SE468250B - Lymphocyte stimulating factor from TRYPANOSOMA BRUCEI - Google Patents

Description

46 10 15 20 25 30 35 250 2 tor is mainly derived from the haemoflagellate Trzpanosoma brucei brucei.

The lymphocyte stimulating factor provided by the present invention is a glycoprotein as determined by proteolytic and glycolytic treatment which abolishes its lymphocyte-stimulating activity.

The factor may have a molecular weight of about 30 kD or a leakage weight which is an integer multiple of about 30 kD.

In accordance with the embodiment of lymphocyte stimulation The determining factor according to the invention is the molecular weight approx 180 kD 1 20 kD.

The trypanosome-derived lymphocyte stimulating factor further defined by its binding and effect on the surface molecule of the lymphocyte membrane - CD8 -, as can be determined by selectively blocking its activity with anti- -CD8 antibodies or soluble CD8.

The lymphocyte stimulating factor of the present invention invention is suitable for use as a vaccine against African sleeping sickness.

According to another aspect of the invention, the above-mentioned defined lymphocyte stimulating factor is used in the development of position of an antiserum useful for combating rican sleeping sickness.

The present invention also includes a method for the treatment of African sleeping sickness, and this procedure includes administration to a mammal in need of .such treatment of an effective amount of an antibody against said lymphocyte stimulating factor, which antibody interacts with said factor released from the parasite and causes said disease.

Finally, according to the invention according to another side of it a procedure for vaccinating a mammals against African sleeping sickness, with the vaccination comprises administering to the mammal an immunological active amount of the lymphocyte stimulating factor according to the invention. 10 15 20 25 30 35 Yes.

CN CO hä LH CD 3 The fact that the lymphocyte stimulating factor in in accordance with the present invention is a specific hot substance that exerts its effect has experimental shown by the use of an antibody directed against said factor. A monoclonal antibody directed against the factor according to the invention thus inhibits the induction of IFN-3 secretion. caused by living trypanosomes or by all of the three peak activity fractions obtained by gel- filtration of killed parasites, and this is a clear indication that such induction of lymphocyte-produced IFN-X by trypanosomes is due to the action of a single and not a complex in vitro interaction between the sites and the mononuclear cells.

According to yet another aspect of the invention, the following actually a monoclonal antibody directed against a lymphocyte stimulating factor as defined above.

The lymphocyte stimulating factor of the present invention invention can thus be used in the treatment of African perhaps sleeping sickness, either as a therapeutic agent or as a means of vaccination against said disorder. Effective effective quantities of said factor may be administered on a living animal body including humans on which preferably, for example orally, such as in capsules or tablets. blisters, parenterally in the form of sterile solutions, suspensions or by pellet implantation, and in some cases intravenously in the form of sterile solutions. Other administrative mode of administration is cutaneous, subcutaneous, buccal, intramuscular and intraperitoneally.

Pharmaceutical preparations are usually prepared from a predetermined quantity of the lymphocyte stimulating factor tower according to the invention, preferably in solid form. So- such formulations may take the form of a powder, elixir, tablets, pills, capsules, pellets or tablets, with el- without, but preferably with any of a variety of pharmaceutically acceptable vehicles or carriers. In mixture with a pharmaceutical vehicle or carrier constitutes the active one component, the lymphocyte stimulating factor according to 10 15 20 25 30 35 468 250 4 the invention, usually from about 0.01 to about 75%, normally from about 0.05 to about 15% by weight of the position. Carriers such as starch, sugar, talc, use synthetic and natural rubbers, water and similar can be used in such preparations. Binder, such as gelatin, and lubricants, such as sodium stearate, can be used to form tablets. Disintegrants, such as sodium bicarbonate, may also be included in terna.

When used for vaccination is provided the factor according to the invention suitably in the form of a sterile solution for injection, such as intramuscular injection tion.

With regard to the amount of active ingredient added administered to the patient in need of treatment, it is necessary that the active ingredient constitutes a effective amount, i.e. such that the appropriate effective dose is which is compatible with the dosage form used. Uppen- several unit dosage forms can be administered in get the same amount of time. The exact individual dose as well the daily dose in a particular case is determined by the in accordance with well-established medical principles per.

