New serum markers for latent toxoplasmosis

Abstract

Here, we report the characterization of BCLA (brain cyst burden-associated antigen), a protein exclusively expressed during the bradyzoite stage of the parasite. In cysts purified directly from mouse brain, the protein is distributed within and on the surface of cysts. ELISA antibody capture using a combination of a serologically reactive BCLA peptide and a recombinantly expressed c-terminal domain (rBCLA) constitutes an efficient seromarker of latent infection, with high sensitivity that correlates specifically and exclusively with the presence of cysts in mouse brain. Antibodies directed against the BCLA antigen have been detected in human patients, with high titers in patients deemed seropositive for Sag1 or tachyzoite-associated antigens. Further correlation between anti-BCLA IgG synthesis and cysts in humans is provided by significantly higher titers recorded in a pathological panel, which strongly correlates with the presence of cysts. Furthermore, newborns with confirmed congenital toxoplasmosis presented significantly higher anti-BCLA IgG at birth when compared with their mothers, suggesting specific intrauterine neosynthesis of such IgG. Thus, the present invention relates to a novel Toxoplasma gondii protein, hereafter referred to as BCLA, which is a novel serum marker whose expression is restricted to latent forms of toxoplasmosis (bradyzoites / cysts). This particular protein and its antigenic fragments can be used to detect autoantibodies in patient serum for the diagnosis of latent forms of toxoplasmosis. The present invention also relates to induced antibodies generated by BCLA immunization that specifically bind to this novel protein.

Description

[Technical Field]

[0001] Field of invention:

[0002] This invention relates to a novel Toxoplasma gondii protein (hereinafter referred to as BCLA (Brain Cystic Load-Associated Antigen)) and is a novel serum marker whose expression is restricted to the latent form of toxoplasmosis (bradyzoite / cyst). The invention also relates to an antibody that specifically binds to this novel protein. This particular protein and its antigenic fragment can be used to detect autoantibodies in a patient's serum for the diagnosis of latent toxoplasmosis.

[0003] Background of the invention:

[0004] The ancient phylum Apicomplexa includes many of the world's most prominent protozoan pathogens. The most deadly to humans is Plasmodium, the pathogen of malaria, which kills nearly 500,000 people annually. T. gondii is the pathogen that causes toxoplasmosis and is one of the most widespread protozoan parasites in livestock, wild animals, and companion animals. Toxoplasmosis is a widespread foodborne infection in humans that poses a significant public health problem and is recognized as a leading cause of foodborne deaths in the United States (Scallan et al., 2015). Toxoplasmosis is usually a mild illness in immune individuals but can be a major threat to immunocompromised patients experiencing life-threatening brain, lung, heart, or disseminated pathologies. Transplacental infections can cause congenital infections with varying degrees of clinical symptoms, ranging from congenital abnormalities (e.g., hydrocephalus, microcephaly, intracranial calcification) to fetal disappearance.

[0005] The cystic enterococcidious parasite T. gondii is transmitted through an alternating two-host life cycle that depends on a specific feline host for sexual transmission, while also undergoing asexual transmission in a diverse range of alternative hosts (including rodents and humans). Throughout its long-term presence in warm-blooded feline metazoans, T. gondii initiates a complex developmental program in response to its surrounding environment (including the host's innate defenses and adaptation to different hosts). Upon initial infection in an intermediate host, the parasite replicates as tachyzoites, dramatically increasing in number before disseminating to numerous tissues throughout the body. Initial infection is generally controlled by a potent Th1-mediated pro-inflammatory host response leading to large-scale disruption of a massive tachyzoite population, while smaller subpopulations of tachyzoites differentiate into a slower-growing bradyzoite stage, which persists throughout the host's life in tissue cysts present in long-lived cells (including neurons and skeletal muscle cells) (Dubey, 1997). The cycle is completed when the feline-specific host ingests the tissue cysts, leading to the shedding of oocysts, which are highly infectious (Dubey, 2001).

[0006] Tissue cysts are a major source of human infection via meat consumption, and as such, a key factor in human disease as a complication of toxoplasmosis is the ability of the bradyzoite to cause irreversible damage while differentiating back to the replicating tachyzoite stage. Indeed, asymptomatic parasitism provides lifelong equilibrium and protection in immune-capable hosts, while persistent immune dysfunction disrupts the parasite's dormancy, and it is known that bradyzoites promote tachyzoite migration and further increase in the tachyzoite population. These combined processes ultimately lead to encephalitis, interstitial pneumonia, retinochoroiditis, or even disseminated toxoplasmosis, as major outcomes in immunocompromised individuals (Dard et al., 2018). Therefore, the strategy of T. gondii as an obligate intracellular parasite is based on the search for nonpathogenicity, that is, the ability to attenuate, but not completely counteract, the host's innate immune response to infection, and thus secure a permanent habitat required to await transmission.

[0007] Despite the importance of histiocysts in the life cycle of T. gondii and their crucial role as reservoirs for toxoplasmosis relapses in immunocompromised hosts, the biology of bradyzoites and the cysts they form remains poorly understood. Cysts are thought to proliferate, disseminate, and perpetuate chronic infections over time, without passing through the intermediate tachyzoite stage, via both the migration of free bradyzoites and the division of bradyzoite cysts (Dzierszinski et al., 2004; Frenkel and Escajadillo, 1987). The concept that bradyzoites within histiocysts are dormant entities has recently been challenged by compelling evidence showing that bradyzoites exhibit periodic, accidental growth within histiocysts in vivo by asynchronous replication using both endogenous binary and endogenous multiple division (Dzierszinski et al., 2004).

[0008] The developmental transition from tachyzoite to bradyzoite is bidirectional, typically represented by dramatic changes in parasite gene expression, leading to major metabolic changes, remodeling of the parasite surface with restricted expression of stage-specific surface antigens, and cyst wall formation. The latter is likely to protect bradyzoite from harsh gastrointestinal environmental conditions and provide a physical barrier against host immune defenses. T. Gondi differentiation has been difficult to test because the stage transition is directed by a complex and still-unknown developmental genetic program, and is also influenced by the physiology of host cells (Lueder and Rahman, 2017). In the laboratory, the conversion from tachyzoite to bradyzoite can be induced by exogenous stress (e.g., alkaline stress, nutritional deficiencies, and drugs) in the absence of host immunity in vitro.

[0009] Transcriptional regulation clearly plays a crucial role in bradyzoite development, as demonstrated by numerous studies showing stage-specific gene expression. While how these changes are regulated at the molecular level remains largely unknown, we and others have provided strong evidence that epigenetic changes are the driving force behind parasite differentiation. Early evidence stemmed from the observation that tachyzoites rapidly recovered from mice during in vivo infection tended to differentiate particularly, gradually losing this "sensitized" state over time. For example, long-term passage of tachyzoites in tissue culture dramatically attenuates the ability of type II strains to generate high cystic load in vivo. Thus, by promoting developmental plasticity—that is, through epigenetic mechanisms involving the manifestation of diverse phenotypes from the same genome—parasites may be able to adapt to thousands of potential intermediate hosts and respond to remarkably different immune systems.

[0010] T. Gondi has evolved sophisticated methods to promote epigenetic changes, such as changes in histone mark activity and chromatin remodeling, that provide zoites with a remarkable ability to undergo stepwise differentiation in response to environmental cues or as part of a developmental program, in order to counter the strategies used by the cells they infect. We were initially interested in post-translational modifications (PTMs) of histones, specifically acetylation (Saksouk et al., 2005), which led us to demonstrate that changes in the rate of histone H4 acetylation near stepwise-specific genes are one of the epigenetic molecular motors driving parasitic differentiation (Bougdour et al., 2009). Core histone acetylation is mediated by histone acetyltransferase (HAT) and in many cases results in relaxation of chromatin structure and transcriptional activation of related genes. Histone deacetylase (HDAC) counteracts HAT activity by catalyzing the removal of the acetyl moiety from lysine residues in histone tails, thereby inducing chromatin condensation and transcriptional repression (Kurdistani and Grunstein, 2003).

[0011] The importance of histone acetylation for differentiation control has been highlighted by the finding that chemical inhibition of TgHDAC3 with low doses of the compound FR235222 induces stepwise conversion from tachyzoites to bradyzoites in vitro (Bougdour et al., 2009; Maubon et al., 2010). Recombinant strains transfected with a TgHDAC3 allele resistant to this compound did not exhibit these effects, confirming the TgHDAC3 specificity of the compound and suggesting that TgHDAC3 activity actively prevents bradyzoite differentiation (Bougdour et al., 2009). This in vitro conversion is accompanied by hyperacetylation of the upstream regions of >350 genes, one-third of which are specific to bradyzoites (Bougdour et al., 2009). TgHDAC3 appears to primarily counteract the action of HATTgGCN5b, which localizes to the promoter of the active gene via ChIP, whereas TgHDAC3 localizes to the promoter of the bradyzoite gene via ChIP (Saksouk et al., 2005). These data represent a step towards understanding the causal relationship between histone acetylation and gene expression in T Gondi and point to a crucial role of TgHDAC3 in stepwise conversion, although these studies were conducted only in pathogenic RH strains that do not readily develop tissue cysts or latent infections in laboratory mice. Ultimately, there is a need to develop new diagnostic methods for latent forms of toxoplasmosis.

[0012] In this study, the inventors re-examined the ability of FR235222 to stimulate tachyzoite-to-bradyzoite conversion in vitro, using a type II-derived strain prone to cystic formation in vivo. Quantitative analysis of the T-gon diproteome response to FR235222 revealed many proteins previously identified as stage-specific proteins (including those recognized as being restricted to bradyzoite). Due to their potential importance to parasitic biology (Hakimi et al., 2017), the inventors chose to focus their attention on novel proteins predicted to be secreted. ~200 putative FR235222-responsive bradyzoite secretion effectors were identified by this approach. One candidate, BCLA (Brain Cystic Load-Associated Antigen), was selected for further study. BCLA is expressed only upon FR235222 treatment, and following its secretion in the vacuolar space, this protein has been shown to accumulate in the parasitic sac membrane (PVM). Under in vivo conditions, BCLA is present in the cystic matrix space as well as in the cystic wall, the latter thought to originate from the PVM during the latent phase. While evaluating its function, we have shown that BCLA deficiency affects the integrity of brain cysts isolated from chronically infected mice, but this protein is essential for proper cystic function, at least in our mouse model of chronic toxoplasmosis.

[0013] Assuming restricted expression of BCLA by bradyzoite and its location in the cyst wall, the inventors then sought to investigate its potential application in serological diagnostics. Here, the inventors found that the C-terminal peptide of BCLA produced by recombinant technology is strongly antigenic and constitutes an excellent antigen candidate for the detection of anti-T Gondi IgG in chronically infected mice. The inventors provide strong data showing a clear correlation between the presence of cysts in the brains of chronically infected mice and the detection of the antigen BCLA in serum. Positive assays using human serum validate the antigenic characteristics of BCLA, paving the way for the use of this antigen for anti-toxoplasma diagnostics, with an intriguing prospect of serological detection of cystic load in chronically infected hosts.

[0014] Summary of the invention:

[0015] The present invention provides an isolated Toxoplasma gondii polypeptide, hereafter referred to as BCLA (Brain Cystic Load-Related Antigen), which comprises amino acid sequence number 1 and an immunogenic peptide fragment.

[0016] The present invention further relates to antibodies generated against the isolated polypeptide of the present invention.

[0017] The present invention further relates to a method for detecting Toxoplasma gondii polypeptide according to the present invention, and / or a method for evaluating its amount in a biological sample, particularly in a solid sample.

[0018] The present invention further relates to a diagnostic method for latent toxoplasmosis, using polypeptides according to the present invention to detect anti-BCLA antibodies in biological samples, particularly in bodily fluid samples.

[0019] Detailed description of the invention:

[0020] By modulating tachyzoite genome expression using epidrugs, the inventors were able to identify genes whose expression is restricted to bradyzoites. In this invention, the inventors report the characterization of BCLA (brain cyst load-associated antigen), a protein that accumulates in vitro on the parasitic cyst membrane when expressed under bradyzoite-inducible conditions. In the mouse brain, this protein is scattered within and on the surface of cysts. Deletion of this gene results in reduced brain cyst load in mice, and the remaining cysts are typically characterized by deformation of their wall surface, ranging from circular loss to a distinctive budding phenotype. Finally, when synthesized as a recombinant protein, BCLA constitutes an efficient serum marker of latent infection with high sensitivity that clearly and exclusively correlates with the presence of cysts in the mouse brain. Using the first ELISA BCLA test developed by the inventors, antibodies directed against the BCLA antigen were detected in human patients with strongly suspected or proven ocular toxoplasmosis, either in serum alone or in both serum and aqueous humor. Serological assays have long been the first-line test for confirming T. Gondi infection, but current serological diagnostics do not always distinguish between acute, latent, and relapsing disease states. Furthermore, current serology does not assess cystic burden in tissues and the subsequent risk of toxoplasmosis relapse in seropositive immunocompromised patients. Some of these limitations have now been overcome with the discovery of BCLA (a significant antigen candidate for serological detection of cysts in chronically infected hosts).

[0021] The initial ELISA test was optimized for the detection of BCLA immunogenic peptides. First, a peptide microarray designed using both the BCLA C-terminal domain and the most conserved internal peptide repeat TgR4 (Figure 12a) was screened using peptide dot blot screening for high-resolution BCLA epitope mapping (Figures 12b and 12c). In contrast to mice, all positive human sera showed robust reactivity against peptides derived from internal repeats that significantly increased test sensitivity once added to rBCLA. Thus, BCLA ELISA was customized based on the most sensitive combination of peptides and polypeptides and proven to be optimal for highly reliable discrimination among humans diagnosed with either ocular toxoplasmosis or confirmed past immunity (Figure 13). By the ELISA test, significant amounts of circulating anti-BCLA antibodies were also detected in sera from immunocompromised patients who had undergone either asymptomatic or symptomatic chronic toxoplasmosis episodes (Figure 13).

[0022] Isolated peptide.<000008>

[0023] The present invention relates to an isolated Toxoplasma gondii polypeptide called BCLA (Brain Cyst Load Associated Antigen) that comprises amino acid sequence number 1.

[0024] The present invention also provides an isolated Toxoplasma gondii polypeptide selected from the group consisting of: (i) the amino acid sequence (sequence number 1) consisting of Toxoplasma gondii polypeptide BCLA; (ii) the amino acid sequence (sequence number 2) consisting of the C-terminal antigen domain (res 1089-1275 of BCLA called rBCLA); (iii) An amino acid sequence consisting of an internal repeat domain of BCLA selected from the group consisting of: 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); (iv) An amino acid sequence substantially homologous to the sequences of (i) to (iii), preferably an amino acid sequence at least 80% identical to the sequences of (i) to (iii); (v) A fragment of at least 9 consecutive amino acids of the sequences of (i) to (iv).

[0025] By using peptide dot blot screening (see Figure 12), it becomes possible to identify the most potent BCLA immunogenic peptides in the C-terminal antigen domain of BCLA (res 1089 - 1275 of BCLA, called rBCLA), as well as in the internal repeat domain of BCLA (res 304 - 924 of BCLA, called TgR1 to TgR13 (SEQ ID NOs: 4 to 16)).

[0026] Thus, in certain embodiments, an isolated toxoplasma gondii polypeptide from the rBCLA polypeptide is selected from the group consisting of: (i) GELQPAEAEEARLLVADLKAV (SEQ ID NO: 32) (ii) VRVEGEAFFRASVDLYEA (SEQ ID NO: 33) (iii) KLRPLTKGELVDVVRQ (SEQ ID NO: 34) (iv) TQIFVQDRASAFLRV (peptide 36 of rBCLA) (SEQ ID NO: 35) (v) AAEQMKAVFAMVEEG (peptide 44 of rBCLA) (SEQ ID NO: 36) (vi) An amino acid sequence substantially homologous to the sequences of (i) to (v), preferably an amino acid sequence at least 95% identical to the sequences of (i) to (v) (vii) A fragment of at least nine consecutive amino acids in the sequence from (i) to (vi).

[0027] In more specific embodiments, the Toxoplasma gondii polypeptide isolated from the rBCLA polypeptide is selected from the group consisting of: (i) GELQPAEAEEARLLV (rBCLA peptide 12) (SEQ ID NO: 37); (ii) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38), (iii) EAEEARLLVADLKAV (rBCLA peptide 14) (SEQ ID NO: 39), (iv)VRVEGEAFFRASVDL (rBCLA peptide 21) (SEQ ID NO: 40), (v)EGEAFFRASVDLYEA(rBCLA peptide 22)(SEQ ID NO: 41); (vi) AFFRASVDLYEAVKN (rBCLA peptide 23) (SEQ ID NO: 42), (vii)KLRPLTKGELVDVVR(rBCLA peptide 30)(SEQ ID NO: 43) (viii) An amino acid sequence substantially homologous to the sequences of (i) to (vii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (vii). (vii) A fragment of at least nine consecutive amino acids in the sequence (i) through (viii).

[0028] Therefore, in certain embodiments, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA is selected from the group consisting of the following: (i) Amino acid sequence consisting of the internal repeat domain of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7) (ii) An amino acid sequence substantially homologous to the sequence of (i), preferably an amino acid sequence that is at least 80% identical to the sequence of (i). (iii) A fragment of at least nine consecutive amino acids in the sequence of (i) to (ii).

[0029] In more specific embodiments, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA is selected from the group consisting of: (i)AAGSMEKEKPVLPGEGEGH(Domain A of TgR4);(Sequence ID 44) (ii) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4), (SEQ ID NO: 45) (iii) An amino acid sequence substantially homologous to the sequences of (i) to (ii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (ii). (iv) A fragment of at least nine consecutive amino acids in the sequence of (i) to (iii).

[0030] In more specific embodiments, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA is selected from the group consisting of: (i) AAGSMEKEKPVLPGE (TgR4 peptide 3); (SEQ ID NO: 46) (ii) GSMEKEKPVLPGEGE (TgR4 peptide 4) (SEQ ID NO: 47) (iii) MEKEKPVLPGEGEGH (TgR4 peptide 5) (SEQ ID NO: 48) (iv)KEKPVLPGEGEGHVL (TgR4 peptide 6) (SEQ ID NO: 49) (v)KPVLPGEGEGHVLPG (TgR4 peptide 7) (SEQ ID NO: 50) (vi) HVLPKHETKPALTDEK (TgR4 peptide 13), (SEQ ID NO: 51) (vii)PKHETKPALTDEKRT(TgR4 peptide 14), (SEQ ID NO: 52) (viii) HETKPALTDEKRTKP (TgR4 peptide 15) (SEQ ID NO: 53) (ix)TKPALTDEKRTKPGG (TgR4 peptide 16) (SEQ ID NO: 54) (x) An amino acid sequence substantially homologous to the sequences (i) through (ix), preferably an amino acid sequence that is at least 95% identical to the sequences (i) through (ix). (xi) A fragment of at least nine consecutive amino acids in the sequence (i) to (x).

[0031] Since BCLA polypeptides have numerous epitopes across different domains (particularly in rBCLA and in the internal repeat domains of BCLA TgR1 to TgR13), combining the BCLA immunogenic peptide fragments of the present invention may be advantageous.

[0032] Therefore, in another embodiment, the isolated polypeptide of the present invention is a fusion of two peptide fragments according to the present invention.

[0033] For the improved ELISA assay, the following BCLA peptides (combining two internal repeat peptides, with at least one fusion peptide) were used in combination with the full-length recombinant BCLA polypeptide (SEQ ID NO: 1).

[0034] Peptide AB_F:MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (a fusion of peptide fragments from repeat motifs present in Tgr4 / Trg12 / Tgr13 and repeat motifs present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID NO: 55)

[0035] Peptide A3_B:AAGSMEKDKLVLPGE (peptide fragment from repeat motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID NO: 56)

[0036] Therefore, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA is selected from the following group: (i)MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (a fusion of peptide fragments from repeat motifs present in Tgr4 / Trg12 / Tgr13 and repeat motifs present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID NO: 55), (ii) AAGSMEEKDKLVLPGE (peptide fragment from repeat motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID NO: 56) (iii) An amino acid sequence substantially homologous to the sequences of (i) to (ii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (ii). (iv) A fragment of at least nine consecutive amino acids in the sequence of (i) to (iii).

[0037] Because BCLA polypeptides have numerous epitopes across different internal repeat domains of BCLA (TgR1 to TgR13), combining amino acid residues from these internal repeat domains can be advantageous.

[0038] Therefore, the present invention also relates to a BCLA polypeptide comprising an internal repeat domain (TgRx) of BCLA having the following sequence: 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 NO: 64) Here, Xaa1 is glutamic acid (E) or there is no amino acid residue. Here, Xaa2 is arginine (R) or serine (S). Here, Xaa3 is proline (P) or glycine (G). Here, Xaa4 is either alanine (A) or glycine (G). Here, Xaa5 is either alanine (A) or there is no amino acid residue. Here, Xaa6 is either glycine (G) or arginine (R). Here, Xaa7 is serine (S), proline (P), or alanine (A). Here, Xaa8 is either lysine (K) or glutamic acid (E). Here, Xaa9 is lysine (K), glutamic acid (E), or aspartic acid (D). Here, Xaa10 is either proline (P) or leucine (L). Here, Xaa11 is leucine (L) or serine (S). Here, Xaa12 is glutamic acid (E) or lysine (K). Here, Xaa13 is either glycine (G) or arginine (R). Here, Xaa14 is valine (V) or alanine (A). Here, Xaa15 is leucine (L) or serine (S). Here, Xaa16 is histidine (H), aspartic acid (D), or alanine (A). Here, Xaa17 is threonine (T), arginine (R), methionine (M), or glutamine (Q). Here, Xaa18 is proline (P), threonine (T), or alanine (A). Here, Xaa19 is aspartic acid (D), glutamic acid (E), or glutamine (Q). Here, Xaa20 is glutamic acid (E) or lysine (K). Here, Xaa21 is lysine (K), glycine (G), or glutamic acid (E). Here, Xaa22 is arginine (R) or valine (V). Here, Xaa23 is lysine (K), glutamic acid (E), or asparagine (N). Here, Xaa24 is glycine (G), valine, or isoleucine (I). Here, Xaa25 is glycine (G) or glutamic acid (E). Here, Xaa26 is arginine (R) or proline (P). Here, Xaa27 is threonine (T) cysteine ​​(C) lysine (K) or methionine (M). Here, Xaa28 is glutamic acid (E) or alanine (A). and a fragment of at least nine consecutive amino acids of the sequence of Sequence ID No. 64.

