New serological markers for latent toxoplasmosis

Abstract

In this invention, the inventors report the characterization of BCLA (brain cyst-loaded antigen), a protein uniquely expressed in the merozoite stage of a parasite. In cysts directly purified from mouse brain, the protein is distributed both internally and on the surface of the cyst. ELISA antibody capture using a combination of serologically reactive BCLA peptide and recombinantly expressed C-terminal domain (rBCLA) constitutes an effective serological marker of latent infection with high sensitivity, definitively and uniquely correlated with the presence of cysts in mouse brain. Antibodies against the BCLA antigen have been detected in human patients. Enrichment titers were detected in patients identified as seropositive for Sag1 or tachyzoite-associated antigen. Further correlation between anti-BCLA IgG synthesis and cysts in humans was demonstrated by significantly stronger recorded titers in the pathology group strongly correlated with the presence of cysts. Furthermore, newborns with confirmed congenital toxoplasmosis showed significantly higher levels of anti-BCLA IgG at birth compared to their mothers, indicating specific intrauterine synthesis of this IgG. Therefore, this invention relates to a novel Toxoplasma gondii protein, hereinafter referred to as BCLA, which is a novel serological marker whose expression is limited to latent toxoplasmosis (schizonts / cysts). This specific protein and its antigenic fragments can be used to detect autoantibodies in patient serum for the diagnosis of latent toxoplasmosis. This invention also relates to derived antibodies that specifically bind to this novel protein, generated through immunization with BCLA.

Description

Technical Field

[0001] This invention relates to a novel *Toxoplasma gondii* protein, hereinafter referred to as BCLA (brain cyst-loaded antigen), a novel serological marker whose expression is limited to latent toxoplasmosis (merozoites / cysts). The invention also relates to antibodies that specifically bind to this novel protein. This specific protein and its antigenic fragments can be used to detect autoantibodies in patient serum for the diagnosis of latent toxoplasmosis. Background Technology

[0002] The ancient phylum Apicomplexa includes many of the world's most prevalent protozoan pathogens. The most deadly to humans is Plasmodium, the pathogen of malaria, which kills nearly 500,000 people annually. Toxoplasma gondii, the pathogen of toxoplasmosis, is one of the most common protozoan parasites in livestock, wild animals, and companion animals. Toxoplasmosis is a common foodborne infection in humans, causing a serious public health problem and is considered a leading cause of foodborne death in the United States (Scallan et al., 2015). Toxoplasmosis is often a mild illness in immunocompetent individuals, but it can become a major threat to immunocompromised patients experiencing life-threatening brain, lung, heart, or disseminated symptoms. Transplacental infection can lead to congenital infections with varying degrees of clinical presentation, ranging from congenital abnormalities (such as hydrocephalus, microcephaly, and intracranial calcification) to miscarriage.

[0003] The cyst-forming enterococcal parasite *Toxoplasma gondii* is transmitted through an alternating two-host lifecycle, which depends on sexual transmission in a feline deterministic host and asexual transmission in a variety of alternative hosts, including rodents and humans. Long-term inhabitants of non-feline warm-blooded metazoans, *Toxoplasma gondii* initiates a complex developmental program in response to environmental factors, including innate host defenses and adaptations to different hosts. During initial infection in an intermediate host, the parasite replicates as a tachyzoite, which proliferates significantly in number before spreading to numerous tissues within the body. Although initial infection is generally controlled by an effective Th1-mediated pro-inflammatory host response leading to the large-scale destruction of the tachyzoite population, smaller subpopulations of tachyzoites differentiate into a slowly growing merozoic stage, which persists throughout the host's lifespan within tissue cysts residing in long-lived cells, including neurons and skeletal muscle cells (Dubey, 1997). The feline deterministic host completes this cycle by taking up tissue cysts, resulting in the shedding of highly infectious oocysts (Dubey, 2001).

[0004] Tissue cysts are a major source of human infection via tachyzoa and are therefore a key contributing factor to human disease, as complications of toxoplasmosis enhance the ability to cause irreversible damage to merozoites while differentiating back to the replicating tachyzoite stage. Indeed, although asymptomatic parasites provide lifelong equilibrium and protection in immune-competent hosts, persistent immune dysfunction is known to disrupt parasite dormancy, promote merozoite-tachyzoite transition, and further tachyzoite population expansion. These combined processes ultimately lead to encephalitis, pneumonia, retinochoroiditis, or even disseminated toxoplasmosis as major outcomes in immunocompromised individuals (Dard et al., 2018). Thus, the strategy of *Toxoplasma gondii* as an obligate intracellular parasite is based on the pursuit of non-toxicity, i.e., weakening but not completely negating the host's innate immune response to infection, thereby ensuring the permanent colonization required for dissemination.

[0005] Despite the importance of tissue cysts in the life cycle of *Toxoplasma gondii* and their crucial role as a reservoir for toxoplasmosis reactivation in immunocompromised hosts, little is known about the biology of the merozoites and cysts they form. Cysts are thought to grow and spread over time to maintain chronic infection, not through an intermediate tachyzoite stage, but through the migration of free merozoites and the division of merozoite cysts (Dzierszinski et al., 2004; Frenkel and Escajadillo, 1987). The idea that merozoites within tissue cysts are dormant entities has recently been challenged by compelling evidence showing that merozoites exhibit periodic, intermittent growth within tissue cysts in vivo through asynchronous replication (Watts et al., 2015), while simultaneously using endogony and endoregenesis (Dzierszinski et al., 2004).

[0006] The developmental transition from tachyzoites to merozoites is bidirectional and characterized by a dramatic change in parasite gene expression, leading to major metabolic alterations, remodeling of the parasite surface with restricted expression of stage-specific surface antigens, and cyst wall formation. The latter is thought to protect merozoites from harsh gastrointestinal conditions and may provide a physical barrier for host immune defense. The differentiation of *Toxoplasma gondii* has been difficult to study because the stage transition is controlled by a complex and still unknown developmental genetic program, but is also influenced by host cellular physiology (Lueder and Rahman, 2017). In the laboratory, in the absence of host immunity in vitro, the conversion from tachyzoites to merozoites can be triggered by exogenous stress (e.g., alkaline stress, nutritional deprivation, and drugs).

[0007] Transcriptional regulation clearly plays a crucial role in merozoite development, as demonstrated by numerous studies showing stage-specific gene expression. How these changes are regulated at the molecular level remains largely unknown; however, we and others have presented strong evidence that epigenetic changes are drivers of parasite differentiation. Early evidence came from the observation that tachyzoites that rapidly recover from infection in mice during in vivo are particularly prone to differentiation and gradually lose this “start-up” state over time. Consequently, long passages of tachyzoites in tissue culture significantly diminish the ability of type II strains to produce high cyst loads in vivo. Thus, epigenetic mechanisms that promote developmental plasticity—i.e., the expression of multiple phenotypes from the same genome—allow parasites to adapt to thousands of potential intermediate hosts and respond to significantly different immune systems.

[0008] *Toxoplasma gondii* has evolved sophisticated ways to promote epigenetic alterations, such as changes in histone marker activity and chromatin remodeling, which compete with the strategies employed by the cells they infect and provide germlines with a significant ability to undergo staged differentiation in response to environmental factors or as part of a developmental program. Our earlier focus on histone post-translational modification (PTM)-specific acetylation (Saksouk et al., 2005) led us to demonstrate that altered rates of histone H4 acetylation near stage-specific genes are one of the epigenetic molecular motors driving parasite differentiation (Bougdour et al., 2009). Acetylation of core histones is mediated by histone acetyltransferases (HATs) and, in many cases, leads to chromatin relaxation and transcriptional activation of related genes. Histone deacetylases (HDACs) counteract HAT activity by catalyzing the removal of the acetyl moiety from lysine residues at the histone tail, thereby inducing chromatin condensation and transcriptional repression (Kurdistani and Grunstein, 2003).

[0009] The importance of histone acetylation in controlling differentiation is highlighted by the following findings: chemical inhibition of TgHDAC3-induced tachyzoan to merozoan transformation in vitro with low doses of the compound FR235222 (Bougdour et al., 2009; Maubon et al., 2010). Recombinant strains transfected with TgHDAC3 alleles resistant to this compound did not exhibit these effects, confirming the TgHDAC3 specificity of the compound and indicating that TgHDAC3 activity actively prevents merozoan differentiation (Bougdour et al., 2009). In vitro transformation was accompanied by hyperacetylation of the upstream regions of >350 genes, one-third of which were merozoan-specific (Bougdour et al., 2009). TgHDAC3 appears to primarily antagonize the effects of HAT TgGCN5b, which locates to the promoters of active genes via ChIP, while TgHDAC3 locates to the promoters of merozoan genes via ChIP (Saksouk et al., 2005). While these data represent a step toward understanding the causal relationship between histone acetylation and gene expression in *Toxoplasma gondii* and point to the crucial role of TgHDAC3 in stage transitions, they were only obtained in virulent *RH* strains that do not readily form tissue cysts or exhibit latent infection in laboratory mice. Finally, further development of novel diagnostics for latent toxoplasmosis remains necessary.

[0010] In this study, the inventors re-examined the ability of FR235222 to stimulate the transformation of tachyzoites into merozoites in vitro using strains of type II origin that readily form cysts in vivo. Quantitative analysis of the Toxoplasma gondii proteome in response to FR235222 revealed many proteins previously identified as stage-specific, including those thought to be confined to merozoites. Due to their potential importance to parasite biology (Hakimi et al., 2017), the inventors chose to focus their attention on predicting novel proteins to be secreted. ~200 putative effectors secreted by FR235222-responsive merozoites were identified using this method. A candidate, BCLA (brain cyst-load-associated antigen), was selected for further investigation. BCLA is expressed only after treatment with FR 235222 and shows accumulation at the parasitic vesicle membrane (PVM) after its secretion in the vacuolar space. Under in vivo conditions, BCLA is located in both the matrix space of the cyst and the cyst wall, the latter believed to originate from the PVM during the latent phase. In assessing its function, the inventors found that BCLA deficiency affected the integrity of brain cysts isolated from chronically infected mice; however, at least in our chronic toxoplasmosis mouse model, the protein was not essential for proper cyst function.

[0011] Given the restricted expression of BCLA by merozoites and its location in the cyst wall, the inventors next explored its potential applications in serological diagnostics. Here, the inventors discovered that the C-terminal peptide of BCLA produced via recombinant technology is highly antigenic and constitutes an excellent antigenic candidate for detecting anti-Toxoplasma gondii IgG in chronically infected mice. The inventors provided strong data demonstrating a clear correlation between the presence of cysts in the brains of chronically infected mice and the detection of antigenic BCLA in serum. Positive assays using human serum confirmed the antigenic characterization of BCLA and pave the way for the use of this antigen in anti-Toxoplasma gondii diagnostics with the interesting perspective of serological detection of cyst load in chronically infected hosts. Invention Overview

[0013] This invention provides isolated Toxoplasma gondii polypeptide, hereinafter referred to as BCLA (brain cyst-loaded antigen), which comprises amino acid SEQ ID NO: 1 and an immunogenic peptide fragment.

[0014] The present invention also relates to antibodies generated against the isolated polypeptides of the present invention.

[0015] The present invention also relates to methods for detecting Toxoplasma gondii polypeptides according to the present invention, and / or assessing their amount in biological samples, particularly in solid samples.

[0016] The present invention also relates to a method for diagnosing latent toxoplasmosis using peptides according to the invention, for detecting anti-BCLA antibodies in biological samples, particularly fluid samples. Invention Details

[0018] By modulating tachyzoite genome expression using epigenetic drugs, the inventors were able to identify genes whose expression is limited to merozoites. In this invention, the inventors report the characterization of the protein BCLA (brain cyst-load-associated antigen), which, when expressed under merozoite-induced conditions, accumulates in vitro on parasitic vesicle membranes. In the mouse brain, the protein is dispersed within and on the surface of cysts. Deletion of the gene results in a reduced cyst load in the mouse brain, with the remaining cysts characterized by a range from loss of roundness to deformation of the wall surface in a distinctive budding phenotype. Finally, when synthesized as a recombinant protein, BCLA constitutes a highly sensitive and effective serological marker of potential infection, definitively and uniquely associated with the presence of mouse brain cysts. Using the first ELISA BCLA test developed by the inventors, antibodies against the BCLA antigen have been detected in patients with strongly suspected or confirmed ocular toxoplasmosis, detected only in serum or in both serum and aqueous humor. Serological assays have long been a first-line test for confirming Toxoplasma gondii infection, but current serological diagnoses do not always differentiate between acute, latent, and reactivated disease states. Furthermore, current serological methods do not assess cyst load in tissues and the subsequent risk of toxoplasmosis reactivation in seropositive, immunocompromised patients. Some of these limitations can now be overcome by the discovery of BCLA, an important antigen candidate for serological detection of cysts in chronically infected hosts.

[0019] The first ELISA assay was optimized to detect BCLA immunogenic peptides. First, peptide dot blot screening was used to screen for high-resolution BCLA epitope mapping of a peptide microarray designed using the BCLA C-terminal domain and the most conserved internal peptide repeat sequence TgR4 (Fig. 12a) (Fig. 12b and Fig. 12c). In contrast to mice, all positive human sera showed strong reactivity to peptides derived from the internal repeat sequence, which significantly increased assay sensitivity once added to rBCLA. Therefore, the BCLAE LISA was customized based on the most sensitive peptide and polypeptide combination, and it was shown to be optimized for high-confidence differentiation between individuals diagnosed with ocular toxoplasmosis or with confirmed past-immunity. Figure 13 The ELISA test also detected significant amounts of circulating anti-BCLA antibodies in the serum of immunocompromised patients who experienced asymptomatic or symptomatic episodes of chronic toxoplasmosis. Figure 13 ).

[0020] isolated peptides

[0021] This invention relates to isolated Toxoplasma gondii polypeptides, known as BCLA (brain cyst-loaded antigen), which contain amino acid SEQ ID NO: 1.

[0022] This invention also provides isolated Toxoplasma gondii polypeptides selected from the following group:

[0023] (i) The amino acid sequence of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1);

[0024] (ii) An amino acid sequence consisting of the C-terminal antigenic domain (residues 1089-1275 of BCLA, referred to as rBCLA) (SEQ ID NO: 2);

[0025] (iii) An amino acid sequence consisting of an internal repeating domain selected from the following group of BCLA: TgR1 (SEQ ID NO:4), TgR2 (SEQ ID NO:5), TgR3 (SEQ ID NO:6), TgR4 (SEQ ID NO:7), TgR5 (SEQ ID NO:8), TgR6 (SEQ ID NO:9), TgR7 (SEQ ID NO:10), TgR8 (SEQ ID NO:11), TgR9 (SEQ ID NO:12), TgR10 (SEQ ID NO:13), TgR11 (SEQ ID NO:14), TgR12 (SEQ ID NO:15), and TgR13 (SEQ ID NO:16);

[0026] (iv) An amino acid sequence that is substantially homologous to the sequences of (i)-(iii), preferably an amino acid sequence that is at least 80% identical to the sequences of (i)-(iii);

[0027] A fragment of at least 9 consecutive amino acids of the sequence (v)(i)-(iv).

[0028] Peptide dot blot screening (see Figure 12) allows the identification of the most potent BCLA immunogenic peptides in the C-terminal antigenic domain of BCLA (residues 1089-1275 of BCLA, referred to as rBCLA) and in the internal repeating domains of BCLA (residues 304-924) referred to as TgR1 to TgR13 (SEQ ID NO: 4-SEQ ID NO: 16).

[0029] Therefore, in one specific implementation, the Toxoplasma gondii peptide isolated from the rBCLA peptide is selected from the group consisting of:

[0030] (i)GELQPAEAEEARLLVADLKAV(SEQ ID N°32)

[0031] (ii)VRVEGEAFFRASVDLYEA(SEQ ID N°33)

[0032] (iii)KLRPLTKGELVDVVRQ(SEQ ID N°34)

[0033] (iv)TQIFVQDRASAFLRV (peptide 36 of rBCLA) (SEQ ID NO. 35)

[0034] (v)AAEQMKAVFAMVEEG (peptide 44 of rBCLA) (SEQ ID N°36)

[0035] (vi) is an amino acid sequence that is substantially homologous to the sequence of (i)-(v), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(v);

[0036] A fragment of at least 9 consecutive amino acids of the sequence (vii)(i)-(vi).

[0037] In a more specific implementation, the Toxoplasma gondii peptide isolated from the rBCLA peptide is selected from the group consisting of:

[0038] (i) GELQPAEAEEARLLV (peptide 12 of rBCLA) (SEQ ID N°37);

[0039] (ii)QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO38),

[0040] (iii) EAEEARLLVADLKAV (peptide 14 of rBCLA) (SEQ ID NO39),

[0041] (iv)VRVEGEAFFRASVDL (peptide 21 of rBCLA) (SEQ ID NO.40)

[0042] (v)EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID N°41);

[0043] (vi)AFFRASVDLYEAVKN(peptide 23 of rBCLA) (SEQ ID NO42),

[0044] (vii)KLRPLTKGELVDVVR(peptide 30 of rBCLA) (SEQ ID NO.43)

[0045] (viii) is an amino acid sequence that is substantially homologous to the sequence of (i)-(vii), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(vii);

[0046] A fragment of at least 9 consecutive amino acids of the sequence (vii)(i)-(viii).

[0047] Therefore, in one specific implementation, the Toxoplasma gondii polypeptide isolated from the internal repeating domain of BCLA is selected from the group consisting of:

[0048] (i) An amino acid sequence consisting of the internal repeating domains of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7);

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

[0050] A fragment of at least nine consecutive amino acids of the sequence (iii)(i)-(ii).

[0051] In a more specific implementation, the Toxoplasma gondii polypeptide isolated from the internal repeating domain of BCLA is selected from the following group:

[0052] (i)AAGSMEKEKPVLPGEGEGH(TgR4 domain A); (SEQ ID N°44)

[0053] (ii)VLPKHETKPALTDEKRTKPGGP(TgR4 domain B), (SEQ ID N°45)

[0054] (iii) An amino acid sequence substantially homologous to the sequences of (i)-(ii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i)-(ii);

[0055] A fragment of at least nine consecutive amino acids of the sequence (iv)(i)-(iii).

[0056] In a more specific implementation, the Toxoplasma gondii polypeptide isolated from the internal repeating domain of BCLA is selected from the following group:

[0057] (i) AAGSMEKEKPVLPGE (peptide 3 of TgR4); (SEQ ID N°46)

[0058] (ii) GSMEKEKPVLPGEGE (peptide 4 of TgR4) (SEQ ID N°47)

[0059] (iii) MEKEKPVLPGEGEGH (peptide 5 of TgR4) (SEQ ID N°48)

[0060] (iv) KEKPVLPGEGEGHVL (peptide 6 of TgR4) (SEQ ID NO.49)

[0061] (v)KPVLPGEGEGHVLPG (peptide 7 of TgR4) (SEQ ID N°50)

[0062] (vi)HVLPKHETKPALTDEK (peptide 13 of TgR4), (SEQ ID N°51)

[0063] (vii)PKHETKPALTDEKRT (peptide 14 of TgR4), (SEQ ID N°52)

[0064] (viii)HETKPALTDEKRTKP (peptide 15 of TgR4) (SEQ ID N°53)

[0065] (ix)TKPALTDEKRTKPGG(peptide 16 of TgR4) (SEQ ID N°54)

[0066] (x) is an amino acid sequence that is substantially homologous to the sequence of (i)-(ix), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(ix);

[0067] A fragment of at least 9 consecutive amino acids of the sequence (xi)(i)-(x).

[0068] Because BCLA peptides have numerous epitopes across different domains (especially in rBCLA and in the internal repeating domains of BCLA TgR1-TgR13), combining the BCLA immunogenic peptide fragments of the present invention can be advantageous.

[0069] Therefore, in another embodiment, the isolated polypeptide of the present invention is a fusion of two peptide fragments according to the present invention.

[0070] For the improved ELISA assay, the following BCLA peptide (a fusion peptide having at least one binding of two internal repeat peptides) is used in combination with the full-length recombinant BCLA polypeptide (SEQ ID N°1).

[0071] Peptide AB_F:MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (a fusion of peptide fragments from repeating motifs present in Tgr4 / Trg12 / Tgr13 and repeating motifs present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID N°55)

[0072] Peptide A3_B:AAGSMEKDKLVLPGE (a peptide fragment derived from a repeating motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID N°56).

[0073] Therefore, the Toxoplasma gondii polypeptides isolated from the internal repeating domains of BCLA were selected from the following group:

[0074] (i)MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (a fusion of peptide fragments from repeating motifs present in Tgr4 / Trg12 / Tgr13 and repeating motifs present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID N°55),

[0075] (ii) AAGSMEKDKLVLPGE (a peptide fragment derived from a repeating motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID N°56)

[0076] (iii) An amino acid sequence substantially homologous to the sequences of (i)-(ii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i)-(ii).

[0077] A fragment of at least nine consecutive amino acids of the sequence (iv)(i)-(iii).

[0078] Because the BCLA polypeptide has a large number of epitopes throughout the different internal repeating domains (TgR1-TgR13) of BCLA, it is advantageous for combining the amino acid residues of the internal repeating domains of BCLA.

[0079] Therefore, the present invention also relates to a BCLA polypeptide comprising a BCLA internal repeating domain (TgRx) having the following sequence:

[0080] M-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5–Xaa6–Xaa7-ME-Xaa8–Xaa9-K-Xaa10-V-Xaa11-PGEG-Xaa12–Xaa13-H-Xaa14-Xaa15 -PK-Xaa16-E-Xaa17-Xaa18-LT-Xaa19-Xaa20-Xaa21-Xaa22-T-Xaa23-P-Xaa24-Xaa25-P-Xaa26-Xaa27-Xaa28(SEQ ID N°64)

[0081] Xaa1 is either glutamic acid (E) or has no amino acid residues.

[0082] Xaa2 is either arginine (R) or serine (S).

[0083] Xaa3 is either proline (P) or glycine (G).

[0084] Xaa4 is either alanine (A) or glycine (G).

[0085] Xaa5 is either alanine (A) or contains no amino acid residues.

[0086] Xaa6 is either glycine (G) or arginine (R).

[0087] Xaa7 can be serine (S), proline (P), or alanine (A).

[0088] Xaa8 is either lysine (K) or glutamic acid (E).

[0089] Xaa9 is either lysine (K), glutamic acid (E), or aspartic acid (D).

[0090] Xaa10 is either proline (P) or leucine (L).

[0091] Xaa11 is either leucine (L) or serine (S).

[0092] Xaa12 is either glutamic acid (E) or lysine (K).

[0093] Xaa13 is either glycine (G) or arginine (R).

[0094] Xaa14 is either valine (V) or alanine (A).

[0095] Xaa15 is either leucine (L) or serine (S).

[0096] Xaa16 can be histidine (H), aspartic acid (D), or alanine (A).

[0097] Xaa17 can be threonine (T), arginine (R), methionine (M), or glutamine (Q).

[0098] Xaa18 can be proline (P), threonine (T), or alanine (A).

[0099] Xaa19 is either aspartic acid (D), glutamic acid (E), or glutamine (Q).

[0100] Xaa20 is either glutamic acid (E) or lysine (K).

