DTIC ADA430048: Comparison of the Protective Efficacy of DNA and Baculovirus-Derived Protein Vaccines for EBOLA Virus in Guinea Pigs

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VirusResearch92(2003)187(cid:1)/193
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Short communication
Comparison of the protective efficacy of DNA and baculovirus-
derived protein vaccines for EBOLA virus in guinea pigs
Jenny L. Mellquist-Riemenschneidera, Aura R. Garrisona, Joan B. Geisberta,
Kamal U. Saikhb, Kelli D. Heidebrinka, Peter B. Jahrlinga, Robert G. Ulrichb,
Connie S. Schmaljohna,*
aDepartmentofMolecularVirology,VirologyDivision,DivisionU.S.ArmyMedicalResearchInstituteofInfectiousDiseases,1301DittoAve.,
Ft.Detrick,Frederick,MD21702,USA
bToxinologyDivision,DivisionU.S.ArmyMedicalResearchInstituteofInfectiousDiseases,1301DittoAve.,Ft.Detrick,Frederick,MD21702,USA
Received21 August 2002; receivedinrevisedform22 November 2002; accepted22 November 2002
Abstract
ThefilovirusesEbolavirus(EBOV)andMarburgvirus(MARV)causeseverehemorrhagicfeverinhumansforwhichnovaccines
areavailable.Previously,aprimingdoseofaDNAvaccineexpressingtheglycoprotein(GP)geneofMARVfollowedbyboosting
with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001.
Vaccine 20, 568(cid:1)593). Todetermine whether asimilar prime-boost vaccine approachwouldbeeffectiveforEBOV, we generated
/
and characterized recombinant baculoviruses expressing full-length EBOV GP (GP ) or a terminally-deleted GP (GPa-) and
1,2
examinedtheirimmunogenicityinguineapigs.Asexpected,cellsinfectedwiththeGPa-recombinantsecretedmoreGP thanthose
1
infected with the GP recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from
1,2
mammaliancellderivedvirionGP,inthattheyhadnocomplex/hybridN-linkedglycansorglycanscontainingsialicacid.Despite
these differences, the baculovirus-derived GPs were able to bind monoclonal antibodies to five distinct epitopes on EBOV GP,
indicatingthattheantigenicstructuresoftheproteinsremainintact.Asameasureoftheabilityofthebaculovirus-derivedproteins
to elicit cell-mediated immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human
dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have the capacity to evoke
cell-mediatedimmuneresponses.Guineapigsvaccinatedwiththebaculovirus-derivedGPsalone,orinaDNAprime-baculovirus
proteinboostregimendevelopedantibodyresponsesasmeasuredbyELISAandplaquereductionneutralizationassays;however,
incompleteprotectionwasachievedwhentheproteinsweregivenaloneorincombinationwithDNAvaccines.Thesedataindicate
that avaccine approachthat waseffectivefor MARV isnoteffectiveforEBOV in guinea pigs.
PublishedbyElsevierScience B.V.
Keywords: Filovirus;Ebolavirus;Prime-boost;Baculovirus;DNAvaccine
The Filoviridae family contains two genera of highly designated,Marburgvirus(MARV),withseveralstrains
pathogenic viruses: Ebola-like viruses, and Marburg- identified. It is likely that as these strains are better
like viruses (IUMS, 2000). Currently four viral species characterized, some of them will be elevated to viral
are listed in the Ebola-like virus genus: Cote d’Ivoire species. All of the viruses in the family except for
Ebola virus, Reston Ebola virus (REBOV), Sudan REBOV are highly pathogenic for humans. ZEBOV
Ebolavirus (SEBOV),andZaire Ebolavirus (ZEBOV). causedtwomajoroutbreaksofEbolahemorrhagicfever
Numerous strains of these viruses have been identified. (EHF) in 1976 and 1995, during which hundreds of
One species of Marburg-like viruses has been officially cases were reported with mortality rates as high as 80(cid:1)
/
90% (Peters et al., 1994). SEBOV caused outbreaks in
Sudanin1976and1979with (cid:1)300casesandmortality
*Correspondingauthor.Fax:(cid:2)/1-301-619-2439. (cid:1)50%. SEBOV appeared in /Uganda in 2000(cid:1)2001
E-mail address: [email protected] (C.S. / /
Schmaljohn). causing (cid:1)/400casesofEHFwithamortalityrateof (cid:1)/
0168-1702/02/$-seefrontmatter.PublishedbyElsevierScienceB.V.
