AU627448B2 - Rhoptry membrane antigen of plasmodium falciparum - Google Patents

Description

OPI DATE 06/09/89 AOJP DATE 05/10/89 APPLN. ID 30699 89 PCT NUMBER PCT/AU89/00056

INTERNA

4 TREATY (PCT) (51)International Patent Classification 4 (11) International Publication Number: WO 89/ 07645 C1.2N 15/00, C12P 19/34 Al C07H 21/04 (43) International Publication Date: 24 August 1989 (24.08.89) (21) International Application Number: PCT/AU89/00056 ANDERS, Robin, Fredric [AU/AU]; 55 Brougham Street, North Melbourne, VIC 3051 KEMP, David, James (22) International Filing Date: 10 February 1989 (10.02.89) [AU/AU]; 309 Belmore Road, North Balwyn, VIC 3103 CULVENOR, Janetta, Gladys [AU/AU]; 52 Hume (31) Priority Application Number: PI 6743 Street, Greensborough, VIC 3088 EDWARDS, Stirling, John (AU/AU]; Flat 2, 9 Walker Street, Northcote, VIC (32) Priority Date: 12 February 1988 (12.02.88) 3070 PYE, David [AU/AU]; RMB 360, Bacchus Marsh Road, Bullengarook, VIC 3437 (AU).

(33) Priority Country: AU (74) Agents: SLATTERY, John, Michael et al.; Davies Collison, (71) Applicant (for all designated States except US): SARAMANE 1 Little Collins Street, Melbourne, VIC 3000 (AU).

PTY. LTD. [AU/AU]; The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, VIC 3052 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, (72) Inventors; and FR (European patent), GB, GB (European patent), IT (Eu- Inventors/Applicants (for US only): PETERSON, Michael, Gre- ropean patent), JP, KR, LU (European patent), NL (Eurogory [AU/AU]; 9 Lowan Avenue, Lower Templestowe, VIC pean patent), SE (European patent), US, 3107 CREWTHER, Pauline, Elizabeth [AU/AU]; 572 Rathdowne Street, North Carlton, VIC 3054 Published SMYTHE, Jason, Arthur [AU/AU]; 204 Station Street, With international search report.

Edithvale, VIC 3196 MARSHALL, Vikki, Maree [AU/ AU]; 13 Giles Retreat, Endeavour Hills, VIC 3801 (AU).

SILVA, Anabel [AU/AU]; 9 Berthandra Court, West Footscray, VIC 3012 (54) Title: RHOPTRY MEMBRANE ANTIGEN OF PLASMODIUMFALCIPARUM (57) Abstract A rhoptry membrane antigen of the asexual blood stages of Plasmodiumfalciparum, is characterised by: being initially synthesized as a polypeptide having a relative molecular mass of approximately 80,000: (ii) being located in the neck region of the rhoptries of merozoites; and (iii) having the solubility characteristics and a hydrophobic domain typical of an integral membrane protein; or an antigenic fragment thereof.

A

i i i i i i i ~Yi ii -~~;IIUIL- W 89/07645 PCT/AU89/00056 RHOPTRY MEMBRANE ANTIGEN OF PLASMODIUM FALCIPARUM This invention relates to the identification of an antigen of the asexual blood stages of Plasmodium falciparum, which is potentially capable of generating an immune response and antibodies which are able to inhibit the growth of the parasite, and to the use of this antigen and antibodies to it in immunization, diagnostic and treatment methods.

The molecular events which occur when a malaria merozoite invades the host erythrocyte are poorly 10 understood. Parasite molecules contained within the rhoptries, flask-shaped secretory organelles at the apical end of the merozoite, are believed to play a critical role in invasion. In the electron microscope the rhoptries appear as membrane-bound electron-dense structures. The -"(jlr 1 tit 'i:i r ii i:- P. *4 WO 89/07645 PCT/AU89/00056 2 invasion process coincides with the depletion of rhoptry contents which presumably are discharged via the apical duct although Bannister et.al. (1986) has suggested that discharge may also occur at the time of schizont rupture.

Electronmicroscopy using tannic acid-containing fixatives has revealed multilamellar membraneous whorls derived from rhoptry secretions. This indicates that there is lipid in the rhoptry secretion and it has been suggested that this may contribute to the formation of the parasitophorous vacuole membrane.

A number of different proteins have been identified in the rhoptries of P.falciparum. Two antigen complexes have been identified. A high molecular weight complex is composed of three antigenic polypeptides of ~Mr 105,000, Mr 135,000 and Mr 145,000 (Campbell et.al. 1984; Holden et.al. 1985; Cooper et.al. 1988 and Lustigman et.al.

1988) and a low molecular weight complex is composed of several polypeptides derived from a Mr 83,000 protein and a doublet of approximate Mr 40,000 (Campbell et.al, 1984; Howard et.al., 1985; Braun-Breton et.al., 1986; Schofield et.al. 1986; Bushell et.al. 1988).

Rhoptry antigens are considered potential vaccine components, initially because a rhoptry antigen from P.Yelii (Mr 235,000) effectively immunized mice against infection (Holder and Freeman, 1981). Subsequently, some monoclonal antibodies that inhibited the growth of P.falciparum in vitro were shown to be directed against rhoptry components (Schofield et.al., 1986). Furthermore, monkeys immunized with purified rhoptry antigens were partially protected against challenge with P.falciparum (Perrin et.al., 1985; Siddiqui et.al., 1987).

According to one aspect of the present invention, there is provided a rhoptry membrane antigen of the asexual blood stages of Plasmodium falciparum, which is characterised by: -3being initially synthesized as a polypeptide having a relative molecul i mass of approximately 80,000 which is rapidly processed to a polypeptide having a relative molecular mass of approximately 62,000; (ii) being detected in schizonts and free merozoites but not in ring stage parasites; (iii) being located in the neck region (peduncle) of the rhoptries of merozoites; and (iv) having the solubility characteristics and a hydrophobic domain typical of an integral membrane protein; or an antigenic fragment thereof.

S 10 Preferably, the antigen is a polypeptide having a primary structure which includes the amino acid sequence set out in Figure 1, or an antigenic o. fragment thereof. The invention also provides a method for actively immunizing a host against Plasmodium falciparum which method comprises administering to the host an antigen according to the present invention, or an antigenic fragment thereof. The invention provides a vaccine comprising an antigen of the present invention, or an antigenic fragment thereof, a pharmaceutically acceptable carrier or diluent, and optionally an adjuvant. The present invention also extends to a recombinant DNA molecule comprising all or a portion of a nucleotide sequence which is capable of being expressed as a r 20 polypeptide having the antigenicity of an antigen as described above, or an antigenic fragment thereof, or a recombinant cloning vehicle or vector, or a i host cell comprising a said recombinant DNA molecule. In this aspect, preferably the recombinant DNA molecule, recombinant cloning vehicle or vector, or host cell comprises all or a portion of a nucleotide sequence as set out in Figure 1.

Finally, the invention extends to a synthetic polypeptide prepared by expression of ail or a portion of a nucleotide sequence as described above, and to a vaccine composition comprising a said synthetic polypeptide.

9l11205jms c.014Jet,699.1e,3 WO 89/07645 PCT/AU89/00056 The present invention relates to a previously unrecognized rhoptry antigen that has the solubility characteristics and primary structural features of an integral membrane protein. This antigen, which has been designated rhoptry membrane antigen-1 (RMA-1), appears to be transported to the merozoite surface in late schizonts and free merozoites. The sequence of RMA-1 has been highly conserved between two isolates of E.falciparum and between P.falciparum and the murine malaria P.chabaudi.

Further features of the present invention will become apparent from the detailed description in the following Example, and the accompanying Figures.

In the Figures: Figure 1 shows the nucleotide sequence of the RMA-1 gene of P,.falciparum isolate D10 (top line) and a RMA-1 cDNA from isolate NF7 (bottom line, the asterisks denote the extent of the sequence). The predicted polypeptide sequence is translated. The cDNA sequence is only shown where it differs from the genomic sequence. Where this also results in an amino acid change, the change is shown in brackets. Two stretches of hydrophobic amino acids are underlined. Cysteine residues are shown in bold.

