Journal of Antimicrobial Chemotherapy Advance Access published July 4, 2015
J Antimicrob Chemother doi:10.1093/jac/dkv176
Dolutegravir maintains a durable effect against HIV replication in tissue culture even after drug washout Nathan Osman1,2, Thibault Mesple`de1, Peter K. Quashie1,2, Maureen Oliveira1, Veronica Zanichelli1 and Mark A. Wainberg1,2* 1
McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada; 2Department of Microbiology and Immunology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada *Corresponding author. E-mail: [email protected]
Objectives: Of the currently approved HIV integrase strand transfer inhibitors (INSTIs), dolutegravir has shown greater efficacy than raltegravir at suppressing HIV-1 replication in treatment-experienced individuals. Biochemical experiments have also shown that dolutegravir has a longer dissociative half-life when bound to HIV integrase than does raltegravir. In order to study the intracellular efficacy of various INSTIs, we asked whether drug removal from INSTI-treated HIV-1-infected cells would result in different times to viral rebound. In addition, we assessed the role of the R263K substitution within the integrase ORF that is associated with low-level resistance to dolutegravir. Methods: HIV-infected MT-2 cells were treated with dolutegravir, raltegravir or a third experimental INSTI (MK-2048) and the drugs were washed out after varying times. Viral replication was monitored by measuring reverse transcriptase (RT) activity in the culture fluids. Results: We observed a significantly slower increase in RTactivity after the removal of dolutegravir compared with raltegravir or MK-2048. The incubation time before the drug was removed also had an impact on the level of RT activity independently of the drug and virus used. The R263K substitution did not significantly impact on levels of RT activity after drug washout, suggesting that dolutegravir remained tightly bound to the integrase enzyme despite the presence of this mutation. Conclusions: These results suggest that the residency time of INSTIs on integrase is a key factor in the activity of these drugs and that the anti-HIV activity of dolutegravir persists more effectively than that of other INSTIs after drug washout.
Introduction The development of combined ART has significantly increased the life expectancy and quality of life of HIV-infected individuals. The latest class of drugs to have been developed against HIV-1 are the integrase strand transfer inhibitors (INSTIs), of which raltegravir and elvitegravir were the first to be approved. Although these compounds are potent,1,2 they have a relatively low genetic barrier for resistance in patients failing therapy.3,4 In contrast, dolutegravir has a high genetic barrier for resistance as well as limited crossresistance to raltegravir and elvitegravir.5 – 7 This notwithstanding, tissue culture selections with dolutegravir have identified an R263K substitution in HIV-1 integrase that confers low-level resistance to this drug.8 In contrast to primary resistance substitutions against raltegravir or elvitegravir, no secondary substitutions that can compensate for the replicative defects associated with R263K have been
identified.9 – 11 This could be due either to the inability of R263Kcontaining dolutegravir-resistant viruses to acquire additional resistance substitutions12 or to the ability of dolutegravir to inhibit HIV genetic evolution.13 The SAILING clinical study reported the presence of this same substitution in two INSTI-naive patients who had failed dolutegravir treatment among a total of 354 patients, the vast majority of whom responded well to treatment.14 In contrast, mutations associated with raltegravir can confer crossresistance to dolutegravir, which was shown in the VIKING trial in which treatment-experienced individuals who had failed raltegravir and who carried raltegravir-associated resistance substitutions were treated with dolutegravir. Seven such subjects subsequently failed dolutegravir-based regimens without acquiring the R263K substitution,15 demonstrating the vulnerability of dolutegravir when used in salvage therapy. In contrast, the SPRING and FLAMINGO clinical trials demonstrated the superiority of dolutegravir compared with efavirenz-based and darunavir-based regimens,
# The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
1 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
Received 22 April 2015; returned 27 May 2015; revised 29 May 2015; accepted 31 May 2015
Osman et al.
respectively, in first-line therapy.16,17 Moreover, no patient who has received dolutegravir in first-line therapy has been shown to possess resistance mutations associated with either dolutegravir itself or the nucleoside compounds with which it has been co-administered during treatment.16 Biochemical experiments have also shown that dolutegravir has a far-longer dissociative half-life for integrase than both raltegravir and elvitegravir.18 Hence, we were interested in knowing whether drug washout from INSTI-treated infected cells would result in differences among these various compounds with regard to the times of viral rebound in tissue culture.
Materials and methods
The generation of the pNL4-3IN(R263K) plasmid has previously been reported.8 Genetically homogeneous viral stocks were produced by transfecting 293T cells with the pNL4-3 and pNL4-3IN(R263K) plasmids using Lipofectamine 2000 (Life Technologies, Burlington, ON, Canada) according to the manufacturer’s instructions. Forty-eight hours after transfection, the cell culture fluids were harvested, treated with Benzonase (Sigma-Aldrich, Oakville, ON, Canada) and filtered at 0.45 mm to remove plasmids and cell debris, respectively. The viruses were then aliquotted and stored at 2808C. Viral stocks were quantified by measuring both the p24 content and the cell-free reverse transcriptase (RT) activity.
Determination of the TCID50/mL
Cells and reagents
Determination of drug concentrations and IC90s in MT-2 cells The susceptibility of HIV to dolutegravir, raltegravir and MK-2048 was measured by the infection of 50 000 MT-2 cells with the same amount
(b) 1 000 000
WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D1 WT 93.75 nM RAL WT 93.75 nM RAL D1 WT 10 nM MK-2048 WT 10 nM MK-2048 D1
100 000 10 000 1000 100
0
5 10 Days post-infection
DTG
1 000 000 RT activity
(a)
The TCID50/mL was determined as described by Johnson and Byington,19 using MT-2 cells and calculated using the Spearman– Karber formula.
WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D1 WT 41.7 nM DTG D2 WT 41.7 nM DTG D3
100 000 10 000 1000 100
15
0
5 10 Days post-infection
15
RAL WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D2 WT 93.75 nM RAL WT 93.75 nM RAL D2 WT 10 nM MK-2048 WT 10 nM MK-2048 D2
100 000 10 000 1000
1 000 000 RT activity
RT activity
1 000 000
100 0
5 10 Days post-infection
10 000 1000 100
15
WT no drug WT 93.75 nM RAL WT 93.75 nM RAL D1 WT 93.75 nM RAL D2 WT 93.75 nM RAL D3
100 000
0
5 10 Days post-infection
15
MK-2048 WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D3 WT 93.75 nM RAL WT 93.75 nM RAL D3 WT 10 nM MK-2048 WT 10 nM MK-2048 D3
100 000 10 000 1000 100 0
5 10 Days post-infection
15
1 000 000 RT activity
RT activity
1 000 000
100 000
WT no drug WT 10 nM MK-2048 WT 10 nM MK-2048 D1 WT 10 nM MK-2048 D2 WT 10 nM MK-2048 D3
10 000 1000 100
0
5 10 Days post-infection
15
Figure 1. Variations among various INSTIs with regard to the durability of anti-HIV effects after drug washout. Viral replication was monitored by measuring RT activity in the culture fluids for up to 2 weeks after drug removal. (a) Comparisons of drug washout after 1, 2 or 3 days (top panel, middle panel and bottom panel, respectively). (b) Comparisons of washout at each of Days 1, 2 and 3 using dolutegravir (top panel), raltegravir (middle panel) and MK-2048 (bottom panel). DTG, dolutegravir; RAL, raltegravir; D1, Day 1; D2, Day 2; D3, Day 3.
2 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
The 293T cell line was obtained from the ATCC (CRL-11268). The MT-2 cell line was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, courtesy of Dr Douglas Richman. The cells were subcultured every 3 – 4 days in DMEM for 293T cells or RPMI medium for MT-2 cells, both supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin and maintained at 378C under 5% CO2. Merck, Inc. kindly provided raltegravir and MK-2048, and dolutegravir was kindly supplied by ViiV Healthcare Inc.
RT activity
Generation of replication-competent genetically homogeneous HIV-1
JAC
Persistence of dolutegravir activity after washout
Table 1. Comparison of viral replication of WT virus after 1, 2 or 3 days of treatment of MT-2 cells with dolutegravir, raltegravir and MK-2048 at 20× the IC90 Dolutegravir
Days to attainment of 50% of maximal RT activity after drug removal Days to attainment of maximal RT activity after drug removal
Raltegravir
MK-2048
1
2
3
1
2
3
1
2
3
8 10
7 9
6 8
3 6
3 5
4 8
7 13
7 9
7 11
In the case of the no-drug control, 50% and maximal levels of RT activity in culture fluids were observed after 3 and 4 days, respectively.
DTG
RT activity
1 000 000
10 000 1000 100
Infection of MT-2 cells with genetically homogeneous HIV-1 and drug washout
0
5 10 Days post-infection
15
RAL 1 000 000
RT activity
Washout experiments were performed as follows. In each instance, 300000 cells were pelleted and resuspended in 1 mL of medium containing the appropriate amount of virus and incubated for 2 h at 378C. The cells were then repelleted and the supernatant was discarded to eliminate any unbound virus. The cells were then resuspended in 2 mL of fresh medium containing the appropriate concentration of drug. Each drug was used at either low or high concentrations corresponding to 5× and 20× the IC90, respectively. To remove the drug from the medium, the cells were diluted in 10 mL of fresh medium and pelleted. The culture fluids were discarded and the cells were resuspended in 10 mL of fresh medium and pelleted again. The cells were then resuspended in 2 mL of fresh medium and observed for viral replication.
WT no drug WT 10.4 nM DTG WT 41.7 nM DTG WT 10.4 nM DTG D3 WT 41.7 nM DTG D3
100 000
WT no drug WT 23.4 nM RAL WT 93.75 nM RAL WT 23.4 nM RAL D3 WT 93.75 nM RAL D3
100 000 10 000 1000 100
0
5 10 Days post-infection
15
MK-2048
Monitoring of HIV-1 infection Infection was monitored by measuring the RT activity in the culture fluids, as previously described.20 To evaluate the number of days needed to reach 50% of maximal RT activity, the upward portions of slopes were fitted to the dose – response stimulation model using GraphPad Prism software, and the replication rate was evaluated as the Hill slope of the fit models.
RT activity
1 000 000
WT no drug WT 2.5 nM MK-2048 WT 10 nM MK-2048 WT 2.5 nM MK-2048 D3 WT 10 nM MK-2048 D3
100 000 10 000 1000 100 0
Results The washout of dolutegravir from infected cells results in a more prolonged anti-HIV effect than does the removal of raltegravir or MK-2048 MT-2 cells were infected with HIV-1 NL4-3IN(WT) and the cells were treated with dolutegravir, raltegravir or MK-2048 at 20× the IC90 for 1, 2 or 3 days. After these times, the cells were washed and fresh medium was added. We observed that viral replication, as measured by levels of RT activity in the culture fluids, was significantly more delayed after the removal of dolutegravir than that of raltegravir (Figure 1 and Table 1). Furthermore, differences of up to 4 days with respect to the attainment of maximal RT activity were noted between raltegravir and dolutegravir when the cells were treated for 1 or 2 days (Figure 1a).
5 10 Days post-infection
15
Figure 2. Concentration-dependent effects of INSTI washout after Day 3. Viral replication was monitored by measuring RT activity in the culture fluids for up to 2 weeks thereafter. Controls are represented by continuous lines. DTG, dolutegravir; RAL, raltegravir; D3, Day 3.
