Induction of Strain-Transcending Immunity against Plasmodium chabaudi adami Malaria with a ...

12 Pages·2005·0.41 MB·English

INFECTIONANDIMMUNITY,May2005,p.2974–2985 Vol.73,No.5
0019-9567/05/$08.00(cid:1)0 doi:10.1128/IAI.73.5.2974–2985.2005
Copyright©2005,AmericanSocietyforMicrobiology.AllRightsReserved.
Induction of Strain-Transcending Immunity against
Plasmodium chabaudi adami Malaria with a
Multiepitope DNA Vaccine
T. Scorza,1,2 K. Grubb,3 P. Smooker,4 A. Rainczuk,3 D. Proll,5,6 and T. W. Spithill2,3*
DepartementdeSciencesBiologiques,Universit´eduQu´ebeca` Montr´eal,1200RueSaintAlexandre,S-2055,
Montr´eal,Qu´ebecH3B3H5,Canada1;CentreforHost-ParasiteInteractions2andInstituteofParasitology,3
McGillUniversity,21111LakeshoreRoad,Ste.-Anne-de-Bellevue,H9X3V9,Quebec,Canada;Department
ofBiotechnologyandEnvironmentalBiology,RMITUniversity,P.O.Box71,3083,Bundoora,Victoria,
Australia4;DepartmentofBiochemistry&MolecularBiology,P.O.Box13D,
D
MonashUniversity,Victoria3800,Australia5;andEijkmanInstitutefor
o
MolecularBiology,JakartaPusat10430,Indonesia6 w
n
Received11October2004/Returnedformodification22November2004/Accepted21January2005 lo
a
d
A major goal of current malaria vaccine programs is to develop multivalent vaccines that will protect e
d
humans against the many heterologous malaria strains that circulate in endemic areas. We describe a

f
multiepitopeDNAvaccine,derivedfromagenomicPlasmodiumchabaudiadamiDSDNAexpressionlibraryof r
o
30,000plasmids,whichinducesstrain-transcendingimmunityinmiceagainstchallengewithP.c.adamiDK. m
Segregation of this library and DNA sequence analysis identified vaccine subpools encoding open reading
h
frames (ORFs)/peptides of >9 amino acids [aa] (the V9(cid:1) pool, 303 plasmids) and >50 aa (V50(cid:1) pool, 56 t
t
plasmids), respectively. The V9(cid:1) and V50(cid:1) plasmid vaccine subpools significantly cross-protected mice p
:
against heterologous P. c. adami DK challenge, and protection correlated with the induction of both specific //
ia
gammainterferonproductionbyspleniccellsandopsonizingantibodies.Bioinformaticanalysisshowedthat
22 of the V50(cid:1) ORFs were polypeptides conserved among three or more Plasmodium spp., 13 of which are i.a
s
predictedhypotheticalproteins.Twenty-nineoftheseORFsareorthologuesofpredictedPlasmodiumfalcipa- m
rum sequences known to be expressed in the blood stage, suggesting that this vaccine pool encodes multiple .
o
blood-stageantigens.Theresultshaveimplicationsformalariavaccinedesignbyprovidingproof-of-principle r
g
that significant strain-transcending immunity can be induced using multiepitope blood-stage DNA vaccines /

