Without an effective vaccine on the horizon, alternative strategies are urgently needed to prevent the further spread of AIDS. Development and efficacy testing of microbicides and other preventive strategies, such as antiretroviral pre-exposure prophylaxis, necessitate animal models [2–5]. Currently, the only surrogate animal model used to study intravaginal HIV transmission are macaques infected with simian immunodeficiency virus (SIV) or SIV/HIV (SHIV) chimeric viruses [6,7]. This model recapitulates several aspects of human infection, but does not support HIV-1 replication. In addition, the expense of non-human primate experiments is high, the availability of animals (especially females) is limited, and variations in host susceptibility along with disease progression occur because non-human primate colonies are outbred. Consequently, there is significant need to develop animal models to investigate measures to prevent intravaginal HIV-1 transmission.

Using humanized mice to address this need is attractive because they allow in vivo studies of HIV-1 infection of human cells. In addition, in vivo preclinical evaluation of potential clinical interventions can minimize human risk [8]. A common aspect of producing humanized mice is transplanting human hematopoietic stem cells (HSC) into one of several immunodeficient mouse strains (reviewed in [8]). The mouse age at transplant and anatomical locations of the transplants vary by model, but in each system, the transplanted HSC engraft the mouse bone marrow. These engrafted HSC then differentiate in situ into human hematopoietic lineages including T, B, myeloid, and dendritic cells [9,10]. It should be noted that in this context, human thymocytes are generated in the mouse thymus and in the absence of human thymic stroma. Alternatively, human thymocytes can develop in the context of human thymus in the SCID-hu thy/liv mouse model [11,12], which has been used to evaluate the efficacy of antiretrovirals, including emtricitabine (FTC), efavirenz, atazanavir, and enfuvirtide [13]. The SCID-hu thy/liv model does not incorporate transplantation of human HSC into the recipient mouse; it involves only implantation of human fetal thymus and liver beneath the kidney capsule of SCID mice. A human thymus complete with human thymocytes is generated, yet systemic reconstitution of SCID-hu thy/liv mice with human hematopoietic cells is sparse and limited to T cells [14].

Humanized bone marrow–liver–thymus (BLT) mice [15] represent advancement beyond these models. Humanized BLT mice are generated by initially implanting human fetal liver and thymus tissue under the kidney capsule of an immunodeficient mouse (as with SCID-hu thy/liv), followed by an autologous human HSC transplant of human fetal liver CD34⁺ cells (similar to other humanization protocols). Thus, humanized BLT mice combine the most desirable attributes of other humanized mouse models into a single system. Namely, in humanized BLT mice, there is human thymic tissue where T cell education occurs, and there is complete systemic reconstitution of all major human hematopoietic lineages, including T, B, monocyte/macrophage, dendritic, and natural killer cells [15]. In addition, BLT mice have been shown to mount robust human immune responses, such as antigen-specific human immunoglobulin G (IgG) production [16] and xenograft rejection [17]. Human T cells in BLT mice can generate human leukocyte antigen class I– and class II–restricted adaptive immune responses to Epstein-Barr virus and are activated by human dendritic cells to mount a potent T cell immune response to superantigens [15]. Particularly relevant to this study is the extensive reconstitution of lymphoid tissue within the gut of humanized BLT mice [15,16]. With this particular finding in mind, we hypothesized that mucosal reconstitution with human lymphoid cells would be systemic and would include the female reproductive tract (FRT), rendering female BLT mice susceptible to intravaginal HIV-1 infection.

