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Abstract：
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.
摘要：
改良的安卡拉痘苗病毒是一种通用的疫苗载体，非常适合转基因传递，具有出色的安全性。然而,某些转基因使重组MVA（rMVA）在遗传上不稳定，导致具有受损的转基因表达的突变rMVA的积累。这代表了扩大和制造rMVA疫苗的主要挑战。为了防止转基因介导的阴性选择，连续禽类细胞系AGE1。修饰CRpIX(CRpIX)以在rMVA生成和扩增期间抑制转基因表达。这是通过在靶向嵌合痘病毒启动子和转基因mRNA上的操纵子元件（tetO）和3'非翻译序列基序的工程化CRpIXPRO抑制细胞中组成型表达四环素阻遏子（TetR）和大鼠衍生的shRNA来实现的。分别。该细胞系有助于在重组介导的编码序列整合到DelIII（CR19M-DelIII）或TK基因座（CR19M-TK）后，产生两个表达猕猴囊状乳头瘤病毒3型（MfPV3）E1E2E6E7人工融合多蛋白的rMVA（分离株CR19）。分别。亲本CRpIX或工程化CRpIXPRO抑制细胞上rMVA的表征揭示了增强的复制动力学，较高的病毒滴度和相当于野生型MVA的焦点形态，当转基因表达被抑制时。rMVA在亲本CRpIX细胞上连续传代10次并通过流式细胞术跟踪E1E2E6E7表达揭示了仅几代后转基因产物的快速损失。PCR分析和下一代测序表明，rMVA在E1E2E6E7开放阅读框（CR19M-TK）内积累了突变或整个转基因盒（CR19M-DelIII）的缺失。相比之下,CRpIXPRO抑制细胞保留了强大的转基因表达多达10代，然而,与DelIII基因座相比,当E1E2E6E7整合到TK中时,rMVA更稳定。总之,CRpIXPRO抑制细胞中转基因表达的持续敲低促进了产生，具有转基因的rMVA的繁殖和大规模制造阻碍了病毒复制。
