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

一种准确、灵敏地检测 HIV 耐药少数变异的方法

• 批准号: 10668218
• 负责人: GARY P. WANG
• 金额: $ 100万
• 依托单位: MEDOSOME BIOTEC, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668218
• 关键词: Accreditation; Anti-Retroviral Agents; Biological Assay; CCR5 gene; CLIA certified; CXCR4 gene; Clinical; Data; Detection; Development; Dose; Drug resistance; Evaluation; Failure; Florida; Frequencies; Genes; Genotype; Guidelines; HIV; HIV drug resistance; HIV resistance; HIV-1; HIV-1 drug resistance; Individual; Integrase; Laboratories; Licensing; Marketing; Measures; Medical Genetics; Methods; Minor; Minority; Minority Groups; Molecular Cloning; Morphologic artifacts; Mother-to-child HIV transmission; NNRTI-resistance; Nevirapine; Patients; Perinatal; Persons; Phase; Population; Pregnant Women; Preparation; Prevalence; RNA; Recommendation; Regimen; Reproducibility; Resistance; Reverse Transcription; Risk; Sampling; Sampling Errors; Sequence Determination; Small Business Technology Transfer Research; Specificity; Specimen; Speed; Techniques; Technology; Testing; Universities; Validation; Variant; Viral; Virus; Work; antiretroviral therapy; assay development; commercialization; cost; design; drug resistant virus; drug testing; falls; genetic testing; improved; in vivo; inhibitor; non-nucleoside reverse transcriptase inhibitors; novel strategies; pressure; prevent; pyrosequencing; receptor; resistance mutation; response; success; treatment response

PROJECT SUMMARY / ABSTRACT 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 guide rational selection of optimal antiretroviral therapy. In Phase I STTR studies, we have 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. We successfully optimized the primers, amplification and sequencing conditions for the PR and RT regions of subtype B HIV-1, and determined the sensitivity, specificity and precision of the assay. We showed that authentic minority variants present at 1% of quasispecies were detected accurately and reproducibly with minimal variations between technical replicates. Building on our success, this Phase II STTR application will complete the development of the assay by expanding to drug resistant loci in the integrase gene and subtype C viruses, and then experimentally validate and commercialize the SVS assay via four Specific Aims: 1) Complete the development of an optimized SVS assay for sensitive quantification of HIV-1 subtype B and C drug resistance minority variants in RT, PR and IN genes, 2) Experimentally validate the SVS assay using well characterized molecular clones and viruses, 3) Conduct pre- market evaluation using real-world clinical samples, and 4) Validate the SVS assay in Medosome’s CLIA certified and CAP accredited Florida licensed clinical genetic testing laboratory. 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.

项目摘要/摘要 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。至.为止 16%的新感染者感染了对至少一种主要抗逆转录病毒类别产生抗药性的艾滋病毒， 病毒抑制不完全和病毒学失败往往与耐药性有关。因此， 目前的卫生部指南建议在开始或更换抗逆转录病毒之前进行耐药性测试 心理治疗。基于群体或批量测序的基因分型分析是最常用的方法 确定艾滋病毒耐药突变。然而，由于艾滋病毒在体内以准种的形式传播，目前 商业化验在检测少数耐药变异株方面不敏感，已知 影响抗逆转录病毒治疗的临床反应。因此，一种准确而灵敏的检测方法 迫切需要能够检测出耐药少数民族人群来指导合理选择 最佳抗逆转录病毒疗法。在第一阶段STTR研究中，我们开发了单变异体测序(SVS) 方法，该方法利用了高吞吐量MiSeq技术的速度和准确性，并且 随机测序标签策略，消除了偏见和已知模糊真值的技术伪像 少数变种的表示法。我们成功地优化了引物、扩增和测序 B亚型HIV-1 PR区和RT区的条件，并测定其敏感性、特异性和 分析的精密度。我们发现，在1%的准种中，真正的少数变种是 准确和可重复性地检测，技术复制之间的差异最小。建立在我们的 成功，这一第二阶段的STTR应用将通过扩展到药物来完成测试的开发 整合酶基因和C亚型病毒中的抗性位点，然后进行实验验证和商业化 SVS法通过四个特定的目的：1)建立一种优化的敏感的SVS法 HIV-1 B、C亚型耐药基因RT、PR和IN少数变异的定量研究(2) 使用特征良好的分子克隆和病毒对SVS分析进行实验验证，3)进行预 使用真实临床样本进行市场评估，以及4)在Medosome的CLIA中验证SVS分析 经认证和CAP认证的佛罗里达州有执照的临床基因检测实验室。准确而灵敏的低气压 考虑到全球艾滋病毒的负担，成本SVS检测将具有巨大的商业潜力 超过3500万艾滋病毒+患者需要至少进行一次耐药性检测。