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Protective antigen in the membranes of mouse erythrocytes infected with Plasmodium chabaudi
Molecular and Biochemical Parasitology, 25 (1987) 195-201 Elsevier
195
MBP 00848
Protective antigen in the membranes of mouse erythrocytes infected with Plasmodium chabaudi Chingchai Wanidworanun, John W. Barnwell and Hannah L. Shear Department of Medical and Molecular Parasitology, New York University Medical Center, New York, NY, U.S.A. (Received 2 February 1987; accepted 5 May 1987)
A malarial antigen, Pc96, in the plasma membrane of erythrocytes infected with Plasmodium chabaudi has been identified. It is synthesized by the parasite and present during most of the growth stages of the intra-erythrocytic cycle as demonstrated by immunofluorescence. The antigen has a molecular weight of approximately 96 000. Monoclonal antibodies raised against this antigen were used to isolate the protein by affinity chromatography. Mice immunized with affinity-purifiedPc96 were partially protected against blood induced-P, chabaudi infection. This result indicates the existence of a protective antigen in the membranes of erythrocytes parasitized by a rodent malaria and encourages the search for analogous antigens in human malaria parasites as possible candidate molecules for malaria vaccination. Key words: Plasmodium chabaudi; Erythrocyte membrane; Protective antigen; Malaria
Introduction
Materials and Methods
The existence of malarial antigens in parasitized erythrocyte m e m b r a n e s has been well docu m e n t e d in Plasmodium knowlesi and Plasmodium falciparum infections [1-5]. T h e r e has been some indirect evidence that specific antig e n - a n t i b o d y reactions on the m e m b r a n e s of parasitized erythrocytes m a y lead to parasite destruction through opsonization, antibody dependent cell-mediated cytotoxicity, or reversal of sequestration of parasitized erythrocytes, which could bring about their destruction in the spleen [6-8]. H e r e we identify a 96 k D a antigen, in the m e m b r a n e s of Plasmodium chabaudi-infected mouse erythrocytes, which is partially protective.
Parasites. Plasmodium chabaudi adami, a non-lethal strain of P. chabaudi, a rodent malaria, was obtained from Dr. Diane Taylor, G e o r g e t o w n University, Washington, D.C., and maintained in SW (Yaconic Farms) and B A L B / c (Timco) mice by intraperitoneal or intravenous inoculation of parasitized blood p r e p a r e d from frozen stabilates [9] or from infected mice.
Correspondence address: Dr. Hannah L. Shear, Department of Medical and Molecular Parasitology, New York University Medical Center, 550 First Avenue, New York, NY 10016, U.S.A. Abbreviations: MoAb, monoclonal antibody; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.
Affinity purification o f parasitized erythrocyte membranes and immunization o f mice. Preparation of the red cell m e m b r a n e s of P. chabaudi-infected erythrocytes was accomplished by affinity chromatography as follows: washed erythrocytes from mice with 20-30% parasitemia were resuspended to 109 m1-1 in isotonic phosphate-buffered saline p H 7.4 (PBS) and incubated with an equal volume of a rabbit anti-mouse erythrocyte antiserum (Cappel Laboratories) at a subagglutinating dilution for 30 min, washed 3 times and resuspended to 109 m1-1 in PBS containing protease inhibitors (1 m M phenylmethylsulfonyl fluoride, 0.2 m M tosyllysine chloromethyl ke-
0166-6851/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
196 tone, 5 mM EDTA, 5 mM EGTA, 5 mM iodoacetamide). The cells were then fragmented by sonication at 80 W for 30 s 3 times on ice, and passed through a 2 ml protein A-Sepharose CL4B column (Pharmacia). After the column was washed exhaustively, the bound material was eluted with 3 M potassium thiocyanate, pelleted by centrifugation at 100 000 × g for 1 h in a Beckman ultracentrifuge, and stored at -20°C until used. For immunization, the pellet was thawed and resuspended in PBS by sonication. BALB/c mice were immunized intravenously with the membrane preparation in PBS and boosted once a week three times. Each inoculum contained membranes of approximately 1.6×107 erythrocytes. Sera were obtained one week after the last injection by retro-orbital venepuncture.
Indirect immunofluorescence.
Glutaraldehydefixed, air-dried erythrocyte monolayers were prepared using the method described by Perlmann [10]. For indirect immunofluorescence, the erythrocytes were incubated sequentially with dilutions of tested antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM and IgG (1:50 dilution, Boehringer Mannheim) for 30 min each. The slides were washed in PBS after each incubation. The parasite nuclei were counterstained for 5-10 min with ethidium bromide (100 ~g ml-l). Occasionally, we used 6.7 ng m1-1 biotinylated goat anti-mouse IgG, or IgM (Boehringer Mannheim) and 20 ~g m1-1 FITClabelled avidin (Sigma) to detect the antigen-antibody reaction.
Monoclonal antibody (MoAb) production. The method described by Kohler and Milstein [11] was used with some modifications. Spleen cells from a BALB/c mouse immunized with the parasitized erythrocyte membrane fraction were used after the third booster. Fusion with P3U1, a myeloma cell line, was achieved using polyethylene glycol. Thirty colonies were positive for the membrane IFA of infected cells. Four colonies were cloned by limiting dilution and implanted into the peritoneal cavities of CD2F1 mice and ascitic fluid was collected. 8F2G9, an IgG1 MoAb, was purified from ascitic fluid by affinity chromatography using Affi-Gel Protein A MoAb Kit (Bio Rad). The
three IgM MoAbs, 6C12E7, 6DllE6, and 7C6E10 were purified by Sephacryl S-300 (Pharmacia) gel filtration. The isotype of each MoAb was determined by double diffusion in agarose using the monoclonal hybridoma culture supernatant fluid against a panel of isotype-specific antibodies. The purity of each MoAb was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. SDS-PAGE was carried out as described by Laemmli [12] with a 5% stacking gel and 7.5% separating gel. For Western protein blotting [13] the proteins resolved in SDS gel were electrophoretically transferred to a nitrocellulose sheet and probed with dilutions of sera or MoAbs. Signals for antigen-antibody binding were provided by the second antibody, 125I-goat anti-mouse IgG or IgM (2 x 10 6 counts min -1 m1-1) with subsequent autoradiography.