Figure 2 shows the nucleotide sequence of the RMA-1 gene of P.chabaudi adami DS. The predicted polypeptide sequence is translated. Two stretches of hydrophobic residues are underlined.

Fiure 3 is a comparison of the predicted amino acid sequences of RMA-1 from P.falciparum isolate D10 (top) and .chabaudi adami DS (bottom). Asterisks denote amino acid identities and dots denote gaps in the sequence, Figure 4 shows the extraction of RMA-1 into Triton X-114. When total infected erythrocytes were fractionated by SDS-PAGE, electroblotted to nitrocellulose and the immunoblots probed with affinity purified rabbit WO 8907645 PCT/AU89/00056 antibodies to the fusion protein produced by clone Ag352.24, two polypeptides were detected: one of Mr 80,000 and the other of Mr 62,000. Infected erythrocytes were fractionated by temperature-dependent phase partitioning with the detergent Triton X-114 to generate three fractions: aqueous phase in which the Mr 80,000 polypeptide was relatively enriched; Triton X-114 phase in which the Mr 62,000 polypeptide was relatively enriched; and insoluble pellet free of either of the RMA-1 polypeptides.

Figure 5 shows indirect immunofluorescence microscopy with antibodies to RMA-1. Antibodies affinity purified on the fusion protein produced by Ag352.24 from the serum of a rabbit immunized with this fusion protein were used to label erythrocytes containing various asexual blood stages of P.falciparum isolate FC27: rhoptry fluorescence on a mature schizont; and rhoptry and merozoite surface labelling; surface labelling on mature merozoites.

Fiure 6 shows the strain and stage specificity of RMA-1. To examine strain specificity erythrocytes infected with nine different isolates of P.falciparum were fractionated by SDS-PAGE, electroblotted to nitrocellulose and probed with rabbit affinity purified antibodies to the fusion protein produced by clone Ag352.24 or the anti-QF3 monoclonal antibody 7H850 Lane 1, uninfected erythrocytes; lane 2, NF7; lane 3, K-l; lane 4, FC27; lane 5, V1; lane 6, FC27 clone D10; lane 7, FC27 clone E12; lane 8, ITG2; lane 9, NF54 clone 3D7; lane 10, CSL2.

To examine stage specificity different asexual blood stages of isolate FC27 were fractionated bv SDS-PAGE, electroblotted to nitrocellulose and probed with rabbit affinity purified antibodies to the fusion protein WO 89/07645 PCT/AU89/00056 6 produced by clone Ag352.24 or the anti-QF3 monoclonal antibody 7H850 Lane 1, uninfected erythrocytes; lane 2, ring-stage trophozoites; lane 3, mature trophozoites; lane 4, schizonts; lane 5, merozoites.

Figure 7 shows the processing of RMA-1. Pulse-chase experiments were-carried out in which mature stages of P.falciparum isolate FC27 were labelled with 35 S-methionine for 15 minutes and then cultured in the absence of 35 S-methionine for 0 minutes (lane minutes (lane 30 minutes (lane 60 minutes (lane The radiolabelled parasites were processed for immunoprecipitation with either rabbit anti-Ag352.24 (A) or the QF3 monoclonal antibody 7H850 as described in the Materials and Methods. The immunoprecipitations were fractionated by SDS-PAGE and the labelled antigens visualized by autoradiography.

Fiuure 8 shows the localization of RMA-1 by immunoelectronmicroscopy. Post-embedding labelling of sections of P.falciparum isolate FC27 with human anti-352 antibodies or with rabbit anti-QF3 antibodies followed by protein A-5nm gold. Labelling occurred over the neck of the rhoptries with anti-352 antibodies and i over the rhoptry body for anti-QF3 antibodies.

Fitre 9 shows the immunogenicity of recombinant RMA-.

ELISA readings on individual rabbit sera, from rabbits designated by number under the graph, are shown. At the top, the responses to the GST-fusion protein are shown.

At the bottom, the responses to RMA-1 expressed in baculovirus are shown. Adjuvants used were: AL alum (aluminium phosphate); FC (or FCA) Freund's complete adjuvant; S/A squalene/Arlacel A (Ciba Geigy); SF SAF-1 (Syntex).

WO §9/07645 PCT/AU89/00056 7

EXAMPLE

Material and Methods Parasites: P.falciparum isolate FCQ27/PNG (FC27), was obtained through collaboration with the Papua New Guinea Institute of Medical Research. D10 and E12 are cloned lines of FC27 (Anders et.al. 1983). The origins of i s P.falciparum isolates NF7, Kl, Vl, ITG2, 3D7 and CSL2 have been described (Peterson et.al., 1988). Parasites were maintained in culture as described by Trager and Jensen (1976).

Antisera: Monoclonal antibody 7H850, specific for the QF3 antigen and rabbit antisera raised by immunization with QF3 antigen affinity-purified on the monoclonal antibody were obtained from the Queensland Institute of Medical Research. Production of the monoclonal antibody and purificntion of QF3 antigen are described elsewhere (Schofield et.al., 1986).

Rabbits were immunized with the fusion polypeptide produced by the clone pGEX-2T 352.24 (Ag352.24), and affinity-purified on glutathione-agarose as described (Smith and Johnson, 1988).

Human and rabbit antibodies were affinity-purified on adsorbents prepared by coupling the Ag352.24 fusion protein to CNBr-activated Sepharose as described (Crewther et.al., 1986). i Immunoelectronmcroscogy: Post-embedding immunolabelling was performed as described in Culvenor et.al. (1986).

Cultured parasites were fixed for ten minutes in 0.25% glutaraldehyde, dehydrated in 70% ethanol and embedded in white resin. Thin sections were incubated on droplets of antibody for 1 hr, washed, then incubated with protein A-5nm colloidal gold (Janssen) and stained with uranyl acetate.

WO 89/07645 PCT/AU&9/00056 i 8 Radiolabellina of parasites and immunoprecipitation: Routinely, parasites were synchronized once with sorbitol (Lambros and Vanderberg, 1979), mature trophozoites and schizonts were washed once in methionine-free RPMI 1640 medium supplemented with human serum and labelled for two hours at 37 0 C in the same medium with 3 5 S-methionine at 200pCi/ml. Similarly, mature parasites were labelled with L-[2,3,5,6- 3 H]tyrosine, L-[2,5- 3 H]histidine, L-[2,3,4,5- 3 H]proline or L-[4,5- 3 H]lysine monohydrochloride in the appropriate amino acid-free medium at 100pCi/ml or with [p9,10(n)- 3 H]myristic acid in regular medium at 100pCi/ml.

For pulse-labelling experiments, parasites were synchronized twice with sorbitol at an interval of 33 hours so that at the start of the experiment only ring-stage parasites 0-4 hours old were present. Aliquots of these synchronous parasite cultures were labelled for mins with 60pCi/ml 3 5 S-methionine at 1,1020,26,29,32, 35,38,41,44 and 48 hours after the second synchronization. After labelling, cells were washed twice in serum-free RPMI 1640 with 1.7mM cold methionine, and the pellets snap frozen. Duplicate cultures were harvested, washed three times in serum-free RPMI 1640 and smeared for subsequent immunofluorescence assay.

In general, pulse-chase labelling experiments were performed with parasites that had received two sorbitol treatments resulting in a 4-5 hour spread of maturation.

These synchronous parasites wre labelled with 100pCi/ml i 35 S-methionine for 15-45 minutes at 37"C. After the labelling period, the cells were harvested and an aliquot removed, washed twice in serum-free RPMI 1640 containing 1.7mM cold methinine and the cell pellet snap frozen. The remaining culture was washed twice in complete medium with ji 'i i1 WO 89/07645 PCT/AU89/00056 9 1.7mM cold methionine and incubated at 37 0 C in this same medium. Further aliquots were subsequently harvested, as described above, after various time intervals.

Giemsa-stained smears were examined at each time point confirmed the stage of maturation of the parasites.

