Sensitive Drug-Resistance Assays Reveal Long-Term Persistence of HIV-1 Variants with the K103N Nevirapine(NVP) Resistance Mutation in Some
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore
Gene Regulation and Chromosome Biology Laboratory, Reverse Transcription and Molecular Biology Section, Basic Research Program, Science Applications International CorporationFrederick, National Cancer InstituteFrederick, Frederick, Maryland
Departments of Paediatrics and Obstetrics and Gynaecology, Makerere University, Kampala, Uganda
Background.
The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)1 variants persisted in women and infants 1 year or more after the administration of SD-NVP.
Methods.
We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 68 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT.
Results.
ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 1224 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay.
Conclusions.
K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.
Short antiretroviral drug regimens can reduce the risk of mother-to-child transmission (MTCT) of HIV-1 in resource-limited settings. In the HIV Network for Prevention Trials (HIVNET) 012 regimen, women receive single-dose nevirapine (SD-NVP) during labor and infants receive SD-NVP shortly after birth. This regimen is simple, safe, inexpensive, and effective for the prevention of MTCT (pMTCT) of HIV-1 [1]. A disadvantage of this regimen is the emergence of NVP resistance (NVPR) after the administration of SD-NVP. In HIVNET 012, NVPR was detected in 25% of women [2] and in 46% of infants 68 weeks after delivery [3]. Emergence of NVPR after the administration of SD-NVP was subsequently observed in other studies. Results of a recent study suggest that women with prior exposure to SD-NVP may have a reduced virologic response to treatment regimens that contain nonnucleoside reverse-transcriptase (RT) inhibitors (NNRTIs) [4]. It is not known whether prior exposure to SD-NVP reduces the efficacy of treatment regimens that contain NVP in HIV-1infected children or the efficacy of SD-NVP for pMTCT in subsequent pregnancies. NVP-resistant variants selected in women by SD-NVP could also potentially be transmitted to infants by breast-feeding or to others in the community.
Few studies have evaluated the persistence of NVPR after the administration of SD-NVP. In HIVNET 012, samples obtained 1224 months after the administration of SD-NVP were available for 11 women and 6 infants who had NVPR at 68 weeks after the administration of SD-NVP. No NVPR mutations were detected in those samples [3]. In the HIVNET 023 trial, variants with NVPR mutations could not be detected in almost all women by 6 months postpartum [5]. However, in a South African cohort, 55 (35%) of 155 women who had NVPR at 7 weeks postpartum still had detectable NVPR at 6 months postpartum [6]. Those studies were performed with assays based on population sequencing that are relatively insensitive for the detection of drug-resistant HIV-1 variants present in conjunction with wild-type (wt) HIV-1. In the present exploratory study, we used sensitive resistance assays to analyze the emergence and persistence of HIV-1 variants with the K103N mutation in a subset of women and infants in the HIVNET 012 cohort.
SUBJECTS, MATERIALS, AND METHODS
Study subjects.
The human experimentation guidelines of the US Department of Health and Human Services and the authors' institutions were followed in the conduct of the research. Women were antiretroviral drug naive and did not receive any other antiretroviral therapy, which is consistent with the standard of care in Uganda at the time the trial was performed. Samples from 9 women and 5 infants who had available samples from long-term follow-up were analyzed. These study subjects had been selected for follow-up in a previous study, after they were found to have NVPR in samples obtained 68 weeks after delivery [3]. Samples obtained before the administration of SD-NVP from 40 additional women in HIVNET 012 were also analyzed.
HIV-1 genotyping and subtyping.
HIV-1 genotyping was performed with the ViroSeq HIV-1 Genotyping System (Celera Diagnostics), and HIV-1 subtyping was performed by phylogenetic analysis of pol region sequences [2]. Some samples were genotyped in studies published elsewhere [3, 7].
Detection of the K103N mutation using the LigAmp assay.
The LigAmp assay involves mutation-specific ligation of 2 oligonucleotides to a DNA template, followed by detection of the ligated product by real-time polymerase chain reaction (PCR) [8] (table 1). The LigAmp assay was optimized for the detection of the K103N mutation in HIV-1 subtypes A and D (figures 1 and 2). The conditions used for analysis are described in the Appendix, in the electronic edition. The limit of detection for the K103N mutation in both subtypes was 0.08% (mean + 3 SDs of the result obtained for wt DNA). The assay cutoff was set at 0.1% mutant. For analysis of plasma samples, PCR products generated with ViroSeq were used as template DNA and were analyzed in triplicate. To permit analysis of the same samples with the TyHRT assay, PCR products were generated without dUTP. A standard curve was included in each experiment and was used to quantify the percentage of K103N mutations in each sample (figure 2C and 2D).
Detection of NVPR mutations using the yeast TyHRT assay.
The TyHRT assay was used to screen libraries of HIV-1 RT-containing variants for RT activity and NVP susceptibility [9]. Plasmids from NVP-resistant variants were sequenced to identify NVPR mutations. A general description of the assay and the conditions used for analysis are provided in the Appendix, in the electronic edition.
