Molecular and Biochemical Parasitology, 54 (1992) 231 246 ,~:) 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

231

MOLBIO 01795

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum S h i r o m a M. H a n d u n n e t t i a' , Brittan L. Pasloske a, M a r i e - R o s e van Schravendijk a, Joao C. A g u i a r a, T h e o d o r e F. Taraschi b, Jill A n n G o r m l e y b a n d Russell J. H o w a r d a aDNAX Research Institute, Palo Alto, CA USA. and bDepartment of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, PA, USA (Received 13 February-1992; accepted 11 May 1992)

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of >200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E 11 (IgM, x), the other by clone 12C11 (IgM, 2). MAb 41El 1 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 4 1 E l l reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41El 1 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41Ell did not immunoprecipitate any cell surface ~25I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecularweight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface ~25I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41El 1. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12Cll was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMPI and is most likely a hitherto unidentified malarial protein. Key words: Plasmodium falciparum; Erythrocyte membrane; Malarial antigen; Monoclonal antibody 33G2; Rat monoclonal antibody

Introduction At least 10 Plasmodium falciparum proteins larger than 200 kDa can be identified by Correspondence address: Russell J. Howard, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304-1104, U.S.A. *Present address." Malaria Research Unit, Faculty of Medicine, Colombo, Sri Lanka. Abbreviations: PfEMP, Plasmodium falciparum erythrocyte membrane protein; PE, parasitized erythrocyte; E, erythrocyte; mAb, monoclonal antibody; Ag, antigen; IFA, immunofluorescence.

immunoprecipitation with human immune sera of biosynthetically radiolabeled extracts from erythrocytes infected with mature asexual stage parasites (PE) and SDS polyacrylamide gel electrophoresis under reducing conditions (e.g., ref. 1). Only a minority of these molecules have been defined unambiguously with specific antibody reagents and often only limited sequence information is available for those that have been named and defined. Interest in this class of malarial proteins stems not only from the fundamental question of the functional roles for such very large molecules, but the observation that several of these molecules are associated with the PE host cell

232

membrane (either under the membrane or exposed at the PE surface) where they may participate in the numerous morphological, antigenic and functional changes in the E membrane that accompany parasite maturation [2,3]. Pf EMP2/MESA/PP300 is an approximately 2 4 0 4 0 0 - k D a P. falciparum protein [4-9] that is defined by specific monoclonal antibodies (mAbs) [4] and complete c D N A sequence [5]. This protein binds to submembrane cytoskeletal components [8] and is localized to the electron dense material under knob protrusions on PE [4,5]. Antigen D260, of similar size to PfEMP2, has different properties of detergent extractibility and differential mAb and polyclonal antibody reactivities [10,11]. The sequence of D260 has not been published. Another very large protein, of approx. 220 kDa, appears to belong to a family of antigenically cross-reactive arginine-rich proteins but has not been extensively characterized due to lack of specific reagents to differentiate it from other antigens in this family [12]. The first very large protein expressed in asexual stage parasites for which partial nucleic acid sequence was obtained, Pfl of approximately 240 kDa [13], has been neglected and little is known of its location in PE or of its significance. Another antigen, Pfll.1, was initially described as a 260-kDa asexual stage protein at the time that a partial gDNA sequence was published [14]. Extensive nucleic acid sequence of the Pfl 1.1 gene was obtained [15] and structural polymorphism studied in different parasites [16]. Subsequent studies concluded that the original g D N A actually corresponded to an asexual stage antigen larger than one megadalton in size [11]. More recently, the M D a protein has been demonstrated to correspond to another malarial gene sequence called 332, and P f l l . l has been described as a high-molecular-weight sexual stage antigen absent from asexual parasites [17]. The asexual stage antigen originally called Pfll.1 probably corresponds to the D260 protein. Another very large malarial protein called PfEMPI [9,18 20] or Sequestrin [21], may contribute to the adherence of PE for

endothelial cells [9,18-21]. Study of PfEMPI has been hindered by lack of specific polyclonal antibody or mAb and lack of any nucleic acid or amino acid sequence. There are considerable difficulties in defining very large malarial antigens. MAbs or affinity-purified antibodies may react with a short, linear amino acid sequence that may occur by chance in numerous malarial proteins (and recombinant proteins). In addition, many malarial proteins share sequences and often the shared sequences are relatively immunogenic and antigenically reactive [22 25]. For example, D260 and Ag332, belong to an antigenically cross-reactive family of proteins including Pfll.1 and Pf155/RESA, all of which have cross-reactive determinants recognized by the human IgM MAb 33G2 [24,25]. The epitope recognized by MAb 33G2 is the repetitive motif EEXXEE [24,25]. Adherence of a knobless P. falciparum parasite to C32 melanoma cells was inhibited by mAb 33G2, indicating that this amino acid repeat motif is expressed on the surface of PE and potentially, that a protein bearing this motif is involved in PE adherence [26]. Since the amino acid motif recognized by mAb 33G2 is shared by many proteins, it is difficult to determine which, if any, of the known proteins might be expressed on the PE surface. The lack of monospecific reagents for definition and study of very large malarial antigens led us to produce monoclonal antibodies against trophozoite/schizont proteins of >200 kDa. We describe production of 2 rat mAbs that react with malarial antigens > 200 kD: m A b 4 1 E l l which recognizes the EEXXEE family of cross-reactive antigens, and, m A b l 2 C l l which defines a distinct set of proteins, including one of approximately 310 kDa. The latter protein is distinct from D260 and PfEMP2. We describe the detergent extraction properties of these antigens, their relative mobilities on SDS-PAGE, the patterns of IFA reactivity of these mAbs with fixed cells, and the stage-specific expression of the antigens.

233

Materials and Methods

Antibodies and proteins.

Mouse IgG mAb 8B7.4 was used as ascites [4]. The human IgM mAb 33G2 [24,25] was generously provided by K. Berzins, as 0.5 mg m1-1 purified mAb and was used at 4 ~tg ml-1 for Western blotting. Control rat IgM myeloma protein, from ascites of clone IR202, was from Zymed Laboratories, South San Francisco, CA (Cat. No. 02-9800). Human hemoglobin was from Sigma Chemical Co, St. Louis, MO (Cat. No. H7379).

