Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1JRCSF or the MSM-derived transmitted/founder (T/F) virus HIV-1THRO within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1JRCSF and HIV-1THRO, respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1JRCSF and 0% (0/6; log rank p = 0.02) for HIV-1THRO. This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides.
Citation: Chateau ML, Denton PW, Swanson MD, McGowan I, Garcia JV (2013) Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice. PLoS ONE 8(3): e60024. https://doi.org/10.1371/journal.pone.0060024
Editor: Gilda Tachedjian, Burnet Institute, Australia
Received: November 21, 2012; Accepted: February 23, 2013; Published: March 20, 2013
Copyright: © 2013 Chateau et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by: U19 AI082637 Combination HIV Antiretroviral Rectal Microbicide (CHARM) Program (Ian McGowan, PI), T32CA009156 and F32AI100775 (MS), AI073146 (JVG) and the UNC Center for AIDS Research P30 AI50410. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Efficacious biomedical HIV prevention interventions could dramatically reduce the number of new HIV infections globally –. Microbicides (also referred to as topical pre-exposure prophylaxis [topical PrEP]) represent one of several classes (e.g. oral PrEP, treatment-as-prevention) of such interventions currently being developed –. There are multiple reasons why microbicides are attractive as tools for HIV prevention: (i) local administration of an antiretroviral gel at the site of exposure will result in higher drug levels at the intended anatomical location than can be achieved using oral PrEP – while reducing the likelihood of experiencing systemic dosing-associated toxicities , ; (ii) the reduced toxicity associated with topical microbicides is expected to increase adherence ; (iii) microbicides are user controlled ; (iv) microbicides are predicted to be cost-effective , ; (v) topical microbicides can be developed with combinations of viral inhibitors ; (vi) an ideal microbicide would be safe and effective in both rectal and vaginal compartments –; and (vii) antiviral microbicides may also protect against viruses other than HIV (e.g. herpes simplex) , .
All microbicide efficacy clinical trials to date have tested the prevention of vaginal HIV transmission , , , –. However, an important driver of the epidemic in both men and women is HIV transmission resulting from anal intercourse – such that rectal microbicide development is also required , –. Proof of concept that administration of an antiretroviral gel rectally can prevent transmission of SIV/SHIV has been demonstrated for tenofovir  and MIV-150 . Tenofovir, UC781, and nonoxynol-9 have been tested for safety and acceptability in Phase 1 rectal microbicide clinical trials and, of these three, only tenofovir is being advanced –, , . Therefore, our in vivo preclinical efficacy study in bone marrow-liver-thymus (BLT) humanized mice was designed to determine the efficacy of topical tenofovir for the prevention of rectal HIV-1 transmission.
BLT mice are the experimental platform of choice for this study for several reasons. For example, BLT mice harbor a de novo generated human immune system distributed throughout each animal –. In the context of this study, an important characteristic of BLT mice is their susceptibility to rectal HIV-1 transmission ,  due to the presence of human CD4+ T cells, macrophages and dendritic cells found throughout BLT mouse intestines, including the rectum , . Previously both topical  and systemic ,  HIV prevention interventions have been extensively tested in BLT mice for their ability to block vaginal transmission of HIV-1. The results obtained from these studies were highly predictive of the clinical trial outcomes , , , , , .
An important and novel aspect of this study is the use of a MSM-derived transmitted/founder (T/F) virus . Typically only one or a few virions (defined as the T/F viruses) are responsible for a mucosal transmission event in humans making T/F viruses physiological relevant for in vivo efficacy studies of HIV prevention interventions , . BLT mice were treated rectally with topical 1% tenofovir and then rectally inoculated with HIV-1JRCSF, a well characterized low passage primary isolate, or the T/F virus HIV-1THRO. We found that rectal transmission of both viruses was efficiently prevented by topical tenofovir.
Materials and Methods
Preparation of BLT Mice and Characterization of Human Reconstitution
BLT mice were prepared essentially as previously described –, , . Briefly, thy/liv implanted  and preconditioned NOD/SCID-gamma chain null (NSG) mice (Jackson Laboratories, Bar Harbor, ME) were transplanted with autologous human fetal liver CD34+ cells (Advanced Bioscience Resources, Alameda, CA) and monitored for human reconstitution in peripheral blood by flow cytometry , , . Mice were maintained at the University of North Carolina at Chapel Hill Division of Laboratory Animal Medicine in accordance with protocols approved by the Institutional Animal Care and Use Committee.
