Antibodies raised against receptor-binding domain of Plasmodiumknowlesi Duffy binding protein inhibit erythrocyte invasion

Agam P. Singh a, Sunil K. Puri b, Chetan E. Chitnis a,*a Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), P.O. Box 10504, Aruna Asaf Ali Marg, New

Delhi 110067, Indiab Central Drug Research Institute (CDRI), Lucknow, India

Received 12 November 2001; received in revised form 8 January 2002; accepted 15 January 2002

Abstract

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium

knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades

rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds

sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite

proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy

binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi a protein, which binds human and

rhesus Duffy antigens, and P. knowlesi b and g proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The

receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here,

we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of

erythrocyte binding proteins. Region II of P. knowelsi a protein (PkaRII), which binds the Duffy antigen, was expressed as a

secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant PkaRII were

tested for inhibition of erythrocyte binding and invasion. Antibodies raised against PkaRII inhibit P. knowlesi invasion of both

human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous

binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Erythrocyte invasion; Erythrocyte binding proteins; Host-parasite interactions; Malaria vaccines

1. Introduction

Malaria parasites have an obligate intra-erythrocytic

stage in vertebrate hosts. Invasion of erythrocytes by

malaria parasites requires specific receptor-ligand inter-

actions [1]. For example, Plasmodium vivax is absolutely

dependent on interaction with the Duffy blood group

antigen to invade human erythrocytes [2]. As a result,

Duffy negative individuals are completely resistant to P.

vivax malaria. Plasmodium falciparum binds sialic acid

residues on glycophorin A as well as alternative

receptors to invade human erythrocytes by multiple

pathways [3�/10]. Like P. vivax , the simian malaria

parasite Plasmodium knowlesi is also absolutely depen-

dent on interaction with the Duffy antigen to invade

human erythrocytes [11]. However, P. knowlesi , like P.

falciparum , uses multiple pathways to invade rhesus

erythrocytes [12]. P. knowlesi can bind the rhesus Duffy

antigen as well as alternative receptors on rhesus

erythrocytes for invasion [12].

Parasite ligands that mediate interaction of P. vivax ,

P. falciparum and P. knowlesi with erythrocyte recep-

Abbreviations: BSA, bovine serum albumin; CD, circular

dichroism; DBL, Duffy-binding-like; EBA-175, 175 kDa P.

falciparum erythrocyte binding antigen; FCS, fetal calf serum; GP67,

67 kDa envelope surface glycoprotein of Autographa californica

nuclear polyhedrosis virus; HSV gD, Herpes simplex virus

glycoprotein D; NTA, nitrilotriacetic acid; OD, optical density; PBS,

phosphate buffered saline; PCR, polymerase chain reaction; PkaRII,

region II of P. knowlesi a protein; PkbRII, region II of P. knowlesi bprotein; PkgRII, region II of P. knowlesi g protein; SDS-PAGE,

sodium dodecyl sulfate-polyacrylamide gel electrohporesis; Sf21,

Spodoptera frugiperda cells.

* Corresponding author. Tel.: �91-11-618-7695; fax: �91-11-616-

2316.

E-mail address: cchitnis@icgeb.res.in (C.E. Chitnis).

Molecular & Biochemical Parasitology 121 (2002) 21�/31

www.parasitology-online.com

0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 0 1 7 - 8

tors described above share similar features and belong

to a family of erythrocyte binding proteins [13]. The

erythrocyte binding protein family includes the 175 kDa

P. falciparum erythrocyte binding antigen (EBA-175),which binds sialic acid residues on glycophorin A, P.

vivax Duffy binding protein, and three related P.

knowlesi proteins referred to as a, b and g [12�/18].

The extracellular regions of each of these proteins

contain two conserved cysteine-rich domains that are

referred to as regions II and VI [13]. The functional

receptor-binding domain of each member of the ery-

throcyte binding protein family lies in the N-terminal,conserved cysteine-rich region, region II [19�/21]. These

conserved, cysteine-rich regions are referred to as Duffy-

binding-like (DBL) domains after the first binding

domain identified from P. vivax Duffy binding protein

[19]. DBL domains of the erythrocyte binding proteins

have diverse binding specificity. Whereas region II of P.

vivax Duffy binding protein binds the human Duffy

antigen, region II of P. knowlesi a protein (PkaRII)binds both human and rhesus Duffy antigens [19,21].

Region II of P. knowlesi b protein (PkbRII) binds sialic

acid residues on rhesus erythrocytes and region II of P.

knowlesi g protein (PkgRII) binds as yet unidentified

Duffy-independent receptors on rhesus erythrocytes

[19,21]. P. knowlesi b and g proteins may be responsible

for the Duffy-independent pathways used by P. knowlesi

to invade rhesus erythrocytes. Region II of P. falci-

parum EBA-175 contains tandem duplication of DBL

domains referred to as F1 and F2 [17]. The receptor

binding domain of EBA-175 lies in region F2 [20]. Genes

encoding paralogs of EBA-175 are present in the P.

falciparum genome. Such EBA-175 paralogs may bind

sialic acid�/glycophorin A independent receptors to

mediate invasion by alternative pathways [22�/25].

