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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, May 2008, p. 1759-1767, Vol. 52, No. 5
0066-4804/08/$08.00+0 doi:10.1128/AAC.01313-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Bristol-Myers Squibb Pharmaceutical Research and Development, Wallingford, Connecticut,1 Monogram Biosciences, South San Francisco, California2
Received 11 October 2007/ Returned for modification 20 December 2007/ Accepted 25 February 2008
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NRTIs are also used to treat chronic HBV infections. Lamivudine (LVD), adefovir, entecavir (ETV), and telbivudine are approved for anti-HBV therapy (16), and emtricitabine (FTC), tenofovir, and clevudine (2'-fluoro-5-methyl-β-L-arabinofuranosyluracil [L-FMAU]) are in late clinical development. Activity against both HIV and HBV has been noted for several NRTIs, including LVD, adefovir, FTC and tenofovir (13). ETV has previously been reported to exhibit highly potent and specific inhibition of HBV, with a 50% effective concentration (EC50) of 4 nM. In contrast, initial assays against HIV-1 yielded an EC50 of >10 µM, with a 50% cytotoxic concentration of 21 µM (10). The combination of weak in vitro activity and a maximum serum concentration (Cmax) of the drug in human plasma of 30 nM following the administration of the 1-mg dose of ETV (23) led to the conclusion that ETV did not exhibit clinically relevant activity against HIV-1. However, a recent report suggested that ETV may exhibit clinically relevant activity against HIV in some patients (15). Three patients coinfected with HIV and HBV showed a reduction of approximately 1 log10 in their circulating HIV-1 RNA levels, in addition to the reductions expected in their HBV levels of several log units while receiving ETV monotherapy. One of the three patients, who had received prior HAART that included LVD, showed an enrichment of an LVD-resistant (M184V) HIV variant over a 6-month treatment period with 1 mg of ETV. The report also noted that in vitro studies using a single-cycle, single-cell reporter assay showed a subnanomolar level of inhibition by ETV of a pseudotype virus carrying the reverse transcriptase (RT) gene from HIV laboratory and patient isolate strains. ETV was also shown to exhibit reduced susceptibility against an HIV-1 variant containing the M184V substitution. In order to gain greater insight into this observation and better understand the basis for the HIV activity in coinfected patients, we performed additional studies to reexamine the in vitro anti-HIV profile of ETV. The results confirm that ETV should not be used for HBV/HIV-coinfected patients without concomitant HAART.
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MATERIALS AND METHODS |
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Viruses.
The HIV-1 B subtype viruses RF, HXB2, SF2, LAI, and IIIB and the proviral DNA clone of NL4-3 were obtained from the NIH AIDS Research and Reference Reagent Program. The NL4-3 M184V virus was constructed at Bristol-Myers Squibb. Titration of viral stocks was performed in the cell lines in which the viruses were originally amplified, using a virus infectivity assay (11), with supernatant RT activity or cytopathic effect (CPE) as endpoints. A recombinant NL-Rluc virus, in which a section of the nef gene from NL4-3 was replaced with the Renilla luciferase gene, was constructed at Bristol-Myers Squibb. NL-RLuc virus was prepared by the cotransfection of two plasmids, pNL-RLuc, containing NL-RLuc DNA, and pVSVenv, expressing the vesicular stomatitis virus (VSV) G envelope protein. Transfection of 293T cells was performed at a 1:3 ratio of pNL-RLuc to pVSVenv, using a Lipofectamine PLUS kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction, and the pseudotype virus generated was titrated in MT-2 cells. A pseudotype virus, HIV-1LAI 
env-luc (LAI-Luc), was prepared at Bristol-Myers Squibb by cotransfecting HEK-293T cells with both a plasmid containing proviral DNA of HIV-1LAI env-luc (firefly luciferase) and a plasmid expressing LAI-env, driven by the HIV long terminal repeat. This pseudotype virus was titrated in HeLa-CD4-CXCR4 cells, using a luciferase reporter gene assay (high sensitivity; Roche) as the endpoint.