Topical Application of Tenofovir and Rectal Exposure of BLT Mice to HIV-1
Stocks of HIV-1JRCSF  and HIV-1THRO  were prepared and titered as we have previously described , . Mice were exposed rectally using 0.6 µg p24 of HIV-1JRCSF (4×106 TCIU, tissue culture infectious units) and 0.7 µg p24 of HIV-1THRO (5×106 TCIU). Topical tenofovir consisted of 1% tenofovir (PMPA; 9-(2-phosphonyl-methoxypropyly)-adenine) in PBS. The vehicle (placebo) control was PBS.
The exposure timeline (Figure 1) consisted of rectal application of vehicle or of 1% tenofovir less than 30 minutes prior to rectal application of virus. Rectal exposures with HIV-1JRCSF and HIV-1THRO were performed essentially as previously described ,  except that all the mucosal exposures were carried out atraumatically and without simulated rectal intercourse . All rectal applications of vehicle or inhibitor as well as virus were performed while mice were anesthetized , . After viral exposure, mice were returned to their housing to recover and were then monitored longitudinally for evidence of HIV-1 infection as indicated below.
BLT mice were utilized to determine the efficacy of topically applied tenofovir to prevent rectal HIV-1 transmission. Rectal HIV-1 exposures were performed within 30 minutes following rectal application of 1% tenofovir. Plasma viral load and real time PCR amplification of tissue associated viral DNA were used as HIV-1 detection strategies to determine whether peripheral blood samples collected at the indicated times and tissues collected at harvest contained HIV-1.
Analysis of HIV-1 Infection of BLT Mice
Infection of BLT mice with HIV-1 was monitored at the indicated time intervals in peripheral blood by determining plasma levels of viral RNA using real time PCR (limit of detection 750 copies/ml) ,  and by monitoring CD4+ T cell percentages by flow cytometry , . At necropsy, tissues were harvested and mononuclear cells isolated as previously described , , , , . Mononuclear cells were washed, enumerated and tested using real time PCR for the presence of HIV-1 DNA (limit of detection 10 copies) , , , .
Sequence analysis was performed on plasma RNA samples in the sole case of breakthrough infection of a tenofovir-treated, HIV-1JRCSF-exposed BLT mouse. The entire reverse transcriptase gene from plasma HIV-1 RNA amplification products was sequenced. No resistance mutations in reverse transcriptase were present –.
All statistical analyses (alpha level: 0.05) were performed using Prism v. 5 (Graph Pad Software). Kaplan-Meier plots indicate the percentage of animals that are HIV-1 positive in the peripheral blood at each time point analyzed. Power analysis calculation for experimental group sample sizes were determined as previously described , . Briefly, we assumed 50 and 65% variance in transmission between our experimental groups for HIV-1JRCSF and HIV-1THRO, respectively. In the case of each viral isolate, the chosen sample sizes were determined to have 90% power to detect statistically significant differences via log rank test analysis in the treatment arm versus the vehicle arm.
Baseline Characterization of BLT Mouse Human PBMC Reconstitution
This study was designed to determine the in vivo efficacy of topical tenofovir for the prevention of rectal HIV-1 transmission. Prior to HIV-1 exposure of the BLT mice, their peripheral blood was characterized by flow cytometry to confirm reconstitution with human cells. All BLT mice used herein (n = 43) had high peripheral blood reconstitution levels of human lymphoid (CD45+) cells (67% mean ±17 SD) and human CD4+ T cells (80% mean ±6 SD) (Summarized in Tables 1 and 2).
Topical Tenofovir Prevents Rectal HIV-1JRCSF Transmission
A total of 29 mice were exposed to HIV-1JRCSF, a CCR5-tropic virus that has been well characterized for its mucosal infection of BLT mice , , , , , , . Seventeen mice received vehicle and 12 mice received topical tenofovir (Figure 2; Table 1). Following viral exposure, peripheral blood from the BLT mice was sampled weekly for the presence of HIV-1 RNA (Figure 1). Eight of the 17 mice in the control arm of the experiment were infected as determined by the presence of viral RNA in plasma (Figure 2A). In contrast, 11 of 12 topical tenofovir treated mice were consistently negative for the presence of plasma viral RNA (Figure 2A). One tenofovir treated mouse was found to have a ‘breakthrough’ infection with readily detectable plasma viral RNA (Figure 2A). No tenofovir resistant mutations from this breakthrough virus were identified when the entire reverse transcriptase gene was sequenced. Over the course of this experiment, we also monitored the levels of CD4+ T cells in peripheral blood. The breakthrough infection mouse and the infected vehicle control mice maintained similar peripheral blood CD4+ T cell levels to the HIV-1 negative mice (Figure 2B), as we have previously observed with this CCR5-tropic HIV-1 isolate in BLT mice , .
