YP total extract; Group 2: 20

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

Humoral

and

cellular

immune

response

of

mice

challenged

with

Yersinia

pestis

antigenic

preparations

Elida

A.

Leal

_,

_Josimar

_D.

_Moreira

_,

_Fernanda

_F.

_Nunes

_,

_Larissa

_R.

_Souza

_,

Janaina

M.

Martins

_,

_Vicente

_P.C.

_Toledo

_,

_Alzira

_M.P.

_Almeida

_,

_Tania

_M.P.

_Guimarães

a_{InstitutoOctavioMagalhães,DivisãodeEpidemologiaeControledeDoenc¸as,Servic¸odeDoenc¸asBacterianaseFúngicas,Belo}

Horizonte,MG,Brazil

b_{UniversidadeFederaldeMinasGerais,FaculdadedeFarmácia,DepartamentodeAnálisesClínicaseToxicológicas,BeloHorizonte,MG,}

Brazil

c_{Fundac¸ãoOswaldoCruz,CentrodePesquisaAggeuMagalhães,DepartamentodeMicrobiologia,Recife,PE,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5April2017

Accepted26September2017

Availableonline12October2017

Keywords: Yersiniapestis

Antigens Mice

Immuneresponse

Vaccines

a

b

s

t

r

a

c

t

Objectives:Theplague,whichisaninfectiousdiseasecausedbyYersiniapestis,still threat-ensmanypopulationsinseveralcountries.Theworldwideincreaseinhumanplaguecases andthepotentialuseofthebacteriaasabiologicalweaponreinforcetheneedtostudythe immunitythatisinducedbypotentialvaccinecandidates.Todeterminethe immunogenic-ityofantigenicpreparationsbasedontheF1proteinandthetotalextractfromY.pestis,we assessedtheroleoftheseantigensininducinganimmuneresponse.

Methods:TheimmunogenicityofantigenicpreparationsbasedontheY.pestis(YP)total extractandtheY.pestisfraction1capsularantigenprotein(F1)wasdeterminedin

Swiss-Webstermiceimmunizedwith40␮gor20␮gforeachpreparation.Immunophenotyping

wasperformedbyflowcytometry.

Results:AnimalsimmunizedwiththeYPtotalextractdidnotelicitdetectableanti-F1 anti-bodies(Ab)inthehemaglutination/inhibition(HA/HI)test.Animalsimmunizedwith40␮g or20␮goftheF1proteinproducedanti-F1Abs,withtitresrangingfrom1/16to1/8132.The averageofCD3+_–CD4+_andCD3+_–CD8+_{Tcellsdidnotdiffersignificantlybetweenthegroups.}

NeitherYPtotalextractnorF1proteininducedasignificantexpressionofIFN-␥andIL-10in CD4+_{Tlymphocytes.Inaddition,F1failedtoinduceIFN-␥expressioninCD8}+_{Tcells,unlike}

theYPtotalextract.

Conclusion:The results showed thatF1 proteinis not animmunogenic T cell antigen, althoughtheYPtotalextract(40␮gdose)favouredCD8+Tcell-mediatedcellularimmunity. ©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](E.A.Leal). http://dx.doi.org/10.1016/j.bjid.2017.09.001

Introduction

Theplague,whichiscausedbyYersiniapestis,isessentially

arodent-flea-transmitteddiseasethataffectsmanandother

mammalianspecies.1_{DuetothepossibilityofusingY.pestis}

forbioterrorismpurposesandthehighmortalityrateofthe

disease,2_{thereisacrucialneedtostudytheimmunityinduced}

bypotentialvaccinecandidatesforfutureuseas

immunopro-phylaxis.Inrecentyears,efforthasfocusedondevelopinga

subunitvaccinebasedonvirulencefactorsofthisbacterium.3

Y.pestisF1capsularantigenprotein(Fraction1orF1)has

been evaluated in several immunization studies that used

experimentalanimals.4–6_{Furthermore,vaccinesthatare}

for-mulatedwithwholecellsmaycontributetotheinductionof

aneffectiveimmuneresponse.7_{Themechanismofprotection}

conferredbythesepreparationshasnotbeenfullyelucidated, butithasalreadybeenshownthataneffectivevaccineagainst

theplaguemustinducebothhumoralimmunityandTh1type

cellularresponse.8

Aimingtodeterminetheimmunogenicityoftheantigenic

preparationsbasedonthetotalextractfromY.pestisandthe F1protein,weassessedtheroleoftheseantigensininducing

theproductionofantibodies,determiningthephenotypeof

splenicT-lymphocytesandstimulatingtheproductionof

IFN-␥andIL-10bysubpopulationsofCD4+andCD8+Tcells.

