HIV-1 subtype C is the most prevalent subtype worldwide and is the dominant subtype in sub-Saharan Africa, where one-half of all infected women and children live [1,2]. Approximately 30% of infants born to untreated HIV-positive women will become infected with the virus, of whom approximately 20% will become infected in utero, 50% intrapartum, and the remaining 30% through breast milk . Little is known about the mechanism of transmission among these distinct groups, but several maternal characteristics are associated with increased rates of mother-to-child transmission (MTCT), including high viral RNA load, advanced disease status, and low CD4+ T-cell count .
One consistent feature of vertical HIV-1 transmission is a viral genetic bottleneck from mother to infant [5–8], whereby the genetic diversity of HIV-1 in the maternal viral population is greater than that in their infected infants. The bottleneck has been attributed to various factors involving selection, including some that are virus specific  and others that are the result of donor/recipient immune responses [9,10]. In contrast to selective mechanisms, vertical transmission could also be a stochastic event that depends solely on the donor's viral burden with chance favoring the most abundant maternal variant for transmission, as suggested by at least one study .
Most previous studies of HIV-1 MTCT have involved small numbers of mother–infant pairs (MIPs); in this study, we used a heteroduplex tracking assay (HTA) and phylogenetic analyses to study the viral diversity of HIV-1 subtype C in 25 intrauterine MIPs, 23 intrapartum MIPs, and 32 nontransmitting HIV-1-positive mothers. In addition, we used a mathematical simulation in an attempt to fit our data to a stochastic model of transmission.
Participants and methods
The HIV-1 seropositive pregnant women and their infants included in this study were participants in the Malaria and HIV-1 in Pregnancy (MHP) prospective cohort [12–14]. The present study was approved by both the Malawi College of Medicine Research Committee and the UNC IRB. Informed consent was obtained from the participants.
Plasma was isolated from maternal blood collected at labor-ward admission from the umbilical cord at delivery and from infant heel-sticks at three time points: within 48 h of birth, 6, and 12 weeks of age. Women and their newborn infants received single-dose nevirapine according to the HIVNET 012 protocol . The timing of HIV-1 transmission was categorized according to the definitions of Bryson et al.  as follows: infants HIV-1 DNA negative by real-time PCR  at 0 and 6 weeks had their mothers defined as nontransmitters; infants HIV-1 DNA positive at birth were defined as in utero infections; and infants HIV-1 DNA negative at birth but DNA positive at 6 weeks were defined as intrapartum infections. Owing to the timing of the infant blood sampling, it is likely that the intrapartum definition cannot resolve late in utero and early breast-feeding transmissions from true intrapartum infections. Four infants who were HIV-1 DNA negative but HIV-1 RNA positive by reverse-transcriptase polymerase chain reaction (RT-PCR) at birth were classified as in utero.
As reported elsewhere , 65 infants were infected in utero, 65 were infected intrapartum (not including those infected postpartum), and 418 were HIV free at 12 weeks. Samples were chosen from these participants on the basis of availability, and after RT-PCR, a total of 32/418 nontransmitters, 25/65 intrauterine, and 23/65 intrapartum samples were included in the data set. Samples were excluded from this study if there was insufficient maternal/infant plasma, the RT-PCR was negative when the DNA was positive or the HTA patterns were not reproducible due to poor sampling of low-abundance variants. Compared with the included samples, the median log10 RNA copies per milliliter was significantly lower in the excluded samples (data not shown).
Genomic DNA was analyzed for the presence of HIV-1 DNA by real-time PCR . Plasma HIV-1 RNA was quantified using Amplicor HIV-1 Monitor version 1.5 (Roche Diagnostics, Indianapolis, Indiana, USA). CD4+ T cells were quantified by FACScan (Becton Dickinson, Franklin Lakes, New Jersey, USA).
Viral RNA isolation
Viral RNA was isolated from peripheral blood plasma using the QIAmp viral RNA kit (Qiagen, Germantown, Maryland, USA). Plasma from six women whose RT-PCR reaction was negative was concentrated by centrifugation and amplicons were obtained from five.
