105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides

Molecular and Biochemical Parasitology, 29 (1988) 251-260 Elsevier

251

MBP 00983

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides J u a n A . C o o p e r 1, L e a n n e T . I n g r a m 1, G i l l i a n R . B u s h e l l 2, C a t h y A . F a r d o u l y s 1, D e b o r a h S t e n z e l 1, L o u i s S c h o f i e l d 3 a n d A l l a n J. S a u l 1 tQueensland Institute of Medical Research, Brisbane, Queensland, Australia, 2School of Science, Griffith University, Brisbane, Queensland, Australia and 3Department of Parasitology, New York University, New York, U.S.A. (Received 30 November 1987; accepted 22 February 1988)

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofiuorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeledparasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent. Key words: Antigenic complex; Monoclonal antibody; Plasmodium falciparum; Rhoptry antigen

Introduction The rhoptries are a pair of tear shaped organelles located at the apical end of plasmodial merozoites and are thought to play a role in the invasion of merozoites into erythrocytes [1]. Several antigens located in the rhoptries of P l a s m o d i u m falciparum have been described including 80/42 k D a [2-8], 150/130 k D a [9] and a series of bands of approximate molecular mass 140, 130 and 105

Correspondence address: Juan A. Cooper, Queensland Institute of Medical Research, Bramston Terrace, Herston, Brisbane 4006, Australia. Abbreviations: MAb, monoclonal antibody; PBS, phosphate buffered saline, TNET; Triton X-100-NaCI-EDTA-Tris buffer; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; IFA, indirect immunofluorescence assay.

k D a [3,10-12]. The nucleotide sequence encoding a portion of a 105 k D a rhoptry protein has been deduced from a c D N A clone using human antisera to screen an expression library [13]. All of these antigens appear to exist as complex spedes in the rhoptries. A rhoptry antigen of 225 k D a has also been reported [14]. Inhibitory monoclonal antibodies (MAbs) have been produced that recognise rhoptry antigens in P. falciparum [5], and in mice, immunisation with a 235 k D a rhoptry antigen was found to protect against parasite challenge in P l a s m o d i u m yoellii [15,16]. Although the function of the rhoptry contents is unknown, the presence of inhibitory M A b s directed against rhoptry antigens suggests an important role in the invasive process. This paper examines the relationship between the m e m b e r s of the 140/130/105 k D a complex of antigens and the ability of M A b directed against the complex to inhibit the development of parasites.

0166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

252 Materials and Methods

Parasite culture. The parasite line FCQ-27/PNG [17] was grown in continuous culture using the method of Trager and Jensen [18] and maintained in a synchronous state by multiple treatment with sorbitol using a modification [19] of the method of Lambros and Vanderburg [20].

Production of monoclonal antibodies. The MAbs used in this study were produced against mature schizonts using procedures similar to those described for other MAbs prepared in this laboratory [21]. The three MAbs 3E10/18, 5Dll/49 and 8D12/49 belonged to the IgG 1 subclass and the fourth MAb, 6F4/53 was of IgG2a subclass. The fusion giving rise to MAb 3E10/18 was against isolate K1 [22], the other fusions used the isolate FCQ27/PNG. Imrnunofluorescence assay and immunoelectron microscopy. Thin films containing mature schizont-stage parasite cultures were air-dried and acetone-fixed. The immunofluorescence assay with MAb was performed essentially as described by Epping et al. [23]. Immunoelectron microscopy on mature schizont-stage parasites was carried out on samples of parasite culture fixed with 0.05% (v/v) glutaraldehyde and embedded in LR White resin, medium grade (London Resin Co. Ltd.) [24]. The sections were etched and immunolabeled using modifications [24] to the methods described by Bendayan and Zollinger [25]. The immunolabeled sections were contrasted using lead citrate and uranyl acetate and examined using a Philips 400 electron microscope.

