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Clinical Science (2013) 124, 77–85 (Printed in Great Britain) doi: 10.1042/CS20120169
Hepatitis B virus: from immunobiology
to immunotherapy
Daniel GRIMM, Maximilian HEEG and Robert THIMME
Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany
Abstract
Owing to the major limitations of current antiviral therapies in HBV (hepatitis B virus) infection, there is a strong
need for novel therapeutic approaches to this major health burden. Stimulation of the host’s innate and adaptive
immune responses in a way that results in the resolution of viral infection is a promising approach. A better
understanding of the virus–host interaction in acute and chronic HBV infection revealed several possible novel
targets for antiviral immunotherapy. In the present review, we will discuss the current state of the art in HBV
immunology and illustrate how control of infection could be achieved by immunotherapeutic interventions.
Key words: hepatitis B virus (HBV), immunity, immunobiology, infection, liver disease.
INTRODUCTION
Infection with HBV (hepatitis B virus) is a major healthcare
problem, with up to 400 million people affected worldwide. The
infection accounts annually for approximately 1 million deaths
from cirrhosis, liver failure and HCC (hepatocellular carcinoma).
Despite the availability of potent antiviral drugs such as NUCs
(nucleoside/nucleotide analogues) (e.g. Entecavir or Tenofovir)
and the application of PEGylated IFN (interferon) α in the treatment of chronic HBV infection, sustained virological response in
terms of HBsAg (HBV surface antigen) loss and anti-HBs (HBV
surface antibody) seroconversion is rarely achieved. Moreover,
each of these therapeutical approaches has additional drawbacks.
For NUC therapy, the optimum duration is still undefined and
long-term therapy is needed. This costly therapy carries the risk
of viral resistance and drug toxicity. Treatment with PEGylated
IFNα has the advantage of a finite duration of treatment. However,
significant side effects and low tolerability must be considered
[1]. Therefore novel therapeutic approaches to this major health
burden are urgently needed. A promising strategy is the development of an immunotherapy for viral control. The potential benefit
of immunotherapy is supported by the facts that: (i) HBV infection is successfully controlled by natural immune responses in
approximately 90 % of the individuals infected as adults; and
(ii) resolution of chronic HBV infection can be achieved by
bone marrow transplantation from an immune donor [2,3]. In
the present review, we will recapitulate the virus–host interaction
in HBV infection and show how a comprehensive understanding
of immunobiology may result in promising immunotherapeutic
approaches.
INNATE IMMUNE RESPONSES AGAINST HBV
The innate immune system has evolved as the host’s first line
of defence against viral infections. By a timely recognition of
viral nucleic acids, viral proteins and tissue damage it has the
capacity to limit viral spread early during the course of infection.
The sensing of viral components for activation of innate immune
responses by PRRs (pattern recognition receptors) is a complex
process that is still not completely defined. Usually, the early
antiviral state consists of three major mechanisms, namely (i)
production of type I IFN, (ii) killing of infected cells via NK
(natural killer) cells and (iii) production of pro-inflammatory
cytokines and chemokines that contribute to the maturation and
recruitment of adaptive immune responses [4,5].
The role of the innate immune response in HBV infection is
still not very well defined. This can be attributed to the fact that the
recruitment of patients in the very early, asymptomatic phase of
acute HBV infection is very difficult. Moreover, the experimental
systems of HBV infection that can be used in analysing innate
immune responses have major drawbacks. Animal models (e.g.
mouse models, woodchucks or chimpanzees) are limited in terms
Abbreviations: cccDNA, covalently closed circular DNA; CTLA-4, cytotoxic T-lymphocyte antigen 4; HBV, hepatitis B virus; HBsAg, HBV surface antigen; HCV, hepatitis C virus; HSC,
hepatic stellate cell; IFN, interferon; IL, interleukin; LCMV, lymphocytic choriomeningitis virus; NK, natural killer; NUC, nucleoside/nucleotide analogue; PD-1, programmed cell death 1;
rcDNA, relaxed circular DNA; TCR, T-cell receptor; TGF-β, transforming growth factor-β; TLR, Toll-like receptor; Treg, regulatory T-cells; WHV, woodchuck hepatitis virus.