The Titan One-Tube RT-PCR system (Roche) or the Stratagene Accuscript RT-PCR system was used to amplify the HIV-1 subtype C V1/V2 region of the env gene as previously described . All samples were RT-PCR amplified in two independent reactions to allow assessment of the quality of sampling.
Heteroduplex tracking assay
The HTA [18–20] was used to document viral diversity as previously described  using a subtype C V1/V2 env probe derived from the DU151 clone [18,22].
ImageQuant TL software (Molecular Dynamics/GE Healthcare, Uppsala, Sweden) was used to quantify the intensity of each heteroduplex band and calculate the percentage abundance. An HTA band was included as an env variant if it was not present in the probe alone lane, on average, it comprised more than 2% of the total viral population, and it was present in both PCR replicates. Reproducibility in sampling of the population of HIV-1 variants was determined using the percentage change between duplicates, as previously described . The maternal replicates had a median 7% (interquartile range: 3, 10) difference; the reproducibility of the replicates among the first positive infant samples was less than 1%, perhaps due to presumed high RNA viral loads and observed low viral complexity among the infants. Validation of proper sampling is done to limit the appearance of population differences where none exists . Infant V1/V2 env variant bands with a corresponding band in the maternal sample (determined by migration in the gel) were defined as ‘detected’. If an infant V1/V2 env variant had no corresponding band in the maternal sample (or the band was below the level of detection, as defined above), the band was classified as ‘undetected’.
Sequencing of V1/V2
RT-PCR products were amplified and cloned into a plasmid vector . V1/V2 sequences (GenBank accession numbers EU374173 to EU374206) from individual clones were manually edited and aligned with MAFFT version 5.8 using the L-INS-i method . A maximum likelihood phylogenetic tree was constructed using Tree-puzzle (version 5.2) with a gamma time-reversible evolutionary model . Phylogenetic trees were subjected to 1000 puzzling steps, with reliability values greater than 0.70 considered significant. According to the Los Alamos HIV geography database, approximately 96% of the HIV-1 sequences deposited from Malawi are subtype C (accessed 11 November 2007). V1/V2 sequences from this study were aligned with the Los Alamos HIV subtype reference sequences (HXB2 coordinates 6555–6980) and a simple neighbor-joining tree was constructed. All of the sequences clustered exclusively with the HIV-1 subtype C reference sequences (data not shown).
Transmission was modeled as a multinomial experiment in which variants were selected from the mother, with replacement, at probabilities equal to their observed frequency in the maternal viral population. For each MIP, the number of viruses sampled was equal to the number of variants observed in the infant. We simulated 10 000 multinomial transmission events for each pair and recorded the probability of each event depending on whether the infant received the mother's most frequent variant. The probability of the infant not receiving the most abundant maternal variant is the binomial probability of 0 successes:
and the probability of receiving it is the probability of one or more successes:
where n is the number of variants in the infant and P the proportion of the mother's most abundant variant. We calculated the joint probability for all intrapartum (or in utero) transmissions as the sum of the log probabilities, as transmission events for each pair are independent. The significance of the observed probability value is equal to the fraction of the random simulations that generated a probability equal to or less than that of the observed data. A low value rejects the hypothesis of stochastic transmission for the observed data. All modeling was conducted in R , and scripts are available on request.
Normally distributed, continuous variables were compared using a one-way ANOVA. Nonnormally distributed, continuous variables were compared using the Mann–Whitney or Kruskal–Wallis statistic. Paired nonnormally distributed continuous variables were compared with the Wilcoxon matched-pair signed-ranks test. A χ 2 or two-sided Fisher's exact statistic was used to compare proportions. All calculations were done using STATA version 8.2.
