EP2499244A1 - Impfstoffe und diagnostika für trypanosomiasis - Google Patents

Impfstoffe und diagnostika für trypanosomiasis

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Publication number
EP2499244A1
EP2499244A1 EP10782574A EP10782574A EP2499244A1 EP 2499244 A1 EP2499244 A1 EP 2499244A1 EP 10782574 A EP10782574 A EP 10782574A EP 10782574 A EP10782574 A EP 10782574A EP 2499244 A1 EP2499244 A1 EP 2499244A1
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EP
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Prior art keywords
tcots
vaccine
proteins
protein
sequences
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EP10782574A
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English (en)
French (fr)
Inventor
Virginie Coustou Linares
Theo Baltz
Nicolas Plazolles
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Centre National de la Recherche Scientifique CNRS
Universite Victor Segalen Bordeaux 2
Universite Bordeaux Segalen
Original Assignee
Centre National de la Recherche Scientifique CNRS
Universite Victor Segalen Bordeaux 2
Universite Bordeaux Segalen
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Application filed by Centre National de la Recherche Scientifique CNRS, Universite Victor Segalen Bordeaux 2, Universite Bordeaux Segalen filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP2499244A1 publication Critical patent/EP2499244A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase

Definitions

  • the present invention relates to a novel genetic material coding for trans-sialidase-like proteins belonging to African trypanosome parasites, and relates to the use of said genes and proteins for vaccinal, therapeutic and diagnostic purposes.
  • the present invention also relates to the immunization of non-human animals and / or humans against trypanosomoses or trypanosomiases.
  • Trypanosomiasis or trypanosomiasis is caused by several species of protozoan parasites of the genus Trypanosoma and African trypanosomes generally refer to trypanosomes belonging to the Salivaria group, which itself comprises three main subgenera: Trypanozoon, Duttonella and Nannomonas.
  • the subgenus Trypanozoon consists of polymorphous trypanosomes (long form and short or stout form), with an optional free flagellum and a small kinetoplast in the subterminal (posterior) position.
  • the species of this subgenus are Trypanosoma (T.) brucei, T. evansi, and T. equiperdum.
  • T. brucei comprises three subspecies: T. b. brucei, T. b. gambiense and T. b. rhodesiense, which are morphologically, antigenically and biochemically very similar, and are distinguished by their infectious characteristics, their pathogenicity and their geographical distribution.
  • T. brucei comprises three subspecies: T. b. brucei, T. b. gambiense and T. b. rhodesiense, which are morphologically, antigenically and biochemically very similar, and are distinguished by their infectious characteristics, their path
  • T. evansi is transmitted to cattle, horses and camels by biting flies other than tsetse flies (Tabanidae) in Africa, South America and South East Asia.
  • T. equiperdum has no invertebrate host (sexual transmission in horses). These last two species largely extend from tsetse flies and are cosmopolitan. Their morphology is similar to that of T. brucei but they are monomorphic (long forms only).
  • Trypanosomes belonging to the subgenus Duttonella have the shape of a club or club, with the rounded and broad posterior end, the body narrowing towards the anterior end.
  • the kinetoplast is bulky, rounded and in a terminal position; the undulating, undeveloped, narrow membrane terminates in free flagellum.
  • T. vivax and T. uniform are parasitic species of wild ruminants and servants. They can be transmitted mechanically or by tsetse flies, whose tubal and proventriculus they colonize exclusively.
  • Trypanosomes of the subgenus Nannomonas are small (8 to 24 ⁇ ), they have no free flagellum at any stage of their development.
  • the mid-sized kinetoplast is in a subterminal or marginal position.
  • the posterior end is rounded and the undulating membrane narrow.
  • Pathogenicity is important for livestock, pigs and dogs in Africa. Tsetse development takes place in the stomach and proboscis exclusively.
  • the main species are T. congolense and T. simiae. These trypanosomes are small, with rounded posterior end, kinetoplast in marginal position, narrow undulating membrane.
  • T. congolense pathogenic trypanosomes
  • T. vivax species of pathogenic trypanosomes
  • T. brucei pathogenic trypanosome species
  • Other animals are infected with another pathogenic trypanosome species, T. evansi, which is responsible for a pathology called Surra. Trypanosomes are characterized by a great genetic diversity, which concerns infectivity, virulence, pathogenicity, transmissibility, and susceptibility to trypanocidal products.
  • T. congolense is the main agent of bovine trypanosomosis in Africa, due to its frequency and pathogenicity. It also adapts to various non-human animal species, and can thus parasitize bovines, pigs, sheep, goats, equines, and canids.
  • T. brucei and in particular the subspecies Trypanosoma brucei gambiense is probably the most well-known because it is responsible for the chronic form of human sleeping sickness in West and Central Africa.
  • the subspecies Trypanosoma brucei brucei is a parasite of domestic and wild animals throughout Africa, but it is not infectious for humans because of the lytic effect on the blood forms of these trypanosomes, the protein Apolipoprotein L present in human serum.
  • the third subspecies is Trypanosoma brucei rhodesiense which is the agent of sleeping sickness in its acute form in Africa.
  • the subspecies T. evansi is transmitted to cattle, horses and camels, and has significant economic repercussions in Africa especially for the cattle and buffalo farms.
  • T. vivax is a parasite essentially ungulates in tropical Africa and transmission is provided by horseflies or tabanids.
  • Trypanosomes have a complex life cycle that includes different morphological forms. They generally have a fusiform body and possess a flagellum which is connected to the body by an undulating membrane. They reproduce asexually by binary fission.
  • the tsetse fly glossina sp.
  • tsetse fly injects into the dermis of the host at the site of the bite, infectious metacyclic forms present in the mouthparts. Parasites multiply in the dermis at the point of inoculation. A local reaction related to the multiplication of parasites in the dermis occurs, and parasites give birth to blood forms. This stage can last from 1 to 3 weeks for example in the case of T. congolense.
  • the parasites invade the blood, the lymphatic system, especially the lymph nodes, as well as various organs, such as the liver, spleen, heart, kidneys, testicles, and important lesions appear then.
  • the tsetse fly becomes infected and feeds on parasitized animals. Once infected, she remains infectious throughout her life.
  • T. brucei and T. congolense the trypanosome undergoes in the insect a complex cycle involving dedifferentiation in the intestine into non-infectious procyclic forms.
  • the trypanosomes turn into epimastigote, adherent forms, which actively multiply. Their differentiation leads to the infectious stage represented by the metacyclic forms, which no longer divide.
  • the T. vivax cycle does not involve a procyclic stage. It begins with the flagellar attachment of the blood forms ingested by the tsetse fly. They differentiate into epimastigote forms, which proliferate and then differentiate into infectious metacyclic forms.
  • the total cycle time in tsetse flies is approximately 5 to 10 days for T. vivax, 18 days for T. congolense, and 30 days for T. brucei.
  • Sources of infection of domestic animals are other domestic animals or diseased wild animals or healthy carriers.
  • the existence of reservoir comes from the fact that some species are not very receptive to infection, and not very sensitive to the disease.
  • T. congolense and T. brucei are exclusively transmitted by biological vectors such as tsetse flies, but T. vivax can also be transmitted by mechanical vectors such as biting flies (tabanids or stomoxes).
  • T. evansi is exclusively transmitted by mechanical vectors. The efficiency of transmission depends on the infection rate of tsetse flies and host-vector interactions. In general, infectious trypanosomes for animals give higher infection rates than trypanosomes infecting humans, which contributes to the very wide distribution of animal trypanosomosis.