[0101] Xaa21 is either lysine (K), glycine (G), or glutamic acid (E).

[0102] Xaa22 is either arginine (R) or valine (V).

[0103] Xaa23 is either lysine (K), glutamic acid (E), or asparagine (N).

[0104] Xaa24 is either glycine (G), valine, or isoleucine (I).

[0105] Xaa25 is either glycine (G) or glutamic acid (E).

[0106] Xaa26 is either arginine (R) or proline (P).

[0107] Xaa27 can be threonine (T), cysteine ​​(C), lysine (K), or methionine (M).

[0108] Xaa28 is either glutamic acid (E) or alanine (A).

[0109] A fragment of at least 9 consecutive amino acids of the sequence SEQ ID N°64.

[0110] As used herein, the term "amino acid" refers to a natural or non-natural amino acid in the D and L stereoisomers of a chiral amino acid. It should be understood to refer to the amino acid and the corresponding amino acid residue, for example, present in the peptide structure. Natural and non-natural amino acids are well known in the art. Common natural amino acids include, but are not limited to, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Uncommon and non-natural amino acids include, but are not limited to, allylglycine, leucine, valine, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), high arginine (hArg), high lysine (hLys), 2-naphthylalanine (2-nal), ornithine (Orn), and pentafluorophenylalanine.

[0111] Amino acids are typically classified into one or more categories based on their side chains, including polar, hydrophobic, acidic, basic, and aromatic. Examples of polar amino acids include those with side-chain functional groups (such as hydroxyl, thiol, and amide groups), as well as acidic and basic amino acids. Polar amino acids include, but are not limited to, asparagine, cysteine, glutamine, histidine, selenocysteine, serine, threonine, tryptophan, and tyrosine. Examples of hydrophobic or nonpolar amino acids include residues with nonpolar aliphatic side chains, such as, but not limited to, leucine, isoleucine, valine, glycine, alanine, proline, methionine, and phenylalanine. Examples of basic amino acid residues include those with basic side chains (such as amino or guanidinium groups). Basic amino acid residues include, but are not limited to, arginine, homolysine, and lysine. Examples of acidic amino acid residues include those with acidic side-chain functional groups (such as carboxyl groups). Acidic amino acid residues include, but are not limited to, aspartic acid and glutamic acid. Aromatic amino acids include those with aromatic side-chain groups. Examples of aromatic amino acids include, but are not limited to, biphenylalanine, histidine, 2-naphthylalanine, pentafluorophenylalanine, phenylalanine, tryptophan, and tyrosine. It should be noted that some amino acids are classified into more than one group; for example, histidine, tryptophan, and tyrosine are classified as both polar and aromatic amino acids. Amino acids can be further classified as uncharged or charged (positively or negatively charged). Examples of positively charged amino acids include, but are not limited to, lysine, arginine, and histidine. Examples of negatively charged amino acids include, but are not limited to, glutamic acid and aspartic acid. Other amino acids classified in the above groups are known to those skilled in the art.

[0112] Peptides that are “substantially homologous” to a reference peptide can be derived from the reference sequence through one or more conserved substitutions. Two amino acid sequences are “substantially homologous” or “substantially similar” when one or more amino acid residues are substituted with biologically similar residues, or when more than 80% of the amino acids are identical, or more than about 90%, preferably more than about 95%, are similar (functionally identical). Preferably, similar, identical, or homologous sequences are identified by alignment using, for example, a GCG (Genetic Computing Group, Manual of Procedure for GCG Package, 7th Edition, Madison, Wisconsin) stacking program or any program known in the art (BLAST, CLUSTAL, FASTA, etc.). Pairwise global alignments based on the Needleman-Wunsch alignment algorithm, such as Needle, and using a BLOSUM62 matrix with a vacancy opening penalty of 10 and a vacancy extension penalty of 0.5, can be used to calculate the percentage of identity to find the best alignment of the two sequences along their full length (including vacancies).

[0113] As used herein, the term "conservative substitution" means that an amino acid residue is replaced by another without altering the overall conformation and function of the peptide, including but not limited to substitution with an amino acid having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, shape, hydrophobicity, aromaticity, etc.). Amino acids with similar properties are well known in the art. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine, a hydrophobic amino acid, can be replaced by leucine, methionine, or valine. Neutral hydrophilic amino acids that can be substituted for each other include asparagine, glutamine, serine, and threonine.

[0114] By "substitution" or "modification", the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.

[0115] Therefore, it should be understood that, in the context of this invention, conservative substitution is considered in the art to be the substitution of one amino acid for another amino acid having similar properties.

[0116] According to the present invention, a first amino acid sequence having at least 80% 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% 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 BLASTP (Karlin and Altschul, 1990).

[0117] In some embodiments, the isolated peptides of the present invention contain up to 1275 amino acids (and at least 9). In some embodiments, the polypeptides of the present invention comprise 1275, 1270, 1265, 1260, 1255, 1250, 1245, 1240, 1235, 1230, 1225, 1220, 1215, 1210; 1205, 1200, 1199, 1198, 1197, 1196, 1195, 1194, 1193, 1192, 1191, 1190, 1189, 1188, 1187, 1186, 1185, 1184, 1183, 1182, 1181, 1180, 1179, 1175, 1174, 1173, 1172, 1171, 1170, 1169, 116 8,1167,1166,1165,1164,1163,1162,1161,1160,1159,1158,1157,1156,1155,1154,1153,1152,1151,1150,1149,1148,1147,1146,1145,1144,1143,1142,1141,1140,1139,1138,1137,1136,1135,1134,1133,1132,1131,1130,1129,1128,1127,1126,1125,1124,1123,1122,1121,1120 ,1119,1118,1117,1116,1115,1114,1113,1112,1111,1110,1109,1108,1107,1106,1105,1104,1103,1102,1101,1100,1099,1098,1097,1096,1095,1094,1093,1092,1091,1090,1089,1088,1087,1086,1085,1084,1083,1082,1081,1080,1079,1078,1077,1076,1075,1074,1073,1072, 1071,1070,1069,1068,1067,1066,1065,1064,1063,10162,1061,1060,1059,1058,1057,1056,1055,1054,1053,1052,1051,1050,1049,1048,1047,1046,1045,1044,1043,1042,1041,1040,1039,1038,1037,1036,1035,1034,1033,1032,1031,1030,1029,1028,1027,1026,1025,1024,1023,1022,1021,1020,1019,1018,1017,1016,1115,1014,1013,1012,1011,1010,1009,1008,1007,1006,1005,1004,1003,1002,1001,1000,999,(….),800,799,798,797,796,795,794,793,792,791,790,789,788,787,786,785,784,783,782,781,780,779,778,777,766,765,764,763,762,761,760,759,758,757,756,755,754,753,752,751,750,749,748,747,746,745,744,743,742,741,740,739,738,737,736,735,734,733,732,731,730,729,728,727,726,725,724,723,722,721,720,719,718,717,716,715,714,713,712,711,710,709,708,707,706,705,704,703,702,701,700,699,698,697,696,695,694,693,692,691,690,689,688,687,686,685,684,683,682,681,680,679,678,677,676,675,674,673,672,671,670,669,668,667,666,665,664,663,662,661,660,659,658,657,656,655,654,653,652,651,650,649,648,647,646,645,644,643,642,641,640,639,638,637,636,635,634,633,632,631,630,629,628,627,626,625,624,623,622,621,620,619,618,617,616,615,614,613,612,611,610,609,608,607,606,605,604,603,602,601,600,599,598,597,596,595,594,593,592,591,590,589,588,587,586,585,584,583,582,581,580,579,578,577,576,575,574,573,572,571,570,569,568,567,566,565,564,563,562,561,560,,559 558,557,556,555,554,553,552,551,550,549,548,547,546,545,544,543,542,541,540,539,538,537,536,535,534,533,532,531,530,529,528,527,526,525,524,523,522,521,520,519,518,517,516,515,514,513,512,511,510,509,508,507,506,505,504,503,502,501,500,499,498,497,496,495,494,493,492,491,490,489,488,487,486,485,484,483,482,481,480,479,478,477,476,475,474,473,472,471,470,469,468,467,466,465,464,463,462,461,460,459,458,457,456,455,454,453,452,451,450,449,448,447,446,445,444,443；442,441,440,439,438,437,436,435,434,433,432,431,430,429,428,427,426,425,424,423,422,421,420,419,418,417,416,415,414,413,412,411,410,409,408,407,406,405,404,403,402,401,400,399,398,397,396,395,394,393,392,391,390,389,388,387,386,385,384,383,382,381,380,379,378,377,376,375,374,373,372,371,370,369,368,367,366,365,364,363,362,361,360,359,358,357,356,355,354,353,352,351,350,349,348,347,346,345,344,343,342,341,340,339,338,337,336,335,334,333,332,331,330,329,328,327,326,325,324,323,322,321,320,319,318,317,316,315,314,313,312,311,310,309,308,307,306,305,304,303,302,301,300,299,298,297,296,295,294,293,292,291,290,289,288,287,286,285,284,283,282,,281,280,279,278,277,276,275,274,,273272,271,270,269,268,267,266,265,264 263,262,261,260,259,258,257,256,255,254,253,,252,251,250,249,248,247,246,245,244,243,242,,241,240,239,238,237,236,235,234,233,232,231,230,229,228,227,226,225,224,223,222,221,220,219,218,217,216,215,214,213,212,211,210,209,208,207,206,205,204,203,202,201,200,199,198,197,196,195,194,193,192,191,190,189,188,187,186,185,184,183,182,181,180,179,178,177,176,175,174,173,172,171,170,169,168,167,166,165,164,163,162,161,160,159,158,157,156,155,154,153,152,151,150,149,148,147,146,145,144,143,142,141,140,139,138,137,136,135,134,133,132,131,130,129,128,127,126,125,124,123,122,121,120,119,118,117,116,115,114,113,112,111,110,109,108,107,106,105,104,103,102,101,100; 99; 98; 97; 96; 95; 94; 93; 92; 91; 90; 89; 88; 87; 86; 85; 84; 83; 82; 81; 80; 79; 78; 77; 76; 75; 74; 73; 72; 71; 70; 69; 68; 67; 66; 65; 64; 63; 62; 61; 60; 59; 58; 57; 56; 55; 5 4; 53; 52; 51; 50; 49; 48; 47; 46; 45; 44; 43; 42; 41; 40; 39; 38; 37; 36; 35; 34; 33; 32; 31; 30; 29; 28; 27; 26; 25; 24; 23; 22; 21; 20; 19; 18; 17; 16; 15; 14; 13; 12; 11; 10 or 9 amino acids. In some embodiments, the polypeptide of the present invention contains fewer than 50 amino acids. In some embodiments, the polypeptide of the present invention contains fewer than 30 amino acids. In some embodiments, the polypeptide of the present invention contains fewer than 25 amino acids. In some embodiments, the polypeptide of the present invention contains fewer than 20 amino acids. In some embodiments, the polypeptide of the present invention contains fewer than 15 amino acids.

[0118] The isolated polypeptides according to the invention can be produced using any method known in the art. They can be produced, for example, as recombinant polypeptides in host cells (e.g., in bacteria, yeast, or eukaryotic host cells), or chemically synthesized (see reviews Kent SBHChem. Soc. Rev., 2009, 38, 338-351 and Bradley L. et al Annu Rev Biophys Biomol Struct. 2005; 34: 91-118 or RBMerrifield (1969). "Solid-phase peptide synthesis." Advances in enzymology and related areas of molecular biology 32: 221-96.; RBMerrifield (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).

[0119] Antibodies according to the present invention

[0120] The inventors have generated specific antibodies against the polypeptides of this invention.

[0121] First, to determine the in situ kinetics of BLCA in *Toxoplasma gondii*, the inventors proposed polyclonal antibodies targeting two synthetic peptides located at the ends of conserved repeat sequences in the BCLA protein (see Example 1 and Figure 2b). Internal antibodies against the two peptides (peptide 1 and 2) contained in these repeats were generated. BCLA expression monitored by Western blotting using the self-made antibodies against the two BCLA-derived peptides showed upregulation of BCLA after treatment with FR235222 (see Figure 2c).

[0122] Secondly, single-domain antibodies (or nanobodies or VHHs) were generated by immunizing mice with the C-terminal antigenic domain (residues 1089-1275) (SEQ ID NO: 2) of the synthetic peptide BCLA. More precisely, the inventors have discovered that antibodies can be screened for their ability to specifically recognize the isolated peptides of the present invention and stain cell lines infected with *Toxoplasma gondii*, as well as brain samples from toxoplasmosis patients (detecting tissue cysts) and from mouse models of toxoplasmosis. The screening steps of the present invention demonstrate that these antibodies are specific for the isolated peptides of the present invention, particularly those possessing the BCLA antigenic domain.

[0123] This invention provides antibodies that specifically bind to the isolated polypeptides of this invention.

[0124] As used herein, the terms “antibody” and “immunoglobulin” have the same meaning and will be used equivalently in this invention. As used herein, the term “antibody” refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that specifically binds to an immune antigen. Therefore, the term antibody covers not only complete antibody molecules but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are interconnected by disulfide bonds, and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains, lambda(1) and kappa(κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different 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 (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine antigen binding recognition and specificity. The constant regions of the light chain (CL) and heavy chain (CH) endow important biological properties, such as antibody chain binding, secretion, transplacental migration, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of variable portions of one light chain and one heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed primarily of residues from the hypervariable region or complementarity-determining region (CDR). Occasionally, residues from the non-hypervariable region or framework region (FR) may participate in the antibody binding site or influence the structure of the entire domain and thus affect the binding site. The complementarity-determining region, or CDR, is the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. Immunoglobulins have three core regions (CDRs) each in their light and heavy chains, designated VL-CDR1, VL-CDR2, VL-CDR3 and VH-CDR1, VH-CDR2, VH-CDR3, respectively. Therefore, the antigen-binding site comprises six CDRs, containing a set of CDRs from each of the V regions of the heavy and light chains. The framework region (FR) refers to the amino acid sequence inserted between the CDRs.

[0125] Antibodies binding to the isolated peptides of the present invention can be determined using conventional methods known in the art. The mature form of the peptides of the present invention is preferably used for determining antibodies binding to the epitopes of the peptides of the present invention. Alternatively, any variant form of the isolated peptides of the present invention that retains binding to nanobody XX can be used. Many different competitive binding assays can be used to determine epitope binding. Immunoassays that can be used include, but are not limited to, competitive assay systems using techniques such as radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, and complement fixation assay. These assays are conventional and well known in the art (see, for example, Ausubel et al., eds., 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). For example, (GE Healthcare, Piscataway, NJ) is one of several surface plasmon resonance assays routinely used for epitope bins 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. Examples of suitable ELISA assays are also described in the following examples.

[0126] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity. Affinity can be measured by K... D To determine. The term "K" used in this article D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a The K of the antibody is obtained and expressed as molar concentration (M). D The value can be determined using methods known in the art. Measurement of antibody K D The method involves using surface plasmon resonance, or using biosensor systems such as system.

[0127] This invention provides antibodies that specifically bind to isolated polypeptides, said isolated polypeptides comprising or consisting of the following:

[0128] (i) The amino acid sequence of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1);

[0129] (ii) An amino acid sequence consisting of the C-terminal antigenic domain (residues 1089-1275 of BCLA) (SEQ ID NO: 2);

[0130] (iii) An amino acid sequence consisting of an internal repeating domain selected from the following group of BCLA: TgR1 (SEQ ID NO:4), TgR2 (SEQ ID NO:5), TgR3 (SEQ ID NO:6), TgR4 (SEQ ID NO:7), TgR5 (SEQ ID NO:8), TgR6 (SEQ ID NO:9), TgR7 (SEQ ID NO:10), TgR8 (SEQ ID NO:11), TgR9 (SEQ ID NO:12), TgR10 (SEQ ID NO:13), TgR11 (SEQ ID NO:14), TgR12 (SEQ ID NO:15), and TgR13 (SEQ ID NO:16);

[0131] (iv) An amino acid sequence that is substantially homologous to the sequences of (i)-(iii), preferably an amino acid sequence that is at least 80% identical to the sequences of (i)-(iii);

[0132] A fragment of at least 9 consecutive amino acids of the sequence (v)(i)-(iv).

[0133] These antibodies can recognize a fragment containing at least nine consecutive amino acids of any of the isolated polypeptides (i)-(v) or an epitope containing at least one amino acid therein.

[0134] Preferably, the epitope is located within a fragment containing or composed of any one of the separated polypeptides (i)-(v).

[0135] Most preferably, the epitope is located within the C-terminal antigenic domain (SEQ ID NO: 2) and the internal repeat domain (residues 304-924) of BCLA, and is referred to as TgR1-TgR13 (SEQ ID NO: 4-SEQ ID NO: 16). These antibodies are characterized by their specific binding to the Toxoplasma gondii BCLA polypeptide of the present invention.

[0136] In one specific implementation, the antibody that specifically binds to the rBCLA peptide specifically binds to an amino acid sequence selected from the group consisting of:

[0137] (i)GELQPAEAEEARLLVADLKAV(SEQ ID N°32)

[0138] (ii)VRVEGEAFFRASVDLYEA(SEQ ID N°33)

[0139] (iii)KLRPLTKGELVDVVRQ(SEQ ID N°34)

[0140] (iv)TQIFVQDRASAFLRV (peptide 36 of rBCLA) (SEQ ID NO. 35)

[0141] (v)AAEQMKAVFAMVEEG (peptide 44 of rBCLA) (SEQ ID N°36)

[0142] (vi) is an amino acid sequence that is substantially homologous to the sequence of (i)-(v), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(v);

[0143] A fragment of at least 9 consecutive amino acids of the sequence (vii)(i)-(vi).

[0144] In a more specific implementation, the antibody that specifically binds to the rBCLA peptide specifically binds to an amino acid sequence selected from the group consisting of:

[0145] (i) GELQPAEAEEARLLV (peptide 12 of rBCLA) (SEQ ID N°37);

[0146] (ii)QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO38),

[0147] (iii) EAEEARLLVADLKAV (peptide 14 of rBCLA) (SEQ ID NO39),

[0148] (iv)VRVEGEAFFRASVDL (peptide 21 of rBCLA) (SEQ ID NO.40)

[0149] (v)EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID N°41);

[0150] (vi)AFFRASVDLYEAVKN(peptide 23 of rBCLA) (SEQ ID NO42),

[0151] (vii)KLRPLTKGELVDVVR(peptide 30 of rBCLA) (SEQ ID NO.43)

[0152] (viii) is an amino acid sequence that is substantially homologous to the sequence of (i)-(vii), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(vii);

[0153] A fragment of at least 9 consecutive amino acids of the sequence (vii)(i)-(viii).

[0154] The present invention further provides antibodies that specifically bind to an amino acid sequence consisting of the BCLA internal repeating domain (residues 304-924) of BCLA, which are called TgR1-TgR13 (SEQ ID NO: 4-SEQ ID NO: 16).

[0155] Therefore, in one specific implementation, the antibody that specifically binds to the internal repeat domain of BCLA binds to an amino acid sequence selected from the group consisting of:

[0156] (i) An amino acid sequence consisting of the internal repeating domains of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7);

[0157] (ii) an amino acid sequence that is substantially homologous to the sequence of (i), preferably an amino acid sequence that is at least 80% identical to the sequence of (i);

[0158] A fragment of at least nine consecutive amino acids of the sequence (iii)(i)-(ii).

[0159] In a more specific implementation, the antibody that specifically binds to the internal repeat domain of BCLA TgR4 binds to an amino acid sequence selected from the following group:

[0160] (i)AAGSMEKEKPVLPGEGEGH(TgR4 domain A); (SEQ ID N°44)

[0161] (ii)VLPKHETKPALTDEKRTKPGGP(TgR4 domain B),(SEQ ID N°45).

[0162] In a more specific implementation, the antibody that specifically binds to the internal repeat domain of BCLA TgR4 binds to an amino acid sequence selected from the following group:

[0163] (i) AAGSMEKEKPVLPGE (peptide 3 of TgR4); (SEQ ID N°46)

[0164] (ii) GSMEKEKPVLPGEGE (peptide 4 of TgR4) (SEQ ID N°47)

[0165] (iii) MEKEKPVLPGEGEGH (peptide 5 of TgR4) (SEQ ID N°48)

[0166] (iv) KEKPVLPGEGEGHVL (peptide 6 of TgR4) (SEQ ID NO.49)

[0167] (v)KPVLPGEGEGHVLPG (peptide 7 of TgR4) (SEQ ID N°50)

[0168] (vi)HVLPKHETKPALTDEK (peptide 13 of TgR4), (SEQ ID N°51)

[0169] (vii)PKHETKPALTDEKRT (peptide 14 of TgR4), (SEQ ID N°52)

[0170] (viii)HETKPALTDEKRTKP (peptide 15 of TgR4) (SEQ ID N°53)

[0171] (ix)TKPALTDEKRTKPGG(TgR4 peptide 16) (SEQ ID N°54).

[0172] In one specific implementation, the antibody specifically binds to the internal repeating domain of BCLA(TgRx) having the following sequence:

[0173] M-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5–Xaa6–Xaa7-ME-Xaa8–Xaa9-K-Xaa10-V-Xaa11-PGEG-Xaa12–Xaa13-H-Xaa14-Xaa15 -PK-Xaa16-E-Xaa17-Xaa18-LT-Xaa19-Xaa20-Xaa21-Xaa22-T-Xaa23-P-Xaa24-Xaa25-P-Xaa26-Xaa27-Xaa28(SEQ ID N°64)

[0174] Xaa1 is either glutamic acid (E) or has no amino acid residues.

[0175] Xaa2 is either arginine (R) or serine (S).

[0176] Xaa3 is either proline (P) or glycine (G).

[0177] Xaa4 is either alanine (A) or glycine (G).

[0178] Xaa5 is either alanine (A) or contains no amino acid residues.

[0179] Xaa6 is either glycine (G) or arginine (R).

[0180] Xaa7 can be serine (S), proline (P), or alanine (A).

[0181] Xaa8 is either lysine (K) or glutamic acid (E).

[0182] Xaa9 is either lysine (K), glutamic acid (E), or aspartic acid (D).

[0183] Xaa10 is either proline (P) or leucine (L).

[0184] Xaa11 is either leucine (L) or serine (S).

[0185] Xaa12 is either glutamic acid (E) or lysine (K).

[0186] Xaa13 is either glycine (G) or arginine (R).

[0187] Xaa14 is either valine (V) or alanine (A).

[0188] Xaa15 is either leucine (L) or serine (S).

[0189] Xaa16 can be histidine (H), aspartic acid (D), or alanine (A).

[0190] Xaa17 can be threonine (T), arginine (R), methionine (M), or glutamine (Q).

[0191] Xaa18 can be proline (P), threonine (T), or alanine (A).

[0192] Xaa19 is either aspartic acid (D), glutamic acid (E), or glutamine (Q).

[0193] Xaa20 is either glutamic acid (E) or lysine (K).

[0194] Xaa21 is either lysine (K), glycine (G), or glutamic acid (E).

[0195] Xaa22 is either arginine (R) or valine (V).

[0196] Xaa23 is either lysine (K), glutamic acid (E), or asparagine (N).

[0197] Xaa24 is either glycine (G), valine, or isoleucine (I).

