T-helper cell responses to HIV envelope peptides in cord blood: protection against intrapartum and breast-feeding transmission
Kuhn, Louise; Coutsoudis, Annaa; Moodley, Dersereea; Trabattoni, Dariab; Mngqundaniso, Nolwandlea; Shearer, Gene M.c; Clerici, Mariob; Coovadia, Hoosen M.a; Stein, Zena
From the Gertrude H. Sergievsky Center, College of Physicians and Surgeons; and Division of Epidemiology, Joseph L. Mailman School of Public Health, Columbia University, New York, New York, USA, the aDepartment of Paediatrics and Child Health, University of Natal, Durban, South Africa, the bCattedra di Immunologia, Universita degli Studi di Milano, Milan, Italy and the cExperimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Received: 28 September 1999;
revised: 26 September 2000; accepted: 10 October 2000.
Sponsorship: Supported in part by grants from Fogarty International Center (TW00231) and NICHD (36177).
Requests for reprints to: L. Kuhn, Columbia University, Sergievsky Center, 630 W 168th Street, New York, NY 10032, USA.
Background: Acquired HIV-specific cell-mediated immune responses have been observed in exposed–uninfected individuals, and it has been inferred, but not demonstrated, that these responses constitute a part of natural protective immunity to HIV. This inference was tested prospectively in the natural exposure setting of maternal–infant HIV transmission in a predominantly breast-fed population.
Methods: Cord blood from infants of HIV-seropositive women in Durban, South Africa, were tested for in vitro reactivity to a cocktail of HIV envelope peptides (Env) using a bioassay measuring interleukin-2 production in a murine cell line. Infants were followed with repeat HIV RNA tests up to 18 months of age to establish which ones acquired HIV-infection.
Results: T-helper cell responses to Env were detected in 33 out of 86 (38%) cord blood samples from infants of HIV-seropositive women and in none of nine samples from seronegative women (P = 0.02). Among infants of HIV-seropositive mothers, three out of 33 with T-helper responses to Env were already infected before delivery (HIV RNA positive on the day of birth), two were lost to follow-up, and none of the others (out of 28) were found to be HIV infected on subsequent tests. In comparison, six out of 53 infants unresponsive to Env were infected before delivery, and eight out of 47 (17%) of the others were found to have acquired HIV infection intrapartum or post-partum through breast-feeding (P = 0.02).
Conclusions: T-helper cell responses to HIV envelope peptides were detected in more than one-third of newborns of HIV-infected women; no new infections were acquired by these infants at the time of delivery or post-natally through breast-feeding if these T-helper cell responses were detected in cord blood.
Naturally acquired protective immunity to HIV is yet to be clearly identified, and failure to demonstrate this is an important impediment to HIV vaccine development and evaluation. Studies of individuals exposed to HIV (some repeatedly and over extended periods) but who escape infection have contributed to identification of immune responses potentially instrumental in acquired resistance to HIV. Studies among a wide range of individuals exposed to HIV have documented acquired HIV-specific cell-mediated immune responses [type 1 T-helper cell responses and cytotoxic T-lymphocyte (CTL) responses] in the absence of systemic HIV-specific humoral immune responses or detectable virus [1–15].
A novel opportunity to test directly the inference that cellular immune responses are protective is provided by the unique circumstances of prospective studies of maternal–infant HIV transmission in breast-feeding populations. Intrauterine exposure to HIV elicits type 1 T-helper responses to HIV that can be measured in cord blood [12,16]. Subsequent infant HIV exposure occurs at delivery and through breast-feeding during which some, but not all, infants acquire infection. Transmission through each of these routes can be quantified, and distinguished from earlier intrauterine transmission with reasonable precision using serial virus-specific tests. Delineation of the timing of infection is crucial because similar immune responses to those identified in exposed–uninfected individuals are observed in some individuals after infection.
In this study, T-helper cell responses to HIV envelope peptides were measured in cord blood of infants of HIV-infected mothers. Among those not already infected in utero, the risk of subsequent transmission of HIV during delivery and throughout the duration of breast-feeding was tested prospectively to determine whether or not the risk was lower among those with cell-mediated immune responses to HIV envelope peptides compared to those without such responses.