  • Trypanosome analysis by electron microscopy shows the existence of a mantle of about 15 nm covering the entire cell body of the parasite.
  • This mantle is present only on the surface of blood and metacyclic forms. It consists essentially of a glycoprotein called VSG (Variable Surface Antigen) and other membrane proteins in small amounts. VSGs thus form a very dense structure constituting a physical barrier between the plasma membrane and the host. The 3D structure predicts that only a small portion of the protein is exposed to the surface of the parasite.
  • VSG Very Surface Antigen
  • the mantle would be to mask the nonvariable membrane antigens of the parasite by presenting some immunodominant motifs to the immune defenses of the host.
  • the mantle also protects the blood forms against lysis by activation of the alternative complement pathway.
  • the Applicant has identified and obtained a new genetic material coding for new trans-sialidase-like proteins, called TcoTS-like 1, 2, and 3, recognized by anti-anti-African trypanosomes.
  • the genetic material can be used to produce proteins and polypeptides for the development of diagnostic tests, the preparation of vaccine or pharmaceutical compositions against infections with African trypanosomes.
  • the protein and any corresponding polypeptide fragment can be used for the production of specific antibodies against the parasite for diagnostic purposes or for passive immunization.
  • Figure 1 represents the nucleotide sequence encoding the trans-sialidase-like protein TcoTS-like 1
  • Figure 2 represents the nucleotide sequence encoding the trans-sialidase-like protein TcoTS-like 2;
  • Figure 3 represents the nucleotide sequence encoding the trans-sialidase-like protein TcoTS-like 3;
  • Figure 4 represents the peptide sequence corresponding to the trans-sialidase-like protein like TcoTS-like 1;
  • Figure 5 represents the peptide sequence corresponding to the trans-sialidase-like protein like TcoTS-like 2;
  • Figure 6 represents the peptide sequence corresponding to the trans-sialidase-like protein TcoTS-like 3;
  • Figure 7 represents a sequence alignment between the transsialidase-like protein (TcoTS-like 2) and a trans-sialidase protein of the parasite Trypanosoma cruzi Y. cruzi TS);
  • Figures 8A and 8B show a schematic of the subfamilies of proteins related to trans-sialidases of the T. congolense parasite; Identity percentages between genes of the same subfamily are shown (Fig. 8A) with a table showing percentages of identity between these proteins (Fig. 8B);
  • Figure 9 Represents a nucleotide sequence encoding TcoTS-A1 protein
  • Figure 10 shows a nucleotide sequence encoding TcoTS-A2 protein
  • Figure 11 shows a nucleotide sequence encoding TcoTS-A3 protein
  • Figure 12 shows a nucleotide sequence encoding TcoTS-B1 protein
  • Figure 13 represents a nucleotide sequence encoding TcoTS-B2 protein
  • Figure 14 shows a nucleotide sequence encoding TcoTS-C protein
  • Figure 15 shows a nucleotide sequence encoding TcoTS-D1 protein
  • Figure 16 shows a nucleotide sequence encoding TcoTS-D2 protein
  • Figure 17 shows a peptide sequence corresponding to TcoTS-A1 protein
  • Figure 18 shows a peptide sequence corresponding to TcoTS-A2 protein
  • Figure 19 shows a peptide sequence corresponding to TcoTS-A3 protein
  • Figure 20 shows a peptide sequence corresponding to TcoTS-B1 protein
  • Figure 21 represents a peptide sequence corresponding to TcoTS-B2 protein
  • Figure 22 shows a peptide sequence corresponding to the TcoTS-1 protein.
  • Figure 23 shows the peptide sequence corresponding to the TcoTS-D1 protein
  • Figure 24 shows the peptide sequence corresponding to the TcoTS-D2 protein
  • Figures 25A and 25B show a sequence alignment between the 1 1 proteins related to trans-sialidases of the parasite Trypanosoma congolense
  • Figure 26 represents a table showing the identity percentages between the proteins related to trans-sialidases of T. congolense and T. brucei parasites.
  • Figure 27 represents a table of the different peptides identified in the immunoprecipitation experiment with anti-TcoTS-A1 serum. Their membership in TcoTS-A1, TcoTS-A2 or TcoTS-A3 (A), TcoTS-like 2 (B) and TcoTS-D2 (C) proteins.
  • Figure 28 represents a table of the different peptides identified in the experiment of T. congolense (A) blood-form membrane preparations, their membership in the TcoTS-A1, TcoTS-A2 or TcoTS-A3 proteins is represented by a +; and a table of peptides belonging to the TcoTS-like 2 protein identified during the immunoprecipitation experiments with anti-peptide 1, anti-peptide 2 or anti-peptide 3 (B) sera.
  • A blood-form membrane preparations
  • Figures 29A and 29B show the measurement of hematocrit (A) and mean survival (B) of mice after immunization with TcoTS-like 2, TcoTS-A1 and TcoTS-B1 proteins or with BSA. The number of mice (n) used during the different immunizations is indicated.
  • African trypanosomes means the protozoan parasites of the genus Trypanosoma belonging to the Salivaria group, which itself comprises three main subgenera: Trypanozoon, Duttonella and Nannomonas, as defined above. These are known as African trypanosomes, but are found today on the African continent as well as in Asia or South America. The most common African trypanosomes are Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi, and Trypanosoma brucei.
  • trypanosomosis and "African animal trypanosomiasis (AAT)” generally refer to non-human animal infections caused by African trypanosomes, while the terms “trypanosomiasis” or “African trypanosomiasis” are used to refer to human infections also caused by African trypanosomes. For the sake of simplification, the terms trypanosomiasis and trypanosomiasis are used interchangeably.
  • the present invention relates to a DNA or RNA molecule encoding novel trans-sialidase-like proteins, called TcoTS-like 1, 2, and 3, and belonging to African trypanosomes.
  • novel DNA or RNA molecules comprise at least one strand comprising a nucleotide sequence chosen from sequences SEQ ID NOs: 1 -3, a sequence complementary, antisense, or equivalent to one of the sequences SEQ ID Nos: 1 -3, and in particular a sequence comprising an identity of at least 70%, with one of the SEQ sequences ID NOs: 1 -3, or a sequence having, on a sequence of 100 contiguous nucleotides, at least 50%, preferably at least 60%, or at least 70%, or at least 80%, 85%, 90% , 91%, 92%, 93%, 94%, or 95% homology with said sequences, or a nucleotide sequence capable of hybridizing with one of the sequences SEQ ID NOs: 1-3 under stringent conditions of hybridization.
  • stringent hybridization conditions hybridization at a temperature of 65 ° C overnight in a solution containing 0.1% SDS, 0.7% skimmed milk powder and 6X SSC, followed by washes at room temperature. room temperature in 2X SSC - 0.1% SDS and at 65 ° C in 0.2X SSC - 0.1% SDS.
  • the invention also relates to DNA or RNA fragments whose nucleotide sequence is identical, complementary, antisense, or equivalent to any one of the following sequences SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and especially DNA or RNA fragments, for any suite of contiguous monomers, at least 50%, preferably at least 60%, or at least 85%, 90%, 91 %, 92%, 93%, 94%, or 95% homology with any of said sequences.
  • nucleotide sequence is meant at least one strand of DNA or its complementary strand, either an RNA strand or its antisense strand or their corresponding complementary DNAs.
