Serological marker for the latent form of toxoplasmosis

Abstract

OF THE INVENTION SEROLOGICAL MARKER FOR THE LATENT FORM OF TOXOPLASMOSIS In the present invention, inventors screened the bradyzoite proteome for immunogenic protein candidates in an unbiased manner and identified the Bradyzoite Serological Marker (BSM) as a second serological biomarker for Toxoplasma chronicity in mice. BSM serology correctly distinguishes cyst-bearing mice with a sensitivity and specificity of 97.96% (IC95: 89.31 - 99.90) and 100.0% (IC95: 86.20 - 100.0) respectively. In humans, both bradyzoite markers BCLA and BSM are in moderate agreement (kappa=0.308) and BSM serology is unexpectedly positive in 30% of patients with past immunity, raising many questions. Nevertheless, bradyzoite serology could provide new reference standards for studying the pathophysiology of chronic toxoplasmosis in humans, especially cyst carriage, which has been poorly described. Thus the invention relates to a new Toxoplasma gondii protein, hereafter referred as BSM, a new serological marker whose expression is restricted to the latent form of Toxoplasmosis (bradyzoite / cyst).

Description

[0001] SEROLOGICAL MARKER FOR THE LATENT FORM OF TOXOPLASMOSIS FIELD OF THE INVENTION:

[0002] The invention relates to a new Toxoplasma gondii protein, hereafter referred as BSM (Bradyzoite Serological Marker), a new serological marker whose expression was restricted to the latent form of Toxoplasma gondii (bradyzoite / cyst). The invention also relates to a method for diagnosis of latent form of Toxoplasmosis.

[0003] BACKGROUND OF THE INVENTION:

[0004] Toxoplasma gondii is considered one of the most successful parasitic pathogens in the world and owes this success to the flexibility of its multi -host life cycle that relies on a final host (Felidae) for sexual reproduction, while asexual multiplication occurs in its intermediate hosts, i.e., any warm-blooded animals, including rodents and humans. Host infection occurs mostly through the fecal-oral and the camivorism routes. Once inside a feline enterocyte, the bradyzoite transforms into a merozoite, clearly initiating the onset of the in vivo sexual cycle. After several rounds of asexual replication, merozoites initiate the formation of gametes, which in turn give rise to postfertilization transmissible oocysts that sporulate when exposed to atmospheric oxygen. After ingestion by an intermediate host, the sporozoites are released from the oocysts and differentiate into tachyzoites, which cause acute infection by multiplying intensively and spreading to complex organs such as the brain or eyes. Subsequently, partly under the influence of a strong immune response, tachyzoites transform into bradyzoites, a zoite that expresses its own repertoire of genes to promote slower metabolism and adaptation to prolonged latency in cystic structures found in long-lived cells, preferably in skeletal muscle, myocardium, brain, and eyes (Dubey J.P et al., 1998, Clin Microbiol Rev). Predation by a cyst-bearing animal is also a way of contaminating homeotherms, and when the predator is a cat, the cycle completes. The infection, which is widespread in livestock, can be particularly damaging, and in small ruminants for example (Stelzer S et al., 2019, Food Waterborne ParasitoT), causes substantial economic losses, and provide a reservoir for transmission to humans (Kijlstra A et al., 2009, Trends ParasitoT). Among the latter, seroprevalence is estimated to exceed 30% worldwide, with considerable geographic variation depending on dietary habits, hygiene practices, and environmental conditions (Bigna J.J et al., 2020, Sci Rep) and age groups (Seeber, F, 2023, IJMM). Although toxoplasmosis is usually benign or even asymptomatic in immunocompetent individuals, primary maternal infection during pregnancy is a significant cause of congenitalinfection, leading to potentially fatal fetal harm. In addition, toxoplasmosis can be particularly severe and life-threatening in immunocompromised individuals, in most cases due to reactivation from cysts and differentiation of bradyzoites into tachyzoites. However, the risk of reactivation from cysts is not limited to immunocompromised individuals, and both congenital and acquired toxoplasmosis can result in ocular toxoplasmosis remote from acute infection (Delair E et al., 2008, Am J Ophthalmol). Thus, toxoplasmosis is one of the main causes of posterior uveitis worldwide (Furtado J.M et al., 2013, Clin Exp Ophthalmol).

[0005] The diagnosis of toxoplasmosis is primarily based on direct methods, mainly the detection of parasite DNA in biological samples, and indirect methods, which include searching for antibodies directed against T. gondii that are produced after exposure to the parasite (Dard C et al., 2016, Trends Parasitol & Robert M.G et al., 2021, Expert Rev Anti Infect Thef). Serology relies on the kinetics of different antibody isotypes, mainly IgG and IgM, to determine among others, infection status and assess the risk of congenital toxoplasmosis, donor-to-recipient transmission after transplantation, or reactivation during immunosuppression and its outcome determines the implementation of the appropriate preventive measures. However, the exact dating of the infection, which may be crucial in certain situations, often remains difficult to determine (Dard C et al., 2016, Trends Parasitol). Much of the existing serologic testing relies on native tachyzoite antigens. However, in an effort to improve the performance and accuracy of serologic diagnosis and to optimize the standardization of antigen preparations, different recombinant antigens have also been studied alone or in combinations (Ybanez R.H.D et al., 2020, Front Cell Infect Microbiol). Although bradyzoites and cysts are responsible for host-to-host transmission, chronicity of infection and its reactivation, bradyzoite-specific antigen is not commonly used in serology. Since there is no simple and noninvasive method to confirm carriage of the chronic form, one usually relies mainly on the presence of IgG directed against tachyzoite stage, paradoxically involved in the acute infection. However, the kinetics of cystogenesis and the duration of persistence remain poorly understood, and the prevailing dogma that any infection leads to the formation of cysts that persist in human for life tends to be challenged (Rougier S et al., 2017, Trends Parasitol). Although the cyst has long been considered a homogeneous and quiescent entity, a recent study shows that it is actually a dynamic entity, that harbors sporadic and sometimes synchronous replication of bradyzoites (Watts E et al., 2015, mBio). Some studies also suggest an association between behavioral changes or mental disorders and chronic T. gondii infection (Johnson H.J et al., 2020, mBio). However, in the absence of a specific method to evaluate the presence of cysts indicative of chronic infection, it is not only difficult to study the pathophysiology but also the impact of the latter. Additionally, none of the clinical therapiestarget chronic stages (Alday and Doggett, 2017, Drug Des Devel Ther), making it currently impossible to eradicate Toxoplasma from the host.

[0006] Although a previous study suggests that &ni\-Toxoplasma antibodies are mainly directed against the tachyzoite and only to a minor extent against cysts (Zhang Y.W et al., 1995, J Clin Pathol), we have recently demonstrated the immunogenicity of the bradyzoite-specific protein BCLA (Brain Cyst Load-associated Antigen), which is localized to the membrane of the parasitophorous vacuole (Dard C et al., 2021, BMC Biol). The importance of BCLA as a reliable serological biomarker for cyst carriage has been established in a murine model with perfectly controlled infection and kinetics. In human sera, BCLA ELISA rates were significantly higher in individuals with past immunity identified by conventional techniques, with particularly high rates in subgroups of patients with clinical forms associated with the presence of cysts, i. e., ocular toxoplasmosis or asymptomatic reactivation (Dard C et al., 2021, C Biol). However, the wide dispersion of titers, the lack of reactivity of some sera from seropositive patients, and the positivity of some sera from seronegative patients remain difficult to interpret because in humans, unlike in mouse models, the only possible comparison of this bradyzoite marker is with tachyzoite serological markers.

[0007] In an attempt to improve and complement bradyzoite antigen-based serology, inventors examined additional serological markers for the bradyzoite stage by using genetically modified parasites that can be induced to express bradyzoite-specific proteins in vitro and compared reactivity patterns (Farhat D.C et al., 2020, Nat Microbiol). This strategy allowed them to identify the protein BSM (Bradyzoite Serological Marker). Inventors then evaluated the diagnostic performance of BCLA and BSM serological markers in humans compared with conventional serological methods.

[0008] SUMMARY OF THE INVENTION:

[0009] The invention provides an isolated Toxoplasma gondii polypeptide, hereafter referred as BSM (Bradyzoite Serological Marker) which comprises the amino acids SEQ ID NO : 1 and immunogenic peptides fragments.

[0010] The invention further relates to a method for detecting Toxoplasma gondii polypeptide according to the invention and / or antibodies directed against a Toxoplasma gondii polypeptide according to the invention, and / or evaluating its amount in a biological sample.

[0011] The invention further relates to a method for diagnosis of latent form of Toxoplasmosis wherein said method comprising detecting the presence of Toxoplasma gondii polypeptideaccording to the invention and / or antibodies directed against a Toxoplasma gondii polypeptide according to the invention, in a biological sample from a subject to be tested.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] The inventors screened the bradyzoite proteome for immunogenic protein candidates in an unbiased manner and identified the Bradyzoite Serological Marker (BSM) as a second serological biomarker for Toxoplasma chronicity in mice. BSM serology correctly distinguishes cyst-bearing mice with a sensitivity and specificity of 97.96% (IC95: 89.31 -99.90) and 100.0% (IC95: 86.20 - 100.0) respectively. In humans, both bradyzoite markers BCLA and BSM are in fair agreement (kappa=0.308) and unexpectedly positive in 30% of patients with past immunity, raising many questions. Nevertheless, they could provide new reference standards for studying the pathophysiology of chronic toxoplasmosis in humans, especially cyst carriage, which has been poorly described.

[0014] Isolated peptides

[0015] The invention provides an isolated Toxoplasma gondii polypeptide selected from the group consisting of:

[0016] (i) the amino acids sequence consisting or comprising of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 1);

[0017] (ii) the amino acids sequence consisting or comprising of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 3);

[0018] (iii) an amino acid sequence substantially homologous to the sequence of (i) preferably an amino acid sequence at least 80% identical to the sequence of (i) or (ii);

[0019] (iv) a fragment of at least 9 consecutive amino acids of the sequence of (i), (ii) or (iii).

[0020] The invention also relates to an isolated Toxoplasma gondii polypeptide referred as BSM (Brain Cyst Load-associated Antigen) which comprises the amino acids SEQ ID NO : 1.

[0021] In one embodiment, BSM polypeptide is encoded by the nucleotide sequence SEQ ID NO : 2.

[0022] In one embodiment, BSM polypeptide is encoded by the gene TGME49 202020. In one embodiment, BSM polypeptide is encoded by the gene TGME49 202020 from the genome reference strain ME49.In one embodiment, the amino sequence SEQ ID NO : 1 is expressed in a Baculovirus as the construct sequence SEQ ID NO : 3.

[0023] SEQ ID NO : 1 : MAAVVTGSDMLLGFELGSQMSVVASGTRDGADRAVNKGGLDKMVARRFIPSSIAY AGKVRLFGEEAEARVNAEPNDTIMELPLWIPVHTVEQAKALETRLAFCPSVQVGLLSDGELLFHVDFNNQQLKIPLP VAVGAFVGRLHTAGLHLSGACSIEQNMSGIDLIRHMHQHRHLPHIGVLVVPSSITEKDLMILTAAQGVEKGPSIMLS RISALINWWCCEKLPAEYESLIRLQSATGASVKEKGVVHVALVDVGFSEISVAVIELRTASIEQDPLVVRQPQKQVS VKVLGSATAGDIGVLDAVNELAKHLRHHIKERFHEEIRTKTARYWRMSKACMKAIRALCDVNQAAIDLKFILKNSD VRYDITRKHFDKLCAPLQAKLRAFLEETFSKAGVAGTNVVAADVLNSLARSAWIHSTIAEAVHCEVKAGKAAKVR WVEDGGAGAALGAVYWAADKKYVDNLVDRNPPYRTDKLLWMTAMETDIQKVEQGELERRYLLAHFESYAFEIQ QAAMTVKEGLVRNAEQLKKEIKDAETALGALHQAPLEEVKHAYENFRSYLMNSEPRLFEAIENERATRIGQAKGID PRRTMDIVVSQLGQESNAKLPNAVLLKRAKRDTEEASQLLTDGQTDLAVYLANQSLGYLSNMDAKTASDADKTA AIELQLHNYLQLAKAQTTEAVEDRAIDYRSTLLTQALDNCNKALAIDSKNADALFQRSAAYMSMNDFSRAKADVD AGLAIVPGSETGKALSAEITERQHSHTA