HIV-seropositive women were enrolled in a randomized clinical trial conducted in Durban, South Africa, to test the effect of vitamin A supplementation in reducing maternal–infant HIV transmission [17,18]. Seronegative women were recruited from the same site. Antiretroviral therapy during pregnancy was not available at the site at the time the study was conducted. Women were counseled about the risks of HIV transmission through breast-feeding and about the health risks of formula-feeding. Consistent with current recommendations , women were encouraged to make their own informed choice of feeding practice. Written informed consent was obtained from all women. The study was approved by the Institutional Review Boards of the University of Natal, Columbia University, and the Office of Protection of Research Risks of the National Institutes of Health.
Immediately after delivery of the placenta, umbilical cord blood was drawn into EDTA tubes for T-helper cell assays. Blood was drawn by cordocentesis to avoid contamination by maternal leukocytes. A venous blood sample was drawn from the infant at 6 months of age for follow-up, repeat T-helper cell assays.
Pregnant HIV-seropositive women were recruited during pregnancy and a blood sample was drawn at enrolment for CD4 and CD8 T-lymphocyte counts, determination of serum retinol levels, and for quantification of HIV RNA in plasma. Clinical newborn and obstetric data were collected at delivery. Mother–child pairs were followed with regular clinical examinations for up to 18 months after delivery. Venous blood was drawn from infants on the day of birth, at 1 week, 6 weeks and 3 months of age, and thereafter every 3 months until 18 months to determine their HIV status. If children were breast-fed beyond 15 months, an additional sample was drawn at least 3 months after complete cessation of breast-feeding. Children of HIV-seronegative control women were not followed after birth.
T-helper cell assays
Type 1 T-helper cell responses in vitro were measured by interleukin (IL)-2 production in response to antigens and mitogen in a bioassay with an IL-2-dependent continuous T-lymphocyte cell line (CTLL-2) as described previously [1,2,11,12,20]. A cocktail of synthetic envelope peptides from T1, T2, TH4, P18 MN, P18 IIIB was used to measure HIV-specific responses. These peptides were identified in previous studies to be broadly immunogenic across MHC haplotypes [21–23], and T-helper cell responses to these peptides have been documented in several independent populations of exposed–uninfected individuals [1,2,6,9,11,12] suggesting a role in protection against primary infection. Response to non-viral cellular antigens was assessed using undepleted allogenic peripheral blood leukocytes (ALLO). ALLO were prepared and aliquoted at the National Cancer Institute from a pool of irradiated (50 Gy) cells from two healthy adult blood donors and was shipped on dry ice to Durban for use. Influenza A (FLU) was used to measure response to recall antigen.
In brief, within 24 h of sample collection, mononuclear cells were separated on Ficoll and cultured with the stimuli at 37°C in a moist, 7% CO2 atmosphere for 7 days. Three replicates of each stimulus and three unstimulated control wells were set up. Anti-IL-2 receptor antibody was added to the cell cultures on the first day to inhibit IL-2 consumption. Culture supernatants were harvested, frozen and stored at −20°C until assayed for IL-2 production. IL-2 production was measured by testing each supernatant for ability to stimulate the proliferation of the IL-2-dependent cell line. Three successive twofold dilutions were performed. Cultures were pulsed with 3H-thymidine and uptake was counted using a LKB Beta-plate spectrometer. Stimulation indices (SI) were calculated as ratios of mean counts per minute (c.p.m.) of triplicate cultures in stimulated to unstimulated cultures. An SI > 3 in any of the three dilutions was considered to be a positive response consistent with previous studies [1,2,11,12,20].
All assays were performed in the on-site laboratory in Durban. Assay results were excluded from the analysis if counts in the unstimulated cultures were high suggesting contamination, or if very weak responses were observed throughout, suggesting cell death. Results of 22 (20%) of 108 cord blood tests from infants of HIV-seropositive and two out of 11 of HIV-seronegative mothers, and 24 (34%) of 70 tests on 6 month samples were discarded. Exclusion of assay results was carried out without knowledge of the HIV RNA results in the infants and occurred equally across infection status. Of 108 cord blood tests from infants of HIV-seropositive mothers, results were excluded for three out of 12 infected in utero, two out of 10 intrapartum/post-natally infected, 14 out of 81 uninfected and three out of five lost-to-follow-up children.
Standard proliferation assays were also performed for a subset of cord blood samples. Peripheral blood mononuclear cell (PBMC) cultures were set up in triplicate using the stimuli described above. PBMC cultures were incubated at 37°C in a moist, 7% CO2 atmosphere for 6 days. The cultures were pulsed with 3H-thymidine and uptake counted. SI > 3 were considered positive [2,20].