The binding domains of malaria parasite proteins thatbind erythrocyte receptors to mediate invasion are

attractive candidates for development of blood-stage

malaria vaccines. Antibodies directed against such

receptor-binding domains may block erythrocyte bind-

ing and invasion by merozoites. Indeed, antibodies

raised against the recombinant sialic acid binding

domain, region II, of EBA-175 expressed as a secreted

protein in insect cells have been shown to blockerythrocyte binding and invasion by P. falciparum

[26�/28]. The binding domain of P. vivax Duffy binding

protein has also been produced in its functional form

[29,30] and antibodies raised against this functional

domain have been shown to block erythrocyte binding

[30].

Here, we have expressed the binding domain of P.

knowlesi a protein, PkaRII, which binds the Duffyblood group antigen, as a secreted protein in insect cells.

Rabbit antibodies raised against recombinant PkaRII

purified from insect cell culture supernatants were tested

for inhibition of erythrocyte binding and invasion by P.

knowlesi . Antibodies raised against PkaRII inhibit

invasion of both human and rhesus erythrocytes by P.

knowlesi . These data suggest that immunization with the

homologous binding domains of P. vivax Duffy bindingprotein and P. falciparum EBA-175 may elicit antibo-

dies that inhibit multiplication of blood-stage P. vivax

and P. falciparum .

2. Materials and methods

2.1. Plasmid construct for expression of recombinant

PkaRII in insect cells

DNA fragments encoding PkaRII, the binding do-

main of P. knowlesi a protein (amino acids 202�/536),

fused to hexa-histidine at the carboxyl end were

amplified by polymerase chain reaction (PCR) and

cloned downstream of the signal sequence of the 67

kDa envelope surface glycoprotein (GP67) and polyhe-

drin promoter of Autographa californica nuclear poly-hedrosis virus in transfer vector pAcGP67B

(Pharmingen) as follows. Primers APM1 (5? CGC

GGA TCC AAT CA AAC TTT TCT TCA A 3?) and

APM4 (5?GAC GAA TTC TTT GTT ATA TTG GTA

GT-3?) were used for PCR with plasmid p1cg [16], which

contains the 5? end of gene encoding P. knowlesi aprotein, as template to amplify a 288 bp DNA fragment

(PCR product 1) encoding the N-terminal region ofPkaRII by PCR. Primers APM3 (5? TGC GAA TTC

TGT AAG GAT AT AAG ATG 3?) and APM2 (5?ATA GTT TAG CGG CCG CTC AGA GAT GAT

GAT GAT GAT GT TCA GTT ATC GGA TTA GA

3?) were used for PCR with plasmid p6D [16], which

contains the 3? end of gene encoding P. knowlesi aprotein, as template to amplify a 764 bp DNA fragment

(PCR product 2) encoding the C-terminal region ofPkaRII fused to a 6-histidine tag. PCR products 1 and 2

were digested with BamHI, EcoRI and EcoRI, NotI

respectively. The transfer vector pAcGP67B (Pharmin-

gen) was digested with BamHI, NotI and used in a

three-way ligation reaction with the two digested PCR

products to yield expression plasmid

pGP67BKADR2.1. Sequencing using an ABI 310 auto-

mated sequencer (Applied Biosystems) was used toconfirm that the DNA fragment encoding PkaRII in

pGP67BKADR2.1 has the correct orientation and no

mutations have been introduced by PCR. Recombinant

transfer plasmid, pGP67BKADR2.1, which contains a

DNA fragment encoding PkaRII as a BamHI�/NotI

fragment fused to the signal sequence of GP67 protein

downstream of the polyhedrin promoter, was used to

obtain recombinant baculovirus for expression of re-combinant PkaRII as a secreted protein in Spodoptera

frugiperda cells (Sf21 insect cells, Pharmingen) as

described below.

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/3122

2.2. Cotransfection for construction of recombinant virus

for expression of secreted, recombinant PkaRII in insect

cells

Sf21 insect cells were co-transfected with

pGP67BKADR2.1 and Baculogold baculoviral genomic

DNA (Pharmingen) as recommended by the manufac-

turer. Isolation of recombinant plaques, plaque assays

and amplification to obtain high titre recombinant virus

(BVGP67PKADR2.1) were performed using standard

procedures.

2.3. Expression and purification of recombinant PkaRII

Sf21 insect cells (4�/106 cells ml�1) adapted forgrowth in suspension cultures in Sf900-II serum-free

medium (GIBCO) were infected with recombinant virus

BVGP67KADR2.1 at multiplicity of infection of 5 and

cultured for 96 h at 27 8C in suspension culture. Culture

supernatant was collected after 96 h, centrifuged to

remove cell debris, dialyzed extensively against dialysis

buffer (50 mM sodium phosphate, 300 mM NaCl, pH

8.0) at 4 8C, filtered through 0.45 mm filter (Millipore)and loaded onto a column containing Ni-nitrilotriacetic

acid (Ni-NTA) matrix (Qiagen) pre-equilibrated with

dialysis buffer. The Ni-NTA column was washed with

20 column volumes of dialysis buffer containing 10 mM

imidazole and 3 column volumes of 50 mM sodium

acetate pH 4.6. Recombinant PkaRII was eluted with

elution buffer containing 50 mM sodium phosphate pH

8.0, 300 mM NaCl, 250 mM imidazole, 10% glycerol.

2.4. Analysis of purified PkaRII by reverse phase

chromatography

PkaRII purified by Ni-NTA chromatography as

described above was loaded on a reverse phase C8

column. 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 with90% Buffer A and 10% Buffer B and reached a

composition of 10% Buffer A and 90% Buffer B in 40

min.

