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. 2014 Sep 4;178(1):86–93. doi: 10.1111/cei.12386

In-utero infection with HIV-1 associated with suppressed lymphoproliferative responses at birth

B Lohman-Payne *,†,‡, T Sandifer §, M OhAinle , C Crudder *, J Lynch †, M M Omenda *, J Maroa *, K Fowke **, G C John-Stewart †,‡,§, C Farquhar †,‡,§
PMCID: PMC4360198  PMID: 24853045

Abstract

In-utero exposure to HIV-1 may affect the immune system of the developing child and may induce HIV-1-specific immune responses, even in the absence of HIV-1 infection. We evaluated lymphoproliferative capacity at birth among 40 HIV-1-uninfected infants born to HIV-1-infected mothers and 10 infants who had acquired HIV-1 in utero. Cord blood mononuclear cells were assayed using [3H]-thymidine incorporation for proliferation in response to HIV-1 p55-gag and the control stimuli phytohaemagglutinin (PHA), Staphylococcus enterotoxin B (SEB) and allogeneic cells. In response to HIV-1 p55-gag, eight (20%) HIV-1-exposed, uninfected (EU) infants had a stimulation index (SI) ≥ 2 and three (30%) in-utero HIV-1 infected infants had SI ≥2. The frequency and magnitude of responses to HIV-1 p55-gag were low overall, and did not differ statistically between groups. However, proliferative responses to control stimuli were significantly higher in EU infants than in infants infected in utero, with a median SI in response to PHA of 123 [interquartile range (IQR) 77–231] versus 18 (IQR 4–86) between EU and infected infants, respectively (P < 0·001). Among infected infants, gestational maturity was associated with the strength of HIV-1 p55-gag response (P < 0·001); neither maternal nor infant HIV-1 viral load was associated. In summary, EU and HIV-1-infected infants mounted HIV-1-specific lymphoproliferative responses at similar rates (20–30%), and although global immune function was preserved among EU infants, neonatal immune responses were significantly compromised by HIV-1 infection. Such early lymphoproliferative compromise may, in part, explain rapid progression to AIDS and death among HIV-1-infected infants.

Keywords: allogenic, Candida, gestational age, p55gag, SEB

Introduction

HIV-1-specific cellular immunity among infants and young children develops from exposure to HIV-1, either as the result of natural infection and continuous exposure to autologous virus or through intermittent contact with maternal virus, which may occur in utero, during delivery or via breastfeeding. It has been proposed that development of an effective immune response may protect against HIV-1 acquisition among exposed, uninfected infants and may reduce HIV-1 disease progression among infected infants 1–3. A South African study found that HIV-specific CD4+ T cell responses in cord blood were more common among children who did not become infected compared to those who became infected 1. Our group also observed that HIV-1-specific CD8+ T cell responses at 1 month of age were associated with lower risk of subsequent HIV-1 acquisition 2. However, the magnitude or breadth of the HIV-1-specific CD8+ T cell responses did not correlate with subsequent disease progression among HIV-infected infants 3.

The prevalence and predictors of HIV-1-specific CD4+ and CD8+ T cell responses among HIV-1-exposed adults and older children are not well defined. In HIV-1-exposed and infected individuals, HIV-specific cellular immune responses may be associated with recent viral exposure 4,5. This is supported by the observation that among HIV-1-infected adults, treatment interruptions of highly active anti-retroviral therapy (HAART) are followed by increased strength and breadth of CD8+ T cell responses during viral rebound 4. Similarly, exogenously exposed, HIV-infected virally suppressed adults with virally suppressed partners on HAART are less likely to mount an HIV-specific immune response than those with viraemic partners 5. Despite the association of greater HIV-1-specific T cell responses with detectable viraemia, this relationship may not continue in a dose-dependent manner, with some studies demonstrating no correlation between viral load and T cell response breadth or magnitude in viraemic patients 6–8.

For infants, exposure to endogenous or maternal virus may also be an important contributing factor to frequency, breadth and magnitude of HIV-1-specific responses 1. In this study, we compared prevalence and correlates of HIV-1-specific T cell responses in cord blood obtained from HIV-1-infected infants and HIV-1-exposed, uninfected infants. In addition, we evaluated infant immunoproliferative responses in both groups to several non-HIV antigens [phytohaemagglutinin (PHA), Staphylococcus enterotoxin B (SEB), allogeneic cells, Candida albicans] in order to understand our results in the context of the global immunoproliferative activity of cord blood from HIV-infected and uninfected infants.

