Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) of presumed infectious and/or autoimmune etiology. While there is clearly immune activation within the CNS in multiple sclerosis, a responsible autoantigen or viral agent has not yet been identified. The CNS and cerebrospinal fluid (CSF) of MS patients contains activated T-cells and B-cells secreting oligoclonal antibodies, but it is not known whether they are directed at autoantigens or viral antigens. The proposed research will employ the most recent developments in molecular biology and immunology to characterize specific antigens against which the immune response in MS might be directed. The proposed study will take advantage of two recently developed recombinant DNA technologies. The first is the ability to construct large, high efficiency cDNA libraries from MS brain and express them as recombinant proteins in phage cloning systems. The second is the utilization of mixed primers in polymerase chain reaction (PCR) amplification to clone human immunoglobulin variable region genes, and then express them in a novel phage vector as recombinant antibodies in E. coli (Barbas et al 1991). This will permit the entire B-cell repertoire in the CNS plaques of MS patients to be cloned and expressed as monoclonal antibodies. CSF oligoclonal antibodies or the recombinant brain immunoglobulins will then be used to identify immunoreactive MS brain antigens, produced as recombinant proteins in cDNA expression libraries generated from brains of MS patients. Immunoreactive antigen clones will then be isolated, subcloned, and sequenced to characterize the target antigens. Antibodies and T-cells in the CSF and blood of patients with MS and normal controls will be tested for immune reactivity to the same antigens to establish the specificity of the interaction for MS. Any reactive antigens, whether an endogenous common component of the nervous system or exogenous viral sequences unique to MS brain, will be isolated. If the antigen is a viral agent, then brain tissue from patients with MS and normal controls will be tested for the same viral sequences by hybridization studies with radiolabeled probes, by PCR amplification with specifically designed primers, or immunocytochemistry. If the target antigen is an autoantigen, its expression, distribution and function in the CNS can be determined. The isolated target protein can be used to study the humoral and cellular immune response in MS, and possibly generate an autoimmune animal model for the disease. Identification of target antigens may help reveal the cause of MS and lead to the development of specific immunomodulatory therapies.
