NZ221115A - An antigen of plasmodium falciparum, antibodies to it, antigenic fragments and pharmaceutical compositions - Google Patents

Description

221 1 1 Priority M Complete Specification Fi!ed:0.\7. ^ Class: ff-j \ r?.fLj.Q. . qpn VC7./.0 .u ^ w.s/®; AWA^fJ/SSl^^ Publication Date: P.O. Journs?, !^r .... ; No.: Date: NEW ZEALAND PATENTS ACT. 1953 IV S oif . 2 1AUGI987' f COMPLETE SPECIFICATION "SMALL MOLECULAR WEIGHT ANTIGEN OF PLASMODIUM FALCIPARUM" %/Wc, SARAMANE PTY LTD, a company incorporated under the laws of the State of Victoria, Australia, of care of The Walter and Eliza Hall Institute of Medical Research, of Royal Parade, Parkville, in the State of Victoria, Commonwealth of Australia, Australia, hereby declare the invention for which £ / we pray that a patent may be granted to rSHB^us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (Followed by page la) 1 ,A 221 1 15 SMALL MOLECULAR WEIGHT ANTIGEN OF PLASMODIUM FALCIPARUM This invention relates to the identification of antigens of the asexual blood stages of Plasmodium falciparum 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 immunizing, diagnostic and treatment methods. other vertebrates, is caused by a unicellular protozoan of the genus Plasmodium and is transmitted by the bite of female Anopheline mosquitoes. Among the four species which infect man, Plasmodium falciparum is by far the most lethal. The pathological consequences 10 of malaria, characterised by febrile paroxysms, anemia, splenomegaly and debilitation, are due to the development and proliferation of asexual blood stages in the vertebrate host.

As is well known, malaria, a widespread disease of man and For most of its life cycle in man, Plasmodium falciparum is 2 22 1 1 1 5 located inside its host cell with the exception of two stages of a brief extracellular existence, the sporozoite and the free merozoite. A number of plasmodial antigens have been identified which appear to be associated with membranes of the parasite or of the infected host 5 cell. Considerable attention has been directed to the sporozoite surface membrane (1), the merozoite surface membrane (2,3) and the plasma membrane of the parasitized erythrocyte (4). One of the intriguing events of the invasion process of the host erythrocyte by the merozoite is the formation of the parasitophorous vacuole. The 10 membrane surrounding the vacuole is a simple membrane bilayer, almost devoid of intramembranous particles (IMPs) at the early stage of infection (5). It has been suggested that components both of the red blood cell membrane and of the invading parasite (merozoite coat and/or rhoptry material) make up the parasitophorous vacuole membrane 15 (6). In the course of schizogony, morphological changes occur in the vacuole membrane such that proteins presumably synthesized by the parrasite become inserted therein. It has long been proposed that the parasitophorous vacuole contains antigenic material. Scaife et.al. (7) have suggested that the Ag5.1 is located within or close to the 2 0 vacuole membrane, while it appears that S antigens accumulate within the vacuole (8,9). investigation of malarial antigens capable of eliciting a protective response. Two approaches have been used to identify such antigens.

One is to compare the antigens recognised by antibodies in sera from individuals exposed to malaria, and correlate particular antigens recognised with the degree of protective immunity exhibited by the sera. A variation on this approach involves the use of human immune sera in the characterisation of antigens cloned in E.coli using recombinant-DNA techniques. The second approach is to use hybridoma technology to produce murine monoclonal antibodies (MAB) which inhibit the growth of malaria in vitro then to identify the corresponding antigen. This approach has led to the production of inhibitory antibodies directed against the sporozoites and asexual and sexual The quest for a vaccine against malaria requires the //* 3 221 blood forms of several species ("Recent Progress in the Development of Malaria vaccines: Memorandum from WHO Meeting". Bulletin of the World Health Organistion §2 (5), 7150727 (1984)).

Holder & Freeman (2 5) have been able to use single antigens of Plasmodium yoelii identified by monoclonal antibodies as vaccines in mice to elicit immunity. In this case the antigen used was purified from infected cells. While it may be possible to prepare sufficient antigen for vaccine trials from cells infected with the human species of malaria, large scale production of vaccines will probably require recombinant DNA technology. This places additional constraints upon the antigen, e.g. it would be difficult to prepare a vaccine based upon a polysaccharide antigen by this route. Thus, protein antigens are more attractive from this viewpoint.

Monoclonal antibodies directed against defined parasitic antigens have been shown to inhibit the development of erythrocytic stages of Plasmodium falciparum in vitro. Perrin et.al. (26), and to prevent release of the merozoites or prevent invasion of the red blood cells by merozoites (in accordance with the present invention).

