3.4 Confirmation of expression of SARS-CoV2-S∆TM by western blot analysis and
3.4.1 Western blot analysis to confirm expression of S∆TM from the recombinant virus
blotting of media and lysate. The immunoblot showed a product of ̴180 kDa corresponding to the S∆TM protein (Figure 3.14) in both the media (lane 7) and lysate (lane 8), with a stronger signal visible in the lysate. The same band was observed for cells transfected with the positive control vector pMEx-SARS-2-S∆TM (lane 9). The cells only control yielded no bands confirming that the observed signal was specific to the protein of interest.
The faint band (lane 7) could be explained by a more dilute product being present in the media as opposed to the more concentrated product in the lysate. When extracting protein, there was only 200 µl lysate as compared to 2 ml media. The presence of the protein in the media strongly suggests that the protein was being secreted from the infected cells as would be expected following the addition of the TPA leader sequence. The successful detection of a band at the expected size confirmed that the protein was being expressed from the recombinant MVA-SARS2-SΔTM.
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Figure 3.14: Validation of SARS-CoV-2-SΔTM expression in infected RK13 cells. Western blot analysis on media and cell lysate to confirm expression of SΔTM in RK-13 cells infected with passage 9 stock of MVA-SARS-2SΔTM.
1, Molecular weight marker; 2, Cells only media; 3, Cells only lysate; 4, Empty lane; 5, Media from cells infected with 20 µl from P9; 6, Lysate from cells infected with 20 µl from P9; 7, Media from cells infected with 50 µl from P9; 8, Lysate from cells infected with 50 µl from P9; 9, pMEx: SARS2-SΔTM lysate as positive control. The cell lysate and media were harvested after 72 hours. Samples were run alongside a 10 µl aliquot of Color Protein Standard Broad Range. The red asterisk indicates the product of interest.
3.4.2 Immunostaining to detect and confirm expression of S∆TM from the recombinant virus
Immunofluorescence was performed to visualize the SARS-CoV-2 SΔTM protein. This would serve as additional confirmation of expression and could give an indication of the cellular distribution of the product. HEK293 cells were infected with MVA-SARS2-S∆TM and fixed as described in Section 2.5.4, before observation under fluorescent light whereby red Cy3 fluorescence was visible (Figure 3.15). The antibody has a red fluorophore therefore the red signal was visualized to signify expression. The presence of red fluorescence in the first and second rows of Figure 3.15 served as confirmation of SΔTM protein expression in cells infected with MVA- SARS2-SΔTM. The protein appeared to have a diffuse distribution in the cells. In row 3, the cells only control yielded no fluorescence, as did the experimental control of secondary antibody (with no primary antibody) in the last row. This confirmed that the SARS-CoV-2-SΔTM protein is expressed by the recombinant MVA.
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3.4.3 Growth of MVA-SARS2-S∆TM in BHK21 and RK13 cells
Growth of recombinant MVA-SARS2-S∆TM was assessed in the two permissive cell lines, BHK21 and RK13 cells. This was conducted to identify a suitable cell line to expand the recombinant for immunogenicity studies. The assessment was made qualitatively based on fluorescence levels only. The two cell lines were infected with MVA-SARS2-S∆TM at MOI 0.01 and observed daily for green fluorescence over a period of five days (Figure 3.16).
On day 1, the virus was growing and spreading well. By day 2, there was significant growth in both cell lines. On day 3, the cells were still attached although most of them had rounded (Figure 3.16). This suggested that the optimum harvest time point for the recombinant is three days post infection. On day 4, cells lifted and exhibit a cytopathic effect. Both BHK21 and RK13 cells proved to be favourable for the growth of the recombinant MVA-SARS2-S∆TM, although the fluorescence differed in the two cell lines. The growth of the recombinant in BHK21 cells was more diffuse whilst in RK13 cells it was more focussed. It was assumed that the level of fluorescence was indicative of the amount of viral growth. Titrations should have been performed to further assess the growth quantitatively but could not be done due to time constraints. Based on the fluorescence levels, it was decided to grow the virus in RK13 cells.
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Figure 3.15: Immunofluorescence to detect expression of antigen S∆TM expression following infection of HEK293 cells with recombinant MVA-SARS2- S∆TM. In the experimental samples (rows 1 and 2) cells were infected with MVA-SARS2-S∆TM and fixed with 4% paraformaldehyde and 100% methanol before being blocked with 2% BSA. Primary rabbit anti-SARS-CoV-2 Spike Glycoprotein S1 antibody was added, followed by secondary donkey-anti-rabbit-Cy3 antibody (red). Both primary and secondary antibodies were used in the first and second rows (MVA-SARS2-S∆TM). Row 3 (uninfected cells) did not have any virus, whilst the row 4 shows uninfected cells treated with secondary antibody only (Zeiss Axio Vert.A1 fluorescent microscope at 40X magnification).
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Figure 3.16: Observation of MVA-SARS2-SΔTM growth in BHK21 and RK13 cells by visualization of eGFP expression at different time points post infection. Cells were infected with MVA-SARS-2-SΔTM. Cells were infected at MOI 0.01 and observed over a series of 5 days to observe fluorescence as an indication of viral growth. (Zeiss Axio Vert.A1 fluorescent microscope at 40X magnification).
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