Shraddha Kumari, Mukesh Samant, Pragya Misra, Prashant Khare, Brijesh Sisodia, Ajit K Shasany ...

12 Pages·2008·0.98 MB·English

Vaccine26(2008)5700–5711
ContentslistsavailableatScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Th1-stimulatory polyproteins of soluble Leishmania donovani promastigotes
ranging from 89.9 to 97.1kDa offers long-lasting protection against
experimental visceral leishmaniasis
ShraddhaKumaria,MukeshSamanta,PragyaMisraa,PrashantKharea,BrijeshSisodiab,
AjitK.Shasanyb,AnuradhaDubea,∗
aDivisionofParasitology,CentralDrugResearchInstitute,Lucknow,India
bProteomicsLaboratory,GeneticResourcesandBiotechnologyDivision,CentralInstituteofMedicinalandAromaticPlants,India
a r t i c l e i n f o a b s t r a c t
Articlehistory: Ourearlierstudiesidentifiedafraction(F2)ofLeishmaniadonovanisolublepromastigoteantigenbelonging
Received4July2008 to97.4–68kDaforitsabilitytostimulateTh1-typecellularresponsesincuredvisceralleishmaniasis
Receivedinrevisedform5August2008 (VL)patientsaswellasincuredhamsters.AfurtherfractionationofF2-fractionintosevensubfractions
Accepted11August2008
(F2.1–F2.7)andre-assessmentfortheirimmunostimulatoryresponsesrevealedthatoutofthese,only
Availableonline30August2008
four(F2.4–F2.7)belongingto89.9–97.1kDa,stimulatedremarkableTh1-typecellularresponseseither
individuallyorinapooledform(P4-7).
Keywords:
Inthisstudythesepotentialsubfractionswerefurtherassessedfortheirprophylacticpotentialincom-
SolubleLeishmaniadonovanipromastigotes
binationwithBCGagainstL.donovanichallengeinhamsters.Optimumparasiteinhibition(∼99%)was
(SLD)
Th1-stimulatoryproteins obtainedinhamstersvaccinatedwithpooledsubfractionsandtheysurvivedfor1year.Theprotection
Immunoprophylaxis wasfurthersupportedbyremarkablelymphoproliferative,IFN-(cid:2)andIL-12responsesalongwithprofound
Hamsters delayedtypehypersensitivityandincreasedlevelsofLeishmania-specificIgG2antibodyasobservedon
MALDI-TOF/MS days45,90and120post-challengesuggestingthatasuccessfulsubunitvaccineagainstVLmayrequire
multiple Th1-immunostimulatory proteins. MALDI-TOF–MS/MS analysis of these subfractions further
revealed that of the 19 identified immunostimulatory proteins, Elongation factor-2, p45, Heat shock
protein-70/83,Aldolase,Enolase,Triosephosphateisomerase,DisulfideisomeraseandCalreticulinwere
themajoronesinthesesubfractions.
©2008ElsevierLtd.Allrightsreserved.
1. Introduction chemotherapyforVLisfarfromsatisfactorybecauseantileishma-
nial drugs are costly with unpleasant side effects. The situation
Visceral leishmaniasis (VL), caused by the intracellular para- hasfurtherworsenedwithemergenceofdrugresistanceinvari-
siteLeishmaniadonovani,L.chagasiandL.infantumischaracterized ousregionsofendemicity[1].Vaccinationwould,therefore,bea
bydefectivecell-mediatedimmunity(CMI)andisusuallyfatalif betteroptionforaneffectivecontrolstrategyforVL.
not treated properly [1]. An estimated 350 million people are at To control Leishmania infections in experimental and human
risk of acquiring infection with Leishmania parasites worldwide VL, the development of an effective CMI, capable of mounting
with approximately 500,000 cases of VL has been reported each Th1-typeofimmuneresponses,playanimportantrole[3–7].This
year.RecentepidemicsofVLinSudanandIndiahaveresultedin is derived from the fact that a large number of people living in
over 100,000 deaths [2]. With the advent of the HIV epidemics, endemic areas have self-resolving subclinical infection and the
thediseasehasemergedasanimportantopportunisticinfection infectedindividualsoncerecoveredafterthetreatmentareimmune
in AIDS patients [1]. In India, high incidence has been reported toreinfection.Thisprovidesarationalefordesigningimmunopro-
from the states of Bihar, Assam, West Bengal and eastern Uttar phylacticstrategiesagainstVL[8].Sofar,successfulimmunization
Pradeshwhereresistanceandrelapseareontheincrease.Available with fractionated and purified antigens reported against murine
VLandCLhasledtoveryfewproteinsthathavebeentakenupfor
preclinical/clinicalevaluation[9–12].Successfulsecondgeneration
vaccineswithexcretedfactors(LiESAp)[13]orpurifiedglycopro-
∗
Correspondingauthor.Tel.:[phone]/212418x4398;
teins(FML)[14,15]areusedintrialswithefficacyandevenlicensed.
fax:[phone]/223938.
There are only few reports in literature deal with vaccines, viz.,
E-mailaddresses:[email protected],
[email protected](A.Dube). FML,FML-QuilASaponin,etc.againstcaninevisceralleishmaniasis
0264-410X/$–seefrontmatter©2008ElsevierLtd.Allrightsreserved.
doi:10.1016/j.vaccine.2008.08.021
S.Kumarietal./Vaccine26(2008)5700–5711 5701
[14,16–20].Amongtherecombinantandnativeantigenstestedin andfedwithstandardrodentfoodpellet(LiptonIndiaLtd.,Bom-
murinemodels,theLACKprotein[21],theLPGAPpeptides[22],the bay) and water ad libitum. The usage of hamsters was approved
LeIFprotein[6]andthedp72glycoproteinofL.donovani[23]were by the Institute’s Animal Ethical Committee (protocol number
protectiveimmunogensformice.Protectionresultsobtainedwith 24/05/Para/IAECdated15September2005).
thethirdgenerationvaccinescomposedofcDNAencodingleishma-
nialantigensclonedintoaneukaryoticexpressionvectorarestill 2.2. Parasites
inpreliminarystage[24].
