AU608096B2 - Rhoptry antigen of plasmodium falciparum - Google Patents

Description

a i rI 1 OPI DATE 05/07/89 AOJP DATE 27/07/89 APPLN. ID 26095 88 PCT NUMBER PCI/AU88/00[63 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classificaion 4: (11) International Publication Number: WO 89/ 05348 C12N 15100, C07K 13/00, 15/12 Al 5Jn 99150.9 C07K 15/14, C12P 21/00 (43) International Publication Date; 15 June 1989 (15,06.89) C07H 21/04, A61K 37/02 (21) International Application Number: PCT/AU88/00463 55 Brougham Street, North Melbourne, VIC 3051

(AU).

(22) International Filing Date: 1 December 1988 (01.12.88) (74) Agents: SLATTERY, John, Michael et al,, Davies Collison, I Little Collins Street, Melbourne, VIC (31) Priority Application Number: PI 5686 3000 (AU).

(32) Priority Date: 1 December 1987 (0 1.12.87) (81) Designated States: AT (European patent), AU, BE (Eu- (33) Priority Country: AU ropean patent), CH (European patent), DE (European patent), DK, FR (European patent), GB, GB (European patent), IT (European patent), JP, KR, LU (71) Applicant for all designated States except US): SARA- (European patent), NL (European patent), SE (Euro- MANE PTY. LTD. [AU/AU]; The Walter and Eliza pean patent), US.

Hall Institute of Medical Research, Royal Parade, Parkville, VIC 3052 (AU).

Published (72) Inventors; and With international search report.