Metabolic labelling of parasitized erythrocytes and immunoprecipitation. Heparinized blood was obtained from a mouse with 30% P. chabaudi parasitemia. To remove fibrinogen, white blood cells and platelets, ADP at 50 ~g ml -~ was added to the blood which was then passed sequentially through columns of glass beads and cellulose powder (CFll). The red cells were then washed three times with RPMI-1640 tissue culture medium (GIBCO) and resuspended to 4x108 cells m1-1 in RPMI-1640 containing 10% fetal calf serum. [3SS]Methionine (50 txCi m1-1) was then added to the cell suspension and incubated for 8 h at 37°C in an atmosphere of 5% O2, 5% CO2, 90% N 2. Afterwards the cells were pelleted and washed three times with chilled PBS and solubilized with 1% Triton X-100 detergent containing protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, tosyllysine chloromethyl ketone, leupeptin, and chymostatin) at a final concentration of 4×108 cells m1-1, centrifuged at 16000 x g for 15 min at 20°C and the supernatant stored in liquid nitrogen until used. For immunoprecipitation, the 1% Triton X-100 extract (200 txl) of the metabolically labelled cells was added to 10 Ixl of MoAb 8F2G9, 7C6E10, and
197 a mixture of two irrelevant IgG1 and IgM MoAbs. The mixture was incubated at 4°C for 18 h and then the immune complexes in each sample were precipitated with 100 txl of 50% suspension of immunoadsorbent (goat anti-mouse IgG or IgM conjugated to Affi-Gel 10) in N E T T buffer (150 mM NaCl, 10 mM E D T A , 50 mM Tris, 0.5% Triton X-100). The immune complex-bound immunoadsorbent was pelleted, washed 5 times with the following: N E T T , N E T T , N E T T + 0.5 M NaC1, NETT, and N E T (NETT without Triton X100). The immune complexes were then eluted with S D S - P A G E sample buffer containing 2mercaptoethanol at 100°C for 5 min and subjected to S D S - P A G E in 7.5/5% slab gel. The gel was soaked in E n H a n c e (New England Nuclear) for 1 h, washed in deionized water for 1 h, dried, and subjected to fluorography at -70°C.
Affinity purification of the P. chabaudi antigen. Six ml of Affi-Gel 10 chromatography matrix (BioRad) was coupled with 12 mg of 6 D l l E 6 , an IgM M o A b , using the coupling method recommended by the manufacturer. Heparinized blood from mice with 20-30% parasitemia was washed three times with PBS and solubilized with deter-
gent solution and protease inhibitors at the following final concentrations: 3 x 109 erythrocytes m1-1, 1% (3-[3-cholamidopropyl]-dimethylammonio)-l-propanesulfonate, 1 mM phenylmethylsulfonyl f u o r i d e , 5 mM iodoacetamide, 5 mM E D T A , 5 mM E G T A , 0.2 mM tosyllysine chloromethyl ketone, 25 ixg m1-1 chymostatin, 25 Ixg m1-1 leupeptin. The mixture was centrifuged at 10000 x g for 1 h to remove particulate materials. The soluble mixture was then passed through the 6DllE6-affinity column and washed exhaustively with PBS. The bound material was eluted with 3 M potassium thiocyanate and was passed immediately through Sephadex G25M (Pharmacia, PD10) pre-equilibrated with PBS for the removal of potassium thiocyanate. This preparation was designated 6 D l l E 6 : A g .
Vaccination of mice with 6DllE6.'Ag. Two groups of five female Swiss Webster mice (4-6 weeks old), were vaccinated intraperitoneally with 50 ixg of 6 D l l E 6 : A g supplemented with 50 ~tg of saponin as the adjuvant [14,15] in PBS. The mice were boosted once a week, for three weeks. The controls received saponin in PBS or PBS alone. Six days after the last booster, they were chal-
Fig. 1. (A,B) Indirect immunofluorescence(IFA) of P. chabaudi-parasitized erythrocytes. Erythrocytes were fixed with 1% glutaraldehyde and air-dried. The fluorescenceon the perimeter of infected-erythrocytesis due to specificantigen-antibody reaction as detected by FITC-goat anti-mouse IgG+IgM. The intra-erythrocyticparasites appear as fluorescencedots due to counterstaining with ethidium bromide. MoAb 6DllE6 was used in panel A; same concentration of normal mouse globulin as the control was used in panel B. Scale bar, 10 ~xm.
198 lenged by intravenous inoculation of 104 P. chabaudi-parasitized erythrocytes. Parasitemia was followed daily by examination of Giemsa-stained blood smears from the tail vein. Results
Demonstration of malarial antigen in the membrane of P. chabaudi-parasitized erythrocytes. Sera of mice immunized with the erythrocyte membrane fraction (prepared from infected erythrocytes) were used to detect the putative malarial antigen(s) associated with the surface of P. chabaudi-parasitized erythrocytes using indirect immunofluorescence (Fig. 1A). The immune sera did not react with normal erythrocytes but produced a uniform membranous fluorescence pattern on the perimeter of parasitized erythrocytes regardless of the maturational stage of the parasite. No fluorescence was observed on parasitized erythrocytes incubated with normal mouse sera (Fig. 1B). These findings demonstrated the presence of a parasite-induced antigen in the eryth-
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E
Monoclonal antibodies for Pc96. Mouse MoAbs were developed [16]. In the following studies we used two MoAbs, 8F2G9 (IgG1,K), and 6 D l l E 6 (IgM,K). Both produce the same immunofluorescence pattern as in Fig. 1A and recognize Pc96 (Fig. 2, lanes E,F). In this and subsequent gels the MoAbs also recognized several lower M, bands which are probably degradative products of Pc96.
