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amplified in BHK cells using DMEM without serum for infection and growth,
before purification over sucrose cushions and titration on CEFs.
bAc recombineering
GalK-based recombineering was done exactly as described by S. Warming et al.
[55], from whom the reagents and strains were obtained (see also http://recom-
bineering.ncifcrf.gov/). Sequencing of PCR products and primer synthesis was
performed by MWG-Biotech AG.
Mouse immunogenicity
Female BALB/c and C57BL/6 mice aged 6 to 8 weeks were obtained from the
Biomedical Services Unit, Oxford University and procedures were conducted ac-
cording to the UK Animals (Scientific Procedures) Act 1986. Mice were anaesthe-
tised with ketamine-dormitor prior to intradermal immunisation with 10
6
pfu of
MVA divided into two 25 µl injections (one per ear). Splenocytes were harvested
as described [58] seven days post-immunisation for flow cytometric analysis or
fourteen days post-immunisation for ELIspot analysis. ELIspots were conducted
using 18-20 h stimulation with 1 µg/mL synthetic peptide (ProImmune) in IVPH
plates (Millipore) coated with anti-IFN-
γ
antibody AN18 (Mabtech). Spots were
developed with R46A2-biotin (Mabtech) followed by streptavidin alkaline phos-
phatase (Mabtech) and substrate kit (BioRad) and enumerated on an automated
reader (Autoimmun Diagnostika). For flow cytometry, cells were stimulated for
6h with 1 µg/mL peptide in the presence of 2 µl/mL Golgi-Plug (BD) and stained
with anti-CD8 pacific blue conjugate and anti-CD4 APC-Alexa-750 conjugate
prior to fixation in 10% neutral buffered formalin (Sigma). Intracellular cytokine
staining was performed using FITC-conjugated anti TNF-
α
, PE-conjugated anti-
IL-2 and Alexa-647-conjugated anti-IFN-
γ
diluted in Cytoperm (BD). All an-
tibodies were obtained from eBiosciences. Data were acquired on a CyAn flow
cytometer (Dako) and analysed using FlowJo (Treestar). PESTLE and SPICE
software were obtained from Dr M. Roederer, Vaccine Research Centre, NIH.
Other statistical analyses were performed using Prism (GraphPad Software, Inc.).
results
Cloning of MVA Genome into BAC
A procedure very similar to that developed by Domi and Moss for VAC-BAC
[53] was used to create BACs containing the genome of MVA. First, a cassette
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