Inventors/Applicants (for US onl) SMYTHE, Jason, Arthur [AU/AU]; 204 Station Street, Edithvale, VIC 3196 COPPEL, Ross, Leon [AU/AU]: 6 Mercer Road, Armadale, VIC 3143 KEMP, David, James [AU/AU]; 309 Belmore Road, North Balwvn, VIC 3103 BROWN, Graham, Vallancey [AU/ AU]; 35 Walsh Street, Balwyn, VIC 3103 AN- DERS, Robin, Fredric [AU/AU]; (54)Title: RHOPTRY ANTIGEN OF PLASMODIUM FALCIPARUM GlyLysLyOLysM,1V01PheTyrAsDLeuTyrLysPrC uG[uAsnGIUSerTyrTyrGluLysLysOInLy4ysGIU GO TAAGAAAAAAATGiTCTTTTATGATTTATACAAGI3CAaiAArAAAATGAATCTTATTATGAAAMAAAACAAAAGAMGA 10 20 30 40 o 60 70 B0 GlsGluGululvsGlGIuLysGlu~elunn y~ie nArMtlusuinlADAnnvluy AGA AAArAG A ACI A PeGAAP A AGT'TGAAC AACM COA TO ACCAAGAAGATAATGAAGAAT 90 100 110 120 130 140 150 160 LysPheG1uGuG0uAsnLvsGluAsLeuLeuA ova I0inOInAsGtuGiuLiuPtoS.rGLuO yLysGnLys ATAALTIOTt GAGAAGAMAT4AAGAAGACCTC TAGATGT CCtACAAOiATGAAOAATTACCGTGAAIA.A..CAAAAA 170 180 190 200 210 220 230 240 VolLysOlyLysSerPneASoDAsn0ulIsLeuAnGI I e0inAsnVoISerAsoVaIHisAloPhe[IeGinLysAso GTAAAAGGAAMT CATTCGATAATGAACATTTAA.4ThAAA 'ACAAATGTIACEiACGT ACAT GCATTATACAAAAOA 250 260 270 2801i 290 300 310 320 MetLysTyrLeuAsgAsLeul eAsGGluGluGInohr IiLysAsoAlaVo TATGAAATATTTAATG07TCTCATAAT0GCAACT ATTAAAGATOCCGTCAAAAAAAOTGCTTATAAACGAAMTA 330 340 300 360 370 360 390 400 LysLmuGlyAsnAsnLysLyzSerGnMeLeuGlI uluGuProGIluGiuAsnPh@GIuOluASOAIOAsDiLu AGMAATTAGGAAATAATAMAAT CACAAATGAT ACTGGAI AOM CCAGA"aAAAATTTTGAAGAGACTGATGAA 410 420 430 44 0 450 460 470 480 GluLeuAsnLyLseuMetGIuIOOu LysAsni leVolA spLysuIulaeLysAsnSerLySAIaASnLysSerAsnLys GAATTAAATAAACTAAT0GAArAG4AAAAAT4 TGTAI ATAAMIAATCAAA TAGTA.AGCAATAAAAGCAACAA 490 000 510 520 5 030 540 500 560 LysLeuGInPheAsnAsn7hrAsnLysQlnAsnLysMet ITyrMtLysAsntuTyrAsfAnLysThrLYSAfASfLys AAAATTACAATTCAATAACACTAATAAACAAAA4MAATG TATATGiAACGAmTATAATAATAAGACAAAAAATMTA 570 080 590 600 610 620 630 640 AsrAlsnLysPheGluGInGin~AsnTyrAsGIuSrTy rM~tASPASPAsrTrGluGi iAldGLUGIuPhtAslriAs AAAACAATAAATTTGMCAACAAITATGATGATCATA TATGGATGATG/ATTATGMAAAAA&TGAAGAATTT1AT 650 660 670 680 690 700 710 720 AlnAsnGinSerGIuASOMtLysGuly,.As,GIuLeuA spLysI.lAsnAsoGtuLuLuthrASDGIGlyPoAlf AATAATCAAAGOC0GAATGAGAAACAATAAlCTG ATA.AAATTAA.ATGAACTATTAACTGGATCGGPACCAAA 730 740 700 760i 770 790 790 800 luAsoThrLeuLcuriuAsOSOAsnLeysI PticAl cx CGAAGATACAT TT TTAGOMMTAAT nT 4AM T AT TCGC 610 820 830 (57) Abstract A rhoptry antigen of the asexual blood stages of Plasmodium falciparum, is characterised by: having an apparent molecular weight of approximately 55kDa; (ii) being a glycoprotein incorporating myristic acid: (iii) being present in a restricted localisation with merozoites consistent with a rhoptry location; and (iv) being extracted into the detergent phase when parasiteantigens are separated by phase partitioning with detergent.

u L~ i- -i

F-

'3 WO 89/05348 PCT/AU88/00463 RHOPTRY 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.

Rhoptry antigens are of particular interest in the study of malarial immunology and the mechanism of host protection to parasites due to their accessibility to host immune mechanisms and because of their likely involvement in the erythrocytic invasion process. A number of rhoptry antigens have been shown to be capable of inducing immune responses that in vivo or in yitro inhibit the growth of the asexual blood stages of E falciparum, i c ,I c- PCT/AU88/00463 WO 89/05348 2 According to one aspect of the present invention, there is provided a rhoptry antigen of the asexual blood stages of Plasmodium falciparum which is characterised by: having an apparent molecular weight of approximately 55 kDa; (ii) being a glycoprotein incorporating myristic acid; (iii) being present in a restricted localisation with merozoites consistent with a rhoptry location; and (iv) being extracted into the detergent phase when parasite antigens are separated by phase partitioning with detergent, for example with the detergent Triton X-114; or an antigenic fragment thereof.

Preferably, the antigen is a polypeptide having a primary structure which includes the amino acid sequence set out in Figure 2, or an antigenic fragment thereof.

The invention also provides a method for actively immunising 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.

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

i; c.-i -i WO 89/05348 PCT/AU88/00463 3

EXAMPLE

In this example, selective partitioning of integral membrane proteins into the detergent phase upon temperature-dependent phase separation of aqueous solutions of the detergent Triton X-114, has been used to enrich for putative integral membrane proteins of P.falciparum. Human antibodies obtained from patients from Papua New Guinea were then affinity-purified on these antigens after they had been blotted onto nitrocellulose. The purified human antibodies were used to identify recombinant clones of Escherichia coli expressing polypeptide fragments corresponding to these antigens.

