GB2099300A

GB2099300A - Antigens from parasites of the genus plasmodium

Info

Publication number: GB2099300A
Application number: GB8214624A
Authority: GB
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1981-05-21
Filing date: 1982-05-19
Publication date: 1982-12-08
Also published as: GB2099300B

Abstract

Protection inducing antigens of parasites of the genus Plasmodium are described. The antigens have an apparent molecular weight of 1.8 to 2.5 x 10<5> and are associated with the membranes of the erythrocytic schizont forms of the parasite. The antigens may be incorporated into vaccines and used for the inducing of immunity into susceptible vertebrate hosts including humans. Methods for the preparation of the antigens are also described.

Description

SPECIFICATION Protozoal antigen The present invention relates to antigenic mater ial, to vaccines containing it and to the use thereof to provide immunity to malaria. The malaria parasites are protozoa belonging to the genus Plasmodium .

Part of their complex life cycle involves cyclic asexial replication within the erythrocytes of the vertebrate host, giving rise to the symptoms of the disease known as malaria. The vertebrate host may, in turn, exhibit a protective immune response which is effective against the blood-borne parasites. It is implied, therefore, that the blood forms synthesize antigens which elicit the production of specific, protective antibodies in the infected or immunized host During the asexual erythrocytic cycle of replication, free parasites (merozoites) recognize and attach specifically to the surface of erythrocytes, and then invade them byinvagination of the plasma mem brane. The infected erythrocyte is known as the trophozoite. During the intadellular differentiation of the parasite the nucleus undergoes repeated divisions, to form the schizont.Nuclear division is followed by segmentation of the cytoplasm to form a number of uninucleate intraerythrocytic merozoites.

Upon lysis of the schizont membranes, the merozoites are released into the plasma and may attach to, and invade, fresh host erythrocytes.

Immunity to malaria is believed to be mediated by antibodies specific for parasite antigens associated with the schizont and/or merozoite forms of the parasite (see for example Cohen, Proc. R. Soc. London B, 1978,203; 323-345; Freeman and Parish, Exp.

Parasitol, 1981,52, 18-24).

A number of parasite antigens with the schizont membrane have been detected by crossed immunoelectrophoresis of solubilised schizont material against hyperimmune serum (see for example Deans, Dennis and Cohen, Parasitology, 1978,77 333-344; Schmidt-Ullrich, Wallach and Lightholder, J. exp. Med. 1979, 150,86-99). In particular the latter group have detected 3 antigens from Plasmodium knowlesi schizont membrane of molecular weight 65,000,90,000 and 125,000. These antigens are precipitated byP.knowlesi and P. falciparum immune serum.The available data on P.knowlesi and P.falciparum do not allow definite conclusions asto the developmental stage(s) at which parasite specific antigens are immonogenic or whether the antigens detected are the ones involved in protective immunity (Schmidt-Ullrich et a/ "The Host-lnvader Interplay", 1980, H. Van den Bossche ed., Elsevier/North Holland, Amsterdam, 117-120).

Schizont antigens ofP.falciparum have also been described (Kilejian, Proc. Nat. Acad. Sci. 1980,77, 3695-3699; J. exp. Med., 1980, 151,1534-1538; Perrin et.al. Trans. Roy, Soc. Trop. Med. Hyg. 1981,75, 163-165). None of these antigens have been purified or shown to be protective antigens.

Antigens derived from schizont membranes and having moleular weights of up to 200,000 have also been referred to in the literature (Freeman et alThe Host-lnvader Interplay, H. Van den Bossche, Ed., Elsevier/North Holland Biochemical Press 1980, 121-124) but the only antibody described, a monoclonal antibody specific for these antigens was shown not to provide passive immunity.

We have now surprisingly found that an antigen recognised by the aforementioned antibody and originating from a schizont membrane is capable of generating protection against malaria and such an antigen associated with schizont forms of Plasmodium parasites has now been isolated and defined.

The antigen so identified is a protection-inducing proteinaceous antigen of parasites of the genus Plasmodium having the following characteristics: (i) a molecular weight in the range of 1.8 x 105 to 2.5x105; (ii) associated with the membranes of the eryt hrocytic schizont form of the parasite; and merozoite forms of the parasites; and (iii) is processed intraerythrocytically to discrete fragments possessing the same antigenic properties, and in which the antigen, or the discrete fragments thereof, are associated with the surface membrane of the merozite form of the parasite, and functional derivatives thereof.

