Journal of Immunological Methods 410 (2014) 3949

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Journal of Immunological Methods

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Review

Mouse models of hepatitis B and delta virus infection

Maura Dandri a,b,, Marc Ltgehetmann a,c
a
I. Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
b
German Center for Infection Research, HamburgLbeckBorstel Partner Site, Germany
c
Institute of Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Liver disease associated to persistent infection with the hepatitis B virus (HBV) continues to be a
Received 29 January 2014 major health problem of global impact. Therapeutic regimens currently available can efficiently
Received in revised form 3 March 2014 suppress HBV replication; however, the unique replication strategies employed by HBV permit
Accepted 4 March 2014 the virus to persist within the infected hepatocytes. As a consequence, relapse of viral activity is
Available online 12 March 2014
commonly observed after cessation of treatment with polymerase inhibitors. Among the HBV
chronically infected patients, more than 15 million patients are estimated to be co-infected with
Keywords: the hepatitis delta virus (HDV), a defective satellite virus that needs the HBV envelope for
Transgenic mice propagation. No specific drugs are currently available against HDV, while nucleos(t)ide analogs
Human liver chimeric mice
are not effective against HDV replication. Since chronic HBV/HDV co-infection leads to the most
Preclinical HBV/HDV infection studies
severe form of chronic viral hepatitis in men, a better understanding of the molecular mechanisms
of HDV-mediated pathogenesis and the development of improved therapeutic approaches is
urgently needed. The obvious limitations imposed by the use of great apes and the paucity of
robust experimental models of HBV infection have hindered progresses in understanding the
complex network of virushost interactions that are established in the course of HBV and HDV
infections. This review focuses on summarizing recent advances obtained with well-established
and more innovative experimental mouse models, giving emphasis on the strength of infection
systems based on the reconstitution of the murine liver with human hepatocytes, as tools for
elucidating the whole life cycle of HBV and HDV, as well as for studies on interactions with the
infected human hepatocytes and for preclinical drug evaluation.
2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
1.1. HBV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
1.2. HDV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2. Mouse models of HBV replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.1. Transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.2. Vector-mediated transduction of HBV in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.3. Hydrodynamic injection of replication competent HBV or HDV genomes . . . . . . . . . . . . . . . . . . . . . . . 42
3. Chimeric mouse models of HBV and HDV infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.1. Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.2. Immune competent human liver chimeric mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Abbreviations: HBV, hepatitis B virus; HDV, hepatitis delta virus; cccDNA, covalently closed circular DNA; HBsAg, HBV surface antigen; HBcAg, HBV core
antigen; HDAg, Hepatitis delta antigen.
Corresponding author at: I. Dept. Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel.: +49 40
7410 52949; fax: +49 40 7410 57232.
E-mail address: m.dandri@uke.de (M. Dandri).

