Human class I major histocompatibility complex alleles determine central nervous system injury versus repair.

BACKGROUND: We investigated the role of human HLA class I molecules in persistent central nervous system (CNS) injury versus repair following virus infection of the CNS. METHODS: Human class I A11+ and B27+ transgenic human beta-2 microglobulin positive (Hß2m+) mice of the H-2 b background were generated on a combined class I-deficient (mouse beta-2 microglobulin deficient, ß2m0) and class II-deficient (mouse Aß0) phenotype. Intracranial infection with Theiler's murine encephalomyelitis virus (TMEV) in susceptible SJL mice results in acute encephalitis with prominent injury in the hippocampus, striatum, and cortex. RESULTS: Following infection with TMEV, a picornavirus, the Aß0.ß2m0 mice lacking active immune responses died within 18 to 21 days post-infection. These mice showed severe encephalomyelitis due to rapid replication of the viral genome. In contrast, transgenic Hß2m mice with insertion of a single human class I MHC gene in the absence of human or mouse class II survived the acute infection. Both A11+ and B27+ mice significantly controlled virus RNA expression by 45 days and did not develop late-onset spinal cord demyelination. By 45 days post-infection (DPI), B27+ transgenic mice showed almost complete repair of the virus-induced brain injury, but A11+ mice conversely showed persistent severe hippocampal and cortical injury. CONCLUSIONS: The findings support the hypothesis that the expression of a single human class I MHC molecule, independent of persistent virus infection, influences the extent of sub frequent chronic neuronal injury or repair in the absence of a class II MHC immune response.

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity.

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.

Naturally Occurring Monoclonal Antibodies and Their Therapeutic Potential for Neurologic Diseases.

IMPORTANCE: Modulating the immune system does not reverse long-term disability in neurologic disorders. Better neuroregenerative and neuroprotective treatment strategies are needed for neuroinflammatory and neurodegenerative diseases. OBJECTIVE: To review the role of monoclonal, naturally occurring antibodies (NAbs) as novel therapeutic molecules for treatment of neurologic disorders. EVIDENCE REVIEW: Peer-reviewed articles, including case reports, case series, retrospective reviews, prospective randomized clinical trials, and basic science reports, were identified in a PubMed search for articles about NAbs and neurologic disorders that were published from January 1, 1964, through June 30, 2015. We concentrated our review on multiple sclerosis, Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis. FINDINGS: Many insults, including trauma, ischemia, infection, inflammation, and neurodegeneration, result in irreversible damage to the central nervous system. Central nervous system injury often results in a pervasive inhibitory microenvironment that hinders regeneration. A common targeted drug development strategy is to identify molecules with high potency in animal models. Many approaches often fail in the clinical setting owing to a lack of efficacy in human diseases (eg, less than the response demonstrated in animal models) or a high incidence of toxic effects. An alternative approach is to identify NAbs in humans because these therapeutic molecules have potential physiologic function without toxic effects. NAbs of the IgG, IgA, or IgM isotype contain germline or close to germline sequences and are reactive to self-components, altered self-components, or foreign antigens. Our investigative group developed recombinant, autoreactive, natural human IgM antibodies directed against oligodendrocytes or neurons with therapeutic potential for central nervous system repair. One such molecule, recombinant HIgM22, directed against myelin and oligodendrocytes completed a successful phase 1 clinical trial without toxic effects with the goal of promoting remyelination in multiple sclerosis. CONCLUSIONS AND RELEVANCE: Animal studies demonstrate that certain monoclonal NAbs are beneficial as therapeutic agents for neurologic diseases. This class of antibodies represents a unique source from which to develop a new class of disease-modifying therapies.

A natural human IgM that binds to gangliosides is therapeutic in murine models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating, fatal neurological disease that primarily affects spinal cord anterior horn cells and their axons for which there is no treatment. Here we report the use of a recombinant natural human IgM that binds to the surface of neurons and supports neurite extension, rHIgM12, as a therapeutic strategy in murine models of human ALS. A single 200 µg intraperitoneal dose of rHIgM12 increases survival in two independent genetic-based mutant SOD1 mouse strains (SOD1G86R and SOD1G93A) by 8 and 10 days, delays the onset of neurological deficits by 16 days, delays the onset of weight loss by 5 days, and preserves spinal cord axons and anterior horn neurons. Immuno-overlay of thin layer chromatography and surface plasmon resonance show that rHIgM12 binds with high affinity to the complex gangliosides GD1a and GT1b. Addition of rHIgM12 to neurons in culture increases α-tubulin tyrosination levels, suggesting an alteration of microtubule dynamics. We previously reported that a single peripheral dose of rHIgM12 preserved neurological function in a murine model of demyelination with axon loss. Because rHIgM12 improves three different models of neurological disease, we propose that the IgM might act late in the cascade of neuronal stress and/or death by a broad mechanism.

Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders.

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long-term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody-mediated cell adhesion, and neurite outgrowth on HIgM12-coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)-positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro-motor disorders including multiple sclerosis.

Deletion of Virus-specific T-cells Enhances Remyelination in a Model of Multiple Sclerosis.

We used transgenic expression of capsid antigens to Theiler's murine encephalomyelitis virus (TMEV) to study how the immune response to VP1 and VP2 influences spinal cord demyelination, remyelination and axonal loss during the acute and chronic phases of infection. Expression from birth of capsid antigen under the ubiquitin promoter resulted in tolerance to the antigen and absence of an immune response to the respective capsid antigen following virus infection. The transgenic mice were crossed to B10.Q mice normally susceptible to demyelination but which, when compared to FVB mice of the same H2 q haplotype, show poor remyelination. The major finding in this study was that VP1+ and VP2+ animals featured more remyelination at all three chronic time points (90, 180 and 270 dpi) than transgene-negative controls. Interestingly, at 270 dpi, remyelination in VP1+ mice tended to be higher and more complete than that in VP2+ mice. Compared with transgene- negative controls, VP1+ and VP2+ animals showed similar demyelination in but less only late in the disease (270 dpi). The number of mid-thoracic axons at the last time point correlated with the levels of remyelination. The increase in number of axons in VP1+ mice with remyelination was driven by counts in medium- and large-caliber axons. This study supports the hypothesis that expression of viral capsid proteins as self and subsequent genetic deletion of capsid-specific T cells influences the extent of spinal cord remyelination following Theiler's virus-induced demyelination. We propose that VP1- and, to a lesser extent, VP2-specific CD8+ T cells limit and/or prevent the naturally occurring process of remyelination. This finding may have relevance to human multiple sclerosis, as targeted removal of CD8+ T cells specific for a yet-to-be-discovered causative peptide may enhance remyelination and prevent axonal loss in patients.

Transgenic expression of viral capsid proteins predisposes to axonal injury in a murine model of multiple sclerosis.

We used transgenic expression of capsid antigens to Theiler's murine encephalomyelitis virus (TMEV) to study the influence of VP1, VP2 or VP2(121-130) to either protection or pathogenesis to chronic spinal cord demyelination, axonal loss and functional deficits during the acute and chronic phases of infection. We used both mice that are normally susceptible (FVB) and mice normally resistant (FVB.D(b) ) to demyelination. Transgenic expression of VP2(121-130) epitope in resistant FVB.D(b) mice caused spinal cord pathology and virus persistence because the VP2(121-130) epitope is the dominant peptide recognized by D(b) , which is critical for virus clearance. In contrast, all three FVB TMEV transgenic mice showed more demyelination, inflammation and axonal loss as compared with wild-type FVB mice, even though virus load was not increased. Motor function measured by rotarod showed weak correlation with total number of midthoracic axons, but a strong correlation with large-caliber axons (>10µm(2) ). This study supports the hypothesis that expression of viral capsid proteins as self influences the extent of axonal pathology following Theiler's virus-induced demyelination. The findings provide insight into the role of axonal injury in the development of functional deficits that may have relevance to human demyelinating disease.

Transgenic expression of the 3D polymerase inhibits Theiler's virus infection and demyelination.

The RNA-dependent RNA polymerase 3D(pol) is required for the elongation of positive- and negative-stranded picornavirus RNA. During the course of investigating the effect of the transgenic expression of viral genes on the host immune response, we evaluated the viral load present in the host after infection. To our surprise, we found that 3D transgenic expression in genetically susceptible FVB mice led to substantially lower viral loads after infection with Theiler's murine encephalomyelitis virus (TMEV). As a result, spinal cord damage caused by chronic viral infection in the central nervous system was reduced in FVB mice that expressed 3D. This led to the preservation of large-diameter axons and motor function in these mice. The 3D transgene also lowered early viral loads when expressed in FVB-D(b) mice resistant to persistent TMEV infection. The protective effect of 3D transgenic expression was not altered in FVB-Rag(-/-).3D mice that are deficient in T and B cells, thus ruling out a mechanism by which the overexpression of 3D enhanced the adaptive immune clearance of the virus. Understanding how endogenously overexpressed 3D polymerase inhibits viral replication may lead to new strategies for targeting therapies to all picornaviruses.

Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.

Axon injury is a major determinant of the loss of neurological function in patients with multiple sclerosis. It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8 T cells and perforin in the Theiler murine encephalomyelitis virus model of multiple sclerosis have used mouse strains resistant to Theiler murine encephalomyelitis virus infection. To test the role of CD8 T cells in axon injury, we established a perforin-deficient mouse model on the H-2 major histocompatibility complex background thereby removing confounding factors related to viral biology in this Theiler murine encephalomyelitis virus-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2 mice, but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediator of axon injury and lend additional support to the hypothesis that CD8 T cells are primarily responsible for axon damage in multiple sclerosis.

Tumor necrosis factor alpha is reparative via TNFR2 [corrected] in the hippocampus and via TNFR1 [corrected] in the striatum after virus-induced encephalitis.

Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor alpha (TNFalpha) in parenchymal brain pathology resolution following virus-induced encephalitis from a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF(-/-) mice (without TNFalpha expression), B6/129 TNFR1(-/-) mice (without TNFalpha receptor 1 expression), and B6/129 TNFR2(-/-) mice (without TNFalpha receptor 2 expression). Normal TNFalpha-expressing controls were TMEV-infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF(-/-) mice following infection. Treating virus-infected mice with active recombinant mouse TNFalpha resulted in less hippocampal and striatal pathology, whereas TNFalpha-neutralizing treatment worsened pathology. T1 "black holes" appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF(-/-) mice. TNFR2 [corrected] mediated hippocampal pathology resolution whereas TNFR1 [corrected] mediated striatal healing. These findings indicate the role of TNFalpha in resolution of sublethal hippocampal and striatal injury.

Human HLA-DR transgenes protect mice from fatal virus-induced encephalomyelitis and chronic demyelination.

We evaluated the participatory role of human HLA-DR molecules in control of virus from the central nervous system and in the development of subsequent spinal cord demyelination. The experiments utilized intracranial infection with Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that, in some strains of mice, results in primary demyelination. We studied DR2 and DR3 transgenic mice that were bred onto a combined class I-deficient mouse (beta-2 microglobulin deficient; beta2m(0)) and class II-deficient mouse (Abeta(0)) of the H-2(b) background. Abeta(0).beta2m(0) mice infected with TMEV died within 18 days of infection. These mice showed severe encephalomyelitis due to rapid replication of virus genome. In contrast, transgenic mice with insertion of a single human class II major histocompatibility complex (MHC) gene (DR2 or DR3) survived the acute infection. DR2 and DR3 mice controlled virus infection by 45 days and did not develop spinal cord demyelination. Levels of virus RNA were reduced in HLA-DR transgenic mice compared to Abeta(0).beta2m(0) mice. Virus-neutralizing antibody responses did not explain why DR mice survived the infection and controlled virus replication. However, DR mice showed an increase in gamma interferon and interleukin-2 transcripts in the brain, which were associated with protection. The findings support the hypothesis that the expression of a single human class II MHC molecule can, by itself, influence the control of an intracerebral pathogen in a host without a competent class I MHC immune response. The mechanism of protection appears to be the result of cytokines released by CD4(+) T cells.

STAT4- and STAT6-signaling molecules in a murine model of multiple sclerosis.

Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4+ T cell responses following Theiler's virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4-/- mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up-regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4-/- immune system. Thus, virus infection of STAT4-/- mice results in a model that may allow for dissection of the immune events predisposing to late-onset demyelination in MS.

A 40-cM region on chromosome 14 plays a critical role in the development of virus persistence, demyelination, brain pathology and neurologic deficits in a murine viral model of multiple sclerosis.

