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. 2021 Jul 28;13(8):1480. doi: 10.3390/v13081480

Figure 7.

Figure 7

Monoclonal antibodies against ASFV CD2v inhibit CD2v-induced NF-κB activation and IFN-β transcription in swine PBMC cultures. (A,B) NF-κB-p65 nuclear translocation in PBMCs. Swine PBMCs were treated for 1.5 h with purified CD2v or purified control preincubated with the anti-CD2v monoclonal antibody mix, anti-ORFV086 antibody or isotype control antibody, and cells were processed for NF-κB-p65 staining as described in Materials and Methods. Control cells were treated with CD2v or the purified control that have not been preincubated with antibodies. (A) Representative confocal images of NF-κB-p65 nuclear translocation. Green, NF-κB-p65; Blue, DAPI. Arrows indicate nuclear NF-κB-p65. Insets show magnified areas of the field. (B) Approximately 2500 cells were counted/slide and results are shown as the mean values from three independent experiments (for % nuclear NF-κB-p65 for the anti-CD2v antibody mix relative to anti-ORFV086, p = 0.01; without preincubation, p = 0.03 and for the isotype control, p = 0.03). (C) IFN-β transcription in PBMCs. Swine PBMCs were treated as in A, total RNA harvested at 6 h pt and IFN-β transcription assessed by RT-PCR. Fold changes are relative to the purified control and data are mean values from five independent experiments. p-value for IFN-β fold induction for anti-CD2v antibody mix compared to anti-ORFV086 is 0.014 and without preincubation is 0.028. (D) Swine PBMCs were treated with the purified CD2v protein or purified control and supernatants collected 24 h post treatment. The supernatant collected from PK15 cells transfected with Poly I:C served as a positive control. PK15 grown in 96 well plates were treated with different dilutions of PBMCs supernatants for 24 h and subsequently infected with VSVGFP (50 PFU/well) for 16 h. Intensity (mean gray value) was measured by ImageJ. Fold changes are relative to the purified control and data are the mean of four independent experiments. p-values relative to purified control for undiluted, 1:2 diluted and 1:4 diluted supernatants were 0.014, 0.033 and 0.017, respectively. (*, p < 0.05 and **, p < 0.01.)