U.S. patent application number 09/955739 was filed with the patent office on 2003-01-09 for urease-based vaccine and treatment for helicobacter infection.
Invention is credited to Blum, Andre, Corthesy-Theulaz, Irene, Davin, Catherine, Haas, Rainer, Kraehenbuhl, Jean-Pierre, Michetti, Pierre, Saraga, Emilia.
Application Number | 20030007980 09/955739 |
Document ID | / |
Family ID | 22741337 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030007980 |
Kind Code |
A1 |
Michetti, Pierre ; et
al. |
January 9, 2003 |
Urease-based vaccine and treatment for helicobacter infection
Abstract
Method of eliciting in a mammalian host a protective immune
response to Helicobacter infection and treatment of Helicobacter
infection by administering to the host an immunogenically effective
amount of a Helicobacter urease or urease subunits as antigen.
Vaccine compositions are also provided.
Inventors: |
Michetti, Pierre; (Lausanne,
CH) ; Corthesy-Theulaz, Irene; (Lausanne, CH)
; Blum, Andre; (Romammotier, CH) ; Davin,
Catherine; (Rule de Moulins, FR) ; Haas, Rainer;
(Tubingen, DE) ; Kraehenbuhl, Jean-Pierre; (Rivaz,
CH) ; Saraga, Emilia; (Lausanne, CH) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
22741337 |
Appl. No.: |
09/955739 |
Filed: |
September 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09955739 |
Sep 18, 2001 |
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08200346 |
Feb 23, 1994 |
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6290962 |
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08200346 |
Feb 23, 1994 |
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08085938 |
Jul 6, 1993 |
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5972336 |
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08085938 |
Jul 6, 1993 |
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07970996 |
Nov 3, 1992 |
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Current U.S.
Class: |
424/190.1 ;
424/234.1 |
Current CPC
Class: |
A61K 2039/6037 20130101;
A61K 2039/541 20130101; Y02A 50/476 20180101; A61K 2039/55505
20130101; A61P 31/04 20180101; Y02A 50/484 20180101; C12N 9/80
20130101; A61K 2039/55544 20130101; A61K 39/105 20130101; A61K
39/00 20130101; Y02A 50/30 20180101; Y02A 50/466 20180101; A61K
2039/542 20130101 |
Class at
Publication: |
424/190.1 ;
424/234.1 |
International
Class: |
A61K 039/02 |
Claims
We claim:
1. A method of treating gastroduodenal disease in a mammal, said
method comprising administering a therapeutically effective amount
of a composition comprising Helicobacter urease peptides.
2. The method of claim 1 wherein said gastroduodenal disease is
gastritis.
3. The method of claim 1 wherein said gastroduodenal disease is
peptic ulcer disease.
4. The method of claim 1 wherein said gastroduodenal disease is
chronic dyspepsia with severe erosive gastroduodenitis.
5. The method of claim 1 wherein said gastroduodenal disease is
refractory non-ulcer dyspepsia.
6. The method of claim 1 wherein said gastroduodenal disease is
intestinal metaplasia.
7. The method of claim 1 wherein said gastroduodenal disease is low
grade MALT lymphoma.
8. The method of claim 1 wherein said gastroduodenal disease is
Helicobacter infection.
9. The method of claim 1 wherein said gastroduodenal disease is
Helicobacter pylori infection.
10. The method of claim 1 wherein said gastroduodenal disease is H.
felis disease.
11. The method of claim 1 wherein said mammal is human.
12. The method of claim 1 wherein said composition comprises
Helicobacter urease.
13. The method of claim 1 wherein said composition comprises the
ure B subunit of Helicobacter urease.
14. The method of claim 1 wherein said composition comprises the
ure B subunit of Helicobacter pylori urease.
15. The method of claim 1 further comprising administering said
composition to a mucosal surface.
16. The method of claim 1 wherein said composition is administered
orally, nasally, rectally, or ocularly.
17. The method of claim 1 further comprising administering said
composition in a dosage ranging from 100 .mu.g to 1 g.
