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Laboratory of Experimental Virology

 

Research Projects

The HBV Specific Cytotoxic T Lymphocyte Response in Infected Patients

The peripheral blood cytotoxic T lymphocyte (CTL) response to the hepatitis B virus (HBV) is vigorous, polyclonal and multispecific in patients with acute viral hepatitis (AVH), it persists indefinitely after recovery, and it is maintained by continued antigenic stimulation by residual virus that persists, apparently harmlessly, in healthy convalescent individuals. In contrast, the CTL response to HBV is relatively weak in patients with chronic hepatitis, except during spontaneous disease flares 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 it is strong, and that it contributes to viral persistence and chronic liver disease when it is weak.

Based on these results, therapeutic induction or augmentation of the CTL response to HBV may lead to viral clearance in chronically infected patients. Experimental CTL vaccines must include several CTL epitopes for each class I allele in order to avoid the selection of CTL escape mutants. We have recently identified several new HBV-specific CTL epitopes that are restricted by multiple class I alleles in the HLA-A2, HLA-A3 and HLA-B7 superfamilies, including a new HLA B51-restricted epitope that is located between HBc residues 19-27. This epitope is particularly interesting because it is contained entirely within a previously described HLA A2-restricted CTL epitope located between HBc residues 18-27. Interestingly, HLA-B57 restricted CTLs specific for HBc19-27 can kill HLA-B51 positive target cells pulsed with the HBc18-27, implying that it can be presented by HLA-B51 as well as by HLA A2 and suggesting that, because of its broad reactivity, the longer peptide should be included in the design of CTL-based vaccines.

The HCV Specific T Lymphocyte Response in Infected Patients

In contrast to HBV infection, the CTL response is relatively strong during chronic HCV infection yet the virus persists, suggesting that the host-virus interactions that determine the outcome of these two infections may be different for each virus. Recently, we showed that the HCV-specific CD4+ T cell response is stronger in convalescent than in chronically infected individuals and that this response is maintained indefinitely after viral clearance. In contrast, HCV-specific CD8+ CTL activity is higher during chronic infection than after viral clearance, although CD8+ T cell lines from chronically infected patients are less likely to produce interferon gamma (IFNg) than those derived from convalescent individuals. These results suggest that the CD4+ T cell response to HCV is more closely associated with HCV clearance than the CD8+ T cell response which is more often associated with viral persistence and chronic liver disease, perhaps because its cytolytic activity is not balanced by its ability to produce IFNg.

In order to define the characteristics of the antiviral T cell response during the early stages of infection, the proliferative T cell response to HCV was studied in subjects who experienced high risk needlestick exposures to HCV. Patients who became infected developed strong proliferative T cell responses to HCV proteins beginning between 4-8 weeks after exposure, corresponding with anti-HCV seroconversion and with the clinical onset of viral hepatitis. Despite these T cell responses, both of these patients became persistently infected. These results demonstrate that the proliferative T cell response to HCV is a relatively early event during HCV infection, and that it coincides with the appearance of antibodies and transaminase elevations but it does not lead to viral clearance.

The T cell Response to HCV in Acutely Infected Chimpanzees

The early events in immune recognition during HCV infection are not well understood; nor is the role of T cell priming in resistance to HCV reinfection. To define these events, we are studying the peripheral and intrahepatic T cell response to HCV in chimpanzees following either intrahepatic transfection of cloned HCV RNA or intravenous infection with monoclonal virus. Our observations thus far can be summarized as follows. First, HCV specific CD4+ T cells appear in the peripheral blood as early as 2 weeks after infection, i.e. soon after the appearance of viral RNA and several weeks before the appearance of HCV-specific CD8+ T cells and antibodies. Second, the HCV-specific T cells appear in the liver after they are detectable in the peripheral blood. Third, the T cell responses have not been associated with any changes in serum HCV RNA titers and they are usually not associated with elevated alanine aminotransferase activity. Fourth, prior infection or exposure to HCV does not protect against infection with homologous or heterologous HCV inocula despite the presence of HCV-specific T cells in the peripheral blood. Fifth, the T cell response to HCV in chronically infected chimpanzees can be as vigorous and multispecific as in animals that clear the virus. These results suggest that the T cell response to HCV may not control viral replication during HCV infection. In addition, the data provide the first evidence that, like the antibody response, T cell priming by prior exposure to HCV does not prevent infection by homologous virus.

