Background of the invention [0002] The impact of African Animal Trypanosomiasis (AAT) is a considerable constraint to animal protection in Africa.

[0003] A number of socio-economic studies outline the impact of this disease and the requirement for new control options [1]. With the combined effects of lower productivity due to AAT and demographic growth, per capita meat and diary availability tends to decrease.

[0004] Large areas of the humid zone can harbor intensive breeding of bovines and small ruminants, given a

rapid elimination of the constraints of AAT and an intensification of livestock breeding.

[0005] Prospective scenarios of the FAO have suggested that 120 million more bovines could be raised in the areas now occupied by the Tsetse fly in the different zoogeographic African zones concerned.

[0006] This figure could even be largely increased should agriculture and livestock breeding become more integrated and intensified.

[0007] The African animal trypanosomiases (AAT) are diseases caused by flagelled protozoans, parasites of the blood, which induce upon sensitive animals a more or less severe anemia which may affect various organs, especially cardiac organs. Left untreated animals often die of the disease.

[0008] These protozoans comprise several pathogenic species for animals and humans: principally Trypanosoma (Nannomonas) congolense, Trypanosoma vivax (Duttonella), Trypanosoma brucei (Trypanozoon), Trypanosoma gambiense species, as well as their sub-species and nosodemes.

[0009] This disease affects principally Africa, but the corresponding animal trypanosomiases exist also in South-America with Trypanosoma vivax viennei and Trypanosoma brucei evansi. Disease caused by the latter also occurs in some countries of Asia.

[0010] In Africa, this disease affecting a human or an animal is present in 36 countries and is transmitted by Glossina species.

[0011] For many years, various publications have stated that it is possible to develop a vaccine in order to protect animals and humans from the destructive effect of Trypanosoma (WO 95/29699).

[0012] However, the various antigens proposed in said vaccine are not efficient.

[0013] It is known that trypanosomes evade the immune responses of their hosts by varying their surface coat protein, the variant surface glycoprotein (VSG).

However, restricted exocytosis and endocytosis is obtained at a specific site: an invagination of the plasma membrane of the trypanosome called"flagellar pocket" (fp).

[0014] Recently, a publication has shown that extracts from flagellar pocket (fp) of Trypanosoma brucei seem to induce a protective immunity in laboratory animals and bovine [2].

[0015] However, the vaccine antigens have not been characterized and thus the published results are incomplete. There is a pressing need for robust Trypanosomiasis control measures of which vaccination would be the preferred option, and the vaccine antigens need to be thoroughly characterized.

[0016] Researches have sought (without success) an effective vaccine against African Trypanosomiasis from the time that the infective parasites were discovered more than one hundred years ago.

[0017] One investigation [3] indicates that protective antigens might reside within the trypanosome flagellar pocket. This specialized region of the trypanosome plasma membrane represents 0.5% of the cellular surface in which membrane turnover occurs at high rates, consistent with its role in endocytotic and exocytotic processes.

[0018] Until now it has been extremely difficult to obtain the segregation of flagellar pocket proteins from surface coat proteins (VSG) of trypanosomes.

[0019] It is known that glycans are involved in the sorting of membrane proteins in polarized cells. It has been described that N-linked glycans containing linear poly-N-acetyllactosamine (pNAL) were only associated with

proteins of the flagellar pocket/endocytic pathway in Trypanosoma brucei and are present only in bloodstream forms of the parasite [4]. The authors show that these glycoproteins bind to tomato lectin (TL), a property that unexpectedly allowed their single-step isolation.

Aims of the present invention [0020] The present invention aims to provide a new method for the recovering and the characterization of an antigenic structure which protects against trypanosomes and related parasites, which does not present the drawbacks of the state of the art and which could be used as a diagnostic tool for identifying infections by said trypanosomes and related parasites or could be used for the prevention and/or the treatment of diseases induced by trypanosomes and related parasites.

Summary of the invention [0021] The present invention is related to a method for the isolation, purification and characterization of a surface immunogenic or antigenic structure of a trypanosome (or another related human or animal parasite), said method comprising the following steps: -purifying membrane glycoproteins located on the surface of the flagellar pocket (fp) of said trypanosome, preferably glycoproteins containing linear poly-N- acetyllactosamine (pNAL), from a biological extract comprising said trypanosome, -deglycosylating said glycoproteins into proteins, -obtaining hybridomas secreting monoclonal antibodies directed against said proteins by techniques which allow the production of specific antibodies or specific active portions thereof (Fab', Fab2', etc.),

-screening the resulting hybridomas for selecting a positive hybridoma which secretes an antibody or a portion thereof able to inhibit trypanosome growth (trypanosome activity in vitro), -obtaining said antibody from said positive hybridoma, -selecting and purifying an immunogenic or antigenic structure among the proteins by a specific binding between said proteins and said monoclonal antibody, and -possibly identifying the amino acid sequence of said antigenic structure and possibly the nucleotide sequence encoding said immunogenic antigenic structure.

