Antigenic regions for detecting anti-toxoplasma gondii IgM antibodies

Abstract

The present invention provides compounds, methods for their preparation, methods for their use and compositions containing them, which are suitable for industrial applications in the fields of medicine and diagnosis / prognosis, in particular for the detection and diagnosis of Toxoplasma gondii infection, and for the treatment and prevention of said infection.

Description

Technical Field

[0001] The present invention provides compounds, methods for their preparation, methods for their use and compositions containing them, which are suitable for industrial applications in the fields of medicine and diagnosis / prognosis, in particular for the detection and diagnosis of Toxoplasma gondii infections, as well as for the treatment and prevention of said infections. Background Art

[0002] The diagnosis of acute toxoplasmosis is mainly based on serological tests for detecting anti-Toxoplasma gondii IgM antibodies. Commercially available kits are usually based on Toxoplasma gondii lysate antigens (TLA) from different sources (i.e. infected mice and / or tissue cultures). The use of Toxoplasma gondii lysate antigens has inherent limitations in terms of performance (sensitivity and specificity, difficult standardization and strong management). In recent years, purified recombinant antigens have become a promising alternative with proven diagnostic utility. However, several problems are associated with its use for serological diagnosis of Toxoplasma gondii infection, such as low inter-assay reproducibility, time-consuming and expensive products, and low immunoreactivity. Therefore, none of the assays based on recombinant antigens have all the features required to replace the Toxoplasma gondii lysate antigens for detecting IgM. Therefore, further work is needed to improve the diagnosis / prognosis of toxoplasmosis, and such improvements will have a direct and positive impact on the clinical management of infected patients. In this sense, in order to achieve such improvements, it is one of the main focuses of the present invention to find new epitopes that can help the clinical management of toxoplasmosis.

[0003] describe

[0004] Serological tests for detecting anti-Toxoplasma gondii IgM are the gold standard for diagnosing acute toxoplasmosis. However, most commercial kits are based on lysate antigens obtained from tachyzoites grown in mice or tissue culture, which have inherent limitations in terms of performance, which provide wrong results to physicians, thus hindering patient management and requiring strict management. In this case, peptide-based antigens have emerged as attractive alternatives to overcome several such problems. In this sense, in the present invention, we focus on designing and synthesizing Toxoplasma gondii IgM-specific chimeric peptides to develop immunoassays for specific detection of anti-Toxoplasma gondii IgM. In addition, we aim to evaluate mouse monoclonal humanized chimeric antibodies as an alternative source of human positive plasma-derived samples currently used to generate reference materials (such as calibrators and controls).

[0005] More specifically, and focusing on T. gondii IgM-specific chimeric peptides, which are used to develop immunoassays for detecting anti-T. gondii IgM. In the present invention, in order to identify new T. gondii epitopes that can be incorporated as diagnostic tools into immunoassays for specific detection of anti-T. gondii IgM, we designed a peptide microarray that displays all peptides covering different antigenic proteins from T. gondii. A complete list of the selected nine proteins identified by UniProt (https: / / www.uniproT.org / ) and the Immune Epitope Database (IEDB) (https: / / www.iedb.org / ) is shown in Table 1 below:

[0006] Table 1. List of nine proteins used to construct peptide microarrays. ID. Identification. aa: amino acid.

[0007]

[0008] From the information in the differential IgM response heat map provided in the examples of the present invention, the top peptide hits exhibited a G1-G2 difference of >400 units, mainly assigned to dense granule protein GRA3, rhabdomerin, granule antigen protein GRA7 and major surface antigen P30.

[0009] Based on the above microarray analysis results, we identified sequences with the best performance potential. We selected them based on the following:

[0010] i) IgM specificity based on the G1-G2 difference and G1 / G2 ratio of different IgM responses,

[0011] ii) homology to the Toxoplasma gondii B cell epitopes found in IEDB, and

[0012] iii) Peptide length.

[0013] Therefore, we designed peptides to include the region within the protein under study that was most specific for IgM but limited the peptide length to about 50-60 residues (this is an approximate threshold, after which the quality of synthetically produced peptides may be affected). 18 selected peptides were synthesized in C-terminal carboxamide form using Fmoc solid phase synthesis, purified to >95% homogeneity using HPLC (high performance liquid chromatography), and appropriately characterized using MS (mass spectrometry). (See Table 2 below).

[0014]

[0015] Table 2: Peptides used in the initial phase of this study. a Theoretical masses (Theo. Mass) were calculated using GPMAW software version 8.10. bThe net charge was calculated using the ExPASy ProtParam website (https: / / web.expasy.org / protparam / ). c Retention time (RT) is the time elapsed from injection to peak detection. d Analytical RP-HPLC was performed using solvent B (0.036% TFA in ACN) at a flow rate of 1 mL / min over 15 min into solvent A (0.045% TFA in H 2 O solution) and was performed with UV detection at 220 nm. e % acetonitrile (ACN) at peptide elution time. f % Purity was calculated using the peak area percentages of the chromatographic profile. g The control peptide (ctrl) does not belong to the T. gondii proteome. ID: Identifier.

[0016] Thus, the microarray study elucidated 18 different peptides that potentially reacted with IgM anti-T. gondii positive samples and did not react with anti-T. gondii IgG positive samples. Furthermore, the results obtained with the multiplex ELISA indicated that the best candidates were peptides 3a, 3b (GRA3) and 6c (ROP1).

[0017] Next, based on the two antigenic regions of GRA3 and ROP1, we evaluated the diagnostic utility of different chimeric peptides. With this in mind, we demonstrated that the combination of 3a and 6c peptides in solution enhanced the assay performance compared to individual peptides, and we designed and produced different synthetic chimeric peptides combining such peptides, and evaluated their performance in a single ELISA. In summary, the results demonstrated that the synthetic chimeric peptide 3a6c can be used in chemiluminescent immunoassays, which replaces the natural protein of the Toxoplasma gondii parasite, and is particularly useful for detecting IgM anti-Toxoplasma gondii in human serum. It is worth mentioning that an interesting feature of peptide 6c is that it is composed of a tandem repeat sequence of 8 residues (PPPNXQEL, where X can be any one of amino acids S or A). In view of the presence of those block tandem sequences in the ROP1 protein, we show in this article that these 8 residues are involved in recognizing IgM. On this basis, we designed and generated other chimeric peptides with more than one repeat of the 6c sequence minus the two N-terminal residues (hereafter referred to as truncated 6c*) as motifs for three different constructs, namely i) a homologous tandem with two consecutive stretches of the truncated peptide 6c (ID 6c*2), ii) a homologous tandem with two truncated 6c peptide sequences separated by a flexible spacer (8-amino-3,6-dioxaoctanoic acid (O2Oc)) (ID 6cO 36c†), and iii) a branched bivalent peptide based on a lysine core from which two truncated 6c peptides (ID(6c*)2K3) branched ( Fig.11 ). The results showed that, as shown in Table 20, all of these antigens provided an AUC greater than 0.80 and were therefore useful for detecting anti- Toxoplasma gondii IgM and for the diagnosis of acute toxoplasmosis (see Table 17).

[0018] Therefore, a first aspect of the present invention relates to an antigen, preferably a recombinant or synthetic antigen, comprising one or more amino acid sequences PPPNXQEL, wherein X can be any of the amino acids S or A, and wherein the antigen is capable of specifically binding to a Toxoplasma gondii-specific IgM antibody. Preferably, the Toxoplasma gondii-specific antibody is extracted from the serum of a subject who has been infected with Toxoplasma gondii.

[0019] In the context of the present invention, "capable of specifically binding to" means that in a mixture of antibodies of various classes, the antigen of the present invention, preferably a recombinant or synthetic antigen, preferentially binds to antibodies of the IgM class, wherein relative to its binding to antibodies of other classes such as IgG antibodies, at least about 50% of the peptides bind to IgM antibodies, more preferably about 70%, more preferably about 80% to 85%, even more preferably about 90%, and most preferably about 95% or more of the peptides bind to IgM antibodies. The terms "selective binding" and "selective binding" mean that in a mixture of antibodies of various classes and other serum or cell proteins and products, the antigen of the present invention, preferably a recombinant or synthetic antigen, preferentially binds to antibodies of the IgM class, wherein relative to its binding to antibodies of other classes, at least about 50% of the antigens bind to IgM antibodies, more preferably about 70%, more preferably about 80% to 85%, even more preferably about 90%, and most preferably about 95% or more of the peptides bind to IgM antibodies.

[0020] In the context of the present invention, the term "linker or spacer" is understood as any molecule that can be added in a linear or branched structure to separate different protein domains. Non-limiting spacers are amino acids, peptides, polyethylene glycol, etc.

[0021] As used herein, "Toxoplasma gondii (T. gondii)" is understood to be an intracellular parasitic protozoan that infects most warm-blooded animal species and causes the disease toxoplasmosis. Toxoplasma gondii can only undergo an entire reproductive cycle in domestic and wild cats, which are the primary hosts of the parasite.

[0022] As used herein, "toxoplasmosis" is understood to be a parasitic disease caused by Toxoplasma gondii. Humans can become infected by any of several routes: eating undercooked animal meat with tissue cysts; eating food or water contaminated by cats or contaminated environmental samples (such as soil contaminated by feces or changing the litter box of a pet cat); through blood transfusion or organ transplantation; or through the placenta from mother to fetus.

[0023] As used herein, "acute toxoplasmosis" is understood to be the body's response to an initial infection with Toxoplasma gondii. Acquired infection with Toxoplasma gondii in immunocompetent humans is usually an asymptomatic infection. However, 10% to 20% of acutely infected patients may develop cervical lymphadenopathy and / or an influenza-like illness. Congenital toxoplasmosis is caused by an acute primary infection acquired by the mother during pregnancy.

[0024] As used herein, a "chimeric peptide" is understood to be a peptide that has been created by joining two or more protein domains that originally encoded separate proteins. The different domains may be separated by a linker or spacer.

