Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodium falciparum antigen Pf155

Molecular and Biochemical Parasitology, 29 (1988) 19-28

19

Elsevier MBP 00959

Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodium falciparum antigen Pf155 Wipaporn Ruangjirachuporn 1, Birgitta W~hlin 1, Hedvig Perlmann 1, Jan Carlsson l, Klavs Berzins 1, Mats Wahlgren l, Rachanee Udomsangpetch 1, Hans Wigzell 2 and Peter Perlmann ~ 1Department of Immunology, University of Stockholm, Stockholm, Sweden and ~Department of Immunology, Karolinska Instituter, Stockholm, Sweden (Received 26 October 1987; accepted 7 January 1988)

Mouse monoclonal antibodies were prepared against a synthetic peptide (EENVEHDA) corresponding to a tandemly repeated sequence in the C-terminus of the Plasmodium falciparum antigen Pf155. One antibody (IgG1) producing bybridoma was studied in detail. The specificity of the antibody was determined by enzyme-linked immunosorbent assays using bovine serum albuminconjugated or free peptides as solid phase antigens and various synthetic peptides for inhibition. The antibody reacted with Pf155 as detected by immunofluorescence and immunoblotting. It was also an efficient inhibitor of merozoite invasion in P. ]?zlciparum in vitro cultures indicating that it defines a biologically important epitope present on the native Pf155 molecule. Key words: Plasmodium falciparum; Monoclonal antibody; Synthetic peptide: Antigen Pfl55

Introduction

Several antigens present in asexual erythrocytic stages of Plasmodium falciparum have been identified and characterized as possible candidates to be included in an anti-malarial vaccine [1,2]. A c o m m o n feature for most of these antigens is the presence of regions of repeated amino acid sequences [3-7]. Pf155 [8,9] which is the same antigen as the ring infected erythrocyte surface antigen, R E S A [3,4], contains two regions of repeated sequences, one in the C-terminus of the molecule consisting mainly of the subunits EEN-

Correspondence address: W. Ruangjirachuporn, Department of Immunology, University of Stockholm, S-106 91 Stockholm, Sweden. Abbreviations: ELISA, enzyme linked immunosorbent assay; KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; EMIF, erythrocyte membrane immunofluorescence.

V E H D A and EENV and one in the middle of the molecule consisting of 11 amino acid subunits [3,4,10]. These repeat regions are immunodominant [11] and are of interest with regard to vaccine development [2]. Various antibodies have been obtained, which react with the repeat regions of Pf155, including rabbit antibodies and mouse monoclonal antibodies to fusion proteins containing the corresponding repeats [11-13], rabbit or mouse antibodies to synthetic peptides corresponding to repeat subunits [9,11,13] and human antibodies affinity purified on synthetic peptides [9~14]. For further structural and functional studies of the Cterminal repeat region of Pf155 we have produced mouse monoclonal antibodies to the synthetic octapeptide E E N V E H D A . We herein report on the reactivity of one of these antibodies with Pf155 as detected by immunofluorescence, immunoblotting and P. falciparum invasion inhibition in vitro. The specificity of the antibody was studied by inhibition experiments in ELISA.

0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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Materials and Methods

Parasites. Parasite material was obtained from P. falciparum in vitro cultures maintained according to the method of Trager and Jensen [15]. Mainly, the Tanzanian strain F32 was used, but in some experiments the Thai 1 strain (Thailand, established by Dr. A. Bj6rkman, Roslagstull Hospital, Stockholm) [24] as well as the clones 7G8 (Brazil) and HB3 (Honduras) were used.