and suggest that both cellular responses and opsonizing antibodies are necessary for optimal protection o
n
againstP.c.adami.
D
e
c
Malaria(Plasmodiumspp.)isoneofthemostseriouspara- single vaccines to protect against malaria populations in the e
m
sitic diseases in the world, but no vaccine exists that protects field,duetoallelicheterogeneityamongantigens.Indeed,re-
b
humansagainstthemultiplicityofstrainsthatcirculateinen- centresultswithahumanvaccinecontainingoneallelicform e
r
demic populations (12, 15, 36, 49). The fact that individuals of MSP2 showed that vaccination selected for occurrence of 4
livinginareaswithstablemalariatransmissionslowlydevelop the alternate MSP2 allele in vaccinated individuals (19). In ,
2
naturally acquired immunity, directed to the blood stage of addition,tobebroadlyprotective,amalariavaccinewillneed 0
infection,suggeststhefeasibilityofdesigningeffectivevaccines to circumvent human HLA genetic diversity. Thus, a large 18
(7,15,36,49).However,thetargetantigensaccountingforthis panelofT-andB-cellepitopesrepresentingasignificantpro- b
protectionarenotfullyknown.Ithasbeensuggestedthatthe portionoftheantigenicrepertoireofthewholeparasiteshould y
g
strain-specificnatureoftheimmuneresponsesinducedfollow-
preferably be included in the vaccine (15, 30, 36, 40). In gen- u
inginfectioncanbereasonsfortheslowdevelopmentofnat- e
eral, combinations of malarial antigens acting synergistically s
urally acquired immunity (7, 14). In general, immunization t
provide the greatest protection to challenge infections (8, 16,
with single malaria vaccine antigens (such as the lead candi-
27,50,59,63).
datesAMA1andMSP1)exclusivelyconfersprotectionagainst
Onestrategytocircumventproblemsassociatedwithallelic
challengewithhomologousparasites(1,13,48,52).Antibodies
polymorphism in malaria antigens is to focus on conserved
to AMA1 show variable levels of cross-inhibition of parasite
antigens/epitopes that can be potentially cross-protective (19,
growth in vitro when tested against heterologous strains of
44). For example, the candidate vaccine antigen MSP4/5 is
Plasmodium falciparum (25, 28, 29), suggesting that existing
highly conserved in 14 strains (isolates) of Plasmodium yoelii
vaccine antigens, of which there are at least 15 blood-stage
(95to100%sequenceidentity),andvaccinationwithMSP4/5
candidates (36, 49), may not be sufficient when delivered as
proteinsfromtwostrains(PlasmodiumyoeliikillickiandPlas-
modium yoelii nigeriensis) cross-protected mice against chal-
lenge with P. y. yoelii YM (20). A lesser degree of cross-
*Correspondingauthor.Mailingaddress:InstituteofParasitology,
protectionagainstP.y.yoeliiwasalsoaffordedbyvaccination
McGill University, 21111 Lakeshore Road, Ste.-Anne-De-Bellevue,
withMSP4/5fromPlasmodiumbergheiANKA(81%sequence
Quebec, Canada H9X3V9. Phone: [phone]. Fax: [phone]
7857.E-mail:[email protected]. identity to P. yoelii), but no protection was observed with
2974
VOL.73,2005 CROSS-PROTECTION WITH MULTIVALENT MALARIA VACCINES 2975
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FIG. 1. Segregation of the P. c. adami DS VR1020 genomic expression library and evaluation of vaccine efficacy. The original library /ia
constructionandevaluationoftheabilityofthelibrarytoinducesurvivalafterhomologouschallengewithvirulentP.c.adamiDSaredescribed
i.
downtothelevelofthe3KAsubpools(46,58).Subpool3KA-4inducedthehighestprotection(63%survival).FollowingDNAsequencingofthe a
733plasmidsinthe3KA-4subpool,thelibrarywassubdividedbasedonthesizeofthepredictedORFin-framewiththeTPAleadersequence. s
m
TheV9(cid:1)poolencodesORFsof(cid:3)9aa:theV50(cid:1)poolencodesORFsof(cid:3)50aa.TheV9(cid:1)andV50(cid:1)poolswereevaluatedforefficacyagainst
.
heterologousP.c.adamiDKchallenge,assessedasreductionincumulativeparasitemia. o
r
g
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o
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MSP4/5fromPlasmodiumchabaudiadamiDS(55%identity) the segregation of this protective library down to subpools of D
(20).Suchresultsprovideevidencethatconservedantigenscan plasmids encoding several conserved sequences and demon- e
c
elicit good cross-strain protection and even partial cross-spe- strate that these sequences induce strain-transcending immu- e
m
ciesprotection.ItisnotablethatseveralantigensfromP.fal- nityinaP.c.adamiheterologouschallengemodel.
b
ciparumhavebeenshowntocross-protectmiceagainstmurine e
malaria,furtherdemonstratingthatcross-speciesprotectionis r
MATERIALSANDMETHODS 4
feasible,presumablyduetoconservationofprotectiveepitopes ,
Creationofplasmidpools.AgenomiclibraryfromP.chabaudiadamiDSwas 2
between species (6, 9, 10, 34, 35, 37, 53, 54): vaccine-induced
producedintheDNAvaccinevectorVR1020asdescribedpreviously(46,58).A 0
partial cross-protection among P. falciparum strains is also 1
totalof10pools(termed3KA-3KJ),eachcomprisingapproximately3,000indi- 8
described(38). vidual clones, were constructed and stored at (cid:2)80°C as glycerol stocks. One
b
Akeychallenge,therefore,istheidentificationofconserved protectivesubpool,3KA-4(733plasmids)(58),wasselectedforDNAsequencing y
antigens/epitopes that could be employed for use as cross- (see below). We evaluated the protective efficacy of plasmids encoding open g
readingframes(ORFs)inframewiththetissueplasminogenactivator(TPA) u
