2014 Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice

2014·0.85 MB·English

Vaccine32(2014)3169–3174

ContentslistsavailableatScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Purified coronavirus spike protein nanoparticles induce coronavirus

neutralizing antibodies in mice

ChristopherM.Colemana,1,YeV.Liub,1,HaiyanMub,JustinK.Taylora,MichaelMassareb,
DavidC.Flye rb ,GregoryM. Gle nn b,Gale E.Smit hb,1, Matth ew B.Friem ana,∗,1

aUniversityofMaryland,SchoolofMedicine,685WestBaltimoreSt,Baltimore,MD21201,USA
bNovavax,I nc .22Firstfie ldRd,G ai thersburg, MD 2085 2,USA

a r t i c l e i n f o a b s t r a c t

Articlehistory: Developmentofvaccinationstrategiesforemergingpathogensareparticularlychallengingbecauseof
Receiv ed15January2014 thesuddenna tu reoftheirem ergencea nd thelongpr ocessneed edf ortraditiona lvaccinede velopme nt.
RAeccceepivteedd i2n Ar epvriilse2d0 1f o4rm 18 March 2014 The refore, t here is a n eed for developm en t of a rap id meth od of va ccin e developm ent tha t can respond
toemergingpathogensinashorttimeframe.
Availableonline13April2014
Theemergenceofsevereacuterespiratorysyndromecoronavirus(SARS-CoV)in2003andMiddle

EastRespiratorySyndromeCoronavirus(MERS-CoV)inlate2012demonstratetheimportanceofcoro-
Keywords:
navirusesasemergingpathogens.Thespikeglycoproteinsofcoronavirusesresideonthesurfaceofthe
MiddleEastRespiratorySyndrome
virionandareresponsibleforvirusentry.Thespikeglycoproteinisthemajorimmunodominantanti-
Coronavirus
CSeovroernea vAicruutse Respiratory Syndrome cgoerno nofa vciorruosneanvtirryusaensd apnrdo mhaost eparnovtiebno dtoy tbaer gaent inexgcoeflliennfet cttaerdgecte lflos.r vaccine designs that seek to block

Neutralizingantibody Vaccinationstrategiesforcoronaviruseshaveinvolvedliveattenuatedvirus,recombinantviruses,non-

Vaccine replicativevirus-likeparticlesexpressingcoronavirusproteinsorDNAplasmidsexpressingcoronavirus

genes.Noneofthesestrategieshasprogressedtoanapprovedhumancoronavirusvaccineintheten

yearssinceSARS-CoVemerged.HerewedescribeanovelmethodforgeneratingMERS-CoVandSARS-

CoVfull-lengthspikenanoparticles,whichincombinationwithadjuvantsareabletoproducehightiter

antibodiesinmice.

©2014ElsevierLtd.Allrightsreserved.

1. Introduction havingemergedfrombats,viacivetcats,toinfecthumans[6,7].

AlthoughtherehavebeennoreportedcasesofSARS-CoVinfection

Coronaviruses infect a range of mammals and birds, causing inhumansafterthis,arecentstudyhasshownthattheparental

respiratory tract and gastrointestinal tract infections. Coron- virusstillexistsinbatsinChina[8].

aviruseswereknowntocausesevereand,therefore,economically Inlate2012,anovelbetacoronavirusnamedMiddleEastRespi-

important diseases in chickens [1] and pigs [2], but, while a ratory Syndrome Coronavirus (MERS-CoV) was identified in a

numberofcoronaviruseswereknowntoinfecthumans,thesymp- sample from a severe respiratory infection patient in The King-

toms are usually mild in healthy adults, akin to a common cold, dom of Saudi Arabia (KSA) [9,10]. Since then, 238 cases have

and only rarely cause more severe pneumonia. In 2003, how- been positively identified, of which 92 have resulted in death

ever,severeacuterespiratorysyndromecoronavirus(SARS-CoV) (www.who.org).Allofthecaseshavebeenlinkedtocountrieson

emerged,causing8273confirmedinfections,ofwhich775resulted orneartheArabianpeninsula(KSA,Jordan,Qatar,Egypt,Omanand

indeath[3–5].MostofthecaseswerelinkedtoChina,HongKong UnitedArabEmirates).Casesinotherpartsoftheworld,notably

andSingapore,withtheonlymajoroutbreakoutsideofthisarea Europe,involvedrecenttravelerstotheMiddleEastregionorwere

occurring in Toronto, Canada. SARS-CoV had a zoonotic origin, closely linked with people who did [11]. Patients infected with

MERS-CoVpresentatthehospitalwithsymptomsconsistentwitha

severelowerrespiratorytractinfectionand,insomecases,develop

kidney failure. MERS-CoV is closely related to bat coronaviruses
∗ Correspondingauthor.Tel.:[phone];fax:[phone]. found i n China , Europe an d Africa, suggesti ng a zo onotic origin,
E-mail address es: mf riema n@ som.umaryl and. edu , [email protected]
similartoSARS-CoV,howeverthereservoirofMERS-CoVhasnot
(M.B.Frieman).
1 Th eseauthorscontributedequallytothiswork. yetbeenidentified.

