Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1998-09-11
2002-06-11
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S192100, C424S197110, C424S234100, C424S236100, C424S239100, C424S247100, C530S350000, C530S403000, C530S820000, C530S825000
Reexamination Certificate
active
06403094
ABSTRACT:
The present application is a U.S. national phase of PCT/GB97/00660, filed Mar. 11, 1997, which claims the benefit of GB 9605222.0, filed Mar. 12, 1996.
The present invention relates to novel peptides capable of eliciting an immunological response that is protective against
Clostridium perfringens
epsilon toxin in man or animals. It relates to the production of these peptides and to pharmaceutical compositions containing them, Preferred agents enable prophylaxis and treatment of
Clostridium perfringens
induced disease states in both humans and other animals.
Clostridium perfringens
(
C. perfringens
) is ubiquitous in the environment and has been found in the soil, decaying organic matter and as part of the gut flora in man and animals. Different strains of
C. perfringens
can be assigned to one of five biotypes (A-E) depending on the spectrum of types produced see McDonel, J. L. (1986); Toxins of
Clostridium perfringens
types A,B,C,D and E.
In Pharmacology of Bacterial Toxins
; F. Dorner and J. Drews, eds. (Oxford: Pergamon Press), pp. 477-517. The epsilon toxin is produced by
C. perfringens
types B and D but not by types A, C or E see Brooks, M. E., Sterne, M., and Warrack, G. H. (1957); A reassessment of the criteria used for type differentiation of
Clostridia perfringens. J. Pathol. Bacteriol
. 74, 185-195
. C. perfringens
types B and D have a limited host range being mainly isolated from goats and cattle and rarely from man, Smith, L. D. and Williams, B. L. (1984);
The pathogenic anaerobic bacteria
(Springfield, Ill.: Charles C. Thomas). They are responsible for producing severe and rapidly fatal enterotoxaemia:
C. perfringens
type B enterotoxaemia infection of lambs causes lamb dysentery while type D enterotoxaemia produces pulpy kidney disease in sheep and lambs. Mortality rates in both cases may be as high as 100%. Neither disease is infectious, but sporadic outbreaks occur when the microbial balance of the gut is disrupted, for example after antibiotic treatment or due to changes in diet. Pulpy kidney disease is often associated with a change from a poor to a rich diet accompanied by excessive over-eating, Bullen, J. J. (1970); Role of toxins in host-parasite relationships.
In Micribial toxins volume
1. S. Ajl, S. Kadis, and T. C. Montie, eds. (New York: Academic Press), pp. 233-276. Such over-eating causes considerable quantities of undigested, starch-rich food to pass from the rumen into the small intestine. The nutritious anaerobic environment this produces allows the multiplication of
C. perfringens
resulting in up to 10
9
cfu per g of ileal contents and high concentrations of epsilon toxin Bullen, J. J. and Scarisbrick, R. (1957); Enterotoxaemia of sheep: experimental reproduction of the disease;
J. Pathol. Bacteriol
. 73, 494-509. Several vaccines exist for the prevention of
C. perfringens
enterotoxaemia. The vaccines are based on formaldehyde-treated cell filtrates or whole cell cultures. The vaccines confer a high degree of protection in animals Stephen, J. and Pietrowski, R. A. (1986); Bacterial toxins (England: van Nostrand Reinhold (UK) Co. Ltd.); however, the immunogenicity of the epsilon toxin in the preparations has been reported to be variable and a more defined and consistent vaccine is preferable. Immunity to a single epitope on the toxin has been shown to be sufficient to protect against purified epsilon toxin and
C. perfringens
infection, Percival, D. A., Shuttleworth, A. D., Williamson, E. D., and Kelly, D. C. (1990), Anti-idiotypic antibody-induced protection against
Clostridium perfringens
type D;
Infect. Immun
. 58, 2487-2492.
