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.

项目摘要 广泛中和抗体（bNAb）是一种有前途的免疫疗法，可以纳入许多免疫治疗。 治疗和/或治愈HIV-1的策略。中和作用和 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尚不清楚。个别临床试验得出了不同的结论 关于在入组之前对参与者的病毒进行体外中和敏感性预筛查的实用性 临床试验主要是由于两个障碍：测定时间的长度和难以获得正确的样品， test. Bosque实验室最近发表了一种超灵敏的方法，可以检测到pg/ml的p24 gag蛋白 水平，并在来自PLWH的离体细胞中验证该测定。在这里，我们将应用这种新的方法， 通过开发一种间接读出中和灵敏度的测定法来解决这两个障碍， 并直接在细胞裂解物中进行。我们将实现这种超灵敏的Env结合测定， 通过为第一个R61阶段开发三个部分：Aim 1将优化捕获和检测抗体， 目标2将测试针对具有已知中和敏感性的各种假病毒的bNAb基质， 通过检测血浆和细胞裂解物中的Env检测来优化生物基质，目标3将确定含量测定 不同病毒准种中敏感和耐药病毒比例的精密度。我们将验证和鉴定 在第二个R33阶段进行的超灵敏Env结合试验分为两部分：目的4将验证试验 通过对经过充分研究的临床试验样本进行事后检测。目标5将实施正确的质量体系， 本试验用于准备CLIA确认。我们的多学科团队拥有实现以下目标所需的所有专业知识： 这些目标，当完成时，导致Env蛋白的超灵敏结合测定，以筛选临床试验 参与者的时间效率高，准确和合格。