptor-mediated process. Once the virion enters the cytoplasm,
it is uncoated; the uncovered nucleocapsid is transported to the nucleus. Following nucleocapsid disassembly, the second strand of the open circular viral genome is completed, and the ends of each strand are
ligated. This process results in production of a covalently closed circular DNA (cccDNA), which is thetranscriptional template of the virus. Transcription by viral polymerase leads to production of viral RNAs
which are transported from the nucleus to the cytoplasm. Transcripts are translated into corresponding
proteins, such as envelope proteins, nucleocapsid proteins, HBeAg, polymerase protein, and X protein.
The envelope proteins traverse the ER membrane as integral membrane proteins. HBcAg and polymerase
proteins assemble around pregenomic RNA (pRNA) to form HBV RNA-containing nucleocapsid, within
which RNA is reverse transcribed to produce the first single-strand viral DNA (ssDNA). ssDNA serves
as the template for second-strand DNA synthesis, producing nucleocapsid containing a partially doublestranded, relaxed circular DNA molecule (rcDNA). At the ER membrane, it interacts with the envelope
proteins that trigger an internal budding reaction. This reaction results in the formation of virion which istransported out of the cell. HBV-specific CTLs recognize and cause death to the HBV-infected cells via
Fas lingad- and perforin/granzyme-induced pathways. Activated CTLs produce cytokines, such as IFN-g,
which causes profound, pleiotropic downregulation of the virus noncytopathically in adjacent infected
cells.

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these mechanisms, CTLs and IFN-g produced by CTLs bring about a very profound downregulation of HBV, most of which is accomplished without destroying hepatocytes.
Helper T-cells (Ths) also have the potential to eliminate HBV, since they are important
producers of IFN-g. In the HBV transgenic mouse model, it was indicated that HBV-specific Ths
also cause HBV downregulation in an IFN-g-dependent manner, but less efficiently, comparedto CTLs.38) On the other hand, Ths are also thought to contribute to controlling HBV infection
by assisting in the induction and maintenance of HBV-specific CTLs by regulating immune
response.38) Indeed, strong HBV-specific Th responses are usually associated with significant CTL
responses in humans and chimpanzees that resolve HBV infection.1,59)
Considering the mechanisms of HBV elimination by adaptive immune responses, it is obviousthat therapeutic vaccination that induces HBV-specific CTLs and Ths has great potential as a
strategy to terminate chronic HBV infection.
Alteration of immune response during chronic HBV infection
Despite the presence of immunological mechanisms of HBV elimination, the virus is rarely
eradicated when the infection becomes chronic. The key features of chronicity of HBV infection
are the impairment of immune responses related to the cellular composition of the liver, and thepresence of excessive viral antigens caused by persistent infection.
The liver is composed of parenchymal hepatocytes and non-parenchymal cells, including
biliary epithelial cells (BECs), hepatic stellate cells (HSCs), sinusoidal endothelial cells (LSECs),
and Kupffer cells (KCs). Importantly, most of these cells do not express enough costimulatory
molecules to induce functional activation of antigen-specific T-cells. Therefore, HBV antigen
presentation by these cells tends to lead T-cells toward tolerance rather than activation.60-62)
Unlike the cell populations mentioned above, dendritic cells (DCs) professional antigenpresenting cells (APCs) do express costimulatory molecules, and play an important role in
the induction of functional T-cells. However, in patients with CHB, decreases in the numberof circulating dentritic cells (DCs) and functional impairment of both myeloid (mDCs) andplasmacytoid DCs (pDCs) have been reported, and they may account for the dysfunction of theadaptive immunity.63-65)
Chronic exposure to excessive antigens leads T-cells to progressive exhaustion, when they lose
the ability to differentiate into memory cells and the effector function, e.g., proliferative capacity, cytotoxicity, and cytokine production (IFN-g, TNF, etc.).66-68) The exhausted T-cells express
inhibitory receptors on their surface, such as programmed death-1 (PD-1), and the interaction
between PD-1 and its ligand, PD-1 ligand 1 (PD-L1) are thought to be the major inhibitory
receptor pathways involved in T-cell exhaustion.66-68) Indeed, PD-1 is shown to be overexpressedon HBV-specific T-cells, which produce only a small amount of IFN-g.67,69) As T-cell exhaustion
progresses along with prolonged exposure to high doses of antigens, it may finally result in theclonal deletion of HBV-specific T-cell populations. In addition, regulatory Foxp3+CD4+ T-cells(Tregs) are known to affect immune responses during chronic infections, and are thought toaffect the exhaustion of antigen-specific T-cells.67) However, the role of Tregs in chronic HBV
infection is not clearly understood.70-73)
Even though our understanding of the role and alteration of immune responses during
chronic HBV infection has been greatly improved, many questions remain to be elucidated. It
is important to continue attempting to answer these questions in order to develop new strategiesof therapeutic vaccination.
Prophylactic HBsAg vaccines for therapeutic vaccination
The first series of therapeutic vaccine trials were conducted with commercially available

