GB1582294A - Immunological agents - Google Patents

Abstract

By bringing a schistosomicidal medicament into contact with a total extract of antigens of the corresponding parasite so that the target antigens bind to the medicament, the antischistosomal immunological agent is obtained. Where appropriate, an antischistosomal separation is carried out.

Description

(54) IMPROVEMENTS IN OR RELATING TO IMMUNOLOGICAL AGENTS (71) We, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - CENTRE DE TECHNOLOGIE BIOMEDICALE I.N.S.E.R.M., a French Body Corporate of 13-17, rue Camille Guerin, 59000 Lille, Franc and INSTITUT PAS TEUR, a French Body Corporate of 20, Bd. Louis XIV, 59000 Lille, France do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: The present invention relates to a method for the preparation of an immunologic agent. It also relates to the product obtained and its application.

In the fight against the major parasitic endemic diseases, few curative or preventive remedies are completely effective, whether they be by chemotherapy or vaccinotherapy.

In the case of chemotherapy, bilharziasis is presently treated solely by chemotherapy, particularly by NIRIDAZOLE which is available under the trade mark "Ambilhar". This product cures the disease but does not protect against subsequent infection. Infestation takes place transcutaneously: infesting larvae, known as cercariae, pass through the skin, change and whilst growing, reach the liver and mature several weeks after the transcutaneous infestation. The males and females mate then the females will lay their eggs in the periintestinal veins (intestinal bilharziasis) or perivesicular capillaries (urinary bilharziasis). These eggs will pass through the intestinal or vesical wall, will be expelled and release a larva which will infest an aquatic mollusc. In this mollusc, this larva multiplies and is transformed into cercariae which will infest humans.

To summarise, chemotherapy does not provide immunity.

In vaccinotherapy the method of protecting an individual against infectious agents, such as bacteria or a virus, generally consists of immunising the individual against the antigens of these infectious agents. However, in the case of parasitic disorders, in particular schistosomiasis, it has never been possible to provide effective and protective immunisation.

In practice, the only weapon currently available is chemotherapy which, although curative, does not prevent re-infection.

Schistosomiasis or bilharziasis currently affects approximately three hundred million individuals. The inventor has tackled the problem of controlling this disease from an immunological angle. It is known that a schistosmicidal drug, i.e. a drug effective for killing schistosmes, will act on particular antigens of the parasite. These antigens are target antigens of the target sites of the parasite, schistosmes with respect to the particular schistosomicidal drug. The target antigens, which are considered to be located at target sites of the parasite, have molecular structures which are essential to the integrity and survival of the parasite.

The present invention is based on the use of a schistosomicidal drug to produce an antischistosomic immunological agent.

According to the present invention there is provided a method of preparing an antischistosomic immunological agent from human or animal schistosomes, the method comprising contacting a schistosomicidal drug with a whole antigen extract from the schistosomes, whereby the drug is bound as a ligand to those antigens of the extract which are target antigens with respect to the schistomicidal drug, and optionally separating the target antigens from the drug.

The immunological agent of the invention may either comprise the target antigens after their separation from the schistomicidal drug or alternatively may comprise the target antigens bound to the drug. The immunological agent of the invention may if desired be formulated with a suitable carrier for purposes of administration of the agent.

The schistosomicidal drug is preferably selected from Niridazole, Thiosianamine, Astiban, Amphotalide and potassium antimony tartrate.

The invention also relates to the use of the immunological agent for the treatment, particularly the preventive treatment, of schistosomiasis. In fact, when the immunological agent is injected into a living body, the target antigens produce an immune response resulting in the formation in the body of antibodies which are effective against the target-antigens and which therefore provide protection from schistosomiasis. It should be noted that hitherto it has not been possible to produce antibodies which provide true protection against the parasite. This is true even if the immunising antigen is a whole antigen extract of schisotomes comprised of the supernatant of pulverised schistosomes in a solution of for example NaC1.

The method of the invention for obtaining the immunological agent may be performed in a number of ways. In one such method, the schistosomicidal drug may be immobilised on an insoluble support, and the whole antigen extract is then used in an affinity chromatography step so that the target antigens become bound to the immobilised drug and are thereby retained. The insoluble support carrying the immobilised drug with its bound target antigens may then be eluted with a suitable solvent to separate the target antigens from the immobilised drug and the target antigens are collected.

