Assay Development and Validation for Precision Antiretroviral Therapy to Combat Drug Resistance Project Summary This application is in response to NOT-AI-21-056 (HIV Drug Resistance Assays and Actionable Data Dissemination Strategies) focusing on assay development and validation to improve care for patients experiencing drug resistance (DR). Most HIV-positive individuals under combination antiretroviral therapy (cART) can have successful viral suppression, although, a subpopulation is unable to keep viral load under control. HIV DR is expected to have a growing impact on the overall effectiveness of cART with the underlying causes being complex and multifaceted. Current genotype and phenotype analyses have limitations in providing consistent and accurate prediction of treatment outcome, and not all patients receiving second- or third-line salvage therapy regimen achieve virological suppression. Therefore, precise and personalized medicines are needed to help care for these misfortunate individuals. We recently established an infectivity assay employing proviral constructs carrying an H2B-mRFP reporter driven by the nef promoter, which highlights the infected cells with a red nucleus allowing for infectivity quantification at a single cell resolution. By comparing the WT and a protease double mutant (V77I/V82T, identified in a patient experiencing indinavir resistance), our phenotype analysis successfully recapitulated the clinical manifestation and defined contributions of each point mutation to DR development showing that mutation 82T predominantly confers indinavir resistance and increases darunavir susceptibility at the same time. Our data support the common consensus that DR can be caused by multiple pathways with each displaying distinct susceptibility towards specific cART regimens, which would provide a therapeutic opportunity to maximize efficacy by selecting antiretroviral drugs based on patient-derived sequences – personalized medicine at point of care. The main objective of this proposal is to determine the technical merit and feasibility of our assay for accurate and consistent DR assessment in guiding precision cART regimen selection. Studies in Aim 1 will improve throughput capacity of the current assay and establish a platform for phenotype analysis of model lymphocytes – the natural targets of HIV infection. Studies in Aim 2 will focus on characterization of protease inhibitor resistance-associated mutations (RAMs) to define the role of individual mutations in DR development and to validate our assay performance and to establish genotype-phenotype correlation baselines. Studies in Aim 3 will establish and validate assay platforms for DR assessment of different cART regimens containing various combinations of protease, reverse transcriptase, and integrase inhibitors in the context of subtype-specific backbones. Results of these proposed studies will generate critical proof-of-concept for our phenotype assay in providing a reliable ...