Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection

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ContentslistsavailableatScienceDirect

Vaccine

jo u rn a l h o m e pa ge : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Nasal

vaccination

with

attenuated

Salmonella

expressing

VapA:

TLR2

activation

is

not

essential

for

protection

against

R. equi

infection

Silvia

Almeida

Cardoso

a,b

_,

_Aline

_Ferreira

_Oliveira

c

_,

_Luciana

_Pereira

_Ruas

a,b

_,

Marcel

Montels

Trevisani

a

_,

_Leandro

_Licursi

_De

_Oliveira

d

_,

_Ebert

_Seixas

_Hanna

a,b

_,

Maria

Cristina

Roque-Barreira

a

_,

_Sandro

_Gomes

_Soares

a,b,∗

a_Departamento_de_Biologia_Celular_e_Molecular_e_Bioagentes_{Patogênicos,}_Faculdade_de_Medicina_de_Ribeirão_Preto,_USP,_14049-900,_Brazil b_Invent_{Biotecnologia,}_14040-900,_Brazil

c_Departamento_de_{Microbiologia}_e_Imunologia,_Instituto_de_Biologia,_Universidade_Estadual_de_Campinas_(UNICAMP),_Campinas_13083-862,_SP,_Brazil d_Departamento_de_Biologia_Geral,_Universidade_Federal_de_Vic¸_osa_(UFV),_Vic¸_osa_36570-000,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6May2013

Receivedinrevisedform15July2013 Accepted25July2013

Available online 7 August 2013 Keywords: Rhodococcusequi Vectoredvaccine VapA AttenuatedSalmonella Toll-likereceptors

a

b

s

t

r

a

c

t

VirulentstrainsofRhodococcusequihavealargeplasmidof80–90kb,whichencodesseveral virulence-associated proteins (Vap), including VapA, alipoprotein highly associated withdisease. We have previouslydemonstratedthatoralimmunisationwithattenuatedSalmonellaentericaTyphimuriumstrain expressingtheantigenVapA(STMVapA+)inducesspecificandlong-termhumoralandcellular immu-nityagainstR.equi.ItwasshownthatVapAactivatesToll-likereceptor2(TLR2)onmacrophagesby establishinganinteractionthatultimatelyfavoursimmunityagainstR.equiinfection.Thepurposeof thisstudywastoevaluatetheimmuneresponsetriggeredbynasalimmunisationwithSTMVapA+and todeterminewhetherTLR2supportsthevaccineeffect.Wedevelopedanoptimisedprotocolforasingle nasalimmunisationthatconferredprotectionagainstR.equiinfectioninmice,whichwasmanifested byefficientR.equiclearanceinchallengedanimals.NasalvaccinationwithSTMVapA+hasalsoinduced protectioninTlr2−/−miceandmicewithnon-functionalTLR4.Moreover,spleencellsofvaccinatedmice augmentedT-betexpression,aswellastheproductionofIL-12,IFN-␥,nitricoxideandhydrogen perox-ide.Notably,thepopulationofCD4+_T_cells_with_memory_phenotype_{significantly}_increased_in_the_spleens

ofvaccinatedmicechallenged1or5monthsafterimmunisation.Intheseanimals,thespleenbacterial burdenwasalsoreduced.WhensimilarexperimentalprocedureswereperformedinTLR2knockout mice,anincreaseinCD4+_T_cells_with_memory_phenotype_was_not_observed._{Consequently,}_we_conclude

thatnasalvaccinationwithattenuatedSalmonellaexpressingtheR.equivirulencefactorVapAconfers long-lastingprotectionagainstexperimentalrhodoccocosisandthatTLR2engagementwasnotcrucial toinducethisprotectionbutmayberequiredforalong-termimmuneresponse.

1. Introduction

Rhodococcusequiisasoilorganismdistributedworldwidethat infectsfoalsaged1–6monthsandcausesseveregranulomatous pneumonia[1].AsR.equisurvivesandreproducesinsidehostcells, pneumoniacausedbythispathogenmustbetreatedwithlipophilic drugsoveraprolongedperiodoftime[2].Currently,novaccines areavailable,althoughseveralimmunisationstrategieshavebeen testedtopreventrhodococcosis[3].