Synthetic peptides. Peptides were synthesized according to the standard method of Merrifield [16] and were purified and analysed for homogeneity by HPLC [9]. The peptides were obtained from different sources as follows: E E N V E H D A and (EENV)2 from Pharmacia, Uppsala, Sweden; K(EENVEHDA)2 and K(EENV)4 from Drs. T. Bartfai and A. Unden, Department of Biochemistry, University of Stockholm; Y(NVEE)4C from Dr. M.E. Patarroyo, Department of Immunobiology, National University of Colombia, Bogot/t, Colombia and K ( D D E H V E E P T V A ) z from Dr. G. Westin Sj6dahl, KabiVitrum AB, Stockholm, Sweden. The peptides Y(SVTEEIAEEDK)2 and (PEEL/vVEEV1/v)zGK were obtained from Dr. O. Mercereau-Puijalon, Pasteur Institute, Paris, and have been described in detail [17]. The purity of the peptides ranged beween 60--90%. Peptides were coupled to bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) at approximate molar ratios 40:1 and 200:1, respectively, using glutaraldehyde as described previously [9]. Monoclonal antibodies'. Mice were immunized i.p. at biweekly intervals with a peptide EENV E H D A - K L H conjugate (20 Ixg peptide per immunization) in Freund's complete adjuvant. Hybridomas were established one week after the fourth immunization by fusion of spleen cells with Sp2/0 mouse myeloma cells as described by Galfre et al. [18]. Screening for production of Pf155 reactive antibodies was performed by indirect immunofluorescence on glutaraldehyde-fixed and air-dried monolayers of P. falciparurn infected erythrocytes [8]. Monoclonality was assessed by analysis in isoelectric focusing and the isotype of the antibodies was determined by double immu-

nodiffusion using mouse IgG subclass specific antisera (Meloy, Springfield, VA).

Irnrnunoglobulin ELISA. Concentrations of mouse immunoglobulin were measured by ELISA using affinity purified rabbit anti mouse IgG antibodies both as solid phase catcher antibodies and as indicator system after conjugation with alkaline phosphatase [19]. Purified mouse IgG was used as standard. Reagents were from Sigma, St. Louis, MO. Indirect immunofluorescence. Two types of immunofluorescence assays were performed: firstly, erythrocyte membrane immunofluorescence (EMIF) on monolayers of glutaraldehyde-fixed and air-dried P. falciparum infected erythrocytes according to the method of Perlmann et al. [8] and secondly, regular parasite immunofluorescence on monolayers of air-dried and methanol-fiixed P. falciparurn infected erythrocytes. Bound antibodies were detected with biotinylated goat antibodies to mouse immunoglobulin and avidin conjugated with fluorescein isothiocyanate (Vector Laboratories, Inc., Burlingame, CA). In inhibition experiments, a fixed concentration of monoclonal antibody (1.4 ixg ml 1) was mixed with different concentrations of synthetic peptides or parasite extracts before adding to the monolayers.

lrnmunoblotting. Immunoblotting was performed as described by Berzins et al. [20]. Three different antigen preparations from P. falciparum in vitro cultures were used in this analysis: (a) a merozoite-enriched fraction [8], (b) spent culture medium collected at the time of sub-cultivation [8] and (c) culture supernatant material eluted from a human erythrocyte glycophorin-aminoethylBioGel resin with 3 M KSCN [21].

Inhibition of P. falciparum invasion in vitro. This assay was performed as described by Wfihlin et al. [22]. The IgG1 monoclonal antibody $2C6, directed against human urinary bladder cell carcinoma [23] was used as a control. For reversion of the invasion inhibition with synthetic peptides, 100 ~xl of a fixed concentration of monoclonal antibodies (60 txg ml 1) was mixed with 10 Ixl of syn-

21 thetic peptide at various concentrations. The mixtures were incubated for 30 min at r o o m temperature before addition of 100 txl of infected erythrocytes. The assay was then p e r f o r m e d as described earlier [22].

Peptide ELISA. Antibody reactivities with synthetic peptides were analysed in m i c r o - E L I S A using as coating antigen either the octapeptide E E N V E H D A conjugated to B S A or the free peptide Y ( N V E E ) 4 C at 5 Ixg ml - l peptide concentration. Control plates were coated with glutaraldehyde treated B S A or poly-L-asparagine (Mol. wt. 5000-15000, Sigma). The coated plates were incubated for 4 h at r o o m temperature with various dilutions of monoclonal antibodies. In inhibition experiments, a mixture of antibodies at 0.4 Ixg m V l and different concentrations of peptides was instead added to the plates at this step. B o u n d antibodies were detected by measuring the absorbance at 405 nm after incubations at r o o m t e m p e r a t u r e first for 4 h with alkaline phosphatase conjugated rabbit anti-mouse I g G antibodies (Sigma) and then for 30 min with p-nitrophenyl phosphate. Results

