S2-peptide conjugated SpyCatcher-Ferritin Nanoparticles elicit broad protection against SARS-CoV-2 variants of concern

Background The continual emergence of SARS-CoV-2 variants highlights the need for vaccine strategies that focus immune responses on structurally conserved viral regions. While most current approaches rely on full-length spike (S) protein, it remains unclear whether minimal conserved epitope peptides alone can induce broadly protective immunity. Here, we investigated whether multivalent presentation of conserved epitope peptides derived from the S2 subunit is sufficient to induce broad and functional antibody responses. To this end, we engineered a ferritin-based nanoparticle vaccine displaying three conserved S2 epitopes (S2.3, S2.5, and the fusion peptide) using SpyTag/SpyCatcher-mediated site-specific conjugation. Results SpyCatcher003-fused ferritin (SC‑FTH) nanoparticles were recombinantly produced in Escherichia coli and efficiently assembled with synthetic S2 peptides containing an N-terminal SpyTag and a miniPEG2 linker, enabling enhanced epitope accessibility. Immunization of mice induced robust spike-reactive antibodies with broad cross-variant binding and rapid recall responses upon boosting, consistent with effective memory B cell activation. Immune sera exhibited multiple antiviral functions, including pseudovirus neutralization, inhibition of spike-mediated cell–cell fusion, and Fc-dependent effector activities. Monoclonal antibody characterization confirmed that conserved S2 epitopes alone can drive broad reactivity and functional diversity. Notably, an antibody targeting the C-terminal region of S2.3 (S2.3.22) showed sub-nanomolar affinity, cross-neutralized both prototype and Omicron-lineage viruses, and potently inhibited membrane fusion, whereas other S2-directed antibodies mediated strong antibody-dependent cellular cytotoxicity despite limited neutralization. Conclusions These findings demonstrate that multivalent display of conserved epitope peptides, even in the absence of the full viral antigen, is sufficient to induce broadly neutralizing and functionally diverse antibody responses. This work establishes a modular “plug-and-display” nanoparticle platform for rapidly targeting conserved epitopes of highly variable viruses and supports the feasibility of epitope-focused vaccine design as a strategy for next-generation coronavirus vaccines.