We then tested the susceptibility of humanized BLT mice to transmission of HIV-1 administered intravaginally. Prior to HIV-1 exposure, we analyzed the peripheral blood of all humanized BLT mice to be used in our study (8 to 12 wk post-transplant) and determined that, on average, slightly more than half (51.9% ± 7.2%) of all circulating peripheral blood cells were of human origin. We inoculated BLT mice (n = 8) with a single dose of cell-free HIV-1 (CCR5-tropic primary isolate JR-CSF [18]). BLT mice that did not receive HIV-1 (n = 6) were used as naive controls. In addition, we assessed intravaginal HIV-1 transmission in BLT mice administered a 7-d course of antiretroviral drugs (n = 5). We used emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) because of potency, daily dosing, and favorable profiles for both toxicity and viral resistance [31]. FTC/TDF was administered 2 d prior to intravaginal inoculation, 3 h prior to inoculation, and for 4 d postinoculation. Whereas 88% (7/8) of BLT mice inoculated with HIV-1 became infected (Figure 2A: Chi square = 7.5, df = 1, p = 0.006), none of the animals (0/5) that received FTC/TDF showed evidence of infection (Figure 2A and ​and22B).

Neither naive nor FTC/TDF-treated BLT mice showed any evidence of plasma antigenemia (Figure 2C and ​and2E).2E). In contrast, HIV-1 antigenemia was evident in the plasma from 7/8 intravaginally inoculated BLT mice as early as 2 wk postinfection (Figure 2D). Infection was corroborated by determining the viral load in the plasma of infected BLT mice. On average, 5.0 × 10⁵ (±1.5 × 10⁵) copies of RNA were detected per milliliter of plasma from the infected mice (Figure 2B). The appearance of HIV-1 in the plasma of infected mice preceded or coincided with a decline in peripheral blood human CD4⁺ T cells (Figure 2D). The levels of CD4⁺ T cells dropped by 30% during the first 3 wk postinfection and remained relatively constant for 7 wk, at which point there was a further 20% decline and an inversion of the ratio of CD4/CD8 cells (Figure 2D). Parallel to the decline of CD4⁺ T cells, there was an increase in the percentage of human CD8⁺ T cells in the periphery of infected BLT mice, which by 11 wk postinfection represented 60% of all the CD3⁺ cells in the periphery (Figure 2D). To eliminate the possibility that the lack of HIV-1 infection in FTC/TDF-treated mice resulted from a deficiency of cells that could be infected by HIV-1 within the mucosal portal of entry; the FRT of FTC/TDF-treated mice were examined for human CD4⁺ cells. The presence of CD4⁺ human cells in the vagina of inoculated mice that received pre-exposure prophylaxis with FTC/TDF rules out a lack of hematopoietic reconstitution of the FRT as responsible for the lack of infection (Figure 2F). Together, these results demonstrate the striking susceptibility of BLT mice to infection by HIV-1 administered intravaginally and highlight the extensive similarity in the course of HIV-1 infection in peripheral blood between BLT mice, humans, and rhesus macaques (infected with R5-tropic SHIV), including plasma viremia and CD4⁺ T cell depletion from peripheral blood [32–34]. Perhaps more important, these data demonstrate that pre-exposure prophylaxis affords complete protection to humanized BLT mice from vaginal HIV-1 transmission.

