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

描述（由申请人提供）：抗逆转录病毒治疗失败一直是感染艾滋病毒-1的非法吸毒者的一个特殊问题。这一失败主要是由于抗逆转录病毒药物治疗方案依从性差。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原发性耐药突变的较高频率可能导致这一群体的不良反应。使用群体测序进行基因型耐药分析已被证明有助于指导在初始治疗后出现病毒复发的个体选择抗逆转录病毒治疗。我们对蛋白酶抑制剂（PI）和非核苷逆转录酶抑制剂（NNRTI）初始IDU中多达10个病毒克隆的初步数据分析表明，IDU受试者访诊中携带PI耐药突变的比例（28%）显著高于非IDU中携带PI耐药突变的比例（8%）。使用标准群体基因分型技术几乎没有检测到这些耐药突变。范德比尔特医学院（Vanderbilt Medical School）的研究人员在一组非注射非法药物使用者中发现了类似的高水平原发性耐药性。该建议假设，在开始HAART之前使用敏感测序技术将预测耐药性的快速发展，因此能够开发个性化和更有效的初始HAART方案。特别是在来自约翰霍普金斯大学和范德比尔特医学院的150名HAART治疗迅速失败的未接受HAART治疗的吸毒者和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20个HIV-1克隆的pol区域的标准克隆分析来确定抗性突变的鉴定是否比来自同一次就诊的群体基因分型更能预测后续治疗失败的风险2)评估通过RT和蛋白酶区相对较短序列（454测序）的高通量克隆分析鉴定耐药突变是否比以下方法更能预测后续治疗失败的风险：i)标准群体基因分型和ii)分析20个克隆（使用Sanger法测序），其中NRTI和NNRTI或PI耐药突变可以在同一个病毒克隆上识别出来。3)使用454测序技术评估PI或NNRTI耐药克隆所需的克隆耐药频率，以预测快速发展的治疗失败风险。这项研究的结果可以为HAART的开始提供一个新的护理标准，并可以大大减少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。这项新技术将允许更好地表征感染个体的病毒株，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.