http://dx.doi.org/10.1016/j.jim.2014.03.002
0022-1759/ 2014 Elsevier B.V. All rights reserved.
40 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
1.1. HBV infection
Characteristic of the hepatitis B virus (HBV), the prototype
member of the Hepadnaviridae family, is its high tissue- and
species-specificity, as well as a unique genomic organization
with asymmetric mechanism of replication (Nassal, 2008).
Because of the narrow host range, HBV infects exclusively
humans, but can be used to experimentally infect chimpan-
zees. Since mice and rats are not permissive for any of the
known Hepadnaviridae, most of the advances in HBV
research have been based on infection studies performed in
chimpanzees and by using infection experiments with
HBV-related hepadnaviruses (such as woodchuck hepatitis
virus and duck hepatitis virus) and their respective hosts
(Mason et al., 1980; Roggendorf and Tolle, 1995). These
studies have been essential for understanding various steps
of the viral life-cycle (Mason et al., 1982) and experiments
performed with the American woodchuck have provided
important insights on the processes involved in infection
persistence and hepatocarcinogenesis (Zoulim et al., 1994;
Dandri et al., 1996; Summers and Mason, 2004; Mason et al.,
2005). Based on the close phylogenetic relationship between
primates and tree shrews (von Weizsacker et al., 2004;
Baumert et al., 2005), the tree shrew species Tupaia belangeri
has been frequently used for infection studies both with HBV
and, more often, with the woolly monkey hepatitis B virus
(WMHV) (Walter et al., 1996). However, because of the
restraints encountered using chimpanzees and animal
models based on HBV-related viruses, it is not surprising
that researchers focused on the development of more
sophisticated systems based both on the generation of
mouse models of HBV replication by using transduced and
transgenic mice harboring over-length HBV-DNA genomes,
and on the development of mice carrying human chimeric
livers. The latter permits to perform in vivo infection studies
and to investigate the complete HBV replication cycle in the
natural target of infection: the human hepatocyte. Never-
theless, important steps of the infection and pathobiology of
HBV have not been clarified. Only recently the discovery of
the cellular receptor has opened up new possibilities to
investigate specific steps of the life-cycle in vitro (Yan et al.,
2012). In this study, Li and colleagues used a synthetic
peptide corresponding to the myristoylated N-terminus of the
HBV preS1 protein to identify the candidate virus receptor from
hepatocyte cultures. By using methods called zero distance
photo-cross-linking and tandem affinity purification, it was
shown that the preS1 peptide of the HBV envelope specifically
interacts with a sodium taurocholate cotransporting polypep-
tide (NTCP), a multiple transmembrane transporter localized
to the basolateral membrane of highly differentiated primary
hepatocytes. NTCP mediates the transport of conjugated bile
acids and some drugs from the portal blood to the liver. Based
on the discovery that NTCP serves as HBV entry receptor,
cell lines susceptible to HBV infection are emerging and first
46
46
studies reported the successful establishment of HBV infection
in a significant proportion of NTCP-transfected HepG2 cells
(Yan et al., 2012; Nkongolo et al., 2013; Yan et al., 2013).
Although large amounts of input viruses (MOI N 1000) are still
necessary to achieve HBV infection in these culture systems,
the availability of in vitro assays permitting investigation of the
early steps of infection, as well as rapid screening of new
anti-HBV agents, is expected to open new opportunities in
HBV research. Nevertheless, despite the importance of such
discovery, additional as-yet-unknown factors may be in-
volved in the HBV infection process, since the production
of limited amounts of HBV particles has been observed.
Intriguingly, murine cells (both hepatoma and primary
hepatocytes) engineered to express the human NTCP have
so far failed to permit the establishment of productive HBV
infection. Whether species-specific differences or the lack of
essential cellular factors within the murine hepatocytes are
responsible for these discrepancies needs further investigations.
1.2. HDV infection
Among the HBV chronically infected patients, more than
15 million patients worldwide are co-infected with the
hepatitis delta virus (HDV) (Hughes et al., 2011), a defective
virus, known to lead to the most aggressive form of chronic
viral hepatitis, since the infection is generally associated with
an accelerated course of fibrosis progression, cirrhosis, and the
highest risk for liver decompensation and death (Rizzetto,
2009; Hughes et al., 2011). HDV is a small single-stranded RNA
virus which hijacks the host DNA-directed RNA polymerase to
propagate its viral genome in a RNA-directed RNA replication
process. Within the hepatocyte, replication leads to accumula-
tion of three HDV RNAs: the circular genomic RNA, a
complementary antigenomic RNA, and a smaller linear
mRNA, which is the template for the translation of the only
known viral protein, the hepatitis delta antigen (HDAg). There
are two HDAg isoforms: a small 24 kDa isoform, which is
essential for virus replication, and a large 27 kDa variant,
necessary for virus morphogenesis, which is generated via a
post-transcriptional RNA-editing event. Moreover, HDV needs
the surface proteins of HBV to generate infectious viral particles
and hence to complete its life-cycle (Sureau, 2006). As a
consequence, infectious HDV virions can only be released from
hepatocytes that are co-infected with HBV. No drugs are
currently available specifically targeting HDV and various
clinical studies have shown that nucleos(t)ide analogs are not
effective against HDV replication (Hughes et al., 2011). This is
not surprising, since polymerase inhibitors efficiently suppress
HBV replication without affecting the transcriptional activ-
ity of the HBV covalently closed circular DNA (cccDNA)
minichromosome, from which all RNA transcripts and HBV
surface proteins (HBsAg) that are needed for HDV assem-
bly, are produced. Furthermore, the lack of robust HBV/HDV
infection models permitting investigation of the full
life-cycle of HDV has hampered our understanding of the
HDV biology, of the consequences of HDV co-infection for HBV
 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949
replication, as well as the development of more efficient
therapeutic strategies.
2. Mouse models of HBV replication
2.1. Transgenic mice
The requirement of convenient inbred animal models to
investigate specific aspects of HBV replication and its oncogenic
