OA18773A - Treatment and detection of Trypanosomes. - Google Patents

Abstract

The present invention relates to methods and compositions for preventing, treating and diagnosing trypanosome infection. It concerns the use of excreted / secreted antigens (exoantigens, secretome) and more specifically the identification of a protein excreted / secreted by trypanosomes, whose inhibition can confer effective protection against infection, development or the spread of trypanosomes, mainly by vaccination. It relates to the use of the protein or of its derivatives or a nucleotide sequence derived therefrom, or of an extract enriched in this protein, as well as that of antibodies directed against them for immunotherapy, diagnosis and monitoring for trypanosome infections.

Description

TREATMENT AND DETECTION OF TRYPANOSOMOSIS

The present invention relates to methods and compositions for preventing, treating and diagnosing a trypanosome infection. It relates in particular to the use of excreted / secreted antigens (exoantigens, secretome) and, more particularly the identification of a protein excreted / secreted by trypanosomes, the neutralization or inhibition of which makes it possible to confer effective protection against infection, development or spread of trypanosomes, primarily by vaccination. The invention allows cross-action against different strains of trypanosomes, and thus provides effective methods and compositions for preventing and combating infections and pathologies induced by trypanosomes in mammals, diagnosing them more precisely, and monitoring the development of the disease. infection after treatment.

INTRODUCTION

Trypanosomes (Trypanosome) are parasitic protozoa mainly infecting mammalian animals, but also humans. The infection causes animals to develop trypanosomiasis, or trypanosomiasis, which can lead to the death of the animal. In humans, human African trypanosomiasis begins with an inoculation canker, followed by a lymphatic-blood phase. with in particular a fever, lymphadenopathy, hepatosplenomegaly, pruritus and edema. A meningocencephalitic phase follows, when the parasite enters the central nervous system, with various neurological signs and in particular sleep disorders (hence its name, sleeping sickness). Chagas disease (human American trypanosomiasis) is caused by Trypanosoma cruzi, transmitted by bedbugs. When T. cruzi penetrates through the skin, there may appear an erysipeloid or pseodofuroncular skin lesion (chagoma). then sometimes sometimes a unilateral bi-palpebral edema (Sign of Romana). The acute phase may go unnoticed, and more rarely, manifest as febrile hepatosplenomegaly. The chronic phase is dominated by the severity of the cardiac forms and the existence of digestive forms with mega organs.

Trypanosomes are characterized by a great genetic diversity, which plays on tropism, virulence, transmissibility, and sensitivity to trypanocides. Among the various groups of trypanosomes, particular mention will be made of the group stercoraria. in which are placed Trypanosoma cruzi, T. theileri, T. lewisi and T muscttli and the group Salivaria, which itself includes three main subgenera: Trypanozoon, Duttonella and Nannomonas. The subgenus Trypanozoon includes species of extracellularly developing trypanosomes that infect animals and humans, while Duttonella and Nannomonas only infect non-human mammals. The subgenus Trypanozoon consists of polymorphic trypanosomes (long form and short or stocky form), with optional free flagellum and small kinetoplast and in subtenninal (posterior) position. The main species in this subgenus are Trypanosoma (T.) brucei, T. evansi and T. equiperdum. T. brucei includes three subspecies: T. b. brucei, T. b. gambiense and T. b. rhodesiense, which are very similar morphologically, antigenically and biochemically, and which are distinguished mainly by their infectious nature, their pathogenicity and their geographic distribution. T. brucei and its subspecies are transmitted by tsetse flies. 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. equiperditm does not have an invertebrate host (sexual transmission in horses), it has been found in Europe, Asia. Africa and America. The trypanosomes of the Duttonella subgender have a club or club shape. The main species are T. vivax and T. uniform, which have a tropism for wild and domestic ruminants. The trypanosomes of the Nannomonas subgenus are small and do not have a free flagellum. The main species are T. congolaise and T. simiae, which have a significant tropism for cattle, pigs and dogs.

In Africa, Congolese T. T. vivax. T. brucei and T. evansi are the main agents responsible for trypanosomiasis. especially in domestic mammals such as ruminants, bovines, pigs, sheep, goats, horses and canids. T. brucei, and in particular the subspecies T. b. gambiense, is probably the best known since it is responsible for the chronic form of sleeping sickness in humans in West and Central Africa. The subspecies Γ. b. rhodesiense is the agent responsible for the acute form of sleeping sickness. T. vivax is a parasite mainly of ungulates in tropical Africa and transmission is ensured by horseflies or tabanids. 7 ’. equiperdum is also present in Africa. The T. evansi subspecies is transmitted to cattle, horses and dromedaries, and has significant economic repercussions throughout the farming regions. Human cases caused by T. evansi are exceptional. Rare cases of trypanosomiasis caused by other species of trypanosomes (lewisi group trypanosomes, T. llieileri) have been reported in humans and animals.

Trypanosomes have a complex life cycle that includes different morphological tonnes, depending on the subspecies. In general, during infection, the tsetse fly (or tsetse fly) injects into the half of the host at the site of the bite the infecting metacyclic forms. The parasites multiply in half at the point of inoculation, giving rise to blood forms. This stage can last from 1 to 3 weeks. Then, the parasites invade the blood, the lymphatic system, as well as various organs, such as the heart or the kidneys, where they cause major lesions. Sources of infection of pets are also

J other infected domestic animals, or sick wild animals or healthy carriers.

At present, control of the disease is mainly based on vector control through the use of insecticides, used in particular for impregnating traps, which has an environmental impact. In South America, a fight against bedbugs, vectors of Chagas disease, is undertaken with persistent insecticides sprayed in the homes, associated with an improvement in the habitat. In infected mammals, the ability of trypanosomes to escape the host's immune defenses by expressing variable antigens on their surface has so far prevented the development of effective vaccine strategies. Only a few trypanocidal molecules are available, but they induce significant side effects and many resistant strains of parasites have appeared. On the diagnostic level, this is generally limited to a suspicion based on the observation of symptoms. To date, however, there are no reliable markers allowing rapid and specific detection of an infection, at reasonable costs.

There is therefore a need in the prior art for effective approaches for the prevention, treatment and detection of trypanosome infections.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for treating and diagnosing a trypanosome infection. It concerns the use of excreted / secreted antigens (exoantigens, secretome) and, more particularly the identification of a protein secreted by trypanosomes, including neutralization (by antibodies acquired by vaccination or by injection) or inhibition (by different molecules) provides effective protection against infection, development or dissemination of trypanosomes. The invention allows cross-action against different strains of trypanosomes, and thus provides effective methods and compositions for combating infections and pathologies induced by trypanosomes in their mammalian hosts. It also allows the detection of this protein and of antibodies directed against it in any sample, as well as the monitoring of the evolution of trypanosomiasis, with or without treatment. It also allows the creation of primers and probes allowing the use of different molecular biology techniques, such as the "polymerase chain reaction" (PCR) applied to the diagnosis of trypanosomiasis.

An object of the invention thus resides in pharmaceutical or veterinary compositions comprising (i) the protein TbKHCl or one or more antigenic peptides thereof, a nucleic acid encoding said protein or said peptide, or an inhibitor of the protein TbKHCl and (ii) a pharmaceutically or veterinally acceptable excipient.

In a particular embodiment, the invention relates to compositions, such as vaccines, comprising (i) the protein TbKHCl or one or more antigenic peptides thereof, or a nucleic acid encoding said protein or said peptide, (ii) a excipient acceptable from the pharmaceutical or veterinary point of view, and (iii) optionally an adjuvant advantageously chosen to reinforce an immune response.

In a particular embodiment, the invention relates to compositions, such as vaccines, comprising (i) the protein TbKHCl, or one or more antigenic peptides thereof, complexed with or in association with one or more other molecules of trypanosomes, (ii) an excipient acceptable from a pharmaceutical or veterinary point of view, and (iii) optionally an adjuvant advantageously chosen to reinforce an antibody response.

In another particular embodiment, the invention relates to compositions comprising (i) an anti-TbKHCl antibody. or a fragment or derivative of such an antibody, and (ii) a pharmaceutically or veterinary acceptable excipient.

The subject of the invention is also a composition as defined above, or the protein TbKHCl or one or more antigenic peptides thereof, or a nucleic acid encoding said protein or said peptide, for its use for vaccinating or immunizing a mammal against trypanosome and / or trypanosomiasis.

The subject of the invention is also a composition as defined above, or the protein TbKHCl or one or more antigenic peptides thereof, or a nucleic acid encoding said protein or said peptide for its use in protecting a mammal against trypanosomoses.

A subject of the invention is also a composition as defined above, or the TbKHCl protein or one or more antigenic peptides thereof, or a nucleic acid encoding said protein or said peptide, or an inhibitor thereof, for use in treating a mammal having trypanosomiasis.

The invention further relates to the use of the protein TbKHCl or one or more antigenic peptides thereof. or a nucleic acid encoding said protein or said peptide, or a secretory extract enriched in this protein, for the preparation of a vaccine to immunize or protect a mammal against the trypanosome.

According to another aspect, the invention also relates to the use of an inhibitor of the TbKHCl protein for the preparation of a medicament for the treatment of a mammal having a trypanosomiasis.

The invention also relates to a method for treating a mammal having trypanosomiasis, comprising inhibiting the protein TbKHCI in said mammal. The inhibition can be obtained by administration of an inhibitor (for example an antibody or a molecule interfering with the binding or the function of this protein in the tissues of mammalian hosts), or by vaccination of said mammal against the protein TbKHCI or a antigen thereof. The invention thus provides new immunotherapies for trypanosomiasis using any anti-TBkHCl antibody, in particular monoclonal, or derivatives of such antibodies or constructs using the amino acid or nucleotide sequence of a part of such antibodies.

