Antiretroviral Preexposure Prophylaxis for Heterosexual HIV Transmission in Botswana

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Abstract

Preexposure prophylaxis with antiretroviral agents has been shown to reduce the transmission of human immunodeficiency virus (HIV) among men who have sex with men; however, the efficacy among heterosexuals is uncertain. We randomly assigned HIV-seronegative men and women to receive either tenofovir disoproxil fumarate and emtricitabine (TDF-FTC) or matching placebo once daily. Monthly study visits were scheduled, and participants received a comprehensive package of prevention services, including HIV testing, counseling on adherence to medication, management of sexually transmitted infections, monitoring for adverse events, and individualized counseling on risk reduction; bone mineral density testing was performed semiannually in a subgroup of participants. A total of 1219 men and women underwent randomization (45.7% women) and were followed for 1563 person-years (median, 1.1 years; maximum, 3.7 years). Because of low retention and logistic limitations, we concluded the study early and followed enrolled participants through an orderly study closure rather than expanding enrollment. The TDF-FTC group had higher rates of nausea (18.5% vs. 7.1%, P<0.001), vomiting (11.3% vs. 7.1%, P=0.008), and dizziness (15.1% vs. 11.0%, P=0.03) than the placebo group, but the rates of serious adverse events were similar (P=0.90). Participants who received TDF-FTC, as compared with those who received placebo, had a significant decline in bone mineral density. K65R, M184V, and A62V resistance mutations developed in 1 participant in the TDF-FTC group who had had an unrecognized acute HIV infection at enrollment. In a modified intention-to-treat analysis that included the 33 participants who became infected during the study (9 in the TDF-FTC group and 24 in the placebo group; 1.2 and 3.1 infections per 100 person-years, respectively), the efficacy of TDF-FTC was 62.2% (95% confidence interval, 21.5 to 83.4; P=0.03). Daily TDF-FTC prophylaxis prevented HIV infection in sexually active heterosexual adults. The long-term safety of daily TDF-FTC prophylaxis, including the effect on bone mineral density, remains unknown. (Funded by the Centers for Disease Control and Prevention and the National Institutes of Health; TDF2 ClinicalTrials.gov number, NCT00448669.).

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Antiretroviral Pre-exposure Prophylaxis Prevents Vaginal Transmission of HIV-1 in Humanized BLT Mice

Paul Denton, Jacob D Estes, Zhifeng Sun … (2008)

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.

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Tenofovir Disoproxil Fumarate for Prevention of HIV Infection in Women: A Phase 2, Double-Blind, Randomized, Placebo-Controlled Trial

Leigh Peterson, Doug Taylor, Ronald Roddy … (2007)