Materials

and

methods

Animals

Female, 6–8-week-old, Swiss-Webster mice (20–24g) were

obtained from the Universidade Federal de Minas Gerais

(UFMG)facilities.Fouranimalspercageweremaintainedwith

temperaturesat21–24◦_{C,a12hlight/darkcycle,andfedwith} pelletizedfoodandwateradlibitum.Duetothepolicyof reduc-ingthenumberofanimalsusedinresearchprotocols,onlythe

minimumnumberofanimalspergroupwasutilized.

PreparationofwholecellextractandY.pestisF1antigen

Y.pestisstrainCYP0579fromtheculturecollection

Fiocruz-CYPwasreactivated byinoculation inbrainheart infusion

brothmedia(Difco, USA)and incubated overnightat37◦_C.

Thepresenceofthe genescaf1,lcr,pla, andirp2,whichare

prominentpathogenicitymarkers,9_{wasdeterminedbyM-PCR}

accordingtoLealandAlmeida.10_{Theculturewasinactivated}

bythe addition of2% formaldehyde (Sigma–Aldrich, USA),

incubatedovernightatroomtemperature,23–25◦_C,andplated onbloodagarbase(Difco,USA)toconfirmbacterialdeath.The

F1antigenwasextracted fromthe Y.pestisstrainA1122 as

previouslydescribed.11

TheY.pestisformaldehyde-killedsuspension(YP)was

frag-mentedbysonicationfor90s(2cyclesof30Hzand2cycles

of60Hzintercalatedwithanicebath).TheYP totalextract

andF1proteinweresubjectedtogammaradiation.The

pro-teinconcentrationofthepreparationswasdeterminedbythe

Lowrymethod12_{andtheproductsweresuspendedinPBSat}

pH7.2–7.4.

Immunization

FourgroupsoffourfemaleSwiss-Webstermicewere

immu-nized with40␮gor20␮goftheYP totalextractand theY.

pestisF1proteinsuspensionsinPBSplus25%(v/v)aluminium

hydroxideadjuvant,administeredintwodoseswitha21-day

interval.Eachanimalwasinjectedintramuscularlyinthe

pos-teriorthighwithatotalvolumeof0.1mL.Thecontrolgroup

receivedthesamevolumeofaluminiumhydroxideadjuvant

in PBS. After primary immunization, on day 42, the mice

receivedaboosterintravenousinjectionatthebaseofthetail

with4␮gor2␮gdoses ofthe antigenswithout adjuvant.13

Onday45afterprimaryimmunization,micewereeuthanized

byanaesthetic overdose.Bloodsamplesand spleensofthe

immunizedandcontrolmicewerecollected.

Hemaglutination/inhibitiontest(HA/HI)

Serawerecollectedfromimmunizedanimalsthroughoutthe

studyandassayedforthepresenceofanti-F1AbsbytheHA/HI

test.11 _{Thetest wasconsideredpositive whentheHA}

end-pointtitrewasdepressedbythreeormoredilutionsinthe

HItest.Atitreof1/16wasconsideredpositive.

Preparationofspleencellsuspensions

Singlecellsuspensionswerepreparedfromeach2/3spleenin

RPMI1640(Gibco,Germany).Eachspleenwasaseptically

col-lected,andthemononuclearcellswerefilteredthroughsterile

nylon.ThecellswerewashedinRPMI1640andspundownat

440×gfor10minat18◦C.Theerythrocyteswerelysedwithan

ammoniumchloridesolution,andthecellswerewashedthree

timesinRPMI1640andresuspendedintoRPMI1640

supple-mentedwith5%heat-inactivatedfoetalcalfserum(Cultilab,

SP,Brazil),penicillin(100UI/mL)andstreptomycin(50pg/mL;

Sigma–Aldrich,USA).