[0022] It is meant by"immunogenic or antigenic structure", a structure made of one or more epitopes, either topographically assembled or linear, which are able to produce an effective immune response against trypanosome or other related human or animal parasites infections, as well as nucleotide sequences (cDNA, RNA sequences), encoding said antigenic structure or said epitopes.

[0023] It is meant by"antibody able to inhibit trypanosome growth (trypanosome activity in vitro)", an antibody able to kill the trypanosome in a biological medium or to reduce its concentration and possibly its multiplication.

[0024] It is meant by"identifying the amino acid sequence or the nucleotide sequence of the antigenic structure according to the invention", obtaining by methods well known by the person skilled in the art, the amino acid sequence of the antigenic structure when said antigenic structure has been purified and when at least a portion of said antigenic structure is characterized. Said identification is preferably correlated with genetic sequences already sequenced for trypanosomes or related parasites.

[0025] Preferably, the step of purifying the glycoproteins located at the surface of the flagellar pocket of the trypanosome according to the method of the invention is made by passing the biological extract containing said trypanosome on a support upon which tomato lectin molecules have been preliminary immobilized in order to retain by binding said glycoproteins to tomato lectin molecules and to obtain affinity complexes between said tomato lectin molecules and said glycoproteins, and by recovering said glycoproteins from said complex, preferably, according to the method described elsewhere [41.

[0026] Furthermore, the purification of said glycoproteins is obtained by applying to said column a solution containing a competitive binder in order to obtain affinity complexes between said tomato lectin molecules and said competitive binder.

[0027] Advantageously, the competitive binder is a solution containing chito-oligosaccharides, preferably tri- N-acetyl chitotriose and tetra-N-acetyl chitotetraose.

[0028] Another aspect of the present invention is related to the antigenic structure recovered, purified and characterized by the method according to the invention, an inhibitor directed against said antigenic structure and their use as a medicament.

[0029] According to the above-mentioned definition of the antigenic structure, an inhibitor directed against said antigenic structure is any natural or synthetic molecule able to interact specifically with said antigenic structure in order to block the expression of said antigenic structure, such as a RNA antisense or a ribozyme able to interact with a RNA or a genomic nucleotide sequence encoding said antigenic structure, or an hypervariable portion of an antibody (monoclonal or

polyclonal) able to interact with said antigenic structure (or one or more of its epitopes), or a cell (or a receptor of a specific cell) able to interact with said antigenic structure and which inhibits the parasite growth or kills the trypanosome directly or increases susceptibility of the organism to killing by a host mechanism thus alleviating disease in infected animals including livestock and people.

[0030] Preferably, said antigenic structure, possibly in combination with said inhibitor, is able to induce an immune response, preferably a cellular humoral and/or local immune response against trypanosomes and related parasites.

[0031] Another aspect of the present invention is related to a diagnostic kit comprising said antigenic structure, nucleotide sequences encoding said antigenic structure or its epitopes, and/or inhibitors directed against said antigenic structure for the detection, the quantification and/or the monitoring of trypanosomes and related parasites inside a patient (cattle or human) and the possible evolution of an infection by said parasites, especially by trypanosomes, through the analysis of biological fluids obtained from said patient.

[0032] Another aspect of the present invention is related to a pharmaceutical composition (preferably a vaccine) comprising an adequate pharmaceutically acceptable carrier or diluent, the antigenic structure according to the invention, the nucleotide sequences encoding said antigenic structure or its epitopes and/or inhibitors directed against said antigenic structure according to the invention, and possibly adjuvants for inducing the immune response (preferably a humoral cellular and/or local immune response) against trypanosomes and related parasites.

[0033] Said vaccine could comprise these elements that can be administrated in vivo or ex vivo to a patient

(a cattle animal and/or a human) suffering from said parasites, especially from said trypanosomes.

[0034] The nucleotide sequences comprising the information for encoding the antigenic structure or the epitopes according to the invention could be introduced as a naked vaccine in a cell or directly in a patient.

[00353 Therefore, a further aspect of the invention is also related to cells transformed by the nucleotide sequences encoding the antigenic structure according to the invention or one or more of its epitopes, such cells could be used for the industrial production of said antigenic structure or could be administrated to the patients when they present said antigenic structure at their surface.

[0036] A further aspect of the present invention is related to a method of prevention and/or treatment of a patient (preferably cattle, but also including the human) suffering from trypanosomes and/or related parasite infections, comprising the administration of a sufficient amount of an antigenic structure, a nucleotide sequence encoding said antigenic structure or its epitopes, inhibitors directed against said antigenic structure or the pharmaceutical composition according to the invention to said patient for inducing a protective immune response against said trypanosomes and related parasites (especially African animal trypanosomiasis, for which the patient is a livestock animal).

[0037] A last aspect of the present invention is related to the use of the antigenic structure according to the invention or the pharmaceutical composition according to the invention for the preparation of a medicament in the prevention and/or the treatment of diseases and infections induced by trypanosomes and related parasites, especially African animal trypanosomiasis, the patient being a cattle animal.

