Single dose of foot-and-mouth disease peptide vaccine fully protects swine and achieves intraserotype crossed neutralization

Fig. 1. Click chemistry synthesis of B2T-TB2. General scheme of the chemoselective click approach used for the synthesis of a B2T-TB2 multiepitopic peptide platform.

Fig. 2. Antibody response elicited by dendrimer peptides. Two groups of 5 animals each were immunized on day 0 with B2T-TB2 (orange circles) or B2T (green circles) and challenged at 25 dpi (down-pointing arrows). A ELISA antibody response in sera from 15, 25, and 36 dpi. B Neutralizing antibody responses at 15, 25, and 36 dpi, determined as detailed in “Methods”. Dotted line indicates the detection limit. Numbers next to each circle indicate pig number. Horizontal lines indicate the geometric mean for each animal group. No spontaneous reactivity was observed in titers at day 0. Statistically significant differences are indicated by asterisks (*) for p < 0.05.

Fig. 3. Cross-neutralizing responses and antigenic differences in FMDV. Top panel shows cross-neutralizing activity against O- and C-type FMDV isolates in sera at 25 dpi. VNTs represent the geometric mean for each animal group immunized with the indicated peptide. Dotted line indicates detection limit. No reactivity observed in VNTs at day 0. Statistically significant differences are indicated by asterisks (*) for p < 0.05, (**) for p < 0.005 and (***) for p < 0.0005. Bottom panel depicts differences (highlighted blue) in B-cell antigenic site residues [VP1 (140–158) for O-type; VP1 (136–154) for C-type C-S8c1] among tested FMDV isolates and the standard O/UK/11/2001 used for challenge.

Fig. 4. Specific T-cell responses to B2T-TB2 and related peptides. Specific T-cell responses measured by an ex vivo IFN-γ ELISPOT at 15 (A) and 25 dpi (B). IFN-γ release by PBMCs stimulated in vitro with either B2T-TB2, B2T, or with single T or B peptide epitopes. The frequency of FMDV-specific IFN-γ secreting cells was determined as detailed in Methods. The box-and-whisker plot shows each individual value as a dot, from the minimum up to the maximum value. The box extends from 25 to 75th percentiles where the dotted line represents the median. The numbers next to each symbol indicate the pig number, as in Table 1. Statistically significant differences are indicated by asterisks (*) for p < 0.05.

Fig. 5. CD4⁺ T-cell cytokine responses. IFN-γ- and TNF-α-producing CD4+ T-cell subsets from immunized pigs upon stimulation with homologous peptide, analyzed by intracellular cytokine staining (ICS) at 25 dpi. A Surface CD4 staining and expression was analyzed (representative plot of pig #1 is shown). B CD4+ T-cells were gated and analyzed for production of a single cytokine (IFN-γ or TNF-α). PBS-inoculated animals (#11 and #12) were stimulated with PHA, thus serving as positive controls. The numbers shown within the plots indicate cell percentages relative to the CD4+ T-cell population. C Percentages of CD4+ T-cell populations from each pig, expressing a single cytokine (IFN-γ or TNF-α) or both.