Soluble Plasmodium falciparum Antigens Contain Carbohydrate

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Apr 6, 1987 - proteins (2, 3). Ramasamy andReese (9) have reported that ... a-D-galactosidase (1.0 U/ml), to the antigens before CIE. 2075 on March 6, 2019 ...
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1987, p. 2075-2079 0095-1137/87/112075-05$02.00/0 Copyright ©D 1987, American Society for Microbiology

Vol. 25, No. 11

Soluble Plasmodium falciparum Antigens Contain Carbohydrate Moieties Important for Immune Reactivity PALLE H. JAKOBSEN,l* THOR G. THEANDER,2 JAMES B. JENSEN,3 KARE M0LBAK,' ANDS0REN JEPSEN' Malaria Research Laboratory, Treponematoses Department, Statens Seruminstitut,' and Lymphocyte Laboratory, Department of Infectious Diseases, Rigshospitalet,2 DK-2300 Copenhagen, Denmark, and Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 488243 Received 6 April 1987/Accepted 3 August 1987

The importance of carbohydrate moieties for the antigenicity of purified soluble Plasmodium falciparum antigens from the asexual blood stage was tested. Digestion of the soluble antigens with a-D-galactosidase clearly affected the ability of the antigen to react with malaria-immune sera from different geographical origins in crossed immunoelectrophoresis and immunoblotting. Antigens of 220, 180, 80, and 74 kilodaltons were affected by the enzyme treatment. Furthermore, the enzyme digestion reduced the ability of the purified soluble antigen to stimulate lymphocytes from malaria-immune donors. The results might have important implications for the strategy of developing a malaria vaccine.

Plasmodium falciparum malaria is a major health problem in large areas of the tropics. Control of malaria has become increasingly difficult, partly because of the development of chloroquine resistance in the P. falciparum parasite. Development of a malaria vaccine, therefore, has high priority. Most vaccine programs focus on parasite proteins and their interaction with the human immune system. Genes for important parasite proteins are cloned into Escherichia coli with the purpose of developing a malaria vaccine. Carbohydrate moieties constitute another target for the immune system. P. falciparum synthesizes several glycoproteins (2, 3). Ramasamy and Reese (9) have reported that carbohydrate moieties contribute to the antigenicity of the malaria parasite. Terminal a-D-galactose residues on glycoprotein antigens of 185, 135, 120, and 75 kilodaltons (kDA) from the asexual blood stage seem to be important carbohydrate targets for antibody reactivity (10). We report here that soluble P. falciparum antigens contain carbohydrate moieties important for the antibody reactivity of African and Indonesian immune sera. Furthermore, these moieties are important for specific stimulation of lymphocytes from clinically malaria-immune individuals. The results might be of importance for the strategy of developing a malaria vaccine. MATERIALS AND METHODS Parasite cultures. P. falciparum isolates F32/Tanzania and K1/Thailand were kept in continuous culture in a modified Trager and Jensen system (12) as described by Jepsen and Andersen (7). Briefly, the cultures were incubated at 37°C with a gas mixture of 10% 02, 5% C02, and 85% N2. The medium, RPMI 1640 supplemented with 21 mM sodium bicarbonate, 25 mM HEPES (N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid) buffer and 10% blood group A human serum, was changed daily. The parasites were grown in 10% (vol/vol) blood group A erythrocytes. Immune sera. Sera were selected from Liberian, Gambian, and Indonesian healthy adult blood donors clinically immune *

Corresponding author.

