WO1999066043A1

WO1999066043A1 - Recombinant production of toxoplasma sag1 antigen

Info

Publication number: WO1999066043A1
Application number: PCT/EP1999/003957
Authority: WO
Inventors: Ralph Biemans; Alex Bollen; Michele Haumont
Original assignee: Smithkline Beecham Biologicals S.A.
Priority date: 1998-06-12
Filing date: 1999-06-08
Publication date: 1999-12-23
Also published as: AR019864A1; AU4510299A; CA2330209A1; CO5070712A1; EP1086228A1

Abstract

The invention provides a method for the production of the toxoplasma antigen SAG1 or a fragment thereof, which comprises: (a) constructing a plasmid comprising DNA encoding SAG1 or a fragment thereof; (b) transforming a P. pastoris host cell with the said plasmid; and culturing the host such that the DNA encoding SAG1 or a fragment thereof is expressed. The invention further provides truncated SAG1 proteins in which the anchor region is absent, particularly truncated SAG1 comprising amino acid residues (48-307) of SAG1, and vaccine compositions comprising the SAG1.

Description

RECOMBINANT PRODUCTION OF TOXOPLASMA SAGl ANTIGEN

Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for toxoplasmosis in warm-blooded animals, including man. Although it is generally clinically asymptomatic in healthy individuals, toxoplasmosis may cause severe complications in pregnant women and immunocompromised patients [1-4]. A live attenuated S48 Toxoplasma strain of the parasite is currently available for vaccination in sheep (Toxovax, Mycofarm) [5], However, this vaccine cannot be administered to humans because of possible reversion to virulent forms. The development of subunit vaccines thus constitutes an alternative way to achieve effective protection of humans against congenital infection and to prevent infection of immunosuppressed individuals. In domestic animals like sheep and pigs, subunit vaccines could also prevent spontaneous abortion and reduce the reservoir of the parasite since tissue cysts in the muscles of these animals is a major cause of human toxoplasmosis.

Antigens which may be important in immunising against toxoplasma gondii are known, for example TG34 as described in WO 92/11366.

SAGl (so called P30), the major surface antigen of T. gondii, is a putative candidate for a subunit vaccine. Indeed, SAGl induces a strong immune response in human and experimental animal models [6-7]. Immunisation of mice with SAGl, purified from tachyzoites and adjuvanted with saponin Quil A or incorporated into liposomes leads to a nearly total protection after challenge [8-9] . This immunity appears to be primarily mediated by CD8⁺ cells specific for SAGl.

The gene encoding SAGl has been cloned and sequenced. It is single copy and contains no introns [10]. Because native SAGl is anchored to the plasma membrane via a glycosylphosphatidylinositol anchor (GPI) [11], its purification from tachyzoites is difficult and time consuming. Expression of T. gondii SAGl antigen in E. coli or mammalian cells has generally been disappointing; indeed, the recombinant protein was either insoluble and misfolded or correctly folded but weakly produced [26-28].

It has been reported that SAGl can be produced in S. cerevisiae (see WO

96/02654), in S. pombe and in insect cells. However the expression level is low and/or a heterogeneous product is obtained.

The present invention provides a method for the production of the toxoplasma antigen SAGl or a fragment thereof, which comprises:

(a) constructing a plasmid comprising DNA encoding SAGl or a fragment thereof;

(b) transforming a P. pastoris host cell with the said plasmid; and

(c) culturing the host such that the DNA encoding SAGl or a fragment thereof is expressed.

The SAGl protein, or fragment thereof, which is produced by the above process may be purified by conventional methods, for instance by a combination of anion exchange (for example Q-sepharose) and gel filtration (for example superdex 75HR) chromatographies.

In one preferred aspect of the invention the DNA encoding the SAGl protein or fragment thereof is positioned downstream from and in frame with a yeast secretion signal sequence, preferably the S. cerevisiae prepro α-mating factor secretion signal sequence (MFα).

In a further preferred aspect of the invention the plasmid comprising DNA encoding the SAGl protein or a fragment thereof is derived from a multicopy P. pastoris expression vector, preferably the vector pPlC9K.

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SUBST1TUTE SHEET (RULE 26) In yet a further preferred aspect of the invention the DNA encoding SAGl or a fragment thereof is expressed under the control of a methanol-inducible promoter, for example the AOX1 promoter.

One advantage of the present invention is that the secreted recombinant SAGl level is at least ten times superior to that observed in s. cerevisiae (see WO 96/02654). Moreover, only two forms of the recombinant protein were secreted.

The Pichia pastoris expression system additionally leads to very high levels of secretion into an almost protein-free medium. As previously reported, the Pichia pastoris expression system is easy for fermentation to high cell density, is genetically stable and can be scaled-up without loss of yield [12-13].

The invention also provides a SAGl protein or a fragment thereof when DNA encoding the said SAGl protein or fragment thereof is expressed in the yeast Pichia pastoris.

In a preferred aspect of the invention the SAGl protein of fragment thereof, in the form produced in P pastoris according to the invention, is purified and when a fragment of the SAGl protein is an immunological derivative of the SAGl protein. The said fragment of SAGl when produced in P pastoris according to the invention is also preferably truncated, especially at the C-terminus. In a preferred aspect the said truncate is an anchor-less SAGl protein, especially one lacking amino acids 308 to 336 of the SAGl protein.

In a further aspect of the invention there is provided a truncated SAGl protein comprising amino acids 48-307 of SAGl, and immunogenic derivatives thereof.

The term "immunogenic derivative" encompasses any molecule such as a truncated or other derivative of the protein which retains the ability to induce an immune response to the protein following internal administration to a human or to an animal or which retains the ability to react with antibodies present in the sera or other biological samples of Toxoplasma gondii-infected humans or animals. Such other derivatives can be prepared by the addition, deletion, substitution or rearrangement of amino acids or by chemical modifications thereof.

The recombinant truncated SAGl protein appears correctly folded since it is recognised by antibodies specific for the native form of SAGl and elicits proliferation of mononuclear cells from seropositive individuals. The recombinant truncated SAGl protein is also capable of inducing a protective immune response against a toxoplasma challenge and in a congenital toxoplasmosis model. The anchor- less SAGl antigen is therefore useful in diagnosis of T. gondii infections and for development of a subunit vaccine.

Accordingly, the invention also provides a vaccine composition comprising the truncated SAGl protein and a method of preventing toxoplasmosis infection which comprises administering to a human subject in need thereof a vaccine composition according to the invention.

The present invention in a further aspect provides a vaccine formulation as herein described for use in medical therapy, particularly for use in the treatment or prophylaxis of toxoplasmosis infections. The vaccine formulation will be useful in the prevention of both horizontal and vertical (congenital) transmission of toxoplasmosis.

The vaccine composition according to the invention will normally comprise a protein according to the invention, as described hereinabove, admixed with a suitable adjuvant and/or carrier.

