Computational identification, characterization and validation of potential antigenic peptide vaccines

25 Pages·2017·27.83 MB·English

RESEARCHARTICLE
Computational identification, characterization
and validation of potential antigenic peptide
vaccines from hrHPVs E6 proteins using
immunoinformatics and computational
systems biology approaches
AbbasKhan1,MuhammadJunaid1,AmanChandraKaushik1,ArifAli1,SyedShujaitAli2,
a1111111111 AamirMehmood1,Dong-QingWei1*
a1111111111
a1111111111 1 StateKeyLaboratoryofMicrobialMetabolism,andCollegeofLifeSciencesandBiotechnology,Shanghai
JiaoTongUniversity,Shanghai,China,2 CenterforBiotechnologyandMicrobiology,UniversityofSwat,
a1111111111
KhyberPakhtunkhwa,Pakistan
a1111111111
*[email protected]
Abstract
OPENACCESS
Citation:KhanA,JunaidM,KaushikAC,AliA,Ali High-riskhumanpapillomaviruses(hrHPVs)arethemostprevalentvirusesinhumandis-
SS,MehmoodA,etal.(2018)Computational easesincludingcervicalcancers.ExpressionofE6proteinhasalreadybeenreportedincer-
identification,characterizationandvalidationof
vicalcancercases,excludingnormaltissues.ContinuousexpressionofE6proteinis
potentialantigenicpeptidevaccinesfromhrHPVs
E6proteinsusingimmunoinformaticsand makingitidealtodeveloptherapeuticvaccinesagainsthrHPVsinfectionandcervicalcan-
computationalsystemsbiologyapproaches.PLoS cer.Therefore,wecarriedoutameta-analysisofmultiplehrHPVstopredictthemostpoten-
ONE13(5):e0196484.https://doi.org/10.1371/
tialprophylacticpeptidevaccines.Inthisstudy,immunoinformaticsapproachwasemployed
journal.pone.0196484
topredictantigenicepitopesofhrHPVsE6proteinsrestrictedto12HumanHLAstoaidthe
Editor:Jinn-MoonYang,NationalChiaoTung
developmentofpeptidevaccinesagainsthrHPVs.ConformationalB-cellandCTLepitopes
UniversityCollegeofBiologicalScienceand
werepredictedforhrHPVsE6proteinsusingElliProandNetCTL.Thepotentialofthepre-
Technology,TAIWAN
dictedpeptidesweretestedandvalidatedbyusingsystemsbiologyapproachconsidering
Received:November12,2017
experimentalconcentration.Wealsoinvestigatedthebindinginteractionsoftheantigenic
Accepted:April13,2018
CTLepitopesbyusingdocking.Thestabilityoftheresultingpeptide-MHCIcomplexeswas
Published:May1,2018 furtherstudiedbymoleculardynamicssimulations.Thesimulationresultshighlightedthe
Copyright:©2018Khanetal.Thisisanopen regionsfrom46–62and65–76thatcouldbethefirstchoiceforthedevelopmentofprophy-
accessarticledistributedunderthetermsofthe lacticpeptidevaccinesagainsthrHPVs.Toovercometheworldwidedistribution,thepre-
CreativeCommonsAttributionLicense,which
dictedepitopesrestrictedtodifferentHLAscouldcovermostofthevaccinationandwould
permitsunrestricteduse,distribution,and
helptoexplorethepossibilityoftheseepitopesforadaptiveimmunotherapyagainstHPVs
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. infections.
DataAvailabilityStatement:Alltherawinput
(sequences)areavailablefromUniprot(http://
www.uniprot.org/)byusingaccessionnumbers
P17386,P06427,P24835,P27228,P21735,
P26554,P36814,P24836,Q547M1,P54667.
Introduction
Funding:Dong-QingWeiissupportedbytheKey
Humanpapillomaviruses(HPVs),cervicalcancercausingagents,areknowntobeinvolvedin
ResearchAreaGrant2016YFA0501703fromthe
MinistryofScienceandTechnologyofChinaand bothmorbidityandmortality.AnnualepidemicsofHPVisapproximately0.5millionwhile
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Highriskhumanpapillomavirusespeptidevaccineprediction
alsograntsfromtheStateKeyLabonMicrobial thedeathrateisabout0.25millionworldwide.Manyotherdisorderssuchasgenital,respira-
Metabolism,andJointResearchFundsforMedical tory,wartsandhyperproliferativeabrasionsareassociatedwiththesesmallDNAviruses[1,2].
andEngineering&ScientificResearchatShanghai
Morethan200differentgenotypesofHPVsarecharacterized.Thephylogeneticreconstruc-
JiaotongUniversity.
tionofthesegenotypes,classifiedthemasAlpha,Beta,Gamma,MuandNu.Alphagenusof
Competinginterests:Theauthorshavedeclared papillomavirusesareknowntobeinvolvedinhumandiseases[3].Amongthecharacterized
thatnocompetinginterestsexist.
speciesofgenusAlphapapillomavirus,mostofthemareassociatedwiththeinfectionofgeni-
taltracts[4,5].Sexualintercourseisoneofthecommonwaysinthetransmissionofthese
viruses.However,fomitetransmissionasanon-majorrouteoftransmissionhasalsobeen
reported[6].
