WO1992018538A1 - A lymphocyte stimulating factor originating from the haemoflagellate trypanosoma - Google Patents

A lymphocyte stimulating factor originating from the haemoflagellate trypanosoma Download PDF

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Publication number
WO1992018538A1
WO1992018538A1 PCT/SE1992/000191 SE9200191W WO9218538A1 WO 1992018538 A1 WO1992018538 A1 WO 1992018538A1 SE 9200191 W SE9200191 W SE 9200191W WO 9218538 A1 WO9218538 A1 WO 9218538A1
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Prior art keywords
stimulating factor
ifn
mnc
lymphocyte stimulating
trypanosomes
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PCT/SE1992/000191
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French (fr)
Inventor
Tomas Olsson
Moiz Bakhiet
Conny Edlund
Bo HÖJEBERG
Krister Kristensson
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Tomas Olsson
Moiz Bakhiet
Conny Edlund
Hoejeberg Bo
Krister Kristensson
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Application filed by Tomas Olsson, Moiz Bakhiet, Conny Edlund, Hoejeberg Bo, Krister Kristensson filed Critical Tomas Olsson
Publication of WO1992018538A1 publication Critical patent/WO1992018538A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a lymphocyte stimulating factor that is useful in the treatment of African sleeping sickness.
  • T.b. is the cause of African sleeping sickness in which polyclonal activation of lymphoid cells and severe immunosuppression are characteristic features.
  • the parasite grows extracellularly in the host and its capacity to induce disease depends both on the strain of the parasite and on the species of the host.
  • the Trypanosoma brucei sub-species rhodiense and gambiense are pathogens for humans with varying patterns of disease, while the subspecies Trypanosoma brucei brucei does not infect humans but grows well in rodents. It has been observed that in both humans and rodents Trypanosoma brucei induces a polyclonal activation of lymphoid cells and a severe immunosuppression. (Askonas, B.A. 1984, Interference in general immune function by parasite infections; African trypanosomiasis as a model system. Parasitology 88:633)
  • trypanosomes secrete a factor that triggers CD8+ lymphocytes resulting in production of interferon-gamma thus providing a growth-stimulus for the trypanosomes and causing immunosuppression of the host.
  • the invention provides for a lymphocyte stimulating factor constituted by a protein which has the ability to stimulate, in a living animal body, CD8+ lymphocytes (T-cells) resulting in the release of interferon-gamma (IFN- ⁇ ) thereby causing immunosuppression in said body.
  • a lymphocyte stimulating factor constituted by a protein which has the ability to stimulate, in a living animal body, CD8+ lymphocytes (T-cells) resulting in the release of interferon-gamma (IFN- ⁇ ) thereby causing immunosuppression in said body.
  • the lymphocyte stimulating factor according to the present invention preferably originates from the haemoflagellate Trypanosoma brucei, said factor being secreted by this parasite, then interacting with CD8+ lymphocytes. Said factor preferably originates from the haemoflagellate Trypanosoma brucei brucei.
  • the lymphocyte stimulating factor provided by the present invention is constituted by a glycoprotein as determined by proteolytic and glycolytic treatments that abolish its lymphocyte stimulating activity.
  • the factor may have a molecular weight of about 30 KD or a molecular weight which is an integer multipel of about 30 KD.
  • the molecular weight is about 180 KD ⁇ 20 KD.
  • the trypanosoma derived lymphocyte stimulating factor is further defined by its binding and action on the lymphocyte membrane surface molecule - CD8 - , as evidenced by selective blocking of its activity with anti CD8 antibodies or soluble CD8.
  • the lymphocyte stimulating factor of the present invention is suitable for use as a vaccine against African sleeping sickness.
  • lymphocyte stimulating factor as defined above can be used in the preparation of an antiserum useful for combatting African sleeping sickness.
  • the present invention also involves a method of treating African sleeping sickness, said method comprising administering to a mammal in need of such treatment an effective amount of an antibody against said lymphocyte stimulating factor, said antibody interacting with said factor released by the parasite causing said sickness.
  • the invention resides in a method of vaccinating a mammal against African sleeping sickness, said vaccination comprising administering to the mammal an immunologically active amount of the lymphocyte stimulating factor of the invention.
  • the lymphocyte stimulating factor in accordance with the present invention is a specific single substance exerting its action has been experimentally shown by the use of an antibody directed against said factor.
  • a monoclonal antibody directed against the factor of the invention does inhibit the induction of IFN- ⁇ secretion by living trypanosomes or by all of three peak activity fractions obtained by gel filtration of killed parasites, and this is a clear indication that such induction of lymphocyte produced IFN- ⁇ by trypanosomes is due to the action of a single molecule and not to a complex in vitro interaction between the parasites and the mononuclear cells.
  • the lymphocyte stimulating factor of the present invention is accordingly useful in the treatment of African sleeping sickness, either as a therapeutic agent or as a means for vaccination against said disorder.
  • Effective quantities of said factor may be administered to a living animal body including humans in anyone of various ways, for example orally as in capsules or tablets, parenterally in the form of sterile solutions, suspensions or by pellet implantation, and in some cases intravenuously in the form of sterile solutions.
  • Other modes of administration are cutaneously, subcutaneously, buccally, intramuscularly and intraperitoneally.
  • compositions are usually prepared from a predetermined quantity of the lymphocyte stimulating factor of the invention, preferably in solid form.
  • Such formulations may take the form of powders, elixirs, solutions, pills, capsules, pellets or tablets, with or without, but preferably with anyone of a large variety of pharmaceutically acceptable vehicles or carriers.
  • the lymphocyte stimulating factor according to the invention usually comprises from about 0.01 to about 75%, normally from about 0.05 to about 15% by weight of the composition.
  • Carriers such as starch, sugar, talc, commonly used synthetic and natural gums, water and the like may be used in such formulations.
  • Binders, such as gelatine, and lubricants, such as sodium stearate, may be used to form tablets.
  • Disintegrating agents, such as sodium bicarbonate may also be included in the tablets.
  • the factor according to the invention is suitably presented in the form of a sterile solution adapted for injection, such as intramuscular injection.
  • the amount of active ingredient administered to the patient in need of treatment it is only necessary that the active ingredient constitutes an effective amount, i.e. such that a suitable effective dosage will be obtained consistent with the dosage form employed.
