2014 Protection from SARS coronavirus conferred by live measles vaccine expressing the spike glycoprotein

2014·1.88 MB·English

Virology452-453(2014)32–41
ContentslistsavailableatScienceDirect
Virology
journal homepage: www.elsevier.com/locate/yviro
Protection from SARS coronavirus conferred by live measles vaccine
expressing the spike glycoprotein
Nicolas Escrioua,c,d,n,1, Benoît Callendreta,c,d, Valérie Lorina,c,d,1, Chantal Combredetb,c,
Philippe Marianneaue, Michèle Févrierb,c, Frédéric Tangyb,c,nn
aInstitutPasteur,UnitédeGénétiqueMoléculairedesVirusàARN,DépartementdeVirologie,F-75015Paris,France
bInstitutPasteur,UnitédeGénomiqueViraleetVaccination,DépartementdeVirologie,F-75015Paris,France
cCNRS,UMR3569,F-75015Paris,France
dUniv.ParisDiderot,Sorbonne,ParisCité,EA302,F-75015Paris,France
eInstitutPasteur,UnitédeBiologiedesInfectionsViralesEmergentes,DépartementdeVirologie,F-69007Lyon,France
a r t i c l e i n f o a b s t r a c t
Articlehistory: TherecentidentificationofanovelhumancoronavirusresponsibleofaSARS-likeillnessintheMiddle-
Received21October2013 EastadecadeaftertheSARSpandemic,demonstratesthatreemergenceofaSARS-likecoronavirusfrom
Returnedtoauthorforrevisions ananimalreservoirremainsacrediblethreat.BecauseSARSiscontractedbyaerosolizedcontamination
7November2013 oftherespiratorytract,avaccineinducingmucosallong-termprotectionwouldbeanassettocontrol
Accepted3January2014
newepidemics.Tothisaim,wegeneratedliveattenuatedrecombinantmeaslesvaccine(MV)candidates
expressingeitherthemembrane-anchoredSARS-CoVspike(S)proteinoritssecretedsolubleectodomain
Keywords: (Ssol). In mice susceptible to measles virus, recombinant MV expressing the anchored full-length S
Coronavirus induced the highest titers of neutralizing antibodies and fully protected immunized animals from
Severeacuterespiratorysyndrome
intranasal infectious challenge with SARS-CoV. As compared to immunization with adjuvanted
Spikeglycoprotein
recombinantSsolprotein,recombinantMVinducedstrongerandTh1-biasedresponses,ahallmarkof
Measlesvaccine
liveattenuatedvirusesandahighlydesirablefeatureforanantiviralvaccine.
&2014ElsevierInc.Allrightsreserved.
Introduction Chinesecivetsandracoondogs(Guanetal.,2003)fromwhichthe
viruswaslikelyintroducedintothehumanpopulation(Kanetal.,
Severeacuterespiratorysyndrome(SARS)isanewlyemerged, 2005; Song et al., 2005). Other SARS-CoV-like viruses sharing
humaninfectiousdiseasethatfirstappearedinChinainlate2002. more than 88% nucleotide identities with SARS-CoV have been
Between November 2002 and July 2003, the virus spread to 29 isolatedfromChinesehorseshoebats,whichhavethereforebeen
differentcountries on5 continentsand wasresponsible for8096 proposed to represent a natural reservoir host of SARS-CoV
clinical cases, leading to 774 deaths (WHO, 2004). WHO case (Lietal.,2005).Todate,endemicbatSARS-CoV-likeviruseshave
management guidelines and restricted travel advices allowed to alsobeendetectedinAfricaandEurope(forreview:Balbonietal.,
bring SARS under control by July 2003. The etiological agent of 2012), and reemergence of a SARS-like disease from an animal
SARS was identified as a novel coronavirus, named SARS- reservoir remains a credible public health threat. An efficient
associated coronavirus (SARS-CoV) (Drosten et al., 2003; Ksiazek vaccine would be the most effective way to control a new
etal.,2003)thatisgeneticallydistinctfrompreviouslycharacter- epidemic.
ized members of the Coronaviridae family (Rota et al., 2003). Similar to other coronaviruses, SARS-CoV is an enveloped,
During the 2002–2003 outbreak, SARS-CoV has been isolated in positive-stranded RNAvirus whose replication takes place in the
cytoplasm of infected cells. Viral particles are composed of four
majorstructuralproteins:thenucleoprotein(N),thesmallenvel-
nCorrespondenceto:UnitédeGénomiqueViraleetVaccination,InstitutPasteur, ope protein (E), the membrane protein (M), and the large spike
25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.:þ33140613901;
fax:þ33140613167. protein (S). The spike protein is a type-I transmembrane glyco-
nnCorrespondenceto:UnitédeGénomiqueViraleetVaccination,InstitutPasteur, proteinof1255aminoacids.Itassemblesintohomotrimersatthe
25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.:þ33145688770; surface of viral particles, and gives the virion its crown-like
fax:þ33140613167. appearance (Neuman et al., 2006). Each monomer (180kDa) is
E-mailaddresses:[email protected](N.Escriou), composed of a signal sequence (a.a.1–14), a large ectodomain (a.a.
[email protected](F.Tangy).
1Presentaddress:InstitutPasteur,UnitédeGénomiqueViraleetVaccination, 15–1190) with 23 potential N-glycosylation sites,a transmembrane
DépartementdeVirologie,F-75015Paris,France. domain (a.a. 1191–1227), and a short cytoplasmic tail of 28 a.a.
0042-6822/$-seefrontmatter&2014ElsevierInc.Allrightsreserved.