INTRODUCTION The HIV epidemic is continuing to grow worldwide [1]. Consistent and correct use of condoms is recommended for prevention of sexually transmitted HIV, but often women are unable to negotiate condom use with their male partners. Safe, effective, and easy to use methods of HIV prevention are urgently needed, especially for women. Tenofovir disoproxil fumarate (TDF) [2], the orally bioavailable prodrug of tenofovir, is metabolized to a competitive inhibitor of viral reverse transcriptase. TDF was selected for clinical development as a treatment for HIV infection because of its (1) potency against wild-type HIV and some nucleoside-resistant strains of HIV [3–6], (2) low potential of selecting for TDF-resistant mutants [7], (3) low likelihood of metabolic/mitochondrial toxicity [8], and (4) pharmacologic profile supporting daily dosing [2]. TDF was licensed for the treatment of established HIV-l infection by the United States Federal Drug Administration in 2001, and the European Commission issued a marketing authorization in 2002 [9]. TDF has since been used worldwide for treatment of HIV infection, accounting for nearly 500,000 patient-years of observation [10]. We investigated the safety and effectiveness of a daily dose of 300 mg of TDF in preventing HIV infection among women at high risk for infection based on the following rationale: (1) initial prevention studies in simian models have provided support for both pre- and post-exposure efficacy of TDF in preventing retroviral infections [11–14]; (2) TDF has been shown to be safe in large numbers of HIV-infected persons [15,16]; (3) TDF is dosed conveniently once a day; (4) TDF has no known interactions with hormonal contraception [17]; (5) a high barrier to resistance was seen in clinical trials of HIV treatment, with the primary mutation identified (K65R) resulting in a reduction of viral replication to almost half that of wild-type [18]; and (6) the drug's sponsor, Gilead Sciences, is supportive of investigating the potential use of TDF as a preventive agent. Moreover, should it prove to be effective for HIV prevention, Gilead Sciences has committed to making TDF available in resource-poor settings for public health use, as they currently do for treatment of HIV, via no-profit pricing and licensing agreements. METHODS Participants Study participants were recruited from areas within each city that were considered high HIV transmission areas, including markets, bars, and hotels. Although we did not specifically ask as part of the clinical trial procedures if the participants were sex workers, most exchanged sex for money. Special ethical considerations were taken into account because of the potential vulnerability of this population. We developed strategies to protect the confidentiality and autonomy of the participants, increase/ensure comprehension of the informed consent and research methods, and promote access to resources and services during and after the trial. We enrolled HIV-antibody-negative women 18 to 35 y old who were at risk of HIV infection by virtue of having an average of three or more coital acts per week and four or more sexual partners per month. Participants had to be willing to use the study drug as directed and participate for up to 12 mo of follow-up. Because TDF has been associated with rare episodes of renal disorders, increased liver enzymes, and hypophosphatemia, participants were also required to have adequate renal function (serum creatinine 2.0 mg/dl) for renal function, grade 3 or 4 AST or ALT elevations (>170 U/l) for hepatic function, and grade 3 or 4 phosphorus abnormalities ( 170 U/l) ALT or AST elevations before product withdrawal, whereas two and three of the 368 participants in the placebo group had grade 3+ ALT and AST elevations, respectively. The percentage of grade 1 or higher (>42 U/l) ALT and AST abnormalities was greater in the TDF group, but the difference did not achieve statistical significance. One participant in the TDF group had a grade 3+ decrease in phosphorus ( 2.0 mg/dl). We did not find significant differences in laboratory abnormalities between treatment groups when the data were stratified by site, although significantly more grade 1 AST and phosphorus abnormalities occurred in Ghana than in Cameroon, and significantly more grade 1 creatinine abnormalities occurred in Cameroon than in Ghana. Among the 56 participants who tested positive for HBsAg, 23 were in the TDF group and 33 in the placebo group. The mean and median ALT and AST levels were not significantly different between groups immediately before or after discontinuation of study drug. Four ALT/AST abnormalities (none over grade 1 [>42 U/l]) occurred within 3 mo after discontinuation of study drug in HBsAg-positive participants; one was in the TDF group and three were in the placebo group. In Cameroon, study drug was stopped before the implementation of the protocol amendment that included HBsAg testing. We therefore monitored liver function for several months after product withdrawal in all participants, regardless of HBV status. The mean and median ALT and AST levels were not significantly different between groups immediately before or after discontinuation of study drug. Twenty ALT/AST abnormalities occurred within 3 mo after discontinuation of study drug; 14 were in the TDF group and six were in the placebo group. With the exception of one, all were grade 1 or 2 events (i.e., less than 85 U/l). One participant (who received TDF) had a grade 3 AST abnormality, which resolved within 1 mo. Adverse events. A total of 320 (75%) women in the TDF group and 310 (72%) women in the placebo group had at least one AE. The most frequently reported AEs (occurring in ≥5% of participants in either treatment group) are summarized in Table 3. There were no significant differences between treatment groups for any AEs within system organ classes. Similar results were obtained among HBsAg-positive participants. Twenty–two SAEs were reported during the study (nine in the TDF group and 13 in the placebo group) in 17 participants. The majority of the SAEs were hospitalizations due to malaria (nine events). No SAEs were considered related to study drug. Two deaths occurred in Cameroon during the course of the study; both occurred approximately 5 mo after study drug dispensation was suspended. One was due to an unspecified condition with anemia (the participant had been randomized