The vaccine composition according to the invention may comprise further components for the treatment or prophylaxis of infections other than toxoplasmosis infections. In particular such further components may be one or more antigens from one or more other pathogens. The vaccine composition according to the invention may comprise one or more additional T. gondii antigens.

The vaccine of the present invention will contain an immunoprotective or immunotherapeutic quantity of the antigen and may be prepared by conventional techniques.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by

Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of protein in the vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-1000 mg of protein, preferably 2-100 mg, most preferably 4-40 mg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.

The proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention. Adjuvants are described in general in Vaccine Design - the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995.

Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes. In the formulation of the invention it is preferred that the adjuvant composition induces a preferential Thl response. However it will be understood that other responses, including other humoral responses, are not excluded.

Preferred Thl -type immunostimulants which may be formulated to form adjuvants suitable for use in the present invention include and are not restricted to the following.

Monophosphoryl lipid A, in particular 3-de-O-acylated monophosphoryl lipid A

(3D-MPL), is a preferred Thl -type immunostimulant for use in the invention. 3D- MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. It can be prepared by the methods taught in GB 2122204 B. A preferred form of 3D-MPL is in the form of a particulate formulation having a small particle size less than 0.2μm in diameter, and its method of manufacture is disclosed in EP 0 689 454.

Saponins are also preferred Thl immunostimulants in accordance with the invention. Saponins are well known adjuvants and are taught in: Lacaille-Dubois,

M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386). For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in US 5,057,540 and "Saponins as vaccine adjuvants" , Kensil, C. R., Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0 362

279 Bl. The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil

A) have been described as potent systemic adjuvants, and the method of their production is disclosed in US Patent No. 5,057,540 and EP 0 362 279 Bl. Also described in these references is the use of QS7 (a non-haemolytic fraction of Quil- A) which acts as a potent adjuvant for systemic vaccines. Use of QS21 is further described in Kensil et al. (1991. J. Immunology vol 146, 431-437). Combinations of QS21 and polysorbate or cyclodextrin are also known (WO 99/10008). Particulate adjuvant systems comprising fractions of QuilA, such as QS21 and QS7 are described in WO 96/33739 and WO 96/11711.

Another preferred immunostimulant is an immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides ("CpG"). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al, J.Immunol, 1998, 160(2): 870-876; McCluskie and Davis, J. Immunol., 1998, 161 (9): 4463 -6). Historically, it was observed that the DNA fraction of BCG could exert an anti-tumour effect. In further studies, synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity. The central role of the CG motif in immunostimulation was later elucidated in a publication by Krieg, Nature 374, p546 1995. Detailed analysis has shown that the CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA. The immunostimulatory sequence is often: Purine, Purine, C, G, pyrimidine, pyrimidine; wherein the CG motif is not methylated, but other unmethylated CpG sequences are known to be immunostimulatory and may be used in the present invention.

In certain combinations of the six nucleotides a palindromic sequence is present. Several of these motifs, either as repeats of one motif or a combination of different motifs, can be present in the same oligonucleotide. The presence of one or more of these immunostimulatory sequences containing oligonucleotides can activate various immune subsets, including natural killer cells (which produce interferon γ and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977). Other unmethylated CpG containing sequences not having this consensus sequence have also now been shown to be immunomodulatory. CpG when formulated into vaccines, is generally administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al. supra ; Brazolot-Millan et al, Proc.Natl.Acad.ScL , USA, 1998, 95(26), 15553-8).

Such immunostimulants as described above may be formulated together with carriers, such as for example liposomes, oil in water emulsions, and or metallic salts, including aluminium salts (such as aluminium hydroxide). For example, 3D- MPL may be formulated with aluminium hydroxide (EP 0 689 454) or oil in water emulsions (WO 95/17210); QS21 may be advantageously formulated with cholesterol containing liposomes (WO 96/33739), oil in water emulsions (WO 95/17210) or alum (WO 98/15287); CpG may be formulated with alum (Davis et al. supra ; Brazolot-Millan supra) or with other cationic carriers.

Combinations of immunostimulants are also preferred, in particular a combination of a monophosphoryl lipid A and a saponin derivative (WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153. Alternatively, a combination of CpG plus a saponin such as QS21 also forms a potent adjuvant for use in the present invention.

Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3D-MPL, together with an aluminium salt. An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched in cholesterol containing liposomes (DQ) as disclosed in WO 96/33739.

A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is another preferred formulation for use in the invention. Another preferred formulation comprises an aluminium salt together with a CpG oligonucleotide.

In a further aspect of the present invention there is provided a method of manufacture of a vaccine formulation as herein described, wherein the method comprises mixing a protein according to the invention with a suitable adjuvant and, optionally, a carrier.

Particularly preferred adjuvant and/or carrier combinations for use in the formulations according to the invention are as follows: i) 3D-MPL + QS21 in DQ ii) Alum + 3D-MPL iii) Alum + QS21 in DQ + 3D-MPL iv) Alum + CpG v) 3D-MPL + QS21 in DQ + oil in water emulsion

There is also provided a diagnostic kit for the diagnosis of toxoplasmosis infection in the blood of mammals which may be infected, which kit comprises an anchor-less SAGl antigen or a fragment thereof.

The invention is illustrated in the accompanying examples and Figures in which the following are depicted:

Figure 1: Immunodetection of SAGl. Part a and b: respectively under reduced

(lane 1 to 3,5 and 7) and nonreduced (lane 4 and 6) conditions; lane 1, 4 and 5: total soluble T. gondii antigens (ARGENE); Lane 2, 6 and 7: recombinant SAGl; lane 3: molecular weight (Rainbow, Amersham).Part c: glycoslyation of SAGl: Lane 1 and 2: respectively incubation without or with N-glycosidase F. Part d: Coomassie blue stain detection of purified SAGl. Lane 1: molecular weight (Rainbow, Amersham), lane 2 and 3: purified SAGl, respectively under reduced and nonreduced conditions. SAGl was detected with antibodies against residues 76-95 of SAGl (part a and c) or with the monoclonal TG5.54 (part b).

Figure 2: Determination of human patients serology by ELISA. Plates were coated with soluble antigen extract from Toxoplasma gondii (A) or with purified recombinant SAGl (B). To simplify the figure, only two negative and five positive sera (respectively DI, D6 and D2, D3, D4, D5 and D7) were represented.

Figure 3 : Proliferative response of PBMC from immune (black box) and nonimmune (white box) individuals to soluble antigen extract from T. gondii (A) or to recombinant SAGl (B). Proliferation was assessed by [³H]Thymidine incorporation. Results are expressed as the means ± standard deviation of 4 experiments.

Figure 4: Challenge and protection of mice immunized with recombinant SAGl.

Groups of 5 mice received two injections of recombinant SAGl before challenge with T. gondii C56 tachyzoites (see text for details). Results are plotted as number of surviving animals according to time (days) post challenge.

Figure 5: Construction of the plasmids for expression of the toxoplasma antigen SAGl in Saccharomyces cerevisiae and in Pichia pastoris. See example 3 for details.