  • the DNA sequence as represented in one of the sequences SEQ ID NOs: 1 -3 corresponds to the sequence of the messenger RNA, it being understood that the thymine (T) in the DNA is replaced by the uracil (U). in RNA.
  • two nucleotide sequences are said to be equivalent to one another, because of the natural variability, in particular spontaneous mutation of the species from which they have been identified, or induced, as well as homologous sequences, the homology being defined below.
  • variability is meant any modification, spontaneous or induced of a sequence, in particular by substitution, and / or insertion and / or deletion of nucleotides and / or nucleotide fragments, and / or extension and / or shortening of the sequence to at least one of the extremities, or a non-natural variability that may result from the genetic engineering techniques used.
  • This variability can result in modifications of any starting sequence, considered as a reference, and which can be expressed by a degree of homology with respect to said reference sequence.
  • Homology characterizes the degree of identity of two nucleotide fragments (or peptides) compared; it is measured by the percentage of identity that is particularly determined by direct comparison of nucleotide (or peptide) sequences, relative to reference nucleotide (or peptide) sequences.
  • the subject of the invention is also proteins, called TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3, having an apparent molecular mass of approximately 85 kDa for the TcoTS-like 1 protein, of approximately 76 kDa. for the TcoTS-like 2 protein, and about 78 kDa for the TcoTS-like 3 protein, and recognized by anti-anti-African Trypanosomes, as well as their antigenic peptide fragments or an immunological equivalent of these proteins or fragments.
  • the amino acid sequences of these proteins are shown in SEQ ID Nos. 4-6 and also include protein sequences homologous to at least 70%, 75%, 80%, 85%, 90%, or at least 95%. %.
  • the proteins newly characterized by the Applicant have in C-terminal a conserved lectin portion intended to allow the attachment on sialic acids of infected animals and N-terminal a catalytic part having a similarity with that of trans-sialidases enzymes and have therefore been designated by the Applicant trans-sialidases-like.
  • immunological equivalent any polypeptide or peptide capable of being immunologically recognized by the antibodies directed against said TcoTS-like proteins 1, 2, and 3.
  • the invention also relates to any fragment of the TcoTS-like 1, 2, and 3 proteins, and more particularly any antigenic peptide fragment specifically recognized by antisera anti-African trypanosomes.
  • proteins and said protein fragments according to the invention may comprise modifications, in particular chemical modifications, which do not alter their immunogenicity.
  • the present invention therefore also relates to one or more peptides, the amino acid sequence of which corresponds to a part of the TcoTS-like 1, TcoTS-like 2, and / or TcoTS-like 3 protein sequence, and which are present alone or in mixtures reactivity with all sera from non-human and / or human animals infected with African trypanosomes.
  • Peptides can be obtained by chemical synthesis, lysis of TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 proteins, or by genetic recombination techniques.
  • the latter relates to a functional expression cassette, in particular in a cell derived from a prokaryotic or eukaryotic organism, allowing the expression of DNA coding for all or a fragment of the proteins.
  • TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 as previously described.
  • a DNA fragment as defined above and placed under the control of the elements necessary for its expression. Said protein or protein fragment thus expressed is recognized by antisera ant-trypanosomes African.
  • any cell derived from a prokaryotic or eukaryotic organism may be used in the context of the present invention.
  • Such cells are known to those skilled in the art.
  • a eukaryotic organism such as cells derived from a mammal, in particular CHO (Chinese Hamster Ovarian) cells; insect cells; cells derived from a fungus, in particular a unicellular fungus or a yeast, in particular from the Pichia, Saccharomyces and Schizosaccharomyces strains, and very particularly selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae,
  • E coli Escherichia coli strain
  • enterobacterial cells may be used, but not limited to.
  • the cell may be wild type or mutant.
  • the mutations are described in the literature accessible to those skilled in the art.
  • an E. coli cell such as for example BL21 (DE3), is used.
  • the expression cassette of the invention is intended for the production of TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 proteins or fragments of these proteins, for example in E. coli, which are recognized by antisera. anti-African trypanosomes. Such antisera come from animals having acquired a recent or old infection by trypanosome species, T. congolense, T. brucei, T. evansi and / or T. vivax, and contain immunoglobulins specifically recognizing TcoTS-like proteins. TcoTS-like 2, and TcoTS-like 3.
  • TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 proteins can be recognized by other antibodies, for example monoclonal or polyclonal antibodies obtained by immunization of various species with the aforementioned natural protein, the recombinant protein, their fragments or peptides.
  • TcoTS-like 1 proteins, TcoTS-like 2, and TcoTS-like 3, or fragment is meant the antigen or antigenic fragment of natural African Trypanosomes, belonging to the species T. congolense, T. brucei, T. evansi and / or T. vivax, produced in particular by the genetic recombination techniques described in the present application, or any fragment or mutant of this antigen provided that it is immunologically reactive with antibodies directed against TcoTS-like 1 proteins, TcoTS-like 2, and TcoTS-like 3 of these parasites.
  • said proteins have an amino acid sequence having a degree of homology of at least 70%, 75%, 80%, 85%, 90%, or minus 95% with respect to the sequences SEQ ID Nos: 4-6.
  • an equivalent can be obtained by deletion, substitution and / or addition of one or more amino acids of the native or recombinant protein. It is within the abilities of those skilled in the art to make these modifications by known techniques without affecting the immunological recognition.
  • the TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 proteins can be modified in vitro, in particular by deletion or addition of chemical groups, such as phosphates, sugars or myristic acids, in order to improve its stability or the presentation of one or more epitopes.
  • chemical groups such as phosphates, sugars or myristic acids
  • the expression cassette according to the invention allows the production of TcoTS TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3, or an antigenic fragment of these proteins, having the amino acid sequences as specified. previously, and fragments of said proteins, may be advantageously fused to an exogenous element that may aid its stability, purification, production or recognition.
  • an exogenous element that may aid its stability, purification, production or recognition.
  • the choice of such an exogenous element is within the reach of those skilled in the art. It may especially be a hapten, or an exogenous peptide.
  • the expression cassette according to the invention comprises the elements necessary for the expression of said DNA fragment in the cell in question.
  • elements necessary for the expression is meant all the elements which allow the transcription of the DNA fragment into messenger RNA (mRNA), such as promoter sequences (for example the CMV promoter) and transcription terminator. , as well as elements allowing the translation of the latter into protein.
  • mRNA messenger RNA
  • promoter sequences for example the CMV promoter
  • transcription terminator for example the CMV promoter
  • the present invention extends to a vector comprising an expression cassette according to the invention. It may also be a plasmid vector with autonomous replication and in particular a multiplier vector. It may be a viral vector and in particular a vector derived from a baculovirus, more particularly intended for expression in insect cells, or a vector derived from an adenovirus for expression in the cells. mammalian cells.
  • the present invention also relates to a cell derived from a prokaryotic or eukaryotic organism, comprising an expression cassette, either in an integrated form in the cellular genome or inserted into a vector.
  • the present invention further relates to a process for preparing one or more proteins selected from TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3, or from antigenic fragments of said protein, according to which: (i) a cell derived from a prokaryotic or eukaryotic organism, including the expression cassette according to the invention; and (ii) recovering the expressed protein from the above organism.
  • the invention relates to monoclonal or polyclonal antibodies obtained by immunological reaction of a non-human animal organism to an immunogenic agent consisting of one or more TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 proteins. , natural or recombinant, and / or their antigenic peptide fragments, as defined above.