These antigenic proteins are known to occur in a numberofmurine malaria protozoa such asPlasmodium yoelii, P. berghei and P. vinckei spp. and in primate malaria parasites, in particularP. fal- ciparum.

By "molecular weight" is meant the apparent molecular weight as determined by polyacrylamide gel electrophoresis and standard molecular weight markers. The molecular weight of the antigenic proteins of the invention may thus be conveniently determined by the techniques described by U.K.

Laemmli, Nature, 1970,227, 680485. Convenient standard molecular weight markers include, for example, spectrin heterodimer (2.2 x 105 molecular weight and 2.4 x105 molecular weight).

The term 'associated' as used herein refers not only to proteinaceous antigenic material originating from the schizont or merozoite forms of malaria parasites but to antigenically identical material of similar or identical amino acid sequence deriving from any other source.

The antigens of the present invention are synthesised only during the later stages of the parasite's intracellular development. Thus forP. falciparum the onset of synthesis of this antigen co-incides with the start of schizogeny and continue right through until the end of the intracellular stage.

It has been found that a characteristic of the parent antigen is that, no matter the parasite from which it derives, it is processed intraerythrocyticallyto dis creetfragments of lower molecularweightwhich possess the same antigenic properties. The moleculay weight of these fragments varies with that of the The drawings originally filed were informal and the print here reproduced is taken from a later filed formal copy.

particular parent antigen but it has been found that the major fragments have molecularweightswhich are respectively 0.3 - 0.4 x 105; 0.7 - 0.8 x 105; 1.0 - 1.4 X 104 lower than that of the parent antigen.

In synchronised cultures of P. falciparum the pro cessing is not a co-translational event, but appears to co-incide with merozoite maturation or release, at the end of the intraerthrocytic development cycle. It is likely that discrete fragment(s) ratherthan the antigen itself are on the surface of merozoites.

It will be appreciated by one skilled in the art that the benefit of the immunogenic properties of the antigens above will be obtained not only from the parent antigens themselves but also immunogenic fragments thereof, including those generated intraerythrocytical ly, and materials incorporating the parent antigens or fragments thereof. All such materials are referred to herein as "functional dervatives".

The antigen proteins may be prepared by any method known in the art forth preparation of such methods comprise either isolation of theantigenic protein from the parasite or chemically or biologically reproducing the antigenic protein.

In one such method the antigenic proteins may be isolated from the schizontformsofthe parasite by means of monoclonal antibodies specific for the antigens of the invention. The technique of antigen separation by means of monoclonal antibodies has not previously been applied to the purification of malaria antigens.

There is thus provided a method for the preparation of an antigenic protein as defined herein which comprises the steps ofr (i) solubilising erythrocytes containing the schiz one forms of a Plasmodium parasite; (ii) contacting the solubilised material with a monoclonal antibody specific for the desired antigenic protein to provide an antibody anti gen complex; and (iii) recovering the antigenic protein from the anti body - antigen complex.

There is also provided an antigenic protein as defined herein when prepared by the above defined method. The schizonts may be solubilised by any method known in the art for effecting such solubilisation. Suitable conditions which solubilise parasite material without extensive proteolysis and denaturation are employed. In particular they may be solubilised by contacting them with a detergent, which may be of the ionic or non-ionic type although non-ionic detergents are preferred. Examples of such detergents include Nonidet P40, Triton X-100, Brij 99 (registered Trade Marks, manufactured respectively by Shell, Rohm and Haas and ICI) and a polyoxyethylene (12) tridecyl ether detergent as known Renex30 (Registered Trade Mark, manufactured by Honeywell Atlas Limited). Detergent is added to a final concentration of between 0.01% and 5% viv.

Monoclonal antibodies for use in the method maybe prepared by any method known in the art for their production (see for example Milstein, Scientific American 1980,243 (4), 56-64). In such a method a mouse is immunised against the parasite concerned, in this case a malaria parasite for example by infec tion with a rodent malaria parasite. Lymphocytes, each ofwhich will have the independent capacity to make an antibody that recognises a different antigen determinant, are then isolated and fused with mouse myeloma cells to provide a "hybridoma". Each hyb ridoma will be capable of expressing the antibody of its parent lymphoyte and of being cioned.Screening of individual hybridomas provides one or more cell lines expressing the antibody, a monoclonal anti body, specific for the antigenic determinant of inter est and the cell-line then used to generate large quantities of the monoclonal antibody which may then be used to separate and purifythe antigenic determinant of interest. Antibodies specific for the antigens defined herein may be readily identified by simple, well known testing procedures.

