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Maraviroc and reverse transcriptase inhibitors combinations as potential preexposure prophylaxis candidates

Herrera, Carolina; Armanasco, Naomi; García-Pérez, Javier; Ziprin, Paul; Olejniczak, Natalia; Alcamí, José; Nuttall, Jeremy; Shattock, Robin J.

doi: 10.1097/QAD.0000000000001043
Basic Science

Objective: Receptive anal intercourse in both men and women is associated with the highest probability for sexual acquisition of HIV infection. As part of a program to develop an effective prevention strategy, we performed an ex-vivo preclinical evaluation to determine the efficacy of multiple double combinations of maraviroc (MVC) and reverse transcriptase inhibitors (RTIs).

Design: The entry inhibitor, MVC, a nucleotide RTI, tenofovir and two non-nucleoside RTIs, UC781 and TMC120 (dapivirine, DPV), were used in double, combinations against a panel of CCR5-using clade B and clade C HIV-1 isolates and against MVC-escape variants. A gel-formulated version of MVC-DPV combination was also tested.

Methods: Indicator cells, cocultures of immature dendritic cells with CD4+T cells, and colorectal tissue explants were used to assess antiviral activity of drug combinations.

Results: All dual MVC-RTI combinations tested inhibited MVC-sensitive and resistant isolates in cellular and colorectal explants models. All the combinations were positive with no reduction in the activity of MVC. In tissue explants, the combinations against all viral isolates tested produced an increase in the activity of MVC. An initial gel-formulation of MVC-DPV combination showed greater and prolonged antiviral activity of MVC in mucosal tissue explants.

Conclusion: This study demonstrates that combinations based on antiretroviral drugs inhibiting HIV transmission at viral entry and reverse transcription have potential as prevention strategies against colorectal transmission of HIV-1 including MVC-resistant isolates. Preclinical evaluation with colorectal tissue explants indicates that a gel-formulation of MVC-DPV is an effective candidate colorectal microbicide.

Supplemental Digital Content is available in the text

aDivision of Cellular and Molecular Medicine, Centre for Infection, St George's University of London

bSection of Infectious Diseases, Faculty of Medicine, St Mary's Campus, Imperial College, UK

cAIDS Immunopathology Unit, National Center of Microbiology, Instituto de Salud Carlos III, Madrid, Spain

dDepartment of Surgery and Cancer, St Mary's Hospital, Imperial College London, UK

eInternational Partnership for Microbicides, Silver Spring, Maryland, USA.

*Both authors contributed equally.

Correspondence to Carolina Herrera, PhD, Section of Infectious Diseases. Faculty of Medicine, Room 460/453 Wright-Fleming Institute, St Mary's Campus, Imperial College. Norfolk Place, London W2 1PG, UK. Tel: +44 0 207 594 2545/5206; fax: +44 0 207 594 2538; e-mail:

Received 28 October, 2015

Revised 24 December, 2015

Accepted 18 January, 2016

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (

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Multiple drug combinations are used in conventional HIV-1 treatment, known as highly active antiretroviral therapy (HAART) [1]. Combinations may also be more effective than single drug formulations as prevention strategies against HIV-1 sexual transmission, including transmission of resistant isolates, which are increasingly prevalent [2,3]. Receptive anal intercourse between serodiscordant couples in both men and women is associated with the highest probability of sexual HIV transmission [4–7] partly because of the abundance of highly activated CCR5+ cells in the colorectal mucosa [8–10]. A limited number of topical prevention strategies, referred to as microbicides, against colorectal transmission have been tested in clinical trials. Furthermore, all completed phase I [11–15] and on-going phase II trials [16] are based on a single antiretroviral drug, specifically an reverse transcriptase inhibitor (RTI).

The majority of HAART regimes combine antiretroviral drugs targeting different steps of the viral replication cycle. Taking into account the predominant transmission of R5-tropic isolates compared with X4-viruses during sexual intercourse [17,18], a good candidate for a combination with an RTI is a CCR5 small molecule inhibitor. Maraviroc (MVC) is the first small-molecule CCR5 inhibitor to have been included in HAART. Formulated for topical application, MVC has shown promising pharmacological results in humans and non-human primates [19,20] and efficacy in non-human primates when tested as a vaginal gel microbicide [21,22]. It has also been formulated as a vaginal ring in combination with an RTI [dapivirine (DPV)] and tested in a phase I clinical trial (IPM-026/MTN013) [23]. The DPV/MVC rings were safe and well tolerated; however, very low levels of MVC were detected in tissue and, therefore, MVC did not block ex vivo challenge of vaginal biopsies.