2.5. Circular dichroism (CD) spectroscopy of

recombinant PkaRII

CD spectra were recorded on a Jasco-J720 spectro-

polarimeter. Spectra of purified PkaRII in 10 mM

phosphate buffer, pH 7.0 were recorded in the far-UV

region from 184 to 260 nm using a cuvette with a pathlength of 0.1 cm and the following instrument para-

meters-instrument sensitivity, 1 mdeg; response time, 2

s; scan speed, 50 nm min�1. Deconvolution of the CD

spectra was performed using the method of Bohm et al.

[31].

2.6. Erythrocyte binding assay with recombinant PkaRII

Blood collected in 10% citrate phosphate dextrose was

stored at 4 8C for up to 4 weeks and washed three times

in RPMI 1640 culture medium (GIBCO) before use.

Duffy phenotypes of erythrocytes were determined by

standard blood typing methods using two antisera (anti-

Fya and anti-Fyb) (Ortho-Clinical Diagnostics). Duffy

positive human erythrocytes and rhesus erythrocytes

were treated with chymotrypsin as described earlier [14].Recombinant PkaRII (10 mg) was incubated for 1 h at

room temperature with normal and chymotrypsin-

treated erythrocytes (100 ml packed erythrocytes) in a

total volume of 600 ml containing 10% fetal calf serum

(FCS) (GIBCO). The reaction mixture was layered over

dibutylpthalate (Sigma) and centrifuged to collect ery-

throcytes. Bound protein was eluted from the erythro-

cytes with 300 mM NaCl. One-third of the elute wasseparated by SDS-PAGE and detected by Western

blotting using a mouse monoclonal antibody raised

against penta-histidine (Qiagen). Purified PkaRII (1 mg)

was used as a positive control for Western blotting.

2.7. Animals and immunization

Rabbits used in this study were procured from theAnimal Facility of the International Center for Genetic

Engineering and Biotechnology, New Delhi, India.

Animals were housed, fed, and used in experiments

according to the guidelines set forth in the National

Institutes of Health manual titled, Guide for the Care

And Use of Laboratory Animals (National Institutes of

Health Publication No. 86-23, US Department of

Health and Human Services, Washington DC). Two16-week-old New Zealand White rabbits were immu-

nized with 400 mg of recombinant PkaRII emulsified in

complete Freund’s adjuvant delivered by the subcuta-

neous route. The rabbits were boosted thrice with 200 mg

of PkaRII formulated in incomplete Freund’s adjuvant

delivered by the subcutaneous route 4, 8 and 12 weeks

after primary immunization. One rabbit was immunized

with adjuvant alone according to the schedule describedabove to provide control serum. Rabbits were bled

before immunization (pre-immune sera) and 2 weeks

after each boost.

2.8. ELISA

Rabbit sera were tested for recognition of recombi-

nant PkaRII by ELISA. Serial dilutions of sera startingwith 1:1000 fold dilution were tested. Briefly, wells of

flat-bottom Immulon-2 plates (Dynatech Laboratories)

were coated with 0.1 mg of PkaRII and blocked with 3%

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/31 23

BSA in PBS (blocking buffer). Antigen-coated wells

were incubated for 90 min at 37 8C with 100 ml of rabbit

serum diluted in 0.1% blocking buffer. Wells were

washed with phosphate-buffered saline (PBS) and0.05% Tween 20 (wash buffer). A total of 100 ml of

horseradish peroxidase-labeled anti-rabbit IgG antibody

(Sigma) diluted 1:2000 fold was then added to each well,

incubated for 60 min at 37 8C and washed three times

with wash buffer prior to development of enzyme

reaction with o-phenylenediamine dihydrochloride as

the chromogen and hydrogen peroxide as the substrate.

The reaction was terminated by addition of sulfuric acidand the absorbance at 490 nm (OD490 nm) was recorded

in each well using an ELISA microplate reader (Mole-

cular Devices). Pre-immune sera as well as control sera

raised against adjuvant alone were used at similar

dilutions.

2.9. Immunoflourescence assay

Rabbit serum raised against recombinant PkaRIIwere tested for recognition of PkaRII, PkbRII and

PkgRII expressed on surface of mammalian COS7 cells

by immunofluorescence assay. Plasmid DNA constructs

pHKADR22, pHKBDR22 and pHKGDR22, which are

designed to express PkaRII, PkbRII and PkgRII on the

surface of COS7 cells, have been described earlier [19].

Briefly, plasmids pHKADR22, pHKBDR22 and

pHKGDR22 contain DNA encoding PkaRII, PkbRIIand PkgRII respectively fused to DNA encoding the

signal sequence of Herpes simplex virus glycoprotein D

(HSV gD) at the 5? end and DNA encoding the

transmembrane segment and cytoplasmic domain

HSVgD at the 3? end cloned in a mammalian cell

expression vector [19,32]. COS7 cells were transfected

with plasmids pHKADR22, pHKBDR22 and

pHKGDR22 using Lipofectin (GIBCO) as describedby the manufacturer. Expression of PkaRII, PkbRII

and PkgRII on the surface of COS7 cells was confirmed

by immunofluorescence assay using murine monoclonal

antibody DL6, which is directed against HSV gD

sequences in the recombinant fusion proteins, as de-

scribed earlier [19,32]. Serial dilutions of rabbit serum

raised against recombinant PkaRII were used in im-

munofluorescence assays with transfected COS7 cellsexpressing PkaRII, PkbRII and PkgRII on the surface

to determine end point titres.