The invention further relates to any antibody specifically binding the TbKHCI protein.

Another subject of the invention also relates to a method of in vitro diagnosis of trypanosomiasis in a mammal, characterized in that it comprises the identification and / or measurement, in a sample of said mammal, of the presence of the protein TbKHCI or antigenic peptides thereof or antibodies to this protein.

Another subject of the invention relates to a method for monitoring the progress of a trypanosome infection in a mammal, characterized in that it comprises the identification and / or the measurement of the amount of TbKHCI protein or of antibodies against this protein in mammalian samples taken at different time intervals.

Yet another object of the invention relates to a method for determining the effectiveness of a treatment against the trypanosome in a mammal, characterized in that it comprises the identification and / or the measurement of the quantity of protein TbKHCI in samples of mammals or of antibodies directed against this protein taken at different time intervals during treatment, and possibly after treatment.

The invention also relates also

- kits for measuring trypanosome in a test sample, characterized in that they comprise at least one antibody as defined above, a medium suitable for the formation of an immune complex with said antibody, and at least one reagent for the detection of an immunological reaction.

- kits for detecting antibodies directed against the TbKHCI protein for the diagnosis of infection using the TbKHCI protein, peptides, or natural or synthetic epitopes derived from this protein.

Another subject of the invention also relates to any diagnostic method using the nucleotide sequence of the TbKHCI protein for the diagnosis of trypanosomiasis or for the precise identification of a species of trypanosome (for example, the preparation of primers or probes allowing the use of different molecular biology techniques such as PCR or hybridization).

The invention can be implemented to prevent, treat, detect or monitor progress after vaccination or treatment of any condition caused by parasites of the genus Trypanosoma. in any mammal, in particular in domestic or farm animals, as well as in humans.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: A TbKHCI inhibitor inhibits parasitic proliferation in vitro.

Figure 2: A TbKHCI inhibitor decreases parasite load in vivo.

Figure 3: Vaccination strategy.

Figure 4: Mouse survival rate (protective effect) to parasitic infection after vaccination according to the invention.

Figure 5: Detection of an infection by measurement of antibodies directed against the TbKHCI protein.

Figure 6: Seroprotection test: The naive mice receive, 24 hours before infection with T. Feo (2000 parasites), 300 μL of serum from mice immunized with the total secretome of T. b. ganibicitsc Fco ("Total PSF serum") or the fraction containing high molecular weights greater than I00 kDa ("H PM serum> 100") or the fraction containing high molecular weights greater than 50 kDa ("HPM serum> 50") or the fraction containing low molecular weights less than 50 kDa ("BPM serum <50") or the fraction containing molecular weights between 50 and 100 kDa ("Serum 100 <PM> 50"). The survival of the mice is monitored as a function of the number of days post-infection with T. Fco.

Figure 7: cross seroprotection test. The mice receive, 24 hours before infection with T. b. bntcei (2,000 parasites). 300 μL of serum from mice immunized with the total secretome of T. b. gcimbiciise Fco ("Total PSF serum") or the fraction containing high molecular weights greater than 50 kDa ("HPM serum> 50") or the fraction containing low molecular weights less than 50 kDa ("BPM serum <50") or serum from naive mice. The survival of the mice is monitored as a function of the number of days post-infection with T. b. britcei.

Figure 8: A: United mouse rate of parasitic infection after vaccination according to the invention: the mice are immunized twice, in the presence of adjuvant (saponin), either by the total secretome of T. b. gambieiise Féo (“PSF Total”) or the fraction containing high molecular weights greater than 50 kDa (“HPM> 50”) or the fraction containing low molecular weights less than 50 kDa (“BPM <50”). Control mice receive the adjuvant alone ("Control"). The mice are infected 2 months later with T. b. brucei (2,000 parasites). The survival of the mice is monitored as a function of the number of days post-infection with T. b. brucei. B: Survival rate of mice to a parasitic infection after vaccination according to the invention: the mice are immunized twice, in the presence of adjuvant (saponin), by the total secretome ("Total PSF") of T. b. brucei or T. b. brucei KO for kinesin. Control mice receive the adjuvant alone ("Control"). The mice are infected 2 months later with T. b. brucei (2,000 parasites). The follow-up of the mice is followed according to the number of days post-infection with T. b. brucei. C: Survival rate of mice to a parasitic infection after vaccination according to the invention: the mice are immunized twice, in the presence of adjuvant (saponin), either by the total secretome of T. evansi ("Total PSF"). Control mice receive the adjuvant alone ("Control"). The mice are infected 2 months later with T. b. brucei (2,000 parasites). The survival of the mice is monitored as a function of the number of days post-infection with T. b. brucei.

Figure 9: Protein profile obtained by electrophoretic migration of 5 pg of each sample in denaturing and non-reducing condition and then stained with Coomassie blue. The boxes identify the protein bands that may contain TbKHCl.

Figure 10: Immunoblot of Ipg equivalent antigen protein after semi-dry transfer 3h30 24mA. Primary antibody (Purified Mabl antibody) diluted to L200th; secondary antibody (anti mouse total IgG) diluted to I / 5000th.

Figure 11: Immunoblot assembly. Deposit l pg of antigen, wet transfer ON 4 ° C 10mA. Primary antibody (anti-PSF serum T. Feo) diluted to / / 200th; secondary antibody (anti mouse total IgG) diluted to l / 5000th.

Figure 12: Immunoblot mounting. Ipg deposit of antigen, wet transfer Y / N 4 ° C 10mA. Primary antibody (anti-HPM50 serum T. Feo) diluted to l / 200th; secondary antibody (anti-total mouse IgG) diluted to l GOOme.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for preventing, treating, diagnosing and monitoring the course of a trypanosome infection based on the neutralization or inhibition of the protein TbKHCl and on its detection or that of antibodies directed against that -this. The invention provides effective protection against infection, development or spread of different strains of trypanosomes, primarily by vaccination. It can be used in any mammal.

Definitions

The term "trypanosomiasis" or "trypanosomiasis" generally refers to all disorders caused by a trypanosome in mammals. The term trypanosomiasis includes in particular Nagana, Surra, Dourine, sleeping sickness, African trypanosomiasis, American trypanosomiasis, Chagas disease, as well as all damage to organs (eg, kidney, heart, liver, testicle) , digestive tract, brain) by a trypanosome infection.

The “treatment” or “treat” outfit means any improvement in a subject's condition. Treatment can be curative or preventive. The curative treatment is intended for an infected mammal and aims to prevent, reduce, slow down or delay the development of a disease in the infected mammal. It includes in particular, in an infected subject, the reduction of the parasitic load, the disappearance of the parasite, the reduction of its proliferation or its transmission, the reduction of disorders caused by the parasite and in particular damage to the organs, reduction of symptoms , or the total eradication of the disease. Curative treatment typically uses an inhibitor of the pathogen (immunotherapy or chemotherapy). Preventive treatment is aimed at a mammal not infected with the parasite and aims to prevent, prevent or reduce infection in a healthy mammal. Preventive treatment usually uses a pathogen antigen, which generates a protective immune response.

Identification of a virulence factor

The invention stems from the identification of the protein TbKHCl, secreted by trypanosomes, the neutralization or blocking of which makes it possible to inhibit the proliferation of the parasite, its transmission, and its virulence. The invention further shows that immunization with a preparation containing the protein TbKHCl produced by different trypanosomes is possible and induces cross-protection against different types of trypanosomes. This protein therefore represents a particularly relevant and attractive target for any therapeutic or diagnostic strategy for trypanosome and trypanosomiasis.

An object of the invention thus resides in the protein TbKHCl, or one or more antigenic peptides thereof, or a nucleic acid encoding said protein or said peptide, or an inhibitor of the protein TbKHCl, for their use for preventive treatment or curative of a trypanosome infection in a mammal. The invention also resides in the use of the protein TbKHCl, or one or more antigenic peptides thereof, or an inhibitor thereof, to treat a trypanosome infection in a mammal. The invention also relates to a method for treating a trypanosome infection in a mammal comprising an inhibition (e.g., reduction, neutralization or blockage) of the protein TbKHCl in the mammal. The inhibition of the protein includes the reduction in the quantity or the inhibition of the activity of the protein and can be obtained for example (i) by immunization or vaccination of the mammal with a preparation containing the protein TbKHCl, for example by administration an immunogenic amount of the TbKHCl protein or one or more antigenic fragments thereof; and'or (ii) by inhibiting the protein TbKHCl present in the mammal, by administering an inhibitor or a competitor thereof.

Thus, within the meaning of the invention, the "inhibit" or "inhibition" behavior of a protein designates any reduction in the quantity or activity of said protein. Inhibition therefore designates in particular a drop or a reduction in the amount of said protein by compounds affecting its synthesis, its secretion, or its structure. Inhibition also designates any reduction in the activity of the protein by compounds (antibodies or derivatives, peptides, chemical molecules) acting directly on it or on one or more of its target molecules in mammalian hosts, in order to interfere with its action.

Within the meaning of the invention, the term “TbKHCl protein” designates a protein comprising the amino acid sequence represented in SEQ ID NO: 2 or any natural variant of this sequence resulting from polymorphisms or variations between species or subgroups of trypanosomes , or any kinesin-type protein secreted by a trypanosome and having a sequence having at least 45% identity with the sequence SEQ ID NO: 2, preferably at least 60%, more preferably at least 70%. Even more preferably, the sequence identity with the sequence SEQ ID NO: 2 is 80% or more, preferably at least 85%, 90%, 95%, 96%, 97%; 98%, 99% or more.

The sequence SEQ ID NO: 2 is shown below. It corresponds to the TbKHC I protein of T. brucei brucer.