http://dx.doi.org/10.1016/j.vaccine.2014.04.016
0264-410X/©2014ElsevierLtd.Allrightsreserved.
3170 C.M.Colemanetal./Vaccine32(2014)3169–3174

Coronavirusesareenvelopedviruseswithlargesingle-stranded SARS-CoV (Urbani) and SARS-CoV (MA15) Spike proteins to be

positivesenseRNAgenomeswhichencode4majorstructuralpro- homologous.

teins: spike (S), membrane (M), envelope (E) and nucleocapsid

(N) [12]. The S protein is a type I trans-membrane glycoprotein
2.2. Recombinantbaculovirus
expressedonthesurfaceofcoronavirusesthatisresponsiblefor

receptorbindingandvirionentrytocells[13].ThelocationofSon
The MERS-CoV S protein sequence was from isolate Al-
thevirionsurface,theroleofSinbindingtocoronavirusreceptors
Hasa12013 with NCBI accession #AGN70962. The SARS-CoV S
andthefindingthatScaninduceneutralizingantibodiesinvivo[14]
protein sequence was from Urbani strain with NCBI accession
havemadeitanattractivetargetforvaccinedevelopmentstrategies
#AAP13441.Thegeneswerecodonoptimizedforoptimalexpres-
[15,16].
sionininsectcellsandbiochemicallysynthesizedforMERS-CoVS
PreviouseffortstocreateavaccineforSARS-CoVhaveutilized
(Genscript,Piscataway,NJ)andSARS-CoVS(GeneartAG,Regens-
anumberofapproaches,butnoneiscurrentlylicensedforuseand
burg, Germany). Full length S genes was cloned between BamHI
arecentstudyoffourputativeSARS-CoVvaccinesyieldednegative
– HindIII sites in pFastBac1 baculovirus transfer vector plasmid
results[17].InitialstudiessuggestedthatwholeinactivatedSARS-
(Invitrogen,Carlsbad,CA)underthetranscriptionalcontrolofthe
CoV could be used as an effective vaccination [18–20], however
AutographacalifornicaMultipleNuclearPolyhedrosisVirus(AcM-
furtherworkhassuggestedthatthelevelofprotectioninducedby
NPV) polyhedrin promoter. Recombinant baculovirus construct
inactivatedSARS-CoVisincompleteandfailstopreventSARS-CoV
wasplaquepurifiedandmasterseedstocksprepared,characterized
symptoms, while also inducing increased eosinophilia in vacci-
foridentity,andusedtoprepareworkingvirusstocks.Thetitersof
nated animals [17,21]. Therefore, the most likely candidates for
baculovirusmasterandworkingstocksweredeterminedbyusing
coronavirusvaccineplatformsarebasedonspikesubunits[22,23],
rapid titration kit (BacPak Baculovirus Rapid Titer Kit; Clontech,
recombinantvirusesexpressingSARS-CoVproteins[24–26],DNA
Mountain View, CA). Recombinant baculovirus stocks were pre-
plasmidsexpressingSARS-CoVproteins[27–29]orvirus-likepar-
paredbyinfectingSf9cellsatalowmultiplicityofinfection(MOI)of
ticle (VLP) based vaccines [30–34], however all of these approaches ≤0.01 pla queform ing unit s( p fu)p ercellandh ar vestedat 68–72 h
comewiththeirownsafetyconcernsandapprovalprocesses.There
postinfection(hpi).
arecurrentlynoapprovedvaccinesforMERS-CoV,butearlystudies

using a modified vaccinia virus and replication deficient MERS-

CoVhavebeenshowntoinduceantibodiescapableofneutralizing 2.3. RecombinantMERSandSARSSprotein

MERS-CoVinvitro[35,36].Ideally,vaccinesforhighlypathogenic

viruses,includingcoronaviruses,shouldbeabletobemaderapidly, MERS-CoVandSARS-CoVSproteinantigenswereproducedin
on dem and and in conjunction with a pp rove d ad juvant s using Sf9cellsat2– 3×1 06cells/m l infected withspe cificr ecombina nt

approvedtechniques[37]. baculovirus.InfectedSf9cellswereincubatedwithcontinuousagi-
Theem ergenceof bothSARS-CoVandMERS-CoVhighlightthe tationat27± 2◦Can dha rves tedat 68–72hpi byc entrifugatio nat
import ance of coro na virus es as pote ntia l human pa thogens t hat 4000× g for 1 5mi n.Sp roteinsw ere extrac ted fro mcellularmem -

can emerge at any time. Therefore, rapid methods for treatment braneswithanon-ionicdetergentandinsolublematerialremoved
and vaccina tio nare requ iredforthi simp ortantgro up ofviruses. by cen trifug a tion at 10 ,000×g fo r 30 min. S p roteins o ligomers

InthisstudywedescribeanovelmethodforcreatingSARS-CoVS werepurifiedusingacombinationofanionexchange,affinityand

andMERS-CoVSnanoparticlesthatinconjunctionwithadjuvant sizeexclusionchromatography.Duringpurificationthemajorityof

areabletoinduceaneutralizingantibodyresponseinmice. thedetergentisremovedallowingStrimerstoformhigherordered

protein–proteinmicellularnanoparticles.PurifiedSnanoparticles
were0.2micron filteredan dstored−80◦C .