  • a suitable effective dosage will be obtained consistent with the dosage form employed.
  • several unit dosage forms may be administered at about the same time.
  • the exact individual dosages as well as daily dosages in a particular case will of course be determined according to well-established medical principles.
  • Fig. 1 shows a diagram on the number of IFN- ⁇ -secreting cells detected as immunospots after exposing lymph node or spleen mononuclear cells (MNC) to different numbers of trypanosomes;
  • Fig. 2 shows the number of IFN- ⁇ -secreting cells detected as immunospots among human peripheral blood MNC after exposure to trypanosomes
  • Fig. 3 is a diagram showing lymph node cells or splenocyte MNC inducted by trypanosomes to IFN- ⁇ secretion;
  • Fig. 4 shows a diagram on number of IFN- ⁇ -secreting cells after exposure of splenocyte MNC to fractions obtained after gelfiltration of disrupted trypanosomes;
  • Fig. 5 shows a diagram on inhibition of IFN- ⁇ secretion
  • Fig. 6 shows inhibition of spleen MNC supported growth
  • Fig. 7 shows selective blocking of Trypanosome induced IFN- ⁇ production
  • Fig. 8 shows a blocking of Trypanosome induced IFN- ⁇ production.
  • DB1 Mouse monoclonal anti-rat IFN- ⁇ (DB1) and a rabbit anti-rat IFN- ⁇ preparation were used (Van der Meide, P.H., M. Dubbeld, K. Vijverberg, T. Kos and H. Schellekens.
  • Tat 1/1 derived from stabilate EATRO 1125, of T.b. brucei (obtained from Dr. Nestor van Meirvenne, Laboratory of Serology, Institute of Tropical Medicine Prins Leopold, Antwerp, Belgium) was passaged once in Sprague-Dawley rats before use. Blood from in fected animals was collected by heart puncture and mixed with phosphate buffer containing 1% blucose and EDTA. To purify the trypanosomes from the blood, the method
  • fractionation experiments were performed. At four different occasions these were performed as follows: The purified parasites (45 ⁇ 10 6 ) were sonicated on ice for 1 min (output 5; 40% duty cycle; cell disruptor B15, Bronson sonifier). Parasite membranes were pelleted by ultra-centrifugation for 3 h 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.
  • rat IFN- ⁇ -SC wells were coated with 100 ⁇ I aliquots of DB1 at 15 ⁇ g/ml at 4°C overnight and washed with PBS, pH 7.4. The microtitre plates were then emptied by suction into a Millititre vacuum filtration holder (Millipore) and 100 ⁇ l aliquots of 1% bovine serum albumin (BSA) in PBS were added for 2 h. The plates were subsequently washed 10 times with PBS by suction in the filtration holder. Aliquots (100 ⁇ l) of cell suspensions were added in triplicates to wells at two different concentrations, 5 and 10 ⁇ 10 6 MNC per ml.
  • BSA bovine serum albumin
  • rat MNC free of CD8+ cells
  • rats were injected i.p. with 1 mg OX8. 24 h later spleen and lymph node MNC were prepared.
  • OX8 a monoclonal anti-T-cell antibodies. Immunohistochemical and functional analysis in normal rats and experimental allergic neuritis. Scand. J. Immunol.
  • MNC from lymph nodes and spleens were cultured at a cell concentration of 2 ⁇ 10 6 per ml medium by applying 200 ⁇ l aliquots in round bottomed polystyrene 96 well microtitre plates (Nunclon, Nunc, Denmark). Triplicate wells received 0, 10, 10 2 , 10 3 and 10 4 parasites. PHA was included as a positive control. 16 h before harvest each well received 10 ⁇ l aliquots containing 1 ⁇ Ci of H 3 labelled thymidin (Amersham, Little
  • MNC were cultivated in round bottomed 96 well microtitre plates (Nunclon) at a cell density of 5 ⁇ 10 /ml culture medium. Both human peripheral blood MNC, and rat spleen and lymph node MNC, from CD8+ depleted and non-depleted animals were used. Purified parasites were added to triplicate wells to achieve an initial density of 10 ⁇ 10 6 trypanosomes/ml culture medium. In some experiments DB-1 (10 ⁇ g/ml), 7-B6-1 (10 ⁇ g/ml ) or the polyclonal anti-rat IFN- ⁇ (final dilution 1/5,000) were added to the wells.
  • each type of in vitro manipulation the experiments were carried out at 3 to 6 different occasions, except the one with addition of human IFN- ⁇ that was carried out once.
  • three wells for each sampling interval that is 2, 4, 8, 12 and 24 h after initiation of the culture, was used to count the number of parasites.
  • each sampling interval contains data from 12 to 18 countings.
  • Means and standard error of the mean (SEM) were calculated. Mann-Whitney's test was used to calculate level of significance.
  • lymph node or spleen MNC were applied in the lower well and trypanosomes in the upper well.
  • the pore size of the membrane restricting exchange between the two chambers was 0.4 ⁇ m.
  • the MNC were washed once in medium, cells rediluted and 100 ⁇ l aliquots, each containing 10 6 MNC, were applied into wells of nitrocellulose bottomed microtitre plates, cultivated for an additional 24 h period for detection of IFN- ⁇ -SC as described above.
  • 10 6 parasites in 100 ⁇ l medium was applied in the upper well, while the lower received 6 ⁇ 10 6 MNC in 600 ⁇ l medium.
  • the parasites were counted as described above in triplicates at 2, 4, 8, 12 and 24 h after initiation of the cultures. Means, SEM and statistics were calculated as described above.
  • Trypanosome induced stimulation of CD8+ lymphocyte IFN- ⁇ secretion and proliferation in vitro As evident from Fig 1 cocultivation of lymph node or spleen MNC with trypanosomes for 24 h caused a striking increase in number of IFN- ⁇ -SC. Also trypanosomes killed by freezing and thawing induced IFN- ⁇ secretion, although less efficiently. For example, 10 4 frozen trypanosomes induced responses of a similar magnitude as 10 2 living trypanosomes. Interestingly, as few as 10 living trypanosomes per well were able to induce a significant response. The trypanosome induced IFN- ⁇ production was completely eliminated when CD8+ depleted MNC suspensions were used.
