Distinct antibody responses of patients with mild and severe leptospirosis determined by whole

17 Pages·2017·1.7 MB·English

RESEARCHARTICLE
Distinct antibody responses of patients with
mild and severe leptospirosis determined by
whole proteome microarray analysis
CarolinaLessa-Aquino1,JanetC.Lindow2,3,ArloRandall4,ElsioWunder2,3,
JozelynPablo5,RieNakajima5,AlgisJasinskas5,JaquelineS.Cruz2,AlcineiaO.Damião2,
N´ıvisonNery2,GuilhermeS.Ribeiro6,FedericoCosta2,6,Jose´E.Hagan3,Mitermayer
GalvãoReis2,AlbertI.Ko2,3,MarcoAlbertoMedeiros1,PhilipL.Felgner5*
a1111111111
1 Fiocruz,Bio-Manguinhos,BrazilianMinistryofHealth,RiodeJaneiro,RJ,Brazil,2 Fiocruz,Gonc¸aloMoniz
a1111111111 ResearchInstitute,BrazilianMinistryofHealth,Salvador,BA,Brazil,3 DepartmentofEpidemiologyof
a1111111111 MicrobialDiseases,YaleSchoolofPublicHealth,NewHaven,CT,UnitedStatesofAmerica,4 Antigen
a1111111111 DiscoveryInc,Irvine,CA,UnitedStatesofAmerica,5 DepartmentofMedicine,DivisionofInfectious
a1111111111 Disease,UniversityofCaliforniaIrvine,Irvine,California,UnitedStatesofAmerica,6 InstituteofCollective
Health,FederalUniversityofBahia,Salvador,BA,Brazil
*[email protected]
OPENACCESS Abstract
Citation:Lessa-AquinoC,LindowJC,RandallA,
WunderE,PabloJ,NakajimaR,etal.(2017)
Distinctantibodyresponsesofpatientswithmild
andsevereleptospirosisdeterminedbywhole
Background
proteomemicroarrayanalysis.PLoSNeglTropDis
11(1):e0005349.doi:10.1371/journal.
Leptospirosisisanimportantzoonoticdiseaseworldwide.Humansusuallypresentamild
pntd.0005349
non-specificfebrileillness,butaproportionofthemdevelopmoresevereoutcomes,such
Editor:JosephM.Vinetz,UniversityofCalifornia
asbymulti-organfailure,lunghemorrhageanddeath.Suchcomplicationsarethoughtto
SanDiegoSchoolofMedicine,UNITEDSTATES
dependonseveralfactors,includingthehostimmunity.Protectiveimmunityisassociated
Received:September14,2016
withhumoralimmuneresponse,butlittleisknownabouttheimmuneresponsemounted
Accepted:January22,2017 duringnaturally-acquiredLeptospirainfection.
Published:January31,2017
Copyright:©2017Lessa-Aquinoetal.Thisisan
openaccessarticledistributedunderthetermsof Methodsandprincipalfindings
theCreativeCommonsAttributionLicense,which
Here,weusedproteinmicroarraychiptoprofiletheantibodyresponsesofpatientswith
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal severeandmildleptospirosisagainstthecompleteLeptospirainterrogansserovarCopen-
authorandsourcearecredited. hagenipredictedORFeome.Wediscoveredalimitednumberofimmunodominantantigens,
DataAvailabilityStatement:Allproteinmicroarray with36antigensspecifictopatients,ofwhich11werepotentialserodiagnosticantigens,
filesareavailablefromtheGEOdatabase identifiedatacutephase,and33werepotentialsubunitvaccinetargets,detectedafter
(accessionnumberGSE86630).
recovery.Moreover,wefounddistinctantibodyprofilesinpatientswithdifferentclinicalout-
Funding:FundingwasprovidedbyNIHgrant comes:intheseveregroup,overallIgMresponsesdonotchangeandIgGresponses
U01AI088752(AIK).Thefundershadnorolein
increaseovertime,whilebothIgMandIgGresponsesremainstableinthemildpatient
studydesign,datacollectionandanalysis,decision
group.Analysesofindividualpatients’responsesshowedthat>74%ofpatientsinthe
topublish,orpreparationofthemanuscript.