  • the polyclonal antibodies according to the present invention can be generated using the TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 (SEQ ID Nos: 4-6) proteins, which are injected into rabbits in order to immunize them as described in Example 2.
  • polyclonal rabbit sera which have been designated respectively anti-peptide 1 antibody, anti-peptide 2 antibody and anti-peptide 3 antibodies, are also part of the present invention since they exhibit a reactivity against their peptide of the invention by indirect ELISA.
  • the subject of the present invention is an active immunotherapeutic composition, especially a vaccine preparation which comprises one or more TcoTS-like 1, TcoTS-like 2, and natural TcoTS-like 3, recombinant proteins and / or their fragments. antigenic peptides, and / or a mixture of one or more TcoTS-like 1, TcoTS-like 2, and TcoTS-like 3 proteins, and / or a mixture of one or more peptide fragments as defined above and optionally an excipient and / or a suitable adjuvant.
  • the vaccine or veterinary compositions according to the invention are intended for the treatment and / or prevention of infection by African trypanosomes in humans and / or non-human animals, particularly against infections with T. congolense, T. brucei, T. evansi and / or T. vivax.
  • African trypanosomiasis result in syndromes of varying severity, ranging from acute lethal infection in 3-4 weeks to chronic infection for months to years.
  • the chronic course characterized by intermittent parasitaemia, is most common in cattle.
  • the disease begins with a hyperthermia phase, then two to three weeks after the infective bite, the number of red blood cells, hemoglobin and hematocrit fall, reflecting anemia, which is the major symptom of trypanosomosis.
  • Chronically infected animals have reduced food consumption, become cachectic, slow growth, and negative reproductive effects.
  • Anemia of trypanosomoses is established in two phases. During the initial phase, anemia is accompanied by parasitaemia and results mainly from extra haemolysis.
  • Vascular Red blood cells are destroyed by the phagocytic system in the spleen, liver, circulation and bone marrow. In the long run, anemia results in a dysfunction of the bone marrow.
  • Said vaccine compositions may be in the form of antigenic vaccine and then comprise a therapeutically effective amount of one or more TcoTS-like 1, TcoTS-like 2, and natural TcoTS-like 3, recombinant proteins, and / or their peptide fragments. antigenic as previously described.
  • the vaccine compositions may be in the form of DNA vaccines and may then comprise an expression cassette, a vector, a cell derived from a prokaryotic or eukaryotic organism as defined above, capable of expressing one or more TcoTS-like proteins. 1, TcoTS-like 2, and TcoTS-like 3, and / or their antigenic peptide fragments, and / or a combination thereof.
  • the DNA vaccines may contain DNA or RNA, modified nucleotide sequences, and preferably one or more expression vectors encoding an antigenic peptide or fragment under the control of a eukaryotic promoter sequence.
  • the vaccines according to the present invention may be monovalent vaccines comprising a therapeutically effective amount of one or more TcoTS-like 1, TcoTS-like 2, and natural TcoTS-like 3, recombinant proteins, and / or their antigenic peptide fragments, such as previously described and / or nucleotide sequences coding for these peptides or peptide fragments.
  • This monovalent vaccine prevents infestation and thus the expression of the disease.
  • anti-disease vaccine does not prevent the infestation but only the expression of the disease, it could be called “anti-disease” vaccine.
  • the use of multivalent vaccines combining the so-called “anti-disease” vaccine with antigens of other trypanosomes and / or Other therapeutic active agents and / or other vaccines commonly used for prophylaxis are particularly advantageous according to the present invention.
  • the vaccines according to the present invention may be monovalent vaccines combining one or more natural, recombinant proteins and / or peptide fragments and / or nucleotide sequence coding for said peptides and peptide fragments of one or more species of trypanosomes, and of preferably derived from one or more species similar or different from trypanosomes.
  • trans-sialidases examples include trans-sialidases of T. cruzi, T. congolense, T. vivax, T. evansi, T. brucei, T. rhodesiense. , and / or T. gambiense. Some trans-sialidases of T.
  • T. cruzi trans-sialidase chains A and B as deposited in GenBank under the numbers Gl: 29726491, Gl: 29726490, Gl: 29726489 and Gl: 29726488. It is also advantageous to use inactive mutated forms of transialidases. In this respect, mention may be made of the T. cruzi mutant trans-sialidases described, for example, in International Application WO2007 / 107488, which have conserved less than 20% of their sialidase and transferase enzymatic activity.
  • tubulin alpha T. brucei (deposited in GenBank accession number AAA30262.1), or tubulin beta (deposited in GenBank accession number AAA30261). .1), T. brucei epsilon tubulin (deposited in GenBank under accession number EAN77544.1), T. brucei epsilon tubulin TREU927 (referenced in NCBI under the numbers XP_822372.1 and XP_829157.1), T. brucei delta tubulin (deposited in GenBank under accession number EAN80045.1), T. brucei zeta tubulin (referenced in NCBI under the number XP 001218818.1), or T. brucei tubulins which are described in the international application WO 2008/134643.
  • flagellar proteins derived from trypanosomes mention may be made of the T. brucei flagellar protein as described in international application WO2002 / 19960, or the T. congolense flagellar protein described in the applicant's French application November 13, 2009 under the number FR09 / 58035. Mention may also be made of the T. brucei TREU927 flagellar protein or the flagella-like proteins (referenced in NCBI under the numbers XP 847376.1; XP_847374.1; XP_847295.1; XP_843961 .1; XP_847377.1), the TB-flagellar protein 44A from T.
  • T. brucei (deposited in GenBank accession number AAZ13310.1), the T. brucei flagellar protein TB-24 (deposited in GenBank accession number AAZ13308.1), the flagellar protein of T. brucei filed in GenBank under accession number AAZ1331 1 .1.
  • proteases examples include trypanosome cysteine proteases, such as T. congolense congopaine or trypanopaine-Tc, rhodesiense rhodesaine, chagasine or T. cruzi cruzipaine.
  • Vaccines according to the present invention may also include adjuvants to increase the antigenic response.
  • adjuvants are well known in the state of the art.
  • adjuvants mention may be made of vitamin E, gels or aluminum salts, such as aluminum hydroxide, aluminum phosphates, metal salts, saponins, polymers of aluminum, polyacrylic acid, such as carbopols®, non-ionic block polymers, fatty acid amines, such as avridine and DDA, dextran-based polymers, such as dextran sulfate, and DEAE dextran liposomes, bacterial immunogens, such as LPS, peptidoglycans, or CDMs.
  • vitamin E gels or aluminum salts, such as aluminum hydroxide, aluminum phosphates, metal salts, saponins, polymers of aluminum, polyacrylic acid, such as carbopols®, non-ionic block polymers, fatty acid amines, such as avridine and
  • Non-human animals that can be treated include, for example, cattle, ovids, felids, suids, camelids, and / or canines.
  • the vaccines may comprise a therapeutically effective amount of a monoclonal or polyclonal antibody as described below.
  • the multivalent vaccines according to the present invention may further contain antigens from other blood parasitoses derived for example from protozoa such as Theirera parva, Babesia bigemina, and B. divergens, T. annulata, for the treatment and / or prevention of trypanosomes. and theileriosis, anaplasmosis, and / or babesiosis.
  • the vaccines according to the present invention are particularly useful for treating and / or preventing trypanosomosis-induced pathogenesis, such as, in particular, anemia, deterioration of the general state, weight loss, and / or immunosuppression of men or women. non-human animals.
