Immunoinformatics-driven Design and in silico Validation of a Multi-epitope Subunit Vaccine Targeting Norovirus

Abstract

Background: Norovirus, a non-enveloped, positive-sense single-stranded RNA virus belonging to the Caliciviridae family, is a leading cause of acute gastroenteritis (AGE) globally, accounting for approximately 19–21 million cases annually in the United States alone. The rapid emergence of genetically diverse variants, including GII.17, poses significant challenges to conventional vaccine development. Therefore, alternative strategies capable of inducing broad and effective immune responses are urgently needed. Methods: An immunoinformatics-driven approach was employed to design a multi-epitope subunit vaccine candidate against Norovirus. Three viral proteins—capsid protein (A7YK10), small protein (A7YK11), and polyprotein (A7YK09)—were selected for epitope prediction using the Immune Epitope Database (IEDB). Predicted B-cell and T-cell epitopes were screened for antigenicity (VaxiJen), allergenicity (AllerTOP), toxicity (ToxinPred), and global population coverage. The finalized construct was evaluated for physicochemical properties (ProtParam), structural integrity (secondary and tertiary structure prediction, Ramachandran analysis, QMEAN scoring), molecular docking interactions with MHC class I, MHC class II, and Toll-like receptor 4 (TLR4), molecular dynamics stability, immune response simulation, and codon optimization for expression feasibility. Results: A 433-amino-acid multi-epitope construct incorporating six B-cell, six cytotoxic T lymphocyte (CTL), and nine helper T lymphocyte (HTL) epitopes was generated. The construct demonstrated high predicted antigenicity and broad global population coverage (98.96%). Structural validation indicated favorable stereochemical quality, with 88.9% residues in the most favored regions of the Ramachandran plot and a QMEAN Z-score of −0.97. Docking analysis revealed strong binding affinity, particularly with TLR4 (HADDOCK score −103.9 ± 11.0), and molecular dynamics simulation confirmed stability of the vaccine–TLR4 complex over 100 ns. Conclusion: The designed multi-epitope vaccine construct exhibits promising immunogenic, structural, and interaction profiles in silico, supporting its potential as a candidate for further experimental validation in vitro and in vivo.