w w w . e l s e v ie r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Assessing
and
screening
for
T-cell
epitopes
from
Mycobacterium
tuberculosis
RD2
proteins
for
the
diagnosis
of
active
tuberculosis
Jiazhen
Chen,
Qiaoling
Ruan,
Yaojie
Shen,
Sen
Wang,
Lingyun
Shao,
Wenhong
Zhang
∗FudanUniversity,HuashanHospital,DepartmentofInfectiousDiseases,Shanghai,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received17August2018
Accepted29October2018
Availableonline5December2018
Keywords:
Mycobacteriumtuberculosis
T-cellepitope
Diagnosticantigen
a
b
s
t
r
a
c
t
TheRegionofDeletion2(RD2)ofMycobacteriumtuberculosisencodesreservedantigens
thatcontributetobacterialvirulence.Amongtheseantigens,Rv1983,Rv1986,Rv1987,and
Rv1989chavebeenshowntobeimmunodominantininfectedcattle;however,their
diag-nosticutilityhasnotbeenevaluatedinhumans.
Inthisstudy,wescreened87overlappingsyntheticpeptidesencodedbyfiveRD2proteins
fordiagnosingtuberculosisepitopesin50activetuberculosis(TB)cases,31non-tuberculosis
patientsand36healthyindividuals.Apoolofpromisingepitopeswasthenassessedfortheir
diagnosticvaluein233suspectedTBpatientsusingawholebloodIFN-␥releaseassay.
Only10peptideswererecognizedbymorethan10%ofactivetuberculosispatients.The
IFN-␥releaseresponsestoRv1986-P9,P15,P16,Rv1988-P4,P11,andRv1987-P11were
signif-icantlyhigherintheactiveTBgroupthaninthecontrolgroups(p<0.05).Thewholeblood
IFN-␥releaseassaybasedontheseepitopesyieldedasensitivityof51%andaspecificityof
85%indiagnosingactivetuberculosis,andthecorrespondingresultsusingtheT-SPOT.TB
assaywere76%and75%,respectively.
Inconclusion,theseresultssuggestthatthesixepitopesfromtheRD2ofM.tuberculosis
havepotentialdiagnosticvalueinTB.
©2018PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileirade
Infectologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Tuberculosis (TB) isthe leading infectious disease,ranking
aboveHIV/AIDS. In 2016, therewere an estimated1.7
mil-lionTBdeathsand6.3millionnewcasesofTBwerereported,
∗ Correspondingauthor.
E-mailaddress:[email protected](W.Zhang).
equivalentto61%oftheestimatedincidenceof10.4million.1
Worldwide,Chinaisthecountrywiththesecondlargest
num-ber of tuberculosis new cases (after India) and carries an
estimated8.6%oftheglobalburdenofnewcasesandan
esti-mated12.1%ofmultidrug-resistantTBcases.1
Afterinfectionwiththetuberclebacillus,activatedCD4+
T-cellsinduceTh1cytokinesbythestimulationofmacrophages
andchangeinnateimmuneresponsestoadaptiveresponses.2
Therefore,theimmunologicaldiagnostictool,tuberculinskin
test (TST), can detect Mycobacterium tuberculosis infection
https://doi.org/10.1016/j.bjid.2018.10.280
1413-8670/©2018PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileiradeInfectologia.Thisisanopenaccessarticle
basedonthe immuneresponse.Astheantigenscontained
intheTSTiscrudeandpoorlyrefinedsupernatantmixtureof
mycobacteria,thewidelyorsometimesmandatorilyusedBCG
vaccinationandtheexposuretoenvironmentalmycobacteria
weakenthespecificityoftheTST.3
Interferongamma (IFN-␥) releaseassays(IGRAs),
includ-ing the commercially available kits QuantiFERON-TB Gold
In-Tube(QFT-GIT;Qiagen,Germany)andT-SPOT.TB® (Oxford
Immunotech, Abingdon, UK) have been recommended for
the immunological diagnosis ofM. tuberculosis infection. A
meta-analysiscomprising27studiesshowedthatthepooled
sensitivity for the diagnosis of active TB was 80% (95%
CI 75-84%) for QFT-GIT and 81% (95% CI 78-84%) for
T-SPOT.TB® in blood fluids. The pooled specificity was 79%
(95% CI 75-82%) and 59% (95% CI 56-62%), respectively.4
Another meta-analysis focusing on mid- and low-income
countriesreportedsimilarresults:pooledsensitivityin
HIV-uninfected persons of 88% (95% CI,81–95%) for T-SPOT.TB
and 84% (95% CI, 78–91%) for QFT-GIT. The pooled
speci-ficitywaslowforbothassays,namely,52%(95%CI41–62%)
and61%(95%CI,40–79%),respectively.5AlthoughbothIGRAs
havehigherdiagnosticsensitivitiesthanTST,theywerestill
not high enough to justify using IGRA as a test to rule
out TB. The specificity limitation in diagnosing active TB
could not be easily solved, which was commonly due to
inability to discriminate between latent and active
tuber-culosisinfection,inaddition tocross-activation withother
environmentalmycobacteria.However,thesensitivitycould
be improved by adding more diagnostic antigens into the
assays, mainly antigensencoded bythe region ofdeletion
2(RD2) ofMTB.6–12 Peptide4 from TB7.7 (Rv2654, encoded
by RD13) was incorporated into the QFT-GIT to improve
sensitivity.13–16
TheRD2ofMTBencodes11 ORF (fromRv1978-Rv1989c)
which contribute to bacterial virulence.17 Previously, we
demonstrated that Rv1978, Rv1981c, and Rv1985c from
RD2 were T-cell antigens and could be used to
discrimi-nate TB-infected individuals from healthy BCG-vaccinated
controls.12,18,19 T-cell responsesagainst peptide pools from
other proteins in RD2, including Rv1983, Rv1986, Rv1987,
and Rv1989c have been tested in M. bovis-infected and
BCG-vaccinatedcattle.8,20 ThepercentagesofpositiveIFN-␥
releasing responders ranged from 41% to62% inM.