[0025] More specifically, the present invention encompasses any antigen or poly (amino acid) sequence comprising one, two or more amino acid sequences consisting of PPPNXQEL, preferably three or more, more preferably four or more, which are capable of specifically binding to Toxoplasma gondii-specific IgM antibodies. It should be noted that the tandem sequences of one or more PPPNXQEL present in the antigen or poly (amino acid) sequence can be optionally separated by a linker sequence within the antigen or poly (amino acid) sequence. More specifically, the present invention covers any antigen or poly (amino acid) sequence comprising, consisting essentially of or consisting of one, two or more amino acid sequences consisting of PPPNXQEL or those defined in Table 17, in particular any antigen or poly (amino acid) sequence comprising, consisting essentially of or consisting of SEQ ID NO 1 (antigen 6c: EVPPPNAQELPPPNSQELPPPNSQELP), SEQ ID NO 2 (6c*: PPPNAQELPPPNSQELPPPNSQELP), SEQ ID NO 3 (6c*2: PPPNAQELPPPNSQELPPPNSQELPPPNAQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 4 (6c*3: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 5 (6c*4: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 6c*5: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP, and / or SEQ ID NO 7 (6c*8: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 8 (6c*9: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 9 (6c*10: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 11 (6c*12: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 13 (6c*14: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 14 (6c*15: PPPNAQELPPPNSQELPPPNSQELPPPNSQELP), and / or SEQ ID NO 15 36c: PPPNAQELPPPNSQELPPPNSQELOOOPPPNAQELPPPNSQELPPPNSQELP, wherein OOO is a flexible spacer (8-amino-3,6-dioxaoctanoic acid), and wherein these sequences are capable of specific binding to Toxoplasma gondii-specific IgM antibodies. It should be noted that the antigen or poly (amino acid) sequence of the present invention may be a peptide or a protein, wherein if the antigen is a peptide, the length of the peptide is preferably between 8 and 150 amino acids (including the upper and lower limits of this range), preferably a maximum length of 75, 100 or 150 amino acids, and if the antigen is a protein, the antigen should preferably have a length of between 75 and 700 amino acids, preferably between 100 and 700 amino acids, more preferably between 150 and 700 amino acids (including the upper and lower limits of this range), preferably a maximum length of 700 amino acids. Preferably, the present invention covers any antigen or poly (amino acid) sequence comprising or consisting of any of the following sequences: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and / or SEQ ID NO 4, which is capable of specific binding to an IgM antibody specific for Toxoplasma gondii, wherein the antigen may be a peptide or protein as described above; or any antigen or poly (amino acid) sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity with any of the sequences SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and / or SEQ ID NO 4, which is capable of specific binding to an IgM antibody specific for Toxoplasma gondii.

[0026] For example, according to the first aspect, the antigen or poly(amino acid) sequence of the invention preferably comprises or consists of the amino acid sequence SEQ ID NO: 1 or the amino acid sequence SEQ ID NO: 2 or the amino acid sequence SEQ ID NO: 3 or the amino acid sequence SEQ ID NO: 4.

[0027] A second aspect of the invention relates to an antigen, preferably a recombinant antigen, or a poly (amino acid) sequence comprising or consisting of SEQ ID NO 5 (3a: FLVAAALGGLAADQPENHQALAEPVTGVGEAGVSPVNEAG) and / or SEQ ID NO 6 (3b: AADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSGVQ), which is capable of specific binding to an IgM antibody specific for Toxoplasma gondii; or any antigen or poly (amino acid) sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to any of the sequences SEQ ID NO 5 and / or SEQ ID NO 6, which is capable of specific binding to an IgM antibody specific for Toxoplasma gondii. Preferably, the Toxoplasma gondii specific antibody is extracted from the serum of a subject that has been infected with Toxoplasma gondii. Also in the context of the second aspect of the invention, it should be noted that the antigen of the invention may be a peptide or a protein, wherein if the antigen is a peptide, the peptide preferably has a length of between 8 and 150 amino acids (including the upper and lower limits of this range), preferably a maximum length of 75, 100 or 150 amino acids, and if the antigen is a protein, the antigen should preferably have a length of between 75 and 700 amino acids, preferably between 100 and 700 amino acids, more preferably between 150 and 700 amino acids (including the upper and lower limits of this range), preferably a maximum length of 700 amino acids.

[0028] The third aspect of the present invention relates to an antigen, in particular a chimeric antigen, preferably a chimeric recombinant antigen, or a poly (amino acid) sequence comprising at least two different antigenic regions, one of these regions being a sequence selected from the first aspect of the present invention and the other being a sequence selected from the second aspect of the present invention, wherein optionally these sequences are separated by a linker. Preferably, such an antigen or poly (amino acid) sequence comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (3a6c) and / or SEQ ID NO 8 (6c3a), which is capable of specifically binding to an IgM antibody specific for Toxoplasma gondii; or any antigen or poly (amino acid) sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity with any one of the sequences SEQ ID NO 7 and / or SEQ ID NO 8, which is capable of specifically binding to an IgM antibody specific for Toxoplasma gondii. Also in the context of the third aspect of the invention, it should be noted that the antigen of the invention may be a peptide or a protein, wherein if the antigen is a peptide, the peptide preferably has a length of between 8 and 150 amino acids (including the upper and lower limits of this range), preferably a maximum length of 75, 100 or 150 amino acids, and if the antigen is a protein, the antigen should preferably have a length of between 75 and 700 amino acids, preferably between 100 and 700 amino acids, more preferably between 150 and 700 amino acids (including the upper and lower limits of this range), preferably a maximum length of 700 amino acids.

[0029] The chimeric antigen of the third aspect of the present invention or the tandem sequence of the first aspect of the present invention can be engineered using known methods. Fusions can be direct (the C-terminal of an amino acid sequence is connected to the N-terminal of another amino acid sequence by a simple covalent bond), or they can adopt a flexible joint domain, or a polypeptide joint consisting of small amino acids such as glycine, serine, threonine or alanine under various lengths and combinations. For example, a joint can be a polyglycine repeat sequence interrupted by serine or threonine at certain intervals.

[0030] In addition, the chimeric antigen or poly (amino acid) sequence of the third aspect of the invention, the antigen or poly (amino acid) sequence as defined in the first aspect of the invention or the antigen or poly (amino acid) sequence as defined in the second aspect of the invention may be tagged with His-His-His-His-His-His (His6) to allow rapid purification by metal chelate chromatography and / or by an epitope accessible to an antibody to allow detection in a biological assay by Western blot, immunoprecipitation or activity reduction / blocking. Other tags are also possible (e.g. SUMO, Strep, etc.)

[0031] In the context of the present invention, the term "antigen of the present invention" or "recombinant antigen or synthetic antigen of the present invention" refers to any one or more of the chimeric antigen of the third aspect of the present invention, the antigen as defined in the first aspect of the present invention and / or the antigen as defined in the second aspect of the present invention, or any combination thereof. It should be further noted that, as used herein, the terms antigen and poly (amino acid) sequence are interchangeable.

[0032] Another object of the present invention is a nucleotide sequence encoding a chimeric antigen according to the third aspect of the present invention, an antigen comprising a tandem sequence according to the first aspect of the present invention, or an antigen as defined in the second aspect of the present invention. Also included within the scope of the present invention are nucleotide sequences that hybridize to any of the above sequences under stringent hybridization conditions, as well as corresponding chimeric recombinant antigens encoded by such hybridized nucleotide sequences.

[0033] Antigens of the present invention can be prepared by cloning and expressing in a prokaryotic or eukaryotic expression system using an appropriate expression vector. Any method known in the art can be used. For example, a DNA molecule encoding an antigen of the present invention is inserted into an appropriately constructed expression vector by techniques well known in the art (see Sambrook et al., 1989). Such vectors are another object of the present invention.

[0034] In order to be able to express the desired protein (in this case, the antigen), the expression vector should also contain a specific nucleotide sequence containing transcription and translation regulatory information, which is connected to the DNA encoding the desired protein in a manner that allows gene expression and protein production. First, in order to transcribe the gene, it must be behind a promoter that can be recognized by RNA polymerase, and the polymerase binds to the promoter and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong promoters and weak promoters).

[0035] For eukaryotic hosts, different transcription and translation regulatory sequences can be used, depending on the nature of the host. They can be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, etc., wherein regulatory signals are associated with specific genes with high expression levels. Examples are the TK promoter of herpes virus, SV40 early promoter, yeast gal4 gene promoter, etc. The transcription initiation regulatory signals that allow inhibition and activation can be selected to regulate the expression of genes. All these hosts are further objects of the present invention.

[0036] The nucleic acid molecules encoding antigens of the present invention can be connected to heterologous sequences so that the nucleic acid molecules of the combination encode fusion proteins. Such nucleic acid molecules of the combination are included in embodiments of the present invention. For example, they can be connected to DNA encoding proteins, which allow the chimeric antigen to be purified by affinity chromatography of only one step. The protein / fusion protein connected can be, for example, glutathione sulfotransferase (GST), to generate fusion products at the carboxyl terminus of the GST protein. The corresponding recombinant protein expressed in the cytoplasm of the transformed Escherichia coli cells can be purified by affinity chromatography using glutathione-agarose resin. Other fusion proteins are also contemplated, to not only simplify purification but also dissolve the chimeric antigen, such as, for example, thioredoxin (trx) or maltose binding protein (MBP).

[0037] The DNA molecule that comprises the nucleotide sequence of encoding any antigen of the present invention can be inserted into the vector, and this vector has the transcription and translation regulatory signal that can be operably connected, and this vector can be integrated into the desired gene sequence in the host cell.Can also select the cell that has been transformed with the DNA stably introduced by introducing one or more following markers, and this marker allows to select the host cell that contains the expression vector.Marker can also provide the phototrophy, biocide resistance to the auxotrophic host, for example, and antibiotic or heavy metal are such as copper etc.Selectable marker gene can be directly connected to the DNA gene sequence to be expressed or be introduced into the same cell by cotransfection.The optimal synthesis of antigen of the present invention may also need extra elements.

[0038] Once a vector or DNA sequence containing a construct for expression has been prepared, the DNA construct may be introduced into a suitable host cell by any of a number of appropriate means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, and the like.

[0039] Host cells can be prokaryotic or eukaryotic. Examples of eukaryotic hosts are mammalian cells, such as humans, monkeys, mice, and Chinese hamster ovary (CHO) cells. Expression in these host cells provides post-translational modifications for protein molecules, including correct folding or glycosylation at the correct site. Yeast cells can also carry out post-translational peptide modifications, including glycosylation. There are many recombinant DNA strategies, which utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence on the cloned mammalian gene product and secretes peptides (i.e., propeptides) with the leader sequence. Examples of prokaryotic hosts are bacteria, such as Escherichia coli.

