9. Author Contributions
JEB: Conceptualization; Methodology; Formal analysis; Investigation; Writing - Original Draft; Visualization
AM: Conceptualization; Methodology; Formal analysis; Resources; Writing - Original Draft
CK: Validation; Investigation
GL: Validation; Investigation, Resources;
MG: Validation; Investigation
IS: Validation; Formal analysis
RG: Validation; Investigation
CS: Resources, Writing Review & Editing
CL: Methodology; Formal analysis; Investigation; Writing - Original Draft
VQ: Methodology; Formal analysis; Investigation; Writing - Original Draft
YD: Conceptualization; Methodology, Resources;
WA: Conceptualization; Methodology; Writing - Review & Editing
IM: Conceptualization; Methodology; Writing - Review & Editing
PZ: Conceptualization; Methodology; Writing - Original Draft; Writing Review & Editing, Visualization; Supervision; Project Administration; Funding adquisition
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Figure 1 . Selection of VLPs dose for vaccination of BALB/c mice. Groups of mice (n=5) were vaccinated with 8, 4, 2, 1, 0.5, 0.3, 0.15 or 0 µg VLPs in PBS at 0 and 21 days and challenged with infective virus at 36 dpv. (A) Abs against FMDV elicited by vaccination with different amounts of VLPs measured by sandwich ELISA at 36 dpv. (B) Percentages of protected animals upon viral challenge. Animals were considered protected if viremia was absent at 24 h post challenge. Protection percentages were calculated as: 100 x [number of vaccinated animals without viremia / number of vaccinated animals].
Figure 2 . Protection upon viral challenge elicited by experimental vaccines. Groups of mice (n=5) were vaccinated with VLPs (0.5 µg), VLPs (0.5 µg)-ISPA, VLPs (0.5 µg)-CA or a Control (+) vaccine, and groups of mice (n=2) were vaccinated with ISPA, CA or PBS alone at 0 dpv and 21 dpv, and challenged with infective FMDV at 36 dpv. Protection was calculated as in Figure 1.
Figure 3 . Abs against FMDV elicited by experimental vaccines in mice. FMDV specific antibody titers were measured by (A) sandwich ELISA at 15, 21 and 36 dpv. Each point represents the mean (n=5) Ab titer ± SD in each group. * Significant differences against VLPs group. ** (p< 0.01); ***(p< 0.001). (B) Isotype profile of vaccinated animals at 36 dpv. Data are expressed as the mean Ab titer ± SD. *** p< 0.001, **(p< 0.01).
Figure 4 . Lymphocyte proliferative response after stimulation with iFMDV measured by CFSE loss, at 36 dpv. Results are expressed as the difference (Δ%) between the percentage of proliferating splenocytes stimulated with inactivated virus and the percentage of proliferating splenocytes without stimuli. Animals were vaccinated with VLPs, VLPs-ISPA, VLPs-CA, control (+) vaccine, ISPA, commercial adjuvant alone or PBS. *** p<0.001; *p< 0.05.
Figure 5 . Humoral response elicited in cattle by experimental vaccines. FMDV-specific antibody titers were measured by Lp ELISA. (A) Kinetics of total anti-FMDV serum Abs. Each bar represents the mean Ab titer ± SD (n= 4) at 15, 30 and 45 dpv. (B) FMDV-specific serum IgG1 and IgG2 response at 45 dpv. Each point represents the serum IgG1 or IgG2 anti-FMDV Ab titers (log10) of each animal. Isotype profiles at 45 dpv, expressed as mean Ab titers ± SD. ** p< 0.01, *p< 0.05.
Figure 6 . IFN-γ production by PBMC from calves inoculated with experimental vaccines at 45 dpv. Cells were stimulated in vitro with iFMDV. Supernatants were tested by ELISA. Each bar represents the IFNγ levels (pg/ml) secreted by PBMCs from each bovine when they were stimulated in vitro with iFMDV, *p< 0.05.
Supplementary Figure 1. Flow cytometry analysis of CD4+/IFNγ+ and CD8+/IFNγ+ cell percentages. At 36 dpv, splenocytes from vaccinated BALB/c were (i) not stimulated stimulated (mock) or stimulated with (ii) 2.5 μg/ml iFMDV or (iii) 5 μg/ml Concanavalin A as positive control. Cells were incubated for 18 h in the presence of brefeldin A. Permeated cells were incubated with anti-mouse INF-γ. Then, cells were stained with anti-mouse CD4 or anti-mouse CD8. Splenocytes were obtained from mice vaccinated with VLPs, VLPs-ISPA, VLPs-CA, Commercial vaccine, ISPA, CA alone or PBS. (A) Percentages of CD4+/IFNγ+ T cells and (B) percentages of CD8+/IFNγ+ T cells from mice spleens. ** p< 0.01; *p< 0.05.