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

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。在单剂量奈韦拉平（sdNVP）后，通过共识基因分型，在20- 69%的母亲和46-87%的婴儿中检测到HIV-1对奈韦拉平的耐药性。更灵敏的试验，包括寡核苷酸结扎试验（OLA），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，抗nvp的突变体降低了后来抗逆转录病毒治疗的疗效，但随着时间的推移，女性体内的突变体衰减到临床无关紧要的水平。很少有关于婴儿nvp耐药的研究。最近，我们在HIV-1人群中使用浓度为e2%的对nvp耐药突变体敏感的OLA，观察到婴儿中有3种nvp耐药模式。那些早在出生前就感染HIV-1的人有频繁的选择，但在6-12个月内突变迅速衰减。在子宫内或产后急性感染的婴儿发生nvp突变的频率较低，但突变持续存在。OLA还显示，与大多数单独服用NVP的妇女相比，在产前和产后服用齐多夫定（ZDV）的妇女很少选择NVP突变。我们建议使用超深焦磷酸测序仔细检查nvp抗性HIV-1的动力学，并将这些结果与OLA进行比较。具体而言，我们将使用焦磷酸测序：(1)通过OLA对约200个病毒模板和1000多个病毒模板/样本的576个密码子进行大规模平行测序（焦磷酸测序），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来估计婴儿和成年妇女中持续超过arv选择压力4-6个月的arv耐药病毒的浓度；(3)确定选择与产前和产后ZDV相关的nvp耐药病毒的妇女是否对ZDV具有低水平耐药性；(4)将OLA和焦磷酸测序技术应用于HIV-1亚型A、AE、B和D。这些研究将有助于深入了解nvp耐药病毒的选择、衰变、持续和传播的动态，并有助于改善妇女和婴儿HIV-1的临床管理。公共卫生相关性：提出了一种敏感点突变寡核苷酸连接试验（OLA）来验证NVP-和zdv -突变体的定量。OLA的基因型将与大量平行焦磷酸测序产生的序列进行比较。将选择标本进行研究，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。

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.