to placebo), and one was suspected to be related to an induced abortion (the participant had been randomized to TDF). Study drug was not discontinued in any participant by the site investigator or study clinician because of an AE or abnormal laboratory result. Effectiveness: HIV Incidence For the primary effectiveness analysis, women in Cameroon, Ghana, and Nigeria contributed 232.6 person-years of follow-up in the TDF group and 241.3 person-years in the placebo group. Eight seroconversions occurred among participants while receiving the study drug. Two HIV infections were diagnosed in participants randomized to TDF (rate = 0.86 per 100 person-years) and six in participants receiving placebo (rate = 2.48 per 100 person-years), yielding a rate ratio of 0.35 (95% confidence interval = 0.03–1.93; p = 0.24). With the exception of two participants (one randomized to TDF and one to placebo), all seroconversions were detected on or after the second monthly follow-up visit. Blood specimens were available from one of the two participants who seroconverted while on TDF; standard genotypic analysis revealed no evidence of drug resistance mutations. An additional six participants seroconverted after study drug was discontinued in Cameroon (four had been randomized to TDF and two to placebo). DISCUSSION Interpretation and Overall Evidence Our data provide an encouraging rationale for additional research to evaluate oral antiretroviral drugs as prophylaxis against HIV infection. No significant differences in safety patterns occurred among participants receiving daily oral TDF compared with those receiving placebo, consistent with results seen in previous treatment studies [2]. No randomized clinical studies have been completed to evaluate the effectiveness of antiretroviral drugs as pre- or post-exposure prophylaxis against HIV infection. In a recent Cochrane review [20], only one case-control study of health-care workers after needlestick injury was identified. HIV-infected workers had significantly lower odds of having taken zidovudine prophylaxis after exposure than those who did not seroconvert (odds ratio = 0.19, 95% confidence interval = 0.06–0.52) [21]. The effectiveness of antiviral prophylaxis after sexual exposure is not known [22]. Non-randomized observations of post-exposure prophylaxis (PEP) are difficult to interpret because there could be underlying and uncontrolled differences between those who seek and use PEP and those who do not, including differences in behavior and access to information and medications. Furthermore, PEP or event-driven dosing is limited by the fact that failures can occur when treatment is not initiated because the risk of the exposure is not recognized [23]. We address this limitation of event-driven dosing in this study by recommending daily dosing for frequently exposed persons—a strategy referred to as pre-exposure prophylaxis. Because public health practice should be guided by evidence, especially in settings having competing demands for scarce public health resources, an urgent need exists for antiviral prophylaxis for HIV to be evaluated in randomized, placebo-controlled trials, as we report here. Further effectiveness studies in populations of women at high risk for acquiring HIV should proceed rapidly. The premature stopping of the study in Cameroon and Nigeria limited the amount of follow-up safety and effectiveness data obtained in this study. Furthermore, AEs and laboratory abnormalities in the TDF group may have been diluted by lower than expected product use due to missed visits, drug stoppage due to pregnancy, and other reasons for non-use of study drug. The overall rate of HIV infection while women were on TDF or placebo in Ghana, Cameroon, and Nigeria was too low to demonstrate a reduction in risk for those assigned to the TDF group. TDF is active against HBV, and is recommended for treatment of HBV infection in Europe and by many experts [24–27]. Transaminase increases have been observed in up to 25% of patients stopping anti-HBV drugs after receiving therapy for clinically important HBV infection, characterized by pre-treatment elevated ALT or AST or signs of liver fibrosis [28]. No flares of ALT or AST were observed among those with HBV infection in this study, although our analysis was limited to 23 TDF-treated women with reactive tests for HBsAg. The rate of HBV flares after withdrawing TDF may be low among people with normal baseline liver tests and no signs or symptoms of advanced liver disease, such as the women enrolled here. Additional data on the use of TDF in persons with circulating HBsAg are needed to confirm the results observed in this study. We expected the HIV incidence in the placebo group to be no less than five per 100 person-years, over twice that we observed in this study. Thus, our power was less than anticipated. The lower than expected HIV incidence may be due in part to at least four factors: (1) the incidence rate was estimated from both our experience in earlier trials in a similar population and from current prevalence data; it was not specifically measured in each population before starting the study; (2) intense and consistent HIV/sexually transmitted infection prevention services and messages were provided to the study participants during the trial; (3) the effect of taking a pill every day for HIV prevention may have served as a timely reminder of imminent HIV risk such that participants modified their behavior to incorporate precautions against infection such as increased use of condoms; and (4) participants who elect to join clinical trials may be more inclined to safer behavior than those in their community who do not participate. Indeed, reductions of risk behavior have been observed in open label studies of PEP [23,29]. Generalizability As a new HIV prevention approach, prophylactic use of TDF could be used with other prevention strategies such as condoms to reduce the number of people who become infected with HIV. Larger phase 3 studies to conclusively determine the safety, effectiveness, and feasibility of using TDF (either alone or in combination with other antiretrovirals) as chemoprophylaxis against HIV infection in both women and men are needed. SUPPORTING INFORMATION CONSORT Checklist Click here for additional data file. Original Trial Protocol Click here for additional data file. Amended Trial Protocol Click here for additional data file.

