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Sensitive Cell-Based Assay for Determination of HumanImmunodeficiency Virus Type 1 Coreceptor Tropism

Jan Weber,a Ana C. Vazquez,b Dane Winner,b Richard M. Gibson,c Ariel M. Rhea,b Justine D. Rose,b Doug Wylie,b Kenneth Henry,c

Alison Wright,d Kevin King,d John Archer,e Eva Poveda,f Vicente Soriano,f David L. Robertson,e Paul D. Olivo,d Eric J. Arts,c

Miguel E. Quiñones-Mateug,h

Institute of Organic Chemistry and Biochemistry, Prague, Czech Republica; Diagnostic HYBRIDS, Inc., Cleveland, Ohio, USAb; Division of Infectious Diseasesc andDepartment of Pathology,g Case Western Reserve University, Cleveland, Ohio, USA; Diagnostic HYBRIDS, Inc., Athens, Ohio, USAd; Computational and EvolutionaryBiology, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdome; Infectious Diseases Department, Hospital Carlos III, Madrid, Spainf;University Hospital Translational Laboratory, University Hospitals Case Medical Center, Cleveland, Ohio, USAh

CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence ofCXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, wehave developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP)based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genomewith the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR productswere introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectorswere then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral popu-lation. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) populationsequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected mi-nority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of >1,000 copies/ml, andfrom B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 ofthe 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. Thedegree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm usedto determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencingusing Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in thetreatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.

Over 15 years ago, a series of seminal studies led to the conclu-sion that the human immunodeficiency virus type 1 (HIV-1)

requires, in addition to the CD4 molecule as the major cellularreceptor (1, 2), a second coreceptor to enter target cells, mainly thechemokine receptors CCR5 and CXCR4 (3–5). HIV-1 was thenclassified, according to its coreceptor usage or tropism, as a CCR5-or CXCR4-tropic virus (R5 or X4, respectively), while HIV-1strains able to use both coreceptors were termed dual tropic (R5/X4) (6). Since then, HIV-1 coreceptor tropism has been associatedwith virus transmission and disease progression, i.e., R5 variantsare commonly associated with the establishment of infection (7–9), while X4 variants seem to emerge in later disease stages andhave been linked to a more rapid CD4� T-cell depletion and pro-gression to AIDS (10–12).

The discovery of new anti-HIV-1 molecules targeting theCCR5 or CXCR4 receptors, better represented by the first CCR5receptor antagonist approved for clinical use (maraviroc; Selzen-try/Celsentri, Pfizer, NY) (13), prompted the development of amultitude of phenotypic and genotypic approaches to determineHIV-1 coreceptor usage or tropism (14). Given their mechanismof action, treatment with CCR5 antagonists requires the priorknowledge of the HIV-1 coreceptor tropism in the patient (12,15). Phenotypic assays usually involve the generation of patient-derived env recombinant viruses to determine their ability to in-

fect reporter cell lines expressing HIV-1 receptors and coreceptors(16–18), while others are based in the quantification of cell-to-cellfusion events (19, 20). On the other hand, genotypic tests based onpopulation (12, 14, 21) or deep sequencing (22–24) take advan-tage of the association of certain regions in the env gene as deter-minants of CCR5 or CXCR4 tropism, mainly in the V3 region ofthe gp120, and their interpretation based on a series of algorithmsand bioinformatic tools to infer the ability of HIV-1 to use any orboth coreceptors to enter host cells (25–28). As expected, bothapproaches have advantages and disadvantages, but particularemphasis has been made on their sensitivity to detect minornon-R5 variants, turnaround time, and, more important, theiraccuracy to determine HIV-1 coreceptor tropism (12, 14, 29).

Received 10 January 2013 Returned for modification 26 February 2013Accepted 28 February 2013

Published ahead of print 13 March 2013

Address correspondence to Miguel E. Quiñones-Mateu, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00092-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00092-13

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In this study, we have developed and characterized a novelphenotypic test to determine HIV-1 coreceptor tropism(VERITROP) based on a novel yeast-based cloning system anda sensitive cell-to-cell fusion assay. We compared this new as-say with several phenotypic and genotypic tests, including deepsequencing that allows for minor variant detection, and showedthat VERITROP is able to detect low levels of non-R5 viruses(0.3%) in plasma samples from HIV-infected individuals.