2.10. Inhibition of erythrocyte binding with rabbit serum

raised against PkaRII

Transfected COS7 cells expressing PkaRII, PkbRII

and PkgRII on the surface were used in erythrocytebinding assays with human and rhesus erythrocytes as

described earlier [19,30]. Erythrocyte binding assays

were performed in the presence of different dilutions

of rabbit serum raised against recombinant PkaRII as

described earlier [30]. Pre-immune rabbit serum and

rabbit serum raised against Freund’s adjuvant alone

were used as control. The number of COS7 cells havingrosettes of bound erythrocytes was scored in 30 fields at

200 fold magnification. Each dilution of serum was

tested in triplicate. The average percent inhibition of

binding (and S.D.) was determined at each dilution.

2.11. Purification of antibodies from rabbit serum

Rabbit antibodies (IgG) were purified using Protein

A-Sepharose (Pharmacia) as recommended by the

manufacturer. Briefly, 5.0 ml Protein A slurry was

loaded in a column, washed with 10 ml PBS and

equilibrated with 10 ml 100 mM Tris pH 8.0. Rabbitserum (4.5 ml) was mixed with 0.5 ml 1 M Tris pH 8.0

and loaded on the Protein A column using gravity flow.

The column was washed with 35 ml 100 mM Tris pH 8.0

and 20 ml 10 mM Tris pH 8.0. Bound IgG was eluted

with 100 mM Glycine pH 3.0. Elutes (0.5 ml each) were

collected in 1.5 ml tubes containing 50 ml 1 M Tris pH

8.0. Purified IgG was separated by SDS-PAGE and

detected by Coomasie-staining. Proteins of expected size(25 and 55 kDa) were detected and the purity of IgG was

�/99%. Purified IgG was dialyzed against RPMI 1640

(GIBCO) prior to use in invasion assays. Concentration

of antibodies was determined by measuring OD280 nm.

2.12. Inhibition of erythrocyte invasion by P. knowlesi

with purified rabbit antibodies raised against PkaRII

Parasitized blood was collected from rhesus monkeys

infected with P. knowlesi H strain by venipuncture when

parasitemia was �/5% and majority of parasites were in

the schizont stage. P. knowlesi schizonts were isolated to�/95% purity by centrifugation of infected blood on

45% Percoll. Purified P. knowlesi schizonts were used

for in vitro erythrocyte invasion assays as described

earlier [33]. Briefly, human or rhesus erythrocytes (1�/

107) were incubated with P. knowlesi schizonts (1�/106)

in the presence of 10% FCS and different dilutions of

purified rabbit antibodies raised against PkaRII or

Freund’s adjuvant alone for 8�/10 h at 37 8C under90% N2, 5% O2, 5% CO2. Different concentrations of

antibodies were tested (2.7, 0.9, 0.3 and 0.1 mg ml�1).

Two thousand erythrocytes were scored for presence of

P. knowlesi rings on Giemsa-stained smears. The

percentage of erythrocytes infected with rings was

calculated to determine invasion rates. Invasion inhibi-

tion efficiencies were determined using invasion rates in

presence of different concentrations (conc a) of anti-PkaRII antibodies (Inv(PkaRII)conc a) and antibodies

raised against adjuvant alone (Inv(Adj.)conc a) as fol-

lows:

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/3124

Inhibition efficiency (%)

�(1�Inv(PkaRII)conc a=Inv(Adj:)conc a)� 100

Invasion assays were performed in triplicate at eachantibody concentration. Average invasion rates and

S.D. are reported. P values were calculated by one-

way ANOVA and non-parametric test for invasion

rates. P values greater than 0.05 were considered not

significant.

3. Results

3.1. Expression and purification of recombinant PkaRII

Sf21 insect cells were infected with recombinant

baculovirus BVGP67KADR2.1 designed to express

recombinant PkaRII as a secreted protein. Recombi-nant PkaRII was purified from culture supernatants of

infected Sf21 cells by metal affinity chromatography on

Ni-NTA columns. Separation by SDS-PAGE indicates

that recombinant PkaRII has an apparent molecular

mass of �/43 kDa, which is slightly higher than the

predicted molecular mass of 40 683 Da (Fig. 1A). Upon

deglycosylation with N-glycanase F, recombinant

PkaRII migrates with the expected molecular mass of�/41 kDa (data not shown). Densitometry scanning of

Coomasie-blue stained SDS-PAGE gels indicates that

purity of recombinant PkaRII is greater than 95%.

Yields of purified PkaRII are �/7 mg l�1 of culture.

3.2. Characterization of recombinant PkaRII

An erythrocyte-binding assay was used to test

whether purified PkaRII is functional. Purified PkaRII

RII was incubated with Duffy positive human and

rhesus erythrocytes as well as chymotrypsin-treatedhuman and rhesus erythrocytes to allow binding.

Erythrocytes with bound protein were collected by

centrifugation. Bound protein was eluted with 300 mM

NaCl separated by SDS-PAGE and detected by Western

blot using a murine monoclonal antibody directed

against penta�/histidine. PkaRII binds Duffy positive

human erythrocytes and rhesus erythrocytes but does

not bind chymotrypsin-treated human or rhesus ery-throcytes, which lack the Duffy antigen (Fig. 1B).