[0024] SEQ ID NO : 2 : ATGGCGGCCGTCGTGACAGGTAGTGATATGTTGCTTGGATTCGAGTTGGGCTCG CAGATGAGCGTCGTCGCAAGTGGCACTCGCGATGGTGCCGATAGAGCGGTGAATAAGGGAGGCTTGGACAAG ATGGTCGCGCGGCGTTTTATCCCTTCCTCTATTGCGTACGCGGGCAAAGTCCGCTTGTTCGGTGAGGAGGCGGA GGCGAGAGTAAACGCGGAACCGAACGATACCATTATGGAATTGCCGTTGTGGATTCCCGTACACACGGTGGAA CAGGCTAAGGCATTGGAAACTCGCCTTGCTTTTTGCCCGAGCGTTCAAGTGGGTCTCCTCAGTGATGGGGAACT GCTTTTTCACGTTGATTTCAACAACCAGCAACTCAAAATCCCCTTGCCAGTAGCCGTTGGAGCGTTCGTAGGTC GGCTGCACACAGCAGGACTGCATCTAAGTGGTGCTTGCTCCACAGAACAGAATATGTCGGGAATCGACTTAAT TCGCCACATGCATCAGCATCGTCATTTACCCCACATCGGCGTTCTGGTCGTACCATCTTCGATTACAGAGAAGG ATTTGATGATACTCACTGCAGCTCAGGGCGTGGAAAAGGGGCCGTCGATAATGCTCTCAAGAATATCTGCGCT AATCAACTGGTGGTGTTGCGAGAAACTTCCGGCGGAGTACGAATCTTTGATTAGGCTACAGTCTGCAACCGGG GCATCTGTGAAGGAAAAGGGAGTGGTCCACGTTGCATTGGTGGACGTTGGCTTCTCCGAAATATCAGTGGCAG TGATTGAGCTCAGAACGGCAAGCACCGAACAAGACCCTTTGGTTGTGAGGCAGCCTCAAAAACAGGTGTCGGT CAAAGTTCTAGGGAGCGCAACGGCGGGAGATATCGGTGTCTTGGACGCTGTCAACGAGCTCGCCAAACACTTG AGACACCACATCAAAGAGAGGTTCCACGAAGAAATCAGAACAAAAACGGCCCGTTATTGGCGCATGAGTAAA GCGTGTATGAAGGCGATTCGTGCTCTTTGTGACGTAAACCAAGCGGCGATCGATTTGAAGTTCATCCTCAAAAA CAGCGACGTGCGCTACGACATAACGCGGAAGCACTTTGACAAGCTGTGTGCGCCGCTGCAGGCCAAGCTCAGG GCATTCCTGGAGGAAACTTTCAGCAAAGCGGGAGTAGCCGGCACGAACGTCGTTGCTGCCGATGTCCTTAATT CCCTTGCGAGAAGTGCTTGGATCCATTCCACCATCGCCGAAGCAGTGCACTGTGAGGTTAAAGCAGGAAAAGC AGCGAAGGTTCGCTGGGTGGAGGACGGGGGAGCTGGAGCTGCTTTGGGAGCGGTGTACTGGGCGGCCGACAA GAAATATGTCGACAACTTAGTCGATAGGAACCCGCCGTACCGGACTGATAAGCTCCTCTGGATGACCGCAATG GAGACTGACATCCAGAAGGTCGAACAGGGCGAATTAGAAAGGAGGTACTTGCTTGCTCATTTCGAGTCGTACG CCTTTGAGATTCAGCAAGCAGCAATGACTGTCAAAGAAGGTCTAGTGCGGAACGCAGAGCAACTGAAGAAAG AAATAAAAGATGCGGAGACTGCCCTTGGTGCACTGCACCAGGCGCCACTAGAGGAAGTGAAGCATGCGTACG AGAATTTCCGTTCGTACTTAATGAACTCGGAACCGAGACTCTTTGAAGCAATCGAGAATGAAAGAGCCACCAG GATCGGCCAAGCTAAAGGAATTGATCCCAGAAGAACAATGGATATCGTTGTCTCTCAGCTCGGCCAAGAGAGC AACGCGAAGCTACCCAATGCGGTATTGCTGAAACGAGCAAAGAGAGACACCGAAGAAGCATCTCAGTTACTC ACCGATGGCCAAACGGACTTGGCGGTGTATCTGGCTAATCAGAGTCTAGGATACCTTTCCAATATGGACGCAA AAACTGCGAGCGACGCAGACAAAACAGCAGCAATCGAGCTCCAGCTACACAACTATCTTCAGCTGGCCAAGGCACAGACAACAGAAGCCGTTGAAGATCGAGCGATCGATTACCGTTCTACACTGCTGACACAAGCACTTGATAA TTGTAACAAAGCCCTGGCTATCGACTCGAAGAATGCAGACGCTCTATTCCAACGAAGCGCCGCGTACATGTCC ATGAACGACTTCAGTCGGGCAAAGGCGGATGTTGACGCAGGCCTTGCGATCTACCGGGTAGTGAAACAGGCAA AGCGTTGTCCGCAGAAATAACCGAGCGGCAGCATTCTCACACAGCTTGA

[0025] SEQ ID NO : 3 : MAAVVTGSDMLLGFELGSQMSVVASGTRDGADRAVNKGGLDKMVARRFIPSSIA YAGKVRLFGEEAEARVNAEPNDTIMELPLWIPVHTVEQAKALETRLAFCPSVQVGLLSDGELLFHVDFNNQQLKIPL PVAVGAFVGRLHTAGLHLSGACSIEQNMSGIDLIRHMHQHRHLPHIGVLVVPSSITEKDLMILTAAQGVEKGPSIML SRISALINWWCCEKLPAEYESLIRLQSATGASVKEKGVVHVALVE)VGFSEISVAVIELRTASTEQDPLVVRQPQKQV SVKVLGSATAGDIGVLDAVNELAKHLRHHIKERFHEEIRTKTARYWRMSKACMKAIRALCDVNQAAIDLKFILKNS DVRYDITRKHFDKLCAPLQAKLRAFLEETFSKAGVAGTNVVAADVLNSLARSAWIHSTIAEAVHCEVKAGKAAKV RWVEDGGAGAALGAVYWAADKKYVDNLVDRNPPYRTDKLLWMTAMETDIQKVEQGELERRYLLAHFESYAFEI QQAAMTVKEGLVRNAEQLKKEIKDAETALGALHQAPLEEVKHAYENFRSYLMNSEPRLFEAIENERATRIGQAKGI DPRRTMDIVVSQLGQESNAKLPNAVLLKRAKRDTEEASQLLTDGQTDLAVYLANQSLGYLSNMDAKTASDADKT AAIELQLHNYLQLAKAQTTEAVEDRAIDYRSTLLTQALDNCNKALAIDSKNADALFQRSAAYMSMNDFSRAKADV DAGLAIVPGSETGKALSAEITERQHSHTAGGHHHHHH

[0026] The invention also provides an isolated Toxoplasma gondii polypeptide selected from the group consisting of:

[0027] (i) the amino acids sequence consisting of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 1);

[0028] (ii) the amino acids sequence consisting or comprising of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 3);

[0029] (iii) an amino acid sequence substantially homologous to the sequence of (i) preferably an amino acid sequence at least 80% identical to the sequence of (i) or ((ii);

[0030] (iv) a fragment of at least 9 consecutive amino acids of the sequence of (i),(ii) or (iii).

[0031] As used herein, the term “amino acid” refers to natural or unnatural amino acids in their D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptidyl structure. Natural and unnatural amino acids are well known in the art. Common natural amino acids include, without limitation, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). Uncommon andunnatural amino acids include, without limitation, allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) and pentafluorophenyl al anine .

[0032] Amino acids are typically classified in one or more categories, including polar, hydrophobic, acidic, basic and aromatic, according to their side chains. Examples of polar amino acids include those having side chain functional groups such as hydroxyl, sulfhydryl, and amide, as well as the acidic and basic amino acids. Polar amino acids include, without limitation, asparagine, cysteine, glutamine, histidine, selenocysteine, serine, threonine, tryptophan and tyrosine. Examples of hydrophobic or non-polar amino acids include those residues having nonpolar aliphatic side chains, such as, without limitation, leucine, isoleucine, valine, glycine, alanine, proline, methionine and phenylalanine. Examples of basic amino acid residues include those having a basic side chain, such as an amino or guanidino group. Basic amino acid residues include, without limitation, arginine, homolysine and lysine. Examples of acidic amino acid residues include those having an acidic side chain functional group, such as a carboxy group. Acidic amino acid residues include, without limitation aspartic acid and glutamic acid. Aromatic amino acids include those having an aromatic side chain group. Examples of aromatic amino acids include, without limitation, biphenylalanine, histidine, 2-napthylalananine, pentafluorophenylalanine, phenylalanine, tryptophan and tyrosine. It is noted that some amino acids are classified in more than one group, for example, histidine, tryptophan and tyrosine are classified as both polar and aromatic amino acids. Amino acids may further be classified as non-charged, or charged (positively or negatively) amino acids. Examples of positively charged amino acids include without limitation lysine, arginine and histidine. Examples of negatively charged amino acids include without limitation glutamic acid and aspartic acid. Additional amino acids that are classified in each of the above groups are known to those of ordinary skill in the art.

[0033] A peptide “substantially homologous” to a reference peptide may derive from the reference sequence by one or more conservative substitutions. Two amino acid sequences are "substantially homologous" or "substantially similar" when one or more amino acid residue are replaced by a biologically similar residue or when greater than 80 % of the amino acids are identical, or greater than about 90 %, preferably greater than about 95%, are similar (functionally identical). Preferably, the similar, identical or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of theprograms known in the art (BLAST, CLUSTAL, FASTA, etc.). The percentage of identity may be calculated by performing a pairwise global alignment based on the Needleman-Wunsch alignment algorithm to find the optimum alignment (including gaps) of two sequences along their entire length, for instance using Needle, and using the BLOSUM62 matrix with a gap opening penalty of 10 and a gap extension penalty of 0.5.

[0034] The term “conservative substitution” as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and function of the peptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine.

[0035] By "substituted" or "modified" the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.

[0036] As such, it should be understood that in the context of the present invention, a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.

[0037] According to the invention a first amino acid sequence having at least 80% of identity with a second amino acid sequence means that the first sequence has 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).

[0038] In some embodiments, the isolated peptide of the invention comprises at most 2291 amino acids (and at least 9). In some embodiments, the polypeptide of the invention comprises less than 2291 amino acids.

[0039] The isolated polypeptides according to the invention may be produced using any method known in the art. They may for example be produced as recombinant polypeptides in a host cell (e.g. in a bacterial, yeast or eukaryotic host cell), or chemically synthesized (see for review Kent S.B.H. Chem. Soc. Rev., 2009,38, 338-351 and Bradley L. et al Annu Rev Biophys Biomol Struct. 2005; 34: 91-118 or R. B. Merrifield (1969). "Solid-phase peptide synthesis." Advances in enzymology and related areas of molecular biology 32: 221-96.; R. B. Merrifield (1969). "The synthesis of biologically active peptides and proteins." JAMA 210(7): 1247-54.and Raibaut, L., O. El Mahdi and O. Melnyk (2015). "Solid Phase Protein Chemical Synthesis." Topics in current chemistry).

[0040] The polypeptide detection may be determined by assaying polypeptide expression with western blot, by assaying polypeptide cellular localization with detection of polyclonal antibodies against polypeptide or by assaying the protein immunogenicity with ELISA test.

[0041] Antibodies according to the invention

[0042] The inventors have generated specific antibodies directed against the polypeptide of the invention and have use the polypeptide of the invention (SEQ ID NO : 1 and SEQ ID NO : 3) in order to detect antibodies directed against the polypeptide of the invention in a biological sample .

[0043] In one embodiment, the invention relates to an antibody that specifically binds to an isolated polypeptide of the invention.

[0044] According to the present invention, “antibody” or “immunoglobulin” have the same meaning, and will be used equally in the present invention. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from thehypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated VL-CDR1, VL-CDR2, VL-CDR3 and VH-CDR1, VH-CDR2, VH-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

[0045] Antibody binding to isolated polypeptide of the invention can be assayed by conventional methods known in the art. The mature form of polypeptide of the invention (SEQ ID NO : 1) is preferably used for assaying antibody binding to epitope of polypeptide of the invention. Many different competitive binding assay format(s) can be used for determining epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such as radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York). For example, the BIACORE® (GE Healthcare, Piscataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed. An example of a suitable ELISA assay is also described in the Example below.

[0046] As used herein, the term "Affinity" refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, the stronger the affinity. Affinity can be determined by measuring KD. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka(i.e. Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A method for determining the KD of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore® system.The invention provides an antibody that specifically binds to an isolated polypeptide comprising or consisting of:

[0047] (i) the amino acids sequence consisting or comprising of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 1);

[0048] (ii) the amino acids sequence consisting or comprising of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 3);

[0049] (iii) an amino acid sequence substantially homologous to the sequence of (i) or ((ii) preferably an amino acid sequence at least 80% identical to the sequence of (i) or (ii);

[0050] (iv) a fragment of at least 9 consecutive amino acids of the sequence of (i), or (ii) or (iii).

[0051] These antibodies can be polyclonal or monoclonal. When the antibodies are monoclonal, they can for example correspond to chimeric, humanized or fully human antibodies, antibody fragment and single domain antibody.

[0052] The term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of an antibody, and a CH domain and a CL domain of a human antibody.

[0053] According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.

[0054] The term "antibody fragment" refers to a fragment of an antibody which contain the variable domains comprising the CDRs of said antibody. The basic antibody fragments include Fab, Fab', F(ab')2 Fv, scFv, dsFv. For example of antibody fragment see also for review, Holliger et al Nature Biotechnology 23, issue 9 1126 - 1136 (2005), which are includes herein by reference.

[0055] The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

[0056] The term “F(ab')2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

[0057] The term “ Fab' “ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. “dsFv” is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

[0058] The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0059] As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also called VHH or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388. The nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody. The low molecular weight and compact size further result in nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. The amino acid sequence and structure of asingle domain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein as "Framework region 1" or "FR1 as "Framework region 2" or "FR2"; as "Framework region 3 " or "FR3"; and as "Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure: FR1 - CDR1 - FR2 - CDR2 -FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http: / / imgt.cines.fr / ).

