AU638216B2 - Allelic variants of plasmodium falciparum merozoite surface antigen - Google Patents

Description

OPI DATE 02/04I/90 PCI' AOJP DATE 10/05/90 APPLN. I D 413090 89 PCT NUMBER PCT/AU89/00388 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent ClassIficatdon 4 C07K 7/06, 13/00, C12N 15/00 C07K 15/12 (11I) International Publication Number: WO 90/02752 (43) International Publication Date: 22 March 1990 (22.03.90) 88 20 Parnell Street, East Killara, NSW 2071 GOSS, Neil.

Howard 118 Campbell Drive, Wahroonga, NSW 19) 2076 (AU), (21) International Application Number: (22) International Filing Date: 12S5 Priority data: PJ 0382 12 Septer P1 5962 24 Augus PCT/AU89/003 eptember 1989 (12.09.8 nber 1989 (12.09,88) P t1989 (24.08.89)

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L;

(71) Applicant (for all designated States except LVS, SA RAMANE I PTY. LTD, 9 Moralla Road. Kooyong. VIC 3144 (A U).

(72) Inventors; and Inventors/Applicants (for US ortlyj SIYTH E, Jason. Ar.

thur 204 Station Street. Edith'.ale, VIC 3196 ANDERS, Robin, Frederic [AL: AUI*; 55 Brougham Street, North Melbourne, VIC 31)51 KEMP.

David, James [AUJ 309 Belmore Road, North 13u1wyn, VIC 3103 COPPEL, Ross, Leon [AU. 6 Mercer Road, Armadale, VIC 3143 (AL'j. SAUL, Allan.

James (AU/AU]; 14 Dejarra Street. The Gap. QLD 4061) JONES, Graham, Lloyd [AL' AU]: 47 Runic Street, Rainworth, QLD 4065 tAU). IRVING. Da~id.

Owen [AU/AU]; 1/43 Mindarie Street. Lane Co~e, NSW 2066 DYER, Sandrai, Leigh [AL: AL'].

5/400 Mowbray Road, Chatswood. NSW 206- (AL), CHEE, David, Wai, Wing [AU 'AUI: (74) Agents: SLAVTERY, John, Michael et al.; Davies Collison, I Little Collins Street, Melbourne, VIC 3000 (AU).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FR (European patent), GB, GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (European patent), Us.

Published 047th international search report.

.63 82o"16 (54)Title; ALLELIC VARIANTS OF PL-ISMODILM FALCIPARUAI MEROZOITE SURFACE ANTIGEN (57) Abstract A merozoite surface antigen of Plamnwdiunifilciparion selected from the class characterised by: having a relative molecular mass, Mr, (determined by SDS-polyacrvlamide gel electrophoresis) in the range of approxtimately 41,000 to 53,000: and (ii) comprising multiple allelic forms of M SA2, having conserved N-terminal and C-terminal sequences; or an antigenic fragment thereof, Particular antigenic frigments include sequence corresponding to repeat sequences and sequences from conserved N-terminal and C-terminal regins. Production of antigens by recombinant DNA techniques as fusion and nonfusion proteins, and use in vaccine compositions is also disclosed, WO 90/02752 PCr/AU189/00388 1 ALLELIC VARIANTS OF PLASODIUM FALCIPARUM MEROZOITE SURFACE ANTIGEN This invention relates to the identification of antigens of the asexual blood stages of Plasmodium falciparu which are capable of generating antibodies which are able to inhibit the growth of the parasite, and to the use of these antigens and antibodies in immunizati.on and treatment methods.

As is described in prior International Patent Specification No. PCT/AU87/00227, merozoite surface 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. Immunity against erythrocytic stages of Plasmodium infections has been demonstrated by simian vaccination studies using merozoite preparations of P.knowlesi(l) and P.falciparum(2,3).

Antigens identified by 12 5 I-surface labelling studies are likely to reside on the merozoite surface membrane whereas the localization of those antigens which are immunoprecipi.tated with immune serum is more WO 90/02752 PCTAU8/00388 2 tenuous. One high Mr antigen of Plasmodium falciparum(9) which is synthesized predominantly in schizonts and processed to generate proteins of Mr 88, 42 and 19 kDa (10,11) upon schizont maturation is the precursor to the major merozoite surface antigen (PMMSA, also called MSA1). This group of antigens provide a degree of immunity to malaria in Saimiri monkeys(12). They have been described in several Plasmodium falciparum strains,(9,13-15) and Plasmodium species(16,17).

International Patent Specification No.

PCT/AU87/00227 describes a second merozoite surface antigen of the asexual blood stages of Plasmodium falciparum which is characterised by having a relative molecular mass, M r (determined by SDS-polyacrylamide gel electrophoresis) in the range of approximately 41,000 to 53,000; (ii) being a glycoprotein incorporating myristic acid; (iii) being present firstly as a diffuse cytoplasmic localisation and in mature schizonts being located on the surface membrane of merozoites; and (iv) being recognised by some monoclonal antibodies against the asexual blood stages of P.falciparum that inhibit parasite growth in vitro.

This second merozoite surface antigen has been given the names GYMSSA, QF122, Mr 45,000 MSA and MSA2. The latter name will be used throughout the present specification to refer to this second merozoite surface antigen. A clone (Ag 513) expressing this antigen has been isolated from a library of X-Amp3 clones expressing P.falciparum cDNA sequences. The nucleotide sequence of Ag513, together with the predicted amino acid sequence is set out in the earlier patent specification, together with experimental detail indicating that the size and the physical characteristics of the antigen encoded by Ag513 WO 90/02752 PCr/A\U89/00388 3 are the same as that of QF122, results which were confirmed by colony immunoassays in which monoclonal antibodies to QF122 reacted strongly with Ag513 but not other P.falciparum antigen-expressing cDNA clones.

The larger merozoite surface antigen, PMMSA or MSA1, has been shown to exhibit extensive antigenic diversity with the corresponding proteins'from different isolates consisting of a series of conserved and variable domains.

Similarly, it has now been shown that the smaller merozoite surface antigen, MSA2, also shows marked antigenic diversity.

The nucleotide sequence and predicted amino acid sequence of Ag513 are shown in Figure 6 of International Patent Specification No. PCT/AU87/00227, the sequence depicted there being derived from the P.falciparum isolate FCQ27/PNG (FC27). Further sequencing work which has now been done on other isolates of P.falciparum has revealed allelic variation of the MSA2, with the sequences derived from various isolates having high homology in regions of 43 amino acids at the N-terminal end and 73 amino acids at the C-terminal end. This work has established that there are at least two "families" of allelic variants of MSA2, and that certain peptides from the conserved regions which are common to all variants are immunogenic and therefore may form the basis of a synthetic peptide vaccine.

In any synthetic vaccine, the antibody to the immunogen must both recognise the authentic molecule and possess the necessary biological properties to confer protection. Malaria has proved a useful system for the study of peptide based vaccines. Antibodies raised to various sized synthetic repeats of the circumsporozoite proteir 4 amino acid subunit (NANP), reacted efficiently with thet native protein in several assay systems, inhibite, the interaction of sporozoites with their target WO 90/02752 PCTAU8900388 4 cells in vitro and neutralized sporozoite infectivity(19). Similarly, antibodies raised against the C-terminal repeat of the blood stage antigen (EENVEHDA) have been shown.to react with the native protein and to inhibit efficiently parasite invasion in Recently, Pattarroyo et.al.(21) have produced a carrier free synthetic peptide based vaccine which appears to protect partially humans against challenge with asexual stages of P.falciparum. These workers used peptides that had been previously demonstrated to induce partial protection in A.triviratus monkeys. Richman and Reese(22) have also shown that a carrier free peptide from the P.falciparum heat shock protein was capable of raising antibodies reactive against the native protein.

According to the present invention, there is provided a class of merozoite surface antigens of P.falciparum which are characterised by: having a relative molecular mass, M r (determined by SDS-polyacrylamide gel electrophoresis) in the range of approximately 41,000 to 53,000; and (ii) comprising multiple allelic forms of MSA2, having conserved N-terminal and C-terminal sequences.

In pirticular, the antigens of the present invention exhibit high homology (greater than 90% homology) in the N-terminal sequence of 43 amino acids and the C-terminal sequence of 74 amino acids, the N-terminal sequence including the signal sequence with a hydrophobic core of approximately 15 residues and the C-terminal sequence including a second very hydrophobic sequence, approximately 17 residues in length and presumed to be a signal for attachment of a.glycosylphosphatidyl inositol membrane anchor. A further feature of the antigens of the present in!vention is the presence of centrally located repetitive sequences that may be diverse in length, number and sequence.

WO 90/02752 PCT/AU89/00388 The present invention also encompasses a vaccine composition comprising at least two allelic forms of MSA2 as broadly described above, or antigenic fragments thereof, together with a pharmaceutically acceptable carrier or diluent, and optionally an adjuvant, as well as a method for actively immunizing a host against P.falciparum, which method comprises administering to the host a vaccine composition as broadly described above.

