a new adjuvanted nanoparticle based h1n1 influenza vaccine induced antigen specific local mucosal and systemic immune responses after administration into the lung

Guzmánc, Vidadi Yusibovb, Katherina Sewalda, Armin Brauna,d,∗ a Fraunhofer Institute for Toxicology and Experimental Medicine, Nikolai-Fuchs-Straße 1, 30625 Hannover, Germany, Biomedical

Vaccine

jo u rn al h om ep a g e :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

A new adjuvanted nanoparticle-based H1N1 influenza vaccine

induced antigen-specific local mucosal and systemic immune

responses after administration into the lung

Vanessa Neuhausa, Jessica A Chichesterb, Thomas Ebensenc, Katharina Schwarza,

Caitlin E Hartmanb, Yoko Shojib, Carlos A Guzmánc, Vidadi Yusibovb,

Katherina Sewalda, Armin Brauna,d,∗

a Fraunhofer Institute for Toxicology and Experimental Medicine, Nikolai-Fuchs-Straße 1, 30625 Hannover, Germany, Biomedical Research in Endstage and

Obstructive Lung Disease Hannover (BREATH), Member of the German Centre for Lung Research (DZL)

b Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Newark, DE 19711, USA

c Helmholtz Centre for Infection Research, Department of Vaccinology and Applied Microbiology, Inhoffenstraße 7, 38124 Braunschweig, Germany

d Institute of Immunology, Hannover Medical School, Hannover, Germany

Article history:

Received 7 November 2013

Received in revised form 31 January 2014

Accepted 1 April 2014

Available online xxx

Keywords:

Influenza vaccine

Plant-based

HAC1

c-di-GMP

Mucosal adjuvant

Silica nanoparticle

a b s t r a c t

(http://creativecommons.org/licenses/by/3.0/)

∗ Corresponding author at: Fraunhofer Institute for Toxicology and

Experimen-tal Medicine, Department of Preclinical Pharmacology and In Vitro Toxicology,

Nikolai-Fuchs-Str 1, 30625 Hannover, Germany Biomedical Research in Endstage

and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre

for Lung Research (DZL) Tel.: +49 511 5350 263.

E-mail address: armin.braun@item.fraunhofer.de (A Braun).

1 Introduction

Annual influenza-associated cases of hospitalization and up

to500,000deathsduringfrequentvirusoutbreaksandsporadic pandemicsillustratetheserioushealthburdenofinfluenzavirus infections [1] The high mutational rate of the virus and fre-quencyofinterspeciestransmissionand/orzoonosisleadingtonew

http://dx.doi.org/10.1016/j.vaccine.2014.04.011

0264-410X/© 2014 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/3.0/ ).

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virussubtypes makes influenza infections highly unpredictable

influenzavaccines

Traditionally, only systemic administration of inactivated

influenzavaccines,mostlyintramuscularly,hasbeenused.In2003

Flumist®,the first nasalinfluenza vaccine withlive attenuated

influenzaviruses,hasbeenapproved in theUS [4],which

pro-tectslocallyatthesiteofvirusentryandinfection.Anadvantage

of delivering vaccines via the respiratory route is, besides the

inductionsoflocalimmuneresponsesatvirussettlement,the

non-invasiveapplicationwhichislikelytoincreasepubliccompliance

However,it has beendescribed that intranasal antigen

admin-istrationinducespoorimmuneresponseswhenappliedwithout

anappropriate mucosaladjuvant[5] Thus,many neweffective

mucosaladjuvantsareinpreclinicaldevelopment(s.review[6])

In2007,bis-(3,5)-cyclicdimericguanosinemonophosphate

(c-di-GMP)wasintroducedasa mucosaladjuvant withpromising

activity[7].Madhunetal.showedthat c-di-GMPimprovedthe

immunogenicityofanintranasallydeliveredsubunitinfluenza

vac-cine,comparedtoantigenonly,byinducingstrongmucosaland

systemicimmuneresponses[8].Additionally,theauthorsshowed

thatintranasaladministrationofthec-di-GMPadjuvantedantigen

inducedprotectiveantibodytitersandcellularimmuneresponses

thatfarexceededtheresponsesinducedbyintramuscular

admin-istrationofthesamevaccine[8].Moreover,Svindlandetal.tested

vaccination with c-di-GMP combined with a second adjuvant,

Chitosan,andshowedthatvaccinationwiththecombination of

thesemolecules can furtherimprove the humoral and cellular

immuneresponsesagainst targetantigens[9].Besides its

adju-vantiveeffects,Chitosanisusedasanintranasaldeliverysystem

Otherdrugdeliverysystemssuchassilicananoparticle(NP)have

alsobeenpreviouslyshowntohaveadjuvantproperties[10,11]