potential in vivo led to the development of transgenic mice
harboring either single viral genes or a terminally redundant
over-length 1.3 HBV-DNA construct (Chisari et al., 1985; Araki
et al., 1989; Kim et al., 1991; Guidotti et al., 1995; Slagle et al.,
1996). The first HBV-replicating transgenic mice were devel-
oped by Chisari and colleagues in 1995 (Guidotti et al., 1995).
Since the transgene construct used contained exclusively viral
promoters and regulatory elements, these studies demonstrat-
ed the feasibility to produce in murine cells, a transgene-driven
production of infectious HBV virions morphologically indistin-
guishable from human-derived virions (Guidotti et al., 1999).
As the immune system of transgenic animals recognizes during
embryonic development the virus as self, these studies
provided the first evidence that HBV replication does not
induce hepatocellular injury and therefore, that HBV-related
pathogenesis must be largely mediated by the host immune
responses (Guidotti and Chisari, 2006). To support this,
induction of acute hepatitis and hepatocellular injury
was demonstrated after adoptive transfer of HBV-antigen
specific CTLs (Moriyama et al., 1990; Guidotti et al., 1994,
1996b). These studies showed that the CTL-mediated release
of cytokines (Guidotti et al., 1996a) can inhibit viral
replication by lowering steady-state levels of HBV-RNAs
also by non-cytolytic mechanisms (Heise et al., 1999) and
that the recruitment of antigen non-specific inflammatory
cells can amplify the severity of liver damage initiated by
antigen-specific CTLs (Sitia et al., 2004). Studies performed
with HBV transgenic mice demonstrated that this small
animal model is also suitable to evaluate the impact of
antiviral treatment strategies on HBV replication. For
instance, the impact of various nucleoside analogs, such as
lamivudine (Weber et al., 2002), adefovir dipivoxil and
entecavir (Julander et al., 2002; Julander et al., 2003) on HBV
replication was first evaluated in HBV-replicating transgenic
mice. Studies involving the analysis of gene expression
profiles also reported the occurrence of IFN-mediated
induction of components of the immune proteasome and
ubiquitin-like proteins in the liver of HBV-replicating
transgenic mice (Wieland et al., 2003). Small interfering
RNAs (siRNAs) specifically recognizing HBV transcripts have
shown to suppress virion productivity in transgenic mice
(Klein et al., 2003; McCaffrey et al., 2003). As an alternative
therapeutic approach aiming at combining both gene
silencing and the induction of interferon responses in the
liver, Protzer and colleagues designed 5-triphosphorylated
small interfering RNAs targeting highly conserved sequences
on HBV RNA transcripts (Ebert et al., 2011). These bi-functional
antiviral molecules suppressed HBV replication in vivo more
efficiently than HBV-specific siRNAs lacking a triphosphate
group, which could trigger antiviral responses upon induction
of the helicase retinoid acid-inducible protein I (RIG-I) (Ebert
et al., 2011).
41
Although HBV transgenic mice provided useful information
related to the mechanisms of HBV pathogenesis, there are
important limitations by using these models that need to be
taken into account. First, the infection step cannot be studied in
transgenic mice, since murine hepatocytes do not allow
investigation of viral entry and spreading steps. Whether the
generation of mice expressing a human-specific cellular
receptor (h-NTCP) will enable the establishment of HBV
infection in mice is currently not clear. Most importantly, in
transgenic mice all viral RNAs are synthesized from a
chromosomally integrated copy of the viral genome and, for
unknown reasons, no cccDNA, the natural template of viral
transcription in human hepatocytes, is normally built in the
liver of transgenic mice. Only HBV transgenic mice crossed to
HNF1 null mice were reported to display low levels of
cccDNA, although these mice showed impaired HBsAg expres-
sion and their use was limited by severe effects due to HNF1
deficiency, such as hepatic and renal dysfunctions, phenylke-
tonuria and infertility (Raney et al., 2001).
Interestingly, first results indicate that murine cells made
transgenic for the human NTCP become fully susceptible for
HDV infection. However, recent studies indicated that restric-
tions likely occurring after viral entry but prior to viral
transcription (i.e. membrane fusion, nuclear entry or cccDNA
formation) hinder the establishment of HBV infection in
murine hepatocytes (Li et al., 2014). Since both HBV and HDV
utilize the same envelope for binding to the cellular NTCP
receptor (Yan et al., 2013), this may be due to species-specific
differences or the lack of cellular factors present only in human
hepatocytes.
The fact that in transgenic mice the HBV genome cannot be
eliminated from the host genome implies that a therapeutically-
mediated complete abrogation of virion productivity, which
requires elimination of the cccDNA and knowledge of its
half-life, cannot be properly addressed in these systems. To
break tolerance to HBV antigens and investigate mechanisms of
viral clearance, alternative mouse models have been developed
which rely on transfecting or transducing the viral genome into
mouse hepatocytes by different means, such as using recombi-
nant adenoviral vectors, or by hydrodynamic injection of naked
DNA in mice.
2.2. Vector-mediated transduction of HBV in mice
Adenoviral vectors containing an over-length HBV genome
(Ad-HBV) have been shown to permit efficient transfer of the
HBV genome into the mouse liver (Sprinzl et al., 2001). After
intravenous injection of adenoviral vectors, HBV replication and
viremia production could be demonstrated for up to 3 months
and mild to moderate liver inflammation with elevated serum
alanine transaminase activities was observed (Isogawa et al.,
2005). After the induction of a B-cell response, anti-HBs
seroconversion and development of neutralizing antibodies
could be determined; this finally led to the clearance of the
infection. Because of the acute, self-limiting character of such
adenoviral-mediated HBV infection, the system offers good
possibilities to investigate the mechanisms of immune-
mediated viral clearance in mice. However, establishment of
long-term viral replication with Ad-HBV vectors is limited
by the immune responses against these vectors, while the
42 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949
occurrence of some adenovirus-mediated cytotoxic effects may
also limit their application.
2.3. Hydrodynamic injection of replication competent HBV or
HDV genomes
Hydrodynamic injection techniques, which involve the fast