Spleencellcultureandflowcytometryanalysis

Mononuclearcellswereisolatedfromthespleensof

immu-nized and control mice by Ficoll-Hypaque (Sigma–Aldrich,

USA) density gradient centrifugation. After counting the

viable lymphocytes, suspensionswere preparedcontaining

1×106spleniccells/mLinRPMIsupplementedwith5%foetal

bovineserum,2mMglutamine,50UIpenicillin,and0.05mg

streptomycin (Sigma–Aldrich, USA). Cell suspensions were

usedforthephenotypicanalysisoflymphocytesandfor intra-cellularcytokineanalysis.

Cellsurfacemarkerstainingwasperformedbyaddingthe

previouslypreparedcellsuspension(0.1mL)toflowcytometry

tubescontainingcombinationsofthefollowing

fluorochrome-labelled Abs: CD3-FITC (145-2C11), CD4-PercpCy55 (RM4-5)

andCD8-APC(53–6.7)(BectonDickinson,USA).Sampleswere

vortexedgentlyandincubatedfor30minatroom

tempera-ture.Thefixingsolutioncontaining4%paraformaldehydewas

addedtothesamples,whichwererefrigerateduntilflow

cyto-metric analysis. Lymphocyteswere specificallyanalyzedby

samefluorochromewasused.Dataanalysiswasperformed usingtheFlowJosoftware.

Forintracellularcytokineanalysis,1×₁₀6_{spleencellsfrom}

each animalwere platedina 24-wellplateand stimulated

with10␮g/mLofYPtotalextractor Y.pestisF1antigenfor 72h.CultureswithoutaddedY.pestisproteinsservedas

con-trols.GolgiPlug(1␮g/mL–BectonDickinson,USA)wasadded

duringthelast4hofincubation.Cellsweresurfacestained

withanti-CD3-FITC (145-2C11),anti-CD4-PercpCy55 (RM4-5)

andanti-CD8-APC(53–6.7) Abs(BectonDickinson,USA)and

thenfixedwith4%paraformaldehydefor10min.After

perme-abilization,cellswerestainedwithanti-IFN-␥-PeCy7(XMG1.2) andanti-IL-10-PE(JES5-16E3)orisotypecontrols(Becton

Dick-inson, USA) and analyzed using flow cytometry (Forteza,

BectonDickinson,USA).

Statisticalanalyses

The difference of the data among groups was compared

by analysis of variance (ANOVA) using the R software14

andprobability valuesof<0.05 wereconsidered significant.

The number of animals per group (N) was determined

using the standard deviation, difference to be detected,

andsignificancelevel15,16_{andwasconfirmedbythewebsite}

http://www.lee.dante.br/esquisa/amostragem/amostra.html.17

Ethics

MicewerehandledfollowingtheGuidefortheCareandUseof

LaboratoryAnimalsguidelines.18_{Theexperimentalprotocols}

wereapprovedbythe EthicsCommitteeonAnimal

Experi-mentation(CETEA)ofUFMG(ProtocolNo.n◦_148/2014).

Results

Y.pestisanti-F1antibody(Ab)inanimalsimmunized withdifferentimmunizationprotocols

Tocharacterizethehumoralresponseinducedby

immuniza-tionwithdifferentdosesoftheYPtotalextractandtheY.pestis

F1protein,anti-F1AbtitresweredeterminedbytheHA/HItest

fromserumsamplesofimmunizedmicethatwerecollected

45days post-immunization.Anti-F1 Abswere notdetected

inthetwogroupsimmunizedwith40␮g(Group1)and20␮g

(Group2)ofYPtotalextractorinthe controlgroup,which

receivedaluminiumhydroxideadjuvantinPBS.Intheanimals

immunizedwiththeY.pestisF1antigen,theanti-F1Abtitres

inHA/HIrangedfrom1/16to≥1/8132inthegroupreceiving

40␮gF1(Group3)andfrom1/64to1/256inthegroupreceiving 20␮gF1(Group4).Itshouldbepointedoutthatthevolumeof serafromsomemiceofgroups2(1),4(2)andcontrol(1)was notenoughfortheHA/HItests(Fig.1).

