Kevin D. Pavelko

Acceleration in the rate of CNS remyelination in lysolecithin-induced demyelination

One important therapeutic goal during CNS injury from trauma or demyelinating diseases such as multiple sclerosis is to develop methods to promote remyelination. We tested the hypothesis that spontaneous remyelination in the toxic nonimmune model of lysolecithin-induced demyelination can be enhanced by manipulating the inflammatory response. In PBS-treated SJL/J mice, the number of remyelinating axons per square millimeter of lesion area increased significantly 3 and 5 weeks after lysolecithin injection in the spinal cord. However, methylprednisolone or a monoclonal antibody (mAb), SCH94.03, developed for its ability to promote remyelination in the Theiler’s virus murine model of demyelination, further increased the number of remyelinating axons per lesion area at 3 weeks by a factor of 2.6 and 1.9, respectively, but did not increase the ratio of myelin sheath thickness to axon diameter or the number of cells incorporating tritiated thymidine in the lesion. After 3 weeks, the number of remyelinating axons in the methylprednisolone or mAb SCH94.03 treatment groups was similar to the spontaneous remyelination in the 5 week PBS control-treated group, indicating that these treatments promoted remyelination by increasing its rate rather than its extent. To address a mechanism for promoting remyelination, through an effect on scavenger function, we assessed morphometrically the number of macrophages in lesions after methylprednisolone and mAb SCH94.03 treatment. Methylprednisolone reduced the number of macrophages, but SCH94.03 did not, although both enhanced remyelination. This study supports the hypothesis that even in toxic nonprimary immune demyelination, manipulating the inflammatory response is a benefit in myelin repair.

Gamma Interferon Is Critical for Neuronal Viral Clearance and Protection in a Susceptible Mouse Strain following Early Intracranial Theiler's Murine Encephalomyelitis Virus Infection

ABSTRACT We evaluated the role of gamma interferon (IFN-γ) in protecting neurons from virus-induced injury following central nervous system infection. IFN-γ−/− and IFN-γ+/+ mice of the resistant major histocompatibility complex (MHC) H-2b haplotype and intracerebrally infected with Theilers murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-γ+/+H-2b mice also cleared virus from the spinal cord white matter, whereas IFN-γ−/−H-2b mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-γ−/− mice of the susceptible H-2q haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-γ−/−H-2q mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-γ−/−H-2q mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-γ−/−H-2q mice. IFN-γ deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-γ−/−H-2q mice was lower than those in IFN-γ+/+H-2q mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-γ exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-γ plays a critical role in protecting spinal cord neurons from persistent infection and death.

Preservation of motor function by inhibition of CD8+ virus peptide-specific T cells in Theiler's virus infection

Central nervous system‐infiltrating CD8+ T cells are potential mediators of neuropathology in models of multiple sclerosis induced by Theilers murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice mount a vigorous cytotoxic T lymphocyte (CTL) response against the immunodominant virus peptide VP2121–130 and clear TMEV infection. Interferon‐γ (IFN‐γ)R‐/‐mice also mount a strong CTL response against the VP2121–130 epitope, but because of genetic deficiencies in critical IFN‐γ signaling pathways, they do not clear TMEV infection and develop prominent neurological deficits within 6 wk. This pronounced disease process, coupled with a defined CTL response, provides an ideal model for evaluating the importance of antiviral CTL activity in the development of severe demyelination and loss of motor neuron function. By administering the VP2121–130 peptide before and during TMEV infection, 99% of the VP2121–130‐specific CD8+ T cell response was inhibited. No decrease in virus infection was observed. Peptide treatment did result in significantly less motor dysfunction, even when no differences in levels of demyelination were observed. Although most investigators focus on the role of CD4+ T cells in demyelinating disease, these studies are the first to demonstrate a clear contribution of antiviral CD8+ T cells in neurological injury in a chronic‐progressive model of multiple sclerosis.