to malaria with precipitating antibodies to soluble P. falciparum antigens in crossed immunoelectrophoresis (CIE). Isolation of P. falkiparum antigens. Supernatants from the daily medium refreshment of the parasite cultures were pooled, and after dialysis they were applied to a CNBrSepharose 4B column containing as a ligand a pool of immunoglobulin G from clinically immune Liberian adults with high titers of precipitating antibodies against soluble P. falciparum antigens. Bound antigens were eluted with 3 M KSCN. The pooled fractions containing eluted antigens were concentrated and tested for the content of soluble antigens by CIE. Plasma from a Danish P. falciparum-infected patient with circulating soluble antigens was also used as an antigen in CIE. Enzymes. cK-D-Galactosidase, P-N-acetylhexosaminidase, a-L-fucosidase, ,B-D-galactosidase, a-D-mannosidase, and neuraminidase were purchased from Sigma Chemical Co., St. Louis, Mo. CIE. a-D-Galactosidase or a corresponding volume of 0.9% NaCl was added to 20 u1l of the affinity-purified antigen preparation, giving a final concentration of 5.0 U of a-Dgalactosidase per ml, and incubated at room temperature for 18 h. In some experiments, protease inhibitors (phenylmethylsulfonyl fluoride, 2 mM; leupeptin, 25 ,ug/ml; chymostatin, 25 ,ug/ml; pepstatin, 25 ,ug/ml [Sigma]) were added to the antigen together with a-D-galactosidase. Twenty microliters of the antigen preparation was run in the first-dimension gel at 10 to 15 V/cm for 30 min. The second-dimension gel was run perpendicular to the first dimension at 2 V/cm for 18 h into a gel containing 400 ,ul of immune serum. The plates were washed, pressed three times, and stained with Coomassie brilliant blue. In a series of experiments, a carbohydrate-splitting enzyme mixture consisting of P-Nacetylhexosaminidase (1.0 U/ml), a-L-fucosidase (0.4 U/ml), P-D-galactosidase (20 U/ml), a-D-mannosidase (8.6 U/ml), and neuraminidase (1.0 U/ml) was added, with or without a-D-galactosidase (1.0 U/ml), to the antigens before CIE. 2075

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saline-0.5% bovine serum albumin-0.5% Tween 20, incubated with immune sera diluted 1:1,000, and washed. The sheets were incubated with peroxidase-conjugated rabbit anti-human immunoglobulin G (Dakopatts, Copenhagen, Denmark) diluted 1:2,000, washed, and stained. MNC isolation and assay. Heparinized blood was collected from clinically malaria-immune individuals (age, 24 to 39 years) from the Gambia and Guinea Bissau. The donors were living where malaria is hyperendemic and had no recent clinical history of malaria and no parasites in their blood at the time of blood collection. Mononuclear cells (MNC) were isolated from peripheral blood by sodium metrizoate Ficoll Lymphoprep density centrifugation (Nyegaard, Oslo, Norway), washed three times in RPMI 1640 medium, cryopreserved, and stored in liquid nitrogen. On the day of use, the MNC were thawed and washed. RPMI 1640 medium supplemented with 15% pooled human serum, L-glutamine (58.4 ,ug/ml), penicillin (20 IU/ml), and streptomycin (20 ,ug/ml) was used as the culture medium. Cell cultures were performed in triplicate in microtiter plates (Nunc, Roskilde, Denmark). Each well contained 5 x 104 MNC in a final volume of 170 ,ul of RPMI 1640 culture medium per well. Antigen-stimulated cell cultures received 20 pil of purified protein derivative (PPD) or 20 pil of soluble antigens that had been incubated with a-D-galactosidase (15 U/ml) or NaCl. Cultures were incubated for 7 days. [3H]thymidine was added 24 h before the cultures were harvested. The cell cultures were collected onto glass filters with a harvesting machine, and [3H]thymidine was measured in a liquid scintillation counter.

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FIG. 1. CIE of a-D-galactosidase-treated, affinity-purified soluble P. falciparum antigens. Electrophoresis was performed in the first dimension with 20 pil of affinity-purified soluble antigens treated with either a-D-galactosidase (5.0 U/ml) or NaCl at 10 to 15 V/cm for 30 min. The second dimension was run perpendicular to the first dimension at 2 V/cm for 18 h (anode at the top). The seconddimension gel contained 300 to 400 ,ul of immune serum. Panels: a, soluble antigens tested against Indonesian serum no. 3; b, soluble antigens tested against Liberian serum no. 9; x, tests without a-D-galactosidase; o, tests with a-D-galactosidase. The numbers indicate the different antigens.

