Best Practice & Research Clinical Gastroenterology

Vol. 22, No. 6, pp. 1049–1061, 2008

doi:10.1016/j.bpg.2008.11.015
available online at http://www.sciencedirect.com

Immunology of hepatitis B and hepatitis C

virus infections

Andre Boonstra * PhD

Project leader HCV Immunology

Andrea M. Woltman PhD

Project leader HBV Immunology

Harry L.A. Janssen MD, PhD

Professor of Hepatology
Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center Rotterdam,
Rotterdam, The Netherlands

Hepatitis B (HBV) and hepatitis C (HCV) viruses are the two major causes of chronic liver
inflammation worldwide. Despite distinct virologic features, both viruses are preferentially
hepatotropic, not directly cytopathic, and elicit liver diseases that share several aspects of their
natural history. HBV and HCV infections also share some important features of the adaptive
antiviral immune response. We describe the innate immune response in the early phase follow-
ing infection, and how these early events may influence the development of the adaptive immune
response in these two important viral infections. The mechanisms by which high levels of viral
antigens, liver immunological features, the presence of regulatory T cells and impaired dendritic
cell functions may maintain the HBV- and HCV-specific immunological failure, characteristic of
chronic hepatitis B and C patients, are also evaluated.

Key words: hepatitis virus; HBV; HCV; immunology.

* Corresponding author. Department of Gastroenterology and Hepatology, Erasmus MC, University Medical
Center Rotterdam, ’s Gravendijkwal 230, Room L-455, 3015 CE Rotterdam, The Netherlands.
E-mail address: p.a.boonstra@erasmusmc.nl (A. Boonstra).
1521-6918/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved.
1050 A. Boonstra et al

IMMUNE RESPONSE TO PATHOGENS

Infection of a host with viruses results in triggering of diverse immune responses in

which the host attempts to prevent and eradicate the virus with minimal damage to
itself. The type of immune response determines whether the virus is eradicated, or
whether chronic infection sets in. However, also many viruses have evolved ways to
evade eradication by the immune system, thus allowing their own survival. Viral infec-
tion is a multiphase process that includes virus entry, genome replication, transcription
and translation of viral products, and the assembly and secretion of virions. Upon viral
infection, the immune system needs to sense the presence of the virus first, and then
rapidly deliver an appropriate response to eliminate the virus as quickly and efficiently
as possible. This requires the precise coordination of several simultaneous and
sequential events in which the infected cells and the immune system each play a critical
role.1,2
In response to viral encounter, infected cells may be triggered by the virus to
produce antiviral cytokines, including type I interferons, that inhibit viral repli-
cation. In addition, these cytokines may activate neighbouring resident immune
cells, but also have the potential to recruit immune cells into the infected tissue
and to activate those cells to acquire effector functions. Activation and attrac-
tion of innate immune cells, including neutrophils, dendritic cells (DC), macro-
phages, natural killer (NK) cells, and perhaps NKT cells constitute a first line of
defence (Figure 1).3,4
For most viruses, the combined action of early non-specific innate immune re-
sponses, and pathogen-specific adaptive immune response, mediated by T cells and
B cells, are required to clear the virus. Antibodies produced by B cells can be ex-
tremely effective in neutralising molecules of the pathogen, leading to eradication. In
addition, cytokines produced by CD4þ T cells as well as cytotoxic effector activity
by CD8þ T cells are also highly effective in clearing virus infected cells. These potent
effector functions of T cells can either directly lyse infected cells or activate other im-
mune cells, such as macrophages in order to kill the pathogen, while others can shut
off the immune system once the pathogen is eradicated. Tight regulation of immune
responses is fundamental since excessive inflammation can lead to extensive tissue
damage of the host.5,6

Figure 1. The complex interactions of multiple leukocytes may lead to elimination of viral infection.
 Immunology of viral hepatitis 1051