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prophylactic HBsAg vaccines.27,74-77) An initial pilot study demonstrated that HBsAg-vaccinationinduced cell-proliferated responses and IFN-g production specific for HBsAg, and reduction ofserum HBV DNA levels in some patients (7 out of 27 patients were positive for proliferative
response to envelope protein).27) Further studies also proved that HBsAg vaccination induced
HBsAg-specific T-ell responses, a decrease of HBV DNA levels, and a significant increase in
HBeAg/antibody (Ab) seroconversion (SC); however, most of these effects were temporal.76,77)
In addition, only a few patients showed HBsAg clearance and development of antibody against
HBsAg in these studies.76,77) Since the ultimate goal of CHB treatment is the clearance of
HBsAg and the induction of HBsAb, which can neutralize viral particles, the use of HBsAg fortherapeutic vaccination seems to be a reasonable choice. However, the results of these studies
clearly indicate that the classical prophylactic vaccines do not have enough antiviral potential,
and that development of novel vaccines or a strategy with greater efficacy is necessary.
Studies also showed that HBsAg vaccines are more effective when used in patients with low
viral loads, suggesting the possibility that the presence of excessive viral antigens lessens theefficacy of vaccination. Therefore, combining antiviral drugs that can suppress viral replication
and reduce the viral antigen load simultaneously through the vaccination is thought to be oneof the better therapeutic approaches.
Combining antiviral drugs to vaccination
Lamivudine (LMV) is the first NA used for the treatment of HBV infection.78,79) LMV itself
does not have a direct immunomodulatory effect, however, LMV treatment is shown to restore
the responses of HBV-specific T-cells during the first few months of the treatment period, andother NAs may have the same effect. This means that reducing the HBV antigen load by antiviraltherapy itself may increase the responsiveness of HBV-specific T-cells,80) probably by restoring
the function of exhausted T-cells. Since the restoration of T-cell responses is weak, transient,and does not lead to an increase of sustained viral response, such as HBeAg/Ab SC, 81) there
have been a number of attempts to combine NA administration to vaccination to gain continuous
activation of HBV-specific T-cell responses (Fig. 4).
Combining LMV to vaccination is the most widely applied method of treatment, and isfound to be well tolerated and safe, without serious adverse effects. This combination therapy isreported to give a significantly higher and earlier rate of viral suppression than LMV or vaccine
monotherapy, and increases the rate of HBeAg/Ab SC to some extent.82-84)
We also conducted a controlled trial composed of 53 patients with CHB, 33 with HBeAg+,
and 20 with HBeAg–, in which participants randomly received either LMV monotherapy or the
combination therapy of LMV and HBsAg vaccine. In the HBeAg– group, LMV treatment byitself was fairly effective, and no difference was seen with regard to HBV DNA levels, the rate
and period of complicating viral breakthrough (BT), or breakthrough hepatitis (BTH) between
the two groups. In the HBeAg+ group, the decrease of serum HBV DNA was faster and therate of HBV DNA negativation was higher in the combination therapy group, while the ratio of
HBeAg/Ab SC did not differ between the two groups. Also, in HBeAg+ patients, the rates ofdeveloping BT and BTH were significantly lower in the combination therapy group than in themonotherapy group. Furthermore, there was a tendency for the time period before developing
BT to be longer in the combination therapy group than in the monotherapy group. However,most of the patients in both groups developed post-treatment relapse after the cessation of LMV
in both groups.85)
Type I IFN is used as an antiviral drug in the treatment of HBV infection, but has thepotential to enhance HBV-specific immunity, as mentioned. In general, it enhances both T- andB-cell response by upregulating the expression of human leukocyte antigen (HLA) class I on the224
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Fig. 4 Balance between immune response and viral load in chronic HBV infection. In patients with perinatally acquired HBV infection, immunotolerant phase is the first phase of infection, when patients are
asymptomatic without significant immune responses and with higher viral load. The second phase is theimmunoactive phase, when patients have liver inflammation with slight increase of immune responses
and slight decrease of viral load. If spontaneous HBeAg/Ab seroconversion (SC) does not occur, liver
damage progresses from chronic hepatitis to cirrhosis. Nucleoside or nucleotide analogue (NA) treatment
can reduce viral load and is thought to increase immune responses probably by restoring the function of
exhausted T-cells. Since increase of immune responses is partial and transient, therapeutic vaccination toincrease immune responses is recognized as a promising measure to achieve sustained control of HBVreplication.