High-riskHPVs(hrHPVs)andlow-riskHPVsarethetwobroadcategoriesofHPV
Viruses.Outofthetotal,99%ofcervicalcancersareassociatedwithHigh-riskHPVs(hrHPVs)
species(HPV16,18,26,31,33,34,35,39,45,51,52,56,58,59,66,68and70)[7–11].Among
thehrHPVs,HPV16and18areresponsibleforapproximately75%ofthetotalcases.However,
low-riskHPVspecies(i.e.,HPV6,7,11,32,42,43,44,54,61,and71)arenotwidelyassociated
withcervicalcancerbutleadtoinfectionlikenon-proliferativewarts[5,12].Despitethediver-
sityinpathogenicity,allHPVssharescommongenomeorganization.Coreandaccessorypro-
teinsarethetwotypesofgenesproductsinpapillomaviruses.Coreproteins,E1andE2,are
reportedtobedirectlyinvolvedintheviralreplicationwhileL1andL2areinvolvedinstruc-
turalassembly.E4,E5,E6andE7areconsideredasaccessoryproteins,whichshowvariability
inbothfunctionalaspectsandinexpressioncontrol.Theaccessoryproteinsarereportedtobe
involvedinvirusreplicationinsideinfectedcell.E6andE7,twoimportantoncoproteins,are
foundtobeexpressedinallpositivecasesofcervicalcancerandareresponsibleforviralentry,
cellularalterationandtumorinduction[13–16].Experimentalresultsprovedthattheexpres-
sionofE6andE7proteinsistheprimarycauseoftheimmortalizationofprimaryhumankera-
tinocytesinagenomewidestudy[17–19].Besidethecarcinogenesis,thedeactivationofthe
tumorsuppressorproteins,suchasp53andtheretinoblastoma(pRb),isduetothecontinuous
expressionofE6proteinsinthecellularenvironment.InteractionofE6proteinswithE6AP
alterthesubstratespecificitysubstratespecificityandpolyubiquitylatesp53,leadingtothein
degradationofp53aidedby26Sproteasome[20,21].Therefore,theimportantroleofE6in
causinganddevelopingcervicalcancerisimportantandclear.Ontheotherhand,E7protein
performthefunctionofdegradationofpRbandp130whichisaproteasome-dependentpro-
cess[12,22].
Predictionanddevelopmentofnovelvaccinecandidatesagainstthecomplexdiseaseshas
sophisticatedlyprovokedthedesiredresponseandhasgreatlyaidedtheworkofmolecularand
chemicalbiologiststoexposesafeandeffectivevaccines[23].Immunologicalmechanismsof
surfacepresentationofantigenalongwithMHCproteindirectstheactivationofcytotoxicT-
lymphocyte(CTL),beinganeffector,tokilltheinfectedcell.UpontheinteractionofCTLson
theinfectedcell,self-destructionorapoptosisisobservedtypically.Usuallythepeptidefrag-
mentofthepathogenconfersthissignalingprocessandthusprovoketheimmunity.The
underlyingmechanismistheattachmentofpeptidefragment,usuallyavirulentfactor,binds
totheMHCmoleculeandispresentedonthesurfaceofinfectedcells.Thisprocessrelyonthe
proteasomalcleavageandtransportationtotheendoplasmicreticulum(ER)alongwithMHC
molecule.Theantigenprocessingchannels(TAP)arerequiredtopresentthepeptide-MHC
complexonthesurfaceofthecellforimmuneresponse.Therefore,consideringthec-terminal
cleavageactivityandTAPefficiencygreatlyhelpintheselectionofeffectivevaccinecandidates
[24–27].
ThepurposeofourpresentstudyistopromotethedesigningofavaccineagainsthrHPVs
speciesusinginsilicomethods,takingthemostimportantproteinE6intoconsideration.The
importantroleofthisproteininthecervicalcancercarcinogenesisisclear.Therefore,we
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Fig1.SchematicoftheworkflowforthepredictionandvalidationofpeptidevaccinesfromhrHPVsE6proteins.Theabovefigureisshowingthewhole
methodologyincludingtheresourcesandresultsobtainedfromtheseanalyses.
https://doi.org/10.1371/journal.pone.0196484.g001
designedanepitope-basedpeptidevaccineagainsthrHPVsthatareimportantbutneglected
bymostoftheresearchers.TodateonlyHPV16&18arestudiedforimmunologicalstudies.
TofillthisgapandstudyotherhrHPVsthismeta-analysiswascarriedouttopredictantigenic
potentialpeptidevaccinesusingimmunoinformaticsapproach.