http://dx.doi.org/10.1016/j.virol.2014.01.002
N.Escriouetal./Virology452-453(2014)32–41 33
(Ksiazeketal.,2003;Rotaetal.,2003).TheSproteinisresponsible Results
forviralentry,bindstothecellularreceptorACE2(Lietal.,2003)and
mediates fusion between the viral and cellular membranes RecombinantMVSchw-SARSvirusesexpresstheSARS-CoVspike
(Petit et al., 2005; Simmons et al., 2005). Structurally, the glycoprotein,secreteitssolubleectodomain,andreplicateefficiently
N-terminal globular head (S1 domain, a.a. 1–680) contains the
receptor-binding region (Wong et al., 2004), and the membrane- Wesynthesizedhumancodon-optimizedgenesencodingthefull-
anchored stalk region (S2 domain, a.a. 727–1255) mediates oligo- length,membraneanchoredSARS-CoVspike(S)proteinanditsentire
merizationandfusion(Petitetal.,2005).Similarlytoothercorona- ectodomain (residues 1–1193, hereafter designed as Ssol), which is
viruses, cleavage of the S protein by proteases into its S1 and S2 expressedinmammaliancellsasasolubleandsecretedpolypeptide
subunits is required foractivation of the membrane fusion domain (Callendret et al., 2007, Callendret et al., unpublished results). Their
following binding to target cell receptors (Matsuyama et al., 2010; lengthrespectsthe“ruleofsix”,whichstipulatesthatthetotalnumber
Simmonsetal.,2005). ofnucleotidesintotheMVgenomemustbeamultipleof6(Calainand
Duetoitscriticalinvolvementinreceptorrecognition,aswell Roux, 1993). MV editing- and polyadenylation-like sequences were
asvirusattachmentandentry,theSproteinisthemostpromising mutated(LambandKolakofsky,2001;Schneideretal.,1997).BothS
andstudiedcandidateantigenforSARS-CoVvaccinedevelopment. andSsolsequenceswereinsertedasanadditionaltranscriptionunit
Itisthemajortargetforneutralizingantibodiesinhumanpatients (ATU) into MV vector (pTM-MVSchw plasmid), which contains an
(Heetal.,2005;Nieetal.,2004)andinanimalmodels(Buchholz infectiousMVcDNAcorrespondingtotheanti-genomeoftheSchwarz
et al., 2004; Tripp et al., 2005). Passive transfer of IgG from vaccine strain(Combredetetal., 2003)(Fig.1A).The resulting pTM-
convalescentSARSpatientsenhancedtherecoveryofacutephase MVSchw-S and pTM-MVSchw-Ssol plasmids were transfected into
patients when administered within 15 days after the onset of helper 293-T7-MV cells as previously described (Combredet et al.,
symptoms(Chengetal.,2005;Yehetal.,2005).Administrationof 2003). The corresponding recombinant measles viruses MV-S and
S-specific antibodies, including monoclonal antibodies, to naïve MV-Ssol were successfully rescued as indicated by the formation of
animals conferred protection against a subsequent SARS-CoV syncytia,andthenpropagated inVerocell culture.We analyzedthe
infection, demonstrating that the antibodies alone can protect replication of MV-S and MV-Ssol viruses on Vero cells by using the
againstSARSinmice(Bishtetal.,2004),hamsters(Robertsetal., sameMOI(0.01)thanforstandardMVstockproduction(Fig.1B).The
2006), ferrets (ter Meulen et al., 2004) and Rhesus macaques growthofMV-Ssolwasonlyslightlydelayed,comparedwiththatof
(Miyoshi-Akiyama et al., 2011). Accordingly, several candidate parentalemptySchwarz MV(MVSchwarz).Thefinalyield,routinely
vaccines relying on the induction of spike-specific neutralizing obtained at 60h post-infection, was high and identical to that of
antibodies, including DNAvaccines (Callendret et al., 2007; Yang parentalMVSchwarz(106TCID50/ml).ViralgrowthandyieldofMV-S
et al., 2004), live viralvectors (Buchholz et al., 2004; Chen et al., weremoreaffectedthanthatofMV-Ssol.Thismaybeduetoreduced
2005; Kapadia et al., 2005), live attenuated vaccines (Lamirande MVbuddingbecauseoftheinsertionoffulllengthSatthesurfaceof
et al., 2008), subunit vaccines (Bishtet al., 2005; He et al., 2006; theinfectedcells,asalreadyobservedforMVexpressingmembrane-
Zhou et al., 2006) and inactivated virus vaccine (Stadler et al., anchoredformsofHIV1gp160(Lorinetal.,2004).
2005;Zhouetal.,2005),havebeenreportedtoinduceaprotective We analyzed the expression of SARS-CoV spike antigens by
immune response in various animal models. Onlya few of them indirectimmunofluorescence(IFA)ofVerocellsinfectedbyrecom-
havebeenevaluatedinphaseIclinicaltrialsand,lackinganatural binant viruses and by immunoblotting of infected-cell lysates. At
challenge, there is no data on efficacy in humans (Roberts et al., 48hpost-infection,IFAperformedonpermeabilizedcellsusingan
2008;RoperandRehm,2009). anti-Shyperimmune mouse asciticfluid revealedastrongexpres-
An ideal vaccine against SARS should induce long-lasting pro- sion of both full-length S and entire ectodomain Ssol of the spike
tectiveresponsesafterasingleadministration,beproducedatlow protein along the compartments of the secretory pathway in
costandscaleduptomillionsofdoses.Liveattenuatedvaccinesare measles-induced syncytia (Fig. 1C, upper panels). When infected
particularlyappropriate for mass vaccination as theyare inexpen- cellswerenotpermeabilizedbeforelabeling,onlythecellsurfaceof
sive tomanufacture andinduceastrongimmunityandlong-term MV-S induced syncytia was readily stained, indicating that the
memory after a single injection. To evaluate such a vaccine membrane-anchored S is efficiently transported to the surface
approach, we previously developed a vector derived from the (Fig. 1C, lower panels). Western blot analysis of cell lysates and
live-attenuated Schwarz strain of measles virus (MV) (Combredet supernatants using rabbit anti-S polyclonal antibodies confirmed
etal.,2003).MVvaccineisalive-attenuatednegative-strandedRNA the expressionofS and Ssol proteinsinrecombinant MV-infected
virus proven to be one of the safest and most effective human Verocellswiththeexpectedapparentmolecularmassof(cid:2)180kDa
vaccines. Produced on a large scale in many countries and dis- (Fig.1D, left panel). Under reducing SDS-PAGE conditions the full
tributedatlowcostthroughtheExtendedProgramonImmuniza- lengthSproteinmigratesasadoublet,whichwasdescribedastwo
tion(EPI),thisvaccineinduceslife-longimmunitytomeaslesafter differentially glycosylated forms (Song et al., 2004). The lighter
oneortwoinjections.WepreviouslyshowedthatMVvectorstably productwassuggestedtobeanER-residentformof theglycopro-
expresseddifferentproteinsfromHIVandflavivirusesandinduced tein and the heavier a Golgi-processed form containing complex
strong and long-term transgene-specific neutralizing antibodies carbohydrates. Other bands of lower molecular weight were also
andcellularimmuneresponses,eveninthepresenceofpreexisting observedthatprobablycorrespondtominordegradationfragments,
immunitytoMV(Brandleretal.,2007,2013;Despresetal.,2005; since they were not present at earlier time points (not shown).