doi:10.1016/S0168-1702(02)00338-6
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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Comparison of the protective efficacy of DNA and baculovirus-derived
5b. GRANT NUMBER
protein vaccines for EBOLA virus in guinea pigs, Virus Rsearch
92:187-193 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Mellquist-Riemenschneider, JL Garrison, AR Geisbert, JB Heidebrink,
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KD Jahrling, PB Schmaljohn, CS
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14. ABSTRACT
The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever in
humans for which no vaccines are available. Previously, a priming dose of a DNA vaccine expressing the
glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein
was found to confer protective immunity to guinea pigs (Hevey et al., 2001. Vaccine 20, 568-593). To
determine whether a similar prime-boost vaccine approach would be effective for EBOV, we generated
and characterized recombinant baculoviruses expressing full-length EBOV GP (GP(1,2)) or a
terminally-deleted GP (GPa-) and examined their immunogenicity in guinea pigs. As expected, cells
infected with the GPa- recombinant secreted more GP(1) than those infected with the GP(1,2)
recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from
mammalian cell derived virion GP, in that they had no complex/hybrid N-linked glycans or glycans
containing sialic acid. Despite these differences, the baculovirus-derived GPs were able to bind monoclonal
antibodies to five distinct epitopes on EBOV GP, indicating that the antigenic structures of the proteins
remain intact. As a measure of the ability of the baculovirus-derived proteins to elicit cell-mediated
immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human
dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have
the capacity to evoke cell-mediated immune responses. Guinea pigs vaccinated with the
baculovirus-derived GPs alone, or in a DNA prime-baculovirus protein boost regimen developed antibody
responses as measured by ELISA and plaque reduction neutralization assays; however, incomplete
protection was achieved when the proteins were given alone or in combination with DNA vaccines. These
data indicate that a vaccine approach that was effective for MARV is not effective for EBOV in guinea
pigs.
15. SUBJECT TERMS
filovirus, Ebola, prime-boost, baculovirus, DNA vaccine, recombinant protein, laboratory animals, guinea
pigs
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
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a. REPORT b. ABSTRACT c. THIS PAGE SAR 7
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Prescribed by ANSI Std Z39-18
188 J.L.Mellquist-Riemenschneideretal./VirusResearch92(2003)187(cid:1)/193
60%. The ecology and epidemiology of EBOV are infection and EHF in humans. Previous studies on
poorly understood and despite attempts to identify the MARV demonstrated that baculovirus-expressed GP
host species that maintains the virus in nature, it offeredpartialprotectiveimmunitytoMARVchallenge
remainsunknown(LeGuenno,1997;Leirsetal.,1999). in guinea pigs (Hevey et al., 1997), and that complete
EBOV particles consist of helical nucleocapsids con- protection could be achieved if a priming dose of a
taining approximately 19 Kb of single-strand, negative- DNA vaccine expressing MARV GP was given prior to
sense RNA. The viral genome encodes seven structural boostswith the baculovirus expression products (Hevey
proteins, one of which, the glycoprotein GP, forms et al., 2001). In this study, we sought to determine if a
spikes on the surface of the virion. GP is a type 1 similarprime-boostvaccineapproachwouldbeeffective
membrane protein, and is highly glycosylated with N- for EBOV. To answer this question, we first prepared
linked and O-linked glycans accounting for approxi- recombinant baculoviruses expressing full-length ZE-
