Serum markers for latent forms of toxoplasmosis

Abstract

To provide a new serum marker whose expression is restricted to the latent form of toxoplasmosis (bradyzoites / cysts). Also provided is an antibody that specifically binds to the new protein.SOLUTION: The present invention relates to a new Toxoplasma gondii protein, hereinafter referred to as BCLA, a new serum marker whose expression is restricted to the latent form of toxoplasmosis. This specific protein and its antigenic fragments can be used to detect autoantibodies in the serum of patients for the diagnosis of latent forms of toxoplasmosis. The invention also relates to derived antibodies generated by BCLA immunization that specifically bind to this new protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of the invention:

[0002] The present invention relates to a novel Toxoplasma gondii protein (hereafter referred to as BCLA (brain cyst burden-associated antigen)), a novel serum marker whose expression is restricted to the latent form of toxoplasmosis (bradyzoites / cysts). The present invention also relates to antibodies that specifically bind to this novel protein. This particular protein and its antigenic fragments can be used to detect autoantibodies in patient serum for the diagnosis of latent forms of toxoplasmosis.

[0003] Background of the invention:

[0004] The ancient phylum Apicomplexa contains many of the world's most prominent protozoan pathogens. The most deadly to humans is Plasmodium, the causative agent of malaria, which causes nearly 500,000 deaths annually. T. gondii is the causative agent of toxoplasmosis, one of the most widespread protozoan parasites of 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 the leading cause of foodborne deaths in the United States (Scallan et al., 2015). Toxoplasmosis is usually a mild disease in immunocompetent humans but can pose a major threat to immunocompromised patients, who experience life-threatening brain, lung, heart, or disseminated pathology. Transplacental infection can result in congenital infection with variable clinical manifestations ranging from congenital anomalies (e.g., hydrocephalus, microcephaly, intracranial calcifications) to fetal loss.

[0005] The cyst-forming intestinal coccidial parasite T. gondii is transmitted through an alternate two-host life cycle, relying on the feline definitive host for sexual transmission, while also undergoing asexual transmission in a variety of alternative hosts (including rodents and humans). Over its long-term residence in warm-blooded feline metazoans, T. gondii initiates a complex developmental program in response to the surrounding environment, including innate host defenses and adaptation to different hosts. Upon initial infection in an intermediate host, the parasite replicates as tachyzoites and dramatically increases in number before disseminating to many tissues throughout the body. While the initial infection is generally controlled by a strong Th1-mediated proinflammatory host response that leads to the extensive destruction of the bulk tachyzoite population, a minor subpopulation of tachyzoites differentiates into a slow-growing bradyzoite stage, which persists throughout the host's lifetime in tissue cysts residing in long-lived cells, including neurons and skeletal muscle cells (Dubey, 1997). Ingestion of tissue cysts by the feline definitive host completes the cycle, resulting in the excretion of oocysts, the latter of which are highly infectious (Dubey, 2001).

[0006] Tissue cysts are the primary source of human infection via carnivory, and as such, a key factor in human disease complications of toxoplasmosis is the ability of bradyzoites to inflict irreversible damage while redifferentiating back to the replicative tachyzoite stage. Indeed, while asymptomatic parasitism provides lifelong equilibrium and protection in immunocompetent hosts, persistent immune dysfunction is known to disrupt parasite dormancy, promoting bradyzoite-to-tachyzoite transition and further tachyzoite population expansion. These combined processes ultimately lead to encephalitis, interstitial pneumonia, retinochoroiditis, or even disseminated toxoplasmosis as major outcomes in immunocompromised individuals (Dard et al., 2018). Thus, the strategy of T. gondii as an obligate intracellular parasite is based on the quest for avirulence, i.e., the ability to attenuate but not completely counteract the host's innate immune response to infection, thus securing a permanent home required to await transmission.

[0007] Despite the importance of tissue cysts in the T. gondii life cycle and their crucial role as reservoirs for reactivation of toxoplasmosis in immunocompromised hosts, the biology of bradyzoites and the cysts they form is poorly understood. Cysts are thought to grow and disseminate over time, without passing through an intermediate tachyzoite stage, through both migration of free bradyzoites and division of bradyzoite cysts, maintaining chronic infection (Dzierszinski et al., 2004; Frenkel and Escajadillo, 1987). The notion that bradyzoites within tissue cysts are dormant entities has recently been challenged by compelling evidence showing that bradyzoites exhibit periodic episodic growth within tissue cysts in vivo by asynchronously replicating using both endogenous binary and endogenous polyfission (Dzierszinski et al., 2004).

[0008] The developmental transition from tachyzoites to bradyzoites is bidirectional and typified by dramatic changes in parasite gene expression, leading to major changes in metabolism, parasite surface remodeling with restricted expression of stage-specific surface antigens, and cyst wall formation. The latter likely protects bradyzoites from harsh gastrointestinal environmental conditions and provides a physical barrier to host immune defenses. T. gondii differentiation has been difficult to study in that stage transitions are directed by complex and still-unknown developmental genetic programs and are also influenced by host cell physiology (Lueder and Rahman, 2017). In the laboratory, tachyzoite-to-bradyzoite conversion can be induced in vitro in the absence of host immunity by exogenous stress (e.g., alkaline stress, nutrient deprivation, and drugs).

[0009] Transcriptional regulation clearly plays a critical role in bradyzoite development, as evidenced by numerous studies demonstrating stage-specific gene expression. How these changes are regulated at the molecular level remains largely unknown, but we and others have provided strong evidence that epigenetic alterations drive parasite differentiation. Initial evidence came from the observation that tachyzoites rapidly recovered from mice during in vivo infection are particularly prone to differentiation, gradually losing this "primed" state over time. As such, long-term passaging of tachyzoites in tissue culture dramatically attenuates the ability of type II strains to generate high cyst burdens in vivo. Thus, epigenetic mechanisms that promote developmental plasticity, i.e., the manifestation of diverse phenotypes from the same genome, may enable the parasite to adapt to thousands of potential intermediate hosts and respond to significantly different immune systems.

[0010] T. gondii has evolved sophisticated methods for promoting epigenetic changes, such as active changes in histone marks and chromatin remodeling, that counteract strategies employed by the cells they infect and provide zoites with a remarkable ability to undergo staged differentiation in response to environmental cues or as part of a developmental program. Our early interest in histone post-translational modifications (PTMs), specifically acetylation (Saksouk et al., 2005), led us to show that changes in the rate of histone H4 acetylation near stage-specific genes are one of the epigenetic molecular motors that drive parasite differentiation (Bougdour et al., 2009). Acetylation of core histones is mediated by histone acetyltransferases (HATs) and, in many instances, leads to relaxation of chromatin structure and transcriptional activation of associated genes. Histone deacetylases (HDACs) counteract HAT activity by catalyzing the removal of acetyl moieties from lysine residues in histone tails, thereby inducing chromatin condensation and transcriptional repression (Kurdistani and Grunstein, 2003).

[0011] The importance of histone acetylation for controlling differentiation is highlighted by the finding that chemical inhibition of TgHDAC3 with low doses of the compound FR235222 induces the stepwise conversion of 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 compound's TgHDAC3 specificity and suggesting that TgHDAC3 activity actively prevents bradyzoite differentiation (Bougdour et al., 2009). This in vitro conversion was 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 oppose the action of HATTgGCN5b, which was localized by ChIP to the promoters of active genes, whereas TgHDAC3 was localized by ChIP to the promoters of bradyzoite genes (Saksouk et al., 2005). While these data represent a step toward understanding the causal relationship between histone acetylation and gene expression in T. gondii and point to a critical role for TgHDAC3 in the staged transformation, they were performed only with the virulent RH strain, which does not readily develop tissue cysts or latent infections in laboratory mice. Finally, there is a need to develop new diagnostic methods for latent forms of toxoplasmosis.

[0012] In this study, we reexamined the ability of FR235222 to stimulate tachyzoite-to-bradyzoite transformation in vitro using a strain of type II origin that is prone to cyst formation in vivo. Quantitative analysis of the T. gondii proteome response to FR235222 revealed many proteins previously identified as stage-specific proteins (including those recognized as restricted to bradyzoites). Due to their potential importance to parasite biology (Hakimi et al., 2017), we chose to focus our attention on novel proteins predicted to be secreted. Approximately 200 putative FR235222-responsive bradyzoite-secreted effectors were identified using this approach. One candidate, BCLA (brain cyst burden-associated antigen), was selected for further study. BCLA was only expressed upon FR235222 treatment, and following its secretion into the vacuolar space, this protein was shown to accumulate in the parasitophorous vacuole membrane (PVM). Under in vivo conditions, BCLA is located in the matrix space of cysts as well as in the cyst wall, the latter believed to originate from the PVM during the latent stage. While assessing its function, we demonstrated that BCLA deficiency affected the integrity of brain cysts isolated from chronically infected mice, indicating that this protein is essential for proper cyst function, at least in our mouse model of chronic toxoplasmosis.

[0013] Given the restricted expression of BCLA by bradyzoites and its location in the cyst wall, we next sought to investigate its potential application in serodiagnosis. Here, we found that a recombinantly produced C-terminal peptide of BCLA is highly antigenic and constitutes an excellent antigen candidate for the detection of anti- T. gondii IgG in chronically infected mice. We provide strong data demonstrating 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 with human serum validate the antigenic characteristics of BCLA and pave the way for the use of this antigen for anti-Toxoplasma diagnosis, with interesting prospects for the serological detection of cyst burden in chronically infected hosts.

[0014] Summary of the Invention:

[0015] The present invention provides an isolated Toxoplasma gondii polypeptide, hereinafter referred to as BCLA (brain cyst burden associated antigen), which comprises amino acid sequence SEQ ID NO: 1 and immunogenic peptide fragments.

[0016] The present invention further relates to antibodies raised against the isolated polypeptides of the present invention.

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

[0018] The present invention further relates to a method for diagnosing latent forms of toxoplasmosis using a polypeptide according to the invention for detecting anti-BCLA antibodies in a biological sample, in particular in a body fluid sample.

[0019] Detailed description of the invention:

[0020] By using epidrugs to modulate tachyzoite genome expression, we were able to identify genes whose expression is restricted to bradyzoites. Here, we report the characterization of BCLA (brain cyst burden-associated antigen), a protein that accumulates in the parasite vacuole membrane in vitro when expressed under bradyzoite-inducing conditions. In mouse brain, this protein is scattered within and on the surface of cysts. Deletion of this gene results in reduced brain cyst burden in mice, and the remaining cysts are typified by deformations of their wall surface, ranging from loss of roundness 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 mouse brain. Using the first ELISA BCLA test developed by us, antibodies directed against the BCLA antigen were detected in serum alone or in both serum and aqueous humor in human patients with highly suspected or proven ocular toxoplasmosis. Although serological assays have long been the test of choice for confirming T. gondii infection, current serodiagnosis does not always distinguish between acute, latent, and relapsing disease states. Furthermore, current serology does not assess the cyst burden in tissues and the subsequent risk of relapse of toxoplasmosis in seropositive immunocompromised patients. Some of these limitations have now been overcome with the discovery of BCLA, a critical antigen candidate for the 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 sequence, TgR4 (Figure 12a), was screened for high-resolution BCLA epitope mapping using peptide dot blot screening (Figures 12b and 12c). In contrast to mouse, all positive human sera showed robust reactivity to peptides derived from the internal repeats, which, once added to rBCLA, significantly increased test sensitivity. Thus, the BCLA ELISA, customized based on the most sensitive peptide and polypeptide combination, proved optimal for reliable discrimination among humans diagnosed with either ocular toxoplasmosis or confirmed past immunity (Figure 13). The ELISA test also detected significant amounts of circulating anti-BCLA antibodies in sera from immunocompromised patients experiencing either asymptomatic or symptomatic episodes of chronic toxoplasmosis (Figure 13).

[0022] Isolated peptides.

[0023] The present invention relates to an isolated Toxoplasma gondii polypeptide, designated BCLA (brain cyst burden associated antigen), comprising amino acid sequence SEQ ID NO:1.

[0024] The present invention also provides an isolated Toxoplasma gondii polypeptide selected from the group consisting of: (i) an amino acid sequence consisting of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1); (ii) the amino acid sequence consisting of the C-terminal antigen domain (res 1089-1275 of BCLA, referred to as rBCLA) (SEQ ID NO: 2); (iii) an amino acid sequence consisting of an internal repeat domain of a 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 that is substantially homologous to a sequence of (i) to (iii), preferably an amino acid sequence that is at least 80% identical to a sequence of (i) to (iii); (v) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (iv).

[0025] By using peptide dot blot screening (see Figure 12), it is possible to identify the most potent BCLA immunogenic peptides in the C-terminal antigenic domain of BCLA (res 1089-1275 of BCLA, referred to as rBCLA) as well as in the internal repeat domain of BCLA (res 304-924 of BCLA, referred to as TgR1 to TgR13 (SEQ ID NO: 4 to SEQ ID NO: 16).

[0026] Thus, in certain embodiments, the 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 a sequence of (i) to (v), preferably an amino acid sequence at least 95% identical to a sequence of (i) to (v); (vii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (vi).

[0027] In more specific embodiments, the isolated Toxoplasma gondii polypeptide from the rBCLA polypeptide is selected from the group consisting of: (i) GELQPAEAEEARLLV (peptide 12 of rBCLA) (SEQ ID NO: 37); (ii) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38); (iii) EAEEARLLVADLKAV (peptide 14 of rBCLA) (SEQ ID NO: 39); (iv) VRVEGEAFFRASVDL (peptide 21 of rBCLA) (SEQ ID NO: 40); (v) EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID NO: 41); (vi) AFFRASVDLYEAVKN (peptide 23 of rBCLA) (SEQ ID NO: 42); (vii) KLRPLTKGELVDVVR (peptide 30 of rBCLA) (SEQ ID NO: 43) (viii) an amino acid sequence substantially homologous to the sequences of (i) to (vii), preferably an amino acid sequence at least 95% identical to the sequences of (i) to (vii); (vii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (viii).

[0028] Thus, in certain embodiments, the isolated Toxoplasma gondii polypeptide from the internal repeat domain of BCLA is selected from the group consisting of: (i) the 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 at least 80% identical to the sequence of (i); (iii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (ii).

[0029] In more particular embodiments, the isolated Toxoplasma gondii polypeptide from the internal repeat domain of BCLA is selected from the group consisting of: (i) AAGSMEKEKPVLPGEGEGH (domain A of TgR4); (SEQ ID NO: 44) (ii) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4), (SEQ ID NO: 45) (iii) an amino acid sequence substantially homologous to the sequence of (i) to (ii), preferably an amino acid sequence at least 95% identical to the sequence of (i) to (ii); (iv) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (iii).

[0030] In more particular embodiments, the isolated Toxoplasma gondii polypeptide 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 (peptide 13 of TgR4), (SEQ ID NO: 51) (vii) PKHETKPALTDEKRT (peptide 14 of TgR4), (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 a sequence of (i) to (ix), preferably an amino acid sequence at least 95% identical to a sequence of (i) to (ix); (xi) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (x).

[0031] Because the BCLA polypeptide has multiple epitopes across different domains (particularly in rBCLA and in the internal repeat domains of BCLA TgR1 to TgR13), it may be advantageous to combine BCLA immunogenic peptide fragments of the invention.

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

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

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

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

[0036] Thus, the isolated Toxoplasma gondii polypeptide from the internal repeat domain of BCLA is selected from the group consisting of: (i) MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (a fusion of a peptide fragment from a repeat motif present in Tgr4 / Trg12 / Tgr13 and a repeat motif present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID NO: 55), (ii) AAGSMEKDKLVLPGE (a peptide fragment from a repeat motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID NO: 56) (iii) an amino acid sequence substantially homologous to the sequence of (i) to (ii), preferably an amino acid sequence at least 95% identical to the sequence of (i) to (ii); (iv) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (iii).

[0037] Because BCLA polypeptides have multiple epitopes across the different internal repeat domains of BCLA (TgR1 to TgR13), it may be advantageous to combine amino acid residues from the internal repeat domains of BCLA.

