Optimized Multi-Epitope Norovirus Vaccines Induce Robust Humoral and Cellular Responses in Mice

Background: Norovirus GII.4 is a major global health threat, yet no licensed vaccines exist due to the virus's rapid evolution and high mutation rates. Objective: To rationally design and experimentally validate multi-epitope vaccine candidates against Norovirus GII.4 using computational immunoinformatics and in vivo evaluation. Methods: We employed reverse vaccinology to screen optimal norovirus GII.4 epitopes and systematically designed four construction strategies to evaluate different epitope topologies and adjuvants. Candidates underwent molecular dynamics simulations and were expressed in E. coli. Immunogenicity was assessed in BALB/c mice via ELISA and ELISPOT to evaluate humoral and cellular responses. Results: Three candidates (NV1, NV4, NV5) were successfully produced and induced cross-reactive antibodies against authentic GII.4 virus-like particles. Notably, the construction strategy influenced the immune response: NV5 (repetitive epitopes and HSP as adjuvant) elicited the highest antigen-specific antibody titers, NV1 (all types of epitopes and TLR as adjuvant) induced the strongest cellular response, and NV4 (repetitive epitopes and TLR as adjuvant) achieved the most rapid immune response. Consistently, in silico analysis showed that the NV1-TLR3 complex exhibits tighter interaction, higher binding energy, and greater structural stability, supporting its superior capacity to trigger cellular immunity. Conclusions: A rational multi-epitope vaccine design workflow successfully realized the translation from computational design to functional vaccines. Optimizing adjuvant selection and epitope construction is critical for eliciting immune responses in next-generation norovirus vaccines.