Chimeric Recombinant Antigens Of Toxoplasma Gondii

Abstract

The invention described herein relates to a method for combining antigen fragments of Toxoplasma gondii proteins, in the form of chimeric fusion products, and their use as diagnostic and immunogenic agents.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 11/885,951 filed on Sep. 7, 2007, which, is the national phase of International Application PCT/EP2006/001760 filed on Feb. 27, 2006, which, in turn, claims priority to European Patent Application No. 05005065.7 filed on Mar. 8, 2005. This application may also be related to U.S. patent application Ser. No. 11/899,754 filed on Sep. 6, 2007. U.S. patent application Ser. No. 11/885,951 is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention described herein relates to the technical field of the preparation of diagnostic means not applied directly to the animal or human body. The invention also furnishes compounds, methods for their preparation, methods for their use and compositions containing them, which are suitable for industrial application in the pharmaceutical and diagnostic field, particularly for the detection and diagnosis of Toxoplasma gondii infections, as well as for the treatment and prevention of said infections.

BACKGROUND OF THE INVENTION

[0003] Early diagnosis is a priority and highly desirable objective in all fields of medicament, particularly because it allows an appreciable improvement in the patient's life and a concomitant saving on the part of health care systems or on the part of the actual patients. In the particular case of the invention described herein, early diagnosis is that of potential or existing Toxoplasma gondii infection in pregnant women, with particular concern for the health of the foetus, and in infected subjects, particularly those with impaired immunity.

[0004] Toxoplasma gondii is an obligate intracellular parasite that infects all mammalian cells, including those of human subjects (McCabe and Remington, N. Engl. J. Med. 1988, 318-313-5). Morphologically, the parasite exhibits three distinct forms of infection: tachyzoite (asexual), bradyzoite (in tissue cysts, asexual) and sporozoite (in oocysts, sexual reproduction). Transmission typically occurs through ingestion of undercooked meat harbouring tissue cysts or vegetables contaminated with oocysts shed by cats. Human infection is generally asymptomatic and self-limiting in immunocompetent hosts. In contrast, in subjects with impaired immunity (particularly those affected by AIDS), toxoplasmosis is a severe opportunist infection, which may give rise to encephalitis with very serious outcomes (Luft, B. J., Remington J. S., 1992, Clin. Infect. Dis. 15, 211-22). Moreover, contracting primary infection during pregnancy may lead to miscarriages or to severe foetal disease in mammals.

[0005] For an extensive overview of the problem of toxoplasmosis the reader is referred to the specific medical literature.

[0006] Diagnosis of T. gondii infection is established by isolating the micro-organism in the blood or body fluids, identifying the parasite in tissues, detecting specific nucleotide sequences with PCR, or detecting specific anti-T. gondii immunoglobulins produced by the host in response to the infection (Beaman et al., 1995 Principles and Practice of Infectious Diseases 4th Ed., Churchill Livingstone Inc., New York, 2455-75; Remington J S et al. 1995, Infectious Diseases of the Fetus and Newborn Infant, W.B. Saunders, Philadelphia, Pa., 140-267).

[0007] Main challenges for clinicians are the diagnosis of primary T. gondii infections in pregnant women and the diagnosis of congenital infection in their newborns/infants. In both cases, to implement suitable therapies in good time and to avoid possible damage to the foetus and newborns/infants, it is very important to establish if the parasitic infection has been contracted before or after conception in pregnant women. Moreover, it is essential determining when the vertical transmission from the mother to the foetus occurred. Finally, for the clinical management of newborns/infants there is an urgent need of a sensitive diagnostic method than can discriminate, early in their life, between infected and uninfected subjects, both born to mothers with primary toxoplasmosis in pregnancy.

[0008] Seroconversion during gestation and diagnosis of congenital infection in neonates are generally done by attempting to detect the presence of the various classes of anti-Toxoplasma immunoglobulins (IgG, IgM, IgA, avidity of IgG), and to compare the immunological profiles of the mother versus her child. However, the available commercial assays do not provide enough sensitivity and specificity to allow a correct diagnosis of infection in all patients. Therefore the availability of specific, sensitive and innovative diagnostic agents is desirable.

[0009] T. gondii antigens have long been known and available, first of all as antigen mixtures obtained in various ways (FR 2,226,468, Merieux; SU 533376, Veterinary Research Institute; JP 54044016, Nihon Toketsu Kanso), then as subsequent isolations of pure antigens (EP 0 082 745, Merieux; EP 0 301 961, INSERM, Pasteur; WO 89/5658, Transgene) and their characterization both as proteins, and of their respective genes (WO 89/08700, U. Leland, Dartmouth Coll.; U.S. Pat. No. 4,877,726, Res. Inst. Palo Alto; WO 89/12683, INSERM, Pasteur; EP 0 391 319, Mochida Pharm.; IT 1,196,817, CNR; EP 0 431 541, Behringwerke; WO 92/01067, CNRS; WO 92/02624, U. Flinders; WO 92/11366, Innogenetics, Smithkline Beecham; U.S. Pat. No. 5,215,917, Res. Inst. Palo Alto; WO 92/25689, FR 2702491, INSERM, Pasteur; WO 96/02654, bioMerieux, Transgene; EP 0 710 724 Akzo; EP 0 724 016, bioMerieux; EP 0 751 147, Behringwerke; U.S. Pat. No. 5,633,139, Res. Inst. Palo Alto; WO 97/27300, Innogenetics; U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto; WO 99/32633, Heska; JP 11225783, Yano; WO 99/61906, Abbott; WO 99/66043, Smithkline Beecham; JP 2000300278, Yano; WO 00/164243, Virsol), and finally, the isolation and characterization of the antigenic regions of Toxoplasma gene products (WO 03/080839, Kenton S. r. l.)

[0010] Numerous studies have found various different antigenic proteins of T. gondii and the gene sequences of these have also been determined.

[0011] Among the most interesting proteins both for diagnostic and therapeutic purposes, in the form of vaccines, we should mention: the microneme proteins (WO 03/080839, Kenton S. r. l.; Beghetto et al., The Journal of Infectious Diseases, 2005, 191:637-645; Beghetto et al., International Journal for Parasitology, 2003, 33:163-173); the surface antigens SAG1 (or P30) (WO 89/08700, Stanford University; WO 89/12683 Pasteur, INSERM; WO 94/17813, WO 96/02654, Transgene, bioMerieux; EP 0 724 016, WO 99/61906, U.S. Pat. No. 5,962,654, Harping et al., Clinical and Diagnostic Laboratory Immunology, May 1996, 355-357) and SAG2 (or P22) (Permley et al., 1992, J. Clin. Microbiol. 30, 1127-33); the dense granule proteins GRA1 (or P24) (EP 0 301 961, Pasteur, INSERM; WO 89/05658, Transgene, Cesbron-Delauw, et al., 1989 P.N.A.S. USA 86, 7537-41), GRA2 (or P28) (WO 93/25689, INSERM, Pasteur; U.S. Pat. No. 5,633,139, U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto; Prince et al., Mol. Biochem. Parasitol., 34 3-14), GRA4 (Mevelec et al., Mol. Biochem. Parasitol. 56, 227-38), GRA6 (or P32) (FR 2,702,491, INSERM, Pasteur; Lecordier al., Mol. Biochem. Parasitol. 70, 85-94), GRA7 (WO 99/61906, Abbott; Jacobs et al., Mol. Biochem. Parasitol. 91, 237-49) and GRA3 (Robben et al. 2002, J. Biol. Chem. 277, 17544-47): the rhoptry antigens ROP1 (or P66) (U.S. Pat. No. 5,976,553, U. Leland; EP 0 431 541, Innogenetics) and ROP2 (or P54) (Sharma et al., J. Immunol., 131, 377-83).

[0012] As described in the above-mentioned references, the antigens were obtained with recombinant cDNA techniques in expression vectors. For example, for the antigen SAG1, WO 98/08700 uses known expression vectors in phage lambda-gt11. WO 98/12683 uses the same phage and transfects E. coli with a proprietary plasmid, or by preparing a special expression cassette, as in WO 96/02654. EP 0 724 016 obtains mimotopes using combinatorial expression libraries of peptides. EP 0 301 961 describes how to obtain excretion-secretion antigens with molecular weights ranging from 20 kDa to 185 kDa. WO 89/05658 describes a protein containing the epitopes of the 24 kDa protein recognized by the antibodies produced against Toxoplasma excretion-secretion antigens; this protein is obtained by transfection of cells by means of expression vectors. WO 03/080839 describes a method based on phage-display technology for identifying antigen fragments of T. gondii proteins and their use as diagnostic and immunogenic agents. The antigen P28 (GRA2) is described in U.S. Pat. No. 5,633,139 and the method of obtaining it is again implemented through expression in phage lambda-gt11. The antigen P32 (GRA6) is described in patent FR 2,702,491, the antigen ROP1 (P66) in U.S. Pat. No. 5,976,553, P35 (or GRAB) in EP 0 431 541, WO 99/57295 and WO 99/61906, and lastly P68 in EP 0 431 541.

[0013] Yang et al. (Parasitol. Res., 2004, 92: 58-64) describe a chimeric protein containing SAG1 and SAG2 and its use to develop immunity against T. gondii in mice.

[0014] Chinese Patent 11 94991C discloses a recombinant fusion protein containing two toxoplasma antigens (GRA6 and p30). No data are reported to show that assays based on this recombinant fusion protein display the required sensitivity in IgG- and IgM-based tests.

[0015] During the last ten years, several studies have reported the use of recombinant antigens for the serological diagnosis of T. gondii infection. Nevertheless, although promising none of the assays based on recombinant antigens displayed all the characteristics required to replace the tachyzoite antigen in IgG- and IgM-based tests, indicating that further work is needed before an immunoassay employing recombinant products will be available for clinical purposes.

[0016] Thus the main aim of the studies in this field is to improve the performance of enzyme-linked immunoassays based on recombinant products, thus improving, for example early diagnosis of congenital toxoplasmosis in newborns/infants.

SUMMARY OF THE INVENTION

[0017] It has now been found that the combination of antigenic regions of Toxoplasma gondii proteins, in the form of recombinant fusion products, retains the antigenic properties of the individual antigen fragments. The corresponding chimeric proteins thus produced can be used for diagnostic and therapeutic purposes.

[0018] The use of said chimeric antigens as diagnostic agents and the related diagnostic aids containing them, for example in the form of enzyme-linked immunoassays or kits, constitute a further object of the present invention.

[0019] Another object of the present invention are the gene sequences coding for the above-mentioned chimeric antigens, their use as medicaments, particularly for the prevention and therapy of Toxoplasma gondii infection, e.g. as gene therapy. The present invention also extends to the gene sequences that hybridize with the sequences of the above-mentioned chimeric antigens in stringent hybridization conditions.

[0020] Another object of the present invention is the use of the chimeric antigens as medicaments, particularly in the form of vaccines, which are useful for the prevention and cure of the infection. The vaccines according to the present invention are suitable for use in humans and other animals (particularly pig, cat, sheep).

[0021] These and other objects will be illustrated here below in detail, also by means of examples and figures.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The main object of the present invention is, therefore, the provision of recombinant chimeric antigens obtained through the fusion of different antigenic regions of Toxoplasma gondii gene products, and the use of the recombinant proteins thus obtained for developing selective diagnostic and therapeutic means.

[0023] The main advantages of the present invention over the other types of antigens or antigen fragments known in the literature as reported above are the following and are evident when these antigens are used in diagnostic immunoassays on sample sera for detection of the infection: [0024] With respect to the use of the entire Toxoplasma gondii antigen, prepared from the parasite as lysed, whole-cell extract, the chimeric recombinant antigens have the advantage of avoiding unspecific reactions due to the presence of other non-proteinaceous material and of providing a better reproducibility. Moreover, some natural protein antigens of the parasite are insoluble and, consequently, are poorly represented in commercial assays employing the lysed, whole-cell extract of T. gondii. [0025] With respect to the use of single antigenic regions or single antigen fragments (as described in WO 03/080839), the recombinant chimeric antigens show the advantage of improving the sensitivity of the assays in which they are used. In other words their use decreases or abolishes the occurrence of false negative responses. [0026] With respect to the use of a mixture or a collection of single antigenic regions (as also envisaged in WO 03/080839), the advantages are least two. From the point of view of the industrial applicability and production is much easier to produce a single engineered construct containing three or more antigen regions rather than separately produce each single fragment and subsequently assemble them by an economic and reproducible method. Secondly, as already said before, the use of the chimeric recombinant antigens of the invention improves the sensitivity of the assays.

[0027] These and other advantages are shown in the Examples section.

[0028] In particular the present invention relates to a chimeric recombinant antigen containing the fusion at least three different antigenic regions of Toxoplasma gondii, wherein said antigenic regions are B-cell epitopes, which bind to Toxoplasma gondii-specific antibodies. Preferably the Toxoplasma gondii-specific antibodies are extracted from sera of subjects who have been infected by Toxoplasma gondii.

[0029] More particularly the present invention covers a chimeric antigen, wherein the three different antigenic regions have an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42. Preferred sequences in the above group are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

[0030] For example the chimeric antigen of the invention comprises the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence of SEQ ID NO: 32.

[0031] The chimeric antigens of the present invention may be engineered using known methods. The fusions may be direct (the C-terminus of one amino acid sequence is linked to the N-terminal of the other through a simple covalent bond) or they may employ a flexible linker domain, such as the hinge region of human IgG, or polypeptide linkers consisting of small amino acids such as glycine, serine, threonine or alanine, at various lengths and combinations. For example the linker may be a polyglycine repeat interrupted by serine or threonine at a certain interval. Preferably, the linker is composed by three glycine residues and two serine residues, giving the aminoacid sequence Ser-Gly-Gly-Gly-Ser (SGGGS linker) (SEQ ID 43).

[0032] Additionally, the chimeric antigens of the invention may be tagged by His-His-His-His-His-His (His6), to allow rapid purification by metal-chelate chromatography, and/or by epitopes to which antibodies are available, to allow for detection on western blots, immunoprecipitation, or activity depletion/blocking in bioassays.

[0033] Another object of the present invention is a nucleotide sequence coding for the chimeric antigen as defined above. According to a preferred embodiment of the invention such nucleotide sequence comprises at least three different nucleotide sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. According to a more preferred embodiment, such nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 27 or the nucleotide sequence of SEQ ID NO: 29 or the nucleotide sequence of SEQ ID NO: 31.

[0034] Also included in the scope of the present invention are nucleotide sequences that hybridizes with any sequence described above under stringent hybridization conditions, as well the corresponding chimeric recombinant antigen encoded by such hybridized nucleotide sequence.

[0035] The chimeric antigens of the present invention may be prepared by cloning and expression in a prokaryotic or eukaryotic expression system, using the appropriate expression vectors. Any method known in the art can be employed.

[0036] For example the DNA molecules coding for the antigens of the invention are inserted into appropriately constructed expression vectors by techniques well known in the art (see Sambrook et al, 1989). Such vectors are another object of the present invention.

[0037] In order to be capable of expressing the desired protein (in this case the chimeric antigens), an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulatory information linked to the DNA coding the desired protein in such a way as to permit gene expression and production of the protein. First in order for the gene to be transcribed, it must be preceded by a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters).

[0038] For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene, which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated. All these hosts are a further object of the present invention.

[0039] Nucleic acid molecules which encode the chimeric antigens of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein. Such combined nucleic acid molecules are included within the embodiments of the invention. For example, they may be joined to the DNA coding for a protein which allows purification of the chimeric antigen by only one step of affinity chromatography. This joined/fused protein may be for example Glutathione Sulpho Transferase (GST) to generate fusion products at the carboxy terminus of GST protein. The corresponding recombinant proteins expressed in the cytoplasm of transformed E. coli cells may be purified by affinity chromatography using a Glutathione-Sepharose resin.

[0040] The DNA molecule comprising the nucleotide sequence coding for the chimeric molecule of the invention is inserted into vector(s), having the operably linked transcriptional and translational regulatory signals, which is capable of integrating the desired gene sequences into the host cell. The cells which have been stably transformed by the introduced DNA can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may also provide for phototrophy to an auxotropic host, biocide resistance, e.g. antibiotics, or heavy metals such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins of the invention.

[0041] Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells, that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0042] Once the vector(s) or DNA sequence containing the construct(s) has been prepared for expression the DNA construct(s) mat be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.

[0043] Host cells may be either prokaryotic or eukaryotic. Example of eukaryotic hosts are mammalian cells, such as human, monkey, mouse, and Chinese hamster ovary (CHO) cells. Expression in these host cells provides post-translational modifications to protein molecules, including correct folding or glycosylation at correct sites. Also yeast cells can carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides). Example of prokaryotic hosts are bacteria, such as Escherichia coli.

[0044] After the introduction of the vector(s), the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired proteins.

[0045] Purification of the recombinant antigens is carried out by any one of the methods known for this purpose, i.e. any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like. A further purification procedure that may be used in preference for purifying the antigens of the invention is affinity chromatography using monoclonal antibodies which bind the target protein and which are produced and immobilized on a gel matrix contained within a column. Impure preparations containing the recombinant protein are passed through the column. The antigens will be bound to the column by the specific antibody while the impurities will pass through. After washing, the antigen is eluted from the gel by a change in pH or ionic strength.

[0046] Another aspect of the present invention is the process for the recombinant production of the chimeric antigen as described above, comprising culturing the host cell transformed with the vector containing the nucleotide sequence of the invention and isolating the desired product.

[0047] A further object of the present invention is a DNA molecule comprising the DNA sequence coding for the above fusion protein, as well as nucleotide sequences substantially the same.

[0048] "Nucleotide sequences substantially the same" includes all other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequence.

[0049] Another object of the present invention is a nucleotide sequence which hybridizes to the complement of the nucleotide sequence coding for the chimeric antigen of the invention under highly stringent or moderately stringent conditions, as long as the antigen obtained maintains the same biological activity, i.e. ability to bind to antibodies against the parasite.

[0050] The term "hybridization" as used here refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethyleneglycol); and the stringency of the washing conditions following hybridization.

[0051] Stringency conditions are a function of the temperature used in the hybridization experiment, the molarity of the monovalent cations and the percentage of formamide in the hybridization solution. To determine the degree of stringency involved with any given set of conditions, one first uses the equation of Meinkoth et al. (1984) for determining the stability of hybrids of 100% identity expressed as melting temperature Tm of the DNA-DNA hybrid: Tm=81.5.degree. C.+16.6 (LogM)+0.41 (% GC)-0.61 (% form)-500/L, where M is the molarity of monovalent cations, % GC is the percentage of G and C nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. For each 1.degree. C. that the Tm is reduced from that calculated for a 100% identity hybrid, the amount of mismatch permitted is increased by about 1%. Thus, if the Tm used for any given hybridization experiment at the specified salt and formamide concentrations is 10.degree. C. below the Tm calculated for a 100% hybrid according to equation of Meinkoth, hybridization will occur even if there is up to about 10% mismatch.

[0052] As used herein, highly stringent conditions are those which are tolerant of up to about 15% sequence divergence, while moderately stringent conditions are those which are tolerant of up to about 20% sequence divergence. Without limitation, examples of highly stringent (12-15.degree. C. below the calculated Tm of the hybrid) and moderately (15-20.degree. C. below the calculated Tm of the hybrid) conditions use a wash solution of 2.times.SSC (standard saline citrate) and 0.5% SDS at the appropriate temperature below the calculated Tm of the hybrid. The ultimate stringency of the conditions is primarily due to the washing conditions, particularly if the hybridization conditions used are those which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency then remove the less stable hybrids. A common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above is hybridization in a solution of 6.times.SSC (or 6.times.SSPE), 5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA at a temperature approximately 20'C to 25.degree. C. below the Tm. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel, 1987-1998).

[0053] The term "nucleic acid molecule" also includes analogues of DNA and RNA, such as those containing modified backbones.

[0054] The nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding antigens of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization). Such antisense molecules, such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J. S., Trends in Pharm. Sci., 10,435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56,560 (1991); Lee et al., Nucleic Acids Res 6,3073 (1979); Cooneyetal., Science 241, 456 (1988); Dervan et al., Science 251, 1360 (1991).

[0055] According to the terminology used herein, a composition containing a compound [.alpha.] is "substantially free of" impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.

[0056] Another aspect of the invention is the use of chimeric antigens described above as medicaments. In particular, one of the main objects of the invention is use of chimeric antigens as active ingredients for the preparation of medicaments for the prevention or treatment of Toxoplasma gondii infections.

[0057] In the case of gene therapy another object of the invention is the use of the nucleotide sequences coding for the antigens of the invention as medicaments, in particular for the preparation of medicaments useful for the treatment and prevention of Toxoplasma gondii infections.

[0058] The pharmaceutical compositions should preferably comprise a therapeutically effective amount of the chimeric antigens of the invention or the corresponding nucleotide sequence. Chimeric antigens of the invention may thus act as vaccines for the prevention or the treatment of Toxoplasma gondii infection.

[0059] For the therapeutic application, where the preparation of medicaments or vaccines comes within the framework of general knowledge for further reference the reader is again referred to the patent literature cited in the present description and, particularly, to Beghetto et al., The Journal of Infectious Diseases, 2005, 191:637-645.

[0060] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.

[0061] The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0062] The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination (s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgment of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

[0063] A pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.

[0064] Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

[0065] Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0066] Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

[0067] Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

[0068] The pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see W098/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means. Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

[0069] Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion.

[0070] Dosage treatment may be a single dose schedule or a multiple dose schedule.

[0071] The method of treating a mammal suffering from Toxoplasma-gondii infection, comprising administering a therapeutically effective amount of the vaccine as described above represents one of the aspects of the present invention.

[0072] A further object of the present invention is the use of chimeric antigens as described above as active agents for the diagnosis of Toxoplasma gondii infections, in particular for the diagnosis of the time of infection.

[0073] Also the kits for the diagnosis of Toxoplasma gondii infection, containing at least one chimeric antigen according are part of the present invention. Such kits may be useful for the diagnosis of an acute and/or chronic Toxoplasma gondii infection.

[0074] The chimeric antigen of the invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. A common feature of all of these assays is that the antigen is contacted with the body component suspected of containing antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strength using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.

[0075] Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

[0076] The immunoassay may be, without limitation, in a heterogenous or in a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation.

[0077] Examples of solid supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride (known as Immulon.TM.) diazotized paper, nylon membranes, activated beads, and Protein A beads. For example, Dynatech Immulon.TM.1 or Immulon.TM.2 microtiter plates or 0.25 inch polysterene beads (Precision Plastic Ball) can be used in the heterogeneous format. The solid support containing the antigenic polypeptides is typically washed after separating it from the test sample, and prior to detection of bound antibodies.

[0078] Both standard and competitive formats are known in the art.

[0079] In a homogeneous format, the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed. Both standard and competitive formats for these assays are known in the art.

[0080] In a standard format, the amount of antibodies forming the antibody-antigen complex is directly monitored. This may be accomplished by determining whether labeled anti-xenogenic (e.g., anti-human) antibodies which recognize an epitope on anti-Toxoplasma gondii antibodies will bind due to complex formation. In a competitive format, the amount of antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex.

[0081] Complexes formed comprising anti-Toxoplasma gondii antibody (or, in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled antibodies in the complex may be detected using a conjugate of antixenogeneic Ig complexed with a label, (e.g., an enzyme label).

[0082] In an immunoprecipitation or agglutination assay format the reaction between the chimeric antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-Toxoplasma gondii antibody is present in the test specimen, no visible precipitate is formed.

[0083] The chimeric antigens of the invention will typically be packaged in the form of a kit for use in these immunoassays. The kit will normally contain in separate containers the combination of antigens (either already bound to a solid matrix or separate with reagents for binding them to the matrix), control antibody formulations (positive and/or negative), labeled antibody when the assay format requires same and signal generating reagents (e.g., enzyme substrate) if the label does not generate a signal directly. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay usually will be included in the kit.

[0084] The diagnostic kits, which are the object of the present invention, are therefore known to the expert in the field. By way of an example, the reader is referred to the patent literature cited above, to which may be added U.S. Pat. No. 6,265,176, WO 01/63283, and WO 03/080839 as further references.

[0085] The invention will now be illustrated in greater detail by means of examples and figures.

DESCRIPTION OF THE FIGURES

[0086] FIG. 1. Plasmid map of the bacterial expression vector pGEX-SN.

[0087] FIG. 2. Schematic representation of the chimeric antigens.

[0088] The DNA sequences of clones Tx-2.a, Tx-1.16, Tx-4.18, Tx-15.11, Tx-1.11 and Tx-11.b, respectively encoding for protein fragments of the T. gondii genes MIC2, MIC3, SAG1, GRA3, GRA7 and M2AP were used for the construction of GST-EC2, GST-EC3 and GST-EC4 fusion proteins.

[0089] FIG. 3. Expression of T. gondii chimeric antigens in E. coli cells.

[0090] SDS-PAGE analysis of purified GST-EC2, GST-EC3 and GST-EC4 fusion proteins. The recombinant proteins were subjected to electrophoresis (0.003 mg/lane) on 12% acrylamide gel. MW, molecular weight markers.

[0091] FIG. 4. Antigenic properties of individual protein fragments within the chimeric antigens.

[0092] Immunoreactivity of individual Tx-2.a, Tx-1.16, Tx-4.18, Tx-15.11, Tx-1.11 and Tx-11.b antigen fragments, and of EC2, EC3 and EC4 chimeric antigens with serum samples from T. gondii infected individuals. Sera were used either as whole speciments (serum) or after depletion of specific antibodies against combinations of antigen fragments (Tx-1.16/Tx-4.18 depletion, Tx-2.a/Tx-4.18 depletion, etc.).

EXAMPLES

[0093] The following Table 1 gives, by way of examples, the DNA sequences used for the construction of recombinant Toxoplasma gondii chimeric antigens:

TABLE-US-00001 TABLE 1 Name Sequence Identification Classification Tx-15.11 GCTGCCTTGGGAGGCCTTGCGGCGGATC GRA3 Dense (SEQ ID 1) AGCCTGAAAATCATCAGGCTCTTGCAGAA granule CCAGTTACGGGTGTGGGGGAAGCAGGA protein GTGTCCCCCGTCAACGAAGCTGGTGAGT CATACAGTTCTGCAACTTCGGGTGTCCAA GAAGCTACCGCCCCAGGTGCAGTGCTCC TGGACGCAATCGATGCCGAGTCGGATAA GGTGGACAATCAGGCGGAGGGAGGTGA GCGTATGAAGAAGGTCGAAGAGGAGTTG TCGTTATTGAGGCGGGAATTATATGATCG CACAGATCGCCCTGGT Tx-1.11 CAGTTCGCTACCGCGGCCACCGCGTCAG GRA7 Dense (SEQ ID 3) ATGACGAACTGATGAGTCGAATCCGAAAT granule TCTGACTTTTTCGATGGTCAAGCACCCGT protein TGACAGTCTCAGACCGACGAACGCCGGT GTCGACTCGAAAGGGACCGACGATCACC TCACCACCAGCATGGATAAGGCATCTGTA GAGAGTCAGCTTCCGAGAAGAGAGCCAT TGGAGACGGAGCCAGATGAACAAGAAGA AGTTCAT Tx-1.16 AGGAGGACTGGATGTCATGCCTTCAGGG MIC3 Microneme (SEQ ID 5) AGAACTGCAGCCCTGGTAGATGTATTGAT protein GACGCCTCGCATGAGAATGGCTACACCT GCGAGTGCCCCACAGGGTACTCACGTGA GGTGACTTCCAAGGCGGAGGAGTCGTGT GTGGAAGGAGTCGAAGTCACGCTGGCTG AGAAATGCGAGAAGGAATTCGGCATCAG CGCGTCATCCTGCAAATGCGAT Tx-4.18 CCATCGGTCGTCAATAATGTCGCAAGGT SAG 1 Surface (SEQ ID 7) GCTCCTACGGTGCAGACAGCACTCTTGG protein TCCTGTCAAGTTGTCTGCGGAAGGACCC ACTACAATGACCCTCGTGTGCGGGAAAG ATGGAGTCAAAGTTCCTCAAGACAACAAT CAGTACTGTTCCGGGACGACGCTGACTG GTTGCAACGAGAAATCGTTCAAAGATATT TTGCCAAAATTAACTGAGAACCCGTGGCA GGGTAACGCTTCGAGTGATAAGGGTGCC ACGCTAACGATCAAGAAGGAAGCATTTCC AGCCGAGTCAAAAAGCGTCATTATTGGAT GCACAGGGGGATCGCCTGAGAAGCATCA CTGTACCGTGAAACTGGAGTTTGCCGGG GCTGCAGGGTCAGCAAAATCGGCT Tx-2.a CCCCAGGATGCCATTTGCTCGGATTGGT MIC2 Microneme (SEQ ID 9) CCGCATGGAGCCCCTGCAGTGTATCCTG protein CGGTGACGGAAGCCAAATCAGGACGCGA ACTGAGGTTTCTGCTCCGCAACCTGGAA CACCAACATGTCCGGACTGCCCTGCGCC CATGGGAAGGACTTGCGTGGAACAAGGC GGACTTGAAGAAATCCGTGAATGCAGTG CGGGGGTATGTGCTGTTGACGCTGGATG TGGCGTCTGGGTT Tx-11.b AACGAACCGGTGGCCCTAGCTCAGCTCA M2AP Microneme (SEQ ID GCACATTCCTCGAGCTCGTCGAGGTGCC protein 11) ATGTAACTCTGTTCATGTTCAGGGGGTGA TGACCCCGAATCAAATGGTCAAAGTGACT GGTGCAGGATGGGATAATGGCGTTCTCG AGTTCTATGTCACGAGGCCAACGAAGAC AGGCGGGGACACAAGCCGAAGCCATCTT GCGTCGATCATGTGTTATTCCAAGGACAT TGACGGCGTGCCGTCAGACAAAGCGGGA AAGTGCTTTCTGAAGAACTTTTCTGGTGA AGACTCGTCGGAAATAGACGAAAAAGAA GTATCTCTACCCATCAAGAGCCACAACGA TGCGTTCATGTTCGTTTGTTCTTCAAATGA TGGATCCGCACTCCAGTGTGATGTTTTCG CCCTTGATAACACCAACTCTAGCGACGG GTGGAAAGTGAATACCGTGGATCTTGGC GTCAGCGTTAGTCCGGATTTGGCATTCG GACTCACTGCAGATGGGGTCAAGGTGAA GAAGTTGTACGCAAGCAGCGGCCTGACA GCGATCAACGACGACCCTTCCTTGGGGT GCAAGGCTCCTCCCCATTCTCCGCCGGC CGGAGAGGAACCGAGTTTGCCGTCGCCT GAAAACAGCGGGTCTGCAACACCAGCGG AAGAAAGTCCGTCTGAGTCTGAATCT

[0094] The sequence Tx-15.11 constitutes a fragment of the gene GRA3 (Bermudes et al., Mol. Biochem. Parasitol., 1994, 68:247-257). Said clone has the amino acid sequence AALGGLAADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSGVQEATA PGAVLLDAIDAESDKVDNQAEGGERMKKVEEELSLLRRELYDRTDRPG (SEQ ID 2) and its use in chimeric antigens is covered by the present invention.

[0095] The sequence Tx-1.11 constitutes a fragment of the antigen GRA7 (Bonhomme et al., J. Histochem. Cytochem., 1998, 46:1411-1421). Said clone has the amino acid sequence ATAATASDDELMSRIRNSDFFDGQAPVDSLRPTNAGVDSKGTDDHLTTSMDK ASVESQLPRREPLETEPDEQEEVHF (SEQ ID 4) and its use in chimeric antigens is covered by the present invention.

[0096] The sequence Tx-1.16 constitutes a fragment of the MIC3 gene (Garcia-Reguet et al., Cellular Microbiol., 2000, 2:353-364). Said clone has the amino acid sequence RRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAE KCEKEFGISASSCKCD (SEQ ID 6) and its use in chimeric antigens is covered by the present invention.

[0097] The sequence Tx-4.18 constitutes a fragment of the antigen SAG1 (Burg et al., J. Immunol., 1988, 141:3584-3591). Said clone has the amino acid sequence PSVVNNVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTG CNEKSFKDILPKLTENPWQGNASSDKGATLTIKKEAFPAESKSVIIGCTGGSPEKHHCT VKLEFAGAAGSAKSA (SEQ ID 8) and its use in chimeric antigens is covered by the present invention.

[0098] The sequence Tx-2.a represents a fragment of the MIC2 gene (Wan et al, Mol. Biochem. Parasitol., 1997, 84:203-214). Said clone has the amino acid sequence PQDAICSDWSAWSPCSVSCGDGSQI RTRTEVSAPQPGTPTCPDCPAPMGRTCVEQG GLEEIRECSAGVCAVDAGCGVWV (SEQ ID 10) and its use in chimeric antigens is covered by the present invention.

[0099] The sequence Tx-11.b represents a distinct fragment of the M2AP gene (Rabenau et al., Mol. Microbiol., 2001, 41:537-547). Said clone has the amino acid sequence NEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKT GGDTSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAF MFVCSSNDGSALQCDVFALDNTNSSDGWKVNTVDLGVSVSPDLAFGLTADGVKVKKL YASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENSGSATPAEESPSESES (SEQ ID 12) and its use in chimeric antigens is covered by the present invention.

Construction of Chimeric Antigens

[0100] EC2 protein product is a chimeric molecule containing the DNA sequences Tx-2.a, Tx-1.16 and Tx-4.18.

[0101] SEQ ID 9 was used as template for DNA amplification of clone Tx-2.a by using oligonucleotides K551 (5'-GGACTAGTCGGCTCCCCCAGGATGCC-3') (SEQ ID 13) and K553 (5'-CATCCAGTCCTGCTACCGCCACCAGACCAGACGCCACATCC AGC-3') (SEQ ID 14). The oligonucleotide K553 contains a sequence encoding for the linker SGGGS (SEQ ID 43), which joins the sequences Tx-2.a and Tx-1.16. PCR condition was 30'' at 94.degree. C., 30'' at 50.degree. C. and 60'' at 72.degree. C. for 20 cycles.

[0102] SEQ ID 5 was used as template for DNA of clone Tx-1.16 by using oligonucleotides K552 (5'-GTGGCGTCTGGTCTGGTGGCGGTAGCAGGACTGGATGTCATGCC-3') (SEQ ID 15) and K555 (5'-TGACGACCGAGCTAC CGCCACCAGAGTTATCGCATTTGCAGGATG-3') (SEQ ID 16). The oligonucleotide K555 contains a sequence encoding for the linker SGGGS (SEQ ID 43), which joins the sequences Tx-1.16 and Tx-4.18. PCR condition was 30'' at 94.degree. C., 30'' at 50.degree. C. and 60'' at 72.degree. C. for 20 cycles.

[0103] SEQ ID 7 was used as template for DNA amplification of clone Tx-4.18 by using oligonucleotides K554 (5'-ATGCGATAACTCTGGTGGCGG TAGCTCGGTCGTCAATAATGTCGC-3') (SEQ ID 17) and K556 (5'-CCGCGGCC GCTAGCCGATTTTGCTGACCCTG-3') (SEQ ID 18). PCR condition was 30'' at 94.degree. C., 30'' at 50.degree. C. and 60'' at 72.degree. C. for 20 cycles.

[0104] The PCR products were purified by means of the "Qiagen Purification Kit" (Qiagen, Calif., USA). 25 ng of DNA amplification products of SEQ ID 9 and SEQ ID 5 were mixed together and used as templates in PCR reaction by using oligonucleotides K551 and K555. PCR condition was 30'' at 94.degree. C., 30'' at 50.degree. C. and 60'' at 72.degree. C. for 20 cycles. 25 ng of the resulting DNA amplification was purified with "Qiagen Purification Kit" (Qiagen, Calif., USA) and then mixed with 25 ng of DNA amplification product of SEQ ID 4. Finally, the DNA mixture was used as template for DNA amplification by using oligonucleotides K551 and K556, following PCR condition of 30'' at 94.degree. C., 30'' at 50.degree. C. and 90'' at 72.degree. C. for 20 cycles.

[0105] EC3 protein product is a chimeric molecule containing the DNA sequences Tx-15.11, Tx-1.11 and Tx-11.b.

[0106] SEQ ID 1 was used as template for DNA amplification of clone Tx-15.11 by using oligonucleotides K563 (5'-GGACTAGTCGGCTGG CTGCCTTGGGAGGCCTTG-3') (SEQ ID 19) and K565 (5'-GCCGCGGTAGCACTACCG CCACCAGACAAACCAGGGCGATCTGTG-3') (SEQ ID 20). The oligonucleotide K565 contains a sequence encoding for the linker SGGGS (SEQ ID 43), which joins the sequences Tx-15.11 and Tx-1.11. The PCR protocol was 30'' at 94.degree. C., 30'' at 48.degree. C. and 60'' at 72.degree. C. for 20 cycles.

[0107] SEQ ID 3 was used as template for DNA amplification of clone Tx-1.11 by using oligonucleotides K564 (5'-GCCCTGGTTTGTCTGGTGGCGGTAG TGCTACCGCGGCCACCGCG-3') (SEQ ID 21) and K567 (5'-CCGGTTCGTTACTACCG CCACCAGAGAAATGAACTTCTTCTTGTTC-3') (SEQ ID 22). The oligonucleotide K567 contains a sequence encoding for the linker SGGGS (SEQ ID 43), which joins the sequences Tx-1.11 and Tx-11.b. The PCR protocol was 30'' at 94.degree. C., 30'' at 48.degree. C. and 60'' at 72.degree. C. for 20 cycles.

[0108] SEQ ID 11 was used as template for DNA amplification of clone Tx-11.b by using oligonucleotides K566 (5'-GAAGTTCATTTCTCTGGTGGCG GTAGTAACGAACCGGTGGCCCTAG-3') (SEQ ID 23) and K568 (5'-CCGCGGCCGC AGATTCAGACTCAGACGGAC-3') (SEQ ID 24). The PCR protocol was 30'' at 94.degree. C., 30'' at 45.degree. C. and 60'' at 72.degree. C. for 20 cycles.

[0109] The PCR products were purified by means of the "Qiagen Purification Kit" (Qiagen, Calif., USA). 25 ng of DNA amplification products of SEQ ID 1 and SEQ ID 3 were mixed together and used as templates in PCR reaction by using oligonucleotides K563 and K567. The PCR protocol was 30'' at 94.degree. C., 30'' at 45.degree. C. and 60'' at 72.degree. C. for 30 cycles. 25 ng of the resulting DNA amplification was purified and then mixed with 25 ng of DNA amplification product of SEQ ID 11. Finally, the DNA mixture was used as template for DNA amplification by using oligonucleotides K563 and K568, following PCR condition of 30'' at 94.degree. C., 30'' at 45.degree. C. and 180'' at 72.degree. C. for 30 cycles.

[0110] EC4 protein product is a chimeric molecule containing the DNA sequences Tx-2.a, Tx-1.16 and Tx-11.b.

[0111] SEQ ID 9 was used as template for DNA amplification of clone Tx-2.a using oligonucleotides K551 and K553.

[0112] SEQ ID 5 was used as template for DNA amplification of clone Tx-1.16 by using oligonucleotides K552 and K572 (5'-CGTTACTACCGC CACCAGAGTTATCGCATTTGCAGGATGA-3') (SEQ ID 25). The oligonucleotide K572 contains a sequence encoding for the linker SGGGS (SEQ ID 43), which joins the sequences Tx-1.16 and Tx-11.b.

[0113] SEQ ID 11 was used as template for DNA amplification of clone Tx-11.b by using oligonucleotides K571 (5'-TAACTCTGGTGGCGGTAGT AACGAACCGGTGGCCCTAGC-3') SEQ ID 26) and K568.

[0114] The PCR products were purified as by using "Qiagen Purification Kit" (Qiagen, Calif., USA). 25 ng of DNA amplification products of SEQ ID 9 and SEQ ID 5 were mixed together and used as templates in PCR reaction by using oligonucleotides K551 and K572. 25 ng of the resulting DNA amplification was purified and then mixed with 25 ng of DNA amplification product of SEQ ID 11. Finally, the DNA mixture was used as template for DNA amplification by using oligonucleotides K551 and K568. PCR conditions for the construction of EC4 were the same that those used for EC2 and EC3 constructs.

[0115] The following Table 2 gives, by way of examples, the DNA sequences of the EC2, EC3 and EC4 chimeric antigens:

TABLE-US-00002 TABLE 2 Name Sequence EC2 ACTAGTCGGCTCCCCCAGGATGCCATTTGCTCGGATTGGTC SEQ CGCATGGAGCCCCTGCAGTGTATCCTGCGGTGACGGAAGC ID 27 CAAATCAGGACGCGAACTGAGGTTTCTGCTCCGCAACCTGG AACACCAACATGTCCGGACTGCCCTGCGCCCATGGGAAGGA CTTGCGTGGAACAAGGCGGACTTGAAGAAATCCGTGAATGC AGTGCGGGGGTATGTGCTGTTGACGCTGGATGTGGCGTCT GGTCTGGTGGCGGTAGCAGGACTGGATGTCATGCCTTCAG GGAGAACTGCAGCCCTGGTAGATGTATTGATGACGCCTCGC ATGAGAATGGCTACACCTGCGAGTGCCCCACAGGGTACTCA CGTGAGGTGACTTCCAAGGCGGAGGAGTCGTGTGTGGAAG GAGTCGAAGTCACGCTGGCTGAGAAATGCGAGAAGGAATTC GGCATCAGCGCGTCATCCTGCAAATGCGATAACTCTGGTGG CGGTAGCTCGGTCGTCAATAATGTCGCAAGGTGCTCCTACG GTGCAGACAGCACTCTTGGTCCTGTCAAGTTGTCTGCGGAA GGACCCACTACAATGACCCTCGTGTGCGGGAAAGATGGAGT CAAAGTTCCTCAAGACAACAATCAGTACTGTTCCGGGACGA CGCTGACTGGTTGCAACGAGAAATCGTTCAAAGATATTTTGC CAAAATTAACTGAGAACCCGTGGCAGGGTAACGCTTCGAGT GATAAGGGTGCCACGCTAACGATCAAGAAGGAAGCATTTCC AGCCGAGTCAAAAAGCGTCATTATTGGATGCACAGGGGGAT CGCCTGAGAAGCATCACTGTACCGTGAAACTGGAGTTTGCC GGGGCTGCAGGGTCAGCAAAATCGGCTAGCGGCCGC EC3 ACTAGTCGGCTGGCTGCCTTGGGAGGCCTTGCGGATCAGC SEQ CTGAAAATCATCAGGCTCTTGCAGAACCAGTTACGGGTGTG ID 29 GGGGAAGCAGGAGTGTCCCCCGTCAACGAAGCTGGTGAGT CATACAGTTCTGCAACTTCGGGTGTCCAAGAAGCTACCGCC CCAGGTGCAGTGCTCCTGGACGCAATCGATGCCGAGTCGG ATAAGGTGGACAATCAGGCGGAGGGAGGTGAGCGTATGAA GAAGGTCGAAGAGGAGTTGTCGTTATTGAGGCGGGAATTAT ATGATCGCACAGATCGCCCTGGTTTGTCTGGTGGCGGTAGT GCTACCGCGGCCACCGCGTCAGATGACGAACTGATGAGTC GAATCCGAAATTCTGACTTTTTCGATGGTCAAGCACCCGTTG ACAGTCTCAGACCGACGAACGCCGGTGTCGACTCGAAAGG GACCGACGATCACCTCACCACCAGCATGGATAAGGCATCTG TAGAGAGTCAGCTTCCGAGAAGAGAGCCATTGGAGACGGAG CCAGATGAACAAGAAGAAGTTCATTTCTCTGGTGGCGGTAG TAACGAACCGGTGGCCCTAGCTCAGCTCAGCACATTCCTCG AGCTCGTCGAGGTGCCATGTAACTCTGTTCATGTTCAGGGG GTGATGACCCCGAATCAAATGGTCAAAGTGACTGGTGCAGG ATGGGATAATGGCGTTCTCGAGTTCTATGTCACGAGGCCAA CGAAGACAGGCGGGGACACAAGCCGAAGCCATCTTGCGTC GATCATGTGTTATTCCAAGGACATTGACGGCGTGCCGTCAG ACAAAGCGGGAAAGTGCTTTCTGAAGAACTTTTCTGGTGAAG ACTCGTCGGAAATAGACGAAAAAGAAGTATCTCTACCCATCA AGAGCCACAACGATGCGTTCATGTTCGTTTGTTCTTCAAATG ATGGATCCGCACTCCAGTGTGATGTTTTCGCCCTTGATAACA CCAACTCTAGCGACGGGTGGAAAGTGAATACCGTGGATCTT GGCGTCAGCGTTAGTCCGGATTTGGCATTCGGACTCACTGC AGATGGGGTCAAGGTGAAGAAGTTGTACGCAAGCAGCGGC CTGACAGCGATCAACGACGACCCTTCCTTGGGGTGCAAGGC TCCTCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGTTTGC CGTCGCCTGAAAACAGCGGGTCTGCAACACCAGCGGAAGA AAGTCCGTCTGAGTCTGAATCTGCGGCCGCGG EC4 ACTAGTCGGCTCCCCCAGGATGCCATTTGCTCGGATTGGTC SEQ CGCATGGAGCCCCTGCAGTGTATCCTGCGGTGACGGAAGC ID 31 CAAATCAGGACGCGAACTGAGGTTTCTGCTCCGCAACCTGG AACACCAACATGTCCGGACTGCCCCGCGCCCATGGGAAGG ACTTGCGTGGAACAAGGCGGACTTGAAGAAATCCGTGAATG CAGTGCGGGGGTATGTGCTGTTGACGCTGGATGTGGCGTCT GGTCTGGTGGCGGTAGCAGGACTGGATGTCATGCCTTCAG GGAGAACTGCCGCCCTGGTAGATGTATTGATGACGCCTCGC ATGAGAATGGCTACACCTGCGAGTGCCCCACATGGTACTCA CGTGAGGTGACTTCCAAGGCGGAGGAGTCGTGTGTGGAAG GAGTCGAAGTCACGCTGGCTGAGAAATGCGAGAAGGAATTC GGCATCAGCGCGTCCTCCTGCAAATGCGATAACTCTGGTGG CGGTAGTAACGAACCGGTGGCCCTAGCTCAGCTCAGCACAT TCCTCGAGCTCGTCGAGGTGCCATGTAACTCTGTTCATGTTC AGGGGGTGATGACCCCGAATCAAATGGTCAAAGTGACTGGT GCAGGATGGGATAATGGCGTTCTCGAGTTCTATGTCACGAG GCCAACGAAGACAGGCGGGGACACAAGCCGAAGCCACCTT GCGTCGATCATGTGTTATTCCAAGGACATTGACGGCGTGCC GTCAGACAAAGCGGGAAAGTGCTTTTTGAAGAACTTTTCTGG TGAAGACTCGTCGGAAATAGACGAAAAAGAAGTATCTCTACC CATCAAGAGCCACAACGATGCGTTCATGTTCGTTTGTTCTTC AAATGATGGATCCGCACTCCAGTGTGATGTTTTCGCCCTTGA TAACACCAACTCTAGCGACGGGTGGAAAGTGAATACCGTGG ATCTTGACGTCAGCGTTAGTCCGGATTTGGCATTCGGACTCA CTGCAGATGGGGTCAAGGTGAAGAAGTTGTACGCAAGCAGC GGCCTGACAGCGATCAACGACGACCCTTCCTTGGGGTGCAA GGCTCCTCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGT TTGCCGTCGCCTGAAAACAGCGGGTCTGCAACACCAGCGGA AGAAAGTCCGTCTGAGTCTGAATCTGCGGCCGCGG

[0116] The chimeric protein EC2 has the amino acid sequence TSRLPQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPAPMGRTC VEQGGLEEIRECSAGVCAVDAGCGVWSGGGSRTGCHAFRENCSPGRCIDDASHENG YTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCDNSGGGSSWN NVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTGCNEK SFKDILPKLTENPWQGNASSDKGATLTIKKEAFPAESKSVIIGCTGGSPEKHHCTVKLEF AGAAGSAKSASGR (SEQ ID 28) and its use as recombinant antigen, containing multiple Toxoplasma gondii protein fragments, is covered by the present invention.

[0117] The chimeric protein EC3 has the amino acid sequence TSRLAALGGLADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSGVQEATAPGA VLLDAIDAESDKVDNQAEGGERMKKVEEELSLLRRELYDRTDRPGLSGGGSATAATAS DDELMSRIRNSDFFDGQAPVDSLRPTNAGVDSKGTDDHLTTSMDKASVESQLPRREP LETEPDEQEEVHFSGGGSNEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTG AGWDNGVLEFYVTRPTKTGGDTSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGED SSEIDEKEVSLPIKSHNDAFMFVCSSNDGSALQCDVFALDNTNSSDGWKVNTVDLGVS VSPDLAFGLTADGVKVKKLYASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENSG SATPAEESPSESESAAA (SEQ ID 30) and its use as recombinant antigen, containing multiple Toxoplasma gondii protein fragments, is covered by the present invention.

[0118] The chimeric protein EC4 has the amino acid sequence TSRLPQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPAPMGRTC VEQGGLEEIRECSAGVCAVDAGCGVWSGGGSRTGCHAFRENCRPGRCIDDASHENG YTCECPTWYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCDNSGGGSNEPV ALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKTGGD TSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAFMFV CSSNDGSALQCDVFALDNTNSSDGWKVNTVDLDVSVSPDLAFGLTADGVKVKKLYAS SGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENSGSATPAEESPSESESAAA (SEQ ID 32) and its use as recombinant antigen, containing multiple Toxoplasma gondii protein fragments, is covered by the present invention.