matelyhalfofitsapparentmolecularweight(Feldmann BOVGP oratruncatedversionofGPfromwhichthe
1,2
et al., 1994). Maturation of GP involves posttransla- carboxy-terminal anchor region of GP is deleted
2
tional cleavage of a precursor by a furin-like enzyme to (GPa-). The full-length GP (GP ) gene contains eight
1,2
yield two fragments, GP and GP , which are disulfide- adenosine residues at the editing site, to ensure produc-
1 2
bonded(GP )andexpressedonthesurfaceofinfected tion of full length GP (Vanderzanden et al., 1998). The
1,2
cells and virions (Volchkov et al., 1998a). GP is also truncated GP gene was constructed by introducing a
1
shed from the surface of infected cells in a soluble form stop codon at amino acid position 651 of GP. Both
and may contribute to the complex pathogenesis of this genes were inserted into the plasmid transfer vector
virus (Volchkov et al., 1998b). pAcUW51 (BD Biosciences), behind the polyhedrin
InadditiontomRNAforGP,transcriptionoftheGP promoter and recombinant baculoviruses were gener-
geneleadstomRNAencodingasecretednon-structural ated by homologous recombination with linear baculo-
glycoprotein, sGP (Volchkov et al., 1995). The sGP virus DNA as described earlier(Schmaljohn et al.,
mRNA is the exact copy of the viral genome, whereas 1983).The GPa- gene is 78 nucleotides shorter than full
the mRNA of the mature GP is generated by transcrip- length GP and encodes a protein that is 26 amino
1,2
tional editing, which results in the addition of a non- acidssmaller.Analysisoftheexpressionproductsofour
templatedadenosineresiduetoarunofsevenadenosine two recombinant baculoviruses by immune-precipita-
residues(Sanchezetal.,1996).AlthoughtheroleofsGP tion and SDS-PAGE revealed that under non-reducing
is not completely understood, a study using a ZEBOV conditions both the full-length GP and the truncated
1,2
infectious clone, showed that in the absence of sGP, GPa- appear as single bands ((cid:3)2ME, Fig. 1). Under
/
much of full length GP synthesis is arrested in the reducingconditions,GP andGP werereadilyobserved
1 2
endoplasmic reticulumorGolgi(Volchkovetal.,2001). in lysates (not shown) or supernatants of cells infected
These data suggest that high-level expression of GP with the GP recombinant ((cid:2)2ME, Fig. 1A).
1,2 /
overwhelms the transport and glycosylation machinery Although GP was also apparent in samples from cells
1
of the host cell, resulting in increased cytopathology. infected with the GPa- recombinant, little of the
ThepresenceofsGPmaytempertheGPexpressionand truncated GP expression product was detected in the
2
cytopathology,allowingthevirustoreplicateforlonger infectedcells(notshown)orthesupernatants(Fig.1A).
periodsoftime(Volchkovetal.,2001).Itwasspeculated AnearlierstudyusingavacciniaT7polymerasesystem,
that sGP might also play a role in pathogenesis by revealed that a similarly truncated ZEBOV GP gene
1,2
inactivating neutrophils and/or acting as a decoy for produced secreted, soluble, disulfide-linked GP ,
1,2
anti-EBOVantibodies(Itoetal.,2001;Maruyamaetal., whereas full-length GP shed predominantly soluble
1,2
1999a; Yang et al., 1998). GP into infected cell supernatants (Volchkov et al.,
1
Studies to define important immune responses for 1998b). In that study, the truncated GP was readily
2
protection from EBOV have indicated that both hu- detectedinthemammaliancellsupernatantsbyimmune
moral and cell-mediated immunity play critical roles in precipitation. We suspect that our differing results with
disease prevention. GP is a target for neutralizing regardtosecreted,truncatedGP relatetodifferencesin
1,2 2
antibodies, which have been shown to be an important the ability of the antibodies to recognize the truncated
element of immunity to EBOV (Jahrling et al., 1996; GP proteins, and/or to glycosylation or other proces-
2
Mikhailov et al., 1994). Moreover, vaccination with sing differences inherent to insect cell propagation that
