Remapping the spatial distribution of neutralizing sites and their immunodominance on the capsid of different topotypes of FMDV serotype O by site-directed competitive ELISA for detection of neutralizing antibodies

ABSTRACT The foot-and-mouth disease virus (FMDV) serotype O contains at least five neutralizing antigenic sites, yet the structural relationship and antibody abundance remain poorly characterized. This study identifies six distinct neutralizing antigenic sites by evaluating 27 host-derived neutralizing antibodies (NAbs) using competitive enzyme-linked immunosorbent assay (cELISA). These sites include the VP1 G-H loop, VP1 C-terminus, site 2, site 4, site 6, and site 7. Notably, classical sites 1 and 5 were reclassified into the VP1 G-H loop and VP1 C-terminus classes. Sites 2 and 4 align with classical classifications, targeting independent epitopes on VP2 and VP3, respectively. We identified two novel sites: site 6, which involves extensive interactions with the G-H loop, C-terminus of VP1 and VP3, and site 7, which interacts with both VP2 and VP3. Sera from cattle, sheep, and pigs immunized with four serotype O lineages (O/SCGH/2016, O/Mya/98, O/Tibet/99, and O/XJ/2017) were used to evaluate the immunodominance of these sites. NAb responses favored site 4 for O/SCGH/2016 and the VP1 G-H loop for O/XJ/2017. Immunization effectiveness varied by strains and host species: O/XJ/2017 and O/Tibet/99 were effective in sheep, while O/Mya/98 showed reduced efficacy; O/Tibet/99 showed good immunogenicity in pigs. No significant differences were observed in cattle. There is a strong correlation (r = 0.8693) between NAb levels at site 6 and virus neutralization tests, suggesting its potential for use in alternative testing methods. This study describes the spatial distribution of neutralizing sites and highlights strain-specific immunodominant epitopes and differential antibody responses across species, providing valuable insights for FMD prevention and control.IMPORTANCEThe antigenic structure of the foot-and-mouth disease virus (FMDV) serotype O is complex, and the immunodominant epitopes among different lineages remain poorly understood. This study classified the capsid surface epitopes into six distinct antigenic sites utilizing 27 neutralizing antibodies (NAbs) by paired competitive ELISAs (cELISAs). High-affinity NAbs were selected for site-directed cELISAs to assess antibody abundance in serum from cattle, sheep, and pigs vaccinated with various inactivated FMDV serotype O vaccines. Additionally, liquid-phase blocking ELISA (LPBE) and virus neutralization test (VNT) were employed to measure total antibody and NAb titers. Results indicated that immunodominant sites vary among different strains, with each strain exhibiting different immunogenicity across the three animal species. Notably, antibody titers from NAb pO18-10, targeting site 6 on VP1 and VP3, correlated strongly with VNT results. This study provides comprehensive insights into the antigenic structure of FMDV serotype O and lays the groundwork for developing new methods to detect NAbs.