ANTIGENS FOR DETECTING TOXOPLASMA INFECTION BY MONITORING CELLULAR IMMUNITY

Abstract

Antigens of Toxoplasma gondii that provide specific and strong delayed type hypersensitivity (DTH) immune response, or which stimulate IFN-.gamma. secretion, are used for testing subjects for infection. Any skin testing format may be adapted for testing for the delayed type hypersensitivity, including a patch, a needle, or a prong. Presence of DTH indicates infection. Alternate methods of detecting a T cell response including monitoring IFN-.gamma. secretion may be used.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 16/347,510, filed May 3, 2019 which was based on International Application number PCT/US2017/059978, filed Nov. 3, 2017 which claims the benefit of U.S. Provisional Application No. 62/417,136, filed Nov. 3, 2016 and U.S. Provisional Application No. 62/550,393, filed Aug. 25, 2017, each of the disclosures of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention is related to the area of clinical testing. In particular, it relates to cellular immune responses including delayed type hypersensitivity reactions and cytokine release, or interferon gamma secretion assays, and their use in diagnosis of toxoplasmosis.

BACKGROUND OF THE INVENTION

[0003] In Toxoplasma gondii, there are three main compartments, called dense granules (GRA proteins), rhoptries (ROP), and micronemes (MIC proteins), which release antigens into the extracellular milieu (Carruthers V B, Sibley L D. 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73:114-123). Although both GRA and MIC compartments release antigens constitutively at low levels, micronemes can be stimulated to release large amounts of antigen in response to certain environmental cues, such as contact with host cells or other host factors (Carruthers V B, Giddings O K, Sibley L D. 1999. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiol 1:225-236.; Carruthers V B, Sibley L D. 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73:114-123.; Carruthers V B, Sibley L D. 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 31:421-428.). Collectively, proteins that are released either constitutively or in a regulated fashion have been defined as "excretory secretory antigens (ESA)."

[0004] The ESA fraction is enriched in secretory microneme (MIC) proteins but also contains constitutively secreted dense granule (GRA) proteins. Several MIC and GRA proteins have been described. Previous studies have shown that MIC2, and its binding partner MIC2 associated protein (M2AP), are abundant components of ESA (Huynh M H, Barenau K E, Harper J M, Beatty W L, Sibley L D, Carruthers V B. 2003. Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex. EMBO J 22:2082-2090.). Additionally, MIC5 (Brydges S D, Sherman G D, Nockemann S, Loyens A, Daubener W, Dubremetz J, Carruthers V B. 2000. Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii. Mol Biochem Parasitol 111:51-66.) and MIC10 (Hoff E F, Cook S H, Sherman G D, Harper J M, Ferguson D J, Dubremetz J F, Carruthers V B. 2001. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Exp Parasitol 97:77-88.) have been studied as soluble micronemal proteins that are immunogenic. Several MIC proteins interact: for example MIC1, MIC4 and MIC6 form a complex involved in recognition of host carbohydrates (Friedrich N, Santos J M, Liu Y, Palma A S, Leon E, Saouros S, Kiso M, Blackman M J, Matthews S, Feizi T, Soldati-Favre D. J Biol Chem. 2010 285:2064-76., Blumenschein T M, Friedrich N, Childs R A, Saouros S, Carpenter E P, Campanero-Rhodes M A, Simpson P, Chai W, Koutroukides T, Blackman M J, Feizi T, Soldati-Favre D, Matthews S. EMBO J. 2007 26:2808-20) Gene deletions of MIC1 or MIC3 alone do not have a profound effect on invasion, but the double mutant is attenuated, indicating these proteins plan complementary roles (Moire N, Dion S, Lebrun M, Dubremetz J F, Dimier-Poisson I. Exp Parasitol. 2009 123:111-7). MIC1 has been used in a variety of immunodiagnostic assays based on detection of antibodies that react to this protein (Holec L, Gasior A, Brillowska-Dabrowska A, Kur J. Exp Parasitol. 2008 119:1-6) or to hybrid proteins containing MIC1 and other parasite antigens (Holec-Gasior L, Ferra B, Drapala D. Clin Vaccine Immunol. 2012 19:1977-9). As well, MIC1 and MIC4 have been used in vaccination studies in mice (Lourenco E V, Bernardes E S, Silva N M, Mineo J R, Panunto-Castelo A, Roque-Barreira M C. Microbes Infect. 2006 8:1244-51). Other studies have shown that the secretory proteins GRA4, GRA6, and GRA7 are targets of the immune response (Mercier C, Cesbron-Delauw M F. 2015. Toxoplasma secretory granules: one population or more? Trends Parasitol 31:60-71.).

[0005] Delayed type hypersensitivity (DTH) responses are driven by cellular immune responses to antigens (Black C A. 1999. Delayed type hypersensitivity: current theories with an historic perspective. Dermatol Online J 5:7.). Typically a test antigen is injected in the skin of the ear, flank, or footpad and swelling measured 24-48 hr later (Allen I C. 2013. Delayed-type hypersensitivity models in mice. Methods Mol Biol 1031:101-107.). The most well-known test uses tuberculin, an extract of purified protein derivative (PPD) from mycobacteria, which is used in a skin test for tuberculosis infection. The skin test is also the basis for many allergy testing protocols. Although previous studies have used skin testing of toxoplasmin in mice and hamsters based on swelling and redness, these assays have not proven to be that specific or sensitive (Frenkel J K. 1948. Dermal hypersensitivity to toxoplasma antigens (toxoplasmins). Proc Soc Exp Biol Med 68:634-639.). Previous studies testing toxoplasmin, a skin test reaction elicited by ESA antigens, showed that it was sensitive and specific for detecting individuals in France that were chronically infected with T. gondii (Rougier D, Ambroise-Thomas P. 1985. Detection of toxoplasmic immunity by multipuncture skin test with excretory-secretory antigen. Lancet 2:121-123.). In those studies, the ES antigen was made from culture supernatants, fixed with formalin, and then dialyzed with a 10 kDa filtration step. In subsequent studies, others have indicated that the active component in toxoplasmin is in the range of 10 kDa to 50 kDa based on filtration (Veprekova. 1978. Approximative molecular weight of the active component in toxoplasmin. Folia Parasitol (Praha) 25:273-275.). It should be noted that proteins may undergo proteolytic processing or breakdown, so this size range does not necessarily indicate the size or identity of the full-length protein. Although these studies refined our knowledge of the active components of ESA, the active components remain undefined at the molecular level. Moreover, there is no way to produce the ESA fraction in large quantities such that it could be made into a commercial product.

[0006] Delayed type hypersensitivity reactions are predominately driven by CD4+ memory T cells that recognize antigen from a previous exposure (Mantoux Test as a model for a secondary immune response in humans. Vukmanovic-Stejic M, Reed J R, Lacy K E, Rustin M H, Akbar A N. Immunol Lett. 2006 10793-101). Upon recognition of their cognate antigen, these memory T cells expand and produce cytokines including interferon gamma (IFN-.gamma.) tumor necrosis factor (TNF) and other chemokines. This initial reaction also results in recruitment of mononuclear (i.e. monocytes) cells and polymorphonuclear (i.e. PMNs) cells from circulation into the tissue. Although the conventional DTH test relies on monitoring induration, and redness that develop at the site of injection, more recent tests have been developed to directly monitor T cells responses to specific antigens. Typically these responses are monitored in circulating T cells obtained from the leukocyte fraction of whole blood. Leukocytes, including antigen-presenting cells and T cells, are mixed in vitro with antigens and the resulting responses monitored by production of IFN-.gamma. or other cytokines. In some applications there are referred to as INF.gamma.-release or IFN-.gamma.-secretion assays, owing the fact that IFN-.gamma. is the primary cytokine thought to drive the DTH response. The advantages of such tests is that they are more quantitative than the traditional skin test, they can be completed with a single office visit, and they often suffer less from cross-reaction to environmental antigens.

[0007] The enzyme-linked immunospot or ELISpot assay was originally developed for detecting B cells that were secreting antigen-specific antibodies (A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. Sedgwick J D, Holt P G. J Immunol Methods. 1983 February 57:301-9). It has seen been modified to detect cytokines secreted by different immune cells. The principle of the assay is that it relies on a sandwich ELISA where a membrane-backed microplate (typically polyvinylidene difluoride) is coated with antibodies to a particular cytokine. Cells from healthy or immune donors are added the plate and incubated overnight in medium under standard culture conditions. Cytokines secreted during this incubation are captured by the antibody-coated membrane. Following the incubation period, the cells are washed off and the captured cytokine is detected by a second antibody that is specific for the protein of interest. Detection is accomplished using an enzyme-linked reagent, either secondary antibody, or streptavidin to detect the biotinylated primary antibody.

[0008] ELISpot assays have previously been used for detection of IFN-.gamma. secretion by T cells in patients that were chronically infected with Toxoplasma gondii (Evolving characteristics of toxoplasmosis in patients infected with human immunodeficiency virus-1: clinical course and Toxoplasma gondii-specific immune responses. Hoffmann C, Ernst M, Meyer P, Wolf E, Rosenkranz T, Plettenberg A, Stoehr A, Horst H A, Marienfeld K, Lange C. Clin Microbiol Infect. 2007 13:510-5). This study focused on immunocompromised patients and used the ELISpot assay as a surrogate for CD4+ T cell responses to whole antigen. Although this study did not evaluate the ELISpot assay as a primary diagnostic tool, it suggests that the degree of immunity in a patient can be inferred from the strength of the response in the ELISpot assay. In this case the ELISpot test was conducted with whole parasite antigen and no attempt was made to define useful antigens that would increase sensitivity or specificity using this assay.

SUMMARY OF THE INVENTION

[0009] According to one aspect of the invention, a Toxoplasma gondii-derived antigen composition is provided. The composition comprises a Toxoplasma gondii-derived antigen selected from the group consisting of: isolated and purified MIC1, MIC3, MIC4, or MIC6; truncated MIC1, MIC3, MIC4, or MIC6; extended MIC1, MIC3, MIC4, or MIC6; a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6; a fusion protein of any of MIC1, MIC3, MIC4, or MIC6 with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and combinations thereof as elements of an antigen or components of a composition. The Toxoplasma gondii-derived antigen composition may alternatively or additionally comprise or consist of any of the antigens shown in Table 1.

[0010] According to another aspect of the invention, a kit is provided. The kit comprises (a) a Toxoplasma gondii-derived antigen composition and (b) an applicator device for administration of the Toxoplasma gondii-derived antigen to a subject. The composition comprises a Toxoplasma gondii-derived antigen selected from the group consisting of: isolated and purified MIC1, MIC3, MIC4, or MIC6; truncated MIC1, MIC3, MIC4, or MIC6; extended MIC1, MIC3, MIC4, or MIC6; a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6; a fusion protein of any of MIC1, MIC3, MIC4, or MIC6 with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and combinations thereof as elements of an antigen or components of a composition. The Toxoplasma gondii-derived antigen composition may comprise or consist of any of the antigens shown in Table 1.

[0011] According to yet another aspect of the invention a method of delivering Toxoplasma gondii-derived antigen to a subject is provided. An applicator device that is loaded with a Toxoplasma gondii-derived antigen composition is contacted with skin of the subject. The Toxoplasma gondii-derived antigen composition is thereby delivered to the skin of the subject. The composition comprises a Toxoplasma gondii-derived antigen selected from the group consisting of: isolated and purified MIC1, MIC3, MIC4, or MIC6; truncated MIC1, MIC3, MIC4, or MIC6; extended MIC1, MIC3, MIC4, or MIC6; a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6; a fusion protein of any of MIC1, MIC3, MIC4, or MIC6 with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and combinations thereof as elements of an antigen or components of a composition. The Toxoplasma gondii-derived antigen composition may comprise or consist of any of the antigens shown in Table 1.

[0012] Yet another aspect of the invention is an applicator device for administering one or more Toxoplasma gondii-derived antigens to a mammal. The applicator device comprises one or more Toxoplasma gondii-derived antigens. The Toxoplasma gondii-derived antigens are selected from the group consisting of: isolated and purified MIC1, MIC3, MIC4, or MIC6; truncated MIC1, MIC3, MIC4, or MIC6; extended MIC1, MIC3, MIC4, or MIC6; a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6; a fusion protein of any of MIC1, MIC3, MIC4, or MIC6 with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and combinations thereof as elements of an antigen or components of a composition. The Toxoplasma gondii-derived antigen composition may comprise or consist of any of the antigens shown in Table 1.

[0013] Yet another aspect of the invention is a method of eliciting and/or monitoring a T cell response in a subject. A Toxoplasma gondii-derived antigen composition is contacted with T cells of the subject. The Toxoplasma gondii-derived antigen composition induces a T cell response, which may involve production or secretion of cytokines. The Toxoplasma gondii-derived antigen composition may be isolated and purified MIC1, MIC3, MIC4, or MIC6; truncated MIC1, MIC3, MIC4, or MIC6; extended MIC1, MIC3, MIC4, or MIC6; a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6; a fusion protein of any of MIC1, MIC3, MIC4, or MIC6 with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and combinations thereof as elements of an antigen or components of a composition. The Toxoplasma gondii-derived antigen composition may comprise or consist of any of the antigens shown in Table 1.

[0014] These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office by request and payment of the necessary fee.

[0016] FIG. 1 shows a luciferase-based secretion assay. Secretion was monitored by release of the reporter protein MIC1-GLuc into the supernatant. Samples include cells that were treated with BAPTA-AM to block secretion (Inhibitor), cells treated only with buffer (Mock), and cells treated with BSA and zaparinast (Stimulated). These later samples contain the fraction that is referred to as ESA.

[0017] FIG. 2 shows an expression system for purification of fusion proteins. SUMO is a ubiquitin-like protein that is highly soluble, aiding in the expression of proteins in E. coli. Proteins can be cleaved by SUMO protease to release tag-free proteins of high purity. The SUMO-fusion protein and the SUMO protease are both tagged with six histidine residues that mediate binding to nickel, allowing one step purification by affinity chromatography.

[0018] FIG. 3 shows luminol responses in mice. FIG. 3A) Animals were injected with antigens in the ear pinne. At 48 hr after injection, animals were imaged after injection of luminol using a Xenogen IVIS 200 instrument. Animals were injected with either ESA or total antigen. Numbers indicate antigen amounts in micrograms. FIG. 3B) Quantification of the images shown in A. Data were processed using Living Image software.

[0019] FIG. 4 Monitoring of lipopolysaccharide (LPS) using the limulus amebocyte assay (LAL). After purification over polymixin B resin, the level of LPS as monitored by the LAL assay was reduced by >50 fold.

[0020] FIG. 5. shows ELISpot assay detecting IFN-.gamma. produced by splenocytes from naive and T. gondii infected mice. Samples treated with PBS or ESA during in vitro culture. Con A serves as a non-specific positive control.

[0021] FIG. 6. shows ELISpot data for IFN-.gamma. secretion by T cell in response to purified ESA proteins. FIG. 6A) Responses from C57/BL6 mice, FIG. 6B) Responses from Balb/C mice. Individual data points represent a result from one mouse, either uninfected (gray) or chronically infected (black). SFC indicates "spot forming cells" that were positive for INF.gamma. secretion.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The inventors have developed a standardized, abundant test antigen composition for use in sensitively and specifically testing individuals for infection by Toxoplasma gondii. Antigens that cause a non-specific reaction (whether the subject has been infected or not) and antigens that cause a specific reaction (only in subject that has been infected) have been identified. The latter have been purified and cloned and modified to form test reagents. The former have been eliminated from test reagents.

[0023] The compositions of antigens preferably contain only antigens that cause a specific reaction and are devoid of antigens that cause a non-specific reaction. Such preparation may be made by any means known in the art, including isolation and purification from, e.g., natural sources, recombinant production, or synthetic production. Carriers for the antigens may be any standardly used, typically a carrier that does not itself cause a DTH reaction or inhibit a DTH reaction by a bona fide antigen. Non-limiting examples of excipients that may be used for the antigen compositions are sucrose, mannitol, trehalose, and Hemaccel.TM. (intravenous colloid). Buffers, salts, sugars, preservatives, isotonic saline solutions, phosphate-buffered saline, can also be used in the compositions. Additional components and excipients include water, polymers, fatty acid esters, parabens. Compositions may be stored as convenient, including without limitation as lyophilized samples, at about or below 4 degrees C., and at about or below -70 degrees C.

[0024] Compositions of antigens may be free of other ESA components such as dense granular proteins (GRA), other microneme proteins, or other components which lead to lower sensitivity and/or specificity. An isolated and purified preparation may be from T. gondii organisms, from a recombinant host cell, or from a synthetic in vitro reaction. The isolated and purified protein may comprise at least at least 1%, at least 5%, 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the protein in a composition.

[0025] Testing for DTH may be used in order to prevent or detect congenital toxoplasmosis, for example by testing women before or during pregnancy, respectively. Primary infection of pregnant women may lead to abortion or severe neonatal malformation. Testing may also be used in immunocompromised patients, in whom a severe form of the disease may be fatal. Testing for DTH might also be performed in healthy adults to determine their infectious status prior to performing a medical procedure as a consequence of which they may become immunocompromised. Detection of infection may be critical in managing the disease. If a positive DTH test occurs, it may be desirable to follow it with a serum test. Because the two types of tests detect different immunological pathways and components, the two types of tests may give complementary information. Serum tests detect antibodies, whereas DTH tests detect cellular immune responses.

[0026] As an alternative, an in vitro reaction may be used to detect a T cell response. The in vitro reaction may be performed on any source of T cells, including whole blood, serum, plasma, and other tissue sources of T cells. The T cells are contacted with one or more of the Toxoplasma gondii-derived antigens or an antigen composition. If the T cells are reactive with the antigens or antigen composition they release a cytokine such as interferon-.gamma. or other cytokines. The presence of interferon-.gamma. or other released cytokine can be detected using any technique known in the art, including but not limited to an antibody or a series of antibodies. The antibodies may be labeled for detection. An antibody may be attached to an enzyme, such as horseradish peroxidase or alkaline phosphatase that produces colored products in the presence of appropriate substrates. An antibody may be fluorescently labeled, as an alternative. The in vitro reaction product may be captured on a solid support or assayed in the reaction fluid.

