Consideration of Viral Resistance for Optimization of Direct Antiviral Therapy of Hepatitis C Virus

17 Pages·2015·1.41 MB·English

RESEARCHARTICLE
Consideration of Viral Resistance for
Optimization of Direct Antiviral Therapy of
Hepatitis C Virus Genotype 1-Infected
Patients
JuliaDietz,SimoneSusser,CaterinaBerkowski,DanyPerner,StefanZeuzem,
ChristophSarrazin*
MedicalDepartment1,GoetheUniversityHospital,Frankfurt,Germany
* [email protected]
Abstract
Differenthighlyeffectiveinterferon-freetreatmentoptionsforchronichepatitisCvirus
OPENACCESS (HCV)infectionarecurrentlyavailable.Pre-existenceofresistanceassociatedvariants
(RAVs)todirectantiviralagents(DAAs)reducessustainedvirologicresponse(SVR)rates
Citation:DietzJ,SusserS,BerkowskiC,PernerD,
ZeuzemS,SarrazinC(2015)ConsiderationofViral by3–53%inhepatitisCvirus(HCV)genotype1infectedpatientsdependingondifferent
ResistanceforOptimizationofDirectAntiviral predictorsandtheDAAregimenused.Frequenciesofsingleandcombinedresistanceto
TherapyofHepatitisCVirusGenotype1-Infected
NS3,NS5AandNS5Binhibitorsandconsequencesfortheapplicabilityofdifferenttreat-
Patients.PLoSONE10(8):e0134395.doi:10.1371/
journal.pone.0134395 mentregimensareunknown.ParallelpopulationbasedsequencingofHCVNS3,NS5A
andNS5Bgenesin312treatment-naïveCaucasianHCVgenotype1infectedpatients
Editor:Ming-LungYu,KaohsiungMedicalUniversity
Hospital,KaohsiungMedicalUniversity,TAIWAN showedthepresenceofmajorresistantvariantsin20.5%(NS3),11.9%(NS5A),and22.1%
(NS5B)withimportantdifferencesforHCVsubtypes.InNS3,Q80Kwasobservedin34.7%
Received:April2,2015
and2.1%ofsubtype1aand1bpatients,respectivelywhileotherRAVstosecondgenera-
Accepted:July8,2015
tionproteaseinhibitorsweredetectedrarely(1.4%).WithinNS5ARAVswereobservedin
Published:August28,2015 7.1%ofsubtype1aand17.6%insubtype1binfectedpatients.RAVstonon-nucleoside
Copyright:©2015Dietzetal.Thisisanopen NS5Binhibitorswereobservedin3.5%and44.4%ofsubtype1aand1bpatients,respec-
accessarticledistributedunderthetermsofthe tively.ConsideringallthreeDAAtargetsallsubtype1aand98.6%ofsubtype1binfected
CreativeCommonsAttributionLicense,whichpermits
patientswerewildtypeforatleastoneinterferonfreeDAAregimencurrentlyavailable.In
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare conclusion,baselineresistancetestingallowstheselectionofatleastoneRAVs-freetreat-
credited. mentoptionfornearlyallpatientsenablingapotentiallycost-andefficacy-optimizedtreat-
DataAvailabilityStatement:Allrelevantdataare mentofchronichepatitisC.
withinthepaperanditsSupportingInformationfiles.