Several mouse hybridomas were established, which produce antibodies against the synthetic octapeptide E E N V E H D A , corresponding to a repeated sequence in the P. falciparum antigen Pf155 [3]. The h y b r i d o m a 1F1 which produced antibodies at high rate was studied in detail. The

antibodies were of IgG1 subclass and were monoclonal as ascertained by isoelectric focusing. The specificity of the monoclonal antibody 1F1 was investigated by p e p t i d e - E L I S A using a conjugate of the synthetic octapeptide E E N V E H D A and BSA as coating antigen (Fig. 1A). The antibody showed no reaction in the controls with either glutaraldehyde-treated BSA or poly-N as coating antigen (not shown). Various synthetic peptides were tested for their capacity to inhibit the reactivity of the antibody in E L I S A . Neither of the free octapeptides E E N V E H D A or (EENV)2 inhibited the antibody binding while the BSA conjugated octapeptides did so, the E E N V E H D A BSA conjugate apparently being a more efficient inhibitor than the (EENV)2-BSA conjugate (Table I). However, the peptide (NVEE)2C in its free form inhibited antibody binding to E E N V E H D A - B S A (Table II). In contrast, dimers of the octapeptide E E N V E H D A and tetramers of the tetrapeptides E E N V and N V E E in their free form all inhibited antibody binding in to E E N V E H D A - B S A (Fig. 1B, Table II), while a dimer of the peptide D D E H V E E P T V A , corresponding to a repeat subunit in the more N-terminally located repeat region of Pf155 was without effect both in its free form (Table II) or when conjugated to BSA (Table I). Similarly, the peptides Y ( S V T E E I A E E D K ) 2 "and (PEEL/vVEEVI/v) 2 G K , corresponding to tandem repeat subunits of the P. falciparurn antigens 332 and 11.1, respectively [17], also did not inhibit the reaction of 1F1 with E E N V E H D A - B S A (Table II). A similar reactivity of antibody 1F1 in peptide-

TABLE I Inhibition with various synthetic peptide-BSA conjugates of the monoclonal antibody 1F1 in peptide ELISA and EMIF Peptidea

ELISAb

EMIFc

EENVEHDA-BSA 16_+2 60_+28 (EENV)2-BSA 130_+30 140-+85 (EENVEHDA)2-BSA 65± 15 200_+0 K(DDEHVEEPTVA)2-BSA Nd N Glutaraldehyde-BSA N N "A similar degree of coupling of the different peptides to BSA has been assumed (see Materials and Methods). bAmounts of peptide-BSA (ixg ml 1) needed for 50% inhibition of ELISA with EENVEHDA-BSA as coating antigen. The values represent the means -+ SD from 2 or 3 experiments in which the samples were tested in duplicate. CAmounts of peptide-BSA (~xgml ~) needed for complete inhibition of immunofluorescence. The values represent the means -+ SD from 2 or 3 independent determinations. dN= no inhibition at the highest concentration used (400 Ixg ml-l).

22

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0.1

4

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Ol

1 lO Peptide (}aM)

0,1

1 10 Peptide ()JM)

IgG (ja9 / rnl)

1.0

1.0

'4405

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0.5

O.~ _

0,t

0.1

o.ol IgG ( p g / m l )

lOO

Fig. 1, Analysis of monoclonal antibody 1F1 in EL1SA using as coating antigen E E N V E H D A - B S A (A,B) or Y(NVEE)4C (C,D). (A,C) Titration curves of the 1F1 antibody. (B,D) Inhibition of the 1F1 antibody reactivity with synthetic peptides, m - - g , K(EENVEHDA)2: • • , K(EENV)4; • •, K(DDEHVEEPTVA)2.