[0027] Kits may comprise an outer package to contain all components as well as optional inner packaging to contain individual components or combinations of components. Optional components include instructions for assembly and/or administration, information on side effects, expiry information, etc. Information may be provided in paper form, on a digital medium, or as an internet address to such information.

[0028] Applicators may be any type as is known in the art for administering an antigen to the skin of a subject and developing a DTH response. These include without limitation patches, needles, multi-needle assemblies, prongs, multi-prong assemblies. Antigens may be administered individually at separate locations or in combination at a single location.

[0029] Fusion proteins can be made using recombinant DNA technology to express two or more proteins or polypeptide portions of proteins as a single expression product. Any suitable technique known in the art for making and expressing such fusion proteins may be used. In some embodiments, a non-T. gondii protein is fused to a T. gondii protein. In other embodiments, two distinct T. gondii proteins are fused together.

[0030] Amounts of antigen composition that may be administered can be empirically determined, but may be between 0.1 and 50 ug, between 0.5 and 25 ug, or between 1 and 10 ug.

[0031] Suitable subjects include, but are not limited to, a human, a livestock animal, a companion animal, a lab animal, and a zoological animal. A subject may or may not be known to have a TDP43-mediated disorder. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a "zoological animal" refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the animal is a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In another preferred embodiment, the subject is a human.

[0032] MIC1 is normally a 456 residue (amino acid) protein that is processed in the parasite remove the N-terminal 16 residues. This leaves a total size of 440 residues. In contrast to this native protein, the form we have expressed, termed here truncated MIC1, is from residues 20-340, for a total size of 320 amino acids. This region of the protein contains a micronemal adhesive repeat that has been shown to bind to host sialic acid residues [Garnett, J.A., et al., Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii. Protein Sci, 2009. 18(9): p. 1935-47.]. We expressed a truncated version of the protein in order to make it soluble, a property that would distinguish it from the native molecule that also contains a C-terminal galectin domain [Saouros, S., et al., A novel galectin-like domain from Toxoplasma gondii micronemal protein 1 assists the folding, assembly, and transport of a cell adhesion complex. J Biol Chem, 2005. 280(46): p. 38583-91.]. It may, as a result, be recognized differently by the immune system.

[0033] MIC3 is normally a 383 residue (amino acids) protein that is processed in the parasite to remove the N-terminal 26 residues. This leaves a mature protein of 357 residues. We expressed a truncated form of MIC3 from residues 134 to 383, for a total size of 250 residues. We expressed a truncated version of the protein in order to make it soluble, a property that distinguishes it from the native molecule. The truncated form of MIC3 lacks most of the N-terminal lectin domain (residues 67-145) but contains the EGF repeats (residues 145-359) described previously (The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin. Cerede O, Dubremetz J F, Bout D, Lebrun M. EMBO J. 2002 21:2526-36).

[0034] MIC4 is normally a 580 residue (amino acid) protein that is processed in the parasite to remove the N-terminal 25 amino acids. This leaves a mature protein of 555 amino acids. Biochemical studies have shown that the full length protein is further processed at the N-terminus between residues 57-58 (VT-SS) and by a C-terminal processing event to generate a 50 kDa and a 15 kDa products (The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. Brecht S, Carruthers V B, Ferguson D J, Giddings O K, Wang G, Jakle U, Harper J M, Sibley L D, Soldati D. J Biol Chem. 2001 276:4119-27). We expressed a truncated form of MIC4 from residues 58 to 231, for a total size of 173 residues, a property that distinguishes it from the native molecule. The region of the protein that we expressed contains the first two Apple domains, but lacks the C-terminal Apple domains 5,6 that mediate binding to host cells (The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. Brecht S, Carruthers V B, Ferguson D J, Giddings O K, Wang G, Jakle U, Harper J M, Sibley L D, Soldati D. J Biol Chem. 2001 276:4119-27).

[0035] MIC6 is a 349 residue (amino acid) protein that is processed in the parasite to remove the first 23 residues. This leaves a mature protein of 326 residues that was expressed as a recombinant protein in E. coli. This full-length form of the protein contains three EGF domains, a single acidic domain and a transmembrane domain near the C-terminus as described previously (Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition. Sawmynaden K, Saouros S, Friedrich N, Marchant J, Simpson P, Bleijlevens B, Blackman M J, Soldati-Favre D, Matthews S. EMBO Rep. 2008 9:1149-55).

[0036] The mixture of ESA proteins, previously referred to as useful for a human skin test [Rougier, D. and P. Ambroise-Thomas, Detection of toxoplasmic immunity by multipuncture skin test with excretory-secretory antigen. Lancet, 1985. 2(8447): p. 121-3] may contain proteins that elicit non-specific responses. By removing these contaminants and focusing on proteins that only give positive responses in infected animals (and individuals) including MIC1, MIC3, MIC4, and MIC6, and truncated and/or fused forms of these proteins, our test achieves properties that are superior to the natural mixture of ESA proteins.

[0037] Previous studies have identified short peptide residues that enhance uptake by dendritic cells and increase the efficiency of antigen presentation [Sioud, M., et al., A novel peptide carrier for efficient targeting of antigens and nucleic acids to dendritic cells. FASEB J, 2013. 27(8): p. 3272-83.]. The receptor to which these peptides bind on host dendritic cells is not known. Nonetheless, it is likely that these short sequences work by enhancing uptake of the antigen and priming the presentation pathway. These steps of antigen uptake, processing, and presentation are critical for the DTH response. MIC1, MIC3, MIC4, and MIC6 and truncated and/or fused forms of these proteins, can be expressed so that these sequences are either at the N-or C-termini. These modified antigens can be purified under conditions that minimize contamination with LPS. Levels of LPS may be reduced to less than 0.5 EU/ml, less than 0.25 EU/ml, less than 0.1 EU/ml, less than 0.05 EU/ml. Any modification described herein for MIC1, 3, 4, or 6 can also be applied to any of the proteins of Table 1 or Table 2.

[0038] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples that are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLES

Example 1

Stimulation of Microneme Secretion

[0039] Freshly isolated tachyzoites of Toxoplasma gondii were stimulated to secrete Excretory-Secretory Antigens (ESA) using procedures similar to those published previously [1]. In brief, high-binding 96-well plates were coated with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) pH 7.4 or PBS alone at 4.degree. C. overnight. The next day, plates were washed with PBS to remove soluble BSA just prior to the secretion assay. Freshly harvested type I strain RH-parasites were purified and resuspended in intracellular buffer (25 mM HEPES pH 7.4, 142 mM KCl, 5 mM NaCl, 1 mM MgCl.sub.2, 2 mM EGTA, 5.6 mM D-Glucose). Control parasites were treated with 50 .mu.M BAPTA-AM to suppress secretion and added to wells washed with PBS (no BSA coating). For stimulated samples, untreated parasites were added to BSA-coated wells in the presence of 500 .mu.M zaprinast. Parasites were allowed to secrete for 10 min at 37.degree. C. prior to collection of the supernatant. Duplicate samples for each treatment were processed for liquid chromatography and tandem mass spectrometry (LC/MS-MS).

Identification of Toxoplasma-Secreted Proteins in ESA by LC-MS/MS

[0040] Secreted proteins were detected by LC-MS/MS as previously described [1]. In brief, ESA samples were reduced with 10 mM Tris-(2-carboxyethyl)phosphine and alkylated with 20 mM iodoacetamide before digestion overnight with 0.5 .mu.g of trypsin. After desalting, the digest was then dried down and resuspended in 15 .mu.l of 5% acetonitrile, 0.1% formic acid. Five microliters was resolved by LC-MS/MS on a NanoLC Ultra (Eksigent Technologies) coupled with an LTQ-Velos Pro Orbitrap (Thermo Scientific) using a 2 hr gradient. Raw data were processed and compared to the predicted proteome of the T. gondii genome to identify the protein components of ESA, as described previously [2]. For comparative semi-quantitative analysis, fold-enrichment was calculated from the frequency of spectral counts for peptides in the BSA-zaprinast stimulated samples compared to the BAPTA-AM treated control (set at 1 where no peptides were detected) for each protein detected in ESA. Proteins were considered candidate ESA proteins if the average fold enrichment was .gtoreq.4 in both of two independent experiments. Additionally, the cell-cycle specific expression profile for each candidate gene was analyzed to determine if it matched the profile of known microneme protein encoding genes [3]. Only proteins that fit both criteria (4 fold enriched and micronemal transcriptional profile) were considered authentic ESA-enriched proteins.

Cloning and Expression of T. gondii Antigens

[0041] Gene sequences encoding T. gondii microneme (MIC) and dense granule (GRA) proteins were obtained from ToxoDB (Protocol: http; Domain: toxodb; Top level domain: org). The coding regions were analyzed for the presence of predicted transmembrane or signal peptides using the ExPASy server (Protocol: https; subdomain: www; second level domain: expasy; Top level domain: org). Proteins were expressed either as full-length proteins or as truncations that were designed to eliminate undesirable hydrophobic regions. Genes were cloned from cDNA produced by Superscript III (Thermo Fischer Scientific) reverse transcriptase priming from polyA mRNA isolated from the type II ME49 strain, according to the manufacturer's recommendations. Primers to the coding regions of interest were designed to contain a BsaI site at the 5 end, and a XbaI site at the 3 end, in order to be compatible with the N-terminal His-tagged SUMO system from LifeSensor (pE-SUMO vector). PCR amplicons containing the region of interest were digested with BsaI and XbaI and cloned into similarly prepared pE-SUMO vector and transformed into competent XL1-Blue E. coli. Ampicillin resistant transformants were checked by PCR amplification and Sanger sequencing to verify plasmid inserts. To express recombinant proteins, pE-SUMO vectors containing inserts of T. gondii genes were transformed into BL21(DE3) Rosetta pLysS E. coli cells grown in terrific broth (TB) or Luria broth (LB). For induction of protein expression, cells were grown as a 5 ml overnight culture, then diluted 1:1,000 in fresh LB or TB and cultured at 37.degree. C. for 4-6 hr followed by addition of IPTG (0.5-1.0 mM) and culture at 15.degree. C. overnight. Protein expression and solubility were tested by lysis of the cell pellet in CelLytic B buffer (Sigma-Aldrich) and separation of pellet and supernatant fractions that were analyzed by SDA PAGE.

[0042] Proteins were purified using His-select nickel affinity columns (Sigma-Aldrich), and eluted in 200-300 mM imidazole, 50 mM sodium phosphate pH 8.0, 300 mM NaCl, according to the manufacturer's instructions. In cases where the SUMO tag was removed, proteins were first bound to nickel beads and then treated with purified SUMO protease U1P1 that was cloned as a N-terminal fusion in pET22b (Novagen) and separately produced in E. coli. The His-tagged SUMO and His-tagged protease were bound to the His-select nickel beads and the eluted fraction was analyzed for enrichment of the cleaved recombinant T. gondii protein. Proteins were checked for purity and concentration by SDS PAGE stained with Coomassie Blue or Syrpo Ruby. Proteins were dialyzed against 150 mM NaCl, 50 mM Tris-HCl, pH 8.0 and stored in aliquots at a concentration of 0.3 .mu.g/.mu.l at -80.degree. C. until used.

[0043] The purified recombinant proteins elicited strong responses in the Limulus amebocyte assay (LAL), the gold standard for monitoring lipopolysaccharide (LPS). To alleviate this problem, we treated the recombinant proteins with polymyxin B resin, an antibiotic that binds LPS (Endotoxin removal from protein solutions. Petsch D, Anspach F B. J Biotechnol. 2000 Jan. 21; 76(2-3):97-119). Recombinant proteins were incubated with Polymyxin B agarose-endotoxin removal resin (Sigma, USA) for 6-12 hrs at 4.degree. C. using gentle end over end mixing to achieve an endotoxin level <0.1 EU/mg. Then, the proteins were eluted using endotoxin-free buffer containing Tris-HCl at pH 6-8 depending on the protein. The amount of residual LPS in the proteins preparation were checked using a LAL assay kit (Pierce, USA) according to the manufacturer's instructions. Purified endotoxin-free proteins were then filter sterilized, quantified and stored at -80.degree. C.

Chronic Mouse Infections

[0044] Specific-pathogen-free mice were obtained from Jackson Laboratories and housed in the Animal Care Facilities at Washington University School of Medicine. Animals were housed and cared for according to the NIH Guide for the Care and Use of Laboratory Animals as approved by the Animal Studies Committee at Washington University.

[0045] Female C57/BL6 or Balb/C mice, age 8-12 weeks, were infected with the type II strains PRU or ME49 by i.p. needle inoculation of tachyzoites grown in vitro, using procedures described previously [4]. Alternatively, naive animals were infected by oral feeding of 5-10 tissue cysts from chronically infected mice, as described previously [4]. To prevent accidental death during acute infection, mice infected with the ME49 strain were given sulfadiazine in the drinking water (0.1-0.2 g/L) 4-10 days post infection. Chronic infections were confirmed by serological analysis of serum obtained 30 days post infection, as described previously [4].

In Vitro Assays for IFN-.gamma. Secretion

[0046] To obtain T lymphocytes for ELISpot analysis, we isolated splenocytes from naive and T. gondii chronically infected mice. Spleens were harvested and splenocytes were isolated by passage through a 70-.mu.m-pore-size nylon cell strainer. Splenocytes were pelleted and red blood cells (RBCs) were removed using RBC lysis buffer (Biolegend, USA) for 5 min at 4.degree. C. Splenocytes were then washed in sterile PBS and HBSS media (Corning, USA). Splenocytes were finally resuspended in CTL-media (CTL, USA) supplemented with 1% L-glutamine and 1.times. Pen-strep antibiotics.

[0047] ELISpot assays were conducting in 96 well plate format using splenocytes isolated as described above. Briefly, 2.5.times.10.sup.5 cells per well were plated in 96-well plate pre-coated with murine IFN-.gamma. capture antibody (Immunospot, CTL, USA) and cultured for 24 hrs with media alone, purified recombinant proteins (2 .mu.g/ml), a recall positive control-ESA (2 .mu.g/ml), a T cell non-specific positive control Con A (2 .mu.g/ml) and purified recombinant control protein-SUMO (2 .mu.g/ml) at 37.degree. C. with 5% CO.sub.2. After washing and developing the plate according to the manufacturer's instructions, the antigen recall response was determined by counting the number of spots (IFN-.gamma. producing cells) per well per treatment. The number of IFN-.gamma. producing T-cells following stimulation with T. gondii antigens, were detected and calculated using an ELISpot reader (Immunospot.RTM.S6 Core, CTL, USA).

Antigen Injection and Monitoring of Luminol Reaction

[0048] Control or chronically T. gondii infected mice were used to test the delayed type hypersensitivity (DTH) response using a previously published protocol for monitoring luminol fluorescence after in vivo injection [5]. Control or chronically infected mice were injected with PBS, ESA proteins (1.5 ug/in 10 .mu.L injection volume). Animals were injected s.c. either in the pinne of the ear (using PBS control on one side and antigen on the other) or s.c. in the back of the animal after it had been shaved to remove fur. At 24, 48, or 72 hr post injection, mice were anesthetized using isoflurane, injected i.p. with luminol (10 .mu.L/gram of body weight of a 20 mg/ml stock) and imaged using an IVSI Spectrum in vivo Imaging System with exposure settings of 1-3 sec. Data were analyzed using the IVIS Living Image software to determine the relative light emission for the region where antigen was injected, compared to a neutral background region or to the PBS control injection. Data were graphed and analyzed using Prism (GraphPad).

