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Molecular and Biochemical Parasitology, 46 (1991) 89-96 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100042P MOLBIO 01511

Definition of the epitope recognized by the Plasmodiumfalciparum-reactive human monoclonal antibody 33G2 Niklas Ahlborg, Klavs Berzins and Peter Perlmann Department of lmmunology, Stockholm University, Stockholm, Sweden (Received 24 September 1990; accepted 19 November 1990)

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pfl 1.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1-8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pfl 1.1 repeats and overlapping heptapeptides corresponding to Pf155fRESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine. Key words: Plasmodiumfalciparum; Monoclonal antibody; Epitope mapping; PEPSCAN

Introduction

One strategy for the selection of antigenic sequences to be included in a subunit vaccine against P.falciparum malaria is to define the epitopes seen by antibodies which have the capacity to interfere with the parasite' s life cycle. Properly presented for the immune system, these epitopes are expected to induce protective antibody responses. With regard to the asexual blood stages of P.falciparum, attention has been given to antibodies which inhibit merozoite reinvasion in vitro [1]. However, antibodies which inhibit cytoadherence of infected Correspondence address: Niklas Ahlborg, Dept. of Immunology, Stockholm University, S- 106 91 Stockholm, Sweden. Abbreviations: mAb, monoclonal antibody; EMIF, erythrocyte membrane immunofluorescence; ELISA, enzyme linked immunosorbent assay; PBS, phosphate-buffered saline; MAP, multiple antigen peptide.

erythrocytes to endothelial cells [2,3] or inhibit rosette formation between uninfected and infected erythrocytes [4] should also be of interest. Such antibodies are expected to interfere in vivo with the sequestration of late-stage infected erythrocytes [2,4]. The human monoclonal antibody (mAb) 33G2, obtained from an Epstein-Barr virus transformed B cell originating from a Liberian P. falciparum-immune donor [5] has several interesting biological properties. It inhibits P.falciparum merozoite reinvasion in in vitro cultures [5] as well as cytoadherence of infected erythrocytes to melanoma cells in vitro [3]. Thus, the mAb has the capacity to interfere with the parasite's erythrocytic life cycle at two potential target sites for protective antibodies in vivo [ 1], making the epitope recognized by the mAb of great interest with regard to vaccine development. The mAb 33G2 was initially selected because of

90 its reactivity with PfI55/RESA as detected by erythrocyte membrane immunofluorescence (EMIF) and immunoblotting [5]; however, subsequent analyses with recombinant fusion proteins and synthetic peptides revealed that the antibody reacted with a family of cross-reacting P. falciparum blood stage antigens, including Pf155/RESA, Pfll.1 and Ag332 [6-8]. A common feature of these antigens is their content of several tandemly repeated amino acid sequences containing regularly spaced pairs of glutamic acid [6,9,10]. These dimers of glutamic acid were suggested to be the structures responsible for the antigenic cross-reactions between the three antigens [6-8]. Inhibition with synthetic peptides of the binding ofmAb 33G2 binding in EMIF showed that peptides corresponding to Ag332 repeat sequences were the most efficient inhibitors, suggesting that Ag332 was the original target for the antibody [8]. In order to define in more detail the epitope specificity of mAb 33G2, we employed the multiple peptide synthesis technique (PEPSCAN) developed by Geysen et al. [ 11 ]. This is a method for simultaneous synthesis of peptides on polyethylene rods where the peptides, still on the rods, can be used directly for analysis of antibody binding by ELISA. By this method it is possible to identify B cell epitopes and to determine their fine specificities. As a basis for our analysis we used the amino acid sequence 1-19 of Ag332 [6], corresponding to the sequence of the most efficient inhibitory peptide for mAb 33G2 binding studied previously [8]. The fine specificity of the antibody was delineated by amino acid omissions and substitutions in the Ag332 pentapeptide showing optimal binding of the antibody. Materials and Methods

Monoclonalantibody.

The mAb 33G2 (IgM) was obtained from an Epstein-Barr virus transformed B cell originating from a Liberian P. falciparum-immune donor [5].

Peptide synthesis.

