The Future of HIV Microbicides: Challenges and Opportunities

The availability of new antiretroviral drugs and formulations, including drugs in new classes, and recent data on treatment choices for antiretroviral-naive and -experienced patients warrant an update of the International AIDS Society-USA guidelines for the use of antiretroviral therapy in adult human immunodeficiency virus (HIV) infection. To summarize new data in the field and to provide current recommendations for the antiretroviral management and laboratory monitoring of HIV infection. This report provides guidelines in key areas of antiretroviral management: when to initiate therapy, choice of initial regimens, patient monitoring, when to change therapy, and how best to approach treatment options, including optimal use of recently approved drugs (maraviroc, raltegravir, and etravirine) in treatment-experienced patients. A 14-member panel with expertise in HIV research and clinical care was appointed. Data published or presented at selected scientific conferences since the last panel report (August 2006) through June 2008 were identified. Data that changed the previous guidelines were reviewed by the panel (according to section). Guidelines were drafted by section writing committees and were then reviewed and edited by the entire panel. Recommendations were made by panel consensus. New data and considerations support initiating therapy before CD4 cell count declines to less than 350/microL. In patients with 350 CD4 cells/microL or more, the decision to begin therapy should be individualized based on the presence of comorbidities, risk factors for progression to AIDS and non-AIDS diseases, and patient readiness for treatment. In addition to the prior recommendation that a high plasma viral load (eg, >100,000 copies/mL) and rapidly declining CD4 cell count (>100/microL per year) should prompt treatment initiation, active hepatitis B or C virus coinfection, cardiovascular disease risk, and HIV-associated nephropathy increasingly prompt earlier therapy. The initial regimen must be individualized, particularly in the presence of comorbid conditions, but usually will include efavirenz or a ritonavir-boosted protease inhibitor plus 2 nucleoside reverse transcriptase inhibitors (tenofovir/emtricitabine or abacavir/lamivudine). Treatment failure should be identified and managed promptly, with the goal of therapy, even in heavily pretreated patients, being an HIV-1 RNA level below assay detection limits.

Introduction HIV, the causative agent of AIDS, is predominantly transmitted by unprotected sexual contact [1]. Currently, women worldwide account for more than half of the estimated 6,800 newly acquired infections every day, with a majority of those transmissions occurring via the vaginal route [1]. Therefore, it is critical that strategies to prevent vaginal transmission of HIV be developed and implemented. 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. To conclusively address these hypotheses, we designed the present study. Our aims were to (1) characterize the reconstitution of the FRT with human lymphoid cells; (2) test the susceptibility of humanized BLT mice to viral transmission following a single intravaginal exposure to cell-free HIV-1; (3) characterize systemic pathogenic effects of HIV-1 transmitted intravaginally and disseminated throughout humanized BLT mice, including effects in the gut-associated lymphoid tissue (GALT); and (4) utilize this small animal model to conduct pre-clinical evaluation of antiretroviral pre-exposure prophylaxis for intravaginal HIV-1 transmission. Materials and Methods Preparation of Humanized BLT Mice, Tissue Harvesting, and Microscopic and Flow Cytometric Analyses Humanized BLT mice were prepared essentially as we have previously described [15,16]. Briefly, thy/liv-implanted mice [12] were transplanted with autologous human fetal liver CD34+ cells (Advanced Bioscience Resources) and monitored for human reconstitution in peripheral blood by flow cytometry as we have previously described [15,16]. Mice were maintained at the Animal Resources Center of University of Texas Southwestern Medical Center (UTSWMC) in accordance with protocols approved by the UTSWMC Institutional Animal Care and Use Committee. Tissues were harvested for both microscopic and flow cytometric analyses. Immunohistochemical and in situ analyses were performed essentially as previously described [15,16]. Specific controls for immunohistochemistry included staining tissue sections from humanized BLT mice with isotype-matched negative control antibodies (mouse IgG1, mouse IgG2a, goat ChromPure IgG, and rabbit ChromPure IgG) to demonstrate that appropriate human lineages were being detected. Conversely, mice never reconstituted with human cells were stained with anti-human CD3, CD4, CD68, and CD11c to rule out the staining of any non-human cells by these antibodies (unpublished data; [15,16]). Single-cell suspensions for flow cytometric analysis of each tissue were prepared essentially as we have previously described [15,16]. Intravaginal Exposure of Humanized BLT Mice to HIV-1 Stocks of HIV-1JR-CSF [18] were prepared, titered, and p24 content determined as we have previously described [19,20]. Prior to inoculation, mice were anesthetized with sodium pentobarbital. Atraumatic intravaginal inoculations were performed essentially as previously described [21] using a total volume of 10 μl (170 ng of p24, ∼9 × 104 tissue culture infectious units). FTC and tenofovir disoproxil fumarate (TDF) (Gilead) were administered intraperitonealy (3.5 mg and 5.2 mg, respectively) once daily for seven