Production of Cross-Neutralizing HIV-1 Antibodies from Single B Cells

从单个 B 细胞生产交叉中和 HIV-1 抗体

• 批准号: 8786133
• 负责人: MIROSLAW K GORNY
• 金额: $ 17.05万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-05 至 2016-03-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786133
• 关键词: AIDS/HIV problem; Affinity; Antibodies; Antibody Formation; Antibody Specificity; Antigens; B-Lymphocytes; Binding; Biological Assay; Blood specimen; Cameroon; Cells; Cellular Immunity; Cloning; Complementary DNA; Complex; Crystallography; Development; Epitope Mapping; Epitopes; Fc Immunoglobulins; Flow Cytometry; Future; Generations; Genes; HIV vaccine; HIV-1; Human; Human Activities; IgG1; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulins; Immunology; Individual; Maps; Measures; Mediating; Memory B-Lymphocyte; Methods; Modeling; Molecular; Monoclonal Antibodies; Nature; Patients; Peptides; Peripheral Blood Mononuclear Cell; Plasma; Production; Proteins; Recombinants; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Serum; Sorting - Cell Movement; Specificity; Specimen; Structure; Surface; Techniques; Testing; Transfection; Vaccine Design; Vaccines; Variant; Viral; Virus; Virus Diseases; Virus-like particle; Work; antibody-dependent cell cytotoxicity; base; cohort; design; env Gene Products; expression vector; human monoclonal antibodies; immunogenic; improved; innovation; monoclonal antibody production; mutant; neutralizing antibody; neutralizing monoclonal antibodies; novel; receptor; transfection/expression vector; vaccine candidate; volunteer

Recent studies using selected patients' sera with cross-clade neutralizing activity revealed that the specificity of at least one-third of the neutralizing activity remains uncharacterized. These results demonstrate the need to identify new epitopes which can guide efforts to develop a promising vaccine. We propose an innovative approach to produce human mAbs using a selected combination of techniques which are not being used together by any other group, i.e., the use of single IgG+ memory B cells, selected with virus-like particles (VLPs) from which recombinant monoclonal antibodies (mAbs) will be generated using highly efficient molecular techniques. A further innovation includes more efficient sorting and selection of B cells specific only for trimeric envelope (Env) proteins. The B cells will be derived from donors infected with diverse HIV-1 subtypes whose plasma Abs cross-neutralize Tier 2 viruses. We hypothesize that these selected volunteers produce neutralizing Abs to new as yet unidentified epitopes that are present on the native trimeric HIV-1 envelope and that reactivity to such epitopes will be detected using VLPs. SPECIFIC AIM 1. Production of recombinant mAbs from single B cells. The blood specimens will come from two well-established cohorts of infected subjects. Three PBMC samples will be provided by the Center for HIV/AIDS Vaccine Immunology (CHAVI) and 10 PBMCs samples will be obtained from Cameroonian subjects whose sera have been shown to mediate cross-clade neutralizing activity. The mAbs will be produced from single Env-specific B cells selected with GFP-tagged VLPs expressing trimeric Env proteins. The immunoglobulin variable genes will be amplified using RT-PCR, cloned into expression vectors, and the genes will be used for the transfection of 293T cells for mAb production. In total, PBMC specimens from 10-15 subjects will be studied; yielding 300-450 mAbs. SPECIFIC AIM 2. Characterization of various functional activities (neutralizing, ADCC and ADCVI) of new mAbs. The purified mAbs will be tested in functional assays for neutralization, ADCC and/or ADCVI activity. The neutralizing activity of mAbs will be screened against pseudoviruses and primary isolates in our lab and selected mAbs will be tested against a standard panel of pseudotyped viruses by a collaborator. New mAbs combining two or three inhibitory functions (neutralization, ADCC and/or ADCVI) will have priority for epitope mapping followed by those mAbs that cross-neutralize only and non-neutralizing mAbs with the FcγR-mediated activity. SPECIFIC AIM 3. Epitope mapping of new monoclonal Abs. Using VLPs for selection of Env-specific B cells will result in production of mAbs against various known epitopes and those which are present on Env trimers. A variety of mapping techniques will be used, including immunochemical and viral assays as well as crystallographic analysis. Mapping will be particularly focused on mAbs to quaternary and newly defined epitopes and that mediate double (or triple) functions. Epitopes of mAbs with potent, cross-neutralizing and varied activities will serve as templates for the future design of immunogens to induce protective Abs.

最近使用选定患者的血清具有交叉中和活性的研究表明特异性 至少三分之一的中和活性仍未表征。这些结果表明需要 确定可以指导开发有前途疫苗的努力的新表位。我们提出了创新的 使用未使用的技术组合产生人物mAb的方法 由任何其他组一起使用，即使用单个IgG+存储B细胞，用病毒样颗粒选择 （VLP）将使用高效产生重组单克隆抗体（mAb） 分子技术。进一步的创新包括更有效的排序和选择B细胞的选择 用于三聚体包膜（Env）蛋白。 B细胞将源自感染不同HIV-1的供体 血浆ABS交叉中和2个病毒的亚型。我们假设这些选定的志愿者 在天然三聚体HIV-1上产生中和的AB 使用VLP将检测到包膜和对此类表位的反应性。特定目标1。生产 来自单个B细胞的重组mAb。血标本将来自两个公认的同类 感染受试者。 HIV/AIDS疫苗免疫学中心将提供三个PBMC样品 （CHAVI）和10个PBMCS样本将从喀麦隆受试者获得，其血清已显示为 介导跨层中和活性。 mAb将由选定的单个ENV特异性B细胞产生 带有GFP标记的VLP表达三聚体env蛋白。免疫球蛋白可变基因将被放大 使用RT-PCR，克隆到表达载体中，这些基因将用于转染293T细胞的转染 mab生产。总共研究了10-15名受试者的PBMC标本；产生300-450 mAb。 特定目的2。新的功能活动的表征（中和，ADCC和ADCVI） mabs。纯化的mAb将在功能测定中进行中和，ADCC和/或ADCVI活性。 MAB的中和活性将针对我们实验室中的假病毒和主要分离株进行筛查，并且 合作者将对选定的mAB进行针对标准的伪型病毒的测试。新的mabs 组合两个或三个抑制功能（中和，ADCC和/或ADCVI）将优先于表位 映射随后是那些仅与FcγR介导的单盘和非中和mAb的mAb 活动。特定目标3。新单克隆ABS的表位图。使用VLP选择ENV特异性B 细胞将导致对各种已知表位的mAb产生，而在Env上存在的表位。 三线。将使用各种映射技术，包括免疫化学和病毒测定法以及 晶体学分析。映射将特别集中在mabs上，以进行第四纪和新定义 表位并介导双重（或三重）函数。具有强大的，交叉中和的mAb的表位 各种活动将作为免疫原子未来设计的模板，以诱导保护性ABS。