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

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

N2 – Introduction A wide variety of microbial pathogens may be transmitted sexually by the oral–anal or genital–anal routes. Inflammatory proctitis caused by an STI may increase the susceptibility and infectivity of HIV[3, 4]. This article aims to provide surgeons with a synopsis of common pathogens, their clinical presentations, diagnostic investigations and treatment regimens. This study aimed to evaluate the outcomes of these surgical procedures in Korean patients, focusing on wound healing and postoperative complications. There was no anal discharge or rectal bleeding on defaecation. 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. In fact, the mucus may be far more infectious than any other fluids either partner might be exposed to.

Copyright: © 2013 Chateau et al. For investigation of the bleeding, he underwent a rigid sigmoidoscopy and a barium enema, results of which were normal. The most common symptom was anorectal pain (57 percent), followed by lumps or warts (28 percent), rectal bleeding (12 percent), discharge (11 percent), and pruritus (6 percent), with 24 percent of patients having multiple complaints. Surgical treatment included lateral internal sphincterotomy in three patients and seton placement in one patient. Follow-up for at least four weeks was obtained in 22 patients. Microbicides (also referred to as topical pre-exposure prophylaxis [topical PrEP]) represent one of several classes (e.g. This should not be a substitute for a faecal specimen for enteric pathogens and parasites.

Cytokines play a central role in the pathogenesis of inflammatory bowel disease and recently Tumor-Necrosis Factor alpha (TNFα)4 has been identified as target molecule for immune intervention in chronic active CD (1). herpes simplex) [27], [28]. Additional symptoms may include fever, weight loss, myalgias, flatulence, urgency, and, in severe cases, melena. vaginalis infections than culture[20]. The true incidence of sexually transmitted diseases of the anorectum remains unclear given their associated stigma. All medical personnel were required to wear a surgical cap, surgical mask, sterilized scrubs (either double or waterproof), double gloves, and footwear protection; surgeons, scrub nurses and anesthesiologists were required to wear eye shields (Fig. The differential diagnosis included an idiopathic anal fissure with a complicating abscess in an HIV-positive patient, an sexually transmitted disease (STD)–associated anogenital ulcer, anorectal abscess from an acutely infected intersphincteric anal gland and anal malignancy (carcinoma, lymphoma or Kaposi sarcoma).

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 [54]–[76]. 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]. When the patient was reviewed in clinic two months later, his anal symptoms had improved considerably and the ulcer had reduced in size by 75%. 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 T/F viruses physiological relevant for in vivo efficacy studies of HIV prevention interventions [79], [80].

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. Twelfe percent of patients required subsequent proctectomy for severe proctitis, 12% developed incontinence and 20% had a recurrence (5). 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]. Enteritis is an inflammatory illness of the duodenum, jejunum, and/or ileum. All C. Lymphogranuloma venereum is caused by infections with LGV serotypes L1, L2, L3 (commonly seen in the tropics and in western populations in homosexual HIV positive men) (5, 6).

The average CD4+ T-cell count was 403/µL (range: 18-1,333/µL), with 12 patients (11/72, 16.7%) showing counts below 200/µL. They tend to present early because of the pain and usually with no constitutional symptoms. 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 [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. 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. 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]. Drainage setons rather than cutting setons should be used to resolve acute sepsis and limit recurrence and, or progression of disease. 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.

The strength of statistical association was determined by proximity to an alpha level of 0.05. Although Gram stains lack sensitivity they are highly specific and provide rapid results. A total of 20 patients presented with anal fistulas, among which 6 had concomitant condyloma acuminata. 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 [89], [90]. 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.

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). 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 [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). If a procedure is to be performed, aggressive preoperative identification of specific pathogens allows more directed antibiotic therapy.

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). Rectal symptoms (OR 2.8, 95% CI 1.0-8.0) and proctitis ( 2.4, 95% CI 0.7-8.7) were weakly associated with M. This is manifested by fever, malaise, arthralgia, weight loss, sore throat, headaches, a maculopapular rash on the trunk and extremities and condyloma lata (gray/ white wart like lesions near the primary chancres). Among these, 23.4% (22/94) were reoperations, and 3.2% (3/94) had complications including 1 case of acute urinary retention following excision of condyloma acuminata, and delayed healing in 2 fistulotomy wounds. (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.
Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice

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. The clinical presentation of pelvic anastomotic dehiscence is variable; in a significant number of cases, patients with radiologically demonstrated leaks remain asymptomatic. Log-rank (Mantel Cox) analysis reveals a statistically significant difference in rectal HIV-1JRCSF transmission between the vehicle and topical tenofovir arms. HIV-1THRO is a CCR5-topic, MSM-derived T/F virus [78]. genitalium identification did not increase the number of cases of proctitis with an identified pathogen. Incubation is 4 to 10 days. The presentation of condyloma acuminata, which results from an infection with the human papilloma virus, differs between HIV-positive and non-HIV patients.

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). The role of omentoplasty in the prevention of anastomotic leakage after colonic or rectal resection has been investigated by the French Association for Surgical Research. 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. That proportion of unknown etiology was similar to the proportion of unknown etiology in our study.

Surgeons need to manage fistulas cautiously as poor wound healing is common (13). 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]–[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]–[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]–[43]. We chose a topical intervention because of the many potential benefits associated with this drug delivery route [14], [17], [19]–[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-1JRCSF in this model [56], [59], [60]. 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). Obviously, proximal diversion is the best option whenever gross fecal contamination has occurred. HIV-1THRO 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]–[80], [94]. The small number of T. 5% imiquimod is applied nightly three times a week (and washed off after 8 hours) for up to 16 weeks. 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 [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. 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. Perianal symptoms typically develop 4 to 21 days after inoculation and are frequently preceded by systemic symptoms (fever, headache, malaise). 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. 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. 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. 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.

You may also like