∗ Correspondingauthorat:InventBiotecnologia,IncubadoraSupera,USP,Av. Bandeirantes,3900,14040-900RibeirãoPreto,SP,Brazil.Tel.:+551681370617.

E-mailaddresses:soares@inventbiotech.com.br,soares@fmrp.usp.br

(S.G.Soares).

As R. equi harbours an 80–90-kb plasmid, proteins (Vaps) encodedbythisplasmidareconsideredgoodcandidatesfor vacci-nalantigens.VapAisthemostinvestigatedimmunogenencoded by this pathogenicity island and hasbeen reported toaccount for R. equi virulence[4,5] and elicit speciﬁc immune responses

[6,7]. In this context, our group has previously demonstrated that oral immunisation with live attenuated Salmonella enter-icaTyphimuriumexpressingVapA conferslong-termprotection againstR.equi.Protectionisduetotheinductionofstrongmucosal, humoralandcell-mediatedTh1-immunity,whichisappropriately regulated,asveriﬁedbytheproﬁleofcytokinesexpressedinthe organs of vaccinated miceafter bacterialchallenge [8,9].These promising resultshavemotivated ustoadvancestudies onthe vaccinalconceptionofVapA.

StudiesontheinnateimmuneresponsetoR.equihaveshown that infected macrophages with viable bacteria undergo NFkB

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S.A.Cardosoetal./Vaccine31 (2013) 4528–4535 4529

translocationandproduceavarietyofpro-inﬂammatory media-tors,includingTNF,IL-12,andnitricoxide(NO).Thisresponsewas attributedtoa pathwaythatis stronglydependentonthe acti-vationofToll-likereceptor2(TLR2)butnotofTLR4.Aspuriﬁed VapAinducesresponsesthataresimilartothosetriggeredbythe wholebacteria,TLR2activationwasattributedtoitsinteraction withVapA.Theseobservationspromptedustoinvestigatewhether TLR2accountsforprotectionconferredbyvaccinationwithVapA.

Inthepresentstudy,westandardisedanewrouteand regi-menofimmunisationwiththeVapAantigeninthecontextofa Salmonella-vectoredvaccineandinvestigatedtherole thatTLR2 playsinthevaccineeffect.Wefoundthatnasalvaccinationofmice withasingledoseofVapA-expressingSalmonellaconfers protec-tionagainstR.equiinfectionviaamechanismthatdoesnotdepend onTLR2activation.Moreover,thevaccinationprocedure stimu-latesmemoryTCD4+_cells_and_promotes_speciﬁc_immune_response

recall.

2. Materialsandmethods 2.1. Experimentalanimals

Groupsof femalemice, 6–8weeksof age,includingBALB/c, C3H/HeJ,C3H/HePAS,Tlr2−/− andwildtype (WT)C57BL/6mice (ﬁvemicepergroup),werehousedunderspeciﬁcpathogen-free conditionsintheAnimalResearchFacilitiesoftheMedicalSchool ofRibeirãoPreto-USP.Thestudieswereconductedasrequiredby theEthicsCommitteeonAnimalResearchoftheUniversityofSão Paulo.

2.2. Bacterialstrains

TheattenuatedS.entericaTyphimurium␹3987strains,carrying eithertheVapAantigen(STMVapA+)ortheemptyvector(control VapA−),weregrownandpreparedfortheimmunisationof ani-malsasdescribedbyOliveiraetal.[9].ThevirulentstrainofR.equi (ATCC33701)wasgrownandthetitreofthechallengeinoculum wascalculatedasdescribedbyOliveiraetal.[8].

2.3. Immunisationandchallengeprotocol

Groupsofanimalswereimmunisedwithasingledoseortwo dosesofattenuatedSalmonellaharbouringtheVapAantigenvia intranasalroute(1×109 _CFU,₅_{␮L/animal).}_As_a_control,_groups

wereinoculatedwithattenuatedSalmonellacarryinganempty vec-toraccordingtothesameschedule.

