In-depth analysis of serum antibodies against Epstein-Barr virus lifecycle proteins, and EBNA1, ANO2, GlialCAM and CRYAB peptides in patients with multiple sclerosis

Abstract

Background: A strong association between multiple sclerosis (MS) and Epstein-Barr virus (EBV) has been established but the exact role of EBV in MS remains controversial. Recently, molecular mimicry between EBNA1 and specific GlialCAM, CRYAB and ANO2 peptides has been suggested as a possible pathophysiological mechanism. The aim of this study was to analyse anti-EBV antibodies in MS patients against (I) EBV lifecycle proteins, (II) putative cross-reactive peptides, and (III) during treatment.

Methods: In this retrospective cross-sectional study, 258 serum samples were included consisting of EBV-negative (n = 25) and EBV-positive (n = 36) controls, 192 MS samples including untreated relapsing-remitting MS (RRMS) with and without relapses, secondary progressive MS (SPMS) and primary progressive MS (PPMS) patients, and 106 patients on 8 different treatment regimens. IgG and IgM antibody titers against EBV docking/fusion proteins (gp350, gh/gp42, gh/gL/gp42), immediate early antigen (BZLF1), early antigens (EA p85, EA P138, EA P54), capsid antigens (VCA P18, VCA P23, VCA gp125) and late antigens (EBNA1) were measured. Specific EBNA1 and GlialCAM, CRYAB and ANO2 peptides were synthesized and also incorporated in our custom magnetic bead based multiplex assay.

Results: We observed significantly elevated IgG antibody titers in EBV-positive controls, RRMS with and without relapse, SPMS and PPMS patients for all lifecycle antigens except for several early antigens when compared to EBV-negative controls. Significantly higher IgG antibody titers were observed in RRMS patients for fusion proteins and EBNA1 peptides when compared to EBV-positive controls. An MS specific response was observed for ANO2 but not for GlialCAM or CRYAB. No significant treatment effects or a specific IgM response were detectable.

Conclusion: The MS-specific, differential antibody response to EBV antigens confirms an altered immunological response to EBV in MS patients. EBV reactivation does not appear to play an important role in MS pathogenesis and no differential antibody signatures were observed between MS disease phases. The MS-specific anti-ANO2 antibody response suggests a potential role for EBNA1 as an antigenic driver, although the exact role of anti-ANO2 antibodies needs to be determined. The precise pathophysiological role of EBV in MS remains uncertain and requires further investigation.

Keywords: EBV nuclear antigen type 1 (EBNA1); Epstein-Barr virus (EBV); alpha B crystallin (CRYAB); anoctamin 2 (ANO2); glial cell adhesion molecule (GlialCAM); multiple sclerosis (MS).

Figure 1

IgG antibody titers against EBV lifecycle peptides. Mean fluorescence intensities (MFI) of the serum antibody response against (A) EBNA1 late antigen, (B) BZLF1 immediate early antigen, (C) gp350/220 docking protein, (D) gH/gp42 and (E) gh/gL/gp42 fusion proteins, (F) EA P85, (G) EA P138, (H) EA P54 early antigens, (I) VCA P18, (J) VCA P23, and (K) VCA gp125 viral capsid antigens are shown for EBV-negative controls, EBV-positive controls, relapsing remitting multiple sclerosis patients during relapse (RRMS RE), untreated and without relapse (RRMS UT), untreated secondary progressive multiple sclerosis (SPMS UT) and untreated primary progressive multiple sclerosis (PPMS) are shown. Kruskal-Wallis test with Dunn’s correction was applied for statistical testing (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The horizontal dotted line indicates the cut-off for IgG seropositivity.

Figure 2

IgG antibody titers against EBNA peptides and potential cross reactivity against related peptides. Mean fluorescence intensities (MFI) of the serum antibody response against (A) EBNA1 full antigen, (B) EBNA1 (Serion), (C) EBNA1 (Aviva), (D) EBNA1 AA386-405, (E) EBNA1 AA393-412, (F) EBNA1 AA425-444, (G) GlialCAM AA370-389, (H) CRYAB AA2-21, and (I) ANO2 AA134-153 are shown for EBV-negative controls, EBV-positive controls, relapsing remitting multiple sclerosis patients during relapse (RRMS RE), untreated and without relapse (RRMS UT), untreated secondary progressive multiple sclerosis (SPMS UT) and untreated primary progressive multiple sclerosis (PPMS) are shown. Kruskal-Wallis test with Dunn’s correction was applied for statistical testing (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The horizontal dotted line indicates the cut-off for IgG seropositivity.

Figure 3

Correlations between EBNA peptides and potentially cross-reactive peptides and correlation matrix. Correlations between (A) EBNA1 AA386-405 and GlialCAM AA370-389, (B) EBNA1 AA393-412 and CRYAB AA2-21, (C) EBNA1 AA425-444 and ANO2, and (D) the correlation matrix between all EBNA1 and related peptides as well as additional patient characteristics are shown. The Spearman correlation test was employed for the purpose of statistical analysis. DMTs, disease-modifying treatments; EDSS, Expanded Disability Status Scale.

Figure 4

Two possible ways by which EBV infection could influence MS pathophysiology (A) Cross-reactivity (molecular mimicry) between EBNA1 and e.g. ANO2 peptides could lead to auto-reactive antibodies that cross the blood-brain-barrier and/or are produced locally by B cells and exert tissue damage (MS lesions). However, the exact pathophysiological role and ability of e.g. anti-ANO2 antibodies to activate CDC and ADCC mediated tissue damage has not been demonstrated yet. Of note, MS relapses do not seem to be associated with elevated ANO2 antibody titers. (B) A certain immune predisposition may influence long term EBV infected B cells that drive autoimmune responses through antigen presentation and thus promote CNS cross-reactivity of T cells. B cell trafficking in and out of the CNS has been demonstrated, with B-cell drainage through the cervical lymph nodes. B cells that have encountered CNS antigens during lesion formation could thus recirculate into the periphery and stimulate auto-reactive T cells. In this context, EBV infected B cells have been shown to efficiently process antigens for the presentation to CD4⁺ T cells via MHC class II (43).