PkaRII thus binds erythrocytes with the same specificity

as P. knowlesi Duffy binding protein indicating that it is

functional.

The mobility of PkaRII by gel filtration chromato-

graphy on a Superdex S200 column is consistent with an

apparent molecular weight of �/43 kDa indicating that

purified PkaRII is monomeric and does not formaggregates or multimers (data not shown). The homo-

geneity of PkaRII was analyzed by reverse phase

chromatography, which can separate different confor-

mers of the same protein based on differences in surface

hydrophobicity. PkaRII elutes as a single symmetric

peak by reverse phase chromatography on a C-8 column

Fig. 1. Expression, purification and functional characterization of

recombinant PkaRII expressed as a secreted protein in insect cells. (A)

Coomasie-stained gel showing expression and purification of PkaRII.

Recombinant PkaRII with a C-terminal 6-histidine fusion was

expressed as a secreted protein in insect cells using recombinant

baculovirus. Recombinant PkaRII was purified from insect cell culture

supernatants by metal affinity chromatography (Ni). MW, molecular

weight markers in kDa; PL, preload-insect cell culture supernatant

used for metal affinity chromatography; Ni elutes, elutes from metal

affinity chromatography. (B) Erythrocyte binding assay. Purified

recombinant PkaRII was tested in functional erythrocyte binding

assays (EBA) as follows. Purified recombinant PkaRII (10 mg) was

incubated with Duffy positive human erythrocytes (Hu), rhesus

erythrocytes (Rh) as well as human and rhesus erythrocytes treated

with chymotrypsin (Chy). Erythrocytes and bound proteins were

separated by centrifugation, bound proteins were eluted with 300

mM NaCl, separated by SDS-PAGE and detected by Western blotting

using a monoclonal antibody against penta-histidine. Recombinant

PkaRII binds Duffy positive human eythrocytes and rhesus erythro-

cytes but not chymotrypsin-treated erythrocytes that have lost the

Duffy antigen. Recombinant PkaRII (1 mg) was directly loaded on the

gel as control for Western blotting. Molecular weight markers are

shown in kDa.

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/31 25

suggesting that it is homogeneous (Fig. 2). CD spectro-scopy was used to probe the secondary structure of

PkaRII. The CD spectrum of PkaRII shows character-

istic a-helical signature minima at 208 and 222 nm (Fig.

3). Deconvolution of the CD spectrum by the method of

Bohm et al. [31] indicates the following distribution of

secondary structure components for PkaRII*/57.1%, a-

helices; 8.8%, b-sheets; 13.2%, b-turns; and 20.3%,

random-coils. The sum of secondary structural elementscalculated from the CD spectrum totals 99.3% showing

confidence in the measurement and deconvolution. The

a-helical content of PkaRII is similar to that reported

previously for the homologous binding domain of P.

vivax Duffy binding protein [30].

3.3. Recognition of PkaRII, PkbRII and PkgRII by

rabbit serum raised against PkaRII

Purified PkaRII formulated in Freund’s adjuvant was

used to immunize two rabbits, R1 and R2. Rabbit R3

was immunized with Freund’s adjuvant alone to provide

control sera. Sera collected following primary and threebooster immunizations were used for all assays. Reac-

tivity of rabbit sera with PkaRII was tested by ELISA.

Sera from rabbits R1 and R2 have ELISA titers of

1:450 000 indicating that PkaRII is highly immunogenic.

Sera from rabbits R1 and R3 were also tested for

recognition of PkaRII, PkbRII and PkgRII expressed

on the surface of transfected COS7 cells by immuno-

fluorescence assay. COS7 cells were transfected withconstructs designed to express PkaRII, PkbRII and

PkgRII on the surface of transfected COS7 cells. Serum

from rabbit R1 reacts with COS7 cells expressing

PkaRII up to a dilution of 1:39 000. PkbRII and

PkgRII are also recognized by serum raised against

PkaRII up to dilutions of 1:2500 and 1:15 625, respec-

tively. Antibodies raised against PkaRII thus cross-react

with homologous DBL domains PkbRII and PkgRII.Control serum from rabbit R3 immunized with Freund’s

adjuvant alone and pre-immune serum from rabbit R1

do not react with COS7 cells expressing PkaRII, PkbRII

and PkgRII.

3.4. Inhibition of erythrocyte binding to PkaRII, PkbRII

and PkgRII with rabbit serum raised against PkaRII

The ability of anti-PkaRII rabbit serum to block

binding of erythrocytes to PkaRII, PkbRII and PkgRII

was tested. Transfected COS7 cells expressing PkaRII,

PkbRII and PkgRII on the surface were incubated with

Duffy positive human erythrocytes and rhesus erythro-cytes in the presence of different dilutions of sera from

rabbits R1 and R3. Following removal of non-adherent

erythrocytes by washing, the number of COS7 cells

Fig. 2. Reverse phase high performance liquid chromatography (RP-HPLC) profile of purified recombinant PkaRII. Purified PkaRII was analyzed

by RP-HPLC on a C-8 column. A linear gradient developed using Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in 90% acetonitrile and

10% water) was used for elution of PkaRII. The column was initially equilibrated with 90% Buffer A and 10% Buffer B and reached a composition of

10% Buffer A and 90% Buffer B in 40 min. PkaRII elutes as a single, symmetric peak indicating that it contains a homogeneous population of

conformers. Fractions collected at 23, 24, 25 and 26 min were separated by SDS-PAGE and detected by Coomasie-staining (Inset). MW, molecular

weight markers (66, 45, 31, 21 and 14 kDa) used for SDS-PAGE are shown.