Correspondence: Professor Robert Thimme (email [email protected]).
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of ethical concerns, costs and difficulties with standardization [6–
9]. Despite recent advances in cell culture models, these models
suffer from a low infection efficacy and poor virus replication
levels [10,11].
HBV: a ‘stealth virus’?
In a prominent study from the chimpanzee model, HBV has been
described as a ‘stealth virus’ that does not directly activate the
host’s innate immune system in the liver [7]. Indeed, by analysing
the intrahepatic gene expression after infection with a monoclonal inoculum, the authors did not observe an induction of a
type I IFN response in the liver in the early infection phase. This
observation has been supported by studies in humans [12,13]. For
example, Dunn et al. [12] showed that circulating type I IFN was
barely detectable throughout the early course of infection when
analysing a cohort of 21 acute HBV patients. From the clinical
point of view, this is in line with the fact that acute HBV infection
usually does not induce ‘flu-like’ symptoms [14]. A possible explanation for the evasion of HBV from innate immunity may be
its replication strategy. After binding of the viral particle to the
hepatocyte, the nucleocapsid is released and transported to
the nucleus. In the nucleus, the partially double-stranded viral
rcDNA (relaxed circular DNA) is repaired and eventually
cccDNA (covalently closed circular DNA) is formed [15,16].
The cccDNA remains in the nucleus of infected cells, and capped
and polyadenylated viral mRNA is produced that resembles the
cellular transcripts. Eventually, the viral replicative genome is sequestered within viral capsids into the cytoplasm. Thus the virus
is almost invisible to the innate sensing machinery [14,17,18]. It
is a hallmark of HBV infection that, owing to the persistence of
cccDNA in the nucleus of infected hepatocytes, the infection can
scarcely be eradicated completely [1,19].
However, the interpretation that HBV completely evades the
innate immune recognition was challenged by various observations. (i) It could be shown that HBV replication in HepaRG cell
lines induced a type I IFN response. The physiological relevance
of this observation remains unclear, as an overexpression system
based on recombinant baculoviruses was used [20]. (ii) A study
in the woodchuck model showed that infection with high doses
of WHV (woodchuck hepatitis virus) has the capacity to induce
intrahepatic gene expression affiliated with innate and adaptive
immune responses [6]. For that study, the rather high dose of
WHV has to be considered, as the size of the viral inocula was discussed to be important for intracellular DNA sensing and outcome
of infection [4,9,21,22]. (iii) In uPA (urokinase-type plasminogen activator)/SCID (severe combined immunodeficiency) mice
harbouring human hepatocytes, baseline levels of human ISGs
(interferon-stimulated genes) were slightly increased in HBVinfected animals when compared with uninfected mice [23]. This
further supports the hypothesis that HBV does not completely
evade innate immune recognition. (iv) Finally, activation of innate
immunity might even be possible without substantial type I IFN
production. Hösel et al. [24] found that, upon infection of primary
human liver cells, IL (interleukin)-6 was produced. Mechanistically, IL-6 was proposed to contribute to early viral control,
limiting adaptive immune responses and preventing hepatocyte
death after HBV infection [24].
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Viral measures to counteract the host’s innate
immune responses
Besides evidence that HBV eludes recognition by the innate
immune system, several studies propose that viral proteins actively counteract the host’s innate immune response. Interference
between the HBV X protein and VISA (virus-induced signalling
adaptor) has been reported as a mechanism for the inhibition of
type I IFN induction [25–27]. Moreover, the HBV polymerase
was shown to inhibit IFNβ induction via interaction with the
transcriptional factor DDX3 (dead-box 3) [28,29]. A hallmark
of HBV infection is the production of a large amount of secretory proteins during the viral replication cycle, namely HBsAg
and HBeAg. A possible evolutionary explanation for this could
be the immunomodulatory effects of these proteins [4]. Indeed,
two studies described an interference of HBV secretory proteins
with TLR (Toll-like receptor)-mediated innate immune responses
[30,31].