IMMUNE RESPONSE TO HBV AND HCV

Infections with HBV and HCV are the most common causes of chronic liver inflamma-
tion worldwide. Although both viruses primarily infect hepatocytes and can establish
chronic infection in the liver, they are virologically distinct and display numerous clin-
ical differences. HBV is a member of the Hepadnaviridae family, and its genome is
a relaxed circular DNA. HCV, on the other hand, is a member of the Flaviviridae family
and is single-stranded RNA virus.
HBV is transmitted by perinatal, percutaneous, and sexual exposure.7 The risk of
developing chronic HBV infection after acute exposure is influenced by age, the
route of infection and the dose of inoculum, and ranges from 90% in neonates to
less than 10% in adults.8–11 Thus, most adults infected with HBV resolve the infec-
tion spontaneously, resulting in lifelong protective immunity. In contrast to HBV,
60–80% of adults infected with HCV develop chronic hepatitis.12,13 These strikingly
different infection outcomes indicate important differences in the immune responses
elicited during HBV and HCV infections. However, the exact underlying mechanisms
to explain the difference in outcome of infection between HBV and HCV are still
largely unknown. The slow progress in this area is caused by the fact that both vi-
ruses have a narrow host specificity with chimpanzees being the only animal that can
be infected experimentally. In this chapter, we intend to discuss mechanisms used by
the immune system to eradicate HBV and HCV, as well as mechanisms that result in
their persistence.

THE EARLY INNATE IMMUNE RESPONSE IN HBV

AND HCV INFECTIONS

The type I interferon response

Immediately following viral infection, the immune system is alerted and evokes
a number of mechanisms that are aimed at eradicating the viral attack. The immune
system senses the presence of the virus by means of receptors that specifically recog-
nise pathogen-derived products, such as double-stranded or single-stranded RNA or
specific DNA motifs (reviewed in Ref. [14]). As a consequence of recognition of viral
components the initial response to viral infection is the rapid release of type I inter-
ferons, IFN-a and IFN-b, which is observed for most viruses studied. Type I IFN
have direct antiviral effects on infected cells by inducing of the expression of multiple
IFN-stimulated genes.15 A number of these genes, including protein kinase R (PKR),
Mx proteins, ISG-15, RnaseL/2,5-OAS and RNA helicases, have been well-character-
ised, and via different mechanisms all have potent antiviral activity.16 Upon stimulation
with IFN-a, expression of hundreds of different IFN-stimulated genes is induced,
suggesting that the host has evolved multiple redundant pathways to make evasion
of specific IFN-induced antiviral responses more difficult. In addition, type I IFN are
immunomodulatory, and are thought to prime multiple immune cells to efficiently
respond to the attack of the host by pathogen.
Indications of the role of type I IFN during viral hepatitis infection are mostly based
on studies in chimpanzees, since this is the only animal that can be infected with HBV
and HCV. Following infection with HCV, the transcription of IFN-a response genes
increases in the liver of chimpanzees as early as 2 days after infection.17 These genes
include OAS1, Mx, ISG-15 and STAT1, which are all involved in the antiviral response
1052 A. Boonstra et al

