evaluation of immune responses elicited in mice against a recombinant malaria vaccine based on...

Vaccine 22 (2004) 3727–3737

Evaluation of immune responses elicited in mice against a recombinantmalaria vaccine based onPlasmodium vivaxDuffy binding protein

Syed Shams Yazdani, Ahmad Rushdi Shakri, Paushali Mukherjee,Sanjeev Kumar Baniwal1, Chetan E. Chitnis∗

Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB),P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India

Received 15 July 2003; accepted 5 March 2004

Available online 10 April 2004

Abstract

Plasmodium vivaxDuffy binding protein (PvDBP) binds the Duffy blood group antigen as the obligate receptor for erythrocyte invasion.We have tested in mice the immunogenicity of recombinantP. vivaxregion II (PvRII), the receptor-binding domain of PvDBP, formulatedwith five adjuvants, namely, Montanide ISA720, AS02A, alum, QS21 and MF59. All the formulations elicited high titer antibodies, withMontanide ISA720 and AS02A yielding the highest titers followed by MF59, QS21 and alum. Sera raised against PvRII formulated withAS02A and Montanide ISA720 followed by alum were most effective at blocking PvRII binding to erythrocytes in a functional assay.Analysis of cellular immune responses indicated that all adjuvant groups induced significant interferon-�, with alum being the highestinterferon-� inducer. These results suggest that recombinant PvRII formulated with human compatible adjuvants is immunogenic in smallanimal models and that Montanide ISA720, AS02A and alum perform better than MF59 and QS21 in terms of their ability to elicit hightiter binding inhibitory antibodies.© 2004 Elsevier Ltd. All rights reserved.

Keywords:Malaria vaccines; Blood-stage malaria vaccines; Erythrocyte invasion; Erythrocyte binding proteins; Adjuvant studies

1. Introduction

Malaria continues to be a major public health problemin many parts of the tropical world. Human malaria iscaused by four Plasmodium species, namely,P. falciparum,P. vivax, P. ovaleandP. malariae. Although P. falciparumis primarily responsible for the mortality associated withmalaria,P. vivax is the most widespread of the four Plas-modium species and is responsible for significant morbidityin South and South-east Asia, Papua New Guinea, LatinAmerica and parts of Africa[1]. The global burden ofmalaria due toP. vivax is ∼70–80 million cases annually[1]. While P. vivaxrepresents around 10% of malaria casesin eastern and sub-Saharan Africa, it accounts for morethan 50% of all malaria cases outside Africa[1]. Vaccinesthat provide protection againstP. falciparumand P. vivax

∗ Corresponding author. Tel.:+91-11-2618-7695;fax: +91-11-2616-2316.

E-mail address:[email protected] (C.E. Chitnis).1 Present address: Biocentre, Goethe University, D-60439 Frankfurt am

Main, Germany.

malaria are urgently needed for worldwide efforts to controlmalaria.

The clinical symptoms of malaria are attributed to theblood-stage of the parasite life cycle resulting from repeatedrounds of erythrocyte invasion, parasite multiplication andlysis of host erythrocytes. The invasion of erythrocytes byPlasmodium sp. merozoites requires specific receptor-ligandinteractions.P. vivaxand the related simian malaria parasite,P. knowlesi, are completely dependent on interaction with theDuffy blood group antigen for invasion of human erythro-cytes[2]. Erythrocytes from Duffy negative individuals arerefractory to invasion byP. vivaxandP. knowlesi. P. knowlesiinvades rhesus erythrocytes by multiple pathways using therhesus Duffy antigen as well as alternative receptors[3]. P.falciparumstrains commonly use sialic acid residues on gly-cophorin A as invasion receptors, although, likeP. knowlesi,P. falciparumcan also use alternative receptors to invadeerythrocytes by multiple pathways[4–6]. Parasite proteinsthat bind erythrocyte receptors to mediate invasion belongto a family of erythrocyte binding proteins (EBP family)[7]. The EBP family includesP. vivaxandP. knowlesiDuffybinding proteins (PvDBP and PkDBP),P. knowlesi� and

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2004.03.030

3728 S.S. Yazdani et al. / Vaccine 22 (2004) 3727–3737

� proteins that bind Duffy-independent receptors on rhesuserythrocytes, andP. falciparumEBA-175 that binds sialicacid residues on glycophorin A[8–10]. The extracellular do-mains of these EBPs contain two conserved cysteine-rich re-gions. The functional receptor-binding domain of each EBPmaps to the N-terminal conserved cysteine-rich region, re-ferred to as region II[11,12]. P. vivax region II (PvRII),the binding domain of PvDBP, is composed of around 350amino acids including 12 conserved cysteines. We proposeto develop a recombinant vaccine forP. vivaxmalaria basedon PvRII. Naturally acquired antibodies against PvRII havebeen shown to block erythrocyte binding by PvDBP[13].Moreover, antibodies raised against the homologous bind-ing domain of PkDBP have been shown to block invasion ofhuman erythrocytes byP. knowlesiin vitro [14], providingsupport for the development of a vaccine based on PvRII,the binding domain of PvDBP.