T A B L E II Inhibition with various free synthetic peptides of the monoclonal antibody 1FI in peptide ELISAs and EMIF Peptide

ELISA"

EMIF'

E E N V E H D A - B S A (NVEE)4C EENVEHDA (EENV)2 (NVEE)2C K(EENVEHDA)2 K(EENV)4 Y(NVEE)4C K(DDEHVEEPTVA)2 Y(SVTEEIAEEDK)2 PEEL/vVEEV1/v)2GK

N~ N 12 +- 1 1 -+ 1 3+_1 3+_2 N N N

N N 11 + 1 2~ 1 5-_ 1 2-+2 N N N

N N 2 4 +- 15 2+2 9-+8 7+-8 N N N

~The micromo/ar concentrations of peptide needed for 50% inhibition of the ELISA, The values represent m e a n s + SD from 2 or 3 experiments in which the samples were tested in duplicate. bThe micromolar concentration of peptide needed for complete inhibition of immunofluorescence. The values represent m e a n s + SD from 2 or 3 i n d e p e n d e n t determinations. ON= no inhibition at the highest concentration used (l//0 IxM).

23

Fig. 2. Indirect immunofluorescence with monoclonal antibody 1Ft on monolayers of glutaraldehyde-fixed and air-dried P. faicip6nun infected erythrocytes. EMIF (A) or monolayers of air-dried and methanol-fixed P. falciparum infected erythrocytes (‘B). R, ring stage; S. Schizont stage.

24

ELISA was seen when the free peptide Y(NVEE)4C was used as coating antigen. In these experiments inhibition of antibody binding with various peptides gave the same pattern as when E E N V E H D A - B S A was the coating antigen (Fig. 1C and D, Table II). Attempts to do similar experiments using the dimer of E E N V E H D A as coating antigen were unsuccessful, probably because of poor adsorbtion of this peptide to the plates. Although made against a synthetic peptide, the IF1 antibody also reacted with P. falciparum infected erythrocytes in immunofluorescence. In EMIF on glutaraldehyde-fixed and air-dried monolayers of infected erythrocytes it gave an immunofluorescence pattern similar to that seen with antibodies to Pf155, i.e. a bright staining of the surface of erythrocytes infected with early stages of the parasite (Fig. 2A). The lowest concentration of antibody giving this type of immunofluorescence was 0.2 ~g ml 1. A similar pattern, but weaker staining, was seen on erythrocytes infected with later stages of the parasite, including late trophozoites, schizonts as well as gametocytes. The same pattern of immunofluorescence was obtained with P. falciparum parasites of different geographic origins, including parasites from Tanzania (F32), Thailand (Thai 1), Brazil (7G8) and Honduras (HB3) [24]. No surface staining of infected erythrocytes was seen unless fixation was undertaken before antibody reaction [8]. Immunofluorescence with monolayers of methanol-fixed infected erythrocytes showed that the 1F1 antibody reacted with all asexual stages of the intraerythrocytic parasite,s as well as with free merozoites (Fig. 1B). Gametocytes were, however, not stained with this antibody in this assay. The specificity of the antibody binding detected by the surface immunofluorescence (EMIF) was studied in inhibition experiments using various synthetic peptides (Table I and II). Similarly to what was found in the ELISA experiments, neither of the free octapeptides E E N V E H D A or (EENV)2 inhibited immunofluorescence even at the highest concentration tested (100 p,M) (Table II), while these peptides conjugated to BSA were efficient inhibitors (Table I). However, dimers of these peptides (16 amino acids) inhibited immu-

nofluorescence in their free form with similar efficiency (Table II). A dimer of the peptide D D E H V E E P T V A , corresponding to a repeat subunit in the second repeat region of Pf155 [4,10] did not inhibit antibody binding (Table 11) and neither did this peptide conjugated to BSA (Table I). Different parasite fractions obtained from P. falciparum cultures were used to inhibit erythrocyte surface immunofluorescence in EMIF. A1-

A

B

C

D

200

155-135 120 116

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Fig. 3. Immunoblotting of P. falciparurn polypeptides probed with 20 ~g ml ~ monoclonal antibody 1F1 (A-C) or a human P. falciparum immune serum at dilution h l 0 0 0 (D). Lanes A and D: merozoite enriched fraction; lane B: spent culture medium from P. falciparum cultures; lane C: culturc supernatant material eluted from a glycophorin-aminoethylBioGel resin.

