Animalresources.med.nyu.edu

Lab Animal
Cell Culture and Animal Models of
Viral Hepatitis. Part I: Hepatitis B
Chandan Guha, MD, PhD, Sankar Mohan, PhD, According to estimates, the hepatitis B virus (HBV) infects ∼2 billion people glob- ally, producing a spectrum of disease including acute and chronic viral hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV and hepatitis C virus (HCV)infections constitute the most common indication for liver transplantation in theUnited States and Europe. A vaccine is now available to prevent acute HBV infec- Despite the existence of a preventative vac-
tion, and routine screening of blood and blood products for HBV reduces post- cine, HBV represents a substantial threat to
transfusion viral hepatitis. Approximately 10% of HBV-infected patients develop public health, suggesting the need for
a chronic disease with devastating consequences, and there is no curative therapyfor the ∼300 million patients worldwide who are infected chronically with HBV.
research to develop new treatments to com-
Thus there is an urgent need for effective treatment strategies to prevent the enor- bat the disease. The authors review the
mous burden of viral hepatitis in worldwide health care.
available in vitro and in vivo models, includ-
Availability of laboratory animal models of HBV infection is obviously of crit- ing recently developed transgenic and
ical importance for the development of effective methods for treatment of thesediseases. Chimpanzees are the only well-tested in vivo animal models for HBV chimeric mouse models.
infection, and development of smaller and less expensive animal models shouldgreatly facilitate this area of research. In this article, we will review the available invitro and in vivo models for studying HBV infection. Cellular and animal modelsfor HCV will be reviewed in a subsequent article.
Life Cycle of HBV
HBV is a hepadnavirus, a class of DNA viruses that causes hepatitis in many species
(reviewed in ref. 1). Each hepadnavirus infects a narrow range of host species. HBV,
which infects humans and chimpanzees, is an enveloped, noncytopathic virus. The
HBV genome consists of plus and minus strands that are incomplete circles but are
stabilized by partial overlapping. The RNA transcript of the viral genome, termed
the pregenomic RNA (pRNA), is reverse-transcribed to the DNA genome during
viral replication2. In this respect, HBV resembles RNA viruses.
The virus seems to enter cells by a receptor-mediated process3. After uncoating of the capsid and nuclear transport, nuclear DNA repair enzymes complete theplus and minus strands of the open circular genomic DNA, generating a cova-lently closed circular DNA (cccDNA) molecule that provides the template for viraltranscription4.
This viral genome is transcribed into four partly overlapping transcripts (3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb in length) that are exported into the cytoplasm, wherethey are translated into viral proteins. The 3.5-kb transcript gives rise to the poly-merase and to the core and precore proteins, and also serves as the pRNA templatefor reverse transcription into the genome of the mature virus5. The 2.4-kb and2.1-kb transcripts generate the envelope proteins including the hepatitis B surfaceantigen (HBsAg). Studies on the woodchuck hepadna virus (WHV) suggest thatthe X protein, which is translated from the 0.7-kb transcript, may be required forinfection initiation6. The precore protein contains a leader sequence that trans- The authors are at Albert Einstein College of ports it into the endoplasmic reticulum (ER), where it is processed and eventual- Medicine, 1300 Morris Park Ave., Bronx, NY. Please ly secreted as hepatitis B e antigen (HbeAg)7.