PhenotypicanalysisofsplenicTcellsafterimmunization

Becausetheresponsetoimmunizationinvolvesactivationat

thecellularlevel,flowcytometricanalysiswasperformedto

investigatewhetherimmunizationresultedinchangesin

cel-lular activation markersor grosschanges ofcell countsof

0

Group3 Group4

Negativ e

Negativ e

Negativ e

Negativ e

Negativ e

Negativ e

Negativ e

1/64 1/256 1/2048 1/8192

1/128

1/6 Negativ e

Negativ e

Negativ e

Control Group2

Group1

Anti-F1 antibody titer

100 200 300 2000 4000 6000 8000 10000

Fig.1–Yersiniapestisanti-F1antibodyinmiceimmunized withdifferentimmunizationprotocols.Group1:40␮gofYP totalextract;Group2:20␮gofYPtotalextract;Group3: 40␮gofY.pestisF1antigen;Group4:20␮gofY.pestisF1 antigen;Control:aluminiumhydroxideadjuvant.n<4 (groups2,4andcontrol):thevolumeofserawas insufficientfortheHA/HItests.

micegivendifferentpreparationsofY.pestis.Therefore,within

the lymphocyte population, the changes inthe percentage

ofsplenicTcellswereevaluatedamongimmunizedgroups.

Nosignificantdifferenceswere observedinthepercentages

ofCD3+_–CD4+_andCD3+_–CD8+_{Tcellsamongthefour}

immu-nizedgroups(p>0.05).However,aclearpredominanceofCD4+

Tcellswasobservedinallgroups(Fig.2AandB).

DetectionofintracellularcytokinesIFN- andIL-10inT

cells

Toinvestigate theresponseofCD4+ _{and CD8}+ _{T cellsafter}

invitrostimulationwithYPtotalextractandY.pestisF1

anti-gen, spleen mononuclear cells from immunizedmice that

werecollected45dayspost-immunizationwereculturedand

assessedfortheproductionofIFN-␥andIL-10byflow

cytom-etry. A higher percentage of CD4+ _{T cells that expressed}

IFN-␥wasobservedamongthegroupthatreceived40␮gYP

total extract compared to the other groups (Fig. 3A). The

levels of CD4+ _{T cells that expressed IL-10 were higher in}

the groupsthatreceived40␮gand20␮gofYPtotalextract

(Fig.3B).However,nosignificantdifferenceswereobservedin

thepercentagesofCD4+_{TcellsthatproducedIFN-␥and}

IL-10cytokinesafterstimulationwithYPtotalextract(p>0.05). ThepercentageofCD4+_{TcellsthatproducedIFN-␥(Fig.3C)}

and IL-10 cytokines (Fig. 3D) was lower after stimulation

withtheY.pestisF1 antigenthanafterstimulationwithYP totalextract.Nosignificantdifferenceswereobservedinthe

percentages ofCD4+ _{T cells thatproduced IFN-␥and IL-10}

cytokines among the groups after stimulation with the Y.

pestis F1 antigen(p>0.05). The percentage ofCD8+ _{T cells}

that producedIFN-␥afterstimulationwithYP totalextract

was significantly higher in the group that received 40␮g

YP total extract (Fig. 3E) compared with the other groups

(p<0.05).Nosignificantdifferenceswereobservedinthe per-centageofCD8+_{TcellsthatexpressedIFN-␥(Fig.3F)among}

the four groups after stimulation withY. pestis F1 antigen

30

25

20

15

10

5 85

80

75

70

65

60

5 Control

Control

Group 4

Group 4

Group 3

Group 3

Group 2

Group 2

Group 1

Group 1

4 3 2 1 5

4 3 2 1 55

A

B

Fig.2–PhenotypicanalysisofsplenicTcellsafterimmunization.A:CD3+_–CD4+_{subpopulations.B:CD3}+_–CD8+

subpopulations.Group1:40␮gofYPtotalextract;Group2:20␮gofYPtotalextract;Group3:40␮gofY.pestisF1antigen; Group4:20␮gofY.pestisF1antigen;Control:aluminiumhydroxideadjuvant.

