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WO1999066043A1 - Production par recombinaison de l'antigene sag1 de toxoplasme - Google Patents

Production par recombinaison de l'antigene sag1 de toxoplasme Download PDF

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Publication number
WO1999066043A1
WO1999066043A1 PCT/EP1999/003957 EP9903957W WO9966043A1 WO 1999066043 A1 WO1999066043 A1 WO 1999066043A1 EP 9903957 W EP9903957 W EP 9903957W WO 9966043 A1 WO9966043 A1 WO 9966043A1
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Prior art keywords
sagl
protein
vaccine composition
adjuvant
fragment
Prior art date
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PCT/EP1999/003957
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English (en)
Inventor
Ralph Biemans
Alex Bollen
Michele Haumont
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Smithkline Beecham Biologicals S.A.
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Publication date
Priority claimed from GBGB9812773.1A external-priority patent/GB9812773D0/en
Priority claimed from GBGB9908564.9A external-priority patent/GB9908564D0/en
Application filed by Smithkline Beecham Biologicals S.A. filed Critical Smithkline Beecham Biologicals S.A.
Priority to AU45102/99A priority Critical patent/AU4510299A/en
Priority to EP99927922A priority patent/EP1086228A1/fr
Priority to CA002330209A priority patent/CA2330209A1/fr
Publication of WO1999066043A1 publication Critical patent/WO1999066043A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/45—Toxoplasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • 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.
  • 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].
  • GPI glycosylphosphatidylinositol anchor
  • the present invention provides a method for the production of the toxoplasma antigen SAGl or a fragment thereof, which comprises:
  • 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.
  • anion exchange for example Q-sepharose
  • gel filtration for example superdex 75HR
  • 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 ⁇ -mating factor secretion signal sequence (MF ⁇ ).
  • a yeast secretion signal sequence preferably the S. cerevisiae prepro ⁇ -mating factor secretion signal sequence (MF ⁇ ).
  • 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.
  • SUBST1TUTE SHEET (RULE 26)
  • 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.
  • 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.
  • 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.
  • the said truncate is an anchor-less SAGl protein, especially one lacking amino acids 308 to 336 of the SAGl protein.
  • a truncated SAGl protein comprising amino acids 48-307 of SAGl, and immunogenic derivatives thereof.
  • 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.
  • 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.
  • 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
  • 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.
  • the adjuvant composition induces a preferential Thl response.
  • other responses including other humoral responses, are not excluded.
  • 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 0.2 ⁇ m 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,
  • CpG immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides
  • 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.
  • 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.
  • a palindromic sequence is present.
  • Several of these 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 ⁇ and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977).
  • natural killer cells which produce interferon ⁇ and have cytolytic activity
  • macrophages Wangrige 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).
  • 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).
  • carriers such as for example liposomes, oil in water emulsions, and or metallic salts, including aluminium salts (such as aluminium hydroxide).
  • 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.
  • a combination of CpG plus a saponin such as QS21 also forms a potent adjuvant for use in the present invention.
  • 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.
  • a method of manufacture of a vaccine formulation as herein described comprising mixing a protein according to the invention with a suitable adjuvant and, optionally, a carrier.
  • 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
  • kits 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.
  • FIG. 1 Immunodetection of SAGl. Part a and b: respectively under reduced
  • 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 [ 3 H]Thymidine incorporation. Results are expressed as the means ⁇ standard deviation of 4 experiments.
  • 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 segment of the alcohol oxidase focus which is necessary for gene replacement.
  • the DH5 ⁇ FTQ 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.
  • Oligonucleotides were synthesised by the solid-phase phosphoramidite method [15] on an Applied Biosystems Synthesizer model 394.
  • PCR amplification was performed on a T. gondii tachyzoites RH strain ⁇ gtl 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 50 ⁇ l reaction mixture containing 10 mM Tris-HCl (pH 8.3), 2mM MgCl 2 , 50 mM KC1, 0.01 % wt/vol gelatin, 200 ⁇ M 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 5 ⁇ ATTCATCATGACCCTGCA3'. 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.
  • 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 ⁇ -mating factor prepro secretion signal sequence of Saccharomyces cerevisiae.
  • 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
  • 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 C 18 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.
  • Rabbits were subcutaneously immunised at one month intervals using 500 ⁇ g of conjugated peptide emulsified in complete Freund's adjuvant for the first injection and Freund's incomplete adjuvant for the second one.
  • 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] .
  • 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.
  • PBMC peripheral-blood mononuclear cells
  • 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 ⁇ -mating factor secretion signal sequence (MF ⁇ ), 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 MF ⁇ 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.
  • 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.
  • 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.
  • GNA lectin Galanthus nivalis agglutinin
  • 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
  • SUBST1TUTE SHEET 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 MF ⁇ 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.
  • Purified recombinant SAGl was further characterised in terms of cellular proliferative capability.
  • polymorphonuclear cells derived from four r.g ⁇ rccf ⁇ ' -seropositive individuals, were isolated then stimulated in vitro either with total soluble antigens of T. gondii or with purified recombinant SAGl.
  • Stimulation Index 4
  • a significant proliferative response 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
  • 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).
  • mice The protective potential of recombinant SAGl was evaluated in a lethal toxoplasmosis mouse model.
  • groups of five BALB/c mice were subcutaneously immunized twice at two weeks intervals with 10 ⁇ g 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.
  • SBASlc adjuvant proprietary composition of SmithKline Biologicals, Rixensart, Belgium
  • mice received adjuvants alone.
  • 15 days after the second injection all mice were challenged with 10 4 tachyzoites of the T. gondii C56 strain administered intraperitoneally.
  • 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 MF ⁇ -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 MF ⁇ - 1 followed by residues 48 to 336 of SAGl for the second one (FIG. 5).
  • the sequence encoding the SAGl hydrophobic carboxy-terminal 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 MF ⁇ -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 ⁇ factor prepro peptide in the P. pastoris expression vector, pIC9K, to give pNIV3488 (FIG. 5).
  • SAGl will be constitutive ly expressed under the control of the ARG3 promotor placed on a 2- ⁇ -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 ⁇ l) 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 .
  • 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 ⁇ l 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.
  • 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 ⁇ - mating factor prepro secretion signal sequence.
  • SAGl was expressed under the control of the ⁇ -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 MF ⁇ .
  • SAGl 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 MF ⁇ 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.
  • the P. pastoris expression system is more efficient for the production of a recombinant SAGl.
  • 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.
  • 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.
  • Example 4 Protective effect of vaccination with recombinant SAGl against congenital toxoplasmosis in Guinea Pig
  • 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.
  • SAGl was produced in Pichia pastoris and purified according to the procedure described in Example 1 above.
  • mice 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.
  • the geometrical mean was 63065 with values between 24226 and 248217.
  • the titers in the mock-immunized group were below the detectable level.
  • 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 5 ⁇ GACAACAATCAGTACTGTTCCGGGAC3' 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 781 bp ZtamHI-Ec ⁇ RI 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.
  • 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.
  • ELISA titer was calculated as the reciprocal dilution giving 50% of the maximal O.D. signal.
  • PBMC Peripheral-blood mononuclear cells
  • 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.
  • UCL Dr Bigaignon
  • IU ELISA VTDAS, Bio-Merieux
  • serum was considered as Toxoplasma seropositive for value > 8 UI.
  • 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.
  • Toxoplasma gondii production of interferon-gamma, inter leukin 2 and strain cross-reactivity. Parasitol. Res. 77: 379-385.

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Abstract

L'invention concerne un procédé de production de l'antigène de toxoplasme SAG1 ou d'un fragment de celui-ci, qui comporte les étapes consistant à: (a) construire un plasmide comportant l'ADN codant pour SAG1 ou pour un fragment de celui-ci ; (b) transformer une cellule hôte de P. pastoris à l'aide dudit plasmide ; et cultiver l'hôte de façon à exprimer l'ADN codant pour SAG1 ou pour un fragment de celui-ci. L'invention a en outre trait à des protéines de SAG1 tronqué dans lesquelles la région d'ancrage est absente, notamment de SAG1 tronqué comportant les résidus d'acides aminés 48-307 de SAG1, et à des compositions de vaccin renfermant SAG1.
PCT/EP1999/003957 1998-06-12 1999-06-08 Production par recombinaison de l'antigene sag1 de toxoplasme WO1999066043A1 (fr)

Priority Applications (3)

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AU45102/99A AU4510299A (en) 1998-06-12 1999-06-08 Recombinant production of toxoplasma sag1 antigen
EP99927922A EP1086228A1 (fr) 1998-06-12 1999-06-08 Production par recombinaison de l'antigene sag1 de toxoplasme
CA002330209A CA2330209A1 (fr) 1998-06-12 1999-06-08 Production par recombinaison de l'antigene sag1 de toxoplasme