address correspondence to J. Roy-Chowdhury at The core and polymerase proteins associate with the pRNA, forming capsids inside which viral DNA is synthesized by reverse transcription. During transla- Volume 33, No. 7 Lab Animal
In Vivo Models
TABLE 1. Models to study HBV infection
Cell culture models
The Chimpanzee as a Model for HBV Infection
The restricted host range of the hepatitis viruses has hampered the • Stable transfection of hepatocyte cell lines• Surrogate models development of suitable animal models. Table 1 provides a partial
Hepadna viruses: Woodchuck hepatocytes for WHV; Currently, one of the few animal models for HBV infection is In vivo models
the chimpanzee. In the wake of the discovery of the Australia anti- gen11,12 and the establishment of its relationship to HBsAg13, as well • Tupaia belangeri sinensis• Transgenic mice as the discovery that Africans had a very high rate of positivity to • Immunodeficient mice or tolerized rats transplanted with human HBsAg, a marker of chronic HBV infection14, there was a search for the presence of HBV infection among chimpanzees caught in the Woodchuck for WHV; Pekin duck for DBV;tamarins for GB wild in Africa. Researchers soon demonstrated that ∼3–6% of the virus A and B (GBV-A, B); ground squirrel for GSHV wild-caught chimpanzees were positive for HBsAg, and ∼50% ofthe older animals were positive for the antibody to HBsAg, a mark-er for resolved HBV infection15. The thinking was that these HBVinfections resulted from the practice of injecting human serum in tion, HBsAg achieves insertion into the ER membrane. Aggregates wild-caught animals to improve their survival during transit and of the envelope protein may assemble with the viral capsid, form- confinement; however, various groups have recently demonstrated ing infectious virions termed ‘Dane particles’, or may emerge as free the presence of a unique chimpanzee HBV strain that was verified HBsAg. A few of the RNA-containing viral capsids may not assem- by sequencing of the entire genome16–18. Chimpanzees inoculated ble with HBsAg but instead return to the nucleus, where they with HBsAg+ chimpanzee plasma developed characteristic hepati- tis, HBs antigenemia, and eventual anti-HBs seroconversion19.
Since these seminal studies, virologists have characterized the biological properties of HBV in chimpanzees. Thomssen et al.20 In Vitro Model Systems
demonstrated that the 42-nm Dane particles were the infectious A major obstacle to the research on the development of drug and virion of HBV. When chimpanzees were immunized with HBsAg, gene-based therapies for HBV infections has been the lack of an all developed high titers of antibodies to HBsAg. Soon researchers efficient cell culture system or a readily available small-animal were using chimpanzees to evaluate the safety and immunogenici- model, permissive for viral infection and replication.
ty of candidate HBV vaccines that had been prepared from the plas- Lack of a robust in vitro cell culture system has seriously ham- ma of infected human chronic carriers21. These studies also proved pered the progress of HBV research. A partial list of existing cell that these plasma vaccines were actually free from infectious virus- culture models appears in Table 1. For reasons that are not clear,
es. Because there were no in vitro assays for detection of infectious infection of primary hepatocytes and established cell lines with HBV, the chimpanzees were the only means of ensuring that batch- hepatitis viruses has produced poor viral replication and low viral es of plasma-derived HBV vaccine did not contain live HBV22.
yields and has suffered from poor reproducibility (reviewed in ref.
Later, chimpanzees also served in the evaluation of the inactivation 8), although the addition of polyethylene glycol to primary hepato- of HBV and HCV for the manufacture of virus-free plasma deriva- cyte cultures maintained in the presence of 2% dimethylsulfoxide tives, such as coagulation Factors VIII and IX preparations23,24. The markedly increases the infection of HBV9. In vitro cell culture mod- use of chimpanzees in testing for the development of new immu- els can at best demonstrate infectivity by the virus but are not suit- nization strategies such as DNA vaccines continues22.
able to study viral life cycle because of very low levels of viral repli- Chimpanzees have contributed immensely in the development cation. They could still prove useful for drug studies.
of HBV vaccines, evaluation of the safety of blood products, and Because infection of hepatocytes with infected sera did not the discovery of HCV. However, their limited availability, expense, always give similar results, researchers generated cell lines in which endangered status, and the lack of chronic liver disease precludes the viral genome was expressed from chromosomally integrated the study of pathogenesis of cirrhosis and HCC. It is also not prac- viral cDNA under the control of constitutive or inducible promot- tical to use chimpanzees for the preclinical evaluation of novel ers. Because stable transfection, unlike natural infection, produces drugs and therapies of viral hepatitis.
virus from an integrated viral transcription template, researchershave also attempted to produce viral infection after infection with The Tupaia as a Model for HBV Infection
recombinant adenoviruses expressing HBV viral genome from an Although nonhuman primate models have helped us learn a great deal about viral infectivity and clearance in a truly unselected pop-ulation, a small-animal system of viral hepatitis infection would be Lab Animal
of great value. Tree shrews (Tupaia belangeri sinensis) are nonro- ducks helped to elucidate the replication scheme of hepadnavirus- dent small animals that are phylogenetically close to primates. They es5,36. Approximately 65–75% of neonatal woodchucks are suscep- adapt easily to a laboratory environment. Apart from the chim- tible to chronic WHV infection37. Almost all chronic carriers of panzee, the tree shrews are the only other animals that HBV can WHV developed HCC within 3 years of infection38,39.
