Enhanced immunogenicity of swine coronavirus PEDV by engineering transcriptional regulatory sequences

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that can cause severe diarrhea and high mortality in neonatal piglets. Therefore, the development of safe and effective vaccines remains a top priority for controlling PEDV infection. Previous studies have found that the transcription regulatory sequence (TRS) of the viral M gene has the strongest regulatory ability for the expression of exogenous gene. In order to investigate whether the transcription level of the S gene is increased after swapping body TRS of the S gene (TRS-S) with the body-TRS of M gene (TRS-M), CRISPR/Cas9 gene editing technology were used to replace the TRS-S of PEDV with TRS-M, and found that replacing TRS-S by TRS-M, as well as the downstream bases of TRS-M, can effectively promote the expression of S protein. Subsequently, the recombinant strain with high expression of S protein was prepared into an inactivated vaccine, which induced higher levels of S-specific immunoglobulin G (IgG), IgA, and neutralizing antibodies in immunized pregnant sows. The newborn spiking pigs also obtained high levels of antibodies through breastfeeding, and after being challenged, it was found that clinical symptoms and microscopic lesions were significantly reduced. In conclusion, these data suggest that replacing TRS-S with the transcriptionally efficient TRS-M effectively promotes PEDV S protein expression, and this modified strain exhibits improved immunogenicity and better protection against viral challenge, which provides new method for the research and development of PEDV vaccines.