In some experiments, 1.0 U of a-D-galactosidase was added to 30 pI of plasma from a malaria-infected individual, giving a final concentration of 33 U of a-D-galactosidase per ml. The mixture was incubated at room temperature overnight and then used as an antigen in CIE. Immunoblotting. A parasite sonic extract for immunoblotting was produced from infected erythrocytes (20 to 30% parasitemia) as described in detail elsewhere (P. H. Jakobsen, S. Jepsen, and R. Agger, Parasitol. Res., in press). A 300-pil volume of supernatant of the parasite sonic extract plus 15 U of aL-D-galactosidase per ml or 0.9% NaCl was incubated at room temperature overnight. The antigens were applied on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis slab gel consisting of an 8% stacking gel and a 10 to 20% separating gel. Electrophoresis was performed at 25 to 40 V for 17 h under reducing conditions with prior heating of the antigens at 100°C for 3 min. The antigens were then electrotransferred to nitrocellulose sheets in a semidry electroblotter A (ANCOS, 01stykke, Denmark). The nitrocellulose sheets were blocked with phosphate-buffered

RESULTS CIE. Immune sera obtained from individuals from various geographic locations precipitated up to seven soluble antigens. Treatment of the soluble antigens with a-D-galactosidase (5.0 U/ml) before electrophoresis diminished or eliminated the reactivity of several antigens (Fig. 1). Treatment with a-D-galactosidase markedly diminished or abolished the reactivity of antigen 1 complex and antigen 3 and significantly lessened the reactivity of antigens 5 and 7. Antigens 2, 4, and 6 were relatively unaffected by the enzyme treatment. Addition of protease inhibitors to the soluble antigen-a-D-galactosidase mixture did not influence the results (data not shown). Most of the experiments were performed with soluble antigens from the F32/Tanzania strain, but soluble antigens from the K1/Thailand strain were similarly affected by a-D-galactosidase treatment (data not shown). Figure 2 shows the results of treatment of the antigens with the mixture of five different carbohydrate-splitting enzymes. a-D-galactosidase alone in very low concentration (1.0 U/ml) did not affect the CIE pattern. The enzyme mixture without a-D-galactosidase had no significant effect on the CIE pattern, whereas the enzyme mixture with a-D-galactosidase at a concentration of 1.0 U/ml strongly affected the reactivity of the soluble antigens. Figure 3 shows the results of CIE of a plasma sample from an acutely infected malaria patient tested against Liberian immune serum. a-D-galactosidase treatment of the antigencontaining plasma eliminated the reactivity of the soluble antigens with Liberian immune serum. Antigen 1 was strongly affected by the enzyme treatment. A similar pattern was seen when Indonesian serum was used (data not

shown).

CARBOHYDRATE CONSTITUENTS OF P. FALCIPARUM ANTIGENS

VOL. 25, 1987

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FIG. 3. CIE of soluble antigens from the plasma of a Danish malaria patient. Electrophoresis was performed with 20 ,ul of plasma from the infected individual treated with a-D-galactosidase (33 U/ml) or NaCI in the first dimension at 10 to 15 V/cm for 30 min. The second dimension was run perpendicular to the first dimension at 2 V/cm for 18 h (anode at the top). The second-dimension gel contained 300 ,ul of Liberian immune serum 10. The plasma was incubated with NaCl (a) or a-D-galactosidase (b). The numbers indicate the different antigens.

antigens treated with a-D-galactosidase showed reduced ability to stimulate MNC from four clinically malariaimmune donors in the proliferation assay as compared with untreated antigens (Table 1). In contrast, a-D-galactosidase

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FIG. 2. CIE of affinity-purified soluble P. falciparum antigens treated with enzyme mixture. Electrophoresis was performed with 30 pil of affinity-purified soluble antigens treated with the following (panel): a, NaCl; b, a-D-galactosidase (1.0 U/ml); c, an enzyme mixture; d, a-D-galactosidase (1.0 U/ml) plus the enzyme mixture as described in Materials and Methods. Note that this a-Dgalactosidase amount is lower than the amount used for CIE in Fig. 1. Electrophoresis was performed in the first dimension at 10 to 15 V/cm for 30 min. The second dimension was run perpendicular to the first dimension at 2 V/cm for 18 h (anode at the top). The second-dimension gel contained 300 ,ul of Liberian immune serum 8. The numbers indicate the different antigens.