ThechallengeswereconductedusinganATCCvirulent,R.equi strain3370130daysaftertheﬁrstimmunisation. IntheBalb/c recallexperiments,theanimalswerechallenged5monthsafter immunisation.Incontrast,intheC57Bl/6recallexperiments,WT andTlr2−/−animalswerechallenged120daysafterimmunisation. Organswerecollected5daysafterchallengeforR.equiclearance evaluationandotheranalysesasdescribedbelow.

2.4. Estimationofbacterialclearanceintheorgansofinfected mice

QuantificationofviableR.equirecoveredfromthespleenand liverofchallengedmicewasperformedonday5post-infectionas reportedelsewhere[8].Briefly,30daysafterthefirst immunisa-tionprocedurewithSTMVapA+,micewereinfectedwith4×106

CFUs(ColonyForming Units)ofvirulent R.equi viaintravenous route.Fivedaysafterthechallenge,spleenandliverwerecollected andasepticallyhomogenised.TodeterminethenumberofCFUs,

aliquotsof100␮LofthehomogenateswereseriallydilutedinPBS, platedontoBHIagarinduplicate,andincubatedat37◦Cfor36h.

2.5. Detectionofcytokinesinspleenhomogenates

Spleen homogenatesampleswereanalysed viaELISAfor IL-12p40,INF-␥,andTNF-␣usingcommerciallyavailablekits(OptEIA set;Pharmingen,SanDiego,CA,USA).Theassayswereperformed accordingtothemanufacturer’srecommendations.

2.6. RealtimequantitativePCRanalysis

TotalcellularRNAwasextractedfromspleencellsamplesusing TRIzolReagent(InvitrogenLifeTechnologies,Carlsbad,CA,USA) accordingtothemanufacturer’srecommendations.RealtimePCR wasperformedaspreviouslydescribedbyOliveiraetal.[9]using primersforGATA-3,T-bet,TLR2,and␤-actin.

2.7. Flowcytometryanalysis

Stainingforﬂowcytometrywasperformedonspleencells iso-latedfromimmunisedand controlmice30days post-infection. Afterharvestingthespleencells,1×106 _cells_were_washed_with

ice-coldPBSandincubatedfor30minat4◦Cwith0.5␮gof anti-CD16/CD32mAb(Fcblock,clone2.4G2,BDPharmingen,SanDiego, CA).Next,thecellswereincubatedforover30minuteswith5_␮gof phycoerythrin-conjugatedanti-CD62L,ﬂuorescein isothiocyanate-conjugatedanti-CD44,anti-TLR2,andPECy5-conjugatedanti-CD3 (BDPharmingen,SanDiego,CA).Washingstepswereperformed withPBS0.5%BSAandthecellswerethensubmittedfor analy-sisonaGuavaﬂowcytometer(Millipore,Billerica,MA,USA)using GuavaCytoSoftversion4.2.1(Millipore,Billerica,MA,USA). 2.8. Statisticalanalysis

Statistical analysis (one-way analysis of variance) was per-formedin conjunctionwiththeTukeymultiplecomparisontest usingGraphPadPrismversion5.00(GraphPadSoftware,SanDiego, CA,USA).TheresultsarepresentedasthemeanandSE.Differences betweengroupswereconsideredsigniﬁcantatthelevelofp<0.05. 3. Results

3.1. NasalimmunisationwithSTMVapA+protectsmiceagainst R.equiinfection

Ourpreviousstudieshavedemonstratedthatoral immunisa-tionofBalb/cmicewithliveattenuatedS.entericaTyphimurium (STM)expressingVapAantigen(STMVapA+)isaneffectivestrategy toinduceprotectionagainstR.equiinfection[8,9].Inthepresent study,weperformednasalimmunisationofBalb/cmicewitha sin-gledoseortwodosesoftheSTMVapA+.Bothproceduresresulted inasigniﬁcantreductionintheR.equiburdeninthespleenandliver ofmicechallenged30daysaftertheﬁrstimmunisationcompared withtheorgansofanimalsinjectedwithcontrolVapA−(negative control)(Fig.1AandB).Becausebothimmunisationregimens pro-motedcomparableR.equi clearance,theprotocolusingasingle nasalinoculationofSTMVapA+waselectedforsubsequent exper-iments.