  • the maximum number of the detectable IFN- ⁇ -SC was approximately 200 per 10 6 MNC, that is 1 out of 5000 MNC. As expected PHA stimulation of parallel wells resulted in an approximately similar number of IFN- ⁇ -SC. Human peripheral blood MNC were triggered by trypanosomes to secretion of IFN- ⁇ (Fig. 2).
  • the IFN- ⁇ inducing factor is released from trypanosomes.
  • CD8+ MNC secretion of a soluble factor causes species restricted trypansome growth.
  • IFN- ⁇ mimicks the MNC derived trypanosome growth promoting factor
  • FIG. 4 shows a diagram on the number of IFN- ⁇ secreting cells after exposure of splenocyte MNC to fractions obtained after gel filtration of disrupted trypanosomes.
  • Fig. 5 shows a diagram on the inhibition of IFN- ⁇ secretion induced by said fractions using a mono clonal antibody against the lymphocyte stimulating factor, in the following Example 6 arbitrarily designated Moiz I.
  • Fig. 6 illustrates the inhibition of spleen MNC supported growth of T.b. brucei in vitro by blocking the trypanosome released lymphocyte stimulating factor with Moiz I.
  • Fig. 7 shows selective blocking of trypanosome induced IFN- ⁇ production using a mouse monoclonal anti-CD8 antibody, designated OX8. Other antibodies are ineffective and this finding indicates the fact that the CD8 molecule is the target for the trypanosome lymphocyte triggering factor.
  • Fig. 8 shows blocking of trypanosome induced IFN- ⁇ production using increasing doses of soluble CD8 molecules added to cultures. This finding illustrated in Fig. 8 indicates the fact that the trypanosome released molecule binds to CD8 and that under these conditions lymphocytes will not be triggered.
  • the fraction with TLTF peak activity (F23 of Fig. 5) was used for immunization of mice.
  • the resulting 40 hybridomas were tested for their ability to inhibit trypanosome induced production of IFN- ⁇ by mononuclear cells in the assay system described above. Of the 40 hybridomas, 4 of them showed such inhibiting activity.
  • the hybridoma with the strongest activity was selected and expanded.
  • the antibody was arbitrarily named Moiz I.
  • the isotype of the antibody was found to be gamma 2B. It has subsequently been purified on protein A and analysed further.
  • the antibody inhibits TLTF in vitro at a concentration down to 0.1 ⁇ g/ml.
  • the antibody acts a low concentration and specifically on trypansome induced IFN- ⁇ induction. It furthermore shows that IFN- ⁇ induction by trypanosomes is due to the action of single molecule and not to a complex in vitro interaction in between parasites and mononuclear cells.
  • Table I Growth characteristics of purified trypanosomes applied into the upper well of a two chamber system separating them from rat mononuclear cells appllied in the lower well.
  • the parasites were applied at an initial density of 10 ⁇ 10 6 /ml medium and each well received 100 ⁇ l.
  • MNC lower well 6x10 6 rat MNC in 600 ⁇ l medium was added (MNC), control cultures received medium alone (O).
  • Data refer to number of trypanosomes ⁇ 10 /ml.
  • m mean values
  • SEM standard error of the mean
  • n number of cultures analysed
  • p level of significance.
  • Table II Growth characteristics of purified trypanosomes subjected to different experimental procedures.
  • the parasites were applied at an initial density of 10 ⁇ 10 6 /ml medium in 100 ⁇ l aliquots.
  • Data refer to numbers of parasites ⁇ 10 6 /ml.

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Abstract

A lymphocyte stimulating factor constituted by a protein having the ability to stimulate, in a living animal body, CD8+ T-cells resulting in release of interferon-η (IFN-η) thereby causing immunosuppression in said body; a method of treating African sleeping sickness using such factor; and a monoclonal antibody directed against said factor.

Description

A LYMPHOCYTE STIMULATING FACTOR ORIGINATING FROM
THE HAEMOFLAGELLATE TRYPANOSOMA
The present invention relates to a lymphocyte stimulating factor that is useful in the treatment of African sleeping sickness.
The parasitic haemoflagellate Trypanosoma brucei
(T.b.) is the cause of African sleeping sickness in which polyclonal activation of lymphoid cells and severe immunosuppression are characteristic features. The parasite grows extracellularly in the host and its capacity to induce disease depends both on the strain of the parasite and on the species of the host. Thus, the Trypanosoma brucei sub-species rhodiense and gambiense are pathogens for humans with varying patterns of disease, while the subspecies Trypanosoma brucei brucei does not infect humans but grows well in rodents. It has been observed that in both humans and rodents Trypanosoma brucei induces a polyclonal activation of lymphoid cells and a severe immunosuppression. (Askonas, B.A. 1984, Interference in general immune function by parasite infections; African trypanosomiasis as a model system. Parasitology 88:633)
In accordance with the present invention it has been surprisingly found that trypanosomes secrete a factor that triggers CD8+ lymphocytes resulting in production of interferon-gamma thus providing a growth-stimulus for the trypanosomes and causing immunosuppression of the host.
Accordingly, the invention provides for a lymphocyte stimulating factor constituted by a protein which has the ability to stimulate, in a living animal body, CD8+ lymphocytes (T-cells) resulting in the release of interferon-gamma (IFN-ɣ) thereby causing immunosuppression in said body.
The lymphocyte stimulating factor according to the present invention preferably originates from the haemoflagellate Trypanosoma brucei, said factor being secreted by this parasite, then interacting with CD8+ lymphocytes. Said factor preferably originates from the haemoflagellate Trypanosoma brucei brucei.
The lymphocyte stimulating factor provided by the present invention is constituted by a glycoprotein as determined by proteolytic and glycolytic treatments that abolish its lymphocyte stimulating activity. The factor may have a molecular weight of about 30 KD or a molecular weight which is an integer multipel of about 30 KD.
In accordance with an embodiment of the lymphocyte stimulating factor of the invention the molecular weight is about 180 KD ± 20 KD.
The trypanosoma derived lymphocyte stimulating factor is further defined by its binding and action on the lymphocyte membrane surface molecule - CD8 - , as evidenced by selective blocking of its activity with anti CD8 antibodies or soluble CD8.
