ABSTRACT
The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and has been reported for several treatment-experienced individuals receiving DTG as part of salvage therapy. The R263K substitution seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG-treated individuals. Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand transfer and 3′ processing activities were measured biochemically. The combination of the R263K and T66I substitutions decreased HIV-1 infectivity, replicative capacity, and strand transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL. These findings suggest that the presence of the T66I substitution will not compromise the activity of DTG and may also help to prevent the additional generation of the R263K mutation. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution.
IMPORTANCE The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus type 1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution, whereas R263K is a signature resistance substitution against dolutegravir. In order to determine how different combinations of integrase resistance mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir therapy who harbor the T66I resistance substitution.
INTRODUCTION
Recent strategies to treat HIV-1 infection involve the use of integrase strand transfer inhibitors (INSTIs), which are the most potent antiretroviral drugs (ARVs) to date and include raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) (1). Despite this, the emergence of drug resistance mutations in integrase (IN) represents a concern for the future use of these drugs, and various resistance mutations against RAL and EVG that are associated with treatment failure have been characterized (2). There is also a high degree of cross-resistance between RAL and EVG, since the major resistance substitutions for RAL are located at positions G140, Y143, Q148, and N155, while those for EVG are located at positions T66, E92, G140, S147, Q148, and N155 (1, 3). Although resistance during initial therapy has not yet been reported for DTG, patients can fail DTG therapy if they were previously treated with RAL or EVG and possess relevant mutations for these drugs (4–6).
In contrast, an R263K substitution was selected in tissue culture with DTG, and this substitution has been reported for several treatment-experienced, INSTI-naive individuals who were not fully suppressed when receiving DTG-based therapy (7). We showed that the R263K mutation alone or in combination with other secondary mutations confers low-level resistance to DTG and that viruses containing the R263K substitution possess significantly reduced viral replication capacity (8–10).
It is also notable that the R263K substitution has been shown to emerge secondary to the T66I substitution during tissue culture selections with EVG (11). Here, we have examined the effect of combining the T66I and R263K substitutions on HIV-1 viral replicative capacity and levels of resistance against various INSTIs, and we have also studied this in the context of the secondary E138K mutation that commonly arises during the emergence of clinically relevant resistance to both RAL and EVG.
MATERIALS AND METHODS
Cells and reagents.
TZM-bl and HEK 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM). PM-1 cells were cultured in Roswell Park Memorial Institute (RPMI) medium. Both DMEM and RPMI medium were supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. Cord blood was obtained from the Department of Obstetrics, Jewish General Hospital, Montréal, Canada. Primary human cord blood mononuclear cells (CBMCs) were isolated from cord blood by using Ficoll-Hypaque (GE Healthcare Life Sciences), and CBMCs were stimulated with phytohemagglutinin. CBMCs were grown in RPMI medium. Cells were maintained at 37°C under 5% CO2. RAL, EVG, and DTG were provided by Merck & Co. Inc., Gilead Sciences, and ViiV Healthcare Inc., respectively.
Generation of replication-competent genetically homogenous virus.
pNL4-3IN(T66I), pNL4-3IN(R263K), pNL4-3IN(T66I/R263K), and pNL4-3IN(T66I/E138K/R263K) were produced by using site-directed mutagenesis. The production of plasmids pNL4-3IN(R263K) and pNL4-3IN(E138K/R263K) was reported previously (10). The primers used for T66I mutagenesis were sense primer 5′-CCAGGAATATGGCAGCTAGATTGTATACATTTAGAAGGAAAAGTT-3′ and antisense primer 5′-AACTTTTCCTTCTAAATGTATACAATCTAGCTGCCATATTCCTGG-3′. All plasmids were verified by sequencing. To produce replication-competent genetically homogenous viruses, 12.5 μg of plasmid pNL4-3IN(WT), pNL4-3IN(T66I), pNL4-3IN(R263K), pNL4-3IN(T66I/R263K), or pNL4-3IN(T66I/E138K/R263K) was used to transfect HEK 293T cells by using Lipofectamine 2000 (Invitrogen). Fresh medium was added at 4 h posttransfection. After 48 h, culture fluids were harvested and passed through a 0.45-μm filter. Quantification of viruses was performed by using p24 and reverse transcriptase (RT) assays as described previously (12).
