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)的耐药性可能会破坏抗逆转录病毒治疗(ART)的临床益处。在单剂奈韦拉平(SdNVP)治疗后，通过一致的基因分型，已在20-69%的母亲和46-87%的婴儿中检测到HIV-1对NVP的耐药性。包括寡核苷酸连接试验(OLA)在内的更敏感的检测方法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耐药突变会降低后来的抗逆转录病毒疗法的疗效，但随着时间的推移，女性中的突变会衰退到临床上微不足道的水平。在婴儿中对NVP耐药的研究较少。最近，利用对NVP耐药突变株敏感的OLA，我们观察到婴儿对NVP耐药的三种模式。那些在出生前很久就感染了HIV-1的人有频繁的选择，但突变在6-12个月内迅速衰退。在宫内或产后受到急性感染的婴儿较少发生NVP突变，但突变持续存在。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)的女性中很少有选择NVP突变的。我们建议使用超深焦磷酸测序技术仔细检查耐NVP的HIV-1的动力学，并将这些结果与OLA进行比较。具体来说，我们将使用焦磷酸测序：(1)通过~200个病毒模板的OLA和超过576个密码子的1,000+病毒模板/样本的大规模平行测序(焦测序)来比较耐NVP和ZDV突变体的浓度；(2)利用HIV-1 Poll序列的焦测序和OLA来估计婴儿和成年妇女中持续4-6个月ARV选择压力的抗逆转录病毒病毒的浓度；(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具有低水平的抗药性；(4)调整OLA和焦磷酸测序分析方法，用于HIV-1 A、AE、B和D亚型。这些研究将深入了解耐NVP病毒的选择、衰退、持续和传播的动态，并导致更好地在妇女和婴儿中对HIV-1进行临床管理。公共卫生相关性：建议通过灵敏的点突变寡核苷酸连接试验(OLA)来验证NVP和ZDV突变的量化。OLA的基因分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艾滋病毒-1临床管理相关的抗药性病毒的动态。

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.