Discovery of acidic epitopes for HIV-1 broadly neutralizing seroantibodies

发现 HIV-1 广泛中和血清抗体的酸性表位

• 批准号: 9788183
• 负责人: Mohammad Mohseni Sajadi
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788183
• 关键词: AIDS prevention; AIDS/HIV problem; Affinity; Affinity Chromatography; Agreement; Antibodies; Antibody Response; Antigens; Binding; Biochemical; Blood; Caring; Cells; Characteristics; Data; Development; Disease; Dissection; Ensure; Epitope Mapping; Epitopes; Fractionation; Genetic; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; HIV-1 vaccine; Health; Healthcare; Human; Humoral Immunities; IgG1; Immune system; Immunoglobulin Variable Region; Impairment; Individual; Infection; Isoelectric Focusing; Isoelectric Point; Knowledge; Length; Life; Light; Maps; Mediating; Memory B-Lymphocyte; Methods; Modeling; Monoclonal Antibodies; Mutagenesis; N-terminal; Patients; Persons; Plasma; Population; Prevention; Preventive vaccine; Property; Reporting; Research; Resources; Savings; Scheme; Selection Bias; Serum; Source; Specificity; Target Populations; Techniques; Testing; Vaccination; Vaccines; Veterans; Work; X-Ray Crystallography; base; cohort; design; improved; interest; mutant; neutralizing antibody; neutralizing monoclonal antibodies; novel; prevent; protein aminoacid sequence; public health relevance; response; success; vaccine candidate; AIDS prevention; AIDS/HIV problem; Affinity; Affinity Chromatography; Agreement; Antibodies; Antibody Response; Antigens; Binding; Biochemical; Blood; Caring; Cells; Characteristics; Data; Development; Disease; Dissection; Ensure; Epitope Mapping; Epitopes; Fractionation; Genetic; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; HIV-1 vaccine; Health; Healthcare; Human; Humoral Immunities; IgG1; Immune system; Immunoglobulin Variable Region; Impairment; Individual; Infection; Isoelectric Focusing; Isoelectric Point; Knowledge; Length; Life; Light; Maps; Mediating; Memory B-Lymphocyte; Methods; Modeling; Monoclonal Antibodies; Mutagenesis; N-terminal; Patients; Persons; Plasma; Population; Prevention; Preventive vaccine; Property; Reporting; Research; Resources; Savings; Scheme; Selection Bias; Serum; Source; Specificity; Target Populations; Techniques; Testing; Vaccination; Vaccines; Veterans; Work; X-Ray Crystallography; base; cohort; design; improved; interest; mutant; neutralizing antibody; neutralizing monoclonal antibodies; novel; prevent; protein aminoacid sequence; public health relevance; response; success; vaccine candidate

DESCRIPTION (provided by applicant): Background/Rationale: HIV-1 infection and its prevention are of particular importance to the health of veterans, with 1 out of 250 veterans known to be HIV-1 infected. Prevention of HIV-1 was specifically mentioned as one of the 3 main goals in President Obama's National HIV/AIDS Strategy in 2010, as well as the Department of Veterans Affairs National HIV/AIDS Strategy Operational Plan in 2011. Our group has focused on a preventive vaccine for HIV-1 through study of potent antibodies directed at HIV-1. A limited number of persons infected with HIV-1 develop circulating plasma antibodies able to potently neutralize a wide variety of HIV-1 isolates representing different genetic subtypes. It is widely held that the characteristics and specificitis of such antibodies can be used to guide the development of HIV-1 vaccine candidates capable of eliciting protective humoral immunity in a target population. Current methods for study of these antibodies include isolation of antibodies from memory B cells, which may not always reflect the antibodies circulating in the blood. Our research has focused on the identification of characterization of the broad neutralizing antibodies directly from patient serum (without potential bias of selection), as neutralizing antibody characteristics seen in multiple individuals are more likely be raised in a broad population. 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 the broad HIV-1 neutralizing response in plasma is due to a shared acidic epitope on the gp120 envelope. 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, and 2) 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. Methods: We have identified 10 patients with broad neutralization, of which 3 will be studies in detail. The affinit 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. These species will be tested for broad neutralization. Upon confirmation, the individual antibody bands will be sequenced de novo using both LC-MS and Edman degradation (N-terminal and internal sequencing). 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 on the other 7 individuals to determine if this epitope is responsible for broad neutralization. Findings: Anticipated results wil be isolation of new mAbs, identification of common characteristics of broadly neutralizing abs, and the identification of a common acidic epitope that can direct broad HIV-1 neutralization. Status: 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 and directly 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. This will have an impact on the designs of an HIV vaccine, which when developed will improve the health of veterans by preventing this life-long disease.

描述（由申请人提供）： 背景/理由：HIV-1感染及其预防对退伍军人的健康尤为重要，其中有250名退伍军人中有1名被称为HIV-1感染。预防HIV-1是2010年奥巴马总统国家艾滋病毒/艾滋病战略中的3个主要目标之一，以及退伍军人事务部国家艾滋病毒/艾滋病战略战略运作计划。我们的小组通过研究针对HIV-1的有效抗体来研究HIV-1的预防性疫苗。受HIV-1感染的人数有限的人会产生循环的血浆抗体，能够有效地中和代表不同遗传亚型的各种HIV-1分离株。人们普遍认为，此类抗体的特征和特异性可用于指导能够在目标群中引起保护性体液免疫的HIV-1疫苗候选者的发展。当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出血液中循环的抗体。我们的研究集中于直接从患者血清中的广泛中和抗体的表征（没有潜在选择的偏见），作为中和抗体特征的表征 在广泛的人口中更有可能养育。我们已经注意到特定的生化特征，该特征指向靶向HIV包膜的共同占主导地位的酸性表位。目的：该提案的具体假设是，血浆中的HIV-1中和反应是由于GP120包膜上的共享酸性表位引起的。该项目的具体目的是1）直接分离并对负责从HIV-1感染个体的血浆进行广泛中和的抗体进行序列，以及2）绘制广泛中和抗体的相应表位，以测试共享酸性表位的假设 GP120的构成广泛的HIV-1中和反应。方法：我们已经确定了10例具有广泛中和的患者，其中3例将详细研究。 Affinit纯化（抗原，亚类和轻链特异性）和分馏（自由流相关 聚焦）方案可以将感兴趣的抗体直接从血浆到个体物种范围缩小。这些物种将进行测试以进行广泛的中和。确认后，将使用LC-MS和EDMAN降解（N末端和内部测序）对单个抗体条带进行测序。将使用ELISA，诱变研究和X射线晶体学进行新的mAb的表位映射。一旦确定了表位，ELISA和诱变研究将用于测试其他7个个体的主动分数，以确定该表位是否负责广泛中和。结果：预期的结果将是新的mAb的隔离，鉴定广泛中和的ABS的共同特征以及可以指导可导致广泛HIV-1中和的常见酸性表位的鉴定。状态：我们已经完成了本文所述的技术（以及每个替代计划）的足够工作，以确保目标是可行的并将完成的。影响：如果我们的假设被证明是正确的，那么结果将是新颖的，直接适用于HIV疫苗的研究。这项研究将对人类中中和反应的广泛中和反应的可能性有更深入的了解，并且还将确定一种天然存在的表位，这可能是针对HIV-1的有效跨层抗体的靶标。这将对艾滋病毒疫苗的设计产生影响，艾滋病毒疫苗的设计将通过防止这种终身疾病来改善退伍军人的健康。