injection of high volume of fluids containing naked DNA into
the tail vein of mice, allow crossing species-specific barriers
and hence permit efficient HBV DNA transfer (Yang et al.,
2002). Hydrodynamic injection of replication-competent HBV
genomes in mice resulted in high levels of HBV replication with
titres up to 1 107 HBV DNA/ml (Yang et al., 2002). Although
HBV replication initiated already 1 day post-injection, replica-
tion levels decreased after one week and HBV was cleared from
blood within 23 weeks, as soon as specific antiviral antibodies
and CD8+ T cells appeared. However, HBV infection persisted
for at least 81 days after hydrodynamic injection of mice
lacking adaptive immune cells and natural killer cells, thus
demonstrating that the outcome of hydrodynamic transfection
of HBV depended on the host immune response (Yang et al.,
2002). Hydrodynamic injection studies also showed that
simultaneous delivery of HBV expressing plasmids and
HBV-specific siRNAs prevented HBV replication (Klein et al.,
2003; McCaffrey et al., 2003), while by injecting modified HBV
DNA plasmids into C57BL/6 mice, a significant immune
clearance of HBV could be achieved (Lin et al., 2010). By
avoiding to raise sufficient cellular immunity against HBcAg,
the authors proposed a model of HBV persistence, which better
resembled the immune tolerant stage of chronic HBV carriers.
Hydrodynamics-based transfection procedures have been
also used for in vivo studies of HDV replication and pathogen-
esis (Chang et al., 2001). A transitory accumulation of HDV RNA
transcripts and appearance of the delta antigens (HDAg) could
be demonstrated in up to 3% of the mouse hepatocytes after
transfection of HDV RNA sequences. Since in the absence of
HBV envelope proteins the newly synthesized HDV genomes
cannot be released, viremia could not be established. To
overcome this problem, a following study used hydrodynamic
injection of HDV-containing plasmids in HBV transgenic mice.
In this case, not only transient development of viremia but also
the in vivo antiviral efficacy of prenylation inhibitors was
reported (Bordier et al., 2003). Despite the relatively short span
of viral replication available, mice transfected by hydrodynam-
ic injection are suitable not only for short-term antiviral studies
but also for testing the consequences of specific mutations
within the viral genomes. In comparison to HBV-transgenic
mice, hydrodynamic-based procedures permit investigation of
immune response emerging during acute infection. Since the
viral genome is not integrated into the host genome as a
transgene, viral clearance, mediated by the immune system or
after antiviral therapy, can be achieved in these systems. A
further advantage of the system is that many different mutant
viruses either cloned from patients, or specifically designed in
vitro, can be injected into mice and analyzed in vivo for their
replication capacity in relatively short time. Nevertheless, it
must be kept in mind that the rapid injection of large volume of
fluids causes strong distress to the animals and significant liver
damage, which may alter cell function and signaling analyses.
Furthermore, viral clearance implies not only virus elimination
of the HBV-transfected cells, but also control of viral spread.
Since re-infection of the mouse hepatocytes is not possible,
viral clearance may be easier to achieve in HBV-transfected
mouse systems than in the course of natural infection, and
therefore, the efficacy of antiviral treatments should be
validated in systems permissive for HBV infection.
3. Chimeric mouse models of HBV and HDV infections
Mouse models of HBV replication do not permit to study
specific steps of the life cycle of human hepatitis viruses,
such as the mechanisms of HBV (and HDV) entry, cccDNA
formation and spreading. Moreover, species-specific differ-
ences and the inability to reproduce the entire infection cycle
of these viruses in murine hepatocytes have hindered our
understanding of the molecular mechanisms by which HBV
and HDV can affect hepatocyte functions and are directly
involved in the carcinogenesis process. Because of these
restraints, in the last 15 years many efforts have concentrat-
ed on the development of models based on the use of the
natural target of HBV and HDV infections: the human
hepatocyte. However, availability of highly viable primary
human hepatocytes is limited, the cannot be propagated in
vitro, and their susceptibility to HBV infection is highly
variable and generally low, since these cells rapidly lose their
high-differentiation status and hence the expression of
hepatocyte-specific factors, such as the NTCP receptor, that
are essential to establish the infection (Yan et al., 2012). The
generation of mice carrying human hepatocytes allows
overcoming most of these limitations. The Trimera mice
represented the first humanmouse chimeric system that
was developed by transplanting human liver fragments
under the kidney capsule of normal BALB/c mice. To
avoid rejection of the implanted tissue, these mice were
preconditioned by total body irradiation and reconstituted
with SCID mouse bone marrow cells (Ilan et al., 1999). Using
this approach and by infecting ex vivo small human liver
specimens with HBV, low levels of viremia could be
determined in most of the implanted animals, where viral
titres remained detectable for at least 20 days. Interestingly,
mice ectopically carrying human liver tissue fragments were
shown to support the engraftment of human peripheral
blood mononuclear cells (PBMC), so that the effects of
polyclonal anti-HBs antibodies against HBV could be assessed
(Bocher et al., 2000; Eren et al., 2000). Nevertheless, due to
the extra-hepatic location of the implanted tissues, human
hepatocytes remained functional only for limited time and in
vivo infection with HBV or other human hepatotropic viruses
could not be established.
To achieve long-term survival of highly differentiated
human hepatocytes permissive for HBV infection in vivo,
major attempts have been made to create mice harboring
human hepatocytes stably integrated in the liver parenchy-
ma. To date and as summarized in Fig. 1, a handful of
different human-liver chimeric mouse models have been
established, even though the urokinase-type plasminogen
activator (uPA) transgenic mouse (Sandgren et al., 1991;
Rhim et al., 1994; Dandri et al., 2001b) and the knockout
fumarylacetoacetate hydrolase (FAH) mouse (Grompe et al.,
1993; Azuma et al., 2007) are the most used and best
characterized. The basic concept of these systems is that the
presence of a transgene-driven hepatocyte damage creates
 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949 43