GP offers protection from lethal EBOV challenge in would result in inefficient complexing of our GP and
1,2 1
rodentmodels(Gilliganetal.,1997;Pushkoetal.,1997, GP . Poor complexing would influence the amount of
2
2000; Sullivan et al., 2000; Vanderzanden et al., 1998; GP that we could detect if our polyclonal antibody
2
Xu et al., 1998). does not specifically recognize GP , but rather predo-
2
Although a variety of vaccine approaches elicit minantly recognizes epitopes on GP . This supposition
1
protective immune responses in rodents, there are is supported by Western blot results in which we were
currently no effective vaccines or therapies for EBOV unable to detect GP in supernatants of insect cells
2
J.L.Mellquist-Riemenschneideretal./VirusResearch92(2003)187(cid:1)/193 189
Fig.1. (A)SDSPAGEofGP andGPa-expressionproductsfromsupernatantsofrecombinantbaculovirus-infectedinsectcellsradiolabeledfrom
1,2
30 to 42 h after infectionwith [35S]cysteine and [35S]methionineand immune-precipitated with anti-EBOV guinea pig sera. The positions of the
disulfidebondedGP andtheindividualGP andGP proteinsareindicatedwitharrows.Aradiolabeledproteinmolecularweightmarker(M)was
1,2 1 2
includedonthegelandtheapparentmolecularweightsareindicatedinkDa.(B)ImmunoblotdetectionofGP secretedintothemediumofinsect
1
cellsatdays3,4,or5afterinfectionwiththerecombinantbaculovirues.Aliquotsofrecombinantbaculovirus-infectedinsectcellsupernatantswere
electrophoresedonSDS-PAGE,thenandwereelectrophoreticallytransferredtomembranesandprobedwithapolyclonalhyperimmuneguineapig
serum.(C)ImmunoblotcomparisonofEBOVvirionproteinstobaculovirus-derivedGP andGPa-products.Twentylitersuspensionculturesof
1,2
Sf9 cells were infected with either the GP or GPa- baculovirus recombinants and supernatants were collected 48 h later. Aliquots of the
1,2
supernatantswereconcentratedbyultrafiltrationandwerecomparedtosucrosegradient-purifiedEBOVbySDS-PAGEandWesternblotusinga
polyclonal anti-EBOV hyperimmune guinea pig serum (left panel) or anti-EBOV monospecific guinea pig sera to GP (right panel). Increasing
concentrationsofpurifiedEBOVwereusedin,lanes1(cid:1)/4(leftpanel).GP1andnucleocapsid(NP,invirionpreparations)areindicatedwitharrows.
(D)ImmuneprecipitationandSDS-PAGEanalysisofbaculovirus-derived,radiolabeledEBOVGP byusingfivedistinctmonoclonalantibodiesto
1,2
EBOVGP.
infectedwitheithertheGP orGPa-recombinantvirus 1A) may be due to differences in the amount of protein
1,2
(Fig.1B).ThetwobandsthatmigratebetweenGP and onthegel,asnomigrationdifferencesareseenwhenthe
1
GP (Fig. 1A) were non-specifically precipitated from proteins are assayed by Western blot (Fig. 1B and C).
2
cell supernatants when testing other baculovirus recom- Comparing the EBOV proteins present in cell super-
binants, including a construct expressing EBOV NP natantsbyWesternblot(Fig.1B)revealedthatapproxi-
using the same polyclonal antibody (data not shown). mately fourfold more GP was secreted from cells
1
Because the GP proteins of the two recombinants infected with the GPa- recombinant as compared those
1
should be processed identically, we expect that they infected with the GP recombinant as determined by
1,2
willhavethesameproperties,includingthesamesizeas densitometry. We theorized that the size differences of
determined by gel electrophoresis. The apparent slight the baculovirus insect cell culture-derived proteins and
migration difference of the GP1 proteins derived from authentic EBOV proteins observed by Western blot
the two recombinants and immune precipitated (Fig. couldbeduetoglycosylationdifferences.Insectcellsare
190 J.L.Mellquist-Riemenschneideretal./VirusResearch92(2003)187(cid:1)/193
known to have limited capability to process complex recognize linear epitopes on GP , but those in groups 4
1
carbohydrates (Jarvis and Finn, 1995) and earlier and5areconformationalepitopes(Wilsonetal.,2000).