Clinical isolates were obtained from the NIH AIDS Research and Reference Reagent Program and were grown in human PBMCs prepared as described above. Virus yields were determined for clinical isolates at 5 to 7 days postinfection by using 
p24 enzyme-linked immunosorbent assay (PerkinElmer Life Sciences) per the manufacturer's instructions. The multiplicity of infection (MOI) was determined by the 50% tissue culture infectious dose method of endpoint dilution (6) with a p24 readout. The 50% tissue culture infectious dose of each viral stock was calculated by the Spearman-Karber statistical method, as described previously (6).
Reagents. ETV and efavirenz (EFV) were synthesized at Bristol-Myers Squibb. LVD was extracted from the commercial formulation of the drug and purified at Bristol-Myers Squibb. Zidovudine (AZT) was purchased from Sigma.
Drug susceptibility and cytotoxicity assays. The susceptibility of viruses to compounds was determined by incubation in the presence of serial dilutions of the compound. The EC50 value was calculated by using the one-ligand-binding-site model with XLFit software for Microsoft Excel, where the percentage of inhibition = 1/[1 + (EC50/drug concentration)m], and m is a parameter that reflects the slope of the concentration response curve. Cytotoxicity assays were performed in parallel for all experiments by exposing uninfected cells to serially diluted compounds and assaying them after 3 to 6 days for cell viability, using MTS [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (5) or XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} (10) assays. The 50% cytotoxic concentration values were calculated using the same equation as described above for EC50 estimation.
Drug susceptibility assays using laboratory strains. Drug susceptibility assays for laboratory isolates were carried out using two slightly different protocols. The chief differences are the volumes in which cells were initially infected with virus and the lengths of time cells were preincubated with virus prior to the addition of antivirals, with protocol 1 being 1 h (+1 h) and protocol 2 generally being 2 to 3 h (+2 to +3 h) with a smaller (more concentrated) virus inoculum. For ease of understanding, both protocols are listed below, and the protocol used in each experiment is also listed in the text.
Protocol 1. Antiviral assays using various laboratory strains were conducted with MT-2 cells in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES buffer (pH 7.55), 2 mM L-glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin. Cells diluted to 2.5 x 105/ml were infected with virus (MOI range, 0.003 to 0.014) for 1 h before the addition of an equal volume (100 µl) of compounds diluted in medium with 2% dimethyl sulfoxide. The extent of virus replication was determined 5 days postinfection by using an RT assay performed in a 96-well scintillation proximity assay (SPA) format. In the RT SPA, 20 µl of the culture supernatant was added to 40 µl of a reaction cocktail [62.5 mM Tris-HCl [pH 7.8], 100 mM KCl, 0.0625% NP-40, 2.5 mM dithiothreitol, 6.25 mM MgCl2, 1 mM EGTA, 6.25 µg/ml poly(rA), 2.5 µg/ml streptavidin SPA beads prebound with biotinylated-dT12-18, and 1 µCi [3H]thymidine triphosphate]. The reaction mixtures were incubated at 37°C for 1.5 h and quenched with 100 µl of 0.25 M EDTA per reaction. The SPA beads were allowed to settle for 2 h prior to counting with a Wallac Trilux, programmed for tritium counting with SPA beads in opaque plates.
Protocol 2. This protocol used a longer preincubation period for virus and cells and a smaller volume than protocol 1. MT-2 cells were centrifuged at 1,200 rpm in a Beckman Allegra centrifuge for 10 min, and virus was added to the cell pellets in a volume of 0.5 ml. A MOI ranging from 0.005 to 0.05 with NL4-3 or NL4-3 M184V virus was used in these experiments. If a preincubation step was not used, the cell-virus mixtures were directly resuspended in culture medium and seeded into 96-well microtiter plates (final density, 1.0 x 105 cells/ml) containing serial dilutions of test compounds. In experiments designed to determine the effect of preincubation on antiviral activity, cell-virus mixtures were first incubated in a small volume (0.5 ml) for 2 to 3 h (+2, +3 h) and then diluted before the addition to plates containing drugs. Cultures were incubated at 37°C in 5% CO2 until CPEs were apparent (5 days), at which time virus yields were quantitated using 20 µl of supernatants in the RT assay, and the remaining suspensions of infected cells were used in a MTS cell viability assay.