Role of NK cells in HBV infection
Numerous studies have been performed in order to elucidate the
role of NK cells in HBV infection. NK cells are abundant in the
cellular infiltrate of the liver. They account for up to 30–40 %
of the resident intrahepatic lymphocytes [32]. A hallmark of NK
cells is that these cells can be activated in an antigen-independent
manner either (i) by a complex interaction between inhibitory
and activating receptors or (ii) via cytokines, for example IL-12,
IL-15 and IL-18 [33]. In the HBV transgenic mouse model it has
been shown that NK cells can inhibit HBV replication upon activation. Interestingly, the role of NK cells in HBV infection has
also been analysed in a cohort of HBV infected patients in the preclinical phase and in a follow-up until resolution of the infection
[12]. In this important study, the authors could show that NK cell
activation and effector functions were suppressed during peak
viraemia. This could be attributed to an increase in IL-10 early
in infection that occurred in parallel with the rapid increase in
HBV viral load [12,34]. In chronic HBV infection, NK cells upregulate the death ligand TRAIL (tumour-necrosis-factor-related
apoptosis-inducing ligand) and have even been described as hypercytolytic, consequently causing liver injury [35,36]. However,
NK cells have an impaired capacity to produce IFNγ in chronic
HBV infection [37]. Importantly, this functional defect could not
be reversed by antiviral therapy, whereas blockade of IL-10 with
or without blockade of TGF-β (transforming growth factor-β)
corrected the functional defect in vitro (Figure 1) [37].
Possible immunotherapeutic approaches
modulating the innate immune response
Overall, the stimulation of the innate immune system by TLRmediated activation has been discussed as a therapeutical approach in HBV infection (Figure 1) [4]. It could be shown in
HBV-transfected hepatoma cell lines and in HBV transgenic
mice that TLR activation can suppress HBV replication [38,39].
Moreover, the therapeutic efficacy of a TLR-7 agonist in woodchucks and chimpanzees has been reported recently [40,41]. The
stimulation of innate immune responses is particularly promising
in HBV infection, since the ability of the virus to counteract innate immune responses seems to be rather weak [42]. In addition, the activation of intrahepatic NK cells directly by cytokines
Hepatitis B virus: from immunobiology to immunotherapy
Figure 1
Possible targets for the modulation of innate immune responses in HBV infection
The use of TLR agonists for the stimulation of innate immune responses in HBV-infected hepatocytes is a very promising
immunotherapeutic approach. Furthermore, effects of antiviral drugs such as IFNα might be increased by a targeted
delivery to infected cells by vehicles functioning as TCR-like antibodies. Inversely, this targeted approach might help to
limit side effects. Apart from direct effects on infected hepatocytes, reconstitution of NK cell function by the blockade of
inhibitory cytokines such as IL-10 might be used for the modulation of the innate immune response to HBV.
(IL-12, IL-18 or IFNα) or indirectly via TLR activation as proposed by Tu et al. [43,44] for immunotherapy may be promising.
Finally, TCR (T-cell receptor)-like antibodies that specifically target HBV-infected cells have been proposed as a vehicle for drug
delivery (Figure 1) [45]. Using such vehicles, the targeted delivery of, for example, IFNα to infected cells could be beneficial
and should be evaluated.
ADAPTIVE IMMUNE RESPONSES AGAINST
HBV
The crucial role of virus-specific T-cells in the control of HBV
infection has been shown in important studies [46,47]. After an
initial quiescence of a few weeks after infection, HBV-specific
CD4 + and CD8 + T-cell responses are detectable. These cell
types are critical for liver disease and viral control [48,49].