Materialsandmethods
E6proteinsequences
TheprimaryaminoacidsequencesofhrHPVs(HPV31,HPV33,HPV35,HPV39,HPV45,
HPV51,HPV52,HPV56,HPV58,HPV68)E6ProteinswereretrievedfromUniversalProtein
Resources(Uniprot)(http://www.uniprot.org/).Thedetailinformationincludingaccession
number,proteinsequencelengthandspeciesusedaregivenintheS1Table.Theschematic
flowofthisworkisgivenintheFig1.
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Highriskhumanpapillomavirusespeptidevaccineprediction
PredictionoflinearB-cellepitopes
InteractionofB-lymphocyteswithantigenB-cellepitopedirectsthedifferentiationofB-lym-
phocytesintomemorycellsandantibodysecreting-plasma[28].Thecharacteristicsproperties
suchasaccessibilityforflexibleregionandhydrophilicnatureareimportantforB-cellepitopes
[29].DifferentinsilicopeptidedevelopmentapproachessuchParkerhydrophilicityprediction
[30],Eminipredictionofsurfaceaccessibility[31],KolaskarandTongaonkar’santigenicity
[32],KarplusandSchulzFlexibilityPredictionwereusedusinganonlineanalysisresourceat
IEDB(http://www.iedb.org/).ElliPro[33](http://tools.immuneepitope.org/toolsElliPro/)isan
integratedtoolinanonlineIEDBserverwhichcanpredictB-cellepitopesusingbothstruc-
turalinformationorproteinsequences.ElliProemploythreedifferentalgorithmsincluding
ProtrusionIndex(PI)ofresidues,proteinshapeapproximationandthefinalneighboringresi-
duesclusteringwhichrelyonPI.
PredictionofpotentialcytotoxicT-lymphocyte(CTL)epitopes
NetCTL.1.2[34](http://www.cbs.dtu.dk/services/NetCTL/)isanonlinemostwidelyusing
serverforthepredictionofCTLepitopes.TheProteomicdatafromallthehrHPVswere
screenedtopredictpotentialT-CD8+(MHCclassIbindingepitopes)epitopesbyusingalgo-
rithmsNetCTLandNetMHC[35,36].NetCTLacceptFASTAsequenceasaninputthatper-
formdifferentanalysissuchaspredictionofMHCclassIbindingaffinity,TAPtransport
efficiencyandC-terminalCleavageactivity.ConcerningtheMHCalleles,thepredictionswere
restrictedto12humanallelesHLA-A(cid:3)0101,HLA-A(cid:3)0201,HLA-A(cid:3)2402,HLA-A(cid:3)2601,
HLA-B(cid:3)0801,HLA-B(cid:3)2705,HLA-B(cid:3)3901,HLA-B(cid:3)4001,HLA-B(cid:3)501,HLA-B(cid:3)1501,HLA-
C(cid:3)0801andHLA-C(cid:3)0202.Theweightmatrixandartificialneuralnetworkwasusedforthe
predictionofMHC-IbindingandproteasomeC-terminalcleavage.
AllergenicandantigenicprofilingofB&T-cellpredictedepitopes
Inordertovalidatethenon-allergenicpotentialofthepredictedB-cellandT-cellepitopesan
onlinewebtoolAlgPred[37](http://crdd.osdd.net/raghava/algpred/)wasutilizedbyusing
multiplealgorithms(SVMc,IgEepitope,ARPsBLASTandMAST)topredicttheallergenic
peptideswithanaccuracyof85%bycombiningthesemethods.Therefore,weusedthepri-
maryaminoacidsequencestotesttheallergenicpotentialofalltheselectedE6proteins.On
theotherhand,tomaptheantigenicindexofourpredictedepitopesANTIGENpro[38]
(http://scratch.proteomics.ics.uci.edu/)wasused.Thisserveraccessfivedifferentmachine-
learningalgorithmsandmultiplerepresentationofprimarysequencestopileuptheantigenic-
ityresultsbyproteinmicroarraydataanalysis.
Peptideslibrariesconstructionandmoleculardocking
The3Dcoordinatesofalltheselectedpeptideswerepredictedbyusinganonlineeffectiveweb
serverPEP-FOLD3.Forsamplingtheconformationsofpredictedpeptidessimulationruns
wasset200[39].sOPEPenergyfunctionintegratedinPEP-FOLD3wasappliedtoclusterthe
diverseconformationalmodels[40].Selectionofspecificepitopefromallthespecieswas
basedonlowpercentilerankandhighC-terminalcleavageactivitywithgoodTAPscore.Shar-
ingofaminoacidsbetweentheB-cellepitopesandT-cellepitopeswerealsoselectedasadock-
ingcriteria.Afterward,thebestpeptidecoordinatesweredockedtotheclassIMHCmolecules
HLA-A(cid:3)0101(PDBID4NQV),HLA-B(cid:3)1501(PDBID1XR9),HLA-B(cid:3)5801(PDBID5IM7)
andHLA-C(cid:3)0801(PDBID4NT6)usingthePatchDockrigid-bodydockingserverbasedon
thedefinedthreshold[41].PatchDockusesageometrybaseddockingalgorithmtofind
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dockingtransformationswithgoodmolecularshapecomplementarity[42].Scoringandrefin-
ingofthedockedcomplexesproducedbyfastrigid-bodydockingwasperformedbyemploy-
ingFireDockserver[43,44].Complexeswithhighglobaldockingenergy,AttractiveVander
WaalEnergyandHydrogenBondingenergyweresubjectedtomoleculardynamicssimulation
[45].