Immunoblotting. In immunoblotting, the immune sera showed several antibody specificities against the supernatant from a parasite sonicate. Treatment of the antigen preparation with a-D-galactosidase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis changed the reactivity. Figure 4 shows paired samples of P. falciparum antigens with and without a-D-galactosidase treatment tested against Liberian, Indonesian, or Gambian immune serum. The reactivity of a 220-kDa antigen was eliminated, the reactivity of 80- and 74-kDa antigens was much diminished, and the reactivity of a 180-kDa antigen was eliminated in some immune sera but unaffected in others. A nonspecific conjugate binding to a 63-kDa antigen was also similarly eliminated by the enzyme treatment. After a-D-galactosidase treatment, most of the immune sera reacted with a new diffuse band of 38 to 40 kDa. There was no difference between Indonesian and African immune sera in their reactivity with enzyme-treated antigens.

MNC proliferation. Affinity-purified soluble P. falciparum

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FIG. 4. Reactivity in immunoblotting of immune sera against a-D-galactosidase- or NaCl-treated parasite sonicate. Marker proteins (Bio-Rad Laboratories, Richmond, Calif.) were Myosin (200 kDa), ,B-galactosidase (116.25 kDa), phosphorylase c (92.6 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Lanes: a, Indonesian serum 1; b, Indonesian serum 2; c, Indonesian serum 3; d, Indonesian serum 4; e, Indonesian serum 5; f. Liberian serum 6; g, Liberian serum 7; h, Liberian serum 8; i, Liberian serum 9; j, Gambian serum 10; k, Danish donor serum pool; 1, buffer; 1, tests with a-D-galactosidase; 2, tests without a-D-galactosidase. Arrows indicates the antigens affected by a-D-galactosidase treatment.

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TABLE 1. [3H]thymidine incorporation of lymphocytes in unstimulated cultures and cultures stimulated with a-D-galactosidase (15 U/ml)-treated and nontreated affinity-purified soluble P. falciparum antigens and PPD Donor no.

No.

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Purified soluble antigens 63.7 (10.0) 33.9 (2.4) 21.0 (3.2) 9.8 (2.1)

Mean (±SEM) cpm (103) incorporated witha: Purified soluble antigèns + ct-D-galactosidase

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treatment of PPD did not reduce its ability to stimulate lymphocytes. DISCUSSION

Soluble P. falciparum antigens may participate in the induction of protective immunity against malaria (4, 6). Moreover, we have previously shown that soluble affinitypurified malaria antigens induced specific proliferative responses of lymphocytes from donors sensitized or immune to malaria, whereas lymphocytes from donors never exposed to malaria did not respond to the antigens (1, 11). Our results show that carbohydrate moieties are important for the antigenicity of soluble antigens from both in vitro cultures and malaria patients. Of the seven different soluble antigens studied, antigen 1 is the most frequently found in plasma of acutely infected malaria-infected children (8). Murine monoclonal antibodies and a polyclonal monospecific human antibody directed against this soluble antigen are inhibitory to the growth of P. falciparum in vitro (Jakobsen et al., in press). a-D-Galactosidase treatment diminished the antigenicity of antigen 1 in CIE and immunoblotting. Thus, carbohydrates may play an important role in the induction of protective immunity. The finding that a-D-galactosidase treatment of parasite sonic extract changed the sonic extract reactivity in immunoblotting confirms the results of Ramasamy and Reese (10) that carbohydrate moieties contribute to the antigenicity of extracted P. falciparum antigens, although we note some differences in the reported molecular weights of these glycosylated antigens. A mixture of ax-D-galactosidase, P-D-galactosidase, a-Lfucosidase, a-D-mannosidase, and ,-N-acetylhexosaminidase had a more pronounced effect on the antigenicity of soluble antigens than did the reaction of a-D-galactosidase alone, whereas a mixture of the same enzymes without aL-D-galactosidase produced no change in antigenicity. These results suggest that when terminal a-D-galactose residues are removed from the soluble antigens other carbohydrates are exposed to enzyme degradation. These results are in agreement with the findings by Ramasamy and Reese (10), who reported the same phenomenon with crude asexual blood stage antigens. Soluble antigens from a Tanzanian, as well as a Thai

isolate of P. falciparum, were used in this study, giving

identical results. It is not clear whether the carbohydrate moieties are associated with variant parasite antigens, constant antigens, or both types of antigens. However, the reactivities of Indonesian, Liberian, and Gambian immune sera were equally affected by the digestion of carbohydrates. The importance of antibody-dependent immunity against

