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Virus Research 128 (2007) 26–33

Immunological evaluation of Escherichia coli expressed E2 protein ofWestern equine encephalitis virus

Dipankar Das a, Les P. Nagata b, Mavanur R. Suresh a,b,∗a Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2N8

b Defence Research and Development Canada-Suffield, P.O. Box 4000, Station Main, Medicine Hat, Alta., Canada T1A 8K6

Received 4 January 2007; received in revised form 29 March 2007; accepted 30 March 2007Available online 11 May 2007

bstract

The Western equine encephalitis virus (WEEV) is a potential Biological Warfare (BW) agent. The WEEV is endemic in western Canada andas caused epidemics of “sleeping sickness” with a mortality rate of 7–9%. The E2 glycoprotein is a structural component of the WEEV and elicitsroduction of neutralizing antibodies against the virus following an infection event. The envelope glycoprotein E2 is considered as the major targetrotein for the development of vaccines because it includes epitopes that elicit neutralizing antibodies. This report describes the successful cloningf the E2 gene of WEEV and expression in Escherichia coli as inclusion bodies. The inclusion bodies were successfully solubilized, refolded andhe immunogenicity of this non-glycosylated protein was assessed in BALB/c mice. Recombinant E2 (rE2) protein was specifically and stronglyecognized by inactivated WEEV-immunized mice serum sample on ELISA, suggesting that E. coli derived rE2 protein retained at least some

unctional characteristics of its native conformation. The immunogenicity of the refolded rE2 protein was demonstrated by strong humoral andell mediated immune (CMI) responses in rE2-immunized BALB/c mice. The current study also demonstrated that rE2-immunized mice could beartially protected from lethal challenge of WEEV.

2007 Elsevier B.V. All rights reserved.

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eywords: WEEV; E2 glycoprotein; Subunit vaccine; Humoral response; Cell

. Introduction

The Western equine encephalitis virus (WEEV) is a poten-ial Biological Warfare (BW) agent, for which no prophylaxisr treatment currently exists for human application. In addition,apid and sensitive methods for clinical or field identificationf WEEV have not yet been developed. Alphaviruses, whichncludes the WEEV, are mosquito-borne enveloped RNA viruseshat cause an acute encephalomyelitis in humans and other mam-

als by infecting neurons in the brain and spinal cord (Griffinnd Mendoza, 1986). The alphaviruses include many human andeterinary pathogens such as eastern, western and Venezuelan

quine encephalitis viruses. The alphavirus genome is approxi-ately 11,700 nucleotides long and encodes four non-structural

roteins (nsP1, P2, P3 and P4), minor structural proteins (E3

∗ Corresponding author at: Faculty of Pharmacy and Pharmaceutical Sciences,niversity of Alberta, Edmonton, Alta., Canada T6G 2N8. Tel.: +1 780 4929233;

ax: +1 780 4921217.E-mail address: msuresh@pharmacy.ualberta.ca (M.R. Suresh).

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168-1702/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2007.03.030

ted immune response

nd 6K) and three major structural proteins (capsid, E1 and E2)Griffin et al., 1997). The E1 and the precursor of E2 (pE2) areransmembrane glycoproteins that heterodimerize in the endo-lasmic reticulum. This heterodimerized protein is processedhrough the Golgi apparatus where pE2 is cleaved to E2 and E3nd then transported to the plasma membrane. At the cell sur-ace, the cytoplasmic tail of E2 interacts with the nucleocapsido initiate budding and release of mature virions from the plasma

embrane of infected cells (Strauss and Strauss, 1994).Alphaviruses have a wide host range and replicate in a variety

f different species ranging from mammals to insects as well asany cell types. The attachment of a virion to a cell surface is

ne of the key steps that can define host range. It appears thathese viruses must use a variety of different molecules for attach-

ent or exploit ubiquitous cell surface molecules for infectionSchlesinger and Schlesinger, 1996). Alphaviruses can infect a

ariety of cell types and probably use more than one cell sur-ace receptor (Frolov et al., 1996). The viral glycoprotein E2 ishe most likely protein that interacts with these cellular receptor

olecules (Schoepp et al., 2002). Epitopes on the surface of

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virus that interact with neutralizing antibodies are importantn protection from viral disease. Many studies have shown thatntibodies directed against E2 glycoprotein are more often neu-ralizing than are antibodies reactive with E1 (Strauss et al.,991; Strauss and Strauss, 1994). Consequently, these proteinsave been the major focus of subunit vaccine research and devel-pment.

