Genetic stability of SIV Gag/Tat gene inserted into Del-II in modified vaccinia virus ankara after serial passage of recombinant vector in pCEFs cells

Faqih, Layla; Vallely, P. J.; Klapper, Paul E.

doi:10.1016/j.jviromet.2022.114651

Cited by 3 publications

(2 citation statements)

References 25 publications

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“…However, depending on the design of the antigen expression cassette, generation may be difficult or even unsuccessful, as we have experienced for an artificial papillomavirus antigen, MfPV3 Ii-E1E2E6E7. Others have reported similar observations ( 23 – 27 , 42 , 43 ) but only few comparative studies were conducted to analyze this systematically ( 23 , 26 , 27 ). Even in cases when generation of recombinant MVA is successful, their propagation and the generation of bulk material for larger preclinical studies or GMP-grade drug substance or drug product for clinical trials may suffer from transgene instability leading to the emergence of mutated recombinant MVA (mrMVA) with reduced or abrogated antigen expression or modification of the transgene product.…”

Section: Discussionmentioning

confidence: 95%

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

Neckermann,

Mohr,

Billmeier

et al. 2024

Front. Immunol.

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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.

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Section: Discussionmentioning

confidence: 95%

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

Neckermann,

Mohr,

Billmeier

et al. 2024

Front. Immunol.

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“…MVA vaccines also employ a costly cell culture, which is one of the main advantages of the plant expression systems described. The issue of transgene stability must be addressed with MVA vaccines, which may affect the development and scaling up of vaccine preparations based on these [ 104 , 105 ].…”

Section: Discussionmentioning

confidence: 99%

The Potential of Plant-Produced Virus-like Particle Vaccines for African Horse Sickness and Other Equine Orbiviruses

Pitchers,

Boakye,

Campeotto

et al. 2024

Pathogens

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African horse sickness is a devastating viral disease of equids. It is transmitted by biting midges of the genus Culicoides with mortalities reaching over 90% in naïve horses. It is endemic to sub-Saharan Africa and is seasonally endemic in many parts of southern Africa. However, outbreaks in Europe and Asia have occurred that caused significant economic issues. There are attenuated vaccines available for control of the virus but concerns regarding the safety and efficacy means that alternatives are sought. One promising alternative is the use of virus-like particles in vaccine preparations, which have the potential to be safer and more efficacious as vaccines against African horse sickness. These particles are best made in a complex, eukaryotic system, but due to technical challenges, this may cause significant economic strain on the developing countries most affected by the disease. Therefore, this review also summarises the success so far, and potential, of recombinant protein expression in plants to reduce the economic strain of production.

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Armored modified vaccinia Ankara in cancer immunotherapy

Atay

Medina-Echeverz

Hochrein

et al. 2023

Viral Vectors in Cancer Immunotherapy

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Genetic stability of SIV Gag/Tat gene inserted into Del-II in modified vaccinia virus ankara after serial passage of recombinant vector in pCEFs cells

Cited by 3 publications

References 25 publications

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

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

The Potential of Plant-Produced Virus-like Particle Vaccines for African Horse Sickness and Other Equine Orbiviruses

Armored modified vaccinia Ankara in cancer immunotherapy

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