Recently,wehaveshownthatacombinationofaplant-produced

recombinanthemagglutinin(HA)antigenfromtheH1N1influenza

virusA/California/04/09(HAC1)withsilica-NP(SiO2)wasableto

recallapreviouslyestablishedimmuneresponseinhumanlung

tissue[12].Manufacturingofrecombinantproteinsinplantsfor

influenzavaccinedevelopmentevolvedas analternativetothe

conventionalegg-basedvaccineproductiontoovercomethe

lim-itationsinquantityandtime consumption[13].Thisbottleneck

ofegg-produced vaccinescanhaveseriousconsequencesduring

influenzapandemics,whentheproductionofsufficientamounts

ofvaccineinanadequatetimeframetoservetheglobalmarket

couldbedifficult

Regardingtheneedof rapidlyproduced vaccinesin timesof

pandemicsandthetimeconsuminglimitationoftheegg-based

vac-cines,theherepresentedstudytestedtherecombinantantigenof

ahighlyimmunogenicH1N1strainresponsibleforthe2009/2010

pandemic.Furthermore,thestudyextendsthepublishedworkwith

HAC1andSiO2andevaluatestheimmunogenicityofthisvaccine

formulationwhencombinedwithc-di-GMPandadministeredat

thesiteofvirusentry.Overall,itshowedthepotentialofthe

c-di-GMP/SiO2double-adjuvantedvaccinetoinducesystemichumoral

andstrongmucosalimmuneresponses,withIgAintheairways

Furthermore,it presentedevidenceofantigen-primedT-cellsin

thelunginintratracheallyvaccinatedmice

2 Materials and methods

2.1 Animals

Femalewild-typeBALB/cmiceaged6–8weeks(CharlesRiver,

Sulzfeld,Germany) were kept at an animal facility under

con-ventionalhousingconditions(22◦C,55%humidity,12-hday/night

cycle)withfoodandtapwateradlibitum.Therandomizedstudy

was approved by a local agency (Application-No 33.9-42502-04-11/0465) and conducted according to the German Animal Protectionlaw

2.2 Media Reagents were, if not stated otherwise, purchased from Sigma–Aldrich (Munich, Germany) Phosphate buffered saline (PBS)withoutCa2+andMg2+,pH7.4,Dulbecco’sModifiedEagle’s Medium/NutrientMixtureF-12HAM(DMEM)withl-glutamine,

15mMHEPESand7.5%w/vsodiumbicarbonatewithoutphenol red,pH7.2–7.4,RPMI1640andEarle’sBalancedSaltSolution(EBSS) wereobtainedfromGibco(Darmstadt,Germany).Cell/tissue cul-tivationmediumwassupplementedwith100U/mLpenicillinand

100␮g/mLstreptomycin

2.3 Vaccineandadjuvants HAC1wasproducedaspreviouslydescribed [14].Briefly,the

HAnucleotidesequence,encompassingaminoacids18–530ofthe A/California/04/09influenzastrain(H1N1,NCBIaccessionnumber ACQ76318.1) wereoptimized forexpression in plantsand syn-thesized.TheoptimizedHA sequencecontains a6× Hisaffinity purificationtagandtheERretentionsignalKDELattheC-terminus ThisgenewasinsertedintothepGRD4launchvectorand trans-formedintoAgrobacteriumtumefaciens.Thetransformedbacterium wasintroducedintohydroponicallygrownNicotianabenthamiana

byvacuuminfiltrationandleaftissueswereharvested, homoge-nized,extracted, filteredandchromatographically purifiedafter

aone-weekgrowingperiod[14].AliquotsofpurifiedHAC1were keptinPBSat−80◦Cuntilusage.Forsilica-NP,DMEMandSiO2

nanopowder(HDK200,WackerChemie,Germany)weremixedand dispersedbyultrasonicsonotrode.Theproductionofc-di-GMPhas beendescribedbefore[15,16].Lyophilizedc-di-GMPwasstored

at−20◦C.Immediatelypriortoimmunization,HAC1wasadmixed

withtheadjuvantand/orsilica-NPandswirled≥10minonan over-headshakertoensurecompletemixing

