Epstein-Barr virus (EBV) - specific antibodies in multiple sclerosis patients, including targeted investigation of a candidate EBV nuclear antigen-1 (EBNA-1) B cell epitope

Multiple Sclerosis (MS) is a very complex disease, with different genetic and environmental factors contributing to its risk. In particular, infection with Epstein-Barr Virus (EBV) has been shown to increase MS risk. This risk is moderately associated with anti-viral capsid antigen (VCA) antibodies, but very strongly associated for anti-EBV nuclear antigen-1 (EBNA-1) antibodies. Focus on EBNA-1 has uncovered that antibody reactivity against a C-terminal part the of the protein (amino acid (aa) positions 385-420) is a stronger risk marker than antibodies against the whole EBNA-1 protein. Recently, in a small study in twins (n=12), a putative B cell epitope within this region (aa 401-411) has been identified as a target for antibodies that are specifically enriched in MS patients compared with healthy controls

Identifying Epstein-Barr virus EBNA-1 sequence variation using 454 FLX technology in Multiple Sclerosis patient samples

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Multiple Sclerosis Patient Sequence Variation In Epstein-Barr Virus Nuclear Antigen 1

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Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis

Background: Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome. Methods: Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins. Results: EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cutoff). In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes (‘AEG’: aa 481–496 and ‘MVF’: aa 562–577), and two putative epitopes between positions 502–543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis. Conclusions: This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of immunogenic regions of EBNA-1 as well as known and novel targets for autoreactive HLA-DRB1*15-restricted T cells within the central nervous system that could arise as a result of cross-reactivity with EBNA-1-specific immune responses

7th Drug hypersensitivity meeting: part two

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FTY720 (fingolimod) modulates the severity of viral-induced encephalomyelitis and demyelination

BACKGROUND: FTY720 (fingolimod) is the first oral drug approved by the Food and Drug Administration for treatment of patients with the relapsing-remitting form of the human demyelinating disease multiple sclerosis. Evidence suggests that the therapeutic benefit of FTY720 occurs by preventing the egress of lymphocytes from lymph nodes thereby inhibiting the infiltration of disease-causing lymphocytes into the central nervous system (CNS). We hypothesized that FTY720 treatment would affect lymphocyte migration to the CNS and influence disease severity in a mouse model of viral-induced neurologic disease. METHODS: Mice were infected intracranially with the neurotropic JHM strain of mouse hepatitis virus. Infected animals were treated with increasing doses (1, 3 and 10 mg/kg) of FTY720 and morbidity and mortality recorded. Infiltration of inflammatory virus-specific T cells (tetramer staining) into the CNS of FTY720-treated mice was determined using flow cytometry. The effects of FTY720 treatment on virus-specific T cell proliferation, cytokine production and cytolytic activity were also determined. The severity of neuroinflammation and demyelination in FTY720-treated mice was examined by flow cytometry and histopathologically, respectively, in the spinal cords of the mice. RESULTS: Administration of FTY720 to JHMV-infected mice resulted in increased clinical disease severity and mortality. These results correlated with impaired ability to control viral replication (P < 0.05) within the CNS at days 7 and 14 post-infection, which was associated with diminished accumulation of virus-specific CD4+ and CD8+ T cells (P < 0.05) into the CNS. Reduced neuroinflammation in FTY720-treated mice correlated with increased retention of T lymphocytes within draining cervical lymph nodes (P < 0.05). Treatment with FTY720 did not affect virus-specific T cell proliferation, expression of IFN-γ, TNF-α or cytolytic activity. FTY720-treated mice exhibited a reduction in the severity of demyelination associated with dampened neuroinflammation. CONCLUSION: These findings indicate that FTY720 mutes effective anti-viral immune responses through impacting migration and accumulation of virus-specific T cells within the CNS during acute viral-induced encephalomyelitis. FTY720 treatment reduces the severity of neuroinflammatory-mediated demyelination by restricting the access of disease-causing lymphocytes into the CNS but is not associated with viral recrudescence in this model

Adaptive immune responses against Epstein-Barr Virus influencing Multiple Sclerosis risk

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Single cell analysis of drug responsive T cells; identification of candidate drug reactive T cell receptors in abacavir and carbamazepine hypersensitivity

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Combining genetic risk factors, gender And Anti-Epstein Barr Virus (EBV) antibody profiles, sing A Novel Anti-EBV Nuclear Antigen-1 (EBNA-1) B Cell Epitope ELISA, to stratify multiple sclerosis risk

Poster presentatio

Investigating Epstein-Barr virus (EBV) antibodies in Multiple Sclerosis patients, and development of an anti-EBV nuclear antigen-1 B cell epitope ELISA

Oral presentatio