Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Patent
1995-08-29
2000-08-22
Housel, James C.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
4241841, 4242341, 435 712, 435 41, 530825, 530820, A61K 3900, A61K 3902, A61K 3910, C12P 100
Patent
active
06106842&
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP94/00597 filed Feb. 28, 1994 which claims priority to foreign application 9304398.0 filed in Great Britain on Mar. 4, 1993.
The present invention relates to a novel process for the isolation of cell proteins having utility as antigenic factors of component or acellular vaccines. In particular, the invention relates to a novel process for the extraction of outer membrane proteins of bacterial organisms, for example the outer membrane protein of Bordetella pertussis, which has a molecular weight of approximately 69,000 Daltons and is generally referred to as the 69 kD protein of Bordetella pertussis, or pertactin.
Whooping cough, or pertussis, is a highly-infectious disease which primarily affects children. In addition to causing respiratory complications, whooping cough may result in nerve damage and a high incidence of mortality, particularly in children from low socioeconomic groups and in newborn infants who do not possess maternal anti-pertussis antibodies. The etiologic agent of pertussis is the Gram-negative coccobacillus, Bordetella pertussis. The bacteria are believed to invade the respiratory tract and induce a toxic state which remains even after their disappearance, several days later.
The disease is currently controlled through immunisation with "whole cell" vaccine prepared by growing the Bordetella pertussis organism in fermenters and then inactivating the resulting cells by heat treatment and/or addition of chemical agents. Although the World Health Organisation presently recommends the immunisation of infants to prevent the incidence and spread of pertussis, concern has arisen over the reported adverse events resulting from various vaccine formulations. The consequent reduced usage of conventional B. pertussis vaccine has resulted in an increase in the incidence of pertussis infections. The need for a pertussis vaccine which avoids the reported adverse events from whole cell vaccine is recognised. Considerable research effort has accordingly been put into the development of an efficaceous acellular vaccine comprising a small number of highly-purified antigenic proteinaceous components.
A number of antigens have been proposed as acellular vaccine components, including for example lymphocytosis promoting factor (LPF), also known as histamine sensitising factor, islet activating protein or, more commonly, pertussis toxin (PT); filamentous hemagglutinin (FHA); and fimbrial agglutinogens.
A further potential antigen is one of the outer membrane proteins of the bacterium, having a molecular weight of approximately 69,000 Daltons (pertactin) found in all virulent strains of B. pertussis. The B. pertussis 69 kD protein is immunologically-related to similar proteins having slight differences in electrophoretic mobility which are produced by the human pathogen B. parapertussis and the animal pathogen B. bronchiseptica. Although the 69 kD protein is secreted in relatively small amounts into the fermentation broth during the cultivation of B. pertussis, the majority of it is found attached to the cell membrane, from which it may be readily extracted. Published procedures for the extraction and purification of the 69 kD protein do not however allow for large-scale commercial production of a highly-purified and stable antigen.
EP-0 162 639 describes acid-glycine extraction of the cells followed by several purification steps culminating in an affinity chromatography separation using a specific monoclonal antibody. The protein obtained has been reported to have both poor stability and adenylate cyclase activity, and the downstream purification procedure is not suitable for large-scale production.
Brennan et al. (Infection and Immunity 56, 3189-3195, 1988) describe a further method whereby protein is released from cells by heat treatment and a protein extract is obtained which is purified by chromatography on fetuin-Sepharose and a monoclonal antibody affinity column.
U.S. Ser. No. 7/308,864 describes extraction and purification involving heat treatment and centrifugation,
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Capiau Carine
Comberbach Martin
Petre Jean
Roelants Piet
Housel James C.
Ryan V.
SmithKline Beecham Biologicals
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