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Antimicrobial Agents and Chemotherapy, July 2008, p. 2544-2554, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01627-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Immunodeficiency Virus Type 1 (HIV-1) Integration: a Potential Target for Microbicides To Prevent Cell-Free or Cell-Associated HIV-1 Infection {triangledown}

Katty Terrazas-Aranda,1,2* Yven Van Herrewege,1 Daria Hazuda,3 Paul Lewi,4 Roberta Costi,5 Roberto Di Santo,5 Andrea Cara,6 and Guido Vanham1,7

Virology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium,1 Virology Laboratory, SELADIS Institute, Faculty of Pharmaceutical and Biochemical Sciences, Universidad Mayor de San Andrés, La Paz, Bolivia,2 Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania,3 University Hospital, Catholic University of Leuven (KUL), Leuven, Belgium,4 Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Università di Roma La Sapienza, Rome, Italy,5 Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy,6 Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium7

Received 19 December 2007/ Returned for modification 29 February 2008/ Accepted 4 May 2008


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ABSTRACT
 
Conceptually, blocking human immunodeficiency virus type 1 (HIV-1) integration is the last possibility for preventing irreversible cellular infection. Using cocultures of monocyte-derived dendritic cells and CD4+ T cells, which represent primary targets in sexual transmission, we demonstrated that blocking integration with integrase strand transfer inhibitors (InSTIs), particularly L-870812, could consistently block cell-free and cell-associated HIV-1 infection. In a pretreatment setting in which the compound was present before and during infection and was afterwards gradually diluted during the culture period, the naphthyridine carboxamide L-870812 blocked infection with the cell-free and cell-associated HIV-1 Ba-L strain at concentrations of, respectively, 1,000 and 10,000 nM. The potency of L-870812 was similar to that of the nucleotide reverse transcriptase inhibitor R-9-(2-phosphonylmethoxypropyl) adenine (PMPA) but one or two orders of magnitude lower than those of the nonnucleoside reverse transcriptase inhibitors UC781 and TMC120. In contrast, the diketo acid RDS derivative InSTIs showed clear-cut but weaker antiviral activity than L-870812. Moreover, L-870812 completely blocked subtype C and CRFO2_AG primary isolates, which are prevalent in the African heterosexual epidemic. Furthermore, the addition of micromolar concentrations of L-870812 even 24 h after infection could still block both cell-free and cell-associated Ba-L, opening the prospect of postexposure prophylaxis. Finally, an evaluation of the combined activity of L-870812 with either T20, zidovudine, PMPA, UC781, or TMC120 against replication-deficient HIV-1 Ba-L (env) pseudovirus suggested synergistic activity for all combinations. Importantly, compounds selected for the study by using the coculture model were devoid of acute or delayed cytotoxic effects at HIV-blocking concentrations. Therefore, these findings provide evidence supporting consideration of HIV-1 integration as a target for microbicide development.


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INTRODUCTION
 
Worldwide, about 33 million people are infected with human immunodeficiency virus (HIV), of which almost 50% are women (34, 43, 54). Heterosexual transmission is the leading route of HIV dissemination (19, 33, 43); however, presently no method under the control of women is available to prevent transmission (40, 48). Therefore, the development of microbicides, topically applied HIV-preventive compounds, represents an attractive protection option for women (1, 30, 31, 48).

The experience with early broad-spectrum candidate microbicides that have reached phase III clinical trial end points (e.g., nonoxynol-9 and cellulose sulfate) showed that products with moderate or low-level potency against HIV in vitro fail to block viral transmission in clinical trials (2, 30, 40). Moreover, this early class of microbicides is coitus dependent (e.g., they need to be applied shortly before high-risk contact), and they may damage epithelia upon frequent use (2, 40). Therefore, more specific and potent anti-HIV agents, which may be coitus independent and should be devoid of toxicity, are being sought. Basically, all early steps of HIV replication are potential targets (3, 40). Since the integration of HIV reverse-transcribed DNA (cDNA) into the cell chromosome is crucial for irreversible and productive infection, integrase (IN) inhibitors (INIs) may be suitable to prevent HIV transmission.

The HIV integration starts in the cytoplasm, shortly after the reverse transcription of the genome. Within a nucleoprotein complex, assembled with viral and cellular proteins and specific cDNA sequences (at the ends of long terminal repeat [LTR] regions), the IN enzyme first removes the 3' dinucleotide ends of both cDNA strands by an endonucleolytic process, leaving recessed-3'-OH at the termini (18, 22, 56). Next, the processed cDNA is translocated to the nucleus, where the IN catalyzes the insertion of the cDNA into the host DNA by a strand transfer mechanism in which the viral cDNA 3'-OH ends are covalently linked to the cellular DNA (22, 45). Subsequently, cellular DNA repair mechanisms fill in the gaps and ligate the viral DNA to the host DNA (18, 45). As a multistep process, integration can be inhibited at various stages by several classes of INIs; for example, (i) Y3 and catechols are inhibitors of the IN 3'-end processing which prevent the formation of the IN-viral DNA complex (18, 44); (ii) L-870810, L-731,988, L-870812, and the compounds MK-0518 (raltegravir) and GS-9137 (elvitegravir), currently in clinical development, are inhibitors of the IN strand transfer process through activity within the enzyme active site (18, 24, 44, 45); and (iii) LEDGF/p75-derived peptides are inhibitors of IN macromolecular complex formation which prevent the contact between IN and host cellular cofactors (21, 32). Until now, however, only the activities of IN strand transfer inhibitors (InSTIs) have been fully validated in biochemical assays (16, 23, 42, 44), in vitro assays (23, 24, 42, 55), experiments with infected rhesus macaques (25), and clinical studies with HIV type 1 (HIV-1)-infected patients (36), demonstrating their potential for therapy. As a result of the outcomes of these studies, raltegravir was approved last year by the U.S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection with multidrug resistance (52).

