Investigating the role of Epstein-Barr virus in the pathogenesis of Multiple Sclerosis

Multiple Sclerosis (MS), a neuronal demyelination disease of the central nervous system, is the most common neurological disease in young adults worldwide, with an unknown cause. It affects over 23,000 people in Australia, and despite effective immune-based treatments, clinical deterioration and disability still occur. Epstein-Barr Virus (EBV), a human herpesvirus, which infects early in life and remains dormant within immune cells, is common in the unaffected population but almost universal in MS patients. Studies have found that MS patients maintain elevated EBV-specific antibody levels, most notably against latent Epstein-Barr Nuclear Antigen-1 (EBNA-1) proteins. The aims of this research were to investigate the significance of EBV immune responses, including those targeting the novel epitope EBNA-1(398-413), previously associated with MS risk in disease-discordant identical twins, and to understand the relationship between EBV-specific serological responses and genetic risk factors in an established MS cohort (n=426) and healthy controls (n=186). This study also investigated the influence of arginine modification (citrullination) on EBNA-1(398-413)-specific antibody responses, as well as the role of EBNA-1-specific IgG subclass bias. Novel in-house assays were compared with commercial ELISAs for EBV viral capsid antigen (VCA) and EBNA-1. MS patients had significantly higher antibodies against all EBV targets. Inclusion of EBNA-1(398-413)-specific antibody levels further discriminated cases and controls in risk analysis, in addition to genetic risk factors. Citrullinated and IgG1-specific EBNA-1 antibody levels were also elevated in MS cases compared to controls, although they did not improve case-control classification in combined statistical models. EBNA-1(398-413)-specific IgG from acute patients showed different protein reactivity from whole serum, as well as differences between progressive MS serum and healthy control plasma. This project contributes to the importance of EBV in the pathogenesis of the complex disease MS, and has identified novel and statistically powerful relationships between environmental and genetic MS risk factors

EBNA-1 sequence variation identified with next-generation sequencing technology, and impact on HLA-DRB1*15 binding affinity within known epitopes.

<p>The two most frequent variants for the A) AEG epitope and B) MVF epitope are printed in bold, the most frequent variant is underlined. All epitopes are predicted to be weak binders (affinity 50nM-500nM).</p

Putative HLA-DRB1*15 binders within autologous EBNA-1 peptide sequences and candidate myelin antigens.

<p>Putative HLA-DRB1*15 binders within autologous EBNA-1 peptide sequences and candidate myelin antigens.</p

Extended axoglial brain protein dataset with HLA-DRB1*1501 predicted brain epitopes overlapping with predicted EBV binders.

<p>Extended axoglial brain protein dataset with HLA-DRB1*1501 predicted brain epitopes overlapping with predicted EBV binders.</p

EBNA-1 sequence conservation and relationship to NetMHCII predicted HLA-DRB1*DR15-restricted epitopes derived from patient MS sequences.

<p>Shown are EBNA-1 amino acid positions 473–593. Predicted epitopes: blue boxes, known epitopes: black underlined, predicted epitope core: grey shade.</p

EBNA-1 quasispecies detected with FLX sequencing.

<p>Minority EBV sequence variants at a level of ≥10% were detected in two samples only. Sequence mixtures present at a ≥5% threshold revealed minority variants in 3.6% of investigated nucleotide positions (27/749 nucleotides sequenced,), increasing to 5.0% at a ≥2% cut-off (37/749 nucleotides,) and 8.3% at a ≥1% cut-off (62/749 nucleotides,). Samples 7, 8 and 13 did not have minority species present at ≥1%.</p

New genetic predictors for abacavir tolerance in HLA-B*57:01 positive individuals.

Abacavir hypersensitivity syndrome (ABC HSS) is strongly associated with carriage of human leukocyte antigen (HLA)-B*57:01, which has a 100% negative predictive value for the development of ABC HSS. However, 45% of individuals who carry HLA-B*57:01 can tolerate ABC. We investigated immune and non-immune related genes in ABC HSS (n = 95) and ABC tolerant (n = 43) HLA-B*57:01 + patients to determine other factors required for the development of ABC HSS. Assignment of phenotype showed that ABC HSS subjects were significantly less likely than tolerants to carry only ERAP1 hypoactive trimming allotypes (p = 0.02). An altered self-peptide repertoire model by which abacavir activates T cells is in keeping with observation that endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes that favour efficient peptide trimming are more common in ABC HSS patients compared to patients who tolerate ABC. Independently, non-specific immune activation via soluble cluster of differentiation antigen 14 (sCD14) may also influence susceptibility to ABC HSS

Age and gender distribution of patients recruited from the Perth Demyelinating Disease Database.

<p>Age and gender distribution of patients recruited from the Perth Demyelinating Disease Database.</p

Phylogenetic tree of C-terminal EBNA-1 Sanger sequences including the reference strains B95-8, AG876, GD1 and HKNPC1.

<p>Phylogenetic tree covering nucleotide positions B95-8: 109135–109815; EBNA-1: 1186–1866; blue triangles: EBV reference strains; red dots: MS cases (n = 53).</p