2.4 Vaccinationandsamplecollection Micewereimmunizedondays0and21witheither5␮gantigen (HAC1),single-or double-adjuvantedvaccine(5␮gHAC1/10␮g SiO2; 5␮gHAC1/7.5␮g c-di-GMP;5␮g HAC1/10␮g SiO2/7.5␮g c-di-GMP)byintratrachealroute(50␮l).Forintratracheal immu-nizationmiceweretilted(∼45◦)andthevaccineadministeredinto

thedeeplungwithsubsequentinsufflationwithanairbolus.A sys-temiccontrolgrouptoensuretheeffectivenessofthevaccination protocol,received1␮gHAC1adsorbedonaluminum hydroxide (Alum)intraperitoneally(200␮l).Bloodwasobtainedby retrob-ulbarsampling and serawere collectedondays 0, 21, 35, and

49todetermineHA-specificantibodyresponseby hemagglutina-tioninhibition(HAI)andenzyme-linkedimmunosorbent(ELISA) assays.On day49,miceweresacrificedwithanintraperitoneal overdosingofpentobarbital-Na(Merial,Hallbergmoos,Germany) andcuttingtheVenacavainferior.BALfluids,agarose-filledlungs, andspleensweresampledandusedforimmunoglobulin(Ig) mea-surementsandre-stimulationassays

2.5 HAIassay Collectedseraweretreatedwithareceptor-destroyingenzyme (Denka Seiken, Japan) HAI assay was performed using 0.75% turkey erythrocytes and A/California/07/09×179A virus (CDC

#2009713114)withaninitialserumdilutionof1:20asdescribed previously[14].HAIendpointtitersweredeterminedasreciprocal

Fig 1. Titers of hemagglutination inhibition (HAI) antibody and antigen-specific IgG in sera of vaccinated mice Mice were vaccinated on days 0 and 21 intratracheally with

5 ␮g HAC1 only, admixed with a single adjuvant (HAC1/SiO2 or HAC1/c-di-GMP), or admixed with two adjuvants (HAC1/SiO2/c-di-GMP) as the double-adjuvanted vaccine The control group received 1 ␮g HAC1 formulated with Alum intraperitoneally (i.p.) On days 0, 21, 35 and 49, blood was drawn retrobulbarly from the vaccinated mice and evaluated for HAI antibody titers (A) as well as HAC1-specific IgG (B) Data are presented as mean ± SEM **p < 0.01 and ***p < 0.001, comparison with vaccination with antigen only; xp < 0.05, comparison of vaccination i.t vs i.p (HAC1/SiO2/c-di-GMP vs HAC1-Alum) using Kruskal–Wallis test and Dunn’s multiple comparison post hoc test (n = 5) HAC1: plant-derived recombinant hemagglutinin protein, SiO2 = silica nanoparticles, c-di-GMP: bis-(3  ,5  )-cyclic dimeric guanosine monophosphate.

ofthehighestserumdilutioncausingcompleteHAI

Seroconver-sioninvaccinatedanimalswasdefinedasanHAIantibodytiterat

aserumdilutionof≥1:40[17,18]

