Clonal amplification and maternal-infant transmission of nevirapine-resistant HIV-1 variants in breast milk following single-dose nevirapine prophylaxis
© Permar et al.; licensee BioMed Central Ltd. 2013
Received: 30 May 2013
Accepted: 6 August 2013
Published: 14 August 2013
Intrapartum administration of single-dose nevirapine (sdNVP) reduces perinatal HIV-1 transmission in resource-limiting settings by half. Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise de novo in the infant.
We performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation.
In this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission.
In 2011, 330,000 infants acquired HIV infection from their mothers . Most of mother-to-child transmissions (MTCT) occur in low- and middle-income countries, where HIV transmission through breastfeeding accounts for 30–50% of infant infections in the absence of antiretroviral (ARV) prophylaxis . In sub-Saharan Africa, formula feeding is not a recommended alternative due to its association with increased morbidity and mortality, caused by malnutrition, respiratory diseases and diarrheal complications . Furthermore, several studies have substantiated the benefit of breastfeeding over formula feeding, despite the risk of breast milk transmission [4–6]. WHO currently recommends exclusive breastfeeding for the first 6 months of life, followed by complementary foods and breastfeeding until 12 months of age, accompanied by postnatal infant or maternal antiretroviral prophylaxis to reduce HIV transmission during breastfeeding . Initial studies showed that intrapartum single-dose nevirapine (sdNVP) prophylaxis reduced the risk of MTCT by half  but lacked efficacy to prevent breast milk HIV transmission. More recently, extended NVP prophylaxis regimens reduced breast milk transmission when compared to sdNVP . Though convenient, inexpensive, and effective, sdNVP prophylaxis selects for NVP-resistant (NVP-R) variants in a high proportion of women (19–75%) and their infected infants (33–87%) [9, 10] and these variants remain detectable for a year or more [10–12]. Moreover, NVP-R variants emerge more frequently and persist longer after exposure to extended NVP prophylaxis [13, 14], while sdNVP administration increases the risk of virologic failure to subsequent ARV treatment .
Although a large fraction of infants harbor NVP-R variants following perinatal NVP administration, it is not clear whether NVP-R variants are transmitted to the infant postnatally or if they arise de novo in the infant [9–11, 16]. Previous studies have not rigorously examined the drug-resistant profile of transmitted/founder (T/F) virus (es) that are responsible for transmission and productive clinical infection in the infant [10, 11, 13, 14, 16]. Understanding the precise origin of drug-resistant strains in MTCT may aide in the design of improved, more broadly effective prophylaxis regimens that will not impair future treatment options for infected infants. Our previous study of chronically HIV-infected Malawian lactating women who received intrapartum sdNVP revealed continual trafficking of blood-derived viral variants into the mammary glands followed by transient local replication of some variants that disproportionally contributed to low-diversity (clonally-amplified) viral populations in breast milk . Here we hypothesized that clonally-amplified variants in breast milk are selected for drug-resistance by NVP and that they are likely to be transmitted to the infant due to their proportional abundance and selective replication fitness in the presence of NVP. To test this hypothesis, we conducted a longitudinal genetic and drug-resistant mutation analysis of HIV variants in plasma and milk of a postnatal-transmitting mother-infant pair following sdNVP administration.
