Scientific Report 2006

Molecular and Integrative Neurosciences

Viral-Immunobiology Laboratory

M.B.A. Oldstone, J.C. de la Torre, S. Kunz, D.B. McGavern, B. Hahm, D. Brooks, A. Capul, R. Clemente, K. Edelmann, L. Garidou, H. Lauterbach, A. Lee, L. Liou, A. Sanchez, M. Trifilo, G. Ying, E. Zuniga, A. Tishon, H. Lewicki, E. Buset, A. Gundersen, P. Borrow,* E. Domingo,** J.E. Gairin,*** R. Kiessling,**** N. Sevilla,** Christina Spiropoulou*****

* Edward Jenner Institute for Vaccine Research, Compton, England
** Universidad Autonoma de Madrid, Madrid, Spain
*** CNRS, Toulouse, France
**** Karolinska Institutet, Stockholm, Sweden
***** Centers for Disease Control and Prevention, Atlanta, Georgia

The Viral-Immunobiology Laboratory encompasses the programs of 4 faculty members: Juan Carlos de la Torre, Stefan Kunz, Dorian B. McGavern, and Michael B.A. Oldstone. Each program is independent, but the interactions between the researchers and the use of different technologies provide an intellectual sum greater than any single part. Our studies of both viral and transmissible spongiform encephalopathies (e.g., prion diseases, scrapie) include basic analysis of the mechanisms by which viruses persist, escape immune recognition, and cause disease. Integral parts of the programs are understanding how viruses infect cells; defining the cellular receptors used by viruses; and mapping the trafficking of viruses into cells and the subsequent viral uncoating, replication, assembly, exit, and spread. Because the immune system has evolved to recognize, attack, and remove these foreign substances, we evaluate the immune response against viruses, probe how viruses subvert this response to provide a selective advantage for their survival, and study how the host can correct this subversion to allow termination of viral persistence.

Other interests include dissecting how viruses and immune cells traffic to the brain and interact there; how viruses are cleared from the brain; and how viruses alter the differentiation processes of cells they persistently infect, thereby disturbing homeostasis and causing disease. We also are investigating how viruses induce autoimmune disease or induce immunosuppression, and we are designing therapies to control viral infections. Because different viruses have different lifestyles, we focus on 3 RNA negative-stranded viruses: Borna disease virus, lymphocytic choriomeningitis virus, and measles virus. We also investigate the mechanism by which infectious agents cause transmissible spongiform encephalopathies.

Resurrection of Nonfunctional T Cells During Persistent Viral Infection

D. Brooks, D.B. McGavern, M.B.A. Oldstone

Persistent viral infections such as HIV disease and hepatitis C are major health problems. A fundamental obstacle in control of these infections is the functional inactivation of antiviral T cells. After a persistent infection is established, both CD4⁺ and CD8+ T cells rapidly lose their antiviral and immunostimulatory functions. Although this phenomenon has been recognized for years, the pertinent question is whether the immune response is programmed to fail or can be fixed to eliminate infection. Also unclear are the molecular events or factors involved.

We found that in contrast to T-cell expansion, which is hardwired during priming, T-cell functional responses are malleable and rely on continuous signals from the cells’ antigenic environment. In accordance with this plasticity, function can be restored to nonresponsive CD4⁺ and CD8⁺ T cells during persistent infection by treatment with the antiviral drug ribavirin. Treatment that reduced the concentration of virus by as little as one log led to the removal of T-cell suppression factors. Removal of IL-10 initiated by viral infection resulted in restoration of T-cell function.

During persistent infection, CD8⁺ T cells with the highest affinity for viral antigens are physically deleted. Removal of these high-affinity cells results in the depletion of the effector population best equipped to fight infection, thereby limiting the breadth, magnitude, and efficacy of the antiviral response. We found that deletion of high-affinity CD8⁺ T cells during persistent viral infection is a direct result of the inactivation of virus-specific CD4⁺ T cells. The deletion of high-affinity CD8⁺ T cells could be averted by therapeutically rescuing CD4⁺ T-cell activity in vivo, resulting in long-term cytolytic activity of CD8⁺ T cells against virus-infected cells and control of infection.