severegrouphadsignificantIgGincreasesovertimecomparedto29%ofpatientsinthe
CompetingInterests:Ihavereadthejournal’s
mildgroup.Additionally,90%ofIgMresponsesdidnotchangeovertimeinthemildgroup,
policyandhavethefollowingconflicts:PLF
receivesincomefromAntigenDiscovery,Inc., comparedto~51%intheseveregroup.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 1/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
whichisdevelopingproductsrelatedtothe Conclusions
researchdescribedinthispaper.Thetermsofthis
Inthepresentstudy,wedetectedantibodyprofilesassociatedwithdiseaseseverityand
arrangementhavebeenreviewedandapprovedby
theUniversityofCalifornia,Irvineinaccordance speculatethatpatientswithmilddiseasewereprotectedfromsevereoutcomesduetopre-
withitsconflictofinterestpolicies.Thisdoesnot existingantibodies,whilepatientswithsevereleptospirosisdemonstratedanantibodypro-
alterouradherencetoallPLOSpoliciesonsharing
filetypicaloffirstexposure.Ourfindingsrepresentasignificantadvanceintheunderstand-
dataandmaterials.
ingofthehumoralimmuneresponsetoLeptospirainfection,andwehaveidentifiednew
targetsforthedevelopmentofsubunitvaccinesanddiagnostictests.
Authorsummary
Leptospirosisiszoonoticdiseaseofglobalimportance,withoveramillioncasesandnearly
60,000deathsannually.Symptomaticdiseasepresentationrangesfromamildfebriledis-
easewithnon-specificsymptomstosevereforms,characterizedbymulti-organfailure,
lunghemorrhage,anddeath.Factorsdrivingsevereoutcomesremainunclear,butthehost
immuneresponselikelyplaysanimportantrole.Inthepresentstudy,weappliedhigh
throughputtechniquestoidentifytheantibodyprofilesofpatientswithsevereandmild
leptospirosis.Wediscoveredalimitednumberofimmunodominantantigens,specificto
patients.Surprisingly,wefoundtheantibodyrepertoirevariesinpatientswithdifferent
clinicaloutcomesandhypothesizedthatpatientswithmildsymptomswereprotectedfrom
severediseaseduetopre-existingantibodies,whiletheprofileofpatientswithsevereout-
comeswasrepresentativeofafirstexposure.Thesefindingsrepresentasubstantialstepfor-
wardintheknowledgeofthehumoralimmuneresponsetoLeptospirainfection,andwe
haveidentifiednewtargetsforvaccineanddiagnostictestdevelopment.
Introduction
Leptospirosiscausesoveronemillioncasesandnearly60,000deathsannually,withthegreat-
estdiseaseburdeninurbanslumsintropicalandsubtropicalcountries[1–3].Tenpathogenic
Leptospiraspecies,over200serovars,andalargenumberofmammalianreservoirs,including
rats,havefacilitatedtheemergenceofleptospirosisasamajor,globalpublichealthproblem.
Humanstypicallybecomeinfectedthroughdirectcontactwithreservoirurine-contaminated
soilorwater,anddevelopabroadspectrumofclinicalmanifestations,includinghepato-renal
failureandpulmonaryhemorrhagesyndromeinseverecases,whichhavehighmortalityrates
[2,4–6].Thefactorscontributingtodiseaseseverityremainpoorlyunderstood,butbacterial
virulence,inoculumdoseandthehostimmuneresponsearethoughttoplayimportantroles
indevelopmentofsevereoutcomes[2,4].
ExperimentalanimalmodelsofLeptospirainfectionhaveprovidedamajorityofevidence
thatantibodiesplayakeyroleinprotectionagainstandclearanceofLeptospirainfection[7–9].
Passivetransferofwholecellleptospiralvaccineandspecificanti-leptospiralantibodies(Ligs)
areprotectiveagainsthomologousinfectioninanimalmodels,demonstratingantibodiesare
sufficientforimmunityagainstexperimentalhomologousinfection[10–13].Additionally,anti-
bodiesagainstLPSareserovar-specific,arecorrelatedwithagglutinatingantibodytiters,and
conferlimitedcross-protectionagainstotherserovars[14,15].Severalstudieshaveshownthat
leptospirosispatientsdeveloparobustantibodyresponseduringinfection,especiallyanti-LPS
antibodies,whichcorrespondtothemajorityoftheantibodiesproduced[12,16,17].
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 2/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
ThelargenumberofpathogenicLeptospiraserovarsandpoorcross-protectionobservedfor
anti-LPSantibodies,havemadetheidentificationofanti-Leptospiraproteinantibodiesahigh
priorityforvaccineanddiagnostictestdevelopment[18,19].Insupportofthis,immunization
withanLPS-deficientLeptospirastraininexperimentalanimalmodelsconferredcross-protec-
tion,implicatinganti-proteinandotherimmuneresponsesinprotectionagainstinfection.