Materials and methods

Study subjects and data collection

Infants included in this study were born to HIV-1-seropositive mothers enrolled between 1999 and 2002 in a Nairobi HIV-1 perinatal transmission trial. Maternal and infant clinical and immunological information from the larger cohort is published elsewhere 2,3,9–15. The current study includes a subset of 40 HIV-1-exposed, uninfected (EU) infants and 10 infants who acquired HIV-1 in utero (HIV IU). Women were recruited during pregnancy and provided with prophylactic zidovudine starting at 34–36 weeks gestation, as per Kenya national guidelines, during the study period 16. The study received human subjects approvals from institutional review boards at the University of Washington and Kenyatta National Hospital in Nairobi, Kenya. Written informed consent was obtained from all participating mothers on behalf of their infants. This study received ethical approval from the institutional review boards of the University of Washington and the University of Nairobi and was conducted according to the guidelines set forth by the United States Department of Health and Human Services.

Clinical and demographic data were collected from mothers at enrolment, 32 weeks gestation and delivery. At 32 weeks, maternal blood and cervical swabs were obtained for HIV-1 RNA viral load and maternal CD4 and CD8 counts were determined. Women were assessed antenatally for sexually transmitted diseases (syphilis, gonorrhoea, chlamydia and trichomonas), as well as bacterial vaginosis and C. albicans infection. At birth, maternal blood was drawn for HIV-1 viral load, and infant cord blood was collected for immune assays. Within 48 h of birth, infants were examined by a study physician for gestational maturity as determined by Dubowitz score, and infant venous blood was drawn for determination of infant HIV-1 status.

Laboratory methods

To determine HIV-1 infection status of infants, nested HIV-1 Gag DNA polymerase chain reaction (PCR) was performed on dried blood filter spots, as described previously 17. Reactions were performed in quadruplicate and ≥ 1 positive result was considered positive for in-utero transmission. In the case of infants who were HIV-1 DNA-negative at birth but HIV-1 DNA-positive at 1 month, samples of plasma collected within 48 h of birth and frozen were used for HIV-1 RNA assays (Gen-Probe, San Diego, CA, USA) to determine timing of infection. Infants who were HIV-1 RNA-positive within 48 h of birth were included in the in-utero transmission group 17. All 40 infants included in the EU infant group remained DNA PCR-negative at 1 month of age and remained uninfected during the study follow-up of 1–2 years.

Cord blood mononuclear cells (CBMC) were prepared from freshly collected, ethylenediamine tetraacetic acid (EDTA)-anti-coagulated cord blood. Plasma was separated by 10 min of centrifugation at 312 × g. The remaining blood was diluted 1:1 with RPMI, layered on a Ficoll gradient, and centrifuged for 30 min at 385 × g. CBMC were collected from the interface and cryopreserved for later use. For proliferation assays, CBMC were thawed, counted and suspended in AIM-V serum-free medium at 2 × 106 cells/ml. To reduce handling, cells were added directly to 96-well round-bottomed plates with 100 μl (2 × 105 cells) per well. After allowing plated cells to rest overnight, antigens were added to test wells to the following final concentrations: 10 μg/ml PHA (Murex, Dartford, UK), 1 μg/ml SEB (Sigma, St Louis, MO, USA), 2 × 105 cells per well allogeneic cells, 10 μg/ml C. albicans antigen (Greer, Lenoir, NC, USA), 10 μg/ml HIV-1 p55-gag antigen (Protein Sciences Corp., Meriden, CT, USA) or left unstimulated. Whole protein antigens were chosen to allow for antigen processing and presentation on major histocompatibility complex (MHC) Class II for stimulation of CD4+ T cells. Allogeneic cells were prepared by mixing PBMC collected from five unrelated individuals of varying ethnicity followed by Caesium151 irradiation to abolish proliferative potential, followed by cryopreservation. Tests were performed in triplicate for each antigen-sample combination. Plates were incubated at 37°C, 5% CO2 for 5 days. On the fifth day, wells were spiked with 1 microCurie per well of [3H]-thymidine. After overnight incubation, cells were harvested onto FilterMats, allowed to dry for at least 4 h, then prepared for scintillation counting using a Wallac MicroBeta Counter (PerkinElmer, Akron, OH, USA).