The objectives of this study were to clone the E2 gene ofEEV along with a C-terminal hexa-histidine fusion protein in

scherichia coli and use the recombinant E2 (rE2) protein as aotential subunit vaccine. In this study, we report the successfulloning of the E2 gene of WEEV, its expression in and purifi-ation from E. coli inclusion bodies and the evaluation of themmunogenicity of rE2 protein in BALB/c mice.

. Materials and methods

.1. Bacterial strain, vector and chemicals

The vector pET22b+, E. coli BL21 (DE3) strain andnti-His6 monoclonal antibody (MAb) were purchased fromovagen Inc. (Madison, USA). The WEEV strain 71V-1658as kindly provided by Dr. Nick Karabatos, Centers for Dis-

ase Control, Fort Collins, CO; WEEV Fleming strain wasurchased from American Type Culture Collection (ATCC,annanas, VA, USA). PCR primers were synthesized by theepartment of Biochemistry, University of Alberta, Edmon-

on, Alta., Canada. The T4 DNA ligase and restriction enzymesere purchased from New England Biolabs (Mississauga,anada). Forty percent acrylamide: bisacrylamide, prestained

ow range protein molecular weight markers and Biorad pro-ein assay reagent were purchased from Bio-Rad Laboratoriestd. (Mississauga, Canada). The Taq DNA polymerase, IPTG

Isopropyl �-d-1-thiogalactopyranoside), Dulbecco’s Modifiedagle’s Medium (DMEM) and fetal bovine serum (FBS)ere purchased from Gibco BRL (Burlington, Canada). Plas-id DNA isolation and gel extraction kits were obtained

rom Qiagen Inc. (Mississauga, Canada). Glutathione (GSHnd GSSG) was purchased from Roche Diagnostics (Laval,anada). The polymyxin B resins, TiterMax Gold adjuvant,TT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

romide)], mitomycin C, l-arginine and Goat anti-mouseorse radish peroxidase (GAM-HRPO) were purchased fromigma (Oakville, Canada). dNTPs, hybond ECL nitrocellu-

ose membrane, X-ray film and the enhanced chemiluminescentECL) Western blotting kit were purchased from Amer-ham BioSciences (BaiedUrfe, Canada). ABTS [Diammonium,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate)] and TMB3,3′,5,5′-Tetramethylbenzidine) peroxidase substrate were pur-hased from Kirkegaard & Perry Laboratory Inc. (Gaithersburg,SA).

.2. Plasmid construction

The standard cloning methods were performed as describedSambrook et al., 1989). The E2 gene was PCR amplified fromhe original 26S clone in pcDWXH-7 vector (Netolitzky et al.,

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ch 128 (2007) 26–33 27

000) using gene specific primers (5′ Primer: 5′-CCT CGAGC CCA GCC GGC CAT GAG CTC CAG CAT TAC CGAGA CTT CAC A-3′ and 3′ Primer: 5′-GGA ATT CAT GGCTC GGG GGC CAA GCT TAG CGT TGG TTG GCC GAAGC A-3′) designed from the published sequence (Hahn et al.,988). The PCR amplified product was electrophoresed through1% agarose/TAE gel and purified using Qiagen gel extrac-

ion kit. The purified PCR product was digested with the SacInd HindIII and ligated to the pET22b+ in the presence of T4NA ligase at 14 ◦C overnight. The ligation mixture was used to

ransform the E. coli Top 10 and the transformants were spreadnto LB agar plates (1% Tryptone, 0.5% Yeast Extract, 0.5%aCl, 1.5% agar, pH 7.5) containing 100 �g/ml carbenicillin.he plasmid DNA was digested with the SacI and HindIII andnalyzed in 1% agarose gel electrophoresis for properly sizedlones.