surface of APCs. It thereby promotes CTL responses by augmenting and maintaining the production of IgG from plasma cells.19,20) Indeed, in haemodialysis patients whose immune responses
were impaired, combining IFN-a to a prophylactic HBsAg vaccine was reported to improve theefficiency of the vaccination.86) There are a few reports describing the efficacy of the combination
therapy of type I IFN and HBsAg vaccine for the treatment of CHB. In these reports, combination
therapy was reported to accelerate the reduction of HBV DNA and to improve the HBeAg/AbSC rate compared to IFN monotherapy, but without significant differences.87,88)
In the studies of therapeutic vaccination combined with either NA or IFN, there is a difficultywhen generalizing the obtained results, since treatment protocols, backgrounds of the subjects,
and the criteria for defining virological responders vary so much. In addition, only a few reports
have analyzed cellular or humoral immune responses specific to HBV, and no obvious association
was found between HBsAg-specific T-cell responses and any clinical or virological parameters,such as HBeAg/Ab SC and HBsAg clearance, in most of the studies. The protocol of treatment
should be deliberately reconsidered, and the immunological analysis should be carefully planned
for further improvement of its efficacy.
Recently, an in vitro study demonstrated that the 50-end triphosphate hepatitis B virus X gene
(HBx)-short interfering RNAs (siRNAs) exerted significantly stronger inhibitory effects on HBVreplication, with a retinoic acid-inducible gene-I (RIG-I) and in a type I IFN-dependent manner.
Moreover, its efficacy was confirmed in HBV carrying mice.89) While there is the unsolved
problem of how to deliver siRNAs as a drug, they may be candidates for antiviral drugs thatcan be combined with vaccination as well as NAs.
Use of adjuvants
Adjuvants have been used for decades to improve the immune response to vaccine antigens.