[0040] After introduction of the vector, host cells are grown in a selective medium that selects for the growth of cells containing the vector. Expression of the cloned gene sequence results in the production of the desired protein.

[0041] Purification of the recombinant antigen is carried out by any of the known methods for this purpose, i.e. any conventional procedures involving extraction, precipitation, chromatography, electrophoresis, etc. A further purification procedure that may be preferably used to purify the antigen of the present invention is affinity chromatography using a monoclonal antibody that binds to the target protein and is produced and immobilized on a gel matrix contained in a column. Impure preparations containing the recombinant protein are passed through the column. The antigen will be bound to the column by the specific antibody, and impurities will pass through. After washing, the antigen is eluted from the gel by changes in pH or ionic strength.

[0042] Another aspect of the invention is a process for the recombinant production of an antigen of the invention as described above, which comprises culturing a host cell transformed with a vector containing a nucleotide sequence of the invention and isolating the desired product.

[0043] A further object of the present invention is a DNA molecule comprising a DNA sequence encoding the above-mentioned fusion protein, as well as substantially identical nucleotide sequences.

[0044] Substantially identical nucleotide sequences include all other nucleic acid sequences that also encode a given amino acid sequence due to the degeneracy of the genetic code.

[0045] The term "nucleic acid molecule" also includes analogs of DNA and RNA, such as those containing modified backbones.

[0046] In another aspect of the present invention, antigens of the present invention can be prepared by any other method such as SPS (solution phase synthesis) or SPPS (solid phase peptide synthesis). Several connection methods can also be used to synthesize antigens of the present invention, such as chemical connection, native chemical connection (NCL), expressed protein connection (EPL) and Staudinger connection. In addition, O-acyl isopeptide (also referred to as "click" or "switch" peptide) method can be used for the chemical assembly of polypeptides with high aggregation tendency. Other molecules or genetic engineering techniques can be used.

[0047] Another aspect of the present invention is the use of the above antigen of the present invention as a medicament, a pharmaceutical composition or the use of the antigen of the present invention in therapy. In particular, one aspect of the present invention is the use of the antigen of the present invention as an active ingredient in the preparation of a medicament or a pharmaceutical composition for preventing and / or treating Toxoplasma gondii infection.

[0048] In the context of gene therapy, another subject of the present invention is the use of a nucleotide sequence encoding an antigen of the present invention as a medicament, in particular for the preparation of a medicament useful for the treatment and prevention of Toxoplasma gondii infections.

[0049] The pharmaceutical composition of the present invention should preferably contain a therapeutically effective amount of the antigen of the present invention or the corresponding nucleotide sequence. Preferably, the antigen of the present invention and any composition comprising it can be used as or as a vaccine for preventing and / or treating Toxoplasma gondii infection.

[0050] For therapeutic applications, where the preparation of medicaments or vaccines is within the framework of the general knowledge, the reader is again referred to the patent literature cited in the present description for further reference, and in particular to Beghetto et al., The Journal of Infectious Diseases, 2005, 191: 637-645.

[0051] As used herein, the term "therapeutically effective amount" refers to the amount of therapeutic agent required to treat, improve or prevent a targeted disease or condition or to exhibit a detectable therapeutic or preventive effect. For any compound, a therapeutically effective dose can be initially estimated in a cell culture assay (e.g., tumor cells) or in an animal model (usually mice, rabbits, dogs or pigs). Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0052] The precise effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, drug combination, reaction sensitivity and tolerance / response to treatment. This amount can be determined by routine experimentation and is within the judgment of the clinician.

[0053] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier for administration of the therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier itself does not induce the production of antibodies harmful to the individual receiving the composition and that it can be administered without excessive toxicity.

[0054] Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles.

[0055] Pharmaceutically acceptable salts may be used therein, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. A comprehensive list of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0056] The pharmaceutically acceptable carrier in the therapeutic composition may additionally contain a liquid such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may be present in such compositions. Such carriers enable the pharmaceutical composition to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by the patient.

[0057] Once formulated, the compositions of the invention can be administered directly to a subject. The subject to be treated can be an animal; in particular, a human subject can be treated.

[0058] The pharmaceutical compositions used in the present invention can be administered by a variety of routes, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous application (e.g., see WO98 / 20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal or rectal means. Gene guns or needleless syringes (hypospray) can also be used to administer the pharmaceutical compositions of the present invention. Generally, the therapeutic composition can be prepared as an injectable, which is a liquid solution or suspension; solid forms suitable for dissolving or suspending in a liquid vehicle before injection can also be prepared.

[0059] Direct delivery of the composition will generally be achieved by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivery to the interstitial space of tissue. The composition may also be administered into a lesion.

[0060] Dosage treatment may be a single dose schedule or a multiple dose schedule.

[0061] A method of treating a mammal suffering from a T. gondii infection, preferably an acute T. gondii infection, comprising administering a therapeutically effective amount of an antigen of the invention by using a vaccine composition, for example as described above, represents one aspect of the present invention.

[0062] A further aspect of the invention relates to the in vitro use of the antigen of the invention for detecting anti-Toxoplasma gondii IgM, in particular for detecting anti-Toxoplasma gondii IgM in a test sample, preferably a biological sample, such as whole blood or a portion of whole blood, e.g. a plasma or serum sample. A preferred embodiment relates to the in vitro use of the antigen of the invention as described above for diagnosing acute toxoplasmosis in a subject in need thereof, preferably in a human subject.

[0063] A further object of the present invention relates to a kit suitable for detecting anti-Toxoplasma gondii IgM in a test sample, preferably a biological sample, such as whole blood or a portion of whole blood, e.g. a plasma or serum sample, comprising at least one antigen according to the present invention. Such a kit can be used for diagnosing acute Toxoplasma gondii infection.

[0064] The antigens of the present invention can be used in almost any assay format that uses antigen to detect IgM antibodies from Toxoplasma gondii infection. The common feature of all these assays is that the antigen is contacted with a body component suspected of containing IgM antibodies under conditions that allow the antigen to bind to any such IgM antibodies present in the component. In a non-limiting manner, such conditions can generally be physiological temperature, pH, and ionic strength using excess antigen. After incubation of the antigen with the sample, immune complexes containing the antigen are detected.

[0065] The design of immunoassays is subject to many variations, and many formats are known in the art. Protocols may, for example, use solid supports or immunoprecipitations. Most assays involve the use of labeled antibodies or polypeptides; the label may be, for example, an enzymatic molecule, a fluorescent molecule, a chemiluminescent molecule, a radioactive molecule, or a dye molecule. Assays that amplify signals from immune complexes are also known; examples are assays utilizing biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

[0066] Immunoassays can be, but are not limited to, heterogeneous or homogeneous formats, and standard or competitive types. In heterogeneous formats, polypeptides, particularly antigens of the invention, are typically bound to a solid matrix or support to facilitate separation of the sample and polypeptide after incubation.

[0067] Examples of solid supports that can be used are nitrocellulose (e.g., in the form of a membrane or a microtiter well), polyvinyl chloride (e.g., in a sheet or a microtiter well), nanoparticles and microparticles of different compositions (metals selected from iron, gold, platinum, silver, titanium, zinc, cerium, iron, copper, thallium and combinations thereof; organic, organometallic, polymeric, quantum dots or carbon structures), polymers selected from latex, polystyrene, alginic acid, gelatin, polylactic acid, chitosan, polylactide-co-glycolide and polycaprolactone (e.g., in beads or microtiter plates, polyvinylidene fluoride (known as Immulon) and polyvinyl chloride (e.g., in a microtiter well). ), diazotized paper, nylon membranes, activated beads, and protein A beads). For example, Dynatech Immulon 1 or Immulon 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in a heterogeneous format. The solid support containing the antigenic polypeptide is usually washed after separating it from the test sample and before detecting the bound antibody. Both standard and competitive formats are known in the art.

[0068] In a homogeneous format, the test sample is incubated with the antigen combination in solution. For example, it can be performed under conditions where any antigen-antibody complexes formed will precipitate. Both standard and competitive formats of these assays are known in the art.

[0069] In the standard format, the amount of antibody that forms an antibody-antigen complex is monitored directly. This can be accomplished by determining whether a labeled anti-xenotype (e.g., anti-human) antibody that recognizes an epitope on an anti-Toxoplasma gondii antibody will bind as a result of complex formation. In the competitive format, the amount of antibody in the sample is inferred by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competitive ligand) in the complex.

[0070] The complex formed containing the anti-Toxoplasma gondii antibody (or in the case of a competitive assay, the amount of competing antibody) is detected by any of a variety of known techniques, depending on the format. For example, a conjugate of an anti-xenogeneic Ig complexed with a label (e.g., an enzyme label) can be used to detect the unlabeled antibody in the complex.

[0071] In the immunoprecipitation or agglutination assay format, the reaction between the chimeric antigen and the antibody forms a network that precipitates from a solution or suspension and forms a visible precipitate layer or film. If there are no anti-Toxoplasma gondii antibodies in the test sample, no visible precipitate is formed. An example of an agglutination immunoassay is a turbidimetric assay using a suspension of uniformly sized polystyrene particles coated with the antigen of the present invention. When a sample containing IgM for Toxoplasma gondii is mixed with a reagent, the coated latex particles agglutinate. The degree of agglutination is proportional to the concentration of IgM in the sample and is measured by measuring the reduction in transmitted light caused by the aggregates (turbidimetric immunoassay). Another example of an immunoassay is a two-step chemiluminescent assay consisting of magnetic particles coated with the antigen of the present invention. After incubation with the sample, magnetic separation and washing steps, the beads are incubated with anti-human IgM antibodies labeled with isoluminol. After the final wash, a reagent that triggers the luminescent reaction is added, and the emitted light is measured as relative light units (RLU). The RLU is proportional to the concentration of anti-T. gondii IgM in the sample.

[0072] The antigens of the present invention will generally be packaged in the form of a kit for use in these immunoassays. The kit generally contains the following combination in separate containers: antigens (already bound to a solid matrix or separated from them and reagents for binding to the matrix), control antibody formulations (positive and / or negative), labeled antibodies when the assay format requires labeled antibodies, and signal generating reagents (e.g., enzyme substrates) if the labeling does not directly generate a signal. Instructions for performing the assay are generally included in the kit.