studies with recombinant baculoviruses expressing These studies suggest that the antigenic structure of the
MARVGPshoweddifferencesinglycosylationbetween baculovirus-derived GPs remains intact.
authentic and baculovirus-derived GP asdetermined by To assess the immunogenicity of the baculovirus-
lectin binding assays (Hevey et al., 1997). Using the derived EBOV proteins, we first performed a pilot
same lectin binding methods, we compared the oligo- vaccine study in which small groups of guinea pigs
saccharide moieties on authentic gradient-purified were vaccinated with various preparations of baculo-
EBOV and the baculovirus expression products. Virion virus-derived EBOV GP and GPa- proteins. We found
GP was found to react most strongly with Datura that expression products from both the full-length and
stramonium agglutinin (DSA), indicating the presence truncated GP genes elicited antibodies to GP and
of complex or hybrid N-linked glycans. In addition, it conferred partial protection from viral challenge (data
reacted, albeit weaker, with Arachis hypogaea (peanut) notshown).Theseresultsweresimilartothoseobtained
agglutinin (PNA), which recognizes unsubstituted ga- with baculovirus-derived MARV proteins (Hevey et al.,
lactose b(1(cid:1)3)N-acetylgalactosamine cores in O-gly- 1997).Todetermineif,asobservedforMARV,aprime-
/
cans, and Maackia amurensis agglutinin (MAA) which boost combination of a DNA vaccine and the baculo-
reactsspecificallywitha(2(cid:1)3)-linkedsialicacid,demon- virus-derived protein could provide better protective
/
strating the presence of unsubstituted O-glycans and immunity than either vaccine alone in the guinea pig
oligosaccharidescontainingsialicacid,respectively.The model,wevaccinatedinbredguineapigsandchallenged
ZEBOV virion GP did not react with Galantus nivalis them with ZEBOV.
agglutinin (GNA), indicating the absence of high The DNA vaccine or control plasmid were precipi-
mannose N-linked glycans. tated onto gold beads and loaded into plastic gene gun
IncontrasttoZEBOVvirionGP,baculovirus-derived cartridges as previously described (Vanderzanden et al.,
GP, either cell-associated or secreted, reacted most 1998).Groupsofsixstrain13guineapigsreceivedDNA
strongly with GNA, indicating modification primarily only, baculovirus-derived ZEBOV GP or GPa- only,
1,2
by high-mannose oligosaccharides. Similar to our ob- or a combination of the two vaccines. For DNA
servation with ZEBOV virion GP, the baculovirus- vaccinations, guinea pigs received 2.5 mg of DNA at
derived GP reacted weakly with PNA, denoting the two inoculation sites delivered by the PowderJect-XR
presence of unsubstituted O-linked oligosaccharides. gene delivery device, as previously described (Vander-
Baculovirus-derived GPs did not react with DSA, zanden et al., 1998). Guinea pigs that received baculo-
MAA, or Sambucus nigra agglutinin (SNA) (which virus-derived antigens were vaccinated subcutaneously
reactsspecificallywitha(2(cid:1)6)-linkedsialicacid)indicat- at two dorsal sites with a total volume of (cid:1)0.5 ml
/ /
ing the absence of complex/hybrid N-linked glycans, or (approximately5mgZEBOVGPorGPa-peranimal).A
those containing sialic acid. Therefore, as demonstrated total of three vaccinations were administered at 4-week
for MARV, the baculovirus derived EBOV GPs have intervals. For the prime-boost vaccinations, groups of
both O-linked and N-linked carbohydrates, but these six guinea pigs received a priming dose of the DNA
carbohydrates differ from those found in ZEBOV, and vaccine followed by two booster doses of the baculo-
thedifferences couldbethereason fortheapparentsize virus products in adjuvant. A control group received a
discrepancies of the insect-cell derived and mammalian DNA vaccine consisting of an empty DNA plasmid.
