Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1990-12-26
2002-02-26
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S252100, C435S320100, C536S023700
Reexamination Certificate
active
06350612
ABSTRACT:
DESCRIPTION
The present invention relates to a cloned and sequenced ECO RI fragment of
Bordetella pertussis
chromosomal DNA containing the genes which code for the five subunits of the pertussis toxin, useful for the preparation of the pertussis toxin or of one or more subunits of the pertussis toxin.
The present invention also relates to a hybrid plasmid containing the cloned and sequenced DNA fragment or further fragments thereof and to a micro-organism transformed by the hybrid plasmid and capable of expressing the cloned DNA fragment or further fragments thereof by synthesis of the pertussis toxin or one or more subunits of the pertussis toxin.
The invention also concerns a method for the preparation of the pertussis toxin or one or more subunits of the pertussis toxin which includes the growth of the micro-organism transformed by the hybrid plasmid in a suitable culture medium.
The pertussis toxin or one or more subunits of the pertussis toxin thus obtained is useful for the preparation of vaccines and diagnostic kits.
Pertussis is an infection of the respiratory tract caused by
Bordetella pertussis
(
B. pertussis
), a Gram-negative coccobacillus which is transmitted directly through the air during a catarrhal or conclusive period from the infirmed to a susceptible healthy individual.
Pertussis may cause respiratory complications, nerve damage and high mortality, particularly in children in low socio-economic groups and in new born babies without maternal, anti-pertussis antibodies. The clinical course of pertussis includes four phases: incubation, cattarhal phase, paroxysmic phase, and a convalescent phase.
During the first two phases there are symptoms comparable to those of a common cold and the
B. pertussis
may be isolated easily from the patients.
During the paroxysmic phase, characterised by the symptoms of pertussis itself, the bacterium is isolated only in 50% of cases.
During the convalescent phase it is no longer possible to isolate
B. pertussis
from the nasopharynx although the patients still have symptoms of pertussis.
It is clear from this that the more violent clinical indications of the illness occur after the disappearance of the bacteria and from this it may be inferred that pertussis is not due to invasion of the respiratory tract by the bacterial but to a toxic state induced by the bacteria but which remains even after their disappearance.
The charge of
B. pertussis
from phase I (virulent) to phase III (non-virulent is accomplished by a loss of capacity to synthesize certain substances such as: the pertussis toxin (PT), haemolysin (hly), adenylcyclase (Adc) and the dermonecrotic toxin (Dmt).
Tests carried out be Munoz. J. J. et al. (1981) (Inf. Immun. 32. 243) have shown that a vaccine constituted by the pertussis toxin alone, suitably detoxified with glutaraldehyde, is capable of protecting mice from death due to the intracerebral administration of bacteria in phase I.
Recent studies (Weiss, A. A. et al. (1983) Inf. Immun 42, 33; Weiss, A. A. et al (1984) J. Inf. Dis. 150, 219) have shown that not all these five substances contribute with equal effect to the virulence of
B. pertussis
. Weiss has succeeded in isolating the mutants which have lost selectively only one of the factors of the virulence by the insertion of a transposable element, a transposon (Tn5), into the genome of
B. pertussis
. From tests carried out in animals, it was found that only the mutants which had lost their capacity to synthesize PT or Adc had, at the same time, lost their virulence.
Hence the pertussis toxin (PT) is the major factor in the virulence of
Bordetella pertussis.
The pertussis toxin a protein with a molecular weight of about 100,000 dalton, is produced and released into the extra cellular environment by
Bordetella pertussis
during phase I.
PT has an enzymatic activity and deactivates ADP-ribosilandol, a GTP-dependent protein which is involved in the deactivation of cellular adenylcylase.
Like other toxins, the pertussis toxin is also constituted by two different fragments: A and B.
The A fragment, which is toxic, comprises a single polypeptide S1 (subunit S1) having a molecular weight of about 28,000 daltons, which can bind an ADP-ribose group to a GTP-binding protein G, which inhibits adenylate cyclase involved in the transmission of signals from the outside to the inside of cells.
The B fragment comprises five polypeptides S2, S3, S4 and S5 (subunits S2, S3, S4, S5) with molecular weights of 23,000, 22,000, 12,000 and 9,000 daltons respectively, disposed as two dimers S2+S4 and S3+S4 and a monomer S5.
The B fragment binds to measure receptors of eucaryotic cells facilitating entry of S1 into the cells.
At present a pertussis vaccine is used which, although giving permanent immunity, has numerous disadvantages.
The vaccine is in fact constituted by virulent bacteria (phase I) treated at 56° C. for 30 minutes to remove a toxin which is heat-labile (dermonecrotic toxin) and killed by merthiolate.
Since the bacteria are not subjected to any detoxification treatment, any toxic substance which withstands 56° C. for 30 minutes is included in the vaccine.
The presence of such toxic substances, particularly from the PT, causes side effects which vary from simple flushing to permanent neurological damage and/or death.
All this has meant that over the last ten years the use of the vaccine has been reduced drastically with a consequent re-explosion of cases of pertussis.
Recently a vaccine has been prepared which is constituted essentially by fibrous haemagglutinin (FHA) and pertussis toxin detoxified with formaldehyde (Sato Y., et al: Lancet Jan. 21. 122 (1984)).
However, this vaccine has disadvantages such as: the presence of side effects, even though less than those of the conventional vaccine; obtaining a product which is too crude to be used such; and the extreme variability of the product from preparation to preparation.
There is thus a need to provide an effective vaccine which can be produced on a large scale and which does not have the disadvantages noted above.
Thus, for example, recent developments in the biochemical filed and in the field of genetic engineering have made it possible to prepare synthetic vaccines and micro-organisms capable of producing proteins useful for the preparation of vaccines with high yields.
In every case a key element for the preparation of the vaccines is a knowledge of the amino acid sequence of the protein and the nucleotide sequence of the gene and/or genes which code for the protein.
Once the gene which codes for a certain protein has been cloned and its nucleotide and amino acid sequences have been determined, the production of these on a large scale and the construction of synthetic vaccines is possible with current techniques.
At present nothing is known of the nature, structure and expression of the gene and/or genes of the pertussis toxin and no data other than the amino acid composition of the individual subunits of the pertussis toxin is available.
Accordingly, by the present invention there has been determined the aminoterminal amino acid sequence of the subunits S1, S2, S3 and S4 of the pertussis toxin and an Eco-RI-fragment of
Bordetella pertussis
chromosomal DNA has been cloned and sequenced, the fragment having 4696 base pairs and containing the genes which code for the five subunits of the pertussis toxin, useful for the preparation of the pertussis toxin or of one or more subunits of the pertussis toxin. Thus a subject of the present invention is a cloned and sequenced 4696-base-pair Eco RI fragment of
Bordetella pertussis
chromosomal DNA containing the genes which code for the five subunits of the pertussis toxin or fragments thereof, useful for the production of the pertussis toxin or of one or more subunits of the pertussis toxin.
Another subject of the invention is a hybrid plasmid containing the cloned and sequenced DNA fragment or further fragments thereof.
A further subject of the present invention is a micro-organism transformed by the hybrid plasmid and capable of expressing the
Arico Maria Beatrice
Nicosia Alfredo
Rappuoli Rino
Blackburn Robert P.
Chiron S.p.A.
Crouch Deborah
Harbin Alisa A.
Paintin Francis A.
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