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Session 137 Poster Abstracts
Vertical Transmission and Antiretroviral Resistance
Friday, 1:30 - 3:30 pm
Hall B


800    
Sensitive Drug Resistance Assays Reveal Long-term Persistence of HIV-1 Variants with the K103N Nevirapine-resistance Mutation in Some Women and Infants after Single-dose NVP: HIVNET 012
Susan Eshleman*1, D Nissley2,3, C Claasen3, D Jones1, C Shi1, L Guay1, P Musoke4, F Mmiro4, J Strathern3, J Jackson1, J Eshleman1, and T Flys1
1Johns Hopkins Med Inst, Baltimore, MD, USA; 2SAIC-Frederick, MD, USA; 3NCI-Frederick, NIH, DHHS, MD, USA; and 4Makerere Univ, Kampala, Uganda

Background:  The HIVNET 012 trial showed that single dose (sd) nevirapine (NVP) can prevent HIV-1 mother-to-child transmission (MTCT). However, NVP-resistant variants are selected in some women and infants who receive this regimen. We tested whether variants with the K103N mutation could persist at low levels in women and infants for a year or more after sdNVP exposure using a sensitive point-mutation assay.

Methods:  Plasma collected before and until 12 to 24 months after sdNVP was available from 9 women and 5 infants in the HIVNET 012 cohort. Samples were genotyped using the ViroSeq system, and the level of K103N-containing variants was quantified using a sensitive resistance assay, LigAmp, which has a lower limit of detection of 0.08%. In the LigAmp assay, a mutation-specific ligation step is followed by a universal real-time PCR detection step. Selected samples were also analyzed with a yeast-based, phenotypic resistance assay, TyHRT. Introduction of HIV-1 reverse transcriptase (RT) domains into hybrid Ty1/HIV retrotransposons in vivo independent of conventional cloning facilitates the assembly of large RT libraries. RT isolates are screened in the presence and absence of NVP to provide a measure of phenotypic NVP susceptibility. The HIV-1 RT region from selected NVP-resistant colonies is sequenced to confirm the presence of NVP resistance mutations.

Results:  At 6 to 8 weeks after NVP, K103N was detected by ViroSeq in 8 of 9 women and 2 of 5 infants, and was detected by LigAmp at a level above 0.1% in 8 of 9 women (mean = 14%) and 4 of 5 infants (mean = 12%). The 2 infants who had the mutation detected by LigAmp only had levels of 0.6% and 0.7%. At 12 to 24 months, K103N was not detected by ViroSeq in any of the samples, but was detected by LigAmp above pre-NVP levels in 3 to 9 women (at 0.8%, 1.3%, and 3.5%) and 1 to 5 infants (at 1.5%). Persistence of K103N in the 12- to 24-month samples was confirmed using the TyHRT assay.

Conclusions:  K103N-containing HIV-1 variants can persist in some women and infants for a year or more after sdNVP. Assays that can quantify HIV-1 variants at low levels may provide new insight into the effect of ART exposure on HIV-1 evolution. Further studies are needed to determine the clinical significance of minority variants with NVP-resistance mutations.

Keywords: resistance; nevirapine; minority variants