cell-derived proteins. Blood samples were collected at the time of each
To determine if the glycosylation differences or other vaccination and 2 weeks after the final vaccination.
modificationsoftheGPproteinsexpressedininsectcell Serum antibody titers to purified ZEBOV were deter-
cultures, affected antigenic changes we analyzed the minedforeachguineapigbyELISA(Heveyetal.,1997;
recombinant proteins by immune-precipitation with a Vanderzanden et al., 1998) and geometric mean titers
panel of monoclonal antibodies that recognize five (GMT) for each vaccine group were calculated (Table
distinctepitopesonGP (Wilsonetal.,2000).Wefound 1).Afterthefirstvaccination,onlyguineapigsreceiving
1
that all five antibodies precipitated the baculovirus- the GP vaccine had detectable antibody responses to
1,2
derivedGP fromtheGP construct(Fig.1D)orfrom ZEBOV. After the second vaccination antibody re-
1 1,2
the GPa- construct (data not shown). As described sponses were detected in all groups except the negative
earlier for these monoclonal antibodies (Wilson et al., controlgroup,andafterthefinalvaccination,allgroups
2000), GP was also precipitated from the GP had GMT by ELISA ]log (data not shown).
2 1,2 / 10
construct, presumably because it remains disulfide Three weeks after the third vaccination, guinea pigs
bonded to GP . No GP was detected in cells infected were challenged with 1000 PFU of guinea pig-adapted
1 2
with the GPa- construct (not shown), consistent with ZEBOV and observed daily for 28 days for signs of
results presented in Fig. 1A. Epitopes bound by the illness and mortality. Seven days after challenge, blood
antibodies in competition groups 1, 2 and 3 (Fig. 1D) samples were collected from the guinea pigs and plaque
J.L.Mellquist-Riemenschneideretal./VirusResearch92(2003)187(cid:1)/193 191
Table1
Vaccinationandchallengeofguineapigs
Primevaccine Boostvaccine Log ELISAtitera nAb/totalb(PRNT ) Survivors/totalc Viremiad(viremic/total) Meandayofdeath(range)
10 80
GPDNA GPDNA 3.0 1/6(1:40) 1/6 3.7(5/6) 12(10(cid:1)/18)
ControlDNA ControlDNA NA 0/6(NA) 0/6 4.4(6/6) 9(8(cid:1)/10)
GPDNA BaculoGP1,2 3.7 1/6(1:40) 0/6 2.7(4/6) 12(10(cid:1)/16)
GPDNA BaculoGPa- 3.9 4/6(1:80) 2/6 3.1(3/6) 12(10(cid:1)/13)
BaculoGP1,2 BaculoGP1,2 3.6 4/6(1:40) 3/6 (0/6) 17(14(cid:1)/19)
BaculoGPa- BaculoGPa- 3.3 4/6(1:60) 1/6 2.8(3/4)e 8(6(cid:1)/12)
a Geometricmeantitersafterthreevaccinations.
b Numberofguineapigsthatdevelopedneutralizingantibodiesafterthreevaccinations/total(nAb/T)andthedilutionofserumthatresultedin
80%plaquereductioninaneutralizingantibodytest.Where ]/2animalswerepositiveanaveragewascalculated.
c Survivors/totalonDay28afters.c.challengewith1000PFUguineapigadaptedEBOV.
d Viremia(Log10PFU/ml)7daysafterchallenge.Where ]/2animalswereviremic,ageometricmeantiterwascalculated.
e Serumwascollectedfromfoursurvivingguineapigs,theothertwohadalreadysuccumbedtoinfection.