Data analysis

Stimulation indices (SI) were calculated for each cord blood sample by dividing the median antigen-stimulated proliferation result by the median control proliferation result, as described by Reimann et al. 18. SIs < 2 were considered negative and thus assigned a value of 1 for rank-based statistics 19. We required a positive response (SI ≥ 2) to at least one positive control stimulus (PHA, SEB or allogeneic cells) to include an infant in our analyses.

Spearman's rank correlations were calculated among the SI results for the five stimuli used in the proliferation assays. Associations between strength of proliferative responses and infant infection status were assessed with Mann–Whitney U-tests. To test for clinical differences between negative and positive p55-gag responders, we used Student's t-tests for continuous clinical variables and Fisher's exact tests for dichotomous clinical variables. The relationships between gestational maturity as measured by Dubowitz score and proliferative response to each stimulus were further examined by single regression with log-transformed stimulation indices. A Dubowitz score <47 was considered indicative of prematurity, as a score of 47 corresponds to an estimated gestational age of 36 weeks 20.

Results

Cohort characteristics

Fifty live singleton births to HIV-1-infected women were selected for inclusion in this study. Forty infants were HIV-1-uninfected at the time of birth and selected randomly from among all infants HIV-1-uninfected at birth, and 10 HIV-1-infected infants were selected randomly from among those infants infected in utero. Mean maternal age was 24·8 years (range 19–37 years) and mean antenatal maternal CD4 count was 468 cells/μl (range 31–949). Mean plasma viral load at delivery was significantly lower than viral load at 32 weeks gestation (4·2 versus 4·9 log10 copies (c)/ml, P value < 0·001) due to prophylactic zidovudine. Mean cervical viral load at 32 weeks gestation was 2·39 log10 copies/ml (range 0·70–4·88 log10 c/ml). Eleven (23%) of 47 mothers were diagnosed with a sexually transmitted infection during pregnancy and 38% of mothers were diagnosed with C. albicans vaginitis at 32 weeks gestation.

Eight per cent of infants were low birth weight, defined as less than 2·5 kg, and 20% of infants were premature, as defined by Dubowitz score < 47. Among the 10 IU-infected neonates, mean viral load at birth was 5·4 log10 c/ml (range 4·2–6·4 log10 c/ml) (Table 1).

Table 1.

Cohort characteristics

Variable Exposed uninfected infants In-utero-infected infants
Maternal/infant characteristics n Mean (s.d.) n Mean (s.d.)
Maternal age (years) 40 24·6 (4·1) 10 25·6 (4·8)
Parity 40 1·3 (1·1) 10 2·0 (1·2)
Maternal pregnancy CD4 count† 39 472·3 (236·7) 10 450·7 (267·5)
Maternal pregnancy CD8 count† 39 958·7 (484·3) 10 1266·8 (665·6)
Maternal viral load at delivery‡ 35 4·1 (1·0) 10 4·6 (0·9)
Maternal viral load at 32 weeks‡ 38 4·7 (0·7) 9 5·0 (0·6)
Cervical viral load at 32 weeks‡ 32 2·2 (0·9) 7 3·3 (1·3)
Days AZT during pregnancy 40 30 (15) 9 15 (20)
Dubowitz score 39 56·0 (7·3) 10 48·8 (9·6)
Birth weight (kg) 40 3·2 (0·4) 10 2·7 (0·7)
Infant viral load within 48 h‡ 40 n.d. 10 5·4 (0·8)
Diagnosed during pregnancy n Proportion n Proportion
History of illness§ 40 0·33 10 0·30
History of malaria 40 0·13 10 0·10
Any STI 38 0·21 9 0·33
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†Units are cells/μl peripheral blood. ‡Units are log10 copies/ml peripheral blood or cervical mucus; n.d. = none detected; s.d. = standard deviation; AZT = zidovudine. §History of fever, cough, diarrhoea, weight loss, thrush or itchy skin rash during pregnancy. Bold-type characteristics were significantly different between EU infants and IU-infected infants at P ≤ 0·05.

