The FlagT4G Vaccine Confers a Strong and Regulated Immunity and Early Virological Protection against Classical Swine Fever

Figures

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  Figure 1. Clinical signs and rectal temperature in pigs during the trial. Individual rectal temperature (A) was recorded in FlagT4G-vaccinated (blue lines and symbols) and unvaccinated (red lines and symbols) pigs throughout the trial. Values above 40 °C (dotted line) were considered as fever. (B) A numeric clinical score value (represented in colorimetric scale) was assigned for each animal after CSFV challenge. Cross symbol indicates an animal that died during monitoring, despite not showing clinical signs.

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  Figure 2. Antibody response after CSFV vaccination and challenge. Sera samples from FlagT4G-vaccinated (blue lines and symbols) and unvaccinated (red lines and symbols) animals were collected and evaluated for antibodies against the E2 (A) and Erns (B) glycoproteins. For the E2 ELISA test, results are expressed as blocking percentage, and values above 40% (red-shaded area) were considered positive. In the case of anti-Erns antibodies, results were expressed as the sample/positive value with samples above 0.5 (green shaded area) determined to be positive. * Euthanized for animal welfare reasons.

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  Figure 3. Cytokine response after CSFV vaccination and challenge. (A) IFN-α concentration in sera samples was evaluated after vaccination and CSFV challenge, using an in-house ELISA test. Individual results for each sampling are represented as the mean value (in units/mL) of duplicates performed for each sample. (B) Sera samples from vaccinated (blue boxes) and unvaccinated (red boxes) animals were also evaluated by Luminex assay for the detection of nine different cytokines. Mean values for each group at every sampling time are represented and cytokine concentrations are shown as pg/mL. Asterisk indicates statistically significant differences between groups on that particular sampling (Kruskal–Wallis non-parametric test; p < 0.05). Differences between a given sampling and the previous one within the same group (Friedman test) are represented by the letters on top of the bar: different letters show statistical difference (p < 0.05). Not represented: IFN-γ concentration in sera (not detected in any sample).

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  Figure 4. Detection of viral RNA by RT-qPCR. Samples from vaccinated and unvaccinated pigs were evaluated by the pan-CSFV (A) and the specific Margarita strain (B) RT-qPCR assays. Results are shown in a colorimetric scale representing the Ct value in green and red for the pan-CSFV and Margarita tests, respectively.

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  Figure 5. IFN-γ response in FlagT4G-vaccinated pigs. PBMCs from all the experimentally infected animals were stimulated by duplicate with the FlagT4G (green bars) and Margarita (Orange bars) viruses and evaluated by ELISPOT assay. Results are represented as the mean value of spots/106 cells for each stimulation at the respective time points. Unvaccinated and challenged animals remained unresponsive to either of the viral stimuli. Differences between a given sampling and the previous one within FlagT4G-vaccinated pigs (Friedman test) are represented by different letters (p < 0.05).

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  Figure 6. Immunological phenotypic profile after CSFV vaccination and challenge. Myelomonocytic and lymphocytic cell surface markers were evaluated in PBMC from vaccinated (blue boxes) and unvaccinated (red boxes) pigs. Mean values and cell subset percentages for each group at every sampling time are represented. Flow cytometry assays were carried out in duplicates. Asterisk indicates statistically significant differences between groups on that particular sampling (Kruskal–Wallis non-parametric test; p < 0.05). Differences between a given sampling and the previous one within the same group (Friedman test) are represented by the letters on top of the bar: different letters show statistical difference (p < 0.05).