Tissue culture selections with RAL or EVG.
CBMCs were infected with NL4.3 virus with wild-type integrase [NL4.3IN(WT)], NL4.3 virus with the T66I substitution in integrase [NL4.3IN(T66I)], or NL4.3IN(E138K/R263K) virus and then grown in the presence of increasing concentrations of RAL or EVG. Viral replication in culture was monitored by an RT assay, and aliquots of culture fluids were collected weekly. Viral RNA was extracted from the aliquots by using an RNA extraction kit (Qiagen) and amplified by RT-PCR (Life Technologies), as previously described (13). The PCR products were then sequenced to detect the emergence of drug resistance mutations.
HIV-1 infectivity and replicative capacity.
HIV-1 infectivity was measured by a short-term TZM-bl assay. Briefly, 30,000 TZM-bl cells/well were infected with serially diluted viruses in a 96-well flat-bottom plate. Cells were lysed 48 h after infection, and luciferase levels were measured to directly monitor short-term infectivity. Fold decreases in infectivity were represented as the relative 50% effective concentration (EC50), which is the amount of virus (previously quantified by using an RT assay) needed for TZM-bl cells to produce half of the maximal level of luciferase in an infection. HIV-1 replicative capacity was measured as counts per minute in PM-1 cells following HIV-1 infection over 21 days. Both assays were described previously (14).
Susceptibility to antiretroviral compounds.
Susceptibilities of virus to ARVs were measured by the addition of serially diluted DTG, RAL, or EVG to TZM-bl cells prior to infection with the viruses described above. Luciferase levels were measured after 48 h of incubation, similar to the protocol for the infectivity assay described above, and 50% inhibitory concentrations (IC50s) were determined.
Generation of plasmids for integrase protein expression and purification.
pET-15b expression plasmids coding for either the wild-type (WT) soluble integrase or the soluble integrases that contained the R263K or E138K/R263K substitutions were generated by using site-directed mutagenesis, as previously described (10). The T66I, T66I/R263K, and T66I/E138K/R263K mutations were produced by using the primers described above. The pET-15b plasmids were then used to express recombinant proteins in BL21(DE3) bacterial cells. The protocol for protein expression and purification of His-tagged integrase was described previously (9).
Cell-free strand transfer assay.
Integrase strand transfer activities of the WT integrase enzyme and integrase proteins containing the T66I, R263K, T66I/R263K, E138K/R263K, or T66I/E138K/R263K substitutions were measured as previously described (15). Briefly, 300 nM processed long terminal repeat (LTR)-DNA duplexes were coated onto Costar 96-well DNA-binding plates (Corning) by incubation overnight at 4°C. The plates were washed once with blocking buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 0.25% bovine serum albumin [BSA]) and then incubated with the same buffer for 30 min at 37°C or overnight at 4°C. Immediately before the strand transfer assay, plates were washed once with phosphate-buffered saline (PBS) (pH 7.4) and assay buffer {50 mM morpholinepropanesulfonic acid (MOPS) (pH 6.8), 0.15% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM NaCl, 30 mM MnCl2, 50 μg/ml BSA}. A total of 400 nM purified integrase proteins was resuspended in assay buffer with 5 mM dithiothreitol (DTT) and added to the microplates for a 30-min incubation at room temperature. Serially diluted biotinylated target DNA (0 to 60 nM) was then added to each well for 1 h at 37°C. The plates were then washed twice with wash buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20, 2 mg/ml BSA). A streptavidin-Eu solution (50 μM diethylenetriaminepentaacetic acid [DTPA], 0.025 μg/ml Eu-labeled streptavidin) diluted in wash buffer was added for 30 min at room temperature. Finally, the plates were washed twice with wash buffer, and 80 μl Wallac enhancement solution (PerkinElmer) was added. Time-resolved fluorescence was read by using a FlUOstar Optima multilabel plate reader (BMC Labtech).