HIV RNA assays
Plasma was separated within 5 h and stored at −70°C for possible subsequent quantitative assay of HIV viral RNA using the PCR (Roche Molecular Systems, Branchburg, New Jersey, USA) . RNA was extracted from 200 μl plasma in the presence of an internal RNA standard, and an aliquot was subjected to reverse transcription–PCR amplification with Thermus thermophilus HB8 (Tth) DNA polymerase in the presence of dUTP and UNG. The relative amounts of the internal standard and HIV-specific PCR products were quantified with a microtiter format enzyme-linked immunosorbent-like assay. The analytic limit of detection of the assay was approximately 10 RNA copies (about 400 copies/ml) with a linear dynamic range of at least 4 log10 and a coefficient of variation of generally 25%. For a few samples collected on the day of birth, an insufficient volume of plasma was obtained and blood spot samples were tested.
Samples collected for HIV RNA testing were stored and were sent for testing towards the end of the study. Samples from children confirmed to be uninfected (two negative ELISA results [Abbott Laboratories, Chicago] at 9 months or older among those either not breast-fed or who had stopped breast-feeding more than 3 months before their last sample) were not sent for HIV RNA testing. Among those sent for HIV RNA testing, the last available sample was tested first, and if this sample was negative, an earlier sample was tested. If the last available sample was positive, the first available sample was tested as well as each sequential sample until two positive results were obtained, or until all available samples had been tested.
Differences in proportions between groups were tested using Fisher's exact test. To take into account differential duration of follow-up, Kaplan–Meier life-table methods for the analysis of longitudinal data were used to estimate the probability of an infant of an HIV-infected mother testing HIV RNA positive over time. Differences in these probabilities between those with and without T-helper cell responses in cord blood were tested with the log-rank test.
T-helper cell responses to HIV envelope peptides and other antigens in cord blood
T-helper cell responses to HIV envelope peptides were elicited in 33 out of 86 (38%) cord blood samples from infants of HIV-seropositive women compared with none of nine (0%) cord blood samples from infants of HIV-seronegative control women (P = 0.02). The median SI (highest value of the three dilutions) was 7.1 (interquartile range, 3.7–10.1) Fig. 1. Responses to non-HIV antigens (FLU, ALLO and phytohemagglutinin) were detected at equal frequency in both groups (Table 1).
T-helper cell responses to HIV in cord blood and subsequent HIV infection status of the infant
Of the 33 infants of HIV-infected mothers responsive to HIV envelope peptides in cord blood, three (9.1%) had HIV RNA detected in venous blood samples collected on the day of birth implying transmission in utero. These responses were presumed to be a result of an established infection, as infection had been acquired in utero before they were tested for helper cell function. A further two of the 33 infants were lost to follow-up before their HIV status could be determined. None of the other infants responsive to HIV envelope peptides (0/28) were found to be HIV infected on subsequent tests. In comparison, six out of 53 (11.3%) infants unresponsive to Env were infected before delivery, and eight out of 47 (17.0%) of the others were found to have acquired HIV-infection either intrapartum or post-partum through breast-feeding on subsequent tests (P = 0.02) (Table 1, Fig. 2). If the criterion for a positive assay was made more stringent to include only those with stimulation indices > 3 in more than two dilutions, none of 17 infants responsive to HIV envelope peptides were found to be HIV infected on later tests compared to eight out of 58 (13.8%) infants unresponsive to Env.
In total, 59 out of 86 (68.6%) women elected to breast-feed their infants. Among the breast-fed, 24 out of 59 (40.7%) elicited a T-helper cell responses to HIV in cord blood. Two out of 24 (8.3%) were found to be infected in utero and no (0/22) subsequent intrapartum or post-natal infections were detected during follow-up through to 18 months. In contrast, among the breast-fed without cord blood reactivity to HIV, three out of 35 (8.6%) were found to be infected in utero and a further eight out of 32 (25%) were subsequently found to be HIV infected through intrapartum or post-natal transmission routes (P = 0.01).
Using Kaplan–Meier lifetable methods to adjust for duration of follow-up, the probability of detecting HIV infection among 35 breast-fed infants without T-helper cell responses to HIV envelope peptides in cord blood increased from 0.09 at birth to 0.336 by 9 months of age. In contrast, among 24 breast-fed infants with T-helper cell responses to HIV envelope peptides in cord blood there was no increase over the course of follow-up above that of 0.08 at birth (log-rank P = 0.04) (Fig. 3).