Although a great number of antigens have been tested for The L. donovani strain (2001) was procured from a patient
protection against the cutaneous disease with in vitro cell or admitted to the Kala-azar Medical Research Centre of the Insti-
mouse models, no effective vaccine against human kala-azar is tuteofMedicalSciences,BHU,Varanasiandwasculturedinvitro
yet available. The reason perhaps being that the cell epitopes as described elsewhere [28]. Promastigotes were grown in L-15
that were protective in the murine model did not elicit an ade- mediumat26◦C(Sigma,USA)in75cm2cultureflasks(Nunc)[34].
quate effective immune response in human [25–27]. Hence, the The strain has also been maintained in hamsters through serial
evaluation of potential leishmanial antigenic proteins for their passage,i.e.fromamastigotetoamastigote[34].
ability to elicit cellular immune responses in humans as well as
in experimental model was considered important [28,29] before 2.3. PreparationofsolubleL.donovanipromastigote(SLD)
theyarefurtherevaluatedfortheirprophylacticpotentialagainst antigen
experimental VL. The golden hamster (Mesocricetus auratus) has
been proven to be the most appropriate experimental model as SLD was prepared as per method described by Gupta et al.
it largely reflects the clinic-pathological features of progressive [33,34].Briefly,latelogphasepromastigotes(109)wereharvested
human VL, including a relentless increase in visceral parasitic from3to4daysofcultureandwashedfourtimesincoldphosphate-
burden,cachexia,hepatosplenomegaly,pancytopenia,hypergam- bufferedsaline(PBS)andresuspendedinPBScontainingprotease
maglobulinemiaandultimatelydeath.Oflate,ithasalsobeenused inhibitors cocktail (Sigma, USA) and subjected to ultrasonication
extensively for vaccination studies [28,30]. Interestingly, while andcentrifugationat40,000×gfor30min.Theproteincontentof
infectionofthehamsterwithL.donovanireproducedthefeatures thesupernatantwasestimated[35]andstoredat−70◦C.
ofhumanVL,themechanismsofdiseaseinthehamsterdifferstrik-
inglyfromthoseobtainedinmice,asuitableexperimentalmodel 2.4. SubfractionationofF2-fractionbycontinuouselutiongel
forearlyinfectionofVLastheinfectiontendstoself-cureinthis electrophoresis
rodentmodel[31].
Themainfocusinvaccinedevelopmenthasbeentheidentifi- SubfractionationofF2-fractionwasdonebycontinuouselution
cationandcharacterizationofdefinedleishmanialantigensaswell SDS-PAGE using Laemmli’s buffer system in a ‘Prep-Cell’ (Model
astheelucidationoftherangeandspecificityofantileishmanial 491;BioRad,Hercules,CA)containingfractioncollector[36].Prior
immune responses. Advances made in clinical proteomic tech- tothis,aresolvinggelconcentrationsuitableforthewholerange
nologieshavefurtherenhancedourmechanisticunderstandingof of proteins was determined by running a series of SDS-PAGE on
leishmanial pathobiology thereby defining novel vaccine targets mini slab gel. Accordingly, 8% resolving gel and 4% stacking gel
[28,32,33].Theevaluationofsuchvaccinetargetsfortheirprophy- werecastintube.Lyophilizedprotein(10mg)wasloadedunder
lacticpotentialwillprovidefurtherleadtowardsthedevelopment standard condition [37] and electrophoresis was carried out as
ofacandidatevaccine(s). perprotocolmentionedinmanufacturer’smanual,Total120frac-
We have further fractionated the Th1-stimulatory fraction of tionswerecollectedandeachwasanalyzedconsequentlyonmini
68–97.4kDa (F2) of soluble protein of L. donovani promastigotes gel slabs and silver stained [38] to visualize the eluted proteins.
bycontinuouselutiongelelectrophoresis(Prep-Cell)onthebasis Wide range molecular weight marker (Bangalore Genei, India)
of molecular weight and there of obtained seven subfractions was used to identify and assess the exact molecular weights of
(F2.1–F2.7)belongingto69.0,74.9,78.4,89.9,94.9,96.9,97.1kDa, protein bands and their number and density were assessed by
respectively[86].Outofsevensubfractionsonlyfoursubfractionsof softwareAlphaImagerTM2200.Fractionnumber40–100displayed
89.9,94.9,96.9,97.1kDastimulatedremarkablecellularresponses, theproteinbandsrangingbetweenthedesiredmolecularweight
i.e.lymphoproliferative,IFN-(cid:2)andIL-12responsesandsuppressed of 68–97.4kDa. The bands with identical molecular weight were
IL-10 cytokine levels in cured/exposed VL patients as well as in pooledinsuchawaysoastoprovidesevendiscretesubfractions
cured Leishmania infected hamsters. Interestingly, all of these as F2.1,F2.2,F2.3,F2.4,F2.5,F2.6andF2.7[86].Thesubfractionswere
pooledsubfractionsyieldedoptimumefficacyascomparedtothe furtherprocessedforSDSremovalbythemethodofWesseland
individualones. Flügge[39].TheremovalofSDSwascheckedbycolorimetricesti-
In this study, these potential antigenic subfractions were mationusingthemethodofArandetal.[40].Proteinquantification
assessed for their prophylactic efficacy alongwith Bacillus Cal- wasdonebyLowry’smethod[35].
metteGuerin(BCG)—anadjuvant,againstL.donovaniinfectionin Themostpotentsubfractions(F2.4–F2.7)weretakenbothindi-
hamsters.Thosesubfractionswhichexhibitedsignificantcellular viduallyaswellasinpooledform(referredasP4-7subfraction)for
responsesaswellasprophylacticpotentialwerefurthercharacter- vaccination studies. In addition, the subfractions F2.1–F2.7 were
izedbyMALDI-TOF/TOF-MSsoastoensuretheproteincontentsas alsopooledintooneandusedasreferenceantigen(F2).
prospectivevaccinetargets.
2.5. Vaccination
2. Materialsandmethods
Ninegroupscontaining12–15hamstersineachwereemployed
2.1. Animalandparasites fortheimmunizationwithdifferentpreparationsofantigenicfrac-
tions.ThehamstersofGroups1–7wereimmunizedintradermally
Laboratory bred male golden hamsters (M. auratus, 45–50g) onthebackwitheachsubfractionsF2.4,F2.5,F2.6,F2.7,P4-7,F2-
from the Institute’s Animal House Facility were used as experi- fractionandSLD(50(cid:3)g/(0.05mlanimal))alongwithequalvolume
mental host. They were housed in climatically controlled room of BCG (0.1mg/(0.05mlanimal)) in emulsified form. The eighth
5702 S.Kumarietal./Vaccine26(2008)5700–5711
groupwasgivenBCGonlyandthelastninthgroupwhichreceived (BARC,India)wasaddedtoeachwellandthentheywereharvested
onlyPBSservedascontrol.Fifteendayslateraboosterdoseofhalf onglassfibremats(Whatman)andcountedinaliquidscintillation
oftheamountofantigensalongwithBCGorPBSwasgivenintra- counter. Results were expressed as stimulation index (SI) which
dermallytoallthehamstersofrespectiveexperimentalgroups(i.e. wascalculatedasmeancountsperminute(cpm)ofstimulatedcul-
Groups1–8)asmentionedabove. ture/meancpmofunstimulatedcontrol.SIvaluesofmorethan2.5
wereconsideredaspositiveresponse.