[0073] The present invention will now be described in more detail by way of examples and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 . Original heatmaps of the Toxoplasma gondii peptide microarray. Heatmaps are represented by a gray color scale: gray indicates a strong antibody response, and black indicates a weak or no antibody response. (A) Heatmap of IgG responses using a goat anti-human IgG (Fc) fluorescent secondary antibody and (B) Heatmap of IgM responses using a goat anti-human IgM (μ chain) fluorescent secondary antibody. (Source: PEPperPRINT GmbH).

[0075] Figure 2 : Heatmap of T. gondii peptide microarrays showing unpaired binary t-test of differential IgM responses. Source molecule name: protein to which each peptide belongs (sequence not shown). G1-mean: mean of all IgM-positive samples. G1-G2 difference: result of binary t-test. Peptides are sorted by decreasing G1-G2 difference.

[0076] Figure 3 : Schematic view of multiplex ELISA. The upper panel shows the spotting pattern within each section of the 96-well ELISA plate (1 to 3 upper rows of the ELISA plate) shown in the lower panel. The first four columns of the ELISA plate are printed with peptides 1a to 3c, columns 4 to 7 are printed with peptides 4a to 5c, and columns 8 to 12 are printed with peptides 6a to 7b. Eight positive and negative samples are evaluated according to the pattern shown in the figure. PC = positive control.

[0077] Figure 4 : Scanned images of sample 4 with 18 peptides. Panel a) corresponds to the scanned image of peptides 1a to 3c. Panel b) corresponds to the scanned image of peptides 4a to 5c. Panel c) corresponds to the scanned image of peptides 6a to 7a. A schematic view of the spotting pattern for each well is shown on the right side of each panel. The reactivity of peptides showing S / CO ≥ 2.2 is emphasized with a hyphenated line graph. (PC: positive control).

[0078] Figure 5 : Schematic view of multiplex ELISA of phase II. Each well of two 96-well plates is printed with a 7×6 spot matrix as shown in the figure. (PC: positive control, Toxoplasma Ag: natural antigen of Toxoplasma gondii).

[0079] Figure 6 : Original images of positive sample 4 before and after treatment. Left image before analysis. Right image after software processing.

[0080] Figure 7: Schematic view of an ELISA plate. ELISA plates coated with a single peptide were originally produced in a single strip removable 96-well plate. Ready-to-use custom ELISA plates can be easily obtained using the test strips in the original plate as needed. C strips refer to control strips, for which we follow the same coating procedure but without peptide.

[0081] Figure 8 : Correlation between multiplex ELISA and singleplex ELISA. For each technique, positive samples (31 and 46) and negative samples (54 and 62) are expressed in their corresponding units (fluorescence units and signal-to-noise ratio (SNR)). All peptides were tested at 100 μg / mL.

[0082] Fig. 9 : Example of a checkerboard template. Peptide concentrations were reduced 1:4 every two columns. The last two columns were used as negative controls. Conjugates were diluted 1:2 every two rows. One column per condition was used to test positive samples (PS) and the other column was used to test negative samples (NS). Sample dilution was fixed at 1 / 50 with sample diluent.

[0083] Fig.10 : Receiver operating characteristic curves for peptides 3a, 3b, 6c, 7b and 3a+6c. The diagnostic performance of each peptide is represented by a different gray intensity line. The solid gray line represents the indiscriminate region of the ROC curve. The curves were generated using the conditional sample set minus sample 49 (n=93).

[0084] Fig.11 : Schematic representation of the original and truncated versions of peptide 6c. The 6c peptide consists of three blocks of 8 amino acids plus tandem repeats of two amino acids at the N-terminus and one amino acid at the C-terminus (27aa). The truncated 6c peptide consists of the same blocks repeated in tandem minus the two amino acids at the N-terminus (25aa). Each different amino acid within a block is named with a different letter. O, O', and O" represent a single amino acid that varies within a block.

[0085] Fig.12 : Schematic representation of chimeric peptides and biotin-labeled peptides. The 6c homo-tandem consists of three 6c peptides in different formats repeated in tandem. The 3a and 6c hetero-tandem consists of tandem repeats of 3a and 6c sequences in different orientations using O2Oc as spacers. The 3a and 6c truncated peptides are biotin-labeled.

[0086] Fig.13 : Receiver operating characteristic curves of chimeric peptides. The diagnostic performance of each peptide is represented by a different gray intensity line. The solid gray line represents the area of ​​no distinction. The conditional sample set minus samples 49 and 31 (n=92) was used to generate the ROC curves for peptides 6c2, 6cO36c and 3a6c. For peptides (6c2)K3 and 6c3a, the conditional sample set minus sample 31 (n=93) was used. Example

[0087] Example 1. Synthetic peptides as antigens: from Toxoplasma gondii peptide microarrays to functional single peptides

[0088] As already mentioned in the background of the invention, the discovery of new epitopes that can aid in the clinical management of toxoplasmosis is one of the focuses of the present invention. It was our starting point to identify which proteins of the Toxoplasma gondii parasite could be used as new potential biomarkers for the diagnosis of the acute phase of the disease, and therefore the first aim of the present invention was to investigate the main antigenic proteins of Toxoplasma gondii. In view of the shortcomings of T. gondii lysate antigens and assays based on recombinant products, we chose a peptide-based approach aimed at identifying epitope sequences with potential application in immunoassays for anti-Toxoplasma gondii IgM detection. To this end, we first used an epitope mapping technology involving peptide microarrays developed by PEPperPRINT GmbH (Heidelberg, Germany) to discover new antigens that are specific for IgM and have no or little reactivity to other Ig types (i.e. IgG).

[0089] In subsequent steps, we used solid phase peptide synthesis (SPPS) to produce amplified, high-quality synthetic versions of the relevant sequences identified as free (i.e., not microarray-bound) soluble peptides in the PEPperPRINT scans for incorporation into diagnostic kits. Finally, we used ELISA to assess the antigenic capacity of the peptides. Initially, we developed a multiplex ELISA to evaluate the feasibility of the synthetic peptides for detecting anti-T. gondii IgM. Once we identified the optimal peptides, we used a monoplex ELISA to validate the results obtained by the multiplex ELISA. Finally, the monoplex ELISA was optimized to improve the performance of the assay for detecting anti-T. gondii IgM-positive samples.

[0090] 1.1. Toxoplasma gondii peptide microarray

[0091] In order to identify new T. gondii epitopes that could be incorporated as diagnostic tools for the specific detection of anti-T. gondii IgM, we initiated a collaboration with PEPperPRINT GmbH to design a peptide microarray that displays all peptides covering the different antigenic proteins from T. gondii. The complete list of the selected nine proteins identified by UniProt (https: / / www.uniproT.org / ) and the Immune Epitope Database (IEDB) (https: / / www.iedb.org / ) is shown in Table 1:

[0092] 1.2. Characterization of microarray sample sets

[0093] To initially evaluate the proteins selected in the epitope mapping microarray experiments, a panel of 13 human serum samples (hereinafter referred to as the microarray serum panel) obtained from the Cerba laboratory and the Biokit biobank was fully characterized for T. gondii IgM and IgG immunoreactivity. Different immunoglobulins, especially IgG and IgM, exhibit different profiles and dynamics during toxoplasmosis, so it is necessary to determine which antibody types are present in the microarray serum panel. Due to the high prevalence of the disease, T. gondii IgG-positive samples lacking IgM are the more common types observed in clinical routine. In contrast, it is difficult to obtain T. gondii IgM-positive samples lacking IgG, which is characteristic of the initial stage of the disease. This is mainly because the early diagnosis of toxoplasmosis is unusual, because immunocompetent patients remain asymptomatic in most cases, or the clinical manifestations are complex and can be confused with a variety of other diseases. Therefore, the most common scenario is that both anti-T. gondii IgG and IgM are usually found together, or only IgG is detected.

[0094] For our microarray evaluation, it was critical that samples containing only IgM were adequately represented. To this end, triplicate samples (MA10, MA11, and MA12) were IgG-depleted to remove anti-T. gondii IgG; therefore, both depleted and non-depleted samples were included for microarray evaluation.

[0095] Characterization of microarray sample sets by commercial BIO-FLASH Toxo IgG and BIO-FLASH The results are shown in Table 3.

[0096]

[0097] Table 3: Characterization of the microarray serum panel. For both assays, the mean of two replicates was interpreted according to the manufacturer's instructions. For the Toxo IgG immunoassay, samples with concentrations ≥10.00 and ≤8.00 IU / mL were considered positive and negative, respectively. In the Toxo IgM assay, signal cutoffs (S / CO) ≥1.00 and ≤0.80 are considered positive and negative, respectively. Subtracted samples are marked with a "d" after the sample ID. ID: Identification.

[0098] Analysis of the microarray serum panel showed that six samples were Toxo IgG immunoassay was positive according to BIOFLASH Toxo IgM was negative (MA1 to MA5 and MA9), four samples were positive for IgM and negative for IgG (MA6 to MA8 and MA13), and three samples were positive for IgG and IgM (MA10 to MA12). The subtracted samples (MA10d to MA12d) showed a 99% reduction in IgG titer relative to the non-subtracted samples. Therefore, they were classified as IgG negative. The subtracting process resulted in a 17-20% reduction in IgM reactivity compared to the non-subtracted.

[0099] 1.3. Evaluation of Toxoplasma gondii peptide microarrays.

[0100] The sequences of nine selected Toxoplasma gondii proteins were evaluated in peptide microarrays and microarray serum panels by PEPperPRINT GmbH. Goat anti-human IgG (Fc) fluorescently conjugated DyLight680 and goat anti-human IgM (μ chain) fluorescently conjugated DyLight800 were used as secondary antibodies to distinguish between either type of antibody response in each group of samples. Heatmaps of unprocessed fluorescence images of IgG and IgM are shown in Figure 2. Figure 1 shown.