We have investigated the inhibitory activity of dual combinations of MVC with an RTI, either a nucleotide RTI tenofovir (TFV) or non-nucleoside RTIs, UC-781 or TMC120 (DPV); as potential candidates for prevention of colorectal transmission, including topical prevention, with MVC and DPV gel-formulated as a rectal microbicide candidate. The antiviral potency of these compounds alone and in combination was evaluated against chronic or transmitted/founder R5-isolates and against MVC-resistant clones using cellular and colorectal tissue explant models.

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Materials and methods

Reagents and plasmids

Base compounds 9-[R-2-(phosphonylmethoxy)propyl] adenine monohydrate (PMPA, or TFV) was donated by Gilead Sciences, Inc. (Foster City, California, USA), UC781 was donated by Biosyn, Inc. (Huntingdon Valley, Pennsylvania, USA), MVC (UK-427,857) and DPV (TMC120) were provided by the International Partnership for Microbicides (IPM) (Silver Spring, Maryland, USA) and by Janssen ID & V (Beerse, Belgium).

MVC 0.10% gel, DPV 0.05% gel, combination MVC 0.10%-DPV 0.05% gel and placebo gel were manufactured by Particle Sciences (Bethlehem, Pennsylvania, USA) for IPM as hypo-osmolar gels (<100 mOsm/kg).

HIV-1 BaL [24] was provided by the NIH AIDS Research & Reference Reagent Program ( Full-length, replication and infection-competent proviral HIV-1 clone, pYU.2 [25,26] was provided by the NIH AIDS Research & Reference Reagent Program ( Transmitted founder clade C isolates, CH042, CH198, CH067, CH162 and CH164 were kindly provided by C. Ochsenbauer and J. Kappes at University of Alabama (Birmingham, Alabama, USA) [27]. The sequences encoding the MVC-sensitive (MVC-Sens), MVC-resistant (MVC-Res) and MVC-Sens with the V3 loop of MVC-Res [MVC-Sens (V3R)] Envs have been previously reported [28]. MVC-Sens and MVC-Res sequences come from the dominant circulating viruses isolated from a patient of the MOTIVATE study before and after MVC therapy, respectively [29]. The V3 loop of MVC-Res Env contains two changes (P308S and G310_P311InsAla) that confer high MVC resistance [28,30,31]. Chimeric viruses carrying the full-length envelope from MVC-Sens, MVC-Res and MVC-Sens (V3R) were generated as previously described [28]. Briefly, the three gp160 described above were digested with KspI and NotI from parental constructions and cloned into the pNL-KspI/Env/NotI vector derived from the HIV-1 proviral clone pNL4–3 to produce replication-competent viruses. The KspI and NotI restriction sites were introduced at the nucleotide positions 6214 and 8796 respectively in pNL4–3 as previously described [28]. DNA sequences of the cloned full-length Envs were confirmed by sequencing.

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Cell and virus culture conditions

All cell cultures were maintained at 37°C in an atmosphere containing 5% CO2. TZM-bl cells [32–34] were grown in Dulbecco's minimal essential medium (Sigma-Aldrich, Inc., St. Louis, Missouri, USA) containing 10% fetal calf serum (FCS), 2 mmol/l L-glutamine and antibiotics (100 U of penicillin/ml, 100 μg of streptomycin/ml). PM-1 cells [35] (AIDS reagent project, National Institute for Biological Standards and Control, United Kingdom) were maintained in RPMI 1640 medium containing 10% FBS, 2 mmol/l L-glutamine and antibiotics (100 U of penicillin/ml and 100 μg of streptomycin/ml). PBMCs were isolated from multidonor buffy coats from healthy HIV-seronegative donors, by centrifugation onto Ficoll-Hypaque, mitogen stimulated as previously described [36], and maintained in RPMI 1640 medium containing 10% FCS, 2 mmol/l L-glutamine, antibiotics (100 U of penicillin/ml, 100 μg of streptomycin/ml), and 100 U of interleukin-2/ml. Immature dendritic cells (iDCs) were grown from PBMC-derived monocytes cultured for 6 days in complete RPMI medium supplemented with 1000 U/ml GM-CSF and 500 U/ml interleukin-4 (R&D Systems, Minneapolis, Minnesota, USA). Monocytes were isolated from PBMCs by autoMACS human CD14 Microbeads (Miltenyi Biotec, United Kingdom) following manufacturer's instructions. iDCs were phenotypically characterized by staining with anti-CD40, anti-CD80, anti-CD86, anti-CD83, anti-CD209, anti-CD123 and anti-CD11c (BD Pharmingen, United Kingdom). Fluorescence-activated cell sorting (FACS) analysis was performed with a BD FACSCanto II flow cytometry system using BD FACSDiva analysis software.

