U.S. patent application number 11/762720 was filed with the patent office on 2008-08-07 for helicobacter pylori adhesin binding group antigen.
Invention is credited to Anna Arnqvist, Thomas Boren, Lennart Hammarstrom, Dag Ilver, Staffan Normark.
Application Number | 20080187541 11/762720 |
Document ID | / |
Family ID | 26662671 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080187541 |
Kind Code |
A1 |
Boren; Thomas ; et
al. |
August 7, 2008 |
HELICOBACTER PYLORI ADHESIN BINDING GROUP ANTIGEN
Abstract
A novel Helicobacter pylori blood group antigen binding (BAB)
adhesin protein was isolated and purified, whereby said protein or
fractions thereof bind specifically to fucosylated blood group
antigens. The protein sequence of said adhesin is disclosed in this
application. Simultaneously the DNA sequences for two genes, babA
and babB, producing highly similar proteins, are disclosed. Said
adhesin and/or DNA is useful for diagnose and therapy and/or
prophylaxis directed against H. pylori induced infections, e.g.
gastritis and acid peptic disease, i.e. active vaccination. A new
immunoglobulin composition, which exhibits specific activity to a
Lewis.sup.b antigen binding Helicobacter pylori adhesin, or
preferably, monoclonal and/or polyclonal antibodies to said adhesin
offer a new and more efficient method of treatment and/or
prevention of gastrointestinal diseases, caused by Helicobacter
pylori or other Helicobacter species, i.e. passive vaccination.
Inventors: |
Boren; Thomas; (Umea,
SE) ; Arnqvist; Anna; (Umea, SE) ;
Hammarstrom; Lennart; (Huddinge, SE) ; Normark;
Staffan; (Stockholm, SE) ; Ilver; Dag; (Umea,
SE) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
26662671 |
Appl. No.: |
11/762720 |
Filed: |
June 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10761201 |
Jan 22, 2004 |
7271251 |
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|
11762720 |
|
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09202178 |
Feb 10, 1999 |
6709656 |
|
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PCT/SE97/01009 |
Jun 10, 1997 |
|
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10761201 |
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Current U.S.
Class: |
424/150.1 ;
424/164.1; 424/234.1; 424/93.2; 435/69.6; 435/7.32; 530/388.1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 2039/51 20130101; A61P 43/00 20180101; C07K 14/205 20130101;
A61K 39/00 20130101; A61P 1/04 20180101 |
Class at
Publication: |
424/150.1 ;
424/234.1; 435/7.32; 530/388.1; 435/69.6; 424/164.1; 424/93.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; G01N 33/569 20060101
G01N033/569; A61P 43/00 20060101 A61P043/00; A61K 35/00 20060101
A61K035/00; C07K 16/18 20060101 C07K016/18; C12P 21/04 20060101
C12P021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 1996 |
SE |
9602287-6 |
Mar 19, 1997 |
SE |
9701014-4 |
Claims
1. A vaccine comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6.
2. A test kit comprising a monospecific antisera that recognizes a
BabA antigen and comprises an immunoglobulin that binds said BabA
antigen via a variable region, wherein said Bab A adhesin protein
is produced using an isolated and purified bacterial blood group
antigen binding protein (BabA) from Helicobacter pylori species,
wherein said BabA protein binds specifically to fucosylated
Lewis.sup.b type I and H-1 blood group antigen-glycoconjugates and,
wherein said BabA protein contains less than 20% bacterial protein
impurities, has a molecular weight in the interval of 73 to 75 kDa
as determined by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), and is not a HopA, HopB, HopC, HopD, or
HopE protein.
3. An isolated monospecific immunoglobulin composition from sera
which recognizes a BabA antigen and binds said BabA antigen via a
variable region and exhibits specific activity to a BabA adhesion
protein from Helicobacter pylori, wherein said adhesin protein
binds Lewis.sup.b and H-1 blood group antigen-glycoconjugates and
is not a HopA, HopB, HopC, HopD or HopE protein.
4. The immunoglobulin composition according to claim 3, wherein
said BabA adhesin protein has a molecular weight in the interval of
about 70 to 77 kDa as determined by SDS-PAGE.
5. The immunoglobulin composition according to any one of claims 3
or 4 wherein said BabA adhesin protein comprises the following
amino acid sequence: EDDGFYTSVGYQIGEAAQMV (SEQ ID NO:5) or
homologues thereof.
6. An isolated monospecific antibody, which recognizes a BabA
antigen and binds said BabA antigen via a variable region and
exhibits specific activity to a BabA adhesion protein from
Helicobacter pylori that binds Lewis.sup.b and H-1 blood group
antigen-glycoconjugates and is not a HopA, HopB, HipC, HopD or HopE
protein.
7. The antibody according to claim 6, wherein said BabA adhesin
protein has a molecular weight in the interval of about 70 to 77 kD
as determined by SDS-PAGE.
8. The antibody according to claim 6, wherein said BabA adhesin
protein comprises the following amino acid sequence:
EDDGFYTSVGYQIGEAAQMV (SEQ ID NO:5) or homologues thereof.
9. The antibody according to claim 6, wherein said antibody is a
monoclonal antibody.
10. A method of manufacturing an immunoglobulin composition
according to claim 3, comprising the following steps: immunizing an
animal with Lewis.sup.b binding protein or fractions thereof,
expressed by Helicobacter pylori, isolating the immunoglobulin
fraction from an excretion of said host animal, and purifying the
immunoglobulin preparation to obtain an isolated monospecific
immunoglobulin composition.
11. The method according to claim 10, wherein said animal is a cow
and the immunoglobulin fraction is isolated from the milk,
preferably the colostrum thereof.
12. The method according to claim 10, wherein said animal is a
chicken and the immunoglobulin fraction is isolated from the egg
yolk thereof.
13. A method of manufacturing an antibody according to claim 6,
wherein the method comprises the following steps: immunizing an
animal with a Lewis.sup.b binding protein (BabA) or fractions
thereof, expressed by Helicobacter pylori, fusing immunised,
immunoglobulin producing cells with a neoplastic cell line,
selecting and growing cells expressing said antibody, and purifying
the antibodies.
14. The method of claim 13, further comprising expressing said
antibody by a culture of viable microorganisms in an expression
system, where said microorganism or organisms are generally
recognized as safe (GRAS) and genetically modified to express said
antibody.
15. The method according to claim 14, wherein said microorganism is
selected from the group consisting of bacteria of the species
Lactobacillus, Staphylococcus and Enteriobacteriacea.
16. A pharmaceutical preparation for treating an individual having
or identified as being at risk of developing Helicobacter pylori
infection, gastric ulcers or acid peptic disease in humans
comprising the immunoglobulin composition according to claim 3.
17. A method of treating an individual having or identified as
being at risk of developing gastric ulcers comprising administering
to a subject in need thereof the immunoglobulin composition
according to claim 3.
18. A method of treating an individual having or identified as
being at risk of developing acid peptic disease comprising
administering to a subject in need thereof the immunoglobulin
composition according to claim 3.
19. A pharmaceutical product for treating an individual having or
being identified at risk of developing Helicobacter pylori
infections, gastric ulcers or acid peptic disease in humans,
comprising the antibody according to claim 6.
20. A pharmaceutical product for treating an individual having or
being identified at risk of developing gastric ulcers comprising
the antibody according to claim 6.
21. A pharmaceutical product for the treating an individual having
or being identified at risk of developing peptic disease comprising
the antibody according to claim 6.
22. The method according to claim 18, wherein said subject is human
and said immunoglobulin composition is administered orally.
23. A method for treating a human having or being identified at
risk of developing Helicobacter pylori infections, comprising
orally administering an effective amount of an antibody according
to claim 6 to said human.
24. A method of treating a human having or being identified at risk
of developing Helicobacter pylori infections, said method
comprising orally administering an effective amount of a culture of
viable microorganisms in an expression system, wherein said
microorganism or organisms are generally recognized as safe (GRAS)
and genetically modified to express an antibody according to claim
8 or 9.
25. An expression system comprising a culture of viable
microorganisms wherein said microorganism or organisms are
generally recognized as safe (GRAS) and genetically modified to
express an antibody according to claim 8 or 9.
26. A method for treating a human having or being identified at
risk of developing Helicobacter pylori infections, said method
comprising orally administering an effective amount of a culture of
viable microorganisms in an expression system, wherein said
microorganisms or organisms are generally recognized as safe (GRAS)
and genetically modified to express an adhesion protein according
to claim 1.
27. The composition according to claim 4, wherein the molecular
weight of said BabA adhesin protein is in the interval of 73 to 75
kDa.
28. The composition according to claim 4, wherein the molecular
weight of said BabA adhesin protein is about 73.5 kDa.
29. The antibody according to claim 6, wherein the molecular weight
of said BabA adhesin protein is in the interval of 73 to 75
kDa.
30. The antibody according to claim 4, wherein the molecular weight
of said BabA adhesin protein is about 73.5 KDa.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/761,201, filed Jan. 22, 2004, which is a divisional of
U.S. patent application Ser. No. 09/202,178 (now issued as U.S.
Pat. No. 6,709,656), which is the U.S. National Stage of
International Application No. PCT/SE97/01009 filed Jun. 10, 1997,
which claims priority to Swedish Application No. 9602287-6 filed
Jun. 10, 1996 and Swedish Application No. 9701014-4 filed Mar. 19,
1997. All of the preceding applications are incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0002] 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.
[0003] 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
[0004] 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.
[0005] 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.sub.2-blockers and the administration of
pH-buffering agents, to mention a few examples.
[0006] 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
diarrhea and other signs of intestinal discomfort, in addition to
destabilising the benign flora in the intestines. Other treatments,
e.g. H.sub.2-blockers often require life-long medication to prevent
the recurrence of disease.
[0007] 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.
[0008] 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
[0009] 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.
[0010] 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.sup.b were described
(Boren 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.sup.b antigens are part of
the blood group antigens that define blood group 0 in the ABO
system.
[0011] 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.
[0012] 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.sup.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.
[0013] 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. pylori titer in the
milk of such magnitude, that daily doses of 0.01-0.1 g/day
immunoglobulin composition, are sufficient for successful therapy.
The claimed interval of 0.01-0.1 g/day is however not supported by
the experiments presented by Ando & Nakamura and so low doses
have hitherto not proven efficient in clinical tests. The doses
actually used in example 5 and 7 are in the order of magnitude of 1
g/day, i.e. 10-fold the upper limit of the given interval.