Role of CD4 + T-cells
In patients with acute HBV infection who ultimately clear the
virus, the CD4 + T-cell response is multispecific and vigorous
(Table 1). In contrast, the CD4 + response is relatively weak in
patients with chronic HBV infection [18,50]. Interestingly, no
effect on viral clearance and liver disease was observed when
CD4 + T-cells were depleted at the peak of infection in the chimpanzee model. However, when CD4 + T-cells were depleted prior
to infection, persistent infection evolved [9]. This indicates that
CD4 + T-cells play a critical role in viral control without having
major direct antiviral effects, for example by supporting efficient
virus-specific CD8 + T-cell responses [9,18,51]. Reconstitution
of CD4 + T-cell failure might be critical for a successful immunotherapy. Thus further understanding of the mechanisms behind
CD4 + T-cell failure in chronic HBV is crucial [52]. The role
of classical FoxP3 + CD4 + Treg (regulatory T-cells) in chronic
HBV is elusive. Treg can suppress HBV-specific T-cell functions
[53,54]. However, this also holds true in patients that successfully control the virus [55]. In addition, an inverse correlation
between Treg frequency and ALT (alanine transaminase) levels
could be shown. This observation gave rise to the hypothesis that
Treg mainly have anti-inflammatory effects without having major
effects on HBV persistence [4].
Role of CD8 + T-cells
Several studies have clearly shown a correlation between vigorous and multi-specific CD8 + T-cell responses and HBV clearance (Table 1). The central role of CD8 + T-cells has been further
confirmed by depletion studies [47]. In acute self-limiting infection, HBV-DNA falls rapidly after the peak of viral replication.
The mechanisms of CD8 + T-cell-mediated viral control are still
debated [52]. It has been shown that non-cytolytic mechanisms,
depending on cytokines such as IFNγ or TNFα contribute to
viral control, for example in chimpanzees, HBV transgenic mice
and in a cell culture model [56,57]. However, the contribution of
cytolytic effects must not be underestimated. This is supported by
the fact that viral clearance seems to occur in the presence of liver
disease in chimpanzees. Cell death might play an important role
in the eradication of cccDNA from the liver [19,58]. For an immunotherapeutic approach to HBV infection, where a large proportion of liver cells is infected, a sequential restoration of noncytolytic and cytolytic mechanisms would probably be beneficial
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Table 1
NK cell and T-cell responses at various stages of HBV and HCV infection
HBV
HCV
Acute/resolving
Chronic
Chronic
NK cells
Suppressed during peak viraemia
Impaired IFNγ production, hypercytolytic
Polarization towards cytotoxicity,
impaired IFNγ production
CD4 + T-cells
Multispecific and vigorous
Weak, monospecific, dysfunctional
Weak or absent, dysfunctional
CD8 + T-cells
Multispecific and vigorous
Weak, monospecific dysfunctional,
Multispecific, but exhausted
to avoid major immunopathology with a potentially fatal outcome
[52,59].
Furthermore, an important role for CD8 + T-cells in the development of liver cirrhosis has been shown. Indeed, it was
demonstrated that CD8 + T-cells contribute to hepatic fibrosis by
activating human HSCs (hepatic stellate cells) in vivo and in vitro
[60]. Thus the interaction between HSCs and T-cells might represent an interesting and promising target for regulating the course
of hepatic fibrosis in HBV infection.
Causes of CD8 + T-cell dysfunction and ways of
reconstitution
HBV persistence is associated with impaired CD8 + T-cell functions, irrespective of the patient’s age at infection [4]. Probably
owing to the long-term exposure of T-cells to viral antigens, HBVspecific T-cells are deleted or functionally inactivated during the
course of infection [61–63]. Specifically, CD8 + T-cells targeting
major HBV epitopes have been shown to be hardly detectable if
viral load exceeds 107 copies/ml [62]. As viral load decreases in
the first months of antiviral therapy, an increase in HBV-specific
T-cell responses has been described [64,65]. However, these responses were rather transient [66].
Many different mechanisms have been suggested for T-cell
failure [3]. An example for a T-cell intrinsic defect is the upregulation of the pro-apoptotic member of the Bcl-2 superfamily Bim in HBV-specific CD8 + T-cells obtained from patients
with chronic HBV infection [3,67,68]. Downstream blockade of
Bim restored multispecific functional responses directly ex vivo
[3,67]. It has been postulated that this pro-apoptotic phenotype
is fostered by tolerogenic activation by antigen-presenting cells
in the liver [3]. Importantly, it could be shown that premature
elimination of T-cells after antigen presentation by T-cells is also
mediated by Bim [3,69,70]. Therefore specific blockade of this
pro-apoptotic pathway could be a promising target in future HBV
immunotherapy [3].