MSDADVKtGT AAGDSVAVPt SWKPDEGRR

SRGESTGGTA AGD7GVPKNI AK7LVYCRLR

PRHKTDFKNG

FCSALMCY3Q

DMLGDLMSAT

PE3SRGHTAL

GFQLVTVSGN

TGÏGKSFTMC

GF.3RVDIHFD

VLRIVSESFS

DlVVKDQRFï

NTTPGQEGII

EQGGVELTGC

DFEAGKLKGK

KFDGAFGDEC

PRSAKLIFDK

SSHVLLSAQE

ITFIDLAGÏE

TQSDIFEAVA IQ3DNARSYE FHRFYRIGND R FS KT GIT H D

7PGITHAFKG V7GQFVQIYR F.R VVT.ATAMt ·; U F I i-IKGFAKG

ΙΟ

IblASLLSLGH gslqfglram ERYNDRREDI SKEIVHLIRE ILASRAtlDTV RAKGVELLAE

TQMVDVSEME RGPSPFDAAR VE.AVETQLAS DVLLETIERQ MSILRLVRES CIRLKNQMLR MLKSLtJEEFE ELLDGKIKMA

VV5CLS5GSR DVKVTAKQSV DRRFEIEMAE Q EHQEAKRRA RLHEHIEVLR VDRLSQECSE TLRVTMQADL NLCSTQRKPP HGVEVPGPYV EHLLN.AARStJ ERKLAAQLAE CGITPCCELP RQSSVIRQ

HIPWRDSKLT HVDYQKLAQK LKRTGASEEQ EMKLAQDLII EQVKELGGVP RLEEINRLRD DEAKAHMREL QKDGTPSPNN PPIKLGFPGS

EEFHRRVICE RDGEDGAEVE DSYNELIERE ERGELVEKKQ LKRFLRRLRS

RILQD3IGGR LQSLLDERDE MLMLREVÏKA AEFQKKLDNA IEEATFPETF ENTQYRAALE AREVEQLKFE TQNENLQRTV APVTSSETDA LQOQMVTAQI ALLEKKDAEL EEELNEQLMC LVTAAHLSRL M

SRTSIÎLTVG RIELLEVQIA EVENLQEQQD REGTNDDLVR LDVGQVEEMR NSGISLÎÎDTD LTATAIPLTA KQLTEQLEFS REPPEDTDMD

QVEDPQNAPP QMKEETILEK QDELL-ARLRS ATEKSQREQI

FSSDHLHETT SRDAERHELM EEFQYREEVY VLKQLSEKDA tJRLEADVQRH DLTEFLSEKR RLRCPPCATA MRERKSLQDR VLLRVKEEEI PVDAIAMDEY ASKAQYAAKL EEEEKHRMQK LEETLRRATQ

Research carried out by the inventors made it possible to identify other TbKHCl proteins within the meaning of the invention from other species of trypanosomes, in particular from T. hrucei ganibicnse (99% identity with SEQ ID NO: 2); T. brttcci rhodcsiense: T. evansi; T. equiperditni; Congolese T. (76% identity with SEQ ID NO: 2); T. vivax (69% identity with SEQ ID NO: 2): T. nntsculi (a parasite of the leuisi group (61% identity with SEQ ID NO: 2). And T. cnizi (61% identity with SEQ ID NO: 2) The sequence of the TbKHCl proteins of these species is shown in SEQ ID NO; 3 (Tiyponosoma bruceigembiense), SEQ ID NO: 4 (Congolese trypanosome), SEQ ID NO: 5 (Trypanosome vivax). SEQ ID NO: 6 (Trypanosome critzi). These proteins represent examples of TbKHCl proteins within the meaning of the invention. Furthermore, a person skilled in the art can, on the basis of the information in the present application and conventional techniques, identify other TbKHCl from other subgroups of trypanosomes. In this context, the "sequence identity" behavior, applied to a nucleic acid or a protein, refers to the quantification (generally in percentage) of residue matches of nucleotides or amino acids between two aligned sequences using a standard algorithm such as the alignment of Smith-Watcnnan (Smith and Watcrman (I98l) J Mol Biol I47: 195-197). CLUSTALW (Thompson et al (1994) Nuclcic Acids Res 22: 46734680; Altschul et al (1997) Nucleic Acids Res I7: 3389-402). or BLAST2 (Nuclcic Acids Res 25: 3389-3402). BI.AST2 can be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and achieve a more meaningful comparison.

The TbKHCl protein can be obtained in different ways. It can be in pure form, of enriched extract (for example an enriched secretion extract), recombinant, synthetic, etc. It can firstly be isolated in the form of a fraction eluted or purified from a culture of trypanosomes. For this, the purified parasites are preferably incubated in a secretion medium (for example of the Ringer Lactate + glucose type), then the secretion products (secretome) are recovered, for example by centrifugation, filtered to sterilize, then passed through a column of affinity comprising an anti-TbKHC l antibody. After washing, the molecules retained on the column are eluted, making it possible to obtain an extract or a fraction comprising the protein TbKHCl and / or its fragments. In a variant, the protein TbKHCl is obtained from the secretome by differential filtration on filters having cutoff thresholds of 50 or 100 kDa, which allow separation of a fraction containing the molecules of high molecular weight (HPM) and a fraction containing the low molecular weight molecules (BPM). The protein TbKHCl is present in the HPM fractions, in particular HPM50 and HPM 100.

The fraction enriched in protein TbKHCl obtained can also comprise proteins or peptides of different nature originating from trypanosome. In particular, the TbKHCl protein having the property of binding and / or transporting other molecules of the trypanosome in particular thanks to its coil-coil structure which promotes its interaction with other molecules, the fraction obtained can comprise the TbKHCl protein, or peptides thereof, in complex or association with other proteins or peptides or molecules of trypanosome. The TbKHCl protein can moreover be concentrated and / or purified further from this fraction, to obtain a purity greater than 90%, for example 95% or more, in particular 98% or more, in particular a TbKHC I protein deprived any other trypanosome protein.

In this regard, the invention also relates to a process for the preparation of an antigenic fraction, comprising obtaining a trypanosome secretome, the differential filtration of the secretome on a filter having a cutoff threshold of 50 or 100 kDa, and the recovery of the high molecular weight fraction. This process has various advantages: it makes it possible to improve the production yield of the fractions enriched in TbKHCl. it improves reproducibility (similar protein assay for the different batches), and it retains the immunogenicity of the antigens (differential filtration alters the antigenic structure less than an acid elution). The invention also relates to the preparation obtained by this process and to its veterinary or pharmaceutical use, as illustrated in the present application.

The TbKHCl protein can also be produced recombinantly, by expressing in a host cell a coding nucleic acid. In this regard, another object of the invention resides in a process for producing TbKHCl protein, comprising the culture of a recombinant cell comprising a nucleic acid encoding TbKHCl under conditions allowing expression and, optionally, secretion of the protein TbKHCl then, recovery and. optionally, the purification of the protein TbKHCl. The cell used can be prokaryotic (for example a bacterium such as E. coli) or eukaryotic (for example a yeast, a mammalian or insect cell). The nucleic acid coding for TbKHCl can be DNA or TARN, and its sequence can be determined by a person skilled in the art depending on the protein sequence to be coded. As an illustration of a sequence coding for the protein of SEQ ID NO: 2, mention may be made of the nucleotide sequence SEQ ID NO: 1. which is shown below:

ATGTCGGATG TCGGTTGTAA GCTGGGGATA CCACGGAACA GATATTGTTG ACACAAAGTG TTTTGCTCAG AATACCACTC ATTCAATCAG GACAACCTTG (3 AJAC A-G A.GGG TTTATGCGCT CCGGAGTCCA GACCCAGAGG CGTTTTAGTA ATCAACGCCT CACATTCCTT AGCCGTACCT AATTCACTGC CATGTGGATT AGAATCAAÎT GAGCGTTACA CTGAAGAGAA GAGGTGGAAA TCAAAGGAGA GAGATGAAAT CGTGAGGGAA ATATTGGCCA

CT3GACGTTG CG T CG T .AAG G CGGTTGGAGG AACAGTGGCA ACTCAGATGG GATGAAGCGA TTAACCGCÀA CGAGGTCCTT CAAAAGGATG AAGCAGCTTA GTTGAGGCTG CCCCCAATCA AGGGAGCCAC GATGTGTTAT GAAGAGTTTC CAGGTGGAAG ATGTCAATTT CGCGATGGAG C AAA G T G b .AA TGCATTCGTC GACTCGTATA CAAGATGAAC ATGCTGAAAT GAGCGCTGTG GCAACGGAAA

CCGATGTGAA AACCAGATGA CCbGTbTbCL AGACTGATTT TGAAGGATCA ATA ATT 1 i b A CGTTGATGTG CTGGCCAAGA ACAATGCGCG GTGACTTGAT GCGTAGAACT TTTACCGCAT GCCGCGGCCA CAGGTAAACT AAA -CTGGTAT CTCTTCTTTC GGCGTGATTC CTATTATTTT ÀGTTTGGTTT ACCAGAAGOT ACT i l ⁽ⁱ Jaag ACGATCGCCG CTGGTGCATC ACCTCCAGGA 1 CbTCCACC i TGGCGCAAGA CAAATGATGA GCCGAGCGAA

GCCAGGTGGA GTGTGGAATT agataaaccg 7TTCATTG.AA TGGATGTTTC AGGCGCACAA CCGCTATTCC CCCCGTTTGA GCACGCCATC CGGAGCAACT TTGAGACGCA AACTTGGTTT CGGAGGATAC TGGAAACAAT ACCGACGCGT ATCCTCAGAA TGCGTTTAGT A G G A T G G C G C AGGAGACCAT TGAAGAACCA ACGAGTTGAT TGTTAGCCAG CACTTAATGA AACTGGTGGA AATCCCAGAG

