Dominant Host Range Selection of Vaccinia Recombinants by Rescue of an Essential Gene

Abstract

We report the rescue of a defective vaccinia virus, forming the basis for a stringent selection protocol to generate replicating recombinant virus without the need for marker cassettes and selection agents. Plaques of recombinant virus could be isolated solely by their ability to grow in wild-type cells normally supporting the growth of vaccinia virus. All growth-competent clones analyzed contained the gene of interest in the intended genomic locus and displayed foreign gene expression to the same levels as was seen with classical recombinants obtained by insertion into the vaccinia virus thymidine kinase locus. The system is based on a defective vaccinia virus, expressing exclusively early genes, termed eVAC-1, and an insertion plasmid vector providing the essential function, the uracil DNA glycosylase gene. In addition, the defective virus is free of selection and color marker genes, thus also representing a basic vector for the generation of defective recombinants.

Immunogenicity and Safety of Defective Vaccinia Virus Lister: Comparison with Modified Vaccinia Virus Ankara

“…A stable lacZ/gpt marker gene cassette was inserted into the MCS of plasmid pER (15); this plasmid has inserts consisting of the D3R, D4R, and D5R genes, and the MCS is located in the D4/D5 intergenic region. The resulting plasmid was designated pDRMa.…”

Defective Vaccinia Virus as a Biologically Safe Tool for the Overproduction of Recombinant Human Secretory Proteins

“…1). Second generation defective viruses lacking any D4R sequences abolish these rare recombination events (9). It is also noteworthy in this context that doses as high as 10 10 pfu of vD4-FVII and of vD4-FXI, respectively, administered intravenously were tolerated by rabbits.…”

“…This system significantly reduces the safety concerns for the work with vaccinia and, more importantly, under complementing conditions growth characteristics of a defective virus lacking D4R do not differ from those of the wild-type VV strain WR (9).…”

Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection

“…For vDD4-mNP-VN, 20 g of pDD4-mH5-mNP-VN plasmid DNA (or pDD4-mH5-5TNT-VN-HA#1, as needed) was transfected into vaccinia virus Lister-infected CV-1 cells by calcium phosphate precipitation and further processed as described previously (16). Plaque isolates were purified three times and expanded for large-scale preparations.…”