Fig. 3. CD spectrum of purified recombinant PkaRII. Minima near

208 and 222 nm and a maximum near 190 nm indicate presence of

significant a-helical content in PkaRII.

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/3126

covered with rosettes of erythrocytes was scored in 30

fields at a magnification of 200. Each dilution of serum

was tested in triplicate wells. Results of one of three

similar experiments are shown in Fig. 4. Binding in the

presence of anti-PkaRII rabbit serum at different

dilutions is shown relative to binding in the presence

of control serum (rabbit R3) raised against adjuvant

alone at 1:600 dilution. Anti-PkaRII rabbit serum

(rabbit R1) blocks binding of human erythrocytes to

PkaRII more efficiently than binding of rhesus erythro-

cytes to PkaRII. Anti-PkaRII serum also blocks binding

of rhesus erythrocytes to PkbRII and PkgRII. Fifty

percent inhibition of binding of rhesus erythrocytes to

PkaRII, PkbRII and PkgRII was achieved at dilutions

of 1:19 200, 1:600 and 1:9600, respectively. Binding of

human erythrocytes to PkaRII was blocked most

efficiently with greater than 50% inhibition at dilutions

up to 1:153 000.

3.5. Inhibition of erythrocyte invasion by P. knowlesi

with antibodies purified from rabbit serum raised against

PkaRII

Antibodies were purified from serum of rabbit R1

following primary and three booster immunizations

with PkaRII formulated in Freund’s adjuvant. Anti-

bodies were also purified from serum of rabbit R3

following primary and three booster immunizations

with Freund’s adjuvant alone. Purified antibodies were

tested for inhibition of invasion of human and rhesuserythrocytes by P. knowlesi . Purified P. knowlesi

schizonts were incubated with uninfected erythrocytes

Fig. 4. Inhibition of erythrocyte binding to PkaRII, PkbRII and PkgRII with rabbit serum raised against recombinant PkaRII. Transfected COS7

cells expressing PkaRII (A, B), PkbRII (C) or PkgRII (D) on the surface were used for binding assays with human (A) or rhesus (B�/D) erythrocytes

in the presence of different dilutions of rabbit serum raised against recombinant PkaRII. Control rabbit serum raised against Freund’s adjuvant

alone (c) was used at a dilution of 1:600. Numbers of COS7 cells with bound rosettes of erythrocytes were scored in 30 fields at 200-fold

magnification. Number of COS7 cells with bound rosettes of erythrocytes in presence of control serum scored in 30 fields at 200-fold magnification

was considered as 100%. Rosettes found in 30 fields in presence of control sera were 9195 for human erythrocytes with PkaRII (A), 135914 for

rhesus eryrthrocytes with PkaRII (B), 429920 for rhesus erythrocytes with PkbRII (C) and 294926 for rhesus erythrocytes with PkgRII (D).

Binding of erythrocytes in presence of different dilutions of rabbit serum raised against PkaRII is shown relative to binding in presence of control

serum.

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/31 27

in the presence of different concentrations of antibodies

purified from rabbits R1 and R3. Invasion rates were

determined by scoring the percentage of erythrocytes

infected with ring-stage parasites following incubation

for 8�/10 h to allow invasion. Invasion inhibition

efficiencies were determined from invasion rates in

presence of antibodies raised against PkaRII (rabbit

R1) and antibodies raised against adjuvant alone (rabbit

R3). Data from two independent experiments is shown

in Table 1. Antibodies raised against PkaRII block

invasion of both human and rhesus erythrocytes by P.

knowlesi (Table 1). Invasion of human erythrocytes is

inhibited more efficiently than invasion of rhesus

erythrocytes (Table 1).

4. Discussion

The receptor-binding domains of members of the

erythrocyte binding protein family of malaria parasites

Table 1

Inhibition of erythrocyte invasion by P. knowlesi with purified rabbit antibodies raised against recombinant PkaRII

Exp. no. Erythrocytes Antibodya Conc. (mg ml�1) Invasion rateb (%) Inhibition efficiencyc (%) P valued