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rocyte membrane of P. chabaudi-infected red cells. To identify the corresponding antigen, the parasitized erythrocytes were subjected to SDSP A G E , transferred onto nitrocellulose sheets and probed with the immune sera. Only one parasite antigen, the 96 kDa band (Pc96) was detected by immune serum from mice immunized with the erythrocyte membrane preparation (Fig. 2, lane A, compared with normal erythrocytes in lane B). In contrast, polyspecific hyperimmune serum detected numerous parasite antigens in the parasitized erythrocyte extract in lane C, compared with the normal erythrocyte extract in lane D.
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Fig. 2. Western blotting of parasitized erythrocytes. SDS-extract of P. chabaudi-parasitized erythrocytes (lanes A,C,E-L) or normal erythrocytes (lanes B,D) was resolved by SDS-PAGE under reducing conditions, transferred onto nitrocellulose sheets and probed with membrane-immune serum (lanes A,B), MoAb 8F2G9 (lanes E,G), MoAb 6DllE6 (lane F), 6DllE6:Ag-immune sera (lanes H-L), or hyperimmune sera from mice repeatedly infected with P. chabaudi (lanes C,D),
199
Biosynthesis of Pc96. To determine the origin of Pc96, in vitro biosynthesis was carried out in erythrocytes from a P. chabaudi-infected mouse incubated in media containing radioactive amino acids. The cells were then solubilized with Triton and immunoprecipitation using M o A b was done. The autoradiograph of the precipitated antigens resolved by S D S - P A G E shown in Fig. 3 demonstrates that the 96 k D a band, corresponding to Pc96, was synthesized during the in vitro culture and precipitated by M o A b 8F2G9 (lane A), and 7C6E10 (lane B), compared with the control in lane C. This indicates that Pc96 is parasite-encoded.
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Affinity-purification of Pc96. Pc96 was purified by affinity chromatography using 6D11E6 as the li-
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Fig. 4. Antigenic analysis of 6DllE6:Ag. 6DllE6:Ag was resolved in SDS-PAGE under reducing conditions and stained with Coomassie blue (lanes A,B). Western blotting of the same material was probed with MoAb 8F2G9 (lanes C,D) or pooled immune sera from mice vaccinated with the same material (lanes E,F).
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Fig. 3. Metabolic labelling and immunoprecipitation of Pc96. P. chabaudi-infected erythrocytes were biosynthetically labelled with [35S]methionine, detergent-solubilized, and immunoprecipitated with MoAb 8F2G9 (lane A), 7C6E10 (lane B), or unrelated IgG1+IgM MoAbs (lane C).
gand. Fig. 4, lanes A and B show the SDS-PAGE of the eluted material (2 different batches), designated 6 D l l E 6 : A g . In addition to the 96 kDa band, Coomassie blue staining of 6 D l l E 6 : A g reveals two other bands of 200 and 75 kDa. To analyze its antigenic components, pooled sera from mice vaccinated with 6 D l l E 6 : A g (below) were used to probe the same material (lanes E,F) in Western blotting. The figure indicates that the major antigenic components of 6 D l l E 6 : A g are bands of 96 and 82 kDa and their degradative products, which is the same pattern recognized by M o A b 8F2G9 (lanes C,D). The 200 and 75 kDa bands were not recognized by the immune sera. Therefore, with the exception of the 82 kDa, which may be a degradative product of the 96 kDa or a cross-reactive antigen, Pc96 is probably the only parasite major immunogen in the 6 D l l E 6 : Ag preparation.
Experimental vaccination using 6DllE6:Ag. Mice were vaccinated with the affinity purified 6DllE6: Ag and challenged six days after the third booster with blood-induced P. chabaudi infection. Fig. 4
200
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J 2° r 1 2 3 4 5 6 7 8 9 10 11 12134415 16 Days After Intravenous Challenge [noculurn with 104 P chabaudi- Parasitized Erythrocytes
Fig. 5. Experimental vaccination with 6DI1E6:Ag. Course of P. chabaudi challenge infection in the mice vaccinated with 6D11E6:Ag batch l+saponin (m), 6DllE6:Ag batch 2+saponin (A), saponin in PBS (o), or PBS alone (e).
shows parasitemia of the vaccinated and control mice. In all groups, patent parasitemia commenced approximately three days after the challenge inoculation and then rose in parallel for a few days. However, in all the vaccinated mice, parasitemia remained less than 10% and then dropped sharply. In contrast, parasitemia in the control groups continued to rise to 40-50% before declining two days later than the vaccinated groups.