[0198] Xaa25 is either glycine (G) or glutamic acid (E).

[0199] Xaa26 is either arginine (R) or proline (P).

[0200] Xaa27 can be threonine (T), cysteine ​​(C), lysine (K), or methionine (M).

[0201] Xaa28 is either glutamic acid (E) or alanine (A).

[0202] A fragment of at least 9 consecutive amino acids of the sequence SEQ ID N°64.

[0203] The present invention also provides an antibody that specifically binds to an amino acid sequence consisting of either peptide 1 or peptide 2 (SEQ ID NO: 17-27) within an internal repeating domain of BCLA, wherein the internal repeating domain of BCLA is referred to as TgR1-TgR13 (SEQ ID NO: 4-SEQ ID NO: 16).

[0204] In one specific implementation, peptides 1 and 2 used in this study are

[0205] Peptide 1: EMERPAAGSMEK (SEQ ID N°21)

[0206] Peptide 2: VLPKHETKPALT (SEQ ID N° 22).

[0207] These antibodies can be polyclonal or monoclonal. When antibodies are monoclonal, they can, for example, correspond to chimeric, humanized, or fully human antibodies, antibody fragments, and single-domain antibodies.

[0208] The term "chimeric antibody" refers to an antibody that contains the VH and VL domains of an antibody and the CH and CL domains of a human antibody.

[0209] According to the present invention, the term "humanized antibody" refers to an antibody having a variable region framework and a constant region derived from a human antibody but retaining the CDR of the previous non-human antibody.

[0210] The term "antibody fragment" refers to a fragment of an antibody containing a variable domain comprising the CDR of the antibody. Basic antibody fragments include Fab, Fab', F(ab')2Fv, scFv, and dsFv. Examples of antibody fragments can also be found in the review, Holliger et al., Nature Biotechnology 23, issue 9 1126-1136 (2005), which is incorporated herein by reference.

[0211] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, wherein in a fragment obtained by treating IgG with the protease papain, approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0212] The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 and antigen-binding activity, in which the antigen-binding activity is slightly greater than that of Fab bound by disulfide bonds through the hinge region, in fragments obtained by treating IgG with the protease pepsin.

[0213] The term "Fab'" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, obtained by cleaving the disulfide bonds in the hinge region of F(ab')2.

[0214] Single-chain Fv (“scFv”) polypeptides are covalently linked VH:VL heterodimers, typically expressed by gene fusions comprising VH and VL encoding genes linked by a peptide linker. “dsFv” is a VH:VL heterodimer stabilized by disulfide bonds. Divalent and multivalent antibody fragments can be spontaneously formed by the binding of monovalent scFvs, or generated by conjugation of monovalent scFvs via peptide linkers (such as divalent sc(Fv)2).

[0215] The terms "biantibody," "triantibody," or "tetraantibody" refer to small antibody fragments containing multiple antigen-binding sites (2, 3, or 4), which are contained within a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on 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 domain is forced to pair with a complementary domain of the other chain, creating two antigen-binding sites.

[0216] As used herein, the term "single-domain antibody" has its general meaning in the art and refers to a type of antibody with a single heavy-chain variable domain that can be found in camel mammals that naturally lack light chains. Such single-domain antibodies are also known as VHH or... For a general description of (single)domain antibodies, reference is also made to the prior art described above and to EP 0368684 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 molecular weight of nanobodies is approximately one-tenth that of human IgG molecules, and the physical diameter of the protein is only a few nanometers. One consequence of this small size is the ability of camel nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins; that is, camel nanobodies can be used as reagents for detecting antigens that are otherwise concealed using classical immunoassay techniques, and can also be used as therapeutic agents. Therefore, another consequence of this small size is that nanobodies can be inhibited by binding to specific sites in the grooves or slits of target proteins, and thus can function more like classical low-molecular-weight drugs than classical antibodies. The low molecular weight and compact size further contribute to the extreme thermal stability of nanobodies, their resistance to extreme pH and proteolytic digestion, and their poor antigenicity. Another result is that nanobodies readily migrate from the circulatory system into tissues, even crossing the blood-brain barrier, and can treat conditions affecting nerve tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. See U.S. Patent Application 20040161738, published August 19, 2004. These characteristics, combined with low antigenicity to humans, indicate significant therapeutic potential. The amino acid sequence and structure of a single-domain antibody can be considered to consist of four frame regions, or “FRs,” referred in the art as “Frame Region 1” or “FR1”; “Frame Region 2” or “FR2”; “Frame Region 3” or “FR3”; and “Frame Region 4” or “FR4”, respectively; these frame regions are interrupted by three complementarity-determining regions, or “CDRs,” referred in the art as “Complementarity-determining Region” or “CDR1”; “Complementarity-determining Region 2” or “CDR2”; and “Complementarity-determining Region 3” or “CDR3”, respectively. Therefore, a single-domain antibody can be defined as having the following general amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3. In the context of this invention, the amino acid residues of a single-domain antibody are numbered according to the universal numbering of the VH domain given by the International Immunogenetic Information System (http: / / imgt.cines.fr / ).

[0217] Several VHH (single-domain antibodies) were generated after immunization, leading to a favorable immune response. The resulting libraries exhibited good size and insertion frequency. Phage display selection on His rBCLA (SEQ ID NO3) yielded numerous good clones, three of which (ERB-1G6, ERB-1B11, and ERB-1A12) showed very good epigenetic affinity, with ERB-1G6 also showing high production levels in E. coli.

[0218] The sequences of the variable heavy chains (VH) of single-domain antibodies, ERB-1F1, ERB-1F2, ERB1H4, ERB-1D7, ERB-1G6, ERB-1B11 and ERB-1A12 VHH, are described in Table 1 below.

[0219] Table 1

[0220]

[0221] Methods for obtaining these antibodies are well known in the art. For example, monoclonal antibodies according to the invention can be obtained by immunizing a non-human mammal with the fragment comprising or consisting of any one of (i)-(vii). Starting with polyclonal antibodies, monoclonal antibodies can then be obtained using standard methods.

[0222] The antibodies of the present invention can be conjugated with detectable labels to form immunoconjugates. Suitable detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels, or colloidal gold. Methods for preparing and detecting such immunoconjugates with detectable labels are well known to those skilled in the art and are described in more detail below.

[0223] The detectable marker can be a radioactive isotope that can be detected by autoradiography. Isotopes particularly suitable for the purposes of this invention are... 3 H, 125 I, 131 I, 35 S and 14 C.

[0224] Immunoconjugates can also be labeled with fluorescent compounds. The presence of fluorescently labeled antibodies is determined by exposing the immunoconjugate to light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescently labeled compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescein.

[0225] Alternatively, immunoconjugates can be detectably labeled by conjugating an antibody to a chemiluminescent compound. The presence of the chemiluminescently labeled immunoconjugate is determined by detecting the presence of light emitted during the chemical reaction. Examples of chemiluminescently labeled compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0226] Similarly, bioluminescent compounds can be used to label the immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems, in which catalytic proteins increase the efficiency of the chemiluminescent reaction. The presence of bioluminescent proteins is determined by detecting the presence of luminescence. Bioluminescent compounds that can be used for labeling include luciferin, luciferase, and jellyfish luminescent proteins.

[0227] Alternatively, immunoconjugates can be detectably labeled by linking a monoclonal antibody to an enzyme. When the enzyme conjugate is incubated in the presence of a suitable substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety, which can be detected, for example, by spectrophotometry, fluorescence, or visual means. Examples of enzymes that can be used for the detectable labeling of multispecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0228] The antibodies of this invention can be labeled with metallic chemical elements (such as lanthanides). Lanthanides offer several advantages over other labels because they are stable isotopes, a large number of them are available (up to 100 or more different labels), they are relatively stable, and they are highly detectable and easily resolved between detection channels when detected using mass spectrometry. Lanthanide labels also provide 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 used in many different settings. Lanthanides are a series of 15 metallic chemical elements with atomic numbers 57-71. They are also known as rare earth elements. Lanthanides can 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, as many parameters as available stable isotope labels.

[0229] Those skilled in the art will recognize other suitable markers that can be used according to the present invention. The binding of the marker portion to the monoclonal antibody can be accomplished using standard techniques known in the art.

[0230] Furthermore, the convenience and versatility of immunochemical assays can be enhanced by using monoclonal antibodies already conjugated with avidin, streptavidin, and biotin.

[0231] Another object of the present invention is a method for using at least one isolated Toxoplasma gondii polypeptide according to the present invention as described above to detect antibodies against the Toxoplasma gondii polypeptide BCLA and / or to assess its amount in biological samples.

[0232] As used herein, the term "biological sample" refers to any biological sample from a subject; a tissue sample or a body fluid sample. In a preferred embodiment of the method for detecting antibodies against the Toxoplasma gondii polypeptide BCLA, the biological sample is the body fluid of the subject. Non-limiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), and aqueous humor.

[0233] More specifically, the body fluid sample is a serum or aqueous humor sample. In a preferred embodiment of the method for detecting antibodies against the Toxoplasma gondii peptide BCLA, the biological sample is a fluid sample, more particularly a brain sample.

[0234] The detection and diagnostic method of the present invention:

[0235] In some embodiments, the method of the present invention is performed in vitro or ex vivo.

[0236] · Methods for detecting the Toxoplasma gondii peptide BCLA

[0237] The purpose of this invention is to provide a method for detecting the Toxoplasma gondii polypeptide BCLA of this invention and / or assessing its amount in biological samples.

[0238] Biological samples refer to, but are not limited to, tissue samples, culture media and cell samples, whole blood samples, serum samples, plasma samples, aqueous humor samples, saliva samples, cerebrospinal fluid samples, muscle samples, or brain tissue samples.

[0239] In a preferred embodiment for detecting Toxoplasma gondii BCLA peptides, the biological sample is a tissue sample, more particularly a muscle sample or a brain sample.

[0240] Detection of the Toxoplasma gondii peptide BCLA can include separating the protein / peptide using: centrifugation based on protein 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 a specific solid phase used. Once separated, the Toxoplasma gondii peptide BCLA can be identified based on a known "separation spectrum," such as the retention time of the protein, and measured using standard techniques. Alternatively, the separated protein can be detected and measured by, for example, mass spectrometry (see the Examples section).

[0241] The species and quantity of the *Toxoplasma gondii* peptide BCLA of the present invention can be determined using standard electrophoresis and immunodiagnostic techniques, including immunoassays (such as competitive assays), direct reactions (such as immunohistochemistry), or sandwich assays. Such assays include, but are not limited to, Western blotting; agglutination assays; enzyme-labeled and mediated immunoassays, such as ELISA; biotin / antibiotin protein assays; radioimmunoassays; immunoelectrophoresis; and immunoprecipitation. Reactions typically involve labeling, such as fluorescence, chemiluminescence, radiolabeled enzymes, or dye molecules, or other methods for detecting the formation of complexes between antigens and antibodies or antibodies reacting with them.

[0242] For example, the amount of Toxoplasma gondii peptide BCLA can be determined using a variety of techniques and methods, using any method known in the art: RIA kits (DiaSorin; IDS, Diasource), ELISA kits (Fujirebio, ThermoFisher, EHTGFBI, R&D DY2935, IDS (manual), IDS (for open analyzers), automated immunochemiluminescence methods (MesoScaleDiscovery, DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (Janssen et al., 2012), Simoa / Quanterix.

[0243] In a specific implementation, the method of the present invention includes contacting a biological sample with a binding partner.

[0244] As used herein, a binding coupler is a molecule capable of selectively interacting with the Toxoplasma gondii polypeptide BCLA of the present invention.

[0245] The binding partner can typically be a polyclonal or monoclonal antibody, with monoclonal antibodies being preferred.

[0246] In another implementation, the binding mate can be an aptamer. Aptamers are a class of molecules that represent alternatives to antibodies in terms of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences capable of recognizing virtually any kind of target molecule with high affinity and specificity. These ligands can be isolated through the phylogenetic analysis of ligands by exponential enrichment (SELEX) of random sequence libraries, as described by Tuerk et al. (1990) Science, 249, 505-510. Random sequence libraries can be obtained through combinatorial chemistry synthesis of DNA. In this library, each member is a linear oligomer that has been chemically modified and has a unique sequence. Possible modifications, uses, and advantages of such molecules were reviewed in Jayaseena 1999. Peptide aptamers are derived from platform proteins (e.g., E. coli thioredoxin A, which are selected from combinatorial libraries by two hybridization methods) (Colas et al. (1996) Nature, 380, 548-50).

[0247] The binding couplers (such as antibodies or aptamers) of the present invention can be labeled with detectable molecules or substances, such as fluorescent molecules, radioactive molecules, or any other labels known in the art. Typically, the labels that provide the signal (directly or indirectly) are known in the art.

[0248] As used herein, the term "labeled" in relation to conjugated aptamers is intended to encompass the direct labeling of antibodies or aptamers by coupling (i.e., physically linking) a detectable substance (such as a radioactive agent or fluorophore, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5)) to an antibody or aptamer, and the indirect labeling of probes or antibodies by their reactivity with a detectable substance. The antibodies or aptamers of the present invention can be labeled with radioactive molecules by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms used in scintillation studies, such as I123, I124, In111, Re186, and Re188.

[0249] The above assays typically involve the binding of a binding partner (i.e., antibody or aptamer) in a solid support. Solid supports that can be used to carry out the invention include matrices such as nitrocellulose (e.g., in the form of membranes or microtiter wells); polyvinyl chloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, etc. More specifically, an ELISA method can be used in which the wells of a microtiter plate are coated with a set of antibodies against the Toxoplasma gondii peptide BCLA. A body fluid sample containing or suspected of containing the Toxoplasma gondii peptide BCLA is then added to the coated wells. After incubation for a period sufficient to form a binding partner-Toxoplasma gondii peptide BCLA complex, the plate can be washed to remove unbound material and labeled secondary binding molecules can be added. The secondary binding molecules are reacted with any captured sample marker proteins, the plate is washed, and the presence of the secondary binding molecules is detected using methods well known in the art.

[0250] As a binding partner, it can be used to label secondary binding molecules.

[0251] The antibody-binding and immunoconjugates of the present invention can be used to detect the anti-Toxoplasma gondii peptide BCLA of the present invention, and / or to assess its quantity in biological samples, particularly tissue samples, culture media and cell samples, whole blood samples, serum samples, plasma samples, cerebrospinal fluid samples, or brain tissue samples. Therefore, they can be used to diagnose all diseases related to Toxoplasma gondii.

[0252] · Methods for diagnosing latent toxoplasmosis (detection of Toxoplasma gondii peptide BCLA)

[0253] Therefore, the detection according to the present invention BCLA (Betula glabrata polypeptide) The method can be used for the in vitro diagnosis of toxoplasmosis from biological samples. In particular, the detection method of the present invention can therefore be used for the in vitro diagnosis of latent toxoplasmosis or latent toxoplasmosis from biological samples. As used herein, the term "biological sample" refers to any biological sample from a subject. Biological sample means, but is not limited to, any tissue sample, culture medium and cell sample, whole blood sample, serum sample, plasma sample, urine sample, saliva sample, or cerebrospinal fluid sample.

[0254] In a preferred embodiment of the method for detecting BCLA peptides in Toxoplasma gondii, the biological sample is a tissue sample, more particularly a brain tissue sample or a muscle tissue sample.

[0255] Another object of the present invention is a method for detecting the Toxoplasma gondii polypeptide BCLA of the present invention and / or assessing its amount in a biological sample, wherein the method comprises contacting the sample with the antibody or immune conjugate of the present invention under conditions that allow the formation of an immune complex between the Toxoplasma gondii polypeptide BCLA and the antibody / immunoconjugate, and detecting or measuring the formed immune complex.

[0256] Another object of the present invention is a method for detecting merozoite cysts in biological samples and / or assessing their quantity, wherein the method comprises contacting the sample with the antibody or immune conjugate of the present invention, and detecting or measuring the formed immune complex, under conditions that allow the formation of an immune complex between the Toxoplasma gondii polypeptide BCLA on the cyst surface and the antibody / immunoconjugate.

[0257] The resulting immune complexes can be detected or measured using standard techniques by a variety of methods, including, as a non-limiting example, enzyme-linked immunosorbent assay (ELISA) or other solid-phase immunoassays, radioimmunoassay, electrophoresis, immunofluorescence, or Western blotting.

[0258] Another object of the present invention is a method for in vitro diagnosis of toxoplasmosis, wherein the method includes detecting the presence of the Toxoplasma gondii polypeptide BCLA as shown above in a biological sample from a subject to be tested.

[0259] The term "toxoplasmosis" has its general meaning in the field and refers to a worldwide zoonotic infection of medical importance in pregnant women and immunocompromised patients. *Toxoplasma gondii* (the causative agent of toxoplasmosis) has co-evolved with its homeothermic hosts (including humans) a persistent strategy that typically functions as a quasi-cryptic population and thus exhibits subclinical signs, thereby optimizing opportunities for transmission to new hosts. During its prolonged residence in warm-blooded animals, the proliferative phase (tachyzoites) transitions to a persistent phase (cyst-closed merozoites), providing the parasite with a unique opportunity to spread to new hosts without undergoing its feline-limited sexual phase. Uncontrolled proliferation of tachyzoite populations, occurring when immune balance is transiently or more persistently disrupted, can lead to life-threatening disease and, in the case of congenital toxoplasmosis, birth defects. Persistence (which depends on the parasite subpopulation’s acquisition of slow replication skills and disruption of the fast replication population) critically requires the IL-12 / IFN-γ immune axis; however, Toxoplasma gondii has uniquely evolved a developmental program of fine-tuned and epigenetic regulation to manipulate the transition phase.

[0260] In some implementations, the toxoplasmosis is congenital toxoplasmosis.

[0261] Therefore, the present invention relates to a method for in vitro diagnosis of congenital toxoplasmosis, wherein the method comprises detecting the presence of the polypeptide according to claim 1 in a biological sample from a subject to be tested.

[0262] As used herein, the term "latent toxoplasmosis" refers to the persistent stage of toxoplasmosis (cysts – closed merozoites). Following the initial infection phase, characterized by tachyzoites proliferating throughout the host, pressure from the host's immune system causes *Toxoplasma gondii* tachyzoites to transform into semi-dormant, slowly dividing merozoites at the parasitic cellular stage. Within the host cell, these clusters of merozoites are called tissue cysts. The cyst wall is formed by a parasitic vesicle membrane. Although tissue cysts containing merozoites can form in virtually any organ, they are primarily formed and persistent in the brain, eyes, and striated muscle (including the heart). However, specific tissue tropisms can vary between intermediate host species; in pigs, most tissue cysts are found in muscle tissue, while in mice, most are found in the brain. The diameter of the cysts is typically 5–50 μm (50 μm is approximately two-thirds the width of an average human hair).

[0263] Furthermore, the present invention provides a kit comprising at least one antibody or fragment thereof of the present invention. The kit of the present invention may contain an antibody conjugated to a solid support, such as a tissue culture plate or beads (e.g., agarose beads). Kits may be provided containing an antibody for the in vitro detection and quantification of the Toxoplasma gondii polypeptide BCLA (e.g., in ELISA or Western blotting). Such an antibody for detection may be labeled, for example, with a fluorescent or radioactive label.

[0264] · Methods for diagnosing latent toxoplasmosis (detection of Toxoplasma gondii peptide BCLA)

[0265] When synthesized as a recombinant protein, the inventors have clearly demonstrated that BCLA constitutes an effective serological marker of latent infection with high sensitivity, and is definitively and uniquely associated with the presence of cysts in the mouse brain. Antibodies against the BCLA antigen have been detected in human patients. Enrichment titers were detected in patients seropositive for Sag1 or tachyzoite-associated antigens. Further correlation between human anti-BCLA IgG synthesis and cysts was demonstrated by significantly stronger recorded titers in the pathological group strongly associated with the presence of cysts. Notably, this was also observed in patients undergoing serological reactivation and those with confirmed ocular toxoplasmosis (see experimental data in Figures 10 and 13 of the Examples). In the latter case, the developed ELISA assay can also detect BCLA antibodies in the aqueous humor and serum of some of these patients. By opening up new diagnostic perspectives, the detection of Toxoplasma gondii antibodies against semi-dormant cysts represents a significant improvement in the serological diagnosis of toxoplasmosis. In fact, at least in commercially available kits, almost no components of the cyst wall or surface merozoites have been identified, and none have shown antigens suitable for serological purposes. Ideally, the antigen should be expressed only during the latent merozoite stage and ideally exposed on the capsule surface; these are two characteristics found in BCLA peptides.

[0266] Furthermore, the inventors have demonstrated that children synthesize specific anti-BCLA IgG before birth. Therefore, compared with... and Compared to titration of Toxo IgG, BCLA responsiveness can further better guide the diagnosis of congenital toxoplasmosis at birth (see Example 3).

[0267] Therefore, detection Autoantibodies against Toxoplasma gondii polypeptides according to the present invention The method can therefore be used for the in vitro diagnosis of toxoplasmosis from biological samples. In particular, the detection method of the present invention can therefore be used for the in vitro diagnosis of latent or congenital toxoplasmosis from biological samples.

[0268] Another object of the present invention is a method for in vitro diagnosis of toxoplasmosis, wherein the method includes detecting, as described above, in a biological sample from a test subject. T autoantibodies against Toxoplasma gondii polypeptides according to the present invention The existence of.

[0269] Therefore, the present invention relates to a method for determining whether a subject has latent toxoplasmosis, the method comprising:

[0270] a) Detecting the immunoreactivity of the *Toxoplasma gondii* polypeptide of the present invention in biological samples from the patient; and optionally...

[0271] b) Inferring from the results of step a) whether the patient has latent toxoplasmosis, and the immunoreactivity of the Toxoplasma gondii polypeptide of the present invention indicates latent toxoplasmosis.

[0272] The present invention also relates to the use of antibodies against latent toxoplasmosis as biomarkers for the diagnosis (or confirmation) of latent toxoplasmosis in patients.

[0273] The present invention also relates to an in vitro method for diagnosing or confirming latent toxoplasmosis in patients who have or are suspected of having latent toxoplasmosis, comprising:

[0274] a) Obtaining biological samples from patients, and

[0275] b) Detecting antibodies against the Toxoplasma gondii polypeptide of the present invention in biological samples;

[0276] The presence of antibodies in the biological sample is used to diagnose or confirm the diagnosis of latent toxoplasmosis in patients.

[0277] Therefore, the present invention relates to a method for determining whether a subject has congenital toxoplasmosis, the method comprising:

[0278] a) Detecting the immunoreactivity of the *Toxoplasma gondii* polypeptide of the present invention in biological samples from the patient; and optionally...

[0279] b) Inferring from the results of step a) whether the patient has congenital toxoplasmosis, and the immunoreactivity of the Toxoplasma gondii polypeptide of the present invention as an indicator of congenital toxoplasmosis.

[0280] The present invention also relates to the use of antibodies against congenital toxoplasmosis as biomarkers for the diagnosis (or confirmation) of congenital toxoplasmosis in patients.