strainvaccines.Itispossiblethatamongthe5,300genesinthe leadersequenceof(cid:3)9residuesinlength(termedtheV9(cid:1)pool)byselectinga e
s
malariagenome,novelconservedantigensexistthathavenot groupof303plasmidsfromthe3KA-4subpool.Fromthese303plasmids,we t
yet been evaluated for protective efficacy (15, 18). In the further evaluated a subset of 56 plasmids encoding ORFs (cid:3)50 residues long
present study, we have used expression library immunization (termed the V50(cid:1) pool; see below). Figure 1 is a flow chart describing the
experimentaldesignusingthislibrary,whichcontainsanaverageinsertsizeof1.5
(ELI) as an approach to identify new combinations of cross-
kb(seereference46fordetails).
protective antigens within the malaria genome, using P. c.
IsolationofplasmidDNAandconstructionofvaccinationcartridges.Prepa-
adamiinmiceasamodeltestsystem.ELIhasbeenshownto rationofplasmidDNAfromthe30KlibraryorfromtheV9(cid:1)andV50(cid:1)pools
be protective against Mycoplasma (2), Leishmania (43), and wasperformedasdescribedpreviously(46,58).DNApurifiedunderendotoxin-
Plasmodium, where we showed that mice were significantly freeconditionswasprecipitatedontogoldmicrocarriersataratioof100(cid:4)g
DNA/50 mg carriers, such that each projectile used for vaccination contains
protectedagainstvirulentP.c.adamiDSinfectionbyvaccina- about1(cid:4)gDNA,asdescribedpreviously(46,58).
tion with a homologous genomic expression library (46, 58). SequencingofplasmidinsertsandBLASTalignmentanalysis.Thesequence
We hypothesized that our library contains multiple antigen ofall733plasmidinsertsinthe3KA-4poolwasdeterminedacrosstheTPA
sequences, many of which could be novel hypothetical se- leadersequenceandcloningsiteintheVR1020vector.Eachplasmidwasgrown
overnight from individual 15% glycerol stocks in 10 ml of LB broth supple-
quences known to exist in the genome (18), and that this
mentedwithkanamycin(50(cid:4)g/ml).PlasmidDNAwasextractedaccordingtothe
multivalent antigen cocktail may elicit good cross-protection
QIAGENmini-prepprocedureandsequenced.Theresultsweretranslatedwith
duetosequenceconservationbetweenstrains.Here,wereport theExpasyDNAtranslationtool(http://us.expasy.org/),andthesizeoftheORF
2976 SCORZA ET AL. INFECT.IMMUN.
inframewiththeTPAsequencewaspredicted.Initialsegregationoftheplasmids expressionlibrary,containingapproximately30,000sequences
intotheV50(cid:1)poolwasperformedin2002beforepublicationofthemalariagenome from P. c. adami DS parasites, protects BALB/c mice against
sequence. Subsequently, BLASTP searches, using the National Center for Bio-
homologouschallengewithvirulentDSparasites(Fig.1)(46,
technology Information (NCBI) database versus the P. falciparum and P. yoelii
genomes(http://www.ncbi.nlm.nih.gov/BLAST/Genome/plasmodium.html)andthe 58). We postulated that since this library contains multiple
PlasmoDB database-NCBI BLASTP 2.1.2 program versus Plasmodium ORFs antigen sequences, it may elicit good cross-protection as a
of (cid:1)50 bp (amino acids [aa]) (http://plasmodb.org/plasmodb/servlet/sv?page multivalent vaccine due to sequence conservation between
(cid:5)blast), were used to compare the amino acid sequences encoded in the
strains of P. c. adami. We evaluated the cross-protective po-
ORFgenestothoseofotherPlasmodiumproteins(expectvaluecutoffof10(cid:2)6)
tentialofthe30KvaccineagainsttheheterologousP.c.adami
(4).BLASTXandBLASTNsearchesusingtheP.chabaudiBLASTServerat
The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast DKstrain(isolate556KA,whichinducesanonlethalinfection
/submitblast/p_chabaudi)wereusedtoidentifysequencespotentiallyuniqueto with moderate parasitemia of 8 to 15% in mice) in BALB/c
theP.chabaudigenome.Thirty-eightoftheORFsof(cid:3)50aashowedsignificant
mice immunized by the i.e.d. (gene gun) or the i.m. route.
similaritywithsequencesinoneormorePlasmodiumspecies.The29ORFsin
theV50(cid:1)poolfoundtohavehomologywithP.falciparum3D7sequenceswere ThreeweeksfollowingthelastDNAdose,30K-vaccinatedand
furtheranalyzedtodeterminetheirlikelyexpressionprofilesusingtheWinzeler control mice were infected with P. c. adami DK parasites.
database(32).ThemaximumhourofexpressionofeachORFwaspredicted Parasitemiawassignificantlylowerinmicevaccinatedwiththe D
according to P. falciparum 3D7 expression data published in the DeRisi Lab o
Malariatranscriptomedatabase(5),wherethemaximumhourofblood-stage 30Klibrarybythei.e.d.routewhencomparedtocontrolmice w
thatreceivedtheemptyVR1020vector(Fig.2A),witha42% n
expressioncorrespondstoaspecificstageofparasitedevelopment(http://ma-
laria.ucsf.edu/).Thenumberofpredictedtransmembranedomains,aswellasthe decrease in mean cumulative parasitemia (total parasite bur- lo
a
presenceofasignalsequence,wasdescribedforeachORFthatwasmostsimilar den during the 9 days of patent infection) (Fig. 2B) (P (cid:8) d
toaP.falciparumorP.yoeliiorthologueusingPlasmoDB(http://plasmodb.org/). 0.001): peak parasitemia was significantly reduced by 48% (P ed
BLASTXanalysiswasperformedusingthePlasmoDBdatabasewiththeNCBI
BLASTX2.1.2programandcomparedwithallPlasmodiumORFsof(cid:3)50bp (cid:8)0.01).Comparableresultswereobtainedinasecondexper- fr
(aa)(http://plasmodb.org/plasmodb/servlet/sv?page(cid:5)BLAST)onthe18inserts iment (Fig. 2C and D), with a 50% reduction in cumulative om
whoseORFsdidnotshowsignificantsimilaritytoPlasmodiumproteins.Sixteen parasitemia and a 52% reduction in peak parasitemia (P (cid:8)