Discussion

Thepresentstudyaimedtoinvestigatetheimmunogenicity

ofthe antigenicpreparationsbased onthe Y.pestisF1

pro-teinand theYP totalextractininducingtheproductionof

anti-F1AbsandindeterminingthephenotypeofsplenicT

lymphocytesandthestimulationoftheproductionofIFN-␥

andIL-10amongthesubpopulationsofCD4+_andCD8+_Tcells

inmice.Theimmunizationprotocol utilized inouranimal

experimentswasbasedonapreviouslyestablishedmodel16

thatevaluatedtheprotectiveefficacyofY.pestisF1proteinand

Vsubunitvaccinesinmiceagainstsubcutaneouschallenges

ofthevirulentY.pestisstrain.Ourresultsshowedthatanimals

immunizedwiththeF1antigenexhibitedastronghumoral

response with high titres ofanti-F1 Ab in the HA/HI test.

Furthermore,itwasobservedthattheanimalsreceivingthe

lowest(20␮g)F1doseshowedtitres>1:16,whichwasthe cut-offpointintheHA/HItest.Thesefindingswereinaccordance

withanotherstudy16_{thatdemonstratedthatadoseof20␮g}

oftheF1antigeninducedsignificantlyhighIgGtitresinmice. TheY.pestisF1andVsubunitvaccineshavebeenthefocusof severalstudies.AlthoughthevalueoftheY.pestisVantigenis

recognizedinitsprotectionagainstnon-encapsulatedstrains

(deficientinF1production),thesignificantcontributionofthe

F1proteinintheinductionofearlyimmuneresponsesisalso

important.Inastudytocharacterizetheimmuneresponse

ofmiceafterimmunizationwiththeF1protein,quantitative

ELISA analysisdemonstrated thatthe levels ofanti-F1 IgM

antibodieswere detectableinserum ofanimalsasearly as

day1post-immunizationandthatanti-F1IgGantibodieswere

detectedafterthreedays.19_{Theabilityofanti-F1Absto}

pro-videeffectiveprotectionagainstthebubonicplaguehasbeen

demonstratedinseveralstudies.19,20 _{Totalprotection(100%)} againstachallengewithavirulentY.pestisstrainwasobserved

inmiceimmunized withthe F1 antigen.19 _{In another}

ani-malmodel,80%survivalratewasdemonstratedwithactive

immunizationwiththerecombinantF1andVantigen.20_The

immunogenicityofrecombinantVandF1proteinsmaybeat

maximumbecausetheirconformationsaresimilartothose

ofnative proteins.21_{Therefore, thisfinding canexplainthe}

hightitresofantibodies observedinourstudy becausethe

antigensusedintheimmunizationexperimentwereobtained

fromY.pestisculturesandnotbyusingrecombinantDNA

tech-niques.Theuseofthealuminiumhydroxideadjuvantcould

also explainthe resultsobtainedforthe humoral immune

response.Furthermore,otherstudieshavereportedthat

sub-unit vaccines comprising F1 and V antigensinaluminium

hydroxideinducedapredominantIgG1responseinbothmice

and humans.16,22 _{Ourresultsshowedthattheanti-F1titres}

werevariableamongindividualsfromthesamegroup,which

can be attributedto the mouse lineage used inour study.

WechosetheexperimentalmodelwithSwiss-Webstermice,

whichisageneticallyheterogeneousoutbredlineage23 _that

respondedindifferentwaystothesamestimulus.The

pur-poseofusingthatmouselinewastoadoptanexperimental

modelthatcoulddemonstratetherealefficacyofourputative

vaccinebecausetheresponsetovaccinationsmayvaryamong

individualsinapopulation.

Due to the policy of reducing the number of animals

inresearchprotocols,onlyexperimentswiththeminimum

number ofanimals pergroup were approvedbythe ethics

committee.Thiswouldbethebestalternative,althoughthe

possibility of loss throughout the experiments, e.g.,

insuf-ficient volume of sera, loss of mononuclear cells when

preparingthe spleenforcell culture,ormicrobial

contami-nationofcultivatedspleencellculture,wasdiscussed.Infact,

thelosswhenworkingwiththeexactnumberofanimalsper

groupdeterminedan“N”lowerthantheminimumcalculated.