[19]Additionally,ourgrouphasappliedaproteinmicroarraymethodologytoevaluatethe
antibodyrepertoiregeneratedinnaturalLeptospirainfectionandidentifiedstrongantibody
responsesinhealthyexposedindividualsaswellasseveralIgGserodiagnosticantigensspecific
topatients[20,21].
Analysesofantibodyimmuneresponsesagainstinfectiousagentsareessentialnotonlyfor
diagnosticandvaccinedevelopment,butalsotoprovidinginsightinthemechanismsinvolved
inpathogenicity[22].Proteinarraysareanexcellentplatformthatallowforthescreeningof
antibodyproteintargetsinahigh-throughputmanner,withhighsensitivityandhighspecific-
ity[22–24].Theseelementsfacilitatetheassessmentofmanyanalytessimultaneouslyand
allowfortheidentification,quantificationandcomparisonofindividualantigenicresponses
followingexposuretomicroorganisms.Ourgrouphasefficientlyemployedhigh-densitypro-
teomearraysinthecharacterizationofantibodysignaturesagainstseveralinfectiousagentsof
humanandveterinaryimportance[25–30],includingLeptospirainterrogansandotherspiro-
chetes[21,31].
Inthecurrentstudy,weusedawholegenomeproteomemicroarrayapproachtodescribe
thefirstcomprehensiveprofileofthehumanantibodyresponsetosymptomaticLeptospira
infection.Weprobed192serumsamplesincludingpatientswithdifferentclinicaloutcomes
andhealthycontrols,andcomparedtheirantibodyprofilesagainstL.interrogansserovar
Copenhageniproteins,theserovarassociatedwith>90%oftheurbanleptospirosiscasesin
Salvador,Brazil[32,33].Weidentifiedpromisingcandidatesforthedevelopmentofnewdiag-
nostictestsandsubunitvaccinesanddiscovereddifferentantibodyprofiles,whichassociated
withdiseaseseverity.Lastly,theantibodykineticssuggestamajorityofpatientswithsevere
leptospirosislikelyhaveaprimaryinfection,whilethosewithmilderdiseasehaveevidenceofa
secondaryinfection.Ourresultsprovidenovelinsightsintothecomplexityoftheimmunityin
naturally-acquiredleptospirosisaswellasnewdiagnostictestcandidates.
Methods
Ethicsstatement
ThestudyprotocolwasapprovedbytheinstitutionalreviewboardcommitteesofYaleUniver-
sityandOswaldoCruzFoundationpriortostudyinitiation.Allparticipantsprovidedwritten
informedconsentintheirnativelanguagepriortosampleanddatacollection.Allsamples
wereanonymizedbeforeresearchuse.
Studyenrollmentandsamplecollection
All61patientsampleswerecollectedduringactivesurveillanceforacuteleptospirosisatthe
HospitalCoutoMaia(31severegrouppatients)andtheSãoMarcosEmergencyClinic(30
mildgroupoutpatients)inSalvador,Brazilbetweenyears2005–2011.Laboratoryconfirmation
wasdefinedaspositivemicroagglutinationtest(seroconversion,four-fourriseintiter,orsin-
gletiter(cid:21)1:800)and/orpositiveELISAand/orpositivePCRforLeptospiraDNAaspreviously
described[32].Serumsamplesfrompatientswithmildorsevereleptospirosiswerecollected
twice:(i)acutesample,collectedatpatientadmittanceatthehealthcareunitand(ii)conva-
lescentsample,collected5–276daysafterthefirstsampling.Controlsconsistedof(i)37sera
fromhealthyLeptospira-unexposed(naïve)volunteersfromCalifornia/USand(ii)37sera
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 3/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
fromhealthyparticipantsenrolledinacohortstudyinahighriskurbanslumcommunityin
Salvador,endemicforleptospirosis.
LeptospiraORFamplificationandhighthroughputcloning
TheentireORFeomeofLeptospirainterrogansserovarCopenhagenistrainFiocruzL1-130was
amplifiedbyPCRandclonedintopXIvectorusingahigh-throughputPCRrecombination
cloningmethoddevelopedbyourgroup[34].Inthisstrategy,clonedORFswereexpressed
withC-terminalhemaglutinin(HA)tagandN-terminalpoly-histidine(His)tag.Geneslarger
than3kbwereclonedassmallersegmentsasdescribedpreviously[20,21]andtheligAand
ligBgenes(LIC10465andLIC10464,respectively)werefragmentedaccordingtotherepeated
Bigdomainspresentinthestructureofeachprotein(LigBRepeats7–12,LigARepeats7–13
andLigA/BRepeats1–6)[35].Afteridentifyingtheseroreactiveantigensonthemicroarrays,
theinsertsinthecorrespondingplasmidswereconfirmedbynucleotidesequencingbythe
Sangermethod.
