Viruses and multiple sclerosis

Multiple sclerosis (MS) is a chronic demyelinating disorder of unknown etiology, possibly caused by a virus or virus-triggered immunopathology. The virus might reactivate after years of latency and lyse oligodendrocytes, as in progressive multifocal leukoencephalopathy, or initiate immunopathological demyelination, as in animals infected with Theiler's murine encephalomyelitis virus or coronaviruses. The argument for a viral cause of MS is supported by epidemiological analyses and studies of MS in identical twins, indicating that disease is acquired. However, the most important evidence is the presence of bands of oligoclonal IgG (OCBs) in MS brain and CSF that persist throughout the lifetime of the patient. OCBs are found almost exclusively in infectious CNS disorders, and antigenic targets of OCBs represent the agent that causes disease. Here, the authors review past attempts to identify an infectious agent in MS brain cells and discuss the promise of using recombinant antibodies generated from clonally expanded plasma cells in brain and CSF to identify disease-relevant antigens. They show how this strategy has been used successfully to analyze antigen specificity in subacute sclerosing panencephalitis, a chronic encephalitis caused by measles virus, and in neuromyelitis optica, a chronic autoimmune demyelinating disease produced by antibodies directed against the aquaporin-4 water channel.     

Demonstration of immunoglobulin in brains of ferrets inoculated with an SSPE strain of measles virus: Use of protein a conjugated to horseradish peroxidase

Summary Measles virus-specific immunoglobulin G (IgG) has been found in the brains of patients with subacute sclerosing panencephalitis (SSPE), a slowly progressing central nervous system (CNS) disease affecting children. IgG/albumin ratios indicate that the antibodies are probably synthesized in the CNS. In a ferret model system, protein A conjugated to horseradish peroxidase (PrAPx) was used to localize Ig's in brains of animals inoculated with a cell associated strain of SSPE. Ig's were found in plasma cells in various stages of antibody production both in perivascular inflammatory lesions and scattered throughout the cerebral cortex. These findings offer corroborative evidence that the Ig found in SSPE ferret brain and CSF is actively synthesized within the CNS. Antibody was also demonstrated in glial and peuronal cell bodies and processes and in postsynaptic profiles. These are the same sites where measles virus antigens are most frequently found and suggests the possibility of immune complex formation.Presented in part at the 41st annual mceting of the Electron Microscopy Society of America, August 8–12, 1983Supported in part by funds from grant S07-RR05838-2 awarded by the Biomedical Research Support Grant Program. Division of Research Resources, NIH (H. R. B.)     

A radioimmunoassay search for measles and distemper antigens in subacute sclerosing panencephalitis and multiple sclerosis brain tissues

Solid-phase direct sandwich radioimmunoassays have been developed which will detect measles antigen in as little as 20 μg of SSPE brain, measles antigen in 5 × 10⁴ pfu/ml of measles virus, and canine distemper antigen in 5 × 10⁵ pfu/ml of this virus. Using these assays, a search was carried out for measles and distemper antigens in CNS autopsy tissue from 3 multiple sclerosis (MS) patients. Even in 1000-fold higher concentrations of MS brain (20 mg), antigens of neither virus were detected. Additional studies were carried out using similar radioimmunoassays but employing IgG from 2 MS patients as probes against MS brain specimens, including IgG and acute plaque material from the same individual. However, no reaction between MS IgG and MS brain was detected. Thus no evidence was found for foreign antigen in the MS brains nor for autoantibody which reacted with brain tissue antigens.It is concluded that either measles or distemper are not present in acute MS lesions, or that they are present in such small amounts that their detection will require techniques even more sensitive than those used in the present study.     

Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: confirmation by non-radioactive in situ hybridization,immunocytochemistry and electron microscopy

Summary As part of continuing multidisciplinary studies on the neuropathogenesis of subacute sclerosing panencephalitis (SSPE), in situ hybridisation, immunocytochemistry and electron microscopy were used to detect measles virus nucleic acid, protein and nucleocapsids in brain perivascular infiltrates of three cases. Perivascular cuffing cells which contained measles virus nucleic acid and antigens were found in all cases. Infected cuffs occurred predominantly in areas of general parenchymal cell infection and in many of these a high proportion of the infiltrating cells were infected. Other cuffs in these areas were either uninfected or contained only a few infected cells. Occasional infected cells were also seen in cuffs in non-infected areas. In contrast, no specific immunocytochemical reactions or in situ hybridisation for measles virus was observed in brain tissue from a patient with herpes encephalitis. By electron microscopy viral nucleocapsid, consistent with measles virus, was found within the cytoplasm of plasma cells in the inflammatory cuffs in SSPE brain tissue. Possible explanations for our results are that infiltrates become infected on arrival in the CNS or alternatively, that the infected infiltrates reflect a generalised infection of the reticuloendothelial system. The frequent presence of uninfected cuffs favours the former explanation.     

Astrocytes and Schwann cells are virus-host cells in the nervous system of rats with Borna disease

Borna disease virus (BDV) replicates only in cells in the central (CNS) and peripheral (PNS) nervous system in adult rats. Infection of the nervous system is associated with a transient, intense mononuclear meningoencephalitis and immunemediated loss of BDV-infected neurons. The identification of BDV antigen in neurons and the accompanying immunologically-specific lysis of these cells led to the prediction that the CNS would be virus-free after the animal had recovered from encephalitis. However, BDV infectivity and antigen persist for the lifetime of the animal. It appeared, therefore, that other neural cells might be hosts for viral replication and provide a reservoir for the virus. Morphological criteria were used to identify astrocytes and Schwann cells which expressed BDV antigens in vivo. Borna disease virus (BDV) infected astrocytes were identified by double labeling tissue sections with combined cell-specific and BDV-specific antibodies in an avidin-biotin immunocytochemical assay. Examination of serial I micrometer-thick cryosections of hippocampus and sciatic nerve preparations revealed several cells that expressed both glial and BDV antigens. Infectious virus was recovered from cultures of Schwann cells from infected rats. Borna disease virus-infected glial elements persisted beyond the period of inflammation and massive neuronal destruction, and represented a major class of infected cells during chronic disease.     

Recombinant antibodies generated from both clonal and less abundant plasma cell immunoglobulin G sequences in subacute sclerosing panencephalitis brain are directed against measles virus

Increased immunoglobulin G (IgG) and intrathecally produced oligoclonal bands (OGBs) are characteristic of a limited number of inflammatory central nervous system (CNS) diseases and are often directed against the cause of disease. In subacute sclerosing panencephalitis (SSPE), the cause of disease and the target of the oligoclonal response is measles virus (MV). The authors previously showed that clonally expanded populations of CD38+ plasma cells in SSPE brain, the likely source of OGBs, are directed against MV. In characterizing the breadth of the plasma cell reactivities, the authors found that a large proportion of the less abundant plasma cells are also directed against MV. The intrathecal response may be useful in determining the causes of other inflammatory CNS diseases, such as multiple sclerosis, Behcet's disease, and neurosarcoidosis.     

Induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in Lewis rats

Intracerebral inoculation of weanling Lewis rats with measles virus led to the development of subacute measles encephalomyelitis (SAME) 4-8 weeks after infection. The disease is characterized pathologically by an intense inflammatory infiltration within both the white and grey matter of the central nervous system (CNS) without apparent demyelination. Both during and after SAME splenic lymphocytes from these animals could be restimulated in vitro to proliferate in the presence of myelin base protein (MBP). MBP-specific class II MHC-restricted T cell lines were isolated from this cell population. They were shown to exhibit no cross-reactivity with measles virus and to induce experimental allergic encephalitis (EAE) in naive syngeneic recipients following adoptive transfer. The clinical and histopathological signs of this T cell-mediated disease were identical to that seen in classical T cell-mediated EAE. A humoral immune response to MBP was only detected in a limited number of those rats with SAME. These results indicate that autoimmune reactions to brain antigen can arise during measles virus infection which may contribute to the pathogenesis of measles virus-associated encephalomyelitis.     

