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Single oral dose of maraviroc does not prevent ex-vivo HIV infection of rectal mucosa in HIV-1 negative human volunteers

Coll, Josepa; Moltó, Joséa; Boix, Jaumeb; Gómez-Mora, Elisabetc; Else, Laurad; García, Elisabetc; Paredes, Rogera,c,e; Ouchi, Danc; Carrillo, Antoniof; Escrig, Rosera; Back, Davidg; Clotet, Bonaventuraa,c,e; Cabrera, Ceciliac

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doi: 10.1097/QAD.0000000000000769
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Abstract

Introduction

Daily oral antiretroviral preexposure prophylaxis (PrEP) with a fixed-dose combination of tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) or with TDF alone can prevent sexual HIV transmission [1–4]. An ‘on demand’ PrEP strategy retains high effectiveness in animal [5] and human [6] studies, improving adherence and reducing side-effects and cost.

Maraviroc (MVC) is a suitable candidate for PrEP. HIV transmission was prevented in humanized RAG-hu mice that received oral or topical MVC gel and were exposed vaginally to HIV [7,8]. Studies in macaques also showed efficacy of vaginal gels, although oral MVC was not able to prevent simian/human immunodeficiency virus (SHIV) infection in macaques, despite high and durable MVC concentrations in rectal tissues were found [9,10]. MVC is being evaluated alone or in association with FTC or TDF as a daily PreP candidate in a phase II, double-blind, randomized clinical trial in men who have sex with men (MSM) [11]; pharmacokinetic studies are being conducted in HIV-negative women [12].

Human mucosal tissue models are a safe and highly informative tool to understand the specific mechanisms of sexual HIV transmission [13–17] and develop interventions to avoid it. To evaluate the efficacy of a single oral dose of MVC to prevent the ex-vivo HIV-1 infection of rectal tissue, we used an ex-vivo challenge assay where in-vivo administration of the drug in healthy volunteers was followed by rectal tissue biopsy, and exposure of the tissue to HIV-1 in the laboratory in the presence or absence of MVC [18–20].

Materials and methods

Study design, ex-vivo challenge and ethics statement

Eight HIV-negative healthy adult MSM volunteers received a witnessed single oral dose of MVC (six volunteers received 300 mg, and two received 600 mg). Additionally, two more volunteers received TDF/FTC 300/200 mg daily for 10 days. Fecal calprotectin, HIV, hepatitis B virus, hepatitis C virus, syphilis, gonococcus and rectal Chlamydia were tested. The ethics committee from the Hospital Universitari Germans Trias i Pujol approved the study (AC-12-078). All volunteers provided written informed consent. The study was registered at ClinicalTrials.gov (NCT01719627).

Blood and rectal tissue collection

Peripheral blood mononuclear cells (PBMCs) and plasma were obtained at baseline and 4 h after MVC dosing. Each participant had two rectoscopies, separated by 7 or 10 days for healing in MVC or TDF/FTC participants, respectively. Between 12 and 20 biopsy specimens were taken by rectoscopy and transported to the laboratory within 5 min of collection.

Ex-vivo infection of rectal biopsies

Two different approaches for infection were used. For volunteers V1–V4 (without and with MVC), two biopsies were placed on gelfoam rafts and infected (R5 HIVNLAD8-D strain, 6 × 104 TCID50, kindly provided by Dr Y. Tsunetsugu-Yokota). After overnight incubation, tissue was thoroughly washed and placed on a fresh gelfoam raft and the infection was followed for 15 days [13,14,17]. For volunteers V5–V10 (without and with drug exposure), the infection was performed by immersion. Two biopsies were incubated with the same amount of the virus for 1 h in a 96-well plate and then thoroughly washed [21,22]. Assays were performed with two to four replicate wells per sample, each well containing two explants. HIV replication was assessed by measuring p24 levels by ELISA (Biorad, Spain) in the supernatants. The same viral stock was used throughout the study.

Maraviroc concentration in plasma and in rectal tissue

Two biopsies of rectal tissue after 4 h of ex-vivo challenge with MVC, and after incubation with the virus (overnight or 1 h) were snap frozen. MVC concentrations were measured by a validated liquid chromatography tandem mass spectrometry assay (MVC limit of detection 2.5 ng/ml) in plasma and in homogenized tissue. Plasma and homogenized rectal tissue samples were extracted using protein precipitation, and a stable isotope internal standard was used (MVC-d5).

CCR5 occupancy

Rectal mononuclear cells were enzymatically isolated and CCR5 occupancy was determined ex vivo using a internalization assay previously described [10,23,24]. PBMCs and rectal cells prior and after MVC exposure were incubated with medium or the chemokine CCR5 (RANTES) (1 μg/ml) during 30 min at 37°C. Cell-surface CD3, CD4 and CCR5 expression was measured by flow cytometry. Analysis was performed using FlowJo software (Tree Star, Inc., Ashland Oregon, USA). Maximum CCR5 internalization was defined in baseline samples as follows: 100-[(%CD3+CCR5+with RANTES/%CD3+CCR5+with medium) X 100] and was used as a reference to estimate the binding of MVC to CCR5.

