Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs

Microglia are the resident macrophage-like population in the CNS. Microglia remain quiescent until injury or infection activates the cells to perform effector inflammatory and APC functions. Our previous studies have shown that microglia infected with a neurotropic strain of Theiler's murine encephalomyelitis virus secreted innate immune cytokines and up-regulated costimulatory molecules and MHC class II, enabling the cells to present viral and myelin Ags to CD4+ T cells. Recently, TLRs have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. We examined TLR expression on brain microglia and their functional responses upon stimulation with various TLR agonists. We report that mouse microglia express mRNA for all of the recently identified TLRs, TLR1-9, used for recognition of bacterial and viral molecular patterns. Furthermore, stimulation of quiescent microglia with various TLR agonists, including LPS (TLR4), peptidoglycan (TLR2), polyinosinic-polycytidylic acid (TLR3), CpG DNA (TLR9), and infection with viable Theiler's murine encephalomyelitis virus, activated the cells to up-regulate unique patterns of innate and effector immune cytokines and chemokines at the mRNA and protein levels. In addition, TLR stimulation activated up-regulation of MHC class II and costimulatory molecules, enabling the microglia to efficiently present myelin Ags to CD4+ T cells. Thus, microglia appear to be a unique and important component of both the innate and adaptive immune response, providing the CNS with a means to rapidly and efficiently respond to a wide variety of pathogens.     

Theiler's virus infection in nude mice: viral RNA in vascular endothelial cells

Infection of athymic (nu/nu) mice with Theiler's murine encephalomyelitis virus results in an acute encephalitis which resembles poliomyelitis. Immunohistochemistry and in situ hybridization were used to delineate the presence of viral proteins and RNA in the nervous systems of nude mice infected with the Daniels strain of Theiler's virus. This system permits the analysis of a viral infection in the absence of an effective immune response. By immunohistochemistry, viral antigen was found in the processes and cell bodies of neurons and glial cells. Besides the presence of viral antigen in these cell types, by in situ hybridization, Theiler's virus RNA was also found in cells associated with vascular endothelium in the brains and spinal cords of these infected mice. Theiler's virus RNA-positive endothelial cells were observed not only near the primary lesions but also away from demonstrable lesions in normal-appearing regions in the central nervous system. Earlier work had suggested an intra-axonal dissemination for this virus (M. C. Dal Canto and H. L. Lipton, Am. J. Pathol. 106:20-29, 1982). Our findings are consistent with this model but also suggest an additional mechanism for virus spread within the central nervous system, i.e., by infecting vascular cells and crossing the blood-brain barrier. Lastly, after Theiler's murine encephalomyelitis virus infection, not only glial cells but also endothelial cells express major histocompatibility complex class II (la) antigen on their surface (M. Rodriguez, M. L. Pierce, and E. A. Howie, J. Immunol. 138:3438-3442, 1987). Our demonstration of Theiler's virus-infected endotheliumlike cells may explain interactions of virus products in stimulating antigen presentation.     

Infection with Theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via NF-kappaB activation: potential role for the initiation of demyelinating disease

Theiler's virus infection in the central nervous system (CNS) induces a demyelinating disease very similar to human multiple sclerosis. We have assessed cytokine gene activation upon Theiler's murine encephalomyelitis virus (TMEV) infection and potential mechanisms in order to delineate the early events in viral infection that lead to immune-mediated demyelinating disease. Infection of SJL/J primary astrocyte cultures induces selective proinflammatory cytokine genes (interleukin-12p40 [IL-12p40], IL-1, IL-6, tumor necrosis factor alpha, and beta interferon [IFN-beta]) important in the innate immune response to infection. We find that TMEV-induced cytokine gene expression is mediated by the NF-kappaB pathway based on the early nuclear NF-kappaB translocation and suppression of cytokine activation in the presence of specific inhibitors of the NF-kappaB pathway. Further studies show this to be partly independent of dsRNA-dependent protein kinase (PKR) and IFN-alpha/beta pathways. Altogether, these results demonstrate that infection of astrocytes and other CNS-resident cells by TMEV provides the early NF-kappaB-mediated signals that directly activate various proinflammatory cytokine genes involved in the initiation and amplification of inflammatory responses in the CNS known to be critical for the development of immune-mediated demyelination.     