The invention will now be further illustrated by the following examples, which, however, may not be considered as restrictive. This illustration by example is done _in connection with the accompanying drawings, wherein Fig. 1 shows a graph of the number of IFN-X secretions cells detected as immunostainers after exposure to lymph node or spleen mononuclear cells (MNC) for different numbers of trypanosomes; Fig. 2 shows the number of IFN-X secreting cells detected as immunoblots among human peripheral blood MNCs after exposure to trypanosomes; Fig. 3 is a diagram showing lymph node cells or splenocyte MNC induced by trypanosomes to IFN-X -secretion; 10 15 20 25 30 35 468 250 5 Fig. 4 shows a diagram of the number of IFN-¶-sec. regenerating cells after exposure to splenocyte MNC for fractions obtained after gel filtration of decomposed trypanosomes; Fig. 5 shows a graph of inhibition of IFN-X -secretion; Fig. 6 shows inhibition of spleen MNC support growth; Fig. 7 shows selective blocking of the trypanosome induced IFN-X production; and Fig. 8 shows a blockade of trypanosome induced IFN-X production.

EXAMPLE 1 The mouse monoclonal anti-rat IFN-X (DB1) and a rabbit anti-rat IFN-X preparation was used (Van der Meide, P.H., M. Dubbeld, K. Vijverberg, T. Kos and H. Schellekens. 1986. The purification of rat gamma interferon by use of two monoclonal antibodies. J.Gen.Virol. 67: 1059). DB1 re- on protein A Sepharose columns from ascites fluid.

Anti-rat CD8 (OX8) (Brideau, R.J., P.B. Carter, W.R.

McMaster, D.W. Mason and A.F. Williams. 1980. Two subsets of rat T lymphocytes defined with monoclonal antibodies.

Eur.J.Immunol. 10: 609) was purified in a similar manner (Holmdahl, R., T. Olsson, T. Moran and L. Klareskog. 1985. In vivo treatment of rats with monoclonal anti-T-cell antibodies.

Immunohistochemical and functional analysis in normal rats and experimental allergic neuritis. Scand.J.Immunol. 22: 157) from culture supernatants of OX8 hybridoma originally obtained from Dr. Alan Williams (Oxford, Stor- Britain). A mouse monoclonal anti-human IFN-X (7-B6-1) (Andersson, G., H.-P. Ekre, G. Alm and P. Perlmann. 1989.

Monoclonal antibody two-site ELISA for human IFN-3.

Adaptation for determinations in human serum or plasma.

J.Immunol.Methods l25: 89) consisted of a gift from Gudrun Andersson (Research and Development Immunobiology, KABI, Biopharma, Stockholm, Sweden). Rabbit polyclonal anti-human .Leeward Ü \ 10 15 20 25 30 35 CO [O (Il 2 6 IFN-X was purchased from Interferon Sciences (New Brunnswick, New York, US). Recombinant rat IFN-3 was prepared and was titrated as previously described (Van der Meide, P.H., M. Dubbeld, K. Vijverberg, T. Kos and H. Schellekens. 1986. The purification of rat gamma interferon by use of two monoclonal antibodies. J.Gen.Virol. 67: 1059). Recombi- human IFN-X was purchased from Genzyme (Boston, M.A., US).

Biotinylated anti-rabbit IgG, rat serum-absorbed biotinyl nylated anti-mouse IgG (Vector Lab., Burlingame, US) and avidin-biotin-peroxidase complex (Vectastain ABC-Elite Kit, Vector Lab.) Was used.

Mononuclear cell suspensions 'Simple cell suspensions from superficial cervicals lymph nodes of normal Sprague-Dawley rats (150-200 g, ALAB, Sollentuna, Sweden) was obtained by grinding through a wire mesh. Spleens were ground in the same manner and erythrocytes mined by osmotic light. The cells were suspended in Iscove's modification of Dulbecco's medium (Flow, Irvine, UK) supplemented with 2 mM glutamine (Flow), 50 IU peni- cillin (Astra, Södertälje, Sweden), 60 pg / ml streptomycin (Flow), 1% (vol / vol) minimum essential medium (Flow) and 5% (v / v) fetal calf serum (FCS; Gibco, Paisley, UK).

Human peripheral blood mononuclear blood cells from four healthy laboratory staff were prepared at Ficoll Hypaque (Lymphoprep, Nygaard, Oslo, Norway). The cell suspensions was washed three times in medium followed by counting of MNC in presence of trypan blue. Percentage of cells excluding trypan blue generally exceeded 90%. The concentration of The MNC in the suspensions was then adjusted to 5 and 10x 106 / ml.

