Investigation of IRES Insertion into the Genome of Recombinant MVA as a Translation Enhancer in the Context of Transcript Decapping

<div><p>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 <i>B8R</i> 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, <i>in trans</i>, 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.</p></div

The effect of IRES insertion on luciferase transcript levels in rMVA.

<p>BHK-21 cells were infected with the two rMVAs at MOI of 1. Cells were either treated with AraC (to assess the early gene transcription) or without AraC treatment (for the overall transcription). 24 h.p.i. the cells were lysed and the total RNA was extracted and used to make cDNA for the qPCR using the ∆∆ Ct method. This method was validated; (A) the Ct values of E9L and rLuc amplicons are presented against RNA template concentration in μg to show the amplification efficiency of these two genes. (B) The ∆ Ct of every RNA dilution is plotted, with slope of 0.094. (C) The ∆∆ Ct method of qPCR was then performed to determine the relative change fold in the rLuc gene expression. The data are representative of two independent experiments.</p

<i>In vivo</i> cellular immunogenicity of rMVA with IRES inserted upstream of the rLuc transgene.

<p>Two groups of female BALB/c mice (n = 4) were immunized with the respective rMVA. Seven days post-immunization, intracellular cytokine staining and flow cytometry were performed to determine the percentage of IFN-γ-secreting CD8⁺ T splenocytes in response to <i>in vitro</i> re-stimulation with (A) pb9 peptide, or (B,D) MVA vector-specific peptides. These values are presented after subtracting the values of (C) unstimulated control cells for every mouse sample. The median of each group is shown. Data are representative of two independent experiments.</p

IRES functionality in a bicistronic expression system.

<p>HEK 293 cells were transfected with a bicistronic vector expressing Influenza H1HA antigen under the control of a CMV promoter, and the Influenza NP-M1 fused antigen under the control of EMCV IRES. (A) H1HA was detected by Western blot (lane 2) using anti-HA antibody. (B) NP-M1 was detected by Western blot (lane 4) using anti-M antibody. Lane 1 and 3 contained cell lysates that were transfected with empty vector. Actin was detected as a control in all lysates. (C) A table explaining the expression and the expected size of the proteins.</p

Schematic representation of the rLuc transgene.

<p>The rLuc transgene consists of the tPA leader sequence, followed by pb9 (the MHC class I H-2K^d epitope of <i>P</i>. <i>berghei</i> circumsporozoite protein), fused to <i>Renilla</i> luciferase (rLuc), under the control of the pB8 promoter and inserted into the <i>B8R</i> locus using the <i>B8R</i> left homology arm (LHA) and right homology arm (RHA) sequences. It also contains the galactokinase (<i>GalK</i>) bacterial selection gene controlled by the prokaryotic EM7 promoter. The transgene is inserted into B8-MVA virus (top), or fused to the encephalitis myocarditis virus’s internal ribosome entry site (EMCV IRES) and inserted into B8-IRES-MVA (bottom). Arrows indicate the used ATG start codons: The ATG start codon of the tPA (Top) and the native 3’ ATG start codon of EMCV IRES (Bottom).</p

The effect of IRES insertion on secreted luciferase expression.

<p>BHK-21 cells were infected with the two rMVAs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127978#pone.0127978.g002" target="_blank">Fig 2</a> at MOI of 1. 24 h.p.i. the supernatants were collected and the cells were lysed, and the level of luciferase expression in the supernatant or cell lysate was measured to determine the effect of IRES insertion on the secretion and the size of expressed luciferase. The <i>Renilla</i> luciferase system, shown on a logarithmic scale, was used (top) and Western blot was also tested (bottom). mH5-MVA as a positive control and non-recombinant wild type MVA (MVA<i>wt</i>) as a negative control were also included. The same samples were taken from the same wells for both assays. The data are representative of two independent experiments. L: Lysate from infected cells. S: Supernatant.</p

<i>In vivo</i> cellular immunogenicity of MVA deletion mutants, lacking fifteen genes, with 85A antigen.

