Fig 1.
Titres of the CCHF virus Turkey-Kelkit06 strain, weight loss curves, body temperatures changes and survival of IFNAR−/−mice.
Titres of the CCHF virus Turkey-Kelkit06 strain recovered in the blood (A), liver (B) and spleen (C) after intraperitoneal infection with 101, 102, 103 or 104 PPFUs of CCHF virus Turkey-Kelkit06 strain. All animals were sacrificed at 2 days post-infection (dpi). The virus was extracted from the blood and indicated organs, and the virus titres were determined by pseudo plaque assay (PPA). Each point represents the mean values of the viral titre of six animals, and the standard deviations are shown as error bars. The IFNAR−/−mice (n = 6 each) were infected with 2.5, 5, 101, 102, 103 or 104 PPFUs of CCHF virus Turkey-Kelkit06 strain. The mice were monitored twice daily for mean weight change (D), body temperature (E), and survival (F). The median lethal dose was calculated by logistical regression. The standard deviations are shown as error bars.
Fig 2.
Confirmation of the antigenicity of the cell culture based vaccine by SDS-PAGE and western blotting.
The unpurified vaccine antigens assessed before sucrose gradient ultracentrifugation and the purified vaccine antigens obtained after the purification and inactivation step were subjected to SDS-PAGE analysis shown in Fig. 2A and 2B, respectively. The purified vaccine samples were immunoblotted with 1:2000 dilution of the hyperimmune serum grown in rabbit against the CCHF virus and followed by goat anti-rabbit AP conjugated antibody to visualize the antigenic bands. (Fig. 2C). The cell culture based vaccine antigens were probed with anti-Gc (Fig. 2D) or anti-NP (Fig. 2E) monoclonal antibodies of the CCHF IbAr10200 virus.
Fig 3.
Humoral immune responses to the cell culture based vaccine against CCHF in IFNAR−/− mice.
Groups of 6 IFNAR−/− mice were immunized three times at three weeks intervals with 5, 20, or 40 μg of the cell culture based vaccine. The sera obtain from the mice at 14, 35, and 56 days were assayed for anti-CCHFV antibody end point titre (A) and neutralizing antibody titres determined by a 50% pseudo plaque reduction neutralization assay (PPRNT) (B). The error bars indicate the standard deviation. Differences in the ELISA titres and virus neutralization titres between groups were analyzed by 2-way ANOVA followed by Bonferroni’s post test, which is denoted by asterisks. *** (p<0.001).
Table 1.
Antibody responses, morbidity and mortality in IFNAR−/− mice intraperiteonally immunized three times at three weeks intervals with 5, 20, or 40 μg of the cell culture based vaccine.
Fig 4.
Protection of IFNAR−/− mice immunized with the cell culture based vaccine against CCHF virus Turkey-Kelkit06 strain challenge.
Groups of 6 IFNAR−/− mice were immunized three times at three weeks intervals with 5, 20, or 40 μg of the cell culture based vaccine. The control group of IFNAR−/− mice (n = 6) was mock immunized with phosphate buffered solution (PBS). All animals were challenged with 1,000 PPFU (400 LD50) of CCHF virus Turkey- Kelkit06 strain two weeks after the last immunization. The mice were monitored twice daily for the cumulative mean symptom scores (A), the daily variations in weight as percentages compared to before the virus challenge (B), body temperature (C), and geometric mean time to death and survival (D). The animals were monitored for three weeks after the challenge. The standard deviations are shown as error bars.
Fig 5.
Determination of virus titres of the vaccinated animals.
Groups of 3 IFNAR−/− mice were immunized three times at three weeks intervals with 5, 20, or 40 μg of the cell culture based vaccine. The control group of IFNAR−/− mice (n = 3) was mock immunized with phosphate buffered solution (PBS). All animals were challenged with 1,000 PPFU (400 LD50) of CCHF virus Turkey-Kelkit06 strain two weeks after the last immunization. All animals were sacrificed at 2 days post-infection. Virus was isolated from the blood (A), liver (B) and spleen (C) as described in the Materials and Methods. Each point represents the mean values of the viral titre of three animals, and the standard deviations are shown as error bars.