consecutive days starting 48 h prior to intravaginal inoculation with HIV-1 [13,22,23]. Analysis of HIV Infection of Humanized BLT Mice Infection of BLT mice with HIV was monitored in peripheral blood by determining plasma viral load (Amplicor; Roche), plasma levels of viral antigenemia (ELISA p24 [sensitivity: 12 pg/ml of diluted mouse plasma]; Coulter) and by determining the levels of human CD4+ and CD8+ T cells in peripheral blood (flow cytometry) essentially as we have previously described [15,16,20]. Analysis of systemic infection was performed by in situ hybridization and flow cytometry, also as we have previously described [15,16,24]. Quantitative real-time PCR for viral DNA was performed using Assays-on-Demand in a 7500 Fast instrument (sensitivity: five copies of JR-CSF; SDS software version 1.3.1.22; Applied Biosystems) following the manufacturer's protocol for universal cycling conditions. ABI custom TaqMan reagents were: Forward primer: 5′-ATCAAGCAGCTATGCAAATGCT-3′; Reverse primer: 3′-CTGAAGGGTACTAGTAGTCCCTGCTATGTC-5′; and MGB probe: 5′-TCAATGAGGAAGCTGCAGAA-3′. Rescue of infectious virus from the indicated tissues was performed by coculture with PHA/IL2-activated PBMC from HIV seronegative donors, and viral spread was monitored by determining p24 levels in the culture supernatant [16]. Human CD4+ T cell depletion was monitored in all tissues indicated essentially as we have previously described using six-color flow cytometry (FACSCanto; BD Biosciences) with analysis performed in FACSDiva software (BD Biosciences) [16]. Statistical analysis using the Student t test and the Kaplan-Meier plot were performed using Prism v. 4 (Graph Pad Software). Results The FRT represents a highly specialized and complex anatomical site where initial infection occurs following intravaginal exposure [25–27]. Therefore, we used immunohistochemistry to determine whether human lymphocytes and other cells important for HIV-1 infection were present in the vagina, ectocervix, endocervix, and uterus after reconstitution of BLT mice with human HSC. All populations of human cells necessary for HIV-1 infection (CD4+ T cells, macrophages, and dendritic cells) were found to be abundant throughout the FRT of BLT mice (Figure 1). Specifically, human CD4+ cells were distributed throughout the FRT. Also, human CD68+ monocyte/macrophage cells and clusters of human CD11c+ dendritic cells were identified throughout the FRT. Together, these data establish that in situ differentiation of human HSC leads to reconstitution of the FRT of BLT mice with the human hematopoietic cells relevant to mucosal HIV-1 transmission [28–30]. Figure 1 Reconstitution of the Female Reproductive Tract of Humanized BLT Mice with Human Hematopoietic Cells Immunohistochemical analysis of the vagina, ectocervix, endocervix, and uterus of female BLT mice for the presence of human hematopoietic lineages (brown cells) (bars indicate 25 μm). Robust reconstitution with cells relevant to HIV-1 infection, including human T cells, monocyte/macrophages, and dendritic cells, was observed in each compartment of the FRT of humanized BLT mice, demonstrating the efficient repopulation of these important mucosal sites. 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 2B). Figure 2 Pre-exposure Prophylaxis Prevents Intravaginal HIV-1 Transmission in Humanized BLT Mice (A) Kaplan-Meier plot of the time course to plasma antigenemia conversion following intravaginal HIV-1 exposure in BLT mice with or without the 7-d pre-exposure regimen of FTC/TDF (administered once daily starting 48 h prior to intravaginal inoculation). (B) Plasma from the seven infected BLT mice and the five FTC/TDF + HIV-1 mice was tested for the presence of HIV-1 RNA. Data presented depict the initial positive viral RNA value for each mouse examined. The dashed line indicates the limit of detection for this assay. (C–E) Shown are the levels of human CD4+ (orange squares) and CD8+ (blue circles) T cells in peripheral blood as well as the levels of HIV p24 antigenemia (black triangles) in plasma for (C) naive control (n = 6), (D) HIV-1 infected (n = 7), and (E) pre-exposure FTC/TDF-treated animals (n = 5). In (D), note that in seven of eight tested BLT mice, a single exposure to HIV-1 led to intravaginal transmission and an initial drop, with subsequent stabilization, in the levels of peripheral blood CD4+ human T cells. In contrast, no changes were observed in either the naive control (C) or BLT mice that received FTC/TDF for pre-exposure prophylaxis (E). In (D) and (E), day 0 is the day of inoculation and is indicated by an arrow. Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 or CD8. (F) Immunohistochemical staining for human CD4+ cells within the vagina of a representative FTC/TDF-treated mouse demonstrating the continued presence of CD4+ T cells in this tissue (left, bar indicates 50 μm; right, bar indicates 12.5 μm; box indicates region magnified in subsequent image). Neither naive nor FTC/TDF-treated BLT mice showed any evidence of plasma antigenemia (Figure 2C and 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 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 3C). Figure 3 Systemic CD4+ T Cell Loss Resulting from Intravaginal HIV-1 Infection in Humanized BLT Mice (A) Comparison of the levels of CD4+ or CD8+ human T cells in the indicated tissues in representative BLT mice that were either naive, HIV-1 infected, or that received FTC/TDF for pre-exposure prophylaxis prior to exposure to HIV-1. Note the HIV-1 induced reduction in the double-positive CD4+CD8+ thymocytes. (B and C) Box plots depicting the levels of CD4+ (B) or CD8+ (C) T cells in the indicated tissues for naive (green), HIV-1 infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. In these plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Data from naive, HIV-1-, or FTC/TDF-treated plus HIV-1–exposed mice were not collected on the same day. Naive (n = 5), HIV-1 infected (n = 4), and FTC/TDF + HIV-1 (n = 3). Flow cytometry gating for this figure was performed as described for Figure 2. BM, bone marrow; LN, lymph node; PB, peripheral blood; Thymic Org., implanted thymic organoid. 