B-lymphocyte requirement for vaccine-mediated protection from Theiler's murine encephalomyelitis virus-induced central nervous system disease.

The role of humoral immunity in the protection of vaccinated SJL/J mice from central nervous system disease induced by the DA strain (DAV) of Theiler's murine encephalomyelitis virus was investigated in B-cell-deficient mice. Mice were depleted of B cells by treatment with a mouse monoclonal antibody specific for immunoglobulin M. DAV-vaccinated, B-cell-deficient mice failed to clear viral infection and were no longer protected from Theiler's murine encephalomyelitis virus-mediated central nervous system disease. CD4+ T cells are required in this model of protection to provide help for the development of an antiviral antibody response in the central nervous system.     

Interleukin-6, produced by resident cells of the central nervous system and infiltrating cells, contributes to the development of seizures following viral infection

Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development.     

Survival of athymic (nu/nu) mice after Theiler''s murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody.

Little or no antiviral immune response is mounted in athymic nude mice infected with the Daniels strain of Theiler's murine encephalomyelitis virus. In these athymic mice, increasing levels of infectious virus could be detected in the central nervous system. Seventy-five percent (9 of 12) of the nude mice were moribund or dead by 4 weeks postinfection. In contrast, treatment of Theiler's virus-infected nude mice with a neutralizing monoclonal antibody (H7-2) against the viral protein VP-1 resulted in a dramatic reduction of infectious virus within the central nervous system. All antibody-treated nude animals survived beyond 4 weeks postinfection. Monoclonal antibody titers could be maintained by passive transfer in treated nude mice at levels comparable to those of polyclonal antibody titers found in heterozygous infected nu/+ littermates. Areas of demyelination were detected in the untreated animals as early as 7 days after infection with little or no remyelination present. In approximately one-half of the antibody-treated nude animals, no demyelinating lesions were found. However, the rest of these treated mice were found to have areas of both demyelination and remyelination. Thus, anti-Theiler's murine encephalomyelitis virus antibody against VP-1 can play a dramatic role in the survival of mice, clearance of virus, limiting viral spread, and altering the pattern of disease in the absence of a functional T-cell response.     

Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler's virus

Microglia are resident central nervous system (CNS) macrophages. Theiler's murine encephalomyelitis virus (TMEV) infection of SJL/J mice causes persistent infection of CNS microglia, leading to the development of a chronic-progressive CD4(+) T-cell-mediated autoimmune demyelinating disease. We asked if TMEV infection of microglia activates their innate immune functions and/or activates their ability to serve as antigen-presenting cells for activation of T-cell responses to virus and endogenous myelin epitopes. The results indicate that microglia lines can be persistently infected with TMEV and that infection significantly upregulates the expression of cytokines involved in innate immunity (tumor necrosis factor alpha, interleukin-6 [IL-6], IL-18, and, most importantly, type I interferons) along with upregulation of major histocompatibility complex class II, IL-12, and various costimulatory molecules (B7-1, B7-2, CD40, and ICAM-1). Most significantly, TMEV-infected microglia were able to efficiently process and present both endogenous virus epitopes and exogenous myelin epitopes to inflammatory CD4(+) Th1 cells. Thus, TMEV infection of microglia activates these cells to initiate an innate immune response which may lead to the activation of naive and memory virus- and myelin-specific adaptive immune responses within the CNS.     

Theiler's murine encephalomyelitis virus-induced cardiac and skeletal muscle disease.

The DA strain of Theiler's murine encephalomyelitis virus, a member of the cardiovirus genus of picornaviruses, induces a restricted and persistent infection associated with a demyelinating process following intracerebral inoculation of mice; both virus infection and the immune response are believed to contribute to the late white matter disease. We now report that intraperitoneal inoculation with DA produces an acute myositis that progresses to a chronic inflammatory muscle disease in CD-1 mice as well as several inbred mouse strains. Some mouse strains also develop central nervous system white matter disease and a focal myocarditis. Infectious virus in skeletal muscle falls to undetectable levels 3 weeks postinoculation (p.i.), although viral genome persists for at least 12 weeks p.i., the longest period of observation. Severe combined immunodeficient animals have evidence of muscle pathology as long as 5 weeks p.i., suggesting that DA virus is capable of inducing chronic muscle disease in the absence of an immune response. The presence in immunocompetent mice, however, of prominent muscle inflammation in the absence of infectious virus suggests that the immune system also contributes to the pathology. T lymphocytes are the predominant cell type infiltrating the skeletal muscle during the chronic disease. This murine model may further our understanding of virus-induced chronic myositis and help to clarify the pathogenesis of human inflammatory myopathies.     