25 though containing Pf155 in relatively large amounts, no inhibition was obtained with merozoite extracts, schizont extracts or spent culture medium. H o w e v e r , a partial inhibition was obtained using culture supernatant components eluted from a glycophorin-aminoethyl-BioGel resin with 3 M KSCN, which gives an antigen preparation enriched in Pf155 [21]. The reactivity of the 1F1 antibody with Pf155 was confirmed in immunoblotting experiments. The 155 k D a polypeptide was the m a j o r component stained in merozoite extracts and P. falciparum culture supernatants (Fig. 3 lanes A,B). H o w e v e r , when culture supernatants were fractionated by adsorption to a glycophorin-aminoethyl-BioGel resin [25], the antibody also stained two parasite derived polypeptides of 135 and 120 kDa in the eluates, the 135 k D a polypeptide being the strongest stained band in some preparations (Fig. 3, lane C) [21]. Pf155 was the m a j o r polypeptide reactive with antibody 1F1 in all stages of asexual erythrocytic parasite development as judged from immunoblotting of lysates of infected erythrocytes taken from synchronized cultures at different phases of development (not shown). The 1F1 antibody also reacted with native P. falciparum proteins, probably Pf155, as sug-

gested by its capacity to inhibit merozoite invasion in P. falciparum in vitro cultures. Invasion inhibition to 50% was obtained at an immunoglobulin concentration of about 45 I~g ml -t when using antibody precipitated with (NHa)2SO 4 from 1F1 culture supernatants (Fig. 4A). Similar resuits were obtained with both F32 and 7G8 parasites. No inhibition was seen with an unrelated control monoclonal antibody of the same subclass. The invasion inhibitory effect of the 1F1 antibody was specific as it could be reversed by the synthetic peptides K ( E E N V E H D A ) 2 and K(EENV)4 while the peptide K ( D D E H V E E P TVA)2 was without effect (Fig. 4B). Discussion We report herein on the reactivity and specificity of a monoclonal mouse antibody to a synthetic octapeptic[e, corresponding to a repeated sequence in the P. falciparum antigen Pf155. The antibody was selected by its reactivity with the surface of glutaraldehyde-fixed and air-dried P. falciparurn infected erythrocytes as detected by immunofluorescence (EMIF), an assay detecting mainly Pf155 [8]. The reactivity of the antibody with Pf155 was confirmed by immunoblotting. Our results indicate that the antibody reacts well 100

100 8CI

80

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40

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20

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20 40 IgG (pg/ml)

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Fig. 4. (A) Analysis of monoclonal antibody 1F1 (o e) in in vitro invasion inhibition of P. falciparum. An unrelated monoclonal antibody (IgG1) was used as control (E3 El). Percent inhibition after 20 h was determined as described by WShlin et al. [22]. The mean parasitemia in the controls was 0.4 _+ 0.1% at time 0 and 1.3 -+ 0.3% after 20 h. Vertical bars indicate standard errors of mean from 3 experiments. (B) Reversion of monoclonal antibody 1F1 invasion inhibition of P. falciparum by synthetic peptides. • m, K(EENVEHDA)2; • • , K(EENV)4; o o, K(DDEHVEEPTVA)2. The mean parasitemia in controls was 0.6 -+ 0.1% at time 0 and 1.8 -+ 0.2% after 20 h. Vertical bars indicate standard deviation of mean from quadruplicate tests.

