AU5075200A

AU5075200A - Recombinant fusion protein, (vaccine) composition containing the same and method for the production thereof

Info

Publication number: AU5075200A
Application number: AU50752/00A
Authority: AU
Inventors: Georg Baljer; Sylvia Franke
Original assignee: Lohmann Animal Health GmbH and Co KG
Current assignee: Lohmann Animal Health GmbH and Co KG
Priority date: 1999-06-04
Filing date: 2000-06-05
Publication date: 2000-12-28
Also published as: WO2000075345A1; EP1057895A1; EP1183374A1; CN1264857C; CN1357047A

Description

1 A recombinant fusion protein, a (vaccine) substance composition containing it, and a method for the preparation thereof The invention relates to a recombinant fusion protein, a (vaccine) substance composition containing the recombinant fusion protein, and a method for the preparation of the recombinant fusion protein. The oedematose of pigs is caused by Shiga toxin forming Escherichia Coli (STEC). The main virulence factor of these pathogenic organisms which is exclusively accountable for of the clinical symptoms is the 2e Shiga toxin (Stx2e) (Mac Leod et al., 1991). Since the disease exhibits a peracute progress in many cases and attempts for a therapy mostly are initiated too late or do not result in the success desired it would be desirable to develop an efficient prophylaxis. It is problematic to produce and thoroughly purify the Stx2e. The B sub-unit of the Stx2e is taken into account as a possible vaccine for various reasons. It is identified by the serums of convaslescent piglets, i.e. it possesses antigeneric determinants. In addition, the B sub-unit of the toxin induces the formation of toxin-neutralizing antibodies after a parental application (Acheson et al., 1996; Boyd et al., 1991). Genetic engineering methods were a successful aid in preparing a recombinant fusion protein which consists of a fragment of the Stx2eB sub-unit and the Glutathion S transferase of Shistosoma Japonicum (Franke et al., 1995). For the oedematose of weaned piglets, both the excretion of the patpigenic organisms and the immunological reaction to the STEC infection was investigated already over a major period of time. The recombinant fusion protein from a fragment of the Stx2eB sub-unit and the Glutathion S transferase, which was used to prove the presence of Stx2e antibodies, is suited very well to indirectly prove the STEC infection and has been hitherto considered to be a potential vaccination antigen for the prophylaxis of the oedematose (Wieler L. H., Franke, Sylvia, Rose M., and Karch, H.: Charakterisierung der Immunantwort bei der Odemkrankheit des Schweines mit einer rekombinanten B-Untereinheit des Shiga-like-Toxins IIe, (Lecture read at the 2 1 st DVG congress at Bad Nauheim (in March, 1995)). .../2 -2 Therefore, it is the object of the invention to provide a recombinant fusion protein suited for vaccination purposes, a plasmid encoding it, a (vaccine) substance composition containing the fusion protein for various applications in conjunction with the oedematose, particularly that of the pigs, and a method for the preparation of the recombinant fusion protein. The object is achieved by a recombinant fusion protein having the features of claim 1, a (vaccine) substance composition having the features of claim 5, an E.coli strain according to the plasmid according to claim 18, and a method having the features of claim 20. Aspects of the invention are indicated in the sub-claims. According to the invention, a recombinant fusion protein and a (vaccine) substance composition containing it are provided which may be used for various applications in conjunction with the oedematose, particularly that of the pigs. Thus, the applications taken into consideration are: - The demonstration of antibodies against Stx2e. - the diagnosis of the oedematose, - the generation of monoclonal antibodies against the toxin of the pathogenic organism causing the oedematose, specifically as a basis of checking the yield in deriving the recombinant fusion protein or as a basis of deriving the holotoxin by immune affinity chromatographic purification, - The immunization against the oedematose, particularly that of the pigs. The recombinant fusion protein is a sub-genic Stx2e fragment of the 2e Shiga toxin in a fusion with a terminal tag the size of which approximately corresponds to the size of the fragment or a fraction of the fragment. The terminal tag is a marked end group in the amino-acid sequence of the protein. Preferably, the sub-genic Stx2e fragment is a B sub-unit (Stx2eB) of the 2e Shiga toxin. The size of the terminal tag is preferably 