[0101] The IgG response heat map shows that the IgG positive samples (MA1 to MA5) have similar fluorescence signal profiles and intensities. For their part, the IgG negative samples (MA6 to MA8) did not show non-specific binding to any of the epitopes evaluated, which together emphasized the obvious differences between the two groups of samples. As expected, the subtracted samples (MA10d to MA12d) did not respond to IgG compared to the non-subtracted samples (MA10 to MA12), which were positive for some epitopes. Although sample MA13 was initially classified as IgM positive, it did not show differences between the IgG and IgM peptide microarrays and was therefore considered an outlier, which was not included in the statistical analysis ( Figure 1 A).

[0102] The IgM response heat map revealed a more complex profile than that of IgG. IgM-positive samples, both IgG-positive and -negative (MA6 to MA8, MA10 to MA12, and MA10d to MA12d), showed significantly stronger responses than IgM-negative samples (MA1 to MA5 and MA9). Sample MA7 had the highest reactivity within the IgM-positive group, consistent with previous BIO-FLASH The results of Toxo IgM (6.6 S / CO) were consistent. Samples MA12 and MA12d with high IgM titers (11.2 and 9.3 S / CO, respectively) in the Toxo IgM characterization showed weaker reactivity ( Figure 1 B).

[0103] For data analysis, the microarray serum panels were divided into the following four different groups: Group 1 (G1): IgM positive (MA6, MA7, MA8, MA10, MA11, MA11d, MA12, MA12d, and MA13); G2: IgG positive (MA1, MA2, MA3, MA4, MA5, MA9, MA10, MA11, and MA12); G3: IgG negative (MA6, MA7, MA8, MA10d, MA11d, and MA12d) and G4: IgM negative (MA1, MA2, MA3, MA4, and MA5).

[0104] Using Statistical Utility for Microarray and Omics data (SUMO) software (Heidelberg, Germany), based on untreated mean spot intensity, under the condition of no data normalization, the obtained results were subjected to statistical analysis. As a noise filter, the minimum spot intensity of 50 fluorescence units was defined (that is, all interactions below the threshold were set to 50 and had no effect on statistical analysis). Unpaired binary classification t-test was carried out to determine: (i) common IgG response (IgG positive for IgG negative sample), (ii) common IgM response (IgM positive for IgM negative sample) (data not shown), and (iii) difference IgM (hereinafter referred to as G1-G2) (IgM positive for IgG positive sample).

[0105] In order to illustrate which epi-position is specific to IgM, differential IgM response was assessed.The mean point intensity of IgM positive serum (MA6, MA7, MA8, MA10, MA11, MA11d, MA12, MA12d and MA13) and IgG positive serum (MA1, MA2, MA3, MA4, MA5, MA9, MA10, MA11 and MA12) is distributed in the response matrix comprising 82 kinds of peptides, wherein the p value is p<1.00E-02.In 82 kinds of peptides, maximum 39 kinds are statistically significant, and therefore raise in IgM positive and IgG positive samples.Based on the information in the differential IgM response heat map, the top peptide hits show the G1-G2 difference of>400 units, which is mainly assigned to dense granule protein GRA3, rod-shaped body protein, granule antigen protein GRA7 and major surface antigen P30 ( Figure 2 ).

[0106] Table 4 shows the previous Figure 2Detailed view of the binary t-test analysis of the 21 statistically significant peptides seen in Figure 3, which exhibit the highest G1 / G2 ratios. The top upregulated peptides (peptides I to VIII) showed a G1-G2 difference of >400 units and almost all showed a G1 / G2 ratio of >5, indicating that they were 5-fold more reactive to IgM-positive samples (G1) compared to IgG-positive samples (G2). The same peptides showed low reactivity to IgG-positive samples, with G2 between 50 and 126, which highlights their specificity for IgM-positive samples.

[0107]

[0108] Table 4: Top hits of differential IgM responses. Protein acronyms (Protein acronyms) refer to the protein to which each peptide belongs. G1 and G2 average () refer to the central tendency of the two groups of samples, and their corresponding standard deviations (σ). G1-G2 (G1-G2 diff.) refers to the result of subtracting G2 from G1. The G1 / G2 ratio is given by dividing the G1 average by the G2 average. The t-values ​​and p-values ​​are from unpaired binary t-tests of IgM responses obtained using SUMO software.

[0109] 1.4. From microarray to synthetic peptides

[0110] The disadvantages of TLAs or recombinant antigens contrast with the proven advantages of synthetic constructs, such as minimized nonspecific interactions, higher reproducibility and the possibility to adjust their chemical structure to enhance their antigenic properties. Therefore, we chose a synthetic peptide-based approach to reproduce the sequences identified in the above screen.

[0111] Based on microarray analysis results, we identified the sequence with the best performance potential.We carry out our selection based on the following: i) IgM specificity of the G1-G2 difference and G1 / G2 ratio based on the difference IgM response, ii) homology with the Toxoplasma gondii B cell epitope found in IEDB, and iii) peptide length.Therefore, we design peptides to include the region that is most specific to IgM in the protein under study but peptide length is limited to about 50-60 residues (this is the approximate threshold), and the quality of the peptide produced by synthesis may be affected after this approximate threshold.Use Fmoc solid phase synthesis to synthesize 18 kinds of peptides selected with C-terminal formamide form, use HPLC (high performance liquid chromatography) to be purified to>95% homogeneity, and use MS (mass spectrum) to suitably characterize (referring to Table 2).

[0112] 1.5. Evaluation of synthetic peptides by ELISA

[0113] Once new synthetic peptides are produced, we hope to evaluate their function using multiple and single indirect ELISA. In order to evaluate the selected peptides, a group of 110 human sera (hereinafter referred to as the screening group) was obtained from several suppliers. AbBaltis, ABOPharma and Biomex provided Toxoplasma gondii IgM positive samples (n=46) as external suppliers. Two IgM positive samples from the microarray group were included to verify the results obtained by the peptide microarray (MA10 and MA11). Sixty samples from Banc de Sangi de Teixits de Catalunya were obtained in two different batches as Toxoplasma gondii IgM negatives.

[0114] For preliminary investigation of cross-reactivity, the Biokit Biobank provided two complementary cytomegalovirus (CMV) positive, T. gondii negative samples, as this virus has been described as a possible cross-reactant by other commercial tests. The composition of the sample set before in-house characterization can be seen in Table 5.

[0115]

[0116] Table 5: Screening panel composition. Samples were obtained from different suppliers (external and internal). (n) Total of each type of sample.

[0117] Samples provided by external vendors were previously characterized for T. gondii IgM using different commercial assays (data not shown). Two negative samples provided by the Biokit biobank were classified as negative based on previous serological information (data not shown). Samples obtained from the blood bank had not been previously tested for toxoplasmosis infection. Three IgM-positive samples belonging to the microarray panel were tested within the present invention. In order to validate the results provided by the vendors and the consistency between the IgM anti-T. gondii detection methods, the screening panel was fully characterized for the presence of IgM using four commercial assays, which were selected taking into account i) assay performance (high sensitivity and specificity), ii) system requirements (i.e., method or specific instrument requirements) and iii) the ability to obtain reagents and technical support. BIO-FLASH was selected Toxo IgM immunoassay (Biokit), bioelisa TOXO IgM (Biokit) and PLATELIATM Toxo IgM (BIO-RAD). In addition, VIDAS for the detection of total anti-Toxoplasma gondii Ig TOXO Competition (BioMérieux) was used as the primary screening method (Table 6).

[0118]

[0119] Table 6: Commercial assays used to characterize the screening panel. Commercial assays are categorized based on the information present in the intended use data sheet.

[0120] To characterize the screening panel, we designed a strategy to use four assays (BIO-FLASH ToxoIgM, bioelisa TOXO IgM, PLATELIATM Toxo IgM and VIDAS TOXO Competition) prioritized the analysis of positive samples from external suppliers (n=46). Finally, from the two microarray serum group samples, due to the available volume of each sample, we only tested MA10 and MA11 samples with two commercial assays. The 60 samples provided by the blood bank (we expect most of them to be negative) were tested using one or two assays, taking into account the sample volume and the detection methods available in-house when receiving the samples. When the result is positive, we use the second or third assay to expand the screening. The first batch consists of 20 samples, tested using two assays (blood bank batch I). Of these 20 samples, one gave a positive result; therefore, it was evaluated by the third assay, which gave a negative result. In the second batch, 40 samples were received and tested using two assays (blood bank batch II). From these, one gave a positive result; therefore, it was tested using the third assay, which gave a negative result. Table 7 details the results of the screening panel characterization.

[0121] The results we obtained with the 46 samples purchased as IgM anti-T. gondii positives varied among the four commercial assays used to characterize the samples, ranging from 45 to 30 positives. TOXO Competition was the assay that classified the largest number of samples as positive (n=45), while BIO-FLASH Toxo classified the lowest number of samples as positive (n=30). The same estimate could not be made for negative samples, as not all samples were evaluated by the same assay.

[0122]

[0123] Table 7: Detailed view of the screening group characterization. The left column categorizes the samples according to the supplier (external supplier, Biokit biobank, microarray panel, and blood bank). The number of samples included in each group is specified in brackets. The blood bank samples (batches I and II) include information for those samples that were initially positive. Each group of samples was categorized according to the manufacturer's instructions as shown in the right column (positive, negative, or uncertain).

[0124] These results show that the four commercial assays are not consistent in the positivity of the samples. Since distinguishing positive samples from negative samples is the key to the present invention, we classified the groups according to the agreement between the assays used to evaluate each sample. The classification is shown in Table 8.

[0125]

[0126] Table 8: Screening set classification. (a) Samples tested by two methods. (b) Samples tested by three methods; they all gave different results.

[0127] Samples showing complete agreement in all methods used were classified as total positive (n=28) or total negative (n=60). Samples purchased as positive from external suppliers, but showing an uncertain result in our characterization process, were classified as total positive conditionality (total pos cond) (n=4), or for samples presenting two uncertain results, were classified as total positive conditionality 2 (total pos cond2) (n=2). This group of samples (total positive, total negative and samples with one or two uncertain results) was named "conditionality" (n=94). The final classification as "excluded" sample group (n=16) corresponds to samples showing different degrees of inconsistency, or the same number of positive and negative results, or even a result of each type (positive, negative and uncertain). These samples were excluded from the study.