2.6. Infection
2.7.3. QuantificationofNOinmacrophagesofhamstersandcell
Twenty-onedaysaftertheboosterdose,allthevaccinatedand lines
unvaccinated control groups were challenged intracardially with Thepresenceofnitrite(NO −)contentwasassessedusingGriess
2
108 latelogphasepromastigotesofL.donovani.Theprophylactic reagent in the culture supernatants of naïve hamster peritoneal
efficacyoftheimmunogenwasassessedinspleen,liverandbone macrophages after the exposure with supernatant of stimulated
marrowofthreevaccinatedhamstersonnecropsyatdifferenttime lymphocyte’s cultures. Briefly, isolated peritoneal macrophages
intervals,i.e.ondays0,45,90,120post-challenge(p.c.).Peritoneal [42]weresuspendedinculturemediumandplatedat106cells/well
exudatescells,inguinallymphnodesandbloodwerealsocollected andexposedtothesupernatantsofabovedescribed5-day-oldanti-
atthesetimepointstoobtaincellsandseraforevaluationofcellular gen stimulated lymphocyte’s cultures from all the study groups.
andantibodyresponses[41]. The supernatants (100(cid:3)l) collected from macrophage cultures
Thecriterionofprophylacticefficacywastheassessmentofpar- 24h after incubation was mixed with an equal volume of Griess
asiteloadasthenumberofamastigotes/1000cellnucleiineach reagent(Sigma,USA)andleftfor10minatroomtemperature.The
organincomparisontotheunvaccinatedcontrolsandthepercent- absorbance of the reaction was measured at 540nm in an ELISA
ageinhibition(PI),wasassessedincomparisontotheunvaccinated reader[43].
controlbyfollowingformula[28]:
2.7.4. RT-PCRofmRNAcytokinesandinducibleNOsynthase
No.ofparasitecountfrominfectedcontrol
(iNOS)
−No.ofparasitefromvaccinatedgroup
PI= ×100 RT-PCRwasperformedtoassesstheexpressionofvariousmRNA
No.ofparasitecountfrominfectedcontrol
cytokinesandiNOSinsplenocytesofmainexperimentalgroupsas
For post-challenge survival animals from both the experimental wellasunvaccinatedcontrolanimals.Threerepresentativeham-
andcontrolgroupsweregivenpropercareandwereobservedfor sters from each experimental group were randomly selected to
theirsurvivalperiodwhichlastedformorethan12monthsp.c.Sur- analyzethespleniccytokineprofile.RNAfromsplenocytesofdif-
vivalofindividualhamsterwasrecordedandmeansurvivalperiod ferentgroupsofhamsterswasisolatedusingTri-reagent(Sigma,
wascalculated. USA) on days 45, 90 and 120 p.c. and quantified by using Gene-
quant(Biorad,USA).TheprimersequencesofcytokineandiNOS
2.7. Immunologicalassays primersasdescribedbyMelbyetal.[44]asmentionedinTable1
wereusedtoamplifytheirrespectivecDNA.HGPRTwasusedasa
2.7.1. Delayedtypehypersensitivity(DTH) housekeepingcontrol.
DTHwasperformedbyinjectingintradermally50(cid:3)g/50(cid:3)lof OnemicrogramoftotalRNAwasusedforthesynthesisofcDNA
SLD in PBS into one footpad and PBS alone into the other foot- usingfirststrandcDNAsynthesiskit(Fermentas).0.5(cid:3)gofcDNA
pad of each vaccinated hamsters and unvaccinated controls. The wasamplifiedbyPCRunderthefollowingcondition:initialdenat-
responsewasevaluated24hlaterbymeasuringthedifferencein urationat95◦Cfor2min,40cyclesofdenaturationstepeachat
footpadswellingbetweeneachofthevaccinatedandcontrolgroups 95◦Cfor30s,annealingat55◦Cfor40s,followedbyextensionstep
ofhamsters[4]. at72◦Cfor40s.Thefinalextensionstepwascarriedoutat72◦C
for10min.Further,thedensitometricanalysisofPCRproductwas
2.7.2. Lymphocyteproliferationassay(LTT) carriedoutusingsoftwareAlphaImagerTM2200(Alfainnotech).The
Lymphocytessuspension(1×106ml−1)ofvaccinatedhamsters samebandareawastakenforbandintensityandwasnormalizedto
was cultured in 96-well flat bottom tissue culture plates (Nunc, HGPRT.Themeanpercentageexpressionvalueswererepresented
Denmark).Thisassaywascarriedoutasperprotocoldescribedby relativetotheircorrespondingHGPRTvalues.
