HIV-2 is related to HIV-1 and was first discovered in West Africa in 1986. HIV-2 is distinguished from HIV-1 by a different species of origin, lower rates of transmission and disease progression, and limited geographic spread.1–3 Despite the lesser pathogenicity of HIV-2, a significant number of those infected develop AIDS and could therefore benefit from antiretroviral therapy.3 However, HIV-2 is susceptible to only a subset of the drugs currently approved for HIV-1 treatment. These include the nucleoside reverse transcriptase inhibitors (NRTIs), some protease inhibitors, and integrase strand transfer inhibitors (INSTIs).4,5
The emergence of drug resistance mutations in HIV-2 can further limit treatment options, analogous to HIV-1. However, HIV-2 resistance has not been as extensively studied as HIV-1. HIV-2 and HIV-1 have approximately 40% heterogeneity at the amino acid level in their pol genes, which encode the protease, RT, and integrase (IN) enzymes. The catalytic sites of HIV-1 and HIV-2 integrase are fairly well conserved, and it seems that the major pathways of resistance to the INSTI raltegravir (RAL), involving integrase mutations N155H, Q148R, and Y143C, are the same in HIV-1 and HIV-2.6–8 Similarly, HIV-2 seems to select for many of the same NRTI resistance mutations as does HIV-1.4,9 However, the frequencies of some emergent mutations, such as K65R and Q151M, are much greater in HIV-2 infection than in HIV-1 infection, where the thymidine analog mutations more commonly develop.10–14 Further characterization of HIV-2 resistance profiles against new and existing drug therapies is necessary for expanding treatment options to HIV-2–infected patients.
The investigational INSTI elvitegravir (EVG) has been coformulated with the pharmacoenhancer cobicistat (COBI) plus NRTIs emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) into a single tablet regimen (the EVG/COBI/FTC/TDF STR). Two phase 3 studies in HIV-1–infected patients have demonstrated that 48 weeks of treatment with EVG/COBI/FTC/TDF is well tolerated and has noninferior efficacy compared with an STR of efavirenz (EFV)/FTC/TDF or ritonavir-boosted atazanavir (ATV/RTV)+FTC/TDF.15,16 In vitro, the antiviral components of EVG/COBI/FTC/TDF, consisting of EVG, FTC, and tenofovir (TFV), have potent activity against both HIV-1 and HIV-2.17–19 Thus, the EVG/COBI/FTC/TDF STR may also have utility in patients infected with HIV-2. Here, HIV-2 susceptibility to EVG, FTC, and TFV and selection of resistance mutations were characterized in tissue culture. Overall, the antiviral potencies and resistance patterns of EVG, FTC, and TFV for HIV-2 were similar to HIV-1.
MATERIALS AND METHODS
Reagents and Cell Lines
The antiretrovirals EVG, RAL, FTC, TFV, zidovudine (AZT), and darunavir (DRV) were synthesized at Gilead Sciences (Foster City, CA). MT-2 and MT-4 cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Germantown, MD). HEK293-T cells were purchased from American Type Culture Collection (Manassas, VA). Wild-type virus stocks HIV-2NIHZ (subtype A) and HIV-1IIIB (subtype B) were purchased from Advanced Biotechnologies, Inc (Columbia, MD). The infectious HIV-2 molecular clone pROD9 (subtype A)20 was obtained from Dr Michael Emerman (Fred Hutchinson Cancer Research Center, Seattle, WA).
Single compound resistance selection experiments with EVG, FTC, and TFV were performed using dose escalation and breakthrough methods. For the dose escalation method, experiments were performed, as previously described, with modifications.21 Briefly, 0.5 × 106 MT-2 cells were infected with HIV-2NIHZ at a multiplicity of infection (MOI) of approximately 0.05 with 5 µg/mL polybrene. At selection initiation, drug was added at a final concentration corresponding to its preliminary antiviral EC50 (1× EC50). Cultures were incubated at 37°C and split as necessary to prevent overgrowth. Viral supernatants were harvested when extensive cytopathic effect (CPE) in the form of cellular syncytia was observed and then concentrated using Lenti-X Concentrator (Clontech, Mountain View, CA). The first harvest was referred to as passage 1 (P1). Subsequent passages (P2–P13) were generated by infecting fresh MT-2 cells with 50–500 µL of the harvested virus from the previous passage and increasing the drug concentration by 2-fold. The duration of each passage ranged from 4 to 77 days. The viral yield for key passages was quantified with the SIV p27 Antigen Capture Assay (Advanced Bioscience Laboratories, Rockville, MD).