Host-Virus Interactions in HBV Transgenic Mice and HBV-infected Chimpanzees

Class I restricted HBV-specific murine CTLs cause a necroinflammatory liver disease in HBV transgenic mice that express all of the viral gene products and replicate the virus in their hepatocytes. In addition to killing the hepatocytes, the CTLs inhibit HBV gene expression and replication in viable hepatocytes by a noncytolytic process that is mediated by IFNg and tumor necrosis factor alpha (TNFa). Importantly, the same noncytolytic antiviral effects can be triggered by the adoptive transfer of HBV-specific, class II restricted CD4+ T cells, as well as by HBV-nonspecific stimuli such as the injection of recombinant IL-12 or during lymphocytic choriomeningitis virus, murine cytomegalovirus and recombinant adenovirus infection. The antiviral effects of these agents are also due to their ability to induce IFNg, TNFa and type I interferon (IFNa/b) in the liver. These observations imply 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 prior to the onset of acute hepatitis in chimpanzees inoculated with serum from the HBV transgenic mice. Collectively, these observations suggest that viral clearance during HBV and, perhaps, other viral infections may be mediated by noncytolytic antiviral signals triggered by inflammatory cytokines including IFNg, TNFa and type 1 IFN in addition to the conventional concept of immune-mediated destruction of infected cells.

We do not know, however, if these cytokines represent the final mediators in this system or if other downstream factors are involved. Nitric oxide (NO) is a highly unstable product of L-arginine metabolism that exerts a wide variety of biological functions including antiviral activities in a paracrine and autocrine fashion. NO is produced by two distinct categories of nitric oxide synthase isoforms. The constitutive forms are present in many different cell types (including endothelial and neuronal cells) that synthesize small amounts of NO. The inducible forms of nitric oxide synthase (iNOS) are present in macrophages and hepatocytes that synthesize large amounts of NO upon activation by cytokines such as IFNg and TNFa. To examine the ability of NO to control HBV replication, we crossed HBV transgenic mice that replicate the virus in their hepatocytes with mice that lack iNOS. Interestingly, iNOS knockout mice replicate HBV in the liver at higher levels than their littermate controls that express iNOS, suggesting that NO present in the normal liver may repress HBV replication. More importantly, HBV-specific CTLs did not inhibit HBV replication in iNOS-deficient mice, despite the fact that they caused a necroinflammatory liver disease and induced IFNg and TNFa in the liver. In contrast, iNOS-deficient mice were not resistant to the antiviral effects of the IFNa/b-inducer polyIC on HBV replication. These results suggest that NO mediates the ability of IFNg and/or TNFa to inhibit HBV replication while the antiviral activity of IFNa/b is NO-independent.

Inhibition of HBV Replication by Activated Macrophages During Murine Malaria

In keeping with the foregoing observations, we speculated that additional HBV-nonspecific proinflammatory events occurring in the liver would inhibit HBV replication. Malaria infection affects the liver and it is widespread in areas where HBV is endemic. We have recently shown that intrahepatic HBV replication is inhibited in mice infected by plasmodium species that cause murine malaria. When injected into susceptible mice, malaria sporozoites infect and multiply in hepatocytes where they develop into merozoites and are released into the circulation. The merozoites are not infectious for hepatocytes; instead, they infect and multiply in erythrocytes that lyse and release new merozoites to establish a massive erythrocytic infection that is ultimately controlled by the immune response. Lysed erythrocytes, hemoglobin and malaria pigment are removed from the blood by splenic and hepatic macrophages (Kupffer cells), resulting in macrophage activation, hyperplasia and the recruitment of inflammatory cells into these organs. In the current study, HBV transgenic mice were infected either with plasmodium sporozoites or merozoite-infected erythrocytes. The animals developed Kupffer cell hyperplasia and a diffuse T cell infiltrate coinciding with parasitemia, the induction of IFNa/b TNFa and IFNg mRNA and the disappearance of HBcAg and HBV replicative intermediates from the liver. Importantly, infection of hepatocytes was not required for this effect since HBV replication was inhibited efficiently when the infection was limited to erythrocytes. These results strongly suggest that, during murine malaria, HBV replication is inhibited by inflammatory cytokines secreted primarily by intrahepatic macrophages that are activated by phagocytosis of merozoite-infected erythrocytes.