bovis-infected cattle, which was higher than the percentages in
BCG-vaccinatedcattle,whichrangedfrom 0%to50%.
Diag-nostic performance and T-cell epitopes may be different
in cattle compared to humans; it is therefore necessary
to identify and verify these promising T-cell epitopes in
individuals. Peptides of Rv1989c have been verified, in 49
TB patients and 38 healthy individuals, but with
unsat-isfying results. Only 16–23% TB patients had a positive
response.7 Cellular epitopes from other proteins remained
unknownexceptforthe epitopesofIL-2 responsesagainst
Rv1986.21
Inthisstudy,weinitiallyscreenedoverlappingsynthetic
peptidesencodedbyRv1983p257-558,Rv1986,Rv1987,Rv1988,
and Rv1989c forTBdiagnostic purposes,and subsequently
pooledsixcandidateepitopesandevaluatedtheirjoint
diag-nosticperformanceforactiveTB.
Materials
and
methods
Participantsandstudydesign
This study includeda retrospective diagnostic section that
screenedpotentialdiagnosticT-cellpeptidesfromRD2
pro-teinsand adiagnostic sectionthatassessedthe prioritized
peptidesforthediagnosisofactiveTBinpatientswith
symp-tomsoftuberculosis(Figure1).
Inthescreeningsection,threegroupsofindividualswere
recruited,including50casesofactiveTB(TB1group),31
non-TBcontrolpatients(NTP1group),and36healthyindividuals
(HCgroup).ThediagnosisofTBwasbasedonthefollowing
criteria:1)suggestiveclinicalsignsandsymptomsincluding
fever,coughandproductivesputum;and2)positiveacid-fast
bacilli (AFB)smearand/or apositivecultureforM.
tubercu-losis.Tominimizetheimpactofanti-TBtreatmentonT-cell
responses,onlythosepatientswithlessthanthreeweeksof
anti-TBchemotherapywereincluded.Non-TBcontrols(NTP1
group)wererecruitedfromtheinpatientsatHuashan
Hospi-tal,Shanghai.Thesepatientsfulfilledthefollowingcriteria:
a)fever;b)negativeacid-fastbacilli(AFB)smearorculture;
or c) alternative diagnosis established and clinical
resolu-tionwithoutanti-tuberculosistreatment.Healthyadults(HC
group)wererecruitedfromamongstudentsandteachersin
FudanUniversity,China.Aquestionnairewasusedtocollect
informationaboutexposuretomycobacteriaandBCG
vacci-nationstatus.IndividualswithahistoryofcontactwithaTB
patientorwithevidenceofTBinfectionwereexcludedfrom
thestudy.AsthereisaconsiderabledegreeoflatentTB
infec-tion inChina,weexcludedindividuals withpossiblelatent
TBinfection.IGRAswereperformedandthosewithpositive
resultswereexcludedfromtheHCgroup.TheNTP1andHC
groupswereusedasthereferencestandardforthespecificity
assessment,andtheTB1groupwasusedforthesensitivity
assessment.
Thediagnosticassessmentwasconductedattwo
hospi-tals inChina. A totalof233 adultpatients withpersistent
feverandothersignsorsymptomsthatsuggestedpulmonary
or extrapulmonary tuberculosisand forwhom IGRAswere
administeredforTBdiagnosis,wereenrolledinthissection
ofthestudy.Patientswhoreceivedanti-tuberculosistherapy
intheprevious12monthsandwhowereHIV-infectedwere
excluded.
Allparticipantsgaveinformedconsentandthestudywas
approved by the Ethics Committee from Huashan
Hospi-tal,FudanUniversity.Thedemographiccharacteristicsofthe
studyparticipantsaredescribedinTables1and2.
Classificationstandard
Classification was based on the results ofthe clinical and
microbiologicalassessmentafterthediagnosticassayswere
performed. Patients were divided into a culture-confirmed
TB group (TB2 group: positive AFB smear or culture for
M. tuberculosis complex,confirmedby strainidentification),
clinically diagnosed TB group (DTB group: chest
active TB group (TB1, N=50)
non-TB patient controls
(NTP1, N=31) healthy control (HC, N=36) 103 peptides screening Screening section Diagnostic assessment
in TB suspected patients (N=233)
Culture-confirmed
TB (TB2, N=26) Clinically diagnosed TB (DTB, N=23) Probable TB (PTB, N=42) Not TB (NTP2, N=91) Indeterminate (excluded from study, N=51) R6 peptides
Figure1–Studydesign.ThestudycontainsascreeningsectiontoscreenpotentialdiagnosticT-cellpeptidesfromRD2 proteinsandadiagnosticsectiontoassesstheprioritizedpeptidesforthediagnosisofactiveTBinpatientswithsymptoms oftuberculosis.