Here, we have tested the immunogenicity of recombi-nant PvRII. Recombinant PvRII was expressed inE. coli,purified from inclusion bodies under denaturing conditions,refolded by rapid dilution and purified to homogeneityby ion-exchange and gel filtration chromatography. Bio-chemical and biophysical characterization confirmed thatrecombinant PvRII was pure, homogenous and functionallyactive in that it bound erythrocytes with specificity. In aneffort to identify an acceptable adjuvant formulation for usein human clinical trials, we tested the immunogenicity ofrecombinant PvRII formulated in five adjuvants that havebeen previously used in human clinical trials, namely, Mon-tanide ISA720, AS02A, MF59, QS21 and alum. Althoughall the formulations tested yield high titer antibodies di-rected against PvRII in BALB/c mice, formulations withMontanide ISA720 and AS02A yield antibodies with mosteffective binding inhibitory activity followed by alum.These studies indicate that recombinant PvRII formulatedin human compatible adjuvants is immunogenic and pro-vide support for the development of a recombinant vaccinebased on PvRII. Recombinant PvRII formulated with ad-juvants Montanide ISA720, AS02A and alum should beconsidered for further development.

2. Materials and methods

2.1. Expression of recombinant P. vivax DBP region II(PvRII)

E. coli BL21(DE3) cells (Novagen) were transformedwith plasmid pET28a+ PvRII, which contains DNA en-coding PvRII fused to a 6-His tag at the carboxyl terminuscloned downstream of the T7 promoter in the expres-sion plasmid pET28a+ (Novagen) as previously described[15]. E. coli BL21(DE3) contains the gene for T7 RNApolymerase under the control of the IPTG induciblelacpromoter. Two ml of an overnight culture ofE. coliBL21(DE3) pET28a+ PvRII grown in Luria broth (LB)

containing kanamycin at 30 mg/ml (Km30) was used to in-oculate 200 ml of semi-defined medium (Na2HPO4, 6.0 g/L;KH2PO4, 3 g/L; NaCl, 0.5 g/L; NH4Cl, 0.2 g/L; yeast ex-tract, 1.0 g/L; glucose, 1.0 g/L; 1.0 mM MgSO4; 0.1 mMCaCl2; kanamycin, 50 mg/L, pH 7.2) and grown to logphase. The log phase culture (200 ml) was used to inoculatea 20 L fermentor (Applikon AG, Netherlands) containing10 L of semi-defined medium at 37◦C. Dissolved oxygen ofthe culture was maintained at 40% by controlling the agita-tion rate and air inlet flow rate. The pH was maintained at6.8 by addition of 2.0N NaOH. Cell density was monitoredby measuring the optical density of the culture at 600 nm(OD600). Expression of PvRII was induced with 1.0 mMIPTG when cell density of the culture reached an OD600of 5. E. coli cells were harvested by centrifugation 4 h af-ter induction. The cell pellet was stored at−80◦C untiluse.

2.2. Refolding and purification of recombinant PvRII

E. colicell pellets were resuspended in chilled lysis buffer(10 mM Tris pH 8.0, 10 mM EDTA, 100 mM NaCl, 5 mMbenzamidine hydrochloride, 10 mM dithiothreitol (DTT),100�g/ml lysozyme) and lysed by sonication. Inclusionbodies were collected from lysed cells by centrifugationat 12,000 g for 45 min at 4◦C, washed in the presence of2% Triton X-100 and 4.0 M urea, and solubilized in 8.0 Mguanidine hydrochloride (Gdn-HCl). Solubilized inclusionbodies were clarified by centrifugation at 12,000× g for45 min at 4◦C and used for purification of PvRII by metalaffinity chromatography using nickel-charged nitrilotri-acetic acid (Ni-NTA) matrix under denaturing conditions.Ni-NTA column loaded with solubilized inclusion bodieswas washed with 10 column volumes of 6.0 M Gdn-HCl pH6.3 and bound PvRII was eluted from the column with 6.0 MGdn-HCl pH 4.3. The elutes were separated by SDS-PAGEto check purity. Fractions containing recombinant PvRIIwere pooled and the concentration of PvRII was adjustedto ∼0.8 mg/ml. Purified PvRII was refolded by performinga 40-fold dilution in refolding buffer (50 mM Tris–HCl pH7.2, 1.0 mM EDTA, 1.0 M urea, 0.5 M arginine, 2.0 mMreduced glutathione and 0.2 mM oxidized glutathione) toachieve a final PvRII concentration of 20�g/ml. The re-folding solution was flushed with N2 and incubated at 10◦Cfor 36 h without stirring. The refolding solution was thendialyzed for 48 h against 50 mM PO4

3− pH 6.5 and 1 Murea to remove arginine. The dialysed sample was loadedon a SP-Sepharose FF column (Amersham Biosciences).Following extensive washing with 50 mM NaCl, refoldedPvRII was eluted with a linear gradient of NaCl from50 mM NaCl to 1.0 M NaCl. Fractions containing PvRIIwere pooled and further purified by gel filtration chro-matography using a preparatory grade Superdex 75 column(Amersham Biosciences). The protein concentration wasmeasured spectrophotometrically, adjusted to 0.5 mg/ml andstored at−80◦C.