Statistical methods

The soft endpoint (SOFT), p24 at day 15, cumulative p24 (CUM), slope (SLOPE) and area under the virus growth curve (AUC) were calculated as previously described [18,19,25,26]. A categorization infected/noninfected in SOFT, cumulative p24 and p24 at day 15 endpoints was performed by imposing the cutpoint 500, 5000 and 100 pg/ml, respectively. Measurements of p24 less than 100 pg/ml were imputed to 100 pg/ml for all analyses. The virus inhibition for each volunteer was calculated at SOFT endpoint by using the formula: [100-(SOFT after ex-vivo challenge × 100/SOFT at baseline)]. Comparisons between paired medians and correlations between continuous variables were performed by means of nonparametric methods (signed-rank test and Spearman's test, respectively). All analyses and graphical representations were done in GraphPad Prism v5.0a (GraphPad Software, Inc., San Diego, California, USA).

Results

Maraviroc concentration in plasma and rectal tissue compartments

MVC was well tolerated and rapidly absorbed, and drug concentrations were detected in plasma and in rectal tissue in all samples analyzed. The median dose-normalized MVC concentration was significantly higher in rectal tissue than in plasma (561.1 ± 282.9 and 155.1 ± 95.62 ng/ml, respectively; P = 0.0078). An association between plasma and rectal tissue MVC levels was observed, although it did not reach statistical significance (Rho = 0.57; P = 0.15).

Ex-vivo HIV-1 infection of rectal tissue

At baseline and at day 7 (4 h after MVC dosing), rectal tissue was infected with a R5 isolate and the kinetics of viral replication were assessed (Fig. 1a and b). HIV infection was observed in baseline biopsies from all volunteers and in all replicates. The ex-vivo challenge of 300 mg of MVC did not result in a consistent viral inhibition in all the participants (Fig. 1a dotted lines). A higher in-vivo intake of MVC (600 mg), although it improved viral suppression, was unable to completely block viral growth in both exposed patients (Fig. 1b red lines). The percentages of virus inhibition using the SOFT endpoint (indicated in Fig. 1 by open symbols) [25] were calculated and only in two participants (V4 and V6, MVC 300 mg) and in one (V7, MVC 600 mg) a inhibition more than 85%, previously reported as the percentage of inhibition considered to be significant, was observed [13]. On the contrary, the ex-vivo challenge of sustained TDF/FTC resulted in a large reduction (>85%) in the viral replication in both patients, compared with baseline biopsies (Fig. 1c).

Fig. 1
Fig. 1:
Kinetics of HIV-1 replication in rectal tissue biopsies.Rectal biopsies from volunteers were obtained at baseline, and 4 hrs after a single oral dose of 300 mg MVC (A) MVC 600 mg (B) or 10 days after sustained treatment with tenofovir/emtricitabine 300/200 mg qd (C). Explants were exposed to the R5 HIVNLAD8-D and culture on gelfoam rafts for 15 days with medium changes every 2 days. HIV infection was determined by p24 antigen release into culture medium. HIV replication in tissue from baseline, without drug exposure (solid lines and circles) and in tissue after MVC or TDF/FTC administration (dotted lines and squares). Data represent the mean ± SEM for the replicates (duplicates to quadruplicates) for all the individual volunteers with and without drug exposure. SOFT is shown by open symbols.

Maraviroc retention in the rectal tissue

The retention of MVC in the rectal tissue was evaluated by measuring MVC concentration before and after virus incubation (overnight or 1 h). Overnight incubation led to a severe loss of the MVC present in the tissue, from a concentration of 1007 and 1018 ng/ml in V1 and V2 just after the biopsy to levels of 13 (98.6% loss) and 67 ng/ml (93.4%), respectively. One hour incubation, in a reduced volume of medium (40 μl), also resulted in the loss of the drug, from 350, 392, 1435 and 809 ng/ml in the tissue just after the biopsy in V5–V8, to 144 (58%), 202 (48%), 392 (72%) and 164 (79%) ng/ml, respectively.

Maraviroc binding to CCR5 in mononuclear cells from blood and rectal tissue

The MVC binding to CCR5 receptor was determined ex vivo in four participants using a RANTES-dependent internalization assay [10,23,24]. The maximum CCR5 internalization in the absence of MVC in baseline samples was 27% (ranging between 23 and 28%) in PBMCs and 16.5% (between 9.3 and 24%) in cells isolated from rectal tissue. A high protection (>80%) from internalization of CCR5 receptors on the surface of the PBMCs following ex-vivo RANTES challenge was found 4 h after a MVC dose in two participants (Fig. 2). However, a partial protection was found in the remaining two participants with approximately 40% of the CCR5 receptors being unoccupied. The binding of MVC to rectal cells, determined just after the biopsy procedure, was highly variable and lower than in PBMCs in all the samples analyzed, with values ranging between 13 and 79%.