Preparation of fractionation of T.b. brucei A variable type An Tat l / 1 derived from stabilized EATRO 1125 by T.b. brucei (obtained from Dr. Nestor van Meirvenne, Laboratory of Serology, Institute of Tropical Medicine Prince Leopold, Antwerp, Belgium) was brought pas- 10 15 20 25 30 35 468 250 7 sera in Sprague-Dawley rats before use. Blood from infected animals were collected by cardiac puncture and mixed with phosphate buffer containing 1% glucose and EDTA. It was used to purify the trypanosomes from the blood method described by Lanham and Godfray (Lanham, S.M., and D.G. Godfrey. 1970. Isolation of salivarian trypanso- mes from man and other animals using DEAE-cellulose.

Exp.Parasitol, 285521), but chromatography was performed on swollen DEAE-Sepharose Fast Flow ion exchanger (Pharmacia, Upp- sala, Sweden). This resulted in a pure population of mobile parasites without contaminating MNC. In some experiments parasites disintegrated by freezing were used and thawing.

To obtain a basis for future purification experiments and to broadly characterize the components of T.b. brucei inducing MNC to IFN-X secretion, fractionation was performed ring experiment. These were performed on four different occasions as follows: the purified parasites (45 x 106) sonicated on ice for 1 minute (output 5; 40% duty cycle; cell disruptor B15, Bronson sonifier). Parasite membrane pel- was centrifuged by ultracentrifugation for 3 hours at 105,000 g (using a 50 Tirotor in a Beckman L8-55 ultra-centrifuge (Beckman Instruments Inc., Palo Alto, California, USA)). The pellet was then dissolved in 50 mM PBS, pH 8.0. The pellet was dissolved in phosphate buffered saline. solution (PBS: 50 mM, pH 8.0), was then subjected to gel filtration. on Sephacryl S-200 superfine (Pharmacia Fine Chemicals) on a 48 x 1.5 cm column, equilibrated with 10 mM Tris-CH1, pH 7.5, at a flow rate of 5.64 ml / hour. 120 fractions of 1.13 ml each were collected.

Molecular weight was determined by comparison with proteins with determined molecular weight (catalase, bovine serum albumin, haemo- globin, ovalbumin, cytochrome c). The parasite fractions lyo- was then filtered and kept at -70 ° C. Immediately before the turn, the lyophilized powder was dissolved in 50 μl sterile water, of which 10 μl was applied to each micro- titer plate well with MNC.

Yes. 10 15 20 25 30 35 C \ 03 BJ cm EXAMPLE 2 Assay for interferon-3 secreting cells The principles of immunospot counting of simple sec- rhetorical cells using nitrocellulose bottomed 96-well microtiter plates (Millititre- -HAM, Millipore Co., Bedford, US) was followed (Czerkinsky, C., G. Andersson, H.-P. Ekre, L.-A. Nilsson, L. Klareskog and Ö. Ouchterlony. Reverse ELI-SPOT assay for clonal analysis of cytokine production. 1988. I. Enumeration of gamma- -interferon secreting cells. J.Immunol.Methods 110: 29; Kabilan L., G. Andersson, F. Lolli, H.-P. Ekre, T. Olsson and M. Troye-Blomberg. 1990). Detection of intracellular expression and secretion of interferon-X at the single- cell level after activation of human T cells with tetanus toxoid in vitro. Eur.J.Immunol. 20: 1085). For detection of straight-IFN-Z-SC the wells were coated with 100 μl aliquots of DB1 at 15 pg / ml at 4 ° C overnight and washed with PBS, pH 7.4. The microtiter plates were then emptied by suction in a Millititre Vaccine Filter Holder (Millipore) and 100 μl aliquots of 1% bovine serum albumin (BSA) in PBS was added for 2 hours. The plates were washed then 10 times with PBS by suction in the filtration well laren. Aliquot amounts (100 μl) of cell suspensions was added in triplicate to the wells at two different concentrations. concentrations, 5 and 10 x 106 MNC per ml. Parasites or other components were added at the concentrations indicated in the character descriptions. After 24 hours culture at 37 ° C and 7% CO 2 in a humid atmosphere, the plates were emptied and washed. was repeated several times with PBS. Aliquot amounts (100 μl) of it polyclonal rabbit anti-rat IFN-3 antibody diluted 1/5,000 was added for 2 hours. After washing was added biotinylated anti-rabbit IgG diluted 1/1000 during more than 2 hours followed by the avidin-biotin peroxidase complex diluted to 1/200 for 1 hour. After peroxidase staining (Kaplow, L.S. 1974. Substitute for benzidine in myelo- peroxidase stains. Am.J.clin.Pathol. 63: 45l), was counted 10 15 20 25 30 35 463 250 9 spots corresponding to cells secreting IFN-β in one dissecting microscope. In specificity control experiments, where the antibody in question has been replaced by an irrelevant one monoclonal mouse antibody or where the polyclonal rabbit the antibody was excluded, no stains appeared. For detection human IFN-X-SC was coated with 7-B6-1, 6 ug / ml, and a rabbit polyclonal anti-human IFN-X diluted 1/500 was used. The variation in the number of spots within the triplicate the cat was always less than 10%. The mean of the triplicate was calculated and expressed as the number of IFN-X-SC per 106 MNC.