Histologically, there was a characteristic absence of cirrhosis in HBV can infect primary hepatocytes isolated from tupaia livers, these animals39. In contrast to humans, HCC is equally prevalent in and the infection results in HBV cccDNA and messenger RNA male and female woodchucks. This may be the result of the unusu- (mRNA) synthesis in hepatocytes and secretion of HBsAg and al circannual reproductive cycle of woodchucks, in which the males HBeAg into the culture medium26. The early steps of HBV infection have abdominal testes for 8 months of the year, resulting in func- in tupaia hepatocytes are very similar to those in human hepato- tional castration40. The WHV viral genome integrates into the host cytes, with both pre-S1 and S antigens being necessary for infec- chromosome, and researchers have cloned the integrated tion27. However, the efficiency of HBV infection is low, and it has sequences41–43. There is extensive rearrangement of the integrated been reported that human serum interferes with HBV binding to viral sequences, and there is incomplete integration of the viral Purification of HBV virions by gradient sedimentation greatly The mechanism of WHV- and HBV-induced HCC seems to enhanced virus binding and infectivity in tupaia hepatocytes28. To consist of many steps. Some have postulated that during chronic circumvent the restrictions posed by infectious serum during viral active hepatitis, viral DNA integration occurs randomly and at a entry, researchers have also infected tupaia hepatocytes with low frequency in hepatocytes42. The increased rate of hepatocyte recombinant adenovirus containing the complete HBV genome29.
proliferation induced by liver necrosis and regeneration during These experiments demonstrate that primary tupaia hepatocytes chronic disease probably stimulates viral genomic integration. The support all steps of HBV replication with formation of cccDNA and integrated viral sequences provide focal points for the generation of secretion of HBsAg, HBeAg, replication-competent nucleocapsids, chromosomal aberrations, which induce HCC. HCC in chronically infected woodchucks occurs more frequently than in ground squir- HBV can also infect tupaias in vivo, resulting in viral DNA repli- rels chronically infected with GSHV38. Because woodchucks can be cation in tupaia livers. The acute infection results in clearance of infected with both hepadnaviruses, one research group experimen- viremia and HBsAg, with appearance of antibodies to HBeAg and tally infected neonatal woodchucks with WHV and GSHV, respec- HBsAg26. This is very similar to the acute, self-limited HBV infec- tively, to determine whether the difference in incidence of cancer is tion in humans. Furthermore, immunization with HBV vaccine secondary to host factors versus viral genetic variations44. By year 2, can prevent the experimental infection of tree shrews with HBV, there was induction of hepatocellular carcinoma in 13 out of 16 with a protection rate of ∼88% (ref. 25). Researchers have examined chronic WHV carriers. In contrast, only 1 out of 16 chronic GSHV in tupaias the hepatocarcinogenic effects of HBV infection alone carriers developed a hepatic adenoma. Thus, it seems that the dif- and in combination with dietary aflatoxins30,31. Chronic infection ference in WHV and GSHV oncogenic capacity may be due to viral induced hepatocellular carcinoma by week 160 (ref. 30). The inci- genetic variations. Because a high percentage of neonatal wood- dence of HCC was significantly higher in the animals that were chucks are susceptible to experimental chronic WHV infection, both infected with HBV and exposed to aflatoxin (52.94%) than in they frequently serve as models for preclinical assessment of antivi- those solely infected with HBV (11.11%) or exposed to aflatoxin B1 ral drugs being developed for treatment of chronic HBV infec- (12.50%)31. In contrast to control animals, precancerous lesions, tion45–48. Thus, the woodchuck model has contributed importantly including liver cell dysplasia and enzyme-altered hyperplastic hepa- to the understanding of viral replication, chronic infection, and tocyte foci, were evident before the occurrence of HCC, and the fre- hepatic carcinogenicity of hepadnaviruses. However, the lack of quency of their appearance correlated well with the incidence of inbred strains and of immunological reagents to study lymphocyte subsets in woodchucks remains a formidable obstacle to investiga-tion of the immunological pathogenesis of viral hepatitis.