Fig. 2
Fig. 2:
Maraviroc binding to CCR5 in rectal and blood cells.Mononuclear cells from blood and rectal tissues collected at baseline and 4 h after MVC dosing were exposed to RANTES or medium. Results are expressed as percentages of MVC bound to CCR5 after MVC administration relative to cells collected at baseline. The kinetics of HIV replication in the analyzed patients are shown in order to observe the correlation between the inhibition of the replication and the level of MVC binding to CCR5. MVC, maraviroc.

Discussion

MVC is a potent CCR5 coreceptor antagonist with many desirable characteristics as a PrEP candidate: it prevents virus entry into target cells, is rapidly absorbed, achieves high concentrations in vaginal and rectal tissues, can be administered once-a-day, is safe and well tolerated, and does not select for resistance to the most frequently used antiretroviral drugs [24,27–29]. However, our study showed that, unlike 10-day TDF/FTC, MVC does not provide consistent protection against rectal HIV infection ex vivo.

We observed a rapid distribution and accumulation of MVC into rectal tissue 4 h after administering a witnessed single oral dose of MVC. This is concordant with previous publications [28,30]. Such high MVC rectal tissue concentrations, however, did not translate into significant and consistent levels of protection. Our findings are in agreement with previous work showing lack of efficacy of oral MVC to prevent rectal SHIV transmission in macaques [10].

One of the main factors that could account for this lack of protection is the existence of suboptimal drug levels in rectal tissue. The drug exposure required for preventing HIV infection at human mucosal surfaces remains unknown; our results suggest that such protective threshold, whichever it might be, is not achieved by a single oral dose of either 300 or 600 mg of MVC.

In addition, MVC was poorly retained in rectal tissue, with more than 60% drug loss after only 1 h of incubation in a minimum volume. These results are in agreement with previous data showing that MVC was easily washed out by seminal plasma or medium, whereas TDF remained in tissues likely because of retention of intracellular tenofovir diphosphate [31].

Although CCR5 occupancy in PBMCs was approximately 80%, similar to that reported in the macaque model [10] and after 5 days of MVC monotherapy in humans [24], most rectal tissue CCR5 receptors remained unoccupied. There was no correlation between MVC binding in rectal tissue and virus replication. Previous in-vitro data showed that viral replication could still be detected in the presence of MVC occupancy close to 95% [10], indicating that few unoccupied receptors are sufficient to sustain productive infection.

In conclusion, despite achieving high MVC concentrations in rectal tissue 4 h after dosing, a single oral dose of MVC (either 300 or 600 mg) does not prevent ex-vivo R5 HIV infection of human rectal mucosa. The lack of prophylactic efficacy observed in our study suggests that a single oral dose of MVC would not prevent rectal HIV transmission in vivo, discouraging this approach as an ‘on demand’ preexposure prophylaxis strategy.

Acknowledgements

The authors would like to thank the participants and also Anna Chamorro and Isabel Bravo for their help with blood sample collection and Nuria Pérez-Alvárez for her help with the statistical analysis. Thanks to Michael Meulbroek, Ferran Pujol and Jorge Saz from BCN Checkpoint for their collaboration in the recruitment and follow-up of the participants.

Authors’ contributions: J.C., J.M., R.P., B.C and C.C designed the study and developed the hypotheses. J.C. recruited and provided clinical care for the participants. J.B. performed the rectoscopies.

E.Go. and E.Ga. performed the ex-vivo assay and the MVC binding assay. L.E. and D.B. performed the measurements of MVC. D.O. performed the statistical analysis. R.E. performed regulatory activities, clinical monitoring and database. E.Go., E.Ga. and C.C. analyzed and interpreted the data. C.C. drafted the article. All authors contributed to the article's preparation and approved its final version.

Source of funding: This work was supported by the PI12/02408 project (Proyectos de Investigación en Salud. Ministerio de Economia y Competitividad). C.C. is a researcher from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the ISCIII and the Health Department of the Catalan Government (Generalitat de Catalunya).

Conflicts of interest

J.B., E.Go., L.E., E.Ga., D.O., A.C., R.E. and C.C. report no disclosures. B.C., D.B., J.M. and R.P have served as a consultant to and/or have received research grant support from Gilead, Janssen, MSD, ViiV, BMS. D.B. and R.P. have served as a consultant to Abbie. J.C. has received a grant from Gilead.

This work has been presented at the Conference on Retroviruses and Opportunistic Infections; 23-26 February 2015.

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Keywords:

‘on demand’ preexposure prophylaxis; ex-vivo challenge; ex-vivo tissue culture; maraviroc; single oral dose

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