In this study, we analyzed plasma from 32 nontransmitting mothers, 25 transmitting MIPs whose infants were infected with HIV-1 in utero, and 23 transmitting MIPs whose infants were infected intrapartum . Baseline characteristics of the subset of mothers of the three groups (nontransmitter, intrauterine, and intrapartum) are outlined in Table 1.
V1/V2 env diversity in mothers
The number of unique HIV-1 variants in each subject was determined using an HTA querying the HIV-1 env variable regions 1 and 2 (V1/V2). Representative HTA autoradiographs are shown in Fig. 1. Among the 80 pregnant women characterized, we detected a median of three V1/V2 env variants per subject (interquartile range: 2, 4.5). There was a weak positive correlation between the number of maternal V1/V2 env variants and log10 HIV-1 RNA copies (correlation coefficient = 0.23, P = 0.05). CD4+ T-cell counts below 200 cells/ml were associated with a greater number of maternal V1/V2 env variants (P = 0.01). Table 1 shows that the transmission groups had a similar number of maternal V1/V2 env variants. This suggests that differences in maternal V1/V2 env diversity are not significantly associated with vertical HIV-1 transmission.
V1/V2 env diversity in infected infants
To characterize the transmission of HIV-1 variants, we compared the V1/V2 env variants present in the maternal plasma at enrollment with those detected in the infant's first HIV-1-positive plasma sample: at birth for the children infected in utero and at 6 weeks for the children infected intrapartum (Table 1). Fewer V1/V2 env variants were detected in the in utero-infected and intrapartum-infected infants than in their mothers (in utero, P = 0.0006; intrapartum, P = 0.005). Thus, during vertical HIV-1 transmission, a restricted number of variants are transmitted from mother to child, representing a genetic bottleneck.
We observed a contrast between the infant V1/V2 env diversity patterns during intrauterine and intrapartum transmissions, suggesting a qualitative difference in the HIV-1 transmission: intrauterine-infected infants tend to be infected with single variants that are more often detected in the maternal plasma, whereas intrapartum-infected infants tend to be infected with multiple V1/V2 env variants typically composed of a mixture of detected and undetected maternal variants (Table 1). Overall, there was no association between the number of variants transmitted and the maternal CD4+T-cell count less than 200 cells/ml (P = 0.2).
To confirm whether the multiple HTA bands in the infant correspond to the transmission of multiple maternal variants, as opposed to the rapid diversification of a single transmitted variant, we created a phylogenetic tree of V1/V2 env region sequences from two MIPs whose infant samples harbored multiple variants (Fig. 2). If multiple maternal variants were transmitted, we would expect multiple branches of intermingled maternal and infant sequences, whereas if transmission of a single maternal variant was followed by outgrowth and diversification in the infant, then the infant samples would cluster together on the same branch. In the MHP-2017 transmission pair, the HTA indicated that the infant was infected with one detected and one undetected maternal variant that composed 86 and 14% of the infant viral population, respectively. In the tree, a majority of the maternal and infant sequences cluster together, likely representing a variant with high abundance, whereas a separate branch at the top of the tree likely represents a low-abundance variant. In MHP-3765, the HTA indicated that the infant was infected with two env variants, composing 84 and 16% of the infant viral population, both detected in the maternal plasma. The phylogenetic tree for this pair shows that maternal and infant sequences are commingled on multiple branches, suggesting transmission of multiple maternal variants. Therefore, in the two MIPs that were sequenced, the phylogenetic trees are consistent with the HTA data and support the transmission of multiple variants.