The laboratory-adapted isolates HIV-1 BaL and YU.2 were passaged through activated PBMCs for 11 days.

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Patients and tissue explants

Surgically resected specimens of colorectal tissue were collected at St George's Hospital, London and St Mary's Hospital, Imperial College London, UK. All tissues were collected after receiving signed informed consent from all patients and under protocols approved by the Local Research Ethics Committee. All patients were HIV negative. On arrival in the laboratory, resected tissue was cut into 2–3 mm3 explants comprising both epithelial and muscularis mucosae as described previously [37]. Colorectal explants were maintained with Dulbecco's minimal essential medium containing 10% FCS, 2 mmol/l L-glutamine and antibiotics (100 U of penicillin/ml, 100 μg of streptomycin/ml and 80 μg of gentamicin/ml) at 37°C in an atmosphere containing 5% CO2.

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Infectivity and inhibition assays

The infectivity of virus preparations was estimated in TZM-bl cells (by luciferase quantitation of cell lysates, Promega, Madison, Wisconsin, USA) and PBMCs (by measure of p24 antigen content in cell culture supernatant). Briefly, TZM-bl cells were seeded at 3 × 103 cells/well 24 h prior to infection with HIV isolates. After incubation for 2 days, the cells were washed with PBS and lysed with 100 μl of luciferase cell culture lysis reagent [38]. Fifty microliters were transferred to a white, opaque assay plate for luciferase quantification in a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc., Burlington, Vermont, USA), using 50 μl of luciferase assay reagent. The extent of luciferase expression was recorded in relative light units. p24 content in supernatant was measured with HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (AIDS Vaccine Program, National Cancer Institute, Frederick, Maryland, USA) following manufacturer's instructions.

All inhibition assays in cellular and tissue explants models were performed using a standardized amount of virus culture supernatant normalized for infectivity. Cells were incubated with serial dilutions of drugs for 1 h at 37°C, and then virus (103.3 TCID50) was added to cells and left for the time of the experiment. iDCs were exposed to virus for 2 h, then washed three times with phosphate-buffered saline (PBS) to remove unbound virus. Infected iDCs were then cocultured with PM-1 cells at a 1 : 2 ratio of infected cells : PM-1 cells (equivalent to 1 × 104 infected iDCs:2 × 104 PM-1 cells) in the presence or absence of drugs alone or in combination. Cultures were maintained for 14 days, with 50% media feeds every 2–3 days. Drugs were kept in the coculture for 2 h, 24 h or continuously during the 14 days of culture. HIV-1 infection was determined by measurement of p24 levels in culture supernatants by ELISA (HIV-1 p24 ELISA, AIDS Vaccine Program, National Cancer Institute, Frederick, Maryland, USA). Alternatively, tissue explants were incubated with drug for 1 h before virus (103 TCID50) was added for 2 h. Explants were then washed four times with PBS to remove unbound virus and drug. Colorectal explants were then transferred onto gelfoam rafts (Welbeck Pharmaceuticals, UK) and cultured for 15 days as previously described [39] in the presence or absence of drug. Explants were cultured for up to 15 days in the presence or absence of drug, and approximately 50% of the supernatants were harvested every 2–3 days and explants were refed with fresh media. The extent of virus replication in tissue explants was determined by measuring the p24 antigen concentration in supernatants (HIV-1 p24 ELISA, AIDS Vaccine Program, National Cancer Institute, Frederick, Maryland, USA).

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Statistical and mathematical analysis

IC50 values were calculated from sigmoid curve fitted (Prism, GraphPad Software, La Jolla, California, USA) fulfilling the criterion of R 2 > 0.7.