The systemic effects of HIV-1 infection in humans are inherently difficult to study. Therefore, we took advantage of the systemic repopulation of BLT mice with human lymphocytes to evaluate the effects of HIV-1 infection in relevant internal organs. Since CD4⁺ T cell depletion is a hallmark of HIV-1 infection, we compared the levels of these cells throughout the body of naive versus HIV-1–infected versus pre-exposure FTC/TDF-treated BLT mice. No statistical difference was observed when CD4⁺ T cell levels of all tissues combined in naive and pre-exposure FTC/TDF-treated BLT mice were compared (% Mean₁ − % Mean₂ [M₁ − M₂] = 1.2 ± 8.8, t = 0.13, df = 65, p = 0.90). However, when comparing HIV-1–infected versus FTC/TDF-treated and HIV-1–exposed mice, statistically significant reductions in CD4⁺ T cells were noted in peripheral blood (M₁ − M₂ = −49 ± 13, t = 3.8, df = 5, p = 0.012), bone marrow (M₁ − M₂ = −52 ± 4.1, t = 13, df = 5, p < 0.001), spleen (M₁ − M₂ = −36 ± 4.7, t = 7.5, df = 5, p < 0.001), lymph nodes (M₁ − M₂ = −28 ± 7.4, t = 3.7, df = 5, p = 0.013), liver (M₁ − M₂ = −34 ± 11, t = 3.2, df = 5, p = 0.024), and lung (M₁ − M₂ = −45 ± 8.1, t = 5.6, df = 5, p = 0.003) in HIV-1–infected mice; no significant difference was noted in the thymic organoid (M₁ − M₂ = 1.8 ± 4.5, t = 0.40, df = 5, p = 0.70) (Figure 3A and ​and3B).3B). Together with the reduction in the levels of CD4⁺ human T cells, there was a concomitant statistically significant increase in the levels of CD8⁺ human T cells comparing HIV-1–infected versus FTC/TDF-treated and HIV-1–exposed mice in all tissues tested, including peripheral blood (M₁ − M₂ = 45 ± 13, t = 3.4, df = 5, p = 0.019), bone marrow (M₁ − M₂ = 46 ± 3.5, t = 13, df = 5, p < 0.001), thymic organoid (M₁ − M₂ = 26 ± 9.9, t = 2.7, df = 5, p = 0.045), spleen (M₁ − M₂ = 29 ± 2.8, t = 10, df = 5, p < 0.001), lymph nodes (M₁ − M₂ = 27 ± 7.8, t = 3.4, df = 5, p = 0.019), liver (M₁ − M₂ = 34 ± 10, t = 3.4, df = 5, p = 0.019), and lung (M₁ − M₂ = 40 ± 6.5, t = 6.2, df = 5, p = 0.002) (Figure 3A and ​and33C).

CCR5 coreceptor expression levels on human lymphocytes vary by tissue, with lower levels on peripheral blood, bone marrow, thymus, spleen, and lymph node lymphocytes and higher levels in liver, lung, and GALT [16,35–37]. Comparison of HIV-1–infected versus FTC/TDF-treated and HIV-1–exposed mice demonstrated a significant reduction of CD4⁺CCR5⁺ T cells in BLT liver (M₁ − M₂ = −15 ± 2.1, t = 7.5, df = 5, p < 0.001) and lungs (M₁ − M₂ = −7.3 ± 0.77, t = 9.4, df = 5, p < 0.001); no significant difference was noted in the peripheral blood (M₁ − M₂ = 0.83 ± 2.1, t = 0.40, df = 5, p = 0.71), bone marrow (M₁ − M₂ = −0.33 ± 2.1, t = 0.16, df = 5, p = 0.88), thymic organoid (M₁ − M₂ = −1.1 ± 0.73, t = 1.5, df = 5, p = 0.19), spleen (M₁ − M₂ = −1.4 ± 0.59, t = 2.4, df = 5, p = 0.060), or lymph nodes (M₁ − M₂ = −1.1 ± 0.47, t = 2.3, df = 5, p = 0.069) (Figure 4A and ​and4B).4B). We also observed a dramatic increase, indicative of a heightened state of immune activation, in the levels of human CD8⁺CCR5⁺ T cells in all tissues in response to HIV-1 infection between HIV-1–infected versus FTC/TDF-treated and HIV-1–exposed mice in peripheral blood (M₁ − M₂ = 52 ± 16, t = 3.3, df = 5, p = 0.022), bone marrow (M₁ − M₂ = 35 ± 7.2, t = 4.9, df = 5, p = 0.005), thymic organoid (M₁ − M₂ = 23 ± 5.0, t = 4.5, df = 5, p = 0.006), spleen (M₁ − M₂ = 24 ± 9.3, t = 2.6, df = 5, p = 0.048), lymph nodes (M₁ − M₂ = 23 ± 7.8, t = 3.0, df = 5, p = 0.031), liver (M₁ − M₂ = 33 ± 12, t = 2.7, df = 5, p = 0.043), and lung (M₁ − M₂ = 22 ± 8.5, t = 2.6, df = 5, p = 0.050) (Figure 4C and ​and4D).4D). Thus, HIV-1 infection altered the proportions of CCR5⁺ T lymphocytes throughout BLT mice. Together, these results demonstrate that intravaginal HIV-1 transmission in humanized BLT mice leads to systemic effects by the virus that closely mimics what is observed in infected humans.

