Division of Translational Vaccine Research, Department of Virology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA.
Vaccine. 2010 Feb 10;28(6):1547-57. doi: 10.1016/j.vaccine.2009.11.056. Epub 2009 Dec 5.
We have engineered recombinant (r) Modified Vaccinia Ankara (MVA) to express multiple antigens under the control of either of two related vaccinia synthetic promoters (pSyn) with early and late transcriptional activity or the modified H5 (mH5) promoter which has predominant early activity. We sequentially passaged these constructs and analyzed their genetic stability by qPCR, and concluded that rMVA expressing multiple antigens using the mH5 promoter exhibit remarkable genetic stability and maintain potent immunogenicity after serial passage. In contrast, rMVA expressing antigens using engineered vaccinia synthetic E/L (pSyn I or II) promoters are genetically unstable. Progressive accumulation of antigen loss variants resulted in a viral preparation with lower immunogenicity after serial passage. Metabolic labeling, followed by cold chase revealed little difference in stability of proteins expressed from mH5 or pSyn promoter constructs. We conclude that maintenance of genetic stability which is achieved using mH5, though not with pSyn promoters, is linked to timing, not the magnitude of expression levels of foreign antigen, which is more closely associated with immunogenicity of the vaccine.
我们已经构建了表达多种抗原的重组（r）改良安卡拉痘苗病毒（MVA），这些抗原受两种相关痘苗合成启动子（pSyn）的控制，这两种启动子具有早期和晚期转录活性，或具有主要早期活性的修饰 H5（mH5）启动子。我们依次传代这些构建体，并通过 qPCR 分析它们的遗传稳定性，得出结论：使用 mH5 启动子表达多种抗原的 rMVA 具有显著的遗传稳定性，并在连续传代后保持强大的免疫原性。相比之下，使用工程化痘苗合成 E/L（pSyn I 或 II）启动子表达抗原的 rMVA 遗传不稳定。抗原丢失变体的逐渐积累导致连续传代后免疫原性降低的病毒制剂。代谢标记，然后冷追踪显示，mH5 或 pSyn 启动子构建体表达的蛋白质稳定性差异不大。我们得出结论，使用 mH5 保持遗传稳定性，尽管 pSyn 启动子不能实现，但与外源抗原表达的时间有关，而与疫苗的免疫原性更密切相关的是表达水平的大小。