Peptides used in all assays were synthesized as described by Geysen et al. [ 11 ] on polyethylene rods on which polymers of polyacrylic acid had been formed by irradiation. Polyethylene rods and Fmoc L-amino acids preformed

as active esters (Cambridge Research Biochemicals, U.K.) were used for synthesis according to instructions of the manufacturer. The N-terminals of all peptides were acetylated. Peptides synthesized were: for epitope mapping, all possible overlapping heptapeptides, hexapeptides, pentapeptides and tetrapeptides corresponding to residues 1-19 (ESVTEEIAEEDKSVIEEAV) of the known amino acid sequence of Ag332 [6]; for omission analog analysis, 8 omission analogs corresponding to amino acid residues 1-8 of Ag332. These peptides differed from the original sequence only by omission of one amino acid residue in each of them; for replacement set analysis (single amino acid substitution assay), octapeptides corresponding to amino acid residues 1-8 of Ag332. Twenty analogs were made for each residue, one containing the original amino acid, and 19 analogs with substitutions. Amino acids used were the 20 most common L-amino acids. In all, 8 parent peptides and 152 single amino acid substitution analogs were made; for peptide analog analysis, pentapeptides corresponding to amino acid residues 3-7 of the Ag332 sequence and analogs in which one or several amino acids had been replaced. Selection of modifications was based on the results from the single amino acid substitution assay; for analysis with Pf155/RESA peptides, overlapping heptapeptides corresponding to amino acid repeats from antigen Pf155/RESA were synthesized. All possible overlapping heptapeptides from the 3'-repeat region, involving the subunits EENVEHDA and EENV, and from the 5'-repeat region, involving the subunits D DEHVEEPTVA and EEHVEEPTVA.

ELISA. Antibody reactivities with peptides were measured by ELISA as described by Geysen et al. [11]. Peptides used as antigens were directly synthesized and tested on polyethylene rods. Supernatant with monoclonal antibody, containing approximately 10/xg IgM m1-1, was diluted 1:20 in the single amino acid substitution assay and 1:100 in all other assays. Peptide rods were washed in PBS with 0.05% Tween 20 between all steps in ELISA. Bound antibodies were detected with a rabbit antihuman IgM alkaline phosphatase conjugate (Sigma, St. Louis, MO) using p-nitrophenyl phosphate, disodium salt (Sigma) as substrate.

91

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Fig. 1. Antibody reactivity, measured in ELISA, with all possible overlapping heptapeptides (A), hexapeptides (B), pentapeptides (C) and tetrapeptides (D), of a sequence correspondingto residue 1-19 of the Ag332 sequence.At the horizontal axis the absorbance at 405 nm is shown.

Results

Epitope mapping and omission analogs. The human mAb 33G2 was tested for reactivity against all possible overlapping heptapeptides, hexapeptides, pentapeptides and tetrapeptides corresponding to amino acid residues 1-19 (ESVTEEIAEEDKSVIEEAV) of the known amino acid sequence of Ag332. The antibody showed reactivity with 4 heptapeptides corresponding to amino acids 1-7 (ESVTEEI), 2-8 (SVTEEIA), 3-9 (VTEEIAE) and 4 - 1 0 (TEEIAEE) (Fig. 1A). When tested against hexapeptides the antibody recognized sequences corresponding to amino acids 2-7 (SVTEEI), 3-8 (VTEEIA) and 4 - 9 (TEEIAE) (Fig. 1B). Reactivity to pentapeptides was restricted to one peptide, corresponding to amino acids 3-7 (VTEEI) (Fig. 1C), while no reactivity was seen

0,6-

o,4

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S

V

T

E

E

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A

Fig. 2. Antibody reactivity, measured in ELISA, to peptide analogs of the sequence ESVTEEIA where one amino acid has been omitted in each analog. Indicated amino acids were omitted. At the vertical axis the absorbance at 405 nm is shown.

92

lli]llllllllllllllllll IIIIIIIIplIIlll,lllflll IIjlIIJIII II. . . ......... . I. . . . . . II. .. ..I.IlfllllJIIIIIIIII A

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Fig. 3. Replacement set analysis of 33G2 reactivity to the octapeptide, ESVTEEIA, corresponding to amino acid residue 1-8 of the known sequence of Ag332. Each residue in the parent octapeptide was substituted by all 19 different ~-amino acids. In order, each set of 20 bars corresponds to amino acids: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, For each set of bars, the parent peptide amino acid is indicated below. The vertical axis shows % antibody binding, measured in ELISA, compared to the parent peptides.

with any of the tetrapeptides (Fig. ID). When tested against 8 heptapeptides corresponding to the sequence ESVTEEIA (amino acids 1-8), where one amino acid residue had been omitted in each peptide, the antibody could not recognize peptides where either V, T, E, E or I had been excluded (Fig. 2).