oftheseinsertsshowedanORFwithasignificanthittoPlasmodiumsequences 0.01): in contrast, i.m. delivery of the 30K vaccine did not ht
inanonexpressedframe,i.e.,theycarrygenomicDNAthatwasinsertedoutof reduce the parasite burden, suggesting that the route of vac- tp
frame into the vector due to the random cloning strategy originally used to :
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generatethelibrary(58).BLASTNanalysisusingNCBIagainstallorganisms cine delivery is important for induction of cross-protection in /ia
(http://www.ncbi.nlm.nih.gov/BLAST/)showedthattheDNAencodingthere- thismodelsystem(Fig.2EandF). i.
mainingtwoORFsof(cid:3)50aawashomologoustoratormousegenomicDNA Vaccination of mice with a subpool of plasmids encoding as
depositedinGenBankin2003:theseORFsarenonsensesequences,sincethey ORFs of nine or more amino acids induces cross-protective m
shownohomologywithanysequenceinBLASTPsearches.Thus,38/56ORFsof
.
(cid:3)50aarepresentauthenticPlasmodiumORFs. immunity. Preliminary experiments using the P. c. adami DS o
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Vaccinationandinfectionofmice.Forintraepidermal(i.e.d.)vaccination,the modelinBALB/cmiceledtotheidentificationofasubpoolof g
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goldparticlescoatedwithDNAweredeliveredinmiceusingaHeliosgenegun 733plasmids(pool3KA-4)withinthe30Klibrarythatelicited o
(Bio-RadLaboratories)withapulseofheliumgasat400lb/in2.Micereceived n
threedosesof1(cid:4)gDNAperdoseseparatedbytwo-weekintervals(46).For significantprotectionintwoindependentexperiments(Fig.1) D
intramuscular(i.m.)vaccination,DNAinphosphate-bufferedsaline(PBS)was (58; P. Smooker and T. W. Spithill, unpublished data). DNA e
deliveredintothetibialisanteriormuscle(threedosesof100(cid:4)gtotalDNAper sequencesofthe733insertsintheseplasmidswereobtainedby c
e
dose,separatedby2-weekintervals).P.c.adamiDKparasites(isolate556KA) sequencing across the cloning site in VR1020, and the se- m
werekindlyprovidedbyD.Walliker(UniversityofEdinburgh).Inbredfemale
quences were analyzed for the presence of an ORF in frame b
BALB/cmice,ages6to8weeksold,wereinoculatedintheperitoneumwith5 e
(cid:6)104infectedredbloodcells(IRBC)obtainedfromasyngeneicinfectedmouse withtheTPAsignalpeptideandthencataloguedaccordingto r