Depletion of CD8 + lymphocytes To obtain rat MNC free from CD8 + cells injected cerades rats i.p. with 1 mg OX8. 24 hours later pre- spleen and lymph node MNC were parried. As previously described joke ((Ho1mdah1, R., T. Olsson, T. Moran and L. Klareskog. 1985. In vivo treatment of rats with anti-T- monoclonal -cell antibodies. Immunohistochemical and functional analysis in normal rats and experimental allergic neuritis. Scand.J.Immunol. 22: l57) results in this action in total depletion of CD8 + cells within 30 minutes lasting for I4 days. In all rats treated on this In this way, the CD8 + depletion was immunohistochemically controlled sections from the spleen as previously described (Bakhiet, M., T. Olsson, P. Van der Meide and K. Kristensson, 1990.

Depletion of CD8 + cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma (IFN-3) production in infected rats. Clin.exp.Immunol. 81: l95).

EXAMPLE 3 Effect of trypanosomes on MNC-IFN-X secretion Immediately after smearing of the spleen and lymphocyte -MNC from CD8 + -depleted and non-depleted rats purified live 10 μl aliquots of trypano- summer to triplicate wells to achieve final numbers of 10, 102 and 103 parasites per well. Other wells exposed JS. 10 15 20 25 30 35 O\ OD BD Uñ CD 10 for frozen and thawed trypanosomes (104 per well) or trypanosome fractions prepared as described above. In each experiment, negative controls consisted of triplicate wells not receiving parasites, below it that positive controls consisted of triplicate receiving optimally diluted phytohemagglutinin (PHA, Difco, Detroit, Mi., US).

Effect of trypanosomes on MNC proliferation In these experiments, MNCs were grown from lymph nodes and spleen at a cell concentration of 2 x 10 6 per ml of medium by applying 200 μl aliquot parts in round bottoms 96-well polystyrene microtiter plates (Nunclon, Nunc, Denmark). Triplicates of wells obtained 0, 10, 102, 103 and 104 parasites. PHA was included as a positive control. 16 hours before harvest, each well received 10 ul of aliquot. containing 1 pCi of H3-labeled thymidine (Amersham, Little Shalfont, UK) in saline solution. In some experiments DB1 (20 pg / ml) was added to parallel wells to inhibit IFN-X produced in vitro. Total cultivation times included 24, 48 and 72 hours at 37 ° C in a humid atmosphere and 7% CO 2. ' Cells were harvested in glass fiber filter strips (Skaltron AS., Lierbyen, Norway) with a multi-channel semi-automated harvesting device (Titertec Skaltron AS) accompanied by repeated the washes in distilled water. Incorporated radioactivity white was counted in a scintillation counter (Marck II, Searle, Analytic, Des Plaines, USA). Mean pulses per minute (cpm) and standard deviation of H3-thymidine incorporation calculations were calculated from the triplicate.

