Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems

Abstract

Background: Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied.

Results: We found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently.

Conclusions: Comparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs.

Keywords: Bacterial artificial chromosome; Norovirus; Recombinant MVA; Self-excising.

Fig. 1

Schematic overview of traditional marker-based method for generation of recMVA. a Construction of a recombinant vaccinia virus by homologous recombination in DF-1 cells infected with WT MVA. b Plaque purification of recMVA. Adapted from Nagel [13] and Rocha [14] with modifications

Fig. 2

Schematic overview of MVA-BAC technique for generation of recMVA. a In E. coli GS1783, following induction at 42 °C, the gene of interest together with I-SceI-Kan fragment is inserted into the DelVI site of the MVA-BAC by homologous recombination. Co-integrated Kan cassette (aphAI) is removed by induction of I-SceI production by arabinose followed by second Red recombination b In DF-1 cells, recMVAs are rescued in present of a helper virus and the BAC backbone including GFP cassette is removed spontaneously upon passaging

Fig. 3

a Schematic map of shuttle vector pIIIH5 Red K1L. b Targeting expression cassette into MVA DelIII region by homologous recombination of adjacent flank sequences FI1 DelIII and FI2 DelIII

Fig. 4

Generation of recMVA-NoV using marker-based approach. a After transfection of the pIIIH5-GII4VP1 into MVA-infected DF-1 cells, mCherry-expressing cells producing recMVAs were picked and plaque purification was carried out. After some rounds of plague picking, a significant increase in the signal was observed. Scale: 100 μm. b Confirmation of gene insertion into the MVA genome by PCR. After extraction of viral DNA from infected DF-1 cells, insertion of the NoV capsid gene was checked by amplifying the DelIII cassette with gene-specific primers. An amplified fragment of 1.6 Kb was expected for the GII.4. DF-1 cells infected with WT-MVA were used as negative control for PCR. c Confirmation of WT-MVA clearance. The presence of the WT-MVA was checked by amplifying DelIII cassette where a PCR product of 446 bp was expected

Fig. 5

Generation of recMVA-NoV BAC plasmid. a Schematic map of the shuttle vector pEP-MVAdVI-PH5. b Linear map of insertion region in the recombinant pEPMVAdVI-PH5VP1. The recombinant plasmid was constructed by insertion of the NoV GII.4 capsid gene VP1 into the shuttle vector downstream of the viral promoter PmH5. c Confirmation of first Red recombination. After first recombination in E. coli 1783, insertion of the I-SecI-Kan-pH 5/VP1 fragment into MVA-BAC genome was confirmed by PCR (expected size: 3.1 kb). d Confirmation of resolution of co-integrated Kan cassette by PCR using DelVI-specific primers (expected size: 2.1 kb)

Fig. 6

Rescue of recMVA-NoV from recBAC plasmid in DF-1 cells. a Confirmation of gene insertion into the MVA genome by PCR. Insertion of the capsid gene was checked by amplifying the DelVI cassette using DelVI primers for the WT (expected size: 498 bp) and gene-specific primers for the recMVA-BAC-GII.4 (expected size: 1.6 kb). Uninfected DF-1 cells were used as negative control. b Confirmation of RFV clearance by PCR using RFV-specific primers with an expected size of 265 bp. RFV-related band was detected in the first rescue sample; it was not detectable from the 4th passage. Purified RFV was used as positive control. c and d BAC self-excision: After transfection of the shuttle vector into RFV-infected DF-1 cells, the BAC backbone and the GFP cassette were spontaneously lost by passaging: (C) Confirmation of removal of BAC backbone by PCR using specific primers for gfp gene. (D) significant reduction in GFP population indicating the loss of BAC cassette from the recMVA genome. Scale: 100 μm

Fig. 7

Biochemical characterization of recMVAs. a Binding assay: Control cells are depicted on the left side. Virus binding to DF-1 cells was visualized by staining with anti-Vaccinia virus antibody. Top: recMVAs derived by the marker-based method; Bottom: BAC-recMVAs. b Electron microscopy with negative staining. Scale: 300 nm. c Expression of NoV VP1 in DF-1 cells infected with recMVAs. Immunoblotting was carried out using human serum from GII-infected patients as primary- and anti-human IgG as secondary antibody. VP1 protein (58 kDa) was detected in both recMVAs. Non-infected- and WT-MVA-infected cells were used as negative controls. Recombinantly expressed GST-tagged GII.4VP1 was used as positive control. d Schematic workflow for recMVA generation using traditional approach (left) and MVA-BAC system (right)