by inhibiting viral replication. The increase in IFN-a related genes coincides with an
increase in HCV RNA levels, suggesting that the increase in viral load is the trigger
for the induction of antiviral response genes. Indeed, it has been demonstrated that
single-stranded RNA is a potent trigger of IFN-a production.18,19 Furthermore,
recently a specific polyuridine motif of the HCV genome was identified that binds
to a sensor molecule present in the cytoplasm of cells, resulting in the induction of
IFN and IFN-response genes.20 This IFN-response occurs in all patients infected
with HCV irrespective of whether the final outcome is a self-limiting resolving infec-
tion, or whether a chronic HCV infection develops.21–24 Although HCV is sensitive to
IFN as shown by numerous in vitro studies,25 HCV replication is not controlled during
the early stages after infection, and in fact the serum HCV RNA levels increase expo-
nentionally in the first weeks after infection. This suggests that HCV has developed
evasion mechanisms to withstand the potent effect of type I IFN. Indeed, in recent
years a number of mechanisms have been identified by which HCV proteins attenuate
the induction or the activity of the antiviral IFN-response, by degradation or inhibition
of crucial molecules such as Cardif, and TRIF.26–29 It is unlikely that HCV is able to
completely block this response, since the products of various IFN-induced genes
are detected. However, reduced efficiency of the IFN-response due to the evolution
of evasion mechanisms by HCV may allow HCV to withstand complete eradication
during the initial stages of the infection. 4–6 weeks after infection, the HCV RNA
levels remain relatively stable, suggesting that a balance has been achieved between
viral replication and control of the replication by immune pressure. It is thought
that this plateau is reached before the adaptive immune system, mediated by
HCV-specific T and B cells, becomes activated. This control is believed to be primarily
exerted by the type I IFN mediated response.
In sharp contrast to HCV, chimpanzees infected with HBV showed a complete lack
in the induction of type I IFN and in IFN-response genes during the early stages of in-
fection, which is an unusual feature when compared to HCV and other viruses.15 Also,
the increase of serum HBV DNA levels occurs much later than as observed for HCV
RNA. In fact, while serum HCV RNA levels increase immediately following infection,
HBV DNA levels start to rise weeks after infection and reach peak levels much later.
During the first weeks when HBV DNA levels are low, hepatocyte apoptosis is not
observed and alanine transferase (ALT) levels are low.17 The lack of induction of
the IFN-system by HBV has also been attributed to the fact that replication of HBV
takes place within nucleocapsid particles, and its genome and replication intermediates
are therefore invisible to the cellular recognition sensors.30

NK cells

Immediately after the exponential phase of HBV expansion, chimpanzees able to control
the virus show a typical acute phase of disease with IFN-g and TNF-a production, caus-
ing rapid inhibition of HBV replication, recruitment of virus-specific and non-specific
cells and triggering of the adaptive immune system.31,32 This initial control of HBV rep-
lication by the host seems to be primarily sustained by NK cells. NK cells recognise tar-
gets expressing abnormal or reduced levels of major histocompatibility complex (MHC)
class I molecules, pathogen-derived proteins and/or stress-induced cellular proteins. NK
cells have been named for their ability to mediate spontaneous cytotoxicity against
transformed and infected cells.33 The mechanism through which NK cells mediate anti-
viral cytotoxicity appears to be organ dependent.34 NK cell cytotoxicity via the perforin/
granzyme pathway is considered to be of less relevance to the liver environment since
 Immunology of viral hepatitis 1053

hepatocytes are relatively resistant to lysis through this pathway.35,36 Receptor-mediated

cell death through ligand-receptor pairs belonging to the TNF superfamily, such as TNF-
related apoptosis-inducing ligand (TRAIL) expressed on infiltrating NK cells interacting
with TRAIL death-inducing receptors on hepatocytes, is likely to play a more important
role in liver damage. However, in recent years it has become evident that their functions
reach far beyond this traditional role. Notably, NK cells can assist in T cell polarisation,
DC maturation, and innate immune control of viral infection by their ability to secrete
immunomodulatory and antiviral cytokines.37 Animals that develop a chronic infection
lack the acute phase response and fail to prime an adequate adaptive response. Thus,
activation of innate components of the immune system seems to represent a key ele-
ment in the control of the initial HBV burst.
A similar response has been reported in patients studied during the incubation
phase of hepatitis B. An increased number of circulating NK cells precedes the peak
of HBV replication, and is followed, 2–4 weeks later, by the appearance of HBV-specific
CD8þ T cells when viral replication has already dropped.38 While virtually all patients
that experience acute hepatitis B resolve infection, development of chronicity is often
associated with absent or mild symptoms of acute hepatitis. In line with these clinical
observations, neonatally infected woodchucks that develop chronicity lack the high
IFN-g and TNF-a production observed in animals that resolve the infection,39–41
and fail to develop an efficient antiviral specific immune response.
The role of NK cells during the early acute response to HCV is considered to be
limited based on three observations. Firstly, control of viraemia occurs in the absence
of an increase in ALT levels, secondly, the HCV envelope protein E2 has been shown to
inhibit NK cell function directly by interacting with CD81, a surface molecule ex-
pressed on different cell lineages including NK cells, and thirdly, expression levels of
IFN regulatory factor-1, a transcription factor essential for NK cell activity in vivo,42
does not change during acute HCV infection. The direct inhibitory role of HCV on
NK cell function may contribute to the absence of liver inflammation, despite the early
signs of HCV replication and the early production of IFN-a, which has been shown to
promote NK cell recruitment into the liver.43