Theiler virus persists and induces immune-mediated demyelination in susceptible mice and serves as a model of multiple sclerosis. Previously, we identified 4 markers--D14Mit54, D14Mit60, D14Mit61, and D14Mit90--in a 40-cM region of chromosome 14 that are associated with demyelination in a cross between susceptible DBA/2 and resistant B10.D2 mice. We generated congenic-inbred mice to examine the contribution of this 40-cM region to disease. DBA Chr.14B10 mice, containing the chromosomal segment marked by the microsatellite polymorphisms, developed less spinal cord demyelination than did DBA/2 mice. More demyelination was found in the reciprocal congenic mouse B10.D2 Chr.14D2 than in the B10.D2 strain. Introduction of the DBA/2 chromosomal region onto the B10.D2 genetic background resulted in more severe disease in the striatum and cortex relative to B10.D2 mice. The importance of the marked region of chromosome 14 is indicated by the decrease in neurological performance using the Rotarod test during chronic disease in B10.D2 Chr.14D2 mice in comparison to B10.D2 mice. Viral replication was increased in B10.D2 Chr.14D2 mice as determined by quantitative real-time RT-PCR. These results indicate that the 40-cM region on chromosome 14 of DBA/2 mice contributes to viral persistence, subsequent demyelination, and loss of neurological function.

Gamma interferon is critical for neuronal viral clearance and protection in a susceptible mouse strain following early intracranial Theiler's murine encephalomyelitis virus infection.

We evaluated the role of gamma interferon (IFN-gamma) in protecting neurons from virus-induced injury following central nervous system infection. IFN-gamma(-/-) and IFN-gamma(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-gamma(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-gamma(-/-) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-gamma(-/-) mice of the susceptible H-2(q) 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-gamma(-/-) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-gamma(-/-) H-2(q) 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-gamma(-/-) H-2(q) mice. IFN-gamma 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-gamma(-/-) H-2(q) mice was lower than those in IFN-gamma(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-gamma exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-gamma plays a critical role in protecting spinal cord neurons from persistent infection and death.

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 intracerebral injection of SJL mice with Daniel's (DA) and BeAn 8386 (BeAn) strains of Theiler's 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.

Membrane lymphotoxin is required for resistance to Theiler's virus infection.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are important in immune system development and function. LT consists of soluble LT-alpha(3), which binds to TNF-R1 and TNF-R2, and membrane LT-alpha(1)beta(2), which binds to LT-beta-R. We investigated the role of LT and TNF in disease induced by Daniel's (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) since the immune response is believed to be important in both resistance to DA infection as well as mediation of virus-induced demyelination. DA persisted and induced inflammatory demyelination in LT-alpha(-/-) (but not TNF(-/-)) weanling mice of a normally resistant haplotype (C57BL/6), suggesting that LT, but not TNF, is critical for resistance to DA infection. This activity of LT depends on membrane LT-alpha(1)beta(2) and not soluble LT-alpha(3), since DA virus persisted and induced inflammatory demyelination in LT-beta-R(-/-), but not TNF-R1(-/-) or TNF-R2(-/-), mice. The LT-alpha(-/-) and LT-beta-R(-/-) mice failed to mount a virus-specific cytotoxic T cell response. Treatment of weanling C57BL/6 mice with LT-beta-R-Ig, which blocks membrane LT activity, failed to increase susceptibility, suggesting that the LT effect is related to its action on immune system development which is fixed by 3 weeks of age. Our data suggest that membrane LT is important in resistance to DA infection (possibly through interference with CD8+ T cell development and function). There was relatively little demyelination associated with inflammation in LT-alpha(-/-) and LT-beta-R(-/-) mice compared to susceptible SJL mice, suggesting the possibility that LT plays a role in mediating demyelination.

ICAM-1 is crucial for protection from TMEV-induced neuronal damage but not demyelination.

Previous work has suggested that the factors protecting mice from Theiler's murine encephalomyelitis virus (TMEV)-induced spinal cord demyelination are distinct from those involved in protection of the brain during the acute encephalitic phase. In this study, we examined the requirement for intercellular adhesion molecule-1 (ICAM-1) in both of these processes. During the acute phase of infection (days 7 to 10 after intracerebral infection with TMEV), no differences in brain or spinal cord pathology or virus burdens were observed between ICAM-1-knockout mice and the infected immunocompetent control mice of a similar background. Examination of brain pathology later in infection (that is, day 45 post infection [p.i.]) revealed that ICAM-1-deficient mice experienced increased levels of pathology in gray matter regions of the brain. We observed an increase in striatal damage and meningeal inflammation in the brains of TMEV-infected ICAM-1-knockout mice compared to C57BL/6J mice. Despite the increase in brain pathology, no immunoreactivity to viral antigens was detected, suggesting that the virus had been cleared by this time. Resistance to demyelination was similar in both groups, indicating that the resulting immune response was sufficient for protection of the spinal cord white matter.