Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice

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.

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 [60,63] due to the presence of human CD4 + T cells, macrophages and dendritic cells found throughout BLT mouse intestines, including the rectum [54,63]. Previously both topical [56] and systemic [59,60] 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 [9,13,56,59,60,77].
An important and novel aspect of this study is the use of a MSM-derived transmitted/founder (T/F) virus [78]. Typically only one or a few virions (defined as the T/F viruses) are responsible for a mucosal transmission event in humans making   [79,80]. BLT mice were treated rectally with topical 1% tenofovir and then rectally inoculated with HIV-1 JRCSF , a well characterized low passage primary isolate, or the T/F virus HIV-1 THRO . We found that rectal transmission of both viruses was efficiently prevented by topical tenofovir.

Preparation of BLT Mice and Characterization of Human Reconstitution
BLT mice were prepared essentially as previously described [54][55][56][57][58][59][60][61]63,76]. Briefly, thy/liv implanted [81] 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 [59,61,63]. 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.
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-1 JRCSF and HIV-1 THRO were performed essentially as previously described [60,63] except that all the mucosal exposures were carried out atraumatically and without simulated rectal intercourse [84]. All rectal applications of vehicle or inhibitor as well as virus were performed while mice were anesthetized [60,63]. 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.

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) [55,56] and by monitoring CD4 + T cell percentages by flow cytometry [59,60]. At necropsy, tissues were harvested and mononuclear cells isolated as previously described [54,56,59,61,63]. Mononuclear cells were washed, enumerated and tested using real time PCR for the presence of HIV-1 DNA (limit of detection 10 copies) [56,57,59,60].
Sequence analysis was performed on plasma RNA samples in the sole case of breakthrough infection of a tenofovir-treated, HIV-1 JRCSF -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 [85][86][87][88].   described [89,90]. Briefly, we assumed 50 and 65% variance in transmission between our experimental groups for HIV-1 JRCSF and HIV-1 THRO , 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 617 SD) and human CD4 + T cells (80% mean 66 SD) (Summarized in Tables 1 and 2).

Topical Tenofovir Prevents Rectal HIV-1 JRCSF Transmission
A total of 29 mice were exposed to HIV-1 JRCSF , a CCR5-tropic virus that has been well characterized for its mucosal infection of BLT mice [56,57,59,60,63,75,76]. 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 [59,60].
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-1 JRCSF transmission in BLT mice.

Rectal Transmission of Transmitted/Founder HIV-1 THRO is Prevented by Topical Tenofovir
HIV-1 THRO is a CCR5-topic, MSM-derived T/F virus [78]. A total of 14 BLT mice were exposed rectally to HIV-1 THRO (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-1 THRO 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-1 THRO exposed mice. The levels of human CD4 + T cells in the infected mice did not change throughout the course of infection ( Figure 4B).
To confirm the lack of HIV-1 infection of the tenofovir treated mice we used real time PCR to determine the presence of cellassociated 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-1 THRO (p = 0.02).

Discussion
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 [91]. Receptive anal intercourse has the highest risk of HIV-1 infection and accounts for most new infections in the US [92,93]. Nevertheless, the vast majority of past and ongoing clinical trials for HIV prevention using topical microbicides have focused on preventing vaginal HIV-1 acquisition [5,9,20,[29][30][31][32][33][34][35][36]. 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 [9,19]. 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 [19,20]. 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 [18][19][20] or dual compartment use [25,26].  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 [37][38][39][40][41][42][43]. We chose a topical intervention because of the many potential benefits associated with this drug delivery route [14,17,[19][20][21][22][23][24][25][26][27][28]. 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-1 JRCSF in this model [56,59,60]. Here when BLT mice were pretreated with topical tenofovir (or vehicle) and then rectally exposed to HIV-1 JRCSF , we found that topical tenofovir efficiently prevents rectal transmission of HIV-1 JRCSF (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-1 THRO . HIV-1 THRO is a MSM-derived T/F virus and therefore its evaluation in the context of rectal transmission is of significant relevance [78]. T/F viruses represent the one or few founder viruses that undergo amplification in local T cells and subsequent systemic dissemination after mucosal exposure [78][79][80]94]. These T/F viruses use CCR5 as a coreceptor for entry and replicate poorly in monocyte/macrophages relative to T cells [78]. Despite their intrinsic relevance, T/F viruses have not been previously used for in vivo transmission studies in animal models. We found that HIV-1 THRO 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 [50]. 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 [50], 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 [14,17,19]. The in vivo preclinical efficacy data presented here together with previous data from NHP [50] 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.