[0038] According to the invention, the trypanosome is a pathogenic trypanosome for human and/or animal, preferably selected from the group consisting of Trypanosoma (Nannomonas) congolense, Trypanosoma vivax (Duttonella), Trypanosoma brucei (Trypanozoon), Trypanosoma <BR> <BR> <BR> <BR> gambiense, Trypanosoma evansi, Trypanosoma eguiperdum species and sub-species, and the nosodemes of said species.

[0039] Other related parasites are Trypanosoma cruzi and various species of Leishmania.

[0040] In the vaccine or pharmaceutical composition according to the invention, the pharmaceutically acceptable carrier can be any compatible non-toxic substance suitable for administering the composition (vaccine) according to the invention. The pharmaceutically acceptable carriers according to the invention are the ones well known by the person skilled in the art such as tablets, coated or non- coated pills, capsules, solutions or syrups. The pharmaceutically acceptable carrier may vary according to the mode of administration (intravenous, intramuscular, parenteral, etc.).

[0041] The vaccine according to the invention may also comprise adjuvants well known by the person skilled in the art, which may increase or regulate the humoral, local and/or cellular response of the immune system against trypanosomes or other related parasites or pathogenic agents.

[0042] The vaccine according to the invention is prepared by the methods generally applied by the person skilled in the art for the preparation of a vaccine, wherein the percentage of active compound/ pharmaceutically acceptable carrier can vary within very large ranges, only limited by the tolerance and the level of acquaintance of the patient to the vaccine. The limits

are in particular determined by the frequency of administration.

[0043] The present invention will be described in detail in the following enclosed and non-limiting examples referring to the accompanying drawings.

Brief description of the drawings [0044] Fig. la shows an elution profile of proteins of T. brucei after tomato lectin chromatography.

[0045] Fig. 1b represents results obtained after SDS-PAGE and silver staining of the corresponding tomato lectin chromatography fractions.

[0046] Fig. lc represents results obtained after SDS-PAGE and autoradiography of the corresponding tomato lectin chromatography fractions.

[0047] Fig. Id represents a Western blot of the corresponding tomato lectin chromatography fractions using different antibodies.

[0048] Fig. le represents the results obtained after SDS-PAGE and autoradiography of precipitated proteins from SDS and CHAPS lysates of T. brucei cells metabolically labelled with 35S.

[0049] Figs. 2a-c show labeling experiments performed on ultrathin sections of bloodstream forms of T. brucei using biotinylated tomato lectin.

[0050] Fig. 2d shows labeling experiments performed on procyclic forms of T. brucei using biotinylated tomato lectin.

[0051] Fig. 2e shows labeling experiments performed on bloodstream forms of T. brucei in the presence of chito-oligosaccharides using biotinylated tomato lectin.

[0052] Fig. 2f shows a direct fluorescence microscopy analysis on T. brucei cells using Texas-Red conjugated tomato lectin.

[0053] Fig. 2g shows a direct fluorescence microscopy analysis on T. brucei cells using FITC- conjugated concanavalin A.

[0054] Fig. 3 shows antibody responses of infection sera from susceptible Boran, trypanotolerant N'Dama and Cape buffalo to different proteins from T. congolese IL 3000.

[0055] Fig. 4a represents an immunization challenge profile of mice after infection with T. brucei (each curve shows the average parasite number in a group containing seven mice at day 0).

[0056] Fig. 4b represents an immunization challenge profile of mice after infection with T. congolese (each curve shows the average parasite number in a group containing seven mice at day 0).

[0057] Fig. 5 represents the screening of a T. congolese IL 3000 Xgtll expression library with sera from mice immunized with N-glycosidase F treated tomato lectin-binding proteins from T. brucei ILTat 1.1.

Detailed description of the invention A. Purification of glycoproteins present in the flagellar pocket of T. brucei [0058] Glycoproteins present in the flagellar pocket of T. brucei were isolated by lectin chromatography of CHAPS lysates of cellular suspensions that contained cells metabolically labelled with 35S or surface-labelled with 3. 251 [0059] The protocol was the following. A tomato lectin was coupled to Affigel 10 (BioRad)-2.0 mg lectin/ml of gel. Cells (107) previously surface-labelled with 125I or metabolically labelled with 35S were added to a suspension of unlabelled cells (2.0 X 10la) and the

combined pellet was extracted by resuspension in 20 ml of TSC (25 mM Tris-Cl pH 7.5,150 mM NaCl, 0.1 mM CaCl2, 1% CHAPS) and protease inhibitors. After 1 h at 0°C, the extract was centrifuged (50,000 X g for 1 h) and the supernatant was dialyzed overnight against 10 volumes of TSC and then applied to a tomato lectin column (-2.2 ml bed volume) equilibrated with TSC. The column was washed before reversing the flow and eluting bound proteins using a mixture of chito-oligosaccharides (20 mg/ml of tri-N- acetyl-chitotriose and tetra-N-acetyl-chitotetraose in TSC; Seikagaku corporation) that specifically compete with linear pNAL for binding to tomato lectin.