Effect of MNC and IFN-X on trypanosome growth MNC was grown in round bottom microtiter plates with 96 wells (Nunclon) at a cell density of 5 x 106 / ml medium. Both human and peripheral blood MNC and rat splenic and lymph node MNCs from CD8 + depleted and odeplated animals were used. Purified parasites were added to a 10 15 20 25 30 .35 4653 250 ll similar wells to achieve an initial density of 10 x 106 trypanosomes / ml culture medium. In some experiments eeftee nB-1 (10 pg / ml), 7-36-1 (10 pg / ml) or peiyklenel anti-IFN-X (final dilution 1/5 000) to well na. In other experiments, wells containing only received trypanosomes different amounts of crude or human recombinant IFN-3. In parallel wells, trypanosomes were grown alone medium. The parasite determinations at different sampling intervals embankments were carried out as follows: the contents of each well mixed thoroughly 10 times with a 100 μl micropipette. l0 μl of the fluid was added to 90 μl of a Trypan blue solution accompanied by thorough mixing. Burker chambers were used for to count the number of mobile parasites that excluded Trypan blue at a x200 magnification. The density of "living" pa- racites per ml of medium present in the well were calculated.

For each type of in vitro manipulation, periment on 3 to 6 different occasions apart that a type with the addition of human IFN-X was performed once.

In each case, three wells were used for each sample. shooting interval, ie. 2, 4, 8, 12 and 24 hours after initiation of culture, to count the number of parasites.

Thus, in total, each sampling interval contains data from 12 to 18 bills. Means and standard errors of means (SEM) was calculated. Mann-Whitney's test was used to calculate the significance level.

Effects of MNC and trypanosome-released factors in one two-chamber system To study about possible signaling between trypanosomes and MNCs mean either direct contact or diffusible molecules used a two-chamber system. Od- plates (24 wells; Transwell, Laboratory Design_ AB, Lidingö, Sweden) was used, in which lymph node or spleen -MNC was applied in the lower well and trypanosomes in the upper well. The pore size of the membrane being limited The yield between the two chambers was 0.4 .mu.m. In studies of trypanosome effects on MNC, 6 x 104 MNC .get O\ OD 10 15 20 25 30 35 250 12 parasites in 100 μl of medium in the upper well. 6 x 106 MNC in 600 μl of medium was applied in the lower well. After 24 hours of culture, MNC was washed once in the medium, the cells was diluted again and 100 μl aliquots, each containing 106 MNC, was applied to wells of nitrocellulose loose-bottomed microtiter plates, were grown under additional 24 hour period for detecting IFN-X-SC as described joke above. In separate experiments on the effect of MNC on trypanosome growth, 106 parasites were applied in 100 μl medium in the upper well, while the lower one received 6 x 106 MNC and 600 ul of medium. The parasites were counted as described above in triplicate at 2, 4, 8, 12 and 24 hours after initiation of the cultures. Averages, SEM and statistics stitches were calculated as described above.

EXAMPLE 4 Study of IFN-X uptake of trypanosomes To study whether T.b. brucei already in it infected values taken up IFN-X were subjected to purification parasites (a) histochemical staining for the detection of rat -IFN-X, b) Western blot to detect approximate leakage weight for colored material. ' a) Purified trypanosomes were air dried on glass plates followed by fixation for 30 seconds in 4% buffered formalin and after washing in PBS for 30 seconds in ice-cooled acetone.

Then the glass plates were washed in PBS, incubated with 2% normal horse serum for 30 minutes. The plates were incubated then with 5 pg / ml DB1 at 4 ° C overnight. After washing appropriate dilutions of were applied in turn biotinylated equine anti-mouse IgG (Vector lab.) diluted in 2% normal rat serum and ABC, accompanied by peroxidase staining (Kaplow, L.S. 1974. Substitute for benzidine in myeloperoxidase stains. Am.J.clin.Pathol. 63: 45l). Som ne- positive controls, a series of irrelevant isotypic matched mouse monoclonal antibodies.

G7 '15 10 15 20 25 30 35 -Surface C7 \ OO PO (fl (D 13 b) Whole purified and otherwise untreated trypanosomes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and transferred by electrophoresis blotting to nitrocellulose membranes (Laemmli, U.K. 1971.

Cleavage of structural proteins during assembly of the head of bacteriphage T4. Nature 227: 680; Towbin, H., T.

Stachelin and J. Gordon. l979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and same applications.