Replacement analysis. The mAb 33G2 was analyzed for reactivity against octapeptides based on the sequence ESVTEEIA, where single amino acid substitutions replaced each residue. Every residue in the parent peptide ESVTEEIA, which corresponds to residues 1-8 of the known sequence of Ag332, was replaced by the most common 20 amino acids (Fig. 3). The first (E), second (S) and last amino acid residue (A) were shown to be replaceable by any other amino acid without abolishing the ability of the monoclonal 33G2 to recognize the peptides. A linear 5-amino acid sequence (VTEEI) was shown to consist of amino acids which were either essential or replaceable mainly by amino acids of similar chemical character. Substitution of valine (V) by C, F, I, L, P, T, W and Y, and threonine (T) by A, C, I, L, P, S and V, gave ELISA absorbance values of 20% or more of those obtained with the parent octapeptides. The 2 glutamic acids (E) contained within the epitope were the most essential residues. The first glutamic acid (E) was totally irreplaceable, while the second glutamic acid (E) could be replaced by aspartic acid (D), a highly conserved replacement, and to some extent by cysteine (C). The last amino acid within the epitope, isoleucine (I), could be replaced by leucine (L) and valine (V), two relatively conserved replacements. It could also

be replaced to some degree by the positively charged amino acid histidine (H).

Peptide analogs. Based on the results in the replacement set analysis, pentapeptides corresponding to residues 3-7 of Ag332 were constructed, in which one or several original amino acids had been replaced simultaneously. The results from this assay showed that it was possible to replace several amino acids within the epitope simultaneously without losing antibody reactivity (Fig. 4). The antibody recognized most of the peptides where the modifications were consistent with the results from the replacement set analysis; ITEEI, VAEEI, IADEI, IADEV, VVEEV and LVEEV. A decrease in antibody reactivity could be seen for some pep-

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Fig. 4. Antibody reactivity, measured in ELISA, to 33G2 epitope analogs. The sequence VTEEI corresponds to residue 3-7 of the Ag332 sequence. The other pentapeptides are sequences where either one or several amino acids have been exchanged. At the vertical axis the absorbance at 405 nm is shown.

93

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0,4

0,6

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1

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Fig. 5. Antibodyreactivity,measuredin ELISA, to overlappingheptapeptides of Pf155/RESA8-met and 4-merrepeats (A) and 11mer repeats (B). At the vertical axis the absorbanceat 405 nm is shown.

tides where several amino acids had been replaced. The antibody did not react with the pentapeptide, YLDEV, indicating that not all of the reactive single amino acid substitutions can be performed simultaneously and still result in reactive peptides.

Reactivity withPf155/RESApeptides. Reactivity of the mAb with overlapping heptapeptides corresponding to Pf155/RESA amino acid repeats was mainly directed to 3 consecutive overlapping peptides from the 5'-repeat region (Fig. 5B). The antibody recognized the peptides PTVAEEH, TVAEE HV and VAEEHVE, which all share the sequence VAEEH. The antibody also reacted, although weakly, with 2 heptapeptides, AEENVEH and VEENVEE, corresponding to sequences in the 3'repeat region ofPf155/RESA (Fig. 5A). Discussion We have previously shown that the human mAb 33G2 reacts with repeat sequences of the three P. falciparum blood stage antigens Pf155/RESA, Pfll.1 and Ag332 [6-8] and that the epitope contained within Ag332 repeats appears to present the optimal structure for mAb binding [8]. Here, we have mapped the mAb 33G2 epitope to amino acids at positions 3-7 (VTEEI) in the Ag332 sequence [6]. The same sequence also appears in the third repeat unit of the antigen comprising amino acids at positions 25-29. Although there are some differ-

ences in the surrounding amino acids, both the 3-7 and 25-29 sequences were able to bind the mAb. These sites were the only ones in the known 101amino acid-long Ag332 sequence [6] which could bind the antibody (unpublished observations). However, the parasite antigen Ag332 appears to be a very large molecule consisting of degenerated repeats (D. Mattei, personal communication) and it is likely that additional mAb 33G2 binding sequences are present. As predicted previously on the basis of the crossreactivity displayed by the antibody [6-8], a dimer of glutamic acid is essential in the epitope for mAb 33G2. While one of these residues could not be replaced by any other amino acid, the second could be replaced by aspartic acid (D) only. The other 3 amino acids within the epitope were replaceable by selected amino acids with similar chemical proper-