2. Materialsandmethods
2.4. Analyticalmethods

2.1. Cellandviruses
MERS-CoVandSARS-CoVSwereanalyzedbySDS-PAGEusing

4–12%gradientpolyacrylamidegels(Invitrogen),stainedwithGel-
Vero E6 cells were originally obtained from the Ameri-
Code Blue stain reagent (Pierce, Rockford, IL) and quantified by
can Type Culture Collection (ATCC) and maintained in Minimal
scanning densitometry using OneDscan system (BD Biosciences,
Eagles Medium (MEM; Corning) supplemented with 10% heat-
inactiv atedFetal Bovine Serum(FB S;SAFC),1%l-G lutam ine(G ibco) Rockville, MD). Purified S proteins were tested for total protein
concentration (BCA bicinchoninic acid protein assay, Pierce Bio-
and1%Penicillin/Streptomycin(GeminiBio-Products).Spodoptera
chemicals) and particle size by dynamic light scattering using
frugiperdaSf9insectcells(ATCCCRL-1711)weremaintainedassus-
ZETASizerNano(MalvernInstruments,PA)usingstandard,man-
pensionculturesinHyQ-SFXinsectserumfreemedium(HyClone,
Logan,U T)at27± 2 ◦C. ufacturer recommended methods.

Mouse adapted SARS-CoV (MA15) has been previously
described [38]andw asgrownin VeroE6c ellsan dstor edat−80◦C. 2.5. Electronmicroscopy

MERS-CoV (Jordan) was obtained from the NIH in conjunction
withAFHS C-GEISan dNA MRU-3,w ithsp ecia lassi sta ncefromDr. Purified MERS-CoV and SARS-CoV S proteins were adsorbed
Moha reb.Allexp erim entswithl ivevi ruswer eperform edun der byflotation for2mino nto afreshlydi sc harged40 0-me shcarbon
biosafety leve l 3 condition s at t he U niver sity o f Maryland, Balti- par lodion-co ate d copp ergr id (Poly-S ciences,Wa rrington,P A).The
more. gridswererinsed withb uffer containing20 mMTris,pH 7.4, and

MERS-CoV(Jordan)Spikeproteinis99.8%identicaltotheMERS- 120mMKClandnegativelystainedwith1%phosphotungsticacid,
CoV(AlHassa 1)Spike prote insequ en ceuse dinthe na nop article then dri ed b y as piration. V irus-like par ticl es were visualize d on
clon ing. Wecon siderM ERS-Co V(AlHas sa1)a nd ME RS-CoV(Jor- aHit achiH -76 00transmi ssionelect ronmicro scope (HitachiH igh
dan)tob eho mologou sintheirS pike protein s.SA RS-CoV(Ur bani) T echnolo giesAme rica,Schaumb urg,IL)o peratingat8 0kVand dig-
Spike p rot ein is 99.9% id entica l to t he SARS- CoV (MA1 5) Spike itallycapture dwithaC CDcameraat 1K ×1Kresol ut ion (A dvan ced
protei n seque nce used in the na no part icle cloning . We co nsider Micro scopyTe chniq u esCo rp.,Dan ve r s, M A ).
C.M.Colemanetal./Vaccine32(2014)3169–3174 3171

2.6. Mouseimmunization Sproteinmoleculeswhenobservedbyelectronmicroscopy(Fig.1C

andD)anddynamiclightscattering(datanotshown).

TheimmunogenicityofpurifiedMERS-CoVandSARS-CoVSpro-

teinnanoparticleswereevaluatedinBALB/cmice(6–8weeksold), 3.2. ImmunogenicityofSproteinnanoparticles

purchasedfromHarlanLaboratories,Inc.(Fredrick,MD).Mice(10

pergroup)received2vaccinationsbyintramuscularinjectioninthe In vaccine development, an effective vaccine should have a

quadricepsmusclesondays0and21.MiceimmunizedwithMERS- strong neutralizing response against the target, i.e. the dilution
CoVSprote inreceiv ed eithe r 1,3o r1 0(cid:2)go fSproteina lone or1or ofanti bodyatwhic hiteffecti velyneu tral izesthe vir ussh ouldbe
3(cid:2)g M ERS-Co VSprote inwit he it he r1 20 (cid:2)g a luminu mhyd rox i de hi gh.Inthes ee xperim en ts,10mice pergroupw ere inocu latedw ith
(A lH ydrogel,Bre n ntagAG )or5 (cid:2)gM atrix M1 (Isconova ,Sweden). MERS -C oVor SARS-CoVS pro teins and serum wa stestedag ainst
Miceimmun izedwith SARS -C o VS protein rec eived3(cid:2)g Sprotein thehomolo go usvirus.
alone orSprotein with either120 (cid:2) galum inumhyd ro xid e (Alum)
or5(cid:2) gM a trixM1 .Sera werec olle cted onday0,2 1and45fo rsero- 3.3. GenerationofneutralizingantibodiestoSARS-CoVinBALB/c

logicalanalysis.Bloodwascollectedviatheretro-orbitalrouteand miceusingSnanoparticles

serumwasobtainedbycentrifugationofwholeblood.

