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 疫苗尚未实现的一个关键目标 发展。 BnAb 10E8 作为疫苗先导物特别受关注，因为它 (1) 非常高 广度，(2) 发展广泛中和所需的突变数量少，(3) 低 自身反应性，(4) 人类初始 B 细胞库中预期前体的高频率，以及 (5) 在被动转移研究中提供令人惊讶的有效体内灭菌保护的能力。 诱导 10E8 样 bnAb 的主要挑战是：(a) HIV 肽的低种系亲和力和 蛋白质，(b) 凹进对 bnAb 接近角度的严格限制， 近膜表位环境和（c）大多数可溶性中不存在 10E8 表位， 类似天然的三聚体（例如 SOSIP 或 NFL 或 UFO）。 启动 10E8 样 bnAb 诱导的一种有前途的策略是种系靶向，其中合适的 使用工程免疫原特异性激活 10E8 类前体，从而选择 BCR 具有在不存在自身反应性的情况下产生广泛中和作用的潜力。这种做法 还将有助于避免与表位凹陷位置相关的空间问题，通过 启动具有已知遗传和结构潜力的前体，使其成熟为与 MPER 空间限制。在这个提案中，我们将设计表位支架免疫原 使用计算设计和定向进化激活类似 10E8 的前体。我们将首先 使用人类原始 B 细胞分选来离体测试免疫原。我们将进一步产生基因敲入小鼠 过度表达 10E8 样前体细胞，我们将使用这些小鼠来测试 B 细胞启动和 体内加强，首先将敲入B细胞过继转移至野生型小鼠体内，以 模拟人类 10E8 bnAb 前体和竞争 B 细胞的频率。 由于已知 bnAb 具有高度突变性，因此在种系初免后疫苗诱导 bnAb 将 可能需要使用其他旨在增强亲和力的免疫原进行顺序免疫 B 细胞受体的成熟。我们将开发不同类别的增强免疫原， 包括具有更多天然表位的表位支架、膜蛋白支架和 膜结合的 Env 变体稳定在 10E8 强烈结合的构象中。我们将 在敲入小鼠中进行连续初免/加强免疫实验，并使用 ELISA、细胞计数法、 单 B 细胞分选、测序和中和测定，以跟踪和优化亲和力 成熟。 总之，这些研究旨在开发新型艾滋病毒候选疫苗，并 将艾滋病毒疫苗研究转向基于战略的还原论方法 bNAb 前体的鉴定，最先进的蛋白质工程开发 种系靶向和增强免疫原，开发人类 Ig 敲入小鼠模型以实现 人类库特异性疫苗的测试，以及疫苗诱导亲和力的深入分析 体内成熟途径指导迭代疫苗优化。