Here, we investigated for the first time the potential roles of INIs, particularly InSTIs, in the prevention of HIV-1 by using our model of in vitro cocultures of dendritic cells and autologous CD4+ T cells, representing primary targets involved in the first steps of sexual HIV transmission (13, 20, 30, 37). We report on (i) the efficacies of InSTIs against replication-competent cell-free or cell-associated HIV-1; (ii) the comparison of the abilities of an entry inhibitor (EI), reverse transcriptase inhibitors (RTIs), and InSTIs to block HIV-1 infection; (iii) the activities of an EI, RTIs, and InSTIs against primary isolates of prevalent subtypes; (iv) the potential efficacies of InSTIs and nucleotide RTIs (NtRTIs) in pre- and postexposure prophylactic interventions; and (v) the potential synergism of InSTIs with an EI and RTIs. Furthermore, possible toxicity toward the primary cells in the cocultures was also evaluated.


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MATERIALS AND METHODS
 
Cell lines and primary cells. For the antiviral screening assay, a human osteosarcoma cell line expressing green fluorescent protein under the control of the HIV LTR promoter and coexpressing the HIV receptor CD4 and the coreceptor CCR5 (the GHOST-CD4-R5 cell line, kindly provided by D. Littman, New York Medical Center, New York) was cultured in Dulbecco's minimal essential medium (BioWhittaker, Verviers, Belgium) containing puromycin (1 µg/ml; Sigma, Bornem, Belgium), hygromycin B (50 µg/ml; Roche, United Kingdom), penicillin (100 U/ml), streptomycin (100 µg/ml), Geneticin (500 µg/ml; Invitrogen, United Kingdom), and fetal calf serum (10%; Biochrom AG, Berlin, Germany) at 37°C and 5% CO2 in a humidified atmosphere and subpassaged by treatment with trypsin (500 mg/liter)-EDTA (200 mg/liter) (both from BioWhittaker, Verviers, Belgium) twice a week as described previously (51).

For the coculture of primary cells, buffy coats (kindly provided by the Antwerp Blood Transfusion Center) from healthy donors were used to obtain monocyte-derived dendritic cells (MDDC) and CD4+ T cells as described previously (58). Briefly, monocytes and lymphocytes were separated by counterflow elutriation and E rosetting. The monocytes were differentiated into MDDC by treatment with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) over 7 days. For the MDDC-CD4+ T cell cocultures, autologous frozen lymphocytes were thawed and CD4+ T cells were positively selected with magnetic beads. For part of the experiments, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation and activated by being cultured for 48 h in complete medium (RPMI 1640 medium [BioWhittaker] containing 10% fetal calf serum [Biochrom AG] and gentamicin at 50 mg/ml [BioWhittaker]) supplemented with phytohemagglutinin (PHA; 2 µg/ml [Remel, Kent, United Kingdom]) and, subsequently, for 24 h in complete medium supplemented with IL-2 (1 ng/ml; Gentaur, Brussels, Belgium).

Single-cycle infectious PV and replication-competent virus strains. The single-cycle infectious HIV-1 pseudovirus (PV) expressing Ba-L env was used for screening the antiviral activities of the compounds (51). Two constructs, the pNL4-3.Luc.R-E reporter construct under the control of the LTR promoter (containing two frameshift mutations in the env and vpr genes which render this clone nonreplicative and carrying a luciferase reporter gene in place of the nef gene) and the pcDNA4/TO Ba-L envelope-expressing construct under the control of the cytomegalovirus promoter (NIH AIDS Research and Reference Reagent Program, Rockville, MD), were used to prepare PV by the cotransfection of 293 T cells according to the calcium phosphate method using the ProFection mammalian transfection system (Promega, Leiden, The Netherlands). At 24 h posttransfection, 1 mM sodium butyrate (Sigma-Aldrich, Bornem, Belgium) was added to the cultures, and 24 h later, supernatants were removed and passed through a 0.45-µm-pore-size filter. The PV infectious titer was determined by the infection of GHOST-CD4-R5 cells. After 48 h of culture, the luciferase substrate (SteadyLite HTS; Perkin Elmer Life Sciences, Zaventem, Belgium) was added to the cells and the resulting signal, expressed as relative light units per second (RLU), was quantified using a luminometer (TriStar LB941; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). For screening experiments, a volume of PV stock diluted to a concentration ultimately resulting in a signal of approximately 105 RLU was used.

The HIV-1 replication-competent subtype B, non-syncytium-inducing, CCR5 coreceptor-using reference strain Ba-L (NIH AIDS Research and Reference Reagent Program, Rockville, MD) was used for experiments with the MDDC and CD4+ T cell cocultures. Additionally, CCR5 coreceptor-using clinical isolates representing subtypes C (VI1358 and VI829; from our own collection) and CRFO2_AG (MP568) (39) were selected. The subtyping of the subtype C isolates was based on the sequencing of the complete env gene, while for the CRFO2_AG isolate, it was based on the sequencing of complete env, pol, gag p24, gag p17, tat, rev, nef, and vpu genes. Stocks of cell-free and cell-associated virus were processed as described previously (51, 58). Briefly, stocks of cell-free virus were prepared, and titers in PHA- and IL-2-activated PBMC were determined. The gag p24 antigen in the supernatant was evaluated using an in-house enzyme-linked immunosorbent assay (ELISA) (4). A stock of cell-associated virus was prepared by the overnight incubation of freshly obtained PBMC with cell-free Ba-L at a multiplicity of infection (MOI) of 10–2 in complete medium without mitogen or cytokines. The next day, infected cells (referred to hereinafter as HIV-infected PBMC or cell-associated virus) were collected, extensively washed, and stored in liquid nitrogen.

