Adaptive Evolution of Serotype O Foot‐and‐Mouth Disease Virus Under Vaccine Pressure: Combined VP1 T142/Q153 Mutations Drive Antigenic Alteration and Immune Evasion

Foot-and-mouth disease virus (FMDV) escapes host immune surveillance via adaptive evolution driven by vaccine-mediated selective pressure, leading to persistent breakthrough infections in immunized animals. In this study, the dominant neutralizing epitope VP1 G-H loop (141-160 aa) was analyzed among 46 serotype O FMDV strains belonging to Southeast Asian (SEA) and Middle East-South Asian (ME-SA) topotypes isolated during 1980-2019. The key amino acid residues at VP1 positions 142 and 153 underwent sequential stepwise evolution across three phases under natural selection. Based on the reverse genetic system of Wt, single-site mutant (T142P, Q153P) and double-site mutant (T142P&Q153P) strains were rescued. These mutation sites were further introduced into an efficient FMDV nanoparticle vaccine to construct four vaccine candidates. Mouse immunization verified all vaccines conferred solid protection against Wt and single-mutant strains, yet protection efficacy was greatly impaired against the double mutant. The double-mutant vaccine elicited high-level neutralizing antibodies against the double-mutant strain (T142P&Q153P) with a titer of 1:426.67, 10-20 folds higher than Wt (1:21.33), T142P (1:32), and Q153P (1:42.67) vaccines. Consistent results were also obtained in pigs immunized with commercial inactivated vaccines. Collectively, combined mutations at VP1 142 and 153 reshape viral antigenicity and act as core drivers of FMDV immune evasion. Integrating such immune escape hotspots into vaccine antigens can broaden neutralizing antibody coverage, offering an experimental basis for clarifying FMDV evolution and developing broad-spectrum vaccines.