JIingjing Zhang ¹ Youfang Yang ² Bingyu Wang ³ Wanting Qiu ⁴ Helin Zhang ⁴ Yuyang Qiu ⁴ Jing Yuan ^4,5,6 Rong Dong ^4,5,6* Zha Yan ^4,5,6*

• ¹ Guizhou Medical University, Guiyang, China
• ² Zunyi Medical University, Zunyi, Guizhou Province, China
• ³ School of Medicine, Guizhou University, Guiyang, Guizhou Province, China
• ⁴ School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, Guangdong Province, China
• ⁵ Guizhou Provincial People's Hospital, Guiyang, Guizhou Province, China
• ⁶ NHC Key Laboratory of Pulmonary Immunological Diseases, Guiyang, Guizhou Province, China

Borna disease virus 1 (BoDV-1) is an emerging zoonotic RNA virus that can cause severe acute encephalitis with high mortality. Currently, there are no effective countermeasures, and the potential risk of a future outbreak requires urgent attention. To address this challenge, the complete genome sequence of BoDV-1 was utilized, and immunoinformatics was applied to identify antigenic peptides suitable for vaccine development. Our study employed antigenicity-focused protein screening and immunoinformatics methods to predict B-cell linear epitopes, B-cell conformational epitopes, and cytotoxic T lymphocyte (CTL) epitopes for developing a vaccine targeting BoDV-1. Five of the six proteins exhibited high antigen scores, and subsequent screening identified eight non-toxic, non-allergenic overlapping epitopes with antigenicity scores greater than 1, selected for vaccine development. The final vaccine design was constructed by linking these epitopes using GPGPG linkers, incorporating β-defensins at the N-terminus to enhance immune response, and incorporating Hit-6 at the C-terminus to improve protein solubility and aid in protein purification. We employed computational tools to predict the immunogenicity, physicochemical properties, and structural stability of the vaccine. Molecular docking analysis was conducted to predict the stability and dynamics of the vaccine in complex with Toll-like receptor-4 (TLR-4) and major histocompatibility complex I (MHC I) receptors. Finally, the vaccine was cloned through in silico restriction to create a plasmid construct for expression in a suitable host. These findings demonstrate the potential of the immunoinformatics-designed multi-epitope vaccines for the prevention and treatment of BoDV-1. Relevant preparations were made in advance for possible future outbreaks and could be quickly utilized for experimental verification.