Materials and Methods Parasites.

The origin of P.falciparum isolate FCQ27/PNG (FC27) used in these studies has been described elsewhere Parasites synchronized by sorbitol treatment were cultured at 0.25% haematocrit with parasitaemias ranging from All samples were washed free of medium by centrifugation and substitution with human tonicity phosphate buffered saline (HTPBS). Cells were then pelleted, and Iml packed cell aliquots were snap frozen and stored at -70 0 C until further processing.

Triton X-114 solubilization and phase separation.

Triton X-114 solubilization and separation of hydrophobic, hydrophilic and insoluble fractions was performed essentially as described by Bordier (4) with the following modifications. Triton X-114 (purchased from Fluka Ag., Switzerland) was precondensed in human tonicity phosphate buffered saline (HTPBS). A 1 ml aliquot of prepacked parasitized cells was solubilized in 15ml of i, L L- ij i WO 89/05348 PCT/AU88/00463 4 Triton X-114 for 90min on ice, with mild vortexing at intervals. A 1 ml sample of the total material.

was removed and snap frozen. The remaining -15ml were then centrifuged at 10,000xg for 15min at 4 0 C to remove insoluble material. The supernatent was removed and this step repeated. The sedimented material (insoluble pellet) was then washed a further three times in 0.5% Triton X-114 before being snap frozen in lml of HTPBS. The detergent-soluble material was then carefully layered over a sucrose cushion of cold 6% sucrose, 0.06% Triton X-114, placed for 5 min in a 37°C waterbath and then centrifuged at 500xg for 5min at 37 0 C. After centrifugation the 15ml detergent-depleted upper layer was collected and chilled on ice. The sucrose cushion was discarded and the detergentenriched pellet (l-2ml) was resuspended on ice with of cold HTPBS. The resuspended detergentenriched phase was again layered over a sucrose cushion, brought to 370 for 5min and re-pelleted by centrifugation. After this second precipitation, the detergent-enriched pellet was resuspended to 5ml in HTPBS and snap frozen. The detergent-depleted upper layer from the sucrose cushion separation was further depleted of hydrophobic proteins by adding lml of 11.4% Triton X-114 on ice, vortexing into solution, warming to 37°C for 5min, then centrifuging and discarding the Triton X-114 pellet. This cycle was repeated three times. The remaining detergent-depleted aqueous solution (aqueous phase) was then snap frozen. All samples were stored at until analysis.

L2 1_1_ WO 89/05348 PCT/AU88/00463 Electrophoresis and immunoblotting.

Samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were processed under reducing conditions and electrophoresed on 10% slab gels. Gels to be analysed for protein were stained with Coomassie brilliant blue. Samples to be analysed by immunoblotting were fractionated on 10% slab gels and electrophoretically transferred to nitrocellulose sheets. After transfer the nitrocellulose was blocked with 5% skim milk powder in HTPBS (Blotto) and probed with sera or affinity purified human antibodies diluted appropriately in Blotto.

Bound antibody was detected by probing with 125 I-protein A followed by autoradiography.

Protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) was iodinated by the chloramine T method to a -1 specific activity of 40pCi/pg-1 Affinity purification of polvclonal monospecific human antibodies.

Antibodies were affinity purified from human serum or plasma on Triton X-114 soluble antigens electrophoretically transferred to nitrocellulose as described previously and modified in this instance as follows. Reduced samples of Triton X-114 extracted membrane antigens were electrophoretically separated and transferred to nitrocellulose.

