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

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.