Parasite culture and purification. Experiments were performed with two culture-adapted parasites, the Malayan Camp (MC) line [27] and clone FCR-3 C-5 [28]. The methods of in vitro culture, selection protocols for maintenance of parasite phenotype and synchronization of MC parasites have been described [29]. These parasites are knob-positive (K+); C32 melanoma cell, CD36 and thrombospondin (TSP) adherence-positive and rosetting-positive ( > 80% of mature asexual stage PE with >2 rosetted E) [29,30]. To study antigen expression by different stages of this parasite, synchronization was performed as follows: trophozoite stage PE were incubated with 100 /~g m1-1 trypsin (Sigma Chemical Co., Cat. No. T9395) in RPMI 1640, at 37°C, for 45 min to disrupt rosettes. Fetal bovine serum was added to 10% (v/v) and the parasites washed with RPMI. PE were purified using gelatin, washed with RPMI 1640 and added back to culture at 6% parasitemia. Upon reinvasion, the parasitemia was 12%. Fresh medium was added and aliquots taken at different times post-invasion. Purification of PE was performed as follows: rosettes were disrupted by treatment of the washed blood for 15 min at 37°C with 100 units m1-1 heparin (Sigma) in PBS [31]. Mature trophozoite stage PE were purified to >85% parasitemia either by centrifugation on a Percotl/sorbitol gradient or unit gravity sedimentation through 0.7% (w/v) gelatin (Sigma; approx. 300 bloom) in RPMI 1640 for 45 min at 37°C [29,32]. FCR-3 C5 parasites were grown in culture

medium comprising RPMI 1640/2 mM glutathione/24 mM NaHCO3/25 mM Hepes/0.44 mM hypoxanthine/21.1 mM glucose/66 #g ml - l gentamicin/10% (v/v) human serum. These parasites are K +, lack detectable rosettes and adhere to C32 cells, CHO cell transformants expressing CD36 or human platelet-derived CD36. Selection of K + PE was performed once every 4~5 days by the gelatin method [33]. To obtain highly synchronous FCR-3 cultures, PE were fractionated on 1% (w/v) gelatin to enrich mature K + PE and synchronized to within 3 h by repeated treatments with 5% (w/v) sorbitol and Percoll step gradient centrifugation.

Reversal of rosetting and parasitized ervthrocyte agglutination assays. The capacity of antibodies to reverse rosettes of E around PE [34] and to cause agglutination of PE [35] was measured by previously described methods.

Detergent extraction.

5 x l0 s PE were sequentially extracted with 1 ml 1% Triton X-100 in PBS and with 1 ml of 2% SDS in PBS for immunoblotting or immunoprecipitation experiments [4,9].

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Proteins were fractionated by SDS-PAGE under reducing conditions [36] using a Mini Protean II Electrophoresis Cell (Bio-Rad Laboratories, Richmond, CA). Apparent Mr was determined by interpolation of a standard curve using prestained standards: myosin, 205 kDa; #galactosidase, 116.5 kDa; phosphorylase B, 106 kDa; bovine serum albumin, 80 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa (Bio-Rad Laboratories). For immunoblotting experiments, parasite extracts from approx. 4 x 107 PE were loaded into a preparative well (72 x 0.75 mm). After SDS-PAGE, proteins were transferred to Immobilon-P (Millipore, Bedford, MA; IPVH 00010) for 1 h at 80 V in transfer buffer (192 mM glycine/25 mM Tris base/20% methanol, pH 8.3) [37] using a Mini Trans-Blot Cell (Bio-

234

Rad). The Immobilon membranes were blocked with 20% fetal calf serum in TBS (10 mM Tris base/150 mM NaC1, pH 8.0) 30 rain, 23°C, incubated with hybridoma supernatant at 1 × concentration or with a 1:500 dilution of mAb 8B7.4 ascites (specific for PfEMP2; refs. 4,9) in TBST (TBS containing 0.05% Tween 20), 30 min, 23°C and washed twice with TBST. The membranes were incubated 30 min, 23°C with an alkaline phosphataseconjugated antibody: either goat anti-mouse IgG (whole molecule) (Cappel/Organon Teknika, West Chester, PA), p-chain specific goat anti-human IgM (Cappel), or, p-chain specific goat anti-rat IgM (Jackson Immunoresearch Laboratories, West Grove, PA) diluted in TBST (1:5,000), washed twice with TBST and stained using the Western Blot Alkaline Phosphatase Conjugate Substrate Kit (BioRad; 170-6432) containing 5-bromo-4-chloro3-indolyl phosphate and Nitroblue tetrazolium.

Analysis of radioactivity on polyacrylamide gels. Gels containing 125I-labeled or biosynthetically radiolabeled malarial proteins were dried and the distribution of radioactivity analysed by autoradiography or fluorography using Amplify (Amersham, Arlington Heights, IL) [9].

Rat monoclonal antibody production. 2% SDS extracts of purified trophozoite/schizont stage PE were fractionated using preparative 5% SDS-PAGE. Gels were stained with Coomassie Blue and the region between the top of the gel and the spectrin 1.1 band excised from several preparative gels in 7 segments of 1-2 mm, taking care to include each of the major stained bands in only one gel segment and to excise each gel in the same manner. The slices were homogenized individually with PBS. Pairs of male Lewis rats were immunized by i.p. injection with the different bands emulsified in Freund's Complete Adjuvant. Animals were reimmunized on day 41 using Freund's Incomplete Adjuvant. Each immunization contained protein derived from 2 x 108 PE. Antisera were collected 8 days after the second

immunization. One rat was immunized a third time on day 150. Four days later the spleen cells from this animal were fused with the murine cell line P3-X63-Ag8.653 [38]. On day 14 postfusion, hybridoma supernatants we~-e assayed for reactivity with PE extracts. Polyvinyl chloride microtiter plates (Dynatech, Chantilly, VA) were coated with sonicated E or trophozoite/schizont PE preparations (2 x 106 cells per well) in carbonate buffer (15 mM Na2CO3/35 mM NaHCO3/0.02% NAN3, pH 9.6) overnight, 4°C. The plates were washed with PBST (PBS containing 0.05% Tween 20), blocked with 1% BSA in PBS, 1 h, 22°C and hybridoma supernatants (diluted 1:3 in PBST), or dilutions of rat sera in PBST, added to the wells for 4 h at 4°C. PBST washing was repeated and alkaline phosphatase conjugated goat anti-rat IgG (whole molecule) (Cappel/ Organon Teknik.a) added, 1:1,000 in 0.1% BSA in PBST, for 1 h, 23°C. Plates were developed using the ELISA Alkaline Phosphatase Substrate Kit (Bio-Rad; 172-1063) containing p-nitrophenyl-phosphate. Reaction was stopped by adding 10 mM EDTA and the absorbance measured at 405 nm. Hybridoma cells producing supernatants that produced ~>2 × the absorbance value with PE extract compared to E extract were expanded and rescreened. Those still positive were expanded in 24 well plates and frozen using fetal bovine serum (J R Scientific, Woodland, CA) and 10% DMSO (Sigma). Selected hybrids were cloned twice by limiting dilution. Hybridoma clones 41Ell and 12Cll were adapted to growth in HB101 serum-free medium (Irvine Scientific) by sequential adaptation to growth in mixtures of RPMI/FCS and HB101 medium. Culture supernatants of these clones were collected by centrifugation at 1000 x g and filtration through a 0.2 pm membrane. Concentrated samples of these culture supernatants were dialyzed against PBS or RPMI and had IgM contents of 1.5 mg m l - i and 2.0 mg m l for mAb 41Ell and mAb 12C11 respectively. Their total protein contents were approximately 1%.

Isotyping of monoclonal antibodies. ELISA

235

plates were coated with sonicated PE extracts (5 × 10 7 PE ml - l ) in PBS, at 4°C, overnight, blocked with 1% BSA (globulin-free, Sigma Cat. No. A7638) in PBS, 1 h, 22°C, and washed with PBST. The monoclonal supernatants were incubated in the wells, 45 rain, 22°C. After washing with PBST, antibody isotypes were determined using the Rat Mono-ID/SP Kit (Zymed Laboratories Inc., South San Francisco, CA). A panel of purified rat myeloma standards (Zymed, Cat. 90-9551) served as controls.

