In a study conducted in Malawi before antiretroviral treatment was available, about 6%-8% of infants born to HIV+ women were found to become infected in utero.1At birth, many infants with HIV DNA (+) cord bloods have quantitative viral levels that are as high as or higher than those in their mothers, indicating sustained in utero replication well preceding parturition.2 Furthermore, although antiretroviral therapy is partially effective when given only at parturition,3 the efficacy is greater when it is given throughout the pregnancy.4 Thus, the mechanisms by which the virus is transmitted in utero are of interest, even though they remain poorly understood. One possible mechanism of HIV in utero transmission is by “transfusion” of HIV-infected maternal blood cells into the infants' circulation during pregnancy. The continuous blood exchange between maternal circulation and fetal circulation during pregnancy has been established,5-7 with blood exchange peaking at the perinatal stage,5 a finding consistent with the highest transmission risk being at the time of delivery.1
We have previously hypothesized that perinatal transmission might occur because of transplacental passage of infected maternal cells (MCs) during delivery. However, using polymerase chain reaction (PCR) testing, we failed to detect statistically significant differences in the likelihood that MCs would be found in umbilical cord blood of perinatally infected infants (cord blood PCR negative but first sample at 6 weeks found to be PCR positive).8 In contrast, another group did find MCs more commonly in perinatally infected infants.9 Furthermore, they reported a marginally significant increase in risk of in utero infection [ie, cord blood PCR HIV (+)], with increasing numbers of MCs in umbilical cord blood at delivery. We, therefore, examined new samples from our study to assess if in utero infection risk might be correlated with the presence of MCs in cord blood.
MATERIALS AND METHODS
Patients and Material
The dried blood spots (DBS) analyzed in this study were collected at a clinical trial in Blantyre, Malawi, in 1994, which enrolled women giving birth to determine rates of HIV transmission with and without vaginal cleansing with chlorhexidine. Details of the study have been published elsewhere.1 HIV infection in the mother was determined by testing umbilical cord blood for passively transmitted antibodies against HIV. HIV infection in the infant was determined by PCR testing of DBS made from the umbilical cord blood or heel sticks at various times after birth. DBS samples were stored in individual sealed plastic envelopes at 4°C. Laboratory methods for sample collection and HIV testing have been reported in previous publications.1,10,11 Infants found to be uninfected postnatally were assumed to be HIV uninfected at all earlier ages, including in utero. Cord blood samples from infants found to be infected on their first postnatal visit were further tested by PCR to determine their HIV status at delivery.
For the current study, we analyzed all available maternal-infant pairs from the 1994 intervention study.1 All infants found to be PCR HIV(+) in the cord blood were assumed to have been infected in utero, an assumption justified by earlier studies of viral levels in umbilical cord blood which showed that HIV levels in infected infants are typically as high or higher as those in the mother.2 For each in utero-infected infant, 2 uninfected controls were obtained. We confirmed that cord blood samples from infants previously found to be HIV negative were indeed negative by retesting cord blood DBS for HIV with a highly sensitive reverse transcription-polymerase chain reaction method that identified sequences specific to HIV.12 Both case and control infants were vaginally delivered singleton infants born at term to untreated HIV-infected women.1 The original study was approved by the University of Malawi Medical School, the Government of Malawi, and the National Institute of Health (USA). The current study was approved by the National Cancer Institute as exempt from further ethical review because it used only anonymous and unlinkable materials collected from a previously approved study.
Previously, we have developed PCR assays to detect the presence of microchimeric cells in the context of a high background of allogeneic cells,13 including the detection of MCs in fetal tissue.7 These assays are based on 2 panels: an insertion/deletion (INDEL) panel consisting of 12 different insertion/deletion polymorphisms first described by Alizadeh et al14 and an HLA-DR panel consisting of 12 HLA-DR alleles (DR1, DR3, DR4, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR15, and DR16). The mother and infant were both typed using these panels. Alleles for which the mother was positive but the infant was negative were termed “informative alleles.” MCs were detected in the cord blood by amplifying for these informative alleles.