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Double combinations of maraviroc and reverse transcriptase inhibitors are more active than individual drugs in TZM-bl cells

To evaluate the potential of MVC, PMPA, UC781 and DPV as part of a microbicide based on entry and RT inhibitors combination, we first tested the inhibitory activity of each compound alone against a panel of clade B R5 isolates in order to design combinations including concentrations of drugs based on a constant ratio of the average IC50 for each individual antiretroviral against wild-type chronic clade B isolates included in the mixture as previously described [37]. The average IC50 of PMPA (ranging from 2.3 to 6.7 μmol/l) was approximately 352 fold higher than that of MVC (ranging from 2.35 to 6.99 nmol/l); hence, to set up the dual combination, PMPA and MVC were combined at a ratio of 352 : 1. Similarly, MVC combinations with UC781 (IC50 between 5.86 and 10.21 nmol/l) and DPV (IC50 ranging from 0.15 to 0.84 nmol/l) were titrated at set ratios of approximately 1 : 1 and 1 : 12, respectively. When establishing a ratio of the IC50 for each compound alone vs. in combination, all combinations resulted in an increase of activity (examples in Supplementary Fig. 1, with a decrease of IC50 for each compound included in the combination (Table 1). For all three MVC-RTI combinations tested, the level of reduction of IC50 values for MVC and RTIs was similar, with ratios IC50 drug A alone/IC50 drug A combined of 2.36 ± 0.43 for MVC and 3.84 ± 1.90 for the three RTIs in average.

Table 1

Table 1

Transmitted/founder (T/F) isolates have been shown to have different CCR5 utilization than chronic viruses in the presence of MVC [40,41]. We then tested the activity of MVC and combinations with the three RTIs against a panel of T/F clade C isolates. The IC50 of the T/Fs was in general a log lower (0.52 ± 0.01 nmol/l for CH042; 0.66 ± 0.26 nmol/l for CH198; 0.21 ± 0.04 nmol/l for CH067 and 0.62 ± 0.08 nmol/l for CH164) than the average IC50 for chronic virus (4.07 ± 1.67 nmol/l); except for one T/F isolate, CH162, whose IC50 was in the range of the chronic viruses (3.27 ± 0.55 nmol/l). Despite the difference in sensitivity to MVC between CH162 and the other T/F isolates, no apparent differences were observed on the level of increase of drug activity when MVC was combined with any of the three RTIs among all the T/Fs. Interestingly, the increase of inhibitory activity for MVC and the RTIs was different between laboratory adapted clade B viruses and clade C T/F isolates. Against clade C transmitted founder isolates, the inhibitory activity of MVC was only slightly increased, with the IC50 for MVC 1.34 ± 0.43 times lower in average when used in combination; however, a greater decrease of IC50 values for the three RTIs was measured when used in combination with MVC against clade C isolates (15.67 ± 13.84 times for TFV, 33.14 ± 36.83 for UC781 and 13.40 ± 10.03 for DPV) (Table 1).

The potential success of any microbicide may be dependent not only on its activity against wild type isolates, but also against possible resistant isolates. A range of mutations can emerge conferring resistance to MVC and prevalence may increase in populations with the wider use of MVC in therapy. We studied the activity of MVC and its combinations with the three RTIs against clonal viruses containing the MVC-resistant Env derived from a patient who developed resistance (MVC-Res) in comparison with the Env derived from the same patient prior to initiation of treatment including MVC (MVC-Sens) [30,31]. We also used a third MVC-resistant chimeric construct from the same individual containing the V3 loop of the MVC-sensitive Env [MVC-Sens (V3R)] [30,31]. In TZM-bl cells, an IC50 was not reached against MVC-Res within the range of MVC concentrations tested (IC50 >> 142 nmol/l); however, with the chimeric construct MVC-Sens (V3R), MVC reached a plateau of 50% of inhibition at around 9 nmol/l in contrast with the MVC-sensitive isolate, against which a dose-response curve was measured for MVC with average IC50 of 0.34 nmol/l and IC95 of 10.02 nmol/l. The different level of resistance to MVC of MVC-Res and MVC-Sens (V3R) reflects the fact that mutations outside the V3 loop further contribute, although to a lesser extent, to the resistance profile. Interestingly, the IC50 of MVC-Sens was in the sub-nmol/l range similar to the T/F isolates. The three RTI-MVC double combinations were then titrated against the MVC-sensitive and resistant isolates. The three isolates were fully sensitive to the RTIs, therefore combinations of MVC with TFV, UC-781 or DPV were able to inhibit infection in TZM-bl cells with all the three isolates tested (Table 1). However, the dose-response curve of the MVC-DPV combination was affected when titrated against MVC-Res, showing a change in slope that resulted in an increase of IC50 for DPV when used in combination with MVC. This resulted in a ratio of IC50s of 0.6 ± 0.74 nmol/l (Table 1), without affecting the maximum level of inhibition reached by DPV alone or in combination at the highest concentration tested (12 nmol/l) (data not shown).