Proc.Natl.Acad.Sci. USA 76: 4350). Approximately 106 trypanosomes was added to each trough in the gel. Electroblotting on nitrocellulose membranes were performed in transblot cell equipment (BioRad, CA, USA) at a constant current of 0.1 Amp in 16 hours at 4 ° C. The procedure means that the parasites breed, their constituents are separated by molecular weight and they exposed to nitrocellulose. Nitrocellulose membranes were blocked in low fat milk to avoid non-specific binding of antibodies. The membrane is incubated was then lined overnight at room temperature with DB1 in one concentration of 5 pg / ml to detect any se- parried IFN-X. After several washes in buffer incubated membranes in a 1/500 dilution of a high affinity purified goat anti-mouse IgG and then subjected to ABC and peroxidase staining according to Kaplow (Kaplow, L.S. 1974. Sub- stitute for benzidine in myeloperoxidase stains.

Am.J.clin.Pathol. 63: 45l).

Calculations and statistics In each experiment it was used unless otherwise stated triplicate. All experiments were repeated 3-8 times and Wilcoxon's rank sum test or Mann Whitney's test an- was used to calculate the significance level.

JE. 10 15 20 25 30 35 G \ OD FJ CF 14 EXAMPLE 5 Trypanosome-induced stimulation of CD8 + lymphocyte-IFN-X- secretion and in vitro proliferation As shown in Fig. 1, co-culture of lymph node or spleen MNC with trypanosomes 24 hours a pronounced increase in the number of IFN-X-SC. Trypanosomes also killed IFN-X secretion induced by freezing and thawing, although less effective. For example, 104 frozen trypanosomes induced responses of an order of magnitude similar to 102 the trypanosomes. As few as 10 living trypanosomes per well could surprisingly induce a significant edge answer. Trypanosome-induced IFN-β production eliminates completely when CD8 + depleted MNC suspensions were used turned. The maximum number of detectable IFN-X-SCs was approx 200 per 106 MNC, i.e. 1 of 5000 MNC. As expected, starved PHA stimulation of parallel wells in one approximately the same number of IFN-X-SC. Human peripheral MNC blood was activated by trypanosomes to secrete IFN-X (Fig. 2).

Measurements of proliferative responses using 3 H-thymidine incorporation revealed, as expected, PHA induced severe proliferation after 24, 48 resp. 72 hours. When trypanosomes in the range of 10 to 104 racites / well were added, a slight proliferative behavior occurred response at 24 hours compared to the control wells. At 48 hours, the response compared to the controls was the same. At However, at 72 hours, 10 and 102 parasites induced MNC proliferation, while 103 did not cause induction. and 104 parasi- The IFN-X inducing factor is released from trypanosomes The use of the two-chamber system as a physical separator The trypanosomes from MNC showed that comparable numbers of IFN-X-SC was induced in this situation as with tryptic nosomes directly co-cultured with MNC. As illustrated in FIG. 3, a two-chamber system was used in this case physically distinguishing T.b. and MNC but permissible exchange of soluble 10 15 20 25 30 35 .get CH CO FO (TI CD 15 mediators. The bindings indicate that the trypanosome-derived the lymphocyte activating factor is released and also acts on distance. This finding shows that the parasites release a soluble mediator that induces MNC to IFN-X secretion.

CD8 + MNC secretion of a soluble factor results in species restriction trypanosome growth A reciprocal relationship between trypanosomes and host-derived cells were evident when the kinetics were analyzed. was calculated by counting the number of parasites in the two-chamber system. with and without MNC in the lower chamber. Thus it was an early increase in the number of parasites in the upper chamber when the lower chamber is supplied with splenic MNC, while the long and decreased in number when exposed to medium only (Table I). It was interesting to note that this plant stimulation was eliminated when CD8 + -depleted MNC was reversed (Table II).

IFN-X mimics the MNC-derived trypanosome growth promoter factor This question was determined by in vitro experiments with antibody growth inhibition and by direct cytokine application. From Table II it is obvious that DB1 and rabbit polyclonal anti-rat IFN-X antiserum completely blocks the growth promotion of rat MNC, while the mouse anti-human IFN-X monoclonal was ineffective (none data showed). Application of recombinant rat IFN-X on cultures with purified parasites resulted in a striking dose dependent growth stimulation (Table II).