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The use of adjuvants with vaccine is aimed at enhancing, accelerating, and prolonging the specificimmune response towards the desired response to vaccine antigens.90) Therefore, adjuvants are
suitable for the purpose of therapeutic vaccination for HBV infection.
The major means by which adjuvants exert themselves are through the upregulation of
antigen/adjuvant uptake to the antigen presentation cells (APCs), or the potentiation of immune
responses, both quantitatively and qualitatively. Alum, which is used for prophylactic HBsAg
vaccine, oil emulsion, and immune stimulating complexes (ISCOMs) (including Quillaia saponins,or liposomes) are used for the former purpose, and monophosphoryl lipid A (MPL) of Toll-likereceptor (TLR) 4 agonist, CpG DNA of TLR9 agonist, poly I:C of TLR3 agonist, Montanide-ISA
51, Montanide-ISA 720, MF59, or QS-21 of saponin derivatives, are used for the latter purpose.91)
Most of the adjuvants listed above have been tested for their ability to enhance the immune
responses to HBV antigens, and have been proven effective. Clinical trials for the application of
the vaccines which include some of these adjuvants, mainly for prophylaxis of HBV infection, arein progress or have been completed, such as “HEPLISAV,” comprised of the HBsAg and TLR9
agonist.92) Therapeutic use of the vaccines containing these adjuvants for the treatment of CHB
is expected to be effective, however, we must wait for the results of appropriate clinical trials.
There have been attempts to use cytokines, such as IL-2 and IL-7, as adjuvants. IL-2 is astrong Th1-inducing cytokine, and is expected to increase the efficacy of therapeutic vaccination by augmenting Th1 responses that help the induction and maintenance of antigen-specific
CTLs. Several trials, including one for HBV infection, have evaluated the efficacy of IL-2 as an
adjuvant of therapeutic vaccine.93) However, the use of IL-2 for treatment should be conductedcarefully because there is a potential concern that IL-2 administration may increase Tregs, whichconstitutively express the IL-2 receptor. IL-7 is essential for the homeostatic proliferation of T
cells in vivo, and may increase the pool of naïve T-cells potentially stimulated by vaccination.94)
A clinical trial combining antiviral treatment with HBsAg vaccine and multiple IL-7 injections
is currently underway.
Blocking the PD-1/PD-L1 inhibitory signals on exhausted T-cells by anti-PD-L1 antibodies
may be an option for enhancing the efficacy of vaccination. Indeed, it has been suggested that
restoration of intrahepatic HBV-specific T-cell responses in CHB patients was possible by blocking
PD-1/PD-L1 signals in vitro.69,95)
In the transgenic mouse model, an anti-CD40 agonistic antibody was reported to inhibit HBVreplication noncytopathically by a process associated with the recruitment of dendritic cells,
macrophages, T-cells, and NK cells into the liver, and the induction of inflammatory cytokines,
such as IFN-g.96) The antibody itself shows an antiviral potential by activating APCs nonspecifically, however, it may also be a candidate of an adjuvant for vaccination, since stimulation of
CD40 on DCs and B-cells has been proven to augment cell-mediated and humoral immunity.97)
We have recently demonstrated that alpha-galactosylceramide (a-GalCer), a ligand for NKT
cells, significantly enhanced the induction and proliferation of HBsAg-specific CTLs by HBsAg
vaccination in the mouse model. Even in HBV transgenic mice which are deeply tolerant to HBVantigens, co-administration of HBsAg and a-GalCer successfully induced HBsAg-specific CTLs.98)
Although this approach could not clear HBsAg from transgenic mice despite the induction of
HBsAg-specific CTLs, we believe that it is one of the promising approaches to augment theefficacy of vaccination.
Other forms of vaccine candidates
Several types of vaccines with different compositions have been proposed with the aim ofincreasing vaccination efficacy.
There have been a number of attempts to use peptide-based vaccines targeting T-cell epitopes