[0049] MIC1 Predicted protein sequence Type II ME49:

TABLE-US-00001 (SEQ ID NO: 1) MGQALFLTVLLPVLFGVGPEAYGEASHSHSPASGRYIQQMLDQRCQEIAA ELCQGGLRKMCVPSSRIVARNAVGITHQNTLEWRCFDTASLLESNQENNG VNCVDDCGHTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASA NKYCNDKFPGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTT WYCPGGRRGTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPP VCLDESGQISRTGGGPPSQPPEMQQPADRSDERGGGKEQSPGGEAQPDHP TKGGNIDLPEKSTSPEKTPKTEIHGDSTKATLEEGQQLTLTFISTKLDVA VGSCHSLVANFLDGFLKFQTGSNSAFDVVEVEEPAGPAVLTIGLGHKGRL AVVLDYTRLNAALGSAAYVV EDSGCSSSEEVSFQGVGSGATLVVTTLGE SPTAVSA

[0050] The form of MIC1 used in the assay (in bold above):

TABLE-US-00002 (SEQ ID NO: 2) His-SUMO-(M)AYGEASHSHSPASGRYIQQMLDQRCQEIAAELCQGGLR KMCVPSSRIVARNAVGITHQNTLEWRCFDTASLLESNQENNGVNCVDDCG HTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASANKYCNDKF PGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTTWYCPGGRR GTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPPVCLDESGQ ISRTGGGPPSQPPEMQQPADRSDERGGGKEQSPGGEAQPDHPTKGGNIDL PEKSTSPEKTPKTEIHGDSTKATLEEGQQLTL

[0051] MIC1 Coding sequence (introns spliced out, coding region in bold) Type II ME49:

TABLE-US-00003 (SEQ ID NO: 3) acctgaaagcgggtgccgcgtcgctaccgtttcctgtggcgtctctagtg cgacatccgaagtaacagtaacgtccggcatggaacgccgacgcgggtgt tccagtcgcctggctccttctactcgcacttcgatgttacgttccttatt ggtgcgacgcggttctcgtgttgctagacgtcgcaccggctgaaagctgt agaaaatttagttattttcctgtcagctagcttgcaggagtgcgtttttg tgtgttggtttcgtctcacatggctgctgatctgttgatgcagctgtgta cacgtgcctcgattctgtagttgacctagaacggatttgcaaagATGGGC CAGGCGTTGTTTCTCACCGTTCTATTGCCGGTGTTATTTGGCGTTGGGCC AGAAGCATATGGAGAAGCGTCGCATTCTCATTCGCCGGCATCGGGACGTT ATATACAACAGATGCTTGACCAACGCTGCCAAGAGATTGCTGCAGAACTC TGCCAAGGCGGACTTCGTAAAATGTGTGTGCCCTCTAGCCGGATAGTAGC TCGAAACGCCGTGGGCATTACTCATCAAAATACACTTGAATGGAGATGCT TTGATACAGCCTCTTTGCTGGAGAGCAATCAAGAAAACAACGGTGTTAAT TGCGTGGACGACTGTGGCCACACGATACCGTGTCCTGGCGGCGTACACCG GCAAAACAGTAATCACGCAACGCGCCATGAGATACTGTCCAAATTGGTCG AAGAAGGAGTACAACGGTTCTGCAGTCCTTATCAAGCATCTGCCAACAAG TACTGTAACGACAAATTTCCAGGGACCATTGCGAGGAGGTCGAAGGGCTT CGGAAACAATGTCGAGGTTGCGTGGAGGTGTTACGAGAAGGCCAGCTTGC TGTACTCGGTTTATGCTGAGTGTGCGAGCAACTGCGGAACAACGTGGTAC TGCCCTGGAGGACGACGAGGGACGTCGACAGAACTAGACAAGCGGCATTA TACAGAAGAGGAAGGAATTCGCCAGGCAATCGGATCCGTCGACAGCCCAT GTTCTGAAGTTGAAGTCTGCCTACCGAAGGATGAGAATCCCCCGGTGTGT TTAGATGAAAGTGGCCAGATTTCACGAACTGGTGGTGGGCCACCGTCACA ACCGCCTGAGATGCAACAGCCCGCCGATCGTTCGGACGAGAGAGGTGGCG GTAAGGAACAGTCGCCTGGAGGAGAAGCTCAGCCGGACCATCCAACGAAG GGTGGTAACATAGACCTGCCTGAGAAATCAACATCTCCCGAGAAGACGCC GAAAACCGAGATCCATGGTGACAGCACGAAAGCGACGCTCGAAGAGGGGC AGCAACTAACGCTCACGTTTATCTCCACTAAACTGGATGTTGCTGTAGGC TCGTGTCATTCACTCGTCGCGAATTTCCTTGATGGATTTTTGAAGTTTCA GACGGGCTCAAATTCGGCGTTCGATGTGGTAGAAGTGGAAGAGCCAGCAG GACCCGCAGTGCTTACGATAGGTCTGGGACACAAAGGCCGTCTCGCTGTT GTCCTCGACTACACCAGGCTCAATGCTGCTTTAGGATCAGCTGCTTACGT GGTCGAAGATTCTGGATGCAGCTCAAGTGAAGAGGTTAGTTTCCAAGGAG TGGGTAGTGGAGCGACGCTCGTGGTGACGACGCTTGGCGAGAGTCCTACG GCCGTCTCTGCTTGAtttatagtactctttggagcatgcttgtggaggaa cgggacaatctcggcaaaatcaggatgaagtttgtgagatacagatcgtt cctgaacagtggaagatgcgtcactattacacctatatgcgtcctggttc ttgtagagttggagttcttgcaggtgtaatgactatgacatacggatata acttcatacggggaactgtg

[0052] Primers used for cloning

[0053] MIC1(20-340) Bsa1-F:

TABLE-US-00004 (SEQ ID NO: 4) ACTGTGGTCTCTAGGTATGGAAGCATATGGAGAAGCGTCGCATTCTCA

[0054] MIC2(20-340) XBA1-R:

TABLE-US-00005 (SEQ ID NO: 5) ACTGTTCTAGATCAGAGCGTTAGTTGCTGCCCCTCTTCGAGCGTCGCTT

[0055] MIC3 Predicted protein sequence type II ME49:

TABLE-US-00006 (SEQ ID NO: 50) MRGGTSALLHALTFSGAVWMCTPAEALPIQKSVQLGSFDKVVPSREVVSE SLAPSFAVTETHSSVQSPSKQETQLCAISSEGKPCRNRQLHTDNGYFIGA SCPKSACCSKTMCGPGGCGEFCSSNWIFCSSSLIYHPDKSYGGDCSCEKQ GHRCDKNAECVENLDAGGGVHCKCKDGFVGTGLTCSEDPCSKRGNAKCGP NGTCIVVDSVSYTCTCGDGETLVNLPEGGQGCKRTGCHAFRENCSPGRCI DDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKEFGISAS SCKCDNGYSGSASATSHHGKGESGSEGSLSEKMNIVFKCPSGYHPRYHAH TVTCEKIKHFALDGAGNHDTTTYVARRRYPASL

[0056] The form of MIC3 used in the assay (in bold above):

[0057] HisSUMO--

TABLE-US-00007 (SEQ ID NO: 51) YHPDKSYGGDCSCEKQGHRCDKNAECVENLDAGGGVHCKCKDGFVGTGL TCSEDPCSKRGNAKCGPNGTCIVVDSVSYTCTCGDGETLVNLPEGGQGC KRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVE GVEVTLAEKCEKEFGISASSCKCDNGYSGSASATSHHGKGESGSEGSLS EKMNIVEKCPSGYHPRYHAHTVTCEKIKHFALDGAGNHDTTTYVARRRY PASL

[0058] Coding sequence (introns spliced out, coding region bold) Type II ME49:

TABLE-US-00008 (SEQ ID NO: 52) tcttctcttcttccgtacttttccctgcatttcacacccctggtatgac tccacaccgcgtgtaaatgtcccttaggtgacacccgcagcagcgcgta ggaggaagtagatgtcagtgtagacgtttttgagatgagagacgataac gtaaaatgccgccgataacttctgcattatacacactctctctccacgc ctaggatgacaggtacggcggcacacggaggaaagtggggggggggggg ggggcgaacagaaaggtcacatggaaggccgctcgactctccactcacg aagtgaaggcttcgtcccgttttgctggacaacgaatgcgaacttcttc actcgcttgtgacacacacaactccagaggcacagagatgtgaagcaga agagtggcgtgtgcgtcgcttctgtcggcggcaagccccgctccgtctc tttggtggcgattctggtgtgcaccgtgtgccaagaagttgcgtgtcac gcgacttttggaaatgcatcaggttcagagtcgttatgttgcgattcag gctctcggcagagaatcatttccctgtaagctagttgaactcgcctttt taaaagcggcagcagtgcccttgtggaaggcctcactgtgcctactttc ctcgtcctgagtttttccgccttcggcctcattttttgctcaccaaaat cgtgtcctaccgtcaagttttgccatagactcctacgggaaaaaacaag ccggtcgacacggacgacgcccgcagggaagcgtcccctccgcagaaat cgggagacaactgtcgttgacggtgctgcgcgaaaggtcacagagtttc cagtgtgttcatcagacctcactgtgcactgttagcggccgctgtcccg cctggtcaacaagtatcacaccctcgtccccgccattggcacggagctc gatgagctgcagtgtcgcttttaggggagtcgtgcaatcacgccgcaac acaggcgtgattcgatcttcaattgctaggtaaccactcgtgcttggta gctctgcaatggctcgagcgacgggggtgatgcaacatgctgctaaaaa ctcgacagacgtgtcaccggaacccacctaaataggagaccacgggtct ctggtgtgtcgcgtcgcattctcgcgtcgcattctcgcgtcgcaatgac cggccagttgctcgacgtcgccagccgggactgaagagcgttcatcgag tcagcagcattgcgtccccttgctcggtgaaaaaagactctctggtcga gtctagctcgtgtcacttctgtttctaacctccttcgttcaccggtaca cctccgatgtgacttttggtacacttgccctgtcgcacgacgcacgctg tcactcaacttgctgctagcgcaatcgataggttccctcgaaccagcca tcacacacacaccttttccgggaagacgtttgcgggcggtgggtcgcag ctcgtcgagagtgcgtttctgtgcatttctgtgggcagtgcagcgcgtt tgcgcgccttactctgtgtgtaacttccttgtccaacactggtaaaaAT GCGAGGCGGGACGTCCGCGCTGTTGCACGCGCTCACCTTCAGTGGGGCC GTGTGGATGTGCACCCCAGCGGAGGCTTTGCCGATTCAGAAGTCTGTGC AGCTGGGCAGCTTTGACAAAGTTGTGCCGAGCCGCGAAGTCGTCTCTGA GAGTCTTGCTCCGTCTTTCGCGGTGACTGAGACTCACTCGTCTGTGCAA TCCCCCAGCAAGCAGGAGACGCAACTCTGTGCTATCTCGAGTGAAGGCA AGCCATGTCGAAACCGTCAGTTGCACACTGACAACGGGTACTTCATCGG GGCCAGTTGCCCCAAGAGCGCTTGCTGCAGCAAGACCATGTGCGGCCCC GGCGGCTGCGGAGAATTCTGCTCCAGCAACTGGATTTTTTGCAGCAGTT CGCTCATCTACCATCCTGACAAAAGCTATGGAGGAGACTGCAGCTGTGA AAAGCAGGGCCATCGGTGCGACAAAAACGCAGAATGCGTCGAAAACTTG GACGCGGGTGGGGGTGTGCACTGCAAGTGCAAAGACGGCTTCGTCGGCA CTGGGTTGACTTGCTCCGAGGATCCTTGTTCAAAAAGAGGGAACGCGAA GTGCGGACCCAACGGGACGTGCATCGTCGTCGATTCAGTCAGCTACACA TGCACCTGCGGCGACGGCGAAACTCTAGTGAACCTCCCGGAAGGGGGAC AAGGATGCAAGAGGACTGGATGTCATGCCTTCAGGGAGAACTGCAGCCC TGGTAGATGTATTGATGACGCCTCGCATGAGAATGGCTACACCTGCGAG TGCCCCACAGGGTACTCACGTGAGGTGACTTCCAAGGCGGAGGAGTCGT GTGTGGAAGGAGTCGAAGTCACGCTGGCTGAGAAATGCGAGAAGGAATT CGGCATCAGCGCGTCATCCTGCAAATGCGATAACGGATACTCCGGATCT GCTTCCGCAACCTCCCACCATGGGAAAGGAGAATCGGGATCCGAGGGGA GCTTGAGTGAAAAAATGAATATTGTCTTCAAGTGCCCCAGTGGCTACCA TCCAAGATACCATGCCCACACCGTGACGTGTGAGAAAATTAAGCACTTT GCCCTTGACGGGGCCGGCAACCACGACACGACTACGTATGTCGCAAGAC GAAGGTACCCAGCGAGTCTCTGAgagcggagatcagcgcaaagacaaga tgcagagtttgactcgagaaacaatagtaacacgaagtaaaaagtctcc acactaagccaaggattgagaatatttcgatttgtgccgctggcaatag tggccttggcctagaaagaagttctgcaacgaagcgatcggctcacacg cggatacacagatgggtttgtaccgagaacgttaggtttgtgaaccgag ttcaggtaaaacaaagtagattgtgcctttacgcagacagcgagggaaa acatgaggacacactgccaactaaagcaagactgcctcactaattacca ccgacacacgacatggttacccccgcgttttgccgcgtgcaaagtttga attctgatggttctcgagtctgaaagcctaaaccgcccaaccatgtatg aaataagaacccatcaaacgtgagacatctctgccgaagtgcctacgaa aagaacgcttctgccactaggaggtgcggcctcttcattctatgagaac ctgctttgtcggtgtcaacctctggggaaatcgcctgcctttacacatt ttgctcgttgtagagcaagggatctgttgctgcgtttactccaatacaa tgatcgccgtttcgctgtaggcaagcgatccgaaaatgtacgttcgagt cagcagctacttgagaagcagccaacgccgacacttgctgcgtttgact gaggtgcactcgcaaacagtctcgtctccccggggcaatttctgagaga aatgcgggaatggacgtaatggtgctcttctgtgagtgctcttccacca atttttcgacaagtgttttcgtgacagtcgagtataccttcttatgtca ttctgtctccgtcagtgctatcggattcttcctattcctctaccctttc tacagtcgcatacaaagctgctgaaacaagacttcctttgtctagggta gttgtacactccacacatatctgactgaaacctacggcaggaagtctgg tcggcactgtgcttccttgttggcttttcgtcgtttctttgtctacgag cttcactgggtccttgacacggcttgtgagcgttgtgctcaatattcga ccagctgtatttgtg

[0059] Primers used for cloning:

[0060] MIC3F (134-383)-BsaI-F

TABLE-US-00009 (SEQ ID NO: 53) ACTGTGGTCTCTAGGTATGATCTACCATCCTGACAAAAGCTATGGAGGA GACT

[0061] MIC3F (64-383)-BsaI-R

TABLE-US-00010 (SEQ ID NO: 54) ACTTGTTCTAGATCAGAGACTCGCTGGGTACCTTCGTCT

[0062] MIC4 Predicted protein sequence type II ME49:

TABLE-US-00011 (SEQ ID NO: 55) MRASLPVHLVVCTQLSAVWFGVAKAHGGHRLEPHVPGFLQGFTDITPAG DDVSANVTSSEPAKLDLSCVHSDNKGSRAPTIGEPVPDVSLEQCAAQCK AVDGCTHFTYNDDSKMCHVKEGKPDLYDLTGGKTASRSCDRSCFEQHVS YEGAPDVMTAMVTSQSADCQAACAADPSCEIFTYNEHDQKCTFKGRGFS AFKERGVLGVTSGPKQFCDEGGKLTQEEMEDQISGCIQLSDVGSMTADL EEPMEADSVGACMERCRCDGRCTHETENDNTRMCYLKGDKMQLYSSPGD RTGPKSCDSSCFSNGVSYVDDPATDVETVFEISHPIYCQVICAANPLCT VFQWYASEAKCVVKRKGFYKHRKTGVTGVTVGPREFCDEGGSIRDREEA DAVGSDDGLNAEATMANSPDFHDEVECVHTGNIGSKAQTIGEVKRASSL SECRARCQAEKECSHYTYNVKSGLCYPKRGKPQFYKYLGDMTGSRTCDT SCLRRGVDYSQGPEVGKPWYSTLPTDCQVACDAEDACLVFTWDSATSRC YLIGSGESAHRRNDVDGVVSGPYTECDNGENLQVLEAKDTE

[0063] The form of MIC4 used in the assay (in bold above):

[0064] HisSUMO--

TABLE-US-00012 (SEQ ID NO: 56) SEPAKLDLSCVHSDNKGSRAPTIGEPVPDVSLEQCAAQCKAVDGCTHET YNDDSKMCHVKEGKPDLYDLTGGKTASRSCDRSCFEQHVSYEGAPDVMT AMVTSQSADCQAACAADPSCEIFTYNEHDQKCTFKGRGESAFKERGVLG VTSGPKQFCDEGGKLTQEEMEDQISG

[0065] Coding sequence (introns spliced out, coding region bold) Type II ME49:

TABLE-US-00013 (SEQ ID NO: 57) ttttctgtgcatctgtgctgcaaaacgggcctctgtgcattatttcccc accaacaattgccgcgtcgatccgggtcccgctcaagctctgcagaact aggctctcgatatagatcagtacaatcattcgcttctgacaatcgcatc gactgagcgacgcgttgatcgtcgactgtcgtgcgtcgcattcgggcat ctcgaaccggtgttgattccctgtgtcattatttcacttccgtccttct ctcgtggcgatctataatacgcgtgtgttgttgcgtgcattgcttgtgt tgttgtggatgtgttttcttttgtgaccgctcacgaacaccccacgcaa aATGAGAGCGTCGCTCCCGGTTCACCTCGTTGTGTGCACGCAGCTAAGT GCCGTTTGGTTTGGAGTGGCTAAAGCCCATGGTGGACACCGACTGGAAC CGCATGTTCCCGGATTCCTGCAAGGCTTCACTGATATCACGCCTGCAGG TGATGACGTTAGTGCCAACGTAACAAGTTCGGAGCCTGCAAAACTTGAT CTCTCTTGTGTGCACTCTGACAATAAGGGATCAAGGGCTCCCACAATAG GCGAGCCAGTGCCAGATGTGTCCCTGGAACAATGTGCTGCGCAATGCAA GGCTGTTGATGGCTGCACACATTTCACTTATAATGACGATTCGAAGATG TGCCATGTGAAGGAGGGAAAACCCGATTTATACGATCTCACAGGAGGCA AAACAGCATCGCGCAGTTGCGATAGATCATGCTTCGAACAACACGTATC GTATGAGGGAGCTCCTGACGTGATGACAGCGATGGTCACGAGCCAGTCA GCGGACTGTCAGGCTGCGTGTGCGGCTGACCCGAGCTGCGAGATCTTCA CTTATAACGAACACGACCAGAAATGTACTTTCAAAGGAAGGGGGTTTTC TGCGTTTAAGGAACGAGGGGTGTTGGGTGTGACTTCCGGGCCGAAACAG TTCTGCGATGAAGGCGGTAAATTAACTCAAGAGGAGATGGAAGATCAGA TCAGTGGCTGCATTCAATTGAGTGACGTTGGATCAATGACTGCTGACCT GGAGGAGCCTATGGAGGCTGATTCTGTTGGCGCTTGTATGGAACGGTGC CGCTGTGATGGAAGATGCACGCACTTCACGTTCAACGATAATACTCGGA TGTGCTACCTCAAAGGTGACAAGATGCAGTTGTACTCATCTCCAGGTGA CAGAACCGGCCCAAAGAGCTGCGATTCAAGCTGCTTCTCGAACGGGGTT TCTTACGTCGATGATCCGGCGACAGATGTTGAGACCGTATTCGAAATTT CACACCCAATTTATTGTCAAGTAATCTGCGCCGCAAATCCGTTGTGTAC AGTGTTTCAGTGGTATGCCTCCGAGGCAAAGTGCGTCGTCAAGAGAAAG GGGTTTTACAAACACAGAAAAACAGGTGTCACGGGAGTCACAGTGGGCC CTCGGGAGTTCTGCGATTTTGGCGGTAGCATCCGCGACCGAGAAGAGGC AGACGCCGTTGGATCAGACGATGGCCTCAACGCGGAAGCAACTATGGCA AATTCTCCTGATTTTCACGACGAAGTAGAATGCGTCCACACGGGCAACA TTGGGTCAAAAGCACAAACCATTGGAGAAGTGAAACGCGCAAGTAGTTT GAGTGAGTGCAGAGCCAGATGCCAAGCGGAGAAAGAATGCAGCCACTAC ACTTACAATGTAAAATCCGGTTTGTGTTATCCAAAAAGAGGAAAGCCTC AATTTTATAAGTATCTTGGCGACATGACGGGATCCAGAACATGTGATAC AAGTTGCCTTAGGAGGGGAGTCGATTACTCACAGGGCCCTGAAGTAGGA AAGCCTTGGTATTCTACGCTGCCGACAGACTGCCAAGTTGCATGCGACG CTGAGGATGCTTGCCTGGTGTTCACCTGGGATTCGGCGACGTCACGATG CTACCTCATCGGCTCAGGTTTCTCGGCACATCGACGGAACGACGTGGAT GGCGTGGTATCTGGACCCTATACTTTCTGTGACAATGGCGAAAACCTTC AGGTGCTTGAAGCGAAAGACACAGAATGAcccaggagggtgccagatac tttgtgtgactgcgacatgcagtcatgtactcaaagtgttgtacatgga caggaggactttttttttaagtcattgcagaggtgcgttttcggagcag cactataactgcgtcagcgactaagcacgccacgtagctgaatgaaacg cagccaccttcgtgtatgtatgcttcgttttttgtcgctgtgcagtttt gaatcatttcccttatgggacatttctgaaaaatgctccccgttcgctt gtagcactatgagaggggccgaagactgcaatggaggtagcgctgcgtt gaaaagacgaggcgctacatttcgcgtagcgacaaggccgtgtagagtt ttgcttttcgcgagacactgctctgagtgtcatatgcatcaaatgcagt ggtagcacacagaggtgagaagaatgatcacctgcgggggaatggcttt gctaaacaacaaggtcgctgtgtgactttacacaacgaaactactgtgg tgagtgctcagttgagtgaaaagaaatgccgcgttatcgtgagttctgg ttcggtggactttgccaccgtagtaaaactcaacctgtaacggaatgcc cagttttactgctctctttaaagggcgtccacgttctctatattcaagc tgtttacccacctgcgtttcggtgcatcgcgcgtgccacatcaaaaatc caggtaacggtgcgggacctatgctacactttatatctctcagaaagca tacacccactgattatggacaacgctgtggtcgcgttgtaccacaatgc aggaatactcagttcaccttgcaagtgttctggtgttcattgcgtgtca gaagtacacgaaaagagacttctttggcctccaagtgatacgtaaccgc ggcagtcatgaacagagtcactcgtgcttctgaaacgcacgtcttctgt acagagacagatgcagtgtgcatacaggaagcccctcgattgttgccgt agcaggtagccagtagaagaaacaaagacacggt