Immunoprecipitation and immunoblotting. Synchronous cultures of FCQ-27/PNG were labeled with [35S]methionine (Amersham, England) and radiolabeled antigen was extracted into 50 mM Tris, 150 mM NaCI, 5 mM EDTA, pH 7.4 containing 1% Triton X-100 (TNET) and immunoprecipitated as described by Schofield et al. [5]. The following mixture of protease inhibitors (0.2 mM phenylmethylsulphonyl fluoride, 10 Ixg ml1-tosylamide-2-phenylethylchloromethyl ketone, and 5 Ixg m 1-1 each of leupeptin, antipain, chy-

mostatin and pepstatin) were used. The precipitated antigen was solubilised in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer, electrophoresed on 7.5% SDS-PAGE [26], fixed and stained in Coomassie blue R250 and impregnated with Amplify (Amersham). The dried gel was autoradiographed at -70°C using Fuji RX film. In a parallel experiment, a portion of the parasite pellet was extracted into 1% (v/v) Triton X114 at 4°C. The extract was then incubated at 37°C for 5 min to allow phase separation and centrifuged to give the detergent and aqueous phases. The two phases were extracted twice again into 1% (v/v) Triton X-114 and the radiolabeled antigens immunoprecipitated in the same way as for the Triton X-100 extracts, as detailed above. Mature schizont stage parasites were harvested and extracted into TNET. This parasite extract was then solubilised in SDS-PAGE sample buffer, under non-reducing conditions (in the absence of [3-mercaptoethanol), and separated on SDSPAGE. The proteins were transferred to nitrocellulose, blocked in 5% (w/v) low-fat milk powder in phosphate-buffered saline (PBS) and immunoblotted with MAb, followed by iodinated goat anti-mouse (KPL) [27] second antibody. The immunoblot was autoradiographed using Fuji RX film.

Pulse chase analysis. Synchronous parasite cultures were metabolically labeled with [35S]methionine at regular time intervals over 48 h. At early ring-stage, the culture was treated with sorbitol to eliminate mature parasites, followed by further treatments at 8 and 24 h after the first treatment to ensure a highly synchronous culture with parasites between 0 and 4 h old [19,20]. Following reinvasion (time zero), the culture was transferred to twenty 10 ml plates and at 0, 12, 20, 24, 28, 32, 36, 40, 44 and 48 h, [35S] methionine (Amersham) was added to the contents of two plates at a concentration of 20 txCi m1-1 to give a final methionine concentration of 0.1 mM. After 2 h pulse labeling, an excess of unlabeled methionine was added to the contents of one of the plates to give a final concentration of 10 mM and the chase allowed to proceed to mature schizont stage. The contents of the other plate were

253 harvested by centrifugation and saponin lysis, as outlined above, and frozen at -70°C. The pulsechased cultures were harvested at 46 h when mature schizonts and early ring stage parasites were both present. Parasite development was monitored at each pulse and harvest interval by the preparation of a Giemsa-stained thin film. In a separate experiment, at 36 and 40 h, [35S]methionine was added to the contents of two plates at a concentration of 50 IxCi m1-1. After 10 rain pulse labeling, the cultures were harvested as described above. The radiolabeled parasite antigens were extracted into TNET, immunoprecipitated with antibody and protein A-Sepharose, separated on SDS-PAGE and fluorographed.

Antigen purification. A parasite culture was harvested at mature schizont stage and extracted into TNET, as outlined above. MAb 3E10/18 was covalently linked to cyanogen bromide-activated Sepharose CL 4B (Pharmacia, Sweden) and the parasite extract was passed through the column. The column was washed extensively with TNET, TNET containing 0.5 M NaCI and finally TNET without Triton X-100, and eluted with 0.1 M glycine, pH 2.8, containing the cocktail of protease inhibitors. The eluted fractions were adjusted to pH 7.0 by adding 2 M Tris to a final concentration of 0.04 M Tris. Peptide mapping. The individual components of the antigen were subjected to digestion with S. aureus V8 protease in the presence of SDS at pH 6.8. Affinity purified antigen was separated on SDS-PAGE and the gel incubated in ice-cold 1 M KC1 for 15 min to visualise the protein bands. After rinsing in ice-cold water and freezing the gel at -70°C for 30 min, the proteins appeared as white bands upon thawing. The protein bands were carefully excised from the gel with a scalpel, incubated.in SDS-PAGE sample buffer for 30 min and placed in the well of an SDS-PAGE gel. An overlay consisting of 5 Izl of S. aureus V8 protease (10 ~g m1-1) prepared in SDS-PAGE sample buffer was placed on top of the gel slices, and the gel was electrophoresed and silver stained.