RESULTS
Detection of NVPR mutations using ViroSeq.
Plasma samples were analyzed using an assay based on population sequencing (ViroSeq) (figures 3A and 4A and table 2). The women and infants selected for the present study all had NVPR mutations detected 68 weeks after the administration of SD-NVP [3].
Samples obtained from women before the administration of SD-NVP and at delivery contained wt HIV-1 (no NVPR mutations were detected) (figure 3A). Genotyping results for 5 women were obtained 1 week after the administration of SD-NVP. None of the samples had HIV-1 variants with the K103N mutation. Samples from 2 women had HIV-1 variants with other NVPR mutations (1 had Y181C and 1 had Y181C and G190A). Samples obtained at 68 weeks after the administration of SD-NVP from 8 of 9 women had HIV-1 variants with the K103N mutation and from 4 of 9 women had HIV-1 variants with other NVPR mutations (3 had K103N and Y181C and 1 had V108I). Samples obtained 1224 months after the administration of SD-NVP from all 9 women had wt HIV-1.
Samples obtained at birth from all 5 infants had wt HIV-1 (figure 4A). Samples obtained at 68 weeks after the administration of SD-NVP from 2 of 5 infants had HIV-1 variants with the K103N mutation, and those from 4 of 5 infants had HIV-1 variants with other NVPR mutations (2 had Y181C, 1 had K103N and Y181C, and 1 had Y181C and Y188C). Samples obtained 1416 weeks after the administration of SD-NVP were available for 4 of 5 infants. One infant had HIV-1 variants with the K103N mutation, and 1 infant had HIV-1 variants with the Y181C mutation. The K103N mutation was not detected in the samples from infants at 1 year after the administration of SD-NVP. The G190A mutation was detected in a sample obtained from 1 infant at 12 months after the administration of SD-NVP but was not detected in samples obtained from that infant at earlier study visits.
Detection and quantification of variants with the K103N mutation using the LigAmp assay.
We used the LigAmp assay to detect and quantify HIV-1 variants with the K103N mutation (figures 3B and 4B). The K103N mutation was selected for analysis, because K103N was the most common NVPR mutation detected by ViroSeq in women in HIVNET 012 68 weeks after exposure to NVP [2] and in a South African cohort 6 months after exposure to NVP [6].
Samples from all 9 women obtained before exposure to NVP and at delivery had frequencies of HIV-1 variants with the K103N mutation below the assay cutoff (< 0.1%) (figure 3B). Samples obtained before exposure to NVP from 40 other women in HIVNET 012 were also analyzed. The frequency of HIV-1 variants with the K103N mutation was below the assay cutoff in 36 (90%) of 40 samples. In the remaining 4 samples, HIV-1 variants with the K103N mutation were detected at frequencies of 0.1%, 0.1%, 0.2%, and 0.3% (data not shown). In 8 of 9 women selected for follow-up, HIV-1 variants with the K103N mutation were detected in the samples obtained 68 weeks after the administration of SD-NVP. The mean percentage of HIV-1 variants with the K103N mutation in the 8 women was 13.9%. In 6 women, the frequency of HIV-1 variants with the K103N mutation declined to <0.1% by 1224 months after the administration of SD-NVP. However, in 3 women, HIV-1 variants with the K103N mutation were detected at a frequency of >0.1% (at 0.8%, 1.3%, and 3.5%) 14 months after the administration of SD-NVP (figure 3B, arrows).
The frequency of HIV-1 variants with the K103N mutation was below the assay cutoff in 4 of 5 infants at birth (figure 4B). In 1 infant, HIV-1 variants with the K103N mutation were detected at a frequency just >0.1%. At 68 weeks after the administration of SD-NVP, HIV-1 variants with the K103N mutation were detected at a frequency of >0.1% in 4 of 5 infants, and the percentage of HIV-1 variants with the K103N mutation in 1 infant (I-545) remained elevated at 1416 weeks after the administration of SD-NVP. In that infant, HIV-1 variants with the K103N mutation were also detected 1 year after the administration of SD-NVP (at a frequency of 1.5%) (figure 4B, arrow).
Comparison of results from the LigAmp and TyHRT assays.
The samples described above were also analyzed using the yeast-based TyHRT assay to confirm the persistence of the K103N mutation in the samples obtained during long-term follow-up (figures 3C and 4C and table 2). The TyHRT assay is a genetic assay that allows libraries of HIV-1 RTcontaining clones to be screened for RT activity and NVP susceptibility.