(A–B) Longitudinal analyses of peripheral blood plasma viral RNA (A) and the percentage of peripheral blood CD3+ T cells also expressing CD4 (B) are presented for vehicle (left) and topical tenofovir (right) -treated BLT mice exposed rectally to HIV-1JRCSF. (C) Real-time PCR analysis of tissues for presence or absence of HIV-1 DNA. Thin dashed lines represent the limit of detection for the respective assays. Error bars indicate standard error of the mean. Open symbols are used to depict data from HIV negative mice and closed symbols are used to depict data from HIV positive mice.
Prior to defining topical tenofovir treated BLT mice as protected from rectal HIV-1 transmission, we tested tissues harvested from these mice for the presence of cell-associated HIV-1 DNA. All mice without plasma viral RNA were also found to be negative for viral DNA in all tissues evaluated (e.g. bone marrow, spleen, human thymic organoid and lymph nodes) confirming the lack of HIV-1 transmission in these animals (Figure 2C; Table 1). The HIV status and time to plasma viremia were then combined to generate a Kaplan-Meier plot of the protection from rectal HIV transmission provided by either the vehicle or topical tenofovir (Figure 3). Log rank analysis (p = 0.03) confirmed that topical tenofovir prevents rectal HIV-1JRCSF transmission in BLT mice.
Kaplan-Meier plot indicates the time to peripheral blood conversion following rectal HIV-1JRCSF exposure in BLT mice pretreated with either vehicle or topical tenofovir. Log-rank (Mantel Cox) analysis reveals a statistically significant difference in rectal HIV-1JRCSF transmission between the vehicle and topical tenofovir arms.
Rectal Transmission of Transmitted/Founder HIV-1THRO is Prevented by Topical Tenofovir
HIV-1THRO is a CCR5-topic, MSM-derived T/F virus . A total of 14 BLT mice were exposed rectally to HIV-1THRO (Figure 4). Eight mice received vehicle and six mice received tenofovir. Five of the mice receiving vehicle were infected as determined by the presence of plasma virus RNA (Figure 4A). In contrast, none of the tenofovir treated BLT mice (0/6) exposed rectally to HIV-1THRO exhibited plasma viremia (Figure 4A). In addition to plasma viremia, we also monitored the levels of human CD4+ T cells in the peripheral blood of all the HIV-1THRO exposed mice. The levels of human CD4+ T cells in the infected mice did not change throughout the course of infection (Figure 4B).
(A–B) Longitudinal analyses of peripheral blood plasma viral RNA (A) and the percentage of peripheral blood CD3+ T cells also expressing CD4 (B) are presented for vehicle (left) and topical tenofovir (right) -treated BLT mice exposed rectally to HIV-1THRO. (C) Real-time PCR analysis of tissues for presence or absence of HIV-1 DNA. Thin dashed lines represent the limit of detection for the respective assays. Error bars indicate standard error of the mean. Open symbols are used to depict data from HIV negative mice and closed symbols are used to depict data from HIV positive mice.
To confirm the lack of HIV-1 infection of the tenofovir treated mice we used real time PCR to determine the presence of cell-associated HIV-1 DNA in tissues obtained from these mice. None of the mice treated with tenofovir had detectable levels of viral DNA in any of the tissues examined (Figure 4C; Table 2). In contrast, the presence of viral DNA in tissues from infected animals was readily confirmed (Figure 4C; Table 2). Log rank analysis of these results presented in a Kaplan-Meier plot (Figure 5) revealed that topical tenofovir administered prior to exposure to BLT mice prevents rectal transmission of the physiologically relevant T/F virus, HIV-1THRO (p = 0.02).
Kaplan-Meier plot indicates the time to peripheral blood conversion following rectal HIV-1THRO exposure in BLT mice pretreated with either vehicle or topical tenofovir. Log-rank (Mantel Cox) analysis reveals a statistically significant difference in rectal HIV-1THRO transmission between the vehicle and topical tenofovir arms.
Mucosal infection after sexual intercourse is the most common route of HIV-1 transmission worldwide which makes the cervicovaginal and rectal mucosa the two most important anatomical sites for viral exposure . Receptive anal intercourse has the highest risk of HIV-1 infection and accounts for most new infections in the US , . Nevertheless, the vast majority of past and ongoing clinical trials for HIV prevention using topical microbicides have focused on preventing vaginal HIV-1 acquisition , , , –. The formulation of tenofovir 1% gel used in the RMP-02/MTN-006 Phase 1 rectal safety study was the same formulation used vaginally in the CAPRISA 004 trial , . Unfortunately, there was a significant increase in gastrointestinal adverse events seen in the RMP-02/MTN-006 study, possibly due to the hyperosmolar nature of the gel , . We therefore elected to evaluate the efficacy of tenofovir directly, in the absence of any type of gel, to make a clear determination of the potential efficacy of tenofovir for the prevention of rectal HIV transmission. Our study supports the choice of tenofovir as an appropriate active pharmaceutical ingredient around which a specifically engineered microbicide can be designed for rectal – or dual compartment use , .