[0038] Thus, the present invention also relates to a BCLA polypeptide comprising the 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) wherein Xaa1 is glutamic acid (E) or no amino acid residue; wherein Xaa2 is arginine (R) or serine (S); wherein Xaa3 is proline (P) or glycine (G); wherein Xaa4 is alanine (A) or glycine (G); wherein Xaa5 is alanine (A) or no amino acid residue; wherein Xaa6 is glycine (G) or arginine (R); wherein Xaa7 is serine (S), proline (P), or alanine (A); wherein Xaa8 is lysine (K) or glutamic acid (E). wherein Xaa9 is lysine (K), glutamic acid (E), or aspartic acid (D); wherein Xaa10 is proline (P) or leucine (L); wherein Xaa11 is leucine (L) or serine (S). wherein Xaa12 is glutamic acid (E) or lysine (K); wherein Xaa13 is glycine (G) or arginine (R); wherein Xaa14 is valine (V) or alanine (A); wherein Xaa15 is leucine (L) or serine (S); wherein Xaa16 is histidine (H), aspartic acid (D), or alanine (A); wherein Xaa17 is threonine (T), arginine (R), methionine (M), or glutamine (Q); wherein Xaa18 is proline (P), threonine (T), or alanine (A); wherein Xaa19 is aspartic acid (D), glutamic acid (E), or glutamine (Q); wherein Xaa20 is glutamic acid (E) or lysine (K); wherein Xaa21 is lysine (K), glycine (G), or glutamic acid (E); wherein Xaa22 is arginine (R) or valine (V); wherein Xaa23 is lysine (K), glutamic acid (E), or asparagine (N). wherein Xaa24 is glycine (G), valine, or isoleucine (I); wherein Xaa25 is glycine (G) or glutamic acid (E). wherein Xaa26 is arginine (R) or proline (P); wherein Xaa27 is threonine (T), cysteine ​​(C), lysine (K), or methionine (M); wherein Xaa28 is glutamic acid (E) or alanine (A); and a fragment of at least 9 consecutive amino acids of the sequence of SEQ ID NO: 64.

[0039] As used herein, the term "amino acid" refers to a natural or unnatural amino acid in its D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and corresponding amino acid residues, for example, as present in peptidyl structures. Natural and unnatural 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 unnatural 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 with 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 with nonpolar aliphatic side chains, such as, but not limited to, leucine, isoleucine, valine, glycine, alanine, proline, methionine, and phenylalanine. Examples of basic amino acid residues include those with basic side chains, such as amino or guanidino groups. Basic amino acid residues include, but are not limited to, arginine, homolysine, and lysine. Examples of acidic amino acid residues include those with acidic side chain functional groups, such as carboxy groups. Acidic amino acid residues include, but are not limited to, aspartic acid and glutamic acid. Aromatic amino acids include those with aromatic side chain groups. Examples of aromatic amino acids include, but are not limited to, biphenylalanine, histidine, 2-naphthylalanine, pentafluorophenylalanine, phenylalanine, tryptophan, and tyrosine. Note that some amino acids fall into multiple groups; for example, histidine, tryptophan, and tyrosine are classified as both polar and aromatic amino acids. Amino acids may be further classified as uncharged or charged (positive or negative). Examples of positively charged amino acids include, but are not limited to, lysine, arginine, and histidine. Examples of negatively charged amino acids include, but are not limited to, glutamic acid and aspartic acid. Additional amino acids classified into each of the above groups are known to those of skill 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 conservative substitutions. Two amino acid sequences are "substantially homologous" or "substantially similar" if one or more amino acid residues are replaced by biologically similar residues, or if more than 80% of the amino acids are identical, or if more than about 90%, preferably more than about 95%, are similar (functionally identical). Preferably, similar, identical, or homologous sequences are identified by alignment, for example, using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or programs known in the art (BLAST, CLUSTAL, FASTA, etc.). The percentage of identity may be calculated by performing a pairwise global alignment based on the Needleman-Wunsch alignment algorithm, e.g., using Needle, using the BLOSUM62 matrix (with a gap opening penalty of 10 and a gap extension penalty of 0.5), and finding the optimal alignment of the two sequences (including gaps) along their entire length.

[0042] As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another amino acid residue without altering the overall conformation and function of the peptide, and includes, but is not limited to, the replacement 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 may be interchangeable. Similarly, isoleucine (a hydrophobic amino acid) may be substituted with leucine, methionine, or valine. Neutral hydrophilic amino acids may be substituted for each other and include asparagine, glutamine, serine, and threonine.

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

[0044] As such, 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 with similar properties.

[0045] According to the present invention, a first amino acid sequence having at least 80% identity to 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 invention comprises up to 1275 amino acids (and at least 9). [Table 1] TIFF2026034816000002.tif249165 TIFF2026034816000003.tif111165 or 9 amino acids. In some embodiments, polypeptides of the invention contain fewer than 50 amino acids. In some embodiments, polypeptides of the invention contain fewer than 30 amino acids. In some embodiments, polypeptides of the invention contain fewer than 25 amino acids. In some embodiments, polypeptides of the invention contain fewer than 20 amino acids. In some embodiments, polypeptides of the invention contain fewer than 15 amino acids.

[0047] The isolated polypeptides according to the present invention can be produced using any method known in the art. They can be produced, for example, as recombinant polypeptides in host cells (e.g., bacterial, yeast, or eukaryotic host cells) or can be chemically synthesized (for reviews, 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] Antibodies of the Invention

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

[0050] First, to assess the in situ dynamics of BLCA in T. gondii, we generated polyclonal antibodies against two synthetic peptides, each located at the terminus of a conserved repeat in the BCLA protein (see Example 1 and Figure 2b). Autologous antibodies were generated against two peptides (peptides 1 and 2) contained within these repeats. Monitoring BCLA expression by Western blot using the homemade antibodies generated against the two BCLA-derived peptides demonstrates upregulation of BCLA after FR235222 treatment (see Figure 2c).

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

[0052] The present invention provides antibodies that specifically bind to the isolated polypeptides of the present invention.

[0053] According to the present invention, "antibody" or "immunoglobulin" have the same meaning and can be used equally in the present invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds to an antigen. As such, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments, and 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 (I) and kappa (K). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine antigen binding recognition and specificity. The constant domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement binding, and Fc receptor (FcR) binding. An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-binding site and an antigenic determinant. An antibody-binding site is primarily composed of residues from hypervariable regions or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable regions or framework regions (FRs) influence the overall domain structure and, therefore, the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated VL-CDR1, VL-CDR2, VL-CDR3 and VH-CDR1, VH-CDR2, VH-CDR3, respectively.An antigen-binding site therefore comprises six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions. Framework regions (FRs) refer to the amino acid sequences interposed between the CDRs.

[0054] Antibodies binding to isolated polypeptides of the invention can be assayed by conventional methods known in the art. Mature forms of the polypeptides of the invention are preferably used to assay for antibodies binding to epitopes of the polypeptides of the invention. Alternatively, any variant form of the isolated polypeptides of the invention that retains Nanobody XX binding 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 radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, fluorescence immunoassays, protein A immunoassays, and complement fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). For example, BIACORE® (GE Healthcare, Piscataway, NJ) 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, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed. Examples of suitable ELISA assays are also described in the Examples below.

[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 regions of the antibody "arms" interact with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity. Affinity is measured by the K D The term "K D " as used herein is intended to refer to the dissociation constant, which is K d Against K a The ratio of (i.e., K d / K a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. D Methods for determining are by using surface plasmon resonance or by using a biosensor system such as a Biacore® system.

[0056] The present invention provides an antibody that specifically binds to an isolated polypeptide comprising or consisting of: (i) an amino acid sequence consisting of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1); (ii) the amino acid sequence consisting of the C-terminal antigen domain (res 1089-1275 of BCLA) (SEQ ID NO: 2); (iii) an amino acid sequence consisting of the 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 sequence of (i) to (iv).

[0057] These antibodies can recognize an epitope that is located within, or includes at least one amino acid located within, a fragment of at least 9 consecutive amino acids of any one of the isolated polypeptides (i) to (v).

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

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

[0060] In certain embodiments, an antibody that specifically binds to an rBCLA polypeptide specifically binds to an amino acid sequence selected from the group consisting of: (i) GELQPAEAEEARLLVADLKAV (domain A of rBCLA) (SEQ ID NO: 32) (ii) VRVEGEAFFRASVDLYEA (domain B of rBCLA) (SEQ ID NO: 33) (iii) KLRPLTKGELVDVVRQ (domain C of rBCLA) (SEQ ID NO: 34) (iv) TQIFVQDRASAFLRV (peptide 36 of rBCLA and domain D of rBCLA) (SEQ ID NO: 35) (v) AAEQMKAVFAMVEEG (peptide 44 of rBCLA and domain E of rBCLA) (SEQ ID NO: 36) (vi) an amino acid sequence substantially homologous to a sequence of (i) to (v), preferably an amino acid sequence at least 95% identical to a sequence of (i) to (v); (vii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (vi). In a more particular embodiment, an antibody that specifically binds to an rBCLA polypeptide specifically binds to an amino acid sequence selected from the group consisting of: (i) GELQPAEAEEARLLV (peptide 12 of rBCLA) (SEQ ID NO: 37); (ii) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38); (iii) EAEEARLLVADLKAV (peptide 14 of rBCLA) (SEQ ID NO: 39); (iv) VRVEGEAFFRASVDL (peptide 21 of rBCLA) (SEQ ID NO: 40); (v) EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID NO: 41); (vi) AFFRASVDLYEAVKN (peptide 23 of rBCLA) (SEQ ID NO: 42); (vii) KLRPLTKGELVDVVR (peptide 30 of rBCLA) (SEQ ID NO: 43) (viii) an amino acid sequence substantially homologous to the sequences of (i) to (vii), preferably an amino acid sequence at least 95% identical to the sequences of (i) to (vii); (vii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (viii).

[0061] The present invention further provides antibodies that specifically bind to an amino acid sequence consisting of the internal repeat domain of BCLA (res304-924), designated TgR1 to TgR13 (SEQ ID NO: 4 to SEQ ID NO: 16).

[0062] Thus, in certain embodiments, antibodies that specifically bind to the internal repeat domain of BCLA bind to an amino acid sequence selected from the group consisting of: (i) the 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 at least 80% identical to the sequence of (i); (iii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (ii). In a more particular embodiment, an 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); (SEQ ID NO: 44) (ii) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4), (SEQ ID NO: 45)

[0063] In a more particular embodiment, an 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 (peptide 13 of TgR4), (SEQ ID NO: 51) (vii) PKHETKPALTDEKRT (peptide 14 of TgR4), (SEQ ID NO: 52) (viii) HETKPALTDEKRTKP (TgR4 peptide 15) (SEQ ID NO: 53) (i) TKPALTDEKRTKPGG (TgR4 peptide 16) (SEQ ID NO: 54)

[0064] In certain embodiments, 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) wherein Xaa1 is glutamic acid (E) or no amino acid residue; wherein Xaa2 is arginine (R) or serine (S); wherein Xaa3 is proline (P) or glycine (G); wherein Xaa4 is alanine (A) or glycine (G); wherein Xaa5 is alanine (A) or no amino acid residue; wherein Xaa6 is glycine (G) or arginine (R); wherein Xaa7 is serine (S), proline (P), or alanine (A); wherein Xaa8 is lysine (K) or glutamic acid (E). wherein Xaa9 is lysine (K), glutamic acid (E), or aspartic acid (D); wherein Xaa10 is proline (P) or leucine (L); wherein Xaa11 is leucine (L) or serine (S). wherein Xaa12 is glutamic acid (E) or lysine (K); wherein Xaa13 is glycine (G) or arginine (R); wherein Xaa14 is valine (V) or alanine (A); wherein Xaa15 is leucine (L) or serine (S); wherein Xaa16 is histidine (H), aspartic acid (D), or alanine (A); wherein Xaa17 is threonine (T), arginine (R), methionine (M), or glutamine (Q); wherein Xaa18 is proline (P), threonine (T), or alanine (A); wherein Xaa19 is aspartic acid (D), glutamic acid (E), or glutamine (Q); wherein Xaa20 is glutamic acid (E) or lysine (K); wherein Xaa21 is lysine (K), glycine (G), or glutamic acid (E); wherein Xaa22 is arginine (R) or valine (V); wherein Xaa23 is lysine (K), glutamic acid (E), or asparagine (N). wherein Xaa24 is glycine (G), valine, or isoleucine (I); wherein Xaa25 is glycine (G) or glutamic acid (E). wherein Xaa26 is arginine (R) or proline (P); wherein Xaa27 is threonine (T), cysteine ​​(C), lysine (K), or methionine (M); wherein Xaa28 is glutamic acid (E) or alanine (A); and a fragment of at least 9 consecutive amino acids of the sequence of SEQ ID NO: 64.

[0065] The present invention further provides antibodies that specifically bind to an amino acid sequence consisting of any of peptide 1 and peptide 2 (SEQ ID NOs: 17 to 27) within the internal repeat domain of BCLA, designated TgR1 to TgR13 (SEQ ID NOs: 4 to 16).

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

[0067] These antibodies can be polyclonal or monoclonal. When 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 comprising the VH and VL domains of an antibody and the CH and CL domains of a human antibody.

[0069] According to the present invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody, but retaining the CDRs of the original non-human antibody.

[0070] The term "antibody fragment" refers to a fragment of an antibody containing the variable domain including the CDRs of said antibody. Basic antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, and dsFv. For examples of antibody fragments, see also the review 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 having a molecular weight of approximately 50,000 and antigen-binding activity, in which approximately the N-terminal half of the H chain and the entire L chain are bound together via disulfide bonds among fragments obtained by treating IgG with the protease papain.

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

[0073] The term "Fab" refers to an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, which can be 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 that are typically expressed from gene fusions containing genes encoding VH and VL linked by a peptide-encoding linker. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. Divalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by linking monovalent scFvs with a peptide linker, e.g., a bivalent sc(Fv)2.

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

[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 the type found in camelid mammals, which naturally lack light chains. Such single-domain antibodies are also called VHHs or "nanobodies." 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 approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of their small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, making them useful as reagents for detecting otherwise cryptic antigens using classical immunological techniques and as potential therapeutic agents. Thus, another consequence of their small size is that nanobodies can bind to specific sites within grooves or narrow clefts of target proteins and thus serve to inhibit them, thus more closely mimicking the function of classical low-molecular-weight drugs than classical antibodies. Their low molecular weight and compact size also result in nanobodies that are extremely thermostable, stable to extreme pH and proteolytic digestion, and poorly antigenic. Another consequence is that nanobodies can easily move from the circulatory system into tissues and even cross the blood-brain barrier to treat disorders affecting nervous tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. See U.S. Patent Application 20040161738, published August 19, 2004. These features, combined with low antigenicity to humans, indicate great therapeutic potential.The amino acid sequence and structure of a single domain antibody can be considered to be composed 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 regions for CDR1," "complementarity-determining regions 2" or "CDR2," and "complementarity-determining regions 3" or "CDR3," respectively. A single domain antibody can therefore be defined as an amino acid sequence with the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 through FR4 refer to framework regions 1 through 4, respectively, and in which CDR1 through CDR3 refer to complementarity-determining regions 1 through 3, respectively. In the context of the present invention, the amino acid residues of single domain antibodies are numbered according to the conventional numbering for VH domains given by the International ImMunoGeneTics information system of amino acid numbering (http: / / imgt.cines.fr / ).

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

[0078] The sequences of ERB-1F1, ERB-1F2, ERB 1H4, ERB-1D7, ERB-1G6, ERB-1B11, and ERB-1A12 VHHs are listed below in Table 1 for the variable heavy chains (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 colloidal gold. 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 that is 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 It is C.

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

[0083] Alternatively, the immunoconjugate can be detectably labeled by coupling the antibody to a chemiluminescent compound. The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0084] Similarly, a bioluminescent compound can be used to label the immunoconjugates of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds useful for labeling include luciferin, luciferase, and aequorin.

[0085] Alternatively, the immunoconjugate can be detectably labeled by linking the monoclonal antibody to an enzyme. When the enzyme conjugate is incubated in the presence of an appropriate substrate, the enzyme reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric, or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

[0086] Antibodies of the present invention may be labeled with metallic chemical elements, such as lanthanides. Lanthanides offer several advantages over other labels in that they are stable isotopes; there are a large number of them available, up to 100 or more different labels; they are relatively stable; they are highly detectable; and when detected using mass spectrometry, they are easily resolved between detection channels. Lanthanide labels also offer a wide dynamic range of detection. Lanthanides exhibit high sensitivity and low sensitivity to light and time, making them very flexible and robust, and they can be used in a number of different settings. Lanthanides are a set of 15 metallic chemical elements with atomic numbers 57 to 71. They are also called rare earth elements. Lanthanides can be detected using CyTOF technology. CyTOF is an inductively coupled plasma time-of-flight mass spectrometry (ICP-MS). CyTOF instruments are capable of analyzing up to 1,000 cells per second for as many parameters as there are stable isotope tags available.

[0087] Those skilled in the art will know of other suitable labels that can be used in accordance with the present invention. The binding of marker moieties to monoclonal antibodies can be accomplished using standard techniques known in the art.

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

[0089] Another object of the present invention is a method for detecting antibodies directed against the T. gondii polypeptide BCLA and / or assessing its amount in a biological sample using at least one isolated Toxoplasma gondii polypeptide according to the invention as described above.