Results from the LigAmp assay (measuring the percentage of variants with the K103N mutation) and the TyHRT assay (measuring the percentage of NVP-resistant variants) were consistent. Seventeen of 20 samples that had HIV-1 variants with the K103N mutation detected by the LigAmp assay also had HIV-1 variants with NVPR detected by the TyHRT assay, and the K103N mutation was detected in the NVP-resistant clones. In the 3 remaining samples, the frequency of HIV-1 variants with the K103N mutation detected in the LigAmp assay was <1%. Failure to detect clones with NVPR in those samples in the TyHRT assay may reflect the lower sensitivity of that assay for the detection of mutants (0.5% mutant). A few samples that did not have HIV-1 variants with the K103N mutation detected by the LigAmp assay had HIV-1 variants with NVPR detected by the TyHRT assay. In those samples, clones isolated in the TyHRT assay did not have the K103N mutation, with the exception of rare clones found in 1 mother-infant pair (1/210 clones from the sample obtained from M-750 before the administration of SD-NVP and 1/190 clones from the sample obtained from I-750 14 weeks after the administration of SD-NVP). In both cases, the K103N mutation was encoded by AAT, which was not probed in the LigAmp assay. The K103N mutation was detected by the LigAmp assay in 4 samples obtained during long-term follow-up, at frequencies of 0.8%, 1.3%, 3.5%, and 1.5% (M-474, M-750, M-830, and I-545, respectively) (figures 3B and 4B, arrows). The percentage of HIV-1 variants with NVPR detected in the TyHRT assay for those samples was 3.4%, 0.6%, 11%, and 0.3%, respectively. Even though relatively few NVP-resistant clones were isolated from those samples, HIV-1 clones with the K103N mutation were detected in all 4 samples. In contrast, samples obtained during long-term follow-up that did not have HIV-1 variants with the K103N mutation detected by the LigAmp assay also did not have HIV-1 variants with the K103N mutation detected by the TyHRT assay. Of note, the only infant sample obtained 12 months after the administration of SD-NVP that had HIV-1 variants with the G190A mutation detected by ViroSeq also had HIV-1 variants with G190A detected by the TyHRT assay. Interestingly, 2 samples (the sample obtained from M-554 1 week after the administration of SD-NVP and the sample obtained from M-847 at delivery) had HIV-1 variants with NVPR detected by the TyHRT assay but did not have known NVPR mutations detected in the NVP-resistant clones. In both cases, substitutions were noted at codon 236 (P236L and P236A, respectively). In HIV-1 subtype B, the P236L mutation is associated with resistance to delavirdine but causes hypersusceptibility (rather than resistance) to NVP.
DISCUSSION
We found that HIV-1 variants with the K103N mutation can persist at frequencies greater than those found before exposure to NVP in some women and infants 1 year after the administration of SD-NVP. This finding is consistent with the results of studies showing that the K103N mutation confers a relatively small fitness cost in vitro [10] and with the results of studies showing persistence of the K103N mutation for years in some patients who are infected with drug-resistant HIV-1 strains [11]. Results of preliminary studies using mutation-specific real-time PCR assays have also shown long-term persistence of NVPR mutations in women with HIV-1 subtype C [12, 13]. It is important to note that the results of our study may not be representative of the entire HIVNET 012 cohort, because the number of women and infants analyzed was small. Furthermore, our study included only women and infants who had HIV-1 variants with NVPR detected by ViroSeq 68 weeks after the administration of SD-NVP. This approach is likely to bias the results in favor of detection of HIV-1 variants with NVPR in samples obtained during long-term follow-up.
The LigAmp assay was particularly useful for quantification of HIV-1 variants with the K103N mutation in our study. The assay has a broad linear range and can quantify variants present at a frequency of 0.1%. The assay can be performed without patient-specific primers and can be performed using PCR products that remain after routine genotyping. The assay is relatively simple to perform and uses very low concentrations of template. The TyHRT assay also offers unique advantages for the analysis of HIV-1 drug-resistance mutations. That assay provides phenotypic selection of drug-resistant variants, which can be further characterized by DNA sequencing. That approach may be particularly useful for analysis of drug-resistant variants in non-B subtypes, because the genetic correlates of antiretroviral drug resistance in HIV-1 subtypes other than subtype B are not well defined.
Further studies are needed to determine whether persistence of HIV-1 variants with NVPR mutations circulating at low levels after the administration of SD-NVP compromises the efficacy of regimens that contain NNRTIs for HIV-1 treatment or pMTCT in subsequent pregnancies. The emergence of NVPR in women and infants receiving regimens for pMTCT can usually be prevented by providing pregnant women with potent combination antiretroviral therapy. However, in resource-poor countries where there is limited access to antiretroviral drugs, simpler regimens are more likely to be used for pMTCT. Some studies have evaluated NVPR in women who received SD-NVP for pMTCT in combination with other antiretroviral drugs. Unfortunately, NVPR often emerges despite the addition of other drugs to the pMTCT regimen [1416]. Further studies to evaluate the clinical significance of NVPR in this setting, and the persistence of low levels of HIV-1 variants with NVPR in women and infants after SD-NVP exposure, are needed. Sensitive resistance assays may be useful for the evaluation of the persistence of drug-resistant variants.