A method for the identification of antigens of the asexual blood stages of the Plasmodium falciparum parasite which can be used to generate antibodies which are able to inhibit the growth of the parasite comprises: (a) growing Plasmodium falciparum isolates In vitro thereby producing erythrocytes infected with the Plasmodium falciparum parasite; (b) purifying these erythrocytes; (c) immunising mice with the purified erythrocytes; (d) fusing spleen cells from the immunised mice with a mouse myeloma cell line to produce hybridomas; (e) testing the monoclonal antibodies produced by the hybridomas to identify those hybridoma lines which secrete monoclonal antibodies against the Plasmodium falciparum parasite; (f) characterising the monoclonal antiparasite antibodies; (g) preparing mouse ascites fluid by injecting mice 4 22 1 1 1 K > O fdK&i.i intraperitoneally with hybridoma cells secreting monoclonal antiparasite antibodies; (h) extracting and purifying monoclonal antibodies from these ascites fluids; (i) adding the purified antibodies to Plasmodium falciparum cultures in vitro and subsequently examining the cultures for a decrease in the multiplication rate of the parasites; (j) characterising the antigen recognised by each inhibitory monoclonal antibody; (k) identifying the cellular distribution of the antigen and the stage of parasite maturation during which it is present.

According to the present invention, there is provided a small 4 molecular weight antigen of the asexual blood stages of Plasmodium falciparum which is characterised by: (i) having an apparent molecular weight in the range of approximately 15 kD and 19 kD; (ii) not showing significant glycosylation by galactose or glucosamine labelling, but being acylated by myristic acid; (iii) being associated with the parasitophorous vacuole membrane 20 and with inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell; and (iv) being recognised by monoclonal antibodies against the asexual blood stages of P.falciparum which inhibit parasite growth in vitro; or an antigenic fragment thereof.

Preferably, the antigen is the antigen QF116 described in detail herein, or an antigenic fragment thereof.

As the small molecular weight antigen described herein is closely associated with both the parasitophorous vacuole membrane and 3 0 vesicles as well as cytoplasmic clefts, it could conceivably be involved in the traffic of proteins.

In another aspect, this invention provides a hybrid cell line which produces an antibody which is specific for the small molecular weight antigen described above, as well as an antibody produced by t N rx o\v ^ \2 2 f AUG 1987 n jj 25 30 JP5—- 21 1.1 < ^ such a hybrid cell line.

Preferably, the antibody is an IgGl, monoclonal antibody, more preferably it is the monoclonal antibody 8E7/55 described in detail herein. Monoclonal antibody 8E7/55 is produced by the hybrid cell line deposited at the European Collection of Animal Cell Cultures, Porton Down, Salisbury, England, on July 10, 1987 under No. 87071009 . - Monoclonal antibody 8E7/55 specifically recognises antigen QF116 as described below.

Monoclonal antibodies of the present invention are characterised in that they are inhibitory, in that they either prevent release of merozoites into the blood stream of an infected individual, prevent these merozoites from invading the red blood cells of such an individual, and prevent intracellular development of parasites in the red blood cells of such an individual.

In another aspect, the present invention relates to an antigen i_ of P.falciparum which is characterised by inclusion of the amino acid sequence Asn-Leu-Val-Ser-Glu-Pro (NLVSGP), or an antigenically active related sequence derived therefrom.

The invention also provides a synthetic peptide comprising an antigenic fragment having an amino acid sequence which comprises or includes the sequence Asn-Leu-Val-Ser-Glu-Pro (NSVSGP), or an antigenically active related sequence derived therefrom by amino acid deletion and/or substitution.

The invention further provides a method of inhibiting the development of asexual blood stages of Plasmodium falciparum which method comprises exposing the blood stages of the parasite to antibodies according to the present invention.

The invention also provides a method for passively immunising a host against Plasmodium falciparum which method comprises administering to the host antibodies according to the present invention.

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. Preferably the antigen or fragment is one recognised by at least one monoclonal antibody according to the invention. More preferably the antigen or fragment is recognised by 8E7/55. Most preferably the antigen is QF116 or an NEW ZEALAND PATENT OFFICE 17MAYI990 received 6 221 1 15 N, antigenic fragment thereof.

The invention provides a vaccine comprising an antigen according to the present invention, preferably antigen QF116, or an antigenic fragment thereof, a pharmaceutically acceptable carrier or ^ diluent and optionally an adjuvant. The invention also provides a passive vaccine comprising antibodies according to the invention, preferably monoclonal antibody 8E7/55, and a pharmaceutically acceptable carrier or diluent.

The invention also provides a reagent for purification of jq Plasmodium falciparum antigens comprising an antibody according to the invention.

In addition the invention provides a diagnostic reagent for « detection of Plasmodium falciparum or antigens derived therefrom comprising an antibody according to the invention.