AllanimalprocedureswereinaccordancewiththeNRCGuide Previousworkhasshownproductionofaprotectiveneutraliz-

for the Care and Use of Laboratory Animals, the Animal Welfare ingantibodyresponseinmiceinoculatedwithchimericSARS-CoVS

Act,andtheCDC/NIHBiosafetyinMicrobiologicalandBiomedical nanoparticlesincombinationwithAlumadjuvant[30].Inthisstudy

Laboratories. wealsousedMatrixM1adjuvanttoassessinductionofneutralizing

antibodyresponsesinmice.
2.7. Coronavirusneutralizationassays When 3(cid:2)gofSA RS -CoVSproteinisusedwithoutadjuvantthe
neutralizi ng an ti body conc en tration in the sera at day 21 po st-

Mouseserawerediluted1:20inVeroE6cellgrowthmediaand inoculationislow(GMT=86)andthisdoesnotchangeatday45
furtherdil uted down to1:4 0960 in atw o-f olds eries.M ERS-C OV- post-inocul ati on ( GMT= 8 0), indic atin g tha t SA RS-CoV S prot ein
Jordan orSARS -CoV( MA 15)was ad de datafin alconc entrationof withoutadjuvan tdoes n otef fectivelyin duce anti-SARS -C oVneu-
3950 T CID /ml and incuba ted f or 30m in at roo m temperatu re. tralizing antibody (Tab le1 andFig.1) .When Alumisused asan
Mock infec5t0edm ouse seraand MER S-C oV- Jor danor SARS-CoVin adjuvant inconjun ctionw i th3 (cid:2)go fSA RS-Co VSpr ote in,th ere is
VeroE 6media serveda sneg ativ eandpositivecontr ols respective ly. a statistic al ly significant 7–1 1 fold in crease in n eutralizi ng ant i-
The i nh ibitory capac ity of each seru m dilut ion was assessed by b ody(forexam ple,at21d ayspo st-in oculation G MT=970;p< 0.01)
TCID assayan dtheser um dilut ionatw hichME RS-C oV-Jordan or andw hen MatrixM 1 ad juvan tisusedinconju nctio n with 3 (cid:2)gof
SARS5-0CoVwa sinh ibi tedby5 0%was rec orded. Thegeometricme an SAR S-CoV S,there is astatistic all ysign ifi cant36–39f oldin c rea se
titer(GMT )for eachgrou po fm icew ascalcula ted andpresen ted± inneutrali zin gant ibo d y(forexamp le,at21da yspost -ino culation,
the9 5%con fide ncei nterva l( CI). GM T=3152;p <0.001),i ndic atingthat M atr ixM 1isamoreeffec-
tivea d juvant t h anAlum .Noneof thev accinat ions w it hSAR S-CoV

S,showedanysignificantdifferenceinneutralizingantibodytiter
2.8. Coronavirus TCID50 at 45days post -inoculatio ncompare dt o21dayspos t-inocula tion,

suggestingthattheneutralizingantibodyresponsedoesnotchange
VeroE6cellswereseededinto96-wellplates(USAscientific)
at1×10 4ce llspe rwel landwe recu lturedo vernig htat3 7◦C.Cells over the vaccination course (Fig. 2).
These data confirm previous observations that Alum signif-
wereinfectedwitha5-folddilutionseriesofviruscontainingmedia
icantly enhances the production of anti-SARS-CoV neutralizing
intriplicateandculturedforafurther48h.Plateswerefixedin4%
antibodiesinmice[30].ThisalsodemonstratesthattheMatrixM1
paraformaldehyde(Sigma–Aldrich)for5minatroomtemperature
adjuvantismoreeffectiveatinducingananti-SARS-CoVneutraliz-
andthenstainedwith0.05%crystalviolet(Sigma–Aldrich)in20%
ingantibodyresponseinmice,suggestingthatMatrixM1canbe
methanol(Sigma–Aldrich)for30minatroomtemperature.Plates
usedasaneffectivevaccineadjuvantwithSARS-CoVS.
werethenwashedtwiceinwaterandcelldeathwasassessedbythe

presence(livecells)orabsence(deadcells)ofcrystalvioletstain.
3.4. GenerationofneutralizingantibodiestoMERS-CoVinBALB/c
Viralloadwascalculatedbasedonthedilutionatwhichthemedia
miceusingSnanoparticles
killed50%ofthecellsusingtheTCID50formula[39].

