Genetic Analysis of Simian Immunodeficiency Virus Expressed in Milk and Selectively Transmitted through Breastfeeding

Breast milk transmission of human immunodeficiency virus (HIV) is a problem of global importance. In many resource-limited areas with high rates of HIV infection, the benefits of breastfeeding often outweigh the risk of transmission (27). In these areas, the use of replacement feeding is often hindered by access to clean water, cost, availability of formula, and cultural expectations to breastfeed (33). Although short-course antiretroviral therapy (ART) given during pregnancy and parturition has significantly reduced the risk of in utero and peripartal HIV transmission (10, 12, 13), ART is a less feasible option for lactating women (16). This is due in part to the long period of breastfeeding, but the risk of infant toxicity and transmission of drug-resistant virus are important concerns that have not been fully addressed (15, 30). Effective intervention strategies that allow HIV-infected mothers to safely breastfeed are urgently needed. To develop these interventions, we must understand the mechanisms involved in transmission as well as the protective factors that allow a large number of HIV-exposed infants to remain uninfected. Studies of HIV-infected women worldwide have established that higher plasma viral load and lower CD4+ T-cell counts increase the risk of mother-to-infant transmission in utero, at parturition, and postnatally through breastfeeding (11, 17, 20, 38). The risk of breast milk transmission is additionally correlated with higher cell-free and cell-associated milk viral load (34). The specific viral and immunological factors contributing to these correlates, however, remain unknown. While maternal plasma viral load is often predictive of transmission, mothers with a wide range of plasma viral loads have transmitted virus to their infants, suggesting that additional viral and immunological factors are responsible for transmission (11, 17). Several studies have shown that viral genotypes transmitted to infants tend to be macrophage tropic, utilize CCR5 as a coreceptor, and have rapid in vitro replication kinetics (25, 28). Phylogenetic analysis of maternal and infant sequences indicates that most infants are infected with a homogeneous virus population, despite the heterogeneous virus population in the mother (1, 14, 26, 37, 45, 46). It has also been shown that HIV isolates from infants are resistant to neutralization by maternal plasma (23). These observations suggest that specific viral genotypes are selectively transmitted from mother to infant. However, other studies have identified infants with heterogeneous virus populations (8, 24, 26, 29, 42, 43). In one report, phylogenetic analysis of maternal and infant sequences did not support selective transmission but suggested that the viral population in the infant reflected the inoculum size and timing of exposure (42). Additional studies are needed to more thoroughly characterize genotypes transmitted from mother to infant. The small number of samples evaluated and the inability to identify the timing of infant infection often limits studies of HIV transmission from mother to infant. Due to the difficulties involved in defining whether transmission occurred during delivery or as a result of breastfeeding, few studies have been able to characterize genotypes transmitted through breastfeeding. Kampinga et al. evaluated HIV genotypes transmitted through breast milk in a small group of Rwandan women that became HIV infected postpartum (22). In this study, five of six breastfed infants seroconverted within the same 3-month period as their mother, and each was infected with a homogeneous virus population that represented the major genotype identified in maternal plasma samples. Multiple viral variants were identified in one infant who seroconverted more than 12 months after the mother. The V3 sequences identified in all of the infants displayed characteristics of macrophage tropism. These limited observations support the hypothesis that selective transmission does occur through breastfeeding. Infection of lactating rhesus macaques with a pathogenic simian immunodeficiency virus (SIV) inoculum just after delivery provides an excellent model of HIV transmission through breastfeeding (3, 4). This natural transmission model allows evaluation of oral infant infection via breast milk in a controlled environment without potential in utero or peripartal exposure. Although the SIV disease course is accelerated and there is a higher rate of transmission compared to that of HIV, infant infection occurs throughout the entire breastfeeding period and is associated with increased and persistent expression of virus in milk, similar to observations in HIV infected mothers. As previously reported, lactating dams were inoculated with SIV/DeltaB670 after normal vaginal delivery of their infant. This primary SIV stock is composed of a quasispecies and has been used in several studies to evaluate the influence of specific viral genotypes on pathogenesis (2, 5, 6, 39-41). In the current studies, 10 of 14 dams inoculated with SIV/DeltaB670 transmitted virus to their infant via breast milk, while four failed to transmit despite disease progression and continued lactation. Eight of the infants became infected during the chronic phase of maternal infection. Although plasma viral load and CD4+ T-cell counts did not predict transmission status, infant infection was correlated with milk viral loads exceeding 500 copies/ml. Total immunoglobulin and SIV envelope-specific antibody were also not predictive of transmission in these animals (35). Two of the infants became SIV positive during the period of peak viremia in the mother (14 days postinoculation). Transmission at this early time point was associated with rapid disease progression, high plasma viral loads, and lack of specific antibody responses in the dam. Results obtained using this model are similar to what has been reported in breastfeeding cohorts of HIV-infected women, providing an animal model to decipher the correlates and mechanisms of breast milk transmission of HIV. In our initial studies, we observed homogeneous viral populations in five macaque infants infected through breastfeeding, despite the presence of a heterogeneous viral population in the milk of each dam. In this report we extend these studies by examining the expression of envelope genotypes in plasma and milk samples obtained from eight transmitting females over the course of disease.