AGAGGGAACG AGGACGGCGG AAAGAATATA. AAbAAb jG i ACGCTTTTAC AGCGGTGGCC CTACGGACAG AGGCATCATT GAGTTATGAA GAGTGCAACT TACCGGTTGC CGGCAATGAC tacagcttta G AA AAA GGG G TACACATGAC ACTTGGTCAC GAAGCTGACG GACTGTTGGG G · - · b.- ¹ b - 1 L .AA? GGCCCASAAG GCAGATCGCT GGAAGAGATT GG AA GAGCAG GCAGCAAGAC T A. TTCGCGAG TCTTATCATT TCTCGTCAGA CGACACGGTG

GGAGATGCGG GCTTGCGGAA ACTCCGCGAC T G A C M C T G A T 1 <jAGriT bGrb ·. CCGGGAGCTG ACTCACAGCC CGCCGCGCGC CCCAAACAAC GGAA7TCAGC ACTTGCTTCG CCCCGGCTCT CGATATGGAT TGAACGGCAG CATTTGTGAG CGCCCCTCCT TCGGGAGTCC GGAGGTGG ACTCGAGAAG GATGCTGCGTAGAGAGGA

GCGGCCGGCG AGCAG.AGGTG bCACGbTGTC GGGTTCCAAC AAGTTTGATG GTCCCTTGCA ACGGGTACAG CCACGGTCCG GTGACAGGAC GGAAGGGACC AGCTCCCATG CGTCGGGTTG GTTCTCCGCA ATTACATTCA AACCCCATTA UTT G1 TCL · ¹ j, T CGG.ATCCTGC CC.AAGT.A'j 1 G GATGTGAAGG CT .AATuAC tctcgtgacg GACAGACGTT ATGCTGAACC gaggagttcc CAGGAGCATC GCGG.AGTTCC GTTTTG .AAGC AGACTCCACG AT AG.AGGAGG AACCGGCTGG GTGG.ATCGTC GAAAACACAC GATTTGACGG ACTCTTCGTG GCGCGGGAGG CGGCTTCGAT AACCTGTGTT ACTCAAAATG ATGCGTGAGA CATGGTGTTG GCACCAGTGA GTGCTGCTCC GAGCACTTGC TTGCAGCAGC CCTGTTGACG UAACgCaAGT GCCCTGiTGG GCGTCGAAGG TGTGGCATCA GAGGAGGAAC GAGGAGGAA.G AGGCAATCCA TTGGTTACGG CTTGAGGAAA

ATTCAGTGGC AGTCTACTGG TTGTTTATTG TAGTGACAGT GTGCTTTTGG T.AACACACGC GTAAGTCTTT CCAAACTTAT AGTTTGTTCA GAGTGGATAT TTCTTCTGAG TAACTGCGAC T CG T ATCAGA T CGACTT.AGC TGAAGGATA GTTT3. i C i .AT ATCACCTCCA T GA.CGGCCAA TCTTGGATGA CAGAAAGACA TTGAGATTGA TGCGTGAAGT AATACAGGGA AGGAAGCGAA

J w 1 AA'T'TGTCCG.A AACATATTGA CGACGTTTCC A. J G C G G A T G T T TTCGCAGCT AATATCGCGC AA.TTCCTTTC T CACCATGC.A TGGAGCAGTT GTCCGCCGTG .AA_AACTTGCA GGAAGTCGCT AGGTTCCGGGAACATGGTGTCATG. i îG-AAT TGGCAGCTCA AGAAGAAGGA CGCAGTAÏGC CACCGTGTTG TGAATGAGCA AAAAGCATCG GCgTCAT1CG CAGCCCACTT CGCTACGACG

CGTTCCCGAG CGGGACAGCT CAGGTTG.AGG AAGCGGGAAT CGACGAATGT ATTTAAAGGT CACTATGTGT TTTCGAC.AAA GATTTACCGT TCACTTCGAC TGCCCAAGAG TGCTATGAAT GAGCCCCAGC A'j'jAT ACuAG GGCGAAGTGC GTA A b b bb LG TGGCGGAAGG CGAA.4CCACA ACAGTCGGTT AAGGGACGAG CGAGTTAATG GATGGCTGAA ATACAAGGCT GGAAGTGTAÏ GCGACGGGCA CG.ACAACGCG AA.AGGACGCC GGTGCTCAGG CG. AAA.CCTTT GCAACGCCAT CTGCTCTGAG C GGATTGGAA TGAGAAGCGC GGCCGACCTT G Am GTTT b AA CGCAACTGCA T.AAACCACCT AAGGACCGTG TCAGGACCGC GCCGTACGTA GGAGGAGGGGGGGTGTGTGTAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGTGTGTGTGTGGTGTGTGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGTGTGTT

AACTGTTCAA GTCGCGATTG TGCAACACAA

GAATTGTTGG ATTGCAAGAT TAAGATGGCC ATGGAAAAAG AAGCAGGTAG CCCGGGTGTG

TTAAAGCGTT TCCTCCGCCG CGTGCGCTCC AACTGA

The nucleic acid sequence can also be optimized for expression in the chosen host cell. Another object of the invention resides in an expression vector comprising a nucleic acid coding for the protein TbKHC 1, preferably under the control of a promoter. Another object of the invention resides in a host cell containing such a vector, or containing a nucleic acid encoding the protein TbKHCl inserted into its genome.

The TbKHCl protein can also be obtained by artificial synthesis, using protein synthesizers. It can also be produced by a combination of these methods.

Within the meaning of the invention, the term “antigenic peptide” or “antigenic fragment” designates a peptide whose sequence or part of the sequence corresponds to part of the sequence of the TbKHCl protein. and which is capable of inducing an immune response against the protein TbKHC l. An antigenic peptide therefore generally comprises at least one specific epitope of the protein TbKHCL making it possible to induce an immune response directed specifically against TbKHCl. A peptide designates within the meaning of the invention a molecule having from 4 to 500 amino acids, for example from 4 to 450 amino acids, for example from 4 to 300 amino acids, or less, such as for example from 4 to 50, 40, or 30, or even less. Examples of antigenic peptides within the meaning of the invention are the peptides comprising at least the lOOO-III residues. 900-Hll, 800-IIIll 700-llli, 687-) H J. or 500-Hll, of the sequence SEQ ID NO: 2 or of natural variants thereof. A particular antigenic peptide is in particular a peptide comprising residues 687-1111 of SEQ ID NO: 2.

The protein or the antigenic peptides according to the invention may include modifications, in particular chemical modifications, which do not alter their immunological specificity. Thus in particular, they can be chemically modified to improve their stability, their tropism, their solubility, or their immunogenicity. Examples of modifications are for example the addition of phosphates, sugars or myristic acids, of polyethylene glycol. In the more specific case of the peptides, they can comprise, in addition to the immunogenic sequence, one or more residues promoting expression, stability, or immunogenicity. Peptides can also be coupled to carrier molecules, or other epitopes, to increase their immunological potential, or complexed or combined with other proteins to increase their immunogenicity. Particular peptides of the invention are peptides consisting of an immunogenic sequence of the TbKHCl protein.

TbKHCl inhibitor

The invention also relates to any TbKHCl inhibitor and its use for the treatment of trypanosome infections. The term "TbKHCl inhibitor" refers to any compound capable of reducing the amount (eg production or secretion) or activity of the TbKHCl protein. It is typically a specific inhibitor, that is to say capable of acting on TbKHCl without direct effect on other proteins produced by the trypanosome or by the infected mammal. The inhibitor compound can be an iigand of the TbKHCl protein, such as for example an antibody or an antibody fragment or derivative, a nucleic acid encoding an antibody or antibody fragment or derivative, an inhibitory nucleic acid (antisense. SiRNA, ribozyme , etc.) inhibiting protein synthesis, a peptide inhibiting the activity of TbKHCl. or a molecule specifically binding to target molecules recognized by TbKHCl at the host level, in particular receptors transmitting the signal normally induced by the TbKHCl protein or component scs, or a combination thereof.

In a particular mode, the inhibitor is a compound capable of specifically binding the TbKHCl protein and neutralizing it. An example of such a compound is an antibody, or a fragment or derivative of antibody or an inhibitor carried by this antibody. Specific binding refers to the fact that the specific inhibitor binds the TbKHCl protein and does not specifically bind other proteins, or with much lower affinity (by a factor of I0 or more). In a particularly preferred manner, the inhibitor is an antibody which binds TbKHCl and which does not bind endogenous proteins from the infected mammal.

The antibody can be a polyclonal, or a monoclonal, or a fragment or derivative of antibody such as the Fab, Fab'2 fragments. CDRs, single-chain antibodies (for example ScFv), nanobodies. human or humanized antibodies, etc. Antibodies can be produced by techniques well known to those skilled in the art, such as, for example, immunizing a non-human animal, and recovering serum or antibody-producing cells. The production of monoclonals can be obtained by obtaining hybridomas using conventional techniques well known to those skilled in the art. By way of examples, antibodies according to the present invention can be generated by injecting the TbKHCl protein or an immunogenic peptide of the invention into animals (for example a rabbit or a mouse), then by collecting the sera or the B cells. The selectivity of the anticoips can then be tested and confirmed by standard tests of the ELISA type. Techniques for producing poly- or monoclonal antibodies, ScFv fragments. human or humanized antibodies are described for example in Harlow et al., Antibodies; A laboratory Manual, CSH Press, 1988; Ward et al .. Nature 341 (I989) 544; Bird et al., Science 242 (1988) 423; WO94 / 02602; US5,223,409; US5,877,293; WO93 / 01288.

