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MINI-REVIEW
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Transgenic technology
and the study of hepatitis
viruses: A review of what
we have learned
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David R Milich PhD
David R Milich. Transgenic technology and the study of hepatitis viruses: A review of what we have learned. Can J
Gastroenterol 2000;14(9):781-787. Because of the absence of
inbred animal models susceptible to infection by the hepatitis B
(HBV), C (HCV) and delta (HDV) viruses, and the inability to
culture these viruses, a number of investigators have produced
transgenic (Tg) mice that express one or all the viral genes. This
review attempts to catalogue and characterize the Tg mice produced to date. The topics addressed are HBV, HCV and HDV
gene expression and regulation; HBV replication models and factors that inhibit replication; HBV pathogenesis models; HBV tolerance and persistence models; modulation of the immune
response to HBV proteins in Tg mice; T cell receptor Tg mice; and
models of hepatocellular carcinoma.
Les techniques transgéniques et l'étude des
virus de l'hépatite : une synthèse de nos
connaissances
RÉSUMÉ : En l’absence de lignées pures de modèles animaux sensibles à
une infection par le virus de l’hépatite B (VHB), C (VHC) et delta
(VHD), et devant l’incapacité de cultiver ces virus, un certain nombre de
chercheurs ont produit des souris transgéniques (Tg) qui expriment un ou
l’ensemble des gènes viraux. La présente synthèse a pour objet de
cataloguer et de caractériser les souris Tg qui ont été produites à date. Les
sujets traités sont l’expression et la régulation des gènes du VHB, VHC et
VHD ; les modèles de réplication du VHB et les facteurs qui inhibent sa
réplication ; les modèles de la pathogenèse du VHB ; les modèles de
persistance et de tolérance du VHB ; la modulation de la réponse immunitaire aux protéines du VHB dans les souris Tg ; les récepteurs des lymphocytes T des souris Tg ; et les modèles de carcinomes hépatocellulaires.
Key Words: Hepatitis B virus; Hepatitis C virus; Hepatitis delta virus; Transgenic mice
P
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rompted by the limited host range of the hepatitis B virus (HBV) (as well as hepatitis C virus [HCV] and hepatitis delta virus [HDV]) and by the lack of in vitro culture
systems to propagate these viruses, a number of investigators
have expressed single viral proteins, combinations of viral
proteins and the complete genome (in the case of HBV) in
transgenic (Tg) mice. It was anticipated that expression of
viral proteins in inbred mice, which possess well characterized immune systems, would allow detailed studies of the immune response to viral proteins in vivo. Use of liver-specific
promoter systems has allowed viral protein expression to be
targeted to the physiologically relevant site – the hepatocyte. These Tg systems have yielded a number of interesting
insights into the biology, immunology and pathogenic potential of these liver-tropic viruses.
EXPRESSION OF VIRAL PROTEINS
IN TG MICE
The first studies using Tg mice focused on the expression of
the HBV envelope proteins in Tg mice (Table 1). These early
studies demonstrated that the envelope proteins could be expressed in the liver by using liver-specific promoters or the
HBV endogenous promoters (1-6). Using the endogenous
HBV promoters resulted in expression in other tissues in addition to the liver. It was also demonstrated that envelope
gene expression is developmentally regulated (5), and is posi-
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This mini-review was prepared from a presentation made at the World Congress of Gastroenterology, Vienna, Austria, September 6 to 11, 1998
Department of Molecular Biology Cal-2, The Scripps Research Institute, La Jolla, California, USA
Correspondence and reprints: Dr David R Milich, The Vaccine Research Institute of San Diego, 3030 Science Park Road, San Diego, California
92121, USA. Telephone 858-587-9505 ext 223, fax 858-587-9208, e-mail dmilich@vrisd.org
Received for publication June 7, 1999. Accepted June 14, 1999
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TABLE 1
Gene expression and regulation in transgenic mice
Table 2
Model of hepatitis B virus (HBV) replication in transgenic
mice
Reference(s)
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Envelope proteins
Expression of the entire HBV genome using
endogenous promoters
1–6
Nucleocapsid proteins
Hepatitis B core antigen
5
Hepatitis B ‘e’ antigen
X protein
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Hepatitis delta virus gene expression
Infectivity of transgenic-derived HBV
9–12
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3,4,25-27
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Inhibition of replication
12–14
Hepatitis B surface antigen-specific
cytotoxic T lymphocytes
15–17
18
Hepatitis C virus gene expression
Structural proteins
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Reference(s)
Hepatitis B virus gene expression
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34
Cytokines
34,55
Heterologous hepatic infections
56,57
Overexpression of the hepatitis B ‘e’ antigen
19–24
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gene products are not directly cytopathic. Both the large and
the small forms of the hepatitis delta antigen (nucleocapsid)
have been expressed in Tg mice, and no biological or histopathological evidence of liver disease was observed, suggesting that neither antigen is directly cytopathic to the
hepatocyte in vivo (18).