[0066] Primers used for cloning:

[0067] MIC4(58-231)-BasI-F

TABLE-US-00014 (SEQ ID NO: 58) ACTGTGGTCTCTAGGTATGAGTTCGGAGCCTGCAAAACTTGATCTCTCT TGTGT

[0068] MIC4(58-231)-XBAI-R

TABLE-US-00015 (SEQ ID NO: 59) ACTGTTCTAGATCAGCCACTGATATGATCTTCCATCTCCTCTTGAGT

[0069] MIC6 Predicted protein sequence type II ME49:

TABLE-US-00016 (SEQ ID NO: 60) MRLFRCCAAAVVAAESLLWLKNGSPFFAFLPGNGEIADNCSGNPCGGTA AGTCINTPSGYDCRCEPGYVLGVENDQVTCMMPSGVPMANFVQLSEKPA ACSSNPCGPEAAGTCNETNSGYICRCNQGYRISLDGTGNVTCIVRQESG CEENGCGPPDAVQSCRRLTGTAGRLCVCKENFIATIDASAHITCKRVPP HYRKPPFEFGKGGHPVDSEPSKRQREDEGESREPESDSTEPGRDQERRT PLEESQEPEGSTPDSQQSRGGSGSDSTESEEQGKEREEGSGHAGAIAGG VIGGLLLLSAAGAGVAYMRKSGSGGGEEIEYERGIEAAEASEVEVLVDL DSKTWD

[0070] The form of MIC6 used in the assay (in bold above):

[0071] His-SUMO

TABLE-US-00017 (SEQ ID NO: 61) SPHAFLPGNGEIADNCSGNPCGGTAAGTCINTPSGYDCRCEPGYVLGVE NDQVTCMMPSGVPMANFVQLSEKPAACSSNPCGPEAAGTCNETNSGYIC RCNQGYRISLDGTGNVTCIVRQESGCEENGCGPPDAVQSCRRLTGTAGR LCVCKENFIATIDASAHITCKRVPPHYRKPPFEFGKGGHPVDSEPSKRQ REDEGESREPESDSTEPGRDQERRTPLEESQEPEGSTPDSQQSRGGSGS DSTESEEQGKEREEGSGHAGAIAGGVIGGLLLLSAAGAGVAYMRKSGSG GGEEIEYERGIEAAEASEVEVLVDLDSKTWD

[0072] Coding sequence (introns spliced out, coding region bold) Type II ME49:

TABLE-US-00018 (SEQ ID NO: 62) cagtccggagcacactcctacaataaacttgatacgtgtcattttgtga aacgacacagcacataaccactcggactgtctcacgaagctgtagggcg gattcaccaatgatctttcgcagccgatccaaaactacttgcccacttc cggtgtacgtacatcgcgcgacatgagaggcattcattgttttccatag aaaacactactggacaaccattcggtagcgcacaagttgagcctctgac aaatctttcctcatcacgtgaatacacgctgcgtgattcgtcagtgact ccactgtggtctttaaccaccatcagagtcctgtaagcatcctttgttt ccgtttaaaatgcctgccagatggcacgacgccgtctggttttgccggc tttctccgagtcctattagactttgatgccttacggcttttttttaaga atggttcttttgagatttgccgactttccagttccgccaccagacgctc ctgttgaactgccaccggcacgatgcagtattccgccacgaaaacgcgc accgcaagctccgctaccattaaacgggtttcgtctgctttagatgttt ccttccgcgtcatcaaggcaaaagcattgccactgatgttaccgaagct ttcccgccatgctgcgcacaatgcccaatcttccgtcacggacctcttc cggtaaccacctaaaggaggattactgggcaacccaaaacgctgcaaca agaagcacagtccaggtgtcgctagattcgagcctgcatggtcgttccg tagctccatacaacaattctctgtgtgacggcgagaggagtaacgcgct agtgtgtgtcagcgacgcggcagtcgatccgatcctgcaacaggcagag gtgtgtcgatgctcagtgatgcgacggcgtatctgaagaggactgtagc tccaccacgaccttcgtgggagcacgaagtgtactctgttgtcgtcggt ctcgtatttttttgagttgtgtacttcgctgcaagaggagggtgagatt cgacatctgtgggcgtttgggatcgtgatgacatcgactgtgctttgat atatgatgtgttttttttcgattggatgagcacattccagtaagcttcc tgccgcgcgtctctgctATGAGGCTCTTCCGGTGCTGTGCTGCGGCCGT TGTGGCGGCCGAATCGTTACTGTGGCTGAAGAACGGCTCCCCGTTTTTT GCCTTTCTTCCTGGGAATGGAGAGATTGCAGACAACTGCTCTGGGAATC CATGCGGTGGCACCGCAGCTGGTACGTGCATAAACACACCATCTGGATA TGATTGCAGGTGCGAACCAGGCTACGTTCTGGGCGTTGAAAATGACCAG GTCACGTGCATGATGCCCTCAGGTGTACCCATGGCTAATTTTGTACAGC TGTCGGAAAAGCCTGCAGCTTGCAGCTCAAACCCTTGTGGACCTGAGGC AGCCGGCACCTGCAACGAGACAAACAGTGGTTACATTTGCCGCTGTAAT CAAGGCTACAGAATATCTCTCGACGGGACAGGAAACGTGACATGTATTG TAAGACAGGAAAGCGGCTGTGAGGAAAACGGGTGTGGGCCGCCAGATGC AGTACAGAGTTGCCGCCGACTAACAGGGACGGCAGGTCGACTATGTGTA TGCAAGGAAAACTTTATAGCGACAATCGACGCCAGTGCCCATATCACCT GCAAGCGTGTGCCTCCCCATTATAGGAAGCCTCCCTTCGAATTTGGCAA GGGAGGTCATCCTGTGGACTCAGAACCATCGAAACGCCAGAGGGAAGAT GAAGGTGAAAGTCGTGAGCCTGAAAGCGACTCAACAGAACCGGGGAGAG ATCAGGAAAGAAGAACACCACTTGAGGAAAGCCAGGAACCGGAAGGAAG CACCCCGGACAGTCAGCAGAGCCGAGGTGGTTCTGGTAGCGACAGTACC GAGAGCGAGGAACAAGGAAAGGAGAGAGAGGAAGGAAGTGGACATGCTG GTGCGATCGCTGGGGGAGTTATTGGAGGCCTGTTACTTCTGAGCGCTGC CGGAGCGGGTGTTGCATACATGAGAAAGAGTGGGAGCGGTGGAGGGGAG GAGATAGAATACGAGAGGGGTATCGAGGCTGCAGAGGCCAGTGAAGTCG AAGTCCTCGTTGATTTGGATAGCAAAACATGGGATTAAcacgttctcgg ctgagacttcacaatgtagggtgtcgctggcagatcagctgcaatgcga gaggtgacgcgagtagtgagcaccgcttcttttaagcgcggacattgtg ctcggtcttctgtcacccccgaatcaaaacacatgtatgataatagttc ctgttgacttcccctgccgacaaagaactgctgtgtcgaggccggcttc tgtgcactcatcccaaatgagatggactgatgttttagagacacctcat cgccgacggaaaccatcagctcccagagaaactatgctgcgtcgttttt taggtgatctgttgcgtaatgcgcaccttcatatcatctgtgtgttgac tgtttggtcgttttccgtttagtcaaatgaatgcagtgaaatgcaggga atttagcagacaccgagaactgtcctcttgttctgtgcgcgagttgttt ttaacgtatagcgatgcgtttgcacttgatattaccctaagccatcagt gggtatttagaggagcccacaggtgatgggggtgatccctgtttcttgt catttggcttgtagggttcgctggaactatctggtgtcacggaagagtg gctttactgtctgtccccaaacgcaaggcatcagtgtaaccccgatagg actctggagacttctgcttcactgccgcgttgcaattttcccgcgtcat gtggcaataacggtaattccacgtgcacgccgcataccggatctttgct cccaggctttcttatgaggtcggcatacgtacagcggcggcgtacctcc gctctagagaagaccggtccaaccgactttgaacagcatgcttgtgaat gagtgcttaaacaccctgaagtgatggtggaatgtagcagtctgggacg gttgatgcgaggatatcaccattagcatagactaccttgctctttagcg aggcgagacaacttatttaggtagccatgaaacacctcgatagtatcaa tgacgacgtgcggttcaccaacttccgtcgctagcgcagaaaacagtcg gaaacacaactcggtgagcacctgaagtgtcagtacacattcgaccgtc gggacccgggattccgcaagtggcacccgctggtccagtagcaggaacc tagttcattcagtataacagatttggggcggcaaagagcaatttgctcg acctaacgcttgc

[0073] Primers used for cloning:

[0074] MICE-(24-349)-BasI-F

TABLE-US-00019 (SEQ ID NO: 63) ACTGTGTGCTCTAGGTATGTCCCCGTTTTTTGCCTTTCTTCCTG

[0075] MICE-(24-349)BasI-R

TABLE-US-00020 (SEQ ID NO: 64) ACTGTTCTAGATTAATCCCATGTTTTGCTATCCAAATCA

[0076] SUMO Protein sequence:

[0077] This sequence is present at the N-terminus of SUMO fusions

[0078] Coding sequence for His tagged SUMO (His-SUMO)

TABLE-US-00021 (SEQ ID NO: 16) ATGGGTCATCACCATCATCATCACGGGTCCCTGCAGGACTCAGAAGTCA ATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCA CATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATC AAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGAC AGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAAT TCAAGCTGATCAGGCCCCTGAAGATTTGGACATGGAGGATAACGATATT ATTGAGGCTCACCGCGAACAGATTGGAGGT

[0079] Predicted protein sequence for His-SUMO:

TABLE-US-00022 (SEQ ID NO: 17) MGHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDI IEAHREQIGG

Example 2

[0080] We have developed an efficient assay where we can control the amplitude of antigen release pharmacologically. We compared constitutive secretion (mock treatment), to conditions where we block or enhance microneme secretion. T. gondii antigens released into the extracellular milieu are likely to stimulate both humoral and cell-mediated immunity. Identification of immunogenic proteins in ESA has led to improved diagnostic reagents for T. gondii infection.

Collection of Excreted/Secreted Antigen (ESA)

[0081] Toxoplasma gondii RH strain parasites were isolated from infected human fibroblasts, filtered, and washed extensively. Parasites were either left untreated (Mock), treated to block (Inhibited), or induce secretion of ES antigens (Stimulated). After 10 min at 37.degree. C., the parasites were chilled to 4.degree. C., centrifuged, and the cell-free supernatant was collected. To evaluate the complexity, samples were separated by SDS-PAGE and the protein composition was assessed by staining. In addition, we used a luciferase-based assay to detect a microneme reporter to specifically determine the level of microneme secretion in each of the fractions (FIG. 1).

LC/MS-MS of ES Antigens

[0082] Samples were processed for mass spectrometry (MS), separated by LC, resolved on an Orbitrap MS/MS instrument, and analyzed using Mascot (Matrix Science, London, UK). Scaffold (Proteome Software Inc., Portland, Oreg.) was used to analyze MS/MS peptides and establish protein identifications by comparison to gene databases.

[0083] We classified ES antigens based on enrichment of peptides in stimulated vs. blocked samples using a cutoff of 4-fold increase in two replicate samples. The proteins were further analyzed for their profile of expression during development to classify those that were bone fide micronemal proteins (the major component of ESA) vs. potential contaminants. The profile of micronemal proteins is highly characteristic and many of the secreted proteins share this transcriptional profile. Based on the fold enrichment and expression pattern, we generated a list of the most abundantly induced proteins in ESA (Table 1).

TABLE-US-00023 TABLE 1 Summary of ESA proteins identified by mass spectrometry. Fold Gene ID Product Description Increase.sup.1 TGME49 267680 microneme protein MIC 12 (MIC 12) 39.0 TGME49 291890 microneme protein MIC 1 (MIC1) 34.7 TGME49 294330 EGF family domain-containing protein 34.0 TGME49 208030 microneme protein MIC4 (MIC4) 28.8 TGME49 201780 microneme protein MIC2 (MIC2) 27.3 TGME49 319560 microneme protein MIC3 (MIC3) 26.1 TGME49 206510 toxolysin TLN4 (TLN4) 25.0 TGME49 214940 MIC2-associated protein M2AP 21.5 TGME49 234380 hypothetical protein (TGME49_234380) 16.5 TGME49 204050 subtilisin SUB1 (SUB1) 16.5 TGME49 218520 microneme protein MIC6 (MIC6) 15.5 TGME49 250710 microneme protein MIC10 (MIC 10) 13.2 TGME49 293440 hypothetical protein (TGME49_293440) 13.0 TGME49 232280 hypothetical protein (TGME49_232280) 11.5 TGME49 204130 perforin-like protein PLP1 (PLP1) 11.5 TGME49 243930 hypothetical protein (TGME49_243930) 5.5 TGME49 277080 microneme protein MIC5 (MIC5) 5.4 TGME49 258870 hypothetical protein (TGME49_258870) 4.5 .sup.1Increase of peptide spectral counts in stimulated fraction vs. control

Example 3

[0084] To express these proteins recombinantly, we are using a fusion system based on the E. coli protein SUMO, which allows for production and purification of soluble, tagged proteins. From this group of initial candidates, we have successfully cloned, expressed, and purified all of the proteins shown in Table 2. These proteins were tested here as fusion proteins with SUMO as a control. However, they can also be purified away form SUMO after protease cleavage by nickel chromatography as shown in FIG. 2.

Example 4

Development of Model for DTH Using Bioluminescence

[0085] We tested an alternative method that relies on light production in the skin. The basis for this method is that recruitment of monocytes and neutrophils to the site of inflammation can be detected using luminol, a substrate that gives off light when converted by myeloperoxidase (Gross S, Gammon S T, Moss B L, Rauch D, Harding J, Heinecke J W, Ratner L, Piwnica-Worms D. 2009. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat Med 15:455-461). This method has been shown to be sensitive for detecting DTH responses in the mouse and for monitoring leukocyte influx to sites of infiltration (Gross, supra).

Example 5

The Luminol DTH Response is Specific to ESA

[0086] We have modified the luminol assay used for monitoring DTH responses in the mouse by injecting antigen in the pinne of the ear. In order to confirm that the DTH responses that we were detecting were in fact due to antigens in ESA, we compared the response for ESA to total parasite antigen or to PBS. The response detected by luminol was highly enriched in ESA sample compared to the PBS control or to total antigen (FIG. 3A, 3B). The response is also only seen in infected animals, confirming that it is due to a specific immune response. This experiment demonstrates that the DTH response is driven by antigens that are enriched in ESA.

Example 6

[0087] We have focused on the ESA antigens defined in Table 1 along with some constitutively secreted dense granule proteins (GRA) that have previously been shown to be immunogenic. We have cloned, expressed, and purified 12 proteins for testing, as shown in Table 2. Test proteins were purified as fusion proteins with SUMO, an E. coli protein that facilitates solubility. We have also purified the SUMO protein as a control. To avoid non-specific responses due to endotoxin (LPS) we purified ESA proteins using polymyxin B, a detergent like molecule that removes endotoxin. The resulting purified proteins showed reduced levels of LPS when examined using the limulus amebocyte assay (FIG. 4).