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 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 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. Figure 4 Changes in CD4+CCR5+ and CD8+CCR5+ Human T Cell Levels Resulting from HIV-1 Infection (A) Comparison of the levels of human CD4+CCR5+ T cells in the indicated tissues in a representative naive BLT mouse, an HIV-1–infected, and an HIV-1–exposed BLT mouse that received FTC/TDF for pre-exposure prophylaxis. Liver and lung were the examined tissues with the greatest constitutive CCR5 expression, and they both showed significant loss of CD4+CCR5+ T cells due to HIV-1 infection. (B) Box plot depicting the levels of CD8+CCR5+ T cells in the indicated tissues for naive (green), HIV-1 infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. (C) Comparison of the levels of human CD8+CCR5+ T cells in the indicated tissues in representative naive, HIV-1 infected and FTC/TDF treated BLT mice. All tissues examined showed increases in CD8+CCR5+ T cells resulting from HIV-1 infection of BLT mice. (D) Box plot depicting the levels of CD8+CCR5+ T cells in the indicated tissues for naive (green), HIV-1–infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. In the box plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Naive (n = 5), HIV-1 infected (n = 4), and FTC/TDF + HIV-1 (n = 3). Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 or CD8 → CCR5. BM, bone marrow; LN, lymph node; PB, peripheral blood; Thymic Org., implanted thymic organoid. 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 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 5D). We also observed a statistically significant reduction in the levels of CD4+ effector memory T cells (CD45RAnegCD27neg) 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. Figure 5 Loss of CCR5+ and Effector Memory CD4+ T Cells from GALT during HIV-1 Infection (A) Comparison of the levels of CD4+ or CD8+ human T cells in the GALT of representative naive and HIV-1–infected mice. (B) Box plot depicting the levels of GALT CD4+ T cells for naive (green) and HIV-1–infected (white) BLT mice. (C) Comparison of the levels of human CD4+CCR5+ T cells in the GALT of naive and HIV-1–infected BLT mice. Significantly fewer GALT CD4+ T cells had detectable CCR5 expression levels following HIV-1 infection. (D) Box plot depicting the levels of CD4+CCR5+ T cells in the GALT for naive (green) and HIV-1–infected (white) BLT mice. (E and F) Comparison of the levels of human CD4+ effector memory T cells in the small intestine intra-epithelial (E) and lamina propria (F) lymphocyte compartments of representative naive and HIV-1–infected BLT mice. HIV-1–infected BLT mice have statistically fewer effector memory CD4+ T cells present in their small intestines. (G) Box plot depicting the levels of CD45RAnegCD27neg effector memory T cells in the small intestine of naive (green) and HIV-1–infected (white) BLT mice. In the box plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Naive (n = 4), HIV-1 infected (n = 4). Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 → CCR5, CD27, or CD45RA. IEL, intraepithelial lymphocytes; LI large intestine; LPL, lamina propria lymphocytes; SI, small intestine. Finally, to confirm the lack of infection in FTC/TDF-treated animals shown in Figures 2, 3, and 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. Figure 6 Pre-exposure FTC/TDF Treatment Resulted in Complete Protection of Humanized BLT Mice from Intravaginal HIV-1 Transmission (A) Box plot depicting real-time PCR levels of HIV-1 viral DNA in the indicated tissues for HIV-1–infected (white) and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. (Viral DNA copies per million CD4+ T cells shown.) HIV-1 infected (n = 2) and FTC/TDF + HIV-1 (n = 4). (B) Box plot depicting virus rescue results from HIV-1–infected (white) and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. Virus rescue data expressed as pg/ml of p24 per 1 × 105 CD4+ T cells cocultured with PHA/IL2-activated peripheral blood lymphocyte from a seronegative donor. HIV-1 infected (n = 2) and FTC/TDF + HIV-1 (n = 4). In the box plots, the middle line indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Dashed lines indicate the limit of detection for each assay. (C) In situ hybridization analysis to determine the presence of productively HIV-1–infected cells in the indicated tissues from HIV-1–infected or FTC/TDF-treated BLT mice (bars indicate 50 μm). Note the lack of HIV-1 in the BLT mice that received pre-exposure prophylaxis with FTC/TDF. HIV-1 infected (n = 4) and FTC/TDF + HIV-1 (n = 3). BM, bone marrow; LPL, lamina propria lymphocytes; Thymic Org., implanted thymic organoid; SI, small intestine. Discussion The present study demonstrates efficient intravaginal HIV-1 transmission in humanized BLT mice that results in a systemic reduction of engrafted human CD4+ T cells and a loss of GALT effector memory human CD4+ T cells, as has been observed in humans [24,38–41]. In addition, we provide evidence of the effectiveness of antiretrovirals for pre-exposure prophylaxis to prevent intravaginal HIV-1 transmission. 