Microarrayprobing
Microarrayfabricationwasperformedasdescribedpreviously[20,21].Briefly,purifiedmini-
preparationsofDNAwereusedforexpressioninE.coliinvitrobasedtranscription-translation
(IVTT)reactionsystem(RTSKit,Roche),followingthemanufacturer´sinstructions.Negative
controlreactionswerethoseperformedintheabsenceofDNAtemplate(“NoDNA”controls).
Proteaseinhibitormixture(Complete,Roche)andTween-20(0.5%v/vfinalconcentration)
wereaddedtothereactions,whichwerethenprintedontonitrocellulosecoatedglassFAST
slidesusinganOmniGrid100microarrayprinter(GenomicSolutions).Multiplenegative
controlreactionsandpositivecontrolspotsofanIgGmixcontainingmouse,ratandhuman
IgGandIgM(JacksonImmunoResearch)wereaddedtothearrays.Proteinexpressionwas
verifiedbyprobingthearraywithmonoclonalanti-polyhistidine(SigmaAldrich)andanti-
hemaglutinin(RocheAppliedScience)aspreviouslydescribed[20,21].
Humanserasampleswerediluted1/100inProteinArrayBlockingBuffer(Whatman)sup-
plementedwith10%v/vE.colilysate10mg/mL(McLab)andincubated30minatroomtem-
perature(RT)withconstantmixingpriortoadditiontothemicroarray.Arrayswereblocked
for30minwithProteinArrayBlockingBufferandthenincubatedwithdilutedsamplesover-
nightat4˚C,withgentlerocking.Washesandincubationwithconjugateantibodieswereper-
formedasdescribedpreviously[20,21].SlideswerescannedinaPerkinElmerScanArray
confocallaserandintensitieswerequantifiedusingQuantArraypackage.
Cloning,expressionandpurificationofrecombinantproteins
SelectedORFswereclonedintopET100-TOPOplasmid(Invitrogen)forHis-taggedrecombi-
nantproteinexpressioninBL21(DE3)StarE.colicells,accordingtothemanufacturer’srec-
ommendations.RecombinantproteinexpressionwasperformedwithEnPressoBsystem
(Biosilta).Briefly,pre-culturedcellswereinoculated1/100into3.5mLofEnPressoBmedium
supplementedwithAmpicilin100μg/mLReagentA1.5U/μLandgrownshaking(160rpm)at
30˚Cfor16–18hsin24-wellcultureblocks.Expressionwasinducedbytheadditionof350μLof
theboosterreagentsupplementedwith15U/μLReagentAand100mMIPTG,for24hat30˚C
under160rpmshaking.Cellswerethenharvestedandlysedwith0.05gofCellyticExpress
(Sigma)foreachmLoffinalculture,for30minatRT.LysateswereappliedtoaNi2+-charged
resin(Qiagen)andrecombinantproteinsweremanuallypurifiedusing20mMTris(pH8.0)buff-
erswithincreasingconcentrationsofImidazole.Washesvariedfrom5mMto40mMImidazole,
dependingontheprotein,andelutionwasperformedwith500mMor1MImidazole.Imidazole
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 4/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
wasremovedbydialysis(ThermoScientificdialysiscassettes)andthepurifiedproteinswere
checkedforhomogeneityin12.5%SDS-PAGE.Proteinconcentrationwasdeterminedbythe
BCAmethod(ThermoScientific)accordingtothemanufacturer’srecommendations.
Multi-antigenprintimmunoassay
Theassaywasperformedasdescribedpreviously[23].Briefly,100ngofeachpurifiedprotein
wasimmobilizedonanitrocellulosemembranestrip.Asemi-automaticmicro-aerolization
devicewasusedtogenerateparallelbandswithnovisiblemarks.Themembranewascutinto
0.5cmwidestripsperpendicularlytotheantigenbands.Thestripswereblockedfor90min
with4%reduced-fatbovinemilkdilutedinPBST(PBS+0.5%Tween20)andthenincubated
for1hatRTwithindividualserumsamplesdiluted1:200inPBST0.25%BSAand5%v/vE.
colilysate20mg/mL.After3washeswithPBST,thestripswereincubatedfor1hourwithalka-
linephosphatase–labeledanti-humanIgGantibody(Sigma-Aldrich)diluted1:30.000inPBST
0.25%BSA.Thestripswerethenwashed3timeswithPBSTandrevealedwithWesternBlue
StabilizedSubstrateforAlkalinePhosphatase(Bio-Rad)for10min.Thereactionwasstopped
withdistilledwater.Stripswereair-driedandscannedimageswereconvertedtograyscale
beforebandintensityquantificationwithImageJsoftware(foundathttp://rsbweb.nih.gov/ij/).
