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

F.V. Chisari, L.G. Guidotti, C. Pasquinelli, C. Ferrar

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. Our laboratory focuses on the immunobiology and pathogenesis of these viruses in infected patients and chimpanzees and in other animal models, including transgenic mice.

Specific Cytotoxic T-Lymphocyte Response in Humans and Chimpanzees Infected With Hepatitis B and C Viruses

K.M Chang, R. Bertoni, F.V. Chisari

The peripheral blood cytotoxic T-lymphocyte (CTL) response to hepatitis B virus (HBV) is vigorous, polyclonal, and multispecific in patients with acute hepatitis B. In patients who recover from the infection, the response persists indefinitely after recovery and is maintained by continued antigenic stimulation by residual virus that persists, apparently harmlessly. In contrast, in patients with chronic hepatitis B, the CTL response to HBV is relatively weak, except during spontaneous flare-up of disease or interferon-induced recovery, when it is readily detectable. Collectively, these results suggest that the CTL response contributes both to viral clearance and to disease pathogenesis in HBV infection when the response is strong and that it contributes to viral persistence and chronic liver disease when the response is weak.

In contrast, the CTL response to hepatitis C virus (HCV) is relatively strong during chronic HCV infection, yet the virus persists. This finding suggests that the host-virus interactions that determine the outcome of HBV and HCV infections may be different for each virus. Even during chronic HCV infection, however, an inverse relationship exists between viral load and the strength of the CTL response, and the class II--restricted T-cell response to HCV is much stronger in patients who have cleared the virus than in those who are chronically infected. Collectively, these observations suggest that although HCV is relatively refractory to immunologic control, it can be at least partially controlled by the T-cell response.

Because these results suggest that therapeutic induction or augmentation of the CTL response to HBV and HCV may lead to viral clearance in chronically infected patients, clinical trials of CTL epitope-based experimental lipopeptides and DNA vaccines are under way. In order to be ethnically unbiased and to avoid the selection of CTL escape mutants, such vaccines must include several CTL epitopes for each class I allele. The search for such epitopes has been facilitated by the discovery that multiple HLA class I alleles (superfamilies) can bind antigenic peptides via common HLA-binding motifs (supermotifs).

Using this information, we identified several new HBV- and HCV-specific CTL epitopes that are restricted by multiple class I alleles in the HLA-A2, HLA-A3, and HLA-B7 superfamilies. We also showed that human class I supertypes and CTL repertoires extend to chimpanzees. Accordingly, we are using these supertype peptides to monitor the kinetics and quality of the CTL response in acutely and chronically infected humans and chimpanzees. Our intent is to determine if vaccines that incorporate these epitopes can terminate chronic HBV and HCV infection in these systems.

Host-Virus Interactions in Hepatitis B Virus Transgenic Mice and in Ducks and Chimpanzees Infected With Hepatitis B Virus

L.G. Guidotti, V. Cavanaugh, U. Schultz, T. Heise, S. Wieland, V. Juillard, V. Pasquetto, Y. Shimizu, A. Franco, F.V. Chisari

Class I--restricted murine cytotoxic T lymphocytes (CTLs) specific for hepatitis B virus (HBV) cause a necroinflammatory liver disease in HBV transgenic mice that contain the complete viral genome, express all the viral gene products, and replicate the virus in their hepatocytes. In addition to killing the hepatocytes, the CTLs inhibit expression and replication of HBV genes in viable hepatocytes by a noncytolytic process that is mediated by IFN- and TNF-. The same noncytolytic antiviral effects can be triggered by the adoptive transfer of HBV-specific, class II--restricted CD4⁺ T cells and by HBV-nonspecific stimuli such as the injection of recombinant IL-12 or infection with lymphocytic choriomeningitis virus, murine cytomegalovirus, or recombinant adenovirus. The antiviral effects of these agents are also due to their ability to cause hepatitis and induce IFN-, TNF-, and IFN type I in the liver.

These observations suggest that HBV may be susceptible to similar regulatory processes during natural infection. This hypothesis is supported by our recent observation that CD4⁺ and CD8⁺ T cells and cytokine markers appear in the liver and that viral DNA disappears from the liver and serum before the onset of acute hepatitis in chimpanzees inoculated with serum from HBV transgenic mice. In separate studies, we showed that replication of a closely related hepadnavirus, duck HBV, in primary duck hepatocytes is also suppressed by IFN- and IFN type I and we have begun to define the susceptible steps in the life cycle of the duck virus that are blocked by these cytokines.

Collectively, these observations suggest that viral clearance during HBV and, perhaps, other viral infections may be mediated by noncytolytic antiviral signals triggered by the immune response in addition to the conventional immune-mediated destruction of infected cells. The results also suggest that the pharmacologic delivery or induction of inflammatory cytokines in the liver may have therapeutic value during chronic HBV infection.

Several studies are under way to define the intracellular molecular basis for the antiviral effects of these cytokines on the expression and replication of HBV. We showed that the cytokines inhibit HBV replication posttranslationally, by a noncytolytic mechanism that eliminates viral nucleocapsids from the hepatocytes. We also showed that the cytokines inhibit intrahepatic HBV RNA content by a posttranscriptional mechanism. Accordingly, we have searched for cellular HBV RNA--binding proteins that might stabilize and/or destabilize the viral RNA.

We recently found that nuclear extracts of transgenic mouse liver contain 3 HBV RNA--binding proteins--p45, p39, and p26--that fluctuate in parallel with the viral RNA when IFN- and TNF- are produced in the liver. All 3 proteins bind to a 17-bp stem-loop structure present in the viral RNA. The proteins p45 and p39 are constitutively expressed together with normal steady-state levels of HBV RNA. After transfer of CTLs, however, p45 disappears from the liver, and p26 is induced concomitantly with the disappearance of HBV RNA. All these events are blocked by antibodies to IFN- and TNF- before CTL transfer. The same events are induced in the cytokine-rich inflamed liver during infection with cytomegalovirus or lymphocytic choriomeningitis virus.

The tight correlation between the disappearance of HBV RNA from the hepatocyte, the appearance of p26, and the disappearance of p45 suggests that these cytokine-inducible HBV RNA--binding proteins may regulate expression of HBV genes during HBV infection. In related studies, using an in vitro ribonuclease activity assay, we showed that RNA oligonucleotides, located just 5´ to the stem-loop binding site of the RNA-binding proteins, are cleaved in a site-specific manner by nuclear RNases present in mouse liver and that cleavage is increased after injection of CTLs, when p45 and viral RNA disappear. These observations suggest a model in which p45 maintains HBV RNA stability by inhibiting access of the RNase to its substrate.