Funding:ThisstudywassupportedbyaDZIF
(DeutschesZentrumfürInfektionsforschung)(http://
www.dzif.de/forschung/hepatitis/)grantentitled
“Geno-&phenotypicNS3,NS5AandNS5Binhibitor
resistanceanalysis”toChristophSarrazinandStefan Introduction
Zeuzem(TTU05.902).Furthersupportwasobtained
DetailedcharacterizationofviralproteinswithcriticalfunctionsinthehepatitisCvirus(HCV)
inpartfromJanssen-CilagGmbH.Thefundershad
replicationcycle,liketheNS3/4Aprotease,theNS5AproteinandtheNS5Bpolymerase
noroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript. togetherwiththeinventionofacellculturereplicationmodelledtothedevelopmentofdirect
PLOSONE|DOI:10.1371/journal.pone.0134395 August28,2015 1/17
ViralResistanceandOptimizationofHepatitisCTherapy
CompetingInterests:Theauthorshavedeclared actingantivirals(DAA)targetingtheseHCVproteins[1,2].Currently,differentinterferon-
thatnocompetinginterestsexists. freecombinationtherapiesfortreatmentofchronichepatitisCvirus(HCV)infectionwith
directantiviralagents(DAAs)areapproved.ForHCVgenotype1infectedpatientscombina-
tiontherapiesofanucleotideNS5Bpolymeraseinhibitor(Sofosbuvir,SOF)witheitheraNS3
proteaseinhibitor(Simeprevir,SMV)oraNS5Ainhibitor(Daclatasvir,DCVandLedipasvir,
LDV)areavailable[3–7].AlternativeoptionsareacombinationofaNS3proteaseandNS5A
inhibitorwithrestrictiontoHCVsubtype1b(Asunaprevir,ASVplusDaclatasvir)oratriple
DAAtherapy(NS3protease-,NS5A-andnon-nucleosideNS5Binhibitor)forallgenotype1
infectedpatients(Paritaprevir,PTV,Ombitasvir,OMVandDasabuvir,DSV)[8–12].Overall,
highratesofsustainedvirologicresponse(SVR)between82%and99%havebeenobservedin
thedifferentunderlyingstudies[3–12].PredictorsofSVRmainlyarenon-responsetoprevious
antiviraltherapyandthepresenceoflivercirrhosis[8,9,11,12].However,alsopre-existence
ofresistanceassociatedvariants(RAVs)wasassociatedwithareductionofSVRratesby
3–53%instudieswithavailabledata[5–8].WhiletheNS5BnucleotideanalogueSofosbuvir
hasahighgeneticbarriertoresistanceandnoclinicalrelevanceofpre-existingL159,S282and
V321variantsforIFN-freetherapieshavebeenshownsofar,forNS3protease-,NS5A-and
non-nucleosideNS5B-inhibitors,RAVswithdifferentlevelsofresistancetothedifferentavail-
ableDAAshavebeendescribedandfoundclinicallyrelevant[13–23].
Inthepresentstudy,frequenciesofRAVstocurrentlyavailableNS3,NS5AandNS5B
inhibitorshavebeenassessedin312CaucasianpatientswithHCVgenotype1infectionbypar-
allelpopulation-basedsequencingfortheexplorationoftherateofpatientswithcoexistenceof
RAVsfordifferentdualandtripleDAAcombinationtherapiescurrentlyavailable.
MaterialsandMethods
Patients
Baselineserumsamplesof312consecutiveCaucasianpatients,withachronicgenotype1hepa-
titisCinfectionwhoweretreatment-andDAA-naïvewereobtainedfrompreviouslycon-
ductedclinicalstudies[24].InvestigationswereperformedaccordingtotheDeclarationof
Helsinkiandapprovaloftheenrollmentintherespectivestudiesaswellastheusageofpatient
bloodsamplesforresearchpurposewasobtainedfromthelocalethicscommittee(Ethikkom-
missionderÄrztekammerdesSaarlandes),andwritteninformedconsentwasobtainedfrom
allpatients.