P. falciparum is well established. However, cell-mediated

and humoral immunity may vary with age and endemicity

(5). T lymphocytes with specificity toward malaria antigens are thought to play a role in both types of immunity. We have reported earlier that cells that respond to soluble antigens are found within the T-helper lymphocyte population (11). The fact that removal of carbohydrate moieties significantly diminished the lymphocyte response to soluble antigens indicates that important P. falciparum-specific Tcell clones of immune individuals recognize these carbohydrate moieties. The results reported here may have important implications for the development of a malaria vaccine. Since many advances in malaria vaccine production rely on using parasite epitopes provided by recombinant DNA techniques using E. coli or synthetic peptides, the antigenicity of glycosylated parasite antigens underscores the importance of further investigations into the role of carbohydrate moieties in the induction of clinical immunity in P. falciparum

malaria. ACKNOWLEDGMENTS Jette Severinsen, Anne Margrethe Olsen, Inge Damgaard Knudsen, and Jette Bendtsen are thanked for excellent technical assistance. Special thanks are given to Stephen Hoffmann, U.S. Navy Medical Research Unit, Jakarta, Indonesia, for supplying Indonesian serum used in this investigation. The work was supported by grants 12-5733, 12-5485, and 12-5810 from the Danish Medical Research Council, grants 104. Dan.8/318 and 104. Dan.8/357 from the Danish International Development Agency, grant TSD-M-347 from the Commission of the European Community, and Public Health Service grant AI 16312 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Bygbjerg, I. C., S. Jepsen, T. G. Theander, and N. 0dum. 1985. Specific proliferative response of human lymphocytes to purified soluble antigens from Plasmodium falciparum in vitro cultures and to antigens in patients' serum. Clin. Exp. Immunol.

59:421-426. 2. Heidrich, H.-G., W. Strych, and P. Prehn. 1984. Spontaneously released Plasmodium falciparum merozoites from culture possess glycoproteins. Z. Parasitenkd. 70:747-751. 3. Howard, R. F., and R. T. Reese. 1984. Synthesis of merozoite proteins and glycoproteins during the schizogony of Plasmodium falciparum. Mol. Biochem. Parasitol. 10:319-334. 4. James, NI. A., I. Kakoma, M. Ristic, and M. Cagnard. 1985. Induction of protective immunity to Plasmodium falciparum in Saimiri scireus monkeys with partially purified exoantigens. Infect. Immun. 49:476-480. 5. Jensen, J. B., S. L. Hoffmann, M. T. Boland, M. A. S. Akood, L. W. Laughlin, L. Karniawan, and H. A. Marwoto. 1984. Comparison of immunity to malaria in Sudan and Indonesia: crisis form versus merozoite-invasion inhibition. Proc. Natl. Acad. Sci. USA 81:922-925. 6. Jepsen, S. 1983. Inhibition of the in vitro growth of Plasmodium falciparum by purified antimalarial human IgG antibodies.

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Scand. J. Immunol. 18:567-571. 7. Jepsen, S., and B. J. Andersen. 1981. Immunosorbent isolation of antigens from the culture medium of in vitro cultivated Plasmodium falciparum. Acta Pathol. Microbiol. Scand. Sect. C 89:99-103. 8. Jepsen, S., and N. H. Axelsen. 1980. Antigens and antibodies in Plasmodium falciparum malaria studied by the immunoelectrophoretic method. Acta Pathol. Microbiol. Scand. Sect. C 88:263-270. 9. Ramasamy, R., and R. T. Reese. 1985. A role for carbohydrate moieties in the immune response to malaria. J. Immunol. 134:

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1952-1955. 10. Ramasamy, R., and R. T. Reese. 1986. Terminal galactose residues and the antigenicity of Plasmodium falciparum glycoproteins. Mol. Biochem. Parasitol. 19:91-101. 11. Theander, T. G., I. C. Bygbjerg, S. Jepsen, M. Svenson, A. Kharazmi, P. B. Larsen, and K. Bendtzen. 1986. Proliferation induced by Plasmodium falciparum antigen and interleukin-2 production by lymphocytes isolated from malaria-immune individuals. Infect. Immun. 53:221-225. 12. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673-675.