Table1–Demographiccharacteristicsofthescreening
studypopulation. Group TB1 HC NTP1 No.ofindividuals n=50 n=36 n=31 Sex(male/female) 32/18 14/22 19/12 Age(median) 16-86(51) 20-58(22) 16-86(45) BCGvaccinationscars 34(68%) 34(94%) 24(77%) PositiveIGRAresults 44(88%) 0(0%) 4(13%) GeographyShanghai/Zhejiang 18/32 36/0 31/0
probable TB group (PTB: clinically suspected TB without confirmatory clues, as above), non-TB group (NTB2: alter-nativediagnosis establishedandclinical resolutionwithout anti-tuberculosistreatment),oranindeterminategroup(ID: any other situation). Those patients who were smear- or culture-positive but were infected with a strain identified as non-tuberculosis Mycobacterium (NTM) were included in the NTP2 group. In the TB2 group, most patients (25/26) hadpulmonaryTBandonehadtuberculouspleurisy.Inthe probable TBgroup there were two probable lumbar verte-bralTBcasesandthe remainingwere probablepulmonary TB.
The TB1 and DTB groups were used as the TB
posi-tive population for sensitivity assessment, and the NTP2 groupwasusedastheTBnegativepopulationforspecificity assessment.
PeptidesandAntigens
One hundred and three overlapping peptides spanning
the complete amino acid sequence of CFP-10, ESAT-6,
Rv1983p257-558, Rv1986, Rv1987, Rv1988 and Rv1989c, were
designed and purchased (GL Biochem Ltd., Shanghai)
(Table S1). The identity ofeach peptide was confirmed by
mass spectrometry and the purity (>90%) was checked by
high-pressureliquid chromatography.Thestock
concentra-tion(20mg/mL)ofthepeptideswaspreparedinDMSO(Sigma),
andthepeptideswerefurtherdilutedtoaworking
concen-trationintissueculturemediumRPIM-1640(LifeTechnology).
AnESAT-6peptidepoolcontainingESAT-6P1-P8(TableS1)was
usedasthecontrolintheepitopescreeningexperiment.
ParallelIGRAtests:QFT-GITandT-SPOT.TB
The QFT-GITassay or T-SPOT.TBassays were performedin
alltheparticipantsoftheretrospectivescreeningstudy.The
T-SPOT.TBassaywasperformedinparallelinallthe
partici-pantsofthestudy.Theassayswereperformedaspreviously
describedandfollowingthemanufacturer’sinstructions.22
T-celldiagnosticpeptidescreening:dilutedwholeblood IFN-releaseassay
All 87 peptides were initially screened in 27 TB1 group
Table2–Demographiccharacteristicsanddiagnosticresultsoftheassessmentstudypopulation. Allpatients (n=233) Culture-confirmed tuberculosis(TB2) (n=26) Clinicallydiagnosed tuberculosis(DTB) (n=23) Probable tuberculosis(PTB) (n=42) Nottuberculosis (NTP2)(n=91) Indeterminate (n=51) Age(median) 18-87(51) 24-80(38) 18-69(41) 18-87(42) 27-86(59) 18-87(57) Male/Female 149/84 18/8 17/6 23/19 57/34 34/17 PositiveforR6 peptidepoolby WBIGRA (Percentage) 79(33.9%) 14(53.8%) 11(47.8%) 24(57.1%) 14(15.4%) 14(27.5%) Positivefor ESAT-6/CFP-10 peptidesbyWBIGRA (Percentage) 109(46.8%) 22(84.6%) 16(69.6%) 31(73.8%) 19(20.9%) 16(31.4%) PositiveT-SPOT.TB assay(Percentage) 108(46.4%) 23(88.5%) 14(60.9%) 31(73.8%) 23(25.3%) 17(33.3%) Indeterminate T-SPOT.TBassay 5 0 2 1 1 1
candidate peptides with T-cell recognition higher than 8% inthe TB1 groupwere furtherevaluated in the remaining individualsintheTB1,NTP1andHCgroups.
Toreducethequantityofpatients’wholebloodthatwas requiredforscreeningthemultiplepeptides,adilutedwhole bloodmethodtomeasureT-cellresponsewasperformedin thestudyasdescribedbyWeiretal.23Briefly,wholebloodwas
collectedviavenipunctureanddrawnintoheparinizedtubes.
Within8h,thewholebloodwasdiluted1:10withRPIM1640
tissue culture medium, and supplemented with 40g/mL
streptomycinand40YU/mLpenicillin(LifeTechnologies,
Pais-ley,UK).Itwasthenculturedin96-welltissuecultureplatesin
atotalvolumeof200Lwith2.5g/mLofPHA,RPIM1640
(neg-ativecontrol)or10g/mLofeachpeptideorthepeptidepool
ofESAT-6.Afterincubationat37◦Cfor5days,supernatants
ofculturedbloodwerecollectedandstoredat−20◦Cbefore
testing.