Extraction of DNA From DBS
Each blood spot representing 50 uL of whole blood was cut out using sterile scissors, immersed in 400 uL of phosphate-buffered saline, and incubated in phosphate-buffered saline overnight at room temperature. A total of 200 uL of Corbett Robotics digest buffer (Sigma, St. Louis, MO) was then added and incubated at 60°C for 2 hours. Both the liquid and paper were transferred to a QIAamp spin column (Qiagen, Valencia, CA) and DNA was extracted, as per the manufacturer's instructions. After elution in water, the samples were dried and reconstituted in 300 uL of Solution A + B.15
HLA-DR and INDEL Typing of Maternal and Cord Blood Samples
Plates for PCR typing were prepared and frozen at −80°C. The 24 × 16 typing plate formats consisted of 12 columns of INDEL PCR assays and 12 columns of HLA-DR PCR assays. Five microliters of DNA were added to 10 uL of PCR mixture consisting of 15 mM KCl, 5 mM Mg, 20 mM Tris-HCl pH 8.0, BSA (0.1 mg/mL), Triton X-100 (1%), 1 mM of dNTPs (Roche Diagnostics GMBH, Mannheim, Germany), 1 uM of each primer (Integrated DNA Technologies, Coralville, IA), SYBR Green (18.75 units/reaction; BioWhittaker Molecular Applications, Rockland, ME), and FastStart Taq (1.5 units/reaction; Roche Diagnostics GMBH, Mannheim, Germany). Real-time PCR was performed with a sequence detection system (Roche LightCycler 480, Roche Diagnostics, Basel, Switzerland) with the following cycle conditions: 1 minute at 95°C followed by 45 cycles of 30 seconds at 95°C, 30 seconds at 64°C, and 45 seconds at 72°C.
Amplification for Microchimerism Detection and Quantification
After the mother and child were both typed with the INDEL and HLA-DR panels, an informative allele was determined for each pair. We used the INDEL allele preferentially. If an informative INDEL allele was not found, we used the HLA-DR allele.
Another cord blood spot was then processed into 300 uL of DNA lysate and amplified for its informative allele. A total of 25 uL of DNA lysate was added to 50 uL of PCR reaction mix and amplified with the above conditions for 4 replicates. To quantify the recovery of genomic DNA, the cord blood samples were amplified with primers specific to a highly conserved region of the HLA DQ alpha locus to measure levels of genomic DNA. To quantify the MCs, 10-fold serial dilutions of quantified standards (ie, peripheral blood mononuclear cells at known concentrations based on replicate determination using a Coulter counter) were amplified along with the clinical samples. Fluorescence was recorded at each extension step and computer software (Roche LightCycler 480, Roche Diagnostics) was used to plot the fluorescence values against cycle number.
We tested DBS samples from 145 mothers and their infants. A total of 31 mother-infant pairs had no informative allele in either INDEL or HLA-DR tests. Of 114 mother-infant pairs with informative alleles, 38 pairs were HIV positive in cord blood [HIV(+)] and 76 pairs were HIV negative in cord blood [HIV(−)].
The mean genomic DNA concentration (copies/50 uL cord blood, ie, the number of nucleated cord blood cells in 1 DBS, assuming a volume of 50 uL in each spot) of cord blood samples for the HIV(+) group was 20,759 human cell equivalents, with a median of 17,200 and a range of 3876-62,220. Similarly, the mean genomic DNA concentration of cord blood samples for the HIV(−) group was 15,055 human cell equivalents, with a median of 13,236 and a range of 768-49,140. (Fig. 1) The genomic DNA concentrations arising from nucleated cord blood cells in the HIV(+) group were significantly higher than those of the HIV(−) group (2-sample T test: P = 0.01).
MCs were detected in the cord blood in 23 of 38 HIV(+) pairs (60.5%) and in 47 of 76 HIV(−) pairs (61.8%). The mean number of MCs in 50 uL of cord blood for the HIV(+) group was 39, with a median of 18 and a range of 6-439. The mean number of MCs for the HIV(−) group was 30, with a median of 12 and a range of 6-289. The difference in the number of MCs in cord blood samples between the HIV(+) group and the HIV(−) group was not statistically significant (2-sample T test: P = 0.65) (Fig. 2).
The proportion of MCs in the cord blood sample was obtained by dividing the quantity of MCs by the quantity of the total cord blood nucleated cells (maternal and fetal). The mean proportion of MCs for the HIV(+)/MC(+) group was 0.33%, with a median of 0.27%, and a range of 0.03%-5.12%. The mean proportion of MCs for the HIV(−)/MC(+) group was 0.09%, with a median of 0.10%, and a range of 0.02%-3.08% (Fig. 3). There was no significant difference in the concentration of MCs in cord blood samples between the HIV(+) group and the HIV(−) group (2-sample T test: P = 0.78).