Frozen cell pellets were extracted for 30 mins at 4 0 C in at least ten volumes of NaCl-EDTA-Tris buffer supplemented with Triton X-100 (T-NET) as described by Kessler (1975) containing a cocktail of protease inhibitors comprising 4mM phenylmethylsulphonyl fluoride (Sigma Chemical Co.), iodoacetamide (BDH), 1mM L-l-tosylamide-2-phenylethylchloromethyl ketone (Sigma Chemical ImM N-a-p-tosyl-L-lysine chloromethyl ketone (Sigma Chemical 25pg/ml leupeptin (hemisulphate salt) (Sigma Chemical 25g/ml antipain (dihydrochloride) (Sigma Chemical 25pg/ml chymostatin (Sigma Chemical Co.) and 25pg/ml pepstatin (Sigma Chemical Insoluble material was removed by centrifugation at 10,000g for mins at 4 0 C. The labelled extracts were precleared using formalir,-fixed Staphylococcus aureus Cowan 1 strains (Commonwealth Serum Laboratories) and Sephatose 4B beads (Pharmacia) for 30 min at 4 0 C. Preclearing agents were i removed by centrifugation for 5 min at 5,000g at 4°C.

Specific antisera were added to aliquots of the labelled extracts and incubated at 4 0 C for 2-4 hours. Immune complexes were precipitated by the addition of Protein 'p A-Sepharose 4B (Pharmacia). The beads were washed four times with T-NET containing protease inhibitors. Culture supernatants collected after labelling of parasites .ere centrifuged for 1 hour at 100,000g prior to immunoprecipitttion.

SDS-PAGE: SDS-PAGE was performed essentially as described by Laemmli (1970). Immunoblotting was carried out as described (Crewther et,al., 1986).

w ,i i i~ WO 89/07645 PCr/AU89/00056 hi Molecular cloning and DNA sequencing; The construction and screening of the P.falciparum NF7 cDNA library in Agtll-Amp3 has been described previously (Stahl et.al. 1984). The P.falciparum partial Sau4AI genomic library in EMBL3 was made as follows: A partial Sau3AI digest of D10 genomic DNA was size fractionated (10-20 kb) on an agarose gel and ligated into BamHI digested, dephosphorylated XEMBL3. The resulting clones were screened with the NF7 cDNA clone, Ag352, using standard procedures (Maniatis et.al. 1982). The P.falciparum partial S1I genomic library in Xgtl0 was made as follows: A partial SsPl digest of P.falciparum genomic DNA was methylated with EcoRI methylase, ligated with EcIE linkers and digested with EcoRI.

Excess linkers were removed by size-fractionation on an agarose gel and the fragments were ligated into EcoRI digested, dephosphorylated )gtl0. The resulting clones were screened with the NF7 cDNA clone, Ag352. The P.chabaudi adami DS sheared genomic library in )gtlO was made as follows: P.chabaudi adami DS genomic DNA was sheared through a 26 gauge needle, methylated with EEQ R methylase and ligated with EcoRI linkers. Excess linkers were removed and the fragments were size-fractionated (4-7 kb) on an agarose gel, then ligated into EcoRI digested, dephosphorylated Xgtl0. The resulting clones were screened with the NF7 cDNA clone, Ag352. Positively hybridizing clones were plaque purified through successive rounds of screening, XDNA prepared and the inserts isolated and subcloned using standard procedures (Maniatis et,al. 1982). DNA inserts were further subcloned into M13 vectors (Messing and Vieira, 1982) and sequenced using the chain termination method (Sanger et.al. 1977).

Expression of the C-terminal 52 amino acids of RMA-1 inL

I

WO 89/07645 PCT/AU89/00056 11 The C-terminal 52 amino acids of RMA-1 Were expressed in E.coli using the pGEX-2T plasmid voLir described by Smith and Johnson (1988). The partially complementary oligonucleotides 5'GATCCGGAAATGCTGAAAATATGATAAAATGGATGAACCACAAC and which encode the complementary strands of the 15 amino acids C-terminal to the transmembrane anchor region where annealed then ligated with a Fokl to EcoRI (linker) fragment (nucleotides 2083-2255, Fig.l) from clone NF7, and BamHI and EcoRI digested pGEX-2T. The ligation was transformed into E.coli JPA101 and the correct recombinant selected. This clone is designated Ag352.24.

Expression of near-full lenoth RMA-1 in E.coli: A segment of RMA-1 consisting of the end filled HinPl. to Rsal site nucleotides 366 to 1833 (from Fig.l) was inserted into the filled BamHl site of Ag352.24, transformed and selected as above. This clone is designated Ag352.2.

Expression of RMA-1 in Baculovirus: The full length RMA-1 gene was removed as a BamHI/B alJ fragment (nucleotides 327-2247 in Fig.l) from a pIC20H subclone (Marsh et.al., 1984) and was inserted into the unique BamHI site of the pAcRP23 (provided by Dr.R.Possee) baculovirus transfection vector behind the baculovirus polyhedrin gene promoter. The pAcRP23/Ag352 DNA was mixed with wild type baculovirus DNA and transfected into Sf9 insect cells. Plaques resulting from the transfection were screened microscopically and those lacking polyhedra were subjected to two further rounds of plaquing. A number of the resultant polyhedron-negative plaques were screened for the presence of the Ag352 gene and all proved to be positive, Virus from recombinant plaques (designated baculovirus 352) was used to infect portion of a nucleotide sequence which is capable of being expressed as a polypeptide having the antigenicity of an antigen according to any one of claims 1 to 3, or an /2 I' 12 fresh cultures of Sf9 cells and production of Ag352 by infected cells examined by SDS-PAGE, Western blot and immunofluorescence.

Immunocenicity: Rabbits were immunized with 1.0ml of antigen formulation I/M in the left back leg. A second first dose. Bleeds were taken on the day of immunization and at two weekly intervals, except that the final bleed was taken 7-8 days after the second dose. For animals in which squalene/Arlacel A (Ciba Geigy nor-MDP formulation) was used as adjuvant, 100pg nor-MDP was used/dose. All antigens were at 200pg/dose.

Structure of RMA-1 and its gene in P.falciparum, Screening a Xgtil-Amp3 cDNA clone library (NF7) with human anti-malarial antibodies (Stahl et.al. 1984) identified a novel cDNA clone designated Ag352. The DNA sequence of Ag352 i shown in Figure 1. To complete the coding sequence, two overlapping genomic fragments of isolate FC27 (a 5' partial Sau3A and a 3' partial Sspl fragment) were cloned and sequenced (Figure 1).

The FC27 genomic sequence extends the 1438 nucleotide NF7 cDNA sequence a further 514 nucleotides 3' and 809 nucleotides A single long open reading frame is present in the FC27 genomic sequence between nucleotides 333 to 2202 encoding a polypeptide of 622 residues with a predicted molecular weight of 71,929 daltons. Based on the subcellular localization results presented below, this polypeptide was termed rhoptty membrane antigen-1 (RMA-1).

The FC27 genomic and the NF7 cDNA sequences are highly homologous with 9 nucleotide differences in 1438 nucleotides and 7 amino acid differences. Strikingly, WO 89/07645 PCT/AU89/00056 S13 only 1 nucleotide difference is silent and 5 of the amino acid differences result in charge changes. These data indicate strong selection, perhaps immunological, on the polypeptide sequence.

Comparison of the sequence of RMA-1 with the Genebank and NBRF databases failed to identify any similar proteins. However, the RMA-1 polypeptide sequence has a number of striking features. Unlike most other blood stage antigens of P.falciparum, RMA-1 lacks repetitive sequences. The antigen has the primary structure expected for an integral membrane protein. It contains 2 hydrophobic stretches, one near the N-terminus, presumably part of a signal peptide, and a second located 55 amino acids from the C-terminus. This consists of 21 predominantly hydrophobic residues and lacks charged residues. It is preceded by a lysine residue and followed by the tripeptide Lys-Arg-Lys, consistent with this region being a membrane spanning domain.

All 17 cysteine residues precede the predicted membrane-spanning domain. The most N-terminal cysteine is presumably removed along with the signal peptide. The remaining 16 residues could form intramolecular disulphide bonds and therefore could be critical for maintaining the secondary structure of the molecule. Consistent with this idea, the mobility of RMA-1 was decreased when electrophoresed under reducing conditions compared to non-reducing conditions (data not shown). The importance of the cysteine residues is supported by the observation that all 16 are found conserved in RMA-1 from P.chabaudi (see below).