Gargetal.[28].About100(cid:3)lofpredeterminedconcentrationof
mitogen-ConA(10(cid:3)g/ml,Sigma,USA)andantigen-SLD(10(cid:3)g/ml 2.7.5. Antileishmanialantibodyresponses
each)wereaddedtothewellsintriplicate.Wellswithoutstimu- Thelevelofantileishmanialantibodyinserasamplesfromham-
lantsservedasblankcontrols. stersofboththesetofexperimentalgroupswasmeasuredasper
Cultureswereincubatedat37◦CinaCO incubator(5%CO )for protocolofVolleretal.[41].Briefly,ELISAplates(Nunc,Denmark)
2 2
3daysinthecaseofConAandfor5daysinthecaseofSLD.Eigh- were coated overnight at 4◦C with 1(cid:3)g/ml SLD antigen diluted
teenhourspriortoterminationofculture,0.5(cid:3)Ciof[3H]thymidine in0.02Mphosphatebuffer(pH7.5)andwerethenblockedwith
Table1
Sequenceofreverseandforwardprimers
S.no. Primer Primersequence Productsize(bp)
1 HGPRT:forward;reverse 5(cid:5)ATCACATTATGGCCCTCTGTG3(cid:5);5(cid:5)CTGATAAAATCTACAGTYATGG3(cid:5) 125
2 iNOS:forward;reverse 5(cid:5)GCAGAATGTGACCATCATGG3(cid:5);5(cid:5)CTCGAYCTGGTAGTAGTAGAA3(cid:5) 198
3 IFN(cid:2):forward;reverse 5(cid:5)GGATATCTGGAGGAACTGGC3(cid:5);5(cid:5)CGACTCCTTTTCCGCTTCCT3(cid:5) 309
4 IL-12:forward;reverse 5(cid:5)GTACACCTGYCACAAAGGAG3(cid:5);5(cid:5)GATGTCCCTGATGAAGAAGC3(cid:5) 430
5 TGF(cid:4):forward;reverse 5(cid:5)CCCTGGAYACCAACTATTGC3(cid:5);5(cid:5)ATGTTGGACARCTGCTCCAC3(cid:5) 310
6 IL-4:forward;reverse 5(cid:5)CATTGCATYGTTAGCRTCTC3(cid:5);5(cid:5)TTCCAGGAAGTCTTTCAGTG3(cid:5) 463
7 IL-10:forward;reverse 5(cid:5)ACAATAACTGCACCCACTTC3(cid:5);5(cid:5)AGGCTTCTATGCAGTTGATG3(cid:5) 432
DegeneratebasesareindicatedbytheappropriateInternationalUnionofPureandAppliedChemistrydesignation(Y=CorT,R=AorG).
S.Kumarietal./Vaccine26(2008)5700–5711 5703
1% BSA in PBS, after washing with PBS containing 0.05% Tween pendentexperiments)wereanalyzedbyone-wayANOVAtestand
20. The optimum dilution of sera for determination of IgG was comparisonswithcontroldataweremadewithDunnett’spost-test
foundtobe1:200andforIgG1andIgG21:100in1%BSA–PBSat usingGraphPadPrismsoftwareprogram.Comparisonsweremade
4◦Cfor1.5h.AfterwashingwithPBSTweentheplateswereincu- betweeninfectedcontrolgroupsandalltheexperimentalgroups.
batedfor3hwithHRP-conjugatedGoatanti-HamsterIgG(H+L) TheupperlevelofsignificancewaschosenasP<0.001.
(1:1000)(Serotec,USA).Parallely,plateswereincubatedovernight
withBiotin-conjugatedmouseanti-HamsterIgG1andmouseanti-
3. Results
Armenianandanti-SyrianhamsterIgG2(BD,Pharmingen)2(cid:3)g/ml
in PBS (100(cid:3)l/well) and incubated for 3h according to manu-
3.1. Vaccinationwithpotentialsubfractionsinpooledform
factures instruction. The plates were developed using the OPD
inducedoptimumprotectionagainstL.donovanichallenge
substrate(o-phenylenediaminedihydrochloride,Sigma)andread
at492nmusinganELISAreader(BioTek,USA).
Thepotentialsubfractionsvaccinatedhamsterswereprotected
from the challenge infection of L. donovani, as observed by their
2.8. MALDIanalysisofpotentTh1-stimulatorysubfractions
weight gains (Fig. 1A) similar to normal hamsters kept simulta-
neouslyforthesametimeperiod,i.e.ondays45,90and120p.c.
The potential subfractions F2.4–F2.7 were run on one-
Hepatosplenomegaly,associatedwithchallengeinfection,wasvir-
dimensional PAGE (12% resolving gel, 1.0mm thick). Bands were
tuallyabsentinthepotentialsubfractionsvaccinatedgroup(Fig.1B
separatedoutandweredigestedin-gelasperprotocoldescribed
andC).Parasiteloadwasdirectlycorrelatedwithweightandsize
earlier [33,45]. Briefly, the coomassie blue stained bands were
of liver and spleens among different groups: significant parasite
washed, in-gel reduced, S-alkylated and digested with trypsin
(Promega, Madison, WI, USA) at 37◦C overnight. Peptides were inhibitionwasobservedinhamstersvaccinatedwithF2.5,F2.6and
F2.7 subfractions, P4-7 subfractions and F2-fraction wherein the
extracted, dried in a Speed-Vac and resolubilized in 0.1% tri-
parasiteloadwasobservedtobe≤2×102.Interestingly,thepro-
fluoroacetic acid. Zip Tips (Millipore) were used to desalt and
phylactic efficacy was more marked in the hamsters immunized
concentratethepeptidemixture.Peptidemassfingerprintingwas
withP4-7subfractions.Therewasprogressivedecreaseinparasite
performedbyspotting0.3(cid:3)loftheextractedproteindigestmixed
loadinspleen,liverandbonemarrowfrom<1×102,amorethan
with(cid:5)-cyano-4-hydroxycinnamicacid(CHCA,Sigma)onaMALDI
96%inhibitionofparasitemultiplicationonday45p.c.toanegli-
targetplate.MSandMS/MSspectrumwereacquiredinthepositive
giblelevelonday120p.c.,renderingthemdifficulttodiscern,i.e.
ionmodeonMALDI-TOF/TOFMassSpectrometer,AppliedBiosys-
∼99%parasiteinhibitionwasobserved(Fig.2A–C).F2andSLDvac-
tems4700ProteomicsAnalyzer(Framingham,MA,USA).
cinatedhamstersharboredmorethan2×102parasitesonday45
Theinstrumentwasoperatedinthedelayedextractionmode
p.c.TheremainingparasiteintheP4-7pooledsubfractionvacci-
with delay time of 200ns. Spectra were obtained by accumula-
natedgroup,whencheckedfortheirvirulenceonday120p.c.by
tionof1000and4000consecutivelasershotsrespectivelyinMS
puttingthesplenic/livertissueandlymphnodesininvitroculture,
and MS/MS mode and laser intensity used were in the range of
didnotconvertintopromastigotesevenafter10daysofculture.
5000–6000.CloseexternalcalibrationforMSwasperformedwith
Allthevaccinatedhamstersimmunizedwiththeindividualsub-
4700CalMix(AppliedBiosystems,USA)astandardmixture.Peak
fractionsF2.5–F2.7andpooledP4-7subfractionssurvivedlonger
harvestingwascarriedoutusing4000SeriesExplorerTMSoftware
afterthelethalchallengeofL.donovaniandremainedhealthyuntil
(AppliedBiosystems,USA).Onlybaselinecorrectionswereapplied
the termination of the experiment at 12 months post-infection.
totherawdata.
