PDF(Pubmed)
Abstract：
The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.
摘要：
重链抗体(VHH)的可变结构域具有用于重定向腺病毒载体的细胞嗜性的潜力。这里,我们试图建立平台,以简化VHHs在整合到腺病毒纤维中时的特异性靶向功能的筛选.家禽腺病毒4(FAdV-4)和猿猴腺病毒1(SAdV-1)都有两种类型的纤维,其中之一对于病毒传播是不必要的，并且是VHH显示的适当位点。中间质粒，pMD-FAV4Fs,被构建为FAdV-4纤维2修饰的起始质粒。来自噬菌体T4的Foldon，三聚化的触发器，用于桥接纤维2和VHH的尾/轴域与人CD16A，自然杀伤（NK）细胞的关键膜标记。通过限制组装的一个步骤，修饰的纤维2被转移到腺病毒质粒，将其线性化并转染到包装细胞中。5种携带GFP基因的FAdV-4病毒最终被拯救并扩增,显示三个VHH。一种重组病毒，FAdV4FC21-EG,几乎不能转导人293或Jurkat细胞。相比之下,当它以每个细胞1000个病毒颗粒的感染复数使用时，表达外源CD16A的293或Jurkat细胞的转导效率达到51%或34%。将这种纤维修饰策略移植到SAdV-1载体上构建SAdV1FC28H-EG,适度转导原代人NK细胞，而亲本病毒没有转导。总的来说，我们改革了将VHH整合到纤维的策略,并建立了筛选VHH的新平台,以构建具有特定向性的腺病毒载体.