6DllE6:Ag-induced monospecific antibody response against Pc96. Sera taken from all the 6D11E6:Ag-vaccinated mice three days before the challenge had very high titers (104-105) of specific antibody for the membranes of P. chabaudi-parasitized erythrocytes. This antibody activity is species specific since the sera did not cross-react with fixed parasitized erythrocytes from mice infected with P. berghei or P. yoelii (data not shown). Furthermore, in Western blot analysis, all the sera recognized only one antigen of P. chabaudi-parasitized erythrocytes (Fig. 2, lanes H-L) with the banding pattern corresponding to Pc96 (lane G). Discussion
These studies demonstrate that Pc96 is a parasite-encoded antigen of Mr 96000 on or in the surface of P. chabaudi-infected erythrocytes. Partial protective immunity against P. chabaudi
could be induced in mice using 6DllE6:Ag, the parasite antigen affinity-purified with Pc96-specific MoAb. Although 6D11E6:Ag contained other proteins in addition to the 96 kDa, the data strongly suggest that Pc96 is the only parasite immunogen in the preparation since mice vaccinated with 6DllE6:Ag produced antibodies only against Pc96 and its fragments. The protective immune mechanisms induced by Pc96 remain to be determined. Passive immunization with MoAb did not protect mice against P. chabaudi (data not shown); however, since MoAbs are epitope specific this does not exclude the possible role of antibodies specific for other epitopes in protection. Studies of passive immunization with sera and T-lymphocyte subsets, should help to elucidate the immune effector mechanisms involved in protective immune response to Pc96. The relationship of Pc96 to parasitized erythrocyte surface antigens of other malaria species is of interest. Since we cannot agglutinate P. chabaudi-parasitized erythrocytes with either membrane-immune sera or MoAb (data not shown) we do not think that this antigen is analogous to the SICA antigen of P. knowlesi [2]. Recently, Perlmann et al. [10] and Coppel et al. [17] independently described an Mr 155 000 protein antigen of P. falciparum. This antigen, designated Pf155 or RESA, is being considered as a possible vaccine candidate since Pf155-specific MoAb inhibited merozoite invasion [18]. Recently, Coppel et al. described MESA, an Mr 250 000 antigen, which is associated with the surface of human erythrocytes containing mature forms of P. falciparum [19]. In rodent systems, antigens of Mr 105000, i.e. Pchl05 (P. chabaudi) [20] and 160000 (P. yoeIii) [21] in the surface of fixed infected erythrocytes have been described. A Pchl05-specific MoAb, generously provided by Drs. P. Perlmann and K. Berzins, recognizes a band of the same M r as the Pc96-specific MoAb (data not shown). Further, this MoAb produces the same pattern of immunofluorescence as the MoAbs that we describe. Interestingly, 7C6E10, a Pc96-specific MoAb produces RESA-type fluorescence with P. falciparum and recognizes a 155 kDa antigen in Western blotting of P. falciparum-infected erythrocytes (Wanidworanun, C., Barnwell, J.W. and
201 Shear, H . L . , in p r e p a r a t i o n ) . T h e r e f o r e , Pc96 a p p e a r s to be the s a m e as P c h l 0 5 a n d an analogue of Pf155 a n d R E S A . In c o n c l u s i o n , successful active i m m u n i z a t i o n with Pc96 in mice s h o u l d offer a useful a n i m a l m o d e l for the s t u d y of m e c h a n i s m s of p r o t e c t i v e i m m u n i t y a n d c o r r o b o r a t e the p o t e n t i a l of Pf155 a n d R E S A as a m a l a r i a vaccine c a n d i d a t e .
9 10
11
Acknowledgements We thank Drs A . H . Cochrane and A. Masuda for their help in the h y b r i d o m a production and the earlier parts of this study; V. N u s s e n z w e i g , R.S. N u s s e n z w e i g , a n d A . F e r r e i r a for r e a d i n g the m a n u s c r i p t a n d h e l p f u l suggestions, M. F e r r e i r a for technical assistance, a n d B. R o b l e s for secretarial assistance. S u p p o r t e d by AI15235 from the N I H a n d D A M D 17-85-C-5175 from the U S Army.
References 1 Howard, R.J. and Barnwell, J.W. (1984) Roles of surface antigens on malaria-infected red blood cells in evasion of immunity. Contemp. Top. Immunobiol. 12, 127-200. 2 Brown, K.N. and Brown, I.N. (1965) Immunity to malaria: antigenic variation on chronic infections of Plasmodium knowlesi. Nature 208, 1286-1288. 3 Howard, R.J., Barnwell, J.W. and Kao, V. (1983) Antigenic variation in Plasmodium knowlesi malaria: Identification of the variant antigen on infected erythrocytes. Proc. Natl. Acad. Sci. USA 80, 4129-4133. 4 Hommel, M., David, P.H., Origino, L.D. and David, J.R. (1982) Expression of strain specific surface antigens on Plasrnodiurn falciparum-infected erythrocytes. Parasite Immunol. 4, 409-419. 5 Leech, J.H., Barnwell, J.W., Miller, L.H. and Howard, R.J. (1984) Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparuminfected erythrocytes. J. Exp. Med. 159, 1567-1575. 6 Celada, A., Cruchaud, A. and Perrin, L.H. (1983) Assessment of immune phagocytosis of Plasrnodium falciparum infected red blood cells by human monocytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes. J. Immunol. Methods 63,263-271. 7 Brown, J. and Smalley, M.E. (1980) Specific antibody-dependent cellular cytotoxicity in human malaria. Clin. Exp. Immunol. 41,423M29. 8 Udeinya, I.J., Miller, L.H., McGregor, I.A. and Jensen, J.B. (1983) Plasmodium falciparum strain specific anti-