[0090] As used herein, the term "biological sample" refers to any biological sample of a subject; tissue sample or body fluid sample.In a preferred embodiment of the method for detecting the antibody directed against T. gondii polypeptide BCLA, the biological sample is the body fluid of the subject.Non-limiting examples of such samples include but are not limited to blood, serum, plasma, urine, saliva, and cerebrospinal fluid (CSF) and aqueous humor.

[0091] More particularly, the body fluid sample is a serum or aqueous humor sample. In a preferred embodiment for the detection of antibodies against T. gondii BCLA polypeptides of the present invention, the biological sample is a body fluid sample, more particularly a brain sample.

[0092] The detection and diagnostic methods of the present invention:

[0093] In some embodiments, the methods of the invention are performed in vitro or ex vivo.

[0094] Methods for detecting T. gondii BCLA polypeptides.

[0095] An object of the present invention is a method for detecting the T. gondii polypeptide BCLA of the invention and / or assessing its amount in a biological sample.

[0096] Biological sample means, but is not limited to, a tissue sample, culture medium and cell sample, whole blood sample, serum sample, plasma sample, aqueous humor sample, saliva sample, cerebrospinal fluid sample, muscle sample, or brain tissue sample.

[0097] In a preferred embodiment for the detection of a T. gondii BCLA polypeptide, the biological sample is a tissue sample, more particularly a muscle sample or a brain sample.

[0098] Detecting the T. gondii polypeptide BCLA can include protein / polypeptide separation; centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid phase affinity based on the protein's affinity for the particular solid phase being used. Once separated, the T. gondii polypeptide BCLA can be identified based on a known "separation profile," for example, the retention time of the protein, as measured using standard techniques. Alternatively, the separated protein can be detected and measured, for example, by mass spectrometry (see the Examples section).

[0099] The detection and quantity of T. gondii polypeptide 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-type assays). Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, e.g., ELISA; biotin / avidin-type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation; and the like. The reaction typically involves revealing a label, e.g., a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme label, or a dye molecule, or other method for detecting the formation of a complex between the antigen and the antibody or antibodies reactive therewith.

[0100] For example, the determination of the amount of T. gondii polypeptide BCLA can be carried out by various techniques and methods, any of which are known in the art: RIA kits (DiaSorin; IDS, Diasource), Elisa kits (Fujirebio, Thermo Fisher, EHTGFBI, R&D DY2935, IDS (manual), IDS (adapted for open analyzers), immunochemiluminescence automated methods (MesoScaleDiscovery, DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (Janssen et al., 2012), Simoa / Quanterix.

[0101] In certain embodiments, the methods of the invention involve contacting a biological sample with a binding partner.

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

[0103] The binding partner may generally be an antibody, which may be polyclonal or monoclonal, preferably monoclonal.

[0104] In another embodiment, the binding partner can be an aptamer. Aptamers are a class of molecules that offer an alternative to antibodies in molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences capable of recognizing virtually 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 random sequence libraries, as described in Tuerk et al. (1990) Science, 249, 505-510. Random sequence libraries are obtainable by combinatorial chemical synthesis of DNA. In this library, each member is ultimately a chemically modified linear oligomer of unique sequence. The possible modifications, uses, and advantages of this class of molecules are reviewed in Jayasena 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins, such as E. coli thioredoxin A, that are selected from combinatorial libraries by a two-hybrid method (Colas et al. (1996) Nature, 380, 548-50).

[0105] Binding partners of the invention, such as antibodies or aptamers, 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 (either directly or indirectly) a signal.

[0106] As used herein, the term "labeled," with respect to a binding partner, is intended to encompass direct labeling of an antibody or aptamer by conjugating (i.e., physically linking) a detectable substance, such as a radioactive substance or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)), to the antibody or aptamer, as well as indirect labeling of a probe or antibody by reactivity with a detectable substance. The antibodies or aptamers of the invention can be labeled with a radioactive molecule by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms for scintigraphic studies, 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 an aptamer) to a solid support. Solid supports that can be used in the practice 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 plates); 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 the T. gondii polypeptide BCLA. A body fluid sample containing or suspected of containing the T. gondii polypeptide BCLA is then added to the coated well. After a period of incubation sufficient to allow the formation of a binding partner-T. gondii polypeptide BCLA complex, the plate is 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] As a binding partner, the secondary binding molecule may be labeled.

[0109] The antibodies and immunoconjugates of the present invention can be used to detect the T. gondii polypeptide BCLA of the present invention and / or assess 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, and thus can be used to diagnose all diseases associated with the Toxoplasma gondii pathogen.

[0110] Method for the diagnosis of latent forms of toxoplasmosis (detection of the T. gondii polypeptide BCLA)

[0111] Therefore, the method for detecting T. gondii 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 from a subject. Biological samples include, but are not limited to, any tissue sample, culture medium and cell sample, whole blood sample, serum sample, plasma sample, urine sample, saliva sample, and cerebrospinal fluid sample.

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

[0113] A further object of the present invention is a method for detecting the T. gondii polypeptide BCLA of the present invention and / or assessing its amount in a biological sample, said method comprising contacting said sample under conditions allowing the formation of an immune complex between the Toxoplasma gondii polypeptide BCLA and said antibody / immunoconjugate, and detecting or measuring the immune complex formed.

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

[0115] The immune complexes formed can be detected or measured in a variety of ways using standard techniques, including, but not limited to, enzyme-linked immunosorbent assay (ELISA) or other solid-phase immunoassays, radioimmunoassay, electrophoresis, immunofluorescence, or Western blot.

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

[0117] The term "toxoplasmosis" has its common meaning in the art and refers to a globally distributed zoonotic disease with medical significance in pregnant women and immunocompromised patients. Toxoplasma gondii, the causative agent of toxoplasmosis, has coevolved with its warm-blooded hosts (including humans) but typically persists as a quasi-latent population, a strategy for persistence with asymptomatic signs, thus optimizing the chances of transmission to new hosts. Over its extended residence in warm-blooded metazoans, the reproductive stage (tachyzoites) switches to a persistence stage (cyst-encapsulated bradyzoites), thereby providing the parasite with a unique opportunity to spread to new hosts without progressing through its sexual stage, which is restricted to felids. Uncontrolled expansion of the tachyzoite population, resulting from a transient or more persistent disruption of immune balance, can lead to life-threatening disease and, in the case of congenital toxoplasmosis, birth defects. Although persistence depends on both the acquisition of slow replicative skills by a subset of parasites and the subversion of fast replicative populations, and critically requires an IL-12 / IFN-γ immune axis, T. gondii has independently evolved a finely tuned, epigenetically regulated developmental program to engineer stage conversion.

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

[0119] Thus, the present invention refers to a method for diagnosing congenital toxoplasmosis in vitro, said method comprising detecting the presence of a polypeptide according to 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 (bradyzoites 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. gondii tachyzoites into bradyzoites (a semi-dormant, slowly dividing cell stage of the parasite). Within host cells, clusters of these bradyzoites are known as tissue cysts. The cyst wall is formed by the parasite vacuole membrane. While tissue cysts containing bradyzoites can form in virtually any organ, they primarily form and persist in the brain, eyes, and striated muscles (including the heart). However, specific tissue tropism may vary between intermediate host species; in pigs, the majority of tissue cysts are found in muscle tissue, whereas in mice, the majority of cysts are found in the brain. Cysts usually range in size between 5 and 50 μm in diameter (50 μm is about two-thirds the width of an average human hair).

[0121] Furthermore, the present invention also provides kits containing at least one antibody or fragment thereof of the present invention. The kits of the present invention can include an antibody bound to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). Kits containing antibodies for in vitro detection and quantification of the Toxoplasma gondii polypeptide BCLA, for example, in ELISA or Western blot, can be provided. Such antibodies useful for detection can be provided with a label, such as a fluorescent or radioactive label.

[0122] Method for the diagnosis of latent forms of toxoplasmosis (detection of autoantibodies to the T. gondii polypeptide BCLA)

[0123] When synthesized as a recombinant protein, the inventors have clearly demonstrated that BCLA constitutes an efficient serum marker of latent infection with high sensitivity, clearly and exclusively correlating with the presence of cysts in mouse brain. Antibodies directed against the BCLA antigen have been detected in human patients. Concentrated titers were detected in patients who qualified as seropositive for Sag1 or tachyzoite-associated antigens. Further correlation between anti-BCLA IgG synthesis and cysts in humans is provided by significantly stronger titers recorded in pathological panels that are strongly associated with the presence of cysts. Notably, in patients experiencing serological flare-ups and in patients with proven ocular toxoplasmosis (see experimental data in Figures 10 and 13 in the Examples section). In the latter case, the developed ELISA assay can also detect BCLA antibodies in the aqueous humor and serum of some of these patients. The detection of Toxoplasma antibodies directed against semi-dormant cysts is a significant improvement for the serodiagnosis of toxoplasmosis, opening new diagnostic perspectives. Indeed, few components of the cyst wall or surface bradyzoite have been identified, none of which have been shown to serve as antigens for serological purposes, at least in commercially available kits. An ideal antigen should be expressed only in the latent bradyzoite stage and ideally exposed on the surface of the cyst, two features found in the BCLA polypeptide.

[0124] Furthermore, we have demonstrated that children specifically synthesize anti-BCLA IgG prenatally. Thus, BCLA reactivity, in comparison with Vidas® and Architect® Toxo IgG titration, can better guide the diagnosis of congenital toxoplasmosis at birth (see Example 3).

[0125] Therefore, the method of detecting autoantibodies to T. gondii polypeptides according to the present invention is consequently useful for the in vitro diagnosis of toxoplasmosis from biological samples, in particular 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, said method comprising detecting the presence of T autoantibodies to a T. gondii polypeptide according to the invention, as indicated above, in a biological sample from a subject to be tested.

[0127] Thus, the present invention relates to a method for determining whether a subject is suffering from a latent form of toxoplasmosis, said method comprising: a) detecting immunoreactivity to a T. gondii polypeptide of the invention in a biological sample from a patient; and, optionally, b) From the results of step a), inferring whether the patient is suffering from a latent form of toxoplasmosis, where immunoreactivity to the T. gondii polypeptide of the present invention indicates a latent form of toxoplasmosis.

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

[0129] The present invention also relates to an in vitro method for diagnosing or confirming the diagnosis of latent toxoplasmosis in a patient suffering from or suspected of suffering from latent toxoplasmosis, comprising: a) obtaining a biological sample from a patient; and b) detecting antibodies against a T. gondii polypeptide of the invention in a biological sample; Here, the presence of antibodies in a biological sample diagnoses or confirms the diagnosis of a latent form of toxoplasmosis in a patient.

[0130] Thus, the present invention relates to a method for determining whether a subject is suffering from congenital toxoplasmosis, said method comprising: a) detecting immunoreactivity to a T. gondii polypeptide of the invention in a biological sample from a patient; and, optionally, b) From the results of step a), inferring whether the patient is suffering from congenital toxoplasmosis, where immunoreactivity to the T. gondii polypeptide of the present invention is indicative of 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 the diagnosis of congenital toxoplasmosis in a patient suffering from or suspected of suffering from congenital toxoplasmosis, comprising: b) obtaining a biological sample from the patient; and b) detecting antibodies against a T. gondii polypeptide of the invention in a biological sample; Here, the presence of antibodies in a biological sample diagnoses or confirms the diagnosis of congenital toxoplasmosis in a patient.

[0133] As used herein, the term " biological sample " refers to any biological sample of a subject.In a preferred embodiment of the method for using the antibody directed against T. gondii BCLA polypeptide to detect, the biological sample is the body fluid of the subject.The 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 a serum or aqueous humor sample.

[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 the method is performed to confirm that the patient does in fact have 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 prior to receiving a transplant), and the method is performed to determine whether the patient is actually suffering from a latent form of toxoplasmosis.

[0138] If a subject presents with signs and symptoms of acute toxoplasmosis, the current treatment for toxoplasmosis is as follows: Pyrimethamine (Daraprim). This medication, typically used for malaria, is a folate antagonist. It can prevent the body from absorbing the B vitamin folate (folic acid, vitamin B-9), especially if patients take high doses over a long period of time. For that reason, taking additional folic acid may be recommended. Other potential side effects of pyrimethamine include bone marrow suppression and liver toxicity. 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, with folic acid (leucovorin). An alternative is pyrimethamine taken with clindamycin (Cleocin).

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

[0141] If the infection occurs before the 16th week of pregnancy, the pregnant woman receives the antibiotic spiramycin. Use of this drug may reduce the baby's risk of neurological problems from congenital toxoplasmosis.

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

[0143] The present invention also provides an in vitro method for selecting a patient suffering from a latent form of toxoplasmosis suitable for treatment with at least one antifolate and / or antibiotic compound, comprising: a) detecting immunoreactivity to a T. gondii polypeptide of the invention in a biological sample from a patient; and, optionally, b) selecting patients suitable for treatment with at least one antifolate (i.e., pyrimethamine) and / or antibiotic compound (i.e., sulfadiazine or spiramycin) if immune reactivity to a T. gondii polypeptide of the present invention is detected;

[0144] The methods for determining whether a patient is suffering from a latent form of toxoplasmosis, the use of antibodies directed against T. gondii polypeptides of the invention as biomarkers for diagnosing (or confirming) a latent form of toxoplasmosis, and the methods for selecting patients suffering from a latent form of toxoplasmosis suitable for treatment with at least one antifolate and / or antibiotic compound of the invention can 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 is immunoreactive to a T. gondii polypeptide of the present invention, which method comprises 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 antifolate (i.e., pyrimethamine) and / or antibiotic compounds (i.e., sulfadiazine or spiramycin), or pharmaceutical compositions comprising said compounds, for use in treating patients suffering from a latent form of toxoplasmosis who are immunoreactive with a T. gondii polypeptide of the present invention.

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

[0148] In a specific embodiment, the protein tested for immunoreactivity is an rBCLA polypeptide.

[0149] In another specific embodiment, the protein tested for immunoreactivity is a peptide fragment of at least 9 consecutive amino acids of BCLA, the rBCLA sequence, or the internal repeat domain of BCLA (res 304-924 of BCLA) (referred to as TgR1 to TgR13 (SEQ ID NO: 4 to SEQ ID NO: 16)).

[0150] In particular, the term "T. gondii polypeptide" according to the present invention, whose immunoreactivity is tested, refers to: (i) an amino acid sequence consisting of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1); (ii) the amino acid sequence consisting of the C-terminal antigen domain (referred to as rBCLA, res 1089-1275 of BCLA) (SEQ ID NO: 2); (iii) an amino acid sequence consisting of the 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 sequence of (i) to (iv).

[0151] Thus, in certain embodiments, the isolated Toxoplasma gondii polypeptide from the rBCLA polypeptide that is tested for immunoreactivity is tested / selected from the group consisting of: (i) GELQPAEAEEARLLVADLKAV (domain A of rBCLA) (SEQ ID NO: 32) (ii) VRVEGEAFFRASVDLYEA (domain B of rBCLA) (SEQ ID NO: 33) (iii) KLRPLTKGELVDVVRQ (domain C of rBCLA) (SEQ ID NO: 34) (iv) TQIFVQDRASAFLRV (peptide 36 of rBCLA and domain D of rBCLA) (SEQ ID NO: 35) (v) AAEQMKAVFAMVEEG (peptide 44 of rBCLA and domain E of rBCLA) (SEQ ID NO: 36) (vi) an amino acid sequence substantially homologous to a sequence of (i) to (v), preferably an amino acid sequence at least 95% identical to a sequence of (i) to (v); (vii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (vi).

[0152] In more specific embodiments, the isolated Toxoplasma gondii polypeptide from the rBCLA polypeptide that is tested for immunoreactivity is selected from the group consisting of: (i) GELQPAEAEEARLLV (peptide 12 of rBCLA) (SEQ ID NO: 37); (ii) QPAEAEEARLLVADL (peptide 13 of rBCLA) (SEQ ID NO: 38); (iii) EAEEARLLVADLKAV (peptide 14 of rBCLA) (SEQ ID NO: 39); (iv) VRVEGEAFFRASVDL (peptide 21 of rBCLA) (SEQ ID NO: 40); (v) EGEAFFRASVDLYEA (peptide 22 of rBCLA) (SEQ ID NO: 41); (vi) AFFRASVDLYEAVKN (peptide 23 of rBCLA) (SEQ ID NO: 42); (vii) KLRPLTKGELVDVVR (peptide 30 of rBCLA) (SEQ ID NO: 43) (viii) an amino acid sequence substantially homologous to the sequences of (i) to (vii), preferably an amino acid sequence at least 95% identical to the sequences of (i) to (vii); (vii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (viii).

[0153] Thus, in certain embodiments, the isolated Toxoplasma gondii polypeptide from the internal repeat domain of BCLA that is tested for immunoreactivity is selected from the group consisting of: (i) the amino acid sequence consisting of the internal repeat domain of TgR4, MERPAAGSMEKEKPVLPGEGEGHVLPKHETKPALTDEKRTKPGGPRTE (SEQ ID NO: 7) (ii) an amino acid sequence that is substantially homologous to the sequence of (i), preferably an amino acid sequence that is at least 80% identical to the sequence of (i). (iii) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (ii).