3′ processing assay.
The 3′ processing activities of the WT integrase enzyme or enzymes containing the T66I, R263K, T66I/R263K, E138K/R263K, and T66I/E138K/R263K substitutions were measured as described previously (16). The 3′ processing assay was similar to the strand transfer assay. Serial dilutions of the unprocessed LTR-DNA duplex with 3′ biotinylation were used to coat the plates at concentrations of between 0 and 40 nM. After the addition of purified integrase proteins, the plates were incubated for 2 h to allow 3′ processing to occur.
Data analysis.
Each experiment was performed at least twice using three or four replicate samples. Data analysis was performed by using GraphPad Prism 5.0.
RESULTS
Emerging substitutions in NL4.3IN(WT), NL4.3IN(T66I), and NL4.3IN(E138K/R263K) under RAL or EVG drug pressure.
To confirm previous findings and verify the possibility of T66I/E138K/R263K triple substitutions, selection studies were performed by using CBMCs infected with NL4.3IN(WT), NL4.3IN(R263K), and NL4.3IN(E138K/R263K) with increasing concentrations of RAL or EVG (Table 1). Together with several substitutions, the T66I substitution emerged from the NL4.3IN(WT) or NL4.3IN(R263K) virus under RAL or EVG pressure, respectively, and from NL4.3IN(E138K/R263K) with both drugs. In contrast, the T66I substitution was not detected when RAL selection experiments were initiated with a virus containing the R263K substitution, nor did it emerge from the WT virus under EVG pressure.
TABLE 1.
New substitutions emerging from NL4.3IN(WT), NL4.3IN(R263K), and NL4.3IN(E138K/R263K) infections of CBMCs under RAL or EVG drug pressure at week 30
| Drug | Concn (μM) | New substitution(s) emerging from virus |
||
|---|---|---|---|---|
| WT | R263K | E138K/R263K | ||
| RAL | 0.05–2.5 | T66I, T97A, G163R | H51N, T66I, T97A, S119R, Y143H | |
| EVG | 1 | N155H, R263K | M50I, T66I | M50I, T66I, S119R, S147G |
Combining the T66I and R263K substitutions impairs viral infectivity.
To determine the effects of the T66I, E138K, and R263K substitutions on viral infectivity, TZM-bl cells were infected with NL4.3IN(WT), NL4.3IN(T66I), NL4.3IN(R263K), NL4.3IN(T66I/R263K), or NL4.3IN(T66I/E138K/R263K) virus (Fig. 1). The NL4.3IN(T66I), NL4.3IN(R263K), and NL4.3IN(T66I/E138K/R263K) viruses showed only slight impairments in infectivity relative to that of the WT (Fig. 1a and c), whereas NL4.3IN(T66I/R263K) displayed a significant defect in infectivity (Fig. 1b). Relative infectivity was decreased by 8-fold by the T66I/R263K combination of substitutions. The addition of E138K to T66I/R263K partially restored infectiousness (1.45-fold decrease in infectivity relative to that of the WT) (Fig. 1d).
FIG 1.
Viral infectivity in TZM-bl cells. (a to c) TZM-bl cells were infected with NL4.3IN(WT), NL4.3IN(T66I), or NL4.3IN(R263K) (a); NL4.3IN(WT) or NL4.3IN(T66I/R263K) (b); or NL4.3IN(WT) or NL4.3IN(T66I/E138K/R263K) (c) virus over 48 h, and luciferase levels were measured. Infectivity of NL4.3IN(WT) virus is represented for comparison. RLU, relative light units. (d) The fold decrease in infectivity was calculated. Error bars indicate means ± standard deviations.
The T66I/R263K combination of substitutions impairs viral replicative capacity.