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body blocks binding of infected erythrocytes to amelanotic melanoma cells. Nature 303,429-431. Meryman, H.T. and Hornblower, M. (1972) A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion 12, 145-156. Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J., Bjorkman, A., Patarroyo, M.E. and Perlmann, P. (1984) Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum. J. Exp. Med. 159, 1686--1704. Kohler, G. and Milstein, C. (1975) Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256,495-497. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Towbin, H.T., Stahelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Holder, A.A. and Freeman, R.R. (1981) Immunization against blood-stage rodent malaria using purified parasite antigens. Nature 294,361-364. McCohn, A.A., Bomford, R. and Dalton, L. (1982) A comparison of saponin with other adjuvants for the potentiation of protective immunity by a killed Plasrnodium yoelii vaccine in the mouse. Parasite Immunol. 4,337-347. Wanidworanun, C., Barnwell, J.W., Masuda, A. and Shear, H.L. (1987) Malarial antigen in the erythrocyte membrane of Plasrnodiurn chabaudi-parasitized red blood cells. In: UCLA Symposium Proceedings Vol. 42, Molecular Strategies of Parasitic Invasion, pp. 355-367, Alan R. Liss, Inc., New York. Coppel, R.L., Cowman, A.F., Anders, R.F., Bianco, A.E., Saint, R.B., Lingelbach, K.R., Kemp, D.J. and Brown, G.V. (1984) Immune sera recognize on erythrocytes a Plasmodiurn falciparum antigen composed of repeated amino acid sequences. Nature 310, 789--792. Udomsangpetch, R., Lundgren, K., Berzins, K., Wahlin, B., Perlmann, H., Troye-Bloomberg, N., Carlsson, J., Wahlgren, M., Perlmann, P. and Bjorkman, A. (1986) Human monoclonal antibodies to Pf 155, a major antigen of malaria parasite Plasmodium falciparum. Science 231, 57-59. Coppel, R.L., Culvenor, J.G., Bianco, A.E., Crewther, P.E., Stahl, H.-D., Brown, G.V., Anders, R.F. and Kemp, D.J. (1986) Variable antigen associated with the surface of erythrocytes infected with mature stages of Plasmodium falciparum. Mol. Biochem. Parasitol. 20, 265-277. Gabriel, J.A., Holmquist, G., Perlmann, H., Berzins, K., Wigzell, H. and Perlmann, P. (1986) Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes. Mol. Biochem. Parasitol. 20, 67-75. Murakami, K. and Tanabe, K. (1985) An antigen of Plasrhodium yoelii that translocates into mouse erythrocyte membrane upon entry into the host cell. J. Cell Sci. 73, 311-320.
195
MBP 00848
Protective antigen in the membranes of mouse erythrocytes infected with Plasmodium chabaudi Chingchai Wanidworanun, John W. Barnwell and Hannah L. Shear Department of Medical and Molecular Parasitology, New York University Medical Center, New York, NY, U.S.A. (Received 2 February 1987; accepted 5 May 1987)
A malarial antigen, Pc96, in the plasma membrane of erythrocytes infected with Plasmodium chabaudi has been identified. It is synthesized by the parasite and present during most of the growth stages of the intra-erythrocytic cycle as demonstrated by immunofluorescence. The antigen has a molecular weight of approximately 96 000. Monoclonal antibodies raised against this antigen were used to isolate the protein by affinity chromatography. Mice immunized with affinity-purifiedPc96 were partially protected against blood induced-P, chabaudi infection. This result indicates the existence of a protective antigen in the membranes of erythrocytes parasitized by a rodent malaria and encourages the search for analogous antigens in human malaria parasites as possible candidate molecules for malaria vaccination. Key words: Plasmodium chabaudi; Erythrocyte membrane; Protective antigen; Malaria
Introduction
Materials and Methods
The existence of malarial antigens in parasitized erythrocyte m e m b r a n e s has been well docu m e n t e d in Plasmodium knowlesi and Plasmodium falciparum infections [1-5]. T h e r e has been some indirect evidence that specific antig e n - a n t i b o d y reactions on the m e m b r a n e s of parasitized erythrocytes m a y lead to parasite destruction through opsonization, antibody dependent cell-mediated cytotoxicity, or reversal of sequestration of parasitized erythrocytes, which could bring about their destruction in the spleen [6-8]. H e r e we identify a 96 k D a antigen, in the m e m b r a n e s of Plasmodium chabaudi-infected mouse erythrocytes, which is partially protective.
Parasites. Plasmodium chabaudi adami, a non-lethal strain of P. chabaudi, a rodent malaria, was obtained from Dr. Diane Taylor, G e o r g e t o w n University, Washington, D.C., and maintained in SW (Yaconic Farms) and B A L B / c (Timco) mice by intraperitoneal or intravenous inoculation of parasitized blood p r e p a r e d from frozen stabilates [9] or from infected mice.
Correspondence address: Dr. Hannah L. Shear, Department of Medical and Molecular Parasitology, New York University Medical Center, 550 First Avenue, New York, NY 10016, U.S.A. Abbreviations: MoAb, monoclonal antibody; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.
Affinity purification o f parasitized erythrocyte membranes and immunization o f mice. Preparation of the red cell m e m b r a n e s of P. chabaudi-infected erythrocytes was accomplished by affinity chromatography as follows: washed erythrocytes from mice with 20-30% parasitemia were resuspended to 109 m1-1 in isotonic phosphate-buffered saline p H 7.4 (PBS) and incubated with an equal volume of a rabbit anti-mouse erythrocyte antiserum (Cappel Laboratories) at a subagglutinating dilution for 30 min, washed 3 times and resuspended to 109 m1-1 in PBS containing protease inhibitors (1 m M phenylmethylsulfonyl fluoride, 0.2 m M tosyllysine chloromethyl ke-
0166-6851/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
196 tone, 5 mM EDTA, 5 mM EGTA, 5 mM iodoacetamide). The cells were then fragmented by sonication at 80 W for 30 s 3 times on ice, and passed through a 2 ml protein A-Sepharose CL4B column (Pharmacia). After the column was washed exhaustively, the bound material was eluted with 3 M potassium thiocyanate, pelleted by centrifugation at 100 000 × g for 1 h in a Beckman ultracentrifuge, and stored at -20°C until used. For immunization, the pellet was thawed and resuspended in PBS by sonication. BALB/c mice were immunized intravenously with the membrane preparation in PBS and boosted once a week three times. Each inoculum contained membranes of approximately 1.6×107 erythrocytes. Sera were obtained one week after the last injection by retro-orbital venepuncture.