Several groups have expressed the structural proteins of
HCV (ie, core, E1, E2) in Tg mice. Three groups reported
that the HCV core protein (19), the core and E2 proteins
(20), and the core, E1 and E2 proteins (21) expressed in the
livers of Tg mice caused no histological or biochemical evidence of liver disease or HCC. In order to examine the immune response to HCV structural proteins, Wakita et al (22)
used the Cre/loxP system to conditionally express the core,
E1 and E2 proteins in Tg mice. Core proteins were detected
in serum seven days after transgene activation, concurrent
with increases in serum alanine aminotransferase levels.
Anticore antibody appeared 14 days after activation of the
transgene. Furthermore, depletion of CD4 and CD8 T cells
normalized the alanine aminotransferase increases as well as
the pathological changes in the liver, suggesting that the immune response rather than the HCV proteins themselves
mediated liver injury. Another group reported that expression of the HCV core protein in the liver of two independent
Tg lineages resulted in progressive hepatic steatosis (fatty
change), which characterizes HCV infection (23). Furthermore, after the age of 16 months, mice of both lineages developed hepatic tumours (24).
In summary, a number of investigators have expressed the
structural proteins of the HBV, HDV and HCV viruses in Tg
mice, and these Tg systems have yielded a number of interesting results, detailed below. However, one important conclusion from these studies is that the viral proteins
themselves are not directly cytopathic within the liver. The
only exceptions to this conclusion are the overexpression of
the HBV large pre-S(1)-containing protein leading to a secretion defect (8) and the expression of the HCV core protein causing hepatic steatosis (23). A secretory defect during
natural HBV infection has not been identified, and the cytotoxic effect of HCV core antigen has not been duplicated in
other Tg lineages.
tively regulated by androgens and glucocorticoids (6). Tg expression of the middle and major envelope proteins led to the
assembly and secretion of 22 nm spherical particles as occurs
during a natural HBV infection (7,8). Inclusion of the gene
encoding the large envelope protein (pre-S[1]-containing)
in the transgene construct resulted in the assembly of long,
branching filamentous hepatitis B surface antigen (HBsAg)
particles (7). If the large envelope protein was overexpressed
in relation to the middle and major proteins by the use of an
exogenous promoter (ie, albumin promoter), the filamentous
HBsAg particles became trapped in the endoplasmic reticulum (ER) and were not secreted by the cell. This secretion defect eventually leads to a dramatic expansion of the ER in the
hepatocyte and severe liver injury (8). It has been suggested
that such hepatocytes are analogous to ‘ground glass’ hepatocytes observed in the liver of chronically infected patients
(8). However, the nature of a putative secretion defect in infected hepatocytes is not clear.