The lymphocyte stimulating factor of the present invention is suitable for use as a vaccine against African sleeping sickness.
According to another aspect of the invention the lymphocyte stimulating factor as defined above can be used in the preparation of an antiserum useful for combatting African sleeping sickness.
The present invention also involves a method of treating African sleeping sickness, said method comprising administering to a mammal in need of such treatment an effective amount of an antibody against said lymphocyte stimulating factor, said antibody interacting with said factor released by the parasite causing said sickness.
Finally, the invention according to another aspect of the invention resides in a method of vaccinating a mammal against African sleeping sickness, said vaccination comprising administering to the mammal an immunologically active amount of the lymphocyte stimulating factor of the invention. The fact that the lymphocyte stimulating factor in accordance with the present invention is a specific single substance exerting its action has been experimentally shown by the use of an antibody directed against said factor. Thus, a monoclonal antibody directed against the factor of the invention does inhibit the induction of IFN- ɣ secretion by living trypanosomes or by all of three peak activity fractions obtained by gel filtration of killed parasites, and this is a clear indication that such induction of lymphocyte produced IFN-ɣ by trypanosomes is due to the action of a single molecule and not to a complex in vitro interaction between the parasites and the mononuclear cells.
According to yet another aspect of the invention there is accordingly provided a monoclonal antibody directed against a lymphocyte stimulating factor as defined above.
The lymphocyte stimulating factor of the present invention is accordingly useful in the treatment of African sleeping sickness, either as a therapeutic agent or as a means for vaccination against said disorder. Effective quantities of said factor may be administered to a living animal body including humans in anyone of various ways, for example orally as in capsules or tablets, parenterally in the form of sterile solutions, suspensions or by pellet implantation, and in some cases intravenuously in the form of sterile solutions. Other modes of administration are cutaneously, subcutaneously, buccally, intramuscularly and intraperitoneally.
Pharmaceutical formulations are usually prepared from a predetermined quantity of the lymphocyte stimulating factor of the invention, preferably in solid form. Such formulations may take the form of powders, elixirs, solutions, pills, capsules, pellets or tablets, with or without, but preferably with anyone of a large variety of pharmaceutically acceptable vehicles or carriers. In admixture with a pharmaceutical vehicle or carrier the ac tive ingredient, the lymphocyte stimulating factor according to the invention, usually comprises from about 0.01 to about 75%, normally from about 0.05 to about 15% by weight of the composition. Carriers, such as starch, sugar, talc, commonly used synthetic and natural gums, water and the like may be used in such formulations. Binders, such as gelatine, and lubricants, such as sodium stearate, may be used to form tablets. Disintegrating agents, such as sodium bicarbonate may also be included in the tablets.
In its use for vaccination the factor according to the invention is suitably presented in the form of a sterile solution adapted for injection, such as intramuscular injection.
In regard to the amount of active ingredient administered to the patient in need of treatment it is only necessary that the active ingredient constitutes an effective amount, i.e. such that a suitable effective dosage will be obtained consistent with the dosage form employed. Obviously, several unit dosage forms may be administered at about the same time. The exact individual dosages as well as daily dosages in a particular case will of course be determined according to well-established medical principles.
The invention will now be further illustrated by the following examples which, however, are not to be construed as limiting. This illustration by examples will be made in conjunction with the appended drawings, wherein
Fig. 1 shows a diagram on the number of IFN-ɣ-secreting cells detected as immunospots after exposing lymph node or spleen mononuclear cells (MNC) to different numbers of trypanosomes;
Fig. 2 shows the number of IFN-ɣ-secreting cells detected as immunospots among human peripheral blood MNC after exposure to trypanosomes;
Fig. 3 is a diagram showing lymph node cells or splenocyte MNC inducted by trypanosomes to IFN-ɣ secretion; Fig. 4 shows a diagram on number of IFN-ɣ-secreting cells after exposure of splenocyte MNC to fractions obtained after gelfiltration of disrupted trypanosomes;
Fig. 5 shows a diagram on inhibition of IFN-ɣ secretion;
Fig. 6 shows inhibition of spleen MNC supported growth;
Fig. 7 shows selective blocking of Trypanosome induced IFN-ɣ production; and
Fig. 8 shows a blocking of Trypanosome induced IFN-ɣ production.
EXAMPLE 1
Mouse monoclonal anti-rat IFN-ɣ (DB1) and a rabbit anti-rat IFN-ɣ preparation were 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 was purified 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 similarly purified (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 the OX8 hybridoma originally obtained from Dr. Alan Williams (Oxford, U.K.). A mouse monoclonal anti-human IFN-ɣ (7-B6-1) (Andersson, G., H.-P. Ekre, G. Aim and P. Perlmann.
1989. Monoclonal antibody two-site ELISA for human IFN-ɣ. Adaption for determinations in human serum or plasma.
J. Immunol.Methods 125:89) was a kind gift from Gudrun Andersson (Research and Development Immunobiology, KABI, Biopharma, Stockholm, Sweden). Rabbit polyclonal anti-human IFN-2C was purchased from Interferon Sciences (New Brunnswick, N.Y. US). Recombinant rat IFN-2 was prepared and titrated as described previously (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). Recombinant human IFN-ɣ was purchased from Genzyme (Boston, M.A., US). Biotinylated anti-rabbit IgG, rat serum absorbed biotionylated anti-mouse IgG (Vector Lab., Burlingame, US) and avidin-biotin peroxidase complex (Vectastain ABC- Elite Kit, Vector Lab. ) were used.
Mononuclear cell suspensions.
Single cell suspensions from superficial cervical lymph nodes of normal Sprague-Dawley rats (150-200 g, ALAB, Sollentuna, Sweden) were obtained by grinding through a wire-mesh. Spleens were similarly grinded and erythrocytes eliminated by osmotic lysis. The cells were suspended in Iscove's modification of Dulbecco's medium (Flow, Irvine, UK), supplemented with 2 mM glutamine
(Flow), 50 IU penicillin (Astra, Södertälje, Sweden), 60 μg/ml streptomycin (Flow), 1% (v/v) minimum essential medium (Flow), and 5% (v/v) fetal calf serum (FCS; Gibco, Paisley, UK). Human peripheral blood mononuclear cells from four healthy laboratory staff members were prepared on Ficoll Hypaque (Lymphoprep, Nygaard, Oslo, Norway). The cell suspensions were washed three times in medium followed by counting of MNC in the presence of trypan blue. The percentage of cells excluding trypan blue generally exceeded 90%. The concentration of MNC in suspensions was then adjusted to 5 and 10 × 106/ml.