Proteinarraydataanalysis
ArraysignalintensitywasquantifiedusingQuantArraysoftware.Spotsintensityrawdatawere
obtainedasthemeanpixelsignalintensitywithautomaticcorrectionforspot-specificback-
ground.DatawasnormalizedbydividingtherawsignalforeachIVTTproteinspotbythe
medianofthesample-specificIVTTcontrolspots(fold-overcontrol[FOC])andthentaking
thebase-2logarithmoftheratio(log2FOC).Conceptually,anormalizedsignalof0.0isequal
tocontrolspotsignal,andanormalizedsignalof1.0is2-foldhigherthancontrolspotsignal.
Whenevaluatingaproteinspotasreactiveornon-reactive,normalizedsignals>1.0were
consideredreactive.Thesedesignationswereusedtoevaluateresponsefrequencyandtoiden-
tifyasubsetofsero-reactiveproteinsforfurtheranalysis.Agivenproteinonthearraywascon-
sideredsero-reactiveifitwasreactiveinatleast60%ofthesamplesinoneormoreofthe
followinggroups:severedisease,acutesample(n=30);severedisease,convalescentsample
(n=30);milddisease,acutesample(n=30);milddisease,convalescentsample(n=30);
endemiccontrols(n=30);naïvecontrols(n=30).Sero-reactiveproteinswereidentifiedsepa-
ratelyusingIgGandIgMresponses.
Foreachsero-reactiveprotein,samplegroupswerecomparedusingt-tests[Rstatspackage]
andtheareaunderreceiveroperatorcharacteristiccurve(AUC)[Rrocrpackage].Proteins
witht-testp-value<0.05aftercorrectionforfalsediscovery[36]andAUC>0.70wereidenti-
fiedasdifferentiallyreactive.
Clinicaldataanalysis
Clinicalfeaturesoftheleptospirosispatientsparticipatinginthisstudyweredescribedusing
frequenciesandmedianswithinterquartile(IQR)rangescalculatedinExcel(Table1).The
FisherExacttestortheMann-Whitneytestwereusedtocompareclinicalpresentationsof
patientswithmildorseverediseaseusingGraphPadPrism5.02software.
Microarraydataaccessionnumber
TherawandnormalizedarraydatausedinthisstudyhavebeendepositedintheGeneExpres-
sionOmnibusarchive(www.ncbi.nlm.nih.gov/geo/),accessionnumberGSE86630.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 5/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
Table1. Clinicalcharacteristicsforpatientswithmildorsevereleptospirosis.
CHARACTERISTICS MILD SEVERE
N Median(IQR)orN(%) N Median(IQR)orN(%) p-value
Demographics
Malesex 30 16.0(53.3) 31 27.0(87.0) 0.003
Age 29 26.5(17.3–36.8) 30 31.0(24.5–48.8) 0.039
ClinicalPresentationa
Daysofsymptomsb
Acutephase 29 5.5(3.0–7.8) 30 7.0(6.0–9.0) 0.085
Convalescentphase 29 44(22.0–69.5) 30 27.0(22.5–61.5) 0.681
Hematocrit(%) 12 38.5(34.5–44.5) 31 34.0(29.0–37.0) 0.027
Plateletcount(1000/μL) 30 217.0(154.3–238.0) 31 73.0(62.5–177.5) 0.011
LaboratoryConfirmation
AgglutinatingAntibodyTiters
Acutephase 30 0(0–175) 31 200(0–2400) 0.013
Convalescentphase 30 300(0–800) 31 3200(1600–6400) 0.0001
Outcomes
Respiratoryfailurec 30 0 31 2(6.5) 0.492
ICUadmission 30 0 31 7(22.5) 0.011
Oliguricrenalfailured 30 0 31 24(77.4) <0.0001
aValuesattimeofhospitalorclinicadmission.
bPriortosamplecollection.
cRespiratoryfailurewasdefinedaspresenceofpulmonaryhemorrhage(>250ml)ormechanicalventilation.
dOliguricrenalfailurewasdefinedasoliguria(<500mLurine/day)oranuria(<50mlurine/day)orpatientreceivedhemodialysis.