2.6 Antibodymeasurement

HA-specificserumIgGantibodiesandnasalIgGandIgAwere

assessedusingELISAassay,aspreviouslydescribed[19].Briefly,

ELISAplateswerecoatedwithinactivatedA/California/07/09virus

and samplesof BAL or serum weretested in series of two- or

four-fold dilutions Antigen-specificIgG and IgA were detected

usinghorseradishperoxidase-conjugatedgoatanti-mouseIgGor

IgAantibody(JacksonImmunoresearchLaboratoriesInc.,PA,USA),

respectively.Endpointtitersweredeterminedasreciprocalserum

dilutionsthatgavemeanopticaldensity(OD)valuesthreetimes

greaterthanthosefrompre-immuneseraata1:100or1:50dilution

forIgGandIgA,respectively

2.7 Splenocyteproliferationassay

Isolated splenocytes (1×105 cells) of vaccinated micewere

incubated in 96-well round bottom plates with RPMI

supple-mentedwith5%fetalbovineserum,containing10␮g/mLHAC1

orPBS(negativecontrol)at37◦Cfor72h.Proliferationwas

mea-suredasdescribedbefore[20].Cellswerepulsedwith5␮Ci/mL

3H-thymidine(AmershamBuchler,Braunschweig,Germany) for

18h and harvested on filter mats (Canberra-Packard, Dreieich,

Germany).ATopcountMicroplateScintillationCounter

(Canberra-Packard, Dreieich, Germany) measured 3H-thymidine-positive

cellsascountsperminute

2.8 Antigenstimulationofprecision-cutlungslices(PCLS)

MurinePCLSwerepreparedasdescribedbefore[21,22].Two

PCLS(approx.300␮mthick)perwellweretreatedwith10␮g/mL

HAC1ormedium(non-stimulated)andculturedundercellculture

conditions(37◦C,5%CO2and95%airhumidity)for24h

Super-natantwascollectedandstoredat−80◦Cuntiluse.

2.9 Cytokinemeasurement Cytokines interleukin (IL)-2, interferon-gamma (IFN-␥), IL-5, and IL-10 in thesupernatant of re-stimulated PCLS were mea-suredusingthemurineTh1/Th2tissueculturekitfromMesoScale Discovery(MSD)Assays(Gaithersburg,MD,USA).Theassaywas performedandresultswereanalyzedaccordingtomanufacturer’s specifications using MSD plates, MSD Sector Imager 2400, and Discoveryworkbenchsoftware.Totalproteinconcentrationswere measuredinPCLSlysatesusingtheBCAProteinAssaykit(Pierce, Rockford,IL,USA)[12].Cytokineswerecorrelatedtototalprotein (ng/mg)and compared tothenon-stimulatedcytokinebaseline levelasfoldinduction

2.10 Statisticalanalysis Statistical analyses were performed by either the Kruskal–Wallis test withDunn’s multiple comparison post hoc testsorbytheMann–WhitneytestusingGraphPad4.03(GraphPad, SanDiego,CA,USA).Datawereexpressedasmean±standarderror

of the mean (SEM) or median±quartiles Differences between treatmentgroupsandcontrolswereconsideredstatistically sig-nificantatp<0.05.Thenumberofmiceisindicatedinthefigure legends

3 Results

3.1 Intratrachealadministrationofdouble-adjuvantedHAC1 withSiO2andc-di-GMPinducedrobustsystemicantibody responses

Asmainreadoutparametersforasystemicantibodyresponse HAI and HAC1-specific IgG titers were analyzed in the blood

of vaccinated mice.The non-adjuvantedgroup vaccinated with HAC1 only did not develop detectable HAI or antigen-specific IgG antibodies in the serum (Fig 1) On the contrary, admin-istration of HAC1 intraperitoneally with Alum served as a positive control and induced very robust HAI (4096±627.1;

Fig.1A)andIgG (286,720±75,248; Fig.1B)antibodytitersafter the second vaccination (day 35) Mice vaccinated with either

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Fig 2. Proliferation index of splenocytes upon re-stimulation with the

recombi-nant HAC1 antigen Spleen cells of vaccinated mice were isolated on day 49 and

re-stimulated with 10 ␮g/mL HAC1 followed by labeling with 3 H-thymidine The

proliferation indexes upon antigen stimulation and in the medium alone were

compared Data are presented as mean ± SEM, **p < 0.01 compared to the

prolifer-ation index of non-stimulated splenocytes from the same animal; Mann–Whitney

test (n = 5) HAC1: plant-derived recombinant hemagglutinin protein, SiO2 = silica

nanoparticles, c-di-GMP: bis-(3,5)-cyclic dimeric guanosine monophosphate,

Alum: Aluminum hydroxide, i.t.: intratracheally, i.p.: intraperitoneally.