Radioactive 14 C high molecular weight markers (Amersham) were used to determine the region of interest. Strips of nitrocellulose corresponding to regions of interest were incubated for 8hrs at 4°C with sera or plasma from individuals exposed to malaria. Sera was removed and the strips washed vigorously over 2 hours with 6 changes of Blotto, i" Y-YYLIII~ WO 89/05348 PCT/AU8800463 6 three changes of HTPBS, followed by a 15 minute wash in borate buffer (0.1 M glycine, 0.15 M sodium chloride, pH 2.6) for 10 minutes. Eluted antibodies were immediately neutralized with 2 M Tris-HCl pH and stored at 4 0 C with 0.05% sodium azide. The affinity-purified antibodies were diluted 1:2 in Blotto to probe immunoblots and XAmp3 cDNA library filters, or concentrated using a Centricon microconcentrator (Amicon) for immunofluorescence assays. Antibodies were also eluted directly from immunopositive XAmp3 clones grown as lawns on nitrocellulose. Filters with lysed lawns were pre-eluted with borate and glycine buffers, incubated with serea, and the monospecific antibodies eluted as above. This second method of affinity purification required the removal of anti E.coli antibodies which was achieved with sonicates and lawns of control XAmp3 clones.

Identification of cDNA clones expressinQ relevant polvpeptide antigens.

Details of the FCQ27/PNG isolate cDNA library, its amplification in XAmp3 and lysogenic expression in E.coli have been described Detection of antigen-expressing clones by in situ colony immunoassay with human antibodies has also been described Positive clones were grown and induced in liquid culture and extracted with sample buffer for electrophoresis as described Samples were electrophoresed on 10% SDS-PAGE slab gels and either stained for protein or immunoblotted and analysed with affinity purified antibodies, to detect the presence or absence of stable fusion polypeptides with -galactosidase.

i, -L i I PCT/AU88/00463 WO 89/05348 7 Antibody depletion.

One ml cultures of immunopositive clones were induced, pelleted by centrifugation, and the liquid medium removed. Pellets were frozen and thawed three times and the lysed cells resuspended in lml HTPBS. A 20pl aliquot of PNG sera was added and incubated with the lysate for 3hrs at 4 0 C. The cellular debris was then spun down at 10,000xg and the supernatant used to probe Triton X-114 extracted and immunoblotted parasite membrane protein antigens in order to determine the degree of antibody depletion attributable to the induced fusion polypeptide.

Subclonina of immunopositive XAmp3 clones.

XAmp3 phage were isolated from immunopositive clones and the cDNA insert extracted by EcoRI digestion as described. The purified insert was subcloned into M13 vectors for single stranded sequence determined by the dideoxy method of Sanger and into pBTA224 (a modification of PUR-290) kindly provided by Gary Coburn (Biotechnology Australia Pty.Ltd.). The plasmid vector pBTA224 is an expressing vector in E,coli cells, and clones were rescreened for immunoreactivity by colony immunoassay.

Indirect immunofluorescence.

Thin blood films of parasitized erythrocytes from asynchronous cultures of P.falciparum were air dried and fixed in 100% acetone at -20 0 C for minutes. Slides were incubated with concentrated affinity-purified monospecific human antibodies, with fluorescein-conjugated sheep antihuman Ig antiserum as second antibody. Parasite nuclei were counterstained with ethidium bromide.

_i I ~rl-L- LYLII WO 8905348 PCT/AU88/00463 WO 89/05348 8 LEGENDS TO FIGURES Fig 1: Triton X-114 extracts of falciparum infected erythrocytes were fractionated on SDS-PAGE, electrophoretically transferred to nitrocellulose, and probed with either pooled adult PNG sera, affinity purified polyclonal human antibodies, or affinity purified monospecific human antibodies.

In and lanes are uninfected erythrocyte control total unfractionated material, aqueous phase, Triton phase, insoluble material.

In human monospecific antibodies were affinity purified from pooled adult PNG sera on a filter lawn of Ag512.(1), control strip of Triton X-114 phase antigens probed with the pooled sera, affinity purified anti Ag512 probed to a duplicate filter strip.

