Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 5 2026 10 05 861 871 Mahdi Arezoumandi Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Sadegh Shojaei Baghini Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Nima Rad Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Elham Taghipour Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Mohammad Hassan Pouriayevali Department of Arboviruses and Viral Hemorrhagic Fevers, National Reference Laboratory, Pasteur Institute of Iran, Tehran, Iran Fateme Frootan Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Mostafa Motallebi Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Ali Hatef Salmanian Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran 2026 05 05 2026 08 24 Background and Objectives: West Nile virus (WNV) is a mosquito-borne flavivirus that represents a significant public health concern, and the development of effective vaccine candidates remains a priority. This study aimed to design a chimeric multi-epitope protein against WNV using immunoinformatics approaches and evaluate its immunogenicity in mice. Materials and Methods: Selected regions of the WNV envelope (E) and non-structural 1 (NS1) proteins were analyzed to identify B- and T-cell epitopes using immunoinformatics tools. The selected epitopes were incorporated into a chimeric construct fused with cholera toxin B subunit, and its antigenicity, allergenicity, structural properties, and TLR3 interaction were evaluated in silico. The optimized construct was expressed in Escherichia coli, purified by Ni-NTA affinity chromatography, and confirmed by SDS-PAGE and western blotting. BALB/c mice were immunized with the recombinant protein plus adjuvant, and immune responses were assessed using ELISA, cytokine assays, and flow cytometry. Results: Immunized mice developed high IgG antibody titers and showed detectable IFN-γ and IL-5 production, suggesting induction of Th1-associated responses with limited Th2-related cytokine production. The chimeric protein also stimulated CD4+ and CD8+ T-cell responses, indicating activation of both humoral and cellular immunity. Conclusion: The designed chimeric protein showed immunogenic potential and induced humoral and cellular immune responses in mice, supporting further evaluation as a candidate for WNV vaccine development. https://ijm.tums.ac.ir/index.php/ijm/article/view/6174 https://ijm.tums.ac.ir/index.php/ijm/article/download/6174/1945