Guerboiset al., 2009; Lorin etal., 2004). In the present study, we Expression levels were similar in lysates of MV-infected Vero-NK
evaluated the immunogenic potential of recombinant MV-SARS cells and SARS-CoV infected VeroE6 cells. As expected, the full-
vectorsexpressingeitherthefull-lengthorthesecretedectodomain lengthSproteinwasonlydetectedincelllysates.Incontrast,Ssol
of the spike glycoprotein of SARS-CoV. In a mouse model of MV was clearly detected both in lysate and supernatant of MV-Ssol
infection,MV-SARSrecombinantvirusesinducedneutralizinganti- infected Vero cells at 40h after infection (Fig. 1D, right panel),
bodies against SARS-CoV and fully protected immunized animals indicating an efficient secretion. Consistently, the Ssol protein
from intranasal challenge with SARS-CoV. Antibody responses secreted in the cell culture medium was heavier than the Ssol
inducedbyMV-SARSvectors werequantitativelyandqualitatively observed within cell lysates, which is in agreement with this
compared to responses induced by a prototype subunit vaccine glycoprotein being synthesized in an immature form in the ER
preparedfromalum-adjuvantedrecombinantSsolprotein. priortotransfertotheGolgi,fromwhichitissecreted.
34 N.Escriouetal./Virology452-453(2014)32–41
7
1193 1255
SP=1-13 TM ml) 6
S-ORF 0/
5
D
CI 5
Ssol-ORF T
Flag er] ( 4
BsiWI eGFP BssHII MV tit 3
T7 N P M F H L T7t [0
g1 2
o
hhR C vR l
V 1
pTM-MVSchw-ATU2 12 36 60 84
hours post-infection
MV-Schwarz MV-S MV-Ssol
Permeabilized
Not
Permeabilized
V
o
MVSchw C MVSchw
S-
S - - + ck R S - + -
Ssol - + - mo SA Ssol - - +
S / Ssol 172 172
110 110
79
79
62
62
Lysates Supernatants
Fig. 1. Characterization of MVSchw-SARS recombinant viruses expressing the full-length transmembrane spike glycoprotein (S) or its secreted ectodomain (Ssol).
(A)SchematicrepresentationofthepTM-MVSchw-ATU2vectorcontainingtheSchwarzMVcDNAwithagreenfluorescentprotein(eGFP)geneasanadditionaltranscription
unit(ATU).Codon-optimizedsyntheticgenescodingforthefull-lengthSproteinorsecretedectodomain(Ssol)wereinsertedintopTM-MVSchwbetweentheBsiWIand
BssHIIsitesoftheATU,inplaceoftheeGFPgene.MVgenesareindicated:N(nucleoprotein),PVC(phoshoproteinandV/Caccessoryproteins),M(matrix),F(fusion),
H(hemaglutinin),L(polymerase).T7:T7RNApolymerasepromoter.hhR:hammerheadribozyme.T7t:T7RNApolymeraseterminator.h∂vR:hepatitisdeltavirus(HDV)
ribozyme.(B)GrowthkineticsofrecombinantMVSchw-SARSviruses.VerocellswereinfectedwiththeparentalMVSchw(opencircles)orrecombinantMV-S(filledsquares)
orMV-Ssol(filledtriangles)virusesatanM.O.I.of0.01.Attheindicatedtimepoints,cellswerecollectedandcell-associatedvirustitersweredeterminedasdescribedin
Materialsandmethods.(C)ImmunostainingofspikepolypeptidesinsyncytiaofMVSchw-SARS-infectedVerocells.Cellswerefixed24–48hafterinfectionwiththe
indicatedvirusesandpermeabilizedwithtritonX-100(upperpanels)ornon-permeabilized(lowerpanels),thenlabeledasdescribedinMaterialsandmethodswithanti-S
mousepolyclonalantibodiesandCy3-conjugatedanti-mouseIgGantibodies.(D)LysatesandsupernatantsofVero-NKcellsinfectedwithMVSchw-SARSviruseswere
analyzedbywesternblotasdescribedinMaterialsandmethodsusingrabbitpolyclonalantibodiesspecificfortheSprotein.Ascontrols,wholecelllysatespreparedfrom
uninfectedorSARS-CoVinfectedVeroE6cellswereanalyzed.Thepositionsoffull-lengthSandSsolpolypeptidesaswellasmolecularweightmarkers(kDa)areshown.
MV-SandMV-SsolinduceTh1-typeimmuneresponseandSARS-CoV to MV infection (Mrkic et al.,1998) and compared to the immuno-
neutralizingantibodiesinmice genicity of purified Ssol polypeptide produced in mammalian cells,
whichconstitutesapotentialsubunitvaccinecandidateagainstSARS
TheimmunogenicityoftherecombinantMVSchwarz-SARSviruses (Du et al., 2008). The CD46-IFNAR mice express CD46, the human
wasinvestigatedingeneticallymodifiedCD46-IFNARmicesusceptible receptor for vaccine MV strains, and lack the INF-α/β receptor. They
N.Escriouetal./Virology452-453(2014)32–41 35
havebeenusedpreviouslyasamodeltoevaluatetheimmunogenicity preimmune sera (not shown) and sera from control animals that
of recombinant MV (Brandler et al., 2007; Combredet et al., 2003; receivedemptyMVSchwremainednegative(Fig.2A).Thesetiters
Despresetal.,2005;Guerboisetal.,2009;Lorinetal.,2004).Eightto werehigherforbothMV-SandMV-Ssolinjectedanimals(average
twelve-week-oldmaleCD46-IFNARmicewereimmunizedwithtwo titerof3.170.3and2.770.1log10,respectively)thanforanimals
intraperitoneal(i.p.)injectionsat4-weekintervalof105TCID ofMV- immunized with Alum-adjuvanted Ssol protein (2.170.3log10
50
S orMV-Ssol recombinantviruses.As controls,agroupofmicewas titer,po10(cid:3)3).Afterthesecondinjection,titerswereboosted10–
injectedwithemptyMVvector(105TCID )andanothergroupwith 20 times for animals immunized with MV vectors. Tallying with
50
2mg of purified Ssol protein adjuvanted with 50mg of aluminum the results observed after the first injection, MV-S induced the
hydroxide (alum), usual doses for small rodents. Mice sera were highest IgG titers (4.470.5log10, po0.1). MV-Ssol induced
collected three weeks after each injection. SARS-CoV- and measles- similartiters(3.870.2log10)thanalum-adjuvantedSsolprotein
specificantibodyresponseswereevaluatedforeachindividualmouse (3.870.3log10). Interestingly, antibodies to MV were raised at
by indirect ELISA against SARS-CoV and MV native antigens, similarlevelsinallmicethatreceivedeitherMVSchworMV-SARS
respectively. viruses(Fig.2B),indicatingthatexpressionof theheterologous S
Significanttitersofanti-SARSIgGwereraisedinallmiceafter proteinbytherecombinantvirusesdidnotaltertheirreplication
the first injection of recombinant MV-SARS viruses, whereas invivonormodifytheirmeasles-specificimmunogenicity.