Postnatal transmission of a single virus and time estimates of infection by HIV sequence diversity analysis in the infant
Clinical and virological data of chronically HIV-infected lactating woman 4403
Sample ID weeks postpartum
Plasma virus load (copies/ml)
Breast milk virus load, left and right (copies/ml)
CD4 count (cells/μl)
Infant blood HIV DNA PCR status
Third trimester of pregnancy
Maternal sdNVP on 05/23/08
Statistics and mathematical model timing estimates of the most recent common ancestor (MRCA) of the infant’s HIV sequences detected at the time of postnatal HIV diagnosis
Total number of sequences
Maximum length of sequence
Mean HD (%)
Poisson estimated days since MRCA (95% CI)
Goodness of fit p-value
HD fit to poisson
43 (32, 54)
38 (31, 46)
37 (30, 44)
The inferred infant T/F HIV genome is closely related to a population of low-diversity variants in maternal breast milk near the predicted time of infection
The transmitted virus is a NVP-R variant that underwent prior selection and clonal amplification in the lactating mother
Discussion and conclusions
Few studies have addressed the origin of NVP-R variants observed in postnatally-infected infants. Studies proposing transmission of NVP-R variants in infants based their conclusion on the results of a first time HIV positive test done at late times during the breastfeeding period under the assumption that NVP had been cleared from tissues [13, 14]. Yet, the time interval between the infant HIV testing is usually broad in these studies, and the existing analyses did not include a time estimate since a MRCA of the variants using sequence diversity. Studies that have proposed transmission of wild-type virus followed by selection of resistant variants that were only detectable by sensitive assays based their interpretation on the timing of infection and likely exposure to NVP [10, 11, 23]. A similar interpretation should follow when population sequencing shows mixed wild-type and resistant viruses, unless both forms were simultaneously transmitted; a scenario less likely given the genetic bottleneck of MTCT of HIV [24, 25] and the fact that most (~80%) HIV infections in heterosexuals [18, 26] and infants via breast milk  result from transmission of a single virus. Thus, our study is important in that it establishes the NVP-R breast milk virus population selected by maternal sdNVP as the origin of the NVP-R virus in this postnatally-infected infant. However, our experimental design has a number of limitations. For instance, we cannot exclude the possibility that infection resulted from transmission of multiple identical viral genomes such as the progeny from a single infected cell in maternal milk, yet this limitation does not change our conclusion that the infant was infected by a NVP-R virus strain. Also, although we cannot exclude that the infecting virus strain in the infant could have been transmitted perinatally, our strict definition for breast milk transmission, i.e., HIV DNA PCR negative at birth and 4–6 weeks, but PCR positive at 12 weeks or later falls within the most likelihood for most cases of postnatal HIV transmission. However, these limitations do not impact the focus of this study which was to clarify whether NVP-R resistance is transmitted or arise de novo in the newly infected child. Antiretroviral treatment of an infant who harbors variants with resistance to a component of the antiretroviral treatment is more likely to fail therapy .
Breast milk HIV quasispecies include multiple independent lineages with evidence of intermixing with plasma virus [17, 29–31], while some variants undergo clonal bursts of replication [17, 30, 31]. Clusters of monotypic and low-diversity variants have also been reported in blood and different body compartments such as the female genital tract , lung , semen , and cerebrospinal fluid . Compartmentalization and local replication have been proposed as an explanation. Here, we propose that upon selection by NVP, blood NVP-R variants traffic into the mammary gland and seed the breast milk compartment. Next, a single NVP-R variant undergoes bursts of local replication, contributing a disproportionately large number of NVP-R virions in the milk virus pool and facilitating transmission of a drug-resistant virus via breastfeeding. The abundance of K103N clonal variants in the mother, and high-level viremia of the subtype C T/F K103N virus in the infant suggest that this mutation has little impact on viral fitness. Indeed, K103N mutation is one of the most common mutations selected by NVP as reported in previous MTCT studies [11, 36]. Moreover, in vitro studies indicated that K103N mutation has no effect on replication fitness, while the Y181C mutation results in a much less fit virus when introduced in the backbone of the subtype C molecular clone MJ4 . Our data demonstrate that a single antiretroviral drug, such as NVP provides a mechanism underlying the clonal amplification of resistant variant(s) in breast milk, thus increasing the transmission risk of drug-resistant viruses in infants. The role of ARV drugs in shifting the viral landscape toward the clonal amplification of drug-resistant variants deserves to be further investigated.
In summary, although we are reporting a single MTCT pair analysis, our data are in line with other studies that emphasize the need to implement MTCT prophylaxis strategies that reduce the risk of development and clonal amplification of drug resistant variants in maternal blood and breast milk to prevent MTCT of drug-resistant HIV variants. Our work suggests that administration of sdNVP prophylaxis can lead to postnatal transmission of selected NVP-R variants, and support the global implementation of effective multidrug maternal/infant prophylaxis strategies to prevent infant acquisition of drug-resistant HIV variants that will complicate pediatric ARV treatment strategies.
We thank the University of Alabama at Birmingham Center for AIDS Research (CFAR) DNA Sequencing core (P30AI027767) for sequencing analyses. This study was supported by the Center for HIV/AIDS Vaccine Immunology (U19 AI067854), and the Doris Duke Charitable Foundation Clinical Scientist Development Award (SP).
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