[0281] The present invention also relates to an in vitro method for diagnosing or confirming latent toxoplasmosis in patients with or suspected of having congenital toxoplasmosis, comprising:

[0282] a) Obtaining biological samples from patients, and

[0283] b) Detecting antibodies against the Toxoplasma gondii polypeptide of the present invention in biological samples;

[0284] The presence of antibodies in the biological sample is used to diagnose or confirm the diagnosis of congenital toxoplasmosis in patients.

[0285] As used herein, the term "biological sample" refers to any biological sample from a subject. In a preferred embodiment of the method for detecting an antibody against Toxoplasma gondii BCLA peptide, the biological sample is the subject's bodily fluids. Non-limiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), and aqueous humor.

[0286] More specifically, the body fluid sample is a serum or aqueous humor sample.

[0287] In a preferred embodiment, the patient to be tested has or is suspected of having toxoplasmosis.

[0288] In another preferred embodiment, the patient to be tested is suspected of having toxoplasmosis, and the method is performed to confirm that the patient actually has latent toxoplasmosis.

[0289] In another implementation, the patient to be tested is a pregnant woman and / or an immunocompromised patient (i.e., an HIV patient or a patient who has undergone immunomodulatory therapy prior to receiving a graft), and the method is performed to determine whether the patient actually has latent toxoplasmosis.

[0290] When a subject presents with signs and symptoms of acute toxoplasmosis, the current treatment for toxoplasmosis is as follows:

[0291] • Pyrimethamine (Daraprim). This drug, typically used for malaria, is a folic acid antagonist. It can prevent the body from absorbing the B vitamin folic acid (folic acid, vitamin B-9), especially when patients take high doses for extended periods. Therefore, supplemental folic acid may be recommended. Other potential side effects of pyrimethamine include bone marrow suppression and hepatotoxicity.

[0292] • Sulfadiazine. This antibiotic is used in combination with pyrimethamine to treat toxoplasmosis.

[0293] For HIV / AIDS patients, the treatment options for toxoplasmosis are pyrimethamine and sulfadiazine with folic acid (formyltetrahydrofolate). Another option is to take pyrimethamine with clindamycin.

[0294] For pregnant women and infants infected with toxoplasmosis:

[0295] If the infection occurs before the 16th week of pregnancy, the pregnant woman receives the antibiotic spiramycin. Using this medication can reduce the risk of the infant developing neurological problems associated with latent toxoplasmosis.

[0296] If the infection occurs after the 16th week of pregnancy, or if a test shows that the unborn child has toxoplasmosis, the pregnant woman can be given pyrimethamine and sulfadiazine, as well as folic acid (formyltetrahydrofolate).

[0297] The present invention also provides an in vitro method for selecting patients with latent toxoplasmosis suitable for treatment with at least one folic acid antagonist and / or antibiotic compound, comprising:

[0298] a) Detecting the immunoreactivity of the *Toxoplasma gondii* polypeptide of the present invention in biological samples from the patient; and optionally...

[0299] b) When an immunoreactivity against the Toxoplasma gondii polypeptide of the present invention is detected, select patients suitable for treatment with at least one folic acid antagonist (i.e., pyrimethamine) and / or an antibiotic compound (i.e., sulfadiazine or spiramycin).

[0300] The method of determining whether a patient has latent toxoplasmosis, the use of antibodies against the Toxoplasma gondii polypeptide of the present invention as biomarkers for diagnosing (or confirming) latent toxoplasmosis, and the method for selecting patients with latent toxoplasmosis suitable for treatment with at least one folic acid antagonist and / or antibiotic compound can be, for example, in vitro or ex vivo methods.

[0301] The present invention also relates to a method for treating a patient infected with latent toxoplasmosis, the patient exhibiting an immune responsiveness to the Toxoplasma gondii polypeptide of the present invention, the method comprising administering to the patient a folic acid antagonist (i.e., pyrimethamine) and / or an antibiotic compound (i.e., sulfadiazine or spiramycin) or a pharmaceutical composition comprising said compound.

[0302] The present invention also provides folic acid antagonists (i.e., pyrimethamine) and / or antibiotic compounds (i.e., sulfadiazine or spiramycin) or pharmaceutical compositions comprising said compounds for treating patients with latent toxoplasmosis exhibiting an immune response to the Toxoplasma gondii polypeptide of the present invention.

[0303] In some embodiments, the Toxoplasma gondii polypeptide of the present invention, which is used to test its immunoreactivity, is the BCLA (brain cyst-loaded antigen) protein (abbreviated as "BCLA"), the C-terminal domain of BCLA (residues 1089-1275, SEQ ID NO2) (abbreviated as "rBCLA"), or the internal repeating domain of BCLA (residues 304-924 of BCLA), which is referred to as TgR1-TgR13 (SEQ ID NO: 4 to SEQ ID NO: 16).

[0304] In the specific implementation plan, the protein used to test its immune reactivity is the rBCLA peptide.

[0305] In another specific embodiment, the protein for which the immunoreactivity is tested is a peptide fragment of at least nine consecutive amino acids of BCLA, the rBCLA sequence, or the internal repeat domain of BCLA (residues 304-924 of BCLA), referred to as TgR1-TgR13 (SEQ ID NO: 4 to SEQ ID NO: 16).

[0306] Specifically, the term "Toxoplasma gondii polypeptide of the present invention for testing its immunoreactivity" refers to:

[0307] (i) The amino acid sequence of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1);

[0308] (ii) An amino acid sequence consisting of the C-terminal antigenic domain (residues 1089-1275 of BCLA, referred to as rBCLA) (SEQ ID NO: 2);

[0309] (iii) An amino acid sequence consisting of an internal repeating domain selected from the following group of BCLA: TgR1 (SEQ ID NO:4), TgR2 (SEQ ID NO:5), TgR3 (SEQ ID NO:6), TgR4 (SEQ ID NO:7), TgR5 (SEQ ID NO:8), TgR6 (SEQ ID NO:9), TgR7 (SEQ ID NO:10), TgR8 (SEQ ID NO:11), TgR9 (SEQ ID NO:12), TgR10 (SEQ ID NO:13), TgR11 (SEQ ID NO:14), TgR12 (SEQ ID NO:15), and TgR13 (SEQ ID NO:16);

[0310] (iv) An amino acid sequence that is substantially homologous to the sequences of (i)-(iii), preferably an amino acid sequence that is at least 80% identical to the sequences of (i)-(iii);

[0311] A fragment of at least 9 consecutive amino acids of the sequence (v)(i)-(iv).

[0312] Therefore, in one specific implementation, the Toxoplasma gondii peptide isolated from the rBCLA peptide for testing its immunoreactivity is selected from the group consisting of:

[0313] (i)GELQPAEAEEARLLVADLKAV(rBCLA domain A) (SEQ ID N°32)

[0314] (ii)VRVEGEAFFRASVDLYEA(rBCLA domain B) (SEQ ID N°33)

[0315] (iii) KLRPLTKGELVDVVRQ(rBCLA domain C) (SEQ ID NO34)

[0316] (iv) TQIFVQDRASAFLRV (peptide 36 of rBCLA and domain D of rBCLA) (SEQ ID NO35)

[0317] (v)AAEQMKAVFAMVEEG(peptide 44 of rBCLA and domain E of rBCLA) (SEQ ID NO36)

[0318] (vi) is an amino acid sequence that is substantially homologous to the sequence of (i)-(v), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(v);

[0319] A fragment of at least 9 consecutive amino acids of the sequence (vii)(i)-(vi).

[0320] In a more specific implementation scheme, the Toxoplasma gondii peptide isolated from the rBCLA peptide for testing immunoreactivity is selected from the group consisting of:

[0321] (i) GELQPAEAEEARLLV (peptide 12 of rBCLA) (SEQ ID N°37);

[0322] (ii)QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO38),

[0323] (iii) EAEEARLLVADLKAV (peptide 14 of rBCLA) (SEQ ID NO39),

[0324] (iv)VRVEGEAFFRASVDL (peptide 21 of rBCLA) (SEQ ID NO.40)

[0325] (v)EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID N°41);

[0326] (vi)AFFRASVDLYEAVKN(peptide 23 of rBCLA) (SEQ ID NO42),

[0327] (vii)KLRPLTKGELVDVVR(peptide 30 of rBCLA) (SEQ ID NO.43)

[0328] (viii) is an amino acid sequence that is substantially homologous to the sequence of (i)-(vii), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(vii);

[0329] A fragment of at least 9 consecutive amino acids of the sequence (vii)(i)-(viii).

[0330] Therefore, in one specific implementation, the Toxoplasma gondii polypeptide isolated from the internal repeating domain of BCLA for testing its immunoreactivity is selected from the group consisting of:

[0331] (i) An amino acid sequence consisting of the internal repeating domains of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7);

[0332] (ii) an amino acid sequence that is substantially homologous to the sequence of (i), preferably an amino acid sequence that is at least 80% identical to the sequence of (i);

[0333] A fragment of at least 9 consecutive amino acids of the sequence (iii)(i)-(ii).

[0334] In a more specific implementation scheme, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA for testing immunoreactivity is selected from the group consisting of:

[0335] (i)AAGSMEKEKPVLPGEGEGH(TgR4 domain A); (SEQ ID N°44)

[0336] (ii)VLPKHETKPALTDEKRTKPGGP(TgR4 domain B), (SEQ ID N°45)

[0337] (iii) An amino acid sequence substantially homologous to the sequences of (i)-(ii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i)-(ii);

[0338] A fragment of at least nine consecutive amino acids of the sequence (iv)(i)-(iii).

[0339] In a more specific implementation scheme, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA for testing immunoreactivity is selected from the group consisting of:

[0340] (i) AAGSMEKEKPVLPGE (peptide 3 of TgR4); (SEQ ID N°46)

[0341] (ii) GSMEKEKPVLPGEGE (peptide 4 of TgR4) (SEQ ID N°47)

[0342] (iii) MEKEKPVLPGEGEGH (peptide 5 of TgR4) (SEQ ID N°48)

[0343] (iv) KEKPVLPGEGEGHVL (peptide 6 of TgR4) (SEQ ID NO.49)

[0344] (v)KPVLPGEGEGHVLPG (peptide 7 of TgR4) (SEQ ID N°50)

[0345] (vi)HVLPKHETKPALTDEK (peptide 13 of TgR4), (SEQ ID N°51)

[0346] (vii)PKHETKPALTDEKRT (peptide 14 of TgR4), (SEQ ID N°52)

[0347] (viii)HETKPALTDEKRTKP (peptide 15 of TgR4) (SEQ ID N°53)

[0348] (ix)TKPALTDEKRTKPGG(peptide 16 of TgR4) (SEQ ID N°54)

[0349] (x) is an amino acid sequence that is substantially homologous to the sequence of (i)-(ix), preferably an amino acid sequence that is at least 95% identical to the sequence of (i)-(ix);

[0350] A fragment of at least 9 consecutive amino acids of the sequence (xi)(i)-(x).

[0351] Because BCLA peptides have a large number of epitopes that span different domains (especially in rBCLA and in the internal repeating domains of BCLA TgR1-TgR13), combining the BCLA immunogenic peptide fragments of the present invention can be advantageous.

[0352] Therefore, in another embodiment, the isolated polypeptide of the present invention for testing its immunoreactivity is a fusion between two immunogenic peptide fragments of the present invention, such as...

[0353] Peptide AB_F:MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (a fusion of peptide fragments from repeating motifs present in Tgr4 / Trg12 / Tgr13 and repeating motifs present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID N°55)

[0354] Peptide A3_B:AAGSMEKDKLVLPGE (a peptide fragment derived from a repeating motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID N°56).

[0355] Therefore, in another embodiment, the polypeptide of the present invention derived from the BCLA internal repeating domain (TgRx) (for testing its immunoreactivity) has the following sequence:

[0356] M-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5–Xaa6–Xaa7-ME-Xaa8–Xaa9-K-Xaa10-V-Xaa11-PGEG-Xaa12–Xaa13-H-Xaa14-Xaa15 -PK-Xaa16-E-Xaa17-Xaa18-LT-Xaa19-Xaa20-Xaa21-Xaa22-T-Xaa23-P-Xaa24-Xaa25-P-Xaa26-Xaa27-Xaa28(SEQ ID N°64)

[0357] Xaa1 is either glutamic acid (E) or has no amino acid residues.

[0358] Xaa2 is either arginine (R) or serine (S).

[0359] Xaa3 is either proline (P) or glycine (G).

[0360] Xaa4 is either alanine (A) or glycine (G).

[0361] Xaa5 is either alanine (A) or contains no amino acid residues.

[0362] Xaa6 is either glycine (G) or arginine (R).

[0363] Xaa7 can be serine (S), proline (P), or alanine (A).

[0364] Xaa8 is either lysine (K) or glutamic acid (E).

[0365] Xaa9 is either lysine (K), glutamic acid (E), or aspartic acid (D).

[0366] Xaa10 is either proline (P) or leucine (L).

[0367] Xaa11 is either leucine (L) or serine (S).

[0368] Xaa12 is either glutamic acid (E) or lysine (K).

[0369] Xaa13 is either glycine (G) or arginine (R).

[0370] Xaa14 is either valine (V) or alanine (A).

[0371] Xaa15 is either leucine (L) or serine (S).

[0372] Xaa16 can be histidine (H), aspartic acid (D), or alanine (A).

[0373] Xaa17 can be threonine (T), arginine (R), methionine (M), or glutamine (Q).

[0374] Xaa18 can be proline (P), threonine (T), or alanine (A).

[0375] Xaa19 is either aspartic acid (D), glutamic acid (E), or glutamine (Q).

[0376] Xaa20 is either glutamic acid (E) or lysine (K).

[0377] Xaa21 is either lysine (K), glycine (G), or glutamic acid (E).

[0378] Xaa22 is either arginine (R) or valine (V).

[0379] Xaa23 is either lysine (K), glutamic acid (E), or asparagine (N).

[0380] Xaa24 is either glycine (G), valine, or isoleucine (I).

[0381] Xaa25 is either glycine (G) or glutamic acid (E).

[0382] Xaa26 is either arginine (R) or proline (P).

[0383] Xaa27 can be threonine (T), cysteine ​​(C), lysine (K), or methionine (M).

[0384] Xaa28 is either glutamic acid (E) or alanine (A).

[0385] A fragment of at least 9 consecutive amino acids of the sequence SEQ ID N°64.

[0386] "A polypeptide having substantially homologous amino acid sequences" refers to a polypeptide having 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 with a full-length polypeptide reference sequence. In the context of this application, the percentage of identity (i.e., comparing two sequences across their full length) is calculated using global alignment. Methods for comparing the identity of two or more sequences are well known in the art. For example, when considering their full length, the Needle program employing the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) can be used to find the best alignment (including gaps) between two sequences. The Needle program is available, for example, at ebi.ac.uk. The identity percentage according to the present invention is preferably calculated using the EMBOSS::needle (global) program and the Blosum62 matrix, where the “vacancy open” parameter is equal to 10.0 and the “vacancy extended” parameter is equal to 0.5.

[0387] As used throughout this application, the expression "immunoreactivity against the target protein" (hereinforcing the Toxoplasma gondii polypeptide of the present invention) means that the sample from the patient being tested contains antibodies against the specific target.

[0388] Therefore, by demonstrating the presence of antibodies against specific target proteins or fragments of such target proteins in the biological sample being tested, the immunoreactivity against the target protein can be readily detected.

[0389] For example, when compared to the full-length protein, the fragment of the target protein may be truncated at the N-terminus or C-terminus, or may lack internal residues. Preferably, the length of the fragment is 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.

[0390] This test can be performed by a person skilled in the art using standard methods such as enzyme-linked immunosorbent assay (“ELISA”), Western blot / dot blot, immunohistochemistry on transfected cells, and Luminex (see reviews Immunodiagnostics: A Practical Approach, R. Edwards Editor, Oxford University Press 2000; Manual of Molecular and Clinical Laboratory Immunology, JD. F. Edwards, R. G. Hamilton, B. Detrick Editors, ASM Press 2006; Immunology and Serology in Laboratory Medicine, MLTurgeon, Mosby Inc, 2008).

[0391] For example, to determine the presence of anti-BCLA antibodies in a sample, the target protein may be the full-length BCLA polypeptide, the C-terminal antigenic domain of rBCLA (residues 1089-1275 of BCLA) referred to as rBCLA (SEQ ID NO:2), the internal repeating domains of BCLA referred to as TgR1 to TgR13 (residues 304-924 of BCLA) referred to as TgR1 to TgR13 (SEQ ID NO:4-SEQ ID NO:16), or fragments thereof. Preferably, the target protein consists of or comprises the following: the C-terminal antigenic domain (residues 1089-1275 of BCLA, referred to as rBCLA (SEQ ID NO:2)), the internal repeating domains of BCLA (residues 304-924 of BCLA), referred to as TgR1 to TgR13 (SEQ ID NO:4-SEQ ID NO:16)), or fragments thereof.

[0392] The term “patient” as used in this article refers to mammals, and more specifically humans.

[0393] In the context of this invention, the term “treatment” is used herein to characterize a treatment method or process intended to (1) slow down or stop the development, aggravation or worsening of symptoms of a disease state or condition to which the term applies; (2) alleviate or improve symptoms of a disease state or condition to which the term applies; and / or (3) reverse or cure a disease state or condition to which the term applies.

[0394] Folic acid antagonists and / or antibiotic compounds used in the above methods or for treating patients with latent toxoplasmosis are provided in pharmaceutically acceptable carriers, excipients, or diluents that do not harm the patient to be treated.

[0395] Pharmaceutically acceptable carriers and excipients that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, hemoproteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0396] As those skilled in the art will understand, the composition is suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral routes, including, for example, intramuscular, subcutaneous, intravenous, intraperitoneal, or local injection. Oral routes may also be used, provided that the composition is in a form suitable for oral administration and can protect the active ingredient from the effects of gastric and intestinal enzymes.

[0397] Furthermore, the amount of folic acid antagonists and / or antibiotic compounds used in the above methods or for treating patients with latent toxoplasmosis is a therapeutically effective amount.

[0398] The exact amounts of the folic acid antagonist and / or antibiotic compound to be used and the composition to be administered will vary depending on the patient's age and weight, disease type, route of administration, frequency of administration, and other components in the composition containing the folic acid antagonist and / or antibiotic compound. These concentrations can typically be determined by those skilled in the art. The amount of compound actually administered is usually determined by the physician based on relevant circumstances, including the condition to be treated, the chosen route of administration, the actual folic acid antagonist and / or antibiotic compound administered, the individual patient's age, weight and response, and the severity of the patient's symptoms.

[0399] Typically, folic acid antagonists and / or antibiotic compounds used for the methods described above or for treating patients with latent toxoplasmosis can be administered within the typical range. Effective doses will also vary depending on the route of administration and the possibility of co-administration with other agents. For example, typical doses.

[0400] The present invention also provides a kit for the above-described method, the kit being used for diagnosing latent toxoplasmosis or for selecting patients with latent toxoplasmosis suitable for treatment with at least one folic acid antagonist and / or antibiotic compound.

[0401] This kit includes a device for detecting antibodies against at least one of the Toxoplasma gondii polypeptides of the present invention.

[0402] Preferably, the kit contains at least a device for detecting antibodies against the BCLA peptide or the C-terminal antigen domain (residues 1089-1275 of BCLA) or fragments thereof.

[0403] Such a device can be a target protein, namely, the *Toxoplasma gondii* polypeptide or fragment thereof of the present invention for testing immunoreactivity against it as described above. For example, when testing immunoreactivity against BCLA, the target protein is a full-length BCLA protein composed of or comprising: a C-terminal antigenic domain called rBCLA (SEQ ID NO:2) (residues 1089-1275 of BCLA), and internal repeating domains of BCLA called TgR1 to TgR13 (SEQ ID NO:4-SEQ ID NO:16) (residues 304-924 of BCLA). Preferably, the target protein is composed of or comprises: a C-terminal antigenic domain called rBCLA (SEQ ID NO:2) (residues 1089-1275 of BCLA), and internal repeating domains of BCLA called TgR1 to TgR13 (SEQ ID NO:4-SEQ ID NO:16) (residues 304-924 of BCLA).

[0404] The apparatus for detecting antibodies against at least one of the Toxoplasma gondii polypeptides of the present invention may further include antibodies that specifically bind to human antibodies (used as “secondary antibodies”, which bind to antibodies from the sample to be tested that specifically bind to the target protein). These antibodies may be labeled with detectable compounds, such as fluorophores or radioactive compounds.

[0405] In a preferred embodiment, the kit according to the invention may further include a control sample containing a known amount of antibody and / or instructions for using the kit to diagnose latent toxoplasmosis or to select patients with latent toxoplasmosis suitable for treatment with at least one folic acid antagonist and / or antibiotic compound.

[0406] The device can be located in, for example, vials or microtiter plates, or attached to a solid support. For example, the target protein can be attached to a membrane or array.

[0407] Another object of the present invention is a method for detecting merozoite cysts and / or assessing their quantity in a subject, wherein the method comprises:

[0408] a) Detecting the immunoreactivity against the Toxoplasma gondii polypeptide according to any one of claims 1 to 2 in the fluid sample of the subject; and optionally

[0409] b) Derive the presence and / or amount of merozoite cysts from the results of step a), indicating the presence and / or amount of merozoite cysts in the subject based on the immunoreactivity of the Toxoplasma gondii polypeptide of the present invention.

[0410] In a preferred embodiment, the biological sample is the subject's bodily fluid. Non-limiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), and aqueous humor.

[0411] More specifically, the body fluid sample is a serum or aqueous humor sample.

[0412] All references cited in this article, including journal articles or abstracts, published patent applications, published patents, or any other references, are incorporated herein in their entirety by reference, including all data, tables, figures, and text presented in the cited references.

[0413] The invention will be further evaluated based on the following embodiments and accompanying drawings. Attached Figure Description

[0414] Figure 1. BCLA is a merozoite-specific gene regulated by TgHDAC3.

[0415] (a) Whole-proteomic analysis by LC-MS / MS after TgHDAC3 inhibition with FR235222 revealed the expression of merozoite-specific proteins in their BCLA. A volcano plot shows the distribution of *Toxoplasma gondii* by comparing untreated (DMSO, 0.1%) vs. FR235222-treated (90 nM) primary human fibroblasts infected with type II (PruΔku80) strain. The log2 ratio (x-axis) of protein counts was obtained by dividing the intensity of the FR235222-treated sample by the intensity of the DMSO-treated sample (control). Downregulated and upregulated proteins are shown as red dots on the left and right sides of the plot, respectively. Vertical black lines represent log2 fold changes. Horizontal black dashed lines distinguish proteins (red dots), showing at least a 2-fold abundance change with a p-value < 0.01. (b) Bar graph showing BCLA gene expression (fractions of transcript per mega-mapped readings / kilobase [FPKM]) in the following processes: acute (tachyzoites) or chronic (cyst-closed merozoites) infection in mice from day 3 to day 7 after oral infection with Toxoplasma gondii cysts (CZ clone H3), in various feline intestinal epithelial stage (EES) samples (EES1: very early EES; EES2: early EES; EES3: mixed EES; EES4: late EES; EES5: very late EES), and in cysts derived from mouse brains and in vitro cultured tachyzoites. BCLA was expressed only in cyst-closed merozoites during the chronic phase and was not detected in feline intestinal epithelial stages (EES) from EES1 to EES5 (Data source: www.ToxoDB.org). (c) A screenshot of the genome browser (IGB) of the BCLA locus (magenta) on chromosome Ib of *Toxoplasma gondii*, showing readings of two histone markers (H3K14ac, H3K9me3), TgHDAC3, TgCRC230, and RNA-seq (expressed as FPKM, black). The y-axis represents the reading density. This figure shows the enrichment of H3K14ac, H3K9me3, TgHDAC3, and TgCRC230 at the BCLA gene. (d) Left panel: CRISPR-mediated TgHDAC3 gene disruption leads to TgHDAC3 signaling inhibition when monitored by immunofluorescence assay. Right panel: CRISPR-mediated TgHDAC3 gene disruption triggers BCLA overexpression when monitored by immunofluorescence assay.