When MERS-CoV S was inoculated into Balb/C mice without
2.9. Statisticalanalysis adjuvant, levelsofne ut raliz ingantibod ytoM ERS-C oV(Jo rdan)at
day21po st-ino cul ationwerel ow(forex am ple1(cid:2)gS GMT=4 9).
Neutralizationtiterswereanalyzedusinganon-parametricone- The se d ata demonstrate that inoc ulat ion with M ERS -C oV S p ro-
wayANOVA.Stati stical signifi cancew asassu m edwhenp<0.0 5. teinal onei snotaneffec tivem ethodford evelo pmentofa ro bust

neutralizingantibodyresponse.Atday45post-inoculationwith-

3. Results outadjuvant,levelsofneutralizationofMERS-CoV-Jordanare3–7

foldhigherthanatday21andarenotstatisticallysignificant(for
3.1. MERSandSARSSproteincharacterization exam ple1(cid:2) gME RS -SG MT =32 0;p >0. 05).Theamo untofMER S-S

proteindidnothaveanysignificantlyeffecttheGMToftheneutral-

Full-length recombinant MERS and SARS S protein were pro- izingresponses,butthereisatrendtomoreconsistentresponses

duced in the baculovirus insect cell expression system. The S withhigherdosesofMERS-S.

proteinswereextractedfrominfectedcellspelletwithnon-ionic WhentheadjuvantAlumisusedinconjunctionwithMERS-CoV

detergentandpurifiedbycolumnchromatographyaspreviously Sforinoculation,thelevelsofneutralizingantibodyatday21post-

described[30].Purifiedfull-lengthMERSandSARSSproteinshad inoculationaresignificantlyhigherthanfoundinserafrommice
an appare nt m olecular weight of 180kD a a nd 16 0 kDa by S DS- inoculated with MERS-S alo ne (for exam ple 1 (cid:2)g ME RS-S alone
PA GE, respe ctively (Fig . 1A an d B). P urifi ed M ERS and SA RS S GMT=49co mpa redto1(cid:2) gMER S-Sw ithAlum GM T =735;p< 0.05),
protei nsformed∼2 5nm dia mete rpa rticlesco nsistin gofm ultip le indic at ing that Alu m p rom otes pr oduc tion of an ti -MER S -C oV-S
3172 C.M.Colemanetal./Vaccine32(2014)3169–3174

Fig.1. Purificationofcoronavirusspikeproteinsandgenerationofspikenanoparticles.(AandB)PurifiedMERS-CoV(A)andSARS-CoV(B)Sproteinmicelle,eachproteinis

loadedonthegelintriplicatelanes.Leftpanel:coomassiebluestain,rightpanel:westernblotusingrabbitanti-MERSS(A)orrabbitanti-SARSS(B)asprimaryantibody.

Proteinmarker:precisionplusproteinmolecularweightmarker(Bio-Rad,Hercules,CA).(CandD)PhosphotungsticacidnegativestainelectronmicroscopyimagesofMERS

(C)andSARS(D)Sproteinmicelle.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

neutralizingantibodies.Thereisnosignificantdifferencebetween thereisnosignificantdifferencebetweenneutralizingantibodiesin
dosing regim ens consis ting o f 1(cid:2) g of MERS -CoV-S or 3(cid:2)g of these ra fro mday21c ompared today45 (forexample 1(cid:2)gMER S-
MERS-C oV-Sinth epresence of Alu m (fo rexample,atd ay 21 ,1 (cid:2)g Spl usA luma tda y2 1GMT=73 5 com par edt oday45 G MT =640;
MERS-Splus Alu mG MT=735 co mpar edto 3(cid:2)gME RS -Spl usA lu m p >0.0 5),ind ica ting th at,in th epr esenceof Alu m,t he vaccin a tion

GMT=368;p>0.05),suggestingthatlowerdosesofMERS-Scanbe coursecausesearlyproductionofneutralizingantibodiesthatare

usedforeffectivevaccinationinthepresenceofAlum.Furthermore, maintained.

Table1

Coronavirusneutralizingantibodytiters.

Vaccine(Sprotein) Dose((cid:2)g) Adjuvant Day GMT 95%LCL 95%UCL

MERS-CoV 1 None 21 49 17 140

45 320 110 934

3 21 43 22 85

45 299 193 461

10 21 65 33 126

45 243 104 567

1 Alum 21 735 419 1,291

45 640 251 1,630

3 21 368 171 792

45 905 505 1,624

1 MatrixM1 21 3,378 1,804 6,325

45 1,940 1,139 3,306

3 21 1,194 542 2,634

45 1,689 831 3,432

SARS-CoV 3 None 21 86 32 228

45 80 38 168

Alum 21 970 497 1,894

45 640 330 1,240

MatrixM1 21 3,152 1,624 6,117

45 3,152 1,864 5,329

Thevaccinegroupsareshownthatweretestedforneutralizingantibodytiter.Dose,adjuvanttype,dayofbleedingandgeometricmeantiterwithconfidenceintervalsare

shownforeachgroup.N=10foreachdosing/adjuvantgroupshown.
C.M.Colemanetal./Vaccine32(2014)3169–3174 3173

Fig.2. Neutralizationtitersofcoronavirusspikevaccinatedmice.SerumfrommicevaccinatedwiththenotedmixofspikeproteinandadjuvantwasanalyzedbyTCID50

assayforneutralizationcapabilityandgeometricmeantiter(GMT)graphedforallgroups(10micepergroup).Errorbarsindicate95%confidenceinterval.Starsdenote

statisticallysignificantdifferences(p<0.05)notedinthetextasdescribedinSection2.

