Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk

Abstract

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10^-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

Keywords: ANO2; Anoctamin 2; Epstein-Barr virus; molecular mimicry; multiple sclerosis.

Fig. 1.

Odds ratios for MS with different combinations of risk factors. Risk factors include ANO2 seropositivity, DRB1*15:01 carrier, A*02:01 noncarrier, and EBNA1-high (anti-EBNA1 antibody levels above median in controls). Red box indicates that the group of individuals is exposed to the risk factor and white box that they are not. The ORs were calculated by comparison with a reference group, carrying none of the risk factors. Total numbers of MS cases and controls in each group and P values are presented in SI Appendix, Table S3. Bars in the graph indicate 95% confidence intervals.

Fig. 2.

Competition assay screening. The amino acid stretch showing high sequence similarity between ANO2 and EBNA1 overlaps with the ANO2 antibody epitope as marked in a schematic overview of the ANO2 and EBNA1 full-length sequences. Illustration was generated with the Protter tool (43), and subsequently modified (A). A heatmap with the mean fold change between not blocking and blocking with peptides, representing ANO2 and EBNA1 (rows), in plasma samples from 7 MS cases across the same peptides (columns). The signal intensity from ANO2 [aa 135 to 149] was affected by 3 EBNA1 peptides [aa 421 to 440], [aa 425 to 444], and [aa 431 to 450] (B). The sequence in common between these 3 EBNA1 sequences is EBNA1 [aa 431 to 440] PGAIEQGPAD, which is the sequence with similarity to ANO2 [aa 140 to 147] PGDIELGPLD.

Fig. 3.

Effects on antibody reactivity to ANO2 and EBNA1 peptides after peptide competition. Fold change between signal intensity without blocking and signal intensity when blocked with each peptide are presented on the y axis. ANO2 [aa 135 to 149], EBNA1 [aa 401 to 420], and EBNA1 [aa 425 to 444] were included in the assay with 82 MS case samples, selected to represent both high and low anti-ANO2 and anti-EBNA1 antibody levels. The x axis presents blocking peptides and P values of the differences between fold changes (Wilcoxon rank-sum test), demonstrating that the signal intensity from ANO2 [aa 135 to 149] is equally affected by ANO2 [aa 135 to 149] and EBNA1 [aa 425 to 444].

Fig. 4.

HLA gene region is genome-wide associated with anti-ANO2 antibody levels. A genome-wide significant signal was detected in the HLA gene region using OmniExpress genotype data (A). Imputed HLA allele data, generated from SNP genotypes from MS replication chip, defined 16 HLA alleles significantly associated with anti-ANO2 antibody levels. The associated alleles represented 3 haplotypes (separated by black lines, SI Appendix, Table S5) (–26). DRB4*01:03 and DRB1*04:01, conferred with the most significant ORs for anti-ANO2 seropositivity (B). Significant P values are marked with asterisks next to error bars (<10⁻⁸ ***, <10⁻⁴ **, and <10⁻² *).