Epsilon toxin is produced by
C. perfringens
types B and D as a relatively inactive prototoxin of 311 amino acids with a molecular weight of 32,700, Worthington, R. W. and Mulders, M. S. (1977); Physical changes in the epsilon prototoxin molecule of
Clostridium perfringens
during enzymatic activation;
Infect. Immun
. 18, 549-551. Proteolytic cleavage of 13 or 14 basic amino acids from the amino terminal of the prototoxin results in the production of the mature toxin with a molecular weight of 31,200 Worthington and Mulders, 1977; Hunter, S. E., Clarke, I. N., Kelly, D. C., and Titball, R. W. (1992); Cloning and nucleotide sequencing of the
Clostridium perfringens
epsilon-toxin gene and its expression in
Escherichia coli; Infect. Immun
. 60, 102-110. Activation also results in a marked shift in pI from 8.02 (prototoxin) to either 5.36 (fully active toxin) or 5.74 (partially active toxin) and a significant change in conformation (Worthington and Mulders, 1977; Habeeb, A. F., Lee, C. L., and Atassi, M. Z. (1973); Conformational studies on modified proteins and peptides, VII; Conformation of epsilon-prototoxin and epsilon-toxin from
Clostridium perfringens
; Conformational changes associated with toxicity;
Biochim. Biophys. Acta
322, 245-250). A complication is that the activation of the prototoxin seems to produce several isoforms with a range of specific activities between that of the prototoxin and the mature toxin (Habeeb, A. F. (1975); Studies on epsilon-prototoxin of
Clostridium perfringens
type D. Physicochemical and chemical properties of epsilon-prototoxin;
Biochim. Biophys. Acta
412, 62-69; Worthington and Mulders, 1977). More recently it has been found that the toxin itself also has several isoforms (Hunter et al., 1992). Thus activation of epsilon prototoxin may be a multi-step process, possibly with multiple proteolytic cleavages and post-translational modifications such as deamination and phosphorylation resulting in the production of the heterogeneous mature toxin (Hunter et al., 1992).
Epsilon toxin is usually obtained from a type D strain of
C. perfringens
and has been purified either individually or in combination by methanol precipitation, ammonium sulphate precipitation, column chromatography, size exclusion and various forms of ion exchange chromatography (Verwoerd, D. W. (1960);
Isolation van die protoksien van Clostidium welchii type D. J. S. Afr. Vet. Med. Assoc
. 31, 195-203; Habeeb, A. F. (1969); Studies on epsilon-prototoxin of
Clostridium perfringens
type D. I. Purification methods: evidence for multiple forms of epsilon-prototoxin;
Arch. Biochem. Biophys
. 130, 430-440; Worthington, R. W., Mulders, M. S., and Van Rensburg, J. J. (1973);
Clostridium perfringens
type D epsilon prototoxin. Some chemical, immunological and biological properties of a highly purified prototoxin;
Onderstepoort. J. Vet. Res
. 40, 143-149; Payne, D. W., Williamson, E. D., Havard, H., Modi, N., and Brown, J. (1994); Evaluation of a new cytotoxicity assay for
Clostridium perfringens
type D epsilon toxin;
FEMS Microbiol. Lett
. 116, 161-167).
Traditionally, the activity of purified epsilon toxin has been determined in mouse lethality tests (Habeeb, A. F. (1969); Studies on epsilon-prototoxin of
Clostridium perfringens
type D. I. Purification methods: evidence for multiple forms of epsilon-prototoxin;
Arch. Biochem. Biophys
. 130, 430-440; Worthington, R. W., Mulders, M. S., and Van Rensburg, J. J. (1973);
Clostridium perfringens
type D epsilon prototoxin. Some chemical, immunological and biological properties of a highly purified prototoxin;
Onderstepoort. J. Vet. Res
. 40, 143-149). The mature toxin is highly toxic with an LD
50
in mice of <100 ng when administered intravenously (Payne, D. W., Williamson, E. D., Havard, H., Modi, N., and Brown, J. (1994); Evaluation of a new cytotoxicity assay for
Clostridium perfringens
type D epsilon toxin;
FEMS Microbiol. Lett
. 116, 161-167). As the basis of an alternative assay for epsilon toxin activity, it has been found that the Madin Darby Canine Kidney (MDCK) cell line was sensitive to C. perfringens type D culture filtrates (Knight, P. A., Burnett, C., Whitaker, A. M., and Queminet, J. (1986); The titration of clostridial toxoids and antisera in cell culture;
Develop. biol. Standard
. 64, 129-136). It was demonstrated that the lethal and dermonecrotic effects of the toxin observed in rabbits and its cytopathic act
Havard Helen L
Oyston Petra C F
Payne Dean W
Titball Richard W
Williamson Ethel D
Navarro Mark
Nixon & Vanderhye P.C.
The Secretary of State for Defence in Her Britannic Majesty&apos
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