The monoclonal antibody is suitable bonded to an inert support material, for example Sepharose (Trade Mark), and the solubilised material passed through the support containing antibody. This may conveniently be effected by means of a column.

The antigenic protein may be recovered from the antibody-antigen complex by disruption ofthe complex. The conditions for effecting this are well known in the art. Suitable conditions which preserve the immunogenicity of the protein include washing with diethylene at high pH, for example pH 11.5 in the presence of a suitable detergent.

The antigens described above may be incorporated into a vaccine for inducing immunity to Malaria in susceptible veterbrate hosts at risk of becoming infected by parasites of the genusPlasmodium. For this purpose the antigen proteins may be presented in association with a pharmaceutically acceptable carrier. The antigenic proteins may be used singly or in combination with other functional derivatives thereof or with other proteins which will provide protection against malaria.

In a further aspect there is provided a vaccine for inducing immunity to Malaria which comprises an antigen as hereinbefore defined or a functional derivative thereof in association with a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers, in this intance, are liquid media suitable for use as vehicles to introduce the antigen into the patient. An example of such a carrier is saline solution. The antigenic protein may be in solution or suspended as a solid in the carrier, or it may be soiubilised by the addition of pharmaceutically acceptable detergent.

The vaccine may also comprise an adjuvantfor stimulating the immune response and thereby enhancing the effect of the vaccine. Convenient adjuvants for use in the present invention include Freunds complete adjuvant and more particularly, saponin, Cor'nebactreium parvum (coparvax) and aluminium hydroxide our a mixture of these or other known adjuvants.

Conveniently the vaccines are formulated to contain a final concentration of antigenic protein in the range of from 0.2 to 5 mg/ml, preferably 0.5 to 2 mg/ml, most preferably 1 mg/ml. After formulation the vaccine may be incorpoated into a sterile con tainerwhich is then sealed and stored at a low temperature, for example 4"C, or may be freeze dried.

In order to induce immunity in veterbrate hosts to malaria one or more doses of the vaccine suitably formulated may be administered. It is recommended that each dose of 0.1 to 2 ml preferably 0.2 to 1 ml, most preferably 0.5 ml of vaccine.

There is in a further aspect provided a method for inducing immunity to malaria in susceptible veterbrate hosts, comprising the administration of an effective amount of a vaccine, as hereinbefore defined, to the host The vaccines may be administered by any conventional methodforthe administration of vaccines including oral and parenteral (eg. subcutaneous or intramuscular) injection. The treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.

The following Examples serve to illustrate the invention but are not intended to limit it in any way.

EXAMPLE 1 Derivation ofHybridoma Line for P.

yoelii antigen Spleen cells from two P. yoelii- immune BALBIc mice were fused with P3-NS1 /1 -Ag4-1 myeloma cells in the presence of polyethylene glycol (by the general method of Galfre, et. al., Nature, 1977,266, 550-552). The cells were dispensed into 144 tissue culture wells in 2 ml volumes of HAT selective medium (Littlefield, Science, 1964, 145,709-710) using RPMI 1640 medium supplemented with 10% foetal calf serum as a base. After 10 days the culture supernatants were tested for P.yoe/ii-specific antibody by indirect immunofluorescence (IIF). Of 143 cultures tested, 38 were positive. Culture number 1 contained an antibody specific for an antigen associated with schizonts but absent from ring-forms and trophozoites.A cloned hybridoma cell line was produced from this culture by growing colonies derived from single cells in semi-solid agar overlayed with medium. The cloned hybridoma line continued to secrete its unique antibody, was designated WIC 25.1. and has been deposited at the Institute Pasteur under the number 1-160 on 16 July 1981. The hybridoma line was further grown as an ascites tumor in BABBLE mice, and ascitic fluid and serum from these mice contained abour 10 mg/ml of the monospecific antibody. A second hydridoma line, WIC 45.1 secretes an antibody specific for the same antigen as antibody 25:1, with the difference that it appears to recognise only the antigen and fragments larger than 0.9 x 105 molecular weight.