Fig, 2: Partial nucleotide sequence of Ag512 with the predicted amino acid sequence of the single open reading frame.

Fig 3: Rhoptry localization of Ag512 with affinity purified monospecific antibodies used in indirect immunofluorescence assays on asynchronous blood films. Pictured is a mature schizont with a punctate pattern of fluorescence characteristic of a rhoptry localized antigen.

1 1 1 1 1 1

II

WO 89/05348 PCT/AU88/00463 9 A subset of P.falciparum antigens partition into Triton X-114.

When sera from individuals repeatedly infected with P.falciparum are used to probe immunoblots of P.falciparum antigens fractionated by phase separation in Triton X-114 several putative integral membrane protein antigens are identified.

The most dominant of these had relative molecular masses (Mr) of 21,000, 35,000, 42,000, 50,000 and 55,000 as determined SDS-PAGE analysis. When the same antigen extracts were probed with antisera raised against or affinity purified on a number of cloned P.falciparum antigens it was shown that the Mr 21,000 Triton X-114 soluble antigen was the circumsporozoite protein related antigen (CRA). This molecule is known to have a 26 amino acid hydrophobic sequence typical of integral membrane proteins confirming that the procedures selects for such molecules. None of the other Triton X-114 soluble antigens appeared to correspond to any of the many P.falciparum antigens that have been previously characterised.

In order to obtain purified antibodies specific for the set of proteins of Mr 35-55,000 Triton X-114 extracts of P.falciparum were electrophoretically fractionated and transferred to nitrocellulose. The appropriate region of the nitrocellulose filter was then excised and used as an adsorbent for affinity purification of the corresponding antibodies from human serum (Materials and Method). The purified antibodies were tested for specificity by reacting them with fractionated P.falciparum proteins. It is clearly evident in Fig.lb that these purified antibodies react almost PCT/AU88/00463 WO 89/05348 exclusively with those Mr 35-55,000 antigens that selectively partition into the Triton X-114 phase.

In order to isolate clones expressing these.

antigens, a library of XAmp3 clones expressing P.falciparum cDNA sequences was screened with Lhe affinity-purified antibodies. Ten positive lysogenic clones that were detected by this procedure were replated for single colonies.

Selected colonies correspond to Triton X-114 soluble antigens.

Antibodies affinity purified on lawns of selected clones were used in colony immunoassays on the array of ten clones and to probe immunoblots of parasite antigens. Seven of the clones were found to be siblings encoding the Mr 55,000 antigen from the Triton X-114 phase. The most immunoreactive was chosen as the type clone and designated Ag512.

Analysis of clones by SDS-PAGE followed by protein staining or immunoblotting showed that all seven sibling clones of Ag512 produced unstable fusion proteins with B-galactosidase. Immuno sera were absorbed with sonicates of the most immunoreactive cDNA clone, Ag512, and then used to probe immunoblots of Triton X-114 extracts of P.falciparum. Sonicates of this cDNA clone totally removed all detectable antibody reactivity to the corresponding parasite antigens. Thus this cDNA clone apparently encodes the dominant naturally immunogenic epitopes in this antigen.

Stage and strain specificity.

Stage-specific parasite preparations were Triton X-114 extracted, fractionated by SDS-PAGE and immunoblots probed with PNG sera. The antigen corresponding to Ag512 was present in all stages of L1; i, i PCT/AU88/00463 WO 89/05348 11 the parasite life cycle, but was least abundant in the asexual ring and tirophozoite stages and most abundant in late schizont preparations. In the late schizont and merozoite stages, the antigen was totally soluble in the detergent Triton X-114, but in ring and trophozoite stages it partitioned into both the Triton X-114 phase and the insoluble pellet.

Triton X-114 extracts were prepared from five strains of P.falciparum giving asychronously in culture. The antigen was present in all strains and there were no apparent strain-related differences in the size or immunoreactivity of the antigen.

Nucleotide and amino acid sequence of Ag512.