The current study was designed to test the hypothesis that a high concentration of MCs in cord blood could reflect more in utero exposure to maternal transplacental microtransfusions and, hence, a greater likelihood of HIV transmission from HIV+ mothers to their infants. To examine this hypothesis, we used a highly sensitive and specific quantitative real-time PCR assay to identify specific maternal DNA sequences, which were then used to quantify MCs in cord blood. This quantitative real-time PCR assay has been applied in many studies, which required a very sensitive assay to detect very low-level chimeric cell populations.13,15,16
We found that about half of the cord blood samples contained a significant number of MCs, which is similar to the same frequency of detection of MCs in cord blood DBS in our previous publication which focused on perinatal HIV transmission.8 When we compared the genomic DNA (amplification of HLA DQ-alpha) recovery from DBS, we found that the genomic DNA concentrations of cord blood samples for the HIV(+) group were significantly higher than those of the HIV(−) group (2-sample T test: P = 0.01), which implies that the HIV(+) cord blood contained a higher number of white blood cells (WBCs) than the HIV(−) cord blood. This finding suggests that in utero HIV infection is associated with leukocytosis, possibly reflecting inflammation or cell activation that results from infection of the fetus.
However, when we compared the detection rates of MCs among cord blood samples from infected and noninfected infants, we found no significant difference between the HIV(+) and the HIV(−) groups (60.5% and 61.8%, respectively, 2-sample T test: P = 0.89). Moreover, the difference in the concentrations of MCs in cord blood and in the proportion of total cord blood leukocytes that were of maternal origin between the HIV(+) and HIV(−) groups was also not statistically significant (2 sample T test: P = 0.65 and 0.78, respectively). These results do not support the hypothesis that the frequency or magnitude of maternal fetal “transfusion” is positively associated with the frequency of in utero HIV transmission. Possibly, infection of the fetus in utero is not necessarily accompanied (and possibly caused) by transplacental movement of MCs carrying HIV and/or by fetal regulatory T-cell responses induced by such cells. Alternatively, some fetuses exposed to infected HIV+ MCs may be able to mount protective immune response against HIV, whereas others cannot. For instance, given recent observations that maternal microchimerism is associated with the development of fetal regulatory T cells that suppress fetal responses against noninherited maternal alleles,16 it is possible that concomitant exposure of the fetus to HIV might also result in the generation of fetal regulatory T cells specific for HIV.17 If so, the fetus might be more susceptible to HIV infection (because protective adaptive antiviral immunity is suppressed) or, alternatively, protected (because pro-inflammatory responses that drive viral replication and spread might be suppressed).
There are several limitations to this study. One is that we do not know when the infants were infected in utero. We presume that the infection must have occurred much earlier than near delivery because HIV levels in cord blood were already as high as in the mother and such levels would likely take several weeks to establish after active viral replication in the fetus.2 Yet, infection may have occurred at varying times, and the concentration of MCs at delivery might not be representative of the levels of transplacentally passed MCs at those times. Hypothetically, some break in the placental barrier might have sent a transient burst (acute “transfusion”) of MCs (and HIV) across the placenta much earlier in the pregnancy, the evidence for which would have been gone by the time of delivery.
Another limitation is that samples used in the current study were stored for a long time, which could affect the quantity and quality of the recovered DNA. Although we were able to recover a reasonable amount of human DNA from the majority of samples, some samples had very low DNA recovery. With optimal and high volume samples, we would have had higher total DNA inputs in the PCR amplifications, which would have enabled detection of MCs in a larger proportion of cord blood samples. However, this limitation should not have undermined our key findings of relatively higher levels of total WBC, but similar levels of chimeric maternal WBC in cord blood DBS eluates from infected and noninfected infants, respectively, because the DBS from both groups were stored and processed under identical conditions and testing was performed under code. We believe that the observation of significantly higher levels of cell genomes in DBS from HIV(+) than HIV(−) infants is valid, albeit unexplained, because the HIV status was unknown at birth and could not have confounded the collection or processing of the blood samples. Additionally, confounding factors that were not available, such as the viral load of maternal HIV and maternal-fetal HLA concordance, should also be taken into consideration,18-21 although we doubt they explain our findings.
In summary, we did not find support for a correlation between MCs found in umbilical cord blood at birth and the risk of HIV infection in utero. Nevertheless, we cannot refute the possibility that in utero infection occurred via transplantal transmission of infected cells earlier in pregnancy that did not result in persistently elevated levels of microchimeric MCs or of HIV in cord blood at delivery.
The authors would like to acknowledge the contributions of Taha E. Taha for the establishment of the cohort and assistance with access to the DBS specimens.
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