Pan-Genomic Entropy Profiling Reveals Conserved Immune Hotspots in the E2 Glycoprotein of Classical Swine Fever Virus

• Entropy mapping reveals conserved cryptic motifs in CSFV E2 glycoprotein • Block 9 identified as a low-entropy region with immune-relevant features • Predicted CTL, HTL, and B-cell epitopes show strong immunogenic potential • Multi-epitope vaccine construct elicits robust humoral and cellular responses • Entropy-guided motif mining enables precision vaccine design for RNA viruses Classical swine fever virus (CSFV) remains a significant threat to swine health, necessitating alternative vaccine strategies that overcome limitations of live-attenuated vaccines. In this study, we present a comprehensive in silico framework that integrates entropy-based conservation analysis, immunoinformatics, structural modeling, and translational feasibility assessment to rationally design a multi-epitope vaccine construct (MEVC) targeting the CSFV E2 glycoprotein. A non-redundant dataset of 312 E2 sequences was analyzed using multiple Shannon entropy profiles, revealing a dominant low-entropy region (Block 9; residues 1–426) that remained stable across multiple entropy thresholds. Residue-level conservation analysis confirmed high consensus across this block, supporting its suitability for epitope mining. Cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and linear B-cell epitopes were predicted within Block 9 and filtered based on binding affinity, conservation, antigenicity, and safety criteria. Epitope density analysis showed significant enrichment within Block 9 relative to the remainder of the E2 protein. The finalized MEVC was structurally modeled and validated using stereochemical and energy-based metrics, and a comparative analysis with an AlphaFold model highlighted structural uncertainty inherent to synthetic multi-epitope constructs. In silico immune simulations were performed to explore qualitative immune response trends. Physicochemical profiling and codon optimization for Escherichia coli expression indicated favorable stability, solubility, and translational feasibility, supported by optimal GC content, codon adaptation index, and absence of rare codon clusters. While all findings are computational and require experimental validation, this study provides a refined, data-driven foundation to guide future experimental development of CSFV epitope-based vaccines.