摘要
Baculovirus expression vector systems are used for the production of an increasing number of licensed commercial subunit vaccines. Though baculoviruses are incapable of replicating outside a narrow host range of lepidopteran insects, a replication-defective baculovirus system or platform provides an additional level of safety without the use of chemical inactivation methods such as binary ethylenimine. Circumventing the need for chemical inactivation protects sensitive proteins from degradation, and use of a live, yet replication-defective baculovirus has the potential to enhance antiviral immune response. These factors together make a replication-defective baculovirus platform highly desirable for subunit vaccine production. Choosing the essential baculovirus envelope protein gp64 as our target, we used recombination to delete gp64 from the baculovirus rendering it replication-defective. We used insect cell based plasmid expression vectors to generate insect cell lines stably expressing gp64 to allow passage of the replication-defective virus.
Porcine epidemic diarrhea virus (PEDV) remains an economically important disease in swine, and a highly effective vaccine remains elusive. To test the capacity of the replication-defective baculovirus system to express an antigenic target of interest, we used recombination to replace the gp64 in the baculovirus genome with a chimeric PEDV spike protein. We then infected Sf9 cells with the PEDV spike expressing replication-defective baculovirus. Analysis of insect cell supernatants indicated that yield of PEDV spike was low in non-complementing cells, but shows an association between the baculovirus capsid VP39 and PEDV spike suggesting that the pseudotyped spike chimera restores budding of replication-defective baculovirus particles in the absence of gp64 suggesting that the system also has potential as a vector for gene delivery.
摘要译文
杆状病毒表达载体系统用于生产越来越多的许可的商业亚基疫苗。尽管杆状病毒不能在狭窄的鳞翅目昆虫宿主范围内复制，但是复制缺陷型杆状病毒系统或平台无需使用化学灭活方法（如二元乙炔亚胺）即可提供更高的安全性。避免化学灭活的需要可保护敏感蛋白免于降解，并使用有生命但仍可复制的缺陷杆状病毒具有增强抗病毒免疫反应的潜力。这些因素共同构成了复制缺陷型杆状病毒平台，非常适合亚单位疫苗的生产。选择必需的杆状病毒包膜蛋白gp64作为我们的目标，我们使用重组从杆状病毒中删除gp64，使其复制（有缺陷）。我们使用基于昆虫细胞的质粒表达载体来产生稳定表达gp64的昆虫细胞系，以使复制缺陷型病毒得以通过。猪流行性腹泻病毒（PEDV）仍然是猪中一种经济上重要的疾病，而高效疫苗仍然难以捉摸。为了测试复制缺陷型杆状病毒系统表达感兴趣的抗原靶标的能力，我们使用重组用嵌合的PEDV刺突蛋白代替杆状病毒基因组中的gp64。然后，我们用表达复制的PEDV尖峰感染Sf9细胞-杆状病毒缺陷。对昆虫细胞上清液的分析表明，非互补细胞中PEDV穗的产量较低，但显示杆状病毒衣壳VP39和PEDV穗之间存在关联，这表明假型穗嵌合体可恢复复制的芽芽-在没有gp64的情况下，杆状病毒颗粒有缺陷。提示该系统还具有作为基因传递载体的潜力。
Ackerman, Scott. Development of a Replication-Defective Baculovirus Platform[D]. US: Iowa State University, 2020