Itmustbenotedthattheabsenceofthehumoralimmune

response to both doses ofthe YP total extract could

indi-catethattheimmunizationschemedidnotinducedetectable

levels ofanti-F1 antibodies. However, it could be that the

YP total extract did not containa sufficient concentration

of F1 protein to induce the humoral immune response in

mice.

Therole ofantibodies inprotectionagainstY.pestishas been verifiedbyseveralstudies.24,25 _{However,it seemsthat}

thehumoralimmuneresponseinducedbysubunitvaccines

alonemaynotbesufficientforcompleteprotectionagainstthe

plague.Itwasobservedthattherewas80%protectionagainst

achallengewithY.pestisinmiceimmunizedwiththeF1andV

antigens;however,20%ofthemdidnotsurvive,eventhough

F

E

D

C

B

A

YP total extract YP total extract

Y. pestis F1 antigen

Y. pestis F1 antigen

Y. pestis F1 antigen

YP total extract

Control

Control

Control Control

Control

Control

Group 2

Group 2

Group 2

Group 2

Group 2

Group 2

Group 4

Group 4

Group 4 Group 4

Group 4

Group 4 Group 3

Group 3

Group 3

Group 3

Group 3 Group 3 Group 1

Group 1

Group 1

Group 1

Group 1

Group 1 0.0

0.0

0.0 0.0

0.0

0.0 0.5

0.5

0.5

0.5

0.5

0.5 1.0

1.0

1.0

1.0

1.0

1.0 1.5

1.5

1.5

% CD4

+

Tcell producing IFN

-γ

% CD4

+

Tcell producing IFN

-γ

% CD8

+

Tcell producing IFN

-γ

% CD8

+

Tcell producing IFN

-γ

% CD4

+

T-cell producing IL-10

% CD4

+

T-cell producing IL-10 1.5

1.5

1.5

Fig.3–DetectionofintracellularcytokinesIFN-␥andIL-10inTcells.(A)CD4+TcellsproducingIFN-␥and(B)IL-10cytokines afterstimulationwithYPtotalextract;(C)CD4+_{TcellsproducingIFN-␥and(D)IL-10cytokinesafterstimulationwithY.}

pestisF1antigen;(E)CD8+_{TcellsproducingIFN-␥afterstimulationwithYPtotalextractand(F)afterstimulationwithY.}

pestisF1antigen.Control:aluminiumhydroxideadjuvant;Group1:40␮gofYPtotalextract;Group2:20␮gofYPtotal extract;Group3:40␮gofY.pestisF1antigen;Group4:20␮gofY.pestisF1antigen.

protectionagainstY.pestisisnotexclusivelymediatedby

anti-bodiesanddependsontheinvolvementofTcellsandtheir

cytokinesecretion.20

TodeterminethechangesinthepercentageofTcellsthat

respondtoantigenic stimulus,weperformed

immunophe-notypic characterization and quantified the mononuclear

spleniccells.Ourfindingsindicatedaclearpredominanceof

CD4+_{Tcellsinallimmunizedgroupsstudied.Anotherstudy}

alsoreportedahigherpercentageofCD4+_{TcellsthanCD8}+_T

cellsinperipheralbloodfromSwissmice.However,our

find-ingsshowednumbersofCD4+ _{Tcellsthatwerehigherthan}

thosepreviouslyreported,24_{whichcanbejustifiedbythefact}

thatourexperimentswere performedinspleenTcells.No

significantdifferenceswereobservedinthepercentageofT

cellsamongmicefromdifferentgroups,whichsuggestedthat

therewasnoTcellproliferationinresponsetoantigenic stim-ulation.