A uPA/SCID mice:
Transplantation in young mice
intrasplenic Reconstitution with
injection human hepatocytes liver injury in newborn mice
reconstitution of mouse liver
with human hepatocytes
2-3 week-old (2 months)
Hepatocyte isolation Alb-uPA/SCID/beige
from adult human livers MUP-uPA/SCID

B intrasplenic Drug withdrawal (NCTB) Reconstitution with FRG and TK-NOG mice:
injection or addition (Gancyclovir) human hepatocytes Transplantation in adult mice
liver injury conditionally activated
reconstitution of mouse liver
with human hepatocytes
Hepatocyte isolation adult Fah-/- (Rag2-IL2rgnull) (2-3 months)
from adult human livers adult TK-NOG

repeated intrasplenic injection

C intrasplenic Reconstitution with human

AFC8hu mice:
Transplantation in adult mice
injection immune cells & hepatocytes
liver injury conditionally activated
partial reconstitution of immune system
(2-3 months)
Hepatic stem cells (HSC) isolation with human cells; limited expansion of
adult AFC8
from human fetal livers the hepatocyte lineage

D intrasplenic
Reconstitution with
human hepatocytes
Reconstitution with human
immune cells after
uPA-NOG mice:
injection treosulfan-conditioning Adult hepatocytes transplantation in
(2-3 months) (2-3 months) young mice
liver injury in newborn mice
reconstitution of mouse liver
2-3 week-old
Hepatocyte isolation with human hepatocytes
Alb-uPA-NOG
from adult human livers partial reconstitution of immune system
with human HSCs

Hepatic stem cells (HSC) isolation

from human fetal livers

Fig. 1. Schematic outlines of the experimental approaches used to generate mice with human chimeric livers. A) One million fresh or cryopreserved hepatocytes
isolated from adult human livers are intrasplenic injected into young immune deficient uPA mice. Reconstitution of the mouse liver with human hepatocytes is
accomplished in 2 months. B) Isolated adult human hepatocytes are transplanted into upon induction of liver injury, which is initiated either by withdrawing the
inhibitor of the tyrosine catabolic pathway NCTB in FRG mice, or by administering gancyclovir to the TK-NOG mice. Transplantation of higher hepatocyte amounts
or a second round of hepatocyte injection was shown to increase levels of human chimerism in FRG mice. C) Hepatic stem cells (HSCs) isolated from a human fetal
liver are injected in adult AFC8 mice to reconstitute mice with cells of both hepatocyte and immune cell lineages. D) Cryopreserved adult hepatocytes were first
transplanted in young uPA-NOG mice to generate chimeric mouse livers. After confirming the presence of human albumin in peripheral blood, mismatched HSCs
derived from a human fetal liver are administered i.v. after 3 days of chemotherapeutic conditioning with treosulfan to dually reconstitute animals with human
hematolymphoid cells and hepatocytes.