MATERIALS AND METHODSCells and viruses. U87.CD4.CCR5 and U87.CD4.CXCR4 cells (30) wereobtained through the AIDS Research and Reference Reagent Program,Division of AIDS, NIAID, NIH, from HongKui Deng and Dan Littman,while the HEK293T cells were obtained from Stanford University (Stan-ford, CA). U87.CD4.CCR5 and U87.CD4.CXCR4 cells were main-tained in Dulbecco’s modified Eagle’s medium (DMEM) with L-glu-tamine (Cellgro; Mediatech) supplemented with 15% fetal bovineserum, 100 U of penicillin/ml, 100 �g of streptomycin/ml, 1 �g/ml ofpuromycin, and 300 �g of G418 (all reagents from Mediatech).HEK293T cells were maintained in DMEM medium/L-glutamine(Gibco), 10% fetal bovine serum (FBS; Cellgro), and penicillin-strep-tomycin (Gibco). The following viruses were obtained from theAIDS Research and Reference Reagent Program, Division of AIDS,NIAID, NIH: HIV-1A-93RW024, HIV-1A-92UG031, HIV-1A-92UG029,HIV-1B-HXB2, HIV-1B-92BR014, HIV-1B-92TH593, HIV-1B-92US727, HIV-1B-92US076, HIV-1C-96USNG58, HIV-1C-93MW959, HIV-1C-98IN022,HIV-1C-92BR025, HIV-1D-92UG021, HIV-1D-92UG024, HIV-1D-94UG114,HIV-1D-92UG038, HIV-1D-93UG065, HIV-1F-93BR20, HIV-1F-93BR29, HIV-1G-RU132, HIV-1G-RU570, HIV-1AE-CMU02, HIV-1AE-CMU06, HIV-1AE-92TH021, HIV-1AE-93TH051, HIV-1AE-95TH001, and HIV-1BF-93BR029.Tissue culture dose for 50% infectivity (TCID50) was determined intriplicate for each serially diluted virus using the Reed and Muenchmethod (31), and viral titers were expressed as infectious units permilliliter (IU/ml). Aliquots of DNA and RNA viruses were obtainedfrom Zeptometrix Corporation, Buffalo, NY (BK virus, BKV; Epstein-Barr virus, EBV; hepatitis B virus, HBV; hepatitis C virus, HCV; hu-man herpesvirus 6, HHV-6; human T-lymphotropic viruses type 1 and2, HTLV-1 and HTLV-2; cytomegalovirus, CMV; herpes simplex virus1 and 2; HSV-1 and HSV-2; and varicella zoster virus, VZV) and Ad-vanced Biotechnologies, Inc., Columbia, MD (human herpesvirus 7,HHV-7; and human immunodeficiency virus type 2, HIV-2).

Clinical samples. Plasma samples for the characterization and verifi-cation of the phenotypic HIV-1 tropism assay were obtained during rou-tine patient monitoring from a well-characterized cohort of HIV-infectedindividuals at the AIDS Clinical Trials Unit (ACTU) at Case WesternReserve University/University Hospitals of Cleveland (Cleveland, OH). Atotal of 76 RNA specimens, derived from plasma samples collected fromHIV-infected individuals enrolled in the (i) maraviroc expanded-accessprogram in Europe or (ii) ALLEGRO trial were obtained from the Hos-pital Carlos III (Madrid, Spain) (32). Phenotypic HIV-1 coreceptor tro-pism was determined at baseline using the original version of the Trofileassay (Monogram Biosciences), which had a reported non-R5 variantdetection limit of 5 to 10% (17). Written informed consent was obtainedfrom the patients before participation in the study as previously described(32, 33). Blood specimens from Cleveland were collected fresh, andplasma samples were processed and stored at �80°C for further analysis.RNA samples from Spain were shipped in dry ice and stored at �80°Cuntil analysis.

Reverse transcription (RT)-PCR amplification and nucleotide (pop-ulation) sequence analysis. Plasma viral RNA was purified from pelletedvirus particles by diluting 1 ml of plasma with 400 �l of phosphate-buff-ered saline (PBS) 1� solution and centrifuging at 20,000 � g for 60 min at4°C, removing 1,260 �l of cell-free supernatant and resuspending thepellet in the remaining 140 �l, to finally extract viral RNA using theQIAamp viral RNA minikit (Qiagen; Valencia, CA). Viral RNA was re-

verse transcribed using AccuScript high-fidelity reverse transcriptase(Stratagene Agilent, Santa Clara, CA) and 0.5 �M the corresponding an-tisense external primer in a 20-�l reaction mixture containing 1 mMdeoxynucleoside triphosphate (dNTPs), 10 mM dithiothreitol (DTT),and 10 units of RNase inhibitor. Viral cDNA was then PCR amplifiedusing a series of external and nested primers with defined cycling condi-tions. The HIV-1 envelope gene was amplified as a 2,302-nucleotide (nt)fragment, that is, all the surface glycoprotein (gp120) and most of thetransmembrane glycoprotein (gp41), missing only 321 nt of the gp41cytoplasmic domain. External PCRs were carried out in a 50-�l mixturecontaining 0.2 mM dNTPs, 3 mM MgCl2, and 2.5 units of Pfu Turbo DNApolymerase (Stratagene). Nested PCRs were carried out in a 50-�l mixturecontaining 0.2 mM dNTPs, 0.3 units of Pfu Turbo DNA polymerase, and1.9 units of Taq polymerase (Denville Scientific, Metuchen, NJ). PCRproducts corresponding to the gp120/gp41-coding regions of HIV-1were purified with the QIAquick PCR purification kit (Qiagen), andthe V3 region was sequenced (population sequence) using the AP Bio-tech DYEnamic ET Terminator cycle with Thermosequenase II (DavisSequencing LCC, Davis, CA). Nucleotide sequences were analyzed us-ing DNASTAR Lasergene Software Suite version 7.1.0 (Madison, WI).