IFN-␥levelsinthesupernatantsweredeterminedbyELISA
usingcommerciallyavailableagents(Mabtech,Sweden),and
performedaccordingtothemanufacturer’sinstructions.The
detectionrangeoftheassaywas4-400Ypg/mL.For
concentra-tionshigherthan400Ypg/mL,thesupernatantswerediluted
10-fold or 100-fold and re-measured. Antigen- or
peptide-specificresponsesareshownasvalues(antigen-stimulated
IFN-␥releaseminusthatintheunstimulatedwell).Positive
recognition was recoded using 17.5Ypg/mL (approximately
mean concentration valueof unstimulated well plus three
timesstandarderrorofalltheparticipants)asthecutofffor
thepeptide-specificIFN-␥levels.
AssessmentofthesixpeptidesfromRD2forTBdiagnosis byawholebloodIFN-releaseassay(WBIGRA)
Sixpromisingpeptides(R6),namely,Rv1986-P9,Rv1986-P15,
Rv1986-P16,Rv1988-P4,Rv1988-P11,andRv1983-P4(all
identi-fiedbyepitopesscreening)werepooledtogethertoassessthe
TBdiagnosispotentialinpatientswithsymptomsof
tubercu-losis.Half-milliliteraliquotsoftheheparinizedbloodfrom233
patientswerestimulatedinthreeseparate1.7mLtubes,each
with10g/mL(finalconcentrationforeachpeptide)ofeither
theR6peptidepool,thepeptidepool ofESAT-6and CFP-10
(EC),orRPIM1640mediumwithDMSO(negativecontrol).The
DMSO thatwasaddedtothenegativecontrolwasequalto
the volumeinthepeptide tubestominimize itsimpacton
IFN-␥releaseassay.Stimulatedbloodwasincubatedat37◦C
for16-24handtheIFN-␥levelwasmeasuredintheplasma
bytheELISAkitmentionedabove.ApositiveIFN-␥response
wasdefinedas(AntigenminusNegative)≥34Ypg/mLand≥1/2
Negativecontrol.Thecutoffvaluewassettooptimize
speci-ficitybyareceiveroperatorcharacteristiccurve(ROC),which
TB2andDTBgroupsweresetasthepositivegroup,andNTP2
groupwassetasthenegativegroup.
Statisticalmethod
TheintensityofIFN-␥releasedamongthe differentgroups
wasanalyzedbytheKruskal-Wallisone-wayANOVAtestand
Dunn’spost-test.Thefrequencyofrecognitionforsingle
pep-tidebetweenTB1groupandthecontrolgroup(NTP+HC)was
analyzedbyChi-squareorFisher’sExacttest.Forthe
diagnos-ticcandidatepeptides,thecut-offvaluetooptimizespecificity
wasdeterminedbyROCcurve,inwhichTB1groupweresetas
thepositivegroup,andNTP1andHCgroupsweresetasthe
negativegroup.SPSSv14andPrism5(GraphpadSoftware5.0,
SanDiego,CA,USA)wereusedfortheanalysis.
Results
ScreeningdiagnosticT-cellepitopesagainstRv1986, Rv1987,Rv1988,Rv1989c,andRv1983intheTB1,NTP1 andHCgroups
Allthepeptideswerefirstscreenedinvitrobythedilutedwhole
bloodIFN-␥releaseassayin11-27individualsoftheTB1group.
Intotal,9/16(56%),8/11(73%),6/11(55%),14/27(52%),and1/16
(6%)ofTB1patientshadpositiveresponsesagainstatleastone
peptideamongRv1983,Rv1986,Rv1987,Rv1988,andRv1989c,
respectively.Twenty-twopeptidesthatwererecognizedbyat
least8%ofTB1patientswerefurtherstudiedinmorepatients
andhealthyindividualsforevaluatingbothdiagnostic
IFN-γ recognition against overlapping Rv1986 peptides in partial TB1 IFN-γ recognition against overlapping Rv1987 peptides in partial TB1
IFN-γ recognition against overlapping Rv1988 peptides in partial TB1 IFN-γ recognition against overlapping Rv1989 peptides in partial TB1
IFN-γ recognition against overlapping Rv1983c peptides in partial TB1 group patients
group patients group patients
group patients group patients
Peptides 86-1 87-1 89-1 89-2 89-3 89-4 89-5 89-6 89-7 89-8 89-9 89-1089-1189-1289-1389-1489-1589-16 87-2 87-3 87-4 87-5 87-6 87-7 87-8 87-9 87-10 87-11 87-12 86-2 86-3 86-4 86-5 86-6 86-7 86-8 86-9 86-1086-1186-1286-1386-1586-1686-17 Peptides Peptides Peptides Peptides 88-1 83-1 83-2 83-3 83-4 83-5 83-6 83-7 83-8 83-9 83-10 83-1183-1283-1383-1483-15 83-1683-1783-1883-1983-20 83-21 83-22 83-2383-24 83-2583-2683-27 88-2 88-3 88-4 88-5 88-6 88-7 88-8 88-9 88-10 88-11 88-1288-1388-1488-15 50% 50% 60% 40% 30% 20% 10% 0% 50% 40% 30% 20% 10% 0% P e rcentage of positiv e recognition 40% 30% 20% 10% 0% P e rcentage of positiv e recognition P e rcentage of positiv e recognition 50% 40% 30% 20% 10% 0% P e rcentage of positiv e recognition 50% 40% 30% 20% 10% 0% P e rcentage of positiv e recognition 17.5-50 pg/m1 17.5-50 pg/m1 17.5-50 pg/m1 17.5-50 pg/m1 17.5-50 pg/m1 >50 pg/m1 >50 pg/m1 >50 pg/m1 >50 pg/m1
A
B
C
D
E
Figure2–PositiveT-cellresponsesagainstRD2peptidesinTB1groups.Dilutedwholebloodfromtheactivetuberculosis grouppatients(TB1)werestimulatedwithpeptidesfromtheRD2proteinofM.tuberculosisandIFN-␥responseswere measured.ThepercentagesofpositiveresponsesagainstRv1986(A),Rv1987(B),Rv1988(C),Rv1989c(D),andRv1983p257-558
(E)inpartialTB1grouppatientsareshown.