Furthermore, it is very unlikely, that unspecific immunoglobulin
mixtures as those manufactured by Ando & Nakamura, would be
effective in claimed doses as similar doses are ineffective against
other gastrointestinal pathogens. The simultaneous administration
of antibiotics, extensively discussed in the description,
underlines the insufficiency of the disclosed immunoglobulins.
[0014] EP 0 469 359 (Cordle & Schaller) likewise describes the
immunization of mammals, preferably pregnant cows, with formalin
killed H. pylori bacteria (ATCC Strain 26695). Anti-H. pylori
polyclonal antibodies were isolated and purified from the milk and
finally fed to piglets, in amounts of about 0.5 g immunoglobulins,
three times daily. The results were assessed by determination of
the number of biopsy specimens, which were positive for
Gram-negative bacteria after the trial. Gram-negative bacteria was
found in 78% of the piglets fed a non-immune nutrient but only
(Sic!) in 35% of the piglets fed a nutrient containing so called
specific anti-H. pylori antibodies.
[0015] Anti-H. pylori polyclonal antibodies, effective to cause
aggregation of H. pylori, have thus been administered orally as a
regimen in the treatment and prevention of H. pylori induced
infections in the gastrointestinal tract. Nevertheless, as also
noted in EP 0 484 148 A1, it is still not clear, how many antigenic
determinants are present on the surface of H. pylori. The
occurrence of a wide variety of H. pylori strains, makes
questionable the practical efficiency of any polyclonal
immunological therapy according to the state of the art.
Immunization using whole bacteria will always trigger a highly
polyclonal immunresponse with a low level of antibodies against a
given antigenic determinant. This is underlined e.g. by the results
presented by Cordle & Schaller, where, although the number of
Helicobacter positive biopsies were reduced, complete cure was not
obtained through the treatment according to their invention.
[0016] It is notable, that the dose of immunoglobulin needed for
oral prophylaxis or therapy has not yet been clearly defined. In a
normal human adult, approximately 5 g IgA is produced and secreted
at mucosal surfaces each day. Obviously, doses of this magnitude
are economically and practically unfeasible for large-scale therapy
or prophylaxis. In studies on the effect of oral immunoglobulin on
rotavirus infection, daily doses in the interval of 600 to 9000 mg
have been tried in clinical tests. Successful intervention has also
been reported when treating H. pylori and cryptosporidial
infections with daily administrations of 3 to 15 g immunoglobulin
from immunized cows (Hammarstrom et al., Immunol Rev, 139 (1994)
43-70). Generally speaking, all studies hitherto point to the
necessity of using high doses of immunoglobulins when trying to
combat an ongoing infection. The need for more specific
immunoglobuline preparations, allowing the use of smaller doses, is
thus an urgent one.
[0017] To maximize the potency of an immunological regimen for the
treatment and prevention of H. pylori, it is of great importance to
find a specific conserved antigenic determinant, which plays a
central role for the pathogenicity of H. pylori. Using such an
antigenic determinant would make it possible to produce highly
specific and therapeutically efficient novel polyclonal and/or
monoclonal immunoglobulin preparations.
SUMMARY OF THE INVENTION
[0018] The above problem of providing specific, cost-efficient and
therapeutically superior immunoglobulin preparations for the
treatment and prevention of H. pylori has now been solved through
the composition and methods according to the attached patent
claims. The present inventors have now surprisingly shown, that
highly specific and therapeutically efficient polyclonal and/or
monoclonal immunoglobulin preparations can be provided through the
immunization of an animal with an adhesin protein, specific for H.
pylori. Said adhesin protein is characterized already in the
priority applications SE 9602287-6 and SE 9701014-4, which hereby
are referred to in their entirety. The invention will now be
described in closer detail with reference to the attached,
non-limiting figures and examples.
[0019] One 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. These objectives were fulfilled through
the protein specified in claim 1, the DNA disclosed in claim 13 and
14 and the methods and materials specified in the subsequent
claims. The DNA sequences are attached as Appendix 1 and 2,
disclosing the babA and babB sequences, respectively. The full
protein sequence is disclosed in Appendix 3.
DESCRIPTION OF THE FIGURES
[0020] FIG. 1 A) illustrates the bacterial binding to soluble blood
group antigens. H. pylori strains were incubated with
.sup.125I-labeled blood group antigen glycoconjugates and bound
.sup.125I-activity was measured (Note the absence of blood group
antigen binding shown for strains MO19 and 26695.),
[0021] FIG. 1 B) illustrates an receptor displacement assay. Strain
CCUG 17875 was first incubated with 10 ng .sup.125I-labeled
Le.sup.b antigen glycoconjugate and the complex was then challenged
(1 h) with an excess of unlabeled Le.sup.b or Le.sup.a
glycoconjugate, before the .sup.125I-activity in the bacterial
pellet was measured. Concentrations of the unlabeled glycoconjugate
ranged from 50 ng to 8 .mu.g and C) shows the results of a
Scatchard analysis of the H. pylori-Le.sup.b antigen interaction.
Bacterial binding to the Le.sup.b glycoconjugate was titrated to an
affinity constant (Ka) value of 8.times.10.sup.-10 M.sup.-1
(13).
[0022] FIG. 2: Upper panel: Prevalence of the BabA adhesin in the
bacterial population. Cells of strain CCUG 17875 were incubated
with biotinylated Le.sup.b (A) or Le.sup.b (B) glycoconjugate.
Bound biotinylated Lewis-conjugate was detected with FITC-labeled
streptavidin (green fluorescence) and bacteria were counterstained
with propidium iodine (red fluorescence). Lower panel: Localisation
of the BabA adhesin. For electron microscopy (15) cells of strain
CCUG 17875 were incubated with biotinylated Le.sup.b (C) or
Le.sup.a (D).
[0023] FIG. 3 shows the characterization of the molecular weight of
the BabA adhesin by the use of receptor overlay analysis (A, B) and
receptor activity directed affinity tagging of BabA (C).
[0024] FIG. 4 shows receptor activity directed affinity tagging and
protein purification of the BabA adhesin.
[0025] FIG. 5 shows the translated amino acid sequences for the
babA and babB genes, corresponding to the N-terminal domain of the
BabA adhesin.
[0026] FIG. 6 shows the procentual inhibition of H. pylori binding
to .sup.125I-labeled Lewis b antigen for different preparations as
a function of the antibody titre.
[0027] FIG. 7 shows a Western blot detection of the BabA adhesin by
the different antibody preparations.
[0028] FIG. 8 shows four Western blot analyses of H. pylori
proteins by the different antibody preparations.
DESCRIPTION OF THE INVENTION
[0029] The blood group antigen binding adhesin, BabA, has now been
biochemically characterized and purified by a novel technique,
receptor Activity Directed Affinity Tagging (Retagging). Two genes,
babA and babB were found to code for two different but very similar
proteins. The present invention thus comprises a novel blood group
antigen binding adhesin according to claim 1 and the subsequent
claims. The DNA sequences are disclosed in appendices 1 (babA) and
2 (babB). The protein sequences is disclosed in appendix 3. The
invention also includes any pharmaceutical composition comprising
said adhesin protein and/or fractions thereof. Examples of such
pharmaceutical compositions are for example medicaments for the
prevention or treatment of Helicobacter pylori induced gastritis,
gastric and duodenal ulcers and gastric adenocarcinoma. Optionally
said pharmaceutical composition additionally encompasses
pharmaceutically acceptable excipients.
[0030] Further, the present invention comprises the BAB-adhesin
gene or genes for expression of an adhesin protein according to the
invention. Said invention also comprises a novel method for the
isolation and purification of said adhesin. The disclosed genes are
contemplated to function as a cassette system, the organism
alternating between these to avoid immunity in the host. It is very
likely, that homologies of the disclosed sequences exist and
additionally supplement said cassette function in other strains of
H. pylori. Also, genes corresponding to a homology of the first 40
amino acids or genes, corresponding to a homology of the last,
about 300 amino acids, can function to this effect. It is further
highly likely, that Helicobacter pylori is able to switch between
several genes, similar to the disclosed genes, in a so-called
cassette system.
[0031] The invention additionally comprises monospecific antisera
produced using the novel adhesin protein and/or fractions thereof.
Said monospecific antisera is preferably produced according to any
suitable, conventional method for producing monospecific antisera
in vitro or in vivo, e.g. by inoculating a suitable animal. Such
methods are familiar to a person skilled in the art. Antibodies
raised in a suitable animal or in the patient to be treated, can
subsequently be administered locally, e.g. orally to the
patient.
[0032] The invention further comprises the use of said monospecific
antisera for the manufacturing of a test kit for quantitative or
qualitative determinations of adhesin protein or fractions thereof
in cells, tissues or body fluids.
[0033] The invention further comprises the use of said adhesin
protein or corresponding DNA for use in therapy or immunisation
and/or in the manufacture of compositions for said uses. The
invention specifically encompasses the use of said DNA for
immunisation therapy and for the manufacture for compositions for
such therapy. Preferably, in an immunisation therapy where said
composition is administered orally to a patient, the adhesin
protein, fractions thereof or said DNA is administered in
combination with a pharmaceutically suitable immunostimulating
agent. Examples of such agents include, but are not limited to the
following: cholera toxin and/or derivatives thereof, heat labile
toxins, such as E. coli toxin and similar agents. The composition
according to the present invention can further include conventional
and pharmaceutically acceptable adjuvants, familiar to a person
skilled in the art of immunisation therapy. Preferably, in an
immunisation therapy using the inventive DNA or fractions thereof,
said DNA is preferably administered intramuscularly, whereby said
DNA is incorporated in suitable plasmide carriers. An additional
gene or genes encoding a suitable immunostimulating agent can
preferably be incorporated in the same plasmide.
[0034] Said immunisation therapies are not restricted to the
above-described routes of administration, but can naturally be
adapted to any one of the following routes of administration: oral,
nasal, subcutaneous and intramuscular. Especially the oral and
nasal methods of administration are promising, in particular for
large-scale immunisations.