Acknowledgments
We thank the HIV Network for Prevention Trials 012 team, for providing the samples; Melissa Allen (clinical trials specialist, Family Health International), for providing assistance; Estelle Piwowar-Manning, Constance Ducar, and the laboratory staff in Uganda, for assisting with the processing of the samples; and Boehringer Ingelheim, for providing nevirapine.
APPENDIX
METHODS FOR DETECTION OF THE K103N MUTATION IN HIV-1 SUBTYPES A AND D USING THE LIGAMP ASSAY
Ligation was performed using 100 pg of plasmid-derived or plasma-derived PCR products. DNA concentrations were determined using a NanoDrop spectrophotometer (ND-1000; NanoDrop Technologies). Gel-purified ligation oligonucleotides (table 1) were purchased from Invitrogen. PCR products were incubated with 1 pmol of the upstream oligonucleotide, 2 pmol of the downstream oligonucleotide, and 4 U of Pfu DNA ligase in 1× Pfu Ligase Buffer (Stratagene). Samples were denatured at 95°C for 1 min, followed by 99 2-step cycles of 95°C for 30 s alternating with 50°C for 4 min. A negative control (no DNA template) was included with each set of ligation reactions and was carried through the real-time detection step. Real-time PCR was performed using a SmartCycler (Cepheid). Each 25-L reaction contained 5 pmol of M13 forward primer and 5 pmol of M13 reverse primer (Invitrogen) (table 1), 5 L of the unpurified ligation reaction, 12.5 L of platinum Quantitative PCR SuperMix-UDG (Invitrogen), and 5 pmol of the LacZ probe (Integrated DNA Technology) (table 1). Reactions were preincubated at 50°C for 2 min and 95°C for 2 min, followed by 45 2-step cycles of 95°C for 10 s alternating with 64°C for 25 s. The cycle threshold was manually set in the middle of the linear range of the amplification curve for each experiment (log scale).
METHODS FOR ANALYSIS OF NNRTI RESISTANCE MUTATIONS USING THE YEAST TYHRT ASSAY
Assay description.
In the TyHRT system, amplified HIV-1 pol region DNA from test samples is cotransformed into yeast with a plasmid containing a TyHRT element with a deletion in the RT region. HIV-1 RT DNA is introduced into the TyHRT element by homologous recombination. Each isolate carries a unique RT domain, and the library of isolates is representative of the RT domains present in the original viral sample. The TyHRT elements carry the reverse-transcription indicator gene his3AI. Expression and reverse transcription of the TyHRT element by the recombined HIV-1 RT results in conversion of the his3AI gene into a functional HIS3 gene. RT activity is detected as the ability to give rise to cells that are able to grow on medium lacking histidine. Selection in the presence of NNRTIs measures the NNRTI susceptibility of individual RT clones. Analysis of the RT activity and NNRTI susceptibility of the isolates present in large libraries of clones makes it possible to detect NNRTI-resistant RT variants that are present at low frequencies.
Conditions used for analysis.
Reverse transcription was performed in yeast strain DG1251 (MAT ura3-167 trp1-hisG spt3-101 his3200). The plasmid pHRT1 has the BsrGI-PvuII fragment of the HIV-1 RT coding region removed, resulting in deletion of HIV-1 RT aa 37250. BsrGI/PvuII linearized, gel-purified pHRT1 (100200 ng) and HIV-1 RT domain PCR product (200500 ng) were cotransformed into DG1251 using a lithium acetate protocol. Transformants were selected on plates that had glucose and synthetic complete medium lacking uracil (SC-URA). Transformants were arrayed using sterile toothpicks in small (100/plate) or large (25/plate) patches on plates that had glucose and SC-URA. After incubation at 30°C overnight, arrayed patches were replica plated with velveteen to plates that had galactose and SC-URA with or without inhibitor and were grown for 2 days at 30°C to induce expression of the hybrid retroelements under the control of the GAL1 promoter. The plates were then replica plated to plates that had synthetic complete medium lacking histidine with or without inhibitor and were grown at 30°C for 34 days to select RT events. NVP was dissolved in dimethylsulfoxide (DMSO) and was used at 80160 mol/L at a final DMSO concentration of 1%.
For DNA sequencing, HIV-1 RT domains in TyHRT elements were obtained by growing isolates in 10 mL of SC-URA at 30°C overnight and preparing DNA with a glass bead/phenol extraction method. DNA was suspended in 50 L of water, and 0.51.0 L of the solution was used in a 50-L PCR to amplify the RT domain. Amplification was performed for 35 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 2 min) using primers A-35 (5-GAACCTCCGAGATCGAAGA-3) and I1097 (5-GCACTGCCTCTGTTAATTGT-3), resulting in a PCR product that included the HIV-1 RT coding region for aa 1367. PCR products were purified with the Qiaquick 96 PCR Purification Kit (Qiagen Sciences) and were sequenced with internal primers B275 (5-AGACTTCTGGGAAGTTCAAT-3), 220F (5-TGGAGAAAATTAGTAGATTT-3), and J801 (5-ATCCCTGGGTAAATCTGACT-3).