[0039] As used herein, the term “amino acid” refers to natural or non-natural amino acids in their D and L stereoisomers for chiral amino acids. For example, it is understood to refer to both amino acids and their corresponding amino acid residues, such as those present in peptidyl structures. 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). Rare non-natural amino acids include, but are not limited to, allylglycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphthylalanine (2-Nal), ornithine (Orn), and pentafluorophenylalanine.

[0040] Amino acids are typically classified into one or more categories (including polar, hydrophobic, acidic, basic, and aromatic) according to their side chains. Examples of polar amino acids include those having side chain functional groups such as hydroxyl, sulfhydryl, and amide, as well as 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 having nonpolar aliphatic side chains, such as, but are not limited to, leucine, isoleucine, valine, glycine, alanine, proline, methionine, and phenylalanine. Examples of basic amino acid residues include those having basic side chains such as an amino group or guanidino group. Basic amino acid residues include, but are not limited to, arginine, homolysine, and lysine. Examples of acidic amino acid residues include those having acidic side chain functional groups such as a carboxyl group. Acidic amino acid residues include, but are not limited to, aspartic acid and glutamic acid. Aromatic amino acids include those having aromatic side chain groups. Examples of aromatic amino acids include, but are not limited to, biphenylalanine, histidine, 2-naphthylalanine, pentafluorophenylalanine, phenylalanine, tryptophan, and tyrosine. Note that some amino acids belong to multiple groups; for example, histidine, tryptophan, and tyrosine belong to both polar and aromatic amino acids. Amino acids can be further classified as uncharged or charged (positive or negative) amino acids. Examples of positively charged amino acids include, but are not limited to, lysine, arginine, and histidine. Examples of charged amino acids include, but are not limited to, glutamic acid and aspartic acid. Additional amino acids belonging to each of the above groups are known to those skilled in the art.

[0041] A peptide that is "substantially homologous" to a reference peptide may be derived from the reference sequence by one or more conserved substitutions. Two amino acid sequences are "substantially homologous" or "substantially similar" if one or more amino acid residues are substituted by biologically similar residues, or if more than 80% of the amino acids are identical, or if more than 90%, preferably more than 95%, are similar (functionally identical). Preferably, similar, identical, or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pile-up program, or a program known in the art (such as BLAST, CLUSTAL, or FASTA). The percentage of identity may be calculated by performing pairwise global alignment based on the Needleman-Wunsch alignment algorithm, for example using Needle and the BLOSUM62 matrix (with a gap opening penalty of 10 and a gap extension penalty of 0.5), and finding the optimal alignment (including gaps) of two sequences along their entire length.

[0042] As used herein, the term “conservative substitution” means the substitution of one amino acid residue with another amino acid residue without altering the overall three-dimensional structure and function of the peptide, and includes, but is not limited to, the substitution of an amino acid 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 interchangeable. Similarly, isoleucine (a hydrophobic amino acid) may be substituted with leucine, methionine, or valine. Neutral hydrophilic amino acids can be substituted with each other and include asparagine, glutamine, serine, and threonine.

[0043] By "replaced" or "modified," the present invention includes amino acids that have been modified or altered from natural amino acids.

[0044] In the context of the present invention, it should be understood that a conservative substitution is recognized in the art as the substitution of one amino acid for another amino acid having similar properties.

[0045] 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 BLAST P (Karlin and Altschul, 1990).

[0046] In some embodiments, the isolated peptide of the present invention contains up to 1275 amino acids (and at least 9). In some embodiments, the polypeptide of the present invention is [Table 1] TIFF0007849292000002.tif249165 TIFF0007849292000003.tif111165 Or it contains 9 amino acids. In some embodiments, the polypeptide of the present invention contains less than 50 amino acids. In some embodiments, the polypeptide of the present invention contains less than 30 amino acids. In some embodiments, the polypeptide of the present invention contains less than 25 amino acids. In some embodiments, the polypeptide of the present invention contains less than 20 amino acids. In some embodiments, the polypeptide of the present invention contains less than 15 amino acids.

[0047] The isolated polypeptide according to the present invention can be produced using any method known in the art. These can be produced, for example, as recombinant polypeptides in host cells (e.g., bacterial, yeast, or eukaryotic host cells) or chemically synthesized (see Kent SBH Chem. Soc. Rev., 2009, 38, 338-351 and Bradley L. et al Annu Rev Biophys Biomol Struct. 2005; 34: 91-118 or RB Merrifield (1969). “Solid-phase peptide synthesis.” Advances in enzymology and related areas of molecular biology 32: 221-96.; RB Merrifield (1969). “The synthesis of biologically active peptides and proteins.” JAMA 210(7): 1247-54. and Raibaut, L., O. El Mahdi and O. Melnyk (2015). “Solid Phase Protein Chemical Synthesis.” Topics in current chemistry).

[0048] The antibody of the present invention

[0049] The inventors have generated specific antibodies directed against the polypeptide of the present invention.

[0050] Firstly, to assay the insights dynamics of BLCA in T. Gondi, we produced polyclonal antibodies against two synthetic peptides located at the ends of conserved repeats of the BCLA protein (see Example 1 and Figure 2b). Autoantibodies were generated against the two peptides (peptides 1 and 2) contained within these repeats. Western blotting monitoring of BCLA expression using the autologous antibodies produced against the two BCLA-derived peptides showed upregulation of BCLA after FR235222 treatment (see Figure 2c).

[0051] Secondly, single-domain antibodies (or nanobodies or VHHs) were produced by immunizing mice with the synthetic peptide, the C-terminal antigen domain of BCLA (res 1089-1275) (SEQ ID NO: 2). More precisely, the inventors found that the antibodies were screened for their ability to specifically recognize the isolated polypeptide of the present invention and stain cell line samples infected with Toxoplasma gondii, as well as brain samples from toxoplasmosis patients (detection of tissue cysts) and mouse models of toxoplasmosis. The screening process of the antibodies of the present invention demonstrates that these antibodies are specific to the isolated polypeptide of the present invention, particularly those with the antigen domain of BCLA.

[0052] The present invention provides an antibody that specifically binds to the isolated polypeptide of the present invention.

[0053] According to the present invention, “antibody” and “immunoglobulin” have the same meaning and can be used interchangeably in the present invention. As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule that includes the immunologically active portion of an immunoglobulin molecule, i.e., an antigen-binding site that binds immunospecifically to an antigen. As such, the term “antibody” includes not only the whole antibody molecule but also antibody fragments, as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda(l) and kappa(k). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain contains two domains, namely a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively called CH). The variable regions of both the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain association, 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 immunoglobulins and consists of a variable region 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 primarily composed of residues from the hypervariable region or complementarity-determining region (CDR). In some cases, residues from the non-hypervariable region or framework region (FR) influence the overall domain structure and, consequently, the binding site. Complementarity-determining regions, or CDRs, refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, which are named VL-CDR1, VL-CDR2, VL-CDR3 and VH-CDR1, VH-CDR2, VH-CDR3, respectively.The antigen-binding site therefore contains six CDRs, including a set of CDRs from the V region of the heavy chain and the light chain, respectively. The framework region (FR) refers to the amino acid sequence inserted between the CDRs.

[0054] Antibodies that bind to the isolated polypeptide of the present invention can be assayed by conventional methods known in the art. The mature form of the polypeptide of the present invention is preferably used to assay antibodies that bind to the epitope of the polypeptide of the present invention. Alternatively, any variant form of the isolated polypeptide of the present invention that retains the binding of nanobody XX can be used. Many different competitive binding assay formats 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. Such assays are routine and well known in the art (e.g., see Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). For example, BIACORE® (GE Healthcare, Piscataway, New Jersey) is one of a variety of surface plasmon resonance assay formats routinely used to epitope bin panels of monoclonal antibodies. In addition, routine cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988. Examples of suitable ELISA assays are also described in the following examples.

[0055] 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 numerous sites through weak non-covalent bonds; the more interactions, the stronger the affinity. Affinity is K D This can be determined by measuring the term "K". D When used herein, " is intended to refer to the dissociation constant, which is K d vs K a The ratio (i.e., K d / K a ) is obtained from and expressed as molar concentration (M). K for antibodies D The value can be determined using methods well established in the art. Antibody K D Methods for determining this include using surface plasmon resonance or using a biosensor system, such as the Biacore® system.

[0056] The present invention provides antibodies that specifically bind to isolated polypeptides comprising or consisting of the following: (i) Amino acid sequence consisting of Toxoplasma gondipeptide BCLA (SEQ ID NO: 1); (ii) Amino acid sequence consisting of the C-terminal antigen domain (res 1089-1275 of BCLA) (SEQ ID NO: 2); (iii) Amino acid sequences comprising the internal repeat domain of BCLA, selected from the group consisting of the following: 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); (iv) An amino acid sequence substantially homologous to the sequences of (i) to (iii), preferably an amino acid sequence that is at least 80% identical to the sequences of (i) to (iii). (v) A fragment of at least nine consecutive amino acids from the sequence (i) to (iv).

[0057] These antibodies can recognize epitopes located within, or containing at least one amino acid located within, any one of the isolated polypeptides (i) to (v), which consist of at least nine consecutive amino acids.

[0058] Preferably, the epitope is located within a fragment containing or consisting of any one of the isolated polypeptides (i) to (v).

[0059] Most preferably, the epitope is located within the C-terminal antigen domain of BCLA (SEQ ID NO: 2) and within the internal repeat domains of BCLA (res 304-924), referred to as TgR1 to TgR13 (SEQ ID NOs: 4 to 16). Such antibodies are characterized in that they specifically bind to the Toxoplasma gondii BCLA polypeptide of the present invention.

[0060] In a particular embodiment, the antibody that specifically binds to the rBCLA polypeptide specifically binds to an amino acid sequence selected from the group consisting of the following: (i) GELQPAEAEEARLLVADLKAV (domain A of rBCLA) (Sequence ID 32) (ii) VRVEGEAFFRASVDLYEA (domain B of rBCLA) (Sequence ID 33) (iii) KLRPLTKGELVDVVRQ (domain C of rBCLA) (Sequence ID 34) (iv)TQIFVQDRASAFLRV(rBCLA peptide 36 and rBCLA domain D))(SEQ ID NO: 35) (v)AAEQMKAVFAMVEEG(rBCLA peptide 44 and rBCLA domain E))(SEQ ID NO: 36) (vi) An amino acid sequence substantially homologous to the sequences of (i) to (v), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (v). (vii) A fragment of at least nine consecutive amino acids in the sequence (i) to (vi). In a more specific embodiment, an antibody that specifically binds to the rBCLA polypeptide specifically binds to an amino acid sequence selected from the group consisting of: (i) GELQPAEAEEARLLV (rBCLA peptide 12) (SEQ ID NO: 37); (ii) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38), (iii) EAEEARLLVADLKAV (rBCLA peptide 14) (SEQ ID NO: 39), (iv)VRVEGEAFFRASVDL (rBCLA peptide 21) (SEQ ID NO: 40), (v)EGEAFFRASVDLYEA(rBCLA peptide 22)(SEQ ID NO: 41); (vi) AFFRASVDLYEAVKN (rBCLA peptide 23) (SEQ ID NO: 42), (vii)KLRPLTKGELVDVVR(rBCLA peptide 30)(SEQ ID NO: 43) (viii) An amino acid sequence substantially homologous to the sequences of (i) to (vii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (vii). (vii) A fragment of at least nine consecutive amino acids in the sequence (i) through (viii).

[0061] The present invention further provides antibodies that specifically bind to amino acid sequences consisting of the internal repeat domain (res304-924) of BCLA, referred to as TgR1 to TgR13 (SEQ ID NOs: 4 to 16).

[0062] Therefore, in a particular embodiment, the antibody that specifically binds to the internal repeat domain of BCLA binds to an amino acid sequence selected from the group consisting of the following: (i) Amino acid sequence consisting of the internal repeat domain of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7) (ii) An amino acid sequence substantially homologous to the sequence of (i), preferably an amino acid sequence that is at least 80% identical to the sequence of (i). (iii) A fragment of at least nine consecutive amino acids in the sequence of (i) to (ii). In a more specific embodiment, the antibody that specifically binds to the internal repeat domain of BCLA TgR4 binds to an amino acid sequence selected from the group consisting of: (i)AAGSMEKEKPVLPGEGEGH(Domain A of TgR4);(Sequence ID 44) (ii) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4), (SEQ ID NO: 45)

[0063] In a more specific embodiment, the antibody that specifically binds to the internal repeat domain of BCLA TgR4 binds to an amino acid sequence selected from the group consisting of: (i) AAGSMEKEKPVLPGE (TgR4 peptide 3); (SEQ ID NO: 46) (ii) GSMEKEKPVLPGEGE (TgR4 peptide 4) (SEQ ID NO: 47) (iii) MEKEKPVLPGEGEGH (TgR4 peptide 5) (SEQ ID NO: 48) (iv)KEKPVLPGEGEGHVL (TgR4 peptide 6) (SEQ ID NO: 49) (v)KPVLPGEGEGHVLPG (TgR4 peptide 7) (SEQ ID NO: 50) (vi) HVLPKHETKPALTDEK (TgR4 peptide 13), (SEQ ID NO: 51) (vii)PKHETKPALTDEKRT(TgR4 peptide 14), (SEQ ID NO: 52) (viii) HETKPALTDEKRTKP (TgR4 peptide 15) (SEQ ID NO: 53) (i) TKPALTDEKRTKPGG (TgR4 peptide 16) (SEQ ID NO: 54)

[0064] In a particular embodiment, the antibody specifically binds to the internal repeat domain of BCLA(TgRx) having the following sequence: 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 NO: 64) Here, Xaa1 is glutamic acid (E) or there is no amino acid residue. Here, Xaa2 is arginine (R) or serine (S). Here, Xaa3 is proline (P) or glycine (G). Here, Xaa4 is either alanine (A) or glycine (G). Here, Xaa5 is either alanine (A) or there is no amino acid residue. Here, Xaa6 is either glycine (G) or arginine (R). Here, Xaa7 is serine (S), proline (P), or alanine (A). Here, Xaa8 is either lysine (K) or glutamic acid (E). Here, Xaa9 is lysine (K), glutamic acid (E), or aspartic acid (D). Here, Xaa10 is either proline (P) or leucine (L). Here, Xaa11 is leucine (L) or serine (S). Here, Xaa12 is glutamic acid (E) or lysine (K). Here, Xaa13 is either glycine (G) or arginine (R). Here, Xaa14 is valine (V) or alanine (A). Here, Xaa15 is leucine (L) or serine (S). Here, Xaa16 is histidine (H), aspartic acid (D), or alanine (A). Here, Xaa17 is threonine (T), arginine (R), methionine (M), or glutamine (Q). Here, Xaa18 is proline (P), threonine (T), or alanine (A). Here, Xaa19 is aspartic acid (D), glutamic acid (E), or glutamine (Q). Here, Xaa20 is glutamic acid (E) or lysine (K). Here, Xaa21 is lysine (K), glycine (G), or glutamic acid (E). Here, Xaa22 is arginine (R) or valine (V). Here, Xaa23 is lysine (K), glutamic acid (E), or asparagine (N). Here, Xaa24 is glycine (G), valine, or isoleucine (I). Here, Xaa25 is glycine (G) or glutamic acid (E). Here, Xaa26 is arginine (R) or proline (P). Here, Xaa27 is threonine (T) cysteine ​​(C) lysine (K) or methionine (M). Here, Xaa28 is glutamic acid (E) or alanine (A). and a fragment of at least nine consecutive amino acids of the sequence of Sequence ID No. 64.

[0065] The present invention further provides an antibody that specifically binds to an amino acid sequence consisting of either peptide 1 or peptide 2 (sequence numbers 17 to 27) within the internal repeat domain of BCLA, referred to as TgR1 to TgR13 (sequence numbers 4 to 16).

[0066] In certain embodiments, peptides 1 and 2 used in this test are Peptide 1: EMERPAAGSMEK (SEQ ID NO: 21) Peptide 2 is VLPKHETKPALT (SEQ ID NO: 22).

[0067] These antibodies can be polyclonal or monoclonal. If the antibodies are monoclonal, they can correspond to, for example, chimeric, humanized or fully human antibodies, antibody fragments, and single-domain antibodies.

[0068] The term "chimeric antibody" refers to an antibody that contains the VH and VL domains of the antibody, as well as the CH and CL domains of the human antibody.

[0069] 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 a previous non-human antibody.

[0070] The term "antibody fragment" refers to a fragment of an antibody containing a variable domain, including the CDR of the antibody. Basic antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, and dsFv. For examples of antibody fragments, see also the review article, Holliger et al., Nature Biotechnology 23, issue 9 1126-1136 (2005), which is incorporated herein by reference.

[0071] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, in which approximately half of the N-terminal side of the H chain and the entire L chain are linked together via disulfide bonds in a fragment obtained by treating IgG with the protease papain.

[0072] The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 and antigen-binding activity, which is slightly larger than Fab, a fragment obtained by treating IgG with the protease pepsin, and is bound via a disulfide bond in the hinge region.

[0073] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0074] Single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers, typically expressed from gene fusions containing VH and VL encoding genes linked by peptide-encoding linkers. "dsFv" are VH::VL heterodimers stabilized by disulfide bonds. Bivalent and multivalent antibody fragments can be spontaneously formed by the association of monovalent scFv or generated by linking monovalent scFv with peptide linkers, such as bivalent sc(Fv)2.

[0075] The terms "diabody," "tribody," or "tetrabody" refer to small antibody fragments containing a multivalent antigen-binding site (2, 3, or 4), which includes a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites.

[0076] As used herein, the term “single-domain antibody” has its general meaning in the art and refers to a single heavy-chain variable domain of an antibody of a type that can be found in the naturally light-chain-deficient camel mammal. Such single-domain antibodies are also called VHH or “nanobody®”. For a general description of (single)domain antibodies, see the prior art cited above, as well as EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388. Nanobodies have a molecular weight about one-tenth that of a human IgG molecule, and the proteins have a physical diameter of only a few nanometers. One consequence of this small size is the ability of camelid nanobodies to bind to antigen sites that are functionally invisible to larger antibody proteins; that is, camelid nanobodies are useful as reagents for detecting antigens that are otherwise latent using classical immunological techniques, and as possible therapeutic agents. Thus, yet another consequence of the small size is that nanobodies can inhibit target proteins by binding to specific sites within grooves or narrow gaps, and therefore can be useful in their ability to more closely resemble the function of classical low molecular weight drugs than the function of classical antibodies. The low molecular weight and compact size further result in nanobodies that are extremely thermally stable, stable to extreme pH and proteolytic digestion, and have insufficient antigenicity. Another consequence is that nanobodies can easily travel from the circulatory system into tissues, and even cross the blood-brain barrier to treat disorders 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, suggest significant therapeutic potential.The amino acid sequence and structure of a single-domain antibody can be considered to consist of four framework regions or "FRs," which are referred to in the art and herein as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4," respectively; these framework regions are interrupted by three complementarity-determining regions or "CDRs," which are referred to in the art as "complementarity-determining region for CDR1"; "complementarity-determining region 2" or "CDR2"; and "complementarity-determining region 3" or "CDR3," respectively. Thus, a single-domain antibody can be defined as an amino acid sequence with the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. In the context of the present invention, the amino acid residues of a single-domain antibody are numbered according to the general numbering for VH domains provided by the amino acid numbering system of the International ImMunoGeneTics information system (http: / / imgt.cines.fr / ).

[0077] Several VHH (single-domain antibodies) were generated after immunization of llamas, resulting in a favorable immune response. The generated libraries exhibited good size and insertion frequency. Phage display selection using His rBCLA (SEQ ID NO: 3) yielded numerous favorable clones, three of which (ERB-1G6, ERB-1B11, and ERB-1A12) showed very good apparent affinity, with ERB-1G6 also exhibiting high production levels in E. coli.

[0078] The sequences of ERB-1F1, ERB-1F2, ERB 1H4, ERB-1D7, ERB-1G6, ERB-1B11, and ERB-1A12VHH are listed below in Table 1 for the variable heavy chain (VH) of single-domain antibodies.

Table 2

[0079] Methods for obtaining such antibodies are well known in the art. For example, monoclonal antibodies according to the present invention can be obtained through immunization of a non-human mammal with the fragment comprising or consisting of any one of (i) to (vi). Starting from polyclonal antibodies, monoclonal antibodies can then be obtained using standard methods.

[0080] The antibodies of the present invention can be conjugated with a detectable label to form an immunoconjugate. Suitable detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, enzyme labels, bioluminescent labels, or gold colloids. Methods for making and detecting such detectably labeled immunoconjugates are well known to those skilled in the art and are described in more detail below.

[0081] The detectable label can be a radioisotope detected by autoradiography. Isotopes that are particularly useful for the purposes of the present invention are 3 H, 125 I, 311 I, 35 S, and 14 C.