Surrogate Animal Models: Woodchuck, Duck, Ground
Immunotherapeutic agents against WHV will not be effective Squirrel
against HBV, and vaccines against HBV can never be tested in Researchers have used naturally occurring HBV-like viruses, such woodchucks. Furthermore, these animals do not develop cirrhosis.
as the ground squirrel hepatitis virus (GSHV) that infects ground Therefore, there are limitations to the types of experiments that squirrels32, the duck hepatitis B virus (DHV) that infects Pekin could be devised to investigate the mechanisms of pathogenesis of ducks33, and the woodchuck hepatitis virus (WHV) that infects woodchucks34, to investigate viral DNA integration and to assessantiviral therapies35. WHV induces HCC in its host34.
Rodent Models of HBV Infection
The mammalian hepadna viruses have a genetic organization Although the natural wild-type animal models are useful for antivi- very similar to that of the HBV genome. Studies of DHV-infected ral studies, inbred strains allow independent manipulation of the Volume 33, No. 7 Lab Animal
host and viral variables that determine infectivity and the to produce HCC65,66. These studies indicate that HBxAg is onco- immunopathological manifestations of the chronic hepatitis.
genic. Experiments in transgenic mice also demonstrated that the Strong evidence indicates a direct link of immune-mediated etiol- HBxAg binds to p53 and completely blocks its entry to the nucle- ogy in chronic liver disease induced by HBV infection. HBV-infect- ed patients undergoing immunosuppressive therapy often have To examine whether the liver disease in viral hepatitis is widespread virus gene expression in the liver and high levels of immune-mediated, researchers generated clones of HBsAg-specific virus in the serum without developing severe liver disease.
cytotoxic T lymphocytes (CTLs) in wild-type naive mice and then However, upon termination of the immunosuppressive therapy used them in adoptive-transfer experiments in transgenic mice.
they often develop severe liver disease49,50. The immune response to Adoptive transfer of HBV-specific CTLs resulted in the clearance of HBV-encoded antigens mediates both the viral clearance and the HBsAg and a spike of alanine transaminase (ALT) in the serum68.
pathogenesis of the HBV-induced liver disease51. The humoral anti- The CTLs induced an immune-mediated acute hepatitis and lysed body response to viral envelope proteins mediates the viral clear- HBsAg-expressing hepatocytes in a major histocompatibility class ance. The cellular immune response to the envelope, nucleocapsid, I–restricted manner. Mice that secreted HBsAg developed a tran- and polymerase antigens eliminates infected hepatocytes. In sient hepatitis with lysis of ∼5% of the hepatocytes. In contrast, patients with acute infection who successfully clear the virus, the T- mice that retained HBsAg developed an acute fulminant hepatitis cell responses to HBV are vigorous, polyclonal, and multispecific.
that caused death in several animals69. A process of several steps, In contrast, weak immune responses to few HBV antigens are char- involving both direct and indirect mechanisms, mediated the CTL- acteristic of infections that become chronic51. With the exception of mediated hepatitis. First, CTLs induced apoptosis among scattered, transgenic mice that overexpress viral proteins52, HBV-induced HBsAg-expressing hepatocytes. Second, there was recruitment of direct cytopathic effects (CPE) do not induce liver disease. Tissue antigen-nonspecific lymphocytes and neutrophils that induced a culture systems that support HBV replication do not develop focal inflammatory response within hours and amplified the local CPE of the CTLs. Finally, the most destructive pathogenetic func- The development of transgenic mice permitted investigators to tion of the CTL is to secrete interferon-γ (IFN-γ) upon encounter- study the replication, gene expression, and immunopathogenesis of ing HBsAg-expressing hepatocytes in vivo. IFN-γ activates the HBV in a laboratory small-animal model. It is important to note intrahepatic macrophage and induces a delayed-type hypersensitiv- that, with some exceptions, most transgenic mouse models are tol- ity response that destroys the liver and kills the mouse69. There is erant to viral antigens and therefore fail to mount an immune increasing evidence from the transgenic mouse models that the response to the virus. Thus, innate and cytokine-mediated immune clearance of HBV viral gene expression and replication can be responses against HBV-infected cells are not amenable to study in achieved by noncytolytic mechanisms that involve secretion of cytokines, such as IFN-γ and tumor necrosis factor-α (TNF-α) (ref.