EXAMPLE2 Preparation of Antigen from P. yoelii Blood forms of Plasmodium yoelii were grown in CD-1 mice to 80-90% parasitaemia and the erthrocytes were harvested and solubilised. The cells were solubilised by lysis at4 C in a buffer of 50mM Tris, SmMethylenediamine tetraacetate (EDTA), 1% Nonidet P40 pH 8.0 containing 1 mM phenylmethyl sulphonyl fluoride (PMSF), 0.1 mM tosyl-L-lysine chloromethyl ketone (TLCK), 5mM ethyleneglycolbis-(amino ethyl ether)N,N'-tetraacetate (EGTA) and 5mM iodoacetamide to inhibit proteolytic activity.

The lysed cells were centrifuged at 100,000 g and the supernatant, containing the erythrocyte soluble proteins and some membrane proteins, together with approximately 70% of the parasite proteins (as estimated from the proportion of35S-methionine label in this fraction after an in vitro incorporation) collected.

Immunoglobulin from the ascites fluid of mice containing hybridoma line WIC 25.1 was purified by binding to Protein A- Sepharose and a single step elution at pH 3.0. The immunoglobulin fraction was coupled to cyanogenbromide activated Sepharose, using 10 mg immunoglobulin/ml swollen gel. The imm unoabsorbant was extremely washed, packed into colums and equilibrated with 10mM Tris, 1mM EDTA, 1 mM EGTA, 1% NP40 pH 8.0.

The supernatant obtained as described above was passed through the immunoabsorbant equilibrated with 10mM Tris, 1 mM EDTA, 1 mM EGTA, 1% NP40 pH 8.0 and washed through with this buffer. Non specifically bound material was removed by washing (5 column volumes) with this buffer containing 0.5M NaCI. The column was then washed with a buffercontaining 10mMTris, 1mM EDTA, 1mM EGTA, 0.1% NP40 pH 8.0. Specific elution was achieved with 50mM diethylamine, 0.1% NP40 pH 11.5.

The eluted material was subjected to a second cycle of immunoabsorbant chromatography. The specific eluate was dialysed against a buffer containing 10mM Tris, 1mM EDTA, 1mM EGTA, 0.1% NP40 pH 8.0. The sample was applied to the column which had been reequilibrated in this buffer, and washed through with 10mM Tris, imM EDTA, imM EGTA, 0.5% sodium deoxycholate pH 8.2. The retained material was eluted with 50mM diethylamine, 0.5% sodium deoxycholate pH 11.5. The eluate was concentrated and dialysed to provide the antigen.

EXAMPLE3 Biochemical Characterization of the Antigen and its proteolytic degrada tion fragments from P. yoelii Antigen was labelled by incorporation of 35S- methionine into the protein by incubating parasitized cells for 2-3 hours in a medium containing this amino acid at high specific activity, the labelled antigenic protein was obtained by immunoprecipitation with the monoclonal antibody 25.1 and Protein A bearing Staphylococcus aureus cells to precipitate the immune complex. This material was subjected to SDS gel electrophoresis and the antigen behaved as species of 2.30 x 105 apparent molecular weight. In addition, a series of degradation products possessing the antigenic determinantwas detected. The principle fragments had apparent molecular weights of 1.97, 1.60, 1.51, 1.26, 0.90, 0.56 and 0.28 x 105.

Minor degradation products of apparent molecular weights2.20,2.13, 1.90, 1.80, 1.74, 1.48, 1.18,1.12 and 1,04 x 105 were also present. The antigen purified by immunoabsorbant column chromatography described in Example 2, displayed a pattern of degradation products identical to that of the immunoprecipitated 35S-methionine labelled material.