Antigenic fragments of particular interest in accordance with the present invention are fragments from the conserved N-terminal and C-terminal regions. In particular, certain peptides from these regions have been found to elicit antibodies which react appropriately on immunofluorescence or immunoblotting analysis with intact MSA2 from different strains of P.falciparum. In this aspect, the present invention provides synthetic peptides capable of eliciting antibodies which recognise intact MSA2, which are characterised in that they consist of or contain an amino acid sequence selected from the group consisting of: SNTFINNA (E71); (ii) QHGHMHGS and (iii) NTSDSQKE (G12); or antigenically active portions thereof.

The present invention also extends to a vaccine composition for eliciting the production of antibodies which recognise intact MSA2, which is characterised in that it comprises at least one synthetic peptide as broadly described above. The vaccine composition may further comprise a pharmaceutically acceptable carrier or diluent, and optionally an adjuvant. The invention also extends to a method of actively immunising a host by eliciting the production of antibodies which recognise intact MSA2, which method comprises the administration to the host of a vaccine composition as described above.

WO 90/02752 PCTAU89/00388 6 In a further aspect of the present invention, there are provided synthetic peptides capable of eliciting specific antibodies against the FC27, Indochina 1 or other variants of MSA2, which are characterised in that they consist of or contain an amino acid sequence corresponding to one of the sequence repeats in MSA2, or are selected from the group consisting of: (iv) MANEGSNT (E87); ANEGSNTN (EBB); (vi) NEGSNTNS (E89); and (vii) SSENPNHN (G34) or antigenically active portions thereof.

These synthetic peptides eliciting specific antibody responses against specific variants of MSA2 may be used alone or in combination with any of the other synthetic peptides described herein in a vaccine composition to generate an immune response against different variants of MSA2.

The synthetic peptides of the present invention may be prepared by any suitable method, 'for example, by chemical synthesis or by expression of an appropriate nucleotide sequence in an appropriate host cell using the well known recombinant DNA techniques.

In order to enhance the immunogenicity of the synthetic peptides of this invention, they may be coupled or conjugated to a suitable carrier protein, such as diphtheria toxoid (DT).

.In general terms, a vaccine composition in accordance with the present invention may be a multicomponent vaccine comprising antigens as broadly described herein from different families of MSA2 allelic variants or antigenic fragments thereof. Alternatively, the vaccine composition may comprise one or more antigenic peptides corresponding to the conserved region or WO 90/02752 PCr/A U89/00388 7 fragments thereof, or the variable regions representative of different MSA2 allelic variants.

The present invention also encompasses 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 one or more allelic forms of MSA2 as broadly described above, or antigenic fragments thereof, or a recombinant cloning vehicle or vector or a host cell comprising a said recombinant DNA molecule. Suitable host cells include, for example, bacteria and yeast, however the present invention also extends to expression in mammalian or insect cell lines using for example recombinant vaccinia virus or recombinant baculovirus. In this aspect, the present invention also extends to a synthetic polypeptide prepared by expression of all or a portion of a nucleotide sequence comprised in a said DNA recombinant molecule, together with a vaccine composition comprising at least one such synthetic polypeptide.

In work leading to the present invention, complete nucleotide sequences of allelic forms of MSA2 have been obtained from five isolates of P.falciparum (FC27, Indochina 1, 3D7, MAD71 and Kl). In addition, the MSA2 of two different isolates has been expressed in E.coli both as a fusion protein and as a non-fused protein, and the immunogenicity of the expressed proteins has been demonstrated.

Further features of the present invention will be apparent from the detailed description in the following Examples.

WO 90/02752 PCT/AU89/00388 8 EXAMPLE 1 DNA corresponding to the MSA2 gene in the Indochina 1, 3D7, MAD71 and K1 isolates was amplified by the method of polymerase chain reaction (PCR) using DNA primers derived from the first and last 30 nucleotides of the Ag513 (FC27 clone) sequence as' shown in Figure 6 of International Patent Specification No. PCT/AU87/00227.

The amplified DNA was then subjected to sequence analysis by known methods.

The polymerase chain reaction (PCR) procedure was based on that described by Saiki et.al.(24), using Taq polymerase isolated from Thermus aquaticus (Perkin Elmer Cetus). Oligonucleotides used as PCR primers corresponded to the first and last 30 nucleotides of the translated FC27 MSA2 gene sequence (nucleotides 94-123 and 859-888) and the gene was amplified through 25 PCR cycles. The reaction mixture contained 100 ng of total genomic parasite DNA, 1pM of each primer, 200pM of each dNTP, in a final vol. of 100 pl of 50 mM KCL, 10 mM Tris. Cl (pH 1.5 mM MgC1 2 0.01% gelatin. The PCR products were gel purified, ligated into the appropriate M13 vectors, and sequenced by the chain termination method as Samples of DNA amplified by the PCR were also dot-blotted onto nitrocellulose and hybridized with oligonucleotide probes as previously described(26). The dephosphorylated oligonucleotide (50 pM) probes were end-labelled by incubation with polynucleotide kinase (Boehringer Mannheim) in the presence of 150 pCi [y- 32 p] ATP (>5000 Ci rmmol-l 1 Amersham). Probes used in the dot-blot hybridization of Figure 7 hereinafter, were: Probe A, 5' conserved region probe corresponding to the first 30 base pairs (bp) of the translated FC27 MSA 2 WO 90/02752 PC/AU9/00388 9 allele, including the initiation codon. Probe B, FC27 repeat-specific probe corresponding to the first 21 bp of the 96 bp repeat of the FC27 MSA2 allele. Probe C, 3D7/Indochina 1 repeat-specific probe (GGTGGTAGTGCTGGTGGTAG) from the MSA2 gene of 3D7 and Indochina 1. Probe D, 3D7 and Indochina 1 repeat-flanking probe corresponding to a 20 bp sequence (GCATCTTGAGTGGGTGGAAC) that is found in the 3D7 and Indochina 1 MSA2 genes but not in the FC27 MSA2 gene.

Probe E, 3' conserved region probe corresponding to the last 30 bp (including the termination codon) of the FC27 MSA2 allele. Probes used in the hybridization of Figure 8 hereinafter were: Probe 1, repeat-specific probe (ATCACAAACTACTACTC) from the MSA2 gene of FC27. Probe 2, repeat-specific probe (GGTGGTAGTGCTGGTGGTAG) from the MSA2 gene of 3D7/Indochina 1. Probe 3, repeat-specific probe (CAGAACCAGCAACA) from the MSA2 gene of MAD71.

The results are shown in the accompanying drawings.

In the drawings: Figure 1 shows the nucleotide sequence and the translated polypeptide for MSA2 of the 3D7 cloned line of P.falciparum, from initiation codon to termination codon.

As depicted, there are two repeat sequences present, a GGSA repeat represented 6 times, and a threonine repeat sequence commencing at base 283.

Figure 2 is the complete nucleotide sequence and the translated polypeptides of the Indochina 1 isolate of P.falciparum, in which the number of GGSA repeats is expanded to 12 copies, and which contains a smaller number of threonine repeats.

Figure 3 is the complete nucleotide sequence and the translated polypeptide for MSA2 of the MAD71 isolate of P.falciparum, which contains a AGAVAGSG repeat sequence represented 6 times.

d Ii ii II 1 i L I 4 il i WO 90/02752 PCT/AU89/00388 Figure 4 is the complete nucleotide sequence and the translated polypeptide for MSA2 of the K1 isolate of P.falciparum, which contains one copy of the 32 amino acid repeat found in MSA2 of isolate FC27 (see Figure 6 of International Patent Specification No. PCT/AU87/00227), and a TTTESNSRSPPI repeat represented 4 times.

Figure 5 is a comparison of the deduced amino acid sequences for the different MSA2 polypeptides.

Figure 6 shows three computer generated "diagon" nucleotide sequence comparisons: fi) a comparison of the 3D7 sequence with itself, showing an upper block of the GGSA repeat and a lower block of the threonine repeat.

a comparison of the Indochina 1 sequence against itself, demonstrating an expansion in the GGSA repeat, and a reduction in the secondary repeat block of t.e threonine residues; and compares the Indochina 1 and the FC27 sequences, showing that these sequences have conserved regions of 43 amino acids and 74 amino acids at the 5' and 3' ends respectively.

Fiure 7 is a summary of dot blots in which oligonucleotide probes based on the conserved N and C-terminal sequences, the FC27 repeat sequence, the 3D7 repeat sequence and a 3D7 repeat flank sequence were hybridized to DNA (amplified by polymerase chain reaction) from 14 different isolates of P.falciparum which had been in long term culture in vitro. These results demonstrate that there are multiple allelic forms of the MSA2. The figure also shows reaction of some of these isolates with monoclonal antibodies 8D88/77, 8G10/48 and 8F6/49 (see International Patent Specification No. PCT/AU87/00227).

These antibodies only react with isolates that hybridize with the probe for the FC27 repeat sequence.

WO 90/02752 PCT/AU89/00388 11 Figure 8 is a summary of dot blots in which oligonucleotide probes based on the FC27, 3D7 and MAD71 repeat sequences were hybridized to DNA amplified by PCR from 16 isolates of P.falciparum recently established in culture in vitro. These results again show that there are different allelic forms of MSA2, and that all isolates react with at least one of the three different repeat probes. Some DNA preparations reacted with more than one probe; this may represent mixed infections (for example KF 1931 or KF 1934), or MSA2 sequences intermediate between the 3D7 and MAD71 repeat types (for example KF 1905).