18. The method of claim 17 further comprising administering said
dosage over three to eight doses for a primary immunization
schedule over one month.
19. The method of claim 1 wherein said composition is administered
in association with a mucosal adjuvant.
20. The method of claim 19 wherein said mucosal adjuvant is
selected from the group consisting of procholeragenoid; cholera
toxin B subunit fungal polysaccharides, including schizophyllan;
muramyl dipeptide; muramyl dipeptide derivatives; phorbol esters;
liposomes; microspheres; non-Helicobacter pylori bacterial lysates;
labile toxin of Escherichia coli; block polymers; saponins; and
ISCOMs.
21. The method of claim 1 wherein said urease peptides are
genetically or chemically linked to a mucosal adjuvant.
22. The method of claim 21, wherein said mucosal adjuvant is the
cholera toxin B subunit.
23. The method of claim 1 wherein said composition is administered
in association with a carrier such that the composition is
delivered in particulate form.
24. The method of claim 23 wherein said carrier is
hydroxyapatite.
25. The method of claim 1 wherein said composition is administered
in association with a microsphere carrier.
26. The method of claim 25 wherein said microsphere carrier is a
polylactide-coglycolide biodegradable microsphere carrier.
27. The method of claim 1 wherein said composition comprises a
recombinant live vector or a recombinant carrier system which
expresses a Helicobacter urease peptide.
28. The method of claim 27 wherein said live vector is selected
from the group consisting of Salmonella typhimurium, Salmonella
typhi, Shigella, Bacillus, Lactobacillus, BCG, Escherichia coli,
Vibrio Cholerae, Campylobacter, Yeast, Herpes virus, Adenovirus,
Poliovirus, Vaccinia, and Avipox.
29. The method of claim 27 wherein said carrier system is selected
from the group consisting of Bluetongue virus-like particles,
Rotavirus virus-like particles and Ty particles.
30. The method of claim 27 wherein said live vector or carrier
system is administered to a mucosal surface.
31. A method of treating a human infected with Helicobacter pylori,
said method comprising orally administering a therapeutically
effective amount of a composition comprising the ure B subunit of
Helicobacter pylori urease, in association with a mucosal adjuvant
selected from the group consisting of procholeragenoid; cholera
toxin B subunit fungal polysaccharides, including schizophyllan;
muramyl dipeptide; muramyl dipeptide derivatives; phorbol esters;
liposomes; microspheres; non-Helicobacter pylori bacterial lysates;
labile toxin of Escherichia coli; block polymers; saponins; and
ISCOMs; said composition administered in particulate form in
association with hydroxyapatite.
32. A method of treating a human infected with Helicobacter pylori,
said method comprising orally administering a therapeutically
effective amount of a composition comprising the ure B subunit of
Helicobacter pylori urease in the form of a fused protein,
genetically linked to the cholera toxin B subunit, said composition
administered in particulate form in association with
hydroxyapatite.
33. A method of treating a mammal infected with Helicobacter, said
method comprising administering a therapeutically effective amount
of a composition comprising peptides that display epitopes
sufficiently homologous to epitopes displayed by Helicobacter
urease such that antibodies that recognize epitopes displayed by
Helicobacter urease will recognize epitopes displayed by said
peptides.
34. A method of treating gastroduodenal disease in a mammal, said
method comprising administering a therapeutically effective amount
of a composition comprising an antibody that recognizes
Helicobacter urease.
35. The method of claim 34 wherein said gastroduodenal disease is
Helicobacter infection.
36. The method of claim 34 wherein said gastroduodenal disease is
Helicobacter pylori infection.
37. The method of claim 34 wherein said mammal is human.
38. The method of claim 34 wherein said antibody is specific for
Helicobacter pylori urease.
39. The method of claim 34 wherein said antibody is specific for
the ure B subunit of Helicobacter pylori urease.
40. The method of claim 34 wherein said antibody is a monoclonal
antibody.
