New Phenotypic HIV-1 Drug Resistance Assay Validation

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DESCRIPTION (Provided by the applicant): Improved sensitivity of phenotypic assays for monitoring HIV-1 drug-resistance in AIDS patients, with an emphasis on early detection of drug-resistant minor species, is needed. We are developing a new assay using a molecular vector system to produce indicator cells that overexpress the genes for CD4, CXCR4, and CCR5, making them permissive to both T-tropic and M-tropic HIV-1 infection so that drug resistance assays can be performed using a direct infection approach with patient sera. These indicator cells, containing a tat-activated molecular switch controlling a GFP reporter gene, provide a sensitive mechanism to detect and quantify virus replication in the presence and absence of drugs. The system allows for viral load assays based on the actual levels of infectious virus present. It should readily detect low levels of drug-resistant virus particles in a mixture with predominantly wild-type virus, since only the resistant virus will replicate in the indicator cells in the presence of drug and trigger the expression of the reporter gene. Since PCR or cloning steps are not involved, this assay measures directly the intrinsic drug resistance of the patient's original virus population. The new assay promises to be a simple, sensitive and high-capacity phenotypic test for HIV-1 drug resistance monitoring and will be applicable to all classes of anti-HIV drugs. This Phase I SBIR-AT-NIAID grant application requests funding to optimize, format, and validate the new assay, according to guidelines issued by the HIV-1 Drug Resistance Collaborative Group (RCG), prior to commercialization. This will require studies of the accuracy, sensitivity, precision, specificity and stability of the assay. It will involve extensive retrospective studies with large numbers of well-characterized HIV-1 clinical isolates (both wild-type and resistant strains) from clinical research collaborators and from repositories at pharmaceutical companies, diagnostic companies, and the NIH and the determination of their relative susceptibilities to currently approved antiretroviral drugs. PROPOSED COMMERCIAL APPLICATION: This research project is for the validation of a significantly improved new phenotypic assay with a very large commercial market potential for anti-HIV drug resistance monitoring in AIDS patients. Its applications include performance of the assay by clinical reference laboratories in support of patient management and clinical trials, development of a diagnostic kit for worldwide distribution, viral load assays based on levels of infectious virus, and high- throughput assays for anti-HIV drug discovery.