Assay Development and Validation for Precision Antiretroviral Therapy to Combat Drug Resistance - 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 and consistent platform for DR assessment. The overall goal of this
project is to validate the use of our phenotype assay with its much-improved resolution and accuracy to predict
cART efficacy from the sequence information found in HIV-infected patients and thereby assist in cART regimen
selection specifically tailored to the individual.
