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首页> 美国卫生研究院文献>Vaccines >Stability of the HSV-2 US-6 Gene in the del II del III CP77 and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells
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Stability of the HSV-2 US-6 Gene in the del II del III CP77 and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells

机译：重组载体在细胞中连续传代后修饰的痘苗病毒安卡拉中的del IIdel IIICP77和I8R-G1L位点中HSV-2 US-6基因的稳定性

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The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the gene locus ( ) and the - intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and sites. Sequencing analysis implicated deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the - site, suggesting better accommodation of transgenes in this intergenic region. Thus, the - intergenic region may be more useful for transgene insertion for enhanced stability.
机译：改良的痘苗病毒安卡拉（MVA）是一种严重减毒的痘苗病毒株，由于其高效的转基因表达和安全性等特性，是用于病毒载体疫苗开发的有前途的载体平台。因此，MVA中的转基因稳定性对于确保免疫原性和效力很重要。 MVA基因组的整体GC含量为33％，据报道，随着MVA载体在细胞中的传递，含有C或G核苷酸序列的富含GC的序列不稳定。根据生产规模，重组MVA疫苗的生产需要在细胞培养中进行许多扩展步骤。我们评估了四个重组MVA载体的广泛传代对富含GC的单纯疱疹2型（HSV-2）基因编码的稳定性的影响，该基因编码插入四个不同基因组位点的病毒糖蛋白D（gD2），包括缺失（del） II和del III位点，基因座（）和基因间区域。我们的数据表明，经过35次传代后，del II，del III和位点的gD2表达有所降低。测序分析暗示缺失和突变事件是造成gD2表达损失的原因。相比之下，85.9％的重组噬菌斑从-位点表达了gD2，表明在该基因间区域中转基因的适应性更好。因此，-基因间区域可能对于转基因插入更有用，以增强稳定性。

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