Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1999-02-10
2004-03-23
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S184100, C424S234100, C435S007320, C435S252100, C530S350000, C800S008000, C514S025000
Reexamination Certificate
active
06709656
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to materials and methods for prevention, treatment and diagnosing of infections caused by
Helicobacter pylori.
More specifically the present invention relates to polypeptides and antibodies useful in vaccines for the treatment and prevention of pathologic infections caused by
Helicobacter pylori
strains. The present invention specifically relates to a bacterial blood group antigen binding adhesin (BAB-adhesin). The present invention further relates to polynucleotides useful for the recombinant production of said polypeptides and for use in immunisation therapies. In addition, it relates to polypeptides, antibodies, and polynucleotides used for the detection of said bacteria.
The present invention further relates to new immunoglobulins, which exhibit specific activity to a blood group binding adhesin, expressed by
Helicobacter pylori,
methods for the production of said immunoglobulins, their isolation and use. The present invention further relates to the treatment and prevention of
H. pylori
induced infections in the gastrointestinal tract.
BACKGROUND OF THE INVENTION
Helicobacter pylori
is a causative agent for acid peptic disease and the presence of this organism is highly correlated to the development of gastric adenocarcinoma. Bacterial adherence to the human gastric epithelial lining was recently shown to be mediated by fucosylated blood group antigens.
Recent research has focused on the role of
Helicobacter pylori
in the development of ulcers in the gastric mucosa. Recent findings show a strong connection between
H. pylori
and chronic, active gastritis and gastric ulcers. Furthermore, there appears to be a strong correlation between ventricular cancer and gastric ulcers. Traditional treatment of gastric ulcers has involved gastric resection, the administration of bismuth compositions, the administration of H
2
-blockers and the administration of pH-buffering agents, to mention a few examples.
More recently, various forms of treatment have been supplemented with the administration of antibiotics. One problem with presently known treatments is the risk for treatment failure. Furthermore, not only do microbes develop antibiotic resistance, the antibiotics administered often upset the natural balance of benign microbes, colonising the intestinal tract. This leads to diarrhoea and other signs of intestinal discomfort, in addition to destabilising the benign flora in the intestines. Other treatments, e.g. H
2
-blockers often require life-long medication to prevent the recurrence of disease.
The foregoing, together with the fact that
H. pylori
is very widely spread among humans—according to a conservative estimate approximately 60% of all adult humans in the industrialised countries are infected—makes the diagnosing, prevention and treatment of
H. pylori
infections an urgent task.
Further, the fact that developing countries frequently lack the resources for conventional treatment of gastric ulcers further underlines the importance of finding new ways of treatment and prevention of
H. pylori
induced infections. It is obvious, for many reasons, that disease prevention with vaccines is a preferable mode. A vaccine would provide an easily administered and economical prophylactic regimen against
H. pylori
infections. An effective vaccine against
H. pylori
is nevertheless presently lacking.
STATE OF THE ART
H. pylori
colonises the human gastric mucosa, in an equilibrium between adherence to the epithelial surface mucous cells and the mucous layer lining the gastric epithelium. Once infected, bacteria seems to colonise for a lifetime. Attachment to the epithelial lining protects the bacteria from the anti-microbial effects of the acidic gastric juice of the stomach lumen, as well as from physical forces such as peristalsis. For survival in this hostile ecological niche,
H. pylori
has developed a battery of virulence factors; such as production of the enzyme urease that buffers the micro-environment around the bacteria and the polar flagellae to ensure high motility, a prerequisite in an ecological niche where the turnover of the mucous layer is in the range of hours. A subset of
H. pylori
strains produces the vacuolating cytotoxin, VacA, and the cytotoxin associated antigen CagA.
Attachment is essential for colonisation of the epithelial lining and bacteria express surface associated adhesion molecules that recognise specific carbohydrate or protein receptors on the cell surfaces or mucous lining. The specificity in this interaction in combination with the genetically regulated receptor distribution results in a restricted range of cell lineages and tissues available for colonisation. Several putative receptor structures have been described for
H. pylori,
such as the hemagglutinin-sialic acid, sulphated glycoconjugates and sulphatides. Recently, the fucosylated blood group antigens H-1 and Lewis
b
were described (Borén et al., Science, 262, 18921993), mediating specific adherence of
H. pylori
to human and rhesus monkey gastric surface mucous cells in situ. The H-1 and Lewis
b
antigens are part of the blood group antigens that define blood group O in the ABO system.
Surface-exposed proteins are often constituents of the outer membrane. The outer membrane has a structural role and acts as a selective barrier, determining what enters the cell and what molecules are secreted. One class of outer membrane proteins are called porins, and create hydrophilic pores through the outer membrane where specific metabolites, such as sugar molecules, can cross. Recently the finding of a number of outer membrane proteins in
H. pylori,
was reported, which proteins were suggested to constitute a family of porin proteins.
The BAB adhesin has previously been identified and shown to be localised on the bacterial surface of
H. pylori
(SE 9602287-6). The blood group binding activity was shown to be pH dependent and the present inventors present evidence that the binding affinity to the Lewis
b
receptor reveals a high equilibrium constant. For the purification of the BAB adhesin, a crosslinker-labelled receptor conjugate was used in order to mediate specific transfer of biotin to the adhesins on the bacterial surface. Thereafter the biotin-labelled adhesin could be extracted by streptavidin coated magnetic beads. Determination of the amino terminal amino acid sequence of the purified BAB adhesin exhibit homologies to outer membrane proteins of
H. pylori
porins.
Intensive research has been directed to the immunological treatment and prevention of
H. pylori
induced infections. EP 0 484 148 (Ando & Nakamura) describes a method for treating and/or preventing upper gastrointestinal disease in mammals, said method comprising orally administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising anti-
Helicobacter pylori
polyclonal immunoglobulins and a pharmaceutically acceptable carrier. Said description further dwells on the combination of said treatment in combination with the administration of antibiotics. As the method of producing said polyclonal antibodies, EP 0 484 148 describes the isolation and purification of anti-
H. pylori
immunoglobulins from the sera and milk of mammals.
H. pylori
itself was not found in the stomachs of cows, goats, sheep, swine or horses, according to EP 0 484 148, but it was assumed that these animal species have colonizing microorganisms with antigenic determinants similar to those of
H. pylori
because they have immunoglobulins which cross-react to strains of
H. pylori
found in humans. Preferably, according to EP 0 484 148, large mammals, e.g. pregnant cows, are immunized with whole cells of
H. pylori
and the immunoglobulins subsequently extracted from the milk or colostrum. In the immunization experiments, NCTC Strain 11362 and clinical isolate
H. pylori
No. 153 were used to trigger the production of immunoglobulins. On the other hand, NCTC Strain 11637 was used for analysing purposes. Immunization is claimed to yield an anti-
H. pyl
Arnqvist Anna
Boren Thomas
Hammarstrom Lennart
Ilver Dag
Normark Staffan
Birch & Stewart Kolasch & Birch, LLP
Portner Ginny Allen
Smith Lynette R. F.
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