.3. Analysis of recombinant functional clones of E2

The recombinant plasmid containing the correctly oriented2 gene was used to transform E. coli BL21 (DE3) for recom-inant protein expression. E. coli BL21 (DE3) transformantsere cultured in 10 ml LB medium containing 100 �g/ml of

arbenicillin and incubated at 37 ◦C with shaking at 250 rpmntil an OD600 of ∼0.4–0.5 was reached. The bacterial cul-ure was induced with 1 mM IPTG and allowed to grow fornother 3 h at 30 ◦C. The culture was then harvested by cen-rifugation at 4000 × g for 10 min at 4 ◦C. The total cell lysateas prepared by addition of Laemmli sample buffer to theellet and heating at 95 ◦C for 5 min. Total cell protein ofnduced and uninduced culture was analyzed by sodium dode-yl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)sing 10% polyacrylamide gel performed according to theublished method (Laemmli, 1970) with a Biorad Mini Pro-ean II apparatus. The protein gel was stained with 0.25%w/v) Coomassie Brilliant Blue R-250 in 10% acetic acid and5% methanol and destained with 10% acetic acid and 30%ethanol.

.4. Immunoblotting

Total cell protein of induced and uninduced culture was elec-rophoresed on SDS-PAGE and electroblotted onto the hybondCL nitrocellulose membrane based on a method (Towbin etl., 1979) using Trans blot apparatus (BioRad) following theanufacturer’s instructions. The ECL membrane was blockedith 5% skim milk in PBST [0.1% Tween 20 in PBS (phos-hate buffered saline), pH 7.3] for 1 h. The membrane wasashed with PBST and incubated for 1 h with the anti-His6Ab that specifically binds to the fusion protein. After wash-

ng four times with PBST, the membrane was incubated with

AM–HRPO (1:10,000 dilution) for 1 h. Finally, the mem-rane was washed four times with PBST and the ECL basedetection was performed according to manufacturer’s instruc-ions.

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.5. Expression of E2 protein and purification

The bacterial cultures (4 L) were propagated and inducedith IPTG as previously described. The periplasmic solublerotein was isolated by resuspending cells in 5% of originalulture volume in periplasmic extraction buffer (50 mM Tris,0% (w/v) sucrose, 1 mM EDTA pH 8.0) followed by incuba-ion on ice for 45–60 min with occasional shaking. The bacterialysate was centrifuged at 12,000 × g for 20 min at 4 ◦C and theupernatant containing periplasmic soluble proteins was col-ected. The remaining insoluble fraction was used to isolatenclusion bodies as described below. The periplasmic solu-le and insoluble proteins were analyzed by Western blot toetermine the localization of expressed protein as describedarlier.

The insoluble fraction was resuspended in PBS (10 ml/g ofell pellet), mixed by vortexing and then passed through a Frenchress. The extract was clarified by centrifugation at 15,000 × gor 30 min at 4 ◦C. The pellet was resuspended in PBS with 2%odium deoxycholate and incubated at RT for 30 min and cen-rifuged at 12,000 × g for 15 min at 4 ◦C. The pellet was furtherashed thrice with PBS. Finally, inclusion bodies were solubi-

ized in denaturing buffer (50 mM Tris pH 8.0, 200 mM NaCl,mM EDTA, 6 M urea) and incubated in ice for 1 h. The insol-ble material and cell debris were separated from the solubleenatured protein by centrifugation at 12,000 × g for 30 min at◦C and the supernatant was collected. Protein was quantifiedy Bradford’s method (Bradford, 1976). The solubilized dena-ured protein was adjusted to 50–100 �g/ml with the TA buffer50 mM Tris pH 8.0, 0.4 M l-arginine) and refolding was donen the presence of 1.0 mM GSH, 0.1 mM GSSG for 3 days at◦C. Finally, dialysis was performed in PBS at 4 ◦C. Refoldedroteins were separated by SDS-PAGE and analyzed by Westernlot as described earlier.

.6. Enzyme linked immunosorbant assay (ELISA)

The ELISA was performed by coating of the rE2 antigen inunc Maxisorb 96-well ELISA microplates. Wells were coatedith rE2 antigen (1 �g/well in 100 �l volume) in PBS or 1%ialyzed BSA in PBS (control) overnight at 4 ◦C. The platesere washed three times with PBST and blocked with 1%ialyzed BSA for 2 h at RT. After incubation, the plates wereashed with PBST. The serially diluted serum samples in PBS

rom individual immunized as well as naı̈ve mice were addednd the plates were incubated for 2 h at RT. The plates wereashed and incubated with the GAM-HRPO (1:10,000 dilution

n 1% dialyzed BSA) for 1 h. After a final wash with PBST,MB peroxidase or ABTS substrate was added and OD650 nm orD405 nm was measured after 20–30 min using an ELISA Vmaxinetic microplate reader (Molecular Devices Corp., California,SA).

