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）的临床获益。奈韦拉平（sdNVP）单次给药后，通过一致基因分型，在20- 69%的母亲和46-87%的婴儿中检测到HIV-1对NVP耐药。更灵敏的检测方法，包括寡核苷酸连接检测（奥拉），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抗性突变体降低了后期ART的疗效，但随着时间的推移，女性中的突变体衰减到临床上不显著的水平。对婴儿NVP耐药性的研究较少。最近，使用浓度为HIV-1人群e2%的对NVP耐药突变体敏感的奥拉，我们在婴儿中观察到3种NVP耐药模式。那些在出生前就感染了HIV-1的人经常被选择，但突变在6-12个月内迅速衰减。在子宫内或产后急性感染的婴儿NVP突变体的频率较低，但突变体持续存在。奥拉还显示，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）的妇女很少发生选择性NVP突变。我们建议使用超深度焦磷酸测序来仔细检查NVP抗性HIV-1的动态，并将这些结果与奥拉进行比较。具体来说，使用焦磷酸测序，我们将：（1）通过~200个病毒模板的奥拉和大规模平行测序来比较NVP和ZDV抗性突变体的浓度（焦磷酸测序）超过576个密码子的1，000+病毒模板/标本;（2）利用HIV-1 pol序列的焦磷酸测序和奥拉（OLA）来估计持续超过ARV 4-6个月的ARV抗性病毒的浓度。（3）确定选择与产前和产后ZDV相关的NVP抗性病毒的妇女是否对ZDV具有低水平抗性;（4）调整奥拉和焦磷酸测序测定法用于HIV-1亚型A、AE、B和D。这些研究将提供对HIV-1亚型A、AE、B和D的选择、衰变、持久性和耐NVP病毒的传播，并导致更好的妇女和婴儿HIV-1临床管理。公共卫生相关性：提出了通过灵敏的点突变寡核苷酸连接试验（奥拉）对NVP和ZDV突变体进行定量的验证。将通过奥拉进行的基因型与通过大规模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.