The nature of the relationship of the polypeptides was revealed by peptide mapping of the major polypeptides and by pulse-chase labelling of P. yoelii schizonts. Homology between the amino acid sequences of the major polypeptides was clearly shown by two dimensional chymotryptic peptide mapping of 35-S-methionine-labelled polypeptides immunoprecipitated by antibody 25.1 and by limited proteolysis peptide mapping in SDS polyacrylamide gels of purified material which had been iodinated using '251-iodine. P. yoelii schizonts were incubated for 10 minutes in the presence of high specific activ ity 35S-methionine which was then chased with non radioactive methionine. At various time intervals after labelling samples were solubilised and immunoprecipitated with antibody 25.1.The label was intially incorporated into the 2.30 x 105 MW polypeptide, then during further incubation it sequentially appeared in the smaller iolypeptides, with a concomitant reduction in the amount of label remaining in the 230,000MW component. Thus the native antigen recognised by antibody 25.1 is a 2.3 x 105 molecular weight protein and the smaller polypeptides are proteolytic fragments of it, each retaining the antigenic determinant recognised by the monoclonal antibody. In vitro, degradation of the native protein commenced soon after its synthesis, and was extensive after7 hours. The results indicate that the degradation occurred during incubation of the intact schizonts ratherthan after cell lysis and solubilisation.

The isoelectric point of the antigen and some of its fragments were determined using a 2-dimensional gel electrophoresis system based on that described byO'Farrell (J.Biol. Chem.250:40074021, 1975) but using either a polyacrylamide or a mixed agarose/polyacrylamide gel in the first dimension. When the pH gradient was generated using pH 3-10 ampholines, the antigen and its fragments had the following approximate isoelectric points; 2.3 x 105 MW, pH 5.35.7; 1.6 x 105 MW, 5.2-5.5; 1.5 x 105 MW, 5.25.5; 0.9 x MW, 6.05.7; 0.56 x 105,5.2-5.4 and 0.30 x 0 MW, 5.0-5.3. The arrangement of the fragments according to isoelectric point was always 0.28 < 0.56 < 1.5, 1.6 < 2.3 < 0.9 x 105 MW.

Standard tests employing periodic acid-Schiffs staining and '251-iodinated Concanavalin A lectin binding to the protein in polyacrylamide gels both suggested that the protein was not glycosylated.

The lack of glycosylation was confirmed by inability to specifically bind the elute the protein from lentil lectin-Sepharose and wheatgerm agglutinin Sepharose, and by the absence of any effect on the size of the protein when synthesised by the parasite in the presence oftunicamycin, a specific inhibitor or protein N-glycosylation.

EXAMPLE4 Localisation of the antigen associated with P. yoelii By IIF using acetone-fixed cells the antigenic portein was localised to the membrane of schizonts and merozoites. Neither antibody 25.1 nor poly-valent antiserum raised against the purified protein reacted with the surface of unfixed intact schizont in suspension. The protein was not labelled by lactoperoxide catalysed iodination of intact schizonts, but was labelled by this technique when the cells were first solubilised by addition of 0.01% (v/v) Nonidet P 40.

Furthermore the addition of trypsin (at 5 uglm I) to the medium did notmodifythe processing of the protein observed during the in vitro incubation of parasitised cells, described in Example 3. It is thus considered that the protein is not exposed on the outer surface of the host cell membrane nor localised at the inner surface of this membrane. The IIF pattern produced by antibody 25.1 suggests that the 2.30 x 105 MW protein of Example 2 is associated specifically with the plasma membrane of the developing intracellular parasite, rather than with the membrane of the host erythrocyte.This interpretation is supported by further evidence provided using a double labelling immunofluorescence technique in which acetone fixed schizonts were incubated with a mixture of antibody 25.1 (a mouse immunoglobulin) and rabbit anti-mouse red cell antiserum. The slides were washed then incubated sequentially with rhodamineconjugated goat anti- rabbit IgG antiserum and with FlTC-conjugated rabbit anti-mouse IgG antiserum. Upon examination it was noted that the P. yoelii schizont antigen (traced with FITC) was localised at a membrane distinct from, and enclosed within, the host red cell membrane (traced with rhodamine).

In early schizonts, prior to the information of the residual body, the antigen appearsto be localised at a spherical membrane enclosing the parasite, but the following segmentation and formation of the residual body, the membrane appears to surround individual merozoites. Thus the 2.3 x 105 MW protein (and/or proteolytic derivatives of it) is evenly distributed on free merozoites as well as being associated with schizonts.

In the IIF test with free merozoites in suspension, antibody 25.1 gave a positive florescense indicating that the antigen is on the surface of the merozoites.