DEVELOPMENT OF PERSISTENT HBV AND HCV INFECTIONS

Self-limiting resolving HBV and HCV infections are observed in about 80% and 10–20%
of infected individuals, respectively. This spontaneous clearance of the virus is most
likely the consequence of the action of an efficient innate immune response, and a rel-
atively strong, sustained, and multi-specific T cell mediated immune response, which
eliminates the virus.44–55 Following clearance of the virus in the acute phase, long-
term memory T cell responses are detected in both HBV and HCV-infected individ-
uals.56–58 Although HBV-specific memory responses are considered to provide lifelong
protection to reinfection, HCV-specific memory responses decline and are eventually
HCV-specific antibodies are lost after 10–20 years.56
There are indications that patients infected with HBV and HCV who eventually
develop chronic infection have a strong T cell response during the initial phase of
infection. However, this response is not sustained, and during chronic infections
HBV and HCV-specific CD4þ and CD8þ responses are difficult to detect in blood
and liver, and are functionally impaired.53–55,59–61 A number of factors have been pro-
posed to explain the weak T cell responses, including effects on NK cells and DC, re-
sulting in incomplete differentiation of effector and memory T cell populations,
1054 A. Boonstra et al

exhaustion of the T cells resulting from persistent high viral loads, and suppression by
regulatory T cells (reviewed in Refs. [62–67]). In addition, also unique immunological
features of the liver environment have been suggested to further contribute to the lim-
ited virus-specific T cell responses as observed in chronic HBV and HCV patients, as
will be discussed below. However, still much needs to be learnt on the mechanisms
resulting in persistence of HBV and HCV. For instance, at present it is not clear
why some patients are able to resolve the infections whereas others do not.
With respect to immunological processes to explain chronic HBV and HCV infec-
tions, parallels can be found in other persistent viral diseases, such as HIV and LCMV
infection in mice. Similar to chronic viral hepatitis infections, also chronic infections
with HIV and LCMV are characterised by the presence of high viral titres for extended
periods, and weak T cell responses to the virus.68 During these persistent infections,
a gradual loss of effector functions is described (CD4þ help, cytotoxicity) accompa-
nied by the reduced production of important cytokines, such as IL-2, IFN-g and
TNF productions following antigenic stimulation.69 Importantly, there are indications
that the functional impairment of T cells is reversible, as shown after therapy following
HBV infection in patients70 or LCMV infection in mice.71,72 Also recently, it was
reported that blockade of negative regulators of T cell responses, such as the immu-
nosuppressive cytokine IL-10 and the surface molecule PD-1, can cure mice persis-
tently infected with LCMV in vivo, and augment virus-specific T cell responses to
HCV and HBV in vitro. These studies clearly demonstrated that immunological control
mechanisms are exploited during persistent viral infections thereby hampering efficient
immune control, and allowing the virus to co-exist with the host.