In an alternative method, the schistosomicidal drug is used in a non-immobilised form for separating the target antigens from the whole antigen extract. In this method, the target antigens are contacted with the drug under conditions in which the drug is in the form of a supersaturated solution containing an insoluble amount of the drug, generally a known, predetermined amount. The target antigens become bound to the insoluble drug which may then be separated and washed with a saturated solution of the drug. The drug-antigen combination thus obtained may be used e.g. as a suspension in a suitable solvent for the treatment of schistosomiasis.

The invention will be further explained with reference to the following description.

Immunochemical analysis has shown that schistosoma are formed of a very complex mixture of antigens. Amongst the numerous methods of study used, immunoelectrophoresis has made it possible to reveal at least 25 precipitating components some of which are of an allergenic nature or possess enzymatic activity. Amongst these antigens are the targetantigens of schistosomicidal drugs some of which have been shown to inhibit the activity of enzymes of the parasite. These drugs are used in the invention to isolate the target-antigens.

The target antigens have been characterised with regard to their enzymatic activities and their location in the parasite as well as their immunogenic power.

Equipment and Methods 1/ Obtaining target-antigens The target antigens are isolated from a whole antigen extract of schistosoma (Schistosoma mansoni) prepared from adult worms collected from the hamster, unless otherwise specified.

3500 freshly collected worms are crushed in 2 ml 0.5 M NaCl, frozen and thawed out by pulverisation with a Potter apparatus, then centrifuged at 40 000g for 30 minutes. The supernatant is used as a whole antigen extract.

Schistosomicidal medicaments used as li ands are NIRIDAZOLE (Ciba 32, 644 Ba), THIOSINAMINE (Fluka), ASTIBAN (Roche), CHLORHYDRATE D'EMETINE (Rhone-Poulenc) and AMPHOTALIDE (6171 Rhone Poulenc).

Affinity Chromatography *Preparation of immobilised ligands The supports known as AH and CH -Sepharose (Pharmacia-Suede; Sepharose is a Registered Trade Mark) facilitate the synthesis of specific absorbants in which the drug which acts, as a ligand for the target antigens is separated from the support by a chain of 6 carbon atoms.

The ligand is immobilised on the support by a coupling reaction with carbodiimide. The CH Sepharose has free carboxylic groups which may be used for coupling ligands with free primary amine groups. The AH - Sepharose has free primary amine groups which may be used for coupling carboxylic groups.The procedure for coupling with carbodiimide recommended by Pharmacia is used. The Niridazole (30mg/10 ml water), the Thiosinamine (30mg/ 10 ml water) the Amphotalide (30mg/20 ml dioxane) are coupled with 2 grams of CH - Sepharose gel. The ASTIBAN (30 mg/15 ml water) and the double tartrate of antimony and potassium (30mg/ 1 Oml water) are coupled with 2 grams of AH- Sephraose gel.

The operating procedure used for each medicament is the same. The lyophilised powder of AH or CH - Sepharose is weighed, then left to swell in an excess of a 0.5 M solution of NaCl.

To eliminate lactose and dextran, the gel is washed with the 0.5 M solution of NaCI, then with distilled water to eliminate the NaCI. The ligand solution is added to the gel. The pH of the suspension is adjusted to between 4.5 and 6. A final proportion of liquid/gel of 2:1 volume by volume is suitable for slight agitation, which takes place at laboratory temperature. The carbodiimide powder is added to a final concentration of 0.1 M. The pH is maintained between 4.5 and 6 for 1 hour. The reaction continues for 24 hours at laboratory temperature.

The gel is washed successively in alkaline and acid buffer solutions containing molar NaCl.If dioxane has been used for dissolving the ligand, it is necessary to wash the gel with this solvent. The gel is finally washed with distilled water.

*Isolation of the target - antigens by means of the immobilised ligands The crude whole antigen extract (3500 worms, equivalent to approx 20mg dry weight) is passed over the column of 2 ml of the gel. The column is washed with a 0.5 M solution of NaCl until the optical density at 280 nm is zero. The fixed antigenic components are eluted with the buffer of glycocoll - 0.2 M HCl, 0.5 M NaCl, pH 2.8. The eluate is immediately neutralised in the molar phosphate buffer, pH 8, then dialysed against water. The quantity of product obtained is evaluated at by optical density measurement at 280 nm.

*Affinity Chromatography on non-immobilised ligands A quantity of 1.5 mg schistosomicidal drug is in an insoluble form under experimental conditions during contact with the whole antigen extract and during washing carried out with saturated solutions of medicament. The whole antigen extract (0.1 ml = 1 mg dry weight) of schistosoma collected from the mouse (for the immunisation of mice) or from the hamster (for the immunisation of rabbits) is placed in contact with 2 mg Niridazole, 5 mg Thiosinamine, 10 mg antimony potassium tartrate, 40 mg Astiban, 15 mg Emetine hydrochloride or 3 mg Amphotalide. After 1 hour of contact at 370C and 30 minutes at laboratory temperature during which procedure 1.5 mg of the drug remains insoluble, the insoluble drug is washed three times with 10 ml of a saturated solution of the drug in question. The drug with its bound target antigens will serve for the immunisation of mice or rabbits 2/ Immunochemical Characterisation and parasitic localisation of target-antigens This study is carried out using the immune sera (I.S.) of the rabbit injected with the various target-antigens.

- Immunisation of rabbits A quantity of insoluble drug, incubated with the whole antigenic extract of schistosoma ( obtained as described previously), is injected into the rabbit according to VAITUKAITIS et. al.

A sample of blood is taken on the 35th day (Si), on the 50th day (S2) and on the 80th day (53).

Before being used immunoelectrophoresis and immunofluorescence, the I.S. were absorbed by host antigens. The absorption was carried out by adding 10 mg lyophilised hamster serum and 10 mg lyophilised hamster liver to 1 ml I.S. After incubation for 2 hours at 37"C, the mixture is left for 1 night at 4"C, then centrifuged. The supernatant represents the absorbed I.S.

- Study of target-antigens by immunoprecipitation Conventional immunoelectrophoresis is carried out according to the method described by CAPRON et. al. using schistosome antigen extract and the various anti-target-antigen immune sera.

Two dimensional immunoelectrophoresis is inspired by the technique described by Laurell. Glass slides measuring 5 x 5 cm are used. The migration of the S. Mansoni antigen (2 mg) in a 1 weight by volume agarose gel with a veronal buffer, pH 8.2 (16 g veronal sodium, 22 ml N HCI water for 1000 ml) takes place for 2 hours with a potential difference of 12.5 vat the edges of the slides. The second migration takes place in an agarose gel containing the I.S.

(agarose 1.3%weight by volume 2.34 ml; I.S. 1.16 ml) for 1 night with a potential difference of 3 volts at the edges of the slides.

Revelation of the enzymatic activities of the target-antigens The enzymatic activities (Lactic dehydrogenase (LDH), Glucose-6-Phosphate Dehydrogenase (G6PDH), Malic dehydrogenase (MDH), Alanine dehydrogenase (A1OH), alpha and beta carboxyl esterases) are studied according to a technique developed by URIEL in the precipitates obtained by double diffusion of the gel between the S. mansoni antigen and the various I.S. target-antigens.

Localisation of the target-antigens in schistosome by immunofluorescence The schistosome samples were prepared and treated in the following manner. After 3 washes in the buffered physiological serum, the worms taken from the hamster were fixed in "BOUIN-HOLLANDE-SUBLIME", dehydrated in baths of ethyl alcohol of increasing concentration, treated with butyl alcohol and immersed in paraffin. The blocks were cut into sections having a depth of 5 to 6 , which were fixed on microscope slides. The paraffin was removed from these worm sections the sections were washed in buffered physiological serum and incubated for 30 minutes at 370C with anti-target-antigen rabbit immunoserum, diluted to 1/20 to 1/160. The slides were then washed in buffered physiological serum and incubated for 30 minutes at 37"C with fluorescent antibodies anti-rabbit immunoglobulins produced in goats (Pasteur Institute) diluted to 1/50. The slides were washed and subjected to countercolouration with Evan's blue. As a control of the specificity, the inhibition of fluorescence was carried out by using the anti-target-antigen immunoserum previously absorbed with the whole antigen of schistosoma.

3/Study of Immunogenicity oftarget-antigens a) Immunisation of rats Fischer rats aged 3 months and weighing 180 g were immunised according to the following method: A J-55 (55 days before the infestation) target antigens were administered by intradermal injection 50 ,ug target-antigens, 40 CL1 diptheria anatoxin of the Pasteur Institute. Freund's complete adjuvant (FCA).

A J-38 Sub-cutaneous injection of the same suspension A J-24 Injection of the same suspension into the plantar pads, A J-0 Infestation by 800 cercariae.

Blood was taken from the rats on day 45 (S1), day 30 (52), day 17 (S3), day 4 (S4), day 22 (85).

Rats receiving the same suspension without target-antigens served as the control.

The worms were collected on day 22 by hepatic perfusion. The male, female and immature worms were counted.

Study of the cytotoxicity of the serums was carried out according to a method used by CAPRON et. al. and described by CLEGG and SMITHERS. b) Immunisation of mice A quantity of insoluble drug incubated with the whole antigen extract of Schistosoma mansoni (1/10th of the insoluble quantity obtained under the conditions described for each mouse) is injected (only 1 injection) into the plantar pads of black C57 mice weighing 18 grams and aged 5 weeks. This dose of antigen-ligand was placed in suspension in 0.2 ml 8 %by weight by volume Nail,0. lug Tween 80 (Tween is a Registered Trade Mark) and 25 1 CFA.

Control mice receive 0. lug Tween 80 and 25 CL1 CFA.Other control mice received the same quantity of original drug without antigen.

The mice were infested 15 days later with 60 cercariae and bled and perfused 70 days later.

The male and female worms collected by hepatic perfusion were counted. c) Passive transfer of anti-target-antigen antibodies in the mouse.

The C57 mice received 0.3 ml IS of rabbit anti-target-antigen intravenously in the tail and 0.25 ml fresh serum from healthy rabbits as a source of complement by intraperitoneal injection. The control mice received 0.3 ml fresh serum from healthy rabbits intravenously and 0.25 ml intraperitoneally. Infestation with 60 cercariae takes place 1 hour after these injections. Blood was drawn from the mice and they were killed 60 days after the infestation.

The worms were counted.

Results 1/ Immunochemical study of the target-antigens a) Isolation 2 ml of Sepharose-ligand gel placed in the presence of 20 mg antigen extract of Schistosoma mansoni make it possible to obtain the following quantities of target-antigens, evaluated by optical density measurement at 280 nm: CH-Sepharose - Niridazole : 160 ijg target-antigen ------ - Thiosinamine 290 ,ag AH-Sepharose - tartrate of anitmoney and potassium : 1.65 mg ------ - Astiban 290 yg CH-Sepharose - Emetine : 260 ,ug ------ ------ - Amphotalide 80 ,ug AH and CH-Sepharose not coupled to ligands do not specifically fix antigens. b) Immunoelectrophoretic chart of the target-antigens The anti-target-antigen immune sera measured by conventional or two-dimensional immunoelectrophoresis against an antigen extract of Schistosoma mansoni revealed distinct pictures comprising one or several precipitating systems. The anti-target-antigen immune sera of amphotalide do not contain any precipitant target-antigens which can be revealed.

Comparatively, the immunoserum anti-S.mansoni whole antigen immune serum reveals more than 40 precipitant systems by two-dimensional immunoelectrophoresis. c) Enzymatic activities of the target-antigens These are given in Table (I) d) Localisation of the target-antigens in the parasite The anti-Niridazole target-antigen immuno sera reveal diffuse fluorescence in the region of the parenchyma of the schistosoma, mainly in the male whose tubercles are also fluorescent.

The anti-target-antigen immuno serum thiosinamine reveals fluorescence limited exclu sively to the cellular seat of the digestive cecum, apart from the cellular nuclei. The anti taret-antigen immuno sera of antimony potassium tartrate reveals a similar fluorescence.

The anti-target-antigen immuno serum of Astiban reveals a fluorescence limited exclusively to the superficial cellular seat of the cecum, not involving the nuclei and of discontinuous type. The anti-target-antigen immuno serum of Emetine reveals complete fluorescence of the superficial cellular seat of the cecum. The anti-target-antigen immuno serum of Amphotalide shows only diffuse fluorescence of the parenchyma.

Table I Immunoserum of Immunoelectrophoresis (IEP)* Enzymatic activities** Localisation of rabbit anti noted in precipitant target-antigens in target-antigens of: Conventional Two-dimensional systems immunofluorescence No. of arcs No. of peaks NIRIDAZOLE 1 1 PARENCHYMA THIOSINAMINE 3 4 G6 PDH CECUM A1DH DOUBLE TARTRATE OF ANTIMONY AND POTASSIUM 4 6 MDH G6PDH CECUM Alpha carboxyl esterase ASTIBAN 3 3 G6 PDH CECUM A1 DH EMETINE 1 3 G6 PDH CECUM AMPHOTALIDE 0 0 0 PARENCHYMA Table 1 *Number of arcs obtained by conventional I.E.P. and number of peaks obtained by twodimensional IEP between a whole antigen extract of Schistosoma mansoni and the antitarget-antigen immuno sera.

* *Number of enzymatic activities revealed in the precipitant systems obtained between the whole antigen extract of schistosoma and the anti-target-antigen immuno sera.