EXAMPLE 2 The MSA2 of the Indochina 1 isolate of P.falciparum has been expressed in E.coli as a fusion protein with the glutathione S-transferase (GST) of Schistosoma laponicum.

The expression construct was prepared by ligating the SspI fragment of the MSA2 gene(27) into the SmaI site of the expression vector, pGEX-3X(28). In so doing, the amino terminal signal sequence and the carboxy terminal hydrophobic domain are excluded from the recombinant molecule. The fusion protein was localised in the soluble fraction of IPTG-induced E.coli cells and was isolated away from bacterial proteins using affinity chromatography(28). The purified protein consisted a major species of Mr~70,000 along with minor components of Mr~45,000 and Mr~30,000 as shown in Fig.9, which depicts a Coomassie blue stained SDS polyacrylamide gel of the purified MSA2 fusion protein (Lane 1, Molecular weight markers; Lane 2, 0.5pg MSA2 fusion protein; Lane 3, 2pg MSA2 fusion protein;. Lane 4, 4pg MSA2 fusion protein).

The Mr-70,000 component, which was estimated to be approximately 90% of the total purified protein, reacted WO 90/02752 PCT/ALU89/00388 12 with antibodies to both MSA2 (Indochina 1 isolate) and glutathione S-transferase, and it therefore concluded that this component corresponds to a polypeptide encoded by the SspI fragment of the MSA2 gene fused to GST. The smaller fragments are proteolytic degradation products of the Mr~70,000 component.

The MSA2 component of this fusion protein lane 3) has been isolated by cleavage of the affinity purified fusion protein with activated Factor X as described (28) and subsequent separation of the cleaved proteins by reverse phase HPLC. The cleaved purified product (Fig.10 lane 2) migrates as a single band of Mr 60,000 on SDS PAGE. This molecule reacts with antibodies to MSA2 but not to glutathione-S-transferase. Definitive proof of the identity of this component was obtained by determining the amino acid sequence of the N-terminus of the purified antigen using an Applied Biosystems gas phase protein sequencer, Model 470A, using a 120A PTA analyser according to the manufacturer's instructions. The sequence obtained was as follows:

GIPIKNESKYSNTF.INN.

This represents the first three amino acids after the Factor X cleavage site of the vector (28) and 14 consecutive amino acids of the Indochina strain of MSA2 commencing at the isoleucine encoded by nucleotides 58-60 in Fig.2. Amino acid analysis was also performed on the cleaved purified protein. This was determined using a Waters Pico-Tag amino acid analysis system according to the manufacturer's instructions. The composition determined agreed, within experimental error, with the expected composition predicted from the amino acid sequence.

In order to express the Indochina 1 isolate of MSA2 in a "near-native" non-fusion form, the full.-length gene WO 90/02752 PCT/A89/00388 13 was subcloned into pKK223/3 (28c) and expressed in IPTG-induced E.coli cells. Translation was initiated from the MSA2 AUG. Western analysis, following SDS-PAGE showed that the product appeared as two bands of Mr 4 6 ,000 and 50,000, reacting with sera from PNG malaria immune individuals. This is shown in the autoradiograph in Fig.9a where Lane 1 represents IPTG induced cells, Lane 2 is uninduced cells and Lane 3 is 14 C-labelled size standards. (The two bands are indicated by arrows.) Using similar techniques, with pGEX-2T(28) as the vector, the Sspl fragment of the MSA2 gene from isolate FC27 has also been expressed as a GST fusion protein in E.coli, and isolated by affinity chromatography. The MSA2 component has been isolated after cleavage of the purified fusion protein with thrombin. MSA2 from the FC27 strain of P.falciparum was also expressed as a non-fusion protein in E.coli. The 666 bp Sspl fragment of Ag 513(25) was isolated and inserted into the Smal site of the vector, pRDB 8 (28a) which contains the 5' and 3' regulatory sequences (including the ATG) of the gene 32 protein of bacteriophage T4 such that the coding sequence of the MSA2 gene was in frame with the ATG of the gene 32 protein gene in pRDB 8. The resulting plasmid was subsequently digested with Clal and Dral. A 922 bp fragment containing the 5' and 3' regions of the gene 32 protein gene flanking the Sspl fragment of the MSA2 sequence was isolated and subcloned into the unique Smal site of pLK57 (28b). This plasmid was then transformed into E.coli cells for expression. This plasmid-host combination is designated BTA 1893 and the antigen expressed termed Ag1609.

Agl609 was purified away from E.coli proteins using a combination of ion exchange chromatography and reverse phase HPLC. The molecule migrated as a single band of Mr 50,000 on SDS PAGE (Fig.10, lane that, on Western blot WO 90/02752 PCT/AU89/00388 14 analysis was immunoreactive with antibodies to MSA2. As with the expressed Indochina 1 MSA2 molecule, N-terminal amino acid sequence analysis was performed and the following sequence was obtained:

XXXXXXXXSXXIKNESKY

where X represents a position to which no amino acid could be confidently ascribed. However, the first 11 amino acids of Ag1609 were expected to have been derived from the cloning vector. The following 7 amino acids match the appropriate 7 amino acids in the FC27 MSA2 sequence described previously(25). Amino acid analysis also confirmed the composition of the recombinant protein as being that expected for the FC27 MSA2.

In addition, the conserved C-terminal region of the MSA2 gene from isolate FC27 (nucleotides 661 to 885 in Figure 3 of ref.25) has been expressed in pGEX-2T, and isolated by affinity chromatography. This nucleotide sequence was first amplified from FC27 genomic DNA by PCR using as priming oligonucleotides AAGGATCCATGGCACCAGAGAATAAAGGTAC and AAGGATCCTATGAATATGGCAAAAGATA, with added BamHl linkers.

The amplified DNA included the entire 74 amino acid conserved region of MSA2 except that the N-terminal residue was mutated to replace alanine with methionine.

The DNA was gel purified, digested with BamHl, and ligated into the BamHl-cut pGEX-2T vector.

EXAMPLE 3 The immunogenicity.of MSA2-GST fusion proteins (both FC27 and Indochina 1 forms.) and MSA2 of FC27 as a non-fusion protein (Agl609) has been tested in rabbits.

Antibody responses were determined by ELISA using as antigens Agl609 (which measured the total response of rabbits immunised with the MSA2 of FC27, and the response WO 90/02752 PCT/AU89/00388 to the constant regions of rabbits immunised with the MSA2 of Indochina 1) and a synthetic peptide (GGSA) 3 coupled to bovine serum albumin (which measured the repeat-specific response of rabbits immunised with the MSA2 of Indochina Monophosphoryl Lipid A/BCG cell wall skeleton/ squalene (RIBI) and Freunds' complete adjuvant (FCA) were used as adjuvants. The results are shown in Figures 11 to 13.

Best responses to the Indochina 1 form of MSA2 (Figures 11 and 12) were obtained when FCA was used as adjuvant; with good responses detected (at 1:10,000 dilution of serum) against Agl609 and high responses (1:100,000) against the GGSA repeat sequence (Figure 12).

With RIBI, the responses measured against Ag1609 were approximately half those obtained with FCA. In contrast, the responses measured against the GGSA peptide were very much lower (by about 30-fold) than those obtained with FCA and were not readily detectable at serum dilutions greater than 1:1000.

Good responses with both versions of the FC27 form of MSA2 were obtained with FCA, being detected at 1:100,000 dilution of serum (data for Ag1609 in Figure 13). With RIBI, the responses to Agl609 were lower than those obtained with FCA for some rabbits but were still detectable in all rabbits at 1:10,000 dilution and detectable in 2 of 4 animals at 1:100,000.

EXAMPLE 4 A. MATERIALS AND METHODS Peptide Synthesis: 41 octapeptides from the N-terminal constant region (E series E65 to E104) as well as 27 octapeptides from the C-terminal constant region and 10 peptides from the variable region of the Indochina 1 sequence (G series -Gl WO 90/02752 PCT/AU89/00388 16 to G27; G28 to G37) were synthesised using the simultaneous multiple peptide synthesis technique originally described by Houghton(29) employing derivatised (tBOC) amino acids (Omni Biochemicals) on a benzhydrylamine resin (Multiple Peptide Systems).

Peptide Sequences Employed in the Inmunogenicity Trials: The sequence of MSA2 from the FC27 isolate (FC27) and the Indochina 1 isolate (IC) is shown in Figure 14 using the one letter amino acid code. The lower case letters at the beginning of the sequence show the common N-terminal leader sequence which is cleaved in the mature protein while the lower case letters at the end of the sequence show a portion of the common acylation site sequence which is believed to be cleaved from the mature protein as it is acylated and anchored in the membrane.

The deduced amino acid sequence of the mature protein is then represented by the upper case letters. The shaded portions of-the sequence show regions from which overlapping or isolated peptides were synthesised and coupled to the carrier protein (DT) for immunogenicity trials. N-terminal peptides (E series) were staggered by one residue at a time as indicated and extended into the variable region of the FC27 sequence. C-terminal peptides (G series) were staggered by two residues at a time and extended into the common cleaved acylation region.

Isolated peptides shaded and indicated were also chosen from the variable region of the Indochina sequence. The FC27 (but not the Indochina 1) sequence contains two copies of the STNS epitope (boxed) recognised by inhibitory Mabs 8G10/48 and 9E3/48 (see International Patent Specification No. PCT/AU87/00227).

