TY - JOUR

T1 - Transgene expression knock-down in recombinant Modified Vaccinia virus Ankara vectors improves genetic stability and sustained transgene maintenance across multiple passages

AU - Neckermann, Patrick

AU - Mohr, Madlen

AU - Billmeier, Martina

AU - Karlas, Alexander

AU - Boilesen, Ditte R.

AU - Thirion, Christian

AU - Holst, Peter J.

AU - Jordan, Ingo

AU - Sandig, Volker

AU - Asbach, Benedikt

AU - Wagner, Ralf

N1 - Funding Information: The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This project has received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and innovation program (E!12151). The project E!12151 was carried out within the framework of the European funding program “Eurostars” and the German partners were funded by the Federal Ministry of Education and Research. The funders had no influence on the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Acknowledgments Publisher Copyright: Copyright © 2024 Neckermann, Mohr, Billmeier, Karlas, Boilesen, Thirion, Holst, Jordan, Sandig, Asbach and Wagner.

PY - 2024

Y1 - 2024

N2 - Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3’ untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

AB - Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3’ untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

KW - Modified Vaccinia Ankara

KW - MVA

KW - poxviral vectors

KW - transgene knock-down

KW - transgene maintenance

KW - transgene stability

KW - vaccine manufacturing

KW - viral vaccines

U2 - 10.3389/fimmu.2024.1338492

DO - 10.3389/fimmu.2024.1338492

M3 - Journal article

C2 - 38380318

AN - SCOPUS:85185307033

VL - 15

JO - Frontiers in Immunology

JF - Frontiers in Immunology

SN - 1664-3224

M1 - 1338492

ER -