HBV transgenic mouse models. Table 2 includes a summary of
70). Administration of monoclonal antibodies to IFN-γ and TNF- the phenotypes of several transgenic mouse lines expressing all α abolished the CTL-mediated clearance of HBsAg and acute open reading frames of HBV or specific HBV proteins from various hepatitis70. These cytokines mediated the virocidal effects of HBV- promoters. Transgenic mice generated in 1985 expressed HBsAg55 specific CTLs by reducing the steady-state levels of most viral RNAs or the HBV proteins HBsAg, and pre-S and X antigens56. The mice in infected hepatocytes71. The cytokines induced three intrahepatic secreted HBsAg in the serum but, because they were immunologi- nuclear proteins that bound to a 91-bp post-transcriptional regula- cally tolerant to the transgenes, they failed to develop any liver dis- tory element in the 5′ end of the HBV RNA and prevented its ease. Researchers also created transgenic mice that support HBV export from the nucleus72. These pathways ultimately destabilized viral replication57–59. In all cases, no liver disease developed, sug- viral RNA and enhanced its degradation, resulting in the elimina- gesting that HBV was not directly cytopathic. However, transgenic tion of HBV nucleocapsid particles and their cargo of replicating mice that overexpressed HBsAg along with pre-S polypeptide retained the proteins in the ER, resulting in its accumulation in the Although these experiments established models of acute fulmi- liver60. Over time, these mice accumulated hepatotoxic amounts of nant hepatitis, the major clinical problem resides with HBV carri- the HBV large-envelope polypeptides in the liver and displayed ers with chronic liver disease. The transgenic mice were immuno- hepatocellular injury, regenerative hyperplasia, chronic inflamma- logically tolerant to HBsAg and developed no evidence of chronic tion, oxidative DNA damage, transcriptional deregulation, and ane- liver disease. To break the tolerance to HBV antigens, researchers uploidy that eventually progressed to HCC52,61,62. These experi- thymectomized, irradiated (9 Gy), and reconstituted these mice ments indicated that chronic liver injury and hepatocellular regen- with bone marrow depleted of T cells before adoptive-transfer eration are prerequisites to the development of HCC. HCC also experiments with splenocytes from syngeneic nontransgenic developed, even in the absence of liver inflammation, in mice that donors that had been previously immunized with HBsAg.
expressed high levels of the HBVx antigen (HBxAg) in the liver63,64.
Vaccinated donor mice produced antibodies to HBsAg and In contrast, transgenic mice expressing low levels of HBxAg failed induced CTLs against HBV. Adoptive transfer of HBV-specific Lab Animal
TABLE 2. Hepatic phenotype of HBV transgenic mice
cytes in these mice resulted inclearance of virus from liver Transgene
Promoter
Phenotype
Reference
same mice, transfer of ∼50 mil-lion naive splenocytes resulted Cytokine- and CTL-induced hepatitis.
mice. Adoptive transfer of naivesplenocytes resulted in eleva- hepatocytes is partly responsi-ble for the acute phase of transfer of splenocytes fromRAG–/– or TCR-α–/– donors did CTLs resulted in the development of acute hepatitis, with the not produce injury, researchers postulated that NKT cells were the clearance of HBsAg, followed by chronic hepatitis. In the setting of effector cells of the innate immune response against HBV.
liver destruction, regeneration, and chronic hepatitis, the bone Consistent with a role of NKT cells, adoptive transfer of NK1.1+- marrow–reconstituted mice developed liver tumors, mostly HCC, depleted splenocytes abolished the early rise of ALT, whereas trans- indicating that the pathogenesis of HCC has an immune-mediat- fer of NK1.1+ cells augmented the liver injury. Thus the first-line ed component73. Nude mice intrahepatically injected with HBV defense against HBV viral infection is probably mediated by NKT DNA developed chronic liver disease74. To develop a transgenic mouse model that is not inherently tolerant to the viral transgenes, Other rodent models of HBV infection. Because rodents cannot
researchers created a HBV transgenic severe combined immunod- be infected with HBV, researchers have offered several strategies to eficiency (SCID) mouse75. These mice supported HBV gene transfect hepatocytes in vivo with plasmids expressing portions of expression and replication. Because they lack T and B lymphocytes the viral genome. One group injected a plasmid expressing the that account for adaptive viral immunity, these mice were not tol- HBsAg genes directly into the liver of adult rats with non-histone erant to HBV proteins. A single adoptive transfer of 10 million chromosomal protein high-mobility group 1 by the hemaggluti- Volume 33, No. 7 Lab Animal
nating B virus of Japan (Sendai virus)–liposome method77. Within The previous reports demonstrate that transplanted human 2 days of injection, HBsAg was detectable in the serum. Multiple hepatocytes can survive for a long time in immunodeficient mice, injections of the plasmid resulted in production of antibodies to without undergoing any significant repopulation of the host liver.