Expression of patient-derived HIV-1 env genes. The HIV-1 gp120/gp41-coding region was introduced into a vector using an innovativeyeast-based cloning technology (34, 35) with minor modifications.Briefly, PCR products spanning the gp120/gp41-coding region of HIV-1were introduced via yeast homologous recombination into the pRECnfl-LEU-�Env(gp120-tatex2)/URA3 vector containing a near-full-lengthHIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacingthe gp120/gp41 HIV-1 coding sequence (Fig. 1A). This construction ex-presses all HIV-1 coding regions, that is, all genes corresponding to theHIV-1NL4-3 strain used as backbone in the vector plus the patient-derivedenv gene; however, it is unable to produce infectious virus since it is miss-ing the 5= long terminal repeat (LTR) region (R. Gibson and E. J. Arts,unpublished data). Following yeast transformation, vector DNA was pu-rified from the entire number of yeast colonies (typically 200 to �1,000individual colonies but not less than 333 colonies to achieve the 0.3%sensitivity) and used to transform Electrocomp TOP10 bacteria (Invitro-gen). Plasmid DNA from all the bacteria preparation, to guarantee thecontinuity of the viral population that may have existed in vivo, was pu-rified from 10 ml of bacteria (QIAprep Spin miniprep kit; Qiagen) andstored at �80°C until further use.

Cell-to-cell fusion assay to determine HIV-1 coreceptor tropism.The ability of HIV-1 to use CCR5 and/or CXCR4 as coreceptors to enterthe host cell was quantified using a modified version of the �-complemen-tation assay for HIV envelope glycoprotein-mediated fusion (19). Briefly,2 �g of the HIV expression vector, carrying the patient-derived env gene,and 2 �g of a vector expressing the � fragment of the �-galactosidase gene(pCMV�) were cotransfected into 7 � 105 HEK293T (donor) cells usingLipofectamine 2000 (Invitrogen). The target cells (U87.CD4.CCR5 orU87.CD4.CXCR4) were transfected with 4 �g of a vector expressing the fragment (pCMV) of the �-galactosidase gene. Forty-eight hours post-transfection, the donor and target cells were washed three times with 1�PBS, removed from the cell culture plates using a 1� solution of PBS and3 mM EDTA, counted, and resuspended in DMEM at a concentration of2 � 106 cells/ml. Fifty microliters (1 � 105) each of donor and target cellswere mixed and added together on a 96-well plate and incubated for 4 h at37°C in 5% CO2 (Fig. 1B). Cell-to-cell fusion events were quantified bymeasuring luminescence related to �-galactosidase activity (relative lightunits [RLU]) using the Galacto-star system (Applied Biosystems, Bed-ford, MA) in a multiwell plate reader (Victor V multilabel reader;PerkinElmer, Waltham, MA). Controls were run in each test, includingmock cells and transfections with plasmid DNA mixtures containing (i)100% and 0%, (ii) 1% and 99%, (iii) 0.3% and 99.7%, and (iv) 0% and100% of vectors expressing the env gene from the X4 HIV-1NL4-3 and theR5 HIV-1BaL strains, respectively. Technical cutoffs for the quantificationof env-mediated cell fusion events were calculated as the mean plus two

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standard deviations (SD) of the �-galactosidase activity detected afterHEK293T cells, transfected with 100% R5 HIV-1BaL or 100% X4 HIV-1NL4-3, were incubated in cell-to-cell fusion experiments withU87.CD4.CXCR4 or U87.CD4.CCR5. cells, respectively.

454 pyrosequencing of the V3 region of env. Ultradeep sequencinganalysis of the V3 region was performed as previously described (36).Briefly, a 232-nt fragment encompassing the V3 region was generated bynested PCR from the same external PCR product used to construct the envexpression vector described above. The nested PCR was carried out withPhusion high-fidelity DNA polymerase (New England BioLabs, Ipswich,MA) in a 50-�l reaction mixture containing 0.2 mM dNTPs, 2.5 mMMgCl2, and 0.2 �M of both the antisense and barcoded sense primers.PCR products were purified with the QIAquick PCR purification kit (Qia-

gen) and quantified with the Quant-iT PicoGreen double-stranded DNA(dsDNA) kit (Invitrogen). Pooled PCR products were clonally amplifiedon capture beads in water-oil emulsion microreactors. A total of 500,000HIV-1 env-enriched DNA beads were deposited in the wells of a 454 GSFLX instrument (454 Life Sciences/Roche, Branford, CT) and pyrose-quenced in the forward direction using 200 cycles in a 10-h sequencingrun. Following the sorting of reads by barcode into sample-derived datasets, the reads were mapped and aligned to the corresponding referencesequence (i.e., global V3 sequence for each sample) using Segminator II(37). Within this software, reads spanning the V3 loop were extracted,truncated, translated, and assembled for genotyping (24, 38).

Genotypic prediction of HIV-1 coreceptor tropism. HIV-1 corecep-tor tropism was predicted from population and 454 V3 sequences using

FIG 1 (A) Strategy to introduce patient-derived env PCR fragments into a proprietary vector via yeast homologous recombination as described previously (34,35). (B) Overview of the novel HIV-1 coreceptor tropism assay (VERITROP). Patient-derived viral amplicons were introduced into a vector lacking thecorresponding HIV-1 env sequence and cotransfected into HEK293T cells with a plasmid encoding the � fragment of the �-galactosidase gene (pCMV�).Cell-to-cell fusion events were quantified by transfecting the target cells (U87.CD4.CCR5 or U87.CD4.CXCR4) with a vector expressing the fragment(pCMV) of the �-galactosidase gene and mixing them with the HIV-1 env- and pCMV�-expressing cells.