(18/27)ofTBpatientsshowedpositiveIFN-␥responsesagainst
thepeptidepoolofESAT-6.
AfterscreeningallindividualsofTB1group,only10of21
peptides (Rv1986-P9,P10, P15, P16, Rv1987-P8,P11,
Rv1988-P4,P11 andRv1983-P4, P10)were recognizedbymorethan
10% ofactive TBpatients, rangingfrom 12% (4/34) to44%
(15/34)oftheTB1patients(Figure3A).Inthe NTP1andHC
groups, 11 peptides were notrecognized byany individual
(Figure3B).
Thefrequencyofresponsestosixpeptides,Rv1986-P9,P15,
P16, Rv1988-P4, P11, and Rv1983-P4in theTB1 group were
significantly greater than in the control groups (NTP+HC)
(p<0.05), suggesting that the peptides had the potential
for diagnosing tuberculosis infection (Figure 4). Although
Rv1987-P8,Rv1987-P11,andRv1983-P10werefrequently
rec-ognizedbyTB1patients,thecross-reactiveresponseamong
the control groups made them unsuitable for diagnostic
purposes.
AlltheenrolledparticipantsagreedtoaparallelIGRAtest
beingperformed.Intotal,45of50(90%)ofTBpatientsand
sixof31(19%)NTP1patientsshowedapositiveIGRAresult,
whichindicateduncompromisedT-cellresponsesagainst
spe-cifictuberculosisantigensamongthestudypopulation.
Inordertobetterevaluatesensitivitiesandspecificitiesfor
theTBdiagnostictests,aROCcurvewasdrawncombiningthe
IFN-␥responsestothesixpromisingpeptides.Theareaunder
thecurvewasonly0.623(95%CI:0.576-0.670,p<0.0001).Using
11.6Ypg/mLastheIFN-␥cut-off,whichproducestheoptimal
specificityof92.7–100%,thesensitivityoftheIFN-␥assayfor
the peptidesRv1986-P9,Rv1986-P15,Rv1986-P16,Rv1988-P4,
Rv1988-P11,andRv1983-P4were24%(10/42),12%(4/34),15%
(5/34),37%(16/43),20%(10/51),and26%(10/39),respectively
(Figure4).
Apparently,thesensitivityofanyofthesixpeptidesalone
wasnotsufficienttodiagnosetuberculosis.Incombinationor
asanadjuncttoRD1epitopestheseepitopescouldbeusedfor
clinicaldiagnosticpurposes.Therefore,weassessedthe
pri-oritizedpeptidepoolinparalleltoRD1antigensfordiagnosis
ofactiveTBinpatientswithsymptomsofthedisease.
AssessmentofthesixpeptidesfromRD2forTBdiagnosis byawholebloodIFN-releaseassay
The diagnostic performance of the RD2 epitope pool was
assessed in 233 patients with suspected TB. As shown in
Table2,26(11.3%)patientswereclassifiedintheTB2group,
23(10%)intheDTBgroup,42(18.3%)inthePTBgroup,and88
(38.3%)intheNTB2group(includingfourwithNTMinfection),
A
B
IFN-γ recognition against the candidate RD2 peptides in TB1 group
IFN-γ recognition against the candidate RD2 peptides in NTP and HC groups Peptides Peptides P ercentage of positiv e recognition P ercentage of positiv e recognition 17.5-50 pg/m1 >50 pg/m1 17.5-50 pg/m1 >50 pg/m1 40% 40% 30% 30% 20% 20% 10% 10% 0% 86-2 86-2 86-10 86-10 86-15 86-15 86-16 86-16 87-1 87-1 87-8 87-8 87-11 87-11 87-12 87-12 88-4 88-4 88-7 88-7 88-11 88-11 88-15 88-15 83-4 83-4 83-6 83-6 83 -7 83-7 83-9 83-9 83-10 83-10 83-11 83-11 83-12 83-12 83-13 83-13 83-24 83-24 86-9 86-9 86-8 86-8 0% 50% 50%
Figure3–PositiveT-cellresponsesagainstcandidateepitopepeptidesinTB1andcontrolgroups.Dilutedwholebloodfrom tuberculosisgrouppatients(TB1)andthecontrolgroupswerestimulatedwith22candidateepitopesandIFN-␥responses weremeasured.ThepercentagesofpositiveresponsesagainstthepeptidesinTB1(A)andthecontrol(HC+NTP1)groups(B) areshown.