2to3weeksfollowingthelastimmunization.Thei.m.experimentwasconducted the length of the encoded peptide. A pool of 303 clones con- 4
,
atthesametimeasthefirsti.e.d.experiment(Fig.2).Parasitemiawasdeter- taining plasmids encoding ORFs of (cid:3)9 aa (large enough to 2
minedasdescribedpreviously(46). 0
InvitrospleencellcultureandELISAforIFN-(cid:2).Splenocytecellcultureswere generate major histocompatibility complex (MHC) binding 1
performedasdescribedpreviously,usingcellscollected15daysafterthefinal peptides [55]) were selected to generate a new vaccine (V9(cid:1) 8
b
DNAvaccination(46).Cellviabilitywasgreaterthan98%,asdeterminedby pool),whichwasassessedagainstacontrolpoolconsistingof y
trypanblueexclusion(Invitrogen).Splenocyteswereplatedatafinalconcentra- 63 plasmids containing out-of-frame (OOF) sequences from g
tionof5(cid:6)106cells/mlin24-wellplatesandwerestimulatedfor72hwith2(cid:6) the P. c. adami DS library: this OOF control pool delivers u
106/mlnoninfectederythrocytesorIRBCobtainedfromasyngeneicmouseat e
malaria DNA but does not encode any ORF (unpublished s
themomentofpeakparasitemia(40to45%forP.c.adamiDSand15to22% t
for P. c. adami DK infections, respectively). Cell culture supernatants were data).Notethatthereisvariationbetweenexperimentsinthe
harvestedandtestedforthepresenceofgammainterferon(IFN-(cid:7))byenzyme- cumulative parasitemias observed in control mice vaccinated
linkedimmunosorbentassay(ELISA)asdescribedpreviously(46).
witheitherVR1020orOOFDNA,andwehavenotobserved
Opsonizationandphagocytosisassays.Phagocytosisassaysusingperitoneal
reproducible differences in parasitemia in mice given these
exudatescellswereperformedasdescribedpreviously(41,46).Adherentcells
wereincubatedfor2hwithP.c.adamiDSorDKIRBC(parasitemiasbetween controlvaccines.IntraepidermaldeliveryoftheV9(cid:1)poolin-
15and30%)previouslyincubatedfor1hwithinactivatedimmuneseraorsera ducedsignificantcross-protection(52%reductioninmeancu-
fromDNA-vaccinatedmice(n(cid:5)4;1:20serumdilution)collectedbeforepara- mulative parasitemia, P (cid:8) 0.001; 60% reduction in peak par-
site challenge. The percentage of macrophages ingesting IRBC (300 macro-
asitemia,P(cid:8)0.05)againstP.c.adamiDKinfectioninBALB/c
phagesperindividualsample)wasthenquantifiedbylightmicroscopy.
mice (Fig. 3A and B). A second experiment confirmed the
protective effect of the V9(cid:1) pool (45% reduction in cumula-
RESULTS
tiveparasitemia,P(cid:8)0.01;45%reductioninpeakparasitemia,
Expression library immunization with genomic sequences P(cid:8)0.01[Fig.3CandD]).Thislevelofprotectioniscompa-
from P. c. adami DS cross-protects BALB/c against heterolo- rabletothatinducedbytheoriginal30Kvaccinepool(Fig.2A
gouschallengewithP.c.adamiDK.TheVR1020/30Kgenomic toD).
VOL.73,2005 CROSS-PROTECTION WITH MULTIVALENT MALARIA VACCINES 2977
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FIG. 2. KineticsofinfectionandcumulativeparasitemiaofmiceimmunizedwiththeVR1020/30KpoolorthecontrolVR1020emptyvector. t
PanelsA-BandC-Drepresenttheresultsoftwoindependentexperiments.Mice(n(cid:5)6)werevaccinatedbythei.e.d.route(A-D)orthei.m.route
(E, F). Two weeks after the third DNA dose, mice were challenged by intraperitoneal infection with 5 (cid:6) 104 P. c. adami DK IRBC. Daily
parasitemiaisshownas%parasitemia(mean(cid:9)standarderrorofthemean[SEM]).Cumulativeparasitemiavaluesrepresentthesumofdaily
parasitemiathroughouttheperiodofpatentinfection.GroupswerecomparedusingaStudentttest:(cid:1),P(cid:8)0.001;(cid:1)(cid:1),P(cid:8)0.01.
VaccinationofmicewithapoolofplasmidsencodingPlas- adami.Asignificantreductioninmeancumulativeparasitemia
modium ORFs of 50 or more amino acids induces cross-pro- (30%reduction;P(cid:8)0.05)wasobservedinvaccinatedmice,and
tectiveimmunity.WithintheV9(cid:1)plasmidpool,weselecteda the effect was apparent following peak parasitemia (Fig. 4A
set of 56 plasmids predicted to encode ORFs of (cid:3)50 aa and B). A second experiment yielded a 27% reduction in
(termed the V50(cid:1) vaccine) and evaluated the protective effi- cumulativeparasitemia(P(cid:8)0.05;Fig.4CandD).Vaccination
cacyofthissubsetinBALB/cmice(i.e.d.route),hypothesizing withtheV50(cid:1)vaccinedidnotinduceasignificantreductionin
that these large ORF genes would encode multiple epitopes, peak parasitemia, an effect that was observed above with the
some of which may be conserved between strains of P. c. 30K and V9(cid:1) vaccines. Since the V50(cid:1) vaccine does not
2978 SCORZA ET AL. INFECT.IMMUN.
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FIG. 3. KineticsofinfectionandcumulativeparasitemiaofmiceimmunizedwiththeVR1020/V9(cid:1)poolorthecontrolVR1020/OOFpoolby m
thei.e.d.route.PanelsA-BandC-Drepresenttheresultsoftwoindependentexperiments.TwoweeksafterthethirdDNAdose,themice(n(cid:5) .
o
6)werechallengedbyintraperitonealinfectionwith5(cid:6)104P.c.adamiDKIRBC.Dailyparasitemiaisshownas%parasitemia(mean(cid:9)standard r
g
error of the mean [SEM]). Cumulative parasitemia represents the sum of daily parasitemia values throughout the period of patent infection. /
GroupswerecomparedusingaStudentttest:(cid:1),P(cid:8)0.001;(cid:1)(cid:1),P(cid:8)0.01. o
n

