Fig 1.
Construction of FMDV serotype Asia1-binding B-cell repertoire.
(A) Schematic diagram of the immunization schedule and workflow for constructing the FMDV Asia1-binding B cell repertoire. PBMCs collected after secondary vaccination were subjected to negative magnetic separation and fluorescence-activated cell sorting (FACS) to isolate Asia1/JS/05-binding B cells. Subsequently, enriched antigen-binding B cells were processed for single-cell BCR and transcriptome sequencing using the 10 × genomics platform with customized porcine BCR primers. Created in BioRender. Chen, Y. (2026) https://BioRender.com/qnwnmh1. (B) Dynamics of serum neutralizing antibody titers against the FMDV Asia1/JS/05 strain at different time points post-vaccination. (C-G) Flow cytometric identification of FMDV-binding B cells using biotinylated Asia1/JS/05 146S antigen. (C) PBMCs were gated (P1) to exclude cell debris based on lower values of FSC-A and SSC-A. (D) Singlets were selected in gate P2 based on the diagonal streak in the FSC-A and FSC-H plot. (E-G) Representative plots showing 146S+ B cells among porcine PBMCs (E), enriched B cells (F), and sorted B cells (G) were identified in gate P3.
Fig 2.
Molecular characterization of FMDV serotype Asia1 binding B-cell repertoire.
(A) UMAP visualization of single-cell transcriptomic profiles of Asia1/JS/05-binding B cells, showing two major clusters corresponding to PBs and memory B cells. (B) Heatmap displaying differentially expressed genes between memory B cells and PBs. (C) Overview of clone sizes, antibody isotypes, and light chain types in the porcine BCRs binding to the Asia1/JS/05 strain. (D) Comparison of SHM frequencies among different antibody isotypes. Statistical analysis was conducted using the non-parametric Mann-Whitney test in R. P < 0.001 indicates an extremely significant difference. (E) Frequency proportion of VH and VL germline gene usage in the Asia1-binding B-cell repertoire. (F) Pairing frequency of VH-VL germline gene usage in the porcine BCR repertoire targeting FMDV serotype Asia1.
Fig 3.
Characterization of porcine mAbs derived from high-frequency clonotypes in the Asia1/JS/05-binding BCR repertoire.
(A) The counts of the top 33 high-frequency clonotypes in the Asia1-binding porcine BCR repertoire. (B) Reactivity of pnAbs against FMDV serotype Asia1 determined by IFA. BHK-21 cells were infected with the Asia1/JS/05 strain and incubated with pnAbs (5 µg/ml), followed by rabbit anti-pig FITC (1: 200 in PBS). Fluorescence signals were observed using an FL Imaging System (Life Technologies, USA). Experiments were independently conducted in triplicate. (C) Reactivity of pnAbs against FMDV serotype Asia1 determined using indirect ELISA. (D) Neutralization titer, clonotype frequency, H_V gene, L_V gene, and SHM of porcine mAbs derived from high-frequency clonotypes in the Asia1/JS/05-binding repertoire. Neutralization activity against the Asia1/JS/05 strain was assessed by microneutralization assay. Values represent virus neutralization titers (VN) in µg/ml and are shown with a color bar on the right.
Table 1.
Porcine neutralizing mAb escape mutants against Asia1/JS/05 strain.
Fig 4.
Cryo-EM analysis of the FMDV-Asia1-PAS5 and FMDV-Asia1-PAS12 complexes.
(A-B) Central cross sections, rendered images, and footprints of the FMDV-Asia1-PAS5 complex (A) or FMDV-Asia1-PAS12 complex (B). The central cross sections obtained from the cryo-EM maps of both complexes display the icosahedral 2-, 3-, and 5-fold axes (Scale bar, 10 nm). In the rendered images, depth cueing with color is used to indicate the radius (< 100 Å, blue; 120-160 Å, from cyan to yellow; > 180 Å, red). The icosahedral 5- and 3-fold axes are represented by pentagons and triangles, respectively. Footprints of PAS5 (A) and PAS12 (B) on the FMDV serotype Asia1 surface show the 2D projections of the viral surface produced using RIVEM [26]. (C-D) The Fourier shell correlation (FSC) curves of the FMDV-Asia1-PAS5 (C) and FMDV-Asia1-PAS12 (D) complexes, indicating the map resolutions at the 0.143 FSC cutoff.
Fig 5.
Structure of the FMDV-Asia1-PAS5 complex and identification of key determinants on VP2 of FMDV serotype Asia1.