[0060] Methods for obtaining such antibodies are well known in the art.

[0061] An antibody of the invention can be conjugated with a detectable label to form an immunoconjugate. Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.

[0062] The detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are3H,125I,1311,35S and14C.

[0063] Immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0064] Alternatively, immunoconjugates can be detectably labeled by coupling an antibody to a chemiluminescent compound. The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.Similarly, a bioluminescent compound can be used to label immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.

[0065] Alternatively, immunoconjugates can be detectably labeled by linking a monoclonal antibody to an enzyme. When the enzyme conjugate is incubated in the presence of the appropriate substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include P-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.

[0066] An antibody of the invention may be labelled with a metallic chemical element such as lanthanides. Lanthanides offer several advantages over other labels in that they are stable isotopes, there are a large number of them available, up to 100 or more distinct labels, they are relatively stable, and they are highly detectable and easily resolved between detection channels when detected using mass spectrometry. Lanthanide labels also offer a wide dynamic range of detection. Lanthanides exhibit high sensitivity, are insensitive to light and time, and are therefore very flexible and robust and can be utilized in numerous different settings. Lanthanides are a series of fifteen metallic chemical elements with atomic numbers 57-71. They are also referred to as rare earth elements. Lanthanides may be detected using CyTOF technology. CyTOF is inductively coupled plasma time-of-flight mass spectrometry (ICP-MS). CyTOF instruments are capable of analyzing up to 1000 cells per second for as many parameters as there are available stable isotope tags.

[0067] Those of skill in the art will know of other suitable labels which can be employed in accordance with the present invention. The binding of marker moieties to monoclonal antibodies can be accomplished using standard techniques known to the art.

[0068] Moreover, the convenience and versatility of immunochemical detection can be enhanced by using monoclonal antibodies that have been conjugated with avidin, streptavidin, and biotin.

[0069] Another object of the invention is a method for detecting antibodies directed against T. gondii polypeptide BSM using at least one isolated Toxoplasma gondii polypeptide according to the invention as described above, and / or evaluating its amount in a biological sample.As used herein, the term "biological sample" refers to any biological sample of a subject; tissue sample or body fluid sample. In a preferred embodiment regarding a method to detect antibody directed against T. gondii polypeptide BSM, the biological sample is a body fluid of said subject. Non-limiting examples of such body fluid samples include, but are not limited to, blood, serum, plasma, urine, saliva, and cerebrospinal fluid (CSF) and aqueous humor.

[0070] More particularly the body fluid sample, is serum or aqueous humor sample. In a preferred embodiment regarding the detection of antibodies directed T. gondii BSM polypeptide of the invention, the biological sample is a fluid sample, more particularly a blood sample.

[0071] The biological sample may be a tissue sample, more particularly a muscle sample or a brain sample.

[0072] As used herein, the term “patient” or “subject” or “individual” refers suffering or may suffer (or not) from Toxoplasma. The In one embodiment, the patient is a mammal. Nonlimiting examples of mammals include rodents, primates, rabbits, dogs, horses, cats, livestock and deer. In one embodiment, the mammal is a human. The subject may be a pregnant woman, an immunocompromised patient or an animal intended for human consumption.

[0073] Detecting and Diagnostic methods of the invention:

[0074] In some embodiments, the method of the present invention are performed in vitro or ex vivo.

[0075] • Method for detecting T. gondii BSM polypeptide and / or antibodies against T.

[0076] gondii BSM polypeptide

[0077] An object of the invention is a method for detecting T. gondii BSM polypeptide of the invention, and / or antibodies against T. gondii BSM polypeptide of the invention and / or evaluating its amount in a biological sample.

[0078] Biological sample, means without limitation a tissue sample, a culture medium and cell samples, a whole blood sample, a serum sample, a plasma sample, aqueous humor sample, a salivary sample, a cerebrospinal fluid sample, muscle sample or a brain tissue sample.

[0079] In a preferred embodiment, the biological sample is a fluid sample, more particularly a blood sample , more particularly a serum sample or a plasma sample.

[0080] In a particular embodiment, the biological sample is a tissue sample, more particularly a muscle sample or a brain sample.Detecting the T. gondii polypeptide BSM and / or antibodies against T. gondii BSM polypeptide may include separation of the proteins / polypeptides: centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the protein's affinity for the particular solid-phase that is use. Once separated, T. gondii polypeptide BSM may be identified based on the known "separation profile" e. g., retention time, for that protein and measured using standard techniques.

[0081] The detection and amount of the T. gondii polypeptide BSM species of the invention and / or antibodies against T. gondii BSM polypeptide of the invention may be determined by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction such as immunohistochemistry, or sandwich type assays. Such assays include, but are not limited to, Western blots; Immunoblot, Immunochromatography (ie ICT (LD Bio) with IgG et IgM anti-Toxoplasma) agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin / avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

[0082] For example, determination of the T. gondii polypeptide BSM amount and / or antibodies against T. gondii BSM polypeptide amount can be performed by a variety of techniques and method any well-known method in the art: RIA kits (DiaSorin; IDS, Diasource) Elisa kits (Fujirebio, Thermo Fisher, EHTGFBI, R&D DY2935, IDS (manual) IDS (adapted on open analyzers) Immunochemiluminescent automated methods (MesoScaleDiscovery, DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (Janssen et al., 2012) Simoa / Quanterix.

[0083] In a particular embodiment, the methods of the invention comprise detection antibodies against T. gondii BSM polypeptide in a biological sample using the T. gondii polypeptide BSM species of the invention.

[0084] In a particular embodiment, the methods of the invention comprise contacting the biological sample with a binding partner.

[0085] As used therein, binding partner refers to a molecule capable of selectively interacting with T. gondii polypeptide BSM of the invention or antibodies against T. gondii BSM polypeptide.

[0086] The binding partner may be generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk et al. (1990) Science, 249, 505-510. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al. (1996) Nature, 380, 548-50).

[0087] The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

[0088] As used herein, the term "labelled", with regard to the binding partner, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example, radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Ini 11, Rel86, Rel88.

[0089] The aforementioned assays generally involve the bounding of the binding partner (i.e., antibody or aptamer) in a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against T. gondii polypeptide BSM. A body fluid sample containing or suspected of containing Toxoplasma gondii polypeptide BSM is then added to the coated wells. After a period of incubation sufficient to allow the formation of binding partner- T. gondii polypeptide BSM complexes, theplate(s) can be washed to remove unbound material and a labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

[0090] As the binding partner, the secondary binding molecule may be labelled.

[0091] Antibodies of the present invention and immunoconjugates can be used for detecting T. gondii polypeptide BSM of the invention, and / or evaluating its amount in a biological sample, in particular a tissue sample a culture medium and cell samples, a whole blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, or a brain tissue sample. Therefore they can be used for diagnosing all diseases associated with Toxoplasma gondii agent.

[0092] The detection of cysts within meats intended for consumption being a challenge for in the meat production sector, the bradyzoite serology could be a benefit for the development of “Toxoplasma-free” meat production. Therefore, BSM could be used to potentially directly detect cysts within meat samples.

[0093] Therefore, the invention further relates a method for detecting T. gondii BSM polypeptide of the invention, and / or evaluating its amount in meat intended for consumption.

[0094] Method of diagnosis of latent form of Toxoplasmosis by detection of antibodies of T. gondii polypeptide BSM

[0095] The present invention relates to a method of detection of antibodies against T. gondii polypeptide (serology) according to the invention and is consequently useful for the in vitro diagnosis of Toxoplasmosis from a biological sample. In particular, the method of detection of the invention is consequently useful for the in vitro diagnosis of latent form of Toxoplasmosis or congenital toxoplasmosis from a biological sample.

[0096] A further object of the invention is a method for in vitro diagnosing a Toxoplasmosis, wherein said method comprising detecting the presence of antibodies against T. gondii polypeptide according to the invention, as indicated above, in a biological sample from a subject to be tested.

[0097] The present invention relates to a method of determining if a subject is afflicted with a latent form of Toxoplasmosis, said method comprising:

[0098] a) detecting in a biological sample of the patient immunoreactivity toward a T. gondii polypeptide of the invention; and optionallyb) deducing from the result of step a) whether the patient is afflicted with latent form of Toxoplasmosis, immunoreactivity toward a T. gondii polypeptide of the invention is indicative of latent form of Toxoplasmosis.

[0099] The present invention also relates to the use of antibody directed against BSM as a biomarker for diagnosing (or confirming) latent form of Toxoplasmosis in a patient.

[0100] The present invention also relates to an in vitro method for diagnosing or confirming a diagnosis of a latent form of Toxoplasmosis in a patient who is suffering, or is suspected to be suffering, a latent form of Toxoplasmosis, comprising:

[0101] a) obtaining a biological sample from the patient, and

[0102] b) detecting, in the biological sample, antibodies toward a T. gondii polypeptide of the invention;

[0103] wherein the presence of antibodies in the biological sample diagnoses or confirms a diagnosis of latent form of Toxoplasmosis in a patient.

[0104] Thus present invention relates to a method of determining if a subject is afflicted with congenital Toxoplasmosis, said method comprising:

[0105] a) detecting in a biological sample of the patient immunoreactivity toward a T. gondii polypeptide of the invention; and optionally

[0106] b) deducing from the result of step a) whether the patient is afflicted with congenital Toxoplasmosis, immunoreactivity toward a T. gondii polypeptide of the invention is indicative of congenital Toxoplasmosis.

[0107] The present invention also relates to the use of antibody directed against congenital Toxoplasmosis as a biomarker for diagnosing (or confirming) congenital Toxoplasmosis in a patient.

[0108] The present invention also relates to an in vitro method for diagnosing or confirming a diagnosis of congenital Toxoplasmosis in a patient who is suffering, or is suspected to be suffering, congenital Toxoplasmosis, comprising:

[0109] b) obtaining a biological sample from the patient, and

[0110] b) detecting, in the biological sample, antibodies toward a T. gondii polypeptide of the invention;

[0111] wherein the presence of antibodies in the biological sample diagnoses or confirms a diagnosis of congenital Toxoplasmosis in a patient.As used herein, the term "biological sample" refers to any biological sample of a subject. In a preferred embodiment regarding a method using the detection of antibody directed against T. gondii BSM polypeptide, the biological sample is a body fluid of said subject. Nonlimiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, and cerebrospinal fluid (CSF) and aqueous humor.

[0112] More particularly the body fluid sample, is a blood sample.

[0113] More particularly the blood sample, is a blood sample is serum sample and / or, plasma serum.

[0114] In one embodiment, the biological sample is a tissue sample, more particularly a muscle sample or a brain sample.

[0115] In a preferred embodiment, the patient to be tested is suffering, or is suspected to be suffering, from Toxoplasmosis.

[0116] In another preferred embodiment, the patient to be tested is suspected to be suffering from Toxoplasmosis and the method is performed to confirm that the patient is actually afflicted with the latent form of Toxoplasmosis disease.

[0117] In another embodiment, the patient to be tested is a pregnant woman and / or an immunocompromised patient (i.e., HIV patient or patient treated by immunomodulatory before receiving a graft) and the method is performed to determine if the patient is actually afflicted with latent form of Toxoplasmosis.

[0118] • Method of diagnosis of latent form of Toxoplasmosis (detection of T. gondii polypeptide BSM)

[0119] In the present study, and as shown in experimental data, inventors shows that BSM serology correctly distinguishes cyst-bearing mice with a sensitivity and specificity of 97.96% (IC95: 89.31 - 99.90) and 100.0% (IC95: 86.20 - 100.0) respectively

[0120] Accordingly, the method of detection of the T. gondii BSM polypeptide according to the invention is consequently useful for the in vitro diagnosis of Toxoplasmosis from a biological sample. In particular, the method of detection of the invention is consequently useful for the in vitro diagnosis of latent form of Toxoplasmosis or congenital toxoplasmosis from a biological sample.

[0121] As used herein, the term "biological sample" refers to any biological sample of a subject. Biological sample, means without limitation any tissue sample, a culture medium andcell samples, a whole blood sample, a serum sample, a plasma sample, an urinary sample, a salivary sample, a cerebrospinal fluid sample.

[0122] In a preferred embodiment regarding a method using the detection of T. gondii BSM polypeptide, the biological sample is a fluid sample, more particularly blood samples, even more particularly a serum sample and / or a plasma sample.

[0123] In a particular embodiment regarding a method using the detection of T. gondii BSM polypeptide, the biological sample is a tissue sample, more particularly a brain tissue sample or muscle tissue sample.

[0124] A further object of the invention is a method for detecting T. gondii polypeptide BSM of the invention, and / or evaluating its amount in a biological sample, wherein said method comprises contacting said sample with an antibody or immunoconjugate of the invention under conditions allowing the formation of an immune complex between Toxoplasma gondii polypeptide BSM and said antibody / immunoconjugate, and detecting or measuring the immune complex formed.

[0125] A further object of the invention is a method for detecting bradyzoite cyst, and / or evaluating its amount in a biological sample, wherein said method comprises contacting said sample with an antibody or immunoconjugate of the invention under conditions allowing the formation of an immune complex between Toxoplasma gondii polypeptide BSM said antibody / immunoconjugate, and detecting or measuring the immune complex formed.