Drug susceptibility assessment using clinical isolates. Antiviral assays for clinical isolates were conducted with stimulated PBMCs. Cells were sedimented for 5 min at 1,200 rpm in a Beckman Allegra centrifuge and resuspended in 1 ml of RPMI 1640 medium supplemented as described above and containing 4 µg/ml PHA and 10 U/ml IL-2. An appropriate volume of virus was added and incubated at 37°C in 5% CO2 for 1.5 h. Infected cells were diluted to 1 x 106 cells/ml with medium supplemented with 2x PHA and 2x IL-2. The diluted cells (100 µl) were immediately added to compounds (100 µl) diluted in medium (plus 2% dimethyl sulfoxide) without PHA and IL-2. Virus yields were determined at 5 days postinfection, using a p24 enzyme-linked immunosorbent assay kit as described by the manufacturer (PerkinElmer Life Sciences). Typically, a 1:1,000 dilution of cell supernatant in medium was used in order to be in the dynamic range of the p24 assay.
Drug susceptibility assessment using pseudotype reporter viruses.
A single-cycle viral infection system was also utilized to determine drug susceptibility (11). For the recombinant NL-RLuc virus expressing the Renilla luciferase protein and pseudotyped with the VSV G protein, antiviral activity was evaluated by measuring the production of luciferase in infected cells at 5 days postinfection, using a Dual luciferase kit (Promega, Madison, WI) with a minor modification. Diluted passive lysis solution was premixed with both the resuspended luciferase assay substrate and the resuspended Stop & Glo substrate (2:1:1 ratio). Fifty microliters of the mixture was added to each aspirated cell monolayer of the assay plates, and luciferase activity was measured immediately on a Wallac TriLux (PerkinElmer Life Sciences). The HIVLAI env-luc virus, pseudotyped with an LAI envelope, was also used to infect HeLa-CD4-CXCR4 and MT-2 cells and PBMCs containing serially diluted compounds. Firefly luciferase activity was monitored at 3 days postinfection, according to the manufacturer's specifications (Luciferase reporter gene assay, high sensitivity; Roche).
Monogram Biosciences pseudovirion (PhenoSense) assay. An ETV stock solution was diluted in DMEM plus 10% FBS (17). Threefold dilutions were used in the range from 600 to 0.03 µM. HEK-293 cells were cotransfected in 10-cm dishes with an HIV-1 genomic vector that contains a luciferase indicator gene cassette in the envelope region and a plasmid that expresses the amphotropic murine leukemia virus envelope. For drug evaluation, two HIV-1 expression plasmids were used, NL4-3 wild type (a drug-sensitive virus) and NL4-3 M184V (an LVD- and FTC-resistant virus). Viruses were harvested from the transfected HEK-293 cells and were used to infect naïve HEK-293 cells. ETV was added to the HEK-293 cells at 24 h prior to viral infection of naïve cells. The ability of the pseudovirions to infect target cells was monitored by the production of luciferase in the infected cells. Just prior to the luciferase readout, cells were evaluated by visual inspection for evidence of toxicity (cell death) or morphological changes (cell growth affected) due to the presence of drug. From an analysis of the measured luciferase signal, the drug concentration required to inhibit viral infection by 50% was calculated for each virus tested. Data points that had visible cell death were omitted in calculating EC50 values.
Analysis of activity in chronically infected cells. MOLT-4/IIIB cells were extensively washed to remove extracellular virus before the compounds were added. Cells (30 ml) were sedimented for 5 min at 1,200 rpm in a Beckman Allegra centrifuge, decanted, and resuspended in 30 ml of RPMI 1640 medium supplemented as described above. This pelleting/washing process was repeated three times. The final cell pellet was resuspended to 2 x 105 cells/ml and was added to an equal volume (0.1 ml) of medium containing compound dilutions. The culture supernatant was assayed for RT activity after 4 days.
Selection of resistant variants.