HBsAg and induction of a focal inflammatory infiltrate in the liver.
Transplanted hepatocytes can, however, repopulate a host liver Recently, researchers developed a murine model for studying under a variety of genetic or acquired conditions (reviewed in ref.
human HBV replication, immunogenicity, and control upon 83). Adult differentiated hepatocytes proliferate extensively and hydrodynamic injection of a sleeping beauty transposon contain- repopulate the host liver after transplantation into the liver of ing the replication-competent, 1.3-kb, linear HBV genome78. The urokinase-type plasminogen activator (uPA)–transgenic84,85 or treatment resulted in synthesis of HBV antigens and replicative fumarylacetoacetate hydrolase–deficient mice86, and in rats treated intermediates and secretion of HBV into the blood. Antiviral anti- with the plant alkaloid, retrorsine87, or subjected to preparative body induction by day 7 resulted in the clearance of HBV antigens.
irradiation of the liver88,89. In all these models, the host liver cells Interestingly, the order of appearance of various HBV-specific are either dying or failing to proliferate in response to the prolifer- antibodies (anti-core antigen → anti-HBeAg → anti-HBsAg) mir- ating stimulus of reduced hepatic mass. Under these circum- rored precisely the sequence observed during a natural HBV infec- stances, normal transplanted hepatocytes undergo preferential tion in humans. HBV transcripts and replicative intermediates dis- proliferation, eventually replacing a large proportion of the host appeared from the liver by day 15, after the appearance of HBV- specific CD8+ CTLs. The hydrodynamic method of HBV transfec- Recently, researchers developed a human-mouse liver chimera tion in vivo might allow investigators to study the role of individ- by exploiting the strengths of two different mouse models, the Alb- ual viral genes in the viral life cycle. Moreover, dissection of com- uPA transgenic and the immunodeficient SCID or RAG-2–defi- ponents of the HBV-specific immune system is possible, allowing cient mice. Transgenic mice expressing uPA from a liver-specific examination of the contribution of innate versus adaptive immune (albumin) promoter show progressive death of hepatocytes84,90. A response. Sprinzl et al.10 constructed adenovirus vectors contain- small number of hepatocytes in heterozygous animals sponta- ing the 1.3-fold-overlength human and duck hepatitis B virus neously lose the transgene at a low frequency, resulting in progres- genomes. The adenovirus-mediated HBV genome transfer initiat- sive proliferation of the transgene-deleted hepatocytes and eventu- ed HBV replication in established cell lines, in primary hepatocytes al repopulation of the mouse liver84. In homozygous mice, trans- from various species, and in the livers of mice. There was release of plantation of normal, wild-type hepatocytes results in extensive high-titer hepatitis B virions into the culture medium of infected proliferation of the donor cells with near-total replacement of the cells and secretion of infectious virions into the sera of mice.