A particular object of the invention resides in an antibody specifically binding the TbKHCl protein. More preferably, the invention resides in an antibody binding an epitope contained in the C-terminal region of the TbKHCl protein. for example an epitope contained in residues 687-11H of the protein TbKHCl. The antibody of the invention is preferably a monoclonal antibody.

Another object of the invention resides in an anti-TbKHCl antibody capable of being obtained by immunization of a non-human mammal with an immunogenic composition comprising a peptide comprising an epitope comprised between residues 687 and 11 11 of the TbKHCl protein (terminals included).

Another object of the invention resides in a Fab or Fab’2 fragment of an antibody as defined above.

Another object of the invention resides in a single-chain antiTbKHCl antibody. It can be nanobodies, ScFv. Tandem antibody, etc.

Another inhibitor and object of the invention is a TbKHCl inhibiting nucleic acid, in particular an antisense, a ribozyme or an interfering RNA specific for TbKHCl. Such nucleic acids comprise a part (generally from 5 to 50 consecutive bases) of the coding sequence of TbKHCl or of its complementary strand, for example a part of the sequence SEQ ID NO: 1 or of its complementary strand, and make it possible to specifically inhibit the expression (transcription or translation) of the protein.

Another inhibitor and particular object of the invention is a molecule inhibiting the effect of TbKHCl at the level of the mammalian host. This is in particular any sc molecule which specifically binds to target molecules recognized by TbKHCl at the level of the host, in particular the receptors of the host transmitting the signal normally induced by the protein TbKHCl or its components. By way of example, mention may be made of sugars, peptides or other molecules (small drugs) blocking the binding of TbKHCl to cellular or humoral receptors of the host.

Veterinary and pharmaceutical compositions

The invention relates to any pharmaceutical or veterinary composition comprising (i) the TbKHCl protein, one or more antigenic peptides thereof, or an inhibitor of the TbKHCl protein, and (ii) a pharmaceutically or veterinary acceptable excipient. Such compositions make it possible to block the action of the protein TbKHCl and thus to prevent or control a trypanosome infection.

According to a first embodiment, the compositions of the invention are of the vaccine type and make it possible to induce very powerful anti-parasitic immunity in mammals. Thus, a particular subject of the invention relates to compositions comprising (i) the protein TbKHCl, or one or more antigenic peptides thereof, (ii) an excipient acceptable from the pharmaceutical or veterinary point of view, and (iii) optionally a adjuvant. Such compositions make it possible to vaccinate or immunize mammals against the protein TbKHCl, and thus to protect the mammal against a trypanosome infection or to treat such an infection.

Vaccines can comprise several antigenic peptides, so as to increase the immunogenic power of the vaccine. Thus, they can comprise several peptides, overlapping or not, comprising from 5 to 100 amino acids each and each comprising an amino acid sequence identical to a domain of the TbKHCl protein preferably comprised between residues 687 and 11 11. Vaccines may include other parasitic molecules associated with TbKHCl.

In a particular embodiment, the vaccine comprises a single antigenic peptide.

In another particular mode, it comprises 2, 3 4, or 5 antigenic peptides distinct from TbKHCl. In this regard, a particular vaccine of the invention comprises antigenic peptides of TbKHCl proteins originating from different strains of trypanosomes, making it possible to increase the potential of the vaccine. The vaccine can thus comprise one or more antigenic peptides of a TbKHCl protein from one or more species of trypanosomes.

In another particular embodiment, the vaccine comprises a whole TbKHCl protein.

In another particular embodiment, the vaccine comprises a nucleic acid encoding said TbKHCl protein or the antigenic peptide (s).

In a particular embodiment, the composition according to the invention comprises the protein of sequence SEQ ID NO: 2 or a natural variant thereof. or a nucleic acid encoding said protein.

I7

In a more particular embodiment, the composition according to the invention comprises a peptide comprising the residues 1000 to 1111 of the sequence SEQ ID NO: 2 or a natural variant thereof, a variant thereof having at least 90 % sequence identity, or a nucleotide sequence encoding said peptide.

Advantageously, in the compositions of the invention, the antigenic protein or peptide (s) or the nucleotide sequence are in pure form or of enriched, reconibinating or synthetic extract.

The veterinary vaccine compositions of the invention advantageously comprise an immunologically effective amount of the protein TbKHCl or of antigenic peptides derived therefrom. as previously described, or of a nucleic acid or expression vector coding or overexpressing the protein TbKHCl or the antigenic peptide (s) thereof.

The vaccines according to the present invention can comprise one or more adjuvants in order to increase their effectiveness. Adjuvants are well known in the art. Examples that may be mentioned include aluminum salts, such as aluminum hydroxide, metal salts, bacterial immunogens such as LPS, CT or LT, adjuvants of classes TLR3, TLR4, TLR5. TLR7. TLR8, or TLR. 9. saponifies and their derivatives, oil in water or water in oil emulsions, polysaccharides, cationic liposomes, virosomes or polyelectrolytes. Other immunomodulators can be used, such as fly saliva proteins, cytokines or heat shock proteins.

The vaccines according to the present invention can be monovalent vaccines (that is to say inducing a response against a single type of pathogen) or multivalent (that is to say capable of inducing a protective response against several types of separate pathogens). In a particular embodiment, the invention thus relates to a multivalent vaccine comprising (i) the protein TbKHCl, or one or more antigenic fragments thereof, (ii) an excipient acceptable from the pharmaceutical or veterinary point of view, (iii) optionally also an adjuvant and (iv) at least one antigen of another parasite. The vaccines according to the present invention can include several other parasitic molecules, in association with TbKHCl. including other kinesins.

According to another embodiment, the compositions of the invention comprise an inhibitor of the protein TbKHCl and make it possible to treat in a powerful and rapid manner a trypanosome infection in mammals. Thus, a particular object relates to a pharmaceutical or veterinary composition comprising (i) an inhibitor of the TbKHCl protein and (ii) an excipient acceptable from the pharmaceutical or veterinary point of view. Such compositions make it possible to block the action of the protein

I8

TbKHCI and thereby prevent or control a trypanosome infection. In a preferred embodiment, the inhibitor is an anti-TbKHCI antibody.

Thus, in a particular embodiment, the invention relates to compositions characterized in that the inhibitor is an anti-TbKHCI antibody. or a fragment or derivative of such an antibody.

In the compositions of the invention, any type of acceptable excipient can be used. In this regard, mention may be made of isotonic solutions, phosphate buffers or other saline solutions and culture media (for example physiological water, PBS, Ringer lactate, medium 199. Ham medium) and stabilizing and preservative excipients (such as acids, sugars , phenoxyethanol, medium I99, albumin, amino acids and derivatives, for example). Furthermore, the compositions of the invention can be in liquid or solid (powder) form. They can be packaged in any suitable container (ampoule, syringe, vial, vials, etc.).

As indicated, the compositions advantageously comprise an effective amount of TbKHCI protein or of antigenic peptide. This quantity can be easily adapted by a person skilled in the art. Generally speaking, an effective amount of TbKHCI protein or peptide designates an amount making it possible to induce an anti-TbKHCI antibody response in the treated mammal. Such an amount is generally between O.lpg and Img / dose, for example between Ipg and 500pg / dose. especially between lOpg and 100 pg / dose.

In the case of a TbKHCI inhibitor, the effective amount denotes an amount making it possible to inhibit by at least 10%, preferably at least 20%, 30%, 40%, 50% or more, the production or the activity of TbKHCI in vitro or in vivo, in the mammal treated. Such an amount is generally between O.lpg and Img of dose inhibitor, preferably between I pg and 500pg / dose.

The compositions of the invention can be used alone, or in combination with other treatments, such as for example trypanocides such as in particular Pentamidine, Eflomithine, Nifurtimox. NECT, Suramin, Melarsoprol, Fexinidazole, Oxaborole Diminazene, Isometamidium. Homidium.

The invention can be used to treat any mammal likely to be infected with a trypanosome, such as for example bovids, ovids. felines, camelids, canids, or humans. The compositions according to the present invention are particularly useful for treating pathologies induced by trypanosomes such as in particular anemia, weight loss, and / or immunosuppression.

I9

Production of anti-trypanosome agents

The present invention also relates to a method for identifying, producing or optimizing anti-trypanosome compounds, comprising a step of evaluating the capacity of a test compound to inhibit the activity or the production (or secretion) of the TbKHCI protein. Compounds with such activity have an important anti-trypanosome action.

The invention also relates to any compound identified, produced or optimized according to the preceding method, for its use for the treatment of a trypanosome infection.

Diagnosis of trypanosome infection

The protein TbKHCI also constitutes a target of interest for the detection, in a mammal, of the presence of trypanosome. This protein being secreted, it can be detected as well as any antibody against it (search for antigen and / or antibody) or any nucleic acid encoding TbKHCI, in any mammalian fluid, in particular in the blood. In addition, both protein and antibodies can not only detect the presence of the parasite, but also monitor the progress of an infection and / or the effectiveness of a treatment.

Thus, an object of the invention also resides in a method of in vitro diagnosis of trypanosomiasis or of detection of the presence of trypanosome in a mammal, characterized in that it comprises the measurement, in a sample of said mammal, or the detection the presence of the protein TbKHCI or of a nucleic acid coding for the protein TbKHC l or of antibodies directed against TbKHC l.

An object of the invention also resides in a method for monitoring the progress of a trypanosome infection in a mammal, characterized in that it comprises the measurement of the amount of TbKHCI protein or of a nucleic acid encoding the protein TbKHCI or antibodies to TbKHCI in mammalian samples taken at different time intervals.

The invention also relates to a method for determining the effectiveness of a treatment against the trypanosome in a mammal, characterized in that it comprises the measurement of the quantity of TbKHCI protein or of a nucleic acid encoding the TbKHCI protein or d antibodies to TbKHCI in mammalian samples taken at different time intervals during treatment.