The balance between co-inhibitory and co-stimulatory signals
during intrahepatic antigen presentation is critical for maintaining
functional CD8 + T-cells. For example, an excess in co-inhibitory
signals can potentially induce Bim-mediated apoptosis of HBVspecific CD8 + T-cells [61,71–74]. Amongst others, co-inhibition
via the PD-1 (programmed cell death 1) pathway has been demonstrated to contribute to HBV-specific CD8 + T-cell failure in
chronic HBV infection. Indeed, PD-1 expression is increased
on HBV-specific CD8 + T-cells [61,71]. Moreover, expression of
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PD-1 ligands is increased on intrahepatic cells in chronic HBV
infection [75]. Thus the blockade of the PD-1 pathway can serve
as a potential target for immunotherapy in chronic hepatitis. Indeed, it was shown that treatment of mice with antibodies to
PD-1 ligand 1 restored CD8 + T-cell functions and reduced virus
replication in the LCMV (lymphocytic choriomeningitis virus)
mouse model [76]. The potential relevance of blocking this inhibitory pathway in HBV was further demonstrated in the HBV
mouse model [77]. Here, treatment of HBV-transgenic mice with
blocking antibodies for PD-1 ligand 1 resulted in an increase in
the number of IFNγ -producing CD8 + T-cells in the liver and in a
delay in suppression of IFNγ -producing CD8 + T-cells (Figure 2).
This approach is particularly promising, as PD-1 blockade
seems to be well tolerated in cancer immunotherapy trials [78].
However, as HBV-specific CD8 + T-cells express multiple inhibitory receptors, a carefully tailored approach might be necessary
for a successful HBV immunotherapy. A more detailed insight
into synergy and redundancy of the multiple pathways is essential
[3,52]. Combined modulation of several co-inhibitory and costimulatory pathways including, for example, 2B4 (CD244) and
CTLA-4 (cytotoxic T-lymphocyte antigen 4) might be beneficial
(Figure 2) [3,4,79,80]. This supposition results from the following observations. (i) In chronic HBV infection, 2B4 and PD-1 are
highly co-expressed on virus-specific CD8 + T-cells. Therefore
blocking 2B4 or its ligand CD48 may contribute in the restoration
of CD8 + T-cell function and might as well be independent of the
PD-1 pathway [80]. However, recent observations in HCV (hepatitis C virus)-specific CD8 + T-cells showed that cross-linking
of 2B4 can lead to inhibition and activation of virus-specific
CD8 + T-cells, depending on expression levels of 2B4 and the
intracellular adaptor molecule SAP [SLAM (signalling lymphocyte activation molecule)-associated protein]. Consequently, a
better understanding of the effects of 2B4 blockade in HBV infection is essential [81]. (ii) CTLA-4 is expressed by HBV-specific
CD8 + T-cells with high levels of Bim. Blocking CTLA-4 resulted in an increased expansion of IFNγ -producing HBV-specific
CD8 + T-cells. Apparently, this pathway is not redundant with
PD-1 [79].
For a functional T-cell response, additive factors such as the
liver microenvironment have to be considered [3]. This includes,
among others, nutritional factors, the intrahepatic cytokine milieu
and cellular constraints. At peak viraemia, HBV-specific CD8 +
T-cells are activated. However, it has been shown that these cells
are exhausted and that the proliferation of these cells is limited
[82]. This effect has been correlated with an increase in IL-10
Hepatitis B virus: from immunobiology to immunotherapy
Figure 2
Mechanisms underlying CD8 + T-cell dysfunction that might be addressed in antiviral immunotherapy
Restoration of CD8 + T-cell dysfunction by interference with co-inhibitory signals is a promising approach in antiviral
immunotherapy. CD8 + T-cell mediated viral control might be achieved by (i) blockade of co-inhibitory receptors such as
PD-1 or 2B4 and (ii) blocking inhibitory cytokines such as TGF-β or IL-10. Reprinted from Journal of Hepatology, 52, Maini,
M.K. and Schurich, A., The molecular basis of the failed immune response in chronic HBV: therapeutic implications,
c (2010), with permission from Elsevier.