[0035] The present inventors have surprisingly shown, that highly
specific and therapeutically efficient polyclonal and/or monoclonal
immunoglobulin preparations can be provided through the
immunisation of an animal with an adhesin protein or fractions
thereof, specific for H. pylori. When considering immunisation
against H. pylori, it is worth noting that the infection is known
to be lifelong despite a vigorous immune response in the gastric
mucosa. An increased local production of IgA in the mucosa is not
necessarily enough and the administration of monospecific
antibodies directed against a central virulens factor, such as the
adhesin according to the present invention, may constitute a more
effective approach.
[0036] The term "immunisation" refers here to a method for inducing
a continuous high level of antibody and/or cellular immunresponse.
The term "animal" here preferentially denotes any member of the
subphylum Vertebrata, a division that includes all animals,
including mammals, which are characterized by a segmented bony or
cartilaginous spinal column. All vertebrates have a functional
immune system and respond to antigens by producing antibodies. The
term "protein" is used here to denote a naturally occurring
polypeptide and the term "polypeptide" is used here in its widest
meaning, i.e. any amino acid polymer (dipeptide or longer) linked
through peptide bonds. Accordingly the term "polypeptide" comprises
proteins, oligopeptides, protein fragments, analogues, muteins,
fusion proteins and the like. The term "antibody" as used in this
context includes an antibody belonging to any of the immunological
classes, such as immunoglobulins A, D, E, G or M. Of particular
interest are nevertheless immunoglobulin A (IgA) since this is the
principle immunoglobulin produced by the secretory system of
warm-blooded animals. However, in cow colostrum, the main antibody
class is IgG 1.
[0037] Boren et al. have recently isolated and characterized a
Lewis.sup.b binding protein with a molecular weight of about 73500
Da (See the priority applications SE 9602287-6 and SE 9701014-4,
which are referred to in their entirety). This adhesin protein is
thought to be a conserved structure and specific for pathogenic
strains of H. pylori. Said protein is specific for at least one of
the H. pylori strains included in the following group: CCUG 17875,
NCTC 11637, A5, P466, G109, G56, Ba 185, Ba 99, 931 and 932.
[0038] This adhesin protein or immunologically effective fractions
thereof are characterized in that the following amino acid sequence
is included:
TABLE-US-00001 EDDGFYTSVGYQIGEAAQMV
or homologues thereof.
[0039] The following DNA sequence or homologues thereof is included
in DNA for expression of said adhesin protein or fractions
thereof:
TABLE-US-00002 5'-GAAGACGACGGCTTTTACACAAGCGTAGGCTATCAAATCGGTGAAGC
CGCTCAAATGGTA-3'
[0040] According to one embodiment of the invention, a pregnant
mammal, preferably a cow or another suitable domestic animal, is
immunised with said Lewis.sup.b binding adhesin protein or
fractions thereof. The adhesin protein or fractions thereof is/are
preferably injected intramuscularly or subcutaneously in the chosen
animal, optionally together with suitable adjuvants. Examples of
such adjuvants include, but are not limited to immunostimulating
agents such as cholera toxin and/or derivatives thereof, heat
labile toxins, such as E. coli toxin and similar, conventional
agents, such as classical adjuvants including mineral and vegetable
oils. Subsequent to the regimen of immunization, comprising a
necessary amount of doses, including so called booster-doses, over
a time span allowing for optimal immunoglobulin expression, milk or
sera is collected from said animal. Preferably the cow colostrum,
which is specially high in immunoglobulins, is collected. The
specific immunoglobulin fraction according to the present invention
is then separated and purified in a conventional manner, e g
including separation of fats, protein precipitation and
concentration by ultrafiltration.
[0041] According to another embodiment of the invention, a bird,
preferably a chicken or another suitable domestic bird, is
immunized with said Lewis.sup.b binding adhesin protein or
fractions thereof. The adhesin protein or fractions thereof is
preferably injected intra-muscularly or subcutaneously in the
chosen bird, optionally together with suitable adjuvants. Examples
of such adjuvants include, but are not limited to immunostimulating
agents such as cholera toxin and/or derivatives thereof, heat
labile toxins, such as E. Coli toxin and similar, conventional
agents, such as classical adjuvants including mineral and vegetable
oils. Subsequent to the regimen of immunization, comprising a
necessary amount of doses, including so called booster-doses, over
a time span allowing for optimal immunoglobulin expression, sera or
eggs is/are collected from said animal. Preferably the egg yolk,
which is specially high in immunoglobulins, is collected. The
specific immunoglobulin fraction according to the present invention
is then separated and purified in a conventional manner, e g
including protein precipitation and ultrafiltration. Alternatively,
the egg yolk being of high nutritional value in addition to
containing a high titer of specific antibodies according to the
present invention, can be administered as such.
[0042] According to a preferred embodiment of the present
invention, monoclonal immunoglobulin is produced by establishing
transgenic animals. Said transgenic animals can be chosen from the
following group of species: mammals, e.g. cow, goat and rabbit, and
birds: e.g. chicken, duck, turkey. The mammal most preferably used
is cow and the most preferable bird is chicken. Further
developments of transgenic animals such as mice and rats could also
offer new possibilities. The choice of animal is naturally governed
by availability and local adaptation.
[0043] According to one embodiment, a stock of transgenic animals
according to the present invention, adapted to the local
conditions, are kept locally, e.g. in villages in developing
countries to function as local units for the production of
immunoglobulins for oral administration. For example transgenic
cows, goats or chicken are suitable for this purpose and preferably
chicken are used. Consumption of the milk or preferably the eggs,
produced by the transgenic animals, can help to eradicate presently
very difficult infectious diseases, e.g. diseases caused by H.
pylori.
[0044] According to yet another embodiment of the present
invention, monoclonal antibodies can be produced using the
hybridoma method. The hybridoma method is well known to a skilled
worker in the field of biochemistry and it is described e.g. in
Galfre, G. And Milstein, C., Preparation of monoclonal antibodies:
strategies and procedures (Methods in Enzymology, 73:3-46, 1981). A
suitable host animal is immunized with the Lewis.sup.b binding
adhesin protein or fractions thereof. When the immunization is
accomplished, the animal is sacrificed, spleen cells collected and
fused with cells from a neoplastic cell line, preferably myeloma
cells. By choosing the growth conditions, the successfully fused
hybridoma cells can be selected. The monoclonal antibodies produced
by the hybridoma cell line can then be administered orally in a
regimen for treatment and/or prevention of H. pylori
infections.
[0045] Preferably the polyclonal and/or monoclonal antibodies are
purified prior to administration and, more preferably, admixed with
pharmaceutically suitable carriers and/or adjuvants. Examples of
suitable carriers are saline, pharmaceutically acceptable fats,
oils, carbohydrates and proteins. The carrier or carriers is/are
preferably chosen so that the solubility and absorption of the
immunoglobulin in the mucus layer lining the stomach is enhanced.
Using suitable adjuvants the stability, therapeutic efficiency and
nutritional value of the composition can be improved. To improve
stability under storage, the immunoglobulin composition can be
lyophilized. Regardless of the exact preparation and formulation,
it is of central importance to avoid denaturating the
immunoglobulins.
[0046] The higher specificity, exhibited by the immunoglobulin
preparation of polyclonal and/or monoclonal antibodies according to
the invention, makes it possible use substantially lower doses
compared to those presently used, thus lowering the cost and
improving the availability of the treatment. The use of specific,
monoclonal antibodies can make it possible to further lower the
doses. The doses are in all cases a function of the antibody titer
of the preparation. A high titer naturally allows the use of lower
doses.
[0047] According to one embodiment of the invention, an
immunoglobulin preparation is manufactured as follows: an animal is
immunized with a Lewis.sup.b binding adhesin protein or fractions
thereof, expressed by Helicobacter pylori, the immunoglobulin
fraction is isolated from a excretion of said animal and
subsequently purified. The purified immunoglobulin composition is
admixed with suitable carriers and adjuvants to form a
immunoglobulin preparation for the prevention or treatment of H.
pylori infections. In cases where the antibody titer is
sufficiently high and the other constituents of the immunoglobulin
composition isolated from the animal are harmless, for example in
the case of colostrum from immunized cows or egg yolk from
immunized chicken, there is always the option of administering the
colostrum or egg yolk to the patient without any further treatment
of the colostrum or egg yolk.
[0048] The immunoglobulin composition according to the invention is
preferably administered orally to the patient, in the smallest
therapeutically or prophylactically effective dose. Presently
conceived are doses in the interval of 0.1 to 1000 mg/day,
preferably in the interval of 0.1 to 100 mg/day. The chosen doses
naturally depend on the antibody titer of the preparation in
question. The exact doses and the regimen of administration can be
chosen by the physician responsible for the patient, infected by
Helicobacter pylori. Routine experimentation and later, with
increasing experience of this method, empirical information will
suffice to establish the required amount. Multiple dosages may be
used, as needed, to provide the desired level of therapeutic or
profylactic effect. The immunoglobulin preparations according to
the present invention can also, being free from adverse side
effects and imposing practically no danger of overdosing, be taken
prophylactically or therapeutically by a person without medical
supervision.
[0049] A therapeutical effect can be attained, except with the
specific antibody according to the present invention, also with at
least two Fab-fragments of said antibody. Said embodiment is also
encompassed by the scope of the present invention.
[0050] According to yet another embodiment, avirulent
microorganisms, preferably bacteria, are used as expression systems
for the specific antibody according to the present invention. An
"avirulent microorganism" in this context is a microorganism which
has the ability to colonize and replicate in an infected
individual, but which does not cause disease symptoms associated
with virulent strains of the same species of microorganism. The
definition inherent in the GRAS (Generally Regarded As Safe)
concept can be applied here. A GRAS-organism is suitable for use
according to the present invention, provided that the organism
externalises the antibody or can be modified to this effect. The
term "microorganism" as used herein includes bacteria, protozoa and
unicellular fungi. Preferably, bacteria are used as expression
systems, e.g. bacteria of the genus Lactobacillus, Streptococcus or
Enterobacteriae. The above mentioned expression system can be
utilised in vitro for the production of the specific antibody
according to the present invention or, according to a further
embodiment of the invention, the micro-organism constituting the
expression system can be administered directly to the patient. The
micro-organisms can be harvested and administered as such, but they
are preferably mixed with a suitable carrier, mixed in a suitable
foodstuff, lyophilised, encapsulated or treated in any other
conventional way, used for the delivery of viable microorganisms to
the gastrointestinal tract.