.7. Immunization of mice

BALB/c female mice (6–8 weeks) were obtained from theealth Sciences Laboratory Animal Services (HSLAS) of the

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ch 128 (2007) 26–33

niversity of Alberta, Edmonton, Canada. The animal treat-ent and care were carried out according to the Canadianouncil on Animal Care (CCAC) and University of Albertauidelines. A polymyxin B immobilized resin was used toemove endotoxin from E. coli expressed and purified rE2ntigen as described previously (Das et al., 2004). Five miceere immunized intraperitoneally with 50 �g of endotoxin-free. coli-expressed rE2 antigen emulsified with equal volumef TiterMax Gold adjuvant. Two weeks following primarymmunization, mice were immunized with the same amountf antigen emulsified with TiterMax Gold adjuvants. After therd and 4th week, mice were further immunized with the samemount of antigen diluted in PBS. Control mice were immu-ized with only TiterMax Gold adjuvants. Serum sample wasollected from each mouse after the 5th week by tail veinleeding.

.8. Antibody and cell mediated immune (CMI) response

Blood samples were collected from each mouse and serumas separated from cells by centrifugation. Antibody titeras measured by direct ELISA as described earlier (Das et

l., 2004).The CMI responses were measured by T-cell proliferation

ethods described previously (Das et al., 2004). Briefly, spleensere removed and a single cell suspension was prepared byacerating the spleens in DMEM medium supplemented withenicillin, Streptomycin and Glutamine (PSG). Cell suspensionsere pelleted by centrifugation at 400 × g for 7 min and cellellet was gently resuspended in 5 ml ACK lysis buffer (0.15 MaCl, 10 mM KHCO3, 0.1 mM EDTA pH 7.3). Cell suspen-

ions were incubated at RT for 5 min, washed three times anduspended in DMEM medium containing 10% FBS, PSG and-mercaptoethanol. Stimulator cells (antigen presenting cells)rom naı̈ve mice were incubated with or without 10 �g/ml endo-oxin free rE2 antigens for 1 h at 37 ◦C in a CO2 incubator.ollowing incubation, 50 �g/ml mitomycin C was added and

he cells were incubated for 20 min at 37 ◦C in a CO2 atmo-phere. Subsequently, cells were washed with DMEM mediumhree times, suspended in DMEM medium and diluted to 106

ells/ml. Responder cells from immunized mice were aliquotedo 96-well tissue cultures and plated in triplicate at 106 cells/well.ne hundred microliters of stimulator cells with or without anti-en were added to each respective well and incubated for 3–4ays at 37 ◦C in a CO2 incubator. Cell survival and cell pro-iferation assays were done by employing a method developedy Mosmann (1983). Sterile, 5 �g/ml MTT solution was added10 �l) to each well on day 4 of culture and the plates werencubated for 4 h at 37 ◦C in a CO2 incubator. Formazan crys-als were formed and solubilized with HCl-isopropanol solutionnd the plates were read at OD570 nm with an OD650 nm as refer-nce. The T-cell proliferation was determined by measuring theD (OD570 nm–OD650 nm) of the formazan generated by growing

ells. The stimulation index (SI) values are also provided for pro-iferation observed in response to rE2 antigen. SI is equal to theD (OD570 nm–OD650 nm) values of T-cells incubated with anti-en presenting cells (APC) and rE2 antigen divided by the OD

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OD570 nm–OD650 nm) values of T-cells with APC but withoutE2 antigen (control).

.9. Virus culture and purification

Minimal essential medium containing 5% fetal calf serum5% MEM) was used to grow Vero (CCL-81) cells (ATCC).eed stocks of WEEV strains were made by inoculation of Veroells with virus suspensions at a multiplicity of infection (MOI)f less than 0.1. The supernatants were clarified by centrifuga-ion, aliquotted and stored at −70 ◦C for further use. All liveirus experiments were carried out in the Defence Researchnd Development Canada (DRDC)-Suffield Biological Level-

containment facilities following recommended guidelinesrom Health Canada and the Canadian Food Inspectiongency.