assays were performed to measure viremia. All of the DNAprimefollowedbytheGPa-boost,withfourofsix
control guinea pigs had high viremias and died between animals having PRNT ]1:40. Nevertheless, there
80% /
eight and 10 days after challenge (Table 1). Five of the was little, if any correlation with protection. One of
six DNA-vaccinated guinea pigs were viremic and died, the two survivors had a reasonably high PRNT titer
80%
although levels of viremia were slightly lower than (1:160), but the other survivor did not have detectable
observed in controls, and death was delayed in most neutralizing antibodies.
animals (Table 1). The guinea pigs that received the The type of immune response needed to protect from
baculovirus-derived GP did not have detectable filovirus challenge is not completely understood, how-
1,2
viremia at 7 days after challenge; however, all of these ever, it is likely that both strong cellular and humoral
guinea pigs became sick later in the study and three of immune responses are needed. Generally, gene gun
sixdiedwithadelayedmeantimetodeathascompared delivered DNA vaccines have been characterized as
tocontrols.Theguineapigsthatreceivedaprimingdose eliciting CD4(cid:2) Th2-type immune response in mice, as
/
of the DNA vaccine followed by two additional reflected by a predominant IgG1 antibody response. In
vaccinations with the baculovirus derived GP or contrast, intramuscular injection of a DNA vaccine
1,2
GPa- in adjuvant had reduced levels of viremia as elicits a mostly Th1-type response (Feltquate et al.,
compared to the controls; however, only two of the 1997;Pertmeretal.,1996).Thisgeneralizationhasledto
twelveguineapigs,bothintheGPa-group,survivedthe a feeling that gene gun delivery is better when the
challenge. The time to death was also delayed for most outcome desired is a strong antibody response than
of these guinea pigs (Table 1). All of the surviving whenastrongcell-mediatedimmuneresponseisneeded.
guinea pigs showed increases in antibody levels when However, we found that gene gun delivered EBOV
assayed by ELISA 44 days after challenge, indicating DNA elicited cytotoxic T lymphocyte responses in mice
thatallhadbeeninfected,andthatsterileimmunitywas (Vanderzanden et al., 1998). Measuring a cell-mediated
not achieved with any of the vaccines. immune response is more difficult in guinea pigs than
Neutralizing antibody responses to EBOV have been mice, and the significance of indirect measures, such
suggestedtoplayaroleinprotectiveimmunity(Jahrling antibodyisotypes,whichcorrelatewithTh1-likeorTh2-
et al., 1996; Maruyama et al., 1999b; Wilson et al., like responses in mice, are not defined for guinea pigs.
2000), although correlation of neutralizing antibody Consequently, as a measure of the ability of the
levels and protection has not been reported. In our baculovirus-derived proteins to elicit cell-mediated im-
study, only one of the guinea pigs in the groups that mune responses, we evaluated the T-cell stimulatory
received the DNA alone or the DNA followed by the capacity of the baculovirus-expression products in
GP ,boosthaddetectableneutralizingantibodies,and cultured human dendritic cells and monocytes as
1,2
neither survived the challenge. In the two groups that described previously (Saikh et al., 2001). Briefly, den-
receivedthebaculovirusproteinsinadjuvant,fourofsix dritic cells or monocytes were incubated with autolo-
guineapigsineachgrouphadPRNT titersof1:40or gousTcellsandsupernatantsfrombaculovirus-infected
80%
greater. However, there was no correlation with protec- cell culturescontaining EBOV GPa-. These T cellswere
tion. For example, the lone survivor in the GPa- group then used as effector cells in a cytotoxicity assay using