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Fig. 6. Delineation of oligopeptide sequencescorresponding to sequencesin the antigens Ag332, Pfl 1.1 and Pf155/RESA previously shown to be reactive with mAb 33G2. Sequences contained within the box correspond to either the mAb 33G2 epitope sequence in Ag332 (VTEEI) or analogs to this sequence where amino acids have been replaced without loss of antibodyreactivity.

94 ties. Several of these substitutions could, furthermore, be performed simultaneously without losing antibody reactivity. Thus, although somewhat weaker, mAb 33G2 also reacted with the two analogs VVEEV and LVEEV. These results explain the basis for the cross-reactivity of the antibody with the antigen Pfl 1.1 [6-8], which contains nonamer repeats with the consensus sequence PEEL/vVEEVI/v [ 10], in which the 2 pentamer sequences mentioned are found. Delineation of the oligopeptides corresponding to sequences in the antigens Ag332, Pfl 1.1 and Pf155/RESA previously shown to be reactive with mAb 33G2 [8] clearly indicates which amino acids are responsible for the antigenic crossreactions (Fig. 6). Our analysis of mAb 33G2 reactivity with overlapping heptapeptides of the sequences from the 3'and 5'-repeat regions of Pf155/RESA [9] suggests that the strong reactivity of the mAb with Pf155/RESA in immunoblotting is mainly due to its reaction with a 5'-repeat sequence. The mAb reacted with 3 adjacent heptapeptides from the 5'-repeat region, all containing the sequence VAEEH. This is consistent with the single amino acid substitution assay with the peptide VTEEI, where T can be replaced by A and I can be replaced by H without any major loss of antibody binding. Two peptides corresponding to sequences in the 3'-repeat region (AEENVEH and VEENVEE) showed a weak binding of the mAb. However, as these sequences are repeated many times in the antigen, multiple antibody binding may be possible and contribute to the strong signal seen in immunoblotting. The reactivity ofmAb 33G2 with the blood stages of P.falciparum is remarkable, as the antibody possesses the capacity to inhibit both merozoite reinvasion into erythrocytes [5] as well as adherence of infected erythrocytes to endothelial cells or melanoma cells in vitro [3]. Thus, the antibody may interfere with the erythrocytic life cycle of the parasite at two different steps essential for parasite development and survival in vivo I1 ]. Due to the cross-reaction of the mAb with several differentP.falciparum antigens [6-8] it is difficult to assess the actual target antigens for the antibody in the 2 different types of inhibitions. The mAb seems to inhibit merozoite reinvasion of all strains or clones of P. falciparum [5,12], probably having Pf155/RESA as well as other cross-reacting antigens as targets (B. W~i hlin,

personal communication). The antibody inhibits the cytoadherence of different P.falciparum strains or isolates to different degrees (R. Udomsangpetch, personal communication), indicating either quantitative differences in the expression of the corresponding target antigen or the presence of an antigenically diverse target molecule. The latter type of target antigen would be in accordance with the antigenically diverse antigen PfEMP 1 being the P.falciparum cytoadherence molecule [ 12]. The relevance of the 33G2 epitope for the immune response in P.falciparum malaria is indicated by the finding that 30-40% of the sera from African blood donors (Gambian and Liberian) react with synthetic peptides corresponding to Ag332 sequences which contain the epitope seen by mAb 33G2 (J. Iqbal, personal communication). Antibodies isolated from Ag332 reactive blood donors by affinity purification on Ag332 related peptides display an epitope specificity resembling that of mAb 33G2 and inhibit merozoite reinvasion in vitro (N. Ahlborg, manuscript in preparation). Furthermore, immunization of rabbits with synthetic peptides corresponding to Ag332 sequences induces mainly antibodies recognizing the same epitope as mAb 33G2. These antibodies also inhibit merozoite reinvasion in vitro (unpublished observations). Properly presented multimers of the mAb 33G2 epitope, either as linear polymers in a recombinant fusion protein [13] or in a multiple antige n peptide system (MAP) [ 14], are expected to give rise to antibodies with specificities and parasite reactivities similar to those of the mAb and should, thus, be suitable components to be included in a subunit vaccine against P.falciparum malaria.