Fourteen(54%)and22(85%)of26TB2patientshada
pos-itiveWBIGRAresultagainstR6andECantigens,respectively
(Table2).Twenty-threeof26(88.5%)patientshadapositive
T-SPOT.TBassayresult.Fourteen(61%),11(48%),and16(70%)of
23DTBpatientshadapositiveT-SPOT.TB,andWBIGRAresults
againstR6andECantigens,respectively.Thirty-one(74%),24
(57%),and31 (74%)of42probableTBpatientshadpositive
T-SPOT.TBand WBIGRAresultsagainstR6andECantigens,
respectively(Table2).
MostpatientsintheNTB2groupwerediagnosedwith
cryp-tococcosis,COPD, lungcancer, viralinfections, candidiasis,
bacterialinfections,andNTM.Twenty-three(25%),16(15.4%),
and19(20.9%)of91NTB2patientshadpositiveT-SPOT.TBand
WBIGRAresultsagainstR6andECantigens,respectively.
Overall,the sensitivityofthe R6 peptideassay was51%
(25/49)fortuberculosis,andthespecificityreached85%(77/91).
For tuberculosis patients, the sensitivities of the EC
pep-tidesandT-SPOT.TBassaywere78%(38/49)and76%(37/49),
respectively,andthe specificitieswere 79%(72/91)and75%
(68/91), respectively. The difference in sensitivity between
theR6peptideassayandT-SPOT.TBwasstatistically
signifi-cant(p=0.023),butthedifferenceinthespecificitywasnot
(p=0.093).
Discussion
Inrecentyears,therehasbeengreatprogressindeveloping
newapproaches forthe immune-diagnosis of tuberculosis,
whichincludesthemeasurementofalternativebiomarkers
secretedaspartofcell-mediatedimmuneresponses,24,25the
identification of novel antigens,12,26,27 monitoring of T-cell
subgroupchanges28,29andthedetectionofhostbiomarkers
ofimmuneresponse.27,30Usingapeptide-basedapproachto
identifynew antigensand epitopesfrom the old
immune-dominant proteins can be an effective way to screen and
improvetheperformanceofthecurrentmethods.
In our screening study, four epitopes from Rv1986, and
two epitopes each from Rv1987, Rv1988,and Rv1983 were
recognized by more than 10% of TB patients (Figure 3A).
Rv1986c,aprobableLysEfamilyproteininvolvedinthe
trans-portoflysine,maycontributetobacilluspersistence,asitis
up-regulatedatlatertimepoints(4-7days)when
mycobac-teriaistreatedwithhypoxia, developingwhatisknownas
the enduring hypoxic response (EHR).31 Rv1986c, together
withotherEHR-inducedantigens,canstimulateanimmune
response that leadsto the secretion ofboth IFN-␥and
IL-2inTB-infectedcattle.32 Oneidentifiedepitoperegion, P15
andP16(aminoacid157-187)wasidentifiedpreviously,21but
the other region P9 and P10 (amino acid 91-120) was not.
ThemostdominantepitopeswereRv1987-P8andRv1987-P11,
withmedianintensityreachedat38Ypg/mLand 12Ypg/mL,
respectively, in the TB1 group. Despite the high intensity,
they were unsuitable for diagnostic purposes due to high
cross-reactivityinthecontrolpopulation.Rv1987encodesa
possible chitinasefamily protein thatis distributed widely
among bacteria, fungi and even animals, and which may
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G
I
J
H
F
D
B
IFN-γ response against peptide Rv1986-P9
IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml) IFN-γ (pg/ml)
IFN-γ response against peptide Rv1986-P15
IFN-γ response against peptide Rv1987-P8
IFN-γ response against peptide Rv1988-P4
IFN-γ response against peptide Rv1983-P4 IFN-γ response against peptide Rv1983-P10 IFN-γ response against peptide Rv1988-P11 IFN-γ response against peptide Rv1987-P11 IFN-γ response against peptide Rv1986-P16 IFN-γ response against peptide Rv1986-P10
TB TB TB TB TB TB TB TB TB TB 300 300 300 300 200 200 200 200 200 200 200 100 100 100 100 60 80 80 80 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 0 0 0 0 0 0 20 20 20 20 20 20 40 40 40 40 40 40 60 60 60 60 60 60 80 80 80 80 80 80 100 100 100 100 100 100 150 150 150 150 150 150 200 200 200 NTP NTP NTP NTP NTP NTP NTP NTP NTP NTP HC HC HC HC HC HC HC HC HC HC Groups Groups Groups Groups Groups Groups Groups Groups Groups Groups ** * *** *** *** *** *** *** **
Figure4–DilutedwholebloodIFN-␥responsesagainst10 epitopesfromRD2amongTB1,NTP1andHCgroups. Dilutedwholebloodfromthetuberculosispatientgroup (TB)andnon-TBpatientgroup(NTP)andBCG-vaccinated healthycontrolgroup(HC)werestimulatedwith10 candidatepeptidesandIFN-␥responsesweremeasured. TheresultoftheIFN-␥responsesinthethreegroups againstpeptidesRv1986-P9(A),Rv1986-P10(B),Rv1986-P15 (C),Rv1986-P16(D),Rv1987-P8(E),Rv1987-P11(F),
Rv1988-P4(G),Rv1988-P11(H),Rv1983-P4(I),and Rv1983-P10(J)areshown.Horizontallinesindicatethe medianofIFN-gammaineachgroup.Forallthepeptides, IFN-gammaresponsesabove11.6´Ypg/mLareconsideredto bepositive,asdeterminedbyROCanalysis(indicatedby thedottedline).*,**and***indicatesstatisticallysignificant differenceamonggroupsbytheKruskal-Wallisone-way ANOVAtestandfollowedby2groupsnonparametric comparison.*,0.01≤p<0.05;**,0.001≤p<0.01;***,p<0.001.