Figure 6: Schematic representation of the recombinant unglycosylated anchor-less SAGl constructs. The details of the construction are described in Example 5.

Figure 7: Construction of unglycosylated anchor-less SAGl expression vector for the methylotrophic yeast P. pastoris. HIS4, P. pastoris histidinol dehydrogenase gene to complement the defective his4 genotype in Pichia SMD1168 host strain. 5 'AOXl, segment of about 1000 bp, including the alcohol oxidase promoter. 3 'AOXl (TT), segment of about 260 bp with the alcohol oxidase transcriptional terminating sequence. 3'AOXl, segment of the alcohol oxidase focus which is necessary for gene replacement.

The invention will now be illustrated by the following examples.

Examples

Example 1: Expression of SAGl (P30) in Pichia Pastoris

Bacterial and Yeast strains

The DH5αFTQ Escherichia coli strain (Bethesda Research Laboratories) was used for bacterial transformation and recombinant plasmid propagation as described by Maniatis et al [14]. P. pastoris strain SMD1 168 (his4, pep4) was purchased from Invitrogen.

Oligonucleotide synthesis

Oligonucleotides were synthesised by the solid-phase phosphoramidite method [15] on an Applied Biosystems Synthesizer model 394.

Polymerase chain reaction (PCR)

PCR amplification was performed on a T. gondii tachyzoites RH strain λgtl 1 cDNA library [16]. The choice of the primers was based on the published sequence [10.] Oligonucleotides 5'GGATCAAGCTTACCATGTTTCCGAAGGCAGTG3' and 5'TGATCGAATTCTCACGCGACACAAGCTGC3' were used to amplify the sequence encoding amino acids 18 to 336 of SAGl. DNA was amplified in a 50μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 2mM MgCl₂, 50 mM KC1, 0.01 % wt/vol gelatin, 200 μM of each deoxynucleoside triphosphate, 20 pmol of each primer, 1U of Taq polymerase (Perkin Elmer Cetus) and cDNA. Samples were amplified for 30 cycles in a DNA thermal cycler (Perkins Elmer Cetus). After an initial 10 min denaturation at 94°C, each cycle consisted of 1 min at 95° C, 2 min at 55°C and 3 min at 72°C. At the end of the 30 cycles of amplification, a primer extension was continued for 10 min at 72°C. The PCR products were analysed after electrophoresis on an 7.5% polyacrylamide gel. Plasmid construction

Amplified DNA fragment was digested by Hindi 11 and EcoRl endonucleases before its insertion in the pUC19 (New England Biolabs) previously opened with the same enzymes, resulting in the plasmid pNIV3418 The resulting plasmid was then opened by Pstl and EcoRl to permit the insertion of the annealed oligonucleotides 5'GGGTCATGATG3' and 5ΑATTCATCATGACCCTGCA3'. The resulting plasmid contains the sequence encoding the amino acids 18 to 307 of SAGl. The sequence of the amplified DNA was confirmed by dideoxy sequencing. Digestion by BamHl and EcoRl generate a 781 bp DNA fragment which was introduced together with annealed oligonucleotides 5'TCGAGAAAAG AGAGGCTGAAGCTTCG3' and 5'GATCCGAAGCTTCAGCCTCTCTTTTC3' to provide the junction between the fragment obtained above and the P. pastoris secretion vector pPIC9 (invitrogen) cut by BamHl, Xhol and EcoRl. The resulting plasmid, pNlV3464 was then cut by BamHl, Xhol and EcoRl to generate a 254 bp BamHl - Xhol DNA fragment and a 807 bp Xhol-ECoRl DNA fragment which were introduced in the pPIC9K previously opened by BamHl and EcoRl. The resulting plasmid pNIV3488 contains the sequence encoding the amino acids 48 to 307 of SAGl downstream to, and in-frame with, the DNA sequence encoding the α-mating factor prepro secretion signal sequence of Saccharomyces cerevisiae.

Transformation of P. pastoris

The plasmid pNIV3488 was introduced into the P. pastoris strain SMD 1168 (his4, pep4) by using the spheroplast transformation method (Invitrogen). Transformants were selected for histidinol dehydrogenase (His⁺) prototrophy by plating on a dextrose-based medium without histidine supplementation. His⁺ cells were then checked for methanol utilisation (Mut⁺) by replica plating on both minimal methanol (MM)( and minimal dextrose (MD). The screening of His⁺ transformants for G418 resistance was realised by pooling and plating them on YPD agar containing increasing concentrations of G418 (0.25, 0.5, 1. 1/5 and 2 mg/ml)[17]. Culture conditions

All cell cultures were performed as described in the manufacturer's instruction leaflet (Invitrogen). Briefly, expression assays were performed by inoculating 40 ml of minimal glycerol medium (MGY) or of buffered glycerol-complex medium (BMGY) with transformants. Cells were grown at 30°C in a rotary shaker at 220 rpm to an A^ of _+ 2, then harvested by centrifugation at 3000 x g for 10 min at room temperature and finally resuspended in 40 ml of minimal methanol medium (MM) or buffered methanol-complex medium (BMMY) to induce expression. Cultures were supplemented with 0.5% methanol every 24 hr.

SDS-PAGE and Western-blotting

Samples of harvested culture medium were analysed on a 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel [18]. Proteins were transferred onto nitrocellulose sheets (Hybond C, Amersham) [19], which were successively incubated with an anti-peptide 76-95 of SAGl, with an anti-rabbit lgG alkaline phosphate conjugate, and finally with 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitroblue tetrazolium (NBT) (Promega). Protein blots were saturated and washed between incubations with phosphate-buffered saline containing 0.05% Tween-20.

Peptides synthesis and purification

Two peptides derived from SAGl were chosen according to predictive algorithms for B-cell epitopes [20-22]: peptides NHFTLKCPKTACTEPPTLAY (aa 76-95) and CNEKSFKDILPKLTEN (aa 238-253). They were synthesised by the Merrified solid phase method on a fully automated peptide synthesiser (AB1 model 430 A, Foster City, CA), according to the tertbutyloxycarbonyl/trifluoroacetic acid (tBoc/TFA) strategy [23]. After synthesis, peptides were deprotected and cleaved from the resin by hydrogen fluoride. The crude peptides were purified by gel- filtration on TSK HW 40s (Merck, Rahway, NJ) and reverse phase HPLC on Nucleosil C₁₈ and thin-layer chromatography, and for identity by amino-acid analysis after total acid hydrolysis. Peptides were conjugated to the tetanus toxoid with coupling agents as carbodiimide for the first peptide and 6-maleimidocaproic acyl N-hydroxysuccinimide ester (MCS) for the second.

Immunisations with peptides

Rabbits were subcutaneously immunised at one month intervals using 500 μg of conjugated peptide emulsified in complete Freund's adjuvant for the first injection and Freund's incomplete adjuvant for the second one.