The nucleotide sequence of AG512 together with the predicted translation is given in Fig.2.

The cDNA clone does not encode the complete coding sequence. It contains one open reading frame which is in frame with B-galactosidase, and the clone produces a fusion polypeptide that is unstable in E. coli. The sequence is 839 base pairs long and contains no lengthy repetitive element. The sequence does not contain a hydrophobic stretch of amino acids. The encoded polypeptide has a predicted molecular weight of 33,534 daltons.

Ao512 corresponds to a putative rhoptry antigen.

Affinity purified human antibodies to Ag512 were prepared as described, concentrated, and used in indirect immunofluorescence assays on asynchronous blood films. The fluorescence pattern observed was consistent with localisation of the antigen in the rhoptry organelles of merozoites.

i ~~I PrT/AI 88/00463 WO 89/05348 12

DISCUSSION

This Example shows the temperature dependent' Triton X-114 detergent separation of integral membrane protein antigens of P.falciparum. One of the antigens that partitioned to the Triton X-114 phase was identified as CRA, a well characterised antigen of known primary structure which is typical of an integral membrane protein. The other dominant antigens that partition into the Triton X-114 clustered into the Mr 40-55,000 range and apparently did not correspond to antigens that had been previously cloned. These proteins, after transfer to nitrocellulose were used to affinity purify polyclonal monospecific human antibodies which were used to isolate a clone designated Ag512, corresponding to a merozoite rhoptry protein, from a XAmp3 cDNA expression library. Antibodies to Ag512 reacted with a Triton X-114 soluble protein of Mr 55,000 which was present in all strains of P.falciparum examined and present in all of the asexual life cycle stages. Indirect immunofluorescent microscopy with these antibodies gave strong staining on mature stages with a "double dot" grape-like pattern on merozoites a pattern characteristic of antibodies to rhoptry antigens.

Part of the primary structure of the Ag512 related antigen has been deduced from the nucleotide sequence of the cDNA insert in clone Ag512.

jl_ I I li I- II WO 89/05348 PCT/A U88/ 00463 13

REFERENCES

1. Holder, V'reeman, A.A. (1981) NaIvjure., 294 :361-364.

2. Perrin, Merkli, Gabra, M.S., Stocker, Chizzolini, Richie, R.

(1985). J.Clin.Inyest. 1718-1721.

3. Chen, Larnont, Elliot, Kidson, Brown, Mitchell, Stace, J. and Alpers, M. (1980). South East Asian J.Trop.

M-ed-P~JH 1t h. 11 :435-440.

4. Bordier, C. (1981). JBiol.Chem. 256, 1604-1607.

Beal, Mitchell, G.F. (1986), J.Immunol. Method$,. 86, 217-223.

6. Kemp, Coppel, Cowman,A.F., Saint, Brown, Anders, R.F, (1983), Proc.Natl.Acad.Sci. USA 3787-3791.

7. Anders, Coppel, Brown, G.V., Saint, Cowman, Lingelbach, K.R., Mitchell, G.F. and Kemp, D.J. (1984). Molec, Biol.Med. Z, 177-192.

8. Messing, J. and Vieira, J. (1982). Gen2e 269-276.

WO 89/05348 PCT/AU88/00463 Sanger, Nicklen, Coulson, A.A.

(1977) Proc.Nptl.Aa.c.UA7 5463-5467.

Hope, Mackay, Hyde, J.E. (1985).

NuclAcids Res. la, 269-279.

