title: Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA).
abstract: The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC) in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20). The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional 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 indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+) T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced 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 higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, 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-concept 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.
authors.affiliation: Wellcome Trust Centre for Human Genetics and The Jenner Institute, University of Oxford, Oxford, United Kingdom. matt.cottingham@well.ox.ac.uk
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Cottingham
authors.forename: Rikke F
authors.initials: RF
authors.lastname: Andersen
authors.forename: Alexandra J
authors.initials: AJ
authors.lastname: Spencer
authors.forename: Saroj
authors.initials: S
authors.lastname: Saurya
authors.forename: Julie
authors.initials: J
authors.lastname: Furze
authors.forename: Adrian V S
authors.initials: AV
authors.lastname: Hill
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D022202:Chromosomes, Artificial, Bacterial; D005818:Genetic Engineering; D051379:Mice; D008807:Mice, Inbred BALB C; D010962:Plasmodium berghei / Q000276:immunology; D011995:Recombination, Genetic; D016896:Treatment Outcome; D014611:Vaccination; D014612:Vaccines; D019444:Vaccines, DNA; D014616:Vaccinia virus / Q000235:genetics*
chemical_list: D014612:Vaccines; D019444:Vaccines, DNA