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Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir

J Garcia-Lerma, Ron A Otten, Shoukat Qari … (2008)

Introduction With an estimated 33.2 million people worldwide living with HIV at the end of 2007 and an estimated 2.5 million new infections in 2007 acquired mostly through sex, HIV/AIDS continues to be a major global health challenge [1]. Currently, no vaccine is available to prevent HIV, and it is unlikely that one will be developed soon. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs is gaining considerable attention as a possible biomedical intervention strategy to prevent sexual transmission of HIV [2–5]. PrEP is a proven concept for other infectious diseases like malaria. Mathematical models estimate that over the next 10 y, an effective PrEP program could prevent 2.7 to 3.2 million new HIV-1 infections in sub-Saharan Africa [6]. This potentially significant public health benefit requires a very high PrEP efficacy and might be lost or substantially reduced with a PrEP efficacy of 60 h) intracellular half-lives in humans [16–18] suggesting the possibility of extended prophylactic activity when administered around virus exposures. We found FTC/tenofovir given intermittently as a two-dose PrEP around each of 14 weekly virus exposures to be as fully protective as the same regimen given daily. Therefore, intermittent PrEP with potent regimens are highly promising modalities. Evaluation of intermittent PrEP with different drug combinations, possibly of different classes, and defining minimal dose requirement and optimal timing relative to virus exposure will all be important. While many biologic similarities exist between rectal and vaginal HIV transmission, some differences in the early events of mucosal infection and dissemination kinetics are possible [8,35]. Therefore, it is important to confirm the PrEP efficacy of these regimens against vaginal transmission in appropriate macaque models. Although daily PrEP with a Truvada-equivalent dosing was highly effective, a regimen with more tenofovir was required to completely block transmission in this model. However, the dose of tenofovir in this regimen would likely be toxic in humans. More work in macaque models could possibly identify two- or three-drug combinations that are fully protective and yet carry low risks of toxicity. The increasing availability of new drugs in different classes such as those that block viral integration or entry through CCR5 will provide additional possibilities. The results of such animal studies may help guide designs of clinical trials that will ultimately measure effectiveness of various PrEP regimens against sexual HIV transmission. Initial macaque studies with tenofovir used single and non-physiologic doses of SHIV or SIV (103 to 105 TCID50) capable of yielding high infection rates in untreated controls [10,11,33,34]. We used a more physiological virus dose that fell within the upper range of viral load observed in human semen during acute HIV-1 infection [20]. The repeated nature of the model has also the advantage of evaluating protection over multiple transmission events. The infection of control macaques after a median of two exposures suggests that treated animals that remain uninfected after 14 challenges were protected over a median of seven transmission events. This model maintains high stringency, increases statistical power, provides improved estimations of risk reductions, and reduces the number of macaques [36]. Although a repeat-challenge model is more relevant to human transmission, which typically requires multiple exposures, a disadvantage of the model is that it is logistically demanding over a long period of time. This limits the ability to do multiple concurrent arms, which raises a potential for bias. However, animal studies with non-concurrent arms can be well controlled. In our study, we have staggered interventions because of logistic feasibility and to prevent unnecessary use of animals. All animal procedures were done under identical conditions by the same personnel and experimental protocol, thus minimizing the potential for