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. More women are being infected by HIV-1 now than at any other time during the AIDS epidemic. The number of infected women worldwide has increased to almost 15.4 [1]. As a female-controlled prevention measure, antiretroviral pre-exposure prophylaxis and/or topical microbicides could provide women with a powerful tool to protect themselves from infection. However, any candidate drug(s) must be safe, especially in individuals without disease, and efficacious and, in order to be successful, must be easy to use [4]. The combination of FTC/TDF appears to meet the criteria for drugs to be used for pre-exposure prophylaxis [31]. In addition, it is one of the few drug combinations that can be administered once daily without food restrictions. In this report, we provide preclinical evidence regarding the potential efficacy of antiretroviral pre-exposure prophylaxis in humans. Our results should provide further impetus for the continued implementation of clinical trials using oral antiretroviral pre-exposure prophylaxis, particularly in parts of the world with highest HIV prevalence, where pre-exposure prophylaxis would be most beneficial and cost effective.

Introduction Ambitious targets have been set for the control of the HIV pandemic [ 1– 3]. While access to life-saving anti-retroviral therapy (ART) for those infected with HIV is increasing rapidly throughout the world, effective programmes to reduce HIV transmission are still needed [ 4], especially in Africa. Many factors influence the risk of acquiring and transmitting HIV [ 5], but where measures intended to reduce transmission have been rigorously tested at a population level, the results have been mixed [ 6, 7]. Furthermore, an effective vaccine will probably not be available for a decade or more [ 8]. There is now clear evidence that male circumcision (MC) significantly reduces female-to-male transmission of HIV. In the first randomized controlled trial (RCT) to report on MC, Auvert and colleagues [ 9] have shown that MC reduces transmission from women to men by 60% (32%−76%; unless otherwise stated ranges are 95% CI). An earlier meta-analysis of observational studies found an adjusted relative risk for HIV in circumcised men of 0.42 (0.34−0.54) [ 10], although a Cochrane review gave a more cautious interpretation [ 11]. In sub-Saharan Africa, estimates of HIV prevalence [ 12] ( Figure 1) are significantly associated with the estimated prevalence of MC (correlation coefficient, ρ = −0.61; p < 0.0001). In countries where fewer than 30% of men are circumcised, the median prevalence of HIV is 17% (IQR: 6% to 27%, n = 9 countries); where more than 90% of men are circumcised it is 2.9% (IQR: 1.5% to 5.5%, n = 13 countries). In a multi-centre study in Africa, Herpes simplex 2 and not being circumcised were independent risk factors for HIV [ 13]; the prevalence of HIV was negatively correlated with the proportion of men who were circumcised (correlation co-efficient, ρ = −0.85; p < 0.001) [ 14]. RCTs in Kenya [ 15] and Uganda [ 16] will provide further information on the impact of MC on HIV and in particular on the possible impact of MC on male-to-female transmission of HIV. The impact of increasing MC coverage on HIV depends on the prevalence of HIV and of MC and we assess this impact using both static and dynamic models. For purposes of illustration, country estimates are aggregated to four regions, but we also present results for South Africa where MC could have the greatest impact and where the estimates of MC and HIV prevalence are reliable. Estimates are made of the reduction in incidence, prevalence, and deaths in each region. Assuming that the prevalence of MC is increased from present levels to full coverage (as defined in Protocol S1 and illustrated in Figure S1) in either 2010 or 2015, we determine the relative impact that MC will have on HIV incidence, prevalence, and related mortality among circumcised and uncircumcised men and on women over the next thirty years. We consider MC independently of the changes that might arise from the implementation of other effective prevention programmes in order to focus on the impact of increasing MC coverage. Methods To estimate the potential impact of MC on HIV prevalence, we first use a static model to estimate Δ J, the reduction in the incidence of infection J. Using the result derived below ( Equations 8–10), the reduction in incidence, if all men were circumcised, would be where π is the reduction in HIV transmission from women to men, χ is the proportion of men who are already circumcised, and the incidence is taken to be one-tenth of the current prevalence, corresponding to a mean life expectancy of ten years in the endemic state. The reduction in incidence, calculated using Equation 1, gives an estimate of the impact of MC on the incidence of HIV but does not account for changes in the long-term dynamics of the epidemic. An immediate reduction in the rate of new infections will only be reflected in the prevalence of HIV and HIV-related deaths some years later, because prevalence depends on cumulative incidence and the mean time from infection to death is about ten years. For this reason, we also develop dynamic models to explore the impact of MC on incidence, prevalence, and deaths over several decades. Although the models are general, we focus the discussion of the model development on parameter values pertaining to South Africa, which has the best national data on HIV and where MC is likely to have the biggest impact. We extrapolate to other countries by adjusting the relevant