High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors
High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors
复制标题
DOI：
10.1128/jvi.01394-18
复制
发表时间：
2019-01-01
影响因子：
5.4
通讯作者：
Agbandje-McKenna, Mavis
中科院分区：
文献类型：
--
作者：
Jose, Ariana;Mietzsch, Mario;Agbandje-McKenna, Mavis
腺相关病毒血清型5(AAV5)正被开发为包括血友病和亨廷顿病在内的多种疾病的基因传递载体，并被证明在肝、肺、骨骼肌和中枢神经系统中具有有效的转导作用。AAV基因传递的一个限制是先前存在的中和抗体，这对载体在治疗应用中的有效性提出了重大挑战。在这里，我们报道了AAV5与新产生的单抗HL2476在3.1埃分辨率下的冷冻电子显微镜(Cryo-EM)和图像重建结构。与其他现有的抗AAV5衣壳抗体不同，ADK5a和ADK5b的表位围绕衣壳的5倍通道，HL2476结合到3倍突起。为了阐明衣壳-抗体的相互作用，对重链和轻链进行了测序，并将它们的坐标与AAV5病毒蛋白一起分配到密度图中。该复合体的高分辨率使人们能够在衣壳的3倍突起鉴定出相互作用的残基，包括R483，它与HL2476的轻链形成两个氢键。产生一组AAV5变异体，并通过天然斑点免疫印迹和转导试验进行分析。基于重组AAVs(RAAVs)的生物制品正日益成为有吸引力的人类基因传递载体，特别是在欧洲批准Glybera和美国批准Luxturna之后。然而，在很大比例的人类群体中，先前存在的针对AAV衣壳的中和抗体限制了这些载体的广泛使用。为了绕过这个问题，必须生成这些抗体无法检测到的隐形载体。这项研究详细描述了AAV5上一个新的抗原区的高分辨率特征，AAV5是一种正在开发的用于多种递送应用的载体。AAV5与新抗体HL2476的结合用低温电子显微镜3.1埃分辨确定。密度图的分辨率使得能够识别衣壳与抗体之间的相互作用残基以及中和的决定因素。因此，本研究获得的信息有助于宿主免疫逃逸载体的生成。
Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington's disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-angstrom resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity.IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit wide-spread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the high-resolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-angstrom resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors.