JPH08512200A - Vaccine composition for cloning, expression and peptide antigen of Toxoplasma gondii - Google Patents

Abstract

(57) Summary The present invention provides a polypeptide corresponding to residues 197 to 216 of the 54 kDa antigen of T. gondii having the amino acid sequence TDPGDVVIEELFNRIPETSV. A method for producing a polypeptide corresponding to the 54 KD antigen of T. gondii or its residues 197 to 216 by expression from a host cell, for example, a host cell composed of vaccinia virus or a CHO cell, is also included in the present invention. It is within the range.

Description

Detailed Description of the Invention Cloning, expression, and response to the peptide antigen of Toxoplasma gondii. Kuching composition   There is a 54 kDa antigen identified and described in WO92 / 11366 Toxoplasma is a parasite responsible for the development of toxoplasmosis in individuals ・ Constituting a potential defense against infections caused by gondii (Toxoplasma gondii) . In WO92 / 11366, T. gondii specific hi T-cell clones have been obtained, one of which is T-cell clone 32 ( The 54 kDa antigen (also known as Tg34) using what is called TCC32) Have been identified.   Identification of the 54KD antigen represents an important advance but still allows for the natural pathway There is a need to present this antigen to the immune system in a manner as close as possible. One In an aspect, the invention relates to an expression system for the 54KD antigen. Another In an aspect, the invention provides an enzyme on the 54KD antigen that is recognized by TCC32. Identification of pitopes and synthesis of polypeptides corresponding to this epitope . In another aspect, the present invention provides a chemical construct incorporating said epitope. It concerns a conjugate or a fusion recombinant protein.   T lymphocytes play a major role in the protective immune response to intracellular pathogens To identify the region of the protein that is recognized by the T cell receptor. A great deal of effort is being put into it. Approaches to designing synthetic vaccines include: Requires proper selection and production of peptides containing immunodominant T cell epitopes To do. Several models have been constructed to predict such antigen segments from the primary sequence. You are a husband.   In order to make the present invention, we have been recognized by human T cell clone 32. Toxoplasma gondii containing an epitope⁽⁷⁾Ku Applying three predictive models to determine the region of the cloned Tg34 protein It was As described in detail below (see Example 1), the epitope is WO92. Of the 54KD antigen of T. gondii described in It was found to correspond to amino acids 197-116.   Therefore, in a first aspect, the invention provides a T. gondii 5 A polypeptide corresponding to residues 197 to 216 of the 4KD antigen ("T cell epitope") I will provide a.   In a related aspect, the invention provides the amino acid sequence TDPGDVVIEELFNRIPETSV. Provide a polypeptide having (where the letters represent standard abbreviations for amino acids) To do. This is amino acid 197 in the 54KD antigen of T. gondii. ~ 216 sequence (see WO92 / 11366).   Preferably, the polypeptide is in purified form, preferably greater than 60% pure, The purity is preferably 75% or more, and 90% or more, for example, 95 to 100. Advantageously, a purity of at least%.   In a preferred embodiment, the polypeptide (T cell epitope) is substantially pure. It is a stylish form.   The polypeptide may also be synthesized by conventional techniques, such as the Merrifield solid phase method. Good.   The epitope identified by the present inventors is a sense of T. gondii. It may form an immunogenic molecule capable of conferring immunity to a disease. In one aspect Is a chemical conjugate that “presents” such epitopes to the immune system in the correct way. It may be a gate.   Accordingly, in another aspect, the invention provides for the chemical attachment of a heterologous molecule. Antibodies against T. gondii after internal administration to humans or animals There is provided a compound consisting of a polypeptide of the invention having the ability to form.   Such chemical conjugates can be prepared using well-known techniques such as Carrent Prototype. Current Protocols in Immunology (1991; By Wiley Intersciences, New York) May be formed.   In another embodiment, recombinant DNA techniques allow the T described herein. It is possible to form hybrid molecules that incorporate cellular epitopes. Literature (Martineau et al. (1991), Bio / Technol ogy)9, 170-179; Rutgers et al. (1988), Bio / Technology Technology)6, 1065-1070), using a technique similar to that already described. To effectively present the T. gondii epitope to the immune system The molecule may be expressed in the form of particles.   Accordingly, the invention comprises a polypeptide of the invention fused to a heterologous antigen, Recombinant proteins capable of forming particles upon expression in a suitable host Also provide.   Preferably, the host is yeast, especially Saccharomyces.  cerivisae).   In a particularly preferred embodiment, the xenoantigen is derived from hepatitis B surface antigen.   The hepatitis B surface antigen is a 226 amino acid sequence optionally containing a pre-S sequence. Mean S protein (For a discussion of the properties of hepatitis B surface antigen, see Ganem, Ganem, D and Barmus, H.E. (1987) Ann Rev. B iochem,55, 651-693).   In a particular embodiment of the invention, the sequence TDPGDVVIEELFNR that follows from the initiation methionine. IPETSV, 13 amino acids from the pre-S2 region of HBsAg and the major HBsAg codon Recombinant protein consisting of 226 amino acids from the coding region (S protein) Quality is provided.   Also, any of the immunologically effective amounts of the present invention mixed with a pharmaceutically acceptable carrier. There is provided a vaccine composition comprising the compound.   The term "immune defense" prevents or alleviates a disease and prevents or slows the spread of the disease. Induce an immune response to T. gondii attack Means the amount needed to   Vaccine manufacturing is generally done by New Torrens & Developments ・ Vaccines (New Trends and Developments invaccines), Voller, etc. (Edit), University Park Press, Mary Baltimore, Land, 1978.   The amount of protein of the invention present in each vaccine dose is At an amount that induces an immune defense response without significant adverse side effects.   Such amount depends on which specific immunogen is used and the vaccine is adjuvanted. It changes depending on whether or not it is done. Generally, each dose is 1-1000 μg, preferably It is expected to consist of 1 to 200 μg of protein. For individual vaccines The optimal dose for standard studies includes observing antibody titers and other responses in subjects. Can be confirmed by. Preferably, after the first vaccination, the subject has about 4 Weekly boost, followed by repeated booths every 6 months while there is a risk of infection Receive   In addition, the present invention needs to prevent T. gondii infection Administration of an immunologically effective amount of the vaccine composition of the present invention to a human or animal To prevent T. gondii infection in humans or animals Provide the law.   As further described in the examples below, the inventors have surprisingly found that WO9 Tea as described herein in a host not described in 2/11366. Expression of 54KD antigen of T. gondii or its T cell epitope Have found that is advantageous. Vaccinia virus as an individual host And M. bovis-BCG. These are raw It has the advantage of being a vehicle. Also, CHO cells are used as host cells. It may be advantageous to be there. These have the advantage of giving stable transformants. Have a point.   Accordingly, in yet another aspect, the invention provides a T. gondii 54KD antigen or its T cell epitope (amino acids 197 to 216). A host cell consisting of a vaccinia virus or M. bovis-BCG By expressing the product and then purifying the product by conventional techniques. 54KD antigen of T. gondii or its T cell epitope (amino acid 1 9 7 to 216) are provided.   The generated antigen may be processed to remove the transmembrane domain. This is It has the advantage that proteins are secreted into the culture supernatant. Full-length protein May produce truncated derivatives in yeast. Typically for transmembrane domain removal Includes removal of most of residues 464-485.   In a related aspect, the invention provides a vaccinia-infected cell, or tea gon. 54 KD antigen of T. gondii or its T cell epitope (amino acid 197 216) to a vector consisting of DNA encoding the 54KD antigen And transformed or transformed M. bovis-BCG host cells. To serve. The individual vectors for this purpose, described below, also form part of the invention.   Hereinafter, the present invention will be described by way of examples and drawings (described below). Real References are listed after the examples.   Example 1: Same Tg34 epitope recognized by human T cell clone 32 Fixed   Berzofsky et al., Called amphipathic helix model⁽¹⁾Who The method is deeply recognized between helper T cell antigenicity and α-helix amphipathicity. Based on mutual relationships. In this model, the antigenic site is one side of the Is recognized by the T cell receptor) and the opposite side where predominance of apolarity (presenting cell surface). Bound to a class I or class II molecule of the major histocompatibility complex on the surface ) It is assumed to be spiral.   Rothbird et al.⁽²⁾The method described in Table 1 shows that most helper and cytotoxic determinants Electric residue or glycine (followed by two or three hydrophobic residues, polar residue Or terminated with glycine).   Kourilsky et al., Called the self-peptidic model^{( 3)} Method of rare tetrapeptides evaluated from the sequence data banks obtained. Based on statistically significant trends of T immunogenic peptides composed of clusters .   Methods and results   1. Prediction of T cell epitopes   Berzovsky's algorithm is adaptable to our computer. Due to some changes, Spouge et al.⁽¹⁾Republished from the publication. The general system of the program is to first convert the amino acid sequence into a hydrophobic ty value) array. Second, the hydrophobic sequence overlaps (residues 7-11 Long). Third, in each part, a regular amphipathic helix structure Examine the matching hydrophobicity periodicity. Fourth, stable amphipathic helix fragments and Identify stable amphipathic helix fragments. In fact, depending on the original program, It was shown that analysis could be performed by selecting windows of 7-11 residues each. Book The inventors used both windows in succession, and the area expected by both windows was Low amphipathic score, predicted from the predicted region and by the window of 11 residues Based on the primary sequence by using different symbols to distinguish from regions with And added the possibility of representing a row of amphipathic fragments.   The Rothbard algorithm consists of four sets of amino acids that make up the proposed form. Was estimated from the publication of (2) above in which The present inventors In the table reporting known epitopes containing either 4 or 5 residues We observed that Tyr may be present at the last position of the motif and therefore contains a polar residue. Tyrosine was added to the fourth set.   In order to implement Kourilsky's algorithm, we first Immune system-related polymorphic protein sequences of T cell receptor, MHC molecule) Protein sequence data van containing only human sequences I made a subset of Ku SWISS PROT. In the second stage, somatic cell self-catalysis of human species This databank subset representing the The number of occurrences for all possible tetrapeptide sequences using the original matrix Recorded. Finally, by submitting the protein sequence to this program, Report to each tetrapeptide found in this sequence by the matrix You have given the corresponding value.   However, different regions of the defined protein are selected by each model. Therefore, we can use the respective prediction methods under the primary sequence of interest. I wrote a program to execute the series of results obtained. This approach uses T cells It increases the likelihood of determinant identification and is expected to fit better with different algorithms. It has been used to focus on those antigenic fragments.   All of the above computer programs are powerful scientific programming languages. Written in FORTRAN.   Submit the primary sequence of the Tg34 protein to this set of 3 programs, Based on the analysis obtained, we found that 3 out of 10 predicted fragments Was chosen to be chemically synthesized. -Fragments 197-216 contain 23.9 and 11 residues for a window of 7 residues Amphiphile predicted by both windows with an amphipathic score of 28.9 for It contains a sex α-helix. Also, it is the two adjacent motifs of Rothbard ( Underlined parts), the first one is a pentamer and the second one is a tetramer. -Fragments 303-410 include 11 of the amphipathic scores of 8.6 and 4.3. Contains two overlapping predicted amphipathic α-helices predicted by residue windows It It also contains two non-adjacent motifs of Rothbird (underlined), and tetra A self-peptide human set of peptides. contains. -Fragments 501-524 are 47.4 for a window of 7 residues, a window of 11 residues The amphipathic α predicted by both windows with an amphipathic score of 54.7 for -Contains helices. In addition, it is three Rothbard motifs (underlined part) , Two of which are contiguous, the first of which is a pentamer and the second of which is Is a tetramer. In addition, the amino-terminal part of this fragment is Give the tetrapeptide with the lowest occurrence in the bank.   2. Synthesis of peptides and conjugates   Fully automatic peptide synthesizer (ABI model 430A, Foss, CA) Merrifield solid phase method using Foster City^(Four)By tert -Butyloxycarbonyl / trifluoroacetic acid (tBoc / TFA) peptide according to strategy Was synthesized. The tBoc-N-a protected amino acid was replaced with tBoc-Ala-OCH₂-Phenylacetamide Methyl (Pam) resin, tBoc-VAl-OCH₂-Pam resin and tBoc-tosyl-Arg-OCH₂-Pam tree The fat was coupled sequentially. Trifunctional amino acids were protected as follows: Ar g (tosyl), Asp and Glu (benzyl ester), Ser and Thr (benzyl ether), Lys (2-chlorobenzyloxycarbonyl), Tyr (di-chloro-benzyl), Trp (formy ), Cys (methylbenzyl) and His (dinitrophenyl). Cleavage at the end of synthesis And thiolysis prior to deprotection (by "low-high" hydrogen fluoride method) The protecting group of His residue was removed by. The crude peptide was added to TSKHW40s (Merc (Me rck), Rahway, NJ, gel filtration and nucleology Nucleosil C₁₈Purified by reverse phase HPLC above. Then, Vyda C) C₁₈Homogeneity by RP-HPLC and thin layer chromatography above , And the peptides for identity by amino acid analysis after complete acid hydrolysis It was   Peptides 197-216, 393-410 and 501-524 are respectively , Dimethyl suberimidate, 1- (3-dimethylaminopropyl) -3-ethyl Carbodiimide and glutaraldehyde^(Five)Bovine serum albumin (BSA ) Is covalently bound to. To remove any uncoupled peptides Characterized the conjugate to determine the peptide / carrier ratio after dialysis into I attached it. For Bio-Rad Protein Assay The amount of protein was evaluated more. During peptide synthesis, we have To determine the number of bound peptides by measuring the total radioactivity of the conjugate To³H-labeled Boc-Leu (Dupont, NEN Research Products (Resear ch Products), Boston, Mass.) were used.   3. T cell proliferation assay   As stated⁽⁶⁾, Allogeneic irradiated PBMC, phytohemagglutinin (PHA) And human T cells by stimulation every 2-3 weeks in the presence of recombinant IL-2. Cell clone 32 was maintained in culture. Antigen presenting cells for proliferation assay⁽⁶⁾age In the presence of cryopreserved cells of all autologous B lymphoblastoid cell lines. T cells were incubated with stock. 3 days later already listed⁽⁶⁾Street To [³Proliferation was assessed by [H] thymidine incorporation.   FIG. 1 shows the dose-response curve of T cell clone 32 obtained with 3 peptides. You As shown therein, the peptide corresponding to residues 197-216 is significantly more significant. 10 growth was detected^-2~ 3 x 10^-2The maximal response of μg / ml was obtained. The other 2 No reaction was observed with this peptide. Fraction F3 or Tg34 lysogen ( Significant growth was observed in the presence of the (positive control) extract, and wild lambda gt11 lysogen No significant growth was observed in the extract of the fungus (negative control). In addition, the proliferation test The conjugate was also tested in the assay. We have found that peptides 197-21 The reaction was observed in the presence of the conjugate corresponding to 6 only (data not shown). Not in).   The amino acid sequence 197 to 216 of the Tg34 protein was determined by T cell clone 32. It can be concluded that it contains a recognized epitope.   Example 2   54kDa Antibodies Limited to Complete or T Cell Epitopes (Amino Acid (aa) 197-216) Construction of Recombinant Vector Retaining Raw (Tg34)   A. Construction of vector for transfer to vaccinia virus   ± 1800 base pairs encoding the 54 kDa antigen (pNIV3401)EcoRI DNA fragment Holds p Bluescript (pBluscript) KS⁺Vector is WO92 / 11366 Example 5 of.   From there, 1556 salts surrounding most of the coding sequence for the antigen BasementNheI-NcoThe I DNA fragment was recovered and the 5'end of the Tg34 DNA sequence was coded for. 59 base pairsBglII-NheI Synthetic double-stranded oligonucleotide (OL1) (Fig. 2A) together with the multi-purpose cloning vehicle pJRD184 (Huter Sprue) Heuterspreute et al., Gene 59,299-304, 1980)BglII-NcoI Department Connected to the rank. The resulting intermediate construct is thenEcoRV andNcoCut with I ,EcoRV (5'-end) andNco3'of Tg34 DNA sequence adjacent to I (3'-end) A second 33 base pair synthetic double stranded oligonucleotide encoding the end (OL2) (Fig. 2A). Therefore, the final construct pNIV3404 has a common 5'start. Array (Kozak, M), Cell44, 283-292, 1986), ATG start codon And the coding sequence for the 54 kDa antigen, including the stop codon (Figure 2B). Carry.   Then, finally, the vaccinia transfer plasmid pULB5213 (standard vaccinia A vector pSC11 derivative; Chakrabati et al., Mol. Cell. Biolog y 5,3404-3409, 1985) which encodes a 54 kDa antigen which is cloned into 164 To recover the 8 base pair DNA module,BglII andEcoRPNIV with V 3404 was cut. The resulting plasmid pNIV3402 is shown in Figure 2C. So This is P_7.5Retains the sequence for Tg34 antigen under the control of the vaccinia promoter To do.   B. Of the transfer plasmid for expression in Mycobacterium bovis-BCG Construction   1. Preparation of starting materials   a) plasmid pNIV2229   pRIB1000 (Thole et al., Infection and Immunity 50, 800-806, 1 985) to 763 base pairsMluI-BamHThe I fragment was isolated. It is P64 (HS P60) contains a putative promoter of Mycobacterium antigen. PUC it 19 plasmidsHincCloning into the II site gave pNIV2229.   b) plasmid pNIV3403   pUC19BamHWith I siteHincPNIV3404 to Tg3 between the II site 4 complete coding sequences of 1600 base pairsBglII-EcoRConverted as V fragment Transferred to obtain pNIV3403.   2. Construction of pNIV3406   PstI-MscThe IDNA fragment was isolated from pNIV2229. It started translating It contains the P64 promoter up to the first base (B) after the ATG. PN it IV3403EcoRWith I siteNheFirst amino acid of Tg34 between I site Was ligated with a synthetic DNA adapter (NATBCG1 / NATBCG2, FIG. 3A) encoding One Come on, Kan from Tn903 (Pharmacia)^RContaining the gene 13 The DNA of 00 base pairs was used as the unique PstI of the resulting plasmid (pNIV3405). Insertion into the site resulted in pNIV3406. This construct is shown in the drawing below (Figure 3B). Enter.   3. Construction of pNIV3407   Temperate mycobacteriophage FRAT1 (in our laboratory Pre-isolated, Haeseleer, Timm and Jacobs bs), 1989, Acta Leprologica 7 (supra 1) 252-253) derived from 1,500 base pairs. SmoothingBamHBy inserting into the unique SmaI site of pNIV3406 of the I fragment , Plasmid pNIV3407 is obtained.   C. Tg34 T cell epitope (aa197-216) and hepatitis B virus (HSV ) Is expressed in yeast as particles obtained by fusion with the pre-S2-S antigen. Construction of vector for   HBsAg as a carrier for presentation of Tg34 T cell epitopes to the immune system Encode Tg34 T cell epitope (aa196-216) for use in particles To the upstream of the DNA encoding the pre-S2 gene (aa135-145) and Fused with the complete sequence for the S protein of HBV (aa 175-400) (Fig. 4A). Amino acid residues 133-145 of the pre-S2 protein and the HBV Sta A 722 base pair DNA fragment corresponding to amino acid residues 175-400 of the protein To recover the plasmid TCR50 (pRIT13220)BamHI andEcoR Digested with I (Cabezon et al., Vaccines 90, 199-203, 1990). Tg DNA encoding the sequence for the 34 T cell epitope (aa196-216) The sequence was reconstructed and the fragment obtained aboveNcoI andEcoRWas cut by I In order to provide a bond between plasmid FF6 (pRIT13717), a synthetic plasmid was added. Rigonucleotides (64mer) were used. The pRIT13717 vector is A Yeast Glyceralde followed by a yeast DNA fragment carrying the RG3 transcription terminator It contains the hydr-3P-dehydrogenase (TDH3) promoter fragment (Fig. 4). B). The 2980 base pair expression cassetteBglII-SalCut out as I fragment , PNIV34 by inserting into yeast shuttle vector OJ10 (pRIT12741) 09 was obtained (FIG. 4C). The pRIT12741 vector is yeast 2-micron DN A and yeast LEU2 as a selectable marker in yeast (harfor Do et al., Postgrad. Med. J. 63, 65-70, 1987). inserted in pNIV3409 The cassette is expected to express a protein of 260 amino acids. The high The brid protein is a Tg34T-cell epitope starting from the initiation methionine. 20 amino acids, 13 amino acids from the pre-S2 region and the major HBsAg It consists of 226 amino acids from the coding region.   D. Vector construction for expression in CHO cells   a) 54KDa antigen   Starting with plasmid pNIV3404 (see Example 2A), digestion with 164 8 base pairsHindIII-EcoRThe V DNA fragment was recovered, and HindIII and Sm pEE14 cleaved with aI (Cockett et al., 1990, Bio / Technology, 8, 662-667) -derived pRIT14073 eukaryotic expression vector. The resulting expression The plasmid pNIV3412 (Fig. 5) was used for human cytomegalovirus (hCMV- MIE) under control of the major immediate-early promoter 54 described in Example 2A. It contains the sequence encoding the KDa antigen.   b) Truncated 54KDa antigen   The amino acid sequence of the 54 KDa antigen is a putative membrane-scaffold domain spanning residues 464-485. Contains mains, which may inhibit secretion of recombinant proteins by host cells. unknown. We have found that the sequence corresponding to residues 464-485 has been deleted. A DNA encoding a form of 54 KDa antigen was constructed. Its construction is as follows went. 1356 base pairsHindIII-SfcThe I DNA fragment was transformed into plasmid pNI. Recovered from V3402 (see Example 2A),SfcI-SmaI synthetic D Together with the NA fragment (Fig. 6a),HindIII andSmaPRIT14 cut with I Introduction into the 073 expression vector gave immediate constructs. Then this construct with SmaI Digest and polymerase chain reaction (primers 1 and 2, pNI as template) DNA of V3402, 151 base pairs obtained by FIG. 6b)HpaI-EcoRI DN The A fragment was accommodated at this site by ligating it. Final construct pNIV34 14 is a truncated form 5 lacking residues 464-485 under the control of the hCMV promoter. It encodes a 4 KDa antigen (Fig. 6c).   Example 3   Limited to complete or T cell epitopes in host cells (aa197-216) Of the expressed 54kDa antigen   A. Expression in eukaryotic cells via vaccinia virus recombinants   The recombinant transfer plasmid pNIV3402 was transfected into vaccinia-infected CV-1 cells. Transfect to a blue color in the presence of bromo-uridine selection and X-gal. Recombinant virus was isolated after basal plaque purification. They are VV3402 Is called. Human H143 fibroblast TK strain is preferred for plaque assay Good The vaccinia virus used to infect the cells is of the WR type (origin : Borysiewicz, UK). The operation is vaccinia Methods already described for obtaining virus recombinants (Macette M (Mac Kett M) and Smith G.L., J. Am. Gen. Virology 67, 2067- 2082, 1986; Macckett M, Smith G.L. And Moss B., J. Virology 49, 857-864, 1984).   Using recombinant vaccinia virus VV3402 in culture at multiplicity of infection 1 CV-1 cells were infected. 18-48 hours after infection, infected cells (approximately 3.10^Five/ Assay) and spent medium (about 2 ml) were collected. Presence of 12% sodium dodecyl sulfate Proteins were separated by existing polyacrylamide gel electrophoresis and After transferring to a Rohs membrane and subjecting it to immunodetection, the presence of the 54kDa antigen was confirmed. I accepted. Details of these techniques are described in WO92 / 11366.   B. Expression in M. bovis-BCG   Plasmids pNIV3406 and pNIV3407 were cloned into M. bovis ) Electroporated into BCG. The first plasmid is its linear form Used as a state (ScaThe second plasmid is used as a circular molecule. I used it. Described in subtitles A13 and A15 of WO92 / 11366 In order to express it by the immunodetection method, M. bovis-BCG recombinants were analyzed.   C. Expression in yeast   1. The plasmid pNIV3409 was used to transform S. cerevisiae. siae) strain DC5 (his3-5, leu2-3, leu2-112, can1-11cir °, Harford and Peeters, Curr. Genet. 11: 315 -319, 1987) to Leu⁺Phenotypically transformed. The transformant was named Y420. . One of them is 40 ml of yeast nitrogen base with 2% glucose as carbon source. Grow to medium exponential growth phase in small medium. Wash cells and collect by centrifugation Then, 300 ml of Tris-HCl 25 mM pH8, 4 mM EDTA, 1% Tween (Tween) 20, 4 mM phenylmethylsulfonyl fluoride, 10% propanol Grinded with glass beads in Lu-2. 30 minutes of the homogenate at 30,000 × g During centrifugation, a crude extract was obtained. 12.5 of supernatant (± 50 μg of total protein) Analyzed on a% SDS-polyacrylamide gel. Then the separated protein Quality of nitrocellulose (Towbin et al., Proc. Natl. Acad. Sci. USA 7 6: 4350-4354, 1979) and blotted onto HBsAg (mAb HBS1, Sumi) SmithKline Beecham or pre-S2 region (mAb For S2.5, SmithKline Beecham The sheet was incubated with the provided monoclonal antibody. Primary antibody test Output was performed by the alkaline phosphatase method. Alkaline Phosphatase and Condyl Conjugated anti-IgG secondary antibody with NBT (nitroblue tetrazolium) and Detected by BCIP (5-bromo-4-chloro-3-indolyl phosphate) did. A protein of approximately 28 kDa that is not present in the negative control (Y1017) Appeared with HBSI and S2.5. From these results, the yeast strain Y420 , The 20 amino acids of the Tg34 T cell epitope starting from the initiation methionine, Les From amino acids 133-145 from the S2 region and the main HBsAg coding region Expresses a fusion protein of 226 amino acids with a molecular weight of 28 kDa.   2) Particle formation   Strain Y42 until late log phase in selective medium (800 ml + 80 μg / ml histidine) 0 was propagated. The cells were collected by centrifugation, washed with cold PBS and washed with 5 ml of 50 mM lysate. Sodium phosphate buffer (pH 8.1), 4 mM EDTA (ethylenediamine tetra Acetic acid and disodium salt), 1% Tween 20 (polyoxyethylene) Sorbitan monolaurate), 4 mM PMSF (phenylmethylsulfonyl) Fluoride) and 10% isopropanol. French pressi The cells were dissociated by passing twice through the jar cell at 20,000 psi. The suspension The liquid was centrifuged at 30,000 xg for 30 minutes. Check the total protein concentration in the supernatant. Bradford dye-binding procedure Bradford M. (1976) Anal. Biochem. 72-248) Measured by the Bio-Rad protein assay (n ° 500-0001) Decided Behringwerke (Marburg, Germany) A more commercially available ELISA kit (Enzygnost-HBsAg Micro (Enzygnost-HBsAg micro)) was used to assay for the presence of HBsAg.   Add 7 ml of the crude cell extract to 1.5 in a Beckman 60Ti rotor. M CsCl, 25mM sodium phosphate buffer (pH 7.4) at 40,000 xg Centrifuged for 68 hours until equilibrated. The collected fractions were analyzed by the above-mentioned ELISA method. Was assayed for the presence of HBsAg. HBsAg is 1.22g / cm³Equilibrate to It has been found. ELISA results confirmed by immunoblot analysis of gradient fractions (FIG. 7A).   0.5 ml of material from the CsCl maximal fraction dialyzed against PBS was added to SW40 lo A speed sucrose gradient was applied for 13 hours at 38,000 g in a water bath. ELISA Enzygnost-HBsAg micro) and mAb HBS Immunoblotting with HBsAg antigenic and hybrid targeting, respectively, Each fraction was analyzed for the presence of proteins (Figure 7B). The result is a hybrid It was shown that the proteins assemble into lipoprotein particles.   C. Expression in yeast   3) plasmid pNIV3417, pNIV3421 and pNIV3423 Use S. cerevisiae strain DC5 to Leu⁺Phenotypically transformed . The transformants were designated YN479, YN478 and YN481, respectively. . Culture and analysis of crude extracts were performed as described above. T in the protein in the medium Precipitated with CA (trichloroacetic acid).   D. Expression in CHO cells (stable transformants)   a) 54kDa antigen   1.25 x 10⁶Plasmid pNIV3 using 20 μg DNA per cell 412 was transfected into CHO-K1 cells by co-precipitation with calcium phosphate. I did. Grow the CHO-K1 cells in GMEM-S medium (Gibco) Let Two days after transfection, 25 μM methionine sulfoximine was added. Transfectants were selected by adding. 10-14 days later, resistant roller Pick up the knee, 96-well plate, then 24-well plate, then 80 cm² Transferred to a flask. G for 54 KDa antigen when cells reached approximately 80% confluence S transfectants were assayed. Cockett et al., Bio / Techno The method described in Bio / Technology 8,662-667 (1990) was followed.   b) Truncated 54KDa antigen   The method for expressing the truncated 54KDa antigen was according to the method described above.   E. FIG. Vector construction for expression of 54KD antigen in yeast   pNIV3402 to 1648 base pairsHindIII-BglII Turn the DNA fragment Bam that has been collected and blunt-ended with the expression vector pRIT13145 (TCM97). The HI site was introduced with blunt ends in the correct orientation for expression (Figure 7). Got Plasmid pNIV3417 is used in Example 2A under the control of the ARG3 promoter. It contains the sequence encoding the described 54KD antigen.   By its own signal sequence from the yeast S. cerevisiae Secretion of a heterologous protein may occur in some (but not all) cases To Was observed. However, the signal sequence of the yeast secreted protein is Its secretion was best achieved by fusion to the coding region (romano). Romanos et al. (1992) Yeast 8: 423-488). Yeast pheromone MFα-1 The signal sequence of has proved to be particularly productive in this respect (Brake ( 1989), Yeast Genetic Engineering 269-280).   In this way, we continue to amino acid residues 28-538 of Tg54. A DNA encoding the signal sequence of MFα-1 was constructed. The construction is as follows I went. The plasmid pNIV3402Rsal andSalDigest with 1 and 470 salt The base pair DNA fragment was recovered. The signal sequence of MFα-1 (19 amino acids) And the fragment obtained above by reconstructing the DNA sequenceHindIII-SalBy 1 Synthetic oligos to confer a bond between the digested plasmid pUC19. Nucleotides (65mer) were used (Fig. 8a). Then from pNIV3402 ofSall-BglII to insert the DNA fragment, the resulting intermediate constructSall andBamHCleavage with I gave pNIV3416 (Fig. 8b). thisHindII I andEcoRThe DNA fragment of 1626 base pairs was recovered by digestion with It was cleaved and introduced into the blunted BamHI site of pRIT13145 (TCM97). It was Therefore, the final construct pNIV3421 regulates the ARG3 promoter. Below, the signal sequence of MFα-1 fused to residues 28-538 of the 54KD antigen was cloned. (Fig. 8c).   The putative membrane-scaffold domain (residues 464-485) of the 54KD antigen is a recombinant tamper protein. In order to suppress the secretion of the cytoplasm, we have determined that residues 464 to 485 of the Tg54 antigen are Encodes a truncated MFα-1-Tg54 antigen lacking the sequence corresponding to The DNA was constructed. Thus, Sal1- from pNIV3414BglII DNA fragment,Sal1 andBamHPNIV3415 cleaved with I was introduced into p NIV3422 was obtained (Figure 9a). Then, the obtained plasmidHindIII Oh And digestion with EcoRI to recover a 1400 base pair DNA fragment and blunt end And blunted pRIT13145 (TCM97).BamHIntroduced at I site did. The resulting expression plasmid, pNIV3423, regulates the ARG3 promoter. MFα-1 following residues 28-465 and 486-538 of the 54KD antigen. It contains a sequence encoding the signal sequence of (Fig. 9b).   References   1. Margalit H., Spouge J. L.), Cornette J.L., Shease K. Bee  K.B.), Delisi C. and Berzovsky J.A. zofsky J.A.) (1987) Prediction of immunodominant helper T cell antigenic sites from primary sequences. Sou (Prediction of immunodominant helper T cell antigenic sites from the primary sequence) J. Immunol. 138, pp 2213-2229.   2. Rothbard J.B. and Taylor W. Taylor W.R. (1988) Sequence patterns common to T cell epitopes (As sequence pattern common to T cell epitopes) EMBO J., 7, pp 93-100.   3. Claverie J-M., Kourillski P rilsky P.), Langlade-Demoyen P., Cha Chalufour-Prochinicka A., Dadaglio Gee ( Dadaglio G.), Tekaia F., Plata F. and Bouguerlet L. (1988) T immunogenic peptides are rare It consists of column patterns. Of the T epitope in the human immunodeficiency virus gag protein Use in identification (T-immunogenic peptides are composed of rare sequence  patterns. Use in the indentification of T epitopes in the human imunode ficiency virus gag protein) Eur. J. Immunol. 18, pp 1547-1553).   4. Kent S.B. (1988) Chemistry of peptides and proteins Chemical synthesis of peptides and proteins Annu. Rev. Biochem., 57, pp 957-985.   5. Van Regenmort M.H.V. , Briand J.P., Muller S. ) And Plaue S. (1988) Synthetic polypeptides as antigens (Synt hetic polypeptides as antigens), Elsevier edition, pp 95-130. 1991) Human T cell clone against Toxoplasma gondii: interferon -Gamma, interleukin-2 production and strain cross-reactivity (Human T-cell clones  againstToxoplasma gondii: Production of interferon-gamma, interleukin 2, and strain cross-reactivity) Parasitol. Res., 77, 379-385.   7. Saarvedra R., De Meuter F., To T cell clones were cloned and expressed in E. coli Toxopraz Identifies a potentially protective 54 kilodalton protein antigen of Ma Gonji ( Human T-cell clone identifies a potentially protective 54-kilodalton pro tein antigen ofToxoplasma gondii cloned and expressed inE. coli.) J. Im munol. 147, 1975-1982 (1991).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI C12N 1/19 8828-4B C12N 1/19 5/10 9637-4B C12P 21/02 C C12P 21/02 9281- 4B C12N 5/00 B // (C12N 1/19 C12R 1: 865) (C12P 21/02 C12R 1:91) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), JP, US (72) Inventor Boran, Alex Belgium 1440 Nibel, Ryu de Landistri No. 24, Servis De Genetic Aprique, Faculté de Suyans, Université Libre du Brussels (72) Inventor Jacob, Paul Baie Guy countries based -1440 Niberu, Ryu de Interview Run District No. 24, Seruvisu de Interview, Jenetiku-applique, Fakyuru Te de Suiyansu, Yuniberushite rib Les du Brussels

Claims (1)

[Claims]   1. Paired with residues 197-216 of the 54 KD antigen of T. gondii Corresponding polypeptide.   2. A polypeptide having the sequence TDPGDVVIEELFNRIPETSV.   3. The polypeptide of claim 1 or claim 2 in substantially purified form.   4. 5 of T. gondii characterized by lacking the transmembrane domain A polypeptide corresponding to the 4KD antigen.   5. It is chemically bonded to a heterologous molecule, and after being administered internally to humans or animals, Tigo 4. The method according to claim 1, which has the ability to form an antibody against T. gondii. A compound comprising the polypeptide according to item 1.   6. A polypeptide according to claim 1, 2, 3 or 4 fused to a heterologous antigen. Recombinant protein having the ability to form particles upon expression in a suitable host quality.   7. The recombinant protein according to claim 6, wherein the heterologous antigen is derived from hepatitis B surface antigen. .   8. The recombinant protein according to claim 6 or 7, which is expressed in yeast.   9. A sequence according to claim 2, which follows from the initiation methionine, the pre-S2 region of HBsAg? 13 amino acids and 226 amino acids from the major HBsAg coding region. The recombinant protein according to claim 8, which comprises noic acid.   10. An immunologically effective amount in admixture with a pharmaceutically acceptable carrier. A vaccine composition comprising the compound according to any one of items.   11. Humans in need of preventing T. gondii infection or By administering to an animal an immunologically effective amount of the vaccine composition of claim 10. A method of preventing T. gondii infection in humans or animals.   12. 54 KD antigen of T. gondii or its T cell epitome (Amino acids 197 to 216) to vaccinia virus or M. bovis (M ． bovis) -expressed from a host cell consisting of BCG, and then the product is subjected to conventional techniques. 54 KD antigen of T. gondii by purification by surgery Was Is a method for producing the T cell epitope (amino acids 197 to 216).   13. 54 KD antigen of T. gondii or its T cell epitome Vector consisting of DNA encoding amino acid (amino acids 197 to 216). Sfected or transformed vaccinia infected cells or M bovis ( M. bovis) -BCG host cells.   14. The 54 KDa antigen of T. gondii or its truncated form is C Expression from HO host cells and then purifying the product by conventional techniques. Method for producing T. gondii 54KDa antigen or truncated form thereof .   15. The 54 KDa antigen of T. gondii or its truncated form CHO cells transfected with a vector consisting of the encoding DNA.   16. The 54 KDa antigen of T. gondii or its truncated form Yeast cells transfected with a coding vector.