5 kDa, as a maximum, and more preferably is 5 kDa. Also preferably, it is an amino terminal His tag. The His tag comprises six histidines. Its size is about 0.66 kDa. ./3 -3 The recombinant fusion protein has substantial antigenic domains of the native protein which substantiate its suitability for various applications in conjunction with the oedematose. It is true that this has also been the theoretical case for the previously known recombinant fusion proteins from a fragment of the Stx2eB sub-unit and the Glutathion S transferase. However, the problem posed here is that as the applicant judges it annoying immunological reactions have to be expected that oppose the use of the generic fusion proteins for therapeutic applications. In contrast, a significant advantage of the inventive fusion protein is that annoying immunological responses are not expected here because of the tag which is especially chosen and, thus, for the first time, fusion proteins will be available that are usable in vaccines. Like for generic fusion proteins, the tag used according to the invention facilitates the derivation of the recombinant fusion protein, particularly its purification, e.g. by an affinity chromatographic method. Oligomers from crosslinked His Stx2eB monomers may form fusion proteins which are particularly efficient. According to an advantageous aspect, the (vaccine) substance composition, in addition to the recombinant fusion proteins, comprises at least one additional antigen. A vaccine substance composition is a formulation of an immunogenic amount of the recombinant fusion protein and an immunogenic amount of at least one additional antigen. This combined vaccine is apt to effect a simultaneous vaccination against the oedematose of the pigs and against a least one further disease. In particular, the (vaccine) substance composition, in addition to the recombinant fusion proteins, may comprise at least one additional antigen which is selected from the group comprising group a Pasteurella multocida bacterin including a cell-bonded toxoid, a Bordetella bronchiseptica bacterin, an Erysipelothrix rhusiopathiae antigen, one or more soluble non-cell toxoids of type D Pasteurella multocida and/or Escherichia coli and/or Clostridium perfringens, disactivated ... /4 -4 whole cells of type A or D Pasteurella multocida, cultures of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli, Clostridium perfringens, Streptococcus suis, Mycoplasma hyopneumoniae as well as Porcine Reproduction and Respiratory Syndrom virus, influenza virus, Pseudorabies virus, and Porcine Circoviruses I and II. The aforementioned antigens are known to cause the diseases which follow: - Pasteurella multocida and Bordetella brochiseptica cause the progressive atrophic rhinitis of the pigs, also called "snuffle disease"; in a pathogenic respect, it is mainly the Pasteurella multocida toxins which play an important part (with the toxoid content being significant in commercial vaccines). - Pasteurella A and D occur in respiratory diseases of the pigs (pneumonia). The Pasteurella multocida D also causes the snuffle disease. - The Erysipelothrix rhusopathiae causes pig erysipelas - The Escherichia coli causes diarrhoea diseases (where the oedematose of the pigs is a special form) (the toxins are decisive) - The Clostridium perfringens causes the necrotizing enteritis of the suckling piglets (the toxins are decisive) - The Actinobacillus pleuropneumoniae causes haemorragically necrotizing pleuropneumonia - The Haemophilus parasuis causes the Glasser disease (fibrinous serositis and arthritis) - The Streptococcus suis causes streptococcal septicaemia - The Mycoplasma hyopneumoniae causes enzootic pneumonia, also called "Piglet influenza" - The Porcine Reproductive and Respiratory Syndrome virus causes respiratory diseases (pneumonia) of piglets and fertility diseases of sows - The influenza virus causes respiratory diseases - The Pseudorabies virus causes the Aujeszky disease of the pig (pseudo-rage) .. 