Construction of DNA Vectors Directing the Expression of Chimeric Antigens as Fusion Products with GST in the Cytoplasm of E. Coli Cells

[0119] DNA fragments encoding for the EC2, EC3 and EC4 chimeric proteins were cloned as fusion products with the protein Glutathione Sulpho Transferase (GST) and expressed as soluble proteins in the cytoplasm of bacterial cells, for the purpose of determining their specificity and selectivity. DNA sequences of EC2, EC3 and EC4 (SEQ ID 27, 29 and 31, respectively) were digested with the restriction enzymes SpeI and NotI. Digested DNA were cloned into vector pGEX-SN (Minenkova et al., International Journal of Cancer, 2003, 106:534-44), which was previously digested with SpeI and NotI endonucleases, to generate fusion products at the carboxy terminus of GST protein. The resulting plasmids were used to transform competent E. coli cells following standard protocols (Sambrook et al., 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

Biochemical Characterisation of the Recombinant Chimeric Antigens

[0120] The recombinant proteins GST-EC2, GST-EC3 and GST-EC4 were expressed in the cytoplasm of transformed E. coli cells and purified by affinity chromatography using Glutathione-Sepharose resin (Amersham Pharmacia Biotech, Sweden), following the manufacturer's instructions. Protein purity and concentration were assessed by SDS-PAGE (Sodium Dodecyl Sulphate-Poly-acrylammide Gel Electrophoresis) analysis and Bradford assay, respectively. The affinity-purified recombinant products were dialyzed against PBS, diluted at a concentration of 1 mg/ml with PBS and stored at -20.degree. C. until use. The yield of purified products were 8 mg/liter of bacterial culture, 5 mg/liter and 4 mg/liter for the chimeric antigens GST-EC2, GST-EC3 and GST-EC4, respectively. The recombinant proteins were subsequently subjected to high-performance liquid chromatography (HPLC) analysis. To this aim a gel filtration was performed using a TSK G4000 SW-XL HPLC-column, by injecting 30 .mu.l of each samples (protein concentration, 2 mg/ml) into the column with a flow rate of 1 ml/min (mobile phase: KH.sub.2PO.sub.4 0.05 M; NaCl 0.3 M pH 7.0). Results from HPLC analysis demonstrated that the chimeric antigens GST-EC2, GST-EC3 and GST-EC4 were purified as homogeneous products in dimeric with a respective molecular weight of 110.4, 152.6 and 130 kDa (Da, Dalton). Protein aggregates of high molecular weight were absent in all of the purified protein preparations.

Immunoreactivity of the Chimeric Recombinant Antigens with IgG Antibodies from Sera of T. gondii Infected Individuals: IgG Rec-ELISA

[0121] The ELISA performance of the GST fusion products was performed by coating Maxisorp-multiwells plates (Nunc) with single antigen fragments or chimeric proteins at a concentration of 5 .mu.g/ml in coating buffer (50 mM NaHCO.sub.3, pH 9.6). After incubation overnight at 4.degree. C. plates were incubated for 1 h at 37.degree. C. with blocking buffer (5% non-fat dry milk, 0.05% Tween-20 in PBS) and subsequently incubated for 1 h at 37.degree. C. with sera from T. gondii seropositive and seronegative individuals, diluted 1:200 in blocking solution. Plates were extensively washed with 0.05% Tween-20 in PBS and anti-human-IgG horse-radish peroxidase-conjugated antibodies (1 mg/ml; Sigma-Aldrich, USA), diluted 1:10000 in blocking solution, was then added to each well. Finally, incubating plates with the chromogenic substrate tetramethylbenzidine (TMB; Sigma-Aldrich, USA) revealed the enzymatic activity. Results were recorded as the difference between the absorbance (Optical Density, OD) at 450 and 620 nm, detected by an automated ELISA reader (Labsystem Multiskan, Finland). For each serum sample the assay was done in triplicate and average values were calculated.

[0122] The following Table 3 shows the IgG reactivity of the single antigen fragments and EC2, EC3 and EC4 chimeric antigens, expressed as GST fusion proteins, by using sera from 36 (T1-T36) T. gondii-seropositive and 27 (N1-N27) T. gondii-seronegative humans.

[0123] The Toxoplasma-specific IgG levels, calculated as International Units (IU) using a commercial assay (VIDAS system, bioMerieux, Marcy-l'Etoile, France) are reported. For every GST-fusion product the cut-off value was determined as the mean plus two times the standard deviation of the absorbency readings obtained from the Toxoplasma IgG negative sera. Normal type, OD<cut-off; bold type, OD>cut-off. The numerical value reported into each cell was calculated as the ratio of the test OD to the cut-off of the corresponding antigen (nd, not determined). Values greater than 1 indicate a positive response.

[0124] Table 3 clearly shows that some of the sera which are positive result to be negative when using single antigenic fragments, whereas they result to be positive (correctly) when using the chimeric antigens of the present invention. Please note that the numerical values of IgG concentrations obtained with the standard assay cannot be compared with the others, because they have been calculated by reference to an International Standard, which cannot be used for the other assays.

TABLE-US-00003 TABLE 3 Serum IgG levels Recombinant GST-fusion proteins sample (IU/ml) Tx-15.11 Tx-1.11 Tx-1.16 Tx-4.18 Tx-2.a Tx-11.b EC2 EC3 EC4 T1 >300 7.4 3.5 9.8 3.7 18.3 12.5 11.0 10.0 7.6 T2 188 8.7 7.4 2.4 5.1 18.8 4.4 26.0 23.5 20.0 T3 265 7.4 2.8 4.6 2.8 8.4 5.6 20.4 17.9 34.0 T4 4090 23.3 27.8 22.4 29.7 25.1 27.4 33.6 43.3 40.5 T5 >300 12.4 9.7 25.7 2.9 nd nd 30.6 34.4 19.5 T6 35 1.2 0.8 1.0 1.3 0.9 1.4 1.4 5.7 1.7 T7 58 1.0 1.3 0.6 2.3 1.2 1.0 4.6 5.1 1.2 T8 101 2.5 0.9 2.5 2.8 11.2 2.9 17.0 4.8 30.9 T9 88 16.2 5.9 2.4 2.7 6.8 6.7 9.4 27.4 29.9 T10 188 1.7 1.2 4.4 3.1 1.7 1.3 4.4 3.6 3.3 T11 530 28.9 12.7 12.1 31.4 32.1 31.2 32.1 42.6 41.5 T12 89 0.9 nd 4.8 nd 1.2 0.5 1.7 2.4 2.1 T13 1095 8.6 2.3 3.4 5.9 nd nd 31.7 23.4 20.3 T14 248 12.6 4.3 2.1 0.9 nd nd 7.4 14.6 9.2 T15 155 17.5 1.0 2.8 3.4 nd nd 6.7 20.1 13.0 T16 427 4.5 3.9 5.0 2.7 nd nd 17.4 37.9 18.9 T17 236 12.1 4.8 6.1 3.7 nd nd 22.0 33.9 12.7 T18 46 2.1 2.0 2.6 2.2 nd nd 1.9 5.4 1.4 T19 247 2.3 2.5 3.6 0.5 nd nd 3.7 3.9 2.3 T20 100 nd nd nd nd 7.5 1.4 13.1 5.2 14.6 T21 27 2.6 0.6 4.1 1.0 nd nd 3.1 4.8 5.5 T22 >300 28.6 5.1 4.6 19.9 nd nd 14.5 14.8 7.4 T23 92 1.7 1.1 1.3 0.8 0.7 0.8 6.9 4.5 6.4 T24 68 0.5 2.1 2.0 2.6 1.3 1.3 2.5 6.6 1.4 T25 63 2.4 2.4 5.5 6.8 8.8 7.7 13.8 16.6 32.9 T26 299 12.1 10.0 6.5 4.6 nd nd 10.0 13.5 6.0 T27 108 3.4 4.0 5.7 3.0 nd nd 4.8 6.2 3.1 T28 68 1.7 2.7 3.3 2.1 2.0 2.3 6.4 6.9 7.7 T29 >300 1.0 1.1 2.8 1.2 4.0 6.3 9.1 13.7 23.5 T30 114 1.8 2.8 19.3 3.8 3.9 2.0 13.0 13.3 13.6 T31 35 3.2 1.2 3.5 0.9 1.5 1.0 3.6 6.3 5.7 T32 300 4.8 16.0 17.3 20.9 nd nd 31.6 33.1 8.4 T33 123 5.6 2.6 3.3 4.5 20.4 2.8 25.8 21.0 36.6 T34 60 nd nd nd nd 6.2 4.2 9.1 7.7 9.3 T35 155 0.7 2.4 0.5 2.3 1.8 1.1 7.3 10.4 9.5 T36 45 0.3 1.6 1.1 0.8 1.2 0.9 3.2 3.2 3.1 N1 0.4 0.4 0.7 0.7 0.5 0.3 0.4 0.7 0.5 N2 0.7 0.5 0.5 0.4 nd nd 0.4 0.5 0.4 N3 nd nd nd nd nd nd 0.9 0.5 0.6 N4 nd nd nd nd nd nd 0.4 0.5 0.7 N5 nd nd nd nd 0.5 0.7 0.4 0.5 0.5 N6 nd nd nd nd nd nd 0.4 0.5 0.5 N7 0.4 0.4 0.6 0.7 nd nd 1.0 0.5 0.7 N8 0.4 0.5 0.5 0.5 nd nd 0.4 0.5 0.6 N9 0.4 0.6 0.7 0.5 nd nd 0.4 0.5 0.4 N10 nd nd nd nd nd nd 0.3 0.5 0.4 N11 nd nd nd nd nd nd 0.4 0.5 0.5 N12 nd nd nd nd nd nd 0.5 0.7 0.8 N13 nd nd nd nd nd nd 0.4 0.7 0.3 N14 0.7 0.5 0.5 0.5 0.9 0.7 0.5 0.5 0.5 N15 0.4 0.6 0.4 1.0 0.5 0.8 0.4 0.5 0.4 N16 0.8 0.2 0.5 0.6 0.5 0.4 0.5 0.7 0.5 N17 0.8 0.5 0.9 0.4 0.6 0.7 0.4 0.5 0.6 N18 0.3 0.4 0.7 0.5 0.6 0.6 0.4 0.6 0.9 N19 0.4 0.5 0.5 0.8 nd nd 0.5 0.8 0.8 N20 0.6 0.6 0.4 0.6 0.7 0.5 0.8 0.7 1.0 N21 0.7 0.7 0.6 0.5 0.5 0.4 0.4 0.7 0.6 N22 0.4 0.5 0.7 0.5 0.5 0.7 0.4 0.6 0.3 N23 0.5 0.5 0.7 0.5 nd nd 0.4 0.6 0.5 N24 nd nd nd nd 0.5 0.5 0.5 0.7 0.7 N25 nd nd nd nd 0.8 0.5 0.8 0.6 0.5 N26 nd nd nd nd 0.5 0.6 0.4 0.6 0.4 N27 nd nd nd nd 0.4 0.6 0.8 1.0 0.4

[0125] The following Table 4 summarizes the results of the ELISA assays based on recombinant proteins, employing serum samples from T. gondii seropositive and seronegative humans. In each column are reported the number and the corresponding percentages of reactive sera. From Table 4 it clearly results that the sensitivity of the assay (see the 2.sup.nd column reporting the occurrence of false negatives) is improved when using the chimeric antigens of the invention. This improvement is evident with respect to both the use of single antigenic fragments and the use of a collection or mixtures (Mix-Tx-1.16/Tx-4.18/Tx-2.a, Mix-Tx-1.16/Tx-4.18/Tx-2.a, Mix-Tx-1.16/Tx-2.a/Tx-11.b) of the single different antigenic fragments.

TABLE-US-00004 TABLE 4 Sera from T. gondii Sera from T. gondii GST-fusion protein infected subjects uninfected subjects Tx-15.11 28/34 (82.4%) 0/15 Tx-1.11 29/33 (87.9%) 0/15 Tx-1.16 31/34 (91.2%) 0/15 Tx-4.18 27/33 (81.8%) 0/15 Tx-2.a 21/23 (91.3%) 0/14 Tx-11.b 18/23 (78.3%) 0/14 Mix-Tx-1.16/Tx-4.18/Tx-2.a 35/36 (97.2) 0/27 Chimera EC2 36/36 (100%) 0/27 Mix-Tx-15.11/Tx-1.11/Tx-11.b 35/36 (97.2%) 0/27 Chimera EC3 36/36 (100%) 0/27 Mix-Tx-1.16/Tx-2.a/Tx-11.b 34/36 (94.4%) 0/27 Chimera EC4 36/36 (100%) 0/27

Immunoreactivity of Single Antigenic Domains within the Chimeric Recombinant Antigens with IgG Antibodies of Sera from T. Gondii Infected Individuals

[0126] To verify that the chimeric antigens retain the immunoreactivity of the single antigen fragments used for their construction, human sera that specifically reacted, in ELISA assays, with single antigen fragments, were adsorbed with different combinations of single antigens and then assayed with the chimeric proteins. To this aim, distinct combinations of the antigen fragments, expressed as GST-fusion products, were coated onto Maxisorb-multiwells plates (Nunc) at a concentration of 10 .mu.g/ml in coating buffer (50 mM NaHCO.sub.3, pH 9.6) and then incubated overnight at 4.degree. C. The plates were extensively washed and subsequently incubated for 30 min. at 37.degree. C. with serum samples (20 .mu.l/well) in blocking solution (5% non-fat dry milk, 0.05% Tween-20 in PBS). The fragment-specific antibody-depleted sera were recovered from each well, added to a new well, incubated for 30 min., and the same procedure was repeated 6 more times. Samples that have been depleted for specific antibodies against a single or multiple antigen fragments were finally analyzed by ELISA assays on the chimeric antigens. For this purpose, the chimeric antigens EC2, EC3 and EC4, as GST-fusion products, were coated overnight at 4.degree. C. onto Maxisorb-multiwells plates at a concentration of 5 .mu.g/ml. The coated plates were blocked and subsequently incubated for 1 h at 37.degree. C. with the antibody-depleted human sera diluted 1:100 in blocking solution. Plates were extensively washed and anti-human-IgG alkaline phosphatase-conjugated antibodies (Sigma-Aldrich, USA), diluted 1:7500 in blocking solution, was then added to each well. Finally, the chromogenic substrate p-nitrophenyl phosphate (Sigma-Aldrich, USA) revealed the enzymatic activity. The results were recorded as the difference between the absorbance at 405 and 620 nm, detected by an automated ELISA reader (Labsystem Multiskan, Finland). For each sample the assay was done in duplicate and average values were calculated.

Biochemical Modification of EC2 and EC3 Chimeric Antigens

[0127] To analyze the immunoreactivity of the chimeric antigens EC2 and EC3 with specific anti-Toxoplasma IgM antibodies in patient sera, the recombinant proteins were chemically modified by biotinilation. To this aim, the purified GST-EC2 and GST-EC3, diluted at a concentration of 1 mg/ml in PBS were incubated in the presence of a five-fold molar excess of sulfosuccinimidyl-6-(biotin-amido)hexanoate (Sulfo-NHS-LC-Biotin from Pierce, USA) for 3 hours on ice. The proteins were then dialyzed overnight against PBS to remove excess of non-reacted and hydrolyzed biotin reagents. Levels of biotin incorporation into chimeric antigens was determined by using "EZ Biotin Quantitation Kit" (Pierce, USA), resulting in 1.4 biotin/molecule for GST-EC2 and 1.3 biotin/molecule for GST-EC3. The biotin-labeled products were finally diluted at a concentration of 0.5 mg/ml and stored at -20.degree. C. until use.

Immunoreactivity of the Biotin-Labeled EC2 and EC3 with Toxoplasma-Specific IqM Antibodies: IqM Rec-ELISA

[0128] To investigate the immunoreactivity of recombinant antigens with Toxoplasma-specific immunoglobulins M, a double-sandwich immunoassay was employed (IgM Rec-ELISA). Maxisorb plates (Nunc, USA) were coated with anti-human IgM antibodies (Sigma-Aldrich, USA) at a concentration of 10 .mu.g/ml in coating buffer (50 mM NaHCO.sub.3, pH 9.6). Plates were blocked with 3% bovine serum albumin in PBS (blocking solution) for 1 h at 37.degree. C. and subsequently incubated for 1 h at 37.degree. C. with h serum samples in blocking solution. Plates were washed and then incubated for 2 h at room temperature with the biotin-labeled GST-fusion proteins, diluted in blocking solution. After being extensively washed the plates were incubated for 1 h at room temperature with horseradish peroxidase-conjugated streptavidin (Pierce, USA) at a concentration of 1 .mu.g/ml in blocking solution. Finally, the enzymatic activity was revealed incubating plates for 30 min. at room temperature with the substrate tetramethylbenzidine (Sigma-Aldrich, USA). Results were recorded as the difference between the absorbance at 450 and 620 nm, detected by an automated ELISA reader (Labsystem Multiskan, Finland). For each sample the assay was done in duplicate and average values were calculated.