Purification of recombinant SAG-1

The Pichia pastoris culture supernatant (400 ml) from a high density fermentation was concentrated by ultrafiltration using YM 10 membrane (cut-off 10 kD) under a pressure of 3 bars. The concentrate was then dialysed against Tris-HCl 20mM, pH 8.5. The sample was then loaded onto a Q-sepharose fast flow (Pharmacia) column equilibrated in the same buffer. Recombinant SAG-1 was eluted with 100 mM NaCl in the same buffer. The sample was then concentrated and applied onto a superdex 75 HR column (Pharmacia LKB) equilibrated in 20 mM Tris-HCl pH 8.5, 150 mM NaCl. The protein content was determined by the method of Lowry with ovalbumin as standard [24] .

N-glycanase treatment

Purified recombinant SAGl was heat-denatured 10 min in presence of 0.05% SDS and 0.1% 2-mercaptoethanol in 50 mM sodium phosphate pH 8. The protein was then digested with 0.3 U N-glycanase F (Boehringer) for 6 hr at 37°C in presence of 0.7% Nonidet P-40. Samples were electrophoresed on a 15% SDS-polyacrylamide gel.

NH2 terminal amino acid sequencing Automated Edman degradation of recombinant SAGl or fragment thereof according to the invention was performed in an Applied Biosystems Model 477A sequencer equipped with a phenythiohydantoin analyser. The samples were prepared after electrob lotting on polyvinylidene fluoride membrane.

Detection of human immune serum reactivity to recombinant SAGl

Heparinised blood from donors were centrifuged. Sera were collected and stored at -20°C until used. The presence of specific anti-SAGl antibodies in sera was detected using an ELISA assay. 96-Microwells plates were coated overnight at 4°C with 0.1 mg/well of purified recombinant SAGl or with 8.3 mg/well of soluble T. gondii lysate (RH strain, ARGENE). Wells were washed four times with Tris- buffered saline (TBS)-0.1% Tween and saturated with TBS-Tween/1 % BSA (bovine serum albumine) (30 min. at 37°C). ELISA plates were then incubated with serial dilutions of sera (45 min. at 37°C). Immune complexes were detected with alkaline phosphatase-labeled goat anti-human lgG antibody (45 min. at 37°C) and N-p- nitrophenyl phosphate. The O.D. value at 415 nm were measured in a microplate reader (Biorad).

Cell Proliferation assay

PBMC were isolated from heparinised blood as previously described [25] . One hundred thousand peripheral-blood mononuclear cells (PBMC) were incubated in the presence of different concentrations of recombinant SAGl or T. gondii lysate

(RH strain, ARGENE). After 4 days at 37°C in a humidified atmosphere containing 5 % CO₂, cells were pulsed with 0.5 μCi of [³H]thymidine (Amersham). The results were expressed as mean counts per minute (c.p.m) ft standard deviation of 4 experiments.

Plasmid construction and expression experiments In a first step, the sequence coding for SAGl (336 amino acids residues) was recovered by PCR amplification from a lambda gtll tachyzoite cDNA library, as described above. This sequence, verified by automatic dideoxy sequencing, carries a 3 ' terminal region coding for a stretch of hydrophobic amino acids (residues 308 to 336) which serves as acceptor of the so-called GP1 group, i.e. a phosphatidylinositol glycolipid. Native SAGl is in fact anchored in toxoplasma membranes via this GP1 group. The SAGl coding sequence was engineered to remove the region specifying amino acids 308 to 336, then inserted, downstream to and in frame with the S. cerevisiae prepro α-mating factor secretion signal sequence (MFα), into the multicopy P. pastoris expression vector pPIC9K. The resulting plasmid, pNIV3488, thus carries, under the control of the methanol-inducible AOXl promoter, the fused sequences of MFα and anchor-less SAGl, together with a kanamycin resistance gene cassette necessary for subsequent selection of multicopy integrants by the antibiotic G418. Plasmid pNIV3488 was linearised with Bg/ll to orient integration events at the AOXl locus P. pastoris recipient cells, strain SMD1168 (his4, pep4) were transformed with linearised plasmid by the spheroplast method.

Altogether 52 clones, obtained after selection of transformants on histidine-deficient medium, were tested for methanol utilisation and then for growth in the presence of G418 (0.25 to 2 mg/ml) in order to identify those carrying multiple copies of the resistance gene and of the gene of interest.

Four transformants resisting to 2 mg/ml G418 were tested for expression; supematants of methanol-induced cultures were analysed, in reducing conditions, on SDS-polyacrylamide gels and proteins were detected by immunoblotting using antipeptide antibodies targetting SAGl residues 76 to 95 and 230 to 253 respectively.

As seen in Figure la lane 2, in all cases, two secreted immunoreactive products were detected, having molecular masses of 34.5 and 31.5 kDa respectively. Since

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SUBST1TUTE SHEET (RULE 26) SAGl is a highly conformational antigen, it was of interest to analyse the recombinant products under non-denaturing conditions using in this case for detection the monoclonal antibody TG5.54 which is specific for native SAGl [29] (gift of Prof. Capron, Lille). Two observations arose from this experiment. First, as expected and already reported, the non-reduced native SAGl antigen migrated with a higher mobility than its denatured equivalent (FIG. la, lane 1 and lb: lane 4, 30 kDa versus 33 kDa). This phenomenon results from the preservation of correct disulfide pairing in the SAGl molecule under non-reducing conditions. Likewise, the two recombinant products secreted by P. pastoris transformants were not only detected by the conformation-specific antibody but migrated also with a higher mobility, banding at apparent molecular masses of 27.5 and 31.5 kDa respectively (FIG. lb, lane 6 and ld;lane 3). These results indicate that recombinant SAGl antigens were secreted under an appropriate conformation for recognition by the monoclonal antibody TG5.54 and suggest that correct disulfide pairing was achieved. The SAGl antigen molecule presents a potential N-Glycosylation site which, in the parasite, is not effectively glycosylated [30]. However, in experiments where SAGl was produced in mammalian cells, glycosylation was observed [28]. We thus checked this possibility for yeast-derived SAGl antigen by treating samples with N-glycanase F. As seen in FIG lc; lane 2, a single immunoreactive band of 31.5 kDa was detected. In another experiment, it was found that the 34.5 kDa form of recombinant SAGl was recognised by the GNA lectin (Galanthus nivalis agglutinin) which identifies mannose residues (data not shown). It appears therefore that P. pastoris achieved N-glycosylation, at least in part, of the SAGl anchor-less antigen and also that this modification had no significant effect on the conformation of the recombinant product since, as said above, it was clearly recognised by the specific monoclonal antibody TG5.54.

Recombinant anchor-less SAGl was then purified to near homogeneity starting from spent culture medium of the highest secreting yeast transformant. The combination of anion exchange (Q-sepharose) and gel filtration (superdex 75HR) chromatographies yielded about 12 mg of > 95 % pure product per litre of culture

(FIG. Id). Both purified 34.5 and 31.5 kDa forms of the recombinant SAGl were

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SUBST1TUTE SHEET (RULE 26) submitted to N-terminal amino acid analysis which indicated the occurrence of two additional amino acid residues. Glu-Ala, on each N-terminal end. This result revealed the incomplete processing of the prepro MFα signal peptide by the dipeptidyl aminopeptidase STE13, a phenomenon already reported in other cases [31-32]. The presence of these excendatary amino acids had obviously no effect on the conformation of the molecule since, as shown above, recognition by the conformation-specific monoclonal antibody TG5.54 was demonstrated.