Cleavage and Extraction of Peptides: Cleavage from each of the peptide resin bags (each bag originally contained 100 mg of benzhydrylamine resin) was performed in a multiple HF cleavage manifold (Multiple WO 90/02752 PC~/AU9/00388 17 Peptide systems) using anhydrous HF:anisole:thioanisoles dimethylsulphide at 0°C for 1 hour. The mixture was then dried under vacuum and the bags washed with cold anhydrous ether, extracted with 10% acetic acid and the crude peptides lyophilised. Yield of crude peptide (60-R5% purity) averaged 30 mg.

Purification of Peptides: Crude synthetic peptides were dissolved in acetonitrile containing 20 mM dithiothreitol and purified by reverse phase HPLC using an acetonitrile gradient with 0.1% trifluoroacetic acid (TFA) as counterion.

Coupling of Peptide to Carrier Protein: Peptides were synthesised with an added N-terminal cysteine to facilitate coupling to the carrier protein and coupled to DT using the heterobifunctional reagent maleimido caproyloxysuccinimide (MCS). High purity DT was obtained from the Commonwealth Serum Laboratories in Melbourne, Australia and derivatized with MCS using the procedure described by Lee et.al.(30). The solution of MCS activated DT protein was added directly to an amount of dry, purified, fully reduced peptide equivalent to 1.4 times the molar equivalent of maleimido groups in the carrier and stirred slowly overnight at room temperature under N 2 The conjugate was then dialysed with several changes against phosphate buffered saline (PBS). The degree of conjugation was determined by titrating the maleimido groups before and after reaction with the peptide using a subtractive Elmans procedure (31).

Peptides were also conjugated to bovine serum albumin (BSA) using the same chemistry.

In both cases the extent of coupling was also monitored by observing the increase in apparent molecular weight (SDS/PAGE 8% gels) of the carrier protein-peptide adduct by comparison with the carrier protein itself(32).

WO 90/02752 PCT/AU89/00388 18 For both E and G series peptides, apparent molar ratio of peptide to carrier protein ranged-from 7-10 for the DT adducts and 8-11 for the BSA adducts.

Immunization with Peptide-Diphtheria Toxoid Conjugates: Female Balb/c mice aged 6-7 weeks were pre-bled and then immunized i.p. with the peptide-DT conjugates at peptide per injection in an emulsion of FCA. One month later, animals received a second injection with incomplete Freund's adjuvant. Two weeks after the final injection animals were bled and the sera inactivated by heating at 56 0 C for one hour. Inactivated sera was serially diluted in 5% milk powder in phosphate buffered saline (PBS) prior to immunological analysis.

Antibody Titration: Wells of flat bottom ELISA plates (Immunolon- Dynatech) were coated overnight with a 5 pg/ml solution of peptide-BSA adducts, blocked with a solution containing skim milk powder in PBS, rinsed in PBS and then incubated either with pre-immunization sera or with sera from mice immunized with the corresponding peptide-DT conjugate serially diluted in.5% milk powder in'PBS. After further rinsing in PBS, anti-mouse globulins conjugated to horse-radish peroxidase and the substrate 2,2'azino-di (3-ethylbenzthiazoline sulphonate) (ABTS) were added successively according to the manufacturer's instructions (Amersham, The absorbance at 410 nm was measured directly at 0 and 30 minutes using an automated microplate reader. Control plates were coated with sham coupled BSA, i.e. BSA that had been reacted with MCS and blocked with p-mercaptoethanol with no added peptide.

SDS-PAGE and Immunoblotting: Parasitized erythrocytes were washed and solubilized with 1% Triton X-100. Protease inhibitors were added pg ml1 each of pepstatin, leupeptin, chymostatin and WO 90/02752 PCTI/AU9/00388 19 antipain). Detergent soluble proteins were resolved on SDS-polyacrylamide gels according to the Laemmli procedure After separation parasite proteins were electrophoretically transferred onto nitrocellulose paper and immunoreacted with mouse antibodies and 125 1-labelled goat anti-mouse antibody or with rabbit anti-serum and 125 1-labelled Protein A, following the procedure of Towbin et.al.(34).

B. RESULTS Anti-Peptide Antibodies: Figure 15(a), and shows the anti-peptide titres for peptides from the conserved N-terminus (E series, Figure C-terminus (G Series, Figure and variable region of the Indochina 1 MSA2 (G series, Figure Results are expressed as the absorbance at 410 nm in the solid phase ELISA assay when the homologous peptide-BSA adduct was used as plate coating antigen and the plates incubated with anti-sera at the indicated dilutions. Most of the peptides are highly immunogenic when presented to Balb/c mice using this protcol. There is however a marked variation in response which is believed to be related to the sequence of the immunising peptide per se since all the sera tested had a high (1/106) titre of anti-DT antibodies when tested against the carrier protein (DT) alone by ELISA. This anti-DT response, although also somewhat variable, did not correlate with the anti-peptide response.

Anti-Linker Antibodies: Figure 16 shows the antibody titre (expressed as

A

410 at 10- 3 sera dilution) when sera'from the N-terminal MSA2 peptides (E series) were tested against sham coupled BSA BSA-MCS). Clearly some of the peptide-DT adducts elicited quite high anti-linker antibodies. This WO 90/02752 P(/AU89/00388 anti-BSA-MCS response could be inhibited by pre-incubation of the sera with a thiosuccinimido cap.oylamide (TSC) analogue of the linker region of the.peptide-DT conjugate. There is no significant correlation between the levels of anti-peptide and anti-linker response (P 0.53) for the E series peptides.

MSA2 Peptides Eliciting an Appropriate Immunofluorescence: Figure 17 shows the peptide-DT conjugates studied which elicited merozoite surface reacting antibodies when incubated with acetone fixed schizonts. Of the constant region peptides tested, only peptide E71 from the N-terminal portion and peptides G5, G7, G12 and G21 from the C-terminal portion elicited surface reactive antibodies against both FC27 and Indochina 1 parasites.

Peptides E87, 88 and 89 elicited IFA positive antibodies against the FC27 parasites only whereas peptides G28 and G34 elicited antibodies against the Indochina 1 parasites only.

MSA2 Peptides Eliciting an Appropriate Immunoblotting Response: Only peptides E71, G5 and G12 from the MSA2 N- and C-terminal constant regions gave rise to antibodies which reacted with the SDS/PAGE separated Western blotted MSA2 from both FC27 and Indochina 1 parasites (Fig.18(a) and Peptide E87 elicited antibodies reactive with FC27 only whereas peptide G34 gave rise to antibodies reactive with only the Indochina 1 MSA2. Sera from animals vaccinated with E71, G5 and G12 also gave a positive immunoblotting response against the recombinant full lengh FC27 MSA2 produced in E.coli. Figure 18 shows immunoblotting patte-rns demonstrated by sera elicited by .vaccination with MSA2 peptide-DT conjugates: probed against FC27 extracts separated on SDS/PAGE; probed against Indochina extracts separated on SDS/PAGE.

WO 90/02752 PCT/AU89/00388 21 Lane 1. 1/100 dilution of sera from E71 vaccinate; Lane 2. 1/100 dilution of sera from G5 vaccinate; Lane 3. 1/100 dilution of sera from G12 vaccinate; Lane 4. 1/100 dilution of sera from G34 vaccinate; Lane 5. Inhibitory Mab 8G10/48 supernatant; and Lane 6. Pre-bled from E71 vaccinate.

WO090/02752 PFA8/08 PCr/AU89/00388 22 1. Mitchell, Butcher, Langhorn, J.

and Cohen, S. (197.7) Clin.Ex..lmunol. 2, 276-279.

2. Mitchell, Richards, Butcher, and Cohen, S. (1977) Lan~etk 1, 1335-1338.

3. Siddiqui, W.A. (1977), Science Mil, 388-389.

4. Freeman, R.R. and Holder, A.A. (1983) J.Ezp.Kezd. 158, 1647-1653.

Heidrich, Strych, and Mrema, 3.E.K. (1983). Z.Parasitenkd jU, 715-725.

6. Reese, Motyl, M.R. and Hofer-Warbinek, R. (1981), Arn.JTropMed.Hyg. 1168-1178 7. Perrin, Dayal, and Reider, H.

(1981) Trans.R.Soc.Trop.Med.Hyg. 11 163-165.

8. Howard, R.F. and Reese, R.T. (1984) Molec.Biochem.Parasitol. U, 319-334.

9. Holder, Lockyer, Odink, K.G., Sandhu, Rivercos-Moreno, Nicholls, Hillman, Davey, Tizard, Schwarz, R.T. and Freeman, R.R.

(1985), Niatu.re, 3_U, 1 270-273.

Holder, A.A. and Freeman, R.R. (1984), 3.x.Md 2.iU. 624-629.

WO 90/02752 PCI/A U89/00388 23 11. Lyon, Geller, Haynes, J.D., Chulay. J.D. ard Weber, J.L. (1986) Pro.Natl.cad.Sci. (USAI. i 1 2989-2993.

12. Perrin, LH., Merkli, Loche, M., Chizzolini, Smart, 3. ard Richie,. R.

(1984) LLZ.xp.Ma. 1,U, 441-451 13. Mackay, M. Goman, Bone, Hyde, J.E., Scaite, Certa, Stunnenberg, H. and Bujard, H. (1985) Embo.J. 1, 3823-3829.