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for the treatment of viral infections as well as cancers, since they are easily produced inlarge amounts and can be safely administered to patients. A lipopeptide-based vaccine named“Theradigm-HBV,” the HLA-A2.1-restricted, dominant CTL epitope, HBV core Ag (HBcAg)
aa18–27, linked to the universal Th epitope tetanus toxoid (TT) has been tested for its ability to
induce CTL responses in healthy individuals and CHB patients. “Theradigm-HBV” was capableof inducing HBV-specific CTL responses in a dose-dependent fashion even in CHB patients;
however, the clinical effects, such as HBV DNA decrease, were not satisfactory. 99,100) Whilethe efficacy of peptide-based vaccine may be further improved with the use of an appropriate
adjuvant, the design of the vaccine construct should be carefully considered. Since the problem
of HLA restriction is inevitable in peptide-based vaccine, an HLA allele targeted to the bindingof epitope peptide should be selected in order to cover a large population of a relevant country
or region. In addition, the emergence of vaccine escape variants should be considered, especiallyin the case of a viral vaccine. We have previously shown that the CTL response to individual
viral epitopes can be markedly polyclonal and multispecific, and that mutational inactivation of asingle site of the epitope will not usually lead to viral escape.101) Even though a viral mutation
which can evade the CTL attack may emerge, simultaneous use of plural epitope peptides may
reduce the risk of disabling vaccine efficacy.
HBsAg-HBsAb immune complexes (IC) with alum as the adjuvant have been developed,
targeting DCs102). In vitro study has demonstrated that IS stimulated DCs to secrete a large
amounts of IL-12, a key cytokine of CD4+ T-cell differentiation into type 1 T helper (Th1)cells that can promote cell-mediated immunity.103) In the phase II trial, a significant virologicaleffect, such as a higher HBeAg/Ab SC rate compared to the control group, was observed in the
IC-treated group.102) However, the immune responses that could explain this therapeutic effect,
were not analyzed. It makes the evaluation of the efficacy of this vaccine rather difficult, aswith other vaccine trials.
It is known that during acute hepatitis, the HBcAg and the polymerase are specific targetsof the immune response as well as envelope antigens.1,104) Therefore, the use of a combinationof recombinant HBsAg and HBcAg, known as ‘‘NASVAC,’’ is a promising therapeutic vaccine
candidate for treating CHB patients. These antigens form virus-like particles, and they work
together in the development of cellular and humoral immune responses. In addition, this novel
vaccine could be administered in a non-invasive way, i.e., intranasally.105,106) Clinical trials areunderway, and the results are awaited.
DNA-based vaccines have been tested for both prophylactic and therapeutic purposes, andencouraging results have been reported in the animal models. The advantage of a DNA vaccine
is that it can induce both humoral and cellular immune responses. The phase I trial administering a DNA vaccine which encodes HBV envelope proteins in CHB patients showed favorable
results.107) DNA vaccination led to the induction of IFN-g-secreting T-cells and CTLs specificto envelope antigens, and to an increase in an NK cell subset known to produce abundantcytokines.108) The clinical trials with DNA vaccination are increasing, and most of them haveshown promising results. However, these trials should be carefully conducted, since there areconcerns about potential side effects, such as possible integration of plasmid DNA into the hostgenome, that result in mutations.
Few studies have reported the use of autologous DCs pulsed with HBV antigens for therapeutic
purposes. One of them is a pilot study with a small number of CHB patients that evaluatedthe safety of HBsAg-pulsed autologous DCs. The administration of DCs was safe with noexacerbation of liver damage; however, the number of patients was too small to evaluate theefficacy of vaccination.109) Another recent trial was conducted using autologous DCs pulsed with
HLA-A2-restricted peptides derived from HBcAg (aa18–27), with re-injection of middle envelope

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Fig. 5 Summary of new strategies for treatment of chronic HBV infection. Strategies for CHB immunotherapy
consist of novel vaccines, adjuvants, and combination of these to antiviral drugs. Details are describedin the text.

protein in CHB patients.108) The treatment was more effective in the patients with HBeAg– or witha low viral load, and some of them had cleared HBsAg. However, in that study, the immune
response-mediated mechanisms accounting for the observed serologic and virological responses
were not reported, raising questions about the specificity of this DC-mediated therapy.110)

CONCLUSION
Therapeutic vaccination is a reasonable approach for the treatment of chronic HBV infection,
considering the natural course of HBV infection and the susceptibility of HBV to the signal of
IFN-g. And there have been a number of attempts at therapeutic vaccination, including thoseusing conventional prophylactic vaccine, those combining antiviral drugs with vaccine, those using
vaccine with novel adjuvant, or those using newly-developed vaccines as introduced in this review(Fig. 5). The efficacies of current therapies are still lower than initially expected. However, theefficacy may be improved thanks to the progress made in understanding the underlying mechanism of immunodysfunction in HBV carriers, and the development of new therapeutic tools for
immunostimulation. The efforts to improve the efficacy of vaccine therapy should be continued
in order to establish an ideal therapeutic vaccination for the treatment of CHB. While most ofthese therapeutic vaccines have been safely used without serious complications, it should be kept
in mind that we should be always careful about possible complications, such as exacerbation ofhepatitis due to the excessive enhancement of immune responses.

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