Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1992-08-07
2001-04-10
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S184100, C424S184100, C424S190100, C424S236100, C424S253100, C424S240100, C424S254100, C530S350000
Reexamination Certificate
active
06214356
ABSTRACT:
The invention relates to vaccines capable of protecting man or animals against lethal infections caused by the Bordetella. It relates, in particular, to the use of vaccinating preparations developed from Bordetella or more particularly from adenylate cyclase produced by these bacteria as protective antigens against the toxic effects of the infections due to Bordetella.
It is known that the Bordetella, more particularly
Bordetella pertussis, Bordetella parapertussis
and
Bordetella bronchiseptica
, are responsible for respiratory diseases in the vertebrates.
Thus, in man,
B.pertussis
is responsible for whooping cough, an infantile disease very wide spread throughout the world.
The vaccination against whooping cough has hitherto been most usually carried out with the aid of inactivated whole bacteria.
However, such vaccines are not always devoid of toxicity in view of the fact that the virulence factors are constituted by proteins secreted by the bacteria and not by the bacteria themselves. The proteins can thus exert serious pathological effects, even after the death of the bacteria.
Among the determinants of virulence of
B.pertussis
, mention should be made of the adhesins such as the agglutinogens (AGG), the filamentous hemagglutinin (FHA) and the pertussis toxin (PTx), a cytotracheal toxin (CTT), a dermonecrotic toxin (DNT) and an adenylate cyclase-hemolysin (AC). The latter is synthesized in the form of a large precursor of 1706 residues. The amino-terminal part of the molecule bears the adenylate cyclase activity and the carboxy-terminal part possesses a strong homology with the Hly gene product of
E.coli
(hemolysin). This adenylate cyclase has the property of being activated by calmodulin.
The research for more suitable methods of prevention has led the inventors to study the role of each of the determinants of virulence and to develop an experimental model of infection mimicking the process of the natural disease.
These studies have made it possible to note the role of adenylate cyclase as cytotoxin and as protective antigen, and to develop a novel use of the vaccinating preparations based on adenylate cyclase or bacterial preparations producing adenylate cyclase.
The invention relates more particularly to the use of a bacterial vaccinating preparation developed from a given bacterium of the Bordetella genus, this use being characterized in that it is used to protect man or animals against the infections and the toxic effects caused by a bacterium of the Bordetella genus but different from that of the vaccinating preparation.
In accordance with another feature, the invention also relates to the use of a vaccinating preparation developed from an adenylate cyclase of a given bacterium of the Bordetella genus in order to protect man or animals against the infections and the toxic effects caused by a bacterium of the Bordetella genus different from that from which the adenylate cyclase is derived.
The bacteria of the Bordetella genus are selected from
B.pertussis, B.parapertussis
, and
B.bronchiseptica.
In a preferred embodiment of the invention, the vaccinating preparation is developed from
B.bronchiseptica
or from the adenylate cyclase of
B.bronchiseptica
and is used to provide protection against the infections and the toxic effects caused by
B.pertussis
or
B.parapertussis.
It should be noted that this crossed protection obtained starting from strains of
B.bronchiseptica
offers the advantage of using strains with more rapid growth than
B.pertussis
or
B.parapertussis
in order to develop vaccines against the infections caused by
B.pertussis
or
B.parapertussis.
According to another embodiment of the invention, the vaccinating preparation is developed from
B.pertussis
or the adenylate cyclase of
B.pertussis
and is used to provide protection against the infections and the toxic effects caused by
B.bronchiseptica
or
B.parapertussis.
According to yet another embodiment of the invention, the vaccinating preparation used is developed from
B.parapertussis
or the adenylate cyclase of
B.parapertussis
and is used to provide protection against the infections and the toxic effects caused by
B.pertussis
or
B. bronchiseptica.
According to yet another embodiment of the invention, the vaccinating preparation used is developed from
B.parapertussis
or the adenylate cyclase of
B.parapertussis
and is used to provide protection against the infections and the toxic effects caused by
B.pertussis
or
B.bronchiseptica.
The adenylate cyclase of the Bordetella to which reference has been made above is an adenylate cyclase such as that obtained by placing a supernatant of a bacterial culture of Bordetella in contact with an Affigel-calmodulin® gel.
More particularly, it is a preparation of adenylate cyclase such as those described in the application FR 2606789 filed on 17.11.86
It will be recalled that these preparations are characterized in that they possess a high purity and are almost completely devoid of contaminating bacterial products, in particular pertussis toxins, lipopolysaccharide (or LPS) and filamentous hemagglutinin (or FHA).
The adenylate cyclase of these preparations exists in a homogeneous form sedimenting on a sucrose density gradient with a coefficient S equal to 3.6. It exists in two structurally related, molecular forms of 45 and 43 kDa respectively.
Such preparations of adenylate cyclase possess an activity which may attain and even exceed 1600 &mgr;mole of cAMP min
−1
mg
−1
.
Such preparations may be prepared from bacterial cultures expressing the AC (adenylate cyclase), more particularly from pathogenic bacteria whose AC is capable of interfering with the AC of eucaryotic cells, by placing a supernatant of bacterial cultures expressing the adenylate cyclase and concentrated beforehand or an extract of these bacteria in contact with calmodulin.
In order to obtain the enzyme in the free form, calmodulin fixed to a support is used, then the absorbed enzyme is recovered with the aid of a denaturing agent which is removed in turn, and the preparation of the free enzyme is recovered.
The support is more particularly constituted by a material inert with respect to the preparation containing the enzyme, and is capable of retaining molecules of high molecular weight, such as a gel or a filtering material.
The filtering material is advantageously made of nitro-cellulose or a plastic material and exhibits a porosity of 0.45 -0.22 &mgr;m.
The denaturing agent is preferably urea and preferably 4 to 8.8 M.
The concentrated supernatant of the bacterial culture is obtained by subjecting a supernatant of bacterial cultures expressing the AC to one or several filtration steps with the aid of nitrocellulose filters or filters of plastic material of porosity advantageously from 0.45 to 0.22 &mgr;m, then by incubating the filters with a detergent in order to release the AC and by removing from the AC preparation the insoluble materials present.
The detergent is, for example, Triton® or NP40®.
The bacterial extract is obtained by treatment of bacterial cells expressing the adenylate cyclase with urea and recovery of the supernatant.
As a variant, an adenylate cyclase is used such as that expressed by the nucleotide sequence given in the only figure with the corresponding amino acid sequence.
It will be obvious that the bases of the nucleotide sequence under consideration may be in an order different from that found in the genes and/or that these bases may be, where appropriate, substituted provided that a probe developed from such a sequence gives a characteristic and unequivocal response with regard to the capactity to recognize the presence of a gene coding for a protein with adenylate cyclase activity.
Any nucleotide sequence which can hybridize with this chain sequence such as that obtainable by reverse enzymatic transcription of the corresponding RNA or also by chemical synthesis is also included in the framework of the invention.
The above vaccinating preparations used may or may not be combined with the FHA and/or the PTx in the s
Finnegan Henderson Farabow Garrett & Dunner
Institut Pasteur
Minnifield Nita
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