[0154] In a more specific embodiment, the isolated Toxoplasma gondii polypeptide from the internal repeat domain of BCLA that is tested for immunoreactivity is selected from the group consisting of: (i) AAGSMEKEKPVLPGEGEGH (domain A of TgR4); (SEQ ID NO: 44) (ii) VLPKHETKPALTDEKRTKPGGP (domain B of TgR4), (SEQ ID NO: 45) (iii) an amino acid sequence substantially homologous to the sequence of (i) to (ii), preferably an amino acid sequence at least 95% identical to the sequence of (i) to (ii); (iv) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (iii).

[0155] In more specific embodiments, the isolated Toxoplasma gondii polypeptide from the internal repeat domain of BCLA that is 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 (peptide 13 of TgR4), (SEQ ID NO: 51) (vii) PKHETKPALTDEKRT (peptide 14 of TgR4), (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 a sequence of (i) to (ix), preferably an amino acid sequence at least 95% identical to a sequence of (i) to (ix); (xi) A fragment of at least 9 consecutive amino acids of the sequence of (i) to (x).

[0156] Because the BCLA polypeptide has multiple epitopes throughout its different domains (particularly in rBCLA, as well as in the internal repeat domains of BCLA TgR1 to TgR13), it may be advantageous to combine BCLA immunogenic peptide fragments of the invention.

[0157] Thus, in another embodiment, the polypeptide of the invention whose immunoreactivity is tested is a fusion between two immunogenic peptide fragments of the invention, e.g. Peptide AB_F: MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (fusion of peptide fragments from a repeat motif present in Tgr4 / Trg12 / Tgr13 and a repeat motif present in Tgr3 / Trg4 / Tgr5 / Tgr6 / Tgr9) (SEQ ID NO: 55) Peptide A3_B: AAGSMEKDKLVLPGE (a peptide fragment from a repeat motif present in Tgr3 / Tgr5 / Tgr6 / Tgr7 / Trg10 / Tgr11) (SEQ ID NO: 56)

[0158] Thus, in another embodiment, the polypeptide of the invention from the internal repeat domain of BCLA (TgRx) to be 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) wherein Xaa1 is glutamic acid (E) or no amino acid residue; wherein Xaa2 is arginine (R) or serine (S); wherein Xaa3 is proline (P) or glycine (G); wherein Xaa4 is alanine (A) or glycine (G); wherein Xaa5 is alanine (A) or no amino acid residue; wherein Xaa6 is glycine (G) or arginine (R); wherein Xaa7 is serine (S), proline (P), or alanine (A); wherein Xaa8 is lysine (K) or glutamic acid (E). wherein Xaa9 is lysine (K), glutamic acid (E), or aspartic acid (D); wherein Xaa10 is proline (P) or leucine (L); wherein Xaa11 is leucine (L) or serine (S). wherein Xaa12 is glutamic acid (E) or lysine (K); wherein Xaa13 is glycine (G) or arginine (R); wherein Xaa14 is valine (V) or alanine (A); wherein Xaa15 is leucine (L) or serine (S); wherein Xaa16 is histidine (H), aspartic acid (D), or alanine (A); wherein Xaa17 is threonine (T), arginine (R), methionine (M), or glutamine (Q); wherein Xaa18 is proline (P), threonine (T), or alanine (A); wherein Xaa19 is aspartic acid (D), glutamic acid (E), or glutamine (Q); wherein Xaa20 is glutamic acid (E) or lysine (K); wherein Xaa21 is lysine (K), glycine (G), or glutamic acid (E); wherein Xaa22 is arginine (R) or valine (V); wherein Xaa23 is lysine (K), glutamic acid (E), or asparagine (N). wherein Xaa24 is glycine (G), valine, or isoleucine (I); wherein Xaa25 is glycine (G) or glutamic acid (E). wherein Xaa26 is arginine (R) or proline (P); wherein Xaa27 is threonine (T), cysteine ​​(C), lysine (K), or methionine (M); wherein Xaa28 is glutamic acid (E) or alanine (A); Or a fragment of at least 9 consecutive amino acids of the sequence of SEQ ID NO: 64.

[0159] A "polypeptide substantially homologous in amino acid sequence" refers to a polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a full-length polypeptide reference sequence. In the context of this application, the percentage of identity is calculated using a global alignment (i.e., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The «needle» program (which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences taking into account their entire length) 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 invention is preferably calculated using the EMBOSS::needle(global) program with the "gap open" parameter equal to 10.0, the "gap extend" parameter equal to 0.5, and the Blosum62 matrix.

[0160] As used throughout this application, the expression "immunoreactive to a target protein" (here a T. gondii polypeptide of the invention) is intended to mean that the sample from the patient being tested contains antibodies specifically directed against the target.

[0161] Thus, immune reactivity against a target protein can be simply detected by demonstrating the presence of antibodies specifically directed against the target protein or a fragment of this target protein in the biological sample being tested.

[0162] Fragments of a target protein may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared to the full-length protein. Preferably, the fragments are at least about 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 250, 300, 350, 400, 450, 500 or more amino acids in length.

[0163] Such tests can be performed by one of ordinary skill in the art using standard methods, such as enzyme-linked immunosorbent assays ("ELISA"), Western blots / dot blots, immunohistochemistry of transfected cells, 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 can be the full-length BCLA polypeptide, the C-terminal antigenic domain (res 1089-1275 of BCLA) (referred to as rBCLA (SEQ ID NO:2)), the internal repeat domain of BCLA (res 304-924 of BCLA) (referred to as TgR1 to TgR13) (SEQ ID NO:4 to SEQ ID NO:16), or a fragment thereof. Preferably, the target protein consists of or comprises the C-terminal antigenic domain (res 1089-1275 of BCLA) (referred to as rBCLA (SEQ ID NO:2)), the internal repeat domain of BCLA (res 304-924 of BCLA) (referred to as TgR1 to TgR13) (SEQ ID NO:4 to SEQ ID NO:16), or a fragment thereof.

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

[0166] In the context of the present invention, the term "treat" is used herein to characterize therapeutic methods or processes that aim to (1) slow or halt the progression, worsening, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) reduce or bring about remission of the symptoms of the disease state or condition to which such term applies; and / or (3) reverse or cure the symptoms of the disease state or condition to which such term applies.

[0167] The antifolate and / or antibiotic compounds used in the above methods or 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 succinate, surfactants used in pharmaceutical dosage forms, such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as 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 substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0169] As will be appreciated 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 can also be used, provided that the composition is in a form suitable for oral administration and can protect the active ingredients from gastric and intestinal enzymes.

[0170] Furthermore, the amount of antifolate and / or antibiotic compound used in the above methods or used to treat a patient suffering from a latent form of toxoplasmosis is a therapeutically effective amount.

[0171] The exact amount of antifolate and / or antibiotic compound used, and 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 the other components in the composition, including the antifolate and / or antibiotic compound. Such concentrations can be routinely determined by one of ordinary skill in the art. The amount of compound actually administered will typically be determined by a physician in light of the relevant circumstances, including the condition being treated, the chosen route of administration, the actual antifolate and / or antibiotic compound being administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, etc.

[0172] Generally, the antifolate and / or antibiotic compounds used in the above methods or used to treat patients suffering from latent forms of toxoplasmosis may be administered in typical ranges. Effective doses may also vary depending on the route of administration and the possibility of concurrent use with other drugs.

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

[0174] Such kits include means for detecting antibodies to at least one T. gondii polypeptide of the present invention.

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

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

[0177] Means for detecting antibodies directed against at least one T. gondii polypeptide of the present invention can also include an antibody that specifically binds to the human antibody (used as a "secondary antibody" that binds to an antibody from the sample being tested that specifically binds to the target protein). 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 invention may further comprise a control sample containing a known amount of antibody and / or instructions for use of said kit in diagnosing a latent form of toxoplasmosis or in selecting patients suffering from a latent form of toxoplasmosis suitable for treatment with at least one antifolate and / or antibiotic compound.

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

[0180] A further object of the present invention is a method for detecting bradyzoite cysts and / or assessing their amount in a subject, said method comprising: a) detecting immunoreactivity against a T. gondii polypeptide according to any one of claims 1 to 2 in a body fluid sample of a subject; and, optionally, b) estimating the presence and / or amount of bradyzoite cysts from the results of step a), where immunoreactivity to the T. gondii polypeptide 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 a bodily 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 a serum or aqueous humor sample.

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

[0184] The invention will be further appreciated in light of the following examples and figures. [Brief explanation of the drawings]

[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 with FR235222 revealed bradyzoite-specific protein expression among BCLA. The volcano plot shows the distribution of T. gondii proteins in FR235222-treated (90 nM) versus untreated (DMSO, 0.1%) primary human fibroblasts infected with the type II (PruΔku80) strain. The log2 ratio (x-axis) for protein counts was obtained by dividing the intensity of the FR235222-treated sample by the intensity of the DMSO-treated sample (control). Down- and up-regulated proteins are indicated by red spots on the left and right sides of the graph, respectively. The vertical black lines indicate the log2 fold change values. Horizontal dashed black lines demarcate proteins (red spots) and indicate a change in abundance of at least 2-fold with a p-value < 0.01. (b) Bar graph showing expression of BCLA genes (fragments per kilobase of transcript per million mapped reads [FPKM] values) during acute (tachyzoites) or chronic (cyst-encapsulated bradyzoites) infection in various feline enteroepithelial stage (EES) samples (EES1: very early EES; EES2: early EES; EES3: mixed EES; EES4: late EES; EES5: very late EES) from days 3 to 7 after oral infection with T. gondii cysts (CZ clone H3) in mice, in cysts from mouse brain, and in tachyzoites cultured in vitro. BCLA is expressed only during the chronic stage of cystic bradyzoites and is not found in feline enteroepithelial stages (EES) from EES1 to EES5 (data source: www.ToxoDB.org). (c) Genome Browser (IGB) screenshot of the BCLA locus (magenta) on chromosome Ib of T. gondii, showing reads for two histone marks (H3K14ac, H3K9me3), TgHDAC3, TgCRC230, and RNA-seq (expressed in FPKM, black). The y-axis depicts read density.The representation shows enrichment of H3K14ac, H3K9me3, TgHDAC3, and TgCRC230 at the BCLA gene. (d) Left panel: CRISPR-mediated gene disruption of TgHDAC3 leads to TgHDAC3 signal suppression as monitored by immunofluorescence assay. Right panel: CRISPR-mediated gene disruption of TgHDAC3 induces BCLA overexpression as monitored by immunofluorescence assay. [Figure 2] Figure 2. BCLA protein reveals a unique structure typified by unstructured tandem repeats. (a) Chart showing disorder scores as a function of protein amino acid position (generated via the IUPred server). Results from the ANCHOR2 and IUPred2 algorithms are displayed in blue and red, respectively. The C-terminal domain end of BCLA (residues 1089 to 1275, hereafter referred to as rBCLA) is predicted to be structured, in contrast to the rest of the protein, which contains a core repeat motif. (b) The BCLA protein encoded by the type II (ME49) T. gondii strain displays 13 repeats in its structure (TgR1 to TgR13). Autoantibodies directed against two peptides contained in these repeats (peptides 1 and 2) were generated by Eurogentec. (c) Monitoring BCLA expression by Western blot using in-house antibodies raised against two BCLA-derived peptides demonstrates upregulation of BCLA after FR235222 treatment compared to DMSO (control). [Figure 3] Figure 3. BCLA is present within the vacuolar space and on the vacuolar membrane upon FR235222 induction. (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 the mean ± standard deviation from two independent experiments; the number of quantified PVs was at least 70. Asterisks indicate statistical significance between each individual FR235222-treated line and the corresponding control (DMSO, 0.1%), as determined by unpaired, two-tailed Student's t-test (Mann-Whitney test) (****p<0.0001; NS, not significant). [Figure 4] Figure 4. BCLA deletion does not dramatically affect parasite growth in vitro or vacuole formation and maturation. (a) Evaluation of the percentage of invasion in HFFs (left panel) and intracellular growth rate (right panel) of in vitro-cultured 76k-GFP-luc-Δbcla tachyzoites compared to the WT strain. The % of HFF invasion is quite similar in both strains, but BCLA deletion induces a 30% decrease in intracellular growth. Results are expressed as the mean ± standard deviation from two independent experiments. Asterisks indicate statistical significance when comparing 76k-GFP-luc-Δbcla and 76k-GFP-luc by Mann-Whitney 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. gondii virulence or cyst burden in mice infected intraperitoneally with tachyzoites. (a) Comparison of the virulence of the 76k-GFP-luc-Δbcla strain and its parental strain, 76k-GFP-luc (WT), in Balb / c and NMRI mice. Balb / c mice (n = 20) and NMRI mice (n = 43) were inoculated by intraperitoneal (ip) injection with 10 and 10 tachyzoites, 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 tachyzoites survived infection over the same time frame as the WT strain (NS, not significant). (b) Evaluation of the ability of the Δbcla strain, compared with the WT strain, to cross the blood-brain barrier and form T. gondii cysts in the brains of mice chronically infected with T. gondii. Brains from NMRI and Balb / c mice surviving the challenge presented in (a) were collected and tested by quantitative PCR ± cyst counts using a microscope to assess parasite burden and cyst number, respectively. Results are presented as mean ± standard deviation from at least two independent experiments. Statistical significance was tested by unpaired, two-tailed Student's t-test (Mann-Whitney test). Mice infected with the Δbcla strain show a trend (but not significant, NS) for a reduction in parasite burden and number of cysts in the brain. [Figure 6]Figure 6. BCLA-deficient cysts are typified by dramatic morphological changes. The cyst morphology of Δbcla bradyzoite-containing cysts was compared with that from the parental 76k-GFP-luc (WT) strain. Brains from NMRI mice that survived the challenge, as shown in Figure 6a, were collected, and cysts were purified using the Percoll gradient method and morphologically characterized under a microscope. (a) Cyst area and (b) GFP fluorescence intensity of Δbcla-containing cysts were measured using ZEN software (Zeiss) and compared with those obtained with WT cysts. Δbcla-containing cysts have 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 between Δbcla-containing cysts and WT cysts, as determined by unpaired, two-tailed Student's t-test (Mann-Whitney test) (***p<0.001). Scale bar, 10 μm. [Figure 7]Figure 7. BCLA deletion did not alter the infectivity or host immune response in mice orally ingested with cysts. Evaluation of the virulence and infectivity of 76k-GFP-luc-Δbcla-containing cysts compared with the 76k-GFP-luc parental strain (WT). C56BL / 6 mice (n = 6) and NMRI mice (n = 20) were orally infected with 46 and 20 cysts of the Δbcla or WT strain, respectively. Acute responses in the ileum were observed in C56BL / 6 mice 8 days postinfection. Chronic responses in the brain were evaluated in NMRI mice 8 to 10 weeks postinfection. (a) Parasite burden in the ileum of C56BL / 6 mice orally infected 8 days prior, quantified by qPCR. Statistical significance between the Δbcla and WT strains was tested using an unpaired, two-tailed Student's t-test (Mann-Whitney test). No significant differences were observed (NS, not significant). (b) qRT-PCR analysis of cytokines (IFNγ, IL-22, IL-18, and IL-1β) and chemokine (CCL2) in the ileum of C56BL / 6 mice orally infected 8 days prior. RNA levels were normalized using TBP levels. Mean ± standard deviation (SD) is shown. Statistical significance between Δbcla and WT was tested by Mann-Whitney test. No significant difference was observed (NS, not significant). (c) Brains from NMRI mice orally infected 8–10 weeks prior were collected and tested by quantitative PCR and microscopic cyst counts to assess parasite burden and cyst number, respectively. Results are presented as mean ± standard deviation (SD) from two independent experiments. Statistical significance between Δbcla and WT was tested by Mann-Whitney test. No significant difference was observed (NS, not significant). Mice infected with the Δbcla strain show a trend (but not significant, NS) for reduced parasite burden and number of cysts in the brain. (d) qRT-PCR analysis of cytokines (TNF-α, IFN-γ, IL-6, IL-22) in the brains of NMRI mice orally infected 8–10 weeks prior. RNA levels were normalized using TBP levels. Mean ± standard deviation is shown. Statistical significance between Δbcla and WT was tested by the Mann-Whitney test. No significant differences were observed (NS, not significant). [Figure 8]Figure 8. rBCLA does not react with acutely infected mouse sera. A single Western blot strip was loaded with 0.5 μg of recombinant rBCLA. The strip was tested with sera collected from mice during the acute phase of toxoplasmosis with various T. gondii strains, routes of infection, and mouse genetic backgrounds. rBCLA does not react with mouse antibodies during the acute phase of infection (days 7–8). (a) Immunoblot with sera from NMRI mice infected by intraperitoneal injection (ip) of 10 tachyzoites of the COUG and COUG-Δmyr1 (atypical haplotype 11) strains for 7 days. Serum does not react with rBCLA. (b) Immunoblot with sera from CBA mice infected ip with 10 tachyzoites of the RH (type I) strain for 7 days. Serum does not react with rBCLA. (c) Immunoblot with sera from C57BL / 6 mice infected orally with 47 cysts of the 76k-GFP-luc or 76k-GFP-luc-Δbcla (type II) strains for 8 days. Serum does not react with rBCLA. [Figure 9]Figure 9. rBCLA is a serum marker of chronic T. gondii infection in a mouse model. A single Western blot strip was loaded with 0.5 μg of rBCLA and tested with sera collected from mice in the subchronic (21-41 days) or chronic (>42 days) phase of toxoplasmosis. rBCLA reacts only with anti-T. gondii IgG antibodies in mice with subchronic or chronic toxoplasmosis following infection with type II cyst-forming strains (PruA7, ME49, or 76k-GFP-luc). (a) Immunoblot with sera from Balb / c mice infected i.p. with 103-106 tachyzoites / mouse with the PruA7 (type II) strain for 42 days. Sera reacted fairly proportionally with rBCLA according to the tachyzoite burden. (b) Immunoblot with serum from CBA mice infected i.p. with 10 tachyzoites / mouse of the ME49 (type II) strain for 80 days. Serum reacts strongly with rBCLA. (c) Immunoblot with serum from NMRI mice infected orally with 20 cysts of the 76k-GFP-luc (type II) strain for 22 months. Serum reacts strongly with rBCLA. (d) Immunoblot with serum from Balb / c mice infected i.p. with 10 tachyzoites / mouse of the 76k-GFP-luc or 76k-GFP-luc-Δbcla (type II) strain for 21 days. Serum from mice infected with 76k-GFP-luc reacts strongly with rBCLA, whereas serum from mice infected with 76k-GFP-luc-Δbcla barely reacts with rBCLA. (e) Immunoblot with serum from CBA mice infected ip with 10 tachyzoites / mouse of the RH (type I) strain for 22 days and followed by pyrimethamine (PYR) or sulfadiazine (Sulfa) treatment. Serum reacts very weakly with rBCLA. (f, g) Immunoblot with serum from NMRI mice infected ip with (f) 10 tachyzoites / mouse with the CTG (type II) strain or (g) the PruΔku80 (type II) strain for 42 days. Serum does not react with rBCLA.(h) Immunoblot of sera from Balb / c mice infected with 10 tachyzoites / mouse of the 76k-GFP-luc (type II) strain for 42 days without ip, bronchial, or corticosteroid treatment. All sera react strongly with rBCLA. [Figure 10] Figure 10. Proteolytic analysis of rBCLA reveals the boundaries of the minimal antigenic region of BCLA. (a) Analysis of proteolytic reactions by SDS-PAGE. Coomassie-stained SDS-PAGE showing input samples for all proteases (trypsin, chymotrypsin, elastase, and papain) and all these time points (10, 20, and 50 minutes). (b) Blot gel incubated with positive mouse serum and revealed with anti-mouse IgG antibody. (c) Blot gel incubated with anti-6his IgG conjugated to peroxidase. The black arrow indicates undegraded rBCLA. The red and blue cursor arrows indicate recurrent N-terminal degradation, demonstrating that rBCLA is rapidly degraded by chymotrypsin and partially degraded by elastase, trypsin, and papain, generating stable fragments around the 17 kDa 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 a human isolate or directly with human serum. (a) Immunoblot with serum from Swiss mice infected ip with amniotic fluid or placenta from women with suspected (clinically suspected, but T. gondii PCR negative in amniotic fluid or placenta) or confirmed (T. gondii PCR positive in amniotic fluid) congenital toxoplasmosis. Serum from infected mice with positive amniotic fluid reacts strongly with rBCLA. (b) Immunoblot 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, Toxoplasma serum assays were performed using the Vidas® (bioMérieux) and Architect® (Abbott) systems (both based on ELISA-derived technology), and clinical status was assessed using each patient's medical record. Notably, Vidas® is based on the rSAG1 antigen, and Architect® is based on the rSAG1 and rGRA8 antigens. The serologic results obtained with rBCLA were compared with each patient's serological and clinical status to assess whether they correlated with specific T. gondii serological and / or clinical status. (α) Sera from patients with proven or suspected ocular toxoplasmosis, (β) relapses of toxoplasmosis during hematopoietic disease (immunosuppression), and (γ) recent primary infections (within 1-2 months) reacted with rBCLA. (δ) Sera from three seropositive patients identified as "past immune" and one serum from a fairly recent infection (2.5 months) did not react with rBCLA. (ζ) All sera tested from seronegative patients did not react with rBCLA, indicating good specificity of this antigen in humans. [Figure 12]Figure 12 | Evaluation of BCLA as a human serum marker. (a) Schematic of epitope mapping regions in both repeat n°4 and rBCLA regions. Peptide coverage is shown as lines representing individual 15aa peptides above or below the peptide sequence, with partial numbering. Regions exhibiting 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 sera. Below are histograms displaying 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 with numbered peptides, performed on a positive human serum. (c) Peptide numbering and peptide dot blot for five positive sera and one negative serum with region coverage. On the right, ELISA titrations for rBCLA and SAG1 (Architect) are shown for these same sera. [Figure 13] Figure 13. BCLA reactivity in human serum. Scatter plot of individual BCLA ELISA titers (UI) grouped within clinical status categories assessed through classical SAG1 serology (Vidas and Architect IgG / IgM) and other medical prerequisites. These groups are: SAG1 seronegative patients (blue dots), previously immune patients (diamonds), active toxoplasmosis in immunocompromised patients (cubes), and asymptomatic serological relapses in immunocompromised patients (triangles) and patients with proven ocular toxoplasmosis (cubes). Histograms display the median and interquartile range of BCLA titers 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 group of seronegative patients. The gray zone (70 to 90 UI) and the positive cutoff line (90 UI) are indicated. [Figure 14]Figure 14. rBCLA immunogenicity correlates with cyst-forming strains during the chronic phase of infection in mice. (a) ELISA serum titration of rBCLA reactivity in mice over time and depending on the T. gondii strain. Individual ELISA measurements given at UI are grouped according to T. gondii strain type, with cyst-forming strains (ME49, PruA7, 76K) indicated by spots, non-cyst-forming strains (RH, PruKU80, CTG) indicated by stars, and ΔBCLA strains (in a 76K or PruKU80 background) indicated by triangles. Time segments after infection are shown to distinguish between acute (≤8 days), subchronic (21-22 days), and chronic (≥42 days) phases. (b) Correlation of rBCLA ELISA reactivity with parasite load, miR-155, and miR-146a expression. Overlay titrations of rBCLA IgG (UI), parasite burden (parasite qPRC counts), and miR-155 / miR-142-a are shown for different mouse strains (NMRI, Balb-C) uninfected or infected with different T. gondii strains, all within the chronic infection time frame (≥11 weeks). Cystic strains (ME49, PruA7, 76K) are indicated by circles, noncystic strains (RH, PruKU80, CTG) are indicated by stars, and ΔBCLA strains (in the 76K or PruKU80 background) are indicated by triangles. [Figure 15] Figure 15: BCLA reactivity in sera from mothers and newborns at risk for congenital toxoplasmosis. (A-B) Violin plots (UI) of BCLA ELISA titrations in sera from 23 mothers and their respective newborns collected at birth (mothers) or between birth and 5.5 months (infants). (C-D) Violin plots of Sag1 titrations (Vidas® and Architect® IgG / IgM). Sera were grouped into clinical status categories: mother-newborn pairs without congenital toxoplasmosis (A and C) and pairs with confirmed congenital toxoplasmosis (B and D). At the top of each panel, mean ± SD values ​​are presented, while differences between medians were calculated using the Mann-Whitney test.