Indirect immunofluorescence assay of fixed parasitized erythrocytes. Blood containing synchronized parasites was washed in PBS and cells resuspended at 5 /~1 ml ~. 35 pl of suspension was added to Cell-Tak (Collaborative Research Inc., Bedford, MA) coated coverslips and the cells allowed to adhere for 60 min at 37°C. Fixation was performed for 120 h using 1% or 2% paraformaldehyde (w/v) in 37.5 mM sodium phosphate with identical results. Quenching was performed by 3 washes with 0.5 mg m l - ' o f NaBH4 in PBS followed by 3 washes in PBS. Alternatively, samples were fixed for 2 min at - 2 0 ° C with 100% acetone. After either fixation, coverslips were incubated for 30 min at 22°C with 5% (v/v) human serum in PBS. Concentrated samples of hybridoma supernatants, described above, were diluted between 1:50 and 1:500 in 5% (w/v) normal human serum in PBS and added to the fixed cells for 60 min incubation at 22°C. After washing 3 times with PBS, secondary antibodies (goat anti-rat IgM, fluorescein isothiocyanate or Texas Red labeled, Fisher Biotechnic Division) at 1:50 or 1:100 dilutions of commercial stocks (1.0 mg m1-1) in PBS containing 5% (w/v) human serum were added for 30 min at 22°C. Samples were then washed 3 times with PBS and mounted for confocal microscopy using 50% glycerol in PBS.

Confocal microscopy. Confocal fluorescence imaging microscopy using a BioRad MRC 600 instrument was performed at excitation wavelength of 488 nm and emission with a 515 nm long-pass filter for FITC labeled antibodies,

or, excitation at 514 nm and emission detection with a 600 nm long-pass filter for Texas Redlabelled antibodies. The spatial resolution was approx. 0.3 pm and z-axis resolution approx. 0.8 ~tm.

Cell-surface iodination, biosynthetic labeling and immunoprecipitation. Mature trophozoite stage PE of the MC strain were purified (85 90% parasitemia), rosettes disrupted with heparin and PE obtained after gelatin selection for surface iodination by the lactoperoxidase mefhod [9,18,19]. In other experiments to biosynthetically label malarial proteins [4,9], PE were cultured from rings to trophozoites with 100/~Ci/ml-l [35S]methionine in medium containing 10% of the normal level of this amino acid. 35S-labeled detergent extracts were immunoprecipitated as described earlier [4,9] except that rabbit anti-rat IgG (whole molecule) and rabbit anti-rat IgM (~t-specific) (Jackson Laboratories) was incubated with the extracts for 30 min, 23°C, before addition of Protein-A Sepharose.

Results

Rat monoclonal antibody production. After immunization of pairs of rats with different gel slices containing proteins of very large size, sera from each animal in one pair reversed rosettes by 30 33% and agglutinated intact PE. These same gel slices included the 125IPfEMP1 band as immunoprecipitated by antiMC Aotus monkey sera. None of the preimmune sera were reactive. The spleen from one of these rats was fused with the P3-X63Ag8.653 murine cell line to produce hybridomas. Nine hybridoma supernatants reacted better by ELISA with a total PE sonicated extract than with an E extract (A > 2 and A < 0.01 respectively). Some supernatants also agglutinated intact PE. Western blotting was performed with each supernatant using sequential Triton X100 and SDS extracts from uninfected E and purified PE. Acrylamide gels were electrophoresed 2 h off the end to allow high resolution of very large proteins. The 9

236

hybridoma supernatants reacted with PE proteins but not with E proteins. Eight hybridoma supernatants gave the same pattern of Western blot reactivity. One of these hybridomas was cloned and designated 41El 1. The remaining supernatant gave a distinct Western blot reactivity and was cloned to yield the 12Cll hybridoma. The monoclonal antibodies (MAbs) produced by these cell lines were M A b 12Cll, an IgM(2) and M A b 4 1 E l l , an IgM(~:).

Tests for agglutination and rosette reversal with monoclonal antibodies. When sera from immune African adults are added to blood containing rosettes of E around PE of the MC strain, large PE agglutinates form. These agglutinates vary in size from 10s to 100s of PE and can include almost all PE in the blood sample. Since > 9 0 % of PE were originally in rosettes with > 2 adherent E/PE, and no rosettes remained among the few non-agglutinated PE seen after addition of such sera, we conclude that all rosettes have been reversed. Both mAbs 12Cll and 4 1 E l l agglutinated PE in samples of MC strain parasites with > 9 0 % of the mature PE in rosettes. These antibodies yielded agglutinates with 10s to 100s of PE tightly apposed in the core of an agglutinate. Only a minority of PE (estimated < 10% of total PE) were agglutinated even with the highest antibody concentrations (up to 500 #g/ml-1). None of the antibodies agglutinated E. Unlike agglutinates with human immune sera, these agglutinates retained some rosetted E, still adherent to PE, especially at the agglutinate periphery. However, in some experiments using the same concentrations of control rat IgM antibodies (either polyclonal IgM derived from rat serum or monoclonal IgMs), PE were agglutinated just as effectively. In view of the occasional agglutination of PE by control as well as test antibodies we did not pursue the agglutination test further. The mAbs were tested for reversal of rosettes in experiments wherein human immune sera at 1:5 dilution reversed 80-90% of the rosettes. Neither m A b affected rosettes during 60 min incubation at 37°C and at

antibody concentrations of 1-500 /tg ml ( > 1 0 % rosette reversal could have been detected). Efforts have also been made to identify reactivity of these mAbs with the surface of intact PE by IFA, using secondary or tertiary fluorescent labels to identify bound mAb. Although weak fluorescence was seen with some PE, this was not reproducible. We also tested whether either mAb blocked adherence of PE to stable C H O cell transformants expressing CD36. In 3 tests, 10-500 #g ml -~ mAb 12Cll blocked PE adherence by 2070%, while mAb 41Ell never had any effect. Control IgM antibodies did not affect adherence. However, in 4 other experiments mAb 12Cll had no effect on adherence. Consequently, we have not obtained reproducible reactivity of mAbs 12Cll or 4 1 E l l with the surface of PE.

Western blot reactivity of mAb 12Cll. Western blotting was performed using Triton X100 and SDS extracts of PE collected at different times during synchronous growth from ring to segmenter stages (Fig. 1). M A b l 2 C I 1 reacted predominantly with 4 malarial proteins of approx. 310, 145, 117, and 95 kDa (designated Ags 12A, 12B, 12C, and 12D, respectively) (Fig. 1). Ag 12A was always a dominant reactive band. In some experiments Ags 12B, 12C and 12D were less intensely labeled. Up to 15 other weakly reactive bands were detected with mAb 12C11, but only in some experiments such as the example in Fig. 1. M A b l 2 C l l did not bind to any highmolecular-weight antigens until the early trophozoite stage (18 h). A mAb 12Cllreactive protein band was detected at approx. 310 k D a in both the Triton X-100 and SDS extracts. For simplicity we will refer to both of these bands as Ag 12A, although it is possible that these two bands are completely different proteins or different forms of the same protein with distinct detergent extraction properties. A g l 2 A was initially expressed at the early trophozoite stage primarily as a Triton X-100 soluble antigen, with minor reactivity in the

237

6

18

27

35

41

6

18

27

41

35

41A •. , - ,

--

,,~

~

~12A

41B 205 --

205 --

T!

