Blood group antigen binding protein and corresponding agents

Details Blood group antigen binding protein and corresponding agents Blood group antigen binding protein and corresponding agents

1998-02-10

2002-06-25

Smith, Lynette R. F. (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C514S04400A

Reexamination Certificate

active

06410719

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 immunization therapies. In addition, it relates to polypeptides, antibodies, and polynucleotides used for the detection of said bacteria.
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.
The foregoing makes the prevention, diagnosing 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 diagnosing, 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
colonizes 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 colonize for a lifetime (Blaser, 1993; Borén and Falk, 1994). Attachment to the epithelial lining protects the bacteria from the antimicrobial 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 (Labigne et al., 1991; Eaton and Krakowka, 1994), that buffers the micro environment around the bacteria and the polar flagellae (Eaton et al., 1992) 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 (Cover et al., 1994, Phadnis et al., 1994; Schmitt and Haas, 1994; Telford et al., 1994), and the cytotoxin associated antigen CagA (Covacci et al., 1993).
Attachment is essential for colonization of the epithelial lining and bacteria express surface associated adhesion molecules that recognize 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 colonization. Several putative receptor structures have been described for
H. pylori
, such as the hemagglutinin-sialic acid (Evans et al., 1988), sulphated glycoconjugates (Ascencio et al., 1993) and sulphatides (Saitoh et al., 1991; Kamisago et al., 1996). Recently, the fucosylated blood group antigens H-1 and Lewis
b
were described (Borén et al., 1993), 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 not seldom 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. Trust and co-workers reported recently about the finding of a number of outer membrane proteins in
H. pylori
which were suggested to constitute a family of porin proteins (Exner et al., 1995).
The BAB adhesin has previously been identified and shown to be localized 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.
THE OBJECTIVE OF THE PRESENT INVENTION
The objective of the present invention was to further purify and characterize the
H. pylori
blood group antigen binding (BAB) adhesin to make possible the development of methods and materials for specific and selective diagnosing and treatment of
H. pylori
induced infections and related diseases and the development of said methods and materials. A further and equally important objective was to determine the DNA sequences of the genes involved in the expression of this protein. The objective was fulfilled through the protein disclosed the DNA disclosed and the methods and materials disclosed. The DNA sequences are attached as SEQ. ID NOS. 1 and 3 disclosing the babA and babB sequences, respectively. The full protein sequence is disclosed in SEQ ID NOS. 2 and 4.


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