Structure of RMA-1 and its gene in P.chabaudi.

A P.chabaudi adami DS sheared genomic library in XgtlO was screened with the P.falciparum NF7 cDNA clone (described above) to identify the homologous gene in this ,i WO 89/07645 PCT/AU89/0056 .14 species, The DNA sequence of the hybridizing region of a positive clone is shown in Figure 2. A single Zong open reading frame is present between nucleotides 556 and 2229 encoding a polypeptide of 558 amino acids with a predicted molecular weight of 63,901 daltons.

The predicted polypeptide of RMA-1 from .chabaudiI and P.falciparum show a remarkable degree of similarity.

Like the P.falciparum homologue, the P.chabaudi RMA-1 polypeptide sequence predicts 2 hydrophobic stretches (underlined in Figure 2) located in very similar positions. These presumably act as an N-terminal signal sequence and a trans-membrane anchor. The sequence lacks repeats, and the 16 cysteine residues, which were such a striking feature of the (predicted) mature P.falciparum molecule are all found conserved in the P.chabaudi molecule. The overall amino acid sequence homology between the 2 species is 43% (Figure 3).

RMA-1 partitions into Triton X-114.

When cell extracts are fractionated by temperature dependent phase partitioning with the non-ionic detergent Triton X-114, integral membrane proteins are recovered in the detergent phase. The solubility characteristics of l RMA-1 were examined using this procedure. Parasite cell pellets from isolate FC27 were solubilized in Triton X-114 and separated into an aqueous phase, a Triton X-114 phase i and an insoluble pellet and fractionated by SDS polyacrylamide gel electrophoresis. An immunoblot of these fractions was probed with antibodies affinity purified (Crewther et.al., 1986) on a glutathione S-transferase fusion protein (Smith and Johnson, 1988) containing only the C-terminal 52 amino acids (571 to 622, Figure 1, Ag352.24). The antibodies reacted with two polypeptides of Mr 80,000 and 62,000 which were present in variable amounts in the total parasite material, in the *rv- WO 89/07645 PCT/AU89/00056 aqueous phase and in the Triton X-114 phase (Figure 4).

The Mr 62,000 polypeptide appears to be a processed fragment of the Mr 80,000 (see below). Both the Mr 80,000 and 62,000 polypeptides are partially soluble in Triton X-114, the smaller fragment being more soluble probably because it has lost hydrophilic N-terminal residues.

RMA-1 is localized to the rhoptries and the merozoite surface.

Indirect immunofluorescence microscopy with affinity-purified human antibodies (described above) stained mature asexual blood forms. Segmented schizonts showed a punctate pattern of immunofluorescence within merozoites (Figure 5a) characteristic of antigens located in the rhoptries (Holder et.al. 1985). This pattern was also evident within some merozoites of disrupted schizonts although other clusters of merozoites gave a "bunch of grapes" fluorescence pattern, characteristic of a merozoite surface location (Holder et.al. 1982) (Figure Patterns pf fluorescence intermediate to these were also evident in some mature schizonts (Figure 5b and where there was merozoite surface fluorescence with "hot-spots" at the apex of each merozoite. RMA-1 is present in all isolates of P.falciparum Nine isolates of P.falciparum of diverse geographic origins were examined by immunoblotting for reactivity with antibodies to RMA-1. Asynchronous parasite preparations solubilized in SDS sample buffer and fractionated on 10% SDS-polyacrylamide gels were probed with affinity purified rabbit anti-Ag352.24 antibodies that had been pre-adsorbed on the Triton X-100-insoluble fraction of normal human erythrocytes. In each isolate Mr 80,000 and 62,000 polypeptides, sometimes resolved as doublets were identified (Figure 6a). In some isolates there was weak reactivity with an additional polypeptide of Mr 70,000.

i i WO 89/07645 PCr/AU89/00056 16 A previously identified rhoptry antigen, QF3, has polypeptide components of similar size to the two polypeptides recognized by anti-Ag35".4 antibodies. The monoclonal antibody 7H850, which recognizes QF3 (Schofield et.al. 1986; Bushell et.al. 1988), when used to probe an immunoblot of the 9 P.falciDarum isolates also recognized polypeptides of -Mr 80,000 and Mr 62,000 that like the polypeptides recognized by anti-Ag352 antibodies were invariant in size among these isolates (Figure 6b).

RMA-1 is found in mature asexual blood-stages.

When synchronized parasites were examined by immunoblotting RMA-1 was detected in schizonts and free merozoites but not in rings or other asexual blood-stages (Figure 6c). The QF3 antigen differed from RMA-1 in that it was clearly present in ring stage parasites in addition to schizonts and merozoites (Figure 6d).

In pulse-labelling studies, synthesis of both Ag352 and QF3 antigens was first detected in mature trophozoites, 29-32 hours old. Maximum synthesis of RMA-1 occurred in mature schizonts (39-41 hours) whereas peak synthesis of QF3 occurred in mid-schizonts, 36 hours after the second synchronization (data not shown). No synthesis of either antigen was detected after the 44 hour time point. Consistent with the immunoblotting studies, affinity-purified rabbit anti-Ag352.24 antibodies specifically immunoprecipitated doublets of Mr 80,000 and 62,000 from Triton X-100 lysates of mature trophozoites and schizonts labelled with 35 S-methionine. The anti-QF3 monoclonal antibody, 7H850, immunoprecipitated Mr 80,000 and 62,000 polypeptides (and a minor Mr 70,000 polypeptide) together with a Mr 39,000 and 38,000 doublet. Similar results were obtained when both antibodies were used to immunoprecipitate the relevant antigens from lysates of parasites labelled with tritiated .cui Ir ii i!:S i; tl :i iu .I 9 g, i: i i i i ^1 :i ji i V" 'T WO 89/07645 PCT/AU89/00056 17 _proline, tyrosine, lysine or histidine. There was no marked difference in the intensity of labelling of the two antigens with any of these amino acids (data not shown).

Repeated clearing of 3 5 S-methionine-labelled parasite lysates with antibodies specific for RMA-1 failed" to diminish the intensity of bands immunoprecipitated with monoclonal antibodies specific for the QF3 antigen. The same was true for the reverse experiment (data not shown).

The RMA-1. MV 80.000 polypeptide is rapidly processed to a M 62.000 form.

Pulse-chase experiments utilizing a labelling period of 15 minutes showed that the Mr 80,000 polypeptide of RMA-1 was rapidly processed to the Mr 62,000 form (Figure Approximately 50% of the antigen synthesized in mature schizonts during a 15 minute pulse-label was processed to the lower molecular weight form over a 1 hour chase period. When the fate of this antigen was followed over a longer chase period, the same rapid processing was seen (data not shown). teither the Mr 80,000 or the 62,000 molecules were detected in new rings after reinvasion. This same pattern of processing was also observed if the antigen was pulse-labelled in mature trophozoites and immature schizonts, indicating that cleavage of the larger molecules is not restricted to a particular life-cycle stage (data not shown). Labelling RMA-1 was not detectable by immunoprecipitation of clarified culture supernatants (data not shown).

In contrast, using the same conditions for Spulse-chase experiments, it was found that the QF3 antigen was synthesized as a short-lived precursor molecule of Mr 84,000, all of which was processed to the Mr 80,000 form over a 1 hour chase period (Figure7t Processing of the Mr 80,000 molecule to the Mr 62,000 form of QF3 was not apparent when mature schizonts were pulse-labelled.

AI

v i i c WO 89/07645 PCT/AU89/00056 18 However, when mature trophozoites and immature schizonts were pulse-labelled and chased for various times, the bulk of this processing was found to occur in mature schizonts during reinvasion. Only the Mr 80,000 molecule of QF3 was carried into rings. None of the molecules of the QF3 complex were detectable by immunoprecipitation of clarified culture supernatants collected after the 12 hour chase period in this experiment.

RMA-1 and OF3 are in different regions of the rhoptry.

Immunogold labelling studies consistently showed localization of the RMA-1 antigen to the neck of the rhoptries of mature merozoites within schizonts (Figure 8a) and, similarly, in free merozites (Figure 8b and c).