41. The method of claim 34 wherein said antibody is an IgA
antibody.
42. A method of treating a human infected with Helicobacter Pylori,
said method comprising administering a therapeutically effective
amount of a composition comprising an IgA monoclonal antibody that
recognizes the ure B subunit of Helicobacter pylori urease.
43. A method of treating a mammal infected with Helicobacter, said
method comprising administering a therapeutically effective amount
of a composition comprising anti-idiotypic antibodies to
Helicobacter urease.
44. A composition useful in the therapeutic treatment of
gastroduodenal disease, said composition comprising Helicobacter
urease peptides.
45. The composition of claim 44 wherein said gastroduodenal disease
is gastritis.
46. The composition of claim 44 wherein said gastroduodenal disease
is peptic ulcer disease.
47. The composition of claim 44 wherein said gastroduodenal disease
is chronic dyspepsia with severe erosive gastroduodenitis.
48. The composition of claim 44 wherein said gastroduodenal disease
is refractory non-ulcer dyspepsia.
49. The composition of claim 44 wherein said gastroduodenal disease
is intestinal metaplasia.
50. The composition of claim 44 wherein said gastroduodenal disease
is low grade MALT lymphoma.
51. The composition of claim 44 wherein said gastroduodenal disease
is Helicobacter infection.
52. The composition of claim 44 wherein said gastroduodenal disease
is Helicobacter pylori infection.
53. The composition of claim 44 wherein said gastroduodenal disease
is Helicobacter felis disease.
54. The composition of claim 44 wherein said mammal is human.
55. The composition of claim 44 wherein said Helicobacter urease
peptides comprise Helicobacter urease.
56. The composition of claim 44 wherein said Helicobacter urease
peptides comprise the ureB subunit of Helicobacter urease.
57. The composition of claim 44 wherein said Helicobacter urease
peptides comprise Helicobacter pylori urease.
58. The composition of claim 44 wherein said Helicobacter urease
comprise the ure B subunit of Helicobacter pylori urease.
59. The composition of claim 44 further comprising a mucosal
adjuvant.
60. The composition of claim 59 wherein said mucosal adjuvant is
selected from the group consisting of procholeragenoid; cholera
toxin B subunit fungal polysaccharides, including schizophyllan;
muramyl dipeptide; muramyl dipeptide derivatives; phorbol esters;
liposomes; microspheres; non-Helicobacter pylori bacterial lysates;
labile toxin of Escherichia coli; block polymers; saponins; and
ISCOMs.
61. The composition of claim 44 wherein said urease peptides are
genetically or chemically linked to a mucosal adjuvant.
62. The composition of claim 61, wherein said mucosal adjuvant is
cholera toxin B subunit.
63. The composition of claim 44 further comprising a carrier such
that the composition can be delivered in particulate form.
64. The composition of claim 44 wherein said carrier is
hydroxyapatite.
65. The composition of claim 44 further comprising a microsphere
carrier.
66. The composition of claim 65, wherein said microsphere carrier
is a polylactide-coglycolide biodegradable microsphere carrier.
67. The composition of claim 44 wherein said composition comprises
a recombinant live vector or a recombinant carrier system which
expresses a Helicobacter urease peptide.
68. The composition of claim 67 wherein said live vector is
selected from the group consisting of Salmonella typhimurium,
Salmonella typhi, Shigella, Bacillus, Lactobacillus, BCG,
Escherichia coli, Vibrio cholerae, Campylobacter, Yeast, Herpes
virus, Adenovirus, Poliovirus, Vaccinia, and Avipox.
69. The composition of claim 67 wherein said carrier system is
selected from the group consisting of Bluetongue virus-like
particles, Rotavirus virus-like particles, and Ty particles.
