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

描述(由申请人提供)：这个探索性/开发性项目结合了康奈尔大学和佛罗里达大学实验室的资源和技能，研究确定3种不同血清型的腺相关病毒(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性质。虽然目前已有几种AAV衣壳的高分辨结构，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，该项目的总体目标是利用小鼠抗AAV1、AAV2和AAV5衣壳的单抗(MAb)来确定抗体与衣壳相互作用的结构基础，这些衣壳代表了人类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克隆。我们已经准备了针对AAV1和AAV5衣壳的单抗面板，这些单抗将作为免疫球蛋白或纤维蛋白进行测试，以结合和中和各种病毒。此外，一些AAV2抗体已经上市。抗体或抗体结构域将被用来选择逃逸突变体，观察到的序列变化将被用来确定衣壳表面表位的性质。冷冻电子显微镜和Fab/衣壳复合体的图像重建也将被用来定义几种抗体与衣壳表面的相互作用，以达到中等高分辨率。这些数据将被用来定义天然衣壳上的主要免疫原性结构，以确定衣壳是否利用共同的表位，并将允许比较每种方法获得的结果。结合的信息将用于指导衣壳突变体的制备，该突变体旨在避免与衣壳的大多数抗体结合或中和，同时保持正常的传染性和细胞或组织嗜性。突变的衣壳基因将被用来产生AAV载体，该载体将被各种免疫球蛋白和免疫球蛋白M单抗以及多克隆抗体进行结合和中和测试。这些衣壳还将被测试其特定的受体结合和转导/趋向性特性。公共卫生意义：抗体是对抗所有脊椎动物病毒的中心防御，但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疑问。AAV的多种血清型以未经修饰或修饰的形式被广泛用作具有不同性质的载体；然而，许多人携带针对各种血清型的中和抗体，而AAV载体的使用可以导致抗体免疫的发展。在这项研究中，我们试图确定病毒的抗原性结构，以便更好地了解抗体对基因治疗试验的不利影响，并可能在未来避免。

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.