[0051] According to yet another embodiment, avirulent
microorganisms, preferably bacteria, are used as expression systems
for the specific adhesin protein according to the present
invention. An "avirulent microorganism" in this context is a
microorganism which has the ability to colonize and replicate in an
infected individual, but which does not cause disease symptoms
associated with virulent strains of the same species of
microorganism. The definition inherent in the GRAS (Generally
Regarded As Safe) concept can be applied here. A GRAS-organism is
suitable for use according to the present invention, provided that
the organism externalises the adhesin protein or can be modified to
this effect. The term "microorganism" as used herein includes
bacteria, protozoa and unicellular fungi. Preferably, bacteria are
used as expression systems, e.g. bacteria of the genus
Lactobacillus, Streptococcus or Enterobacteriae. The above
mentioned expression system can be utilised in vitro for the
production of the specific adhesin according to the present
invention or, according to a further embodiment of the invention,
the micro-organism constituting the expression system can be
administered directly to the patient. The micro-organisms can be
harvested and administered as such, but they are preferably mixed
with a suitable carrier, mixed in a suitable foodstuff,
lyophilised, encapsulated or treated in any other conventional way,
used for the delivery of viable micro-organisms to the
gastrointestinal tract.
[0052] The exact doses and the regimen of administration of said
micro-organisms can be chosen by the physician responsible for the
patient, infected by Helicobacter pylori. Routine experimentation
and later, with increasing experience of this method, empirical
information will suffice to establish the required amount. Multiple
dosages may be used, as needed, to provide the desired level of
therapeutic or prophylactic effect. The avirulent micro-organism
expressing the antibody or adhesin protein according to the present
invention can also, being free from adverse side effects and
imposing practically no danger of overdosing, be taken
prophylactically or therapeutically by a person without medical
supervision. A preferred carrier in this specific application is a
foodstuff, e.g. a fermented product such as fermented cereal or
dairy product.
[0053] The creation of previously mentioned expression systems and
still earlier mentioned methods of creating hybridomas and
transgenic animals can include steps involving recombinant DNA
techniques. Recombinant DNA techniques are now sufficiently well
known and widespread so as to be considered routine. In very
general and broad terms, recombinant DNA techniques consist of
transferring part of the genetic material of one organism into a
second organism, so that the transferred genetic material becomes a
permanent part of the genetic material of the organism to which it
is transferred. Methods for achieving this are well known and the
mere choice of specific methods for achieving the objectives, set
out in the present description and claims, fall under the scope of
the invention.
[0054] It is possible, that H. pylori alone or together with
related slow-acting bacteria are involved in the genesis and
aggravation of other chronic inflammatory diseases in the
gastrointestinal tract. It is obvious for a skilled practitioner
how to modify the present invention, within the scope of the
claims, to gain utility in the treatment and/or prevention of such
diseases. Examples of such diseases are ulcerative colitis, Crohn's
disease, sarcoidosis, Wegener's granulomatosis and other
vasculithic disorders, as well as various neoplasms, including
carcinomas of the colon, pancreas and prostate.
EXAMPLES
[0055] H. pylori strain CCUG 17875 was obtained from CCUG,
Goteborg, Sweden. Strain A5, a gastric ulcer isolate, from Astra
Arcus, Sodertalje, Sweden. Strains P466 and MO19 were described
previously (Boren et. al, Science, 262, 1892 (1993)). Strain 26695
came from Dr. K. A. Eaton, The Ohio State University and its genome
was recently sequenced by TIGR, Rockville, Md., USA. The panel of
45 H. pylori clinical isolates came from the University Hospital in
Uppsala, Sweden. Bacteria were grown at 37.degree. C. in 10% CO2
and 5% O2 for 48 h.
[0056] All blood group antigen glycoconjugates used, i.e.
semi-synthetic glycoproteins constructed by the conjugation of
purified fucosylated oligosacharides to serum albumin were from
IsoSep AB, Tullinge, Sweden. The RIA was performed according to
Falk et al. (Meth. Enzymol., 236, 353, 1994) with some
modifications; the H-1, Le.sup.b, Le.sup.a, H-2, Le.sup.x and
Le.sup.y glycoconjugates were 125I-labeled by the Chloramine T
method. 1 ml of bacteria (A600=OD 0.10) was incubated with 300 ng
of 125I-labelled conjugate (i.e. an excess of receptors) for 30
min. in phosphate buffered saline (PBS), 0.5% albumin, 0.05%
Tween-20 (BB-buffer). After centrifugation, 125I-activity in the
bacterial pellet was measured by gamma scintillation counting.
[0057] In this study the present inventors' first biochemically
characterized and identified the H. pylori blood group antigen
binding adhesin, BabA. H. pylori strains were analysed for binding
to soluble .sup.125I-labeled fucosylated blood group antigens (FIG.
1A). Binding of these strains to the soluble blood group antigens
correlate with adherence in situ. The prevalence of blood group
antigen binding (BAB)-activity was assessed among 45 clinical H.
pylori isolates and the majority of the isolates, 71%, express
Le.sup.b antigen binding properties (data not shown). In contrast,
none of the reference strains (FIG. 1A), or strains from the panel
of 45 clinical isolates, bind to the Le.sup.a, H-2, Le.sup.x, or
Le.sup.y antigens. These results support our previous findings of
high receptor specificity for the Le.sup.b and H-1 blood group
antigens and demonstrate the high prevalence of BAB activity among
clinical isolates.
[0058] Based on the presence or absence of virulence factors such
as the Cytotoxin associated gene A (CagA) and the Vacuolating
cytotoxin A (VacA), H. pylori strains are classified as type I or
type II strains. H. pylori isolates from patients with duodenal
ulcers most often express the VacA and the CagA-proteins, i.e.
type-I strains. By definition, type II strains express neither
markers. Twenty-one clinical isolates previously defined for
expression of CagA and VacA were analysed for Le.sup.b antigen
binding properties. Expression of CagA was found to correlate with
bacterial binding to the Le.sup.b antigen (Table 1). The cagA gene
belongs to a 40 kb pathogenicity island that encodes components of
secretion and transport systems. These findings could indicate
functional crosstalk between the cag pathogenicity island and the
BabA adhesin gene, for the correct presentation of the BabA adhesin
protein in the bacterial outer membrane.
[0059] To further characterize BabA, the present inventors
determined the affinity constant (K.sub.a) between BabA and the
Le.sup.b antigen. Since K.sub.a-values are based on equilibrium
conditions (13), the present inventors first analysed the
interaction by performing receptor displacement analysis. H. pylori
CCUG 17875 (positive for Le.sup.b binding, FIG. 1 A) was first
incubated with .sup.125I-labeled Le.sup.b glycoconjugate. Then
unlabeled Le.sup.b glycoconjugate was added in a dilution series.
The unlabeled Le.sup.b conjugate displaced the bound
.sup.125I-labeled Le.sup.b glycoconjugate efficiently (FIG. 1 B).
The results demonstrate that the receptor-adhesin complex formed is
in a true state of equilibrium. An equivalent excess of Le.sup.a
glycoconjugate did not dissociate the Le.sup.b-BabA complex,
verifying the high receptor specificity (FIG. 1B). The
K.sub.a-value for the Le.sup.b-BabA complex of strain CCUG 17875
was titrated with Le.sup.b glycoconjugate in concentrations from 10
ng to 260 ng/ml and determined to be of an high affinity close to
1.times.10.sup.10 M.sup.-1, (FIG. 1C). The number of Le.sup.b
glycoconjugate molecules bound to BabA on the bacterial cell
surface was calculated to be around 500 per cell. This number is
similar to the number or fimbriae organelles on the surface of E.
coli (14). However, for the BabA adhesin, the calculations are
based on the assumption that the majority of bacterial cells in the
experiment exhibit an equal number of adhesin molecules with
Le.sup.b antigen binding properties.
TABLE-US-00003 TABLE 1 BAB activity among H. pylori Type I and Type
II strains Type Strain BAB Activity Type I CCUG 17874 - CagA.sup.+,
VacA.sup.+ G39 - G11 - G20 - G27 + G56 + G106 - G109 + 932 + Bal85
+ 87A300 + Type Ia 931 + CagA.sup.+, VacA.sup.- Ba99 + Ba179 +
Ba194 + Type Ib G12 - CagA.sup.-, VacA.sup.+ TypeId G104 -
.DELTA.cagA, VacA.sup.+ Tx30 - Type II G21 - CagA.sup.-, VacA.sup.-
G50 - G198 -
[0060] To determine the prevalence of BabA in the bacterial
population, strain CCUG 17875 was incubated with Le.sup.b or
Le.sup.a antigens, and bacterial binding activity was visualised by
confocal fluorescence microscopy (FIG. 2, upper panel). The
analyses demonstrate the high prevalence of BabA binding activity
in the bacterial population to the Le.sup.b antigen (FIG. 2A, green
staining) and the complete lack of binding to the Le.sup.a antigen
(FIG. 2B, red counter staining).
[0061] Next, the localisation and density of BabA on the bacterial
cell surfaces was investigated by immunogold electron microscopy.
The Le.sup.b antigen binding activity of the adhesin localised gold
particles to the bacterial outer membrane (FIG. 2C). Individual
bacterial cells exhibit an equal number of gold particles (data not
shown). When the Le.sup.b antigen was substituted with the Le.sup.a
antigen (lacking receptor activity), no gold particles were
detected (FIG. 2D).
[0062] The molecular weight of BabA was characterized by receptor
overlay analysis. A protein extract of strain CCUG 17875 was
separated on SDS-PAGE and blotted to a membrane. The membrane was
incubated with biotinylated Le.sup.b glycoconjugate, followed by
detection with streptavidin and enhanced chemiluminescence. The
BabA adhesin activity corresponds to a single 74 kDa band (FIG.
3A). The 40 kDa band is presumably endogenous peroxidase activity
since it stains independently of the Le.sup.b conjugate overlay
(lane 3). BabA was very heat stable and could regain some activity
after heating to 97.degree. C. (FIG. 3A, lane 2). The panel of
strains exhibited the same molecular weight of BabA (FIG. 3B).
[0063] To purify BabA, a novel technique was developed, Receptor
Activity Directed Affinity Tagging (ReTagging). Multi-functional
crosslinking agents with radiolabeled donating tags have been
previously used for receptor-ligand characterization studies.