70. A composition useful in the therapeutic treatment of
Helicobacter pylori infection of a human, said composition
comprising the ure B subunit of Helicobacter pylori urease, a
mucosal adjuvant selected from a group consisting of
procholeragenoid; cholera toxin B subunit fungal polysaccharides,
including schizophyllan; muramyl dipeptide; muramyl dipeptide
derivatives; phorbol esters; liposomes; microspheres;
non-Helicobacter pylori bacterial lysates; labile toxin of
Escherichia coli; block polymers; saponins; and ISCOMs, and further
comprising hydroxyapatite.
71. A composition useful in the therapeutic treatment of
Helicobacter pylori infection of a human, said composition
comprising the ure B subunit of Helicobacter pylori urease in the
form of a fused protein, genetically linked to the cholera toxin B
subunit and hydroxyapatite, in particulate form.
72. A composition useful for the therapeutic treatment of a mammal
infected with Helicobacter, said composition comprising peptides
that display epitopes sufficiently homologous to epitopes displayed
by Helicobacter urease such that antibodies that recognize epitopes
displayed by Helicobacter urease will recognize epitopes displayed
by said peptides.
73. A composition useful for the therapeutic treatment of a mammal
infected with Helicobacter said composition comprising
anti-idiotypic antibodies to Helicobacter urease.
74. A composition useful in the therapeutic treatment of
gastroduodenal disease, said composition comprising an antibody
that recognizes Helicobacter urease.
75. The composition of claim 74 wherein said gastroduodenal disease
is Helicobacter infection.
76. The composition of claim 74 wherein said gastroduodenal disease
is Helicobacter pylori infection.
77. The composition of claim 74 wherein said gastroduodenal disease
is Helicobacter felis disease.
78. The composition of claim 74 wherein said mammal is human.
79. The composition of claim 74 wherein the antibody is specific
for Helicobacter pylori urease.
80. The composition of claim 74 wherein the antibody is specific
for the ure B subunit of Helicobacter pylori urease.
81. The composition of claim 74 wherein the antibody is a
monoclonal antibody.
82. The composition of claim 74 wherein the antibody is an IgA
antibody.
83. A composition useful in the therapeutic treatment of
gastroduodenal disease, said composition comprising an IgA
monoclonal antibody that recognizes the ure B subunit of
Helicobacter pylori urease and a mucosal adjuvant.
84. A method of preventing Helicobacter pylori infection of a
human, said method comprising orally administering a
pro-phylactically effective amount of a composition comprising
Helicobacter pylori urease, in association with a mucosal adjuvant
selected from the group consisting of procholeragenoid; cholera
toxin B subunit; fungal saccharides, including schizophyllan;
muramyl dipeptide; muramyl dipeptide derivatives; phorbol esters;
liposomes; microspheres; non-Helicobacter pylori bacterial lysates;
labile toxin of Escherichia coli; block polymers; saponins; and
ISCOMs, said composition administered in particulate form in
association with hydroxyapatite.
85. A method of preventing Helicobacter pylori infection of a
human, said method comprising orally administering a
prophylactically effective amount of a composition comprising
Helicobacter pylori urease, in association with a mucosal adjuvant
selected from the group consisting of procholeragenoid; cholera
toxin B subunit; fungal saccharides, including schizophyllan;
muramyl dipeptide; muramyl dipeptide derivatives; phorbol esters;
liposomes; microspheres; non-Helicobacter pylori bacterial lysates;
labile toxin of Escherichia coli; block polymers; saponins; and
ISCOMs, said composition administered in particulate form in
association with hydroxyapatite.
86. A method of preventing Helicobacter pylori infection of a
human, said method comprising orally administering a
pro-phylactically effective amount of a composition comprising
Helicobacter pylori urease in the form of a fused protein,
genetically linked to the cholera toxin B subunit, said composition
administered in particulate form, in association with
hydroxyapatite.
87. A composition useful in preventing Helicobacter pylori
infection of a human, said composition comprising Helicobacter
pylori urease, in association with a mucosal adjuvant selected from
the group consisting of procholeragenoid; cholera toxin B subunit;
fungal polysaccharides, including schizophyllan; muramyl dipeptide;
muramyl dipeptide derivatives; phorbol esters; liposomes;
microspheres; non-Helicobacter pylori bacterial lysates; labile
toxin of Escherichia coli; block polymers; saponins; and ISCOMs,
said composition present in particulate form in association with
hydroxyapatite.