The infectiousness of both viral stocks in our model was measured by the incubation of cell-free virus with MDDC at several MOIs or of various proportions of HIV-infected PBMC and MDDC. After 2 h, MDDC were washed and cocultured with autologous CD4+ T cells for 14 days. Viral replication was determined by the detection of the HIV-1 gag p24 antigen in culture supernatants.

INIs and other anti-HIV agents. The INIs RSD1624, RSD1625, RSD1996, RSD1997, RSD2196, and RSD2197 (all diketo acid derivatives, kindly provided by the Dipartimento di Studi Farmaceutici, Università di Roma La Sapienza, Rome, Italy), L-731,988 (a diketo acid, kindly provided by C. Pannecouque, Rega Institute for Medical Research, Leuven, Belgium), and L-870812 (a naphthyridine carboxamide, kindly provided by the Department of Antiviral Research, Merck Research Laboratories, West Point, PA), which selectively block the IN strand transfer mechanism, were included in this study (see Fig. 1). The EI T20 (a gp41 fusion inhibitor) was obtained from the NIH AIDS Research and Reference Reagent Program, Rockville, MD; the NtRTI R-9-(2-phosphonylmethoxypropyl) adenine (PMPA) and the nonnucleoside RTI (NNRTI) UC781 were kindly provided by J. Balzarini, Rega Institute, Leuven, Belgium, whereas the NNRTI TMC120 was kindly donated by Tibotec BVBA, Mechelen, Belgium. All compound stocks were prepared by dissolution in dimethyl sulfoxide (DMSO) to obtain compound concentrations of 10, 25, or 100 mM, except for T20 and L-870812, which were dissolved in phosphate-buffered saline (BioWhittaker, Verviers, Belgium) to obtain a concentration of 10 mM. To avoid a toxic influence of DMSO, the final DMSO concentration in the experiments was at least fivefold lower than the minimum nontoxic concentration for cells, as determined in preliminary experiments by the WST-1 tetrazolium reduction assay (data not shown).


Figure 1
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  		FIG. 1. Chemical structures of the integrase inhibitors. Structural representations of the diketo acid derivative (RDS derivative and L-731988) and the naphthyridine carboxamide (L-870812) InSTIs are shown.

Single-cycle infectious PV assay for screening compound-mediated antiviral activity. Aliquots (50 µl) of diluted PV stock (ultimately resulting in a signal of 105 RLU) were preincubated with 50-µl samples of either culture medium or a compound dilution series in a 96-white-well, transparent-bottom plate (Greiner; Bio-One GMBH, Germany). After 30 min at 37°C and 5% CO2 in saturating humidity, GHOST-CD4-R5 cells (1.2 x 105/ml; 100 µl) were added and the cultures were subsequently incubated for 48 h. Each condition was evaluated in triplicate. To determine the degree of infection, GHOST-CD4-R5 cells were lysed by the addition of substrate buffer and the luciferase activity was quantified in a luminometer. The compound's antiviral activity was expressed as the percent inhibition of replication relative to the level of replication in the untreated controls. The percent inhibition was plotted against the compound concentration, and a linear regression analysis was applied to calculate the 50% effective concentration (EC50).

MDDC-CD4+ T cell coculture model. Aliquots (50 µl) containing 2, 20, or 200 50% tissue culture infective doses (a MOI of 10–4, 10–3, or 10–2 to MDDC, respectively) of cell-free virus or 2 x 104 cells of cell-associated virus were preincubated with 50-µl samples of culture medium (control) or a compound dilution series at 37°C and 5% CO2 in saturating humidity. After 30 min, MDDC (0.2 x 106/ml; 100 µl) were added and the cultures were further incubated for 2 h at 37°C and 5% CO2 in saturating humidity. Subsequently, cultures were exhaustively washed with medium and autologous CD4+ T cells (106/ml; 100 µl) and either medium or a compound (100 µl) were added. During 14 days of primary culture, medium was refreshed every 3 to 4 days by replacing 100 µl of the culture supernatant with 100 µl of complete medium (without compounds). On day 14, the supernatant was removed and PHA-IL-2-activated PBMC (0.5 x 106/ml; 200 µl) were added to amplify possible subliminal or latent infection. After an additional incubation for 7 days (secondary culture), supernatant was collected and HIV-1 replication was analyzed by the detection of gag p24 antigen.