HAC1/SiO2 orHAC1/c-di-GMPdeveloped lowtiters ofHAI

anti-bodiesafterthesecondvaccination(43±30and12±7;Fig.1A),

aswell as modestserum IgG titers following the boosterdose

(205±81 and 2980±1419; Fig 1B) The group receiving the

double-adjuvantedvaccine,HAC1/SiO2/c-di-GMP,developedhigh

HAI titers (770±470; Fig 1A) and antigen-specific IgG titers

(43,840±23,923;Fig.1B)

3.2 Additionofc-di-GMPadjuvantwasnecessarytoinduce

antigen-specificsplenocytes

Tofurtherevaluatethesystemicimmune responsefollowing

intratrachealvaccination,theproliferation index of splenocytes

uponantigenicre-stimulationwasassessed(Fig.2).Splenocytes

isolatedfromimmunizedmicewerere-stimulatedinvitrowith

HAC1followed by 3H-thymidinelabeling Thecell proliferation

levelwascomparedtonon-stimulatedsplenocytesfromthesame

animal.Results showednodetectableantigen-induced

prolifer-ationof splenocytes inthe non-adjuvanted,positivecontrol, or

the HAC1/SiO2 groups (HAC1: 1.2±0.1; HAC1-Alum: 1.5±0.2;

HAC1/SiO2:1.2±0.2)

Incontrast,inthesingle-adjuvanted group(HAC1/c-di-GMP)

thelevelofproliferationwastwo-foldcomparedtonon-stimulated

splenocytes(2.2±0.4)andthedouble-adjuvantedvaccineinduced

thehighestlevelofsplenocyteproliferation(4.4±1.7)uponHAC1

re-stimulation

3.3 Thedouble-adjuvantedvaccineinducedlocalIgGandIgA

antibodyresponsesintheBAL

Localimmuneresponsesinthelungwereassessedby

measur-ingHA-specificIgGorIgAtitersinBALsamples(Fig.3AandB).The

non-adjuvantedgroupvaccinatedwithHAC1onlydidnotdevelop

detectableIgGorIgAintheBAL(baselineIgG/IgAlevel25;Fig.3A

andB).Incontrast,thepositivecontrolgroup(HAC1-Alum)showed

antigen-specific IgG titers in the BAL (115±37) comparable to

thedouble-adjuvantedgroup,whileIgAlevelswereundetectable

HAC1/SiO2orHAC1/c-di-GMPdidnotinducedetectableIgGorIgA

intheBALofimmunizedmice.However,additionofc-di-GMPto

HAC1/SiO2didinducedetectablelevelsofIgGin2/5mice(115±73;

Fig.3A)andinonemousedetectablelevelsofIgA(Fig.3B)

Fig 3. Titers of antigen-specific IgG and IgA in the bronchoalveolar lavage (BAL) fluid of vaccinated mice BAL from mice vaccinated with 5 ␮g HAC1 or 10 ␮g HAC1 was collected on day 49 and analyzed for HAC1-specific IgG and IgA (A and B) Data are presented as Boxplot and whiskers median ± quartiles (n = 5) and com-pared using the Kruskal–Wallis test and Dunn’s multiple comparison post-hoc test.

*p < 0.05 and **p < 0.01, comparison to vaccination with antigen only HAC1: plant-derived recombinant hemagglutinin protein, SiO2 = silica nanoparticles, c-di-GMP: bis-(3,5)-cyclic dimeric guanosine monophosphate, Alum: Aluminum hydroxide, i.t.: intratracheally, i.p.: intraperitoneally.

InordertoensurethattheinductionofmucosalIgAinthesingle positivemousewasaresultofvaccination,micewereimmunized withahigherantigenconcentration(10␮gHAC1)andtheBALwas examinedforthepresenceofHAC1-specificIgGandIgA(Fig.3Aand B).Thenon-adjuvantedgroup(10␮gHAC1)showednoincreased localIgGorIgAtiters(Fig.3AandB).OnemousegivenHAC1/SiO2 developedmucosalIgGtitersabovebaseline(30±5vs.25)while twomicedevelopeddetectableIgA(titer45±15vs.25) HAC1/c-di-GMPinducedelevatedtitersofmucosalIgG(135±68)andIgA (385±172) withpositive titers in 80% of the vaccinated mice MicereceivingHAC1/SiO2/c-di-GMPdevelopedenhancedlevelsof mucosalIgG(540±271)andIgA(490±283)in100%ofvaccinated mice.Additionally,doublingtheantigendoseincreasedIgGby 4.3-fold(Fig.3A)

