A chimeric trivalent Echovirus vaccine designed by loop substitution elicits cross-neutralizing immunity against serotypes 11, 18, and 30.

Echoviruses, members of the Enterovirus B genus in the Picornaviridae family, comprise numerous serotypes that frequently cause aseptic meningitis, myocarditis, and severe neonatal infections. The coexistence of multiple antigenically distinct serotypes poses a significant challenge for vaccine development, underscoring the clinical need for a broad-spectrum multivalent vaccine. In this study, we focused on three prevalent serotypes-Echovirus 11 (E11), 18 (E18), and 30 (E30)-as representative models to explore multivalent vaccine design. We first predicted and experimentally identified dominant B-cell epitopes within the VP1 proteins of these viruses. Guided by these analyses, a chimeric trivalent antigen (VP1-III) was rationally constructed by substituting epitope-containing loops from E18 and E30 into the VP1 backbone of E11. Structural modeling confirmed that VP1-III preserved native VP1 conformation while displaying multitype epitopes. Both VP1-III recombinant protein and mRNA-lipid nanoparticle vaccines induced potent immunogenicity in mice, characterized by cross-neutralizing antibodies and Th1-biased cellular responses. These findings support structure-guided epitope integration as a promising strategy for developing broadly protective multivalent Echovirus vaccines.IMPORTANCEEchoviruses cause widespread outbreaks and severe neonatal disease, yet vaccine development remains limited by extensive antigenic diversity among serotypes. Here, we rationally engineered a chimeric trivalent VP1 antigen by grafting dominant epitopes from E11, E18, and E30 through structure-guided loop substitution. Both recombinant protein and mRNA formulations induced robust cross-neutralizing and Th1-biased immune responses in mice, demonstrating that strategic epitope substitution can effectively overcome serotype barriers and providing a promising path toward broad-spectrum Echovirus vaccines.