In order to better understand the relationships between the drugs with regard to their antiviral effect, the data in Figure 1(a) were replotted in Figure 1(b) so that direct comparisons among the INSTIs that were used are obvious in each case. It is clear that more profound drug effects were observed as the time of exposure to the drug increased (Figure 1b and Table 1). Although viral rebound after drug washout occurred at similar levels regardless of the INSTI employed, the use of dolutegravir
3 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
of either WT or R263K-containing viruses, based on RT activity, in the presence of 1: 2 serial dilutions of the drugs. After 5 days, the activity of RT was quantified in the culture fluids and the IC90 values of the different drugs were calculated using GraphPad Prism software. The drug concentrations that were chosen for this study corresponded to 5× and 20× the IC90 for each drug evaluated in order to ensure that there would be an optimal suppression of viral replication prior to drug washout.
Osman et al.
Table 2. Comparison of viral replication of WT virus after 3 days of treatment with different concentrations of dolutegravir, raltegravir and MK-2048 Dolutegravir
Days to attainment of 50% of maximal RT activity after drug removal Days to attainment of maximal RT activity after drug removal
Raltegravir
MK-2048
10.4 nM
41.7 nM
23.4 nM
93.75 nM
2.5 nM
10 nM
4 8
6 8
2 4
4 8
3 6
7 11
In the case of the no-drug control, 50% and maximal levels of RT activity in culture fluids were observed after 3 and 4 days, respectively. For each drug used, the concentrations used represent 5× and 20× the IC90, respectively.
DTG RT activity
1 000 000
WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D3 R263K no drug R263K 41.7 nM DTG R263K 41.7 nM DTG D3
100 000 10 000 1000 100
0
5 10 Days post-infection
15
RAL
RT activity
Next, we compared two different concentrations of each of dolutegravir, raltegravir and MK-2048 in our washout protocols (Figure 2). The results show that viral replication resumed earlier after the removal of the drug when a lower concentration of the drug was employed, independent of the drug used (Table 2). A difference of 2.4 days to reach 50% of maximal RT activity was observed in a comparison of dolutegravir and raltegravir, with a corresponding value of 4.6 days for MK-2048. The data show that the lowest drug concentrations of dolutegravir, raltegravir and MK-2048 that were used in this work as controls were suboptimal as the virus was able to replicate even when drug was not removed from the medium.
10 000 1000
In order to evaluate the impact of the R263K substitution on the ability of HIV to rebound after drug washout, we infected MT-2 cells with HIV-1 NL4-3IN(WT) or NL4-3IN(R263K) viruses. The cells were treated with dolutegravir, raltegravir or MK-2048 at 20× the IC90 for 3 days. The results show that very similar RT activity levels were seen with the WT and R263K viruses in the presence of either dolutegravir or MK-2048 (Figure 3 and Table 3). In the case of raltegravir, R263K virus replication reached 50% of maximal activity after 3 additional days compared with WT; in addition, a significantly higher maximal RT activity was attained for the R263K virus, i.e. 72% in the presence of raltegravir compared with WT in the absence of the drug, compared with 26% for WT in the presence of the drugs (P,0.05; Student’s t-test). This is consistent with previous observations that the R263K substitution increases the susceptibility of HIV to raltegravir.8
4 of 6
0
5 10 Days post-infection
15
MK-2048 1 000 000
WT no drug WT 10 nM MK-2048 WT 10 nM MK-2048 D3 R263K no drug R263K 10 nM MK-2048 R263K 10 nM MK-2048 D3
100 000 10 000 1000 100
The R263K resistance substitution does not affect viral rebound after washout of either dolutegravir or MK-2048 and delays viral rebound after washout of raltegravir
WT no drug WT 93.75 nM RAL WT 93.75 nM RAL D3 R263K no drug R263K 93.75 nM RAL R263K 93.75 nM RAL D3
100 000
100
RT activity
The washout effect of INSTIs is dependent on drug concentration
1 000 000
0
5 10 Days post-infection
15
Figure 3. Effect of the R263K substitution on levels of antiviral activity in drug washout experiments. Drug washout was after 3 days and viral replication was monitored by measuring RT activity in the culture fluids for up to 2 weeks thereafter. Controls are represented by continuous lines. DTG, dolutegravir; RAL, raltegravir; D3, Day 3.
Discussion We have shown that the superior binding of dolutegravir to integrase–DNA complexes, which has previously been demonstrated in biochemical assays,18 correlates with a longer action of dolutegravir than raltegravir after its washout in tissue culture. These results provide the first evidence that dolutegravir possesses a very long effective half-life within cells. The slower dissociation of dolutegravir than raltegravir from integrase – DNA complexes, along with the shift of RT activity
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
delayed the rebound in each instance more efficiently than did either raltegravir or MK-2048. Based on these results, we conjectured that the time of treatment before drug washout might be important, since nonintegrated forms of viral DNA might be able to attain integration if the drug were removed at early timepoints. Consistent with this notion, the results in Table 1 show that the recovery of viral replication seems to have occurred more quickly after washout of raltegravir than of either MK-2048 or dolutegravir.
JAC
Persistence of dolutegravir activity after washout
Table 3. Comparison of viral replication of WT and R263K-containing viruses after 3 days of treatment with dolutegravir, raltegravir and MK-2048 at 20× the IC90 Dolutegravir
Days to attainment of 50% of maximal RT activity after drug removal Days to attainment of maximal RT activity after drug removal
Raltegravir
MK-2048
WT
R263K
WT
R263K
WT
R263K
6 8
6 8
4 8
6 8
7 11
6 8
In the case of the no-drug control, 50% and maximal levels of RT activity in culture fluids were observed after 3 and 4 days, respectively.
Funding This work was supported by the Canadian Institutes of Health Research and by the Reseau SIDA of the Fonds de Recherche du Quebec.
Transparency declarations None to declare.