[0060] The elution profile of trypanosomal proteins thus obtained is shown on Fig. la. Peak 1 corresponds to fractions of non-binding material and peak 2 corresponds to fractions of binding material. It can be seen that most proteins did not bind to tomato lectin but a small amount of material was retained and specifically eluted with tomato lectin-competing chito-oligosaccharides.

[0061] Samples of the material loaded onto the column (L) and the combined flow-through fractions of peak 1 (FT) along with the individual fractions (28-31) and pooled material from peak 2 (TL pool) were subjected to SDS-PAGE and silver staining. The L, FT and TL pool samples were applied at 2 g/lane. Results are presented on Fig. lb. The positions of molecular mass markers are shown on the left (St ; BenchMark protein ladder, GibcoBRL).

Silver staining demonstrated a significant enrichment in the binding fraction of a group of proteins ranging in molecular weight from 30 to over 100 kDa.

[0062] Fractions representing non-binding (peak 1) and binding (peak 2) material were pooled and subjected to SDS-PAGE and autoradiography. Results are presented on

Fig. Ic. An arrowhead indicates the VSG protein. For comparison, the Concanavalin A-binding fraction from cells metabolically labelled with 35S [5] is shown on the right. Autoradiography confirmed this result with silver staining and demonstrated that most proteins, including the major surface glycoprotein VSG, did not bind to tomato lectin.

The pattern of 35S-labelled proteins in the binding fraction was very similar to that identified by silver staining. Some proteins were also labelled by 125,, indicating a surface location. A very different group of proteins, including VSG, was observed in the Concanavalin A-binding fraction.

[0063] When the tomato lectin-binding fraction was digested overnight with N-glycosidase F and reapplied to the tomato lectin column, all of the proteins were recovered in the non-binding fraction.

[0064] These results were obtained with a cloned variant (MIT at 1.2) in which the VSG contains an N-linked but branched pNAL oligosaccharide [6].

[0065] This directly confirmed the specificity of tomato lectin for linear N-linked pNAL.

[0066] Identical results were obtained using different VSG cloned variants.

B. Characterization of the tomato lectin-binding glycoproteins [0067] The tomato lectin binding fraction was further characterized using antibodies against flagellar pocket and lysosomal proteins (Figure 4c), for example, the expression-site-associated genes (ESAGs) 6 and 7, which encode subunits of the transferrin receptor [7-9], the 145 kDa LDL-binding protein [10] and invariant glycoproteins Igloo and CB1-gp [11]. The anti CB1-gp antibody was obtained from Cedar lane Laboratories Hornby, Ontario,

Canada; antibodies against LDL-binding protein were a gift from P. Courtoy (ICP, Brussels).

[0068] All of these proteins bound to tomato lectin, whereas other proteins, which are uniformly distributed over the trypanosomal surface [12], such as VSG and ISG70, did not.

[0069] Because the VSG expression site contains additional genes or ESAGs thought to encode surface proteins [12], Western blots were used to test whether the products of these ESAGs also bound to tomato lectin and, of those examined (ESAGs 2-4 and 8), only that encoded by ESAG 2 bound (Figure ld).

[0070] The protein encoded by ESAG 2 migrated at -120 kDa, which is higher than the molecular mass predicted from the gene sequence (-46 kDa) [12], but this decreased to 60 kDa after treatment with N-glycosidase F.

[0071] Silver staining and 35S labeling also showed the presence of a protein in the tomato lectin binding fraction (Figures la, c) that was similar in size to a 42 kDa protein recently identified in the flagellar pocket [13].

[0072] Interestingly, CB1-gp initially appeared as a 100 kDa glycoform, which then underwent elongation of N- glycans to generate the larger sized (-150-180 kDa) mature form of the protein [11]. This elongation does not occur in procyclic forms and it was proposed that the enzymes responsible are not active during this stage of the parasite life cycle [11]. These data support this view and suggest that the elongation of the CB1-gp N-glycans reflects the addition of linear pNAL chains that interact with tomato lectin.

[0073] In order to determine whether proteins bound to tomato lectin directly or by association with bound

glycoproteins, tomato lectin precipitations were performed using denatured (SDS) or native (CHAPS) lysates of cells metabolically labelled with 35S (Figure le). More precisely, biotinylated tomato lectin (40 jug) and streptavidin-agarose were used to precipitate proteins from SDS and CHAPS lysates (4 X 107 cell equivalents) of cells metabolically labelled with 35S [5]. After extensive washing, the precipitated proteins were eluted using the chito-oligosaccharide mixture and subjected to SDS-PAGE and autobiography.

[0074] These experiments showed that several proteins, most notably those migrating below 40 kDa, were not precipitated from denatured lysates, which suggested a non-covalent association with glycoproteins that bind to tomato lectin.

[0075] Results similar to those outlined above were found for other species of African trypanosomes, namely T. congolese and T. vivax. These data show that: (i) in all of these trypanosomes N-glycans containing linear pNAL are restricted to proteins of the flagellar pocket and endocytic pathway, (ii) these proteins can also be purified using tomato lectin, (iii), that antibodies against deglycosylated forms of these proteins from one species cross react with the native proteins purified from another species and (iv) that immunizing with deglycosylated forms of these proteins from one species generates cross reacting antibodies and cross protection in other species.