Modeling the genetic bottleneck at vertical transmission
We determined the relative abundance of each maternal V1/V2 env variant within the sample population and used that information to determine if our data were consistent with a stochastic mechanism of transmission. Transmitted variants undetected in the maternal peripheral plasma viral population were assigned an abundance of 1%. As seen in Fig. 3a and b, both high-abundance and low-abundance maternal variants were detected in the first positive infant sample, suggesting that variant abundance was not strongly associated with either intrauterine or intrapartum transmission (intrauterine, P = 0.6; intrapartum, P = 0.6). The probability of intrauterine or intrapartum transmission of the observed variants, according to their abundance in maternal plasma, was compared with a set of 10 000 simulated transmissions in which the maternal variants were sampled on the basis of abundance (Fig. 3c and d). When the observed data are compared with the simulated data sets, they do not support the bottleneck being generated by random sampling of plasma-associated maternal variants on the basis of abundance; in other words, the observed data correspond to an uncommon outcome (intrauterine, P = 0.003; intrapartum, P = 0.007). To exclude the possibility that our observed transmission pattern was skewed by the inclusion of the undetected maternal variants, we repeated the simulation using only the detected maternal variants. Similar to the previous simulation, the observed transmission pattern remained an uncommon outcome (intrauterine, P = 0.02; intrapartum, P = 0.006), providing further support for a nonstochastic bottleneck mechanism.
Umbilical cord plasma
Finally, we used the V1/V2 env HTA to determine whether HIV-1 isolated from umbilical cord plasma more closely resembles the infant or the maternal viral population. Umbilical cord plasma samples from the six nontransmitter women examined were V1/V2 env RT-PCR negative (data not shown). Similarly, for three infants infected intrapartum, the cord blood V1/V2 env RT-PCR reaction was negative (Fig. 4). In contrast, in four infants infected in utero, the cord blood sample had a viral population that was indistinguishable from the infant birth sample but distinct from the mother's sample. These results show that cord blood plasma represents the HIV-1 population present in the infant and suggests that cord blood plasma could be a readily accessible source of the HIV-1 population present at birth in intrauterine-infected infants.
In this study, we describe the relationship between genetic diversity in the HIV-1 env V1/V2 region and subtype C HIV-1 MTCT in nontransmitter mothers and intrauterine-transmitting and intrapartum-transmitting MIPs. Although we found no relationship between the amount of maternal env diversity and the rate of MTCT, we did observe a significant genetic bottleneck between the matched maternal and infant infections. The pattern of transmitted V1/V2 variants differed by the timing of HIV-1 transmission: infants infected in utero frequently harbored single variants, which were detected in the maternal plasma, and infants infected intrapartum frequently harbored multiple variants, which were a mixture of detected and undetected maternal variants. Finally, modeling of our data showed that on average MTCT did not favor transmission of the most abundant env variants present in maternal plasma, arguing against a stochastic model of vertical transmission.
These conclusions are based on the data generated with a HTA against the env V1/V2 region, which could have several limitations. First, although the HTA cannot reliably sample genomic variants comprising less than 1% of the viral population, sampling of these low-abundance variants with DNA sequencing would require a minimum of 300 cloned env genes per sample. Second, it could be argued that a measure of HIV-1 diversity should sample larger regions than the approximately 400 base pairs sampled with our assay. Nevertheless, the HTA is most sensitive to sequence and size changes in genomic regions of this size, and this region is one of the most heterogeneous in the HIV-1 genome . These limitations must be balanced against the resources required to generate similar data through DNA sequencing, and owing to this constraint, we have chosen to sample a larger number of MIPs, in a hypervariable region of the env gene, rather than report diversity of longer regions of env in fewer MIPs. Finally, any misclassification of population diversity derived from using the V1/V2 region as a surrogate for actual diversity is likely to be nondifferential and unlikely to bias our comparisons.
The observation of similarity in HIV-1 env diversity in women in this study is different from the findings of Dickover et al. , who examined HIV-1 subtype B env diversity (using a heteroduplex mobility assay approach). Dickover et al. observed that women who transmitted in utero had lower V3/V4 diversity (and lower CD4+ T-cell counts), suggesting that women who transmit in utero have poor immunologic control of their HIV-1. In our study, women in all groups had similar diversity. There are, however, many differences between the US-based cohort and our Malawi-based cohort, such as coinfections, the region of the env gene examined, the sensitivity of the HTA compared with the HMA, and subtype B versus C HIV-1 .