For the viral breakthrough method, 0.5 × 106 MT-4 cells were infected with HIV-2NIHZ at an MOI of 0.05 with 5 µg/mL polybrene, nutated for 2 hours at 37°C, and washed with phosphate-buffered saline to remove unbound virus. Cells were then resuspended in fresh medium and cultured at 37°C in the presence of constant drug concentrations, which ranged from 10- to 250-fold above the preliminary EC50 values of EVG, FTC, and TFV. Every 4–10 days, the cultures were split to prevent overgrowth, and aliquots of supernatant were collected. Virus production was monitored by p27 quantification and CPE. Fresh MT-4 cells were added when extensive cell killing was observed. The breakthrough selections lasted up to 64 days: cultures were either ended once observable virus production was achieved or maintained beyond initial viral breakthrough.
For preparation of viral DNA for population and clonal sequencing, viral supernatants were treated with DNase I (New England Biolabs, Ipswich, MA), and RNA was extracted using the EZ1 Virus Mini Kit v2.0 with the BioRobot EZ1 Workstation (QIAGEN, Valencia, CA). Viral cDNA was synthesized with Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Buckinghamshire, UK) using primers KA49N (5′-CTGATGGCTCTTCTT-3′) or JR47 (5′-ATTACCCTGCTGCAAGTCCACC-3′).22 Polymerase chain reaction (PCR) of the viral cDNA was then performed to amplify the RT and IN coding regions of the HIV-2 pol gene either separately or as a 2839 base pair viral DNA fragment spanning nucleotide 99 of the protease coding region to nucleotide 151 of the vif gene. The primers used for PCR and sequencing of HIV-2NIHZ viruses were modified from previously described HIV-2ROD primers.20,22 Primers RTCKA (5′-AGCAGGAATAGAGTTAGGGA-3′) and JR46N (5′-ATGCCCACCCCACCTTATGATG-3′) generated the 2839 base pair RT-IN amplicon, RTCKA and RT2KA (5′-CCTGACCAGTGGTGGGGTAGA-3′) were used for amplification of the RT coding region, and H2Mp9N (5′-GGATGATATCTTAATAGCCAG-3′) and JR46N were used to amplify the IN coding region.
For population sequencing of viral pools, PCR products were purified (QIAquick PCR purification kit, QIAGEN) and sequenced through the RT and IN coding regions on both DNA strands (ELIM Biopharmaceuticals, Inc, Hayward, CA). The primers used for sequencing RT were RTCKA, RT4KA (5′-TCTCTCTCTACTGGTAGGTGAA-3′), RT6 (5′-GATGTCATTGACTGTC-3′), RT5KA (5′-ACTGTACTAGATGTAGGGGAT-3′), and RT3KA (5′-AAGCCAGGGAAAGATGGACC-3′). The primers used for sequencing IN were JR46N, JR45N (5′-TATGTTGCGTGGGTCCCAGC-3′), AV33N (5′-GTGAAAATGGTAGCATGGTGG-3′), JR48N (5′-GTTCTATACCTACCCACC-3′), and JR44N (5′-GAAACTTTCTACACAGATGG-3′).
For clonal sequencing, amplicons generated by PCR as described above were cloned into the pCR-Blunt II-TOPO plasmid (Invitrogen, Carlsbad, CA). Plasmid DNAs were isolated from 50 to 60 transformed bacterial colonies and sequenced through the RT and IN coding regions on both DNA strands using primers RT4KA, RT6, RTCKA, JR45N, JR46N, and JR48N (TACGen, Richmond, CA).
