Antigenic structure of adeno-associated virus capsids and antibody escape mutants

腺相关病毒衣壳和抗体逃逸突变体的抗原结构

• 批准号: 7314575
• 负责人: Colin R. Parrish
• 金额: $ 19.81万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-21 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7314575
• 关键词: Adverse effects; Antibodies; Antibody Diversity; Antigenic Diversity; Binding; Binding Sites; Capsid; Cells; Common Epitope; Complementarity Determining Regions; Complex; Cryoelectron Microscopy; Data; Dependovirus; Development; Epitopes; Escape Mutant; Expression Library; Florida; Future; Genes; Goals; Human; Human Cloning; Immunity; Immunoglobulin G; Immunoglobulin M; Infection; Laboratories; Lead; Libraries; Light; Maps; Methods; Modeling; Monoclonal Antibodies; Mus; Normal Cell; Numbers; Preparation; Process; Property; Proteins; Public Health; Range; Resolution; Resources; Screening procedure; Serotyping; Site; Structure; Surface; Testing; Tropism; Universities; Variant; Vertebrate Viruses; Viral; Virus; Work; Yeasts; adeno-associated viral vector; base; cross reactivity; design; gene therapy; image reconstruction; immunogenic; improved; mutant; neutralizing antibody; novel strategies; polyclonal antibody; prevent; receptor binding; response; skills; tissue tropism; tissue/cell culture; vector

DESCRIPTION (provided by applicant): This Exploratory/Developmental project combines the resources and skills of laboratories at Cornell University and the University of Florida in studies to define the antigenic structures of the capsids of 3 different serotypes of adeno-associated virus (AAV). We would use the information obtained to prepare variant viruses with improved antigenic properties for applications in corrective human gene therapy as well as to understand the general properties of antibody binding and neutralization of viruses by antibodies. Although the high resolution structures of several AAV capsids are now available, there is still little information about their general or specific antigenic structures. The overall goals of this project are therefore to define the structural bases of antibody-capsid interactions using mouse IgG or IgM monoclonal antibodies (MAb) to the AAV1, AAV2, and AAV5 capsids, which are representative of the antigenic range of AAV serotypes in humans. We would also isolate clones of human antibody sequences that recognize the capsids by screening a yeast expression library of human antibody VH and VL sequences. We have already prepared panels of MAb against AAV1 and AAV5 capsids which will be tested as IgGs or Fabs for binding and neutralization of the various viruses. In addition, a number of AAV2 antibodies are already available. Antibodies or antibody domains will be used to select escape mutants, and the sequence variation observed will be used to define properties of the epitopes on the capsid surfaces. Cryo-electron microscopy and image reconstruction of Fab/capsid complexes will also be used to define to moderately high resolution the interactions of several antibodies with the capsid surfaces. These data will be used to define the dominant immunogenic structures on the native capsids, to determine if the capsids utilize common epitopes, and will allow comparison of the results obtained with each of the methods. The combined information will used to guide the preparation of capsid mutants designed to avoid binding or neutralization by most of the antibodies to the capsids, while retaining normal infectivity and cell or tissue tropisms. Mutant capsid genes would be used to generate AAV vectors which would be tested for binding to and neutralization by the various IgG and IgM MAbs and also by polyclonal antibodies. These capsids would also be tested for their specific receptor binding and transduction/tropism properties. Public health significance: Antibodies are central defenses against all viruses of vertebrates, but questions remain about the structural bases of antibody binding to the capsids, the bases of antigenic diversity and variation, as well as the mechanisms of neutralization. The many serotypes of AAV are being widely used in unmodified or modified forms as vectors with distinct properties; however many people carry neutralizing antibodies against various serotypes, and the use of AAV vectors can lead to the development of antibody immunity. In this study we seek to define the antigenic structures if the viruses so that the adverse effects of antibodies on gene therapy trials can be better understood, and perhaps avoided in the future.

描述（由申请人提供）：该探索/开发项目结合了康奈尔大学和佛罗里达大学实验室在研究中的资源和技能，以确定3种不同血清型腺相关病毒（AAV）衣壳的抗原结构。我们将利用所获得的信息来制备具有改进的抗原特性的变异病毒，用于矫正人类基因治疗，以及了解抗体结合和抗体中和病毒的一般特性。虽然现在可以获得几种AAV衣壳的高分辨率结构，但关于它们的一般或特异性抗原结构的信息仍然很少。因此，本项目的总体目标是使用针对AAV 1、AAV 2和AAV 5衣壳的小鼠IgG或IgM单克隆抗体（MAb）定义抗体-衣壳相互作用的结构基础，这些抗体代表人类AAV血清型的抗原范围。我们还将通过筛选人抗体VH和VL序列的酵母表达文库来分离识别衣壳的人抗体序列的克隆。我们已经制备了针对AAV 1和AAV 5衣壳的MAb组，其将作为IgG或Fab测试各种病毒的结合和中和。此外，许多AAV 2抗体已经可用。抗体或抗体结构域将用于选择逃逸突变体，并且观察到的序列变异将用于定义衣壳表面上的表位的性质。冷冻电子显微镜和Fab/衣壳复合物的图像重建也将用于以中等高分辨率定义几种抗体与衣壳表面的相互作用。这些数据将用于定义天然衣壳上的显性免疫原性结构，以确定衣壳是否利用共同表位，并将允许比较用每种方法获得的结果。组合的信息将用于指导衣壳突变体的制备，所述衣壳突变体被设计成避免大多数抗体与衣壳结合或中和，同时保留正常的感染性和细胞或组织向性。突变体衣壳基因将用于产生AAV载体，其将被测试与各种IgG和IgM MAb以及多克隆抗体的结合和中和。还将测试这些衣壳的特异性受体结合和转导/向性特性。公共卫生意义：抗体是脊椎动物抵抗所有病毒的核心防御手段，但关于抗体与衣壳结合的结构基础、抗原多样性和变异的基础以及中和机制仍存在问题。AAV的许多血清型以未修饰或修饰的形式作为具有不同性质的载体被广泛使用;然而，许多人携带针对各种血清型的中和抗体，并且AAV载体的使用可导致抗体免疫的发展。在这项研究中，我们试图定义病毒的抗原结构，以便更好地了解抗体对基因治疗试验的不良影响，并可能在未来避免。