3.4 AnimalsimmunizedwithHAC1/SiO2/c-di-GMPshoweda T-cellimmuneresponseuponantigenicre-stimulation

To determine the local antigen-specific T-cell-mediated immune response at the cytokine level, PCLS from vaccinated

Fig 4. Secreted cytokine profile by PCLS from different vaccinated mouse groups upon re-stimulation with the recombinant HAC1 antigen PCLS from vaccinated mice were prepared 28 days post the last vaccination, re-stimulated with 10 ␮g/mL HAC1 and cultured for 24 h The secretion of IFN-␥ (A), IL-2 (B), IL-5 (C) and IL-10 (D) was measured upon antigen stimulation Data are presented as mean ± SEM fold induction compared to re-stimulation with medium alone of PCLS from the same animal using the Mann–Whitney test (n = 3) PCLS: precision-cut lung slices, HAC1: plant-derived recombinant hemagglutinin protein, SiO2 = silica nanoparticles, c-di-GMP: bis-(3  ,5  )-cyclic dimeric guanosine monophosphate, Alum: Aluminum hydroxide, i.t.: intratracheally, i.p.: intraperitoneally, IL: Interleukin, IFN-␥: Interferon-gamma.

mice were re-stimulated with HAC1 Cytokine secretion upon

antigen stimulation was compared to the non-stimulated

cytokine baseline level and expressed as fold induction The

non-adjuvanted group (HAC1 only) showed no altered IL-2

or IFN-␥ expression upon antigen-stimulation compared to

non-stimulated PCLS (fold induction≤2; Fig 4A and B) The

positivecontrolmice,however,secretedlowlevelsofIL-2

com-pared with non-stimulated samples (fold induction 37±35)

but showed no increase in IFN-␥ production (27±27) Results

also showed that in contrast to HAC1/SiO2, re-stimulation

with HAC1/c-di-GMP did induce antigen-specific cells

pro-ducing IL-2 and IFN-␥ (155±60 and 244±118, respectively)

Additionally, re-stimulation of PCLS from HAC1/SiO2/c-di-GMP

vaccinatedmicealsoinducedIL-2andIFN-␥(262±132-foldand

275±138-fold)

No induction of IL-5 secretion was detected in the

non-adjuvanted,positivecontrolorHAC1/SiO2vaccinatedgroupsupon

antigenicre-stimulation(foldinduction≤2;Fig.4C).However,the

c-di-GMP-adjuvantedHAC1antigeninducedcellstosecretslightly

elevatedlevelsof IL-5 upon HAC1re-stimulation (2.2±0.1 and

2.4±0.1 for single- and double-adjuvanted, respectively)

com-paredtonon-stimulatedPCLS

Thereleaseoftheanti-inflammatorycytokineIL-10wasat base-linelevelsinPCLSfromthenon-adjuvantedandpositivecontrol groups(foldinduction≤2;Fig.4D)aswellasHAC1/SiO2 immu-nizedmice.Incontrast,IL-10levelswereenhancedinPCLSsamples fromHAC1/c-di-GMPaswellasHAC1/SiO2/c-di-GMPvaccinated mice,when re-stimulated withHAC1(12±4and 7±2, respec-tively)

4 Discussion

Thepresentstudyevaluatedthesystemicandlocal immuno-genicityofadouble-adjuvantedinfluenzavaccine(HAC1/SiO2 /c-di-GMP)deliveredviatherespiratorytract.Thevaccineisintended

tobeusedasaninhalableneedle-freevaccinetargetingtheupper andlowerrespiratorytract.However,fortheworkdescribedhere,

weadministeredthevaccineintratracheally asa practical alter-nativetoevaluateeffectsofthevaccineinthedeeperlungbefore conductinganinhalationstudypriortothechallengeexperiments Minneandcolleaguesdescribedtheimpactofvaccinedeliverysite

ontheimmuneresponsesandconcludedthattargetingthelower lungsforaninhaledinfluenzavaccinationcaninducesystemicand localimmuneresponsesmostefficiently[23]