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Combinations are active against trans-infection between immature dendritic cells and T cells

The inhibitory potency of MVC-RTI combinations were further evaluated in a cellular model of coculture of PM-1 CD4+ T cells with infected iDC, mimicking the potential cell-associated transmission of HIV-1 from virus-exposed iDC to uninfected CD4+ T cells that occurs during the local expansion of infection following establishment of the initial foci of infection in mucosal tissues [42,43]. Initial studies exposing the coculture to MVC in a range of concentrations between 10 and 0.0001 μmol/l, for a 2 h pulse did not result in a robust inhibition of trans-infection (data not shown), indicating that the effective level of MVC required to block local mucosal expansion should be higher. Hence, we tested longer drug exposure times mimicking repeated dosing (24 h pulse) or sustained release (continuous exposure). The three RTIs and MVC were able to inhibit iDC-facilitated infection of PM-1 cells when added during a pulse of 24 h or kept continuously during the 14 days of coculture (as shown in Table 2 and Supplementary Fig. 2, ). As expected, the plateau of maximum inhibition was reached at lower concentrations with sustained exposure to compounds. The inhibitory activity of the drugs was increased when titrated in MVC-RTI double combinations (Table 2 and Supplementary Fig. 2, with a decrease of the IC50 for at least one of the compounds included in all the MVC-RTI combinations tested.

Table 2

Table 2

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Inhibitory activity of maraviroc-reverse transcriptase inhibitors dual combination in tissue explants

Based on the results obtained with TZM-bl cells and iDC-PM-1 cocultures, the double combinations were also titrated against clade B HIV-1 BaL in colorectal tissue explants to assess the potential of such combinations as colorectal microbicides. In colorectal explants we have previously described that MVC reaches the maximum level of inhibition earlier than RTIs, where after 11 days of culture the % of inhibition measured for MVC at the highest concentration tested, 1 μmol/l, decreased from ∼ 85% to less than 80% at day 15 (Fletcher et al., submitted). Hence, when assessing the effect of dual combinations on antiviral activity we compared days 11 and 15 of culture. A positive shift (to the left) in the dose-response curve for all mixed compounds was seen at both days (Fig. 1) with a reduction of the IC50 of each drug when used in combination. For all combinations tested, the IC50 values of all drugs combined showed a similar reduction at day 11 and 15 (Table 3). This is probably because of the slight loss of inhibitory potency between day 11 and day 15 observed for MVC. With the MVC-UC781 combination, both drugs had their activity increased to similar proportions, however, when MVC was combined with TFV or DPV, the % of reduction in the IC50 was greater for MVC than for the two RTIs. This reflects a higher contribution by TFV and DPV to the activity of the dual combinations.

Fig. 1

Fig. 1

Table 3

Table 3

Based on the encouraging results obtained with the screening in TZM-bl against the MVC-resistant isolates [MVC-Res and MVC-Sens (V3R)], we tested the three MVC-RTI double combinations against MVC-Res in colorectal tissue explants. As expected, the maximum level of inhibition reached at the highest concentration tested for MVC against this isolate was significantly reduced, but an IC50 could be calculated within the range of concentration tested (as shown in Supplementary Fig. 3, As in TZM-bl cells, MVC-Res was sensitive to the three RTIs and the dual combinations with MVC resulted in an increase of antiviral potency. The ratios of IC50s for the three RTIs were similar to those observed when the drugs alone and in combination were titrated against the MVC-sensitive virus (Table 3). However, the reduction in IC50 for MVC against the MVC-resistant isolate was, as predicted, greater than against wild-type virus, resulting in higher ratios of IC50s (Table 3) and reflecting, once more, the activity of the more potent drug in the combination, in this case the RTIs (either TFV, UC-781 or DPV).

