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Pairs of recombinant MVA (Modified Vaccinia Ankara) and FPV (Fowlpox Virus) expressing the same transgene are reasonable candidates for prime/boost regimens, because cross-reacting immune responses between the two vectors, both non-replicative in mammalian hosts, are very limited. The acceptor virus FPD-Red, a derivative of FPV, carrying a red fluorescent protein gene flanked by the homology regions of MVA deletion III, was constructed. The same MVA Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III locus, can therefore be used to transfer transgenes into both acceptor viruses MVA-Red and FPD-Red with the described recently Red-to-Green gene swapping method. Cells infected by either recombinant virus can be sorted differentially by a simple and reliable FACS-based purification protocol. The procedure is carried out in primary chick embryo fibroblasts grown in serum-free media and was applied to the production of three rMVA/rFPV pairs expressing the H5N1 avian influenza antigens M1, M2 and NP. The viral genes were human codon-optimized and expressed at high levels in both chick and mammalian cells. Both single-step and multiple-step growth analyses showed no significant differences in growth due to the transgenes in either rMVA or rFPV derivatives.

Modified vaccinia virus Ankara (MVA) is employed as a human vaccine vector for the high expression of heterologous genes and the lack of replication in mammalian cells. This study demonstrates that cells infected by recombinant viruses can be obtained by fluorescence-activated cell sorting. Recombinant viruses are generated by a swapping event between a red fluorescent protein gene in the acceptor virus and a plasmid cassette coding for both a green fluorescent marker and a transgene. To prevent the carry-over of parental virus, due to superinfection of the cells harbouring recombinant viruses, the sorting is performed on cells infected at low m.o.i. in the presence of a reversible inhibitor of viral particle release. Terminal dilution cloning is then used to isolate both green and marker-free recombinant viruses, which can be identified by whole-plate fluoroimaging. The differential visualization of all the viral types involved allows a stepwise monitoring of all recombinations and leads to a straightforward and efficient flow cytometry-based cell sorting purification protocol. As an example of the efficacy of this sorting procedure, the construction of rMVA's coding for the rat nuclear protein HMGB1 and H5N1 influenza A virus hemagglutinin is reported. The entire recombinant MVA production process is carried out in serum-free media employing primary chicken embryo fibroblasts (CEF), which are certified for the preparation of human vaccines. This rMVA production method is faster, simpler and more reliable than any other available procedure for obtaining safe vaccine stocks for human use.

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental and human vaccine vector for its lack of replication in mammalian cells and high expression of heterologous genes. Recombinant MVA technology can be improved greatly by combining transient host-range selection (based on the restoration in MVA of the deleted vaccinia gene K1L) with the differential expression of fluorescent proteins. Recombinant virus results from swapping a red protein gene (in the acceptor virus) with a cassette of the transfer plasmid comprising the transgene and the green marker K1Lgfp (a chimeric gene comprising K1L and EGFP). Recombinant selection is performed in the selective host RK13. Finally, in the non-selective host BHK-21, a single crossover between identical flanking regions excises the marker gene. The three types of viruses involved (red parental, green intermediate and colourless final recombinant) are visualized differentially by fluorescence microscopy or fluoro-imaging of terminal dilution microcultures, leading to a straightforward and efficient purification protocol. This method (Red-to-Green gene swapping) reduces greatly the time needed to obtain marker-free recombinant MVA and increases the reliability of the construction process.