Kineticssimulationforthevalidationofpredictedepitopes
Acomputationalsystemsbiologyworkbench[46]wasusedtodesignandexecuteaninsilico
biochemicalpathwaytoconfirmtheantigenicpotentialofthepeptides.Kineticssimulationor
pharmacokineticssimulation[47]isusefultooltodescribethesufficientdoseofatestingdrug.
Thepharmacokineticsofmostdrugsisfirstorderattherapeuticdoses.Thisnon-linearkinet-
icsschemefollowsimpleMasskineticsequationorTypically,Michaelis–Mentenequation
[48]asshownbelow;
ðVmaxÞ:ðSÞ
V¼ ðiÞ
KmþS
Thisequationcanbetransformedto
ðCmaxÞ:ðDÞ
C¼
KmþD
Or
d½P(cid:138) ðAmaxÞ:ðDÞ
V¼ ¼ ðiiÞ
dt KmþD
HereCrepresentsthesteadystateconcentration,C thetheoreticalmaximumforC,A
max
theamountabsorbed,A thetheoreticalmaximumforA,andDthedose.
max
TheliteraturesurveywasdonetocollectthenecessaryinformationforthehrHPVs.Aphar-
macokineticspathwaywasestablishedtovalidatethepeptidesfortheirpotentialaction.Nodes
inthepathwayrepresenttheentities,andedgesrepresenttheconnectivityofonenodeto
anothernode,whichiscloselyrelatedtoeachother.Inordertocarryoutpharmacokinetic,
concentrationdoses(0.2μm)wereassignedfromavailableresearch[49].
Epitopeclusteranalysis
Clusteringofepitopesintogroupsbasedonidentityamongtheselectedproteinssequences
wascarriedoutwiththeaidofanonlineEpitopeClusterAnalysistool(http://tools.
immuneepitope.org/main/index.html).Inthecurrentstudy,aclusterisagroupofsequences
sharingaminimumof80%ofthesequenceidentityisknowntobeacluster.
Moleculardynamicssimulations
MDsimulationsofalltheselectedcomplexeswerecarriedoutbyusingAMBER14molecular
dynamicspackage[50].ToneutralizethesystemscounterNa+ionsandhydrogenswere
added.ThetleappackageofAmberwasutilizedtoperformthisprocess.ATIP3Pwaterboxof
8.0A˚wasused.Atwostagesenergyminimization,eachof6000steps,ofthecomplexesusing
theSANDERmoduleofAMBER14,wasperformedtoremovetheconstraintsallatomsinthe
systemsexceptthosefromthewatermolecules.PMEMD.cuda[51]unitofAMBER14was
usedtoaccomplishMDsimulationsoftheminimizedcomplexes.Forlong-terminteractions,
theSHAKEalgorithmandParticle-MeshEwald(PME)methodwasusedandanon-bond
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Highriskhumanpapillomavirusespeptidevaccineprediction
contactscutoffradiusof10A˚waskept.UsingtheLangevintemperature310Kandconstant
pressure(1atm)withisotropicmolecule-basedscalingwasconsideredforequilibrationof
10,000pstime,followedbyatotalof20nssimulationwascarried.SamplingofMDtrajectories
wascarriedoutafterevery2.0pstimescale.AnalysissuchasRMSDandHydrogenbonding
analysiswascarriedoutbyusinganintegratedprogramsCPPTRAJandPYTRAJ[52]in
AMBER14.Thefollowingequationwassolvedtocalculatethestabilityofthecomplexesafter
20ns.
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
N ½m (cid:3)ðX (cid:3)YÞ2(cid:138)
RMSD¼ i¼0 i i i
M
WhereNisthenumberofatoms,m isthemassofatomi,x isthecoordinatevectorfortar-
i i
getatomi,Y isthecoordinatevectorforreferenceatomi,andMisthetotalmass.Ifthe
i
RMSDisnotmass-weighted,allm =1andM=N.
i
Hydrogenbondinganalysis
Hydrogenbondsareanimportantnon-covalentstructuralforceinmolecularsystems.They
areformedwhenasinglehydrogenatomiseffectivelysharedbetweentheheavyatomitis
covalentlybondedto(thehydrogenbonddonor)andanotherheavyatom(thehydrogenbond
acceptor).Here,weanalyzedthehydrogenbondsbetweenalltheselectedcomplexes.Hydro-
genBondswereanalyzedatthreedifferentstages.Thebondswerecheckedbeforethesimula-
tionandafterthesimulation.AftertheminimizationandproductioninthePDBcoordinated
ofthecomplexesweresavedfromthe.rstfilesandwereanalyzedbyusinganonlineserver
PDBePISA[53]UCSFChimera[54]andPyMOL[55]visualizationsoftware.