The GALT is a major site of HIV-1 replication and CD4⁺ T cell depletion during HIV disease in humans [38]. Therefore, we isolated intraepithelial (IEL) and lamina propria (LPL) lymphocytes from both small and large intestine (SI and LI, respectively) of BLT mice for analysis. Consistent with what has been observed during the course of human HIV-1 infection, we also observed a dramatic reduction in CD4⁺ T cells in the SI IEL (M₁ − M₂ = 56 ± 8.0, t = 7.0, df = 6, p < 0.001), SI LPL (M₁ − M₂ = 40 ± 4.9, t = 8.3, df = 6, p < 0.001), LI IEL (M₁ − M₂ = 23 ± 5.3, t = 4.3, df = 6, p = 0.005), and LI LPL (M₁ − M₂ = 49 ± 9.3, t = 5.2, df = 6, p = 0.002) (Figure 5A and ​and5B).5B). As described above for liver and lung, HIV-1 infection resulted in a significant reduction of CD4⁺CCR5⁺ T cells in BLT mouse SI IEL (M₁ − M₂ = 21 ± 4.1, t = 5.1, df = 6, p = 0.002), SI LPL (M₁ − M₂ = 22 ± 8.5, t = 2.6, df = 6, p = 0.042), LI IEL (M₁ − M₂ = 38 ± 8.9, t = 4.3, df = 6, p = 0.005), and LI LPL (M₁ − M₂ = 48 ± 4.4, t = 11, df = 6, p < 0.001) (Figure 5C and ​and5D).5D). We also observed a statistically significant reduction in the levels of CD4⁺ effector memory T cells (CD45RA^negCD27^neg) from the SI IEL (M₁ − M₂ = 41 ± 11, t = 3.7, df = 6, p = 0.010) and SI LPL (M₁ − M₂ = 36 ± 12, t = 3.1, df = 6, p = 0.021) of infected BLT mice (Figure 5E–5G). These findings are in agreement with studies in humans and macaques regarding memory T cell loss in GALT by HIV-1 and SIV/SHIV [24,38–41], and highlight the usefulness of the BLT model for studying HIV-1 pathogenesis, particularly in GALT.

Finally, to confirm the lack of infection in FTC/TDF-treated animals shown in Figures 2, ​,3,3, and ​and4,4, three additional approaches were utilized. DNA isolated from cells obtained from different organs of either HIV-1–infected or FTC/TDF-treated BLT mice was analyzed by quantitative real-time PCR for HIV-1 viral DNA. Whereas the tissues from HIV-1–infected mice were clearly positive for viral DNA, samples from pre-exposure FTC/TDF-treated mice were consistently negative (Figure 6A). Cells isolated from multiple organs of HIV-1–infected or FTC/TDF-treated BLT mice were cocultured with PHA/IL2-activated peripheral blood lymphocytes from a seronegative donor. Virus was readily rescued from cells isolated from tissues obtained from the HIV-1–infected mice (Figure 6B). In contrast, no virus was rescued from any of the tissues obtained from the BLT mice treated with FTC/TDF. Last, we used in situ hybridization to determine the presence of productively infected cells in HIV-1–infected or FTC/TDF-treated BLT mice. Productively HIV-1–infected cells were readily observed in tissues from the HIV-1–infected BLT mice (Figure 6C). In contrast, no productively infected cells were found in any of the tissues from the FTC/TDF-treated mice. These results verify the protection afforded by this pre-exposure prophylaxis approach to the prevention of intravaginal transmission of HIV-1 in BLT mice.