26 with denatured Pf155, in the way the antigen is presented in the immunofluorescence or immunoblotting assays. Although there is a complex pattern of antigenic cross-reactions between distinct P. falciparum antigens involving epitopes within the C-terminal repeat region of Pf155 [12,17], the monoclonal antibody 1F1 reacts only with Pf155 and its proteolytic break-down products as detected by immunoblotting [17]. Thus, Pf155 is the probable target antigen also in the invasion inhibition mediated by antibody 1F1 and this suggests reactivity of the antibody with native Pf155 antigen as well. The monoclonal antibody 1F1 was obtained after immunization with the octapeptide EENV E H D A coupled to K L H . Antibody binding to this octapeptide coupled to BSA was inhibited by the homologous E E N V E H D A - B S A and, less efficiently, by ( E E N V ) : - B S A . In contrast, free E E N V E H D A or (EENV)2 could not inhibit the binding of the antibody to either octapeptide-BSA in E L I S A or Pf155 in E M I F whereas a dimer of the octapeptide as well as a tetramer of the peptide E E N V inhibited this antibody binding efficiently. The inhibition obtained with the short peptide (NVEE)2 C (Table II) probably reflected dimerisation mediated by its C-terminal cysteine. Very similar results were obtained when a free peptide (NVEE)4C without carrier protein was used for coating the plates. Taken together the results indicate that the antibody reacts preferentially with the E E N V sequence of the octapeptide as presented when the peptide is coupled Nterminally to a carrier protein or when it is a sequence in a longer peptide. It may also be noted that no inhibition was obtained with any one of the three related peptides containing similar doublets of glutamic acid as well as valine (or isoleucine) (Table II). However, these peptides did not contain asparagine, suggesting that this amino acid may be an important constituent of the epitope seen by antibody 1F1. For a more exact delineation of the epitope recognized by this monoclonal antibody, an epitope mapping will have to be performed using peptides with overlapping sequences [26]. Anders et al. recently described a series of monoclonal antibodies against recombinant fusion proteins containing Pf155/RESA repeats,

which all reacted with a tetramer of E E N V but some of them also with the octapeptide EENV E H D A as analysed by E L I S A [12]. A similar pattern of specificity was reported by Masuda et al. for a monoclonal antibody to P. falciparum gametocytes and reactive with a 150 kDa polypeptide present in gametocytes as well as in all stages of the asexual erythrocytic parasite [27]. We have previously shown that at least three different antigenic epitopes are present in the C-terminal repeat region of Pf155 as defined by human antibodies formed against the antigen after exposure to P. falciparum [9,14,28]. One population of antibodies was specific for the peptide (EENV)2 and one was specific for the peptide E E N V E H D A while a third population reacted with both peptides. The reactivity of the monoclonal antibody 1F1 is most closely related to the specificity of the cross-reacting antibody population. In addition to reacting with Pf155 in immunoblotting, the 1F1 antibody also reacted with polypeptides of 135 and 120 kDa from P, falciparum culture supernatants. These two polypeptides as well as Pf155 can be enriched on a glycophorinaminoethyl-BioGel resin [21,29], giving an eluate containing the different polypeptides in varying relative amounts in different preparations. Preliminary results with proteolytic digestions indicate that these polypeptides represent products of proteolytic break-down of Pf155 (Ruangjirachuporn, unpublished data). It cannot, however, be excluded that a part of the 135 kDa polypeptide as detected by immunoblotting could correspond to the glycophorin binding protein of 130 kDa described by Perkins [25]. This protein contains the sequence EEN in a couple of sites [30], which could be expected to constitute parts of cross-reactive epitopes with the C-terminal repeat region of Pf155. It should be pointed out that we have confirmed the results of Van Schravendijk et al. [31] that the binding of Pf155 and the 135 kDa and 120 kDa polypeptides to the glycophorin substituted resin is not entirely dependent on glycophorin, as the same pattern of binding polypeptides, although quantitatively less, is obtained with aminoethyl-BioGel alone or with a fetuin substituted resin (unpublished results). The IF1 antibody adds to the series of differ-

27

ent antibodies reactive with the C-terminal repeats of Pf155 displaying a high capacity to inhibit P. falciparum invasion in vitro. These other antibodies include rabbit antibodies to the synthetic peptides EENVEHDA and (EENV)2 [9,11,14], rabbit antibodies to the product of a gene construct, coding for four octapeptide repeats [13] and human antibodies affinity purified on synthetic peptides [9,14]. As the reinvasion inhibition assay is thought to reflect the protective capacity of antibodies, the monoclonal 1F1 antibody defines an epitope of great interest with regard to vaccine development. This antibody should constitute an important tool in the dissec-

tion of the structure and function of the C-terminal repeat region and of its role in the merozoite invasion process.

Acknowledgements

This work was supported by grants from the UNDP/World Bank/World Health Organization, Special Programme for Research and Training in Tropical Diseases, the Swedish Medical Research Council, the Rockefeller Foundation Great Neglected Diseases Network and the National Board for Laboratory Animals.

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