The combination of marker gene swapping and fluorescence-activated cell sorting improves the efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use

Joint production of prime/boost pairs of Fowlpox Virus and Modified Vaccinia Ankara recombinants carrying the same transgene

“…The plasmid pIII-sP-Red (Di Lullo et al, 2010) was digested with SpeI, blunted by Klenow fragment and digested again with EcoRI to obtain a 1808-bp fragment containing the HcRed1.1 gene (under the synthetic VV promoter sP) flanked by the MVA-deletion-III homology regions. The fragment was inserted into the EcoRV and EcoRI sites of pBlueScript SK(-) to obtain the pSK(-)Red intermediate, from which the HindIII-SmaI 1836 bp fragment was derived and inserted into the HindIII and NruI sites within the FPV fp9.046 gene 3-β-hydroxysteroid dehydrogenase 5-delta 4 isomerase, using the recombinant pFP128 plasmid after excision of the gene inserted previously ( [Pozzi et al, 2009] and [Radaelli et al, 2007]).…”

“…( [Pozzi et al, 2009] and [Radaelli et al, 2007]). The lysate derived from such infection/transfection was diluted 1/50 and used to infect CEF in the presence of 1 mM cytochalasin D (Di Lullo et al, 2010). Single-red cells were sorted onto CEF microcultures and the recombinant virus FPV-Dual-Red (FPD-Red) was cloned by two rounds of terminal dilution in microcultures.…”

“…Wild-type and modified genes were cloned into the pTH plasmid to yield plasmids pTH-wtM1, pTH-wtM2, pTH-wtNP, pTH-coM1, pTH-coM2, pTH-coNP. The human codon-optimized genes were then PCR-elongated after inserting BamHI and AscI restriction sites and subcloned into the BamHI-AscI sites of Transfer Plasmid Green (TPG) (Di Lullo et al, 2010) to yield plasmids TPG-coM1, TPG-coM2 and TPG-coNP.…”

“…A quick and reliable method to produce marker-free rMVA by swapping green and red fluorescence genes combined with fluorescence-activated cell sorting has been described previously (Di Lullo et al, 2010). To facilitate the production of pairs of recombinant MVA and FPV expressing the same transgene, the method was extended to the production of rFPVs.…”

“…To achieve this, an acceptor virus FPD-Red, a derivative of FPV containing an active red fluorescent protein gene flanked by the homology regions of MVA deletion III within the fp9.046 gene, was constructed. Consequently, the same Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III borders by homology recombination (Di Lullo et al, 2010) can be used to transfer transgenes into both MVA-Red and FPD-Red acceptor viruses.…”