[0416] Figure 2. The BCLA protein reveals a unique structure characterized by unstructured and tandem repeat sequences.

[0417] (a) The chart shows the symptom score as a function of protein amino acid position (generated via the IUPred server). Results from the ANCHOR2 and IUPred2 algorithms are shown in blue and red, respectively. The C-terminal domain of BCLA (residues 1089-1275, hereinafter referred to as rBCLA) is predicted to be structured, in contrast to the rest of the protein containing the core repeat motif. (b) The BCLA protein encoded by type II (ME49) Toxoplasma gondii strain shows 13 repeats (TgR1 to TgR13) in its structure. Internal antibodies against two peptides (peptide 1 and 2) contained in these repeats were prepared by Eurogentec. (c) BCLA expression monitored by Western blot using self-made antibodies against the two BCLA-derived peptides, compared to DMSO (control), showed upregulation of BCLA after treatment with FR23522.

[0418] Figure 3. BCLA induced by FR235222 is located in the vacuolar space and at the vacuolar membrane.

[0419] (a) Quantification of BCLA intensity in each PV after FR235222 stimulation. Each symbol indicates the BCLA density of a single PV. Results are expressed as mean ± standard deviation of two independent experiments; the number of PVs quantified was at least 70. An asterisk indicates statistical significance when comparing each individual FR235222-treated strain with the corresponding control (DMSO, 0.1%), as determined by an unpaired two-tailed Student's t-test (Mann-Whitney test) (****p<0.0001; NS, not significant).

[0420] Figure 4. BCLA deletion does not significantly affect parasite in vitro growth or vacuolar formation and maturation.

[0421] (a) The percentage of 76k-GFP-luc-Δbcla tachyzoites in HFF cultured in vitro (left panel) and intracellular proliferation rate (right panel) were assessed compared with the WT strain. HFF invasion percentages were very similar in both strains, but BCLA deletion induced a 30% reduction in intracellular proliferation. Results are presented as mean ± standard deviation of two independent experiments. An asterisk indicates statistical significance when comparing 76k-GFP-luc-Δbcla and 76k-GFP-luc by the Mann-Whitney test (unpaired two-tailed Student's t-test), **p < 0.01; NS, not significant.

[0422] Figure 5. BCLA deletion does not significantly alter the virulence or cyst burden of Toxoplasma gondii in mice infected with tachyzoites in the peritoneum.

[0423] (a) Comparison of virulence of the 76k-GFP-luc-Δbcla strain with its parent strain 76k-GFP-luc (WT) in Balb / c and NMRI mice. Balb / c mice (n=20) and NMRI mice (n=43) were administered 10 kJ of 76k-GFP-luc Δbcla via intraperitoneal (ip) injection. 4 and 10 6 (a) Inoculation with tachyzoites and monitoring survival over 35 days. Significance was assessed using the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. Mice infected with Δbcla tachyzoites survived to the same timeframe as the WT strain (NS, not significant). (b) Assessment of the ability of the Δbcla strain to migrate across the blood-brain barrier and form Toxoplasma gondii cysts in the brain of chronically infected mice compared to the WT strain. Brains of NMRI and Balb / c mice presented in (a) that survived the attack were harvested and parasite load and cyst number were assessed, respectively, by quantitative PCR ± cyst count using microscopy. Results are expressed as mean ± standard deviation of at least two independent experiments. Statistical significance was assessed by unpaired two-tailed Student's t-test (Mann-Whitney test). Mice infected with the Δbcla strain showed a trend toward reduced parasite load and cyst number in the brain (but not significantly, NS).

[0424] Figure 6. The capsule of the BCLA defect is characterized by abrupt morphological changes.

[0425] The morphology of cysts containing Δbcla merozoites was compared with that of cysts from the parental 76k-GFP-luc (WT) strain. Brains of challenge-surviving NMRI mice, as shown in Figure 6a, were harvested, and cysts were purified using the Percoll gradient method and morphologically characterized under a microscope. (a) Cyst area and (b) GFP fluorescence intensity of Δbcla-containing cysts were measured using ZEN software (Zeiss) and compared with those obtained from WT cysts. Cysts containing Δbcla exhibited significantly smaller size and lower GFP intensity than WT cysts. Results are expressed as mean ± standard deviation of at least two independent experiments. An asterisk indicates statistical significance when comparing cyst areas of Δbcla-containing and WT cysts, as determined by an unpaired two-tailed Student's t-test (Mann-Whitney test) (***p<0.001). Scale bar, 10 μm.

[0426] Figure 7. BCLA deletion does not alter infectivity or host immune response in mice orally fed with encapsulated protein.

[0427] The virulence and infectivity of cysts containing 76k-GFP-luc-Δbcla were evaluated compared to the 76k-GFP-luc parental strain (WT). C56BL / 6 mice (n=6) and NMRI mice (n=20) were orally infected with 46 cysts of Δbcla and 20 cysts of WT strains, respectively. Acute responses in the ileum were observed in C56BL / 6 mice at 8 days post-infection. Chronic responses in the brain were assessed in NMRI mice at 8–10 weeks post-infection. (a) Parasite load in the ileum of orally infected C56BL / 6 mice 8 days prior was quantified by qPCR. Statistical significance between Δbcla and WT strains was tested by an unpaired two-tailed Student's t-test (Mann-Whitney test). No significant differences were observed (NS, not significant). (b) qRT-PCR analysis of cytokines (IFNγ, IL-22, IL-18, and IL-1β) and chemokines (CCL2) in the ileum of C56BL / 6 mice orally infected 8 days prior. RNA levels were normalized using TBP levels. Mean ± standard deviation is shown. Statistical significance between Δbcla and WT was assessed using the Mann-Whitney test. No significant difference was observed (NS, not significant). (c) Brains of NMRI mice orally infected 8–10 weeks prior were collected, and parasite load and cyst number were evaluated using microscopy by quantitative PCR and cyst counting tests, respectively. Results are presented as mean ± standard deviation of two independent experiments. Statistical significance between Δbcla and WT was assessed using the Mann-Whitney test. No significant difference was observed (NS, not significant). Mice infected with the Δbcla strain showed a trend toward reduced parasite load and cyst number in the brain (but not significantly, NS). (d) qRT-PCR analysis of cytokines (TNF-α, IFNγ, IL-6, IL-22) in the brains of NMRI mice orally infected 8–10 weeks prior. RNA levels were normalized using TBP levels. Mean ± standard deviation is shown. Statistical significance between Δbcla and WT was tested using the Mann-Whitney test. No significant difference was observed (NS, not significant).

[0428] Figure 8. rBCLA does not react with serum from acutely infected mice.

[0429] Single protein blot bands were loaded with 0.5 μg of recombinant rBCLA. Serum blots were tested from mice with various Toxoplasma gondii strains, routes of infection, and mouse genetic backgrounds during the acute phase of toxoplasmosis. rBCLA did not react with mouse antibodies during the acute phase of infection (7–8 days). (a) Bands were tested from 10 mice infected via intraperitoneal injection (ip) of COUG and COUG-Δmyr1 (atypical haplotype 11). 4Immunoblots of serum from NMRI mice infected with tachyzoites for 7 days. The serum did not react with rBCLA. (b) Immunoblots of serum from 10 NMRI mice infected with RH (type I) strain via intraperitoneal injection. 3 (c) Immunoblotting of serum from CBA mice infected with tachyzoites for 7 days. The serum did not react with rBCLA.

[0430] Figure 9. rBCLA is a serological marker of chronic Toxoplasma gondii infection in a mouse model.

[0431] Single protein blots were loaded with 0.5 μg rBCLA and tested on serum collected from mice during the subchronic (21–41 days) or chronic (>42 days) phase of toxoplasmosis. rBCLA reacted only with anti-Toxoplasma gondii IgG antibodies in mice with subchronic or chronic toxoplasmosis following infection with type II cystoplasmosis strains (PruA7, ME49, or 76k-GFP-luc). (a) 10 μg rBCLA samples were collected from mice infected with type II cystoplasmosis strains via intraperitoneal injection during the 42-day period. 3 Up to 10 6 Immunoblots of serum from Balb / c mice infected with tachyzoites / mice. Serum reacted proportionally to rBCLA based on tachyzoite load. (b) Immunoblots of serum from 10 mice infected with ME49(II) strain via intraperitoneal injection over 80 days. 6 Immunoblotting of serum from CBA mice infected with tachyzoites / mice. The serum reacted strongly with rBCLA. (c) Immunoblotting of serum from 20 NMRI mice orally infected with 76k-GFP-luc(type II) strain during 22 months. The serum reacted strongly with rBCLA. (d) Immunoblotting of serum from 10 NMRI mice orally infected with 76k-GFP-luc or 76k-GFP-luc-Δbcla(type II) strain during 21 days. 6 Immunoblot analysis of serum from Balb / c mice infected with tachyzoites / mice. Serum from mice infected with 76k-GFP-luc reacted strongly with rBCLA, while serum from mice infected with 76k-GFP-luc-Δbcla showed almost no reaction with rBCLA. (e) Immunoblot analysis of serum from 10 RH (type I) strains obtained by intraperitoneal injection. 3Immunoblots were taken from serum of CBA mice infected with tachyzoites / mice and subsequently treated with pyrimethamine (PYR) or sulfadiazine (Sulfa) for 22 days. Serum showed very mild reaction with rBCLA. (f, g) 10 serotypes were obtained from CBA mice infected with (f) CTG (type III) strain or (g) PruΔku80 (type II) strain via intraperitoneal injection. 6 Immunoblots of serum from NMRI mice infected with tachyzoites / mice for 42 days. The serum did not react with rBCLA. (h) Immunoblots of serum from 10 tachyzoites / mice infected with 76k-GFP-luc (type II) strain via intraperitoneal injection. 5 Immunoblots of serum from Balb / c mice infected with tachyzoites and reactivated over 42 days or without corticosteroid treatment. All sera reacted strongly with rBCLA.

[0432] Figure 10. Proteolytic analysis of rBCLA reveals the boundary of the minimal antigenic region of BCLA.

[0433] (a) Analysis of proteolytic reactions by SDS-PAGE. Coomassie staining shows SDS-PAGE of all these time points (10, 20, and 50 min) for the input sample and each protease (trypsin, chymotrypsin, elastase, and papain). (b) Blot gels incubated with positive mouse serum and visualized by anti-mouse IgG antibody. (c) Blot gels incubated with anti-6his IgG conjugated to peroxidase. Black arrows indicate undegraded rBCLA. Red and blue cursor arrows indicate recurrent N-terminal degradation, indicating that rBCLA is rapidly degraded by chymotrypsin and partially degraded by elastase, trypsin, and papain, producing stable fragments around the 17-kDa marker.

[0434] Figure 11. Evaluation of rBCLA as a serological biomarker in humans.

[0435] Single protein blots were loaded with 0.5 μg rBCLA and tested on mouse serum infected with human strains or directly on human serum. (a) Immunoblotting of serum from Swiss mice infected via intraperitoneal injection with amniotic fluid or placenta from women with suspected (clinically suspected but amniotic fluid or placental Toxoplasma gondii PCR negative) or confirmed (amniotic fluid Toxoplasma gondii PCR positive) congenital toxoplasmosis. Serum from mice with positive amniotic fluid reacted strongly with rBCLA. (b) Immunoblotting of serum (S) or aqueous humor (HA) from human patients with or without Toxoplasma gondii infection. Human serum and aqueous humor were randomly selected from the biobank of the Parasitology-Mycology Clinical Laboratory at Glennbuzepa University Hospital. For each sample, [the following was performed using...]. (bioMérieux) and (Abbott) systems were used for serological determination of Toxoplasma gondii. Both systems were based on ELISA-derived techniques and the medical records of each patient were used to evaluate the clinical status. It should be noted that is based on the rSAG1 antigen, while is based on the rSAG1 and rGRA8 antigens. The serological results obtained with rBCLA were compared with the serology and clinical status of each patient to evaluate whether they were associated with specific Toxoplasma gondii serology and / or clinical status. Sera from patients with (α) proven or suspected ocular toxoplasmosis, (β) reactivation of toxoplasmosis during hematological diseases (immunosuppression), and (γ) recent primary infection (1 - 2 months) reacted with rBCLA. (δ) Sera from 3 seropositive patients identified as "previously immunized" and sera from 1 patient with a fairly recent infection (2.5 months) did not react with rBCLA. (ζ) All test sera from seronegative patients did not react with rBCLA, indicating good specificity of this antigen in humans.

[0436] Figure 12. Evaluation of BCLA as a human serological marker.

[0437] (a) Schematic representation of the epitope mapping regions in repeat sequence n°4 and the rBCLA region. Peptide coverage is shown as lines representing individual 15aa peptides above or below the peptide sequence, with the partial numbers shown. Regions showing significant or strong reactivity are highlighted in full boxes or dashed boxes respectively, and each individual peptide fragment is labeled with (* or **). (b) Epitope mapping of BCLA-positive sera. The lower figure is a bar graph showing the relative reactivity of peptides on the core repeat region and the rBCLA region, calculated using 5 different positive blots and negative background subtraction. The upper figure is an example of the spot blot membrane imaging pattern with numbered peptides on positive human sera. (c) Peptide spot blot for 5 positive sera and 1 negative serum, with the peptide numbers and regions covered. On the right, the ELISA titrations of rBCLA and SAG1 (Architect) for these same sera are shown.

[0438] Figure 13BCLA reactivity in human serum. Scatter plot of individual BCLA ELISA titers (in UI) grouped into clinical status categories assessed by classic SAG1 serology (Vidas and Architect IgG / IgM) and other medical prerequisites. These groups are as follows: SAG1 seronegative patients (blue dots), previously immunized patients (diamonds), immunocompromised patients with active toxoplasmosis (cubes), immunocompromised patients with asymptomatic serological reactivation (triangles), and confirmed ocular toxoplasmosis (cubes). Bar plots show the median BCLA titer and interquartile range for each group. Statistical significance was calculated using the Kruskal-Wallis nonparametric test, followed by Dunn's post-test, to compare all the latter groups individually with the seronegative patient group. Indicates the gray area (70–90 UI) and the positive cutoff line (at 90 UI).

[0439] Figure 14. Correlation between rBCLA immunogenicity and cyst-causing strains during chronic infection in mice. (a) Serological titration of mouse rBCLA reactivity over time and depending on *Toxoplasma gondii* strains using ELISA. Individual ELISA measurements were given according to the *Toxoplasma gondii* strain type grouping UI, where cyst-causing strains (ME49, PruA7, 76K) were shown as spots, non-cyst-causing strains (RH, PruKU80, CTG) as stars, and ΔBCLA strains (in a 76K or PruKU80 background) as triangles. Post-infection time segmentation is shown to distinguish between the acute phase (≤8 days), subchronic phase (21–22 days), and chronic phase (≥42 days). (b) Correlation between rBCLA ELISA reactivity and parasite load, miR-155, and miR-146a expression. The overlapping titrations of rBCLA IgG (expressed as UI), parasite load (expressed as parasite qPRC count), and miR-155 / miR-142-a are shown in different mouse strains (NMRI, Balb-C) that were uninfected or infected with different Toxoplasma gondii strains across all chronic infection timeframes (≥±11 weeks). Cystogenic strains (ME49, PruA7, 76K) are shown in circles, non-cystogenic strains (RH, PruKU80, CTG) are shown in stars, and ΔBCLA (in the 76K or PruKU80 background) are shown in triangles.

[0440] Figure 15: BCLA reactivity in the serum of mothers and newborns at risk of congenital toxoplasmosis.

[0441] (AB) Violin plots of BCLA ELISA titration (in UI) in serum of 23 mothers and corresponding newborns collected at delivery (mothers) or from birth to 5.5 months of age (infants). (CD) Sag titration ( and Violin plots of IgG / IgM. Serum samples were divided into mother-infant pairs (A and C) without congenital toxoplasmosis and mother-infant pairs (B and D) diagnosed with congenital toxoplasmosis. The mean ± SD values ​​are shown at the top of each subplot, while the difference in medians was calculated using the Mann-Whitney test. Detailed Implementation

[0442] Materials and methods

[0443] Host cell and parasite cultures. HFF primary cells (Bougdour et al., 2009), RAW264.7, L929, HCT116, A549, and HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fischer Scientific, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 10 mM (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (HEPES) buffer pH 7.2, 2 mM L-glutamine, and 50 μg / ml penicillin and streptomycin (Thermo Fisher Scientific). Cells were incubated at 37°C in 5% CO2. The following Toxoplasma gondii strains were used in this study: type I (RH, GT1), type II (ME49), type III (CTG), atypical (COUG), and Neospora caninum; RHΔku80 (Huynh and Carruthers, 2009), PruΔku80 (Fox et al., 2011), PruA7 (Saeij et al., 2007), COUGΔmyr1 (Hakimi, unpublished), and PruΔku80Δbcla, PruΔku80-HF-BCLA, and 76k-GFP-luc-Δbcla obtained in this study. All parasitic strains were maintained in vitro by continuous passage on a monolayer of HFF.

[0444] Toxoplasma gondii transfection was performed. Using a BTX ECM 630 instrument (Harvard Apparatus), Toxoplasma gondii RHΔku80, PruΔku80, and 76k-GFP-luc vectors in cell mixing buffer (120mM KCl, 0.15mM CaCl2, 10mM K2HPO4 / KH2PO4, pH 7.6, 25mM HEPES, pH 7.6, 2mM MEGTA, 5mM MgCl2) were electroporated. Electroporation was performed in 2mm cuvettes at 1.100V, 25Ω, and 25μF. Stable transgenic parasites were selected with 1μM pyrimethamine, cloned by limiting dilution in 96-well plates, and validated by immunofluorescence assay.

[0445] Cas9-mediated C-terminal labeling and gene disruption in *Toxoplasma gondii*. The plasmid pTOXO_Cas9-CRISPR was described by (Sangaré et al., 2016). The target gene (GOI) was BCLA (TGME49_209755), used for C-terminal labeling (HA-Flag (HF)) and gene disruption (KO) using the CRISPR / Cas9 system. Four oligonucleotides corresponding to BCLA were cloned using the Golden strategy. Briefly, primers TgBCLA-CRISP_FWD and TgBCLA-CRISP_REV containing sgRNA targeting the TgBCLA genomic sequence were phosphorylated, annealed, and ligated into a linearized pTOXO_Cas9-CRISP plasmid with Bsal, yielding pTOXO_Cas-CRISPR::sgTgBCLA. *Toxoplasma gondii* tachyzoites were then transfected with the plasmid and grown on HFF cells for 18–36 hours.

[0446] The clonal oligonucleotides used in this study are:

[0447] TgBCLA-KO-CRISP-FWD:

[0448] 5'-AAGTTGATCACTATTCGTGAAGAAGG-3'(SEQ ID N°28)

[0449] TgBCLA-KO-CRISP-REV:

[0450] 5'-AAAACCTTCTTCACGAATAGTGATCA-3'(SEQ ID N°29)

[0451] TgBCLA-HF-CRISP-FWD:

[0452] 5'-AAGTTGGAACGGCGGTACGGCGACCG-3'(SEQ ID N°30)

[0453] TgBCLA-HF-CRISP-REV:

[0454] 5'-AAAACGGTCGCCGTACCGCCGTTCCA-3'(SEQ ID N°31)

[0455] FR235222 treatment and induction. FR235222 was obtained from Astellas Pharma Inc. (Osaka, Japan) and dissolved in DMSO as described by Bougdour et al., 2009, with a final concentration in the culture medium of 25 ng / mL or 50 ng / mL. From 24 hours to 7 days after infection, culture medium containing FR235222 was added to infected HFF cells.

[0456] Mice and Experimental Infections. Six-week-old BALBC / c, CBA, NMRI, or Swiss mice were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France). Mouse care and experimental procedures were performed under pathogen-free conditions according to established institutional guidelines and approved protocols from Institutional Animal Care and the Use Committee of the University Grenoble Alpes (Protocol No. B 3851610006). Female mice were used for all studies. For intraperitoneal (ip) infection, tachyzoites were grown in vitro and extracted from host cells using a 27-gauge needle, washed three times in PBS, and quantified using a hemocytometer. The parasites were diluted in Hank balanced salt solution (Life) and mice were inoculated via ip with 200 μl of tachyzoites for each strain using a 28-gauge needle. For oral feeding of infectious cysts, brains from chronically infected mice (76k-GFP-luc and 76k-GFP-luc-Δbcla) were crushed in PBS, cyst counts were determined under a microscope, and mice were force-fed 100 μl of brain homogenate containing 20–40 cysts using a bulb-tipped feeding needle. Blood was collected via tail puncture or intracardiac puncture when mice were euthanized. Euthanasia was performed in an approved CO2 chamber. For histological analysis of the ileum and immunomarking of brain sections, the ileum and brain were removed from the mice, completely embedded in paraffin blocks, and sliced ​​into 5 μm thick layers using a microtome under a microscope. Statistical analysis of mouse survival data was performed using the Mantel-Cox and Gehan-Breslow-Wilcoxon tests.

[0457] Enzyme purification. Enzymes were isolated from mouse brains chronically infected with 76k-GFP-luc or 76k-GFP-luc-Δbcla strains for at least 6 weeks, using the Percoll gradient method as previously described (Cornelissen et al., 1981), or directly by treating the cysts with 10 μl pipettes for dye experiments to avoid deterioration of the cyst wall for permeability studies. Neither saponins nor trypsin were added at the end of the experiments.

[0458] Cyst quantification. Five to 12 weeks post-infection, the brain of each recipient mouse was homogenized in 2 ml PBS. The number of cysts in three or ten aliquots (20 μl each) of the brain suspension was counted under a microscope. The total number of cysts was determined by multiplying the number of cysts in each 20 μl aliquot by 100. For statistical analysis of differences in cyst quantification between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, the nonparametric Wilcoxon-Mann-Whitney test was used.

[0459] Encapsulation characterization. Images of purified cysts were acquired between a slide and a coverslip using a fluorescence ZEISS ApoTome.2 microscope. Encapsulation area and GFP intensity were measured using ZEN software (Zeiss). The nonparametric Wilcoxon-Mann-Whitney test was used to statistically analyze the differences in encapsulation area and GFP intensity between 76k-GFP-luc and 76k-GFP-luc-Δbcla cysts.

[0460] Quantitative PCR. Following DNA extraction (QiAmp DNA mini kit, Qiagen), the parasite load in the brain or ileum was quantified using quantitative PCR targeting a Toxoplasma gondii-specific 529 bp repeat element (Reischl et al., 2003). For statistical analysis of the difference in parasite load between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, the nonparametric Wilcoxon-Mann-Whitney test was used.