Measles virus antibodies in serum and cerebrospinal fluid in patients with multiple sclerosis and other neurological disorders,with special reference to measles antibody synthesis within the central nervous system

Measles virus hemagglutination-inhibiting (HI) and gel precipitating (GP) antibodies were determined in sera and cerebrospinal fluids (CSF) from 65 patients with multiple sclerosis (MS) and 65 patients with other neurological diseases. The serological results were correlated to content of immunoglobulin-G (IgG) and electrophoretic patterns of sera and CSF.Measles GP antibodies, identified as directed against measles virus ribonucleoprotein antigens, were detected in sera and in CSF from a significantly higher proportion of MS than of non-MS patients. No significant difference between the 2 groups of patients was found for measles HI antibodies.Reduced serum/CSF HI and/or GP antibody ratios were found in about one half of the MS patients and in 2 patients with chronic myelopathy. All patients with reduced antibody ratios had evidence of IgG synthesis within the central nervous system (CNS), as inferred from oligoclonal IgG patterns of the CSF. Reduced ratios of measles GP antibodies were 3 times as common as reduced ratios of HI antibodies. Immuno-electrophoretic assays indicated that the CSF GP antibodies were electrophoretically restricted in a number of MS patients.The results indicate that measles virus may be an active immunogen within the CNS in many MS patients and in some patients with chronic myelopathy, giving rise to an oligoclonal IgG antibody response.     

Antibodies in oligoclonal immunoglobulins in CSF from patients with acute cerebrovascular disease

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) of CSF and serum, and subsequent immunofixation with viral and structural brain components followed by autoradiography revealed in eight out of nine selected patients with oligoclonal CSF IgG and cerebrovascular disease local synthesis within the CNS of antibodies against one or more of the viruses tested: six patients against measles, five against herpes simplex virus type 1, and two against varicella virus. This finding may reflect a polyclonal B cell activation secondary to brain damage and elaboration of certain structural brain components. None of the patients had local synthesis of antibodies against the other viruses tested (mumps, rubella and cytomegalovirus), or against structural brain components (crude saline, lipid-proteolipid, myelin basic protein extracts from human brain and purified bovine myelin basic protein).     

Immunocytochemical studies for the localisation of measles antigens in multiple sclerosis plaques and measles virus-infected CNS tissue.

Actively demyelinating central nervous system (CNS) lesions from a patient with acute multiple sclerosis (MS) were tested for measles antigens using peroxidase-conjugated antimeasles antibody. No evidence of measles antigens was found. Similarly reacted tissue from 2 patients with chronic MS also revealed no evidence of measles antigens. Identically treated and simultaneously tested measles-infected CNS cultures and human SSPE brain tissue stained strongly for measles antigens. The possible reasons underlying the failure to detect measles antigens in MS are discussed.     

Peptide reactivity between multiple sclerosis (MS) CSF IgG and recombinant antibodies generated from clonally expanded plasma cells in MS CSF

We employed 19 recombinant antibodies (rAbs) generated from clonally expanded plasma cells, and native IgG from cerebrospinal fluid (CSF) of three multiple sclerosis (MS) patients for panning with phage displayed random peptide libraries. Specific peptide epitopes/mimotopes were identified and characterized. Importantly, peptide-antibody interactions were shared by rAbs and native IgG from the same patient. Three peptides strongly interacted with at least one other MS CSF, but not to inflammatory CNS controls. Database searches revealed several protein candidates including stress proteins, cell surface proteins, and neuronal proteins. Peptides derived from the candidate proteins were recognized by rAbs. Identification of peptide epitopes/mimotopes in MS may provide clues regarding disease-relevant antigens.     

