PROJECT SUMMARY/ABSTRACT Infection with Epstein-Barr Virus (EBV) has been epidemiologically demonstrated to be a pre-requisite for developing multiple sclerosis (MS), as essentially 100% of MS patients become infected with EBV prior to MS onset. We recently identified molecular mimicry between the EBV transcription factor EBNA1 and the glial cellular adhesion molecule GlialCAM in 20 - 25% of MS patients, and this is likely a critical mechanism underlying the development of MS in this subset of patients. This R01 will take our investigation of the role of EBV in mediating the pathogenesis of MS to the next level, through: (i) analysis of dysregulated transcriptional pathways in EBV- transformed B cells derived from MS patients, (ii) investigation of the ability of EBV-transformed B cells to mediate autoreactive T cell activation, and (iii) characterization of molecular mimicry-related EBV and CNS autoantibodies in MS. We will test the hypothesis that in MS, EBV activates B cells to circumvent B cell tolerance, and thereby drive autoantibody production, autoreactive T cell activation, and MS pathology. We further hypothesize that different reactivities to self-antigens, to EBV, and to molecular mimics are pathognomonic for distinct MS disease subtypes with different HLA/genetic backgrounds and disease characteristics. Aim 1 will analyze dysregulated transcriptional pathways in EBV+ B cells derived from MS vs. controls. Aim 2 will express MS EBV+ B cell-encoded monoclonal antibodies and identify their EBV and CNS targets. Aim 3 will investigate the ability of EBV-transformed B cells to mediate autoreactive T cell activation. Aim 4 will perform serologic analysis to characterize molecular mimicry-related EBV and CNS autoantibodies in sera from large cohorts of MS and comparator patients. Success of the proposed studies would elucidate the mechanisms by which EBV causes MS, which would transform our understanding of MS and could lead to fundamental therapies.