However, the use of affinity donating tags, such as biotin residues
presented on flexible spacer structures, adds a new dimension to
the applicability of crosslinker technology. An affinity tag,
biotin, is transferred to the adhesin protein by the receptor
activity and is used for further identification and for affinity
purification of the adhesin part of the interaction, by
streptavidin (FIG. 4A, B).
[0064] A multi-functional crosslinking agent with a biotin donating
handle was attached to the Le.sup.b glycoconjugate. The receptor
activity of the Le.sup.b glycoconjugate subsequently directed the
targeted biotin tagging of the BabA adhesin protein (FIG. 4A, B).
After crosslinking, the bacterial protein from strains A5, P466,
and CCUG 17875 were separated on SDS-PAGE. Immunodetection with
streptavidin demonstrated a biotin tagged protein, with the
molecular weight of 74 kDa (FIG. 3C) (28), These results support
the estimates of the molecular weight from the previous overlay
analyses (FIG. 3B). Strain MO19 devoid of Le.sup.b antigen binding
properties (FIG. 3B) (FIG. 1A), was negative for binding also in
this set of analyses (FIG. 3C).
[0065] The high specificity in the ReTagging technique provided a
method for purification of the adhesin protein. Strains CCUG 17875
and A5, that both express the BabA adhesin (FIG. 1A) were processed
by the ReTagging technique using crosslinker labelled Le.sup.b
receptor conjugate as the biotin donor. After crosslinking,
bacteria were suspended in SDS sample buffer. Streptavidin coated
magnetic beads were subsequently added to the solubilised proteins,
and biotin tagged BabA was extracted (FIG. 4C). The N-terminal 20
amino acid sequences of the BabA adhesins from strains CCUG 17875
(Australia) and A5 (Sweden) were found to be identical, indicating
a biologically conserved protein (FIG. 5). Recently, a series of
outer membrane proteins from H. pylori were characterized. These
proteins, HopA-E, are homologous in their N-terminal sequences to
BabA (17), possible indicating a motif for a common secretion
mechanism. The biotin tagged BabA adhesin was purified more than
3000-fold from the cell extract, and the yield was calculated to
20%. However, based on data from the Scatchard plots, the level of
available BabA adhesin would be about 5-times higher, i.e.
approximately 1 mg adhesin/750 mg bacterial protein, which
nevertheless could be the reason for the high signal to noise ratio
(FIG. 3B). The purification of BabA via the ReTagging technique
indicates the potential of this technique for the purification of
lectins in complex receptor-ligand interactions, such as the
selectin family of cell adhesion molecules.
[0066] To clone the gene encoding BabA, the N-terminal 20 aa
sequence was utilised for the construction of degenerate primers
(18). Two sets of clones were identified which both encode two
different but very similar proteins. Both genes code for proteins
having almost identical N-terminal domains and identical C-terminal
domains, complicating the identification of the functional BabA
gene. (FIG. 5). To identify the corresponding gene, the BabA
adhesin was purified in large scale by ReTagging. This provided
enough protein for an extended amino terminal sequence. 41 amino
acids were identified and these residues unambiguously
discriminated between the two genes by the differences in
aa-positions 28, 35, 37, 38 and 41 (FIG. 5). The gene encoding BabA
was named babA and correspond to a basic protein with a pI of 9.4
and a molecular weight of 78 kDa, i.e. of slightly higher molecular
weight than that predicted from the SDS PAGE analyses (FIG. 3). The
other gene, babB, corresponds to a protein of a calculated
molecular weight of 75.5 kDa. In contrast to babA, the babB gene
contains a predicted translational initiation codon (FIG. 5). This
could indicate the existence of a third bab gene in the genome or
mechanisms for recombination activities. Interestingly, the
bab-genes were also detected in strains lacking Lewis b binding
properties (data not shown). Gene cassette systems have been shown
to promote antigenic variation in Neisseria gonorrhoeae (19).
Another possibility would be the presence of similar genes coding
for adhesins with differences in receptor specificity/host tissue
tropism (20). Gene inactivation experiments targeting the bab-genes
could aid in understanding this complex gene organisation.
[0067] Immunisation experiments with adhesins from Bordetella
pertussis (21) indicate the potential for outer membrane proteins
to act as vaccine candidates (discussed in ref. 22). In a mouse
model for persistent H. pylori infection, oral immunisation with H.
pylori antigens proved protective against H. pylori infection (10).
However, results from animal models are difficult to evaluate for
human specific pathogens, such as H. pylori and Polio virus. For
Polio, an animal model has been achieved by expressing the virus
receptor in transgenic mice (23). A similar strategy was taken for
H. pylori. A transgenic mouse was constructed by the use of an
.alpha.1,3/4-fucosyltransferase, driving the synthesis of the human
specific Le.sup.b antigen in the gastrointestinal tract (24). The
Lewis b mouse can be useful for the evaluation of the role of the
BabA adhesin as a colonisation/virulence factor and in addition for
the evaluation of BabA as a vaccine candidate against acid peptic
disease and gastric adenocarcinoma.
[0068] In the present study the ReTagging technique was used for
the purification of the adhesin part of the microbial
receptor-ligand interaction. By the use of purified
adhesin/lectin-protein, the ReTagging technique could, in addition,
be used to further study the receptor part of the interaction.
Identification of the biologically active receptor structure,
carrying Le.sup.b oligosaccharides, would aid in the understanding
of the mechanisms supporting the chronic H. pylori infection.
[0069] Inhibition of H. pylori binding to .sup.125I-labeled Lewis b
antigen by preparations is presented graphically, as a function of
antibody concentration (mg/ml) in FIG. 6: 1 ml aliquots of H.
pylori bacteria (A.sub.600=OD 0.10) were pre-incubated with
dilution series of antibody preparations, in 0.01-10 mg/ml for 2
hours in phosphate buffered saline (PBS), 0.5% albumin, 0.05%
Tween-20. Then 500 ng of .sup.125I-labeled conjugate (i.e. an
excess of receptor structure) was added and incubated for 30
minutes. After centrifugation, .sup.125I-activity in the bacterial
pellet was measured by gamma scintillation counting. The Lewis b
blood group antigen glycoconjugates used, i.e. semi-synthetic
glycoproteins constructed by the conjugation of purified
fucosylated oligosaccharides to serum albumin were from IsoSep AB,
Tullinge, Sweden.
[0070] Western blot detection of the BabA adhesin by the different
antibody preparations is presented in FIG. 7: Molecular weight
rainbow marker (2 .mu.L) from Amersham, Buckinghamshire, England,
was dissolved in SDS sample buffer (lane 1). Approx. 100 ng of
purified BabA adhesin (approx. 74 kDa with degradation product of
approx. 55 kDa) was dissolved in SDS sample buffer (lane 2). SDS
solubilized protein extracts of strain CCUG 17875 were prepared by
dissolving the bacterial pellet corresponding to 0.15 ml of
bacteria (A.sub.600=OD 0.10) by SDS sample buffer (lane 3). The 3
protein samples were then boiled at 100.degree. C. for 5 minutes.
The proteins were separated on SDS-PAGE, and transferred to a
PVDF-membrane for Western blot immuno analysis. Five sets of
PVDF-membranes were prepared. The PVDF membranes were
blocked/incubated overnight with 4% human sera/plasma, in phosphate
buffered saline, from a patient with no H. pylori infection, i.e.
with no serum antibodies against H. pylori. The membrane was then
washed in phosphate buffered saline (PBS), 0.5% albumin, 0.05%
Tween-20, followed by the addition of the antibody preparations.
The sets of membranes were incubated with the following 5 antibody
preparations; 1) pooled human sera from H. pylori infected
patients, diluted 1:500. 2) Chicken antibodies (positive) 1 mg/ml
diluted 1:100.times., 3) Bovine I preparation of antibodies, 1
mg/ml diluted 1:100.times.. 4) Bovine II preparation of antibodies,
1 mg/ml diluted 1:100.times.. 5) Bovine III preparation of
antibodies, 1 mg/ml diluted 1:100.times. (indicated in the figure).
These antibodies were incubated with the membrane for 2 hours
followed by extensive washings in phosphate buffered saline (PBS),
0.05% Tween-20, followed by the addition of secondary anti-human,
anti-chicken, and anti-bovine antibodies labeled with
HRP-peroxidase (from DAKO, Denmark), all diluted 1:2000.times..
Membranes were incubated for 1 hour, followed by extensive washings
in phosphate buffered saline (PBS), 0.05% Tween-20. The membranes
were developed with enhanced chemoluminescens (ECL) from Amersham.
The results show, that the antigenic response against the adhesin
is strongly enhanced in the bovine preparations. This finding is
also supported by the inhibition data in FIG. 6.
[0071] Western blot analyses of H. pylori proteins by the different
antibody preparations are shown in FIG. 8. 2 clinical isolates
(1-2) from Dr. Lars Engstrand, Department of Clinical Microbiology
and Cancerepidemiology, University Hospital, Uppsala, Sweden and
strain CCUG 17875 (3), from Culture Collection, University of
Goteborg, Department of Clinical Bacteriology, Goteborg, Sweden,
and strain 52 (4) from Prof. Torkel Wadstrom, Dept. Medical
Microbiology, Lunds University, were prepared for SDS-PAGE
electrophoresis. Bacterial pellets corresponding to 0.15 ml of
bacteria (A.sub.600=OD 0.10) were dissolved in SDS sample buffer
and heated to 100.degree. C. for 5 minutes. The proteins were
separated on SDS-PAGE, and transferred to PVDF-membranes for
Western blot immuno analysis. The western blot analyses were as
described above, i.e. the sets of membranes were incubated with the
following 4 antibody preparations; 1) pooled human sera from H.
pylori infected patients, diluted 1:500. 2) Chicken antibodies
(positive) 1 mg/ml diluted 1:100.times., 3) Bovine I preparation of
antibodies, 1 mg/ml diluted 1:100.times.. 4) Bovine III preparation
of antibodies, 1 mg/ml diluted 1:100.times. (indicated in the
figure). These antibodies were incubated with the membrane for 2
hours followed by extensive washings in phosphate buffered saline
(PBS), 0.05% Tween-20, followed by the addition of secondary
anti-human, anti-chicken, and anti-bovine antibodies labeled with
HRP-peroxidase (from DAKO, Denmark), all diluted 1:2000.times..
Membranes were incubated for 1 hour, followed by extensive washings
in phosphate buffered saline (PBS), 0.05% Tween-20. The membranes
were developed with enhanced chemoluminescens (ECL) from Amersham.
