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. 2022 Dec 8;13:1063679. doi: 10.3389/fimmu.2022.1063679

Figure 1.

Figure 1

Characterization of BBV154 vaccine candidate. Growth kinetics of BBV154 in HEK cells was studied by infecting HEK cells with 0.25, 0.5, 1, 2 and 3 MOI of BBV154 virus. Samples were collected at 12 hours intervals and were subjected to qPCR analysis to estimate the genome copies of the virus (A). SDS-PAGE and silver staining of BBV154 (drug product) and Ad5 reference reagent from ATCC (B). DNA was extracted from three batches of BBV154 and primers flanking the expression cassette were used to detect its presence. Amplification of 5889 bp PCR product indicates the presence of the spike expression cassette. The DNA extracted from ChAd vector was used as negative control which amplified a 2069 bp PCR product which indicates the absence of the spike expression cassette. Marker: Gene ruler SM03111, Thermoscientific (C). The HEK cells were infected with 5 MOI of three different batches of BBV154 or were left uninfected. Twenty-four hours post-infection the cells were harvested and lysed. The samples were subjected to western blotting with rabbit polyclonal antibody against RBD and anti-rabbit peroxidase conjugated antibody. Inactivated and purified SARS-CoV2 was used as positive control and Hfimmu.2022.1063679EK cell lysate was used as negative control (D). The cells were either left uninfected (E) or infected with 10-2 dilution of BBV154 (F). 48 hours post infection, the cells were fixed and probed with rabbit polyclonal S1 antibody followed by anti-rabbit IgG peroxidase. The peroxidase activity was detected by 3-Amino-9-Ethylcarbazole. BBV154 characterization assays were repeated at least three times using three batches of BBV154 drug substances.