TABLE-US-00024 TABLE 2 Constructs Clone Molecular ESA Protein (aa) Vector Strain weight (kD) MIC 10 Full length pE- Rosetta 23.1 SUMO (DE3) GRA7 Full length pE- Rosetta 26 SUMO (DE3) GRA6 Full length pE- Rosetta 25 SUMO [DE3] M2AP Full length pE- Rosetta 34.6 SUMO (DE3) MIC5 Full length pE- Rosetta 19.9 SUMO [DE3] MIC6 Full length pE- Rosetta 36.7 SUMO (DE3) Hypothetic Protein 2 Full length pE- Rosetta 30.2 (TGME49_232280) SUMO (DE3) GRA4 21-247 pE- Rosetta 38 SUMO (DE3) MIC1 20-340 pE- Rosetta 48.6 SUMO (DE3) MIC3 134-383 pE- Rosetta 40.5 SUMO (DE3) MIC4 58-231 pE- Rosetta 63.0 SUMO (DE3] Hypothetic Protein 1 89-347 pE- Rosetta 38.7 (TGME.234380) SUMO (DE3)

Example 7

An In Vitro Method to Monitor Antigen Presentation

[0088] This method is based on the ability of specialized immune cells (dendritic cells and macrophages) to present antigen to memory T-cells that in turn produce interferon gamma (IFN-.gamma.). T-cells that produce IFN-.gamma. in response to recall antigens are one of the primary drivers of the DTH response (Black C A. 1999. Delayed type hypersensitivity: current theories with an historic perspective. Dermatol Online J 5:7.). However, instead of injecting antigens into the skin, we monitored the production of IFN-.gamma. using a technique called ELISpot to specifically detect IFN-.gamma. producing T-cells following antigen presentation in vitro. Following incubation of splenocytes with specific antigens or controls, IFN-.gamma. is captured by an antibody on the membrane and then detected using an enzyme-linked immuno-assay (the blue spots represent positives). As shown in FIG. 5, this assay measures robust responses of splenocytes from T. gondii infected mice incubated with ESA, while there is minimal response in naive animals. ConA is used as a non-specific stimulus as it evokes responses from all T-cells regardless of specific antigen presentation. We have adapted this assay for monitoring recombinant ESA proteins and peptides that are synthetically produced.

Example 8

Using the ELISpot Assay to Monitor IFN-.gamma. Secretion to Individual ESA Proteins

[0089] The ELISpot assay was also used to examine the response of uninfected and chronically infected mice to individual ESA proteins that were produced recombinantly. Initially, the ESA fraction was compared to ConA as a positive control and SUMO as a negative control. High numbers of spot forming cells (SFC) were detected using an IFN-.gamma. specific ELISpot assay as shown in FIG. 6. Individual ESA proteins were used at 3 microgams per well in combination with 10.sup.5 splenocytes. Specific responses were detected to MIC1, MIC3, MIC4 and MICE, but not to M2AP or to MIC10 as shown in FIG. 6. Similar responses were seen in infected C57/BL6 mice (FIG. 6A) and Balb/C mice (FIG. 6B).

REFERENCES

[0090] The disclosure of each reference cited below and throughout this application is expressly incorporated herein.

References for Example 1



[0091] Brown, K. M., S. Lourido, and L. D. Sibley, Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii. J Biol Chem, 2016. 291(18): p. 9554-65.

[0092] 2. Etheridge, R. D., et al., ROP18 and ROP17 kinase complexes synergize to control acute virulence of Toxoplasma in the mouse. Cell Host Microbe, 2014. 15: p. 537-550.

[0093] 3. Behnke, M., et al., Coordinated progression through two subtranscriptions underlies the tachyzoite cycle of Toxoplasma gondii. Plos One, 2010. 5: p. e12354.

[0094] 4. Khan, A., et al., Geographic separation of domestic and wild strains of Toxoplasma gondii in French Guiana correlates with a monomorphic version of chromosome 1a. Plos Negl. Trop. Dis., 2014. 8: p. e3182.

[0095] 5. Gross, S., et al., Bioluminescence imaging of myeloperoxidase activity in vivo. Nat Med, 2009. 15(4): p. 455-61.

References for Examples 2-4

[0095]

[0096] 1. Carruthers V B, Sibley L D. 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73:114-123.

[0097] 2. Carruthers V B, Giddings O K, Sibley L D. 1999. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiol 1:225-236.

[0098] 3. Carruthers V B, Moreno S N J, Sibley L D. 1999. Ethanol and acetaldehyde elevate intracellular [Ca2+] calcium and stimulate microneme discharge in Toxoplasma gondii. Biochem J 342:379-386.

[0099] 4. Carruthers V B, Sibley L D. 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 31:421-428.

[0100] 5. Huynh M H, Barenau K E, Harper J M, Beatty W L, Sibley L D, Carruthers V B. 2003 Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex. EMBO J 22:2082-2090.

[0101] 6. Brydges S D, Sherman G D, Nockemann S, Loyens A, Daubener W, Dubremetz J, Carruthers V B. 2000. Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii. Mol Biochem Parasitol 111:51-66.

[0102] 7. Hoff E F, Cook S H, Sherman G D, Harper J M, Ferguson D J, Dubremetz J F, Carruthers V B. 2001. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Exp Parasitol 97:77-88.

[0103] 8. Mercier C, Cesbron-Delauw M F. 2015. Toxoplasma secretory granules: one population or more? Trends Parasitol 31:60-71.

[0104] 9. Black C A. 1999. Delayed type hypersensitivity: current theories with an historic perspective. Dermatol Online J 5:7.

[0105] 10. Allen I C. 2013. Delayed-type hypersensitivity models in mice. Methods Mol Biol 1031:101-107.

[0106] 11. Rougier D, Ambroise-Thomas P. 1985. Detection of toxoplasmic immunity by multipuncture skin test with excretory-secretory antigen. Lancet 2:121-123.

[0107] 12. Veprekova. 1978. Approximative molecular weight of the active component in toxoplasmin. Folia Parasitol (Praha) 25:273-275.

[0108] 13. Frenkel J K. 1948. Dermal hypersentsitivity to Toxoplasma antigens (toxoplasmins). Proc Soc Exp Biol Med 68:634-639.

[0109] 14. Gross S, Gammon S T, Moss B L, Rauch D, Harding J, Heinecke J W, Ratner L, Piwnica-Worms D. 2009. Bioluminescence imaging of myeloperoxidase activity in vivo.

[0110] Nat Med 15:455-461

References for Examples 5-6

[0110]

[0111] 1. Carruthers V B, Sibley L D. 1997. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73:114-123.

[0112] 2. Carruthers V B, Giddings O K, Sibley L D. 1999. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiol 1:225-236.

[0113] 3. Carruthers V B, Moreno S N J, Sibley L D. 1999. Ethanol and acetaldehyde elevate intracellular [Ca2+] calcium and stimulate microneme discharge in Toxoplasma gondii. Biochem J 342:379-386.

[0114] 4. Carruthers V B, Sibley L D. 1999. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 31:421-428.

[0115] 5. Huynh M H, Barenau K E, Harper J M, Beatty W L, Sibley L D, Carruthers V B. 2003. Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex. EMBO J 22:2082-2090.

[0116] 6. Brydges S D, Sherman G D, Nockemann S, Loyens A, Daubener W, Dubremetz J, Carruthers V B. 2000. Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii. Mol Biochem Parasitol 111:51-66.

[0117] Hoff E F, Cook S H, Sherman G D, Harper J M, Ferguson D J, Dubremetz J F, Carruthers V B. 2001. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIClO. Exp Parasitol 97:77-88.

[0118] 8. Mercier C, Cesbron-Delauw M F. 2015. Toxoplasma secretory granules: one population or more? Trends Parasitol 31:60-71.

[0119] 9. Black C A. 1999. Delayed type hypersensitivity: current theories with an historic perspective. Dermatol Online J 5:7.

[0120] 10. Allen I C. 2013. Delayed-type hypersensitivity models in mice. Methods Mol Biol 1031:101-107.

[0121] 11. Rougier D, Ambroise-Thomas P. 1985. Detection of toxoplasmic immunity by multipuncture skin test with excretory-secretory antigen. Lancet 2:121-123.

[0122] 12. Veprekova. 1978. Approximative molecular weight of the active component in toxoplasmin. Folia Parasitol (Praha) 25:273-275.

[0123] 13. Frenkel J K. 1948. Dermal hypersensitivity to Toxoplasma antigens (toxoplasmas]. Proc Soc Exp Biol Med 68:634-639.

[0124] 14. Gross S, Gammon S T, Moss B L, Rauch D, Harding J, Heinecke J W, Ratner L, Piwnica-Worms D. 2009. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat Med 15:455-461.

References For Examples 7

[0124]

[0125] 1. Huynh M H, Barenau K E, Harper J M, Beatty W L, Sibley L D, Carruthers V B. 2003. Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex. EMBO J 22:2082-2090.

[0126] 2. Brydges S D, Sherman G D, Nockemann S, Loyens A, Daubener W, Dubremetz J, Carruthers V B. 2000. Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii. Mol Biochem Parasitol 111:51-66.

[0127] 3. Hoff E F, Cook S H, Sherman G D, Harper J M, Ferguson D J, Dubremetz J F, Carruthers V B. 2001. Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIClO. Exp Parasitol 97:77-88.

[0128] 4. Mercier C, Cesbron-Delauw M F. 2015. Toxoplasma secretory granules: one population or more? Trends Parasitol 31:60-71.

[0129] 5. Black C A. 1999. Delayed type hypersensitivity: current theories with an historic perspective. Dermatol Online J 5:7.

[0130] 6. Allen I C. 2013. Delayed-type hypersensitivity models in mice. Methods Mol Biol 1031:101-107.

[0131] 7. Rougier D, Ambroise-Thomas P. 1985. Detection of toxoplasmic immunity by multipuncture skin test with excretory-secretory antigen. Lancet 2:121-123.

[0132] 8. Veprekova. 1978. Approximative molecular weight of the active component in toxoplasmin. Folia Parasitol (Praha) 25:273-275.

[0133] 9. Frenkel J K. 1948. Dermal hypersensitivity to Toxoplasma antigens (toxoplasmins). Proc Soc Exp Biol Med 68:634-639.

[0134] 10. Gross S, Gammon S T, Moss BL, Rauch D, Harding J, Heinecke J W, Ratner L, Piwnica-Worms D. 2009. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat Med 15:455-461.

[0135] 11. Philpott D J, Girardin S E. 2004. The role of Toll-like receptors and Nod proteins in bacterial infection. Molec Immunol 41:1099-1108.

[0136] 12. Jacobs D M, Morrison D C. 1977. Inhibition of the mitogenic response to lipopolysaccharide (LPS) in mouse spleen cells by polymyxin B. J Immunol 118:2127.

[0137] 13. Nielsen M, Lund O, Buus S, Lundegaard C. 2010. WIC class II epitope predictive algorithms. Immunology 130:319-328.

[0138] 14. Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol 4:el000048.

[0139] 15. Erskine C L, Krco C), Hedin K E, Borson N D, Kalli K R, Behrens M D, Heman-Ackah S M, von Hofe E, Wettstein P J, Mohamadzadeh M, Knutson K L. 2011. MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells. J Immunol 187:316-324.

Sequence CWU 1

1

641456PRTToxopolasmosis gondii 1Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Glu Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 4552321PRTToxopolasmosis gondii 2Met Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala Ser Gly Arg1 5 10 15Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile Ala Ala Glu 20 25 30Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser Ser Arg Ile 35 40 45Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr Leu Glu Trp 50 55 60Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln Glu Asn Asn65 70 75 80Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro Cys Pro Gly 85 90 95Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His Glu Ile Leu 100 105 110Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser Pro Tyr Gln 115 120 125Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly Thr Ile Ala 130 135 140Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala Trp Arg Cys145 150 155 160Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu Cys Ala Ser 165 170 175Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg Gly Thr Ser 180 185 190Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly Ile Arg Gln 195 200 205Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu Val Cys Leu 210 215 220Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser Gly Gln Ile225 230 235 240Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu Met Gln Gln 245 250 255Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu Gln Ser Pro 260 265 270Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly Asn Ile Asp 275 280 285Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys Thr Glu Ile 290 295 300His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln Gln Leu Thr305 310 315 320Leu31920DNAToxopolasmosis gondii 3acctgaaagc gggtgccgcg tcgctaccgt ttcctgtggc gtctctagtg cgacatccga 60agtaacagta acgtccggca tggaacgccg acgcgggtgt tccagtcgcc tggctccttc 120tactcgcact tcgatgttac gttccttatt ggtgcgacgc ggttctcgtg ttgctagacg 180tcgcaccggc tgaaagctgt agaaaattta gttattttcc tgtcagctag cttgcaggag 240tgcgtttttg tgtgttggtt tcgtctcaca tggctgctga tctgttgatg cagctgtgta 300cacgtgcctc gattctgtag ttgacctaga acggatttgc aaagatgggc caggcgttgt 360ttctcaccgt tctattgccg gtgttatttg gcgttgggcc agaagcatat ggagaagcgt 420cgcattctca ttcgccggca tcgggacgtt atatacaaca gatgcttgac caacgctgcc 480aagagattgc tgcagaactc tgccaaggcg gacttcgtaa aatgtgtgtg ccctctagcc 540ggatagtagc tcgaaacgcc gtgggcatta ctcatcaaaa tacacttgaa tggagatgct 600ttgatacagc ctctttgctg gagagcaatc aagaaaacaa cggtgttaat tgcgtggacg 660actgtggcca cacgataccg tgtcctggcg gcgtacaccg gcaaaacagt aatcacgcaa 720cgcgccatga gatactgtcc aaattggtcg aagaaggagt acaacggttc tgcagtcctt 780atcaagcatc tgccaacaag tactgtaacg acaaatttcc agggaccatt gcgaggaggt 840cgaagggctt cggaaacaat gtcgaggttg cgtggaggtg ttacgagaag gccagcttgc 900tgtactcggt ttatgctgag tgtgcgagca actgcggaac aacgtggtac tgccctggag 960gacgacgagg gacgtcgaca gaactagaca agcggcatta tacagaagag gaaggaattc 1020gccaggcaat cggatccgtc gacagcccat gttctgaagt tgaagtctgc ctaccgaagg 1080atgagaatcc cccggtgtgt ttagatgaaa gtggccagat ttcacgaact ggtggtgggc 1140caccgtcaca accgcctgag atgcaacagc ccgccgatcg ttcggacgag agaggtggcg 1200gtaaggaaca gtcgcctgga ggagaagctc agccggacca tccaacgaag ggtggtaaca 1260tagacctgcc tgagaaatca acatctcccg agaagacgcc gaaaaccgag atccatggtg 1320acagcacgaa agcgacgctc gaagaggggc agcaactaac gctcacgttt atctccacta 1380aactggatgt tgctgtaggc tcgtgtcatt cactcgtcgc gaatttcctt gatggatttt 1440tgaagtttca gacgggctca aattcggcgt tcgatgtggt agaagtggaa gagccagcag 1500gacccgcagt gcttacgata ggtctgggac acaaaggccg tctcgctgtt gtcctcgact 1560acaccaggct caatgctgct ttaggatcag ctgcttacgt ggtcgaagat tctggatgca 1620gctcaagtga agaggttagt ttccaaggag tgggtagtgg agcgacgctc gtggtgacga 1680cgcttggcga gagtcctacg gccgtctctg cttgatttat agtactcttt ggagcatgct 1740tgtggaggaa cgggacaatc tcggcaaaat caggatgaag tttgtgagat acagatcgtt 1800cctgaacagt ggaagatgcg tcactattac acctatatgc gtcctggttc ttgtagagtt 1860ggagttcttg caggtgtaat gactatgaca tacggatata acttcatacg gggaactgtg 1920448DNAToxopolasmosis gondii 4actgtggtct ctaggtatgg aagcatatgg agaagcgtcg cattctca 48549DNAToxopolasmosis gondii 5actgttctag atcagagcgt tagttgctgc ccctcttcga gcgtcgctt 496198PRTToxopolasmosis gondii 6Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 1957198PRTToxopolasmosis gondii 7Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 1958960DNAToxopolasmosis gondii 8ttgtcggata gaacacatca aaaatttttt ctgtttcatc tgtcacgtgc gtccttttgg 60tggaagcgac gcgtcgtcgc ggccctgttg ggggtagtca atatttcagt aagcgtttct 120agcgacaccc gctacacaga acactcggtt gaagcactgt gatgctggcg tttctgtccg 180ctgtctttct gcagaatggc gctttcttct ttgaacaata tcaggccttt tagcgggttg 240ctgggttgcg gcctgctgtt tggcgccctt gtggtcgtgg tggcatgtgt tttcagcgtt 300cctgtggaag caggcgttct tcgtaaggta gcaggtgcag gaagtctcca ggcgtctatt 360ggagagcacg atttttttaa cgattacgat caggacgagg aatacaggaa gcgccagcaa 420gaactgcaga atcagagtcc agaagaagtc gaggaagcga aacgcaaata ccacgaagag 480ctgagacgga aagcagaaga agatgcagag acgaaacgta agcaagaagc agtcattcaa 540gaactgaaag aggtggcaaa gaaaagagga cttcgtgaag ccgctgagcg tgaggagaag 600cgcattgatg agcagcaggc taattacgag caacgacaac aagaactgag agacatggat 660tcagcaatgg aggagaggct tatgcagcag agaaaaaaag accaggaaga gagagaactt 720gcaagaaaaa acagcgataa ggtcatggag gagctcaaag agaaactcgc aagacgcagg 780aaatcaatgt agaagaaagg caagttctga ctagtgtatg aagtgtagca gcggtccgtt 840attaccgtgt atacagggag atcgtcgttc agtttgaagt gtttattccg tgttgaaaga 900agcgtttgtg taatgcagat gatcctgaga tgatagtgcg ggaaatcgaa tgactcattg 960941DNAToxopolasmosis gondii 9actgtggtct ctaggtatgg cgctttcttc tttgaacaat a 411038DNAToxopolasmosis gondii 10actgttctag atcacattga tttcctgcgt cttgcgag 3811236PRTToxopolasmosis gondii 11Met Ala Arg His Ala Ile Phe Phe Ala Leu Cys Val Leu Gly Leu Val1 5 10 15Ala Ala Ala Leu Pro Gln Phe Ala Thr Ala Ala Thr Ala Ser Asp Asp 20 25 30Glu Leu Met Ser Arg Ile Arg Asn Ser Asp Phe Phe Asp Gly Gln Ala 35 40 45Pro Val Asp Ser Leu Arg Pro Thr Asn Ala Gly Val Asp Ser Lys Gly 50 55 60Thr Asp Asp His Leu Thr Thr Ser Met Asp Lys Ala Ser Val Glu Ser65 70 75 80Gln Leu Pro Arg Arg Glu Pro Leu Glu Thr Glu Pro Asp Glu Gln Glu 85 90 95Glu Val His Phe Arg Lys Arg Gly Val Arg Ser Asp Ala Glu Val Thr 100 105 110Asp Asp Asn Ile Tyr Glu Glu His Thr Asp Arg Lys Val Val Pro Arg 115 120 125Lys Ser Glu Gly Lys Arg Ser Phe Lys Asp Leu Leu Lys Lys Leu Ala 130 135 140Leu Pro Ala Val Gly Met Gly Ala Ser Tyr Phe Ala Ala Asp Arg Ile145 150 155 160Leu Pro Glu Leu Thr Glu Gln Gln Gln Thr Gly Glu Glu Pro Leu Thr 165 170 175Thr Gly Gln Asn Val Ser Thr Val Leu Gly Phe Ala Ala Leu Ala Ala 180 185 190Ala Ala Ala Phe Leu Gly Met Gly Leu Thr Arg Thr Tyr Arg His Phe 195 200 205Ser Pro Arg Lys Asn Arg Ser Arg Gln Pro Ala Leu Glu Gln Glu Val 210 215 220Pro Glu Ser Gly Lys Asp Gly Glu Asp Ala Arg Gln225 230 23512236PRTToxopolasmosis gondii 12Met Ala Arg His Ala Ile Phe Phe Ala Leu Cys Val Leu Gly Leu Val1 5 10 15Ala Ala Ala Leu Pro Gln Phe Ala Thr Ala Ala Thr Ala Ser Asp Asp 20 25 30Glu Leu Met Ser Arg Ile Arg Asn Ser Asp Phe Phe Asp Gly Gln Ala 35 40 45Pro Val Asp Ser Leu Arg Pro Thr Asn Ala Gly Val Asp Ser Lys Gly 50 55 60Thr Asp Asp His Leu Thr Thr Ser Met Asp Lys Ala Ser Val Glu Ser65 70 75 80Gln Leu Pro Arg Arg Glu Pro Leu Glu Thr Glu Pro Asp Glu Gln Glu 85 90 95Glu Val His Phe Arg Lys Arg Gly Val Arg Ser Asp Ala Glu Val Thr 100 105 110Asp Asp Asn Ile Tyr Glu Glu His Thr Asp Arg Lys Val Val Pro Arg 115 120 125Lys Ser Glu Gly Lys Arg Ser Phe Lys Asp Leu Leu Lys Lys Leu Ala 130 135 140Leu Pro Ala Val Gly Met Gly Ala Ser Tyr Phe Ala Ala Asp Arg Ile145 150 155 160Leu Pro Glu Leu Thr Glu Gln Gln Gln Thr Gly Glu Glu Pro Leu Thr 165 170 175Thr Gly Gln Asn Val Ser Thr Val Leu Gly Phe Ala Ala Leu Ala Ala 180 185 190Ala Ala Ala Phe Leu Gly Met Gly Leu Thr Arg Thr Tyr Arg His Phe 195 200 205Ser Pro Arg Lys Asn Arg Ser Arg Gln Pro Ala Leu Glu Gln Glu Val 210 215 220Pro Glu Ser Gly Lys Asp Gly Glu Asp Ala Arg Gln225 230 235131120DNAToxopolasmosis gondii 13ttactttctt cggattacat tcttccacta aaagctggtt ttgtccagta tccattcgtc 60gctaccgttg cgcagtcacg ttgaattttg cagcggcaaa acatcttgtg taaaattcga 120gttttgttga tgattgaagt accctgtatt ggggcttgct aacgttttgt attaaaaggg 180tttactgcgg cgtctcattt ccaaaatggc ccgacacgca atttttttcg cgctttgtgt 240tttaggcctg gtggcggcgg ctttgcccca gttcgctacc gcggccaccg cgtcagatga 300cgaactgatg agtcgaatcc gaaattctga ctttttcgat ggtcaagcac ccgttgacag 360tctcagaccg acgaacgccg gtgtcgactc gaaagggacc gacgatcacc tcaccaccag 420catggataag gcatctgtag agagtcagct tccgagaaga gagccattgg agacggagcc 480agatgaacaa gaagaagttc atttcaggaa gcgaggcgtc cgttccgacg ctgaagtgac 540tgacgacaac atctacgagg agcacactga tcgtaaagtg gttccgagga agtcggaggg 600caagcgaagc ttcaaagact tgctgaagaa gctcgcgctg ccggctgttg gtatgggtgc 660atcgtatttt gccgctgata gaattctgcc ggaactaaca gagcagcaac agacaggcga 720agaaccccta accaccggcc agaatgtgag cactgtgtta ggcttcgcag cgcttgctgc 780tgccgcagcg ttccttggca tgggtctcac gaggacgtac cgacattttt ccccacgcaa 840aaacagatca cggcagcctg cactcgagca agaggtgcct gaatcaggca aagatgggga 900ggatgcccgc cagtaggata tgggggctaa taaaagtgag taggagctcg aggacagtgt 960cccgaacgcg cctgagaggc agacagacac agaagagtga agaaaaacaa catggtatta 1020cgtgcggtga gtgtttgctg tcacgtgttt tttgcgccac aaagacagct tgtgttgtat 1080gcatgggatc gacagttcat ggacggcgct acccagagag 11201437DNAToxopolasmosis gondii 14actgtgagga ctcaggtatg gctttgcccc agttcgc 371535DNAToxopolasmosis gondii 15actgttctag atcactggcg ggcatcctcc ccatc 3516324DNAToxopolasmosis gondii 16atgggtcatc accatcatca tcacgggtcc ctgcaggact cagaagtcaa tcaagaagct 60aagccagagg tcaagccaga agtcaagcct gagactcaca tcaatttaaa ggtgtccgat 120ggatcttcag agatcttctt caagatcaaa aagaccactc ctttaagaag gctgatggaa 180gcgttcgcta aaagacaggg taaggaaatg gactccttaa gattcttgta cgacggtatt 240agaattcaag ctgatcaggc ccctgaagat

