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, PNAS 99 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-γ, TNF-α 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.
Highlights
Modified Vaccinia virus Ankara (MVA) is a replication-deficient attenuated poxvirus that was derived from Vaccinia virus by Mayr et al through more than 500 blind passages in chick embryo fibroblast (CEF) cell culture [1]
A procedure very similar to that developed by Domi and Moss for VAC-bacterial artificial chromosome (BAC) [53] was used to create BACs containing the genome of MVA
With an efficiency similar to that observed in the case of VAC-BAC, we generated four MVA-BAC clones, which were identical by restriction map and had indistinguishable CD8+ T cell immunogenicity in two strains of mice
Summary
Modified Vaccinia virus Ankara (MVA) is a replication-deficient attenuated poxvirus that was derived from Vaccinia virus by Mayr et al through more than 500 blind passages in chick embryo fibroblast (CEF) cell culture [1]. Attention has focussed on MVA as a possible ‘ generation’ smallpox vaccine [15,16,17], and as a recombinant vaccine vector, with particular value in combination with other vectors (e.g. DNA, adenovirus, or an avian poxvirus) as a component of heterologous prime-boost regimens designed to elicit high frequencies of antigen-specific T cells [18] This approach is undergoing development and clinical trial for diseases such as HIVAIDS [19,20], tuberculosis [21,22] and malaria [23,24], where protective T cell responses are required for vaccine efficacy. There is a need to identify recombinant vaccine vectors with greater immunogenicity, either by using novel vectors (e.g. simian adenoviruses [27]) or by improving existing platforms
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