  • Monovalent or multivalent vaccines may also be administered in combination with anti-parasitic agents, anti-infective agents, and / or anti-symptomatic agents.
  • the antiparasitic agents are, for example, trypanocides, such as diamidine (pentamidine or pentamidine mesilate, diminazene or diminazene aceturate), arsenical derivatives such as melarsoprol®, melarsomine, eflornithine or DMFO, arsobal, MelBdm nitrofuran derivatives such as nifurtimox or 5-nitrofuran, ornithine analogues (eflornithine® or difluoromethylornithine), phenanthridrin (isometamidium or homidium®), a polysulfone naphthaea such as suramin®, an antimalinic such as quinapyramine, buthionine sulfoximine (BSO), azaserine, 6-diazo-5-oxo-norleucine (DON), and / or acivicin.
  • vaccines When vaccines are administered in combination with antiparasitics, these are preferably administered before and / or simultaneously and / or after the mono or multivalent vaccines previously described.
  • Other non-specific trypanosome antiparasitics are well known in the art, and are administered before and / or simultaneously and / or after the vaccines according to the invention. These include avermectins (ivermectin, abamectin, doramectin, eprinomectin and selamectin), pyrethrins (deltamethrin, etc.), and / or anthelmintic antiparasitics (oxybendazole, piperazine, flubendazole).
  • antibiotics such as ⁇ -lactams, fosfomycin, glycopeptides or polypeptides with antibiotic activity, bacitracin, aminoglycosides, macrolides, lincosamides, streptogramins , tetracyclines, phenicolis, fusidanides, or quinolones.
  • Anti-symptomatic agents are for example anti-anemic agents, such as iron, vitamin B12, folic acid, levofolinate calcium; hepatoprotective agents, such as flavonoids complexes (sylymarin silybin, etc.), turmeric, desmodium ascendens, and / or chrysanthellum Americanum (charcoal).
  • anti-anemic agents such as iron, vitamin B12, folic acid, levofolinate calcium
  • hepatoprotective agents such as flavonoids complexes (sylymarin silybin, etc.), turmeric, desmodium ascendens, and / or chrysanthellum Americanum (charcoal).
  • Non-steroidal anti-inflammatory drugs can be, among others, oxicams (meloxicam, piroxicam, and / or tenoxicam), salicylated derivatives (methyl salicylate and lysine acetylate), 2-arylpropionic acids (profenes), derivatives thereof. indolic sulfonamides, selective cox-2 NSAIDs (celecoxib, etoricoxib, etc.), phenylbutazone, niflumic acid, and / or phenamic acids.
  • oxicams meloxicam, piroxicam, and / or tenoxicam
  • salicylated derivatives methyl salicylate and lysine acetylate
  • 2-arylpropionic acids profenes
  • indolic sulfonamides selective cox-2 NSAIDs (celecoxib, etoricoxib, etc.)
  • phenylbutazone niflumic acid,
  • the subject of the present invention is probes or primers specific for African Trypanosomes, and their uses in diagnostic tests.
  • probe refers to DNA or RNA comprising at least one strand having a nucleotide sequence allowing hybridization to nucleic acids having at least one nucleotide sequence as represented in the SEQ sequences ID Nos: 1 -3, or a complementary sequence, or antisense, or equivalent to said sequence, and in particular a sequence having, 5 to 100 contiguous nucleotides, at least 50%, preferably at least 60%, or at least 85% homology to the sequences SEQ ID Nos: 1 -3, or to a synthetic oligonucleotide allowing such hybridization, unmodified or comprising one or more modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine , dimethylamino-5-deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any other modified base.
  • modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine , dimethylamino-5-
  • these probes can be modified at the level of the sugar, namely the replacement of at least one deoxyribose with a polyamide or at the level of the phosphate group, for example its replacement by esters, especially chosen from diphosphate, dialkyl and arylphosphonate and phosphorothioate esters.
  • the probes can be much shorter than the sequences identified in the sequences SEQ ID Nos: 1 -3.
  • such probes comprise at least 5 nucleotides, advantageously between 5 and 50 nucleotides, preferably around 20 nucleotides possessing hybridization specificity under determined conditions to form a hybridization complex with the DNA or the nucleotide.
  • the probes according to the invention can be used for diagnostic purposes, as a capture and / or detection probe.
  • the primers according to the invention comprise a sequence of 5 to 30 monomers chosen from the sequences SEQ ID Nos: 1 -3, and have a specificity of hybridization under predetermined conditions, for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), in an elongation method such as sequencing, in a reverse transcription method or the like.
  • an amplification technique such as PCR (Polymerase Chain Reaction)
  • elongation method such as sequencing
  • reverse transcription method or the like.
  • the present invention relates to a reagent for the detection and / or monitoring and a method and kits for the diagnosis of infections with African trypanosomes, including T. congolense, T. brucei, T. evansi and / or T. vivax.
  • the reagents for the detection or trypanosome diagnostic kits comprise as reactive substance at least one monoclonal or polyclonal antibody as described above.
  • the reagents for the detection or diagnostic kit of trypanosomes may comprise a probe and / or a primer as defined above, for detecting and / or identifying African trypanosomes in a biological sample, in particular a capture probe and a probe. detection, one and / or the other as defined above.
  • the above reagent can be attached directly or indirectly to a suitable solid support.
  • the solid support may be in particular in the form of a cone, a tube, a well, a ball, or the like.
  • the term "solid support” as used herein includes all materials on which a reagent may be immobilized for use in diagnostic tests.
  • Natural, synthetic, chemically modified or non-chemically modified materials can be used as solid supports, especially polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose; polymers such as vinyl chloride, polyethylene, polystyrenes, polyacrylate or copolymers such as polymers of vinyl chloride and propylene, polymers of vinyl chloride and vinyl acetate, styrene-based copolymers, natural fibers such as cotton and synthetic fibers such as nylon.
  • polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose
  • polymers such as vinyl chloride, polyethylene, polystyrenes, polyacrylate or copolymers such as polymers of vinyl chloride and propylene, polymers of vinyl chloride and vinyl acetate, styrene-based copolymers, natural fibers such as cotton and synthetic fibers such as nylon.
  • the attachment of the reagent to the solid support can be carried out directly or indirectly.
  • two approaches are possible: either by adsorption of the reagent on the solid support, that is to say by non-covalent bonds (mainly of hydrogen, Van der Walls or ionic type), or by establishment of covalent bonds between the reagent and the support.
  • an anti-TcoTS-like antibody 1, 2 and 3 provided that it is immunologically reactive with a part of the protein different from that involved in the antibody recognition reaction of the antibodies.
  • a ligand-receptor system for example by grafting on proteins TcoTS-like 1, 2, and 3 a molecule such as a vitamin, and immobilizing on the solid phase the corresponding receptor (for example the biotin-streptavidin system).
  • indirect means is also meant the prior grafting or fusion by genetic recombination of a protein, or a fragment thereof, or of a polypeptide, at one end of the TcoTS-like 1 proteins, TcoTS-like 2, and TcoTS-like 3, and the immobilization of the latter on the solid support by passive adsorption or covalence of the grafted or fused protein or polypeptide.
  • the capture probes can be immobilized on a solid support by any appropriate means, that is to say directly or indirectly, for example by covalence or passive adsorption.