[0461] qRT-PCR analysis of interleukins in the brain and ileum. Total RNA was isolated from the brain or ileum using TRIzol (Thermo Fisher Scientific). cDNA was synthesized using a high-capacity RNA-to-cDNA kit (Applied Biosystems) with random hexamers. Samples were analyzed by real-time quantitative PCR using appropriate probes (brain: TNF-α, INF-γ, IL-6, IL-22β; ileum: INF-γ, CCL2, IL-22β, IL-18, and IL-1β) using TaqMan Gene Expression Master Mix (Applied Biosystems). RNA levels were normalized using TBP levels. qRT-PCR was repeated for three independent bioreplicas of each sample, and the results were averaged. For statistical analysis of RNA levels between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, the nonparametric Wilcoxon-Mann-Whitney test was used.

[0462] Immunofluorescence microscopy. In vitro immunofluorescence assays of parasites were performed as previously described (Braun et al., 2013). Briefly, HFF cells infected with *Toxoplasma gondii* and grown on coverslips or cysts purified from mouse brain were fixed in 3% formaldehyde for 20 min at room temperature, infiltrated with 0.1% (v / v) Triton X-100 for 15 min, and blocked in phosphate-buffered saline (PBS) containing 3% (v / v) bovine serum albumin (BSA). To perform immunolabeling on brain tissue sections, brain layers spotted on slides were first dewaxed with toluene for 3 x 10 min and then with anhydrous ethanol for 3 x 10 min. The slides were then treated with citrate buffer pH 6, heated at 100°C for 1 h, rinsed with water 2 x 10 min, and blocked in PBS containing 3% (v / v) bovine serum albumin (BSA). Then, the cells or brain layers were incubated with the primary antibody shown in the attached diagram for 1 hour, followed by the addition of a secondary antibody conjugated to Alexa Fluor 488 or 594 (molecular probe) at a 1:1,000 dilution for 1 hour. The nuclei of host cells and parasites were stained with 2 μg / ml Hoechst 33258 in PBS for 10 minutes at room temperature. Coverslips were mounted on slides with Mowiol mounting medium, and images were acquired using a fluorescence ZEISS ApoTome.2 microscope and processed using ZEN software (Zeiss).

[0463] Antibodies. Primary antibodies: rabbit anti-BCLA (Eurogentec), mouse anti-HA (Roche, RRID: ab_2314622), rat anti-flag (SIGMA), mouse anti-CC2 (gift from Pr. Louis Weiss), mouse anti-GRA1, mouse anti-GRA5, and mouse anti-GRA7. The Western blot secondary antibodies were conjugated to alkaline phosphatase (Promega), while the immunofluorescent secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 494 (Thermo Fisher Scientific).

[0464] Western blotting. Proteins were isolated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Immobilon-P; EMP Millipore) via liquid transfer. The protein blots were detected using an appropriate primary antibody followed by a phosphatase-conjugated goat secondary antibody (Promega). The signal was detected using NBT-BCIP (Amresco).

[0465] DBA lectin labeling on FR235222 parasites and isolated cysts in vitro. HFF cells infected with *Toxoplasma gondii* and grown on coverslips or cysts purified from mouse brain were fixed in 3% formaldehyde for 20 min at room temperature, infiltrated with 0.1% (v / v) Triton X-100 for 15 min, and blocked in phosphate-buffered saline (PBS) containing 3% (v / v) bovine serum albumin (BSA). Infected cells or cysts were stained with 1:100 diluted Dolichos lectin for 30 min. The stained vacuoles or cysts were examined using a fluorescence ZEISS ApoTome.2 microscope, and images were processed using ZEN software (Zeiss).

[0466] Capsule wall permeability. Capsules isolated from mouse brain using 76k-GFP-luc and 76k-GFP-luc-Δbcla were incubated with different dyes of varying sizes (dextran, Texas Red, or Cascade Blue, 3000-40000 Da, with neutral or anionic lysine fixation) (Promega) at a 1:100 dilution. After incubation at room temperature for 20 minutes, images were acquired using a fluorescence ZEISS ApoTome.2 microscope and processed using ZEN software (Zeiss). At least five capsules were analyzed for each different dye. Capsules incubated without the dye served as negative controls.

[0467] Recombinant expression of the C-terminal domain of BCLA (Cter-BCLA)

[0468] Design and Cloning. Disorder tendency search (using Dis-EMBL or IUPred) predicted that BCLA was highly disordered in most of its sequence, including the core repetitive motif. However, the C-terminus (approximately from aa 1100–1275) was predicted to be structured and could form a separate domain. To recombine this domain, the N-terminal boundary was selected at methionine 1089, and the original C-terminus was conserved. DNA synthesis was performed via Genscript to generate a fusion construct consisting of Cter-BCLA (1089–1275) and a TEV-cleavable N-terminal His-tag (Fig. 1b). Codon optimization for E. coli was performed, and the gene was cloned via Genscript in the pet30-(a) vector (Addgene) using the NdeI and XhoI sites.

[0469] Recombinant expression. Transformation was performed using BL21(DE3)-CodonPlus-RIL chemocompetent *E. coli* (Stratagene), which was incubated with 1 μg of pet30-(a)Cter-BCLA plasmid on ice for 10 min, heat-shocked at 42°C for 45 s, pre-incubated in LB agar at 37°C for 45 min, and then plated onto LB agar plates containing kanamycin (Kan) and chloramphenicol (Chlo) and incubated for 12 h. Single colonies were then selected and inoculated into 50 ml of LB / Kan / Chlo pre-culture that had grown for 16 h. 5 ml of the grown pre-culture was then inoculated into 1 L flasks containing Chlo / Kan Terrific broth. The cultures were grown at 37°C until an OD600 of 0.5–0.8 was achieved, then induced by the addition of 0.7 mM IPTG (VWR) and further incubated at 18°C ​​ON. After incubation, the cells were centrifuged at 3000G for 25 minutes, the supernatant was discarded, and the precipitate was rapidly frozen in liquid nitrogen and kept at -80°C.

[0470] Lysis. Three 1L culture pellets were purified and each resuspended in 50 mL lysis buffer containing 600 mM NaCl, 50 mM Tris pH 8, 5 mM Beta-mercaptoethanol (BME), 0.2% w / v N-LaurylSarkozine, and 1 Complete antiprotease mixture (Roche) tag. Lysis was performed on ice using 10-minute pulsed sonication (15 sec on, 30 sec off) at 50° amplitude, with the lysates never exceeding 13°C. After sonication, the lysates were centrifuged at 15000 G for 1 hour at 4°C and the pellet was discarded. All subsequent steps were performed at 4°C. The clarified lysates were supplemented with 30 mM imidazole before incubation with 5 mL of pre-equilibrated Ni-NTA resin. Incubation was performed in batches at 4°C for 30 min with gentle stirring. After incubation, the resin was retained on a vertical column and washed with 3 x 20 ml wash buffers containing 600 mM NaCl, 50 mM Tris pH 8, 5 mM BME, 0.2% w / v N-Lauryl Sarkosine, and 30 mM imidazole. Then, direct elution with 1.5 ml of fractionation buffer containing 300 mM NaCl, 50 mM Tris pH 8, 5 mM BME, and 300 mM imidazole was performed. The fractions of interest were then combined (Figure 8) and dialyzed using a 10 kDa cutoff dialysis kit (Thermo Scientific) in 50 mM NaCl, 50 mM Tris pH 8, and 5 mM BME.

[0471] Ion exchange and size exclusion chromatography were performed. All samples were then pumped directly onto a 5 mL HL-Mono-Q (GE Healthcare) column pre-equilibrated with the same buffer as for dialysis using a chromatography system (Akta Pure, GE Healthcare). The column was washed with two column volumes (CV) and then eluted with a salt gradient (50 mM – 2 M NaCl) for 40 mL, 1.5 mL fractions, with absorbance monitored at 280 nm throughout the elution. SDS-PAGE analysis (Figure 3) during elution showed that the sample was purified in the later stages of the gradient elution, and that the early elution fractions contained most of the bacterial contaminants visible at higher molecular weights. The desired fractions were collected, pooled, and concentrated to 600 μL using a 10 kDa cutoff concentrator (Amicon-Ultra, Millipore). After concentration, the sample was injected into an S75 (GE Healthcare) containing a running buffer of 150 mM NaCl, 50 mM Tris pH 8, and 5 mM BME, and eluted with a heterogeneous peak consistent with the multimeric state, eluting more than 3 ml starting from near the pore volume. All eluted fractions were collected to produce the final sample.

[0472] Ammonium sulfate precipitation. To avoid nucleic acid contamination, ammonium precipitation was performed by adding 15% w / v ammonium sulfate (Sigma), gently rolling at 4°C for 1 hour, followed by centrifugation at 10000*G for 30 minutes. The supernatant was discarded, and the precipitate was resuspended in the same initial volume of buffer. To remove all ammonium sulfate, the sample was dialyzed in a buffer with the same size exclusion ratio.

[0473] Limited proteolysis was employed to identify antigenic subfractions within Cter-BCLA. To recover highly antigenic subfractions of Cter-BCLA, the purified sample was subjected to limited proteolysis using trypsin, chymotrypsin, elastase, and papain (all from Sigma-Aldrich). Reactions were performed in 30 μl reaction volumes at 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM BME, and 0.5 mM MgCl2. In each reaction, 3 μg of Cter-BCLA was digested with 100 ng of protease (1 / 30 w / w) at 37 °C for 50 min. At each time point, the reaction was stopped by adding 10 μl of SDS-PAGE loading buffer, followed by heating at 95 °C for 5 min, and then holding on ice until loaded onto a gel.

[0474] Western blot BCLA serological assay. Single protein blot bands were prepared using 15-well 4-12% NuPage gels (Life Technologies) loaded with 5 μl of 0.1 mg / ml sample. The gel was run in MES buffer at 185 V for 40 min, followed by electrotransfer at 105 V for 1.5 h on a PVDF membrane. The transferred lanes were then cut into individual bands. The bands were then blocked in TTBS containing 5% milk powder (w / v) for 1 h. Serological testing was then performed in TTBS at 4 °C for 1 h at a 1 / 400 serum dilution. The bands were then washed three times in TTBS and further incubated for 1 h with a 1 / 7500 dilution of a secondary antibody targeting mouse IgG or human IgG and conjugated with phosphatase alkaline enzyme (Promega). After three TTBS washes, the blots were visualized on RT (Invitrogen) by adding a chromogenic substrate. Bands in positive serum appeared within 1–5 min. Parallel to the serum test, a single band was always used as an internal antigen control for each blot group. After blocking, the band was incubated for 1 hour with peroxidase-conjugated anti-polyhistidine monoclonal antibody (Sigma) diluted 1 / 2000 in TTBS. After three washes in TTBS, the blot was visualized using SigmaFast DAB with a metal enhancer (Sigma). For each series of mice infected by IP or oral administration, Western blot analysis of the IgG immune response was performed using the commercially available LD bio Toxoplasma gondii mouse IgG kit (LD bio), using the same anti-mouse IgG-alkaline phosphatase conjugate and chromogenic substrate previously described for BCLA, with serum tested for Toxoplasma gondii antibodies.

[0475] Human serum. Human serum was retrospectively selected from the biobank collection of the Clinical Laboratory of Parasitology and Mycology at Grenoble Alpes University Hospital, France. This biobank is registered with the French Ministry of Health under number DC-2008-582. Selected sera were stored between January 1, 2014, and May 1, 2018, for routine Toxoplasma gondii serological analysis. The analyses of Toxo IgM and IgG (bioMérieux, France) and Architect Toxo IgG and IgM (Abbott, Germany) were performed in the Parasitology-Mycology Clinical Laboratory at Grenoble Alpes University Hospital.

[0476] Proteomics analysis based on protein purification, Western blotting, and mass spectrometry. Host HFF cell extracts infected with PruΔku80-BCLA-HAFlag containing Flag-tagged proteins were incubated with anti-FLAG M2 affinity gels (Sigma-Aldrich) at 4°C for 1 hour. Beads were washed with 10 column volumes of BC500 buffer (20% glycerol, 20 mM Tris-HCl pH 8.0, 500 mM KCl, 0.05% NP-40, 100 mM PMSF (phenylmethylsulfonyl fluoride), 0.5 mM DTT, and 1X protease inhibitor). Bound peptides were eluted stepwise with 250 g / ml FLAG peptides (Sigma-Aldrich) diluted in BC100 buffer. Protein bands were excised from colloidal blue-stained gels (Thermo Fisher Scientific), treated with DTT and iodoacetamide to alkylate cysteine, and then digested in the gel using modified trypsin (Sequencing grade; Promega). Peptides obtained from individual bands were analyzed using a 25-minute gradient analysis on an online nanoLC-MS / MS (UltiMate 3000 coupled with LTQ-Orbitrap Velos Pro; ThermoFisher Scientific). Peptides and proteins were identified and quantified using MaxQuant (version 1.5.3.17) through a companion search against ToxoDB (version 20151112) and a database of frequently observed contaminants embedded in MaxQuant. The minimum peptide length was set to 7 amino acids. The minimum number of peptides, razor+unique peptides, and unique peptides was all set to 1. The maximum false discovery rate for both peptide and protein levels was set to 0.01.

[0477] BCLA repeat sequence and rBCLA epitope mapping. Custom-synthesized dot-blot peptide assays were performed on cellulose membranes with an N-terminal N-acetyl moiety using JPT peptide technology. Two sets of membranes were screened: 1) covering the rBCLA region (residues 1089-1275) with a total of 59 peptides, each 15 amino acids long, with 12 overlaps and 3 offsets; 2) covering repeat sequence 4 (residues 446-493) with a total of 18 peptides, each 15 amino acids long, with 12 overlaps and 3 offsets. Dot-blot analysis was performed as described by the manufacturer. Briefly, the membrane was first activated in 100% ethanol for 5 min, then washed three times in DPBS-Tween for 3 min each time. It was then blocked ON in DPBS-Tween 0.5% milk powder at 4°C, followed by washing three times in DPBS-Tween for 3 min each time. The test serum was diluted to 1 / 400 in DPBS-Tween 0.1% BSA and incubated with the membrane at room temperature for 3 h. After washing with DPBS-tween for 3*3 min, the membrane was incubated with anti-IgG peroxidase-coupled Ab (Sigma A0170) diluted to 1 / 100,000 for 2 h at room temperature. After washing with DPBS-tween for 3*3 min, the membrane was briefly immersed in freshly prepared SuperSignal West Pico chemiluminescent substrate (ThermoFisher) and visualized using a C-Digit (Licor) scanner. ImageJ integrated spot intensities were used. For data analysis of independent dot blots, the baseline integrated intensity of peptide 59, which had never reacted with any serum, was used. (p＝59) The integral intensity from each peptide point [I] (p) Normalized to enrichment factor Fe (p) The following can be expressed by the following equation:

[0478] Fe (p) =I (p) / I (p＝69)

[0479] Where p represents the peptide number.

[0480] To increase the reactivity score of several independent positive serological blots marked (+), Fe was... (p) Enrichment scores are summed and nonspecific reactivity is subtracted. The same summing and subtraction are performed on the same number of negative serum peptides marked (-). Peptide reactivity scores can be expressed by the following equation:

[0481] Rs (p) =[∑(Fe (p) ) (+) -∑(Fe (P) ) (-) ]

[0482] Where Rs (p) It is the total reactivity score at a specific peptide location.

[0483] BCLA ELISA. Peptide synthesis: The following BCLA peptides were synthesized via Genscript with an N-terminal acetyl group:

[0484] AB_F:

[0485] Nter-MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP-Cter(SEQ ID N°55)

[0486] A3_B:Nter-AAGSMEKDKLVLPGE-Cter(SEQ ID N°56)

[0487] Plate preparation: Midisorp plates (Nunc) were coated at 100 μl / well with 2 μg / ml rBCLA, peptide AB_F, and A3_B in 100 mM calcium carbonate buffer (pH 9.6) at 4°C. After coating, the plates were washed twice with 350 μl of 0.05% Tween 20 DPBS (DPBS / Tween), then blocked with 300 μL of Superblock blocking buffer (ThermoFisher) for at least 2 hours. The buffer was then removed, and the plates were inverted and dried. Once dried, the plates can be stored at 4°C for extended periods without loss of serological reactivity.

[0488] Sample preparation: All serum diluents were prepared in DPBS 0.05% Tween 20, 0.1% BSA no more than 2 hours prior to assay. For mouse and human test sera, 1 / 400 dilutions were prepared. Eleven standards were also freshly prepared for both assays, consisting of 10 serially diluted positive cryopreserved serum solutions set at 100 UI. The following titration points were prepared starting at a 1 / 200 dilution and in 3 / 4 dilution increments: 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). OUI standards were prepared using serum-negative serum diluted 1 / 400.

[0489] Assay: All subsequent steps were performed on the Gemini ELISA automated platform (Stratec), but could also be performed manually at room temperature. First, the dried plate was washed twice with 350 μl of DPBS / Tween. Then, the test serum and standard diluents were dispensed in duplicate into the plate at 100 μl / well. The plate was then incubated at room temperature for 1 hour. After incubation, the plate was washed four times with 350 μl of DPBS / Tween, and then 100 μl of peroxidase-conjugated secondary antibody dilution (1 / 50,000 anti-mouse IgG or 1 / 60,000 anti-human IgG, Sigma Aldrich ref A0168 and A0170, respectively) was rapidly dispensed into all wells in DPBS 0.05% Tween 20, 0.1% BSA. After 1 hour at room temperature, the plate was washed four times in DPBS Tween. The reaction was initiated precisely at room temperature for 20 minutes by adding 100 μl of TMB substrate (Thermofisher ref 34029), followed by termination with 50 μl of 0.2 M H2SO4, and then mixed for 30 seconds to allow for color development. The absorbance was then measured at 450 nm using a Gemini integrating spectrophotometer.

[0490] Data processing: Blank subtraction was performed in duplicate blank wells. No primary antibody / serum was processed in the wells, but all subsequent steps (washing, secondary antibody, substrate) were performed. The average of the standard serum dilutions was taken and fitted with a 4-parameter logistic regression, where the upper asymptote value (D... i The value is fixed at 2.5 AU and allows fitting of all other variables (A). i B i C i From this regression, if a coefficient of variation greater than 10% is observed in duplicate measurements, the apparent UI of the diluted duplicates can be calculated and averaged, and the samples are then retested. All ELISA data presented in this work were obtained in several independent titrations.

[0491] result

[0492] Quantitative analysis of the proteome in tachyzoites responding to FR235222 identified BCLA as a novel merozoite-specific protein.

[0493] The specific inhibition of TgHDAC3 by the cyclic peptide FR235222 revealed a disruption of histone H4 acetylation across the de-repressed homeostatic levels of the Toxoplasma gondii genome, inducing phase-specific genes (Bougdour et al., 2009; Sindikubwabo et al., 2017). We have leveraged the properties of FR235222 to develop an in vitro cyst-forming system capable of producing large quantities of proteins required for large-scale proteomic studies (Farhat D et al., manuscript preparation). Quantitative proteomics studies following low-dose and short-duration treatment of cyst-inducing type II (PruΔku80) strains revealed a significant enrichment of phase-specific proteins in the FR235222-treated proteome, including those considered confined to the merozoite stage (Fig. 1a). Based on this analysis, we found that the protein TGME49_209755 (hereinafter referred to as BCLA (brain cyst-load-associated antigen)) was significantly induced after FR235222 treatment in the same manner as several proteins involved in the chronic infection phase (Fig. 1a). This is consistent with its expression profile, which has been reported as being limited to the fission subset dataset (Figure 1b, source: ToxoDB).

[0494] Other evidence supports the epigenetic regulation of BCLA expression. We recently reported transiently repressed H3K14ac and H3K9me3 PTMs bookmarked genes awaiting parasite stage differentiation for stage-specific expression (Sindikubwabo et al., 2017). In tachyzoites, the BCLA locus showed enrichment of this dual PTM, which discriminately marks 'balanced' stage-specific genes (Fig. 1c). Furthermore, a recent TgHDAC3 ChIP-seq analysis (Farhat D et al., manuscript preparation) revealed the presence of histone deacetylases at the BCLA locus (Fig. 1c). CRISPR-mediated gene disruption of BCLA-induced TgHDAC3 in transfected tachyzoites provided definitive genetic evidence that TgHDAC3 is involved in its regulation (Fig. 1d), mimicking the role of FR235222 in the enzyme. From these data, we conclude that BCLA belongs to the merozoite gene family regulated by TgHDAC3, and that its surrounding heterochromatin, represented by so-called bivalent chromatin domains, can silence developmental genes while maintaining their balance for rapid activation after cell differentiation (Sindikubwabo et al., 2017).

[0495] BCLA is secreted into PV and associates with PVM containing merozoites transformed in vitro.

[0496] BCLA is a single open reading frame encoding a 140-kDa protein with a predicted N-terminal signal peptide and a conserved C-terminal region of ~150 residues adjacent to a central core domain represented by a motif repeated 13 times with 48 amino acids (Fig. 2a), the composition and frequency of which have evolved through the subclass Coccidia and in the lineage of *Gnaphalium tigrinum* (Fig. 2b). While BCLA homologs are poorly conserved in *Neospora canis*, they share a generally similar structure, with shorter repeat sequences exhibiting characteristics common to BCLA repeat sequences (data not shown). Disorder tendency searches (using dis-embl or IUPred) predict that BCLA is highly disordered in most of its sequence, including the core repeat motif. However, the C-terminus (approximately from aa 1100–1275) is predicted to be structured and could form a separate domain (Fig. 2a).

[0497] Although BCLA was definitively and specifically identified by mass spectrometry in FR235222-treated samples (Fig. 1a), the protein dynamics and subcellular distribution during infection remain poorly investigated. To further investigate the dynamics of BLCA in *Amoebae gargarizans* in situ, we generated polyclonal antibodies against two synthetic peptides located at the ends of conserved repeat sequences (Fig. 2a). We first validated the proteomic data by showing that cell exposure to FR235222 significantly increased the BCLA signal intensity, which was a protein band of the expected size of approximately 140 kDa, otherwise undetectable in untreated tachyzoites (Fig. 2c).

[0498] In fibroblasts containing tachyzoites, which express bradyzoite-specific markers in the form of a C-terminal HA-Flag tag, BCLA was clearly detected in the vacuolar space upon stimulation with FR235222 and accumulated clearly at the PVM, with its expression consistent with the induction of merozoite markers ENO1 and LDH2 (data not shown). Conversely, BCLA was no longer detected in cells infected with tachyzoites genetically engineered to lack BCLA (Δbcla, Table 2), thus confirming the specificity of the endogenous antibody (data not shown). Finally, when we monitored BCLA kinetics in type I (RHΔku80) and type II (PruΔku80) lines expressing endogenous proteins fused with the HA-Flag tag, we showed that HA-tagged BCLA proteins target both the vacuolar space and the membrane upon stimulation with FR235222, regardless of strain type (data not shown). Therefore, the presence of the C-terminal fusion tag does not affect the subcellular localization of BCLA, as it is similar to that observed when using anti-BCLA serum in untagged strains.