[0082] The immunoconjugate can also be labeled with a fluorescent compound. The presence of the fluorescently labeled antibody is determined by exposing the immunoconjugate to light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0083] Alternatively, immunoconjugates can be detectably labeled by conjugating antibodies to chemiluminescent compounds. The presence of a chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence produced during the course of a chemical reaction. Examples of chemiluminescent-labeled compounds include luminols, isoluminols, aromatic acridinium esters, imidazoles, acridinium salts, and oxalic acid esters.

[0084] Similarly, the immunoconjugates of the present invention can be labeled using bioluminescent compounds. Bioluminescence is a type of chemiluminescence found in biological systems in which catalytic proteins increase the efficiency of chemiluminescent reactions. The presence of bioluminescent proteins is determined by detecting the presence of luminescence. Useful bioluminescent compounds for labeling include luciferin, luciferase, and aequorin.

[0085] Alternatively, the immunoconjugate can be detectably labeled by linking a monoclonal antibody to the 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 that can be detected, for example, by spectrophotometric, fluorescence-spectrometric, or visual means. Examples of enzymes that can be used to detectably label multispecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0086] The antibodies of the present invention may be labeled with metal chemistry elements, such as lanthanides. Lanthanides offer several advantages over other labels in that they are stable isotopes, and there are a large number of them available, up to 100 or more different labels, they are relatively stable, they are highly detectable, and when detected using mass spectrometry, they are easily separated between detection channels. Lanthanide labeling also provides detection with a wide dynamic range. Lanthanides exhibit high sensitivity and low sensitivity to light and time, and are therefore very flexible and robust and can be used in a large number of different settings. Lanthanides are a set of 15 metal chemistry elements with atomic numbers 57–71. They are also called 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 for as many parameters as available stable isotope tags.

[0087] Those skilled in the art will know of other suitable labels that can be used in accordance with the present invention. Binding of the marker moiety to a monoclonal antibody can be achieved using standard techniques known in the art.

[0088] Furthermore, the convenience and versatility of immunochemical detection can be enhanced by using monoclonal antibodies conjugated with avidin, streptavidin, and biotin.

[0089] Another object of the present invention, as described above, is a method for detecting an antibody directed against T gondipeptide BCLA using at least one isolated toxoplasma gondipeptide according to the present invention, and / or for evaluating its amount in a biological sample.

[0090] As used herein, the term “biological sample” refers to any biological sample of the subject; a tissue sample or a body fluid sample. In a preferred embodiment of the method for detecting antibodies directed against T-gondipolypeptide 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, and cerebrospinal fluid (CSF) and aqueous humor.

[0091] More specifically, the body fluid sample is a serum or aqueous humor sample. In a preferred embodiment of the present invention relating to the detection of antibodies against T-Gondi BCLA polypeptide, the biological sample is a body fluid sample, more specifically a brain sample.

[0092] Detection and diagnostic method of the present invention:

[0093] In some embodiments, the method of the present invention is carried out in vitro or ex vivo.

[0094] A method for detecting T-gondi BCLA polypeptide.

[0095] The object of the present invention is a method for detecting the T-gondi polypeptide BCLA and / or evaluating its amount in a biological sample.

[0096] Biological samples include, 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.

[0097] In a preferred embodiment relating to the detection of T-Gondi BCLA polypeptide, the biological sample is a tissue sample, more specifically a muscle sample or a brain sample.

[0098] Detection of T-gonid polypeptide BCLA may involve protein / polypeptide separation; 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 to the specific solid phase used. Once separated, T-gonid polypeptide BCLA can be identified based on a known "separation profile," e.g., retention time measured using standard techniques for that protein. Alternatively, the separated protein can be detected and measured, for example, by mass spectrometry (see the Examples section).

[0099] The detection and quantity of the Tgondipolypeptide BCLA species of the present invention can be determined by using standard electrophoretic and immunodiagnostic techniques (including immunoassays, e.g., competitive, direct reaction, e.g., immunohistochemistry, or sandwich assays). Such assays include, but are not limited to, Western blotting; agglutination tests; enzyme-labeled and mediated immunoassays, e.g., ELISA; biotin / avidin assays; radioimmunoassays; immunoelectrophoresis; and immunoprecipitation. These reactions generally involve identifying labels, e.g., fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, or dye molecules, or other methods for detecting the formation of complexes between an antigen and an antibody that reacts with it, or between multiple antibodies.

[0100] For example, the determination of T-gondipolypeptide BCLA quantity can be carried out by a variety of techniques and methods, including any well-known methods in the art: RIA kits (DiaSorin; IDS, Diasource), Elisa kits (Fujirebio, Thermo Fisher, EHTGFBI, R&D DY2935, IDS (manual), IDS (adapted with open analyzer), immunochemiluminescence automated methods (MesoScaleDiscovery, DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (Janssen et al., 2012), Simoa / Quanterix.

[0101] In certain embodiments, the method of the present invention includes bringing a biological sample into contact with a binding partner.

[0102] As used herein, a binding partner refers to a molecule capable of selectively interacting with the Tgondipolypeptide BCLA of the present invention.

[0103] The binding partner is generally a polyclonal or monoclonal antibody, preferably one that can be monoclonal.

[0104] In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecules that serve as alternatives to antibodies in terms of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the ability to recognize substantially any class of target molecules with high affinity and specificity. Such ligands can be isolated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) of a random sequence library, as described in Tuerk et al. (1990) Science, 249, 505-510. Random sequence libraries are available by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of a unique sequence, ultimately chemically modified. The possible modifications, uses, and advantages of this class of molecules are outlined in Jayasena 1999. Peptide aptamers consist of a structurally constrained antibody variable region presented by a platform protein, such as E. coli thioredoxin A, selected from a combinatorial library using two hybrid methods (Colas et al. (1996) Nature, 380, 548-50).

[0105] The binding partner of the present invention, such as an antibody or aptamer, may be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art. Labels are known in the art and generally provide a signal (either directly or indirectly).

[0106] As used herein, the term “labeled” is intended to encompass both direct labeling of an antibody or aptamer by binding (i.e., physically linking) a detectable substance, such as a radioactive substance or fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5)), to the antibody or aptamer with respect to its binding partner, and indirect labeling of a probe or antibody by reactivity with a detectable substance. The antibodies or aptamers of the present invention may be labeled with radioactive molecules by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms for scintigraphy testing, such as I123, I124, In111, Re186, Re188, etc.

[0107] The aforementioned assays generally involve the binding of a binding partner (i.e., an antibody or aptamer) to a solid support. Solid supports that can be used in the implementation of the present invention include substrates such as nitrocellulose (e.g., in the form of a membrane or microtiter well); polyvinyl chloride (e.g., a sheet or microtiter well); polystyrene latex (e.g., beads or microtiter plate); polyvinylidine fluoride; diazotized paper; nylon membrane; 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 Tgondipolypeptide BCLA. A body fluid sample containing or suspected to contain Tgondipolypeptide BCLA is then added to the coated wells. After a sufficient incubation period to allow the formation of a binding partner-Tgondipolypeptide BCLA complex, the plate can be washed to remove unbound material, and a labeled secondary binding molecule can be added. The secondary binding molecule can react with any captured sample marker protein, the plate is washed, and the presence of the secondary binding molecule is detected using methods well known in the art.

[0108] The secondary bonding molecule may be labeled as a bonding partner.

[0109] The antibodies and immunoconjugates of the present invention can be used to detect the T-gondi polypeptide BCLA of the present invention and / or to evaluate its amount 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. Accordingly, they can be used to diagnose all diseases associated with the Toxoplasma gondii pathogen.

[0110] Diagnostic methods for latent toxoplasmosis (detection of T-gondipolypeptide BCLA)

[0111] Therefore, the method for detecting T-Gondi BCLA polypeptide according to the present invention is consequently useful for the in vitro diagnosis of toxoplasmosis from biological samples. In particular, the detection method of the present invention is consequently useful for the in vitro diagnosis of latent forms of toxoplasmosis or congenital toxoplasmosis from biological samples. As used herein, the term “biological sample” refers to any biological sample of interest. A 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.

[0112] In a preferred embodiment of the method using the detection of T-Gondi BCLA polypeptide, the biological sample is a tissue sample, more specifically, a brain tissue sample or a muscle tissue sample.

[0113] A further object of the present invention is a method for detecting and / or evaluating the amount thereof in a biological sample, the method comprising contacting the sample with Toxoplasma gondipeptide BCLA under conditions that allow for the formation of an immune complex between the antibody / immunoconjugate and the sample, and detecting or measuring the formed immune complex.

[0114] A further object of the present invention is a method for detecting and / or evaluating the amount thereof in a biological sample, the method comprising contacting the sample with the antibody or immunoconjugate of the present invention under conditions that allow for the formation of an immune complex between Toxoplasma gondii polypeptide BCLA and the antibody / immunoconjugate on the surface of the cyst, and detecting or measuring the formed immune complex.

[0115] The formed immune complexes can be detected or measured by a variety of methods using standard techniques (including, but not limited to, enzyme-linked immunosorbent assays (ELISA) or other solid-phase immunoassays, radioimmunoassays, electrophoresis, immunofluorescence, or Western blotting).

[0116] A further object of the present invention is a method for diagnosing toxoplasmosis in vitro, the method comprising detecting the presence of Toxoplasma gondii polypeptide BCLA in a biological sample from a subject to be tested, as shown above.

[0117] The term "toxoplasmosis" has its general meaning in this field and refers to a globally distributed zoonotic disease of medical importance in pregnant women and immunocompromised patients. Toxoplasma gondii, the pathogen of toxoplasmosis, has co-evolved with its homeothermic hosts (including humans), but usually persists as a quasi-latent population, thus a strategy for persistence with asymptomatic signs, and therefore optimizing the possibility of transmission to new hosts. During its long-term residency in warm-blooded metazoans, the proliferation stage (tachyzoite) switches to a persistence stage (bradyzoite enclosed in cysts), thereby giving the parasite a unique opportunity to spread to new hosts without progressing through its sexual stage, which is limited to felines. Uncontrolled amplification of the tachyzoite population resulting from a temporary or more persistent disruption of the immune balance can lead to life-threatening diseases, and in the case of congenital toxoplasmosis, birth defects. Persistence depends on both the acquisition of slow replication skills by a subset of the parasite and the destruction of rapid replication populations, and critically requires the IL-12 / IFN-γ immune axis, but T. Gondi has independently developed a finely tuned, epigenetically regulated developmental program to manipulate the stepwise transition.

[0118] In some embodiments, toxoplasmosis is congenital toxoplasmosis.

[0119] Thus, the present invention relates to a method for diagnosing congenital toxoplasmosis in vitro, the method comprising detecting the presence of the polypeptide described in claim 1 in a biological sample from a subject to be tested.

[0120] As used herein, the term “latent form of toxoplasmosis” refers to the persistent stage of toxoplasmosis (bloodizoids encapsulated in cysts). Following an initial period of infection characterized by tachyzoite proliferation throughout the body, pressure from the host's immune system converts T gonditachyzoites into bloodyzoites (a semi-dormant, slowly dividing cellular stage of the parasite). Within host cells, clusters of these bloodyzoites are known as histiocysts. The cyst wall is formed by the parasitic cyst membrane. While bloodyzoite-containing histiocysts can form in virtually any organ, they primarily form and persist in the brain, eyes, and striated muscle (including the heart). However, specific histotropy can vary between intermediate host species; in pigs, the majority of histiocysts are found in muscle tissue, while in mice, the majority of cysts are found in the brain. Cysts typically range in size from 5 to 50 μm in diameter (50 μm is about two-thirds the width of an average human hair).

[0121] Furthermore, the present invention also provides a kit comprising at least one antibody or fragment thereof. The kit of the present invention may include an antibody conjugated to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). A kit comprising an antibody for in vitro detection and quantification of Toxoplasma gondii polypeptide BCLA, for example, in ELISA or Western blotting, can be provided. Such antibodies useful for detection may be provided labeled, such as fluorescent or radiolabeled.

[0122] Diagnostic method for latent toxoplasmosis (detection of autoantibodies of T-gondipolypeptide BCLA)

[0123] When synthesized as a recombinant protein, the inventors have clearly demonstrated that BCLA constitutes an efficient serum marker of highly susceptible latent infection, clearly and exclusively correlated with the presence of cysts in mouse brains. Antibodies directed against BCLA antigens have been detected in human patients. Enriched titers were detected in patients qualified as seropositive for Sag1 or tachyzoite-related antigens. Further correlation in humans between anti-BCLA IgG synthesis and cysts is brought about by significantly stronger recorded titers in pathological panels strongly associated with the presence of cysts. Notably, in patients experiencing serological relapses and those with proven 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 some aqueous humor and serum of these patients. Detection of toxoplasma antibodies directed against semi-dormant cysts represents a significant improvement to the serological diagnosis of toxoplasmosis, opening up new diagnostic prospects. In fact, a few components of cyst wall or surface bradyzoites have been identified, but none have been shown to be useful as antigens for serological purposes, at least in commercially available kits. The ideal antigen should be expressed only at the latent bradyzoite stage, and ideally exposed to the cyst surface, which are two features found in BCLA polypeptides.

[0124] Furthermore, the inventors have demonstrated that children specifically synthesize anti-BCLA IgG before birth. Thus, the reactivity of BCLA can further guide the diagnosis of congenital toxoplasmosis at birth, compared with titrations of Toxo IgG by Vidas® and Architect® (see Example 3).

[0125] Therefore, the method for detecting autoantibodies of T-gondipolypeptides according to the present invention is consequently useful for the in vitro diagnosis of toxoplasmosis from biological samples. In particular, the detection method of the present invention is consequently useful for the in vitro diagnosis of latent forms of toxoplasmosis or congenital toxoplasmosis from biological samples.

[0126] A further object of the present invention is a method for diagnosing toxoplasmosis in vitro, the method comprising detecting the presence of a T autoantibody of the T gondipeptide according to the present invention in a biological sample from a subject to be tested, as shown above.

[0127] Thus, the present invention relates to a method for determining whether or not a subject is suffering from a latent form of toxoplasmosis, the method comprising the following: a) To detect immunoreactivity to the T-gondipolypeptide of the present invention in a patient's biological sample; and optionally b) Based on the results of step a), it can be estimated whether or not the patient has latent toxoplasmosis, and the immunoreactivity to the T-gondipolypeptide of the present invention indicates latent toxoplasmosis.

[0128] The present invention also relates to the use of antibodies directed against latent toxoplasmosis as biomarkers for diagnosing (or confirming) latent toxoplasmosis in patients.

[0129] 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, and includes the following: a) Obtaining biological samples from patients, and b) Detecting antibodies against the T-gondipolypeptide of the present invention in a biological sample; Here, the presence of antibodies in a biological sample is used to diagnose or confirm latent toxoplasmosis in a patient.

[0130] Thus, the present invention relates to a method for determining whether or not a subject is suffering from congenital toxoplasmosis, the method comprising the following: a) To detect immunoreactivity to the T-gondipolypeptide of the present invention in a patient's biological sample; and optionally b) Based on the results of step a), it can be estimated whether or not the patient has congenital toxoplasmosis, and the immunoreactivity to the T-gondipolypeptide of the present invention indicates congenital toxoplasmosis.

[0131] The present invention also relates to the use of antibodies directed against congenital toxoplasmosis as biomarkers for diagnosing (or confirming) congenital toxoplasmosis in patients.

[0132] The present invention also relates to an in vitro method for diagnosing or confirming congenital toxoplasmosis in patients who have or are suspected of having congenital toxoplasmosis, and includes the following: b) Obtaining biological samples from patients, and b) Detecting antibodies against the T-gondipolypeptide of the present invention in a biological sample; Here, the presence of antibodies in a biological sample is used to diagnose or confirm congenital toxoplasmosis in a patient.

[0133] As used herein, the term “biological sample” refers to any biological sample of the subject. In a preferred embodiment of the method of using the detection of antibodies directed against T. Gondi BCLA polypeptide, the biological sample is a body fluid of the subject. Non-limiting examples of such samples include, but are not limited to, blood, serum, plasma, urine, saliva, and cerebrospinal fluid (CSF) and aqueous humor.

[0134] More specifically, the body fluid sample is either serum or aqueous humor.

[0135] In a preferred embodiment, the patient being tested has or is suspected of having toxoplasmosis.

[0136] In another preferred embodiment, the patient being tested is suspected of having toxoplasmosis, and this method is performed to confirm that the patient actually has a latent form of toxoplasmosis.

[0137] In another embodiment, the patient being tested is a pregnant woman and / or an immunocompromised patient (i.e., an HIV patient or a patient treated with immunomodulation before transplantation), and this method is performed to determine whether the patient actually has a latent form of toxoplasmosis.

[0138] The current treatment for toxoplasmosis in subjects exhibiting signs and symptoms of acute toxoplasmosis is as follows: • Pyrimethamine (Daraprim). This medication is typically used for malaria and is a folate antagonist. It can interfere with the body's absorption of folate (folic acid, vitamin B-9), a B vitamin, especially if the patient is taking high doses over a long period. For this reason, supplemental folate intake may be recommended. Other potential side effects of pyrimethamine include bone marrow suppression and hepatotoxicity. • Sulfadiazine. This antibiotic is used with pyrimethamine to treat toxoplasmosis.

[0139] For HIV / AIDS patients, the treatment of choice for toxoplasmosis is also pyrimethamine and sulfadiazine, accompanied by folic acid (leucovorin). An alternative is pyrimethamine taken with clindamycin (creosin).

[0140] Regarding pregnant women and infants infected with toxoplasmosis:

[0141] If the infection occurs before 16 weeks of gestation, the pregnant woman will receive the antibiotic spiramycin. The use of this drug may reduce the risk of the infant developing neurological problems from congenital toxoplasmosis.

[0142] If the infection occurs after 16 weeks of gestation, or if testing indicates that the fetus has toxoplasmosis, the pregnant woman may be given pyrimethamine, sulfadiazine, and folic acid (leucovorin).

[0143] The present invention also provides an in vitro method for selecting patients suffering from a latent form of toxoplasmosis suitable for treatment with at least one folate antagonist and / or antibiotic compound, which includes: a) To detect immunoreactivity to the T-gondipolypeptide of the present invention in a patient's biological sample; and optionally b) If an immunoreactivity to the T-gondipolypeptide of the present invention is detected, select patients who are suitable to be treated with at least one folate antagonist (i.e., pyrimethamine) and / or an antibiotic compound (i.e., sulfadiazine or spiramycin).

[0144] Methods for determining whether a patient has latent toxoplasmosis, the use of antibodies directed against the T-gondipolypeptide of the present invention as a biomarker for diagnosing (or confirming) latent toxoplasmosis, and methods for selecting patients with latent toxoplasmosis suitable for treatment with at least one folate antagonist and / or antibiotic compound of the present invention may be, for example, in vitro or ex vivo methods.

[0145] The present invention also relates to a method for treating a patient infected with a latent form of toxoplasmosis that exhibits immunoreactivity to the T-gondipolypeptide of the present invention, the method comprising administering to the patient a folate antagonist (i.e., pyrimethamine) and / or an antibiotic compound (i.e., sulfadiazine or spiramycin), or a pharmaceutical composition containing said compound.

[0146] The present invention also provides a folate antagonist (i.e., pyrimethamine) and / or an antibiotic compound (i.e., sulfadiazine or spiramycin), or a pharmaceutical composition comprising such compound, for use in the treatment of patients suffering from latent forms of toxoplasmosis who exhibit immunoreactivity to the T-gondipolypeptide of the present invention.

[0147] In some embodiments, the T-gondipolypeptide of the present invention, which is tested for immunoreactivity, is the BCLA (brain cystic load-associated antigen) protein (abbreviated as "BCLA"), the C-terminal domain of BCLA (res 1089 to 1275, SEQ ID NO: 2) (abbreviated as "rBCLA"), or the internal repeat domain of BCLA (res 304 to 924 of BCLA) (referred to as TgR1 to TgR13 (SEQ ID NOs: 4 to 16)).

[0148] In certain embodiments, the protein whose immunoreactivity is tested is the rBCLA polypeptide.

[0149] In another specific embodiment, the protein being tested for immunoreactivity is a peptide fragment of at least nine consecutive amino acids of BCLA, an rBCLA sequence, or the internal repeat domain of BCLA (res 304-924 of BCLA) (referred to as TgR1 to TgR13 (sequences 4 to 16)).

[0150] In particular, the term "T-gondipolypeptide" of this invention, which is tested for immunoreactivity, refers to the following: (i) Amino acid sequence consisting of Toxoplasma gondipeptide BCLA (SEQ ID NO: 1); (ii) The amino acid sequence consisting of the C-terminal antigen domain (called rBCLA, res 1089-1275 of BCLA) (SEQ ID NO: 2); (iii) Amino acid sequences consisting of the internal repeat domain of BCLA, selected from the group consisting of the following: 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); (iv) An amino acid sequence substantially homologous to the sequences of (i) to (iii), preferably an amino acid sequence that is at least 80% identical to the sequences of (i) to (iii). (v) A fragment of at least nine consecutive amino acids from the sequence (i) to (iv).

[0151] Therefore, in a particular embodiment, the Toxoplasma gondii polypeptide isolated from the rBCLA polypeptide to be tested for immunoreactivity is tested / selected from the group consisting of: (i) GELQPAEAEEARLLVADLKAV (domain A of rBCLA) (Sequence ID 32) (ii) VRVEGEAFFRASVDLYEA (domain B of rBCLA) (Sequence ID 33) (iii) KLRPLTKGELVDVVRQ (domain C of rBCLA) (Sequence ID 34) (iv)TQIFVQDRASAFLRV(rBCLA peptide 36 and rBCLA domain D)(SEQ ID NO: 35) (v)AAEQMKAVFAMVEEG (rBCLA peptide 44 and rBCLA domain E) (SEQ ID NO: 36) (vi) An amino acid sequence substantially homologous to the sequences of (i) to (v), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (v). (vii) A fragment of at least nine consecutive amino acids in the sequence from (i) to (vi).