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Gene Regulation and Chromosome Biology Laboratory, Reverse Transcription and Molecular Biology Section, Basic Research Program, Science Applications International CorporationFrederick, National Cancer InstituteFrederick, Frederick, Maryland
Departments of Paediatrics and Obstetrics and Gynaecology, Makerere University, Kampala, Uganda
Background.
The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)1 variants persisted in women and infants 1 year or more after the administration of SD-NVP.
Methods.
We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 68 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT.
Results.
ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 1224 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay.
Conclusions.
K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.
Short antiretroviral drug regimens can reduce the risk of mother-to-child transmission (MTCT) of HIV-1 in resource-limited settings. In the HIV Network for Prevention Trials (HIVNET) 012 regimen, women receive single-dose nevirapine (SD-NVP) during labor and infants receive SD-NVP shortly after birth. This regimen is simple, safe, inexpensive, and effective for the prevention of MTCT (pMTCT) of HIV-1 [1]. A disadvantage of this regimen is the emergence of NVP resistance (NVPR) after the administration of SD-NVP. In HIVNET 012, NVPR was detected in 25% of women [2] and in 46% of infants 68 weeks after delivery [3]. Emergence of NVPR after the administration of SD-NVP was subsequently observed in other studies. Results of a recent study suggest that women with prior exposure to SD-NVP may have a reduced virologic response to treatment regimens that contain nonnucleoside reverse-transcriptase (RT) inhibitors (NNRTIs) [4]. It is not known whether prior exposure to SD-NVP reduces the efficacy of treatment regimens that contain NVP in HIV-1infected children or the efficacy of SD-NVP for pMTCT in subsequent pregnancies. NVP-resistant variants selected in women by SD-NVP could also potentially be transmitted to infants by breast-feeding or to others in the community.
Few studies have evaluated the persistence of NVPR after the administration of SD-NVP. In HIVNET 012, samples obtained 1224 months after the administration of SD-NVP were available for 11 women and 6 infants who had NVPR at 68 weeks after the administration of SD-NVP. No NVPR mutations were detected in those samples [3]. In the HIVNET 023 trial, variants with NVPR mutations could not be detected in almost all women by 6 months postpartum [5]. However, in a South African cohort, 55 (35%) of 155 women who had NVPR at 7 weeks postpartum still had detectable NVPR at 6 months postpartum [6]. Those studies were performed with assays based on population sequencing that are relatively insensitive for the detection of drug-resistant HIV-1 variants present in conjunction with wild-type (wt) HIV-1. In the present exploratory study, we used sensitive resistance assays to analyze the emergence and persistence of HIV-1 variants with the K103N mutation in a subset of women and infants in the HIVNET 012 cohort.
SUBJECTS, MATERIALS, AND METHODS
Study subjects.
The human experimentation guidelines of the US Department of Health and Human Services and the authors' institutions were followed in the conduct of the research. Women were antiretroviral drug naive and did not receive any other antiretroviral therapy, which is consistent with the standard of care in Uganda at the time the trial was performed. Samples from 9 women and 5 infants who had available samples from long-term follow-up were analyzed. These study subjects had been selected for follow-up in a previous study, after they were found to have NVPR in samples obtained 68 weeks after delivery [3]. Samples obtained before the administration of SD-NVP from 40 additional women in HIVNET 012 were also analyzed.
HIV-1 genotyping and subtyping.
HIV-1 genotyping was performed with the ViroSeq HIV-1 Genotyping System (Celera Diagnostics), and HIV-1 subtyping was performed by phylogenetic analysis of pol region sequences [2]. Some samples were genotyped in studies published elsewhere [3, 7].
Detection of the K103N mutation using the LigAmp assay.
The LigAmp assay involves mutation-specific ligation of 2 oligonucleotides to a DNA template, followed by detection of the ligated product by real-time polymerase chain reaction (PCR) [8] (table 1). The LigAmp assay was optimized for the detection of the K103N mutation in HIV-1 subtypes A and D (figures 1 and 2). The conditions used for analysis are described in the Appendix, in the electronic edition. The limit of detection for the K103N mutation in both subtypes was 0.08% (mean + 3 SDs of the result obtained for wt DNA). The assay cutoff was set at 0.1% mutant. For analysis of plasma samples, PCR products generated with ViroSeq were used as template DNA and were analyzed in triplicate. To permit analysis of the same samples with the TyHRT assay, PCR products were generated without dUTP. A standard curve was included in each experiment and was used to quantify the percentage of K103N mutations in each sample (figure 2C and 2D).
Detection of NVPR mutations using the yeast TyHRT assay.
The TyHRT assay was used to screen libraries of HIV-1 RT-containing variants for RT activity and NVP susceptibility [9]. Plasmids from NVP-resistant variants were sequenced to identify NVPR mutations. A general description of the assay and the conditions used for analysis are provided in the Appendix, in the electronic edition.