Construction of Site-Directed HIV-2 Mutant Viruses
Because no molecular clone of HIV-2NIHZ was available, RT and IN mutations were introduced into the infectious wild-type HIV-2 DNA clone of a closely related HIV-2 subtype A virus, pROD9,23,24 by site-directed mutagenesis using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). After sequence confirmation, the XhoI-SapI fragment of pROD9 containing mutated RT and IN coding regions was cloned back into wild-type pROD9 plasmids and transformed into MAX Efficiency Stbl2 competent cells (Invitrogen) for DNA preparation and sequencing. Mutant virus stocks were generated by transfection of 293-T cells using TransIT-293 reagent (Mirus Bio Corporation, Madison, WI). Eight hours posttransfection, cells were washed with phosphate-buffered saline containing 2% fetal bovine serum and fresh medium was added. The cell culture supernatants containing HIV-2ROD9 viruses were harvested 3 days after transfection, filtered, and concentrated with Lenti-X Concentrator (Clontech). Virus yields were quantified by p27 assays and genotyped.
The phenotypes of HIV-1 and HIV-2 were determined using a 5-day antiviral assay in MT-4 cells with a luciferase readout as previously described.21 For each virus/viral pool tested, 1.5 × 106 MT-4 cells were preincubated with virus for 1 hour at 37°C with 5 µg/mL polybrene to produce a signal-to-noise ratio (uninfected cells to infected cells) in the range of 4–7, which was equivalent to an MOI of approximately 0.0012 for wild-type HIV-2NIHZ. Infected MT-4 cells were then transferred in triplicate to 96-well assay plates (approximately 5400 cells per well) along with equal volumes of drug dilutions in 1% DMSO. After 5 days of incubation at 37°C, CellTiter-Glo reagent (Promega, Madison, WI) was added, and luminescence was measured using a Wallac EnVision 2104 Multilabel Reader (PerkinElmer, Shelton, CT). The data were converted to percent cell death, and the antiviral dose response was analyzed by curve fitting using GraphPad Prism (San Diego, CA) to determine the effective concentration to inhibit 50% of viral replication (EC50). Calculation of mean EC50 values and standard deviation used arithmetic mean in all cases. Statistical significance of the fold changes for the mutants compared with the wild-type control was determined using the 2-tailed Student's t-test.
Genotypic and Phenotypic Characterization of Wild-Type HIV-2NIHZ
Genotypic analysis of the HIV-2NIHZ stock virus IN and RT coding regions by clonal sequencing was performed to identify viral quasispecies present at the start of the resistance selection experiments. Substitutions occurring in at least 2 clones were considered part of the baseline viral population. The most prevalent quasispecies (occurring in >20% clones analyzed) in IN were A41T, I72V, D163N, and I200L and in RT were K20R, K22R, S69N, H162Y, and R173K. RT quasispecies E39D, I189V, and V263I were observed in 6%–16% of viral clones analyzed, and additional IN and RT quasispecies were present at a frequency of <5% (for list of HIV-2NIHZ quasispecies, see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A368). Many of these observed quasispecies corresponded to naturally occurring polymorphisms that have been identified among other HIV-2 subtype A viruses.9,22,25,26 No mutations associated with resistance to EVG, FTC, or TFV were detected in the virus stock.
The antiviral activities of the INSTIs EVG and RAL and NRTIs FTC and TFV against HIV-2NIHZ were determined and compared with HIV-1IIIB (Table 1 ). EVG and RAL were potent inhibitors, with EC50 values in the nanomolar range for both HIV-1 and HIV-2. The potency of TFV was also similar for HIV-1 and HIV-2. The EC50 of FTC against HIV-2 was approximately 4-fold higher than that of HIV-1, which is in accordance with previously reported susceptibilities of other subtype A HIV-2 viruses.11,18,27
Resistance Selections With EVG, FTC, and TFV in HIV-2
EVG Resistance Selections
The HIV-2NIHZ dose escalation resistance selection with EVG commenced at a concentration of 1.2 nM, just below the EVG EC50 of 1.6 nM, and proceeded in 2-fold increments (Fig. 1A). At passage 6 (P6, 38.4 nM EVG) after 62 days in culture, an E92G mutation in the catalytic core domain of IN was observed by sequence analysis and persisted throughout the duration of the selection. At passage 7 (P7), a mixture of glycine and glutamine at position 92 (E92G/Q) was observed. As the selection progressed, E92Q was only transiently detected by population sequencing, but clonal sequence analysis revealed its continued low-level presence through the end of the selection at passage 13 on day 166 (P13, 4915.2 nM EVG) (Table 2 ). Additional IN substitutions corresponding to quasispecies of the NIHZ stock virus were also present during the selection.