Results
AntigenicB-cellepitopesprediction
TheantigenicepitopesweredeterminedbyusingTongaonkar’smethod[32]usingthephysio-
chemicalpropertiesofaminoacids.Experimentalprecisionforthismethodisobservedtobe
75%[32].FourantigenicpeptidesineachE6proteinofHPV31,HPV35,HPV45,andHPV68
werepredictedthatrangefrom7–15aminoacids.Moreover,HPV39,HPV51,HPV52and
HPV58possessfiveantigenicepitopes,rangingfrom6–14aminoacidsrespectively.HPV56
containthreeantigenicepitopeswhileHPV33possesssixantigenicepitopes.Therangeof
HPV56epitopesis9–14aminoacidswhileHPV33peptidesrangefrom6–14aminoacids.The
B-cellepitopespredictedareshownintheTable1.
TopredictthemaximumresidualscoreforeachaminoacidintheE6ofhrHPVsspecies
KolaskarandTongaonkar’swasused.Proteinswithresidualscore>1werequantified.Among
thetotalselectedproteinslargenumberofresidueswithscoregreaterthan1werefound,
whichisshowingtheantigenicpotentialofE6protein.Thegraphicalillustration,giveninS1
Fig,ofpredictedantigenicpropensity,maximumandminimumresidualscoreandnumberof
residueswithresidualscore>1aregivenintheS2Table.
SurfaceaccessibilityforE6proteins
Thesurfaceprobabilityofeachresiduewaspredictedusingathreshold1.0.Aminoacidswith
thescoregreaterthan1hasthehighestprobabilitytobefoundonthesurface[31].Themini-
mumsurfaceprobabilityscore0.05forHPV31fromaminoacidposition(ICDLLI ),
96-101
0.032forHPV33fromaminoacidposition(IRCIIC ),0.034forHPV35fromaminoacid
101-106
position(ICLNCV ),0.035forHPV39fromaminoacidposition(IRCMCC ),0.032
26-31 103-108
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Table1. PredictedantigenicB-cellepitopesofhrHPVsE6proteins. Atotalof44antigenicsiteswereidentifiedfromalltheE6proteins.ResiduessharedbybothB-cell
andT-cellepitopesaregiveninbold.
Specie No Start Stop PeptideSequence Length
HPV31 1 1 13 MFKNPAERPRKLH 13
2 36 42 QLTETVL 7
3 109 120 PLCPEEKQRHLD 12
4 1 13 MFKNPAERPRKLH 13
HPV33 1 1 11 MFQDTEEKPRT 11
2 35 42 KPLQRSEV 8
3 55 60 REGNPF 6
4 82 95 SVYGNTLEQTVKKP 14
5 110 119 LCPQEKKRHV 10
6 130 137 GRWAGAAC 8
HPV35 1 1 11 MFQDPAERPYK 11
2 56 61 EGQPYG 6
3 83 91 VYGETLEKQ 9
4 110 120 LCPVEKQRHLE 11
HPV39 1 4 15 FHNPAERPYKLP 12
2 38 44 PLQQTEV 7
3 57 64 RDGEPLAA 8
4 83 88 DSVYAT 6
5 132 137 GSYTGQ 6
HPV45 1 2 16 ARFDDPKQRPYKLPD 15
2 39 45 LERTEVY 7
3 83 93 NSVYGETLEKI 11
4 110 122 KPLNPAEKRRHLK 13
HPV51 1 1 11 MFEDKRERPRT 11
2 55 61 RDNNPYA 7
3 81 86 RSVYGT 6
4 107 120 QRPLGPEEKQKLVD 14
5 130 135 GRWTGQ 6
HPV52 1 1 11 MFEDPATRPRT 11
2 36 42 ELQRREV 7
3 55 60 RDNNPY 6
6 86 96 KTLEERVKKPL 11
7 110 121 LCPEEKERHVNA 12
HPV56 1 1 14 MEPQFNNPQERPRS 14
2 86 94 VYGATLESI 9
3 110 121 QSPLTPEEKQLH 12
HPV58 1 2 11 FQDAEEKPRT 10
2 36 41 TLQRSE 6
3 55 60 RDGNPF 6
4 83 90 LYGDTLEQ 8
HPV68 1 5 14 HNPEERPYKL 10
2 59 65 GVPFAAC 7
3 83 88 ESVYAT 6
4 91 96 ETITNT 6
https://doi.org/10.1371/journal.pone.0196484.t001
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forHPV45fromaminoacidposition(IACVYC ),0.025forHPV51fromaminoacidposi-
30-35
tion(VVCVYC ),0.021forHPV52fromaminoacidposition(VCIMCL ),0.041for
28-33 62-67
HPV56fromaminoacidposition(VCRVCL ),0.003forHPV58fromaminoacidposition
65-70
(IRCIIC )and0.065forHPV68fromaminoacidposition(IDCVYC )wascalculated
101-106 30-35
asthesurfaceaccessibilityscore.Ontheotherhand,themaximumsurfaceprobabilityscore
5.234(PEEKQR ),6.016(PQEKKR ),5.15(KPTRRE ),7.349(KREDRR ),
112-117 112-117 141-146 145-150
5.289(RRRRET ),5.536(KRERPR ),5.353(PEEKER ),4.74(RKYRYY ),6.653
151-156 5-10 112-117 77-82
(RPRRRQ )and7.205(KREDRR )waspredictedforallHPVsfromHPV31to
141-146 145-150
HPV68.S2FigisshowingthegraphicalillustrationofpredictedSurfaceaccessibilityofE6Pro-
teinsofhrHPVs.