Occasional gold particles were found around the periphery of merozoites (Figure 8b and c) and over micronemes which were of similar electron density to the rhoptry neck (Figure 8a). Sections derived from the same resin block of FC27 incubated with rabbit anti-QF3 antibodies showed i labelling over the body of the rhoptry, not in association with the neck or the plasma membrane (Figure 8d).

Expression of RMA-1 in baculovirus; Baculovirus expressed RMA-1 as a protein doublet of Mr 84-87,000 with secondary bands of Mr 67-77,000. The protein was recognized on immunoblots of baculovirus RMA-1 infected cells by rabbit antisera to RMA-1 and also by human antibodies. The protein was detectable as a minor band on Coomassie-stained gel (data not shown).

Immunogenicity of recombinant RMA-1 in rabbits: S" To examine the immunogenicity of the near-full length RMA-1 expressed in E.coli (Materials and Methods) and of the RMA-1 expressed in baculovirus (see above), rabbits were immunized with lml of an antigen formulation, boosted after four weeks and bled 7-8 days later (Fig.9).

It is clear from the ELISA readings Freund's complete j 19 19 i adjuvant gave, on average, a greater response than the !I nor-MDP adjuvant, and RMA-1 expressed in baculovirus gave a greater response than when expressed as a fusion protein.

DISCUSSION

Previous studies have identified two antigen complexes in the rhoptries of P.falciparum merozoites reviewed in Anders (1988). In addition to these antigen complexes at least two other rhoptry antigens have been described, one an Mr 55,000 polypeptide with the solubility characteristics of an integral membrane protein (Smythe et.al., 1988) and another, a Mr 240,000 molecule which is processed to an Mr 225,000 fragment (Roger et.al., 1988). The results reported here show that RMA-1 is unrelated to any of these antigens and thus represents a previously uncharacterized rhoptry antigen. RMA-1 is initially synthesized as an Mr 80,000 polypeptide which is rapidly processed to an Mr 62,000 form. Coincidentally, two of the major components of the lower molecular weight rhoptry complex (QF3) (Schofield et.al. 1986; Bushell et.al. 1988) have the same relative molecular masses but the different locations within the rhoptry, the different processing pattern seen in pulse-chase experiments, and different co-precipitating proteins indicate that the RMA-1 and QF3 polypeptides are different.

RMA-1 appears to be located first in the rhoptry and then exported to the merozoite surface around the time of schizont rupture. Within the rhoptry RMA-1 appears to be particularly localized in the neck (or peduncle). One other rhoptry antigen, the Mr 240,000 polypeptide described by Roger et.al.(1988) is also in this location but the other rhoptry antigens that have been studied using immunoelectronmicroscopy are located in the body of the rhoptry. The heterogenous distribution of rhoptry WO 89/07645 PCT/AU89/00056 contents indicated by these observations has not been explained but it is of some interest that RMA-1, located in the rhoptry neck, is an integral membrane protein whereas the Mr 105,000 component of the high molecular weight rhoptry complex, which is located in the body of the rhoptry (Culvenor et.al., 1986) lacks any structural or solubility characteristics of an integral membrane protein (H.Brown, unpublished results). Thus, as there is good evidence of lipid within the rhoptry secretion it may be that integral membrane proteins are associated with lipid in the rhoptry neck. Presumably RMA-1 becomes associated with the mature merozoite surface after being released, possibly together with lipid, via the apical duct of the merozoite. Consistent with this, others have suggested that some release of rhoptry contents may occur at the time of schizont rupture (Bannister et.al., 1986).

The presence of RMA-1 within rhoptries and then apparently associated with the merozoite surface provides strong support for RMA-1 as a potential vaccine component. RMA-1 is a natural immnunogen which induces antibodies in individuals infected with P.falciDarum.

L "vmV 4, NWO 89/07645 PCr/AU89/00056 1. Anders, R.F. et.al. (1983), Proc.Natl.Acad.Sci.USA U&f: 6652-6656.

2. Anders, R.F. (1988). In: The Biology of Parasitism. (Eds.P.T.Englund and A.Sher), pp.201-204. Alan R.Liss Inc., New York.

3. Bannister, L.H. et.al. (1986). ParaSitolo~qy iZ:291- 303.

4. Braun-Breton, C. et.al. (1986). Molec.biochem.

£.arasJ.±Q. 2&:33-43.

P~aai±.o. 2&~:105-112.

6. Campbell, G.H. et.al. (1984). IAm.J.TroD.MedHyg.

7. Cooper, J.A. et.al.(1988). Mol.Biochem. Parasitol.

.Z2: 251-260.

8. Crewther, P.E. et.al. (1986).

9. Culvprnor, J.G. et.al. (1986).

.EZ:58 -67.

Holder, A.A. and Freeman, R.R.

361-364.

11. Holder, A.A. and Freeman, R.R.

J.Immunol. Methods.

Exp.Parasitol.

(1981). Nature 22A: (1982). a.EX]p.Med.

WO 89/07645 PCf/AU89/00056 ~tq <7 22 12. Holder, A.A. et.al. (1985) Molec.Biochem.- Parasitol. 1A.:293-303.

13. Howard, R.F. et.al. (1984). Am.Jrov,,edyg. Ua: 1055-1059.

14. Kessler, S.W. (1975). Jtnnuzxol. 112:1482.

Laenunli, J.K. (1970). NatLure 22a:680.

16. Lambros, C. and Vanderberg, J.P. (1979).

J.Parasitol. 41.8-420.

17. Lustigman, S. et.al. (1988) Mol.Biochem. Parasitol.

U: 217-224.

18. Maniatis, T. et.al. (1982). Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory.

19. Marsh, J.L. et.al. (1984). Gen U:481-485.

Messing, J. and Vieira, J. (1982). Gen 1.2: 269-276, 21. Perrin, L.H. et.al. (1985). J.Ciin.Invest.

1718-1721.

22. Peterson, M.G. et.al. (1988). Molec. Cell Biol. 2664-2667.

23. Roger, N. et.al. (1988). Molec.Biochen.Parasitol.

2a6. 135-142.

24. Sanger, F. et.al. (1977). Proc.Naf1I.Acad Sci.S .74.:5463-5467i

I

WO 89/07645 WO 8907645PCT/AU89/00056 Schofield, L. et.al. (1986). Molec,.Biochem.

Parasi±Q1. 183.-195.

26. Siddiqui, W.A. et.al. (1987). Proc.Natl.Acad.Sci.

MS .A:3014-3018.

27. Smith, D.B. and Johnson, K.S. (1988). Gen L:31-40.

28. Smythe, J. et.al. (1988). Proc.Natl.Acad.Sci. USA Us: 5195-5199.

29. Stahl, H.D. et.al. (1984). Proc.Natl.Acad.Sci. USA BIi.2456-24 Trager, W. and Jensen, J.B. (1976). sience Ma:678- 675.

pii

Claims (9)

1. A rhoptry membrane antigen of the asexual blood stages of Plasmodium falciparum, which is characterised by. being initially synthesized as a polypeptide having a relative molecular mass of approximately 80,000 which is rapidly processed to a polypeptide having a relative molecular mass of approximately 62,000; (ii) being detected in schizonts and free merozoites but not in ring stage parasites; 10 (iii) being located in the neck region (peduncle) of the rhoptries of merozoites; and (iv) having the solubility characteristics and a hydrophobic domain typical of an integral membrane protein; or an antigenic fragment thereof,

2. An antigen according to claim 1, which is antigen RMA-I described herein, or an antigenic fragment thereof.

3. An antigen according to claim 1, having a primary structure which 20 includes the amino acid sequence set out in Figure 1, or an antigenic fragment thereof.

4. A vaccine composition comprising an antigen according to any one of claims 1 to 3, or an antigenic fragment thereof, and a pharmaceutically acceptable carrier or diluent.

A composition according to claim 4, further comprising an adjuvant.