1 Human Adj. 2.7 2.2990.10 �/ Ns

PkaRII 2.7 0.4490.04 80.79 B0.0001

Adj. 0.9 2.4790.18 �/ Ns

PkaRII 0.9 1.4190.09 42.91 0.0005

Adj. 0.3 2.7090.04 �/ Ns

PkaRII 0.3 2.0190.06 25.55 0.0021

Adj. 0.1 2.8590.09 �/ Ns

PkaRII 0.1 2.4090.35 15.79 Ns

Nonee �/ 2.8690.24 �/ �/

Rhesus Adj. 2.7 4.3690.13 �/ Ns

PkaRII 2.7 2.2390.16 48.85 B0.0001

Adj. 0.9 4.8290.09 �/ Ns

PkaRII 0.9 3.5990.23 25.52 0.0003

Adj. 0.3 5.0590.17 �/ Ns

PkaRII 0.3 4.3390.33 14.26 0.0032

Adj. 0.1 5.2290.14 �/ Ns

PkaRII 0.1 4.7090.12 9.96 0.0048

None �/ 6.0590.50 �/ �/

2 Human Adj. 2.7 4.7090.09 �/ 0.0391

PkaRII 2.7 0.8690.08 81.81 B0.0001

Adj. 0.9 4.7690.21 �/ Ns

PkaRII 0.9 2.4990.06 47.79 B0.0001

Adj. 0.3 5.0090.16 �/ Ns

PkaRII 0.3 3.6890.20 26.40 0.0002

Adj. 0.1 5.1190.24 �/ Ns

PkaRII 0.1 4.3290.15 15.46 0.0038

None �/ 5.2590.25 �/ �/

Rhesus Adj. 2.7 12.9690.94 �/ Ns

PkaRII 2.7 6.7090.21 48.30 B0.0001

Adj. 0.9 13.0790.39 �/ Ns

PkaRII 0.9 9.0590.59 30.76 B0.0001

Adj. 0.3 13.4890.17 �/ Ns

PkaRII 0.3 11.4190.15 15.36 B0.0001

Adj. 0.1 13.4590.11 �/ Ns

PkaRII 0.1 12.1790.33 9.52 0.0396

None �/ 14.5390.41 �/ �/

a Antibodies purified from rabbits immunized with either recombinant PkaRII formulated in Freund’s adjuvant or Freund’s adjuvant alone (Adj.)

were tested for inhibition of erythrocyte invasion by P. knowlesi .b Invasion rates were determined by scoring percent of erythrocytes infected with ring-stage parasites following incubation of purified P. knowlesi

schizonts with uninfected erythrocytes to allow invasion.c Inhibition efficiency at different concentrations (conc a) of antibodies raised against PkaRII is calculated from invasion rates in presence of

antibodies raised against PkaRII (Inv(PkaRII)conc a) and invasion rates in presence of antibodies raised against adjuvant alone (Inv(Adj.)conc a) as

follows:

Inhibition efficiency (%)�(1�Inv(PkaRII)conc a=Inv(Adj:)conc a)�100

d P values were calculated by one way ANOVA and non-parametric test; Ns, not significant (P �0.05).e Invasion rates measured in absence of antibodies.

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/3128

lie in conserved N-terminal cysteine-rich regions referred

to as region II [19�/21]. Here, we have produced

recombinant PkaRII, region II of P. knowlesi a protein,

which binds the Duffy antigen on human and rhesuserythrocytes, as a secreted protein in insect cells.

Recombinant PkaRII was purified from culture super-

natants of insect cells by metal affinity chromatography

and characterized to show that it is pure, homogenous

and functional. Mobility of recombinant PkaRII on

SDS-PAGE gels suggests that it has an apparent

molecular mass of �/43 kDa, which is higher than the

predicted molecular mass of 40 683 Da. PkaRII containsthree putative N-glycosylation sites namely, NQT, NNT

and NES. Treatment with N-glycanase F results in

reduction of the apparent molecular mass to �/41 kDa

indicating that recombinant PkaRII is glycosylated. The

CD spectrum of recombinant PkaRII indicates presence

of significant a-helical content (57.1%), which is similar

to the a-helical content reported previously for the

homologous binding domain from P. vivax Duffybinding protein [30] and as predicted by sequence

analysis of DBL domains [34]. Importantly, recombi-

nant PkaRII is functional in that it binds normal Duffy

positive human erythrocytes and rhesus erythrocytes but

not chymotrypsin-treated human or rhesus erythrocytes

that have lost the Duffy antigen.

Recombinant PkaRII was used to immunize rabbits.

Antibodies raised against this functional receptor-bind-ing domain were tested for inhibition of erythrocyte

binding and invasion by P. knowlesi in vitro. Results of

these experiments are of direct relevance for vaccine

development efforts based on the homologous receptor-

binding domains of P. vivax Duffy binding protein and

P. falciparum EBA-175. Recombinant PkaRII is highly

immunogenic and elicits high titre antibodies that

recognize PkaRII expressed on surface of COS7 cells.Rabbit serum raised against PkaRII cross-reacts with

the homologous DBL domains PkbRII and PkgRII to

varying extents. Anti-PkaRII rabbit serum was tested

for inhibition of erythrocyte binding to PkaRII, PkbRII

and PkgRII expressed on the surface of COS7 cells.

Anti-PkaRII serum inhibits binding of both human and

rhesus erythrocytes to PkaRII. Binding of human

erythrocytes to PkaRII is inhibited more efficientlythan binding of rhesus erythrocytes. Anti-PkaRII serum

also inhibits binding of rhesus erythrocytes to PkbRII

and PkgRII although less efficiently than it inhibits

binding to PkaRII. Immunofluorescence titres of anti-

PkaRII serum for recognition of PkaRII, PkbRII and

PkgRII correlate with the efficiency of inhibition of

erythrocyte binding to these domains.

Antibodies raised against recombinant PkaRII havegreater cross-reactivity with PkgRII than PkbRII. Pair

wise comparison of amino acid sequences of PkaRII

with amino acid sequences of PkbRII and PkgRII

indicates that the percent sequence identity between

these DBL domains is similar, around 70%. One would

therefore not predict that antibodies raised against

PkaRII would have greater cross-reactivity with

PkgRII than PkbRII. The cross-reactivity data suggeststhat PkaRII shares some cross-reactive epitopes with

PkgRII that are not present in PkbRII. Sequence

analysis does reveal the presence of a number of

hydrophilic amino acid residues that are unique to

PkaRII and PkgRII. Such residues are likely to be

exposed and may be responsible for presence of cross-

reactive epitopes shared by PkaRII and PkgRII.