Both HBV nucleocapsid proteins – the particulate hepatitis B core antigen (HBcAg) and the nonparticulate hepatitis B ‘e’ antigen (HBeAg) – have been expressed in Tg mice
(Table 1). HBeAg is efficiently secreted into the blood, and
HBcAg accumulates in the nucleus of hepatocytes (9-14).
Expression of high levels of HBcAg revealed that intact
HBcAg particles cannot traverse the nuclear membrane in
either direction (11). The nonstructural HBV X protein has
also been expressed in Tg mice (15-17). The X protein displays transcriptional transactivation properties, and it has
been suggested that expression of this protein in the liver
may play a role in the induction of hepatocellular carcinoma
(HCC), which is associated with chronic HBV infection. In
support of this hypothesis, high level liver-specific expression of the HBV X protein has led to HCC in Tg mice
(15,16). However, other investigators have not observed the
induction of HCC in independently derived X gene Tg mice
(17).
Simultaneous infection with HDV and HBV, which provides the viral envelope necessary for HDV, is often associated with severe liver disease. This may be due to the
additive effects of the immune response to both infections or
to direct cytotoxic effects of HDV proteins because HBV
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TABLE 3
Transgenic models of hepatitis B virus (HBV) pathogenesis
and liver injury
HBV REPLICATION IN TG MICE
Several laboratories have used constructs containing the entire HBV genome and HBV-derived regulatory sequences to
produce Tg mice capable of viral replication (Table 2). Interestingly, HBV replication occurred in the kidney as well as
the liver, and the supercoiled form of HBV DNA (cccDNA)
has not been observed in any lineage of Tg mouse (3,4,2527). In several Tg lineages, high level replication is sustained
in 20% to 30% of primarily centrilobular hepatocytes, although virtually all hepatocytes express nuclear HBcAg
(27). This suggests that hepatocytes infected in a natural infection may not all be equally permissive for HBV replication. High level replication and HBV gene expression are not
associated with liver pathology (3,4,25-27). One group has
successfully infected chimpanzees with viral particles derived
from Tg mice (28). This model of HBV replication has provided the opportunity to examine the influence of viral and
host factors on HBV replication, pathogenesis and clearance,
including the effects of the immune response. For example, a
number of factors have been shown to be capable of inhibiting HBV replication in the Tg model (Table 2). These and
other approaches are discussed in the following sections.
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Reference(s)
Hepatitis B surface antigen (HBsAg)-specific CD8+
cytotoxic T lymphocytes (CTL) mediate liver injury
Acute hepatitis
29,30
Fulminant hepatitis
31
Chronic hepatitis and hepatocellular carcinoma
32
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HBsAg-specific CD8+ CTL suppress HBV gene expression
Cytokine-mediated suppression
HBsAg-specific CD8+ CTL Inhibit HBV replication
35–37
34
HBsAg-specific CD4+ T helper cells suppress HBV gene
expression
40,41
HBe/HBcAg-specific CD4+ T helper cells mediate liver
injury
42
HBV envelope Tg mice (32). This was accomplished by
transferring HBsAg-specific CTL into thymectomized, irradiated, bone marrow-reconstituted envelope-Tg recipients.
In addition to chronic hepatitis, the Tg recipients eventually
(17 months) developed HCC, suggesting that a prolonged
inflammatory immune response to an HBV protein can
cause liver cancer (32), consistent with a number of observations in chronically infected humans (33).
In addition to the direct hepatocyte injury triggered by
HBsAg-specific CTL, a second noncytolytic mechanism has
been described in which the cytokines secreted by the
HBsAg-specific CTL profoundly suppress hepatocellular
HBV gene expression and HBV replication in Tg mice
(34,35). The cytokines responsible for these noncytolytic
antiviral effects were CTL-derived interferon (IFN)-gamma
and CTL-induced tumour necrosis factor (TNF)-alpha (34).
