Functional Evaluation of Neutralizing Antibodies Against Foot-and-Mouth Disease Virus Serotype O Using a Luciferase-Based Surrogate Neutralization Assay

Foot-and-mouth disease (FMD) is a highly contagious viral disease that seriously threatens livestock health. Protective immunity induced by vaccination is primarily associated with the generation of neutralizing antibodies; however, conventional virus neutralization tests are time-consuming and require the handling of live virus, limiting their suitability for rapid and repeated evaluation in routine settings. In this study, we established a surrogate neutralization assay for the functional evaluation of neutralizing antibody activity against FMD virus (FMDV) serotype O. The assay measures changes in luciferase-based luminescent signals generated following exposure of LgBiT-expressing cells to HiBiT-tagged virus-like particle (VLP) preparations. FMDV serotype O-derived VLPs exhibited stable capsid protein expression, assembly characteristics, and morphological integrity similar to those of virus particles, as confirmed by sucrose gradient fractionation and electron microscopy. In the presence of neutralizing antibodies, a reduction in luminescent signals was observed, enabling functional discrimination of antibody activity. The surrogate assay results showed a strong correlation with those of the conventional virus neutralization test for FMDV serotype O (R2 = 0.9068). This study demonstrates the feasibility of the surrogate neutralization assay for assessing the functional activity of neutralizing antibodies and its potential applicability to vaccine immunogenicity assessment and functional antibody analysis.