5 5
A IgG titer] 4 SA IgG titer] 4
LIS ELI
V E us
S-Co 3 es vir 3
R sl
A a
S e
anti- 2 nti-M 2
[10 [a0
g 1
o g
l o
l
1 1
S1 S2 S1 S2 S1 2S S1 S2 PI S1 S2 S1 S2 S1 S2
I I I I I I I I I I I I I I
MVSchw MV-S MV-Ssol MVSchw MV-S MV-Ssol
Measles virus Protein Measles virus
4
er] 5
e tit
A isotyp 4 gA titer]
ELIS SA I 3
CoV 3 V ELI
S- Co
AR S-
nti-S 2 SAR 2
g [a10 og [10
o l
l
1
G1 G2a G1 G2a G1 G2a G1 G2a 1
Ig Ig Ig Ig Ig Ig Ig Ig IS2 IS2 IS2 IS2
MVSchw MV-S MV-Ssol MVSchw MV-S MV-Ssol
Measles virus Protein Measles virus Protein
Fig.2. AntibodyresponseinIFN-α⧸βR(cid:3)/(cid:3)CD46þ/(cid:3)miceimmunizedwithMVSchw-SARSrecombinantviruses.Groupsof6IFN-α⧸βR(cid:3)/(cid:3)CD46þ/(cid:3)micewereinjectedtwice
intraperitoneallyatfour-weekintervalwith105TCID50oftheindicatedrecombinantMVSchw-SARSmeaslesvirusesorwithparentalMVSchw,ascontrol.Anothergroupof
micewasimmunizedwithtwointramuscularinjectionsof2mgofpurifiedSsolpolypeptideadjuvantedwith50mgAlum.Serawerecollectedbeforeimmunization(PI)or
3weeksaftereachinjection(IS1andIS2,respectively).SARS-CoV-specific(A)orMV-specific(B)IgGantibodytitersweredeterminedbyindirectELISA,asdescribedin
Materialsandmethods.(C)SARS-CoV-specific,IgG1(filledcircles)andIgG2a(opencircles)isotypetitersweredeterminedforeachIS2serumbyindirectELISA.(D)SARS-
CoV-specificIgAantibodytitersweredeterminedforeachIS2serumbyindirectELISA.Valuesobtainedforeachindividualmousearerepresentedwithcircles,withmeans
foreachgroupofmiceshownbyhorizontalbars.Detectionlimitsoftheassaysareindicatedbydottedlines.
36 N.Escriouetal./Virology452-453(2014)32–41
Additionally, the quality of the humoral response induced by wereinoculatedintranasallywith105pfuofSARS-CoVfiveweeks
the various immunogens was studied on sera collected 3 weeks after the second immunization. Mice inoculated with empty
after the second injection by IgG and IgA isotype analysis and MVSchwwereusedascontrols.Toevaluatethelevelofprotection
neutralization assay. We first determined the specific IgG1 and against viral replication, we quantified the SARS-CoV infectious
IgG2aisotypetiterstotheSARS-CoVantigensbyanti-SARSELISA titers in lung homogenates prepared two days after challenge
(Fig. 2C). The immunizations with the alum-adjuvanted Ssol (Fig. 4). Control mice immunized with empty MVSchw were all
protein almost exclusively induced IgG1, indicating that the infected except one, SARS-CoV replicating in their lungs at titers
inducedimmuneresponsesarepredominantlyofTh2-typeaswe upto106pfu/lung.ThisshowsthatCD46-IFNARmicearesuscep-
previouslyobservedinBALB/cmice(Callendretetal.,unpublished tibletointranasalSARS-CoVinfection,inagreementwithprevious
results).Contrariwise,MV-SandMV-Ssolvirusesinducedsignifi- observations describing a self-limited bronchiolitis with mild or
cantlyhighertitersofIgG2athanIgG1antibodies,particularlyfor moderatepneumonitisin129/Svmice(Hoganetal.,2004)andno
MV-S(averageratioIgG2aoverIgG1of14.7and2.7respectively), increaseinsusceptibility,pathogenesisandhistologicaloutcomes
reflecting a predominant Th1-type immune response induced by in IFNAR mice of the same genetic background (Frieman et al.,
the live recombinant measles vector. Interestingly, MV-Ssol and 2010).
MV-S viruses also induced moderate (2.470.3log10) to high On the contrary, all mice immunized with MV-S were fully
titers (3.270.3log10) of anti-SARS IgA antibodies respectively protected from challenge. The absence of SARS-CoV replication
(Fig. 2D), whereas sera from animals immunized with alum- wasconfirmedbyquantitativereal-timeRT-PCR,whichevidenced
adjuvantedSsolproteinremainednegative(log10titero1.7). more than a 1000-fold reduction of SARS-CoV RNA levels in the
Wethenfurtherexaminedwhethertheantibodyresponsehad lungsofMV-S-immunizedmice(log10geq/lungso3.8,belowthe
SARS-CoV neutralizing activity, which represents the unique detection level) compared to MVSchw-immunized mice
correlate of protective immunity against SARS (Enjuanes et al., (7.671.9log10 geq/lungs). Mice immunized with MV-Ssol or
2008;Robertsetal.,2008).Neutralizingtitersweredeterminedby adjuvanted Ssol were also protected, albeit less efficiently; one
usinganinfectivityreductionassayasthehighestserumdilution mouse in either group had low residual viral titers in the lungs
thatsuppressedSARS-CoVcytopathiceffectinatleast50%ofthe (Fig.4)andSARS-CoVRNAremaineddetectablealbeitatstrongly
inoculated wells of cultured FRhK-4 cells (Fig. 3). Immunization reduced levels in the lungs of half of the immunized mice (not
withMV-Ssolandalum-adjuvantedSsolproteininducedcompar- shown).
