Plasmodium Antibody


An Invasion Inhibitory Human Monoclonal Is Directed against a Malarial Glycolipid Antigen



*Stockholm University, Department of Immunology, S-106 91 Stockholm, Sweden, and fKarolinska Institutet, Department of Immunology, S-104 01 Stockholm, Sweden SJ~BERG, K., HOSEIN, Z., WAHLIN, B., CARLSSON, J., WAHLGREN, M., BLOMBERG, M., BERZINS, K., AND PERLMANN, P. 1991. Plasmodiumfalciparum:


An invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen. Experimental Parasitology 73, 317-325. A Plasmodium falciparum malaria blood stage antigen was detected using a human monoclonal antibody (MAb A52A6) obtained from a clinically immune donor. Immunofluorescence analysis showed that the MAb reacted with the intracellular parasite throughout the asexual blood stage cycle as well as with gametocytes. The MAb also reacted with the surface of erythrocytes containing late stage P. falciparum parasites. The antigen seen by the MAb was species- but not strain- or isolatespecific. At rupture of the infected erythrocytes, antigenic material was deposited on the membrane of uninfected cells surrounding the parasite. At merozoite invasion MAb reactive material was present on the invaginating erythrocyte membrane, indicating an involvement of the antigen in the invasion process. This was also indicated by the high capacity of the MAb to inhibit merozoite invasion in vitro. The antigen appears to be a phosphoglycolipid, sensitive to phospholipase and present in lipid extracts of P. falciparum-infected erythrocytes. 0 1991 Academic Press. Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Plasmodium falciparum; Protozoa, parasitic; Malaria, human; Human monoclonal antibody; Glycolipid antigen; Immunoflourescence; Epstein-Barr Virus (EBV); Monoclonal antibody (MAb); Immunoglobulin M (IgM); Enzyme-linked immunosorbent assay (ELISA); High-performance thin-layer chromatography (HPTLC); Radioimmunoassay (RIA); Ethylenediamine tetraacetic acid (EDTA); Bovine serum albumin (BSA); Phosphate-buffered saline (PBS); Sodium dodecyl sulfate (SDS); Polyacrylamide gel electrophoresis (PAGE); Kilodalton (kDa).

(Lundgren et al. 1983). Furthermore, using lymphocytes from a donor considered clinically immune to P. falciparum malaria, we have established several monoclonal cell lines producing antibodies reactive with different P. falciparum antigens present in the erythrocytic stages of the parasite (Udomsangpetch et al. 1986). One such antibody showing reactivity with a family of cross-reacting antigens, including the vaccine candidate antigen PflSS/RESA, has been described in detail (Udomsangpetch et al. 1986, 1989a; Ahlborg et al.


Knowledge about the molecular mechanisms behind the invasion of malaria parasites into erythrocytes and identification of antigens involved in this process is of importance for the selection of components suitable for inclusion in a vaccine against malaria. Analysis of inhibitory effects of monoclonal antibodies (MAbs) on merozoite invasion into erythrocytes has frequently been used to identify and characterize antigens of interest. We have earlier reported on the production of human MAbs by cell lines obtained by EBV-transformation of B-cells from individuals acutely infected with Plasmodium


We now report on a MAb produced by B-cells derived from the same P. falciparum immune donor which efficiently in317 0014-4894/91$3.00 Copyright All rights

8 1991 by Academic Press, Inc. of reproduction in any form reserved.



hibits merozoite invasion in vitro. The antibody reacts with a P. fulciparum antigen of glycolipid character. MATERIAL


Establishment and selection of a human monoclonal anti-P. falciparum antibody. Blood was obtained from a Liberian donor considered clinically immune to P. falciparum malaria. The lymphocytes were isolated as described by Perlmann et a/. (1976) and the T-cells

were removed by rosetting with sheep erythrocytes (Jonsdottir et al. 1976). The enriched B-cell fraction was collected and transformed with Epstein-Barr virus, propagated, cloned, and recloned as previously described (Lundgren et al. 1983). All clones were initially screened in indirect immunofluorescence using glutaraldehyde-fixed and air-dried monolayers of P. falciparum-infected erythrocytes (EMIF, see below). The concentration of immunoglobulin secreted into the culture medium was determined by ELISA (Engvall and Perlmann 1971). Among several positive clones, one designated A52A6, was selected for further characterization. A52A6 was recloned twice and its monoclonality was assessed by isoelectric focusing (Lundgren et al. 1983). The cell line has secreted 30-80 kg antibody (IgM)/ml/106 cells/72 hr for over 1 year. Purification

