Avoidance of breastfeeding by HIV seropositive mothers in resource limited settings where prophylaxis is not available reduces the risk of HIV transmission but does not increase the overall survival of their children; breastfeeding protects these children from infections that result in diarrhea, pneumonia and sepsis 
. In this study, we first established that human hematopoietic cells generated in situ
are capable of repopulating the oral cavity and upper GI tract of humanized BLT mice. Specifically, these important mucosal tissues of BLT mice are repopulated with the types of human cells that have been identified to be important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4+
T cells). Our results demonstrate that the presence of these human cells renders BLT mice susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV results in systemic infection of lymphoid and non-lymphoid tissues that is characterized by a gradual decline of CD4+
T cells in peripheral blood. In addition, infection of the oral cavity results in virus shedding into saliva, recapitulating the human condition 
. Our data also offers the first in vivo
demonstration that human breast milk can inhibit oral transmission of cell-free and cell-associated HIV. Furthermore, oral transmission of HIV can be prevented with systemic FTC/TDF PrEP.
Previous studies utilizing NOD/SCID and NOD/SCID/β2
mice reconstituted with human peripheral blood leukocytes (hu-PBL mice) failed to demonstrate oral transmission of cell-free HIV following an oral exposure to CXCR4 and CCR5 tropic strains, including HIV-1JR-CSF
. Therefore, our work represents a significant advance since we demonstrated, for the first time, highly reproducible oral transmission of multiple HIV strains in BLT mice. Specifically, in this study we demonstrate oral transmission of the well characterized CCR5-tropic HIV-1 isolate JR-CSF and of several T/F viruses. Our data revealed that the efficiency of oral transmission varied among T/F viruses, suggesting that intrinsic properties of these viruses may contribute to their transmission in vivo
. Recent studies of mother-infant transmission pairs indicate that the replicative fitness as well as the length of the variable loop and number of n-linked glycosylation sites in the envelope protein may influence which maternal variant(s) are preferentially transmitted 
. Our results indicate that BLT mice could be used to study the molecular and biological properties of HIV strains that provide a selective advantage for oral transmission during breastfeeding. A comparison of viruses transmitted at early, mid and late stages of lactation would indicate if the composition of breast milk, which changes during lactation, influences which viruses are preferentially transmitted.
During breastfeeding, HIV is transmitted from mother-to-child in the context of human breast milk. In the absence of antiretroviral therapy, the breast milk of most HIV-infected women possesses cell-free and/or cell-associated HIV 
. Although elevated levels of both cell-free and cell-associated HIV in the breast milk of HIV-infected mothers have been associated with an increased risk for HIV transmission during breastfeeding 
, it is currently not known if both cell-free and cell-associated HIV are transmitted to infants. Studies illustrating that HAART administered to HIV-infected mothers during pregnancy or post-partum significantly decreases the amount of cell-free but not cell-associated HIV in breast milk 
, combined with reports demonstrating that maternal HAART significantly decreases but does not eliminate HIV transmission during breastfeeding, suggest that both cell-free and cell-associated HIV may be transmitted 
. As the importance of cell-associated HIV in breast milk transmission becomes increasingly more appreciated, antiretroviral drugs and preventative strategies may be needed that reduce the burden of HIV-infected cells in the breast milk of HIV-infected women and/or directly inhibit transmission of cell-associated HIV in infants during breastfeeding. Our data demonstrating oral transmission of both cell-free and cell-associated HIV is of high relevance since it will make possible the future evaluation of novel prophylactic strategies aimed at preventing oral transmission of both cell-free and cell-associated HIV. However, even though infected cells were used for the exposures, we cannot rule out the possibility that actual transmission across the mucosal surface could occur with cell-free virions released from infected cells.
Paradoxically, although breastfeeding can be attributed to a significant number of HIV infections in children, breast milk has been shown to potently inhibit HIV infectivity and to possess several innate factors with in vitro
anti-HIV inhibitory activity 
. Our results offer the first in vivo
evidence that human breast milk can strongly inhibit oral transmission of both cell-free and cell-associated HIV.