host liver90. To allow xenotransplantation, researchers have bred the Human-mouse chimeric liver models of viral hepatitis. The
uPA-transgenic mice with immunodeficient mice (SCID/beige or long-term engraftment of human hepatocytes in the mouse would RAG-2 gene knockout mice) and have exploited the uPA-scid or the be useful for the study of viral hepatitis. The transgenic mouse uPA-RAG-2 knockout mice models to allow orthotopic engraft- models of HBV do not allow a complete viral life cycle, especially ment and repopulation by woodchuck hepatocytes that can be the early stages of viral binding and entry into hepatocytes. The infected with WHV91 and human hepatocytes that can be infected viral DNA in transgenic mice is stably integrated, and the produc- tion of an episomal transcriptional genomic template does not Initial efforts of several laboratories to repopulate the livers of occur. After intrasplenic transplantation, transplanted hepatocytes immunodeficient Alb-uPA-transgenic mice by transplanting engraft and integrate into the host liver, maintain normal function, human hepatocytes yielded disappointing results, leading to the and survive permanently79,80. To create a chimeric human-mouse notion that human hepatocytes might not be able to respond to liver, Brown et al.81 transplanted immortalized human hepatocytes, the mitogenic stimulus within mouse livers. There are several con- permanently transfected with an HBV genome, intrasplenically ditions that determine successful engraftment and repopulation of into recombination activation gene-2 (RAG-2)–deficient immuno- human hepatocytes in these animals. First, the quality of the compromised mice. The transplanted human hepatocytes human hepatocytes is critical. Hepatocytes, isolated from livers remained observable in the mouse liver for as long as 8 months, and that had undergone only short periods of ischemia and were >80% the engrafted cells secreted 3 × 107–3 × 108 virions/ml of blood. In viable, could be transplanted successfully92. Second, the hepatocyte another approach, researchers achieved long-term engraftment of repopulation was less in uPA-heterozygous mice than in the primary hepatocytes transplanted in a matrix under the kidney homozygous counterparts. Human hepatocytes replaced as much capsule of immunodeficient nonobese diabetic (NOD)/SCID mice as 15% of the mouse liver hepatocytes in immunodeficient uPA- with the administration of an agonistic antibody against c-Met, the heterozygous mice92. In contrast, there was as much as 50% hepatocyte growth factor receptor82. These mice were susceptible to replacement of the host liver in uPA-homozygous mice93. The HBV infection and supported the completion of the viral life cycle.
human hepatocytes survived for as long as 35 weeks, and upon Furthermore, the investigators demonstrated a superinfection of infection with HCV-infected serum there were persistent high lev- the HBV-infected mice with hepatitis delta virus (HDV).
els of viremia. The virus could be serially passaged to new mice.
Lab Animal
Maintenance of homozygous Alb-uPA–immunodeficient mice HBV core antigen96. It is not clear whether the HBV-infected liver is difficult and requires special expertise. Of the newborns, ∼35% xenograft in the trimera mice exhibited chronic liver injury, as seen die from bleeding disorder. The high neonatal mortality of the Alb- in chronic viral hepatitis. Because the xenograft is placed under the uPA–homozygous mice is a problem that could limit the usefulness kidney capsule and not in its normal anatomical position, this of this model to investigators. It would be interesting to examine model may not allow investigations of immune-mediated chronic whether the use of hepatotoxic transgenes, such as that encoding liver damage that is seen in viral hepatitis.
uPA, could be circumvented using other regimens of liver repopu- Researchers have achieved specific immune tolerance to human lation such as retrorsine87 or preparative hepatic irradiation88,89.
hepatocytes in rats without generalized immune suppression by The applicability of the human-mouse liver chimeric models could injecting primary human hepatocytes into the peritoneal cavity of extend beyond the field of human-specific infectious diseases, into fetal rats, 17 days after conception97. Within 24 hours after birth, the areas of liver-directed gene therapy vectors, drug metabolism, they transplanted tolerized rats with human hepatocytes intrasplenically, and then infected them with HBV 7 days later.
To permit xenografts, researchers develop most of the human- HBV-infected mice produced HBsAg within 3 days of infection, mouse chimeric models in immunodeficient mice. Thus no one has and serum HBV DNA remained detectable for as long as 15 weeks yet shown whether HBV can infect and replicate in normal human after infection. The liver and serum of these animals contained hepatocytes in an immunocompetent mouse model.
cccDNA, reflecting HBV replication. Because the follow-up was Immunodeficient mouse models of viral hepatitis are useful to test limited to 15 weeks, it was not possible to assess whether the infect- the direct antiviral effect of drugs or gene therapy, but they cannot ed rats acquired chronic HBV infection. The number of engrafted address the issues of immune-mediated clearance of the virus or human hepatocytes was small and, therefore, the level of viremia CTL-mediated hepatocellular damage in chronic viral hepatitis. It was low. A further improvement on this model would be a prepar- is feasible to engraft human hematopoietic stem cells into sub- ative regimen, such as irradiation of the host liver98, that would lethally irradiated NOD/SCID mice, thereby establishing all human allow repopulation of the host liver by the transplanted human hematopoietic lineage cells in the peripheral blood. Thus, one could hepatocytes. Experiments are in progress to examine whether the envision transplanting human bone marrow cells in the SCID/Alb- transplanted human hepatocytes could proliferate and repopulate uPA–transgenic mice before or after human hepatocyte transplan- the host liver in xenotolerant animals.
tation to permit the investigation of immune effects of viral hepati-tis infection.