[0186] Example 1:

[0187] Materials and Methods

[0188] Host cell and parasite culture. HFF primary cells (Bougdour et al., 2009), RAW264.7, L929, HCT116, A549, and HEK293 cells were cultured in Dulbecco's modified Eagle's 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% CO. The following Toxoplasma strains were used in this study: type I (RH, GT1), type II (ME49), type III (CTG), atypical (COUG), and Neospora caninum; RHΔku80 (Huynh and Carruthers, 2009), PruΔku80 (Fox et al., 2011), PruA7 (Saeij et al., 2007), COUGΔmyr1 (Hakimi, unpublished), PruΔku80Δbcla, PruΔku80-HF-BCLA, and 76kJ-GFP-luc-Δbcla (obtained in this study). All parasite strains were maintained in vitro by serial passage on monolayers of HFFs.

[0189] Transfection of T. gondii. T. gondii RHΔku80, PruΔku80, and 76k-GFP-luc were electroporated with vectors in cytomix buffer (120 mM KCl, 0.15 mM CaCl, 10 mM KHPO / KHPO, pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl) using a BTX ECM 630 machine (Harvard Apparatus). Electroporation was performed in a 2 mm cuvette at 1.100 V, 25 Ω, and 25 μF. Stable transgenic parasites were selected with 1 μM pyrimethamine, single cloned in 96-well plates by limiting dilution, and verified by immunofluorescence.

[0190] Cas9-mediated C-terminal tagging and gene disruption in T. gondii. The plasmid pTOXO_Cas9-CRISPR was described by (Sangare et al., 2016). The 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 genomic sequence were phosphorylated, annealed, and ligated into the linearized pTOXO_Cas9-CRISP plasmid using Bsal, resulting in pTOXO_Cas-CRISPR::sgTgBCLA. T gondii tachyzoites were then transfected with the plasmids and grown on HFF cells for 18–36 h.

[0191] The cloning oligonucleotides used in this study were: TgBCLA-KO-CRISP-FWD: 5'-AAGTTGATCACTATTCGTGAAGAAGG-3' (SEQ ID NO: 28) TgBCLA-KO-CRISP-REV: 5'-AAAACCTTCTTCACGAATAGTGATCA-3' (SEQ ID NO: 29) TgBCLA-HF-CRISP-FWD: 5'-AAGTTGGAACGGCGGTACGGCGACCG-3' (SEQ ID NO: 30) TgBCLA-HF-CRISP-REV: 5'-AAAACGGTCGCCGTACCGCCGTTCCA-3' (SEQ ID NO: 31)

[0192] FR235222 treatment and induction. FR235222 was provided by Astellas Pharma Inc. (Osaka, Japan) and dissolved in DMSO as described by Bougdour et al., 2009, with a final concentration of either 25 ng / mL or 50 ng / mL in the medium. 16 hours postinfection, medium containing FR235222 was added to infected HFF cells 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 performed under pathogen-free conditions in accordance with established institutional guidelines and approved protocols from the Institutional Animal Care and Use Committee of the University of Grenoble Alpes (agreement number B3851610006). Female mice were used for all studies. 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 with a hemocytometer. Parasites were diluted in Hank's balanced salt solution (Life's), and mice were inoculated ip with tachyzoites of each strain (in 200 μl) using a 28-gauge needle. For oral gavage of infectious cysts, brains from chronically infected mice (76k-GFP-luc and 76k-GFP-luc-Δbcla) were ground in PBS. The number of cysts was quantified microscopically. Mice were force-fed with 100 μl of brain homogenate containing 20 to 40 cysts using a bulb-tipped feeding needle. Blood was collected by caudal puncture or intracardiac puncture at the time of euthanasia. Animal euthanasia was completed in an approved CO2 chamber. For histological analysis of the ileum and immunolabeling in histological sections of the brain, the ileum and brain were removed from the mice, completely embedded in paraffin wax blocks, and cut into 5-μm-thick layers using a microtome. For statistical analysis of mouse survival data, Mantel-Cox and Gehan-Breslow-Wilcoxon tests were used.

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

[0195] Cyst quantification. Five to 12 weeks after infection, each brain from recipient mice was homogenized in 2 ml of PBS. The number of cysts in three or ten aliquots (20 μl each) of the brain suspension was counted microscopically. The total number of cysts was determined by counting the cysts in the 20 μl aliquots and multiplying by 100. For statistical analysis of the difference in cyst quantification between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, a nonparametric Wilcoxon-Mann-Whitney test was used.

[0196] Cyst characterization. Images of purified cysts were captured between slides and slipcovers using a fluorescent ZEISS ApoTome.2 microscope. Cyst area and GFP intensity were measured using ZEN software (Zeiss). For statistical analysis of the differences in cyst area and GFP intensity between 76k-GFP-luc and 76k-GFP-luc-Δbcla cysts, a nonparametric Wilcoxon-Mann-Whitney test was used.

[0197] Quantitative PCR. Parasite burden in the brain or ileum was quantified following DNA extraction (QiAmp DNA Mini Kit, Qiagen) using quantitative PCR targeting the Toxoplasma-specific 529-bp repeat element (Reischl et al., 2003). For statistical analysis of the difference in parasite burden between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, a nonparametric Wilcoxon-Mann-Whitney test was used.

[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 with random hexamers using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems). Samples were analyzed by real-time quantitative PCR using TaqMan Gene Expression Master Mix (Applied Biosystems) for appropriate probes (brain: TNF-α, INF-γ, IL-6, IL-22β; ileum: INF-γ, CCL2, IL-22β, IL-18, and IL-1β). RNA levels were normalized using TBP levels. qRT-PCR was repeated for three independent biological replicates of each sample, and the mean values ​​of the results were used. For statistical analysis of RNA levels between mice infected with 76k-GFP-luc and 76k-GFP-luc-Δbcla, a nonparametric Wilcoxon-Mann-Whitney test was used.

[0199] Immunofluorescence microscopy. Immunofluorescence assays with in vitro 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, permeabilized with 0.1% (v / v) Triton X-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 solvent-degreased using toluene for 3 x 10 minutes and absolute alcohol for 3 x 10 minutes. The slides were then treated with citrate buffer (pH 6), heated at 100°C for 1 hour, rinsed with water for 2 x 10 minutes, and blocked in PBS containing 3% (v / v) bovine serum albumin (BSA). Cells or brain layers were then incubated with the indicated primary antibody for 1 hour, followed by the addition of a 1:1,000 dilution of secondary antibody conjugated to Alexa Fluor 488 or 594 (Molecular Probes) for 1 hour. Nuclei of both host cells and parasites were stained with 2 μg / ml Hoechst 33258 in PBS for 10 minutes at room temperature. Coverslips were mounted on slides using Mowiol mounting medium, and images were acquired using a fluorescent 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 (a gift from Dr. Louis Weiss), mouse anti-GRA1, mouse anti-GRA5, mouse anti-GRA7. Western blot secondary antibodies were conjugated to alkaline phosphatase (Promega), whereas immunofluorescence secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 494 (Thermo Fisher Scientific).

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

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

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

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

[0205] Design and Cloning. Disorder-proneness searches (using Dis-EMBL or IUPred) predict that BCLA is highly disordered throughout most of its sequence, including the core repeat motif. However, the C-terminus (approximately aa 1100 to 1275) is predicted to be structured and may constitute a separate domain. To recombinantly express this domain, the N-terminal boundary was chosen at methionine 1089, while the native C-terminus was preserved. 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 into the pet30-(a) vector (Addgene) using the NdeI and XhoI sites.

[0206] Recombinant expression. Transformation was performed using BL21(DE3)-CodonPlus-RIL chemically competent E. coli (Stratagene) with 1 μg of the pet30-(a)Cter-BCLA plasmid, incubated on ice for 10 min, heat-shocked at 42°C for 45 min, preincubated in LB at 37°C for 45 min, and then spread onto LB agar plates containing kanamycin (Kan) and chloramphenicol (Chlo) and incubated for 12 h. A single colony was then picked and inoculated into a 50 ml preculture of LB / Kan / Chlo that had been grown for 16 h. Five ml of the grown preculture was then used to inoculate a 1 L flask of Terrific Broth medium (Formedium) containing Chlo / Kan. The culture was grown at 37°C until an OD600 of 0.5–0.8 was reached, then induced by adding 0.7 mM IPTG (VWR) and further incubated overnight at 18°C. After incubation, the cells were centrifuged at 3000 g for 25 min, the supernatant was discarded, and the pellet was flash frozen in liquid nitrogen and kept at -80°C.