Although it is not possible to distinguish early breast-feeding infections from intrapartum infections, ‘late’ post-natal infections, occurring with prolonged breast-feeding after 6 weeks of age, can be identified among infected infants with negative PCR tests at 6 weeks. Two such breast-fed infants had HIV diagnostic tests consistent with ‘late’ post-natal transmission. One child had a negative HIV RNA assay at 6 months of age and a positive HIV RNA assay at 7 months of age. The second child had a negative HIV RNA assay at 6 weeks of age and a positive one at 3 months. Both were unresponsive to HIV envelope peptides in cord blood. When re-tested at 6 months of age, the first child (pre-infection at 6 months) was unresponsive; the second child (post-infection at 6 months) was responsive to HIV peptides.
T-helper cell responses to non-HIV stimuli and transmission
T-helper responses to non-HIV stimuli were not associated with the risk of transmission. T-helper cell responses to FLU (detected among infants of both HIV-infected and uninfected control mothers) unlike responses to HIV envelope peptides, did not distinguish between those children of HIV-infected mothers who acquired HIV infection themselves from those who did not (Table 1). There was a borderline association between a positive T-helper cell response to ALLO and lack of HIV transmission (Table 1). However, this association appeared to be explained by a correlation between responses to ALLO and to HIV peptides. In the subgroup lacking responses to HIV envelope peptides, the association between transmission and ALLO responses, although in the same direction, was no longer significant.
Proliferation assay results
Using a standard proliferation assay, similar associations with HIV transmission were obtained. Six out of 47 (12.8%) uninfected children of HIV-seropositive mothers with proliferation assay results had positive lymphoproliferative responses to HIV envelope peptides, whereas none of seven infected children were reactive in cord blood (P = 0.58) (Fig. 4). The proliferation assay was less sensitive to HIV-specific responses than the IL-2 assay: three out of 16 with positive responses on the IL-2 assay had positive responses on the proliferation assay. The proliferation assay also appeared to detect some non-IL-2 proliferation as three out of six children with positive proliferation assay results did not respond on the IL-2 assay.
Associations between T-helper cell responses to HIV and other risk factors for transmission
Whether the strong association observed between T-helper cell responses to HIV and lack of HIV transmission might be explained by other risk factors for maternal–infant HIV transmission, particularly maternal viral load, was investigated. Maternal viral load was an independent predictor of HIV transmission to the infant: mean (SD) log10 HIV RNA copy number in maternal plasma was 4.84 (0.83) among infected children and 4.26 (0.80) among uninfected children (P = 0.005). However, there was no evidence that maternal viremia may have accounted for associations observed between transmission and T-helper cell responses to HIV. Mean (SD) log10 HIV RNA copy number was 4.39 (1.01) among those with T-helper responses to HIV peptides and 4.31 (0.82) among those without (P = 0.70). T-helper cell responses to HIV were detected across the full range of maternal HIV RNA copy numbers (Fig. 5). It was also investigated whether an unequal distribution of other known risk factors for maternal–infant HIV transmission could have accounted for the observed association between T-helper cell responses to HIV and transmission. There was no evidence of an unequal distribution of maternal CD4 T-lymphocyte counts, CD4 : CD8 ratios, or serum retinol levels, or of infant birthweight and gestational age or of mode of delivery between those with and without T-helper cell responses to HIV. T-helper cell responses to HIV were slightly, but not significantly, more frequent among cord blood samples from infants of HIV-infected women assigned to the placebo group (47%), than among those assigned to the vitamin A supplementation group (28%), but the relationship between T-helper cell responses to HIV and child HIV status was similar in the two groups.
T-helper cell responses at 6 months of age
If sample volumes permitted, T-helper cell assays were repeated on venous blood samples collected from children at 6 months of age. T-helper cell responses to HIV peptides at 6 months were detected in three out of four children known to be infected by that age and in 23 out of 41 (56%) children with no evidence of HIV infection (Table 2). The proportion of uninfected children with positive T-helper cell responses to HIV in samples collected at 6 months was marginally but non-significantly higher in breast-fed (60%) than in formula-fed (45%) children.
Of four infected children with T-helper cell assays at 6 months, three had T-helper cell assay results available for cord blood: none of these showed a response to HIV peptides in cord blood. Of 41 uninfected children with T-helper cell assays at 6 months, 27 had T-helper cell assay results available for cord blood. Seven out of 11 (64%) with positive responses to HIV in cord blood had persistently positive responses at 6 months, and nine out of 16 (56%) with negative responses in cord blood had newly detected positive responses to HIV at 6 months.
