Modification of N-glycosylation site in the spike protein enhances the neutralizing antibody response against PEDV.

Abstract

Porcine epidemic diarrhea virus (PEDV) is a pathogenic enteric coronavirus of swine that causes severe diarrhea and high mortality in neonatal piglets. The extensively glycosylated spike (S) protein is essential for viral entry and vaccine design. For comprehensively characterizing N-glycosylation modifications of S protein, we analyzed the S proteins of 152 PEDV strains, revealing that the average number of N-glycosylation sites per S protein ranged from 27.5 to 30. To elucidate the function of N-glycosylation in the S protein, we generated 26 recombinant PEDV GX4/2021 strains harboring single-point mutations at individual N-glycosylation sites. The N873 glycosylation site of S protein was essential for viral viability, while mutations at N351, N264, N324, N688, N118, N425, N787, N726, N1249, N1261, N1273, N1278, and N1308 significantly reduced viral titers. In vivo mouse studies demonstrated that a PEDV mutant harboring the N324 mutation in S protein increased neutralizing antibody titers against PEDV, positioning it as a promising candidate for inactivated vaccine. Additionally, single-site mutations at the N324 or N873 site of S protein enhanced the capacity of recombinant Sendai virus (SeV) encoding the PEDV S protein to induce neutralizing antibodies against PEDV, suggesting that S proteins containing these mutations are promising candidate immunogens for engineered vector-based vaccines against PEDV. This study demonstrates that S protein N-glycosylation is critical for PEDV replication and immunogenicity, providing a foundation for novel vaccine design.