Thermal Stability of the Biotin-Labeled Chimeric Antigens

[0129] In order to determine the thermal stability of the biotin-labeled GST-EC2 and GST-EC3, recombinant products were diluted at a concentration of 5 .mu.g/ml in the commercial buffer "Stabilzyme" (SurModics, USA) and stored at +4'C until use. After different interval times (up to 80 days), the immunoreactivity of recombinant proteins in the IgM Rec-ELISA analysis was assessed and results obtained analyzing the corresponding products maintained frozen at -20.degree. C. were compared. The IgM Rec-ELISA was performed as described above, using the biotin-labeled GST-EC2 and GST-EC3 antigens at a final concentration of 500 ng/ml in blocking solution (3% BSA in PBS) and human sera diluted 1:100 in blocking solution. For each sample the assay was done in duplicate and average values were calculated. The ID50, calculated as day-limit when the 50% of toxoplasma-specific IgM-immunoreactivity was measured, were 189 days and 97 days for GST-EC2 and GST-EC3, respectively. These findings clearly indicate that the chimeric antigens of the invention are stable in diluted solutions for a long time, which a fundamental requisite for the commercial usefulness of a recombinant product.

Expanded Evaluation of IgM Rec-ELISA

[0130] The biotin-labeled GST-EC2 and GST-EC3 chimeric antigens were assayed with IgM antibodies in sera from T. gondii infected individuals and the results of the IgM Rec-ELISAs were compared with those obtained with commercial assays employing lysed, whole-cell Toxoplasma antigen (VIDAS system from bioMerieux, France; ETI-TOXOK-M Reverse-PLUS from DiaSorin, Italy). To this aim, serum samples from women who acquired primary toxoplasmosis during gestation and referred for post-natal follow-up at the Center for Perinatal Infection of Campania Region, Italy, were assayed. The bioMerieux VIDAS Toxo IgG and IgM assays were used to select three groups of serum samples for the Toxoplasma IgM Rec-ELISA performance evaluation. Group A (n=22) was composed of samples negative for T. gondii-specific IgM and IgG antibody as measured by the VIDAS Toxo IgM and IgG assays. Group B (n=18) was composed of samples with a serological profile consistent with a chronic infection (presence of T. gondii-specific IgG antibody and absence of T. gondii-specific IgM as measured by the VIDAS Toxo IgM and IgG assays, respectively). Group C (n=50) was composed of samples with a serological profile consistent with an acute infection (presence of T. gondii-specific IgM and IgG antibodies as measured by the VIDAS Toxo IgM and IgG assays). IgM Rec ELISA was performed as described above and for each serum sample the assay was done in duplicate and average values were calculated.

[0131] The following Table 5 shows the IgM reactivity of the biotin-labeled GST-EC2 and GST-EC3 chimeric antigens, compared to the results obtained with commercial assays (VIDAS and ETI-TOXO-K), by using sera from group A (A1-A22), group B (B1-B18) and group C(C1-C50). The Toxoplasma-specific IgG levels, calculated as International Units (IU) are also reported. For each biotin-labeled GST-fusion product the cut-off was determined as the mean plus two times the standard deviation of the absorbency readings obtained from the T. gondii-specific IgM negative sera (groups A and B, n=40). Cut-off values for VIDAS IgM, ETI-TOXOK-M Reverse-PLUS, GST-EC2 and GST-EC3 were 0.650, 0.500, 0.343 and 0.378, respectively. Values typed in bold indicate a positive response.

TABLE-US-00005 TABLE 5 Toxo-IgG VIDAS ETI-TOXOK-M IgM Rec-ELISA Serum (UI/ml) IgM Reverse-PLUS GST-EC2 GST-EC3 A1 0 0.05 0.397 0.268 0.256 A2 4 0.22 0.317 0.263 0.270 A3 0 0.18 0.252 0.264 0.237 A4 0 0.05 0.375 0.324 0.241 A5 2 0.17 0.272 0.298 0.222 A6 0 0.03 0.288 0.270 0.234 A7 0 0.19 0.210 0.215 0.379 A8 0 0.10 0.108 0.203 0.296 A9 0 0.06 0.324 0.314 0.291 A10 0 0.09 0.339 0.325 0.286 A11 0 0.05 0.193 0.223 0.271 A12 2 0.08 0.134 0.268 0.378 A13 4 0.12 0.115 0.309 0.335 A14 0 0.23 0.115 0.221 0.286 A15 0 0.06 0.230 0.281 0.374 A16 2 0.08 0.132 0.317 0.269 A17 1 0.18 0.123 0.277 0.281 A18 0 0.35 0.097 0.316 0.279 A19 0 0.28 0.346 0.274 0.272 A20 0 0.09 0.054 0.259 0.132 A21 0 0.61 0.206 0.24 0.312 A22 0 0.06 0.127 0.238 0.233 B1 24 0.06 0.239 0.255 0.189 B2 10 0.09 0.076 0.283 0.304 B3 44 0.14 0.124 0.265 0.261 B4 44 0.28 0.195 0.298 0.216 B5 25 0.1 0.131 0.273 0.185 B6 57 0.12 0.164 0.296 0.293 B7 12 0.22 0.185 0.257 0.194 B8 58 0.12 0.148 0.255 0.248 B9 56 0.4 0.174 0.232 0.268 B10 19 0.09 0.068 0.290 0.194 B11 56 0.16 0.136 0.179 0.221 B12 45 0.12 0.139 0.235 0.181 B13 87 0.12 0.096 0.207 0.218 B14 27 0.15 0.144 0.196 0.174 B15 33 0.46 0.242 0.285 0.378 B16 13 0.04 0.064 0.161 0.177 B17 67 0.13 0.111 0.177 0.213 B18 53 0.27 0.170 0.238 0.165 C1 28 5.37 1.04 0.350 0.548 C2 255 3.86 1.49 0.546 0.498 C3 78 3.01 1.38 0.471 0.867 C4 1358 2.28 1.17 0.464 0.453 C5 178 2.31 1.27 0.598 0.406 C6 155 2.00 0.97 0.993 0.720 C7 109 3.20 1.76 0.794 0.642 C8 99 3.16 1.78 0.572 0.389 C9 103 2.28 1.34 1.056 1.222 C10 85 2.22 1.44 0.930 0.704 C11 70 1.01 0.79 0.416 0.376 C12 26 1.34 0.93 0.392 0.461 C13 36 1.22 0.86 0.532 0.499 C14 156 0.99 0.58 0.534 0.833 C15 204 0.93 0.90 0.810 0.710 C16 133 1.13 0.85 0.465 0.322 C17 183 1.14 0.82 0.320 0.327 C18 242 0.90 0.71 0.497 0.500 C19 80 1.00 0.79 0.444 0.706 C20 258 1.40 0.88 2.678 0.484 C21 278 1.69 1.06 0.703 0.509 C22 246 1.25 0.76 1.094 0.780 C23 59 1.23 0.71 0.495 1.499 C24 38 0.78 0.87 0.584 0.455 C25 130 0.76 0.92 0.562 0.545 C26 262 0.84 0.65 0.649 0.551 C27 168 0.96 0.85 1.439 0.938 C28 126 0.78 0.80 2.475 1.160 C29 197 1.38 0.61 0.544 0.358 C30 127 0.86 0.52 0.847 0.531 C31 72 1.28 0.93 1.756 0.891 C32 130 0.77 0.71 0.505 0.381 C33 439 1.00 0.66 0.834 0.464 C34 83 0.66 1.32 1.162 0.989 C35 178 0.89 0.87 0.694 0.487 C36 560 0.86 0.69 0.817 0.628 C37 223 0.96 0.73 0.531 0.819 C38 242 0.98 0.41 0.379 0.318 C39 118 1.16 0.84 0.420 0.380 C40 232 1.39 1.01 0.490 0.467 C41 213 1.03 1.05 0.750 0.822 C42 243 1.06 0.97 0.534 0.502 C43 154 0.75 0.73 0.455 0.337 C44 35 1.90 1.51 0.383 1.008 C45 667 0.85 1.01 0.366 0.285 C46 275 0.95 0.99 0.411 0.544 C47 157 1.93 1.36 0.382 0.464 C48 1037 1.08 0.51 0.385 0.301 C49 92 1.31 0.68 0.537 0.427 C50 255 0.69 0.69 0.354 0.801

[0132] The following Table 6 shows the performance characteristics of the commercial assays (VIDAS IgM and ETI-TOXOK-M Reverse PLUS), compared to the results obtained with the biotin-labeled EC2 and EC3 chimeric antigens (IgM Rec-ELISA). From Table 6 it clearly results that both specificity and positive predictive values of the assays (see the 3.sup.rd column reporting the occurrence of false positives) reached the maximum (100%) when using the chimeric antigens of the invention. With regard to sensitivity and agreement, both the commercial test ETI-TOXOK-M employing lysed, whole-cell Toxoplasma antigen and the IgM rec-ELISA with the chimeric antigen EC2 display identical performance characteristics, with both values very close to 100%.

TABLE-US-00006 TABLE 6 Diagnostic Sensitivity Specificity Agreement PPV* NPV* test (%) (%) (%) (%) (%) VIDAS IgM 100 100 100 100 100 ETI-TOXOK-M 98.0 100 98.9 100 97.6 EC2-IgM 98.0 100 98.9 100 97.6 Rec-ELISA EC3-IgM 84.0 100 91.1 100 83.3 Rec-ELISA *PPV, positive predictive value; NPV, negative predictive value.

[0133] Finally, the immunoreactivity of the biotin-labeled GST-EC2 and GST-EC3 antigens with IgM antibodies in sera from infants with congenital toxoplasmosis was investigated. In a retrospective study, sera from 30 infants of mothers with primary T. gondii infection during pregnancy were analyzed. Twenty infants had congenital toxoplasmosis and ten was uninfected, as demonstrated by the persistence or disappearance of Toxoplasma-specific IgG antibodies after 12 months of age, respectively. Within the infected patient cohort, the gestational age at the time of maternal infection was the second trimester in 6 mothers and the third trimester in 14 mothers. 30 serum samples from infected and uninfected infants were analyzed by IgM Rec-ELISA, and results obtained with commercial assays employing the whole-cell Toxoplasma antigen (ELFA-IgM and ETI-TOXOK-M Reverse PLUS) were compared. Specific levels of anti-Toxoplasma IgG ranged from 28 to 1147 IU/ml for sera from infected infants and from 19 to 170 IU/ml for sera from uninfected subjects. For every GST-fusion product the cut-off value was determined as the mean plus 2SD of the absorbency readings obtained with sera from uninfected infants. In Table 7 are summarized the results of the IgM Rec-ELISAs with individual sera from infected infants. Overall, the number of IgM-reactive sera ranged from 70% (14/20) to 50% (10/20) using the GST-EC2 and GST-EC3 antigens, respectively. In contrast, only 7 out of 20 infected infants (35%) had positive results when ELFA-IgM or ETI-TOXOK-M assays were employed. Among uninfected infants, none of the sera recognized GST-EC2 and GST-EC3 antigens in the IgM Rec-ELISA or resulted to be positive using commercial assays.

[0134] In conclusion, these results demonstrate that the use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected from uninfected individuals, having comparable or even better assay performance with respect of using the whole-cell tachyzoite antigen, and could provide the basis for standardized commercial immunoassays for toxoplasmosis serodiagnosis.

TABLE-US-00007 TABLE 7 Toxoplasma-specific IgM reactivity of serum samples from 30 infants born to mothers with primary T. gondii infection acquired during pregnancy .sup.a Time IgM Rec-ELISA after IgG ELFA- ETI- cutoff .sup.d Patient birth levels IgM ToxoM GST- GST- no. (wk) Onset .sup.b (IU/ml) cutoff .sup.c cutoff .sup.c EC2 EC3 T1 1 B 169 6.41 2.66 2.479 0.542 T2 2 B 988 0.73 0.15 0.360 0.270 T3 2 Sub 300 0.09 0.13 0.212 0.209 T4 3 Sub 57 0.05 0.23 0.206 0.211 T5 3 Sub 124 0.13 1.62 0.641 1.103 T6 4 Sub 218 0.04 0.09 0.452 0.269 T7 4 S 157 2.61 1.62 1.522 0.225 T8 4 S 172 3.98 1.50 1.804 0.353 T9 5 S 1147 0.07 0.10 0.519 0.206 T10 5 B 47 0.11 0.12 0.272 0.276 T11 6 Sub 28 0.10 0.18 2.617 0.731 T12 6 Sub 136 0.07 0.11 0.314 0.216 T13 7 S 209 0.88 0.47 0.683 0.217 T14 8 Sub 43 0.06 0.07 0.196 0.213 T15 8 B 160 0.82 0.08 0.206 0.219 T16 8 B 64 0.02 0.40 0.231 0.228 T17 8 Sub 145 0.31 0.57 0.985 0.314 T18 9 Sub 300 6.37 1.30 0.548 0.315 T19 12 Sub 196 0.05 0.17 0.463 0.268 T20 12 Sub 75 0.05 0.07 0.237 0.222 N1 5 90 0.38 0.06 0.237 0.218 N2 5 170 0.06 0.07 0.204 0.200 N3 5 66 0.23 0.09 0.238 0.235 N4 3 44 0.07 0.12 0.176 0.209 N5 9 41 0.05 0.05 0.209 0.193 N6 5 66 0.07 0.06 0.194 0.196 N7 8 13 0.09 0.06 0.193 0.201 N8 9 13 0.14 0.06 0.208 0.231 N9 6 19 0.28 0.07 0.184 0.215 N10 5 20 0.13 0.05 0.189 0.240 Notes to Table 7 .sup.a Serum samples from T. gondii infected (T1-T20) or uninfected children (N1-N10) were analyzed by IgM Rec-ELISAs with GST-EC2 and GST-EC3 antigens or by commercial assays (ELFA-IgM and ETI-TOXO-M). .sup.b Severity of clinical onset: S. severe; B. benign; Sub. subclinical. .sup.c Cutoff values for the ELFA-IgM and ETI-TOXO-M assays were 0.65 and 0.41 as indicated by manufacturers. respectively. Bold type. values > cutoff. .sup.d Cutoff values for the IgM Rec-ELISA using GST-EC2 and GST-EC3 antigens were 0.25 and 0.26. respectively. Bold type. values > cutoff.

[0135] The paper copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entirety.

