GB1592535A - Proteinase from trypanosoma its preparation use and antibodies thereto - Google Patents

Description

(54) PROTEINASE FROM TRYPANOSOMA, ITS PREPARATION, USE, AND ANTIBODIES THERETO (71) We, BEHRINGWERKE AKTIENGESELLSCHAFT, a body corporate organised according to the laws of the Federal Republic of Germany, of D-3550 Marburg/Lahn, Federal Republic of Germany, 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 an antigen obtainable from Trypanosoma, a process for its preparation and its use.

The present invention provides a proteinase which may be obtained from Trypanosoma cruzi, and which is characterised by the following parameters: a) an average molecular weight of 200 000 e 40 000 b) an isoelectric point at a pH value of 5.3 + 0.2 c) a Michaelis constant with the substrate a-N-benzoyl-argininep-nitroanilide of 1.22 x 10-4 M + 0.05 x 10-4M d) a reaction optimum with a-N-benzoyl-arginine p-nitroanilide as substrate at a pH value of 8.5 1 1.5 and a temperature of 40"C + 10 C.

This enzyme can be used for early immunological and serologic diagnostics of infection with Chagas' disease and also as the main constituent of a Chagas vaccine.

Depending on the methods used to determine the molecular weight, different values were obtained: A molecular weight within the range of from 1.8 to 2.3 x 105 was obtained by way of molecular sieve filtration using as carrier a dextran cross-linked with epichlorohydrin, (Sephadex G 150 of Messrs. Pharmacia, Upsala, "Sephadex" being a Trade Mark). As standard substances for this determination there were used the enzyme catalase, the proteins ovalbumin and gammaglobulin, and, furthermore, cytochrome C.

A molecular weight in the range of from 1.6 to 2.1 x 105 was determined by means of electrophoresis using polyacrylamide as carrier and a buffer containing 2% (w/v) sodium dodecylsulphate.

The gradient-gel electrophoresis showed a molecular weight in the range of from 1.45 to 1.9 x 105.

Accordingly, from the three different methods described above, an average molecular weight of 200 000 + 40 000 has been calculated.

In determining the isoelectric point, the protein prealbumin was used as reference.

The enzyme of the invention consists of about 98% of amino acids. The enzyme prepared in accordance with the Example given below showed the following composition of amino acids, which was determined according to the process of S. Moore, D.H. Speckman, W.H.

Stein, Anal. Chem. 30, page 185, (1958).

Amino acid Frequency in % lysine 5.47 histidine 1.52 ammonia (18.14) arginine 4.57 aspartic acid 9.47 threonine 6.00 serine 5.09 glutamic acid 10.26 proline 4.83 glycine 8.16 alanine 8.71 cystine /2 2.33 valine 8.16 methionine 2.85 isoleucine 4.79 leucine 9.11 tyrosine 1.54 phenylalanine 4.62 tryptophan 2.09 l 100.00 Each of the individual values shown is subject to a permissible variation of + 10 % due to the method of determination.

The chromatographic analysis of the hydrolized enzyme reveals the presence of five sugar structural units, i.e. glucose, galactose, mannose, xylose and glucosamine. There could not be detected ribose and desoxyribose.

In order to determine the specificity, the p-nitroanilides of a-N-benzoyl-arginine, a-N-acetyl-tyrosine, alanine, glutamine, glycine, leucine, lysine, phenylalanine, proline and a-N-benzoyl-lysine were subjected to a reaction with the protease. a-N-Benzoyl-arginine p-nitroanilide and a-N-benzoyl-lysine-p-nitroanilide are split. In order to determine the specificity more precisely, the following oligopeptides were examined: Ala-Ile His-Lys Ser-Gly Arg-Leu Leu-Ala Asp-Leu Lys-Gly Thr-Phe Lys-Lys-Lys Gly-Leu Met-Asp Trp-Gly Gly-His-Gly Phe-Leu Tyr-Leu Glu-Tyr Pro-Leu Val-Gly Of these peptides, Arg-Leu, Trp-Gly and Trp-Lys are split. This means that the enzyme splits those peptide bonds in which the carboxyl groups of arginine, tryptophan and a-amino-substituted lysine are involved.

