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. 2011 Dec 12:8:529.
doi: 10.1186/1743-422X-8-529.

Selection of recombinant MVA by rescue of the essential D4R gene

Patricia S Ricci  1 , Birgit Schäfer, Thomas R Kreil, Falko G Falkner, Georg W Holzer
Affiliations

Selection of recombinant MVA by rescue of the essential D4R gene

Patricia S Ricci et al. Virol J. .

Abstract

Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant.

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Figures

Figure 1
Figure 1
Outline of the D4R-dominant host range selection system. a. Schematic representation of the plasmid pDM-Zgpt for the construction of the D4R-deleted dMVA-ZG, and the corresponding insertion locus in the genome of MVA. b. Plasmid pDM-D4R-YF and the defective parental virus dMVA-ZG. MVA sequences are represented by boxes. The MVA genes D3R, D4R, D5R are marked grey. Foreign genes cassettes inserted into MVA are symbolized by hatched boxes. Arrows indicate the binding sites of the PCR primers that were used for confirmation of the structure and purity of dMVA-ZG.
Figure 2
Figure 2
Plaque formation of dMVA-ZG and wild-type MVA in avian DF-1 cells. cDF-1 (a, b) and wild-type DF-1 (c, d) cells were infected with 100 pfu/well of dMVA-ZG (a, c) or MVA-wt (b, d). Four days post infection, immunostaining was performed with anti-vaccinia virus antibodies. Photographs show immune-stained plaques.
Figure 3
Figure 3
Growth kinetics of MVA and dMVA-ZG. cDF-1 cells were infected with the defective virus dMVA-ZG at moi = 0.05. For comparison, infections of DF-1 cells dMVA-ZG and with wild-type MVA were performed in parallel. Cells were harvested at the indicated time points and the viral titers were determined by TCID50 titration. Mean values ± SEM are shown (n = 6).
Figure 4
Figure 4
PCR analysis of the D4R deletion in the defective virus dMVA-ZG. Genomic DNA of dMVA-ZG was subjected to PCR amplification using a primer pair that frames the D4R deletion (Lane 5). Negative controls were run without any template (Lane 2), with uninfected cDF-1 (Lane 3) cells and with the recombination plasmid pDM-Zgpt (Lane 4). Spike controls were performed with dMVA-ZG containing MVA-wt at 0.001-1% (Lanes 6-9). 1% corresponds to 1 000 pfu virus. A reaction with MVA-wt alone is shown on lane 10.
Figure 5
Figure 5
Growth kinetics of the recombinants rMVA-YF/D4 and rMVA-YF/del3. DF-1 cells were infected with rMVA-YF/D4 and rMVA-YF/del3 at moi = 0.05. Cells were harvested at the indicated time points and the viral titers were determined by TCID50 titration. Mean values ± SEM are shown (n = 6).
Figure 6
Figure 6
YFV antigen expression from a non-purified virus stock. Western blot analysis of yellow fever prME protein expression was performed at different passages of an unselected rMVA-YF/D4 stock, using a polyclonal anti YFV guinea pig serum that detects the YFV E protein as a ~50 kDa band. The blot shows the primary virus crude stock following infection of DF-1 cells with dMVA-ZG and transfection with pDM-D4R/YF (lane 2), and harvest from the first (lane 3), the second (lane 4) and the third passage (lane 5) of this stock. In parallel, the plasmid pd3-YFprMEco that lacks the D4R gene was transfected into dMVA-ZG infected DF-1 cells (lane 6) and passaged once (lane 7). As a positive control, clonally purified rMVA-YF/del3 was used (lane 8). Uninfected cells are shown on lane 9.
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