Antibodies purified from rabbit serum raised againstPkaRII were tested for inhibition of invasion of human

and rhesus erythrocytes by P. knowlesi in vitro. Anti-

bodies raised against PkaRII inhibit invasion of human

erythrocytes by around 80% at the highest concentration

tested (2.7 mg ml�1) in two independent experiments.

Like P. knowlesi , P. vivax is also completely dependent

on interaction with the Duffy antigen for invasion of

human erythrocytes. The observation that antibodiesdirected against PkaRII block invasion of human

erythrocytes by P. knowlesi efficiently suggests that

antibodies elicited by a recombinant vaccine based on

the binding domain of P. vivax Duffy binding protein

should block multiplication of blood-stage P. vivax

effectively.

Antibodies raised against PkaRII also inhibit inva-

sion of rhesus erythrocytes by P. knowlesi . However,inhibition of invasion of rhesus erythrocytes (around

48% at antibody concentration of 2.7 mg ml�1) is less

efficient than inhibition of invasion of human erythro-

cytes (around 80% at antibody concentration of 2.7 mg

ml�1). Anti-PkaRII serum blocks binding of rhesus

erythrocytes to PkaRII less efficiently than binding of

human erythrocytes. Moreover, while P. knowlesi is

completely dependent on the interaction of a proteinwith the Duffy antigen for invasion of human erythro-

cytes, it can use alternative pathways mediated by b and

g proteins to invade rhesus erythrocytes. Although

antibodies raised against PkaRII cross-react with

PkbRII and PkgRII, they inhibit binding of rhesus

erythrocytes to PkbRII and PkgRII less efficiently

than binding of human erythrocytes to PkaRII. The

availability of multiple pathways for invasion ofrhesus erythrocytes and the lower efficiency for inhibi-

tion of binding of rhesus erythrocytes to PkaRII,

PkbRII and PkgRII compared to human erythro

cytes may be responsible for the lower inhibition of

invasion of rhesus erythrocytes compared to human

erythrocytes.

Like P. knowlesi , P. falciparum also uses multiple

pathways to invade human erythrocytes [3�/10]. Para-logs of P. knowlesi a protein, namely b and g proteins,

mediate Duffy-independent alternative invasion path-

ways. Similarly, EBA-175 paralogs, which contain

receptor-binding DBL domains, may bind sialic acid�/

A.P. Singh et al. / Molecular & Biochemical Parasitology 121 (2002) 21�/31 29

glycophorin A independent receptors to mediate inva-

sion by alternative pathways [22�/25]. The observations

made here with P. knowlesi suggest that antibodies

raised against the binding domain of EBA-175 maycross-react with binding domains of EBA-175 paralogs

to inhibit invasion by alternative pathways, although the

inhibition may not be very efficient. Indeed, it has been

observed that antibodies raised against recombinant

region II of EBA-175 expressed as a secreted protein in

insect cells inhibit invasion by P. falciparum isolates that

use multiple invasion pathways [28]. The efficiencies

reported for inhibition of P. falciparum invasion byantibodies raised against recombinant EBA-175 region

II [28] are similar to the efficiencies described here for

inhibition of P. knowlesi invasion of rhesus erythrocytes

by antibodies raised against PkaRII.

In conclusion, antibodies raised against the receptor-

binding domain of P. knowlesi a protein, which binds

the Duffy antigen, block invasion of human erythro-

cytes by P. knowlesi efficiently. By analogy, antibodiesraised against the receptor-binding domain of P. vivax

Duffy binding protein should effectively block erythro-

cyte invasion by P. vivax . This provides support for the

development of a vaccine based on region II of P. vivax

Duffy binding protein. Antibodies raised against

PkaRII also inhibit P. knowlesi invasion of rhesus

erythrocytes although at reduced efficiency compared

to inhibition of invasion of human erythrocytes. Thisobservation suggests that in case of P. falciparum , which

like P. knowlesi uses multiple invasion pathways,

antibodies raised against the binding domain of EBA-

175 should block invasion of human erythrocytes,

although at reduced efficiency. It has been shown that

immunization of Aotus monkeys by a variety of prime-

boost combinations using EBA-175 region II DNA

immunization constructs as well as recombinant EBA-175 region II produced in insect cells results in reduction

of parasite densities and partial protection against

blood-stage P. falciparum challenge [35]. These data

support the inclusion of the binding domain of EBA-175

in a vaccine for P. falciparum malaria. However, it may

be necessary to combine EBA-175 with other parasite

proteins that play functional roles in invasion to block

erythrocyte invasion efficiently and provide significantprotection against P. falciparum malaria.

Acknowledgements

We thank Drs Gary Cohen and Roselyn Eisenberg for

providing plasmid pRE4 and monoclonal antibody

DL6, Amit Sharma and Virander S. Chauhan for

comments on the manuscript, and Andrew Lynn forCD spectroscopy. This investigation was supported by a

grant from the European Commission (IC18CT980369)

to SKP and CEC and an International Research

Scholar’s grant from the Howard Hughes Medical

Institute to CEC.

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