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News and Publications
Division of Experimental Pathology
Francis V. Chisari, M.D., Division Head
Molecular Biology of Hepatitis B and C Viruses and the Immune Response to Their Antigens
Hepatitis B and C viruses cause acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. More than 500 million persons worldwide are chronically infected with these viruses. In the Division of Experimental Pathology, we focus on the immunobiology and pathogenesis of these viruses in infected humans and chimpanzees and other animal models, including transgenic mice.
Viral Clearance and Persistence During Acute Hepatitis C Virus Infection in Humans and Chimpanzees
R. Thimme, H.C. Spangenberg, S. Wieland, A. Brideau, K.M. Chang, F.V. Chisari
Cellular immune responses most likely determine the outcome of hepatitis C virus (HCV) infection, but the dynamics of such responses and their relationship to viral clearance and persistence are poorly understood. Thus far, the early cellular immune events during the incubation phase of HCV infection in humans have not been defined. We analyzed the HCV-specific T-cell response immediately after exposure to HCV and during the evolution of asymptomatic or symptomatic acute viral hepatitis in 5 healthcare workers.
In 4 of the 5 subjects, acute hepatitis developed and progressed to a chronic infection; in the other subject, the virus was cleared. In all subjects, viremia was first detectable within 12 weeks of inoculation, 1 month or more before the appearance of virus-specific T cells. The subject who cleared the virus experienced a prolonged episode of acute hepatitis that coincided with a CD38+ IFN-g CD8+ T-cell response to HCV and a small reduction in viremia. Subsequently, a strong CD4+ T-cell response emerged, and the CD8+ T cells became CD38 and began producing IFN-g in response to HCV, coinciding with a rapid 100,000-fold decrease in viremia that occurred without a corresponding surge of disease activity.
In the 4 subjects in whom chronic infection developed, 2 did not have a significant T-cell response, and 2 had initially strong CD4+ T-cell responses that ultimately waned. In all subjects, viremia was higher at the peak of acute hepatitis than it was when the disease began, and the disease abated during the viremia.
These results suggest that the vigor and quality of the antiviral T-cell response determines the outcome of acute HCV infection; that the ability of HCV to outpace the T-cell response may contribute to the tendency of the virus to persist; that disease pathogenesis and viral clearance are mediated by different CD8+ T-cell populations that control HCV by both cytolytic and noncytolytic mechanisms; and that asymptomatic and symptomatic acute HCV infections have different pathways to viral persistence.
In related studies, we compared the course of viremia with the
peripheral and intrahepatic T-cell response and intrahepatic cytokine
profile in 6 acutely infected chimpanzees. Three very different
outcomes of acute infection occurred after peak viral titers were
reached: viral clearance, viral control, and uncontrolled persistent
viral infection. The results indicate that the spread of HCV greatly
outpaces the accumulation of antiviral T cells in the liver and
that HCV rapidly induces but is not controlled by IFN-a/b.
The data also suggest that control of the infection depends on
the entry and accumulation of HCV-specific T cells that produce
IFN-g , especially CD8+
T cells, in the liver and that viral clearance may depend on the
induction of IFN-g in the liver
and not require the destruction of infected cells. This hypothesis
is supported by our recent finding that replication of an HCV
replicon in HuH-7 cells is profoundly inhibited by IFN-g
.
Host-Virus Interactions in Mice Transgenic for Hepatitis B Virus
L.G. Guidotti, S. Wieland, K. Kakimi, K. Kimura, G. Sitia, I. Campbell,* F.V. Chisari
* Department of Neuropharmacology, TSRI
We previously showed that replication of hepatitis B virus (HBV) is inhibited noncytopathically in the liver of HBV transgenic mice by the intrahepatic induction of cytokines such as IFN-a/b and IFN-g , by HBV-specific cytotoxic T lymphocytes (CTLs), and by other stimuli (e.g., viral and parasitic infections, a -galactosylceramide) that induce the local production of IFN-g , TNF-a , and/or IFN-a/b by effector cells of the innate and adaptive immune system. To begin to identify the pathways inducible by IFN that could play a role in our system, we crossed HBV transgenic mice with mice that lacked the genes for interferon regulatory factor 1, RNAse L, or the double-stranded RNA-activated protein kinase, 3 well-characterized IFN-inducible genes that mediate antiviral activity.