WhenMatrixM1adjuvantisusedinconjunctionwithMERS- These data demonstrate that there is only limited, if any,

CoVSforinoculation,theincreaseinlevelsofneutralizingantibody cross-protectioninmouseseracontainingneutralizingantibodies

atday21post-inoculationaresignificantlyhighercomparedtothe inducedagainstMERS-SorSARS-S.

serafrommiceinoculatedwithMERS-Salone(27–68fold)and3–4

foldhigherthanserafrommiceinoculatedwithMERS-SplusAlum
(for exampl e1(cid:2) gME RS-S alone GMT=49c ompa redto1 (cid:2)gM ERS- 4. Discussion
Swi thAlum G MT =735co mpare dto 1 (cid:2)g MERS-Sw ith M at rixM1

GMT=3378; p<0.001), indicating that Matrix M1 promotes pro- There is a need in vaccine production for a rapid process of

duction of anti-MERS-CoV-S neutralizing antibodies higher than manufacturing vaccines to emerging pathogens [37]. The recent

thatseenwhenAlumisusedastheadjuvant.Thereisnosignifi- emergencesofSARS-CoV,in2003,andMERS-CoV,in2012,have
cant diffe renceb etwe en dosin gm ice with1(cid:2)g ofME R S-C oV-Sor demonstrate d the import an ce of t he C oronavirida e v irus fa mily
3(cid:2)g of MERS-C oV-S in t he pre sence of M at rix M 1 (for examp le, as an importa nt g roup of eme rg ing p athogens. Pre vious efforts
at da y2 1,1(cid:2)gMERS -S plu sMatrixM 1 GMT=3 378 com paredto to dev elopvaccin estoS AR S-CoVhav eencompas sedmany meth-
3(cid:2) gM ERS -S pl usMatrix M1 GMT=1 194 ;p>0 . 05),a gainsugge st- od s, but ha ve yielde d variable re sults. In this study we de scribe

ingthatlowerdosesofMERS-Scanbeusedforeffectivevaccination a novel method for development of protein nanoparticles carry-

inthepresenceofMatrixM1.Furthermore,thereisnosignificant ing the spike proteins from SARS-CoV and MERS-CoV that were

differencebetweenneutralizingantibodiesintheserafromday21 abletoinducesignificantneutralizingantibodyresponsesinmice.
compared today45 (forexamp le1(cid:2)gMER S- Sp lusM atrix M1 at The na noparti cles contai n only MERS -S or SAR S-S and n o other

day21GMT=3378comparedtoday45GMT=1940;p>0.05),sug- potentially immunogenic viral or insect proteins. The formation

gestingthat,inthepresenceofMatrixM1,thevaccinationcourse ofamphiphilicmembraneproteinsaggregates(proteinnanoparti-

causesearlyproductionofneutralizingantibodiesthataremain- cles),usedinthisstudy,whicharerelativelyhomogeneousinsize,

tained.ThesedatademonstratethattheMERS-Svaccineplatform solubleinaqueousmedia,andusedasaplatformforvaccineswas

can be used to induce a neutralizing antibody response in mice. firstdescribedbySimonsetal.[40].Recentlyarespiratorysyncytial

We also demonstrate that the Matrix M1 adjuvant significantly virus(RSV)fusion(F)glycoproteinnanoparticlevaccinewasshown

enhances the level of neutralizing antibody produced compared tobehighlyimmunogenic,inducingprotectiveimmuneresponses

toAlum. againstconformationalepitopes[41].Inaphase1humanstudythe

RSVFnanoparticlevaccinewaswelltoleratedandinducedneutral-

izingantibodiesatlevelsassociatedwithdecreasedhospitalization

3.5. Cross-protectionactivityofanti-MERS-CoVand fromRSVinchildren[42].

anti-SARS-CoVsera We have shown that mice vaccinated with coronavirus S

nanoparticles produced high levels of neutralizing antibodies

Todetermineifthereiscross-neutralizationofSARS-CoVSanti- against the homologous virus, but did not offer cross-protection

seraagainstMERS-CoVinfection,andviceversa,wetestedserathat againsttheheterologousvirus.Thesedatasuggestthatthecoro-

offeredstrongprotectionagainstthehomologousvirusagainstthe navirus S nanoparticles are able to elicit specific anti-SARS-CoV
heterol ogousv irus.There fore,ser afr ommiceinoc ulate dwith3 (cid:2)g or MER S- CoV antibody resp onse s in mice , but th at there is not

ofMERS-SorSARS-SincombinationwithMatrixM1adjuvantwere a general anti-coronavirus response that can be used to pro-

testedagainstSARS-CoVandMERS-CoVrespectively. tect against multiple coronaviruses. Given that, so far, only one
The serafro mmiceino cula tedwith3(cid:2) gMERS-Sincombination coro navirus hasemer gedatatime, thissh ouldb e suffi cient, but

withMatrixM1wasunabletoneutralizeSARS-CoV,asnoneofthe furtherworkwilldetermineifnanoparticlevaccinescanbeusedin

dilutionstestedhadanyeffectonthetiterofSARS-CoV.Thesera combination.
frommic einocu lated wit h3(cid:2)g SAR S-S inco mb inationwit hM atrix Wealsoinvestigatedtheuseofadjuvantstoboostproduction