of MAb from



Spent medium from cell cultures was collected and the IgM was precipitated with ammonium sulfate and then purified on concanavalin A-Sepharose (Pharmacia, Uppsala, Sweden) as described (Kleine et al. 1979). Indirect immunojluorescence assays. Antibody reactivity with antigens associated with the membrane of P. falciparum-infected erythrocytes was analyzed on glutaraldehyde-fixed and air-dried monolayers of infected erythrocytes (Perlmann et al. 1984). Antibody reactivity with intraerythrocytic parasite antigens was analyzed on unfixed, air-dried monolayers of infected erythrocytes. The antibody to be tested was added in serial dilutions and incubated for 30 min. Bound antibodies were detected by biotinylated goat anti-human immunoglobulins and avidin conjugated with fluorescein isothiocyanate (Vector Laboratories Inc., Burlingame, CA). Fixed slides were counterstained with ethidium bromide to visualize parasites inside the erythrocytes (Perlmann et al. 1984).Slides with monolayers of erythrocytes infected with P. vivax or different simian parasites (P. knowlesi, P. gonderi, P. brazilianum, P. fragile, P. coatney. and P. reichenowi) were kindly supplied by Dr. P. Ngyen-Dinh, (CDC, Atlanta, GA). Enzyme treatment of infected erythrocytes. Monolayers of infected erythrocytes were treated with enzymes either before or after glutaraldehyde fixation

and air drying as described by Perlmann et a/. (1984). Enzymes used were trypsin (bovine pancreas type III, 9440 U/mg; Sigma Chemical Co, St Louis, MO) and pronase (nonspecific protease, Streptomyces griseus type XIV, 5.8 U/mg, Sigma) in concentrations of 100 and 400 pg/ml, neuraminidase (Clostridium petfringens, Sigma) in a concentration of 0.02 U/ml and phospholipase A2 (lecithinase A, bee venom, 1300 U/mg, Sigma) in concentrations up to 200 kg/ml. One drop of the enzyme solution was added to each well for 30 min or 1 hr at 37°C. Parasites. For screening and most of the immunofluorescence studies the F32 strain (Tanzania) of P. falciparum cultured in vitro (Trager and Jensen 1976) was used. Additional strains tested were Thai 1 (Thailand); FVO (Vietnam); IMTM 22, clone7G8 (Brazil); Kl (Thailand); Palo Alto (Uganda); HB3 clone of l/CD3 strain (Honduras); and several isolates from P. falciparum patients. In vitro merozoite invasion inhibition. This assay was performed in micro-culture plates with P. falciparum culture, as described (Wlhlin et al. 1984). The values given are the means from 40,000 erythrocytes screened and the 50% inhibition titers were calculated using at least four different concentrations of MAb A52A6 or a control human IgM MAb directed against a tumor antigen. Preparation of parasite fractions and extraction of lipids. Two different fractions of parasite material were prepared: P. falciparum-infected erythrocytes

containing late parasite stages (trophozoites, schizants) enriched by gradient centrifugation on Percoll (Pharmacia) as described (Troye-Blomherg et al. 1983) and a merozoite-enriched fraction obtained from spent culture medium (Perlmann et al. 1984). Normal human erythrocyte membranes (Dodge et al. 1963)were used as control material. Lipids were extracted from the different fractions according to the method of Folch et al. (1957). Briefly, antigen material (containing 15 mg of protein) was extracted in IO0 ml chloroform/ methanol (2:l) and 20 ml of 0.9% NaCl was added. The phases were separated overnight in a separatory funnel and the chloroform phase was evaporated to dryness in a rotary evaporator. The dried material was dissolved in ethanol for use in dot immunoblotting and ELISA. For the HPTLC method, lipids were extracted according to a method by Svennerholm and Fredman (1980). Packed erythrocytes (normal or P. falciparum infected) or merozoites were homogenized in 20 vol of chloroform:methanol:water (C:M:W) 4:8:3 v/v. After mixing for 1 hr at room temperature, the homogenate was centrifuged (2000 t-pm for 10 min) and the supernatant was collected. The residue was reextracted with 20 vol of C:M:W and mixed overnight at room temperature. After centrifugation, the supematants were pooled and adjusted to C:M:W 4:8:5.6 with dou-