N o

m

~ '~ 12B 116.5 -

~

~

12C

80 -

N

,~

,,,.,,,=

,,,=~

4laID

.,..~

m

-.N

41C 41D

-"

41E

116.5 -

80 MAb 1 2 C l l

MAb 41 E11

Fig. 1. Western blot analysis of P../ah'iparum antigens that react with mAbs 12Cll or 41El 1. Samples of blood were taken from a synchronous in vitro culture of MC strain parasites (12% parasitemia) at increasing times after merozoite reinvasion (h 6, 18, 27, 35 and 41 indicated above the gel lanes) and extracted sequentially with 1% Triton X-100 and 2% SDS. These extracts, labeled T and S respectively above each gel lane, were electrophoresed on a 5% acrylamide gel (the dye front was run to the bottom of the gel) and transferred for blotting with m A b 12C11 or mAb 41El 1. Prominent antigens reactive with these mAbs are indicated as 12A-12D or 41A-41E as appropriate. Positions of migration of standard proteins are indicated in k Da on the left of the immunoblots.

SDS extract. As parasites matured, the proportion of total mAb 12C11-reactivity in the SDS extract increased. Ag 12B (145 kDa) and Ag12C (117 kDa) were detected mainly as Triton X-100 soluble products whereas Ag 12D (95 kDa) was Triton X-100 insoluble. On 15% acrylamide gels of segmeriter parasites we also detected a quantitatively minor PE antigen doublet (45 and 47 kDa) recognized by mAb 12C11, designated Ag12E (Fig. 2). Agl2E was not detected with uninfected normal E (Fig. 2). During the asexual cycle Agl2E was initially expressed at the midtrophozoite stage (data not shown). Agl2E was not extracted by Triton X100 but was extracted with SDS. MAb12C11 also reacted with two normal E antigens of 40 and 30 kDa which were detected on 15% acrylamide gels. These two antigens, (designated Ag12F and Ag12G, respectively), were evident in the Triton X-100 extracts of normal E as well as with PE of all parasite stages, and are therefore not malarial proteins

(Fig. 2). Commercially-derived, purified hemoglobin was tested for reactivity with mAb 12C11. A Western blot of human hemoglobin with m A b l 2 C l l produced a strongly reactive band at 30 kDa that comigrated with Ag12G (Fig. 2). Agl2G is presumably either hemoglobin dimer or an adduct that is not dissociated by SDS (it was colored brown), or another copurified protein derived from the E cytoplasm. The monomeric hemoglobin band at 16 kDa reacted very weakly, even though it was in great excess compared to the 30 kDa Ag12G band.

Western blot reactivity of mAb 41E11. With mature trophozoite-infected PE, MAb41Ell reacted primarily with 5 antigens of > 1000, 320, 175, 160 and 138 kDa (designated Ags 41A, 41B, 41C, 41D, and 41E, respectively) (Fig. 1). Ag 41 B detected by Western blotting comigrated with Ag 41B immunoprecipitated by mAb 41Ell (results not shown). In some Western blots with mAb 41E11, up to 25 other

238

33G2 41E11 ~ /

6 h 41h I~-~1 ~----~ N Hb

/41A

106 49.5 - ~

J ,-~

12E

- 40

32.5-N 27.5 - M ~

12A & PfEMP2 '41B

12F

205

~i~!~i~i)!!i!i~ii~ !~

41D

18,5 .

.

.

.

I-Ib

Fig. 2. Western blot reactivity of m A b l 2 C l l with nonmalarial erythrocyte antigens. Triton X-100 (T) or SDS (S) extracts of blood containing ring stage PE (6 h) or schizont/ segmenter PE (41 h) from the same experiment as in Fig. 1 were electrophoresed on a 15% acrylamide gel. Normal E solubilized in 1% Triton X-100 (lane N) were electrophoresed in parallel, together with a sample of human hemoglobin (lane Hb). The position of migration of monomeric hemoglobin is indicated (Hb). A fine doublet of bands specific for the very mature PE sample is labeled 12E. mAb 12Cll reactivities with 30 and 40 kDa components shared by PE, E and the human hemoglobin preparation are labeled 12F and 12G, respectively.

weakly reactive bands were detected (Fig. l). m A b 4 1 E l l did not react with detergent extracts from uninfected E, nor did it recognize any antigen below Ag 41E of 138 kDa (Fig. 1 and data not shown). Western blotting was performed on adjacent gel lanes with mAb 41El 1 and the human IgM mAb 33G2 [24-26] (Fig. 3). The antigens identified by these mAbs were identical in mobility except for a major mAb 33G2 antigen migrating between Ag 41A and Ag 41B that was lacking from the mAb 41E 11 pattern. A summary of the designations of antigens reactive with mAbs 12C11 and 41El 1 and their detergent solubilities is listed in Table I. Immunoprecipitation with mAbs 1 2 C l l and 4 1 E l l . mAbs 12Cll and 41Ell were tested

Fig. 3. Comparison of the Western blot reactivity of rat mAb 4 1 E l l with the human IgM mAb 33G2 [25]. Sequential SDS extracts from the MC strain were electrophoresed on a 7.5% acrylamide gel and adjacent gel lanes analysed by Western blotting with the two antibodies. An antigen that reacts only with mAb 33G2 is indicated by an asterisk.

for immunoprecipitation of radioiodinated PE surface proteins, using both the Triton X-100 extract and SDS extract from Triton X I00insoluble material. In experiments that yielded specific immunoprecipitation of ]25I-iabeled PfEMP1 by human immune sera or Aotus monkey sera, neither mAb immunoprecipitated 125I-PfEMP1 (results for 41Ell in Fig. 1). Coomassie Blue staining revealed that both the primary (rat IgM) and secondary (rabbit IgG) antibodies were adsorbed onto the Protein ASepharose during these experiments. Immunoprecipitation was also performed with [35S]methionine biosynthetically radiolabeled proteins. MAb 12C11 failed to immunoprecipitate any labeled proteins. MAb 41Ell specifically immunoprecipitated a radiolabeled band of >1000 kDa from the SDS extract (designated Ag 41A) and another of 320 kDa from the Triton X100 detergent extract (designated Ag 41B; Fig. 4). Other bands of lower Mr apparent in Fig. 4 were not

239

TABLE

I

P. falciparum a s e x u a l s t a g e a n t i g e n s r e a c t i v e w i t h r a t m A b s 12C11 a n d 4 1 E l l mAb

Antigen

Apparent Mr a (kDa)

Detergent extractibility 6h

12C11

41El1

12A 12B 12C 12D 12E 12 F c 12G ~ 41A

41B 41C 41D 41E

310 145 117 95 45/478 40 30 2.5 × 103d 320 175 160 138

18h

41 h

TX-100

SDS

TX-100

SDS

TX-100

SDS

. .

-

+ + + + + +

+ + -

+ + + + +

+ + + + + +

. .

. .

. .

. .

+

-

+

-

+

-

+

-

+

-

+

+

+ + + + +

+ +++ + + +

+ + ++ + + + -

. ++ + + + + .

.

.

.