This study was designed to test the a priori hypothesis that T-helper cell responses to HIV envelope peptides (a response previously identified in several cross-sectional studies of exposed, uninfected individuals) when measured prior to on-going HIV exposure would, on prospective follow-up, predict who acquired HIV infections and who did not. The study population, predominantly breast-fed infants of HIV-infected women, offered a ‘natural experiment’ in which T-helper cell responses to HIV could be measured in humans in the face of HIV challenge, and subsequent risks of HIV transmission quantified in responsive and unresponsive newborns. It wasobserved that T-helper cell responses to HIV envelope peptides could be detected in more than one-third of uninfected newborns of HIV-infected women, and no child with these responses acquired HIV infection during subsequent HIV exposure at delivery or post-natally through breast-feeding.
The strong and significant association observed between T-helper cell responses to HIV envelope peptides in cord blood and lack of subsequent HIV infection was not explained by other risk factors for maternal–infant HIV transmission, including maternal viral load. The association was confined to response to HIV peptides, and no significant associations were observed between HIV transmission and T-helper cell responses to non-HIV antigens. The borderline association observed between ALLO responses and transmission needs to be interpreted cautiously. Responsivity to ALLO was somewhat lower than may be expected based on other studies [12,25]; this could be explained in part by the shipping of the ALLO stimulus on dry ice to the site for use, or by other geographic differences.
Subtype C HIV predominates in the study population  but appears not to have affected responses to the cocktail of HIV envelope peptides used. Compared to North American populations, a similar proportion of HIV-exposed (but not unexposed) children responded to the cocktail. Because of limited cell volumes, responses to only few stimuli could be tested. Further investigation of responses to other HIV proteins which have been associated with reduced viral burden in HIV-infected adults  is warranted.
The study design required that infections in utero could be distinguished from infections acquired later – during delivery or through breast-feeding. The use of serial HIV PCR tests for this purpose is reasonable  although not definitive. Detection of HIV within 48 h of birth has been proposed as the working definition of infection in utero. The longer the delay between birth and testing, the greater the likelihood of incorrectly classifying infections actually acquired during delivery as ‘intrauterine', as virus may become detectable rapidly. Testing in the present study was on the day of birth, thus misclassification of this nature is likely to be small. Breast-feeding complicates delineation of the timing of infection as early breast-feeding infections cannot be distinguished from intrapartum infections. However, almost all intrapartum infections in non-breast-fed populations are detectable by PCR or viral culture by 1 month of age [30–33] hence any infections occurring thereafter this can be identified as ‘late’ post-natal infections.
Memory T-cell responses were detected in fetal cord blood implying they were primed via intrauterine experiences rather than via the too recent intrapartum experience. Despite the placental barrier, HIV has been detected in fetal tissue from elective and spontaneous abortions, supporting the notion that HIV crosses the placenta during gestation. An unusual aspect of the fetal studies is that the proportion of fetuses presumed infected is often higher than the transmission rate expected in surviving infants [34–38]. We hypothesize that a more common outcome of exposure to HIV in utero may be induction of fetal immune responses and not necessarily infection. The apparent rarity of viral clearance  does not preclude other, more common, immunologic manifestations of HIV exposure.
Mechanisms involved in facilitating the development of these responses need to be investigated. One such possibility is mother–child HLA discordance which was shown in one study to protect against perinatal transmission [40,41]. Other genetic factors may also be important although it is unlikely that the Δ32 mutation in CCR5 gene [42–47] is relevant because this mutation is very rare in Africans [44–48].
Immunologically immature newborns appear to be able to elicit apparently protective T-helper cell responses. Further investigation of whether in utero exposure to other viruses may prime similar cell-mediated immune responses is needed. Acquired cellular immune responses to human T cell leukemia virus-1 have been detected among exposed–uninfected newborns .
We observed, in a natural exposure setting, a strong and significant association between a newborn cellular immune response and protection against HIV transmission. The protective mechanisms through which these responses operate require further investigation. T-helper cell responses may support effective CTL responses  or may operate independently of CTL, such as through production of type 1 cytokines (e.g. IL-2, interferon-γ, and tumor necrosis factor-α) or β-chemokines , or possibly in providing T-helper signals for generating CD8-mediated anti-HIV factors.
The authors thank E. Spooner and K. Uebel of McCord Hospital, Durban, for assistance with patients, and G. Sinclair of the National Institutes of Virology, Johannesburg, and S. Cassol from the University of Ottawa, Otario for HIV RNA tests.
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