Preparation and fractionation of T.b. brucei.
A variable type An Tat 1/1, derived from stabilate EATRO 1125, of T.b. brucei (obtained from Dr. Nestor van Meirvenne, Laboratory of Serology, Institute of Tropical Medicine Prins Leopold, Antwerp, Belgium) was passaged once in Sprague-Dawley rats before use. Blood from in fected animals was collected by heart puncture and mixed with phosphate buffer containing 1% blucose and EDTA. To purify the trypanosomes from the blood, the method
described by Lanham and Godfrey (Lanham, S.M., and D.G. Godfrey. 1970. Isolation of salivarian trypansomes from man and other animals using DEAE-cellulose. Exp. Parasitol. 28:521) was used, but chromatography was performed on pre-swollen DEAE Sepharose Fast Flow ion exchanger (Pharmacia, Uppsala, Sweden). This resulted in a pure population of mobile parasites without any contaminating MNC. In some experiments parasites disrupted by freezing and thawing were used.
To get a basis for future purification attempts and to grossly characterize the component(s) of T.b. brucei that induce MNC to IFN-ɣ secretion, fractionation experiments were performed. At four different occasions these were performed as follows: The purified parasites (45 × 106 ) were sonicated on ice for 1 min (output 5; 40% duty cycle; cell disruptor B15, Bronson sonifier). Parasite membranes were pelleted by ultra-centrifugation for 3 h 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 dissolved in phosphate buffered saline (PBS; 50 mM, pH 8.0) was then subjected to gel filtration on Sephacryl S-200 superfine (Pharmacia Fine Chemicals) on a 48 × 1.5 cm column, equilibrated with 10 mM Tris-CHl, pH 7.5, at a flowrate of 5.64 ml/h. 120 fractions of 1.13 ml each were collected. Molecular weight was estimated by comparison with proteins with established m.w. (catalase, bovine serum albumin, hemoglobin, ovalbumin, cytochrome c). The parasite fractions were then lyophilized and kept at -70°C. Immediately before use the lyophilized powder was dissolved in 50 μl of sterile water of which 10 μl was applied into each microtitre plate well with MNC. EXAMPLE 2
Assay for interferon-ɣ secreting cells.
The principles for immunospot enumeration of single secretory cells utilizing 96 well nitrocellulose-bottomed microtitre plates (Millititre-HAM, Millipore Co., Bedford, US) were followed (Czerkinsky, C., G. Andersson, H.-P.
Ekre, L.-Å. 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-ɣ at the single-cell level after activation of human T cells with tetanus toxoid in vitro. Eur.J.Immunol. 20:1085). For detection of rat IFN-ɣ-SC wells were coated with 100 μI aliquots of DB1 at 15 μg/ml at 4°C overnight and washed with PBS, pH 7.4. The microtitre plates were then emptied by suction into a Millititre vacuum filtration holder (Millipore) and 100 μl aliquots of 1% bovine serum albumin (BSA) in PBS were added for 2 h. The plates were subsequently washed 10 times with PBS by suction in the filtration holder. Aliquots (100 μl) of cell suspensions were added in triplicates to wells at two different concentrations, 5 and 10 × 106 MNC per ml. Parasites or other components were added at concentrations indicated in figure legends. After 24 h culture at 37°C and 7% CO2 in humid atmosphere plates were emptied and washed several times with PBS. Aliquots (100 μl) of the rabbit polyclonal anti-rat IFN-ɣ antibody diluted 1/5,000 were added for 2 h. After washing, biotinylated anti-rabbit IgG diluted 1/1,000 was added for another 2 h followed by the avidin-biotin peroxidase complex diluted
1/200 for 1 h. After peroxidase staining (Kaplow, L.S.
1974. Substitute for benzidine in myeloperoxidase stains. Am. J.clin.Pathol. 63:451), spots which corresponded to cells that had secreted IFN-ɣ were enumerated in a dissection microscope. In specificity control experiments, in which the capture antibody was replaced by an irrelevant mouse monoclonal antibody or in which the rabbit polyclonal, antibody was omitted, no spots appeared. For detection of human IFN-ɣ-SC, the plates were coated with 7-B6-1, 6 μg/ml, and a rabbit polyclonal anti-human IFN-ɣ, diluted 1/500 was used. The variation in numbers of spots within triplicates was always less than 10%. The mean value of the triplicates was calculated and expressed as number of IFN-ɣ-SC per 106 MNC.
Depletion of CD8+ lymphocytes.
To obtain rat MNC, free of CD8+ cells, rats were injected i.p. with 1 mg OX8. 24 h later spleen and lymph node MNC were prepared. As shown previously ((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) this treatment results in a total depletion of CD8+ cells within 30 min, lasting for 14 days. In all rats treated in this way the CD8+ depletion was checked immunohistochemically in sections from the spleen as described before (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-ɣ) production in infected rats. Clin.exp. Immunol.
81:195).
EXAMPLE 3
Effect of trypanosomes on MNC IFN-ɣ secretion
Immediately after plating of spleen and lymphocyte MNC, from CD8+ depleted and non-depleted rats, purified living 10 μl aliquots of trypanosomes were added to triplicate wells to reach final numbers of 10, 10 2 and 103 parasites per well. Other wells were exposed to frozen and thawed trypanosomes (104 per well) or to trypanosome fractions prepared as described above. In each experiment negative controls consisted of triplicate wells receiving no parasites, while positive controls consisted of triplicates receiving optimally diluted phytohemagglutinin (PHA, Difco, Detroit, Mi., US).
