Thursday, April 3, 2025
4/3/2025
Ultrasensitive Env Detection Assay for Broadly Neutralizing Antibody Screening - 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.
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