[0152] In a more specific embodiment, the Toxoplasma gondii polypeptide isolated from the rBCLA polypeptide to be tested for immunoreactivity is selected from the following group: (i) GELQPAEAEEARLLV (rBCLA peptide 12) (SEQ ID NO: 37); (ii) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38), (iii) EAEEARLLVADLKAV (rBCLA peptide 14) (SEQ ID NO: 39), (iv)VRVEGEAFFRASVDL (rBCLA peptide 21) (SEQ ID NO: 40), (v)EGEAFFRASVDLYEA(rBCLA peptide 22)(SEQ ID NO: 41); (vi) AFFRASVDLYEAVKN (rBCLA peptide 23) (SEQ ID NO: 42), (vii)KLRPLTKGELVDVVR(rBCLA peptide 30)(SEQ ID NO: 43) (viii) An amino acid sequence substantially homologous to the sequences of (i) to (vii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (vii). (vii) A fragment of at least nine consecutive amino acids in the sequence (i) through (viii).

[0153] Therefore, in a particular embodiment, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA, which is to be tested for immunoreactivity, is selected from the group consisting of: (i) Amino acid sequence consisting of the internal repeat domain of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7) (ii) An amino acid sequence substantially homologous to the sequence of (i), preferably an amino acid sequence that is at least 80% identical to the sequence of (i). (iii) A fragment of at least nine consecutive amino acids in the sequence of (i) to (ii).

[0154] In more specific embodiments, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA, which is tested for immunoreactivity, is selected from the group consisting of: (i)AAGSMEKEKPVLPGEGEGH(Domain A of TgR4);(Sequence ID 44) (ii) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4), (SEQ ID NO: 45) (iii) An amino acid sequence substantially homologous to the sequences of (i) to (ii), preferably an amino acid sequence that is at least 95% identical to the sequences of (i) to (ii). (iv) A fragment of at least nine consecutive amino acids in the sequence of (i) to (iii).

[0155] In a more specific embodiment, the Toxoplasma gondii polypeptide isolated from the internal repeat domain of BCLA, which is to be tested for immunoreactivity, is selected from the group consisting of: (i) AAGSMEKEKPVLPGE (TgR4 peptide 3); (SEQ ID NO: 46) (ii) GSMEKEKPVLPGEGE (TgR4 peptide 4) (SEQ ID NO: 47) (iii) MEKEKPVLPGEGEGH (TgR4 peptide 5) (SEQ ID NO: 48) (iv)KEKPVLPGEGEGHVL (TgR4 peptide 6) (SEQ ID NO: 49) (v)KPVLPGEGEGHVLPG (TgR4 peptide 7) (SEQ ID NO: 50) (vi) HVLPKHETKPALTDEK (TgR4 peptide 13), (SEQ ID NO: 51) (vii)PKHETKPALTDEKRT(TgR4 peptide 14), (SEQ ID NO: 52) (viii) HETKPALTDEKRTKP (TgR4 peptide 15) (SEQ ID NO: 53) (ix)TKPALTDEKRTKPGG (TgR4 peptide 16) (SEQ ID NO: 54) (x) An amino acid sequence substantially homologous to the sequences (i) through (ix), preferably an amino acid sequence that is at least 95% identical to the sequences (i) through (ix). (xi) A fragment of at least nine consecutive amino acids in the sequence (i) to (x).

[0156] Because BCLA polypeptides have numerous epitopes throughout their different domains (particularly in rBCLA and in the internal repeat domains of BCLA TgR1 to TgR13), combining the BCLA immunogenic peptide fragments of the present invention can be advantageous.

[0157] Therefore, in another embodiment, the polypeptide of the present invention whose immunoreactivity is to be tested is a fusion of two immunogenic peptide fragments of the present invention, for example. 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 NO: 55) Peptide A3_B:AAGSMEKDKLVLPGE (peptide fragment from repeating motifs present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID NO: 56)

[0158] Therefore, in another embodiment, the polypeptide of the present invention derived from the internal repeat domain (TgRx) of BCLA, which is tested for immunoreactivity, has the following sequence: 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 NO: 64) Here, Xaa1 is glutamic acid (E) or there is no amino acid residue. Here, Xaa2 is arginine (R) or serine (S). Here, Xaa3 is proline (P) or glycine (G). Here, Xaa4 is either alanine (A) or glycine (G). Here, Xaa5 is either alanine (A) or there is no amino acid residue. Here, Xaa6 is either glycine (G) or arginine (R). Here, Xaa7 is serine (S), proline (P), or alanine (A). Here, Xaa8 is either lysine (K) or glutamic acid (E). Here, Xaa9 is lysine (K), glutamic acid (E), or aspartic acid (D). Here, Xaa10 is either proline (P) or leucine (L). Here, Xaa11 is leucine (L) or serine (S). Here, Xaa12 is glutamic acid (E) or lysine (K). Here, Xaa13 is either glycine (G) or arginine (R). Here, Xaa14 is valine (V) or alanine (A). Here, Xaa15 is leucine (L) or serine (S). Here, Xaa16 is histidine (H), aspartic acid (D), or alanine (A). Here, Xaa17 is threonine (T), arginine (R), methionine (M), or glutamine (Q). Here, Xaa18 is proline (P), threonine (T), or alanine (A). Here, Xaa19 is aspartic acid (D), glutamic acid (E), or glutamine (Q). Here, Xaa20 is glutamic acid (E) or lysine (K). Here, Xaa21 is lysine (K), glycine (G), or glutamic acid (E). Here, Xaa22 is arginine (R) or valine (V). Here, Xaa23 is lysine (K), glutamic acid (E), or asparagine (N). Here, Xaa24 is glycine (G), valine, or isoleucine (I). Here, Xaa25 is glycine (G) or glutamic acid (E). Here, Xaa26 is arginine (R) or proline (P). Here, Xaa27 is threonine (T) cysteine ​​(C) lysine (K) or methionine (M). Here, Xaa28 is glutamic acid (E) or alanine (A). Or a fragment of at least nine consecutive amino acids of the sequence of SEQ ID NO: 64.

[0159] "Polypeptides with substantially homologous amino acid sequences" means polypeptides 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 respect to a full-length polypeptide reference sequence. In the context of this application, the percentage of identity is calculated using global alignment (i.e., two sequences are compared over their full lengths). Methods for comparing the identity of two or more sequences are well known in the art. The ≪needle≫ program (which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences, taking their entire lengths into account) can be used, for example. The needle program is available, for example, on the ebi.ac.uk worldwide website. The percentage of identity according to the present invention is preferably calculated using the EMBOSS :: needle (global) program with a “gap open” parameter equal to 10.0, a “gap extended” parameter equal to 0.5, and a Blosum62 matrix.

[0160] As used throughout this application, the expression “immunoreactivity to target protein” (here, T-gondipolypeptide of the present invention) is intended to mean that a sample from the patient being tested contains antibodies specifically against the target.

[0161] Therefore, immunoreactivity to a target protein can be easily detected by demonstrating the presence of antibodies specifically against the target protein or a fragment of this target protein in the biological sample being tested.

[0162] The target protein fragment may be cleaved at the N-terminus or C-terminus, or may lack internal residues compared to, for example, the full-length protein. Preferably, the fragment has an amino acid length of 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.

[0163] Such tests can be performed by one of the usual skills in the art, using standard methods such as enzyme-linked immunosorbent assay ("ELISA"), Western blot / dot blot, immunohistochemistry of transfected cells, and Luminex (for reviews, see Immunodiagnostics: A Practical Approach, R. Edwards Editor, Oxford University Press 2000; Manual of Molecular And Clinical Laboratory Immunology, JD Folds RG Hamilton, B. Detrick Editors ASM Press 2006; Immunology and Serology in Laboratory Medicine, ML Turgeon, Mosby Inc, 2008).

[0164] 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 antigen domain (BCLA res 1089-1275) (referred to as rBCLA (SEQ ID NO: 2)), the internal repeat domain of BCLA (BCLA res 304-924) (referred to as TgR1 to TgR13) (SEQ ID NOs: 4 to 16), or a fragment thereof. Preferably, the target protein consists of, or includes, the C-terminal antigen domain (BCLA res 1089-1275, referred to as rBCLA (SEQ ID NO: 2)), the internal repeat domain of BCLA (BCLA res 304-924) (referred to as TgR1 to TgR13) (SEQ ID NOs: 4 to 16), or a fragment thereof.

[0165] As used herein, the term “patient” means a mammal, more specifically, a human.

[0166] In the context of the present invention, the term “to treat” is used herein to characterize a treatment method or process aimed at (1) slowing or halting the progression, exacerbation, or worsening of the symptoms of a disease state or condition to which such term applies; (2) reducing or relieving the symptoms of a disease state or condition to which such term applies; and / or (3) reversing or curing the symptoms of a disease state or condition to which such term applies.

[0167] The folate antagonists and / or antibiotic compounds used in the above-described method, or used to treat patients with latent forms of toxoplasmosis, are provided in a pharmaceutically acceptable carrier, excipient, or diluent that is not harmful to the patient being treated.

[0168] pharmaceutically acceptable carriers and excipients that may 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 da-tocopherol polyethylene glycol 1000 succinic acid, surfactants used in pharmaceutical dosage forms, such as Tweens or other similar polymer delivery matrices, serum proteins, such as human serum albumin, buffering substances, such as phosphoric acid, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and lanolin.

[0169] As will be understood by those skilled in the art, compositions are appropriately formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral routes, such as 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 that the active ingredient can be protected from gastric and intestinal enzymes.

[0170] Furthermore, the amounts of folate antagonists and / or antibiotic compounds used in the above-described method, or used to treat patients suffering from latent toxoplasmosis, are therapeutically effective.

[0171] The exact amount of folate antagonist and / or antibiotic compound used, as well as the composition administered, may vary depending on the age and weight of the patient being treated, the type of disease, the mode of administration, the frequency of administration, and other components in the composition containing the folate antagonist and / or antibiotic compound. Such concentrations can be routinely determined by those skilled in the art. The actual amount of compound administered may typically be determined by a physician in light of the relevant circumstances, including the condition being treated, the chosen route of administration, the actual folate antagonist and / or antibiotic compound administered, the individual patient's age, weight, and response, and the severity of the patient's symptoms.

[0172] Generally, folate antagonists and / or antibiotic compounds used in the above-described methods, or used to treat patients with latent toxoplasmosis, may be administered within a typical range. Effective doses may also vary depending on the route of administration and the possibility of concomitant use with other drugs.

[0173] The present invention further provides a kit useful in the above method for diagnosing latent toxoplasmosis or for selecting patients suffering from latent toxoplasmosis suitable for treatment with at least one folate antagonist and / or antibiotic compound.

[0174] Such a kit includes means for detecting an antibody against at least one Tgondipolypeptide of the present invention.

[0175] Preferably, the kit includes means for detecting antibodies directed at at least the BCLA polypeptide, or the C-terminal antigen domain (res 1089-1275 of BCLA) or a fragment thereof.

[0176] Such means may be a target protein, i.e., a T-gondipolypeptide of the present invention whose immunoreactivity is being tested, or a fragment thereof as described above. For example, when immunoreactivity to BCLA is being tested, the target protein is a full-length BCLA protein consisting of or comprising a C-terminal antigen domain called rBCLA (SEQ ID NO: 2) (BCLA res 1089-1275) and internal repeat domains of BCLA called TgR1 to TgR13 (SEQ ID NO: 4 to SEQ ID NO: 16) (BCLA res 304-924). Preferably, the target protein consists of or comprises a C-terminal antigen domain called rBCLA (SEQ ID NO: 2) (BCLA res 1089-1275) and internal repeat domains of BCLA called TgR1 to TgR13 (BCLA res 304-924).

[0177] The means for detecting an antibody directed against at least one T-gondipolypeptide of the present invention may also include an antibody that specifically binds to a human antibody (used as a “secondary antibody” that specifically binds to the target protein and binds to an antibody from the sample being tested). Such an antibody can be labeled with a detectable compound, such as a fluorophore or a radioactive compound.

[0178] In a preferred embodiment, the kit according to the present invention may further include a control sample containing a known amount of antibody and / or instructions for the use of the kit in diagnosing latent toxoplasmosis or in selecting a patient suffering from latent toxoplasmosis who is suitable to be treated with at least one folate antagonist and / or antibiotic compound.

[0179] The means may be present, for example, in a vial or microtiter plate, or attached to a solid support. For example, the target protein can be attached to a membrane or array.

[0180] A further object of the present invention is a method for detecting bradyzoite cysts and / or evaluating their quantity in a subject, the method comprising: a) To detect immunoreactivity to the Tgondipolypeptide described in any one of claims 1 to 2 in the target body fluid sample; and optionally b) The presence and / or amount of bradyzoite cysts can be estimated from the results of step a), and the immunoreactivity to the T-gondipolypeptide of the present invention indicates the presence and / or amount of bradyzoite cysts in the subject.

[0181] In a preferred embodiment, 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, and cerebrospinal fluid (CSF) and aqueous humor.

[0182] More specifically, the body fluid sample is either serum or aqueous humor.

[0183] All references cited herein include journal articles or abstracts, published patent applications, issued patents, or other references, which are incorporated herein in whole by reference and include all data, tables, figures, and text presented in the cited references.

[0184] The present invention will be further evaluated with reference to the following examples and figures. [Brief explanation of the drawing]