WhilehamstersimmunizedwithF2.4subfraction,F2-fractionand
Database searching for protein identifications was performed
SLDsurvivedonlytill6months,allthehamsterswhichwereimmu-
with mass spectrometry data (MS or MS/MS) using Global Pro-
nizedwithBCGaloneaswellasunimmunizedonessuccumbedto
teome Server v3.5 software (Applied Biosystems, USA) equipped
virulentL.donovanichallengewithin2–2.5months.
withMASCOT(MatrixScience)searchengine.Onlymonoisotopic
masseswereusedforsearchingagainsttheSwiss-ProtandNCBInr
databases with a minimum number of matched masses set at 3.2. PotentialsubfractionsstimulatesubstantialDTHand
4. The maximum peptide precursor tolerance was set at 40ppm mitogenicandLeishmania-specificcellularresponsesin
and MS/MS fragment tolerance was defined as 0.2Da. At most immunizedhamsters
one missed cleavage for tryptic peptides was allowed, and the
modificationsacceptedwerecarbamidomethylcysteinesasfixed Tocharacterizethefateofcell-mediatedimmuneresponsefol-
modification and methionine oxidation as variable modification. lowingimmunizationwehaveinvestigatedthepotentsubfractions
Tandem MS was performed only in the cases where identifica- (F2.4–F2.7)inducedDTHresponsesinhamsterschallengedwithL.
tionappearedambiguouswithMALDI-TOF–MSdata.Thecriteria donovaniandthecapacityoftheircellstoproliferateinresponseto
usedtoacceptidentificationsforpeptidemassfingerprintincluded mitogenandleishmaniaantigen-SLDondays0,45,90and120p.c.
the probabilistic protein score-based confidence interval %, the Fig.3Ashowsthatthehamstersreceivingpotentsubfractionseither
extentofsequencecoverage,thenumberofpeptidesmatchedand individuallyorinpooledformdisplayedsignificantlystrongerDTH
whetherLeishmaniaspp.orTrypanosomaproteinappearedastop responseascomparedwiththeothergroups,viz.,infectedcontrol
candidatesduringthefirstsearch,whennorestrictionwasapplied and BCG vaccinated hamsters at these time points. Immuniza-
to the species of origin. Identification criteria with MS/MS data tionwithP4-7inducedsignificantly(P<0.001)higherlevelofDTH
werethatpeptidescountshouldbenotlessthantwoormoreand response,i.e.eightfoldincreaseascomparedtoinfectedcontrolon
confidenceinterval%forthebestionscoreshouldbeabove95[33]. day45p.c.thatremainedincreasedsignificantlythroughday90till
day120p.c.
2.9. Statisticalanalysis Invitrostimulationofthelymphocyteswiththemitogen-Con
A, showed proliferative responses with no differences between
Resultswereexpressedasmean±S.D.Threesetsofexperiments control and antigen immunized groups at pre-challenge time
were performed for vaccination studies and in each experiment point(Fig.3BandC).Followingchallenge,thevaccinatedgroups
10–15animalswereused.Theresults(pooleddataofthreeinde- showed intact responses to Con A but, on the other hand, it
5704 S.Kumarietal./Vaccine26(2008)5700–5711
Fig.1. Bodyweight(A),weightofspleen(B)andliver(C)ingondays45,90and120p.c.ofvaccinatedhamsterswithpotentsubfractions(F2.4–F2.7),pooledsubfraction
P4-7,F2-fractionandSLD.Eachbarrepresentsthepooleddata(mean±S.D.value)ofthreereplicates.
was lowered in the infected control groups. In antigen-specific 3.3. ImmunizationwithP4-7subfractionelicitsprominent
re-stimulation assays, performed after immunization, there was Th1-typecytokineresponseinprotectedgroupofhamstersby
significant(P<0.001)stimulatoryresponseinthecellsofhamsters RT-PCR
vaccinatedwithpotentsubfractionsF2.4–F2.7incombinationwith
BCGonday45p.c.,whichwas∼10–20foldshigherascompared Impairment of CMI response during active VL is reflected by
totheinfectedcontrolgroup(Fig.3C).Moreover,maximumlym- marked T-cell anergy specific to Leishmania antigens [30]. Since
phoproliferativeresponsewasnoticedonday45p.c.inthecells optimumprotectiveefficacywasobservedinP4-7vaccinatedham-
ofanimalsimmunizedwithP4-7subfractionwhichwas∼27folds sters, the expression of Th1 and Th2 cytokines was evaluated in
higherandtheresponsesincreasedprogressivelyondays90and this group only and compared with infected and normal control
120p.c.Ontheotherhand,therewasnoproliferativeresponsein groupofanimals.AcomparativeRNAcytokineprofileofspleno-
animalsvaccinatedwithBCGaloneaswellasunvaccinatedinfected cytes,analyzedondays45,90,and120p.c.,showedthatamongthe
control(Fig.3C). threegroupsofhamstersexpressionofIFN-(cid:2)andIL-12transcripts
Similarly, we have observed that macrophages isolated from wassuppressedininfectedgroup,butwassignificantlyhigherby
naïvehamsters,whenincubatedwithstimulatedsupernatantsof two and three folds, respectively (P<0.001) in vaccinated group
lymphocytesfromP4-7subfractionvaccinatedhamsters,produced (Fig. 4A). Higher expression of two to three folds of iNOS tran-
significantamountofNOwhichwas∼7foldsmorethantheinfected scriptwasobservedinP4-7vaccinatedhamstersascomparedto
controlsonday45p.c.TheNOlevelwasfurtherincreasedincredibly theinfectedcontrolson45daysp.c.(Fig.4B).Levelofexpressionof
bydays90and120p.c.Incontrast,littleamountofNOwaspro- Th1suppressivecytokines,TGF-(cid:4),IL-10andIL-4whichwereup-
ducedbythecellsofhamstersbelongingtounvaccinatedcontrol regulatedininfectedgroup,weresignificantlydown-regulatedin
groupandBCGonly(Fig.3D). P4-7vaccinatedhamstersbydays45through120p.c.(Fig.4B).
S.Kumarietal./Vaccine26(2008)5700–5711 5705
Fig.2. Parasiteburden(no.ofamastigotesper1000cellnuclei)ondays45,90and120p.c.in(A)spleen,(B)liverand(C)bonemarrowofvaccinatedhamsterswithpotent
subfractions(F2.4–F2.7),pooledsubfractionP4-7,F2-fractionandSLD.Eachbarrepresentsthepooleddata(mean±S.D.value)ofthreereplicates.