The antiviral regulatory potential of inflammatory cytokines
was confirmed by the fact that administration of recombinant TNFa (36), interleukin (IL)-2 and to a lesser extent
IFNa and IFNb (37) also inhibited HBV gene expression in
HBV envelope Tg mice. Furthermore, these cytokine effects
were mediated by a post-transcriptional mechanism involving the degradation of cytoplasmic HBV mRNA (38,39). Interestingly, systemic treatment of HBsAg-Tg mice in vivo
with IFNg did not affect HBV gene expression, whereas
CTL-delivered IFNg did (37). This suggested that systemic
IFNg may have multiple effects, some of which may be crossregulatory in terms of inhibition of HBV gene expression.
Using these Tg models, it has been shown that all of the viral
gene products, including the viral replicative intermediates,
are susceptible to the effects of these CTL-derived inflammatory cytokines, suggesting that HBV is exquisitely sensitive to this effect. Recently, the source of the inflammatory
cytokines responsible for inhibition of HBV gene expression
has been extended to include CD4+ T helper (Th) 1 cells
(40,41). Furthermore, it was demonstrated that the HBsAg+
specific CD4 Th cells could be elicited by DNA immunization in HBsAg-Tg mice. We recently produced T cell recep+
tor (TCR) Tg mice in which the majority of the CD4 Th
TG MODELS OF HBV PATHOGENESIS
AND LIVER INJURY
+
Studies employing the adoptive transfer of CD8 , HBsAgspecific cytotoxic T lymphocyte (CTL) into HBV envelopeexpressing Tg mice have demonstrated that CTL can induce
an acute necroinflammatory liver disease similar to that of
natural acute HBV infection (Table 3). The investigators described a three-step process through which the liver disease
progresses. The first step involves the attachment of the donor CTL to HBsAg-positive hepatocytes, which are triggered
to undergo apoptosis. Thereafter, between 4 and 12 h after
injection, the CTLs recruit host-derived antigen-nonspecific
inflammatory cells (ie, polymorphonuclear cells) that amplify the effects of the CTL (step 2) (29,30). This process results in necroinflammatory foci in which hepatocellular
necrosis extends well beyond the location of CTL, suggesting
that most hepatocytes are killed by cells other than the donor
CTL. In these studies, the liver injury in most Tg lineages is
transient and is confined to no more than 5% of hepatocytes.
However, in recipient Tg mice that overexpress and accumulate HBsAg filaments (see above), the disease process proceeds to step 3, in which approximately half of the mice die of
liver failure within 24 to 72 h of CTL transfer (31). The investigators suggest that this process resembles the histopathological features of HBV-induced fulminant hepatitis in
humans, characterized by widespread necrosis of HBsAgladen hepatocytes and diffuse lymphomononuclear inflammatory cell infiltrate and Kupffer cell hyperplasia (31). However, this model does not explain what the trigger for
progression to step 3 may be in the absence of overexpression
of large envelope protein, which has not been described in
natural infection.
