Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA Toritse Orubu, Naif Khalaf Alharbi, Teresa Lambe, Sarah C. Gilbert, Matthew G. Cottingham* The Jenner Institute, University of Oxford, Oxford, United Kingdom Abstract CD8 + T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8 + T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens. Citation: Orubu T, Alharbi NK, Lambe T, Gilbert SC, Cottingham MG (2012) Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA. PLoS ONE 7(6): e40167. doi:10.1371/journal.pone.0040167 Editor: Bruno Verhasselt, Ghent University, Belgium Received January 25, 2012; Accepted June 2, 2012; Published June 27, 2012 Copyright: ß 2012 Orubu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by a Grand Challenges in Global Health grant to Prof. Adrian V. S. Hill, Jenner Institute, University of Oxford administered by the Foundation for the National Institutes of Health. MGC and TL are supported by the Oxford Martin School. SCG is a Jenner Investigator. The authors are grateful to the Jenner Institute’s Vector Core Facility, funded by the Wellcome Trust, for technical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: MGC is a named inventor on a patent application relating to the method described (United Kingdom Patent Application No. 1006405.3 ‘‘Poxvirus expression system’’). SCG is a named inventor on a patent relating to prime-boost vaccination (WO9856919 ‘‘Methods and reagents for vaccination which generate a CD8 T cell immune response’’). This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.. * E-mail: matt.cottingham@ndm.ox.ac.uk Introduction The year 1983 saw the first descriptions of viral vectored vaccines, employing recombinant vaccinia virus to express foreign genes and elicit immune responses against various target pathogens [1–3]. Two different methods for expression of a cloned cDNA in vaccinia virus were employed in these inaugural studies, though both relied on insertion by homologous recombination in virus-infected cells. Whereas B. Moss’ group fused the cDNA of interest to a promoter prior to incorporation into vaccinia virus [2,3], E. Paoletti and colleagues relied on endogenous transcrip- tional activity near the viral insertion locus [1,4]. The disadvantage of using an endogenous promoter in the 1980s was that transgene expression by recombinants could be detected even when not adjacent to a transcriptional regulatory sequence [1,5]. Using the technology and knowledge of the day, it was hard to avoid inserting extra sequence upstream of the transgene, so this phenomenon was attributed either to accidental presence of a sequence with weak promoter activity in the inserted sequence, or to formation of a fusion protein [5]. The linking of a cloned viral promoter and ORF of interest prior to insertion therefore became the standard method for generation of recombinant vaccinia virus [6]. Essentially the same technique, with variations, has been applied to other poxviruses [7]. Despite its sterling service in the eradication of smallpox, vaccinia virus lacks the improved human safety profile of attenuated derivatives such as NYVAC and MVA [8] or of avian poxviruses [9]. Yet as recombinants, these too have typically employed the p7.5 promoter, as in 1983, or one of a small number of other promoters with early/late activity, for example, H5 (previously called H6) [10], modified H5 (mH5) [11] and the short synthetic promoter (SSP) [12] to drive transgene expression. The cassette is generally still inserted into the traditional thymidine kinase (TK) locus, or into one of a similarly limited number of alternative loci: in MVA, into one of the sites of the large genomic deletions [13], or more recently into an intergenic region [14–15]. The application of BAC recombineering technology to cloned poxviral genomes [16–18], coupled with recent transcriptomic studies [19–21], has allowed us to revisit the endogenous promoter PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e40167