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

 描述(由申请人提供)：对艾滋病毒的抗药性是实现艾滋病毒+患者长期病毒抑制的主要威胁。高达16%的新感染者感染了对至少一种主要抗逆转录病毒类别具有耐药性的艾滋病毒，病毒抑制不完全和病毒学失败往往与耐药性有关。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测试。基于群体或批量测序的基因分型方法是目前检测HIV耐药突变最常用的方法。然而，由于艾滋病毒在体内以准种的形式传播，目前的商业检测方法在检测少数耐药变异方面不敏感，众所周知，这些变异会影响抗逆转录病毒治疗的临床反应。因此，迫切需要一种能够准确、灵敏地检测耐药少数群体的检测方法，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方案。这一阶段STTR应用的目标是开发一种灵敏的分析方法，能够准确地定量HIV-1少数变异。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，它利用了高通量MiSeq技术的速度和准确性，以及随机测序标签策略，消除了已知的偏见和技术假象，掩盖了少数变异体的真实代表。通过开发自动分析耐药电话的生物信息学管道，他的实验室应用了这种SVS方法来量化临床样本中与丙型肝炎病毒耐药相关的轻微变异。Medosome Biotec和它在密歇根大学的研究伙伴假设，这种SVS方法可以被优化，以准确地量化抗药性HIV-1的少数群体。在第一阶段，该团队将通过追求两个具体目标来测试这一假设：1)优化SVS方法，以敏感和准确地量化HIV-1耐药少数变异；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耐药性测试的SVS方法是创新的，因为它将利用MiSeq个人测序仪的速度、准确性和长对端读取能力，以及随机测序标签方法来产生一种新的HIV-1耐药性检测方法，用于定量检测HIV-1微小变异。如果成功，SVS检测将在第二阶段研究中得到验证和开发。考虑到全球艾滋病毒的负担，准确和灵敏的低成本SVS检测将具有巨大的商业潜力，超过3500万艾滋病毒+患者至少需要进行一次耐药性检测。

 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.