Sequence CWU 1

1

431297DNAToxoplasma gondiiCDS(1)..(297) 1gct gcc ttg gga ggc ctt gcg gcg gat cag cct gaa aat cat cag gct 48Ala Ala Leu Gly Gly Leu Ala Ala Asp Gln Pro Glu Asn His Gln Ala1 5 10 15ctt gca gaa cca gtt acg ggt gtg ggg gaa gca gga gtg tcc ccc gtc 96Leu Ala Glu Pro Val Thr Gly Val Gly Glu Ala Gly Val Ser Pro Val 20 25 30aac gaa gct ggt gag tca tac agt tct gca act tcg ggt gtc caa gaa 144Asn Glu Ala Gly Glu Ser Tyr Ser Ser Ala Thr Ser Gly Val Gln Glu 35 40 45gct acc gcc cca ggt gca gtg ctc ctg gac gca atc gat gcc gag tcg 192Ala Thr Ala Pro Gly Ala Val Leu Leu Asp Ala Ile Asp Ala Glu Ser 50 55 60gat aag gtg gac aat cag gcg gag gga ggt gag cgt atg aag aag gtc 240Asp Lys Val Asp Asn Gln Ala Glu Gly Gly Glu Arg Met Lys Lys Val65 70 75 80gaa gag gag ttg tcg tta ttg agg cgg gaa tta tat gat cgc aca gat 288Glu Glu Glu Leu Ser Leu Leu Arg Arg Glu Leu Tyr Asp Arg Thr Asp 85 90 95cgc cct ggt 297Arg Pro Gly299PRTToxoplasma gondii 2Ala Ala Leu Gly Gly Leu Ala Ala Asp Gln Pro Glu Asn His Gln Ala1 5 10 15Leu Ala Glu Pro Val Thr Gly Val Gly Glu Ala Gly Val Ser Pro Val 20 25 30Asn Glu Ala Gly Glu Ser Tyr Ser Ser Ala Thr Ser Gly Val Gln Glu 35 40 45Ala Thr Ala Pro Gly Ala Val Leu Leu Asp Ala Ile Asp Ala Glu Ser 50 55 60Asp Lys Val Asp Asn Gln Ala Glu Gly Gly Glu Arg Met Lys Lys Val65 70 75 80Glu Glu Glu Leu Ser Leu Leu Arg Arg Glu Leu Tyr Asp Arg Thr Asp 85 90 95Arg Pro Gly3231DNAToxoplasma gondiiCDS(1)..(231) 3gct acc gcg gcc acc gcg tca gat gac gaa ctg atg agt cga atc cga 48Ala Thr Ala Ala Thr Ala Ser Asp Asp Glu Leu Met Ser Arg Ile Arg1 5 10 15aat tct gac ttt ttc gat ggt caa gca ccc gtt gac agt ctc aga ccg 96Asn Ser Asp Phe Phe Asp Gly Gln Ala Pro Val Asp Ser Leu Arg Pro 20 25 30acg aac gcc ggt gtc gac tcg aaa ggg acc gac gat cac ctc acc acc 144Thr Asn Ala Gly Val Asp Ser Lys Gly Thr Asp Asp His Leu Thr Thr 35 40 45agc atg gat aag gca tct gta gag agt cag ctt ccg aga aga gag cca 192Ser Met Asp Lys Ala Ser Val Glu Ser Gln Leu Pro Arg Arg Glu Pro 50 55 60ttg gag acg gag cca gat gaa caa gaa gaa gtt cat ttc 231Leu Glu Thr Glu Pro Asp Glu Gln Glu Glu Val His Phe65 70 75477PRTToxoplasma gondii 4Ala Thr Ala Ala Thr Ala Ser Asp Asp Glu Leu Met Ser Arg Ile Arg1 5 10 15Asn Ser Asp Phe Phe Asp Gly Gln Ala Pro Val Asp Ser Leu Arg Pro 20 25 30Thr Asn Ala Gly Val Asp Ser Lys Gly Thr Asp Asp His Leu Thr Thr 35 40 45Ser Met Asp Lys Ala Ser Val Glu Ser Gln Leu Pro Arg Arg Glu Pro 50 55 60Leu Glu Thr Glu Pro Asp Glu Gln Glu Glu Val His Phe65 70 755219DNAToxoplasma gondiiCDS(1)..(219) 5agg agg act gga tgt cat gcc ttc agg gag aac tgc agc cct ggt aga 48Arg Arg Thr Gly Cys His Ala Phe Arg Glu Asn Cys Ser Pro Gly Arg1 5 10 15tgt att gat gac gcc tcg cat gag aat ggc tac acc tgc gag tgc ccc 96Cys Ile Asp Asp Ala Ser His Glu Asn Gly Tyr Thr Cys Glu Cys Pro 20 25 30aca ggg tac tca cgt gag gtg act tcc aag gcg gag gag tcg tgt gtg 144Thr Gly Tyr Ser Arg Glu Val Thr Ser Lys Ala Glu Glu Ser Cys Val 35 40 45gaa gga gtc gaa gtc acg ctg gct gag aaa tgc gag aag gaa ttc ggc 192Glu Gly Val Glu Val Thr Leu Ala Glu Lys Cys Glu Lys Glu Phe Gly 50 55 60atc agc gcg tca tcc tgc aaa tgc gat 219Ile Ser Ala Ser Ser Cys Lys Cys Asp65 70673PRTToxoplasma gondii 6Arg Arg Thr Gly Cys His Ala Phe Arg Glu Asn Cys Ser Pro Gly Arg1 5 10 15Cys Ile Asp Asp Ala Ser His Glu Asn Gly Tyr Thr Cys Glu Cys Pro 20 25 30Thr Gly Tyr Ser Arg Glu Val Thr Ser Lys Ala Glu Glu Ser Cys Val 35 40 45Glu Gly Val Glu Val Thr Leu Ala Glu Lys Cys Glu Lys Glu Phe Gly 50 55 60Ile Ser Ala Ser Ser Cys Lys Cys Asp65 707393DNAToxoplasma gondiiCDS(1)..(393) 7cca tcg gtc gtc aat aat gtc gca agg tgc tcc tac ggt gca gac agc 48Pro Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr Gly Ala Asp Ser1 5 10 15act ctt ggt cct gtc aag ttg tct gcg gaa gga ccc act aca atg acc 96Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro Thr Thr Met Thr 20 25 30ctc gtg tgc ggg aaa gat gga gtc aaa gtt cct caa gac aac aat cag 144Leu Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln Asp Asn Asn Gln 35 40 45tac tgt tcc ggg acg acg ctg act ggt tgc aac gag aaa tcg ttc aaa 192Tyr Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu Lys Ser Phe Lys 50 55 60gat att ttg cca aaa tta act gag aac ccg tgg cag ggt aac gct tcg 240Asp Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp Gln Gly Asn Ala Ser65 70 75 80agt gat aag ggt gcc acg cta acg atc aag aag gaa gca ttt cca gcc 288Ser Asp Lys Gly Ala Thr Leu Thr Ile Lys Lys Glu Ala Phe Pro Ala 85 90 95gag tca aaa agc gtc att att gga tgc aca ggg gga tcg cct gag aag 336Glu Ser Lys Ser Val Ile Ile Gly Cys Thr Gly Gly Ser Pro Glu Lys 100 105 110cat cac tgt acc gtg aaa ctg gag ttt gcc ggg gct gca ggg tca gca 384His His Cys Thr Val Lys Leu Glu Phe Ala Gly Ala Ala Gly Ser Ala 115 120 125aaa tcg gct 393Lys Ser Ala 1308131PRTToxoplasma gondii 8Pro Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr Gly Ala Asp Ser1 5 10 15Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro Thr Thr Met Thr 20 25 30Leu Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln Asp Asn Asn Gln 35 40 45Tyr Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu Lys Ser Phe Lys 50 55 60Asp Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp Gln Gly Asn Ala Ser65 70 75 80Ser Asp Lys Gly Ala Thr Leu Thr Ile Lys Lys Glu Ala Phe Pro Ala 85 90 95Glu Ser Lys Ser Val Ile Ile Gly Cys Thr Gly Gly Ser Pro Glu Lys 100 105 110His His Cys Thr Val Lys Leu Glu Phe Ala Gly Ala Ala Gly Ser Ala 115 120 125Lys Ser Ala 1309237DNAToxoplasma gondiiCDS(1)..(237) 9ccc cag gat gcc att tgc tcg gat tgg tcc gca tgg agc ccc tgc agt 48Pro Gln Asp Ala Ile Cys Ser Asp Trp Ser Ala Trp Ser Pro Cys Ser1 5 10 15gta tcc tgc ggt gac gga agc caa atc agg acg cga act gag gtt tct 96Val Ser Cys Gly Asp Gly Ser Gln Ile Arg Thr Arg Thr Glu Val Ser 20 25 30gct ccg caa cct gga aca cca aca tgt ccg gac tgc cct gcg ccc atg 144Ala Pro Gln Pro Gly Thr Pro Thr Cys Pro Asp Cys Pro Ala Pro Met 35 40 45gga agg act tgc gtg gaa caa ggc gga ctt gaa gaa atc cgt gaa tgc 192Gly Arg Thr Cys Val Glu Gln Gly Gly Leu Glu Glu Ile Arg Glu Cys 50 55 60agt gcg ggg gta tgt gct gtt gac gct gga tgt ggc gtc tgg gtt 237Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly Val Trp Val65 70 751079PRTToxoplasma gondii 10Pro Gln Asp Ala Ile Cys Ser Asp Trp Ser Ala Trp Ser Pro Cys Ser1 5 10 15Val Ser Cys Gly Asp Gly Ser Gln Ile Arg Thr Arg Thr Glu Val Ser 20 25 30Ala Pro Gln Pro Gly Thr Pro Thr Cys Pro Asp Cys Pro Ala Pro Met 35 40 45Gly Arg Thr Cys Val Glu Gln Gly Gly Leu Glu Glu Ile Arg Glu Cys 50 55 60Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly Val Trp Val65 70 7511678DNAToxoplasma gondiiCDS(1)..(678) 11aac gaa ccg gtg gcc cta gct cag ctc agc aca ttc ctc gag ctc gtc 48Asn Glu Pro Val Ala Leu Ala Gln Leu Ser Thr Phe Leu Glu Leu Val1 5 10 15gag gtg cca tgt aac tct gtt cat gtt cag ggg gtg atg acc ccg aat 96Glu Val Pro Cys Asn Ser Val His Val Gln Gly Val Met Thr Pro Asn 20 25 30caa atg gtc aaa gtg act ggt gca gga tgg gat aat ggc gtt ctc gag 144Gln Met Val Lys Val Thr Gly Ala Gly Trp Asp Asn Gly Val Leu Glu 35 40 45ttc tat gtc acg agg cca acg aag aca ggc ggg gac aca agc cga agc 192Phe Tyr Val Thr Arg Pro Thr Lys Thr Gly Gly Asp Thr Ser Arg Ser 50 55 60cat ctt gcg tcg atc atg tgt tat tcc aag gac att gac ggc gtg ccg 240His Leu Ala Ser Ile Met Cys Tyr Ser Lys Asp Ile Asp Gly Val Pro65 70 75 80tca gac aaa gcg gga aag tgc ttt ctg aag aac ttt tct ggt gaa gac 288Ser Asp Lys Ala Gly Lys Cys Phe Leu Lys Asn Phe Ser Gly Glu Asp 85 90 95tcg tcg gaa ata gac gaa aaa gaa gta tct cta ccc atc aag agc cac 336Ser Ser Glu Ile Asp Glu Lys Glu Val Ser Leu Pro Ile Lys Ser His 100 105 110aac gat gcg ttc atg ttc gtt tgt tct tca aat gat gga tcc gca ctc 384Asn Asp Ala Phe Met Phe Val Cys Ser Ser Asn Asp Gly Ser Ala Leu 115 120 125cag tgt gat gtt ttc gcc ctt gat aac acc aac tct agc gac ggg tgg 432Gln Cys Asp Val Phe Ala Leu Asp Asn Thr Asn Ser Ser Asp Gly Trp 130 135 140aaa gtg aat acc gtg gat ctt ggc gtc agc gtt agt ccg gat ttg gca 480Lys Val Asn Thr Val Asp Leu Gly Val Ser Val Ser Pro Asp Leu Ala145 150 155 160ttc gga ctc act gca gat ggg gtc aag gtg aag aag ttg tac gca agc 528Phe Gly Leu Thr Ala Asp Gly Val Lys Val Lys Lys Leu Tyr Ala Ser 165 170 175agc ggc ctg aca gcg atc aac gac gac cct tcc ttg ggg tgc aag gct 576Ser Gly Leu Thr Ala Ile Asn Asp Asp Pro Ser Leu Gly Cys Lys Ala 180 185 190cct ccc cat tct ccg ccg gcc gga gag gaa ccg agt ttg ccg tcg cct 624Pro Pro His Ser Pro Pro Ala Gly Glu Glu Pro Ser Leu Pro Ser Pro 195 200 205gaa aac agc ggg tct gca aca cca gcg gaa gaa agt ccg tct gag tct 672Glu Asn Ser Gly Ser Ala Thr Pro Ala Glu Glu Ser Pro Ser Glu Ser 210 215 220gaa tct 678Glu Ser22512226PRTToxoplasma gondii 12Asn Glu Pro Val Ala Leu Ala Gln Leu Ser Thr Phe Leu Glu Leu Val1 5 10 15Glu Val Pro Cys Asn Ser Val His Val Gln Gly Val Met Thr Pro Asn 20 25 30Gln Met Val Lys Val Thr Gly Ala Gly Trp Asp Asn Gly Val Leu Glu 35 40 45Phe Tyr Val Thr Arg Pro Thr Lys Thr Gly Gly Asp Thr Ser Arg Ser 50 55 60His Leu Ala Ser Ile Met Cys Tyr Ser Lys Asp Ile Asp Gly Val Pro65 70 75 80Ser Asp Lys Ala Gly Lys Cys Phe Leu Lys Asn Phe Ser Gly Glu Asp 85 90 95Ser Ser Glu Ile Asp Glu Lys Glu Val Ser Leu Pro Ile Lys Ser His 100 105 110Asn Asp Ala Phe Met Phe Val Cys Ser Ser Asn Asp Gly Ser Ala Leu 115 120 125Gln Cys Asp Val Phe Ala Leu Asp Asn Thr Asn Ser Ser Asp Gly Trp 130 135 140Lys Val Asn Thr Val Asp Leu Gly Val Ser Val Ser Pro Asp Leu Ala145 150 155 160Phe Gly Leu Thr Ala Asp Gly Val Lys Val Lys Lys Leu Tyr Ala Ser 165 170 175Ser Gly Leu Thr Ala Ile Asn Asp Asp Pro Ser Leu Gly Cys Lys Ala 180 185 190Pro Pro His Ser Pro Pro Ala Gly Glu Glu Pro Ser Leu Pro Ser Pro 195 200 205Glu Asn Ser Gly Ser Ala Thr Pro Ala Glu Glu Ser Pro Ser Glu Ser 210 215 220Glu Ser2251326DNAArtificial SequencePrimer K551 13ggactagtcg gctcccccag gatgcc 261444DNAArtificial SequencePrimer K553 14catccagtcc tgctaccgcc accagaccag acgccacatc cagc 441544DNAArtificial SequencePrimer K552 15gtggcgtctg gtctggtggc ggtagcagga ctggatgtca tgcc 441645DNAArtificial SequencePrimer K555 16tgacgaccga gctaccgcca ccagagttat cgcatttgca ggatg 451745DNAArtificial SequencePrimer K554 17atgcgataac tctggtggcg gtagctcggt cgtcaataat gtcgc 451831DNAArtificial SequencePrimer 556 18ccgcggccgc tagccgattt tgctgaccct g 311933DNAArtificial SequencePrimer K563 19ggactagtcg gctggctgcc ttgggaggcc ttg 332045DNAArtificial SequencePrimer K565 20gccgcggtag cactaccgcc accagacaaa ccagggcgat ctgtg 452144DNAArtificial SequencePrimer K564 21gccctggttt gtctggtggc ggtagtgcta ccgcggccac cgcg 442246DNAArtificial SequencePrimer K567 22ccggttcgtt actaccgcca ccagagaaat gaacttcttc ttgttc 462346DNAArtificial SequencePrimer K566 23gaagttcatt tctctggtgg cggtagtaac gaaccggtgg ccctag 462430DNAArtificial SequencePrimer K568 24ccgcggccgc agattcagac tcagacggac 302540DNAArtificial SequencePrimer K572 25cgttactacc gccaccagag ttatcgcatt tgcaggatga 402639DNAArtificial SequencePrimer K571 26taactctggt ggcggtagta acgaaccggt ggccctagc 3927894DNAArtificial SequenceFirst chimeric antigen 27act agt cgg ctc ccc cag gat gcc att tgc tcg gat tgg tcc gca tgg 48Thr Ser Arg Leu Pro Gln Asp Ala Ile Cys Ser Asp Trp Ser Ala Trp1 5 10 15agc ccc tgc agt gta tcc tgc ggt gac gga agc caa atc agg acg cga 96Ser Pro Cys Ser Val Ser Cys Gly Asp Gly Ser Gln Ile Arg Thr Arg 20 25 30act gag gtt tct gct ccg caa cct gga aca cca aca tgt ccg gac tgc 144Thr Glu Val Ser Ala Pro Gln Pro Gly Thr Pro Thr Cys Pro Asp Cys 35 40 45cct gcg ccc atg gga agg act tgc gtg gaa caa ggc gga ctt gaa gaa 192Pro Ala Pro Met Gly Arg Thr Cys Val Glu Gln Gly Gly Leu Glu Glu 50 55 60atc cgt gaa tgc agt gcg ggg gta tgt gct gtt gac gct gga tgt ggc 240Ile Arg Glu Cys Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly65 70 75 80gtc tgg tct ggt ggc ggt agc agg act gga tgt cat gcc ttc agg gag 288Val Trp Ser Gly Gly Gly Ser Arg Thr Gly Cys His Ala Phe Arg Glu 85 90 95aac tgc agc cct ggt aga tgt att gat gac gcc tcg cat gag aat ggc 336Asn Cys Ser Pro Gly Arg Cys Ile Asp Asp Ala Ser His Glu Asn Gly 100 105 110tac acc tgc gag tgc ccc aca ggg tac tca cgt gag gtg act tcc aag 384Tyr Thr Cys Glu Cys Pro Thr Gly Tyr Ser Arg Glu Val Thr Ser Lys 115 120 125gcg gag gag tcg tgt gtg gaa gga gtc gaa gtc acg ctg gct gag aaa 432Ala Glu Glu Ser Cys Val Glu Gly Val Glu Val Thr Leu Ala Glu Lys 130 135 140tgc gag aag gaa ttc ggc atc agc gcg tca tcc tgc aaa tgc gat aac 480Cys Glu Lys Glu Phe Gly Ile Ser Ala Ser Ser Cys Lys Cys Asp Asn145 150 155 160tct ggt ggc ggt agc tcg gtc gtc aat aat gtc gca agg tgc tcc tac 528Ser Gly Gly Gly Ser Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr 165 170 175ggt gca gac agc act ctt ggt cct gtc aag ttg tct gcg gaa gga ccc 576Gly Ala Asp Ser Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro 180 185 190act aca atg acc ctc gtg tgc ggg aaa gat gga gtc aaa gtt cct caa 624Thr Thr Met Thr Leu Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln 195 200 205gac aac aat cag tac tgt tcc ggg acg acg ctg act ggt tgc aac gag 672Asp Asn Asn Gln Tyr Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu 210 215 220aaa tcg ttc aaa gat att ttg cca aaa tta act gag aac ccg tgg cag 720Lys Ser Phe Lys Asp Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp Gln225 230 235 240ggt aac gct tcg agt gat aag ggt gcc acg