HCVRNAextraction,reversetranscriptionandPCR
HCVRNAwasextractedfrom140μLserum(QIAampViralRNAMini-Kit,Qiagen,Hilden,
Germany)andcomplementaryDNA(cDNA)wassynthesizedusingSuperScriptIIIReverse
Transcriptase(Invitrogen)aspreviouslydescribed[25].Forallamplificationsoftherespective
HCVregions,weconductednestedPCRsusing1/10ofcDNAorouterPCRproductrespec-
tivelybyapplyingtheFastCyclingPCRKit(Qiagen).AllPCRswerecarriedoutbyusingthe
primersforbothsubtypes(1aand1b)incombination.TheHCVNS3proteasedomainwas
amplifiedintheouterPCRwithprimersdescribedpreviously[26]:U376_1bc_F,ATGGAGACC
AAGATCATCACCTGGG;U3276_1a_F,ATGGAGACCAAGCTCATCACGTGGG;D4421_1b_R,
CCGTCGGCAAGGAACTTGCCATAGGTGGAandD4421_1a_R,ACCCGCCGTCGGCAAGGAA
CTTGCCGTA.First,PCRmixtureswereincubatedat95°Cfor5minutes,followedby35cycles
at96°Cfor5seconds,60°Cfor5secondsand68°Cfor1minuteandafinalelongationoccurred
at72°Cfor1minute.TheinnerPCRwasperformedwith3420_1b_F,AGGGCATTTAAATAG
CCACCATGGCGCCCATCACGGCCTACTCCCAACAGAC;3420_1a_F,AGGGCATTTAAATAGCC
ACCATGGCGCCCATCACGGCGTACGCCCAGCAGAC;4038_1b_R,AAAAAGCGGCCGCAGCC
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ViralResistanceandOptimizationofHepatitisCTherapy
GGCACCTTAGTGCTCTTGCCGCTGCC;4038_1a_R,AAAAAGCGGCCGCAGCCGGGACCTT
GGTGCTCTTACCGCTGCCandthesamecyclingconditionsasfortheouterPCR.NS5Awas
amplifiedusingNS5A_1a_6279_F,ATCTGGGACTGGATATGC;NS5A_1b_6279_F,
GTTTGGGACTGGATATGC;NS5A_1a_6599_R,AGACACCCTCCACAGandNS5A_1b_6650_R,
CGTCACGTAGTGGAAATCintheouterPCR.ThecyclingconditionsforNS5Awerethesame
asfortheamplificationofNS3,butanannealingtemperatureof50°Caswellasa30second
elongationstepat68°Cwereused.FortheinnerPCR,NS5A_1a_6285_F,GACTGGATATGCG
AGGTG;NS5A_1b_6318_F,ACCTGGCTCCAGTCCAAG;NS5A_1a_6590_R,CCACAGCGCGAA
CKTATAG;andNS5A_1b_6618_R,CCTCCACRTACTCCTCAGwereused.Besidestheanneal-
ingtemperatureof54°C,thePCRprofilewasthesameasfortheouterNS5APCR.Forthe
semi-nestedamplificationofNS5B,intheouterPCR,NS5B_1a_8445_F,AGCGGCGTACTGAC
AAC;NS5B_1b_8460_F,ACTAGCTGCGGCAACACC;NS5B_1a_1702_R,GGGCATGAGACAC
GCTGTGandNS5B_1b_1700_R,GCACGAGACAGGCTGTGprimerswereused.TheinnerPCR
amplificationwasperformedwithbothreverseprimersoftheouterPCRincombinationwith
NS5B_1a_8467_F,GTGGTAACACCCTCACTTGandNS5B_1b_8522_F,GCTCCAGGACTGCAC
AATGforwardprimers.ThetemperatureprofileofbothouterandinnerNS5BPCRswasthe
sameasfortheamplificationofNS3,butanannealingtemperatureof52°Cwasappliedforthe
innerandouterNS5BPCRs.TheresultingPCRproductswereanalyzedforcorrectsizeon1%
agarosegelsstainedwithethidiumbromideandweregel-purifiedusingtheQIAquickGel
ExtractionKit(Qiagen).
SequencinganalysisofHCVNS3,NS5AandNS5Bgenes
ThepurifiedPCRproductsofNS3,NS5AandNS5Bwerepopulation-basedsequenced.NS3of
GT1aand1bwassequencedwiththeinnerforwardPCRprimerandNS5Asequencing
occurredforGT1awiththeinnerforwardandforGT1bwiththeinnerreversePCRprimer.
NS5Bwassequencedusingoneforwardprimerforbothgenotypes(NS5B_1a/1b_1213F,
GTCAATTCCTGGCTAGGC)andspecificreverseprimersforGT1aandGT1bwhichwere
alsousedfortheamplificationofNS5B.Allsequencinganalyseswereperformedaccordingto
themanufacturer´sprotocol(BigDyeTerminatorv1.1CycleSequencingKit,AppliedBiosys-
tems)onanABIPrism3130xlGeneticAnalyzer(AppliedBiosystems).Population-based
sequencinggeneratesaconsensussequenceoftheviralquasispeciesandhasasensitivityof
approximately20%forminorityvariants.Inaccordancewithanotherstudy[27]allRAVs
observedattherespectivepositionsintheelectropherogramweretakenintoaccount.This
includedalsominorityvariantswhichwererecognizedasmixedpeaksinthesequence.All
caseswithminorityvariantsarelistedinthesupplementaryinformation(S1Table).After
proofreading,allsequenceswerealignedusingBioEdit.version7.2.3[28].