Tooptimisethequantityof thevaccineinoculum,groups of Balb/cmicewereimmunisedwithdifferentdosesofSTMVapA+ (102_–109₎_and _challenged_with_R._equi ₃₀_days _after

immunisa-tion.AsigniﬁcantreductioninsplenicR.equiburdenwasobserved whentheinoculumoftheVapA+vaccinestrainwasatleast107

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Fig.1. NasalimmunisationwithSTMVapA+reducesR.equiburdenininfectedmice.Balb/cmicewereimmunisedwithasingledoseortwodosesofattenuatedSalmonella entericaTyphimuriumcarryingVapAantigen(STMVapA+).Allanimalswereeuthanisedonday5afterinfection,andR.equiburdenwasevaluatedinthespleen(A)and liver(B).PanelCshowstherecoveringofR.equifromthespleenoftheanimalsthatwereimmunisedwithasingledoseofvariousvaccineinoculum.AttenuatedSalmonella carryingemptyvectorwasusedasanegativecontrol(Control).Datarepresentthemean±SE,n=5animals.#_p_<_0.05.

3.2. NasalimmunisationwithSTMVapA+inducesTh1immunity Toevaluatewhethertheprotectionconferredwiththenovel vaccinationprocedurewasassociatedwiththedevelopmentofTh1 immunityaspreviouslyveriﬁedfororalvaccination,weassessed thelevelofsomemediatorsinthespleensofmiceimmunisedwith theSTMVapA+strainandchallengedtheanimalswithR.equi.IL-12 (Fig.2A),nitricoxide(Fig.2B)andhydrogenperoxidelevels(Fig.2C) weresigniﬁcantlyhigherinthespleentissueofVapA+-immunised mice,whilethelevelsofTNF-␣remainedlower(Fig.2D).

Tobettercharacterisethe immune responseinduced bythe vaccinationprocedure, wedetermined themRNAlevelsforthe transcriptionfactorsGATA-3andT-bet30daysafter immunisa-tion.Comparedwiththenegativecontrols,i.e.,miceinjectedwith controlVapA−orPBS,miceimmunisedwithSTMVapA+displayed higherlevelsofT-betmRNA(Fig.2E)andlowerlevelsofGATA-3 mRNA(Fig.2F).Similaranalyseswereperformed5daysafter chal-lenging,andnodifferenceinthelevelsofT-betmRNAwasobserved amongallgroups(Fig.2E).GATA-3mRNAlevelsremainedlowerin vaccinated/challengedanimals(Fig.2F).

3.3. NasalimmunisationwithSTMVapA+inducesmemoryTcells andimmuneresponserecall

ToevaluatetheinductionofTcellswithmemoryphenotype vianasalvaccinationwithSTMVapA+,wehaveassessedthe fre-quencyofcellsco-expressingCD3,CD62LandCD44(high)markers, whichcharacteriseTcellswithmemoryphenotype,30daysafter immunisation.Thispopulation,accordingtotheﬂowcytometric analysis,waslargerinthespleensofvaccinatedmicecompared withthecontrolgroups(Fig.3A).Wealsoinvestigatedwhetherthe

expansionofthepopulationofcellswasassociatedwithimmune response recall. Accordingly, Balb/c mice were immunised and challenged5monthslater.Next,spleenandliverwerecollected foranalysis5daysafterchallenge.Ourresultsdemonstratea sig-niﬁcantdecreaseinR.equiburdeninthegroupimmunisedwith STMVapA+(Fig.3BandC).

