Europaisches Patentamt (19) European Patent Office Office europeeneen des brevets EP 0 892 054 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) |nt C|.6: C12N 15/31, A61 K 39/08, 20.01.1999 Bulletin 1999/03 C07K 1 4/33, C1 2N 1/21 (21) Application number: 98202032.3 (22) Date of filing: 17.06.1998 (84) Designated Contracting States: • Waterfield, Nicolas Robin AT BE CH CY DE DK ES Fl FR GB GR IE IT LI LU Cherry Hinton, Cambridge CB1 4YR (GB) MC NL PT SE • Frandsen, Peer Lyng Designated Extension States: 2840 Holte (DK) AL LT LV MK RO SI • Wells, Jeremy Mark Fen Ditton, Cambridge CB5 8ST (GB) (30) Priority: 20.06.1997 EP 97201888 (74) Representative: (71) Applicant: Akzo Nobel N.V. Ogilvie-Emanuelson, Claudia Maria et al 6824 BM Arnhem (NL) Patent Department Pharma N.V. Organon (72) Inventors: P.O. Box 20 • Sergers, Ruud Philip Antoon Maria 5340 BH Oss (NL) 5831 MZ Boxmeer (NL) (54) Clostridium perfringens vaccine (57) The present invention relates to detoxified im- genes encoding such p-toxins, as well as to expression munogenic derivatives of Clostridium perfringens p-tox- systems expressing such p-toxins. Moreover, the inven- in or an immunogenic fragment thereof that have as a tion relates to bacterial expression systems expressing characteristic that they carry a mutation in the p-toxin a native p-toxin. Finally, the invention relates to vaccines amino acid sequence, not found in the wild-type p-toxin based upon detoxified immunogenic derivatives of amino acid sequence. The invention also relates to Clostridium perfringens p-toxin, and methods for the preparation of such vaccines. < o CM O) 00 o a. LU Printed by Jouve, 75001 PARIS (FR) EP 0 892 054 A1 Description The present invention refers to detoxified immunogenic derivatives of Clostridium perfringens p-toxin, DNA en- coding such derivatives, Clostridium perfringens bacteria comprising DNA encoding such derivatives, Grampositive 5 bacterial expression systems comprising DNA encoding such derivatives, non-Clostridium perfringens-based Gram- positive bacterial expression systems expressing wild-type p-toxin, vaccines for combating Clostridium perfringens based thereon, methods for the preparation of native Clostridium perfringens p-toxin, methods for the preparation of detoxified immunogenic derivatives of Clostridium perfringens p-toxin and to methods for the preparation of vaccines for combating Clostridium perfringens. Clostridium perfringens (also known as C. welchii) is a species of the large 10 genus Clostridium. All bacteria belonging to this genus are spore-forming anaerobic Grampositive bacilli. The species C. perfringens can be subdivided in subspecies. Five subspecies have been described. These subspecies are generally known as "type" A-E. All subspecies produce several toxins, both major and minor toxins. The four major toxins are the a, p, e and x toxin. All C. perfringens types produce the a-toxin. The p-toxin is produced by C. perfringens types B and C. In addition, a range of minor toxins is produced by all C. perfringens types It is mainly due to the presence of is one or more of these various toxins in the five C. perfringens types, that all C. perfringens species are pathogenic. Type A is known to be pathogenic for both man and pigs. Type B is mainly pathogenic for lamb, sheep and goat, and causes "lamb dysenteria" and haemorragic enteritis. Type C is pathogenic for man, sheep, calf, lamb, pig, and bird. It is the cause of "struck1, haemorragic enteritis, necrotic enteritis and enterotoxemia. As mentioned above, both C. perfringens type B and type C are known to produce the p-toxin. The p-toxin is known 20 to play the major role in the pathogenesis of necrotic enteritis in both man and animal. In man, this disease has been termed pigbel (Johnson et al.: Lancet ii: 496-500, (1987)). In animals, necrotic enteritis has been described in calves, lambs and pigs (Hauschild, A.H.W. in S. Kadis, T.C.Montie, (ed.) Microbial toxins, p. 159-188. Academic press, Inc, New York (1971) and Willis, T.A. Anaerobic bacteriology: clinical and laboratory practice, 3rd ed. Butterworths, London (1977). 25 The p-toxin from Clostridium perfringens has been isolated in pure form (Sakurai et al., Infect. & Immun. 1 8: 741 -745 (1977), Sakurai et al., Toxicon 25: 1301-1310 (1987)). Much is still unknown about the biophysical properties of the toxin and thus about its mode of action. Due however to the fact that it could be obtained in a purified form the toxicity of p-toxin could be clearly demonstrated in animals. The lethal toxicity of purified p-toxin for e.g. mice and guinea-pigs was shown by Sakurai et al. (Infect. & Immun. 21: 678-680 (1978), Sakurai et al., Toxicon 25: 1301-1310 (1987)). 