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Inter-serotype cross-reactivity of foot-and-mouth disease (FMD) antibody enzyme-linked immunosorbent assays (ELISAs) can exceed 50%, leading to incorrect serotyping of outbreaks with implications for vaccine selection. In this study, synthetic peptides that mimic the hypervariable G-H loop of FMD viruses (FMDVs) that currently circulate in East Africa (O, A, SAT1, and SAT2) were evaluated as capture antigens in ELISAs (pELISAs). A panel of monovalent bovine sera was tested using these novel assays in parallel with separate ELISAs that utilized stabilized virus-like particles (VLPs). Virus neutralization tests using the same viruses were used to benchmark the status of the sera, which revealed evidence of cross-reactivity for the serotype O and SAT2 antigens (encompassing 2/19 and 3/19 of the heterologous sera, respectively). Equivalent diagnostic serotype sensitivity was observed for prototype peptide and VLP ELISAs for serotype O and SAT1 antigens (86% and 100%, respectively), while there was higher diagnostic serotype sensitivity for the VLP ELISAs targeting serotypes A and SAT2 compared to the corresponding pELISAs (86% vs 71% and 100% vs 86%, respectively). The serotype specificity of these tests ranged from 71% to 79% and 52% to 89% for the pELISA and VLP ELISA formats, respectively. Peptides offer a simple, biosafe, and cost-effective approach to present FMDV-specific epitopes, and these initial findings suggest that peptide ELISAs could be a promising approach to develop serological ELISA assays to present authentic epitopes in comparison to ELISAs that use full capsid VLPs.

Introduction: Foot-and-mouth disease virus (FMDV) has a hypervariable G-H loop region within the VP1 capsid protein. This structure is associated with virus neutralisation and contains the virus attachment motif (RGD) which binds to the cellular integrin receptor facilitating virus entry for all seven FMDV serotypes. Methods: Six monoclonal antibodies (Mabs) were tested against 10 peptides representing the wild-type G-H loops of serotypes O, A, SAT1, and SAT2. D9 and B2 Mabs were raised against serotype O and tested against three more sets of peptides: (1) nine overlapping peptides with one amino acid difference, (2) alanine scanning peptide, both for O1K strain and (3) four mutated peptides one for A22 and three for SAT2 strains in the enzyme-linked immunosorbent assay format with correlation to virus neutralisation test. Results: The D9 Mab was bound to peptides corresponding to the G-H loops of serotype O, A, and SAT1 strains, but only neutralised serotype O and SAT1 strains in the virus neutralisation test. The B2 Mab is also bound to serotype O and SAT1 but only neutralised serotype O. Using a set of overlapping peptides, the binding region for the D9 Mab was confirmed as amino acid positions 144, 147 and 148. An additional critical amino acid residue at position 145R was identified using a set of alanine scanning peptides. The binding region for B2 Mab appears to be upstream of RGD as B2 showed lower binding to peptides lacking the first three amino acids of the GH-loop peptides. These critical amino acids were further confirmed by designing modified SAT2 and A peptides at these positions, which led to a significant improvement in the binding ability of D9 but not B2. Conclusion: These findings help to map cross-reactive epitopes within the G-H loop which may contribute to the inter-serotypic cross-reactivity observed in diagnostic serological assays giving insights that motivate the design of custom peptides that have improved serotype specificity.