[0499] When different parasitic strains of *Amoeba gruntii* were exposed to FR235222, we eventually found that the BCLA signal intensity varied greatly with the infected strain, from very strong induction in type II strains (PruΔku80, ME49, 76K-GFP-Luc), to rather mild in type I (GT1 and RHΔku80) and haplotype II (COUG) strains, and surprisingly, a weak (if absent) signal was detected in cells infected with type III (CTG) strains (Figure 3a and data not shown). This difference can be explained below by the strain's ability to readily produce tissue cysts.

[0500] BCLA is located in the capsule matrix and capsule wall in vivo.

[0501] Glycosylated cyst walls, where Bifidobacterium lectin (DBA) binds, are a key structural feature promoting persistence and oral delivery in *Amoebae gessoides* (Tomita et al., 2013). Here, we provide strong evidence (data not shown) of co-staining BCLA with DBA only around the membrane of merozoites transformed in vitro, strongly suggesting that BCLA accumulates over time in immature cyst walls after delivery into the vacuolar space (based on thin DBA-positive cyst walls).

[0502] However, considering that in vitro merozoite development in tissue culture does not lead to fully mature cysts, we re-examined the localization of BCLA in merozoite-containing cysts isolated from mice chronically infected with Amoeba spp. type II. In chronically infected mice, the internal antibody against BCLA stained the cyst wall and the matrix space surrounding the merozoite (data not shown). Immunofluorescence results did not allow for an unambiguous determination of whether BCLA was located in the inner or outer layer of the cyst wall; however, interestingly, the impermeable detached cysts were readily stained by the antibody, indicating the external location of the protein (data not shown) and thus its exposure to the host cell cytoplasm. No signal was detected on cysts containing Δbcla merozoites (data not shown), thus confirming the in vivo specificity of the anti-BCLA antibody (Fig. 4d).

[0503] There is little evidence of extravagal function of BCLA, but occasionally the protein appears to be exported outside the vacuolar membrane into the cytoplasm of the host cell (data not shown). Unfortunately, despite numerous attempts, we have not found a specific case where BCLA export extends beyond the PVM to further investigate its function in the host cell (if any), as we have with other effectors (Hakimi et al., 2017). However, we were able to show that BCLA export is Myr1-independent (data not shown), thus requiring no amoeba translocation of the exporting protein (Franco et al., 2016). An elegant way to explain the accumulation of the protein in the cytosol of infected cells is post-processing release at the PVM, possibly under the control of host proteases, but this remains to be demonstrated. The BCLA-associated proteome of infected and FR235222-stimulated host cells will be analyzed to determine whether interactions (if any) between BCLA and host cell proteins (including proteases) occur on the outward-facing side of the PVM, or even in the cytoplasm of the infected cell when BCLA is delivered there.

[0504] BCLA is capable of allocating appropriate capsule function within the body.

[0505] To determine the function of BCLA in merozoite cysts, we created two parasitic lines in which the coding region of BCLA was deleted (PruΔku80Δbcla) or interrupted via DHFR box using Cas9-mediated gene editing (76K-GFP-LucΔbcla) (Table 2). We then investigated pathogenesis and cyst formation. First, the BCLA-deficient strains did not show a significant growth phenotype under in vitro tachyzoite conditions compared to their parental strains. Figure 4A (And data not shown). BCLA mutations do not impair the expression of PV-resident proteins or PVM-related proteins, nor do they impair the localization of PV-resident proteins or PVM-related proteins previously identified as being involved in PV formation and maturation (i.e., GRA1, GRA5, GRA7; data not shown). As shown in vacuoles containing Δbcla labeled with lectin DBA after FR235222 stimulation, no differences were detected in the ability of BCLA-deficient parasites to transform into merozoite stages and form cysts in vitro (data not shown).

[0506] BCLA is not necessary for inducing infection in tachyzoites.

[0507] To investigate the role of BCLA in vivo during acute infection, we compared the parasitic process in BALB / c or NMRI mice infected intraperitoneally (ip) with WT or BCLA-deficient parasites from a type II background, with an inoculum concentration of 1 × 10⁻⁶.4 -1×10 6 Within the tachyzoite range. Five to eight days post-infection, all mice infected with type II BCLA-deficient tachyzoites began to exhibit signs of infection (i.e., weight loss and feathering) and survived infection within the same timeframe as the parental strain 76K, regardless of the inoculum or genetic background of the mice (Fig. 5a). Therefore, BCLA appears to be unnecessary for in vivo growth and pathogenesis during the acute phase of infection in mice. Serological responses to parasite antigens in animals surviving challenge by Western blot assay at 10 weeks post-infection were subsequently observed (data not shown). Clearly, BCLA deficiency does not impair infectivity, as all mice exhibited anti-Toxoplasma gondii IgG in the same pattern, regardless of the parasite strain (data not shown).

[0508] BCLA deficiency affects the integrity of brain cysts isolated from chronically infected mice.

[0509] Examination of the brains of mice infected with the Δbcla mutant demonstrated that cyst formation still occurred in the presence of the mutant strain (Fig. 6b). However, the BCLA-deficient mutant produced a considerably reduced parasite load in the CNS of chronically infected mice compared to the parental strain, though the difference was not statistically significant (Fig. 6b and data not shown), demonstrating that BCLA is at least not distributable for the establishment and maintenance of cysts during chronic infection. However, thorough examination of the cysts revealed that those isolated from mice infected with the mutant parasite were relatively small (Fig. 6a) and contained fewer merozoites, resulting in a “lower packing density” (Watts et al., 2015) and an overall decrease in GFP fluorescence (Fig. 6b), which was entirely consistent with the slight decrease in parasite load measured in the total brain (Fig. 6b). In addition to these quantitative indicators, Δbcla-containing cysts are characterized by significant deformation of their cyst wall surface leading to a loss of roundness, and to some extent exhibit a unique “budding” and “segmentation or rupture” phenotype (Figure 6a and data not shown), suggesting a possible role of BCLA in cyst growth, maintenance and / or stability.

[0510] We then assessed whether surface malformations made the cysts brittle, a phenotype previously reported in Δcst1-containing brain cysts (Tomita et al., 2013). Although the cysts were subjected to mechanical stress during their isolation to release them from the brain tissue for purification by isodense centrifugation (see Methods), we did not observe that Δbcla-containing cysts were more brittle than WT cysts during this harsh procedure (data not shown); however, very few of them broke apart, independent of genetic background.

[0511] BCLA deficiency does not impair the wall staining of difluorophosphate choline lectin (DBA) in cysts isolated from the brains of chronically infected mice (data not shown). Therefore, as concluded on tachyzoites treated with FR235222, BCLA does not directly imply galactosylation of the cyst wall. The viability of merozoites within cysts is contingent on the permeability of the wall to nutrients from host cells, which is very limited; the wall acts as a sieve to avoid components of the immune response. To test whether wall permeability is altered in the absence of BCLA, we monitored the entry of fluorophores of different sizes, representing 3–40 kDa, into the cysts. Intact cysts (without parasite leakage) were observed and examined only under a microscope. The permeability between WT and BCLA-deficient cysts with 3-kDa (diffusion pattern throughout the cyst matrix) or 10-kDa (diffusion pattern with punctate locations) staining was remarkably similar. Interestingly, fluorescent tracers with higher molecular weights (40-kDa) did not penetrate the capsule wall effectively, as previously reported (Lemgruber et al., 2011). Furthermore, the weak labeling varied even between strains, possibly because capsules containing Δbcla were more “loose” and permeable than those containing the parental strain, and were filled with merozoites surrounded by a less permeable, well-defined, and continuous capsule wall (data not shown). Overall, our results suggest that BCLA is allocable to appropriate capsule function in vivo; however, the protein plays a structural role in the capsule wall, which can lead to a defective capsule wall permeability phenotype.

[0512] BCLA is not necessary for effective oral infection of Toxoplasma gondii containing merozoite cysts.

[0513] To examine the in vivo functional outcomes of BCLA-dependent cyst deformation, we fed mice with cysts containing either Δbcla or the parental strain and assessed virulence and infectivity in two different mouse genetic backgrounds. C57BL / 6 mice were orally infected with 46 cysts of either the 76k-GFP-luc-Δbcla or 76k-GFP-WT strains, and the invasion and spread dynamics of the parasite in the intestine and the local immune response induced by the parasite were investigated. On day 8 of infection, Toxoplasma gondii-specific IgG levels in mouse serum were very similar (data not shown), and they did not differ significantly in parasite load in the ileum (Fig. 7a). Histological analysis of the ileum revealed an overall loss of intestinal epithelial structures with altered crypt-villus morphology (data not shown) and inflammatory loci (data not shown), independent of strain genetic background. Cytokine profiling showed the same pattern, with a significant increase in pro-inflammatory cytokines (IFN) and chemokines (CCL2) in the ileum, but in a BCLA-independent manner (Fig. 7b). We then orally infected NMRI mice with 20 cysts to assess the ability of Δbcla cysts to spread into the bloodstream and form new cysts in deep tissues. All orally infected mice exhibited signs of disease (weight loss) and seroconversion during the acute infection phase (data not shown). At 10 weeks, no significant differences were detected between strains in terms of cyst number and parasite load for all mice (Fig. 7c). These data suggest that BCLA-deficient cysts can spread infection orally and establish a chronic infection characterized by a mild inflammatory state in mice. A profile of pro-inflammatory cytokines in the brains of chronically infected NMRI mice suggested that inflammation in Δbcla was less severe than in wild-type mice; however, this was not statistically significant and may be due to the low sample size (3 mice per disease; Fig. 7d). This relatively mild inflammation in the brain may be a consequence of the relatively small number of cysts in Δbcla-infected mice; however, this must be determined.

[0514] High-level expression and purification of BCLA chimeric peptide for serological diagnostics

[0515] The humoral and cellular defenses of the innate immune system are the body's first line of defense against Toxoplasma gondii. Antibodies have been reported to help clear the parasite during acute infection and mediate resistance to secondary Toxoplasma gondii infection (Sayles et al., 2000). Therefore, once immunity is established, IgG protects the fetus from vertical transmission during pregnancy. Although the serological distinction between acute and chronic infection is clinically and epidemiologically relevant, there is currently no merozoite-specific serological assay for toxoplasmosis to accurately estimate the time of infection and the presence of cysts. Furthermore, reactivation can occur in both fully immune patients (e.g., retinochoroiditis) and immunocompromised patients, and the presence of cysts in the brain has recently been suspected as being associated with several neuropsychiatric disorders. Therefore, detecting Toxoplasma gondii antibodies against semi-dormant cysts would be a significant improvement in the serological diagnosis of toxoplasmosis by opening up new diagnostic perspectives. However, at least in commercial kits, few components of the cyst wall or surface merozoites are identified, and none show antigens suitable for serological purposes. Ideally, antigens should be expressed only in the latent merozoite stage and ideally exposed on the surface of the capsule. Two characteristics found in BCLA prompted us to test its antigenicity.

[0516] To obtain the high purity and large quantity of BCLA required for serum WB testing, we selected the C-terminal domain of recombinant BCLA (residues 1100-1275, hereinafter referred to as rBCLA), which was predicted to be structured, in contrast to the rest of the protein containing the core repeating motif (Fig. 2a). Thus, rBCLA was expressed in *E. coli* as a chimeric protein with an N-terminal polyhistidine tag. Although efficiently expressed, it is natively insoluble or isolated as an insoluble inclusion body, but can be dissolved using 0.2% N-Lauryl Sarkoside during the lysis step. After lysis and centrifugation, rBCLA was first pulled down with nickel affinity resin (data not shown). At a theoretical Mw of 20.9 kDa and a pI of 4.7, BCLA was observed migrating between 17-25 kDa molecular weight markers on an SDS-PAGE gel; similarly, in pH:8 buffer, BCLA would be strongly negatively charged. Therefore, anion exchange chromatography was used to effectively remove *E. coli* contaminants (data not shown). Finally, rBCLA eluted from size exclusion chromatography in a soluble form (data not shown), although polydisperse due to the wide elution range and forming multiple oligomers when the elution volume approached the void volume of the S75 column. After combining the elution fractions, a final stage of ammonium sulfate precipitation and dialysis was performed to remove nucleic acid contaminants (data not shown). Following the final purification stage, tachyzogenic antigens of the RH strain (LD bio) and rBCLA were analyzed separately by SDS-PAGE, followed by immunoblotting with mouse antiserum induced by different states of toxoplasmosis to allow for parallel analysis of antigen recognition via immunoglobulins G, M, and A.

[0517] rBCLA does not react with the serum of acutely infected mice, but constitutes an excellent antigen for detecting Toxoplasma gondii from chronically infected mice.

[0518] We first performed immunoblotting on serology collected from mice during the acute infection phase. rBCLA protein apparently did not react with serology from mice acutely infected with atypical (COUG, monomeric type II), virulent (RH, type I), or cystic (76K, type II) strains (Fig. 8a-c), but did react with serology from mice exposed to all *Toxoplasma gondii* strains, regardless of their genetic background (NMRI, CBA, C57BL / 6) or route of infection (intraperitoneal or oral) seroconversion (Fig. 8a-c, data not shown). However, rBCLA reacted strongly with *Toxoplasma gondii* IgG antibodies in mice that developed latent toxoplasmosis after infection with type II cystic strains (Pru, ME49, or 76K) (Fig. 9a-c). rBCLA was detected in mouse serology during subchronic (>21 days, Fig. 9d) or chronic (>42 days, Fig. 9a-c) infection phases, with extremely strong signals observed in serology from mice continuously infected for 22 months (Fig. 9c). When serum from mice uninfected or chronically infected with BCLA-deficient strains was measured, no reactivity was detected, indicating that IgG antibodies are specifically anti-BCLA in vivo (Fig. 9d). Since the selection process occurs during antibody affinity maturation (Eisen, 2014), we infer that the rBCLA antigen can be detected by comparing IgG during acute infection with IgG during chronic infection. Clearly, rBCLA does not react with anti-Toxoplasma gondii IgM or IgA (data not shown). Therefore, these findings strongly support the ability of rBCLA to distinguish the parasitic stage in infected mice, exhibiting preferential IgG reactivity for latent infection.

[0519] Only rBCLA was detected in the serum of mice persistently infected with the cyst-causing strain.

[0520] The results showed that rBCLA exhibits a specific reactivity to cystic strains that readily induce latent infection (Figs. 9a-d). However, to maintain this argument, it must be shown that non-cystic strains do not produce a specific antibody response against rBCLA. Serological analysis of animals first infected with a non-cystic virulent strain RH and subsequently treated with pyrimethamine or sulfadiazine to overcome acute toxoplasmosis showed enrichment levels of anti-tachyzoite-specific antibodies (22 days post-infection; Fig. 9e, bottom), while rBCLA was barely detectable (Fig. 9e, top). Since we could not rule out that the treatment altered the transmission of the parasites in deeper tissues and thus altered their differentiation into merozoites, we monitored the IgG response to rBCLA in mice persistently infected with CTG, a type III strain that causes non-lethal chronic latent infection characterized by a favorable positive serology (Fig. 9f, right). No reactivity to rBCLA was observed 42 days post-inoculation (Fig. 9f). The main difference from type II infection (Figs. 9a–d) was the lower (if any) number of cysts in the brains of mice chronically infected with CTG (Cannella et al., 2014), suggesting a possible relationship between cyst load and rBCLA antibody levels. Similarly, serum from mice persistently infected with type II (PruΔku80) strains, which typically produce lower cyst numbers, did not react with rBCLA (Fig. 9g), indicating that the mouse antibody response to the rBCLA antigen occurs rapidly after subchronic infection (>21 days pi) and appears to be regulated, at least in the mouse model, by the presence of cysts. Reactivation of latent type II infection with immunosuppressive therapy (corticosteroids) did not enhance the antibody response against rBCLA (Fig. 9h), ruling out the hypothesis of an immune response in response to release into the merozoite cycle.

[0521] Limited proteolysis to discover antigenic subfractions within rBCLA

[0522] To recover the highly antigenic subfraction of rBCLA, limited proteolysis of the purified sample was performed using trypsin, chymotrypsin, elastase, and papain. Proteolysis analysis by SDS-PAGE (Fig. 10a) showed that rBCLA was rapidly degraded by chymotrypsin and partially degraded by elastase, trypsin, and papain, producing a stable fragment around the 17-kDa marker. When Western blotted against positive mouse IgG serum (Fig. 10b) and His tag (Fig. 10c) using the same protocol as described above, most degradation was observed at the C-terminus, as they remained positive in the His tag blot. These same degradations presented as low-intensity bands in anti-mouse IgG WB, indicating that further truncation of the construct did not increase the specificity or sensitivity of mouse IgG in Western blot analysis.

[0523] rBCLA also reacts with human serum, however, the positive pattern is still under investigation.

[0524] We then showed that, unlike mice infected with qPCR-negative amniotic fluid or placenta, mice infected with positive amniotic fluid from pregnant women who were first infected during pregnancy and confirmed to have congenital toxoplasmosis clearly responded to rBCLA ( Figure 11A Therefore, rBCLA is a suitable serological marker for predicting the cyst-causing properties of clinical isolates. Following the evaluation of anti-rBCLA immunoglobulin detection in a mouse model, our aim was to assess the pattern of anti-rBCLA detection in humans based on patient serological and clinical status (Table 3). Antibodies against the rBCLA antigen were detected in three patients with strongly suspected or confirmed ocular toxoplasmosis, either in serum only or in both serum and aqueous humor (Figure 11b). These clinical cases were due to reactivation of *Toxoplasma gondii* cysts in the retina, rather than primary infection, as IgM was not detected. In the same vein, three patients with reactivated toxoplasmosis due to hematologic-associated immunosuppression also had anti-rBCLA IgG, but the labeling in the protein blot was weaker compared to patients with ocular toxoplasmosis, despite the use of... and Antibody levels were quite high (Table 3). Even though the relatively small sample size limited broad generalization, the reactivity of rBCLA to human serum induced by toxoplasmosis reactivation provided further evidence for our mouse model, in which we correlated the presence of rBCLA as a serological marker and cyst load. Surprisingly, three recently seroconverted sera from pregnant women and one serum from a child with congenital toxoplasmosis also reacted with rBCLA (Figure 11b). While this is difficult to prove, recent primary infection may generate *Toxoplasma gondii* cysts in peripheral tissues, thereby triggering a humoral anti-BCLA immune response. In any case, rBCLA was undetectable in the sera of all patients identified as *Toxoplasma gondii* seronegative, indicating good specificity of this antigen for patients with toxoplasmosis (Figure 11b).

[0525] Table 2. Toxoplasma strains used in this invention

[0526]

[0527] Table 3. Reactions of rBCLA antigen with some human serum and aqueous humor samples

[0528]

[0529]

[0530] Manufacturer recommends using and Cutoff values ​​for interpreting serological values

[0531] IgG (IU / mL): Negative <4; Gray area: 4.0 ≤ x < 8.0; Positive: ≥ 8.0

[0532] IgM (index): Negative < 0.55; Gray area: 0.55 ≤ x < 0.65; Positive: ≥ 0.65

[0533] IgG (IU / mL): Negative <1.6; Gray area: 1.6 ≤ x < 3.0; Positive: ≤ 3.0

[0534] IgM (index): Negative < 0.50; Gray area: 0.50 ≤ x < 0.60; Positive: ≥ 0.60

[0535] Epitope mapping in rBCLA-positive patients shows consistent reactivity within numerous antigenic regions and repeat regions of rBCLA.

[0536] The specific immunogenicity of rBCLA was demonstrated by Western blotting in a range of sera from different clinical categories. One of the main objectives was to develop an ELISA-based assay for screening large sera populations in a cost-effective, robust, and rapid manner. However, to properly establish this assay based almost entirely on chemically synthesized peptides, a more precise understanding of the local epitope immunogenicity of BCLA is needed. To this end, we designed and synthesized cellulose-printed peptide arrays covering repeat regions and rBCLA domains (Figure 12a). These arrays were designed using 15 amino acids (AA) peptides with 3 AA gaps between peptides. We then tested several sera that were definitively positive for rBCLA by Western blotting, and took an equal number of negative sera to proportionally subtract nonspecific reactivity. When analyzed, the total reactivity scores obtained for each peptide in the repeat region and on rBCLA (Fig. 12b) provided two key observations: First, despite having several highly reactive regions, particularly near peptides 13, 22, 30, and 43, rBCLA possesses numerous epitopes throughout its domain, and different sera will react very differently with different regions (Fig. 12c). This underscores the requirement to maintain rBCLA as a recombinant protein in ELISA assays. Furthermore, since the rBCLA domain is predicted to be structured, structured epitopes will only be available through recombinant protein strategies, further emphasizing its usefulness. Second, the repeat motif was found to react consistently in almost all tested human sera in two separate regions (peptides 3–7 as motif A and 13–16 as motif B). This feature underscores the importance of including one or more peptides covering these motifs to obtain more sensitive ELISA techniques. Therefore, based on these results, we designed an ELISA combining full-length recombinant BCLA protein with chemically synthesized repeat motifs.

[0537] ELISA titration using rBCLA and repeating peptides confirmed that BCLA seropositivity was higher in individuals with both acute and chronic infections.

[0538] Strict rules were established to classify and differentiate different clinical profiles. 123 serum samples (all from different individuals) were tested using the developed BCLA-ELISA assay. Patients' ELISA scores, expressed in International Units (UI), were displayed based on their clinical profiles. Figure 13 The following is a list:

[0539] 1) "Seronegative" regroups all patients (healthy or with other pre-existing conditions) with negative SAG1 IgG / IgM serology.

[0540] 2) "Previous immunity" refers to the regrouping of all patients (healthy or with other conditions) into SAG1-positive IgG but not SAG1-reactive IgM, and they do not belong to the following three categories.

[0541] 3) "Active toxoplasmosis in immunocompromised patients" regroups all SAG1 IgG positive and immunocompromised patients with confirmed symptomatic toxoplasmosis (recombinant disseminated, cerebral, and primary toxoplasmosis).

[0542] 4) "Asymptomatic serological reactivation in immunocompromised patients" regroups all immunocompromised patients who have undergone serological reactivation but have no visible symptoms.

[0543] 5) "Ocular toxoplasmosis", which regroups patients with SAG1 positive serology and confirmed ocular toxoplasmosis.

[0544] Several observations emerged from this analysis. First, all groups showed a significant increase in median BCLA titer and a much higher positivity rate compared to the seronegative group. This demonstrates a direct correlation between SAG1 seropositivity and the ability to develop BCLA positivity in humans. It also reveals the current difference between SAG1-negative serology and BCLA serology, which still shows a false positive rate of approximately 10%. This can be explained in some sera by nonspecific interactions with different BCLA epitopes, highly immunogenic exogenous bacterial contamination co-purified with rBCLA, and potentially true BCLA-positive patients with negative SAG1 serology. A second major observation was that some clinical features tend to produce a stronger immunogenic response, most notably the "asymptomatic serological reactivation in immunocompromised patients" group, where BCLA serological titers significantly exceeded the median positive BCLA serology in the "previously immunized" group. Finally, a few groups that should always be expected to be BCLA-positive, such as those with "active toxoplasmosis in immunocompromised patients" and "ocular toxoplasmosis," remained serologically negative or below the positive cutoff value. This observation highlights the lack of sensitivity in ELISA tests, or may indicate that BCLA serology can become negative during immunosuppression.

