Scientific Report 2008

Immunology & Microbial Science

Laboratory of Experimental Virology

Molecular Biology of Hepatitis B and C Viruses and the Immune Response to Their Antigens

Hepatitis B and C viruses are noncytopathic DNA and RNA viruses that cause acute and chronic hepatitis and hepatocellular carcinoma. More than 500 million people worldwide are chronically infected, and more than 2 million people die of these infections every year. The focus of our research is to unravel the life cycle of these viruses, discover the roles played by the innate and adaptive immune responses in the control of the infections, and elucidate the mechanisms responsible for viral clearance and disease pathogenesis. Our goal is to devise novel strategies to prevent and cure these infections.

Impact of Intrahepatic Antigen Recognition on Priming of the CD8⁺ T-Cell Response

M. Isogawa, F.V. Chisari

The CD8⁺ T-cell response contributes to the pathogenesis of liver disease and viral clearance during infection with hepatitis B virus (HBV), and failure to induce and/or sustain that response results in viral persistence, chronic hepatitis, and hepatocellular carcinoma. To delineate the mechanisms that regulate the CD8⁺ T-cell response to HBV, we are using T-cell receptor transgenic mice that have CD8⁺ T cells specific for the HBV core and envelope proteins. When adoptively transferred into HBV transgenic mice, naive CD8⁺ T cells proliferate vigorously intrahepatically before they appear in lymphoid tissues but do not develop antiviral effector functions, suggesting that intrahepatic T-cell priming induces functionally defective T-cell responses. Intravital imaging suggests that HBV-specific naive T cells are primed by antigen-presenting cells in the hepatic sinusoids of the HBV transgenic mice rather than by the animals' hepatocytes or in peripheral lymphoid organs as widely assumed. These studies provide insight into previously unknown early immunologic events that occur in response to HBV infection. Currently, we are identifying the intrahepatic antigen-presenting cells that prime the T-cell response and the nature of the signal that prevents functional maturation of the T cells.

Interaction Between Platelets and Virus-Specific Cytotoxic T Lymphocytes Within the Hepatic Microcirculation

M. Iannacone, G. Sitia, M. Isogawa, F.V. Chisari, Z.M. Ruggeri,* L.G. Guidotti

* Department of Molecular and Experimental Medicine, Scripps Research

Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs) and normal inbred mice infected with hepatotropic, replication-deficient adenoviruses, we recently showed that platelets play a crucial and previously unrecognized role in viral pathogenesis. Indeed, upon activation, platelets contribute to liver disease and viral clearance by promoting the recruitment of virus-specific CTLs into the liver. As indicated by ex vivo experiments under flow, this remarkable effect most likely depends on specific interactions between platelets and CTLs. Thus, platelet-CTL interactions occurring within the hepatic microcirculation may direct CTLs to extravasate, reach parenchymal cells (i.e., hepatocytes), and perform pathogenetic and/or antiviral effector functions.

Intrahepatic visualization and mechanistic understanding of platelet-CTL interactions are major goals of our research program. Through the use of virus-specific CTLs, HBV transgenic mice, and mice genetically deficient in various platelet molecules, we plan to define in vivo the molecular basis of platelet-CTL interactions. Through the use of confocal and intravital microscopy, we plan to visualize where such interactions take place within the hepatic venous microvasculature.

The hepatic venous microvasculature consists of postsinusoidal venules and sinusoids, whose hemodynamic conditions, anatomy, and function can vary greatly between different compartments and with time or injury. Furthermore, sinusoidal endothelial cells are morphologically unique, characterized by an absence of tight junctions between cells, a lack of basal membrane, and the presence of open fenestrations. The low-flow sinusoidal environment coupled with the peculiar anatomic and molecular features of this vascular bed may allow leukocyte extravasation through nonconventional adhesion or migration mechanisms (e.g., integrin- or selectin-independent mechanisms). Studying leukocyte extravasation across the hepatic microvasculature in inflammatory conditions may unravel not only new pathways that promote diapedesis but also novel therapeutic approaches for treating chronic hepatitis and other inflammatory diseases.