[0128] 1.6.Multiple ELISA

[0129] Microarray studies elucidated 18 different peptides that potentially reacted with IgM anti-T. gondii positive samples and did not react with anti-T. gondii IgG positive samples. The sequences of the selected peptides were synthesized by SPPS. In order to determine which reacted with the greatest number of IgM positive samples in a fast and efficient way, and taking into account the lack of sample volume, we designed a multiplex ELISA in collaboration with InfYnityBiomarkers.

[0130] Phase I. To elucidate which are the best peptides, we initially need to design an optimal assay with appropriate assay conditions. To establish the optimal peptide concentration, three peptide dilutions (5, 50, and 100 μg / mL) were spotted in duplicate and organized into a 6 × 6 array in each well of a 96-well plate ( Figure 3). In addition, positive controls (PC) were spotted in triplicate to check reactivity in each well and array spatial orientation. Two different conjugate dilutions were tested (1 / 1500 and 1 / 3000). Eight total positive (4, 5, 24, 31, 36, 37, 40, and 46) and eight total negative (52, 54, 55, 56, 58, 60, 61, and 62) samples were selected from the conditional set and diluted 1 / 50. The ELISA plate design is shown in Figure 3 .

[0131] The net fluorescence intensity (average of replicates) of the 16 samples included in the study was determined. The average of the negative samples was used to establish a separate cutoff for each peptide (Table 9A). Almost all cutoffs were below 600 units. Peptides 6d, 7a, and 7b showed higher reactivity (672, 723, and 625, respectively). Both peptides showed cutoffs 2 and 3 times higher than the other peptides (1545 for peptide 1a and 3953 for 5b).

[0132]

[0133] Table 9A: Cut-off values ​​for each peptide. Cut-offs are expressed in relative fluorescence units. ID: ID.

[0134] Table 9B below shows further information associated with each peptide in Table 9A above:

[0135]

[0136] Table 9B.

[0137] The net intensity of each peptide of the positive or negative samples was converted into positive or negative reactivity according to the cutoff value. The cutoff value signal (S / CO) for each sample is shown in Table 10. In addition, the final S / CO was calculated by subtracting the average negative value from the average positive value. Samples with an S / CO value of ≥1 were considered positive, and samples with an S / CO of <1 were considered negative. Peptides showing a final S / CO value>1 were considered to be specific against Toxoplasma gondii IgM-positive samples.

[0138] Positive samples were reactive to almost all peptides (1a, 2a, 3a, 3b, 3c, 4a, 4b, 4c, 5a, 5b, 5c, 6b, 6c, 6d and 7b). Peptides 3a, 3b, 3c, 6c and 7b showed higher final S / CO (≥3,5). Peptides 5a and 5c showed final S / CO ≥1. The final S / CO of peptide 4a was equal to 1. Peptides 5b, 6a and 7b reacted more to negative samples than to positive samples. This result indicates that these peptides are non-specific to IgM positive samples.

[0139]

[0140] Table 10: Peptide screening via multiplex ELISA (Phase I). S / CO values for each peptide of each sample (#). Final S / CO is shown using color coding (red indicates reactivity (S / CO > 1) and black indicates non-reactivity (S / CO ≤ 1)).

[0141] Examples of putting the present invention into practice are shown below.

[0142] The reactivity of 18 peptides of sample 4 can be visually explored in Figure 4 and correlated with the numerical cut-off values presented in Table 11. For each peptide, since non-specific results can be reported when precipitation occurs, all scanned images were visually inspected and compared with their net intensity values. Peptide 6c is one of the best-performing peptides (along with 3a), marked in Figure 4 . 6c visually presents the strongest spot (which correlates with its S / CO of 25.8), the highest among all peptides (see Table 11). When comparing peptides 5b and 5c, an example of non-specific results was observed.

[0143] Visually, the spot intensity of 5b is higher than that of 5c. In contrast, the cut-off value of peptide 5c is higher (2.8 S / CO and 2.3 S / CO respectively). This example illustrates the importance of comparing the numerical results obtained by software and giving priority to the visual exploration of images to avoid misinterpretation of results. Multiplex ELISA Phase I allowed us to establish the optimal assay conditions (peptide concentration of 100 μg / mL, sample of 1 / 50, and conjugate of 1 / 1500). The results obtained showed that for the eight positive samples analyzed, peptides 3a and 6c were reactive against IgM anti-Toxoplasma gondii, and for the eight negative samples, peptides 3a and 6c were non-reactive against IgM anti-Toxoplasma gondii. In contrast, peptides 1a, 5b, and 7a showed cross-reactivity with the negative samples, which may indicate that they are non-specific for IgM anti-Toxoplasma gondii. Even so, the study of the 18 peptides was extended to Phase II, using the assay conditions established in Phase I and a higher number of samples (a complete sample set consisting of 110 samples).

[0144]

[0145] Table 11: Summary of results for positive sample 4 (Phase I). Net intensity, cut-off, and S / CO for each peptide are presented. The Interpretation column shows the classification of each peptide of sample 4. Peptides with an S / CO ≥ 2.3 are interpreted as reactive. Peptides with an S / CO < 2.3 are interpreted as non-reactive. ID: Identification.

[0146] Phase II. As explained above, Phase I allowed us to establish appropriate assay conditions for the 18 peptides. In Phase II, our goal was to identify which peptides had the best antigenic properties. To do this, we analyzed the performance of each peptide—those samples that were correctly classified as positive or negative—by using the assay conditions established in Phase I with the full screening panel. Peptides were spotted in duplicate at 100 μg / mL and organized into 7×6 arrays in each well of a 96-well plate ( Figure 5 ). In addition, native antigen of T. gondii produced by Biokit was included as an additional control. Biokit Toxo antigen was spotted in duplicate at three different concentrations (5, 50 and 100 μg / mL).

[0147] In phase II, InfYnity Biomarkers changed the plate reader and the software for capturing and analyzing the image from 96 orifice plates.New software sets up the cutoff level according to the overall data.Then, according to the cutoff value set up by the software, the net intensity of each antigen of positive and negative samples is transformed into positive or negative reactivity.Although complete screening group (n=110) has been used, only condition group (referring to Table 8) (characterized as total positive, total pos cond, total pos cond 2 and total negative sample) (n=94) is carried out data analysis.In order to minimize the possibility of false positive or false negative including the possibility of causing data misunderstanding and selecting non-specific peptides, only consider the sample of reporting high homogeneity in the mensuration for characterizing sample and analyze.Result is as shown in table 12.

[0148]

[0149] Table 12: Peptide screening by multiplex ELISA (Phase II). The average values ​​of all positive and negative samples included in the conditional group are shown. The result column was calculated by subtracting the average neg sample from the average pos sample. The interpretation column was completed according to the following criteria; S / CO ≤3 is non-reactive and S / CO>10 is reactive. (Toxo Ag: natural antigen of Toxoplasma gondii, AU: absorbance unit).

[0150] The reactive peptides showing the highest S / CO were 3a, 3b, 6c. The native antigen of T. gondii was positive only when tested at 50 and 100 μg / mL. Even so, the mean values ​​for negative samples in the study were the highest, followed by peptide 5b, which could indicate that using the native antigen of T. gondii is not a good method for detecting IgM anti-T. gondii positive samples. Specific examples such as Figure 6 and as shown in Table 13. The raw images obtained with sample 4 were processed using the software according to the established parameters. Numerical results were then obtained. Interpretations were provided based on the established cutoffs and uncertain areas were proposed by the software.

[0151]

[0152] Table 13: Summary of results for positive sample 4 (Phase II). The cutoff signal (S / CO) and interpretation are shown. Interpretation was done according to the following criteria; S / CO ≤3 is non-reactive and S / CO>10 is reactive. An indeterminate (Tx Ag: Toxoplasma gondii natural antigen) was established between >3 and ≤10.

[0153] First, the aim was to find out if we could reproduce the results obtained with the multiplex ELISA during the collaboration with InfYnity Biomarkers. Then, if the first aim was achieved, the second was to establish an in-house protocol to perform an indirect ELISA. The new indirect ELISA will use synthetic peptides as antigens, thus validating the functionality of the selected peptides as new diagnostic tools for the detection of T. gondii IgM-positive samples. The multiplex parameters were reproduced in a monoplex ELISA.

[0154] During the collaboration with InfYnity Biomarkers, we showed that the selected peptides could be attached directly to the solid surface of a 96-well plate, rather than being used as a conjugate (which is a common approach used by other commercially available immunoassays, as shown in Table 8). Therefore, we decided to design an indirect ELISA to validate the results obtained by the multiplex ELISA in both Phase I and II. The indirect approach allowed us to simplify the method because we did not have to label all peptides with peroxidase.

[0155] Several ELISA plates were coated with the five selected peptides at 100 μg / mL following a standard in-house coating protocol using a single strip 96-well plate. The single strip 96-well plate allows us to easily reassemble the ELISA plate and customize the final ELISA plate for functional assays (see Figure 7 ).

[0156] To validate the results obtained by multiplex ELISA, two positive and two negative samples tested in phase I were selected. Figure 7 In the ELISA format shown, 1 / 50 sample and 1 / 1000 conjugate dilutions were evaluated. Once the ELISA protocol was completed, the optical density (OD) of each plate well was read using a 630 nm filter. Relative absorbance units were calculated by subtracting the blank result (read at 450 nm with a blank well). The cutoff was calculated as the average of the absorbance of all negative samples (0.187 OD). The signal-to-noise ratio (SNR) was then calculated based on the absorbance value of each positive sample divided by the cutoff value. The results are shown in Figure 8 shown.

[0157] The results of the monoplex ELISA showed good correlation with the results of the multiplex ELISA developed in collaboration with InfYnity Biomarkers. Peptides 3a, 3b and 6c showed higher fluorescence units and SNR, followed by 3c and 7b. Since the units of the two technologies were not comparable, we calculated the standard deviation and extrapolated the results to percentage values ​​relative to the highest values ​​(data not shown). In the process of doing so, we observed an interesting fact. In the multiplex ELISA, the standard deviation of the 3a, 3b and 6c peptides of sample #31 was 979, representing 14% relative to the highest reactivity value (6912). The same determination in the monoplex ELISA gave a standard deviation of 3.7, i.e. 28% relative to the highest reactivity value (13.3). This difference was not observed in sample 46 (twice as high in the monoplex than in the multiplex); the difference can be attributed to sample degradation or operational errors.