[0207] Lysis. Purification was performed on three pellets from a 1 L culture, each 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 sarcosine, and 1 tab / 50 ml of Complete antiprotease cocktail (Roche). Lysis was performed on ice using 10 minipulse sonications (15 seconds on, 30 seconds off) at 50° amplitude, ensuring that 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 incubations were performed at 4°C for 30 minutes with gentle agitation. After incubation, the resin was retained on a vertical column and then washed with 3 x 20 ml of wash 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 with 1.5 ml fractions was then performed using a buffer containing 300 mM NaCl, 50 mM Tris pH 8, 5 mM BME, and 300 mM imidazole. The fractions of interest (Figure 8) were then pooled and dialyzed in 50 mM NaCl, 50 mM Tris pH 8, 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 through a chromatography system (Akta Pure, GE Healthcare) directly onto a 5 ml HL-Mono-Q (GE Healthcare) column pre-equilibrated with the same buffer as used for dialysis. The column was washed with two column volumes (CV) and then eluted with a salt gradient (50 mM to 2 M NaCl) over 40 ml in 1.5 ml fractions. Absorbance monitoring at 280 nm was performed throughout the elution. During the elution, SDS-PAGE analysis (Figure 3) revealed that the sample was purified in the later stages of the gradient elution, with the early elution fractions displaying the majority of the higher molecular weight, visible bacterial contaminants. 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 an S75 (GE Healthcare) using a running buffer containing 150 mM NaCl, 50 mM Tris pH 8, and 5 mM BME. It eluted in a heterogeneous peak consistent with a multimeric state, starting near the void volume and spanning over 3 ml. All eluted fractions were pooled to generate the final sample.

[0209] Ammonium sulfate precipitation. To avoid nucleic acid contamination, ammonium precipitation was performed by adding 15% w / v ammonium sulfate (Sigma), gentle rotation for 1 hour at 4°C, followed by centrifugation at 10,000*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 as for size exclusion.

[0210] Limited proteolysis to identify antigenic subfragments within Cter-BCLA. To recover highly antigenic subfragments of Cter-BCLA, limited proteolysis of purified samples was attempted using trypsin, chymotrypsin, elastase, and papain (all from Sigma-Aldrich). Reactions were performed in 30 μl reaction volumes in 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM BME, and 0.5 mM MgCl. In each reaction, 3 μg of Cter-BCLA was digested with 100 ng of protease (1 / 30 w / w) over the course of 50 minutes at 37°C. Reactions were stopped at each time point by adding 10 μl of SDS PAGE loading buffer, followed by heating to 95°C for 5 minutes, and then kept on ice until loading onto the gel.

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

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

[0213] Protein purification, immunoblotting, and mass spectrometry-based proteomic analysis. PruΔku80-BCLA-HAFlag-infected host HFF cell extracts containing Flag-tagged proteins were incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich) for 1 h at 4 °C. The beads were washed with 10 column volumes of BC500 buffer (20% glycerol, 20 mM Tris-HCl pH 8.0, 500 mM KCl, 0.05% NP-40, 100 mM PMSF (phenylmethylsulfonyl fluoride), 0.5 mM DTT, and 1x protease inhibitors). Bound peptides were stepwise eluted with 250 μg / ml FLAG peptide (Sigma-Aldrich) diluted in BC100 buffer. Protein bands were excised from colloidal blue-stained gels (Thermo Fisher Scientific) and treated with DTT and iodoacetamide to alkylate cysteines before in-gel digestion using modified trypsin (sequencing grade; Promega). The resulting peptides from individual bands were analyzed by online nanoLC-MS / MS (UltiMate 3000 coupled to an 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) through simultaneous searches against ToxoDB (version 20151112) and a frequently observed contaminant database embedded within MaxQuant. The minimum peptide length was set to 7 amino acids. The minimum number of peptides, razor+ unique peptides, and unique peptides were all set to 1. The maximum false discovery rate was set to 0.01 at the peptide and protein levels.

[0214] Epitope mapping of BCLA repeats and rBCLA. Dot blot peptide assays were custom synthesized by JPT Peptide Technology on cellulose membranes with N-acetyl moieties on the N-terminus. Two sets of membranes were screened: 1) covering the rBCLA region (res 1089-1275) with a total of 59 peptides, each 15 aa long, with 12 overlaps and 3 offsets; and 2) covering repeat 4 (res 446-493) with a total of 18 peptides, each 15 aa long, with 12 overlaps and 3 offsets. Dot blot assays were performed as described by the manufacturer. Briefly, membranes were first activated in 100% ethanol for 5 min, then washed three times for 3 min in DPBS-Tween. They were blocked overnight at 4°C in DPBS-Tween 0.5% milk powder, and then washed again for 3 min in DPBS-Tween. Tested sera were diluted 1 / 400 in DPBS-tween 0.1% BSA and incubated with the membrane for 3 h at room temperature. Following 3 x 3 min DPBS-tween washes, the membrane was incubated with anti-IgG peroxidase-conjugated Ab (Sigma A0170) diluted 1 / 100,000 for 2 h at RT. Following 3 x 3 min washes 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 data analysis of independent dot blots, the integrated intensities from all peptide dots [I(p)] were normalized to an enrichment 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, symbolized as (+), the Fe(p) enrichment scores were summed together, and to subtract nonspecific reactivity, the same summation was performed with the same number of negative serum peptides, symbolized as (-), and subtracted. The peptide reactivity score can be expressed through the following equation:

number

[0216] BCLA ELISA. Peptide synthesis: The following BCLA peptides were synthesized by Genscript with an N-terminal acetyl group: AB_F:Nter-MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP-Cter (SEQ ID NO: 55) A3_B: Nter-AAGSMEKDKLVLPGE-Cter (SEQ ID NO: 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, plates were washed twice with 350 μl of DPBS 0.05% Tween 20 (DPBS / Tween) and 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, plates could be stored at 4°C for long periods without loss of seroreactivity.

[0218] Sample preparation: All serum dilutions were prepared in DPBS 0.05% Tween 20, 0.1% BSA within 2 hours prior to the assay. A 1 / 400 dilution was prepared for both mouse and human tested sera. Eleven standards were also prepared fresh for both tests, consisting of 10 serial dilutions of a positive frozen stock serum set at 100 UI. Starting with a 1 / 200 dilution and following 3 / 4 dilution increments, the following titration points were prepared: 200 UI (1 / 200), 150 UI (1 / 266), 112.5 UI (1 / 356), 84.4 UI (1 / 474), 63.3 UI (1 / 632), 47.5 UI (1 / 843), 35.6 UI (1 / 1124), 26.7 UI (1 / 1498), 20 UI (1 / 1998), 15 UI (1 / 2663). A 0 UI standard was prepared using seronegative serum diluted 1 / 400.

[0219] Assay: All subsequent steps were performed on a Gemini ELISA automation platform (Stratec) but can also be performed manually at RT. The dried plate was first washed twice with 350 μl of DPBS / Tween. Dilutions of the tested sera and standards were dispensed into the plate in series of duplicates with 100 μl per well. The plate was then incubated for 1 h at RT. After the incubation period, the plate was washed four times with 350 μl of DPBS / Tween, and 100 μl of peroxidase-conjugated secondary antibody dilution (1 / 50,000 anti-mouse IgG or 1 / 60,000 anti-human IgG, Sigma Aldrich refs. A0168 and A0170, respectively) in DPBS 0.05% Tween 20, 0.1% BSA was then rapidly dispensed into all wells. After 1 h at RT, the plate was washed four times with DPBS / Tween. The development reaction was carried out for exactly 20 min at RT by adding 100 μl of TMB substrate (Thermofisher ref 34029), then the reaction was stopped with 50 μl of H2SO4 0.2 M followed by mixing for 30 s. Absorbance measurements of the wells were then performed at 450 nm using a Gemini integrated spectrophotometer.

[0220] Data processing: Blank subtraction was performed on duplicate blank wells; no primary antibody / serum was placed in the wells, but all subsequent steps (washing, secondary Ab, substrate) were performed. Standard serum dilutions were averaged and fitted using a four-parameter logistic regression, with the upper asymptote (Di) fixed at 2.5 AU and all other variables (Ai, Bi, Ci) allowed for fitting. From this regression, the apparent UIs of the tested dilutions could be calculated and averaged. If a coefficient of variation greater than 10% was observed in duplicate measurements, then the sample could be retested. All ELISA data presented in this study were obtained several times in independent titrations.

[0221] result

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

[0223] Specific inhibition of TgHDAC3 by the cyclopeptide FR235222 has been shown to disrupt steady-state levels of histone H4 acetylation across the T. gondii genome and induce derepression of stage-specific genes (Bougdour et al., 2009; Sindikubwabo et al., 2017). Taking advantage of the properties of FR235222, we developed an in vitro cyst formation system capable of producing the quantities of protein required for large-scale proteomic studies (Farhat D et al., manuscript in preparation). Following low-dose and short-term treatment of an cyst-forming type II (PruΔku80) strain, we performed quantitative proteomic studies and found that the FR235222-treated proteome was significantly enriched in stage-specific proteins, including those recognized as restricted to the bradyzoite stage (Figure 1a). From this analysis, we found that the protein TGME49_209755 (hereafter referred to as BCLA, or brain cyst burden-associated antigen) was significantly induced upon FR235222 treatment, in the same manner as several proteins involved in the chronic phase of infection (Fig. 1a), consistent with its expression profile reported as being restricted to the bradyzoite dataset (Fig. 1b, source ToxoDB).

[0224] Other evidence supports epigenetic regulation of BCLA expression. We recently reported that H3K14ac and H3K9me3 PTMs bookmark transiently repressed genes, awaiting parasite stage differentiation for stage-specific expression (Sindikubwabo et al., 2017). In tachyzoites, the BCLA locus exhibits this dual PTM enrichment, which differentially marks "balanced" stage-specific genes (Figure 1c). Furthermore, recent TgHDAC3 ChIP-seq analysis (Farhat D et al., manuscript in preparation) revealed the presence of histone deacetylases at the BCLA locus (Figure 1c). Definitive genetic evidence underlying the involvement of TgHDAC3 in this regulation was provided by CRISPR-mediated gene disruption of TgHDAC3, which led to BCLA induction in transfected tachyzoites (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, the surrounding heterochromatin of which is typified by so-called bivalent chromatin domains capable of silencing developmental genes while balancing them for rapid activation during cell differentiation (Sindikubwabo et al., 2017).

[0225] BCLA is secreted into the PV and associates with the PVM of bradyzoite-containing vacuoles transformed in vitro.

[0226] BCLA is a single open reading frame encoding a 140 kDa protein with a conserved C-terminal region of ~150 residues bounded by a predicted N-terminal signal peptide and a central core domain typified by a 48-amino acid motif repeated 13 times (Figure 2a). Its composition and frequency have evolved among T. gondii lineages throughout the coccidia subclass (Figure 2b). While BCLA-homologous proteins are poorly conserved in Neospora caninum, they share the same overall architecture, with short repeats sharing characteristics with BCLA repeats (data not shown). Disorder-prone 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 to 1275) is predicted to be structured and may constitute a separate domain (Figure 2a).

[0227] Although BCLA was unambiguously and exclusively identified by mass spectrometry in FR235222-treated samples (Fig. 1a), the protein's dynamics and subcellular distribution during infection remain to be studied. To further explore the dynamics of BLCA in T. gondii in situ, we generated polyclonal antibodies against two synthetic peptides, each mapping to the termini of a conserved repeat (Fig. 2a). We first validated the proteomic data by showing that exposure of cells to FR235222 significantly increased BCLA signal intensity as a protein band of the expected size of ~140 kDa, which was otherwise undetectable in untreated tachyzoites (Fig. 2c).

[0228] In fibroblasts hosting tachyzoites expressing a C-terminal HA-Flag-tagged version of the bradyzoite-specific marker, BCLA was clearly detected in the vacuolar space upon stimulation with FR235222 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 detectable in cells infected with tachyzoites genetically engineered to lack BCLA (Δbcla, Table 2), thereby confirming autoantibody specificity (data not shown). Finally, when we monitored BCLA dynamics in type I (RHΔku80) and type II (PruΔku80) strains expressing the endogenous protein in fusion with an HA-Flag tag, we showed that once stimulated with FR235222, the HA-tagged BCLA protein was targeted to the vacuolar space and at the membrane, regardless of strain type (data not shown). Thus, the presence of the C-terminal fusion tag does not affect the subcellular localization of BCLA, as it is similar to that observed using anti-BCLA serum in untagged strains.

[0229] During exposure of different parasite strains of T. gondii to FR235222, we finally found that BCLA signal intensity varied greatly depending on the infecting strain, ranging from very strong induction in type II (PruΔku80, ME49, 76K-GFP-Luc) strains to moderate induction in type I (GT1 and RHΔku80) and haplogroup 11 (COUG) strains; surprisingly, faint (if absent) signals were detected in cells infected with type III (CTG) strains (Fig. 3a and data not shown). This discrepancy, which may be explained by the ability of the strains to readily develop tissue cysts, is discussed below.

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

[0231] The glycosylated cyst wall, to which the lectin Lablab bean agglutinin (DBA) binds, is a key structural feature that facilitates T. gondii persistence and oral transmission (Tomita et al., 2013). Here, we provided strong evidence of co-staining of BCLA and DBA exclusively in the membrane surrounding in vitro transformed bradyzoites (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, given that in vitro bradyzoite development in tissue culture does not result in fully mature cysts, we reexamined the localization of BCLA in bradyzoite-containing cysts isolated from mice chronically infected with T. gondii type II strains. In chronically infected mice, autoantibodies raised against BCLA stained the cyst wall as well as the matrix space surrounding the bradyzoites (data not shown). Although immunofluorescence results did not allow us to unequivocally determine whether BCLA was located in the inner or outer layer of the cyst wall, interestingly, non-permeabilized ex vivo cysts were readily stained by the antibody, suggesting the protein's external location (data not shown) and, therefore, its exposure to the host cell cytoplasm. No signal was detected in cysts hosting Δbcla bradyzoites (data not shown), thus confirming the specificity of the anti-BCLA antibody in vivo (Fig. 4a).

[0233] There is little evidence for extravacuolar functions of BCLA, but occasionally the protein appears to be transported across the vacuolar membrane into the host cell cytoplasm (data not shown). Unfortunately, despite numerous attempts, we did not find any ad hoc conditions underlying BCLA export beyond the PVM to further examine its function in host cells in more detail, as we have done with other effectors (Hakimi et al., 2017). Nevertheless, we were able to show that BCLA export was Myr1-independent (data not shown) and, as such, did not require the T. gondii translocon for exported proteins (Franco et al., 2016). An elegant way to explain the protein's accumulation in the cytosol of infected cells would be its release after possible processing at the PVM, likely under the control of host proteases, but this has not yet been demonstrated. Analysis of the BCLA-associated proteome of infected, FR235222-stimulated host cells will be performed to determine if, in any case, BCLA interactions with host cell proteins (including proteases) occur on the outward-facing side of the PVM or even in the cytoplasm of infected cells when BCLA is delivered there.

[0234] BCLA is not required for proper cyst function in vivo.

[0235] To determine the function of BCLA in bradyzoite tissue cysts, we generated two parasite strains in which the BCLA coding region was deleted (PruΔku80Δbcla) or disrupted by a DHFR cassette using Cas9-mediated gene editing (76K-GFP-LucΔbcla) (Table 2). We then examined pathogenesis and cyst formation. First, BCLA-deficient strains did not exhibit a clear growth phenotype compared to their parental strains in vitro under tachyzoite conditions (Figure 4a and data not shown). BCLA mutations did not impair the expression or localization of PV-resident or PVM-associated proteins previously identified as involved in PV formation and maturation (i.e., GRA1, GRA5, and 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 that positively labeled with lectin DBA following FR235222 stimulation (data not shown).

[0236] BCLA is not essential for initiating in vivo infection with tachyzoites.

[0237] To examine the importance of BCLA in vivo during acute infection, we compared the course of infection in BALB / c or NMRI mice infected intraperitoneally (ip) with either WT or BCLA-deficient parasites from a type II background, using an inoculum of 1 × 10 4 From 1×10 6The range of tachyzoites was 100%. Between days 5 and 8 postinfection, all mice infected with type II BCLA-deficient tachyzoites began to show signs of infection (i.e., weight loss and ruffled coat) and survived infection within the same time frame as the parental strain 76K, regardless of the inoculum and genetic background of the mice (Fig. 5a). Thus, BCLA appears dispensable for in vivo proliferation and pathogenesis during the acute phase of infection in mice. Animals that survived challenge were subsequently tested for serum responses to parasite antigens by Western blot at 10 weeks postinfection (data not shown). Clearly, BCLA deletion does not impair infectivity, as all mice expressed IgG against T. gondii with the same pattern, regardless of the parasite strain (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 could still occur in the mutant context (Figure 6b). However, the BCLA-deficient mutant produced significantly reduced parasite burdens in the CNS of chronically infected mice compared with the parental strain, although the difference did not reach statistical significance (Figure 6b and data not shown), demonstrating that BCLA is not dispensable, at least for establishing and maintaining cysts during chronic infection. Thorough examination of cysts, however, revealed that those isolated from mice infected with the mutant parasite were relatively smaller (Figure 6a) and contained fewer bradyzoites, resulting in a "lower packing density" (Watts et al., 2015) and an overall reduction in GFP fluorescence (Figure 6b), which is quite consistent with the slight reduction in parasite burden measured in the whole brain (Figure 6b). Beyond these quantitative indicators, Δbcla -containing cysts were uniquely typified by significant deformation of their cyst wall surface leading to loss of circularity and, to some extent, by distinctive “budding” and “segmentation or fissure” phenotypes ( Fig. 6 a and data not shown), revealing a possible role for BCLA in cyst growth, maintenance, and / or stability.

