“Linking EBV infection status to the genetic risk variant BAFF-var in multiple sclerosis

Linking EBV infection status to the genetic risk variant BAFF-var in multiple sclerosis C.Meloni1, L. Benincasa1, P. Valentino2, S. Martire2, C.I. Bava2, L. Giorgi2, G.M. Abdel Azim2, A. Di Sapio2, Clara Ballerini3, Anna Repice4, I. De Bartolo5,6, G. Leodori 6,7, G. Ferrazzano7, A. Conte 6,7, C. Mancosu8, M. Pitzalis9, J. Frau8, E. Cocco8, C. Veroni1 1Department of Neuroscience, Istituto Superiore di Sanità, Rome, Italy 2 CRESM (Multiple Sclerosis Regional Reference Center), Neuroscience Institute Cavalieri Ottolenghi (NICO) and AOU San Luigi Gonzaga, Orbassano (TO), Italy 3 Department of Neuroscience, Drug and Child Health (NEUROFARBA), University of Florence, Italy. 4 Department of Experimental and Clinical Medicine (DMSC), University of Florence, Italy. 5Department of radiological sciences, oncology and anatomical pathology, Sapienza University of Rome, Italy 6IRCSS Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy 7Departement of Human Neurosciences, Sapienza University of Rome, Italy 8Department of Medical Science and Public Health, University of Cagliari, MS Centre, Binaghi Hospital, Cagliari, Italy. 9Institute for Genetic and Biomedical Research, National Research Council, Cagliari, Italy Introduction: MS results from the interplay between genetic and environmental factors. Among these, Epstein-Barr virus (EBV) infection is a prerequisite for MS development. The genetic variant BAFF-var of the TNFSF13B gene, encoding for the B-cell activating factor (BAFF), is linked to an increased MS risk, elevated serum BAFF levels and expansion of memory B cells, the main EBV reservoir. Aim: To explore the interplay between EBV and BAFF-var in MS by assessing EBV status in the peripheral blood of People with MS (PwMS) and healthy donors (HD) and the impact of BAFF-var on EBV biology. Methods: Peripheral blood mononuclear cells (PBMC) and sera were collected from 149 therapy-naïve PwMS and 105 HD. BAFF-var prevalence was assessed by SNP-genotyping. EBV DNA was quantified by droplet-digital PCR, while the expression of EBV latency/lytic transcripts and BAFF/B cell-related genes were analyzed using enhanced real time RT-PCR. Soluble BAFF, EBNA1 IgG and VCA IgG levels in serum were measured by ELISA. Statistical analyses used non-parametric tests Results: BAFF-var was present in 30% of PwMS (26% heterozygotes, 4% homozygotes) and 22% of HD (20% heterozygotes, 2% homozygotes), while 70% of PwMS and 78% of HD were wild-type (WT). As expected, BAFF-var carriers had higher serum BAFF levels (p<0.001). In addition, BAFF-var PwMS showed significantly increased expression of the B cell marker CD20 and key BAFF pathway genes (BAFF, BAFF-R, APRIL, TACI) in PBMC, indicating a broader effect on B cell regulation. PwMS had higher levels of EBNA1 IgG (p<0.001) and VCA IgG (p=0.03) compared to HD. In PwMS, VCA IgG titers were higher in BAFF-var carriers than in WT (p=0.02), suggesting potential EBV reactivation. Both EBV DNA and RNA were more frequently detected in PwMS than in HD, with PwMS showing higher viral DNA load (p=0.004) and prevalence of transcripts associated with EBV latency disruption and lytic reactivation (p<0.001). While viral load did not differ between BAFF-var and WT PwMS, BAFF-var carriers had higher expression of EBV genes involved in latent (EBNA3A) and lytic (BZLF1) infection. Conclusions: These results support EBV dysregulation in MS and suggest that elevated BAFF levels associated with BAFF-var might promote EBV latency disruption/reactivation in PwMS. Although confirmation in an independent cohort is needed, this study reinforces the idea that impaired control of EBV infection is related to MS and influenced by genetic risk factors, helping identify new disease biomarkers and more targeted therapies. Funding: This study is funded by Italian Multiple Sclerosis Foundation (grant FISM GRANT 2022/R/11) and co-funded by European and Union’s Horizon Europe Research and Innovation Actions under grant no. 101137235 (BEHIND-MS)