S.S. Yazdani et al. / Vaccine 22 (2004) 3727–3737 3729

2.3. Reverse phase chromatography with recombinantPvRII

Recombinant PvRII was tested for homogeneity byreverse phase high pressure liquid chromatography(RP-HPLC) using a C-8 column (Supelco). The gradientused for elution was developed using buffer A (0.05%triflouroacetic acid in water) and Buffer B (0.05% tri-flouroacetic acid in acetonitrile). The column was initiallyequilibrated in 90% Buffer A, 10% Buffer B and reached10% Buffer A, 90% Buffer B in 40 min.

2.4. Erythrocyte binding assay with recombinant PvRII

The functional activity of PvRII was tested using anerythrocyte-binding assay as described earlier[15]. Briefly,refolded PvRII was incubated with Duffy positive humanerythrocytes and chymotrypsin-treated human erythrocytesto allow binding. Erythrocytes with bound protein wereseparated by centrifugation and the bound protein waseluted with 300 mM NaCl, separated by SDS-PAGE, anddetected by Western blotting using mice polyclonal seraraised against PvRII.

2.5. Testing for endotoxin levels

Endotoxin levels were measured with Limulus amebocytelysate (LAL) kit using gel clot assay as recommended bythe manufacturer (Salesworth).

2.6. Formulation of PvRII with adjuvants

PvRII was formulated with the following adjuvants, Mon-tanide ISA720, AS02A, alum, QS21 and MF59 according tomanufacturer’s instructions. Briefly, formulations with dif-ferent adjuvants were prepared as follows. In case of Mon-tanide ISA720, 2.3 parts Montanide ISA720 (Seppic) weremixed with one part of antigen (v/v). For AS02A, 1.3 partsof AS02A (GlaxoSmithKline, Belgium) were mixed withone part of antigen (v/v). In cases of both QS21 (Antigenics)and MF59 (Chiron), one part of each adjuvant was addedto one part of antigen (v/v). The contents in each case weremixed by vortexing for 2–3 min. For formulation with alumhydroxide, the pH of alum (18 mg/ml) was first adjusted to7.0 with NaOH and then mixed with PvRII in the ratio of onepart adjuvant:one part antigen (v/v). The formulation wasincubated for 4 h at 4◦C on a gently rocking nutator, cen-trifuged at 3000 rpm for 10 min. The pellet was resuspendedin 0.9% NaCl. The supernatant was collected and tested forthe presence of any un-adsorbed protein. Greater than 90%of PvRII was adsorbed by the alum. Final concentration ofPvRII in each of the formulations was 100�g/ml.

2.7. Immunization of BALB/c mice

Immunogenicity of PvRII formulated with MontanideISA720, AS02A, alum, QS21 and MF59 was tested in

BALB/c mice. Groups of five BALB/c mice were immu-nized intramuscularly with 25�g of PvRII formulated withthe different adjuvants described above. Control groupsof three mice were administered with adjuvant and buffer(50 mM Tris, pH 7.2; 150 mM NaCl) alone. The total vol-ume used per mouse at each immunization was 250�l(125�l administered in each leg). Priming was followed bytwo booster immunizations on days 21 and 42. Sera werecollected on days-1, 14, 35, 56 and 70. All sera were storedat −80◦C until used.

2.8. Enzyme-linked immunosorbent assay (ELISA) andimmunofluorescence assay (IFA)

Sera collected at each time point from individual mice(test and control groups) were tested for recognition ofPvRII by ELISA. Flat-bottom microtitre ELISA plates(MaxiSorp, Nunc) were coated with PvRII (0.2�g/well) at4◦C overnight. The plates were washed three times withphosphate buffered saline (PBS), 0.05% Tween 20 in PBS(PBST) and blocked with 200�l of 1% skimmed milk pow-der in PBS for 2 h at 37◦C. Test sera were diluted 1:40,000in 0.25% skimmed milk powder in PBST and were incu-bated in duplicate wells (100�l/well) overnight at 4◦C. Serafrom control groups were diluted 1:200. The wells werewashed three times with PBST. Anti-mouse immunoglobu-lin (IgG) goat antibodies conjugated to horseradish peroxi-dase (HRP) (Sigma) were diluted 1:10,000 added to wellsand incubated for 1.5 h at 37◦C. ELISA was developed atroom temperature with 100�l/well of o-phenylenediaminedihydrochloride (1.0 mg/ml, Sigma) as chromogen and hy-drogen peroxide as the substrate. The reaction was stoppedwith 2.0N sulfuric acid. The absorbance was measured at490 nm (A490) using an ELISA plate reader (Molecular De-vices). MeanA490 values and standard errors of the meanfor each group were calculated. Sera from individual micein each group were pooled for determination of ELISA endpoint titers. Dilutions of pooled sera at whichA490 is 1.5times the background (A490 using sera from day-1, blankwells, or sera from adjuvant controls) were considered tobe the ELISA end point titers.