D
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includeplasmidsencodingshorterORFs((cid:8)49aa)itispossible sequencesthathadknownP.falciparumorthologuesreported ce
m
thattheseORFsmaybecontributingtotheprotectioninduced inPlasmoDB(Table1).Inaddition,32/38ORFs(84%)shared
bythe30KandV9(cid:1)vaccines. significant similarity to P. yoelii proteins (Table 1). Interest- be
SequenceanalysisofplasmidsencodingORFsof50ormore ingly, 22/38 ORFs (57%) aligned significantly with predicted r
4
amino acids. Using the published P. falciparum and P. yoelii proteins from 3 or more Plasmodium species, suggesting that ,

2
genomesequences,weanalyzedthepredictedORFsofthe56 these sequences encode polypeptides that are conserved dur-
0
inserts encoding 50 or more residues in a BLASTP and ingspeciationofPlasmodium(Fig.5A).SevenORFscontaina 1
8
BLASTX alignment search (PlasmoDB and NCBI data predicted signal sequence, and 27 ORFs exhibit one or more
b
source) to identify similarities with Plasmodium sequences predicted transmembrane anchor domains (Table 1). The y

withinGenBankdatabases,usingafalse-positiveexpection(E) stage of expression, level of expression, and peak time of ex- g
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10(cid:2)6(4).Thirty-eightORFsexhibitedsignificantidentitywith pression during blood-stage infection of each of the ORFs e
s
sequencesfromoneormorePlasmodiumspecies(Table1;Fig. homologous with sequences in P. falciparum were analyzed t
5A), of which 16 ORFs (42%) matched with known Plasmo- usingthedatafromLeRochetal.(32)andBozdechetal.(5)
diumproteins(erythrocytemembrane-associatedantigen,von (Table1;Fig.5C)(notethatfiveoftheseORFswerenotfound
Willebrand factor (cid:10)-domain-related protein, putative PLP- intheBozdechetal.database).Ofthe31ORFsforwhichdata
transportATPase,karyopherinbeta,betadyneinheavychain, existintheWinzelerdatabase,30ORFsareexpressedinblood
ubiquitin-activating enzyme e11, putative eukaryote transla- stagesand29ORFsareexpressedineachoftheblood,game-
tion eIF-1a, PfEMP3, clustered asparagine-rich protein, sub- tocyte,andsporozoitestages.ThepeaktimesofORFexpres-
tilisin-like protease 2, transportin/putative bromodomain pro- sionweredistributedthroughoutthebloodstage,andmanyof
tein, putative tubulin-tyrosine ligase family, CCAAT box- theseORFsareexpressedatarelativelyhighlevelintheblood
bindingproteinsubunitB,putativebir1protein)(Fig.5B);20 stageofP.falciparum(Table1;Fig.5C).Thesedatashowthat
ORFs (52%) showed similarity with various Plasmodium hy- a high proportion of the cross-protective V50(cid:1) vaccine se-
potheticalproteins,and2ORFsshowedhomologytounknown quencesorthologoustoP.falciparumarepotentialblood-stage
sequences from P. chabaudi (Fig. 5B). Overall, by BLASTP antigens.
analysis,29/38ORFs(76%)wereorthologuesofP.falciparum Cross-protection elicited by multivalent DNA vaccines cor-
proteins and a further 3 ORFs were orthologous to P. yoelii relates with opsonizing antibodies and IFN-(cid:2) production by
VOL.73,2005 CROSS-PROTECTION WITH MULTIVALENT MALARIA VACCINES 2979
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FIG. 4. KineticsofinfectionandcumulativeparasitemiaofmiceimmunizedwiththeVR1020/V50(cid:1)poolorthecontrolVR1020emptyvector i.a
bythei.e.d.route.PanelsA-BandC-Drepresenttheresultsoftwoindependentexperiments.TwoweeksafterthethirdDNAdose,themice(n s
(cid:5)6)werechallengedbyintraperitonealinfectionwith5(cid:6)104P.c.adamiDKIRBC.Dailyparasitemiaisshownas%parasitemia(mean(cid:9) m
.
standard error of the mean [SEM]). Cumulative parasitemia represents the sum of daily parasitemia values throughout the period of patent o
infection.GroupswerecomparedusingaStudentttest:(cid:1),P(cid:8)0.05. rg
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antigen-stimulated splenic cells. Humoral and cellular im- antigens expressed by both homologous and heterologous c
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muneresponsesbothplayaroleinthecontrolandresolution IRBC.
m
ofmurinemalariainfections(31,33,42,60,61,62).Wehave b
previously shown a role for both opsonizing antibodies and e
r
cellular responses in immunity induced by the 30K vaccine DISCUSSION 4
,
againstP.c.adamiDSchallenge(46).Inordertoassessarole 2
for antibody in vaccine-mediated cross-protection, the opso- Inthepresentstudy,wehavedemonstratedthatnewcross- 0
1
nizingcapacityofthesera(n(cid:5)4)frommicevaccinatedwith protectiveantigens/epitopescanbeidentifiedfromthemalaria 8
the30Klibrary,thepoolofV9(cid:1)plasmids,ortheV50(cid:1)plas- genome using a genomic screening approach and that high b
y
midswasassessedusinghomologousP.c.adamiDSandhet- levels of cross-protection against heterologous blood-stage P.
g
erologous P. c. adami DK IRBC. All sera significantly opso- c. adami challenge can be achieved in mice with multivalent u
e
nized both P. c. adami DS and DK IRBC in a manner DNA vaccines comprising 38 to 303 sequences. We initially s
comparable to immune sera from mice that had recovered showedthatthe30KP.c.adamiDSvaccineinducessignificant t
from infection with either the virulent DS or avirulent DK reductions in mean cumulative and peak parasitemia in
strains (Fig. 6), suggesting that the DS and DK strains share BALB/c mice challenged with heterologous P. c. adami DK
antigenic cross-reactivity at the surface of the infected eryth- parasites. One protective pool of 733 plasmids (3KA-4) de-
rocyte. rivedfromthe30Klibrarypreviouslyshowntoprotectagainst
Specific IFN-(cid:7) responses by splenic cells from vaccinated P.c.adamiDSchallenge(58)wasselectedforsequenceanal-
miceinresponsetonativeantigensfrombothP.c.adamiDS ysis to identify ORF genes encoding protective antigens or
andDKIRBCwereinducedbyboththeV9(cid:1)andtheV50(cid:1) epitopes.Wecombined303clonescontainingplasmidsencod-
vaccines(Fig.7),consistentwithearlierobservationswiththe ing predicted ORFs of nine or more aa and showed that this
30KvaccineinthehomologousP.c.adamiDSmodel(46).We V9(cid:1)poolalsoprotectedBALB/cmiceagainstchallengewith
didnotdetectresponsestononinfectederythrocytesinanyof heterologousP.c.adamiDKparasitesinamannercomparable
thegroupsevaluated(datanotshown).Theseresultsshowthat to the 30K vaccine, with a significant 42 to 52% reduction in
