A method for accurate and sensitive detection of HIV drug-resistant minority variants

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PROJECT SUMMARY / ABSTRACT Drug resistance to HIV is a major threat to achieving long-term viral suppression in HIV+ individuals. Up to 16% of newly infected individuals acquire HIV with resistance to at least one of the major antiretroviral classes, and incomplete viral suppression and virologic failure are often associated with drug resistance. Therefore, current DHHS guideline recommends drug resistance testing before beginning or changing antiretroviral therapy. Genotypic assay based on population or bulk sequencing is the most commonly used method to determine HIV drug resistance mutations. However, because HIV circulates as quasispecies in vivo, current commercial assays are not sensitive in detecting minority drug resistant variants, which are known to compromise clinical response to antiretroviral therapy. Therefore, an accurate and sensitive assay that is capable of detecting drug resistant minority populations is urgently needed to guide rational selection of optimal antiretroviral therapy. In Phase I STTR studies, we have developed a Single Variant Sequencing (SVS) approach, which takes advantage of the speed and accuracy of the high-throughput MiSeq technology, and a random sequencing tags strategy that removes biases and technical artifacts known to obscure true representations of minority variants. We successfully optimized the primers, amplification and sequencing conditions for the PR and RT regions of subtype B HIV-1, and determined the sensitivity, specificity and precision of the assay. We showed that authentic minority variants present at 1% of quasispecies were detected accurately and reproducibly with minimal variations between technical replicates. Building on our success, this Phase II STTR application will complete the development of the assay by expanding to drug resistant loci in the integrase gene and subtype C viruses, and then experimentally validate and commercialize the SVS assay via four Specific Aims: 1) Complete the development of an optimized SVS assay for sensitive quantification of HIV-1 subtype B and C drug resistance minority variants in RT, PR and IN genes, 2) Experimentally validate the SVS assay using well characterized molecular clones and viruses, 3) Conduct pre- market evaluation using real-world clinical samples, and 4) Validate the SVS assay in Medosome’s CLIA certified and CAP accredited Florida licensed clinical genetic testing laboratory. An accurate and sensitive low cost SVS assay will have tremendous commercialization potential, given the global burden of HIV with more than 35 million HIV+ individuals requiring resistance testing at least once.