6. A method for actively immunising a host against Plasmodium falcipanm, which method comprises administering to the host an antigen according to any one of claims 1 to 3, or an antigenic fragment thereof, or a vaccine composition according to claim 4 or claim 1 911205jmscii .O14.]et,30699.1t,24 L A. -AM LY~: i i::a r ili I rl;- ~i V/0 89/0764 PCT/AU89/00056 i ii i 'i ii I i i

7. A recombinant DNA molecule comprising all or a portion of a nucleotide sequence which is capable of being expressed as a polypeptide having the antigenicity of an antigen according to any one of claims 1 to 3, or an antigenic fragment thereof, or a recombinant cloning vehicle or vector, or a host cell comprising a said recombinant BNA molecule.

8. A recombinant DNA molecule, recombinant cloning vehicle or vector, or host cell according to claim 7, wherein said nucleotide sequence is a sequence as set out in Figure i.

9. A synthetic polypeptide prepared by expression of all or a portion of a nucleotide sequence accordingsto claim 7 or claim 8. A vaccine composition comprising a synthetic polypeptide according to claim 1. GENE GATCTAATAACGGATATAT CTATTTTTTTTAATGEACAAGAAAAAAAAAAAAAGAAAAAA ATATTTAATATATTTATATATTATAAAAAAAAGGAAAAAAAAAAAAAAAAAAAAAAA TTAI GENE GTAATATATTTTTTATTTTAATATAATTTTAAAAACCTAATAATTTATTTGATAATTTTT. C: L SA FE FT YM I NFGRG (IN YW E GENE TTGAGCGCCTTTGAGTTT.ACATATATGATAAAETTTGGAAGAGGACAGAATTATTGGGAA CE Y (I Y P L H Q E H T Y a, (I E 0 S G E 0 ZZ mI GENE GAATACCAATATCCATTACACCAGGAACATACATACCAAEAAGAAGATTCAGGAGAAGAC E1 aQN L FSS I E I VER SN Y MGN P GENE GAACAAAATTTATTTTCAAGCATTGAAATAGTAGAAAGAAGTAATTATATGGGTAATCCA DL GEOA E V AGTQlaY RL PS GK C 'iE GATTTAGGAGAAGATGCTGAAGTAGCTGGAACTCAATATAGACTTECCATEAGGGAAATGT GENE GCTACGGGAAATCAATATTTAAAAGATGGAGGTTTTGCTTTTCC TCCAACAGAAC CTCTT Fig.1A. !AAAAAAGAACAAGGGAAAAAAGAAGAAAAATGGAAATTAAAATT ITACAC TTATTAATAA 120 -ACCATATTTAATGGTTTTATTTTTTTATTTTTTTAAAAAAAAAAATTGAGAATATAAATT 240 KMATTA MRK LY CV L L 9 CAAATAAGTATTGTTATAAATTGTACAAAAATGAGAAAATTATACTGCGTATTATTA 360 Ln H P Y Q K SO0 V Y H P IN E H R E H P K 49 CATCCATATCAAAAAAGTGATGTGTATCATCCAATCAACGAACATAGGGAAEATCCAAAA 480 'E NT L QHA YPI DOHE GA E P AP Q 89 N\) u-ICGAAAATACATTACAACACGCATATCCAATAGACCACGAAGGTGCCGAACCCGCACCACAA 600 W T E Y M A K Y 0 I E E V H G3 S G3 I R V 129 rnrnTGGACGGAATATATGGCAAAATATGATATTGAAGAAGTTCATGGTTCAGGTATAAGAGTA 720 P V F G3K G I[II ENS N T T F L T P V 169 CCAGTATTTGGTAAAGGTATAATTATTGAGAATTCATCATTTACCGT 840 cONA START* M S P M T L 0 E M R H F Y K 0 N K Y V K 209 ATGTCACCAATGACATTAGATG AAATGA(3ACATTTTTATAAAGATAATAAATATG TAAAA 960 0 Fig. 18. N L 0 E L T L C S R H AG N M I P ODN D GENE AATTTAGATcAATTGA[TTTAIGTTCAAG"LACATGCAGGAAATATGATTC CAGATAATGAT cONA L Y I A AQGE N N G PR Y C N KOD E S K GENE TTATATATTGCAGCTCAAGAAAATAATGGTCCTAGATATTGTAATAAAGACGAAAGTAAA cDNA LSK N VVDN W E KVC P RK N LQlN GENE TTAAGTAAAAATGTAGTTGATAACTGGGAAAAAGTTTG[CCTAGAAAGAATTTAEAGAAT J -4cNA G (Q to in S A! D L F E CN!K LV F EL S ASODQ GENE TCAGCAATTGATCTTTTTGAATGTAATAAATTAGTTTTTGAATTGAGTGCTTEGGATCAAI inDcNA A (I to N) K NA S MI K SA F LPT GA F KAD R GENE AAGAACGCTAGTATGATCAAAAGTGCTTTTCTTCCCACTGGTGCTTTTAAAGCAGATAGA WfNA ElJ F NV KP T CL I NN SS Y I AT T GENE GAAATTTTTAATGT CAAACCAACATGTTTAATTAAC AATTECAT CATACATTGC TAC TACT cDNA Fig. 1iC. 'Ii CC AK N S N Y K Y P A V Y E 0 K 0 K K N H F 329 ACAAAATTCGAAATTATAATCATAAATTTTAATAAAATAAATGAATAT 130 R N S M F G F L P A K I S F Q N Y T Y 329 AO rEAAAACATTTTTGTATTTAACAOAAAAAAATATTAAAACTTCATAT 1240 Y*A 5 K F Y LW V DO N Y ED I PH V NE F432 (iCTAAAATTCATTATGGTCOATOAATTOTAAATATACACATOTCAAAAAATTT 1320 4 ET (H to)0 P K QS E H LT YE K IK OF FPC K N 3649 1 CCTAAATCAATCAATGAAACAATTTCATTTAACAATTATOAAAATAAATTAATAA 1440 A (H to N)H) Fig. 16. I M K E I E R E S K R I K L N 0 N 0 0 E GENE AT AATGAAAGAAAT CGAAAG AGAAT CAAAA CGAATTAAAT TAAAT GATAAT GAT GAT GAA cONA C PC O P ElI V S NS T CN F F V CK C GENE TG CC CAT GTGACC CTGAAATTGTAAGT AATAGTA CATGTAATTT CT TTGTATGTAAATGT cDNA 0 (I to M) CG (N to R) OE YA D I P EH KP T YODK MKI I I c GENE GATGAATATGCAGATATTCCTGAACATAAACCAACTTATGATAAAATGAAAATTATAATT cJNA GENE AAAGGAAATQCTGAAAAATAT'GATAAAATGGATGAACCACAACATTATGGGAAATCAAAT cDNA S H TT P V LM E K P YY* GENE T[ACATAC.AACACCAGTTCTG'ATGGAAAAAC CATACTATTAAAATGTGAAC TATAATAAT cONA GENE ATTTATAAATATATTTATATATATTTATATTTATATTTCTATGjATTT CTTAATATTTTTC 0 GENE TATTTTCTTTATAAACTAAATGATTTTCACCTTCATTTTTTGTATTAATAATAAACGAAC GENE TGTATATACTAATAT CAGCATGTA'TTTATTTTTAACOAATAAAAGTGGACACAAGGAAA GENE TAAATGGTAATTTCT CAATATT Fg.i 0 C -J In C w "I nsf C -I 'U' U, z Pt Eu .4 G N KKI IA PR I F I SOODKODS L K GGGAATAAAAAAATTATAGC TCCAAGAATTTTTATTTCAGATGATAAAGACAGTTTAAAA V E RR AEV TS N NE VV VKE EY K GTAGAAAGAAG 10C AGAAGTAAC AT CAAATAATGAAGTTG TAG TTAAAG AAGAATATAAA A S SA AVA V LAT I L MV YL Y KR GCATCATCAGCTGCTGTC GCTGTATTAGCAACT.ATTTTAATGGTTTATCTTTATAAAAGA S R N E M LD PEA S FW GEE KR A TCAAGAAATGATGAAATGTTAGATCC TGAGGCATCT TTTTGGGGOAAGAAAAAAGAGCA TTCAACGTCTGATATAATCAGCTTCTCTTTTATGCTAAAAAAAAAAAAATATATATATAT END cONA TATGCTCATCGGATATTTCATTTTTTCTTTGAI3TTGATAAATTTCCATCTTGAATTAATT TAAAAGATG TCATATACGATTTTGAACATAAAAAAAAOAC ACAGGAATTTAATAAAATTA AAMAATGAACAATAAAAT3ATTTGACATACTATACATATGCATTAAAAAAGAGTAACTAA Fig. 1F 489 1800 529 1920 569 2040 609 2160 622 2280 2400 2520 2640 2662 :273:1. WO 89/07645 PI/U9/05 GO AT CCAAATA C T TAATTTC AATG TOT OT GOAAC TTAT AAOGGAAAAAAAGGGGAAAAAAC3TACTTCTACACATTGT, TACA CAT TTT AC T CATAGAAG AAGGTTAT AT AAT TA TAT A I N L GNC S E GT DK I ATAAACTTGGGTAATTGTT CCOAAGOTAC AGATAAAATTA, E NQODY R I PSOG K C P GGAAAATC AAGAT TAT AGAATAC C TTC AGOTAAATO TC CA HK E I L A L NODMS L C AC CAT AAAGAAAT ATT GOCAT TAAA TGATATOT C AT TGT N Q PF C F T P E K KOD E AAT CAAC CATTTTG TT TTA CAC CAG AAAAAAAAGAC OAAT N QI V F N E S A SO0P TAACCAAATAGTTTTTAACGAAAGTGCTTCTGATCAACCT2 K T K KCYI F N KK P T AAAAAACCAAAAAATGTTACATTTT CAATAAAAAAC CAAC K FP R I F I SOODK ES AAATTCCCTAGGATATTTATTTCAGATGA CAAAGAGAOTT I1I1 I L I A T GAI LA' AATTATTATAATTTTGATTGCTACTGGAGCTATATTAGCC P V L M E K P Y Y CACCAGTTTTGATGGAAAAACCATACTATTAAAAAATATA Fig. 2A. I SUBSTITUTE SHEET j