As a result of applying the general techniques described above, a range of monoclonal antibodies have been identified as able to inhibit the growth of malaria in vitro, and one of these inhibitory monoclonal antibodies have been investigated in greater detail as outlined below. The immunisation regime and fusion leading to the 2Q production of monoclonal antibody 8E7/55 were performed essentially as described in Schofield et.al. (14) and Saul et.al. (8). A mouse was 7 immunised with 10 red cells infected with mature schizonts of the PNG isolate FCQ-27/PNG. These cells were purified from culture by the method of Saul, et.al. (12). The cells were injected intraperitoneally 25 with 0.1 ml Freund's complete adjuvant. The mice were boosted 4 weeks later by an intravenous injection of 5 x 10^ schizont infected red cells in saline. A similar boost was given after a further 2 weeks. Four days after the last injection, the spleen cells were harvested and fused. 2Q Monoclonal antibodies were purified by affinity chromatography on Protein A Sepharose (Pharmacia) and were tested for inhibition of invasion using a similar method to that described in Schofield (14) • Briefly, purified monoclonal antibodies in 1640 medium were added to synchronised parasites at ring stage and 4 1 AUG1987*// 221115 incubation of the parasites continued to allow reinvasion. [ S] 3 Methionine (3-4|iCi) or [ H]-hypoxanthine (3-4jl.Ci) was added to each well and growth of the parasite allowed to proceed to trophozoite stage. The samples were harvested, protein precipitated with ice cold 10% trichloro-acetic acid onto glass fibre filters and the filters were washed and the radioactivity remaining was measured in a scintillation counter. The class and subclass of the monoclonal antibodies were determined by immunodiffusion in agar using rabbit antisera specific for mouse IgM, IgGl, IgG2a, IgG2b and IgG3.

Isoelectric points were determined by isoelectric focussing of hybridoma culture supernatant in thin layer agarose gels. Following the focussing, the gels were fixed, washed in phosphate buffered 125 saline, soaked in I-labelled antibody to mouse Ig, washed, dried and the position of the radioactive label determined by ]_5 autoradiography.

Preliminary characterisation of the antigen (antigen QF116) recognised by the monoclonal antibody 8E7/55 as described in detail below revealed the following: Size: The antigen is detectable on Western blots of FCQ-27/PNG.with the mo'noclonal antibody described giving a band of J.9 kD. On immunoprecipitation a major band at 19 kD is detected; occasionally weak bands of higher molecular weight are seen. Immunoprecipitation 3 of antigen from parasites biosynthetically labelled with H-myristic 25 acid also gives a 19 kD band, consistent with acylation of the protein. Cellular location and stacre specificity: The antigen is detectable in all blood stages. The fluorescence appears over the whole asexual parasite, sometimes localised outside the parasite but inside the host red cell. On 30 immunoelectron microscopy at the schizont stage the antigen is associated with the parasitophorous vacuole membrane and with vesicles. Monoclonal: The antigen is recognised by the monoclonal antibody 8E7/55 which is an IgG^ class antibody with a pi of 7.05 on -isoelectric 35 focussing. - i | 2 J AUG1987 • v Z ■■■& a 2211 15 o Inhibition; The monoclonal 8E7/55 gives 30-55% inhibition at 0.06-0.6 o mg/mL ([ H]-hypoxanthine assay).

Further details of the characterisation of antigen QF116 and monoclonal antibody 8E7/55 are set out in the following Example.

EXAMPLE MATERIALS AND HETTHODS Parasite cultures. Parasite strains FCQ-27 from Papua Hew Guinea CIO3 and FCR-3K+ from West Africa were groun in synchronous cultures as previously described 10 Cll]. Parasite infected erythrocytes at different stages of development were 1 harvested on Percoll gradients [12], Monoclonal antibody. The 8E7/55 hybridoma line used in this study was derived from mice immunized with schizonts of the FCQ-27 PNG strain and was cloned by limiting dilution [133. The monoclonal antibody o£ the IgGl class used for 15 immunoelectron microscopic studies was purified from ascites fluid by affinity chromatography on Protein A-Sepharose (Pharmacia Chemicals).

Inhibition of parasite growth in vitro. Affinity purified antibody vas added in varying concentrations to quadruplicate cultures of synchronised FCQ-27 in %- well plates. The wells contained 45 pi PJ>HI 1640/TES. 10*s pooled human serum, with a 5* haematocrit and 1-2* schizont parasitemia,-and 5 fil of test antibody.

Control wells were identical in all respects except that they contained either affinity purified normal .mouse IgG or medium alone. The cultures were incubated overnight at 37'C. Following merozoite release and subsequent invasion 100 pi medium was added containing. 10* pooled human serum and supplemented uith 30 (£i ~1 3 ml [ H3hypoxanthine (Amersham). The parasites were allowed to develop for 40 h to the mature schizont stage, and harvested by pipetting well contents onto Whatman glass fibre discs which were washed in trichloroacetic acid (TCA) and ethanol. The dry discs were immersed in 3 ml toluene scintillant and counted in a Packard Scintillation Counter. .-'V* b ' 0 h % *2 1 AUG 19871 9 22 I 1 1 Affinity purification of antigen. MAb 8E7/55 was covalently linked to cyanogen bromide-activated Sepharose (Pharmacia) according to the manufacturer's instructions, and a 1* Triton X-114 extract of FCQ-27 was passed through the column. After extensive washings the bound antigen was eluted with 0.2 M glycine, pH 2.8.

