Rescue of broadly neutralizing mAbs using native trimer

使用天然三聚体拯救广泛中和的单克隆抗体

基本信息

批准号：
8459997
负责人：
JAMES M BINLEY
金额：
$ 42.77万
依托单位：
TORREY PINES INST FOR MOLECULAR STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8459997
关键词：
Address Antibodies Antigens B-Lymphocytes Binding Biological Assay Chimera organism Data Development Enzyme-Linked Immunosorbent Assay Epitope Mapping Epitopes Exhibits Genes Growth HIV HIV-1 Heating Immune Sera Immunity Immunoglobulin G Individual Infection Knowledge Label Maps Memory B-Lymphocyte Methods Nature Parents Patients Population Reagent Recovery Research Personnel Reverse Transcriptase Polymerase Chain Reaction Sampling Serum Site Solutions Specificity Structural Biologist Technology Testing Therapeutic Transfection Ursidae Family Vaccination Vaccine Design Vaccines Virion Virus Virus-like particle abstracting base design env Glycoproteins high throughput screening immunogenicity indexing innovation insight member neutralizing antibody neutralizing monoclonal antibodies novel prophylactic public health relevance response screening success vaccine development

项目摘要

Project Summary/Abstract Broadly neutralizing antibodies (bNAbs) may be crucial component of the protective immunity conferred by an effective HIV-1 vaccine. Ab responses in natural HIV-1 infection are overwhelmingly non-neutralizing. However, a fraction of patients do eventually develop exceptionally broad and potent bNAb responses. We hypothesize that a better understanding of the specificities that underlie this broad serum neutralization will enable us to identify the most desirable targets and develop a full spectrum of vaccine design strategies. New epitopes may be particularly amenable to design or attractive by being unusually broad or potent. Researchers have recently accessed various monoclonal bNAbs (mbNAbs) by high throughput functional screening and/or by targeted selection of memory B cells from infected donors who exhibit broad serum NAb responses. However, these efforts remain challenging, in part due to the rarity of desirable memory B cells and the lack of authentic baits to select new specificities. Based on the premise that native Env trimers are the exclusive targets of NAbs, here we propose using virus-like particles (VLPs) bearing only native Env trimers as baits to retrieve novel mbNAbs that will illuminate novel sites of vulnerability. Our Specific Aims are: Specific Aim 1: To investigate monoclonal bNAb relationships on native trimer VLPs. We will determine the binding relationships of various mbNAb pairs by trimer VLP ELISA. Any antagonistic or synergistic combinations we identify could impact the therapeutic or prophylactic applications of mbNAb combinations. Selected mbMAb combinations will be further investigated in neutralization synergy assays. Specific Aim 2: To map the specificities of broadly neutralizing sera using native trimer VLPs. We will evaluate the ability of a panel of broadly neutralizing HIV+ sera to inhibit trimer VLP binding by the panel of mbNAbs used in Aim 1. This will allow us to prioritize B cell selections (Aim 3) from donor PBMCs whose sera exhibit novel NAb specificities. Specific Aim 3: To rescue mbNAbs using fluorescent trimer VLPs as baits. With an expert collaborator, we will develop methods to use fluorescently labeled trimer VLPs as baits and probe donor PBMCs to label and retrieve mbNAb clones responsible for broad serum neutralization. We will prioritize donors whose sera appear to target unique epitopes from mapping studies in Aim 2. Specific Aim 4: To characterize new mbNAbs. We will determine the specificity, breadth and potency of new mbNAbs. We will examine their binding relationships with other mbNAb specificities on the native trimer, as in Aim 1. To better understand their ontogeny, we will also examine their sequences, segment usage and divergence from nearest germline.

项目概要/摘要广泛中和抗体（bNAb）可能是保护性免疫的重要组成部分通过有效的 HIV-1 疫苗。自然 HIV-1 感染中的抗体反应绝大多数是非中和性的。然而，一小部分患者最终确实产生了异常广泛且有效的 bNAb 反应。我们假设更好地了解这种广泛血清的特异性中和将使我们能够确定最理想的目标并制定全方位的疫苗设计策略。新表位可能特别适合设计或通过被设计而具有吸引力异常广泛或有效。研究人员最近通过高通量获得了各种单克隆 bNAb (mbNAb) 通过功能筛选和/或从受感染的捐赠者中定向选择记忆 B 细胞表现出广泛的血清 NAB 反应。然而，这些努力仍然具有挑战性，部分原因是稀缺性理想的记忆 B 细胞以及缺乏选择新特异性的真实诱饵。基于这样的前提天然 Env 三聚体是 NAb 的唯一靶标，在这里我们建议使用带有病毒样颗粒 (VLP) 仅使用天然 Env 三聚体作为诱饵来检索新的 mbNAb，从而阐明新的漏洞位点。我们的具体目标是：具体目标 1：研究单克隆 bNAb 与天然三聚体 VLP 的关系。我们将确定通过三聚体 VLP ELISA 分析不同 mbNAb 对的结合关系。任何拮抗或协同作用我们确定的组合可能会影响 mbNAb 组合的治疗或预防应用。选定的 mbMAb 组合将在中和协同测定中进一步研究。具体目标 2：利用天然三聚体 VLP 绘制广泛中和血清的特异性。我们将评估一组广泛中和 HIV+ 血清抑制三聚体 VLP 结合的能力目标 1 中使用的 mbNAb。这将使我们能够优先从其血清的供体 PBMC 中选择 B 细胞（目标 3）表现出新颖的 NAb 特异性。具体目标 3：使用荧光三聚体 VLP 作为诱饵来拯救 mbNAb。与专家合作，我们将开发使用荧光标记的三聚体 VLP 作为诱饵并探针供体 PBMC 进行标记的方法并检索负责广泛血清中和的 mbNAb 克隆。我们将优先考虑血清的捐赠者似乎针对目标 2 中的作图研究中的独特表位。具体目标 4：表征新的 mbNAb。我们将确定新方案的特异性、广度和效力 mbNAb。我们将检查它们与天然三聚体上其他 mbNAb 特异性的结合关系，如目标 1. 为了更好地理解它们的个体发育，我们还将检查它们的序列、片段使用和与最近种系的分歧。