Dynamics of transgene expression in the MVA vaccine vector under the control of the p11, p13.5, pLEO160, p7.5 and mH5 promoters, independence of the transgene expression level from the insertion locus

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The modified vaccinia virus Ankara (MVA), characterized by high immunogenicity and proven safety, is considered a promising vector for vaccine development. An undeniable advantage of the MVA vector is its high capacity and the ability to incorporate several transgenes into different loci of the viral genome, enabling the creation of multivalent vaccines that encode multiple antigens simultaneously. This study examined the expression of a transgene encoding influenza virus protein epitopes after its integration into five loci of the MVA genome. The results demonstrated that the level of transgene expression is independent of the integration locus. Additionally, the dynamics of the expression of a reporter gene encoding Enhanced Green Fluorescent Protein (EGFP) were determined under the control of the vaccinia virus promoters p11, p13.5, pLEO160, p7.5, and mH5 upon insertion of the expression cassette into the F13L gene locus of MVA. The highest expression level, though with a delayed onset of protein synthesis, was achieved with the late promoter p11. Using the p13.5 promoter resulted in earlier synthesis of EGFP in cells and higher gene expression levels compared to the promoters pLEO160, p7.5, and mH5, which provided similar levels and dynamics of reporter gene expression. These findings may be useful for developing multi-antigenic MVA-vectored vaccines.

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作者简介

O. Orlova

Centre for Strategic Planning and Management of Biomedical Health Risks, Federal Medical Biological Agency

编辑信件的主要联系方式.
Email: oorlova@cspmz.ru
俄罗斯联邦, Moscow

D. Glazkova

Centre for Strategic Planning and Management of Biomedical Health Risks, Federal Medical Biological Agency

Email: oorlova@cspmz.ru
俄罗斯联邦, Moscow

О. Sidorova

Centre for Strategic Planning and Management of Biomedical Health Risks, Federal Medical Biological Agency

Email: oorlova@cspmz.ru
俄罗斯联邦, Moscow

F. Urusov

Centre for Strategic Planning and Management of Biomedical Health Risks, Federal Medical Biological Agency; Izmerov Research Institute of Occupational Health

Email: oorlova@cspmz.ru
俄罗斯联邦, Moscow; Moscow

G. Shipulin

Centre for Strategic Planning and Management of Biomedical Health Risks, Federal Medical Biological Agency

Email: oorlova@cspmz.ru
俄罗斯联邦, Moscow

Е. Bogoslovskaya

Centre for Strategic Planning and Management of Biomedical Health Risks, Federal Medical Biological Agency

Email: oorlova@cspmz.ru
俄罗斯联邦, Moscow

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2. Fig. 1. a – Scheme of the arrangement of loci 007/008, F13L, TK, D4R, 136/137 in the MVA genome. GOI – analyzed gene. b – Scheme of the shuttle vector for the F13L selection system. Left arm – left arm of homology with the F13L locus. Promoter – location of one of the five promoters (p7.5; mH5; p13.5; p11; pLEO160). EGFP – sequence encoding the EGFP protein. Transcription termination site. Right arm – right arm of homology with the F13L locus; BamHI, NheI and EcoRI restriction sites are shown. c – Scheme of insertion of shuttle vectors into the F13L locus of the rMVAΔF13L vector.

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3. Fig. 2. Gating strategy to detect EGFP-positive cells among HEK293FT cells transduced with rMVA-p7.5-EGFP.

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4. Fig. 3. Western blot analysis of k2 polypeptide synthesis in HEK293FT cells infected with rMVA-F13L-k2, rMVA-D4R-k2, rMVA-007/008-k2, rMVA-136/137-k2, rMVA-tk-k2 or wild-type (WT) MVA. WT was used as a negative control for rMVA-infected cells. The mass of k2 polypeptide is 31 kDa.

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5. Fig. 4. a – Dynamics of EGFP synthesis under the control of different promoters. Content (%) of EGFP+ cells in dynamics. b–c – Fluorescence intensity in infected cells in the EGFP+ population (b) and in the entire cell population (c), normalized to the MFI of cells at the time of infection with each rMVA. Shown are the mean values ​​and standard deviations calculated from the results of three experiments.

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6. Fig. 5. Alignment of p7.5 and mH5 promoters. Vaccinia – fragment of the vaccinia virus genomic sequence (GeneBank accession number NC_006998). Dai L., 2014 and Wyatt L.S., 1996 – promoter sequences used in the corresponding publications.

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