Epitope Discovery via Nanocluster Presentation

描述（由申请人提供）：本提案的目的是开发一种多功能纳米颗粒平台，用于在金纳米簇上进行表位呈递，并进行免疫学分析，以创建靶向免疫系统的纳米技术。基于纳米颗粒的疫苗的未来发展需要产生忠实地再现病原微生物的保护性抗原的构象的蛋白质结构。具有讽刺意味的是，我们在逻辑上设计具有保护性的重组或合成蛋白质抗原的能力相当有限。由于天然蛋白质上的大多数表位可能是组装的，因此表位测定工作面临的最重大挑战之一是鉴定构象组装的表位。我们假设，在金纳米团簇表面上的靶蛋白环结构的呈现将呈现潜在的环构象的统计上显著的多样性，从而为发现致病毒素的未知表位的新方法提供基础。为了证明这种方法的可行性，我们正在创建一个表位标记的纳米簇，模拟炭疽杆菌的保护性抗原（PA）。在这个提议中，我们的目标是通过以下具体目标来完善和扩展我们在纳米簇上的表位呈递的概念策略：1.在金纳米团簇表面设计、合成和组装线性和构象表位。2.确定哪些构象肽环构建体忠实地代表天然蛋白质中天然存在的免疫原性结构。3.筛选小鼠和人类抗体库，以及使用纳米簇上的表位的患者血清，致力于开发诊断微阵列。4.优化多功能纳米颗粒在小鼠体内的免疫应答。纳米技术的具体挑战将是在纳米颗粒表面上创建特定的子结构，这些特定构象的免疫识别，多功能纳米颗粒的创建，以及作为纳米颗粒尺寸和曲率的函数的肽环结构的相关性。生物学和医学的具体变化包括未映射的线性和构象表位的发现，免疫系统对功能化纳米颗粒的反应，构象表位的抗体库的筛选，以及最终发展为构象表位标记的纳米颗粒作为潜在的疫苗。

DESCRIPTION (provided by applicant): The purpose of this proposal is to develop a multifunctional nanoparticle platform for epitope presentation on gold nanoclusters with immunological analysis for the creation of nanotechnologies that target the immune system. The future development of nanoparticle-based vaccines requires the generation of protein structures that faithfully recapitulate the conformation of protective antigens of pathogenic microorganisms. Ironically, our ability to logically design recombinant or synthetic protein antigens that are protective is quite limited. As most epitopes on native proteins are likely to be assembled, one of the most significant challenges facing epitope determination efforts is the identification of conformationally assembled epitopes. We hypothesize that the presentation of target protein loop structures on the surface of gold nanoclusters will present a statistically significant diversity of potential loop conformations to provide the foundation of a new method for discovering unknown epitopes of pathogenic toxins. To demonstrate the feasibility of this approach, we are creating an epitope-labeled nanocluster that mimics the protective antigen (PA) of Bacillus anthracis. In this proposal, our goal is to refine and extend our conceptual strategies for epitope presentation on nanoclusters via the following specific aims: 1. Design, synthesize, and assemble linear and conformational epitopes on the surface of gold nanoclusters. 2. Determine which of the conformational peptide loop constructs faithfully represent the naturally occurring immunogenic structures in native protein. 3. Screen mouse and human antibody libraries, and patient sera using epitopes presented on nanoclusters working towards the development of a diagnostic microarray. 4. Optimize the immunological response of multifunctional nanoparticles in mice. Specific challenges in nanotechnology will be the creation of specific substructures on a nanoparticle surface, immunorecognition of these specific conformations, creation of multifunctional nanoparticles, and correlation of peptide loop structure as a function of nanoparticle size and curvature. Specific changes in biology and medicine include the discovery of unmapped linear and conformational epitopes, the immune system response to functionalized nanoparticles, the screening of antibodies libraries for conformational epitopes, and ultimate development towards conformational epitope-labeled nanoparticles as potential vaccines.