parameters to fit the data on national trends in HIV prevalence and the local prevalence of MC. Parameter Estimates for South Africa Early in the epidemic, the prevalence of HIV in South Africa increased exponentially at a rate r = 0.55 ± 0.16/year, giving an intrinsic doubling time d = 1.26 ± 0.37 years [ 17]. The life expectancy after infection with HIV and without ART, standardized to a mean age at infection of 27 years, is τ = 9.8 ± 0.5 years [ 18, 19]. The average mortality rate δ = 1/τ is then 0.102 ± 0.005/year and the case reproduction number R 0 = ( r + δ)/ δ is 6.4 ± 1.6 [ 20], where we have used Monte Carlo sampling from normal distributions to estimate the standard deviations [ 21]. Asymmetry in Sexual Transmission An infected man is about twice as likely, per contact, to infect a susceptible female partner, as an infected woman is to infect a susceptible male partner. In two European studies, the transmission ratio was 2.3 (1.1−4.8) [ 22] and 1.9 (1.1−3.3) [ 23], respectively, although the ratio was significantly lower for sex during menses and higher for anal sex and for sex involving older women [ 22]. The results of the RCT [ 9] suggest that MC will increase the transmission ratio by a further factor of 2.5 (1.5−4.2). Relative Prevalence of HIV in Men and in Women To determine the relative prevalence of HIV in men and women as a function of the transmission parameters, we use a two-sex, susceptible–infected model. We let i f and i m be the prevalence of HIV in women and men. We assume that HIV-related mortality, δ, is independent of time since infection to facilitate the development of analytical expressions that can be used to parameterize the different effects on men and women in terms of an overall effect averaged over men and women. In the detailed analysis below ( Equations 12–15), we represent HIV-related mortality using a Weibull survival curve [ 24]. Although viral load is high during primary infection and near the end of life, we assume here that infectiousness is independent of time since infection. Then assuming an effective contact rate c, and probabilities of infection per contact of φ m for female-to-male transmission and φ f for male-to-female transmission, the model is Early in the epidemic, i f and i m are both much less than 1, the initial growth rate is the ratio of the prevalence in women to that in men in the early stages of the epidemic is while the case reproduction number is In the endemic state, the ratio of the prevalence in women to that in men is For the South African epidemic, if no men were circumcised, we estimate that R 0 = 7.2 ± 1.8 so that with δ = 0.102 ± 0.005/y and φ f /φ m = 2.0 ± 0.5 Equation 6 gives cφ m = 0.52 ± 0.16/year and cφ f = 1.04 ± 0.29/y. From Equation 7 the proportion of infected adults who are women is 52% ± 1%. (A more detailed examination of the proportion of infected adults who are women is given in Protocol S1 and Figure S2.) Collapsing the Two-Sex Model to a One-Group Model To determine rates of infection, averaged over men and women, we replace Equations 2 and 3 by where i is the population prevalence averaged over men and women, and so that R 0 for Equation 8 is the same as in Equation 6. It follows that reducing φ m by a factor of 1 − π is equivalent to reducing both φ m and φ f by a factor of . To allow for the fact that not all men are circumcised, we let χ be the proportion of men who are circumcised and π be the reduction in female-to-male infectiousness when men are circumcised. Then assuming random mixing the average female-to-male infectiousness is reduced to If the proportion of men who are already circumcised is χ, the effect of circumcising all men will be to reduce φ m to In Protocol S1 and Figures S4 and S5, we analyse a three-group model (distinguishing women, circumcised men, and uncircumcised men), and show that under certain assumptions it can be reduced to a one-group model with errors of less than 2.5% in the endemic prevalence for parameter values of interest. The Effect of Heterogeneity in Sexual Activity Levels of sexual activity are highly skewed [ 25, 26]. If men and women have contact rate distributions with means and, and variances and , and mixing is random, then Equation 6 still holds [ 27] but with so that heterogeneity in sexual activity increases R 0. However, MC only affects the transmission probabilities φ m and φ f and not the contact rates c m and c f, so that Equations 6–10 still hold. With R 0 ≈ 6, the predicted steady state prevalence, ( R 0 − 1)/ R 0 ≈ 83%, is much higher than is generally observed. In some models this is allowed for by assuming that people have either zero risk or a fixed risk, and then varying the risk-free fraction to fit the steady-state prevalence [ 28]. If we assume, more realistically, that there is a continuous distribution of risk, those at greater risk will tend to be infected earlier, and the risk of infection for uninfected people will fall as prevalence rises. Here we assume (and corroborate with South African data) that the average contact rate for those who are uninfected declines exponentially with prevalence (see Protocol S1 and Figure S6). The Aggregate Model Based on the above arguments, we constructed an aggregate model in which we considered the HIV prevalence, incidence, and related deaths averaged over circumcised and uncircumcised men. The model was fitted to the available time series of HIV prevalence for each country and used to make projections. We let S( t) and I( t) be the number of susceptible and infected adults over the age of 15 years at time t, with N( t) = S( t) + I( t) , and i( t) = I( t)/ N( t). Adults enter the population at risk at a rate b times the population 15 years earlier so that the total population growth rate matches the reported values for each country [ 29]. To allow for heterogeneity in sexual behaviour and to fit the observed asymptotic prevalence of infection, the transmission parameter takes the value λ 0 at the start of the epidemic and declines exponentially at rate α times the prevalence of infection. The background death rate is μ. We use a Weibull survivorship, W( t) , with a median of 9.8 years and a shape parameter of 2.25 to capture the dependence of HIV-related mortality on time since infection [ 24]. The model is then where o× indicates convolution (see Protocol S1) and D is number of HIV-related deaths per unit time. For those few countries, such as Uganda, where there is evidence that the prevalence of HIV is falling, we allow the transmission parameter to decline with mortality, D/N, to account for behavioural changes in response to the epidemic. To do this, we multiply λ 0 in Equations 12–14 by e −ɛD/N and vary ɛ to fit the data. The dynamic model was fitted to the UNAIDS estimates of national HIV prevalence in adults by least-squares. Epidemics were projected 30 years into the future assuming that the coverage of MC increases logistically (see Protocol S1 and Figure S1) from present levels to full coverage over ten years. Changing MC coverage is allowed for by scaling λ 0 as in Equations 9 and 10. Three scenarios were considered for the impact of MC on female-to-male transmission of HIV corresponding to the best estimate and to the lower and upper 95% confidence limits from the RCT [ 9]. Data The data on circumcision ( Table 1) are taken from a study by Murdock in 1967 [ 30] updated by Bongaarts et al. in 1989 [ 31] and adjusted for ethnic groups by Wendell and Werker in 2004 [ 32]. We updated these estimates further using data from Demographic and Health Surveys (DHS) carried out by Macro International (Calverton, Maryland, United States) in Kenya, Tanzania, Mozambique, Uganda, Burkina Faso, Cameroon, and Ghana [ 33], and on national population–based surveys carried out in Botswana [ 34] and South Africa [ 35]. A study in Tanzania [ 36] where 41% of men were circumcised, found that self-reported MC had a sensitivity of 94% and a specificity of 72%, so self-reported rates may overestimate true rates. The most important change to the earlier data arises from the assumption that men in a particular ethnic group, bordering Ghana and Côte d'Ivoire, are not circumcised. The DHS survey for Ghana suggests that 95% of men are circumcised in contrast to the earlier estimate of 42% [ 32]. A study carried out in Abidjan [ 37] suggests that in Côte d'Ivoire the true rate of circumcision is 93%. We used these values here. The estimates of HIV prevalence are from UNAIDS [ 12], the estimates of population size and growth rates are from the United Nations Development Programme [ 29]. Data for the time trend in the prevalence of HIV in South Africa were obtained from the series of 14 annual antenatal clinic surveys [ 38]. Results In West Africa MC is common and the prevalence of HIV is low ( Figure 1 and Figure 2A and 2B), while in southern Africa the reverse is true. Using these values in our static model ( Equation 1), circumcising all uncircumcised men in Africa would reduce the incidence of HIV as shown in Figures 2C and 2D. When incidence is measured as an absolute reduction in the number of incident infections expressed as proportion of the adult population, the potential reduction in HIV transmission is greatest in southern Africa ( Figure 2); when measured as a reduction in the total number of incident cases, the region of greatest impact extends to parts of East and Central Africa, as well as Ethiopia and Nigeria, which have large populations ( Figure 2). In South Africa alone, increasing MC coverage has the potential to avert up to 174,000 new infections each year ( Table 1). While useful and immediate, the results in Figure 2 do not provide estimates of the differential impact of MC on the risk of infection in circumcised and uncircumcised men and in women, and do not capture the temporal dynamics of the impact on incidence, prevalence, and deaths. Using a two-sex model ( Equation 7), we first determine the ratio of the prevalence of HIV in women to men at the steady state. In South Africa, if no men were circumcised, we predict that 52% ± 4% of HIV-positive adults would be women, whereas if all men were circumcised this proportion would increase to 58% ± 4%. Using a three-group model ( Protocol S1), the expected prevalence of HIV in circumcised men is close to 80% of that in uncircumcised men regardless of the overall proportion of men who are circumcised (see Figure S3). We note that these results assume random mixing and use a simplified description of heterogeneity in risk behaviour; where this assumption is not true the relative prevalence in the two groups of men may be different. Whereas MC provides greater long-term benefits to circumcised than to uncircumcised men, and to men than to women, the differences in the steady-state prevalence among the three groups are not large and suggest that the use of a simplified one-group model will be acceptable. In developing a one-group model to fit country-level data and to project changes in HIV incidence, prevalence, and deaths, we note ( Equation 6) that reducing female-to-male transmission by a proportion π, without changing male-to-female transmission, is equivalent to reducing transmission in the one-group model by factor of . In particular, if MC reduces female-to-male