EXAMPLES immunisation of mice with an antigen ofPlasmodium yoelii, purified using monoclonal antibody25. 1: antibody response Groups of 5 BALBIc mice were immunised with the antigenic protein obtained from Example 2 or with control preparations according to the following schedule: Group 1: 50,ug antigen 25.1 emulsified in Freund's Complete Adjuvant (FCA) and injected intraperitioneally (i.p.) in a volume of 0.2 ml on day 0.

20 pug antigen 25.1 suspended in 0.2 mis normal mouse serum and injected intravenously on day 26.

25,ag antigen 25.1 suspended in saline and injected i.v. on day 39.

Group2: FCA only, given i.p. on day 0 NMS only, given i.v. on day 18 Saline only, given i.v. on day 42.

On day 49, serum samples were taken for titration and examination by indirect immunofluorescence (I IF). The results are shown below.

Group Mouse llF titre antiserum specificity 1 1 > 1:5120 schizonts and merozoites 2 > 1:5120 schizonts and merozoites 3 > 1:5120 schizonts and merozoites 4 > 1:5120 schizonts and merozoites 5 > 1:640 schizonts and merozoites 2 1 < 1:40 - 2 < 1:40 3 < 1:40 - 4 < 1:40 - 5 < 1:40 - The pattern of fluorescent staining using the serum from the mice immunised with antigen 25.1 was indistinguishable from that produced using monoclonal antibody 25.1. Staining was restricted to the schizont and merozoite forms of P. yoelii. This result confirmed the purity and immunogemic nature of antigen 25.1 prepared as described in Example 2.

EXAMPLE6 Immunisation ofmice with an antigen ofPlasmodium yoelii, purified using monoclonal antibody 25. 7: protection against challenge infection Groups of 5 BALBIc mice were immunised with the antigenic protein obtained from Example 2, or with control preparations according to the following schedule: Group 1: 12 g antigen 25.1 emulsified in FCA and injected i.p. in a volume of 0.2 mls on day 0.

12,ag antigen 25.1 in FCA i.p. on day 35.

20 clog antigen 25.1 in 0.1 ml saline i.v. on day 50.

Group 2: 2,ag antigen 25.1 in FCA i.p. on day 0.

2,ug antigen 25.1 in FCA i.p. on day 35.

20 pug antigen 25.1 in 0.1 ml saline i.v. on day 50.

Group3: Saline in FCA i.p. on day 0.

Saline in FCA i.p. on day 35.

0.1 ml saline given i.v. on day 50.

On day 60, serum samples were taken for IIF titration of antibody, and for analysis by immunoprecipitation. on day 61 all mice were challenged i.v. with 104 P. yoelii YM-parasitised erythrocytes. The antibody response of the groups on immunised mice are given in Table 1. The results demonstrate that under the conditions used, immunisation with 12,(b9 of antigen 25.1 was more effective in inducing an antibody response than was immunisation with 2 clog of antigen 25.1.

Table 1: Antibodyresponse ofmiceimmunised with antigen 25.1 Group IIF titre antiserum specfficity 1 > 1:10240 schizonts and merozoites 2 > 1:5120 schizonts and merozoites 3 < 1:40 nonspecific The protection against challenge with P. yoelli provided to each group shown in Figure 1. The results show that the group with the higher antibody titre were more effectively protected against challenge.

The control group of mice all died within 8 days of challenge. All mice immunised with antigen 25.1 survived.

The antigen specificity of serum from the immunised mice was checked by immunopresipitation which demonstrated that the antibody response was directed against the 230,000 m.w. schizont antigen (and its degradation frangments) used for immunisation.

EXAMPLE7 Identification of the Antigen from P.

falciparum A schizont-enriched fraction of erythrocytes, parasitised with P. falciparum prepared by centrifugation through a layer of Percoll, was labelled with 35S- methionine during a period of 2 hours. The cells were harvested and solubilised by lysis at4 as described in Example 2. After centrifugation the supernatant was used for immunoprecipitation with an antibody specific for P. falciparum and derived from a hybridoma prepared by the method of Example 1. The specific precipitate was subjected to SDS gel electrophoresis and the antigen behaved as a species of 1.95 x 105 apparent molecular weight. In addition, a number of discrete degradation products were observed.The principle fragments had molecular weights of 1.53, 1.50 and 0.83 x 105 and additional minor fragments of 1.12, 1.10 and 1.08 x 105 molecu larweightwere also present.