14. McBride, Newbold, and Anand, R.

(1985) J.Exp.Mad. 1ii, 160-180.

Weber, Leininger, W.M. and Lyon, J.A.

(1986) Nucl.AcidzRr,. J, 3311-3323.

16. Holder, A.A. and Freeman, R.R. (1981) Nature 22A, 361-364.

17. Epstein, Miller, Kaushel, D.C., Udeinya, Rener, Howard, R., Asofsky, Aikawa, M. and Hess, R.L., (1981), J.mmunl. 221, ;12-217.

18. Kilejian, A. (1980), Proc.Natl.Acad.Sci.

(USA) 21, 3695-3699.

WO 90/02752 PCT/AL)89/00388 24 257-259.

Berzins, K. et.al. (1986). ProcNatl.Acad.Sci (USA) 1065-1069.

21. Pattarroyo, M.E. et.al. (1980). 1LattUx& aa2.

158-161.

22. Richmnan, and Reese, R.T. (1988). Proc. Natl.

Acad.Sci (UJSA) BI. 1662-1666.

23. Saul, A. et.al. In: "Technological Advances in Vaccine Development", Alan Liss Inc., New York (in press).

24. Saiki, R. et.al. (1985). Sience2~ 1350-1354.

Smythe, J.A. et.al. (1988). ProcNatl.Acad.Sci (USA)IBI 5195-5199.

26. Peterson, M.G. et-al. (1988). o1. Cell Biol.a 2664-2667.

27. Smythe, et.al. (1989). In: Vaccines '89, Cold Spring Harbor Laboratory, USA.

28. .Smith, D.B. and Johnson, K.S. (1988). Grn 67 3 1-40.

28a. Duvoisin, R.M. et.al. Gen 45 193-201.

28b. Botterman, J. and Sabeau, M. (1987). DN~A 83-59 1.

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Claims (12)

1. A vaccine composition comprising at least two allelic forms of a merozoite surface antigen of Plasmodium falciparum selected from the class characterised by: having a relative molecular mass, (determined by SDS-polyacrylamide gel electrophoresis) in the range of approximately 41,000 to 53,000; and (ii) comprising multiple allelic forms having conserved N- terminal and. C-terminal sequences, or antigenic fragments thereof, and a pharmaceutically acceptable carrier or diluent.

2. A vaccine composition according to claim 1, wherein each of said allelic forms has an amino acid sequence set out in Figure 1, Figure 2, Figure 3 or Figure 4, or an antigenic fragment thereof.

3. A vaccine composition comprising a peptide antigen which comprises or is derived from the conserved N- terminal and/or C-terminal regions of a merozoite surface antigen of Plasmodium falciparum selected from the class characterised by: having a relative molecular mass, Mr, (determined by SDS-polyacrylamide gel electrophoresis) in the range of approximately 41,000 to 53,000; and (ii) comprising multiple allelic forms having conserved N- terminal and C-terminal sequences, and a pharmaceutically acceptable carrier or diluent.

4. A vaccine composition according to any one of claims 1 to 3, further comprising an adjuvant.

5. A method of actively immunising a host against P.falciparum which comprises administering to the host a vaccine composition according to any one of claims 1 to 4,

6. A synthetic peptide capable of eliciting antibodies which recognise intact MSA2 of P.falciparum, characterised in that it consists of or contains an amino acid 930322,p:\opcr\jms,43090/89,26 %rc sequence selected from the group consisting of: SNTFINNA; (ii) QHGHMHGS; and (iii) NTSDSQKE; or antigenically active portions thereof.

7. A synthetic peptide capable of eliciting specific antibodies against the FC27, Indochina 1 or other variants of MSA2 of P.falciparum, characterised in that it consist of or contains an amino acid sequence corresponding to one of the repeat sequences in MSA2, or selected from the group consisting of: (iv) MANEGSNT; ANEGSNTN; (vi) NEGSNTNS; and (vii) SSENPNHN; or antigenically active portions thereof.

8. A vaccine composition comprising at least one synthetic peptide according to claim 6 or claim 7, and a pharmaceutically acceptable carrier or diluent.

9. A vaccine composition according to claim 8, wherein said at least one synthetic peptide is coupled or conjugated to a carrier protein. A vaccine composition according to claim 9, wherein said carrier protein is diphtheria toxoid.