ablelevelofneutralizingantibodies(2.470.3and2.670.3log10 Altogether, these experiments demonstrate that recombinant
titer, respectively), whereas MV-S induced significantly three- to MV-SARSvirusesexpressingeitherthefull-lengthorthesecreted
four-foldhigherneutralizingtiters(3.070.4log10titer,po0.1). ectodomainofthespikeproteininducedneutralizingantibodiesto
SARS-CoV at titers that were sufficient to protect animals from
experimental intranasal infection. Noticeably, the efficiency of
ImmunizationwithrecombinantMV-SARSprotectsfromintranasal protection followed the hierarchy observed for neutralization
experimentalchallenge antibody levels, MV-S conferring complete and better protection
TodeterminewhetherimmunizationbyrecombinantMV-SARS
could induce protection from an experimental challenge, mice
7
4
er] s) 6
body tit U/lung 5
nti PF
utralizing a 3 oV titer] ( 4
e C
V n S-
o R 3
C 2 A
S- S
AR [10
[S0 log 2
1
g
o
l
1
1
IS2 IS2 IS2 IS2 MVSchw MV-S MV-Ssol
MVSchw MV-S MV-Ssol Measles virus Protein
Measles virus Protein Fig. 4. Protection of MVSchw-SARS-immunized mice from intranasal challenge
withSARS-CoV.FiveweeksafterthesecondinjectionofMVSchw-SARSrecombi-
Fig.3. NeutralizingantibodyresponseinIFN-α⧸βR(cid:3)/(cid:3)CD46þ/(cid:3)miceimmunized nantvirusesorSsolpolypeptide,miceweresubjectedtointranasalchallengewith
with MVSchw-SARS recombinant viruses. SARS-CoV neutralizing antibody titers 105pfuofSARS-CoV.SARS-CoVinfectioustiters(expressedaslog10pfu/lungsfor
weredeterminedforeachIS2serumasthereciprocalofthehighestdilutionof individual mice) were dermined in lung homogenates collected two days after
serumwhichcompletelypreventedSARS-CoVcytopathiceffectin50%ofthewells. challenge,usingplaqueassayonVerocells.Valuesforeachindividualmouseare
Values obtained for each individual mouse are represented with circles, with representedwithfilledcircles,andmeansforeachgroupbyhorizontalbars.The
meansforeachgroupofmiceshownbyhorizontalbars.Detectionlimitsofthe detectionlimitsoftheassaysareindicatedbydottedlines.Datashownarefrom
assaysareindicatedbydottedlines. oneexperimentrepresentativeoftwo.
N.Escriouetal./Virology452-453(2014)32–41 37
from virus replication in mice lungs than both MV-Ssol and the Usually given intramuscularly, measles vaccine protects very
referenceadjuvantedsubunitvaccine. efficiently against measles disease, which is contracted by aero-
solized contamination of the upper respiratory tract, similarly to
SARS. Our data demonstrate that parenteral immunization with
Discussion MV-SARS vector protected mice lungs from SARS-CoV intranasal
infection. A similar approach was previously reported by Liniger
Theobjectiveofthisstudywastoevaluatetheproof-of-concept etal.(2008)thatalsodemonstratedtheinductionofneutralizing
ofanewSARS-CoVvaccinestrategybasedonastandardmeasles antibodies in mice to SARS-CoV spike expressed bya MV vector.
vaccine engineered to express the SARS-CoV spike protein. This However, this study did not evaluate the efficacy of vaccine
strategymightprovideasaferecombinantvaccinetoprotectfrom preparationtoprotectimmunizedmicefromSARS-CoVchallenge.
SARS-CoVintheregionsthatmightbecomeaffectedbySARS-CoV Our observation that MV-SARS induced a strong Th1 response
re-emergence. appears of particular importance given the recently reported
Due toits critical roleinviralentry, the S protein is the most safety concerns by Tseng et al., which described Th2-type lung
promising candidate antigen for SARS-CoV vaccine development immunopathologyuponchallengeofmicevaccinatedwithinacti-
(Enjuanes et al., 2008; Roberts et al., 2008; Roper and Rehm, vatedwholevirus,SARSVLPoralum-adjuvantedSprotein(Tseng
2009).Wedesignedhumancodon-optimizedgenesencodingthe et al., 2012). They suggested that most previously described
full-lengthSARS-CoVspike(S)proteinanditssolubleectodomain vaccine-inducedimmunopathologymightproceedfromthesame
(Ssol). These sequences were inserted into MV vector. Recombi- Th2-type immunopathology with prominent eosinophil infiltra-
nant viruses were produced by reverse genetics and grew at tion upon SARS-CoV challenge. Such effects will likely not occur
standardtiters.TheyexpressedhighlevelsofSARS-CoVfull-length withlivevaccinessincetheyinducepotentTh1-biasedresponses.
spikeproteinatthecellmembraneandledtoefficientsecretionof Inagreementwiththishypothesis,itwasrecentlyreportedthata
thesolubleectodomain.ImmunizationofmicesusceptibletoMV live attenuated SARS-CoV lacking E protein expression provided
with recombinant MV-S and MV-Ssol viruses induced specific long-term protection against lethal challenge in an aged-mice
antibodies that neutralized SARS-CoV infection in vitro. The model in the absence of any sign of vaccine-induced immuno-
induction of measles-specific immunity was not altered by the pathology(Fettetal.,2013).
expression of the transgenes. Immunization primed a SARS-CoV- Live attenuated RNAvirus vaccines like those against mumps,
specificmemoryresponsethatwasvigorouslyboostedbyasecond measles, polio or rubella viruses induce long-term cell-mediated
injection. The anchored full-length form of the spike protein andhumoralimmunityafteroneortwoinjections.UsingMVasa
induced the highest titers of neutralizing antibodies, as we vaccination vector presents a number of advantages: this highly
previouslyobservedwithHIVgp160(Lorinetal.,2004),andalso efficient and most safe vaccine is easily produced at large scale,
amorerobustresponsethanaprototypesubunitvaccineprepared vaccine strains are genetically stable, MV does not recombine or
from adjuvanted recombinant Ssol protein. As compared to integrategeneticmaterial,vaccinedoesnotpersistordiffuse.The
immunization with adjuvanted Ssol protein, both recombinant Schwarz/Moraten strain from which we derived our vector
MV-SARS viruses induced higher titers of IgG2a than IgG1 anti- (Combredet et al., 2003) is currently the most attenuated and
bodies, indicating a Th1 biased response, a hallmark of live the most widely used measles vaccine. Measles vector expresses
attenuated viruses and a highly desirable feature for an antiviral verystablylargeamountsofheterologousgeneticmaterial,likely
vaccine. Remarkably, both recombinant MV-SARS vectors also due to the absence of geometric constraints on the size of
induced SARS-CoV specific IgA, whereas the subunit reference helicoidal nucleocapsids in this pleiomorphic virus (Tangy and
vaccine did not. This raises an interesting point since several Naim, 2005). Although the stability of expression of the specific
clinical studies already evidenced that parenteral administration SARStransgeneremainstobedetermined,theremarkablegenetic
ofliveattenuatedmeaslesvaccinetochildrenstimulatessecretory stabilityofadded, expressed ORFshas alreadybeen described by
IgAresponsesin nasal washes (Bellanti et al., 2004; Simon etal., us and others for a variety of transgenes inserted into measles
2011).AlthoughwedidnotassesstheabilityofrecombinantMV- vector and other members of the Mononegavirales. The measles
SARS to induce secretory IgA in the lungs and upper respiratory vector demonstrated a strong capacity to stimulate both cellular
tract of immunized mice, this result is strongly indicative of the andhumoralneutralizingimmunityagainstanumberofantigens
potential of MV-SARS to induce SARS-CoV specific secretory IgA andprovidedprotectionfromexperimentalchallengebothinmice
responses. and primates (Brandler et al., 2007, 2012; Despres et al., 2005;
To evaluate the efficacy of recombinant MV-SARS vectors, we Lorinetal.,2004;TangyandNaim,2005).Ameasles-HIVvaccine
relied on challenge with wt human SARS-CoV and evaluation of candidate currently under clinical development has been evalu-
lungvirustiterssince,unfortunately,mouse-adaptedSARS-CoVis ated in phase I clinical trial, demonstrating the GMP preclinical
not lethal in 129/Sv mice (Frieman et al., 2010). After two andclinicalsafetyandimmunogenicityofthisvector(Lorinetal.,
successiveimmunizationswithrecombinantMV-Svirus,allmice 2012;Stebbingsetal.,2012).