SurfaceflexibilityforE6proteins
TemperatureorBfactorisusedtodemonstratethebackandforthmotionofatomswithin
aproteincoordinates.TocalculatethemotionofatomsKarplusandSchulz’sflexibility
methodwasimplemented.Atomswithprofoundlysystematizedstructureappearedtohave
lowB-factorwhilethedistortedappearedhigher[56].HPV31,HPV35,HPV52andHPV56
showedaminimumflexibilityscoreof0.901forheptapeptidesLIRCITC ,LLIRCIT ,
100-106 99-105
QVVCVYC andRLSCVYC respectively.TherestHPV33,HPV39,HPV45,HPV51,
27-33 30-36
HPV58andHPV68showedaminimumuniformscoreof0.885to0.889formoreordered
structureofheptapeptidesLIRCIIC ,LIRCMSC ,CIAYAAC ,VCIMCLR ,
100-106 102-108 59-65 62-68
LIRCIIC andLIRCMSC respectively.Ontheotherhand,maximumsurfaceflexi-
100-106 102-108
bilityscoreforE6proteinsfromHPV31-68showingamoreorderedstructurewithasequence
ofheptapeptidesPEEKQRH ,CPQEKKR ,YREGQPY ,YRDGEPL ,
112-118 111-117 54-60 56-62
DDPKQRP ,PEEKQKL ,PEEKERH ,RQTSREP ,CPQEKKR and
5-11 112-118 112-118 144-150 111-117
RIRQETQ werefound1.089,1.088,1.091,1.067,1.084,1.095,1.076,1.101,1.088and
151-157
1.077respectively.S3FigisshowingthegraphicalrepresentationofpredictedSurfaceflexibil-
ityofE6ProteinsofhrHPVs.
ParkerhydrophilicitypredictionforE6protein
Hydrophilicityofthepredictedpeptidewascalculatedbasedonretentiontimesofapeptide
duringHPLCusingreversedphasecolumn.Here,weusedParkerhydrophilicityprediction
methodtopredictthewaterlovingpotentialofthepredictedantigenicpeptides.Immunologi-
calstudiesreportedthedirectassociationofhydrophilicregionwiththeantigenicsites[30].
S4FigisshowingthegraphicalillustrationofpredictedParkerHydrophilicityofE6Proteins
ofhrHPVsonthebasisofthex-axisisshowingthepositionoftheaminoacidsandy-axisis
plottingthehydrophilicity.Amongtheselectedspecies,thelowesthydrophilicitywascalcu-
latedas-5.086fromalltheE6proteinofHPV35andHPV58fromaminoacidposition
LCHLLIR andILIRCII .TheseregionswerepredictedtoactasactiveT-cellepitopes.
96-102 99-105
Ontheotherhandthemaximumhydrophilicityscore6.371wascalculatedforHPV35E6pro-
teinforthepeptidesequenceQDTEEKP .Moreover,themaximumandminimumhydro-
3-9
philicityscoreforalltheotherHPVspeciesareshownintheS3Table.
AntigenicT-cellepitopesprediction
CytotoxicT-lymphocyte(CTL)epitopeswereexploredfromtheE6proteinsoftheselected
hrHPVs.NetCTL1.2server[34]wasutilizedtopredicttheCTLepitopes.MHCbindingaffin-
ity,proteasomalC-terminalcleavage,TAPtransportaffinityandpotentialMHCligandswere
recognizedbyfollowing>0.75000thresholdascriteria.Atotalof38peptidesequencesfrom
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Table2. PredictedCTLepitopesfrom(HPV31toHPV68)E6protein. Predictionscorethresholdwassetat>0.75000.Boldindicatesaminoacidsthatwerealsopre-
dictedasantigenicsites.