3.4. TLR-2expressionisupregulatedafterimmunisationand downregulatedafterchallenge

It is wellestablishedthat VapA canactivate Toll-like recep-tor2;thisinteractionisconsideredamajormechanisminvolved intriggeringspeciﬁcimmuneresponsestoR.equi.Therefore,the expressionlevelsofTLR2inthespleen cellsofmicevaccinated withSTMVapA+wereevaluatedviarealtimePCR(Fig.4A)and ﬂowcytometry(Fig.4B),whichwascompared withthe expres-sionlevelsinthecellsofmiceinjectedwiththecontrolsVapA− orPBS.Thirtydaysafterimmunisation,micespleencellsdisplayed anincreaseinTLR2expressionlevels,asdemonstratedbyelevated mRNA(Fig.4A)andproteinlevels(Fig.4B).Fivedaysafterchallenge withR.equi,murinespleencellsdisplayedreducedTLR2expression levelssimilartothatdetectedinthenegativecontrolgroup. 3.5. ProtectionagainstR.equiconferredbySTMVapA+isnot dependentonTLR2

WefurtherinvestigatedtherolethatTLR2playsintheVapA+ vaccine-inducedprotectionagainstR.equiinfection.Accordingly, groupsofTLR2knockoutmice(C57Bl/6Tlr2−/−)orwildtypemice (WT)weretreatedwiththestandardvaccinationand challenge protocols. Asexpected, among all WT vaccinated mice, R. equi

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Fig.2. NasalimmunisationwithSTMVapA+inducesTh1-immunity.Balb/cmicewereimmunisedwithasingledoseofSTMVapA+andthelevelsofIL-12(A),nitricoxide (B),hydrogenperoxide(C)andTNF-␣(D)inthespleenwereevaluated.TherelativemRNAexpressionlevelsofT-bet(E)andGATA-3(F)werealsoanalysed.Attenuated Salmonellacarryingemptyvectorwasusedasnegativecontrol.Datarepresentthemean±SE,n=5animals.#_p_<_0.05.

burdeninthespleen(Fig.5A)andliver(Fig.5B)waslessthanthat detectedintheorgansofnegativecontrolmiceinjectedwiththe controlsVapA−orPBS.Nonetheless,similarresultswereobtained withtheTlr2−/−group,therebyindicatingthatTLR2isnotessential fortheprotectionconferredbyourVapA+_method_of_vaccination

usingattenuatedSalmonella.Thisindicationwasreinforcedbythe factthatsimilarlevelsofIL-12(Fig.5C),IFN-␥(Fig.5D)andTNF␣ (Fig.5E)weredetectedinthespleentissueoftheTlr2−/−andWT animals.Additionally,theseresultsshowthatSTMVapA+ vaccina-tionofC57BL/6andBalb/cmiceisabletoconferprotectionagainst R.equiinfectionandinducesTh1immuneresponseasdenotedby increasedproductionofIL-12andIFN-␥.

Similarimmunisationandchallengeswereperformedinmice withnon-functionalTLR4(C3H/HeJ).Intheseanimals,R.equi clear-ance was comparable to that observed in the spleen (Fig. 5F) andliver(Fig.5G)ofvaccinatedmiceexpressingfunctionalTLR4 (C3H/HePAS).Thisresultindicatesthattheprotectionconferredby vaccinationwiththeSTMVapA+strainisalsoindependentofTLR4.

3.6. HighfrequencyofmemoryphenotypeCD4Tcellsis maintainedintheabsenceofTLR2

TounderstandthefunctionofTLR2inthenasal immunisation-mediated response, we investigated whethertheemergence of memoryTcellsinducedbyvaccinationcouldbeaffectedbythe absenceofTLR2.ThefrequencyofCD4+_or_CD8+_T_cells

express-ingCD44high_/CD62L_was_measured_in_the_spleen_of_WT_and_Tlr2−/−

mice30daysafterimmunisation.AsshowninFig.6A,vaccination ledtoanincreasedcellpopulationwithaCD4+_CD62L+_CD44+high

phenotype.Notably,thisincreasewassimilarforWTandTlr2−/− mice.Incontrast,thepopulationofCD8+_CD62L+_CD44+high_cells_was

elevatedonlyinthespleensofvaccinatedWTmice,thereby indi-catingthatvaccinationhadnoeffectonthefrequencyofthesecells inTlr2−/−animals(Fig.6B).

