Title |
Recombination-Mediated Genetic Engineering of a Bacterial Artificial Chromosome Clone of Modified Vaccinia virus Ankara (MVA)
|
---|---|
Published in |
PLOS ONE, February 2008
|
DOI | 10.1371/journal.pone.0001638 |
Pubmed ID | |
Authors |
Matthew G. Cottingham, Rikke F. Andersen, Alexandra J. Spencer, Saroj Saurya, Julie Furze, Adrian V. S. Hill, Sarah C. Gilbert |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
United States | 3 | 2% |
United Kingdom | 1 | <1% |
Unknown | 145 | 97% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 30 | 20% |
Student > Master | 21 | 14% |
Student > Ph. D. Student | 18 | 12% |
Student > Bachelor | 17 | 11% |
Student > Postgraduate | 7 | 5% |
Other | 27 | 18% |
Unknown | 29 | 19% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 28 | 19% |
Biochemistry, Genetics and Molecular Biology | 13 | 9% |
Medicine and Dentistry | 11 | 7% |
Immunology and Microbiology | 9 | 6% |
Social Sciences | 6 | 4% |
Other | 45 | 30% |
Unknown | 37 | 25% |