15 -5 - The Porcine Circovirus I and II causes the post-weaning multisystemic wasting syndrome. Preferably, the at least one additional antigen is chosen so as to refer to a disease which typically attacks the pig at approximately the same age as the oedematose does. This is largely the case for the above mentioned antigens. The vaccine substance composition will then make possible a particularly operative combined vaccination. The vaccine composition preferably contains the recombinant fusion protein and/or the at least one additional antigen each in an immunogenic amount for the vaccination of pigs against the oedematose of the pigs and other viral and/or bacterial infections. In addition, the invention relates to vaccine substance compositions the compositions and/or amounts of which are chosen so as to make achievable an immunization of the animal concerned against the at least one disease by sequential and/or simultaneous vaccination with the vaccine compositions. The choice of the adjuvant is of particular significance for the vaccine (substance) composition. For instance, a W/O/W emulsion (e.g. ISA 206), a W/O emulsion (e.g. an iFA incomplete Freund adjuvant, an aqueous suspension (e.g. aluminum hydroxide) or an O/W emulsion may be employed. According to the inventive method for the recombinant preparation of a sub genic fragment of the 2e Shiga toxin (Stx2e) in a fusion with a terminal tag, a suitable vector system of a sub-unit is cloned from the Stx2e operon, the resultant recombinant plasmid is transformed into an E.coli strain, the resultant expression system is induced, and the fusion protein is expressed and purified. The gen of the B sub-unit of the 2e Shiga toxin (Stx2eB) was cloned into various expression vectors. The recombinant plasmides thus formed were used for transforming various E.coli K12 laboratory-scale strains. All transformants were tested under varying conditions (temperature, level of induction, duration of ... /6 -6 induction) in expression studies for the formation of the recombinant B sub-unit. The transformant or clone having the largest yield of recombinant protein as compared to the cell protein overall content was determined. A purification method was developed for the fusion protein formed in this strain, comprising the mature B sub-unit with an N terminal His tag (His-Stx2eB), and was tested at a laboratory scale. FPLC which uses appropriate buffer systems is contemplated for implementing the purification method at a large scale. Example Preparation of the recombinant B sub-unit of the Stx2e The strain E.coli Cux-Stx2eB, DSM No. 12721 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig) is used for the preparation of the recombinant B sub-unit. This E.coli laboratory-scale strain contains the plasmid pHIT-24 which clones the B sub-unit of the Stx2e. A seed lot system was set up from this strain, was filled into 2 ml cryo vials, and was stored at -78 0 C. For the production of the recombinant B sub-unit, 1 ampulla of working seed (2 ml) is defrosted for the growth of a pre-culture 1. The pre-culture 1 is prepared under the following conditions: Medium: 150 ml sterile standard I nutrient broth + 0.01 % Ampicillin in a 300 ml Erlenmeyer flask Incubation: for 15 hours at 37 'C, upright stationary culture A "Biostat B" fermenter having a 5-litre culture vessel is used to prepare the main substance. This vessel is filled with 4 litres of standard I nutrient broth + 0.01 % Ampicillin and was autoclaved as a unit for 25 minutes at 121 'C. The pre-culture 1 is placed in this medium and is cultivated for 6 hours under the following conditions: Temperature: 37 'C ./7 -7 pH = 7.0 to 7.1 Stirring speed: from 100 to 150 rpm Air supply: 2 litres/min The regulation of the pH is ensured by an automatic feed of a sterile 10 % NaOH solution. Induction was initiated by adding 0.25 mM of an IPTG solution* after a cultivation of 6 hours and a pH leap from 7.1 to 7.5. The induction period was abt. 3.5 hours. Subsequently, the culture was pumped into a 10-litre harvesting container and was hydroextrated in a centrifuge of 2500 x g. The supernatant substance was discarded, the pellet was received in 200 ml of an 8 M urea buffer and was kept in a refrigeration room (at 4 to 8 C) for about 15 hours. The resuspended pellet was then treated with ultrasonic sound (for 4 x 15 minutes at 190 Hertz at pulses of 0.3 seconds) and was centrifuged at 10,000 x g subsequently. The supernatant substance was cautiously removed and served for further processing; the pellet obtained in this step was discarded. Subsequently to this, the solution was restricted in volume from 200 ml to 80 ml by means of an ultrafiltration ("Pellicon XL"). The protein solution thus obtained then underwent further processing by means of affinity chromatography (FPCL "Aktaexplorer"). The material containing the recombinant target protein was fractioned at 3 ml each, was applied and was fed over a column loaded with a metal-chelat matrix (NI NTA, Qiagen) (volume: 8 ml) This matrix specifically bonds the His tag of the recombinant protein. .../8 -8 The target protein is retained by the metal and is washed under denaturizing conditions (8 M of urea, 0.1 M of NaH 2