The invention also relates to a method of in vitro diagnosis of trypanosomiasis in a mammal, characterized in that it comprises measurement.

in a sample of said mammal, of the presence of the protein TbKHCl, of a fragment thereof, of a nucleotide sequence coding for TbKHCl, or of antibodies directed against TbKHCl or against one or more antigenic peptides thereof.

The invention further relates to a method for monitoring the progress of a trypanosome infection in a mammal, characterized in that it comprises measuring the amount of protein TbKHCl, of a fragment thereof. of a nucleotide sequence encoding TbKHCl, or of antibodies directed against TbKHCl, in samples of the mammal taken at different time intervals.

The invention also relates to a method for determining the effectiveness of a treatment against the trypanosome in a mammal, characterized in that it comprises measuring the amount of protein TbKHCl, a fragment thereof, a nucleotide sequence encoding TbKHCl, or of antibodies directed against TbKHCl. in mammalian samples taken at different time intervals during treatment.

The determination of the presence or (relative) quantity of TbKHCl protein or of anticotps can be carried out by any technique known per se, such as in particular by means of a specific ligand, for example an antibody or a fragment or derivative of antibody, or the protein or a fragment thereof or an epitope or a mimotope. Preferably, the ligand is an antibody specific for the polypeptide, or a fragment of such an antibody (for example a Fab, Fab ', CDR, etc.), or a derivative of such an antibody (for example a simple antibody). chain. ScFv), or the protein or a fragment thereof or an epitope or a mimotope. The ligand is typically immobilized on a support, such as a blade, ball, column, plate, etc. The presence or the quantity of protein of interest or of its fragments or of antibodies in the sample can be detected by the detection of a complex between the target and the ligand, for example by using a labeled ligand, by using a second labeled revealing ligand, etc. Immunological techniques which can be used and which are well known are ELISA, RIA, etc. If necessary, the quantity of polypeptide detected can be compared with a reference value, for example a median or average value observed in human patients or non-human mammals which are not infected, or with a value measured in parallel in a control sample.

All immunological techniques based on antigen-antibody reactions can be implemented, using either the TbKHCl protein or its fragments or natural or synthetic derivative compounds or an epitope, a mimotope as antigen, or molecules specifically recognizing the TbKHCl protein or its fragments or natural or synthetic derivative compounds (eg, antibodies, Fab, Fab ', CDR fragments, or derivatives of an antibody or nanobody). Basic immunological techniques, for example, agglutination,

2I precipitation, enzyme immunoassay, immunoblotting, western blot are suitable.

In another variant, the invention detects the presence of nucleic acid encoding TbKHCI. This detection can be carried out by techniques known per se to those skilled in the art, such as in particular by Northern Blot, selective hybridization, use of supports coated with probe oligonucleotides, selective amplification of nucleic acid such as for example by RT-PCR, Quantitative PCR or ligation-PCR, etc. These methods may include the use of a nucleic acid probe (for example an oligonucleotide) capable of selectively or specifically detecting the target nucleic acid in the sample. Amplification can be carried out according to various methods known per se to those skilled in the art, such as PCR, LCR, transcription-mediated amplification (TMA), strand displacement amplification (SDA), NASBA, the use of allele specific oligonucleotides (ASO), allele specific amplification, LAMP (Loop-mediated isothennal amplification), RPA (Recombinase Polymerase Amplification), Southern blot, SSCA conformational analysis, in situ hybridization (eg, FISH), gel migration, heteroduplex analysis, etc.

According to a preferred embodiment, the method comprises the detection of the presence or absence (or the (relative) amount) of a nucleic acid encoding TbKHCI by selective hybridization or selective amplification.

Selective hybridization is typically carried out using nucleic acid probes, preferably immobilized on a support, such as a solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic probes. Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, etc. The nucleic acid probes can be any nucleic acid (DNA, RNA, PNA, etc.), preferably single-stranded. comprising a specific sequence of a nucleic acid encoding TbKHCI. The probes typically comprise from 5 to 400 bases, preferably from 8 to 200, more preferably less than 100, and even more preferably less than 75, 60, 50, 40 or even 30 bases. The probes can be synthetic oligonucleotides, produced on the basis of the sequence SEQ ID NO: 1 according to conventional synthesis techniques. Such probe oligonucleotides typically comprise from 10 to 50 bases, preferably from 20 to 40, for example approximately 25 bases. The probes can be synthesized beforehand and then deposited on the support, or synthesized directly in situ, on the support, according to methods known per se to those skilled in the art. The probes can also be manufactured by genetic techniques, for example by amplification, recombination, ligation, etc.

The probes thus defined constitute another object of the present application, as well as their uses (essentially in vitro) for the detection of a trypnanosome infection in a subject.

Hybridization can be carried out under conventional conditions, known to the person skilled in the art and adjustable by the latter (Sambrook, Fritsch, Maniatis (I989) Molecular Cloning, Cold Spring Harbor Laboratory Press). In particular, hybridization can be carried out under conditions of high, medium or low stringency, depending on the level of sensitivity sought, the amount of material available, etc. For example, suitable hybridization conditions include a temperature between 55 and 63 ° C for 2 to 18 hours. Other hybridization conditions, suitable for high density supports, are for example a hybridization temperature between 45 and 55 ° C. After hybridization, various washings can be carried out to remove the non-hybridized molecules, typically in SSC buffers comprising SDS, such as a buffer comprising 0.1 to 10 × SSC and 0.5-0.01% SDS. Other wash pads containing SSPE, MES, NaCI or EDTA can also be used.

A particular object of the invention thus resides in a method for detecting the presence of a trypanosome in a mammal, or for evaluating the response to a treatment against the trypanosome, comprising contacting, under conditions allowing hybridization between sequences. complementary, nucleic acids from a mammalian sample and a TbKHCl-specific nucleic probe, the formation of a hybrid being indicative of the presence of trypanosome.

The selective amplification is preferably carried out using a primer or a pair of primers allowing the amplification of all or part of a nucleic acid encoding TbKHCl. The primer can be specific for a coding sequence (for example SEQ ID NO: 1), or for a region flanking the coding sequence. The primer typically comprises a single-stranded nucleic acid, of a length advantageously between 5 and 50 bases, preferably between 5 and 30. Such a primer constitutes another object of the present application, as well as its use (essentially in vitro) for the detection of the presence of trypanosome in a subject.

In this regard, another object of the invention lies in the use of a nucleotide primer or of a set of nucleotide primers allowing the amplification of all or part of a TbKHCl gene to detect the presence of trypanosome in a mammal.

Another particular object of the invention resides in a method for detecting the presence of trypanosome in a mammal, comprising bringing into contact, under conditions allowing amplification, the nucleic acids derived from a sample of the mammal and from a d primer specific for TbKHCI, the existence of an amplification product being characteristic of the presence of trypanosome in this mammal.

The detection method is applicable to any biological sample from the mammal tested. As such, generally means by "sample" in the sense of the invention any sample comprising nucleic acids or polypeptides. Mention may advantageously be made of a sample of blood, plasma, platelet, ganglion, saliva, urine, stool, etc., more generally any tissue, organ or, advantageously, biological fluid comprising nucleic acids or polypeptides or antibodies. In a preferred and particularly advantageous embodiment, the sample used for the detection method is a sample derived from blood, for example a sample of blood, serum or plasma. The sample can be obtained by any technique known per se. for example by sampling, by non-invasive techniques, from collections or banks of samples, etc. The sample can also be pre-treated to facilitate accessibility of the protein or its nucleic acid, for example by lysis (mechanical, chemical, enzymatic, etc.), purification, centrifugation, separation, etc. The sample can also be labeled, to facilitate the determination of the presence of the target molecules (fluorescent, radioactive, luminescent, chemical, enzymatic labeling, etc.). The nucleic acids in the sample can also be separated, processed, enriched, purified, reverse transcribed, amplified, fragmented, etc. In a particular mode, the nucleic acids of the sample are DNAs or RNAs, in particular the or mRNAs of the sample. In a very particular mode, the nucleic acids are the product of amplification of RNA, in particular of mRNA; or the / cDNAs prepared from the RNAs, in particular the mRNAs of the sample.

The presence of the protein (or a nucleic acid encoding the protein) TbKHCI in the sample is indicative of the presence of trypanosome in the mammal considered.

Kits

Another object of the invention relates to a kit for detecting or measuring trypanosomes in a test sample, characterized in that it comprises at least one ligand specific for the protein TbKHCI, and at least one reagent for detecting a reaction between the ligand and the TbKHCI protein. Advantageously, the ligand is an anti-TbKHCI antibody and the reagent allows the detection of an immune complex. The kit may include a suitable support (for example a plate, column, chip, etc.) on which the ligand is immobilized, allowing easy detection of the formation of a complex. The detection reagent can be a second ligand (e.g, antibody) binding the TbKHCI protein or binding the first ligand. It can be any other reagent that can reveal the formation of a complex (enzyme, dye, etc.).

Another object of the invention relates to a kit for detecting or measuring trypanosomes in a test sample, characterized in that it comprises at least one ligand specific for an anti-TbKHCI anticotps, and at least one reagent for the detection of 'a reaction between the ligand and the antibody. Advantageously, the ligand is a TbKHCI protein or a TbKHCI antigenic peptide, or a synthetic product, and the reagent allows the detection of an immune complex. The kit may include a suitable support (for example a plate, column, chip, etc.) on which the ligand is immobilized, allowing easy detection of the formation of a complex. The detection reagent can be a second ligand (e.g. antibody) binding the antibodies. It can be any other reagent that can reveal the formation of a complex (enzyme, dye, etc.).

Another object relates to a kit for detecting or measuring trypanosome in a test sample, characterized in that it comprises at least one antibody according to claim 12 or the TbKHCI protein or a peptide thereof, a medium suitable for formation of an immune complex, and at least one reagent for the detection of an immunological reaction.

