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. 2012;8(6):e1002732.
doi: 10.1371/journal.ppat.1002732. Epub 2012 Jun 14.

Human breast milk and antiretrovirals dramatically reduce oral HIV-1 transmission in BLT humanized mice

Angela Wahl  1 , Michael D Swanson, Tomonori Nochi, Rikke Olesen, Paul W Denton, Morgan Chateau, J Victor Garcia
Affiliations

Human breast milk and antiretrovirals dramatically reduce oral HIV-1 transmission in BLT humanized mice

Angela Wahl et al. PLoS Pathog. 2012.

Abstract

Currently, over 15% of new HIV infections occur in children. Breastfeeding is a major contributor to HIV infections in infants. This represents a major paradox in the field because in vitro, breast milk has been shown to have a strong inhibitory effect on HIV infectivity. However, this inhibitory effect has never been demonstrated in vivo. Here, we address this important paradox using the first humanized mouse model of oral HIV transmission. We established that reconstitution of the oral cavity and upper gastrointestinal (GI) tract of humanized bone marrow/liver/thymus (BLT) mice with human leukocytes, including the human cell types important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4⁺ T cells), renders them susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV resulted in systemic infection of lymphoid and non-lymphoid tissues that is characterized by the presence of HIV RNA in plasma and a gradual decline of CD4⁺ T cells in peripheral blood. Consistent with infection of the oral cavity, we observed virus shedding into saliva. We then evaluated the role of human breast milk on oral HIV transmission. Our in vivo results demonstrate that breast milk has a strong inhibitory effect on oral transmission of both cell-free and cell-associated HIV. Finally, we evaluated the effect of antiretrovirals on oral transmission of HIV. Our results show that systemic antiretrovirals administered prior to exposure can efficiently prevent oral HIV transmission in BLT mice.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reconstitution of the oral cavity of humanized BLT mice with human hematopoietic cells.
The oral mucosa, salivary glands, CLN and NALT were harvested from BLT mice and stained with the appropriate antibodies to verify the presence of human leukocytes (CD45+) including dendritic cells (CD11c+), macrophages (CD68+), B cells (CD20+), T cells (CD3+), CD4+ T cells (CD4+) and CD8+ T cells (CD8+). Positive cells appear brown. Scale bars = 100 µm.
Figure 2
Figure 2. The upper GI tract of humanized BLT mice is repopulated with HIV target cells.
The esophagus, stomach and duodenum were harvested from BLT mice for IHC analysis to determine if these potential sites for HIV transmission following an oral exposure possess HIV target cells. The tissues harvested were stained with the appropriate antibodies to verify the presence of human leukocytes (CD45+) including dendritic cells (CD11c+), macrophages (CD68+) and T cells (CD3+), specifically, CD4+ T cells (CD4+). Positive cells appear brown. Scale bars = 100 µm.
Figure 3
Figure 3. Oral transmission of cell-free HIV in humanized BLT mice.
(A) BLT mice (n = 10) were exposed orally to the CCR5-tropic HIV-1 isolate JR-CSF. Infection was monitored weekly by measuring the plasma viral load and percentage of human CD4+ T cells in peripheral blood. A two-tailed Mann-Whitney U test was used to compare the percentages of CD4+ T cells in peripheral blood pre-exposure (week 0) and post-exposure (p values<0.05 are indicated with an asterisk). (B) Saliva and peripheral blood (PB) were collected from five BLT mice 5–8 weeks following oral HIV exposure. Saliva was collected on the same day as peripheral blood or one week later. The corresponding saliva and peripheral blood viral loads for each mouse are shown with the same shape. The limit of detection for the assay is illustrated with a dashed line.
Figure 4
Figure 4. Susceptibility of BLT mice to HIV infection after administration via gavage.
To determine if the upper GI tract of BLT mice is susceptible to HIV transmission, we evaluated HIV acquisition after a single direct administration of virus to the upper GI tract via gavage. Infection was monitored in peripheral blood by determining the levels of viral load in BLT mice (n = 4) receiving cell-free HIV-1JR-CSF directly into the stomach by gavage. The viral load (RNA copies/ml) for each mouse is indicated and the limit of detection for the assay is illustrated with a dashed line.
Figure 5
Figure 5. Infection of the oral cavity and upper GI tract following oral HIV transmission.
(A) Tissues were harvested from the oral cavity and upper GI tract of infected BLT mice following oral HIV exposure and stained with an antibody directed against HIV p24 Gag. (B) HIV p24 Gag staining also identified infected cells in peripheral lymphoid, mucosal and non-mucosal tissues isolated from infected BLT mice outside of the oral cavity and upper GI tract. HIV-infected cells appear brown. Scale bars = 100 µm.
Figure 6
Figure 6. Oral transmission of cell-associated HIV in humanized BLT mice.
(A) BLT mice (n = 7) were exposed orally to HIV-1JR-CSF infected PBMCs. Transmission was monitored weekly by measuring the plasma viral load and percentage of human CD4+ T cells in peripheral blood. A two-tailed Mann-Whitney U test was used to compare the percentages of CD4+ T cells in peripheral blood pre-exposure (week 0) and post-exposure (p values<0.05 are indicated with an asterisk). (B) Saliva and peripheral blood (PB) were harvested from five BLT mice following oral HIV exposure with cell-associated HIV-1JR-CSF. Saliva and peripheral blood were collected from BLT mice on the same day and the corresponding saliva and peripheral blood viral loads for each mouse are shown with the same color and shape. (C) HIV transmission of cell associated virus administered via gavage into the stomach of BLT mice. Shown is the viral load in the peripheral blood of BLT mice (n = 4) receiving a single dose of HIV-1JR-CSF infected PBMCs directly into the stomach by gavage.
Figure 7
Figure 7. Oral Transmission of HIV in the presence of human breast milk.
(A) In vitro inhibitory activity of human breast milk from five different donors on infection with cell-free HIV. Infection was normalized to that of cells infected with virus in the absence of milk (positive control). Note the strong inhibition observed for all 5 human breast milk samples tested. (B) Concentration dependence of the in vitro inhibitory activity of human breast milk on infection with cell-free HIV. HIV infection was normalized to that of cells infected with virus in the absence of milk (positive control) and compared to that of cells infected with virus in the presence of different amounts of whole human breast milk (at the indicated dilutions). (C and D) Human breast milk potently inhibits cell-free and cell- associated HIV transmission in vivo. HIV transmission was examined by exposing two groups of mice orally to cell-free (C) or cell-associated (D) HIV-1JR-CSF in the presence of breast milk or RPMI medium. The peripheral blood viral load of mice was monitored weekly by real-time PCR. The mean peripheral blood viral load is shown for each exposure group. The limit of detection for the assay is illustrated with a dashed line.
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