Further on details in Figs. 4 to 8 As indicated earlier, Fig. 4 shows a diagram above the number of IFN-X secreting cells after exposure to splenocyte MNC for fractions obtained after gel filtration of decomposed trypanosomes. At the chart, these were three peak activities approximate molecular weights of 185, 70 and 30 kD, and these activities correspond to fractions 23, 46 4ss_2so 10 15 20 25 30 35 16 and 79 shown in the diagram in Fig. 5. As also Fig. 5 shows a diagram of the inhibition of IFN-X secretion induced by said fractions during use of a monoclonal antibody against the lymphocyte stimulating factor, in the following Example 6 arbitrarily cartoon Moiz I.

Fig. 6 illustrates the inhibition of splenic MNC support growth of T.b. brucei in vitro by blocking it trypanosome-released lymphocyte-stimulating factor with Moiz IN.

Furthermore, Fig. 7 shows selective blocking of trypan induced IFN-X production using a mono- clonal mouse anti-CD8 antibody, designated OX8. Other anti- bodies are inefficient, and this finding indicates that CD8 molecules the target is the target of the trypanosome lymphocyte activating factor tower.

Finally, Fig. 8 shows blockade of the trypanosome induced IFN-X production using ascending doses of soluble CD8 molecules added to the cultures.

This finding, illustrated in Fig. 8, indicates that the the nosome-released molecule binds to CD8 and that under these conditions lymphocytes are not activated.

EXAMPLE 6 Principle description of the anti-TLTF antibody, Moiz I The fraction with TLTF peak activity (F23 in Fig. 5) was used was used to immunize mice. Nine days later it was used regional lymph node cells for the production of hybridomas according to standard technology. The resulting 40 hybridomas were tested by looking at its ability to inhibit trypanosome-induced production of IFN-X mononuclear cells as described above assay system. Of 40 hybridomas, 4 of them showed such inhibitory activity. The hybridoma with the strongest activity was selected and expanded. The antibody was called arbitrarily Moiz I. The isotype of the antibody was found to be gamma 2B. It has since been purified on protein A and analyzed rats further. The antibody inhibits TLTF in vitro at a 'x 10 15 20 25 30 35 .Ia CR CD PO (11 CD 17 concentration down to 0.1 pg / ml. It does not inhibit induction to IFN-X production by other means, such as PHA indu- certified IFN-3 production. The antibody thus acts at low concentration and specifically of trypanosome-induced IFN-β -induction. It further shows that IFN-X induction by trypanosomes depend on the action of a single molecule and not a complex in vitro interaction between parasites and mono- nuclear cells.

When using Moiz I for immunohistochemical staining parasites, trypanosomes are specifically stained by the lesion but not by a series of control antibodies.

TABLE I Time after initiation of cultivation (h) 2 4 12 24 Experiment- 0 m 11.3 5.5 3.3 0.4 tell process- SEM 0.3 0.2 0.2 0.1 dur n 12 12 12 12 MNC m 18.6 15.5 6.5 2.6 SEM 2.1 0.5 0.4 0.6 n 12 12 12 12 p 0.001 0.09 0.001 0.005 JE 10 15 20 25 30 35 68 250 18 TABLE II Time after initiation of cultivation (h) Experimental procedure 2 4 8 12 24 0 m 10.8 11.8 4.8 3.1 0.7 SEM 0.8 0.9 0.28 0.2 0.05 n 18 18 18 18 18 'MNc M 29.3 17 9.7 6.9 1.4 SEM 2 0.4 1.1 0.3 0.17 n 18 18 18 18 18 p 0.001 0.002 0.001 0.003 0.01 cna * - m 11.1 11.1 3.3 2.1 0.5 -deple- SEM 1.1 0.8 0.2 0.2 0.2 0.2 terad n 18 18 18 18 18 MNC p compared n.s. n.s. n.s. n.s. n.s. with 0 p compared 0.001 0.002 0.003 0.002 0.002 with 106 MNC _DB1 + m 5.6 6.4 5.6 2 0.2 MNC SEM 0.2 0.2 0.15 0.1 0.01 n 12 12 12 12 12 p compared n.s. n.s. n.s. n.s. n.s. with 0 p compared 0.001 0.002 0.003 0.002 0.002 with 106 MNC cont. 10 15 20 25 30 35 468 250 19 cont.

Time after initiation of cultivation (h) Experimental procedure 2 4 8 12 24 Rabbit- m 8.8 7.2 8 1.6 -anti- SEM 0.3 0.2 0.2 0.2 0.1 _ -rått n 12 12 12 12 12 12 + MC p compared n.s. n.s. n.s. n.s. n.s.