The indirect immunofluorescence assay was performedwith acetone-fixed slides made withP. vivaxinfected humanblood (kindly provided by Dr. Myriam Arevalo, Universidaddel Valle, Cali, Colombia and Dr. T. Adak, Malaria ResearchCentre, Delhi). Test sera (day 70 mice bleed) from AS02Aand Montanide ISA720 groups were diluted with PBS con-taining 1% BSA and tested in immunofluorescence assaysat dilutions of 1:10, 1:50, 1:100, 1:250, 1:500 and 1:1000.Pre-immune sera diluted at 1:10 and 1:50 were used as con-trols. Twenty microliters of each dilution from test and con-trol sera was incubated with slides ofP. vivaxinfected bloodfor 1 h at RT in a humidified chamber. Slides were washedthree times with PBS and incubated for 1 h at RT with 20�lof anti-mouse FITC conjugated goat antibodies (Sigma) di-luted 1:100 in PBS containing 1% BSA. Slides were washed


four times with PBS, sealed with Antifade Fluoroguard (Bio-Rad) and observed using an fluorescence microscope.

2.9. ELISA assay to determine isotypes of PvRII specificIgG

Microtiter plates (Maxisorp, Nunc) were coated with100�l of PvRII (10�g/ml) in bicarbonate coating bufferovernight at 4◦C. Plates were washed with PBST andblocked with 1% milk in PBS (200�l/well) for 2.0 hrsat 37◦C. After blocking, the plates were washed and serawere added at different dilutions from 1:300 to 1:1,000,000in 0.25% milk in PBST. Following overnight incubation at4◦C, the plates were washed and incubated for 1.0 h at 37◦Cwith polyclonal goat anti-mouse IgG isotype specific anti-bodies diluted 1:2000. After washing, plates were incubatedwith anti-goat HRP-conjugated antibodies diluted 1:10,000.The ELISA was developed with 100�l of o-phenyldiamineand hydrogen peroxide.A490 was measured on a microplatereader.

2.10. Inhibition of binding of erythrocytes to PvRIIexpressing COS-7 cells with sera directed againstrecombinant PvRII

2.10.1. Culture and transfection of COS-7 cellsCOS-7 cells (African green monkey kidney cell line)

were cultured in 35mm diameter 6-well plates to a con-fluency of ∼60% in complete Dulbecco’s Modified EagleMedium (DMEM + 10% fetal calf serum (FCS)) at 37◦C,5% CO2. The plasmid construct pHVDR22 was used toexpress PvRII on surface of COS-7 cells[11]. The plasmidpHVDR22 contains DNA encoding PvRII fused to the signalsequence ofHerpes simplexvirus glycoprotein D (HSV gD)at the amino-terminus, and the trans-membrane region andcytoplasmic domain of HSV gD at the carboxyl-terminusin a mammalian expression vector[11,16]. COS-7 cellswere transfected with plasmid pHVDR22 using Lipofec-tamine (Invitrogen) as per instructions of the manufacturer.Transfection efficiency was determined by an immunoflu-orescence assay using a mouse monoclonal antibody, DL6,specific to HSV gD sequences in the fusion protein (pro-vided by R. Eisenberg and G. Cohen, University of Penn-sylvania, USA), as described earlier[11].

2.10.2. Inhibition of erythrocyte binding to PvRII withantisera raised against PvRII

Sera raised against recombinant PvRII in BALB/c miceusing five adjuvants described above were tested for inhibi-tion of red cell binding to COS-7 cells expressing PvRII ontheir surface. Pooled sera from the third bleed (day 56 bleed)from each group were diluted 1:200, 1:600, 1:1800, 1:5400,1:16,200 and 1:48,600 with DMEM. Diluted sera (1 ml perwell) were incubated with transfected COS-7 cells for 1.0 hat 37◦C. Sera raised against adjuvants alone were used at

1:200 dilution as control. Duffy positive erythrocytes (10�lpacked volume per well) washed with incomplete RPMIwere added to the COS-7 cells and incubated for 1.5 h at37◦C. COS-7 cells were then washed 2–3 times with incom-plete DMEM and rosettes containing eight or more bounderythrocytes were scored in 60 fields using an inverted mi-croscope at a magnification of 40. The percent inhibition (ascompared to control sera) was plotted as a function of seradilution and a linear best-fit curve of the data was used todetermine 50% inhibition titers.

