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Recombinant modified vaccinia virus Ankara (MVA) has been used to deliver vaccine candidate antigens against infectious diseases and cancer. MVA is a potent viral vector for inducing high magnitudes of antigen-specific CD8+ T cells; however the cellular immune responses to a recombinant antigen in MVA could be further enhanced by increasing transgene expression. Previous reports showed the importance of utilizing an early poxviral promoter for increasing transgene expression and therefore enhancing cellular immune responses. However, the vaccinia D10 decapping enzyme is reported to target and decap vaccinia virus early transcripts - a mechanism that could limit the usefulness of early promoters in MVA viral vectors if this enzyme shows the same activity in this closely related virus. Therefore, we attempted to increase transgene expression in recombinant MVA by inserting the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) upstream of a transgene sequence that is controlled by the B8R early promoter, and assessed D10 enzyme decapping activity in MVA. The aim of the IRES element was to initiate translation of the transgene transcript (after the removal of the cap structure by the D10 decapping protein) in a cap-independent manner. Here, we report that overexpression of the D10 decapping protein, in trans, in MVA reduced growth and transgene expression; however, the IRES element was not able to compensate for the negative effect of the D10 decapping protein. Recombinant MVA with EMCV IRES induced levels of both gene expression and transcription that were similar to the control recombinant MVA, encoding the same transgene but without the IRES element. Both viruses were tested in BALB/c mice and induced similar magnitudes of epitope-specific CD8+ T cells. This work indicates that the MVA version of the D10 decapping enzyme, overexpressed using a plasmid, is functional, but its negative effect on transgene expression by recombinant MVA cannot be overcome by the use of the EMCV IRES inserted upstream of the transgene initiation codon.

Original publication




Journal article


PLoS One

Publication Date





Antigens, Viral, Genome, Viral, Internal Ribosome Entry Sites, Luciferases, Mutagenesis, Insertional, Protein Biosynthesis, RNA Caps, RNA, Messenger, Recombination, Genetic, Transgenes, Vaccinia virus