Thwarting HIV evasion of antibody avidity with novel antibody architectures

利用新型抗体结构阻止 HIV 逃避抗体亲合力

• 批准号: 8702139
• 负责人: Pamela J Bjorkman
• 金额: $ 80.19万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702139
• 关键词: AIDS/HIV problem; Affinity; Affinity Chromatography; Antibodies; Antibody Avidity; Antibody Formation; Architecture; Avidity; Binding; Binding Proteins; Binding Sites; Coupled; Cross-Linking Reagents; DNA; Geometry; HIV; HIV Infections; Highly Active Antiretroviral Therapy; Human; Immunity; Lateral; Length; Libraries; Link; Linker DNA; Mediating; Methodology; Mutation; Passive Immunization; Pharmaceutical Preparations; Proteins; Public Health; Reagent; Site; Testing; Vaccines; Virus; abstracting; base; crosslink; density; design; ds-DNA; gene therapy; monomer; neutralizing antibody; novel; prevent

DESCRIPTION Abstract: Despite decades of effort, no current vaccine elicits neutralizing antibodies at concentrations blocking HIV infection. In addition to structural features of HIV's envelope spike that facilitate antibody evasion, we propose that the low density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The HIV spike trimer geometry does not allow intra-spike cross-linking by naturally-occurring bivalent antibodies, but we can construct proteins capable of high-avidity trivalent binding to a spike for gene therapy and/or passive immunization. We will design, express, and test trimeric intra-spike cross-linking reagents that bind to two or three non-overlapping sites per spike monomer (6-9 sites per trimer). Choosing HIV-binding proteins and how to link them will be done combinatorially starting with a library of 15-30 HIV spike-binding proteins coupled to double-stranded DNA identifying tags. These will be linked using variable-length DNA to form bispecific reagents separated by different distances. Pooled bispecific reagents will separated by affinity chromatography to isolate the tightest binding pairs, which will be PCR amplified/sequenced to determine the two protein components and the linker length. Upon identifying the optimal length for the linker, the DNA is replaced with a comparable-length protein linker, and the two-binding-protein reagent is linked to a third binding protein from the HIV-binding library. Optimal two- or three-component HIV binding proteins will be trimerized by attaching a trimerization motif first via DNA linkers to determine the optimal length, then using protein-based linkers. The trimeric reagent’s intra-spike cross-linking would reduce the concentration required for sterilizing immunity, making HIV’s low spike density irrelevant. Analogous to using ~3 drugs during HAART therapy, simultaneous binding to several different sites/monomer would reduce/abrogate sensitivity to spike mutations.

描述 抽象的： 尽管经过数十年的努力，目前的疫苗还没有能够在阻止HIV感染的浓度下引发中和抗体。除了HIV包膜刺突的结构特征有利于逃避外，我们还认为HIV刺突的低密度和有限的横向移动性阻碍了抗体的二价结合。产生的主要单价结合最大限度地减少了亲和力，从而实现了高亲和力结合和有效的中和，从而扩大了允许抗体逃避的HIV突变范围。HIV刺突三聚体几何结构不允许刺突内交联。天然存在的二价抗体，但我们可以构建能够与刺突高亲和力三价结合的蛋白质，用于基因治疗和/或被动免疫。我们将设计、表达和测试与两个刺突内结合的三聚体内交联试剂。或每个刺突单体三个非重叠位点（每个三聚体 6-9 个位点）的选择以及如何连接它们将从 15-30 个 HIV 的文库开始组合完成。与双链 DNA 识别标签偶联的刺突结合蛋白将使用可变长度 DNA 连接以形成间隔不同距离的双特异性试剂，将通过亲和层析分离出最紧密的结合对。 PCR 扩增/测序以确定两种蛋白质成分和接头长度。确定接头的最佳长度后，用长度相当的蛋白质接头替换 DNA，然后连接两种结合蛋白质试剂。最佳的二元或三元 HIV 结合蛋白将通过首先通过 DNA 接头连接三聚基序来三聚化，以确定最佳长度，然后使用基于蛋白质的接头。内尖峰 交联会降低免疫消毒所需的浓度，从而降低艾滋病病毒感染率 类似于在 HAART 治疗期间同时使用 ~3 种药物。 与几个不同位点/单体的结合会降低/消除对刺突突变的敏感性。