References 1 Grinsztejn B, Nguyen BY, Katlama C et al. Safety and efficacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in treatment-experienced patients with multidrug-resistant virus: a phase II randomised controlled trial. Lancet 2007; 369: 1261 –9. 2 Shimura K, Kodama E, Sakagami Y et al. Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137). J Virol 2008; 82: 764– 74. 3 Delelis O, Thierry S, Subra F et al. Impact of Y143 HIV-1 integrase mutations on resistance to raltegravir in vitro and in vivo. Antimicrob Agents Chemother 2010; 54: 491–501. 4 Maiga AI, Malet I, Soulie C et al. Genetic barriers for integrase inhibitor drug resistance in HIV type-1 B and CRF02_AG subtypes. Antivir Ther 2009; 14: 123–9. 5 Kobayashi M, Yoshinaga T, Seki T et al. In vitro antiretroviral properties of S/GSK1349572, a next-generation HIV integrase inhibitor. Antimicrob Agents Chemother 2011; 55: 813–21. 6 Lenz JC, Rockstroh JK. S/GSK1349572, a new integrase inhibitor for the treatment of HIV: promises and challenges. Expert Opin Investig Drugs 2011; 20: 537–48. 7 Prada N, Markowitz M. Novel integrase inhibitors for HIV. Expert Opin Investig Drugs 2010; 19: 1087– 98. 8 Quashie PK, Mesplede T, Han YS et al. Characterization of the R263K mutation in HIV-1 integrase that confers low-level resistance to the second-generation integrase strand transfer inhibitor dolutegravir. J Virol 2012; 86: 2696– 705. 9 Mesple`de T, Quashie PK, Osman N et al. Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure. Retrovirology 2013; 10: 22.
Acknowledgements
10 Mesple`de T, Osman N, Wares M et al. Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity. J Antimicrob Chemother 2014; 69: 2733– 40.
We would like to thank Said Hassounah and Vincent Cutillas for fruitful discussions. This work was largely performed by Nathan Osman in partial fulfilment of the requirements for a PhD degree, Faculty of Graduate Studies and Research, McGill University, Montreal, Canada.
11 Wares M, Mesplede T, Quashie PK et al. The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness. Retrovirology 2014; 11: 7.
5 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
measured in tissue culture, as seen here in washout studies, suggests that dolutegravir is able to remain bound to the integrase– DNA complexes for an extended period of time compared with raltegravir. This results in a longer block of the strand transfer step, thus delaying the time at which viral replication can resume. This effect is stronger with higher drug concentrations since higher concentrations provide for better saturation of the integrase catalytic site. Hence, a longer period of time is needed after drug washout for the drug to dissociate from the integrase–DNA complexes. Longer treatment times can of course also result in a more protracted block of the strand transfer step. In this circumstance, more linear viral DNA originating from the 3′ -processing step might be degraded, thereby compromising the ability of the virus to resume replication after drug washout. Consistent with our findings, recent work suggests that 2-LTR circles, resulting from the accumulation of unintegrated viral DNA that is caused by a block of the strand transfer step, can be used by the integrase enzyme as a substrate for strand transfer after the washout of raltegravir.21 We hypothesize that the delayed resumption of replication after withdrawal of the drug, as shown here over long incubation times, can be explained by the fact that the amount of linear viral DNA available for integration is decreasing over time. We hope to confirm this hypothesis in future studies by measuring linear viral DNA and 2-LTR circles by quantitative PCR; such work is in progress. Our data also suggest that the time of residence of INSTIs on integrase is a key factor in the activity of these drugs. Both dolutegravir and MK-2048 are apparently more durable after drug washout than is raltegravir. MK-2048 is no longer being developed for use in the clinic and the reason for this seems to be the poor pharmacokinetic profile of this drug rather than any failings with regard to either its potency or its duration of association with the integrase enzyme. Our studies were internally controlled and indicate that the effective residency times for dolutegravir in cells infected by either WT or R263K-containing viruses were longer than those of raltegravir against these same viruses. Our data also help to explain why dolutegravir possesses such a high genetic barrier to the development of drug resistance. Moreover, our findings in tissue culture point to the durability and long-acting effect of dolutegravir.
Osman et al.
12 Oliveira M, Mesple`de T, Quashie PK et al. Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors. AIDS 2014; 28: 813– 9.
17 Molina JM, Clotet B, van Lunzen J et al. Once-daily dolutegravir is superior to once-daily darunavir/ritonavir in treatment-naı¨ve HIV-1-positive individuals: 96 week results from FLAMINGO. J Int AIDS Soc 2014; 17: 19490.
13 Mesple`de T, Moı¨si D, Oliveira M et al. Dolutegravir inhibits HIV-1 Env evolution in primary human cells. AIDS 2015; 29: 659–65.
18 Hightower KE, Wang R, Deanda F et al. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes. Antimicrob Agents Chemother 2011; 55: 4552 –9.
14 Cahn P, Pozniak AL, Mingrone H et al. Dolutegravir versus raltegravir in antiretroviral-experienced, integrase-inhibitor-naive adults with HIV: week 48 results from the randomised, double-blind, non-inferiority SAILING study. Lancet 2013; 382: 700–8. 15 Eron JJ, Clotet B, Durant J et al. Safety and efficacy of dolutegravir in treatment-experienced subjects with raltegravir-resistant HIV type 1 infection: 24-week results of the VIKING Study. J Infect Dis 2013; 207: 740–8.
6 of 6
20 Quan Y, Brenner BG, Marlink RG et al. Drug resistance profiles of recombinant reverse transcriptases from human immunodeficiency virus type 1 subtypes A/E, B, and C. AIDS Res Hum Retroviruses 2003; 19: 743– 53. 21 Thierry S, Munir S, Thierry E et al. Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 DNA initiate de novo integration. Retrovirology 2015; 12: 24.
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
16 Raffi F, Rachlis A, Stellbrink HJ et al. Once-daily dolutegravir versus raltegravir in antiretroviral-naive adults with HIV-1 infection: 48 week results from the randomised, double-blind, non-inferiority SPRING-2 study. Lancet 2013; 381: 735–43.
19 Johnson VA, Byington RE. Infectivity assay (virus yield assay). In: Aldovini A, Walker BD, eds. Techniques in HIV Research. New York: Stockton Press, 1990; 71– 6.