PO

4 , 10 mM of Tris/HCL, pH = 8). After the contaminating proteins are removed the recombinant protein is desorbed by the affinity matrix by a pH leap (8 M of urea, 0.1 M of NaH 2

PO

4 , 10 mM of Tris/HCL, pH =3) and is collected at the exit of the column. The purified protein is subjected to concentration by means of cross-flow filtration (pore size 5 kDa). After the purity and yield are checked (via an SDS gel electrophoresis, western blotting, Elisa, protein determination) the urea puffer is exchanged against a physiological buffer solution (PBS, pH = 7.2). Exchange is performed by means of cross-flow filtration (pore size 5 kDa). The recombinant protein was present at a concentration of 300 ptg/ml. * IPTG: Isoproplybeta-D-thiogalactopyranosite Description of the recombinant fusion protein The target protein is encoded by the gen fragment Stx2eB. The size of this sub-genic fragment of the B sub-unit of Stx2e is 228 bp. A test was made of the following properties of the recombinant protein: 1. Molecular weight size The target protein has a molecular weight determined in the SDS gel electrophoresis of abt. 7.5 kDa. 2. Check of the recombinant protein in the Immunoblot with serums of fallen-ill piglets The purified antigen was examined in the Immunoblot with serums of piglets fallen ill with the oedematose. The animals concerned were piglets from pig .../9 -9 breeding companies in which clinically manifest diseases occurred with Stx2e E.coli strains More than 90 % of these serums reacted positively with the recombinant protein. In order to exclude wrongly positive results, the examinations were verified with the B sub-unit coupled to the Glutathion S transferase of Schistosomajaponicum and were verified. 3. Check of the recombinant protein with monoclonal antibodies against Stx2eB In order to find out whether the conformation of the recombinant B sub-unit is similar to the wild-type protein the recombinant Stx2eB was examined with the Dot-Blot method. For this purpose, the monoclonal antibody BC5BB12 was usefd which specifically recognizes die B sub-unit of Stx2 and also cross reacts with the B sub-unit of Stx2e. The Stx2e holotoxin was carried along as a positive check. A raw toxin preparation of Stx1 served as a negative check. The monoclonal antibody BC5BB12 reacted with both the Stx2e holotoxin and the recombinant Stx2eB protein, but did not react with the Stx 1. 4. Test of the recombinant protein for cytotoxicity in the Verocell test The cytotoxicity of the recombinant protein was tested on verocells, helacells, and MDBK cells in the cytotoxicity test. To this effect, concentrations of from 0.3 pg/ml to 100 ptg/ml were employed on recombinant Stx2eB. Even in the lowest stages of dilution, no significant difference from the negative check was found to exist in any one of the cell lines examined. These results confirm that the recombinant Stx2eB is not cytotoxic per s6. 5. Demonstration of the immunogeneity of the recombinant Stx2eB in the rabbit test Two male rabbits of the "White New Zealander" race at the age of abt. 12 months were immunized with the recombinant Stx2eB. 100 ptg of antigen were subcutaneously injected in the 14 vaccination while adding the incomplete ... /10 - 10 Freund adjuvant (iFA). Boostering was subcutaneously done with 50 pig of recombinant Stx2eB, also with iFA, six weeks later. The serums obtained prior to and after the vaccination were examined in the Immunoblot. A specific serum conversion was proved to exist in the two rabbits. Description of how to prepare vaccine formulations (Examples) F. How to prepare a W/O/W vaccine formulation The antigen is continuously added to the adjuvant (e.g. Montanide ISA 206) under sterile conditions as an aqueous phase (at a temperature of 22 C) while being stirred (at a speed of < 2,000 r.p.m.). Subsequently, the emulsion is homogenized for 10 minutes at abt. 2,000 r.p.m. The vaccine formulation undergoes a new homogenization after a storage period of 24 h at 8 'C. The phase position is tested microscopically and in a dyeing test. 2. How to prepare a W/O vaccine formulation The antigen is continuously added to the adjuvant (e.g. an incomplete Freund adjuvant) under sterile conditions as an aqueous phase (at a temperature of 22 C) while being stirred (at a speed of < 2,000 r.p.m.). and is emulsified. The phase position is tested microscopically and in a dyeing test. 3. How to prepare an aqueous suspension The aqueous antigen is continuously added to the aqueous adjuvant (e.g. aluminum hydroxide) under sterile conditions while being stirred (e.g. using a magnetic stirrer) and is stirred. The vaccine is tested with respect to the pH and tonicity parameters. 4. How to prepare an O/W emulsion - 11 The aqueous antigen is continuously added to the adjuvant and is emulsified. The phase position is tested microscopically and in a dyeing test. After the vaccine formulations are prepared they are stored in a refrigerator at temperatures of from +4 'C to +8 'C prior their further use. Example of how to prove the immunogenic action of the recombinant Stx2eB in the pig as a target animal by using various vaccine formulations Object of the test An examination is made on the question: Can the recombinant Stx2eB protein prepared by a genetic engineering method (using various adjuvants) induce an immunogenic response in the weaned piglet ? The test was made on 8 weaned piglets at the age of 6 weeks. 