both the V9(cid:1) and V50(cid:1) vaccine pools of recombinant plas- meancumulativeparasitemiaand45to60%reductioninpeak
midsprimedcellularimmuneresponsestonativeblood-stage parasitemia.ThiscontrastswiththefailureoftheAMA1vac-
2980 SCORZA ET AL. INFECT.IMMUN.
hD
M es es eseseseses es es eseses eses eseses es eses eseses eseseses
T Y Y YYYYY Y Y YYY YY YYY Y YY YYY YYYY
e
alnc
ne
Sigsequ Yes YesYes Yes Yes Yes Yes
Maxtimeofexpressiong(h) 36 1712452834433012 1 NA NA111NA101 112929 20 2227 31NANA44 35221237 e.
oit
Proteinfcluster 14 11jNANA10NA15NANA 14 NA NANA12NANA2 7NANA NA NA11 3532 1010NANA a.sporoz
atp
dS
Blood-stageeexpression 123.4 59.2217.0510.251,216.3571.95381.657763,378.35 49.35 4,221.35 8.6139.65241.3155.6229.10 7,093.944.4911.05 443.65 25463.25 32.83,591.8529.9302.55 181.35112.7585.3567.75 expressionametocyte, Dow
P.c.adamiTABLE1.SequenceanalysisofORFsfromDSDNAlibrary Size%%No.CloneofE-valueE-valueExpressionacPfPyProteincandidate(PforPy)Gene()IdentityGene()IdentityofOrthologuedPfPyno.ORF()()stagesbPfPy()()spp.(aa) (cid:3)2242230Pf/PyHypproteinsPF11_01583.00E-7461PY007881.00E-121885PF11_0158RTSMGSp(cid:3)2424224Py1betadyneinheavychainPY047391.00E-113861(cid:3)2744204Pf/PyHypproteinsPF13_02354.00E-5556PY012341.00E-102874PF13_0235RTSMGSp(cid:3)2339232Pf/PyHypproteinsPFL1815c4.00E-1928PY050991.00E-98715PFD0320cRTSMGSp(cid:3)2608297Pf/PyHypproteinPFL2135c2.00E-2031PY064073.00E-84632PFL2135cRSMGSp(cid:3)3668190Pf/PyHypproteinsPF14_04123.00E-4555PY019129.00E-84753PF14_0412RTSMGSp3633211PfHyp/PyunknownproteinsPFE1315w2.00E-6153chrPyl_026063.00E-73864RTSMGSp(cid:3)2260250Pf/Pysubtilisin-likeprotease2PF11_03811.00E-2544PY012221.00E-67534PF11_0381RSMGSp(cid:3)2261169Pf/PyHypproteinsPF14_04193.00E-0625PY071968.00E-65733(PF11_0333)RTSMGSp(cid:3)3602118PfHypprotein/PyAsnrichMAL6P1.1981.00E-1634PY034989.00E-63873RTSMGSpprotein(cid:3)2333125Pf/Pyerythrocytememb.PF10_01771.00E-4460PY069595.00E-62855RSGantigen(cid:3)2423118PfPLP-transportATPase/PyPFL1125w1.00E-3261PY064833.00E-59863PFL1125wRTSMGSpATPase(cid:3)2622116Pf/PyHypproteinsPF08_00905.00E-3358PY046837.00E-59894PF08_0090RS(cid:3)2207135Pf/PyHypproteinsPFL1735c1.00E-3246PY042292.00E-53854RTSMG(cid:3)2519107PfHypprotein/PyPFEMP3PFI0225w4.00E-1134PY022703.00E-48823(MAL6P1.23)RTSMGSp(cid:3)284792PfVWfactor/Pymicronprot.PF08_0136(b)4.00E-3369PY076811.00E-42884RTSMGSp(cid:3)263098PfHypprotein/PyPFEMP3PF13_00411.00E-1040PY000324.00E-40762PF13_0041RTSMGSp265581Pytubulin-Tyrligasefamily,PY024126.00E-38891PF11_0481GSpputative(cid:3)267270Pf/PykaryopherinbetaPFE1195w5.00E-2780PY001555.00E-351005PFE1195wRTSMGSp(cid:3)252668Pf/PyHypproteinsPF14_05047.00E-1872PY049479.00E-34924PF14_0504RTSMG(cid:3)238468Pyputativebromodomain/PfMAL6P1.1518.00E-0635PY006842.00E-29912(MAL6P1.131)RTSMGSptransportin(cid:3)273560Pf/PyHypproteinPFL1795c7.00E-1779PY055388.00E-29962RTSMGSp(cid:3)220664PyCCAATbox-bindingprot.PY009135.00E-28691subunitB(cid:3)287193PfClpproteinase/PyunknownX952764.00E-1860chrpyl_016972.00E-26772(cid:3)363453Pf/PyHypproteinMAL13P1.782.00E-0950PY046192.00E-24903MAL13P1.78RTSMSp270361PfHypprot./Pyubiquitinact.PF13_01821.00E-1466PY064136.00E-24914PF13_0182RTSMGSpenz.e11(cid:3)2851214Pf/PyHypproteinPF14_02521.00E-11089PY057954.00E-23983PF14_0252RTSMG(cid:3)365764Pf/Pyeuk.translationelF-1aPF11_04473.00E-2887PY019735.00E-34975PF11_0447RTSMGSp277355PyHypproteinPY053993.00E-14872PFL1020wRTSMGSp(cid:3)280894PyHypproteinPY020572.00E-12432PF10_0374RTSMGSp(cid:3)2549117Pyputativebir1proteinPY023688.00E-103422786186Pyunknownchrpyl_007199.00E-086023603248PfHypproteinPF14_05523.00E-56514RTSGSp(cid:3)2677110PfHypproteinPF08_00511.00E-19593RTSSp(cid:3)273362PfHypproteinPFC0760c7.00E-12502RTSMGSp(cid:3)254078PfHypproteinPFE1555c2.00E-08441RTSGj69Pchabunique(contig7620,24302E-41,95%)i53Pchabunique(contig7574,36786.20E-99,97%) aP.falciparum;P.yoelii;Pf,Py,act.,activating;enz.,enzyme;euk.,eukaryotic;Hyp,hypothetical;memb.,membrane;prot.,protein.bP.falciparum,P.yoelii,P.berghei,P.knowlesi,P.vivax.Speciesanalyzed:andcP.falciparumPyoeliiP.falciparumorthologueofthesequenceannotatedinPlasmoDB.Parenthesesindicatethatthissequencewasusedfortheblood-stagedRecordedifpositivebyeithersorbitolortemperaturesynchronization;WinzelerdatabaseinPlasmoDB;Rring;Ttrophozoite;Sschizont;Mmerozoite,GgeRecordedastheaveragetotalvalueoftheRTSMexpressionlevelusingsorbitolandtemperaturesynchronizationmethods;WinzelerdatabaseinPlasmoDB.fProteinclusterdefinedinWinzelerdatabase.gMaximumhourofblood-stageexpressioninDeRisidatabaseinPlasmoDB.hTransmembranedomainpredictionsinPlasmoDBrecordedusingfourtools:TMAP,TMHMM2,TMPRED,andTOPPRED2.iP.chabaudiHomologoustotheindicatedcontig.jNA,notavailable. on December 4, 2018 by guesthttp://iai.asm.org/nloaded from
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FIG. 5. Sequencehomologyandblood-stageexpressionlevelsofthe38ORFsorthologoustoPlasmodiumsequencesintheV50(cid:1)vaccine.A:
homologywithotherPlasmodiumspecies;B:theannotationofthesesequences;C:stackedbarchartshowingtheblood-stageexpressionpatterns
(inP.falciparum)of31sequencesdemonstratingsignificantorthologywithP.falciparumdefinedbythemaximalhourofexpression(seereference
5)andbytheoveralllevelofblood-stageexpression,using150unitsasthecutoffvaluetodifferentiatebetweenhighandlowexpression(32;Le
RochandWinzeler,personalcommunication).Nosequenceshowedmaximalexpressioninthemerozoitestage.Twenty-eightsequencesshowed
directorthologytoP.falciparumbyBLASTPanalysis(nodatawereavailableforgeneX95276).Threeadditionalsequenceswereorthologousto
aP.yoeliisequencethatwasdefinedasorthologoustoP.falciparuminPlasmoDB(seeTable1).
2981
2982 SCORZA ET AL. INFECT.IMMUN.
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FIG. 6. PhagocytosisofP.c.adamiIRBCpreincubatedwithserafrommiceimmunizedwiththeVR1020/30K,VR1020/V50(cid:1),orVR1020/V9(cid:1) em
vaccinesandfrommicethathadreceivedthecontrolVR1020emptyvector.A.UptakeofP.c.adamiDSIRBC.B.UptakeofP.c.adamiDK
b
IRBC.C.Inaseparateexperiment,serafrommicevaccinatedwiththeV9(cid:1)plasmidswereassessedusingP.c.adamiDSandDKIRBC.DS e
immuneandDKimmune:serafromimmunemicethathadresolvedaprimaryinfectionwithP.c.adamiDSandDKparasiteswereincludedas r

