Modified vaccinia virus Ankara (MVA) is an attenuated vaccinia virus that belongs to the genus Orthopoxviridae. MVA is growth incompetent in humans, has a well established safety record and elicits a broad immune response.
In this study, a new technique for the generation of MVA recombinants on the basis of vaccinia virus D4R rescue is presented. The rescue procedure is based on a growth defective MVA, that lacks an essential gene, and reintroduction of this gene for selection. The D4R gene is essential for MVA and deletion of this sequence leads to defective viruses that can only replicate in an engineered cell line that complements the deleted D4R gene product. In parental D4R-defective viruses, foreign genes can be introduced together with the essential D4R gene that restores the growth competent phenotype (rescue). In the resulting recombinants, the foreign genes are integrated into the D4R / D5R intergenic region, while the wild type MVA backbone is not modified by integration of additional marker cassettes. To grow defective parental virus, a D4R complementing cell line was generated by retroviral transduction of wild type avian DF-1 cells. In this cell line D4R-defective MVA was created by homologous recombination. Application of this defective MVA as the parental virus for generation of MVA recombinants was examined with the example of a yellow fever (YF) virus surface antigen. The resulting recombinants were fully growth competent and could be isolated in wild type, non-complementing cells where parental defective virus growth is inhibited. By applying this rescue technique, 100% of the recombinants showed adequate structure, purity and foreign gene expression after only one plaque purification round.
Thus, the MVA D4R rescue technique established in this study is a useful tool for rapid and stringent selection of recombinant MVA. Parental D4R defective viruses are permanently inhibited in wild type cells allowing rapid and efficient selection of recombinant replicating virus. For experimental purposes, MVA stocks that efficiently express foreign genes can thus also be generated without any plaque purification solely by passage following recombination.
Rescue of the original D4R gene enables the integration of target genes into the viral genome without further selection markers. Absence of marker sequences is a requirement for state of the art vaccine vectors.