The influence of various inhibitors on the activity of the enzyme is indicated as follows: Substance Concentration (M) (50% inhibition) a-N-benzoyl-arginine 7.0 x 10-2 CdSO4 3.4 x 10-3 iodoacetamide (IAA) 8.0 x 10-3 N-ethylmaleinimide (NEM) 2.5 x 10-4 iodoacetic acid 4.8 x 10-4 CuCI2 5.2 x 10-4 ZnCI2 8.0 x 10-4 sodium salt of p-(ethylmercuric) mercaptobenzenesulfonic acid 8.0 x 10-5 (Thiocid) HgCl2 2.9 x 10-6 p-chloromercuric benzoate (pCMB) 8.0 x 10-6 tosyl-lysine chloromethyl ketone 4.5 x 10-8 (TLCK) The inhibition of the enzyme by the sodium salt of p-(ethylmercuric)mercaptobenzenesulfonic acid (Thiocid) and by tosyl-lysine chloromethyl ketone (TLCK) is not competitive.

Gaseous oxygen shows an inhibiting influence on the enzyme activity; the presence of 2 for 30 minutes inhibits 60 No of the original activity. This inhibition is reversible. A concentration of 5 mmoles of mercaptoethanol neutralizes the inhibition almost completely.

If phenylmethane-sulfonylfluoride (PMSF) is added to the enzyme solution in a concentration of 0.1 FM/ml or iodoacetamide (IAA) is added in a concentration of 10 IlM/ml, these substances have indeed no influence on the activity of the protease, however, they protect the latter from the inactivating action of oxygen.This fact, together with the results of the inhibition, indicates the presence of sulfhydryl groups in the enzyme molecule.

The strong inhibition of the protease by tosyl-lysine chloromethyl ketone indicates the presence of a catalytic histidine in the active center of the enzyme molecule.

The invention also provides a process for the preparation of the enzyme from Trypanosoma, preferably from the culture form of Trypanosoma cruzi.

Culture forms of Trypanosoma cruzi are obtained, for example, in the following manner: An epimastigote culture form of Trypanosoma cruzi, Brasil strain, is cultivated on diphasic blood agar (modified according to Senekjie) for 4 to 5 days at 280 C. Subsequently the liquid phase with the Trypanosoma is filtered through sterile gauze. The Trypanosoma suspension thus obtained serves as inoculum for the reproduction in the LiT-medium (Canargo, E.P., Rev. Inst. Med. Trop. Sao Paulo, 6, pages 93 - 100 (1964)). Following an incubation at 280 C over five days the suspension is centrifuged, and the sediment is washed twice with a physiological sodium chloride solution. Thereafter the Trypanosoma are disintegrated, generally mechanically, preferably using ultrasonics. Autolysis of the organisms is also possible.

Whereas the supernatant culture fluid does not show any proteolytic activity, a suspension of the disintegrated Trypanosoma does show proteinase activity. Following a high-speed centrifugation the entire proteolytic activity set free is found in the supernatant.

Thus, the product concerned is an enzyme which is soluble in the aqueous medium and is not particle-bound.

The process of the invention accordingly comprises causing trypanosomes in suspension to disintegrate, and subjecting the resulting liquid phase to one or more processes suitable for isolating a protein having an average molecular weight of 200,000 + 40,000 and an isoelectric point at a pH value of 5.3 + 0.2.

The water-soluble proteinase obtainable from disrupted trypanosomes, especially Trypanosoma cruzi may be isolated by using any one or more of the following techniques, in any order: (a) the precipitation of the proteinase by means of a salt used in protein chemistry for precipitating proteins with subsequent dissolution of the precipitate, if appropriate; (b) molecular sieve fractionation; (c) adsorption on an ion exchanger with subsequent elution.