.10. Immunization of mice with subunit vaccine and wholenactivated WEEV

Female BALB/c mice (8 mice in each group), 17–25 g, werebtained from the pathogen-free mouse breeding colony atRDC Suffield, with the original breeding pairs purchased fromharles River Canada (St. Constant, Que., Canada). The use of

hese animals was reviewed and approved by Animal Care Com-ittee at DRDC Suffield and the care and handling of the mice

ollowed the guidelines set out by the CCAC. A dose of 30 �gE2 protein in a volume of 50 �l or 50 �l of SALK WEE inacti-ated vaccine was diluted to a final volume of 100 �l/mouse. Forhe initial dose, the inoculums were diluted 1:1 in TiterMax Goldnd subsequent immunizations were diluted in Hanks balancedaline solution (HBSS). A 27 g needle was used to deliver two0 �l doses of diluted inoculum intramuscularly to each hindeg of a mouse. The purified rE2 protein or SALK WEE inac-ivated vaccine in PBS, was administered to 17–25 g BALB/cemale mice. The control mice were injected with saline only.he immunization schedule was 0, 14 and 28 days, followed byirus challenge at day 49.

.11. Protection studies

Live virus working stocks were prepared by diluting.5 × 103 Plaque Forming Units (PFUs) of WEEV in 50 �lBSS and was administered to the mice intranasally. Sodiumentobarbital (50 mg/kg body weight) was used intraperi-oneally to anaesthetize the mice. When the animals werenconscious, they were carefully supported by hand with theirose up and the virus suspension in HBSS gently applied withmicropipette intranasally. The applied volume was then nat-

rally inhaled into the lungs. Infected animals were observedaily, for up to 14 days post-infection. The survival rates ofhe treatment and non-treatment control groups were compared

sing a two-tailed t-test and one-way analysis of variance, usingraph-Pad Prism ver. 2.0 (GraphPad Software, San Diego,A). Differences were considered statistically significant at< 0.05.

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ch 128 (2007) 26–33 29

. Results

.1. Plasmid construction and expression of recombinant2 (rE2) protein

The E2 glycoprotein gene of WEEV was successfully PCRmplified and cloned into the pET22b+ vector, resulting in aexa-histidine fusion construct. The rE2 clones were confirmedy restriction enzyme digestion fragment mapping. The plas-id pET22b+ containing the E2 gene was transformed into the. coli expression host BL21 (DE3) and stable transformantsere screened for the expression of rE2 protein. SDS-PAGE

howed that although the different clones were expressing dif-erent levels of rE2 protein, all had MW bands at the desired55 kDa (data not shown). The E2/11 clone was expressing rE2

rotein at the highest level and was chosen for further analysisFig. 1A). Expression of the rE2 protein was confirmed by West-rn blot analysis using the anti-His6 MAb (Fig. 1B). This cloneas selected for medium scale production and purification of

E2 protein from a bulk 4 L culture.

.2. Purification of inclusion bodies and refolding

The expression of the rE2 protein was found almost exclu-ively in inclusion bodies. The periplasmic extract of the inducedulture and the total protein of the uninduced culture did notave the ∼55 kDa band in Western blot (data not shown), whichndicated that the majority of the rE2 protein was contained innclusion bodies. Similar data were obtained earlier with WEEV1 protein (Das et al., 2004). Following extraction, solubiliza-

ion and refolding of the insoluble protein, the refolded rE2rotein was analyzed by SDS-PAGE (Fig. 1C) and the anti-His6Ab showed strong binding to the rE2 protein in Western blot

Fig. 1D). The results showed that the rE2 purified protein is sub-tantially pure with a single band in the SDS-PAGE and Westernlot analysis. The purified inclusion bodies and refolded rE2 pro-ein were analyzed together on the Western blot using anti-His6

Ab and there was no change in size or mobility of the refoldedE2 protein (Fig. 1E).

