Tabriz University of Medical Sciences BioImpacts 2228-5652 15 1 2025 01 19 Immunoinformatics design of an mRNA vaccine against classical swine fever virus using conserved E2 protein and NS3 T-lymphocyte epitopes 30778 30778 10.34172/bi.30778 EN Edward C. Banico https://orcid.org/0009-0009-9838-9315 Ella Mae Joy S. Sira https://orcid.org/0000-0001-9802-0242 Lauren Emily Fajardo https://orcid.org/0009-0002-0350-1532 Fredmoore L. Orosco https://orcid.org/0000-0002-8861-7923 Journal Article 10.34172/bi.30778 2024 09 26 2025 01 21 Introduction: The classical swine fever virus (CSFV) causes significant economic losses in the livestock industry. While the existing E2 marker vaccine offers protection against infections, it is characterized by delayed immunity and reduced effectiveness over time. Optimizing the existing vaccine is crucial to better control CSFV outbreaks worldwide. This study aimed to improve the existing E2 marker vaccine for CSFV by integrating NS3 T lymphocyte-inducing epitopes into the conserved E2 protein sequence and using mRNA technology for vaccine delivery. Methods: The design and evaluation of the vaccine were carried out exclusively through in silico methods. T lymphocyte epitopes were identified from the CSFV NS3 protein using multiple epitope prediction tools. A vaccine construct was formed after linking the predicted NS3 epitopes, E2 protein, and an immunogenic adjuvant. Molecular docking and dynamics simulations were performed to analyze the interaction between the adjuvant used and its immune receptor. Signal peptides were incorporated into the design, and mRNA sequences with varying codon usage biases were generated using LinearDesign. The mRNA sequence with minimum free energy (MFE) and codon adaptation index (CAI) closest to the controls was selected as the final design. Results: Twenty epitopes with high binding affinity to major histocompatibility complexes (MHCs) were identified from the CSFV NS3 protein. The vaccine construct with swine CD154 adjuvant demonstrated high antigenicity, making it the optimal choice for the final vaccine design. Molecular docking and dynamics simulations confirmed the adjuvant's strong affinity and stable interaction with its canonical receptor, swine CD40. Moreover, the final vaccine design exhibited higher populations of lymphocytes and antibodies compared to the components of the commercialized E2 marker vaccine in immune simulation. The final mRNA vaccine sequence exhibited a higher MFE and CAI than the two licensed mRNA vaccine controls. Conclusion: The mRNA vaccine designed in this study serves as a potential CSFV vaccine candidate. In vivo and in vitro validation is needed to confirm its efficacy.