Effect of trypanosomes on MNC proliferation
In these experiments MNC from lymph nodes and spleens were cultured at a cell concentration of 2 × 106 per ml medium by applying 200 μl aliquots in round bottomed polystyrene 96 well microtitre plates (Nunclon, Nunc, Denmark). Triplicate wells received 0, 10, 10 2, 103 and 104 parasites. PHA was included as a positive control. 16 h before harvest each well received 10 μl aliquots containing 1 μCi of H3 labelled thymidin (Amersham, Little
Shalfont, UK) in saline. In some experiments DB1 (20 μg/ml) was added to parallel wells to inhibit in vitro produced IFN-ɣ. Total culture times included 24, 48 and 72 h in 37°C humid atmosphere and 7% CO2.
Cells were harvested onto glass fibre filter strips (Skaltron AS., Lierbyen, Norway) with a multichannel semiautomated harvesting device (Titertec Skaltron AS) followed by repeated washings in distilled water. The incorporated radioactivity was counted in a scintillation counter
(Marck II, Searle, Analytic, Des Plaines, USA). The mean counts per min (cpm) and standard deviation of H3 thymid. incorporations were calculated from the triplicates.
Effect of MNC and IFN-ɣ on trypanosome growth
MNC were cultivated in round bottomed 96 well microtitre plates (Nunclon) at a cell density of 5 × 10 /ml culture medium. Both human peripheral blood MNC, and rat spleen and lymph node MNC, from CD8+ depleted and non-depleted animals were used. Purified parasites were added to triplicate wells to achieve an initial density of 10 × 106 trypanosomes/ml culture medium. In some experiments DB-1 (10 μg/ml), 7-B6-1 (10 μg/ml ) or the polyclonal anti-rat IFN-ɣ (final dilution 1/5,000) were added to the wells. In other experiments wells containing only trypanosomes received different amounts of rat or human recombinant IFN-ɣ. In parallel wells, trypanosomes were grown in medium alone. The parasite countings at different sampling intervals were performed as follows: The content of each well was thoroughly mixed, 10 times, with a 100 μl micropipette. 10 μl of the fluid was added to 90 μl of a Trypan blue solution followed by thorough mixing. Burker chambers were used to count the number of mobile parasites that excluded Trypan blue at a x200 magnification. The density of "living" parasites per ml medium present in the well was calculated.
For each type of in vitro manipulation the experiments were carried out at 3 to 6 different occasions, except the one with addition of human IFN-ɣ that was carried out once. At each occasion three wells for each sampling interval, that is 2, 4, 8, 12 and 24 h after initiation of the culture, was used to count the number of parasites. Thus in total, each sampling interval contains data from 12 to 18 countings. Means and standard error of the mean (SEM) were calculated. Mann-Whitney's test was used to calculate level of significance.
Effects of MNC and trypanosome released factors in a two chamber system.
To study if the possible signalling between trypanosomes and MNC involved either direct contact or diffusible molecules a two chamber system was utilized. Cultivation plates (24 wells; Transwell, Laboratorie Design AB,
Lidingö, Sweden) were used in which lymph node or spleen MNC were applied in the lower well and trypanosomes in the upper well. The pore size of the membrane restricting exchange between the two chambers was 0.4 μm. In studies of trypanosome effects on MNC 6 × 104 MNC parasites in 100 μl of medium was applied in the upper well. 6 × 106 MNC in 600 μl medium was applied in the lower well. After 24 h of culture the MNC were washed once in medium, cells rediluted and 100 μl aliquots, each containing 106 MNC, were applied into wells of nitrocellulose bottomed microtitre plates, cultivated for an additional 24 h period for detection of IFN-ɣ-SC as described above. In separate experiments on the effects of MNC on trypanosome growth, 106 parasites in 100 μl medium was applied in the upper well, while the lower received 6 × 106 MNC in 600 μl medium. The parasites were counted as described above in triplicates at 2, 4, 8, 12 and 24 h after initiation of the cultures. Means, SEM and statistics were calculated as described above.
EXAMPLE 4
Studies of IFN-ɣ uptake by trypanosomes
To study if T.b. brucei already in the infected host had taken up IFN-ɣ, purified parasites were subjected to a) histochemical staining for detection of rat IFN-ɣ, b) Western blot to detect approximal m.w. of any stained material.
a) Purified trypanosomes were airdried onto glass slides followed by fixation for 30 sec in 4% buffered formalin and, after washing in PBS, for 30 sec in icecold acetone. Thereafter, the slides were washed in PBS, incubated with 2% normal horse serum for 30 min. The slides were then incubated with 5 μg/ml of DB1 at 4°C overnight. After washing, appropriate dilutions of biotinylated horse anti-mouse IgG (Vector lab.) diluted in 2% normal rat serum and ABC were applied sequentially, followed by peroxidase staining (Kaplow, L.S. 1974. Substitute for benzidine in myeloperoxidase stains. Am.J.clin.Pathol. 63:451). As negative controls a series of irrelevant isotype matched mouse monoclonal antibodies were used. b) Whole purified and otherwise nontreated trypanosomes were separated by sodium-dodecyl-sulphate polyacry - amide gel electrophoresis (SDS PAGE) and transferred by electroblotting 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. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and same applications.
Proc.Natl.Acad.Sci. USA 76:4350). Approximately 106 trypanosomes were added to each trough in the gel. Electroblotting onto nitrocellulose membranes was performed in transblot cell equipment (BioRad, CA, USA) at a constant current of 0.1 Amp for 16 hours at 4°C. The procedure implies that the parasites become lyzed, their constituents are separated accordig to m.w. and these are blotted onto the nitrocellulose. The nitrocellulose membranes were blocked in low fat milk to avoid nonspecific binding of antibodies. The membrane was then incubated overnight at room temperature with DB1 at a concentration of 5 μg/ml to detect any separated IFN-ɣ. After several washings in buffer the membranes were incubated 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. Substitute for benzidine in myeloperoxidase stains. Am. J.clin.Pathol. 63:451).
Calculations and statistics
In each experiment, unless otherwise indicated, triplicates were used. All experiments were repeated 3-8 times and Wilcoxon's rank sum test or Mann Whitney's test were used to calculate level of significance.
