Antibody Content against Epstein-Barr Virus in Blood Extracellular Vesicles Correlates with Disease Activity and Brain Volume in Patients with Relapsing-Remitting Multiple Sclerosis

Abstract

We aimed to analyze whether EVs carry antibodies against EBV antigens and the possibility that they could serve as diagnostic and disease activity blood biomarkers in RRMS. This was a prospective and observational study including patients with RRMS with active and inactive disease and healthy controls. Blood EVs were isolated by precipitation. Titers of antibodies against nuclear (anti-EBNA1) and capsid (anti-VCA) EBV antigens in EVs and in plasma, as well as content of myelin antibodies in EVs were determined by ELISA. An exploratory analysis of correlations with clinical and radiological data was performed. Patients with RRMS had higher titers of anti-VCA inside EVs and free in plasma than healthy controls. Patients with active disease showed higher levels of anti-EBNA1 in EVs, but not in plasma, than patients with inactive disease. EV anti-VCA levels correlated with disease duration and with decreased brain volume structures-total brain, white matter, gray matter, cerebellum, hippocampus, -but not with T2/FLAIR lesion volume or EDSS, SDMT, or 9HPT. In addition, EV anti-VCA correlated with EV anti-MBP. The anti-VCA and anti-EBNA1 content in EVs could represent diagnostic and disease activity blood biomarkers, respectively, in RRMS.

Keywords: Epstein–Barr virus; antibodies; biomarker; extracellular vesicles; multiple sclerosis.

Figure 4

Relation of VCA antibody content in EVs and the time from diagnosis and brain structure volume. Correlation between anti-VCA levels in EVs and (A) disease duration, (B) total brain volume, (C) white matter volume, (D) gray matter volume, (E) volume of the cerebellum, (F) hippocampus volume, (G) volume of the thalamus, (H) caudate nucleus volume, and (I) volume of the putamen.

Figure 1

EV characterization. (A) Electron microscopy showing EVs of <200 nm. (B) CD9, CD81, CD63, and Alix markers present in EVs. (C) Graph showing EV sizes ranging 100–200 nm. The sample was loaded three times, shown in left panel (red, yellow, and orange lines). The mean (black line) and SD (red line) are shown in the right panel. (D) EVs as seen by NTA.

Figure 2

EVB antibody content in MS patients. (A) Comparison of EVB titers for VCA and EBNA1 in plasma and EVs between MS patients and healthy controls. (B) EVB antibodies for VCA and EBNA1 in plasma and EVs between MS patients according to disease activity. * = p ≤ 0.05.

Figure 3

Relation between EBV antibody levels and disease status. (A) Plasma anti-VCA antibody levels. (B) EVs anti-VCA content. (C) Plasma anti-EBNA levels. (D) EVs anti-EBNA content.

Figure 5

Relationship between EBV antibody content and anti-myelin antibodies in EVs and plasma. Correlation between anti-VCA levels in EVs and (A) anti-MBP, (B) anti-MOG, and (C) albumin in EVs. (D) Correlation between anti-VCA antibodies in plasma with anti-MBP and (E) anti-MOG in plasma. Correlation between anti-EBNA1 levels in EVs and (F) anti-MBP, (G) anti-MOG, and (H) albumin in EVs. (I) Relationship between anti-EBNA1 antibodies in plasma with anti-MBP and (J) anti-MOG in plasma.