In order to evaluate the seroreactivity of recombinant SAGl, sera from T. gondii- positive and negative individuals were tested in an ELISA format where either total soluble T. gondii antigens or purified recombinant SAGl were coated onto microtiter plates. All sera has been characterised beforehand by commercial immunoassays (anti-Toxoplasma immunoglobulin M (IgM) and IgG detection kit from Behring) for the presence of IgG and IgM. All 20 sera tested were IgM negative, 8 were clearly IgG-positive, 4 probably IgG-positive and 8 IgG-negative. The absence of IgM and the presence of IgG indicates that the individuals were chronically infected by T. gondii. As seen in FIG.2, all positive samples reacted with recombinant SAGl indicating recognition of the antigen by IgG antibodies. Having no IgM-containing sera, it is impossible at this stage to evaluate the seroreactivity of recombinant SAGl in instances of acute infection.

Purified recombinant SAGl was further characterised in terms of cellular proliferative capability. To this end, polymorphonuclear cells, derived from four r.gørccfø^'-seropositive individuals, were isolated then stimulated in vitro either with total soluble antigens of T. gondii or with purified recombinant SAGl. As seen in FIG.3, a significant proliferative response (Stimulation Index =4) was observed with the recombinant protein. This result strengthens the interest of yeast-derived SAGl as a putative antigen for the preparation of a toxoplasmosis vaccine.

Example 2: protection against a Toxoplasma challenge Mouse immunization and parasite challenge

Female BALB/c (8-week-old) were subcutaneously immunized twice at two weeks intervals with 10 μg of purified recombinant SAGl combined either with the SBASlc adjuvant (10 μg of 3D-MPL, 10 μg of DQ) or with 100 μg of aluminium hydroxide; 1 dose = 100μl. Two weeks after the last injection, all mice were injected interperitoneally with 10⁴T. gondii C56 strain tachyzoites.

Parasites

The T. gondii C56 strain (kindly donated by Darde, Centre Hospitalier Regional et Universitaire de Limoges, France) was maintained by serial passage in the peritoneal cavities of BALB/c mice. Tachyzoites were collected from the peritoneal cavity of infected mice as previously described (Saavedra et al, 1991 b).

Results

The protective potential of recombinant SAGl was evaluated in a lethal toxoplasmosis mouse model. To this end, groups of five BALB/c mice were subcutaneously immunized twice at two weeks intervals with 10 μg of recombinant SAGl combined either with the SBASlc adjuvant (proprietary composition of SmithKline Biologicals, Rixensart, Belgium), which induces a Thl-type response or with aluminium hydroxide known to induce a Th2-type response. Control groups of mice received adjuvants alone. 15 days after the second injection, all mice were challenged with 10⁴ tachyzoites of the T. gondii C56 strain administered intraperitoneally. As seen in Figure 4, and as expected, all mice from control groups died within 8 days post challenge. In addition, all mice immunized with SAGl adjuvanted with aluminium hydroxide also died in this time span, indicating that a Th2-type response is inappropriate to confer protection. In contrast, an overall protection of 60% was observed in the group of mice immunized with recombinant SAGl combined to the Thl-type inducing adjuvant. Although two mice in this group died, their survival time was nevertheless significantly extended with respect to that of control mice (13 and 18 days respectively versus 8 days). These results thus indicate that immunization with recombinant SAGl, properly adjuvanted to induce a Thl-type immune response can protect mice against a lethal challenge. The level of protection obtained here is of the same magnitude (60% versus 67%) as observed before (Khan et al, 1991) in bred A/J mice immunized with purified parasite-derived SAGl adjuvanted with Quil A.

In conclusion, the set of data presented here strengthens the interest of yeast-derived SAGl as a potential vaccine antigen.

Example 3: Expression of the toxoplasma antigen SAGl in Saccharomyces cerevisiae and in Pichia pastoris. Comparison between the two systems.

The DNA sequence coding for SAGl with its native sequence signal or with the signal sequence of the yeast pheromone MFα-1 was introduced in the S. cerevisiae expression plasmid TCM97 (pRIT13145). The resulting plasmids respectively pNIV3433 and pNIV3435, contain, under the control of the ARG3 promoter, the sequence encoding the residues 18 to 336 of SAGl for the first one and the sequence encoding the 19 amino acids of the signal sequence of MFα- 1 followed by residues 48 to 336 of SAGl for the second one (FIG. 5). In order to obtain secretion of SAGl by yeast cells, the sequence encoding the SAGl hydrophobic carboxy-terminal (residues 308 to 336) was deleted to prevent addition of the GPI group. The resulting plasmids, pNIV3448 and pNIV3441, contain respectively the sequence encoding the residues 18 to 307 of SAGl and the sequence encoding the 19 amino acids of the signal sequence of MFα-1 followed by residues 48 to 307 of SAGl (FIG. 5). The sequence coding for the residues 48 to 307 of SAGl was also introduced downstream of the α factor prepro peptide in the P. pastoris expression vector, pIC9K, to give pNIV3488 (FIG. 5).

21 -

SUBST1TUTE SHEET (RULE 26) In S. cerevisiae, SAGl will be constitutive ly expressed under the control of the ARG3 promotor placed on a 2-μ-based high copy plasmid TCM97 with dLEU2 selection maker. Complementation of the leucine auxotrophy requires a higher copy number since the expression level of the dLEU2 gene is low due to its deleted promoter.

The transformed S. cerevisiae strains were grown for 72 hours in 40 ml YNB at 30°C and 200 rpm shaking. Cells were harvested by centrifugation, lysed and the soluble protein extracts and culture medium (20 μl) were analysed for the presence of SAGl by proteins separation on SDS-PAGE and transfer onto nitrocellulose membrane. However, immunodetection of SAGl was only observed after TCA (trichloroacetic acid) precipitation of proteins from 40 ml of culture medium. Indeed, two proteins of about 33 and 36kDa were detected in immunoblot but not visualised by silver-staining detection, confirming the very low secretion of SAGl . Curiously, these bands were principally observed with the yeast cells producing the GPI-anchored SAGl (ρNIV3433 plasmid). Soluble forms of a variety of GPI- anchored proteins occurring extracellularly were recently reported (Wang et al, Biochemistry, 36: 14583-14592 (1997)). However, the molecular mechanisms governing their release are not entirely clear. An intracellular form of SAGl was also observed but only in a 8 M urea extract of the insoluble fraction.

In conclusion in S. cerevisiae, we observed a very low production of SAGl. It is possible that overproduction conditions represent a stress situation for the yeast cells leading to a growth advantage for cells with reduced expression and thus reduced copy number.