This same group of investigators has developed a model of
prolonged chronic immune-mediated (CTL) hepatitis in
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TABLE 4
Transgenic models of hepatitis B virus persistence and/or
tolerance
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Secreted hepatitis B ‘e’ antigen (HBeAg) induces
CD4+ T cell tolerance in utero
Tolerance to the HBeAg is variable and major
histocompatability complex dependent
45
Secreted HBeAg can delete HBe/HBcAg-specific
T helper 1 cells in adult mice
50
HBeAg-specific CD4+ T helper 2 cells can survive
in HBeAg+ transgenic mice
48
The nonsecreted HBcAg is variably tolerogenic in
transgenic mice
9,10
Tolerance to the hepatitis B surface antigen can be
broken in transgenic mice
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Studies in HBeAg-Tg mice revealed that the level of Th
cell tolerance is dependent on the major histocompatibility
complex (MHC) background and the Th cell site recognized
by the Tg murine strain. The Th cells of HBeAg-Tg mice on
an H-2s background (residue 120-131-specific) are very susceptible to tolerance induction by the neoself HBeAg,
b
whereas the Th cells of HBeAg-Tg mice on an H-2 background (residues 129-140-specific) are significantly less tolerant to the same serum levels of HBeAg (45). Incomplete
Th cell tolerance may also explain the ability of HBsAg-Tg
mice to generate anti-HBs antibodies after immunization
with HBsAg particles (46,47). The HBeAg-Tg model has
provided the opportunity to examine the immunoregulatory
properties of circulating HBeAg. For example, because the
Th cells of HBeAg-Tg mice on an H-2b background are not
completely tolerized, a single injection of an HBeAg-derived
Th cell site (peptide 129-140) leads to sufficient anti-HBe
‘autoantibody’ production to complex with and mask the detection of serum HBeAg. This system serves as a model of
HBeAg/anti-HBe seroconversion. The finding of residual
and functional HBeAg-specific Th cells in the periphery of
HBeAg-Tg mice suggests the possibility that similar HBeAgspecific Th cells are present and can be activated in chronically infected HBV patients. Subsequent studies revealed
that the HBeAg-specific Th cells that evade tolerance and
mediate anti-HBe autoantibody production in HBeAg-Tg
mice are significantly ‘altered’ by their coexistence with the
circulating HBeAg. The HBeAg-self-reactive Th cells surviving in HBeAg-Tg mice exhibit a unique fine specificity
that can be distinguished from the HBeAg-specific Th cell
repertoire of non-Tg mice and are comprised predominantly
of Th2-like cells (48). The preferential survival of HBeAgspecific Th cells of the Th2-type in HBeAg-Tg mice is of
particular interest because of the suggestive serological evidence that an imbalance in HBe/HBcAg-specific Th1/Th2
cell function may contribute to the induction and/or maintenance of persistent HBV infection (49). Cumulatively,
these data suggest that conservation of secretion of the
HBeAg may be a viral strategy to guarantee persistence following vertical transmission of HBV, which is the major
source of chronic infection in endemic areas.
Secretion of the HBeAg is also conserved in the avian hepadna viruses in which in utero tolerance mechanisms described previously are not relevant. Furthermore, adult
infection with the HBeAg-negative mutant virus is often associated with a fulminant course of infection rather than the
relatively benign acute course that characterizes most adultonset infections with wild-type HBV. These observations
suggest that the HBeAg may function to modulate the immune response during chronic HBV infection in the adult in
addition to its effects on neonatal tolerance. Because the
HBeAg is a secreted protein, its effects on the HBe/HBcAgspecific Th cell repertoire can be mediated within the thymus and/or in the peripheral lymphoid compartment. We
are particularly interested in the effects of circulating
HBeAg on Th cells in the periphery because of the possible
implications for HBV infection in the adult. We, therefore,
Reference(s)
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HBcAg Hepatitis B core antigen
cells were specific for the HBe/HBcAgs of HBV. Adoptive
+
transfer of CD4 from these TCR-Tg mice polarized toward
the Th1 subset into HBe/HBcAg-expressing Tg recipients
results in liver injury. The degree and kinetics of liver injury
depend on the affinity and specificity of the transferred Th
cells and whether the recipient expresses HBeAg or HBcAg
(42).
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TG MODELS OF PERSISTENCE
AND/OR TOLERANCE
Infants born to HBeAg-positive HBV carrier mothers invariably become persistently infected. To investigate the role
of immunological tolerance mechanisms in chronic infection of the newborn, HBeAg-expressing Tg mice were generated (13). HBeAg-Tg mice represent a model system to
examine the consequences of in utero exposure to HBeAg on
HBc/HBeAg-specific immune responses (Table 4). Characterization of tolerance in HBeAg-Tg mice and mice rendered
neonatally tolerant indicated that T cells but not B cells were
rendered tolerant by HBeAg present in the serum at a concentration of 10 to 100 ng/mL; that T cell tolerance elicited
by HBeAg also extends to HBcAg-specific T cells; that Tg
mice produced anti-HBc but not anti-HBe antibodies upon
immunization; that the immunoglobulin (Ig) G but not the
IgM anti-HBc response was diminished in HBeAg-Tg mice;
and that the T-cell tolerance induced by a single neonatal exposure to HBeAg was reversible and persisted for 12 to 16
weeks (13).
