PROJECT SUMMARY HIV-infected individuals harbor viral quasispecies that differ in genetic sequences and susceptibility to broadly neutralizing antibodies (bNAbs) targeting the HIV envelope (Env). Due to difficulties with current assays in confirming viral sensitivity to bNAbs, most clinical trials enrolled participants without knowledge of their viral susceptibility. Thus, an accurate and sensitive assay with quick turnaround (1-2 weeks) is needed. We propose to approach this aim with two prongs: Aim 1, a phenotypic (Env function) assay for bulk Env neutralization assessment and Aim 2, a genotypic (Env sequence) algorithm for high-throughput prediction of neutralization susceptibility. We propose 10 HIV bNAbs targeting 5 distinct gp120 sites for assay development, with 2 non-HIV human IgG1 antibodies as negative controls. Current assays are difficult because of two labor-intensive and time-consuming procedures: Env single-genome amplification (SGA) and individual Env cloning. Aim 1 will address the latter procedure required because the current TZM-bl neutralization assay cannot tease out bulk Env clones that differ in bNAb susceptibility. Aim 1 will apply DNA barcoding to simultaneously track bNAb sensitivity of hundreds of Env variants in a high-throughput manner. This will be achieved by a novel Env- expressing lentiviral system in which each Env is linked to a unique barcode sequence. Aim 2 will address Env sequencing, for which SGA is necessary because Sanger sequencing cannot sequence bulk templates. We propose in Aim 2 to apply a third-generation sequencing technology, Nanopore sequencing, for effective bulk template sequencing and develop machine learning models for a fast and reliable prediction algorithm. Combining with the ample bNAb neutralization data from literature and Aim 1 of this project, trained computational models can be fast, accurate, and high-throughput to predict bNAb susceptibility. Since the detection of a low yield of functional Env sequences and minority variants from participant’s HIV DNA reservoir is essential, we propose in Aim 3 to investigate whether a virion capture assay could be applied to enrich functional Envs. We will also apply a DNA analysis to enrich minor variants from the viral reservoir. We have biospecimens stored onsite and access to a wide distribution of HIV subtypes from the AIDS Clinical Trials Group (ACTG) and HIV Vaccine Trials Network (HVTN) biorepositories for assay development and validation. All proposed R61 Aims have clear milestones defined and the R61 assays will be developed with streamlined and standard operating procedure (SOP) for adaptation to clinical labs. The Columbia Clinical Trials Support Laboratory is fully equipped and staffed to perform the SOPs and provide input and validation on assay performance. Successful development of an accurate and sensitive bNAb susceptibility assay with quick turnaround will alter the enrollment for bNAb trials and greatly improve clinical develo...