“Investigating the interaction between the genetic risk variant baff-var and ebv reactivation in multiple sclerosis: implications for immune dysregulation”

INVESTIGATING THE INTERACTION BETWEEN THE GENETIC RISK VARIANT BAFF-VAR AND EBV REACTIVATION IN MULTIPLE SCLEROSIS: IMPLICATIONS FOR IMMUNE DYSREGULATION C.Meloni1, L. Benincasa1, P. Valentino2, A., S. Martire2, C.I. Bava2, L. Giorgi2, G.M. Abdel Azim2, A. Di Sapio2, A.Repice3, C. Ballerini4, I. De Bartolo5,6, G. Leodori 6,7, G. Ferrazzano7, A. Conte 6,7, C. Mancosu8, M. Pitzalis8, 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 4Department of Experimental and Clinical Medicine (DMSC), University of Florence, Italy 5 Department of radiological sciences, oncology and anatomical pathology, Sapienza University of Rome, Italy 6 IRCSS Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy 7Departement of Human Neurosciences, Sapienza University of Rome, Italy 8 Department of Medical Science and Public Health, University of Cagliari, MS Centre, Binaghi Hospital, Cagliari, Italy. Background: Both genetic and environmental factors contribute to Multiple Sclerosis (MS). Previous infection with the B-lymphotropic Epstein-Barr virus (EBV) is a prerequisite for MS, yet the underlying mechanisms remain under investigation. A variant of the TNFSF13B gene (BAFF-var), encoding the B-cell growth factor BAFF, has been associated to increased MS risk and is more prevalent in Sardinians, a high-MS-incidence population. BAFF-var leads to elevated soluble serum BAFF levels and increased number of circulating memory B cells, the primary EBV reservoir. This raises the hypothesis that BAFF-var may promote the survival of EBV-infected memory B cells, facilitating viral reactivation and leading to immune homeostasis disruption. The aim of this study is to investigate this link by assessing how BAFF-var influences EBV biology and immune regulation in MS. Methods: Peripheral blood mononuclear cell (PBMC) and serum samples were collected from 150 therapy-naïve people with MS (PwMS) across four Italian MS centers, including one in Sardinia. BAFF-var prevalence was assessed by SNP-genotyping while EBV DNA in PBMCs was quantified by droplet-digital PCR. EBV latency/lytic transcripts together with panel of immune-related genes were analysed using enhanced real time RT-PCR. Levels of serum BAFF and anti-EBV antibodies (anti-EBNA1, anti-VCA IgG) were measured by ELISA. Statistical analyses used non-parametric tests. Results: BAFF-var was found in 27% of PwMS as heterozygous and in 4% as homozygous, while 69% carried the wild-type (WT) alleles. Consistent with previous studies, BAFF-var was more frequent in Sardinians than in mainland Italians (p<0.001) and BAFF-var carriers had significantly higher levels of serum soluble BAFF (p<0.001). Titers of anti-VCA IgG, but not anti-EBNA1 IgG, were elevated in BAFF-var PwMS compared to WT (p=0.015), suggesting a shift towards EBV reactivation. EBV DNA load did not differ between the two groups. However, although EBV RNA was detected in only 29% of PBMC samples, BAFF-var PwMS exhibited higher level of transcripts encoding EBV antigens involved in immune recognition, such as EBNA3A, a major target of CD8⁺ T-cell response, and BZLF1, a key driver of lytic activation. Additionally, BAFF-var PwMS showed increased expression of immune-related genes associated with BAFF pathway, NK/T-cell activation and anti-viral type-I IFN signalling. Conclusions: Our findings support the hypothesis that elevated soluble BAFF levels associated with BAFF-var may favour EBV latency disruption and reactivation, altering viral- host immune system interactions, ultimately contributing to MS pathogenesis. Further studies in larger PwMS cohorts are needed due to the low BAFF-var frequency. Patient recruitment is ongoing to validate these findings.