I mmuno f luorescence microscopy. I FAT was performed on fixed and unfixed parasites. For the former, thin films of parasitized blood were methanol-fixed and incubated with monoclonal antibody and FITC-labeied, affinity purified goat-anti-mouse IgG as described elsewhere [14]. IFA on unfixed parasites was performed using 200 j/1 of parasite culture at a defined stage. Schizont infected erythrocytes were treated with anti-red blood cell antibody (CSL) plus complement for 60 minutes at 37'C. A two hour incubation of parasites with monoclonal antibody at 37* C was followed by a one hour incubation with FITC-labeled secondary antibody at 37" C. Parasites were gently but extensively washed with medium after each incubation step. Parasites were mounted on microscope slides and examined by fluorescence microscopy.

Immunoe 1 ectron microscopy. Immunoelectron microscopy was carried out on thin sectioned parasites at the ring or schizont stage using colloidal gold as an electron-dense marker. Samples of parasite culture were fixed with 0.5-i glutaraldehyde for 10 minutes at room temperature. Cells were washed three times in 0.1 H cacodylate buffer, dehydrated in 50% ethanol followed by 70*< ethanol. infiltrated with LR-White resin (London Resin Co. Ltd.) and polymerized at 50° C for 24 hours. Thin sections were mounted on nickel grids, rinsed with water, blocked with S'< BSA, 0.05* Tween 20 in 20 mM Tris-buffer. and incubated with monoclonal antibody for 60 minutes. One hour incubations uith secondary antibody (rabbit anti-mouse) and tertiary antibody (goat anti-rabbit, colloidal gold conjugated, 10-15mm; Janssen Pharmaceutica, Belgium) followed. Sections were washed extensively after each incubation step, before being contrasted with lead citrate and uranyl acetate and viewed with a Philips EM400 M / ■. •' o' electronmicroscope. t'-V 221115 Biosynthetic labeling studies. Malarial proteins were radiolabeled by uptake of 50 fiCi ml * [2-^H]glycine, 25 fiCi ml * [U-^C]leucine or 100 /^Ci ml 3^ C "S]methionine in RPHI medium. Glycoproteins uere labeled by incorporation of 50 fiCi ml 1 D-[6-3H]glucosamine hydrochloride or 25 jiCi ml-1 D-[U-14C]galactose.

All radiochemicals were purchased from Amersham Radiochemical Centre. Metabolic labeling with fatty acid was performed in RPHI medium losing 50 fiCi ml-1 [9,10-3 H]myristic acid coupled to BSA. Synchronous cultures at ring stage were labelled for up to 20 hours depending on the precursor used with the (~exception of [^S] methionine which was added to schizont stage cultures. Parasitized cells were washed and parasites extracted by saponin treatment (15). Protease inhibitors (5 mg ml each of pepstatin, leupeptin, chymostatin and antipaiir) were added and detergent solubilized proteins were used for immunoprecipitation.

Immunoprecipitaton. Aliquots of total parasite or Triton X-114 extracts from radiolabeled schizonts were incubated with ascites fluid of monoclonal antibody overnight at 4°C. Immune complexes were then precipitated with rabbit anti-mouse IgG/Protein A-Sepharose. The precipitate was washed in PBS/Triton and the 2q immune complex was eluted with Laemmli sample buffer as described for use in SDS-PAGE [16].

SDS-PAGE and autoradiography. Radiolabeled proteins were analysed on 12* or 15* SDS-polyacrylamde gels according to the Laemmli procedure [17]. Following 25 electrophoresis gels uere fluorographed uith Amplify (Amersham) for 30 minutes, dried and autoradiographed on Kodak XAR 5 film at -70°C.

Q o 11 221115 Immunoblotting. After separation on SDS-PAGE parasite proteins uere electrophorectically transferred onto nitrocellulose paper, immunoreacted with 125 HAb 8E7/55 and I-labeled goat anti-mouse antibody, follouing the Western blotting procedure of Towbin et.al. [18]. Human serum used in immunoblotting was affinity-purified on the recombinant protein expressed by Ag61 [27 ] .

Scanning. Scanning for antibody-reactive peptides using the predicted seguence of the polypeptide coded for by the insert of Ag61 was performed as described by Geysen et.al. [283 - Synthetic peptides were coupled via a peptide-like spacer to polyethylene rods to which a linear polymer of acrylic acid had been attached by radiation grafting. Overlapping octapeptides were synthesized consecutively using solid phase peptide synthesis. Following removal of the t-butyloxy carbonyl group from the final amino acid, peptides were acylated and de-protected to provide material of sufficient purity to ~ enable ELISA testing for the specific binding of monoclonal antibody to each octapeptide [29]• > /* jfj nl! ft 1 AUG 1987' 12 FIGURE LEGENDS 2 2 1 1 15 Fig. 1. Indirect immunofluorescence of fixed P. falciparum asexual blood stages reacted with HAb 8E7/55. Fluorescence micrograph shows ring (r). trophozoite (t) and schizont (s) stage parasites in methanol-fixed smears of isolate FCQ-27. 5 The antibody reacts with inclusions (arrows) inside the host cell of schizont r infected erythrocytes.