Indirect immunofluorescence.
Glutaraldehydefixed, air-dried erythrocyte monolayers were prepared using the method described by Perlmann [10]. For indirect immunofluorescence, the erythrocytes were incubated sequentially with dilutions of tested antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM and IgG (1:50 dilution, Boehringer Mannheim) for 30 min each. The slides were washed in PBS after each incubation. The parasite nuclei were counterstained for 5-10 min with ethidium bromide (100 ~g ml-l). Occasionally, we used 6.7 ng m1-1 biotinylated goat anti-mouse IgG, or IgM (Boehringer Mannheim) and 20 ~g m1-1 FITClabelled avidin (Sigma) to detect the antigen-antibody reaction.
Monoclonal antibody (MoAb) production. The method described by Kohler and Milstein [11] was used with some modifications. Spleen cells from a BALB/c mouse immunized with the parasitized erythrocyte membrane fraction were used after the third booster. Fusion with P3U1, a myeloma cell line, was achieved using polyethylene glycol. Thirty colonies were positive for the membrane IFA of infected cells. Four colonies were cloned by limiting dilution and implanted into the peritoneal cavities of CD2F1 mice and ascitic fluid was collected. 8F2G9, an IgG1 MoAb, was purified from ascitic fluid by affinity chromatography using Affi-Gel Protein A MoAb Kit (Bio Rad). The
three IgM MoAbs, 6C12E7, 6DllE6, and 7C6E10 were purified by Sephacryl S-300 (Pharmacia) gel filtration. The isotype of each MoAb was determined by double diffusion in agarose using the monoclonal hybridoma culture supernatant fluid against a panel of isotype-specific antibodies. The purity of each MoAb was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. SDS-PAGE was carried out as described by Laemmli [12] with a 5% stacking gel and 7.5% separating gel. For Western protein blotting [13] the proteins resolved in SDS gel were electrophoretically transferred to a nitrocellulose sheet and probed with dilutions of sera or MoAbs. Signals for antigen-antibody binding were provided by the second antibody, 125I-goat anti-mouse IgG or IgM (2 x 10 6 counts min -1 m1-1) with subsequent autoradiography.
Metabolic labelling of parasitized erythrocytes and immunoprecipitation. Heparinized blood was obtained from a mouse with 30% P. chabaudi parasitemia. To remove fibrinogen, white blood cells and platelets, ADP at 50 ~g ml -~ was added to the blood which was then passed sequentially through columns of glass beads and cellulose powder (CFll). The red cells were then washed three times with RPMI-1640 tissue culture medium (GIBCO) and resuspended to 4x108 cells m1-1 in RPMI-1640 containing 10% fetal calf serum. [3SS]Methionine (50 txCi m1-1) was then added to the cell suspension and incubated for 8 h at 37°C in an atmosphere of 5% O2, 5% CO2, 90% N 2. Afterwards the cells were pelleted and washed three times with chilled PBS and solubilized with 1% Triton X-100 detergent containing protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, tosyllysine chloromethyl ketone, leupeptin, and chymostatin) at a final concentration of 4×108 cells m1-1, centrifuged at 16000 x g for 15 min at 20°C and the supernatant stored in liquid nitrogen until used. For immunoprecipitation, the 1% Triton X-100 extract (200 txl) of the metabolically labelled cells was added to 10 Ixl of MoAb 8F2G9, 7C6E10, and
197 a mixture of two irrelevant IgG1 and IgM MoAbs. The mixture was incubated at 4°C for 18 h and then the immune complexes in each sample were precipitated with 100 txl of 50% suspension of immunoadsorbent (goat anti-mouse IgG or IgM conjugated to Affi-Gel 10) in N E T T buffer (150 mM NaCl, 10 mM E D T A , 50 mM Tris, 0.5% Triton X-100). The immune complex-bound immunoadsorbent was pelleted, washed 5 times with the following: N E T T , N E T T , N E T T + 0.5 M NaC1, NETT, and N E T (NETT without Triton X100). The immune complexes were then eluted with S D S - P A G E sample buffer containing 2mercaptoethanol at 100°C for 5 min and subjected to S D S - P A G E in 7.5/5% slab gel. The gel was soaked in E n H a n c e (New England Nuclear) for 1 h, washed in deionized water for 1 h, dried, and subjected to fluorography at -70°C.
Affinity purification of the P. chabaudi antigen. Six ml of Affi-Gel 10 chromatography matrix (BioRad) was coupled with 12 mg of 6 D l l E 6 , an IgM M o A b , using the coupling method recommended by the manufacturer. Heparinized blood from mice with 20-30% parasitemia was washed three times with PBS and solubilized with deter-
gent solution and protease inhibitors at the following final concentrations: 3 x 109 erythrocytes m1-1, 1% (3-[3-cholamidopropyl]-dimethylammonio)-l-propanesulfonate, 1 mM phenylmethylsulfonyl f u o r i d e , 5 mM iodoacetamide, 5 mM E D T A , 5 mM E G T A , 0.2 mM tosyllysine chloromethyl ketone, 25 ixg m1-1 chymostatin, 25 Ixg m1-1 leupeptin. The mixture was centrifuged at 10000 x g for 1 h to remove particulate materials. The soluble mixture was then passed through the 6DllE6-affinity column and washed exhaustively with PBS. The bound material was eluted with 3 M potassium thiocyanate and was passed immediately through Sephadex G25M (Pharmacia, PD10) pre-equilibrated with PBS for the removal of potassium thiocyanate. This preparation was designated 6 D l l E 6 : A g .