1528-1538.- WO 89/07645 PCT/AU89/00056 8/18 TTT TTTTTTTTTT AT AAAATAOCT AT TTTT ATAT TTOAT T TAT TT AGCAAT AAAA COOAOAACAAAT CCTATrAAATATAT AOTOTOTOGTAT AT AAOTAT TOTATOOTAATTOTTTTATAA I SE N G D0V K F D LI P K TTT CAOAAAATOOTO ACOTTAAATTTOGATT TAATTCC AAA VMOGKOG I T I Q NS K V OTT ATOOGGAAAAGOT AT AACTATT CAAAATT CTAAOOTTT A K HA S F Y V POGT N V TGCAAAACATGCCT CAT TT TATOTTC CAGGTACAAATGTC Y 'K N L S Y L T K N L RED0 AT AAAAATTT AT CC TATTT AACTAAA AATTTO C OT AAOA K Q YEK H LED0T A K I AAACAATATOAAAAA CAT C TAGAGGATA CTO C CAAAATT C C L I N O K O F V AT T A ATOTT TAATTAATOATAAOOATTTTOTCOCAACAACTOCT LN C P C E P TQGLT GQST TAAATTGTCCATOTGAACC TACACAATTGACTCAAAOCAC S L L I F Y F F K S N K P TCTCTATTAAT CTT TTAT TT CTTTAAOAGTAACAAACCAG GC CAT TAAATAATT AAGTAATTATAATTAA Fig. 26. ",UTITUTE SHEET WD 89107645 Wf) 8907645PCTIAU89/00056 lj 9/18 ATO CATGC AA C ATTST AA AAT AAAAAT AAT AT AATTTTT AAT TT TT ATOC CTAT AAT ATAATS TO TOTACAC AAAAAAA AT TAAATTT TT AAAAAAC AT TAT T CTAT TTT ATAATT T T EN T ER S H K L I N P W OSAAAATACTGAAAGAAGT CATAAACTAATTAAT CCATGG S F L T R VA T O N Q KV R CAT TT TTAACA C AG TA C TAC TOST AATC A AAA OTAAO' 'NT A YR H P A VY 0 K S AATACAOCATATASS CATC CCGCTGTTTATGACAAAAOTA W ET S C P N K S I Q NA TTOOOAAACCAGTTO CCCTAATAAAAGTATACAAAATOCT R R G I V 0 R N G K L I G E' GAC GAGOSAATTGSTAO ATAS OAATSS TAAACT TAT AGOTGA L S S L EE G P QE S F P TTATC AA TT TAGAAOAASSC C CT CAAOAAASTTT CC CTT C K F F V C N C V E K RQ0 TTO TA AAT TTT TTST ATGT AAT TOTST AGAG AAAA SAC AA G 0 0 Y 0 K Mi S Q A 0 T Y G GTGACGATTATGATAAAATGGGACAAGCAGATAC CTATGG Fig2 C I SBSTUTE SHEEJ' WO 89/07645 PC/AU89/O056 1~ 10/18 AT TTTTT ACA CAT.ATTTTO TTTTTTTTTT ACT CAAAATTG TAATTTAAATTATAATTTT ATAATAATTTT CAT TTTT AT T M K AAATT AA AAAT AT T A AT AAAAT AAT AG C TAT AAAA ATG0AA E KF M E KY D I E KV H OAAAAOT T C ATi 0 GAAAAAT AT GAT AT AG AAAAGOTG C AT G E GG L A F PQG T DV N I AOAAGOAOOGATT AGCATTT CC TCAAACAOATOTAAAC AT T N KT C Y I L YV AA QE N AT AAAACTTG T TACA TTTT GTAT GTAGCAOCA CAAGAAAA K FG V W VODGY C S E Y AAATT TGGAGT TT 0GTTG AT G C TATT OTT C AG AAT AT C A LL P'I G S Y R AODQ V AG CTT TAT TAC CAATAGGG TCATAT AGAGCAOGAT CAAGTT C DI Y K K K I A E E I KV GT GAT AT ATATAAAAAGAAAATAG CC GAAOAAATTAAAGT F IS E N N EV E I KOD E TTT AT AT C T GAG AATAATOAGGTTGAAATAAAAGAC GAG T K AQ S R KODE ML 0 P E TAAAG CC CAAAG CA GAAAA GAT GAAATG CTAGAC C CAGAA 4 F. 2D. I SUBSTITUTE SHEET WA) 89107645 PC/u 89/00056 I, 11/18 TGCTTTTTTTTTAGTGCACATGAAAAAAAAAAGGAAAAAT TTTTATT GTTTTTTTAATTGGAAAT AATT AAAAGT GATTT E I YY IV I L C S L Y L AGAAATATATTATATTGTAATTTTG'TG CT CT CTTTAT CTA G S G I RV DL G E DA RYV GTTCTGGTATTAGAGTCGACTTAGGTGAAGATGCAC OTOT 200 400 600 800 S P IT I DONL KL MY KOD TCTCCTATAACAATTGACAATCTCAAACTAA.TGTATAAAG 1000 MOGP RY C SN E E ON E TATGGOTCCAAGATATTGTAOCAATQAGGAAGACAATGAG 1 200 Q KK EV HODN K TLL E C AAAAGAAAGAAGTTCATGATAATAAAACCTTATTAOAATG 1 400 K S KG K GY NW ANY DOK AAAAGTAAAGOAAAGGGATATAACTGGGCAAATTATGATA 1 600 M N VN RN NN GNO0T I CATGAACGTGAACCGAAACAATAATGGAAATGATACCATT 1 800 F KS E Y ES P1I NQ RM L TTAAAAGTGAATATGAAAOTCCAATAAATCAGAGAATGTT 2000 V S FWG EDK RA S H T T OTGTCATTTTGGGGTGAAGATAAAAGAciCATC.ACACACAA 2200 22,70 Aic. ZE. L SUBSTITUTE SHEET 20 MRKLYEVLLL SAFEFTYMIN MKEIYYIVIL CSL... .YLIN 130 140 EVHGSGIRVD LGEDAEVAGT KVHGSGIRVD LGEDARVENQ 250 260 DDKDKKCHIL YlAAQENNGP 4 m 30 FGRGQNYWEH LG-...NCSEG 150 QYRLPSG KCP DYRIPSGKrp 270 RYCNKDESKR RYCSNEEDNE 390 PTGAFKAIJRY PIGSYRAOQV TCNFFVCKCIV TC.KFFVCNCV 40 PYQIKSDVYH P T. DK. IS EN. G D0 OKSNKTCYI.L 370 EIKIKEGFKNK AKIRRGIVDR 490 I SDDKDSLKC ISDDKESLNC 610 SFWGEEKRAS SFWGEDKRAS YVAAQENMG P 380 NASMIKSAF L NGKLI GEALL 500 PCDPE. LVSNS PC'EPTQLTQS 620 HTTPVLMEKP HTTPVLMEKP 50 INEHREHPKE 160 VFG KGI II EN VMGKGITIQN 280 NSMFCFRPAK NQPFCFTPEK 400 'KS HGKG YNWG KSKGKGYNWA 520 ERRAEVTSNN EKRQFISENN 170 SNTTFLTPVA SK VS FLTR VA 290 015 FQNYTYL KDEYKNLSYL 41 0 NYNTETQKCE NYDKKTKKCY 530 EVVVKEE.YKO EVELKOEFKS YQIYPLHQEHT 180 TGNQYLKDt3 T-GNQKVREGG 30 0 ,SKNVVDNWE K TKNLREDWET 4.2 0 I FNVKPTCLI I FNKKPTC LL I EYAO1 PEHKP EY. ESPIN.. *00 4~ C C C 0~ 630 640 650 660 ,Fig. 3A. T'r In !i YQQEDSGEOE 19e' FAFPPTEPLM LAFPQTDVN I 31 0 VCPRKNLQNA SCPNKSIGt'A 430 NNSSYIAlTTA 80 NTLQH-AYPID SPMTLOEMRH SPITIDNLKL 320 KFGLWVOGNC KFGVIWVOGYC 440 LSHPI EVEHN 90 HEGAEPAPQE 21 0 FYkANKYVN MYKDHKE ILA 330 EDIPHVNEFS SEYQKKEVHD 450 FPCSLYKNEI 100 QNLFSSIEIV 22 0 LDE LT L CSRH L-N-OMSLCAKH 340 AIDLFECNKL NKT LLECNI 460 MKE IERESKR 110 E RS N Y M W ERSHKLI NPW 230 AGNMIPDNI( AS FY\PGTNV VFE LSASDQP