Many characteristics of immune tolerance found in
HBeAg-Tg mice parallel the long term immunological status
of neonates born to HBeAg-positive HBV carrier mothers,
suggesting that the aberrant immunological responses of
neonates born to carrier mothers may result from in utero exposure to HBeAg, as occurs in the Tg model. In support of
the possibility that maternal HBeAg may traverse the placenta, non-Tg littermates born to HBeAg-Tg mothers were
tolerant to HBc/HBeAg (13). Furthermore, HBeAg has
been detected in the neonatal cord serum of infants born to
HBeAg-positive HBV carrier mothers (43,44).
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TABLE 5
Modulation of the immune response to hepatitis B virus
proteins in transgenic (Tg) mice
bred HBeAg-Tg (H-2 ) mice with Fas-defective lpr/lpr mice
(50). Thymic T cell deletion (ie, negative selection) is not
Fas-mediated, whereas peripheral clonal deletion appears to
be Fas-mediated and impaired in lpr/lpr mice. Therefore, the
availability of HBeAg-Tg mice on Fas-expressing or Fasdeficient backgrounds enabled us to examine the ability of
secretory HBeAg to deplete HBeAg-specific Th cells in the
periphery and determine whether Th1 or Th2 cells were
preferentially affected by this mechanism. The results of the
study in HBeAg-Tg/lpr mice suggested that circulating
HBeAg preferentially depletes HBeAg-specific Th1-like
cells in the periphery via Fas-mediated apoptosis and that
HBeAg-specific Th2-like cells survive this process to a
greater degree. This study suggested a mechanism by which
the HBeAg may maintain or induce chronicity even during
an adult infection. The fact that HBeAg is secreted and
widely disseminated coupled with its ability to deplete
HBe/HBcAg-specific Th1-like cells and spare Th2-like cells,
which are more resistant to peripheral depletion mechanisms (ie, Fas-mediated apoptosis), make it an ideal candidate to promote viral persistence (50).
To determine the tolerogenic potential of a nonsecreted
intracellular form of the HBeAg, namely the HBcAg, Tg
mice expressing the HBcAg in the liver were produced. Exs
pression of the HBcAg at birth in mice of an H-2 MHC
background resulted in Th cell tolerance (10). In another
HBcAg-expressing Tg system, the serum amyloid P promoter
was used and HBcAg expression in the liver was delayed until three to four days after birth. In these HBcAg-Tg mice,
spontaneous anti-HBc antibody production occurred, indicating a lack of Th cell tolerance (9). Therefore, the time of
developmental expression of a transgene is a critical factor in
determining the degree of self-tolerance.
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Reference(s)
Seroconversion in hepatitis B ‘e’ antigen (HBeAg)-Tg
mice is major histocompatability complex (MHC)
dependent
Inhibition of seroconversion in HBeAg-Tg mice
Blockade of B7 costimulation
MHC blockade with envelope T cell sites
Interleukin-12 (T helper 2 Ú T helper 1 switch)
Seroconversion in hepatitis B core antigen-Tg mice
Seroconversion in hepatitis B surface antigen-Tg mice
Protein immunization
Recombinant vaccinia virus
DNA immunization
Non-Tg dendritic cells
45
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45
52
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40
53
envelope proteins circulate in tremendous excess of the
HBeAg. Therefore, ‘intraviral’ protein competition for
MHC binding sites may be a viral strategy to saturate MHC
molecules and prevent less abundant, and possibly more relevant, T cell recognition sites from gaining access to antigen
presentation. Seroconversion in HBcAg-Tg mice has been
accomplished by transferring primed non-Tg CD4+ T cells
into HBcAg-Tg recipients (50).
