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）失败一直是HIV-1感染的非法药物使用者的一个特殊问题。这一失败主要归因于对抗逆转录病毒药物治疗方案的依从性差。该实验室的研究表明，我们在注射吸毒者（IDU）中记录的较高病毒突变频率以及由此导致的pol原发性耐药突变频率较高可能导致该组的应答较差。使用群体测序的基因型耐药性分析已被证明在指导初始治疗后出现病毒复发的个体选择抗逆转录病毒治疗方面是有用的。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）初治IDU的多达10个病毒克隆进行分析的初步数据表明，与非IDU中发现的低得多的比例（8%）相比，IDU受试者访视中携带PI耐药突变的比例显著高得多（28%）。使用标准人群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。该提案假设在开始HAART之前使用敏感的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和范德比尔特医学院的150名HAART初治药物使用者的队列中，这些药物使用者快速地失败了HAART，并且150名治疗成功的可比较的受试者，我们将1）通过使用桑格测序方法研究来自20名HIV-1的pol区域的标准克隆分析来确定是否鉴定耐药突变。1来自单次访视的克隆比来自同一次访视的群体基因分型更好地预测后续治疗失败的风险2）评估是否通过来自RT和蛋白酶区域的相对短序列的高通量克隆分析鉴定耐药突变（454测序）预测后续治疗失败的风险优于：i）标准群体基因分型和ii）20个克隆的分析（使用桑格法测序），其中可以在同一病毒克隆上鉴定NRTI和NNRTI或PI抗性突变，和3）使用454测序技术评估，具有PI或NNRTI抗性的克隆所需的克隆抗性频率，以预测治疗失败的快速发展的风险增加。这项研究的结果可以为HAART的启动提供一个新的护理标准，并可以大大减少HAART失败的经济和社会影响。这项研究旨在评估新技术，使抗HIV-1药物治疗更有效。这项新技术将允许更好地表征感染个体的病毒株，以便治疗可以特异性地针对这些病毒。

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.