-+ .

+++ + .

.

/ -

N o m i n a l Mr, for c o m p a r a t i v e p u r p o s e s o n l y , u n l e s s o t h e r w i s e n o t e d , b a s e d o n r e s u l t s o f 5 % S D S - P A G E u n d e r r e d u c i n g c o n d i t i o n s a n d i n t e r p o l a t i o n e x t r a p o l a t i o n o f a s t a n d a r d c u r v e (see M a t e r i a l s a n d M e t h o d s ) . b Mr of a doublet derived from 15% SDS-PAGE under reducing conditions and interpolation of a standard curve using lowmolecular-weight markers. c A g 12F a n d A g 12G a r e i n c l u d e d in t h i s t a b l e e v e n t h o u g h t h e y are n o t P. falciparum a n t i g e n s b u t r e p r e s e n t m A b 1 2 C l lreactive constituents of normal erythrocytes. d D e t e r m i n e d b y P e t e r s e n et al. [10] for a n a s e x u a l s t a g e p r o t e i n c a l l e d P f l 1.1 [11] a n d n o w c a l l e d A g 332 [17].

consistently immunoprecipitated. PfEMP2, defined by immunoprecipitation with murine mAb 8B7.4 [4] (Fig. 4), was distinct from Ag 41B: on 7.5% acrylamide gels Ag 41B had slightly higher mobility than PfEMP2; Ag 41B was also distinguished by recovery from the Triton X100 extract rather than the SDS extract (Fig. 1).

Effect of acrylamide concentration on the relative mobilities of mAb41Ell-reactive antigens. SDS extracts of trophozoite PE were fractionated by 4% and 7.5% acrylamide SDSPAGE and immunoblotted with mAbs 12C11, 41El 1 and 8B7.4 (anti-Pf EMP2). In both gels, PfEMP2 had a slightly faster mobility than Agl2A (Fig. 4) and the mobility of PfEMP2 relative to Agl2A was unchanged at 1.1 (Table II). However, as the acrylamide concentration decreased, the mobilities of the mAb41Ellreactive antigens were retarded compared to the mobility of Agl2A. On 7.5% gels Ag41B was faster than both PfEMP2 and Agl2A (Fig. 5). On 4% gels, Ag41B migrated behind PfEMP2 and Agl2A. The relative mobilities

of Ags 41A and 41B compared to Agl2A increased 2-5-fold when the acrylamide concentration was changed from 4 to 7.5% (Table II).

Indirect immunofluorescence of fixed infected erythrocytes. Indirect immunofluorescence assay (IFA) was performed after acetone or paraformaldehyde fixation using confocal fluorescence imaging microscopy. Figs. 6 and 7 show results for K + C + R + MC parasites. Identical results were obtained with the K+RFCR-3 C-5 clone. Neither mAb reacted by IFA with uninfected E. Controls lacking either primary mAb or secondary antibody were negative. There were no significant differences if paraformaldehyde or acetone were used. Reactivity of mAb 41Ell was observed in young ring-stage parasites within 4 h postinvasion, especially within the parasite (Fig. 6). The host E membrane was also labelled. In some cells there was diffuse labelling over the entire cytoplasm of the host E, while in others a patchy and intense fluorescence distribution

240 TABLE II

8B7.4 41 E11 I

I

1251

35S

Effect of acrylamide concentration on mobilities of mAb 41 E11-reactive antigens and PfEMP2 compared to Agl 2A

f Aotus (~-MC I

Antigen

Mobility index a Ratio of mobility (malarial antigen: indices 7.5%: Agl2A) 4.0% acrylamide Percentage acrylamide

1251

+

12A 12B PfEMP2 41A 41B 41D Myosin marker

200--

P2

97.4

4.0

7.5

1.0 2.1 1. I 0.2 0.9 1.8 1.6

1.0 5.6 1.1 0.8 1.8 5.6 4.4

2.7 1.0 4.7 2.0 3.1 2.8

a The distance travelled by each antigen was measured in mm from the top of the running gel to the front of the immunoreactive band. Relative mobilities, normalized to that of antigen 12A, were calculated and denoted the mobility index. The distance travelled by each antigen was divided by the distance travelled by Ag 12A on the same gel.

Strong mAb 41Ell reactivity on extended structures that appear to be between the parasite and the outer cell membrane (12 h, Fig. 6) represents folds in the PE plasma membrane. Late trophozoites and early schizonts exhibited a marked increase in the

i--

0

,,,

~

m

0

~

Fig. 4. Immunoprecipitation of cell surface 125I-labeled detergent extracts or 35S]methionine biosynthetically labeled extracts of PE (trophozoite stage) using m A b 41El 1. PE were extracted sequentially with Triton X-100 (T) or SDS (S) for immunoprecipitation and antigens separated by SDS-PAGE on a 5% acrylamide gel. The entire gel was processed for fluorography. For comparison, immunoprecipitation was performed with murine m A b 8B7.4 (specific for PfEMP2) or serum from an Aotus monkey infected repeatedly with the MC strain (Aotus ~-MC: to identify PfEMP1). The positions of migration of PfEMPI, PfEMP2 and Ag 41B are shown, together with standard proteins (kDa).

-41A

--41B -- 12A \

PfEMP2 715%

was noted, particularly at the E membrane. At the late ring stage/early trophozoite stage (1218 h) the majority of PE had foci of antigenic reactivity around the cell periphery, in the host E cytoplasm and at the E membrane. These foci ranged in size from 0.24).5 /~m diameter.

m ~.

4%

Fig, 5. Effect of acrylamide concentration on relative mobilities of very-high-Mr malarial antigens detected by ELISA reactivity with Western blots. An SDS extract from trophozoite stage PE was submitted to SDS-PAGE on a 7.5% or 4% acrylamide gel and immunoblotted with mAbs 8B7.4, 12C11, or 41EI 1. These mAbs identify PfEMP2, Ag 12A and Ags 41A and 4lB. The location of the top of the resolving gel is indicated by the arrowheads.

241

MAb 41 E11

4hr

12hr

29hr

MAb 12C11

4hr

15hr

27hr

40hr

Fig. 6. Indirect immunofluorescence reactivity of mAbs 12C11 and 41E 11 with paraformaldehyde-fixed PE of the MC strain. Samples of blood were taken from a synchronous in vitro culture and analyzed by confocal fluorescence microscopy at the times indicated. The bars represent 5#m. White arrowheads indicate fluorescent-labelled folds of the PE plasma membrane.

number and size of antigenic foci in the host E cytoplasm. The pattern of evenly distributed labelling of the outer cell membrane seen first with ring-stage parasites decreased with mature parasites (29 h in Fig. 6). Reactivity with mAb 41El 1 decreased in mature schizonts and segmenters, persisting at the parasites and focal points at the outer membrane (not shown). MAb 12Cll IFA reactivity was also seen with early rings (4 h post-invasion), but in contrast to M A b 41Ell at this stage, mAb 12C1 l only labelled the parasite (Fig. 6). As parasites matured, diffuse staining of the host cell cytoplasm was detected and antigen foci appeared between the parasite and the outer cell membrane (15 h, Figs. 6 and 7). This can be seen clearly in Fig. 7 wherein the bright field and fluorescence appearance of the same cell are compared. These foci were approx. 0.2~).5

#m in diameter. In trophozoite-infected cells the number of foci in the host cell cytoplasm increased, but their number and size (0.2-0.3 #m diameter) were less than seen with MAb 41El I (27 h, Fig. 6). Of particular interest with m A b l 2 C l l was the presence of a granular fluorescence reactivity in many very small foci. These small foci appeared to be of 0.2 ~m diameter but may have been smaller. Fig. 7 shows these small foci at high magnification. These foci occurred outside the parasite and appeared over the entire PE, extending to the outer cell membrane. Similar foci were not seen with mAb 41El 1.