Dealing in more detail with these techniques: a) The proteinase is precipitable with salts that are commonly used in protein chemistry for protein precipitation, for example, it is precipitated by ammonium sulphate at a saturation concentration of 50% (w/v) (about 2 moles/l). Instead of ammonium sulphate there may, of course, be used any other salt suitable for protein precipitation, if it is added to the proteinase-containing solution up to the appropriate concentration.

b) By molecular sieve fractionation, the enzyme can be separated from accompanying impurities. In view of the molecular weight of the enzyme, the most suitable molecular sieves are those which are able to concentrate proteins having a molecular weight of about 200,000, whether substances of this nature filter through the molecular sieve, i.e. they pass through, or they are held back by the sieve, i.e. they are set free with delay. When using the dextran cross-linked with epichlorohydrin, which is sold by Messrs. Pharmacia, Upsala under the name of Sephadex G 200, ("Sephadex" being a Trade Mark), the enzyme is held back i.e. when this Sephadex is used to charge a chromatography column, and a solution containing the enzyme is introduced into the column, impurities of a higher molecular weight are eluted from the column before the enzyme and lower molecular weight impurities are eluted after the enzyme.

The fractions obtained are tested for their proteolytic activity using a suitable substrate, for example, a-N-benzoyl-arginine p-nitroanilide under the conditions mentioned above.

The proteolytically active fractions are collected; they contain the concentrated enzyme.

c) The proteinase from Trypanosoma is bound by exchange resins especially by anion exchange resins, from low conductivity solutions containing the proteinase. Preferred are ion exchangers with aminoethyl, diethylaminoethyl or triethylaminoethyl groups, and especially preferred are ion exchangers carrying these groups on a cellulose or cross-linked dextran carrier material. As mentioned above, when there is used an ion exchanger of this kind, preferably a diethylaminoethyl ion exchanger, the enzyme is adsorbed from a solution thereof having a low salt concentration, and a large part of the accompanying impurities are not bound. By using an elution solution having a higher salt concentration and/or conductivity, the enzyme may be desorbed from the ion exchanger. This may be effected simply by using a salt solution having a linear concentration gradient, for example, a sodium chloride solution in a gradient of from 0 to 1 mole/litre. As mentioned above for molecular sieve fractionation, the activity of the resulting fractions is measured, and those fractions which show a proteolytic activity with an appropriate substrate are collected.

It is, of course, possible to use any of the procedures mentioned above or any other suitable procedure either alone or in any suitable combination with one another, in order to prepare the proteinase with the desired degree of purity from a solution which has been obtained by disintegrating Trypanosoma and which still contains undesirable accompanying substances, for example, if a solution containing the proteinase is subjected first to chromatography, especially on a diethylaminoethyl ion exchanger, and subsequently to a molecular sieve fractionation, the specific activity with regard to the cleavage of a-N-benzoyl-arginine p-nitroanilide (BAPA test) can be concentrated from about 10 to 20 units/ml (cf. the Example for the definition) to about 700 to 1000 units/ml of the solution.

Generally, the enzyme activity of the proteinase of the invention can be stabilised by adding a substance that contains SH groups, for example, mercaptoethanol or diethiothreitol.

The proteinase from Trypatiosonia cruzi which can be obtained thus has proved to be immunogenic: when it is administered parenterally in a sufficient amount to vertebrate animals, for example, test animals, for example, rabbits, specific antibodies are formed against the enzyme.

It has been shown that the serum obtained from animals immunised with the proteinase forms precipitation bands with the proteinase when tested using the techniques of immunodiffusion and/or immuno-electrophoresis. By inhibition tests it has been demonstrated that the activity of the proteinase can be inhibited by approximately 100% by the addition of immune serum.

It has also been shown that permanent cultures of the cell line HeLa can be protected against the invasion of infectious Trypanosoma by adding the specific anti-enzyme to the HeLa culture fluid.

The above findings indicate that it is possible to protect animals and persons from the intracellular reproduction of the infectious Trypanosoma, and thus from the occurrence of pathological changes, by active immunization with the enzyme of the invention and/or by passive immunisation, that is to say, by the administration of antibodies to the proteinase, for example, as an immune serum or another antibody preparation.