  • the detection probes are labeled with a marker chosen from radioactive isotopes, enzymes chosen in particular from peroxidase and alkaline phosphatase, and those capable of hydrolyzing a chromogenic, fluorogenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorogenic or luminescent compounds, nucleotide base analogs, and biotin.
  • the probes of the present invention used for diagnostic purposes can be implemented in all known hybridization techniques, and in particular the so-called “Dot-Blot”, Southern blot, and Northern Blot techniques, which is a technique identical to the Southern blot technique but uses RNA as a target, the sandwich technique.
  • the method for detecting and / or monitoring an infection with African trypanosomes in a biological sample is to in contact with said sample and a reagent as defined above, in conditions permitting a possible immunological reaction, and then detecting the presence of an immune complex with said reagent.
  • the method of detection by the ELISA technique in one or more steps, which consists in reacting a first monoclonal or polyclonal antibody specific for a desired antigen, fixed on a solid support, with the sample, and to highlight the possible presence of an immune complex thus formed by a second antibody labeled with any suitable marker known to those skilled in the art, including a radioactive isotope, an enzyme, for example peroxidase or phosphatase alkaline or the like; by the so-called competition techniques well known to those skilled in the art.
  • the method for the selective detection of African trypanosomes in a biological sample, and the diagnosis of trypanosomoses consists in taking a blood sample, exposing the DNA extracted from the sample and optionally denatured to at least one probe as defined above. and hybridization of said probe is detected.
  • the subject of the present invention is a kit for veterinary use for the diagnosis of trypanosomiasis in a biological sample comprising a probe or a primer as described above, or an antibody as previously described, as well as a reagent for detection of an immunological reaction.
  • kits according to the present invention comprise at least one compartment for a possibly sterile packaging comprising a therapeutically effective amount of a reagent as described above, as well as an instruction sheet concerning the protocol for implementing the veterinary diagnosis according to the invention. 'invention.
  • the subject of the present invention is the sequences related to trans-sialidases-like in T. congolense. More specifically, eleven genes encoding sialidase-related sequences have been characterized and classified into 5 subfamilies according to their sequence homologies (FIGS. 8A and 8B):
  • the first subfamily Trans-sialidases-like comprises the 3 previously described genes designated TcoTS-like 1, 2, and 3, which have 17 to 24% identity with each other ( Figures 1 to 6).
  • the second subfamily has been named subfamily A and comprises three genes designated A1, A2, and A3 and whose nucleotide sequences are given respectively in SEQ ID Nos: 7, 8, and 9.
  • the genes A1, A2, and A3 have 94 to 97% identity with each other ( Figures 9 to 1 1) and encode respectively three proteins TcoTS-A1, TcoTS-A2, and TcoTS-A3, whose amino acid sequences are provided respectively in SEQ IDs. Nos .: 15, 16, and 17 ( Figures 17-19).
  • the third subfamily designated B comprises two genes designated hereinafter B1 and B2, whose nucleotide sequences are respectively given in SEQ ID Nos: 10 and 11, and which have 76% identity with each other (FIGS. 12 and 13). ).
  • Both genes B1 and B2 encode trans-sialidases TcoTS-B1 and TcoTS-B2 whose peptide sequences are shown in SEQ ID Nos: 18 and 19 (FIGS. 20 and 21).
  • the fourth subfamily designated C comprises a single gene designated C, whose nucleotide sequence is represented in SEQ ID NO: 12 (FIG. 14), and which encodes the TcoTS-C protein whose peptide sequence is provided in SEQ. ID NO: 20 ( Figure 22).
  • subfamily D comprises two genes named D1 and D2, whose nucleotide sequences are provided in SEQ ID Nos: 13 and 14 ( Figures 15 and 16). These two genes D1 and D2 have indeed 96% identity between them. They encode the TcoTS-D1 and TcoTS-D2 proteins whose amino acid sequences are provided in SEQ ID Nos. 21 and 22 (FIGS. 23 and 24).
  • the subject of the present invention is therefore new nucleotide sequences coding for new trans-sialidase-like proteins called TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS.
  • This novel DNA or RNA molecule comprises at least one strand comprising a nucleotide sequence chosen from the sequences SEQ ID NOs: 7-14, a complementary sequence, antisense, or equivalent to one of the sequences SEQ ID Nos: 7-14, and in particular a sequence comprising an identity of at least 70%, with one of the sequences SEQ ID NOs: 7-14, or a sequence having, on a sequence of 100 contiguous nucleotides, at least 50%, preferably at least 60%, or at least 70%, or at least 80% homology with said sequences, or a nucleotide sequence capable of hybridizing with one of the sequences SEQ ID NOs: 7-14 in stringen conditions Hybridization tests, as defined above.
  • the invention also relates to DNA or RNA fragments whose nucleotide sequence is identical, complementary, antisense, or equivalent to any one of the SEQ ID NOs: 7-14 sequences, and in particular the fragments of DNA or RNA, for any sequence of contiguous monomers, at least 50%, preferably at least 60%, or at least 85% homology with any one of said sequences.
  • the invention relates to the so-called TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2 proteins, as well as the peptide sequences. of these proteins as represented respectively in the sequences SEQ ID Nos: 15-22, and any amino acid sequences having a homology of at least 70%, 75%, 80%, 85%, 90%, or from minus 95% with the peptide sequences SEQ ID Nos: 15-22.
  • the invention also relates to all antigenic peptide fragments of TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2 proteins, specifically recognized by antisera. anti-African trypanosomes, as well as all immunological functional equivalents of these proteins which may be immunologically recognized by the antibodies directed against TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C proteins, TcoTS-D1, and TcoTS-D2 of the present invention.
  • the proteins and said antigenic peptide fragments according to the invention may comprise modifications, in particular chemical modifications, which do not alter their immunogenicity.
  • a peptide antigenic fragment according to the present invention may be the peptide PKNIKGSWHRDRLQLWLTD (SEQ ID NO: 24) belonging to the TcoTS-B1 protein or peptides homologous to at least 70%, 75%, 80%, 85%. %, 90%, or at least 95% with said fragment.
  • the present invention further relates to the combination or a mixture of one or more proteins selected from TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS -B2, TcoTS-C, TcoTS-D1, and TcoTS-D2, and / or one or more antigenic peptide fragments of these proteins, and / or one or more immunological functional equivalents of these proteins.
  • These techniques for producing proteins, fragments, functional equivalents, and combinations are performed by chemical synthesis, lysis of proteins, or by genetic recombination techniques. They are well known to those skilled in the art, and have been described above.
  • the invention relates to monoclonal or polyclonal antibodies obtained by immunological reaction of a non-human animal organism to an agent.
  • immunogen consisting of one or more TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2 natural or recombinant proteins and their peptide fragments as previously described.
  • a vaccine composition comprising a mixture of one or more proteins selected from TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS -B2, TcoTS-C, TcoTS-D1, and TcoTS-D2, and / or one or more antigenic peptide fragments of these proteins, and / or one or more immunological functional equivalents of these proteins and / or a combination of said proteins, fragments or functional equivalents.
  • TbTS gene family (AF310231.1) in T. brucei. It describes, in particular, a truncated version of the TbTS gene, namely TbTSsh, the genes B and C coding for TbSA B and TbSA C transialidases of T. brucei, and finally the genes D1, D2 and E coding for trans-sialidases. like T. brucei.
  • TbTSsh a truncated version of the TbTS gene
  • the genes B and C coding for TbSA B and TbSA C transialidases of T. brucei and finally the genes D1, D2 and E coding for trans-sialidases.