Recently, researchers have developed two models that allow the In vivo models of HBV based on cell culture generally involve pri- investigation of the host-viral interactions in an immunocompe- mary hepatocytes or cell lines derived from hepatocytes. However, tent mouse model. First to be developed was a human-mouse infection of these cells with HBV has produced poor viral replica- chimera, the trimera system, in which functional human lympho- tion and low viral yields. Therefore, although these cell culture sys- cytes were engrafted in normal strains of mice that had been made tems demonstrate infectivity by the virus and may be useful for immunodeficient by lethal total body irradiation, followed by sal- some drug studies, they are inefficient models for studying the vage SCID mouse bone marrow transplantation94. Following trans- viral life cycle. Although HBV can be generated from integrated plantation in the chimeric mice, human lymphocytes colonize HBV genome into host cell chromosomes, in this model the mode mouse lymphatic organs and secrete human antibodies. Trimera of viral replication is different from that in natural infection.
mice engrafted with lymphocytes from human donors positive for Among the in vivo models, chimpanzees are natural hosts for HBV.
antibodies to HBsAg mounted a strong memory response to Chimpanzees develop acute hepatitis after HBV infection and HBsAg94. In another variation of the trimera mouse, researchers mount immune responses, but they do not develop chronic liver created a human liver chimera by transplanting human liver under disease. Limited availability, endangered status, and expense fur- the kidney capsule in mice with ablated bone marrow, reconstitut- ther limit the routine use of these animals. Recently, researchers ed with SCID bone marrow. There was prolonged viremia in these have shown that a strain of tree shrews (T. belangeri sinensis) devel- mice when the transplanted liver was infected ex vivo with HBV95.
ops acute and, in some cases, chronic hepatitis after infection with Because the liver specimen was transplanted en bloc, the human HBV. Because of the paucity of natural animal hosts for HBV, hepatocytes would retain some physiological relationship with researchers have thoroughly studied surrogate animals (e.g., wood- extracellular matrix and nonparenchymal cells. Investigators have chuck, duck, and ground squirrel) that host hepadna viruses spe- used these trimera mouse models to examine the efficacy of antivi- cific to their species. Unfortunately, these animals do not develop ral agents in the clearance of viral loads; in addition, they have ana- cirrhosis and thus are not useful for testing anti-HBV vaccines.
lyzed therapeutic vaccination strategies against HBV in this Transgenic mouse models expressing all or some of the HBV open model96. Transfer of antigen-loaded dendritic cells together with reading frames have allowed investigators to study the replication, autologous peripheral blood mononuclear cells from HBV-naive gene expression, and immunopathogenesis of HBV. Transgenic donors induced a strong primary T-helper cell response against mouse models are tolerant to HBV and do not naturally mount an Volume 33, No. 7 Lab Animal
10. Sprinzl, M.F., Oberwinkler, H., Schaller, H. & Protzer, U. Transfer of immune response to it or develop hepatitis. However, investigators hepatitis B virus genome by adenovirus vectors into cultured cells have used various methods to reconstitute their lymphocyte pool and mice: crossing the species barrier. J. Virol. 75(11), 5108–5118
with anti-HBV CTLs or NKT cells to generate immune-mediated 11. Blumberg, B.S., Alter, H.J. & Visnich, S. A “new” antigen in leukemia hepatitis, resembling human HBV hepatitis, permitting the identi- sera. JAMA 191, 541–546 (1965).
fication of lymphocyte subsets responsible for causing HBV 12. Blumberg, B.S., Gerstley, B.J., Hungerford, D.A., London, W.T. & hepatitis. Although HBV proteins are not generally cytopathic, Sutnick, A.I. A serum antigen (Australia antigen) in Down’s syndrome,
leukemia, and hepatitis. Ann. Intern. Med. 66(5), 924–931 (1967).
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Cancer Society: RPG-00-066-01-CCE (to C.G.) and the following 21. Prince, A.M., Vnek, J., Brotman, B., Hashimoto, N. & Van den Ende, grants from NIH: RO1-DK 46057 (to J.R.C.), RO1-DK 39137 (to M.C. in Viral Hepatitis (eds. Vyas, G.N., Cohn, S.N. & Schmid, R.) N.R.C.), and the Liver Research Core Center grant DK-P30-41296.
507–523 (Franklin Institute Press, Philadelphia, 1978).
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