[0240] We next assessed whether surface deformation could render the cysts fragile, a phenotype previously reported for brain Δcst1-containing cysts (Tomita et al., 2013). While we subjected cysts to mechanical stress during their isolation, releasing them from brain tissue, and purified them by isopycnic centrifugation (see Methods), we did not observe that Δbcla-containing cysts were more fragile than WT cysts during this harsh procedure (data not shown), although a small number of them broke apart, regardless of genetic background.

[0241] Deletion of BCLA did not impair wall staining with Lablab bean lectin (DBA) in cysts isolated from the brains of chronically infected mice (data not shown). Therefore, and as previously concluded with FR235222-treated tachyzoites, BCLA is not directly involved in GalNAc glycosylation of the cyst wall. The survival of bradyzoites within cysts is conditioned by the permeability of the wall to nutrients coming from the host cell, but the latter is very limited, and the wall functions as a sieve to avoid components of the immune response. To test whether wall permeability was altered in some way in the absence of BCLA, we monitored the penetration of fluorophores, typically represented by different sizes ranging from 3 to 40 kDa, into cysts. Only intact cysts (without parasite leakage) were visualized and examined under a microscope. Permeability was very similar between WT and BCLA-deficient cysts using either a 3 kDa (diffuse pattern throughout the cyst matrix) or a 10 kDa (diffuse pattern with cross-sectional areas) dye. Interestingly, fluorescent tracers with higher molecular weights (40 kDa) failed to efficiently penetrate the cyst wall, as previously reported (Lemgruber et al., 2011). Furthermore, weak labeling even differed between strains. This is likely because Δbcla-containing cysts were "looser" and more permeable than cysts containing the parental strain, and were less permeable and more filled with bradyzoites surrounded by a well-defined, continuous cyst wall (data not shown). Overall, our results indicate that BCLA is dispensable for proper cyst function in vivo, but that the protein has a structural role in the cyst wall, which may lead to the cyst wall permeability-deficient phenotype.

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

[0243] To verify the in vivo functional consequences of BCLA-dependent cyst transformation, we fed mice with cysts containing either Δbcla or the parental strain and evaluated virulence and infectivity in two different mouse genetic backgrounds. C57BL / 6 mice were orally infected with 46 cysts of the 76k-GFP-luc-Δbcla or 76k-GFP-WT strains to examine the kinetics of parasite invasion and dissemination in the intestine, as well as the local immune response elicited by the parasite. On day 8 postinfection, the levels of T. gondii-specific IgG in mouse serum were quite similar (data not shown), and there was no significant difference in the parasite burden in the ileum (Figure 7a). Histological analysis of the ileum showed a global loss of intestinal epithelial structure with altered crypt-villus morphology (data not shown), accompanied by inflammatory areas (data not shown), regardless of the strain genetic background. The cytokine profile showed the same pattern, with a clear increase in proinflammatory cytokines (IFNγ) and chemokine (CCL2) in the ileum, but in a BCLA-independent manner (Fig. 7b). We next orally infected NMRI mice with 20 cysts and evaluated the ability of Δbcla cysts to disseminate into the bloodstream and form new cysts in deep tissues. All orally infected mice showed signs of illness (loss in body weight) throughout the acute phase of infection and seroconverted (data not shown). After 10 weeks, no significant differences in cyst numbers or parasite burdens were detected between strains for all mice (Fig. 7c). These data indicate that BCLA-deficient cysts can transmit infection by the oral route and cause chronic infection in mice, typified by a mild inflammatory state. Profiling of proinflammatory cytokines in the brains of chronically infected NMRI mice suggested that inflammation was less severe in Δbcla than in wild-type mice, although statistical significance was not achieved, likely due to the low sample size (three mice for each condition; Fig. 7d). This relatively mild inflammation in the brain could be the result of the relatively low number of cysts in Δbcla-infected mice, but this remains to be determined.

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

[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 parasite clearance during acute infection and mediate resistance to secondary Toxoplasma infection (Sayles et al., 2000). As such, once immunity is established, IgG protects the fetus from vertical transmission during pregnancy. Although the serological distinction between acute and chronic infection has clinical and epidemiological relevance, there are currently no bradyzoite-specific serum assays for toxoplasmosis to accurately estimate the time of infection and the presence of cysts. Furthermore, because relapses can occur in both fully immunocompetent (e.g., retinochoroiditis) and immunocompromised patients, and the presence of cysts in the brain has recently been suspected to be associated with some neuropsychiatric disorders, detecting Toxoplasma antibodies directed against semi-dormant cysts could be a significant improvement to the serodiagnosis of toxoplasmosis by opening new diagnostic perspectives. However, few components of the cyst wall or surface bradyzoites have been identified, and none have been shown to serve as antigens for serological purposes, at least in commercially available kits. An ideal antigen should be expressed exclusively in the latent bradyzoite stage and ideally exposed on the surface of the cyst, two features found in BCLA that motivated us to test its antigenicity.

[0246] To obtain the highly purified and abundant amounts of BCLA required for serum WB testing, we chose to recombinantly express the C-terminal domain end of BCLA (res 1100–1275, hereafter referred to as rBCLA), which is predicted to be structured, in contrast to the remainder of the protein, which contains a 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 naturally insoluble or sequestered in insoluble inclusion bodies; however, it can be solubilized using 0.2% N-lauryl sarcoside during the lysis step. 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 is observed to migrate between molecular weight markers of 17 and 25 kDa on SDS-PAGE gels. Furthermore, in a pH 8 buffer, BCLA is strongly negatively charged. E. coli contaminants can therefore be efficiently removed using anion exchange chromatography (data not shown). Finally, rBCLA elutes in a soluble form from size-exclusion chromatography (data not shown). However, it is polydisperse due to the wide elution volume range and forms multiple oligomers because the elution volume is close to the void volume of the S75 column. When pooling eluted fractions, a final step of ammonium sulfate precipitation and dialysis is performed to remove nucleic acid contaminants (data not shown). After this final step of purification, tachyzoite antigens of both the RH strain (LD bio) and rBCLA were separated by SDS-PAGE and then probed by immunoblot with mouse antisera elicited by different states of toxoplasmosis, allowing parallel analysis of antigen recognition by immunoglobulins G, M, and A.

[0247] rBCLA does not react with sera from acutely infected mice, but constitutes an excellent antigen for the detection of anti- T. gondii IgG from chronically infected mice.

[0248] We first performed immunoblotting with sera collected from mice during the acute phase of infection. While rBCLA protein apparently did not react with sera from mice acutely infected with atypical (COUG, haplotype 11), virulent (RH, type I), or cyst-forming (76K, type II) strains (Fig. 8a–c), all T. gondii-exposed mice seroconverted, regardless of genetic background (NMRI, CBA, C57BL / 6) or route of infection (intraperitoneal or oral) (Fig. 8a–c and data not shown). However, rBCLA reacted strongly with T. gondii IgG antibodies from mice that developed latent toxoplasmosis following infection with type II cyst-forming strains (Pru, ME49, or 76K) (Fig. 9a–c). rBCLA was detected by sera from mice at the subchronic (>21 days, Figure 9d) or chronic (>42 days, Figures 9a–c) stages of infection, with a very strong signal in sera from mice persistently infected for 22 months (Figure 9c). Reactivity was not detected when sera from uninfected or chronically infected mice with the BCLA-deficient strain were assayed, demonstrating that IgG antibodies are specifically directed against BCLA in vivo (Figure 9d). Because a selection process occurs during antibody affinity maturation (Eisen, 2014), we reasoned that rBCLA antigen could be detected by IgM during acute infection compared with IgG during chronic infection. Notably, rBCLA did not react with anti-T. gondii IgM or IgA (data not shown). These findings therefore strongly support that rBCLA was able to distinguish between parasite stages infecting mice, with preferential IgG reactivity for latent infection.

[0249] rBCLA is detected exclusively in the serum of mice persistently infected with the cyst-forming strain.

[0250] rBCLA was shown to have specific reactivity with encysting strains prone to latent infection (Fig. 9a–d). To sustain the argument, however, it would be necessary to demonstrate that nonencysting strains are unable to generate specific antibody responses directed against rBCLA. Initially, serum analysis of animals infected with the nonencysting virulent strain RH and treated continuously with pyrimethamine or sulfadiazine to overcome acute toxoplasmosis revealed concentrated levels of antitachyzoite-specific antibodies (22 days postinfection; Fig. 9e, lower panel), whereas rBCLA was barely detectable (Fig. 9e, upper panel). Because we could not exclude that treatment altered the dissemination of parasites in deep tissues and, consequently, their differentiation into bradyzoites, we monitored the IgG response to rBCLA in mice persistently infected with CTG, a type III strain that causes a nonlethal, chronic latent infection characterized by appropriate positive sera (Fig. 9f, right panel). At 42 days postinoculation, no reactivity to rBCLA was observed (Figure 9f). A major difference from type II infection (Figure 9a–d) was that mice chronically infected with CTG had fewer (if not more) cysts in their brains (Cannella et al., 2014), suggesting a likely relationship between cyst burden and rBCLA antibody levels. Similarly, sera from mice persistently infected with type II (PruΔku80) strains, which typically produce fewer cysts, did not react with rBCLA (Figure 9g), indicating that mouse antibody responses to rBCLA antigens occur soon after subchronic infection (>21 days p.i.) and appear to be conditioned by the presence of cysts, at least in this mouse model. Immunosuppressive treatment (corticoids), which produces relapses of latent type II infection, did not enhance antibody responses to rBCLA, ruling out the hypothesis of an immune response in response to the release of bradyzoites into the circulation (Figure 9h).

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

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

[0253] rBCLA also reacts with human sera, however the pattern of positivity is still under investigation.

[0254] We next demonstrated that mice infected with positive amniotic fluid from pregnant women who were primarily infected during pregnancy with evidence of congenital toxoplasmosis clearly reacted with rBCLA, in contrast to mice infected with qPCR-negative amniotic fluid or placenta (Fig. 11a). rBCLA is therefore a suitable serum marker for predicting the cystogenic properties of clinical isolates. Following the evaluation of anti-rBCLA immunoglobulin detection in mouse models, we aimed to evaluate the pattern 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 highly suspected or proven ocular toxoplasmosis (Fig. 11b). These clinical cases were due to recurrence of T. gondii cysts in the retina, but were not primary infections, as IgM was not detected. Similarly, three patients with toxoplasmosis flare-ups due to immunosuppression associated with hematological disease also had anti-rBCLA IgG; however, labeling in Western blots was weaker than those with ocular toxoplasmosis, although antibody levels were significantly higher using Vidas® and Architect® (Table 3). Even if broad generalization is limited by the relatively small sample size, the reactivity of rBCLA to human sera from toxoplasmosis flare-ups provides further evidence for our mouse model, in which we correlated the presence of rBCLA as a serum marker with cyst burden. Unexpectedly, three sera from pregnant women with recent seroconversion and one serum from a child with congenital toxoplasmosis also reacted to rBCLA (Figure 11b). Although this is difficult to prove, it is possible that recent primary infection produces T. gondii cysts in peripheral tissues, which in turn elicit a humoral anti-BCLA immune response. In any case, rBCLA was not detected in all sera of patients identified as seronegative for T. gondii, indicating good specificity of this antigen for patients with toxoplasmosis (Fig. 11b). [Table 3] [Table 4] TIFF2026034816000009.tif181165 Manufacturer-recommended cutoffs for interpretation of serological values ​​using Vidas® and Architect® Vida® IgG (IU / mL): Negative <4; Gray zone: 4.0≦x<8.0; Positive: ≧8.0 Vida® IgM (index): 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 (index): negative <0.50; gray zone: 0.50≦x<0.60; positive: ≧0.60

[0255] Epitope mapping of rBCLA-positive patients reveals consistent reactivity within multiple antigenic and repeat regions within rBCLA.

[0256] The specific immunogenic qualities of rBCLA have been demonstrated by Western blot analysis of a series of sera from different clinical categories. One of the main objectives was to develop an ELISA-based assay to screen larger serum cohorts in a cost-effective, reliable, and rapid manner. However, to accurately configure 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 15-aa peptides with a 3-aa gap step between peptides. We then tested several sera that were clearly positive by Western blot analysis against rBCLA and took an equal number of negative sera, proportionally subtracting nonspecific reactivity. When analyzed, the total reactivity scores for each peptide obtained for both the repeat region and rBCLA (Figure 12b) provide two important observations: First, although rBCLA has several zones of stronger reactivity, particularly near peptides 13, 22, 30, and 43, it possesses numerous epitopes throughout the domain, and different sera may react quite differently to different zones (Figure 12c). This emphasizes the requirement for maintaining rBCLA as a recombinant protein within ELISA testing. Furthermore, because the rBCLA domain is predicted to be structured, structured epitopes can only be provided by a recombinant protein strategy, further highlighting its use. Second, the repeat motif was found to consistently react in two separate zones (peptides 3 to 7 as motif A and 13 to 16 as motif B) in nearly all tested human sera. This feature emphasizes the importance of including one or more peptides covering these motifs to obtain a more sensitive ELISA technique. Based on these results, we therefore designed an ELISA combining the full-length BCLA recombinant protein and a chemically synthesized repeat motif.

[0257] ELISA titrations using rBCLA and repeat peptides demonstrate that BCLA seropositivity is higher in acutely and chronically infected individuals.

[0258] In establishing strict rules for classifying and distinguishing different clinical profiles, 123 sera (all taken from different individuals) were tested using the developed BCLA-ELISA test. The ELISA scores, expressed in International Units (UI), are presented according to the patient's clinical profile, which are listed as follows (Figure 13): 1) "Seronegative", regrouping all patients (healthy or with other pre-existing conditions) with SAG1 IgG / IgM negative sera. 2) "Pre-immune", regrouping all patients (healthy or with other conditions) classified as SAG1-positive IgG but without SAG1-reactive IgM and not falling into the following three categories: 3) "Active toxoplasmosis in immunocompromised patients", regrouping all SAG1 IgG-positive and immunocompromised patients with proven symptomatic toxoplasmosis (regrouping disseminated, cerebral and primary toxoplasmosis). 4) "Asymptomatic serologic flare-up in immunocompromised patients," regrouping all immunocompromised patients who experience a serologic flare-up but without visible symptoms. 5) "Ocular toxoplasmosis," regrouping patients with proven ocular toxoplasmosis with SAG1-positive serology.

[0259] Several observations can be made from this analysis. First, all groups exhibited a significant increase in median BCLA titers and much higher positivity rates compared with the seronegative group. This demonstrates a direct correlation between SAG1 seropositivity and the ability to develop a BCLA-positive state in humans. This also demonstrates the current discrepancy 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, strongly immunogenic exogenous bacterial contaminants copurified with rBCLA, and potentially true BCLA-positive patients with negative SAG1 sera. The second major observation is that some clinical profiles have a tendency to generate much stronger immunogenic responses, most notably the "asymptomatic serological relapse in immunocompromised patients" group, where BCLA sera titrated well above the median value of positive BCLA sera in the "previously immunized" group. A final observation is that for some groups where BCLA positivity should always be expected, e.g., in the cases of "active toxoplasmosis in immunocompromised patients" and "ocular toxoplasmosis," a small number of sera remain negative or are below the positivity cutoff. This observation may highlight the lack of sensitivity from the ELISA test or potentially illustrate the fact that BCLA sera can become negative during immunosuppression.

[0260] The ELISA test is also consistent in relating positive BCLA sera to proportional cyst burden in mice.

[0261] Overall, semiquantitative analysis of anti-rBCLA antibody titers identified BALB / c and NMRI mice likely bearing WT cysts as highly responsive to BCLA, with increasing titers over time (Figure 14A). In combination with quantitative PCR on brain-associated T. gondii 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 of rBCLA as a reliable antigen for serologically detecting T. gondii bradyzoite-laden cysts over long-term protozoan persistence in rodents (Figure 14B). These results are particularly interesting because the semiquantitative nature of the ELISA test significantly differentiates responses of cyst-forming T. gondii strains over time.