88. A composition useful in preventing Helicobacter pylori
infection of a human, said composition comprising Helicobacter
pylori urease in the form of a fused protein, genetically linked to
the cholera toxin B subunit, said fused protein present in
particulate form, in association with hydroxyapatite.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/085,938, filed Jul. 6, 1993, which is a
continuation-in-part application of U.S. application Ser. No.
07/970,996, filed Nov. 3, 1992, the whole of which applications
(including drawings) are hereby incorporated by reference.
[0002] The present invention relates to the prevention and
treatment of gastric infection in mammals, including humans. More
particularly, the present invention relates to a vaccine suitable
for use in the prevention and treatment of Helicobacter infection
in mammals, including humans, and to a method of treatment of
humans suffering from gastric infection, its consequences such as
chronic gastritis or peptic ulcer, and prevention of gastric
cancer.4
BACKGROUND OF THE INVENTION
[0003] Helicobacter infections of human gastric epithelium cause
gastritis, are a major factor in the development of peptic ulcers
and gastric lymphoma, and may be a risk factor for the development
of gastric cancer. (Blaser, M. J. "Gastric Campylobacter-like
Organisms, Gastritis and Peptic Ulcer Disease" Gastroenterology,
vol. 93, 371-383 (1987); Graham, D. Y. "Campylobacter pylori and
Peptic Ulcer Disease" Gastroenterology, vol. 196, 615-625 (1989);
Parsonnet, J. et al. "Helicobacter pylori Infection in Intestinal
and Diffuse-Type Gastric Adenocarcinomas" J. Natl. Cancer Inst.,
vol. 93, 640-643 (1991); Wotherspoon, A. C., et al., "Regression of
Primary Low-Grade B-Cell Gastric Lymphoma of Mucosa-Associated
Lymphoid Tissue Type After Eradication of Helicobacter pylori,"
Lancet, vol. 342, 575-577 (1993)). The most frequent infection
agent is Helicobacter pylori, followed at a much lower frequency by
Helicobacter heilmanii. Helicobacter pylori is a slender S-shaped
gram negative microorganism, which is routinely recovered from
gastric biopsies of adults and children with histologic evidence of
gastritis or peptic ulceration. Evidence for a causal relationship
between Helicobacter pylori and gastroduodenal disease comes from
studies in human volunteers, patients with ulcers and gastric
cancer, gnotobiotic pigs, and germ-free rodents. Regarding
etiology, Koch's postulates were satisfied by creating
histologically confirmed gastritis in previously uninfected
individuals following consumption of viable microorganisms.
(Marshall, B. J. et al. "Attempt to Fulfill Koch's Postulate for
pyloric Campylobacter" Med. J. Aust., vol. 142, 436-439 (1985);
Morris, A. et al. "Ingestion of Campylobacter pyloritis Causes
Gastritis and Raised Fasting Gastric pH" Am. J. Gastroenterol.,
vol. 82, 192-199 (1987); Engstrand, L. et al. "Inoculation of
Barrier-Born Pigs With Helicobacter pylori: A Useful Animal Model
for Gastritis Type B" Infect. Immun., vol. 53, 1763-1768 (1990);
Fox, J. G. et al. "Gastric Colonization by Campylobacter pylori
Subsp. mustelae in Ferrets" Infect. Immun., vol. 56, 2994-2996
(1988); Fox, J. G. et al. "Helicobacter mustelae-Associated
Gastritis in Ferrets: An Animal Model of Helicobacter pylori
Gastritis in Humans" Gastroenterology, vol. 99, 352-361 (1990);
Lee, A. et al. "A Small Animal Model of Human Helicobacter pylori
Active Chronic Gastritis" Gastroenterology, vol. 99, 1315-1323
(1990); Fox, J. G. et al. "Helicobacter Felis Gastritis in
Gnotobiotic Rats: An Animal Model of Helicobacter pylori Gastritis"
Infect. Immun., vol. 59, 785-791 (1991); Eaton, K. A. et al.