RESULTS
Detection of NVPR mutations using ViroSeq.
Plasma samples were analyzed using an assay based on population sequencing (ViroSeq) (figures 3A and 4A and table 2). The women and infants selected for the present study all had NVPR mutations detected 68 weeks after the administration of SD-NVP [3].
Samples obtained from women before the administration of SD-NVP and at delivery contained wt HIV-1 (no NVPR mutations were detected) (figure 3A). Genotyping results for 5 women were obtained 1 week after the administration of SD-NVP. None of the samples had HIV-1 variants with the K103N mutation. Samples from 2 women had HIV-1 variants with other NVPR mutations (1 had Y181C and 1 had Y181C and G190A). Samples obtained at 68 weeks after the administration of SD-NVP from 8 of 9 women had HIV-1 variants with the K103N mutation and from 4 of 9 women had HIV-1 variants with other NVPR mutations (3 had K103N and Y181C and 1 had V108I). Samples obtained 1224 months after the administration of SD-NVP from all 9 women had wt HIV-1.
Samples obtained at birth from all 5 infants had wt HIV-1 (figure 4A). Samples obtained at 68 weeks after the administration of SD-NVP from 2 of 5 infants had HIV-1 variants with the K103N mutation, and those from 4 of 5 infants had HIV-1 variants with other NVPR mutations (2 had Y181C, 1 had K103N and Y181C, and 1 had Y181C and Y188C). Samples obtained 1416 weeks after the administration of SD-NVP were available for 4 of 5 infants. One infant had HIV-1 variants with the K103N mutation, and 1 infant had HIV-1 variants with the Y181C mutation. The K103N mutation was not detected in the samples from infants at 1 year after the administration of SD-NVP. The G190A mutation was detected in a sample obtained from 1 infant at 12 months after the administration of SD-NVP but was not detected in samples obtained from that infant at earlier study visits.
Detection and quantification of variants with the K103N mutation using the LigAmp assay.
We used the LigAmp assay to detect and quantify HIV-1 variants with the K103N mutation (figures 3B and 4B). The K103N mutation was selected for analysis, because K103N was the most common NVPR mutation detected by ViroSeq in women in HIVNET 012 68 weeks after exposure to NVP [2] and in a South African cohort 6 months after exposure to NVP [6].
Samples from all 9 women obtained before exposure to NVP and at delivery had frequencies of HIV-1 variants with the K103N mutation below the assay cutoff (< 0.1%) (figure 3B). Samples obtained before exposure to NVP from 40 other women in HIVNET 012 were also analyzed. The frequency of HIV-1 variants with the K103N mutation was below the assay cutoff in 36 (90%) of 40 samples. In the remaining 4 samples, HIV-1 variants with the K103N mutation were detected at frequencies of 0.1%, 0.1%, 0.2%, and 0.3% (data not shown). In 8 of 9 women selected for follow-up, HIV-1 variants with the K103N mutation were detected in the samples obtained 68 weeks after the administration of SD-NVP. The mean percentage of HIV-1 variants with the K103N mutation in the 8 women was 13.9%. In 6 women, the frequency of HIV-1 variants with the K103N mutation declined to <0.1% by 1224 months after the administration of SD-NVP. However, in 3 women, HIV-1 variants with the K103N mutation were detected at a frequency of >0.1% (at 0.8%, 1.3%, and 3.5%) 14 months after the administration of SD-NVP (figure 3B, arrows).
The frequency of HIV-1 variants with the K103N mutation was below the assay cutoff in 4 of 5 infants at birth (figure 4B). In 1 infant, HIV-1 variants with the K103N mutation were detected at a frequency just >0.1%. At 68 weeks after the administration of SD-NVP, HIV-1 variants with the K103N mutation were detected at a frequency of >0.1% in 4 of 5 infants, and the percentage of HIV-1 variants with the K103N mutation in 1 infant (I-545) remained elevated at 1416 weeks after the administration of SD-NVP. In that infant, HIV-1 variants with the K103N mutation were also detected 1 year after the administration of SD-NVP (at a frequency of 1.5%) (figure 4B, arrow).
Comparison of results from the LigAmp and TyHRT assays.
The samples described above were also analyzed using the yeast-based TyHRT assay to confirm the persistence of the K103N mutation in the samples obtained during long-term follow-up (figures 3C and 4C and table 2). The TyHRT assay is a genetic assay that allows libraries of HIV-1 RTcontaining clones to be screened for RT activity and NVP susceptibility.