The highly inducible and stringently regulated methanol oxidase gene (AOXl) promoter was used for the production of SAGl in P. pastoris. Recombinant SAGl was easily detected after proteins separation from 20 μl of the culture medium (40 ml) on SDS-PAGE followed by coomassie or silver staining. Two proteins of about 31.5 and 34.5 were immunodetected using antipeptides targetting SAGl residues 76 to 95 and 230 to 253 respectively. The combination of anion exchange (Q-sepharose) and gel filtration (superdex 75HR) chromatographies yielded about 12 mg of -0-95 % pure product per litre of culture.

It has been reported that SAGl has been produced in S. cerevisiae (see WO 96/02654). The DNA sequence encoding the amino acids 48 to 316 of SAGl was also placed downstream to, and in-frame with, the DNA sequence encoding the α- mating factor prepro secretion signal sequence. SAGl was expressed under the control of the α-mating factor promoter. The dURA3 gene was used as selection marker and the KEX2 gene used in order to circumvent an eventual problem of incomplete processing of the prepro region of MFα. The recombinant SAGl was secreted under a heterogeneous form suggesting an incomplete processing by KEX2 and/or heterogeneous glycosylation of the protein (SAGl and the pro region of MFα possess respectively one and three potential site of N-glycosylation). Expression level of SAGl seems to be low: secreted SAGl production obtained in WO 96/02654 in Schizosaccharomyces pombe, S. cerevisiae or in insect cells is between 0.1 mg/1 to 0.3 mg/1. In Schizosaccharomyces pombe, only a major protein of about 35 kDa (28 kDa if the N-glycosylation site is mutated) was observed in the culture medium.

In conclusion, the P. pastoris expression system is more efficient for the production of a recombinant SAGl. Indeed, the secreted recombinant SAGl level is at least ten times superior to the one observed in WO 96/02654 in S. cerevisiae, in S. pombe and in insect cells. Moreover, only two forms of the recombinant protein were secreted in P. pastoris in contrast to a heterogeneous product in S. cerevisiae. The preferred mode of expression in P. pastoris is by chromosomal integration using one of the integrative plasmids. There are several advantages for using integrative transformation which include expression cassette stability, generation of multicopy strains, control of site of integration and ability to engineer different modes of integration using appropriately cleaved DNA. Both S. cerevisiae and P. pastoris have a majority of N-linked glycosylation of the high-mannose type; however, the length of the oligosaccharide chains added post- translationally to proteins in Pichia (average 8-14 mannose residues per side chain) is much shorter than those in S. cerevisiae (50-100 mannose residues).

Example 4: Protective effect of vaccination with recombinant SAGl against congenital toxoplasmosis in Guinea Pig

Materials and methods T.gondii

The C56 medium-virulent strain of Toxoplasma gondii (Supplied by ML Darde, CHU Limoges), maintained by passage of infective brain homogenate in the peritoneum of BalbC mice, was used for experimental infections in Durkin-Hartley guinea pigs.

Vaccination procedure

Groups of 20 guinea pigs were immunized three times at 3 weeks intervals using either lOμg of recombinant SAGl formulated with SBASlc adjuvant (lOμg of 3D- MPL, lOμg of DQ) or SBASlc alone (SC route). SAGl was produced in Pichia pastoris and purified according to the procedure described in Example 1 above.

Measurement of antibody response

Two weeks after the last immunization, animals were bled and sera were checked for the presence of anti-SAGl IgG antibodies in an enzyme-linked immunosorbent assay (ELISA) using recombinant SAGl as coating reagent (lOOng/well). Serial dilutions of each serum were tested and titer was expressed as the reciprocal of the dilution giving one-half the maximal optical density. Congenital infection model

Before antigen injection, all guinea pigs were monitored for absence of seroreactivity against toxoplasma. Females were mated with males for breeding three weeks after the last immunization ; challenge infection using 5 x 10³ tachyzoites (ID route) followed 7 weeks later.

Infectious status of pups delivered from guinea pigs was evaluated in a mouse assay : pups were sacrified within 48 hours following delivery, each brain was homogenized in 1ml of PBS and intra peritoneally injected into two female BalbC mice (0.5 ml each). Mice that did not survive from 21 days onwards after brain homogenate injection were considered infected and their mortality indicated the infection status of the pups ; it was assessed that a pup was infected once one of the two injected mice died.

Results

IgG response of guinea pigs immunized with rSAGl

The geometrical mean was 63065 with values between 24226 and 248217. The titers in the mock-immunized group were below the detectable level.

Congenital transmission

After challenge, 18 SAGl- and 17 mock-immunized guinea pigs produced respectively 69 and 59 pups ; of those 19 and 17 respectivally were excluded from further analysis because, in a precedent experiment, we observed that stillborn pups or pups retrieved from dead mothers were always negative in the mouse assay even when they originated from the mock-immunized group, probably due to parasite inactivation.

After exclusion of the 36 pups, 50 and 42 pups, originated from 17 and 15 litters respectivelly, were analysed.

Protection against vertical transmission was observed and summarized in Table 1.

Table 1

Similar results were obtained in a further experiment.

Example 5: Expression of unglycosylated SAGl protein in P. pastoris

The SAGl gene encodes a consensus N-linked glycosylation site (Asn-X-Ser/Thr) which is not used by the parasite (Odenthal-Schnittler et al, 1993, Biochem. J. 291: 713-721). Elimination of the consensus N-glycosylation site can prevent glycosylation of SAGl by the yeast. To this end, the asparagine at the potential N- linked glycosylation site (amino acid 259) was mutated to glutamine.

The sequence encoding the unglycosylated anchor-less SAGl was obtained as follows: to change the Asn in position 250 to glutamine the following mutagenic oligonucleotide was synthesized 5'AGCGTGGCACCCTTATCACTCGAAGCTTGA CCCTG3' and used as antisense primer with the sense oligonucleotide 5ΑGACAACAATCAGTACTGTTCCGGGAC3' to amplify a 129 bp DNA fragment. The DNA sequence (pNIV3418) encoding SAGl was used as template. The amplified DNA fragment was then digested by Seal and Banl endonuc leases. A 642 bp HindUI-Scal and a 149 bp Banl-EcoRL DNA fragments were recovered from plasmid pNIV3418 and inserted with the digested PCR fragment in the pUC19 previously opened with Hindi^'l and EcoRl to give pNIV4710 (FIG. 6).

A 781 bp ZtamHI-EcøRI DNA fragment was recovered from plasmid pNIV4710 and introduced together with annealed oligonucleotides 5 'TCGAGAAAAGAGAGGCTGAAGCTTCG3 ' and 5 'GATCCGAAGCTTCAGCCT CTCTTTTC3' to provide the junction between the fragment obtained above and the P. pastoris secretion vector pPIC9 (Invitrogen) cut by Xhol and EcoRl (FIG. 7). The resulting plasmid, pNIV4729 contains as the plasmid pNIV3488 the sequence encoding the amino acids 48 to 307 of SAGl except that the Asn in position 259 was mutated to glutamine. The sequence encoding the unglycosylated SAGl was also introduced in the P. pastoris vector pPIC9K to give pNIV4732.