A single cycle of infection does not always capture replicative defects. Therefore, we also assessed the long-term replicative capacity of different viruses that contained the T66I substitution using PM-1 cells. Infections were carried out with NL4.3IN(WT), NL4.3IN(T66I), NL4.3IN(R263K), NL4.3IN(T66I/R263K), or NL4.3IN(T66I/E138K/R263K) over 21 days (Fig. 2). RT activity was measured in culture fluids at days 3, 7, 14, and 21. Similar to the results of the TZM-bl infectivity assay, we found that the T66I substitution alone had little effect on viral replicative capacity (Fig. 2a), and the R263K substitution decreased viral replication to a similar extent, as previously reported (12). In contrast, the NL4.3IN(T66I/R263K) virus showed a major defect in replicative capacity (Fig. 2c). Although the T66I/R263K-containing virus yielded levels of RT activity at day 3 that were similar to those of the other viruses tested, replication gradually decreased over the subsequent 18 days, while the other viruses attained higher levels of replication at or after day 7. In particular, the NL4.3IN(T66I/E138K/R263K) virus showed a partially restored replicative capacity in comparison to that of the NL4.3IN(T66I/R263K) virus.
FIG 2.
Viral replicative capacity in PM-1 cells. PM-1 cells were infected with NL4.3IN(WT) or NL4.3IN(T66I) (a), NL4.3IN(R263K) (b), NL4.3IN(T66I/R263K) (c), and NL4.3IN(T66I/E138K/R263K) (d) viruses over 21 days. The replicative capacity of the above-mentioned viruses was normalized to RT levels of the NL4.3IN(WT) virus at day 7. Supernatants were collected at days 3, 7, 14, and 21, at which time RT levels were measured as counts per minute. Error bars indicate means ± standard deviations.
Strand transfer activities of recombinant integrase containing the T66I, E138K, and/or R263K substitution.
To determine whether the deficits in replicative capacity observed with mutated viruses in PM1 cells were caused by changes in integrase activity, cell-free biochemical strand transfer assays were performed by using purified recombinant integrases containing the T66I, E138K, and/or R263K substitution. Maximal enzyme activity (Vmax) and the amount of target LTR-DNA used to reach half Vmax (1/2MaxDNA) were calculated for each of the recombinant integrase enzymes. The relative Vmax was measured for each recombinant integrase, and maximal strand transfer activity of the WT integrase was arbitrarily set at 100% (Table 2). The results show that the presence of the T66I substitution increased the 1/2MaxDNA value by 2.4-fold while decreasing the Vmax to 67% of the WT level. Similarly, the R263K substitution increased the 1/2MaxDNA value by 2-fold, and the E138K/R263K substitutions in tandem decreased the Vmax to 38.7% of the WT level. For T66I/R263K substitutions, the 1/2MaxDNA value was increased by 2.3-fold, and the Vmax decreased to 13% of the WT level. The three substitutions T66I, E138K, and R263K together resulted in a slightly decreased 1/2MaxDNA value (1.2-fold), but the Vmax was only 17% of the WT level.
TABLE 2.
Strand transfer activity of recombinant subtype B integrase enzymes containing the T66I, E138K, and/or R263K substitutiona
| Virus genotype | Relative Vmax (%) | 95% CI for relative Vmax (%) | 1/2MaxDNA (nM) | 95% CI for 1/2MaxDNA (nM) |
|---|---|---|---|---|
| WT | 100 | 91.6–108.5 | 10.15 | 7.5–12.8 |
| T66I | 67.3 | 56.4–78.2 | 24.4 | 15.5–33.2 |
| R263K | 89.1 | 74–104.3 | 20.1 | 12.1–28.2 |
| T66I/R263K | 13.0 | 10.4–15.6 | 23.2 | 12.9–33.6 |
| E138K/R263K | 38.7 | 32–45.5 | 5.15 | 2.0–8.3 |
| T66I/E138K/R263K | 17.1 | 13.9–20.2 | 8.4 | 3.5–13.3 |
CI, confidence interval.