2.11. T cell proliferation

Spleens were collected aseptically from immunizedmice on day 70. The splenocytes (3× 105 cells/well) werestimulated with either PvRII or ConA (0.01–10�g/ml) in200�l of RPMI 1640 (Invitrogen) supplemented with 10%FCS, 0.5 mM 2-mercaptoethanol, 100 U of penicillin perml, 50�g of streptomycin per ml in 96 well flat bottomplates (Nunc). Stimulated cultures were incubated for 96 hat 37◦C in 5% CO2 and pulsed with 0.5�Ci per well of[3H]-thymidine (NEN Life Science) for 16 h to measureproliferative cellular immune responses. Plates were har-vested onto glass fiber filters for scintillation counting (Betaplate).

2.12. Cytokine analysis

Cytokine profiles of mice immunized with recombinantPvRII formulated with different adjuvants were determinedby measuring levels of interferon-� (IFN-�), IL-10, IL-4and IL-2 in supernatants of splenocytes stimulated with re-combinant PvRII in vitro by ELISA. Culture supernatantsof splenocytes stimulated with different concentrations ofPvRII were collected at 24 or 48 h (for IL-2 and IL-4), and60 h (for IL-10 and IFN-�) and frozen at−80◦C until usedfor analysis. Frozen supernatants were thawed and cytokinelevels were determined using commercial kits for quantita-tive detection of IL-4, IL-10 and IFN-� (Duo Set kits fromR&D Systems) and IL-2 (BD OptEIATM ELISA kits fromBD Pharmingen). Concentrations of cytokines in the sam-ples were calculated using standard curves generated withknown concentrations of recombinant cytokines. Results areexpressed in picograms per milliliter (pg/ml). Cytokine lev-els below 25 pg/ml were considered negative.

3. Results

3.1. Expression, refolding and purification of recombinantPvRII

Batch fermentation ofE. coliBL21(DE3) pET28a+ PvRIIat 10 L scale using conditions described inSection 2yielded∼250 g of wet biomass. Purification of recombinant PvRII


from solubilized inclusion bodies under denaturing condi-tions by metal affinity chromatography yielded∼3.0 mg ofPvRII per gm of wet biomass. Following purification underdenaturing conditions by metal affinity chromatography, re-combinant PvRII was refolded by rapid dilution in presenceof oxidized and reduced glutathione to allow disulfide bondformation and co-solvents arginine and urea as describedearlier [15]. Following removal of arginine by dialysis, re-folded PvRII was purified by ion-exchange chromatographyusing Sepharose SP and gel filtration chromatography usingSuperdex 75. Final yield of refolded and purified PvRII was∼8.0 mg per liter ofE. coli culture.

3.2. Characterization of recombinant PvRII for purity,homogeneity, correct folding and endotoxin content

Refolded and purified PvRII migrates on a reducing SDS-PAGE gel with the expected mobility of∼39 kDa (Fig. 1A).Densitometry scanning of silver-stained SDS-PAGE gels

Fig. 1. Characterization of recombinant, refolded PvRII. (A) Silver stained SDS-PAGE gel of refolded and purified PvRII. Different quantities (0.25, 0.5,0.75 and 1.0�g) of refolded and purified PvRII were separated by SDS-PAGE under non-reducing conditions and detected by silver-staining. Molecularweight markers in kDa are shown. (B) Mobility of refolded and purified PvRII by SDS-PAGE before and after reduction. Refolded PvRII has slowermobility by SDS-PAGE after reduction with dithiothreitol (+DTT), indicating the presence of disulfide linkages. Molecular weight markers in kDa areshown. (C) RP-HPLC profile of refolded and purified PvRII. Refolded and purified PvRII was analyzed by reverse phase chromatography on a C8column. The gradient used for elution was developed using Buffer A (0.05% trifluoroacetic acid in water) and Buffer B (0.05% trifluoroacetic acid in 90%acetonitrile, 10% water). The column was initially equilibrated with 90% Buffer A and 10% Buffer B and reached a composition of 10% Buffer A and90% Buffer B in 40 min. Refolded PvRII elutes as a single, symmetric peak, indicating that it contains a single, homogeneous population of conformers.

indicates the existence of single band corresponding to thesize of recombinant PvRII. The difference in mobility ofPvRII on SDS-PAGE gels before and after reduction withDTT indicates presence of disulfide bonds in refolded PvRII(Fig. 1B). Reverse phase chromatography can be used toseparate different conformers of the same protein based ondifferences in surface hydrophobicity. Recombinant PvRIIelutes in a single symmetric peak upon separation by reversephase chromatography on a C-8 column indicating pres-ence of a homogenous population of conformers (Fig. 1C).The purity of recombinant PvRII is estimated to be ap-proximately 98% by reverse phase chromatography. Uponseparation by analytical gel filtration chromatography usingSuperdex 75, purified PvRII migrates as a single peak withthe expected retention time corresponding to a protein of∼39 kDa indicating that it is monomeric (data not shown).Refolded PvRII binds Duffy positive human erythrocytesbut not chymotrypsin-treated human erythrocytes that havelost the Duffy antigen indicating that it is functional and