Our goal was to evaluate the in vivo efficacy of a rectal microbicide candidate for inclusion into a rectal microbicide to prevent HIV-1 acquisition. We focused on rectal HIV transmission because this route of virus spread continues to be a major contributor to the number of men and women becoming infected with HIV –. We chose a topical intervention because of the many potential benefits associated with this drug delivery route , , –. BLT mice were chosen as the experimental platform for this evaluation because previous studies have shown that FDA approved drugs prevent mucosal HIV transmission of the human primary virus isolate HIV-1JRCSF in this model , , . Here when BLT mice were pretreated with topical tenofovir (or vehicle) and then rectally exposed to HIV-1JRCSF, we found that topical tenofovir efficiently prevents rectal transmission of HIV-1JRCSF (Figures 2 and 3; Table 1).
To extend and expand on this observation we also evaluated the protective effect of tenofovir using a second virus, HIV-1THRO. HIV-1THRO is a MSM-derived T/F virus and therefore its evaluation in the context of rectal transmission is of significant relevance . T/F viruses represent the one or few founder viruses that undergo amplification in local T cells and subsequent systemic dissemination after mucosal exposure –, . These T/F viruses use CCR5 as a coreceptor for entry and replicate poorly in monocyte/macrophages relative to T cells . Despite their intrinsic relevance, T/F viruses have not been previously used for in vivo transmission studies in animal models. We found that HIV-1THRO transmits rectally in BLT mice and that its transmission can be efficiently prevented by pretreatment with rectally applied tenofovir (Figures 4 and 5; Table 2).
Analysis of the data from two HIV-1 isolates indicates that 1 of 18 BLT mice became infected despite treatment with topical 1% tenofovir prior to rectal HIV-1 exposure, while 13 of 25 vehicle treated BLT mice became infected (p = 0.002 Fisher’s exact test) (Tables 1 and 2). In an in vivo study using non-human primates (NHP), 2 of 6 macaques became infected despite treatment with topical 1% tenofovir 15 minutes prior to rectal SIV exposure, while 3 of 4 vehicle treated macaques became infected . The conclusion reached by the authors of the macaque study and our conclusion of the study presented here are the same – topical tenofovir can inhibit rectal transmission of SIV , primary HIV-1 (Figure 3) and T/F HIV-1 (Figure 5).
Topical microbicides are of significant interest in HIV prevention because they achieve high local drug concentrations capable of preventing HIV transmission with reduced risk for toxicity , , . The in vivo preclinical efficacy data presented here together with previous data from NHP  show that topical tenofovir can efficiently block rectal transmission. The incorporation of a physiologically relevant T/F HIV-1 into this study of rectal HIV prevention increases its translational value. The results presented here show the importance of animal models for the evaluation of HIV-1 prevention strategies and demonstrate the potential for efficacy of tenofovir-based rectal microbicides in humans. Future studies will leverage the results from this work and the BLT model to perform dose-ranging tenofovir studies, evaluate rectal-specific gel formulations containing tenofovir and evaluate other topical rectal microbicide agents for efficacy.
We thank P. Anton and C. Dezzutti for their critical comments regarding this manuscript. We thank Drs. I. Chen and John Kappes for providing pJRCSF and pTHRO.c/2626, respectively, via the AIDS Research and Reagent Program. We would like to thank former and current lab members and veterinary technicians at UNC Division of Laboratory Animal Medicine for their assistance with various technical aspects of this work.
Conceived and designed the experiments: MLC PWD MDS IM JVG. Performed the experiments: MLC PWD MDS. Analyzed the data: MLC PWD MDS IM JVG. Wrote the paper: MLC PWD JVG.
- 1. Cohen MS, Gay C, Kashuba AD, Blower S, Paxton L (2007) Narrative review: antiretroviral therapy to prevent the sexual transmission of HIV-1. Ann Intern Med 146: 591–601.
- 2. Abbas UL, Anderson RM, Mellors JW (2007) Potential impact of antiretroviral chemoprophylaxis on HIV-1 transmission in resource-limited settings. PLoS ONE 2: e875.
- 3. Feinberg J (2012) Truvada PrEP: Why I Voted “Yes”. Ann Intern Med.