AnalysisofbaselineRAVs
WeinvestigatedtheoccurrenceofbaselineRAVswithinsamplesof312HCVGT1-infected
DAA-naïvepatientsforwhichparallelsequencesforNS3,NS5AandNS5Bwereobtained
(n=170GT1a,n=142GT1b).RAVsforcurrentlyapprovedforIFN-freetreatmentofchronic
hepatitisC(SMV,ASV;PTV,DCV,LDV,OMV,DSV)wereconsideredasrelevant,ifthey
weredescribedinpreviousstudiestobeassociatedinvivowithtreatmentfailureand/orhave
beenshownininvitrophenotypicassaystoconferamorethan2-foldchangeddrugsuscepti-
bilityincomparisontothewildtypereferencestrain.Basedonthis,RAVswereanalysedatthe
followingpositionsincomparisonwiththerespectiveHCVreferencestrain(GT1a:H77[29],
GT1b:Con1[30]):NS3:F43/I/L/S//V,Y56H,Q80K/R,S122R,R155K/G,A156G/S/Tand
D168A/C/E/G/H/N/T/V/Y;NS5A:L/M28A/T/V,Q30E/H/RandR30Q(secondarysite),L31F/
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I/M/V,P32L,Q54H(secondarysite),H58DandP58S(secondarysite),Y93C/F/H/N/S;NS5B:
C316H/N/Y,S368T,Y448C/H,S556G/R,D559R[17–22,31–36].Table1showstheEC50val-
uesofvariantswhichweredetectedinourcohort.Sequencingofvariantsassociatedwithresis-
tancetosofosbuvirinvitroandinvivo(L159F,S282T,V321A)wasnotperformedasthese
variantswerenotdetectedbeforetreatmentinitiation(S282T)[15,16]and/or(L159F,
V321A)werenotassociatedwithreducedsusceptibilitytosofosbuvir[16,22,37,38].AllHCV
subtypesweredeterminedbysequencinganalysisoftheNS3proteasedomain.Wereferan
HCVregionaswildtypewhentherespectiveregiondoesnotcontainRAVsaccordingtoour
definition.WeclassifiedidentifiedRAVsonthebasisoffoldchangecomparedtothewildtype
replicondeterminedinpreviousstudiesinlow-levelresistant(2-10-foldchange),intermediate
resistant(11-100-foldchange)andhigh-levelresistant(>100-foldchange)variants(Table1).
Results
Patientcharacteristicsofthecohort
ThebaselineclinicalcharacteristicsoftheinvestigatedpatientsareshowninTable2.The
majorityofpatientsweremale(n=207,66.4%)andthemedianagewas47years.Themedian
HCVviralloadwas1.4x106IU/mLandapproximatelyhalfofthepatientswereinfectedwith
HCVsubtype1aand1b,respectively.InformationonIL28Bgenotypeinformationwasavail-
ablefor273individualsandaCCgenotypewasfoundin94patients(34.4%)andalivercirrho-
siswasdiagnosedin17.6%(n=48)ofpatients.
BaselineNS3proteaseRAVs
TheHCVNS3proteasesequenceswereanalyzedatpositionswhichhavepreviouslybeen
reportedtoberesponsiblefortheemergenceofRAVsduringtreatmentwithproteaseinhibitors
(PIs).ForthemacrocyclicPIssimeprevir,asunaprevirandparitaprevir,whichareapprovedfor
aninterferon-freetreatmentofchronichepatitisC,theseprimaryresistancemutationswere
detectedatpositionsQ80,R155andD168[39].Atbaseline,wewerenotabletodetecttheinter-
mediateresistanceconferringR155KmutationandalsoR155Gwasnotobserved.Onlytwo
GT1b-infectedpatients(1.4%)displayedaD168EvariantwhichisresistanttoSMV,ASVand
PTV(SMV:43-fold,ASV:78-fold,PTV:4-foldchangeinEC ,Table1).TheQ80Kvariant
50
conferringlow-levelresistancetoSMV(inGT1aand1b)andASV(inGT1b)occurredatbase-
linein19.9%(n=62/312)ofGT1-infectedpatientsandwasdetectedin34.7%(n=59)of
GT1a-andin2.1%(n=3)ofGT1b-infectedindividuals(Fig1).However,Q80Kconfersonly
minimalresistancetoPTV[34].Atposition80,onlyoneindividualwithaGT1binfectionhada
Q80Rvariant(0.7%).TheoveralloccurrenceofNS3RAVsinGT1was20.5%.