MT-2 cells at a density of 2 x 105 cells/ml were infected with the NL4-3 strain at an MOI of 0.005. At the time of virus exposure, 0.1 µM or 0.4 µM ETV (
two- or sevenfold the EC50 value) was added to the cultures. As a control, LVD resistance was also selected with a starting concentration of 0.2 µM (fivefold the EC50 value). The cultures were incubated until a CPE was observed, at which time the culture supernatant was harvested. Twenty-five microliters of this supernatant was used to infect a fresh culture of MT-2 cells, to which double the initial concentration of compound was added. Cultures were again incubated until a CPE was observed, at which time the entire process was repeated, with drug concentrations doubling at each dose. Consequently, at passage eight, the concentration of ETV was 12.8 µM or 51.2 µM. For selected passages, the NL4-3 RNA was extracted from the culture supernatants, using a viral RNA extraction kit (Qiagen). The viral RNA served as a template for reverse transcription-PCR (First-Strand Synthesis and Platinum high-fidelity DNA polymerase kits; Invitrogen) using primers that annealed to HIV-1 gag and vpr. The resulting DNA products were subjected to fluorescent dye-terminator nucleotide sequencing (ABI) using HIV-specific primers, and the resulting data were analyzed using DNASTAR version 6 software (Lasergene).
The ability of LVD and ETV to selectively enrich for the M184V HIV-1 virus from a mixture of M184V/wild-type viruses was also examined. The two NL4-3 viruses were mixed at a ratio of 1/100 (M184V/wild type) and used to infect 105 MT-2 cells/ml at a total MOI of 0.005 (with no preincubation step). Parallel cultures were then incubated in the presence of 2.5 and 10 µM ETV (45-fold and 179-fold the EC50 values) or 2.5 µM LVD (63-fold the EC50 value). After a 6-day incubation (when CPE was evident), supernatants were harvested, viral RNA was isolated, and sequence analyses were performed as described above.
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RESULTS |
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Assessment of ETV activity using pseudotype HIV-1. Routine screening for inhibitory activity and resistance has been improved through the use of pseudotype viruses (17). These recombinant viruses may contain the G glycoprotein from VSV within their envelope (the LAI pseudotype used here was pseudotyped with the LAI envelope protein). The G glycoprotein improves the stability and infectivity of the virus population, enabling greater ease of use and sensitivity of the assay. In addition, the pseudotype virus may contain a reporter gene, such as luciferase, the expression of which can be readily measured and quantitated. ETV activity was assayed with two such pseudotype virus assays derived from either the HIV-1 NL4-3 or LAI viral backbones. The NL-RLuc virus contains the Renilla luciferase gene, while the LAI-Luc virus contains the firefly luciferase gene, both in lieu of the nef gene. In addition, ETV was also tested with a PhenoSense assay (Monogram Biosciences) to determine the activity of ETV against that of their NL4-3 pseudotype virus stock, relative to other antiretrovirals. The results show EC50 values for ETV in the µM range regardless of the pseudotype strain, cell type, or assay used (Table 1). Examination of the NL4-3 pseudotypes against LVD, AZT, and EFV produced EC50 values within the range of expected values.
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Impact of assay conditions on observed potency of ETV. The results described above suggest that ETV activity against HIV-1 is highly variable and is likely to be dependent on multiple factors. The wide range of susceptibilities to ETV observed with the panel of laboratory strains differed from the narrower range of activities observed with the approved HIV drugs included as controls. Also, only weak inhibitory activity was observed against pseudotype viruses (Table 1), while certain clinical isolates exhibited a high degree of susceptibility to ETV. In an attempt to better understand the underlying variables that contribute to the range of susceptibilities observed, a series of investigational studies was performed that further evaluated the conditions of infection, MOI, and assay readout methodologies.
The effects of MOI and preincubation time of cells with virus prior to drug exposure were studied using the laboratory strain NL4-3. At various MOIs, ranging from 0.005 to 0.05, cells were infected in a small volume for 2 h prior to the addition of the drug (+2 h), or the drug was added at the time of virus inoculation (zero h). Parallel experiments were performed with ETV, LVD, AZT, and EFV, and results are summarized in Table 3 and presented in Fig. 1. MOI and the virus/cell preincubation step were key variables, significantly affecting the measured susceptibilities to all four drugs, although the observed effects were more dramatic with ETV than with the other control drugs. Increasing the virus inoculum from an MOI of 0.005 to 0.05 in the absence of a virus/cell preincubation step resulted in an increase in the EC50 values (decreased potency) for EFV, AZT, and LVD of 3.5-, 4.3-, and 9.3-fold, respectively (Fig. 1). For ETV, the EC50 at an MOI of 0.005 was 0.056 µM, rising to 6.45 µM at an MOI of 0.05, a 115-fold increase. The largest increases were observed between MOIs of 0.01 to 0.02, where the ETV EC50 increased 4.7-fold, and from 0.02 to 0.05, where the EC50 increased an additional 14.4-fold. The underlying mechanism responsible for this observation is unknown at this time, but clearly, decreased MOI has a greater effect on the potency of ETV than on the other established HIV RT inhibitors examined.