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Evaluation of a gel-formulated combination of maraviroc and dapivirine

To optimize formulation and taking into account the greater inhibitory potency of DPV, the MVC-DPV combination gel was prepared with MVC at 0.10% and DPV at 0.05%, and not at an equipotent ratio (based on the IC50 values of each drug). Hence, the activity of the gels for each individual drug or the combination was first evaluated in TZM-bl cells against a panel of clade B, clade C T/F and MVC-resistant isolates. The placebo gel had no inhibitory activity per se (data not shown) and importantly, the gel-formulated MVC and DPV alone and in combination showed no cytotoxic effect by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide viability assay (data not shown). The DPV gel and the MVC gel were active against all isolates tested, although, in general, there was an increase of the IC50s of the DPV gel against all isolates tested compared with the base compound. The effect of the combination on the activity of DPV was positive against all isolates tested with an average reduction of IC50 against the clade B MVC-sensitive viruses of 10.56 ± 6.59 times, of 2.30 ± 1.32 times for the MVC-resistant clade B isolates and of 18.13 ± 12.20 times for clade C isolates (Table 1). MVC was also more potent against MVC-sensitive clade B viruses when tested in the combination gel, with IC50 values on average 1.88 ± 0.93 times lower than when titrated alone. An increase in activity was also seen for MVC in combination against the clade C isolates CH162 and CH164; however, a slight increase in the IC50 values for MVC was seen against CH042, CH198 and CH067 resulting in ratio of IC50s < 1 (Table 1). This increase had no effect on the maximum level of inhibition reached by the combination gel in comparison with the MVC gel at the highest concentration tested (142 nmol/l) (data not shown) and therefore reflected a slight change in the slope of the dose-response curve. The activity of the gel-formulated drugs was also assessed against the two MVC-resistant isolates. DPV gel was active against MVC-Res and MVC-Sens (V3R); however, an IC50 could not be calculated for MVC gel even at the highest concentration tested. The combination gel was active against both isolates with an increase in activity reflected in ratios of IC50s > 1 for both drugs (Table 1).

The gels were next tested in the mucosal tissue explant model. In colorectal explants, formulated MVC was more potent, reaching higher levels of inhibition, than base compound against the clade B virus BaL. Furthermore, the MVC-DPV gel combination was fully inhibitory at days 11 and 15 (Fig. 2). Both drugs were more potent in the combinatorial gel with a similar reduction in the IC50 values for MVC (IC50 4.91 ± 0.66 times lower in combination than alone, as average of day 11 and 15) and for DPV (IC50 values 2.61 ± 0.50 times lower in average at both time points) (Table 3). The activity of the MVC gel against the MVC-resistant isolate MVC-Res reached a plateau at around 50% of inhibition at the highest concentration tested, but the MVC-DPV combination gel was able to inhibit this isolate with a decrease in the value of IC50 for MVC and for DPV, resulting in ratios of IC50s > 1 for both drugs (Table 3).

Fig. 2

Fig. 2

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To date, the majority of microbicide trials have tested the safety and/or efficacy of single antiretroviral drugs, and in the specific case of colorectal microbicides, all the trials have assessed an RTI [44]. Only a phase I vaginal microbicide trial has assessed the pharmacokinetics and safety of a combination of MVC with DPV formulated as a vaginal ring [23]. Previous preclinical studies have shown that topical use of antiretroviral combinations could be effective against HIV-1 transmission during receptive anal intercourse [37,45]. In this study, we have evaluated, side by side, the activity of MVC and three RTIs (TFV, UC781 and DPV) alone or in combination in two cellular models and in colorectal explants. The order of potency of the four antiretroviral drugs was the same in all models; however, the IC50 of MVC was affected by the level of expression of CCR5 on the cell surface of each model and by the ability of the virus to recognize MVC-bound CCR5. Indeed, the average IC50 of MVC against chronic R5-clade B isolates in TZM-bl was greater (4.07 ± 1.67 nmol/l) (Table 1) than that previously described in the literature when tested in PBMCs (1.2 nmol/l) [46]. This is because of the reported high levels of CCR5 in TZM-bl cells, which with stable transfection, express >2 logs more CCR5 than PBMC [47]. The differences of IC50s for MVC in TZM-bl cells observed between T/F and laboratory adapted chronic isolates (Table 1) have also previously been described in two consistent studies were T/F isolates were more sensitive to MVC than chronic viruses on cells expressing high levels of CCR5 [40,41]. This is because of the capacity of R5-chronic isolates to infect cells despite MVC being bound to CCR5. Among the T/F isolates tested one of them, CH162, had an IC50 (3.27 ± 0.55 nmol/l) in the range measured for chronic isolates (average IC50 of 4.07 ± 1.67 nmol/l). The V3 loop of CH162 has greatest homology with BaL among the T/F tested [27].