<p>Four groups of female BALB/c mice (n = 4) were immunized (i.m.) with the respective MVAs at the dose of 2x10⁶pfu/ml. Seven days post immunization, intracellular cytokine staining and flow cytometry was performed to determine the percentage of splenic IFN-γ-secreting T cells in response to <i>in vitro</i> re-stimulation with MVA vector-specific peptide (<b>A</b> and <b>B</b>), with CD8⁺ T cell specific 85A peptide pool <b>(C)</b>, or with CD4⁺ T cell specific 85A peptide pool <b>(D)</b>. Two individual peptides were incubated for 18 hours to determine the CD8⁺<b>(E)</b> or CD4⁺ T <b>(F)</b> cell responses by ELISpot. These values are presented after subtracting the values of unstimulated cells for every mouse (sample). The mean of each group with the SEM error bars are shown. Data is representative of two independent experiments. There was no statistical significant difference in any of MVA mutant groups as compared to MVA85A or MVA-BAC85A groups, using Kruskal-Wallis test with Dunn's multiple comparisons. <b>MVA<i>wt</i>:</b> MVA wild type, <b>MVA85A:</b> MVA expressing TB 85A antigen, <b>MVA-BAC85A:</b> MVA, containing BAC DNA and expressing TB 85A antigen, and <b>∆15-MVA-85A:</b> MVA mutant, containing BAC DNA and expressing TB 85A antigen, with 15 gene deleted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.t001" target="_blank">Table 1</a>).</p

The effect of IRES insertion on luciferase expression of rMVA.

<p>(A) BHK-21 (permissive) cells were infected at MOI of 1 with rMVA which either contain the rLuc transgene under the control of B8 endogenous promoter, or the B8 promoter with IRES inserted upstream of the transgene (B8-MVA and B8-IRES-MVA, respectively). Cells were either treated with AraC (to assess the early gene expression) or without AraC treatment (for the overall promoter activity). 24 h.p.i. the cells were lysed, and added to the supernatant and the total level of luciferase expression was measured to determine the effect of IRES insertion on the transgene expression, using the <i>Renilla</i> luciferase system. Non-recombinant wild type MVA (MVA<i>wt</i>) as a negative control was also included. (B) The same experiment was repeated with different MOI; and <b>(C)</b> was also repeated in HEK 293 (non-permissive) cells. All data, shown on a logarithmic scale, represent the mean of 4 wells with SEM error bars. Data are representative of more than five independent experiments.</p

MVA deletion mutants.

<p>MVA deletion mutants used in this study. Different clusters of ORFs were deleted from the mutants, encoding proteins with different functions (mentioned with references). In a few cases, there were no known functions associated with ORFs. The mutants express the 85A or TIP antigens that were inserted at the TK locus. All mutants were made using MVA-BAC recombineering, therefore, all contain BAC DNA and GFP (green fluorescent protein) marker.</p><p>^aTK: Thymidine kinase locus, used as an insertion site for the recombinant antigens.</p><p>^bVACV COP: Vaccinia virus Copenhagen strain.</p><p>^cWR: Vaccinia virus Western Reserve strain.</p><p>^dThe ORF <i>B15R</i> is the <i>MVA184R</i> gene. It is named <i>B15R</i> in VACV WR while it is <i>B16R</i> in VACV COP. This made inconsistency in reporting this ORF as well as the downstream B fragment ORFs in the literature. Here, we report <i>MVA184</i> as the <i>B15R</i>, consistent with our previous work [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref022" target="_blank">22</a>], and in accordance with another report <i>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref011" target="_blank">11</a>]</i>. However, it was reported as <i>B16R</i> in another study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref013" target="_blank">13</a>]. This ORF encodes IL-1β binding protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref011" target="_blank">11</a>].</p><p>MVA deletion mutants.</p

<i>In vivo</i> cellular immunogenicity of MVA deletion mutants, lacking fifteen genes, with TIP antigen.

<p>Two groups of female BALB/c mice (n = 10) were immunized (i.m.) with MVA<i>wt</i> with TIP model antigen (MVA-TIP), or MVA deletion mutant (lacking 15 genes) ∆15-MVA-TIP at the dose of 1x10⁶ pfu/ml. <b>Seven days</b> post immunization (top), <i>ex vivo</i> ELISpot was performed to determine the percentage of IFN-γ-secreting CD8⁺ splenocytes in response to <i>in vitro</i> re-stimulation with pb9 peptide <b>(A)</b>, or with MVA vector-specific peptides <b>(B and C)</b> (all three peptides are CD8⁺ T cell specific). <b>28 days</b> (middle), or <b>84 days</b> (bottom) post immunization, five mice were sacrificed and spleens collected for intracellular cytokine staining and flow cytometry to determine the percentage of IFN-γ-secreting CD8⁺ T splenocytes in response to <i>in vitro</i> re-stimulation with pb9 peptide <b>(A)</b>, or with MVA vector-specific peptides <b>(B and C)</b>. These values are presented after subtracting the values of unstimulated cells for every mouse (sample). The mean of each group with the SEM error bars are shown. Data is representative of two independent experiments. * <i>P</i> = 0.0331 using Mann Whitney test.</p