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These data demonstrate that the variables and underlying mechanisms of ETV inhibition of HIV-1 replication likely differ considerably from those of LVD, AZT, and EFV. Unlike these other drugs, ETV exhibits significant antiviral activity only under conditions of reduced viral challenge. Enhancing the infection by increasing the virus inoculum or by preincubating the cells with virus during the first cycle of a multicycle infection (+2 h) greatly reduces ETV potency. The +2-h virus/cell incubation step could also enable virus replication to proceed prior to the phosphorylation of ETV to the active triphosphate (ETV-TP), although the preincubation of cells with ETV prior to infection did not appear to substantially alter the EC50s of ETV (data not shown).
The conditions of infection also affect the shape of inhibition curves for ETV. Lowering the virus challenge appears to be associated with decreased EC50 values and flatter titration curves (Fig. 2). To enable a direct comparison of ETV potency levels under various assay conditions, the experiments described in Table 3, using the concentrated viral inoculum of protocol 2, with and without the 2-h virus/cell (zero h and +2 h), were repeated along with an arm utilizing a more diluted inoculum for protocol 1 with a 1-h preincubation step (+1 h). The ability of ETV to inhibit NL4-3 growth was examined at an MOI of 0.006, using RT as an endpoint. Figure 2 shows the inhibition curves for the three conditions tested in parallel. The zero-h sample produced the lowest EC50 of 0.151 µM (compared to 0.056 µM at an MOI of 0.005, as shown in Table 3 and Fig. 1), while the EC50 using the +1-h virus/cell incubation was 0.287 µM. As noted in Table 3, preincubation with cell pellets for a longer time (+2 h) resulted in much lower susceptibility to ETV inhibition, this time producing an EC50 of 13.5 µM. The shapes of the inhibition curves also differed between the conditions. The zero-h inhibition curve was much flatter than the +2-h curve (both protocol 2), while the shape of the +1-h curve with diluted virus (protocol 1) was somewhat intermediate (Fig. 2). The flat-shaped inhibition curves may indicate a plateau or threshold of inhibition.
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Activity of ETV against HIV-1 containing the M184V LVD resistance substitution. Extensive studies and analyses of clinical samples from HBV-infected patients treated with ETV have demonstrated that HBV has decreased susceptibility to ETV when the M204V substitution is present and that clinical resistance requires the presence of M204V in HBV with additional ETV-specific substitutions (4, 18). The M204V substitution in the HBV RT is analogous to the M184V substitution in the HIV-RT, as they are both part of the YMDD motif (7). The recent case of an HIV/HBV-coinfected patient who exhibited an enrichment of HIV variants containing the M184V substitution while receiving ETV monotherapy (15) was of particular interest because of the overlapping resistance profiles of ETV and LVD when the equivalent M204V substitution was present in HBV (12). Therefore, the activity levels of ETV, LVD, AZT, and EFV against paired NL4-3 viruses with or without an M184V substitution were compared. Infection was performed at a low MOI (0.005) but without a virus/cell preincubation step in order to maximize ETV antiviral activity. The results, shown in Table 5, indicate that the presence of the M184V substitution reduced the virus' susceptibility to both ETV and LVD by >178-fold and >250-fold, respectively, while having no significant impact on AZT and EFV susceptibilities. A similar result was obtained with the PhenoSense assay performed at Monogram Biosciences, although the dynamic range of the assay was limited by the weak activity exhibited by ETV in the pseudotype assay. These data provide clear evidence that ETV can have antiviral activity against HIV-1 through the inhibition of HIV-1 RT and that the virus can escape via the LVD resistance pathway.