The results show, that the chicken antibodies and the bovine
preparations reacts nearly identically against all four strains,
indicating conserved properties in strains of different
geographical origin.
[0072] Although the invention has been described with regard to its
preferred embodiments, which constitute the best mode presently
known to the inventors, it should be understood that various
changes and modifications as would be obvious to one having the
ordinary skill in this art may be made without departing from the
scope of the invention which is set forth in the claims appended
hereto.
REFERENCES AND NOTES
[0073] 1. J. R. Warren, Lancet, i, 1273, 1983, B. Marshall, Lancet,
i, 1273, 1983. [0074] 2. A. Dubois, Emerging Infectious Diseases 1,
79 (1995). [0075] 3. M. J. Blaser, Sci. Amer. 2, 92 (1996). [0076]
4. 3. M. J. Blaser, Trends Microbiol. 7, 255 (1993), D. E.
Kirschner and M. J. Blaser, J. Theor. Biol. 176, 281 (1995). [0077]
5. P. Falk, T. Boren, and S, Normark, Meth. Enzymol. 236, 353
(1994). [0078] 6. D. G. Evans, D. J. Evans Jr., J. J. Moulds and D.
Y. Graham, Infect. Immun. 56, 2896 (1988), S. Hirmo, M. Utt, M.
Ringner and T. Wadstrom, FEMS Immunol. and Med. Microbiol. 10, 301
(1995), T. Saitoh, et al FEBS Lett. 282, 385 (1991). [0079] 7. T.
Boren, P. Falk, K. A. Roth, G. Larson, and S, Normark, Science.
262, 1892 (1993), P. Falk, et al Proc. Natl. Acad. Sci. U.S.A. 90,
2035 (1993). [0080] 8. H. pylori strain CCUG 17875 was obtained
from CCUG, Goteborg, Sweden. Strain A5, a gastric ulcer isolate,
came from Astra Arcus, Sodertalje, Sweden. Strains P466 and MO19
were described previously (7). Strain 26695 came from Dr. K A.
Eaton, The Ohio State University, and its genome was recently
sequenced by The Institute for Genomic Research (TIGR), Rockville,
Md. (J.-F. Tomb, et al, abstract 3B: 059, IX International Workshop
on Gastroduodenal Pathology and Helicobacter pylori, Copenhagen,
Denmark, 1996). The panel of 45 H. pylori clinical isolates came
from the University Hospital in Uppsala, Sweden. Bacteria were
grown at 37(C in 10% CO.sub.2 and 5% O.sub.2 for 48 h. [0081] 9.
All blood group antigen glycoconjugates used, i.e. semi-synthetic
glycoproteins constructed by the conjugation of purified
fucosylated oligosaccharides to serum albumin (7, 25), were from
IsoSep AB, Tullinge, Sweden. The RIA was performed according to
ref. 26 with some modifications; The H-1, Le.sup.b, Le.sup.a, H-2,
Le.sup.x, and Le.sup.y glycoconjugates were .sup.125I-labeled by
the Chloramine T method. 1 ml of bacteria (A.sub.600=OD 0.10) was
incubated with 300 ng of .sup.125I-labeled conjugate (i.e. an
excess of receptors) for 30 min. in phosphate buffered saline
(PBS), 0.5% albumin, 0.05% Tween-20 (BB-buffer). After
centrifugation, .sup.125I-activity in the bacterial pellet was
measured by gamma scintillation counting. [0082] 10. A. Covacci, et
al, Proc. Natl. Acad. Sci. U.S.A. 90, 5791 (1993), M. Marchetti, et
al, Science 267, 1655 (1995). [0083] 11 S. Censini et al, Proc.
Natl. Acad. Sci. U.S.A. 93, 14648, (1996). [0084] 12. Z. Xiang, et
al, Infect. Immun. 63, 94 (1995). [0085] 13. A. G. Scatchard, Ann.
N.Y. Acad. Sci. 51, 600 (1949). [0086] 14. O. Mol, and B. Oudega,
FEMS. Microbiol. Reviews, 19, 25 (1996). [0087] 15. Confocal
microscopy was performed on a Nikon/Multiprobe 2001 instrument
(Molecular Dynamics, Sunnyvale, Calif.). Electron microscopy was
performed on a JEOL 100 CX instrument. [0088] 16. J. Brunner,
Trends in Cell Biol. 6, 154 (1996), J. D. Bleil and P. M.
Wassarman, Proc. Natl. Acad. Sci. U.S.A. 87, 5563, (1990). [0089]
17. M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock,
Infect. Immun. 63, 1567 (1995), P. Doig, M. M. Exner, R. E. W.
Hancock and T. J. Trust, J. Bacteriol. 177, 5447 (1995). [0090] 18.
The BabA N-terminal sequence analysis was used to make degenerate
oligonucleotides, which were used in PCR to obtain an amplified
fragment from the chromosome of the babA gene. A 59 bp fragment was
identified and used as probe for the screening of a low-copy
plasmide (pACYC184) library of Sau3A partially digested chromosomal
DNA from strain CCUG 17875. [0091] 19. P. Hagblom, E. Segal, E.
Billyard, and M. So, Nature, 315, 156 (1985), R. Haas and T. F.
Meyer, Cell, 44, 107 (1986). [0092] 20. A.-B. Jonsson, D. Ilver, P.
Falk, J. Pepose, and S, Normark, Mol. Microbiol, 13, 403 (1994), N.
Stromberg, P. G. Nyholm, I. Pascher, and S, Normark, Proc. Natl.
Acad. Sci. USA 88, 9340 (1991). [0093] 21. A. Kimura, K. T.
Mountzouros, D. A. Relman, S. Falkow, J. L. Cowell, Infect. Immun.
58, 7 (1990). [0094] 22. T. Boren, and P. Falk, Sci. Amer., Sci.
& Med. 4 (1994), L. S. Tompkins and S. Falkow, Science 267,
1621 (1995). [0095] 23. R. B. Ren, et al, Cell 63, 353 (1990).
[0096] 24. P. G. Falk, L. Bry, J. Holgersson, and J. I. Gordon,
Proc. Natl. Acad. Sci. U.S.A. 92, 1515 (1995). [0097] 25. P. D.
Rye, Nature Biotechnology. 2, 155 (1996). [0098] 26 P. Falk, T.
Boren, D. Haslam, M. G. Caparon, Meth. Cell Biol. 45, 161 (1994)
[0099] 27. Cell extracts were prepared in SDS sample buffer without
mercapto ethanol and heated at 37.degree. C. or 97.degree. C. for
10 min. before separation on SDS-PAGE. Proteins were blotted onto a
PVDF membrane. The membrane was incubated with 1 .mu.g/ml
biotinylated Le.sup.b glycoconjugate or biotinylated albumin
(negative control) overnight, labelled as described in ref. 7.
After washing in PBS/0.05% Tween-20, the biotinylated structures
bound by the BabA band were probed by HRP-streptavidin and detected
using ECL reagents (Amersham, Buckinghamshire, England). [0100] 28.
The bacterial suspension was incubated with Le.sup.b
glycoconjugate, to which the Sulfo-SBED crosslinker (Pierce,
Rockville, Ill.) had been conjugated by the N-hydroxysuccinimide
ester (NHS), according to the manufacturers specifications. The
aryl azide crosslinker group was activated by UV irradiation (360
nm). Bacteria were washed with PBS pH 7.6, 0.05% Tween-20 and
protease inhibitors (EDTA and benzamidine) under reducing
conditions with 50 mM dithiothreitol (DTT). Bacterial proteins were
separated on SDS-PAGE, and the biotin tagged BabA protein was
detected by immunodetection (PVDF membrane/HRP-streptavidin and
ECL) (FIG. 3C). [0101] 29. Strains CCUG 17875 and A5 were first
processed by crosslinking and DTT treatment, as above (28),
followed by solubilisation in SDS sample buffer. The biotin tagged
BabA protein was then extracted with streptavidin coated magnetic
beads (Advanced Magnetics Inc., Cambridge, Mass.). The beads were
boiled in SDS sample buffer, and bound proteins were eluted and
alkylated. The protein preparation was further fractionated by
preparative SDS-PAGE (Prep-Cell 491, BioRad, Hercules, Calif.).
Fractions with the biotin tagged protein, i.e. the BabA fractions,
were identified by immunodetection using streptavidin/ECL. The
pooled BabA preparation was then separated on SDS-PAGE and
transferred to PVDF membrane. The BabA band was excised and the
BabA protein was N-terminally sequenced using a Procise.TM. 494
instrument (Applied Biosystems, Foster City, Calif.).