J Antimicrob Chemother doi:10.1093/jac/dkv176
Dolutegravir maintains a durable effect against HIV replication in tissue culture even after drug washout Nathan Osman1,2, Thibault Mesple`de1, Peter K. Quashie1,2, Maureen Oliveira1, Veronica Zanichelli1 and Mark A. Wainberg1,2* 1
McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada; 2Department of Microbiology and Immunology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada *Corresponding author. E-mail: [email protected]
Objectives: Of the currently approved HIV integrase strand transfer inhibitors (INSTIs), dolutegravir has shown greater efficacy than raltegravir at suppressing HIV-1 replication in treatment-experienced individuals. Biochemical experiments have also shown that dolutegravir has a longer dissociative half-life when bound to HIV integrase than does raltegravir. In order to study the intracellular efficacy of various INSTIs, we asked whether drug removal from INSTI-treated HIV-1-infected cells would result in different times to viral rebound. In addition, we assessed the role of the R263K substitution within the integrase ORF that is associated with low-level resistance to dolutegravir. Methods: HIV-infected MT-2 cells were treated with dolutegravir, raltegravir or a third experimental INSTI (MK-2048) and the drugs were washed out after varying times. Viral replication was monitored by measuring reverse transcriptase (RT) activity in the culture fluids. Results: We observed a significantly slower increase in RTactivity after the removal of dolutegravir compared with raltegravir or MK-2048. The incubation time before the drug was removed also had an impact on the level of RT activity independently of the drug and virus used. The R263K substitution did not significantly impact on levels of RT activity after drug washout, suggesting that dolutegravir remained tightly bound to the integrase enzyme despite the presence of this mutation. Conclusions: These results suggest that the residency time of INSTIs on integrase is a key factor in the activity of these drugs and that the anti-HIV activity of dolutegravir persists more effectively than that of other INSTIs after drug washout.
Introduction The development of combined ART has significantly increased the life expectancy and quality of life of HIV-infected individuals. The latest class of drugs to have been developed against HIV-1 are the integrase strand transfer inhibitors (INSTIs), of which raltegravir and elvitegravir were the first to be approved. Although these compounds are potent,1,2 they have a relatively low genetic barrier for resistance in patients failing therapy.3,4 In contrast, dolutegravir has a high genetic barrier for resistance as well as limited crossresistance to raltegravir and elvitegravir.5 – 7 This notwithstanding, tissue culture selections with dolutegravir have identified an R263K substitution in HIV-1 integrase that confers low-level resistance to this drug.8 In contrast to primary resistance substitutions against raltegravir or elvitegravir, no secondary substitutions that can compensate for the replicative defects associated with R263K have been
identified.9 – 11 This could be due either to the inability of R263Kcontaining dolutegravir-resistant viruses to acquire additional resistance substitutions12 or to the ability of dolutegravir to inhibit HIV genetic evolution.13 The SAILING clinical study reported the presence of this same substitution in two INSTI-naive patients who had failed dolutegravir treatment among a total of 354 patients, the vast majority of whom responded well to treatment.14 In contrast, mutations associated with raltegravir can confer crossresistance to dolutegravir, which was shown in the VIKING trial in which treatment-experienced individuals who had failed raltegravir and who carried raltegravir-associated resistance substitutions were treated with dolutegravir. Seven such subjects subsequently failed dolutegravir-based regimens without acquiring the R263K substitution,15 demonstrating the vulnerability of dolutegravir when used in salvage therapy. In contrast, the SPRING and FLAMINGO clinical trials demonstrated the superiority of dolutegravir compared with efavirenz-based and darunavir-based regimens,
# The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
1 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
Received 22 April 2015; returned 27 May 2015; revised 29 May 2015; accepted 31 May 2015
Osman et al.
respectively, in first-line therapy.16,17 Moreover, no patient who has received dolutegravir in first-line therapy has been shown to possess resistance mutations associated with either dolutegravir itself or the nucleoside compounds with which it has been co-administered during treatment.16 Biochemical experiments have also shown that dolutegravir has a far-longer dissociative half-life for integrase than both raltegravir and elvitegravir.18 Hence, we were interested in knowing whether drug washout from INSTI-treated infected cells would result in differences among these various compounds with regard to the times of viral rebound in tissue culture.
Materials and methods
The generation of the pNL4-3IN(R263K) plasmid has previously been reported.8 Genetically homogeneous viral stocks were produced by transfecting 293T cells with the pNL4-3 and pNL4-3IN(R263K) plasmids using Lipofectamine 2000 (Life Technologies, Burlington, ON, Canada) according to the manufacturer’s instructions. Forty-eight hours after transfection, the cell culture fluids were harvested, treated with Benzonase (Sigma-Aldrich, Oakville, ON, Canada) and filtered at 0.45 mm to remove plasmids and cell debris, respectively. The viruses were then aliquotted and stored at 2808C. Viral stocks were quantified by measuring both the p24 content and the cell-free reverse transcriptase (RT) activity.
Determination of the TCID50/mL
Cells and reagents
Determination of drug concentrations and IC90s in MT-2 cells The susceptibility of HIV to dolutegravir, raltegravir and MK-2048 was measured by the infection of 50 000 MT-2 cells with the same amount
(b) 1 000 000
WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D1 WT 93.75 nM RAL WT 93.75 nM RAL D1 WT 10 nM MK-2048 WT 10 nM MK-2048 D1
100 000 10 000 1000 100
0
5 10 Days post-infection
DTG
1 000 000 RT activity
(a)
The TCID50/mL was determined as described by Johnson and Byington,19 using MT-2 cells and calculated using the Spearman– Karber formula.
WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D1 WT 41.7 nM DTG D2 WT 41.7 nM DTG D3
100 000 10 000 1000 100
15
0
5 10 Days post-infection
15
RAL WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D2 WT 93.75 nM RAL WT 93.75 nM RAL D2 WT 10 nM MK-2048 WT 10 nM MK-2048 D2
100 000 10 000 1000
1 000 000 RT activity
RT activity
1 000 000
100 0
5 10 Days post-infection
10 000 1000 100
15
WT no drug WT 93.75 nM RAL WT 93.75 nM RAL D1 WT 93.75 nM RAL D2 WT 93.75 nM RAL D3
100 000
0
5 10 Days post-infection
15
MK-2048 WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D3 WT 93.75 nM RAL WT 93.75 nM RAL D3 WT 10 nM MK-2048 WT 10 nM MK-2048 D3
100 000 10 000 1000 100 0
5 10 Days post-infection
15
1 000 000 RT activity
RT activity
1 000 000
100 000
WT no drug WT 10 nM MK-2048 WT 10 nM MK-2048 D1 WT 10 nM MK-2048 D2 WT 10 nM MK-2048 D3
10 000 1000 100
0
5 10 Days post-infection
15
Figure 1. Variations among various INSTIs with regard to the durability of anti-HIV effects after drug washout. Viral replication was monitored by measuring RT activity in the culture fluids for up to 2 weeks after drug removal. (a) Comparisons of drug washout after 1, 2 or 3 days (top panel, middle panel and bottom panel, respectively). (b) Comparisons of washout at each of Days 1, 2 and 3 using dolutegravir (top panel), raltegravir (middle panel) and MK-2048 (bottom panel). DTG, dolutegravir; RAL, raltegravir; D1, Day 1; D2, Day 2; D3, Day 3.
2 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
The 293T cell line was obtained from the ATCC (CRL-11268). The MT-2 cell line was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, courtesy of Dr Douglas Richman. The cells were subcultured every 3 – 4 days in DMEM for 293T cells or RPMI medium for MT-2 cells, both supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin and maintained at 378C under 5% CO2. Merck, Inc. kindly provided raltegravir and MK-2048, and dolutegravir was kindly supplied by ViiV Healthcare Inc.
RT activity
Generation of replication-competent genetically homogeneous HIV-1
JAC
Persistence of dolutegravir activity after washout
Table 1. Comparison of viral replication of WT virus after 1, 2 or 3 days of treatment of MT-2 cells with dolutegravir, raltegravir and MK-2048 at 20× the IC90 Dolutegravir
Days to attainment of 50% of maximal RT activity after drug removal Days to attainment of maximal RT activity after drug removal
Raltegravir
MK-2048
1
2
3
1
2
3
1
2
3
8 10
7 9
6 8
3 6
3 5
4 8
7 13
7 9
7 11
In the case of the no-drug control, 50% and maximal levels of RT activity in culture fluids were observed after 3 and 4 days, respectively.
DTG
RT activity
1 000 000
10 000 1000 100
Infection of MT-2 cells with genetically homogeneous HIV-1 and drug washout
0
5 10 Days post-infection
15
RAL 1 000 000
RT activity
Washout experiments were performed as follows. In each instance, 300000 cells were pelleted and resuspended in 1 mL of medium containing the appropriate amount of virus and incubated for 2 h at 378C. The cells were then repelleted and the supernatant was discarded to eliminate any unbound virus. The cells were then resuspended in 2 mL of fresh medium containing the appropriate concentration of drug. Each drug was used at either low or high concentrations corresponding to 5× and 20× the IC90, respectively. To remove the drug from the medium, the cells were diluted in 10 mL of fresh medium and pelleted. The culture fluids were discarded and the cells were resuspended in 10 mL of fresh medium and pelleted again. The cells were then resuspended in 2 mL of fresh medium and observed for viral replication.
WT no drug WT 10.4 nM DTG WT 41.7 nM DTG WT 10.4 nM DTG D3 WT 41.7 nM DTG D3
100 000
WT no drug WT 23.4 nM RAL WT 93.75 nM RAL WT 23.4 nM RAL D3 WT 93.75 nM RAL D3
100 000 10 000 1000 100
0
5 10 Days post-infection
15
MK-2048
Monitoring of HIV-1 infection Infection was monitored by measuring the RT activity in the culture fluids, as previously described.20 To evaluate the number of days needed to reach 50% of maximal RT activity, the upward portions of slopes were fitted to the dose – response stimulation model using GraphPad Prism software, and the replication rate was evaluated as the Hill slope of the fit models.
RT activity
1 000 000
WT no drug WT 2.5 nM MK-2048 WT 10 nM MK-2048 WT 2.5 nM MK-2048 D3 WT 10 nM MK-2048 D3
100 000 10 000 1000 100 0
Results The washout of dolutegravir from infected cells results in a more prolonged anti-HIV effect than does the removal of raltegravir or MK-2048 MT-2 cells were infected with HIV-1 NL4-3IN(WT) and the cells were treated with dolutegravir, raltegravir or MK-2048 at 20× the IC90 for 1, 2 or 3 days. After these times, the cells were washed and fresh medium was added. We observed that viral replication, as measured by levels of RT activity in the culture fluids, was significantly more delayed after the removal of dolutegravir than that of raltegravir (Figure 1 and Table 1). Furthermore, differences of up to 4 days with respect to the attainment of maximal RT activity were noted between raltegravir and dolutegravir when the cells were treated for 1 or 2 days (Figure 1a).
5 10 Days post-infection
15
Figure 2. Concentration-dependent effects of INSTI washout after Day 3. Viral replication was monitored by measuring RT activity in the culture fluids for up to 2 weeks thereafter. Controls are represented by continuous lines. DTG, dolutegravir; RAL, raltegravir; D3, Day 3.
In order to better understand the relationships between the drugs with regard to their antiviral effect, the data in Figure 1(a) were replotted in Figure 1(b) so that direct comparisons among the INSTIs that were used are obvious in each case. It is clear that more profound drug effects were observed as the time of exposure to the drug increased (Figure 1b and Table 1). Although viral rebound after drug washout occurred at similar levels regardless of the INSTI employed, the use of dolutegravir
3 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
of either WT or R263K-containing viruses, based on RT activity, in the presence of 1: 2 serial dilutions of the drugs. After 5 days, the activity of RT was quantified in the culture fluids and the IC90 values of the different drugs were calculated using GraphPad Prism software. The drug concentrations that were chosen for this study corresponded to 5× and 20× the IC90 for each drug evaluated in order to ensure that there would be an optimal suppression of viral replication prior to drug washout.
