Table 1.
Antigens selected for evaluation of immunogenicity.
Fig 1.
Protein expression by ASFV constructs.
The expression and authenticity of the ASFV antigens encoded by the generated recombinant constructs was evaluated by immunocytochemistry and western blot analysis. Panels: A) HEK-293A cells infected with recombinant adenoviruses and probed with anti-HA mAb; and B) HEK-293A cells infected with recombinant adenoviruses and probed with gamma-irradiated ASFV-specific convalescent serum. Negative controls are mock infected HEK-293A cells. C) A western blot of the affinity purified ASFV proteins probed with the convalescent serum. The molecular weights are expressed in kDa. The arrow points to the faint band detected for B438L (the signal intensity of the band increased with longer exposure times).
Fig 2.
Mean antigen-specific IgG responses post-priming and post-boost.
Antigen-specific IgG response was evaluated post-prime and post-boost by ELISA. A) Sera from week 4 post-prime were evaluated at a 1:100 dilution. B) Sera from week 2 post-boost were evaluated at a 1:8,000 dilution (to prevent the absorbance values from going out of range). The error bars represent the SEM. The asterisks denote a significant difference between the mean response of the treatment and control animals. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Antigen-specific end-point IgG titers.
Antigen specific antibody titers, determined by ELISA, in sera from treatment group animals (T) collected two weeks post-boost. The dilution of the sera at which the absorbance reading was higher than that of the cognate pre-bleed +3 standard deviations is reported as the end-point titer. The ASFV-specific convalescent serum was titrated similarly and is represented by the red star symbol (S). Data is represented as the reciprocal of the end-point sera dilution x 106. For antigen, B119L the sera from control group animals was also titrated to gauge background reactivity to host-cell and vector-derived antigens. An average of the titers of the control group animals was then subtracted from the titer of each treatment group animal to give B119L-specific titers. For the remaining antigens, the post-boost sera from the control group animals showed no reactivity as seen in Fig 2B.
Fig 4.
Antibodies primed by the Ad-ASFV cocktail recognized ASF virus.
Analysis of sera from two weeks post-boost by Indirect Fluorescence Antibody assay (IFA) and western blot showed that the antibodies primed by the Ad-ASFV cocktail recognized the parental ASFV infected cells and ASFV-derived antigens. Panel A) Vero cells infected with ASFV Georgia 2007/1 probed with sera from treatment and negative control animals. ASFV specific convalescent serum was used as the positive control and normal swine serum served as the negative control. Data for three animals (that gave the strongest reaction) from the treatment group and one animal from the control group is shown. A summary of IFA results for all animals is presented in Table 2. B) Lysates from Vero cells infected with ASFV Georgia 2007/1 isolate were blotted and probed with sera from all animals. Normal swine serum was the negative control and ASFV-specific convalescent serum was the positive control. Differences in coloration are due to actual band intensities; darker color is higher concentration of antibody bound to antigen (antigen concentration is constant).
Table 2.
Summary of IFA results.
Fig 5.
ASFV antigen-specific IFN-γ response post-prime and post-boost.
The frequency of antigen-specific IFN-γ-secreting cells in PBMCs collected post-prime and post-boost was evaluated by IFN-γ ELISPOT assays. Data for A) One week post-prime; and B) three weeks post-boost is shown. The response is presented as IFN-γ Spot Forming Cells (SFC)/106 PBMCs. The mean response of the treatment group (T) was compared to the mean response of the control group (C) using an unpaired t-test with Welch’s correction. ** represents p<0.01, * represents p<0.05 and ‘ns’ stands for a non-significant difference.