HIV-1 acidic epitope discovery from broadly neutralizing seroantibodies

从广泛中和血清抗体中发现 HIV-1 酸性表位

• 批准号: 9182870
• 负责人: Mohammad Mohseni Sajadi
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182870
• 关键词: Affinity; Affinity Chromatography; Agreement; Antibodies; Antibody Response; Antigens; Binding; Biochemical; Blood; Cells; Characteristics; Chronic; Data; Development; Dissection; Ensure; Epitope Mapping; Epitopes; Fractionation; Gene Family; Genetic; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; HIV-1 vaccine; Human; Humoral Immunities; IgG1; Immune system; Immunoglobulin Family Gene; Individual; Isoelectric Focusing; Isoelectric Point; Light; Maps; Mediating; Memory B-Lymphocyte; Methods; Monoclonal Antibodies; Mutagenesis; Patients; Persons; Plasma; Property; Reporting; Research; Resources; Scheme; Selection Bias; Source; Specificity; Target Populations; Techniques; Testing; Vaccination; Vaccines; Work; X-Ray Crystallography; base; cohort; interest; mutant; neutralizing antibody; neutralizing monoclonal antibodies; novel; prevent; public health relevance; response; success; vaccine candidate

 DESCRIPTION (provided by applicant): Background/Rationale: A limited number of persons infected with HIV-1 develop circulating plasma antibodies that potently neutralize a wide variety of HIV-1 isolates. The characteristics and specificities of such antibodies can guide the development of HIV-1 vaccine candidates. Current methods for the study of these antibodies include isolation of antibodies from memory B cells, which may not always reflect the circulating antibodies. Our research has focused on the identification and characterization of broad neutralizing antibodies directly from patient plasma (without potential bias of selection). We have noted particular biochemical signatures that point to a common dominant, acidic epitope that is targeted on the envelope of HIV. Objectives: The specific hypothesis of this proposal is that a shared acidic epitope on the gp120 envelope binds a shared immunoglobulin gene family giving rise to the broad HIV-1 neutralizing response in plasma. The specific aims of the project are to 1) Directly isolate and sequence the antibodies responsible for the broad neutralization from the plasma of HIV-1 infected individuals with broad neutralization, 2) Define the gene family usage of neutralizing versus non-neutralizing antibodies to test the hypothesis that a shared immunoglobulin gene family is responsible for the broad HIV-1 neutralization response in plasma, 3) Map the corresponding epitope(s) of the broad neutralizing antibodies to test the hypothesis that a shared acidic epitope of gp120 is responsible for the broad HIV-1 neutralization response in plasma. Methods: We have identified 10 patients with broad neutralization, of which 3 will be studied in detail. The affinity purification (antigen, subclass, and light chain specific) and fractionation (free-flow isoelectric focusing) scheme can narrow the antibodies of interest, directly, from the plasma to individual species, which will be tested for broad neutralization. Upon confirmation, the individual antibody bands will be sequenced de novo using LC-MS. Epitope mapping of the new mAbs will be undertaken with Elisa, mutagenesis studies, and X-ray crystallography. Once the epitope is identified, Elisa and mutagenesis studies will be used to test the active fraction o the other 7 individuals to determine if this epitope is responsible for broad neutralization. We wil also define the gene family usage of neutralizing versus non-neutralizing antibodies in 20 patients. Anticipated results will be isolation of new mAbs and the identification of a common acidic epitope that can direct broad HIV-1 neutralization. We have completed enough work on the techniques described herein (as well as each alternative plan) to ensure that the aims are feasible and will be completed. Impact: If our hypothesis proves correct, then the results will be novel in its method, and directl applicable to the study of HIV vaccines. This study will provide a deeper understanding of possibilities of the broad HIV-1 neutralizing response in humans, and it will also have identified a naturally occurring epitope(s) that can be the target of potent cross-clade antibodies against HIV-1 as well as the gene family(ies) responsible.

 描述（由申请人提供）： 背景/理由：有限数量的 HIV-1 感染者产生循环血浆抗体，可有效中和多种 HIV-1 分离株。此类抗体的特性和特异性可以指导 HIV-1 候选疫苗的开发。目前研究这些抗体的方法包括从记忆 B 细胞中分离抗体，这可能并不总是反映循环抗体。我们的研究重点是直接从患者血浆中鉴定和表征广泛的中和抗体（没有潜在的选择偏差）。我们注意到特定的生化特征，这些特征指向针对 HIV 包膜的共同显性酸性表位。 目的：该提议的具体假设是 gp120 包膜上的共享酸性表位结合共享的免疫球蛋白基因家族，从而在血浆中产生广泛的 HIV-1 中和反应。该项目的具体目标是 1) 直接从 HIV-1 感染者血浆中分离并测序负责广泛中和的抗体，2) 定义中和抗体与非中和抗体的基因家族用途，以检验共享免疫球蛋白基因家族负责血浆中广泛 HIV-1 中和反应的假设，3) 绘制相应的图谱 广泛中和抗体的表位，用于测试共享酸性表位的假设 gp120 负责血浆中广泛的 HIV-1 中和反应。 方法：我们已经确定了 10 名具有广泛中和作用的患者，其中 3 名将进行详细研究。亲和纯化（抗原、亚类和轻链特异性）和分级分离（自由流动等电聚焦）方案可以直接将目标抗体从血浆缩小到单个物种，然后对其进行广泛中和测试。确认后，将使用 LC-MS 对各个抗体条带进行从头测序。新单克隆抗体的表位作图将通过 Elisa、诱变研究和 X 射线晶体学进行。一旦确定了表位，Elisa 和诱变研究将用于测试其他 7 个个体的活性部分，以确定该表位是否负责广泛中和。我们还将在 20 名患者中定义中和抗体与非中和抗体的基因家族使用情况。预期结果将是分离出新的单克隆抗体，并鉴定出可以指导广泛的 HIV-1 中和作用的常见酸性表位。我们已经对本文描述的技术（以及每个替代计划）完成了足够的工作，以确保目标可行并将完成。 影响：如果我们的假设被证明是正确的，那么结果将在方法上是新颖的，并且可以直接应用于艾滋病毒疫苗的研究。这项研究将更深入地了解人类广泛的 HIV-1 中和反应的可能性，并且还将鉴定出一个天然存在的表位，该表位可以成为针对 HIV-1 的有效跨进化枝抗体以及相关基因家族的目标。