For the breakthrough selection experiments with EVG, cells were infected with HIV-2NIHZ and cultured under constant drug concentrations ranging from 10- to 250-fold above the EC50 of EVG (Table 3 ). In the selections at 10× and 50× the EVG EC50, the HIV-2NIHZ virus was never fully suppressed after the initial infection, as indicated by detectable levels of p27 at days 4 and 6 postinfection (Fig. 2). This may be because of incomplete inhibition of integration associated with the relatively high MOI of the infection. These viruses continued to be cultured in the presence of drug for 43 days with no emergent resistance detected by population sequencing. At the completion of the experiment, clonal sequence analysis was also performed. In the 50× viral pool, an S147N mutation was observed at a frequency of 11% (3 of 28 clones). For the remaining selections, either viral breakthrough did not occur (100× and 250× cultures), or no mutations associated with EVG resistance were detected in the viral pool (200× culture). The A286T substitution present in the selection at 200× was not a pre-existing HIV-2NIHZ viral quasispecies, however, A286T is a polymorphism found in up to 20% of subtype A viruses.22,25 Furthermore, the viral pool harvested from the final EVG 200× culture on day 27 remained fully susceptible to both EVG and RAL (data not shown).
FTC Resistance Selections
The HIV-2NIHZ dose escalation resistance selection using FTC was initiated at 0.5 µM, which corresponded to 0.5× the EC50 of FTC (Fig. 1B). By passages 8 and 9 (P8, 64 µM; P9, 128 µM FTC), the M184I mutation, located within the YMDD motif of the RT catalytic site, was observed in viral pools. At the completion of the selection at passage 9 on day 59, M184I was the dominant mutation, detected at a frequency of 100% (41 of 41 clones) by clonal sequence analysis (Table 2).
For the breakthrough selections with FTC, resistance mutations at the M184 residue emerged at every drug concentration tested (Table 3). M184V was selected in the 10× and 50× cultures, whereas M184I was selected in the 20×, 40×, and 250× cultures. Pre-existing, transient, or polymorphic substitutions were also detected in both dose escalation and breakthrough selections with FTC.
TFV Resistance Selections
The dose escalation selection with TFV commenced at a drug concentration of 5 μM, approximately 1.4-fold above the EC50 of TFV (Fig. 1C). After 145 days in culture, at passage 5 (P5, 80 μM TFV), the K65R mutation was observed. At the following passage (P6, 160 μM TFV), the Y115F mutation in RT was detected in addition to K65R, and the selection was terminated after 173 days. Clonal sequencing was performed on the final viral pool from P6, and K65R and Y115F, as well as viral quasispecies, were observed in 100% of the 38 of viral clones analyzed, possibly the result of a founder effect in which a new population is established from the survival of a few resistant viral variants (Table 2).
For the breakthrough selections with TFV, the only emergent resistance occurred in the 10× culture, where clonal sequence analysis revealed the low-level presence of the K65R mutation (1 of 29 clones) (Table 3 and Fig. 2). No viral breakthrough was detectable by p27 assay or observable CPE in the remaining cultures from the TFV breakthrough selections. However, viral DNA could be amplified from the supernatant of the 50× culture on days 33 and 43, and population sequence analysis indicated the presence of the RT substitutions K103R and an HIV-2NIHZ quasispecies present at the start of the selection (R173K). K103R has been identified as a polymorphism occurring in HIV-2 subtype A patient isolates and is not associated with NRTI resistance.14,26
Phenotypic Analysis of HIV-2 With Mutant IN and RT
HIV-2NIHZ viral pools from the resistance selections were tested for phenotypic resistance to EVG, FTC, TFV, and RAL (Table 1). To characterize the effect of selected mutations on drug susceptibility independent of other amino acid substitutions, IN and RT site-directed mutants were constructed in the HIV-2ROD9 plasmid. Three IN mutants represent the EVG selections: E92G, E92Q, and S147N. Two RT mutants represent the FTC selections (M184I and M184V) and 3 RT mutants represent the TFV selections (K65R, Y115F, and K65R + Y115F) (Table 1).
