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

Giulia Di Lullo, Elisa Soprana, Maddalena Panigada, Alessio Palini, Alessandra Agresti, Claudio Comunian, Adelaide Milani, Ilaria Capua, Volker Erfle, Antonio G. Siccardi

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)195-204
Number of pages10
JournalJournal of Virological Methods
Volume163
Issue number2
DOIs
Publication statusPublished - Feb 2010

Fingerprint

Vaccinia virus
Flow Cytometry
Vaccines
Viruses
Genes
HMGB1 Protein
H5N1 Subtype Influenza A Virus
Superinfection
Serum-Free Culture Media
Influenza A virus
Hemagglutinins
Nuclear Proteins
Transgenes
Virion
Genetic Recombination
Organism Cloning
Chickens
Plasmids
Embryonic Structures
Fibroblasts

Keywords

  • Flow cytometry sorting/cloning
  • Fluorescent proteins
  • Marker gene swapping
  • Recombinant MVA

ASJC Scopus subject areas

  • Virology

Cite this

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. / Di Lullo, Giulia; Soprana, Elisa; Panigada, Maddalena; Palini, Alessio; Agresti, Alessandra; Comunian, Claudio; Milani, Adelaide; Capua, Ilaria; Erfle, Volker; Siccardi, Antonio G.

In: Journal of Virological Methods, Vol. 163, No. 2, 02.2010, p. 195-204.

Research output: Contribution to journalArticle

Di Lullo, Giulia ; Soprana, Elisa ; Panigada, Maddalena ; Palini, Alessio ; Agresti, Alessandra ; Comunian, Claudio ; Milani, Adelaide ; Capua, Ilaria ; Erfle, Volker ; Siccardi, Antonio G. / 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. In: Journal of Virological Methods. 2010 ; Vol. 163, No. 2. pp. 195-204.
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