Residue PeptideSequence MHCBinding RescaleBinding C-terminalCleavage Transport PredictionScore MHC-I
No Affinity Affinity Affinity Affinity Binding
15 LSSALEIPY 0.4620 1.9614 0.6850 2.7980 2.2040 Yes
37 LTETEVLDF 0.5417 2.3000 0.8056 2.3240 2.5371 Yes
39 ETEVLDFAF 0.2203 0.9355 0.0724 2.1750 1.0551 Yes
47 FTDLTIVYR 0.1915 0.8130 0.8104 1.3460 1.0019 Yes
73 VSEFRWYRY 0.5615 2.3842 2.3842 2.9840 2.6666 Yes
39 RSEVYDFAF 0.2390 1.0147 0.1258 2.6260 1.1649 Yes
73 ISEYRHYNY 0.5954 2.5278 0.8555 2.9450 2.8033 Yes
2 FQDPAERPY 0.2014 0.8549 0.8643 2.7650 1.1228 Yes
73 ISEYRWYRY 0.5572 2.3657 0.8670 2.9450 2.6430 Yes
41 QTEVYEFAF 0.3068 1.3027 0.0842 2.3330 1.4320 Yes
45 YEFAFSDLY 0.1254 0.5323 0.9668 2.9280 0.8237 Yes
49 FSDLYVVYR 0.1983 0.8419 0.8143 1.4080 1.0345 Yes
72 YAKIRELRY 0.2013 0.8546 0.9407 2.8500 1.1382 Yes
81 YSDSVYATT 0.3351 1.4228 0.0616 0.9130 1.3863 Yes
95 NTKLYNLLI 0.1752 0.7439 0.7623 0.5580 0.8862 Yes
18 CTELNTSLQ 0.2302 0.9776 0.0277 -0.2640 0.9686 Yes
37 ATLERTEVY 0.3216 1.3654 0.9669 3.1660 1.6687 Yes
41 RTEVYQFAF 0.3261 1.3848 0.2831 2.4460 1.5495 Yes
72 YSRIRELRY 0.3333 1.4151 0.8846 2.8890 1.6922 Yes
95 NTELYNLLI 0.4589 1.9483 0.8040 0.4160 2.0897 Yes
39 RADVYNVAF 0.2034 0.8635 0.3223 2.6480 1.0442 Yes
89 EAITKKSLY 0.1378 0.5852 0.9040 2.8900 0.8653 Yes
47 FTDLRIVYR 0.1636 0.6948 0.5907 1.2400 0.8454 Yes
73 ISEYRHYQY 0.5593 2.3746 0.9645 2.9450 2.6666 Yes
76 YRHYQYSLY 0.1668 0.7084 0.9696 2.9740 1.0025 Yes
73 YSKVRKYRY 0.3031 1.2871 0.8705 2.8380 1.5596 Yes
99 LCDLLIRCY 0.1708 0.7250 0.8642 2.7240 0.9909 Yes
35 KTLQRSEVY 0.2565 1.0890 0.8691 3.1290 1.3758 Yes
39 RSEVYDFVF 0.2152 0.9138 0.2751 2.6260 1.0864 Yes
68 RLLSKISEY 0.1342 0.5698 0.9665 3.2540 0.8774 Yes
73 ISEYRHYNY 0.5954 2.5278 0.9588 2.9450 2.8188 Yes
76 YRHYNYSLY 0.1608 0.6829 0.9673 2.9740 0.9767 Yes
20 TLDTTLHDV 0.1793 0.7613 0.7970 0.2690 0.8943 Yes
41 RTEVYEFAF 0.2257 0.9584 0.1397 2.5640 1.1075 Yes
49 FSDLCVVYR 0.1559 0.6619 0.2967 1.4080 0.7768 Yes
72 YAKIRELRY 0.2013 0.8546 0.9002 2.8500 1.1322 Yes
91 ETITNTKLY 0.3233 1.3728 0.8601 2.8160 1.6426 Yes
95 NTKLYNLLI 0.1752 0.7439 0.8751 0.5580 0.9031 Yes
https://doi.org/10.1371/journal.pone.0196484.t002
E6ProteinsofalltheselectedhrHPVswerepredictedasCTLepitopeswhosepredictionscore
were>0.75000(Table2).
MoleculardockingofE6proteinswithHLA-A(cid:3)0101
Amongthetotal38epitopes,only9epitopes,weredockedtoMHCclassIHLA-A(cid:3)0101,2epi-
topesagainsttheHLA-B(cid:3)1501,1epitopeagainstHLA-B(cid:3)5801and3epitopesagainst
PLOSONE|https://doi.org/10.1371/journal.pone.0196484 May1,2018 9/25
Highriskhumanpapillomavirusespeptidevaccineprediction
Table3. BindingaffinityagainsteachalleleisdeterminedintermsofpercentilerankbyIEDBforMHCclassI. Basedonthelowpercentilerank(2%)theseepitopes
weredockedagainstthespecificallele.