11. A vaccine composition according to claim 8 or claim 9, further comprising an adjuvant.

12. A method of actively immunising a host against P.falciparum which comprises administering to the host a vaccine composition according to any one of claims 8 to 11. Dated this 22nd day of March, 1993 Saramane Pty.Ltd. By its Patent Attorneys, DAVIES COLLISON CAVE 930322,p:\oper\jms,43090/89,27 MetLysVaileLysThrLeuSerlieIieAsflPhePhelePheVaiThrPheAsf I~e ATGAAGGTAATTAAAACATTGTCTATTATAAATTTC TTTATTTTTGTTACCTTTAAIATT 20 30 4.0 50 LysAsnGluSerLysTyrSerAslThrPheleAsflAslAiaTyI'AsfMetSelteArg AAAAATGAAAGTAAATATAGCAACACATTCATAAACAATGCTTATAATATGAGTATAAGG 80 90 100 110 120 Ar gSe rMe tAt aSluSer LysPr oSerlh rS IyAla~ly ly Th rAlaGlyGlySeriAla AGAAGTATGGCAGAAAGTAAGC CTTCTACTGGTGCTcjGTGGTACTGCTGGTGGTAGTGCT 130 140 150 160 170 180 Gly 01 ySerA~aGlyGlySerAIQGIYGIYSerAQGG[YOySe-AI aOLySerOL yAsp N GGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTTCTGGTGAT 190 200 210 220 230 240 G1yAsnOtyAaAspA~aG~ugtySe-SeSeThrPoA1QTh-ThrThiThfThrLys GGTAATf3OTG CAGATS CTGAGGGAAGTTCAAGTACT CCCGCTACTAC CACAACTAC CAAA 250 260 270 280 290 300 r ThrThrThrThrThrThrThrThrAsnAspAtaGtuAIQSei-ThrSerThrSerSerGlu ACT IACCACAACTACCACAACTACTAATGATGCAGAAGCATCTALCAGTACCTCTTCAGAA 310 320 330 340 350 360 L AsnProAsnHisLysAsnAlaGIuThrAsnProLYSG LYSGt YG[uV I GflG t uPro F 1(a AATC CAAATCATAAAAATGCCGAAACAAATC CAAAAGGTAAAGGAGAAGTT CAAGAAC CA gD 370 380 390 400 410 420 r 00) C AsnG[nAlaAsnLysGtulhrG[ 'nAsnAsnSerAsnVa[G tnG InAspSerG InThrLys AATCAAGCAAATAAAGAAACTCAAAATAAC TCAAATOTTC AACAAGAC TCTCAAACTAAA 430 440 ''450 460 470 480 SerAsnVat ProProThrGlnAspAlaAspThrLysSerProThrAt aGlnProG luGin TCAAATOTTCCACCCACTCAAGATGCAGACACTAAAAGTCCTACTOCACAACCTGAACAA 490 500 510 520 530 540 At aG IluAsnSer A[ aPr oThr At aG I uGi nThr~luSer Pro~tuLeuG I nSerA IaPro GCTGAAAATTCTGCTC CAACAGCCGAACAAACTGAATCCCCCGAATTACAATCTGCACCA 550 560 570 580 590 600 G1.uAsnLysGlylhr~ly~lnHisGtyHi sMetHisGtySerArgAsnAsnHisProGiln GAGAATAAAGGTACAGGACAACATGGACATATGCATGGTTCTAGAAATAAT[ATCCACAA 610 620 630 640 650 660 AsnThrSerAspSerGtnLysGIuCysThrAspG~yAsnLysGtuAsnCysG~yAtQAta AATACTTCTGATAGTCAAAAAGAATGTACCGATGGTAACAAAGAAAACTGTGGAGCAGCA 670 680 690 700 710 720 L ThrSerLeuLeuAsnAsnSerSerAsnlhleAlaSerlteAsnLysPheVatVatLeutle c ACAT CCCTCTTAAATAACTCTAGTAATATTGCTTCAATAAATAAATTTGTTGTTTTAATT 730 740 750 760 770 780 mi SerAlaThrLeuVatLeuSerPheAcl IePhelle*** Pu-/h TCAGCAACACTTGTTTTATCTTTTGCCATATTCATAIAA r ?h790 800 810 m IICO MetLysVa1IeLysThrLeuSerltelleAsnPhePhelte~heVallhrPheAsnlte ATGAA~fTAATTAAAACATTGTCTATTATAAATT TCTTTATTTTTGITACCTTTAAIATT 20 30 40 50 LysAsnGluSerLysT.yrSerAsnThrPhel1.eAsnAsnAlaTyrAsnMetSerlteArg AAAAATGAAAGTAAATATAGCAACACATTCATAAACAATGCTTATAATATGAGTATAAGG 80 90 100 110 120 Ar gSe rMe tThrG IuSer AsnPr oProTh rG~yA~aSerG I.ySerAt aGlyGlySe rAira AGAAGTATGACAGAAAGTAATCCTC CTACTGGTGCTAGTGGTIAIJTGCTGGTGGTAGTGCT 130 140 150 160 170 180 G IyG IySerAraG I yG IySerAl~aGI3yG IySerA I iGyG LySerA IaG IyG IySerAtcl GGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCTGGTGGTAGTGCT 190 200 210 220 230 240 61 yGI ySerA~raGlyG~ySerAtcIG~yGlySerAtaGlyG~ySerA[aGtyGtySerAta Nj GGTGGTAGTGCTGGTGGTAGTG CTGGTGGTAGTG~CTGGTGGTAGTGCTGGTGTAGTGCT 250 260 270 280 290 300 01 GL 01ySerAlaGl ySerG[ AspOIyAsnGl yAlaAsnProGI yA~aAspAla(uArg GTGGTAGTGCTGGTTC TGG T GG AATGT GCTAATCCTGG TGCAGAT GCTGAGAGA K 310 320 330 340 350 360 n SerProSerThrProA~ciThrThrThrThrThrThrThrThrAsnAspAta~tuAlaSer r AGTCCAAGTACTC CCG CTACTACCACAACTAC CACAACTACTAATGATGCAGAAG CAT CT 370 380 390 400 4+10 420 ThrSerThrSerSerGiuAsnProAsnli sAsnAsnAlQGluThrAsnG~nALQAsnLys Fig.2(ai ACCAGTACCTCTTCAGiAAAAT CCAAAT CATAATAATGCCGAAACAAATCAAG CAAATAAA 430 4+40 4 50 460 470 480 C'-)c -4 GluTh rG InAsnAs nSer As nVal.UlnGt nAspSer6 I.nThr LYsSer AsnVQI. Pro Pro OAAACTCAAAATAACT CAAACGTTCAACAAGACT CT CAAACTAAATCAAATGTTC CACCC 490 500: 510 520 530 540 ThrG~nAspAtaAspThrArgSerProThrAQGflProGuGlflAlG[uAsflSerAIQ ACTCAAGATGCAGACACTAGAAGTCCTACTGCACAACCTGAACAAGCTGAAAATTCTGCT 550 1560 570 580 590 600 ProThrAtaOI~uGI nThrGtuSerProGluLeuGlflSerAtQProGIuAsflLysGiyThr CCAACAOC CGAACAAACTGAATCCC CC GAATTACrAATCTGCACCAGAGAATAAAGGTACA 610 620 630 640 650 660 GIyG~ nHisGtyHi sMetHisGySerArgAsnAsflHisProGtflAsfThrSerAspSer GGACAACATGGACATATG CATGGTTCTAGAAATAATCATC CACAAAATACTTCTGATAGT 670 680 690 700 710 720 G~nLysGLuCysThrAspGyAsflLysGuAslCysGIyAQAQThlSerLeuLeuAsf CAAAAAGAATGTACCGATGGTAACAAAGAAAACTGTGGAf3CAGCAACATCCCTCTTAAAT 730 740 750 760 770 780 AsnSerSerAslhLeAlaSerlIt eAsnLysPheVat Vat Leu~IeSe rAt aThr Le uVQt AACTCTAGTAATATTG CTTCAATAAATAAATTTGTTQTTTTAATTTCAGCAACACTTGTT_ 790 800 810 820 830 840 LeuSerPhe~lallePhelte