I

Claims (4)

1. 6. again 1. A rhoptry antigen of the asexual blood admin stages of Plasmodium falciparun, which is anyon characterised by: there having an apparent molecular weight of 4 or approximately (ii) being a glycoprotein incorporating myristic
2. 7. acid; a por (iii) being present in a restricted localisation of be: with merozoites consistent with a rhoptry antig( location; and claim: (iv) being extracted into the detergent phase recoml when parasite antigens are separated by comprJ phase partitioning with detergent; or an antigenic fragment thereof.
3. 8. clonir 2. An antigen according to claim 1, which is claim antigen Ag5l2 described herein, or an antigenic sequer fragment thereof.
4. 9. 3. An antigen according to claim 1, having a expres primary structure which includes the amino acid sequen sequence set out in Figure 2, or an antigenic fragment thereof. polype 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. PCT/AU88/00463 WO 89/05348 16 6. A method for actively immunising a host against Plasmodium falciparum, which method comprises administering to the host an antigen according to anyone of claims 1 to 3, or an antigenic fragment thereof, or a vaccine composition according to claim 4 or claim 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 DNA 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 2. 9. A synthetic polypeptide prepared by expression of all or a portion of a nucleotide sequence according to claim 7 or claim 8. A vaccine composition comprising a synthetic polypeptide according to claim 9. -doom 7 7- WO 89/05348 WO 8905348PCT/ AU88/00463 WO 89/( 1/4 17' "I I I AI= ti IM A 0 0 04 SUBSTITUTE SHEET WO 89/05348 WO 8905348PCT/AU88/00463 2/4 cGyLysLysLysMetVaiPheTyrAspLeuTyrLysProG O OTAAOAAAMAAATOOTC TTTTATGATTT ATACAAG CCAG 20 30 AGAAAAAGAAGAGAAAGAAGAGAAAGAACAOTTTO MC 100 110 120 LysPheGtuGtuG LuAsnLysGtuAspLeuLeuAspVa ATAAATTTGAAGAMGAAAATAAAGAAGACCTTCTAGATG T 170 180 190 200 Vat I Ly sGly Ly sSe rPheAspAs nO uH i sLeu A snO lu I OTAAAAOOAAAAT CATT COAT AATGAACATTTGAATGAAA 250 260 270 280 Met LysTy rLeuAspAspLeul I eAspG tuGi( uGl[nThr TATGAAATATTTAGATGAT CT CATAGATGAAGAGCAAACT 330 340 350 360 LysLeuGtyAsnAsnLysLysSerGtnMetl teLeuGt AGAAATT AGO AAATAATAAAAAAT CAC AAATOAT ACTOGA 410 420 430 440 G[uLeuAsnLysLeuMetGtuGlnGtuLysAsnlte\![A GAATTAAATAAACTAATOOAACAAGAAAAAAATATTOTAG 490 500 510 520 LysLeuGlnPheAsnAsnThrAsnLysGlnAsnLysMet AAAATTACAATT CAATAACACTAATAAACAAAACAAAATO 570 580 590 600 AsnAsnLysPheO tuG lnGtnAsnTyrAspGtuSerTy AAAACAATAAATTTGAACAACAAAATTATGATOMATCATA 650 660 670 680 As nAsnOIn SerG IuAspMet LysG IuTh rAsnOLu LeuA AATAATCAAAOCOAAOATATGAMOGAAACAAATGAACTC 0 730 740 750 760 GluAspThrLeuLeu~tuAsnAsnAsnLysIlePheAta COGAAOATAC ATTAT TA GAAAATAATAAT AA:ATT TTCOC 810 820 830 Ap. SUBSTI-iUTE s-UEET WO 89/05348 PCT/AU88/00463 3/4 [uG[ uAsnG tuSerTyrTyrGt uLysLysO tnLysLysGtu AAGAAAATGAATCTTATTA TGAAM GAMCAAAAGAAAGA s0 60 70 LysOtnAsnAspMetGtuAspGriGIuAspAsnGtuG[uTyr AAACAAAACGATATGGAAGACCAAGAAGATAATGAAGAAT 130 140 150 160 [GtnO tnAspGluGtuLeuProSerG[uGtyLysGtnLys CCAACAAGATGAAGAATTACCAAGTGAAGGAAAACAAAAA 210 220 230 240 I eGI nAsnVat Se rAspVaIH isAl cPhell eQ InLy sAsp TACAAAJ4TGTTAG C ACGTACATGCATTTATAC AAAAAGA 290 300 310 320 I I eL ysAspA QVQ[ L ysLy sSerA[ aTy rLy sG yAsnL ys ATTAAAGATOCCGT CAAAAAAAGTGCTTATMAAGGAAATA 370 380 390 400 