Ultrasensitive Env Detection Assay for Broadly Neutralizing Antibody Screening

用于广泛中和抗体筛选的超灵敏包膜检测分析

• 批准号: 10676393
• 负责人: Alberto Bosque
• 金额: $ 45.02万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-12 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676393
• 关键词: Address; Aliquot; Antibodies; Binding; Biological; Biological Assay; CLIA certified; Cells; Clinical Trials; Cloning; Combined Modality Therapy; Consumption; Data; Detection; Documentation; Enrollment; Epitopes; Equipment; HIV; HIV Antibodies; HIV-1; Hour; Immunotherapy; In Vitro; Individual; Infusion procedures; Length; Measurement; Measures; Methodology; Methods; Minority; Nature; Participant; Performance; Peripheral Blood Mononuclear Cell; Persons; Phase; Phenotype; Plasma; Proviruses; Publishing; Qualifying; Quality Control; Reagent; Reporting; Resistance; Sampling; Signal Transduction; Specific qualifier value; System; Technology; Testing; Therapeutic; Time; Variant; Viral; Virion; Virus; Virus Replication; antibody detection; clinical development; clinical efficacy; clinical predictors; clinical trial participant; clinically relevant; detection assay; detector; env Gene Products; env Genes; gag Gene Products; improved; multidisciplinary; neutralizing antibody; novel; resistant strain; screening; treatment strategy

PROJECT SUMMARY Broadly neutralizing antibodies (bNAbs) are a promising immunotherapy that can be incorporated in many strategies for the treatment and/or cure of HIV-1. There is a clear association between the neutralization sensitivity of virus and how effective bNAbs are in suppressing virus replication during clinical trials, but the precise nature of this relationship is still not clear. Individual clinical trials have reached different conclusions about the utility of pre-screening participants’ virus for in vitro neutralization sensitivity before enrollment into clinical trials mainly due to two obstacles: length of assay time and difficulty in obtaining the correct samples to test. The Bosque lab has recently published an ultrasensitive method to detect p24 gag protein down to the fg/ml level, and the assay was validated in ex vivo cells from PLWH. Here we will apply this novel methodology to address these 2 obstacles by developing an assay that gives an indirect readout of neutralization sensitivity but in a very short amount of time and directly in cell lysates. We will achieve this ultra-sensitive Env binding assay by developing it in three parts for the first R61 phase: Aim 1 will optimize the capture and detector antibodies by testing a matrix of bNAbs against diverse pseudoviruses with known neutralization sensitivities, Aim 2 will optimize biological matrices by testing Env detection in plasma and cell lysates, and Aim 3 will determine assay precision for ratios of sensitive and resistant virus in a diverse viral quasispecies. We will validate and qualify this ultra-sensitive Env binding assay during the second R33 phase in two parts: Aim 4 will validate the assay through post-hoc testing of well-studied clinical trial samples. Aim 5 will implement correct quality systems for this assay to prepare for CLIA qualification. Our multi-disciplinary team has all the expertise necessary to achieve these aims that, when completed, result in an ultra-sensitive binding assay for Env protein to screen clinical trial participants that is time-efficient, accurate and qualified.

项目概要 广泛中和抗体 (bNAb) 是一种有前途的免疫疗法，可应用于许多领域 HIV-1 的治疗和/或治愈策略。中和作用之间存在明显的关联 病毒的敏感性以及 bNAb 在临床试验中抑制病毒复制的有效性，但 这种关系的确切性质仍不清楚。个别临床试验得出不同结论 关于在入组之前预筛选参与者病毒的体外中和敏感性的效用 临床试验主要由于两个障碍：检测时间长和难以获得正确的样本 测试。 Bosque 实验室最近发布了一种超灵敏方法，可检测低至 fg/ml 的 p24 gag 蛋白 水平，并且该测定在来自 PLWH 的离体细胞中得到验证。在这里，我们将应用这种新颖的方法 通过开发一种可以间接读出中和灵敏度的检测方法来解决这两个障碍，但是 在很短的时间内直接在细胞裂解液中。我们将实现这种超灵敏的 Env 结合测定 通过将其分为三个部分来开发第一个 R61 阶段：目标 1 将通过以下方式优化捕获和检测抗体： 测试 bNAb 基质针对已知中和敏感性的多种假病毒，目标 2 将 通过测试血浆和细胞裂解物中的 Env 检测来优化生物基质，Aim 3 将确定检测结果 多种病毒准种中敏感病毒和耐药病毒比率的精确度。我们将验证并确定资格 这种超灵敏的 Env 结合测定在第二个 R33 阶段分两部分进行：目标 4 将验证该测定 通过对经过充分研究的临床试验样本进行事后测试。目标 5 将实施正确的质量体系 该测定是为了准备 CLIA 资格。我们的多学科团队拥有实现目标所需的所有专业知识 这些目标完成后，可对 Env 蛋白进行超灵敏的结合测定，以筛选临床试验 时间高效、准确且合格的参与者。