Fi'2(b)V TTAT CTTTTGCCATATTCATATAA 850 860 C C" 0D En 1l1u/'L U U RECEIVED DEC 1989 5/22 MetLysVa I eLysThirLeuSerl lel IeAsnPhePhel LePhe ATGAAGOTAATTAAAACATTGTC TATTATAAATTTC TTTATTTTT 20 30 IleAsnAsnAtaTyrAsnMefSerlle~rgArgSerMetGluGluI ATAAACAATGC TTATAATATGAGTATAAG3AGAAGTATGGAAGAA .1100 110 120 130 G1 ySercl yAlaOl yAlaVa IA[aGIySerG[ yAlaGlyAtaVa tI GGTTCTOGTGCTOOTG CTGTTGC TG3GTTCTOGTGCTGOTGCTGT T 190 200 210 220 Va tAtaGlySerG ty~tatyAtaVatAtaGl ySer~lyAtaVat GTTGCTGGTTCTGGTGCTGGTGiCTGTTGCTGGTTCTGGTGCTGT T 260 290 300 310 GtuArgGtyProSerThrProAtaThrTh rTh rThrThrThrTh ri GAGAGAGGTC EAAGTACTCCCI3CTAC CACAACTACCACCACAACT' 370 360 390 400 AsnHi sAsnAsnAtaGtuTh rAsnProLysGl yLysGtlGtuVa t AAT CATAATAATG CCGAAACAAAT CCAAAAGGTAAAf3GAGAAGTT 460 470 480 490 Va(G Ini31nAspSerG tnThrLysSerAsnVa tProArgThr~t n GTTCAACAAGACTCTCAAACTAAATCAAATGTTCCACGCACTCAA -550 560 570 580 AsnSe rAta ProTh rAt aGtuG t nT hrGt nSe rP ro G u LeuGt in AATTCTGCTCCAACAGCCGAACAAACTGAATCCCCCGAATTACAA 640 650 660 670 Gt ySe rArgAsnAs nHis ProGl nAsnTh rSe rAs pSerIn Lys GGTTCTAGAAATAATCATCCACAAAATACTTCTGATAGTCAAAAA 730 740 750 760 LeuLeuAsnAsnSerSerAsnlI eAlaSerI teAsnLysPheVa t CT CT TAAATAACT CTA GTAATA TTGC TTC AATAAAT AAAT TTG TT 820 630 640 650 Fig. 3(a) SUBSTITUTE SHIEET O2CEIVE D DE C 1989 6/22 Va iThr PheAsnlt eLy sAsnG uS er Ly sTy rSe rAsnTir Phe OT TAC CTTT AATA.TT AAAAAT GAAAGT AAAT ATAG CA ACACATTC s0 60 70 80 SerLysProP--roTh rG Iy AtaVaIA[ aG IySe rG IyA a G IyA Ia ,AGTAAGCC TCCTACTGGTGCTGTTGCTGGTTCTGGTGCTGGTGCT 14V 150 160 170 180 A taG ivSe rGtyAt aGtyAtaVa tA.t aGlySe rG tyAtaG yA ta G CTGGTTCTG GTOL:TGGTGCTGTTG CTGGTTCTGGTGCTGGTGCT 230 240 250 260 270 A I aG I yS e rG I yA I aG I yAsnG I yA I a As riP r oG ty AlaAs pAl a IGC TO TT CTGGTG CTGGT AATGGTGC TAATCC TGGTIGCAGATGCTI Th rA snAs p A t a GI uA I a Se rT h rS e rT h rS erS e r G IuA s nA rg AC TA ATGATG CAG CAG CAT CTACC AGTACC TCTT CA GAAAA TCG A 410 420 430 440 450 'GlnLysProAsnGtnAtaAsnLysf~luThrGtnAsnAsnSerAsn C AAAAA C CAAAT CAAG CAAA TAAAGAAAC TC AAAAT AAC TCAA AT 500 510 520 530 540 As pA IaAspTh r LysS e rP roT hrAIa G In Pr oG Iu G InA Ia6I u GATGCAGACACTAAAAGTCCTACTGCACAACCTGAACAA~fCTGAA. 59 0 600 610 620 630 Se rAta Pr oG t uAnLy sG tyTh rG I n H isG (y H isMe tH Is TCTG CACCAGAGAATAAAGGTACAGGACAACATGGACATAT OCAT 680 690 700 710 720 GluCysThrAspGtyAsnLysGtuAsnCysGlyAla.AtaThrSer 770 780 790 800 810 Val Leul teSerA I aT hrLeuVa [L euSe rPheAtal tePhel Ie** GTTTTAATTTCAGCAACACTTGTTTTATCTTTTGCC ATATTCATATAA 860 8 70' 880 890 900 Fig. 3(b) SUBSTITUTE sHScur RECEIVED -1DEC 1989 7/22 MetLysVaitleLyshLeuSe(eleAsnPhePheltePhe ATGAAGGTAATTAAAACATTGTC TATTATAAATTTC TTTATTTTT 20 30 It eAsnAsnA(QlyrAsnMetSer I eArgArgSerMetAlaAsn ATAAACAATGCTTATAATATGAGTATAAGGAGAAGTATGG CAAAT 100 110 120 130 AspThrlleAlQSerGtySerGtnSerSerlhrAsnSerAlaSer f GATAC TATTGCTAGTGGAAGTCAAAGTAG TACAAATAGTGCAAGiT 190 200 210 220 AtaAtQAspThrProThrAtaThrGtuSerAsnSerArgSerPro) OC TO C TATAC CC CTAC TO CTACAGAAAO TAATTCA CGTCAC CA 280 290 300 310 ThrThrGluSerAsnSerArgSerProProIlelhrThrThrGtu AC TA CAAAAGTAA TT CACO TTC AC CACC CAT CACT ACTA CAGAA 370 380 390 400 Ar gSe rPr oPr ol eTh rThr ThrGOI.uSe rSerSe rSerGlyAsn CGTTCACCACCCATCACTACTACAGAAAOTICAAOTTCTGGCAAT 460 470 480 490 AsnGt uLeuAs nO LuSer Th rO uGluGt yPr oLysAL aPr oOIn, AATOAATTAAATGAATCAACTGAAOAAGGACCCAAAGCTCCACAA 550 560 570 580 LysGtylhr~lyGlnHisGlyHisMetHis ,G'ySerArgAsnAsn AAAOOTACAOOACAACATGOAC ATATOCATOOGTTCTAOAAATAAT 640 650 660 670 AsnL y sO uAsnCy sO yAt QA .alhr Se L euLeu AsnAsnSer AACAAAOAAAACTGTGGAGCAGCAACATCCCTCTTAAATAACTCT 730 740 750 760 ThrLeuVal LeuSerPheAt allePhel Ie*** AC AC TTO TTTTATC TT TTGC CA TATlCAT ATAA 820 830 840 Fig. 4 SUBSTITUTE SHIEET RECEIVED VJ V. 1 DE C 1989 8/22 VaiThrPheAsnlteLysAsnG[ySerLysTyrSerAsnThrPhe GTTA C CT TTAA TAT TAAAAAT GGAAG TAAATATAGC AAC ACATTC 60 70 80 GtuGtySerAsnThrLysSerVI.Gt yAtoAsnAtaProLysAla GAAGGTTCTAATACTAAGAGTGTAGGTGCAAATGiCTCLCAAAAGCT 140 150 160 170 180 *ThrSerThrThrAsnAsnGtyG~uSerGL nThrThrThrProThr ACTAGTACTACTAATAATGOAGAATCACAAACTACTACTCCTACC 230 240 250 260 270 ProJ teThrThrThrGtuSerAsnSerArgSerProProt telhr C C CATCA CT ACTA CAG AAAG TAAT T C CG TT CAC CAC CCAT CACT 320 330 340 350 360 SlerAsnSerArgSerProProl IeThrThrThrGluSerA-snSer I AGTAATT CAC OTTCAC CAC C CATCAC TAC TACAGAAAGTAATTCA 410 420 430 440 450 AtQiProAsnLysThrAspG[ yLysGtIyGI uGI uSerGtuLysGi n GC AC CAAATAAAACAGACGGTAAAGGA~iAAGAGAGTGAAAAACAA S00 510 520 530 540 GluProGtnThrAciGtuAsnGtuAsnProAQA.QProGuAsfl GAACCTCAAACGGCAGAAAATGAAAATCCTGC FGCACCAGAGAAT 590 600 610 620 630 Hi sProGInAsnThrSerAspSerGtnLys~tuCysThrAspG1 y CATC CACAAAATACTTCTGATAGTCAAAAAGAA TGTACCGATGGT