6 animals were treated with vaccine preparations, 2 animals were administered a placebo. The vaccine was applied twice at an interval of 3 weeks. Blood specimens were taken of each animal. 1. prior to the 1st immunization 2. 14 days after the 1 st immunization 3. Directly prior to the 2"d immunization (21 days after the 1 St immunization) 4. 14 days after the 2 "d immunization 5. 21 days after the 2 "d immunization The serums were examined in the Elisa for the presence of specific antibodies which are directed against the recombinant Stx2eB. In addition, the compatibility and safety of the vaccines were assessed. ... /12 - 12 General testing data Animals Type of animal: Pig Category of animal: Weaned piglet Age: 6 weeks (at the time of 1 st vaccination) Sex: mixed Immunity status of the animals at the start of tests: Stx2eB antibodies, negative Way of keeping: in groups Feeding scheme: ad libitum Water supply: ad libitum from a water piping Fodder additives used: no use of fodder additives Vaccine administration parameters Manner of application: by injection Path of application: intramuscular Period between the two vaccine applications: 3 weeks Pre-treatment of the vaccine administered: nil Pre-treatment of the animals being tested: nil Number of animals being vaccinated: 8 Number of control animals: 2 Study design: randomized, blank Vaccine dosage, animal identification, vaccine use The definite test scheme is shown in Table 1. Adjuvant key Adjuvant A - ISA 206 ... /13 - 13 Adjuvant B - iFA Adjuvant C - Montanide Course of tests Side effects encountered: After 1 " vaccination - Slight effect on the general condition and fodder acceptance Animals 7 and 8 showed a slight increase in body temperature + slight diarrhoea After 2 nd vaccination- No further side effects, apart from increase in body tempera ture, for animal 8 Number of animals which were withdrawn from the test: Weaned piglet No. 2 because E.coli caused an intestimal inflammation Diseases which occurred, but were not due to the vaccination: None except for the disease of the weaned piglet No. 2 Treatment made with other medicines: nil Results Compatibility and safety of vaccine formulations: The vaccine formulations can be considered to be generally compatible and safe although a slight disturbance of the general state of health, a short-time adverse effect on fooder acceptance, and an increase in the body temperature combined with ... /14 - 14 a slight diarrhoea occurred for the animals 7 and 8 after the 1 st immunization. The 2 "d immunization was stood with no appearance of clinical symptoms. Only the animal No. 8 reacted to the new vaccine application by an increase in body temperature. Local tissue reactions - a slight oedema detectable by palpation - only occurred at the injection point of animals 5 and 6 after the 1 s" vaccination. After the piglets were slaughtered macroscopically detectable inflammations were proved to exist at the points of injection around the injection channel, which were filled with necrotic material, except for the animals 1 and 3. When the piglets 1 and 3 were histologically examined only a slight connective-tissue proliferation (angioplasts with infiltered lymphozytes and histiocytes) was identified whereas the injection channel filled with necrotic material was surrounded by a connective-tissue capsule in all of the other piglets. An inflammation with infiltered lymphozytes and histiocytes was observed in the connective-tissue capsule. Effectiveness of the vaccine formulations This test demonstrated that the recombinant Stx2eB, after an intramuscular application in 6 weeks old weaned piglets, is identified by the immunity system of the animals and will induce an immunizing response, the production of specific immune globulins. The existence of such antibodies was demonstrated by Elisa and Immunoblot. The intensity of the immunizing response seems to be dependent on the choice of the vaccine formulation used. The best results amongst the testing conditions chosen were achieved by a W/O emulsion (e.g. using iFA). The results are reported in detail in Table 2. ... /115 >4 >N (N. C14 C1 4 (N (N4 (N (N C1 CI C )0 C o 4 4) c O"4) C 0 th u OILo Cd0. oa " ca 0 -0 4. d QE > uC .a E .0 :3. 0 X *9 , 0 F - ;- - 7:1 4) ( 0 >) 4 u C4- 4) V 0) 0 - s - a - - - w -c 4) - S en C4 l*t 10 C-cc C/) 0%0 C) n 00 00 % 0 +C>+ Ci- 00 + +~ + 'c n1 o r- 60 1% CnoT cn< en %0 ~ - ~ + %0' + Cs en + + 0 - i= C- + + + + 4o -~ ~ 00 0 ( 4)~ ~ 1 100 0+ % ' 0 < -C --- - CD -- - - 00L) # I~+ ++ + + + <D0e '-q -4 IC C% -CI -'4 4- %0 <.C. 00 ~c~N 0 Ln + -~ 00 -. + m C.) '-~00 oo B -~ + 4- < <04 -d > ~.O fl UC.) ~-C- -17 Deposited microorganism The E.coli strain Cux-Stx2eB was deposited under the original designation Cux-SLT-IIe-B with the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Mascheroder Weg Ibm D - 38124 Braunschweig It was given the receipt No. DSM 12721 by the office of lodgement. ... /18