Intracellularcytokineanalysisshowedthatthehigherdose ofYPtotalextract(40␮g)inducedhigherexpressionofIFN-␥ inCD4+_{Tcells(group1).Althoughnosignificantdifferences}

wereobserved,thisfindingmayindicatethatTcellactivation

ishigherinanimalsimmunizedwithahigherdose of

bac-terialtotalextract.Therefore,increasingthedoseofantigen

candidatesforthevaccineoradministeringadditionaldoses

mayrepresentanalternativetoimprovethecellularimmune

responsetoY.pestis.

Our study demonstrated that YP total extract induced

differencewasnotsignificant.AhigherpercentageofCD4+

Tcells thatexpressedIL-10ingroupsimmunizedwiththe

YPtotalextract(40␮gand20␮g)wasobserved.Theseresults

maysuggest aTh2response,which couldbeconfirmed by

determiningthepresenceofothercytokines,suchasIL-4,IL-5

andIL-6.GiventheimportanceofTh1andTh2inprotecting

againstY.pestisandthepossibleuseofappropriateadjuvants todirecttheadaptiveimmuneresponse,itishighlynecessary

tofindnewco-stimulatorymoleculesthatcaninduceamixed

responsebasedoncytokinesandantibodyproduction,which

wouldrepresentanimportantinnovationinvaccineresearch

fieldfortheplague.

Nosignificantdifferenceswereobservedinthepercentage

ofTcellactivationinresponsetotheF1antigen.

Addition-ally,ourfindings revealed thattheTcell responseinduced

bytheF1proteinwaslowerthan thatofthegroups

immu-nizedwiththeYPtotalextract.Thisfindingisconsistentwith apreviousstudy26_{thatdemonstratedthatF1antigenisunable}

toinduceastrongcellularresponseinanimalsimmunized

withthe liveattenuated EV76 vaccine, suggestingthat the

F1proteinmay notrepresentadominantantigeninTcell

activation.Our results alsoshowed that the percentage of

CD8+ _{Tcells that expressedIFN-␥washigher inthegroup}

thatreceived40␮gYPtotalextractthanintheothergroups, whichmaysuggesttheroleofYPtotalextractinelicitingaTh1 responsemediatedbythiscellularsubpopulation.Thisfinding

ishighlyimportantbecausetheproductionofIFN-␥and

TNF-␣byCD8+Tcellsplaysacrucialroleinactivatingmacrophages

andneutrophils,which,inturn,exhibitanincreased

produc-tionofreactiveoxygenintermediatesthatactinthecontrol

ofintracellularbacteria.27 _{ItwasdemonstratedthatY.pestis} cannotreplicateinmacrophagesofmiceactivatedbyIFN-␥.28

MicewhoseTcells couldnotproduceIFN-␥andTNF-␣ did

notsurvivealethalpulmonaryY.pestisinfection.29 _Anti-F1

andanti-VAbsprotectmiceagainstexperimentalpneumonic

plague,butthisprotectionishighlydependentonIFN-␥and

TNF-␣production.Therefore,animalsthatcanproduceand

respondeffectively to cytokinesseem to protectagainst Y.

pestisinfections.30_{Ourintracellularcytokinesanalysis}

exhib-itedadifferentiatedresponseofanimalsfromgroup1,which

received40␮gYPtotalextract.Thesefindingsmaysuggestthe

activationofCD8+ _{TcellsbyY.pestisimmunodominant}

epi-topes.Toconfirmthishypothesis,furtherstudiesareneeded

toidentifytheproteinsintheYPtotalextractandtheir

con-tributionstoinducingthecellularimmuneresponse.

Inbrief,weshowedthattheimmunizationregimenbased

ontheF1antigeninducedastronghumoralimmuneresponse,

whereas 40␮gand 20␮gofYP totalextractdidnot induce

anti-F1Ab productioninmice.Furthermore,immunization

withtheYPtotalextractandtheY.pestisF1antigenneither

promotedCD4+_orCD8+_{Tcellproliferationnorinducedthe}

expressionofIFN-␥andIL-10byCD4+_{Tcells;however,itdid}

stimulatetheproductionofIFN-␥byCD8+_{Tcells.Therefore,}

wecansuggestthatY.pestisF1proteinisnotanimmunogenic antigentoTcells.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsthankFAPEMIG,UFMGandFUNEDfortechnical

andfinancialsupports.

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