Fig. 2. Indirect immunofluorescence of unfixed P. falciparum asexual blood stages reacted with HAb 8E7/55. Fluorescein staining occurs on schizont stage D w 2.0 parasites (s) and ruptured schizonts (rs) ot isolate FCQ-27 (A and B) and FCR-3K+ (C).

Fig. 3. Immunoelectronmicrograph showing ring stage infected erythrocytes from P. falciparum labeled with MAb 8E7/55 and colloidal gold (lOnm) IgG complex. 15 The inset shows ring stage parasite surface labeling using 15nm gold particles. The bar equals 0.5 pm.

Fig. 4. Immunoelectronmicrograph of falciparum infected erythrocytes labeled with monoclonal antibody and colloidal gold IgG complex. Sections of schizonts 20 were reacted with MAb 8E7/S5. rabbit anti-mouse IgG and colloidal gold (10 mm) labeled goat anti-rabbit antibody. Nu. nucleus: M, merozoite: PM. plasma membrane: PVM, parasitophorous vacuole membrane: R, rhoptry; RBCM, erythrocyte plasma membrane. The bar equals lum.

© O .7£ k i ^ J J Fig. 5. Immunoelectronmicrographs of P. falciparum infected erythrocytes labeled with monoclonal antibody and colloidal gold IgG complex. Sections of infected erythrocytes were reacted with antibodies as described in Fig. 4 (A and B). schizont infected erythrocyte showing distinct separation of PVM from PM with antibody labeling restricted to the vacuole membrane: (C). Membrane bound cleft and multimembranous vesicle showing antibody labeling. fD). Dense '3^ ptibody labeling around membrane bound EDM free vesicle. The bar equals 0.5 pm. o 13 2211 1 5 Fig. 6. SDS-PAGE analysis (12*) and fluorography of amino acid labeled, detergent extracted proteins from FCR-3K+ (A) and FCQ-27 strains (B) of P. falciparum. Immunoprecipitation uith HAb 8E7/55 of parasites labeled with (a) 14 3 35 C C] leucine; (b) [ H3 glycine acid (c) [ S] methionine. Migration positions of molecular weight standards (in kD) are shown. The high molecular weight bands in c can be attributed to immunoglobulin.

Fig. 7. (A), SDS-PAGE analysis (12*) and fluorography of fatty acid labeled, detergent extracted proteins of Pj_ falciparum. Immunoprecipitation with MAb 2o 8E7/S5 of (a) myristic acid labeled FCR-3K+ strain parasites; (b) myristic acid labeled FCQ-27 strain parasites. (B), SDS-PAGE analysis (15*) and fluorography of affinity purified vacuole antigen from a FCR-3K+ strain of P. falciparum." (a). Silver stain of purified antigens, (b), Immunoblot of purified antigen 1°5 using MAb 8E7/5S and I-labeled goat anti-mouse IgG. The positions of 15 molecular weight standards (in kD) are marked.

Fig. 8. Results of scanning the predicted sequence of the polypeptide coded for by the insert of Ag61, using monoclonal antibody 8E7/55.

" ^ //V I 2 1 AUG 1987 ^ /j 14 2211 15 RESULTS jmmunof 1 uorescence with HAb 8E7/55. The reactivity of methanol-fixed ring, trophozoite and schizont infected erythrocytes of the PNG FCQ-27 and the Gambiar. FCR-3K+ isolates with MAb 8E7/55 is illustrated in Fig 1. Both isolates reacted strongly with the monoclonal antilbody at all stages, showing fluorescence around the parasite inside the infected red blood cell. The antigen recognized by the antibody was also present in inclusions located in the cytoplasm of the host cell. Similar results were obtained in the indirect fluorescence test using unfixed parasites after release by treatment with anti-erythrocyte antibody and complement, as shown in Fig 2 A-C. Thus fluorescence microscopic " examination suggests a location of the antigen either in the parasitophorous vacuole (membrane) or in the plasma membrane of the parasite. This antigen has been designated QF116.

Immunogold labeling studies. A more precise determination of the antigen site was obtained by electronmicroscopic examination. To disciminate between location of the antigen in the parasitophorous vacuole, the parasitophorous vacuole membrane (PVM) or the plasma membrane (PM). thin sections of parasite infected erythocytes were subjected to immunogold labeling. The antigen was just detectable at the ring stage located mainly around the parasite (Fig. 3).- while at the early and late schizont stages the antigen was found to be closely associated with the parasitophorous vacuole membrane, with a very lew background of non-specific gold labeling, occuring most frequently over the nucleus (Fig. 4). At this stage both membranes, the PVM and the PM. were morphologically distinctly separated (Fig. SA and B) allowing clear identificiation o£ the localization of antigen QF116 in the PVM. Dense antigen distribution was also seen in membrane bound vesicles and inclusions residing in the cytoplasm of the host cell (Fig. 5C and D), as well as close to cytoplasmic clefts.