Acknowledgements This work was supported by grants from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Swedish Medical Research Council. The support from KabiVitrum AB and SmithKline Beecham Biologicals is gratefully acknowledged. We thank Dr. Rachanee Udomsangpetch for culture supernatant containing mAb 33G2.

95

References 1 Anders, R.F. (1985) Candidate antigens for an asexual blood-stage vaccine. Parasitol. Today 1,152-155. 2 Howard, R.J. (1988) Malarial proteins at the membrane of Plasmodium falciparum-infected erythrocytes and their involvement in cytoadherence to endothelial cells. Prog. Allergy 41,98-147. 3 Udomsangpetch, R., Aikawa, M., Berzins, K., Wahlgren, M. and Perlmann, P. (1989) Cytoadherence of knobless Plasmodiumfalciparum-infected erythrocytes and its inhibition by a human monoclonal antibody. Nature 338, 763765. 4 Carlson, J., Holmquist, G., Taylor, D.W., Perlmarm, P. and Wahlgren, M. (1990) Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodiumfalciparurn erythrocyte rosettes. Proc. Natl. Acad. Sci. USA 87,2511-2515. 5 Udomsangpetch, R., Lundgren, K., Berzins, K., W~hlin, B., Perlmann, H., Troye-Blomberg, M., Carlsson, J., Wahlgren, M., Per/mann, P. and BjOrkrnan, A. (1986) Human monoclonal antibodies to Pf155, a major antigen of malaria parasite Plasmodium falciparurn. Science 231, 57-59. 6 Mattei, D., Berzins, K., Wahlgren, M., Udomsangpetch, R., Perlmann, P., Griesser, H.W., Scherf, A., M~iller-Hill, B., Bonnefoy, S., Guillotte, M., Langsley, G., Pereira da Silva, L. and Mercereau-Puijalon, O. (1989) Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens. Parasite Immunol. 11, 15-30. 7 Mercereau-Puijalon, O., Langsley, G., Mattei, D., Guillotte, M., Blisnick, T., Berzins, K., Griesser, H.W., Scherf, A., Miiller-Hill, B. and Pereira da Silva, L. (1987) Presence of cross-reacting determinants on several blood-stage anti-

gens ofPlasmodiumfalciparum. UCLA Symp. Mol. Cell. Biol. 42, 343-354. 8 Udomsangpetch, R., Carlsson, J., W~lhlin, B., Holmquist, G., Ozaki, L.S., Scberf, A., Mattei, D., Mercereau-PuijaIon, O., Uni, S., Aikawa, M., Berzins, K. and Perlmann, P. (1989) Reactivity of the human rnonoclonal antibody 33G2 with repeated sequences of three distinct Plasmodiumfalciparum antigens. J. Immunol. 142, 3620-3626. 9 Favaloro, J.M., Coppel, R.L., Corcoran, L.M., Foote, S.J., Brown, G.V., Anders, R.F. and Kemp, D.J. (1986) Structure of the RESA gene of Plasmodiumfalciparum. Nucleic Acids Res. 14, 8265-8277. I0 Scherf, A., Hilbich, C., Sieg, K., Mattei, D., MercereauPuijalon, O. and MUller-Hill, B. (1988) The 11-1 gene of Plasmodiumfalciparum codes for distinct fast evolving repeats. EMBO J. 7, 1129-1137. 11 Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G. and Schoofs, P.G. (1987) Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102, 259-274. 12 Perlmann, H.K., Berzins, K., W~dalin,B., Udomsangpetch, R., Ruangjirachuporn, W., Wahlgren, M. and Perlmann, P.H. (1987) Absence of antigenic diversity in Pf155, a major parasite antigen in membranes of erythrocytes infected with Plasmodiumfalciparum. J. Clin. Microbiol. 25, 2347-2354. 13 St~hl, S., Sj61ander, A., Hansson, M., Nygren, P.-~. and Uhl6n, M. (1990) A general strategy for polymerization, assembly and expression of epitope-carrying peptides applied to the Plasmodiumfalciparum antigen Pf155/RESA. Gene 89, 187-193. 14 Tam, J.P. (1988) Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 85, 54095413.

Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2.

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitr...
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