Discussion

In view of evidence for expression of very large malarial proteins on the surface of PE

242

I 0

g

~ 4

b

I

Fig. 7. Indirect immunofluorescence of trophozoite-stage MC parasitized erythrocytes with mAb 12C11 showing small foci of fluorescence visible at high magnification and labeling external to the parasite cytoplasm. Bars represent 2 pm. (A) Bright field light microscopy showing a single PE surrounded by uninfected E. Within the PE the parasite cytoplasm can be visualised around the central pigment granule. (B) Fluorescence light microscopy of m A b 12C11 reactivity with the same field as A. Uninfected E are non-fluorescent. (C) Fluorescence light microscopy at higher magnification. Large roughly spherical areas of fluorescence reactivity are seen external to the parasite. Many small foci of fluorescence are also seen external to the parasite and extending to the outer plasma-membrane of the PE. The majority of PE at the trophozoite stage exhibited these small foci. Such loci of fluorescence reactivity were not detected with mAb 41El 1.

243

[9,18-20] and their involvement in PE adherence properties [18,19,21], two important questions arise. First, are mAbs 12Cll and 41Ell reactive with antigens on the surface of unfixed, intact PE? We performed standard tests for antibody reactivity with the PE surface: agglutination, I FA, rosette reversal, adherence blockade and immunoprecipitation of 125I-surface proteins. Although some experiments gave internally consistent data suggesting specific mAb reactivity with the cell surface, these results were not reproducible. We assume that weak or irreproducible effects reflect preferential mAb reactivity with partially denatured proteins, parasite phenotypic variation, or non-specific cell binding properties of some rat IgMs. These mAbs are inappropriate reagents to resolve the question of whether the target antigens are present on the cell surface. However, they have permitted cloning of gDNA clones (data not shown), which will allow production of polyclonal antisera against recombinant protein fragments to enable resolution of this question. The second important question concerns the relationship, if any, of Agl2A to PfEMP1. PfEMP1 is in the same size range and has been defined as a trypsin-sensitive, surface-iodinated protein immunoprecipitated by human or Aotus immune sera [9,18-20]. The properties of PfEMP1 have not been studied using Western blotting. M A b l 2 C l l repeatedly failed to immunoprecipitate any surface ~25Iprotein or [35S]methionine biosynthetically labeled proteins, despite use of diverse secondary antibodies and affinity adsorbents, mAb 12Cll appears restricted to recognition of linear epitopes reactive after SDS-PAGE and Western blotting, but inaccessible with undenatured Agl2A. This is not unexpected since m A b l 2 C l l was elicited by immunization with excised SDS-PAGE gel slices from an SDS extract. Agi2A, detected by Western blotting with mAb 12Cll, was not affected by treatment of intact PE with trypsin, under conditions that quantitatively cleaved ~25I-PfEMP1 (results not shown). Clearly the bulk of Agl2A detected with MAb 12Ci 1 cannot correspond

to cell surface-expressed PfEMPI. Agl2A and 125I-PfEMP1 of MC parasites comigrate when ~25I-PfEMP1 is immunoprecipitated by polyspecific serum, the sample electrophoresed on a 5% SDS-gel, Western blotted and the gel lane probed with mAb 12Cll (results not shown). However, Agl2A is very similar in apparent M r in MC and FVO strains of P. falciparum (results not shown), whereas 125I-PfEMP1 is very different in mobility in these parasites [9]. Taken together, these results suggest that Agl2A is a different protein to PfEMP1. Agl2A does not correspond to PfEMP2, D260/Ag 41B, or other known very large malarial proteins. Ag 12A was the largest antigen recognized by mAb 12C11 on Western blots (approximately 310 kDa), with mobility close to D260/Ag 41B, PfEMP2 and the PfEMPI protein of MC strain parasites. AgI2A was clearly resolved from D260/Ag 41B and PfEMP2 by SDS-PAGE. D260/Ag 41B was also distinguished from AgI2A by its predominance in the Triton X-100 extract. PfEMP2 was distinguished not only by unique SDS-PAGE mobility but by recovery exclusively from the SDS extract of Triton X-100insoluble material. Agl2A was present in both Triton X-100 and SDS extracts. mAb 12Cll also reacted on Western blotting with several lower-Mr antigens that must also represent malarial proteins since they were absent from E extracts (Ags 12B 12E). Ags 12B-12E may correspond to proteolytic degradation products of Agl2A. Alternatively, these antigens may be different gene products which cross-react with m A b l 2 C l l . We have proven that mAb 12Cll reacts specifically with epitopes on malarial protein by two criteria. First, immunoprobing a )~gtll malarial genomic DNA expression library with mAb 12Cll identified clones that contain malarial nucleic acid sequences (according to Southern and Northern blotting with the insert DNA) and which express mAb 12Cll-reactive fusion proteins. Second, mAb 12Cll has been shown to react with linear peptides derived from the deduced amino acid sequence of one of the gDNA clones. Efforts are underway to test whether

244

these clones are related to previously identified malarial proteins and to Ags 12A-12E. A curious feature of mAb 12Cll was its Western blot reactivity with protein constituents of normal human E of M r 40 and 30 kDa (Ags 12F and 12G). Ag 12G may correspond to hemoglobin dimer or an adduct with hemoglobin. In contrast, mAb 12Cll failed to react by IFA with hemoglobin in paraformaldehyde or acetone-fixed normal E or PE, reacting only with parasite-dependent structures. MAb 41Ell recognizes a quite different group of antigens on Western blotting, similar to those recognized by the human mAb 33G2 [24,25]. Except for the presence of one antigen detected with mAb 33G2 and not with mAb 41El 1, mAbs 41El 1 and 33G2 share reactivity with antigens of identical mobilities and detergent solubility characteristics. We have used MAb 41Ell to clone a gene fragment from a 2gtll malarial g D N A expression library which has strong homology to the published DNA sequence of Pfll.1 (data not shown and 15). Pfll.1 is a sexual blood stage antigen [17], a member of a group of P. falciparum antigens bearing the EEXXEE amino acid repeat motif recognized by the human IgM MAb 33G2 [24,25]. MAb 41Ell also reacted strongly on ELISA testing with the peptide (EENV)6, a consensus repeat structure within the RESA/PfI55 protein (D. Baruch, personal communication). This peptide contains the EE dipeptide shown to be the mAb 33G2 epitope [24,39] and also reacts strongly with mAb 33G2 by ELISA (D. Baruch, personal communication). Considering all these data, including the observation that Pf155/RESA can migrate as a doublet [25] and that it is predominantly Triton X-100insoluble [40], we conclude that Ags 41A, 41B, and 41C plus 41D correspond to Ags 332, D260 and Pf155/RESA, respectively. These results are summarized in Table III. As the acrylamide concentration of SDSPAGE gels varied between 4% and 7.5%, several of the mAb 41Ell-reactive antigens had large differences in relative mobilities with respect to Agl2A and PfEMP2. Agl2A, in the

