Quantification and dynamics of HIV-1 drug resistant mutants by pirosequencing

通过吡咯测序对 HIV-1 耐药突变体进行定量和动态分析

• 批准号: 7893793
• 负责人: Lisa M Frenkel
• 金额: $ 24.89万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893793
• 关键词: Address; Adult; Age; Alleles; Anti-Retroviral Agents; Archives; Binding; Biological Assay; Birth; Breast Feeding; Child; Clinical; Clinical Management; Codon Nucleotides; Collaborations; Communities; Complex; Consensus; DNA; Data; Databases; Detection; Dose; Drug resistance; Failure; Genetic Polymorphism; Genome; Genotype; Goals; HIV-1; HIV-1 drug resistance; Individual; Infant; Infection; Kenya; Lead; Learning; Ligation; Methods; Monitor; Mothers; Mozambique; Mutation; Nevirapine; Oligonucleotides; Pathogenesis; Pattern; Plasma; Point Mutation; Population; Positioning Attribute; Postpartum Period; Preparation; Prevalence; Prevalence Study; Prevention; Procedures; Prophylactic treatment; RNA; Reagent; Regimen; Research; Resistance; Resources; Risk; South Africa; Specimen; Temperature; Testing; Thailand; Time; Treatment Failure; Treatment Protocols; Validation; Variant; Viral; Viral Load result; Virus; Woman; Zidovudine; Zidovudine resistance; base; clinically relevant; design; drug resistant virus; efavirenz; improved; in utero; insight; interest; mutant; nevirapine resistance; non-nucleoside reverse transcriptase inhibitors; pressure; prevent; public health relevance; transmission process; uptake

DESCRIPTION (provided by applicant): HIV-1 resistance to antiretrovirals (ARV) has the potential to undermine the clinical benefits of antiretroviral treatment (ART). Following single-dose nevirapine (sdNVP) HIV-1 resistance to NVP has been detected in 20- 69% of mothers and 46-87% of infants by consensus genotyping. More sensitive assays, including an oligonucleotide ligation assay (OLA), detect resistant viruses in an even greater proportion of mothers and infants. Others have observed that NVP-resistant mutants diminish the efficacy of later ART, but that over time mutants in women decay to clinically insignificant levels. Fewer studies have been done of NVP-resistance in infants. Recently, using an OLA sensitive to NVP-resistant mutants at concentrations of e2% of the HIV-1 population, we observed 3 patterns of NVP-resistance in infants. Those who acquired HIV-1 well before birth had frequent selection but rapid decay of mutations over 6-12 months. Infants infected acutely in utero or postpartum had NVP-mutants less frequently, but mutants persisted. OLA also showed few women who take zidovudine (ZDV) pre- and postpartum select NVP mutations compared to most women who take NVP alone. We propose to examine closely the dynamics of NVP-resistant HIV-1 using ultra-deep pyrosequencing, and to compare these results to OLA. Specifically, using pyrosequencing we will: (1) Compare the concentration of NVP- and ZDV-resistant mutants by OLA of ~200 viral templates and massive parallel sequencing (pyrosequencing) of 1,000+ viral templates/specimen over 576 codons; (2) Utilize pyrosequencing and OLA of HIV-1 pol sequences to estimate the concentration of ARV-resistant viruses persisting beyond 4-6 months of ARV-selective pressure in infants and adult women; (3) Determine whether women who select NVP-resistant viruses in association with pre- and postpartum ZDV have low-level resistance to ZDV; (4) Adapt the OLA and pyrosequencing assays for use with HIV-1 Subtypes A, AE, B and D These studies will provide insight into the dynamics of the selection, decay, persistence, and transmission of NVP-resistant viruses, and lead to better clinical management of HIV-1 in women and infants. PUBLIC HEALTH RELEVANCE: Validation of quantification of NVP- and ZDV-mutants by a sensitive point-mutation oligonucleotide ligation assay (OLA) is proposed. Genotypes by OLA will be compared to sequences generated by massive parallel pyrosequencing. Specimens will be selected for study so as to provide insight into the dynamics of drug- resistant viruses relevant to clinical management of HIV-1 in women and infants.

描述（由申请人提供）：HIV-1 对抗逆转录病毒药物（ARV）的耐药性有可能破坏抗逆转录病毒治疗（ART）的临床益处。通过共识基因分型，在单剂量奈韦拉平 (sdNVP) 后，20-69% 的母亲和 46-87% 的婴儿发现 HIV-1 对 NVP 产生耐药性。更灵敏的检测，包括寡核苷酸连接检测（OLA），可以在更大比例的母亲和婴儿中检测到耐药病毒。其他人观察到 NVP 抗性突变体会降低后来 ART 的疗效，但随着时间的推移，女性中的突变体会衰减到临床上不显着的水平。关于婴儿 NVP 耐药性的研究较少。最近，使用对 NVP 抗性突变体敏感的 OLA，其浓度为 HIV-1 群体的 e2%，我们观察到婴儿中 3 种 NVP 抗性模式。那些在出生前就感染了 HIV-1 的人会经历频繁的选择，但突变在 6-12 个月内会迅速衰减。在子宫内或产后急性感染的婴儿出现 NVP 突变的频率较低，但突变仍然存在。 OLA 还显示，与大多数单独服用 NVP 的女性相比，产前和产后服用齐多夫定 (ZDV) 的女性很少出现选择 NVP 突变。我们建议使用超深焦磷酸测序仔细检查 NVP 耐药 HIV-1 的动态，并将这些结果与 OLA 进行比较。具体来说，使用焦磷酸测序，我们将：（1）通过约 200 个病毒模板的 OLA 和超过 576 个密码子的 1,000 多个病毒模板/样本的大规模并行测序（焦磷酸测序）来比较 NVP 和 ZDV 抗性突变体的浓度； (2) 利用 HIV-1 pol 序列的焦磷酸测序和 OLA 来估计婴儿和成年女性中抗逆转录病毒药物选择压力超过 4-6 个月后持续存在的抗逆转录病毒药物浓度； (3)确定选择与产前和产后ZDV相关的NVP耐药病毒的女性是否对ZDV具有低水平的耐药性； (4) 调整 OLA 和焦磷酸测序测定法以用于 HIV-1 亚型 A、AE、B 和 D 这些研究将深入了解 NVP 抗性病毒的选择、衰变、持久性和传播的动态，并导致更好地对妇女和婴儿中的 HIV-1 进行临床管理。公共卫生相关性：建议通过敏感点突变寡核苷酸连接测定 (OLA) 来验证 NVP 和 ZDV 突变体的定量。 OLA 的基因型将与大规模并行焦磷酸测序产生的序列进行比较。将选择样本进行研究，以便深入了解与妇女和婴儿 HIV-1 临床管理相关的耐药病毒动态。