Vaccination of mice with 6DllE6.'Ag. Two groups of five female Swiss Webster mice (4-6 weeks old), were vaccinated intraperitoneally with 50 ixg of 6 D l l E 6 : A g supplemented with 50 ~tg of saponin as the adjuvant [14,15] in PBS. The mice were boosted once a week, for three weeks. The controls received saponin in PBS or PBS alone. Six days after the last booster, they were chal-
Fig. 1. (A,B) Indirect immunofluorescence(IFA) of P. chabaudi-parasitized erythrocytes. Erythrocytes were fixed with 1% glutaraldehyde and air-dried. The fluorescenceon the perimeter of infected-erythrocytesis due to specificantigen-antibody reaction as detected by FITC-goat anti-mouse IgG+IgM. The intra-erythrocyticparasites appear as fluorescencedots due to counterstaining with ethidium bromide. MoAb 6DllE6 was used in panel A; same concentration of normal mouse globulin as the control was used in panel B. Scale bar, 10 ~xm.
198 lenged by intravenous inoculation of 104 P. chabaudi-parasitized erythrocytes. Parasitemia was followed daily by examination of Giemsa-stained blood smears from the tail vein. Results
Demonstration of malarial antigen in the membrane of P. chabaudi-parasitized erythrocytes. Sera of mice immunized with the erythrocyte membrane fraction (prepared from infected erythrocytes) were used to detect the putative malarial antigen(s) associated with the surface of P. chabaudi-parasitized erythrocytes using indirect immunofluorescence (Fig. 1A). The immune sera did not react with normal erythrocytes but produced a uniform membranous fluorescence pattern on the perimeter of parasitized erythrocytes regardless of the maturational stage of the parasite. No fluorescence was observed on parasitized erythrocytes incubated with normal mouse sera (Fig. 1B). These findings demonstrated the presence of a parasite-induced antigen in the eryth-
AB CD
20
11
E
Monoclonal antibodies for Pc96. Mouse MoAbs were developed [16]. In the following studies we used two MoAbs, 8F2G9 (IgG1,K), and 6 D l l E 6 (IgM,K). Both produce the same immunofluorescence pattern as in Fig. 1A and recognize Pc96 (Fig. 2, lanes E,F). In this and subsequent gels the MoAbs also recognized several lower M, bands which are probably degradative products of Pc96.
G
F
H
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K
L
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11 9
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6
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96
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rocyte membrane of P. chabaudi-infected red cells. To identify the corresponding antigen, the parasitized erythrocytes were subjected to SDSP A G E , transferred onto nitrocellulose sheets and probed with the immune sera. Only one parasite antigen, the 96 kDa band (Pc96) was detected by immune serum from mice immunized with the erythrocyte membrane preparation (Fig. 2, lane A, compared with normal erythrocytes in lane B). In contrast, polyspecific hyperimmune serum detected numerous parasite antigens in the parasitized erythrocyte extract in lane C, compared with the normal erythrocyte extract in lane D.
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Fig. 2. Western blotting of parasitized erythrocytes. SDS-extract of P. chabaudi-parasitized erythrocytes (lanes A,C,E-L) or normal erythrocytes (lanes B,D) was resolved by SDS-PAGE under reducing conditions, transferred onto nitrocellulose sheets and probed with membrane-immune serum (lanes A,B), MoAb 8F2G9 (lanes E,G), MoAb 6DllE6 (lane F), 6DllE6:Ag-immune sera (lanes H-L), or hyperimmune sera from mice repeatedly infected with P. chabaudi (lanes C,D),
199
Biosynthesis of Pc96. To determine the origin of Pc96, in vitro biosynthesis was carried out in erythrocytes from a P. chabaudi-infected mouse incubated in media containing radioactive amino acids. The cells were then solubilized with Triton and immunoprecipitation using M o A b was done. The autoradiograph of the precipitated antigens resolved by S D S - P A G E shown in Fig. 3 demonstrates that the 96 k D a band, corresponding to Pc96, was synthesized during the in vitro culture and precipitated by M o A b 8F2G9 (lane A), and 7C6E10 (lane B), compared with the control in lane C. This indicates that Pc96 is parasite-encoded.
AB
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EF
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~
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116--
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92--
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Affinity-purification of Pc96. Pc96 was purified by affinity chromatography using 6D11E6 as the li-
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--96
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+++,+~
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Fig. 4. Antigenic analysis of 6DllE6:Ag. 6DllE6:Ag was resolved in SDS-PAGE under reducing conditions and stained with Coomassie blue (lanes A,B). Western blotting of the same material was probed with MoAb 8F2G9 (lanes C,D) or pooled immune sera from mice vaccinated with the same material (lanes E,F).
ABC
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++++++
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Fig. 3. Metabolic labelling and immunoprecipitation of Pc96. P. chabaudi-infected erythrocytes were biosynthetically labelled with [35S]methionine, detergent-solubilized, and immunoprecipitated with MoAb 8F2G9 (lane A), 7C6E10 (lane B), or unrelated IgG1+IgM MoAbs (lane C).
gand. Fig. 4, lanes A and B show the SDS-PAGE of the eluted material (2 different batches), designated 6 D l l E 6 : A g . In addition to the 96 kDa band, Coomassie blue staining of 6 D l l E 6 : A g reveals two other bands of 200 and 75 kDa. To analyze its antigenic components, pooled sera from mice vaccinated with 6 D l l E 6 : A g (below) were used to probe the same material (lanes E,F) in Western blotting. The figure indicates that the major antigenic components of 6 D l l E 6 : A g are bands of 96 and 82 kDa and their degradative products, which is the same pattern recognized by M o A b 8F2G9 (lanes C,D). The 200 and 75 kDa bands were not recognized by the immune sera. Therefore, with the exception of the 82 kDa, which may be a degradative product of the 96 kDa or a cross-reactive antigen, Pc96 is probably the only parasite major immunogen in the 6 D l l E 6 : Ag preparation.