3′ processing activity of recombinant integrase enzymes containing the T66I, E13K8, and/or R263K substitution.
3′ processing is a rate-limiting step in HIV-1 integration, and a 3′ processing assay was performed to determine the effects of the T66I, E138K, and R263K substitutions on enzyme activity. The results show that no significant differences were observed among the various recombinant integrase enzymes that were tested (Table 3).
TABLE 3.
3′ processing activity of recombinant subtype B integrase containing the T66I, E138K, and/or R263K substitution
| Virus genotype | Relative Vmax (%) | 95% CI for relative Vmax (%) | 1/2MaxDNA (nM) | 95% CI for 1/2MaxDNA (nM) |
|---|---|---|---|---|
| WT | 100 | 81.9–118.1 | 1.3 | 0.63–1.9 |
| T66I | 70.85 | 55.8–85.9 | 0.95 | 0.33–1.58 |
| R263K | 69.2 | 55.7–82.6 | 0.99 | 0.41–1.6 |
| T66I/R263K | 106.7 | 77–135.7 | 1.7 | 0.53–2.9 |
| E138K/R263K | 126.2 | 98.1–154.4 | 1.83 | 0.85–2.8 |
| T66I/E138K/R263K | 97.6 | 76.2–114.9 | 1.26 | 0.55–1.97 |
Virus with the T66I/R263K combination of substitutions confers significant resistance to EVG but remains susceptible to DTG.
The T66I substitution in integrase was previously reported to confer major resistance to EVG while increasing HIV-1 susceptibility to DTG. Previously, we showed that the R263K substitution, alone or in combination with several secondary mutations, conferred moderate-level resistance to EVG and low-level resistance against DTG (8–10). Here, we conducted resistance assays using TZM-bl cells to determine the effects of the T66I substitution combined with the R263K and/or the E138K secondary substitution on resistance to each of the drugs DTG, RAL, and EVG (Table 4). Compared to the IC50 of the WT, and in agreement with data from previous studies (17), the T66I substitution alone increased susceptibility to DTG by 1,000-fold and conferred low-level resistance against RAL (2.4-fold) and higher-level resistance against EVG (10-fold). Viruses containing the T66I/R263K combination of substitutions were susceptible to DTG (0.089-fold), slightly resistant to RAL (1.6-fold), and more resistant to EVG (22-fold). The T66I/E138K/R263K virus was susceptible to DTG (0.027-fold), slightly resistant to RAL (2.5-fold), and highly resistant to EVG (164-fold).
TABLE 4.
Susceptibility of NL4.3IN(WT), NL4.3IN(T66I), NL4.3IN(R263K), NL4.3IN(T66I/R263K), and NL4.3IN(T66I/E138K/R263K) viruses to DTG, RAL, and EVG relative to the NL4.3IN(WT) virusa
| Virus genotype | DTG |
RAL |
EVG |
||||||
|---|---|---|---|---|---|---|---|---|---|
| IC50 (nM) | 95% CI (nM) | FC | IC50 (nM) | 95% CI (nM) | FC | IC50 (nM) | 95% CI (nM) | FC | |
| WT | 0.3 | 0.2–0.5 | 1 | 1.3 | 0.3–5.3 | 1 | 6.4 | 2.5–16.6 | 1 |
| T66I | 0.0004 | 0.0002–0.0007 | 0.001 | 3.2 | 1.3–7.8 | 2.4 | 61 | 42.6–87.6 | 10 |
| R263K | 1.5 | 1.2–1.7 | 4.8 | 1.8 | 0.8–4.2 | 1.3 | 28 | 16.3–47 | 4 |
| T66I/R263K | 0.03 | 0.02–0.04 | 0.09 | 2.2 | 0.5–8.9 | 1.6 | 141 | 95–209.3 | 22 |
| T66I/E138K/R263K | 0.008 | 0.004–0.02 | 0.03 | 3.4 | 0.9–12.6 | 2.5 | 1,054 | 881.6–1,261 | 164 |
FC, fold change.
