3.1 Construction of a mammalian expression plasmid encoding SARS-CoV-2 S∆TM
3.2.2 Generation of recombinant MVA by homologous recombination
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healthy. Cells in rows E and F were round and lifted due to the infection with wild-type MVA virus. These did not fluoresce because of the absence of the transfer vector that encodes eGFP.
Cells from A and B were used for isolation of the recombinant MVA, which is summarized in Figure 3.11. The presence of the K1L gene in the recombinant allowed for growth of the recombinant only, in RK13 cells as described in Section 3.2.1.
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Figure 3.10: Expression of eGFP in BHK21 cells infected with wildtype MVA and transfected with pMVA- FNK2 plasmid encoding the S∆TM protein. A) BHK cells infected with MVA at an MOI of 0.5 and transfected with pMVA-FNK1. B) BHK cells infected with MVA at an MOI of 1 and transfected with pMVA-FNK1. C) Transfection control comprising BHK cells transfected with pMVA-FNK2 only. D) Cells only control comprising uninfected and untransfected cells. E) Infection control comprising BHK cells infected with MVA only at an MOI of 0.5. F) BHK cells infected with MVA only at an MOI of 1. Cells were visualized under phase contrast using transmitted light to visualize cell morphology (left panel), fluorescent light to show eGFP expression (central panel), and a merge of the two (right panel). Images are shown at 3 days post infection. D-F showed no green fluorescence
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ad therefore only images viewed under transmitted light are shown (Zeiss Axio Vert.A1 fluorescent microscope at 5X magnification). Scale bars represent 200 µm.
Recombinant MVA was purified in RK13 cells through a series of passages where cells infected with putative recombinant were lysed by freeze/thawing and the lysate was used to infect fresh RK13 cells. Cell lysate from passages 1-3 containing multiple foci was used to infect cells, whilst cell lysate from wells with a single fluorescing focus was used at passage 4 and carried forward to passage 5 to purify the recombinant. Cells were infected with cell lysate from passage 5, and passaging continued until passage 9 where the recombinant was completely purified from parent MVA (Figure 3.11). PCR was performed at passages 7 and 9 to confirm that the recombinant was isolated from the parent MVA, whilst western blot analysis was conducted at passage 9 to confirm that the spike protein was being expressed.
Figure 3.11: Flow diagram outlining the process used to generate and purify recombinant MVA-SARS2-SΔTM in mammalian cells. BHK21 cells were infected with parent MVA and transfected with pMVA-FNK1 to generate the recombinant MVA, MVA-SARS2-S∆TM. Crude cell lysate containing putative recombinant virus was used to infect RK13 cells to purify the desired recombinant from undesired parent virus. Cell lysate was used from passages 1-3 whilst lysate from wells with a single fluorescing focus was used to purify the recombinant in passages 3 and 4.
Purification in RK13 cells continued using cell lysate in passages 5-9. The diagram outlines the tissue culture plates used for each passage, and whether cell lysate from wells with multiple green foci or lysate from a single focus was used for the next infection. Stages at which PCR and Western Blot were performed are also shown. Figure was created using BioRender.com.
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Many fluorescent cells were visible after the first passage in RK13 cells, (P1); at this stage the fluorescence could still be due to transient expression of eGFP in MVA-infected cells. By P2 the fluorescence was clearly present in clumps of cells as opposed to individual cells, suggesting a recombinant virus had been generated and was spreading from a single infected cell (Figure 3.12).
At P3 the virus continued to spread forming more clumps of fluorescent cells. Vaccinia viruses produce a vaccinia virus growth factor (VGF) which stimulates proliferation of cells thus facilitating the spread of infection, which causes clustering of cells as virus particles spread to neighbouring cells [239], [240]. The clumping could also be due to cell apoptosis as a response to, and a way to control the spread of viral infection. At passage 4, RK13 cells were seeded into a 96- well tissue culture plate to isolate single foci. The cell lysate from P3 was serially diluted in order to completely dilute out the parental virus that could still be present yet undetectable, and to isolate a single fluorescent focus. A single fluorescing focus was visible at P4, and this was used going forward. The non-fluorescing foci were no longer visible in most wells by P4, which suggested that residual parental MVA had largely been eliminated. The wells without any non-fluorescing foci were used. By passage 7 the green fluorescence had spread and there was no indication that parent MVA was present. At passage 9 only fluorescing foci were present, which suggested the successful purification of recombinant MVA.
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Figure 3.12: Enrichment of MVA-SARS-2-S∆TM by serial passage in RK13 cells. P1, P2, P3, P4, P7 and P9 refer to passages 1, 2, 3, 4, 7 and 9 respectively. eGFP is expressed from the recombinant virus. Cells were visualized under UV light at 3 days post infection. At each passage images were taken using transmitted light, fluorescent light and merged light. (Zeiss Axio Vert.A1 fluorescent microscope at 5X magnification. Scale bars represent 200 µm).
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3.3 Validation and characterization of recombinant MVA-SARS-2-S∆TM by PCR and