Project 1. Design of immunogens that bind to the UCA and to IAs and mature DH511 bnAbs in optimal affinities

项目 1. 设计以最佳亲和力与 UCA 和 IAs 以及成熟 DH511 bnAb 结合的免疫原

• 批准号: 10132977
• 负责人: S. Munir ALAM
• 金额: $ 32.5万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-09 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132977
• 关键词: ART protein; Affinity; Animal Model; Animals; Antibodies; Antigens; Avidity; B-Cell Activation; B-Cell Antigen Receptor; B-Lymphocytes; Binding; Biological Assay; Cell Separation; Complement; Complex; Cryoelectron Microscopy; Crystallization; Cytometry; Development; Directed Molecular Evolution; Distal; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Epitopes; Feedback; Genetic Structures; Glycoproteins; Goals; HIV; HIV vaccine; HIV-1; Head; Health Priorities; Human; Immunity; Immunization; In Vitro; Knock-in; Knock-in Mouse; Lead; Length; Lipid Binding; Lipids; Liposomes; Location; Masks; Membrane; Membrane Proteins; Methods; Molecular Conformation; Mus; Mutate; Mutation; Passive Immunization; Pathway interactions; Peptides; Polysaccharides; Protein Engineering; Proteins; Regimen; Resolution; Scaffolding Protein; Specificity; Structure; Techniques; Testing; Vaccination; Vaccine Research; Vaccines; Variant; Viral; Virus; X-Ray Crystallography; Yeasts; autoreactivity; base; cross reactivity; design; env Gene Products; experimental study; global health; immunogenicity; improved; in vivo; interest; member; mouse model; nanodisk; nanoparticle; neutralizing antibody; next generation sequencing; novel; reconstitution; response; restraint; scaffold; simian human immunodeficiency virus; vaccine candidate; vaccine development

Induction of broadly neutralizing antibodies (bnAbs) is a critical unmet goal of HIV vaccine development. BnAb DH511 is of high interest as a vaccine lead due to its very high breadth and the high in vivo protective potency of MPER bNAbs. Key challenges for inducing DH511-like bnAbs are: (a) the low affinity of DH511- like precursors for HIV peptides and proteins, (b) the restriction on bnAb angle of approach imposed by the recessed, membrane-proximal epitope environment and (c) the absence of the DH511 epitope from most soluble, native-like trimers. A promising strategy to initiate DH511-like bnAb induction is germline targeting, in which suitable DH511-class precursors are specifically activated using engineered immunogens, thus selecting BCRs with the potential to develop broad neutralization in the absence of autoreactivity. This approach will also help circumvent steric problems associated with the recessed location of the epitope, by priming precursors with known genetic and structural potential to mature into bnAbs compatible with MPER steric restraints. In this project, which is Project 1 of a multi-project collaborative proposal, we will engineer epitope-scaffold immunogens that bind with high affinity to and activate DH511-like precursors, using computational design and directed evolution. As known bnAbs are highly mutated, vaccine induction of bnAbs following a germline-targeting prime will likely require sequential immunization with other immunogens designed to shepherd affinity maturation of the B-cell receptor. We will develop different classes of boosting immunogens, including epitope- scaffolds with more native epitopes, membrane-protein scaffolds and membrane-bound Env variants stabilized in a conformation to which DH511 binds strongly. Structural studies of soluble and membrane- bound immunogens in complex with DH511 lineage members will guide immunogen development. The Animal Core of this collaborative proposal will generate knock-in mice that express DH511-like precursors, and Project 2 will use those mice to test B cell priming and boosting in vivo. Project 2 will conduct sequential prime/boost immunization experiments in knock-in mice and use ELISA, cytometry, single B cell sorting and sequencing and neutralization assays to track and optimize affinity maturation, providing experimental feedback to Project 1 to allow for iterative improvement of immunogens. In summary, these studies seek to develop novel HIV vaccine candidates and also to shift HIV vaccine research towards a reductionist approach based on state-of-the-art protein engineering to develop germline-targeting and boosting immunogens, development of human Ig knock-in mouse models to enable testing of human-repertoire-specific vaccines, and in-depth analysis of vaccine-induced affinity maturation pathways in vivo to guide iterative vaccine optimization.

广泛中和抗体（BNAB）的诱导是HIV疫苗发育的关键未得到的目标。 BNAB DH511由于其很高的宽度和高体内保护性而引起了疫苗的高度兴趣 MPER BNABS的效力。诱导DH511样BNAB的主要挑战是：（a）DH511-的低亲和力 - 像HIV肽和蛋白质的前体一样，（b）对BNAB接近角度的限制 嵌入式，膜 - 膜的表位环境和（c）大多数DH511表位的不存在 可溶性，类似于本地的三聚体。 启动类似DH511的BNAB诱导的有前途的策略是种系靶向，其中合适 DH511级的前体是使用工程免疫剂特异性激活的，从而选择了BCR 在没有自动反应性的情况下发展广泛中和的潜力。这种方法也将有所帮助 通过启动前体的启动前体 已知的遗传和结构潜力，使其成熟成与MPER空间约束兼容的BNAB。在这个 Project是多项目协作提案的项目1 使用计算设计，与高亲和力结合并激活类似DH511的前体的免疫原子 和指向进化。 由于已知的bnab是高度突变的，因此在靶向种系的prime之后诱导BNAB的疫苗 可能需要对旨在牧羊人亲和力成熟的其他免疫原子进行顺序免疫 B细胞受体的。我们将开发不同类别的增强免疫原，包括表位 - 带有更多本地表位，膜 - 蛋白质脚手架和膜结合的Env变体的脚手架 DH511强烈结合的构象稳定。可溶性和膜的结构研究 与DH511谱系成员复合物中结合的免疫原子将指导免疫原。 该协作提案的动物核心将产生表达DH511的敲门型小鼠 前体和项目2将使用这些小鼠在体内测试B细胞启动和增强。项目2将 在敲入小鼠中进行顺序素/增强免疫实验，并使用ELISA，细胞术， 单个B细胞分类，测序和中和测定，以跟踪和优化亲和力成熟， 提供对项目1的实验反馈，以允许迭代化免疫原子。 总而言之，这些研究试图开发新的HIV疫苗候选物并转移HIV疫苗 基于最先进的蛋白质工程的还原主义方法的研究 生殖线靶向和增强免疫原，开发人IG敲击小鼠模型以启用 测试人类替代特异性疫苗，并深入分析疫苗诱导的亲和力成熟 体内途径可指导迭代疫苗优化。