Ultrasensitive Env Detection Assay for Broadly Neutralizing Antibody Screening

项目总结 广谱中和抗体(BNAbs)是一种很有前途的免疫疗法，可用于许多 治疗和/或治愈艾滋病毒-1的战略。在中和之间有明显的联系 在临床试验期间，病毒的敏感性以及bNAbs在抑制病毒复制方面的有效性，但 这种关系的确切性质仍不清楚。个别临床试验得出了不同的结论。 关于在注册前对参与者的病毒进行体外中和敏感性预筛选的效用 临床试验主要是由于两个障碍：化验时间长和难以获得正确的样本 测试。博斯克实验室最近发布了一种超灵敏的方法，可以检测p24 Gag蛋白，精确度可达fg/ml 水平，并在PLWH的体外细胞中验证了该方法。在这里，我们将把这种新颖的方法应用于 解决这两个障碍的方法是开发一种间接读出中和灵敏度的分析方法，但是 在很短的时间内，直接在细胞裂解物中。我们将实现这种超灵敏的环境病毒结合分析 通过将其开发为第一个R61阶段的三个部分：Aim 1将通过以下方式优化捕获和检测抗体 测试具有已知中和敏感性的针对不同伪病毒的bNAbs矩阵，Aim 2将 通过检测血浆和细胞裂解物中的环境病毒检测来优化生物基质，目标3将决定检测 在不同的病毒准物种中，敏感和耐药病毒比率的精确度。我们将验证和鉴定 在第二个R33阶段，这一超灵敏的Env结合分析分两部分进行：AIM 4将验证该分析 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将为以下项目实施正确的质量体系 本次化验为CLIA资质做准备。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 这些目标是在完成后，产生一种超灵敏的环境蛋白结合分析来筛选临床试验 参赛者要做到省时、准确、合格。

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.