680. 690 700 710 720 SerAsnI~eA~aSerlteAsnLysPheVa(VaILeuI~eSerAIQ AGTAATATTGCTTCAATAAATAAATTTGTTGTTTTAATTTCAGCA 770 780 790 800 810 Fig. 4(b). SUBSTITUTE SHEET,. FC2 7 Ki 307 I c MAD71 FC27 Ki 307 1 c MA07 1 FC27 Ki 307 I c MA 07 1 MKVIKtLSlII MKVIKTLSI I M KVIKTLSI 1 KTLSI I MKVIKTLSI I NSVGANAPNA KS VGA NA PKA GAGGTAGGSA GASGSAGGSA GAVAGSGAGA SQRST NSAST NSRSPPI TTT .SOD GGS. .AGSO OiSOAVAGSGA NFF IFVTFNI NFF IFVTFNJ NFFI FVTFNI NFFI FVTFNI NFFI FVTFNI OT IASGSQRS DTIASGSQSS GGSAGGSAGG GO SAG S AG GSGAGAVAGS STTNNGESQT ESNSRSPP IT ONGA. .DA O NOANPOADA O NOANPOADA K NES KYS NT F KNGSKYSNTF KNESKYSNTF K NE SKYS NTF KNESKYSNTF TNSASTS TTN TNSASTSTTN SAGGSAG SAGGSAGGSA GAG AVAG SGA TTPTAAOTPT TTESNSRSPP EGSSST PATT ERSPST PATT ERG PST PATT INNAYNMSIR INNAYNMSIR I NNAYNMSI R I NNAYNMSI P I NNAYNMSIR NO ES QTTT PT NO ESQT TT PT ITTTESNSRS TTT KTTTTTT TTTTTT... TTTT TT... RSMANEGSNT R SMANEOS N T RS MAE SKPS T RS MT E SNPP I RSMEE SKPPT AADT. ASG AADT PlATES SAGO SAG GSA VAOSOAGAVA .TESIS PPITTTESNS TTNDAEASTS S. NOAFASTS NOAEASTS r\) 100 C -i rn rh -I 150 o toC I I FC2 7 Ki 307 I c MAD71 FC2 7 Ki 307 I c MAD71 FC27 Ki 307 Ic MAD71 FC27 Ki 307 It: MAO71 PSPPI TTTES RSPPITTTES TSSE N PNH KN TSSENPNHNN TS SE NRNH NN QOAOTRSPTA QODTKSPTA NHPQNTSOSO NHPQNTSOSQ NHPQNT SOSO NHPQNTSOSQ NHPQNTSDSQ SKFWQCTNKT SSSGNAPNKT AETNPKGKGE AETN... AETNPKGKGE PQTAENENP PQTAENENP OPE QA E NSAP QPEQLAENSAP GPE QAEN SAP KECTOONKEN KECTOGNKEN KECTOONKEN KECTOGNKEN KECTDGNKEN OGKGEESEKQ OGKGEESEKQ VQEPNQANKE QANKE A TAEQTESPEL TAEOTESPEL CGAATSLLSN CGAATSL LNN CGAATSLLNN CGAATSL LNN CGAATSLLNN NELNESTEEG NEL NE STE EG T QN NSNVQQD TO NNSN VQ000 TQNNSNVQQ.D *..APENKGTG APENKGTG QSAPENKGT S QSAPENKGTG OSAPENKGTG SSNIASI NKF SSNIASI NKF SSNIASI NKF SSNIASINKF SSNIASINKF PKAPQE... PKAPQE SOaTK SNV PPT SQTKSNVPPT SOT KSNVPRT aHS H MH6S SRN OH GH MHGSSRN QHGHMHGSRN QHGHMHGSRN O[HGHMHG SRN VVLI SAT LVL VVLI SATLVL VVLI SATLVL VVL I SAT LV L VVLI SAT LVL 200 250 300 S FA IF I 'SFA IF I* SFAIF I SFA I F I SFAIFI 307 (b) rl C M EIVED E DC 1989 111/zz SUBSTITUTE SKEET rut/AU. 9 0 0 3 8 8 RECF1VED DEC 989 12/22 PROBE OLIGONUCLEOTIDE MONOCLONAL ANTIBODY ISOLATE A B C D E 8D88/77 8G10/48 8F6/49 Palo Alto K1 CSL2 NO ND ND NF7 IfG2 V1 010 HB3 IMR143 NO NO ND IMR144 ND 'ND NO ND ND ND IMR147 ND ND NO FC27 MA071 ND NO ND OLIGONUCLEOTIDE PROBES: A.N' TERMINAL PROBE B FC27 REPEAT PROBE C 3D7 -REPEAT PROBE D 307 REPEAT FLANK PROBE E C' TERMINAL PROBE MONOCLONAL ANTIBODY PROBES: 80D88/77 REACTS WITH THE SEQUENCE SNTN 8010148 REACTS WITH THE SEQUENCE STNS 8F6/49 REACTS WITH THE SEQUENCE DTPTATE ND NOT DONE Fig. 7 SUBSTITUTE SHE.;" POT/A, 0 o 00 38 '.2CEIVED I DEC 1989 13/22 ISOLATE KF1703 KF1776 KF1934 K F19 16 K F1935S KF1905 KF1787 K F193 3 KF1785 KF1931 KF19 917 KF18 89 8 K F19 18 KF1925 K F193 0 KF1775 PROBE 2 OLIGONUICLEOTIDE 1 =32 AMINO ACID 2 =4 AMINO ACID 3 =8 AMINO ACID PROBES REPEAT PROBE REPEAT PROBE REPEAT PROBE Fig.8. SUBSTITUTE SKEET 0:06638 -1DEC 1989 14/22 Mr x Mr x 63 69 1 23 1 23 4 Fig. 9. Fig. 9a. Mr x 10-3 39 Mr x -97 -67 -43 31 21 1 2 3 4 Fig. I SUBSTITUTE SKEET I D DEC 1989 15/22 ABSORBANCE (414nm) AT 1 -1000 DILUT/Ok RIB! ADJUVANT FCA /IF/A PREBLEED RESPONSE TO RESPONSE 703 1sf DOSE 2nd DOSE ABSORBANCE (414nm) AT 1: 10,000 DILUTION 2-51 12-5 160 161 400 162 389 394 RIB1 ADJUVANT FCA F/A PR.EBLEED RESPONSE TO RESPONSE TO lsf DOSE 2nd DOSE Fig. ii. SUBSTITUTE SKEET 4:~tlVtL) i Ult 1~f 16/22 ABSOi84NCE (414nm) AT 1:1000 DILUTV m Go w PREBLEED RESPONSE TO RESPOSE TO 1sf DOSE 2nd DOSE 161 400 RIBI ADJUVANT PREBLEED ez RESPONSE 70 1st DOSE 389 394 FCA FIA RESPONSE TO 2nd DOSE ABSORBANCE (414nm) AT 1:O,000 DILUTION 2-51 p~ p I J j 160 161. 400 RII ADJ/ANT 162 389 FCA IFIA Fg.12. SUBSTITUTE Stir--EE PREBLEED RESPONSE TO RESPONSE TO 1st DOSE 2nd DOSE ,C1L U u u RECEIVE I DEC 1989 17/22 ABSORBANCE A 1: 10,000 DILUTION 811 815 819 8 R/8I ADJUVANT PREBLEED RESPONSE TO 1st DOSE RESPOVSE 73 2nd DOSE ABSORBANCE AT 1: 1,000 DILUTION I F4.9 Ma I I 810 814 822 817 816 819 823 FCA FIA RIBI ADJIJUVANT PREBLEED RESPONSE TO RESPQVSE TO 1st DOSE 2nd DOSE ABSORBANCE AT 1: 100,000 DILUTION -A 810 814 822 817 816 819 823 FCA FIA RI/I/ ADJUVANT Sa a PREBLEED RESPONSE TO RESPWSE TO 1st DOSE 2nd DOSE Fig. 13. etc to E104 F c2 7m kv i kI s i i n ff i f vt f n IKES*.KYS-MT$t4*AN& RR "NEG N R'PNETJA Ic mkvi ktlsi inffi tvtfniKNESKYSNTFINNAYNMSIRRSMES.4PP ASGSAG3SA3GSAG3S G337 Fc27 ORST NSAST ST TNN1E SQT TTPTAAT IASGSQIRSTNSASTSTT NNGE SQTTT PTAAOT PTA I c AGGS AGOSAOSA G SAG GSAGSAGGSA3OSAGSA3SGD3N13AH ARS EST PATT T G36 Fc27 TE SIS PSPSITTMESS KFWQC TNKTDG KGEES EKQNEL NESTE EG PKAPQEPQTAENENPA Ic G327 G28 LG 3 3ih 3 2 3113 Ic A-Q--P:EOQ-AEfN-SA PTAEQTEPELS 12-] etc to G327 Fc iK-N sa t Iv I s f i a f I Ic APENKOTGQIHGHMHOSRNNHPQINTSDSQKECTOGNKENCGAATSLLNNSSNIASINKfvvtisaI~vtsfiafi< L Fig. 14. C., ISUBSTITUTE SHLl C ru"LCb1- .I 3 u J U 0)L "..CE1VED D E DC 19 89 19/22 2.000- S 1.500- CIO 0.500n 0.000 1MnIIinll MI, n E 65 67 69 71 73 75 78F 82~ K 86 9 94698 m E66 687 2 76 79 883 857 90 9395 97101103 Fig. 15 (a) 26 26 22.23 21__ 19__ 18 1 I 16 15__ 12 1211 43 2 .000 0aso0 1.00 1.500 2.0400 10-6 ILU77ON -A410 Fig. SUBSTITUTE SHEET PCT/ArU 9 0 3 8 8 RECEIVED DEC 1989 20/22 scr~, ~crm~ i 0.000 0.500 1.000 1.500 2.000 i-6 ILU77 ABSORBANCE 41Onm Fig. Ill 67 69 71 3 75 78 80 82 86 89 92 94 96 9810 104 66 68 70 72 74 76 79 81 83 85 87 90 93 95 97 101 103 Fig. 16. SUBSTITUTE SHEET E71 E87 Fc27mkv i k is i i n f f if vtifn i KNESKY-I YNM SI R R GA~~S~fNVANAPNADT IASGS Ic mkviktlsi inffifvtfniKNESKYSNTFINNAYNMSIRRSMTESNPPTGASGSAGGSAGGSAGGS Fc27 QRST NSASTSTTNNGESQTTTPTAADTIIASGSQ RSTNSASTSTTNNGESQTTT PTAAOTPTA Ic AGGSAGGSAGGSAGGSAGO.SAGGSAGGSAGGSAGGSAGSGDGNGANPGADAERSESTPATTT Fc27TES ISPS PSITTTESS KFWQCTNKTDGKGEES EKQNEL NES TEEG PKAPQEPQTAENENPA Ic TTTTTNDAEASTSTSS.N- .,.HtNAETNQANKETQNNSNVQOISCTKSNVPPTQDA DTRSPT G34 I% G28 I c AQ PEQAEN SA PTA EOTIRRE-US GS G~77 G12 G21 Fc 27AP ENKGT f~MiiRtHQt~Q~TDN K ENC0AT $SSNI ASI NKfvv I i sa t Ivis f ia f i c~ I c APENKGTGQLHGHMHGSRNNHPQNTSDSQKECTDGNKENCGAATSLLNNSSNI ASINKf vv I ita t Iv Is fiaf im FigqlZ co RECEIVED, 1DEC 1989 22/22 LIb 6 1. 2 Fig .18. SUBSTITUTjjj..:J INTERNATIONAL SEARCH REPORT International AppL tion No. PCT/AU 89/00388 I. CLASSIFICATION OF StUBJET MATTER (if several classification symbols apply, indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC Int. Cl. C07K 7/06 C07K 13/00, C12N 15/00, C07K 15/12 II. FIELDS SEARCHED finimum Documentation Searched 7 Classification System I Classification Symbols IPC VPI and PIL USPA Keyword: Plasmodi u falcipanm Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched 8 AU: IPC CO7K 7/06, C07K 13/00, C12N 15/00, C07K 15/12 Chemical Abstracts; keyword: Plasmodium falcipanru III. DOCMENTS CONSIDRED TO BE RELEVANT 9 Category* Citation of Document, with indication, where appropriate, I Relevant to I of the relevant passages 12 Claim No 13 S I AU,A, 76998/87 (Saramane Pty Ltd and The Walter and Eliza Hll 1-11, 13-18 Y Institute of Medical Research) 17 March 1588 (17.03.88) 12 See claims and page 3, line 20 page 4, line 31 X Molecular and Biochemical Parasitology, Vol 28 No 1, 1988 1-11, 14-18 Y (Elsevier N.Y) Epping et al. "An epitope recognised b? iLhibitory 12 mcnoclanal antibody that reacts with a 51 Kilodaltcn merozoite surface antigen in Plamodium falciparum." See pages 1-10 especially page 1, colum 2, page 6, coluun 2 page 7 CONTINUED Special categories of cited documents: 10 Later document published after the international filing date or priority date A" document defining the general state of the and not in conflict with the application but art which is not considered to be of cited to understand the principle or theory particular relevance underlying the invention earlier document but published on or document of particular relevance: the after the international filing date claimed invention cannot be considered novel document which may throw doubts on priority or cannot be considered to involve an claim(s) or which is cited to establish the inventive step publication date of another citation or document of particular relevance; the other special reason (as specified) claimed invention cannot be considered to "0 document referring to an oral disclosure, involve an inventive step when the document use, exhibition or other means is combined with one or more other such document published prior to the documents, such combination being obvious to international filing date but later than a person skilled in the art. the priority date claimed document member of the same patent family IV. CERICATION Date of the Actual Completion of the Date of Mailing of this International International Search I Search Report 7 DCE ER 1989 (07.12.89) 1 13 of uthi International Searching Authority j Signature of Authorized Officer Australian Patent Office ";~t~ji~e Form PCT/ISA/210 (second sheet) (January 1985) International App'*,'ation No. PCr/hU 89/00388 M MR~ flMMIkATION CrN~TNM~ FWM~ THE SEMMN MT X Molecular and Biochemical Parasitology, Vol 32, No 1 1989 2 I C-lsvier Claric et al., '46-53 Kilodalton glycoprortein fromI the surface of Plasmodium falcipan= merozoites." See pages 15-24 especialy Coun2 page 2.3. XP jUS,A, 4835529 (Scrimos Clinic and Research Foundation) 1 lMay 1989 (30.05.89) See claim. I A JilS, 4767622 (University of IlIlinois) 30 August 1988 (30.08.89) See whole docismnt. IV. OBSMATIONS ZXRE CERTAIN aAfl WME FDWT UN7A4RADL= I This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: Claim numbers because they relate to subject matter not required to be searched by this Authority, namely: Claim numbers because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out. specificaLLyt LEI2 Claim numbers because they are dependent claims and are not drafted in accordance with the second and third sentences of PCT Rule 6.4 V1. OBSERVATIONS WHR~E UNITY OF INVENTION IS LAt 2 IThis International Searching Authority found multiple inventions in this international application Ias follows: I1.[ 3 As aLL required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the international application. 3 As only some of the required additionaC search fees were timely paid by the applicant, this international search report covers only those claims of the international application for which fees were paid, specifically claims: I3.[ No required additional search fees were timely paid by the applicant. Consequently, this Iinternational search report is restricted to. the invention f irst mentioned in the claims; Iit is covered by claim numbers: 2 As all searchable claims could be searched without effort justifying an additional fee, Ithe international Searching Authority did not invite payment of any additional fee. Remark on Protest I The additional sear~ch fees were accompanied by applicant's protest. [3 No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (supplemental sheet (January 1985)