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in the case of Vaccinia virus protein N1 that preservation of structure and func-
tion can occur in the absence of significant sequence similarity [52]. An unbiased
experimental approach is therefore required for identification of MVA genes that
negatively affect its immunogenicity. In order to expedite this approach, we have
constructed a bacterial artificial chromosome (BAC) clone of the MVA genome
using the elegant method devised by A. Domi and B. Moss for Vaccinia virus [53],
with slight modifications. Recombination-mediated genetic engineering (recom-
bineering) of this construct [54], [55] permits more rapid generation of mutants
for analysis than can be achieved by traditional methods, with the possible excep-
tion of host-range selection on rabbit cells using K1L [56], [57]. To test this con-
cept, we deleted five MVA genes using recombineering of a fully sequenced BAC
clone, rescued the constructs to infectious virus, and here demonstrate a modest
but significant improvement in immunogenicity of MVA lacking B15R, support-
ing and extending published work [51]. For large-scale biomanufacturing of an
MVA-based vaccine for clinical use, the ability to use clonal purifed BAC DNA
as the starting point, rather than a conventional plaque-purified recombinant, is
likely to be of considerable value. With this aim in mind, as well as pre-clinical
studies, we demonstrate immunogenicity of a recombinant antigen inserted at the
traditional thymidine kinase locus of MVA-BAC by recombineering.
Materials and Methods
Recombinant MVA Construction
Figure 1 shows the plasmid construct used to generate a recombinant virus (re-
ferred to as MVA-BAC-parent) containing the entire sequence of the BAC vec-
tor pBELO-BAC11 at the Deletion III locus of MVA (between the remnants of
A51R and A56R) together with a GFP reporter gene driven by the p4B late pro-
moter from Fowlpox virus. The fragments required were amplified by PCR with
primers designed to introduce restriction sites for assembly by standard methods.
The flanking regions for recombination correspond to positions 148412-149083
and 149340-149855 of MVA genome sequence U94848 [5]. The plasmid was
linearised with PacI and transfected into MVA-infected primary chick embryo
fibroblast (CEF) cells (Institute for Animal Health, Compton, UK). The cells
were trypsinised and GFP-positive cells were sorted by flow cytometry on a Dako
Cytomation MoFlo prior to plaque picking of the recombinant virus to purity,
amplification and titration on CEFs. Cells were maintained in Dulbecco's modi-
fied Eagle medium (DMEM) supplemented with 10% FCS or with 2% FCS for
viral growth. Identity and purity were confirmed by PCR. MVA expressing the
recombinant antigen TIP was prepared as described (A. Spencer et al., manuscript
in preparation).
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