Glycosylated Foot‐And‐Mouth Disease Virus‐Like Particles Produced in Pichia Pastoris Enhance Stability and Immunogenicity

Figures

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  FIGURE 1. Schematic of the pPink‐SPVP0, pPink‐GHH and screening of signal peptides for VP0. (A) Schematic representation of the plasmids employed in this study. (B) SDS‐PAGE analysis of background proteins in untransformed yeast. Lane M: Marker, Lane 1–3: Untransformed strains. (C) SDS‐PAGE and WB analysis of the expression levels of VP0 mediated by different signal peptides. The pPink‐GHH was constructed with a His6 inserted between the 136th and 137th of VP1, and (GGGGS)2 linker sequences added on both ends of His6. (D) Relative expression level analysis of VP0. The relative secretion levels were quantified using ImageJ. Data are presented as mean ± standard deviation of 3 biological replicates.

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  FIGURE 2. Secretory and intracellular expression analysis of FMDV structural proteins in P. pastoris. SDS‐PAGE analysis of VP3 (A) and VP1 (B) secretory expression. (C) SDS‐PAGE and WB analysis of glycosylated VP1. Glyco‐VP1: Glycosylated VP1. (D) SDS‐PAGE analysis of glycosylated VP1 treated with PNGase F. (E) The position of His6 in the 5S and 12S after being inserted at residue 136 of VP1. The images were generated using PyMOL, PDB: 7enp. The insert position is blue, VP0 is magenta, VP1 is green, VP3 is yellow. (F) Purification and characterisation of VLPs derived from pPink‐GHH. Lane M: Marker, Lane 1: Supernatant after disruption, Lane 2: Flow‐through fraction, Lane 3: 20 mM imidazole eluent, Lane 4: 300 mM imidazole eluent, and Lane 5: Ultrafiltration concentrates. (G) WB analysis of VLPs derived from pPink‐GHH.

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  FIGURE 3. Assembly and Characterisation of WT VLPs and glycosylated VLPs. (A) TEM observation of VLPs. Scale bar shows 100 nm. (B) Analysis of VLPs by DLS. (C) Statistical data of the VLPs' size. The size was measured using Nano Measurer 1.2 and statistically analysed using GraphPad Prism 8. (D) Evaluation of VLPs assembly efficiency. (E) Analysis of VLPs stability by ELISA. VLPs with different amino acid changes in VP1 were incubated to assess stability and the intact VLPs were quantified by ELISA. (F) The location of G166T amino acid change formed the N‐glycosylation motif ‘FNYT’ within the 5S.

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  FIGURE 4. N‐glycan species analysis of the G166T VP1. (A) Ion chromatogram of N‐glycans released from G166T after PNGase F treatment. The ion peaks of the 18 glycans identified are labeled. (B) Structure and types of the 18 N‐glycans identified. A, Branch on the core structure of tri‐mannose; F, Fucose; G, Galactose (Gal); Glc, Glucose; M, Mannose (Man); S, Sialic acid. (C) Classification of these 18 N‐glycans identified in G166T VP1.

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  FIGURE 5. Uptake of VLPs by DCs. WT VLPs, L51T VLPs, P160N VLPs, and G166T VLPs were incubated in DCs for 1 h (A), 2 h (B), and 6 h (C), after which IFA was conducted using anti‐FMDV polyclonal antibody and FITC‐labelled goat anti‐pig IgG as the secondary antibody. The cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI), and the DAPI and FITC fluorescence images were integrated into a merged image. The control group was incubated with PBS, the WT group was incubated with WT VLPs, the L51T group was incubated with L51T VLPs, the P160N group was incubated with P160N VLPs, and the G166T group was incubated with G166T VLPs, 1 bar = 100 μm. Representative data shown are mean ± SD from one of three independent experiments (*p < 0.05).

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  FIGURE 6. Evaluation of the immune response of glycosylated VLPs in mice. Blood samples were collected at 14, 28 and 42 dpi for antibody detection. Specific antibody levels were quantified by sandwich ELISA at 14 dpi (A), 28 dpi (B) and 42 dpi (C). (D) Neutralising antibody levels in serum of mice at 28 dpi (measured by microplate neutralising assay). (E) Detection of lymphocyte proliferation levels in mice at 28 dpi. (F) Detection of IFN‐γ levels at 28 dpi using ELISA kits. The statistical significance of differences was evaluated (*p < 0.05, **p < 0.01). Groups nomenclature are as follows: PBS: Negative control; PC, Positive control; L51T: L51T VLPs; P160N: P160N VLPs; G166T: G166T VLPs.

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  FIGURE 7. Evaluation of the immune effect of glycosylated VLPs in pigs. (A) The level of specific antibodies against FMDV was detected by sandwich ELISA in pigs. (B) The level of neutralising antibodies was detected by microplate neutralising assay in pigs at 28 dpi. PBMCs were isolated at 28 dpi, and CD4+ and CD8+ T cells in PBMCs were detected using flow cytometry (C and D). (E) The levels of IFN‐γ, TNF‐α, IL‐4 and IL‐1β induced by VLPs in serum samples from pigs at 28 dpi were detected using ELISA kits. The error bars indicate the standard deviation. Significant differences were calculated (*p < 0.05, **p < 0.01, ***p < 0.001). (F) Four weeks after immunisation, pigs were challenged and the protection was monitored.