Finally,weassessedthefrequencyofTcellswithmemory phe-notypeinchallengedanimals(120dayspost-immunisation).Inthis particular case, the CD4+_CD62L+_CD44+high _population_remained

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Fig.3. NasalimmunisationwithSTMVapA+increasesthesplenicpopulationofTcellswithmemoryphenotypeandtherecallofimmuneresponse.VaccinatedBalb/cmice withSTMVapA+werechallengedwithR.equi30daysafterimmunisation,andthefrequencyofCD3+_/CD62L+_/CD44high_cells_(A)_was_analysed._A_second_group_of_Balb/c_mice

waschallenged5monthsafterimmunisationandevaluatedintermsofspleen(B)andliver(C)R.equiburden.AttenuatedSalmonellacarryingemptyvectorwasusedasa negativecontrol.Datarepresentthemean±SE,n=5animals.#_p_<_0.05.

elevatedin WT,but notinTlr2−/− mice(Fig.6C).Regardingthe CD8+_CD62L+_CD44+high_cells,_no_noticeable_difference_was_detected

amongallgroupsofanimals(Fig.6D).

4. Discussion

Variousstrategieshavebeenproposedforthedevelopmentofa safeandeffectivevaccineagainstrhodococcosis.Forinstance,the passiveimmunisationwithhyperimmuneplasma[10],vaccination procedureswithinactivatedR.equistrains[11],VapADNAvaccine

[12]and,morerecently,attenuatedR.equivaccine[13]havebeen reported.However,noneoftheseapproacheshaveprovidedan efﬁ-cientmethodtocontrolthedisease.Amongthedifferentstrategies ofvaccinationforintracellularpathogens,a promisingapproach maybetheuseofliveoralvaccines.Inthiscontext,ourgrouphas

demonstratedthatoralimmunisationwithanattenuatedS.enterica TyphimuriumstrainexpressingtheVapAproteininduces protec-tionagainstexperimentalrhodococcosisinmice[8].Morerecently, wehavedemonstratedthattheoralimmunisationprotocolinduces a strongandspecifichumoral andcell-mediated Th1-immunity againsttheheterologousantigen,withanappropriateregulatory responseandalong-termprotectionagainstR.equiinfection[9].In thepresentwork,wereportthatnasaldeliveryisaneffectiveroute forimmunisation,whichconfersprotectionwithasingledoseof thevaccine.Wehavealsoverifiedthatnasalvaccinationinduces aTh1immuneresponse,whichisasprotectiveandspecificasthe oralimmunisation[9].

Nasalroutehasbeenproposedasanalternativeroutefor vacci-nationduetoeaseoftheprocessofimmunisationandthepresence of nasopharynx-associatedlymphoidtissue (NALT).Aswiththe Peyer’s patches,NALTaccomplishestherequirementstoinduce

Fig.4.NasalvaccinationwithSTMVapA+enhancesTLR2+expressionlevelsinspleencells,whichisrevertedbyR.equichallenge.TherelativeexpressionlevelsofTLR2 mRNAinthespleencellsofvaccinatedBalb/cmiceeitherunchallengedorchallengedorwithR.equiisshowninpanelA.PanelBshowstherelativefrequencyofspleen cellspositiveforTLR2stainingasdetectedviaﬂowcytometry.AttenuatedSalmonellaharbouringemptyvectorwasusedasanegativecontrol.In(A),cDNAcontentwas normalisedonthebasisofpredetermined␤-actinlevels.Datarepresentthemean±SEM(n=5animals).#_p_<_0.01._In_(B),_data_represent_the_mean_±_SE.#_p_<_0.05.

