Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
1995-05-02
2001-06-19
Chin, Christopher L. (Department: 1641)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S234100, C424S184100, C435S006120, C435S069100
Reexamination Certificate
active
06248330
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to immunogenic compositions for inducing protective antibodies against Helicobacter spp. infection. It also relates to proteinaceous material derived from Helicobacter, and to nucleic acid sequences encoding them. Antibodies to these proteinaceous materials are also included in the invention.
H. pylori
is a microorganism, which infects human gastric mucosa and is associated with active chronic gastritis. It has been shown to be an aetiological agent in gastroduodenal ulceration (Peterson, 1991), and two recent studies have reported that persons infected with
H. pylori
had a higher risk of developing gastric cancer (Nomura et al., 1991; Parsonnet et al., 1991).
In vivo studies of the bacterium, and consequently, work on the development of appropriate preventive or therapeutic agents, has been severely hindered by the fact that
Helicobacter pylori
only associates with gastric-type epithelium from very few animal hosts, none of which are suitable for use as laboratory models.
A mouse model of gastric colonization has been developed using a helical bacterium isolated from cat gastric mucus (Lee et al., 1988, 1990) and identified as a member of the genus Helicobacter. It has been named
H. felis
(Paster et al., 1990).
To date, only limited information concerning
H. felis
and the extent of its similarities and differences with
H. pylori
is available. The reliability of the mouse model for the development of treatments for
H. pylori
infection is, therefore, uncertain. Recently, it was shown that
H. pylori
urease is a protective antigen in the
H. felis
/mouse model (Davin et al., 1993; Corthesy-Theulaz et al., 1993).
It is, therefore, an aim of the present invention to provide therapeutic and preventive compositions for use in Helicobacter infection, which furthermore can be tested in laboratory animals.
It is known that
H. pylori
expresses urease activity and that urease plays an important role in bacterial colonization and mediation of certain pathogenic processes (Ferrero and Lee, 1991;
Hazel et al., 1991).
The genes coding for the urease structural polypeptides of
H. pylori
(UreA (SEQ ID NO:22), UreB (SEQ ID NO:26)) have been cloned and sequenced (Labigne et al., 1991; and French Patent Application FR 8813135), as have the genes coding the “accessory” polypeptides necessary for urease activity in
H. pylori
(International patent application WO 93/07273).
Attempts have been made to use nucleic acid sequences from the
H. pylori
urease gene cluster as probes to identify urease sequences in
H. felis.
However, none of these attempts have been successful. Furthermore, the establishment and maintenance of
H. felis
cultures in vitro is extremely difficult, and the large quantities of nucleases present in the bacteria complicates the extraction of DNA.
SUMMARY OF THE INVENTION
The present inventors have, however, succeeded in cloning and sequencing the genes of the urease structural polypeptides of
H. felis
, and of the accessory polypeptides. This has enabled, in the context of the invention, the comparison of the amino acid sequence data for the
H. felis
Ure gene products with that for Helicobacter pylori, and a high degree of conservation between the urease sub-units has been found. An immunological relationship between the two ureases exists, and protective antibodies to Helicobacter infection can be induced using the urease sub-units or fragments thereof as immunogens.
Indeed, to elucidate the efficiency of individual urease subunits to act as mucosal immunogens, the genes encoding the respective urease sub-units (UreA (SEQ ID NOS:20,22) and UreB (SEQ ID NOS:21,26)) of
Helicobacter pylori
and
Helicobacter felis
have been cloned in an expression vector (pMAL) and expressed in
Escherichia coli
cells as translational fusion proteins. The recombinant UreA (SEQ ID NOS:20,22) and UreB (SEQ ID NOS:21,26) proteins have been purified by affinity and anion exchange chromatography techniques, and have predicted molecular weights of approximately 68 and 103 kDa, respectively. Western blotting studies indicated that the urease components of the fusion proteins are strongly immunogenic and are specifically recognized by polyclonal rabbit anti-Helicobacter sera. Orogastric immunization of mice with 50 &mgr;mg of recombinant
H. felis
UreB (SEQ ID NO:21), administered in combination with a mucosal adjuvant (cholera toxin), protected 60% (n=7; p<0.005) of mice from gastric colonization by
H. felis
bacteria at over 4 months. This compared with a value of 25% (n=8; p>0.05) for the heterologous
H. pylori
UreB (SEQ ID NO:26) antigen. For the first time, a recombinant subunit antigen has been shown to induce an immunoprotective response against gastric Helicobacter infection.
The inventors have also identified, in the context of the invention, new heat shock proteins or chaperonins in Helicobacter, which have an enhancing effect on urease activity. Use of the chaperonins in an immunogenic composition may induce therefore an enhancement of protection.
Indeed, the genes encoding each of the HspA (SEQ ID NO:29) and HspB (SEQ ID NO:30) polypeptides of
Helicobacter pylori
have been cloned, expressed independently as fused proteins to the Maltose-Binding-Protein (MBP), and purified on a large scale. These proteins have been used as recombinant antigens to immunize rabbits, and in Western immunoblotting assays as well as ELISA, to determine their immunogenicity in patients infected with HP (HP+). The MBP-HspA (SEQ ID NO:29) and MBP-HspB (SEQ ID NO:30) fusion proteins have been shown to retain their antigenic properties. Comparison of the humoral immune response against HspA (SEQ ID NO:29) and/or HspB (SEQ ID NO:30) in (HP+) patient sera demonstrated that not only HspB (SEQ ID NO:30) but also HspA (SEQ ID NO:29) was recognized by (HP+) patient sera (29/38 and 15/38, respectively). None of the 14 uninfected patients had antibodies reacting with the Hsps.
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Labigne, A et al, Am. Gastroenterol. Hepatol. (France) Mar.-Apr. 1992, 28(2) pp. 93-97 (abstract only in English).*
Labigne, A et al, Bull. Acad. Natl. Med. (France), Jun.-Jul. 1991, vol. 175(6), pp. 791-800 (abstract only in English).*
Pallen, M.J. et al, Lancet, 1990, vol. 336/8708, (186-187).*
Ferrero et al, Gastroenterology, vol. 104, (4), Apr. 1993, p. A699.*
Allan D. Pronovost et al., “Evaluation of a New Immunodiagnostic Assay forHelicobacter pyloriAntibody Detection: Correlation with Histopathological and Microbiological Results,” Journal of Clinical Microbiology, vol. 32, No. 1, Jan. 1994, pp. 46-50.
Steven J. Czinn et al., “Protection of germ-free mice from infection byHelicobacter felisafter active oral or passive IgA immunization.”, Vaccine, vol. 11, Issue 6, pp. 637-642, 1993.
C. Stewart Goodwin, Overview ofHelicobacter PyloriGastritis, Peptic Ulcer, and Gastric Cancer and the Possible Development of anH. Pylori0 Vaccine, Helicobacter Pylori Biology and Clinical Practice, Chapter 25 pp. 431-444, 1993.
E.G. Fox, et al. “Comparison of Two New Immunodiagnostic Assays forHelicobacter Pyloriwith Established Clinical and Histopathologic Findings”, Gastroenterology, vol. 100, No. 5, Part 2, p. A66.
R.L. Ferrero et al., “Molecular Evidence Demonstrating Significant Homology Between the Urease Polypeptides ofHelicobacter felisand andHelicobacter pylori,”Gastroenterology
Ferrero Richard L.
Labigne Agnes
Suerbaum Sebastien
Thiberge Jean-Michel
Chin Christopher L.
Finnegan Henderson Farabow Garrett & Dunner
Institut Pasteur
Portner Ginny Allen
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