Structural basis of a conserved, broad antigenic region surrounding the five-fold axis of foot-and-mouth disease virus revealed by swine neutralizing antibodies

Figures

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  Fig 1. Screening and characterization of porcine-derived monoclonal antibodies against FMDV. (A) Workflow for screening FMDV-specific porcine monoclonal antibodies (mAbs). Peripheral blood mononuclear cells (PBMCs) were subjected to negative magnetic selection and fluorescence-activated cell sorting (FACS) to isolate antigen-specific B cells. Enriched cells were then processed for single-cell B cell receptor (BCR) and transcriptome sequencing using the 10 × Genomics platform with customized porcine BCR primers. Antibody expression plasmids were constructed, transfected into 293F cells, and purified to obtain recombinant antibodies. Created in BioRender. zhao, Z. (2026) https://BioRender.com/7dik6qh. (B) The four mAbs were evaluated for neutralization potency and breadth against representative strains of FMDV serotypes O, A and Asia1 using microneutralization assays. Neutralization titers (VN; µg/ml) are shown and color-coded. The germline gene segments (VH, DH, JH, Vλ, Jλ) corresponding to each mAb were identified and characterized. (C) Competitive ELISA was performed to assess epitope overlap among six mAbs (pO18-10, pO18-17, pO18-52, pO18-53, C4 and B82), using biotinylated pO18-10 as…

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  Fig 2. Cryo-EM structure of the FMDV-O18-pO18-10 complex. (A) A representative cryo-EM micrograph of the FMDV-O18-pO18-10 complex, Scale bar, 50 nm. (B) Selected two-dimensional class averages illustrating the distribution of bound antibodies on the viral capsid surface, Box size, 46.5 nm. (C) Central cross sections obtained through cryo-EM maps are shown with icosahedral 2-, 3-, and 5-fold axes. (D) Rendered images of the FMDV-O18-pO18-10 complex. Depth cueing with color is used to indicate the radius (< 130 Å, yellow; 140-150 Å, from green to cyan; > 160 Å, blue). The icosahedral 5- and 3-fold axes are represented by pentagons and triangles, respectively. (E) Footprint of pO18-10 on the FMDV surface were produced using RIVEM [46]. (F) Fourier shell correlation (FSC) of the final 3D reconstruction after gold-standard refinement using RELION. The resolution corresponding to an FSC of 0.143 is shown. FSC curves before (gray) and after (pink) masking, as well as the post-correction curve (purple) accounting for mask effects using phase randomization, are plotted. (G) Representative structures of antibody-FMDV complexes available in the PDB, including serotype-specific and cross-serotype…

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  Fig 3. Interactions between the pO18-10 and FMDV-O18. (A) Cartoon representation of two adjacent protomers showing the interaction interface between pO18-10 scFv and the capsid of the O/18074 strain. The heavy chain and light chain of pO18-10 are in light blue and purple, respectively. Capsid proteins VP1, VP2, VP3 and VP4 are shown in blue, green, orange and earthy yellow, respectively. (B-C) Expanded views of the interaction interface highlighting the VP1 C-terminal and VP3 G-H loop within protomer 1 (B) as well as the VP1 E-F loop within protomer 2 (C). Hydrogen bonds and salt bridges in the interaction interface are marked separately by black and orange dashed lines. (D) The epitope footprint of pO18-10 on the surface of two protomers from two adjacent pentamers of FMDV. One protomer (comprising VP1-VP4) is circled in a grey line. The epitope includes residues VP1 197, 198, 202, 204, 205 and 210, and VP3 173 from one protomer, and VP1 95 and 99 from the adjacent protomer, was marked in pink.

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  Fig 4. Functional analysis of pO18-10 and related bnAbs against wild-type and mutant FMDV strains. (A) Neutralization efficacy of pO18-10 against the wild-type (O/18074) and mutant viruses (VP1 E95A, VP1 D99A, VP1 S197A, VP1 T198A, VP1 K204A, VP1 K210A, and VP3 D173A) was assessed by microneutralization assay. (B) Neutralization efficacy of pO18-10 against the wild-type (A/WH/CHA/09) and mutant (VP1 D99A, VP1 K203A, VP1 K209A, and VP3 D174A) viruses was assessed by microneutralization assay. (C) Sequence alignment of VP1 and VP3 regions from representative FMDV strains of serotypes O, A and Asia 1. (D) Full-length amino acid sequences of available FMDV serotypes O, A, and Asia1 were downloaded from the National Center for Biotechnology Information (NCBI) database as of June 30, 2025. Sequence logos were generated for the VP1 (serotype O, n = 5,974; A, n = 2,119; Asia1, n = 805) and the VP3 (O, n = 1,660; A, n = 826; Asia1, n = 219). (E to G) Neutralization efficacy of pO18-17 (E), pO18-52 (F), and pO18-53 (G) against the wild-type (O/18074) and the same panel of site-directed mutants as in A. The neutralization concentration represented the lowest antibody concentration required to fully…

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  Fig 5. Identification of cross-serotype neutralization mechanisms of pO18-10 against FMDV serotypes O and A. (A-F) The inhibition effect of pO18-10 on viral attachment to BHK-21 cells. Different amounts of pO18-10 were respectively pre-incubated with FMDV serotype O (O/18074 strain) (A, C, E) or serotype A (A/WH/CHA/09 strain) (B, D, F). The viruses were quantified through detecting VP1 protein by IFA (A, B) and Western blotting (C, D) and 3D gene by qRT-PCR (E, F). The experiments were performed in triplicate. The data differences between conditions with virus only and different bnAb treatments were assessed using unpaired T-test (Holm-Sidak method, α = 0.05) in GraphPad Prism 9.5.1.*** Indicates an extremely significant difference at P < 0.001. ** Indicates a very significant difference at P < 0.01. NS indicates no significant difference. (G, H) Thermostability assay of FMDV serotypes O and A in the presence of four porcine-derived bnAbs. The Y-axis indicates the derivate of raw fluorescence representing the level of viral RNA release. PBS was used as a blank control.

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  Fig 6. Prophylactic and therapeutic efficacy of pO18-10 against FMDV infection in suckling KM mice. (A) Prophylactic efficacy of pO18-10 against lethal challenge with O/18074. Clinical scores (left) and survival (right) curves of suckling KM mice (n = 5 per group) treated with 10, 5, 1, 0.1, or 0.01 mg/kg pO18-10, or pO18-36 (10 mg/kg) 24 h before lethal challenge are shown. (B) Therapeutic efficacy of pO18-10 against lethal challenge with O/18074. Clinical scores (left) and survival (right) curves of suckling KM mice (n = 5 per group) treated with 20, 10, 5, 1, or 0.1 mg/kg pO18-10, or pO18-36 (20 mg/kg) 24 h after lethal challenge are shown. Upper panel: study outline. Lower panel: clinical score and survival. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, limb paralysis; 4, death. For survival data, the Log-rank (Mantel-Cox) test was used to evaluate significance. *** Indicates an extremely significant difference at P < 0.001. ** Indicates a very significant difference at P < 0.01. * Indicates a significant difference at P < 0.05. All error bars represent SEM.