Dissecting the Molecular Role of CD4⁺ and CD8⁺ T Cells in Control of Acute Viral Infections

A. Tishon, H. Lewicki, K. Edelmann, M.B.A. Oldstone

Measles virus is one of the most infectious of human pathogens and today still infects more than 30 million persons each year, killing more than 500,000 persons annually. Death is primarily due to secondary microbial infections associated with immunosuppression or to CNS disease.

We used a transgenic mouse model to express receptors for measles virus in neurons in the CNS. Infecting the transgenic mice with measles virus in concert with depleting and reconstituting individual T-cell subsets and B cells alone or in combination revealed that neither CD8⁺ nor CD4⁺ nor B cells alone can control acute measles virus infection. Combinations of either (1) CD4⁺ cells and B cells or (2) CD4⁺ and CD8⁺ T cells were required, but CD8⁺ T cells with B cells were not effective. Both IFN-γ and neutralizing antibodies, but not perforin or TNF-α, were associated with clearance of the virus. Interestingly, lack of IFN-γ but not lack of TNF-α led to persistent measles virus infection.

Influenza virus remains a major concern for a returning infection with potential devastating results for the human population. To better understand how to control influenza virus infection of the lung, in collaboration with Y. Kawaoka, University of Wisconsin, Madison, we used reverse genetics to insert the known H-2^b–restricted immunodominant CD8⁺ T-cell epitope (GP 33–41) and the CD4⁺ T-cell epitope (GP 61–80) of lymphocytic choriomeningitis virus (LCMV) into the neuraminidase gene for WSN influenza virus. We then adoptively transferred fluorescently labeled LCMV-specific GP 33 CD8⁺ cells or GP 61 CD4⁺ T cells alone or in combination and used fluorescence methods to identify and measure the trafficking of these specific T cells to the lung (Fig. 1). We also examined the effects of various therapies on trafficking of these cells to the lung and control of the resultant immunopathologic injury.

Fig. 1. Top, The genomic structure of LCMV and its immunodominant CD8+ (GP 33) and CD4+ (GP 65) epitopes. The GP 33 and GP 65 sequences are inserted into the neuraminidase gene of influenza WSN virus. Bottom, Infiltration into the lung of fluorescently labeled GP 33–specific T cells after intranasal inoculation with 1 x 105 plaque-forming units of WSN-LCMV influenza virus.

Prion-Induced Amyloid CNS and Heart Disease With High Levels of Infectivity in Blood and a Transgenic Model for Chronic Wasting Disease

M.J. Trifilo, G. Ying, M.B.A. Oldstone

Transmissible spongiform encephalopathies, or prion diseases, are a group of infectious diseases due to abnormal folding of the normal cellular protein PrP. More than 98% of PrP exists as a membrane-bound, glycosylphosphatidylinositol-anchored protein. In collaboration with B. Chesebro, Rocky Mountain Laboratories, Hamilton, Montana, we produced transgenic mice in which the C-terminal 21 amino acids of PrP are not transcribed; in these mice more than 98% of PrP exists in an anchorless, non–membrane-bound form.

When these transgenic mice were inoculated intracerebrally with the agent that causes murine scrapie, a dramatic accumulation of abnormally folded prion protein, PrPres, occurred within the brain (Fig. 1A). PrPres was infectious and formed large amyloid plaques in the absence of overt clinical disease during observation times of up to 720 days. However, the infected mice had learning and memory deficits, including an inability to perform cued learning tests and a failure to induce long-term potentiation. In other studies, we showed that inhibition of learning and memory was associated with PrPres binding, upregulating, and signaling through the γ-aminobutyric acid receptor. These results indicate for the first time that PrP can function as a ligand for this receptor.

Fig. 1. Deposition of PrPres in the frontal cortex (A) and around endothelial cells (B) of the brain in transgenic mice with anchorless, non–membrane-bound PrP infected with the agent that causes murine scrapie. Both PrPres and infectious material enter the blood and are deposited in several extraneural tissues, including the heart (C and D). Deposits of PrPres (C) colocalize with amyloid deposits (D), preventing proper function of the heart.

PrPres deposits in the brains of the infected transgenic mice also occurred within and around endothelial cells lining blood vessels in the brain (Fig. 1B). Examination of blood indicated that both infectivity and PrPres could be readily detected. These findings were the first demonstration of PrPres in the blood and indicate that a way exists to determine the precise blood components involved and to detect or remove PrPres to safeguard blood supplies.