The IU-infected infant group differed from the EU infant group by maternal cervical viral load, duration of zidovudine use during pregnancy, Dubowitz score and birth weight (Table 1). Cervical viral load at 32 weeks gestation was higher in mothers of IU-infected infants (mean 3·3 log10 versus 2·2 log10 c/ml in IU versus EU infants, P = 0·01). Maternal peripheral viral load at 32 weeks and at delivery were also higher in the infected group, but differences did not reach statistical significance. Duration of zidovudine use was shorter among mothers of IU-infected infants compared to mothers of EU infants, averaging 15 days therapy versus 30 days (P = 0·01). IU-infected infants had a lower mean Dubowitz score (49 versus 56, P = 0·01) and a lower mean birth weight (2·7 versus 3·2 kg, P = 0·01). Together these data present the impact of in-utero infection with HIV on the birth characteristics of infants in this cohort that may have a direct impact on the ability to mount immune responses.

Proliferative responses to positive control antigens and C. albicans

All 50 infant CBMC samples had a positive response (SI ≥ 2) to at least one of the positive control antigens, and 88% of infants had a positive response to all three positive control antigens (36 of 40 EU infants and eight of 10 IU-infected infants, P = 0·59). Forty-four (88%) infants responded to C. albicans, and this proportion did not differ based on the diagnosis of maternal C. albicans in pregnancy (88% of infants with maternal C. albicans in pregnancy versus 86% of infants without had a positive C. albicans immune response, P = 1·0). Although the proliferative responses were above the cut-off, the IU-infected infant group had lower proliferative responses to all three positive control antigens than the EU infant group (Table 2). Median IU-infected CBMC stimulation indices for each positive control antigen were less than 20% of those produced by EU CBMC: 18 versus 123 for PHA, 7 versus 57 for SEB and 7 versus 135 for allogeneic cells (P = 0·002, P = 0·04, P < 0·001, respectively). Response to C. albicans was also significantly lower among IU infants than EU infants (median SI of 4 versus 8, P = 0·04) (Table 2). If this finding, that antigen-specific clonal proliferation of cord blood mononuclear cells is reduced by 80% in HIV-1 infected infants, is generalizable, this reduced ability to respond to antigen stimulation may, in part, explain rapid disease progression in observed in untreated paediatric HIV infection.

Table 2.

Lymphoproliferative responses by infection status

Antigens Exposed uninfected infants In-utero-infected infants
n Median (IQR) stimulation index n Median (IQR) stimulation index P-value†
PHA 40 123 (77–231) 10 18 (4–86) 0·002
SEB 40 57 (11–188) 10 7 (3–35) 0·04
Allogeneic cells 40 135 (54–278) 10 7 (4–10) <0·001
Candida 40 8 (3–18) 10 4 (2–7) 0·04
HIV-1 p55 40 1·2 (0·8–1·7) 10 1·4 (0·9–2·1) 0·66
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†

Calculated by Mann–Whitney U-test, P-values ≤ 0·05 in bold type. PHA = phytohaemagglutinin; SEB = Staphlyococcus enterotoxin B.

Among the group of 40 EU infants, responses to PHA, SEB and allogeneic cells were correlated significantly. Spearman's correlation coefficients between these three stimuli were all ≥ 0·51, with P-values ≤ 0·001 (Table 3). Among the group of 10 IU-infected infants, allogeneic and C. albicans responses were correlated significantly (0·82, P = 0·004), while PHA and SEB responses were correlated (Spearman's correlation coefficient of 0·72), but did not reach significance. Proliferative responses to allogeneic cells did not correlate with PHA or with SEB in this group (Table 3). Within the HIV EU infants, the correlation between various strong proliferative signals was consistent with the high SIs observed in this group; however, there were fewer correlated responses evident in the HIV IU infants.

Table 3.

Correlations† between antigen response stimulation indices in cord blood mononuclear cells (CBMC)

Exposed uninfected infants
PHA SEB Allogeneic cells Candida
SEB 0·57 (<0·001) *** *** ***
Allogeneic cells 0·70 (<0·001) 0·51 (<0·001) *** ***
Candida 0·07 (0·67) 0·001 (0·99) −0·02 (0·89) ***
HIV-1 p55-gag 0·21 (0·20) 0·09 (0·56) 0·19 (0·24) 0·20 (0·21)
In-utero-infected infants
PHA SEB Allogeneic cells Candida
SEB 0·72 (0·02) *** *** ***
Allogeneic cells 0·22 (0·53) −0·02 (0·96) *** ***
Candida 0·06 (0·88) −0·18 (0·62) 0·82 (0·004) ***
HIV-1 p55-gag −0·32 (0·36) −0·31 (0·39) −0·23 (0·52) −0·34 (0·34)
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†

Shown are Spearman's rank correlation coefficients with P-values in parentheses. PHA = phytohaemagglutinin; SEB = Staphlyococcus enterotoxin B.

