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In this study, the dominant neutralizing epitope VP1 G\u2013H loop (141\u2013160 aa) was analyzed among 46 serotype O FMDV strains belonging to Southeast Asian (SEA) and Middle East\u2013South Asian (ME\u2010SA) topotypes isolated during 1980\u20132019. 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\u2010site mutant (T142P, Q153P) and double\u2010site mutant (T142P&amp;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\u2010mutant strains, yet protection efficacy was greatly impaired against the double mutant. The double\u2010mutant vaccine elicited high\u2010level neutralizing antibodies against the double\u2010mutant strain (T142P&amp;Q153P) with a titer of 1:426.67, 10\u201320 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\u2010spectrum vaccines.<\/jats:p>","DOI":"10.1155\/tbed\/4808637","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T07:07:37Z","timestamp":1783926457000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Adaptive Evolution of Serotype O Foot\u2010and\u2010Mouth Disease Virus Under Vaccine Pressure: Combined VP1 T142\/Q153 Mutations Drive Antigenic Alteration and Immune 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