Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1994-06-17
2004-03-30
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S254100, C424S185100, C530S350000
Reexamination Certificate
active
06713072
ABSTRACT:
The present invention relates to immunologically active polypeptides with no or reduced toxicity useful for the production of an antipertussis vaccine.
The invention also relates to a method for the preparation of said polypeptides and to an antipertussis vaccine comprising a therapeutically effective amount of at least one of said polypeptides.
Pertussis is a respiratory system disease caused by
Bordetella pertussis
(
B. pertussis
), a bacillus the transmission of which occurs during the catarrhal and convulsive phase from a sick person to a healthy predisposed individual through the respiratory system.
A vaccine effective against said disease is particularly desirable since pertussis may cause convulsions, cerebral damages and, sometimes, death, principally in tender age children and in newborn babies lacking maternal antipertussis antibodies obtained passively.
At present, it is employed an antipertussis vaccine comprising virulent bacteria killed with merthiolate and treated at 56° C. that, even if it confers a permanent protection, it is not, however, completely satisfactory, either for the presence of undesired side effects or for the numerous problems deriving from the preparation and purification thereof.
This results in the necessity of preparing an antipertussis vaccine lacking of the aforementioned drawbacks.
It is known that
B. pertussis
has per se, no virulence and that its toxicity is corrolated with the synthesis, during the phase I (virulent), such substances as: hemolysin (Hls), adenylcyclase (Adc), dermonecrotic toxin (Dnc), filamentary hemagglutinin (Fha) and pertussis toxin (PT). The latter, in particular, represents not only the major virulence factor caused by
B. pertussis
(Weiss A. et al. (1983) Infect, Immun. 42, 333-41; Weiss A. et al. (1984) J. Inf. Dis. 150, 219-222but also one of the major protective antigens against infections caused by said bacterium.
Anti-PT antibodies, in fact, have been found in individuals immunized by the cellular vaccine (Ashworth L. A. E. et al. (1983) Lancet. October 878-881) and a protective immunity has been obtained in mice infected, via aereosol or intracerebrally, using formaldehyde-detoxified PT (Sato Y. et al. (1983) Inf. and Imm. 41, 313). Even if the pertussis toxin represents an essential component in the preparation of new antipertussis vaccines, its use is limited by the numerous drawbacks deriving from its toxicity.
The PT, in fact, induces undesirable pathophysiologic effects such as: lymphocytosis, histamine sensitivity, hypoglycemia, insensitivity to the hyperglycemic effect of epinephrine and activation of the islands of Langerhans.
Furthermore, it has been found that the PT presence in the vaccine now employed is the principal cause of such side effects as: fever, pomphus, neurologic alteration and death which have led, in recent years, to drastically reducing the use of the vaccine with the consequent new outbreak of pertussis cases.
The PT detoxification treatment by means of formaldehyde though allowing to get an immunogenic protein without toxicity (Sato et al. reference reported above), presents some drawbacks deriving from the fact that said protein is not obtainable in pure, reproducible and stable form.
According to that, polypeptides have now been found which are able to overcome the prior art drawbacks and are obtainable in pure form by means of a simple and economically feasible method. One object of the present invention, therefore, consists of immunologically active polypeptides with no or reduced toxicity useful for the preparation of an antipertussis vaccine.
A further object of the present invention consists of a method for the preparation of said polypeptides.
Another object of the present invention is a vaccine comprising a therapeutically effective amount of at least one of said polypeptides.
Further objects of the present invention will become apparent from a reading of the following description and examples.
The pertussis toxin is a protein comprising five different subunits the toxicity of which is due to ADP-ribosylation of proteins which bind GTP involved in the transmission of messages through eukaryotic cells membranes.
Said PT comprises two fractions with different functionality: A comprising the S1 subunit and B comprising S2, S3, S4 and S5 subunits placed in two dimers D1(S2+S4) and D2 (S3+S4) linked to each other by the S5 subunit.
The A fraction represents the enzymatically active and therefore, toxic part of PT, whereas the B fraction is linked to the eukaryotic cells membrane receptors and allows the introduction of the S1 subunit therein.
In the copending Italian patent application No. 19208-A/86, now filed in the United States as U.S. application Ser. No. 07/006,438, filed Jan. 23, 1987, the cloning, sequencing and expression of the genes which code for said subunits have been described and claimed and it has been shown that said genes are grouped in a sole operon.
Furthermore, the ADP-ribosylation activity of the S1 subunit has been determined, by cultivating a microorganism transformed with the hybrid plasmid PTE225, and it has been found that said subunit possesses an enzymatic activity comparable to that of PT.
According to that and to the end of obtaining a protein having the immunologic and protective properties of the pertussis toxin but with no or reduced toxicity, the positions and the fundamental aminoacids for the enzymatic activity of the protein have been identified.
In particular, the following positions and aminoacids have been found:
tyrosine(8), arginine(9), phenylalanine (50), threonine(53), glutamic acid (129), glycine (121), alanine (124), aspartic acid (109), glycine (99), arginine (135), threonine (159) and tyrosine (111).
The substitution of one or more of said amino acids with any aminoacid different from the one which is bound to be changed, allows to obtain a protein with altered toxicity.
According to that, in accordance with the present invention, polypeptides have been synthesized containing S1 subunits of the modified pertussis toxin by means of direct mutagenesis substituting, in one or more positions of the S1 region comprised between the 1-180 amino acids, one aminoacid with another capable of destroying or reducing its enzymatic activity without altering the immunologic properties thereof.
In particular, polypeptides have been synthesized containing the S1 subunit of the pertussis toxin modified by substituting:
the tyrosine in position 8 and arginine in position 9 with aspartic acid and glycine;
the phenylalanine in position 50 and the threonine in position 53 with glutamic acid and isoleucine;
the glutamic acid in position 129 with glycine;
the glycine in position 121 with glutamic acid;
the alanine in position 124 with aspartic acid;
the aspartic acid in position 109 and the alanine in position 124 with glycine and aspartic acid respectively;
the glycine in position 99 with glutamic acid;
the aspartic acid in position 109 with glycine;
the arginine in position 135 with glutamic acid;
the threonine in position 159 with lysine;
the tyrosine in position 111 with glycine and insertion of Asp Thr Gly Gly amminoacids in position 113.
In particular, the polypeptides according to the present invention have been prepared by a method which comprises:
a) modifying by means of direct mutagenesis the gene which codes for the S1 subunit of the pertussis toxin substituting, in one or more sites of the DNA molecule, the bases sequence which codes for a predetermined aminoacid with a bases sequence which codes for the aminoacid of interest;
b) constructing a hybrid plasmid linking a cloning vector with the DNA fragment containing the modified S1;
c) transforming a host microorganism with a hybrid plasmid obtained as reported in b);
d) cultivating in a suitable culture medium, in presence of carbon, nitrogen and mineral salts sources a transformed microorganism and then,
e) recovering the polypeptide containing the modified S1 subunit from the culture medium or from the cells.
According to the present invention and to the end of identi
Bartoloni Antonella
Pizza Mariagrazia
Rappuoli Rino
Blackburn Robert P.
Chiron S.r.l.
Harbin Alisa A.
Hoscheit Dale H.
Mosher Mary E.
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