C. Localization of the tomato lectin binding glycoproteins in trypanosomes [0076] Biotinylated tomato lectin was used to investigate the localization of tomato lectin binding sites in trypanosomes. More precisely, ultra thin sections of

bloodstream forms of T. brucei (Fig. 2a-c), of procyclic forms of T. brucei (Fig. 2d) and of bloodstream forms of T. brucei in the presence of chito-oligosaccharides (20 mg/ml) (Fig. 2e) were incubated with biotinylated tomato lectin followed by gold (10 nm)-conjugated goat antibiotin antibody. In these figures, scale bars represent 0,5 Rm.

[0077] All elements of the endocytic pathway were labelled, including the lumen and ectoplasmic face of membranes of the flagellar pocket (fp), endocytic vesicles, collecting tubules (tv) and a large perinuclear digestive vacuole (DV) (Figures 2a-c). No other regions of the plasma membrane (pm) or intracellular compartments were labelled.

No labeling occurred in procyclic cells (Figure 2d). These data indicate that N-glycans that bind tomato lectin are developmentally regulated, which might reflect the morphological differences between the flagellar pocket endocytic pathway in bloodstream and insect forms of the parasite and the significant reduction in endocytic activity in the latter [143.

[0078] Labeling was abolished in bloodstream forms when competing chito-oligosaccharides were present (Figure 2e, where f is the abbreviation for flagellum).

[0079] Identical results were obtained with Texas- Red-conjugated tomato lectin, which specifically labelled the cellular region associated with endocytosis, that is, the region between the nucleus and the kinetoplast (Figure 2f). Again, no labeling was observed in procyclic forms nor in the presence of chito-oligosaccharides.

[0080] In contrast, the distribution of FITC- conjugated concanavalin A (Con A) was widespread (Figure 2g), a result consistent with the broader specificity of Con A.

D. Ability of tomato lectin-binding material of trypanosomes to be recognized by antibodies of Cape buffalo [0081] It has been evidenced that Cape buffalo that were bred in tsetse-free conditions from trypanosomiasis-free parents and submitted then to experimental infections, develop only one of few parasitemic waves before permanently suppressing parasitemia to a level of one to ten trypanosomes/ml blood [15,16]. This property is related to the presence of trypanosome growth-inhibitory antibodies in the immune Cape buffalo serum, as evidenced by the inventors and shown in table 1.

100823 Furthermore, a large portion of the growth inhibitory antibodies elaborated by the Cape buffalo were specific for flagellar pocket proteins and could be selectively removed from immune serum by adsorption on said proteins that were immobilized by covalent association with Sepharose 4B, as shown in table 2.

[0083] In contrast to infected Cape buffalo, similarly infected cattle did not generate trypanosome growth inhibitory antibodies as evidenced by the inventors. An analysis of responses in pools of matched immune sera from infected cattle and Cape buffalo was performed by Western blotting on lysates and TL-purified components of T. congolese IL 3000 after reducing SDS-PAGE. Cattle used were the highly trypanosomiasis-susceptible Boran breed and the relatively trypanosomiasis-resistant N'dama breed.

These animals were infected with T. congolese IL 1180 and T. brucei ILTat 1.1 at the same time as the Cape buffalo [16] but unlike the buffalo, developed recurring waves of parasitemia and did not develop trypanosome growth- inhibitory antibodies. Sera that were tested spanned up to 90 days after infection and were used at a dilution of 1: 200. Results are presented in Figure 3. It should be noticed that strips were probed with pre-infection sera

(lanes 1) and for Boran sera from 22,35 and 82 days post- infection (dpi) (lanes 2,3 and 4) for N'Dama 22,36 and 83 dpi (lanes 2,3 and 4) and for Cape buffalo 11,40 and 90 dpi (lanes 2,3 and 4).

[0084] Western blotting on total trypanosome lysates showed that immune Cape buffalo sera recognized higher molecular weight material than the immune cattle sera (panel A). Both the cattle and Cape buffalo made antibodies that reacted with TL-purified trypanosome components (panel B), but Cape buffalo responses were stronger than those of the cattle. Boran cattle did not make antibodies that reacted with deglycosylated TL-purified material (panel C), N'dama mounted a weak response against lower molecular weight components of the deglycosylated polypeptides and Cape buffalo mounted a stronger response than either of the cattle breeds and antibodies were predominantly directed against higher molecular weight components. The Cape buffalo response was sustained even although parasitemia was cryptic. Similar results were obtained using total lysates and TL-purified components of T. brucei GUTat 3.1.

These data suggest that a link may exist between the ability of infected animals to suppress trypanosome parasitemia and their ability to make antibodies against polypeptide components of TL-purified trypanosome material.

E. Removal of antibodies that react with TL-binding trypanosome components reduces the T. brucei growth- inhibitory activity of post-infection Cape buffalo serum.