Role of Platelets in Controlling Hemorrhage and Effecting Cytotoxic T-Lymphocyte— Dependent Viral Clearance During Lymphocytic Choriomeningitis Virus Infection

M. Iannacone, G. Sitia, M. Isogawa, J.K. Whitmire,* P. Marchese,** F.V. Chisari, Z.M. Ruggeri,** L.G. Guidotti

* Department of Molecular and Integrative Neurosciences, Scripps Research
* Department of Molecular and Experimental Medicine, Scripps Research

Mice infected with lymphocytic choriomeningitis virus (LCMV) have an IFN-α/β—dependent platelet dysfunction, and if the level of platelets is less than a critical threshold number, this dysfunction results in severe bleeding and acute anemia and is often followed by death. Moreover, the decreased platelet count and function are linked to a reduction in the virus-specific cytotoxic T-lymphocyte response in blood and infected organs such that viral clearance cannot occur. Platelets therefore play a key role in the progression and severity of LCMV infection in mice, because the cells are essential to control the propagation of the pathogen and the consequences of the pathogen's presence. To perform these functions, platelets require activation; inhibitors of such processes impair the ability of the cells to promote viral clearance and prevent hemorrhage. Although hemorrhage is an expected consequence of reduced platelet function, the effect on a cytotoxic T lymphocyte—dependent process required to block spreading of a virus was previously unknown.

Our results indicate that induction of IFN-α/β, with its consequences on platelet number and function, is the key pathogenetic event in this murine model of infection by a member of the Arenaviridae family. Exceptionally high levels of circulating IFN-α are found in humans infected by Junin virus, also an arenavirus, and patients who progress to a fatal outcome often have marked thrombocytopenia associated with platelet dysfunction, mucocutaneous hemorrhage, impaired cellular immunity, and lack of viral clearance. Because fully functional platelets can prevent death in platelet-depleted LCMV-infected mice, even when the number of platelets is less than normal, transfusion of normal platelets should be considered, along with neutralization of IFN-α/β activity, in the treatment of life-threatening arenavirus infections in humans.

Size of the Viral Inoculum and the Kinetics, Quality, and Magnitude of the T-Cell Response and the Outcome of Infection With Hepatitis B Virus

S. Asabe, S.F. Wieland, R.H. Purcell,* F.V. Chisari

* National Institutes of Health, Bethesda, Maryland

We previously showed that low-dose viral inocula do not induce early peripheral CD4⁺ T-cell responses and lead to persistent infection in chimpanzees experimentally infected with hepatitis B virus (HBV). We have now discovered that persistent HBV infection is characterized by the delayed onset of a poorly synchronized, functionally defective intrahepatic CD8⁺ T-cell response that is highly activated and that can be surprisingly strong but ineffective. These results suggest that early priming by subviral antigens in high-dose inocula prepares the T-cell response for rapid expansion and functional maturation when the virus appears in the liver. In contrast, when priming occurs intrahepatically after the virus has spread to most of the hepatocytes after a low-dose infection, a poorly synchronized and functionally impaired CD8⁺ T-cell response is triggered and a prolonged or persistent infection ensues. These results suggest a hitherto unappreciated role for the superabundant noninfectious subviral antigens present in serum in the outcome of HBV infection.

To test the hypothesis that an early CD4⁺ T-cell response is required for clearance of HBV, we infected control and CD4⁺ T cell—depleted chimpanzees with the same HBV inoculum and monitored the course of infection. As expected, in the control animals, an acute self-limited HBV infection associated with early peripheral CD4⁺ T-cell responses occurred and was followed by a highly synchronized, IFN-γ—producing peripheral CD8⁺ T-cell response that efficiently cleared the infection. In contrast, in chimpanzees depleted of CD4⁺ T cells, no CD4⁺ T-cell response to HBV occurred, and a persistent infection developed that was associated with weak CD8⁺ T-cell responses with no production of IFN-γ similar to the responses induced by the low-dose infections described earlier. Importantly, depletion of CD4⁺ T cells at the peak of infection 6 weeks after inoculation had no effect on the kinetics of viral clearance, whereas depletion of CD8⁺ T cells at that time prolonged the infection until CD8⁺ T-cell numbers returned to baseline. These results indicate that an early CD4⁺ T-cell response is required to induce an effective CD8⁺ T-cell response to terminate HBV infection.