Fig. 2. Erythrocyte binding assay with refolded and purified PvRII.Refolded PvRII was incubated with Duffy-positive (Fy(a+b+)) andDuffy-negative (Fy(a−b−)) human erythrocytes to allow binding. Ery-throcytes and bound protein was collected by centrifugation. Bound PvRIIwas eluted with 300 mM NaCl, separated by SDS-PAGE, and detected byWestern blotting using mouse antiserum raised against refolded and puri-fied PvRII. Refolded PvRII specifically binds Duffy-positive (Fy(a+b+))but not Duffy-negative (Fy(a−b−)) human erythrocytes.

correctly folded (Fig. 2). The endotoxin content of purifiedPvRII was less than 25 EU per 25�g of PvRII, the doseused per immunization, as determined using standard LALassay.

3.3. Humoral response in BALB/c mice immunized withrecombinant PvRII formulated in five different adjuvants

Refolded and characterized PvRII was formulated withfive different adjuvants, Montanide ISA720, AS02A, alum,QS21 and MF59, as described inSection 2and used to im-munize BALB/c mice (25�g PvRII/dose) as per the sched-ule shown inFig. 3A. Each adjuvant test group comprisedof five mice and the control group, which received adjuvantalone, comprised of three mice. Sera were collected priorto start of immunization and 2 weeks after each immuniza-tion from all mice. A final bleed was collected 4 weeksafter second boost. Sera collected at different time pointsfrom each mouse were diluted 1:40,000-fold and tested forrecognition of PvRII by ELISA. Pre-immunization sera andadjuvant control sera tested at 1:2500-fold dilution yieldedA490 values that were comparable to background and wereused as controls. A significant increase inA490 is seen withsera from all adjuvant test groups after first boost (data notshown). However, no further increase is observed after sec-ond boost. TheA490 values with sera from mice immunizedwith Montanide ISA720 and AS02A are clearly higher thanother test adjuvants. SinceA490 values determined by ELISAfor individual mice from the same test groups showed min-imal variation and small standard errors of mean (data notshown), the sera from mice belonging to the same groupwere pooled for determination of end-point titers and otherassays described below.

The end-point titers for reactivity with PvRII were deter-mined by ELISA using pooled sera for each adjuvant group(Fig. 3B). There is a significant increase in end-point titersof all adjuvant groups after the first boost but no further in-crease in end-point titers is observed upon second boost inany of the adjuvant test groups. End point titer of the AS02Agroup is highest 2 weeks after both first and second boosts(days 35 and 56, respectively). However, the end-point titerof the AS02A group drops by day 70, 4 weeks after secondboost, so that the Montanide ISA720 group has the highestend-point titer on day 70. Formulations of PvRII with Mon-tanide ISA720 and AS02A were more immunogenic andyielded ELISA titers that were higher than formulations ofPvRII with alum, QS21 and MF59.

Sera collected from different adjuvant groups on day70 were analyzed for presence of different IgG subclasses(Fig. 3C). Montanide ISA720 and AS02A groups hadsignificantly higher total IgG on day 70. The MontanideISA720 group had significant levels of IgG1 followed byIgG3 whereas AS02A had high levels of IgG2b. The alumand MF59 groups had higher levels of IgG1 compared toother subtypes.

Sera from the Montanide ISA720 and AS02A groups,which yielded highest ELISA titers were tested for recogni-tion of the native Duffy binding protein by IFA using slidesmade withP. vivax-infected blood. Both, Montanide ISA720and AS02A reacted withP. vivax schizonts in a typicalpunctate pattern at sera dilutions of 1:50 (data not shown).Both groups had immunofluorescence end-point titers of1:250.

3.4. Binding inhibitory activity of sera from miceimmunized with recombinant PvRII formulated withdifferent adjuvants

Sera raised in BALB/c mice against recombinant PvRIIformulated in five different adjuvants were tested for theirability to block erythrocyte binding byP. vivaxDuffy bind-ing protein. The binding domain, PvRII, ofP. vivaxDuffybinding protein was expressed on the surface of COS-7cells and tested for binding to Duffy positive human ery-throcytes in presence of different dilutions of sera raisedagainst recombinant PvRII formulated in different adju-vants. The number of COS-7 cells with rosettes of bounderythrocytes was scored and the percent inhibition wasdetermined using binding in presence of sera from miceimmunized with adjuvant alone as control (Fig. 4). Serafrom the Montanide ISA720 and AS02A groups inhibitedbinding more efficiently than the other three adjuvants. The50% inhibition titers for Montanide ISA720 and AS02Awere highest at∼1:16,000. Addition of recombinant PvRII(50�g/ml) completely reversed the binding inhibitory ac-tivity of sera from the Montanide ISA720 and AS02Agroups tested at dilution of 1:4000 indicating that the in-hibition of binding was specific. Moreover, preimmunesera of Montanide ISA720 and AS02A groups had no


