Adaptive Evolution of Serotype O Foot‐and‐Mouth Disease Virus Under Vaccine Pressure: Combined VP1 T142/Q153 Mutations Drive Antigenic Alteration and Immune Evasion

Figures

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  Figure 1. Sequence variations in the G–H loop domain of VP1 protein among representative strains of SEA and ME‐SA topotypes of serotype O FMDV, arranged chronologically by their dates of isolation and identification. This study specifically analyzed amino acid variations at positions 142 and 153 of the VP1 protein in ME‐SA and SEA subtype strains of serotype O FMDV, with a focus on their temporal correlation with isolation dates. Three evolutionary phases: 1980–2010: VP1 position 142 was dominated by threonine (T), and position 153 by glutamine (Q). 2010–2013: All strains exhibited a T → P substitution at position 142, while only a small proportion of strains carried a Q → P substitution at position 153. 2014–2019: The T → P substitution at position 142 became fully fixed, and the Q → P substitution at position 153 was uniformly established across all strains. The key determinants identified at positions 142 and 153 are highlighted in green (T), blue (P), and red (Q) respectively.

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  Figure 2. Spatial localization and conformational change prediction of key mutation sites in VP1 G–H loop. (A) Spatial distribution of all structural proteins in FMDV. (B) Spatial distribution of the VP1 protein on FMDV virions. (C) Spatial localization and conformational change prediction of T142P&Q153P mutations in FMDV VP1 structural protein.

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  Figure 3. Construction, expression and purification of self‐assembling nanoparticle proteins based on amino acid site mutants of FMDV 3D protein T‐cell epitopes and VP1 G–H loop B‐cell epitope. (A) Schematic diagram of the construction of 3D‐LS‐LOOP mutant self‐assembling nanoparticle protein. (B) SDS‐PAGE and western blot analysis of 3D‐LS‐LOOP (Wt) nanoparticle proteins. (C) SDS‐PAGE and western blot analysis of 3D‐LS‐LOOP (T142P) nanoparticle protein. (D) SDS‐PAGE and western blot analysis of 3D‐LS‐LOOP (Q153P) nanoparticle protein. (E) SDS‐PAGE and western blot analysis of 3D‐LS‐LOOP (T142P&Q153P) nanoparticle protein. SDS‐PAGE analysis was performed by Coomassie brilliant blue staining (left); western blot analysis was performed using a FMDV mAb (pO18‐39) as the primary antibody and horseradish peroxidase‐labeled goat anti‐mouse antibody as the secondary antibody (right). 3D: T‐cell epitope of FMDV 3D protein; LS: lumazine synthase; LOOP: B‐cell epitope of FMDV VP1 G–H loop protein. The 3D‐LS‐LOOP nanoparticle protein can self‐assemble into 60‐mer polymers in vitro.

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  Figure 4. Analysis of virological characteristics of FMDV O/MYA/01/1998 (Wt) strain following key amino acid substitutions in the VP1 G–H loop (T142P, Q153P, and T142P&Q153P). (A) Analysis of Sanger sequencing chromatograms for the VP1 G–H loop of Wt and rescued mutant viruses. (B) One‐step growth kinetics analysis of Wt and rescued mutant strains BHK‐21 monolayer cells were seeded in 24‐well plates and infected with Wt and mutant strains at a multiplicity of infection (MOI) of 1.0, respectively. Samples were collected at 0, 4, 8, 12, 16, 20, and 24 h postinfection, and the average log10 titers at each time point were determined by the TCID50 assay. (C) Plaque assay to determine the viral titers (PFU/mL) of Wt and mutant strains. (D) Plaque phenotypes of Wt and mutant strains in BHK‐21 cells at 48 h postinfection (h.p.i.)., with their sizes correlated to cytopathic effect (CPE) patterns. (E) Indirect Immunofluorescence Assay (IFA) identification and analysis of Wt and rescued mutant strains. BHK‐21 cells were infected with viruses (including Wt and all mutant strains) at an MOI of 1.0. At 10 h postinfection, the cells were assayed using a porcine anti‐VP1 monoclonal antibody (pO18‐39) as…