Several groups have also induced seroconversion to the
HBsAg in Tg mice. Immunization of HBsAg-Tg mice with
HBsAg in adjuvant induced low level anti-HBs production,
approximately 500-fold less than in non-Tg littermates
(46,47). Similarly, multiple immunizations with an HBsAgvaccinia recombinant virus elicited low level anti-HBs seroconversion (47). Induction of seroconversion to the HBeAg,
the HBcAg or the HBsAg has not resulted in liver injury, indicating that antibodies specific for these viral antigens are
not pathogenic. Recently, one group immunized HBsAg-Tg
mice with a DNA construct coding for the HBsAg and reported anti-HBs seroconversion, CD4+ Th cell priming and
inhibition of HBsAg gene expression probably due to IFNg
production (40). Finally, one group has suggested that the
lack of anti-HBs production by HBV chronic carriers and in
HBsAg-Tg mice is due to defective function of antigenpresenting cells rather than immune tolerance (53). It was
suggested that circulating HBsAg reduced MHC class II and B
7.2 (CD86) expression on dendritic cells in HBsAg-Tg mice.
Treatment with IFNg or replacement of Tg dendritic cells
with dendritic cells from non-Tg mice allowed Th cells from
HBsAg-Tg mice to mediate anti-HBs production in vitro.
MODULATION OF THE IMMUNE RESPONSE
TO HBV PROTEINS IN TG MICE
As noted previously, tolerance to the HBeAg in Tg mice is
MHC dependent. Tolerance to HBeAg is complete in
HBeAg-expressing Tg mice on an H-2s genetic background,
b
and incomplete on HBeAg-Tg mice on an H-2 background.
Therefore, a population of functional 129-140 peptide-specific Th cells coexist with circulating HBeAg in B10HBeAg-Tg mice. The Th cells that evade deletion in
HBeAg-Tg mice are quiescent unless activated by a dose of
the 129-140 peptide as low as 0.6 µg, which induces anti-HBe
seroconversion (45). This model has been useful to test reagents that may modulate anti-HBe seroconversion (Table 5).
Soluble CD 152 (cytotoxic T lymphocyte antigen-4) has
been shown to suppress anti-HBe seroconversion in this
model (51). Similarly, IL-12 suppresses anti-HBe ‘autoantibody’ production and skews the Th cells toward the Th1 subset (52). Finally, injection of an envelope (pre-S[2]) T cell
site peptide that binds the same MHC class II molecule as
129-140 peptide inhibits anti-HBe seroconversion by competitively binding to IAb and preventing the activation of
129-140 peptide-specific Th cells (45). This may have important implications during a natural HBV infection because
HBV-SPECIFIC TCR-TG MICE
Another application of the Tg technology to the study of HBV
is the development of TCR-Tg mice in which a majority of
CD4+ or CD8+ T cells are specific for an HBV protein. These
mice are a source of monoclonal CTL or Th cells that can be
used, much like monoclonal antibodies, for a variety of in vitro studies (Table 6). Furthermore, the TCR-Tg mice can be
crossed with HBV protein-expressing Tg mice to produce
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TABLE 6
Hepatitis B virus (HBV)-specific T cell receptor (TCR)transgenic (Tg) mice
TABLE 7
Transgenic mice as a model for hepatocellular carcinoma
Reference(s)
Source of monoclonal, naive T cells
T cell activation
T cell specificity
T cell-antigen presenting cell interactions
T cell-B cell interactions
T helper 1/T helper 2 polarization
HBV-Tg × TCR-transgenic ‘double Tg mice’
Tolerance (ie, clonal deletion) mechanisms
Pathogenesis
Overexpression of the hepatitis B virus (HBV)
pre-S(1) protein
High level expression of the HBV X protein
Long term chronic hepatitis
Hepatitis C virus core expression
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of HCC in independently derived X gene-Tg mice (17). This
discrepancy may relate to the level of expression or the genetic backgrounds of the mice. The Tg mice that developed
HCC were produced on a CD1 background, which displays a
high spontaneous rate of HCC. Finally, the development of
HCC in two independent lineages of mice Tg for the HCV
core gene was recently reported (24). These HCV core-Tg
mice develop hepatic steatosis early in life and after the age of
16 months develop hepatic tumours that first appear as adenomas. Thereafter, more poorly differentiated HCC develops from within the adenomas. This closely resembles the
histopathological characteristics of the early stage of HCC in
patients infected with HCV. Although this observation has
not been repeated in other Tg mice expressing the HCV core
protein, these particular Tg mice may be useful in determining the molecular events in hepatocarcinogenesis associated
with HCV infection.