The invention therefore also provides a vaccine which comprises the proteinase of the invention. The vaccine, which may be lyophilized is preferably in unit dosage form, one unit dose comprising an amount proteinase in a physiologically tolerable medium which is sufficient for the dvelopmcnt of antibodies. The vaccine may be used as indicated above as a medicament, i.e. for the development of immunity to Chagas disease, or to stimulate antibody production for the preparation of antisera. The antibodies which are obtained in this manner are, as indicated above, active ingredients of important medicaments against Trypanosoma. Finally, the proteinase itself is an antigen which is a valuable agent for diagnostic detection and for the determination of the enzyme antibodies in vitro and in vivo.

For this purpose there may be used any immunological techniques for the determination of antigens and/or antibodies.

The invention accordingly provides not only the enzyme of the invention and vaccines comprising it, but also antibodies directed against this proteinase and pharmaceutical preparations which comprise the antibodies in admixture with a pharmaceutically suitable carrier. The latter may be lyophilized and/or in unit dosage form. The invention further provides a method for producing the antibodies of the invention, wherein the proteinase of the invention is administered to a mammal then, when sufficient time has elapsed for antibody formation, the mammal is bled and a suitable antibody preparation, for example, an antiserum, is prepared from the whole blood.

The invention further provides a method of determining the proteinase itself or antibodies thereto in a sample of fluid, which comprises subjecting the fluid to an immunological test using as an antigen or antibody reagent, as appropriate, the proteinase of the invention or antibodies thereto. Any immunological technique may be used, for example, any immuno-diffusion or immunoelectrophoresis method, and the antigen (proteinase) or antibody may be labelled e.g. with a fluorescent or radioactive marker. The antigen or antibody in the sample may be assayed qualitatively or quantitatively, and competitive binding assays may be used. The antigen or antibody may be determined directly or by a "sandwich" technique. The sample of fluid is generally a body fluid.

Immunological tests may also be carried out in vivo.

The enzyme of the invention is therefor a useful test in the fight against Chagas disease, enabling the disease to be diagnosed at an early stage and treated by passive immunization with antibodies, and for prevention of the occurrence of the disease by active immunisation.

The following Example illustrates the invention.

Example: 19.5 Liters of a completely grown fermenter culture with a total of 2.9 x 1011 Trypanosoma cruzi (culture form D1) are centrifuged (Sorvall RC 28, rotor for continuous flow SS 34, 39 000 x g), and the supernatant is rejected. The Trypanosoma sediment is dissolved twice in 200 ml of 0.9 So (w/v) NaCI solution and is centrifuged again (Sorvall Superspeed, model RC 2B, rotor GSA, 8000 x g/30 minutes). The sediment is dissolved in 50 ml of 0.1 molar trishydroxymethylaminomethane-HC1 buffer of pH 7.5 + 5 mmoles/l of mercaptoethanol and is treated with ultra-sonics, while cooling at 40C (Bransone Sonifier B-12; 6 x 30 seconds, output 10 "Bransone" is a Trade Mark). The homogenized product is centrifuged (16 300 x g/60 minutes), and the sediment is rejected. The proteolytic activity of the supernatant is determined in the BAPA test. The total activity is 6071.1 TU (TU = 1 trypsin unit = extinction achieved by 10 Rg of trypsin within 20 minutes at 40 C and at a pH value of 8.5 with 1 mmol/l of BAPA in a final volume of 7 ml at 405 nm). The specific activity is 13.2 TU/mg of protein.

This supernatant is introduced into a DEAE Sephadex A 50 ion exchangerchromotography column (column = 8 x 10 cm) and is eluted with an elution buffer of 50 mmoles/l of trishydroxy-methylaminomethane-HC1 buffer of pH 7.5 + 5 mmoles of mercaptoethanol with a linear gradient of from 0 to 1 mole/l of Nail. The protein determination is effected by means of a flow photometer of the type Uvicord with a wave length of 280 nm. The fraction volume is 25 ml. The elution of the proteolytically active fractions is performed at a concentration of 0.2 to 0.3 mole/l of NaCI. The elution period of the chromatography is about 20 hours at room temprature. The proteinase activity of the individual fractions is determined in the BAPA test. The active fractions are combined and concentrated by ultra-filtration (Amicon membrane UF-100). The determination of the activity of the concentrate shows a total activity of 5413.1 TU, and the specific activity is 239.7 TU/mg of protein.