VFNESASDQ[P 4 '70 IKLNONDDEG 120 TEYMAKYDI E EKFMEKYD LE 24+0 N SNY(Y PA VY NTAYRH PAVY 360 KQYEQH LTOY KQYEKHLEDT 480 NKKI IAPRIF NOTIKFPRIF 600 RNDEMLOPEA RKOEMLOPEV 720 i68 NDKDFVATTA LSSLEEGPQE SFPCOIYKKK 1AEE1T..KV MNVNRNNN.A3 550 TYDKMKI IIA. **W .QRMLIIII 560 SSAAVAVLAT LI ATGAI LAS 570 I LMVYLY KRK LLI FYFFKSN 580 GN.AEKYOKMO KPGDDYDKMG 590 EPOQHYG KS'NS 0.4 OTYG KAQS? 670 680 690 700 71,0 Fig. 3B. f PCr/AU89/00056 WO 89/07645 14/18 123 4 100 Fig.4. Fig. -S S I TUTE S AErI -DL-. WO0 89/07645 PC/AU89/00056, 15/18 200-,, A 43- Mr X 200- 1 2 3 4 5 6 7 8 9 93- 67- omm Mao -A B 43- Mr X 200- 9 3- 67- 43- 2 3 4 5 6 7 1 8 9 234A Fig. 6. FUSST ITUTE SHEET 4 ~I I WO 89/07645 PCr/AU89/ 00056 16/18 Mrx1O 3 200- V. 0* 93- 67- 43- Al1 2 3 4 Bi1 2 3 4 Fig. 7. I, ~,ij~TTL1T~SKEET WO 89/07645 PCTIAU89/0005 6 17/18 Fig. 8. WO 89/07645 8907645PCr/AU89/00056 18/18 2-2- 2- 18- 1-6- 1.4- 12- I- 0-8" 0-6- 0-4- 0-2- n. I- 2~i. 659S/A 6615/A 6635/A 666FCA 668FCA 670FCA ANIMAL NUMBER/ADA2U VANT U PREBLEED M st DOSE El 2nd DOSE ABSORBANCEAT 1:10.000 DIWT/ON 14A L I 154L 164L 13FC I BC 19SF I 22SF 17FC RABBWr NUMBER/ADJUVANT PREBLEED C. 1st DOSE 2nd DOSE Fig.9 SUBSTITUTE SHEET r INTERNATIONAL SEARCH REPORT International, Application NO PCT/AU 89/00056 1. CLASSIFICATION OF SUBJECT MATTER (ot moveral classirication symois apply, inulet all) According to International Patent Classiicaslon (iPC) or to both National Classification and IPC Int. cl 4 C12N 15/00, C12P 19/34, C07H 21/04 It. FIELDS SEARCHED Minimum Documentation Searched F Classification System Classification Symbols I Pc 4 I C07H 21/04, C07K 13/00 WPIL, WPI, USPA (Derwent Database) I Keywords: Plasmodium, falciparun Documentation Searched other than Minimum Documentaion to the Extent that such Documents are Included In the Fields Searched AU IPC as above Chem. Abs. Keywords: Plasmodiumi, falciparum l1t. DOCUMENTS CONSIDERED TO BE RELEVANT'________ Categ-ory Citation of Document, 11 with Indication. where appropriate. of the relevant passages Is Relevant to Claim No. 1i X Molecular and Biochemical Parasitology, 18: 183-195 (1,4-6) (1986) Schofield et al., A Rhoptry Antigen of Plasmodium falciparum Contains Conserved and Variable Epitopes Recognized by Inhibitory Monoclonal Antibodies, Elsevier Science Pubs. Y Am. J. Trop. Med. Hyg. 33(6): 1051-1054 (1984)" (1-3) Campbell et al., Monoclonal Antibody Characterization, of Pl asmodi um fal cipa rum. Antigens. Y Am. Trop. Med. Hyg. 33(6): 1055-1059 (1984) (1-3) Howard et al., Proteins Responsible for a Punctate Fluorescence Pattern. in Plasmodium falciparum Merozol tes. Y Molecular and Biochemical Parasitology, 20: 33-43 (1-3) (1986) Braun-Breton et al., In vivo time course of synthesis and processing of major schizont membrane polypeptides in Plasmodium falciparum, Elsevier Sci ence Pubs. A R.F. Anders, "Antigens of Plasmodium falciparum and their potential as components of a Malaria vaccine" In: The Biology of Parasitism, Published 1988 by 7Alan R. Lisj, Inc. New York, Englund P. and Sher A. pp 201-224. *Special categories of cited documental Is IT" later document published ahtr the International riling date "A dcuet efnigth gnea sat o hear w~h s o of priority daie and not in conflict wit h them appiication but '"dcnsidrnn t h e oriuar stelev heance ic s o cited to undeiltInd the principle or theory underlying, the con~dcrd t beof artiula reevac "Invention earlier document but published on or alter the International docmen of particular relevance; the claimed Invention fi ling date cannot be considered novel or cannot be considered to dociument which may throw doubts on priority claimOs Or Involve an Inventive step which Is cited to es tablish the publication date ot another OY" document of particular relevance,' the claimed Invenition citation or other special reason las speciied) cannot be considered to Involve en Inventive step w.hon the document referring to an oral disclosure, use, exhibition or document is combined willh one or more other such docu*, other means nients. such conioinetion bein2 obvious lio a parson skilled document Published pilor to the initrnational iling date but I4 ocn th art. oftearn aot 'm laitr than the priority date claimed""douetmbrofteervpantaml IV, CERTIFlCAT 7 Date of the Actual Completion it' International search May. 1989 (05,05.89) Dole of Melting ol this International Search Report [International Searching Authority SI Australian Patent Office form PCT11SAW~O tsecond sr',yrll tJanuitry 19t)l