TABLE Ill Correspondence between mAb 41El 1-reactive antigens and previously characterized P. Jalciparum antigens that bear EEXXEE amino acid motifs mAb 41 E11 antigen

Mr ~L(kDa)

Previous identification b

Reference

41A 41B 41C 41D

2.5 x l03 320 175 160

332 D260 PfI55/RESA PfI55/RESA

[17] [11] [24,25] [24.25]

From Table 1. b Provisional identification based on the combined characteristics of Mr, detergent extractibility and comigration with mAb 33G2 reactive antigens.

same size range, did not exhibit changes in relative mobility compared to PfEMP2. Our IFA results provide information on the locations of the distinct groups of antigens recognized by the rat mAbs but cannot provide information on the location of individual antigen bands. The IFA pattern of mAb 33G2 with fixed PE has been described [25]. Our results with mAb 41Ell were identical, confirming the similarity in specificity of these antibodies apparent on Western blotting. With both mAbs 12Cll and 41Ell there was evidence for export of malarial antigens from the parasite to the PE periphery. As parasites matured, increasing IFA reactivity was within the cytoplasm of the PE and later adjacent to the outer PE membrane. This reactivity may correspond to membranous structures or electron-dense material identified in the host cell cytoplasm by transmission electron microscopy [41,42] and shown in several immunoelectron microscopy studies to bear malarial antigens (e.g., refs. 4,5). The subcellular distributions of antigens recognized by IFA with mAbs 12Cll and 41El 1 were different, mAb 12C11 gave weaker fluorescence intensity and smaller antigen aggregates in the host cell cytoplasm. Furthermore, with mAb 12C11 we noted many very small aggregates of relatively weak fluorescence reactivity entirely absent with mAb 41Ell. Since these aggregates are 0.2 /~m or less in diameter and knob protrusions are approx. 0.1 /~m in diameter, we speculate that

245

mAb 12Cll reacts with knobs. Indeed, immunoelectron microscopy with LR gold resinembedded material has revealed reactivity of mAb 12Cll, but not 41Ell, with knobs (K-I. Nakamura et al., unpublished results). To conclude, we have characterized antigens defined by 2 IgM rat mAbs, with particular emphasis on very large malarial antigens of approx. 300 kDa that have been so difficult to study systematically. One of these mAbs, 41Ell, reacts with a group of proteins that have been studied previously [24,25]. mAb 12C11 defines a hitherto undescribed group of malarial protein antigens, called here Ags 12A, 12B, 12C, 12D and 12E, with approximate M r of 310, 145, 117, 95 and 47/45 kDa. Agl2A is a new very high-molecular-weight malarial antigen. The antigens identified by mAb 12Cll appear sufficiently novel to warrant attempts at molecular cloning of the relevant malarial gene(s) by antibody screening of expression libraries.

Acknowledgements We thank Dr. Klavs Berzins, Stockholm, for providing mAb 33G2. We are also grateful to Cynthia Ma and Kerstin Moorehead for technical assistance. SMH was supported in part by a Rockefeller Foundation Fellowship. BLP was supported in part by a fellowship from the Medical Research Council of Canada and a research allowance from the Alberta Heritage Foundation for Medical Research. This work was supported by grants to RJH from The Malaria Immunity Vaccine Development Program, Agency for International Development (Grant DPE-0453-G-SS-804900) and by the WHO /UNDP/World Bank Special Programme for Research and Training in Tropical Diseases (Grants 880122 and 880123), together with grant AI 27247 to TET from the National Institutes of Health. The DNAX Research Institute of Molecular and Cellular Biology is supported by ScheringPlough Corporation.

References 1 Brown, G.V., Anders, R.F., Mitchell, G.F. and Heywood, P.F. (1982) Target antigens of purified human immunoglobulins which inhibit growth of PlasmoaTumfalciparum in vitro. Nature 297, 591 593. 2 Howard, R,J. (1988) Malarial proteins at the membrane of Plasmodium falciparum-infected erythrocytes and their involvement in cytoadherence to endothelial cells. Prog. Allergy 41, 98 147. 6753 3 Berendt, A.R., Ferguson, D.J.P. and Newbold, C.I. (1990) Sequestration in Plasmodium JLtlciparum malaria: sticky cells and sticky problems. Parasitol. Today 6, 247 254. 4 Howard, R.J., Lyon, J.A., Uni, S., Saul, A.J., Aley, S.B., Klotz, F., Panton, L.J., Sherwood, J.A., Marsh, K., Aikawa, M. and Rock, E.P. (1987) Transport of an Mr approx. 300000 Plasmodium falciparum protein (PfEMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic l:ace of the host cell membrane. J. Cell Biol. 104, 1269-1280. 5 Coppel, R.L. (1992) Repeat structures in a Plasmodium falciparum protein (MESA) that binds human erythrocyte protein 4.1. Mol. Biochem. Parasitol. 50, 335 348. 6 Howard, R.F., Stanley, H.A. and Reese, R.T. (1988) Characterization of a high-molecular-weight phosphoprotein synthesized by the human malarial parasite Plasmodiumfalciparum. Gene 64, 65 75. 7 Petersen, C., Nelson, R., Magowan, C., Wollish, W., Jensen, J. and Leech, J. (1989) The mature E surface antigen of Plasmodium JLllciparum is not required for knobs or cytoadherence. Mol. Biochem. Parasitol. 36, 61 66. 8 Coppel, R.L., Lustigman, S., Murray, L. and Anders, R.F. (1988) MESA is a Plasmodium .falciparum phosphoprotein associated with the erythrocyte membrane skeleton. Mol. Biochem. Parasitol. 31, 223 232. 9 Howard, R.J., Barnwell, J.W., Rock, E.P., Neequaye, J., Ofori-Adjei, D., Maloy, W.L. and Saul, A. (1988) Two approximately 300 kiloDalton Plasmodium falciparum proteins at the surface membrane of infected erythrocytes. Mol. Biochem. Parasitol. 27, 207-224. 10 Petersen, C., Wollish, W. and Leech, J. (1990) A newly identified 260 kD protein of intraerythrocytic Plasmodium falciparum parasites cross-reacts with the 11.1 protein and Pf155-RESA. Proc. 38th Meeting Am. Soc. Trop. Med. Hyg. Abstract 24. 11 Petersen, C., Nelson, R., Leech, J., Jensen, J., Wollish, W. and Scherf, A. (1990) The gene product of the Plasmodium falciparum 11.1 locus is a protein larger than one megadalton. Mol. Biochem. Parasitol. 42, 189 196. 12 Stahl, H.D., Bianco, A.E., Crewther, P.E., Burkot, T., Coppel, R.L., Brown, G.V., Anders, R.F. and Kemp, D.J. (1986) An asparagine-rich protein from blood stages of Plasmodium Jalciparum shares determinants with sporozoites. Nucleic Acids Res. 14, 3089-3102. 13 Irving, D.O., Morry, M.J., Haas, L.O.C., Clayton, C.E., Wallach, M.G. and Cross, G.A.M, (1987) Characterization of a histidine-rich protein related gene from Plasmodium falciparum. Molecular Strategies of Parasitic Invasion, Alan R.Liss, Inc. pp 37 46. 14 Koenen, M., Scherf, A., Mercereau, O., Langsley, G., Sibilli, L., Dubois, P., Pereira da Silva, L. and MullerHill, B. (1984) Human antisera detect a Plasmodium falciparum genomic clone encoding a nonapeptide