.3. Antibody response against rE2 antigen using serumrom mice immunized with inactivated WEE vaccines

Mice immunized with inactivated WEEV (Long et al., 2000)ere completely protected against intranasal challenge with

ither WEEV 71V-1658 or WEEV Fleming strains. Serumamples were taken from individual mice immunized with inac-ivated WEEV vaccine 2 days before viral challenge and wereitrated using ELISA against fixed concentrations of rE2 antigen.he samples exhibited high anti-rE2 titers (Fig. 2) compared to

he whole inactivated WEEV as an antigen (data not shown). TheE2 protein was specifically recognized by serum from mousemmunized with inactivated WEEV by ELISA. The magnitude

f the anti-rE2 serum titers suggests that the E. coli derived agly-osyl rE2 protein retained significant characteristics of its nativeonformation. The ELISA result also demonstrates that the rE2ntigen could be useful as a diagnostic reagent for monitoring

30 D. Das et al. / Virus Research 128 (2007) 26–33

Fig. 1. Analysis of E2 gene expression in E. coli. Total cell protein (10 �g) analysis by SDS-PAGE and Western blot. The gel was stained with Coomassie BrilliantBlue (A). Proteins transferred to nitrocellulose membrane were subsequently probed with anti-His6 MAb followed by Goat anti-mouse-HRPO (B). Lane M: standardp d cultb ed rEW 1: inc

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restained molecular weight markers, Lane 1: induced culture, Lane 2: uninducelot (D). Lane M: standard prestained molecular weight markers, Lane 1: refoldestern blot (E). Lane M: standard prestained molecular weight markers, Lane

ntibody titers in serum of infected individuals. The cross reac-ivity of rE2 protein with immune sera from other alphavirusesas not investigated.

.4. Immunogenicity analysis of rE2 protein in naı̈ve mice

The endotoxin-free rE2 antigen when injected into micelicited both antibody and CMI responses. Serially diluted serumamples from each mouse were titrated for anti-E2 antibodies

sit

ure. Analysis of the refolded rE2 protein (1 �g) by SDS-PAGE (C) and Western2 protein. Analysis of the purified inclusion bodies and refolded rE2 protein bylusion bodies, Lane 2: refolded rE2 protein.

sing an ELISA with the purified rE2 protein adsorbed to theolid phase. All mice immunized with the rE2 protein developedtrong polyclonal antibody responses that showed strong reac-ivity against the rE2 antigen in comparison with control micenjected with only adjuvant (Fig. 3).

The CMI response was demonstrated by in vitro, antigen-pecific T-cell proliferation assays. The splenocytes of five micen the group were specifically stimulated by the in vitro presen-ation of rE2 antigen, as indicated by specific T-cell proliferation

D. Das et al. / Virus Research 128 (2007) 26–33 31

Fig. 2. The mouse antibody response employing whole inactivated WEEV vac-cine detected by rE2 protein. The ELISA plates were coated with 10 �g/mlof rE2 antigen and incubated with different dilutions of mouse serum samplesimmunized with whole inactivated virus. Control serum samples were takenfrom individual mice 2 days before live virus challenge at a single time point.The data points are expressed as mean values from a group of seven mice. Bind-ia

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Fig. 4. The cell mediated immune responses to WEEV rE2 protein. The spleno-cytes were isolated from immunized and naı̈ve mice. Responder spleen cellsfrom immunized mice were aliquoted to 96-well tissue cultures plate in trip-licate at 106 cells/well. Stimulator cells (100 �l, 106 cells/ml) ± antigen wereadded to each well and incubated for 3–4 days at 37 ◦C in a CO2 incubator.Sterile, 5 �g/ml solution of MTT was added (10 �l/well) to each well at day 4of culture and plates were incubated for 4 h at 37 ◦C in a CO2 incubator. For-mazan crystals were solubilized by the addition of acid–alcohol solution andvigorous mixing. Optical density was taken at OD570 with an OD650 reference.Error bar indicate the standard deviation (n = 3). Asterisks indicate *P < 0.05 and*

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ng was detected with Goat anti-mouse-HRPO and followed by ABTS solutionnd OD was measured at 405 nm.

