Background:  Breast milk transmission of HIV remains an important source of pediatric HIV infection with ≤40% of HIV-infected children in Africa acquiring HIV through this route. Breast milk is a distinct immunologic compartment, however, data on virologic compartmentalization are limited and conflicting.

Methods:  The gp160 coding sequences of paired blood and breast milk from the right (BMR) and left breast (BML) of 9 women participating in the Zambia Exclusive Breastfeeding Study were analyzed. To avoid virus resampling, both limiting dilution and multiple independent polymerase chain reaction (PCR) amplifications were performed. An average of 18 breast milk samples (9 right breast, 9 left breast), 11 plasma, and 5 peripheral blood mononuclear cell (PMBC) clones were sequenced for each patient. To determine compartmentalization, maximum-likelihood trees based on the V1-V5 region (excluding ambiguously aligned regions and hypermutated sequences) were calculated and Slatkin-Maddison analysis of phylogenetic affinities was performed. Co-receptor usage was predicted using position-specific scoring matrices (PSSM).

Results:  Statistically significant compartmentalization between blood and breast milk was observed in 5 of 9 women. In 2 of 4 women without significant compartmentalization, phylogenetic trees strongly suggest compartmentalization will be evident upon sequence analysis of additional clones. In fact, compartmentalization was detected in 1 woman between the plasma and BML only (p = 0.003). The 2 women without compartmentalization had very low CD4 counts (55 and 94 cells) indicating compromised immunity, 1 of whom had a near homogenous viral population similar to that observed in acute infection. Interestingly, BMR and BML variants were indistinguishable in 7 of 7 women. The number of potential N-linked glycosylation (PNG) sites in V1-V5 was also examined and breast milk virus showed fewer PNG than plasma in 3 of 5 cases. Syncitia-inducing variants were predicted by PSSM in the breast milk, plasma, and PBMC of 3 of 5 women.

Conclusions:  These data demonstrate distinct compartmentalization of breast milk viral variants from those circulating in the plasma, indicating profound selective pressure within the lactating breast. The uniform milieu between geographically distinct breasts on the same woman can be attributed to either the trafficking of infected cells to the lactating breast or to factors within the breast epithelium or milk that restrict the outgrowth of specific clones.