Antigenic preparations and isolation of such preparations

Details Antigenic preparations and isolation of such preparations Antigenic preparations and isolation of such preparations

1999-06-17

2001-04-03

Smith, Lynette R. F. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S234100, C424S240100, C424S253100, C424S184100, C530S350000, C530S825000

Reexamination Certificate

active

06210685

ABSTRACT:

The present invention relates to antigenic preparations for use in acellular vaccines against
Bordetella pertussis
, and to a method for the isolation of such preparations.
Bordetella pertussis
causes a serious and debilitating disease in humans, children being particularly susceptible, which is kept under control in the developed countries by large scale immunisation programmes. It has been found that immunisation is a very important factor in the reduction of the disease and that failure to vaccinate can lead to increased incidence of the disease. In practically all areas, immunisation is effected using a whole cell
B. pertussis
vaccine which has been found to be relatively effective in preventing the disease. However, it has been recognised that whole cell vaccines may suffer from several draw-backs. Thus, for example, in about 1 in every 10,000 children inoculated, clinical symptoms occur which may include fever, local reactions and persistent screaming. Further, it would appear that some batches of whole cell vaccine provide no protection at all while still being associated with the possibility of undesirable side-effects.
With the currently low occurrence of the disease in developed countries with immunisation programmes, the benefit/risk ratio is poorly defined, and many clinicians believe that the risks of inoculation outweigh the benefits gained by immunisation. As a result, many children are not inoculated and there is then a serious risk of a pandemic of whooping cough. Considerable research effort has, therefore, been directed toward the development of improved pertussis vaccines and especially acellular vaccines which lack the components associated with the toxic effects of the whole cell vaccines hitherto used whilst incorporating those components necessary to protect against the disease.
The search for a safer, effective, acellular
B. pertussis
vaccine has been hampered in the past by the paucity of information regarding the identity and mechanisms of action of the pathogenic, toxic and protective moieties of
B. pertussis
contained in the whole cell vaccines. Work has, therefore, been concentrated on isolating and purifying the 20 or more surface antigens of the
B. pertussis
organism and characterising their ability to induce immune reactions (see, for example, J. Am. Med. Soc., 248 (1) 22-23). Examples of antigens that have been suggested for investigation include lymphocytosis promoting factor (pertussis toxin/LPF) filamentous haemagglutinin (FHA), lipopolysaccharide (LPS), agglutinogens, dermonecrotic toxin (DNT), heat labile and heat stable toxins, polymorphonuclear leukocyte inhibitor factor, adenylate cyclase and other surface components (Pertussis Vaccine Workshop, Feb. 11, 1982, Bureau of Biologics, U.S.A.). Other proposed candidate antigens for investigation include tracheal cytotoxin and various outer membrane proteins.
An early extract vaccine was developed by L. Pillemer (Proc. Soc. Exp. Biol. Med. (1950) 75, 704-705) which was based on disrupted
B. pertussis
cells and found to provide protection but was not adopted commercially in view of the toxicity of the preparation.
Examples of more recent
B. pertussis
extract vaccines that have been suggested include those described in U.K. Patent Specification 2 083 358A (Takeda) involving removal of endotoxin from culture supernatants; French Patent Specification 2 047 886 (Institut Merrieux) involving extraction of a microbial suspension with an anionic surfactant; and Japanese Patent Specification 58-222032 (Teijin) which comprises a sub-unit protein based on pertussis toxin (LPF).
Much of the work carried out on acellular pertussis vaccines is concentrated on the possibility of basing such a vaccine on LPF. However, it is believed that most (if not all) of the adverse effects hitherto observed to be associated with pertussis vaccination are related to the toxin. In combination with tetanus or diptheria toxoid and LPS, it is able to induce experimental encephalopathy in susceptible mice (L. Steinman, et al. Nature (1982) 299, 738-740; Redhead et al., Workshop on
B. pertussis
, Nat. Instl. of Biol. Standards & Controls, Holy Hill, Hampstead, London, 1983). Thus, LPF may, possibly, be responsible for brain damage should such complications occur after vaccination.
It has now been discovered that certain proteinaceous material, associated with adenylate cyclase activity, as hereinafter described, found in the cultures of
B. pertussis
, is capable of providing protection against challenge by
B. pertussis
when administered to experimental animals. This discovery that the proteinaceous material usually associated with adenylate cyclase activity is a major protective antigen against
B. pertussis
permits the preparation of vaccine formulations comprising antigenic preparations which are free from, or contain reduced amounts of, other known
B. pertussis
components which may be responsible for the toxic side-effects demonstrated by whole cell vaccines.
The term ‘proteinaceous material associated with adenylate cyclase activity (abbreviated to ‘ACAP’ hereinafter) is used herein to refer to proteinaceous material which is extracted together with adenylate cyclase activity when extraction of the adenylate cyclase activity is performed using an aqueous, acidic (pH3) solution of glycine (0.25 M). The ACAP as defined above may comprise the adenylate cyclase enzyme per se or a binding protein for the enzyme.
Adenylate cyclase activity was assayed by the method of Hewlett, E., and Wolff, J. (J. Bacteriol. (1976) 127, 89-898).
In a first feature of the present invention is provided a vaccine formulation for protection against
B. pertussis
which includes an antigenic preparation derived from
B. pertussis
comprising ACAP, optionally toxoided e.g. using formalin, gluteraldehyde or &bgr;-propiolactone, together with a pharmaceutically acceptable carrier therefor.
In more detail the ACAP may be detected by isoelectric focussing as two bands, one having an isoelectric point (pI) of, about 7.0, the other (diffuse) band having an isoelectric point of 7.2-7.4. Adenylate cyclase activity was associated almost entirely with the neutral band (pI=7.0) but monoclonal antibodies to ACAP bound both bands strongly.
The ACAP in the above-mentioned preparations generally has a relative molecular weight of about 67,000 to 73,000, particularly 69,000, and an isoelectric point of 7.0-7.4 under preparative conditions as described infra.
By “relative molecular weight” is meant the apparent molecular weight as determined by 12% (w/w) polyacrylamide gel electrophoresis and standard molecular weight markers. The molecular weight of the antigenic proteins of the invention may thus be conveniently determined by the techniques described by U.K. Laemmli,
Nature,
1970, 227, 680-685. Convenient standard molecular weight markers include, for example, bovine serum albumin, chymotrypsinogen A and ribonuclease.
Amino acid analysis has also shown that ACAP contains an unusually high proportion of proline, such that the proline: glutamic acid ratio is about 1:1 and this feature serves to distinguish ACAP from other
B. pertussis
proteins. A further distinguishing characteristic of ACAP is the fact that it cannot be detected by radio-iodination of its tyrosine residues by either the Chloramine T or the Iodogen methods.
According to a preferred embodiment of the present invention the above-mentioned ACAP is proteinaceous material which is characterised as having one or more of the following properties:
(i) a ratio of proline to glutamic acid of substantially 1:1;
(ii) the tyrosine residues are not iodinatable;
(iii) substantially free from intracellular,
B. pertussis
material;
(iv) a relative molecular weight of 67,000 to 73,000;
(v) an isoelectric point of 7.0 to 7.4, and
(vi) being acid-labile below a pH of about 3.
The above-mentioned antigenic preparations for use in the vaccine formulations according to the invention may, if desired, contain minor quantities of other antigenic compounds, in addition to the ACAP, for exampl

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