Fig. 4). CMI responses were significantly higher than the con-rol sample as analyzed by the Student’s t-test (P < 0.05). Inuture studies additional controls should be examined to com-are inactivated WEEV along with free rE2 protein, polymyxin-treated rE2 protein, and uninfected Vero cell lysate. It was

emonstrated that both the antibody and the CMI responses weretrongly elicited against purified rE2 antigen in BALB/c mice.

ig. 3. The individual mouse antibody response to WEEV rE2 protein. TheLISA plates were coated with 100 �l of 10 �g/ml of rE2 antigen, blockednd incubated with different dilutions of mouse (n = 5) serum samples immu-ized with rE2 antigen. The serum samples were collected after the 5th weeky tail vein bleeding. The data points are expressed as the mean of tripli-ate values. Binding was detected with Goat anti-mouse-HRPO followed byMB-peroxidase substrate and OD was measured at 650 nm.

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.5. Protection studies

Groups of eight female BALB/c mice were immunized intra-uscularly with the rE2 protein or inactivated WEEV. Two

ooster injections were administered on day 14 and 28. At day9, vaccinated mice were challenged by the intranasal route withethal doses (1.5 × 103 PFUs) of two different WEEV strains.ontrol animals were injected with only saline. None of the con-

rol mice challenged with WEEV survived at the end of the day1. Although the rE2 vaccinated mice resulted in only three sur-ivals out of eight when challenged with the WEE 71V-1658Table 1), only one mouse survived when challenged with the

EE Fleming strain. Since the E2 gene was cloned from thetructural gene 26S of the WEE 71V-1658, the result suggestshat some of the epitopes of the E2 which elicited protectivemmunity were likely virus strain specific. Although the puri-ed rE2 protein did not elicit complete protective immunity asffectively as the inactivated WEEV (Figs. 5 and 6), the data indi-

ate that the purified aglycosyl rE2 protein could elicit protectivemmunity in some mice.

able 1hallenge of BALB/c mice immunized with rE2 protein or parental inactivatedEE virus

accine Dose Challenge virus Survivors/total

E2 antigen 30 (�g/ml) WEEV 71V-1658 3/8WEEV Fleming 1/8

EEV vaccine 1:10 of SALK WEEV 71V-1658 8/8Vaccine WEEV Fleming 5/6

aline control None WEEV 71V-1658 0/8WEEV Fleming 0/7

32 D. Das et al. / Virus Resear

Fig. 5. Evaluation of rE2 vaccine in mice challenged with the WEE 71V-1658virus strain. The mice were vaccinated with 30 �g of the rE2 protein, inactivatedWEEV or PBS three times. The mice were then challenged with 1.5 × 103 PFUsof WEEV 3 weeks after the last vaccination. Results were plotted as percentsurvival for each vaccination group (n = 8 per group).

Fig. 6. Evaluation of the rE2 subunit vaccine against the WEE Fleming viruschallenge. The mice were vaccinated with 30 �g of rE2 protein, inactivatedWPp

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EEV or PBS three times. The mice were later challenged with 1.5 × 103

FUs of WEEV 3 weeks after the last vaccination. Results were plotted as theercent survival for each vaccination group (n = 8 per group).

. Discussion

The currently available and widely used investigational vac-ine for the WEEV is a formalin-inactivated whole virus vaccineTurell et al., 2003). Inactivation of the virus using formalin is

common procedure, but can result in the loss or modifica-ion of the epitopes. The live-attenuated virus vaccine offers

any advantages over the use of inactivated immunogens; how-ver a major concern with its use is the potential for spread ofither the vaccine or a pathogenic revertant in susceptible hostsPedersen et al., 1972). Subsequently, it is very important to con-ider the risk of introducing a virulent pathogen when using aive-attenuated virus as vaccine. Recombinant subunit vaccinesre desirable due to their safety compared with whole attenuatedr inactivated virus. The rapid development of biotechnology,mmunology and protein biochemistry have allowed differenttrategies for the construction of subunit vaccines based on viralr bacterial recombinant constructs and peptides or plasmid vec-ors (Ertl and Xiang, 1996). Recombinant chimeric vaccinesave also been described to protect against a lethal virus chal-

enge in mice (Schoepp et al., 2002). Two monoclonal antibodiesirected against the E2 glycoprotein of Semliki Forest Virusould protect mice from a lethal viral challenge (Boere et al.,983), and antibodies against E2 glycoprotein helped recovery