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RecentresultswiththeNP-admixedantigeninahumanlung

tis-suemodelshowedthatHAC1/SiO2wasabletore-activateformerly

primedT-cells[12].EventhoughHAC1/SiO2 hada re-activating

potentialinhumanPCLS,vaccinationofmiceintratracheallywas

barelyabletoinduceseroprotection(HAItiter>1:40).Moreover,it

didnotinduceanylocalimmuneresponse,suchasantigen-specific

IgsecretionorT-cellinductionuponre-stimulation,when

admin-isteredatalowerantigendose(5␮gHAC1).However,additionof

themucosaladjuvantc-di-GMPtoHAC1/SiO2inducedHAIandIgG

antibodiesand T-cellsthatareconsideredpotentialmarkersfor

systemicandlocalprotectiveimmuneresponsesagainstinfluenza

infection.Importantly,noadversesideeffectsorclinicalsignsof

decreased well-being ofthestudy animalswere observedafter

intratracheal administration of the double-adjuvanted vaccine

Theseincreasedantigen-specificimmuneresponsesdemonstrated

thesynergisticeffectofthecombinationofnontoxicconcentrations

ofSiO2 andc-di-GMPandwereinlinewiththeworkof

Svind-landetal.[9].AlthoughmucosalIgGandIgAwereinducedbythe

single-adjuvantedvaccineHAC1/c-di-GMP,ahigherantigendose

wasrequired.Withregardtotheshortageofvaccinesupply

dur-inginfluenzapandemicsandthefocusofnewvaccinestrategies

onantigendose-sparingformulations,thedouble-adjuvanted

vac-cineshowedpromisingresultsasitprovidedadosesparingeffect

However,10␮gofantigenwererequiredtoinducelocalIgGand

IgAin100%ofthevaccinatedmice.Atafirstview,systemic

vaccina-tionseemedtobemoreeffectivethanlocalvaccinationregarding

theantigendoserequiredtoinducesystemicHAIandIgGtiters

Onthecontrary,1␮gHAC1givensystemicallywasnotsufficient

toinducelocalIgAtiters.Infact,thisstudywasnotdesignedto

comparedose-sparingeffectsoflocalversussystemicapplications,

butrathertoevaluateanadditiveeffectofcombinedadjuvants

Thesystemicadministrationwasonlyusedasacontrolforthe

vac-cinationprotocolaswellasantigenstabilityandnotmeantasa

comparativegrouptoevaluatesuperiorefficacyoftherespiratory

vaccinationtothesystemicvaccination

TheimportanceofmucosalIgAduringinfluenzainfectionandits

abilitytoneutralizevirusininfectedepithelialcellshaspreviously

beenshown[24,25].AlsotheroleofIgAincross-protectionagainst

driftedvirusstrainshasbeenshowntocontributetoprotection,

albeititisnotessential[26,27].Newinsightsintoimmune

protec-tionhavealteredsecondgenerationinfluenzavaccinesfrombeing

designedtoinducesystemicIgGtowardtheinductionofbroader

cross-protectiveresponsesagainstthevirus,includingother

anti-bodyisotypes,suchasIgA.Thisnewprotectionstrategycombines

theinductionofsystemicandlocalaswellashumoraland

cellu-larimmuneresponses[25].Inthisstudy,thedouble-adjuvanted

vaccinedemonstrated the ability toinduce systemic functional

antibodyresponses as wellas local cellularimmune responses

suggestingtheadvantageofcombiningproperadjuvantsandthe

relevanceofimmunizingatthesiteofinfection

Eventhoughachallengestudywould benecessarytoprove

that the local and systemic immune responses observed here

canprovideprotectionagainstinfluenzavirusinfection,thereis

convincingevidenceintheliteraturethatthemeasuredimmune

responsesdiscussed above have been linked toprotective

ofimmunizationand theireffectonlocaland systemicimmune

responses and combined this with lung protection against an

influenzainfection[29].Theirresultsregardingtheinductionof

mucosal IgA, serum IgG and systemic HAI titers after vaccine