SeveralotherpositionswithvaryingrelevancefordifferentPIshavebeenreportedandvari-
antsatpositionsF43andA156conferresistanceinvitrotoSMVandASVbutwerenot
observedinvivoatbaselineorinassociationwithtreatmentfailure[17,27,31,32,40,41].
Thesevariants,theSMVintermediate-resistantS122RvariantaswellastheY56Hmutation
whichoccurredincombinationwithD168AinPTV-treatedpatients[11]andisknowntobe
relevantfortreatmentwithMK-5172[27]werenotdetectableinthepresentcohortbybaseline
population-basedsequencing.
BaselineNS5ARAVs
ResistancemutationswithinNS5Awhichweredescribedtoconferresistancetodaclatasvir,
ledipasvirandombitasvirarelargelyoverlapping.Theseincludetheprimaryvariantsatposi-
tionsM28,Q30,L31,andY93[42].Secondarysitemutationsweredescribedfordaclatasvir
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Table1. EC50valuesofbaselineRAVswithinNS3,NS5AandNS5Bdetectedinthepresentcohort.
Position Variant HCVregion EC50[fold-change]1(subtype) ResistanceLevel DAA References
Q80 K NS3 9.3(1a),7.7(1b) low SMV [31]
3(1a),6.5(1b) ASV [32]
3(1a) PTV [34]
R NS3 13(1a),6.9(1b) low-intermediate SMV [31]
4(1b) ASV [32]
2(1a) PTV [34]
D168 E NS3 26(1a),43(1b) low-intermediate SMV [31]
58(1a),78(1b) ASV [32]
14(1a),4(1b) PTV [34]
M28 V NS5A 1.3(1a) intermediate DCV [19]
n.d. LDV n.d.
58(1a) OMV [18]
Q30 H NS5A 1477(1a) low-high DCV [19]
73(1a) LDV [21]
3(1a) OMV [36]
L31 M NS5A 341(1a),3(1b) low-high DCV [19]
140(1a),2.5–100(1b) LDV [21,22]
2(1a),0.9(1b) OMV [33]
F NS5A 5(1b) low DCV [20]
n.d. LDV n.d.
10(1b) OMV [18]
Y93 C NS5A 1864(1a) high DCV [19]
327(1a) LDV [21]
1675(1a) OMV [18]
F NS5A n.d. low-intermediate DCV n.d.
2.5–100(1a) LDV [22]
n.d. OMV n.d.
H NS5A 5432(1a),24(1b) intermediate-high DCV [19]
3309(1a),1319(1b) LDV [21]
41383(1a),77(1b) OMV [36]
N NS5A 47477(1a) high DCV [19]
>100(1a) LDV [22]
66739-fold(1a) OMV [18]
C316 H NS5B 229(1b) high DSV [33]
N NS5B 5(1b) low DSV [35]
Y NS5B 1472(1a),1569(1b) high DSV [35]
Y448 H NS5B 975(1a),46(1b) intermediate-high DSV [35]
S556 G NS5B 30(1a),11(1b) intermediate DSV [35]
N NS5B 29(1a) intermediate DSV [35]
R NS5B 261(1a) high DSV [33]
C316N+S556G NS5B 38(1b) intermediate DSV [33]
1comparedtoWTreplicon
n.d.:notdetermined
doi:10.1371/journal.pone.0134395.t001
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Table2. Patientcharacteristicsatbaseline.