Parasite inhibition assay. The assay of inhibition of erythrocyte invasion and in vitro growth was a modification [23] of the method of Schofield et al. [51. A positive control antibody, 3A10/ll [28], was included in the inhibition assays. Results

Indirect immunofluorescence assay and immunoelectron microscopy. The localisation of antigens recognised by this group of MAbs was accomplished using an indirect immunofluorescence assay (IFA) and immunoelectron microscopy. All four MAbs reacted by IFA on acetone-fixed slides, giving an intense double dot fluorescence pattern, suggestive of the paired rhoptry organelles, identical to patterns obtained for other rhoptry antigens [3-5,7,10,12-14,29] (data not shown). This pattern was only observed in mature schizonts and newly released merozoites.

Fig. 1. Immunoelectronmicroscopyof P. falciparum using MAb 8D12/49. Sectionsof mature schizontswere reactedwith MAb, rabbit anti-mouse antibodyand goat anti-rabbit antibody labeled with colloidalgold (10 nm). R, rhoptry. (Bar equals 0.5 ~m.)

254 A m o r e precise localisation involved immunogold labeling of thin sections of fixed mature schizonts and examination under an electron microscope. Although labeling with all four M A b s was a t t e m p t e d only M A b 8D12/49 gave specific labeling (Fig. 1) confirming that the epitope recognised by 8D12/49 is present in the rhoptries. Presumably the epitopes recognised by the other M A b s were destroyed by the embedding process.

Immunoprecipitation and immunoblotting. Parasite cultures were metabolically labeled with [35S]methionine, and all four M A b s i m m u n o p r e cipitated radiolabeled bands at 140, 130, and 105 k D a (Fig. 2). The lower molecular weight band was less intense than the other two bands. When

1

2

3

4

2 0 0 5

9 2-5 -

69-

46-

5

200-

30-

o

9 2 "5

1

~

8

-

69--

Fig. 3. SDS-PAGE and immunoblotting. A Triton X-100 extract of mature schizonts was run on SDS-PAGE, in the absence of 13-mercaptoethanol, and immunoblotted with MAbs 3E10/18 (lane 1), 5Dll/49 (lane 2), 8D12/49 (lane 3) and 6F4/53 (lane 4).

•

46-

30Fig. 2. Immunoprecipitation of antigen. A culture of P. falciparum was labeled with [35S]methionine at trophozoite stage and harvested at mature schizont stage. The radiolabeled antigen was extracted into TNET and immunoprecipitated with MAb and protein A-Sepharose before solubilising in SDSPAGE sample buffer, containing 13-mercaptoethanol, and separation on SDS-PAGE. Lanes 1-4, immunoprecipitated with MAbs 3E10/18, 5Dll/49, 8D12/49 and 6F4/53, respectively; lane 5, immunoprecipitated with MAb 3E10/18, followed by separation on SDS-PAGE in the absence of 13-mercaptoethanol.

[3-mercaptoethanol was omitted from the SDSP A G E sample buffer, there was a small decrease in apparent size of the protein bands with the MAbs immunoprecipitating bands at 135, 125, and 98 k D a (Fig. 2). Rabbit sera raised against the fusion polypeptide Ag44 of Coppel et al. [13] also immunoprecipitated bands at 140, 130 and 105 k D a (data not shown). After extraction of antigens with Triton X-114, at a t e m p e r a t u r e of 4°C, the M A b s immunoprecipitated radiolabeled bands at 140, 130 and 105 k D a from the aqueous phase but not the detergent phase (data not shown), suggesting that this antigen is not an integral m e m b r a n e protein. The majority of the antigen was also released during freeze/thaw lysis of mature schizonts (data not shown). When 1% Triton X-100 extracts of mature schizonts were run on S D S - P A G E , in the absence of [3-mercaptoethanol and immunoblotted

255 with the four rhoptry MAbs, MAb 5 D l l / 4 9 recognised a band at 125 kDa and 8D12/49 gave a band at 98 kDa (Fig. 3, lanes 2 and 3). These bands correspond to the 130 and 105 kDa bands, respectively, seen in immunoprecipitates electrophoresed in the presence of reducing agent. These MAbs failed to immunoblot when antigens were electrophoresed under reduced conditions. MAbs 3E10/18 and 6F4/53 failed to immunoblot under any conditions (Fig. 3, lanes 1 and 4). No antibody was found which recognised the 140 kDa