EXAMPLE 5
Trypanosome induced stimulation of CD8+ lymphocyte IFN-ɣ secretion and proliferation in vitro. As evident from Fig 1 cocultivation of lymph node or spleen MNC with trypanosomes for 24 h caused a striking increase in number of IFN-ɣ-SC. Also trypanosomes killed by freezing and thawing induced IFN-ɣ secretion, although less efficiently. For example, 104 frozen trypanosomes induced responses of a similar magnitude as 102 living trypanosomes. Interestingly, as few as 10 living trypanosomes per well were able to induce a significant response. The trypanosome induced IFN-ɣ production was completely eliminated when CD8+ depleted MNC suspensions were used. The maximum number of the detectable IFN-ɣ-SC was approximately 200 per 106 MNC, that is 1 out of 5000 MNC. As expected PHA stimulation of parallel wells resulted in an approximately similar number of IFN-ɣ-SC. Human peripheral blood MNC were triggered by trypanosomes to secretion of IFN-ɣ (Fig. 2).
Measurements of proliferative responses employing 3H thymidin incorporation revealed as expected that PHA induced a vigorous proliferation after 24, 48 and 72 h, respectively. When trypanosomes in the range of 10 to 104 parasites/well were added, a mild proliferative response occurred at 24 h compared to control wells. At 48 h the response compared to controls was equivocal. At 72 h, however, 10 and 102 parasites consistently induced MNC proliferation, while 10 3 and 104 parasites did not.
The IFN-ɣ inducing factor is released from trypanosomes.
The use of the two chamber system that physically separated the trypanosomes from the MNC, showed that comparable numbers of IFN-ɣ-SC were induced in this situation as with trypanosomes directly cocultivated with MNC. As illustrated in Fig. 3 a two-chamber system was used in this case, physically separating T.b. and MNC but allowing exchange of soluble mediators. The bindings indicate that the trypanosome derived lymphocyte triggering factor is released and acts also on distance. This finding shows that the parasites release a soluble mediator that induces the MNC to IFN-ɣ secretion.
CD8+ MNC secretion of a soluble factor causes species restricted trypansome growth.
A reciprocal relationship between trypansomes and host derived cells was evident when the kinetics were analyzed by counting the number of parasites in the two chamber system with and without MNC in the lower chamber. Thus, there was an early increase in number of parasites in the upper chamber when the lower chamber had been supplied with spleen MNC, whereas they declined slowly in number when exposed to medium alone (Table I). Interestingly, this growth stimulation was abrogated when CD8+ depleted MNC were used (Table II).
IFN-ɣ mimicks the MNC derived trypanosome growth promoting factor
This question was assessed by in vitro antibody growth inhibition experiments and by direct cytokine application. From Table II it is evident that DB1 and polyclonal rabbit-anti-rat IFN-ɣ antiserum completely block the growth promotion by rat MNC, while the mouse monoclonal anti-human IFN-ɣ was ineffective (data not shown). Application of recombinant rat IFN-ɣ to cultures with purified parasites resulted in a striking dose-dependent growth stimulation (Table II).
Further concerning details of Figs. 4 to 8.
As indicated earlier Fig. 4 shows a diagram on the number of IFN-ɣ secreting cells after exposure of splenocyte MNC to fractions obtained after gel filtration of disrupted trypanosomes. In the diagram there are three peak activities of approximate molecular weights of 185, 70 and 30 kD, and these activities correspond to fractions 23, 46 and 79 given in the diagram of Fig. 5. As also previously indicated Fig. 5 shows a diagram on the inhibition of IFN-ɣ secretion induced by said fractions using a mono clonal antibody against the lymphocyte stimulating factor, in the following Example 6 arbitrarily designated Moiz I.
Fig. 6 illustrates the inhibition of spleen MNC supported growth of T.b. brucei in vitro by blocking the trypanosome released lymphocyte stimulating factor with Moiz I.
Furthermore, Fig. 7 shows selective blocking of trypanosome induced IFN-ɣ production using a mouse monoclonal anti-CD8 antibody, designated OX8. Other antibodies are ineffective and this finding indicates the fact that the CD8 molecule is the target for the trypanosome lymphocyte triggering factor.
Finally, Fig. 8 shows blocking of trypanosome induced IFN-ɣ production using increasing doses of soluble CD8 molecules added to cultures. This finding illustrated in Fig. 8 indicates the fact that the trypanosome released molecule binds to CD8 and that under these conditions lymphocytes will not be triggered. EXAMPLE 6
Principle description of the anti-TLTF antibody, Moiz I
The fraction with TLTF peak activity (F23 of Fig. 5) was used for immunization of mice. Nine days later regional lymph node cells were used for production of hybridomas according to standard techniques. The resulting 40 hybridomas were tested for their ability to inhibit trypanosome induced production of IFN-ɣ by mononuclear cells in the assay system described above. Of the 40 hybridomas, 4 of them showed such inhibiting activity. The hybridoma with the strongest activity was selected and expanded. The antibody was arbitrarily named Moiz I. The isotype of the antibody was found to be gamma 2B. It has subsequently been purified on protein A and analysed further. The antibody inhibits TLTF in vitro at a concentration down to 0.1 μg/ml. It does not inhibit induction to IFN-ɣ production by other means such as PHA induced IFN-ɣ production. Thus, the antibody acts a low concentration and specifically on trypansome induced IFN-ɣ induction. It furthermore shows that IFN-ɣ induction by trypanosomes is due to the action of single molecule and not to a complex in vitro interaction in between parasites and mononuclear cells.
When using Moiz I for immunohistochemical staining of parasites, trypansomes are specifically stained by the molecule but not by a series of control antibodies.
Table I.
Time after Initiation of culture (h) 2 4 12 24
Experimental 0 m 11.3 5.5 3.3 0.4 procedure SEM 0.3 0.2 0.2 0.1 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
Table II .