Chemokine expression in the central nervous system of mice with a viral disease resembling multiple sclerosis: roles of CD4+ and CD8+ T cells and viral persistence

During the first 45 days after intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV), the levels of mRNAs encoding chemokines MCP-1/CCL2, RANTES/CCL5, and IP-10/CXCL10 in the central nervous system (CNS) are closely related to the sites of virus gene expression and tissue inflammation. In the present study, these chemokines were monitored during the latter 135 days of a 6-month course of TMEV-induced disease in susceptible (PLJ) or resistant (C57BL/6) mice that possessed or lacked either CD4+ or CD8+ T cells. These data were additionally correlated to mouse genotype, virus persistence in the CNS, antiviral antibody titers, mortality, and the severity of neurological disease. Surprisingly, the major determinant of chemokine expression was virus persistence: the factors of susceptible or resistant genotype, severity of neuropathology, and presence or absence of regulatory T cells exerted minimal effects. Our observations indicated that chemokine expression in the CNS in this chronic viral disorder was intrinsic to the CNS innate immune response to infection and was not governed by elements of the adaptive immune system.     

Monoclonal anti-I-A antibody reverses chronic paralysis and demyelination in Theiler's virus-infected mice: critical importance of timing of treatment

Susceptibility to demyelination caused by the WW isolate of Theiler's murine encephalomyelitis viruses is linked to class II genes of the major histocompatibility complex. SJL/J (H-2s) mice, expressing only I-As class II gene products of the major histocompatibility complex, are highly susceptible to Theiler's murine encephalomyelitis virus infection with the WW virus isolate, with chronic paralysis and severe inflammation and demyelination in the central nervous system. The effect of in vivo administration of anti-I-As monoclonal antibodies on Theiler's murine encephalomyelitis virus infection was observed. SJL/J mice were treated in various protocols pre- or postinfection. Anti-I-As monoclonal antibody reversed chronic paralysis and reduced inflammation and demyelination when given after the establishment of persistent infection. The effect was long lasting, but clinical signs, inflammation, and demyelination recurred 2 months after treatment ceased. Anti-I-As antibodies had no effect on viral titers within the central nervous system. The timing of the administration of monoclonal antibodies was critical. Administration of anti-I-As before the establishment of the persistent infection resulted in fatal encephalitis.     

Mode of spread to and within the central nervous system after oral infection of neonatal mice with the DA strain of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus is a neurotropic enterovirus known to cause biphasic neural disease after intracerebral inoculation into adult mice. The present study characterizes a neonatal mouse model with a high disease incidence for the study of the acute phase of the pathogenesis of the DA strain of Theiler's murine encephalomyelitis virus after oral infection. The route of viral spread to and within the central nervous system (CNS) was determined by examining the kinetics of viral replication in various organs and by performing histopathological analysis. Viral antigen was detected widely in the neonatal CNS, mainly in the gray matter, and it was asymmetrical and multifocal in its distribution, with considerable variation in lesion distribution from animal to animal. Necrotizing lesions appeared to expand by direct extension from infected cells to their close neighbors, with a general disregard of neuroanatomical boundaries. The diencephalon showed particular susceptibility to viral infection. Other areas of the CNS, including the cerebellum and dentate gyrus of the hippocampus, were consistently spared. Neurons with axons extending peripherally to other organs or receiving direct input from the peripheral nervous system were not preferentially affected. The kinetics of viral replication in the liver, spleen, and CNS and the histopathological findings indicate that viral entry to the CNS is via a direct hematogenous route in orally infected neonatal mice and that the disease then progresses within the CNS mainly by direct extension from initial foci.     