bias. Likewise, it is not known if repeated exposures to the virus can ultimately alter susceptibility to infection. The similar infection rates observed among previously or newly exposed animals suggest that the impact of repeated exposures on susceptibility to infection is minimal [12,37]. Several important observations were made from the longitudinal analysis of the breakthrough infections. The finding that wild-type SHIV initiated all six infections suggests that PrEP failure in these animals is due to residual virus replication in cells not protected by drugs, rather than a rapid selection of a drug-resistant virus. Of the four animals infected during FTC treatment, only one selected resistant viruses, while an FTC-resistant virus emerged in one of two animals that failed FTC/TDF PrEP. The absence of tenofovir resistance in both macaques is consistent with clinical observations showing resistance to FTC and not tenofovir as the most frequent pathway of resistance to Truvada [38]. The two macaques in which FTC-resistant mutants emerged had the highest peak viremias, suggesting that selection of drug resistance may be facilitated by higher virus replication. Thus, lower acute viremias may have diminished the risk of resistance during extended treatment with FTC or FTC/TDF. Similar blunted viremias during early infection have been noted in macaques failing PrEP with an orally administered CCR5 inhibitor [39]. These data underscore the potential differences in virus load and drug resistance dynamics during PrEP failures from those in mono- or dual drug therapy of established infections [21,22,40,41]. The attenuated acute viremias may have additional clinical and public health implications. It is well established that massive depletion of CD4+ T cells, specifically CD4+ memory T cells, begins in the acute stage of SIV as well as HIV-1 infection in the gut and other lymphoid tissues, and is generally proportional to the degree of virus replication [42–44]. Substantial reductions in acute viremia may conceivably reduce CD4+ T cell depletion, help preserve immune function, and attenuate the course of HIV infection. In humans, reduction in virus set points by 1 log10 have been estimated to double the time to progression to HIV disease [45]. Reductions in virus loads in the animals that failed PrEP were apparent at the first time points before seroconversion and were sustained under continued drug treatment after all the animals seroconverted. Blunted viremia during acute infection in persons who fail PrEP will likely depend on the period of drug exposure and the potency of the PrEP regimen. PrEP-treated populations will likely be monitored by serologic testing for infection to minimize drug exposure and reduce the risks of drug resistance. The period of drug exposure will thus depend on the frequency of serologic testing but will inevitably be at least several weeks long, enough to affect early CD4+ T cell depletion. Individuals with acute infections who have very high virus loads may also play a key role in the epidemic spread of HIV-1 because they are more infectious than individuals with chronic infections who have lower virus loads [46–48]. Therefore, reductions in acute viremia during PrEP treatment may contribute to decreases in HIV-1 transmissibility at the population level and could add to the overall effectiveness of PrEP. The data from this study demonstrate the potential high effectiveness of daily or intermittent PrEP against sexual HIV transmission, support expanding PrEP trials in humans and identify promising PrEP modalities. Supporting Information Alternative Language Abstract S1 Translation of the Abstract into French by Dominique Rollin and Walid Heneine (23 KB DOC) Click here for additional data file. Alternative Language Abstract S2 Translation of the Abstract into Spanish by J. Gerardo García-Lerma (43 KB DOC) Click here for additional data file. Table S1 Macaques, Interventions, and Outcome of the Challenge Series (51 KB DOC) Click here for additional data file. Text S1 Description of Current and Historic Macaques and Susceptibility to Infection (28 KB DOC) Click here for additional data file.