Time course assay for the evaluation of pre-EP and post-EP in MDDC-CD4+ T cell cocultures. Aliquots (50 µl) of cell-free virus (at a MOI of 10–3 to MDDC) or cell-associated virus (2 x 104 cells at a ratio of 1:1 to MDDC) were preincubated with 50-µl samples of culture medium with or without samples from a compound dilution series (preexposure prophylaxis [pre-EP] setting) or with medium alone (postexposure prophylaxis [post-EP] setting) for 30 min at 37°C and 5% CO2 in saturating humidity. Afterwards, MDDC (0.2 x 106/ml; 100 µl) were added and the cultures were incubated for 2 h. Subsequently, the cultures were exhaustively washed with medium, and the cells were cocultured with autologous CD4+ T cells (106/ml; 100 µl). For the pre-EP setting, culture medium or a compound (100 µl) at an appropriate concentration was added to the MDDC-CD4+ T cell cocultures and the cultures were incubated for 14 days as mentioned above. For the post-EP setting, MDDC-CD4+ T cell cocultures were incubated with medium only for 2, 4, or 24 h and, at those time points, 100 µl of either medium or a compound was added, after which the cocultures were incubated for 14 days as described above.

Determination of viral replication and compound-mediated activity in MDDC-CD4+ T cell cocultures. Viral replication was assessed by the detection of the gag p24 HIV-1 antigen in culture supernatants by using an in-house ELISA (4). Compound-mediated antiviral activity was expressed as the percent inhibition of viral replication relative to the level of viral replication in infected but untreated cultures and was reported as the arithmetic mean ± the standard deviation of the results obtained from a respective number of experiments. The percent inhibition was plotted against the compound concentration, and the amount required to reduce viral production by 50% (the EC50) was calculated by a linear regression analysis. The EC50s are reported as the geometric means of the results obtained from sets of independent experiments (51, 57).

Assay for evaluation of synergistic activities. Combinations of compounds were evaluated for synergism in the single-cycle PV assay according to the fixed-ratio or ray design method described by Straetemans et al. (50). For these experiments, dilution series of two compounds (compound A and compound B) were prepared containing 100, 87.5, 75, 50, 25, 12.5, and 0% of the EC50 of compound A or compound B. Both sets of dilutions were combined at a 1:1 ratio with the corresponding fraction (in master mixes) (i.e., compound A containing 75% of EC50 with compound B containing 25% of EC50). Next, from these master mixes, a dilution range was prepared (twofold dilution series) and, subsequently, compounds were tested individually or in combination with at least six replicates. Fifty microliters of each master mix dilution was preincubated with 50 µl of HIV-1 Ba-L (env) PV. After 30 min, 100 µl of GHOST-CD4-R5 target cells (1.2 x 105/ml) was added, and subsequently, the culture was incubated for 48 h. Afterwards, cells were lysed in the luciferin substrate and processed as described for the antiviral screening assay. For each combination, the EC50 was determined and used for the calculation of combination indices (CI) as described previously (50). A synergistic effect of combined compounds was defined as a CI significantly below 1. An additive effect of combinations was defined as a CI equal to 1. Similarly, an antagonistic effect of combined compounds was defined as a CI significantly above 1.

Evaluation of compound-mediated cytotoxicity in MDDC-CD4+ T cell cocultures. Cocultures of MDDC (0.4 x 106/ml; 50 µl) and CD4+ T cells (1 x 106/ml; 100 µl) were incubated with culture medium (50 µl) (control) or samples from a compound dilution series (50 µl) at 37°C and 5% CO2 in saturating humidity for 14 days. During this period, the cultures were refreshed every 3 to 4 days by replacing 100 µl of the supernatant with 100 µl of culture medium (without compounds). Acute toxicity was evaluated after the first 3 days of culture, whereas the delayed toxicity effect was assessed after 7 and 14 days of culture. At each time point, cells were carefully washed with medium and incubated with WST-1 tetrazolium salt (Roche Diagnostic, Mannheim, Germany) for 4 h, the resulting formazan product was quantified in a microplate reader (Bio-Rad, Tokio, Japan) at 450 nm, and the percentage of viable cells relative to those in untreated controls was calculated.


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RESULTS
 
INIs show potent antiviral activity in a single-cycle infectious PV assay. A set of INIs (Fig. 1) with strand transfer IN-inhibitory activities (InSTIs), including RDS1624, RDS1625, RDS1996, RDS1997, RDS2196, and RDS2197 (all quinolinone-based diketo acid derivatives) (5, 7, 14, 15, 45), L-731,988 (a styrylquinoline diketo acid derivative) (6, 16, 18, 24, 44), and L-870812 (a naphthyridine carboxamide) (18, 25, 45), was evaluated in a single-cycle Ba-L (env) PV assay. The potencies of these compounds were compared to those of the fusion inhibitor T20, the nucleoside RTI (NRTI) zidovudine (AZT), the NtRTI PMPA, and the NNRTIs UC781 and TMC120 (Table 1). RDS derivatives showed EC50s ranging between 313 and 4,588 nM, while the EC50 of L-731,988 was 1,382 nM and that of L-870812 was 20 nM. The antiviral activities of the EI and the RTIs were in the range of those described in previous reports (EC50s of 1 to 1,112 nM) (Table 1) (51, 57).


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  		TABLE 1. Activities of individual anti-HIV-1 compounds in the single-cycle Ba-L (env) PV assay

INIs completely block cell-free and cell-associated HIV-1 infection in primary target cells. Various InSTIs were evaluated for their capacities to block both cell-free and cell-associated virus infection by using a coculture model of autologous MDDC and CD4+ T cells, which represent mucosal target cells involved in sexual HIV transmission (8, 20, 26). Since microbicides will be applied preferably before high-risk exposure, we mimicked a pretreatment setting with a single application in our in vitro system. In this setup, the compound was present prior to and during the infection and cocultivation of MDDC and CD4+ T cells with cell-free or cell-associated HIV-1 Ba-L. The compound was gradually diluted during the coculture period by replacing half of the supernatant with culture medium free of compounds twice a week. An early evaluation of INIs in the coculture model allowed the selection of the compounds most active in this system (L-870812, RDS2197, RDS1997, and RDS1624). Although RDS1996 showed a favorable EC50 in this system, the initial microscopic observation and the more formal cytotoxicity evaluation (the WST-1 test) (see below) indicated that RDS1996 activity was related to cell toxicity at that concentration; therefore, this compound was not further included in the study (data not shown).