In the absence of an effective vaccine or topical microbicide, alternative preventative measures are desperately needed to help block the spread of AIDS. Antiretroviral drugs have considerable potential for preventing HIV-1 transmission [42]. The expectation for pre-exposure prophylaxis is that antiretroviral drugs taken appropriately can prevent HIV infection [4]. There is as yet no clinical evidence for the effectiveness of this approach [43–46]. However, precedent for the administration of antiretrovirals to large populations of individuals at high risk for infection is exemplified by the widespread use of nevirapine for the prevention of mother-to-child transmission of HIV [47,48]. Similarly, if proven safe and effective, pre-exposure prophylaxis together with other behavioral interventions could provide protection to men and women at risk of HIV infection by preventing sexual transmission. Therefore, it is critical to evaluate new prevention methods aimed at the populations at highest risk. Despite the urgency to develop and implement novel approaches capable of preventing HIV transmission, this process has been hindered by the lack of adequate animal models readily available for pre-clinical efficacy and safety testing [49].

We investigated the possibility that BLT mice might serve as an efficient, relatively fast, and cost-effective small animal model of intravaginal HIV-1 infection. We demonstrate that the female reproductive tract of BLT mice is populated with in situ–generated human cells critical for the transmission and dissemination of HIV-1. We observed that a single intravaginal exposure to HIV-1 results in infection in 88% of the exposed humanized BLT mice, demonstrating their susceptibility to vaginal transmission. These observations distinguish the BLT system (with its self-renewing, hematopoietic stem cell–based systemic human reconstitution, including throughout the female reproductive tract) from SCID mice injected with human peripheral blood lymphocyte (SCID-hu PBL) with respect to vaginal HIV-1 transmission [50,51]. The systemic nature of BLT human reconstitution facilitated examination of the pathogenic effects caused by infection in BLT mice. Our analyses revealed that HIV-1 disseminates from the vaginal mucosa to cause systemic CD4⁺ T cell loss, including GALT CD4⁺ effector memory T cell loss, as in humans [38]. Thus, humanized BLT mice represent a useful model for HIV-1 intravaginal transmission, systemic spread, and pathogenesis.

We utilized the fact that BLT mice are susceptible to intravaginal HIV-1 infection to demonstrate that this system is well suited for the preclinical evaluation of pre-exposure prophylactic regimens to prevent intravaginal HIV-1 transmission. Our results show that the BLT model can serve as a relatively fast and simple system to test whether pre-exposure prophylaxis can prevent vaginal HIV-1 transmission. Using this system, we found that FTC/TDF can afford complete protection from vaginal HIV-1 transmission. These results suggest that the BLT model could also be suitable for testing topical microbicides. Our results serve as preclinical evidence for the potential success of this approach aimed at preventing the further spread of AIDS.

As with all animal models of human disease, there are limitations to this study. Although our findings are consistent with findings from non-human primate research regarding the potential of pre-exposure prophylaxis to prevent HIV-1 transmission [2,42], neither model has been shown to predict efficacy or safety in humans. This is due to the lack of any kind of data from similar pre-exposure prophylaxis in humans. Therefore, an important limitation is that the BLT model currently has no known predictive value for clinical medicine. It is essential that this and future BLT studies be validated against data from human clinical trials, some of which are ongoing. Variables between humanized BLT mice and humans include possible differences in drug concentrations, in adherence, in renal and liver biology, virus dosage, and coinfections with viruses such as hepatitis B virus. Although many aspects of HIV-1 GALT pathogenesis are recapitulated in BLT mice, we have not determined whether there is a direct and/or indirect pathologic effect of HIV-1 on enterocytes, as seen in humans. Many of these limitations can be addressed in future studies. In the interim, our data support the potential for antiretrovirals in general and FTC/TDF in particular to function as a pre-exposure prophylaxis measure against the spread of HIV/AIDS in humans.