[0185] [Figure 1]Figure 1. BCLA is a bradyzoite-specific gene regulated by TgHDAC3. (a) Quantitative proteome-wide analysis by LC-MS / MS after TgHDAC3 inhibition using FR235222 revealed the expression of bradyzoite-specific proteins among BCLAs. The volcano plot shows the distribution of T gondii protein when comparing primary human fibroblasts treated with FR235222 (90 nM) to untreated cells (DMSO, 0.1%) infected with type II (PruΔku80) strain. The log2 ratio (x axis) for protein count was obtained by dividing the intensity of the FR235222-treated sample by the intensity of the DMSO-treated sample (control). Downward and upward regulated proteins are shown as red spots on the left and right sides of the graph, respectively. The vertical black line shows the value of the log2 change. The horizontal black dashed lines distinguish the protein (red spots), showing at least a twofold change in abundance with a p value < 0.01. (b) Bar graphs showing BCLA gene expression (kilobases of transcript fragments per million mapped reads [FPKM]) between acute (tachyzoite) and chronic (bradyzoite encapsulated in cysts) infections in mice, in various feline intestinal epithelial stage (EES1: very early EES; EES2: early EES; EES3: mixed EES; EES4: late EES; EES5: very late EES) samples from 3 to 7 days after oral infection with T Gondi cysts (CZ clone H3), and in cysts from mouse brains and in vitro cultured tachyzoites. BCLA is expressed only during the chronic phase of cystic-encapsulated bradyzoites and is not found in the feline intestinal epithelial stage (EES1 to EES5) (data source: www.ToxoDB.org). (c) Genome Browser (IGB) screenshot of the BCLA locus (magenta) on chromosome Ib of T. Gondi, showing reads for two histone marks (H3K14ac, H3K9me3), TgHDAC3, TgCRC230, and RNA-seq (expressed in FPKM, black). The y-axis depicts read density.This display shows enrichment of H3K14ac, H3K9me3, TgHDAC3, and TgCRC230 in the BCLA gene. (d) Left panel: CRISPR-mediated disruption of TgHDAC3 leads to suppression of TgHDAC3 signaling when monitored by immunofluorescence assay. Right panel: CRISPR-mediated disruption of TgHDAC3 induces BCLA overexpression when monitored by immunofluorescence assay. [Figure 2] Figure 2. The BCLA protein reveals a unique structure typically represented by unstructured tandem repeats. (a) Chart showing the impairment score as a function of protein amino acid positions (generated via the IUPred server). Results from the ANCHOR2 and IUPred2 algorithms are presented in blue and red, respectively. The C-terminal domain of BCLA (residues 1089 to 1275, hereafter 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 the type II (ME49) T Gondi strain presents 13 repeats in its structure (TgR1 to TgR13). Autoantibodies directed against the two peptides (peptides 1 and 2) contained in these repeats were produced by Eurogentec. (c) Western blotting monitoring of BCLA expression using autologous antibodies produced against the two BCLA-derived peptides shows upregulation of BCLA after FR235222 treatment compared to DMSO (control). [Figure 3] Figure 3. BCLA during FR235222 induction is present within the vacuolar space and on the vacuolar membrane. (a) Quantification of BCLA intensity in each PV following FR235222 stimulation. Each symbol marks the BCLA density of a single PV. Results are expressed as mean ± standard deviation from two independent experiments; the number of PVs quantified was at least 70. Asterisks indicate statistical significance when comparing each individual FR235222-treated strain with the corresponding control (DMSO, 0.1%), as determined by an unpaired two-sided Student's t-test (Mann-Whitney U test) (****p<0.0001; NS, not significant). [Figure 4] Figure 4. BCLA deletion does not dramatically affect parasite growth or vacuole formation and maturation in vitro. (a) Evaluation of the percentage of invasion in HFF (left panel) and intracellular proliferation rate (right panel) of 76k-GFP-luc-Δbcla tachyzoite cultured in vitro compared with WT strains. The % of HFF invasion was quite similar in both strains, however, BCLA deletion induced a 30% decrease in intracellular proliferation. Results are presented as mean ± standard deviation from two independent experiments. Asterisks indicate statistical significance when comparing 76k-GFP-luc-Δbcla and 76k-GFP-luc by Mann-Whitney U test (unpaired two-tailed Student's t-test). **p<0.01; NS, not significant. [Figure 5]Figure 5. BCLA deletion does not significantly alter T. Gondi pathogenicity or cystic load in mice infected intraperitoneally with tachyzoite. (a) Comparison of pathogenicity of the 76k-GFP-luc-Δbcla strain and its parent strain 76k-GFP-luc (WT) in Balb / c mice and NMRI mice. Balb / c mice (n=20) and NMRI mice (n=43) were inoculated with 104 and 106 tachyzoites by intraperitoneal (ip) injection, respectively, and survival was monitored for 35 days. Significance was tested using the log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. Mice infected with Δbcla tachyzoite survived infection within the same time frame as the WT strain (NS, not significant). (b) Evaluation of the ability of the Δbcla strain to migrate across the blood-brain barrier and form T. Gondii cysts in the brains of mice chronically infected with T. Gondii, compared to the WT strain. Brains of NMRI and Balb / c mice that survived the challenge, as presented in (a), were collected and tested by quantitative PCR ± cyst count using a microscope to assess parasite load and cyst number, respectively. Results are expressed as mean ± standard deviation from at least two independent experiments. Statistical significance was tested by an unpaired two-sided Student's t-test (Mann-Whitney U test). Mice infected with the Δbcla strain showed a trend (but not statistically significant, NS) in reduced parasite load and number of cysts in the brain. [Figure 6]Figure 6. BCLA-deficient cysts are typically represented by dramatic morphological changes. The cyst morphology of Δbcla bradyzoite-containing cysts was compared to that of the parental 76k-GFP-luc (WT) strain. Brains of NMRI mice that survived the challenge, as presented in Figure 6a, were collected, and the 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 to those obtained from WT cysts. Δbcla-containing cysts had significantly smaller size and lower GFP intensity than WT cysts. Results are presented as mean ± standard deviation from at least two independent experiments. Asterisks indicate statistical significance when comparing cyst area of ​​Δbcla-containing cysts and WT cysts, as determined by an unpaired two-sided Student's t-test (Mann-Whitney U test) (***p<0.001). Scale bar, 10 μm. [Figure 7]Figure 7. BCLA deletion did not alter the infectivity of mice orally ingesting cysts or the host immune response. Evaluation of pathogenicity and infectivity of 76k-GFP-luc-Δbcla-containing cysts 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 and 20 cysts of either the Δbcla strain or the WT strain, respectively. An acute response in the ileum was observed in C56BL / 6 mice 8 days post-infection. A chronic response in the brain was evaluated in NMRI mice 8 to 10 weeks post-infection. (a) Parasite load in the ileum of C56BL / 6 mice orally infected 8 days prior, quantified by qPCR. Statistical significance between the Δbcla strain and the WT strain was tested by an unpaired two-sided Student's t-test (Mann-Whitney U test). No significant difference was 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 standardized using TBP levels. Mean ± standard deviation is shown. Statistical significance between Δbcla and WT was tested by the Mann-Whitney U test. No significant difference was observed (NS, not significant). (c) Brains of NMRI mice orally infected 8-10 weeks prior were collected and tested by quantitative PCR and microscopic cyst count to assess parasite load and cyst number, respectively. Results are expressed as mean ± standard deviation from two independent experiments. Statistical significance between Δbcla and WT was tested by the Mann-Whitney U test. No significant difference was observed (NS, not significant). Mice infected with the Δbcla strain show a trend (but not significant, NS) in reduced parasite load and number of cysts in the brain. (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 standardized using TBP levels. Mean ± standard deviation is shown. Statistical significance between Δbcla and WT was tested by the Mann-Whitney U test. No significant difference was observed (NS, not significant). [Figure 8]Figure 8. rBCLA did not react with acutely infected mouse serum. A single Western blot strip was loaded with 0.5 μg of recombinant rBCLA. The strip was tested with serum collected from mice in the acute phase of toxoplasmosis, with various T Gondi strains, routes of infection, and the genetic background of the mice. rBCLA did not react with mouse antibodies during the acute phase of infection (7-8 days). (a) Immunoblot with serum from NMRI mice infected by intraperitoneal injection (ip) of 104 tachyzoites of COUG strain and COUG-Δmyr1 (atypical haplotype 11) strain over 7 days. Serum did not react with rBCLA. (b) Immunoblot with serum from CBA mice infected by ip with 103 tachyzoites of RH (type I) strain over 7 days. Serum did not react with rBCLA. (c) Immunoblotting of serum from C57BL / 6 mice infected orally with 76k-GFP-luc strain or 76k-GFP-luc-Δbcla (type II) strain in 47 cysts over an 8-day period. Serum did not react with rBCLA. [Figure 9]Figure 9. rBCLA is a serum marker for chronic T. Gondi infection in a mouse model. 0.5 μg of rBCLA was loaded onto a single Western blot strip and tested with serum collected from mice in the subchronic (21–41 days) or chronic (>42 days) phase of toxoplasmosis. rBCLA reacts only with anti-T. Gondi IgG antibodies in mice with subchronic or chronic toxoplasmosis following infection with type II cystic strains (PruA7, ME49, or 76k-GFP-luc). (a) Immunoblots with serum from Balb / c mice infected with ip in 103–106 tachyzoites / mouse of PruA7 (type II) strain over 42 days. Serum reacts fairly proportionally with rBCLA according to tachyzoite loading. (b) Immunoblots with serum from CBA mice infected with 106 tachyzoites / mouse of the ME49 (type II) strain via ip over 80 days. The serum reacted strongly with rBCLA. (c) Immunoblots with serum from NMRI mice orally infected with 20 cysts of the 76k-GFP-luc (type II) strain over 22 months. The serum reacted strongly with rBCLA. (d) Immunoblots with serum from Balb / c mice infected with 106 tachyzoites / mouse of the 76k-GFP-luc strain or the 76k-GFP-luc-Δbcla (type II) strain via ip over 21 days. Serum from mice infected with 76k-GFP-luc reacted strongly with rBCLA, while serum from mice infected with 76k-GFP-luc-Δbcla reacted very little with rBCLA. (e) Immunoblotting of serum from CBA mice infected with 103 tachyzoite / mouse RH(type I) strain for 22 days, followed by pyrimethamine (PYR) or sulfadiazine (Sulfa) treatment. Serum showed very slight reaction with rBCLA. (f, g) Immunoblotting of serum from NMRI mice infected with 42 days by ip with (f) 106 tachyzoite / mouse, followed by ip with CTG(type II I) strain or (g) PruΔku80(type II) strain. Serum did not react with rBCLA.(h) Immunoblotting with serum from Balb / c mice infected with 105 tachyzoites / mouse of the 76k-GFP-luc(type II) strain for 42 days without ip, burn, or corticosteroid treatment. All serums reacted strongly with rBCLA. [Figure 10] Figure 10. Proteolytic analysis of rBCLA reveals the boundary of the minimal antigenic region of BCLA. (a) Analysis of proteolytic reactions by SDS-PAGE. Coomassie-colored SDS-PAGE showing samples with all proteases (trypsin, chymotrypsin, elastase, and papain) and all of these time points (10, 20, and 50 min). (b) Blot gel incubated with positive mouse serum and revealed by anti-mouse IgG antibody. (c) Blot gel incubated with peroxidase-bound anti-6his IgG. Black arrows indicate undegraded rBCLA. Red and blue cursor arrows indicate recurrent N-terminal degradation, showing that rBCLA is rapidly degraded by chymotrypsin and partially degraded by elastase, trypsin, and papain, generating stable fragments around the 17kDa marker. [Figure 11]Figure 11. Evaluation of rBCLA as a serum marker in humans. A single Western blot strip was loaded with 0.5 μg of rBCLA and tested with mouse serum infected with strains isolated from humans or directly with human serum. (a) Immunoblots with serum from Swiss mice infected with ip in the amniotic fluid or placenta from women with suspected (clinically suspected, but T-Gondi PCR negative in amniotic fluid or placenta) or confirmed (T-Gondi PCR positive in amniotic fluid) congenital toxoplasmosis. Serum from mice infected with positive amniotic fluid reacted strongly with rBCLA. (b) Immunoblots with serum (S) or aqueous humor (HA) from human patients with or without toxoplasmosis infection. Human serum and aqueous humor were randomly selected from the biobank of the Parasitology-Mycology Clinical Laboratory at Grenoble Alpes University Hospital. For each sample, a toxoplasmosis seroassay was performed using the Vidas® (bioMerieux) and Architect® (Abbott) systems (both based on ELISA-derived technology), and the clinical status was assessed using each patient's medical record. Notably, Vidas® is based on the rSAG1 antigen, and Architect® is based on both the rSAG1 and rGRA8 antigens. Serological results obtained with rBCLA were compared to the serological and clinical status of each patient to assess whether they correlated with the serological and / or clinical status of a specific T. Gondi. (α) Serum from patients with proven or suspected ocular toxoplasmosis, (β) relapses of toxoplasmosis between hematopoietic disorders (immunosuppression), and (γ) recent primary infections (between 1 and 2 months) reacted with rBCLA. (δ) Serum from three seropositive patients identified as "past immunization" and one serum from a fairly recent infection (2.5 months) did not react with rBCLA. (ζ) All serum tested from serologically negative patients did not react with rBCLA, demonstrating good specificity of this antigen in humans. [Figure 12]Figure 12 | Evaluation of BCLA as a human serum marker. (a) Schematic diagram of epitope mapping regions in both repeat n°4 and rBCLA regions. Peptide coverage is presented as lines representing individual 15aa peptides above or below the peptide sequence, with partial numbering. Regions showing significant or strong reactivity are highlighted in solid or dashed boxes, respectively, and each individual peptide fragment is marked with (* or **). (b) Epitope mapping of BCLA-positive serum. The following are histograms showing the relative reactivity of peptides in both the core repeat region and rBCLA region, calculated using five different positive blots with negative background subtraction. Above is an example of the revealed pattern of a dot blot membrane using numbered peptides, performed with positive human serum. (c) Peptide dot blots for five positive and one negative serum with peptide numbering and region coverage. On the right, ELISA titrations for rBCLA and SAG1(Architect) are shown for these same serum samples. [Figure 13] Figure 13. BCLA reactivity in human serum. Scatter plots of individual BCLA ELISA titrations (UI) grouped into clinical status categories assessed through classical SAG1 serum (Vidas and Architect IgG / IgM) and other medical prerequisites. These groups are: active toxoplasmosis in SAG1 seronegative patients (blue dots), previously immune patients (diamonds), immunocompromised patients (cubes), asymptomatic serological relapse in immunocompromised patients (triangles), and patients with proven ocular toxoplasmosis (cubes). Histograms show the median and interquartile ranges of BCLA titration values ​​per group. Statistical significance was calculated using the Kruskal-Wallis nonparametric test followed by Dunn's post-hoc test, comparing all latter groups individually with the seronegative patient group. The gray zone (70 to 90 UI) and positive cutoff line (90 UI) are shown. [Figure 14]Figure 14. rBCLA immunogenicity correlates with cystic strains in the chronic phase of infection in mice. (a) ELISA serum titration of rBCLA reactivity in mice, time-dependent and T. Gondii strain-dependent. Individual ELISA values ​​given in UI are grouped according to T. Gondii strain type, with cystic strains (ME49, PruA7, 76K) shown as spots, non-cystic strains (RH, PruKU80, CTG) as stars, and ΔBCLA strains (76K or PruKU80 in the background) as triangles. Post-infection time segments are presented to distinguish between the acute phase (≤8 days), subchronic phase (21-22 days), and chronic phase (≥42 days). (b) Correlation of rBCLA ELISA reactivity with parasite load, miR-155 and miR-146a expression. The superimposed titrations of rBCLA IgG (UI), parasite load (parasite qPRC count), and miR-155 / miR-142-a are shown for different mouse strains (NMRI, Balb-C) that were either uninfected or infected with different T Gondi strains (all within the chronic infection timeframe (≧11 weeks)). Cystic strains (ME49, PruA7, 76K) are shown as circles, non-cystic strains (RH, PruKU80, CTG) as stars, and ΔBCLA strains (76K or PruKU80 in the background) as triangles. [Figure 15] Figure 15: BCLA reactivity in the serum of mothers and neonates at risk of congenital toxoplasmosis. (AB) Violin plots (UI) of BCLA ELISA titrations in the serum of 23 mothers and their respective neonates collected at birth (mother) or between birth and 5.5 months (infant). (CD) Violin plots of Sag1 titrations (Vidas® and Architect® IgG / IgM). Serums were classified into clinical status categories of “mother-neonatal” pairs without congenital toxoplasmosis (A and C) and pairs with confirmed congenital toxoplasmosis (B and D). The mean ± SD values ​​are shown at the top of each panel, while the difference between medians was calculated using the Mann-Whitney U test.

[0186] Example 1:

[0187] Materials and methods

[0188] Host cell and parasite culture. Primary HFF cells (Bougdour et al., 2009), RAW264.7 cells, L929 cells, HCT116 cells, A549 cells, and HEK293 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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 strains were used in this study: Type I (RH, GT1), Type II (ME49), Type III (CTG), Atypical (COUG), Neospora caninum; RHΔku80 (Huynh and Carruthers, 2009), PruΔku80 (Fox et al., 2011), PruA7 (Saeij et al., 2007), COUGΔmyr1 (Hakimi, unpublished), PruΔku80Δbcla, PruΔku80-HF-BCLA, and 76k-GFP-luc-Δbcla (obtained in this study). All parasitic strains were maintained in vitro by continuous passaging on a monolayer of HFF.

[0189] Transfection of T. gondii. T. gondii RHΔku80, PruΔku80, and 76k-GFP-luc were electroporated using a vector in cytomix buffer (120mM KCl, 0.15mM CaCl2, 10mM K2HPO4 / KH2PO4, pH 7.6, 25mM HEPES, pH 7.6, 2mM EGTA, 5mM MgCl2) using a BTX ECM 630 machine (Harvard instrument). Electroporation was performed in a 2mm cuvette at 1,100V, 25Ω, and 25μF. Stable transgenic parasites were selected with 1μM pyrimethamine, single-cloned in a 96-well plate by limiting dilution, and validated by immunofluorescence.

[0190] Cas9-mediated C-terminal tagging and gene disruption in T. Gondi. The plasmid pTOXO_Cas9-CRISPR was described by Sangare et al., 2016. The target gene of interest (GOI) was BCLA (TGME49_209755) for both C-terminal tagging (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 sgRNAs targeting the TgBCLA genome sequence, were phosphorylated, annealed, and ligated into the linearized pTOXO_Cas9-CRISP plasmid using Bsal to lead to pTOXO_Cas-CRISPR::sgTgBCLA. T gonditaxoite was then transfected using a plasmid and grown on HFF cells for 18–36 hours.

[0191] The cloning oligonucleotides used in this study are as follows: TgBCLA-KO-CRISP-FWD:5'-AAGTTGATCACTATTCGTGAAGAAGG-3'(Sequence ID 28) TgBCLA-KO-CRISP-REV:5'-AAAACCTTCTTCACGAATAGTGATCA-3'(Sequence ID 29) TgBCLA-HF-CRISP-FWD:5'-AAGTTGGAACGGCGGTACGGCGACCG-3'(Sequence ID 30) TgBCLA-HF-CRISP-REV:5'-AAAACGGTCGCCGTACCGCCGTTCCA-3'(Sequence ID 31)

[0192] FR235222 treatment and induction. FR235222 was supplied by 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 either 25 ng / mL or 50 ng / mL. The culture medium containing FR235222 was added to infected HFF cells 16 hours post-infection for 24 hours to 7 days.

[0193] Mice and experimental infection. 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 carried out under pathogen-free conditions in accordance with established facility guidance and approved protocols (agreement number B3851610006) from the University Grenoble Alpes Institutional Animal Care and Use Committee. Female mice were used for all tests. For intraperitoneal (ip) infection, tachyzoites were grown in vitro, extracted from host cells by passage through a 27-gauge needle, washed three times in PBS, and quantified using a hemocytometer. Parasites were diluted in Hanks equilibrium salt solution (Life), and mice were inoculated via the ip route using a 28-gauge needle with tachyzoites (200 μl) of each strain. For oral forced administration of infectious cysts, brains from chronically infected mice (76k-GFP-luc and 76k-GFP-luc-Δbcla) were pulverized in PBS, the number of cysts was microscopically quantified, and mice were force-fed 100 μl of brain homogenate containing 20 to 40 cysts using a spherical-tipped feeding needle. Blood was collected by caudal or intracardiac puncture at the time of euthanasia of the mice. Euthanasia of the animals was completed in an approved CO2 chamber. For histological analysis of the ileum and immunolabeling of histological sections of the brain, or, the ileum and brain were removed from the mice, fully embedded in paraffin wax blocks, and cut into 5 μm thick layers using a microtome. Mantel-Cox and Gehan-Breslow-Wilcoxon tests were used for statistical analysis of mouse survival data.

[0194] Cyst purification. Cysts were isolated directly from the brains of mice chronically infected with the 76k-GFP-luc or 76k-GFP-luc-Δbcla strain for at least 6 weeks using the previously described Percoll gradient method (Cornelissen et al., 1981). To avoid altering the cyst wall for permeability testing, a 10 μl pipette was used for dye experiments. Neither saponins nor trypsins were added at the end of the experiment.

[0195] Cyst quantification. Five to twelve weeks post-infection, the brains of each recipient mouse were homogenized in 2 ml of PBS. The number of cysts in three or ten aliquots (20 μl each) of the brain suspension was microscopically counted. The total number of cysts was determined by counting the cysts in the 20 μl aliquot and multiplying by 100. A non-parametric Wilcoxon-Mann-Whitney U test was used for statistical analysis of differences in cyst quantification between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla.

[0196] Cyst characterization. Images of purified cysts were acquired between slides and slipcovers using a fluorescence ZEISS ApoTome.2 microscope. Cyst area and GFP intensity were measured using ZEN software (Zeiss). Nonparametric Wilcoxon-Mann-Whitney U test was used for statistical analysis of cyst area and the difference in GFP intensity between 76k-GFP-luc and 76k-GFP-luc-Δbcla cysts.

[0197] Quantitative PCR. Parasite load in the brain or ileum was quantified following DNA extraction (QiAmp DNA minikit, Qiagen) using quantitative PCR targeting of Toxoplasma-specific 529 bp repeat elements (Reischl et al., 2003). Nonparametric Wilcoxon-Mann-Whitney U test was used for statistical analysis of differences in parasite load between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla.

[0198] 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 in a random hexamer using a high-volume RNA-to-cDNA kit (Applied Biosystems). Samples were analyzed by real-time quantitative PCR for 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 standardized using TBP levels. qRT-PCR was repeated for three independent biological replicas of each sample, and the mean of the results was used. Nonparametric Wilcoxon-Mann-Whitney U test was used for statistical analysis of RNA levels between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla.

[0199] Immunofluorescence microscopy. In vitro immunofluorescence assays with parasites were performed as previously described (Braun et al., 2013). Briefly, T. Gondii-infected HFF cells grown on coverslips or purified cysts from mouse brains were fixed in 3% formaldehyde at room temperature for 20 minutes, permeable with 0.1% (v / v) TritonX-100 for 15 minutes, and blocked in phosphate-buffered saline (PBS) containing 3% (v / v) bovine serum albumin (BSA). For immunolabeling on brain tissue sections, brain layers spotted on glass slides were first degreased using toluene for 3*10 minutes and anhydrous alcohol for 3*10 minutes. The slides were then treated with citrate buffer pH 6, heated at 100°C for 1 hour, rinsed with water for 2*10 minutes, and blocked in PBS containing 3% (v / v) bovine serum albumin (BSA). Cells or brain layers were then incubated with the primary antibody shown in the figure for 1 hour, followed by the addition of a 1:1,000 dilution secondary antibody conjugated to Alexa Fluor 488 or 594 (Molecular Probes) for 1 hour. Both host cell and parasite nuclei were stained with 2 μg / ml Hoechst 33258 in PBS at room temperature for 10 minutes. Cover slips were mounted on glass slides using Mowiol mounting medium, and images were acquired using a fluorescence ZEISS ApoTome.2 microscope. Images were processed using ZEN software (Zeiss).

[0200] Antibodies. Primary antibodies: Rabbit anti-BCLA (Eurogentec), mouse anti-HA (Roche, RRID: ab_2314622), rat anti-flag (SIGMA), mouse anti-CC2 (donated by Pr. Louis Weiss), mouse anti-GRA1, mouse anti-GRA5, mouse anti-GRA7. Western blot secondary antibodies were conjugated to alkaline phosphatase (Promega), while immunofluorescence secondary antibodies were conjugated to Alexa Fluor488 or Alexa Fluor494 (Thermo Fisher Scientific).

[0201] Western blot. Proteins were separated by SDS-PAGE and transferred by liquid transfer onto polyvinylidene fluoride membranes (Immobilon-P; EMP Millipore). Western blots were probed using appropriate primary antibodies followed by phosphatase-conjugated goat secondary antibodies (Promega). Signals were detected using NBT-BCIP (Amresco).

[0202] In vitro FR235222 parasites and DBA lectin labeling in ex vivo cysts. T gondii-infected HFF cells grown on coverslips or cysts purified from mouse brains were fixed in 3% formaldehyde for 20 min at room temperature, permeabilized with 0.1% (v / v) Triton X-100 for 15 min, and blocked in phosphate-buffered saline (PBS) containing 3% (v / v) bovine serum albumin (BSA). Infected cells or cysts were stained with diluted (1:100) Dolichos lectin for 30 min. Stained vacuoles or cysts were examined with a fluorescence ZEISS ApoTome.2 microscope, and images were processed with ZEN software (Zeiss).

[0203] Permeability of the cyst wall. 76k-GFP-luc and 76k-GFP-luc-Δbcla isolated cysts purified from mouse brains were incubated with different dyes (dextran, Texas Red, or Cascade Blue, 3000 to 40 000 Da, neutral or anionic lysine-fixable) diluted 1:100 (Promega). After 20 min incubation at room temperature, images were acquired using a fluorescence ZEISS ApoTome.2 microscope, and images were processed with ZEN software (Zeiss). At least five cysts were analyzed for each different dye. Cysts incubated in the absence of dye were used as negative controls.

[0204] Recombinant expression of the C-terminal domain of BCLA (Cter-BCLA).

[0205] Design and cloning. Disorder tendency analysis (using Dis-EMBL or IUPred) predicted that BCLA would be highly disordered throughout most of its sequence, including the core repeat motif. However, the C-terminus (approximately aa 1100–1275) was predicted to be structured and capable of forming a separate domain. To recombinate expression of this domain, the N-terminal boundary was selected at methionine 1089, and the original C-terminus was conserved. DNA synthesis was performed using Genscript to generate a fusion construct consisting of Cter-BCLA (1089–1275) with a TEV-cleavable N-terminal His tag (Figure 1b). Codon optimization for E. coli was performed, and the gene was cloned using Genscript in the pet30-(a) vector (Addgene) with the NdeI and XhoI sites.

[0206] Recombinant expression. Transformation was performed using BL21(DE3)-CodonPlus-RIL chemically competent Escherichia coli (Stratagene), incubated with 1 μg of pet30-(a)Cter-BCLA plasmid on ice for 10 minutes, subjected to heat shock at 42°C for 45 minutes, pre-incubated in LB at 37°C for 45 minutes, then spread onto LB agar plates containing kanamycin (Kan) and chloramphenicol (Chlo) and incubated for 12 hours. A single colony was then selected and inoculated into 50 ml of LB / Kan / Chlo pre-culture grown for 16 hours. Next, 5 ml of the grown pre-culture was inoculated into a 1 L flask of Terrific Broth medium (Formedium) containing Chlo / Kan. The culture was grown at 37°C until the OD600 reached 0.5-0.8, then induced by adding 0.7 mM IPTG (VWR), and further incubated overnight at 18°C. After incubation, the cells were centrifuged at 3000G for 25 minutes, the supernatant was discarded, and the pellet was rapidly frozen in liquid nitrogen and kept at -80°C.

[0207] Dissolution. Purification was performed on three pellets of 1 L culture, and each was resuspended in 50 ml of lysis buffer containing 600 mM NaCl, 50 mM Tris pH 8, 5 mM beta-mercaptoethanol (BME), 0.2% w / v N-lauryl sarcodin, and 1 Complete anti-protease cocktail (Roche) tab / 50 ml. Dissolution was performed on ice using 10 mini-pulse sonication at 50° amplitude (15 sec ON, 30 sec OFF), and the lysate never reached a temperature above 13°C. After sonication, the lysate was centrifuged at 15,000 G for 1 hour at 4°C, and the pellet was discarded. All subsequent steps were performed at 4°C. 30 mM imidazole was added to the clarified lysate before incubation with 5 mL of pre-equilibrated Ni-NTA resin. Batch incubation was performed at 4°C for 30 minutes with gentle stirring. After incubation, the resin was retained on a vertical column and then washed with 3 x 20 ml of washing buffer containing 600 mM NaCl, 50 mM Tris pH 8, 5 mM BME, 0.2% w / v N-lauryl sarcosine, and 30 mM imidazole. Direct elution using a 1.5 ml fraction was then performed using a buffer containing 300 mM NaCl, 50 mM Tris pH 8, 5 mM BME, and 300 mM imidazole. The target fraction (Figure 8) was then pooled and dialyzed in 50 mM NaCl, 50 mM Tris pH 8, and 5 mM BME using a 10 kDa cutoff dialysis cassette (Thermo Scientific).

[0208] Ion exchange and size exclusion chromatography. The entire sample was then pumped directly onto a 5 ml HL-Mono-Q (GE Healthcare) column, pre-equilibrated with the same buffer used for dialysis, through a chromatography system (Akta Pure, GE Healthcare). The column was washed with two-column volume (CV), and then eluted in 1.5 ml fractions of over 40 ml using a salt gradient (50 mM to 2 M NaCl), with absorbance monitoring at 280 nm performed throughout the elution. During elution, SDS-PAGE analysis (Figure 3) revealed that the sample was purified in the later stages of gradient elution, and that the initial eluted fraction presented the majority of visible bacterial contamination 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 onto S75 (GE Healthcare) using electrophoresis buffer containing 150 mM NaCl, 50 mM Tris pH 8, and 5 mM BME. Elution was performed within a heterogeneous peak corresponding to the polymer state, starting near the void volume and continuing for more than 3 ml. All eluted fractions were pooled to produce the final sample.