Plasmid pNIV4729 or pNIV4732 was introduced into the P. pastoris strain SMD1168 (his4, pep4) by using the spheroplast transformation method. Cell culture were performed as described for the N-glycosylated anchor-less SAGl.

Supematants were collected every 24 hr and analyzed for protein expression by silver-stained SDS-PAGΕ and western blot. Elimination of the N-glycosylation site did not affect the secretion of SAGl . Indeed, a protein of an apparent molecular mass of 31.5 kDa was detected with antibodies targeting SAGl residues 76-95 and 230-253 respectively. Weaker proteins with slower mobility at apparent molecular masses of 36 and 40 kDa were also detected suggesting an incomplete processing of the prepro MFa signal peptide. Under non-reducing conditions, only a single protein at apparent molecular mass of 27.5 kDa was detected with conformation- specific antibodies as the monoclonal antibodies TG5.54 (gift of Prof. Capron, Lille) or GIL 9 (ARGENE), suggesting a correct disulfide pairing. Unglycosylated recombinant SAG-1 purification

The P. pastor is culture supernatant from a high density fermentation was concentrated by ultrafiltration using YM 10 membrane (cut-off, 10 kDa, Amicon) under a pressure of 3 bar. The concentrate was desalted onto a sephadex G-25 column (2.6 x 35 cm, Pharmacia) equilibrated in 20 mM citrate bufer, pH 3.3. The sample was then directly applied onto a macroprep S column (2.6 x 10 cm, Bio-rad) conditioned in the citrate buffer. Unglycosylated recombinant SAG-1 was eluted with 400-500 mM NaCl in the same buffer. Enriched SAG-1 fractions were pooled, concentrated by ultrafiltration and loaded onto a superdex 75 HR column (1 x 30 cm, Pharmacia) equilibrated in phosphate-buffered saline (PBS) pH 7.3. Unglycosylated recombinant SAG-1 migrated on SDS-PAGE as a molecule of approximately 30 kDa; the protein was recognized as a single band on western blot by antipeptide antibodies directed to SAG-1 residues 230-253. The final yield was about 16mg of purified unglycosylated recombinant SAG-1 per liter of yeast culture. The product was estimated more than 95% pure.

Example 6: Recognition of Recombinant SAGl by Sera and PBMC

Human seroreactivity to SAG-1

Heparinized blood from donors was centrifuged; the presence of specific anti-SAGl antibodies was detected in the supernatant using an enzyme-linked immunosorbent assay (ELISA). 96 Microwell plates were coated overnight at 4°C with either 100 ng per well of purified recombinant SAGl produced as described in example 1 or with unglycosylated SAGl produced as described in example 5. Wells were washed five times with Tris-buffered saline (TBS)-0.1 % Tween 80 and saturated in the same buffer supplemented with 1 % bovine serum albumin (BSA) for 1 hour at 37°C. Plates were then incubated with serial dilutions of sera (1 hour at 37°C). Plates were washed as described above and alkaline phosphatase-labelled goat anti- human IgG antibody diluted 1 :7500 was used as the secondary antibody (1 h at 37 °C). After washing, immune complexes were developed with p-nitrophenyl phosphate as chromogenic substrate. Absorbance at 415 nm was measured in a microplate reader (Biorad).

ELISA titer was calculated as the reciprocal dilution giving 50% of the maximal O.D. signal.

Cell proliferation assay

Peripheral-blood mononuclear cells (PBMC) were isolated from heparinized blood. Two hundred thousand PBMC were resuspended in RPMI 1640 medium supplemented with 10% human serum AB ( PAA laboratories ), 25 mM HEPES (N- 2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid), 2mM L-glutamine, 1 mM sodium pyruvate, 5.10^"5mM β-mercaptoethanol and 50 IU/ml penicilline- streptomycine. Cells were incubated in the presence of different concentrations of glycosylated and unglycosylated recombinant SAGl (50, 5, 0.5 and 0.05 μg/ml) . After 4 days at 37 °C, cells were pulsed with 1 μCi per well of (³H)-thymidine. 16 hours later, they were harvested and incorporated radioactivity was determined using a scintillation counter (Wallac, 1450 Micro Beta). T cell proliferation was expressed as the stimulation index (SI), calculated as the ratio of mean cpm in SAGl stimulated wells to control wells, in the presence of 5 μg/ml of SAGl. A positive response was defined as an SI > 2

(a) coating antigen, prepared as described in example 5

(b) coating antigen, prepared as described in example 1

(c) stimulating antigen, prepared as described in example 5

(d) stimulating antigen, prepared as described in example 1

(e) IgG anti-Toxoplasma ELISA was performed by Dr Bigaignon (UCL) and expressed in IU (ELISA VTDAS, Bio-Merieux), serum was considered as Toxoplasma seropositive for value > 8 UI. Like SAGl described in example 1, the unglycosylated form of SAGl was able to induce proliferative response of T lymphocytes from Toxoplasma seropositive donors and was also recognized by antibodies from the same donors.

REFERENCES

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4. Luft, B.J., Remington, J.S. (1992) Toxoplasmic encephalitis in AIDS. Clin. Infect Dis. 15: 211-222.

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J.S. (1983) Western blot analysis of the antigens of Toxoplasma gondii recognised by human IgM and IgG antibodies. J. Immunol.131: 977- 983.

8. Bϋlow, R. and Boothroyd, J.C. (1991) Protection of mice from fatal

Toxoplasma gondii infection by immunisation with P30 antigen in liposomes. J. Immunol. 147: 3496-3500. . Khan, I.A., Ely K.H. and Kasper, L.H. (1991) A purified parasite antigen (p30) mediates CD8⁺ T-cell immunity against fatal Toxoplasma gondii infection in mice. J. Immunol. 147: 3501-3506.

10. Burg, J.L., Perelman, D. , Kasper, L.H., Ware, P.L. and Boothroyd, J.C. (1988) Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. J. Immunol. 141: 3584-3591.

11. Nagel, S.D. and Boothroyd, J.C. (1989) The major surface antigen,

P30, of Toxoplasma gondii is anchored by a glycolipid. J. Biol, Chem. 264: 5569-5574.

12. Cregg, J.M., Velvick, T.S., Rasche, W.C. (1993) Recent advances in the expression of foreign genes in P. pastoris. Bio/ Technology 11:905-

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Harbor, NY).

15. Beaucage, S.L., Caruthers, M.H. (1981) Deoxynucleoside phosphoramidites, a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22: 1859-1862.

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SUBST1TUTE SHEET (RULE 26) 16. Saavedra R., De Meuter F. , Decourt J-L and Herion P. (1991) Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli. J. Immunol. 147: 1975-1982.

17. Scorer C.A., Clare J.J., McCombie W.R., Romanos M.A. and Sreekrishna K. (i994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Bio/technol. 12: 181-184.

18. Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970).