Proliferative response to HIV-1 p55-gag

Overall, 11 (22%) of 50 infants had a positive response (SI ≥ 2) to HIV-1 p55-gag antigen. Eight (20%) of 40 EU infants had an SI ≥ 2 in response to gag antigen, while three (30%) of 10 IU-infected infants had an SI ≥ 2 in response to gag antigen (P = 0·67). The rank distributions of gag stimulation indices also did not differ (P = 0·66) (Table 2).

To test for the possibility that the strength of proliferative responses to HIV-1 p55-gag or C. albicans was limited by general immunosuppression, we compared PHA, SEB and allogeneic cell stimulation indices between positive and negative responders to p55-gag and C. albicans antigens separately in the 40 EU and 10 IU-infected infants. We did not observe associations between positive p55-gag response and PHA, SEB or allogeneic stimuli-induced proliferative responses in either EU- or IU-infected infants. However, we found significantly higher allogeneic responses among C. albicans responders than non-responders in IU-infected infants (median SI 9 versus 4, P = 0·05), but no associations with PHA or SEB. No similar associations were seen among the EU infants.

Clinical correlates of HIV-1 p55-gag response differed by infant HIV infection status (Table 4). Among the group of 10 IU-infected infants, Dubowitz score trended towards an inverse association with positive p55-gag response. The average Dubowitz score was 52·4 among the non-responders and 40·3 among the p55-gag responders (P = 0·06). All three IU-infected gag responders were premature, compared to two of seven IU-infected gag non-responders. We did not observe a significant association between infant viral load and positive p55-gag response (5·2 log10 c/ml among seven non-responders versus 5·9 log10 c/ml among three responders, P = 0·20). For all infants there was a trend towards a lower maternal CD4 count between HIV-1 p55-gag responders and non-responders. Mean maternal pregnancy CD4 counts of HIV IU-infected infant responders and non-responders were 270 versus 571 cells/μl, P = 0·06. Similarly, maternal CD4 counts of HIV EU infant responders and non-responders were 363 versus 504 cell/μl, P = 0·1. Dubowitz score, maternal viral load and other potential correlates were not associated with proliferative responses to stimuli other than p55-gag (data not shown).

Table 4.

Characteristics of infants by proliferative response to HIV-1 p55

Characteristic Responders Non-responders
HIV-1 infection Status n Mean (s.d.) n (%)† n Mean (s.d.) n (%) P-value†
HIV-1-infected IU
 Dubowitz score 3 41 (2·1) 7 57 (1·8) 0·001
 Prematurity† 3 3 (100) 7 2 (28) 0·05
 Maternal pregnancy CD4 count 3 270 (50) 7 571 (114) 0·06
 Infant viral load at 48 h 3 5·9 (.65) 7 5·2 (.89) 0·2
HIV-1 exposed uninfected
 Dubowitz score 8 56 (2·1) 31 56 (1·3) 1·0
 Prematurity† 8 1 (13) 31 4 (13) 1·0
 Maternal pregnancy CD4 count 8 363 (62) 31 500 (44) 0·10
 Infant VL n.a. n.a.
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†

Prematurity defined as Dubowitz score <47; n.a. = not applicable; s.d. = standard deviation.

Discussion

We found substantial differences in global lymphoproliferative capacity between HIV-exposed uninfected and in-utero HIV-infected infants. Marked compromise of global lymphoproliferative responses has not been observed in adult HIV-1 infection; most studies comparing HIV-1-infected adults with seronegative adults find modest to minimal differences in proliferative responses to PHA 21–23. When observed, the degree of diminished response varies with different stimuli, from moderate defects in response to allogeneic stimuli to severe defects in response to recall antigen 24,25. HIV-1-infected infants have a bimodal distribution of disease progression, with ∼25% of infants progressing to clinical disease within the first year of life 26,27. The patterns of mortality and peak and set-point viraemia also differ between infants and adults; infants experience higher peak and set-point viral loads after infection than adults, and mortality rates of ∼50% in the first 6 months of life if the infection is untreated 11,28,29. We and others have investigated factors that may influence disease progression, including the phenotype of the transmitted virus 30, the increased number of target cells available for infection 31 and the ability of the infant's immune system to respond to infection 3,15,32–39. Lower lymphoproliferative function in general and low HIV-specific lymphoproliferative responses may partially explain the accelerated course of infant HIV-1 infection when compared with adult infection. Maternal cytokine environment may affect fetal lymphocyte development and function, facilitating fetal viral replication.