Parameter Patients
Age(years)[median(range)] 47(19–79)
Sex
Male[n(%)] 207(66.4%)
Female[n(%)] 105(33.6%)
Bodymassindex(kg/m2)[median(range)] 24.5(16.6–45.5)
IL28BCCgenotype
CC[n(%)] 94(34.4%)
non-CC[n(%)] 179(65.6%)
n.d.[n] 39
Liverenyzmes
ALT(U/l)[median(range)] 78(14–551)
AST(U/l)[median(range)] 56(5–398)
GGT[median(range)] 63(2–720)
Cirrhosis
Patientswithcirrhosis[n(%)] 48(17.6%)
Patientswithoutcirrhosis[n(%)] 225(82.4%)
n.d.[n] 39
HCVinfection
HCVviralload(IU/mL)[median(range)] 1.4x106(5.2x103–3.6x107)
HCVGT1a[n(%)] 170(54.5%)
HCVGT1b[n(%)] 142(45.5%)
doi:10.1371/journal.pone.0134395.t002
withR30QandP58Senhancingtheresistanceofprimarymutationsbutwhicharenotthem-
selvesresistantandQ54Hwasalsoreportedassecondarymutation,notincreasingtheresis-
tanceofprimaryresistancemutations[20].However,M28A/T,L31VandH58Dvariantswere
notdetectedinourcohort.ForGT1a,thesevariantswereshowntoconferresistancetowards
DCV,LDVandOMVinstudieswithavailabledata[18–20,22,36].
InGT1a-infectedpatients,NS5Ainhibitorresistantvariantsweredetectedrarely(7.1%)
withtheM28VvariantconferringintermediateresistancetowardsOMV(nottoDCVand
LDV)occurringmostfrequently(3.5%)followedbyDCVandLDVhighlevelresistantL31M
(1.2%)mutation.FurtherdetectedvariantswereY93F/N,Q30HandL31M,Y93H(each0.6%)
(Fig2A).InGT1b-infectedpatients,theprevalenceofRAVs(17.6%)washigherincomparison
toGT1aandtheintermediatetohighlevelY93Hvariantwastheprimarymutationwhich
emerged(Y93H:14.1%),whichwasfrequentlyaccompaniedbyDCVsecondarymutationsat
positionsR30,P58andQ54(Fig2B).InGT1thetotalnumberofNS5ARAVswas11.9%.
Besidesthis,L31F/MvariantswererarelyfoundinGT1b(L31F:1.4%,L31Mandorwith
R30Q,Q54H:2.1%).L31MconfersnoresistancetowardsOMV[36]andtheresistanceofL31F
towardsLDVisunknownsofar.
Baselinenon-nucleosideinhibitorNS5BRAVs
pt?>WithinNS5B,weanalyzedresistantvariantsknowntobeassociatedwithdasabuvirtreat-
mentfailure:C316H/N/Y,Y448H/C,S556G/N/RandD559G[11,33,35,43].TheS368Tmuta-
tionishighlevelresistantinvitroinGT1bagainstDSV[35]butwasnotfoundinvivosofar
andwasalsonotdetectableinthepresentstudy.WeobservedNS5BRAVsin22.1%ofGT1
patients.Interestingly,theprevalenceofDSV-resistantRAVswaslowinGT1a-infected
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Fig1.BaselineNS3RAVsinDAA-naïvepatients.A)GT1a-andB)GT1b-infectedpatients.
doi:10.1371/journal.pone.0134395.g001
individuals(3.5%).Twopatientseachcarriedhigh-levelresistantC316Yorintermediateresis-
tantS556N(1.2%)andoneindividualeachhadintermediateresistantS556Gorhighlevel
resistantS556Rmutation(0.6%).Bycontrast,RAVsappearedfrequentlyin44.4%ofGT1b
infectedpatientsandthedominatingvariantswereC316N(22.5%),S556G(7.0%)aswellas
C316N+S556G(12.7%)(Fig3).However,thesemutationshavebeendescribedtoconfera
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Fig2.OccurrenceofRAVswithinNS5AinGT1a-(A)andGT1b-(B)infectedpatients.
doi:10.1371/journal.pone.0134395.g002
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relativelylowresistancewitha5-fold(C316N)and11-fold(S556G)resistance,respectively
comparedtoaGT1bwildtypereplicon.Acombinationofbothmutationsyieldedina38-fold
resistance[33,35].
RAVswithrespecttodifferenttreatmentregimens
Next,weanalyzedtheproportionofpatientswhoarefavourablysuitedtoreceiveinterferon-
freetreatmentregimensundertheconsiderationofbaselineRAVs.Forthetreatmentwith
SMV/SOF,weconsideredallNS3proteasemutationsasrelevantshowninFig1.NoRAVs
(wildtype)weredetectedin65.3%ofGT1a-and96.5%ofGT1b-infectedpatients,potentially
enablingaSMV/SOF-basedtherapy(Fig4).