Results from the LigAmp assay (measuring the percentage of variants with the K103N mutation) and the TyHRT assay (measuring the percentage of NVP-resistant variants) were consistent. Seventeen of 20 samples that had HIV-1 variants with the K103N mutation detected by the LigAmp assay also had HIV-1 variants with NVPR detected by the TyHRT assay, and the K103N mutation was detected in the NVP-resistant clones. In the 3 remaining samples, the frequency of HIV-1 variants with the K103N mutation detected in the LigAmp assay was <1%. Failure to detect clones with NVPR in those samples in the TyHRT assay may reflect the lower sensitivity of that assay for the detection of mutants (0.5% mutant). A few samples that did not have HIV-1 variants with the K103N mutation detected by the LigAmp assay had HIV-1 variants with NVPR detected by the TyHRT assay. In those samples, clones isolated in the TyHRT assay did not have the K103N mutation, with the exception of rare clones found in 1 mother-infant pair (1/210 clones from the sample obtained from M-750 before the administration of SD-NVP and 1/190 clones from the sample obtained from I-750 14 weeks after the administration of SD-NVP). In both cases, the K103N mutation was encoded by AAT, which was not probed in the LigAmp assay. The K103N mutation was detected by the LigAmp assay in 4 samples obtained during long-term follow-up, at frequencies of 0.8%, 1.3%, 3.5%, and 1.5% (M-474, M-750, M-830, and I-545, respectively) (figures 3B and 4B, arrows). The percentage of HIV-1 variants with NVPR detected in the TyHRT assay for those samples was 3.4%, 0.6%, 11%, and 0.3%, respectively. Even though relatively few NVP-resistant clones were isolated from those samples, HIV-1 clones with the K103N mutation were detected in all 4 samples. In contrast, samples obtained during long-term follow-up that did not have HIV-1 variants with the K103N mutation detected by the LigAmp assay also did not have HIV-1 variants with the K103N mutation detected by the TyHRT assay. Of note, the only infant sample obtained 12 months after the administration of SD-NVP that had HIV-1 variants with the G190A mutation detected by ViroSeq also had HIV-1 variants with G190A detected by the TyHRT assay. Interestingly, 2 samples (the sample obtained from M-554 1 week after the administration of SD-NVP and the sample obtained from M-847 at delivery) had HIV-1 variants with NVPR detected by the TyHRT assay but did not have known NVPR mutations detected in the NVP-resistant clones. In both cases, substitutions were noted at codon 236 (P236L and P236A, respectively). In HIV-1 subtype B, the P236L mutation is associated with resistance to delavirdine but causes hypersusceptibility (rather than resistance) to NVP.
DISCUSSION
We found that HIV-1 variants with the K103N mutation can persist at frequencies greater than those found before exposure to NVP in some women and infants 1 year after the administration of SD-NVP. This finding is consistent with the results of studies showing that the K103N mutation confers a relatively small fitness cost in vitro [10] and with the results of studies showing persistence of the K103N mutation for years in some patients who are infected with drug-resistant HIV-1 strains [11]. Results of preliminary studies using mutation-specific real-time PCR assays have also shown long-term persistence of NVPR mutations in women with HIV-1 subtype C [12, 13]. It is important to note that the results of our study may not be representative of the entire HIVNET 012 cohort, because the number of women and infants analyzed was small. Furthermore, our study included only women and infants who had HIV-1 variants with NVPR detected by ViroSeq 68 weeks after the administration of SD-NVP. This approach is likely to bias the results in favor of detection of HIV-1 variants with NVPR in samples obtained during long-term follow-up.
The LigAmp assay was particularly useful for quantification of HIV-1 variants with the K103N mutation in our study. The assay has a broad linear range and can quantify variants present at a frequency of 0.1%. The assay can be performed without patient-specific primers and can be performed using PCR products that remain after routine genotyping. The assay is relatively simple to perform and uses very low concentrations of template. The TyHRT assay also offers unique advantages for the analysis of HIV-1 drug-resistance mutations. That assay provides phenotypic selection of drug-resistant variants, which can be further characterized by DNA sequencing. That approach may be particularly useful for analysis of drug-resistant variants in non-B subtypes, because the genetic correlates of antiretroviral drug resistance in HIV-1 subtypes other than subtype B are not well defined.
Further studies are needed to determine whether persistence of HIV-1 variants with NVPR mutations circulating at low levels after the administration of SD-NVP compromises the efficacy of regimens that contain NNRTIs for HIV-1 treatment or pMTCT in subsequent pregnancies. The emergence of NVPR in women and infants receiving regimens for pMTCT can usually be prevented by providing pregnant women with potent combination antiretroviral therapy. However, in resource-poor countries where there is limited access to antiretroviral drugs, simpler regimens are more likely to be used for pMTCT. Some studies have evaluated NVPR in women who received SD-NVP for pMTCT in combination with other antiretroviral drugs. Unfortunately, NVPR often emerges despite the addition of other drugs to the pMTCT regimen [1416]. Further studies to evaluate the clinical significance of NVPR in this setting, and the persistence of low levels of HIV-1 variants with NVPR in women and infants after SD-NVP exposure, are needed. Sensitive resistance assays may be useful for the evaluation of the persistence of drug-resistant variants.