transmission by 60%, then this is equivalent to reducing transmission in the one-group model by 37%, so that MC would have a population-level impact equivalent to an intervention that reduces transmission in both directions by 37%. We fitted this model to UNAIDS estimates of HIV prevalence over time for each country in sub-Saharan Africa to determine the model parameters, as illustrated for South Africa in Figure 3. The centre panel ( Figure 3B) shows the prevalence of HIV in South Africa obtained from national antenatal clinic data [ 38], adjusted to match UNAIDS estimates for the adult population [ 12]. We fit the model to the prevalence data, from which incidence ( Figure 3A) and deaths ( Figure 3C) follow directly. We then allow MC coverage to increase from 35% in 2005 to full coverage in 2015 and in 2010 (see Protocol S1) as illustrated in the top three panels and bottom three panels of Figure 3, respectively. We first examine the impact on HIV if full coverage of MC is reached in 2015. The incidence of HIV infection responds immediately to the intervention ( Figure 3A) and by 2015 is close to its new asymptotic value. Prevalence responds more slowly and only approaches its new asymptotic value in 2025, while the reduction in mortality is slower still. In South Africa ( Figure 3 and Table 2), MC could avert 0.5 (0.3−1.0) million infections but only 0.1 (0.0−0.1) million deaths in the first decade of the program, 0.9 (0.5−1.8) million new infections and 0.7 (0.4−1.3) million deaths in the following decade, and 1.0 (0.5−2.0) million new infections and 1.2 (0.6−2.3) million deaths in the decade after that. The percentage reductions from the baseline scenario (with no increase in MC) as shown in Table 2 are much less in West Africa than in the other regions, but, because of the large population, the number of cases or deaths averted in West Africa is almost as great as in Central Africa, although still much less than in East and southern Africa. Over the 20 years from 2005 to 2025, in the whole of sub-Saharan Africa, MC could avert 5.7 million new cases and 3.0 million deaths, while reducing the number of people infected with HIV in 2025 by 4.1 million ( Table 2). We note that in South Africa the prevalence of HIV is high and the prevalence of MC is low, but the population is large, and approximately one-quarter of all infections prevented and deaths averted could be in South Africa. Figure 3D– 3F repeats Figure 3A– 3C, but with the intervention being introduced twice as quickly and reaching full coverage in 2010. While the incidence declines much more rapidly ( Figure 3D and 3A, respectively), there is little change in the rate at which prevalence and deaths respond ( Figure 3E and 3B; Figure 3F and 3C; respectively), reflecting the low incidence and slow progression to death for people infected with HIV. Discussion Our analysis uses a simple, parsimonious model to evaluate the potential impact of MC, and further empirical research is needed to support more detailed models. However, this analysis shows that MC could avert nearly six million new infections and save three million lives in sub-Saharan Africa over the next twenty years. Especially in southern Africa this could go some way to meeting the 2001 United Nations General Assembly Special Session on AIDS targets, the Millennium Development Goals, and the objectives set by bilateral donors, such as the US President's Emergency Plan for AIDS Relief. Many questions remain to be answered. Better data are needed on the national prevalence of HIV in Africa, as well as on the associated uncertainties. UNAIDS gives plausibility bounds for estimates of national HIV prevalence that are typically about ±30% [ 12], so the absolute values of the estimates presented here are also uncertain to this extent, although the trends should be more reliable. Better data are also needed on the prevalence of MC in Africa and on the age at circumcision, preferably at the subnational level. Most of the currently available data on the prevalence of MC are several decades old, while several of the recent studies were carried out as adjuncts to demographic and health surveys and were not designed to determine the prevalence of MC [ 33]. Without such data it is difficult to make reliable estimates of the overall uncertainty in the potential impact of MC on HIV in Africa. Data are also needed on current circumcision practices, especially with regard to safety, on the acceptability of MC, on the cost of MC, and on the feasibility of making it available in places where it is not routinely done. A detailed study is needed of the cost effectiveness of MC as a way of managing the HIV epidemic in Africa using a dynamic model of transmission, accounting for the cost of MC and allowing for the savings that follow reductions in AIDS-related morbidity and the need for ART. In addition, this analysis is based on the result of just one RCT; it will be necessary for the results of that trial to be confirmed before it is clear how accurate these estimates of future infections are. While the models presented here are a first step towards predicting the impact of MC on HIV in Africa, more detailed models are needed to explore the effect of MC on the age-specific incidence and prevalence of HIV among men and women and on the relative benefits of initially targeting men in certain age groups or in high risk occupations, such as truck drivers or mine workers. Synergies with other potential interventions, including HIV vaccines, should also be explored, as well as possible synergies acting through the impact of MC on other sexually transmitted