Design and testing of germline-targeting and boosting immunogens to elicit 10E8-like broadly neutralizing antibodies against HIV

设计和测试种系靶向和增强免疫原，以引发针对 HIV 的 10E8 样广泛中和抗体

• 批准号: 10188410
• 负责人: WILLIAM R. SCHIEF
• 金额: $ 90.46万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-08 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188410
• 关键词: ART protein; Adoptive Transfer; Affinity; Animal Model; Antibodies; Antigens; Avidity; B cell differentiation; B-Cell Activation; B-Cell Antigen Receptor; B-Lymphocytes; B-cell receptor repertoire sequencing; Binding; Biological Assay; Biophysics; Cell Separation; Cells; Cytometry; Data; Development; Directed Molecular Evolution; Engineering; Environment; Enzyme-Linked Immunosorbent Assay; Epitopes; Exhibits; Flow Cytometry; Frequencies; Funding; Generations; Genetic Structures; Glycoproteins; Goals; HIV; HIV vaccine; HIV-1; Health Priorities; Human; Immune response; Immunity; Immunization; Immunoglobulin Somatic Hypermutation; In Vitro; Individual; Induced Mutation; Knock-in; Knock-in Mouse; Lead; Liposomes; Location; Mammalian Cell; Masks; Membrane; Membrane Proteins; Messenger RNA; Methods; Modeling; Molecular Conformation; Mus; Mutate; Mutation; Passive Immunization; Pathway interactions; Peptides; Physiological; Polysaccharides; Protein Engineering; Proteins; RNA; Regimen; Replicon; Resistance; Scaffolding Protein; Sorting - Cell Movement; Specificity; Structure; Techniques; Testing; Vaccination; Vaccine Research; Vaccines; Variant; Viral; Virus; Virus-like particle; Wild Type Mouse; Yeasts; autoreactivity; base; cross reactivity; design; env Gene Products; experimental study; global health; glycosylation; in vivo; interest; mouse model; nanoparticle; neutralizing antibody; next generation sequencing; novel; overexpression; protein reconstitution; prototype; 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 10E8 is of particular interest as a vaccine lead due to its (1) very high breadth, (2) low number of mutations required to develop broad neutralization, (3) low auto-reactivity, (4) expected high frequency of precursors in the human na'ive B-cell pool and (5) ability to provide surprisingly potent in vivo sterilizing protection in passive transfer studies. Key challenges for inducing 10E8-like bnAbs are: (a) the low germline affinity by HIV peptides and proteins, (b) the severe restriction on bnAb angle of approach imposed by the recessed, membrane-proximal epitope environment and (c) the absence of the 10E8 epitope from most soluble, native-like trimers (e.g. SOSIP or NFL or UFO). A promising strategy to initiate 10E8-like bnAb induction is germline targeting, in which suitable 10E8-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 proposal, we will engineer epitope-scaffold immunogens that activate 10E8-like precursors, using computational design and directed evolution. We will initially test immunogens ex vivo using human na'ive B cell sorting. We will further generate knock-in mice that over-express 10E8-like precursors, and we will use those mice to test B-cell priming and boosting in vivo, first adoptively transferring knockin B cells to wild- type mice in order to mimic frequencies of 10E8 bnAb precursor and competitor B cells in humans. As known bnAbs are highly mutated, vaccine induction of bnAbs following a germline 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 10E8 binds strongly. We 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. In summary, these studies seek to develop novel HIV vaccine candidates and also to shift HIV vaccine research towards a reductionist approach based on strategic identification of bNAb precursors, 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.

诱导广泛中和抗体（BNABS）是HIV疫苗的关键未得到的目标 发展。 BNAB 10E8由于其（1）非常高，特别是疫苗铅 宽度，（2）发展广泛中和所需的突变数少，（3）低 自动反应性，（4）人类na'ive B细胞池中的前体的高频和（5） 在被动转移研究中提供令人惊讶的有效体内消毒保护保护的能力。 诱导10e8样bnab的关键挑战是：（a）HIV肽和 蛋白质，（b）严重限制嵌入式嵌入式接近角度的bnab角度， 膜 - 透明体表位环境和（c）最可溶的10e8表位， 类似天然的三聚体（例如SOSIP或NFL或UFO）。 启动10E8样BNAB诱导的有前途的策略是种系靶向，其中合适 使用工程免疫剂专门激活10E8级前体，从而选择BCR 在没有自动反应性的情况下，有可能发展广泛的中和。这种方法 还将有助于规避与表位置嵌入位置相关的空间问题， 具有已知遗传和结构潜力的启动前体，以使BNAB成熟与与 MPER空间约束。在此提案中，我们将设计表位表位型免疫剂 使用计算设计和定向演化激活10E8样前体。我们最初会 使用人Na'ive B细胞分类测试免疫原子。我们将进一步产生敲击小鼠 那个过表达的10e8样前体，我们将使用这些小鼠测试B细胞启动和 在体内提升，首先将敲击蛋白B细胞转移到野生型小鼠中，以便 在人类中模仿10E8 BNAB前体和竞争者B细胞的频率。 由于已知的bnab是高度突变的，因此在生殖线素将诱导BNAB的疫苗 可能需要对旨在牧羊人亲和力的其他免疫原子进行顺序免疫 B细胞受体的成熟。我们将开发不同类别的增强免疫原， 包括具有更多本地表位，膜蛋白支架和 膜结合的ENV变体以10E8结合的构象稳定。我们将 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。 总而言之，这些研究试图开发新型的HIV疫苗候选者，也是 将艾滋病毒疫苗研究转移到基于战略的简化主义方法 识别BNAB前体，最先进的蛋白质工程 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 体内成熟途径，以指导迭代疫苗优化。