Hierarchy of effects of the MHC and T cell receptor beta-chain genes in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible mice with Theiler's murine encephalomyelitis virus induces a demyelinating disease that is similar to human multiple sclerosis. This murine model for human multiple sclerosis is apparently immune-mediated and the genes involved in the immune response influence the outcome of disease susceptibility as observed with human multiple sclerosis. These genes include the MHC and TCR genes. However, the functional relationships among these genes on the disease susceptibility has not yet been studied. In this study, we demonstrate that the effect of the H-2s genotype from susceptible SJL/J mice overrides the resistant effect of the BALB/c TCR beta-chain gene in CXJ recombinant-inbred and BALB.S congenic mice. These results strongly suggest the presence of a hierarchy of genes involved in the immune response in Theiler's murine encephalomyelitis virus-induced demyelinating disease.     

Proteins induced in tissue culture by four isolates of Theiler''s murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

Lack of correlation of central nervous system inflammation and neuropathology with the development of seizures following acute virus infection

Infection of C57BL/6 mice by the intracerebral route with the Daniels (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) resulted in acute behavioral seizures in approximately 50% of the mice. By titration, the viral dose correlated with the percentage of mice developing seizures; however, neuropathological changes were similar over the dose range, and viral clearance from the brains occurred uniformly by day 14 postinfection (p.i.). Other TMEV strains and mutants (GDVII, WW, BeAn 8386 [BeAn], DApBL2M, H101) induced seizures in C57BL/6 mice to various degrees. The BeAn strain and DApBL2M mutant were similar to the DA strain in the percentages of mice developing seizures and neuropathological changes and in the extent of infected cells. The GDVII and WW strains caused 100% mortality by days 5 and 6 p.i., respectively, at which time neuropathological changes and neuronal infection were extensive. The H101 mutant induced seizures and caused 100% mortality by day 7 p.i.; however, only minor neuropathological changes and few infected cells were observed. Thus, in H101 mutant infections, it appears that elevated levels of cytokines, rather than neuronal cell death, play the dominant role in seizure induction.     

Clonal expansion of infiltrating T cells in the spinal cords of SJL/J mice infected with Theiler's virus

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus results in immune-mediated inflammatory demyelination in the white matter and consequent clinical symptoms. This system has been utilized as an important virus model for human multiple sclerosis. Although the potential involvement of virus-specific Th cells has been studied extensively, very little is known about the nature of T cells infiltrating the CNS during viral infection and their role in the development of demyelinating disease. In this study, the clonal nature of T cells in the spinal cord during the disease course was analyzed using size spectratyping and sequencing of the TCR beta-chain CDR3 region. These studies clearly indicate that T cells are clonally expanded in the CNS after viral infection, although the overall TCR repertoire appears to be diverse. The clonal expansion appears to be Ag-driven in that it includes Th cells specific for known viral epitopes. Interestingly, such restricted accumulation of T cells was not detectable in the infiltrates of mice with proteolipid protein peptide-induced experimental autoimmune encephalomyelitis. The initial T cell repertoire (7-9 days postinfection) seems to be more diverse than that observed in the later stage (65 days) of virally induced demyelination, despite the more restricted utilization of Vbeta subfamilies. These results strongly suggest continuous stimulation and clonal expansion of virus-specific T cells in the CNS of Theiler's murine encephalomyelitis virus-infected mice during the entire course of demyelinating disease.     

Anticapsid immunity level, not viral persistence level, correlates with the progression of Theiler's virus-induced demyelinating disease in viral P1-transgenic mice

Intracranial infection of Theiler's murine encephalomyelitis virus (TMEV) induces demyelination and a neurological disease in susceptible SJL/J (SJL) mice that resembles multiple sclerosis. While the virus is cleared from the central nervous system (CNS) of resistant C57BL/6 (B6) mice, it persists in SJL mice. To investigate the role of viral persistence and its accompanying immune responses in the development of demyelinating disease, transgenic mice expressing the P1 region of the TMEV genome (P1-Tg) were employed. Interestingly, P1-Tg mice with the B6 background showed severe reductions in both CD4(+) and CD8(+) T-cell responses to capsid epitopes, while P1-Tg mice with the SJL background displayed transient reductions following viral infection. Reduced antiviral immune responses in P1-Tg mice led to >100- to 1,000-fold increases in viral persistence at 120 days postinfection in the CNS of mice with both backgrounds. Despite the increased CNS TMEV levels in these P1-Tg mice, B6 P1-Tg mice developed neither neuropathological symptoms nor demyelinating lesions, and SJL P1-Tg mice developed significantly less severe TMEV-induced demyelinating disease. These results strongly suggest that viral persistence alone is not sufficient to induce disease and that the level of T-cell immunity to viral capsid epitopes is critical for the development of demyelinating disease in SJL mice.     

