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

项目概要/摘要 对艾滋病毒的耐药性是实现艾滋病毒+个体长期病毒抑制的主要威胁。高达 16%的新感染者感染艾滋病毒时对至少一种主要的抗逆转录病毒药物具有耐药性， 并且不完全的病毒抑制和病毒学失败通常与耐药性有关。因此，我们认为， 目前的DHHS指南建议在开始或改变抗逆转录病毒药物之前进行耐药性测试 疗法基于群体或批量测序的基因型测定是最常用的方法， 确定艾滋病毒耐药性突变。然而，由于艾滋病毒在体内作为准种传播， 商业测定在检测少数耐药变异体方面不敏感，已知这些变异体 危及抗逆转录病毒治疗的临床反应。因此，一个准确和灵敏的测定， 迫切需要能够检测耐药少数群体的方法来指导合理选择 最佳抗逆转录病毒治疗在I期STTR研究中，我们开发了单变异体测序（SVS） 该方法利用了高通量MiSeq技术的速度和准确性， 随机测序标签策略，消除了偏见和技术工件，已知掩盖真正的 少数民族的代表性。我们成功地优化了引物、扩增和测序 HIV-1亚型B的PR和RT区的条件，并确定了灵敏度、特异性和 测定的精密度。我们表明，在1%的准种中存在真正的少数变体， 准确且可重复检测，技术重复之间的变化最小。充分发挥两国 如果成功，该II期STTR应用将通过扩展到药物， 整合酶基因和C亚型病毒中的耐药位点，然后进行实验验证和商业化 通过四个特定目的的SVS测定：1）完成用于灵敏度测定的优化SVS测定的开发。 RT、PR和IN基因中HIV-1亚型B和C耐药少数变异体的定量，2） 使用充分表征的分子克隆和病毒实验验证SVS测定，3）进行预测定。 使用真实世界临床样品进行市场评价，以及4）在Medosome的CLIA中验证SVS测定 认证和CAP认可的佛罗里达许可的临床基因检测实验室。准确灵敏的低 考虑到全球艾滋病毒的负担， 超过3500万艾滋病毒阳性者需要至少一次耐药性检测。

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.