Structure based design of trimer interface epitope focused universal influenza vaccines

本申请中的“免疫计算模型”项目侧重于开发和 流感血凝素(HA)蛋白新型结构设计工具的实现 三聚体界面特异性抗体或疫苗抗原。这些项目将利用关于 人抗HA头部三聚体界面中和抗体的结构和功能 我们已经拥有或将会发现，以设计新的抗体或疫苗在硅胶。我们 可以从一组先前自然感染的受试者中获取外周血细胞， 或暴露于编码季节性HA分子的疫苗的实验性接种中 疫苗和不寻常的实验性流感亚型，包括H3变种、H5、H6、H7、H9和 H10病毒。这些人的免疫B记忆细胞群是理想的起点 分离不寻常的异型抗体的材料。最近，我们确定了医管局的头部修剪分子 接口是通用流感抗体和候选抗体的主要新易感部位 疫苗。在这里，我们将研究现有的并分离出更多广泛异型的人 抗HA头部三聚体界面的抗体。我们将测定人类的免疫组学 利用高通量下一代抗体测序反应异亚型克隆 包括所分离的大多数异型抗体的克隆谱系的基因谱系。 一旦分离出具有异常广度或活性的抗体，这些抗体的结构 将与结构核心中纯化的HA分子形成络合物，使用 结晶学和单粒子电子显微镜(EM)研究。这样的结构将提供 使用Rosetta进行建模实验的坐标。我们将在硅胶成熟的人类中 增加对特定病毒类型的HA抗原的亲和力的抗体并使用多状态 设计以最大限度地扩大广度，即创建识别所有分支的HAS的抗体， 子类型、组甚至类型。然后我们将合成并表达这些新的抗体 并测定所设计的中和活性、结合亲和力和竞争结合基团 抗体，使用多样化的HA面板和伪型病毒与所有A型都有本质上的。这个 这些人抗体与HA的共晶结构将成为硅胶设计中的模板 结构稳定的表位聚焦免疫原。我们将首先验证这些设计 通过测试与目标人类抗体的相互作用来获得免疫原。此外，这些 免疫原将通过评估免疫反应进行实验测试。然后，我们将使用 用于从自然暴露于流感的对象中分离新抗体的新型免疫原，以 表明免疫原呈现可被自然免疫反应识别的抗原。

The “Computational Models of Immunity” projects in this application focus on development and implementation of new structure-based design tools for influenza hemagglutinin (HA) protein trimer interface specific antibodies or vaccine antigens. These projects will use knowledge about the structure and function of human neutralizing antibodies to the trimer interface of the HA head that we have in hand or will discover, in order to design new antibodies or vaccines in silico. We have access to peripheral blood cells from a diverse panels of subjects with prior natural infection, or exposure to experimental inoculation with vaccines encoding HA molecules with both seasonal vaccines and unusual experimental influenza subtypes, including H3variant, H5, H6, H7, H9, and H10 viruses. The immune B memory cell populations from these individuals are the ideal starting materials to isolate unusual heterosubtypic antibodies. Recently, we identified the HA head trimer interface as a major new site of vulnerability for universal influenza antibodies and candidate vaccines. Here, we will study existing and isolate additional broadly heterosubtypic human antibodies to the trimer interface of the HA head. We will determine the immunome of the responding heterosubtypic clones using high-throughput next generation sequencing of antibody gene repertoires that comprise the clonal lineages of the most heterosubtypic antibodies isolated. Once antibodies with unusual breadth or activity are isolated, the structure of these antibodies will be determined in complex with purified HA molecules in the Structural Core using crystallography and single particle electron microscopy (EM) studies. Such structures will provide the coordinates for the modeling experiments using Rosetta. We will in silico mature human antibodies to increase affinity for the HA antigen of specific virus types and use multi-state design to maximize breadth, i.e., create antibodies that recognize HAs of all clades, subtypes, groups, or even types. We then will synthesize and express these novel antibodies and determine neutralization activity, binding affinity, and competition binding groups of designed antibodies, using a diverse HA panel and pseudotyped viruses with all type A HAs in nature. The co-crystal structure of these human antibodies with HA will be the template for in silico design of structurally stable epitope-focused immunogens. We will first validate these designed immunogens by testing the interaction with the target human antibodies. Further, these immunogens will be experimentally tested by evaluating immune responses. Then, we will use the novel immunogens to isolate new antibodies from subjects naturally exposed to influenza, to show that the immunogens present antigens recognized by natural immune responses.