Fig. 4. Pulse-chase analysis of a synchronous P..falciparum culture. Synchronous cultures were pulse-labeled with [35S]methionine for 2 h and either harvested or chased to the mature schizont stage. The radiolabeledparasite antigens were extracted into TNET, immunoprecipitated with 3E10/18 and protein A-Sepharose, separated on SDS-PAGE and fluorographed. The cultures were pulse-labeled at 28 h (lane 1), 32 h (lane 2), 36 h (lane 3), 40 h (lane 4) and 44 h (lane 5), pulsechased at 28 h (lane 6), 32 h (lane 7), 36 h (lane 8) and 40 h (lane 9), and harvested at 46 h. Synchronised cultures were pulse-labeled for 10 rain with [35S]methionineand harvested immediately. The radiolabeled parasite antigens were extracted into TNET, immunoprecipitated with 3E10/18 and protein A-Sepharose, separated on SDS-PAGE and fluorographed. Cultures were pulse-labeled at 36 h (lane 10) and 40 h (lane 11).

protein on an immunoblot of parasite antigens. These data indicate that the individual components of the antigen represent three distinct proteins. The anti-Ag44 rabbit sera of Coppel et al. [13] immunoblotted the 105 kDa protein (data not shown).

Pulse-chase labeling of antigen. Pulse-chase labeling of synchronous cultures throughout a complete 48 h cycle of the parasite was used to study the time of synthesis of antigen. At regular intervals, the culture was pulse-labeled with [35S]methionine and after 2 h, half the parasites were harvested and the other half chased with unlabeled methionine until the mature schizont stage before harvesting. The antigen was immunoprecipitated with 3E10/18 and analysed by SDSP A G E and fluorography (Fig. 4). At labeling times up to 28 h after invasion, no antigen was detected by immunoprecipitation, but at 32 h, the antigen appeared distinctly labeled. When compared with Giemsa-stained thin smears prepared at the same time interval, the antigen appeared to be synthesised at mid-trophozoite stage. Throughout the following intervals up to the mature schizont stage, the amount of labeled antigen remained constant; however, the 140 and 105 kDa proteins did decrease in amount during the chase with unlabeled methionine, which may indicate some degradation during parasite maturation. The short pulse-labeling experiment, using a pulse-labeling interval of only 10 min, failed to show the presence of any rapidly processed precursor proteins (Fig. 4). However, under these conditions the 130 kDa protein was not observed after immunoprecipitation.

Antigen purification. Antigen was affinity purified using a 3E10/18-Sepharose column, separated by S D S - P A G E and silver stained (Fig. 5, lane 1). Proteins of 140, 130, 105 and 98 kDa were observed, in the presence of [~-mercaptoethanol. Unlike the results obtained by metabolic labeling with [35S]methionine, silver stained gels of purified antigen indicate that the 105 kDa protein is present in greater quantities than the 140 and 130 kDa proteins.

256

1

2

3

4

Peptide mapping. The individual components of the antigen were subjected to digestion with S. aureus V8 protease, after first excising the bands from an SDS-PAGE gel (Fig. 5, lanes 2-5). The 140, 130, and 105 kDa proteins gave very different peptide maps; however, the band patterns from the 105 and 98 kDa proteins were very similar. The 7 kDa difference between the 105 and 98 kDa proteins was not seen in the peptide maps, possibly due to the very small peptides not being resolved in this gel system.

5

220-

9~

8

6760-

m 43-

8 3630-

Fig. 5. S D S - P A G E analysis and S. aureus V8 protease digest. Affinity-purified antigen was separated on S D S - P A G E and the individual protein bands were excised and placed in the well of a second gel. The gel slices were overlayed with S. aureus V8 protease in S D S - P A G E sample buffer, subjected to digestion during electrophoresis and the gel was silver stained. The wells were loaded as follows: untreated antigen (lane 1), 140 kDa (lane 2), 130 kDa (lane 3), 105 kDa (lane 4) and 98 kDa (lane 5).

