Figure 3
Characterization of VEE-SARS-CoV-2 viral vectors. (A–C) The effects of structural gene and package signal sequence on transduction activity of VEE-SARS-CoV-2 vectors prepared by co-transfection of 293T cells with all but lacking one structural gene or package signal sequence. The viral vectors were prepared and titrated by qPCR and examined for transduction activity in ACE2-TMPRSS2-293T cells. (D) Cell type-dependent transduction activity of VEE-SARS-CoV-2 viral vector. Same number of cells (2 × 105 /well) were transduced with equal number of VEE-SARS-CoV-2-Luc vectors. The transduction activity was determined by luciferase assay at 48 h post-transduction. (E) Dose-dependent transduction of VEE-SARS-CoV-2 viral vectors. Same number of ACE2-TMPRSS2-293T cells (2 × 105 /well) were transduced with increasing amount of VEE-SARS-CoV-2-Luc vectors and luciferase activity was determined. (F) Cytotoxicity assessment of VEE-SARS-CoV-2-Luc vectors in ACE2-TMPRSS2-293T cells. Lactate dehydrogenase (LDH) activity in the supernatant of transduced cells was determined 48 h post-transduction. Cell lysate was used as the positive control for total release of LDH. (G) Luciferase gene expression as function of MOI. (H) VEE-SARS-CoV-2-Luc vector stability under different conditions. All luciferase activity in transduced cells was measured 24 h post-transduction except for (D) and (F). Data are presented as the mean ± SD of 3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey Post Hoc tests. ***p < 0.001.