Compared with their respective controls, none of the experimental
animals were resistant to the antiviral effect of the IFN-a/b
inducer polyinosinic-polycytidylic acid complex or recombinant
murine IFN-g on HBV replication.
These results indicate that interferon regulatory factor 1, RNAse
L, and RNA-activated protein kinase do not mediate per se the
IFN-a/b-
or the IFN-g dependent inhibition
of HBV replication that occurs in the liver in HBV transgenic
mice. This finding also indicates that in order to inhibit HBV
replication, either expression of the genes for these proteins
must be simultaneously induced in hepatocytes or other IFN-inducible
genes mediate the antiviral effect of interferons in our transgenic
mouse model.
Recently, we evaluated the ability of activated intrahepatic
antigen-presenting cells (APCs) to inhibit HBV replication in
HBV transgenic mice. Intrahepatic APCs were activated by the administration
of an anti-CD40 agonistic monoclonal antibody. We showed that
a single intravenous injection of the antibody was sufficient
to inhibit HBV replication noncytopathically. This effect was
associated with the recruitment of dendritic cells, macrophages,
T cells, and natural killer cells into the liver and the intrahepatic
induction of cytokines such as IL-12, IL-18, IFN-a/b,
IFN-g , and TNF-a.
The antiviral effect was mostly dependent on activated APCs and
required the production of IL-12, IFN-g
and TNF-a .
These results indicate that activated APCs can play a role in the control of HBV replication and that they do so by producing antiviral inflammatory cytokines in the liver. The data also suggest that therapeutic activation of APCs may be a new strategy for the treatment of chronic HBV infection.
Recruitment of host-derived antigen-nonspecific inflammatory cells into the liver is associated with the intrahepatic production of chemokines and most likely contributes to the pathogenesis of liver disease. Indeed, we recently found that blocking the chemokines CXCL9 and CXCL10 reduced the intrahepatic recruitment of host-derived lymphomononuclear cells and the severity of liver disease that occur when HBV-specific CTLs are transferred into HBV transgenic mice. Because the severity of the CTL-initiated liver disease is also ameliorated by the depletion of neutrophils, most likely neutrophils also contribute to the pathogenetic process.
Interestingly, depletion of neutrophils did not affect the intrahepatic migration and antiviral activity of CTLs but profoundly inhibited the recruitment of all antigen-nonspecific cells, including lymphomononuclear cells. This effect occurred despite high intrahepatic levels of expression of chemokine genes, suggesting that neutrophil-dependent functions other than chemokine production are necessary for the recruitment process. These functions may include the production of matrix metalloproteinases that facilitate leukocyte trafficking through the endothelial barrier and the extracellular matrix. A variety of these metalloproteinases are strongly and rapidly induced in the liver of mice after transfer of CTLs. In future experiments, we will define the pathogenetic role of these enzymes in our system.
Intracellular Pathways and Mechanisms That Inhibit Hepatitis B Virus Replication
S. Wieland, M. Robek, L.G. Guidotti, T.G. Sutcliffe,* P. Schultz,** F.V. Chisari
* Department of Molecular Biology, TSRI, and Digital Gene Technologies, La Jolla, CA
** Department of Chemistry, TSRI, and Novartis Institute for Functional Genomics, San Diego, CA
The intracellular pathways responsible for the cytokine-dependent antiviral activity are still poorly understood. We recently began to identify the cytokine-activated cellular genes that encode proteins that interrupt the HBV life cycle. We used 2 independent gene expression profiling methods: robotic differential display analysis, in collaboration with T.G. Sutcliffe and scientists at Digital Gene Technologies, and gene chip analysis, in collaboration with P. Schultz and scientists at the Novartis Institute for Functional Genomics.
The cytokine-activated gene expression profiles in the livers of HBV transgenic mice and in immortalized hepatocytes were analyzed under conditions in which HBV replication was suppressed. The results indicated that a small number of hepatocellular genes are tightly associated with the cytokine-mediated inhibition of HBV replication and may therefore contribute mechanistically to the antiviral effect. Immunoproteasome subunits are prominent among the proteins encoded by this small family of genes. On the basis of this information, we recently showed that inhibitors of cellular proteasome activity can block the antiviral effect. These results suggest that a critical component of the IFN-induced antiviral response may be the proteasome-dependent degradation of viral or cellular proteins that are required for HBV replication.