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several bioinformatics tools. In the case of population sequences, nucleo-tide mixtures were considered when the second highest peak in the elec-tropherogram was above 25%, and then these nucleotide mixtures weretranslated into all possible permutations. The algorithms used to inferHIV-1 tropism from V3 amino acid sequences were (i) Geno2Pheno (25),with false-positive rates (FPR; predicted frequency of classifying an R5sequence as a non-R5 virus) based on optimized cutoffs associated withthe analysis of clinical data from MOTIVATE (2.5% and 5.75%), 3.5%FPR as previously described (39–41), or the recommendation from theEuropean Consensus Group on clinical management of HIV-1 tropismtesting (10%) as described on the Geno2Pheno website (http://coreceptor.bioinf.mpi-inf.mpg.de/index.php); (ii) webPSSM using the subtype Bx4r5 matrix (26); and (iii) the 11/24/25 charge rule (27, 28) implementedwithin our analysis pipeline (36). Finally, using the 454 pyrosequencingdata, plasma samples were classified as containing non-R5 viruses if atleast 0.3%, corresponding to the analytical sensitivity of the phenotypictests VERITROP and enhanced-sensitivity Trofile assay (ESTA) (42), or2% (39, 40) of the individual sequences were predicted to be non-R5.

Statistical analyses. Descriptive results are expressed as median valuesand interquartile ranges. Pearson correlation coefficient was used to de-termine the strength of association between categorical variables. All dif-ferences with a P value of 0.05 were considered statistically significant.All statistical analyses were performed using GraphPad Prism version 5.01(GraphPad Software, La Jolla, CA) unless otherwise specified.

RESULTSCharacterization of the RT-PCR amplification step. A 2,302-ntfragment of the HIV-1 env gene, containing the entire surfaceglycoprotein (gp120) and most of the transmembrane glycopro-tein (gp41), was RT-PCR amplified to be used in the cell-to-cellfusion assay (Fig. 1). Amplifying these large PCR products can bechallenging, particularly using clinical specimens with low viralloads. Thus, sensitivity of the RT-PCR amplification step wastested by analyzing 106 plasma samples obtained from two differ-ent clinical sources (i.e., the ACTU, Cleveland, OH, and the Hos-pital Carlos III, Madrid, Spain). Blood samples from HIV-infectedindividuals with plasma viral loads ranging from 50 to 10,000copies of viral RNA/ml were used to PCR amplify the env frag-ment. RT-PCR products of the correct size were consistently ob-tained (96%; 77/80) in plasma samples with �1,000 copies/ml ofHIV RNA (Table 1).

Highly reproducible success in RT-PCR amplification of thespecified product was obtained when testing 18 plasma sampleswith different viral loads. Details of these tests using two differentoperators, with different lots of critical reagents, and over a 7-dayperiod are described in Table S1 in the supplemental material.Finally, the specificity of the RT-PCR primers and reactions wasanalyzed using nucleic acids from a series of RNA and DNA vi-

ruses (i.e., HBV, HCV, HIV-2, HTLV-1, HTLV-2, BKV, EBV,HHV-6, HHV-7, CMV, HSV-1, HSV-2, and VZV). As expected,no cross-reactivity was observed with any of these viruses, as allRT-PCRs failed to generate any detectable amplicons (data notshown).

Cell-to-cell fusion assay to determine HIV-1 coreceptor tro-pism: proof of concept. Unlike previous approaches that utilizeligation-based cloning techniques (17, 42) or homologous recom-bination in mammalian cells (16, 18) to clone HIV-1 env genes,here we used a yeast-based recombination/gap repair method tointroduce patient-derived HIV-1 env fragments into a vector withthe final goal of determining HIV-1 tropism in a modified versionof the �-complementation assay for HIV envelope glycoprotein-mediated fusion (19). To test the system, mixtures of the env genefrom X4 HIV-1NL4-3 and R5 HIV-1BaL viruses (i.e., 100% and 0%,1% and 99%, 0.3% and 99.7%, and 0% and 100%, respectively)were introduced into the HIV expression vector and cotransfectedwith a vector expressing the � fragment of the �-galactosidasegene (pCMV�) into the HEK293T donor cells. The target cells(U87.CD4.CCR5 or U87.CD4.CXCR4) were transfected with avector expressing the fragment (pCMV) of the �-galactosidasegene (Fig. 1). As shown in Fig. 2, and in each further experiment,cells transfected with vectors containing 100% NL4-3 or 100%BaL were used to calculate the cutoffs defining a query virus asCCR5 or CXCR4 tropic, respectively. Importantly, our assay wasable to detect cells expressing X4 env genes at 0.3% of a populationcomprised mostly of R5-env-expressing cells (Fig. 2). Finally, aseries of controls ruled out the possibility of nonspecific lumines-cence levels contributing to the quantification of X4 or R5 expres-sion. False positives due to nonspecific cell-to-cell fusion and/or �and fragment complementation events were discarded by per-forming cell-to-cell fusion experiments between (i) HEK293Tcells expressing � fragment but not HIV-1 env and U87 cells withno fragment and (ii) quantifying the background luminescencelevels of HEK293T cells expressing � fragment and NL4-3 or BaLenv in the absence of U87 cells (Fig. 2).