  • T. brucei The identity percentages between the sequences identified in T. congolense and T. brucei are presented in Figure 26. Montagna et al.
  • trans-sialidases are expressed in vivo in insect or procyclic forms, and probably play an important role in the transfer of sialic acid to the parasite membrane, thus ensuring parasite protection and survival when transported by insect vectors.
  • Montagna et al. does not describe the possibility of detecting these trans-sialidases in sufficient quantities in the blood forms of the parasites, that is to say in the infected animals and thus to use them as vaccines or diagnoses.
  • the Applicant has furthermore demonstrated during the immunization protection experiment on murine models (Example 5, FIGS. 29A and 29B) that the TcoTS-A1, TcoTS-B1 and TS-like 2 antigenic proteins made it possible to obtain a protective effect. higher in terms of average survival of animals as well as in relation to hematocrit. This protection can even be total (absence of development of parasitaemia and normal hematocrit) in some cases: 3 mice out of 12 in the case of TcoTS-like2 and 1 out of 9 in the case of TcoTS-B1.
  • the present invention relates to vaccine or veterinary compositions for the treatment and / or prevention of infection with African trypanosomes in a non-human animal, particularly against T. congolense, T. brucei infections. , T. evansi and / or T. vivax.
  • These veterinary vaccine compositions may be in the form of antigenic vaccine and then comprise a therapeutically effective amount of one or more proteins selected from TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-A2.
  • said vaccine or veterinary compositions comprise at least one protein selected from TcoTS-A1, TcoTS-B1, and TcoTS-like 2. Even more preferably, said vaccine or veterinary compositions comprise at least TcoTS-like protein. 2, and / or an antigenic peptide fragment, and / or an immunological functional equivalent of TcoTS-like 2.
  • the vaccine compositions may comprise a therapeutically effective amount of a monoclonal or polyclonal antibody directed against one or more proteins selected from TcoTS. -like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2.
  • TcoTS monoclonal or polyclonal antibody directed against one or more proteins selected from TcoTS.
  • TcoTS TcoTS-like 1
  • TcoTS-like 3 TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2.
  • trypanosomosis-induced pathogenesis such as, in particular, anemia, general state deterioration, weight loss, and / or immunosuppression in non-human animals.
  • the present invention relates to a reagent for detection and / or monitoring and a method and kits for the diagnosis of infections with African trypanosomes, including T. congolense, T. brucei, T. evansi and / or T. vivax.
  • the detection reagents or trypanosome diagnostic kits comprise as reactive substance at least one monoclonal or polyclonal antibody directed against one or more TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS proteins. -C, TcoTS-D1, and TcoTS-D2.
  • the reagents for the detection or trypanosome diagnostic kits comprise as reactive substance at least one monoclonal or polyclonal antibody directed against one or more proteins selected from TcoTS-A1, TcoTS-A2, TcoTS-A3, and TcoTS- like 2.
  • the method for detecting and / or monitoring an infection with African trypanosomes in a biological sample is to contacting said sample and a reagent as defined above, under conditions allowing a possible immunological reaction, and then detecting the presence of an immune complex with said reagent.
  • the method of detection by the ELISA technique in one or more steps, which consists in reacting a first monoclonal or polyclonal antibody specific for a desired antigen, fixed on a solid support, with the sample, and to highlight the possible presence of an immune complex thus formed by a second antibody labeled with any suitable marker known to those skilled in the art, including a radioactive isotope, an enzyme, for example peroxidase or phosphatase alkaline or the like; by the so-called competition techniques well known to those skilled in the art.
  • kits for veterinary use for the diagnosis of trypanosomosis in a biological sample comprising an antibody as described above and a reagent for the detection of an immunological reaction.
  • kits according to the present invention comprise at least one compartment for a possibly sterile packaging comprising a therapeutically effective amount of a reagent as described above, as well as an instruction sheet concerning the protocol for implementing the veterinary diagnosis according to the invention. 'invention.
  • TcoTS-A1 protein was produced in the yeast Pichia pastoris.
  • the X33 strain was transformed by the PICZ vector (Invitrogen) containing the coding sequence for the TcoTS-A1 protein lacking its first 29 amino acids.
  • Protein secreted into the culture supernatant after 4 days of induction of methanol expression was purified by successive ion exchange chromatography. First, the culture supernatant was dialyzed against 20mM NaAc buffer pH4.5 for 16 hours, centrifuged for 30 minutes at 10000g, and then chromatographed on 1 HP HiTrap SP HP column (GE Healthcare).
  • This purified recombinant protein was then used to immunize Balb-c mice or rabbits. 20 g of recombinant protein were injected into the mice at 4 times spaced 15 days or 100 g of recombinant protein were injected into the rabbits at a rate of 4 times spaced 15 days. For the first injection the recombinant protein was mixed as an emulsion with complete Freund's adjuvant and then for subsequent injections with incomplete Freund's adjuvant. The serum of the immunized animals was collected at the end of the experiment (anti-TcoTS-A1 serum) and its reactivity against the recombinant protein was verified by indirect ELISA test.
  • Example 2 Production of polyclonal antibodies against peptides derived from sequences related to sialidases.
  • C-RTSIDYHLIDTVAKYSADDG SEQ ID NO: 23
  • C-PKNIKGSWHRDRLQLWLTD SEQ ID NO: 24
  • C-PVSAQGQDHRYEAANAEHT SEQ ID NO: 25
  • peptides 1, 2 and 3 were coupled through cysteine Nter to a carrier protein (KLH) activated by a maleimide function and used to immunize rabbits at 5 injections of 100 g spaced 20 days.
  • KLH carrier protein
  • the recombinant protein was mixed as an emulsion with complete Freund's adjuvant and then for subsequent injections with incomplete Freund's adjuvant.
  • the polyclonal sera obtained were designated respectively as anti-peptide 1, anti-peptide 2, and anti-peptide 3 antibodies, were harvested at the end of the experiment and checked for their reactivity against their respective peptide by indirect ELISA.
  • the IgGs were then incubated for 2 hours at room temperature with BrCN-activated Sepharose (Sigma) previously prepared according to the supplier's recommendations. After centrifugation for 1 minute at 1000 g, the resin was washed with the previous buffer and then saturated by adding Tris-HCl 0.1 M pH8 for 2 hours at room temperature. After centrifugation for 1 minute at 1000 g, the resin was washed successively with Tris-HCl buffer pH8 0.5M NaCl and then 0.1 M NaCl pHM NaCl 0.5M buffer.
  • the resin thus ready for use for an immunoprecipitation experiment was equilibrated with OLB buffer (100 mM KCl, 17% glycerol, 1 mM MgCl 2 , 2.25 mM CaCl 2 0.5% NP40, 10 mM Tris -HCl pH8).
  • OLB buffer 100 mM KCl, 17% glycerol, 1 mM MgCl 2 , 2.25 mM CaCl 2 0.5% NP40, 10 mM Tris -HCl pH8.
  • 10 9 cell of the IL3000 strain were lysed in the OLB buffer for 1 hour at 4 ⁇ C and then centrifuged 10 minutes at 20,000 g. The supernatant was incubated with the resin previously prepared for 16 hours at 4 ° C. The resin was then centrifuged for 1 minute at 1000 g and then rinsed with OLB buffer The IgG-bound antigens were eluted with boiling SDS 2%.