Molecular Basis for Inhibition of HBV Replication by Inflammatory Cytokines

Several studies are underway to define the intracellular molecular basis for the antiviral effects of IFNg and TNFa on HBV gene expression and replication. One study focuses on the mechanism whereby the cytokines eliminate cytoplasmic HBV capsids and replicative intermediates from the cell. To determine whether this is due to translational inhibition of HBV mRNA we examined the translational status of HBV transcripts in the liver before and after HBV replication had been blocked by inflammatory cytokines. The polysomal distribution of HBV specific RNAs did not change after administration of IL-12 or the IFN type I inducer poly I/C, despite the disappearance of capsids and replicative intermediates, suggesting that capsid elimination is a post-translational event. Analysis of the kinetics of elimination of encapsidated RNA and DNA from the livers of poly I/C-treated mice revealed that RNA-containing capsids were eliminated before capsids containing immature HBV DNA, and the immature DNA-containing capsids disappeared before capsids containing mature HBV DNA. These results suggest that the cytokines inhibit an early step in HBV replication between translation and capsid maturation rather than by degrading preformed particles, inhibiting capsid maturation or accelerating the export of capsids from the cell. This was supported in BrdU-labeling experiments demonstrating that the kinetics of HBV replication and release of virions are unaffected when cytoplasmic capsids and HBV replicative intermediates are disappearing from the cell. It was further supported by demonstrating that the kinetics of HBV clearance from the serum of poly I/C-treated mice parallels the half life of virions in the serum of untreated mice. Collectively, these results suggest that inflammatory cytokines inhibit encapsidation of HBV pgRNA while the preformed viral capsids and replicative intermediates are depleted from the cell by normal viral maturation and secretion.

We have also demonstrated that the cytokines eliminate HBV RNA by a post-transcriptional mechanism. While examining the molecular basis of this process, we recently showed that the cellular La protein and its cleavage products bind a predicted RNA stem loop structure between nts 1275 and 1291 of HBV RNA. The presence of the full-length La protein in liver nuclear extracts is associated with normal levels of HBV RNA in the liver of HBV transgenic mice. In contrast, cytokine-induced suppression of HBV RNA content is associated with the proteolytic cleavage of La. Recently, we identified endonucleolytic cleavage sites within the viral RNA close to the La-binding site. Preliminary results indicate that recombinant La can reduce the cleavage of HBV RNA by cellular RNases. Taken together, these results suggest that hepatocellular HBV RNA is stabilized by full-length La by preventing RNA cleavage and that cytokine-mediated proteolytic processing of La abrogates this activity and destabilizes HBV RNA by exposing specific endoribonucleolytic cleavage sites close to the La binding site.

Thymic Tolerance to One Viral Protein Reduces Viral-Induced Immunopathology

Under certain circumstances, cytokine activation in virus-infected tissues can also be harmful and it can lead to immunopathology, as occurs during LCMV infection of perforin-deficient mice. Perforin-deficient mice do not clear LCMV infection and they usually die within few weeks of the infection. In these animals, death is associated with severe immunopathology in various organs in which high numbers of viral-specific T cells are constantly activated and produce inflammatory cytokines. We have recently tested in this model whether it is possible to specifically dampen the immune response and therefore reduce the immunopathology and prolong the survival of the animals. To do so, we crossed perforin-deficient mice with LCMV-transgenic mice that are immunologically tolerant to a single LCMV antigen that is expressed in the thymus. Upon LCMV infection, the perforin-deficient LCMV-transgenic mice show reduced immunopathology and in most cases survive the infection for several months, thus indicating that sometimes it can be beneficiary for the host to specifically dampen the immune response.

TSRI > IMS > Exp Vir