Fig. 3. Immunization schedule and analysis of antibody responses by ELISA. PvRII was formulated in five adjuvants, namely, Montanide ISA720,AS02A, alum, QS21 and MF59. A dose of 25�g was administered per BALB/c mouse. (A) Immunization schedule. Mice were primed on day 0 andreceived boosts on days 21 and 42. The mice were bled on days 1, 14, 35, 56 and 70. (B) End point ELISA titers of mice sera. Sera from five micecollected at days 14, 35, 56 and 90 in each group were pooled and tested for recognition of recombinant PvRII at various dilutions by ELISA. End pointtiters were considered as the dilution of sera that yields an absorbance value that is 1.5 times the background absorbance observed with pre-immune seraat 1:200 dilution. (C) Profile of IgG subclass in sera from mice immunized with PvRII formulated with different adjuvants. Mice were immunized withrecombinant PvRII formulated with Montanide ISA720, AS02A, alum, QS21 and MF59. Sera collected after 4 weeks of second boost were analyzed forpresence of PvRII-specific IgG1, IgG2a, IgG2b, and IgG3 subclass antibodies by ELISA.

significant binding inhibitory activity when tested at dilu-tion of 1:1000. The 50% inhibition titers for alum, QS21and MF59 were∼1:8000,∼1:6000 and∼1:4000, respec-tively. These data suggest that refolded PvRII formulatedin AS02A and Montanide ISA720 is highly immuno-genic and can elicit high titer antibodies that will inhibitbinding of P. vivax Duffy binding protein to erythrocytesefficiently.

3.5. Cytokine profile of proliferating splenocytesstimulated with recombinant PvRII

Splenocytes from mice immunized with PvRII formu-lated with Montanide ISA720, AS02A and alum showedmaximum proliferative responses (data not shown). QS21also resulted in significant proliferation but only at higherantigen concentrations. MF59 yielded poorest proliferation


Fig. 4. Inhibition of erythrocyte binding to PvRII on COS-7 cells by mice sera raised against recombinant PvRII formulated with different adjuvants.Transfected COS-7 expressing PvRII on the surface were tested for binding to human erythrocytes in presence of different dilutions of sera raised againstPvRII formulated with five adjuvants. Number of erythrocyte rosettes bound to COS-7 cells was scored in 60 fields at 40× magnification. Relativebinding compared to binding in presence of sera raised against adjuvant alone (1:200 dilution) is shown. (A) Montanide ISA720, (B) AS02A, (C) alum,(D) QS21 and (E) MF59.

response. Splenocytes from mice immunized with adjuvantalone did not show any proliferation when stimulated withPvRII demonstrating specificity of the proliferation ob-served. Splenocytes from all the groups responded to ConA(data not shown).

Cytokine profiles of mice immunized with recombinantPvRII formulated with different adjuvants were determinedby measuring levels of IFN-�, IL-10, IL-4 and IL-2 in super-natants of splenocytes stimulated with recombinant PvRIIin vitro (Fig. 5). Splenocytes from mice immunized withPvRII formulated with alum yielded highest IFN-� levels.Splenocytes from the Montanide ISA720 and AS02A groups

produced detectable levels of IFN-� only at the highest anti-gen concentrations used for stimulation. Splenocytes fromthe Montanide ISA720 and AS02A groups produced signifi-cant levels of IL-10 and IL-4. Splenocytes from all adjuvantgroups produced IL-2 upon stimulation with PvRII.

4. Discussion

P. vivax is completely dependent on interaction of theP.vivax Duffy binding protein with the Duffy antigen for in-vasion of human erythrocytes[17]. The functional binding


Fig. 5. Cytokine profiles of mice immunized with PvRII formulated with various adjuvants. Cytokines produced by splenocytes collected from miceimmunized with PvRII formulated with various adjuvants following restimulation with PvRII were measured. Concentrations of cytokines detected insplenocyte supernatants upon stimulation with different amounts PvRII are shown. Graphs show concentrations of (A) IFN-�, (B) IL-10, (C) IL-4 and(D) IL-2 in picograms per ml (pg/ml).

domain ofP. vivaxDuffy binding protein lies in a conserved,N-terminal, cysteine-rich region,P. vivaxregion II (PvRII)[11,18]. We propose to develop a recombinant vaccine forP. vivaxmalaria based on PvRII. Immunization with recom-binant PvRII may elicit antibodies that block the interac-tion of P. vivaxDuffy binding protein with erythrocytes andthereby block invasion. Here, we have produced recombi-nant PvRII in its correctly folded, functional conformationand studied the immunogenicity of PvRII formulated in fivedifferent adjuvants in BALB/c mice.

