Recombinant MVA was constructed by first generating a transfer vector, pMVA-FNK1, which is diagrammatically depicted in Figure 2.1. The cloning strategy employed is shown in Figure 3.6 of the Results section together with a description of why specific genes were chosen. pMVA-FNK1 was designed with the mH5 vaccinia virus promoter driving expression of the antigen of interest, SARS-CoV-2-SΔTM, the early/late p7.5 promoter driving expression of eGFP as a fluorescent marker gene and the synthetic poxvirus promotor pSS driving expression of the K1L host range gene. These genes were placed between sequences homologous to the 3’ ends of the convergent MVA open reading frames (ORFs) I8R on the left flank and G1L on the right flank (Figure 2.1).
These promoters are regulated by pox polymerases and transcription of the genes is only possible in the presence of the polymerase.
Figure 2.1: Diagram showing essential features of transfer vector pMVA-FNK1. I8R = MVA left flank, G1L = MVA right flank, promotor eGFP = green fluorescent protein, K1L = host range gene, p7.5 = early/late vaccinia virus promotor, pSS = synthetic vaccinia virus promotor, mH5 = vaccinia virus modified H5 promotor. This is not drawn to scale and the plasmid vector backbone is located between the 18R and G1L flanking sequences.
BHK21 cells, permissive for MVA, were infected with wild type MVA virus and transfected with pMVA-FNK1. Homologous recombination could take place between the identical regions of the
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virus genome and the transfer vector, resulting in generation of recombinant MVA expressing the antigen of interest and the other genes located between the I8R and G1L flanking regions (Figure 2.2).
A)
B)
Figure 2.2: Schematic of experimental procedure to generate recombinant Modified Vaccinia Ankara (MVA) virus. A) BHK21 cells were infected with wild-type parent MVA virus and then transfected with a transfer vector (pMVA-FNK1) containing the modified SARS-CoV-2 spike glycoprotein gene (S∆TM). This was cloned between the MVA flanking sequences that corresponded to the ends of the open reading frames, I8R and G1L. The identical flanking regions between the transfer vector and wild-type MVA are able to recombine by a process of homologous recombination resulting in insertion of the foreign genes (K1L selection gene, eGFP marker gene, and SARS-CoV-2- S∆TM.) into the MVA genome thus generating recombinant MVA, MVA-SARS-2-S∆TM. Figure created using BioRender.com. B) A line diagram showing the integrated coding region. The K1L gene functions to positively select for the recombinant MVA in RK13 cells and subsequently allows for purification of the recombinant from the wildtype MVA virus which cannot grow in this cell line. Additionally, eGFP was used as a fluorescent marker to identify recombinant virus.
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2.4.1 Infection of BHK21 cells with wild type MVA and transfection with pMVA-FNK1 A 6-well plate was seeded with 2 ml per well of BHK21 cells, at a concentration of 0.3 x 10⁶ cells/ml and incubated overnight at 37 °C in a CO2 incubator under standard growth conditions (Section 2.2.3). After 24 hours, cells were washed with 2 ml cDMEM before infection with 10 µl of MVA at a MOI of 0.5 and 1. Cells were incubated under standard growth conditions for 2 hours.
After incubation, the media was removed and replaced with 2 ml fresh cDMEM.
Cells were then transfected as outlined in Section 2.3.1. Control wells of plasmid only, MVA only (MOI 0.5 and 1) and uninfected BHK21 cells were also prepared. The plate was incubated for 48 hours and viewed using a Zeiss Axio Vert.A1 fluorescent microscope to observe green fluorescence from eGFP expression. Green fluorescence is driven by the control of a poxvirus specific promotor p7.5. Therefore, only when a transfected cell is infected with a poxvirus (MVA), will there be eGFP expression.
2.4.2 Purification of recombinant MVA-SARS2-SΔTM
MVA-SARS2-SΔTM was passaged in RK13 cells to purify the recombinant virus from unwanted parental virus. Cells were freeze-thawed for three cycles to release the virus from the infected BHK21 cells. Freeze cycles were conducted at -80 °C for 30 minutes and thaw cycles were performed at 37 °C for 15 minutes. The cell lysate was mixed and transferred to a 2 ml cryovial tube to prepare for infection of RK13 cells. At passage 1, a concentration of 2 x 10⁵ cells/ml was seeded into two 12-well tissue culture plates: one for MOI 1 and the other for MOI 0.5. Each well was infected with 10 – 200 µl of lysate. Plates were incubated for 3-5 days under standard growth conditions to allow for growth of the virus and the plates were observed daily using a Zeiss Axio Vert.A1 fluorescent microscope to monitor green fluorescence. This lysate was used for passage 2. For subsequent rounds of infections (passages 2 – 9), lysate of different volumes ranging from 20 µl to 200 µl were used to infect cells. At passages 3 and 4 the virus was serially diluted such that a single focus could be isolated in a single well of a 96-well plate. Each well containing a single focus was marked and those cell lysates were used for further passages in preparation for subsequent experiments. To expand the foci, subsequent passages were performed using 6-well and 12-well plates. PCR was performed using DNA isolated from cells in passage 7 to determine whether any residual parent MVA was present in the purified recombinant. PCR was performed
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again using DNA derived from cells at passage 9, this time to confirm integration of the entire gene cassette into the MVA genome.