The identification of the 1.95 x 105 molecular weight component as the primary protein, from which the specific fragments were subsequently derived, was confirmed by labelling the cells for a short period of 20 minutes. Immunoprecipitates from extracts of this material using the P. falciparum antibody contained all of the 35S-methionine label in the 1.95 x 105 molecular weight antigen.

This antigen (and its specific fragments) is strongly represented in the material recognised by human immune serum toP. falciparum as assessed by immunoprecipitation and SDS gel electrophoresis.

The amino acid sequence relatedness of the major fragments and the 1.95X105 M.W. species was confirmed by peptide mapping of the 355-methionine- containing peptides. The isoelectric points of the antigen and its fragments were determined as approximately 5.8(1.95X105), 5.2(1.53 and 1.50X105), 6.0(1.1 0X1 0'), and 6.2-6.5(0.83X105).

Example 8 Preparation of antigen from P. fat- ciparum P. falciparum parasites grown in culture in human erythrocytes, were harvested and solubilised, as described in Example 2. The solubilised material was passed down a column of Sepharose coupled with immunoglobuiin obtained from the ascites fluid of mice containing hybridoma line WIC 89.1. When the column was washed and eluted with 50 mM dieth ylamine,0.1%NP40, pH 11.5, as described in Example 2 the eluate contained predominantly the 195 x 105 MW antigen and the 0.83 x 105 MW fragment.

Example 9 Synthesis and processing ofthe antigen from P. falciparum The synthesis and processing of the protein during the intracellular development of the parasite was investigated during one 48 hour cycle in an in vitro culture of P. falciparum which had been synchronised by two treatments with sorbitol 33 hours apart.

The stage of development was monitored by morphological examination of Giemsa stained parasites, every 3 hours after the second sorbitol treatment.

only ring forms were observed for the first 12 hours, afterwhichtimetrophozoitesbegunto appear and were present maximally at 24 hours. Schizonts were first observed at27 hours and predominated at39 hours. At 39 hours a small number of new ring forms were visible, indicating that some merozoite release had occured, although most ring forms did not appear until 45 - 48 hours.

At six hour intervals the polypeptides synthesised during a 30 minute pulse with 35S-methionine were investigated. Maximal protein synthesis occured during schizogeny, at36 hours. The time of synth esis of many proteins was restricted during the development cycle. One of the abundant proteins synthesised only during schizogeny was the 1.95 x 105 MW protein.

When the proteins synthesised at each time immunoprecipitated with pooled immune human serum. A clear pattern was observed. During the ring and trophozoite stages only two polypeptides of 1.6 x 105 MW and 0.71 x 105 MW were recognised by human immune serum and precipitated. During schizogeny a number if polypeptides were recognised by human immune serum. The predominant species (larger than 0.3 x 105) had molecular weights of 0.37, OAO, 0A6, 0.62,0.72,0.80,0.84, 0.89, 1.04, 1.20. 1.34, 1 A6 (doublet), 1.78, 1.95,2.08,2.20 and 2.35 x 105. The synthesis of several of these proteins was at specific times during schizogeny. the 1.95 x 105 MW antigen was recognised by the immune serum and was synthesised throughout schizogeny.

The monoclonal antibody 89.1 also precipitated the 1.95 x 105 MW protein throughout schizogeny.

The processing of the protein subsequent to its synthesis, was investigated by performing immunoprecipitations on the solubilised pulse labelled material and on solubilised material from cells which had been pulse labelled and then chased with unlabelled methioninefor60 or 120 min.At30 h into the parasite development cycle (immature schizons) the 120 min chase resulted in no apparent shift in the label to lower molecular weight bands. At42 h most of the radioactivity was incorporated during the pulse record period into the 195,000MW band but after 60 min chase the 1.53 and 1.50 x 105 MW bands were more apparent and the 0.83 x105 MW band appeared. After 120 min chase the 0.83 x 105 MW band increased in intensity and some of the label was associated with a 0.6 x 105 MW polypeptide.

After 45 h of parasite development, processing of the 195,000 MW protein to its discrete fragments was moch more extensive during the chase period. (The immunoprecipitate at 48 h showed that some processing of the 195,000 MW protein through to the 60,000 MW fragment had occurred even within the 30 minuted pulse period). The onset of processing was concomitant with the appearance of new rings forms in the culture and, therefore, also with the maturation of schizonts and release of infective merozoites.