P. fakiparum:


ble-distilled water. The resulting layers were allowed to separate and the upper layer was collected. The lower layer was washed with 1.5 vol of methanol and 1 vol of 0.01 M KCl. The upper layers were pooled, evaporated to dryness, and redissolved in C:M:W 60:30:4.5. After centrifugation, the material was dialyzed exhaustively against distilled water, dried, and redissolved in a small volume of C:M:W 60:30:4.5. The lower phase was dried by rotary evaporation and redissolved in an equivalent volume of C:M:W 60:30:4.5. Dot immunoblotting. Total lipid extracts of infected and uninfected erythrocytes were applied onto nitrocellulose strips (Bio-Rad). The strips were incubated with MAb A52A6 or a control IgM MAb with specificity for a P. falciparum protein antigen (Udomsangpetch et al. 1986) and then with an alkaline phosphatase-conjugated anti-immunoglobulin antibody. Finally, the nitrocellulose was stained for alkaline phosphatase (Berzins et a/. 1983). Lipid ELISA. Lipid preparations from the different parasite fractions were used as antigens in ELISA. The method was modified from a RIA described by Smolarsky (1980). Briefly, 50 pl of the antigen diluted in ethanol were aliquoted in a %-well plate (Dynatech micro-ELISA) to give a concentration of 5 &ml. The ethanol was evaporated in 45°C for 1 hr and the plates were washed three times with 1 mM EDTA in PBS containing 0.3% gelatin. Culture supematants containing the MAb (80 &ml) were serially diluted with 0.3% gelatin-EDTA solution, added to the plates, and incubated in room temperature for 3 hr. After washing, the plates were incubated overnight with alkaline phosphatase-conjugated anti-human immunoglobulin diluted in 1% BSA in PBS, washed again, and then incubated with substrate. The OD,, values were measured in a spectrophotometer. HPTLC. The method was performed as described by Magnani et al. (1982). In brief, samples of lipid extracts (equivalent to 1 mg protein of parasite material) were chromatographed on aluminium-backed high-performance thin-layer chromatography plates (silica gel 60, Merck, Darmstadt, Germany) in a solvent system containing chloroform:methanol:0.25% KC1 (5:4:1 by volume). The dried plates were then soaked for 1 min in 0.1% poly-iso-butyl methacrylate (Polysciences Inc., Warrington, PA) in hexane for 90 sec. After air drying the plates were sprayed with Trisbuffered saline (0.05 M Tris HCl, 0.15 M NaCl, pH 7.4) containing 1% bovine serum albumin and 0.1% sodium azide and immediately soaked in the same buffer for 30 min. The plates were then removed and placed horizontally. Antibodies diluted in the buffer were layered on the plates and incubated for 1 hr. The plates were washed four times in PBS followed by overlaying with buffer containing 2 x lo6 cpm/ml of ‘251-labeled anti-human IgM antibodies. After 1 hr of




incubation, four washings, and air drying the plates were exposed to X-ray film (XR-5, Eastman Kodak, Rochester, NY). The control antibody was an IgM obtained from a patient with macroglobulinemia WaldenStrom myeloma. Immunoblotting. Parasite fractions and erythrocyte membranes separated in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions were transferred to nitrocellulose and then probed with antibodies as described (Berzins et al. 1983). Immunoprecipitation. ls5S]methionine metabolically labeled parasite antigens were analyzed in immunoprecipitation mainly as described by Petersen et al. (1990). Immune complexes were collected with rabbit anti-human IgM coupled to Sepharose 4B and analyzed by SDS-PAGE and autoradiography. RESULTS

