In this study we systematically evaluated correlates of HIV-1−specific CD8+
T-cell IFN-γ responses among breastfeeding HIV-1−uninfected infants with ongoing exposure to maternal HIV-1. We found induction of HIV-1−specific T cells in uninfected breastfeeding infants was positively associated with HIV-1 in breast milk. Mother−infant pairs with breast milk HIV-1 above the median (250 copies/mL) at 1 month of age were more likely to have positive infant responses at 3 months of age. Higher HIV-1 concentrations in breast milk were also associated with increased risk of a positive infant assay at the subsequent visit. Thus, our data provide direct evidence that HIV-1−specific responses among exposed, uninfected individuals are related to HIV-1 exposure. These findings validate predictions from other studies that have demonstrated an association between positive HIV-1−specific CTL assays in exposed, uninfected female sex workers and factors that would be expected to increase exposure to virus over time [5
]. Furthermore, these data suggest that systemic responses can be induced by HIV-1 exposure at oral mucosal surfaces, an observation made recently by Perez et al [10
] that may be relevant to development of mucosally administered vaccines.
We did not observe a contemporaneous association between exposure and immune responses. Month 1 breast milk viral RNA levels were not associated with positive Elispot responses in samples collected from infants at the same visit (data not shown). That breast milk virus levels only impact later responses is biologically plausible and consistent with development of a cellular response over the course of 1–2 weeks, as has been demonstrated in studies examining HIV-specific CD8+
T-cell responses in animal models [11
]. In a study of primary HIV infection among adults, CTL responses using Elispot assays also developed well after exposure at a median of 22 days after onset of symptoms [12
]. The relevant predictor of a positive Elispot response in 1-month-old infants would therefore be breast milk virus from an earlier time point, one closer to the time of delivery, which was not collected in this study.
Other important correlates were clinical mastitis and infant sex. The trend for an association between clinical mastitis and a positive IFN-γ response was not present after we adjusted for HIV-1 concentrations in breast milk, suggesting that mastitis influenced infant HIV-specific CD8+
T-cell activity by increasing breast milk viral load. Female sex remained predictive of a positive infant response independent of breast milk HIV-1 RNA. The observation that female infants were more likely than male infants to mount a positive response at 3 months of age is intriguing because several investigators have found risk for HIV-1 acquisition in utero to be higher for girls compared with boys. Mechanisms for this association have not been defined, and hormonal, genetic, and immunologic factors may play a role [13
]. In addition, humoral and cellular responses to a variety of vaccines differ between females and males [14
], and differences in Toll-like receptor results in increased CD8 immune activation in females [15
In conclusion, we found breast milk HIV-1 levels and infant sex to be associated with HIV-1−specific CD8+ T-cell responses among exposed, uninfected breastfeeding infants. There has been speculation that such responses among HIV-1−uninfected individuals are not directed at HIV-1 antigens but target cross-reacting non-HIV epitopes. Our findings indicate that these responses are related to HIV-1 exposure and are less likely to be the result of chance events or nonspecific T-cell responses. These data support the hypothesis that HIV-1−specific T-cell responses in exposed uninfected infants result from HIV-1 exposure and exposure across mucosal surfaces, and may provide insight into the design of vaccines targeting promotion of CTL responses in infants, including orally administered vaccines.