positivecontrols.Eachanalysisusedserafromfourindividualmice.Ineachpanel,theopsonizationobservedwiththecontrolVR1020serumis 4
,
comparedindividuallywitheachvaccineserum(30K,V50(cid:1),orV9(cid:1))usinganonparametricMann-Whitneyttest.(cid:1),P(cid:8)0.03. 2
0
1
8

b
cine from P. c. adami DS to cross-protect mice against chal- orthologous to P. falciparum sequences, many of which are y

lengewiththeDKstrain(13). expressedathighlevelsinthebloodstage,suggestingthatthe g
u
WithintheV9(cid:1)plasmidpool,weselectedasubsetofplas- multivalentcross-protectiveV50(cid:1)(andV9(cid:1))vaccinescontain e
midsencodingORFsof(cid:3)50aa(V50(cid:1)vaccine),hypothesizing conserved blood-stage antigens. The V50(cid:1) pool contains 20 st
that these large ORFs would contain a variety of T-cell or ORFsorthologoustohypotheticalproteins,and16ORFsgave
B-cell epitopes which may elicit cross-protection. The V50(cid:1) significantalignmentscoreswithknownPlasmodiumproteins,
vaccine induced significant protection in BALB/c mice, al- such as the erythrocyte membrane-associated antigen
thoughthiswasmoderatewhencomparedtothehighprotec- PfEMP3,anerythrocytemembraneprotein,thesubtilisin-like
tioninducedbythe30KvaccineorbytheV9(cid:1)vaccine.These protease2,theubiquitin-activatingenzymee11,andothers.Of
differences suggest that shorter ORFs of (cid:8)49 aa, excluded thesevenORFsexhibitingpredictedsignalsequences,fourare
fromtheV50(cid:1)pool,maysynergizewiththelargerORFsand hypothetical proteins with predicted transmembrane domains
contribute to cross-protection against blood-stage infection. and one is an erythrocyte membrane-associated antigen: this
We are currently evaluating the immunogenic and synergistic may suggest that the V9(cid:1) and V50(cid:1) pools encode several
propertiesassociatedwithORFsofsmallersize. putativemembrane-associatedantigens,whichmaycontribute
Sequence analysis of the ORFs of (cid:3)50 aa showed that 38 tothecross-protectionobserved.
ORFscorrespondedtoauthenticPlasmodiumpolypeptidese- AnalysisoftheimmuneresponsesinducedbytheV9(cid:1)and
quences: 22 ORFs aligned with sequences from 3 or more V50(cid:1) vaccine pools showed that mice primed with the P. c.
Plasmodium species, and 29 ORF genes encoded sequences adami DS vaccine sequences exhibited opsonizing antibodies
VOL.73,2005 CROSS-PROTECTION WITH MULTIVALENT MALARIA VACCINES 2983
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FIG. 7. IFN-(cid:7) secretion by splenocytes from individual BALB/c mice vaccinated i.e.d. with the gene gun. Mice were immunized with the s
VR1020/V9(cid:1) and VR1020/V50(cid:1) vaccines. Control values (CTRL) represent splenocyte cultures from mice that received the VR1020 empty m
vector.Splenocyteswerekepteitherunstimulated(UNST.)orstimulatedwith2(cid:6)106IRBC(P.c.adamiDKand/orDS)for17h.Thesupernatants .o
fromthesplenocytecultureswereharvested,andIFN-(cid:7)wasmeasuredbyELISA.TheresponsesinspleniccellsfrommicegivenemptyVR1020 rg
plasmidDNAstimulatedwithIRBC(CTRL(cid:1)IRBC)werecomparedwiththeresponseswithspleniccellsfrommicegivenvaccineplasmidDNA /
alsostimulatedwithIRBC(V9(cid:1)orV50(cid:1)IRBC),usinganonparametricMann-Whitneyttest.PanelA:(cid:1),P(cid:5)0.0159.PanelB:(cid:1),P(cid:5)0.0079. o
n
PanelC:(cid:1),P(cid:5)0.0002.PanelD:(cid:1),P(cid:5)0.0286).
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and IFN-(cid:7)-producing splenic cells that cross-reacted with na- mediated phagocytosis in the protection transferred passively b
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tiveP.c.adamiDKantigensexpressedinIRBC.Comparables byantibodies(51,62,65). r

4
resultswereobtainedinourearlierstudythatassessedthe30K The results suggest that the cross-protection observed here ,

library in three different targeting vectors: only the vaccine withthemultivalentvaccinesresultsfromresponsestooneor 2
0
poolsinducingboththehumoralandcellulararmsofimmunity more sequences within the P. c. adami genome that are con- 1
(opsonizing antibodies and IFN-(cid:7), respectively) were capable servedbetweentheDSandDKstrains.Sincevaccinationwith 8
b
of protecting against lethal P. c. adami DS infection (46). known highly protective antigens, such as AMA1 and MSP1, y

Opsonizing antibodies in our model system promote the up- doesnotinducecross-protectioninmousemalariamodelsys- g
u
take of IRBC by macrophages via antibody binding to CD16/ tems (13, 48, 52), one interpretation of the data is that cross- e
Fc(cid:7)IIandCD32/Fc(cid:7)IIIreceptorsonmacrophages(46).Since protection results from the synergistic or additive activity of s
t
IFN-(cid:7)hasbeenshowntoenhanceexpressionofFcRonmac- responsestothemultipleconservedantigenspresentintheDS
rophages,itispossiblethatproductionofIFN-(cid:7)enhancesthe vaccinepools.ThesequenceanalysisoftheV50(cid:1)pooldirectly
protectionconferredbyopsonizingantibodiesinthecontrolof showsthat29/38Plasmodiumorthologuesinthepoolexhibita
blood-stagemalaria(21,22,47,64).Antibody-dependentcel- high degree of conservation with other Plasmodium species
lular inhibition is involved in protection against blood-stage (e.g.,EvalueinBLASTPof(cid:8)3(cid:6)E-14;sequenceidentityof
malaria in humans, and vaccine-induced antibody-dependent 53 to 100% with P. yoelii; Table 1). Twenty of the protective
cellularinhibitionmaybeausefulstrategyforamalariablood- sequences(cid:3)50aainlengthencodepreviouslyuntestedhypo-
stage vaccine (3, 23, 36, 56, 57). It has been suggested that theticalproteins,and14oftheseproteinsshowedconservation
antibody binding to FcR on macrophages triggers the release across several malaria species, suggesting that conserved hy-
of mediators that inhibit the replication of malaria parasites potheticalproteinsmaybecontributingtotheobservedcross-
within opsonized erythrocytes (3, 36). Although FcR do not protection. This is analogous to results of Melby et al. (39),
seemtoplayaroleintheprotectionconferredbyantibodiesin whoidentifiedpoolsofprotectivehypotheticalproteinswithin
the P. yoelii 17XL infection model, data using the P. berghei aLeishmaniaELIvaccine,andHaddadetal.(24),whoiden-
XAT nonlethal model suggest an important role for FcR- tifiedhypotheticalproteinsinaP.yoeliiliver-stageexonDNA