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2- or 8-fold increase in EC50 value), or LVD, starting at 0.2 µM (5-fold increase in EC50 value). At each subsequent passage, the concentration of the drug was doubled. Population sequencing of the HIV RT gene was performed at selected passages to monitor for sequence changes. For the selection starting at 0.1 µM ETV, virus passaged for seven cycles (30 days) in the presence of ETV (final concentration, 6.4 µM) contained a G-to-A mutation at nucleotide 552 of the RT gene (which encodes the M184I substitution), representing 20% of the total population. The prevalence of this mutation increased to 50% by passage eight (34 days; final concentration, 12.8 µM). For the selection starting at 0.4 µM ETV, this nucleotide change appeared at passage six (27 days; final concentration, 12.8 µM) in 50% of the virus population. Consequently, this mutation progressed into 80 to 90% of the virus population at passage seven (30 days; final concentration, 25.6 µM) and 100% of the virus population at passage eight (34 days; final concentration, 51.2 µM). For virus passaged with LVD, this same nucleotide change was observed at passage six (34 days; final concentration, 3.2 µM) in 30% of the virus population. This mutation increased to 100% of the virus population at passage seven (39 days; final concentration, 6.4 µM). During the selection of LVD resistance in patients, the first substitution usually detected is M184I, which under continued selection is reported to progress to the M184V change (9, 21). Susceptibility assays were performed with the starting NL4-3 wild-type strain and passage eight (34 days) virus, at which time the ETV final concentration was 51.2 µM and the M184I substitution was detected in 100% of the virus population. The M184I substitution induced a change in EC50 value from 0.75 µM to >10 µM (average of four assays). While the M184V substitution was not detected in this selection experiment, it is evident that the M184I substitution alone provides sufficient escape from drug pressure so that further evolution to M184V is likely to be unnecessary in vitro (3). Nonetheless, this result further confirms that ETV can inhibit HIV RT and that at very high concentrations, it has the potential to select for resistance in a manner similar to that of LVD. An additional study was conducted to determine if ETV could cause an enrichment of the M184V variant in a mixed population. A parallel study using LVD as the selective agent served as a control. In this experiment, the M184V variant and wild-type HIV-1 NL4-3 were mixed at a ratio of 1:100 (total MOI, 0.005) in the presence of 2.5 or 10 µM ETV (45-fold and 179-fold EC50, respectively) or 2.5 µM LVD (63-fold increase in the EC50) and incubated for 6 days, by which time CPE was observed. Sequence analyses of the harvested supernatants revealed that the treatment with the 2.5 µM dose of LVD resulted in an approximately 60-fold increase in the prevalence of the M184V variant in the population. Surprisingly, detectable levels of M184V were not observed for either of the ETV arms by population sequencing for reasons which remain unexplained at this time.
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These previously conducted studies conflict with a recent report by McMahon et al. (15), which indicated that ETV has potent activity (EC50, 0.1 nM) against HIV in a unique single-cycle, single-cell-based pseudovirus assay (24) with CD4+ lymphocytes using a green fluorescent protein reporter fluorescence-activated cell sorter assay as the endpoint. In their report, they also described that ETV monotherapy for HIV/HBV-coinfected patients resulted in a decrease in HIV levels of approximately 1 log10 in three patients and that enrichment of the M184V LVD resistance substitution occurred in one of these patients who had received prior HAART that included LVD. Our repeated assays utilizing both multicycle whole virus and pseudovirus single-cycle conventional methodologies failed to confirm such potent activity against HIV. These same assays and methodologies have accurately predicted the clinical activity of previously approved antiretroviral agents. Investigational studies into possible explanations for these differences identified MOI (the concentration of virus used to infect each cell) as a primary variable that could help explain the differences observed. While all of the HIV NRTIs show enhanced antiviral activity when lower MOIs are utilized, ETV appears to be particularly sensitive to this variable, giving a >100-fold increase in potency when the MOI is lowered by 10-fold, for reasons which remain unexplained (Table 3 and Fig. 1). The cell culture assay employed in the McMahon et al. study (15) involved measuring inhibition in individual cells, perhaps resulting in greater sensitivity to such a variable. Nonetheless, reducing the MOI, either directly or indirectly by eliminating the virus/cell preincubation step, clearly resulted in enhanced ETV activity. However, the titration curves become somewhat flatter, and results were more variable under these altered conditions. These flatter titration curves may indicate that ETV activity reaches a plateau or threshold. At this time, it is unclear what mechanism(s) is responsible for the unusual MOI-dependent boost in potency observed with ETV versus other HIV NRTIs, using a low viral inoculum.