[0158] Multiplex ELISAs allowed us to establish an optimal working range for each assay condition studied (peptide, sample, and conjugate concentrations) and to identify which were the best performing peptides. Next, we converted the assay conditions established with the multiplex ELISA into a single-plex format. However, due to methodological differences between multiplex and single-plex, such as coating of the plate (peptide printing vs. carbonate buffer precipitation, etc.), we decided to examine the assay conditions established by the multiplex ELISA more deeply and optimize them to single-plex ELISAs, with the goal of improving the performance of the latter. To this end, we performed a checkerboard titration. In all immunoassays, it is important to optimize the concentration of the sample and the antigen or antibody used to capture or detect the sample. If the sample or antigen / antibody is too concentrated, there is a risk of saturation. Conversely, if the sample or antibody is not concentrated enough, the signal is weak and difficult to detect. Checkerboard titration can be used to evaluate two variables simultaneously: in this case, we decided to study the concentration of the peptide that will be used to coat the ELISA plate to capture IgM anti-Toxoplasma gondii, and the conjugate concentration. By running each well with different ratios of peptide and conjugate, not only the optimal concentration of each can be found, but also the optimal ratio of the two concentrations.

[0159] An example of a chessboard titration is Fig. 9 As shown. Each of columns 1-12 corresponds to a different peptide dilution factor in descending order, and rows AH also correspond to conjugate dilution factors in descending order. Table 14 summarizes the concentration range evaluated for each peptide, the peptide concentration finally selected, the absorbance and SNR of the positive and negative samples used. For all peptides, the optimal dilution of the conjugate was 1 / 2000. Peptide 6c required the lowest peptide concentration, yet it showed the highest SNR. In contrast, peptide 7b required the highest peptide concentration to achieve an SNR of 1.9.

[0160]

[0161] Table 14: Summary of results of chessboard optimization of original peptides. a Peptide 3a was tested from 10 to 0.6 μg / mL, and peptide 6c was tested from 0.2 to 0.05 μg / mL. b Selected concentrations of 3a and 6c peptides, respectively.

[0162] Once the optimal assay conditions for the indirect ELISA were established, a conditional panel was evaluated with peptides 3a, 3b, 6c and 7b (see Table 8). Sample 49 was excluded from the study due to low volume (n=93). Peptide 3c was excluded due to low reactivity shown during the checkerboard (SNR of 1.2).

[0163] For evaluation, three 96-well plates were coated with each peptide (3a, 3b, 6c, 7b) at the selected peptide concentration. Each sample, including the blank, was evaluated in duplicate. The optical density of each well was read using a 630 nm filter. Relative absorbance units were calculated by subtracting the blank result (read at 450 nm). The net absorbance of each sample was reported, and its status (negative or positive) was used to analyze the sample using Analyseit The software generates a receiver operating characteristic curve (ROC curve). A ROC curve is a graphical plot that illustrates the diagnostic power of a binary classification system as its discrimination threshold is varied. Thus, a ROC curve is created by plotting the true positive proportion (TPP, also known as sensitivity) against the false positive proportion (FPP, also known as the probability of a false alarm or false positive, calculated as 1 - specificity) at various threshold settings.

[0164] In addition, we wanted to evaluate whether the combination of the two peptides could have a synergistic effect (synergy), thereby increasing the SNR obtained individually. Peptide 7b was discarded because of its low reactivity to IgM-positive samples. Peptide 6c was selected because it was the best candidate so far. In order to decide between peptides 3a and 3b, we established a tentative cutoff (0.21) and analyzed the number of true positives, true negatives, false positives, and false negatives obtained after screening with the conditional group (data not shown). The number of false positive and false negative results was consistent, although peptide 3a showed an additional two true positive results and an additional true negative result. Therefore, we decided to test the combination of peptides 3a and 6c. A chessboard was performed with a mixture of the two peptides at different concentrations. The chessboard revealed that when the two peptides were used as antigens together, a high SNR could be obtained, with the 3a concentration being nearly 10 times lower than when 3a was used alone (see Table 14). Once the assay conditions were established, the conditional group minus sample 49 (see Table 8) (n=93) was evaluated with a combination of peptides 3a and 6c. (See Fig.10 ).

[0165] from Fig.10 As can be seen in Table 15, peptide 7b itself is not a good candidate because the line is close to the indiscriminate region (random classification) at some points, indicating that 7b cannot reliably classify samples as positive or negative. In addition, its area under the curve (AUC) is the lowest, at 0.741, with a 95% confidence interval (CI) of 0.624 to 0.859 (see Table 15). Peptides 3a, 3b, and 6c showed better diagnostic performance because their ROC curves are closer to the upper left corner (see Fig.10 ). This angle is also called 0.1 point or perfect classification, representing 100% sensitivity (no false negatives) and 100% specificity (no false positives). In addition, in the case of peptide 6c and the combination of 3a and 6c, their AUC values ​​were around 0.85 or even higher, both of which were 0.89, with 95% CI of 0.82 to 0.95. (See Table 15).

[0166]

[0167] Table 15: Area under the curve (AUC) of ROC curves For each peptide, the AUC and its 95% confidence interval (CI) were calculated according to the Wilcoxon-Mann-Whitney test.

[0168] ROC curves allow us to establish the optimal threshold or cutoff level where each peptide achieves its best diagnostic performance. The software calculated estimates of accuracy, sensitivity (Se) and specificity (Sp) for each peptide. We determined that the combination of peptides 3a and 6c acted synergistically in terms of sensitivity.

[0169]

[0170] Table 16: Accuracy estimates for each peptide. Cutoffs are expressed in relative absorbance units (RAU). TP: True Positive, FP: False Positive, TN: True Negative, FN: False Negative. TPP: True Positive Proportion, also known as Sensitivity (Se), TNP: True Negative Proportion, also known as Specificity (Sp). FPP: False Positive Proportion and FNP: False Negative Proportion.

[0171] Example 2. Peptide chimeras to enhance sensitivity

[0172] In the previous embodiment, we have shown that the combination of two peptides in solution enhances reactivity compared to the separate use of the two peptides. In view of this, the logical next step is to design and evaluate the chimeric peptides of the sequences of the preferred candidates identified in combination Example 1. Therefore, the basic principles for designing chimeric peptides are three aspects: i) increase IgM specificity, ii) control the peptide orientation on the solid surface (i.e., polystyrene plate or magnetic particles), and assess whether one can improve the performance of the assay if a peptide that reacts not only to IgM positive but also to IgG positive samples is used. The polymeric peptides that display several B cells and / or T cell epitopes on a single molecule scaffold have been shown to have a wide range of biomedical applications as tools in drug design, targeted delivery, serodiagnosis, oncology, and vaccinology. These chimeric molecules have demonstrable advantages because they are multifunctional, highly stable (i.e., compared to native proteins), easy to produce at moderate cost, and have good immunogenicity.

[0173] To do this, we first selected the two best performing sequences from the microarray study in Example 1. Next, we generated synthetic versions of these peptides using SPP. We also included peptide 7b after having initially discarded it because it showed specific reactivity to IgG-positive samples in the microarray study. Finally, we labeled some of the chimeric peptides with biotin to assess whether the orientation of the peptides on the solid surface could affect the assay performance.

[0174] We used a single-plex ELISA to evaluate the performance of the new chimeric constructs with IgM anti-T. gondii positive samples. We chose the same approach as in Example 1 to establish the optimal assay conditions for the new peptides.

[0175] 2.1. Design and synthesis of chimeric peptides based on the top-forming peptides 6c and 3a

[0176] Many immunoassays rely on attaching antigens to solid surfaces such as polystyrene plates or magnetic particles to detect molecules of interest. Short synthetic peptides that are easy to produce by chemical synthesis are commonly used antigens because of the high specificity they confer. However, many of them show less desirable ability to bind to solid surfaces. Multimeric peptide constructs, particularly those with dendritic (branched) arrangements, tend to be much better than linear constructs in overcoming such limitations. This may be because those peptides tend to adopt a more extended spatial organization than linear species, which provides increased surface binding properties and enhanced sensitivity, thereby becoming more effective in disease diagnosis.

[0177] Multi-epitope linear peptides contain more than one repeating sequence of a given epitope in a juxtaposed manner. For its part, multi-epitope dendrimer peptides have a branching structure with high molecular organization and stability. In dendrimers, molecular scaffolds are constructed near the core matrix, and different branches are tethered to the core matrix. Different amino acids are used for core formation, but lysine (Lys) is preferentially used because its two amino groups (α, ∑) can be used as branching points to generate diversity. In this work, we designed and produced two types of multi-epitope peptides, i.e., linear and branched versions of constructs with multiple repeating sequences of the same epitope (chimeric homologous tandem), and constructs with more than one repeat of different epitopes, which are always in a linear manner. In addition, we generate peptide constructs to optimize peptide immobilization by biotin labeling. All peptides were synthesized in C-terminal carboxamide form using Fmoc solid phase synthesis, purified to> 95% homogeneity using HPLC, and appropriately characterized using MS.

[0178] 2.2.Embedding the same source in series

[0179] It is worth mentioning that an interesting feature of peptide 6c is that it consists of a tandem repeat sequence of eight residues (PPPNXQEL, where X can be either amino acid S or A). Given the presence of those block tandem sequences in the ROP1 protein, we hypothesized that these eight residues might be involved in recognizing IgM. On this basis, we designed and generated other chimeric peptides with more than one repeat of the 6c sequence minus the two N-terminal residues (hereafter referred to as truncated 6c*) as the motif for three different constructs, namely i) a homologous tandem with two consecutive stretches of truncated peptide 6c (ID 6c*2), ii) a homologous tandem with two truncated 6c peptide sequences separated by a flexible spacer (8-amino-3,6-dioxaoctanoic acid (O2Oc)) (ID 6cO 3 6c†), and iii) a branched bivalent peptide based on a lysine core from which two truncated 6c peptides (ID(6c*)2K3) branched ( Fig.11 ).

[0180] 2.3. Chimeric heterologous tandem

[0181] To evaluate whether the combination of peptides 3a and 6c in a single construct could enhance the reactivity achieved with the original sequences tested together in solution (see Example 1 ), we designed and generated two chimeric peptides as follows: i) a heterotandem construct combining the truncated 6c and 3a sequences using 8-amino-3,6-dioxaoctanoic acid (O2Oc) as a flexible spacer between them (ID 6c3a ), and ii) a reverse heterotandem version in which we changed only the sequence order of the truncated 6c and 3a peptides (ID 3a6c ). Fig.12 ). In Table 17, we show therein each of the peptides shown in Sections 2.2, 2.3 and 2.4.

