Neckermann P, Mohr M, Billmeier M, Karlas A, Boilesen DR, Thirion C, Holst PJ, Jordan I, Sandig V, Asbach B, and Wagner R
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., Competing Interests: PH is an inventor on a patent detailing the use of the invariant chain as an adjuvant for virally delivered vaccines owned by the University of Copenhagen. The right to use the patent for treating HPV is licensed to InProTher ApS where author PH is the founder, major shareholder, board member, and employee. DB was employed by the company InProTher ApS when the study was carried out. CT is the founder and shareholder of SIRION Biotech GmbH and a board member of InProTher ApS. PN, BA, RW, DB, PH, AK, IJ, VS, and CT are inventors of a patent application detailing the use of the MfPV3 antigen EP21191940.2. AK, IJ, and VS are employed by ProBioGen AG. IJ and VS are listed as inventors on patents that protect the avian cell line and the novel MVA strain WO 2005/042728 A3 and WO 2014/048500 A1. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Neckermann, Mohr, Billmeier, Karlas, Boilesen, Thirion, Holst, Jordan, Sandig, Asbach and Wagner.)