Discrepancy of Serological and Molecular Patterns of Circulating Epstein-Barr Virus Reactivation in Primary Sjögren's Syndrome

Abstract

Primary Sjögren's syndrome (pSS) is characterized by B cell hyperactivation, production of autoantibodies and increased risk of B cell lymphomas. Serological profile of Epstein-Barr virus (EBV) reactivation and increase EBV DNA levels in exocrine glands are observed in pSS, but whether these abnormalities are accompanied with disturbed systemic EBV control or have any association with pSS activity remains to be investigated. In this observational study, we initially explored anti-EBV antibodies and cell-free DNA in 395 samples from a cross-sectional plasma collection of pSS patients included in ASSESS French national cohort. Results were assessed in relation with disease activity. Further, to assess cell-associated EBV DNA we organized a case-control study including 20 blood samples from pSS patients followed in University Hospital Center of Montpellier. Results were compared with matched controls. Robust response against EBV early antigen (EA) was observed in pSS patients with anti-SSA/B (Sjögren's syndrome A and B) and anti-SSA autoantibodies compared to anti-SSA/B negatives (P < 0.01 and P = 0.01, respectively). Increased beta-2 microglobulin, kappa and lambda light chains, and immunoglobulin G levels were more frequently observed in anti-EA seropositive pSS subjects compared to anti-EA negative subjects (P < 0.001; P = 0.001; P = 0.003, respectively). Beta-2 microglobulin was independently associated with anti-EA positivity in multivariate analysis (P < 0.001). Plasma cell-free EBV DNA and EBV cellular reservoir was not different between pSS patients and controls. We conclude that serological evidence of EBV reactivation was more frequently observed and more strongly associated with anti-SSA/B status and B cell activation markers in pSS. However, serological profile of EBV reactivation was not accompanied by molecular evidence of systemic EBV reactivation. Our data indicated that EBV infection remains efficiently controlled in the blood of pSS patients.

Keywords: EBV DNA; Epstein-Barr virus; anti-EA antibodies; autoantibodies; beta-2 microglobulin; primary Sjögren's syndrome.

Figure 1

Schematic representation of evaluation of EBV infection in ASSESS cohort. Plasma samples from ASSESS cohort of pSS patients were initially evaluated for cell-free EBV DNA. After molecular testing the available plasma underwent to serological assessment of anti-EBV antibodies. Results were analyzed in comparison with pSS activity markers. ASSESS, the Assessment of Systemic Signs and Evolution in Sjögren's syndrome; pSS, primary Sjögren's syndrome.

Figure 2

Plasma anti-EBV antibodies in pSS patients from ASSESS cohort. The positivity rates and levels of anti-VCA (A,B), anti-EBNA-1 (C,D) and anti-EA (E,F) IgG antibodies in anti-SSA/B autoantibody positive, anti-SSA positive and anti-SSA/B autoantibody negative patients. EA, Early Antigen; EBNA-1, EBV Nuclear Antigen 1; pSS, primary Sjögren's syndrome; SSA and B, Sjögren's syndrome A and B antigens; VCA, Viral Capsid Antigen.

Figure 3

PSS activity markers in anti-EA positive and anti-EA negative patients. Abnormally increased beta-2 microglobulin levels (A), kappa and lambda light chain ratios (B), summary kappa and lambda light chain levels (C), and total immunoglobulin G levels (D) are compared between anti-EA positive and negative pSS samples. Each column represent the percentage of cases having higher value than the threshold written on the top of each graph.

Figure 4

ESSDAI disease severity score (A) and pSS treatment history (B) in anti-EA positive and negative samples of ASSESS cohort.

Figure 5

EBV DNA in circulating compartment in pSS and controls. EBV DNA load in peripheral blood mononuclear cells (A), enriched B cells (B) and in unstimulated B cell supernatants (C). Cell-free EBV is represented as EBV DNA IU per ml of supernatant, while cell-associated EBV is quantified as EBV DNA IU per million of human cell Genome Equivalents (GE). pSS, primary Sjögren's syndrome.