Additionally, multiple extraneural tissues, including the heart, had PrPres deposition (Fig. 1C). Studies indicated that similar to deposits in the brain, deposits of PrPres in the heart formed amyloid (Fig. 1D) that was infectious. In catheterization studies done in collaboration with K. Knowlton, University of California, San Diego, we found that the deposition of amyloidogenic PrPres within the hearts of infected transgenic mice caused significant alterations in both systolic (reduced compliance) and diastolic (stiffening of the heart) functions of the heart.

These results provided the first evidence that prion-mediated disease could occur outside the CNS. Last, in collaboration with Dr. Chesebro, we inserted the gene for normal deer PrP into mouse genes behind the PrP promoter and introduced this construct into mice in which the gene for mouse PrP had been inactivated. When such transgenic mice were inoculated intracerebrally or orally with the agent that causes deer scrapie, clinical and pathologic evidence of prion disease developed and normal cellular deer PrP was biochemically converted into deer PrPres. Thus, we now have an animal model that can be used to investigate the pathogenesis and mechanism of spread of chronic wasting disease in deer. Both of these characteristics are currently unknown, although chronic wasting disease is a major economic problem for those who hunt or breed deer and an unknown public health risk for humans.

Publications

Brooks, D.G., McGavern, D.B., Oldstone, M.B.A. Reprogramming of antiviral T cells prevents inactivation and restores T cell activity during persistent viral infection. J. Clin. Invest. 116:1675, 2006.

Homann, D., Dummer, W., Wolfe, T., Rodrigo, E., Theofilopoulos, A.N., Oldstone, M.B.A., von Herrath, M.G. Lack of intrinsic CTLA-4 expression has minimal effect on regulation of antiviral T-cell immunity. J. Virol. 80:270, 2006.

Kunz, S., Rojek, J.M., Kanagawa, M., Spiropoulou, C.F., Barresi, R., Campbell, K.P., Oldstone, M.B.A. Posttranslational modification of α-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding. J. Virol. 79:14282, 2005.

Oldstone, M.B.A. Molecular and cellular mechanisms, pathogenesis, and treatment of insulin-dependent diabetes obtained through study of a transgenic model of molecular mimicry. Curr. Top. Microbiol. Immunol. 296:65, 2005.

Oldstone, M.B.A. Molecular mimicry, microbial infection, and autoimmune disease: evolution of the concept. Curr. Top. Microbiol. Immunol. 296:1, 2005.

Oldstone, M.B.A. Viral persistence: parameters, mechanisms and future predictions. Virology 344:111, 2006.

Oldstone, M.B.A., Dales, S., Tishon, A., Lewicki, H., Martin, L. A role for dual viral hits in causation of subacute sclerosing panencephalitis. J. Exp. Med. 202:1185, 2005.

Rhode, A., Pauza, M.E., Barral, A.M., Rodrigo, E., Oldstone, M.B., von Herrath, M.G., Christen, U. Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes development. J. Immunol. 175:3516, 2005.

Tishon, A., Lewicki, H., Andaya, A., McGavern, D., Martin, L., Oldstone, M.B.A. CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective. Virology 347:234, 2006.

Trifilo, M.J., Hahm, B., Zuniga, E.I., Edelmann, K.H., Oldstone, M.B.A. Dendritic cell inhibition: memoirs from immunosuppressive viruses. J. Infect. Dis., in press.

Trifilo, M.J., Yajima, T., Gu, Y., Dalton, N., Peterson, K.L., Race, R.E., Meade-White, K., Portis, J.L., Masliah, E., Knowlton, K.U., Chesebro, B., Oldstone, M.B.A. Prion-induced amyloid heart disease with high blood infectivity in transgenic mice. Science 313:94, 2006.

Zuniga, E.I., Edelmann, K.H., Oldstone, M.B.A. Viruses and dendritic cells: a prominent mechanism for subverting the immune response. In: Microbial Subversion of the Host Immune Response. Lachmann, P., Oldstone, M.B.A. (Eds.). Horizon Scientific Press, London, 2006, p. 211.