M1wasonlyminimallyabletoneutralizeMERS-CoVaboveback- ofneutralizingantibodytocoronavirusS.WhenweusedAlumor

ground,with9outof10miceshowinglowlevelneutralizingeffects MatrixM1,neutralizingantibodylevelsweresignificantlyboosted

againstMERS-CoV,butnoneshowingcompleteneutralizationand (15foldwithAlumand68foldwithMatrixM1)overlevelsofneu-

allneutralizingtoamuchlowerextentthananti-SARS-CoVsera tralizingantibodyproducedfromSnanoparticlesalone.Alumisa

againstSARS-CoVoranti-MERS-CoVseraagainstMERS-CoV(data commonadjuvantthathasbeenusedpreviouslytoboostneutraliz-

notshown). ingantibodiestoSARS-CoVinaVLPvaccinestrategy[30],buthere
3174 C.M.Colemanetal./Vaccine32(2014)3169–3174

weshowthatAlumalsosignificantlyboostsresponsestoSARS-CoV [19] QinE,etal.Immunogenicityandprotectiveefficacyinmonkeysofpurified
Sna nopa rticle sand also toMERS-Co VSnan oparticles in mice. inac tiv ate dV ero-cellSARSvac cine .Vaccine2 006;24(7 ):1 028–34.
[20] Lin JT, et a l. Safety and immuno genicity from a phase I trial of inactiv-
NovMavatarxix, AMB1( foisr maa lslyapIosncoinn obvaaseAdB ,aUdjpupvsaanlat mSwaenduefanc)t.uQreudil labjya 2at0e0 d7 ;s1e 2v(e7 r)e:1 a 1c0u7t–e1 3re .spir atory syndrome c orona vi rus vac ci ne. A nti vir Ther