the space and the regenerative stimulus necessary for the
transplanted hepatocytes to expand and reconstitute the
diseased mouse liver, while the absence of murine adaptive
immune cells and NK cells permits the engraftment and survival
of transplanted xenogenic hepatocytes for the life-span of the
mice.
The Alb-urokinase-type plasminogen activator (uPA) trans-
genic mouse was the first model where the strong regeneration
capacity of healthy endogenous and transplanted hepatocytes
was reported. In this system, over-expression of the hepatotoxic
transgene, which is driven by the mouse albumin promoter,
induces high levels of uPA in plasma, hypo-fibrinogenemia, and
subacute liver failure in young mice (Sandgren et al., 1991).
Soon after birth, somatic deletion of the uPA transgene takes
place in a small fraction of mouse hepatocytes, so that these
cured cells suddenly have a strong selective growth advantage
over the transgene-carrying diseased hepatocytes. Since
inactivation of both copies of the uPA transgene within the
same cell is a rare event, reconstitution of the mouse liver with
healthy recipient-derived hepatocytes is very inefficient and
mortality of homozygous young adult uPA mice is high if the
animals are not transplanted with normal mouse hepatocytes in
the first month of life (Brezillon et al., 2008). To reconstitute the
liver of these animals with xenogenic hepatocytes, the uPA mice
need to be backcrossed with immunodeficient mouse strains,
such as the RAG2/, the Severe Combined Immune Deficient
(SCID), or SCID/beige mice, which lack functional B, T and
NK-cells (Rhim et al., 1995; Petersen et al., 1998; Dandri et al.,
2001b; Mercer et al., 2001). Following a single injection of one
million human hepatocytes into the mouse spleen, a proportion
of the transplanted cells migrates via the splenic and portal
veins to the liver where cells can integrate into the liver
parenchyma. A few days post-transplantation, isolated single
clusters of engrafted human hepatocytes start to proliferate, as
shown by the presence of cell-proliferation markers (PCNA and
Ki67), to form larger regenerative nodules which eventually
merge together to replace most of the diseased liver parenchy-
ma (Fig. 2). Reconstitution of the mouse liver takes eight to ten
weeks, meanwhile the levels of human chimerism can be
estimated by determining the concentration of human serum
albumin in mouse blood (Meuleman and Leroux-Roels, 2008;
Dandri and Petersen, 2012). Since the transplanted human
44 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949
hepatocytes reside in their natural environment, the liver, they
appear to maintain normal metabolic functions (Tateno et al.,
2004). The use of highly viable cryopreserved-thawed hepato-
cytes (Dandri et al., 2001a) gives the possibility to generate on
demand human chimeric mice, thus overcoming previous
limitations due to the narrow time frame available for
transplantation. Nevertheless, high levels of human chime-
rism are only achieved in homozygous uPA immunodeficient
recipients. Since these mice suffer from infertility, also the
breeder pairs need to be rescued by transplantation with
normal mouse hepatocytes (Brezillon et al., 2008). To
attenuate liver injury in new-born mice and hence delay
the production of the toxic transgene which makes trans-
plantation procedures necessary in the first month of life,
alternative mouse models, such as mice where the expres-
sion of the uPA transgene is inducible (Song et al., 2009) or is
regulated by the MUP (major urinary protein) promoter
have been developed (Tesfaye et al., 2013).
In 2007, two groups reported the establishment of an
alternative human-liver chimeric model, based on the use of
fumaryl acetoacetate hydrolase-deficient (FAH) mice, where
hepatocyte injury can be induced, at will, in adult mice
(Azuma et al., 2007; Bissig et al., 2007; Bissig et al., 2010).
FAH plays a crucial role in the tyrosine breakdown pathway
and its deficiency leads to hypertyrosinemia and liver failure.
A B
C D
E F
Fig. 2. Human chimeric livers generated after human hepatocyte transplan-
tation of immunodeficient (SCID/beige) uPA transgenic mice. Immunostain-
ing with human specific CK-18 antibody (brown) shows a close-up of a
human hepatocyte cluster (A) as well as an example of a cross-section from
a highly repopulated mouse liver. CF) Immune fluorescence staining of
different viral hepatitis antigens demonstrating susceptibility of repopulated
chimeric mice to a broad spectrum of hepatotropic viruses (HBV, HDV and
HCV). C) Establishment of HBV infection (HBcAg = green) and (D) HBV/
HDV co-infection (HBcAg = green; HDAg = red, hCK18 = blue) in human
hepatocytes. E) Close-up of HDV positive human hepatocytes in HDV
monoinfected mice (HDAg = red, hCK18 = blue) and F) HCV infected cells
(HCV core antigen = green, human calnexin = red) within the mouse liver.
However, accumulation of toxic metabolites can be avoided
by treating the mice with a pathway inhibitor, the drug NTBC
(2-(2-nitro-4-trifluoromethylbenzyol)-cyclohexane-1,3-dio-
ne), so that the animals are protected from the occurrence of
liver injury until drug administration is withdrawn. More
recently, mice lacking the Fah-gene, the recombination
activating gene 2 [Rag2] and the gamma-chain of the receptor
for IL-2, were successfully reconstituted with human hepato-
cytes, so that by transplanting higher amounts of human
hepatocytes, or by performing hepatocyte transplantation
twice, high rates of human hepatocyte chimerism could be
achieved in these so-called FRG mice (Bissig et al., 2010) (Fig. 1,
B). In an additional humanized mouse model that was lately
developed (Kosaka et al., 2013), human hepatocytes were also
successfully transplanted in mice expressing the herpes
simplex type-1 thymidine kinase (TK) transgene that were
backcrossed with NOG (NOD/SCID/gamma(c)(null)) mice.
Even in this case, human hepatocyte transplantation can be
performed in adult mice and at will, since mouse liver cells
expressing the TK-transgene can be selectively destroyed upon
administration of ganciclovir (Hasegawa et al., 2011).
Regardless of the mouse systems employed, the human
cells residing in the mouse liver have been shown to
maintain susceptibility of infection with human hepatotropic
viruses (Dandri et al., 2001b; Mercer et al., 2001; Bissig et al.,
2010; Lutgehetmann et al., 2011; Kosaka et al., 2013). From
the first successful transplantation of human hepatocytes
into uPA/RAG2 mice and establishment of de novo infection
with HBV (Dandri et al., 2001b), several groups demonstrat-
ed the establishment of HBV infection (Tsuge et al., 2005;
Meuleman and Leroux-Roels, 2008; Tsuge et al., 2010), as
well as of infection with other human hepatitis viruses in
these humanized mice (Fig. 2) (Mercer et al., 2001;
Meuleman and Leroux-Roels, 2008; Lutgehetmann et al.,
2012). Notably, after intra-peritoneal inoculation of HBV
infectious serum or cell culture derived virions, infection
establishment is first achieved in a small minority of human
