Mass Spectrometric Detection of Drug-Resistant HIV

DESCRIPTION (provided by applicant): There are an estimated one million people in the U.S. infected with HIV-1, with 40,000 new cases reported each year. While most of these patients greatly benefit from highly active anti-retroviral therapy (HAART), they often develop multi-drug resistance associated with mutations in viral genes which cause problems for long-term treatment. Knowledge of the spectrum of mutations in HIV-1 genes is extremely important in order to effectively adjust the drug treatment for these patients and also for drug-nave patients. DNA sequencing is the most accurate and comprehensive method currently available for detecting these mutations and genotyping mutant viruses. However sequencing is both expensive and most importantly does not easily detect subpopulations of drug-resistant strains below 20%. It is also difficult to link specific mutations to individual viral species. The principal objective of this proposal is to develop a highly sensitive, cost-effective and high throughput system for detecting drug-resistant strains of the HIV-1 virus based on mass spectrometry of in vitro expressed proteins (MASSIVE-PRO). During Phase I, MASSIVE-PRO was developed and evaluated using WT and drug-resistant forms of HIV-1 genes. Several important goals were achieved including: i) Elimination of proteolysis of expressed polypeptides, ii) Demonstration of the ability to detect drug resistance mutations in subpopulations of HIV-1 down to the 5% level and iii) Sequence- based determination of drug-resistance mutations. In Phase II, a comprehensive HIV-1 genotyping drug resistance test based on MASSIVE-PRO will be developed. A key aspect of this test will be the ability to routinely detect and characterize drug- resistance mutations in the PR and RT genes down to the 5% level. Dr. Richard D'Aquila's laboratory at Vanderbilt Medical School, recognized internationally for its expertise on HIV-1 drug-resistance, will serve as a subcontractor, participating in assay development and providing samples to AmberGen for validating the MASSIVE-PRO assay. Quest Diagnostics will also advise on assay development to ensure compatibility with large-scale commercial laboratory practices. Public Narrative There are an estimated one million people in the U.S. infected with HIV-1, with 40,000 new cases reported each year. Optimal treatment of these patients requires a low-cost but highly sensitive diagnostic method of detecting drug-resistance mutations in HIV-1 genes in patients initiating or undergoing drug therapy. Currently, DNA sequencing of the mutant virus cannot easily detect subpopulations of drug-resistant strains below 20% which can lead to failure of treatment. Recent progress by AmberGen demonstrates that this limitation can be overcome by using a newly developed method based on mass spectrometry of in vitro expressed proteins (MASSIVE-PRO). During Phase II, a HIV-1 genotyping drug-resistance test with sensitivity for drug resistant subpopulations of virus under 5% will be developed and extensively tested.