saponinsareextractedfromthebarkofthetreeQuillajasaponaria [21] BollesM,etal.Adouble-inactivatedsevereacuterespiratorysyndromecoro-
naviru sva cc ine p rovidesincomplete protec tionin miceandin ducesincr eased
Molina and two fractions, A and C, are independently formu- eosinop hilic pro inflamm atory pulm onary resp on se up on challeng e. J Virol
latedwithphospholipidsandcholesteroltocreatematrixparticles 2011;85(23) :12201–15.
(∼40 nmca gelikestructu res) .MatrixM1 isc ompri sedoft wocom- [22] LiJ,etal.Immunogenicityandprotectionefficacyofmonomericandtrimeric
pone nts, Matr ix-A and Matrix- C at an 85/ 15 ratio [43] a nd has been Irme cmo mun boinl a2n0t1 S3A;2R6S( 2co):r1o2n6a–v 3ir2u.s spike prote in subun it vaccine cand ida tes. Viral
shown to be a potent adjuvant in clinical trials [44]. The data in [23] HeY,etal .Receptor-bindingdomainofSARS-CoVspikeproteininduceshighly
thisstu dy dem onstrate thatMat rix M1ism oreef fectiv etha nAlu m pot en t neu tralizingantibod ies:imp lic ationford evelo pingsu bunitva ccine.
at b oostin g the neutra lizin g antib ody re spon se to cor onavi rus S [24] BShioimcheB mS, eBtioapl.hSyusb Rlien sg Cuoamlimmmunu 2n 0iz0a4t;io3n24w(2it)h: 7r7e3c –o8m1b.inanta denoviru sencod-
nanoparticlesinmice,especiallywithMERS-CoVspike. ingSA RS -Co V spikeprote ininducessyst emic andmucosal immunityw ithout
Further stu di es wi ll be neede d to determine if coronavirus S red irectionof thevi rustoth ebrain. VirolJ20 12;9 :215.
nanopartic levaccin ation sb lockcor ona virusreplic at ionandpatho - [25] BishtH,et al. Sev erea cu ter espirat orys y ndromecoronavirusspikeprotein
expre sse db ya ttenuat edvac ciniavirusp rotectively immunizesm ice.P rocNatl
gmeondeeslisc uinr rveinvtol,y aesx aisntism,aaslM mEoRdSe-lCso aVred odeesvnelootpreedp.l iNcaot esminamll iacne,imfera-l [26] AFecnagd QSc,ie tUa Sl. AB a2c0u0lo4v;1ir0u1s( s1u7r)f:a6c6e4d1 i–s6p.la yofSARScor onavirus(SA RS-C oV)s pike
rets,or hamsters .[45,46 ]. Wehavede mons trat edthatco ro navir usS prote in an di mmunogeni cityoft hedispl ay edpro teininmice models.DN ACell
Biol200 6;25 (12):668–73.
ninanmoipcea,rtaincliems aproer atabnlet tfior ssttismteuplattoew naerudtsravlaizcicning eandteivbeoldoyp rmesepnot.nses [27] pYraontg e cZtYiv, eeitm aml. uAn DitNyAin vmaciccein.Ne aintudruec2e0s 0S4A;R42S 8c(o6r9o8n2a)v:5ir6u1s– n4e.utralization and
[28] MartinJE, etal.ASA RS DNA vaccine inducesneutralizingantibodyandcel-
lularim m un ere sp onses inh ealthya dultsin aPhaseIclin icaltrial. Vac cine
Acknowledgement 2008 ;26(50):6 338–43.
[29] LuB,etal.Humoralandcellularimmuneresponsesinducedby3aDNAvaccines
ThisprojecthasbeenfundedinpartbyasupplementtoNIHRO1 aga in st se vereacut eres pirator ysyndrom e(SARS) orSARS- lik ec oron avirusin
mice.C linVacc ineIm munol200 9;16(1):73 –7.
AI095569 (MBF). [30] Liu Y V, e t al. Ch imeric se vere acute respiratory syndrome coronavirus
(SA RS-C oV) Sg lycoprotei nandin fluenz amatrix1ef ficientlyfo rmvirus-like
References particles(V LP s)thatprotec tmi ceagainst challen g ewithSAR S-CoV .Vaccine
2011;29( 38):66 06–1 3.
[31] Lu X, et al. Immune responses against severe acute respiratory syn-
[[12]] DJSaoicsnk g2w0o1Do2,d;5 MP6a(Wr4k).: 6RB3e.4vi–eP4wo1r .coifn ienfeecptiidoeums bicrondciahrirtihso veiarusv iarruosu:nda thceo mwporreldh.e Anvsiiavne 2d0ro 0m7;e1 2c2o (r4o)n:4 a9v6ir–u5s0 2in.d uced by v irus-like particle s in m ice. Immun ology
r2e0v1i2e w;4 4o( 2f ):m16o7l e–c7u5 la.r epide miology, d iagnosis, a nd vac cin es. Virus Genes [32] pdBrraioit tiBeceicnte sal fllsr. o.VPmilro udSsi-fOfleinkreee n2pt0a o0rrt8iig;c3lien(7ss ) od:efe 2mS6Ao8Rn5Ss.-tlriaktee csotirmonualavtiirnugs afoctrimvietyd ibny h mumemanb draenne-
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[7] 2nPo0eso0e n5 ;h7oL9 rL:s,2 e 0se0ht 1 o–ea 9lb.. atIsd .e Pnrtoific cNaatitol nA caodf Sa c i Un oSv Ael 2 0c 0o5ro;1n0a2v(ir3u9s) :1i4n0 4b 0a–ts5.. J V irol [35] 2MA0lim1d3da;zl4ea( nE5 a)F:s,e te0 tr0 ea6sl5p. 0Eir–na7gto1inr3ye. esryinngd rao rmepe liccoartoionna-vciroums paest ean vt:a cpcrionpea gcaantidoind-adteef. emctBivioe
[36] SongF,etal.MiddleEastrespiratorysyndromecoronavirusspikeproteindeliv-
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[12] 2M0a0s6te;6rs6 :1P9S3. –2T9h2e. molecular biology of coronaviruses. Adv Virus Res cinostetoctn crae tllss. PfoLor mSO pnreot2e0in1 2n; 7a(n1o1p)a:ert5i0cl 8e5s2 t.ha t indu ce protective immunity in
[[1134]] tsBQhpeieuilkoi rMeu nzp, eaerurotd ttar elSa.i ,nlA ie.zn tVat atiibirlo.uo Mnsdeyaesc cr 2thei0svap1nito2iisen;m4ss.(es6Ms )o t:ifoc1 cr 0ioon1brd1oei–nvs3iaId3nvu.ifreaucl stp 2croe0tl0le 5ein;n7st( ro5yf– Sm6A)e:Rd8S8i ac2to–erd9o. nbayv tihrues v airnadl [42] r2Ge0lse1pn3inr;3a t1Go(Mr3y), : 5es2ty 4na–c l3y. 2tSi.aal f etvyir uasn df uismiomn unporogteeniinc itnya noof paa rStifc9l ei nvseacctc inceel. l-Vdaecricvinede
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[18] r2Sup0sr0 uv6ta;h2c c4Mi(n5,e )e :s6tt 5iam2l.– uA6la 1dt.eosu nbeleu-tirnaalicstiinvga taendd wprhootleec tvivireu as nctaibnoddidya rtees pSoAnRsSe sc.o Vroacncaivnie- 2ce0p1t3ib;9le5 : t4o0 8th– e1 2 M.id dle E ast R espi ratory Syndrom e Corona virus. J G en V irol