Assessment on Different Vaccine Formulation Parameters in the Protection against Heterologous Challenge with FMDV in Cattle

Figures

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  Figure 1. Progression of foot-and-mouth disease (FMD) clinical signs after heterologous challenge. Each line depicts the clinical scores registered for individual animals after infection with FMD virus (FMDV) A/Arg/01. The score was determined by assigning 1 point for hyperthermia > 40.0 °C and 1 point for each limb with vesicular lesions, reaching a maximum daily score of 5 points. Mild hyperthermia (≥39.5 °C and ≤40.0 °C) in the absence of other FMD-related signs was scored as 0.5 points.

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  Figure 2. Total FMDV-specific antibodies induced in each experimental group at 30 days post-primary vaccination (dpv). Total anti-FMDV antibody titers measured by liquid-phase blocking ELISA (LPBE) against the A24/Cruzeiro, A/Arg/01, C3/Indaial, and O1/Campos FMDV strains. Results are expressed as the log10 of the mean LPBE titer in each experimental group at 30 dpv before heterologous challenge. Bars represent mean values of each experimental group ± SD. Dotted and black lines depict the titers corresponding to the expected percentage of protection ≥ 75% (EPP75) for A24/Cruzeiro (1.90) and A/Arg/01 (2.20) strains, respectively. Asterisks denote statistical differences between experimental groups (ANOVA) * p ≤ 0.05; *** p ≤ 0.001.

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  Figure 3. Neutralizing antibodies against FMDV A24/Cruzeiro and A/Arg/01 strains at 30 dpv. Serum neutralizing antibodies against FMDV A24/Cruzeiro (a) and A/Arg/01 (b) strains were measured by virus neutralization test (VNT) for all vaccinated animals. Results represent the mean of four independent experiments and are expressed as the log10 of the resulting mean titer in each experimental group at 30 dpv. Bars represent the mean value of each experimental group ± SD. Dotted lines depict VNT titers corresponding to the EPP75 for A24/Cruzeiro strain (1.35) and A/Arg/01 strain (1.43) in the corresponding charts. Asterisks denote statistical differences between experimental groups (ANOVA) * p ≤ 0.05 and ** p ≤ 0.01.

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  Figure 4. Total and neutralizing antibody responses against FMDV A/Arg/01 strain in protected and non-protected animals against the heterologous challenge at 30 dpv. Total FMDV-specific antibodies were measure by LPBE (a) and neutralizing antibodies were measure by VNT (b). Results are expressed as the log10 of the mean titers against heterologous strain, A/Arg/01. Dotted lines depict the titers corresponding to the EPP75 for A/Arg/01 strain for each serological assay. Bars represent mean value ± SD.

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  Figure 5. FMDV-specific IgG isotype responses in vaccinated animals at 30 dpv. (a–d) FMDV-specific IgG1 and IgG2 measured by ELISA for each experimental group against A24/Cruzeiro (a,c) and A/Arg/01 (b,d) strains. Results are expressed as the log10 of the mean titer for each vaccine group at 30 dpv. (e,f) IgG isotype responses in animals protected and non-protected to the heterologous challenge. Results are expressed as the log10 of the mean titers for IgG1 (e) or IgG2 (f) against the A/Arg/01 strain. (g) Ratio between mean IgG1 and IgG2 titers obtained by ELISA for protected and non-protected steers against the A/Arg/01 strain. (h,i) IgG isotype responses against FMDV A/Arg/01 strain according to the antigenic payload of the formulations. Animals were grouped as 10 μg/dose (including the A24 10 µg group) or 40 μg/dose (including the A24/C3I/O1C and A24 40 µg groups). FMDV-specific IgG1 (h), and IgG2 (i) antibodies against the A/Arg/01 strain at 30 dpv were measured by ELISA, expressed as the log10 of the mean titers. For all panels, bars represent mean values of each group ± SD. Asterisks denote statistical differences between experimental groups * p ≤ 0.05 (Mann–Whitney test) and ** p ≤…

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  Figure 6. Avidity of the IgG antibodies against the FMDV A/Arg/01 at 30 dpv. The avidity of the FMDV-specific IgG antibody responses against the A/Arg/01 strain was measured by Avidity ELISA. (a) Avidity indexes (AI) for all vaccinated animals following an increasing order. Non-protected animals are indicated as clear shaded bars. (b) Mean AI in animals protected and non-protected against heterologous challenge at 30 dpv. Bars represent the mean value ± SD. Statistical differences were determined by Mann–Whitney test.

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  Figure 7. Mean avidity of the FMDV-specific IgG antibodies against the A24/Cruzeiro and A/Arg/01 strains at 30 dpv. The avidity for FMDV-specific whole IgG (a), IgG1 (b), and IgG2 (c) antibodies were measured by avidity ELISA against A24/Cruzeiro (light bars) and A/Arg/01 strains (dark bars) for experimental group. Results are expressed as the AI and bars represent the mean AI ± SD. Values in parentheses represent the corresponding ELISA mean antibody titers expressed as log10 for each experimental group. Statistical differences were determined by a paired t-test.

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  Figure 8. Mean avidity of the FMDV-specific total IgG antibodies against the A24/Cruzeiro and O1/Campos strains at 30 dpv. Animals were grouped according to the results in the in vivo FMDV A/Arg/01 challenge at 30 dpv. Mean AI of the FMDV-specific IgG antibody responses against the A24/Cruzeiro (a) and O1/Campos (b) strains were measured by Avidity ELISA. Results are expressed as the mean AI and bars represent the mean value ± SD. Significant statistical differences between protected and non-protected steers are indicated by the corresponding p values (Mann–Whitney test).

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  Figure 9. Correlation of avidity indexes against different FMDV strains in serum samples from vaccinated animals at 30 dpv. The AI of the FMDV-specific IgG antibodies measured against the A24/Cruzeiro, A/Arg/01 and O1/Campos strains in each vaccinated animal were analyzed by pairs using a Pearson’s correlation test, as indicated in each panel. White circles denote non-protected animals (#84, #104, #195), and the corresponding Pearson’s r values are shown in each graph (p value < 0.0001).