246 repeat. Nature 31 l, 382-385. 15 Scherf, A., Hilbich, C., Sieg, K., Mattei, D,, MercereauPuijalon, O. and Muller-Hill, B. (1988) The I 1-1 gene of Plasmodium falciparum codes for distinct fast evolving repeats. EMBO J. 7, 1129 1137. 16 Kahane, B., Sibili, L., Scherf, A., Jaureguiberry, G,, Langsley, G., Ozaki, L.S., Guillotte, M., Muller-Hill, B., Pereira da Silva, L. and Mercereau-Puijalon, O. (1987) The polymorphic 11.1 locus of Plasmodiurn Jillciparum. Mol. Biochem. Parasitol. 26, 77 86. 17 Mattei, D. and Scherf A. (1992) The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes. Gene, 110, 71 74. 18 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 Jalciparum-infected erythrocytes. J. Exp. Med. 159, 1567 1575. 19 Aley, S.B., Sherwood, J.A. and Howard, R.J. (1984) K n o b - p o s i t i v e and K n o b - n e g a t i v e Plasmodium falciparum differ in expression of strain-specific malarial antigen on the surface of infected erythrocytes. J. Exp. Med. 160, 1585 1590. 20 Magowan, C., Wollish, W., Anderson, L. and Leech, J. (1988) Cytoadherence by Plasmodium falciparuminfected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes. J. Exp. Med. 168, 1307 1320. 21 Ockenhouse, C.F., Klotz, F.W., Tandon, N.N. and Jamieson, G.A. (1991) Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by antiidiotype antibodies. Proc. Natl. Acad. Sci. USA 88, 3175-3179. 22 Anders, R.F. (1986) Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol. 8, 529 539. 23 Kemp, D.J., Coppel, R.L. and Anders, R.F. (1987) Repetitive proteins and genes of malaria. Annu, Rev. Microbiol. 41, 181 189. 24 Mattei, D., Berzins, K., Wahlgren, M., Udomsangpetch, R., Perlmann, P., Griesser, H.W., Scherf, A., Muller-Hill, B., Bonnefoy, S., Guillotte, M., Langsley, G., Pereira da Silva, L. and Mercereau-Puijalon, O. (1989) Cross-reactive antigenic determinants present on different Plasmodiurn falciparum blood-stage antigens. Parasite Immunol. I1, 15-30. 25 Udomsangpetch, R., Carlsson, J., Wahlin, B., Holmquist, G., Ozaki, L.S., Scherf, A., Mattei, D., Mercereau-Puijalon, O., Uni, S., Aikawa, M., Berzins, K. and Perlmann, P. (1989) Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J. Immunol. 142, 3620 3626. 26 Udomsangpetch, R., Aikawa, M., Berzins, K. Wahlgren, M. and Perlmann, P. (1989) Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody. Nature 338, 763 765. 27 Degowin, R.L. and Powell, R.D. (1965) Drug resistance of a strain of Plasmodiumfalciparum from Malaya. Am. J. Trop. Med. Hyg. 14, 519 528. 28 Green, T.J., Gadsden, G., Seed, T., Jacobs, R., Morhardt, M. and Brackett, R. (1985) Cloning and characterization of Plasmodium falciparum FCR-3/ F M G strain. Am. J. Trop. Med, Hyg. 34, 24 30. 29 Handunnetti, S.M., Gilladoga, A.D., Van Schraven-

dijk, M-R., Nakamura, K-I., Aikawa, M. and Howard, R.J. (in press) Purification and in vitro selection of rosette-positive (R +) and rosette-negative ( R - ) phenotypes of knob-positive Plasmodium falciparum parasites. Am. J. Trop. Med. Hyg. 46, 371 381. 30 Handunnetti, S.M., Hasler, T.H. and Howard, R.J. (In Press) Plasmodium falciparum infected erythrocytes do not adhere well to C32 melanoma cells or CD36 unless rosettes with uninfected erythrocytes are first disrupted. Infect. Immun. 31 Udomsangpetch, R., Wahlin, B., Carlson, J., Berzins, K., Torii, M., Aikawa, M., Perlmann, P. and Wahlgren M. (1989) Plasmodium Jitl~iparum-infected erythrocytes form spontaneous erythrocyte rosettes. J. Exp. Med. 169, 1835 1840. 32 Handunnetti, S.M., Gilladoga, A.D. and Howard, R.J. (1990) A new cytoadherence property of Plasmodium Jalciparum-infected erythrocytes: rosetting with uninfected erythrocytes, Cellular and Molecular Biology of Normal and Abnormal Erythroid Membranes, pp. 249 265. Alan R. Liss, New York. 33 Jensen, J.B. (1978) Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium.[alciparum. Am. J. Trop. Med. Hyg. 27, 1274-1270. 34 David P.H., Handunnetti, S.M., Leech, J.H., Gamage~ P. and Mendis, K.N. (1988) Rosetting: a new cytoadherence property of malaria-infected erythrocytes. Am. J. Trop. Med. Hyg. 38, 289 297. 35 Marsh, K., Sherwood, J.A. and Howard, R.J. (1986) Analysis of antigens induced on Plasmodium falciparum-infected erythrocytes from Gambian patients by indirect immunofluorescence and microagglutination assays. J. Immunol. Methods 91, 107 115. 36 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. 37 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. 38 Abrams, J.S. and Pearce, M.K. (1988) Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro. J. lmmunol. 140, 131 137. 39 Ahlborg, N., Berzins, K. and Perlmann, P. (1991) Definition of the epitope recognized by the Plasmodium Jalciparum-reactive human monoclonal antibody 33G2. Mol. Biochem. Parasitol. 46, 89 96. 40 Brown, G.V., Culvenor, J.G., Crewther, P.E., Bianco, A.E., Coppel, R.L., Saint, R.B., Stahl, H.D., Kemp, D.J. and Anders, R.F. (1985) Localization of the ringinfected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes. J. Exp. Med. 162, 774-779. 41 Aikawa, M., Uni, Y., Andrutis, A.T. and Howard, R.J. (1986) Membrane-associated electron-dense material of the asexual stages of Plasmodiumfalciparum: evidence for movement from the intracellular parasite to the erythrocyte membrane. Am, J. Trop. Med. Hyg. 35.30,36. 42 Howard, R.J,, Uni, S., Lyon, J.A,, Taylor, D.W., Daniel, W. and Aikawa, M. (1987) Export oI Plasmodium falciparum proteins to the host erythrocyte membrane: Special problems of protein trafficking and topogenesis. In: Host-Parasite Cellular and Molecular Interactions in Protozoan Diseases. NATO ASI series. Vol Hl l Chang K.-P., Snary, D., eds.), pp. 281 296. Springer-Verlag, Berlin.