Viral pools from the dose escalation selection with EVG contained IN mutations at the E92 residue. The P10 viral pool containing an E92G/Q mixture had moderately reduced susceptibility to both EVG and RAL of 5.3- and 2.2-fold, respectively. The E92G site-directed mutant displayed 3.7-fold reduced susceptibility to EVG and no cross-resistance to RAL. The E92Q mutant was more resistant to EVG with 16-fold reduced susceptibility and low-level cross-resistance to RAL (2.5-fold). HIV-2 viruses with E92G/Q remained fully susceptible to FTC, TFV, AZT, and DRV (fold change compared with wild-type of <2). The S147N mutation was observed at low-level in one breakthrough selection. Despite multiple attempts, the S147N mutant was unable to replicate in cell culture and thus yielded no phenotypic data.
Both dose escalation and breakthrough selections with FTC resulted in the development of M184V/I mutations in RT. The P9 viral pool with M184I was highly resistant to FTC with 78-fold reduced susceptibility. The M184I site-directed mutant yielded 34-fold reduced susceptibility to FTC. The M184V site-directed mutant was also highly resistant to FTC, with >1000-fold reduced susceptibility. HIV-2 viruses with M184V/I had no cross-resistance to the INSTIs, DRV, or the other NRTIs tested.
Selection with TFV led to emergence of the K65R mutation in RT. The P5 viral pool of the TFV dose escalation selection containing K65R had 7.1-fold reduced susceptibility to TFV and 25-fold reduced susceptibility to FTC. The K65R site-directed mutation conferred moderate resistance to both TFV and FTC with 2.2- and 9.1-fold reduced susceptibilities, respectively. The TFV P6 pool contained K65R and Y115F. The Y115F single mutant was fully susceptible to all drugs tested. However, the virus with Y115F and K65R together was more resistant to both FTC and TFV, with 36- and 5.4-fold reduced susceptibilities, respectively. The HIV-2ROD9 K65R + Y115F double mutant also showed minor cross-resistance to AZT with a 2-fold increase in EC50; however, this was not statistically significant. The K65R, Y115F, and K65R + Y115F mutants all remained fully susceptible to EVG, RAL, and DRV.
Current treatment options for HIV-2–infected individuals are limited. HIV-2 is intrinsically resistant to several HIV-1 inhibitors, including the entire class of nonnucleoside RT inhibitors. The EVG/COBI/FTC/TDF STR may become the first STR suitable for treatment of HIV-2, as other STRs currently on the market contain a nonnucleoside RT inhibitor. This study characterized the susceptibility and resistance profile of HIV-2 against EVG, FTC, and TFV. EVG, FTC, and TFV had similar antiviral potencies against wild-type HIV-2 and HIV-1, confirming previous reports.5,17–19 In this study, only HIV-2 subtype A viruses were tested, however, the HIV-2 subtype B strain EHO has also been found to be equally susceptible to EVG and TFV and more sensitive to other NRTIs when compared with HIV-1 and HIV-2 subtype A viruses.5,17 Individual HIV-2 resistance selection experiments with EVG, FTC, and TFV resulted in the emergence of IN and RT mutations and phenotypic resistance similar to previous observations for HIV-1 with no cross-class drug resistance.