Allele Length Peptide Methodused Percentilerank
HLA-A(cid:3)0101 9 ETEVLDFAF Consensus(ann/smm) 0.8
HLA-A(cid:3)0101 9 RSEVYDFAF Consensus(ann/smm) 0.95
HLA-A(cid:3)0101 9 FQDPAERPY Consensus(ann/smm) 1.0
HLA-A(cid:3)0101 9 QTEVYEFAF Consensus(ann/smm) 0.75
HLA-A(cid:3)0101 9 ATLERTEVY Consensus(ann/smm) 0.8
HLA-A(cid:3)2601 9 ATLERTEVY Consensus(ann/smm) 1.45
HLA-B(cid:3)1501 9 ATLERTEVY Consensus(ann/smm) 1.9
HLA-A(cid:3)2601 9 EATIKKSLY Consensus(ann/smm) 0.75
HLA-A(cid:3)0101 9 FTDLRIVYR Consensus(ann/smm) 0.65
HLA-A(cid:3)0101 9 LCDLLIRCY Consensus(ann/smm) 0.65
HLA-B(cid:3)5801 9 KTLQRSEVY Consensus(ann/smm) 0.9
HLA-B(cid:3)1501 9 KTLQRSEVY Consensus(ann/smm) 1.3
HLA-A(cid:3)0101 9 KTLQRSEVY Consensus(ann/smm) 1.3
HLA-A(cid:3)2601 9 ETITNTKLY Consensus(ann/smm) 0.1
HLA-A(cid:3)0101 9 ETITNTKLY Consensus(ann/smm) 0.55
HLA-C(cid:3)0801 9 FTDLRIVYR Consensus(ann/smm) 0.4
HLA-C(cid:3)0801 9 FQDPAERPY Consensus(ann/smm) 0.6
HLA-C(cid:3)0801 9 ATLERTEVY NetMHCpan 1.8
https://doi.org/10.1371/journal.pone.0196484.t003
HLA-C(cid:3)0801fromalltheselectedHPVspeciesasshownintheTable3.Initially,epitopespre-
dictedagainstthe12humanalleleswithgoodpercentilerank,TAPtransportefficiencyand
C-terminalCleavageactivitywereselectedtobeanalyzedforthebindingefficiency[57]
approach.A2%percentilewasusedinthepresentstudy.Becauseithasbeenreportedthat
usingadefinedthresholdofpercentilerankandMHCbindingaffinity,mostofthepredicted
epitopesprovokedtheimmuneresponseinexperimentalcondition.Theglobalandattractive
vanderWaalsenergy(vdW)werecomputedrangingfrom-22.25to-54.31kcal/moland
-17.52to-31.80todeterminethebindingefficiencyofeachepitoperespectively.Thedocking
scoresalongwiththebondingpatternispresentedintheTable4.Amongtheessentialfeatures
Asn77,Tyr99,Arg114andArg156residuesfromtheMHC-Igrooveweremostabundantly
involvedinbondingwithdifferentpredictedpeptides.Amongthetotal10epitopesdocked
againstHLA-A(cid:3)0101(ETEVLDFAF,RSEVYDFAFandKTLQRSEVY)showedthehighest
bindingaffinitiesandhighestnumberofhydrogenbondingwithin3Å.However,theother
epitopesalsoshowedgoodaffinities.Thisanalysisestablishedagoodinteractionofthemod-
eledantigenicpeptideswiththeMHC-Imolecules.Nevertheless,residuessharedbyMHC-Iin
bondingwithdifferentpeptidesarealsoreportedbyMirza,Rafiqueetal.[58]inthesame
computationalstudywhilepredictingantigenicepitopes.Furthermore,residuesfromdifferent
epitopessuchas(Arg5,Glu3andThr4)werepredictedtoactasantigenic.Thegraphicalrepre-
sentationofthesedockedcomplexesaregivenintheFig2.Thehydrogenbondswithlength
lessthan3Åweremostfrequentlyfoundinallcomplexes.Overallstabilityofthedockedcom-
plexseemstobewellpreservedbytheformationofhydrogenbonds.
MoleculardockingofE6proteinswithHLA-B(cid:3)1501andHLA-B(cid:3)5801
EpitopeATLERTEVYwasfoundtobegoodagainstHLA-B(cid:3)1501withthepercentilerank1.9.
WhileepitopeKTLQRSEVYwithpercentilerank1.3and0.9restrictedtobothHLA-B(cid:3)1501
andHLA-B(cid:3)5801showedgoodaffinity.Theglobalenergiesofeachoftheseepitopesrestricted
PLOSONE|https://doi.org/10.1371/journal.pone.0196484 May1,2018 10/25