hepatocytes, so that several weeks are needed to accomplish
viral spreading (Dandri and Petersen, 2012). After that,
nearly all human hepatocytes stain HBcAg-positive and
viremia reaches a stable plateau which, to a certain extent,
correlates with the levels of human chimerism.
The use of patient serum as virus inoculum allows the
functional analysis of distinct HBV genotypes, naturally
occurring variants and drug-resistance mutants. Moreover,
genetically engineered viruses produced in cell cultures can
be used to investigate the role of distinct viral proteins in
human infected hepatocytes and in the contest of physiolog-
ical levels of viral replication. Using this type of approach,
experiments with HBV strains lacking the precore protein
proved that the HBeAg is dispensable for viral infection and
replication in vivo (Tsuge et al., 2005), while the expression
of the regulatory HBx protein provided in trans was shown to
be essential for HBV replication in infected human hepato-
cytes, where viral transcription is exclusively driven by the
cccDNA minichromosome (Tsuge et al., 2010). Although
studies focusing on investigating how HBV may affect cellular
pathways and innate immune responses of the human
hepatocytes are still limited (Lutgehetmann et al., 2011),
highly repopulated mice have proved to be useful for the
study of drug metabolism (Katoh et al., 2007). In an attempt
 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949
to study the effects of viral infection on the host genome,
recent studies in human liver chimeric mice indicated that
HBV and HCV can contribute to the induction and accumu-
lation of aberrant DNA methylation in human hepatocytes
through the activation of NK-cell-dependent innate immune
responses (Okamoto et al., 2014).
3.1. Preclinical studies
Since mice are well-characterized small laboratory animals,
mice with humanized livers offer unique opportunities to
evaluate the in vivo efficacy of antiviral compounds targeting
different steps of the HBV life-cycle. Besides evaluating the
effect of clinically approved HBV polymerase inhibitors, such as
lamivudine (Tsuge et al., 2005), adefovir (Dandri et al., 2005)
and entecavir (Lutgehetmann et al., 2011), chimeric models
have been also employed to assess the in vivo efficacy of novel
polymerase or capsid inhibitors (Brezillon et al., 2011; Volz et
al., 2012). Humanized uPA mice served to assess the in vivo
efficacy of viral entry inhibitors, derived from the large
envelope protein of HBV, to prevent de novo HBV (Petersen
et al., 2008) and HDV infection (Lutgehetmann et al., 2012),
as well as to investigate the ability of the most clinically
advanced entry inhibitor, Myrcludex-B, to block HBV spreading
post-infection (Volz et al., 2013). In this study, Myrcludex-B
could block HBV dissemination from initially infected human
hepatocytes also when treatment was started 3 weeks
post-infection, at a time when mice already displayed detect-
able levels of circulating virions. Notably, after 6-weeks of daily
treatment not only the amount of HBcAg-positive hepatocytes
but also of intrahepatic cccDNA loads remained comparable to
values found in mice sacrificed 3-weeks post-infection, thus
indicating that not only intrahepatic spreading but also
intracellular cccDNA amplification mostly relayed on the
secretion of progeny viruses and their binding to target
hepatocytes (Volz et al., 2013).
The passage of infected tupaia (Lutgehetmann et al., 2010)
or human hepatocytes (Allweiss et al., 2013) isolated from
chimeric mice and transplanted into new uPA/SCID recipient
mice has also shown to permit investigation of the impact of
hepatocyte proliferation on cccDNA stability and activity in
vivo. The drastic reduction of intrahepatic cccDNA loads
induced by cell division even in the absence of antiviral therapy
underscores a potentially interesting weak point in HBV
persistence that deserves further investigations. Because of
the absence of adaptive immune cells in these chimeric mice,
immune-mediated elimination of the infected hepatocytes
cannot occur. Nevertheless, these systems offer unique oppor-
tunities to investigate factors that can affect the stability and/or
activity of the cccDNA minichromosome, as well as the direct
antiviral effects of cytokines and immune modulatory sub-
stances, such as interferons. Human hepatocytes residing in the
mouse liver were shown to maintain a functional innate
immune system, since they properly respond to stimuli
induced by exogenously applied interferon. To this regard,
human liver chimeric mice have been used to investigate
whether HBV can circumvent the induction of antiviral defence
mechanisms (Lutgehetmann et al., 2011). Upon administration
of regular human IFN, HBV was shown to inhibit the nuclear
accumulation of STAT-1, thus providing a potential mecha-
nism for the reduced induction of interferon stimulated
45
genes (ISGs) determined in HBV-infected human hepato-
cytes (Lutgehetmann et al., 2011). On the other hand,
studies in these mice also provided evidence that IFN can
inhibit HBV transcription by mediating epigenetic repres-
sion of the cccDNA minichromosome (Belloni et al., 2012),
while studies in uPA/SCID/beige mice demonstrated that
repeated administrations of the longer-active pegylated IFN
(peg-IFN) could breach the impairment of HBV-infected
hepatocyte responsiveness and induce sustained enhancement
of human interferon stimulated genes (ISGs) (Allweiss et al.,
2013; Allweiss et al., 2014). Because of the capacity of IFN to
induce epigenetic suppression of cccDNA-driven transcrip-
tion of both pregenomic RNA (pgRNA) and subgenomic HBV
RNAs, the stronger antiviral effects of peg-IFN exerted on the
human hepatocytes were shown to trigger a substantial decline
of circulating HBsAg and HBeAg levels in chimeric mice,
without claiming the involvement of immune cell responses
(Allweiss et al., 2014). Altogether, these studies demonstrate
that mice with humanized livers permit not only the
exploration of the direct antiviral effects of distinct sub-
stances but also the simultaneous and comparative analysis
of the distinct innate immune responses that can be induced
in human and mouse hepatocytes within the same liver. To
this regard, it was recently reported that type I, II and III IFNs
are differently induced in murine and human hepatocytes
by treating chimeric mice with complexes comprising
hepatotropic cationic liposomes and synthetic double-
stranded RNA analogs (Nakagawa et al., 2013). In contrast
to mouse hepatocytes, human hepatocytes appeared to
express higher levels of IFN- receptor-encoding genes,
while IFN- and IFN- were produced at lower levels in
these cells. Thus, these studies showed that expression and
function of the distinct IFNs may differ between animal
species, thus underlying the importance of validating results
obtained in murine systems also by employing primary
human hepatocytes.
Human liver chimeric mice can be super-infected or
simultaneously infected with different human hepatotropic