The use of EBV-transformed B-cells from a clinically immune blood donor with high serum titers of anti-P. falciparum antibodies for production of human MAbs against the erythrocytic stages of the parasite resulted in several positive clones producing MAbs with different antigenic specificities (Udomsangpetch et al. 1986; and unpublished results). One of the clones, A52A6, showed a distinct pattern in immunofluorescence against P. falciparuminfected erythrocytes and was a stable antibody producer. This MAb was selected for further studies. When the culture supernatant from the clone was tested in an indirect immunofluorescence assay on unfixed air-dried infected erythrocytes, all stages of the parasite were stained, including gametocytes (Fig. 1). Free merozoites (strain F32) were diffusely stained all over but with a brighter fluorescence in their apical end (Fig. la). Trophozoites were stained with a speckled pattern over the parasite (Fig. lb). Schizonts were very brightly stained and late stage schizonts showed, in addition, a patchy staining outside the infected erythrocytes probably reflecting membrane traces from surrounding uninfected erythrocytes (Fig. lc). Furthermore, staining of small vesicular structures surrounding rup-



FIG. 1. Indirect immunofluorescence on asexual blood stages of Plasmodium falciparum using MAb A52A6. Merozoite (a), trophozoites (b), schizont (c), ruptured schizont (d), and gametocytes (e).

turing schizonts was frequently seen (Fig. Id). MAb A52A6 also showed reactivity with unfixed air-dried gametocytes, giving a granular staining all over the parasite (Fig. le). MAb A52A6 also reacted with the surface of glutaraldehyde fixed and air-dried infected erythrocytes, staining mainly late stage infected erythrocytes (Fig. 2~). MAb reactive material was, however, also detected on the invaginating membrane at the moment of merozoite invasion into the erythrocyte (Fig. 2a) as well as on the sur-

face of newly infected erythrocytes mainly at the site where the merozoite appeared to have entered (Fig. 2b). At late stages, antigenie material was detected on the surface of uninfected erythrocytes surrounding the infected one, giving a rosette-like pattern of staining (Fig. 2d), which became more extensive when the schizonts ruptured (Fig. 2e). The different staining patterns in immunofluorescence were seen on all P. f&iparum strains tested: F32, Thai I, 7G8, Kl, HB3, FVO from in vitro cultures as well as

P. fdCifX2W?Z:





FIG. 2. Indirect immunofluorescence on glutaraldehyde fixed and air-dried monolayers of Plasmodium falciparum-infected erythrocytes probed with MAb A52A6. Invading merozoite (a), early ring

stage (b), trophozoite (c), schizont (d), and ruptured schizont (e).

fluorescence. The antigenic activity on several isolates from infected individudetected on glutaraldehyde fixed and airals in Liberia and Colombia. In contrast, dried monolayers was sensitive to treatMAb A52A6 did not react with several ment with phospholipase A2 but not to proother species of Plasmodium: P. vivax, P. nase, trypsin, nor neuraminidase. In conknowlesi, P. gonderi, P. braziliensis, P. trast, pronase treatment increased the fragile, P. coatney and P. reichenowi. brightness of the fluorescence and more inHowever, a weak cross-reaction was seen fected cells appeared to give surface stainwith P. chabaudi. When immunofluorescence was per- ing. The antigenic activity detected with formed on intact infected erythrocytes, ei- MAb A52A6 on unfixed infected erythrocytes was, however, resistant to phosphother in suspension or on glutaraldehydelipase A2 digestion while pronase treatment fixed monolayers, the fluorescence pattern was different from what was seen with air- gave a totally different immunofluoresdried cells. Instead of a distinct membrane cence pattern (speckled and much more instaining, patches of the infected cell were tense) as compared to untreated erythrocytes. Fixation of the infected erythrocytes faintly and diffusely stained (not shown). with methanol completely abolished the anThe nature of the antigenic activity detected by MAb A52A6 was studied by di- tigenic activity while acetone fixation gave gestion of infected erythrocytes with en- a weaker but less diffuse staining of the parzymes and subsequent analysis in immuno- asite, as compared to unfixed cells.