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Fig.5.ProtectioninducedbynasalvaccinationwithSTMVapA+isindependentofTLR2activation.WTandTlr2−/−_C57BL/6_mice_were_challenged_with_R._equi₃₀_days_after

vaccinationwithSTMVapA+.Fivedaysafterchallenge,R.equiburdeninthespleen(A)andliver(B)wasevaluated.Thespleentissuewasalsoanalysedforthelevelsof IL12p40(C),IFN-␥(D),andTNF-␣(E).SimilarexperimentalprocedureswereperformedwithC3H/HePASandC3H/HeJmice;R.equiburdenwasassessedinthespleen(F) andliver(G)samples.AttenuatedSalmonellacarryingemptyvectorwasusedasanegativecontrol.Thebarsrepresentthemean±SEM(n=5animals).#_p_<_0.05.

andregulateaprotectiveimmuneresponse,whichinvolvesMcells, APCs,TcellsandBcells[14].Nonetheless,NALTischaracterisedas aTh0environment, whichcanberevertedtoeitherTh1orTh2 proneambiance[15].Althoughtheoraladministrationisa nat-uralroutefor Salmonella-basedvaccines,theintranasaldelivery

allowsfor theimmunogentoreachthesame organs,including thelungsandPeyer’spatchesand,asaconsequence,caninduce serologicandproliferativeresponses[16].Currently,Kimetal.[17]

havedemonstratedthatattenuatedSalmonellaTyphimuriumisan efﬁcientcarrierofheterologousantigens,whichisabletoinvolve

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Fig.6. NasalimmunisationwithSTMVapA+inducesincreasedfrequencyandrecallofCD4+_T_cells_with_memory_phenotype_via_a_{TLR2-dependent}_mechanism._Immunised

WTandTlr2−/−_C57BL/6_mice_were_challenged_with_R._equi₃₀_days_(panels_A_and_B)_or₁₂₀_days_(panels_C_and_D)_after_vaccination._The_frequencies_of_CD4+_/CD62L+_/CD44high

(AandC)andCD8+_/CD62L+_/CD44high_cells_(B_and_D)_were_assessed_via_ﬂow_cytometry_in_spleen_samples._Attenuated_Salmonella_carrying_empty_vector_was_used_as_a_negative

control.Thebarsrepresentthemean±SEM(n=5animals).#_p_<_0.05.

Mcellsinnasopharynxandtracheobronchiallymphoidtissueand inducelocalandsystemic-speciﬁcimmuneresponses.Such ﬁnd-ingshavemotivatedseveralgroupstodevelopSalmonella-vectored vaccinesforadministeredvianasalrouteinmice[18,19]andmares

[20].

Darrah and co-workers [21] have demonstrated that the lipoproteinVapAactivatesmacrophageTLR2,areceptorthatisa majormediatoroftheinnateresponsetoR.equi.Therefore,along withtheknowledgethatTLR2activationfavoursthe differentia-tionofTh1cells,it islikely thatthis receptorcouldplaya role intheprotectionconferredbySalmonella-expressingVapAused herein.Thisideawasreinforcedwiththefactthatvaccination ele-vatedTLR2expressionlevelsonmurinemacrophages.However, thehighTLR2expressionlevelswerenotmaintainedwhenthe immunisedmicewerechallenged withR. equi.Withregardsto thisﬁndings,theymightberelatedto(i)asupposedTLR2 inter-nalisation,whichaccompaniestheinternalisationofR.equior(ii) triggeringasignalviaTLR2activation,therebyinducinganegative feedbackmechanismabletocontrolinﬂammationafterchallenge. ThelatterassumptionissupportedbytheobservationthatTNF-␣ levelsalsodecreasedafterR.equichallenge.