Sequence CWU 1
1
913340DNAHelicobacter pylori 1tttcagtcaa gcccaaagct atgcgcaaaa
cgcttatgct aaagagaatt tacaagcaca 60gccgtccaag tatcaaaaca gcgtgcctga
aatcaatatt gatgaagaag aaatcccctt 120taagggttaa aattaaggag
acattatgga aagaaaacgc tattcaaaac gctattgcaa 180atacactgaa
gctaaaatca gctttattga ctataaagat ttggacatgc tcaagcacac
240gctatcagag cgctataaaa tcatgccaag gaggttgaca ggcaatagca
aaaagtggca 300agagagggtg gaagttagcg atcaaaagag cccgccacat
ggctttaatc ccctacattg 360tggataggaa aaaagtcgtg gatagccctt
ttaaacagca ctgaattttt gattagggct 420aatagggggc atgcctttta
atcttgttta atcttggctc tatttttgtt aaacatcggt 480tataaaagcg
ttaaaagcac ttttaaaatc caattaaaag cgttcaaaag taacgcaaaa
540aatcaaaaaa atgacaaaat ttttaagaaa atgacaaaaa aaaaaaaaac
gctttatgct 600ataatattcc aaatacattc taatgcaaat gcattctaat
gcaaatgtat aatgaatgta 660tgaaatccct aatattcaat ccaatttaat
ccaaaaagga gaaaaaacac atcctttcat 720taactttagg ctcgctttta
gtttccactt tgagcgctga agacgacggc ttttacacaa 780gcgtaggcta
tcaaatcggt gaagccgctc aaatggtaac aaacaccaaa ggcatccaag
840atctttcaga caactatgaa aacttgagca aacttttgac ccgatacagc
accctaaaca 900cccttatcaa attgtccgct gatccgagcg cgattaacgc
ggcacgtgaa aatctgggcg 960cgagcgcgaa gaacttgatc ggcgatacca
aaaattcccc cgcctatcaa gccgtgcttt 1020tggcgatcaa tgcggcggta
gggttttgga atgtcttagg ctatgctacg caatgcgggg 1080gtaacgctaa
tggtcaagaa agcacctctt caaccaccat cttcaacaac gagccagggt
1140atcgatccac ttccatcact tgcagtttga acaggtataa gcctggatac
tacggcccta 1200tgagcattga aaatttcaaa aagcttaacg aagcctatca
aatcctccaa acggctttaa 1260ataaaggctt acccgcgctc aaagaaaaca
acggaacggt cagtgtaacc tacacctaca 1320catgctcagg ggaagggaat
gataactgct cgaaaaaagc cacaggtgta agtgaccaaa 1380atggcggaac
caaaactaaa acccaaacca tagacggcaa aaccgtaacc accacgatca
1440gttcaaaagt cgttgatagt caggcaaaag gtaatacaac aagggtgtcc
tacaccgaaa 1500tcactaacaa attagacggt gtgcctgata gcgctcaagc
gctcttggcg caagcgagca 1560cgctcatcaa caccatcaac acggcatgcc
cgtattttag tgtaactaat aaaagtggtg 1620gtccacagat ggaaccgact
agagggaagt tgtgcggttt tacagaagaa atcagcgcga 1680tccaaaagat
gatcacagac gcgcaagagc tggtcaatca aacgagcgtc attaacgagc
1740atgaacaatc aaccccggta ggcggtaata atggcaagcc tttcaaccct
ttcacggacg 1800caagcttcgc tcaaggcatg ctcgctaacg ctagtgcgca
agccaaaatg ctcaatctag 1860cccatcaagt ggggcaaacc attaaccctg
acaatcttac cgggactttt aaaaattttg 1920ttacaggctt tttagccaca
tgcaacaaca aatcaacagc tggcactagt ggcacacaag 1980gttcacctcc
tggcacagta accactcaaa ctttcgcttc cggttgcgcc tatgtggagc
2040aaaccataac gaatctaaac aacagcatcg ctcattttgg cactcaagag
cagcagatac 2100agcaagctga aaacatcgct gacactctag tgaatttcaa
atctagatac agcgaattag 2160ggaatactta taacagcatc actactgcgc
tctccaaagt ccctaacgcg caaagcttgc 2220aaaacgtggt gggaaaaaag
aataacccct atagcccgca aggcatagaa accaattact 2280acttgaatca
aaactcttac aaccaaatcc aaaccatcaa ccaagaatta gggcgtaacc
2340cctttaggaa agtgggcatc gtcagttctc aaaccaacaa tggtgccatg
aatgggatcg 2400gtatccaggt gggctacaag caattctttg ggcaaaaaag
gaaatggggt gcaagatact 2460acggcttttt tgattacaac catgcgttca
ttaaatccag cttcttcaac tcggcttctg 2520acgtgtggac ttatggtttt
ggagcggacg ctctttataa cttcatcaac gataaagcca 2580ccaatttctt
aggcaaaaac aacaagcttt ctgtggggct ttttggcggg attgcgttag
2640cgggcacttc atggcttaat tctgaatacg tgaatttagc caccatgaat
aacgtctata 2700acgctaaaat gaacgtggcg aacttccaat tcttattcaa
catgggagtg aggatgaatt 2760tagccagatc caagaaaaaa ggcagcgatc
atgcggctca gcatggcatt gagttagggc 2820ttaaaatccc caccattaac
acgaactact attcctttat gggggctgaa ctcaaatacc 2880gcaggctcta
tagcgtgtat ttgaattatg tgttcgctta ctaaaaacta aaaatccttt
2940gtggaactcc ctttttaagg ggtttctttt aaagccttta tttttttttg
gaggggttta 3000atttttttga aacctttgtt tttgaattct ctttttaatg
ggtttctttt ttgaactctt 3060tgttttgaac tccttttttt gaactccctt
ttttaaaccc tttctttttt aaaattctct 3120tttttggggg gtttgatgaa
aaatcctttt ttagcgtttt ggtattggtt agtggaaaac 3180ttgatactaa
tttaagcgat agtttttaaa aagtgcttct ttaatatagg gggtttaagt
3240tggtgattaa aaggggggaa tggtttcaaa gcgcttccta tccctttaag
aaaataaaat 3300aaaactttaa taaaatgagt tttacaacaa aatgagatcc
334022781DNAHelicobacter pylori 2catttgatcg cattggattt caaagaaggg
cgttttgtga aaggctttgg tcaagcttat 60gatattttag gcgacaaaat cgcttatgtt
gggggtaaag gcaacccaca caatttcgct 120cacaagaaat aaactttctc
acccataagg ggcaaacgcc cccaaaagag tgctttttaa 180agaggttaag
gcaaaatcaa gctctttagt atttaatctt aaaaaatact aaaagccttt
240ttatgggcta acaccacaca aaaagcgtca aaatcaaaaa aatgacaaaa
ttttccccaa 300atgacaaaaa aaaaaaaaaa cgattttatg ctatattaac
gaaatcttgt gataagatct 360tattctttta aaagatttac ctaaccattt
taatttcaag gagaaaacat gaaaaaaaac 420ccttttactc tctctctctc
tctctcgttt ttgctccacg ctgaagacga cggcttttac 480acaagcgtag
gctatcaaat cggtgaagcc gctcaaatgg taaccaacac caaaggcatc
540caacagcttt cagacaatta tgaaaagctg aacaatcttt tgaataatta
cagcacccta 600aacaccctta tcaaattatc cgctgatccg agtgcgatta
acgacgcaag ggataatcta 660ggctcaagtg ctaagaattt gcttgatgtt
aaaaccaact ccccggccta tcaagccgtg 720cttttagcgt tgaatgcggc
ggtggggttg tggcaagtta caagctacgc ttttactgct 780tgtggtcctg
gcagtaacga gagcgcaaat ggaggtatcc aaacttttaa taatgtgcca
840ggacaaaaga cgacaaccat cacttgcaat tcgtattatc aaccaggaca
tggtgggcct 900atatccactg caaactatgc aaaaatcaat caagcctatc
aaatcattca aaaggctttg 960acagccaatg aagctaatgg agatggggtc
cccgttttaa gcgacaccac tacaaaactt 1020gatttcacta ttcaaggaga
caaaagaacg ggtggccgac caaatacacc taaaaagttc 1080ccatggagtg
atgggaaata tattcacacc caatggattg acacaacacc acaatcaaca
1140gaaacaaaga tcaacacaga aaataacgct caagagcttt taaaacaagc
gagcatcatt 1200atcactaccc taaatgaggc atgcccaaac ttccaaaatg
gtggtagcgg ttattggcaa 1260gggataagcg gcaatgggac aatgtgtggg
atgtttaaga atgaaatcag cgctatccaa 1320ggcatgatcg ctaacgcgca
agaagctgtc gcgcaaagta aaatcgttag tgaaaatgcg 1380caaaatcaaa
acaacttgga tactggaaaa ccattcaacc ctttcacgga cgctagcttc
1440gctcaaagca tgctcaaaaa cgctcaagcc caagcagaga ttttaaacca
agccgaacaa 1500gtggtgaaaa actttgaaaa aatccctaaa aattttgtat
cagactcttt aggggtgtgt 1560tatgaagagc aagggggtga gcgtaggggc
accaatccag gtcaggttac ttctaacact 1620ttcgcttccg gttgcgccta
tgtggagcaa accataacga atctaaacaa cagcatcgct 1680cattttggca
ctcaagagca gcagatacag caagctgaaa acatcgctga cactctagtg
1740aatttcaaat ctagatacag cgaattaggg aatacttata acagcatcac
tactgcgctc 1800tccaaagtcc ctaacgcgca aagcttgcaa aacgtggtgg
gaaaaaagaa taacccctat 1860agcccgcaag gcatagaaac caattactac
ttgaatcaaa actcttacaa ccaaatccaa 1920accatcaacc aagaattagg
gcgtaacccc tttaggaaag tgggcatcgt cagttctcaa 1980accaacaatg
gtgccatgaa tgggatcggt atccaggtgg gctacaagca attctttggg
2040caaaaaagga aatggggtgc aagatactac ggcttttttg attacaacca
tgcgttcatt 2100aaatccagct tcttcaactc ggcttctgac gtgtggactt
atggttttgg agcggacgct 2160ctttataact tcatcaacga taaagccacc
aatttcttag gcaaaaacaa caagctttct 2220gtggggcttt ttggcgggat
tgcgttagcg ggcacttcat ggcttaattc tgaatacgtg 2280aatttagcca
ccatgaataa cgtctataac gctaaaatga acgtggcgaa cttccaattc
2340ttattcaaca tgggagtgag gatgaattta gccagatcca agaaaaaagg
cagcgatcat 2400gcggctcagc atggcattga gttagggctt aaaatcccca
ccattaacac gaactactat 2460tcctttatgg gggctgaact caaataccgc
aggctctata gcgtgtattt gaattatgtg 2520ttcgcttact agaaactaaa
aatcctttgt ggaactccct ttttaagggg tttcttttaa 2580agcctttatt
tttttttgga ggggtttaat ttttttgaaa cctttgtttt tgaattctct
2640ttttaatggg tttctttttt gaactctttg ttttgaactc ctttttttga
actccctttt 2700ttaaaccctt tcttttttaa aattctcttt tttggggggt
ttgatgaaaa atcctttttt 2760agcgttttgg tattggttag t
27813744PRTHelicobacter pyloriPEPTIDE(24)..(64)N-terminal domain of
the BabA adhesin 3Ser Lys Lys Glu Lys Lys His Ile Leu Ser Leu Thr
Leu Gly Ser Leu 1 5 10 15Leu Val Ser Thr Leu Ser Ala Glu Asp Asp
Gly Phe Tyr Thr Ser Val 20 25 30Gly Tyr Gln Ile Gly Glu Ala Ala Gln
Met Val Thr Asn Thr Lys Gly 35 40 45Ile Gln Asp Leu Ser Asp Asn Tyr
Glu Asn Leu Ser Lys Leu Leu Thr 50 55 60Arg Tyr Ser Thr Leu Asn Thr