Analysis of parasite extracts in immunoblotting with MAb A52A6 only gave a broad diffuse band migrating ahead of the protein front, indicating a reactivity with lipids (not shown). However, MAb A52A6 gave three bands (approximate molecular weights 200, 150, and 40 kDa) in immunoprecipitation with extracts of parasites metabolically labeled with [35S]methionine (not shown). As the results above indicated that the antigen detected by MAb A52A6 was of lipid nature, the reactivity of the MAb with lipid fractions of infected erythrocytes, merozoites, and normal erythrocyte membranes was analyzed. In dot immunoblotting the MAb reacted with lipids from the parasite preparations, but not with lipids from normal erythrocytes (not shown). This reactivity was confirmed in ELISA using the lipid fractions as coating antigens (Fig. 3). MAb A52A6 reacted strongly with lipids from infected erythrocytes and merozoites but not with normal erythrocyte lipids. Attempts to characterize the lipid components recognized by MAb A52A6 were

done with lipids separated on HPTLC. The MAb stained several bands in the area towards the gel front where the phospholipid standards migrate (Fig. 4). These bands were, however, also seen in the lipid fraction from normal erythrocyte membranes and were also stained by the control IgM and the reactivity was therefore considered nonspecific. However, close to the origin, a clearly specific band was detected by MAb A52A6. This band was only seen in the lipid fractions from parasite material and was not seen in lipids from normal erythrocyte membranes, nor was it stained with the control IgM. The band appeared as a doublet in the merozoite fraction and as a single band in the lipids from infected erythrocytes. It was detected in both the organic



23456 7 8 9 4. Immunostaining of lipid extracts separated on HRTLC and probed with MAb A52A6 (a) and control IgM (b). Lanes l-3 represent standards of ganghosides (lane I), neutral glycolipids (lane 2) and phospholipids (lane 3). Reactivity patterns against lipid extracts from normal erythrocytes obtained in the organic solvent phase (lane 4) or in the water phase (lane 5), from infected erythrocytes organic solvent phase (lane 6) and water phase (lane 7), and from merozoites organic solvent phase (lane 8) and water phase (lane 9). The arrow indicates the position of the band specifically recognized by MAb A52A6. FIG.

I II25






FIG. 3. Reactivity of MAb A52A6 in ELISA against lipid extracts from enriched infected erythrocytes (O), from merozoites (O), and from uninfected erythrocytes (0).







and the water phases, probably due to in- fulcipurum immune donor (Udomsangcomplete separation. The band was not petch et al. 1986). This MAb showed reacidentified with the standards used (i.e., tivity with a family of cross-reacting P. fulphospholipids, gangliosides, neutral gly- cipurum protein antigens, including Pf155/ colipids). When lipids from infected cells RESA, Pfl 1.1, and Ag332 (Udomsangpetch metabolically labeled with [3H]glucosamine et al. 1989a;Ahlborg et al. 1991), and it was or 32P-labeled inorganic phosphorous were shown to inhibit the merozoite invasion in separated by HPTLC, a labeled band was vitro (Udomsangpetch et al. 1986) as well obtained at the same site as the MAb reac- as the cytoadherence of P. fulcipurumtive band. infected erythrocytes to endothelial cells In order to investigate if the antigen de- (Udomsangpetch et al. 1989b). Here we retected by MAb A52A6 is involved in the port on another merozoite invasion inhibimerozoite invasion process, the MAb was tory MAb, A52A6, derived from the same donor. The major P. fulcipurum antigen tested for its capacity to inhibit P. fulciparum invasion in in vitro cultures (Table recognized by this new MAb appears to be I). MAb A52A6 both in crude culture su- of lipid nature and is species specific but pematant as well as purified, inhibited par- is present in all P. fulcipurum strains anaasite invasion with a 50% inhibition titer of lyzed so far. 20 p,gIgM/ml. A control human monoclonal MAb A52A6 showed reactivity with all IgM antibody of an irrelevant specificity parasite stages throughout the asexual did not have any effect in this assay. erythrocytic cycle, as well as with gametocytes. It is, however, not known if it is the DISCUSSION same antigen recognized in all parasite We have earlier reported on a human stages. The immunoprecipitation of three MAb, 3362, derived from a clinically P. parasite polypeptides from extracts of infected erythrocytes indicates that the MAb might cross-react with such antigens or, TABLE I Inhibition of P. fulciparum merozoite invasion more likely, that the lipid antigen is associwith MAb A52A6 ated with these polypeptides in the extracts. Using glutaraldehyde-fixed and airPercentage dried monolayer preparations, the MAb reinvasion Percentage MAb acted with the membrane of infected cells, inhibition parasitemiad whl preferentially on cells containing late stages A52A6” of the parasite. This is in contrast with what 55 0.77 f 0.04 22 16 1.44 + 0.05 is seen with antibodies to Pfl55/RESA, 11 1.70 k 0.05 0 5.5 which give a similar staining pattern but AS2A6b mainly on erythrocytes infected with early 41 1.01 * 0.04 23 parasite stages (Perlmann et al. 1984; 1.38 + 0.05 20 11.5 Udomsangpetch et al. 1986). Moreover, 0 1.74 2 0.08 5.8 noninfected cells surrounding a late stage GlB3’ 0 1.73 2 0.04 16 parasite-infected erythrocyte were also 0 1.72 2 0.04 8 stained, giving a rosette-like pattern which 0 1.71 5 0.05 4 became most extensive when the schizont burst. a MAb A52A6 in culture supematant. b MAb A52A6 purified (see Materials and Methods). Taken together, the immunofluorescence c Control IgM MAb in culture supematant. data suggest that the antigenic material recd Percentage parasitemia k standard deviation of ognized by MAb A52A6 is mainly synthethe means from quadruplicate tests. Time of incubasized late in schizogony and is then present tion 20 hr, percentage parasitemia at time 0 is 0.4 + in merozoites and is also shed from the in0.04 and at time 20 is 1.72 * 0.03.