The dose escalation selection with EVG led to the development of a predominant E92G population and a minor E92Q subpopulation in HIV-2 IN. E92Q is a well-characterized EVG primary resistance mutation selected by HIV-1 in vitro and in vivo.17,28,29 E92G is also associated with EVG resistance in HIV-1 and has previously emerged under in vitro selective pressure from a metabolite of EVG21 and been observed clinically with EVG.21,28 In HIV-1, E92G and E92Q reduce susceptibility to EVG in the range of 30- to 40-fold, and only E92Q shows moderate cross-resistance to RAL,17,21,29,30 similar to the pattern of resistance displayed here by HIV-2ROD9 E92G/Q mutants. Additionally, the E92G/Q mutations are involved with resistance to RAL in HIV-2 infected patients,6,31 and a recent study has shown another HIV-2ROD9 E92Q mutant confers similar fold-changes in RAL and EVG EC50s as reported here.32 An S147N IN mutation developed at low levels in one EVG breakthrough selection after continuous viral replication in the presence of EVG. The S147 residue lies within the flexible loop of the catalytic core domain of IN, and S147G was previously identified as another primary resistance mutation of HIV-1 selected by EVG in vitro and in vivo.17,28 The S147N mutation, however, is not a true EVG breakthrough mutation given that in this experiment, 50× EVG failed to completely suppress the initial infection. Furthermore, the S147N mutation did not replicate in tissue culture when studied in isolation, suggesting that the viral fitness of this variant in the context of HIV-2 is likely very poor.
FTC rapidly and consistently selected for the M184V/I mutations in HIV-2 RT. M184V and M184I are the primary resistance mutations that emerge in vitro and in vivo in HIV-2 and HIV-1 under selective pressure from FTC or lamivudine (3TC).11,33–37 HIV-1 viruses with M184V/I are highly resistant to FTC (>100-fold),36–38 as were the HIV-2 viruses with M184V/I reported here.
Selection of HIV-2 with TFV resulted in the initial development of the K65R mutation in RT followed by the addition of Y115F under higher drug pressure. In HIV-1, K65R is the primary mutation associated with TFV resistance, emerging during in vitro resistance selections with TFV and in patients undergoing treatment with TDF.14,34,39–42 The K65R mutation has also developed in HIV-2–infected patients on TDF containing regimens14 but thus far has not been reported to arise in HIV-2 under in vitro selective pressure with TFV.33 The Y115F mutation is often observed together with K65R in HIV-1 RT as a secondary NRTI resistance mutation and has previously been selected in vitro and in vivo in addition to K65R under TFV/TDF plus 3TC pressure.13,43,44 The Y115F mutation is also associated with NRTI resistance in the context of HIV-2.9,14 In HIV-1, K65R alone causes moderate resistance to TFV (approximately 3-fold) and higher resistance to FTC (approximately 10-fold), Y115F alone is fully susceptible to TFV and several other NRTIs, and K65R + Y115F has greater resistance to TFV (6-fold) and FTC (26-fold).34,45 Therefore, the susceptibilities of HIV-1 and HIV-2 viruses harboring K65R and Y115F to TFV are very similar.
These genotypic and phenotypic results indicate that the antiviral potency and resistance patterns of EVG, FTC, and TFV as individual agents are similar in HIV-2 and HIV-1. This suggests that the STR of EVG/COBI/FTC/TDF may be an effective treatment option to be studied in those infected with HIV-2 and would likely select for known resistance mutations in cases of virologic failure. A recent review of HIV-2 management for low-resource settings recommends TDF/FTC as a first- or second-line NRTI backbone regimen for HIV-2 treatment.46 The INSTI RAL has also been used successfully to treat HIV-2 infected patients,47 but its utility in regions with the greatest HIV-2 burden is limited by its high cost.46 Currently, however, no antiretroviral inhibitors are specifically approved for treatment of HIV-2, and there have been no randomized trials to assess optimal therapy for those infected. Further clinical studies are necessary to verify the safety and efficacy of the EVG/COBI/FTC/TDF STR for use in HIV-2 infection.
The authors would like to thank Michael Emerman for providing the HIV-2 pROD9 infectious molecular clone. They would also like to acknowledge Nicolas Margot, Rima Kulkarni, Michael Abram, Damian McColl, Rebecca Hluhanich, Hongmei Mo, and Christian Callebaut for helpful suggestions.
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