Experimental vaccination using 6DllE6:Ag. Mice were vaccinated with the affinity purified 6DllE6: Ag and challenged six days after the third booster with blood-induced P. chabaudi infection. Fig. 4
200
I
4oi
t'-
?O-
J 2° r 1 2 3 4 5 6 7 8 9 10 11 12134415 16 Days After Intravenous Challenge [noculurn with 104 P chabaudi- Parasitized Erythrocytes
Fig. 5. Experimental vaccination with 6DI1E6:Ag. Course of P. chabaudi challenge infection in the mice vaccinated with 6D11E6:Ag batch l+saponin (m), 6DllE6:Ag batch 2+saponin (A), saponin in PBS (o), or PBS alone (e).
shows parasitemia of the vaccinated and control mice. In all groups, patent parasitemia commenced approximately three days after the challenge inoculation and then rose in parallel for a few days. However, in all the vaccinated mice, parasitemia remained less than 10% and then dropped sharply. In contrast, parasitemia in the control groups continued to rise to 40-50% before declining two days later than the vaccinated groups.
6DllE6:Ag-induced monospecific antibody response against Pc96. Sera taken from all the 6D11E6:Ag-vaccinated mice three days before the challenge had very high titers (104-105) of specific antibody for the membranes of P. chabaudi-parasitized erythrocytes. This antibody activity is species specific since the sera did not cross-react with fixed parasitized erythrocytes from mice infected with P. berghei or P. yoelii (data not shown). Furthermore, in Western blot analysis, all the sera recognized only one antigen of P. chabaudi-parasitized erythrocytes (Fig. 2, lanes H-L) with the banding pattern corresponding to Pc96 (lane G). Discussion
These studies demonstrate that Pc96 is a parasite-encoded antigen of Mr 96000 on or in the surface of P. chabaudi-infected erythrocytes. Partial protective immunity against P. chabaudi
could be induced in mice using 6DllE6:Ag, the parasite antigen affinity-purified with Pc96-specific MoAb. Although 6D11E6:Ag contained other proteins in addition to the 96 kDa, the data strongly suggest that Pc96 is the only parasite immunogen in the preparation since mice vaccinated with 6DllE6:Ag produced antibodies only against Pc96 and its fragments. The protective immune mechanisms induced by Pc96 remain to be determined. Passive immunization with MoAb did not protect mice against P. chabaudi (data not shown); however, since MoAbs are epitope specific this does not exclude the possible role of antibodies specific for other epitopes in protection. Studies of passive immunization with sera and T-lymphocyte subsets, should help to elucidate the immune effector mechanisms involved in protective immune response to Pc96. The relationship of Pc96 to parasitized erythrocyte surface antigens of other malaria species is of interest. Since we cannot agglutinate P. chabaudi-parasitized erythrocytes with either membrane-immune sera or MoAb (data not shown) we do not think that this antigen is analogous to the SICA antigen of P. knowlesi [2]. Recently, Perlmann et al. [10] and Coppel et al. [17] independently described an Mr 155 000 protein antigen of P. falciparum. This antigen, designated Pf155 or RESA, is being considered as a possible vaccine candidate since Pf155-specific MoAb inhibited merozoite invasion [18]. Recently, Coppel et al. described MESA, an Mr 250 000 antigen, which is associated with the surface of human erythrocytes containing mature forms of P. falciparum [19]. In rodent systems, antigens of Mr 105000, i.e. Pchl05 (P. chabaudi) [20] and 160000 (P. yoeIii) [21] in the surface of fixed infected erythrocytes have been described. A Pchl05-specific MoAb, generously provided by Drs. P. Perlmann and K. Berzins, recognizes a band of the same M r as the Pc96-specific MoAb (data not shown). Further, this MoAb produces the same pattern of immunofluorescence as the MoAbs that we describe. Interestingly, 7C6E10, a Pc96-specific MoAb produces RESA-type fluorescence with P. falciparum and recognizes a 155 kDa antigen in Western blotting of P. falciparum-infected erythrocytes (Wanidworanun, C., Barnwell, J.W. and
201 Shear, H . L . , in p r e p a r a t i o n ) . T h e r e f o r e , Pc96 a p p e a r s to be the s a m e as P c h l 0 5 a n d an analogue of Pf155 a n d R E S A . In c o n c l u s i o n , successful active i m m u n i z a t i o n with Pc96 in mice s h o u l d offer a useful a n i m a l m o d e l for the s t u d y of m e c h a n i s m s of p r o t e c t i v e i m m u n i t y a n d c o r r o b o r a t e the p o t e n t i a l of Pf155 a n d R E S A as a m a l a r i a vaccine c a n d i d a t e .
9 10
11
Acknowledgements We thank Drs A . H . Cochrane and A. Masuda for their help in the h y b r i d o m a production and the earlier parts of this study; V. N u s s e n z w e i g , R.S. N u s s e n z w e i g , a n d A . F e r r e i r a for r e a d i n g the m a n u s c r i p t a n d h e l p f u l suggestions, M. F e r r e i r a for technical assistance, a n d B. R o b l e s for secretarial assistance. S u p p o r t e d by AI15235 from the N I H a n d D A M D 17-85-C-5175 from the U S Army.
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