[0545] ELISA tests also consistently correlated positive BCLA serology in mice with proportional capsule load.

[0546] In summary, semi-quantitative analysis of anti-rBCLA antibody titers identified BALB / c and NMRI mice that may carry WT capsules highly responsive to BCLA, with yields increasing over time. Figure 14ABy combining quantitative PCR of brain-associated Toxoplasma gondii DNA with the quantification of brain-associated miR-155 and miR-146 microRNAs, which are reported to be specifically induced during slow-onset disease (Cannella et al., 2014), we provide evidence that rBCLA is a reliable antigen for serological detection of Toxoplasma gondii merozoite-loaded cysts in rodents with long-term protozoan presence. Figure 14B These results are particularly interesting because the semi-quantitative nature of the ELISA assay clearly distinguishes the response of cystic Toxoplasma gondii strains over time.

[0547] discuss

[0548] Infection with *Toxoplasma gondii* leads to an acute systemic phase through which spores rapidly establish themselves and complete their developmental program, such as merozoites enclosed in cysts surrounded by thick walls that persist in the brain, heart, and skeletal muscle (Jeffers et al., 2018). The host immune response can rapidly control the expansion of the tachyzoite population, leading to lifelong immunity, represented by seroconversion. However, because the developmental transition from tachyzoite to merozoite is entirely bidirectional, any impairment of immune function (e.g., in AIDS patients, hematologic disorders, and immunosuppressive therapy) can lead to reactivation of latent infection, which can cause encephalitis and focal brain injury, lung disease, or disseminated disease.

[0549] Diagnosis of acute and chronic toxoplasmosis in immune-active subjects relies primarily on serology, and because infection is often asymptomatic, serological diagnosis is retrospective in many cases, as it is based on evidence of seroconversion, for example, during pregnancy or in the graft setting (Robert-Gangneux and Darde, 2012). Elevated IgM and IgA antibody levels are serological markers of primary / acute infection, while high IgG affinity excludes primary infection, and persistent and steady-state IgG levels in the absence of IgM represent latent infection (Dard et al., 2016). However, even for trained specialists, the interpretation of serological results remains challenging. Current challenges to overcome include: (i) differentiating between recent and distant infections; (ii) diagnosing congenital toxoplasmosis in infants and reactivation in immunocompromised patients; and (iii) determining the source of infection, i.e., oocysts vs. cysts. While numerous methods have been developed over the past few decades to improve the accuracy and sensitivity of serological assays, they have struggled to address the aforementioned problems. The obvious reason is that many (if not all) commercial serological test kits are detecting lysates or recombinant antigens that are universally expressed in the tachyzoite stage (e.g., SAG1) or co-expressed in both infection stages of the parasite (e.g., GRA8).

[0550] Currently, although there is no reliable merozoite-specific serological assay for toxoplasmosis to estimate the worldwide source of infection, nor can it accurately distinguish between acute and latent infections, progress has been made. In fact, recent proteomic studies have elucidated all the components of sporophyte-specific proteins (Fritz et al., 2012; Possenti et al., 2013), revealing that CCP5A, as a serological marker, can distinguish the parasitic stages infecting chickens, pigs, and mice, with a specific reactivity to animals infected with oocysts (Santana et al., 2015).

[0551] However, although earlier studies reported that specific merozoite antigens, including BAG1, contribute to stimulating humoral (Munet et al., 1999) and cell-mediated immunity against Toxoplasma gondii infection (Di Cristina et al., 2004), merozoite / cyst antigens are not currently considered potential markers of latent infection in diagnostic tests. Research on merozoite / cyst-specific markers is somewhat limited by the ability to harvest sufficient mouse brain cysts to analyze the latent proteome. In this study, we discovered a method to circumvent this limitation by depressorizing merozoite genes in cell cultures while simultaneously manipulating tachyzoite chromatin states with epigenetic drugs. Consequently, hundreds of merozoite-restricting proteins, including BCLA, were identified.

[0552] This indicates that the protein BCLA is not essential for initiating or maintaining latent infection; however, BCLA deficiency results in a rather singular phenotype characterized by deformed and lost-roundness of brain cysts in a mouse model. To date, two cyst wall-associated proteins, BPK1 and CST1, have been involved in the structural integrity of *Toxoplasma gondii* cysts (Jeffers et al., 2018). In the Δbpk1 strain, cysts are smaller and more sensitive to pepsin-acid treatment, and unlike BCLA, the Δbpk1 strain exhibits reduced ability to induce oral infection (Buchholz et al., 2013). CST1 is responsible for the lentinan (DBA) lectin binding properties of *Toxoplasma gondii* cysts. CST1 deficiency leads to a reduced number of cysts and a fragile brain cyst phenotype characterized by thinning and rupture of the lower cyst wall (Tomita et al., 2013). Glycosylation defects may also explain the deformability of Δbcla cysts. In fact, we have preliminary interaction data indicating that BCLA was co-purified with a jasmonic acid-binding protein (data not shown), which is a lectin that binds to GalNAcα1-Ser / Thr oligosaccharides and covers the PVM around the merozoite (Tomita et al., 2017). Further investigation is needed to determine whether this interaction is responsible for the BCLA-deficiency-mediated specific phenotype.

[0553] Regardless of the route and timing of infection, there is no clearly defined BCLA-associated phenotype in mice, and we are directed toward investigating the tendency of BCLA to exert an immunogenic effect. Therefore, we achieved another milestone by producing rBCLA in a highly purified recombinant protein form, which provides the opportunity for standardized serological testing and, to some extent, reduces production costs, provided the antigen proves meaningful for serology. Indeed, we provide strong data supporting that rBCLA is antigenic and constitutes an excellent antigenic candidate for detecting anti-Toxoplasma gondii IgG in chronically infected mice. Notably, we clearly correlated strong detection of antigenic rBCLA in serum with cyst load in the brains of all mice potentially infected with type II cystic strains. Similar studies have claimed that MAG1 antibody levels are associated with brain cyst load, but their experimental settings were somewhat biased using an unrelated model of chronic type I (GT1) infection, which required anti-Toxoplasma gondii chemotherapy to control tachyzoite proliferation and avoid animal death in the acute phase (Xiao et al., 2016).

[0554] Notably, rBCLA did not respond to IgM or IgA (data not shown), biomarkers typically associated with acute infection but only with IgG and subchronic infection. This result is consistent with observations of a significant IgM response in tissue-encapsulated mice on day 10. This contrasts sharply with and reinforces the idea of ​​a humoral response against BCLA during the incubation period. Similarly, mice inoculated with tissue cysts via oral feeding did not develop antibodies against BCLA at acute infection (Fig. 8c), indicating that the host immune response against BCLA does not originate from the initial exposure to merozoites and cyst proteins released from the ingested parasite in the gastrointestinal tract during primary infection. This contrasts sharply with the humoral responses against BAG1 and MAG1 that occur very early post-infection (Di Cristina et al., 2004; Mun et al., 1999). Our findings regarding the humoral immune response to BCLA-containing tissue cysts contradict the view that *Toxoplasma gondii* cysts are primarily located in immune-exempt sites such as the brain and skeletal muscle. Further work in mice will be needed to elucidate the contribution of the humoral immune response during chronic toxoplasmosis, and BCLA may provide a powerful tool for studying these processes.

[0555] Finally, anti-rBCLA antibodies have been detected in the serum of some patients with ocular toxoplasmosis during toxoplasmosis reactivation associated with immunosuppression or congenital toxoplasmosis. These findings are consistent with conclusions drawn from mouse models that rBCLA is an excellent serological marker for the presence of tissue cysts in chronically infected hosts.

[0556] Example 2 (VHH Production)

[0557] Immunization

[0558] LamasSEL005 and SEL006 were immunized with four injections via Eurogentec on days 0, 14, 28, and 35. Serum was obtained on days 0, 28, and 43. Peripheral blood mononuclear cells (PBMCs) were obtained from a large blood volume on day 43.

[0559] Immune response

[0560] The immune response of SEL005 and SEL006 was assessed by evaluating the presence of rBCLA-specific antibodies in serum on day 43. MaxiSorp plates were coated overnight at 4°C with 200 ng of antigen per well. After washing three times with PBS containing 0.05% Tween-20, the plates were blocked with 4% milk powder in PBS (MPBS). Serial dilutions of serum in 1% MPBS were then added to the wells and incubated for 1 hour. Unbound antibodies were removed during PBS-Tween washing. Binding antibodies were subsequently detected using rabbit anti-VHH (clone K1216) and HRP-conjugated donkey anti-rabbit. Antibody binding was quantified by a colorimetric reaction of O-phenylenediamine (OPD) at 490 nm in the presence of H2O2. Llamas SEL005 and SEL006 showed very good responses to His rBCLA.

[0561] Library construction for day 43 of SEL005 and day 43 of SEL006

[0562] RNA isolation and cDNA synthesis

[0563] Peripheral blood lymphocytes were isolated from a large volume of blood on day 43, and RNA was isolated from them at Eurogentec. The precipitated RNA was dissolved in RNase-free MQ and the RNA concentration was measured. To assess RNA quality, 5 μl of dissolved RNA was analyzed on a gel. Figure 2A The complete 28S and 18S rRNA are clearly visible, indicating proper RNA integrity.

[0564] Approximately 40 μg of RNA (4 reactions, 10 μg each) was transcribed into cDNA using a reverse transcriptase kit (Thermo Fisher Scientific). The cDNA was purified on a Macherey Nagel PCR cleansing column. Variable domains of the heavy chain (regular and heavy chain only) fragments were amplified using primers annealed at the leader and CH2 regions. 5 μl was loaded onto a 1% TBE agarose gel as an amplification control.

[0565] Following this control, the remaining sample was loaded onto a 1% TAE agarose gel, and a 700b fragment was excised and purified from the gel. A total of 80 ng of isolated PCR product was used as a template for nested PCR (final volume 800 μl) to introduce SfiI and Eco91I restriction sites at either end of the VHH gene. The amplified VHH fragment was washed on a Macherey Nagel PCR washing column and eluted with 120 μl. The eluted DNA was digested first with SfiI and then with Eco91I. As a restriction digestion control, 4 μl of this mixture was loaded onto a 1.5% TBE agarose gel.

[0566] After restriction digestion, the sample was loaded onto a 1.5% TAE agarose gel. A 400bp fragment was excised from the gel and purified on a Maccherry Nagel gel extraction column. The purified 400bp VHH fragment (~330ng) was ligated into the pUR8100 phage vector (~1μg) and transformed into TG1 Escherichia coli.

[0567] Library size

[0568] Titrate the transformed TG1 with a 10-fold dilution. Spot 5 μl of the dilution onto an LB agar plate supplemented with 100 μg / ml ampicillin and 2% glucose. Count the number of transformants from the spotted dilution of the transformed TG1 culture (remember the final transformation volume is 8 ml). Calculate the total number of transformants by counting the highest dilution and using the following formula to calculate the library size:

[0569] Library size = (amount of colonies) × (dilution) × 8 (ml) / 0.005 (ml; sample volume)

[0570] The VHH insertion frequency in phage vectors was determined by selecting 24 different clones and performing colony PCR. A band of approximately 700 bp indicates a successfully cloned VHH fragment. A band of approximately 300 bp indicates an empty plasmid. The insertion frequency of library SEL005 was 100% on day 43. The insertion frequency of library SEL006 was almost 95% on day 43 (Figure 4), which was sufficient for continued phage panning.

[0571] bacteriophage production and selection

[0572] Phage generation from the library is as follows: *E. coli* TG1 containing day 43 of libraries SEL005 and SEL006 were diluted from glycerol stock solution to an OD600 of 0.05 in 2xYT medium containing 2% glucose and 100 μg / ml ampicillin. The inoculum must contain at least 10 times the size of the library (>10 inoculum). 9(100 bacteria). The culture was incubated at 37°C for 2 hours to achieve an OD600 of ~0.5. Subsequently, approximately 7 ml of the culture was infected with helper phage VCSM13 at 100 MOI (multiple of infection), and incubated at 37°C for 30 minutes. The infected bacteria were centrifuged and resuspended in 50 ml of fresh 2xYT medium supplemented with ampicillin (100 μg / ml for phage particles) and kanamycin (25 μg / ml for M13 phage), and incubated overnight at 37°C with shaking. The generated phage was precipitated from the culture supernatant using PEG-NaCl precipitation. The titer of the generated phage was calculated by serial dilution of the phage and infection of *E. coli* TG1. The titer of the generated phage for SEL005 on day 43 was 3 × 10⁻⁶. 11 / ml, for SEL006 on day 43 it was 6×10 11 / ml, that's enough to continue selecting.

[0573] For the first round of panning / selection, 20 μl of precipitated phage (~10 10 One phage (>100-fold greater than the library diversity) was applied to wells coated with His rBCLA. In short, 100 μl of antigen was coated onto MaxiSorp at two concentrations, 5 μg / ml and 0.5 μg / ml, and incubated overnight. As a negative control, one well was incubated with PBS only. The next day, after removing unbound antigen, the plates were washed three times with PBS and blocked with 4% milk powder in PBS (MPBS). Simultaneously, freshly precipitated phages were pre-blocked with 2% MPBS for 30 minutes. The pre-blocked phages were incubated with directly coated His rBCLA for 2 hours. After thorough washing with PBS-Tween and PBS, bound phages were eluted with 0.1 M TEA solution, followed by neutralization with 1 M Tris / HCl pH 7.5. The eluted phages were serially diluted and then used to infect TG1 bacteria. The phages were spotted onto LB agar plates supplemented with 2% glucose and 100 μg / ml ampicillin and incubated at 37°C.

[0574] For the second round of selection, new phages from the selected rescue output were generated on 5 μg / ml His rBCLA (maximum concentration). The overnight-grown rescue output was diluted 100-fold in 5 ml of fresh 2xYT medium supplemented with 2% glucose and 100 μg / ml ampicillin and grown for 2 hours until the logarithmic phase. Then, 1 μl of helper phage VCS M13 was added and incubated at 37°C for 30 minutes. The culture was allowed to incubate overnight at 37°C to generate phages. The generated phages were precipitated from the culture supernatant using PEG-NaCl precipitation.

[0575] Subsequently, for the second round of panning / selection, 1 μl of precipitated phage was applied to wells coated with His rBCLA as follows: antigen was coated on MaxiSorp plates overnight at three concentrations (5 μg / ml, 0.5 μg / ml, and 0.05 μg / ml). As a negative control, one well was incubated with PBS only. The next day, after removing unbound antigen, the plate was washed three times with PBS and blocked with 4% MPBS. Simultaneously, the freshly precipitated phage was pre-blocked in 2% MPBS for 30 minutes as described above. The pre-blocked phage was incubated with directly coated His rBCLA for 2 hours. After thorough washing with PBS-Tween and PBS, the bound phage was eluted with 0.1 M TEA solution, followed by neutralization with 1 M Tris / HCl pH 7.5. The eluted phages were serially diluted and then used to infect TG1 cells. The cells were spotted onto LB agar plates supplemented with 2% glucose and 100 μg / ml ampicillin and incubated overnight at 37°C.

[0576] Screening after 2 rounds of phage display selection

[0577] The rescue output selected in the second round on His rBCLA was plated to select individual clones. For the master plate ERB-1, a total of 92 individual clones were selected in a 96-well plate.

[0578] To screen for His rBCLA-binding agents, the master plate ERB-1 was used to produce a periplasmic extract containing monoclonal VHH. The master plate was cultured at 37°C in 2xYT medium supplemented with 2% glucose and 100 μg / ml ampicillin, and stored at -80°C after adding glycerol to a final concentration of 20%. To produce the periplasmic extract, the master plate ERB-1 was replicated into deep-well plates containing 1 ml of 2xYT medium supplemented with 0.1% glucose and 100 μg / ml ampicillin and grown at 37°C for 3 hours, followed by the addition of 1 mM IPTG to induce VHH expression. VHH expression was allowed to proceed overnight at room temperature. Bacteria were collected by centrifugation, and the precipitate was resuspended in 120 μl PBS and subjected to one freeze-thaw cycle to prepare the periplasmic extract. The bacteria were centrifuged to separate the VHH-containing soluble periplasmic fraction from the cell debris (precipitate). To test the binding specificity of monoclonal VHH by ELISA, His rBCLA (100 ng / well, in PBS) was coated onto MaxiSorp plates overnight at 4 °C. The coated plates were washed and then blocked with 4% MPBS. The blocked wells were incubated with 10 μl of peritoneal extract and 40 μl of 1% MPBS at room temperature for 1 h. Unbound VHH was removed by washing with PBS containing 0.05% Tween-20. Subsequently, bound VHH was detected using rabbit anti-VHH (clone K976) and HRP-conjugated donkey anti-rabbit. VHH binding was quantified by a colorimetric reaction of OPD at 490 nm in the presence of H2O2. All clones of the mainboard ERB-1 were able to specifically bind His rBCLA. There was no difference between the two libraries used.

[0579] Sequence analysis of His rBCLA combined with VHH

[0580] Based on the ELISA results, 17 clones (ERB-1A1, ERB-1F1, ERB-1A2, ERB-1E2, ERB-1F2, ERB-1G2, ERB-1B3, ERB-1H4, ERB-1A5, ERB-1G6, ERB-1D7, ERB-1F7, ERB-1G8, ERB-1E9, ERB-1E10, ERB-1B11, and ERB-1A12) were selected for sequencing. These clones were selected based on the bindings observed in the ELISA results, and they should represent the majority of clones selected from different outputs.

[0581] Cloning and production of VHH selected on His rBCLA

[0582] From all sequenced clones, seven clones (ERB-1F1, ERB-1F2, ERB-1H4, ERB-1G6, ERB-1D7, ERB-1B11, and ERB-1A12) were selected as good representatives of the discovered VHH sequences. These VHHs were then subcloned from the phage vector into the expression vector pMEK222 using SfiI and Eco91I restriction enzymes. Recloning into pMEK222 also added FLAG and His- tags to the C-terminus of the VHHs, allowing for detection and affinity purification. For production, a preculture was prepared by growing bacteria containing the plasmid and the selected VHHs overnight at 37°C in 8 ml of 2xYT medium supplemented with 2% glucose and 100 μg / ml ampicillin. The preculture was diluted in 800 ml of fresh 2xYT, which was preheated at 37°C and supplemented with 100 μg / ml ampicillin and 0.1% glucose. The bacteria were grown at 37°C for 2 hours, followed by induction of VHH expression with 1 mM IPTG. VHH expression was carried out at 37°C for 4 hours, and the bacteria were harvested by centrifugation. The bacterial pellet was resuspended in 30 ml of PBS and frozen at -20°C.

[0583] Purification and Analysis of VHH

[0584] The frozen bacterial pellet was thawed at room temperature, and cell debris was removed by centrifugation. VHH was purified from the supernatant (soluble fraction) using His-tag affinity with cobalt-bound agarose beads (immobilized metal affinity chromatography using TALON beads (IMAC)). Bound VHH was eluted with 150 mM imidazole and dialyzed with PBS.

[0585] Protein concentration was measured using absorbance at 280 nm and corrected for molar extinction coefficients and molecular weights of different VHHs.

[0586] As a quality check, 1 μg of purified VHH was loaded onto an SDS-PAGE.

[0587] The binding of purified VHH to immobilized His rBCLA was analyzed by ELISA. MaxiSorp plates were coated overnight in PBS with 200 ng / well of antigen at 4 °C. After blocking the wells with 4% MPBS, serially diluted VHH was added to the coated wells and incubated at room temperature for 1 h. After washing away unbound VHH, the binding of VHH was detected using mouse anti-labeled (clone M2) and HRP-conjugated donkey anti-mouse. Binding was quantified by measuring the colorimetric reaction of OPD + H2O2 at 490 nm. ERB-1G6, ERB-1B11, and ERB-1A12 showed sub-nanomolar apparent affinity for immobilized His rBCLA. ERB-1F1 and ERB-1F2 showed low nanomolar affinity. ERB-1H4 and ERB-1D7 showed molar apparent affinity for His rBCLA.

[0588] in conclusion

[0589] Immunization with llamas SEL005 and SEL006 elicited a good immune response. The resulting libraries had good size and insertion frequency. Phage display selection on His rBCLA has yielded many good clones, three of which (ERB-1G6, ERB-1B11, and ERB-1A12) showed very good epigenetic affinity, with ERB-1G6 also showing high production levels in E. coli.

[0590] Table 4: Useful amino acid sequences for carrying out the present invention

[0591]

[0592]

[0593]

[0594]

[0595] Example 3:

[0596] In this longitudinal study, it is conceivable that we have shown that the detection of BCLA antibodies, when properly combined, could further improve the sensitivity of current tests. We performed the BCLA test in cases of maternal-infant congenital toxoplasmosis. Only 10 pairs of mothers / children were tested in each group, and the results should be accounted for accordingly. Two groups were compared: one group confirmed congenital toxoplasmosis through persistent Sag1 IgG titers in the child's serum over a long period after birth, and the other group excluded congenital toxoplasmosis when the child's serum became Sag1 negative over time (Lebech M et al., 1996).

[0597] If passed and Comparative titrations of Toxo IgG showed that at birth, infants in both groups shared comparable titers with no discernible distribution. Figure 15C -D). This can be explained by the mother transmitting anti-Sag1 IgG across the placental barrier, thus making a definitive biological conclusion impossible at birth. The separation between congenital toxoplasmosis and excluded congenital toxoplasmosis is clearer when BCLA ELISA titration is observed. Newborn serum showed significantly greater BCLA titration reactivity than that of their mothers or those in the excluded congenital toxoplasmosis group (Figures 15A-B).

[0598] These observations indicate that infants synthesize new, specific anti-BCLA antibodies before birth, and suggest that strong BCLA reactivity could further guide the diagnosis of congenital toxoplasmosis at birth.

[0599] References:

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Claims

1. An isolated polypeptide selected from the group consisting of: (i) An amino acid sequence consisting of residues 1089-1275 of the C-terminal antigenic domain BCLA shown in SEQ ID NO: 2; (ii) An amino acid sequence consisting of internal repeating domains of BCLA selected from the following groups: TgR1 shown in SEQ ID NO:4, TgR2 shown in SEQ ID NO:5, TgR3 shown in SEQ ID NO:6, TgR4 shown in SEQ ID NO:7, TgR5 shown in SEQ ID NO:8, TgR6 shown in SEQ ID NO:9, TgR7 shown in SEQ ID NO:10, TgR8 shown in SEQ ID NO:11, TgR9 shown in SEQ ID NO:12, TgR10 shown in SEQ ID NO:13, TgR11 shown in SEQ ID NO:14, TgR12 shown in SEQ ID NO:15, and TgR13 shown in SEQ ID NO:

16.

2. An isolated polypeptide selected from the group consisting of: (i) MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP, as shown in SEQ ID NO:55 (ii) AAGSMEKDKLVLPGE as shown in SEQ ID NO:

56.

3. The isolated polypeptide according to any one of claims 1-2, which is used as an antigen.

4. An antibody that specifically binds to the isolated peptide of any one of claims 1-3 or to a polypeptide consisting of an amino acid sequence of the Toxoplasma gondii polypeptide BCLA shown in SEQ ID NO:1, wherein the antibody is a single-domain antibody having a variable heavy chain sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:

63.

5. A kit comprising the antibody according to claim 4.

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