Adenovirus hexon and its role in virus interaction with the host

描述(申请人提供)：腺病毒载体(Ad)是美国临床试验中第二常用的载体，用于治疗包括癌症在内的许多先天和获得性人类疾病。虽然到目前为止还没有发现治疗播散性转移性肿瘤疾病的方法，但目前公认的是，播散性转移性肿瘤可以通过全身输送途径，如血管系统，进入转移肿瘤可能存在的所有身体部位进行治疗。然而，当使用这种途径实现全身腺病毒传递时，超过90%的给药载体剂量被肝脏迅速隔离，导致病毒灭活，降低肝外基因转移的效果，并引发全身先天免疫和炎症反应。虽然体外建立的Ad细胞感染模型假定了Ad纤维和五角体蛋白在介导病毒进入细胞中的关键作用，但我们的体内分析表明，血管内递送后，主要的Ad衣壳蛋白-六邻体-在驱动病毒在肝脏中的隔离和肝细胞转导方面发挥了主要的机制作用。重要的是，我们的初步研究强烈表明，循环抗体与溶剂暴露的高可变六邻体环的特定相互作用机械地定义了病毒与Kupffer细胞的相互作用，导致病毒在肝脏中捕获和灭活。此外，我们的初步研究还表明，只有同时灭活腺病毒与肝细胞、肝窦内皮细胞和库普弗细胞的相互作用，才能使病毒在血管内输送后逃脱隔离在肝脏中。虽然已经描述了在肝细胞转导或与Kupffer细胞相互作用时减弱的Ad载体，但到目前为止，还没有发表的研究提供直接和明确的证据表明这种载体在血管内注射后不久就逃脱了肝隔离。基于不同肝细胞隔室在从血液中隔离Ad的等同功能的新概念，在这项提议中，我们将填补我们对Ad Hexon在引导病毒在血管内传递后的生物分布和感染性方面的作用的认识的空白。通过结构低温电子显微镜(Cryo-EM)和计算分析方法和定点突变的结合，在这项建议中，我们将1)确定腺病毒六邻体与低亲和力天然抗体(IgM)和高亲和力鼠和人抗体(IgG)相互作用的表面区。我们还将2)确定Hexon HVR1环变异在病毒感染、复制和Kupffer细胞捕获中的作用。最后，我们将使用一组独特的带有修饰的五六子和六边形的载体，3)开发新型的六邻体突变病毒，这种病毒将避免Kupffer细胞捕获，并在血管内传递后抵抗病毒特异性抗体的中和。我们的假设和本申请中提出的数据驱动的研究将极大地促进我们对体内Ad Hexon-宿主细胞和因子相互作用的理解，并最终导致防止Ad从血液中隔离的新策略的实验验证。这些策略的概念和实验验证将代表着朝着开发安全有效、系统适用的Ad载体迈出的重要一步，这些载体可用于人类的多种治疗应用。

DESCRIPTION (provided by applicant): Adenovirus vectors (Ad) are the second most frequently used vectors in clinical trials in the US to treat numerous inborn and acquired human diseases, including cancer. Although no cure so far is found for disseminated metastatic tumor disease, it is currently accepted that disseminated metastases can potentially be treated through a systemic delivery routes, such as vasculature, to allow for access to all body sites were metastatic tumors may reside. However, upon using this route to achieve systemic adenovirus delivery, over 90% of the administered vector dose is rapidly sequestered by the liver, leading to virus inactivation, reducing the efficacy of extra-hepatic gene transfer, and triggering systemic innate immune and inflammatory responses. Although the in vitro-derived model of Ad cell infection postulates key roles for Ad fiber and penton proteins in mediating virus entry into cells, our in vivo analyses demonstrate that after intravascular delivery, the major Ad capsid protein - hexon - plays the principal mechanistic role in driving virus sequestration in the liver and hepatocyte transduction. Importantly, our preliminary studies strongly suggest that specific interactions of circulating antibodies with solvent-exposed hyper-variable hexon loops mechanistically define virus interaction with Kupffer cells, leading to virus trapping in the liver and inactivation. Furthermore, our preliminary studies also demonstrated that only simultaneous inactivation of adenovirus interactions with hepatocytes, sinusoid endothelial cells, and Kupffer cells allows for virus escape from being sequestered in the liver after intravascular delivery. Although Ad vectors that are attenuated at either hepatocyte transduction or interaction with Kupffer cells have been described, to date, there are no studies published that provide direct and definitive evidence that such vectors escape liver sequestration shortly after intravascular injection. Based on the novel concept of equifunctional role of different hepatocellular compartments in sequestering Ad from the blood, in this proposal we will fill the gap in our knowledge of the role of Ad hexon in guiding virus bio- distribution an infectivity after intravascular delivery. Through a combination of structural cryo-electron- microscopy (cryo-EM) and computational methods of analysis and site-directed mutagenesis, in this proposal we will 1) determine the surface regions of adenovirus hexon that interact with low affinity natural antibodies (IgM) and high affinity mouse and human antibodies (IgG). We will also 2) determine the role of the hexon HVR1 loop variation in virus infection, replication, and Kupffer cell trapping. Finally, using a set of unique vectors with modified pentons and hexons, we will 3) develop novel hexon-mutated viruses that will avoid Kupffer cell trapping and resist neutralization with virus-specific antibodies after intravascular delivery. Our hypothesis and data-driven studies proposed in this application will greatly advance our understanding of Ad hexon - host cell and factor interactions in vivo and should ultimately lead to the experimental validation of novel strategies to prevent Ad sequestration from the blood. Conceptual and experimental validation of these strategies would represent a major step toward the development of safe and effective systemically-applicable Ad vectors for numerous therapeutic applications in humans.