[0085] Xanthine oxidase-depleted and heat-inactivated post-infection serum from Cape buffalo 7810, which had the highest titre of trypanosome growth-inhibitory activity of the three infected Cape buffalo tested, was adsorbed on TL- purified trypanosome components conjugated to Sepharose 4B following which it was examined for its impact on

trypanosome growth in vitro. Control adsorptions used Sepharose 4B alone. Adsorptions were carried out for 1 hour at 4°C using 1 ml post-infection serum per 200 p1 Sepharose 4B bearing 250 ag covalently-associated TL-purified trypanosome material. Adsorption on immobilized TL-purified proteins depleted serum of binding antibodies as determined by Western blotting on TL-purified proteins after SDS-PAGE.

[0086] Results presented in Table 2 show that the T. brucei growth-supporting capacity of post-infection Cape buffalo serum was largely restored by adsorption on immobilized TL-purified trypanosome material. It has been demonstrated that residual trypanosome growth-inhibitory activity of the absorbed serum is due to antibodies against other trypanosome components.

[0087] In conclusion, the above-mentioned data suggest that the long-term suppression of parasitemia in T. brucei ILTat 1. 1-infected Cape buffalo is likely to be mediated by antibodies against TL-binding trypanosome material. Because the post-infection serum had a higher titre of growth- inhibitory activity against T. brucei ILTat 1.1 than any of the other test organisms, it is considered that the infection-induced protective antibodies in Cape buffalo are mainly trypanosome-species-and serodeme-restricted, a serodeme being defined as a group of antigenic variant trypanosomes derived from a cloned organism.

F. Immunization of mice with deglycosylated tomato lectin binding trypanosome material induces trypanosome growth- inhibitory antibodies.

[0088] Mice (BALB/c) were immunized by intraperitoneal injection of 25 yg tomato lectin purified T. brucei ILTat 1.1 protein that was deglycosylated using N-glycosidase F as described [4] and emulsified in Freund's complete adjuvant (0.2 ml/mouse). The mice were boosted twice with

So Ag protein in Freund's incomplete adjuvant with 2 weeks between each immunization. One group of control mice was immunized with ovalbumin using the same immunization protocol as the tomato lectin immunized group, and a second control group was not treated. Blood for serum preparation was collected 2 weeks after the second boost and screened for antibodies against the deglycosylated tomato lectin purified trypanosome material by Western blotting after SDS-PAGE.

[0089] Between 5 and 12 bands could be detected, dependent on the percentage of gel used (data not shown).

[00901 Groups of 7 immunized and control mice were challenged by intraperitoneal injection of 5 x 102 T. brucei GUTat 3.1, a different serodeme to the one from which the receptors were purified, or the same number of T. congolese IL 1180 which are of a different species.

(00911 All control mice became parasitemic. Those infected with T. congolese IL 1180 were all dead by day 20 post-infection, whereas those infected with T. brucei GUTat 3.1 developed an undulating parasitemia and survived up to 80 days post-infection.

[0092] In contrast to the control animals, 6 out of 7 of the mice immunized with the deglycosylated tomato lectin purified T. brucei ILTat 1.1 material did not develop patent parasitemia after challenge with T. congolese IL 1180.

[0093] Parasites were also not detected in 3 out of 7 mice that were challenged with T. brucei GUTat 3.1.

[0094] Mice in the experimental group that became parasitemic did not display any improved parasitemia control and succumbed to infection in the same manner as the controls.

[0095] Differences in the level of protection obtained against T. congolese and T. brucei may reflect

the parasite life styles. T. congolense resides solely within the vascular system, whereas T. brucei also invades lymph, tissue fluids and the central nervous system. As a result of their tissue location it may be necessary to induce production of higher concentrations of antibody, or different classes of antibody, or both, to fully protect against infection with T. brucei.

[0096] Tomato lectin protein immunized mice that did not develop patent parasitemia were euthanized by C02 on day 80 post-infection and sera collected. Mouse serum has low xanthine oxidase activity and there is no need for its depletion prior to testing for any impact of the serum on trypanosome growth in vitro. Pooled sera from the immunised mice that resisted infection, and from control mice that had not been immunized, were heat-inactivated and tested for growth-inhibitory activity on T. brucei ILTat 1. 1, T. brucei GUTat 3.1, T. congolese IL1180 and T. congolese IL3338 in vitro.

[0097] Results are presented in Table 3 and show that there was complete inhibition of trypanosome growth in the presence of 20% immune, but not control mouse serum.

[0098] Some variation in growth inhibition of the different clones was observed at the lower concentrations of immune sera, with T. congolese 3338 displaying complete inhibition down to 2.5%. This may be due to variation between tomato lectin binding proteins of the different clones, levels of expression of these proteins, binding efficiency of the antibodies or a combination of these.