[0126] The immune complex formed can be detected or measured by a variety of methods using standard techniques, including, by way of non-limitative examples, enzyme-linked immunosorbent assay (ELISA) or other solid phase immunoassays, radioimmunoassay, electrophoresis, immunofluorescence, or Western blot.

[0127] A further object of the invention is a method for in vitro diagnosing a Toxoplasmosis, wherein said method comprising detecting the presence of Toxoplasma gondii polypeptide BSM, as indicated above, in a biological sample from a subject to be tested.

[0128] The term "Toxoplasmosis " has its general meaning in the art and refers to a worldwide distributed zoonotic infection with medical importance in pregnant women and immunocompromised patients. Toxoplasma gondii, the etiologic agent of toxoplasmosis, has co-evolved with its homeothermic hosts, including humans, strategies to persist usually as a quasi - cryptic population and accordingly with subclinical signs, hence optimizing the chance of transmission to new hosts. Over its prolonged residence in warm blooded metazoans, the proliferative stage (tachyzoite) switches into a persistent stage (cyst-enclosed bradyzoites) that affords the parasite a unique opportunity to spread to new hosts without proceeding through itssexual stage, which is restricted to felids. Uncontrolled amplification of tachyzoite population as it occurs when the immune balance is transiently or more sustainably ruptured can lead to life-threatening disease, and in the case of congenital toxoplasmosis, to birth defects. Persistence, which depends both on the acquisition of slow replicative skills by a subset of parasites and the destruction of the fast-replicative population, critically requires the IL-12 / IFN-y immune axis, yet T. gondii has singularly evolved a finely tuned and epigenetically regulated developmental program to operate stage conversion.

[0129] In some embodiment, the toxoplasmosis is congenital toxoplasmosis.

[0130] Thus, the invention refers to a method for in vitro diagnosing a congenital toxoplasmosis, wherein said method comprising detecting the presence of polypeptide, according to claims 1, in a biological sample from a subject to be tested.

[0131] As used herein, the term “latent form of Toxoplasmosis” or “chronic toxoplasmosis” refer to persistent stage (cyst-enclosed bradyzoites) of the Toxoplasmosis disease. Following the initial period of infection characterized by tachyzoite proliferation throughout the body, pressure from the host's immune system causes T. gondii tachyzoites to convert into bradyzoites, the semi-dormant, slowly dividing cellular stage of the parasite. Inside host cells, clusters of these bradyzoites are known as tissue cysts. The cyst wall is formed by the parasitophorous vacuole membrane. Although bradyzoite-containing tissue cysts can form in virtually any organ, tissue cysts predominantly form and persist in the brain, the eyes, and striated muscle (including the heart). However, specific tissue tropisms can vary between intermediate host species; in pigs, the majority of tissue cysts are found in muscle tissue, whereas in mice, the majority of cysts are found in the brain. Cysts usually range in size between five and 50 pm in diameter.

[0132] Furthermore, the invention also provides kits comprising at least one antibody of the invention or a fragment thereof or at least one T. gondii polypeptide BSM of the invention or a fragment thereof (in order to detect in a biological sample antibodies directed against T. gondii polypeptide BSM of the invention)

[0133] . Kits of the invention can contain an antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain antibodies for detection and quantification of Toxoplasma gondii polypeptide BSM in vitro, or T. gondii polypeptide BSM of the invention or a fragment thereof in order to detect in a biological sample antibodies directed against T. gondii polypeptide BSM of the invention e.g. in an ELISA or aimmunoblot. Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.

[0134] The current treatment for toxoplasmosis, when a subject presents signs and symptoms of acute toxoplasmosis, is the following:

[0135] • Pyrimethamine This medication, typically used for malaria, is a folic acid antagonist. It may prevent the body from absorbing the B vitamin folate (folic acid, vitamin B-9), especially when patient take high doses over a long period. For that reason, it may recommend taking additional folic acid. Other potential side effects of pyrimethamine include bone marrow suppression and liver toxicity.

[0136] • Sulfadiazine. This antibiotic is associated with pyrimethamine to treat toxoplasmosis.

[0137] For HIV / AIDS patients, the treatment of choice for toxoplasmosis is also pyrimethamine and sulfadiazine, with folic acid. An alternative is pyrimethamine taken with clindamycin

[0138] For pregnant women and neonates infected with toxoplasmosis:

[0139] If infection occurred before the 14th week of pregnancy, pregnant women receive the antibiotic spiramycin. Use of this drug may reduce the risk of transmission to the fetus. v If infection occurred after the 14th week of pregnancy, or if tests show that the unborn child has toxoplasmosis, pregnant women may be given pyrimethamine and sulfadiazine and folic acid.

[0140] The present invention also provides an in vitro method for selecting a patient afflicted with a latent form of Toxoplasmosis suitable to be treated with at least one folic acid antagonist and / or antibiotic compound, comprising:

[0141] a) detecting in a biological sample of the patient immunoreactivity toward a T. gondii polypeptide of the invention ; and optionally

[0142] b) selecting the patient as suitable to be treated with at least one folic acid antagonist (i.e. Pyrimethamine) and / or antibiotic compound (i.e. Sulfadiazine or spiramycin; Sulfadiazine or sulfamethoxazole) when immunoreactivity toward a T. gondii polypeptide of the invention is detected.

[0143] The method of determining if a patient is afflicted with latent form of Toxoplasmosis, the use of antibody directed against a T. gondii polypeptide of the invention as a biomarker fordiagnosing (or confirming) latent form of Toxoplasmosis, and the method of selecting a patient afflicted with latent form of Toxoplasmosis suitable to be treated with at least one folic acid antagonist and / or antibiotic compound of the invention may be, for instance, in vitro or ex vivo methods.

[0144] Therapeutic application :

[0145] The invention also concerns a method for treating a patient infected with latent form of Toxoplasmosis who shows immunoreactivity toward a T. gondii polypeptide of the invention, which method comprises administering to the patient folic acid antagonist (i.e. Pyrimethamine) and / or antibiotic compound (i.e. Sulfadiazine or spiramycin), or a pharmaceutical composition comprising said compounds.

[0146] The invention also provides folic acid antagonist (i.e., Pyrimethamine) and / or antibiotic compound (i.e., Sulfadiazine and / or spiramycin), or a pharmaceutical composition comprising said compounds, for use in the treatment of a patient suffering from latent form of Toxoplasmosis who shows immunoreactivity toward a T. gondii polypeptide of the invention.

[0147] In particular, the terms T. gondii polypeptide of the invention against which immunoreactivity is tested refer to:

[0148] (i) the amino acids sequence consisting of Toxoplasma gondii polypeptide BSM (SEQ ID NO :1) ;

[0149] (ii) the amino acids sequence consisting or comprising of Toxoplasma gondii polypeptide BSM (SEQ ID NO : 3);

[0150] (iii) an amino acid sequence substantially homologous to the sequence of (i) or ((ii) preferably an amino acid sequence at least 80% identical to the sequence of (i) or (ii)

[0151] (iv) a fragment of at least 9 consecutive amino acids of the sequence of (i),(ii) or (iii).

[0152] By “polypeptide with amino acid sequence substantially homologous” is meant a polypeptide that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a full-length polypeptide reference sequence. In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The « needle » program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk world wide web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS: rneedle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

[0153] As used throughout the present application, the expression “Immunoreactivity toward a target protein” (here T. gondii polypeptide of the invention) is intended to mean that the sample from the patient to be tested comprises antibodies specifically directed against the target.

[0154] Therefore, immunoreactivity toward a target protein can be easily detected by demonstrating in the biological sample to be tested the presence of antibodies specifically directed against the target protein or a fragment of this target protein.

[0155] Fragments of the target proteins may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full-length protein. Preferably, said fragments are at least about 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 250, 300, 350, 400, 450, 500 or more amino acids in length.

[0156] Such a test can be performed by one of ordinary skill in the art by using standard methods, for instance Enzyme-linked immunosorbent assay (“ELISA”), Western Blot / Dot Blot, Immunohistochemistry on transfected cells, Luminex (see for review Immunodiagnostics: A Practical Approach, R. Edwards Editor, Oxford University Press 2000; Manual of Molecular And Clinical Laboratory Immunology, J. D. Folds R. G. Hamilton, B. Detrick Editors ASM Press 2006; Immunology and Serology in Laboratory Medicine, M. L. Turgeon, Mosby Inc, 2008).

[0157] As used herein, the term “patient” denotes a mammal and more particularly a human being.

[0158] In the context of the invention, the term "treating" is used herein to characterize a therapeutic method or process that is aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) alleviating or bringing about ameliorations of the symptoms of the disease state or condition to which such term applies; and / or (3) reversing or curing the disease state or condition to which such term applies.

[0159] The folic acid antagonist and / or antibiotic compound used in the above recited method or use for treating patients afflicted with latent form of Toxoplasmosis are provided in apharmaceutically acceptable carrier, excipient or diluent which is not prejudicial to the patient to be treated.

[0160] Pharmaceutically acceptable carriers and excipient that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0161] As appreciated by skilled artisans, compositions are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral route, including for instance intramuscular, subcutaneous, intravenous, intraperitoneal or local injections. The oral route can also be used, provided that the composition is in a form suitable for oral administration, able to protect the active principle from the gastric and intestinal enzymes.

[0162] Further, the amount of folic acid antagonist and / or antibiotic compound used in the above recited method or use for treating patients afflicted with latent form of Toxoplasmosis is a therapeutically effective amount.

[0163] The exact amount of folic acid antagonist and / or antibiotic compound to be used and the composition to be administered will vary according to the age and the weight of the patient being treated, the type of disease, the mode of administration, the frequency of administration as well as the other ingredients in the composition which comprises the folic acid antagonist and / or antibiotic compound. Such concentrations can be routinely determined by those of skilled in the art. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual folic acid antagonist and / or antibiotic compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, etc.

[0164] Generally, the folic acid antagonist and / or antibiotic compound used in the above recited method or use for treating patients afflicted with latent form of Toxoplasmosis may beadministered in the typical range. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.

[0165] Kit and Method of diagnosis of latent form of Toxoplasmosis (detection of T. gondii BSM and BCLA)

[0166] The invention further provides kits that are useful in the above methods for diagnosing latent form of Toxoplasmosis or for selecting a patient afflicted with latent form of Toxoplasmosis suitable to be treated with at least one folic acid antagonist and / or antibiotic compound.

[0167] Such kits comprise means for detecting antibodies directed toward at least one T. gondii polypeptide of the invention.

[0168] Preferably, the kit comprises at least means for detecting antibodies directed toward at least one T. gondii polypeptide of the invention.

[0169] Means for detecting antibodies directed toward at least one T. gondii polypeptide of the invention may also include an antibody specifically binding to human antibodies (used as a “secondary antibody” which binds to antibodies from the sample to be tested specifically binding to the target protein) an antibody specifically binding to mammal antibodies. Such antibodies can be labeled with detectable compound such as fhiorophores or radioactive compounds.

[0170] In a preferred embodiment, the kit according to the invention may further comprises a control sample comprising a known amount of antibodies and / or instructions for the use of said kit in diagnosing latent form of Toxoplasmosis or in selecting a patient afflicted with latent form of Toxoplasmosis suitable to be treated with at least one folic acid antagonist and / or antibiotic compound.

[0171] The means may be present, e.g., in vials or microtiter plates, or be attached to a solid support. For instance the target protein can be attached to a membrane or to an array.

[0172] A further object of the invention is a method for detecting bradyzoite cyst, and / or evaluating its amount in a subject, wherein said method comprises

[0173] a) detecting in a biological sample of the subject immunoreactivity toward a T. gondii polypeptide; and optionally

[0174] b) deducing from the result of step a) the presence and / or amount of bradyzoite cyst, immunoreactivity toward a T. gondii polypeptide of the invention is indicative of presence and / or amount of bradyzoite cyst in said subject.In a preferred embodiment the biological sample is a body fluid of said subject. Nonlimiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, and cerebrospinal fluid (CSF) and aqueous humor.

[0175] More particularly the body fluid sample, is a blood sample.

[0176] In one embodiment, the biological sample is a tissue sample, more particularly a muscle sample or a brain sample.

[0177] In one embodiment, the detecting and diagnostic methods according to the invention are suitable for detecting ocular toxoplasmosis, cerebral toxoplasmosis and / or asymptomatic reactivations.

[0178] • Method of diagnosis of latent form of Toxoplasmosis (detection of T, gondii BSM and BCLA)

[0179] The inventors have recently demonstrated the immunogenicity of the bradyzoite-specific protein BCLA, which is localized to the membrane of the parasitophorous vacuole (Dard C et al., 2021, BMC Biol). In human sera, BCLA ELISA rates were significantly higher in individuals with past immunity with particularly high rates in subgroups of patients with clinical forms associated with the presence of cysts, i. e., ocular toxoplasmosis or asymptomatic reactivation (Dard C et al., 2021, BMC Biol). Thus, the inventors demonstrated that BCLA may be used as a biomarker.

[0180] Therefore, the methods according to the invention may comprise a detection of both biomarkers BSM and BCLA or antibodies against both biomarkers BSM and BCLA.

[0181] Detecting the T. gondii polypeptide BCLA or antibodies against T. gondii polypeptide BCLA may include separation of the proteins / polypeptides: centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the protein's affinity for the particular solid-phase that is use. Once separated, T. gondii polypeptide BCLA or antibodies against T. gondii polypeptide BCLA may be identified based on the known "separation profile" e. g., retention time, for that protein and measured using standard techniques.

[0182] T. gondii polypeptides BCLA or antibodies against T. gondii polypeptide BCLA and Method of diagnosis of latent form of Toxoplasmosis using these markers are described in W02021 / 094308 which is incorporated by reference.

[0183] In particular embodiment the detection of the antibodies against T. gondii polypeptide BCLA may be performed with the “mini BCLA” recombinant protein (SEQ ID NO : 4)

[0184] SEQ ID NO : 4 :MHHHHHHENLYFQGMPEITIREEEEELFPSLDDVLDTSPFPARLWMGPGEKQATESH TPATIPTAYKSTPGLSATVTGGEDGSAKMVGMDVAKKPVKVSVKKEEENKDVEANE DGWDYIVSKGVPGKIPATVMDEARKADVVADGEAKPAMREAQERRKPWETQEEKI LVLPKVQRILALPKEEKKHVSTAKGEEPF S SKEEERHVLLNGEERKPVVPRAGREQP A VPRQEEQKLVLQKTERKPVLPEEDQKPVLPETGAKHVLPEIATKSTLTQKEVTKPVET RQDMERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTEMERPA APIEGEKGVVSSEEEKPVSPKEATRRILPKEGKESLGTRKEEVKPIVRRAKRGRRIAQK GKEKQIAPKEGKKPAVPKEGEERPAEPTEGEERPVGPKEGEERPVGPKEGEERPVVPD VDKEKPVVPEGDKEKPVVPEGDKDHPALPEQDEEKHATWEKEMIPGVGDKTEASVL DSIENAVQKVLENLLKAAAGELQPAEAEEARLLVADLKAVVDTAEQVRVEGEAFFR ASVDLYEAVKNLRDSEEKLRPLTKGELVDVVRQFLATQIFVQDRASAFLRVFERLAE LLAAEQMKAVFAMVEEGVSSSERVARVAGELVPMMKKDRERRYGDLVAVTSWFM RRMEHI.

[0185] The detection and amount of the T. gondii polypeptide BCLA species of the invention and / or antibodies against T. gondii polypeptide BCLA species may be determined by using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction such as immunohistochemistry, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin / avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

[0186] For example, determination of the amount of T. gondii polypeptide BCLA and / or amount of antibodies against T. gondii polypeptide BCLA can be performed by a variety of techniques and method any well-known method in the art: RIA kits (DiaSorin; IDS, Diasource) Elisa kits (Fujirebio, Thermo Fisher, EHTGFBI, R&D DY2935, IDS (manual) IDS (adapted on open analyzers) Immunochemiluminescent automated methods (MesoScaleDiscovery, DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (lanssen et al., 2012) Simoa / Quanterix.

[0187] The invention will be further illustrated by the following figures and examples. However, the examples and figures should not be interpreted in any way as limiting the scope of the present invention.FIGURES

[0188] Figure 1.

[0189] Graphs show the distribution of the size of visible plaques representing the lytic cycle of 76K-GFP-luc and ^hsm parasites. Confluent HFFs were infected with 76K-GFP-luc or / bsm strains. After 7 days, the cells were fixed and stained with Coomassie blue to detect the presence of plaques. Statistical analyses were performed using Mann-Whitney test.

[0190] Figure 2. Bsm deletion does not affect virulence-and Bradyzoite-specific ELISA serologies correlate with parasite load in mouse brain.

[0191] A. NMRJ (n = 15) mice were given 5.104tachyzoites of the 76K-GFP-luc WT and Absm strains intraperitoneally and their survival was monitored. Significance was tested using log-rank Mantel-Cox and Gehan-Breslow-Wilcoxon tests (p = 0.3496). B. Parasitic load in parasite per brain (qPCR count), C. miR-155, and miR-146a expression in mouse brain infected with 76K-GFP-luc and bsm strains. Statistical significance was calculated using a non-parametric Mann-Whitney test. D. Parasitic load in mice from different background (NMRI, Balb / C, CD1) and infected by cystogenic strains (76K, ME49), low-cystogenic strains (CTG, Pru ku80), 76K bsm, 76K or Pru ku80 AZ>c / aE. BSM and BCLA ELISA serology in mice, displayed with the same color code as in panel Statistical significance was calculated using a non-parametric Mann-Whitney test. F. ROC curves of BSM ELISA serology and BCLA ELISA serology.

[0192] Figure 3. Bradyzoite-specific serology in human sera. BSM ELISA serology (A), and BCLA ELISA serology (B) in seronegative (n = 134) or previously immunized patients against T. gondii infection (n = 222), and according to the clinical context (grid histograms). Statistical significance was calculated using a non-parametric Mann-Whitney test. C. ROC curves of BSM ELISA serology and BCLA ELISA serology. D. BSM and BCLA ELISA serology titers in the course of past (n = 134) and acute infection (n = 74). Statistical significance was calculated using a non-parametric Mann-Whitney test. E. BSM and BCLA ELISA serology titers at birth in children bom to mothers who had seroconverted during pregnancy and for whom the diagnosis of congenital toxoplasmosis (CT) had been made (n = 15, green histogram) or excluded (n = 11, orange histogram).

[0193] Figure 4: Recombinant purification steps. A. Size Exclusion Chromatography using a S200 (10 / 300 Gl) combined to a Multi-Angle Laser Light Scattering analysis. B. Schematic representation of rBCLA (1st generation of the antigene) and miniBCLA (this work) proteins.

[0194] C. Size exclusion chromatography of the purified miniBCLA antigen, 280 mm absorbance isshown as a function of volume. Peak elution fractions F5 to F10 were analyzed by coomassie blue stained 4-12% NuPAGE.

[0195] EXAMPLES

[0196] Material & Methods

[0197] Parasites and human cell culture

[0198] Human primary fibroblasts (HFFs, ATCC® CCL-171™) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 10 mM (4-(2 -hydroxy ethyl)- 1 -piperazine ethanesulphonic acid) (HEPES) buffer pH 7.2, 2 mM L-glutamine, and 50 pg / mL of penicillin and streptomycin (Invitrogen). Cells were incubated at 37 °C in 5% CO2. The Toxoplasma strains used in this study and listed in table 2. were maintained in vitro by serial passage on monolayers of HFFs.

[0199] Reagents

[0200] The following primary antibodies were used in the immunofluorescence, and immunoblotting assays: rabbit anti-TgHDAC3 (RRID: AB 2713903), mouse anti-TgBAGl, mouse anti-HA tag (Roche, RRID: AB 2314622), anti-QRS (van Rooyen JM et al. 2014), rabbit anti-BCLA (Dard C et al., 2021, BMC Biol). Immunofluorescence secondary antibodies were coupled with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific). Secondary antibodies used in Western blotting were conjugated to alkaline phosphatase (Promega). We also commissioned Eurogentec to produce polyclonal serum from rabbits immunized against the full-length BSM recombinant protein and used it for immunofluorescence and immunoblotting assays.

[0201] Mouse infection and experimental survey

[0202] Six-week-old NMRI, CD1 or Balb / C mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France). Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with established institutional guidance and approved protocols from the Institutional Animal Care and Use Committee of the University Grenoble Alpes (APAFIS#4536-2016031 017075121 v5). Female mice were used for all studies. For intraperitoneal (i.p.) infection; tachyzoites were grown in vitro and extracted from host cells by passage through a 27-gauge needle, washed three times in phosphate-buffered saline (PBS), and quantified with a hemocytometer. Parasites were diluted in Hank’s Balanced Salt Solution (Life), and mice were inoculated by the i.p. route with tachyzoites of each strain (in 200 pl volume) using a 28-gauge needle. Blood was collected by intracardiac puncture when the micewere euthanized. Animal euthanasia was completed in an approved CO2 chamber. For immunolabeling on histological sections of the brains, the brains were removed from mice, entirely embedded in a paraffin wax block and cut in 5-pm-thick layers using microtome. For statistical analysis of mouse survival data, the Mantel-Cox and Gehan-Breslow-Wilcoxon tests were used.

[0203] Auxin-induced degradation

[0204] Depletion of MORC-mAID-HA was achieved with 3-indoleacetic acid (IAA, Sigma-Aldrich # 45533) used at 500 pM final concentration from a 500-mM stock solution prepared in EtOH, as described by Farhat et al., 2020 (F arhat D.C et al., 2020, Nat Microbiol). To monitor the degradation of AID-tagged proteins, parasites grown in HFF monolayers were treated with auxin for 24 to 48h at 37 °C before parasites were harvested and analyzed by immunofluorescence or Western blotting.

[0205] HDAC3 inhibition using FR235222

[0206] FR.235222 was provided by Astellas Pharma Inc. (Osaka, Japan) and dissolved into DMSO, and the final concentration in the culture medium was 50 ng / mL. Fifteen hours after infection of HFF monolayers, FR235222 was added to the medium and cells cultivated for 48h.

[0207] Immunofluorescence microscopy

[0208] T. gondii-m cteA HFF monolayers grown on coverslips were fixed in 3% formaldehyde for 20 min at room temperature, permeabilized with 0.1% (v / v) Triton X-100 for 15 min, and blocked in PBS containing 3% (w / v) bovine serum albumin (BSA). For immunolabeling on histological sections of the brains, the brain layers spotted on glass slides were first solvent-dewaxed using toluene for 3 times 10 min and absolute alcohol for 3 times 10 min. The slides were then treated with citrate buffer pH 6, heated at 100 °C during 1 h, rinsed extensively with water and blocked in PBS containing 3% (v / v) BSA. The infected cells or brain layers were then incubated for 1 h with the primary antibodies indicated in the figures followed by the addition of secondary antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes) at a 1:1000 dilution for 1 h. The nuclei of both host cells and parasites were stained for 10 min at room temperature with Hoechst 33258 at 2 pg / mL in PBS. After four washes in PBS, coverslips were mounted on a glass slide with Mowiol mounting medium; images were acquired with a fluorescence ZEISS ApoTome.2 microscope and processed with the ZEN software (Zeiss).

[0209] Western blot

[0210] Immunoblot analysis of protein was performed as followed: ~ 107cells were lysed in 50 pl lysis buffer (10 mM Tris-HCl, pH 6.8, 0.5% SDS [v / v], 10% glycerol [v / v], and 1 mM EDTA and protease inhibitors cocktail) and sonicated. Proteins were separated by SDS-PAGEand transferred to a polyvinylidene fluoride membrane (PVDF, Immobilon-P; EMD Millipore) by liquid transfer 2h at 110 V; blotted membranes were then probed using appropriate primary antibodies followed by alkaline phosphatase secondary antibodies (Life technologies). Band revelation was detected using NBT-BCIP (Amresco).

[0211] Cyst observation

[0212] Fifty-nine days post-infection, the brain of each of the recipient mouse was homogenized in 2 mL of PBS. Images of cysts were acquired between slide and slip cover with a fluorescence ZEISS ApoTome.2 microscope.

[0213] Plaque assays

[0214] Confluent HFFs were infected with freshly egressed tachyzoites. Cultures were grown at 37°C for 7 days, fixed, and stained with Coomassie blue staining solution (0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid). The size of the plaques was measured using ZEN 2 lite 9 software (Carl Zeiss, Inc.) and plotted using GraphPad Prism 8.

[0215] Quantitative PCR

[0216] The parasite loads in the brain were quantified following DNA extraction (QiAmp DNA mini kit, Qiagen) using the quantitative PCR targeting of the Tox ptoma-specific 529-bp repeat element (Reischl U et al., 2003, BMC Infect Dis'). For statistical analysis of parasitic load differences between mice infected with 76K-GFP-luc and 76K-GFP-luc-A / zs77?, the nonparametric Wilcoxon-Mann-Whitney test was applied.

[0217] Quantitative RT-PCR analysis of interleukins in the brain

[0218] Total RNA was isolated from brains using TRIzol (Thermo Fisher Scientific). First-strand cDNA was reverse transcribed from 50-100 ng small RNA using TaqMan microRNA reverse transcription kit (Applied Biosystems) and TaqMan probes (Applied Biosystems) for miR-155 (ID 002623), miR-146a (ID 000468). Quantitative PCR analyses of miRNAs were performed using Taqman miRNA expression (Applied Biosystems) assays according to the manufacturer’s protocols in the ABI 7500 real-time PCR system (Applied Biosystems). Murine U6 snRNA / RNU6B (ID 001093) and RNU24 (ID 001001) were used as endogenous controls for normalization. Relative quantities of miRNA were analyzed by using the delta Ct method (Livak K.J et al., 2001, Methods).

[0219] Toxoplasma gondii transfection

[0220] T. gondii strains were electroporated with vectors in cytomix buffer (120 mM KC1, 0.15 mM CaCh, 10 mM K2HPO4 / KH2PO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCh) using a BTX ECM 630 machine (Harvard Apparatus). Electroporation was performed in a 2 mm cuvette at 1.100 V, 25 Q and 25 pF. Drug selection was performed usingpyrimethamine (3 pM). Single-clone of stable transgenic tachyzoites were obtained by limiting dilution in 96-well plates and verified by immunofluorescence assay or genomic analysis.

[0221] Plasmid construction

[0222] The plasmids and primers for the genes of interest (GOI) used in this work are listed below. To construct the vector pLIC-GOI-HAFlag, the coding sequence of GOI was amplified using primers LIC-GOI-Fwd and LIC-GOI-Rev using T. gondii genomic DNA as template. The resulting PCR product was cloned into the pLIC-HF-dhfr vector using the ligation-independent cloning (LIC) cloning method (Bougdour A el al., 2013, Cell Host Microbe).

[0223] For BSM disruption, the plasmid pTOXO_Cas9-CRISPR was described previously (Sangare L.O el al., 2016, Nat Commuri). Twenty mer-oligonucleotides corresponding BSM were cloned using the Golden Gate strategy. Briefly, primers BSM-gRNA-Fwd and BSM-gRNA-Rev containing the sgRNA targeting BSM genomic sequence were phosphorylated, annealed, and ligated into the pTOXO_Cas9-CRISPR plasmid linearized with Bsal, leading to pTOXO_Cas9-CRISPR: : sgB SM.

[0224] Gene synthesis for recombinant expression of Y JSM

[0225] Gene synthesis for insect cell codon optimized constructs was provided by Genscript. The original T. gondii T BSM construct (aa 1-763) was designed with a non-cleavable C-terminal 6His tag. The TgBCLA construct consisted of a 686 amino acid sequence designed to cover the N-terminal part followed by a conserved repetition, a degenerated repetition, and the C -terminal part of the BCLA protein encoded by TGME49 209755. The construct was also flanked by an N-terminal non-cleavable 6His tag. Both constructs were separately cloned between BamHI and Hindlll sites into the pFastBacl vector (Invitrogen).

[0226] Generation of baculovirus

[0227] For both BSM and BCLA, bacmid cloning steps and baculovirus generation were performed using EMBacY baculovirus (kindly gifted by Imre Berger), which contains a YFP reporter gene in the virus backbone. The established standard cloning and transfection protocols setup within the EMBL Grenoble eukaryotic expression facility were used. Baculovirus synthesis (V0) and amplification (to VI) were performed with SF21 cells cultured in ESF921 media (Expression system), large-scale expression cultures were performed with Hi-5 cells cultured in ESF921 media (Expression system) and infected with 0,5% vol / vol of generation 2 (VI) baculovirus suspensions and harvested 72h post-infection.

[0228] Protein expression and purification

[0229] • BSMFor purification, the cell pellets of approximately 900 mL of Hi-5 culture were resuspended in 40 mL of lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl and 4 mM P-mercaptoethanol (P-ME)) in the presence of an anti-protease cocktail (Complete EDTA free, Roche) and 1 ul benzonase (MERK Millipore 70746). Lysis was performed on ice by sonication for 3 min (30 sec on / 30 sec off, 45° amplitude). After the lysis step, 5% of glycerol was added. Clarification was then performed by centrifugation for Ih at 12,000 xg and 4°C. Twenty mM imidazole was then added to the supernatant and incubated with 3 mL of Ni-NTA resin (Qiagen) with a stirring magnet at 4°C for 30 min. All further purification steps were then performed at room temperature. After flowing through the lysate, the resin was washed with 10 column volumes of lysis buffer containing 20 mM imidazole. Elution was then performed by increasing the imidazole content to 300 mM in a buffer system containing 200 mM NaCl, 50 mM Tris pH 7.5, 2 mM BME and 5% glycerol. Eluted fractions were pooled based on an SDS PAGE gel analysis and flown directly through a previously equilibrated (in 250 mM NaCl, 50 mM Tris pH 7.5, 2 mM BME and 5% glycerol) S200 column connected to an AKTA© pure system. Peak fractions were pooled and concentrated using a 50 kDa Amicon ultra (Sigma Aldrich) concentrator. Glycerol concentration was adjusted to 20% before being frozen in liquid nitrogen and stored long-term at -80°C.

[0230] • BCLA

[0231] The protein expression, cell lysis and Ni-NTA purification processes were identical for BCLA and BSM. After Ni-NTA purification, BCLA eluted fractions were pooled and dialyzed overnight in 50 mM NaCl, 50 mM Tris pH 7.5, 2 mM BME and 5% glycerol before injection on a Mono-Q column (GE healthcare) preequilibrated with the same buffer as for dialysis. The column was eluted by a salt gradient (50 mM to 2 M NaCl) and peak fractions were pooled and injected on an S200 column as mentioned above for BSM.

[0232] SEC-MALLS

[0233] The MALLS run was performed using an S200 Increase SEC column (10 / 300 GL, GE Healthcare). Sample injection and buffer flow were controlled by a Hitachi L2130 pump. The SEC column was followed by an L-2400 UV detector (Hitachi), an Optilab T-rEX refractometer (Wyatt technologies), and a DAWN HELEOS-II multi-angle light scattering detector (Wyatt technologies). Injections of 50 pL were performed using protein samples concentrated at a minimum of 4 mg.mL1, a constant flow rate of 0.5 mL.min1was used. Accurate MALLS mass prediction was performed with the Astra software (Wyatt Technologies). The curve was plotted using Graphpad (Prism).

[0234] Plate preparationMidisorp plates (Nunc) were coated overnight (O.N) at 4 °C with either recombinant BSM or BCLA at 2 pg / mL in 100 mM calcium carbonate buffer pH 9.6 with 100 pl per well. After coating, plates were washed twice with 250 pl of DPBS 0.05% Tween 20 (DPBS / Tween) then blocked for at least 2h with 300 pl Superblock blocking buffer (Thermo Fisher) after which the buffer was removed and the plates dried upside down. Once dried, the plates could be stored for extended periods of time at 4 °C with no loss in serological reactivity.

[0235] Sample preparation

[0236] All serum dilutions were prepared in DPBS 0.05% Tween 20, 0.1% BSA no more than 2 h prior to the assay. For mouse tested sera, 1:400 dilutions were prepared. Eleven standards were also freshly prepared, consisting of 10 serial dilutions of a positive frozen stock serum set at 100 UI. Starting at a dilution 1:200 and following a % dilution increment, the following titration points were prepared: 200 UI (1:200), 150 UI (1:266), 112.5 UI (1:356), 84.4 UI (1:474), 63.3 UI (1:632), 47.5 UI, (1:843) 35.6 UI (1:1124), 26.7 UI (1:1498), 20 UI (1:1998), and 15 UI (1:2663). A 0 UI standard was prepared with a seronegative serum diluted at 1:400. For BCLA serology in humans, 1:400 sera dilutions were prepared. Six standards were also freshly prepared, consisting of five serial dilutions of an anti-His-tag chimeric human monoclonal antibody (Sigma-Aldrich, Saint Louis, USA). The following titration points were prepared: 200 UI, 100 UI, 50 UI, 25 UI, 12.5 UI. A 0 UI standard was prepared with a seronegative serum diluted at 1:400. For BSM serology in humans, sera were diluted at 1:200. Ten standards were also freshly prepared, consisting of nine serial dilutions of an anti-His-tag chimeric human monoclonal antibody (Sigma-Aldrich, Saint Louis, USA). The following titration points were prepared: 100 UI, 66.67 UI, 44.44 UI, 29.63 UI, 19.75 UI, 13.17 UI, 8.78 UI, 5.85 UI, 3.9 UI. A 0 UI standard was prepared with a dilution buffer only. The reactivity of a sample prepared with a dilution of anti-His-tag chimeric human monoclonal antibody (Sigma-Aldrich, Saint Louis, USA) at 10 UI was measured in each ELISA plate. BSM serology was calculated for each sample as the ratio between its own optical density (OD) and the OD of the cutoff from the same plate.

[0237] Assay

[0238] All the subsequent steps were implemented on the Gemini ELISA automation platform (Stratec). Dried plates were first washed twice with 350 pl of DPBS / Tween. Dilutions of the tested sera and standards were then distributed in the plates as row duplicates with 100 pl per well. Plates were then incubated Ih at RT. After the incubation period, plates were washed 4 times with 350 pl of DPBS / Tween, 100 pl of peroxidase coupled secondary antibody dilution (1:50,000 anti-mouse IgG or 1:60,000 anti-human IgG, Sigma Aldrich ref A0168 and A0170respectively) in DPBS 0.05% Tween 20, 0.1% BSA were then rapidly distributed in all wells. For human BSM serology, purified recombinant protein A / G peroxidase conjugated (ThermoScientific) stored at -20°C at 87Units / mL was used diluted at 1:50,000 instead of antihuman IgG. After Ih at RT, plates were washed 4 times in DPBS-Tween. Revelation reaction was performed by adding 100 pl of TMB Substrate (Thermo Fisher ref 34029) for 20 min precisely at RT then stopping the reaction with 50 pl of H2SO4 O.2M followed by 30 s of mixing. Well absorbance measurement was then performed using the Gemini integrated spectrophotometer at 450 nm.

[0239] Data treatment

[0240] Blank subtractions were performed on duplicate blank wells for which where primary antibody / sera were omitted but treated similarly as the others for all subsequent steps (washes, secondary Ab, substrate). Standard serum dilutions were averaged and fitted with a 4-parameter logistic regression with the upper asymptote value (77) fixed at 2.5 AU and all other variables (At, Bi, Ct) allowed to fit. For mouse and human BCLA serology and BSM mouse serology, tested dilution duplicates could have their apparent UI calculated and averaged from this regression; if in a duplicate measurement the coefficient of variation was observed above 20%, then the sample would be re-tested. All the ELISA data presented in this work was obtained several times in independent titrations.

[0241] Human sera

[0242] This non-interventional, multicentric retrospective study involving data and samples from human participants was conducted at the Grenoble Alpes University Hospital using serum samples collected from both the Grenoble Alpes University Hospital and the Hospices Civils de Lyon, in accordance with French current regulation. The principal investigator (Dr Marie- Pierre Brenier-Pinchart, MD, PhD) has signed a commitment to comply with Reference Methodology n°MR004 issued by French Authorities (CNIL). Subjects were all informed and did not oppose; written consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The raw data supporting the conclusions of this article will be made available by the authors within respect of General Data Protection Regulation, without undue reservation.

[0243] All the sera were collected among sera received at the laboratory of the Grenoble Alpes University Hospital or the laboratory of the Hospices Civils de Lyon for toxoplasmosis serological routine analysis between December 1, 2011, and February 1, 2022, in agreement with the respective institutions and after patients’ information and non-objection. Clinical data (age, sex, clinical and immune context) were collected and stored according to local ethicprocedures. In Grenoble, analyses were performed using Vidas® Toxo IgM and IgG (bioMerieux, France) and Architect Toxo IgG and IgM (Abbott, Germany) in the Parasitology- Mycology Clinical Laboratory of the Grenoble Alpes University Hospital. In Lyon, analyses were conducted using Architect Toxo IgG and IgM (Abbott, Germany) and Vidas® Toxo IgG (bioMerieux, France) in the Parasitology-Mycology Clinical Laboratory of the Hospices Civils de Lyon. Briefly, the antibody titers for IgG were quantitatively expressed in lU / mL whereas IgM were expressed as an index. The cutoffs defined by Vidas®, bioMerieux manufacturer were as follows: (i) IgG (lU / mL): negative < 4.0; 4.0 < equivocal (gray zone) < 8; > 8 positive; (ii) IgM (index): negative <0.55; 0.55 < equivocal (gray zone) <0.65; >0.65 positive. The cutoffs defined by Architect®, Abbott manufacturer were as follows: (i) IgG(IU / mL): negative < 1.6; 1.6 < equivocal (gray zone) <3.0; >3.0 positive; (ii) IgM (index): negative <0.50; 0.50 < equivocal (gray zone) < 0.60; > 0.60 positive. BCLA and BSM titers were measured on 357 selected patients’ sera corresponding to two different serological status for toxoplasmosis: non-immunized patients against toxoplasmosis (seronegative, n= 134 including 76 from Grenoble and 58 from Lyon) and patients with past immunity (chronic toxoplasmosis, n = 222 including 113 from Grenoble and 109 from Lyon). Among the patients with past immunity, those with a clinical context related primarily to reactivation from cysts were categorized into the subgroups "proven ocular toxoplasmosis" (n = 23) "cerebral toxoplasmosis" (n = 5) and "asymptomatic reactivation" (n = 6). In addition, 115 sera from consecutive samples collected from 39 pregnant women who seroconverted during pregnancy and one man presenting with an acute infection were used to establish the kinetics of anti-bradyzoite serology. Of these precisely dated sera, 74 were collected within four months of infection and were assigned to the category "recent infection”. Furthermore, BSM and BCLA ELISA serology titers was measured in sera collected at birth in children bom to mothers who had seroconverted during pregnancy and for whom the diagnosis of congenital toxoplasmosis had been made (n = 15) or excluded (n = 11) The absence of T. gondii immunity was concluded when the IgM- and IgG-specific antibody levels measured using Architect Toxo® IgG and Toxo® IgM assays were negative. Past immunity was considered when IgG were above the threshold of positivity with at least one method, and the IgM were negative or weakly positive (Architect® and Vidas®). Proved ocular toxoplasmosis (OT) was confirmed by detection of either Toxoplasma DNA using PCR and / or a local production of IgG and / or IgA antibodies by Western blot (LDBIO Diagnostics, Lyon, France) (Greigert V et al., 2019, mSphere). Cerebral toxoplasmosis was diagnosed by PCR in cerebrospinal fluid in immunocompromised patients presenting acquired immunodeficiency syndrome (Brenier-Pinchart M.P et al., 2022). These patients had clinicalsymptoms and radiological evidence of active disease. Asymptomatic serological reactivations were observed in immunocompromised patients during the serological follow-up (Dard C et al., 2018, Expert Rev Anti Infect Ther, Robert-Gangneux F et al., 2018, Emerg Infect Dis). In these patients, an increase of IgG levels compared to previous serological results were observed; furthermore, the Toxoplasma-PC . performed were negative, and these patients did not develop any clinical signs of toxoplasmosis (Fricker-Hidalgo H et al., 2009, Clin Infect Dis). Seroconversion during pregnancy was objectified by the appearance of anti-Toxoplasma antibodies or by a significant increase in IgG levels between 2 consecutive sera (Villard O et al., 2016, Diagn Microbiol Infect Dis). Congenital toxoplasmosis in children has been confirmed by the presence of IgM, neosynthesized antibodies or persistence of IgG beyond one year.

[0244] Results

[0245] Identification of Immunogenic Markers in Bradyzoite Proteins Enriched from MORC Knockdown Extracts.

[0246] MORC collaborates with histone deacetylase HDAC3 in tachyzoites to limit chromatin access to the transcription machinery at genes that are expressed exclusively during the sexual and chronic stages (Antunes AV et al., 2023, Nature; Farhat D.C et al., 2020, Nat Microbiol). Depletion of MORC results in significant transcriptional changes, leading to the expression of a large repertoire of bradyzoite-specific genes, encompassing up to 50% of the chronic sub-transcriptome. Inhibition of HDAC3 by the HD AC inhibitor FR235222 phenocopies the depletion of MORC, inducing a transition from tachyzoites to bradyzoites (Farhat D.C et al., 2020, Nat Microbiol, Bougdour A et al., 2009, J Exp Med). The identification of new immunogenic bradyzoite proteins has been impeded by the challenge associated with cultivating large quantities of these specialized zoites in vitro. However, these obstacles related to insufficient starting materials can now be addressed through the overexpression (e.g. BFD1; (Waldman. B.S et al., 2020, Cell)) or suppression (e.g. MORC / HDAC3; (Farhat D.C et al., 2020, Nat Microbiol)) of genetic regulators that control bradyzoite development. Proteomic analysis following MORC knockdown indicated that the strong mRNA expression of the bradyzoite genes was mirrored by an increased presence of the corresponding proteins (Farhat D.C et al., 2020, Nat Microbiol), including BAG1 and BCLA / MAG2, a hallmark of bradyzoites and latent tissue cysts, respectively (Data not shown). We hypothesized that reducing MORC levels could be an effective strategy for identifying previously unknown immunogenic bradyzoite markers. Consequently, we examined the overall immunoreactivity in sera from chronically infected mice, which tested positive for BCLA (Dard C et al., 2021, BMC Biol), across fractions derived fromfive conventional chromatographic steps used to separate and concentrate MORC-depleted protein extracts (Data not shown). Despite substantial immunological responses, only the decrease in MORC levels exposed a specific protein band, approximately 80 kDa in size, which was consistent across various genetic backgrounds of the parasite in the sera of chronically infected mice (Data not shown). Following precipitation with 30 % w / v ammonium sulfate, the MORC-depleted protein extract underwent further separation through gel filtration chromatography and spin filtration using a Centricon column with a 30 kDa cut-off (Data not shown). Silver stain analysis of the final chromatographic step showed a nearly homogeneously purified polypeptide(s) with a size of 80 kDa (Data not shown), which elicited a strong and specific reaction with sera from chronically infected mice (Data not shown). Mass spectrometry (MS)-based proteomic analysis confidently identified the proteins TGME49 216140 and TGME49 202020, based on their abundance, predicted molecular weight of 62 and 83 kDa, respectively, and their restricted expression during the bradyzoite stage (Data not shown).

[0247] Notably, both proteins were previously reported to be up-regulated in MORC-depleted cells (F arhat D.C et al., 2020, Nat Microbiol).

[0248] TGME49 202020, a Bradyzoite-Expressed Protein with Immunogenic Properties.

[0249] To better document the silencing of their expression in tachyzoites by MORC, we endogenously Flag-tagged TGME49 216140 and TGME49 202020 in the RH MORC-mAID-HA KD lineage. As anticipated, treatment of these edited parasites with IAA led to the near elimination of MORC, followed by the accumulation of the FLAG-tagged version of the TGME49 216140 and TGME49 202020 chimeric proteins (Data not shown). Both proteins were then immunopurified from MORC-depleted extracts using anti-Flag affinity chromatography, resolved by SDS-PAGE, and analyzed by Western blot to assess their specific humoral response to infected mouse sera. While TGME49 216140 was clearly nonimmunogenic (Data not Shown), TGME49_202020 was specifically detected by sera from mice chronically infected with type II strains that carry brain cysts (ME49 and 76K), but not by sera from NMRI mice infected with CTG, a type III strain characterized by a low cyst load at the limit of detection (Data not shown) (Dard C et al., 2021, C Biol). Given its restricted expression to bradyzoites (Data not shown) and its reactivity with sera from mice with brain cysts, we henceforth refer to TGME49_202020 to as the Bradyzoite Serological Marker (BSM).

[0250] This protein has also been independently identified as DnaK-TPR, referring to its heat shock protein (DnaK) and tetratri copeptide repeat (TPR) domains (Ueno A et al., 2011, Exp ParasitoT) (Data not shown). The structure of BSM, as predicted by AlphaFold2 and sourced from the EBI / AlphaFold repository, features the previously mentioned DnaK and TPR domainsintricately fold against each other (Data not shown), suggesting potential allosteric regulation of client protein binding by the TPR domain. The closest experimentally determined structural homologue to the DnaK domain is a bacterial Hsp70 from E. coli K12, as identified by the Foldseek structural search engine (Kempen M et al., 2023, Nat Biotechnol) with a Tm score of 0.71 (a Tm score above 0.5 indicates significant homology), despite only 10.5% sequence homology. The TPR domain, on the other hand, partially aligns with segments of human FKBP38 with a Tm score of 0.55 and 7.3% sequence identity. Overall, these structural predictions confidently suggest that this DnaK-TPR protein functions as an ATP-dependent chaperone, potentially involved in bradyzoite-specific protein folding pathways.

[0251] BSM Accumulates in the Cytoplasm of Bradyzoites Converted In Vitro or Found in Brain Cysts from Mice.

[0252] To further investigate the subcellular location of BSM, we have developed polyclonal antibodies against the protein. Following MORC -depletion or HDAC3 inhibition, BSM was detected exclusively in the cytoplasm of in vitro induced bradyzoites (Data not shown), as evidenced by a clear accumulation of Dolichos biflorus agglutinin (DBA) lectin at the MORC-depleted vacuoles, probably as a consequence of the induction of CST1-SRS44 - a gene known to encode a cyst wall protein that is responsible for the reactivity of DBA (Tomita T et al., 2013, PLOS Pathogens). No BSM was detected in the 76K strain treated with FR235222, which was genetically modified to lack BSM (Nbsm), confirming the specificity of our in-house antibody (Data not shown). Meanwhile, the expression of BCLA in the vacuolar space and at the parasite vacuolar membrane (PVM) remained unaffected by the absence of BSM when induced by FR235222 (Data not shown). Considering that full maturation of bradyzoites and complete cyst biogenesis of T. gondii cannot be achieved in vitro (Milligan-Myhre K et al., 2016, Mol Microbiol), we also examined the presence of BSM in histological brain sections from mice chronically infected with a cystogenic strain and confirmed the cytoplasmic localization of BSM in fully differentiated bradyzoites (Data not shown).

[0253] / ISV / -deficient Parasites Exhibit no Growth Defect in vitro and Maintain Chronic Infection Capability in Mice.

[0254] / iS'A7-deficient tachyzoites were able to grow and form plaques in vitro similarly to the parental strain (Figure 1), aligning with a fitness score of -0.12 (source ToxoDB). The consequences of BSM genetic deletion were further assessed in vivo by intraperitoneal inoculation of 5.104tachyzoites of the 76K Nbsm parasite line into 8 NMRI mice, alongside a control group of 8 NMRI mice infected with the 76K wild-type strain. Both groups exhibited similar clinical symptoms such as weight loss and ruffled fur, but ultimately survived infection, indicating thatthere is no significant difference in virulence between the strains over a two-month period (Mantel-Cox test and Gehan-Breslow-Wilcoxon test p=0.3496) (Figure 2A). All mice, except one that was excluded from the study, demonstrated a strong adaptive immune response as indicated by IgGII Western blots (LDBIO Diagnostics, Lyon, France)) (Data not shown).

[0255] Cystogenesis remained unaffected in mice challenged with / iS'Af-deficient parasites, with cysts displaying a normal round morphology consistent with that of the parental strain (Data not shown). However, parasite load was significantly higher in the brains of mice infected with 76K ABSM (Figure 2B), corroborated by increased expression levels of miR-146a and miR-155 (Figure 2C), two host microRNAs linked to Toxoplasma persistence in the mouse brain (Cannella D et al., 2014, Cell Rep . This elevated parasite load was likely missed by Yang et al. (Yang J et al., 2017, Front Microbiol) due to their earlier termination of the experiment at 30 days, as opposed to the 2-month period in our study. These findings suggest that BSM may function as a pro-host effector that limits cyst burden.

[0256] Bradyzoite Serology Using BSM and BCLA Proxies Accurately Identifies Mice Harboring Cysts.

[0257] To confirm the selective immunogenicity of BSM, we produced the recombinant BSM protein tagged with a C-terminal histidine tag using the baculovirus expression system. Unlike expression in prokaryotic cells, eukaryotic systems produce proteins surface post-translational modifications that more closely mimic those of the native protein (Chambers A.C et al., 2018, Curr Protoc Protein Sei). Additionally, they offer the benefit of co-purifying fewer immunogenic contaminants typically associated with E. coli-^asQ expression strategies. After affinity purification on Ni-NTA resin (Data not shown) and size exclusion chromatography, we obtained highly purified recombinant BSM protein in substantial quantities (Figure 4A), allowing us to assess its immunogenicity. Concurrently, to improve our original ELISA assay against BCLA (Dard C et al., 2021, BMC Biol), we expressed a new internally truncated version of BCLA that includes all the immunogenic regions of the protein in insect cells (Figure 4B). This revised version of the recombinant protein is natively soluble, stable and straightforward to purify, unlike the bacterially produced C-terminus, which required partial denaturation for solubilization. It includes both the N-terminal start, a minimized immunogenic repeat regions with two full repeats, and the predicted structured C-terminus. The total predicted molecular weight of the protein is 75.6 kDa (Figures 4B, 4C). Previously, ELISAs were performed with a partial fragment of BCLA produced in bacteria alongside synthetic peptides (Dard C et al., 2021, BMC Biol),- this update has standardized the antigen production process and minimized non-specific contaminants (Figure 4B). Subsequently, an ELISA assay was developed and used to measure anti-BSM andanti-BCLA antibody titers in sera from 83 NMRJ, CD1 and Balb / c mice, either uninfected or chronically infected with different parasite strains (Figures 2D, 2E).

[0258] BSM serology specifically identified mice infected with cystogenic strains (76K and ME49) that exhibited positive cerebral parasite loads, except for mice infected with the 76K ABSM strain, underscoring the specificity of the assay (Figure 2E). In contrast, mice infected with strains characterized with low cystogenic potential (culture-attenuated PruA T / SO and CTG) and had very low or undetectable cerebral parasite loads exhibited negligible or no anti -BSM titers (Figure 2D). Similarly, BCLA serology effectively identified cyst-bearing mice, including those infected with the 76KABSM strain but not those infected with noncystogenic strains (Figure 2E). However, the performance of BCLA antigen in discriminating cyst carriage was slightly reduced compared to the BSM antigen (area under the ROC curves: 0.9256 (IC95: 0.8651 -0.9861) and 0.9923 (IC95: 0.9765 - 1), respectively, p=0.0365) (Hanley. J. A et al., 1983) (Figure 2F). The ELISA cutoff values were set at 15.85 and 3.286 Ul / ml for BSM and BCLA respectively, optimized for specificity. The sensitivity and specificity of BSM ELISA were 97.96% (IC95: 89.31 - 99.90) and 100.0% (IC95: 86.20 - 100.0), respectively, whereas the BCLA ELISA demonstrated a sensitivity of 76.47% (IC95: 63.24 - 86.00) and a specificity of 100.0% (IC95: 79.61 - 100.0). The agreement between the two serological tests was quantified by a kappa coefficient of 0.736, corresponding to substantial agreement (Landis J.R et al., 1977, Biometrics).

[0259] Bradyzoite-Specific Serology in Human Patients with Different Forms of Toxoplasmosis.

[0260] The next challenge was to determine whether BSM can be validated as a serological biomarker for detecting cysts during chronic infection in humans. To address this, we performed side-by-side ELISA assays to compare the reactivity of BCLA and BSM with human sera from two French toxoplasmosis biobank (Figures 3A, 3B). Initially, we compared bradyzoite serology results between two groups: patients with negative conventional serology (n=134) and those previously identified as immunized (n=222). Although antibody titers can decrease below the detection limit of conventional serology over time in some subjects (Burrells A et al., 2016, Parasit Vectors; Rougier S et al., 2017, Trends Parasilol). it is not clear if this correlates with cyst disappearance. Additionally, positive tachyzoite serology does not necessarily coincide with the presence of cysts. However, it is reasonable to hypothesize that T. gondii cysts are commonly present in individuals with positive conventional serology, and rare or absent in those with negative serology.