The activities of InSTIs against cell-free Ba-L were dependent on viral input. At a relatively low viral dose (a MOI of 10–4), a 50,000 nM concentration of either RDS1624 or RDS1997 inhibited, respectively, 35 and 80% of viral replication whereas RDS2197 and L-870812 at 50,000 and 1,000 nM, respectively, completely blocked viral infection (Fig. 2A). At an increased dose of cell-free Ba-L (a MOI of 10–3), complete blocking activity was achieved only by using 10,000 nM L-870812 whereas 50,000 nM RDS2197 and RDS1997 inhibited, respectively, 60 and 35% of viral replication. In contrast, RDS1624 failed to inhibit viral infection in this setting (Fig. 2B).


Figure 2
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  		FIG. 2. Activity of INIs against cell-free or cell-associated HIV-1 in a pre-EP setting in the MDDC-CD4+ T cell coculture model. INIs were present prior to and during the infection of MDDC with cell-free virus at a MOI of 10–4 (A) or 10–3 (B) or with cell-associated virus (HIV Ba-L-infected PBMC at a 1:1 ratio to MDDC) (C). Subsequently, MDDC were cocultured with CD4+ T cells for 14 days. During this period, compounds were gradually diluted by the refreshment of the supernatants with medium (without compounds) twice weekly. At day 14, cultures were washed and subliminal infection was revealed by the addition of activated PBMC, and 1 week later, gag p24 HIV antigen was measured in an ELISA. Data shown are the percentages of viral inhibition relative to the level of replication in untreated controls and are the means ± the standard deviations of results from three independent experiments for cell-free virus (except for RDS2197 and L-870812, for which results from one and eight experiments, respectively, are reported) and two independent experiments for cell-associated virus (except for RDS2197, for which results from a single experiment are reported) in which each condition was tested with six replicates.

Testing the InSTIs RDS2197 and L-870812 against cell-associated HIV-1 (at a ratio of HIV-PBMC to MDDC of 1:1) showed that 50,000 nM RDS2197 reduced viral production by 83%, whereas 1,000 nM L-870812 sufficed to completely block infection with cell-associated virus (Fig. 2C). Thus, INIs could inhibit both cell-free and cell-associated virus infection, which is a prerequisite to considering them as candidate microbicides in further studies.

Potencies of INIs compared to those of the reference EI and RTIs against cell-free and cell-associated HIV-1 infection in primary cell cultures. In the experiments to investigate the potencies of the INIs, we used the same reference EI and RTIs as in the PV assay, as well as two selected RDS derivatives (RDS1997 and RDS2197) and L-870812. In a pretreatment setting in the MDDC-CD4+ T cell cocultures with the gradual dilution of compounds, it was evident that the activity of the InSTI L-870812 against infection with cell-free Ba-L virus at a MOI of 10–3 was similar to those of the fusion inhibitor T20, the NRTI AZT, and the NtRTI PMPA (EC50s of 671, 724, 880, and 1,498 nM, respectively). Moreover, against cell-associated virus also, L-870812 showed activity similar to those of AZT and PMPA (EC50s of 128, 400, and 314 nM, respectively), whereas T20 was much less active (EC50 > 10,000 nM), as previously reported (51). Both RDS derivatives 2197 and 1997 were less potent than the other compound against cell-free virus and cell-associated virus infections than the other compounds. The NNRTIs UC781 and TMC120 were clearly the most active, as previously reported (17, 51, 57) (Table 2). In looking at the compounds' capacities to block viral replication, we found that T20, AZT, PMPA, and L-870812 all blocked cell-free Ba-L at a MOI of 10–3 at a concentration of 10,000 nM but that a 1,000 nM concentration of UC781 or a 10 nM concentration of TMC120 sufficed (Fig. 3). In contrast, when cell-associated Ba-L was used, only PMPA, L-870812, UC781, and TMC120 completely blocked viral infection, at concentrations of 10,000, 1,000, 100, and 10 nM, respectively (Fig. 3).


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  		TABLE 2. Antiviral and cytotoxic activities of individual anti-HIV compounds in MDDC-CD4+-T-cell cocultures


Figure 3
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  		FIG. 3. HIV-1 replication-blocking capacities of EIs, RTIs, and INIs. The capacities of the EI T20 (A), the RTIs AZT, PMPA, UC781, and TMC120 (B), and the INIs L-870812 and RDS2197 (C) to block the replication of cell-free Ba-L (MOI of 10–3 relative to MDDC) or cell-associated Ba-L (HIV-infected PBMC at a 1:1 ratio to MDDC) in a pre-EP setting were compared. Compounds were present prior to and during the infection of MDDC. Subsequently, MDDC were cocultured with autologous CD4+ T cells for 14 days. During this period, compounds were gradually diluted by refreshing the culture medium (without compounds) twice weekly. Afterwards, cultures were washed and activated PBMC were added to reveal subliminal infection. After 1 week of additional culture, gag p24 was measured. Data shown are the percentages of viral inhibition relative to the level of replication in infected but untreated cultures at day 21 and are the means ± standard deviations of results from two independent experiments (except for AZT against cell-free virus and T20, AZT, UC781, and RDS2197 against cell-associated virus, for which the results of a single experiment are reported) in which each condition was tested with five replicates.