3.4. PotentsubfractionscausedecreaseinLeishmania-specific 3.5. Characterizationofthefourpotentsubfractionsby1-DGE
IgGandIgG1isotypeandincreaseinIgG2isotypeantibody andMALDI-TOF–MS
responsesinvaccinatedhamsters
Inordertoassessthecomponentsofthefourpotentsubfractions
Wehaveassessedtheleishmanialantigen-specificIgGandits theircharacterizationbyMALDI-TOF–MSwascarriedout.Major-
isotype(IgG1andIgG2)levelsintheseraofallthegroupsofvac- ity of the proteins was detected around 4–8 pI acidic to neutral
cinated hamsters through ELISA. As shown in Fig. 5C the groups pHrange.MassesandpIwerecalculatedbysoftware;measuresof
ofhamstersvaccinatedwithpotentialsubfractionsdevelopedan theconfidenceoftheidentificationonthebasisofnumberofpep-
effectiveimmuneresponsebyshowingsubstantiallyhigherlevels tidesmatchedandsequencecoveragewhichwasdeterminedusing
ofIgG2antibody,whichisameasureofCMI(Fig.5C).Ahighlysig- MASCOT.Theidentifiedspotsmatchedto168databaseentries.Of
nificantdifference(twofolds)wasfoundinIgG2levelbetweenP4-7 spotsanalyzedbyMALDI-TOFandMS/MS,41%wereclearlyiden-
vaccinatedandinfectedcontrolgroupsofhamsters(P<0.001).In tifiedbytheirhomologywiththoseofL.major.Theproteinsthus
contrast,therewasdecreasedlevelsofIgGandIgG1(P<0.5)inP4-7 identifiedarelistedinTable2.Theanalysisrevealedthesimilar-
vaccinatedhamstersascomparedtotheinfectedcontrols(Fig.5A ityofproteinpatternasobservedwith2DgelmapofF2-fraction
andB). [33]. In all, a total of 19 proteins were identified by 1-DGE and
5706 S.Kumarietal./Vaccine26(2008)5700–5711
Fig.3. Cellularresponsesondays45,90and120p.c.inhamstersvaccinatedwithpotentfractionhamsterswithpotentsubfractions(F2.4–F2.7),pooledsubfractionP4-7,
F2-fractionandSLDalongwithBCG.Datafornormalandunvaccinatedinfectedgroupshavebeenrepresentedascontrolgroups,respectively.(A)DTHresponse(mm)to
SLDinhamsterswasmeasuredasfootpadswellingat24h,(BandC)LTTresponse(SIvalue)againstConAandSLD.SIvaluesofmorethan2.5wereconsideredaspositive
response,and(D)NOproduction((cid:3)g/ml).Eachbarrepresentsthepooleddata(mean±S.D.value)ofthreereplicates.
Fig.4. AnalysisofTh1andTh2mRNAcytokineprofileinnormal,infectedandP4-7vaccinatedhamstersondays45,90and120p.c.byRT-PCR.(A)SpleniciNOSandcytokine
expression.Eachbandrepresentsoneoutofthreerepresentativehamstersfromeachexperimentalgroup.(B)Densitometryanalysisshowingtherelativemean%changein
iNOSandcytokinemRNAexpression±S.D.incomparisontocontrol(HGPRT).Significancevaluesindicatethedifferencebetweenvariousanimalgroupsandnormalgroup
(*P<0.05and***P<0.001).
S.Kumarietal./Vaccine26(2008)5700–5711 5707
Fig.5. Antileishmanialantibodylevels(ODvalue)inhamstersvaccinatedwithpotentsubfractions(F2.4–F2.7),pooledsubfractionP4-7,F2-fractionandSLDofLeishmania
donovanialongwithBCGondays45,90and120p.c.DataforBCGvaccinated,unvaccinatedcontrolandnormalhamstersrepresentedaspositiveandnegativecontrolgroups,
respectively(A)IgGanditsisotype(B)IgG1or(C)IgG2.Eachbarrepresentsthepooleddata(mean±S.D.value)ofthreereplicates.
MALDI-TOF–MSinsubfractionsF2.4–F2.7,includingfivehypothet- of lymphocytes in vitro and the release of very high amount of
icalproteins/unknowns.Amongthese,mostoftheseproteinswere Th1-typecytokines,viz.,IFN-(cid:2)andIL-12p40,wereobserved.Again,
ofknownimmunostimulatoryorimmunogenictypeorhavebeen theexcellentcellularresponseswereobtainedwhenallthefour
evaluatedasvaccinecandidatessuchasElongationfactor-2,p45, subfractions were pooled together, i.e. P4-7. On the other hand,
HSP-70, HSP-83, Fructose-1,6-Bisphosphate Aldolase (Aldolase), theTh2-typecytokineIL-10wasfoundtobesuppressedincured
Enolase, Triosephosphate isomerase (TPI), Protein Disulfideiso- patientsaswellasinendemiccontactsagainstallthesubfractions
merase and Calreticulin. Some of the other proteins including [86].
someenzymesfromenergymetabolism,phosphorylationpathway, Basedontheirimmunostimulatoryproperties,thefourpotent
aminoacidmetabolismpathwaysandfromdiversemetabolicroute individualsubfractionsaswellaspooledonewerefurtherevalu-
havealsobeenreportedaspotentialdrugtargets,viz.,Adenosyl- atedfortheirimmunoprophylacticpotentialwithBCGinhamsters.
homocysteinase,Cofactor-independentphosphoglyceratemutase, BCGhadbeenusedasanadjuvantduetoitspropertyofactivating
Trypanothione reductase, and NAD-dependent deacetylase SIR2 macrophagesforinducingNO[46,47]andelicitinglong-lastingcel-
homolog. lularandhumoralimmuneresponses[48,49].Althoughsignificant
parasiteinhibitionwasnoticedinhamstersimmunizedwitheither
4. Discussion ofF2.5–F2.7subfractionsorF2-fraction,theefficacywasremark-
ableinhamstersreceivingP4-7subfractions.Theparasiteloadinall
Previous studies from this laboratory have hinted toward the visceralorgansdecreasedprogressivelyreachinganegligiblelevel
prophylacticpotentialofF2-fractionofsolublepromastigoteanti- byday120p.c.,renderingthemdifficulttodiscerndemonstrating
genbelongingto68–97.4kDainexperimentalVLmodel[28].Using its strong vaccine potential. Moreover, post-challenge mean sur-
theconventionalscreeningapproachfoursubfractionsbelongingto vivalofhamstersinP4-7subfractionvaccinatedgroupalongwith
molecularweightof89.9,94.9,96.9,97.1kDawereidentifiedwhich F2.5–F2.7vaccinatedhamstersbeingmorethan12monthsfurther
gaveconsiderablygoodcellularresponse,viz.,lymphoproliferative strengthen the evidence that combination of all the four potent
aswellasNOreleaseagainstallthecuredhamsters.However,the subfractionsarerequiredtoinduceoptimumprophylacticefficacy.