Innate Control of Hepatitis C Virus Infection: Antiviral and Evasion Mechanisms

G. Cheng, F.V. Chisari

Most of the details of the hepatitis C virus (HCV) life cycle and the host-virus interactions that determine the outcome of infection are poorly understood, largely because of the lack of tractable cell culture and animal models of HCV infection. Thanks to the recent development of a cell culture model of HCV infection, analysis of all aspects of the HCV life cycle is now possible.

Using that model, we recently found that despite strongly inducing type 1 interferon or interferon-stimulated genes in infected liver, HCV does not induce interferon or interferon-stimulated genes in infected cells. We also found that the viral NS3/4A protease blocks double-stranded RNA signaling by cleaving a key intermediate in the double-stranded RNA signaling pathway. In addition, we discovered a novel NS3/4A-independent mechanism whereby HCV evades the innate host response in infected cells. We also found that interferon rapidly modulates the expression of numerous cellular microRNAs, several of which have sequence-predicted targets within the HCV genome. Synthetic microRNA mimics corresponding to some of these interferon-regulated microRNAs reproduced the antiviral effects of interferon on HCV infection, whereas neutralization of these microRNAs had the opposite effect. These findings indicate a previously unsuspected effector arm of the interferon response that appears to contribute to the control of HCV infection.

Hepatitis C Virus Infection and Very Low-Density Lipoprotein

P. Gastaminza, F.V. Chisari

Intracellular infectious particles of hepatitis C virus (HCV) and precursors of very low-density lipoprotein (VLDL) have a higher buoyant density than their secreted counterparts outside the cell. These biophysical differences suggest that both VLDL and HCV particles acquire lipids while leaving the cell. VLDL synthesis involves the acquisition of cholesteryl esters and neutral lipids by intracellular apolipoprotein B in a process catalyzed by the microsomal transfer protein.

In recent studies, we showed that an inhibitor of microsomal transfer protein and apolipoprotein B—specific short hairpin RNAs prevent assembly of infectious HCV particles and the subsequent maturation and secretion of low-density HCV particles in infected cells. We also showed that as in VLDL biogenesis, presecretory degradation of intracellular infectious HCV precursors via a nonproteasomal mechanism regulates secretion of low-density infectious HCV particles. These findings suggest that assembly and secretion of HCV particles are tightly regulated by the VLDL metabolic machinery and that only mature, low-density HCV particles are secreted, whereas most newly assembled high-density infectious particles are degraded, implying that acquisition of apolipoprotein B and cellular lipids by HCV imparts a selective advantage as HCV adapts to its natural host.

In ongoing research, we are using mass spectrometry to determine if cell-derived proteins, including components of VLDL and apolipoproteins B and E, are structural components of intracellular and/or extracellular infectious HCV particles. We are also testing the hypothesis that apolipoproteins B and E and other virus-associated cellular factors identified by mass spectrometry play a role in viral entry. We are also studying the role of host cell apolipoprotein E in assembly and egress of HCV particles. Overall, these experiments will provide insight into the role of host cell factors in the HCV life cycle by illuminating the cellular mechanisms that regulate entry, assembly, and egress of infectious viral particles.