- 4. US-FDA (2012) Truvada for PrEP Fact Sheet: Ensuring Safe and Proper Use. Silver Spring, MD: Available: http://www.fda.gov/downloads/NewsEvents/Newsroom/FactSheets/UCM312279.pdf. Accessed 2012 Nov 12.
- 5. Cutler B, Justman J (2008) Vaginal microbicides and the prevention of HIV transmission. Lancet Infect Dis 8: 685–697.
- 6. Fauci AS, Johnston MI, Dieffenbach CW, Burton DR, Hammer SM, et al. (2008) HIV vaccine research: the way forward. Science 321: 530–532.
- 7. Landovitz RJ (2007) Recent efforts in biomedical prevention of HIV. Top HIV Med 15: 99–103.
- 8. McGowan I (2010) Microbicides for HIV prevention: reality or hope? Curr Opin Infect Dis 23: 26–31.
- 9. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, et al. (2010) Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 329: 1168–1174.
- 10. Baeten JM, Donnell D, Ndase P, Mugo NR, Campbell JD, et al. (2012) Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N Engl J Med 367: 399–410.
- 11. Cohen MS, Baden LR (2012) Preexposure prophylaxis for HIV–where do we go from here? N Engl J Med 367: 459–461.
- 12. Cohen MS, Chen YQ, McCauley M, Gamble T, Hosseinipour MC, et al. (2011) Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med 365: 493–505.
- 13. Grant RM, Lama JR, Anderson PL, McMahan V, Liu AY, et al. (2010) Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med 363: 2587–2599.
- 14. Thigpen MC, Kebaabetswe PM, Paxton LA, Smith DK, Rose CE, et al. (2012) Antiretroviral preexposure prophylaxis for heterosexual HIV transmission in Botswana. N Engl J Med 367: 423–434.
- 15. Van Damme L, Corneli A, Ahmed K, Agot K, Lombaard J, et al. (2012) Preexposure prophylaxis for HIV infection among African women. N Engl J Med 367: 411–422.
- 16. Hendrix CW (2012) The clinical pharmacology of antiretrovirals for HIV prevention. Curr Opin HIV AIDS Early online publication.
- 17. Shattock RJ, Rosenberg Z (2012) Microbicides: Topical Prevention against HIV. Cold Spring Harb Perspect Med 2: a007385.
- 18. Anton PA, Saunders T, Elliott J, Khanukhova E, Dennis R, et al. (2011) First phase 1 double-blind, placebo-controlled, randomized rectal microbicide trial using UC781 gel with a novel index of ex vivo efficacy. PLoS ONE 6: e23243.
- 19. Anton PA, Cranston RD, Kashuba A, Hendrix CW, Bumpus NN, et al. (2012) RMP-02/MTN-006: A Phase 1 Rectal Safety, Acceptability, Pharmacokinetic, and Pharmacodynamic Study of Tenofovir 1% Gel Compared with Oral Tenofovir Disoproxil Fumarate. AIDS Res Hum Retroviruses 28: 1412–1421.
- 20. McGowan I (2012) Rectal microbicide development. Curr Opin HIV AIDS 7: 526–533.
- 21. Walensky RP, Park JE, Wood R, Freedberg KA, Scott CA, et al. (2012) The cost-effectiveness of pre-exposure prophylaxis for HIV infection in South African women. Clin Infect Dis 54: 1504–1513.
- 22. Williams BG, Abdool Karim SS, Karim QA, Gouws E (2011) Epidemiological impact of tenofovir gel on the HIV epidemic in South Africa. J Acquir Immune Defic Syndr 58: 207–210.
- 23. McGowan I (2009) Microbicides. In: Mayer KH, Pizer HF, editors. HIV Prevention: A Comprehensive Approach: Academic Press. 85–106.
- 24. Balzarini J, Van Damme L (2007) Microbicide drug candidates to prevent HIV infection. The Lancet 369: 787–797.
- 25. Dezzutti CS, Rohan LC, Wang L, Uranker K, Shetler C, et al. (2012) Reformulated tenofovir gel for use as a dual compartment microbicide. J Antimicrob Chemother 67: 2139–2142.
- 26. Dezzutti CS, Shetler C, Mahalingam A, Ugaonkar SR, Gwozdz G, et al. (2012) Safety and efficacy of tenofovir/IQP-0528 combination gels - A dual compartment microbicide for HIV-1 prevention. Antiviral Res 96: 221–225.
- 27. Abdool Karim Q, Abdool Karim SS (2010) Safety and effectiveness of 1% Tenofovir Vaginal Microbicide Gel in South African Women: Results of the CAPRISA 004 Trial. XVIII International AIDS Conference: Viena, Austria TUSS05.
- 28. Tan D (2012) Potential role of tenofovir vaginal gel for reduction of risk of herpes simplex virus in females. Int J Womens Health 4: 341–350.
