The High Immunity Induced by the Virus-Like Particles of Foot-and-Mouth Disease Virus Serotype O

Figures

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  Figure 1. Identification of FMDV capsid protein expression, purification and SUMO cleavage. (A) Analysis of the FMDV capsid protein expression in E. coli with different permutation after purification with Ni2+ resin. M: protein marker; lanes 1–6: the recombinant proteins of Mya98-VP103, -VP130, -VP013, -VP031, -VP301, and -VP310. (B) SDS-PAGE analysis of Mya98-VP031 after SUMO removal. Lane 1: the protein treated with Ulp 1 protease; lane 2: the proteins (from the sample of line 1) purified by Ni2+ resin.

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  Figure 2. Characterization of FMDV VLPs. (A) Protein purification by gel filtration chromatography. (B) SDS-PAGE analysis of peak a, and peak b proteins obtained from gel filtration chromatography. Lane 1: peak a; lane 2: peak b. (C) DLS analysis of the size distribution of proteins in peaks a and b. (D) Protein visualization of peak a by TEM, the red arrow indicating the VLPs. (E) Protein visualization of peak B by TEM; the black arrow indicated the unassembled proteins. Magnification of 42,000×, the scale bar indicates 50 nm.

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  Figure 3. Analysis of FMDV VLPs by gel filtration chromatography. Lines in black, red, and blue represent the SUMO protease activity of 20, 10, and 5 units, respectively.

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  Figure 4. The solution competitive ELISA methods for quantitative antigenicity analysis of VLPs and unassembled proteins. The inhibition ability of mAbs F21-48, F21-64, F21-58, and F21-41 to VLPs (●) and unassembled proteins (■) were shown in (A–D), respectively.

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  Figure 5. FMDV-specific immune responses in pigs. FMD immune efficacy studies. Pigs (n = 5) in three groups were immunized with inactivated FMDV serotype O vaccine (●) or FMD VLPs of 25 (▼) and 100 (▲) μg/dose, and five PBS-inoculated pigs in a control group (■). Serum samples were collected preimmune and weekly up to 10 weeks postimmunization for FMDV-specific antibody response detected by LPBE. The LPBE titers of PBS-inoculated pigs were all lower than 1:8. Data are expressed as the mean ± standard deviation (SD). The one-way ANOVA test was applied to state the differences on antibody titers among the three groups in each time point after immunization.

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  Figure 6. ELISpot assays of FMDV serotype O–specific IFN-γ after immunization with VLPs. (A) Representative IFN-γ ELISpot wells for the PBS, inactivated serotype O vaccine, and FMDV VLPs (25 and 100 μg/dose). The numbers in the upper left corners indicate counted spots of IFN-γ forming cells per 3 × 105 responder PBMCs at 24 h after stimulation. (B) FMDV serotype O–specific IFN-γ of all immunized pigs; the concentrations of VLPs for stimulation were 0 μg/well (●), 1.0 μg/well (■), and 1.0 μg/well (▲). The PBMCs were collected at 28 dpi. Data are expressed as the mean ± standard deviation (SD). The one-way ANOVA test was applied to state the differences on IFN-positive cell frequency among the three groups (different concentrations of the VLPs during stimulation).