optimumcellularresponseswereobservedwhenallthefoursub- Mostoftheassaysinthisstudyweredonebetween45and120
fractions were pooled together, i.e. P4-7. The responses of these dayspost-challengeasthediseaseprogressionreachesitspeakby
identifiedsubfractionsinhamsterswerefurthervalidatedincured thistime.Successfulvaccinationofhumansandanimalsisoften
patients/endemiccontactsandanalogousresults,i.e.proliferation relatedtoantigeninducedDTHresponsesinvivoandT-cellstim-
5708 S.Kumarietal./Vaccine26(2008)5700–5711
Table2
ImmunostimulatoryproteinsidentifiedinpotentsubfractionF2.4,F2.5,F2.6andF2.7ofF2MALDI-TOF–MS/MS
SFsa Identifiedproteinsb Sp.c Acc.no.d kDa/pIe Pm/Ms/Sc%f FCg Remarksh
Cofactor-independentphosphoglyceratemutase Lmx 28400787 61/5.4 10/151/20 5 DT[72]
Trypanothionereductase Lmj 7677022 53/5.8 12/117/32 5 DT[73]
HypotheticalproteinL5769.02 Lmj 12311865 29/6.8 10/100/25 ??i ?j
NAD-dependentdeacetylaseSIR2homolog Lmj *SIR2LEIMA 43/5.64 16/75/14 4 DT[74]
Adenosylhomocysteinase Ld 1710837 48/5.7 17/98/21 5 DT[75]
F2.4 Proteinofunknownfunction Tc 32401138 38/5.22 15/114/31 ??i ?j
Fructose-1,6-BisphosphateAldolase Lmx 5834626 47/7.9 18/152/14 1–3 VC,DT[58,59,76]
Elongationfactor-2 Lmj 11244578 75/7.2 24/124/32 4 Th1[68]
Enolase Lmj 8388689 46/5.6 21/124/38 1–4 IGP[59,63,76]
Heatshock70-relatedprotein1precursor Lmj 50299857 69/5.5 19/115/32 1 Th1[65–67]
NAD-dependentdeacetylaseSIR2homolog Lmj *SIR2LEIMA 43/5.7 12/63/28 4 DT[74]
Adenosylhomocysteinase Ld 1710837 48/5.7 10/102/23 5 DT[75]
Proteinofunknownfunction Tc 32401138 38/5.22 15/121/36 ??i ?j
Enolase Lmj 8388689 46/6.6 16/167/27 1–4 IGP[59,63,76]
F2.5 Fructose-1,6-BisphosphateAldolase Lmx 5834626 47/7.5 16/155/38 1–3 VC,DT[58,59,76]
dnaK-typemolecularchaperonehsp70.4 Lmj 7441842 70/5.5 13/89/39 1 Th1[65–67]
DisulfideisomerasePDI Lmj 25990151 52/5.2 9/100/25 1 VF,DT,VC[70]
Elongationfactor-2 Lmj 11244578 73/7.2 15/114/29 4 Th1[68]
Hypotheticalprotein5769.02 Lmj 12311865 29/6.8 11/86/34 ??i ?j
Elongationfactor-2 Lmj 11244578 73/7.2 15/114/29 4 Th1[68]
Enolase Lmj 8388689 46/6.6 16/104/27 1–4 IGP[59,63,76]
Fructose-1,6-BisphosphateAldolase Lmx 5834626 41/7.0 16/156/32 1–3 VC,DT[58,59,76]
Proteinofunknownfunction Tc 32401138 38/5.22 15/121/36 ??i ?j
Heatshock70-relatedprotein1precursor Lmj 50299857 69/5.5 9/128/29 1 Th1[65–67]
F2.6 DisulfideisomerasePDI Lmj 25990151 55/5.2 9/100/25 1 VF,DT,VC[70]
Hypotheticalprotein,unlikely Tb 25992853 53/5.5 17/86/33 ??i ?j
p45 Lmj 6274526 44/6.6 15/156/37 5 T-cellst[68]
Calreticulin Lmj 5263289 33/4.7 16/87/25 1 VF,IGP[20,69]
Triosephosphateisomeraseglycosomal Tc *TPISTRYCR 76/6.6 15/142/38 1–3 Th1,VC[61,62]
Heatshockprotein-90 Ld 323030 53/5.6 24/128/34 1 Th1[65–67]
Calreticulin Lmj 5263289 33/4.7 8/74/21 1 VF,IGP[20,69]
Triosephosphateisomerase,glycosomal Tc *TPISTRYCR 76/6.6 21/212/31 1–3 Th1,VC[61,62]
Hypotheticalprotein Lmx 2131001 47/7.1 12/125/31 ??i ?j
HypotheticalproteinL2385.08 Lmj 12311835 84/5.4 14/121/37 ??i ?j
Heatshock70-related Lmj 50299857 69/5.5 21/137/32 1 Th1[65–67]
F2.7 Elongationfactor-2 Lmj 11244578 73/7.2 15/114/29 4 Th1[68]
DisulfideisomerasePDI Lmj 25990151 52/5.2 15/110/25 1 VF,DT,VC[70]
Enolase Lmj 8388689 46/6.6 19/127/39 1–4 IGP[59,63,76]
Fructose-1,6-BisphosphateAldolase Lmx 5834626 45/7.9 16/102/27 1–3 VC,DT[58,59,76]
p45 Lmj 6274526 41/6.6 12/126/46 5 T-cellst[68]
Hsp83protein Ldi 362545 81/5.1 21/141/25 1 Th1[65–67]
Theproteinspotsindicatedinthistablewereidentifiedusingpeptidemassfingerprinting.
a Subfractionno.
b Nameoftheprotein.