INIs block infection with subtype C and CRFO2_AG HIV-1 clinical isolates. We evaluated the activities of RTIs and INIs against three HIV-1 clinical isolates, two subtype C isolates (VI1358 and VI829) and one CRFO2_AG isolate (MP568), representative of the subtypes which are the most predominant and rapidly spreading in southern and western Africa, respectively. All compounds were more active against subtype C and CRFO2_AG isolates than against the reference subtype B Ba-L virus, even though a 10-fold-higher viral input (a MOI of 10–2) was used (Table 3). The EC50s of RTIs ranged from <1 to 128 nM for subtype C isolates and from <1 to 241 nM for the subtype CRFO2_AG virus. In comparison, the EC50s of RDS1997 and RDS2197 ranged from 3,630 to 30,637 nM for subtype C and from 11,151 to 45,646 nM for subtype CRFO2_AG. Notably, the EC50 of L-870812 ranged from 58 to 103 nM for subtype C isolates, while an EC50 of 261 nM for the CRFO2_AG isolate was found (Table 3). RDS1997 blocked infection with subtype C isolates (VI1358 and VI829) at 10,000 to 50,000 nM, while concentrations of 100 to 1,000 nM of L-870812 sufficed for these isolates (VI1358 and VI829). Similarly, 1,000 to 10,000 nM concentrations of L-870812 blocked infection with the CRFO2_AG HIV-1 isolate, whereas 50,000 nM concentrations of RDS1997 and RDS2197 inhibited, respectively, 95 and 45% of viral infection. Consequently, both RTIs and INIs were efficient at blocking infection with subtype C and CRFO2_AG clinical isolates, providing another argument for further evaluation as candidate microbicides.


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  		TABLE 3. Activities of anti-HIV compounds against cell-free B and non-B subtypes in the MDDC-CD4+ T cell cocultures

The InSTI L-870812 and the NtRTI PMPA show similar HIV-blocking activities in an in vitro setting of post-EP. The efficacy of the InSTI L-870812 in blocking the transmission of cell-free or cell-associated virus when added postexposure was evaluated in comparison to the efficacy in the preexposure setting. Since PMPA is known for its post-EP activity, even in the macaque model (53), and showed potency similar to that of L-870812 in the pre-EP setting (Fig. 3B and C), PMPA and L-870812 were evaluated side by side (Fig. 4). At 10,000 nM, both PMPA and L-870812 blocked infection with cell-free Ba-L at a MOI of 10–3 in a pre-EP setting, as well as in settings in which they were added 2 or 4 h postinfection. When added 24 h postinfection, a 25,000 nM concentration of either compound was needed to completely block the virus (Fig. 4A). In the setup with cell-associated virus, 10,000 nM PMPA sufficed to block infection in both the pre-EP setting and the 4-h post-EP setting. However, 25,000 nM PMPA was needed to achieve blocking activity when the compound was added 24 h after infection (Fig. 4B). L-870812 proved to be slightly more effective than PMPA; at 1,000 nM, it blocked cell-associated virus in both the pre-EP setting and the 4-h post-EP setting, whereas 10,000 nM L-870812 was needed to block cell-associated virus when the compound was added 24 h after infection (Fig. 4B). Our results suggest that INIs may be efficient at blocking viral infection in both pre- and postexposure settings, which is an additional advantage for a candidate microbicide.


Figure 4
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  		FIG. 4. Pre-EP and post-EP against cell-free and cell-associated HIV infection. The RTI PMPA and the INI L-870812 against cell-free virus (Ba-L; MOI of 10–3) (A) or cell-associated virus (HIV-infected PBMC at a 1:1 ratio to MDDC) (B) were evaluated in a preexposure or postexposure in vitro setting by using the MDDC-CD4+ T cell coculture model. MDDC were infected in the presence (pre-EP) or absence (post-EP) of compounds and cocultivated with CD4+ T cells for 14 days. In the postexposure setting, compounds were added at the indicated hours after HIV infection of MDDC and subsequent coculture with CD4+ T cells. Compounds were gradually diluted by refreshing supernatants with medium free of compounds twice weekly. On day 14 of culture, cells were washed and activated PBMC were added to reveal subliminal infection. After a week of additional culture, gag p24 antigen was measured in an ELISA. Data shown are the percentages of viral inhibition relative to the level of replication in infected but untreated cultures. The mean values for six replicates from a single experiment are represented.

The InSTI L-870812 shows synergistic activity in combination with a fusion inhibitor or RTIs. The combined activity of L-870812 with T20, PMPA, AZT, UC781, or TMC120 was evaluated in the single-cycle PV assay using a fixed-ratio design in which several proportions of two compounds were combined (50). Clearly, all combinations at all proportions tested consistently showed CI values below 1, implying that synergistic effects may be obtained when combinations of an InSTI with T20 or an RTI are used (Table 4).


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  		TABLE 4. Combined activity of L-870812 with an EI and RTIs in the single-cycle Ba-L (env) PV assaya

The EI, RTIs, and INIs show limited direct or delayed toxic effects on MDDC-CD4+ T cell cocultures. Direct cytotoxic effects (acute toxicity) or the possibility of delayed toxic effects of compounds on MDDC-CD4+ T cell cocultures was evaluated in a WST-1 tetrazolium-based assay. Compounds were tested at concentrations up to a maximum of 10,000 nM for the EI and RTIs (except PMPA, tested at up to 25,000 nM) and 50,000 nM for INIs (except L-870812, tested at up to 25,000 nM). For compounds dissolved in DMSO, the maximal concentration corresponded to fivefold the minimum dilution that could be used in the cultures to avoid stock solvent toxicity (data not shown).