Hydrodynamic Injection of Viral DNA: A Mouse Model of Acute Hepatitis B Virus Infection
P.L. Yang, F.V. Chisari
Hepatitis B virus (HBV) is a prototype for liver-specific pathogens in which the failure of the immune system to mount an effective response leads to chronic infection. The absence of appropriate experimental models has impeded our understanding of the immune response to HBV. We developed a new murine model for studying human HBV replication, immunogenicity, and control. After transfection of hepatocytes in vivo with a replication-competent, over-length, linear HBV genome, viral antigens and replicative intermediates were synthesized and virus was secreted into the blood. Viral antigens disappeared from the blood as early as 7 days after transfection, coincident with the appearance of antiviral antibodies. HBV transcripts and replicative intermediates disappeared from the liver by day 15, after the appearance of antiviral CD8+ T cells.
Thus, the outcome of hydrodynamic transfection of immunocompetent mice is comparable to that of a natural infection. These experimental methods will enable us to dissect the immune response to HBV in transgenic and knockout mice, examine viral dynamics in a controlled system, and use mutagenesis methods to study HBV. In addition, this method is a prototype for the study of other known and to-be-discovered liver-specific pathogens.
PUBLICATIONS
Bukh, J., Thimme, R., Govindarajan, S., Forns, X., Satterfield, W., Eder, G., Chang, K.-M., Yanagi, M., Emerson, S.U., Chisari, F.V., Purcell, R.H. Monoclonal hepatitis C virus infection in chimpanzees. In: Viral Hepatitis and Liver Disease. Margolis, H.W., et al. (Eds.). International Medical Press, Atlanta, 2002, p. 336.
Guidotti, L.G., Morris, A., Mendez, H., Koch, R., Silverman, R.H., Williams, B.R., Chisari, F.V. Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice. J. Virol. 76:2617, 2002.
Kakimi, K., Isogawa, M., Chung, J.S., Sette, A., Chisari, F.V. Immunogenicity and tolerogenicity of hepatitis B viral structural and nonstructural proteins: implications for immunotherapy of persistent viral infections. J. Virol. 76:8609, 2002.
Kakimi, K., Lane, T., Wieland, S.F., Asensio, V.C., Campbell,
I., Chisari, F.V., Guidotti, L.G. Blocking chemokine responsive
to g-2/interferon (IFN)-g-inducible
protein and monokine induced by IFN-g
activity in vivo reduces the pathogenetic but not the antiviral
potential of hepatitis B virus-specific cytotoxic T lymphocytes.
J. Exp. Med. 194:1755, 2001.
Kakimi, K., Lane, T.E., Chisari, F.V., Guidotti, L.G. Cutting edge: inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver. J. Immunol. 167:6701, 2001.
Mosier, D.E., Chisari, F.V. GB virus C and mortality from HIV infection. N. Engl. J. Med. 346:377, 2002.
Pasquetto, V., Wieland, S.F., Uprichard, S.L., Tripodi, M., Chisari, F.V. Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures. J. Virol. 76:5646, 2002.
Robek, M.D., Wieland, S.F., Chisari, F.V. Inhibition of hepatitis B virus replication by interferon requires proteasome activity. J. Virol. 76:3570, 2002.
Thimme, R., Bukh, J., Pemberton, J., Guidotti, L.G., Purcell, R.H., Chisari, F.V. T-cell responses and intrahepatic cytokine profiles in HBV- and HCV-infected chimpanzees. In: Viral Hepatitis and Liver Disease. Margolis, H.W., et al. (Eds.). International Medical Press, Atlanta, 2002, p. 175.
Thimme, R., Bukh, J., Wieland, S., Pemberton, J., Steiger, C., Ghrayeb, J., Reimann, K.A., Purcell, R.H., Chisari, F.V. Viral and immunological determinants of HCV clearance, persistence, and disease. Proc. Natl. Acad. Sci. U. S. A., in press.
Thimme, R., Oldach, D., Chang, K.M., Steiger, C., Ray, S., Chisari, F.V. Determinants of viral clearance and persistence during acute HCV infection. J. Exp. Med. 194:1395, 2001.
Yang, P.L., Althage, A., Chung, J., Chisari, F.V. Hydrodynamic transfection: a mouse model of acute hepatitis B virus infection. Proc. Natl. Acad. Sci. U. S. A., in press.
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