[0209] Ammonium sulfate precipitation. To avoid nucleic acid contamination, ammonium precipitation was performed by adding 15% w / v ammonium sulfate (Sigma), gently rotating at 4°C for 1 hour, followed by centrifugation at 10000*G for 30 minutes. The supernatant was discarded, and the pellet was resuspended in the same initial volume of buffer. To remove all ammonium sulfate, the sample was dialyzed in the same buffer used for size exclusion.

[0210] Limited proteolysis to identify antigenic subfragments within Cter-BCLA. To recover highly antigenic subfragments of Cter-BCLA, limited proteolysis was attempted in purified samples using trypsin, chymotrypsin, elastase, and papain (all Sigma Aldrich). The reaction was carried out in 30 μl reaction volumes in 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) over 50 minutes at 37°C. The reaction was stopped at each time point by adding 10 μl of SDS-PAGE loading buffer, followed by heating at 95°C for 5 minutes, and then kept on ice until loading onto the gel.

[0211] Western blot BCLA serum test. Single Western blot strips were prepared using a 15-well 4-12% NuPage gel (Life Technologies) loaded with 5 μl of 0.1 mg / ml sample. The gel was run in MES buffer at 185 V for 40 minutes, then electrotransferred on a PVDF membrane at 105 V for 1.5 hours. The transferred lanes were then cut into individualized strips. The strips were then blocked in TTBS with 5% w / v milk powder for 1 hour. The serum test was then performed in TTBS with a 1 / 400 dilution of serum at 4°C for 1 hour. The strips were then washed three times in TTBS and incubated for a further 1 hour with a 1 / 7500 dilution secondary antibody targeting either mouse IgG or human IgG and conjugated to phosphatase alkaline enzyme (Promega). Following three TTBS washes, the blots were revealed by adding a chromogenic substrate (Invitrogen) at RT. The band in positive serum appeared within 1 to 5 minutes. In parallel with serum testing, a single strip was always used as an internal antigen control for each blot set. After blocking, this strip 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 revealed using SigmaFast DAB (Sigma) with a metal enhancer. For each series of mice infected with ibuprofen or orally, serum from at least the mice in each series was checked for toxoplasma antibodies using Western blot analysis of the IgG immune response with the commercially available LD bio Toxoplasma mouse IgG (LD bio) kit, using the same anti-mouse IgG-alkaline phosphatase conjugate and chromogenic substrate previously described for BCLA.

[0212] Human serum. Human serum was retrospectively selected from the biobank collection of the Parasitology-Mycology Clinical Laboratory at the University Hospital of Grenoble Alpes, France. This biobank is registered under French Ministry of Health number DC-2008-582. The selected serum was stored for routine toxoplasmosis serum analysis between January 1, 2014 and May 1, 2018. Analysis using Vidas® Toxo IgM and IgG (bioMerieux, France) and Architect Toxo IgG and IgM (Abbott, Germany) was performed at the Parasitology-Mycology Clinical Laboratory at the University Hospital of Grenoble Alpes.

[0213] Proteomic analysis based on protein purification, immunoblotting, and mass spectrometry. Extracts of PruΔku80-BCLA-HAFlag-infected host HFF cells containing Flag-tagged proteins were incubated with anti-FLAG M2 affinity gel (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 1× protease inhibitor). The bound peptides were eluted stepwise with 250 g / ml FLAG peptide (Sigma-Aldrich) diluted in BC100 buffer. Protein bands were excised from a colloidal blue-stained gel (Thermo Fisher Scientific) and treated with DTT and iodoacetamide to alkylate cysteine ​​before in-gel digestion using modified trypsin (Sequencing grade; Promega). The resulting peptides from individual bands were analyzed by online nanoLC-MS / MS (UltiMate3000 conjugated to LTQ-Orbitrap Velos Pro; Thermo Fisher Scientific) using a 25-minute gradient. Peptides and proteins were identified and quantified using MaxQuant (version 1.5.3.17) with simultaneous searches against ToxoDB (version 20151112) and a database of frequently observed contaminants embedded in MaxQuant. A minimum peptide length was set to 7 amino acids. The minimum number of peptides, razor + unique peptide, and unique peptide were all set to 1. The maximum false positive rate was set to 0.01 at both the peptide and protein levels.

[0214] Epitope mapping of BCLA repeats and rBCLA. Dot blot peptide assays were custom synthesized using JPT peptide technology on cellulose membranes with an N-acetyl moiety at the N-terminus. Two sets of membranes were screened: 1) rBCLA region (res 1089-1275) covered with a total of 59 peptides, each 15 aa in length with 12 overlaps and 3 offsets; 2) Repeat 4 (res 446-493) covered with a total of 18 peptides, each 15 aa in length with 12 overlaps and 3 offsets. Dot blot assays were performed as described by the manufacturer. Briefly, the membranes were first activated in 100% ethanol for 5 minutes, then washed three times in DPBS-tween for 3 minutes each. Blocked overnight at 4°C in 0.5% milk powder DPBS-Tween, then washed again in DPBS-tween for 3 times each for 3 minutes each. The tested serum was diluted 1 / 400 in 0.1% BSA in DPBS-tween and incubated with the membrane at room temperature for 3 hours. Following a 3*3 minute DPBS-tween wash, the membrane was incubated with 1 / 100,000 diluted anti-IgG peroxidase-conjugated Ab (Sigma A0170) at RT for 2 hours. Following a 3*3 minute wash in DPBS-tween, the membrane was briefly immersed in freshly prepared SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher) and revealed using a C-Digit (Licor) scanner. Dot intensities were integrated using ImageJ. For independent dot blot data analysis, the integrated intensities from all peptide dots [I(p)] were standardized to a concentration factor Fe(p) using the baseline integrated intensity of peptide 59 [I(p = 59)], which never reacts with any serum. The following can be expressed using the following equation:

number

[0215] To increase the reactivity score across several independent positive serum blots, represented by the symbol (+), the Fe(p) enrichment scores were summed together. To subtract nonspecific reactivity, the same sum was performed on the same number of negative serum peptides, represented by the symbol (-), and these were subtracted. The peptide reactivity score can be expressed through the following equation:

number

[0216] BCLA ELISA. Peptide synthesis: The following BCLA peptides were synthesized using Genscript with an N-terminal acetyl group: AB_F:Nter-MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP-Cter(Sequence ID 55) A3_B:Nter-AAGSMEKDKLVLPGE-Cter(Sequence ID 56)

[0217] Plate preparation: Midisorp plates (Nunc) were coated with rBCLA, peptides AB_F and A3_B (all 2 μg / ml) in 100 mM calcium carbonate buffer pH: 9.6 (100 μl / well). After coating, the plates were washed twice with 350 μl of DPBS 0.05% Tween 20 (DPBS / Tween), then blocked with 300 μl of Superblock blocking buffer (ThermoFisher) for at least 2 hours, after which the buffer was removed and the plates were dried upside down. Once dried, the plates can be stored for extended periods at 4°C without loss of serum reactivity.

[0218] Sample preparation: All serum diluents were prepared within 2 hours prior to the assay in DPBS 0.05% Tween 20, 0.1% BSA. 1 / 400 diluents were prepared for both mouse and human tested serum. In addition, 11 standards were newly prepared for both tests and consisted of 10 series dilutions of positive frozen stock serum set to 100 UI. Starting with a 1 / 200 dilution, the following titration points were prepared with 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). A 0 UI standard was prepared using seronegative serum diluted to 1 / 400.

[0219] Assay: All subsequent steps were performed on the Gemini ELISA automated platform (Stratec), however, they can also be performed manually in RT. The dried plate was first washed twice with 350 μl of DPBS / Tween. The tested serum and standard dilutions were dispensed into the plate as a series of duplicates with 100 μl / well. The plate was then incubated in RT for 1 hour. After the incubation period, 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) in DPBS 0.05% Tween 20, 0.1% BSA was rapidly dispensed into all wells. After 1 hour in RT, the plate was washed four times in DPBS tween. The color development reaction was carried out by adding 100 μl of TMB substrate (Thermofisher ref 34029) and performing the reaction under RT for exactly 20 minutes. The reaction was then stopped with 50 μl of 0.2 M H2SO4, followed by mixing for 30 seconds. Next, the absorbance of the wells was measured at 450 nm using a Gemini integrated spectrophotometer.

[0220] Data processing: Blank subtraction was performed in duplicate blank wells where the primary antibody / serum was not placed in the wells, but all subsequent steps (washing, secondary Ab, substrate) were performed. The standard serum dilutions were averaged and fitted using a four-parameter logistic regression with an asymptote upper limit value (Di) fixed at 2.5 AU and all other variables (Ai, Bi, Ci) that could be fitted. From this regression, the apparent UIs of the tested dilutions could be calculated and averaged by duplicates. In duplicate measurements, if the coefficient of variation was observed to exceed 10%, then the sample could be retested. All ELISA data presented in this study were obtained several times in independent titrations.

[0221] Results

[0222] Quantitative analysis of the proteomic response to FR235222 in tachyzoites identified BCLA as a novel bradyzoite-specific protein.

[0223] Specific inhibition of TgHDAC3 by the cyclopeptide FR235222 disrupts the steady-state level of histone H4 acetylation across the T gondii genome, inducing the desuppression of stage-specific genes (Bougdour et al., 2009; Sindikubwabo et al., 2017). Utilizing the properties of FR235222, we developed an in vitro cyst formation system capable of producing the amount of protein required for large-scale proteomic testing (Farhat D et al., manuscript in preparation). Following low-dose and short-term treatment of cystic type II (PruΔku80) strains, we performed quantitative proteomics tests and revealed that the FR235222-treated proteome was significantly enriched in stage-specific proteins (including those recognized as being limited to the bradyzoite stage) (Figure 1a). From this analysis, we found that the protein TGME49_209755 (hereinafter referred to as BCLA (Brain Cystic Load-Related Antigen)) is significantly induced upon FR235222 treatment in the same way that several proteins are involved in the chronic phase of infection (Figure 1a). This is consistent with its expression profile reported as being limited to the bradyzoite dataset (Figure 1b, source ToxoDB).

[0224] Other evidence supports the epigenetic regulation of BCLA expression. We recently reported that H3K14ac and H3K9me3 PTMs bookmark transiently repressed genes, which then await parasite differentiation for stage-specific expression (Sindikubwabo et al., 2017). In tachyzoites, the BCLA locus exhibits this dual PTM enrichment, discriminantly marking "balanced" stage-specific genes (Figure 1c). Furthermore, recent TgHDAC3 ChIP-seq analysis (Farhat D et al., manuscript in preparation) revealed the presence of histone deacetylase at the BCLA locus (Figure 1c). The definitive genetic evidence underlying TgHDAC3's regulatory involvement was obtained by CRISPR-mediated disruption of TgHDAC3 in transfected tachyzoites, which induces BCLA induction (Figure 1d), thereby mimicking the effect of FR235222 on the enzyme. From these data, we concluded that BCLA belongs to a family of bradyzoite genes regulated by TgHDAC3, and is typically represented by a so-called bivalent chromatin domain that can silence developmental genes while maintaining equilibrium in the surrounding heterochromatin for rapid activation during cell differentiation (Sindikubwabo et al., 2017).

[0225] BCLA is secreted during PV and associates with the PVM of the bradyzoite-containing vacuole that has been converted in vitro.

[0226] BCLA is a single open reading frame encoding a 140 kDa protein with a central core domain typically represented by a predicted N-terminal signal peptide and a 48-amino acid motif repeated 13 times, bordered by a conserved C-terminal region of ~150 residues (Figure 2a). Its composition and frequency have evolved among the T. Gondi lineages through subclasses of Coccidium (Figure 2b). BCLA homologous proteins are poorly conserved in Neospora caninum, while they have the same overall architecture with short repeats that share common characteristics with BCLA repeats (data not shown). Disorder tendency searches (using dis-embl or IUPred) predict that BCLA is highly disordered throughout most of its sequence, including the core repeat motif (Figure 2a). However, the C-terminus (approximately aa 1100–1275) is predicted to be structured and may constitute a separate domain (Figure 2a).

[0227] BCLA was clearly and exclusively identified by mass spectrometry in FR235222-treated samples (Figure 1a), but the protein's dynamics and intracellular distribution during infection remain unstudied. To further explore the dynamics of BCLA in T. Gondi in insights, we produced polyclonal antibodies against two synthetic peptides positioned at the ends of a conserved repeat, respectively (Figure 2a). We first validate the proteomic data by exposing cells to FR235222, demonstrating a significant increase in BCLA signal intensity as a protein band of expected size ~140 kDa, which would otherwise be undetectable in untreated tachyzoites (Figure 2c).

[0228] In fibroblasts hosted by tachyzoites expressing a C-terminal HA-Flag tagged version of the bradyzoite-specific marker, BCLA was clearly detected upon FR235222 stimulation in the vacuolar space and clearly accumulated in the PVM, while its expression coincided with the induction of the bradyzoite 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 autoantibody specificity (data not shown). Finally, when the inventors monitored BCLA dynamics in type I (RHΔku80) and type II (PruΔku80) strains expressing endogenous proteins in fusions with the HA-Flag tag, they demonstrated that, once stimulated with FR235222, the HA-tagged BCLA protein is targeted in the vacuolar space and at the membrane, regardless of the strain type (data not shown). Thus, the presence of the C-terminal fusion tag does not affect the intracellular localization of BCLA, because it is similar to what is observed when using anti-BCLA serum in untagged strains.

[0229] When different parasitic strains of T. gondii were exposed to FR235222, the inventors ultimately discovered that BCLA signal intensity varied significantly depending on the infected strain, ranging from very strong induction in type II (PruΔku80, ME49, 76K-GFP-Luc) strains to moderate levels in type I (GT1 and RHΔku80) strains and haplogroup 11 (COUG) strains, and, surprisingly, faint (or absent) signals were detected in cells infected with type III (CTG) strains (Figure 3a and data not shown). This inconsistency, which can be explained by the ability of certain strains to readily induce tissue cysts, is discussed below.

[0230] BCLA localizes to the cyst matrix and cyst wall in vivo.

[0231] The glycosylated cyst wall to which lectin Himalayan hyacinth agglutinin (DBA) binds is a key structural feature that promotes the persistence and oral transmission of T. gondii (Tomita et al., 2013). Here, we obtained strong evidence of co-staining of BCLA and DBA exclusively in the membrane surrounding converted bradyzoite in vitro (data not shown), strongly indicating that BCLA accumulates over time in the wall of immature cysts following its delivery into the vacuolar space (based on thin DBA-positive cyst walls).

[0232] However, considering that in vitro bradyzoite development in tissue culture does not result in fully mature cysts, we re-examined the localization of BCLA in bradyzoite-containing cysts isolated from mice chronically infected with T. Gondi type II strain. In chronically infected mice, the cyst wall and the matrix space surrounding the bradyzoites were stained by autoantibodies produced against BCLA (data not shown). Immunofluorescence results did not clearly determine whether BCLA was located in the inner or outer layer of the cyst wall, but interestingly, impermeable ex vivo cysts were readily stained by the antibody, suggesting the external location of the protein (data not shown) and therefore its exposure to the cytoplasm of host cells. The signal was not detected in cysts hosting Δbcla bradyzoites (data not shown), thus confirming the specificity of the anti-BCLA antibody in vivo (Figure 4a).

[0233] There is little evidence for the extra-vacuolecular function of BCLA, however, occasionally the protein appears to be transported across the vacuolar membrane into the cytoplasm of host cells (data not shown). Unfortunately, despite numerous attempts, the inventors have not found the ad-hoc conditions underlying BCLA export beyond PVM to further investigate, in some cases, its function in host cells, as they have done with other effectors (Hakimi et al., 2017). Nevertheless, the inventors were able to show that BCLA export was Myr1-independent (data not shown), but as such, it does not require the T-gon detranslocon of the exported protein (Franco et al., 2016). An elegant way to explain the accumulation of the protein in the cytosol of infected cells is its release after treatment, which may occur in PVM, likely under the control of host proteases, but this has not yet been demonstrated. Analysis of the BCLA-related proteome of infected, FR235222-stimulated host cells would be performed to determine, if any, whether BCLA interactions with host cell proteins (including proteases) occur on the outward side of the PVM, or even in the cytoplasm of the infected cell if BCLA is delivered there.

[0234] BCLA is unnecessary for suitable cystic function in vivo.

[0235] To determine the function of BCLA in bradyzoite tissue cysts, two parasitic lineages were created using either deletion of the BCLA coding region (PruΔku80Δbcla) or Cas9-mediated gene editing (76K-GFP-LucΔbcla), and then interrupted by a DHFR cassette (Table 2). The inventors then investigated pathogenesis and cyst formation. First, the BCLA-deficient lineages did not exhibit a clear proliferation phenotype compared to their parental lineages in vitro under tachyzoite conditions (Figure 4a and data not shown). The BCLA mutation did not impair the expression or localization of PV commensal or PVM-related proteins that were previously recognized in studies as being involved in PV formation and maturation (i.e., GRA1, GRA5, GRA7; data not shown). No difference was detected in the ability of BCLA-deficient parasites to convert to the bradyzoite stage and form cysts in vitro, as indicated by Δbcla-containing vacuoles positively labeled with lectin DBA following FR235222 stimulation (data not shown).

[0236] BCLA is not required to initiate in vivo infection with taxoids.

[0237] To investigate the importance of BCLA in vivo during acute infection, the inventors compared the parasitic processes in BALB / c mice or NMRI mice infected intraperitoneally (ip) with either WT parasites or BCLA-deficient parasites from a type II background, using an inoculation dose of 1 × 10⁻⁶. 4 From 1 x 10 6The tachyzoite range was observed. 5–8 days post-infection, all mice infected with type II BCLA-deficient tachyzoites began to show signs of infection (i.e., weight loss and wavy coat) and survived to infection within the same timeframe as the parent strain 76K, regardless of the inoculation and the mice's genetic background (Figure 5a). Thus, BCLA appears to be unnecessary for in vivo proliferation and pathogenesis during the acute phase of infection in mice. Animals that survived the challenge were then tested for serological response to parasitic antigens by Western blotting at 10 weeks post-infection (data not shown). Clearly, BCLA deletion does not impair infectivity, because all mice, regardless of the parasitic strain, showed IgG against T. gondii with the same pattern (data not shown).

[0238] BCLA deficiency affects the integrity of brain cysts isolated from chronically infected mice.

[0239] Examination of the brains of mice infected with the Δbcla mutant demonstrated that cyst formation can still occur even in the mutant (Figure 6b). However, the BCLA-deficient mutant produced a significantly reduced parasitic load in the CNS of chronically infected mice compared to the parent strain, although the difference did not reach statistical significance (Figure 6b and data not shown), demonstrating that BCLA is not unnecessary, at least for establishing and maintaining cysts during chronic infection. Thorough examination of the cysts, however, revealed that those isolated from mice infected with the mutant parasite were relatively small (Figure 6a), contained fewer bradyzoites, and resulted in a “lower packing density” (Watts et al., 2015) and an overall decrease in GFP fluorescence (Figure 6b), which is fairly consistent with a slight decrease in parasitic load measured throughout the brain (Figure 6b). Beyond these quantitative indicators, Δbcla-containing cysts are uniquely and typically represented, to some extent, by significant deformation of their cyst wall surface leading to loss of roundness, as well as by distinctive “budding” and “segmentation or cracking” phenotypes (Figure 6a and data not shown), revealing a possible role of BCLA in cyst growth, maintenance, and / or stability.

[0240] The inventors then evaluated whether surface deformation could make the cysts fragile (the phenotype previously reported for brain Δcst1-containing cysts (Tomita et al., 2013)). During their isolation, the cysts were subjected to mechanical stress to release them from brain tissue and purified by isodense centrifugation (see Methods). The inventors did not observe that Δbcla-containing cysts were more fragile than WT cysts during this harsh procedure (data not shown), however, a small number of them were fragmented regardless of genetic background.

[0241] The absence of BCLA does not impair wall staining with hyacinth lectin (DBA) of cysts isolated from the brains of chronically infected mice (data not shown). Therefore, as already concluded with tachyzoites treated with FR235222, BCLA is not directly involved in GalNAc glycosylation of the cyst wall. The survival rate of bradyzoites within cysts is conditioned on the permeability of the wall to nutrients coming from host cells, however, the latter is very limited, and the wall acts as a sieve to avoid components of the immune response. To test whether wall permeability was altered in any way in the absence of BCLA, we monitored the entry of fluorophores into cysts, typically represented by different sizes ranging from 3 to 40 kDa. Only intact cysts (without parasitic leakage) were visualized and examined under a microscope. Permeability was remarkably similar between wild-type (WT) and BCLA-deficient cysts, using either a 3kDa (diffusion pattern across the entire cyst matrix) or 10kDa (diffusion pattern with cross-sectional areas) dye. Interestingly, fluorescent tracers with higher molecular weights (40kDa) were unable to efficiently pass through the cyst wall, as previously reported (Lemgruber et al., 2011). Furthermore, weaker labeling differed even between strains. This is likely because Δbcla-containing cysts were "looser" and more permeable than cysts containing the parent strain, and were more filled with bradyzoites surrounded by a well-defined, continuous cyst wall (data not shown). Overall, our results suggest that BCLA is not necessary for proper cystic function in vivo, however, the protein plays a structural role in the cyst wall, thereby leading to a phenotype of cyst wall permeability deficiency.

