Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA).
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 and Moss, 2002, PNAS99 12415-20). The genomic BAC clone was 'rescued'...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
| Language: | English |
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Public Library of Science
2008
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| _version_ | 1797050224399613952 |
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| author | Cottingham, M Andersen, R Spencer, A Saurya, S Furze, J Hill, A Gilbert, S |
| author_facet | Cottingham, M Andersen, R Spencer, A Saurya, S Furze, J Hill, A Gilbert, S |
| author_sort | Cottingham, M |
| collection | OXFORD |
| description | 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 and 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. |
| first_indexed | 2024-03-06T18:02:00Z |
| format | Journal article |
| id | oxford-uuid:0016ced2-2abb-4bd1-85ab-7b549e698201 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:02:00Z |
| publishDate | 2008 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | oxford-uuid:0016ced2-2abb-4bd1-85ab-7b549e6982012022-03-26T08:27:44ZRecombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA).Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:0016ced2-2abb-4bd1-85ab-7b549e698201EnglishSymplectic Elements at OxfordPublic Library of Science2008Cottingham, MAndersen, RSpencer, ASaurya, SFurze, JHill, AGilbert, SThe 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 and 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. |
| spellingShingle | Cottingham, M Andersen, R Spencer, A Saurya, S Furze, J Hill, A Gilbert, S Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). |
| title | Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). |
| title_full | Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). |
| title_fullStr | Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). |
| title_full_unstemmed | Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). |
| title_short | Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). |
| title_sort | recombination mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus ankara mva |
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