Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold

Figures

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  Figure 1. Schematic of HBcAg presentation methods. (A) HBc-native VLP, (B) HBcN15: HBc VLP with the N-terminal 15 amino acids of FMDV VP4 (purple) inserted, allowing this sequence to be presented on the major immunodominant region. (C) HBcPepN15: HBc-native VLP decorated with VP4N15 peptides via a peptide tag with affinity for the HBc spike.

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  Figure 2. Formation of VP4N15 HBcAg VLPs (A) Purified HBc-C61A VLP, (B) HBcN15 VLP and (C) HBcPepN15 were visualised by negative stain TEM. The scale bars denote 100 nm. (D) HBc-Native and HBcN15 recombinant proteins were confirmed as having the expected molecular weights and reactivity by Western blot with anti-HBcAg antibody. The positions of molecular weight markers are shown to the left. Arrows on the right indicate the expected sizes of HBc protein. (E) HBc-native VLP only (native), VP4N15 peptide only (N15) or VLPs + VP4N15 peptide (N15 + native) before and after washing to remove free peptide. Samples were compared by Coomassie stained SDS-PAGE. The ladder and molecular weight are shown to the left. Arrows on the right indicate the expected sizes of HBc protein and the peptide (21 kDa and 4 kDa).

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  Figure 3. Immune responses to HBcAg VLPs displaying FMDV VP4N15. (A) Balb/c mice were immunised with 10 μg of immunogen and boosted over the course of 42 days and test bleeds were collected. ELISAs were used to detect antibodies against (B) HBc-native VLPs and (C) VP4N15 peptide in pooled terminal sera for each group. The data plotted represent the mean of triplicate wells ± SD and are representative of multiple experiments. (D) Peptide ELISAs were used to follow the VP4N15-specific immune response in the HBcN15 group over the course of the immunisations. Arrows indicate booster immunisations.

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  Figure 4. Anti-HBcN15 serum does not neutralise FMDV but does recognise virus particles (A) Plaque reduction neutralisation assays were carried out to assess the effect of anti-HBcN15 serum on virus infectivity. Data plotted represent mean of triplicate wells and error bars indicate standard deviation. (B) Sandwich ELISAs using bovine αvβ6 integrin as the capture were used to confirm that serum against HBcN15 recognised FMDV virus particles.