Performance of the novel HIV-1 coreceptor tropism assay.The success of any anti-HIV therapy based on a CCR5 antago-nist depends on the ability of the HIV-1 tropism assay to detectminority non-R5 viruses within the HIV-1 population. For thatreason, we evaluated extensively the analytical sensitivity of ournovel HIV-1 tropism test to quantify non-R5 variants in mix-tures of cells expressing R5 and X4 env genes. We first mixedplasmid DNA (R5 HIV-1BaL and X4 HIV-1NL4-3) at differentproportions prior to transfection of the HEK293T cells, whichhas been the standard approach used to determine the sensitiv-ity of phenotypic HIV-1 tropism tests. As expected, our novelassay detected X4 env clones when they constituted 0.3% of theplasmid population and R5 env clones when present at 1% ofthe population (Fig. 3A). Next, we mixed yeast colonies priorto plasmid DNA isolation (used to transfect HEK293T cells)and obtained similar results, i.e., 0.3% sensitivity detecting X4-expressing cells, but we were able to also quantify R5 env clonespresent at 0.3% of the population (Fig. 3B). Finally, and moreimportantly, we mixed BaL and NL4-3 DNA prior to PCR am-plification (one of the first steps of the assay; Fig. 1B), mimick-ing the presence of R5 and X4 cDNA after RT-PCR, and againdetected 0.3% of cells expressing the X4 or R5 env genes inmixtures of cells mostly expressing R5 or X4 env genes, respec-tively (Fig. 3C).

TABLE 1 Sensitivity of RT-PCR amplification of the env gene (2,302 nt)

Viral load (copies/ml)

% positive samples by RT-PCR(no. of positive samples/totalno. of samples tested)a

50b 0 (0/11)50–1,000 27 (4/15)1,001–5,000 93 (26/28)5,001–10,000 93 (13/14)�10,000 100 (38/38)a RT-PCR amplification of patient-derived env fragments was performed with plasmasamples (n � 106) from HIV-infected individuals with viral loads ranging from 50 to�10,000 copies of viral RNA/ml as described in Materials and Methods.b The plasma viral loads of some of these samples may have been zero.

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Reproducibility of the novel HIV-1 tropism assay was firstevaluated by testing DNA from three viruses with different andknown HIV-1 coreceptor usage (X4 HIV-1NL4-3, R5 HIV-1BaL,and dual/mixed HIV-110-172), 10 times (10� replicates), by threeoperators, with distinct lots of critical reagents over a 4-week pe-riod. VERITROP accurately and repeatedly identified the virusesas X4, R5, and dual mixed (Fig. 4A). More importantly, goodreproducibility of the entire assay (from RNA extraction to cell-to-cell fusion) was found when over a 14-day period, two differentoperators analyzed two separate aliquots of 15 plasma samplesfrom HIV-infected individuals using different lots of critical re-agents (Fig. 4B).

Finally, although VERITROP was originally developed usingsubtype B HIV-1 strains, predominant in North America and Eu-rope (http://www.who.int/hiv/pub/global_report2010/en/index.html), it was important to test the ability of the assay to work withmore worldwide prevalent non-B HIV-1 variants. For that, thephenotypic HIV-1 tropism of 26 viruses was assayed, includingthree subtype A (HIV-1A-93RW024, HIV-1A-92UG031, and HIV-1A-92UG029), four subtype B (HIV-1B-92BR014, HIV-1B-92TH593,HIV-1B-92US727, and HIV-1B-92US076), four subtype C (HIV-1C-96USNG58, HIV-1C-93MW959, HIV-1C-98IN022, and HIV-1C-92BR025),five subtype D (HIV-1D-92UG021, HIV-1D-92UG024, HIV-1D-94UG114,HIV-1D-92UG038, and HIV-1D-93UG065), two subtype F (HIV-1F-93BR20 and HIV-1F-93BR29), and two subtype G (HIV-1G-RU132

and HIV-1G-RU570) viruses and six circulating recombinant forms(HIV-1AE-CMU02, HIV-1AE-CMU06, HIV-1AE-92TH021, HIV-1AE-93TH051, HIV-1AE-95TH001, and HIV-1BF-93BR029). A 100% con-cordance was observed between the HIV-1 tropism determined byVERITROP with the predetermined coreceptor tropism knownfor each one of these viruses (see Table S2 in the supplementalmaterial). Interestingly, although inconclusive results were ob-tained with HIV-1 group O viruses, HIV-2, and simian immuno-deficiency virus (SIV), VERITROP was able to determine the tro-pism of an HIV-1 group N strain (data not shown).

