Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia Virus Ankara (MVA) - Genetic Engineering: Recent Developments in Applications - page 40

(Domi & Moss, 2002, PNAS 99 12415-20). The genomic BAC clone was
'rescued' back to infectious virus using a Fowlpox virus helper to supply tran-
scriptional machinery. We apply here a similar approach to the attenuated
strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe
non-replicating recombinant vaccine vector in mammals, including humans.
Four apparently full-length, rescuable clones were obtained, which had indis-
tinguishable immunogenicity in mice. One clone was shotgun sequenced and
found to be identical to the parent. We employed GalK recombination-medi-
ated genetic engineering (recombineering) of MVA-BAC to delete five selected
viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly af-
fect CD8 + T cell immunogenicity in BALB/c mice, but deletion of B15R en-
hanced specific CD8 + T cell responses to one of two endogenous viral epitopes
(from the E2 and F2 proteins), in accordance with published work (Staib et
al., 2005, J. Gen. Virol. 86, 1997-2006). In addition, we found a high-
er frequency of triple-positive IFN- γ , TNF- α and IL-2 secreting E3-specific
CD8 + T-cells 8 weeks after vaccination with MVA lacking B15R. Further-
more, a recombinant vaccine capable of inducing CD8 + T cells against an
epitope from Plasmodium berghei was created using GalK counterselection to
insert an antigen expression cassette lacking a tandem marker gene into the
traditional thymidine kinase locus of MVA-BAC. MVA continues to feature
prominently in clinical trials of recombinant vaccines against diseases such as
HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-con-
cept experiments that MVA-BAC recombineering is a viable route to more
rapid and efficient generation of new candidate mutant and recombinant
vaccines based on a clinically deployable viral vector.
introduction
Modified Vaccinia virus Ankara (MVA) is a replication-deficient attenuated pox-
virus that was derived from Vaccinia virus by Mayr et al. through more than 500
blind passages in chick embryo fibroblast (CEF) cell culture [1]. With the notable
exception of the Syrian hamster cell line BHK-21 [2], MVA is unable to replicate
productively in mammalian cells, though genome replication, late gene expres-
sion and immature virion formation usually occur [3], [4]. During its attenua-
tion, MVA acquired large genomic deletions totalling about 30 kb, resulting in
the complete loss of 26 open reading frames (ORFs), together with truncation or
fragmentation of a further 21 ORFs and numerous smaller scale mutations [5].
MVA does not therefore express many of the known poxviral immune evasion
and virulence factors [6]-[8], though how this determines its abortive phenotype
in mammalian cells is not well understood [9]. Despite these features, MVA is at