Design and preclinical evaluation of a multi-epitope circular RNA vaccine targeting the spike protein of canine coronavirus genotype II.

Abstract

The rapid evolution and immune evasion of the Spike (S) protein in canine coronavirus type II (CCoV-II) necessitate the development of vaccines with enhanced durability and broad-spectrum efficacy. Circular RNA (circRNA) offers a superior platform to linear mRNA due to its covalently closed structure, which resists exonuclease degradation and ensures sustained antigen expression. In this study, we designed a multiepitope circRNA vaccine targeting conserved CTL, HTL, and LBL epitopes of the CCoV-II S protein, encapsulated in lipid nanoparticles (LNPs). The LNP-circRNA formulations exhibited high physicochemical stability and robust S protein expression in vitro. In both murine and canine models, the vaccine elicited potent S-specific IgG and neutralizing antibody titers, significantly enhancing T follicular helper (Tfh) cell and germinal center B (GC) cell responses. Crucially, vaccinated dogs challenged with virulent CCoV-II showed markedly reduced viral shedding, attenuated clinical symptoms, and preserved intestinal integrity without systemic inflammatory adverse effects. These results demonstrate that the multiepitope circRNA vaccine induces robust, long-term immune protection, establishing it as a highly effective next-generation candidate for the prevention of CCoV-II infection.