Comparison of HIV-1 tropism data obtained withVERITROP to other phenotypic or genotypic HIV-1 tropismassays. As described above, several phenotypic and genotypicHIV-1 coreceptor tropism methods have been developed, withthe ESTA version of the phenotypic Trofile assay (42) and ge-notypic tests based on population sequencing (21, 43) beingwidely used in the clinical setting. Here, the coreceptor tropismof viruses obtained from 76 HIV-infected individuals, screenedto be treated with a maraviroc-containing regimen, was ana-lyzed using two phenotypic (VERITROP and Trofile) and twogenotypic (population and 454 sequencing, together with a se-ries of algorithms to infer HIV-1 tropism from V3 sequences)assays. Hierarchical clustering analysis grouped the differentHIV-1 coreceptor tropism determinations based not only onthe sequencing method (population versus 454) but also on thealgorithm (11/24/25, Geno2Pheno, or webPSSM) and thresh-old used for the detection of individual non-R5 sequenceswithin the population (0.3% versus 2%) (Fig. 5A). HIV-1 tro-pism determinations based on 454 sequencing clustered to-gether by algorithm, regardless of the threshold for the detec-tion of individual non-R5 sequences, with the exception of thedata obtained using Geno2Pheno at 3.5% (65.8%) and 10%(68.4%) FPR with a 0.3% threshold that clustered withVERITROP (Fig. 5A). On the other hand, the original Trofileassay showed a better overall correlation (79.3%) with populationsequencing using either Geno2Pheno or PSSM (Fig. 5A). Interest-ingly, a 73.7% (56/76) concordance was observed between the twophenotypic tests, VERITROP and Trofile, with 19 of the 20 dis-cordant results corresponding to non-R5 variants detected byVERITROP and not by the original Trofile assay (Fig. 5A).

As described above, this cohort of 76 HIV-infected individ-uals was screened for the presence of CXCR4-tropic virusesusing the original Trofile assay before entering a maraviroc-containing regimen (32). Non-R5 viruses were detected in 27patients (who were not treated with maraviroc), while 9 failed

FIG 2 HIV-1 coreceptor tropism test: proof of principle. The new assay was tested by mixing different amounts of env amplicons from R5 (HIV-1BaL) and X4(HIV-1NL4-3) prior to cloning into the HIV expression vector. Cell-to-cell fusion events were quantified, including controls to take into account potentialnonspecific luminescence expression, i.e., (i) mock transfection, (ii) transfecting HEK293T cells with the � fragment but no env amplicon and target (U87) cellswith no fragment, and (iii) HEK293T cells expressing HIV-1 env (R5 BaL or X4 NL4-3) with the � fragment in the total absence of target (U87) cells. Technicalcutoffs for the quantification of env-mediated cell fusion events (green and blue dashed lines) were calculated as the means plus two standard deviations (SD) ofthe �-galactosidase activity detected after HEK293T cells transfected with 100% R5 HIV-1BaL or 100% X4 HIV-1NL4-3 were incubated in cell-to-cell fusionexperiments with U87.CD4.CXCR4 or U87.CD4.CCR5 cells, respectively. The bar denoting the minimal amount of non-R5 (X4) env detected with statisticalsignificance (0.3%) is enclosed by a red dashed line. RLU, relative light units.




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to respond to the therapy (i.e., plasma viral load above 400copies/ml at week 12), presumably due to the presence of un-detected non-R5 viruses. Thus, the ability to predict the successof the maraviroc-based regimen by a given HIV-1 tropism testwas calculated as the percentage of patients identified as carry-ing non-R5 viruses at baseline relative to the patients who werenot able to enter (n � 27) or failed to respond to the maraviroc-based treatment (n � 9), i.e., 27/36 or 75% in the case of Trofile(Fig. 5B). Based on this calculation, HIV-1 tropism assaysbased on population sequencing showed the lowest predictionability (44.4% to 58.3%) followed by the test based on 454

sequencing (55.6% to 86.1%), depending on the algorithm andthreshold used (Fig. 5B). Interestingly, HIV-1 tropism analysisbased on 454 sequencing using Geno2Pheno at a 10% FPR anda 0.3% threshold (31/36; 86.1%) and the phenotypic testVERITROP (29/36, 80.6%) seems to have more accurately pre-dicted the success of the maraviroc-based therapy (Fig. 5B).

DISCUSSION

The discovery of the chemokine receptors CCR5 and CXCR4 asthe main HIV-1 coreceptors led to a myriad of studies aimed to (i)identify the viral signatures responsible for coreceptor tropism,

FIG 3 Analytical sensitivity. The ability of the novel HIV-1 coreceptor tropism assay to detect minority non-R5 viruses within the HIV-1 population wasevaluated by mixing at different proportions: plasmid DNA (HIV-1BaL and HIV-1NL4-3) prior to transfection of the HEK293T cells (A), yeast colonies prior toplasmid DNA isolation (used to transfect HEK293T cells) (B), and HIV-1BaL and HIV-1NL4-3 DNA prior to PCR amplification (C). R5 and X4 technical cutoffswere calculated as described in the legend to Fig. 1 and Materials and Methods. Bars denoting the minimal amount of non-R5 (X4) env detected with statisticalsignificance (0.3%) are enclosed by a red dashed line. RLU, relative light units.

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(ii) understand the role of HIV-1 coreceptor tropism in virustransmission and disease progression, and (iii) explore the poten-tial use as therapeutic targets (reviewed in references 12, 14, and44–46). Following the identification of the env gene, primarily theV3 region of gp120, as the main determinant for HIV-1 coreceptortropism, a multitude of cell-based (phenotypic) and sequencing-based (genotypic) approaches have been developed to estimateand quantify the ability of the virus to use any or both receptors toenter the host cell (reviewed in references 12, 14, 15, 21, 23, and47). Here, we have developed a novel phenotypic test to determineHIV-1 coreceptor tropism (VERITROP) based on an innovativeyeast-based cloning strategy (34, 35) and a sensitive cell-to-cellfusion assay (19).