[0262] Consideration

[0263] Infection with Toxoplasma gondii leads to an acute systemic phase in which zoites rapidly colonize and further complete their developmental program as bradyzoites, encapsulated in thick-walled cysts that persist in the brain, heart, and skeletal muscle (Jeffers et al., 2018). The host immune response can rapidly control the proliferation of tachyzoite populations, leading to lifelong immunity typified by seroconversion. However, because the developmental transition from tachyzoites to bradyzoites is fully bidirectional, any impairment of immune function (e.g., AIDS patients, hematological disorders, and immunosuppressive treatments) can result in reactivation of latent infection, which can cause encephalitis and focal brain lesions, pulmonary disease, or disseminated disease.

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

[0265] While there are currently no reliable bradyzoite-specific serum assays for toxoplasmosis to estimate the source of infection worldwide or accurately distinguish acute from latent infection, progress is being made. Indeed, recent proteomic studies have shed light on the repertoire of sporozoite-specific proteins (Fritz et al., 2012; Possenti et al., 2013), revealing CCp5A as a serum marker capable of distinguishing parasite stages infecting chickens, pigs, and mice, with specific reactivity for oocyst-infected animals (Santana et al., 2015).

[0266] However, despite early studies reporting that specific 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. gondii 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 the specific proteome of the latent stage. 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, several hundred bradyzoite-restricted proteins were identified, including BCLA.

[0267] The protein BCLA has been shown to be dispensable for initiating or maintaining latent infection. However, BCLA deficiency results in a highly distinctive phenotype, typified by the deformation and loss of circularity of brain cysts in mouse models. Previously, two cyst wall-associated proteins, namely BPK1 and CST1, were implicated in the structural integrity of T. gondii cysts (Jeffers et al., 2018). In Δbpk1 strains, cysts are smaller and more sensitive to pepsin acid treatment, and unlike BCLA, Δbpk1 strains have a reduced ability to cause oral infection (Buchholz et al., 2013). CST1 is involved in the binding of Lablab bean agglutinin (DBA) lectin, characteristic of T. gondii cysts. CST1 deletion results in a fragile brain cyst phenotype characterized by reduced cyst numbers and thinning and destruction of the basal region of the cyst wall (Tomita et al., 2013). Defective glycosylation may also explain the deformed Δbcla cysts. Indeed, we have preliminary interactome data showing BCLA copurified with Jacaline-binding protein (Jacaline), a lectin that binds to GalNAcα1-Ser / Thr oligosaccharides covering the PVM surrounding bradyzoites (data not shown) (Tomita et al., 2017). Further studies are required to confirm whether this interaction is involved in the unique phenotype mediated by BCLA deficiency.

[0268] The lack of a clear BCLA-associated phenotype in mice, regardless of the route and time of infection, led us to focus our research on the propensity of BCLA to play an immunogenic role. We therefore reached another milestone by producing rBCLA as a recombinant protein with a high degree of purity, which would provide an opportunity to standardize serum testing and reduce production costs to a certain extent if this antigen proves to be interesting for serology. Indeed, we provided strong data confirming that rBCLA is antigenic and constitutes an excellent antigen candidate for the detection of anti-T. gondii IgG in chronically infected mice. Remarkably, we clearly correlated the strong detection of the antigen rBCLA in serum with the cyst burden in the brains of all mice latently infected with a type II cyst-forming strain. Similar studies have correlated MAG1 antibody levels with brain cyst burden, however, it has been argued that their experimental setup was somewhat biased by the use of an unrelated model of chronic type 1 (GT1) infection, which requires anti-T. gondii chemotherapy to control tachyzoite proliferation during the acute phase and avoid animal death (Xiao et al., 2016).

[0269] Notably, rBCLA did not react with IgM or IgA (markers frequently associated with acute infection) (data not shown), but reacted only with IgG, and exclusively in subchronic infection. This result contrasts sharply with the observation that mice fed tissue cysts showed significant IgM responses at day 10 (Doskaya et al., 2018), reinforcing the idea of ​​a humoral response to BCLA during the latent phase of infection. Similarly, mice inoculated by oral gavage with tissue cysts did not produce antibodies directed against BCLA during acute infection (Figure 8c), indicating that the host immune response to BCLA was not due to initial exposure to bradyzoites and cyst proteins released from ingested parasites in the gastrointestinal tract during primary infection. This contrasts sharply with the humoral responses to BAG1 and MAG1, which occur 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 and contrast with the notion that T. gondii cysts are found primarily in immune-privileged sites, such as the brain and skeletal muscle. Although elucidating the contribution of the humoral immune response during chronic toxoplasmosis may require further studies in mice, BCLA likely represents an excellent tool for studying these processes.

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

[0271] Example 2 (VHH production)

[0272] immunization

[0273] Llamas SEL005 and SEL006 were immunized via Eurogentec via 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 a bleed on day 43.

[0274] immune response

[0275] The immune response of SEL005 and SEL006 was tested by assessing 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% milk powder in PBS (MPBS). Serial dilutions of serum in 1% MPBS were then added to the wells and incubated for 1 hour. Unbound antibodies were removed during washes with PBS-Tween. Bound antibodies were then detected with rabbit anti-VHH (clone K1216) and donkey anti-rabbit conjugated to HRP. Antibody binding was quantified by the colorimetric reaction of O-phenylenediamine (OPD) in the presence of H2O2 at 490 nm. Llama SEL005 and SEL006 show excellent responses to His rBCLA.

[0276] SEL005, day 43 and SEL006, day 43 library construction.

[0277] RNA isolation and cDNA synthesis

[0278] Peripheral blood lymphocytes were isolated from a large blood draw 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 the dissolved RNA was analyzed on a gel. Figure 2A shows that intact 28S and 18S rRNA were clearly visible, indicating adequate RNA integrity.

[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 with a Macherey Nagel PCR cleanup column. The variable domains of the heavy chain (both conventional and heavy chain only) fragments were amplified using primers annealing in the leader sequence region and in 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, and a 700 bp fragment was excised and purified from the gel. A total of 80 ng of the isolated PCR product was used as a template for a nested PCR (final volume 800 μl) to introduce SfiI and Eco91I restriction sites at either end of the VHH gene. The amplified VHH fragment was washed on a Macherey Nagel PCR wash column and eluted in 120 μl. The eluted DNA was first digested with SfiI followed by Eco91I. As a restriction digestion control, 4 μl of this mixture was loaded onto a 1.5% TBE agarose gel.

[0281] After restriction 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 the pUR8100 phagemid vector (~1 μg) and transformed into TG1 E. coli.

[0282] Library Size

[0283] The transformed TG1 was titrated using 10-fold dilutions. 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 spotted dilutions of the transformed TG1 culture (keeping in mind that the final volume of transformation was 8 ml). The total number of transformants, and therefore the library size, was calculated by counting the colonies in the highest dilution and using the following formula:

[0284] Library size = (amount of colonies) * (dilution) * 8 (ml) / 0.005 (ml; spot volume)

[0285] The VHH insertion frequency of the phagemid vector was determined by picking 24 different clones and performing colony PCR. A band of ~700 bp indicates a successfully cloned VHH fragment. A band of ~300 bp indicates an empty plasmid. For library SEL005, day 43, the insertion frequency is 100%. For library SEL006, day 43, the insertion frequency is nearly 95% (Figure 4), which is sufficient to continue phage panning selection.

[0286] Phage production and selection.

[0287] Phage were produced from the libraries as outlined below: E. coli TG1 containing libraries SEL005, day 43, and SEL006, day 43, were diluted from a glycerol stock to an OD of 0.05 in 2xYT medium containing 2% glucose and 100 μg / ml ampicillin. The number of bacteria in this inoculum was at least 10x the library size (>10 bacteria in the inoculum). This culture was grown at 37°C for 2 hours to reach an OD of ~0.5. Approximately 7 ml of the culture was then left stationary at 37°C for 30 minutes and infected with helper phage VCS M13 using an MOI (multiplicity of infection) of 100. The infected bacteria were spun down and resuspended in 50 ml of fresh 2xYT medium supplemented with both ampicillin (100 μg / ml for phagemids) and kanamycin (25 μg / ml for M13 phage), and grown overnight at 37°C with shaking. The phage produced was precipitated from the culture supernatant using PEG-NaCl precipitation. The titers of the phage produced were calculated by serial dilution of the phage and infection of E. coli TG1. The titers of the phage produced were 3 x 10 / ml for SEL005, day 43, and 6 x 10 / ml for SEL006, day 43, respectively, which were sufficient to continue with selection.

[0288] For the first round of panning / selection, 20 μl of precipitated phage (∼10 phage, >100-fold the library diversity) was 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, freshly precipitated phage was preblocked with 2% MPBS for 30 min. The preblocked phage was incubated with directly coated His rBCLA for 2 h. Upon extensive washing with PBS-Tween and PBS, bound phage 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, which 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 generated from the rescue output from the selection with 5 μg / ml His rBCLA (the highest concentration). The rescue output, grown overnight, was diluted 100-fold in 5 ml of fresh 2xYT medium supplemented with 2% glucose and 100 μg / ml ampicillin and grown to logarithmic phase for 2 hours. 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. Produced phages were precipitated from the culture supernatant using PEG-NaCl precipitation.

[0290] Then, for the second round of panning / selection, 1 μl of precipitated phage was applied to wells coated with His rBCLA as follows: antigen was coated overnight on a MaxiSorp plate 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, freshly precipitated phage was preblocked for 30 minutes in 2% MPBS as described above. The preblocked phage was incubated with directly coated His rBCLA for 2 hours. Upon extensive washing with PBS-Tween and PBS, bound phage were 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, which were spotted onto LB agar plates supplemented with 2% glucose and 100 μg / ml ampicillin and incubated overnight at 37°C.

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

[0292] Rescue outputs of the second round of selection on His rBCLA were plated out for single clone selection. For the master plate ERB-1, a total of 92 single clones were picked into 96-well plates.

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

[0294] Sequence analysis of His rBCLA-binding VHHs

[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 chosen based on binding in ELISA and should represent the majority of clones selected from different production runs.

[0296] Cloning and production of His rBCLA-selected VHHs.

[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 VHH sequences found. These VHHs were then subcloned from the phagemid vector into the expression vector pMEK222 using SfiI and Eco91I restriction enzymes. Recloning into pMEK222 also adds FLAG and His tags to the C-terminus of the VHHs, allowing for detection and affinity purification. For production, precultures were prepared by growing bacteria containing plasmids with the selected VHHs overnight at 37°C in 8 ml of 2xYT medium supplemented with 2% glucose and 100 μg / ml ampicillin. This preculture was diluted into 800 ml of fresh 2xYT pre-warmed to 37°C and supplemented with 100 μg / ml ampicillin and 0.1% glucose. Bacteria were grown at 37°C for 2 hours before inducing VHH expression with 1 mM IPTG. The VHH was expressed at 37°C for 4 hours and bacteria were harvested by centrifugation. The bacterial pellet was resuspended in 30 ml of PBS and frozen at -20°C.

[0298] Purification and analysis of VHHs.

[0299] The frozen bacterial pellet was thawed at room temperature, and cell debris was spun down by centrifugation. VHHs were purified from the supernatant (soluble fraction) using affinity chromatography of the His tag on cobalt-charged Sepharose beads (immobilized metal affinity chromatography (IMAC) using TALON beads). Bound VHHs were 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 the different VHHs.

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

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

[0303] conclusion

[0304] Immunization of llamas SEL005 and SEL006 resulted in a good immune response. The resulting libraries were of good size and insert frequency. Phage display selection with His rBCLA yielded many good clones, three of which (ERB-1G6, ERB-1B11, and ERB-1A12) showed very good apparent affinity, with ERB-1G6 also showing high production levels in E. coli. [Table 5] TIFF2026034816000011.tif244165 TIFF2026034816000012.tif251165 TIFF2026034816000013.tif44165

[0305] Example 3:

[0306] In the current longitudinal study, we suggest that the detection of BCLA antibodies, if appropriately combined, could potentially improve the sensitivity of the current test. We have experience testing BCLA in the context of maternal-fetal 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: in one, congenital toxoplasmosis was confirmed through persistent Sag1 IgG titers in the child's serum long after birth, and in the other, congenital toxoplasmosis was excluded if the child's serum became negative for Sag1 over time (Lebech M et al., 1996).

[0307] At birth, infants in both groups share comparable titers without distinguishable profiles, as shown by comparative titration of Toxo IgG by Vidas® and Architect® (Figures 15C-D). This is explained by the fact that mothers transmit anti-Sag1 IgG across the placental barrier; 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 even clearer. The sera of the infants at birth exhibit much more reactive BCLA titers than the sera of their mothers or those of the excluded congenital toxoplasmosis group (Figures 15A-B).

[0308] This observation implies that children specifically neosynthesize anti-BCLA IgG before birth and indicates that strong BCLA reactivity can further guide the diagnosis of congenital toxoplasmosis at birth.

[0309] References:

[0310] Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. [Table 6] TIFF2026034816000015.tif248165 TIFF2026034816000016.tif235165 TIFF2026034816000017.tif199165

Claims

1. 1. An isolated polypeptide comprising: (i) an amino acid sequence consisting of the Toxoplasma gondii polypeptide BCLA (SEQ ID NO: 1); (ii) an amino acid sequence (SEQ ID NO: 2) consisting of the C-terminal antigen domain (res 1089-1275 of BCLA); (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 that is substantially homologous to a sequence of (i) to (iii), preferably an amino acid sequence that is at least 80% identical to a sequence of (i) to (iii); (v) a fragment of at least 9 consecutive amino acids of the sequence of (i) to (iv).

1. An isolated polypeptide selected from the group comprising, or consisting of:

2. 2. The isolated polypeptide of claim 1, which consists of a fusion between two peptide fragments of any of the sequences (i) to (v) of claim 1.

3. 3. The isolated polypeptide of claim 1 or 2, comprising: (i) MERPAAGSMEKEKPVLPGEGEGLPKHETKPALTDEKRTKPGGP (SEQ ID NO: 55); (ii) AAGSMEKDKLVLPGE (SEQ ID NO: 56); (iii) an amino acid sequence that is substantially homologous to a sequence of (i) to (ii), preferably an amino acid sequence that is at least 95% identical to a sequence of (i) to (ii); (iv) a fragment of at least 9 consecutive amino acids of the sequence of (i) to (iii). An isolated polypeptide selected from the group consisting of:

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

5. An antibody that specifically binds to the isolated peptide of any one of claims 1 to 4.

6. The antibody of claim 5, wherein the antibody is a single domain antibody.

7. A kit comprising the antibody of any one of claims 3 to 5.

8. 10. A method for detecting a polypeptide according to claim 1 and / or assessing its amount in a biological sample.

9. 9. The method of claim 8, wherein the method comprises contacting the sample with an antibody according to any one of claims 5 to 6.

10. 10. A method according to claim 8 or 9 for the in vitro diagnosis of latent forms of toxoplasmosis.

11. 10. A method for diagnosing toxoplasmosis in vitro, said method comprising detecting the presence of the polypeptide of claim 1 in a biological sample from a subject to be tested.

12. The method of claim 11 , wherein the sample is a tissue sample.

13. 1. An in vitro method for determining whether a subject is suffering from a latent form of toxoplasmosis, comprising: a) detecting immune reactivity against a polypeptide according to any one of claims 1 to 3 in a biological sample of said patient; and, optionally b) predicting from the results of step a) whether the patient is suffering from a latent form of toxoplasmosis and whether immune reactivity to a polypeptide described in any one of claims 1 to 3 indicates a latent form of toxoplasmosis.

14. 1. An in vitro method for diagnosing or confirming the diagnosis of latent toxoplasmosis in a patient suffering from or suspected of suffering from latent toxoplasmosis, comprising: a) obtaining a biological sample from said patient; and b) detecting in said biological sample an antibody against a T. gondii polypeptide according to any one of claims 1 to 3, wherein the presence of antibodies in said biological sample diagnoses or confirms the diagnosis of a latent form of toxoplasmosis in the patient.

15. 1. An in vitro method for diagnosing or confirming the diagnosis of congenital toxoplasmosis in a patient suffering from or suspected of suffering from a latent form of toxoplasmosis, comprising: a) obtaining a biological sample from said patient; and b) detecting in said biological sample antibodies against a T. gondii polypeptide according to any one of claims 1 to 3; wherein the presence of antibodies in said biological sample diagnoses or confirms the diagnosis of congenital toxoplasmosis in the patient.

16. 1. A method for detecting bradyzoite cysts and / or assessing their abundance in a subject, comprising: a) detecting in a body fluid sample of a subject immunoreactivity against a T. gondii polypeptide according to any one of claims 1 to 3; and, optionally, b) from the results of step a), inferring that the presence and / or amount of bradyzoite cysts, and that immune reactivity to a T. gondii polypeptide described in any one of claims 1 to 3, indicates the presence and / or amount of bradyzoite cysts in the subject.

17. 17. Any of the methods of claims 13 to 16, wherein the sample is a body fluid sample.

18. A method for treating a patient infected with a latent form of toxoplasmosis who exhibits immune reactivity to the T. gondii polypeptide of claim 1, 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.

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