Rational design and sequence optimization of an mRNA-LNP vaccine elicits protective immunity against infectious...

Infectious bronchitis virus (IBV) severely threatens the global poultry industry due to constant genetic variation and broad serotype diversity. Traditional live-attenuated vaccines often lack sufficient cross-protection and carry the risk of virulence reversion, necessitating safer alternatives. Here, we developed and evaluated a novel lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccine against IBV. To maximize avian expression, we identified a chicken-derived Hsp70 5'-untranslated region that strongly drives translation. Using this backbone, we stabilized the spike (S) protein in its prefusion conformation by introducing diproline substitutions (2P) and mutating the furin cleavage site (FCSmut). We then optimized the mRNA secondary structure using the LinearDesign algorithm, which yielded superior in vitro translation efficiency and structural stability. Intramuscular vaccination of chickens rapidly elicited potent humoral and cellular immune responses, characterized by high-titer neutralizing antibodies and robust expansion of antigen-specific CD8⁺ T cells. Following homologous challenge with a virulent IBV strain, the vaccine effectively alleviated clinical symptoms and reduced tracheal ciliary damage. It also drastically reduced viral replication in primary target organs and lowered respiratory viral shedding. Integrating avian-specific regulatory elements, conformational stabilization, and structural RNA optimization provides a highly effective framework for next-generation avian coronavirus vaccines.