doi:10.1371/journal.pntd.0005349.t001
Results
Patientclinicalandlaboratorycharacteristics
Toidentifyantigensassociatedwithsymptomaticleptospirosisandseveredisease(requiring
hospitalization),weenrolled31patientshospitalizedwithsuspectedleptospirosis,30individu-
alstreatedatanurgentcarefacilityforsuspectedleptospirosis,30individualslivinginthe
samecommunitiesasenrolledpatients(hyperendemiccontrols),and30unexposedcontrols
(naïvecontrols).Allpatientssurvivedandprovidedpairedacuteandconvalescentserasam-
ples.Table1describespatientcharacteristicsforclinicalandbiochemicaltestsperformeddur-
inghospitalizationoroutpatienttreatment.Hospitalizedpatientspresentedwithmoresevere
disease:77.4%hadoliguricrenalfailure,6.5%hadrespiratoryfailure,and22.5%requiredICU
admission,whilenoneoftheseoutcomeswereobservedinoutpatients.Additionally,the
agglutinatingantibodytitersforhospitalizedpatientsweresignificantlyhigherduringacuteill-
nessandconvalescencecomparedtopatientswithmildleptospirosis(p=0.011;p=<0.0001).
However,whilehospitalizedpatients(severedisease)wereolder(p=0.039)andpredomi-
nantlymale(p=0.03),therewerenosignificantdifferencesindaysofsymptomsatacuteor
convalescentsamplecollectionsbetweenpatientswithmildandsevereleptospirosis(acute
p=0.085;convalescentp=0.681).Therefore,anydifferencesobservedinoutcomeswerenot
duetodurationofillnessorsamplingtimes.
Identificationofpotentialleptospirosisserodiagnosticantigens
Inordertodeterminewhetherthereisanantibodysignaturespecifictosymptomaticdisease,
weprobedtheproteinarrayswithacollectionof192serasamples,includingleptospirosis
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 6/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
Fig1.OverallIgMandIgGantibodyrecognitionofleptospiralproteins.(A)VenndiagramsofIgMand
IgGleptospiralproteinsrecognizedbyhumans.OverlapofIgM(B)orIgG(C)sero-reactiveantigensidentified
inpatientswithmildandsevereleptospirosis.
doi:10.1371/journal.pntd.0005349.g001
patientsandhealthyindividualslivinginareaswithorwithoutendemictransmissionoflepto-
spirosis.IgMandIgGprobingrevealedasetof478reactiveantigensforbothacuteandconva-
lescentphases,correspondingto12.5%ofall3819proteinsandsegmentsincludedonthe
arrays.Ofthese,255werespecificforIgM,128werespecificforIgGand95wererecognized
bybothantibodies(Fig1A).Interestingly,wedetectedamajorityoftheIgMandIgGantigens
inpatientswithmilddisease(Fig1Band1C).Toidentifyantigensspecifictopatientswith
confirmedleptospirosis(serodiagnosticantigens),wethencomparedantigensfromtheseraof
patientswiththosefromhealthyindividualsandfound36antigenswithsignificantlyhigher
IgGreactivityinleptospirosispatientsthaninhealthyvolunteersfromUnitedStatesorhealthy
individualslivinginahighlyendemicareainBrazil.Ofthese,12(33%)wereidentifiedduring
acuteleptospirosis(S2Table)and33(92%)duringconvalescence(S3Table).
Earlyantigendetectionduringinfectioniscriticalforthedevelopmentofanewdiagnostic
testforleptospirosis.Therefore,wefirstfocusedonserodiagnosticantigensidentifiedduring
acutephaseinpatientswithmildorseveredisease.Surprisingly,wefoundonlyalimitedsub-
setofalltheseroreactiveantigensweresignificantlyrecognizedbyIgGsinpatientsrelativeto
endemicandnaïvecontrolvolunteers:11ofthe128inthemildpatientgroupand28ofthe55
intheseveregroup(Fig2A).Oftheseonly5ofthe11and9ofthe28werepresentduring
acuteillnesss.Forthemildgroup,theLigproteinsweretheantigenswithhighestaccuracy,
especiallyLigA/B1–6,with90%sensitivity,86%specificityandAUCof0.916.Todetermine
whetherwecouldincreasebothsensitivityandspecificitybycombiningtheantigens,wecon-
structedReceiverOperatingCharacteristic(ROC)curvesforcombinationsofthe5antigensto
assessantigensdiagnosticperformance(Fig2A).Wefoundthatcombiningthetoptwoanti-
gensLigA/B1–6andLigA8–13yieldedslightlyhighersensitivity(86%)andspecificity(91%)
thantheothercombinations(Fig2A).Weperformedsimilaranalysesforthe9antigensspe-
cifictotheseveregroup.Again,thebestdiagnosticaccuracywasachievedwithLigA/B1–6
(AUC=0.935,87%sensitivity,100%specificity)followedbyLIC20276(AUC=0.901,84%
sensitivity,92%specificity).Whenwecombinedbothantigens,sensitivityreached94%,and
specificitywas100%(Fig2B).Fortheremainingantigens,sensitivityrangedfrom77%to90%
andspecificityrangedfrom77%to92%.Again,othercombinationsdidnotyieldbettercom-
binedsensitivityandspecificity(Fig2B).Ourresultsindicatethatwehaveidentifiedcandi-
datesfornewleptospirosisdiagnostictestsandhavediscoveredthattheremaybealimited
dominantantigenantibodyresponsetoLeptospirainfection.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 7/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
Fig2.Serodiagnosticantigensidentifiedforpatientswithmildandsevereleptospirosis.Histogramsplottheaveragenormalizedintensity
(Yaxis)ofeachantigen(Xaxis)forhyperendemiccontrols(darkgraybars)andpatientswithmild(A)orsevere(B)disease(lightgraybars),with
thefrequencyofresponsiveindividuals(blackline,secondaryaxis).ErrorbarsindicateS.E.Singleormulti-antigensROCcurvesoftheidentified
serodiagnosticantigensformild(A)orsevere(B)groupsareshownwithsensitivityandspecificityrates.