[0242] BCLA is not essential for efficient oral infection by toxoplasma bradyzoite-containing cysts.

[0243] To validate the in vivo functional outcomes of BCLA-dependent cystic deformation, we administered Δbcla or parental strain-containing cysts to mice and evaluated their pathogenicity 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 strain, and the dynamics of parasitic invasion and dissemination in the intestines, as well as the parasitic-induced local immune response, were examined. On day 8 of infection, levels of T Gondi-specific IgG in mouse serum were fairly similar (data not shown), and they did not show significant differences in parasitic load in the ileum (Figure 7a). Histological analysis of the ileum revealed a general loss of intestinal epithelial structure with altered crypt-villous morphology (data not shown) and inflammatory sites (data not shown), regardless of the genetic background of the strain. The cytokine profiles exhibited the same pattern, with a clear increase in pro-inflammatory cytokines (IFNγ) and chemokines (CCL2) in the ileum, but in a BCLA-independent manner (Figure 7b). We then orally infected NMRI mice with 20 cysts, disseminated them into the bloodstream, and evaluated the ability of Δbcla cysts to form new cysts in deep tissue. All orally infected mice showed signs of disease (loss of body weight) throughout the acute phase of infection and underwent serological conversion (data not shown). After 10 weeks, no significant differences in cyst number or parasite load were detected between strains in any of the mice (Figure 7c). These data suggest that BCLA-deficient cysts can transmit infection via the oral route and cause chronic infection in mice, typically manifested as a mild inflammatory state. Profiling of pro-inflammatory cytokines in the brains of chronically infected NMRI mice suggested that inflammation was less severe in Δbcla than in wild-type mice, but statistical significance was not achieved, likely due to the small sample size (3 mice for each condition; Figure 7d). This relatively mild inflammation in the brain may be a result of a relatively small number of cysts in Δbcla-infected mice, however, this must be determined.

[0244] High-level expression and purification of BCLA chimeric peptides for serological diagnostics.

[0245] The humoral and cellular defenses of the innate immune system are the body's first line of defense against T. gondii. Antibodies have been reported to aid in the elimination of parasites during acute infection and to mediate resistance to secondary toxoplasmosis infection (Sayles et al., 2000). As such, once immunity is established, IgG protects the fetus from vertical transmission during pregnancy. While the serological distinction between acute and chronic infections has clinical and epidemiological relevance, there is currently no bradyzoite-specific serological assay for toxoplasmosis to accurately estimate the time of infection and the presence of cysts. Furthermore, because relapses can occur in both fully immune patients (e.g., retinochoroiditis) and immunocompromised patients, and because the presence of cysts in the brain has recently been suspected to be linked to certain neuropsychiatric disorders, detecting Toxoplasma antibodies directed against semi-dormant cysts could represent a significant improvement to the serological diagnosis of toxoplasmosis by opening up new diagnostic prospects. However, few components of the cyst wall or surface bradyzoite have been identified, and at least in commercially available kits, none have been shown to serve as antigens for serological purposes. An ideal antigen should be expressed exclusively in the latent bradyzoite stage and ideally exposed to the cyst surface, two features found in BCLA that motivated the inventors to test its antigenicity.

[0246] To obtain the high purity and abundant quantity of BCLA required for serum WB testing, we chose to recombinantly express the C-terminal domain of BCLA (res 1100-1275, hereafter referred to as rBCLA), which is expected to be structured, in contrast to the rest of the protein containing the core repeat motif (Figure 2a). rBCLA was therefore expressed in E. coli as a chimeric protein with an N-terminal polyhistidine tag. Although efficiently expressed, it is insoluble in nature or sequestered in insoluble inclusions, however, it can be solubilized using 0.2% N-lauryl sarcoside during the lysis process. After lysis and centrifugation, rBCLA was first pulled down using nickel affinity resin (data not shown). With a theoretical Mw of 20.9 kDa and a pI of 4.7, BCLA was observed to migrate between molecular weight markers of 17–25 kDa on the SDS-PAGE gel, and in a pH 8 buffer, BCLA would be strongly negatively charged. E. coli contaminants can therefore be efficiently removed using anion exchange chromatography (data not shown). Finally, rBCLA is eluted in soluble form from size exclusion chromatography (data not shown), but is polydisperse due to its broad volume elution range and forms multi-oligomers because the elution volume is close to the void volume of the S75 column. When pooling the eluted fraction, ammonium sulfate precipitation and dialysis are performed as the final steps to remove nucleic acid contaminants (data not shown). Following this final purification step, the tachyzoite antigens of both the RH strain (LD bio) and rBCLA were isolated by SDS-PAGE and then probed using immunoblotting with mouse antiserum induced by different states of toxoplasmosis, enabling parallel analysis of antigen recognition by immunoglobulins G, M, and A.

[0247] rBCLA does not react with serum from acutely infected mice, but it constitutes an excellent antigen for detecting anti-T Gondi IgG from chronically infected mice.

[0248] The inventors initially performed immunoblotting on serum collected from mice in the acute phase of infection. The rBCLA protein did not appear to react with serum from mice acutely infected with atypical (COUG, haplotype 11), pathogenic (RH, type I), or cystic (76K, type II) strains (Figure 8a-c), while all T Gondhi-exposed mice underwent seroconversion regardless of genetic background (NMRI, CBA, C57BL / 6) or route of infection (intraperitoneal or oral) (Figure 8a-c and data not shown). However, rBCLA reacted strongly with T Gondhi IgG antibodies from mice that developed latent toxoplasmosis following infection with type II cystic strains (Pru, ME49, or 76K) (Figure 9a-c). rBCLA was detected in the serum of mice in the subchronic (>21 days, Figure 9d) or chronic (>42 days, Figure 9a-c) stages of infection, with a very strong signal in the serum of mice persistently infected for 22 months (Figure 9c). Reactivity was not detected when assaying serum from mice not infected with BCLA-deficient strains or chronically infected mice, but this demonstrates that IgG antibodies are specifically directed against BCLA in vivo (Figure 9d). Since the selection process occurs during antibody affinity maturation (Eisen, 2014), we concluded that the rBCLA antigen may be detectable by IgM during acute infection compared to IgG during chronic infection. Clearly, rBCLA does not react with anti-T Gondi IgM or IgA (data not shown). The above findings therefore strongly support the idea that rBCLA could distinguish the parasitic stages of infection in mice, with preferential IgG reactivity for latent infection.

[0249] rBCLA is exclusively detected in the serum of mice persistently infected with cystic strains.

[0250] rBCLA was shown to exhibit a specific reactivity to cystic strains that tend to produce latent infections (Figure 9a-d). To continue the discussion, however, it would be necessary to show that non-cystic strains cannot produce a specific antibody response directed against rBCLA. First, serological analysis of animals infected with the non-cystic pathogenic strain RH and sequentially treated with pyrimethamine or sulfadiazine to overcome acute toxoplasmosis revealed levels of enrichment of anti-tachyzoite-specific antibodies (22 days post-infection; Figure 9e, lower panel), while rBCLA was barely detectable (Figure 9e, upper panel). Since we could not rule out that the treatment altered the dissemination of the parasite in deep tissues and, consequently, their differentiation into bradyzoites, we monitored the IgG response to rBCLA in mice persistently infected with CTG, a non-lethal, chronic latent infection-causing type III strain characterized by appropriate positive sera (Figure 9f, right panel). No response to rBCLA was observed 42 days post-inoculation (Figure 9f). The main difference from type II infection (Figure 9a-d) was that mice chronically infected with CTG had fewer (or none) cysts in their brains (Cannella et al., 2014), suggesting a likely relationship between cyst load and rBCLA antibody levels. Similarly, serum from mice persistently infected with a type II (PruΔku80) strain that normally produces fewer cysts did not react with rBCLA (Figure 9g), indicating that the mouse antibody response to rBCLA antigen occurs immediately after subchronic infection (>21 pi), and appears to be conditioned by the presence of cysts, at least in the mouse model. Immunosuppressive therapy (corticoids) that produces relapses of latent type II infection does not enhance the antibody response to rBCLA, but it also rules out the hypothesis of an immune response in response to the release of bradyzoite into the circulation (Figure 9h).

[0251] Limited proteolysis to identify antigenic subfragments within rBCLA.

[0252] To recover highly antigenic subfragments of rBCLA, limited proteolysis was performed in purified samples using trypsin, chymotrypsin, elastase, and papain. Analysis of the proteolysis reactions by SDS-PAGE (Figure 10a) showed that rBCLA is rapidly degraded by chymotrypsin and partially degraded by elastase, trypsin, and papain, generating a stable fragment around the 17kDa marker. When blotting against positive mouse IgG serum (Figure 10b) and His tag (Figure 10c) according to the same protocol mentioned above, it can be observed that most of the degradation occurs within the C-terminus, as they remain positive in the his tag blot. These same degradations present a weaker intensity chromogenic band in anti-mouse IgG WB, suggesting that further truncation of the construct does not increase the specificity or sensitivity of mouse IgG in Western blotting analysis.

[0253] rBCLA also reacts with human serum, however, the pattern of positive reactions is still under investigation.

[0254] The inventors then demonstrated that mice infected with positive amniotic fluid from pregnant women who were primaryally infected with evidence of congenital toxoplasmosis during pregnancy reacted clearly with rBCLA, in contrast to mice infected with qPCR-negative amniotic fluid or placenta (Figure 11a). rBCLA, therefore, is a suitable serum marker for predicting their cystic characteristics in clinical isolates. Following the evaluation of anti-rBCLA immunoglobulin detection in the mouse model, the inventors aimed to evaluate patterns of anti-rBCLA detection in humans according to the patient's serological and clinical status (Table 3). Antibodies directed against the rBCLA antigen were detected in serum alone or in both serum and aqueous humor in three patients with strongly suspected or proven ocular toxoplasmosis (Figure 11b). These clinical cases were due to recurrence of T-Gondi cysts in the retina, but were not primary infections, as no IgM was detected. Similarly, three patients with toxoplasmosis relapses due to immunosuppression associated with hematological disorders also possessed anti-rBCLA IgG; however, labeling on Western blot was weaker compared to those with ocular toxoplasmosis, although antibody levels were considerably higher using Vidas® and Architect® (Table 3). Even though broad generalization is limited by relatively small sample sizes, the reactivity of rBCLA to human serum in toxoplasmosis relapses provides further evidence for our mouse model in which we correlated the presence of rBCLA as a serum marker with cystic load. Unexpectedly, three serums with recent seroconversion in pregnant women and one serum from a child with congenital toxoplasmosis also reacted to rBCLA (Figure 11b). Although difficult to demonstrate, it is possible that recent primary infections lead to the formation of T. Gondii cysts in peripheral tissues, thereby triggering a humoral anti-BCLA immune response. In any case, rBCLA was not detected in the serum of any patient identified as seronegative for T. Gondi, demonstrating good specificity of this antigen for patients with toxoplasmosis (Figure 11b). [Table 3] [Table 4] TIFF0007849292000009.tif181165 Manufacturer-recommended cutoffs for interpreting serological values ​​using Vidas® and Architect®. Vidas(registered trademark) IgG (IU / mL): Negative < 4; Gray zone: 4.0 ≤ x < 8.0; Positive: ≥ 8.0 Vidas® IgM (indicator): Negative < 0.55; Gray zone: 0.55 ≤ x < 0.65; Positive: ≥ 0.65 Architect® IgG (IU / mL): Negative < 1.6; Gray zone: 1.6 ≤ x < 3.0; Positive: ≤ 3.0 Architect® IgM (indicator): Negative < 0.50; Gray zone: 0.50 ≤ x < 0.60; Positive: ≥ 0.60

[0255] Epitope mapping in rBCLA-positive patients reveals consistent reactivity within numerous antigenic and repeating regions of the rBCLA.

[0256] The specific immunogenicity of rBCLA has been demonstrated by Western blotting in a range of serums from different clinical categories. One of the main objectives was to develop an ELISA-based assay to screen a larger serum cohort in a cost-effective, reliable, and rapid manner. However, to accurately set up such an assay, which is almost entirely based on chemically synthesized peptides, a more precise understanding of the local epitope immunogenicity of BCLA was required. To do so, we designed and synthesized cellulose-printed peptide arrays covering both the repeat region and the rBCLA domain (Figure 12a). These arrays were designed using 15aa of peptides with 3aa gap steps between the peptides. We then tested several serums that were clearly positive by Western blotting for rBCLA, took the same number of negative serums, and proportionally subtracted nonspecific reactivity. When analyzed, the total reactivity scores for each peptide obtained in both the repeat region and rBCLA (Figure 12b) provide two important observations: First, rBCLA has numerous epitopes throughout its domain, despite having several more strongly reactive zones, particularly near peptides 13, 22, 30, and 43, and different sera can react quite differently to different zones (Figure 12c). This highlights the requirement for retaining rBCLA as a recombinant protein within ELISA testing. Furthermore, since the rBCLA domain is expected to be structured, structured epitopes are only provided by recombinant protein strategies, which may further emphasize their use. Second, the repeat motif has been found to consistently react in two separate zones (peptides 3-7 as motif A and 13-16 as motif B) in almost all tested human sera. This feature highlights the importance of including one or more peptides covering these motifs to obtain more sensitive ELISA techniques. Based on these results, the inventors therefore designed an ELISA combining full-length recombinant BCLA protein and chemically synthesized repeat motifs.

[0257] ELISA titration using rBCLA and repeated peptides demonstrates that BCLA seropositivity is higher in individuals with acute and chronic infections.

[0258] In establishing rigorous rules for classifying and distinguishing different clinical profiles, 123 serum samples (all taken from different individuals) were tested using the developed BCLA-ELISA test. ELISA scores are expressed in International Units (UI) and presented according to the patient's clinical profile, as listed below (Figure 13). 1) Regroup all patients with "seronegative" or SAG1IgG / IgM-negative serum (healthy or with other prior conditions). 2) Regroup all patients (healthy or in other conditions) who are classified as having "past immunity" as SAG1-positive IgG, but who do not have SAG1-reactive IgM and do not fall into any of the following three categories: 3) “Active toxoplasmosis in immunocompromised patients”: Regroup all SAG1 IgG-positive and immunocompromised patients with proven symptomatic toxoplasmosis (regrouping disseminated, cerebral, and primary toxoplasmosis). 4) "Asymptomatic serological relapse in immunocompromised patients": Regroup all immunocompromised patients who are experiencing a serological relapse but do not present with visible symptoms. 5) Regroup patients with "ocular toxoplasmosis," specifically those with SAG1-positive serum and proven ocular toxoplasmosis.

[0259] This analysis allows for several observations. First, all groups exhibit a significant increase in the median BCLA titration and a much higher positive rate compared to the seronegative group. This demonstrates a direct correlation in humans between the ability to develop SAG1 seropositivity and BCLA positivity. This also highlights the current inconsistency between SAG1-negative sera and BCLA sera, which still exhibit a false-positive detection rate of approximately 10%. This can be explained by nonspecific interactions with different BCLA epitopes in some sera, highly immunogenic exogenous bacterial contaminants co-purified with rBCLA, and potentially true BCLA-positive patients with negative SAG1 sera. The second key observation is that some clinical profiles tend to produce a much stronger immunogenic response, most notably in the "asymptomatic serological relapse in immunocompromised patients" group, where BCLA sera titrations well above the median positive BCLA sera in the "previous immunity" group. The final observation is that, in certain groups where BCLA positivity should always be predicted, such as in "active toxoplasmosis in immunocompromised patients" and "ocular toxoplasmosis," a small number of serum samples remain negative or are below the positive cutoff. This observation may highlight the lack of sensitivity from the ELISA test or potentially illustrate the fact that BCLA serum can be negative during immunosuppression.

[0260] ELISA testing has also been consistent in linking positive BCLA serum in mice to proportional cystic load.

[0261] Overall, semi-quantitative analysis of anti-rBCLA antibody titers identified BALB / c and NMRI mice, which are likely to have WT cysts, as highly reactive to BCLA with increasing yields over time (Figure 14A). In combination with quantitative PCR of brain-associated T Gondi DNA and quantification of brain-associated miR-155 and miR-146 microRNAs, which have been reported to be specifically induced during bradygenesis (Cannella et al., 2014), we provided evidence for rBCLA as a reliable antigen for serological detection of T Gondi bradyzoite-loaded cysts over long-term protozoan persistence in rodents (Figure 14B). These results are particularly interesting because the semi-quantitative nature of the ELISA test allows for significant differentiation of the response of cystic T Gondi strains over time.

[0262] Consideration

[0263] Infection with Toxoplasma gondii leads to an acute systemic phase in which tachyzoites rapidly colonize and further complete their developmental program as bradyzoites encapsulated in cysts surrounded by persistent thick cystic walls in the brain, heart, and skeletal muscle (Jeffers et al., 2018). The host immune response can rapidly control the proliferation of the tachyzoite population, leading to lifelong immunity typically expressed by seroconversion. However, because the developmental transition from tachyzoite to bradyzoite is entirely bidirectional, any impairment of immune function (e.g., AIDS patients, hematological disorders, and immunosuppressive treatments) can lead to a reactivation of latent infection, potentially causing encephalitis and focal brain lesions, lung disease, or disseminated disease.

[0264] The diagnosis of acute and chronic toxoplasmosis in immunocompromised subjects relies primarily on serology because the infection is often asymptomatic. Serological diagnosis is often retrospective because it is based, for example, on evidence of seroconversion during pregnancy or in the transplant environment (Robert-Gangneux and Darde, 2012). Increased levels of IgM and IgA antibodies are serological indicators of primary / acute infection, while high IgG binding affinity rules out primary infection, and persistent and steady-state IgG levels in the absence of IgM typically represent latent infection (Dard et al., 2016). However, interpreting serological diagnostic results remains difficult even for well-trained specialists. The current challenges to overcome are: (i) distinguishing recent infections from more distant ones; (ii) diagnosing congenital toxoplasmosis in infants and relapses in immunocompromised patients; and (iii) confirming the origin of the infection, i.e., cysts in contrast to oocysts. Many methods have been developed over the past few decades to improve the accuracy and sensitivity of serum assays, but they have only adequately addressed the aforementioned concerns. The obvious reason is that many, if not all, commercially available serum test kits detect lysates or recombinant antigens that are predominantly expressed at the tachyzoite stage (e.g., SAG1) or common to both infection stages of the parasite (e.g., GRA8).

[0265] Currently, there is no reliable bradyzoite-specific seroassay for toxoplasmosis that can estimate the source of infection worldwide or accurately distinguish between acute and latent infections, although progress is being made. In fact, recent proteomics studies have shed light on the repertoire of sporozoite-specific proteins (Fritz et al., 2012; Possenti et al., 2013), which has revealed CCp5A as a seromarker that can distinguish between parasitic stages infecting chickens, pigs, and mice with specific reactivity for oocyst-infected animals (Santana et al., 2015).

[0266] However, despite early studies reporting that certain bradyzoite antigens, including BAG1, contribute to the stimulation of both humoral immunity (Mun et al., 1999) and cellular immunity (Di Cristina et al., 2004) against T. Gondi infection, bradyzoite / cyst antigens are not currently considered as potential markers of latent infection in diagnostic tests. The search for bradyzoite / cyst-specific markers has been somewhat limited by the ability to collect enough mouse brain cysts to analyze specific proteomes in the latent phase. In this study, we found a way to circumvent this problem by derepressing bradyzoite genes in cell culture while manipulating the chromatin state of tachyzoites with epidrugs. Thus, hundreds of bradyzoite restriction proteins were identified, including BCLA.

[0267] Although the protein BCLA has been shown not to be essential for initiating or maintaining latent infection, BCLA deficiency has resulted in a highly specific phenotype, typically represented by deformation and loss of roundness of brain cysts in mouse models. To date, two cyst wall-associated proteins, namely BPK1 and CST1, have been associated with the structural integrity of T. Gondii cysts (Jeffers et al., 2018). In the Δbpk1 strain, cysts are smaller, more susceptible to pepsic acid treatment, and, unlike BCLA, the Δbpk1 strain has a reduced ability to cause oral infections (Buchholz et al., 2013). CST1 is involved in the binding of hyacinth agglutinin (DBA) lectin, which is characteristic of T. Gondii cysts. CST1 deletion results in a phenotype of fragile brain cysts characterized by reduced cyst number and thinning and destruction of the basal region of the cyst wall (Tomita et al., 2013). Glycosylation deficiency may also explain the deformation of Δbcla cysts. In fact, we have preliminary interactome data showing BCLA co-purified with jacaline-binding protein, a lectin that binds to the GalNAcα1-Ser / Thr oligosaccharide covering the PVM surrounding bradyzoite (data not shown) (Tomita et al., 2017). Further research is needed to confirm whether this interaction is involved in the BCLA deficiency-mediated peculiar phenotype.

[0268] Because mice do not exhibit a clear BCLA-related phenotype regardless of the route and timing of infection, the inventors directed their research on the tendency of BCLA to play an immunogenic role. The inventors thus reached another milestone by producing rBCLA as a recombinant protein with a high degree of purity, which would provide an opportunity to standardize serum tests and reduce manufacturing costs to some extent if this antigen proves to be of interest to serology. In fact, the inventors obtained strong data confirming that rBCLA is antigenic and constitutes an excellent antigen candidate for the detection of anti-T Gondi IgG in chronically infected mice. Notably, the inventors clearly correlated the strong detection of the antigen rBCLA in serum with cystic load in the brains of all mice latently infected with type II cystic strains. Similar studies have shown that MAG1 antibody levels correlate with brain cyst load; however, it has been argued that these experimental settings are somewhat biased due to the use of an unrelated model of chronic type I (GT1) infection requiring anti-T Gondi chemotherapy to control tachyzoite proliferation during the acute phase and avoid animal death (Xiao et al., 2016).