DISCUSSION
The T66I substitution was originally described as a change in integrase selected in tissue culture under EVG pressure. It was later shown to be common in the genomes of viruses isolated from individuals failing EVG treatment (18). Other substitutions that are associated with treatment failure under EVG-based therapy include E92Q, G140S/A, S147G, Q148H/R/K, and N155H (18), the latter of which also emerged from the WT virus under EVG pressure in this study (Table 1). The T66I substitution can also be found in viruses from individuals who have experienced treatment failure with RAL although more rarely (19). This may be due to the high versus low levels of resistance conferred by this substitution against EVG and RAL, respectively (17), an observation that we have confirmed here (Table 4). In addition, we have confirmed that the T66I substitution does not confer resistance against DTG but significantly increases HIV-1 susceptibility to this drug. Structural models derived from the crystal structure of the prototype foamy virus integrase protein suggest that the T66I substitution might increase susceptibility to DTG by disrupting an electrostatic interaction between T66 and N155 (15). No single integrase substitution besides R263K has ever been shown to confer significant levels of resistance against DTG (17, 20), helping to explain the prevalence of the R263K substitution in some treatment-experienced, INSTI-naive individuals who experienced DTG-based treatment failure (7).
We have shown previously that the R263K substitution is also associated with decreases in viral DNA integration and viral replication capacity (8), suggesting that the development of resistance both in tissue culture and in vivo involves a balance between levels of resistance and replicative capacity. The emergence of the T66I/R263K combination of substitutions in tissue culture selections with EVG has been documented (11, 21), and we show here that this combination severely impaired both integrase strand transfer activity and HIV-1 replicative capacity (Table 2 and Fig. 2, respectively). In contrast, the T66I/R263K combination of substitutions has not been observed in the presence of RAL (19, 22), suggesting that the low levels of resistance against RAL that are associated with this combination are not sufficient to compensate for deficits in replication capacity, which are related to decreased strand transfer integrase activity but not 3′ processing activity (Tables 2 and 3).
The positive effect of the E138K substitution on strand transfer activity seems to be due to an improvement in DNA-binding activity, as shown by decreases in 1/2MaxDNA values when this substitution was present (Table 2). In contrast, the E138K substitution had little effect on maximal strand transfer activity. These findings correlate with the E138K-associated partial compensation for defects in infectivity and replicative capacity that were observed with the T66I/R263K combination of mutations (Fig. 1 and 2). The addition of the E138K substitution to the T66I/R263K combination also increased the levels of resistance against RAL and EVG by 1.5- and 7.5-fold, respectively (Table 4).
Importantly, the addition of R263K to the T66I mutation did not confer resistance against DTG, although it may have moderated the increase in susceptibility to this drug that was associated with the T66I mutation alone (Table 4). Furthermore, the T66I/R263K combination of substitutions severely impaired viral replicative capacity (Fig. 2). This suggests that patients who experience EVG-based treatment failure with an emergent T66I substitution can be successfully treated with DTG and may not be able to develop the R263K substitution in combination with T66I. Given the high prevalence of the latter substitution in individuals who have failed EVG (18), our results provide additional justification for the use of DTG in second-line therapy after development of the T66I mutation.
In this study, we also tested the ability of E138K, a secondary substitution that has been observed together with the R263K substitution in tissue culture, to act together with the T66I/R263K combination of mutations to modulate strand transfer activity and replicative capacity (Table 2 and Fig. 1 and 2). However, viruses that contain the T66I/E138K/R263K combination of substitutions remained highly susceptible to DTG (Table 4).
ACKNOWLEDGMENTS
J.L. performed experiments, analyzed the data, and wrote the initial manuscript. T.M. designed and performed experiments, analyzed the data, and corrected the manuscript. M.O. and K.A. performed experiments. M.A.W. supervised the project and revised the manuscript. All authors read and approved the final version of the paper.
This work was supported by the Canadian Institutes for Health Research (CIHR).
We declare that we have no conflicts of interest.
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