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

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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.