were fully protected from intranasal infectious challenge with RecombinantMVvaccinesmightbeusedtoimmunizeboththe
SARS-CoVandneitherinfectiousvirusnorresidualviralRNAcould pediatric and adult/adolescent populations in case of SARS-CoV
be recovered from the lungs of the challenged animals. Interest- outbreaks.Thepresenceofanti-MVimmunityinnearlytheentire
ingly, despite a lower replication rate in cell culture, MV-S adulthumanpopulationmightrestricttheuseofrecombinantMV
conferred a better protection from SARS-CoV replication in mice to naïve infants, an already worthy goal in any event. However,
lungsthanMV-Ssol.Thismayreflectbetterimmunogenicityofthe numerous studies have shown that revaccinating immunized
native anchored-form of S or suggest structural dissimilarities individuals results in a boost of anti-MV antibodies, suggesting
between the monomeric soluble Ssol ectodomain and its corre- that the attenuated live vaccine replicates and expresses its
spondingmembrane-associatedtrimericform.Inthatrespect,the proteins in spite of preexisting immunity (Dilraj et al., 2000;
absence, or inefficient presentation, of antibody epitopes on Rager-Zismanetal.,2003;Sepulveda-Amoretal.,2002).Likewise,
truncated soluble immunogens derived from functional virion- the presence of maternal anti-MV antibodies has been shown to
associated glycoproteins has already been evidenced for filovirus limit the induction of anti-measles antibodies but notthe induc-
attachment proteins (Sullivan et al., 2006) and HIV gp160 tion of specific T-cell responses in infants given measles vaccine
(Davenportetal.,2011;Kovacsetal.,2012). duringthefirstyearoflife(Gansetal.,2003,2001).Moreover,we
38 N.Escriouetal./Virology452-453(2014)32–41
previously demonstrated that recombinant MV vectors were with the FLAG tag was producedand purified byimmunoaffinity
immunogenic and induced protection in the presence of MV chromatography (Sigma-Aldrich) from the supernatant of stable
preexisting immunity in animal models (Brandler et al., 2013; mammaliancelllines(Callendretetal.,unpublishedresults).
Lorin et al., 2004), which opens the possibility of using recombi-
nantMVvectortoimmunizeadolescentsandadults,althoughthis ConstructionandrescueofrecombinantMVSchw-SARSviruses
pointneedstobeevaluatedinhumantrials.Measlesappearsvery
difficult to eliminate and still causes 160,000 deaths annually Ahumancodon-optimizedgeneencodingtheSARS-CoVspike
worldwide, mostly in poorly developed countries (Centers for (S)proteinof the♯031589 specimen(Callendretetal., 2007)was
Disease and Prevention, 2009). Europe also recently experienced chemically synthesized by Geneart (Regensburg, Germany) and
severe measles outbreaks (Cottrell and Roberts, 2011). Improving subcloned into the pCI mammalian expression vector (Promega),
and maintaining measles vaccination for decades is essential to yielding plasmid pCI-Ssynth. In addition to codon bias optimiza-
contain this most contagious disease. In this context, the use of tion,regionsofveryhigh(480%)orlow(o30%)GCcontentwere
recombinant MV designed to immunize the pediatric or adult avoided whenever possible. Furthermore, cis-acting sequence
populationsappearsdesirable. motifs such as internal TATA-boxes, chi-sites, ribosomal entry
In conclusion, we have produced new recombinant MV-SARS sites, ARE, INS, and CRS sequence elements, as well as repetitive
virusesabletoinduceneutralizingantibodiestoSARS-CoVandfull sequences,RNAsecondarystructuresandsplicedonorandaccep-
protection from intranasal challenge, thus making the proof-of- tor sites, were avoided. The resulting optimized gene had an
concept of this strategy for SARS vaccine development and more increased GC content (61.7% from 38.9%). The sequence of the
generally for severe respiratory infectious diseases. These char- codon-optimized S gene is available upon request. A codon-
acterized SARS vaccine candidates deserve to be evaluated in a optimized gene encoding the soluble and secreted spike ectodo-
much more adapted non-human-primate model, in which the main (Ssol) was obtained by PCR amplification using the
cross-protective potential of the induced immune responses pCI-Ssynth plasmid as a template and oligonucleotides 50-
against zoonotic SARS isolates/variants could be addressed. ACTAGCTAGC GGATCCACCA TGTTCATCTT CCTG-30 containing
Indeed, several studies have underlined the need to induce Nhe-1restrictionsite(underlined)and50-AGTATCCGGACTTGATG-
broadly neutralizing antibodies able to protect against heterolo- TAC TGCTCGTACT TGC-30 containing BspE1 restriction site. The
gous viral variants, which may arise during independent emer- PCRfragmentwasinsertedintopCI-Ssolwhichcontainsthewild-
gence events (Bolles et al., 2011; Sheahan et al., 2011). Such typenon-optimizedgeneencodingSsol(Callendretetal.,unpub-
antigenicvariantshavealreadybeenisolatedfromanimalsduring lisheddata),yieldingplasmidpCI-Scube.
