Computational design and in vitro validation of a multiepitope vaccine candidate against severe fever with thrombocytopenia syndrome virus.

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) is an emerging tick-borne pathogen associated with high mortality ad the absence of approved vaccines or specific antiviral therapies. The present study aimed to design and experimentally validate a multiepitope vaccine candidate targeting the SFTSV membrane glycoprotein using an integrative immunoinformatics and in vitro experimental approach. Immunoinformatics tools were employed to identify cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes with high antigenicity, strong HLA-binding affinity, and non-allergenic and non-toxic properties. Selected epitopes were assembled into a multiepitope vaccine construct incorporating human β-defensin-3 as an adjuvant, a PADRE sequence to enhance helper T-cell responses, and suitable linkers to promote structural stability and efficient antigen processing. Structural modeling, molecular docking with Toll-like receptor 4 (TLR4), molecular dynamics simulations, immune simulations, and disulfide engineering were performed to evaluate the structural stability and immunogenic potential of the designed construct. The codon-optimized gene sequence was cloned into the pET-28a(+) vector, expressed in Escherichia coli, and purified using Ni-NTA affinity chromatography. Experimental characterization demonstrated efficient recombinant expression of an approximately 21.8 kDa protein with high purity and strong antigenic reactivity. Furthermore, the purified protein exhibited minimal cytotoxicity in mammalian cell models and significantly stimulated innate immune signaling pathways, cytokine production, and antibody responses. These findings suggest that the designed multiepitope vaccine construct is structurally stable and immunogenic in vitro, supporting its potential for further evaluation in animal models and preclinical studies against SFTSV infection.