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21. Chow P.Y., Fasman G.D. (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. 47: 45-57.

22. Hopp, T.P., Woods, K.R. (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA

78: 3824-3828.

23. Kent, S.B. (1988) Chemical synthesis of peptides and proteins. Annu. Rev. Biochem. 57: 957-985. 24. Lowry O.H. Roseburgh N.J., Farr A.L., Randall R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-

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Toxoplasma gondii: production of interferon-gamma, inter leukin 2 and strain cross-reactivity. Parasitol. Res. 77: 379-385.

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S-transferase fusion protein. Jpn. J. Parasitol. 40: 344-351.

27. Xiong C, Grieve R.B., Boothroyd J.C. (1993) Expression of Toxoplasma gondii P30 as fusions with glutathione S-transferase in animal cells by Sindbis recombinant virus. Mol. Biochem. Parasitol.

61: 143-148.

28. Kim K., Bulow R., Kampmeier, Boothroyd J.C. (1994) Conformationally appropriate expression of the Toxoplasma antigen SAGl (p30) in CHO cells. Infect. Immun. 62: 203-209.

29. Rodriguez, C, Afchan, D., Capron, D., Dissous, C, Santoro, F. (1985) Major surface protein of Toxoplasma gondii (P30) contains an immunodominant region with repetitive epitopes. Eur. J. Immunol. 15: 747-749.

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31. Romanos, M.A., Scorer, C.A., Clare J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8: 423-488. Brake, A., Merryweather J.P., Coit, D., Heberlein, U.A., Masiarz,

F.R., Mullenbach, G.T., Urdea, M.S., Valenzuela, P., Barr, P.J. (1984) α-factor directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81: 4642-4646.

- 36 -

SUBST1TUTE SHEET (RULE 26)

Claims

1. A method for the production of the toxoplasma antigen SAGl or a fragment thereof, which comprises: (a) constructing a plasmid comprising DNA encoding SAGl or a fragment thereof; (b) transforming a P. pastoris host cell with the said plasmid; and culturing the host such that the DNA encoding SAGl or a fragment thereof is expressed.

2. A method as claimed in claim 1 which further comprises the step of purifying the SAGl protein or fragment thereof from the culture medium.

3. A method as claimed in claim 1 or claim 2 in which the DNA encoding the SAG 1 protein or a fragment thereof encodes a truncated SAGl protein lacking the anchor domain (lacking amino acids 308 to 336 of the SAGl protein).

4. A method as claimed in claim 3 in which the DNA encoding the truncated SAGl protein encodes amino acids 48-307 inclusive of the SAGl protein.

5. A method as claimed in any one of claims 1 to 4 in which the DNA encoding the SAGl protein or fragment thereof is positioned downstream from and in frame with a yeast secretion signal sequence.

6. A method as claimed in any one of claims 1 to 5 in which the said plasmid is derived from a multicopy P. pastoris expression vector.

7. A method as claimed in any one of claims 1 to 6 in which the DNA encoding SAGl or a fragment thereof is expressed under the control of a methanol-inducible promoter.

8. Plasmid pNIV3488.

9. A SAGl protein or fragment thereof when expressed in P. pastoris.

10. A SAGl protein or fragment as claimed in claim 9, which is a truncated SAGl.

11. A truncated SAGl protein as claimed in claim 10 in which the anchor region of SAGl is absent.

12. A truncated SAGl protein as claimed in claim 10 or claim 11 which comprises amino acid residues 48-307 of SAGl, and immunogenic derivatives thereof.

13. A vaccine composition comprising the protein of any one of claims 9 to 12 in combination with a suitable adjuvant and/or carrier.

14. A vaccine composition as claimed in claim 13 wherein the adjuvant is a Thl- type inducing adjuvant.

15. A vaccine composition as claimed in claim 14 wherein the adjuvant comprises at least one immunostimulant chosen from the group consisting of 3D-MPL, QS21 and CpG.

16. A vaccine composition as claimed in claim 14 or claim 15, formulated with at least one carrier chosen from the group consisting of an oil in water emulsion, an aluminium salt and cholesterol-containing liposomes.

17. A vaccine composition as claimed in claim 15 or claim 16, wherein the adjuvant comprises 3D-MPL.

18. A vaccine composition as claimed in any one of claims 15 to 17, wherein the adjuvant comprises a saponin such as QS21.

19. A vaccine composition as claimed in claim 18 wherein the adjuvant is 3D- MPL. in combination with QS21 in a cholesterol-containing liposome.

20. A vaccine composition as claimed in any one of claims 17 to 19, further comprising alum.

21. A vaccine composition as claimed in claim 18 wherein the adjuvant is 3D- MPL and QS21 in an oil in water emulsion.

22. A vaccine composition as claimed in claim 15 or claim 16 wherein the adjuvant comprises CpG and alum.

23. A truncated SAGl protein in which the anchor region of SAGl is absent.

24. A truncated SAGl protein as claimed in claim 23 which comprises amino acid residues 48-307 of SAGl, and immunogenic derivatives thereof.

25. A vaccine composition comprising the protein of claim 23 or claim 24 in combination with a suitable adjuvant and/or carrier.

26. A vaccine composition as claimed in claim 25 wherein the adjuvant is a Thl- type inducing adjuvant.

27. A vaccine composition as claimed in claim 26 wherein the adjuvant comprises at least one immunostimulant chosen from the group consisting of 3D-MPL, QS21 and CpG.

28. A vaccine composition as claimed in claim 26 or claim 27, formulated with at least one carrier chosen from the group consisting of an oil in water emulsion, an aluminium salt and cholesterol-containing liposomes.

29. A vaccine composition as claimed in claim 27 or claim 28, wherein the adjuvant comprises 3D-MPL.

30. A vaccine composition as claimed in any one of claims 27 to 29, wherein the adjuvant comprises a saponin such as QS21.

31. A vaccine composition as claimed in claim 30 wherein the adjuvant is 3D- MPL, in combination with QS21 in a cholesterol-containing liposome.

32. A vaccine composition as claimed in any one of claims 29 to 31 further comprising alum.

33. A vaccine composition as claimed in claim 30 wherein the adjuvant is 3D- MPL and QS21 in an oil in water emulsion.

34. A vaccine composition as claimed in claim 27 or claim 28, wherein the adjuvant comprises CpG and alum.

35. A vaccine composition as claimed in any one of claims 25 to 34 which additionally comprises another Toxoplasma gondii antigen.

36. A vaccine composition as claimed in any one of claims 25 to 35 wherein the truncated SAGl is expressed in yeast, such as P. pastoris.

37. Use of a protein according to any of claims 9 to 12, 23 and 24 in the manufacture of a medicament for the prevention or treatment of toxoplasmosis infections in mammals.

38. The use according to claim 37, for the prevention of congenital toxoplasmosis infections.

39. A diagnostic kit for the diagnosis of toxoplasmosis infection in the blood of mammals which may be infected, which kit comprises an anchor-less SAGl antigen or a fragment thereof.