Recombinant PvRII was expressed inE. coli, pu-rified from inclusion bodies under denaturing condi-tions by metal affinity chromatography, refolded bymethod of rapid dilution and purified to homogene-ity by ion-exchange chromatography and gel filtration

chromatography. Characterization of refolded PvRII demon-strated that it is pure, homogenous and correctly folded inits functional conformation. Analysis by reverse phase chro-matography suggests that refolded PvRII contains a singlepopulation of conformers. Recombinant PvRII is 98% purebased on reverse phase chromatography (Fig. 1C). Im-portantly, recombinant PvRII binds Duffy positive humanerythrocytes with specificity suggesting that it is correctlyfolded (Fig. 2). Testing for presence of endotoxins indicatedthat each immunization dose of purified PvRII (25�g)contains less than 25 EU.

In an effort to down select a formulation for further devel-opment, we have tested in mice the immunogenicity of PvRIIformulated with five different adjuvants, Montanide ISA720,AS02A, alum, QS21 and MF59, which are compatible for


use in humans. Sera from mice immunized with PvRII for-mulated with these adjuvants were tested for recognition ofPvRII by ELISA. Sera from mice immunized with PvRII for-mulated with Montanide ISA720 and AS02A had the highestELISA titers followed by sera from mice immunized withformulations made with QS21, MF59 and alum (Fig. 3B).Significant boosting of antibody titers was observed with alladjuvants after first boost however the second boost did notlead to further increase in antibody titers (Fig. 3B). A longertime interval between the first and second boosts may berequired to get further boosting of antibody titers. A mixedpopulation of IgG isotypes was detected in sera from all theadjuvant groups. The IgG1 subtype was most prevalent incase of Montanide ISA720, alum and MF59 whereas IgG2bwas most prevalent in case of AS02A (Fig. 3C). No sin-gle isotype was more prevalent than others in case of PvRIIformulated with QS21. From the cytokine profiles it ap-pears that AS02A and Montanide ISA720 elicit a balancedTh1/Th2 response by inducing IFN-�, IL-2, IL-10 and IL-4while alum, QS21 and MF59 mainly induce IFN-� (Fig. 5).

P. vivaxcannot be cultured in vitro. It is therefore not pos-sible to test the sera raised against PvRII for inhibition oferythrocyte invasion byP. vivax. Sera raised against PvRIIwere tested for inhibition of binding ofP. vivaxDuffy bind-ing protein to erythrocytes. The binding domain ofP. vi-vax Duffy binding protein was expressed on the surface ofCOS-7 cells and tested for binding to erythrocytes in pres-ence of sera raised against recombinant PvRII. Sera raisedwith PvRII formulated with Montanide ISA720 and AS02Awere most effective at blocking binding of erythrocytes toPvRII-expressing COS-7 cells, with 50% inhibition at di-lutions of ∼1:16,000 (Fig. 4). This is consistent with theobservation that Montanide ISA720 and AS02A yield sig-nificantly higher ELISA titers compared to the other adju-vants tested. Sera raised with PvRII formulated with alumyielded lowest antibody titers. However, sera from the alumgroup had better binding inhibitory activity than the QS21and MF59 groups.

In summary, we have shown that recombinant PvRII for-mulated with human compatible adjuvants is highly im-munogenic in mice. In addition, we have demonstrated thatrecombinant PvRII formulated with adjuvants MontanideISA720, AS02A and alum can elicit high titer antibodies inmice that can efficiently block the binding ofP. vivaxDuffybinding protein to erythrocytes. These antibodies should beable to block erythrocyte invasion byP. vivax. Based on thedata presented here we recommend that formulations of re-combinant PvRII with Montanide ISA720, AS02A and alumbe considered for further development.

Acknowledgements

We thank Drs. Roselyn Eisenberg and Gary Cohen, Uni-versity of Pennsylvania, Philadelphia, USA, for providingplasmid pRE4 and monoclonal antibody DL6, Joe Cohen,

GlaxoSmithKline Biologicals, Rixensart, Belgium, for pro-viding AS02A, Charlotte Kensil, Antigenics Inc., Boston,USA for providing QS21, Rino Rappouli, Chiron SPA,Siena, Italy for providing MF59, and Ghislaine Ionkoff,Seppic, Paris, France for providing Montanide ISA720. Wealso thank Joe Cohen, Charlotte Kensil and Filip Dubovskyfor comments on the manuscript. This work was supportedby grants from WHO-TDR Programme for Research andTraining in Tropical Diseases, Indo-US Vaccine Action Pro-gram and the Malaria Vaccine Initiative, PATH. CEC is anInternational Research Scholar of Howard Hughes MedicalInstitute, USA and International Senior Research Fellow ofThe Wellcome Trust, UK.

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