The antigen was also localised on free membranes found associated j with intact merozoites following the rupture of schizont-infected 221115 Growth inhibition. Purified monoclonal antibody 8E7/55 inhibited the in vitro multiplication of FCQ-27 in a concentration dependent manner as determined bv Microscopic examination of parasites cultured in the presence of purified 8E7/55 antibody showed both fewer parasitized red blood cells and intracellular death of those parasites in red blood cells.

Biosvnthetic labeling with amino acids and carbohydrate. The Papua New Guinean and Gambian isolates uere cultured in vitro uith one or mere of three amino acids, glycine and leucine from the ring to schizont stage,and methionine through the schizont stage.

Schizont infected cells uere then harvested, parasites isolated and detergent soluble material immunoprecipitated uith HAb 8E7/SS. A single band of Mr- IS kD uas detected by SDS-PAGE of immunoprecipitated material using the Gambian isolate and a Mr 19 kD band uas detected using the PNG isolate (Fig. 6A and B). Immunoprecipitates of carbohydrate labeled parasite extracts did not shou significant glycosylation of the protein as judged by either galactose or glucosamine incorporation.

Hetabolic labeling uith fatty acid. While the molecule did not appear to be glycosylated it did shou acylation by myristic acid, strongly suggesting that it could be associated uith membranes. When immunoprecipitating myristic acid labeled antigen from the Gambian strain parasites uith HAB 8E7/55. a broad band of 15. kD as uell as free fatty acid and/or glycolipid could be detected in SDS- * PAGE, uhereas the FCQ-27 strain gave a 19 kD band (Fig. 7A).

Western blot transfer studies. After SDS-PAGE of detergent solubilized parasite extracts, proteins uere electrophoretically blotted onto nitrocellulose filter paper. Immunodetection by incubation uith hybridoma culture supernatant of HAb 8E7/55 and subsequently with iodinated goat-anti-mouse antibody is positive for total parasite extracts as well as for detergent soluble material giving a band at Mr 19kD. By contrast, immunoblotting using a human serumVaffinity-purified on the recombinant protein expressed by Ag6i gave a band of Mr 23kD. Using affinity purified antigen a 15kD band was identified by blotting in the case of the FCR-3K+---;^ C H]hypoxanthine uptake. 55*; inhibition uas achieved at Q.t. mg ml-' antibody. strain as shown in Fig.7B. 16 221 1 1 Identification of epitope recognised bv 8E7/55. On scanning of the predicted amino acid sequence of the polypeptide coded for by the „ "insert of Ag61, three octapeptides at the C-terminal end of the sequence produced strong positive ELISA signals using 8E7/55 (Fig.8). The epitope recognised by this monoclonal antibody is contained within the sequence common to these three octapeptides, namely NLVSGP, and may be a sequence derived from the common sequence, for example VSGP.

DISCUSSION Thus far the chemical characteristics of the antigen described in this study mark it a3 a membrane bound protein of small apparent molecular ueight varying in size between strains. Its location can be attributed to the parasitophorous vacuole membrane of P. falciparum infected erythrocytes as veil as clefts and vesicles uithin the cytoplasm of the host cell as shown by immunofluorescence and immunoelectron microscopy. While the molecule is . apparently not glycosylated, it does contain myristic acid in its lipid moiety. The finding that a number of P. falciparum proteins aire myristyiated poses the question as to the purpose of this kind of protein modification. Me^-brane proteins most commonly are anchored in the membrane by a transmembrane spanning hydrophobic amino acid sequence. Its replacement by a complex containing fatty acid, phosphatidyl choline, and carbohydrate has been found in the variant surface antigen (VSG) of African trypanosomes [19]. The merozoite surface coat of P^ falciparum may be constructed and function in some respects in a similar way to the trypanosome coat. The major mferozoite surface glycoprotein, which contains both carbohydrate and myristic acid, may show a VSG-like type of linkage of the lipid tail to the peptide chain [16], thus enabling the parasite to shed its coat during invasion of the host cell, possibly by means of an endogenous phopholipase. In contrast, this parasitophorous vacuole membrane antigen, associated uith a membrane inside the host cell, evidently lacks the carbohydrate moiety, and its fatty acid residue may be bound to the peptide ^backbone in an ester or amide linkage as described fcr tumour virus proteins[20]. o\ TTi 2211 15 The scan of the Ag61 insert sequence shows that antibody 8E7/55 can bind to an epitope coded for by this sequence. The Ag61 sequence from FCQ-27/PNG is almost identical to the Ag5.1 sequence reported by Hope et.al. [34] from the K1 isolate of P.falciparum, and on the basis of the epitope scanning Ag5.1 would also contain this epitope. However, the predominant antigen according to the present invention is smaller than that reported for the native antigen in FCQ-27/PNG corresponding to the Ag61 clone sequence (19kD vs. 23kD) .