ttggacatgg aggataacga tattattgag 300gctcaccgcg aacagattgg aggt 32417108PRTToxopolasmosis gondii 17Met Gly His His His His His His Gly Ser Leu Gln Asp Ser Glu Val1 5 10 15Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr 20 25 30His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys 35 40 45Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys 50 55 60Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile65 70 75 80Arg Ile Gln Ala Asp Gln Ala Pro Glu Asp Leu Asp Met Glu Asp Asn 85 90 95Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly 100 10518456PRTToxopolasmosis gondii 18Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Glu Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45519456PRTToxopolasmosis gondii 19Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Glu Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45520456PRTToxopolasmosis gondii 20Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Glu Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45521456PRTToxopolasmosis gondii 21Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Glu Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45522456PRTToxopolasmosis gondii 22Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45523456PRTToxopolasmosis gondii 23Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile

35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45524456PRTToxopolasmosis gondii 24Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45525456PRTToxopolasmosis gondii 25Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45526456PRTToxopolasmosis gondii 26Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45527456PRTToxopolasmosis gondii 27Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Gln Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45528456PRTToxopolasmosis gondii 28Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu

Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Asp Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45529456PRTToxopolasmosis gondii 29Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45530456PRTToxopolasmosis gondii 30Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45531456PRTToxopolasmosis gondii 31Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Gly Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Glu Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Val Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45532456PRTToxopolasmosis gondii 32Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45533456PRTToxopolasmosis gondii 33Met Gly Gln Ala Leu Phe Leu Thr Val Leu Leu Pro Val Leu Phe Gly1 5 10 15Val Gly Pro Glu Ala Tyr Gly Glu Ala Ser His Ser His Ser Pro Ala 20 25 30Ser Gly Arg Tyr Ile Gln Gln Met Leu Asp Gln Arg Cys Gln Glu Ile 35 40 45Ala Ala Glu Leu Cys Gln Ser Gly Leu Arg Lys Met Cys Val Pro Ser 50 55 60Ser Arg Ile Val Ala Arg Asn Ala Val Gly Ile Thr His Gln Asn Thr65 70 75 80Leu Gln Trp Arg Cys Phe Asp Thr Ala Ser Leu Leu Glu Ser Asn Gln 85 90 95Glu Asn Asn Gly Val Asn Cys Val Asp Asp Cys Gly His Thr Ile Pro 100 105 110Cys Pro Gly Gly Val His Arg Gln Asn Ser Asn His Ala Thr Arg His 115 120 125Glu Ile Leu Ser Lys Leu Val Glu Glu Gly Val Gln Arg Phe Cys Ser 130 135 140Pro Tyr Gln Ala Ser Ala Asn Lys Tyr Cys Asn Asp Lys Phe Pro Gly145 150 155 160Thr Ile Ala Arg Arg Ser Lys Gly Phe Gly Asn Asn Val Glu Val Ala 165 170 175Trp Arg Cys Tyr Glu Lys Ala Ser Leu Leu Tyr Ser Val Tyr Ala Glu 180 185 190Cys Ala Ser Asn Cys Gly Thr Thr Trp Tyr Cys Pro Gly Gly Arg Arg 195 200 205Gly Thr Ser Thr Glu Leu Asp Lys Arg His Tyr Thr Glu Glu Glu Gly 210 215 220Ile Arg Gln Ala Ile Gly Ser Val Asp Ser Pro Cys Ser Glu Val Glu225 230 235 240Val Cys Leu Pro Lys Asp Glu Asn Pro Pro Leu Cys Leu Asp Glu Ser 245 250 255Gly Gln Ile Ser Arg Thr Gly Gly Gly Pro Pro Ser Gln Pro Pro Glu 260 265 270Met Gln Gln Pro Ala Asp Arg Ser Asp Glu Arg Gly Gly Gly Lys Glu 275 280 285Gln Ser Pro Gly Gly Glu Ala Gln Pro Asp His Pro Thr Lys Gly Gly 290 295 300Asn Ile Asp Leu Pro Glu Lys Ser Thr Ser Pro Glu Lys Thr Pro Lys305 310 315 320Thr Glu Ile His Gly Asp Ser Thr Lys Ala Thr Leu Glu Glu Gly Gln 325 330 335Gln Leu Thr Leu Thr Phe Ile Ser Thr Lys Leu Asp Val Ala Val Gly 340 345 350Ser Cys His Ser Leu Val Ala Asn Phe Leu Asp Gly Phe Leu Lys Phe 355 360 365Gln Thr Gly Ser Asn Ser Ala Phe Asp Val Val Glu Val Glu Glu Pro 370 375 380Ala Gly Pro Ala Val Leu Thr Ile Gly Leu Gly His

Lys Gly Arg Leu385 390 395 400Ala Val Val Leu Asp Tyr Thr Arg Leu Asn Ala Ala Leu Gly Ser Ala 405 410 415Ala Tyr Val Val Glu Asp Ser Gly Cys Ser Ser Ser Glu Glu Val Ser 420 425 430Phe Gln Gly Val Gly Ser Gly Ala Thr Leu Val Val Thr Thr Leu Gly 435 440 445Glu Ser Pro Thr Ala Val Ser Ala 450 45534198PRTToxopolasmosis gondii 34Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19535198PRTToxopolasmosis gondii 35Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19536198PRTToxopolasmosis gondii 36Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19537198PRTToxopolasmosis gondii 37Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19538198PRTToxopolasmosis gondii 38Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19539198PRTToxopolasmosis gondii 39Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19540198PRTToxopolasmosis gondii 40Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19541198PRTToxopolasmosis gondii 41Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19542198PRTToxopolasmosis gondii 42Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Thr Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19543198PRTToxopolasmosis gondii 43Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Ala 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Thr Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19544167PRTToxopolasmosis gondii 44Met Leu Val Phe Leu Ser Ala Val Phe Leu Gln Asn Gly Ala Phe Phe1 5 10 15Phe Glu Gln Tyr Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp 20 25 30Tyr Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn 35 40 45Gln Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu 50 55 60Leu Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu65 70 75 80Ala Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg 85 90 95Glu Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn 100 105 110Tyr Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu 115 120 125Glu Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu 130 135 140Ala Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu145 150 155 160Ala Arg Arg Arg Lys Ser Met 16545198PRTToxopolasmosis gondii 45Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19546198PRTToxopolasmosis gondii 46Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20