inhealthyindividualswasinagreementwithapreviousstudy
withM.bovis-infectedcattle:57%recognitionininfected
cat-tle and 50%in BCG-vaccinatedcattle.8 Rv1983 (PEPGRS35),
whichbelongstothePE/PPEproteinfamilyofMycobacterium,is
anotherantigenthatisdefinitivelyrecognizedinbothinfected
andBCG-vaccinatedcattle.8Inordertoavoidcross-reactivity
maximally,the regionofPEPGRS35p1–p256,whichhad
com-monmotifssharedamongthePE/PPEfamilymembers,was
not chosen forepitope screening. In the screening
experi-ments,56%(9/16)ofTBpatientshadapositiveresponseto
atleastonepeptideofRv1983.Thepercentagethatwas
rec-ognizedwasequivalenttothatinthecattlestudy(59%).8TB
patientspreferentially recognizedRv1983-P4,P6-P13(amino
acid312-408),andP24,butthepercentagethatwasrecognized
waslowforanysinglepeptide.Amongthem,twowere
demon-stratedtobeimmune-dominantepitopesandone,Rv1983-P4
(P290-309),hadpotentialdiagnosticvalue.Rv1988encodesan
intrinsic macrolide-resistantgene namederm,37,33 probably
throughitsmethyltransferasefunction.Inthisstudy,Rv1988
wasrecognizedby52%ofTBpatientsandidentifiedasa
T-cellantigen.Twodominantepitopes,Rv1988-P4andP11,had
relatively high recognitionin TBpatients but less or none
inNTP1orhealthycontrols,whichmakesthem potentially
usefulforthediagnosisofTB.Toourknowledge,thisisthe
first timetheyhavebeenstudied. Itislesscommon foran
antibiotic-resistantgenetobeidentifiedasadiagnostic
anti-genwithhighspecificity;however,ithasbeenfoundthata
highsimilarityhomologoftheermfamilyismainlyrestricted
tothetuberculosiscomplexandlimitedspeciesof
Mycobac-terium,includingMycobacteriumabscessus andMycobacterium
bolletii.34
In this study, T-cellresponses were measured by using
adilutedwholeblood assayandtheincubationperiodwas
lengthenedto5daysinsteadofovernighttoensurethe
pro-ductionofasufficientamountofsecretedIFN-␥.35Although
theantigen-specificcytokineswerereportedtoprogressively
decreasewhenwholebloodwasdiluted,theassayrequired
muchless blood thanexpected, consideringthatscreening
included 87 peptide subgroups for each patient. The data
showedthattheamountofcytokinesecreted from20Lof
bloodwassufficienttodeterminetheT-cellepitopes.Allbut
threeofthe117NTP1individualshadpositiveIFN-␥responses
tothepositivecontrolantigen,PHA,andtheresultsranged
from 10 to>400Ypg/mL, with the median value exceeding
400Ypg/mL.Similarly,allbutoneparticipanthadapositive
T-cellresponsetoPHAinanundilutedparallelIGRAtest.The
patient who hadnegativeresponses toPHAhad apositive
responsetoTBantigen,suggestingthatmostpatientswere
immune-competentatacellularlevel.However,wenotedthat
the5-dayincubationwasnotsuitableforclinicaldiagnosis.
Therefore,overnightincubationandanundilutedwholeblood
assay were performed in the assessmentstudy. Prolonged
incubationcanstimulatemorememoryT-cells,butshort-time
incubationmainlystimulateseffector T-cells.Inaddition,a
longerstimulationduration(6days)wasshowntobe
neces-saryfortheoptimalinductionofIFN-␥andTNF-,andwas
practicallyconvenientforthedetectionofIL-10,IL-1-,TNF-␣,
andIL-6.36
In the assessment study, it is inspiring that a pool of
activeTBpatients, althoughthisislower than thenumber
detectedbytheT-SPOT.TBassay.Atotalof57%ofthepatients
alsohad positiveresultsforthe peptidesinthePTBgroup.