This difference in size is real since immunoblotting of parasite extracts with human antibodies purified on the Ag61 clone product gives the reported size of 23kD in the system described herein.. The size of the predominant antigen recognised by 8E7/55 in the FCR-3K+ isolate is even smaller than that in the FCQ-27/PNG isolate, and is . . much smaller than that of the Ag5.1 antigen reported for any isolate."Taken together, these data show that although the 8E7/55 monoclonal antibody could theoretically bind to the Ag5.1 protein, the dominant antigen (designated QF116 herein) recognised in parasite extracts is distinctly smaller.

The results from the growth inhibition experiments suggest that antibody 8E7/55 may kill parasites by two mechanisms. Firstly, by aggregating membranes released during rupture of the schizont-infected red blood cells thereby blocking invasion of merozoites. Secondly, the antibody has an unexpected direct effect on intraerythrocytic parasites resulting in death. A plausible mechanisum is the interference of trafficking of membranous structures from the parasitophorous vacuole to the surface, and vice versa.

Whether the growth inhibition is caused by binding of 8E7/55 monoclonal antibody to Ag5.1, QF116 or to some other as yet undescribed antigen, the investigation of the specificity of this monoclonal antibody shows that such inhibition will depend upon the presence of the sequence NLVSGP, or some closely related antigenically active sequence. '2 2 1 AUG 1987- 18 221 1 15 References 1. Nardin. E.H.. Nus3enzueig, V., Hussenzweig, R.S.. Collins. W.E..

Harinasuta. K.T.. Tapchaisri, P. and Chomcham. Y. (1982) Circumspcrczoite proteins of human malaria parasites Plasmodium falciparum and Plasmodium vivax. J. Exp. Med. 156, 20-30. 2. Heidrich, H.G., Strych, W. and Mrema, J.E.K. (1983) Identification of surface and internal antigens from spontaneously released Plasmodium falciparum merozoites by radio-iodination and metabolic labelling, Z. Parasiten k. 69, 71S-72S. 3. Freeman, P.R. and Holder, A.A. (1983) Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000 mol wt form expressed on the surface of Plasmodium falciparum merozoites. J. Exp. Med. 158. 1647-1653. 4. Brown, G.V., Culvenor, J.G., Creuther, P.E., Bianco, A.E.. Coppel, R.L., Saint, R.B., Stahl, H.D., Kemp, D.J. and Anders. R.F. (1985) Localisation of the ring-infected erythrocyte surf ace'antigen (RESA) of Plasmodium falciparum in merozoites and ring infected erythrocytes. J. Exp. Hed. 162. 774-779. ' i . McLaren, D.J., Bannister, L., Trigg, P. and Butcher. G. (1979) Freeze fracture studies on the interaction between the malaria parasite and host erythrocyte in Plasmodium knowlesi infections. Parasitology 79. 125-139. 6. Sherman, J.W. (1985) Membrane structure and function of malaria parasites and the infected erythrocyte. Parasitology 91, 609-645. - m O- 19 221 1 15 7. Hope. I.A., Hall, R., Simmons. D.L.I., Hyde. J.E. and Scaife. 3.G. C19S4! Evidence for immunological cross-reaction betueen sporozoites and blocd stages of a human malaria parasite. Mature (London) 308, 191-193. 8. Saul, A.. Myler, P.. Schofield. L. and Kidson, C. (1984) A high molecular weight antigen in Plasmodium falciparum recognized by inhibitory monoclonal antibodies. Parasite Immunology 6, 39-50. 9. Saul, A., Cooper, J.. Ingram, L., Anders. R.F. and Broun, G.V. (1985) Invasion of erythrocytes in vivo by Plasmodium falciparum can be inhibited by monoclonal antibody against an S-antigen. Parasite Immunology 7, 587-593.

. Chen, P., Lamont, G. Elliot, T. Kidson, C., Broun, G.. Mitchell, G., Stace, J. and Alpers, M. (1980) Plasmodium falciparum strains from Papua New . Guinea: culture characteristics and drug sensitivity. Southeast Asian J. Trop. Med. Public Health 11; 43,5-440. 11. Lambros, C. and Vanderberg, J.P. (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65. 413-420. 12. Saul, A., Myler, P., Elliot, T. and Kidson, C. (1982) Purification of mature schizonts of Plasmodium falciparum on colloidal silica gradients.

Bull. WHO 60, 755-759. 13. Anders, R.F., Broun. G.V. and Edwards, A. (1983) Characterisation of an S antigen synthesized by several isolates of Plasmodium falciparum. Proc.

Natl. Acad. Sci. U.S.A. 80, 6652-6656. ^ . -V % 2 ■\ 'AUG1987 9 221 1 15 Schofield, L.. Saul, A,, Myler, P. and Kidson, C. (19S2) Antigenic differences between isolates of Plasmodium falciparum demonstrated by monoclonal antibodies. Infect. Immun. 38, 893-897.

Schofield, L., Bushell, G.R.. Cooper, J.A., Saul. A., Upcroft, J.A. and Kidson, C. (1986) A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies. Mol. Biochem. Parasitol. 18, 183-195.