Type III IFN interleukin-28 mediates the antitumor efficacy of oncolytic virus VSV in immune-competent mouse models of cancer

Innate immune effector mechanisms triggered by oncolytic viruses may contribute to the clearance of both infected and uninfected tumor cells in immunocompetent murine hosts. Here, we developed an in vitro tumor cell/bone marrow coculture assay and used it to dissect innate immune sensor and effector responses to intratumoral vesicular stomatitis virus (VSV). We found that the type III IFN interleukin-28 (IL-28) was induced by viral activation of innate immune-sensing cells, acting as a key mediator of VSV-mediated virotherapy of B16ova melanomas. Using tumor variants which differentially express the IL-28 receptor, we showed that IL-28 induced by VSV within the tumor microenvironment sensitizes tumor cells to natural killer cell recognition and activation. These results revealed new insights into the immunovirological mechanisms associated with oncolytic virotherapy in immune-competent hosts. Moreover, they defined a new class of tumor-associated mutation, such as acquired loss of responsiveness to IL-28 signaling, which confers insensitivity to oncolytic virotherapy through a mechanism independent of viral replication in vitro. Lastly, the findings suggested new strategies to manipulate immune signals that may enhance viral replication, along with antitumor immune activation, and improve the efficacy of oncolytic virotherapies.

Promotion of remyelination by polyclonal immunoglobulin in Theiler's virus-induced demyelination and in multiple sclerosis

Spontaneous remyelination occurs in experimental models of demyelination and in patients with multiple sclerosis, although to a limited extent. This enables the search for factors that promote remyelination. Using the Theilers virus model of central nervous system demyelination, promotion of remyelination was observed after passive transfer of CNS-specific antiserum and transfer of CNS-specific purified immunoglobulin. Mouse polyclonal immunoglobulin from normal non-syngeneic mice, comparable with the human immunoglobulin preparation, also promotes CNS remyelination in the Theilers virus model of multiple sclerosis. These experimental findings further bridge the gap with a pilot study that suggests clinical improvement after polyclonal immunoglobulin administration, possibly due to remyelination, in some multiple sclerosis patients with stable, steroid-unresponsive optic neuritis. In view of these experimental and clinical data, the physiological role of myelin in demyelination and remyelination is discussed, and the role of IgG solely as a deleterious molecule in the pathophysiology of multiple sclerosis and experimental demyelination is addressed.

Direct Comparison of Demyelinating Disease Induced by the Daniel's Strain and BeAn Strain of Theiler's Murine Encephalomyelitis Virus

We compared CNS disease following intracere‐bral injection of SJL mice with Daniels (DA) and BeAn 8386 (BeAn) strains of Theilers murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.

Transgenic Expression of Theiler's Murine Encephalomyelitis Virus Genes in H-2b Mice Inhibits Resistance to Virus-Induced Demyelination

ABSTRACT We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2b ) congenic mice expressing three independent contiguous coding regions of the Theilers murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8+ T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8+ T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis

Theilers murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L‐dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV‐induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H‐2b mice. CD40L‐mediated interactions were also necessary for development of protective H‐2b‐restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody‐ and cell‐mediated protective immune responses in distinct phases of TMEV‐mediated pathology.

Theiler's Murine Encephalomyelitis Virus as a Vaccine Candidate for Immunotherapy

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theilers murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.

Genetic Deletion of a Single Immunodominant T‐cell Response Confers Susceptibility to Virus‐induced Demyelination

An important question in neuropathology involves determining the antigens that are targeted during demyelinating disease. Viral infection of the central nervous system (CNS) leads to T‐cell responses that can be protective as well as pathogenic. In the Theiler’s murine encephalomyelitis virus (TMEV) model of demyelination it is known that the immune response to the viral capsid protein 2 (VP2) is critical for disease pathogenesis. This study shows that expressing the whole viral capsid VP2 or the minimal CD8‐specific peptide VP2121‐130 as “self” leads to a loss of VP2‐specific immune responses. Loss of responsiveness is caused by T cell‐specific tolerance, as VP2‐specific antibodies are generated in response to infection. More importantly, these mice lose the CD8 T‐cell response to the immunodominant peptide VP2121‐130, which is critical for the development of demyelinating disease. The transgenic mice fail to clear the infection and develop chronic demyelinating disease in the spinal cord white matter. These findings demonstrate that T‐cell responses can be removed by transgenic expression and that lack of responsiveness alters viral clearance and CNS pathology. This model will be important for understanding the mechanisms involved in antigen‐specific T‐cell deletion and the contribution of this response to CNS pathology.