In the evaluation of acute toxicity, in which cells were incubated with a compound for 3 days, cell viability was above 90% in cultures with the EI, RTIs, or INIs at the highest tested concentrations, except for cultures with RDS1996, which showed levels of viable cells of 61 and 28%, respectively, at RDS1996 concentrations of 25,000 and 50,000 nM. To evaluate the impact of compounds present in the cultures for a longer period, but in a scheme of gradual dilution (a delayed toxic effect), cells were incubated with PMPA, UC781, TMC120, RDS1997, RDS2197, or L-870812 for 14 days and every 3 to 4 days the supernatant was diluted with medium free of compounds. None of the compounds showed detectable delayed negative effects on cell viability after 7 or 14 days of culture (cell viabilities were above 90% at the highest tested concentrations). The 50% cytotoxic concentrations (CC50s) for acute toxicity were 31,059 nM for RDS1996, above 25,000 nM for RDS1624, and above 50,000 nM for both RDS1625 and RDS2196. The CC50s of compounds included further in the microbicide study in settings of both acute and delayed toxicity were above the maximal tested concentrations (Table 2).


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DISCUSSION
 
A set of INIs, including diketo acid compounds (RDS derivatives and L-731,988) and a naphthyridine carboxamide (L-870812) which selectively block the IN strand transfer activity, was investigated for blocking activity against HIV-1 infection in various settings. The initial screening in a single-cycle Ba-L (env) PV assay showed that all INIs were highly active at nano- and micromolar concentrations, as previously shown (5, 16, 18, 24, 25, 45). Next, the activities and toxic effects were further explored in a physiologically more relevant coculture model of MDDC and CD4+ T cells, which represent primary target cells in sexual HIV transmission (8, 35, 37, 61). As CCR5 coreceptor-using strains are strongly associated with the sexual transmission of HIV (1, 13, 26), we focused this study on R5-phenotype viruses.

Our results clearly demonstrate that INIs, particularly the InSTI L-870812, consistently block the transmission of replication-competent cell-free virus of B and non-B HIV-1 subtypes. We found that L-870812 protected primary target cells with potency similar to that of the NtRTI PMPA but that it was less active than the NNRTIs UC781 and TMC120. Moreover, like NtRTIs or NNRTIs, the InSTI also blocked infection driven by cell-associated HIV-1, which was more difficult to control by using the fusion inhibitor T20 or the NRTI AZT than by using PMPA, UC781, TMC120, or L-870812.

Microbicides are developed for preexposure intervention (11, 12, 17, 31); therefore, we explored the preventive capacities of InSTIs in a pretreatment setting under stringent conditions to mimic those under which the virus has a maximal opportunity to infect target cells (for example, when the mucosal epithelium is physically or inflammatorily damaged and HIV has easy access to mucosal dendritic and T cells). Whereas L-870812 blocked viral replication irrespective of the viral dose or source (cell-free or cell-associated virus), the RDS derivatives were more susceptible than L-870812 to differences in the viral dose (for example, RDS1997 and RDS2197 were less active against high-dose cell-free virus than against low-dose cell-free virus) but remained active against cell-associated HIV. The difference in potency, apart from the respective structure-activity relationship (a naphthyridine carboxamide versus a diketo acid), probably reflects the cellular-level activities of various drug transporters of the ATP-binding cassette (ABC) family that may restrict cellular penetration and result in a reduced intracellular antiviral effect, as has been described previously for some protease inhibitors (49) and INIs (7).

The limited efficacies and low levels of safety of early nonspecific candidate microbicides provided a strong rationale for studying HIV-specific EIs and RTIs as new microbicides (2, 9, 27, 60). Although it has been shown previously that EIs can block cell-free virus infection in cervical tissue (27) and in the macaque model (60), we observed, consistent with the results of our previous study (51), that the fusion inhibitor T20 might be ineffective against cell-associated virus but that the InSTIs consistently blocked it. Considering that semen or cervicovaginal fluids from HIV-infected persons contain both cell-free and cell-associated HIV (9, 30, 35) and that both can be transmitted (26, 28, 30), the capacity of InSTIs to block cell-associated virus may provide a better profile for this novel class of anti-HIV agents.

After virus enters target cells, RTIs are candidates to block further viral infection. In fact, several RTIs (PMPA, UC781, and TMC120) are currently in clinical trials for safety and efficacy as microbicide candidates (1, 3, 30). We included in our study representatives of the three classes of RTIs currently available, the NRTIs, NtRTIs, and NNRTIs. Our findings show that the NRTI AZT, even though it efficiently blocked cell-free virus, had limited effects on cell-associated virus. Probably its requirement for three phosphorylation steps to obtain its bioactive form impaired its efficiency in our infection model of nonactivated primary target cells (10). Consistent with the findings in previous reports, the NtRTI and NNRTIs potently blocked HIV-1 infection, regardless of the dose and source of virus (17, 51, 57, 59). Interestingly, both L-870812 and PMPA showed similar prevention capacities against infections driven by either cell-free virus or cell-associated virus. The NNRTIs were clearly more potent than InSTIs (by one or two logs); however, due to emerging resistance, NNRTIs may be less efficient in preventing infection if similar compounds from the same NNRTI class are considered for both therapy and prevention (30). Using different classes of antivirals, e.g., NNRTIs for treatment and InSTIs for prevention or a combination of both, may provide better protection. Also, NNRTIs may fail to prevent infections with HIV-1 group M and HIV-2 (10), whereas InSTIs have been shown previously to be similarly active against these viruses and those in the present study (23, 45, 47; S. Fransen, S. Gupta, E. Paxinos, W. Huang, C. Chappey, C. Petropoulus, and N. Parkin, presented at the XV International HIV Drug Resistance Workshop, 2006, and personal communication).

