PROJECT 2:Structural studies of SOSIP trimers

项目2摘要 项目2将生成结构信息，以验证和改进新一代SOSIP的设计 与项目1和核心B密切互动的三聚体项目2将采取高度集成的方法，包括 冷冻和负染色电子显微镜(NS-EM)、X射线结晶学和其他一系列生物物理 工具(SEC-MALS、ITC、DSC和SPR)，以提供对三聚体和 三聚体-bNab络合物。数据将与项目1共享，以提高稳定性、抗原性和 新SOSIP三聚体设计的免疫原性。伊恩·A·威尔逊博士将在斯克里普斯研究中心指导项目2 研究所和安德鲁·B·沃德博士将共同领导。具体目标是： 目的1：鉴定新的SOSIP三聚体的生物物理和结构特征，包括生殖系 瞄准SOSIP三聚体。我们将通过低温EM或X射线结晶学产生高分辨率结构 使NS-EM能够进一步设计SOSIP三聚体和低分辨率结构，以提供快速反馈 关于SOSIP三聚体的质量。我们还将使用NS-EM在压力测试后评估三聚体的质量 模拟疫苗的储存和配制条件，如佐剂混合的效果。这些化验是至关重要的 在动物免疫研究之前，确保只对高质量的三聚体进行评估。 目的2：确定新城疫病毒感染、疫苗诱导的高分辨结构 NABS，或用GT-SOSIP三聚体分离的幼稚前体抗体。我们将解决以下问题的结构 从三聚体免疫和新城疫病毒感染的动物中分离的中和抗体(NAB)，以评估它们与 已知的bNAbs及其发展广度的潜力。我们的目标是进一步优化 SOSIP三聚体上的bNab表位，并有助于设计一组或一系列三聚体，以引导反应向 模拟感染的优质-增强-增强策略中的bNAbs。我们将解决SOSIP三聚体络合物的结构 与胚系前体抗体比较亲和力成熟的bNAbs。我们的目标是设计SOSIP三聚体 用于引导前体抗体走向广度的助推剂。 目的3：设计、优化和确定SOSIP三聚体的结构 纳米颗粒平台。我们将在各种蛋白质纳米颗粒(NPs)中加入SOSIP三聚体， 例如，通过基于结构的设计和测试的迭代循环，双组分自组装NPN。这个 目的是保留SOSIP三聚体理想的抗原性，同时获得免疫学益处 颗粒抗原的呈递。与项目1和核心B合作，我们将评估一系列设计和 从而产生一批将在动物身上进行测试的SOSIP-NP免疫原库。这些项目的结果 研究将进一步指导新的SOSIP-NP结构的设计。

Project 2 Abstract Project 2 will generate structural information to validate and improve the design of new generations of SOSIP trimers in close interaction with Project 1 and Core B. Project 2 will take a highly integrative approach involving cryo- and negative-stain-electron microscopy (NS-EM), X-ray crystallography and a range of other biophysical tools (SEC-MALS, ITC, DSC and SPR) to provide a comprehensive biophysical understanding of trimers and trimer-bnAb complexes. The data will be shared with Project 1 to improve the stability, antigenicity and immunogenicity of new SOSIP trimer designs. Dr. Ian A. Wilson will direct Project 2 at The Scripps Research Institute and Dr. Andrew B. Ward will be co-Leader. The Specific Aims are: Aim 1: To biophysically and structurally characterize new SOSIP trimers, including germline targeting SOSIP trimers. We will generate high-resolution structures by cryo-EM or X-ray crystallography to enable further engineering SOSIP trimers, and low-resolution structures by NS-EM to provide rapid feedback about the quality of SOSIP trimers. We will also use NS-EM to assess the quality of trimers after stress tests that mimic vaccine storage and formulation conditions such as the effect of adjuvant mixing. These assays are critical prior to animal immunization studies, to ensure only high-quality trimers are evaluated. Aim 2: To determine high-resolution structures of SHIV infection-elicited, vaccine-induced NAbs, or naïve precursor antibodies isolated with GT-SOSIP trimers. We will solve structures of neutralizing Abs (NAbs) isolated from trimer-immunized and SHIV-infected animals to assess their similarity to known bNAbs and their potential for developing breadth. The goals are to further optimize the presentation of bNAb epitopes on SOSIP trimers and facilitate the design of sets or series of trimers for steering responses toward bnAbs in prime-boost-boost strategies that mimic infection. We will solve structures of SOSIP trimer complexes with germline precursor antibodies to compare with affinity-matured bnAbs. The goal is to design SOSIP trimer boosts for guiding precursor antibodies toward breadth. Aim 3: To design, optimize and determine structures of SOSIP trimers displayed on nanoparticle platforms. We will incorporate SOSIP trimers into various proteinaceous nanoparticles (NPs), such as the two-component self-assembling NPs, via an iterative cycle of structure-based design and testing. The goal is to preserve the desirable antigenic properties of SOSIP trimers, while gaining the immunological benefits of particulate antigen presentation. Working with Project 1 and Core B, we will evaluate a range of designs and thereby generate an arsenal of SOSIP-NP immunogens that will be tested in animals. The outcomes of those studies will further guide the design of new SOSIP-NP constructs.