The ability of human breast milk to inhibit cell-associated HIV transmission in BLT mice is in contrast to in vitro
studies suggesting that milk does not inhibit cell-associated infection 
. This apparent discrepancy may be explained by the use of whole human breast milk for our experiments. Specifically, the in vitro
experiments comparing breast milk inhibition of cell-free and cell-associated HIV infection utilized the skim milk fraction of breast milk. Further in vitro
analysis comparing inhibition of cell-free HIV infection in the presence of whole breast milk or the skim milk fraction will be needed to address this issue. However, since most children at risk of HIV infection via breast milk do not receive skim milk, the potential relevance of this in vitro
observation may be questionable. Nevertheless, inhibitory factors present in breast milk may differ in their ability to inhibit cell-free versus cell-associated HIV infection. While the skim milk fraction of human breast milk possesses proteins with HIV inhibitory activity (i.e. mucin, lactoferrin, bile salt-stimulated lipase and secretory leukocyte protease inhibitor [SLPI]) 
, the lipid fraction may contain additional factors that can inhibit transmission of cell-free and/or cell-associated HIV. For example, increased concentrations of certain long-chain polyunsaturated fatty acids (LCPUFAs) in breast milk are associated with a decreased risk of HIV breastfeeding transmission 
. Although their ability to inhibit cell-free versus cell-associated HIV infection has not been experimentally tested to our knowledge, it has been hypothesized that LCPUFAs may inhibit HIV infection by inactivating the virus' envelope, suppressing the release of HIV virions from the host cell membrane and/or enhancing the viability of infected CD4+
T cells 
. All together, our results highlight the protective role of human breast milk against HIV transmission and suggest that components in both the skim milk and lipid fractions may contribute to its HIV inhibitory activity.
Despite ingesting liters of breast milk over a span of several months to years, the majority of infants born to HIV-infected HAART naïve women (~85%) do not acquire HIV during breastfeeding 
. This observation is in agreement with our data demonstrating the potent in vivo
inhibitory activity of human breast milk on oral transmission of both cell-free and cell-associated HIV following a single oral exposure. Oral transmission of HIV in the presence of human breast milk may require multiple exposures over time. Furthermore, although increased levels of HIV in breast milk have been associated with an increased risk for HIV transmission during breastfeeding, several other maternal and infant factors have been associated with breastfeeding transmission. Additional maternal factors include seroconversion during lactation, CD4+
T cells counts below 500 cells per mm3
, poor breast health (mastitis, nipple bleeding, etc), and decreased levels of alpha-defensins in breast milk. Infants that receive both breast milk and other food (mixed-feeding) are also more susceptible to HIV transmission during breastfeeding as are infants with oral thrush and decreased levels of salivary SLPI [reviewed in 
]. One remaining question is, therefore, whether or not there are differences in the babies or mothers or in the breast milk in the cases where mother-to-child transmission does occur. In the future, it will be important to compare the inhibitory activity of breast milk obtained from HIV-infected mothers who transmit HIV to that of HIV-infected mothers that do not transmit HIV in vivo
. In addition, in vivo
experiments evaluating transmission in the presence of saliva obtained from infants with low and high levels of salivary SLPI will help assess the contribution of the inhibitory activity of infant salivary SLPI on oral transmission of cell-free and cell-associated HIV.
Collectively, our results demonstrate that BLT mice are an attractive small animal model that can be utilized to study key aspects of oral HIV transmission and to test the efficacy of HIV vaccines, antiretroviral therapies and other preventative measures aimed at reducing mother-to-child transmission of HIV during breastfeeding. In addition, our data demonstrating the presence of human immune cells in the oral cavity and GI tract of BLT mice indicate that BLT mice may be utilized to study other human pathogens that are transmitted orally and/or infect these tissues (i.e. HCMV and EBV) and to answer fundamental questions about human oral and gastrointestinal immunity.