Claims

1. A recombinant fusion protein including a sub-genic Stx2e fragment of the 2e Shiga toxin (Stx2e) in a fusion with a terminal tag the size of which approximately corresponds to the size of the fragment or a fraction of the fragment.

2. The recombinant fusion protein according to claim 1 wherein the sub-genic Stx2e fragment is a B sub-unit (Stx2eB) of the 2e Shiga toxin.

3. The recombinant fusion protein according to claim 1 or 2 wherein the size of the terminal tag is 1 kDa, as a maximum.

4. The recombinant fusion protein according to any one of claims 1 to 3 which has an amino terminal His tag.

5. The recombinant fusion protein according to any one of claims 1 to 4 which has a plurality of crosslinked fusion proteins.

6. A (vaccine) substance composition for various applications in conjunction with the oedematose of animals, particularly those of mammals, specifically pigs, having a sub-genic fragment of the 2e Shiga toxin in a fusion with a terminal tag the size of which approximately corresponds to the size of the fragment or a fraction of the fragment B.

7. The (vaccine) substance composition according to claim 6 wherein the sub genic Stx2e fragment is a B sub-unit (Stx2eB) of the 2e Shiga toxin. ... /19 - 19

8. The (vaccine) substance composition according to claim 6 or 7 wherein the size of the terminal tag is 1 kDa, as a maximum.

9. The (vaccine) substance composition according to any one of claims 6 to 8 which has an amino terminal His tag.

10. The (vaccine) substance composition according to any one of claims 6 to 9 which has a plurality of crosslinked fusion proteins.

