Method of separating protective components of Bordetella pertuss

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...

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530412, 530414, 530415, 530416, 530418, A61K 3910, C07K 100, C07K 114, C07K 136

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060512407

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BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to a method of separating protective components of Bordetella pertussis. The pertussis component vaccine can be produced by suitably mixing the protective components separated by the method of the present invention.


BACKGROUND ART

Vaccines are widely used to prevent communicable diseases. Pertussis, a communicable respiratory disease caused by infection with Bordetella pertussis, is likely to severely affect patients, especially infants, due to apneic cough with occasional spasm. To cope with this disease, it has been common practice to use whole cultured cells of Bordetella pertussis after inactivation (inactivated vaccine). However, localized reactions at the site of vaccination and side reactions, such as fever, have been reported, creating a social urge to solve this problem. To solve this problem, there have been a large number of attempts of using protective components separated from Bordetella pertussis as vaccine. For example, acellular pertussis vaccine (ACP vaccine), prepared by extracting protective proteins, such as pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), from Bordetella pertussis cells, and removing endotoxin (ET), is being into practical application, but is not fully satisfactory, due to the drawbacks described below.
Pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), all protective components of Bordetella pertussis already in practical application with validated efficacy, are separated by respective methods.
Pertussis toxin (PT) can be separated by affinity chromatography using human haptoglobin as a ligand [Biochimica et Biophysica Acta, Vol. 580, p. 175 (1979)]. However, human haptoglobin can be contaminated with hepatitis virus, because it is collected from human blood; the same applies when animal sera are used. Another available method is affinity chromatography using denatured ceruloplasmin as a ligand (Japanese Patent Unexamined Publication No. 62135/1987). Although this method is free of the problem of viral contamination, some problems arise, including vaccine contamination with ceruloplasmin and the high toxicity and potential body retention of sodium thiocyanate and other eluents having protein-denaturing effect.
As for pertussis filamentous hemagglutinin (FHA), a purification method using hydroxyapatite gel is available [Infection and Immunity, Vol. 41, p. 313 (1983) and EP-A-231083, EP-A-427462, EP-A-462534; Japanese Patent Unexamined Publication Nos. 234031/1987, 169893/1992, 368337/1993). However, it takes long time for column operation, and is uneconomic due to the high cost of hydroxyapatite.
As for pertactin (PRN, 69K-OMP), affinity chromatography using a mouse serum as a ligand is available [Infection and Immunity, Vol. 56, p. 3189 (1988)], but has the same drawbacks as above.
As for pertussis fimbriae (FIM), Bordetella pertussis cell extract is purified by salting-out: with ammonium sulfate and magnesium chloride [Infection and Immunity, Vol. 48, p. 442 (1985)], but this method is poor in vaccine production efficiency due to low yield.
There is a method of preparing Gram-negative bacterial vaccine by adosorbing with the aluminum hydroxide gel (WO 93/10216). This method needs the large amount of the aluminum hydroxide gel, which adsorbs both the protective components and the endotoxin originated in Gram-negative bacteria. The vaccine obtained by the method of WO93/10216 has a danger of side effects, such as fever and endotoxin-shock, by the endotoxin released into body because of the diluted vaccines.
As for the pertussis vaccine production included as the components mixture without separating each protective component originated in Bordetella pertussis, a method of using calcium phosphate gel is available (EP-A-291968, Japanese Patent Unexamined Publication No. 52726/1989). However, this method formed the calcium phosphate in the presence of a 1M sodium chloride does not abso

REFERENCES:
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patent: 4784589 (1988-11-01), Robinson et al.
patent: 4849358 (1989-07-01), Chazano et al.
patent: 5101014 (1992-03-01), Burns et al.
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The Journal of Medical Microbiology, vol. 35, No. 3, 1991, p. 187.
Zhang et al., "Purification and Characterization of Fimbriae Isolated from Bordetella pertussis", Infection and Immunity, vol. 48, No. 2, May 1985, p. 422-427.
Brennan et al., "Identification of a 69-Kilodalton Nonfimbrial Protein As an Agglutinogen of Bordetella pertussis", Infection and Immunity, vol. 56, No. 12, Dec. 1988, p. 3189-3195.
Sato et al., "Separation and Purification of the Hemagglutinins from Bordetella pertussis", Infection and Immunity, vol. 41, No. 1, Jul. 1983, p. 313-320.

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