Another object relates to a kit for detecting or measuring trypanosome in a test sample, characterized in that it comprises at least one nucleic probe specific for TbKHCI, a medium suitable for the formation of hybridization, and at least one reagent for the detection of a hybridization reaction.

Another subject of the present application relates to a product comprising a support on which is immobilized at least one ligand specific for the TbKHCI protein. Preferably, the ligand is an antibody or a fragment or derivative of anti-TbKHC 1 antibodies.

Another subject of the present application relates to a product comprising a support on which is immobilized at least one TbKHCI protein or an antigenic peptide thereof.

Another subject of the present application relates to a product comprising a support on which is immobilized at least one nucleic probe specific for TbKHCI. Preferably, the nucleic acid probe is a single-stranded DNA molecule, from 10 to 200 nucleotides in length, having a sequence complementary to the gene coding for the protein TbKHCI.

The support can be any solid or semi-solid support having at least one surface, planar or not (that is to say in 2 or 3 dimensions), allowing the immobilization of nucleic acids or polypeptides. Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as in particular glass, silica, plastic, fiber, metal, polymer, polystyrene, teflon, etc. The reagents can be immobilized on the surface of the support by known techniques or, in the case of nucleic acids, synthesized directly in situ on the support. Immobilization techniques include passive adsorption (Inouye et al., J. Clin. Microbiol. 28 (1990) 1469). covalent bond. Techniques are described for example in W090 / 03382. WO99 / 46403. The reagents immobilized on the support can be ordered according to a pre-established scheme, to facilitate the detection and identification of the complexes formed, and according to a variable and adaptable density. The products of the invention typically include control molecules, making it possible to calibrate and - or normalize the results.

Other aspects and advantages of the invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.

EXAMPLES

Materials & Methods

Animals

Female Swiss mice 25-30 gr (Charles River, Domaine des Oncins. 69592 L’arbresle codex) maintained in the laboratory according to animal protection regulations.

Parasites

The following parasite strains were used:

Trypanosoma bntcei bnicei (Antat l.l E)

Trypanosoma bnicei gambîense Fco (ITM AP 1893)

Trypanosoma bnicei gambîense "Biyamina" (MHOM'SD 82),

Trypanosoma bntcei bntcei EATRO 1125

Trypanosoma muscidi "Partinico 11"

Trypanosoma bnicei rhodesiense (State i.2'R)

Trypanosoma evansi (Mantecal EC8)

Trypanosoma congolense (E325)

Trypanosoma cruzi (MN cl2)

Secretome preparation

The parasites are purified by ion exchange chromatography (DEAE cellulose) from the blood of infected mice, and incubated for 2 hours in a secretory medium (Ringer Lactate + glucose 50 mM) at 37 ° C. The secretion products are recovered by centrifugation (1200 g, 10 minutes 4 ° C). This supernatant is filtered over 0.22pm, aliquoted and stored at -80 ° C. The amount of protein present is measured by the Bradford technique. This secretome, also called PSF (Parasite Soluble Factor) or secretion product, containing the protein TbKHCl, can be used as it is.

Production of an anti-TbKHCl antibody

BALB'c mice were immunized with a preparation containing the protein TbKHCl to make hybridomas by fusion. The resulting monoclonal antibodies were used to screen a T. b. Expression library. gambiense. The recognized clones were then used for the production of recombinants. The selected monoclonal antibody, Mabl, fixes the C-terminal region of kinesin, which is predicted to be a coiled region.

Preparation of a fraction enriched in TbKHCl protein by affinity chromatography

The parasites are purified by ion exchange chromatography (DEAE cellulose) from the blood of infected mice, and incubated for 2 hours in a secretory medium (Ringer Lactate + glucose 50 mM) at 37 ° C. The secretion products (secretome) are recovered by centrifugation (1200 g. 10 minutes 4 ° C). This supernatant is filtered through a 0.22 μm filter, aliquoted and stored at -80 ° C. The amount of protein present is measured by the Bradford technique.

The Mabl monoclonal antibody in buffered solution (carbonate OJM'NaCl 5M, pH 8.3) is grafted onto a chromatography column (Sephadex CNBr). After 48 hours at 4 ° C, and 5 washes in carbonate buffer, the secretion products are deposited on this column. After passage and successive washings in secretion medium, the molecules hooked by the antibody are eluted by successive additions of glycine buffer IM, 'HCl pH3 and of glycine buffer IM'NaOH, pHl L The pH of the eluted fraction is adjusted to pH8 . After dialysis in 0.015 M PBS, the enriched fraction is aliquoted in tubes stored at -80 ° C. The quantity obtained is measured according to the Bradford technique. The content of the enriched fraction is analyzed by gel electrophoresis and by Western blot.

Preparation of a fraction enriched in TbKHCl protein by differential filtration

In this example, the vaccine fractions were prepared from the secretion products by differential filtration. The parasites were purified on an ion exchange column (DEAE cellulose) and allowed to secrete (200 × 10 ⁶ / ml of secretion medium for 2 hours). The secretion products (PSF) of the strains of the following trypanosome species were thus obtained:

- T. brucei brucei (T. b. B);

- T. brucei brucei KO for the two alleles of TbKHCl (T. b. B KO);

- 7 ’. bruceigambiense {T. Féo);

- T. evcmsi (T. e).

The PSFs were then fractionated by differential filtration allowing separation in high weights (HPM) and low molecular weights (BPM). Different cutoff thresholds were used for the filtrations: 50kDa, which gives the HPM 50 and BPM 50 fractions; and 100kDa, which gives the fractions HPM 100 and BPM I00. The intermediate fraction results from the passage of the PSF first on the filter having the cut-off threshold of 50 kDa, then then on the filter having a cut-off threshold of 100 kDa, and corresponds to the molecules having a PM> 50 kDa, but <100 kDa. The secretome is placed on a filter having a cutoff threshold of 50 or 100 kDa; after centrifugation (4000g, 1 hour, 4 ° C), the fraction containing the molecules having a molecular weight lower than the cut-off threshold (BPM, low molecular weight) is separated from that containing the molecules having a molecular weight higher than the cut-off threshold (HPM, high molecular weight). The TbKHCl protein is present in the HPM fractions. The fractions are aliquoted in tubes stored at -80 ° C. The amount obtained in each fraction is measured according to the Bradford technique.

EXAMPLE 1 Inhibition of the Growth of the Parasites by a Monoclonal Antibody Directed Against Kinesin

In vitro, the monoclonal antibody Mabl was added to parasites in co-culture with feeder layers (concentration of Mabl in the culture 4pg / mL). Compared to the control, it inhibits parasite growth while the IgG2b control isotype (concentration 4pg / mL) has no effect (Figure 1).

In vivo, the injection of Mabl (200 pg in 200 pL of PBS intraperitoneally) into mice parasitized for 2 days inhibits the development of parasitaemia (expressed in log10 of the number of parasites per ml of blood), the control isotype IgG2b (200 pg in 200 pL of PBS intraperitoneally) has no effect (Figure 2).

EXAMPLE 2 In Vivo Protection Against Parasites by Vaccination

The fraction enriched in TbKHCl protein is injected (10-20 pg / mouse) twice, 30 days apart (D0 and D30), by subcutaneous route to the mouse, with or without adjuvant (saponin, 25 pg per mouse) . Control mice receive the medium alone with or without adjuvant.

The mice are infected I, 2, or 3 months after the last injection (see Figure 3).

The parasitaemia of vaccinated mice and controls (medium alone + adjuvant) is evaluated daily for 25 days after infection, then once a week thereafter. The results are presented in Figure 4,

Control mice that received medium with or without adjuvant die to 7-8 ^u post infection day ^myself. Remarkably, 22 mice out of 26 (84.6%) having received 2 injections of TbKHCl protein + adjuvant survive. The addition of the adjuvant to the protein TbKHCl increases the survival rate of the vaccinated mice and the duration of effectiveness of the vaccine.

Example 3: Vaccination with TbKHCl induces cross-protection

The mice are immunized with the protein TbKHCl, then infected two months later either with the same trypanosome or with a trypanosome of another species.

Lots Origin of TbKHCl for immunization Number of subcutaneous injections + saponin Infection 2 months after the last injection + adjuvant Parasitaemia and survival study Lot l (I4 mouse) T. brucei gambiense 2 T. b. gambiense No parasites detected in mice, all survive 50 days Lot 2 (14 mice) T. brucei gambiense 2 T. b. brucei No parasites detected in mice, all survive 50 days

These results show cross-protection, the TbKHCl protein of T. brucei gambiense being capable of inducing protection against infection by T. brucei brucei.

Example 4: Diagnosis of infected patients

Presence of anti-TbKHC 1 antibodies in the serum of patients with human African trypanosomiasis and their absence in the serum of control subjects from the same endemic region.

The results are presented in FIG. 5. They show that anti-kinesin antibodies TbKHCl were detected in all the patients tested suffering from human African fever. These results thus illustrate the possibility of discriminating infected patients by measuring antibodies directed against TbKHC I.

EXAMPLE 5 Protection Trial by Serotherapy

Serotherapy tests carried out on batches of 5.8 or 10 mice have demonstrated that the sera of mice having received two injections spaced three weeks apart from total T. Feo PSF (30 μg / injection), or from HPM fractions 50 (20pg / injection) or HPM 100 (20pg / injection), in the presence of saponin (25pg), effectively protect (100% protection) the naive mice experimentally infected with T. Fëo (2,000 parasites under the skin) ( Figure 6). On the other hand, the sera of mice having received two injections spaced one month apart from BPM50 or intermediate fractions (IOO <PM> 5O) have no protective effect in infected naive mice. Mice receiving normal mouse serum before infection (not shown in Figure 6) die 7 days after infection.