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  Figure 5. Evaluation of serum neutralizing antibody‐mediated cross‐protection against prevalent FMDV O/MYA/01/1998 (AKH05287.1) and FMDV O/TZ/CHA/2010 (AEC48727.1) strain strain of serotype O FMDV after immunization of C57BL/6 mice with 3D‐LS‐LOOP nanoparticle vaccines based on Wt and double‐mutant strain amino acid sites in the FMDV VP1 G–H loop. (A) Schematic diagram of immunization protocol and dosage for C57BL/6 mice. C57BL/6 mice were prime/boost‐vaccinated with vaccines at 0 day and 21 days. Blood was collected at 0, 14, 28, 35, and 42 dpi. All mice were euthanized at 47 dpi. (B) Determination of nAb titers against the Wt strain (FMDV O/MYA/01/1998) in the serum of each immunized mouse at specified time intervals. (C) Determination of nAb titers against the double‐mutant strain (FMDV O/TZ/CHA/2010) in the serum of each immunized mouse at specified time intervals. (D–F) Detection of FMDV‐specific IgG, IgG1 and IgG2a antibodies levels at different time points by Indirect ELISA. (G) The ratio of FMDV‐specific IgG1 and IgG2a antibodies titers at 42 dpi by Indirect ELISA. Data in subparts B–G are presented as mean ± SD. Statistical differences were analyzed via one‐way ANOVA followed by…

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  Figure 6. Evaluation of cross‐protection mediated by serum neutralizing antibodies against foot‐and‐mouth disease virus VP1 G–H loop isogenic mutant rescued strains after immunization of C57BL/6 mice with 3D‐LS‐LOOP nanoparticle vaccines based on VP1 G–H loop amino acid site mutations (T142P&Q153P). (A) Schematic diagram of immunization protocol and dosage for C57BL/6 mice. C57BL/6 mice were prime/boost‐vaccinated with vaccines at 0 day and 21 days. Blood was collected at 0, 14, 28, 35, and 42 dpi. All mice were euthanized at 47 dpi. (B) Determination of neutralizing antibody (nAb) titers against the FMDV (Wt) strain in the serum of each immunized mouse at specified time intervals. (C) Determination of nAb titers against the FMDV single mutant (T142P) strain in the serum of immunized mice at specified time intervals. (D) Determination of nAb titers against the FMDV single mutant (Q153P) strain in the serum of each immunized mouse at specified time intervals. (E) Determination of nAb titers against the FMDV double mutant (T142P&Q153P) strain in the serum of each immunized mouse at specified time intervals. (F) The cross‐protective efficacy of the 3D‐LS‐LOOP nanoparticle vaccines based on…

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  Figure 7. Evaluation of cross‐protective efficacy of neutralizing antibodies against FMDV rescue strains with different amino acid site mutations in the VP1 protein G–H loop with multiple FMDV vaccines. (A) Schematic diagram of immunization protocol and dosage for swine. The primary/booster immunization were conducted respectively on Day 0 and Day 21. Blood samples were only collected at 0 and 40 dpi. All swine were euthanized at 42 dpi. (B–F) Evaluation of cross‐protective efficacy of neutralizing antibodies against FMDV rescue strains with different amino acid site mutations in the VP1 protein G–H loop at 40 dpi of swine with multiple FMDV commercial inactivated vaccines [(B‐D) FMDV serotypes O/A commercial inactivated vaccine A‐C] and FMDV nanoparticle vaccines [(E) 3D‐LS‐LOOP (T142P&Q153P) and (F) 3D‐LS‐LOOP (Wt)]. (G) The cross‐protective efficacy of the multiple FMDV vaccines in inducing swine serum NAbs titers against prevalent FMDV Wt strain (GenBank: AEC48727.1 and AFE84737.1) and FMDV rescue strains. Data in subparts B–F are presented as mean ± SD. Statistical differences were analyzed via one‐way ANOVA followed by Tukey’s multiple comparisons test. All experiments were performed…