[0182] Table 17

[0183]

[0184] *minus residues EV at the N-terminus; PPPNXQEL repeats only (X=A, S)

[0185] † O=O 2 Oc, 8-amino-3,6-dioxaoctanoic acid ( )

[0186] 2.4. Biotinylated peptides

[0187] In many cases, proteins or peptides are randomly immobilized by simple adhesion to immunoassay solid surfaces, which results in the loss of functional epitopes responsible for conferring specificity to the molecules we aim to capture. Biotin labels have been used for decades for protein and peptide orientation and many laboratory research techniques (i.e., labeling, detection, and purification). The biotin moiety has a very strong affinity for streptavidin (Kd <10-10M), and therefore biotinylation is an effective method for specifically binding peptides to streptavidin-coated surfaces. Biotinylation can be performed at the N-terminus or the C-terminus. At the N-terminus, it can be performed directly on the primary terminal amino group, which is the strategy we chose.

[0188] We synthesized and purified different biotinylated peptides to investigate how different peptide orientations on the immunoassay surface could affect the reactivity of the final assay; i) biotinylated 3a sequence (ID 3ab), ii) biotinylated truncated 6c peptide (ID 6c*b), iii) 3a6c biotinylated heterologous tandem (ID 3a6c*b) and iv) biotinylated 7b peptide (ID7bb).

[0189] 2.5. Evaluation of chimeric peptides by ELISA

[0190] Once we have synthesized chimeric peptides, we evaluate their ability to detect IgM compared to the original peptides. Initially, we established the optimal assay conditions. To this end, we reproduced the procedure for the original sequence (see Example 1). Since we know the optimal assay conditions for the original sequence of the chimeric peptides we designed, we established different concentration ranges to be tested based on previous data. Therefore, we use 0.2 to 8 × 10-4 μg / mL for all chimeric peptides. For common IgM-IgG peptides, since they are tested for the first time, we evaluated a wider concentration range (from 50 to 0.2 μg / mL) (Table 18).

[0191] The checkerboard evaluation revealed that the signal-to-noise ratio (SNR) of the chimeric peptides was almost doubled compared to the signal-to-noise ratio (SNR) obtained by combining the 3a and 6c peptides in solution (Table 18). The chimeric peptide that showed the lowest SNR ratio was (6c*2)K3. In contrast, 3a6c* showed the highest SNR. The common IgM-IgG sequences (peptides 8a and 9a) showed lower SNRs compared to the original sequences tested in Example 1, indicating that they are not good candidates for use as antigens.

[0192]

[0193] Table 18. Summary of chessboard results for chimeric peptides. a Peptide 3a was tested from 10 to 0.6 μg / mL, and peptide 6c was tested from 0.2 to 0.05 μg / mL. b Selected concentrations for 3a and 6c peptides, respectively.

[0194] Once we evaluated the chimeric peptides using the checkerboard, we analyzed their performance using a conditioned sample set (n=94).

[0195] The use of chimeric peptides did not increase assay performance compared to the peptide combination in solution (3a+6c), with the exception of 3a6c (see Table 19), which achieved the same number of true positives (TPs) as 3a+6c, one true negative (TN), and one less false positive (FP) and false negative (FN) than 3a+6c.

[0196]

[0197] Table 19: Accuracy estimates for each chimeric peptide. Cutoffs are expressed in relative absorbance units (RAU). TP: True Positive, FP: False Positive, TN: True Negative, FN: False Negative. TPP: True Positive Proportion, also known as Sensitivity (Se), TNP: True Negative Proportion, also known as Specificity (Sp). FPP: False Positive Proportion and FNP: False Negative Proportion. *n=93 due to insufficient sample size.

[0198] Fig.13 The ROC curves for all chimeric peptides are presented. The 6cO36c peptide runs at a point in the indiscriminate region, indicating that it is unable to classify the sample according to its true state (91% sensitivity, 63% specificity). Although this sensitivity is the same as that of the 6c2, 6c3a, and (6c2)K3 peptides and the specificity is similar to that of the 6c2, 6c3a, and (6c2)K3 peptides, none of the other ROC curves cross the indiscriminate line, indicating that for this particular threshold, only the 6cO36c peptide misclassifies the sample.

[0199] We also calculated the area under the curve (AUC) for each ROC, with a 95% confidence interval (Table 20). Peptide 3a6c showed the highest AUC (0.90), followed by peptide 6c3a (0.88).

[0200]

[0201] Table 20: Area under the curve (AUC) of the ROC curves. For each chimeric peptide, the AUC and its 95% confidence interval (CI) were calculated using the Wilcoxon-Mann-Whitney test.

[0202] 2.6. Evaluation of biotinylated peptides via ELISA

[0203] So far, we have seen that the 3a6c chimeric peptide, which combines the sequences of peptides 3a and 6c within the same molecule, increased the assay performance compared to that obtained by testing the same peptides (3a + 6c) together in solution. As mentioned above, peptides randomly immobilized on the surface of an ELISA plate are prone to loss due to occlusion of functional epitopes, which has a direct impact on assay performance. Therefore, we wanted to clarify whether we could obtain better assay performance by controlling the orientation using biotinylated peptides. Although we knew that the best candidate so far was the 3a6c chimeric peptide, we continued to evaluate the biotinylated truncated 6c peptide (6c*b). We performed checkerboards to establish the optimal assay conditions. Since we already knew the optimal conditions for its non-labeled analogue, we started with a peptide concentration of 2.5 μg / mL on streptavidin-coated 96-well plates.

[0204] We performed this experiment using standard ELISA plates. When we did so, the absorbance of the blank was as expected, and the absorbance of the wells containing 2.5 μg / ml of the 6c*b peptide, although lower than that obtained with the 6c peptide, was three times higher than that of the blank (data not shown).

Claims

1. An antigen comprising an antigenic region of Toxoplasma gondii, wherein the antigenic region of Toxoplasma gondii comprises one or more amino acid sequences of SEQ ID: PPPNXQEL, wherein X can be any of amino acids S or A, and wherein the antigen is capable of specifically binding to a Toxoplasma gondii-specific IgM antibody.

2. The antigen of claim 1, wherein the antigenic region of Toxoplasma gondii comprises two or more amino acid sequences of SEQ ID: PPPNXQEL, wherein X can be any of the amino acids S or A, and wherein the C-terminus of each amino acid sequence is linked to the N-terminus of the subsequent amino acid sequence by a simple covalent bond, or they can employ a flexible linker domain, or polypeptide linkers consisting of small amino acids such as glycine, serine, threonine or alanine in various lengths and combinations.

3. The antigen according to any one of claims 1 or 2, wherein the antigenic region comprises any one of the following sequences: SEQ ID NO 1 (antigen 6c), SEQ ID NO 2 (6c*), SEQ ID NO 3 (6c*2) and / or SEQ ID NO 4 (6cO36c), and wherein each of these sequences is capable of specifically binding to Toxoplasma gondii-specific IgM antibodies and preferably has a maximum length of 150 amino acids.

4. The antigen according to claim 1, wherein the antigen is a chimeric antigen comprising two or more different antigenic regions, one of which is a sequence selected from any one of claims 1 or 2, and the other antigenic region is selected from any one of SEQ ID NO 5 (3a) and / or SEQ ID NO 6 (3b), and wherein the C-terminus of the first antigenic region is linked to the N-terminus of the other antigenic region by a simple covalent bond, or they can use a flexible linker domain, or a polypeptide linker composed of small amino acids such as glycine, serine, threonine or alanine in various lengths and combinations.

5. The chimeric antigen according to claim 4, comprising an amino acid sequence of SEQ ID NO: SEQ ID NO: 7 (3a6c), wherein this sequence is capable of specifically binding to a Toxoplasma gondii-specific IgM antibody and preferably has a maximum length of 150 amino acids.

6. The chimeric antigen according to claim 4, comprising the amino acid sequence of SEQ ID NO 8 (6c3a), wherein the sequence is capable of specifically binding to a Toxoplasma gondii-specific IgM antibody and preferably has a maximum length of 150 amino acids.

7. The chimeric antigen according to claim 4, wherein the antigen is selected from the group consisting of SEQ ID NO: 7 (3a6c) and / or SEQ ID NO 8 (6c3a).

8. In vitro use of any one of the antigens according to any one of claims 1 to 7 for detecting anti-Toxoplasma gondii IgM in an isolated test sample, preferably a biological sample, such as whole blood or a portion of whole blood, for example a plasma or serum sample.

9. Use of any one of the antigens according to any one of claims 1 to 7 for the in vitro diagnosis of acute toxoplasmosis in an isolated test sample or biological sample collected from a subject in need thereof.

10. A kit for diagnosing acute Toxoplasma gondii infection comprising at least one antigen according to any one of claims 1 to 7.

11. A kit for detecting Toxoplasma gondii-specific IgM antibodies in an isolated test sample or biological sample, comprising at least one antigen according to any one of claims 1 to 7.

12. An antigen according to any one of claims 1 to 7 for use in therapy.

13. An immunoassay device or kit for detecting IgM antibodies from Toxoplasma gondii infection using an antigen, wherein the immunoassay format contains at least one antigen according to any one of claims 1 to 7.

14. The immunoassay device or kit according to claim 13, wherein the antigen is labeled, or the device further comprises a labeled antibody; wherein the label is selected from enzymatic, fluorescent, chemiluminescent, radioactive or dye molecules.

15. An immunoassay device or kit according to any one of claims 13 or 14, wherein the device comprises a solid support bound to the antigen / antigens.

16. A kit according to claim 10 or 11 or an immunoassay according to any one of claims 13 to 15, wherein the kit contains in separate containers a combination of: antigens (already bound to a solid matrix or separated from reagents for binding them to the matrix), control antibody preparations (positive and / or negative), labeled antibodies when the assay format requires labeled antibodies and signal generating reagents (e.g., enzyme substrates) when the label does not directly generate a signal, and optionally instructions for performing the assay will typically be included in the kit.

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