Early phenotypic methods to determine HIV-1 tropism usedHIV-1 isolates and their ability to induce syncytium formation(48) or to infect indicator cell lines (49–51). A problem with thesemethods is that prolonged culturing during virus isolation usuallyleads to changes in the original HIV-1 quasispecies population

(52). Thus, a viable alternative has been the use of env recombi-nant viruses and reporter cell lines expressing HIV-1 receptor andcoreceptors to evaluate HIV-1 coreceptor usage (16, 18, 53), in-cluding the only commercially available assay (Trofile; Mono-gram Biosciences, South San Francisco, CA) (17, 42). VERITROPis based on the introduction of patient-derived env fragments intoa noninfectious vector using yeast-based cloning (34, 35) and amodified version of the �-complementation assay for env-medi-ated cell-to-cell fusion (19). Yeast-based cloning is approximately100-fold more efficient than bacterium-based restriction enzymecloning or mammalian cell-based recombination, providing alarger number of unique clones and thereby better representationof the intrapatient HIV-1 population than other cloning method-ologies (34). Similarly, although numerous cell-to-cell fusionmethods have been used to study the env gp120-mediated entryand fusion process (54–56), the �-complementation system issimple, accurate, and well suited for high-throughput testing (19).

Our new VERITROP assay provided sensitive and reproduc-ible HIV-1 coreceptor tropism data. A 96% overall amplificationsuccess of the gp120/gp41 fragment from plasma samples with�1,000 copies/ml of HIV RNA, and the use of proprietary univer-sal primers, not only ensured successful amplification of samplesof diverse HIV-1 subtypes but also avoided amplification of non-specific products from endogenous or any of the related virusestested. Moreover, the subtype B backbone (HIV-1NL4-3) used toclone the patient-derived gp120/gp41 region was compatible notonly with env fragments from subtype B wild-type and multidrug-resistant strains but also with that from all non-B HIV-1 group Msubtypes analyzed (35). Furthermore, the VERITROP assay is ef-ficient and reproducible, with a turnaround time (11 days) com-parable to other phenotypic HIV-1 tropism tests, such as the en-hanced-sensitivity Trofile assay (16 days) (42), the RVAToulouse-TTT (9 days) (57), and TROCAI (21 to 42 days) (53).

The use of CCR5 antagonists in antiretroviral regimens hasrelied on the ability of HIV-1 tropism assays to detect virusescapable of using CXCR4 to enter the host cell (29, 58–60). As aconsequence, assay sensitivity (i.e., percentage of the non-R5 mi-nority variants that can be detected consistently) has played a ma-jor role on the success of clinical trials of CCR5 antagonists (13, 58,59, 61, 62). Here, we have consistently shown that VERITROP hasa 0.3% sensitivity threshold for detecting CXCR4-using minorvariants, which is similar to the sensitivity reported for the widelyused ESTA (42). This analytical sensitivity was determined notonly by using the standard approach of mixing plasmid DNA fromR5 and X4 viruses prior to transfection of the producer and targetcells but by mixing proviral DNA prior to the RT-PCR step, anal-ogous to what occurs in the analysis of clinical samples. Moreimportant, the 0.3% sensitivity for VERITROP and ESTA is (i)lower than the reported sensitivity for other phenotypic methodsto assess HIV-1 coreceptor tropism, e.g., 0.5% for the RVA Tou-louse-TTT (57) and 1% for the RVA Madrid (63); (ii) at least50-fold lower than the 15% to 20% sensitivity reported for popu-lation-based sequencing, independently of the bioinformatic toolused for predicting HIV-1 tropism (14, 21, 60); and (iii) in thesame range of more recent genotypic HIV-1 tropism assays basedon deep sequencing (22–24, 36, 39, 40). The 0.3% sensitivity es-tablished by ESTA, and now matched by VERITROP, continues tobe the reference standard for the detection of minor CXCR4-usingviruses (42). Although a sensitivity of 2% has been suggested instudies using deep sequencing as the clinically meaningful thresh-

FIG 4 Reproducibility of the HIV-1 coreceptor tropism assay. (A) env PCRproducts from three viruses with different and known HIV-1 coreceptor usage(X4, HIV-1NL4-3; R5, HIV-1BaL; and dual/mixed, HIV-110-172) were analyzed10 times (10� replicates), by three operators, with distinct lots of criticalreagents over a 4-week period. The coefficient of variation (%) is indicatedabove each bar. R5 and X4 technical cutoffs were calculated as described in thelegend to Fig. 1 and Materials and Methods. D/M, dual/mixed. (B) Reproduc-ibility of the entire assay, starting from RNA extraction from 15 plasma sam-ples from HIV-infected individuals, as determined by two different operatorswith different lots of critical reagents over a 14-day period. Pearson correlationcoefficient was used to determine the strength of association between the twodeterminations; r, correlation coefficient; p, two-tailed P value.