didnothavedetectableneutralizingantibodies,andonly HLA-matchedor-unmatchedBcelltargetcellsthathad
oneofthreesurvivorsintheGP ,grouphaddetectable beenpulsedwithsupernatantsfrombaculovirusinfected
1,2
neutralizing antibodies. The best neutralizing antibody cell cultures containing EBOV GPa-, an unrelated viral
responses were obtained in guinea pigs given the GP , antigen,ornoantigen.AlthoughweassayedbothGP
1,2 1,2
192 J.L.Mellquist-Riemenschneideretal./VirusResearch92(2003)187(cid:1)/193
Table2
T-cellstimulatorycapacityofGPa-inculturedhumandendriticcellsa
Percentcytotoxicity
DCactivatedCTL Monocytes
Effector(Tcell):target(BSMcell)b
1:1 10:1 1:1 10:1
Targets(Experiment1)
BSM,noantigenc 30 26 26 28
BSM(cid:2)/GPa- 29 53 16 38
BSM(cid:2)/controlsupernatant 12 2 0 2
K562d(cid:2)/GPa- 8 24 3 18
Targets(Experiment2)e
BSM,noantigen 15 20 N/Df N/D
BSM(cid:2)/Gpa- 30 64 N/D N/D
BSM(cid:2)/controlantigen 11 12 N/D N/D
a AdherentmonocytesfromaHLA-A2donorwereculturedwithIL-15for60htotransformthemintodendriticcells(DC).Cellswerewashed
extensivelytoremoveIL-15andnonadherentcells.MonocytesculturedwithoutIL-15remainasmonocytesthroughoutculture.AutologousTcells
andAntigens(10mg/ml)wereaddedtotheculture.After6days,Tcellswerecollectedandusedaseffectorcellsincytotoxicityassays.
b Effectorcell(Tcell)totargetcell(BSMcell)ratio.
c BSM (B cell, HLA-A2) were pulsed with Ebola Gpa- (10 mg/ml) culture supernatants or supernatants from cells infected with a wild-type
baculovirus(controlinExperiment1),orculturesupernatantfromabaculovirusexpressinganunrelatedprotein(Experiment2)for1hat378Cand
usedastargetcells.
d non-HLAmatchedcontroltargetcells(K562cells).
e Experiment1andExperiment2aresimilarassaysexceptthatDC-activatedeffectorCTLsforexperiment1weremadebyculturingautologousT
cellswithDCandEbolaantigenwherasinexperiment2autologousTcellswereculturedwithDCinfectedwithaVEErepliconexpressingEBOV
GP.
f Notdetermined(N/D).
and GPa- concentrated from insect cell culture super- T cell responses in human cells, and presumably also in
natants,thebackgroundreactivityoftheGP prepara- guinea pigs.
1,2
tions was toohigh toachieve valid results. Thisislikely
duetothelowerconcentrationoftheexpressionproduct
in the supernatant as compared to that in supernatants Acknowledgements
from cells infected with the GPa- baculovirus.
In two independent assays performed with the GPa- The views, opinions and/or findings contained in this
expression product, we observed zero to twofold in- report are those of the authors and should not be
creases in cytotoxicity in HLA-matched cells when we construed as an official Department of the Army
used an effector:target ratio of 1:1 (Table 2). When the position, policy, or decision unless so designated by
effector:target ratio was increased to 10:1, a twofold other documentation. The authors would like to thank
increaseincytotoxicitywasmeasuredinoneexperiment Dr Mary Kate Hart for providing the monoclonal
and a greater than threefold increase was seen in the antibodies to EBOV GP and the VEE replicon expres-
secondexperiment.Noincreaseincytotoxicitywasseen sing EBOV GP. We would like to thank the USAM-
in pulsed HLA-unmatched cells or with a control RIID Veterinary Medicine Division Small Animal
antigen at either effector:target ratio. Monocyte acti- Sectiontechniciansforassistanceinanimalexperiments.
This work was performed while Jenny Mellquist-Rie-
vatedCTLswerealsoevaluatedinoneoftheassaysand
menschneider held a National Research Council fellow-
revealedthatEBOVGPa-induceda1.4-foldincreasein
ship at USAMRIID.
cytotoxicity over background at an effector:target ratio
of10:1.Anenhancedcytotoxicityofmonocyteactivated
CTLswasnotseenatthe1:1effector:targetratio(Table
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