double Tg mice, which are useful for examining tolerance
mechanisms (ie, clonal deletion) and pathogenesis in vivo.
We recently developed several lineages of TCR-Tg mice
harbouring CD4+ Th cells specific for the HBe/HBcAg. The
TCR genes were derived from T cell hybridomas produced
from either wild-type mice immunized with HBeAg or
HBeAg-Tg mice. The CD4+ Th cells present in the TCR-Tg
lineages derived from wild-type mice were never exposed to
the HBe/HBcAgs, are of relatively high affinity and represent naive populations similar to an HBe/HBcAg-specific
repertoire during an acute infection. In contrast, the CD4+
Th cells present in the TCR-Tg mice derived from HBeAgTg mice are HBe/HBcAg-specific populations that were selected in the presence of circulating HBeAg, are of relatively
low affinity and may serve as a model for the CD4+ repertoire
during chronic infections. The acute-like TCR-Tg lineages
differ markedly from the chronic-like TCR-Tg lineages with
respect to affinity and kinetics of T cell activation; fine
specificity for either HBeAg or HBcAg; in vitro and in vivo
immune responsiveness; Th1/Th2 subset differentiation;
clonal deletion as analyzed in double Tg mice; and the kinetics and severity of liver injury occurring after adoptive transfer into HBe and/or HBcAg-Tg recipients (42). It is
anticipated that these TCR-Tg and TCR-Tg × HBe/HBcAgdouble Tg systems will provide unique insights into the complex interactions between HBV and the host immune response.
100
95
SUMMARY
The advantages of Tg technology for the study of hepatitis virus biology and the immune response to viral proteins are obvious from the literature reviewed herein. However, the
limitations of this technology are not as obvious and should
also be appreciated. Until virus receptor-Tg mice are developed, these systems are not models of infection and, therefore, are somewhat limiting. Furthermore, very high level
gene expression and/or inappropriate tissue expression can
lead to artifacts that are not relevant to the natural infection.
These limitations are inherent in ‘reductionist’ model systems but can be ameliorated by interpreting the results obtained in these Tg systems in the context of what is known
about the natural infection. In this way, we can avoid making
the Tg model a disease rather than a model for the disease.
TG MICE AS A MODEL FOR HCC
Several investigators have reported the occurrence of cancerous lesions in the livers of mice Tg for various viral proteins
(Table 7). As stated earlier, Tg mice expressing large
amounts of the HBV pre-S(1)-containing large envelope
protein demonstrate an ER storage disorder, leading to massive hepatocyte death, causing a secondary inflammatory and
regenerative response that eventually leads to HCC in mice
strains that have a low incidence of spontaneous HCC (54).
Although excess pre-S(1) protein is unlikely to accumulate
in natural infection, a link between chronic liver injury and
HCC has been demonstrated in a number of systems, including a recent Tg model of immune-mediated chronic hepatitis
by these same investigators (32). High level expression of the
HBV X protein can also lead to HCC in Tg mice (15,16), although other investigators have not observed the induction
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