25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19547198PRTToxopolasmosis gondii 47Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19548198PRTToxopolasmosis gondii 48Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19549198PRTToxopolasmosis gondii 49Met Ala Leu Ser Ser Leu Asn Asn Ile Arg Pro Phe Ser Gly Leu Leu1 5 10 15Gly Cys Gly Leu Leu Phe Gly Ala Leu Val Val Val Val Ala Cys Val 20 25 30Phe Ser Val Pro Val Glu Ala Gly Val Leu Arg Lys Val Ala Gly Ala 35 40 45Gly Ser Leu Gln Ala Ser Ile Gly Glu His Asp Phe Phe Asn Asp Tyr 50 55 60Asp Gln Asp Glu Glu Tyr Arg Lys Arg Gln Gln Glu Leu Gln Asn Gln65 70 75 80Ser Pro Glu Glu Val Glu Glu Ala Lys Arg Lys Tyr His Glu Glu Leu 85 90 95Arg Arg Lys Ala Glu Glu Asp Ala Glu Thr Lys Arg Lys Gln Glu Ala 100 105 110Val Ile Gln Glu Leu Lys Glu Val Ala Lys Lys Arg Gly Leu Arg Glu 115 120 125Ala Ala Glu Arg Glu Glu Lys Arg Ile Asp Glu Gln Gln Ala Asn Tyr 130 135 140Glu Gln Arg Gln Gln Glu Leu Arg Asp Met Asp Ser Ala Met Glu Glu145 150 155 160Arg Leu Met Gln Gln Arg Lys Lys Asp Gln Glu Glu Arg Glu Leu Ala 165 170 175Arg Lys Asn Ser Asp Lys Val Met Glu Glu Leu Lys Glu Lys Leu Ala 180 185 190Arg Arg Arg Lys Ser Met 19550383PRTToxopolasmosis gondii 50Met Arg Gly Gly Thr Ser Ala Leu Leu His Ala Leu Thr Phe Ser Gly1 5 10 15Ala Val Trp Met Cys Thr Pro Ala Glu Ala Leu Pro Ile Gln Lys Ser 20 25 30Val Gln Leu Gly Ser Phe Asp Lys Val Val Pro Ser Arg Glu Val Val 35 40 45Ser Glu Ser Leu Ala Pro Ser Phe Ala Val Thr Glu Thr His Ser Ser 50 55 60Val Gln Ser Pro Ser Lys Gln Glu Thr Gln Leu Cys Ala Ile Ser Ser65 70 75 80Glu Gly Lys Pro Cys Arg Asn Arg Gln Leu His Thr Asp Asn Gly Tyr 85 90 95Phe Ile Gly Ala Ser Cys Pro Lys Ser Ala Cys Cys Ser Lys Thr Met 100 105 110Cys Gly Pro Gly Gly Cys Gly Glu Phe Cys Ser Ser Asn Trp Ile Phe 115 120 125Cys Ser Ser Ser Leu Ile Tyr His Pro Asp Lys Ser Tyr Gly Gly Asp 130 135 140Cys Ser Cys Glu Lys Gln Gly His Arg Cys Asp Lys Asn Ala Glu Cys145 150 155 160Val Glu Asn Leu Asp Ala Gly Gly Gly Val His Cys Lys Cys Lys Asp 165 170 175Gly Phe Val Gly Thr Gly Leu Thr Cys Ser Glu Asp Pro Cys Ser Lys 180 185 190Arg Gly Asn Ala Lys Cys Gly Pro Asn Gly Thr Cys Ile Val Val Asp 195 200 205Ser Val Ser Tyr Thr Cys Thr Cys Gly Asp Gly Glu Thr Leu Val Asn 210 215 220Leu Pro Glu Gly Gly Gln Gly Cys Lys Arg Thr Gly Cys His Ala Phe225 230 235 240Arg Glu Asn Cys Ser Pro Gly Arg Cys Ile Asp Asp Ala Ser His Glu 245 250 255Asn Gly Tyr Thr Cys Glu Cys Pro Thr Gly Tyr Ser Arg Glu Val Thr 260 265 270Ser Lys Ala Glu Glu Ser Cys Val Glu Gly Val Glu Val Thr Leu Ala 275 280 285Glu Lys Cys Glu Lys Glu Phe Gly Ile Ser Ala Ser Ser Cys Lys Cys 290 295 300Asp Asn Gly Tyr Ser Gly Ser Ala Ser Ala Thr Ser His His Gly Lys305 310 315 320Gly Glu Ser Gly Ser Glu Gly Ser Leu Ser Glu Lys Met Asn Ile Val 325 330 335Phe Lys Cys Pro Ser Gly Tyr His Pro Arg Tyr His Ala His Thr Val 340 345 350Thr Cys Glu Lys Ile Lys His Phe Ala Leu Asp Gly Ala Gly Asn His 355 360 365Asp Thr Thr Thr Tyr Val Ala Arg Arg Arg Tyr Pro Ala Ser Leu 370 375 38051249PRTToxopolasmosis gondii 51Tyr His Pro Asp Lys Ser Tyr Gly Gly Asp Cys Ser Cys Glu Lys Gln1 5 10 15Gly His Arg Cys Asp Lys Asn Ala Glu Cys Val Glu Asn Leu Asp Ala 20 25 30Gly Gly Gly Val His Cys Lys Cys Lys Asp Gly Phe Val Gly Thr Gly 35 40 45Leu Thr Cys Ser Glu Asp Pro Cys Ser Lys Arg Gly Asn Ala Lys Cys 50 55 60Gly Pro Asn Gly Thr Cys Ile Val Val Asp Ser Val Ser Tyr Thr Cys65 70 75 80Thr Cys Gly Asp Gly Glu Thr Leu Val Asn Leu Pro Glu Gly Gly Gln 85 90 95Gly Cys Lys Arg Thr Gly Cys His Ala Phe Arg Glu Asn Cys Ser Pro 100 105 110Gly Arg Cys Ile Asp Asp Ala Ser His Glu Asn Gly Tyr Thr Cys Glu 115 120 125Cys Pro Thr Gly Tyr Ser Arg Glu Val Thr Ser Lys Ala Glu Glu Ser 130 135 140Cys Val Glu Gly Val Glu Val Thr Leu Ala Glu Lys Cys Glu Lys Glu145 150 155 160Phe Gly Ile Ser Ala Ser Ser Cys Lys Cys Asp Asn Gly Tyr Ser Gly 165 170 175Ser Ala Ser Ala Thr Ser His His Gly Lys Gly Glu Ser Gly Ser Glu 180 185 190Gly Ser Leu Ser Glu Lys Met Asn Ile Val Phe Lys Cys Pro Ser Gly 195 200 205Tyr His Pro Arg Tyr His Ala His Thr Val Thr Cys Glu Lys Ile Lys 210 215 220His Phe Ala Leu Asp Gly Ala Gly Asn His Asp Thr Thr Thr Tyr Val225 230 235 240Ala Arg Arg Arg Tyr Pro Ala Ser Leu 245523837DNAToxopolasmosis gondii 52tcttctcttc ttccgtactt ttccctgcat ttcacacccc tggtatgact ccacaccgcg 60tgtaaatgtc ccttaggtga cacccgcagc agcgcgtagg aggaagtaga tgtcagtgta 120gacgtttttg agatgagaga cgataacgta aaatgccgcc gataacttct gcattataca 180cactctctct ccacgcctag gatgacaggt acggcggcac acggaggaaa gtgggggggg 240gggggggggc gaacagaaag gtcacatgga aggccgctcg actctccact cacgaagtga 300aggcttcgtc ccgttttgct ggacaacgaa tgcgaacttc ttcactcgct tgtgacacac 360acaactccag aggcacagag atgtgaagca gaagagtggc gtgtgcgtcg cttctgtcgg 420cggcaagccc cgctccgtct ctttggtggc gattctggtg tgcaccgtgt gccaagaagt 480tgcgtgtcac gcgacttttg gaaatgcatc aggttcagag tcgttatgtt gcgattcagg 540ctctcggcag agaatcattt ccctgtaagc tagttgaact cgccttttta aaagcggcag 600cagtgccctt gtggaaggcc tcactgtgcc tactttcctc gtcctgagtt tttccgcctt 660cggcctcatt ttttgctcac caaaatcgtg tcctaccgtc aagttttgcc atagactcct 720acgggaaaaa acaagccggt cgacacggac gacgcccgca gggaagcgtc ccctccgcag 780aaatcgggag acaactgtcg ttgacggtgc tgcgcgaaag gtcacagagt ttccagtgtg 840ttcatcagac ctcactgtgc actgttagcg gccgctgtcc cgcctggtca acaagtatca 900caccctcgtc cccgccattg gcacggagct cgatgagctg cagtgtcgct tttaggggag 960tcgtgcaatc acgccgcaac acaggcgtga ttcgatcttc aattgctagg taaccactcg 1020tgcttggtag ctctgcaatg gctcgagcga cgggggtgat gcaacatgct gctaaaaact 1080cgacagacgt gtcaccggaa cccacctaaa taggagacca cgggtctctg gtgtgtcgcg 1140tcgcattctc gcgtcgcatt ctcgcgtcgc aatgaccggc cagttgctcg acgtcgccag 1200ccgggactga agagcgttca tcgagtcagc agcattgcgt ccccttgctc ggtgaaaaaa 1260gactctctgg tcgagtctag ctcgtgtcac ttctgtttct aacctccttc gttcaccggt 1320acacctccga tgtgactttt ggtacacttg ccctgtcgca cgacgcacgc tgtcactcaa 1380cttgctgcta gcgcaatcga taggttccct cgaaccagcc atcacacaca caccttttcc 1440gggaagacgt ttgcgggcgg tgggtcgcag ctcgtcgaga gtgcgtttct gtgcatttct 1500gtgggcagtg cagcgcgttt gcgcgcctta ctctgtgtgt aacttccttg tccaacactg 1560gtaaaaatgc gaggcgggac gtccgcgctg ttgcacgcgc tcaccttcag tggggccgtg 1620tggatgtgca ccccagcgga ggctttgccg attcagaagt ctgtgcagct gggcagcttt 1680gacaaagttg tgccgagccg cgaagtcgtc tctgagagtc ttgctccgtc tttcgcggtg 1740actgagactc actcgtctgt gcaatccccc agcaagcagg agacgcaact ctgtgctatc 1800tcgagtgaag gcaagccatg tcgaaaccgt cagttgcaca ctgacaacgg gtacttcatc 1860ggggccagtt gccccaagag cgcttgctgc agcaagacca tgtgcggccc cggcggctgc 1920ggagaattct gctccagcaa ctggattttt tgcagcagtt cgctcatcta ccatcctgac 1980aaaagctatg gaggagactg cagctgtgaa aagcagggcc atcggtgcga caaaaacgca 2040gaatgcgtcg aaaacttgga cgcgggtggg ggtgtgcact gcaagtgcaa agacggcttc 2100gtcggcactg ggttgacttg ctccgaggat ccttgttcaa aaagagggaa cgcgaagtgc 2160ggacccaacg ggacgtgcat cgtcgtcgat tcagtcagct acacatgcac ctgcggcgac 2220ggcgaaactc tagtgaacct cccggaaggg ggacaaggat gcaagaggac tggatgtcat 2280gccttcaggg agaactgcag ccctggtaga tgtattgatg acgcctcgca tgagaatggc 2340tacacctgcg agtgccccac agggtactca cgtgaggtga cttccaaggc ggaggagtcg 2400tgtgtggaag gagtcgaagt cacgctggct gagaaatgcg agaaggaatt cggcatcagc 2460gcgtcatcct gcaaatgcga taacggatac tccggatctg cttccgcaac ctcccaccat 2520gggaaaggag aatcgggatc cgaggggagc ttgagtgaaa aaatgaatat tgtcttcaag 2580tgccccagtg gctaccatcc aagataccat gcccacaccg tgacgtgtga gaaaattaag 2640cactttgccc ttgacggggc cggcaaccac gacacgacta cgtatgtcgc aagacgaagg 2700tacccagcga gtctctgaga gcggagatca gcgcaaagac aagatgcaga gtttgactcg 2760agaaacaata gtaacacgaa gtaaaaagtc tccacactaa gccaaggatt gagaatattt 2820cgatttgtgc cgctggcaat agtggccttg gcctagaaag aagttctgca acgaagcgat 2880cggctcacac gcggatacac agatgggttt gtaccgagaa cgttaggttt gtgaaccgag 2940ttcaggtaaa acaaagtaga ttgtgccttt acgcagacag cgagggaaaa catgaggaca 3000cactgccaac taaagcaaga ctgcctcact aattaccacc gacacacgac atggttaccc 3060ccgcgttttg ccgcgtgcaa agtttgaatt ctgatggttc tcgagtctga aagcctaaac 3120cgcccaacca tgtatgaaat aagaacccat caaacgtgag acatctctgc cgaagtgcct 3180acgaaaagaa cgcttctgcc actaggaggt gcggcctctt cattctatga gaacctgctt 3240tgtcggtgtc aacctctggg gaaatcgcct gcctttacac attttgctcg ttgtagagca 3300agggatctgt tgctgcgttt actccaatac aatgatcgcc gtttcgctgt aggcaagcga 3360tccgaaaatg tacgttcgag tcagcagcta cttgagaagc agccaacgcc gacacttgct 3420gcgtttgact gaggtgcact cgcaaacagt ctcgtctccc cggggcaatt tctgagagaa 3480atgcgggaat ggacgtaatg gtgctcttct gtgagtgctc ttccaccaat ttttcgacaa 3540gtgttttcgt gacagtcgag tataccttct tatgtcattc tgtctccgtc agtgctatcg 3600gattcttcct attcctctac cctttctaca gtcgcataca aagctgctga aacaagactt 3660cctttgtcta gggtagttgt acactccaca catatctgac tgaaacctac ggcaggaagt 3720ctggtcggca ctgtgcttcc ttgttggctt ttcgtcgttt ctttgtctac gagcttcact 3780gggtccttga cacggcttgt gagcgttgtg ctcaatattc gaccagctgt atttgtg 38375353DNAToxopolasmosis gondii 53actgtggtct ctaggtatga tctaccatcc tgacaaaagc tatggaggag act 535439DNAToxopolasmosis gondii 54acttgttcta gatcagagac tcgctgggta ccttcgtct 3955580PRTToxopolasmosis gondii 55Met Arg Ala Ser Leu Pro Val His Leu Val Val Cys Thr Gln Leu Ser1 5 10 15Ala Val Trp Phe Gly Val Ala Lys Ala His Gly Gly His Arg Leu Glu 20 25 30Pro His Val Pro Gly Phe Leu Gln Gly Phe Thr Asp Ile Thr Pro Ala 35 40 45Gly Asp Asp Val Ser Ala Asn Val Thr Ser Ser Glu Pro Ala Lys Leu 50 55 60Asp Leu Ser Cys Val His Ser Asp Asn Lys Gly Ser Arg Ala Pro Thr65 70 75 80Ile Gly Glu Pro Val Pro Asp Val Ser Leu Glu Gln Cys Ala Ala Gln 85 90 95Cys Lys Ala Val Asp Gly Cys Thr His Phe Thr Tyr Asn Asp Asp Ser 100 105 110Lys Met Cys His Val Lys Glu Gly Lys Pro Asp Leu Tyr Asp Leu Thr 115 120 125Gly Gly Lys Thr Ala Ser Arg Ser Cys Asp Arg Ser Cys Phe Glu Gln 130 135 140His Val Ser Tyr Glu Gly Ala Pro Asp Val Met Thr Ala Met Val Thr145 150 155 160Ser Gln Ser Ala Asp Cys Gln Ala Ala Cys Ala Ala Asp Pro Ser Cys 165 170 175Glu Ile Phe Thr Tyr Asn Glu His Asp Gln Lys Cys Thr Phe Lys Gly 180 185 190Arg Gly Phe Ser Ala Phe Lys Glu Arg Gly Val Leu Gly Val Thr Ser 195 200 205Gly Pro Lys Gln Phe Cys Asp Glu Gly Gly Lys Leu Thr Gln Glu Glu 210 215 220Met Glu Asp Gln Ile Ser Gly Cys Ile Gln Leu Ser Asp Val Gly Ser225 230 235 240Met Thr Ala Asp Leu Glu Glu Pro Met Glu Ala Asp Ser Val Gly Ala 245 250 255Cys Met Glu Arg Cys Arg Cys Asp Gly Arg Cys Thr His Phe Thr Phe 260 265 270Asn Asp Asn Thr Arg Met Cys Tyr Leu Lys Gly Asp Lys Met Gln Leu 275 280 285Tyr Ser Ser Pro Gly Asp Arg Thr Gly Pro Lys Ser Cys Asp Ser Ser 290 295 300Cys Phe Ser Asn Gly Val Ser Tyr Val Asp Asp Pro Ala Thr Asp Val305 310 315 320Glu Thr Val Phe Glu Ile Ser His Pro Ile Tyr Cys Gln Val Ile Cys 325 330 335Ala Ala Asn Pro Leu Cys Thr Val Phe Gln Trp Tyr Ala Ser Glu Ala 340 345 350Lys Cys Val Val Lys Arg Lys Gly Phe Tyr Lys His Arg Lys Thr Gly 355 360 365Val Thr Gly Val Thr Val Gly Pro Arg Glu Phe Cys Asp Phe Gly Gly 370 375 380Ser Ile Arg Asp Arg Glu Glu Ala Asp Ala Val Gly Ser Asp Asp Gly385 390

