Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

• Published in: PloS one 2008-02, Vol.3 (2), p.e1638
• Main Authors: Cottingham, Matthew G, Andersen, Rikke F, Spencer, Alexandra J, Saurya, Saroj, Furze, Julie, Hill, Adrian V S, Gilbert, Sarah C
• Format: Article
• Language: English
• Subjects: Acquired immune deficiency syndrome; AIDS; AIDS vaccines; Animals; Antigenic determinants; Antigens; Artificial chromosomes; Bacteria; Bacterial artificial chromosomes; Bacterial genetics; CD8 antigen; CD8-Positive T-Lymphocytes - immunology; Chemokines; Chromosomes, Artificial, Bacterial; Clinical trials; Clonal deletion; Cloning; Deoxyribonucleic acid; DNA; Epitopes; Expression vectors; Fowlpox; Gene expression; Genetic Engineering; Genetically modified organisms; Genomes; Genomics; Hepatitis; HIV; Homeostasis; Human immunodeficiency virus; Immunogenicity; Immunology; Immunology/Immune Response; Influenza; Interferon; Interleukin 2; Kinases; Leishmania major; Lymphocytes; Lymphocytes T; Malaria; Medical research; Mice; Mice, Inbred BALB C; Mutagenesis; Plasmodium berghei - immunology; Proteins; Recombination; Recombination, Genetic; Replication; T cells; Thymidine; Thymidine kinase; Transcription; Treatment Outcome; Tuberculosis; Vaccination; Vaccines; Vaccines, DNA; Vaccinia; Vaccinia virus - genetics; Vector-borne diseases; Vectors (Biology); Virology; Virology/Immune Evasion; Virology/Vaccines; Viruses; γ-Interferon
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0001638

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.