We found proliferative responses to HIV-1 p55-gag antigen in approximately one-quarter of infants, regardless of HIV-1 infection status, and this prevalence is comparable to that seen in other studies 2,40,41. In adult HIV-1 infection, HIV-1-specific CD4 responses are common as a result of exposure to endogenous virus 42. Our findings are consistent with other studies noting that neonates infected with HIV-1 appear less able to mount a prompt effective HIV-1-specific cellular immune response than adults. Ramduth et al. observed that 20% of acutely infected South African infants produced interferon (IFN)-γ in response to HIV-1 gag 38, while Thobakgale et al. observed no appreciable IFN-γ production by CD4 T cells in response to gag peptides in HIV-1 infected infants aged 2 months 37. Although detection of HIV-1 specific responses in neonates has previously been associated with protection from vertical transmission 1, we did not observe that HIV-1-specific lymphoproliferative responses were more common in EU infants protected from infection compared to IU-infected infants. This may be due to differences in the assay readout: interleukin (IL)-2 secretion quantified by proliferation of an IL-2-dependent cell line compared with tritiated thymidine uptake. Alternatively, the induction of HIV-specific cellular responses may occur in late gestation, and be reduced in the HIV EU infants due to a greater reduction in viral load associated with duration of zidovudine prophylaxis initiated at the 32nd week of pregnancy.

This study had several limitations, including sample size and lack of CD4 count per individual. Whole protein antigens were used for preferential stimulation of CD4+ T cells, and while CD8+ T cells present in cord blood could potentially respond in the assay, the majority of responding T cells are expected to express CD4. The reduction in proliferative capacity, due to a reduction in T cell number or reduction in function, is a broader defect in immune capacity than previously appreciated.

Most infants had responses to C. albicans; while IU-infected infants had significantly lower SIs in response to C. albicans than EU infants, both groups' response frequency and strength were relatively high. Pabst et al. found that only 15% of cord blood samples from healthy, normal newborns had a stimulation index ≥ 2 to C. albicans, with maximum response around SI = 4 43. While not directly comparable, it appears that the EU infant C. albicans responses in our study may be substantially stronger than the HIV-1-unexposed infants in the study by Pabst et al. This may be due to increased antigen exposure resulting from more frequent and intense C. albicans infections in HIV-1-positive mothers, or to increased immune activation postulated in HIV-1-exposure or anti-retroviral drug exposure 44–46. While lymphoproliferative response to Candida is not clinically important for controlling invasive Candida infection in newborns, which relies upon lymphocyte adhesion functionality 47, these results offer a specific antigen comparison for the HIV-1 p55-gag results. Allogeneic stimulus responses were associated with C. albicans responses among the IU-infected infants, suggesting that immunosuppression may contribute to the lower C. albicans proliferative responses in IU-infected infants. The correlation between the two responses may reflect increased exposure to maternal antigens due to poor placental health.

The negative association between gestational maturity and lymphoproliferative response to p55-gag only in IU-infected infants was unanticipated. It is possible that IU infants with prematurity may have had longer fetal duration of infection leading to the higher immune responses. Earlier HIV-1 infection in utero would provide a longer window for HIV-specific immune responses to develop, while also increasing risk of preterm delivery.

In conclusion, this study presents novel findings on lymphoproliferative responses to HIV-1-specific antigen and global immune stimuli by HIV-1-exposed, uninfected and HIV-1-infected neonates. These findings suggest that neonates infected with HIV-1 in utero may be born with lymphoproliferative defects which, in turn, may contribute to their accelerated disease progression.

Acknowledgments

This work was supported by US National Institutes of Health (NIH) grants HD23412 (G. J. S.) and HD41879 (C. F). M. O., C. C., M. M. and J. M. were trainees in the AIDS International Training and Research Program, which is supported by the NIH/Fogarty International Center grant D43 TW000007. We thank the women and infants of the CTL study for their participation. We thank Sandy Emery for the infant HIV-1 diagnosis The University of Washington Center For AIDS Research grant AI 027757 provided support for specimen storage and data analysis.

Disclosure

The authors have no financial or commercial conflicts of interest.

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