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

 描述（由申请人提供）：对HIV的耐药性是实现HIV+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，病毒抑制不完全和病毒学失败通常与耐药性有关。因此，目前的DHHS指南建议在开始或改变抗逆转录病毒治疗之前进行耐药性检测。基于群体或批量测序的基因型检测是确定HIV耐药突变的最常用方法。然而，由于HIV在体内作为准种传播，目前的商业检测方法在检测少数耐药变异体方面不敏感，已知这些变异体会损害抗逆转录病毒治疗的临床反应。因此，迫切需要一种能够检测耐药少数群体的准确和灵敏的检测方法，以确定HIV微小变异对病毒抑制的影响，并指导合理选择最佳抗逆转录病毒治疗。这项I期STTR申请的目的是开发一种灵敏的检测方法，可以准确定量HIV-1少数变异体。最近，Wang博士（PI，佛罗里达大学）的实验室开发了一种单变异体测序（SVS）方法，该方法利用了高通量MiSeq技术的速度和准确性，以及一种随机测序标签策略，该策略消除了已知模糊少数变异体真实表达的偏见和技术伪影。通过开发一种自动分析耐药性呼叫的生物信息学管道，他的实验室已经应用这种SVS方法来量化临床样本中与HCV耐药性相关的微小变异。Medosome Biotec及其在UF的研究合作伙伴假设，这种SVS方法可以优化，以准确量化耐药HIV-1的少数群体。在第一阶段，该团队将通过追求两个特定目标来测试这一假设：1）优化SVS方法，以灵敏和准确地定量HIV-1耐药少数变异体，以及2）使用实验室HIV毒株和临床分离株对优化的SVS方法进行初步测试。用于HIV耐药性检测的SVS方法是创新的，因为它将利用MiSeq个人测序仪的速度、准确性和长配对末端读取能力，以及随机测序标签方法来产生新的HIV-1耐药性检测方法，用于定量检测HIV-1微小变异体。如果成功，将在II期研究中验证和开发SVS测定。鉴于全球HIV负担超过3500万HIV+个体需要至少一次耐药性检测，准确且灵敏的低成本SVS检测将具有巨大的商业化潜力。

 DESCRIPTION (provided by applicant): 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 determine the impact of HIV minor variants on viral suppression and guide rational selection of optimal antiretroviral therapy. The objective of this Phase I STTR application is to develop a sensitive assay that can accurately quantify HIV-1 minority variants. Recently, Dr. Wang's (PI, University of Florida) laboratory 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. By developing a bioinformatics pipeline that automates analysis of drug resistance calls, his laboratory has applied this SVS approach to quantify minor HCV resistance-associated variants in clinical samples. Medosome Biotec and its research partner at UF hypothesize that this SVS approach can be optimized to quantify minority populations of drug resistant HIV-1 accurately. In Phase I, the team will test this hypothesis by pursuing two Specific Aims: 1) Optimize the SVS method for sensitive and accurate quantification of HIV-1 drug resistance minority variants, and 2) Conduct initial testing of the optimized SVS method using laboratory HIV strains and clinical isolates. The SVS approach for HIV resistance testing is innovative because it will leverage the speed, accuracy, and the long paired-end read capability of MiSeq personal sequencer, and the random sequencing tag method to produce a new HIV-1 resistance assay for quantitative detection of HIV-1 minor variants. If successful, the SVS assay will be validated and developed in Phase II studies. 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.