Acknowledgments
We thank the HIV Network for Prevention Trials 012 team, for providing the samples; Melissa Allen (clinical trials specialist, Family Health International), for providing assistance; Estelle Piwowar-Manning, Constance Ducar, and the laboratory staff in Uganda, for assisting with the processing of the samples; and Boehringer Ingelheim, for providing nevirapine.
APPENDIX
METHODS FOR DETECTION OF THE K103N MUTATION IN HIV-1 SUBTYPES A AND D USING THE LIGAMP ASSAY
Ligation was performed using 100 pg of plasmid-derived or plasma-derived PCR products. DNA concentrations were determined using a NanoDrop spectrophotometer (ND-1000; NanoDrop Technologies). Gel-purified ligation oligonucleotides (table 1) were purchased from Invitrogen. PCR products were incubated with 1 pmol of the upstream oligonucleotide, 2 pmol of the downstream oligonucleotide, and 4 U of Pfu DNA ligase in 1× Pfu Ligase Buffer (Stratagene). Samples were denatured at 95°C for 1 min, followed by 99 2-step cycles of 95°C for 30 s alternating with 50°C for 4 min. A negative control (no DNA template) was included with each set of ligation reactions and was carried through the real-time detection step. Real-time PCR was performed using a SmartCycler (Cepheid). Each 25-L reaction contained 5 pmol of M13 forward primer and 5 pmol of M13 reverse primer (Invitrogen) (table 1), 5 L of the unpurified ligation reaction, 12.5 L of platinum Quantitative PCR SuperMix-UDG (Invitrogen), and 5 pmol of the LacZ probe (Integrated DNA Technology) (table 1). Reactions were preincubated at 50°C for 2 min and 95°C for 2 min, followed by 45 2-step cycles of 95°C for 10 s alternating with 64°C for 25 s. The cycle threshold was manually set in the middle of the linear range of the amplification curve for each experiment (log scale).
METHODS FOR ANALYSIS OF NNRTI RESISTANCE MUTATIONS USING THE YEAST TYHRT ASSAY
Assay description.
In the TyHRT system, amplified HIV-1 pol region DNA from test samples is cotransformed into yeast with a plasmid containing a TyHRT element with a deletion in the RT region. HIV-1 RT DNA is introduced into the TyHRT element by homologous recombination. Each isolate carries a unique RT domain, and the library of isolates is representative of the RT domains present in the original viral sample. The TyHRT elements carry the reverse-transcription indicator gene his3AI. Expression and reverse transcription of the TyHRT element by the recombined HIV-1 RT results in conversion of the his3AI gene into a functional HIS3 gene. RT activity is detected as the ability to give rise to cells that are able to grow on medium lacking histidine. Selection in the presence of NNRTIs measures the NNRTI susceptibility of individual RT clones. Analysis of the RT activity and NNRTI susceptibility of the isolates present in large libraries of clones makes it possible to detect NNRTI-resistant RT variants that are present at low frequencies.
Conditions used for analysis.
Reverse transcription was performed in yeast strain DG1251 (MAT ura3-167 trp1-hisG spt3-101 his3200). The plasmid pHRT1 has the BsrGI-PvuII fragment of the HIV-1 RT coding region removed, resulting in deletion of HIV-1 RT aa 37250. BsrGI/PvuII linearized, gel-purified pHRT1 (100200 ng) and HIV-1 RT domain PCR product (200500 ng) were cotransformed into DG1251 using a lithium acetate protocol. Transformants were selected on plates that had glucose and synthetic complete medium lacking uracil (SC-URA). Transformants were arrayed using sterile toothpicks in small (100/plate) or large (25/plate) patches on plates that had glucose and SC-URA. After incubation at 30°C overnight, arrayed patches were replica plated with velveteen to plates that had galactose and SC-URA with or without inhibitor and were grown for 2 days at 30°C to induce expression of the hybrid retroelements under the control of the GAL1 promoter. The plates were then replica plated to plates that had synthetic complete medium lacking histidine with or without inhibitor and were grown at 30°C for 34 days to select RT events. NVP was dissolved in dimethylsulfoxide (DMSO) and was used at 80160 mol/L at a final DMSO concentration of 1%.
For DNA sequencing, HIV-1 RT domains in TyHRT elements were obtained by growing isolates in 10 mL of SC-URA at 30°C overnight and preparing DNA with a glass bead/phenol extraction method. DNA was suspended in 50 L of water, and 0.51.0 L of the solution was used in a 50-L PCR to amplify the RT domain. Amplification was performed for 35 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 2 min) using primers A-35 (5-GAACCTCCGAGATCGAAGA-3) and I1097 (5-GCACTGCCTCTGTTAATTGT-3), resulting in a PCR product that included the HIV-1 RT coding region for aa 1367. PCR products were purified with the Qiaquick 96 PCR Purification Kit (Qiagen Sciences) and were sequenced with internal primers B275 (5-AGACTTCTGGGAAGTTCAAT-3), 220F (5-TGGAGAAAATTAGTAGATTT-3), and J801 (5-ATCCCTGGGTAAATCTGACT-3).
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