the2002–2003epidemicsandfromsporadichumancasesduring Measles recombinant vector was derived from plasmid pTM-
the 2003–2004 reemergence (Deming et al., 2006; Yang et al., MVSchw-ATU2whichcarriesaninfectiouscDNAcorrespondingto
2005), and are likely to emerge again from the large heteroge- the anti-genome of the Schwarz MV vaccine strain and an
neous zoonotic reservoir of SARS-like viruses, should a newout- additional transcription unit containing unique restriction sites
break occur. The recent identification of a novel human for the insertion of foreign sequences downstream of the P gene
betacoronavirus,MERS-CoV,astheagentofaSARS-likeillnessin (Combredetetal.,2003)(Fig.1A).Thefull-length(S)andsecreted
the Middle-East, has raised great concerns about the pandemic (Ssol)spikesequenceswereamplifiedbyPCRusingthepCI-Ssynth
potential of the disease (Perlman, 2013; WHO, 2013; Zaki et al., or pCI-Scube plasmids as templates and oligonucleotides
2012). Adecade after the SARS pandemic, it has renewed world- 50-ATAGGATCCC GTACGACCAT GTTCATCTTC CTGCTGTTC-30 con-
wide attention on coronaviruses and stressed the need of con- taining BsiWI restriction site (underlined) and 50-ATAGGATCCG
tinued research and investment in the development of safe and CGCGCTTATC AGGTGTAGTG CAGCTTCAC-30 or 50-ATAGGATCCG
effectivevaccinesagainsthighlypathogeniccoronaviruses. CGCGCTCATT ATTTATCGTC GTCATCTTTA TAATC-30 containing
BssHII restriction site (underlined). After digestion with BsiWI
andBssHIIrestrictionenzymes,theresultingDNAfragmentswere
Materialsandmethods inserted into the corresponding sites of pTM-MVSchw-eGFP. The
sequences inserted into the MV vector respect the “rule of six”,
Celllines,virusesandantigens which stipulates that the number of nucleotides of the MV
genome must be a multiple of 6 (Calain and Roux, 1993;
FRhK-4 (Fetal Rhesus monkey Kidney) and Vero-NK (African Schneider et al., 1997). The resulting plasmids pTM-MVSchw-S
GreenMonkeyKidney)cellsweregrownat371Cunder5%CO in andpTM-MVSchw-Ssolwere usedtorescue recombinantviruses
2
completeDMEM[Dulbecco0smodifiedEaglemediumwith4.5mg/ using a helper-cell-based system as previously described
ml L-glucose, 100U/ml penicillin and 100mg/ml streptomycin], (Combredet et al., 2003). Single viral clones were amplified on
supplemented with 5% heat-inactivated fetal calf serum (FCS) Vero-NK cells.Allviralstockswerestoredat (cid:3)801Candtitrated
(DMEM-5). Helper 293-T7-MV cells stably expressing T7 RNA by an endpoint limiting dilution assay on Vero-NK cell mono-
polymeraseandNandPgenes fromSchwarz MVweregrownin layers. Growth curves of recombinant and parental viruses were
completeDMEMsupplementedwith10%FCS(DMEM-10). determined on Vero-NK cells infected at an M.O.I. of 0.01, as
SARS-CoV FFM-1 strain (Drosten et al., 2003) was kindly described(Combredetetal.,2003).
provided by Dr. H.W. Doerr (Institute of Medical Virology, Frank-
furt University Medical School, Germany). Viral stocks were Analysisofspikeproteinexpressionininfectedcells
produced and titrated as described previously (Callendret et al.,
2007). All work involving infectious SARS-CoV was performed in For indirect immunofluorescence assays, monolayers of Vero-
anenhancedbiosafetylevel3containmentlaboratorywithrigor- NKcellsplatedon20mmglasscoverslipsina12-wellplatewere
oussafetyproceduresaccordingtoWHOguidelines. infected with the recombinant MVSchw-SARS or parental MV-
Crude cell lysates prepared from SARS-CoV infected VeroE6 Schw viruses at a multiplicity of infection (M.O.I.) of 0.05. When
cells and inactivated frozen by gamma irradiation, as described syncytiawere clearly visible but notyet confluent (24–48h after
(Callendret et al., 2007), were used as SARS native antigens for infection) cells were washed in D-PBS, fixed with PBS-4% paraf-
ELISA. A soluble monomeric spike protein, termed Ssol and ormaldehyde for 15min and in some instances permeabilized
correspondingtotheentireSectodomain(residues1–1193)fused with PBS-0.2% triton X-100 for 10min. Coverslips were then
N.Escriouetal./Virology452-453(2014)32–41 39
incubated for 60min with anti-Ssol hyperimmune mouse ascitic the highest dilution of each serum, which suppressed CPE in at
fluid diluted 1/1000 in PBS-1% donkey serum (DKS). After sub- least2outof4wells.
sequent incubation with a Cy3-conjugated anti-mouse IgG sec-
ondary antibody (Jackson Immunoresearch), the samples were
ChallengeinfectionofanimalswithSARS-CoV
mounted on slides with DAPI-containing Vectashield (Vector
laboratory) and analyzed under an Axioplan 2 epifluorescence
Fiveweeksafterthesecondimmunization,animalswereligh-
microscope(Zeiss).PictureswereacquiredwithanAxiocamMRm tly anesthetized with Isoflurane (Mundipharma) and inoculated
cameraandprocessedwiththeAxiovisionsoftware(v4.2,Zeiss). intranasally with 105pfu of SARS-CoV in 40ml PBS. Mice were
For western blot assays, cytosolic cell extracts were prepared
euthanized48hafterchallengeinfection.Lunghomogenateswere
fromVero-NKcellsinfectedwiththerecombinantMVSchw-SARS preparedin500mlDMEMsupplementedwith2%FCSandtitrated
or parental MV-Schw viruses, essentially as described previously
for infectious virus on VeroE6 monolayers, as described
(Lorin et al., 2004). Proteins were separated by 8% SDS-poly-
(Callendretetal.,2007).ViralRNAwasextractedusingtheQIAamp
acrylamide gels and transferred onto a PVDF membrane prior to Viral RNA Mini kit (Qiagen) from 100ml of tissue homogenates
immunoblotting with rabbit anti-S antibodies, as described accordingtothemanufacturer0srecommendations,elutedin60ml
(Callendretetal.,2007).