Fordaclatasvirandledipasvirtheresistanceprofilesweresimilar(seeabove).Thevast
majorityofGT1a-infectedindividualscontainnoRAVswithinNS5AsupportingaDCV/SOF
(97.1%)orLDV/SOFtherapy(96.5%).TheY93FvariantdetectedinoneGT1apatientis
knowntoconferresistanceonlytowardsLDV[22]andL31FinGT1bislow-levelresistant
towardsDCVandOMVbutwasnotdescribedforLDV[18,20].Thereforetheresistancepro-
filesofthesesubstancesareslightlydeviatingand82.4%and83.8%ofGT1b-infectedpatients
wereeligibletoreceiveDCV/SOForLDV/SOFrespectively(Fig4).
Asthecombinationtherapyconsistingofasunapreviranddaclatasvir(ASV/DCV)showed
reducedefficacyagainstGT1a[44],thistherapyisapprovedonlyforGT1b.81.0%ofGT1b-
infectedindividualshadnoNS3orNS5ARAVsconferringresistancetoASVorDCV(Fig4).
Ofthe27patientswithRAVs,threepersonscarriedRAVswithinbothNS3andNS5A(not
shown)andthemajorityofRAVscanbeattributedtotheNS5AY93Hmutation(Fig2).
Forparitaprevir,ombitasviranddasabuvirtripletherapy,combinedRAVswithinNS3,
NS5AandNS5Bwereinvestigatedandwildtypevariantsforallthreeregionswereidentifiedin
91.8%ofGT1aand46.5%ofGT1bpatients(Fig4).InGT1aRAVsmainlyconsistedofmuta-
tionswithinNS5A(M28V,Q30H,Y93C)orNS5B(C316Y,S556G/N/R).Ofnote,RAVswithin
bothNS5AandNS5BweredisplayedbyoneGT1aand10GT1b-infectedpatients(datanot
shown).InGT1b,Y93HwithinNS5AaswellasC316NandS556GwithinNS5Bwerethedom-
inatingmutations(Figs2and3).TheonlyNS3RAVrelevantforthistherapywasD168E
(n=2,GT1b)(Fig1).
Furthermore,ourinterestwastoidentifypatientswithoutanyrestrictionstoreceivethe
describedtherapyregimensundertheconsiderationofRAVs.Wildtypevariantswithrespect
toalltreatmentregimensweredisplayedby60.6%ofpatientswithGT1aand45.1%withGT1b
(Fig4).Thesepatientsareinprinciplesuitedforalltherapies.InGT1a,RAVsmainlyconsisted
oftheNS3Q80KmutationrelevantforSMV/ASV(34.7%,Fig1A)andtoamuchlesserextend
oftheOMV-relevantM28VvariantwithinNS5A(3.5%,Fig2A).NS5BRAVsweredetected
rarelyinGT1a-infectedindividuals.ViceversainGT1b,RAVswithinNS3occurredrelatively
infrequentlyandtheNS5AY93Hmutation(14.1%,Fig2B)aswellastheNS5BC316N/S556G
mutationsdominated(42.3%,Fig3B).
Anotherimportantpointwastheevaluationofpatientswhoarenoteligibletoreceiveany
approvedIFN-freetreatmentregimenwhenbaselineRAVsareconsidered.Therefore,thepro-
portionofpatientswhoarewildtypeforatleastonetherapywasdetermined.AllGT1aand
98.6%ofGT1b-infectedpatientsdisplayedwildtypevariantsforatleastonetherapyoption
(Fig4).OnlytwoindividualswithGT1bhadRAVswithrelevanceforallsubstances.Both
patientsexhibitedNS3RAVsatposition80(Q80K,Q80R+D168E)aswellastheNS5AY93H
mutationwhichareassociatedwithresistancetoalloftheabovetreatmentregimens.When
thesingletherapyregimensarecompared,DCV-andLDV-basedtherapiesarethetreatment
PLOSONE|DOI:10.1371/journal.pone.0134395 August28,2015 9/17
ViralResistanceandOptimizationofHepatitisCTherapy
Fig3.PrevalenceofNS5BRAVsinGT1a-(A)andGT1b-(B)infectedpatients.
doi:10.1371/journal.pone.0134395.g003
PLOSONE|DOI:10.1371/journal.pone.0134395 August28,2015 10/17