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).
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.
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.
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.
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.
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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