30 Recently, the nucleic acid sequence of the Clostridium perfringens p-toxin has been elucidated by Hunter et al. (Infect. & Immun. 61 : 3958-3965 (1 993)). The nucleic acid sequence revealed the size of the p-toxin protein to be about 35 kD. Due to the fact that the role of Clostridium perfringens p-toxin from type B and C in pathogenesis is of such paramount importance, much effort has been put in the development of immunity against this toxin. Immunity against the p-toxin is sufficient to protect against Clostridium perfringens type B and type C infection. The only way of inducing immunity 35 against the p-toxin is, to administer the toxin to the animal to be protected. It is however obvious that the toxin must be given in a detoxified form, since otherwise administration would lead to severe illness or death of the animal to be vaccinated. Vaccines based on detoxified p-toxin, also called p-toxoid, were available already around 1960 (e.g. G.B.Pat. No. 901 ,433, and U.S.Pat. No 3,288,680). 40 All currently available vaccines based on inactivated Clostridium perfringens p-toxin have however several important drawbacks. In the first place, all p-toxin-based vaccines comprise chemically detoxified, mainly formalin-detoxified, p-toxin and it was shown through the years that it is very difficult to standardise these chemical detoxification processes. The classical chemical methods for detoxification of proteins have the disadvantage that they alter the overall structure of the protein 45 in a fairly random manner. And as a result, during the process of chemical detoxification the immunogenic properties of the p-toxin also rapidly decrease. This can be seen e.g. from graph 1 and 2: in graph 2 it is shown, that at least 1% formalin, a very commonly used inactivation-compound, is necessary to detoxify the p-toxin under certain standard conditions. From graph 1 and 2, it can be seen however, that the antigenicity titre decreases dramatically with an 50 55 2 EP 0 892 054 A1 formalin vs titre 100 1 — — !■- ' — f- 70 f r P 10 75 Jf- 20 3% 2% 1% 0,5% 0,25% %formalin 25 • 30 Mg/ml - 6 Mg/ml • 1 .2 |jg/ml 0.24 pg/ml Graph 1 30 35 40 45 50 55 3 EP 0 892 054 A1 % dead animals in the groups 100 sa 80 CO E E 60 CO "O CD 20 0 3% 2% 1% 0,5% 0,25% formalin % I 30 Mg/ml 6 Mg/ml 1.2 Mg/ml ^VJ 0.24 Mg/ml Graph 2 increasing amount of formalin. A full titre can not be obtained anyway, because even the lowest amount of formalin needed for detoxification (graph 2) already gives a decrease of the titre. This implicates that there is only a very narrow band, in which both detoxification and a reasonable titre and thus immunogenicity can be obtained. Given this very narrow band, and the fact that there are at least three variables; time of detoxification, temperature and precise formalin- concentration, it is clear that it is very difficult to reproducibly produce detoxified p-toxin. Another approach for the detoxification of p-toxin is therefore highly wanted. No acceptable alternative has been found until now, for the following reason: the delicate balance between a sufficient level of detoxification and remaining immunogenicity implicates a close link between, on the one hand, the structure of the protein and, on the other hand, the biological properties of the protein. It could therefore not be expected to be possible to change the protein structure thereby detoxifying the protein, without at the same time significantly impairing the immunogenic characteristics of the protein. It is one of the merits of the present invention that it discloses for the first time a way to avoid the above-mentioned problem: using genetical manipulation techniques, specific and relatively large amino acid regions were surprisingly found, that can be mutated to provide the desired non-toxic derivatives of p-toxin without significantly impairing its necessary immunogenic properties. Thus, in one embodiment, the invention relates to detoxified immunogenic derivatives of Clostridium perfringens P-toxin or an immunogenic fragment thereof, carrying at least one mutation in its amino acid sequence, not found in wild-type p-toxin. An immunogenic fragment thereof is understood to be a fragment that, although not comprising the full length amino acid sequence of the derivative of the p-toxin, still comprises those regions of the derivative that are capable of inducing a protective immune response in the host animal. A mutation is understood to be a change in the amino acid sequence of the derivative p-toxin in comparison to the amino acid sequence of the wild-type p-toxin. The mutation is a substitution, deletion, insertion or inversion, or a combination thereof.