Osman et al.
Table 2. Comparison of viral replication of WT virus after 3 days of treatment with different concentrations of dolutegravir, raltegravir and MK-2048 Dolutegravir
Days to attainment of 50% of maximal RT activity after drug removal Days to attainment of maximal RT activity after drug removal
Raltegravir
MK-2048
10.4 nM
41.7 nM
23.4 nM
93.75 nM
2.5 nM
10 nM
4 8
6 8
2 4
4 8
3 6
7 11
In the case of the no-drug control, 50% and maximal levels of RT activity in culture fluids were observed after 3 and 4 days, respectively. For each drug used, the concentrations used represent 5× and 20× the IC90, respectively.
DTG RT activity
1 000 000
WT no drug WT 41.7 nM DTG WT 41.7 nM DTG D3 R263K no drug R263K 41.7 nM DTG R263K 41.7 nM DTG D3
100 000 10 000 1000 100
0
5 10 Days post-infection
15
RAL
RT activity
Next, we compared two different concentrations of each of dolutegravir, raltegravir and MK-2048 in our washout protocols (Figure 2). The results show that viral replication resumed earlier after the removal of the drug when a lower concentration of the drug was employed, independent of the drug used (Table 2). A difference of 2.4 days to reach 50% of maximal RT activity was observed in a comparison of dolutegravir and raltegravir, with a corresponding value of 4.6 days for MK-2048. The data show that the lowest drug concentrations of dolutegravir, raltegravir and MK-2048 that were used in this work as controls were suboptimal as the virus was able to replicate even when drug was not removed from the medium.
10 000 1000
In order to evaluate the impact of the R263K substitution on the ability of HIV to rebound after drug washout, we infected MT-2 cells with HIV-1 NL4-3IN(WT) or NL4-3IN(R263K) viruses. The cells were treated with dolutegravir, raltegravir or MK-2048 at 20× the IC90 for 3 days. The results show that very similar RT activity levels were seen with the WT and R263K viruses in the presence of either dolutegravir or MK-2048 (Figure 3 and Table 3). In the case of raltegravir, R263K virus replication reached 50% of maximal activity after 3 additional days compared with WT; in addition, a significantly higher maximal RT activity was attained for the R263K virus, i.e. 72% in the presence of raltegravir compared with WT in the absence of the drug, compared with 26% for WT in the presence of the drugs (P,0.05; Student’s t-test). This is consistent with previous observations that the R263K substitution increases the susceptibility of HIV to raltegravir.8
4 of 6
0
5 10 Days post-infection
15
MK-2048 1 000 000
WT no drug WT 10 nM MK-2048 WT 10 nM MK-2048 D3 R263K no drug R263K 10 nM MK-2048 R263K 10 nM MK-2048 D3
100 000 10 000 1000 100
The R263K resistance substitution does not affect viral rebound after washout of either dolutegravir or MK-2048 and delays viral rebound after washout of raltegravir
WT no drug WT 93.75 nM RAL WT 93.75 nM RAL D3 R263K no drug R263K 93.75 nM RAL R263K 93.75 nM RAL D3
100 000
100
RT activity
The washout effect of INSTIs is dependent on drug concentration
1 000 000
0
5 10 Days post-infection
15
Figure 3. Effect of the R263K substitution on levels of antiviral activity in drug washout experiments. Drug washout was after 3 days and viral replication was monitored by measuring RT activity in the culture fluids for up to 2 weeks thereafter. Controls are represented by continuous lines. DTG, dolutegravir; RAL, raltegravir; D3, Day 3.
Discussion We have shown that the superior binding of dolutegravir to integrase–DNA complexes, which has previously been demonstrated in biochemical assays,18 correlates with a longer action of dolutegravir than raltegravir after its washout in tissue culture. These results provide the first evidence that dolutegravir possesses a very long effective half-life within cells. The slower dissociation of dolutegravir than raltegravir from integrase – DNA complexes, along with the shift of RT activity
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
delayed the rebound in each instance more efficiently than did either raltegravir or MK-2048. Based on these results, we conjectured that the time of treatment before drug washout might be important, since nonintegrated forms of viral DNA might be able to attain integration if the drug were removed at early timepoints. Consistent with this notion, the results in Table 1 show that the recovery of viral replication seems to have occurred more quickly after washout of raltegravir than of either MK-2048 or dolutegravir.
JAC
Persistence of dolutegravir activity after washout
Table 3. Comparison of viral replication of WT and R263K-containing viruses after 3 days of treatment with dolutegravir, raltegravir and MK-2048 at 20× the IC90 Dolutegravir
Days to attainment of 50% of maximal RT activity after drug removal Days to attainment of maximal RT activity after drug removal
Raltegravir
MK-2048
WT
R263K
WT
R263K
WT
R263K
6 8
6 8
4 8
6 8
7 11
6 8
In the case of the no-drug control, 50% and maximal levels of RT activity in culture fluids were observed after 3 and 4 days, respectively.
Funding This work was supported by the Canadian Institutes of Health Research and by the Reseau SIDA of the Fonds de Recherche du Quebec.
Transparency declarations None to declare.