Haldar, K., Ferguson. M.A.J, and Cross. G.A.M. (1985) Acylation of a "" Plasmodium falciparum merozoite surface antigen via sn-l,2-diacylglycerol. J. Biol. Chem. 260, 4972-4974.

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Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer o£ proteins from polyacrylamide gels to nitrocellulose sheets: procedure.and some applications. Proc. Natl. Acad. Aci. U.S.A. 76, 4350-4354.

Ferguson, M.A.J, and Cross, G.A.M. (1984) Myristylation of the membrane form of a Trypanosoma brucei variant surface glycoprotein. J. Biol. Chem. 259, 3011-3015.

Pellman, D., Garber. E.A., Cross, F.R. and Hanafusa, H. (1985) An H-terminal peptide from p 60 " can direct myristylation and plasma membrane localisation uhen fused to heterologons proteins. Nature'314, 374-376. 221 1 1 5 21. Aikaua, H., Miller, L.H. and Rabbage, J. (1975) Careola Vesicle complexes in the plasma lemma of erythrocytes infected by Plasmodium viva:-: and P. cvnomolai Am. J. Pathol. 79, 285-300. 22. Aikaua, M. (1971) Plasmodium: The fine structure of malarial parasites. Exp. Parasitol. 30, 284-320. .23. Aikaua, M., Uni, Y., Aridritis, A.T. and Houard. R.J. (1986). Membrane associated electron-dense material of the asexual stages o? Flasxcdiuz: falciparum evidence for movement from the intracellular parasite to the erythrocyte membrane. Am. J. Trop. Med. Hyg. 35. 30-36. 24. Elford, B.C., Haynes, J.D., Chulay, J.D. and Wilson, R.J.M. (1985) - Selective stage-specific changes in the permability to small hvdrophilic solutes of human erythrocytes infected uith Plasmodium falciparum. Mol. Biochem. Parasitol. 16, 43-60. 22111 5 22 o . Holder, A.A. and Freeman, R.R. (1981). Immunization against blood-stage rodent malaria using purified parasite antigen. Nature (London) 294, 361. 26. Perrin, L.H., Ramirex, E., Lambert, P.H. and Miescher, P.A. (1981). Inhibition of P.falciparum growth in human erythrocytes by monoclonal antibodies. Nature (London) 289. 301. 27. Coppel, R.L., Favaloro, J.M., Crewther, P.E., Burkott, T.R., Bianco, A.E., Stahl, H.D., Kemp, D.J., Anders, R.F. and Brown, G.V. (1985). A blood stage antigen of Plasmodium falciparum shares determinants with the sporozoite coat protein. Proc. Natl.Acad.Sci.USA £2, 5121-5125. .28. Geysen, H.M., Meloen, R.H., and Barteling, S.J. (1984).

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Claims (13)

WHAT WE CLAIM IS:

1. A small molecular weight antigen, in substantially pure form of the asexual blood stages of Plasmodium falciparum whicn is characterised by: (i) having an apparent molecular weight in the range of substantially 15 kD to 19 kD as detected by SDS-PAGE; (ii) not showing significant glycosylation by galactose or glucosamine labelling, but being acylated by myristic acid; (iii) being associated with the parasitophorous vacuole membrane and with inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell; and (iv) being recognised by monoclonal antibodies against the asexual blood stages of P.falciparum which inhibit parasite growth in vitro; or an antigenic fragment thereof.

2. An antigen according to.claim 1, in substantially pure form, which is antigen QF116 described herein; or an antigen fragment tnereof.

3. A hybrid cell line which produces an antibody which is specific for the small molecular weight antigen according to claim 1.

4. A hybrid cell line according to claim 3 which is the cell line deposited under ECACC No. 87071009.

5. An antibody produced by a hybrid cell line according to claim 3.

6. An antibody according to claim 5, which is the monoclonal antibody 8E7/55 described herein.

7. An antigen —— of Plasmodium falciparum , in substantially pure form, which is characterised by the inclusion of the amino acid sequence Asn-Leu-Val-Ser-Glu-Pro (NLVSGP), or an antigenically active related sequence derived therefrom by amino acid deletion and/or substitution, i. m 24 22 I 1 ( S* antigenic fragment thereof.

8. A passive vaccine composition comprising an antibody according to claim 5/ and a pharmaceutically acceptable carrier or diluent. K

9. A composition according to claim 8 , wherein the antibody is monoclonal antibody 8E7/55. u O NEW ZEALAND PATENT OFflCE 17MAYIW0 RECEIVED

10. A vaccine composition comprising an antigen according to claim 1 or claim 7, or an antigenic fragment thereof, and a pharmaceutically acceptable carrier or diluent.

11. A composition according to claim 1^ , further comprising an adjuvant.

12. A composition according to claim 1Q, wherein the antigen is antigen QF116, or an antigenic fragment thereof.

13. A synthetic peptide comprising an antigenic fragment having an amino acid sequence which comprises or includes the sequence Asn-Leu-Val-Ser-Glu-Pro (NLVSGP), or an antigenically active related sequence derived therefrom, by amino acid deletion and/or substitution. " h.gWVi-