cta acg atc aag aag gaa 768Gly Asn Ala Ser Ser Asp Lys Gly Ala Thr Leu Thr Ile Lys Lys Glu 245 250 255gca ttt cca gcc gag tca aaa agc gtc att att gga tgc aca ggg gga 816Ala Phe Pro Ala Glu Ser Lys Ser Val Ile Ile Gly Cys Thr Gly Gly 260 265 270tcg cct gag aag cat cac tgt acc gtg aaa ctg gag ttt gcc ggg gct 864Ser Pro Glu Lys His His Cys Thr Val Lys Leu Glu Phe Ala Gly Ala 275 280 285gca ggg tca gca aaa tcg gct agc ggc cgc 894Ala Gly Ser Ala Lys Ser Ala Ser Gly Arg 290 29528298PRTArtificial SequenceSynthetic Construct 28Thr Ser Arg Leu Pro Gln Asp Ala Ile Cys Ser Asp Trp Ser Ala Trp1 5 10 15Ser Pro Cys Ser Val Ser Cys Gly Asp Gly Ser Gln Ile Arg Thr Arg 20 25 30Thr Glu Val Ser Ala Pro Gln Pro Gly Thr Pro Thr Cys Pro Asp Cys 35 40 45Pro Ala Pro Met Gly Arg Thr Cys Val Glu Gln Gly Gly Leu Glu Glu 50 55 60Ile Arg Glu Cys Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly65 70 75 80Val Trp Ser Gly Gly Gly Ser Arg Thr Gly Cys His Ala Phe Arg Glu 85 90 95Asn Cys Ser Pro Gly Arg Cys Ile Asp Asp Ala Ser His Glu Asn Gly 100 105 110Tyr Thr Cys Glu Cys Pro Thr Gly Tyr Ser Arg Glu Val Thr Ser Lys 115 120 125Ala Glu Glu Ser Cys Val Glu Gly Val Glu Val Thr Leu Ala Glu Lys 130 135 140Cys Glu Lys Glu Phe Gly Ile Ser Ala Ser Ser Cys Lys Cys Asp Asn145 150 155 160Ser Gly Gly Gly Ser Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr 165 170 175Gly Ala Asp Ser Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro 180 185 190Thr Thr Met Thr Leu Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln 195 200 205Asp Asn Asn Gln Tyr Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu 210 215 220Lys Ser Phe Lys Asp Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp Gln225 230 235 240Gly Asn Ala Ser Ser Asp Lys Gly Ala Thr Leu Thr Ile Lys Lys Glu 245 250 255Ala Phe Pro Ala Glu Ser Lys Ser Val Ile Ile Gly Cys Thr Gly Gly 260 265 270Ser Pro Glu Lys His His Cys Thr Val Lys Leu Glu Phe Ala Gly Ala 275 280 285Ala Gly Ser Ala Lys Ser Ala Ser Gly Arg 290 295291258DNAArtificial SequenceSecond chimeric antigen 29act agt cgg ctg gct gcc ttg gga ggc ctt gcg gat cag cct gaa aat 48Thr Ser Arg Leu Ala Ala Leu Gly Gly Leu Ala Asp Gln Pro Glu Asn1 5 10 15cat cag gct ctt gca gaa cca gtt acg ggt gtg ggg gaa gca gga gtg 96His Gln Ala Leu Ala Glu Pro Val Thr Gly Val Gly Glu Ala Gly Val 20 25 30tcc ccc gtc aac gaa gct ggt gag tca tac agt tct gca act tcg ggt 144Ser Pro Val Asn Glu Ala Gly Glu Ser Tyr Ser Ser Ala Thr Ser Gly 35 40 45gtc caa gaa gct acc gcc cca ggt gca gtg ctc ctg gac gca atc gat 192Val Gln Glu Ala Thr Ala Pro Gly Ala Val Leu Leu Asp Ala Ile Asp 50 55 60gcc gag tcg gat aag gtg gac aat cag gcg gag gga ggt gag cgt atg 240Ala Glu Ser Asp Lys Val Asp Asn Gln Ala Glu Gly Gly Glu Arg Met65 70 75 80aag aag gtc gaa gag gag ttg tcg tta ttg agg cgg gaa tta tat gat 288Lys Lys Val Glu Glu Glu Leu Ser Leu Leu Arg Arg Glu Leu Tyr Asp 85 90 95cgc aca gat cgc cct ggt ttg tct ggt ggc ggt agt gct acc gcg gcc 336Arg Thr Asp Arg Pro Gly Leu Ser Gly Gly Gly Ser Ala Thr Ala Ala 100 105 110acc gcg tca gat gac gaa ctg atg agt cga atc cga aat tct gac ttt 384Thr Ala Ser Asp Asp Glu Leu Met Ser Arg Ile Arg Asn Ser Asp Phe 115 120 125ttc gat ggt caa gca ccc gtt gac agt ctc aga ccg acg aac gcc ggt 432Phe Asp Gly Gln Ala Pro Val Asp Ser Leu Arg Pro Thr Asn Ala Gly 130 135 140gtc gac tcg aaa ggg acc gac gat cac ctc acc acc agc atg gat aag 480Val Asp Ser Lys Gly Thr Asp Asp His Leu Thr Thr Ser Met Asp Lys145 150 155 160gca tct gta gag agt cag ctt ccg aga aga gag cca ttg gag acg gag 528Ala Ser Val Glu Ser Gln Leu Pro Arg Arg Glu Pro Leu Glu Thr Glu 165 170 175cca gat gaa caa gaa gaa gtt cat ttc tct ggt ggc ggt agt aac gaa 576Pro Asp Glu Gln Glu Glu Val His Phe Ser Gly Gly Gly Ser Asn Glu 180 185 190ccg gtg gcc cta gct cag ctc agc aca ttc ctc gag ctc gtc gag gtg 624Pro Val Ala Leu Ala Gln Leu Ser Thr Phe Leu Glu Leu Val Glu Val 195 200 205cca tgt aac tct gtt cat gtt cag ggg gtg atg acc ccg aat caa atg 672Pro Cys Asn Ser Val His Val Gln Gly Val Met Thr Pro Asn Gln Met 210 215 220gtc aaa gtg act ggt gca gga tgg gat aat ggc gtt ctc gag ttc tat 720Val Lys Val Thr Gly Ala Gly Trp Asp Asn Gly Val Leu Glu Phe Tyr225 230 235 240gtc acg agg cca acg aag aca ggc ggg gac aca agc cga agc cat ctt 768Val Thr Arg Pro Thr Lys Thr Gly Gly Asp Thr Ser Arg Ser His Leu 245 250 255gcg tcg atc atg tgt tat tcc aag gac att gac ggc gtg ccg tca gac 816Ala Ser Ile Met Cys Tyr Ser Lys Asp Ile Asp Gly Val Pro Ser Asp 260 265 270aaa gcg gga aag tgc ttt ctg aag aac ttt tct ggt gaa gac tcg tcg 864Lys Ala Gly Lys Cys Phe Leu Lys Asn Phe Ser Gly Glu Asp Ser Ser 275 280 285gaa ata gac gaa aaa gaa gta tct cta ccc atc aag agc cac aac gat 912Glu Ile Asp Glu Lys Glu Val Ser Leu Pro Ile Lys Ser His Asn Asp 290 295 300gcg ttc atg ttc gtt tgt tct tca aat gat gga tcc gca ctc cag tgt 960Ala Phe Met Phe Val Cys Ser Ser Asn Asp Gly Ser Ala Leu Gln Cys305 310 315 320gat gtt ttc gcc ctt gat aac acc aac tct agc gac ggg tgg aaa gtg 1008Asp Val Phe Ala Leu Asp Asn Thr Asn Ser Ser Asp Gly Trp Lys Val 325 330 335aat acc gtg gat ctt ggc gtc agc gtt agt ccg gat ttg gca ttc gga 1056Asn Thr Val Asp Leu Gly Val Ser Val Ser Pro Asp Leu Ala Phe Gly 340 345 350ctc act gca gat ggg gtc aag gtg aag aag ttg tac gca agc agc ggc 1104Leu Thr Ala Asp Gly Val Lys Val Lys Lys Leu Tyr Ala Ser Ser Gly 355 360 365ctg aca gcg atc aac gac gac cct tcc ttg ggg tgc aag gct cct ccc 1152Leu Thr Ala Ile Asn Asp Asp Pro Ser Leu Gly Cys Lys Ala Pro Pro 370 375 380cat tct ccg ccg gcc gga gag gaa ccg agt ttg ccg tcg cct gaa aac 1200His Ser Pro Pro Ala Gly Glu Glu Pro Ser Leu Pro Ser Pro Glu Asn385 390 395 400agc ggg tct gca aca cca gcg gaa gaa agt ccg tct gag tct gaa tct 1248Ser Gly Ser Ala Thr Pro Ala Glu Glu Ser Pro Ser Glu Ser Glu Ser 405 410 415gcg gcc gcg g 1258Ala Ala Ala30419PRTArtificial SequenceSynthetic Construct 30Thr Ser Arg Leu Ala Ala Leu Gly Gly Leu Ala Asp Gln Pro Glu Asn1 5 10 15His Gln Ala Leu Ala Glu Pro Val Thr Gly Val Gly Glu Ala Gly Val 20 25 30Ser Pro Val Asn Glu Ala Gly Glu Ser Tyr Ser Ser Ala Thr Ser Gly 35 40 45Val Gln Glu Ala Thr Ala Pro Gly Ala Val Leu Leu Asp Ala Ile Asp 50 55 60Ala Glu Ser Asp Lys Val Asp Asn Gln Ala Glu Gly Gly Glu Arg Met65 70 75 80Lys Lys Val Glu Glu Glu Leu Ser Leu Leu Arg Arg Glu Leu Tyr Asp 85 90 95Arg Thr Asp Arg Pro Gly Leu Ser Gly Gly Gly Ser Ala Thr Ala Ala 100 105 110Thr Ala Ser Asp Asp Glu Leu Met Ser Arg Ile Arg Asn Ser Asp Phe 115 120 125Phe Asp Gly Gln Ala Pro Val Asp Ser Leu Arg Pro Thr Asn Ala Gly 130 135 140Val Asp Ser Lys Gly Thr Asp Asp His Leu Thr Thr Ser Met Asp Lys145 150 155 160Ala Ser Val Glu Ser Gln Leu Pro Arg Arg Glu Pro Leu Glu Thr Glu 165 170 175Pro Asp Glu Gln Glu Glu Val His Phe Ser Gly Gly Gly Ser Asn Glu 180 185 190Pro Val Ala Leu Ala Gln Leu Ser Thr Phe Leu Glu Leu Val Glu Val 195 200 205Pro Cys Asn Ser Val His Val Gln Gly Val Met Thr Pro Asn Gln Met 210 215 220Val Lys Val Thr Gly Ala Gly Trp Asp Asn Gly Val Leu Glu Phe Tyr225 230 235 240Val Thr Arg Pro Thr Lys Thr Gly Gly Asp Thr Ser Arg Ser His Leu 245 250 255Ala Ser Ile Met Cys Tyr Ser Lys Asp Ile Asp Gly Val Pro Ser Asp 260 265 270Lys Ala Gly Lys Cys Phe Leu Lys Asn Phe Ser Gly Glu Asp Ser Ser 275 280 285Glu Ile Asp Glu Lys Glu Val Ser Leu Pro Ile Lys Ser His Asn Asp 290 295 300Ala Phe Met Phe Val Cys Ser Ser Asn Asp Gly Ser Ala Leu Gln Cys305 310 315 320Asp Val Phe Ala Leu Asp Asn Thr Asn Ser Ser Asp Gly Trp Lys Val 325 330 335Asn Thr Val Asp Leu Gly Val Ser Val Ser Pro Asp Leu Ala Phe Gly 340 345 350Leu Thr Ala Asp Gly Val Lys Val Lys Lys Leu Tyr Ala Ser Ser Gly 355 360 365Leu Thr Ala Ile Asn Asp Asp Pro Ser Leu Gly Cys Lys Ala Pro Pro 370 375 380His Ser Pro Pro Ala Gly Glu Glu Pro Ser Leu Pro Ser Pro Glu Asn385 390 395 400Ser Gly Ser Ala Thr Pro Ala Glu Glu Ser Pro Ser Glu Ser Glu Ser 405 410 415Ala Ala Ala311183DNAArtificial SequenceThird chimeric antigen 31act agt cgg ctc ccc cag gat gcc att tgc tcg gat tgg tcc gca tgg 48Thr Ser Arg Leu Pro Gln Asp Ala Ile Cys Ser Asp Trp Ser Ala Trp1 5 10 15agc ccc tgc agt gta tcc tgc ggt gac gga agc caa atc agg acg cga 96Ser Pro Cys Ser Val Ser Cys Gly Asp Gly Ser Gln Ile Arg Thr Arg 20 25 30act gag gtt tct gct ccg caa cct gga aca cca aca tgt ccg gac tgc 144Thr Glu Val Ser Ala Pro Gln Pro Gly Thr Pro Thr Cys Pro Asp Cys 35 40 45ccc gcg ccc atg gga agg act tgc gtg gaa caa ggc gga ctt gaa gaa 192Pro Ala Pro Met Gly Arg Thr Cys Val Glu Gln Gly Gly Leu Glu Glu 50 55 60atc cgt gaa tgc agt gcg ggg gta tgt gct gtt gac gct gga tgt ggc 240Ile Arg Glu Cys Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly65 70 75 80gtc tgg tct ggt ggc ggt agc agg act gga tgt cat gcc ttc agg gag 288Val Trp Ser Gly Gly Gly Ser Arg Thr Gly Cys His Ala Phe Arg Glu 85 90 95aac tgc cgc cct ggt aga tgt att gat gac gcc tcg cat gag aat ggc 336Asn Cys Arg Pro Gly Arg Cys Ile Asp Asp Ala Ser His Glu Asn Gly 100 105 110tac acc tgc gag tgc ccc aca tgg tac tca cgt gag gtg act tcc aag 384Tyr Thr Cys Glu Cys Pro Thr Trp Tyr Ser Arg Glu Val Thr Ser Lys 115 120 125gcg gag gag tcg tgt gtg gaa gga gtc gaa gtc acg ctg gct gag aaa 432Ala Glu Glu Ser Cys Val Glu Gly Val Glu Val Thr Leu Ala Glu Lys 130 135 140tgc gag aag gaa ttc ggc atc agc gcg tcc tcc tgc aaa tgc gat aac 480Cys Glu Lys Glu Phe Gly Ile Ser Ala Ser Ser Cys Lys Cys Asp Asn145 150 155 160tct ggt ggc ggt agt aac gaa ccg gtg gcc cta gct cag ctc agc aca 528Ser Gly Gly Gly Ser Asn Glu Pro Val Ala Leu Ala Gln Leu Ser Thr 165 170 175ttc ctc gag ctc gtc gag gtg cca tgt aac tct gtt cat gtt cag ggg 576Phe Leu Glu Leu Val Glu Val Pro Cys Asn Ser Val His Val Gln Gly 180 185 190gtg atg acc ccg aat caa atg gtc aaa gtg act ggt gca gga tgg gat 624Val Met Thr Pro Asn Gln Met Val Lys Val Thr Gly Ala Gly Trp Asp 195 200 205aat ggc gtt ctc gag ttc tat gtc acg agg cca acg aag aca ggc ggg 672Asn Gly Val Leu Glu Phe Tyr Val Thr Arg Pro Thr Lys Thr Gly Gly 210 215 220gac aca agc cga agc cac ctt gcg tcg atc atg tgt tat tcc aag gac 720Asp Thr Ser Arg Ser His Leu Ala Ser Ile Met Cys Tyr Ser Lys Asp225 230 235 240att gac ggc gtg ccg tca gac aaa gcg gga aag tgc ttt ttg aag aac 768Ile Asp Gly Val Pro Ser Asp Lys Ala Gly Lys Cys Phe Leu Lys Asn 245 250 255ttt tct ggt gaa gac tcg tcg gaa ata gac gaa aaa gaa gta tct cta 816Phe Ser Gly Glu Asp Ser Ser Glu Ile Asp Glu Lys Glu Val Ser Leu 260 265 270ccc atc aag agc cac aac gat gcg ttc atg ttc gtt tgt tct tca aat 864Pro Ile Lys Ser His Asn Asp Ala Phe Met Phe Val Cys Ser Ser Asn 275 280 285gat gga tcc gca ctc cag tgt gat gtt ttc gcc ctt gat aac acc aac 912Asp Gly Ser Ala Leu Gln Cys Asp Val Phe Ala Leu Asp Asn Thr Asn 290 295 300tct agc gac ggg tgg aaa gtg aat acc gtg gat ctt gac gtc agc gtt 960Ser Ser Asp Gly Trp Lys Val Asn Thr Val Asp Leu Asp Val Ser Val305 310 315 320agt ccg gat ttg gca ttc gga ctc act gca gat ggg gtc aag gtg aag 1008Ser Pro Asp Leu Ala Phe Gly Leu Thr Ala Asp Gly Val Lys Val Lys 325 330 335aag ttg tac gca agc agc ggc ctg aca gcg atc aac gac gac cct tcc 1056Lys Leu Tyr Ala Ser Ser Gly Leu Thr Ala Ile Asn Asp Asp Pro Ser 340 345 350ttg ggg tgc aag gct cct ccc cat tct ccg ccg gcc gga gag gaa ccg 1104Leu Gly Cys Lys Ala Pro Pro His Ser Pro Pro Ala Gly Glu Glu Pro 355 360 365agt ttg ccg tcg cct gaa aac agc ggg tct gca aca cca gcg gaa gaa 1152Ser Leu Pro Ser Pro Glu Asn Ser Gly Ser Ala Thr Pro Ala Glu Glu 370 375 380agt ccg tct gag tct gaa tct gcg gcc gcg g 1183Ser Pro Ser Glu Ser Glu Ser Ala Ala Ala385 39032394PRTArtificial SequenceSynthetic Construct 32Thr Ser Arg Leu Pro Gln Asp Ala Ile Cys Ser Asp Trp Ser Ala Trp1 5 10 15Ser Pro Cys Ser Val Ser Cys Gly Asp Gly Ser Gln Ile Arg Thr Arg 20 25 30Thr Glu Val Ser Ala Pro Gln Pro Gly Thr Pro Thr Cys Pro Asp Cys 35 40 45Pro Ala Pro Met Gly Arg Thr Cys Val Glu Gln Gly Gly Leu Glu Glu 50 55 60Ile Arg Glu Cys Ser Ala Gly Val Cys Ala Val Asp Ala Gly Cys Gly65 70 75 80Val Trp Ser Gly Gly Gly Ser Arg Thr Gly Cys His Ala Phe Arg Glu 85 90 95Asn Cys Arg Pro Gly Arg Cys Ile Asp Asp Ala Ser His Glu Asn Gly 100 105 110Tyr Thr Cys Glu Cys Pro Thr Trp Tyr Ser Arg Glu Val Thr Ser Lys 115 120 125Ala Glu Glu Ser Cys Val Glu Gly Val Glu Val Thr Leu Ala Glu Lys 130 135 140Cys Glu Lys Glu Phe Gly Ile Ser Ala Ser Ser Cys Lys Cys Asp Asn145 150 155 160Ser Gly Gly Gly Ser Asn Glu Pro Val Ala Leu Ala Gln Leu Ser Thr 165 170 175Phe Leu Glu Leu Val Glu Val Pro Cys Asn Ser Val His Val Gln Gly 180 185 190Val Met Thr Pro Asn Gln Met Val Lys Val Thr Gly Ala Gly Trp Asp 195 200 205Asn Gly Val Leu Glu Phe Tyr Val Thr Arg Pro Thr Lys Thr Gly Gly 210 215 220Asp Thr Ser Arg Ser His Leu Ala Ser Ile Met Cys Tyr Ser Lys Asp225 230 235 240Ile Asp Gly Val Pro Ser Asp Lys Ala Gly Lys Cys Phe Leu Lys Asn 245 250 255Phe Ser Gly Glu Asp Ser Ser Glu Ile Asp Glu Lys Glu Val Ser Leu 260 265 270Pro Ile Lys Ser His Asn Asp Ala Phe Met Phe Val Cys Ser Ser Asn 275 280 285Asp Gly Ser Ala Leu Gln Cys Asp Val Phe Ala Leu Asp Asn Thr Asn 290 295 300Ser Ser Asp Gly Trp Lys Val Asn Thr Val Asp Leu Asp Val Ser Val305 310 315 320Ser Pro Asp Leu