*** Localisation of target-antigens carried out with the various anti-target antigen immuno sera.

2/ Immunobiological Study a) Study of Immunological phenomena in immunised rats - Study in vitro of the cytotoxicity of the serums.

The serums (S, to Sg) were collected separately to test their lethal effect on schistosoma and schistosomules. Each test was carried out in duplicate. The various serums had no lethal effect on the adult schistosoma. The serums of the rats (S5) immunized with the targetantigens of Emetine or Thiosinamine had a mortality rate of respectively 40 and 27.5%. The infested control specimens and healthy control specimens had a mortality rate of only 20 and 7% respectively.

- Notation of worms collected by hepatic perfusion.

The number of male, female and immature worms is noted. The results are given in Table II Table II Males Females Immature Total Control rats 19.50#3 1.6#3.74 2.75#1.71 38.25#5.38 (4)* Rats immunized with target-antigens of: DOUBLE TARTRATE OF ANTIMONY AND POTASSIUM (4)* 3.25#3.20 3#2.16 1.25#0.96 7.50#5.07 AMPHOTALIDE (4)* 7.50#3.32 5.50#5.07 1.75#1.71 14.75#9.60 Table II: Average (variation given as #) of male, female, immature and total worms, collected by hepatic perfusion from rats immunised with the various target-antigens and infested for 22 days.

*Number of Animals Student's test, applicable after checking the spreading test, makes it possible to obtain highly significant values for the reduction in the number of worms in rats immunised with the target-antigens of antimony potassium tartrate or Amphotalide. t = 8.32; ddl = 6; p < 0.001 and t = 4.26; ddl = 6; p < 0.05) b) Study of biological phenomena in immunised mice Notation of worms collected by hepatic perfusion.

The number of male and female worms was noted. The number of worms from drugcontrol mice was not significantly different from the number of worms from control mice. The student test does not show significantly different values between the number of worms from immunised mice and that from control mice. c) Study ofbiologicalphenomena in the mouse subjected to the passive transfer ofantibodies The number of male and female worms is noted in Table III.

Table III males females Total Control mice receiving serum from a healthy rabbit (5)* 10.60#7.80 11#10.77 21.60#17.81 Mice receiving anti-target-antigen of rabbits - THIOSINAMINE (4) 5.75#4.27 7.75#9.54 13.50#13.72 - TARTRATE OF ANTIMONY AND POTASSIUM (3) 3.67#4.04 3.61#5 8.67#7.51 - ASTIBAN (6) 4.50#5.50 3.83#3.71 8.33#8.69 - EMETINE (7) 2#3.61 1.43#2.51 3.43#6.11 Table In: Average () of the number of male, female and total worms from the mice having received the anti-target-antigen immunoserum of the drugs.

*Number of animals.

Claims (9)

The Student test shows highly significant differences between the number of worms from the control mice and that of the mice having received the rabbit anti-target-antigen immunoserum of Emetine. t = 2.59; d.d.l = 10; P < 0.001 WHAT WE CLAIM IS:

1. A method of preparing an antischistosomic immunological agent from human or animal schistosomes, the method comprising contacting a schistomicidal drug with a whole antigen extract from the schistosomes, whereby the drug is bound as a ligand to those antigens of the extract which are target antigens with respect to the schistomicidal drug, and optionally separating the target antigens from the drug.

2. A method as claimed in claim 1 wherein the schistosomicidal drug to be contacted with the whole antigen extract is immobilised on an insoluble support, said contacting in an affinity chromatographic step effected to retain the target antigens bound to the immobilised drug, and the target antigens are separated from the immobilised drug.

3. A method as claimed in claim 2 wherein said solvent is an acid buffer.

4. A method as claimed in claim 1 wherein said contacting is performed under conditions in which the schistosomicidal drug is in supersaturated solution with there being present an insoluble amount of the drug whereby the target antigens become bound to the insoluble drug.

5. A method as claimed in claim 4 wherein the insoluble drug having the target antigens bound thereto is separated and washed with a saturated solution of the drug.

6. A method as claimed in any one of claims 1 to 5 wherein the schistosomicidal drug is selected from Niridazole, Thiosinamine, Astiban, Emetine, Amphotalide, or potassium antimony tartrate.

7. A method of preparing an antischistosomic immunological agent substantially as hereinbefore described.

8. An immunological agent prepared by the process of any one of claims 1 to 7.

9. An antischistosomic composition comprising an immunological agent as claimed in claim 8, and an acceptable carrier.