The concentrate is charged into a Sephadex G-200 column (column = 48 x 5 cm) and is eluted with 0.05 mole of trishydroxy-methyl-aminomethane-HC1 buffer of pH 7.5 + 5 mmoles/l of mercaptoethanol. The protein determination is again performed at 280 nm in the flow photometer. The fraction volume is 25 ml. The elution period of the chromotography is about 24 hours at room temperature. The proteolytic activity is determined in the BAPA test, and the active fractions are combined and concentrated. The activity of the pure enzyme yields a total of 4699.1 TU, and the specific activity is 721.0 TU/mg.

Thus, the enzyme is isolated in this Example with a purification factor of 55, with a yield of about 80% of the activity originally present in the decomposition phase.

Claims (18)

WHAT WE CLAIM IS:

1. A proteinase obtainable from Trypanosoma which is characterised by the following parameters: a) an average molecular weight of 200 000 + 40 000; b) an isoelectric point at a pH value of 5.3 1 0.2; c) a Michaelis constant with the substrate a-N-benzoyl-argininep-nitroanilide of 1.22 x 10-4 M 1 0.05 x 10-4M; d) a reaction optimum with a-N-benzoyl-argininel-p-nitroanilide as substrate at a pH value of 8.5 1 1.5 and a temperature of 40"C + 10 C.

2. A process for the preparation of a proteinase as claimed in claim 1, which comprises causing trypanosomes in suspension to disintegrate, and subjecting the resulting liquid phase to one or more processes suitable for purifying a protein having an average molecular weight of 200 000 1 40 000 and an isoelectric point at a pH value of 5.3 + 0.2.

3. A process as claimed in claim 2, wherein the trypanosomes are Trypanosoma cruzi.

4. A process as claimed in claim 2 or claim 3, wherein the purification process comprises any one or more of the following steps, carried out in any order: (a) the precipitation of the proteinase by means of a salt used in protein chemistry for precipitating proteins with subsequent dissolution of the precipitate, if appropriate; (b) molecular sieve fractionation (c) adsorption on an ion exchanger with subsequent elution.

5. A process as claimed in claim 4 (c), wherein the ion exchanger has aminoethyl, diethylaminoethyl or triethylaminoethyl groups.

6. A process as claimed in claim 5, wherein the groups are carried on a cellulose or cross-linked dextran carrier material.

7. A process as claimed in any one of claims 3 to 6, wherein the liquid phase is subjected to ion exchange chromatography followed by molecular sieve fractionation.

8. A process as claimed in claim 7, wherein the ion exchange chromatography is carried out on a diethylaminaoethyl ion exchanger.

9. A process as claimed in claim 2, carried out substantially as described in the Example herein.

10. A proteinase as claimed in claim 1, whenever prepared by a process as claimed in any one of claims 2 to 9.

11. A vaccine which comprises a proteinase as claimed in claim 1 or claim 10.

12. A vaccine as claimed in claim 11, in lyophilized form.

13. A vaccine as claimed in claim 11 or claim 12, in unit dosage form.

14. An antibody directed against a proteinase as claimed in claim 1 or claim 10.

15. A method of producing an antibody as claimed in claim 14, which comprises administering to a mammal a proteinase as claimed in claim 1 or claim 10, withdrawing a sample of whole blood therefrom after antibody formation has occurred, and separating an antibody containing fraction from the whole blood.

16. A pharmaceutical preparation which comprises an antibody as claimed in claim 14 in admixture or conjunction with a pharmaceutically suitable carrier.

17. A method of determining a proteinase as claimed in claim 1 or an antibody as claimed in claim 14 in a sample of fluid, wherein the fluid is subjected to an immunological test using as an antigen reagent or an antibody reagent, an antigen as claimed in claim 1 or an antibody as claimed in claim 14 respectively.

18. A method as claimed in claim 17, wherein the fluid is a body fluid.