Inhibition assays. The effect of the presence of monoclonal antibody during the invasion step on the subsequent incorporation of either [3H]hypoxanthine or [35S]methionine is shown for 5 experiments in Table I. Significant inhibition of incorporation was found in 4/5 experiments although the degree of inhibition was quite small. The counts from the medium control and test antibody for each experiment were used as the blocking factor in a two-way analysis of variance (data not shown). In each experiment MAb 3A10/ll was included as an example of a MAb giving low levels of inhibition (inhibition of the order of 20-30% at 300 txg ml-1). Control MAbs included at up to 700 txg m1-1 failed to inhibit incorporation of label into the parasite. The other MAbs directed against this antigen failed to inhibit the incorporation of label.

TABLE I Inhibition of P. f a l c i p a r u m growth and erythrocyte invasion by M A b cpm × 10 -3 Experiment

Antibody concentration (~,g m1-1)

n

Medium control

Test antibody

Percentage inhibition P

1a 2a 3a 4b 5b

760 211 694 694 694

3(8) 4(4) 3(6) 4(4) 4(4)

104"-- 14 294--- 14 307-+19 484---34 398---31

9 6 +- 18 247--- 13 221-+6 399--- 18 260--- 13

8 16 28 18 35

~1 <0.01 <0.001 <0.01 <0.001

MAb 3E10/18 was added to synchronous cultures at schizont stage and after merozoite release and erythrocyte invasion, the uptake of either [35S]methionine (a) or [3H]hypoxanthine (b) was determined after harvesting the culture at mature schizont stage. n is the number of replicate assays in each experiment (the figures in brackets from the control assays). P is the level of significance of the difference between the control and test values determined using a classical t-test.

257 Discussion

The four MAbs used in this study all immunoprecipitated bands at 140, 130, and 105 kDa, from [35S]methionine-labeled parasites. The antigen recognised by these MAbs was found to be located in the rhoptries of merozoites using both IFA and electron microscopy with immunogold labeling. Although four MAbs immunoprecipitated this antigen, only two of these MAbs immunoblotted antigen from parasite extracts. These latter MAbs each recognised only a single band, one of 125 kDa and the other of 98 kDa, respectively. These molecular masses correspond to the 130 and 105 kDa proteins since the immunoblotting was performed on nonreduced parasite antigen, resulting in a change in apparent molecular mass. The epitopes recognised by these MAbs are considered to be conformationally constrained since no immunoblot was obtained from parasite extracts processed under reducing conditions. The epitopes recognised by the other two MAbs are also presumed to be non-contiguous since these MAbs did not immunoblot. It seems likely that the antigen under study is the same antigen described by Campbell et al. (145, 135, and 114 kDa) [3], Holder et al. (155, 140 and 110 kDa) [10] and Siddiqui et al. (143 and 132 kDa) [11,12]. Holder et al. showed the 155 and 140 kDa proteins to be unrelated since rabbit antisera produced against the 140 kDa protein failed to react with the 155 kDa protein [10]. These polypeptides also show unrelated two-dimensional peptide maps. The 105 kDa protein identified by Coppel et al. [13] appears to be related to this antigen since rabbit sera raised against the fusion polypeptide reacts with a 105 kDa protein by immunoblot and immunoprecipitates the 140/130/105 kDa antigen in agreement with results reported by Lustigman et al. (manuscript in preparation). The 150/130 kDa rhoptry antigen described by Uni et al. [9] was recognised by a single MAb which immunoblotted both proteins. It is not clear whether the 150/130 kDa proteins are the same as the apparently unrelated 140 and 130 kDa proteins discussed in this report. A comparison of antibodies from different laboratories was carried out by Dayal et al. [29] and MAbs supplied by Campbell et al. [3], Holder