"Campylobacter pylori Virulence Factors in Gnotobiotic Piglets"
Infect. Immun., vol. 57, 1119-1125 (1989)), and by treatment to
eradicate Helicobacter pylori, with resolution of the gastritis
and, in patients with peptic ulcer disease, a decrease in the
recurrence rate. (Peterson, W. L. "Helicobacter pylori and Peptic
Ulcer Disease" N. Engl. J. Med., vol. 324, 1043-1048 (1991)).
[0004] Gastroduodenal diseases thought to be associated with
Helicobacter infection include acute, chronic, and atrophic
gastritis, peptic ulcer disease including both gastric and duodenal
ulcers, gastric cancer, chronic dyspepsia with severe erosive
gastroduodenitis, refractory non-ulcer dyspepsia, intestinal
metaplasia, and low grade MALT lymphoma. Helicobacter infection is
also the principle cause of asymptomatic chronic gastritis.
[0005] In spite of in vitro susceptibility to many antimicrobial
agents, in vivo eradication of established Helicobacter pylori
infections with antimicrobial agents is often difficult to achieve.
(Czinn, S. J. and Nedrud, J. G. "Oral Immunization Against
Helicobacter pylori" Infect. Immun., vol. 59, 2359-2363 (1991)).
The microorganism is found within the mucous coat overlying the
gastric epithelium and in gastric pits. These are locations which
do not appear to allow for adequate antimicrobial levels to be
achieved even when antibiotics are given orally at high doses. At
the present time, most authorities recommend a "triple therapy",
namely a bismuth salt in combination with drugs such as
tetracycline and metronidazole for 2-4 weeks. However, the
effectiveness of this or other chemotherapeutic regimens remains
suboptimal. Recently, a National Institutes of Health panel of
medical experts recommended a triple therapy with bismuth,
tetracycline and metronidazole, administered for two weeks for
treatment of peptic ulcers (Cimons, M., "Drug Combination Found
Effective on Peptic Ulcers," L.A. Times at A14 (Feb. 10, 1994)).
However, this treatment is commonly associated with diarrhea and it
may produce serious adverse drug reactions. (See, Dick-Hegedus, E.
and Lee, A., "Use of a Mouse Model to Examine Anti-Helicobacter
pylori Agents," Scand. J. Gastroenterol., vol. 26, 909-915 (1991)).
Treatment with antibiotics also may not solve the problem of
reinfection and there is evidence for a high incidence of
reinfection in some studies (Coelho, L. G., et al., "Duodenal Ulcer
and Eradication of H. pylori in a Developing Country: An 18-Month
Follow-Up Study," Scand. J. Gastroenterol. vol. 27, 362-66 (1992)).
Therefore there is a great need for a vaccine that can be used to
treat infection and to prevent future infections.
[0006] At the present time little is known regarding the role of
the mucosal immune systems in the stomach. The distribution of
immunoglobulin (Ig) producing cells in the normal gastric antrum
indicates that IgA plasma cells make up 80% of the total plasma
cell population. In addition, the number of plasma IgA cells
present in the gastric antrum is comparable to other mucus
membranes. (Brandtzaeg, P. "Role of J Chain and Secretory Component
in Receptor-Mediated Glandular and Hepatic Transport of
Immunoglobulins in Man" Scand. J. Immunol., vol. 22, 111-146
(1985); Brandtzaeg, P. et al. "Production and Secretion of
Immunoglobulins in the Gastrointestinal Tract" Ann. Allergy, vol.