Regulatory T cells

Regulation of the HBV and HCV-specific T cells also occurs via the activity of regula-
tory CD4þ T cells. Regulatory T cells may limit viral clearance in chronic infections by
inhibiting the proliferation and effector function of T cells and other immune cells.5,6
We and others showed that patients with a chronic HBV infection have increased per-
centages of CD4þ CD25þ FoxP3þ regulatory T cells in their peripheral blood com-
pared to healthy controls and individuals who have resolved their HBV infection.73–76
Regulatory T cells isolated from peripheral blood of these patients are capable of in-
hibiting the HBV-specific CD4þ and CD8þ T cell response in vitro.73,74,77 Further-
more, we showed that reduction of the viral load by potent antiviral therapy
resulted in a decrease of the frequency of peripheral blood regulatory T cells.78 Similar
findings have been demonstrated in patients chronically infected with HCV.79–83 An im-
portant role for regulatory T cells has been described in a number of infections, and
have been shown crucial to limit immune-mediated pathology, suggesting their impor-
tance to limit liver damage during chronic viral hepatitis. However, it is difficult, if not
impossible, to determine if regulatory T cells also play a role in the establishment or
the maintenance of chronic HBV and HCV infections in patients.
Dendritic cells

Other immune cells that are implicated in persistence of viral hepatitis are dendritic
cells (DC). These cells are central players in an immune response, since they take up
pathogen products, and present these products to T cells, which results in activation
of these T cells. DC are also potent cytokine producing cells, and in this way they not
only determine the quantity of the T cell response, but also the type of T cells that
 Immunology of viral hepatitis 1055

develop.84,85 In this, the production of IL-12 by conventional DC, and IFN-a by plas-
macytoid DC are important to induce the development of IFN-g producing Th1
cells, which are important during antiviral responses.86 Although conflicting studies
have been reported, numerous studies observed reduced numbers of DC in periph-
eral blood of patients chronically infected with hepatitis viruses (reviewed in Ref.
[87]). Whether these reduced numbers may (partially) explain the weak anti-
HBV/HCV immune response as observed in chronic HBV and HCV patients is doubt-
ful, since active depletion of DC or DC precursors by HBV or HCV has not been
convincingly shown. Therefore, the reduced circulating DC number highly likely re-
flects accumulation of DC at the site of infection, as suggested in both HBV and
HCV-infected patients.88–92 More important for the regulation of the host immune
response is probably the modulation of DC function by both HBV and HCV. A crucial
factor disturbing the antigen presenting functions of conventional DC seems to be
the actual expression and modulatory effect of HBV and HCV proteins. Expression
of HCV proteins by at least 5% of conventional DC is required to impair presenta-
tion of antigen to CD4þ T cells.93 Similarly, uptake of HBsAg by part of the conven-
tional DC population impairs allogeneic T cell activation.94 Also the HBV precore
protein (HBeAg) is believed to be important in the persistence of HBV infection.
HBV is able to produce large amounts of non-infectious particles containing the
HBeAg that induces Th2 cell development and deletion of IFN-g producing Th1
cells.95,96 In addition, HBV-specific B cells are deleted by HBeAg.97 These immuno-
modulatory effects of HBeAg are believed to be responsible for the stronger antiviral
Th1 responses in HBV chronic carriers who seroconvert from HBeAg to HbeAb,
thus explaining the favourable clinical outcome.98
Also, HCV proteins have been shown to modulate the activity of immune cells.
HCV core protein selectively inhibits IL-12 production after interacting with the
gC1q-receptor on the surface of DC.99,100 In addition, HCV core protein has been
shown to suppress the proliferation of T cells either directly or indirectly.101,102 In
line with this, it was reported that HCV NS3 and NS4 protein augments the produc-
tion of the immunosuppressive cytokine IL-10 by monocytes, which resulted in sup-
pression of IFN-g production by CD4þ T cells in leucocytes of HCV patients.102,103
These data indicate that the expression of HBV and HCV proteins in/by DC impairs
their immunostimulatory function, causing insufficient/ineffective priming of CD4þ
T cells, possibly contributing to the persistence of viral hepatitis viruses.

