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same pathways may negate the effect of the deletion mutants. Indeed, poxviruses
appear to have taken a grapeshot-like approach to disruption of the TLR/IL-1R
signalling pathway [6], though MVA's gun has been spiked by deletion of many
components, including A52R, which like A46R can inhibit NF-
κ
B activation,
albeit by a distinct subset of stimuli [46].
The deletion of these genes also did not impair the CD8+ T cell responses
to the vector, suggesting that, as well as being dispensable for growth in BHK
cell culture, they are not required for circumvention of host defence mechanisms
operating in vivo (though since MVA is non-replicating in mammals, this con-
clusion does not extend to poxviral pathogenesis). If multiple MVA genes were
to be deleted, in order to overcome putative functional redundancy, one might
assume that at some point the virus would be so crippled as to be only very poorly
immunogenic, for example by an antiviral response occurring early during the
infection cycle, which normally proceeds to immature virion formation even in
non-permissive cells. On the other hand, the major evolutionary driving force
for acquisition of immunomodulators by poxviruses is more likely productive
replication of a virulent virus, rather than evasion of adaptive responses (i.e. anti-
body or cytotoxic T cell responses). The MVA-BAC recombineering system will
hopefully be versatile and rapid enough to allow these subtleties to be addressed,
especially as it is likely to speed up the production of 'revertant' viruses in order
to confirm the genetic specificity of a novel phenotype. Furthermore, the ability
to make multiple modifications by 'recycling' the GalK dual-selectable marker is
likely to be of particular value.
Other applications of MVA-BAC include production of viruses carrying re-
combinant antigens and/or molecular adjuvants for pre-clinical evaluation and
clinical trial. The use of BAC DNA and an inactivated clinical grade helper virus
as the input to a manufacturing process could reduce the burden of traceability
currently required by regulatory authorities, though a method for efficient re-
moval of the BAC cassette would be needed (see Figure 1). In this setting, the
issues of construct stability and helper virus contamination would doubtless re-
quire more rigorous investigation than described here. Insertion of antigen ex-
pression cassettes by recombineering additionally circumvents the requirement
for a reporter gene or selectable marker and its removal by transient-dominant
selection [70] when a markerless product is required. In this regard, the difficulty
of insertion of the TIP antigen by GalK counterselection is rather disappointing,
though there is very likely room for improvement, for example by using longer
homology arms in the targeting DNA, or electroporating maximal quantities of
DNA. When removing GalK without concomitant insertion of DNA (i.e., from
a deletion locus), we have achieved much higher recombineering efficiencies of
up to 95% (data not shown). Alternatively, recombination in RecA+ E. coli [71]
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