The bottleneck of population diversity during vertical HIV-1 transmission seen here has been previously reported [5–8]. In addition to the reduction in viral diversity in infants, we found that the pattern of transmitted V1/V2 variants differed in the timing of HIV-1 transmission, with intrauterine transmission more often representing a single variant and intrapartum transmission more often involving multiple variants. The results agree with that of a previous study of 10 intrauterine-infected infants and their mothers by Fischetti et al. , where the majority of infants harbored a homogeneous virus population that consisted of both major and minor maternal variants. A confounder of this result could be that the first positive sample from infants infected in utero was collected within 48 h of single-dose nevirapine treatment, which may have lowered viral RNA load and created an artificial bottleneck. Nevertheless, the turnover rate of HIV-infected cells, the slow decline of viral RNA in the presence of a single dose of nevirapine relative to the timing of sampling , and the relative ease of HIV amplification in these samples from small volumes of infant plasma suggest high viral RNA loads. Nevirapine could also have confounded intrapartum transmission through lowered maternal diversity prior to delivery. Given the greater diversity in intrapartum-infected infants compared with intrauterine and the frequent transmission of low-abundance maternal variants, we suggest a NVP effect would dampen the difference between the groups, which only emphasizes the suggested difference in the mechanism of transmission intrauterine versus intrapartum.
Among the 48 transmission events examined in this study, nearly 50% included the transmission of variants we were unable to detect in the mother's blood plasma. Although the origin of these undetected variants is unknown, there are several possibilities including a compartmentalized HIV-1 population that was not in equilibrium with the sampled peripheral blood, low-abundance maternal variants, or variants that arose in the infant de novo, as the virus evolved in response to the single dose nevirapine exposure or its new environment. If infants are being infected with compartmentalized viruses, it is possible that the transmitted viruses were the most abundant variants in those compartments. Regardless, on average, the most abundant maternal variant observed in the blood plasma was not the most frequently transmitted variant in the infant (intrauterine or intrapartum). Therefore, the most plausible mechanisms for the bottleneck are either transmission of many variants followed by selective amplification of the detected variants or selective transmission . In the selective amplification model, maternal antibodies, antiretroviral drugs, or the infant immune response could all restrict outgrowth of some variants in the infant or be involved in selection. Distinguishing between these mechanisms is difficult, as variants in the infant need to undergo amplification to be detected.
Despite the strong bottleneck, more than one variant was often seen in infant viral populations. Multiple mechanisms could account for the presence of multiple variants in infant infections, including multiple transmissions of a single variant, a single transmission of multiple variants, a single transmission event with a multiple-infected cell, or a single transmission event with rapid diversification, or evolution, between the transmission event and the time of population sampling. We examined the potential for early evolution after transmission by comparing the viral population in the first positive infant sample with the subsequent samples collected at 6-week intervals (data not shown). Using changes in env diversity as a measure of viral evolution, we observed diversification in many of the intrauterine-infected and intrapartum-infected children following transmission. To begin to address the question of infection by multiple variants generating apparent diversity, we selected two MIPs, whose infant's viral populations were comprised of two variants and subjected the viral populations to sequence analysis. Although we cannot exclude rapid diversification in the infant after transmission, the phylogenetic trees for these two transmission pairs are most consistent with transmission of multiple variants from the mother.
In summary, these findings argue for a MTCT model involving selection or selective outgrowth. These results are similar to the subtype B data published by Dickover et al. , thus extending the MTCT paradigm to subtype C.
We thank the Malawian mothers and infants for their participation; the MHP Nursing Staff and technicians for excellent logistical and technical support; Milloni Patel and Janera Harris for help with sample extraction. This work was presented, in part, at the 13th Conference on Retroviruses and Opportunistic Infections and the Malawi College of Medicine 11th Annual Research Dissemination Conference. This research was supported by NIH awards to JJK (K99-HD056586), SRM (R01-AI49084; R21-AI065369), RS (R37-AI44667), and by the UNC CFAR (P30-AI50410).
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