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GB9812773.1 1998-06-12
GBGB9812773.1A GB9812773D0 (en) 1998-06-12 1998-06-12 Vaccine
GBGB9908564.9A GB9908564D0 (en) 1999-04-15 1999-04-15 Novel compounds and process
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EP0988862A3 (fr) * 1998-09-21 2001-06-20 Allergy Therapeutics Limited Formulation pour une utilisation en immunisation
WO2001043768A3 (fr) * 1999-12-13 2002-03-07 Smithkline Beecham Biolog Nouvelle composition de vaccin
WO2002095361A3 (fr) * 2001-05-22 2003-08-14 Harvard College Identification d'agents anti-protozoaires
WO2003028760A3 (fr) * 2001-10-01 2004-03-11 Glaxosmithkline Biolog Sa Vaccin
JP2006501826A (ja) * 2002-10-02 2006-01-19 アボット・ラボラトリーズ 遺伝子操作したｐ３０抗原、改善された抗原カクテル及びそれらの使用
WO2007051271A3 (fr) * 2005-11-01 2007-07-26 Fundacao Oswaldo Cruz Construction d'un adenovirus recombinant avec des genes codant pour sag1, sag2 et sag3
US7718178B2 (en) 1997-04-05 2010-05-18 Allergy Therapeutics Limited Allergen formulation
US7790187B2 (en) 2005-03-08 2010-09-07 Kenton S.R.L. Chimeric recombinant antigens of Toxoplasma gondii
US8470331B2 (en) 2000-01-14 2013-06-25 Allergy Therapeutics (Uk) Limited Composition of antigen and glycolipid adjuvant for sublingual administration
WO2020056229A1 (fr) * 2018-09-14 2020-03-19 Prommune, Inc. Compositions immunologiques antiparasitaires
CN116179569A (zh) * 2021-08-13 2023-05-30 郑州伊美诺生物技术有限公司 编码弓形虫sag抗原的核酸及其应用、该抗原的制备方法

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WO1996002654A1 (fr) * 1994-07-13 1996-02-01 Transgene S.A. Cassette d'expression d'une proteine p30 de toxoplasma gondii

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KAMI KIM ET AL.: "Conformationally appropriate expression of the Toxoplasma antigen SAG1 (p30) in CHO cells", INFECTION AND IMMUNITY, vol. 62, no. 1, January 1994 (1994-01-01), WASHINGTON US, pages 203 - 209, XP002117866 *
RALPH BIEMANS ET AL.: "The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation", JOURNAL OF BIOTECHNOLOGY, vol. 66, no. 2/3, 11 December 1998 (1998-12-11), pages 137 - 146, XP004154147 *
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Cited By (20)

* Cited by examiner, † Cited by third party
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US8105605B2 (en) 1997-04-05 2012-01-31 Allergy Therapeutics (Uk) Ltd. Allergen formulation
US7718178B2 (en) 1997-04-05 2010-05-18 Allergy Therapeutics Limited Allergen formulation
US7815920B2 (en) 1998-09-21 2010-10-19 Allergy Therapeutics (UK) Ltd Method of preparing an antigen-containing formulation
US6440426B1 (en) 1998-09-21 2002-08-27 Allergy Therapeutics Limited Antigen-containing formulation and methods of use thereof
EP0988862A3 (fr) * 1998-09-21 2001-06-20 Allergy Therapeutics Limited Formulation pour une utilisation en immunisation
WO2001043768A3 (fr) * 1999-12-13 2002-03-07 Smithkline Beecham Biolog Nouvelle composition de vaccin
US8470331B2 (en) 2000-01-14 2013-06-25 Allergy Therapeutics (Uk) Limited Composition of antigen and glycolipid adjuvant for sublingual administration
WO2002095361A3 (fr) * 2001-05-22 2003-08-14 Harvard College Identification d'agents anti-protozoaires
US7067315B2 (en) 2001-05-22 2006-06-27 President And Fellows Of Harvard College Identification of anti-protozoal agents
WO2003028760A3 (fr) * 2001-10-01 2004-03-11 Glaxosmithkline Biolog Sa Vaccin
JP2006501826A (ja) * 2002-10-02 2006-01-19 アボット・ラボラトリーズ 遺伝子操作したｐ３０抗原、改善された抗原カクテル及びそれらの使用
US7314924B2 (en) 2002-10-02 2008-01-01 Abbott Laboratories Polynucleotide encoding a genetically engineered P30 antigen
US7824908B2 (en) 2002-10-02 2010-11-02 Maine Gregory T Genetically engineered P30 antigen, improved antigen cocktail, and uses thereof
EP1556404A4 (fr) * 2002-10-02 2006-07-26 Abbott Lab Antigene p30 genetiquement modifie, combinaison d'antigenes amelioree et utilisations associees
US7790187B2 (en) 2005-03-08 2010-09-07 Kenton S.R.L. Chimeric recombinant antigens of Toxoplasma gondii
US7867503B2 (en) 2005-03-08 2011-01-11 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. Chimeric recombinant antigens of Toxoplasma gondii
WO2007051271A3 (fr) * 2005-11-01 2007-07-26 Fundacao Oswaldo Cruz Construction d'un adenovirus recombinant avec des genes codant pour sag1, sag2 et sag3
WO2020056229A1 (fr) * 2018-09-14 2020-03-19 Prommune, Inc. Compositions immunologiques antiparasitaires
US11911464B2 (en) 2018-09-14 2024-02-27 Prommune, Inc. Anti-parasitic immunological compositions
CN116179569A (zh) * 2021-08-13 2023-05-30 郑州伊美诺生物技术有限公司 编码弓形虫sag抗原的核酸及其应用、该抗原的制备方法

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AR019864A1 (es) 2002-03-20

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