References 1 Grinsztejn B, Nguyen BY, Katlama C et al. Safety and efficacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in treatment-experienced patients with multidrug-resistant virus: a phase II randomised controlled trial. Lancet 2007; 369: 1261 –9. 2 Shimura K, Kodama E, Sakagami Y et al. Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137). J Virol 2008; 82: 764– 74. 3 Delelis O, Thierry S, Subra F et al. Impact of Y143 HIV-1 integrase mutations on resistance to raltegravir in vitro and in vivo. Antimicrob Agents Chemother 2010; 54: 491–501. 4 Maiga AI, Malet I, Soulie C et al. Genetic barriers for integrase inhibitor drug resistance in HIV type-1 B and CRF02_AG subtypes. Antivir Ther 2009; 14: 123–9. 5 Kobayashi M, Yoshinaga T, Seki T et al. In vitro antiretroviral properties of S/GSK1349572, a next-generation HIV integrase inhibitor. Antimicrob Agents Chemother 2011; 55: 813–21. 6 Lenz JC, Rockstroh JK. S/GSK1349572, a new integrase inhibitor for the treatment of HIV: promises and challenges. Expert Opin Investig Drugs 2011; 20: 537–48. 7 Prada N, Markowitz M. Novel integrase inhibitors for HIV. Expert Opin Investig Drugs 2010; 19: 1087– 98. 8 Quashie PK, Mesplede T, Han YS et al. Characterization of the R263K mutation in HIV-1 integrase that confers low-level resistance to the second-generation integrase strand transfer inhibitor dolutegravir. J Virol 2012; 86: 2696– 705. 9 Mesple`de T, Quashie PK, Osman N et al. Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure. Retrovirology 2013; 10: 22.
Acknowledgements
10 Mesple`de T, Osman N, Wares M et al. Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity. J Antimicrob Chemother 2014; 69: 2733– 40.
We would like to thank Said Hassounah and Vincent Cutillas for fruitful discussions. This work was largely performed by Nathan Osman in partial fulfilment of the requirements for a PhD degree, Faculty of Graduate Studies and Research, McGill University, Montreal, Canada.
11 Wares M, Mesplede T, Quashie PK et al. The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness. Retrovirology 2014; 11: 7.
5 of 6
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
measured in tissue culture, as seen here in washout studies, suggests that dolutegravir is able to remain bound to the integrase– DNA complexes for an extended period of time compared with raltegravir. This results in a longer block of the strand transfer step, thus delaying the time at which viral replication can resume. This effect is stronger with higher drug concentrations since higher concentrations provide for better saturation of the integrase catalytic site. Hence, a longer period of time is needed after drug washout for the drug to dissociate from the integrase–DNA complexes. Longer treatment times can of course also result in a more protracted block of the strand transfer step. In this circumstance, more linear viral DNA originating from the 3′ -processing step might be degraded, thereby compromising the ability of the virus to resume replication after drug washout. Consistent with our findings, recent work suggests that 2-LTR circles, resulting from the accumulation of unintegrated viral DNA that is caused by a block of the strand transfer step, can be used by the integrase enzyme as a substrate for strand transfer after the washout of raltegravir.21 We hypothesize that the delayed resumption of replication after withdrawal of the drug, as shown here over long incubation times, can be explained by the fact that the amount of linear viral DNA available for integration is decreasing over time. We hope to confirm this hypothesis in future studies by measuring linear viral DNA and 2-LTR circles by quantitative PCR; such work is in progress. Our data also suggest that the time of residence of INSTIs on integrase is a key factor in the activity of these drugs. Both dolutegravir and MK-2048 are apparently more durable after drug washout than is raltegravir. MK-2048 is no longer being developed for use in the clinic and the reason for this seems to be the poor pharmacokinetic profile of this drug rather than any failings with regard to either its potency or its duration of association with the integrase enzyme. Our studies were internally controlled and indicate that the effective residency times for dolutegravir in cells infected by either WT or R263K-containing viruses were longer than those of raltegravir against these same viruses. Our data also help to explain why dolutegravir possesses such a high genetic barrier to the development of drug resistance. Moreover, our findings in tissue culture point to the durability and long-acting effect of dolutegravir.
Osman et al.
12 Oliveira M, Mesple`de T, Quashie PK et al. Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors. AIDS 2014; 28: 813– 9.
17 Molina JM, Clotet B, van Lunzen J et al. Once-daily dolutegravir is superior to once-daily darunavir/ritonavir in treatment-naı¨ve HIV-1-positive individuals: 96 week results from FLAMINGO. J Int AIDS Soc 2014; 17: 19490.
13 Mesple`de T, Moı¨si D, Oliveira M et al. Dolutegravir inhibits HIV-1 Env evolution in primary human cells. AIDS 2015; 29: 659–65.
18 Hightower KE, Wang R, Deanda F et al. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes. Antimicrob Agents Chemother 2011; 55: 4552 –9.
14 Cahn P, Pozniak AL, Mingrone H et al. Dolutegravir versus raltegravir in antiretroviral-experienced, integrase-inhibitor-naive adults with HIV: week 48 results from the randomised, double-blind, non-inferiority SAILING study. Lancet 2013; 382: 700–8. 15 Eron JJ, Clotet B, Durant J et al. Safety and efficacy of dolutegravir in treatment-experienced subjects with raltegravir-resistant HIV type 1 infection: 24-week results of the VIKING Study. J Infect Dis 2013; 207: 740–8.
6 of 6
20 Quan Y, Brenner BG, Marlink RG et al. Drug resistance profiles of recombinant reverse transcriptases from human immunodeficiency virus type 1 subtypes A/E, B, and C. AIDS Res Hum Retroviruses 2003; 19: 743– 53. 21 Thierry S, Munir S, Thierry E et al. Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 DNA initiate de novo integration. Retrovirology 2015; 12: 24.
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on July 15, 2015
16 Raffi F, Rachlis A, Stellbrink HJ et al. Once-daily dolutegravir versus raltegravir in antiretroviral-naive adults with HIV-1 infection: 48 week results from the randomised, double-blind, non-inferiority SPRING-2 study. Lancet 2013; 381: 735–43.
19 Johnson VA, Byington RE. Infectivity assay (virus yield assay). In: Aldovini A, Walker BD, eds. Techniques in HIV Research. New York: Stockton Press, 1990; 71– 6.
