Nanovaccine based on chitosan oligosaccharide-polyethyleneimine packaged circRNA encoding sigma C proteins elicits protective immunity against novel duck reovirus in ducks.

The promise of mRNA vaccines lies in their design flexibility and efficient immune activation. Emerging as a next-generation platform, circular RNAs (circRNAs) offer superior molecular stability and lower immunogenicity than their linear mRNA counterparts. However, the feasibility of a circRNA vaccine against avian viral diseases remains unexplored. To address this, we devised a novel nanovaccine based on the natural biomacromolecule chitosan oligosaccharide conjugated polyethylenimine (CS-PEI) packaged circRNA platform for avian species and evaluated its efficacy and protective potential. We engineered the circRNA expression platform by constructing a series of plasmids for preparing circRNAs encoding a reporter gene (Nanoluciferase) or the sigma C protein of novel duck reovirus (NDRV). Purified circRNAs successfully transfected and mediated exogenous protein expression in both HEK293T and duck embryo fibroblast (DEF) cells by transfection reagent and CS-PEI, as confirmed by Nanoluc luciferase assays, fluorescence imaging, and indirect immunofluorescence. The CS-PEI encapsulated with circRNA encoding sigma C (circRNA-σC) into nanoparticles approximately 170 nm in diameter and fit for delivery in ducklings. The immunogenicity and protective efficacy of this CS-PEI-circRNA-σC vaccine were comprehensively assessed. Results demonstrated that the vaccine induced robust humoral and cellular immune responses and provided effective protection against a subsequent NDRV challenge. These findings provide a foundational framework and valuable technical support for developing next-generation vaccines against avian viral infections, with potential applications for other viral diseases.