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

基本信息

批准号：
8852065
负责人：
PHOEBE L STEWART
金额：
$ 58.75万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8852065
关键词：
Ablation Adenovirus Vector Adenovirus hexon capsid protein Adenoviruses Affinity Antibodies Applications Grants Area Attenuated Automobile Driving Biology Blood Blood coagulation Capsid Capsid Proteins Cells Clinical Clinical Trials Complement Receptor Complex Computing Methodologies Cryoelectron Microscopy Data Development Discontinuous Capillary Disease Dose Drug Kinetics Endothelial Cells Ensure Epitopes Excision Fiber Gene Transfer Goals Health Hepatic Hepatocyte Histocompatibility Testing Human Human body Immune response Immunity Immunoglobulin G Immunoglobulin M In Vitro Individual Infection Inflammatory Response Injection of therapeutic agent Integration Host Factors Knowledge Kupffer Cells Lead Link Liver Liver Circulation Malignant Neoplasms Mediating Modeling Molecular Mus Mutate Mutation Neoplasm Metastasis Peptides Play Proteins Publishing RGD (sequence)Resolution Risk Role Route Serotyping Site Site-Directed Mutagenesis Solvents Structural Models Surface Therapeutic Therapeutic Intervention Tissues Validation Variant Virion Virus Virus Diseases Virus Inactivation adenovirus penton protein base cell type cellular transduction hepatic sinusoid human disease in vivo macrophage neoplastic cell novel novel strategies particle prevent receptor scavenger receptor vector

项目摘要

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.

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后天性人类疾病，包括癌症。尽管迄今为止还没有找到治愈播散性转移性肿瘤疾病的方法，但目前人们普遍认为，播散性转移瘤可以通过全身输送途径（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，在使用这种途径实现全身腺病毒递送时，超过90%的施用载体剂量被肝脏迅速隔离，导致病毒失活，降低肝外基因转移的功效，并引发全身先天免疫和炎症反应。尽管 Ad 细胞感染的体外衍生模型假设 Ad 纤维和五邻体蛋白在介导病毒进入细胞中发挥关键作用，但我们的体内分析表明，在血管内递送后，主要的 Ad 衣壳蛋白 - 六邻体 - 发挥主要机制作用驱动病毒在肝脏中的隔离和肝细胞转导。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超变六邻体环的特异性相互作用从机制上定义了病毒与库普弗细胞的相互作用，导致病毒被捕获在肝脏中并失活。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，病毒才能在血管内递送后逃脱被隔离在肝脏中的情况。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此类载体在血管内注射后不久就逃脱了肝隔离。基于不同肝细胞区室在从血液中隔离 Ad 方面发挥同等作用的新概念，在本提案中，我们将填补 Ad 六邻体在血管内递送后指导病毒生物分布和感染性作用方面的知识空白。通过结合结构冷冻电子显微镜 (cryo-EM) 和分析计算方法以及定点诱变，在本提案中，我们将 1) 确定与低亲和力天然抗体 (IgM) 相互作用的腺病毒六邻体的表面区域以及高亲和力小鼠和人类抗体 (IgG)。我们还将 2) 确定六邻体 HVR1 环变异在病毒感染、复制和库普弗细胞捕获中的作用。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，该病毒将避免库普弗细胞捕获并在血管内递送后抵抗病毒特异性抗体的中和。我们在本申请中提出的假设和数据驱动的研究将极大地促进我们对 Ad 六邻体 - 宿主细胞和体内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的系统适用的 Ad 载体迈出的重要一步，该载体可用于人类的众多治疗应用。