- 29. Peterson L, Nanda K, Opoku BK, Ampofo WK, Owusu-Amoako M, et al. (2007) SAVVY (C31G) gel for prevention of HIV infection in women: a Phase 3, double-blind, randomized, placebo-controlled trial in Ghana. PLoS ONE 2: e1312.
- 30. Feldblum PJ, Adeiga A, Bakare R, Wevill S, Lendvay A, et al. (2008) SAVVY vaginal gel (C31G) for prevention of HIV infection: a randomized controlled trial in Nigeria. PLoS ONE 3: e1474.
- 31. Halpern V, Ogunsola F, Obunge O, Wang CH, Onyejepu N, et al. (2008) Effectiveness of cellulose sulfate vaginal gel for the prevention of HIV infection: results of a Phase III trial in Nigeria. PLoS ONE 3: e3784.
- 32. McCormack S, Ramjee G, Kamali A, Rees H, Crook AM, et al. (2010) PRO2000 vaginal gel for prevention of HIV-1 infection (Microbicides Development Programme 301): a phase 3, randomised, double-blind, parallel-group trial. Lancet 376: 1329–1337.
- 33. Nunn A, McCormack S, Crook AM, Pool R, Rutterford C, et al. (2009) Microbicides Development Programme: design of a phase III trial to measure the efficacy of the vaginal microbicide PRO 2000/5 for HIV prevention. Trials 10: 99.
- 34. Skoler-Karpoff S, Ramjee G, Ahmed K, Altini L, Plagianos MG, et al. (2008) Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomised, double-blind, placebo-controlled trial. Lancet 372: 1977–1987.
- 35. Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, et al. (2002) Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 360: 971–977.
- 36. Van Damme L, Govinden R, Mirembe FM, Guedou F, Solomon S, et al. (2008) Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med 359: 463–472.
- 37. Jansen IA, Geskus RB, Davidovich U, Jurriaans S, Coutinho RA, et al. (2011) Ongoing HIV-1 transmission among men who have sex with men in Amsterdam: a 25-year prospective cohort study. AIDS 25: 493–501.
- 38. Misegades L, Page-Shafer K, Halperin D, McFarland W (2001) Anal intercourse among young low-income women in California: an overlooked risk factor for HIV? AIDS 15: 534–535.
- 39. Mosher WD, Chandra A, Jones J (2005) Sexual behavior and selected health measures: men and women 15–44 years of age, United States, 2002. Adv Data: 1–55.
- 40. Gorbach PM, Manhart LE, Hess KL, Stoner BP, Martin DH, et al. (2009) Anal intercourse among young heterosexuals in three sexually transmitted disease clinics in the United States. Sex Transm Dis 36: 193–198.
- 41. Karim SS, Ramjee G (1998) Anal sex and HIV transmission in women. Am J Public Health 88: 1265–1266.
- 42. Lane T, Pettifor A, Pascoe S, Fiamma A, Rees H (2006) Heterosexual anal intercourse increases risk of HIV infection among young South African men. AIDS 20: 123–125.
- 43. Kalichman SC, Simbayi LC, Cain D, Jooste S (2009) Heterosexual anal intercourse among community and clinical settings in Cape Town, South Africa. Sex Transm Infect 85: 411–415.
- 44. Hendrix CW, Cao YJ, Fuchs EJ (2009) Topical microbicides to prevent HIV: clinical drug development challenges. Annu Rev Pharmacol Toxicol 49: 349–375.
- 45. Abner SR, Guenthner PC, Guarner J, Hancock KA, Cummins JE Jr, et al. (2005) A Human Colorectal Explant Culture to Evaluate Topical Microbicides for the Prevention of HIV Infection. J Infect Dis 192: 1545–1556.
- 46. Patterson KB, Prince HA, Kraft E, Jenkins AJ, Shaheen NJ, et al. (2011) Penetration of Tenofovir and Emtricitabine in Mucosal Tissues: Implications for Prevention of HIV-1 Transmission. Sci Transl Med 3: 112re114.
- 47. Phillips D, Zacharopoulos V (1998) Nonoxynol-9 enhances rectal infection by herpes simplex virus in mice. Contraception 57: 341–348.
- 48. Rohan LC, Moncla BJ, Kunjara Na Ayudhya RP, Cost M, Huang Y, et al. (2010) In vitro and ex vivo testing of tenofovir shows it is effective as an HIV-1 microbicide. PLoS ONE 5: e9310.
- 49. Sudol KM, Phillips DM (2004) Relative safety of sexual lubricants for rectal intercourse. Sex Transm Dis 31: 346–349.