Experimental Time after initiation of culture (h) procedure 2 4 8 12 24
O 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 20.3 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
CD8+ depleted m 11.1 11.1 3.3 2.1 0.5
MNC SEM 1.1 0.8 0.2 0.2 0.2 n 18 18 18 18 18 p compared n.s. n.s n.s. n.s. n.s. to 0
p compared 0.001 0.002 0.003 0.002 0.002 to 106 MNC
DB1 + MNC m 5.6 6.4 5.6 2 0.2
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.β. to 0
p compared 0.001 0.002 0.003 0.002 0.002 to 106 MNC
Rabbit anti-rat m 8.8 7.2 8 1.6 1
+ MNC IFN-ɣ SEM 0.3 0.2 0.2 0.1 1 n 12 12 12 12 12 p compared n.s. n.s. n.s. n.s. n.s. to 0
p compared 0.002 0.001 0.03 0.001 0.03 to 106 MNC
Human MNC m 3 2.3 2.9 1.2 1
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 rat m 2.1 26 13 8 4.8
IFN-ɣ SEM 2.1 4.2 1.1 0.7 0.5 n 12 12 12 12 12 p 0.001 0.001 0.001 0.001 0.2
2000 U rat m 14 18 11. 8 6.4 3 IFN-ɣ SEM 1. 1 3. 1 0.8 0.6 0.2 Table II continued
n 12 12 12 12 12 p 0.08 0.001 0.003 0.001 0.008
200 U rat m 11.2 12 5 3.8 1.8
IFN- ɣ SEM 0.8 0.6 0.2 0.1 0.1 n 12 12 12 12 12 p 0.06 0.001 0.01 0.08 0.13
20000 U human m 5.6 8 9.6 2.4 2.2
IFN- ɣ SEM 0.1 0.1 0.2 0.1 0.07 n 3 3 3 3 3 p n.d. n.d. n.d. n.d. n.d.
2000 U human m 5.6 8 7.2 3.2 1.2
IFN- ɣ SEM 0.1 0.1 0.1 0.17 0.05 n 3 3 3 3 3 p n.d. n.d. n.d. n.d. n. d
LEGENDS TO TABLES
Table I. Growth characteristics of purified trypanosomes applied into the upper well of a two chamber system separating them from rat mononuclear cells appllied in the lower well. The parasites were applied at an initial density of 10×106/ml medium and each well received 100 μl. In the lower well 6x106 rat MNC in 600 μl medium was added (MNC), control cultures received medium alone (O). Data refer to number of trypanosomes × 10 /ml. m = mean values, SEM = standard error of the mean, n = number of cultures analysed, p = level of significance.
Table II. Growth characteristics of purified trypanosomes subjected to different experimental procedures. The parasites were applied at an initial density of 10×106/ml medium in 100 μl aliquots. Data refer to numbers of parasites × 106/ml. The procedures were as follows: medium alone (0), cocultivation with 106 rat MNC (MNC), cocultivated with CD8+ depleted rat MNC (CD8+ depleted MNC), cocultivated with 106 rat MNC and 20 μg/ml of DBl added (DB1+MNC), cocultivated with rat MNC and rabbit polyclonal anti-rat IFN-ɣ added (rabbit anti-rat IFN-ɣ + MNC), cocultivated with human MNC (human MNC), different quantities of recombinant rat or human IFN-ɣ. m = mean values, SEM = standard error of the mean, n = number of cultures analysed, p = level of significance in comparison with cultures with medium alone, unless otherwise indicated, n.s. = not significant, n.d. = not determined.

Claims

1. A lymphocyte stimulating factor constituted by a protein having the ability to stimulate, in a living animal body, CD8+ T-cells resulting in release of interferon- ɣ (IFN-ɣ) thereby causing immunosuppression in said body.
2. A lymphocyte stimulating factor according to claim
1 originating from the haemoflagellate Trypanosoma
brucei.
3. A lymphocyte stimulating factor according to claim
2 originating from the haemoflagellate Trypanosoma brucei brucei.
4. A lymphocyte stimulating factor according to claim 1, 2 or 3 having a molecular weight of about 30 KD or an integer multiple thereof.
5. A lymphocyte stimulating factor according to claim 4 having a molecular weight of about 180 KD ± 20 KD.
6. A lymphocyte stimulating factor according to any preceding claim for use as a vaccine against African sleeping sickness.
7. A lymphocyte stimulating factor according to any of claims 1 to 5 for use in the preparation of an antiserum useful for combatting African sleeping sickness.
8. A method of treating African sleeping sickness, comprising administering to a mammal in need of such treatment an effective amount of an antibody against the lymphocyte stimulating factor of any of claims 1 to 5, said antibody interacting with said factor released by the parasite causing said sickness.
9. A method of vaccinating a mammal against African sleeping sickness, comprising administering to the mammal an immunologically active amount of the lymphocyte stimulating factor of any of claims 1 to 5.
10. A monoclonal antibody directed against the lymphocyte stimulating factor according to any of claims 1 to 5.
PCT/SE1992/000191 1991-04-11 1992-03-25 A lymphocyte stimulating factor originating from the haemoflagellate trypanosoma WO1992018538A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004588A1 (en) * 1996-07-26 1998-02-05 Sbl Vaccin Ab Lymphocyte stimulating factor from trypanosoma
WO2007091580A1 (en) * 2006-02-07 2007-08-16 Nippon Biologicals, Inc. Novel vaccine carrier

Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0041189A1 (en) * 1980-05-22 1981-12-09 Stiftung Deutsches Krebsforschungszentrum Process for the production of Interferon II and stimulated clones for carrying out the process

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0041189A1 (en) * 1980-05-22 1981-12-09 Stiftung Deutsches Krebsforschungszentrum Process for the production of Interferon II and stimulated clones for carrying out the process

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Title
DIALOG INFORMATION SERVICES, file 154, Medline 85-92, Dialog Accession No. 07445854, BAKHJET M et al.: "Depletion of CD8+ T Cells Suppresses Growth of Trypanosoma Brucei and Interferon-Gamma Production in Infected Rats"; & CLIN EXP IMMUNOL, Aug 1990, 81 (2), p 195-9. *
DIALOG INFORMATION SERVICES, file 154, medline 85-92, Dialog Accession No. 07462019, OLSSON I et al.: "Autoreactive T Lymphocytes in Multiple Sclerosis Determined by Antigeninduced Secretion of Interfero-Gamma; & J CLIN INVEST, Sep 1990, 86 (3), p. 981-5. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004588A1 (en) * 1996-07-26 1998-02-05 Sbl Vaccin Ab Lymphocyte stimulating factor from trypanosoma
US5891439A (en) * 1996-07-26 1999-04-06 Sbl Vaccin Ab, Stockholm, Se Lymphocyte stimulating factor
WO2007091580A1 (en) * 2006-02-07 2007-08-16 Nippon Biologicals, Inc. Novel vaccine carrier

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