doi:10.1371/journal.pntd.0005349.g002
Identificationofpotentialsubunitvaccinecandidatesinrecovered
leptospirosispatients
Weanalyzedtheresponsesfromconvalescentseratodeterminewhetherthereweremajor
shiftsinantibodyresponsestospecificantigenswithtime.Patientsrecoveringfrommilddis-
easehadsignificantlyhigherIgGtitersfor10antigenscomparedtoendemiccontrols,while
thenumberofantigensnearlytripledforpatientswithsevereclinicalpresentation(S3Table).
Antigensidentifiedatconvalescentphaseaccountedfor~92%ofalldiagnosticantigens(33in
36totalIgGantigens)andLigA/B1–6andLigB8–12weretheantigenswithbestdiagnostic
performanceforpatientswithsevereandmilddisease,respectively.Whiletheseantigensdo
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 8/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
nothavediagnosticpotential,theydorepresentpossiblesubunitvaccinecandidatesasrobust
antibodyresponsesweregeneratedoverthedurationofillness.
ProteinmicroarrayvalidationbyMAPIA
Toconfirmthediagnosticandsubunitvaccinepotentialofthesero-reactiveantigensdetected
onthemicroarraychips,wepurifiedsixproteinsfromE.coliBL21invitro(Fig3B),andprinted
ontonitrocellulosemembranes.Weprobedtheimmunostripswithserumfrom8endemiccon-
trolsand20acute-phasepatients,ofwhich10hadmilddiseaseand10hadseveredisease.Serum
fromleptospirosispatientsshowedgreaterreactivitythanserumfromcontrols,especiallyserum
fromseverepatientsatconvalescentphase(Fig3A).Toassesstheabilityofthesesixantigensto
distinguishbetweenpatientsandcontrols,amulti-antigenROCcurvewasgenerated(Fig3C),
anddemonstratedthatthesixselectedantigensyieldedaspecificityof100%andasensitivityof
60%foracutemildgroupand90%fortheremaininggroups.
Distinctantibodyprofilesassociatedwithdiseasepresentation
Asthereislimitedknowledgeofthefactorscontributingtoleptospirosisseverediseaseout-
comes,wecomparedtheantibodykineticsofpatientstodeterminewhethertherearediffer-
encesinantibodyresponsesbasedondiseaseseverity.WefirstcomparedtheglobalIgGand
IgMreactivitiesagainstall478reactiveantigensidentifiedinthemicroarraysbycomparing
thesummedaveragesignalintensitiesforeachantigenduringacuteillnesswiththatatconva-
lescence.WedetectedatrendingincreaseinIgGreactivityinpatientswithsevereleptospirosis,
Fig3.ValidationofmicroarrayresultsbyMAPIA.(A)MAPIAstripsprobedforspecificIgGinleptospirosispatientsandendemiccontrols.Stripsare
groupedbydiseaseseverity,inacuteandconvalescentphases.(B)SDSPAGEofthe6purifiedrecombinantproteinsthatwereappliedtoMAPIAstrips.(C)
ROCcurvesofthecombinationofall6antigensareshownformildandseveregroupsatbothacuteandconvalescentphases.
doi:10.1371/journal.pntd.0005349.g003
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 9/17
Antibodyprofileofpatientswithmildandsevereleptospirosis
whichreachedstatisticalsignificancewhenweanalyzedthesignalsfromthe36patient-specific
antigens(p<0.05)(S2Fig).Wedidnotobservethistrendinpatientswithmilddisease.For
IgM-specificantigens,weobservednosignificantdifferencesforeitherpatientgrouporanti-
genset(S2Fig).Thus,weidentifiedsignificantIgGresponsesincreasesonlyinthesevere
patientgroupovertime.