To assess the combinatorial activity (synergy/additivity/antagonism) of drugs, it was not possible to use the equation of Chou-Talalay [48] included in the analysis software Calcusyn. To apply this equation correctly, the slopes of all the titration curves compared must be parallel and the activity of the drug must cover the full range between 0 and 100% of inhibition. However, donor-to-donor variation of the explant model, assessment of antiretroviral drugs with different mechanisms of action and use of RTI-resistant isolates makes this impossible to achieve. Hence, to provide a quantitative indication of the potential increase in activity we chose a similar concept to the ‘dose reduction’ [49] and calculated for each drug the ratio of IC50 of drug alone vs. IC50 of drug in combination with another drug. Although this does not provide a numerical indication (combination index) of the combinatorial effects, it does allow classification of combinations as being ‘positive or negative combinations’.

In addition to determining the efficacy of individual antiretroviral drugs and MVC-RTI dual combinations against wild type isolates, assessment of their activity against resistant strains is of critical importance. Different sets of mutations have been associated with resistance to MVC [31,50], in this study, we chose a MVC-resistant isolate obtained from a subject who commenced HAART including MVC in a phase III trial and experienced virologic failure because of resistance to MVC [31]. The MVC-resistant and sensitive Envs were isolated to obtain two viral clones, MVC-Res and MVC-Sens, respectively. A third clone was prepared by replacing the V3 loop of MVC-Sens with the one of MVC-Res. All three dual MVC-RTI combinations restored the activity against MVC-Res and MVC-Sens (V3R). Interestingly, the dose-response curve of the MVC-DPV combination in TZM-bl cells was affected when titrated against MVC-Res, showing a change in slope that resulted in an increase in IC50 for DPV in the combination (Table 1). Importantly, the increase in IC50 did not affect the maximum level of inhibition attained, reflecting a change of slope in the dose–response curve. This highlights the importance of other parameters in addition to the IC50 when evaluating the inhibitory profile of an antiretroviral.

A first hypo-osmolar gel-formulated version of MVC-DPV was tested in this preclinical study showing greater inhibitory activity than the gels containing MVC or DPV alone against all isolates. In colorectal explants, two important effects were observed with the MVC-gel formulation. A higher level of inhibition was obtained for MVC and the activity was maintained between days 11 and 15 with the formulated drug (Fig. 2). This indicates that formulation of MVC as a gel for topical application could promote the inhibitory potency of MVC in the mucosal environment. In addition, and more importantly, this combination was able to fully inhibit MVC-resistant isolates in all the models tested.

The results with resistant-isolates demonstrate the importance of considering combinations of compounds with different inhibitory mechanisms and/or targeting different steps of the viral replication cycle in the design of microbicides. Furthermore, an initial gel-formulation of MVC-DPV shows encouraging results for further development of this combination as a colorectal microbicide. This study further validates the use of multiple preclinical models including mucosal tissue explants tailored to the antiretroviral drugs considered and that reflect the multiple aspects affecting the potential in vivo efficacy of the candidate antiretroviral-based microbicide. The results obtained with these chosen efficacy models (including IC50, slope of the dose-response curve and maximum percentage of inhibition) will have to be correlated with safety and pharmacokinetic studies in humans to better predict the in vivo potential of any colorectal microbicide candidate.

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We thank Mr Haggar, Mr Melville and the Colorectal Surgery Team, St. George's Hospital, London, for their assistance in obtaining human colorectal tissue. We thank the International Partnership for Microbicides (IPM) for donation of Maraviroc base compound and gel-formulated Maraviroc and DPV. This work was funded by IPM, as well as the European Commission 7th Framework ‘Combined Highly Active Anti-Retroviral Microbicides’ (CHAARM) program.

Authors’ contribution: the project was conceptualized and supervised by C.H., J.N. and R.J.S. The study was designed by C.H. as well as the data analysed and interpreted. The resistant isolates were prepared by J.G.P and J.A. Human gastrointestinal surgeries were performed by P.Z. The experiments were performed by C.H., N.A. and N.O.

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Conflicts of interest

There are no conflicts of interest.

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antiretrovirals; combination; explant; mucosal; prevention

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