Example 10 Locallsation of the antigen associated with P. flaciparum and cross-reaction with a polyvalent serum raised against the P. yoelAi antigen.

By IIF using acetone-fixed cells the antigen protein was localised to the membrane of schizonts and merozoites, the distribution being identical to the antigen described in Example 4. Immunological cross-reaction between the antigen of P. yoelii and that of P. falciparum was demonstrated by raising high tire polyvalentantiserum in mice to the P.

yoelii antigen, which had been prepared as described in Example 2. When this antiserum was assayed by immunofluorescence with P. yoelii infected mouse cells, the pattern of fluorescent stain ing was indistinguishable from that produced using monoclonal antibody 25.i and the antiserum had a litre of 1 in 20,000. When this antiserum was assayed against P. falciparum infected human red cells, it gave a staining pattern identical to that of antibody 89.1, with a IIF titre of 1 in 1,600. This indicated an immunological cross-reaction between the two anti gens which is probably related to their structural similarities.

Example 11 Vaccines Vaccines for use in immunisation may be prepared by conventional techniques with the following con stitutents: Formulation A Antigen 2 mg Physiological Saline to 1 ml Formulation B Antigen 1 mg Al hydrogel 1 mg Physiological Saline to 1 ml

Claims

1. A protein-inducing proteinaceous antigen of parasites of the genusPlasmodium characterised by: (i) having an apparent molecular weight in the rangeof1.8x105to2.5x105; (ii) being associated with the membranes of the erythrocytic schizont forms of the parasite; and (iii) being processed intraethrocyticallyto dis crete fragments possessing the same antigen properties and wherein the antigen, or the discrete fragments thereof, are associated with the surface membrane of the merozoite; and functional derivatives thereof.

2. An antigen as claimed in claim 1 characterised in that the apparent molecular weight of the antigen is from 1.90to2.30 x 105.

3. An antigen as claimed in claim 1 or claim 2 characterised in that the parasite is a humanPlasmodium parasite.

4. An antigen as claimed in claim 3 wherein the molecular weight is about 1.95 x 105.

5. An antigen as claimed in any one of claims 1 to 4 wherein the discrete fragments include fragments having a molecular weight of 0.3-0.4 x 05; 0.7-0.8 x 105 and 1.0-1.4 x 105 lower than that of the parent antigen.

6. An antigen as claimed in claim 4 wherein the discrete fragments include fragments having a molecularweightof1.53-1.5x105; 1.5x105; 1.10x 105 and 0.9 x105.

7. An antigen as claimed in any one of claims 1 to 6 wherein the parasite isPlasmodium falciparum.

8. An antigen as claimed in any one of claims 1 to 7 having an isoelectric point of about 5.8.

9. An antigen as claimed in any one of claims 1 to 8 wherein the discrete fragments include fragments having isoelectric points of about 5.2, about 6.0 and 6.2-6.5

10. An antigen as claimed in any one of claims 1 to 9 characterised in that it is prepared by the steps of: (i) solubilising erythrocytes containing the schiz ont form of a Plasmodium parasite; (ii) contacting the solubilised materials with a monoclonal antibody specific for the desired anti gen to provide an antibody-antigen complex; and (iii) recovering the antigen from the antibody antigen complex.

11. An antigen as claimed in any one of claims 1 to 9 characterised in that it is prepared by a chemical or biological reproduction.

12. A method for the preparation of an antigen as defined in any one of claims 1 to 9 which method comprises: (a) isolating the antigen from the erythrocytic schizontfrom of the parasite; or (b) chemical or biological reproduction of the antigen.

13. A vaccine which vaccine comprises an antigen as defined in any one of claims 1 to 11 together with a pharmaceutically acceptable carrier therefor.

14. A vaccine as claimed in claim 13 suitable for inducing immunity in humans to Plasmodium falciparum.

15. An anitgen, as defined in any one of claims 1 to 11, for use in inducing immunity in a susceptable vetebrate host

16. A method for inducing immunity to malaria in a susceptable vetebrate host comprising administration to the host of an antigen, as defined in any one of claims 1 toll.

17. An antigen as claimed in claim 15, in the form of a vaccine.

18. A method as claimed in claim 16 wherein the antigen is in the form of a vaccine.