Adenovirus hexon and its role in virus interaction with the host

腺病毒六邻体及其在病毒与宿主相互作用中的作用。 项目总结/摘要： 腺病毒载体（Ad）是美国临床试验中第二常用的载体， 包括癌症在内的许多先天和后天人类疾病。虽然目前还没有找到治愈的方法 对于播散性转移性肿瘤疾病，目前公认播散性转移可 可以通过全身递送途径（例如脉管系统）进行治疗，以允许进入所有的 身体部位可能存在转移性肿瘤。然而，当使用这种途径来实现系统性时， 在腺病毒递送中，超过90%的施用载体剂量被肝脏迅速隔离， 病毒灭活，降低肝外基因转移的功效，并引发全身先天性 免疫和炎症反应。尽管体外来源的Ad细胞感染模型假设 Ad纤维和五邻体蛋白在介导病毒进入细胞中的关键作用，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-起主要作用。 在驱动肝脏中的病毒隔离和肝细胞转导中的机制作用。重要的是我们的 初步研究强烈提示，循环抗体与溶剂暴露的 高变六邻体环在机制上限定了病毒与枯否细胞相互作用，导致病毒 滞留在肝脏中并灭活。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， 库普弗细胞允许病毒在血管内感染后从被隔离在肝脏中逃逸 交付.尽管在肝细胞转导或与肝细胞相互作用时减毒的Ad载体， 库普弗细胞已被描述，迄今为止，没有研究发表，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 不同肝细胞隔室在隔离Ad中的等功能作用的新概念 血液，在这个建议中，我们将填补我们的知识差距的差距的作用，广告六邻体在引导病毒生物学。 在血管内递送后的分布和传染性。通过结构低温电子- 显微镜（cryo-EM）和分析和定点诱变的计算方法，在此 建议我们将1）确定腺病毒六邻体的表面区域，其与低亲和力的天然 抗体（IgM）和高亲和力小鼠和人抗体（IgG）。我们还将确定 病毒感染、复制和枯否细胞捕获中的六邻体HVR 1环变异。最后，使用一组 我们将开发新型的六邻体突变病毒， 将避免枯否细胞捕获和抵抗中和病毒特异性抗体后，血管内 交付.我们在本申请中提出的假设和数据驱动的研究将大大推进我们的研究。 了解Ad六邻体-宿主细胞和因子在体内的相互作用，并最终导致 实验验证新的策略，以防止从血液中的广告隔离。概念和 这些策略的实验验证将是朝着安全发展的重要一步。 和有效的全身可应用的Ad载体，用于人类的多种治疗应用。

Adenovirus hexon and its role in virus interaction with the host. PROJECT SUMMARY/ABSTRACT: 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 and 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.