Fig. 3.

The expression of 3E8 with VEEV-VRP-3E8 delivery in vitro a) Schematic representation of the packaging system of VEEV-VRP-3E8 including the VEEV-rep-3E8, and two helper RNAs as described previously.

b) BHK-21 cells were infected with VEEV-VRP-3E8 at different MOIs (0.1/1/10), the cells were fixed and stained for the expression of 3E8 mAb (red) and VEEV-nsP1 (green) using Alexa Fluor™ 568 conjugated goat anti-human IgG (H + L) and anti-VEEV nsP1 mouse serum respectively at 24 h post-infection (hpi). The mock-infected BHK-21 cells were used as a negative control. Nuclei were stained with DAPI (blue). Scale bars represent 50 μm.

c) Quantification of 3E8 expression levels by hACE2-specific ELISA using the supernatants collected at 48 hpi from the VEEV-VRP-3E8 infected BHK cells with different MOIs (0.1/1/10) and mock treated BHK-21 cells. The antibody titers were determined in triplicate. Statistical analysis was performed by Ordinary one-way ANOVA. *P < 0.05, **P < 0.01.

d) Quantification of 3E8 expression levels by antigen (hACE2)-specific ELISA using the supernatants collected at 48 hpi from the VEEV-VRP-3E8 infected different cells (BHK/293T/Huh7). The antibody titers were determined in triplicate. Statistical analysis was performed by Ordinary one-way ANOVA. *P < 0.05, ***P < 0.001.

e) The VEEV-VRP-3E8 infected BHK-21 cells (MOI = 1) and the mock treated cells were lysed with RIPA at 48 hpi and subjected to western blotting using the HRP conjugated goat anti-human IgG (H + L) and the anti-β-actin antibody.