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ch 128 (2007) 26–33

rom alphavirus encephalitis in a mouse model (Griffin et al.,997). Furthermore, DNA vaccination against WEEV protected0–100% of the mice from a lethal challenge of three differentEEV strains and elicited CMI responses to the rE2 protein as

hown in a T-cell proliferation assay (Nagata et al., 2005).In this report, we evaluated the expression of the E2 enve-

ope antigen of WEEV in E. coli for immunological studies.part from high levels of expression, the production of recom-inant proteins as bacterial inclusion bodies has several otherdvantages. The protein is present in a nearly pure concen-rated form, it is resistant to bacterial protease and its insolubilityllows for easy purification provided a successful refolding strat-gy can be developed. One disadvantage is that viral proteinsuite often form aggregates (Yurkova et al., 2004) or partic-lates (Valenzuela et al., 1982) during their expression in theecombinant system, yet in some cases these forms retain theunctionality of the parent protein.

Most recombinant proteins are expressed in E. coli as inclu-ion bodies and different strategies have been reported toxtract and renature proteins from inclusion bodies (Das et al.,004; Yurkova et al., 2004). The non-glycosylated E2 proteinxpressed and purified in E. coli as inclusion bodies were suc-essfully solubilized in urea and renaturated in TA buffer inresence of GSH/GSSG. The final yield of refolded rE2 pro-ein was 5–6 mg/l of initial bacterial culture. The serum samplesf the mice immunized against whole inactivated WEEV weretrongly reactive against the rE2 protein by ELISA. AlthoughE2 protein was derived as a folded non-glycosylated protein,he results demonstrate that rE2 protein still exhibits signif-cant reactivity to antibodies from mice immunized with thenactivated whole virus vaccine.

In the present study, vaccination with the rE2 protein alone inALB/c mice generated strong antibody and CMI responses byntigen-specific T-cell proliferation assay in mice. This suggestshat the rE2 antigen has both the B-cell and the T-cell epitopes;owever the relative magnitude of the CMI responses were notompared with the cytokine levels when inactivated WEEV wassed as the antigen. The prophylactic ability of the rE2 proteinor mouse protection against a lethal challenge was also inves-igated. The rE2 protein was used to vaccinate the mice and thenimals were subsequently challenged with the two strains ofhe WEEV. Only 37.5% mice survived the challenge with the

EEV 71V-1658 strain; however, 12.5% survival was observedmong the rE2-vaccinated group challenged with the WEEVleming strain. The group of mice vaccinated with inactivatedhole virus as a positive control, exhibited complete protec-

ion with WEEV 71V-1658 and 83% protection with the WEEVleming strain. The low stimulation of the immune responseslicited by the rE2 could be due to the lack of glycosylation ofhe rE2 antigen when expressed in E. coli. Adoptive transfer ofhe antibodies from the rE2 and inactivated virus was not per-ormed to assess whether protection of naı̈ve mice challengedith the WEEV would occur; however, plans are underway. The

fficacy of the rE2 protein as a subunit vaccine with regardo cross protection with other alphaviruses was also not eval-ated. A combined immunization strategy using both the rE1nd rE2 protein could be an alternative approach to augment

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he partial response against WEEV infection. This study how-ver demonstrated that the non-glycosylated rE2 protein coulde successfully employed as a potential serological diagnosticeagent. Our DNA vaccine against WEEV 71V-1658 elicited aood CMI response, but no detectable antibody response to rE1r rE2 protein, yet it completely protected mice against WEEV1V-1658 (Nagata et al., 2005).

In summary our results demonstrated that the non-lycosylated rE2 retains significant antigenic functionality afterefolding. The results suggest that rE2 protein can be useful asn antigen for screening serum levels for specific antibodies inmmunized or infected animals as a simple diagnostic procedure.owever, cross reactivity of rE2 protein has not been evaluatedith immune sera to other alphaviruses.

cknowledgements

This work was supported by the DND-NSERC grant. MRShanks the CIHR-Industry Chair for salary support. We wouldike to thank George Rayner and Christine Chan for their excel-ent technical assistance and Jany Zhanz for reading of the

anuscript. Thanks are due to Dr. Sandra Marcus for help withhe French Press facility, Department of Chemistry, Universityf Alberta, Edmonton, Alta., Canada.

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