[0099] Immunization challenge profiles corresponding to these experiments are presented on Fig. 4a-b. The profiles show the survival of challenged animals for the duration of the infection. For both T. brucei and T. congolese challenges, two non-immunized control mouse strains were used, Balb/c and Swiss. Whereas Balb/c mice are inbred,

Swiss mice are out bred and are thought to be somewhat more resilient to infection. In case of the T. brucei challenge (Fig. 4a), all ovalbumin-immunized animals were dead by day 80 post-infection and there was one survivor in the non- immunized control group, although this animal was weak and was euthanized on day 85 post-infection. For the animals that were immunized with the deglycosylated TL-binding proteins of ILTat 1.1, four displayed a detectable infection and were dead by day 80 post-infection. Three animals did not show an infection and these remained healthy for the duration of the infection. In case of the T. congolese challenge (Fig. 4b), all non-immunized and ovalbumin-immunized mice were dead by day 18 post-infection whereas the 6 of the Balb/c mice immunized with TL-binding proteins of ILTat 1.1 survived for the duration of the experiment. The surviving animals were maintained till day 85 post infection and never displayed parasitemia. They were then euthanized and sera collected. A comparison between pre-infection and post-infection sera from these animals on Western blots of proteins from T. brucei and T. congolese showed a boosting of responses against a subset of the TL-binding proteins of both parasite species following the challenge infection, showing that these animals had been infected, but had the ability to control it. Interestingly, the sera from mice that were protected against challenge infection stained trypanosomes the same way as the tomato lectin in immunofluorescence experiments.

[0100] The challenge infection experiment was repeated on Balb/C mice immunized three times with each injection two weeks apart using (1) 20 ug of TL purified material from T. brucei that was deglycosylated (2) 20 Ug of ovalbumin and (3) no antigen. The results on animals that were infected and succumbed were, from the T. brucei challenges:

(1) 4/7 (i. e. 3 protected) (2) 6/6 (3) 7/7 and from the T. congolese challenges: (1) 1/7 (i. e. 6 protected) (2) 7/7 (3) 7/7 No parasites could be detected in any of the protected animals.

G. Derivation and testing of monoclonal antibodies [0101] Two BALB/c inbred mice were immunized over a 15-day period with a total of 25 micrograms per mouse of deglycosylated TL-purified trypanosome (T. brucei) flagellar pocket receptors. The immunization was performed in the absence of adjuvant using a method that allows immune responses against both major and minor immunogens in a mixture. Antigen (25 micrograms) was dissolved in 0.5 ml (per mouse) saline and 50 microlitres of stock antigen was injected per mouse leg (subcutaneous bilateral injection in the diffuse adipose tissue between the calcaneous tendon and the posterior aspect of the tibia). Five injections were given at three-day intervals. Three days after the last injection, the superficial and deep inguinal, femoral and popliteal lymph nodes were removed, single cell suspensions were made and the lymphocytes were fused with a selected clone of X63-Ag-8.6.5.3 parental myeloma cells.

One-step growth, HAT selection and cloning of hybridomas were performed using a specially formulated methylcellulose growth medium containing recombinant growth factors (ClonaCellTM-HY ; Stemcell Technologies Inc, Vancouver, B. C). After 10 days incubation, 2000 clones had grown and 576 were picked and transferred into growth medium in 96 well plates. After 4 days of incubation the supernatants

were tested for antibodies to the deglycosylated receptors in an enzyme-linked immunosorbent assay (ELISA). After screening of 61 positive supernatants against human transferrin (to eliminate non-specific sticky antibodies) 48 were chosen for further analysis and were grown into larger volumes for cryopreservation and for production of tissue culture supernatants for testing in a variety of assays.

[0102] Sterile tissue culture supernatants from each of the 48 selected hybridomas were isotyped using standard antigen-capture enzyme-linked immunosorbent assay. Nine mAbs were IgM, 38 were IgGi and one was IgG2b. The supernatants were also tested for their effects on growth of T. brucei brucei clone 1-bloodstream forms, in vitro. In addition, the supernatants were tested on T. brucei brucei clone 22, which expresses a different variant surface glycoprotein (VSG) than that expressed by clone 1. Two different individuals repeated the growth inhibitory experiments. Supernatants were also tested independently on the clone 1 bloodstream form parasites.

[0103] The results agreed in all experiments and a total of 31 hybridoma supernatants were found to inhibit the growth of trypanosomes to varying degrees with 19 showing strong effects. Parasite growth was inhibited without prejudice to the VSG type.

H. Screening of trypanosome libraries with antisera against deglycosylated tomato lectin purified trypanosome material.

[0104] Recombinant expression libraries of T. congolese IL3000 cDNA cloned in kgtll were screened with the Cape buffalo infection sera and serum from immunized mice.

[0105] It has been evidenced that Cape buffalo that were bred in tsetse-free conditions from trypanosomiasis- free parents and submitted then to experimental infections, develop only one of few parasitemic waves before permanently suppressing parasitemia to a level of one to ten trypanosomes/ml blood [15,16].

[0106] Approximately 200,000 plaques were screened and 20 positive plaques were selected for secondary screening of which 15 were confirmed as positive. A representative result is shown on figure 5.