Ala Phe Gly Leu Thr Ala Asp Gly Val Lys Val Lys 325 330 335Lys Leu Tyr Ala Ser Ser Gly Leu Thr Ala Ile Asn Asp Asp Pro Ser 340 345 350Leu Gly Cys Lys Ala Pro Pro His Ser Pro Pro Ala Gly Glu Glu Pro 355 360 365Ser Leu Pro Ser Pro Glu Asn Ser Gly Ser Ala Thr Pro Ala Glu Glu 370 375 380Ser Pro Ser Glu Ser Glu Ser Ala Ala Ala385 39033115PRTToxoplasma gondii 33Ser Gly Gly Thr Gly Gln Gly Leu Gly Ile Gly Glu Ser Val Asp Leu1 5 10 15Glu Met Met Gly Asn Thr Tyr Arg Val Glu Arg Pro Thr Gly Asn Pro 20 25 30Asp Leu Leu Lys Ile Ala Ile Lys Ala Ser Asp Gly Ser Tyr Ser Glu 35 40 45Val Gly Asn Val Asn Val Glu Glu Val Ile Asp Thr Met Lys Ser Met 50 55 60Gln Arg Asp Glu Asp Ile Phe Leu Arg Ala Leu Asn Lys Gly Glu Thr65 70 75 80Val Glu Glu Ala Ile Glu Asp Val Ala Gln Ala Glu Gly Leu Asn Ser 85 90 95Glu Gln Thr Leu Gln Leu Glu Asp Ala Val Ser Ala Val Ala Ser Trp 100 105 110Gln Asp Glu 1153453PRTToxoplasma gondii 34Tyr Ser Ser Pro Arg Ile Val Val Leu Ile Arg Tyr Cys Phe Phe Ser1 5 10 15Thr Tyr Arg Leu Thr Met Phe Ala Val Lys His Cys Leu Leu Trp Ala 20 25 30Val Gly Ala Leu Val Asn Val Ser Val Arg Ala Ala Glu Phe Ser Gly 35 40 45Trp Asn Gln Gly Pro 503535PRTToxoplasma gondii 35Glu Asn Pro Val Arg Pro Pro Pro Pro Gly Phe His Pro Ser Val Ile1 5 10 15Pro Asn Pro Pro Tyr Pro Leu Gly Thr Pro Ala Gly Met Pro Gln Pro 20 25 30Glu Val Pro 353690PRTToxoplasma gondii 36Ala Pro Thr Gln Ser Glu Met Lys Glu Phe Gln Glu Glu Ile Lys Glu1 5 10 15Gly Val Glu Glu Thr Lys His Glu Asp Asp Pro Glu Met Thr Arg Leu 20 25 30Met Val Thr Glu Lys Gln Glu Ser Lys Asn Phe Ser Lys Met Ala Lys 35 40 45Ser Gln Ser Phe Ser Thr Arg Ile Glu Glu Leu Gly Gly Ser Ile Ser 50 55 60Phe Leu Thr Glu Thr Gly Val Thr Met Ile Glu Leu Pro Lys Thr Val65 70 75 80Ser Glu His Asp Met Asp Gln Leu Leu His 85 9037149PRTToxoplasma gondii 37Val Met Ala Ser Asp Pro Pro Leu Val Ala Asn Gln Trp Thr Cys Pro1 5 10 15Asp Lys Lys Ser Thr Ala Ala Val Ile Leu Thr Pro Thr Glu Asn His 20 25 30Phe Thr Leu Lys Cys Pro Lys Thr Ala Leu Thr Glu Pro Pro Thr Leu 35 40 45Ala Tyr Ser Pro Asn Arg Gln Ile Cys Pro Ala Gly Thr Thr Ser Ser 50 55 60Cys Thr Ser Lys Ala Val Thr Leu Ser Ser Leu Ile Pro Glu Ala Glu65 70 75 80Asp Ser Trp Trp Thr Gly Asp Ser Ala Ser Leu Asp Thr Ala Gly Ile 85 90 95Lys Leu Thr Val Pro Ile Glu Lys Phe Pro Val Thr Thr Gln Thr Phe 100 105 110Trp Gly Cys Ile Lys Gly Asp Asp Ala Gln Ser Cys Met Val Thr Val 115 120 125Thr Val Gln Ala Arg Ala Ser Ser Trp Asn Asn Val Ala Arg Cys Ser 130 135 140Tyr Gly Ala Asp Ser1453898PRTToxoplasma gondii 38Gly Leu Ser Gln Arg Val Pro Glu Leu Pro Glu Val Glu Pro Phe Asp1 5 10 15Glu Val Gly Thr Gly Ala Arg Arg Ser Gly Ser Ile Ala Thr Leu Leu 20 25 30Pro Gln Asp Ala Val Leu Tyr Glu Asn Ser Glu Asp Val Ala Val Pro 35 40 45Ser Asp Ser Ala Ser Thr Pro Ser Tyr Phe His Val Glu Ser Pro Ser 50 55 60Ala Ser Val Glu Ala Ala Thr Gly Ala Val Gly Glu Val Val Pro Asp65 70 75 80Cys Glu Glu Gln Gln Glu Gln Gly Asp Thr Thr Leu Ser Asp His Asp 85 90 95Phe His39135PRTToxoplasma gondii 39Leu Asn Pro Ile Asp Asp Met Leu Phe Glu Thr Ala Leu Thr Ala Asn1 5 10 15Glu Met Met Glu Asp Ile Thr Trp Arg Pro Arg Val Asp Val Glu Phe 20 25 30Asp Ser Lys Lys Lys Glu Met Ile Ile Leu Ala Asp Leu Pro Gly Leu 35 40 45Gln Lys Asp Asp Val Thr Ile Glu Val Asp Asn Gly Ala Ile Val Ile 50 55 60Lys Gly Glu Lys Thr Ser Lys Glu Ala Glu Lys Val Asp Asp Gly Lys65 70 75 80Thr Lys Asn Ile Leu Thr Glu Arg Val Ser Gly Tyr Phe Ala Arg Arg 85 90 95Phe Gln Leu Pro Ser Asn Tyr Lys Pro Asp Gly Ile Ser Ala Ala Met 100 105 110Asp Asn Gly Val Leu Arg Val Thr Ile Lys Val Glu Asp Ser Gly Gly 115 120 125Ala Lys Gln Gln Ile Ser Val 130 13540144PRTToxoplasma gondii 40Pro Cys Pro Ile Asn Ala Thr Cys Gly Gln Phe Glu Glu Trp Ser Thr1 5 10 15Cys Ser Val Ser Cys Gly Gly Gly Leu Lys Thr Arg Ser Arg Asn Pro 20 25 30Trp Asn Glu Asp Gln Gln His Gly Gly Leu Ser Cys Glu Gln Gln His 35 40 45Pro Gly Gly Arg Thr Glu Thr Val Thr Cys Asn Pro Gln Ala Cys Pro 50 55 60Val Asp Glu Arg Pro Gly Glu Trp Ala Glu Trp Gly Glu Cys Ser Val65 70 75 80Thr Cys Gly Asp Gly Val Arg Glu Arg Arg Arg Gly Lys Ser Leu Val 85 90 95Glu Ala Lys Phe Gly Gly Arg Thr Ile Asp Gln Gln Asn Glu Ala Leu 100 105 110Pro Glu Asp Leu Lys Ile Lys Asn Val Glu Tyr Glu Pro Cys Ser Tyr 115 120 125Pro Ala Cys Gly Ala Ser Cys Thr Tyr Val Trp Ser Asp Trp Asn Lys 130 135 14041194PRTToxoplasma gondii 41Leu Arg Gly Tyr Arg Phe Gly Val Trp Lys Lys Gly Arg Cys Leu Asp1 5 10 15Tyr Thr Glu Leu Thr Asp Thr Val Ile Glu Arg Val Glu Ser Lys Ala 20 25 30Gln Cys Trp Val Lys Thr Phe Glu Asn Asp Gly Val Ala Ser Asp Gln 35 40 45Pro His Thr Tyr Pro Leu Thr Ser Gln Ala Ser Trp Asn Asp Trp Trp 50 55 60Pro Leu His Gln Ser Asp Gln Pro His Ser Gly Gly Val Gly Arg Asn65 70 75 80Tyr Gly Phe Tyr Tyr Val Asp Thr Thr Gly Glu Gly Lys Cys Ala Leu 85 90 95Ser Asp Gln Val Pro Asp Cys Leu Val Ser Asp Ser Ala Ala Val Ser 100 105 110Tyr Thr Ala Ala Gly Ser Leu Ser Glu Glu Thr Pro Asn Phe Ile Ile 115 120 125Pro Ser Asn Pro Ser Val Thr Pro Pro Thr Pro Glu Thr Ala Leu Gln 130 135 140Cys Thr Ala Asp Lys Phe Pro Asp Ser Phe Gly Ala Cys Asp Val Gln145 150 155 160Ala Cys Lys Arg Gln Lys Thr Ser Cys Val Gly Gly Gln Ile Gln Ser 165 170 175Thr Ser Val Asp Cys Thr Ala Asp Glu Gln Asn Glu Cys Gly Ser Asn 180 185 190Thr Ala42169PRTToxoplasma gondii 42Ser Ala Asn Val Thr Ser Ser Glu Pro Ala Lys Leu Asp Leu Ser Cys1 5 10 15Ala His Ser Asp Asn Lys Gly Ser Arg Ala Pro Thr Ile Gly Glu Pro 20 25 30Val Pro Asp Val Ser Leu Glu Gln Cys Ala Ala Gln Cys Lys Ala Val 35 40 45Asp Gly Cys Thr His Phe Thr Tyr Asn Asp Asp Ser Lys Met Cys His 50 55 60Val Lys Glu Gly Lys Pro Asp Leu Tyr Asp Leu Thr Gly Gly Lys Thr65 70 75 80Ala Pro Arg Ser Cys Asp Arg Ser Cys Phe Glu Gln His Val Ser Tyr 85 90 95Glu Gly Ala Pro Asp Val Met Thr Ala Met Val Thr Ser Gln Ser Ala 100 105 110Asp Cys Gln Ala Ala Cys Ala Ala Asp Pro Ser Cys Glu Ile Phe Thr 115 120 125Tyr Asn Glu His Asp Gln Lys Cys Thr Phe Lys Gly Arg Gly Phe Ser 130 135 140Ala Phe Lys Glu Arg Gly Val Leu Gly Val Thr Ser Gly Pro Lys Gln145 150 155 160Phe Cys Asp Glu Gly Gly Lys Leu Thr 165435PRTArtificial SequencePeptide Linker SGGGS 43Ser Gly Gly Gly Ser1 5

Claims

1-31. (canceled)

32. A chimeric recombinant antigen containing a fusion of at least three different antigenic fragments of Toxoplasma gondii polypeptides, wherein said antigenic fragments are B-cell epitopes which bind to Toxoplasma gondii-specific antibodies and wherein the three different antigenic regions have an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42.

33. The chimeric antigen of claim 32, wherein the Toxoplasma gondii specific antibodies are extracted from sera of subjects who have been infected by Toxoplasma gondii.

34. The chimeric antigen of claim 32, wherein the at least three different antigenic regions are linked by a covalent bond or by a peptide linker.

35. A nucleotide sequence coding for the chimeric recombinant antigen of claim 32.

36. The nucleotide sequence according to claim 35, comprising at least three different nucleotide sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 AND SEQ ID NO:11.

37. The nucleotide sequence according to claim 35, comprising the nucleotide sequence of SEQ ID NO:27.

38. The nucleotide sequence according to claim 35, comprising the nucleotide sequence of SEQ ID NO:29.

39. The nucleotide sequence according to claim 35, comprising the nucleotide sequence of SEQ ID NO:31.

40. A nucleotide sequence that hybridizes under stringent hybridization conditions with a nucleotide sequence coding for the chimeric recombinant antigen of claim 32.

41. The chimeric recombinant antigen encoded by the nucleotide sequence of claim 40.

42. The nucleotide sequence of claim 35, which is a DNA sequence.

43. A vector comprising the DNA sequence of claim 42.

44. A host cell transformed with the vector of claim 43.

45. The chimeric recombinant antigen according to claim 32, obtained by a process comprising culturing a host cell transformed with a vector comprising a nucleotide sequence that codes for the chimeric recombinant antigen of claim 32 or that hybridizes under stringent hybridization conditions with a complement of a nucleotide sequence coding for the chimeric recombinant antigen of claim 32, and isolating the chimeric recombinant antigen from the cultured host cells.

46. A method for diagnosing a Toxoplasma gondii infection in a subject, the method comprising: contacting a chimeric recombinant antigen according to claim 32 with a body component of the subject for a time and under condition to allow the chimeric antigen to form an antigen/antibody immune complex; and detecting the antigen/antibody immune complex comprising the chimeric antigen.

47. The method according to claim 46, wherein the Toxoplasma gondii infection is a congenital toxoplasmosis and the subject is an infant.

48. The method according to claim 46, wherein the detecting of the antigen/antibody immune complex is indicative of a time of infection.

49. A kit for diagnosis of a Toxoplasma gondii infection, containing at least one chimeric recombinant antigen according to claim 32.

50. The kit of claim 49, wherein the kit is for the diagnosis of an acute and/or chronic Toxoplasma gondii infection.