Use of clonal genotyping to predict resistance development in ART-naive IDU

描述(由申请人提供)：抗逆转录病毒治疗(ART)的失败一直是艾滋病毒-1感染的非法吸毒者中的一个特别问题。这一失败主要归因于抗逆转录病毒药物方案依从性差。该实验室的研究表明，我们在注射吸毒者(IDU)中记录的较高的病毒突变频率以及由此导致的POL原发耐药突变的较高频率可能是导致这一群体反应较差的原因之一。使用群体测序的基因耐药分析已被证明在指导在最初治疗后出现病毒复发的个体中选择抗逆转录病毒治疗是有用的。我们对来自蛋白酶抑制剂(PI)和非核苷类逆转录酶抑制剂(NNRTI)的10个病毒克隆的初步数据分析表明，与非IDU中发现的低得多的比例(8%)相比，IDU患者中携带PI耐药突变的比例显著和显著高(28%)。使用标准的群体基因分型技术几乎没有检测到这些耐药突变。在一群非注射非法吸毒者中也发现了类似的高水平的初级耐药性，随后是范德比尔特医学院的调查人员。该建议假设，在启动HAART之前使用敏感的测序技术将预测耐药性的快速发展，从而使个性化和更有效的初始HAART方案的开发成为可能。具体地说，在来自约翰霍普金斯医学院和范德比尔特医学院的150名快速未能通过HAART的初治吸毒者和150名治疗成功的类似受试者的队列中，我们将1)通过使用Sanger测序方法研究来自一次访问的20个HIV-1克隆的Pol1区的标准克隆分析来确定耐药性突变的鉴定是否比同一次访问的人群基因分型更好地预测后续治疗失败的风险2)评估通过来自RT和蛋白酶区域(454测序)的相对较短序列的高通量克隆分析来鉴定耐药性突变是否更好地预测后续治疗失败的风险：i)基因分型和ii)对20个克隆的分析(使用Sanger方法测序)，其中可以在同一病毒克隆上识别出NRTI和NNRTI或PI抗性突变，以及3)评估，使用454测序技术，具有PI或NNRTI抗性的克隆所需的克隆性耐药性的频率，以预测快速发展为治疗失败的风险增加。这项研究的结果可以为HAART的启动提供一个新的护理标准，并可以极大地减少HAART失败的经济和社会影响。这项研究旨在评估将使抗HIV-1药物治疗更有效的新技术。这项新技术将能够更好地描述感染个人的病毒株的特征，从而使治疗能够专门针对这些病毒。

DESCRIPTION (provided by applicant): Failure of antiretroviral therapy (ART) has been a particular problem among HIV-1 infected illicit drug users. This failure has primarily been attributed to poor compliance with antiretroviral drug regimens. Studies from this laboratory have indicated that a higher viral mutation frequency that we have documented in injection drug users (IDU) and the resulting higher frequency of primary resistance mutations in pol might contribute to the poor response in this group. Genotypic resistance analysis using population sequencing has proved useful in guiding selection of antiretroviral therapy in individuals who have developed viral relapse after initial treatment. Our preliminary data analyzing up to 10 viral clones from protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) naive IDU have indicated that a strikingly and significantly high proportion of IDU subject-visits (28%) carry PI resistance mutations compared to the much lower proportion (8%) found in non-IDU. Almost none of these resistance mutations were detected using standard population genotyping techniques. Similar high levels of primary resistance have been found in a cohort of non-injection illicit drug users followed by investigators at Vanderbilt Medical School. This proposal hypothesizes that use of sensitive sequencing techniques prior to initiation of HAART will be predictive of rapid development of resistance, therefore enabling the development of personalized and more effective initial HAART regimens. Specifically in cohorts from the Johns Hopkins and Vanderbilt Schools of Medicine of 150 HAART-naive drug users who rapidly failed HAART and 150 comparable subjects for whom therapy was successful we will 1) Determine whether identification of resistance mutations by standard clonal analysis using the Sanger sequencing method to study the pol region from 20 HIV-1 clones from a single visit predicts risk of subsequent therapy failure better than population genotyping from that same visit 2) evaluate whether identification of resistance mutations by high throughput clonal analysis of relatively short sequences from the RT and protease regions (454 sequencing) predicts risk of subsequent therapy failure better than: i) standard population genotyping and ii) analysis of 20 clones (sequenced using the Sanger method) in which both NRTI and NNRTI or PI resistance mutations can be identified on the same viral clone and 3) Evaluate, using 454 sequencing technology, the frequency of clonal resistance needed for clones with PI or NNRTI resistance to predict increased risk of rapid development of therapy failure. The results of this study could provide a new standard of care for initiation of HAART and could greatly reduce the financial and social impact of HAART failure. This study is designed to evaluate new technologies that will render anti-HIV-1 drug therapy more effective. This new technology will permit better characterization of the viral strains that are infecting an individual so that the therapy can be specifically targeted to those viruses.