Patientsinthisgroupallhad symptoms,radiographicsigns
orhistologicalevidencethatsuggestedahigh possibilityof
TBinfection,while 60% (25/42)ofthem received empirical
anti-tuberculosistherapyandmostshowedclinical
improve-ment.Positivedetectioninthisgroupisalsomeaningfuland
instructiveasmicrobiologicalinvestigationfailedtoprovide
conclusiveresults.Inthepreliminaryscreen,only10peptides
wererecognizedbymorethan10%ofTBpatients.Thepeptides
withlessfrequentrecognitionwouldbeusefulfordiagnostic
purposesandwouldhavepotentialtoincreasesensitivity,if
therecognitionpatterntothesepeptideswere
complemen-tary to the more frequently recognizedpeptides. However,
consideringthebudgetlimitationandworkload,the
recog-nitionvalueinTB1groupwassetto8%,whichincluded22
peptides (25%) in the latter screen. Although the
sensitiv-ityisnothighenoughtoallowthemtobeusedalone,the
peptides haveshown potential diagnostic value,and to be
adjunctiveantigenstothecurrentimmunologicaldiagnostic
methods.
In the NTP2 group and the ID group, in which TBwas
lesslikely, thepositive resultsforthe R6assay were lower
than in the highly likelyTB groups, as expected.
Interest-ingly,patientsdiagnosedwithcancer,especiallylungcancer,
were themajor reasons forfalsepositive resultsinthe R6
assayintheNTP2group.Intotal,10of16patientswithfalse
positiveresultsbytheR6assayhadcancer.Afterexcluding
those patients whose condition was complicated by
can-cer,thespecificitycouldbeimprovedto90.3%(56/62).Lung
cancermaycauseanautoimmunedisorderthatresultsin
fre-quentpositiveIGRAresults37;therefore,oneshouldbevery
carefulwheninterpretingIGRApositiveresultsfromlung
can-cerpatients. Fortheremainder,falsepositiveresultsinthe
NTP2groupandlatentTBinfectioncould bethe most
fea-sibleexplanation.PeptideRv1988-P4,whichwasrecognized
bycontrolindividualsinscreening assay(Figure4G),could
be another reason for reducing the specificity of the
pep-tide pool. As all the patients are from China, which is a
highTBepidemiccountry,the10-20% positiveIGRA results
in the NTP group are reasonable and close to the
previ-ouslyreportedpositiverateinhealthyindividualsinChina.38
AlthoughweassumedthatlatentTBinfection couldbethe
reasonforfalsepositivityinthestudypopulation, asthese
antigens are not specifically expressed in latent infection,
wedidnotdesignagrouptotest whetherlatentTBcould
berecognizedby the R6 assayor any different recognition
betweenactive and latentTBindividuals, whichis worthy
investigatinginthefuturestudy.Fourpatientswere
culture-positive but confirmed to have NTM disease by selective
PNBculturemedia.NonehadanypositiveresultsfortheR6
assay,suggestingthatthepeptidesmayalsobesuitablefor
differentiatingNTMfromTB.Despitethis,morecasesneedto
beevaluated.
FortheparallelT-SPOT.TBassay,thesensitivityand
speci-ficitywere76%(37/49)and 75%(68/91),respectively, forthe
twogroups.Unliketheassessmentsinhealthycontrolsorin
someretrospectivestudies,theunsatisfactoryresultsreflect
thereal-lifediagnosticchallengeinhighlyepidemicregions.
ThediagnosingperformanceinactiveTBwasclosetothat
summarizedinmeta-analysisstudies,4,5 and reinforcesthe
conclusion that the sensitivity is not high enough to rule
outTB.LikeWBIGRAfortheR6peptides, morethanhalfof
thefalsepositiveresults(13/23)intheNTP2groupcouldbe
attributedto thediagnosis ofcancer. Ifpatients diagnosed
withcancerwereexcluded,thespecificityimprovedto83.9%
(52/62).
Conclusions
Inthisstudy,thediagnosticperformanceofthesixpromising
epitopes, Rv1986-P9, P15, P16, Rv1988-P4,P11, and
Rv1983-P4, was assessed on a panel of 233 patients suspected
of having TB for the first time. Our data suggest that
theseepitopesfromRD2ofM.tuberculosismayhave
poten-tial diagnostic value in the immunological diagnosis of
TB.
Conflict
of
interest
Theauthorshaveappliedapatentforfivepeptides,including
Rv1986-P9,Rv1986-P15,Rv1986-P16,Rv1988-P4,Rv1988-P11,in
TBdiagnosis.
Acknowledgments
ThisworkwassponsoredbytheMajorProjectoftheTwelfth
Five-Year Plan (2013ZX10003001-002), National Natural
Sci-ence Foundation of China (81101226) and the Shanghai
ScienceandTechnologyCommission(12DZ1941402).
Appendix
A.
Supplementary
data
Supplementary data associated with this article can
be found, in the online version, at https://doi.org/10.
1016/j.bjid.2018.10.280.
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