INTRAHEPATIC IMMUNE REGULATION

Since hepatocytes are the main target cell for HBV and HCV, the microenvironment of the
liver takes a central place in the antiviral response. HBV and HCV do not kill the hepato-
cytes it infects, but trigger an immune-mediated inflammatory response, which in turn
causes damage to the liver. Interest in the local immune responses of the liver has focussed
on intrahepatic lymphocytes, whereas other intrahepatic cells have received much less at-
tention. However, almost two thirds of the total cell population of the liver are hepato-
cytes. In addition, also antigen-presenting cells (APC), such as liver sinusoidal
endothelial cells (LSEC), liver macrophages (Kupffer cells) and DC are present in large
numbers in the liver. These resident cells can all produce cytokines upon stimulation, pres-
ent antigen and activate T cells, although this is not entirely clear for hepatocytes.104–106
Under non-inflammatory conditions, it is generally believed that the liver is an
immunotolerant organ, because of the local production of cytokines, such as
1056 A. Boonstra et al

TGF-b and IL-10. These cytokines are known to modulate APC function, which may
result in suppression of T cell responses. However, the source of these cytokines
within the liver is still largely unknown. Moreover, the consequence of the presence
of these cytokines on intrahepatic immune responses are not clear, since all data re-
ported to date on the effects of TGF-b and IL-10 have studied this in peripheral DC
or macrophages.107 A detailed analysis of individual intrahepatic cell populations, and
their cytokine production, is critical to understand what happens during chronic
HBV and HCV infections. Since these viruses infect predominantly hepatocytes,
these cells are likely to play a crucial role during the course of the persistent liver
infection, but the interaction with Kupffer cells, LSEC and T cells is likely as impor-
tant. Virus-specific CD4þ and CD8þ T cells are abundantly present in the liver of
chronically infected patients, but it is unclear if these cells are functional and able
to control viral replication in the liver. It is possible that intrahepatic virus-specific
CD8þ T cells are inactivated if antigen is presented by hepatocytes in the absence
of costimulatory signals in the liver, which leads to partial activation of T cells. As
a consequence these T cells have weak effector functions. Although it hampers
the antiviral immune response, the limited T cell response in the liver may have ad-
vantages. Since HBV and HCV are non-cytopathic viruses, collateral liver damage by
excessive inflammation or elimination of all infected hepatocytes would have detri-
mental consequences for the patient. In addition, the immunosuppressive environ-
ment in the liver of patients with chronic viral hepatitis characterised by high
numbers of regulatory T cells and immunosuppressive cytokines may further limit
the ability of intrahepatic T cells to eradicate HBV and HCV.108–110

CONCLUSION

Our understanding of the immune response elicited by HBV and HCV infection has
improved immensely in recent years. However, many fundamental questions are still
unanswered. We still don’t understand why some individuals are able to resolve
HBV or HCV infections spontaneously whereas others become chronically infected.
Furthermore, although a number of mechanisms have been described, at present it
is unclear how we can reactivate the weak virus-specific immune response in order
to revert chronic infections. These questions need to be addressed in order to
pave the way to develop strategies to re-invigorate the T cell response, thereby pos-
sibly clearing the virus. However, since both liver injury and viral control are mediated
by the immune system of the infected host, a better understanding is crucial before
immunotherapy of chronic viral hepatitis should be attempted.

Research agenda

 Investigate the role of the intrahepatic immune system in the establishment and
maintenance of chronic viral hepatitis.
 Study the factors that determine why the majority of HCV-infected individuals
develop a chronic infection, whereas only a minority of HBV infected individ-
uals become chronic.
 Investigate the mechanisms why the immune response is unable to control
chronic HBV and HCV infections.
 Develop a vaccine for HCV infections.
 Immunology of viral hepatitis 1057

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