- 50. Cranage M, Sharpe S, Herrera C, Cope A, Dennis M, et al. (2008) Prevention of SIV rectal transmission and priming of T cell responses in macaques after local pre-exposure application of tenofovir gel. PLoS Med 5: e157.
- 51. Singer R, Derby N, Rodriguez A, Kizima L, Kenney J, et al. (2011) The nonnucleoside reverse transcriptase inhibitor MIV-150 in carrageenan gel prevents rectal transmission of simian/human immunodeficiency virus infection in macaques. J Virol 85: 5504–5512.
- 52. Tabet SR, Surawicz C, Horton S, Paradise M, Coletti AS, et al. (1999) Safety and toxicity of nonoxynol-9 gel as a rectal microbicide. Sex Transm Dis 26: 564–571.
- 53. McGowan I, Hoesley C, Andrew P, Janocko L, Dai J, et al.. (2012) MTN-007: A Phase 1 Randomized, Double-blind, Placebo-controlled Rectal Safety and Acceptability Study of Tenofovir 1% Gel. 19th Conference on Retroviruses and Opportunistic Infections, Seattle, Washington Paper #34LB.
- 54. Denton PW, Nochi T, Lim A, Krisko JF, Martinez-Torres F, et al. (2012) IL-2 receptor gamma-chain molecule is critical for intestinal T-cell reconstitution in humanized mice. Mucosal Immunol 5: 555–566.
- 55. Denton PW, Olesen R, Choudhary SK, Archin NM, Wahl A, et al. (2012) Generation of HIV Latency in BLT Humanized Mice. J Virol 86: 630–634.
- 56. Denton PW, Othieno F, Martinez-Torres F, Zou W, Krisko JF, et al. (2011) One Percent Tenofovir Applied Topically to Humanized BLT Mice and Used According to the CAPRISA 004 Experimental Design Demonstrates Partial Protection from Vaginal HIV Infection, Validating the BLT Model for Evaluation of New Microbicide Candidates. J Virol 85: 7582–7593.
- 57. Wahl A, Swanson MD, Nochi T, Olesen R, Denton PW, et al. (2012) Human Breast Milk and Antiretrovirals Dramatically Reduce Oral HIV-1 Transmission in BLT Humanized Mice. PLoS Pathog 8: e1002732.
- 58. Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, et al. (2012) Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes. Retrovirology 9: 44.
- 59. Denton PW, Estes JD, Sun Z, Othieno FA, Wei BL, et al. (2008) Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice. PLoS Med 5: e16.
- 60. Denton PW, Krisko JF, Powell DA, Mathias M, Kwak YT, et al. (2010) Systemic Administration of Antiretrovirals Prior to Exposure Prevents Rectal and Intravenous HIV-1 Transmission in Humanized BLT Mice. PLoS ONE 5: e8829.
- 61. Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, et al. (2006) Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med 12: 1316–1322.
- 62. Chang H, Biswas S, Tallarico AS, Sarkis PT, Geng S, et al. (2012) Human B-cell ontogeny in humanized NOD/SCID gammac(null) mice generates a diverse yet auto/poly- and HIV-1-reactive antibody repertoire. Genes Immun 13: 399–410.
- 63. Sun Z, Denton PW, Estes JD, Othieno FA, Wei BL, et al. (2007) Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1. J Exp Med 204: 705–714.
- 64. Dudek TE, No DC, Seung E, Vrbanac VD, Fadda L, et al. (2012) Rapid Evolution of HIV-1 to Functional CD8+ T Cell Responses in Humanized BLT Mice. Sci Transl Med 4: 143ra198.
- 65. Hu Z, Yang YG (2012) Human lymphohematopoietic reconstitution and immune function in immunodeficient mice receiving cotransplantation of human thymic tissue and CD34(+) cells. Cell Mol Immunol 9: 232–236.
- 66. Jaiswal S, Pazoles P, Woda M, Shultz LD, Greiner DL, et al. (2012) Enhanced humoral and HLA-A2-restricted dengue virus-specific T-cell responses in humanized BLT NSG mice. Immunology 136: 334–343.
- 67. Kalscheuer H, Danzl N, Onoe T, Faust T, Winchester R, et al. (2012) A model for personalized in vivo analysis of human immune responsiveness. Sci Transl Med 4: 125ra130.
- 68. Kim SS, Peer D, Kumar P, Subramanya S, Wu H, et al. (2010) RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther 18: 370–376.
- 69. Kitchen SG, Levin BR, Bristol G, Rezek V, Kim S, et al. (2012) In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells. PLoS Pathog 8: e1002649.
- 70. Lan P, Tonomura N, Shimizu A, Wang S, Yang YG (2006) Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood 108: 487–492.