Higher amounts of infecting virus are needed for the cell survival assays (XTT and MTS) than for the RT assay, likely reflecting the fact that XTT and MTS assays require significant cell death, while RT assays require only virus to be present in supernatants. This observation, along with the impact of virus/cell preincubation, likely provides a further basis for the reduced potencies observed in previous HIV assays using the cell survival endpoint. In addition, assays that used pseudotyped virus inoculum, including the commercial phenotyping assay (PhenoSense), also resulted in reduced ETV potency, presumably as a result of more efficient infection.
The current data confirmed that ETV has limited activity against HIV-1 (generally in the µM range), using traditional assay systems or a commercial phenotypic assay (PhenoSense). The observed ETV anti-HIV activity under reduced viral input appears to be directly related to the inhibition of the HIV RT, since assays using chronically infected (postintegration) cells failed to exhibit any antiretroviral activity. Importantly, ETV can select for the precursor M184I substitution in cell passage studies, and variants encoding the M184V substitution exhibited >178-fold reduced susceptibility to ETV. A recent study by Domaoal et al. (8) confirms that HIV-1 RT can utilize ETV-TP and that the M184V enzyme has lower affinity to ETV-TP than the wild-type enzyme.
The interpretation of these data is challenging, since standard assay methodologies predict that ETV should have no significant clinical activity against HIV under dosing conditions where the Cmax achieved in human blood is only 0.030 µM (23). However, the clinical data may tell a different story, as a patient taking the 1-mg ETV dose exhibited a selection/enrichment of the HIV-1 M184V variant. Treating HBV in HBV/HIV coinfection has been shown to reduce HIV-RNA levels, either by an indirect immunologic effect or potentially by diminishing some synergistic effect of coinfection such as the action of the HBV viral protein X, which has (at least in vitro) been shown to enhance the transactivation of HIV (1, 20). Similar reductions in HIV-1 RNA levels were noted in patients coinfected with HIV and herpes simplex virus (HSV) who received valacyclovir (14). However, the enhancement of the M184V substitution while a patient receives ETV therapy can occur only due to direct inhibitory pressure on HIV replication, and the enzyme study by Domaoal et al. supports this (8).
These results have important implications for drug discovery and profiling of antiviral compounds. The significant enhancement of ETV activity against HIV under conditions of reduced viral inoculum was not predicted using the standard assay conditions that have been optimized for robustness and reproducibility. In fact, little interest would be given to compounds that worked only under the modified conditions described here, since effective drugs need to demonstrate that they can work against high virus challenge. The lesson learned from the current studies is that non-HIV compounds need to be tested under reduced viral challenge to fully evaluate their degree of antiviral activity prior to being administered to HIV-infected patients.
ETV has been shown to be a potent agent for treating HBV in HIV/HBV-coinfected patients in whom HAART is currently being used to control their HIV infection (R. Colonno, R. Rose, C. Baldick, K. Pokornowski, B. Eggers, M. Plym, J. Fang, J. Yang, E. Ledesma, and D. Tenney, presented at the 13th Conference on Retroviruses and Opportunistic Infections, Denver, CO, 2006). The clinical relevance and the magnitude of the anti-HIV effect of ETV in coinfected patients cannot be assessed outside the context of a clinical study. However, due to its relatively weak activity against HIV, ETV should not be considered for use as an anti-HIV agent, and the use of ETV for the treatment of chronic HBV infection in HIV/HBV-coinfected patients in the absence of effective HAART is not recommended.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 3 March 2008.
Present address: Presidio Pharmaceuticals, 1700 Owens St., Suite 585, San Francisco, CA 94158.
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), or 2-h virus incubation with concentrated cells (
). MOI was 0.006 for each titration. Each point of the curves represents the mean value of triplicate RT activity. The percentage of control was calculated from the maximum RT activity under each condition.