Leu Ile Lys Leu Ser Ala Asp Pro Ser 65 70 75 80Ala Ile Asn Ala Ala
Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu 85 90 95Ile Gly Asp Thr
Lys Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala 100 105 110Ile Asn
Ala Ala Val Gly Phe Trp Asn Val Leu Gly Tyr Ala Thr Gln 115 120
125Cys Gly Gly Asn Ala Asn Gly Gln Glu Ser Thr Ser Ser Thr Thr Ile
130 135 140Phe Asn Asn Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys
Ser Leu145 150 155 160Asn Arg Tyr Lys Pro Gly Tyr Tyr Gly Pro Met
Ser Ile Glu Asn Phe 165 170 175Lys Lys Leu Asn Glu Ala Tyr Gln Ile
Leu Gln Thr Ala Leu Asn Lys 180 185 190Gly Leu Pro Ala Leu Lys Glu
Asn Asn Gly Thr Val Ser Val Thr Tyr 195 200 205Thr Tyr Thr Cys Ser
Gly Glu Gly Asn Asp Asn Cys Ser Lys Lys Ala 210 215 220Thr Gly Val
Ser Asp Gln Asn Gly Gly Thr Lys Thr Lys Thr Gln Thr225 230 235
240Ile Asp Gly Lys Thr Val Thr Thr Thr Ile Ser Ser Lys Val Val Asp
245 250 255Ser Gln Ala Lys Gly Asn Thr Thr Arg Val Ser Tyr Thr Glu
Ile Thr 260 265 270Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gln Ala
Leu Leu Ala Gln 275 280 285Ala Ser Thr Leu Ile Asn Thr Ile Asn Thr
Ala Cys Pro Tyr Phe Ser 290 295 300Val Thr Asn Lys Ser Gly Gly Pro
Gln Met Glu Pro Thr Arg Gly Lys305 310 315 320Leu Cys Gly Phe Thr
Glu Glu Ile Ser Ala Ile Gln Lys Met Ile Thr 325 330 335Asp Ala Gln
Glu Leu Val Asn Gln Thr Ser Val Ile Asn Glu His Glu 340 345 350Gln
Ser Thr Pro Val Gly Gly Asn Asn Gly Lys Pro Phe Asn Pro Phe 355 360
365Thr Asp Ala Ser Phe Ala Gln Gly Met Leu Ala Asn Ala Ser Ala Gln
370 375 380Ala Lys Met Leu Asn Leu Ala His Gln Val Gly Gln Thr Ile
Asn Pro385 390 395 400Asp Asn Leu Thr Gly Thr Phe Lys Asn Phe Val
Thr Gly Phe Leu Ala 405 410 415Thr Cys Asn Asn Lys Ser Thr Ala Gly
Thr Ser Gly Thr Gln Gly Ser 420 425 430Pro Pro Gly Thr Val Thr Thr
Gln Thr Phe Ala Ser Gly Cys Ala Tyr 435 440 445Val Glu Gln Thr Ile
Thr Asn Leu Asn Asn Ser Ile Ala His Phe Gly 450 455 460Thr Gln Glu
Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu465 470 475
480Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser
485 490 495Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu
Gln Asn 500 505 510Val Val Gly Lys Lys Asn Asn Pro Tyr Ser Pro Gln
Gly Ile Glu Thr 515 520 525Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn
Gln Ile Gln Thr Ile Asn 530 535 540Gln Glu Leu Gly Arg Asn Pro Phe
Arg Lys Val Gly Ile Val Ser Ser545 550 555 560Gln Thr Asn Asn Gly
Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr 565 570 575Lys Gln Phe
Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly 580 585 590Phe
Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser 595 600
605Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn
610 615 620Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn
Lys Leu625 630 635 640Ser Val Gly Leu Phe Gly Gly Ile Ala Leu Ala
Gly Thr Ser Trp Leu 645 650 655Asn Ser Glu Tyr Val Asn Leu Ala Thr
Met Asn Asn Val Tyr Asn Ala 660 665 670Lys Met Asn Val Ala Asn Phe
Gln Phe Leu Phe Asn Met Gly Val Arg 675 680 685Met Asn Leu Ala Arg
Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln 690 695 700His Gly Ile
Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr705 710 715
720Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val
725 730 735Tyr Leu Asn Tyr Val Phe Ala Tyr 7404707PRTHelicobacter
pyloriPEPTIDE(46)..(59)Corresponding to the N-terminal domain of
the BabA adhesin 4Met Lys Lys Asn Pro Phe Thr Leu Ser Leu Ser Leu
Ser Phe Leu Leu 1 5 10 15His Ala Glu Asp Asp Gly Phe Tyr Thr Ser
Val Gly Tyr Gln Ile Gly 20 25 30Glu Ala Ala Gln Met Val Thr Asn Thr
Lys Gly Ile Gln Gln Leu Ser 35 40 45Asp Asn Tyr Glu Lys Leu Asn Asn
Leu Leu Asn Asn Tyr Ser Thr Leu 50 55 60Asn Thr Leu Ile Lys Leu Ser
Ala Asp Pro Ser Ala Ile Asn Asp Ala 65 70 75 80Arg Asp Asn Leu Gly
Ser Ser Ala Lys Asn Leu Leu Asp Val Lys Thr 85 90 95Asn Ser Pro Ala
Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala Ala Val 100 105 110Gly Leu
Trp Gln Val Thr Ser Tyr Ala Phe Thr Ala Cys Gly Pro Gly 115 120
125Ser Asn Glu Ser Ala Asn Gly Gly Ile Gln Thr Phe Asn Asn Val Pro
130 135 140Gly Gln Lys Thr Thr Thr Ile Thr Cys Asn Ser Tyr Tyr Gln
Pro Gly145 150 155 160His Gly Gly Pro Ile Ser Thr Ala Asn Tyr Ala
Lys Ile Asn Gln Ala 165 170 175Tyr Gln Ile Ile Gln Lys Ala Leu Thr
Ala Asn Glu Ala Asn Gly Asp 180 185 190Gly Val Pro Val Leu Ser Asp
Thr Thr Thr Lys Leu Asp Phe Thr Ile 195 200 205Gln Gly Asp Lys Arg
Thr Gly Gly Arg Pro Asn Thr Pro Lys Lys Phe 210 215 220Pro Trp Ser
Asp Gly Lys Tyr Ile His Thr Gln Trp Ile Asp Thr Thr225 230 235
240Pro Gln Ser Thr Glu Thr Lys Ile Asn Thr Glu Asn Asn Ala Gln Glu
245 250 255Leu Leu Lys Gln Ala Ser Ile Ile Ile Thr Thr Leu Asn Glu
Ala Cys 260 265 270Pro Asn Phe Gln Asn Gly Gly Ser Gly Tyr Trp Gln
Gly Ile Ser Gly 275 280 285Asn Gly Thr Met Cys Gly Met Phe Lys Asn
Glu Ile Ser Ala Ile Gln 290 295 300Gly Met Ile Ala Asn Ala Gln Glu
Ala Val Ala Gln Ser Lys Ile Val305 310 315 320Ser Glu Asn Ala Gln
Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro Phe 325 330 335Asn Pro Phe
Thr Asp Ala Ser Phe Ala Gln Ser Met Leu Lys Asn Ala 340 345 350Gln
Ala Gln Ala Glu Ile Leu Asn Gln Ala Glu Gln Val Val Lys Asn 355 360
365Phe Glu Lys Ile Pro Lys Asn Phe Val Ser Asp Ser Leu Gly Val Cys
370 375 380Tyr Glu Glu Gln Gly Gly Glu Arg Arg Gly Thr Asn Pro Gly
Gln Val385 390 395 400Thr Ser Asn Thr Phe Ala Ser Gly Cys Ala Tyr
Val Glu Gln Thr Ile 405 410 415Thr Asn Leu Asn Asn Ser Ile Ala His
Phe Gly Thr Gln Glu Gln Gln 420 425 430Ile Gln Gln Ala Glu Asn Ile
Ala Asp Thr Leu Val Asn Phe Lys Ser 435 440 445Arg Tyr Ser Glu Leu
Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu 450 455 460Ser Lys Val
Pro Asn Ala Gln Ser Leu Gln Asn Val Val Gly Lys Lys465 470 475
480Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn
485 490 495Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu
Gly Arg 500 505 510Asn Pro Phe Arg Lys Val Gly Ile Val Ser Ser Gln
Thr Asn Asn Gly 515 520 525Ala Met Asn Gly Ile Gly Ile Gln Val Gly
Tyr Lys Gln Phe Phe Gly 530 535 540Gln Lys Arg Lys Trp Gly Ala Arg
Tyr Tyr Gly Phe Phe Asp Tyr Asn545 550 555 560His Ala Phe Ile Lys
Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp 565 570 575Thr Tyr Gly
Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys 580 585 590Ala
Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe 595 600
605Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val
610 615 620Asn Leu Ala Thr Met Asn Asn Val Tyr Asn Ala Lys Met Asn
Val Ala625 630 635 640Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg
Met Asn Leu Ala Arg 645 650 655Ser Lys Lys Lys Gly Ser Asp His Ala
Ala Gln His Gly Ile Glu Leu 660 665 670Gly Leu Lys Ile Pro Thr Ile
Asn Thr Asn Tyr
Tyr Ser Phe Met Gly 675 680 685Ala Glu Leu Lys Tyr Arg Arg Leu Tyr
Ser Val Tyr Leu Asn Tyr Val 690 695 700Phe Ala
Tyr705520PRTHelicobacter pylori 5Glu Asp Asp Gly Phe Tyr Thr Ser
Val Gly Tyr Gln Ile Gly Glu Ala 1 5 10 15Ala Gln Met Val
20660DNAHelicobacter pylori 6gaagacgacg gcttttacac aagcgtaggc
tatcaaatcg gtgaagccgc tcaaatggta 60741PRTHelicobacter pylori 7Glu
Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala 1 5 10
15Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Asp Leu Ser Asp Asn
20 25 30Tyr Glu Asn Leu Ser Lys Leu Leu Thr 35 40820PRTHelicobacter
pylori 8Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu
Ala 1 5 10 15Ala Gln Met Val 20941PRTHelicobacter pylori 9Glu Asp
Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala 1 5 10
15Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln Leu Ser Asp Asn
20 25 30Tyr Glu Lys Leu Asn Asn Leu Leu Asn 35 40
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