- 71. Long BR, Stoddart CA (2012) Alpha interferon and HIV infection cause activation of human T cells in NSG-BLT mice. J Virol 86: 3327–3336.
- 72. Ma SD, Yu X, Mertz JE, Gumperz JE, Reinheim E, et al. (2012) An Epstein-Barr virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model. J Virol 86: 7976–7987.
- 73. Marsden MD, Kovochich M, Suree N, Shimizu S, Mehta R, et al. (2012) HIV latency in the humanized BLT mouse. J Virol 86: 339–347.
- 74. Murooka TT, Deruaz M, Marangoni F, Vrbanac VD, Seung E, et al. (2012) HIV-infected T cells are migratory vehicles for viral dissemination. Nature 490: 283–287.
- 75. Wheeler LA, Trifonova R, Vrbanac V, Basar E, McKernan S, et al. (2011) Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras. J Clin Invest 121: 2401–2412.
- 76. Chateau M, Swanson MD, Garcia JV (2012) Inefficient vaginal transmission of tenofovir resistant HIV-1. J Virol: epub ahead of print.
- 77. Denton PW, Garcia JV (2012) Mucosal HIV-1 transmission and prevention strategies in BLT humanized mice. Trends Microbiol 20: 268–274.
- 78. Ochsenbauer C, Edmonds TG, Ding H, Keele BF, Decker J, et al. (2012) Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages. J Virol 86: 2715–2728.
- 79. Keele BF, Giorgi EE, Salazar-Gonzalez JF, Decker JM, Pham KT, et al. (2008) Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A 105: 7552–7557.
- 80. Salazar-Gonzalez JF, Salazar MG, Keele BF, Learn GH, Giorgi EE, et al. (2009) Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J Exp Med 206: 1273–1289.
- 81. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, et al. (1988) The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241: 1632–1639.
- 82. Koyanagi Y, Miles S, Mitsuyasu RT, Merrill JE, Vinters HV, et al. (1987) Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 236: 819–822.
- 83. Wei BL, Denton PW, O’Neill E, Luo T, Foster JL, et al. (2005) Inhibition of lysosome and proteasome function enhances human immunodeficiency virus type 1 infection. J Virol 79: 5705–5712.
- 84. Berges BK, Akkina SR, Folkvord JM, Connick E, Akkina R (2008) Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2−/− gammac −/− (RAG-hu) mice. Virology 373: 342–351.
- 85. Gu Z, Gao Q, Fang H, Salomon H, Parniak MA, et al. (1994) Identification of a mutation at codon 65 in the IKKK motif of reverse transcriptase that encodes human immunodeficiency virus resistance to 2′,3′-dideoxycytidine and 2′,3′-dideoxy-3′-thiacytidine. Antimicrob Agents Chemother 38: 275–281.
- 86. Johnson VA, Calvez V, Gunthard HF, Paredes R, Pillay D, et al. (2011) 2011 update of the drug resistance mutations in HIV-1. Top Antivir Med 19: 156–164.
- 87. Wainberg MA, Miller MD, Quan Y, Salomon H, Mulato AS, et al. (1999) In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA. Antivir Ther 4: 87–94.
- 88. White KL, Margot NA, Wrin T, Petropoulos CJ, Miller MD, et al. (2002) Molecular mechanisms of resistance to human immunodeficiency virus type 1 with reverse transcriptase mutations K65R and K65R+M184V and their effects on enzyme function and viral replication capacity. Antimicrob Agents Chemother 46: 3437–3446.
- 89. Hudgens MG, Gilbert PB (2009) Assessing vaccine effects in repeated low-dose challenge experiments. Biometrics 65: 1223–1232.
- 90. Hudgens MG, Gilbert PB, Mascola JR, Wu CD, Barouch DH, et al. (2009) Power to detect the effects of HIV vaccination in repeated low-dose challenge experiments. J Infect Dis 200: 609–613.
- 91. WHO-UNAIDS (2011) Progress report 2011: Global HIV/AIDS response. Geneva, Switzerland: Available: http://www.who.int/hiv/pub/progress_report2011/en/index.html.Accessed 2012 Nov 12.
- 92. Boily MC, Baggaley RF, Wang L, Masse B, White RG, et al. (2009) Heterosexual risk of HIV-1 infection per sexual act: systematic review and meta-analysis of observational studies. Lancet Infect Dis 9: 118–129.
- 93. US-CDC (2010) HIV/AIDS Surveillance Report Volume 22. Atlanta, GA: US-DH&HS and US-CDC. 40 p.
- 94. Haase AT (2010) Targeting early infection to prevent HIV-1 mucosal transmission. Nature 464: 217–223.