[0107] Positive clones are analyzed to identify duplicates and grouped according to whether they are recognized by one or both sera. Similar screening of T. brucei cDNA expression libraries are being undertaken to identify positive clones.

[0108] After classification according to size, uniqueness and frequency of occurrence, the largest of each of the individual positive clones from the most to the least abundant will be sequenced using standard methods.

[0109] Multiple cloning strategies can be used in order to ensure that as many as possible of the genes for proteins from the flagellar pocket are identified. In particular, the monoclonal antibodies showing growth inhibitory activity on T. brucei are used to identify their target antigens in 2D-electrophoresis gels of TL-purified flagellar pocket extracts. Microsequencing of these antigens using mass spectrometry allows the design of specific oligonucleotide primers for gene amplification from genomic or cDNA libraries, by polymerase chain reaction. Alternatively, the sequence obtained by microsequencing is searched in the current T. brucei genome database, which is estimated to be about 50% complete (http://www. tigr. org/tdb/mdb/tbdb/).

[0110] Once identified these genes are used for the construction of a recombinant vaccine against trypanosomes.

[0111] These genes as well as monoclonal antibodies specific for their gene products may also be used for diagnostic purposes.

Table 1 Impact of pre-and post-infection Cape buffalo serum on trypanosme growth in vitro. Trypanosomes (x104)/ml culture medium* supplemented with Cape buffalo IgG at concentrations Indicated Test Test IgG 2 mg/ml 1 mg/ml 0.5 mg/ml 0.25 mg/ml 0 mg/m] Parasite T. brucei Post-Dead 10 98 263 256 ILTat 1.1 infection Pre-120 198 246 256 infection T. brucei Post-64 201 192 GUTat infection Pre-196 185 216 infection T. congo Post-58 116 112 IL 1180 infection Pre-106 121 107 infection T. congo Post-63 101 98 97 101 IL 3338 infection Pre-114 98 116 104 117 infection * Data are the mean of duplicate cultures seeded with 5x10 trypanosomes/ml medium and counted after 120 hours. Variability between duplicate cultures was <5%.

Table 2 Effect of adsorption of trypanosome post- infection Cape buffalo serum on immobilized TL- purified trypanosome material. Trypanosomes (x104)/ml culture medium* supplemented with intact and receptor-depleted immune Cape buffalo serum at % indicated Test Test serum 25% 12. 5% 6. 25% 3. 12% 1. 56% 0% Parasite T. brucei antigen-5 40 87 108 138 130 ILA4 absorbed post- infection serum mock-absorbed Dead Dead 2 33 lu4 13 post-infection serum * Data are the mean of duplicate cultures seeded with 3xlO'trypanosomes/ml medium and counted after 54 hours

Table 3 Impact of immune and non-immune murine serum on trypanosome growth. Trypanosomes (x104)/ml culture medium* supplemented with mouse ser at % vol. indicated Target Parasite Test 20% 10% 5% 2. 5% 1.25% 0% 0% Serum T.brucei ILTatl. 1 Immune Dead 28 168 214 Non-172 225 198 203 immune T.brucei GUT 3.1 Immune Dead 17 140 192 Non-198 220 195 217 immune T. congo IL1180 Immune Dead 87 117 92 Non-62 110 102 95 immune T. congo IL3338 Immune Dead Dead Dead Dead 64 96 Non-74 116 94 97 122 112 immune * Data are the mean of duplicate cultures seeded with 10'trypanosomes/ml medium and counted after 120 hours. Variability between duplicate cultures was <5%.

REFERENCES [1] KRISTJANSON P. M. et al., Agricultural Systems (1999) 59: 79-98.

[2] MKUNZA et al., Vaccine (1995) 13: 151-154.

[3] OLENICK J. G. et al., Infection and Immunity (1988), 56: 92-98.

[4] NOLAN D. P. et al., Current Biol. (1999), 9 : 1169- 1172.

[5] NOLAN D. P. et al., J. Biol. Chem. (1997), 272 : 29212- 29221.

[6] ZAME S. E. et al., J. Biol. Chem. (1991), 266 : 20244- 20261.

[7] SALMON D. et al., Cell (1994), 78 : 75-86.

[8] LIGTENBERG M. J. L. et al., EMBO J. (1994), 13 : 2565- 2573.

[9] STEVERDING D. et al., J. Cell. Biol. (1995), 131 : 6443-6447.

[10] BASTIN P. et al., Mol. Biochem. Parasitol. (1996), 76 : 43-56.

[11] KELLEY R. J. et al., Mol. Biochem. Parasitol (1995), 74 : 167-178.

[12] PAYS E. & NOLAN D. P.; Mol. Biochem. Parasitol (1998), 91 : 3-36.

[13] HOMANN M. & GERINGER H. U.; Nucleic Acids Res. (1999), 9 : 2006-2014.

[14] LANGRETH S. G. & BALBER A. E.; J. Protozool. (1975), 22 : 40-53.

[15] REDUTH D. et al., J. Eukaryot. Microbiol. (1994), 41 95-103.

[16] WANG Q. et al., Infect. Immun. (1999), 67 : 2797-2803.