viruses (Fig. 2). Whereas such studies indicated an absence of
interference between HBV and HCV (Hiraga et al., 2009)
co-infection with the delta hepatitis virus (HDV) appeared
to hinder the kinetics of HBV infection and replication
(Lutgehetmann et al., 2012). This latter study showed that
establishment of HDV infection is highly efficient in naive
chimeric mice, since the majority of human hepatocytes
stained HDAg-positive long before HBV spreading was com-
pleted, thus confirming that HDV can replicate intrahepatically
also in the absence of HBV infection. Furthermore, in the setting
of HBV/HDV simultaneous infection the increase of HBV
viremia and intrahepatic cccDNA loads appeared significantly
slower than in HBV mono-infected mice, while HDV inocula-
tion in HBV stably infected mice led to a light reduction
(median 0.6log) of HBV viremia.
The intrahepatic latency of large delta antigen (HDAg) has
been detected following liver transplantation (Samuel et al.,
1995; Mederacke et al., 2012). To investigate whether HDV
can persist intrahepatically in the absence of HBV and be
rescued at later time points from HBV infection, humanized
mice have been recently infected with cell culture derived
HDV particles and the presence of HDAg-positive human
hepatocytes was determined after several weeks of HDV
46 M. Dandri, M. Ltgehetmann / Journal of Immunological Methods 410 (2014) 3949
inoculation to demonstrate establishment and maintenance
of intrahepatic HDV mono-infection (Giersch et al., 2014).
Although intrahepatic amounts of large HDAg and edited
HDV-RNA forms increased over time in HDV mono-infected
livers, HBV super-infection led to prompt viremia develop-
ment even after 6 weeks of latent mono-infection and the
infectivity of the rescued HDV virions was verified by serial
passage in naive chimeric mice (Giersch et al., 2014).
Altogether, these in vivo studies indicated that conversion
of a latent HDV infection to a productive HBV/HDV
co-infection may contribute to HDV persistence even in
patients with low HBV replication and in the setting of liver
transplantation.
Although the transplantation of mice is technically chal-
lenging and good quality primary human hepatocytes are
needed, most of the human-liver chimeric models available
have shown great potential for the study of human hepatitis
virus infection and preclinical drug investigation. Moreover, by
using hepatocytes from different donors, possible host varia-
tions and the impact of genetic polymorphisms of the donor on
the efficiency of antiviral therapy can be investigated. By
employing chimeric mice carrying human hepatocytes with
different IL28B gene polymorphisms, it could be shown that
viral replication is suppressed with comparable efficiency upon
peg-IFN administration, thus indicating that such genetic
variations mostly affect the responsiveness of the immune cells
rather than those of the infected cells (Watanabe et al., 2014).
3.2. Immune competent human liver chimeric mice
Human hepatocytes can be very abundant within the
chimeric livers but non-parenchymal cells, such as sinusoidal
endothelial cells and Kupffer cells, are of murine origin. As a
consequence, development of fibrosis and pathophysiologic
processes that are commonly associated with chronic viral
hepatitis infections but involve a crosstalk between the
hepatocytes and other liver resident cells is not observed in
the above-mentioned systems. Moreover, since these chime-
ric animals are genetically immune deficient they are not
suited for vaccine studies and for evaluation of adaptive
immune responses. To circumvent, at least in part, this
problem, adoptive transfer of antibodies or lymphocytes can
be applied. In the HCV field, for instance, these methods were
used to analyze neutralizing activity of antibodies (Law et al.,
2008; Meuleman et al., 2012) or of activated lymphocytes
(Ohira et al., 2009). Partially haploidentical human periph-
eral blood mononuclear cells (PBMC) were also recently
transferred in uPA/SCID chimeric mice after the establish-
ment of HBV infection (Okazaki et al., 2012). Notably,
infiltrating human immune cells caused severe hepatocyte
degeneration, while treatment with anti-Fas antibodies or
depletion of dendritic cells prevented the death of human
hepatocytes.
A different attempt to generate a humanized mouse
model harboring both human liver cells and a human immune
system was the development of the AFC8 model (Washburn
et al., 2011). These mice were obtained by crossing
BALB/c-RAG2/c/mice, which lack functional B, T and
natural killer cells, with mice carrying a liver-specific suicidal
transgene with inducible activity based on the induction of
caspase 8 in mouse hepatocytes. These animals were used to
transplant simultaneously human fetal hepatocytes and
hematopoietic stem cells that were obtained by the digestion
of human fetal liver tissues (1518 weeks of gestation period)
(Fig. 1). Since the fetal liver provides both types of cells,
reconstitution of both cell lineages is syngeneic, hepatocyte
rejection by the human immune system is not expected.
Although HBV infection studies were not performed, after
inoculation of hepatitis C virus, low levels of intrahepatic viral
replication, as well as T-cell responses and development of
fibrosis could be determined. Despite partial success and the
ethical restrains encountered by employing human fetal
tissues, the generation of chimeric systems equipped with
both a human liver and a functional human immune system,
with a matched major histocompatibility complex, still
remains a major challenge. The latest advances reported by
Gutti et al. showed the feasibility to reconstitute a uPA-NOG
mouse strain with both adult human hepatocytes and
hematolymphoid cells (Gutti et al., 2014). In this study, dual
reconstitution was achieved by transplanting fetal or even
adult hepatocytes and mismatched hematopoietic stem cells
(CD34+ HSCs) derived from either a fetal liver or umbilical cord
blood. As an alternative and well-tolerated procedure to total
body irradiation, mice received 3 days of a treosulfan-based
chemotherapeutic conditioning before HSC injection. The
presence of CD8+ and CD4+ human cells and of human
hepatocyte clusters was observed in the liver of these animals.
No cell-mediated rejection but also no evidence of cell
interactions were determined in animals reconstituted with
mismatched HSCs. Although the applicability of these ap-
proaches for the study of HBV and HDV associated immune
pathological processes needs further research, these technical
advances have paved the way for the development of dually
reconstituted humanized systems.
Acknowledgment
We thank our laboratory team, particularly Dr. Tassilo
Volz, Dr. Lena Allweiss & Katja Giersch, for their excellent
work and dedication to the model, as well as the German
Research Foundation (SFB841 (project A5 and A8) and
DZIF-BMBF (TTU05.804)) for continuous financial support.
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