Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8
Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8
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DOI：
10.1128/jvi.00218-12
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发表时间：
2012-08-01
影响因子：
5.4
通讯作者：
Agbandje-McKenna, Mavis
中科院分区：
文献类型：
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作者：
Gurda, Brittney L.;Raupp, Christina;Agbandje-McKenna, Mavis
腺相关病毒（Adeno-associated viruses，AAV）是一种小的单链DNA病毒，可以包装和递送非基因组DNA用于治疗性基因递送。AAV 8是一种嗜肝性载体，已显示出治疗血友病A和B的巨大前景。然而，与其他AAV载体一样，宿主抗衣壳免疫应答是治疗成功的阻碍。为了表征该载体的抗原结构，使用冷冻电子显微镜和图像重建（冷冻重建）结合分子遗传学、生物化学和体内方法来定义中和单克隆抗体ADK 8的AAV 8衣壳表面上的抗原表位。将AAV 8和通用Fab的晶体结构对接到AAV 8-ADK 8复合物的冷冻重建中鉴定了位于二十面体对称衣壳的3重轴侧面的突出突起上的足迹。诱变和细胞结合研究以及体外和体内转导测定沿着显示，主要ADK 8表位由AAV可变区VRVIII（氨基酸586至591 [AAV 8 VPI编号]）形成，其位于面向3倍轴的突起表面上。该区域在AAV 2和AAV 8细胞转导中起作用。巧合的是，细胞结合和运输试验表明，ADK 8影响成功将病毒运输到细胞核所需的后步骤，表明可能的中和机制。因此，这种用于表征AAV的抗原区域的结构导向策略可以产生有用的信息，以帮助重新设计逃避宿主中和并因此更有效的载体。
Adeno-associated viruses (AAVs) are small single-stranded DNA viruses that can package and deliver nongenomic DNA for therapeutic gene delivery. AAV8, a liver-tropic vector, has shown great promise for the treatment of hemophilia A and B. However, as with other AAV vectors, host anti-capsid immune responses are a deterrent to therapeutic success. To characterize the antigenic structure of this vector, cryo-electron microscopy and image reconstruction (cryo-reconstruction) combined with molecular genetics, biochemistry, and in vivo approaches were used to define an antigenic epitope on the AAV8 capsid surface for a neutralizing monoclonal antibody, ADK8. Docking of the crystal structures of AAV8 and a generic Fab into the cryo-reconstruction for the AAV8-ADK8 complex identified a footprint on the prominent protrusions that flank the 3-fold axes of the icosahedrally symmetric capsid. Mutagenesis and cell-binding studies, along with in vitro and in vivo transduction assays, showed that the major ADK8 epitope is formed by an AAV variable region, VRVIII (amino acids 586 to 591 [AAV8 VPI numbering]), which lies on the surface of the protrusions facing the 3-fold axis. This region plays a role in AAV2 and AAV8 cellular transduction. Coincidently, cell binding and trafficking assays indicate that ADK8 affects a postentry step required for successful virus trafficking to the nucleus, suggesting a probable mechanism of neutralization. This structure-directed strategy for characterizing the antigenic regions of AAVs can thus generate useful information to help re-engineer vectors that escape host neutralization and are hence more efficacious.