Neuronal surface glycolytic enzymes are autoantigen targets in post-streptococcal autoimmune CNS disease

Infection with the Group A Streptococcus (GAS) can result in immune mediated brain disease characterised by a spectrum of movement and psychiatric disorders. We have previously described anti-neuronal antibodies in patients that bind to a restricted group of brain antigens with molecular weights 40 kDa, 45 kDa (doublet) and 60 kDa. The aim of this study was to define these antigens using 2-dimensional electrophoresis or ion exchange and hydrophobic interaction chromatography, followed by mass spectrometry. The findings were confirmed using commercial antibodies, commercial antigens and recombinant human antigens. The autoantigens were neuronal glycolytic enzymes--NGE (pyruvate kinase M1, aldolase C, neuronal-specific and non-neuronal enolase). These are multifunctional proteins that are all expressed intracellularly and on the neuronal cell surface. On the neuronal plasma membrane, NGE are involved in energy metabolism, cell signalling and synaptic neurotransmission. Anti-NGE antibodies were more common in the 20 unselected post-streptococcal CNS patients compared to 20 controls. In vitro experiments using cultured neurons showed that commercial anti-NGE antibodies induced apoptosis compared to blank incubation and control anti-HuD antibody. GAS also expresses glycolytic enzymes on cell surfaces that have 0-49% identity with human NGE, suggesting molecular mimicry and autoimmune cross-reactivity may be the pathogenic mechanism in post-streptococcal CNS disease.     

Subtractive expression cloning reveals high expression of CD46 at the blood-brain barrier

A subtractive expression cloning methodology was used to identify proteins having enriched expression at the blood-brain barrier (BBB) in comparison to liver and kidney tissues. A bovine brain capillary COS-1 cell cDNA expression library was screened with a BBB-specific antiserum. This strategy revealed that the membrane cofactor protein CD46, which is a regulator of complement activation in vivo and is also a potential measles virus receptor, is highly expressed at the BBB. The selective CD46 expression in brain at the BBB was confirmed by Northern blot analysis and confocal microscopy. The finding of selective expression of CD46 at the BBB is consistent with an important role played by the microvasculature in the immune surveillance of the brain.     

Optic neuritis and distribution of genetic markers of the HLA system

Thirty patients with optic neuritis (ON), in which neither multiple sclerosis (MS) nor any other etiology could be discriminated, were reexamined after a mean observeation period of 5 years. Eleven patients revealed oligoclonal IgG in the CSF and in five of them a measles virus antibody response within the CNS was demonstrable. the remaining 19 patients did not display oligoclonal CSF IgG and no local antibody production was detectable.
The occurrence of the HLA antigens A3 and B7 in ON did not correlate to the presence of oligoclonal IgG in CSF. the frequencies did not differ from those found in controls. the HLA-B7 linked lymphocyte defined antigen HLA-Dw2 occurred in ON at increased frequency, which was intermediate to that observed in MS and controls. an association was found in ON between oligoclonal IgG in CSF and Dw2. This association was of the same magnitude as in 22 MS patients who had ON as their first symptom of MS. In ON without oligoclonal CSF IgG the frequency of Dw2 was similar to that of controls.
No association was observed between the occurrence of the HLA antigens A3, B7 and Dw2, and increased measles antibody titers in serum or a measles virus antibody response in the CNS.
The occurrence of oligoclonal IgG in CSF in patients with ON may be assumed to increase the risk of developing MS.     

Decreased CNS inflammation and absence of clinical exacerbation of disease after six months oral administration of bovine myelin in diseased SJL/J mice with chronic relapsing experimental autoimmune encephalomyelitis

Murine chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) is a model of inflammatory demyelinating disease of the central nervous system (CNS) with similarity to multiple sclerosis (MS) in humans. Mice with confirmed neurologic deficits from CR-EAE were treated by oral administration of whole bovine myelin to investigate the effect of long-term oral delivery of myelin antigens on clinical disease and on the inflammatory response in the CNS. EAE-positive mice were fed doses of 1 mg, 10 mg, or 20 mg of bovine myelin every other day for 6 months. We found that prolonged oral delivery of neuroantigen suppressed inflammatory and demyelination foci in the CNS of myelin-treated mice with no exacerbation of clinical disease status compared with the control group. Analysis of histologic sections of brain and spinal cords with hematoxylin-eosin (H&E) and Luxol fast blue (LFB) staining showed a decrease in the inflammatory cell infiltration and active centers of demyelination, respectively. Furthermore, after 6 months of treatment, there was no increased sensitization to myelin antigens seen, as measured by antimyelin basic protein (MBP) or anti-proteolipid apoprotein (PLP) antibodies. These results demonstrate that prolonged oral administration of myelin antigens in diseased animals has an ameliorating effect on the pathologic process and supports its potential long-term use in humans with MS. © 1996 Wiley-Liss, Inc.     

Autoreactive T lymphocytes in multiple sclerosis: pathogenic role and therapeutic targeting

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), leading to demyelination. Accumulating evidence suggests that MS is an autoimmune disease, mediated by autoreactive T cells with specificity for myelin antigens. The identity of the brain antigens, which are the primary targets of the autoimmune process remains unknown, but myelin basic protein (MBP) is a likely candidate. We will overview some of the experimental evidence, suggesting that MBP reactive T cells hold a central position in the pathogenesis of MS, and discuss how these autoreactive T cells can be therapeutically targeted by T cell vaccination.     

Determination of autoantibody for myelin antigens in the serum of patients HLA-DQB1*0602 with multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the human central nervous system (CNS) mediated by autoimmune Th1 lymphocytes. We determined the serum levels of autoantibodies for myelin basic protein (MBP), proteolipid (PLP) and myelin oligodendrocyte glycoprotein sequence MOG 92-106 in a group of 54 healthy individuals and 26 MS patients expressing or not HLA-DQB1*0602. Regardless expression of the susceptibility allele DQB1*0602, MS patients presented marked (p<0.0001) IgG antibody production for MBP and MOG92-106. Yet, significant (p<0.0001) IgA antibody levels were mainly observed for PLP and MOG antigens. Our results suggest that other HLA class II alleles may be conferring susceptibility to MS in this population and influencing the pattern of immune recognition of encephalitogen antigens. Furthermore, distinct IgG and/or IgA autoantibody production may be contributing to the control or maintenance of the CNS inflammatory reaction.     

Virus-specific and autoreactive T cell lines isolated from cerebrospinal fluid of a patient with chronic rubella panencephalitis

Using a recently described technique for expanding of human T lymphocyte populations from cerebrospinal fluid (CSF), we investigated the local cellular immune response in a patient with chronic rubella panencephalitis. A total of 328 T cell lines (TCLs) was established by seeding CSF cells at limiting dilution into histoplates in the presence of irradiated feeder cells and phytohemagglutinin (PHA)-containing conditioned medium. 80% of TCLs expressed the CD4+CD8-, 5% the CD4-CD8+ phenotype and 15% of TCLs contained different proportions of CD4+ and CD8+ cells. Of 191 TCLs analyzed, 85 were cytotoxic, as shown by their lectin-dependent cytotoxicity against allogeneic uninfected target cells. Eight of them demonstrated specificity for the autologous, rubella virus-infected target cells. When tested for antigen-specific proliferative activity, 26 TCLs responded to rubella antigen, 16 TCLs reacted to myelin basic protein (MBP), four TCLs to proteolipid protein (PLP), four to galactocerebrosides and two to actin. Fourteen out of 16 MBP-specific TCLs also responded, to a minor degree, to rubella antigen and/or actin. The results showed that the persisting rubella infection had given rise to autoreactive T cells. Virus-induced autoreactivity to brain antigens may be an important pathogenetic mechanism in other chronic inflammatory disorders of the CNS.     

Antibodies to viral and non-viral antigens in subacute sclerosing panencephalitis and multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing,antigen immunofixation and autoradiography

Antibody activity in IgG zones separated by thin-layer polyacrylamide gel isoelectric focusing (PAGIEF) was determined in 3 patients with subacute sclerosing panencephalitis (SSPE), 4 patients with multiple sclerosis (MS) and 4 subjects with psychosomatic disorders, using antigen immunofixation and autoradiography. Viral (measles, herpes simplex type 1, mumps) and non-viral (purified bovine myelin, bovine myelin basic protein, bovine oligodendrocytes, MS and normal human brain extract) were used as antigens. All oligoclonal and some of the polyclonal CSF IgG zones in the patients with SSPE contained measles virus antibodies, as did some of the oligoclonal and polyclonal CSF IgG zones in 3 of the patients with MS. No antibodies were detectable in CSF or serum IgG zones against any of the non-viral antigens tested.