395 400Leu Asn Ala Glu Ala Thr Met Ala Asn Ser Pro Asp Phe His Asp Glu 405 410 415Val Glu Cys Val His Thr Gly Asn Ile Gly Ser Lys Ala Gln Thr Ile 420 425 430Gly Glu Val Lys Arg Ala Ser Ser Leu Ser Glu Cys Arg Ala Arg Cys 435 440 445Gln Ala Glu Lys Glu Cys Ser His Tyr Thr Tyr Asn Val Lys Ser Gly 450 455 460Leu Cys Tyr Pro Lys Arg Gly Lys Pro Gln Phe Tyr Lys Tyr Leu Gly465 470 475 480Asp Met Thr Gly Ser Arg Thr Cys Asp Thr Ser Cys Leu Arg Arg Gly 485 490 495Val Asp Tyr Ser Gln Gly Pro Glu Val Gly Lys Pro Trp Tyr Ser Thr 500 505 510Leu Pro Thr Asp Cys Gln Val Ala Cys Asp Ala Glu Asp Ala Cys Leu 515 520 525Val Phe Thr Trp Asp Ser Ala Thr Ser Arg Cys Tyr Leu Ile Gly Ser 530 535 540Gly Phe Ser Ala His Arg Arg Asn Asp Val Asp Gly Val Val Ser Gly545 550 555 560Pro Tyr Thr Phe Cys Asp Asn Gly Glu Asn Leu Gln Val Leu Glu Ala 565 570 575Lys Asp Thr Glu 58056173PRTToxopolasmosis gondii 56Ser Glu Pro Ala Lys Leu Asp Leu Ser Cys Val His Ser Asp Asn Lys1 5 10 15Gly Ser Arg Ala Pro Thr Ile Gly Glu Pro Val Pro Asp Val Ser Leu 20 25 30Glu Gln Cys Ala Ala Gln Cys Lys Ala Val Asp Gly Cys Thr His Phe 35 40 45Thr Tyr Asn Asp Asp Ser Lys Met Cys His Val Lys Glu Gly Lys Pro 50 55 60Asp Leu Tyr Asp Leu Thr Gly Gly Lys Thr Ala Ser Arg Ser Cys Asp65 70 75 80Arg Ser Cys Phe Glu Gln His Val Ser Tyr Glu Gly Ala Pro Asp Val 85 90 95Met Thr Ala Met Val Thr Ser Gln Ser Ala Asp Cys Gln Ala Ala Cys 100 105 110Ala Ala Asp Pro Ser Cys Glu Ile Phe Thr Tyr Asn Glu His Asp Gln 115 120 125Lys Cys Thr Phe Lys Gly Arg Gly Phe Ser Ala Phe Lys Glu Arg Gly 130 135 140Val Leu Gly Val Thr Ser Gly Pro Lys Gln Phe Cys Asp Glu Gly Gly145 150 155 160Lys Leu Thr Gln Glu Glu Met Glu Asp Gln Ile Ser Gly 165 170573121DNAToxopolasmosis gondii 57ttttctgtgc atctgtgctg caaaacgggc ctctgtgcat tatttcccca ccaacaattg 60ccgcgtcgat ccgggtcccg ctcaagctct gcagaactag gctctcgata tagatcagta 120caatcattcg cttctgacaa tcgcatcgac tgagcgacgc gttgatcgtc gactgtcgtg 180cgtcgcattc gggcatctcg aaccggtgtt gattccctgt gtcattattt cacttccgtc 240cttctctcgt ggcgatctat aatacgcgtg tgttgttgcg tgcattgctt gtgttgttgt 300ggatgtgttt tcttttgtga ccgctcacga acaccccacg caaaatgaga gcgtcgctcc 360cggttcacct cgttgtgtgc acgcagctaa gtgccgtttg gtttggagtg gctaaagccc 420atggtggaca ccgactggaa ccgcatgttc ccggattcct gcaaggcttc actgatatca 480cgcctgcagg tgatgacgtt agtgccaacg taacaagttc ggagcctgca aaacttgatc 540tctcttgtgt gcactctgac aataagggat caagggctcc cacaataggc gagccagtgc 600cagatgtgtc cctggaacaa tgtgctgcgc aatgcaaggc tgttgatggc tgcacacatt 660tcacttataa tgacgattcg aagatgtgcc atgtgaagga gggaaaaccc gatttatacg 720atctcacagg aggcaaaaca gcatcgcgca gttgcgatag atcatgcttc gaacaacacg 780tatcgtatga gggagctcct gacgtgatga cagcgatggt cacgagccag tcagcggact 840gtcaggctgc gtgtgcggct gacccgagct gcgagatctt cacttataac gaacacgacc 900agaaatgtac tttcaaagga agggggtttt ctgcgtttaa ggaacgaggg gtgttgggtg 960tgacttccgg gccgaaacag ttctgcgatg aaggcggtaa attaactcaa gaggagatgg 1020aagatcagat cagtggctgc attcaattga gtgacgttgg atcaatgact gctgacctgg 1080aggagcctat ggaggctgat tctgttggcg cttgtatgga acggtgccgc tgtgatggaa 1140gatgcacgca cttcacgttc aacgataata ctcggatgtg ctacctcaaa ggtgacaaga 1200tgcagttgta ctcatctcca ggtgacagaa ccggcccaaa gagctgcgat tcaagctgct 1260tctcgaacgg ggtttcttac gtcgatgatc cggcgacaga tgttgagacc gtattcgaaa 1320tttcacaccc aatttattgt caagtaatct gcgccgcaaa tccgttgtgt acagtgtttc 1380agtggtatgc ctccgaggca aagtgcgtcg tcaagagaaa ggggttttac aaacacagaa 1440aaacaggtgt cacgggagtc acagtgggcc ctcgggagtt ctgcgatttt ggcggtagca 1500tccgcgaccg agaagaggca gacgccgttg gatcagacga tggcctcaac gcggaagcaa 1560ctatggcaaa ttctcctgat tttcacgacg aagtagaatg cgtccacacg ggcaacattg 1620ggtcaaaagc acaaaccatt ggagaagtga aacgcgcaag tagtttgagt gagtgcagag 1680ccagatgcca agcggagaaa gaatgcagcc actacactta caatgtaaaa tccggtttgt 1740gttatccaaa aagaggaaag cctcaatttt ataagtatct tggcgacatg acgggatcca 1800gaacatgtga tacaagttgc cttaggaggg gagtcgatta ctcacagggc cctgaagtag 1860gaaagccttg gtattctacg ctgccgacag actgccaagt tgcatgcgac gctgaggatg 1920cttgcctggt gttcacctgg gattcggcga cgtcacgatg ctacctcatc ggctcaggtt 1980tctcggcaca tcgacggaac gacgtggatg gcgtggtatc tggaccctat actttctgtg 2040acaatggcga aaaccttcag gtgcttgaag cgaaagacac agaatgaccc aggagggtgc 2100cagatacttt gtgtgactgc gacatgcagt catgtactca aagtgttgta catggacagg 2160aggacttttt ttttaagtca ttgcagaggt gcgttttcgg agcagcacta taactgcgtc 2220agcgactaag cacgccacgt agctgaatga aacgcagcca ccttcgtgta tgtatgcttc 2280gttttttgtc gctgtgcagt tttgaatcat ttcccttatg ggacatttct gaaaaatgct 2340ccccgttcgc ttgtagcact atgagagggg ccgaagactg caatggaggt agcgctgcgt 2400tgaaaagacg aggcgctaca tttcgcgtag cgacaaggcc gtgtagagtt ttgcttttcg 2460cgagacactg ctctgagtgt catatgcatc aaatgcagtg gtagcacaca gaggtgagaa 2520gaatgatcac ctgcggggga atggctttgc taaacaacaa ggtcgctgtg tgactttaca 2580caacgaaact actgtggtga gtgctcagtt gagtgaaaag aaatgccgcg ttatcgtgag 2640ttctggttcg gtggactttg ccaccgtagt aaaactcaac ctgtaacgga atgcccagtt 2700ttactgctct ctttaaaggg cgtccacgtt ctctatattc aagctgttta cccacctgcg 2760tttcggtgca tcgcgcgtgc cacatcaaaa atccaggtaa cggtgcggga cctatgctac 2820actttatatc tctcagaaag catacaccca ctgattatgg acaacgctgt ggtcgcgttg 2880taccacaatg caggaatact cagttcacct tgcaagtgtt ctggtgttca ttgcgtgtca 2940gaagtacacg aaaagagact tctttggcct ccaagtgata cgtaaccgcg gcagtcatga 3000acagagtcac tcgtgcttct gaaacgcacg tcttctgtac agagacagat gcagtgtgca 3060tacaggaagc ccctcgattg ttgccgtagc aggtagccag tagaagaaac aaagacacgg 3120t 31215854DNAToxopolasmosis gondii 58actgtggtct ctaggtatga gttcggagcc tgcaaaactt gatctctctt gtgt 545947DNAToxopolasmosis gondii 59actgttctag atcagccact gatatgatct tccatctcct cttgagt 4760349PRTToxopolasmosis gondii 60Met Arg Leu Phe Arg Cys Cys Ala Ala Ala Val Val Ala Ala Glu Ser1 5 10 15Leu Leu Trp Leu Lys Asn Gly Ser Pro Phe Phe Ala Phe Leu Pro Gly 20 25 30Asn Gly Glu Ile Ala Asp Asn Cys Ser Gly Asn Pro Cys Gly Gly Thr 35 40 45Ala Ala Gly Thr Cys Ile Asn Thr Pro Ser Gly Tyr Asp Cys Arg Cys 50 55 60Glu Pro Gly Tyr Val Leu Gly Val Glu Asn Asp Gln Val Thr Cys Met65 70 75 80Met Pro Ser Gly Val Pro Met Ala Asn Phe Val Gln Leu Ser Glu Lys 85 90 95Pro Ala Ala Cys Ser Ser Asn Pro Cys Gly Pro Glu Ala Ala Gly Thr 100 105 110Cys Asn Glu Thr Asn Ser Gly Tyr Ile Cys Arg Cys Asn Gln Gly Tyr 115 120 125Arg Ile Ser Leu Asp Gly Thr Gly Asn Val Thr Cys Ile Val Arg Gln 130 135 140Glu Ser Gly Cys Glu Glu Asn Gly Cys Gly Pro Pro Asp Ala Val Gln145 150 155 160Ser Cys Arg Arg Leu Thr Gly Thr Ala Gly Arg Leu Cys Val Cys Lys 165 170 175Glu Asn Phe Ile Ala Thr Ile Asp Ala Ser Ala His Ile Thr Cys Lys 180 185 190Arg Val Pro Pro His Tyr Arg Lys Pro Pro Phe Glu Phe Gly Lys Gly 195 200 205Gly His Pro Val Asp Ser Glu Pro Ser Lys Arg Gln Arg Glu Asp Glu 210 215 220Gly Glu Ser Arg Glu Pro Glu Ser Asp Ser Thr Glu Pro Gly Arg Asp225 230 235 240Gln Glu Arg Arg Thr Pro Leu Glu Glu Ser Gln Glu Pro Glu Gly Ser 245 250 255Thr Pro Asp Ser Gln Gln Ser Arg Gly Gly Ser Gly Ser Asp Ser Thr 260 265 270Glu Ser Glu Glu Gln Gly Lys Glu Arg Glu Glu Gly Ser Gly His Ala 275 280 285Gly Ala Ile Ala Gly Gly Val Ile Gly Gly Leu Leu Leu Leu Ser Ala 290 295 300Ala Gly Ala Gly Val Ala Tyr Met Arg Lys Ser Gly Ser Gly Gly Gly305 310 315 320Glu Glu Ile Glu Tyr Glu Arg Gly Ile Glu Ala Ala Glu Ala Ser Glu 325 330 335Val Glu Val Leu Val Asp Leu Asp Ser Lys Thr Trp Asp 340 34561326PRTToxopolasmosis gondii 61Ser Pro Phe Phe Ala Phe Leu Pro Gly Asn Gly Glu Ile Ala Asp Asn1 5 10 15Cys Ser Gly Asn Pro Cys Gly Gly Thr Ala Ala Gly Thr Cys Ile Asn 20 25 30Thr Pro Ser Gly Tyr Asp Cys Arg Cys Glu Pro Gly Tyr Val Leu Gly 35 40 45Val Glu Asn Asp Gln Val Thr Cys Met Met Pro Ser Gly Val Pro Met 50 55 60Ala Asn Phe Val Gln Leu Ser Glu Lys Pro Ala Ala Cys Ser Ser Asn65 70 75 80Pro Cys Gly Pro Glu Ala Ala Gly Thr Cys Asn Glu Thr Asn Ser Gly 85 90 95Tyr Ile Cys Arg Cys Asn Gln Gly Tyr Arg Ile Ser Leu Asp Gly Thr 100 105 110Gly Asn Val Thr Cys Ile Val Arg Gln Glu Ser Gly Cys Glu Glu Asn 115 120 125Gly Cys Gly Pro Pro Asp Ala Val Gln Ser Cys Arg Arg Leu Thr Gly 130 135 140Thr Ala Gly Arg Leu Cys Val Cys Lys Glu Asn Phe Ile Ala Thr Ile145 150 155 160Asp Ala Ser Ala His Ile Thr Cys Lys Arg Val Pro Pro His Tyr Arg 165 170 175Lys Pro Pro Phe Glu Phe Gly Lys Gly Gly His Pro Val Asp Ser Glu 180 185 190Pro Ser Lys Arg Gln Arg Glu Asp Glu Gly Glu Ser Arg Glu Pro Glu 195 200 205Ser Asp Ser Thr Glu Pro Gly Arg Asp Gln Glu Arg Arg Thr Pro Leu 210 215 220Glu Glu Ser Gln Glu Pro Glu Gly Ser Thr Pro Asp Ser Gln Gln Ser225 230 235 240Arg Gly Gly Ser Gly Ser Asp Ser Thr Glu Ser Glu Glu Gln Gly Lys 245 250 255Glu Arg Glu Glu Gly Ser Gly His Ala Gly Ala Ile Ala Gly Gly Val 260 265 270Ile Gly Gly Leu Leu Leu Leu Ser Ala Ala Gly Ala Gly Val Ala Tyr 275 280 285Met Arg Lys Ser Gly Ser Gly Gly Gly Glu Glu Ile Glu Tyr Glu Arg 290 295 300Gly Ile Glu Ala Ala Glu Ala Ser Glu Val Glu Val Leu Val Asp Leu305 310 315 320Asp Ser Lys Thr Trp Asp 325623394DNAToxopolasmosis gondii 62cagtccggag cacactccta caataaactt gatacgtgtc attttgtgaa acgacacagc 60acataaccac tcggactgtc tcacgaagct gtagggcgga ttcaccaatg atctttcgca 120gccgatccaa aactacttgc ccacttccgg tgtacgtaca tcgcgcgaca tgagaggcat 180tcattgtttt ccatagaaaa cactactgga caaccattcg gtagcgcaca agttgagcct 240ctgacaaatc tttcctcatc acgtgaatac acgctgcgtg attcgtcagt gactccactg 300tggtctttaa ccaccatcag agtcctgtaa gcatcctttg tttccgttta aaatgcctgc 360cagatggcac gacgccgtct ggttttgccg gctttctccg agtcctatta gactttgatg 420ccttacggct tttttttaag aatggttctt ttgagatttg ccgactttcc agttccgcca 480ccagacgctc ctgttgaact gccaccggca cgatgcagta ttccgccacg aaaacgcgca 540ccgcaagctc cgctaccatt aaacgggttt cgtctgcttt agatgtttcc ttccgcgtca 600tcaaggcaaa agcattgcca ctgatgttac cgaagctttc ccgccatgct gcgcacaatg 660cccaatcttc cgtcacggac ctcttccggt aaccacctaa aggaggatta ctgggcaacc 720caaaacgctg caacaagaag cacagtccag gtgtcgctag attcgagcct gcatggtcgt 780tccgtagctc catacaacaa ttctctgtgt gacggcgaga ggagtaacgc gctagtgtgt 840gtcagcgacg cggcagtcga tccgatcctg caacaggcag aggtgtgtcg atgctcagtg 900atgcgacggc gtatctgaag aggactgtag ctccaccacg accttcgtgg gagcacgaag 960tgtactctgt tgtcgtcggt ctcgtatttt tttgagttgt gtacttcgct gcaagaggag 1020ggtgagattc gacatctgtg ggcgtttggg atcgtgatga catcgactgt gctttgatat 1080atgatgtgtt ttttttcgat tggatgagca cattccagta agcttcctgc cgcgcgtctc 1140tgctatgagg ctcttccggt gctgtgctgc ggccgttgtg gcggccgaat cgttactgtg 1200gctgaagaac ggctccccgt tttttgcctt tcttcctggg aatggagaga ttgcagacaa 1260ctgctctggg aatccatgcg gtggcaccgc agctggtacg tgcataaaca caccatctgg 1320atatgattgc aggtgcgaac caggctacgt tctgggcgtt gaaaatgacc aggtcacgtg 1380catgatgccc tcaggtgtac ccatggctaa ttttgtacag ctgtcggaaa agcctgcagc 1440ttgcagctca aacccttgtg gacctgaggc agccggcacc tgcaacgaga caaacagtgg 1500ttacatttgc cgctgtaatc aaggctacag aatatctctc gacgggacag gaaacgtgac 1560atgtattgta agacaggaaa gcggctgtga ggaaaacggg tgtgggccgc cagatgcagt 1620acagagttgc cgccgactaa cagggacggc aggtcgacta tgtgtatgca aggaaaactt 1680tatagcgaca atcgacgcca gtgcccatat cacctgcaag cgtgtgcctc cccattatag 1740gaagcctccc ttcgaatttg gcaagggagg tcatcctgtg gactcagaac catcgaaacg 1800ccagagggaa gatgaaggtg aaagtcgtga gcctgaaagc gactcaacag aaccggggag 1860agatcaggaa agaagaacac cacttgagga aagccaggaa ccggaaggaa gcaccccgga 1920cagtcagcag agccgaggtg gttctggtag cgacagtacc gagagcgagg aacaaggaaa 1980ggagagagag gaaggaagtg gacatgctgg tgcgatcgct gggggagtta ttggaggcct 2040gttacttctg agcgctgccg gagcgggtgt tgcatacatg agaaagagtg ggagcggtgg 2100aggggaggag atagaatacg agaggggtat cgaggctgca gaggccagtg aagtcgaagt 2160cctcgttgat ttggatagca aaacatggga ttaacacgtt ctcggctgag acttcacaat 2220gtagggtgtc gctggcagat cagctgcaat gcgagaggtg acgcgagtag tgagcaccgc 2280ttcttttaag cgcggacatt gtgctcggtc ttctgtcacc cccgaatcaa aacacatgta 2340tgataatagt tcctgttgac ttcccctgcc gacaaagaac tgctgtgtcg aggccggctt 2400ctgtgcactc atcccaaatg agatggactg atgttttaga gacacctcat cgccgacgga 2460aaccatcagc tcccagagaa actatgctgc gtcgtttttt aggtgatctg ttgcgtaatg 2520cgcaccttca tatcatctgt gtgttgactg tttggtcgtt ttccgtttag tcaaatgaat 2580gcagtgaaat gcagggaatt tagcagacac cgagaactgt cctcttgttc tgtgcgcgag 2640ttgtttttaa cgtatagcga tgcgtttgca cttgatatta ccctaagcca tcagtgggta 2700tttagaggag cccacaggtg atgggggtga tccctgtttc ttgtcatttg gcttgtaggg 2760ttcgctggaa ctatctggtg tcacggaaga gtggctttac tgtctgtccc caaacgcaag 2820gcatcagtgt aaccccgata ggactctgga gacttctgct tcactgccgc gttgcaattt 2880tcccgcgtca tgtggcaata acggtaattc cacgtgcacg ccgcataccg gatctttgct 2940cccaggcttt cttatgaggt cggcatacgt acagcggcgg cgtacctccg ctctagagaa 3000gaccggtcca accgactttg aacagcatgc ttgtgaatga gtgcttaaac accctgaagt 3060gatggtggaa tgtagcagtc tgggacggtt gatgcgagga tatcaccatt agcatagact 3120accttgctct ttagcgaggc gagacaactt atttaggtag ccatgaaaca cctcgatagt 3180atcaatgacg acgtgcggtt caccaacttc cgtcgctagc gcagaaaaca gtcggaaaca 3240caactcggtg agcacctgaa gtgtcagtac acattcgacc gtcgggaccc gggattccgc 3300aagtggcacc cgctggtcca gtagcaggaa cctagttcat tcagtataac agatttgggg 3360cggcaaagag caatttgctc gacctaacgc ttgc 33946344DNAToxopolasmosis gondii 63actgtgtgct ctaggtatgt ccccgttttt tgcctttctt cctg 446439DNAToxopolasmosis gondii 64actgttctag attaatccca tgttttgcta tccaaatca 39

Claims

1. A Toxoplasma gondii-derived antigen composition, wherein the composition comprises a Toxoplasma gondii-derived antigen selected from the group consisting of: a. isolated and purified MIC1, MIC3, MIC4, or MIC6, b. truncated MIC1, MIC3, MIC4, or MIC6, c. extended MIC1, MIC3, MIC4, or MIC6, d. a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6, e. a fusion protein of any of MIC1, MIC3, MIC4, or MICE with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and f. combinations thereof.

2. The antigen composition of claim 1 which is produced in bacteria and comprises less than 0.1 EU/ml lipopolysaccharide.

3. A kit comprising (a) a Toxoplasma gondii-derived antigen composition of claim 1 and (b) an applicator device for administration of the Toxoplasma gondii-derived antigen to a subject.

4. The kit of claim 3 wherein the Toxoplasma gondii-derived antigen composition is separately packaged within the kit.

5. The kit of claim 3 wherein the applicator device is separately packaged within the kit.

6. The kit of claim 3 wherein the applicator device comprises a patch.

7. The kit of claim 3 wherein the applicator device comprises a needle.

8. The kit of claim 3 wherein the applicator device comprises a prong.

9. The kit of claim 8 wherein the applicator device delivers the composition percutaneously.

10. A method of delivering Toxoplasma gondii-derived antigen to a subject comprising: contacting an applicator device which is loaded with the Toxoplasma gondii-derived antigen composition of claim 1 with skin of the subject, whereby the Toxoplasma gondii-derived antigen composition is delivered to the skin of the subject.

11. The method of claim 10 wherein the applicator device comprises a patch.

12. The method of claim 10 wherein the applicator device comprises a needle.

13. The method of claim 10 wherein the applicator device comprises a prong.

14. The method of claim 13 wherein the applicator device delivers the composition percutaneously.

15. An applicator device for administering one or more Toxoplasma gondii-derived antigens to a mammal, comprising one or more Toxoplasma gondii-derived antigens, wherein the Toxoplasma gondii-derived antigens are selected from the group consisting of: a. isolated and purified MIC1, MIC3, MIC4, or MIC6, b. truncated MIC1, MIC3, MIC4, or MIC6, c. extended MIC1, MIC3, MIC4, or MIC6, d. a fusion protein comprising any two or more of MIC1, MIC3, MIC4, or MIC6, e. a fusion protein of any of MIC1, MIC3, MIC4, or MICE with a moiety that enhances or facilitates purification, recombinant production, or immune cell stimulation, and f. combinations thereof.

16. The applicator device of claim 15 which comprises a plurality of prongs to which a plurality of Toxoplasma gondii-derived antigens has been applied.

17. The applicator device of claim 15 wherein the applicator comprises one or more prongs which are configured to receive a liquid comprising Toxoplasma gondii-derived antigen and to puncture skin to deliver the Toxoplasma gondii-derived antigen percutaneously.

18. The applicator device of claim 15 wherein the Toxoplasma gondii-derived antigens are devoid of GRA7.

19. The applicator device of claim 15 which comprises a patch for transdermal administration.

20. The applicator device of claim 15 which comprises a needle.

21. A method of eliciting a T cell response in a subject comprising: contacting a Toxoplasma gondii-derived antigen composition of claim 1 with T cells of the subject, whereby the Toxoplasma gondii-derived antigen composition induces a T cell response.

22. The method of claim 21 wherein the response is a delayed type hypersensitivity response.

23. The method of claim 21 wherein the response is release of a cytokine.

24. The method of claim 21 wherein the response is release of interferon-.gamma..

25. The method of claim 21 wherein the T cells are in peripheral blood mononuclear cells.

26. The method of claim 21 wherein the T cells are in a blood sample.

27. The method of claim 21 wherein the contacting is performed in vitro.

28. The method of claim 21 further comprising the step of detecting or quantifying release of a cytokine.

29. The method of claim 21 further comprising the step of detecting or quantifying release of interferon-.gamma..