et al. [10] and Hall et al.[30] immunoprecipitated a 145, 135 kDa doublet from [35S]methionine-labeled antigen. MAb 3E10/18 was also tested in this study and was reported to immunoprecipitate a 36 kDa protein. We have been consistently unable to reproduce this result, and the reason for this discrepancy has not been established. In the present study, peptide maps prepared with S. a u r e u s V8 protease, of the individual proteins of 140,130, 105 and 98 kDa showed that the 140,130 and 105 kDa proteins are unrelated. The 98 kDa protein appears to be closely related to the 105 kDa protein, with similar peptide maps from both proteins and the 98 kDa protein may be a degradation product of the 105 kDa protein. In addition, no 130 kDa protein was observed in the immunoprecipitation of the 10 min labeled culture. Since the peptide maps of the 140 and the 105 kDa proteins are so different to that of the 130 kDa protein, neither of these can have precursor/product relationships to the 130 kDa protein. The lack of labeled 130 kDa protein in this case could reflect the way in which these antigens form a complex. Since the antibody used for the immunoprecipitations, 3E10/18, presumably only reacts with one member of the complex, the lack of immunoprecipitation of the 130 kDa protein following the 10 min labeling could be due to a slow association of the 130 kDa protein with the 140 and 105 kDa proteins. If this hypothesis is correct it would suggest that MAb 3E10/18 recognises either the 140 kDa protein, the 105 kDa protein or the binary complex of these two antigens. Because this antigen is precipitated as a complex, it is not possible to determine definitively when in the parasite cycle each antigen commences to be synthesized. However, the results of the pulse-chase experiments indicate that they are all being synthesized by the mid-trophozoite stage and that the component recognised by 3E10/18 is not present in an immunologically active form before the synthesis of the other members of the antigenic complex starts. Significantly, this complex is present in immunoprecipitates from parasites before rhoptries are clearly detectable. This complex formation therefore cannot be dependent upon the maturation of the rhoptries. We cannot rule out the possibility

258 that the formation of this complex occurs spontaneously and artifactually upon extraction of the parasites, although the immunofluorescence data suggest their common localisation, and the lack of labeled 130 kDa protein in the complex following a 10 min pulse would suggest that this is unlikely. Therefore, it appears as though this complex forms soon after synthesis of the proteins, initially through a 140 and 105 kDa binary complex followed by the further association of the 130 kDa protein. The native protein is also considered to be a soluble complex. Further work involving pulse-chase analysis with the other MAbs is needed to completely resolve when this complex is formed. Other antigens have been described which also exist as stable complexes in malarial parasites [2,5-8,31-33]. The antigenic complex reported by McBride et al. [31] consists of at least six components related to the 185 kDa polymorphic schizont antigen and a series of MAbs recognise epitopes both on the individual proteins and shared between them. MAbs conveying transmission blocking immunity in gametocytes have been shown to bind to a stable membrane bound complex found on the surface of the gametocyte [32,33]. Whilst these antigenic complexes seem to be associated with parasite surface membranes, another complex has been discovered in the rhoptries of merozoites [2,5-8]. The antigen discussed by Schofield et al. [5] and Bushell et al. [8] consists of proteins of 80, 66 and 42 kDa, the 80/66 kDa proteins having a precursor/product relationship. After surface immunogold labeling of intact merozoites, MAb 7H8/50 (directed against the 80 kDa component) clearly labeled membraneous material extruded from the merozoite [8]. The rhoptries have been shown to consist of multilamellar structures of possibly lipidic bilayers [34,35] and the secretion of this membraneous material may contribute to the formation of the parasitophorous vacuole membrane.

One MAb which recognises this antigen was found to inhibit parasite growth and/or subsequent merozoite invasion of erythrocytes in a weak but statistically significant manner. The finding of at least some inhibition suggests an important role for this antigen in the invasive process. The small degree of inhibition obtained is in accord with the kinetic constraints placed upon the binding of antibody to a transiently exposed antigen during the rapid invasion process [36]. Immunisation of monkeys with the 143, 132, and 102 kDa rhoptry proteins by Siddiqui et al. [12], conferred partial protection upon subsequent challenge. Although the mechanisms mediating immunity were not discussed, this finding emphasises the importance of these antigens in parasite development. Whether or not this antigen proves to be important for vaccine production, the further study of this antigenic complex is likely to make an important contribution to our understanding of the molecular basis of parasite invasion.

Acknowledgements This work was supported by grants from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases, the National Health and Medical Research Council of Australia, the Australian National Biotechnology Grants Scheme and the Australian Industrial Development Corporation. The authors would like to thank Diana Battistutta for statistical interpretation of the inhibition assay results. The supply of parasites used in part of this work by Margaret B. Moloney, Commonwealth Serum Laboratories, Melbourne, Australia is gratefully acknowledged. We also thank Ross L. Coppel, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia for the gift of rabbit sera.

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