  • the eluate was dialyzed against water and then freeze-dried
  • the gel was then stained with silver nitrate and the bands thus revealed were cut and analyzed by mass spectrometry using MSMS technology.
  • 109 cells of the IL3000 strain were lysed in 1 ml of hypotonic buffer (5 mM Na2HP04, 0.3 mM KH2P04) for 30 minutes at 4 ⁇ € then centrifuged 10 minutes at 20,000 g. The pellet was subjected to the same treatment 3 times in a row. The last pellet is taken up at 4 ° C. in 100 ⁇ l of this same hypotonic lysis buffer, to which 0.5 ml of the following buffer are then added: 2 mM EDTA, 15.4 mM NaOH, 0.2 mM dithiothreitol. After 10 minutes of incubation, the mixture is centrifuged for 10 minutes at 20000 g.
  • hypotonic buffer 5 mM Na2HP04, 0.3 mM KH2P04
  • the supernatant is recovered (soluble fraction) and the pellet (insoluble fraction) is taken up in 50 ⁇ l of water to which are then added 50 ⁇ l of 2% SDS.
  • mice 2 batches of Balb-c mice are injected intraperitoneally with 20 g of BSA (negative control mouse lot) or with a TcoTS-like 1 recombinant protein (batch of immunized mice) at a rate of 4 times spaced 15 days apart. Then, the mice are infected with 10 4 parasites of the T. congolense strain IL3000. Hematocrit and parasitaemia are measured every 2 days on both lots of mice.
  • the average hematocrit over the duration of the parasitaemia was calculated: it is 43.311, 2% for the mice immunized with TcoTS-like2 and 37.0 + 0.7% for the control mice immunized with BSA ( Figure 28)
  • mice survival was also determined, it is 453 + 81 hours for the TcoTS-like2 immunized mice and 267 + 23 hours for the BSA immunized control mice.
  • mice 2 batches of Balb-c mice are injected intraperitoneally with 20 g of BSA (batch of negative control mice) or with the recombinant protein TcoTS-like 3 (batch of immunized mice) at a rate of 4 times spaced 15 days. Then, the mice are infected with 10 4 parasites of the T. congolense strain IL3000. Hematocrit and parasitaemia are measured every 2 days on both lots of mice.
  • Example 5.4 TocTS-A1 vaccination trials
  • mice were injected intraperitoneally with 20 g of BSA (8 negative control mice) or TcoTS-A1 recombinant protein (5 mice) at a rate of 4 times spaced 15 days apart. Then, the mice were infected with 10 4 parasites of the T. congolense strain IL3000. Hematocrit and parasitaemia were measured every 2 days.
  • the average hematocrit over the duration of the parasitaemia was calculated: it is 41.4% for the TcoTS-A1 immunized mice and 37.0 ⁇ 0.7% for the immunized mice. of the BSA ( Figure 28)
  • mice survival was also determined, it is 299 ⁇ 14 hours for the TcoTS-A1 immunized mice and 267 ⁇ 23 hours for the BSA immunized control mice.
  • mice were injected intraperitoneally with 20 g of BSA (8 negative control mice) or TcoTS-B1 recombinant protein (4 mice) 4 times spaced 15 days apart. Then, the mice were infected with 10 4 parasites of the strain IL3000 from T. congolense. Hematocrit and parasitaemia were measured every 2 days.
  • the average hematocrit over the duration of the parasitaemia was calculated: it is 41.4 ⁇ 0.5% for the mice immunized with TcoTS-B1 and 37.0 ⁇ 0.7% for the control mice immunized with of the BSA ( Figure 28)
  • Example 5.6 Vaccination assays with one or more proteins selected from TcoTS-A2, TcoTS-A3, TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2.
  • mice 2 batches of Balb-c mice are injected intraperitoneally with 20 g of BSA (negative control mouse lot) or with one or more recombinant proteins selected from the TcoTS-A2 proteins, TcoTS-A3, TcoTS-B2, TcoTS- C, TcoTS-D1, and TcoTS-D2 (batch of immunized mice) at 4 times spaced 15 days. Then, the mice are infected with 10 4 parasites of the T. congolense strain IL3000. Hematocrit and parasitaemia are measured every 2 days on both lots of mice.
  • 2 batches of cattle are injected subcutaneously with one or more antigens such as TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2 , TcoTS-C, TcoTS-D1, and TcoTS-D2, mixed with two types of additives Quil A (saponin) 1 mg / ml and Adjuphos (colloidal aluminum phosphate) volume to volume in a final volume of 1 mL or just with the mixture of adjuvants (control). 3 injections at 3-week intervals are carried out with respectively 100 ⁇ g, 50 ⁇ g and 25 ⁇ g of antigen (s).
  • antigens such as TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-A2, TcoTS-A3, TcoTS-B1 TcoTS-B2 , TcoTS-C, TcoTS-D1, and
  • the animals are infected with T. congolense strain IL3000 3 weeks after the last injection at the rate of 1000 parasites per animal intradermally.
  • Daily blood samples are taken until all the animals are recognized as infected, the determination of the parasitemia being done on buffy-coat.
  • weekly blood samples are taken to monitor parasitaemia and anemia, the weight of the animals is monitored monthly. The kinetics of response to immunization and infection is followed by ELISA on the different immunizing antigens.
  • the antigens used in this immunization experiment may be TcoTS-like 1, 2 or 3 or TcoTS-A1 or TcoTS-B1, alone or in combination in any combination possible.
  • Example 7 Example of diagnostic tests on blood of infected animals.
  • This test is carried out by detection of circulating antigens such as TcoTS-A1, TcoTS-A2, TcoTS-A3, and TcoTS-like 2 by the sandwich ELISA method.
  • the capture of said antibody is adsorbed to the wells of a 96 well plate by incubation overnight at 4 ⁇ C of 1 to 10 .mu.g / ml capture antibody diluted in 100 ⁇ ⁇ - NaHC0 3 buffer 50 mM pH 9 6.
  • the plate is then emptied and then washed 3 times with 200 ⁇ l per well of a solution of PBS-Tween (3.2 mM Na 2 HPO 4, 0.5 mM KH 2 PO 4, 1.3 mM KCl, 135 mM NaCl, pH 7, 4, 0.05% Tween 20). Then 100 ⁇ l of a blocking solution (PBS-Tween 0.2% gelatin) are added to each well and incubated for 30 minutes at room temperature. The plates are emptied then 100 ⁇ of sera of test animals are deposited in the wells and incubated for 2 hours at 37 ⁇ ⁇ . The plate is then emptied and then washed 3 times with 200 ⁇ l per well of a solution of PBS-Tween.
  • PBS-Tween 3.2 mM Na 2 HPO 4, 0.5 mM KH 2 PO 4, 1.3 mM KCl, 135 mM NaCl, pH 7, 4, 0.05% Tween 20.
  • the capture antibody used may be either an immunopurified polyclonal serum against one or a mixture of T. congolense sialidase-like proteins, such as TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS -A2, TcoTS-A3, TcoTS-B1 TcoTS-B2, TcoTS-C, TcoTS-D1, and TcoTS-D2, or a monoclonal antibody recognizing an epitope present on one or more of these T. congolense sialidase-like proteins. .
  • the second antibody is a monoclonal antibody different from the capture antibody which recognizes an epitope different from one or more of the TcoTS-like 1, TcoTS-like 2, TcoTS-like 3, TcoTS-A1, TcoTS-2 sialidase-like proteins.

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