In addition, the mice having received serum from mice immunized with the PSFs of T. Feo or with the HPM50 fraction of T. Feo are also protected against infection with T. b. b (cross protection) (Figure 7).

EXAMPLE 6 Test for Protection by Vaccination

6.1. Vaccination with a fraction of T. b. gambiense (Féo)

Naive mice receive two injections 3 weeks apart, in the presence of saponin (25pg ^/ mouse). fractions from T. b. gambiense (Féo) and, more precisely, total PSF (30pg / injection), HPM50 (20pg / injection). or BPM50 (20pg / injection). The mice are then challenged by live parasites T. b. b (2,000 per mouse) two months after administration of the second immunization.

The results presented in FIG. 8A show:

- that the protective antigens are present mainly in the high molecular weights of the PSF of T. Féo, and

- cross-protection is obtained against infection with T. b. brucci.

6.2. Vaccination with a fraction of T.b. brucci

The mice receive two injections of total PSF (50 pg) of T. b. brucci or T. b. brucci KO for kinesin 30 days apart (D0 and D30), subcutaneously with adjuvant (saponin, 25 pg per mouse). The "control" mice receive the adjuvant alone. The mice are infected 2 months after the last injection (D30) by 2000 live T. b. brucci.

The results presented in FIG. 8B show that all the mice having received the PSF of T. b. brucci survive, while mice given the T. b. brucci KO for kinesin died 8 days after infection, along with the "control" mice.

6.3. Vaccination with a fraction of T. evansi

The mice receive two injections of total PSF (50 μg) from T. evausi, 30 days apart (D0 and D30). subcutaneously with adjuvant (saponin. 25 pg per mouse). The "control" mice receive the adjuvant alone. The mice are infected 2 months after the last injection with T. evausi (2000 parasites).

The results presented in FIG. 8C show an improved survival of the mice having received the PSF of T. evansi, while all the controls die.

EXAMPLE 7 Proteomic Analyzes

7.1. Protein profiles

The protein profiles, obtained after migration under denaturing and non-reducing conditions of the secretory or of secretome fractions (HPM or HBM), show the presence of a band of high molecular weight (around 125 kDa- boxed in Figure 9) sufficiently present in pathogenic trypanosome species (T. Feo; T. b. brucci; T. rhodesiense; T. evansi) to be detected after blue staining

Coomassie. This band corresponds to the molecular weight of the TbKHCl protein. On the other hand, this band seems to be absent or in too small a quantity in T. b. brucei KO for kinesin. Significantly, this band is well present in the HPM fractions and little revealed in the BPM fractions.

7.2. Mass spectrometry

All the samples were analyzed by nano flow HPLC (Ultimate 3000, Dionex) coupled to a mass spectrometer having a nanoelectrospray source (Orbitrap Elite, Thermo Fisher Scientific). The peptides were separated on a capillary column (reverse phase Cl8, NanoViper, Dionex) according to a gradient of 0-40% B in 60 min (run of 105 min) (A = 0.1% formic acid, 2% acetonitrile ; B = 0.1% formic acid in acetonitrile) at a flow rate of 300 nl / min. The spectra were recorded using Xcalibur software (Thermo Fisher Scientific). The spectral data were analyzed using MaxQuant 1.5.0.0 software then retreated with Perseus 1.5.3.0 software after using the Leading v2.2 script developed by Oana Vigy. We used as database: Uniprot_Trypanosoma-all_2016_01 .fasta with the following modifications: Carbamidomethylation (C) as fixed and Oxidation (M) as variable.

This technique made it possible to identify the presence of TbKHCl in the protective samples of T. Feo (PSF Feo and HPM50) and its absence in the BPM 50 fraction.

7.3. Analysis of the immunological profile by Western blot

After differential filtration of PSF secretion products. the HPM50 and HPM 100 fractions concentrate the protective antigens. The serum of the mice immunized with these protected fractions made it possible to analyze the antigenic targets of the protective antibodies. Molecular weight markers (MM) are used to estimate the molecular weight of antigens revealed by sera.

Western blot revealed by the purified Mabl (Figure 10):

The purified monoclonal antibody Mabl targets Kinesin TbKHCl. It recognizes high molecular weight proteins and in particular a protein with an apparent molecular weight of around 125 kDa as well as a protein of 59 kDa which seems to be common to all the species of trypanosomes studied. Our data show that the 125 kDa antigen corresponds to the kinesin TbKHCl (125.89kDa). The 59kDa protein corresponds to a fragment of the protein. This recognition for the 125 kDa protein is very marked in the HPM 100 Féo and HPM50 Féo samples: lighter for the PSF Féo, PSF T. b. brucci and weak or even nonexistent for PSF T samples. b. britcei KO.

Western blot revealed by the anti-PSF serum féo (Figure l I):

Two major immunogenic complexes, between 198 and! 20kDa and around 55kDa, are revealed by the anti-PSF serum Féo.

Western blot revealed by the anti-HPM50 serum Féo (Figure 12):

The anti-HPM 50 serum selectively targets the antigen corresponding to a molecular weight of 125 kDa: Differential filtration> 50 therefore concentrates this antigen well, which corresponds to the kinesin protein TbKHCl (! 25.89kDa), The 125 antigen kDa is practically no longer recognized by this serum in the PSF 7 '. b. bntcci KO for TbKHCl. This confirms that the 125 kDa antigen corresponds well to the kinesin TbKHCl.

Western blot revealed by anti-HPM 100 serum Féo:

Anti-HPM 100 serum also preferentially targets an antigen of 25 kDa: Differential filtration> 100 concentrates this antigen which corresponds to the kinesin protein TbKHCl (125.89kDa).

SHORT

The present invention relates to methods and compositions for preventing, treating and diagnosing trypanosome infection. It relates to the use of excreted ^ secreted antigens (antiantigens, secretome) and more particularly the identification of a protein excreted / secreted by trypanosomes, the inhibition of which makes it possible to confer effective protection against infection, development or spread of trypanosomes, mainly by vaccination. It relates to the use of the protein or its derivatives or a nucleotide sequence derived therefrom, or of an extract enriched in this protein, as well as that of antibodies directed against them for immunotherapy, diagnosis and monitoring trypanosome infections.

Claims (17)

1. Pharmaceutical or veterinary composition comprising (i) the TbKHCI protein or one or more antigenic peptides thereof; a nucleic acid encoding said protein or said peptide; or an inhibitor of the protein TbKHCI, and (ii) a pharmaceutically or veterinary acceptable excipient. 2. Composition according to claim I, characterized in that it comprises (i) the protein TbKHCI or one or more antigenic peptides thereof, or a nucleic acid encoding said protein or said peptide, (ii) an acceptable excipient on the pharmaceutical or veterinary plan, and (iii) optionally an adjuvant. 3. Composition according to claim l or 2, characterized in that it comprises the protein of sequence SEQ ID NO: 2 or a natural variant thereof, or a nucleic acid encoding said protein. 4. Composition according to claim l or 2, characterized in that it comprises a peptide comprising the residues 1000 to 1111 of the sequence SEQ ID NO: 2 or of a natural variant thereof, a variant thereof having at least 90% sequence identity, or a nucleotide sequence encoding said peptide. 5. Composition according to any one of the preceding claims, characterized in that the protein or the antigenic peptide (s) or the nucleotide sequence are in pure form or of enriched, recombinant or synthetic extract. 6. Composition according to claim l, characterized in that the inhibitor is an anti-TbKHCl antibody, or a fragment or derivative of such an antibody. 7. Composition according to any one of claims 2 to 5, for its use for vaccinating or immunizing a mammal against the trypanosome. 8. Composition according to any one of claims 2 to 5, for its use for protecting a mammal against a trypanosomiasis. 9. Composition according to any one of claims 1 to 6, for its use for treating a mammal having a trypanosomiasis. 10. Use of the protein TbKHCI, of one or more antigenic peptides thereof, or of a nucleic acid encoding said protein or said peptide, or of a secretion extract enriched in this protein, for the preparation of a vaccine to immunize or protect a mammal against the trypanosome. 11. Use of a TbKHCI protein inhibitor for the preparation of a medicament for treating a mammal having trypanosomiasis. 12. Use according to claim 11, characterized in that the inhibitor is an antibody specifically binding the TbKHCI protein. 13. A method of in vitro diagnosis of trypanosomiasis in a mammal, characterized in that it comprises measuring, in a sample of said mammal, the presence of the protein TbKHCl, a fragment thereof, of a sequence nucleotide encoding TbKHCl, or antibodies directed against TbKHCl or against one or more antigenic peptides thereof. 14. Method for monitoring the evolution of a trypanosome infection in a mammal, characterized in that it comprises measuring the amount of TbKHCl protein, a fragment thereof, a nucleotide sequence coding for TbKHCl , or anticoips directed against TbKHCl, in samples of the mammal taken at different time intervals. 15. Method for determining the effectiveness of a treatment against the trypanosome in a mammal, characterized in that it comprises measuring the amount of protein TbKHCl. of a fragment thereof, of a nucleotide sequence coding for TbKHCl, or of antibodies directed against TbKHCl, in samples of the mammal taken at different time intervals during the treatment. 16. Kit for detecting or measuring trypanosome in a test sample, characterized in that it comprises at least one antibody specifically binding the TbKHCI protein or the TbKHCl protein or a peptide thereof, an appropriate medium for the formation of a immune complex, and at least one reagent for detecting an immunological reaction. 17. Kit for detection or measurement of trypanosome in a test sample, characterized in that it comprises at least one nucleic probe specific for TbKHCl, a medium suitable for the formation of hybridization, and at least one reagent for the detection of 'a hybridization reaction.