fected erythrocyte, resulting in binding to the surface of surrounding erythrocytes. At merozoite invasion, MAb reactive material appears to be concentrated in the invagination of the erythrocyte membrane, suggesting that the antigen is a part of the parasite lipid material participating in the moditication of the erythrocyte membrane and formation of the parasitophorous vacuole (Mikkelsen et al. 1988). The invasion inhibitory capacity of the MAb also indicates an involvement of the antigen in the invasion process. Our results suggest that the major antigen recognized by MAb A52A6 is a phosphoglycolipid, but we do not know whether the antibody is directed against the carbohydrate or the lipid portion of the antigen. However, with the methods used for lipid extraction, antibody reactivity with contaminating peptidic components cannot be fully excluded. The malaria parasite infection has been shown to induce profound changes in the phospholipid composition of the erythrocyte (Joshi and Gupta 1988; Schwartz et al. 1987; Vial et al. 1982). Glycolipids synthesized by P. falciparum have been identified and characterized (Sherwood et al. 1986) but nothing is known about their immunogenic or antigenic properties. Malarial lipid antigens have been described to be present in extracts of P. berg&-infected rat erythrocytes but their molecular identity was not determined (Frankenburg et al. 1984; Rosen et al. 1987). A direct correlation was found between levels of anti-lipid antibodies in sera from convalescent and immune rats and progress of infection (Frankenburg et al. 1984). Furthermore, lipid associated antigens were recently detected among exoantigens of P. falciparum, P. yoelii, and P. berghei asexual bloodstages (Taverne et al. 1990). The antigens induced secretion of tumor necrosis factor (TNF) both in vitro from human macrophages and in vivo in mice. The antigens gave rise to T-cell independent antibody responses and, in contrast to the lipid antigen recognized by

MAb A52A6, the TNF-inducing lipid antigen of the different plasmodial species cross-reacted (Taverne et al. 1990). The lipid nature of the antigen recognized by MAb A52A6 may limit its use as a component in a vaccine, such antigens mainly giving rise to T-cell-independent antibody responses with short immunological memory. However, using anti-idiotypic antibodies or mimotopic peptides in conjunction with proper malarial T-cell epitopes should circumvent these problems. ACKNOWLEDGMENTS

This work was supported by grants from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases, the Swedish Medical Research Council, and the Rockefeller Foundation Great Neglected Diseases Network. The support from Kabi Vitrum AB and SmithKline Beecham Biologicals is gratefully acknowledged. REFERENCES

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Plasmodium falciparum: an invasion inhibitory human monoclonal antibody is directed against a malarial glycolipid antigen.

A Plasmodium falciparum malaria blood stage antigen was detected using a human monoclonal antibody (MAb A52A6) obtained from a clinically immune donor...
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