Glycosylated Foot-And-Mouth Disease Virus-Like Particles Produced in Pichia Pastoris Enhance Stability and Immunogenicity.

Despite the availability of vaccines, foot-and-mouth disease (FMD) remains a significant concern in many developing countries, causing severe economic losses and affecting local farming communities. Virus-like particle (VLP) vaccines are highly regarded for their safety and efficacy. N-glycosylation for stabilisation and recognition by antigen-presenting cells has been a widely adopted strategy, particularly in enveloped viruses. Here, FMD virus (FMDV) VLPs were employed as a model for artificial glycosylation. N-glycosylation was introduced by mutating the potential glycosylation site of VP1 and then N-glycosylated FMDV VLPs were successfully produced in Pichia pastoris. Glycan profiling revealed that the majority of associated glycans (72.93%) were of the high-mannose type, with additional hybrid type (4.16%) and complex type (22.92%) detected. Functional analyses demonstrated that glycosylation significantly enhanced the stability of VLPs and facilitated the uptake by antigen-presenting cells. Animal experiments further revealed that glycosylation could induce a higher cellular immune response compared to WT VLPs, offering a reference for the glycosylation design of VLP vaccines.