Tounderstandthedifferencesinantibodykineticsinpatientsinmoredetail,wenextcom-
paredtheantibodyresponsestothe36differentiallyattheacuteandconvalescenttimepoints
foreachindividualbytwowayt-test.Basedontheresultsofeacht-testtheindividualswere
categorizedas:(i)increasing,whenaverageresponsetothe36differentiallyreactiveantigens
higheratconvalescenttimepointthanacute,andp-value<0.05),(ii)nochange(p-value>
0.05)or(iii)decreasing,whenaverageresponsetothe36differentiallyreactiveantigenslower
atconvalescenttimepointthanacute,andp-value<0.05.Thiscomparisonyieldedvastly
differentprofilesforpatientswithmilddiseaseandseveredisease.WhenanalyzingIgGre-
sponses,wecategorized74.4%ofpatientswithintheseveregroupas“increasing”versusonly
29.6%ofpatientsinthemildgroup(Fig4A).WhenanalyzingIgMresponses,wecategorized
32.3%ofpatientsintheseveregroupas“increasing”versusonly3.3%inthemildgroup(Fig
4B).Additionally,90.0%ofIgMresponsesdidnotchangeovertimeinthemildgroup,com-
paredto51.6%intheseveregroup.Altogether,thesedataclearlydemonstratethatleptospiro-
sispatientswithdifferentclinicalpresentationsgeneratedistinctantibodyprofiles.
Inourkineticantibodyanalyses,weenrolledfivepatientswithmildleptospirosis,which
hadantibodyprofilesthatresembledthoseofpatientswithsevereleptospirosis:allhad
increasesinIgGlevelsovertimefor10antigens(Fig4CandS2).Thoughthesefivepatients
clearlydevelopedanantibodyresponsemorerepresentativeofpatientswithseveredisease(S3
Fig),includingahigherconvalescentagglutinatingantibodytiter(400–12800),theydidnot
presentwithanysevereclinicaloutcomeswemeasured.Allotherclinicalandlaboratoryfea-
turesweresimilartothe25patientswithmildleptospirosis(S4Table).
Discussion
Leptospirosisisadiseasewithabroadspectrumofclinicalmanifestationsrangingfrom
asymptomaticandnonspecificacutefebrileillnessestolife-threateningrenalfailureorpulmo-
naryhemorrhagesyndrome[2,37].Overamillioncasesofsevereleptospirosisoccurevery
year.Thisfigurerepresentsonlyafaction(potentially5–15%)ofthetotalmildleptospirosis
cases,whichusuallyarenotidentifiedbysurveillancesystems.Themechanismsinvolvedin
poordiseaseprogressionremainpoorlydefined,butpathogenrelatedandhostfactorslikely
contributetothisheterogeneity[2,4].Here,weidentified12specificIgGantigensthatdiffer-
entiateacutesymptomaticdiseasefromuninfectedindividualsinendemicregionsandthere-
forerepresentpromisingdiagnosticcandidatesforanearlylaboratorytestforthediagnosisof
leptospirosis.Wealsoidentifiedpatient-specificantigensduringconvalescence,whichare
putativesubunitvaccinecandidates.Lastly,weshowedthatpatientswithdifferentclinicalpre-
sentationsgeneratedistinctantibodykineticprofiles,andwehypothesizethatsinceantibodies
areprotective,diseaseseverityandtheantibodysignaturesmayindicateprimaryandsecond-
aryinfections.
Weidentified12IgGserodiganosticantigensforacuteleptospirosis.Amongthemarethe
well-knownsero-reactiveproteinsLigA/B1–6,LigA8–13,LigB8–12andLIC10973(OmpL1).
SeveralpublishedstudiesusedtheLigsasdiagnosticmarkersforleptospirosis[35,38–41]as
wellasOmpL1,especiallyincombinationwithLipL21,LipL32orLipL41[42].Ourgrouphas
previouslyidentifiedLIC10486(hypotheticalprotein)andLIC12544(DNAbindingprotein)
usingtheproteinmicroarrayplatform[21].Theremaining6proteinsLIC10024(adenylate/
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005349 January31,2017 10/17