Gender bias in Theiler's virus-induced demyelinating disease correlates with the level of antiviral immune responses

Multiple sclerosis is an immune-mediated disease of the CNS and shows a sex-biased distribution in which 60-75% of all cases are female. A mouse model of multiple sclerosis, Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, also displays a gender bias. However, in the C57L/J strain of mice, males are susceptible to disease whereas females are completely resistant. In this study we determined the gender differences in the TMEV-specific immune response, which may be responsible for the gender bias in clinical disease. Our data clearly demonstrate that female C57L/J mice induce significantly higher levels of TMEV-specific neutralizing Ab as well as a stronger peripheral T cell response throughout the course of viral infection. In contrast, male mice have a higher level of TMEV-specific CD4(+) and CD8(+) T cell infiltration into the CNS as well as viral persistence. These results suggest that a higher level of the initial antiviral immune response in female mice may be able to effectively clear virus from the periphery and CNS and therefore prevent further disease manifestations. Male mice in contrast do not mount as effective an immune response, thereby allowing for eventual viral persistence in the CNS and continuous T cell expansion leading to clinical symptoms.     

Viral delivery of an epitope from Haemophilus influenzae induces central nervous system autoimmune disease by molecular mimicry

Multiple sclerosis (MS) is an autoimmune CNS demyelinating disease in which infection may be an important initiating factor. Pathogen-induced cross-activation of autoimmune T cells may occur by molecular mimicry. Infection with wild-type Theiler's murine encephalomyelitis virus induces a late-onset, progressive T cell-mediated demyelinating disease, similar to MS. To determine the potential of virus-induced autoimmunity by molecular mimicry, a nonpathogenic neurotropic Theiler's murine encephalomyelitis virus variant was engineered to encode a mimic peptide from protease IV of Haemophilus influenzae (HI), sharing 6 of 13 aa with the dominant encephalitogenic proteolipid protein (PLP) epitope PLP(139-151). Infection of SJL mice with the HI mimic-expressing virus induced a rapid-onset, nonprogressive paralytic disease characterized by potent activation of self-reactive PLP(139-151)-specific CD4(+) Th1 responses. In contrast, mice immunized with the HI mimic-peptide in CFA did not develop disease, associated with the failure to induce activation of PLP(139-151)-specific CD4(+) Th1 cells. However, preinfection with the mimic-expressing virus before mimic-peptide immunization led to severe disease. Therefore, infection with a mimic-expressing virus directly initiates organ-specific T cell-mediated autoimmunity, suggesting that pathogen-delivered innate immune signals may play a crucial role in triggering differentiation of pathogenic self-reactive responses. These results have important implications for explaining the pathogenesis of MS and other autoimmune diseases.     

Preferential induction of IL-10 in APC correlates with a switch from Th1 to Th2 response following infection with a low pathogenic variant of Theiler's virus

Theiler's murine encephalomyelitis virus induces immune-mediated demyelination in susceptible mice after intracerebral inoculation. A naturally occurring, low pathogenic Theiler's murine encephalomyelitis virus variant showed a single amino acid change within a predominant Th epitope from lysine to arginine at position 244 of VP1. This substitution is the only one present in the entire viral capsid proteins. In this paper, we demonstrate that the majority of T cells specific for VP1(233-250) and VP2(74-86) from wild-type virus-infected mice are Th1 type and these VP1-specific cells poorly recognize the variant VP1 epitope (VP1(K244R)) containing the substituted arginine. In contrast, the Th2-type T cell population specific for these epitopes predominates in variant virus-infected mice. Immunization with UV-inactivated virus or VP1 epitope peptides could not duplicate the preferential Th1/Th2 responses following viral infection. Interestingly, the major APC populations, such as dendritic cells and macrophages, produce IL-12 on exposure to the pathogenic wild-type virus, whereas they preferentially produce IL-10 in response to the low pathogenic variant virus. Thus, such a spontaneous mutant virus may have a profoundly different capability to induce Th-type responses via selective production of cytokines involved in T cell differentiation and the consequent pathogenicity of virally induced immune-mediated inflammatory diseases.     

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 Theiler's 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.