616–619, production (Figure 2) [82]. Moreover, this phenomenon has also
been discussed as being related to an arginase release by hepatocytes, consequently leading to a lack of arginine in the inflamed
liver. This was shown to interfere with CD8 + T-cell receptor signalling indirectly [83,84]. Consequently, this leads to a decrease
in IL-2 production and in a defective T-cell proliferation. Therefore supplementation of IL-2 has been proposed as a therapeutic
approach. However, data from the LCMV mouse model show
that timing of IL-2 administration and differentiation status of
T-cells are critical parameters in designing IL-2 therapies [85].
In addition, TGF-β has been discussed for HBV immunotherapy [86]. This cytokine can be produced by HBV-specific CD8 +
T-cells and seems to contribute to the auto-immunosuppression
of these cells [3,87]. Data from the LCMV mouse model raise
hope that selective blockade of TGF-β might contribute to CD8 +
T-cell restoration (Figure 2) [88]. The recent observation that
IL-21 correlates with viral control defined another important cytokine that might have implications in the therapeutic augmentation of immune responses to HBV [89].
Further immunotherapeutic approaches based on
adaptive immune responses
On the basis of the results discussed above, reconstitution of
CD4 + and CD8 + T-cell functions are very promising for HBV
immunotherapy. Focusing on the reconstitution of virus-specific
CD8 + T-cells, we have discussed the possible role of (i) the
downstream blockade of Bim, (ii) the modulation of co-inhibitory
and co-stimulatory receptors, and (iii) the modulation of inhibitory and stimulatory cytokines (Figure 3). Besides the restoration
of T-cell defects, other experimental approaches have been proposed for immunotherapy in HBV infection. Several efforts have
been made to develop a therapeutic vaccination in HBV infection
(Figure 3). This strategy aims to eliminate viral infection by stimulation the host’s immune response. The state of the art of this
antiviral approach has been reviewed elsewhere [90]. Gehring
et al. [91] described the engineering of functional HBV-specific
T-cells by T-cell receptor gene transfer that could potentially be
used in immunotherapy (Figure 3). Another promising strategy
for viral control is an immunological approach that does not
rely on the restoration of HBV-specific CD8 + T-cells. In the
HBV mouse model, a recombinant HBV carrying influenza epitopes was generated. These epitopes were selectively expressed
in HBV-infected cells. Thus HBV-infected hepatocytes were
targeted by functional influenza-specific memory T-cells. This
system was shown to be effective in viral control without causing
major liver injury in mice [92].
Moreover, a TCR-like antibody has been described that specifically targets HBV-infected cells (Figure 3) [45]. Thus a potential application of this TCR-like antibody could be the delivery
of antiviral drugs directly to virus-infected cells [45].
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D. Grimm, M. Heeg and R. Thimme
Figure 3
Overview of therapeutic interventions that might help to achieve viral control in HBV infection
Each single approach might contribute to viral control. However, a combination of interventions is most promising. Rational
combinations of immunomodulatory and antiviral therapeutics need to be tested. pegIFN-α, PEGylated IFN-α.
CONCLUSIONS
Comprehensive studies of innate and adaptive immune responses
have highlighted several potential targets for immunotherapeutic
approaches in HBV infection. The first pre-clinical and clinical
trials are already ongoing in order to evaluate effects and side effects of novel therapies and have shown promising results [93,94].
Overall, the field of HBV immunotherapy shows nicely how the
results of basic research can be used in order to improve patient
care.
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Received 3 April 2012/20 June 2012; accepted 5 July 2012
Published on the Internet 12 September 2012, doi: 10.1042/CS20120169
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