[0261] As expected, significantly higher titers of anti-BCLA and anti-BSM antibodies were observed in the “past immunity” group with medians, 25% et 75% percentiles of 9.87, 5.15 and16.9 vs 20.2, 12.6 and 42.2 UI / mL in the “seronegative” and “past immunity” group respectively with BCLA ELISA and 0.945, 0.730 and 1.41 vs 1.30, 0.876 and 2.05 (index ratio) with BSM ELISA (p < 0.0001 for both ELISA tests) (Figure 3A). However, results in the “past immunity” group were widely scattered, with several patients showing low titers (Figures 3A, 3B). Based on the serological results between these two groups, ROC curves were generated for each test to select appropriate thresholds (Figure 3C). The cutoff values were set at 34.52 UI / mL for BLCA and 1.697 (index ratio) for BSM. These thresholds allowed identification of patients with past immunity with a sensitivity and specificity of 31.08% (IC95: 25.36 - 37.45) and 97.76% (IC95: 93.62 - 99.39) for BCLA and 32.43% (IC95: 26.62 - 38.84) and 83.58% (IC95:76.39 - 88.90) for BSM. Subsequently, among patients with past immunity, we isolated the clinical contexts mainly related to cyst reactivation, namely ocular toxoplasmosis (n=23), cerebral toxoplasmosis (n=5), and asymptomatic reactivations detected during the serological screening of immunocompromised patients (n=6). Despite the limited number of patients in these subcategories, the bradyzoite serological titers were significantly higher than those in the seronegative group (Figures 3A, 3B).

[0262] Agreement between the two tests was measured using Cohen’s kappa coefficient, resulting in a value of 0.308, which can be interpreted as “fair agreement” (Landis J.R et al., 1977, Biometrics). While only 17.16% of patients in the seronegative group had at least one positive bradyzoite serological test, this proportion reached 46.84% in the "past immunity" group. This suggests a more pronounced immune response to one marker or the other depending on the individual, rather than a systematic detection of both markers together. Nine patients with clinical conditions typically associated with cyst reactivation had negative serology for both antigens.

[0263] Assessing the Effectiveness of Cyst and Bradyzoite Serology in Differentiating Acute and Past Infections and identifying congenital infections.

[0264] One component of the prevention strategy for congenital toxoplasmosis is maternal serologic screening and, in some countries, regular serologic follow-up of seronegative expectant mothers (Van der Giessen J et al., 2021, Parasite Epidemiol Control). When screening relies on a single sample, and even sometimes with consecutive samples, dating the infection to determine fetal risk can be complex due to the possible persistence of IgM and atypical serologic profiles (Fricker-Hidalgo H et al., 2013, J Clin Microbiol,' Gras L et al., 2004, Epidemiol Infect). Therefore, a key challenge in improving serology is optimizing the determination of the date of infection to distinguish between recent and past infections. Bradyzoite antigen-based serology could be a valuable complementary approach, as these antigens appear later compared to tachyzoite antigens. To investigate the value of bradyzoite serology in dating infection, weevaluated the titers of bradyzoite serologies in 115 iterative sera that allowed dating of infection in 39 pregnant women who seroconverted during pregnancy, and one man presenting with an acute infection. Of these, 74 sera could be accurately dated within 4 months of infection and were included in the "acute infection” group. Anti-BSM and anti-BCLA antibody levels were compared with those of sera from the “past immunity” group, whose IgG titers measured in Architect were above 20 Ul / ml, suggesting a sustained immune response, with the assumption that some of the patients with lower reactivity to tachyzoites could potentially have been infected too long ago and have eliminated their cysts and bradyzoites. Anti-BSM and anti-BCLA antibody titers were significantly different between the " past infection " and " acute infection " groups (p=0.0303 and 0.0009 respectively) (Figure 3D).

[0265] Another challenging issue with current toxoplasmosis diagnostic techniques concerns the diagnosis of congenital toxoplasmosis in children bom to mothers who seroconverted during pregnancy. IgG can be transmitted from the mother, and the absence of IgM at birth does not rule out infection in the child. We therefore assessed the value of BSM and BCLA serology at birth in children bom to mothers who had seroconverted during pregnancy and for whom the diagnosis of congenital toxoplasmosis had been made (N=15) or excluded (N=l 1) based on regular postnatal serological monitoring during their first year. While BSM serology was not significantly different between the 2 groups (p=0.5063), BCLA serology was significantly higher in the congenital toxoplasmosis group (p=0.0077) (Figure 3E).Table 1 : Useful sequences for practicing the invention

[0266]

[0267]

[0268] REFERENCES

[0269] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0270] Alday H and Doggett J. S., 2017, Drug Des Devel Ther, DOI: 10.2147 / DDDT.S60973 Antunes AV et al., 2023, Nature, DOI : 10.1038 / s41586-023-06821-y

[0271] Bigna J.J et al., 2020, Sci Rep, DOI : 10.1038 / s41598-020-69078-9

[0272] Bougdour A et al., 2013, Cell Host Microbe, DOI : 10.1016 / j.chom.2013.03.002 Bougdour A et al., 2009, J Exp Med, DOI : 10.1084 / jem.20082826

[0273] Brenier-Pinchart M.P et al., 2022, J Mol Diagn, DOI : 10.1016 / j.jmoldx.2022.03.009 Burrells A et al., 2016, Parasit Vectors, DOI : 10.1186 / sl3071-016-1610-6

[0274] Cannella D et al., 2014, Cell Rep, DOI : 10.1016 / j.celrep.2014.02.002

[0275] Chambers. A.C et al., 2018, Curr Protoc Protein Sci, DOI : 10.1002 / cpps.47

[0276] Dard C et al., 2016, Trends Parasitol, DOI : 10.1016 / j ,pt.2016.04.001

[0277] Dard C et al., 2018, Expert Rev Anti Infect Ther, DOI : 10.1080 / 14787210.2018.1483721 Dard C et al., 2021, BMC Biol, DOI : 10.1186 / sl2915-021-00959-9

[0278] Delair. E et al., 2008, Am J Ophthalmol, DOI : 10.1016 / j.ajo.2008.06.027

[0279] Dubey J.P et al., 1998, Clin Microbiol Rev, DOI : 10.1128 / CMR.l 1.2.267

[0280] Farhat D.C et al., 2020, Nat Microbiol, DOI : 10.1038 / s41564-020-0674-4

[0281] Fricker-Hidalgo. H et al., 2009, Clin Infect Dis, DOI : 10.1086 / 595709

[0282] Fricker-Hidalgo H et al., 2013, J Clin Microbiol, DOI : 10.1128 / JCM.00169-13 Furtado J.M et al., 2013, Clin Exp Ophthalmol, DOI : 10.1111 / j.1442-9071.2012.02821.x Gras L et al., 2004, Epidemiol Infect, DOI : 10.1017 / s0950268803001948

[0283] Greigert V et al., 2019, mSphere, DOI : 10.1128 / mSphere.00636-19

[0284] Hanley. J.A et al., 1983, Radiology, DOI : 10.1148 / radiology.148.3.6878708

[0285] Johnson H.J et al., 2020, mBio, DOI : 10.1128 / mBio.02164-19

[0286] Kempen M et al., 2023, Nat Biotechnol, DOI : 10.1038 / s41587-023-01773-0

[0287] Kijlstra A et al., 2009, Trends Parasitol, DOI : 10.1016 / j.pt.2008.09.008

[0288] Landis J.R et al., 1977, Biometrics, PMID : 843571

[0289] Livak. K.J et al., 2001, Methods, DOI : 10.1006 / meth.2001.1262

[0290] Milligan-Myhre K et al., 2016, Mol Microbiol, DOI : 10.1111 / mmi.l3538

[0291] Reischl U et al., 2003, BMC Infect Dis, DOI : 10.1186 / 1471-2334-3-7Robert. M.G et al., 2021, Expert Rev Anti Infect Ther, DOI : 10.1080 / 14787210.2021.1941867 Robert-Gangneux F et al., 2018, Emerg Infect Dis, DOI : 10.3201 / eid2408.180045 Rougier S et al., 2017, Trends Par asitol, DOI : 10.1016 / j ,pt.2016.10.007

[0292] Sangare L.O et al., 2016, Nat Commun, DOI : 10.1038 / ncommslll91

[0293] Seeber, F, 2023, IJMM, DOI : 10.1016 / j.ijmm.2023.151592

[0294] Stelzer. S et al., 2019, Food Waterborne Parasitol, DOI : 10.1016 / j.fawpar.2019.e00037 Tomita T et al., 2013, P LOS Pathogens, DOI : 10.1371 / journal.ppat.1003823

[0295] Ueno A et al., 2011, Exp Parasitol, DOI : 10.1016 / j. exppara.2011.01.015

[0296] Van der Giessen J et al., 2021, Parasite Epidemiol Control, DOI : 10.1016 / j.parepi.2021.e00205 Villard O et al., 2016, Diagn Microbiol Infect Dis, DOI : 10.1016 / j. diagmicrobio.2015.09.009 Waldman. B.S et al., 2020, Cell, DOI : 10.1016 / j. cell.2019.12.013

[0297] Watts E et al., 2015, mBio, DOI : 10.1128 / mBio.Ol 155-15

[0298] Yang J et al., 2017, Front Microbiol, DOI : 10.3389 / fmicb.2017.02210

[0299] Ybanez R.H.D et al., 2020, Front Cell Infect Microbiol, DOI : 10.3389 / fcimb.2020.00204 Zhang Y.W et al., 1995, J Clin Pathol, DOI : 10.1136 / jcp.48.10.908

Claims

CLAIMS :

1. An isolated polypeptide selected from the group comprising or consisting of:(i) the amino acids sequence consisting of Toxoplasma gondii polypeptide BSM (SEQ IDNO :1) ;(ii) an amino acid sequence substantially homologous to the sequence of (i) preferably an amino acid sequence at least 80% identical to the sequence of (i);(iii) a fragment of at least 9 consecutive amino acids of the sequence of (i), or (ii).

2. A method for detecting the polypeptide according to claim 1 and / or antibodies against the polypeptide according to claim 1, and / or evaluating its amount in a biological sample.

3. A method for detecting bradyzoite cyst, and / or evaluating in a subject, wherein said method comprises:a) detecting in a biological sample of the subject the polypeptide according to claim 1 or the immunoreactivity toward the polypeptide according to claim 1; and optionally b) deducing from the result of step a) the presence and / or amount of bradyzoite cyst, polypeptide according to claim 1 or immunoreactivity toward the polypeptide according to claim 1 is indicative of presence and / or amount of bradyzoite cyst in said subject.

4. The method according to claim 3 wherein the biological sample is a meat intended for consumption.

5. The method according to claim 3 which comprises contacting the biological sample with a binding partner.

6. The method according to claim 5 wherein the binding partner is an antibody.

7. A method for in vitro diagnosing a Toxoplasmosis, wherein said method comprising detecting the presence of polypeptide, according to claim 1, and / or antibodies against the polypeptide according to claim 1, in a biological sample from a subject to be tested.

8. A method of determining if a subject is afflicted with a latent form of Toxoplasmosis, said method comprising:a) detecting in a biological sample of the patient polypeptide, according to claim 1, and / or immunoreactivity toward a polypeptide according to claims 1; and optionallyb) deducing from the result of step a) whether the patient is afflicted with latent form of Toxoplasmosis, polypeptide, according to claim 1, and / or immunoreactivity toward a polypeptide according to claim 1 is indicative of latent form of Toxoplasmosis.

9. The methods according to claims 2 to 8 wherein the biological sample is a fluid sample or a tissue sample.

10. The methods according to claims 2 to 8 wherein the biological sample is a blood sample, a muscle sample or a brain sample.

11. A method for treating a patient infected with latent form of Toxoplasmosis who shows immunoreactivity toward the polypeptide according to claim 1 comprising administering to the patient folic acid antagonist (i.e. Pyrimethamine) and / or antibiotic compound (i.e. Sulfadiazine or spiramycin), or a pharmaceutical composition comprising said compounds.

12. A method for detecting bradyzoite cyst according to claim 3 to 6 wherein said method comprises further step :a) detecting in a biological sample of the subject a BCLA polypeptide or the immunoreactivity toward a BCLA polypeptide; and optionallyb) deducing from the result of step a) the presence and / or amount of bradyzoite cyst, BCLA polypeptide or immunoreactivity toward BCLA polypeptide is indicative of presence and / or amount of bradyzoite cyst in said subject.

13. A method for in vitro diagnosing a Toxoplasmosis according to claim 7, 9 and 10, wherein said method further comprising detecting the presence of BCLA polypeptide, / or antibodies against the BCLA polypeptide, in a biological sample from a subject to be tested.

14. A method of determining if a subject is afflicted with a latent form of Toxoplasmosis, according to claim 8 to 10 said method further comprising:a) detecting in a biological sample of the patient BCLA polypeptide and / or immunoreactivity toward a BCLA polypeptide according to claims 1; and optionallyb) deducing from the result of step a) whether the patient is afflicted with latent form of Toxoplasmosis, BCLA polypeptide and / or immunoreactivity toward a BCLA polypeptide is indicative of latent form of Toxoplasmosis.

15. The method for according to claim 12 to 14 wherein BCLA polypeptide used to detect immunoreactivity toward a BCLA polypeptide, is BCLA polypeptide of SEQ ID NO : 4.

Images