11. The (vaccine) substance composition according to any one of claims 6 to 10 which comprises one additional antigen or several additional antigens.

12. The (vaccine) substance composition according to claim 11 wherein the one additional antigen or the several additional antigens are selected from the group comprising a Pasteurella multocida bacterin including a cell-bonded toxoid, a Bordetella bronchiseptica bacterin, an Erysipelothrix rhusiopathiae antigen, one or more soluble non-cell toxoids of type D Pasteurella multocida and/or Escherichia coli and/or Clostridium perfringens, disactivated whole cells of type A or D Pasteurella multocida, cultures of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli, Clostridium perfringens, Streptococcus suis, Mycoplasma hyopneumoniae as well as Porcine Reproduction and Respiratory Syndrom virus, influenza virus, Pseudorabies virus, and Porcine Circoviruses I and II.

13. The (vaccine) substance composition according to any one of claims 6 to 12 which comprises the 2e Stx-IIe fragment of the 2e Shiga toxin (Stx2e) in a fusion with a terminal tag and/or which comprises at least one additional ... /20 - 20 antigen each in an immunogenic amount for the vaccination of pigs against the oedematose of the pigs or against the oedematose of the pigs and other viral and/or bacterial infections.

14. (Vaccine) substance compositions according to any one of claims 6 to 13 in compositions and amounts such that if pigs are vaccinated sequentially or simultaneously they immunize them against oedematose of the pigs or against the oedematose of the pigs and other viral and/or bacterial infections.

15. A (vaccine) substance composition having a sub-genic Stx2e fragment of the 2e Shiga toxin (Stx2e) in a fusion with a terminal tag, particularly according to any one of claims 6 to 12, in a W/O/W emulsion, in an O/W emulsion, in a W/O emulsion or in an aqueous suspension.

16. The vaccine) substance composition according to claim 15 including an incomplete Freund adjuvant (iFA).

17. A plasmid containing DNA which encodes a fusion protein according to any one of claims 1 to 6.

18. An E.coli strain transformed by a plasmid according to claim 17.

19. An E.coli strain according to claim 18, deposited with the DSZM - Deutsche Sammlung von Mikroorganismen und Zellkulturen under the number DSM

12721.

20. A method for the recombinant preparation of a sub-genic fragment of the Shiga toxin (Stx2e) in a fusion with a terminal tag wherein a sub-unit from the Stx2e ...

/21 -21 operon is cloned into a suitable vector system, the resultant recombinant plasmid is transformed into an E. coli strain, the resultant expression system is induced, and the fusion protein is expressed and purified. 21. The method according to claim 20 wherein the sub-genic fragment is a B sub unit (Stx2eB) of the 2e Shiga toxin.

22. The method according to claim 20 or 21 wherein the size of the terminal tag is 1 kDa, as a maximum.

23. The method according to claim 20 to 22 wherein the terminal tag is an amino terminal His tag.

24. The method according to any one of claims 20 to 23 wherein the expression culture is subjected to a lytic buffer treatment.

25. The method according to any one of claims 20 to 24 wherein the expression culture is subjected to a treatment in a French Press or by means of ultrasonic sound.

26. The method according to any one of claims 20 to 25 wherein the expression culture, after being treated by the French Press or by ultrasonic sound and/or a lytic buffer, is submitted to an affinity chromatographic purification.

27. The method according to claim 26 wherein purification is performed by means of a FPLC. .../22 - 22

28. The method according to any one of claims 20 to 27 wherein the purified fusion protein undergoes crosslinking.

29. A method according to any one of claims 20 to 28 wherein the fusion of spleen cells of mice immunized by using the recombinant fusion protein with myelom cells is used for producing hybridoma clones for the preparation of anti-Stx2eB immune globulins.

30. The method according to claim 29 where the antibodies produced by means of the hybridoma clones are employed for the in-process control for the production of the recombinant fusion proteins.

31. The method according to claim 29 or 30 wherein the antibodies produced by the hybridoma clones are used for an affinity chromatographic purification method for the Stx2e holotoxin. .../23