[0269] Notably, rBCLA did not respond to either IgM or IgA (markers frequently associated with acute infection) (data not shown), but did respond only to IgG, and exclusively in subchronic infection. This result is in stark contrast to the observation that mice given histological cysts showed a significant IgM response on day 10 (Doskaya et al., 2018), and reinforces the idea of ​​a humoral response to BCLA during the incubation phase of infection. Similarly, mice inoculated by forced oral administration using histological cysts did not produce antibodies directed against BCLA at the time of acute infection (Figure 8c), indicating that the host immune response to BCLA was not attributable to initial exposure to the release of bradyzoites and cyst proteins from ingested parasites in the gastrointestinal tract during primary infection. This is in sharp contrast to the humoral response to BAG1 and MAG1 that occurs very early after infection (Di Cristina et al., 2004; Mun et al., 1999). Our findings suggest a humoral immune response to BCLA-containing tissue cysts, in contrast to the idea that T. Gondii cysts are primarily found in immune-privileged sites, such as the brain and skeletal muscle. While elucidating the contribution of humoral immune responses during chronic toxoplasmosis may require further testing in mice, BCLA may be an excellent tool for investigating these processes.

[0270] Finally, anti-rBCLA antibodies have been detected in some human serum from patients with ocular toxoplasmosis following a toxoplasmosis relapse, during immunosuppression or relapses of toxoplasmosis associated with congenital toxoplasmosis. These findings are consistent with conclusions drawn from mouse models that rBCLA is a remarkable serum marker for the presence of tissue cysts in chronically infected hosts.

[0271] Example 2 (VHH production)

[0272] immunization

[0273] Lama SEL005 and SEL006 were immunized via Eurogentec by four injections on days 0, 14, 28, and 35. Serum was obtained on days 0, 28, and 43. Peripheral blood mononuclear cells (PBMCs) were obtained from large blood collection on day 43.

[0274] Immune response

[0275] The immune response of SEL005 and SEL006 was tested by evaluating the presence of rBCLA-specific antibodies in serum on day 43. MaxiSorp plates were coated with 200 ng of antigen per well overnight at 4°C. After washing three times with PBS containing 0.05% Tween-20, the plates were blocked with 4% powdered milk in PBS (MPBS). Next, serial dilutions of serum in 1% MPBS were added to the wells and incubated for 1 hour. Unbound antibodies were removed during the PBS-Tween wash. Subsequently, bound antibodies were detected with rabbit anti-VHH (clone K1216) and donkey anti-rabbit conjugated with HRP. Antibody binding was quantified by a colorimetric reaction of O-phenylenediamine (OPD) in the presence of H2O2 at 490 nm. Lama SEL005 and SEL006 show a very good response to His rBCLA.

[0276] Library construction for SEL005, day 43, and SEL006, day 43.

[0277] RNA isolation and cDNA synthesis

[0278] Peripheral blood lymphocytes were isolated from a large blood sample on day 43, and RNA was isolated from them using Eurogentec. The precipitated RNA was dissolved in RNase-free MQ, and the RNA concentration was measured. To assess the quality of the RNA, 5 μl of lysed RNA was analyzed on a gel. Figure 2A shows that intact 28S and 18S rRNA are clearly visible, indicating the proper integrity of the RNA.

[0279] Approximately 40 μg of RNA (four reactions of 10 μg each) was transcribed into cDNA using a reverse transcriptase kit (Thermo Fisher Scientific). The cDNA was purified using a Macherey-Nagel PCR cleanup column. Variable domains of heavy chain fragments (both conventional and heavy chain-only) were amplified using primers that annealed at the leader sequence region and the CH2 region. 5 μl was loaded onto a 1% TBE agarose gel for amplification control.

[0280] After this control, the remainder of the sample was loaded onto a 1% TAE agarose gel, a 700 bp fragment was excised, and purified from the gel. A total of 80 ng of isolated PCR products were used as templates for nested PCR (final volume 800 μl), and SfiI and Eco91I restriction sites were introduced to either end of the VHH gene. The amplified VHH fragment was washed on a Macherey-Nagel PCR washing column and eluted in 120 μl. The eluted DNA was digested first with SfiI, followed by Eco91I. As a control for restriction digestion, 4 μl of this mixture was loaded onto a 1.5% TBE agarose gel.

[0281] After restricted digestion, the sample was loaded onto a 1.5% TAE agarose gel. A 400 bp fragment was excised from the gel and purified using a Machery-Nagel gel extraction column. The purified 400 bp VHH fragment (~330 ng) was ligated into a pUR8100 phagemide vector (~1 μg) and transformed into TG1 E. coli.

[0282] Library size

[0283] Transformed TG1 was titrated using a 10-fold dilution. 5 μl of the dilution was spotted onto an LB agar plate supplemented with 100 μg / ml ampicillin and 2% glucose. The number of transformants was calculated from the spot dilutions of the transformed TG1 culture (remembering that the final volume of transformation is 8 ml). The total number of transformants and, consequently, the library size was calculated by counting the colonies in the highest dilution and using the following formula:

[0284] Library size = (Colony volume) * (Diluent) * 8 (ml) / 0.005 (ml; spot volume)

[0285] The VHH insertion frequency of the phagemide vector was determined by selecting 24 different clones and performing colony PCR. Bands of ~700 bp indicate successfully cloned VHH fragments, while bands of ~300 bp indicate empty plasmids. For library SEL005, the insertion frequency was 100% at day 43. For library SEL006, the insertion frequency was approximately 95% at day 43 (Figure 4), which is sufficient to continue phage panning selection.

[0286] Phage production and selection.

[0287] Phages were produced from the library as outlined below: E. coli TG1, including libraries SEL005, 43 days old, and SEL006, 43 days old, was diluted from glycerol stock to an OD600 of 0.05 in 2×YT medium containing 2% glucose and 100 μg / ml ampicillin. The number of bacteria in this inoculum was at least 10× library size (>10⁹ bacteria in the inoculum). This culture was incubated at 37°C for 2 hours to reach an OD600 of ~0.5. Subsequently, approximately 7 ml of the culture was allowed to stand at 37°C for 30 minutes and infected with helper phage VCS M13 using an MOI (Moment of Infection) of 100. Infected bacteria were spun down and resuspended in 50 ml of fresh 2×YT medium supplemented with both ampicillin (100 μg / ml for phagemids) and kanamycin (25 μg / ml for M13 phages), and grown overnight at 37°C with shaking. The produced phages were precipitated from the culture supernatant using PEG-NaCl precipitation. The titer of the produced phages was calculated by serial dilution of the phages and infection with E. coli TG1. The titers of the produced phages were 3 × 10¹¹ / ml for SEL005 on day 43 and 6 × 10¹¹ / ml for SEL006 on day 43, respectively, which were sufficient for selective and continued growth.

[0288] For the first round of panning / selection, 20 μl of precipitated phages (~1010 phages, >100 times the library diversity) were applied to wells coated with His rBCLA. Briefly, 100 μl of antigen was coated overnight on MaxiSorp at two concentrations, 5 μg / ml and 0.5 μg / ml. As a negative control, one well was incubated with PBS alone. The following day, after removal of unbound antigen, the plate was washed three times with PBS and blocked with 4% milk powder in PBS (MPBS). Simultaneously, fresh precipitated phages were pre-blocked with 2% MPBS for 30 minutes. Pre-blocked phages were incubated with directly coated His rBCLA for 2 hours. During heavy PBS-Tween and washing with PBS, bound phages were eluted with 0.1 M TEA solution, which was then neutralized with 1 M Tris / HCl pH 7.5. The eluted phages were serially diluted and then used to infect TG1 bacteria. They were spotted onto LB agar plates supplemented with 2% glucose and 100 μg / ml ampicillin and incubated at 37°C.

[0289] For the second round of selection, new phages were produced from rescue products selected with 5 μg / ml His rBCLA (highest concentration). The rescue products, grown overnight, were diluted 100-fold in 5 ml of fresh 2×YT medium supplemented with 2% glucose and 100 μg / ml ampicillin, and grown for 2 hours until the logarithmic growth phase. Then, 1 μl of helper phage VCSM13 was added and incubated at 37°C for 30 minutes. The culture was allowed to produce phages overnight at 37°C. The produced phages were precipitated from the culture supernatant using PEG-NaCl precipitation.

[0290] Subsequently, for a second panning / selection, 1 μl of precipitated phage was applied to wells coated with His rBCLA as shown below: the antigen was coated overnight on MaxiSorp plates 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 alone. The following day, after removal of unbound antigen, the plate was washed three times with PBS and blocked with 4% MPBS. Simultaneously, fresh precipitated phage was pre-blocked in 2% MPBS for 30 minutes as described above. The pre-blocked phage was incubated directly with His rBCLA for 2 hours. During PBS-Tween and extensive washing with PBS, bound phage was eluted with 0.1 M TEA solution and then neutralized with 1 M Tris / HCl pH 7.5. The eluted phages were serially diluted and then used to infect TG1 cells. They were spotted onto LB agar plates supplemented with 2% glucose and 100 μg / ml ampicillin and incubated overnight at 37°C.

[0291] Screening after 2 rounds of phage display selection.

[0292] The rescue products from the second round of selection at His rBCLA were plated out to select single clones. For the master plate ERB-1, a total of 92 single clones were selected into 96-well plates.

[0293] To screen master plate ERB-1 for His rBCLA binder, periplasmic extracts containing monoclonal VHH were produced. This master plate was cultured at 37°C in 2×YT medium supplemented with 2% glucose and 100 μg / ml ampicillin, and stored at -80°C after adding glycerol to a final concentration of 20%. For periplasmic extract production, master plate ERB-1 was replicated in deep-well plates containing 1 ml of 2×YT medium supplemented with 0.1% glucose and 100 μg / ml ampicillin, and grown at 37°C for 3 hours before adding 1 mM IPTG to induce VHH expression. VHH expression was performed overnight at room temperature. Periplasmic extracts were prepared by centrifugation, resuspending the pellet in 120 μl of PBS, and collecting the bacteria by one freeze-thaw cycle. The bacteria were centrifuged, and the soluble periplasmic fraction containing VHH was separated from the cell debris (pellet). To test the binding specificity of monoclonal VHH by ELISA, His rBCLA (100 ng / well in PBS) was coated overnight onto MaxiSorp plates at 4°C. The coated plates were washed and then blocked with 4% MPBS. The blocked wells were incubated with 10 μl of periplasmic extract and 40 μl of 1% MPBS at room temperature for 1 hour. Unbound VHH was removed by washing with PBS containing 0.05% Tween-20. Subsequently, bound VHH was detected with rabbit anti-VHH (clone K976) and donkey anti-rabbit bound to HRP. VHH binding was quantified by a colorimetric reaction of OPD in the presence of H2O2 at 490 nm. All clones from master plate ERB-1 were able to specifically bind to His rBCLA. There was no difference between the two libraries used.

[0294] Sequence analysis of His rBCLA-bound VHH

[0295] 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 binding in ELISA, but should represent the majority of clones selected from different yields.

[0296] Cloning and production of VHH selected by His rBCLA.

[0297] 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 found VHH sequences. These VHHs were then subcloned from the phagemide 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, enabling detection and affinity purification. For production, pre-cultures were prepared by growing bacteria containing plasmids with the selected VHHs overnight at 37°C in 8 ml of 2×YT medium supplemented with 2% glucose and 100 μg / ml ampicillin. The pre-culture was preheated to 37°C and diluted in 800 ml of fresh 2×YT containing 100 μg / ml ampicillin and 0.1% glucose. The bacteria were grown at 37°C for 2 hours before induction of VHH expression using 1 mM IPTG. VHH was expressed at 37°C for 4 hours, and the bacteria were collected by centrifugation. The bacterial pellet was resuspended in 30 ml of PBS and frozen at -20°C.

[0298] Purification and analysis of VHH.

[0299] Frozen bacterial pellets were thawed at room temperature, and cell fragments were spun down by centrifugation. VHH was purified from the supernatant (soluble fraction) using immobilized metal affinity chromatography (IMAC) with TALON beads to determine the affinity of the His tag to cobalt-charged Sepharose beads. Binding VHH was eluted with 150 mM imidazole and dialyzed against PBS.

[0300] Protein concentrations were measured using absorbance at 280 nm and corrected according to the molar extinction coefficient and molecular weight of different VHHs.

[0301] As a quality check, 1 μg of purified VHH was loaded onto an SDS-PAGE.

[0302] The binding of purified VHH to immobilized His rBCLA was analyzed by ELISA. MaxiSorp plates were coated with 200 ng / well antigen in PBS overnight at 4°C. After blocking the wells with 4% MPBS, serial dilutions of VHH were added to the coated wells and incubated at room temperature for 1 hour. After washing off unbound VHH, bound VHH was detected using mouse anti-flag (clone M2) and donkey anti-mouse conjugates bound to HRP. Binding was quantified by measuring the colorimetric reaction of OPD + H2O2 at 490 nm. ERB-1G6, ERB-1B11, and ERB-1A12 show sub-nanomolar apparent affinity to immobilized His rBCLA. ERB-1F1 and ERB-1F2 show low nanomolar affinity. ERB-1H4 and ERB-1D7 show molar apparent affinity to His rBCLA.

[0303] conclusion

[0304] Immunization of Lama SEL005 and SEL006 yielded a favorable immune response. The generated libraries were of good size and insertion frequency. Phage display selection in His rBCLA yielded many good clones, three of which (ERB-1G6, ERB-1B11, and ERB-1A12) showed very good and clear affinity, with ERB-1G6 also showing high production levels in E. coli. [Table 5] TIFF0007849292000011.tif244165 TIFF0007849292000012.tif251165 TIFF0007849292000013.tif44165

[0305] Example 3:

[0306] In the current longitudinal study, the inventors believe that the detection of BCLA antibodies, when appropriately combined, could potentially enhance the sensitivity of the current test. The inventors have experience testing for BCLA in association with maternal-child congenital toxoplasmosis. For the time being, only 10 couples per mother / child group were tested, and therefore the results should be considered accordingly. Two groups were compared: one in which congenital toxoplasmosis was confirmed through persistent Sag1 IgG titers in the child's serum throughout the postnatal period, and the other in which congenital toxoplasmosis was ruled out when the child's serum became negative for Sag1 over time (Lebech M et al., 1996).

[0307] As shown by comparative titrations of Toxo IgG by Vidas® and Architect®, at birth, infants in both groups share equivalent titers without distinguishable profiles (Figure 15C-D). This is explained by the fact that the mother transmits anti-Sag1 IgG through the placental barrier, and therefore, definitive biological conclusions are not possible at birth. When looking at BCLA ELISA titrations, the distinction between congenital toxoplasmosis and excluded congenital toxoplasmosis becomes clearer. The serum of children at birth presents a much more reactive BCLA titration than the serum of their mothers or the excluded congenital toxoplasmosis group (Figure 15A-B).

[0308] This observation suggests that children specifically synthesize anti-BCLA IgG before birth, indicating that strong BCLA reactivity can further guide the diagnosis of congenital toxoplasmosis at birth.

[0309] References:

[0310] Throughout this application, the latest technology relating to the present invention is described by various references. The disclosures of these references are incorporated into this disclosure by reference. [Table 6] TIFF0007849292000015.tif248165 TIFF0007849292000016.tif235165 TIFF0007849292000017.tif199165

Claims

1. Isolated polypeptide: (i) Amino acid sequence consisting of the C-terminal antigen domain (res 1089-1275 of BCLA) (SEQ ID NO: 2); (ii) an amino acid sequence consisting of an internal repeat domain of BCLA selected from the group consisting of 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); (iii) Below: (1) GELQPAEAEEARLLVADLKAV (Sequence ID 32), (2) VRVEGEAFFRASVDLYEA (Sequence ID 33), (3) KLRPLTKGELVDVVRQ (Sequence ID 34), (4) TQIFVQDRASAFLRV (peptide 36 of rBCLA) (SEQ ID NO: 35), (5) AAEQMKAVFAMVEEG (peptide 44 of rBCLA) (SEQ ID NO: 36), (6) GELQPAEAEEARLLV (rBCLA peptide 12) (SEQ ID NO: 37), (7) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38), (8) EAEEARLLVADLKAV (rBCLA peptide 14) (SEQ ID NO: 39), (9) VRVEGEAFFRASVDL (rBCLA peptide 21) (SEQ ID NO: 40), (10) EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID NO: 41), (11) AFFRASVDLYEAVKN (peptide 23 of rBCLA) (SEQ ID NO: 42), (12) KLRPLTKGELVDVVR (rBCLA peptide 30) (SEQ ID NO: 43), (13) AAGSMEKEKPVLPGEGEGH (domain A of TgR4) (Sequence ID 44), (14) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4) (Sequence ID 45), (15) AAGSMEKEKPVLPGE (TgR4 peptide 3) (SEQ ID NO: 46), (16) GSMEKEKPVLPGEGE (TgR4 peptide 4) (SEQ ID NO: 47), (17) MEKEKPVLPGEGEGH (TgR4 peptide 5) (SEQ ID NO: 48), (18) KEKPVLPGEGEGHVL (TgR4 peptide 6) (SEQ ID NO: 49), (19) KPVLPGEGEGHVLPG (TgR4 peptide 7) (SEQ ID NO: 50), (20) HVLPKHETKPALTDEK (TgR4 peptide 13) (SEQ ID NO: 51), (21) PKHETKPALTDEKRT (TgR4 peptide 14) (SEQ ID NO: 52), (22) HETKPALTDEKRTKP (TgR4 peptide 15) (SEQ ID NO: 53), (23) TKPALTDEKRTKPGG (TgR4 peptide 16) (SEQ ID NO: 54), (24) MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (Sequence No. 7) Fragments selected from the group consisting of An isolated polypeptide selected from the group consisting of the following.

2. An isolated polypeptide comprising a fusion between two peptide fragments of any sequence from (i) to (iii) as described in claim 1.

3. (i) MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (Sequence No. 55), (ii) AAGSMEKDKLVLPGE (Sequence No. 56), An isolated polypeptide selected from the group consisting of the following.

4. An isolated polypeptide according to any one of claims 1 to 3 for use as an antigen.

5. An antibody that specifically binds to a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) comprising Toxoplasma gondii polypeptide BCLA, The aforementioned antibody is a single-domain antibody, The antibody is a single-domain antibody having a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NO: 57 (VH ERB-1G6), SEQ ID NO: 58 (VH ERB-1B11), SEQ ID NO: 59 (VH ERB-1A12), SEQ ID NO: 60 (VH ERB-1F1), SEQ ID NO: 61 (VH ERB-1F2), SEQ ID NO: 62 (VH ERB-1H4), and SEQ ID NO: 63 (VH ERB-1D7).

6. A kit comprising the antibody described in claim 5.

7. An in vitro method for detecting a polypeptide according to any one of claims 1 to 3 or a polypeptide consisting of an amino acid sequence of Toxoplasma gondipeptide BCLA (SEQ ID NO: 1) in a biological sample, and / or for evaluating the amount thereof in a biological sample.

8. An in vitro method for detecting a polypeptide according to SEQ ID NO: 3 or a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) comprising Toxoplasma gondipeptide BCLA in a biological sample, and / or for evaluating the amount thereof in a biological sample, comprising contacting the sample with the antibody according to Claim 5.

9. The method according to claim 7 or 8 for providing data for the in vitro diagnosis of latent toxoplasmosis.

10. A method for providing data for in vitro diagnosis of toxoplasmosis, comprising detecting the presence of the polypeptide described in claim 1 or a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) of Toxoplasma gondipeptide BCLA in a biological sample from a subject to be tested.

11. The method according to claim 10, wherein the sample is a tissue sample.

12. An in vitro method for providing data to determine whether or not a subject is infected with latent toxoplasmosis: a) The process involves detecting the immunoreactivity of the target biological sample to the polypeptide described in any one of claims 1 to 3 or to a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) comprising Toxoplasma gondii polypeptide BCLA, A method wherein the immunoreactivity to the polypeptide described in any one of claims 1 to 3 or to a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) comprising Toxoplasma gondii polypeptide BCLA indicates a latent form of toxoplasmosis.

13. An in vitro method for providing data to diagnose or confirm latent toxoplasmosis in patients who have or are suspected of having latent toxoplasmosis: a) Obtaining a biological sample from the patient, and b) The biological sample includes detecting an antibody against the polypeptide described in any one of claims 1 to 3 or an antibody against a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) comprising Toxoplasma gondii polypeptide BCLA, Herein, a method for diagnosing or confirming latent toxoplasmosis in a patient based on the presence of antibodies in the biological sample.

14. An in vitro method for providing data to diagnose or confirm the diagnosis of congenital toxoplasmosis in patients who have or are suspected of having latent toxoplasmosis: a) Obtaining a biological sample from the patient, and b) The biological sample includes detecting an antibody against the polypeptide described in any one of claims 1 to 3 or an antibody against a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) comprising Toxoplasma gondii polypeptide BCLA, Herein, a method for diagnosing or confirming congenital toxoplasmosis in a patient based on the presence of antibodies in the biological sample.

15. An in vitro method for detecting bradyzoite cysts in a subject and / or evaluating their quantity in a subject. a) To detect immunoreactivity in a target body fluid sample to the polypeptide described in any one of claims 1 to 3 or to a polypeptide consisting of an amino acid sequence (SEQ ID NO: 1) of Toxoplasma gondii polypeptide BCLA; and, b) A method comprising estimating the presence and / or amount of bradyzoite cysts from the results of step a), wherein the immunoreactivity to the polypeptide described in any one of claims 1 to 3 or the polypeptide consisting of an amino acid sequence of Toxoplasma gondipeptide BCLA (SEQ ID NO: 1) indicates the presence and / or amount of bradyzoite cysts in the subject.

16. The in vitro method according to any one of claims 12 to 15, wherein the sample is a bodily fluid sample.

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