c Species:Lmx,Leishmaniamexicana;Lmj,Leishmaniamajor;Li,Leishmaniainfantum;Ld,Leishmaniadonovani;Ldf,Leishmaniadonovaniinfantum;Ldc,Leishmaniadonovani
chagasi;Tb,Trypanosomabrucei;Tbr,Trypanosomabruceirhodesiense;Tc,Trypanosomacruzi.
d AccessionnumbersaccordingtoNCBIand*Swiss-Protaccessionnumber.
e PredictedMrandpI.
f No.ofpeptidesmatched/MOWSEscore/sequencecovered%.
g Identifiedproteinsfellintothefollowingmajorsixfunctionalcategories;withsomeofthemfallingintotwoormoregroups:1.stressresponse;2.cytoskeletonandcell
membrane;3.energymetabolismandphosphorylation;4.cellcycleandproliferation;5.aminoacidmetabolism.
h Remarks:VC,vaccinecandidate;Th1,Th1stimulatory;T-cellst,T-cellstimulatoryproteins;VF,virulencefactor;DT,drugtargetmolecule;IGP,immunogenicprotein;
IDP,immunodiagnosticprotein.
i ?Notpreviouslydescribed.
j ??Unknowns/hypotheticalfunctionoftheproteinarenotknown.
ulation with antigen in vitro [4,50]. It has been further reported regardingtheup-regulationofinducibleNOsynthase(iNOS,NOS2)
that a major factor that is believed to contribute to healing in byTh1cellassociatedcytokinesandconfirmsthatNOmediated
leishmaniasis is the development of strong CMI responses like macrophageeffectormechanismiscriticalinthecontrolofparasite
DTH responses, T-cell responses and NO production [28,51–53]. replicationintheanimalmodel[28].
Notably, the hamsters vaccinated with P4-7 subfraction elicited ThepresenceofacomparableexistenceofTh1andTh2clones
strongestDTHreaction,LTTresponsesandNOproduction,among producingIL-12andIFN-(cid:2)aswellasIL-10obtainedfrompatients
alltheexperimentalgroupssuggestingagoodcorrelationbetween cured of VL encouraged us to assess whether the protective
CMIandresistancetoinfectioninthismodel.Inaddition,allthe responsewhichwasutmostelicitedbyP4-7vaccinationinham-
hamstersvaccinatedwithpotentsubfractionschallengedwithL. sterscanreflectthisfeatureofclinicalfindings[54–56].Following
donovani have a specific active T-cell response that was severely vaccination with P4-7 subfractions in the hamster transcripts of
impededininfectedunvaccinatedandBCGvaccinatedhamsters. iNOS, IL-12 and IFN-(cid:2) showed manifold increase and the syner-
The generation of NO in these cultures also support the view gismofIL-12withIFN-(cid:2)mighthaveanadditionalparamounteffect
S.Kumarietal./Vaccine26(2008)5700–5711 5709
onleishmanicidalactivitiesofL.donovani[30].Wefoundthatsig- Theoretically,linkingtheabovestatedantigenicproteinsmight
nificant iNOS transcript production in P4-7 vaccinated hamsters increase the number of epitopes available for inducing Th1-type
correlatedproportionallywithNOgenerationthatwasconsider- immune responses and protective immunity in a heterogeneous
ably higher with SLD stimulation. Possibly the cumulative effect human population with diverse major histocompatibility com-
of IFN-(cid:2) and IL-12 alongwith iNOS mediated the parasite killing plextypes.Thus,thisclearlyindicatethatwhileidentificationof
[30,51,57,58]. The levels of Th-2 cytokines IL-4, IL-10 and TGF-(cid:4) antigens recognized by T cell is an important step in defining a
mRNA, on the other hand, were observed to be down-regulated protective immunogens, empirical immunization studies in vac-
inthevaccinatedhamsters.ThestrongpresenceofIL-4,IL-10and cinemodelsarecrucialindefiningaleishmanialvaccine,therefore,
TGF-(cid:4)ininfectedhamstersarereportedtobethemajorimmuno- emphasizingthatasuccessfulsubunitvaccinemayrequiremultiple
suppressivecytokinesinexperimentalandhumanVL[44,58–62]. immunogenic/Th1immunostimulatoryproteinsthatarealsopro-
Apartfromdiminishedcellularresponses,VLisassociatedwith tectiveagainstVL.Extendedstudiessuchascloningandexpression
theproductionofhighlevelofantibody,observedpriortodetection ofthebestantigenictargets,asdeterminedbytheirimmunoprotec-
ofparasite-specificT-cellresponse[4].Unlikeinmice,whereinIL-4 tivepotential,togetherwiththeirspecificassociationanddefinite
andIL-12,IFN-(cid:2),thetwocytokinesthatdirectIgGclassswitching allocationarerequiredtocharacterizethesenewproteinsfurther.
ofIgG1andIgG2a,respectively,therearenosuchdistinctclassi-
ficationsofIgGinhamsters[30].ItisbelievedthathamsterIgG2
Acknowledgements
correspondstomouseIgG2a/IgG2bandhamsterIgG1corresponds
tomurineIgG1[30].Ithasbeenwell-establishedthattheincrease
We express our sincere gratitude to the Directors CDRI and
ofIgGandIgG1antibodiestitreisindicativeoftheL.donovaniload
CIMAP for their keen interest and for providing facilities for the
[30]. These antibodies were quiet low in P4-7 vaccinated group
experiments. Our grateful acknowledgement is due to Dr. Nikhil
which reflected the decreased parasite burden. In contrast, the
Kumar,forcriticallyreviewingthemanuscriptandforhisencour-
levelofIgG2significantlyincreasedonlyinP4-7vaccinatedanimals
agingsuggestions.WearealsothankfultoDrRajeevSingh,Scientist
furthersupporttheviewsthatprotectionagainstleishmaniasisis
and Mr. Malaya Sahoo, research fellow for their kind support.
inducedbyastrongT-cellresponseandthispatternwasalsoseen
Financial support to this work from DBT, New Delhi and for
inclinicalaswellasexperimentalVL[4,28,30,49,63–65].
SeniorResearchFellowshiptoMsSKandMr.MSfromCSIR,New
To examine the molecular basis of the immune responses
Delhi is gratefully acknowledged. This has CDRI communication
elicited during Leishmania infection, recent efforts have been
no7555.
focused on evaluating responses to defined parasite T-cell epi-
topesasvaccinetargetsusingproteomicsapproaches[33].Since
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