infections. Since R 0 for HIV is on the order of 5–10, the 37% reduction in overall transmission associated with MC could make a significant contribution towards reaching the target of reducing R 0 to 1 in the areas where few men are currently circumcised. Combined with other interventions to reduce transmission, this raises the possibility of reducing the prevalence of HIV to such low levels that it is no longer a major public health problem. The impact of MC in South Africa may also be mediated by its impact on other sexually transmitted infections; the results of the other two RCTs of MC, currently being conducted in Kenya [ 15] and Uganda [ 16] where the prevalence of other sexually transmitted infections may be different, should throw light on this. The impact of MC on HIV should also be considered in the context of the increasing availability of ART. To the extent that ART reduces transmission, it will also reduce R 0 and act synergistically with MC. Many studies have shown that ART leads to substantial declines in plasma viral load [ 39] and may reduce the risk of transmission for those on ART [ 40]. However, if people in Africa start ART late in the course of their HIV infection, the provision of ART is unlikely to reduce overall transmission significantly [ 41, 42]. As a cautionary note, increases in risk-taking behaviour among circumcised men could reduce the benefit of MC. The RCT [ 9] on which these models are based followed men for an average of 18 months, so that the effects of short-term behaviour changes have been accounted for. Community or population level studies of MC are now needed to determine the likelihood of behavioural disinhibition and to assess its impact on transmission in the long term. While MC confers greater direct benefits on men than on women, women benefit indirectly through the reduction in the prevalence of HIV among their male sexual partners. Nevertheless, it is already the case that in Africa more women than men are infected with HIV [ 12], and additional methods that help to protect women, such as the development of effective vaginal microbicides, are still needed. A trial under way in Uganda [ 16] has been designed to measure the impact of MC on male-to-female transmission of HIV. The earlier observational studies and the recent RCT all suggest that MC will have a long-term, population-level impact on HIV transmission. However, this assumption needs to be tested at the population level, and a large-scale, community-based programme to implement MC as widely as possible should be implemented and carefully monitored to determine the population level impact of MC directly. This analysis makes it clear that MC could have an immediate impact on HIV transmission, but the full impact on prevalence and deaths will only be apparent about ten to fifteen years later. The reason is that circumcision averts infections some years into the future among people who would have died ten years later, on average. The same argument applies, of course, to other prevention methods because reductions in illness and death will only be manifested a decade or more after their introduction. The need to keep HIV-positive people alive through the provision of ART remains the most immediate priority while ways are found to reduce transmission using MC and other interventions. Supporting Information Figure S1 The Increase of MC in South Africa Starting from a Coverage of 35% and Reaching Full Coverage in 2015 (Red Line) and 2010 (Blue Line) (365 KB JPG) Click here for additional data file. Figure S2 The Percentage of All HIV Cases That Occur in Women, as a Function of Circumcision Parameters (A) Circumcision is assumed to have no protective benefit for women ( π f = 0). (B) Circumcision coverage is 100% ( χ = 1). Other parameters were chosen with reference to the South African HIV epidemic as discussed in the main text: δ = 0.102 yr −1, cφ m = 0.52 y −1, cφ f = 1.04 y −1. (2.7 MB JPG) Click here for additional data file. Figure S3 Ratio of the Prevalence of HIV in Circumcised to Uncircumcised Men at the Steady State Assuming an Average Infectious Period of 9.8 Years The relationship is almost completely independent of the circumcision coverage, χ, and the possible protective effect for women, π f . Other parameters: χ = 1, π f = 0, cφ m = 0.52/y, and cφ f = 1.04/y. (490 KB JPG) Click here for additional data file. Figure S4 Comparison of Three-Group (Blue Line) and the Equivalent One-Group (Red Line) Models Parameter values: χ = 0.35, π m = 0.60, π f = 0, cφ m = 0.52/y, and cφ f = 1.04/y, δ = 0.102/y, and p = 0.29. (7.8 MB TIF) Click here for additional data file. Figure S5 Absolute Difference between the Percent Prevalence as Predicted by the One-Group (Collapsed) Model and the Three-Group Model for Different Levels of MC Coverage The lines give different levels of protective efficacy π m : green, 0.76; red, 0.60; black, 0.32. Other parameters are the same as in Figure S4. (431 KB JPG) Click here for additional data file. Figure S6 Force of Infection, Scaled to One at Zero Prevalence, as a Function of Prevalence The red line is estimated from survey data for Carletonville, South Africa. The green line assumes that the risk declines exponentially with prevalence, the blue line that the risk has the same value for all those who are at risk as assumed in the EPP model. The curves are scaled to pass through the point where the curve based on the Carletonville data passes through the prevalence among men in the survey (21%). (587 KB JPG) Click here for additional data file. Protocol S1 MC and HIV in Africa (5.1 MB DOC) Click here for additional data file.