(A) Cartoon representation of the interaction interface between the PAS5-scFv and the viral capsid of the Asia1/JS/05 strain within a single protomer. The heavy and light chains of PAS5 are colored cyan and purple, respectively. The viral capsid proteins VP1 to VP4 are colored blue, green, orange, and yellow, respectively. (B-D) Expanded views of the PAS5-FMDV interface highlighting the VP2 βΒ strand, B-C loop (B), H-I loop (C), and βC strand (D). Presumptive hydrogen bonds and salt bridges are marked by black and orange dashed lines, respectively. (E) Binding affinity of PAS5 to the inactivated 146S antigen (FMDV Asia1/JS/05 strain) determined by biolayer interferometry (BLI). (F) Neutralization potency of PAS5 against the wild-type (WT, Asia1/JS/05 strain) and its single-substitution mutants, evaluated by microneutralization assay. Neutralization concentration represented the minimum antibody concentration required to completely prevent CPE. The experiment was performed in triplicate. Statistical analysis was conducted by One-Way ANOVA followed by Dunnett’s multiple comparison test with a 95% confidence interval using GraphPad Prism 8.0. *, **, *** indicate significant differences from WT at P < 0.05, P < 0.01, P < 0.001, respectively. ns indicates no significant difference. (G) Sequence conservation analysis of the VP2 B-C loop (residues 66-80) across 209 available FMDV Asia1 sequences.
Fig 6.
Structure of the FMDV-Asia1-PAS12 complex and key determinants on VP2 and VP3 of FMDV serotype Asia1.
(A) Cartoon representation of the interaction interface between the PAS12-scFv and the viral capsid of the Asia1/JS/05 strain within a single protomer. The heavy and light chains of PAS12 are colored cyan and purple, respectively. The viral capsid proteins VP1 to VP4 are colored blue, green, orange, and yellow, respectively. (B-D) Expanded views of the PAS12-FMDV interface highlighting the VP2 B-C and H-I loops (B), the VP3 βΒ strand (C) and VP3 B-B knob (D). Presumptive hydrogen bonds and salt bridges are marked by black and orange dashed lines, respectively. (E) Binding affinity of PAS12 to the inactivated 146S antigen (FMDV Asia1/JS/05 strain) determined by BLI. (F) Neutralization potency of PAS12 against the wild-type (WT, Asia1/JS/05 strain) and its single-substitution mutants, evaluated using a microneutralization assay. The neutralization concentration represented the minimum antibody concentration required to completely prevent CPE. The experiments were independently conducted in triplicate. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s multiple-comparisons test with a 95% confidence interval in GraphPad Prism 8.0. *, **, and *** indicate significant difference compared with WT at P < 0.05, P < 0.01, P < 0.001, respectively. ns indicates no significant difference. (G) Sequence conservation analysis of the VP2 B-C loop (residues 70-76) and VP3 B-B knob (residues 56-62) among available FMDV Asia1 strains (VP2 sequences = 209; VP3 sequences = 210).
Fig 7.
Identification of immunodominant antigenic sites on FMDV serotype Asia1.
(A) Footprints of four distinct antigenic sites on FMDV serotype Asia1 identified by pnAbs. The capsid proteins VP1, VP2, and VP3 are colored blue, green, and orange, respectively. The residues of the “RGDL” motif in the VP1 G-H loop are marked in pink, and the key determinants 206 and 209 in the VP1 C-terminus are shown in magenta. The common residues recognized by PAS5 and PAS12 are shown in red. The rest of PAS5-interacting residues on VP2 are marked in purple, including three key residues 68, 72 and 77. The rest of PAS12-interacting residues are marked in cyan, including residues 73 on VP2 and 59 on VP3. (B) Characterization of pnAbs targeting four distinct antigenic sites on FMDV serotype Asia1. (C) Structural modeling of steric hindrance between PAS5 and PAS12 bound to FMDV Asia1/JS/05. VP1, VP2, VP3, and VP4 are colored blue, green, orange, and yellow, respectively. PAS5 and PAS12 are colored in purple and cyan, respectively. Black dashed circles indicate significant clashes between PAS5 and PAS12. (D) Schematic diagram of the immunization and sampling schedule for 10 pigs immunized with the inactivated Asia1/JS/05 vaccine. Created in BioRender. Chen, Y. (2026) https://BioRender.com/vathqjk. (E) Dynamics of neutralizing antibody titers against the Asia1/JS/05 strain in serum samples collected at different time points post-vaccination. (F-G) Competitive ELISA detection of antibody titers against different antigenic sites in serum samples collected at 28 dpi (F) and 56 dpi (G) from the 10 pigs. Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s multiple-comparison test with a 95% confidence interval in GraphPad Prism 8.0. ns indicates no significant difference. *, **, *** indicate significant difference at P < 0.05, P < 0.01, P < 0.001, respectively.