of RNAse-free water and analyzed by real-time RT-PCR, as
describedbelow.Challengeinfectionofallanimalsandsubsequent
analysis involving infectious materials were done in the “Jean
Miceexperimentsandcharacterizationofhumoralimmuneresponses Merieux” biosafety level 4 containment laboratory. Statistical
analysiswasperformedonthelog10oftheviraltitersmeasured
Allexperimentswereapprovedandconductedinaccordanceto for individual mice using the Student0s independent t-test,
the Pasteur Institute guidelines in compliance with European
withtheassumptionsusedforsmallsamples(normaldistribution
animal welfare regulations. To obtain CD46þ/(cid:3) IFNα/ßR(cid:3)/(cid:3) mice
of the variables and same variance for the populations to be
permissive for measles vaccine (Mrkic et al., 1998), FVB mice
compared).
heterozygous for the measles vaccine CD46 receptor transgene
werebackcrossedto129/SvmicelackingthetypeIFN(Combredet
etal.,2003).Aftermorethan10generationsofbackcrossinginour SARS-CoVgenomeRNAquantificationbyreal-timeRT-PCR
breedingcolony,theresultingCD46-IFNARlineacquiredauniform
129/Svbackground. SARS-CoV genome RNA levels in tissue RNA samples were
Six- to nine-week-old CD46-IFNAR mice were used to assess quantified by real-time RT-PCR with the PRISM 7700 sequence
the immune response induced by recombinant MVSchw-SARS. detection system. Briefly, RNA samples (2ml) were reverse-
Groups of 6 mice were injected intraperitoneally (i.p.) with 105 transcribedandamplifiedwiththeTaqMansOneStepPCRMaster
TCID of recombinant MVSchw-SARS or parental MVSchw. Con- Mix Reagents Kit (Applied Biosystems) according to the man-
50
trol groups of mice were injected intramuscularly with formula- ufacturer0srecommendations,incombinationwithprimersetand
tions containing 2mg of purified Ssol polypeptide and 50mg flurorogenicprobespecificforthenucleoprotein(N)gene(Drosten
aluminum hydroxide (Alu-Gel-S, Serva). Booster injections were et al., 2004). Reactions were performed in thermo-fasts 96-well
administered 4 weeks thereafter. Serum samples were collected reaction plates (Abgene) with 25ml mixtures containing 2ml of
one week before immunization and 3 weeks after each injection templateRNA,300nMconcentration(each)ofprimersSANS1(50-
(PI,IS1andIS2sera). TGGACCCAC AGATTCAAC TGA-30) and SANPAs2 (50-GCTGTGAAC
WemeasuredtheinductionofSARS-CoVspecificantibodiesin CAAGACGCAGTAT-30)(Eurogentec),anda100nMconcentrationof
immunized mice by indirect ELISA and neutralization assay, as probe SANP1 (6-FAM-TAACCAGAATGGAGGACGCAATGG-TAMRA)
described previously (Callendret et al., 2007). Briefly, microtiter (Eurogentec). The cycling conditions were 451C for 15min, fol-
plates were coated with SARS-CoV infected or mock-infected lowedby951Cfor5minandthen45cyclesof951Cfor15sand
VeroE6 crude cell lysates and incubated with serial dilutions of 601Cfor60s.Fluorescencewasreadatthecombinedannealing-
thetestsera.Boundantibodieswererevealedwithmouse-specific extension stepat601C.Absolutequantification ofRNAwas done
anti-IgG (1/2000, Amersham) secondary antibody coupled to by using in vitro transcribed RNA standards prepared from the
horseradish peroxidase and TMB (3,30-5,50-tetramethylbenzidine, cloned SARS-CoV N gene and quantified spectrophotometrically.
KPL). Readings from wells coated with mock lysates were sub- Analyticalsensitivityofthisreal-timeNRT-PCRassaywasexperi-
stracted from wells with SARS-CoV lysates and the SARS-CoV mentally determined by using limiting serial dilutions of the N
specificIgGtiterswerecalculatedasthereciprocalofthehighest RNAstandard.Usingprobitnon-linearregressionanalysis,the95%
dilution of individual serum, giving an absorbance of 0.5. The detectionlimitoftheRT-PCRwascalculatedat40copiesofinput
isotype determination of the antibody responses was performed RNAperreaction,whichcorrespondedto3.8log10virusgenome-
using isotype-specific (IgG1 and IgG2a) secondary antibodies equivalent (geq) per mouse tissue, assuming 100% efficiency in
coupled to horseradish peroxidase (Southern Biotech). IgA titers RNApreparation.
weresimilarlymeasuredusingmouse-specificanti-IgAsecondary
antibody (1/2000, Southern Biotech) and calculated as the reci-
procal of the highest dilution of individual serum, giving an Acknowledgments
absorbanceof0.2.MVspecificantibodiesweresimilarlymeasured
using ELISA plates coatedwith purified measles antigens (Trinity We thank Prof. Dr. H.W. Doerr (Institute of Medical Virology,
Biotech,USA).Forneutralization,two-foldserialdilutionofheat- Frankfurt University Medical School, Germany) for providing us
inactivated serum samples were incubated at 371C for 60min withtheSARS-CoVFFM-1strain.WealsothankDr.AnnetteMartin
with 100 TCID50 of SARS-CoV and added to a subconfluent forcriticalreadingofthemanuscriptandhelpfulsuggestions,Prof.
monolayer of FRhK-4 cells in a 96-well microtiter plate. Each SylvievanderWerfforcontinuoussupportandPhilippeLothfor
dilutionofserumwastestedinquadruplicateandcytopathiceffect experttechnicalassistanceinperformingtheSARS-CoVchallenge
(CPE) endpoints were read up to 7 days after inoculation. Neu- experiment. This work was supported in part by Grants from
tralizing antibody titers were determined according to the Reed GlaxoSmithKline Biologicals. B.C. was supported by a fellowship
andMuenchmethod(ReedandMuench,1938)asthereciprocalof from GlaxoSmithKline Biologicals. The funders had no role in
40 N.Escriouetal./Virology452-453(2014)32–41
study design, data collection and analysis, decision topublish, or Dilraj,A.,Cutts,F.T.,Bennett,J.V.,FernandezdeCastro,J.,Cohen,B.,Coovadia,H.M.,
preparationofthemanuscript. 2000.Persistenceofmeaslesantibodytwoyearsafterrevaccinationbyaerosol
orsubcutaneousroutes.Pediatr.Infect.Dis.J.19,1211–1213.
Drosten,C.,Chiu,L.L.,Panning,M.,Leong,H.N.,Preiser,W.,Tam,J.S.,Gunther,S.,
Kramme,S.,Emmerich,P.,Ng,W.L.,Schmitz,H.,Koay,E.S.,2004.Evaluationof
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