administrationinto thelower airwaysof thelung were inline

withtheresultspresentedabove.Theydetectedonlyintheprimed

intrapulmonaryimmunizationmucosalsIgAinthelung,butnot

theintramuscularadministration.Furthermore,theyobservedthe

highestnasalandlungIgGtitersinmiceprimed(andboosted)via

themucosalroute[29].Ofnote,thechallengestudyperformedby

Liuetal.demonstratedthattheimmuneresponsesinducedbythe adjuvantedintrapulmonaryvaccinationweresufficienttoprotect againstinfluenzavirusinfectionandreducethevirustitersinthe lungpostinfectiontolevelsbelowdetection[29]

It hasbeen shown previouslythat intranasal administration

of c-di-GMP as an adjuvant for influenza vaccines can induce multifunctionalinfluenza-specificCD4+Th1cellsinthespleenof immunizedmice[8,9].Furthermore,multifunctionalTh1cellshave alsobeenshowntobepresentinthebloodofvaccinatedhuman volunteersand in thenon-inflamednormal humanlung tissue,

as determinedby theirpotentialto produceIL-2, IFN-␥and/or TNF-␣ upon re-activation[31,32] Consistentwith thecytokine profileofinfluenza-specificmultifunctional Th1cells,ourstudy showedincreasedIL-2andIFN-␥levelsinantigenre-stimulated PCLSofmicevaccinatedwithHAC1/c-di-GMP.TheinductionofTh1 cytokinesinre-stimulatedPCLSindicatesthattheantigenwas rec-ognizedbyHAC1-specificmemoryT-cells.Theseresultsareinline withthehypothesisbyJul-Larsenandcolleagueswhodiscussed thatadditionofanadjuvantimprovestheefficacyofHAC1toward theinductionofarobustT-cellresponse[32]

Additionally, our results aligned with previous studies on intranasallyadministeredc-di-GMPshowinganinductionofa Th1-biasedcytokineprofileinre-stimulatedsplenocytesagainsttarget antigen[8,9,33].Yet,ourstudyalsoshowedamildinductionofthe Th2cytokineIL-5andtheanti-inflammatorycytokineIL-10in re-stimulatedPCLSofintratracheallyc-di-GMP-vaccinatedmice.The foldinductionoftheTh1cytokinesforthedouble-adjuvanted vac-cinatedmice,however,farexceededthelevelofTh2cytokinesthat wereinduced(IFN-␥:IL-5,about119-fold;IFN-␥:IL-10,about 39-fold).Nevertheless,thedouble-adjuvantedvaccine,aswellasthe c-di-GMPadmixedantigen,inducedIL-10secretioninPCLSupon antigenicre-stimulationwhich exceededthenon-stimulated

IL-10baselinelevel.Amongothercytokines,IL-10canbereleasedby influenza-specificCD4+memoryT-cellsandhasbeendescribedas havingaputativelycrucialroleinregulatinginflammationduring acuteinfluenzainfection[34].Thefactthatthedouble-adjuvanted vaccineinducedIL-10-competentcellsmightalsocontributetoa reducedlevelofinflammationinthelungswithrepeatedexposure

totheviruspostvaccination

Overall,thedatapresentedinthecurrentstudydemonstrate thatthedouble-adjuvantedHAC1vaccineisimmunogenicinthe mousemodelwhenadministeredintratracheally.Eventhoughthe protectiveefficacyofthedouble-adjuvantedHAC1vaccineneeds

tobe evaluated in a relevant animal model, thepresent study demonstratesthatthedouble-adjuvantedHAC1inducessystemic functionalantibodyresponseaswellaslocalhumoraland cellu-larimmuneresponseswhenadministeredviatherespiratorytract, indicatingpotentialforfutureneedle-freevaccineapplications

Acknowledgments

TheauthorswouldliketothankOlafMacke,SabineSchild,Sarah DunkerandOlgaDanovfortheirtechnicalassistance.Theauthors wouldliketothankDr.NatashaKushnirforcriticalreadingofthe manuscript.WearegratefultoDr.R.Kellnerforstatisticaladvice ThestudywaspartoftheFraunhoferGesellschaftPROFIL“Mucosal Nano-VaccineAgainstInfluenza”

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