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FIG 5 Comparison of the novel HIV-1 coreceptor tropism assay with another phenotypic (Trofile) and two genotypic (population and 454 sequencing) assays.Plasma samples from 76 HIV-infected individuals prior to treatment with the CCR5 antagonist maraviroc were analyzed. HIV-1 coreceptor tropism wasdetermined using two phenotypic assays (Trofile and VERITROP) and inferred from population and 454 sequences using a series of algorithms as described inMaterials and Methods. (A) Hierarchical clustering analysis was used to group the HIV-1 coreceptor tropism determinations by similarity. Dendrograms werecalculated using the Euclidean distance and Complete cluster methods with 100 bootstrap iterations as described (http://www.hiv.lanl.gov/content/sequence/HEATMAP/heatmap.html). Green and red blocks indicate the absence or presence of non-R5 (X4) viruses, respectively, as determined by each assay. G2P,Geno2Pheno; PSSM, position-specific scoring matrices. Bootstrap values above 55% are indicated. (B) The ability of the different HIV-1 coreceptor tropism teststo predict the success of a maraviroc-based therapy was determined as the percentage of patients where non-R5 viruses were detected at baseline (no. of non-R5viruses) from the total number of patients who were not able to enter (due to the presence of non-R5 viruses at baseline as determined by Trofile) or failed torespond to the maraviroc-based regimen (n � 36), i.e., % treatment success prediction � (no. of patients with non-R5 viruses/no. of patients not entering orfailing the maraviroc-based regimen [n � 36]) � 100. Values for the tests with the three highest percentages (best capability to predict the success ofmaraviroc-based therapy) are indicated.



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old (39, 40), the limit above which minor non-R5 variants lead tovirological failure in patients on CCR5 antagonist-based therapyremains unclear.

Several studies have compared the efficacy of phenotypic andgenotypic HIV-1 tropism assays to detect non-R5 variants (64–68). In general, population-based sequencing tests are less sensi-tive and less specific than phenotypic assays (14, 29), although afew studies have shown significant concordance and similar pre-dictive values (40, 68, 69). On the other hand, more sensitive deepsequencing methods for HIV-1 coreceptor tropism assays resultedin the detection of minor variants, which correlated well with bothphenotypic assays (39, 40, 57) and virological response to mara-viroc (39, 40). Here, VERITROP and the original Trofile assay,which had a reported non-R5 variant detection limit of 5 to 10%(17), showed good correlation (73.7%), with most of the discor-dant results related to the higher number of non-R5 variants de-tected by VERITROP than the original Trofile. Interestingly, andin support of these findings, VERITROP results correlated betterwith data obtained using 454 sequencing and Geno2Pheno pre-diction, with the most stringent 0.3% threshold for the detectionof individual non-R5 sequences. On the other hand, the less sen-sitive original Trofile showed a higher concordance with popula-tion sequencing. Furthermore, both the phenotypic VERITROPassay and a genotypic test based on 454 sequencing usingGeno2Pheno at a 10% FPR and a 0.3% threshold predicted betterthe potential success of a maraviroc-based antiretroviral regimen.

In summary, we have developed, characterized, verified, andprevalidated a novel phenotypic test to determine HIV-1 corecep-tor tropism (VERITROP) based on the analysis of patient-derivedHIV-1 env fragments using an innovative yeast-based cloning sys-tem coupled with a sensitive cell-to-cell fusion assay. With a fastturnaround time, VERITROP is specific and sensitive, capable ofdetecting 0.3% minority non-R5 variants in clinical samples. Webelieve that with additional studies to further validate our assay,VERITROP will improve the management of HIV-infected indi-viduals prior to and/or during treatment with CCR5 antagonists.

ACKNOWLEDGMENTS

We thank Benigno Rodriguez and the Case Western Reserve University/University Hospitals Center for AIDS Research (NIH P30 AI036219) forproviding access to the clinical samples. We also thank Nathaniel R. Lan-dau (NYU School of Medicine, New York, NY) for providing the expres-sion vectors related to the �-complementation assay for HIV envelopeglycoprotein-mediated fusion and for reviewing and providing valuablecriticism on the manuscript. We thank James F. Demarest (ViiV Health-care, Research Triangle Park, NC) and Robert Burnside (Pfizer, New Lon-don/Norwich, CT) for providing clinical data and statistical support. Weare grateful to Hendrik Poinar (McMaster University, Hamilton, Canada)for his support and access to his laboratory to perform the 454 sequencingruns. J.A. was funded by BBSRC project grant BB/H012419/1.

M.E.Q.-M. designed the study, collected and assembled the data, andwrote and drafted the manuscript. J.W., A.C.V., D.W., R.M.G., A.M.R.,J.D.R., D.W., and A.W. performed cell culture and molecular experi-ments. J.W., K.H., and J.A. performed and analyzed deep sequencing.J.W., J.A., D.L.R., and M.E.Q.-M. contributed to the analysis of the data.E.P., K.K., V.S., P.D.O., and E.J.A. provided access to material, technol-ogy, and critical advice. All authors read and approved the final manu-script.

Most of the experiments described in this study were performed atDiagnostic Hybrids, Inc., a Quidel Company. Diagnostic Hybrids, Inc.,acknowledges the contribution of the State of Ohio, Department of De-velopment and Third Frontier Commission, which provided funding in

support of the Platform for Antiviral Resistance Testing and Vaccine De-velopment project. This publication was prepared with financial supportfrom the State of Ohio. The content reflects the views of Diagnostic Hy-brids, Inc., and does not purport to reflect the views of the State of Ohio.

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