To our knowledge, this is the first demonstration of an association between breast milk cellular immune responses and protection from vertical transmission. Breast milk responses were independently associated with an approximately 70% lower odds of infant HIV infection at 1 month of life. Importantly, the breadth of breast milk response most significantly influenced transmission: infant infection was reduced by a third for each additional HIV-gag
peptide pool targeted, which suggests that a broad response against multiple epitopes may be more protective against peripartum transmission than a narrow response. The level of breast milk MIP-1β, an effector molecule secreted by IFN-γ-producing CD8+
T cells [28
], was also independently associated with protection from infection. Lastly, detection of these responses in women was not associated with detection of responses in HIV-exposed uninfected infants, or reduced viral replication in HIV-infected infants, suggesting their effect is concentrated in breast milk. Together, these data suggest a role for breast milk cell-mediated immunity in preventing vertical transmission of viruses and may explain why only a minority of infants exposed to HIV through breast milk become infected.
Breast milk IFN-γ responses could potentially decrease transmission by several mechanisms. Breast milk HIV-specific cytotoxic T cells (CTLs) may facilitate destruction of HIV-infected cells or lead to decreased production of infectious virus; this could occur either in the breast and/or at infant mucosal surfaces. This mechanism would be best validated by measuring the association between breast milk IFN-γ responses and cell-associated viral load. We observed correlations of breast milk IFN-γ with breast milk HIV-1 RNA, suggesting antigenic induction of IFN-γ responses rather than IFN-γ reduction of viral levels; however, we did not measure breast milk proviral DNA in this study. Our study supports a second potential mechanism by which breast milk HIV-specific CD8+
T cells could reduce transmission through secretion of HIV co-receptor binding chemokines that block infection. CD8+
T-cell secretion of MIP-1β has been associated with slower HIV disease progression [30
]. We previously found breast milk MIP-1β levels to be associated with reduced risk of overall postpartum transmission during the first year of life [25
]. We found MIP-1β detection in breast milk was associated with lower risk of infant HIV transmissions between delivery and 1 month postpartum, independent of breast milk viral load and breast milk IFN-γ responses. A large proportion of activated CD8+
T cells secrete both IFN-γ and MIP-1β [29
], consistent with the correlation observed in our study between breast milk IFN-γ responses and MIP-1β levels. MIP-1β could protect against HIV entry by binding to CCR5 on target cells in either the breast milk and/or the infant gut. A third mechanism by which breast milk cellular immune responses may protect transmission is via passive transfer of immune cells to the infant. Animal studies have suggested that ingested breast milk cells can cross the neonatal gut and modify immune responses within the infant [33
]. We addressed this hypothesis by evaluating HIV-specific immune responses in exposed uninfected infants and found no difference in the percentage of responses in uninfected children of mothers with breast milk responses versus those without. We also found no evidence to support the hypothesis that ingested breast milk cells in the infant circulation may affect HIV replication: HIV set-point was similar between HIV-infected infants of mothers with detectable and those with undetectable breast milk immune responses.
Three main factors were identified as correlates of breast milk IFN-γ responses: breast milk HIV viral load, chemokine levels, and subclinical mastitis (Na : K ratio). The association between breast milk viral load and breast milk HIV-specific IFN-γ responses was significant, although a low ρ
value suggests viral load alone is not responsible for expansion of antigen-specific breast milk cells. Notably, breast milk HIV-specific responses were detected in some women with undetectable breast milk viral load in our study, which is consistent with the finding of a previous study [9
]. The association between subclinical mastitis and breast milk IFN-γ responses could be explained by several mechanisms, including exudation of peripheral blood lymphocytes into the milk secondary to increased membrane permeability, proliferation of HIV-specific T cells in response to increased breast milk viral load, or increased homing of T cells to the milk in response to increased chemokine production during inflammation. As previously reported in this cohort, subclinical mastitis was associated with higher breast milk viral load and also with higher MIP-1α and MIP-1β chemokine levels [25
This work complements our previous study examining breast milk HIV-specific responses in 53 women from the same cohort using recombinant vaccinia virus (rVV) expressing HIV-gag
]. We note several important distinctions from our earlier work. First, overall prevalence of both breast milk and blood responses was greater in the current study. We hypothesize that the use of peptide pools resulted in higher sensitivity to detect low-level responses as opposed to rVV vectors, which require infection, antigen processing, and presentation. Also, the current study was designed to have adequate power to detect an effect of immune responses on transmission and focused on infections occurring during the first month of life, when the rate of transmission, the cellularity of breast milk, and, therefore, the relevance of protective breast milk responses were expected to be greatest.
Limitations of this study include inability to definitively distinguish intrapartum transmission from early breast milk transmission, low cell numbers recovered in breast milk, and the inability to evaluate late transmission events due to the combined effect of decreased breast milk cellularity and rarity of late infections. Colostrum and early milk are highly cellular, whereas the number of cells in mature breast milk are approximately 1 log10
lower than those in early milk [14
]; we, thus, speculated that if breast milk T-cell responses were associated with protection from HIV transmission, their effects would be most discernable in the early postpartum period. Although HIV DNA and RNA were undetectable in all infants at birth, some transmissions could have occurred either late in utero
or intrapartum; since breast milk T cells would not be expected to affect these routes of transmission, our data may underestimate the protective effect of HIV-specific T-cell responses. Although the measurement of IFN-γ secretion in response to one HIV protein does not fully describe the anti-HIV cellular immune response present in milk, HIV-gag
peptide pools were used exclusively due to limited cellularity; previous studies have correlated breadth of IFN-γ gag
responses with viral control [39
]. Finally, because PBMCs include CD4 T and natural killer cells, we can determine neither the source of IFN-γ nor the proportion of CD8+
T cells capable of MIP-1β and IFN-γ secretion. Regardless of the cellular source, HIV-specific IFN-γ-secretion in the breast milk was associated with reduced HIV transmission.
In conclusion, in this large study, HIV-infected mothers frequently had HIV-specific IFN-γ responses detected in breast milk that were associated with decreased infant HIV infection. Cellular immune responses in breast milk may play a role in protecting infants from mucosal exposures to viral pathogens. More generally, these data support the relevance of the HIV-gag-specific responses, and the breadth of responses, in the development of an HIV vaccine.