ActivationofTLRsdirectlymodulatesTcellbiology[22–24].In thiscontext,alterationofTLR2expressionlevelsbythesecellscould affectvaccineefﬁciencyandthefrequencyofTcellpopulations. Surprisingly,theresultsofprotectionwiththeSTMVapA+vaccine weresomewhatsimilarforboththeWTandtheTlr2−/−animals, asshownbyasimilarreductioninR.equiburdeninthespleenand liver.Furthermore,thepopulationofCD4+ _T_cells_with_memory

phenotypewassimilarlyexpandedinTlr2−/−andWTvaccinated mice.ThefrequencyofCD8+_T_cells_with_memory_phenotype;

how-ever,wasonlygreaterinWTmiceaftervaccination.Theseresults are supportedby thestudy of Cottalorda and co-workers [25], whichdemonstratedthatTLR2playsacriticalroleinthe gener-ationofmemoryCD8+_T_cells._Our_results_indicate_that_CD4+_T_cells

playamajorroleinR.equiimmunity.Thisisinagreementwith thefactthatactivatedCD4+_T_cells_express_TLR3_and_TLR9_but_not

TLR2andTLR4[24].Followingvaccination,activationofCD4+_T

cellsoccursindependentoftheirdirectinteractionwithVapAor Salmonellacomponents.

OurresultsdonotexcludethepossibilitythatTLR2might par-ticipateintheprotectiveresponseagainstR.equiinducedbySTM VapA+.Acloserexaminationofourresultsrevealsthatvaccinated WTmicedisplayedareducedbacterialburdenofalmost3logs, whilethevaccinatedTlr2−/− micedisplayedareducedburdenof approximately1.5logs.Thus,theVapAantigen,inthecontextof theSalmonellacell,isabletoinduceaprotectiveimmuneresponse viaapathwaythatisindependentofTLR2activation. Neverthe-less,TLR2seemstoactsynergisticallytomountastrongprotective responseagainstR.equi.

TheexactmechanismbywhichtheVapA+antigenispresented bytheattenuatedSalmonellaremainsunclear.S.entericaserovars survive inside the hostcells. Although attenuated strains were shown tobeunable tocausedisease,theyretain theability to colonisethehosttissueandtriggercertainsignalsinthecontextof thehostcells.Likewise,R.equiisanintracellularpathogenand nat-uralinfectionshouldbemimickedviavaccinationwithSTMVapA+, whichhastheadvantageofprovidingbetterconditionsfor presen-tationoftheVapAantigen.Suchpresentationseemstobecrucial, as VapA ishighly associated withR. equi virulenceand thus is extensivelydescribedasagoodantigenforvaccination.Currently, manystrategieshavebeenproposedbasedontheimmunity devel-opedagainstVapA,butonlyfewofstrategieswereconsideredto havepotentialforvaccineapplication.Theresultspresentedherein suggestthatthe␹3987attenuatedstrainofSalmonellaisan efﬁ-cientcarrierfororalornasalimmunisationwithVapA.Thevaccine stimulatesantigen-speciﬁcCD4+_T_cells_without_the_{participation}

ofTLR2and leadstoa proﬁleof immuneresponsethat confers resistancetotheR.equiinfection.Mostimportantly,theefﬁcient

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clearanceofR.equifromtheorgansofvaccinatedmiceandthe long-termrecallofimmunityindicatethatthestrategyusedhereinmay beapplicableforvaccinationagainstrhodococcosis.

Acknowledgments

WewouldliketothankSandraO.Thomazand Patricia Ven-druscolofortechnicalsupport.Thisstudywasinpartsupported bygrantsfromFAPESP(05/50460-1)andCNPq(154963/2006-2). S.A.C.wasaPhDfellowofCAPES,andMCRBisseniorfellowofCNPq. Contributors:S.A.C.andS.G.S.designedexperiments,performed assays,interpreteddataandwrotethemanuscript.A.F.O.designed experimentsandperformedassayswithTLR2KOmice.M.M.T. per-formedassayswithTLR4-deﬁcientanimals.L.P.R.performedPCR andﬂowcytometryanalysis.L.L.O.,M.C.R.B.andE.S.H.interpreted dataandcontributedtothemanuscript.Competinginterests:The technologydescribedinthispaperissubjecttopendingpatents. Thedata andmaterials described hereinadheretothe Vaccine journalpoliciesforacademic,non-commercialresearch.

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