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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.
- Source :
-
Journal of virological methods [J Virol Methods] 2010 Feb; Vol. 163 (2), pp. 195-204. Date of Electronic Publication: 2009 Sep 22. - Publication Year :
- 2010
-
Abstract
- 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.<br /> (2009 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Cell Culture Techniques methods
Cells, Cultured
Chick Embryo
Chickens
Culture Media, Serum-Free
Fibroblasts virology
Flow Cytometry methods
Green Fluorescent Proteins genetics
HMGB1 Protein genetics
Influenza A Virus, H5N1 Subtype genetics
Luminescent Proteins genetics
Rats
Staining and Labeling methods
Vaccinia virus genetics
Red Fluorescent Protein
Genes, Viral
Recombination, Genetic
Smallpox Vaccine
Vaccinia virus growth & development
Vaccinia virus isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0984
- Volume :
- 163
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of virological methods
- Publication Type :
- Academic Journal
- Accession number :
- 19778556
- Full Text :
- https://doi.org/10.1016/j.jviromet.2009.09.016