Discovery and Development of Small-Molecule Inhibitors of Hepatitis C Virus Infection

P. Gastaminza, S. Pitram,* A. Montero,* L. Krasnova,* M.R. Ghadiri,* V.V. Fokin,* K.B. Sharpless,* F.V. Chisari

* Department of Chemistry, Scripps Research

Using a simple screening assay that reproduces the entire life cycle of hepatitis C virus (HCV) in a miniaturized format and enables simultaneous analysis of the antiviral activity and toxicity of hundreds of compounds, we are screening compound libraries for antiviral activity against HCV. We have discovered 2 novel families of small molecules that profoundly inhibit HCV infection in the low-micromolar range. The nature of the molecules and the click chemistry method used for their synthesis allow rapid preparation of multiple derivatives for analyzing structure-activity relationships. In addition, we discovered a family of peptides that form amphipathic nanotubes that selectively and efficiently inhibit early stages of HCV infection. The identification of novel antiviral molecules and the characterization of their mode of action provide chemical tools to unravel currently unknown aspects of HCV infection and new compounds for possible development into therapeutic drugs.

Publications

Bobardt, M.D., Cheng, G., de Witte, L., Selvarajah, S., Chatterji, U., Sanders-Beer, B.E., Geijtenbeek, T.B.H., Chisari, F.V., Gallay, P.A . Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc. Natl. Acad. Sci. U. S. A. 105:5525, 2008.

Brown, B.D., Sitia, G., Annoni, A., Hauben, E., Sergi, L.S., Zingale, A., Roncarolo, M.G., Guidotti, L.G., Naldini, L. In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance. Blood 109:2797, 2007.

Cheng, G., Montero, A., Gastaminza, P., Whitten-Bauer, C., Wieland, S.F., Isogawa, M., Fredericksen, B., Selvarajah, S., Gallay, P., Ghadiri, M.R., Chisari, F.V. A virocidal amphipathic α -helical peptide that inhibits hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. U. S. A. 105:3088, 2008.

Gastaminza, P., Cheng, G., Wieland, S., Zhong, J., Liao, W., Chisari, F.V. Cellular determinants of hepatitis C virus assembly, maturation, degradation and secretion. J. Virol. 82:2120, 2008.

Iannacone, M., Sitia, G., Isogawa, M., Whitmire, J.K., Marchese, P., Chisari, F.V., Ruggeri, Z.M., Guidotti, L.G. Platelets prevent IFN-α /β -induced lethal hemorrhage promoting CTL-dependent clearance of lymphocytic choriomeningitis virus. Proc. Natl. Acad. Sci. U. S. A. 105:629, 2008.

Iannacone, M., Sitia, G., Narvaiza, I., Ruggeri, Z.M., Guidotti, L.G. Antiplatelet drug therapy moderates immune-mediated liver disease and inhibits viral clearance in mice infected with a replication-deficient adenovirus. Clin. Vaccine Immunol. 14:1532, 2007.

Iannacone, M., Sitia, G., Ruggeri, Z.M., Guidotti, L.G. HBV pathogenesis in animal models: recent advances on the role of platelets. J. Hepatol. 46:719, 2007.

Law, M., Maruyama, T., Lewis, J., Giang, E., Tarr, A.W., Stamataki, Z., Gastaminza, P., Chisari, F.V., Jones, I.M., Fox, R.I., Ball, J.K., McKeating, J.A., Kneteman, N.M., Burton, D.R. Broadly neutralizing human monoclonal antibodies protect against hepatitis C virus quasispecies challenge. Nat. Med. 14:25, 2008.

Pedersen, I.M., Cheng, G., Wieland, S., Volinia, S., Croce, C.M., Chisari, F.V., David, M. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 449:919, 2007.

Sidney, J., Peters, B., Moore, C., Pencille, T.J., Ngo, S., Masterman, K.A., Asabe, S., Pinilla, C., Chisari, F.V., Sette, A. Characterization of the peptide-binding specificity of the chimpanzee class I alleles A*0301 and A*0401 using a combinatorial peptide library. Immunogenetics 59:745, 2007.

Sitia, G., Iannacone, M., Müller, S., Bianchi, M.E., Guidotti, L.G. Treatment with HMGB1 inhibitors diminishes CTL-induced liver disease in HBV transgenic mice. J. Leukoc. Biol. 81:100, 2007.