Defining Epstein-Barr virus reservoirs and EBV-regulated gene expression in multiple sclerosis - Recent work suggests that infection with the B-lymphotropic herpesvirus, Epstein-Barr virus (EBV), is a key early event in the etiology of multiple sclerosis (MS). However, the molecular basis for how EBV promotes MS is currently unknown. EBV infects most human in the first decade of life and by adulthood, nearly 95% are latently infected. Given this very high prevalence of EBV in the general population, it is unclear why only 1:300 people in the US are afflicted with MS. Despite the well-studied, yet heterogeneous, genetic risk factors associated with MS, this apparent disconnect might be explained by a mechanism whereby EBV infects a particular B cell subset that accumulates in individuals at high genetic risk for MS. The expansion of such a pathogenic, potentially autoreactive and pro-inflammatory B cell subset has been described, yet little is known about the mechanism of how EBV plays a role in the expansion or activity of these cells. It is our ultimate goal to define the role of EBV in MS pathogenesis. In this proposal, we aim to characterize the temporal and spatial expression of EBV and EBV-regulated genes within a unique B cell subset. It is our central hypothesis that EBV persists in atypical memory B cells which display markers consistent with CNS trafficking ability and pro-inflammatory cytokine production. We have formulated our central hypothesis based on extensive preliminary data characterizing an EBV de novo infection signature in T-bet+/CXCR3+ B cells and observing this EBV-associated signature in clinically isolated syndrome (CIS) and MS patient samples. However, the capture of EBV-specific transcripts are outstanding in these clinical samples, which is a critical need to determine the putative role of EBV in MS pathogenesis. The rationale for this proposed research is that defining the role of EBV and its effects in B cells of CIS/MS patients will provide important insight into MS pathogenesis as well as new diagnostic and prognostic indicators of disease. Our laboratories are well positioned to pursue these studies as they have complementary expertise in EBV and MS biology and immunology as well as access to and experience with cutting edge single cell resolution techniques for studying viral and host gene expression in clinical samples. We plan to test our hypothesis and complete the objectives outlined in this proposal through the following two specific aims: 1) Validate and extend an EBV-associated peripheral atypical B cell gene expression signature in CIS/MS patients and 2) In situ transcriptomic analysis of ABC and EBV status in MS CNS tissue. The proposed study constitutes a comprehensive approach to precisely define the peripheral and CNS-resident cells that harbor EBV in patients with MS. In conjunction with our prior work, the data generated herein will yield invaluable insight into EBV- mediated loss of immune tolerance associated with CNS pathology. Completion of this project will directly guide future mechanistic studies of EBV involvement in neuroimmune dysregulation and inform clinically actionable biomarkers and therapeutic targets for the treatment and management of MS, thereby providing a springboard for a more expansive proposal.
