Armen Sanosyan

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.

Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log₁₀ versus 3.08 log₁₀ IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log₁₀; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log₁₀ IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.