uG lGuProG IuGI uAs nPheGI uGIuAspAIQAspG Iu AGAAGAACCAGAAGAAAATTTTGAAGAMOATGCTGATGAA 450 460 470 480 spLysi.u IIeLysAsnSerLysAlaiAsnLysSerAsnLys ATAAAGAAATCAAAAATAOTAAAGCAAATAAAAGCAACAA 530 54+0 550 560 Ty rMetLysAsnGl uTyrAsnAsnLysThrLysAsnAsnLys TATATGAMAAACGAATATMATAATAAGACAAAAAATAA FA 610 620 630 640 rMetAspAspAspTyrGl TATOGATGATGATTA TGAC AAAATGAAGAATT TAATGAT 690 700 710 720 spLysl leAsnAspO IuLeuLeuThrAspG [nt yPr'oAsn A TAAAATTAATGATOAAC TATTAACTGATCAMOGAC CAAA 770 780 790 800 Fig. SUBSTITUTE SHEET PCT/AU88/00463 WO 89/05348 4A~ Fig.2 SUBSTITUTE SHEET INTERNATIONAL SEARCH REPORT Inlernational Application No PCT/AU 88/00463 I. CLASSIFICATION OF SUBJECT MATTER (it several classificatlon symbols apply, Indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC 4I C12N 15/00, C07K 13/00, 15/12, 15/14, C12P 21/02, C07H 21/04, A61K 37/02 II. FIELDS SEARCHED Minimum Documentation Searched Classification System Claasification Symbols IPC WPI, WPIL, USPA Derwent database keywords: "PLASMODIUM FALCIPARUM" Documentation Searched other than Minimum Documentallon to the Extent that such Documents are Included In the Fields Searched AU: C12N 15/00, C07G 7/00, C07K 15/12, 13/00, 15/14, CO7C 103/52 Chemical Abstracts Keywords as above III. DOCUMENTS CONSIDERED TO BE RELEVANT' Crtegory Citation of Document. I with Indication, where approprlate, of the relevant passagesl Relevant to Claim No. A Proceedings of National Academy of Science U.S.A. Volume 83 pp 6093-6097 issued 1986 (Washington, Harold A. Stanley et al, "Plasmodium falcip .'um polypeptides associated with the infected erythrocyte plasma membrane". A Nature, Volume 332, pages 158-161, issued 10 March 1988, (London, England), M.E. Patarroyo et al, "A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria". A Nature, Volume 328, pages 629-632, issued 13 August 1987, (London, England), M.E. Patarroyo et al, "Induction of protective immunity against experimental infection with malaria using synthetic peptides". A Vaccines 87 Modern Approaches to New Vaccines pages 117-124, published: Cold Spring Harbor Laboratory 1987 M.E. Patarroyo et al, "Protective Synthetic Peptides against Experimental Plasmodium falciparum-induced Malaria". Sp.cial categories of cited documents: later document published after the International filing date or priority data and not in conflict with the application but document defining the general state of the an which Is not cited to understand the principle or theory underlying the considered to be of particular relevance Invention earlier document but published on or after the International document of particular relevance; the claimed invention filing dale cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an Inventive step which Is cited to establish the publicatlon date of another document of particular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to involve an Inventive step when the "0 document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvlous to a person skilled document published prior to the international filing date but In the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 3 March.1989 (03.03.89) q MA CU-l jq ,q 0 .C3S 5 International Searching Authority Slgnatur of A forlltd Ofice Australian Patent Office J.H. CHAN Form PCTIISA/210 (second sheet) (January 1985) C L. i iw -e I