IFN-X with 0 p compared 0.002 0.001 0.03 0.001 0.03 with 106 MNC Human m 3 2.3 2.9 1.2 1 MNC SEM 0.28 1.37 1.7 0.7 0.6 n 12 12 12 12 12 p 0.001 0.001 0.008 0.003 0.12 20000 U m 2.1 26 13 8 4.8 raw SEM _ 2.1 4.2 1.1 0.7 0.5 IFN-X n 12 12 12 12 12 p 0.001 0.001 0.001 0.001 0.2 2ooo U m 14 18 11.8 6.4 3 crude SEM 1.1 3.1 0.8 0.6 0.2 0.2 IFN-X n 12 12 12 12 12 p 0.08 0.001 0.003 0.001 0.008 200 U m 11.2 12 5 3.8 1.8 crude SEM 0.8 0.6 0.2 0.1 0.1 0.1 IFN-β 12 12 121 -12 12 1 p 0.06 0.001 0.01 0.08 0.13 cont. 468 250 10 15 20 25 30 35 'well received 100 pl. In the lower well, 6x10 was added 20 cont.

Time after initiation of cultivation (h) Experimental procedure 2 4 8 12 24 20000 U m 5.6 8 9.6 2.4 2.2 human SEM 0.1 0.1 0.2 0.1 0.07 IFN-X n 3 3 3 3 3 p n.d. n.d. n.d. n.d. n.d. 2000 U m 5.6 8 7.2 3.2 1.2 human SEM 0.1 0.1 0.1 0.17 0.05 IFN-X n 3 3 3 3 3 p n.d. n.d. n.d. n.d. n.d.

EXPLANATION OF THE TABLES Table I. applied in the upper well of the two-chamber system below Growth characteristics of purified trypanosomes separation of the same from rat mononuclear cells cerade in the lower well. The parasites were applied at an initial density of 10x106 / ml medium and each 6 rat MNC in 600 μl medium (MNC), control cultures received medium only (0). Data refer to the number of trypanosomes x 106 / ml. m = mean values, SEM = standard error in mean values de, n = number of analyzed cultures, p = level of significance.

Table II. Growth characteristics of purified trypanosomes subjected to various experimental procedures. The parasites was applied at an initial density of 10x106 / ml medium. dium in 100 μl aliquots. Data refer to the number of parasi- ter x 106 / ml. The procedures were as follows: medium only x) f; 1D 10 15 20 25 30 35 468 259 21 (0), co-culture with 106 rat MNC (MNC), co-culture with CD8 + - -depleted rat MNC (CD8 + -depleted MNC), co-cultured with lo6 rat-Mnc aan zo pg / ml mal added (nan-Mus), co-cultured with 106 rat MNC and 20 μg / ml added nB1 (DB1 + MNc), combined culture with rat MNC and polyclonal rabbit anti-rat IFN-X added (rabbit anti-rat IFN-3 + MNC), co-culture with human MNC (human MNC), different quantities of recombinant rat averages, SEM = standard error in mean, n = number of cultures analyzed, p = significant or human IFN-3. m = chance level in comparison with cultures with only medium, if not otherwise stated, n.s. = not significant, n.d. = not determined.

Claims (7)

Jb. 10 15 20 25 30 35 O \ 03 I \ 'J (J'1 3 22 PATENTKRAV Lymphocyte-stimulating factor, characterized by a protein derived from the haemoflagellate Trypanosoma brucei capable of stimulating CD8 + T cells in a living animal body resulting in the release of interferon-X (IFN-X), so that there is immunosuppression occurs in said body. Lymphocyte stimulating factor according to claim 1 derived from the haemoflagellate Trypanosoma brucei brucei. A lymphocyte stimulating factor according to claim 1 or 2 having a molecular weight of about 30 kD or an integer multiple thereof. A lymphocyte stimulating factor according to claim 3 having a molecular weight of about 180 kD in 20 kD. Lymphocyte stimulating factor according to any one of the preceding claims for use as a vaccine against African sleeping sickness. Lymphocyte stimulating factor according to any one of claims 1 to 4 for use in the manufacture of an antiserum useful in the control of African sleeping sickness. Monoclonal antibody directed against the lymphocyte stimulating factor according to any one of claims 1 to '40 f)