Adenovirus hexon and its role in virus interaction with the host

描述（由申请人提供）：腺病毒载体（Ad）是美国临床试验中第二常用的载体，用于治疗多种先天性和获得性人类疾病，包括癌症。尽管迄今为止还没有发现播散性转移性肿瘤疾病的治愈方法，但目前公认的是，播散性转移可以通过全身递送途径（例如脉管系统）进行治疗，以允许进入转移性肿瘤可能存在的所有身体部位。然而，在使用该途径实现全身性腺病毒递送时，超过90%的施用载体剂量被肝脏快速隔离，导致病毒失活，降低肝外基因转移的功效，并引发全身性先天免疫和炎症反应。虽然在体外衍生的模型的Ad细胞感染假定的关键作用，Ad纤维和五邻体蛋白介导的病毒进入细胞，我们在体内的分析表明，血管内输送后，主要的Ad衣壳蛋白-六邻体-在驱动病毒隔离在肝脏和肝细胞转导的主要机制的作用。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变六邻体环的特异性相互作用机制上定义了病毒与枯否细胞的相互作用，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、窦状内皮细胞和枯否细胞的相互作用，才能使病毒在血管内递送后逃离被隔离在肝脏中的状态。尽管已经描述了在肝细胞转导或与库普弗细胞相互作用时减弱的Ad载体，但迄今为止，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脏隔离。基于不同肝细胞区室在从血液中隔离Ad中的等功能作用的新概念，在该提议中，我们将填补我们对Ad六邻体在引导病毒生物分布和血管内递送后的感染性中的作用的知识中的差距。通过结构冷冻电子显微镜（cryo-EM）和分析的计算方法以及定点诱变的组合，在该提议中，我们将1）确定与低亲和力天然抗体（IgM）和高亲和力小鼠和人抗体（IgG）相互作用的腺病毒六邻体的表面区域。我们还将2）确定六邻体HVR 1环变异在病毒感染、复制和枯否细胞捕获中的作用。最后，使用一组具有修饰的五邻体和六邻体的独特载体，我们将3）开发新型六邻体突变病毒，其将避免枯否细胞捕获并在血管内递送后抵抗病毒特异性抗体的中和。本申请中提出的我们的假设和数据驱动的研究将极大地推进我们对体内Ad六邻体-宿主细胞和因子相互作用的理解，并最终导致实验验证新策略以防止Ad从血液中隔离。这些策略的概念和实验验证将代表朝着开发安全有效的全身适用的Ad载体以用于人类的许多治疗应用迈出的重要一步。

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.