Wuhan-ancestral multi-antigen SARS-CoV-2 virus-like particles protect against the omicron sublineage JN.1 variant.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve through mutations in the spike (S) protein, leading to antigenically divergent variants such as the Omicron sublineage JN.1 that markedly reduce vaccine-induced neutralizing antibody activity. These limitations highlight the need for vaccine strategies that can induce multilayered protective immunity rather than relying solely on S protein-targeted immunity. Here, we developed a Wuhan-lineage-based multi-antigen virus-like particle (VLP) vaccine incorporating S, envelope (E), membrane (M), and nucleocapsid (N) proteins using a baculovirus dual-expression system. Mice were primed intramuscularly and boosted via either the intramuscular or intranasal route, and humoral, mucosal, and cellular immune responses were evaluated. Both vaccination regimens induced robust systemic IgG responses, whereas mucosal IgA responses were selectively enhanced following intranasal boosting. Sera from VLP-immunized mice efficiently neutralized the homologous Wuhan-lineage strain but showed no detectable neutralizing activity against the JN.1 variant. Despite this, VLP immunization elicited antigen-specific CD4+ and CD8+ T cell responses in both pulmonary and systemic compartments. In a highly susceptible K18-hACE2 mouse model, VLP immunization completely prevented mortality following challenge with both the ancestral Wuhan-lineage strain and the JN.1 variant and markedly suppressed viral replication in respiratory tissues. Histopathological analysis further showed minimal interstitial lung lesions with organized perivascular lymphocytic infiltration in vaccinated mice, while pulmonary cytokine analysis revealed qualitatively distinct immune response patterns depending on the challenge strain and vaccination status. Our findings show that a Wuhan-lineage-based multi-antigen VLP vaccine can provide cross-protection against an antigenically divergent SARS-CoV-2 variant that is not fully explained by detectable serum neutralizing activity alone, suggesting the importance of integrated immune responses involving humoral, cellular, and local immune mechanisms.