In general, all RTIs and InSTIs were more potent toward cell-free subtype C and CRFO2_AG clinical isolates, representing the most prevalent HIV-1 clades in the epidemic (29, 39), than the reference subtype B Ba-L. However, this tendency for increased blocking activity may be due to the relatively low replicative capacities that these primary isolates show in vitro in comparison to that of culture-adapted virus. Results from susceptibility studies of InSTIs against clinical isolates, including B and non-B subtypes, concur in showing similar ranges of susceptibility among subtypes (47; Fransen et al., workshop presentation and personal communication). Therefore, the broad activities of InSTIs against non-B subtype HIV-1 as well as HIV-2 (22) could be further exploited to provide potency to the early-class microbicide candidates that have been shown to be ineffective against non-B subtypes, like Carraguard, which in vitro lacks effectiveness against subtype C isolates (12).

In a real-life setting, women will be tempted to use microbicides after high-risk sexual contact; thus, it is important to estimate potential post-EP effects. In fact, the success of postexposure intervention in preventing occupation-related transmission or mother-to-child transmission has proven that HIV infection can still be blocked soon after exposure (38, 41, 46, 54). Furthermore, the efficacy of post-EP after intravaginal infection has been evaluated in the macaque model (41). Testing L-870812 in our coculture model of autologous resting cells, which represent physiological conditions, we were able to demonstrate blocking activity against cell-free and cell-associated virus infection, even 24 h after viral exposure, with potency similar to that of the NtRTI PMPA. The observed postexposure blocking activity probably is related to the delayed infection process that takes place in resting cells in comparison to activated ones. The low level of cytoplasmic deoxynucleoside triphosphate in resting cells limits the rate of HIV reverse transcription and, therefore, delays the viral replication process. This delayed infectious process may provide a window of opportunity to InSTIs for an efficient postponed intervention. This feature may give InSTIs an advantage over early microbicide candidates or HIV EIs that need to be present during viral exposure. In addition, targeting viral fusion or reverse transcription along with integration in a combination strategy proved to have a significant synergistic effect that may be further explored to avoid a potentially limited effect or resistance selection.

Safety in the microbicide field is paramount. All compounds evaluated in the MDDC-CD4+ T cell cocultures were active in the absence of acute or delayed toxic effects on primary target cells at all tested concentrations.

Although we succeeded in blocking HIV-1 infection in primary target cells by using INIs and, therefore, provided a proof of concept that integration may be a suitable target for microbicides, obviously, much more work needs to be done before INIs can be considered for preventive use. In the present work, we studied the effects of compounds on the production of replicating virus. We should complement these studies by measuring the effects on proviral DNA integrated into cellular DNA versus nonintegrated viral DNA and two-LTR circles. In addition, as for any candidate microbicide, consistent activity in other in vitro systems (e.g., cervical explants) needs to be shown. Next, the best formulation for in vivo application needs to be found. Extensive investigations of in vivo toxicity (in rabbits, macaques, and healthy women) have to be performed. Pharmacokinetics needs to be studied, and true protection against vaginal challenge in macaques needs to be shown (11, 13).

In conclusion, we demonstrated that even by interfering with the last step before the establishment of an irreversible cellular HIV infection, INIs could efficiently protect susceptible target cells by blocking (i) cell-free virus, (ii) cell-associated virus, and (iii) subtype C and CRFO2_AG clinical isolates and could have an effect in both pre- and postexposure settings without cytotoxic activity. Therefore, integration may be considered as a potential target for the development of HIV-1 microbicides either alone or in combination with EIs or RTIs.


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ACKNOWLEDGMENTS
 
We thank the Antwerp Blood Transfusion Center of Antwerp University Hospital for providing us with buffy coats. We thank the NIH AIDS Research and Reference Reagent Program for providing the pNL4-3.Luc.R-E and pcDNA4/TO constructs, the reference strain Ba-L, and the fusion inhibitor T20. We also thank the Fondation Dormeur for donating important equipment for these studies.

This work was financially supported by grants from the European Microbicides Project (EMPRO 6th framework program), the Agence Nationale de Recherche sur le SIDA (ANRS), the International Partnership on Microbicides (IPM), the Ministero della Sanità, Istituto Superiore di Sanità, "VI Programma Nazionale di ricerca sull'AIDS" (grant no. 30G.9), and the Italian MIUR (PRIN 2006). K.T.-A. holds a Ph.D. fellowship from EMPRO.


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FOOTNOTES
 
* Corresponding author. Mailing address: Virology Unit, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen, Belgium. Phone: 32 3 247 6489. Fax: 32 3 247 6333. E-mail: kterrazas{at}itg.be Back

{triangledown} Published ahead of print on 12 May 2008. Back


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Antimicrobial Agents and Chemotherapy, July 2008, p. 2544-2554, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01627-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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