59, 21-39 (November, 1987)). A number of studies in humans (Wyatt,
J. I. et al. "Local Immune Response to Gastritis Campylobacter in
Non-ulcer Dyspepsia" J. Clin. Path., vol. 39, 863-870 (1986)), and
in animal models (Fox, J. G. et al. "Helicobacter
mustalae-Associated Gastritis in Ferrets: An Animal Model of
Helicobacter pylori Gastritis in Humans" Gastroenterology, vol. 99,
352-361 (1990); Fox, J. G. et al. "Helicobacter felis Gastritis in
Gnotobiotic Rats: An Animal Model of Helicobacter pylori Gastritis"
Infect. Immun., vol. 59, 785-791 (1991); Fox, J. G. et al. "Local
and Systemic Immune Responses in Murine Helicobacter felis Active
Chronic Gastritis," Infect. & Immun., vol. 61, 2309-15 (1993)),
have demonstrated specific IgG and IgA responses in serum and in
gastric secretions in response to Helicobacter infection. However,
the observation that Helicobacter pylori infection persists as a
chronic infection for years, despite inducing a local and systemic
immune response, is not encouraging the development of immunization
strategies.
[0007] Lee et al. have reported the ability to infect germ-free
rodents with Helicobacter felis, a bacterium closely related to
Helicobacter pylori, and reproducibly document histologic
gastritis. (Lee, A. et al. "A Small Animal Model of Human
Helicobacter pylori Active Chronic Gastritis" Gastroenterology,
vol. 99, 1315-1323 (1990); Fox, J. G. et al. "Helicobacter felis
Gastritis in Gnotobiotic Rats: An Animal Model of Helicobacter
pylori Gastritis" Infect. Immun., vol. 59, 785-791 (1991)). Since
then, this bacterium-host pairing has been accepted as a good model
to study Helicobacter-mediated gastritis and its initiating
factors. (Lee, A. et al. "Pathogenicity of Helicobacter pylori: A
Perspective" Infect. Immun., vol. 61, 1601-1610 (1993)). Infection
of mice with H. felis results in a similar pathologic response to
that found in humans infected with H. pylori; both types of
infections result in active, chronic gastritis. (Lee et al.,
Gastroenterology, vol. 99, pp. 1315-1323 (1990)). Researchers have
found that Helicobacter felis has the same susceptibility to
antimicrobial therapy as Helicobacter pylori, and the H.
felis/mouse model has been used to develop new treatments against
H. pylori infection. (Dick-Hegedus, E. and Lee, A., "Use of a Mouse
Model to Examine Anti-Helicobacter pylori Agents," Scand. J.
Gastroenterol., vol. 26, 909-915 (1991); Chen et al., "Immunization
Against Gastric Helicobacter Infection in a Mouse/Helicobacter
felis Model," Lancet, vol. 339, p.1120 (1992)). Czinn et al. have
shown that repetitive oral immunization with a crude lysate of
Helicobacter pylori plus cholera toxin adjuvant induces a vigorous
gastrointestinal IgA anti-Helicobacter pylori response in mice and
ferrets. (Czinn, S. J. and Nedrud, J. G. "Oral Immunization Against
Helicobacter pylori" Infect. Immun., vol. 59, 2359-2363 (1991)). In
addition, Chen et al. and Czinn et al. have recently reported that
oral immunization with a crude lysate of H. felis induced
protection against H. felis infection in mice. (Chen, et al.
"Immunization Against Gastric Helicobacter Infection in a
Mouse/Helicobacter felis Model, " (letter) Lancet, vol.
339,1120-1121 (1992); Czinn, S. et al. "Oral Immunization Protects
Germ-Free Mice Against Infection from Helicobacter felis"
Proceedings of the DDW, American Gastroenterological Association,
1321, A-331 (May 10-13, 1992); Czinn et al., Vaccine, vol. 11,
637-42 (1993)). The exact nature of the antigen(s) responsible for
the induction of this protection, however, had not been determined,
and no information suggested that the protective antigen(s) of H.
felis that induced protection against this pathogen would induce a
cross-reactive protection extending to another Helicobacter
species.
[0008] We have demonstrated for the first time that Helicobacter
pylori and H. felis shared antigenic determinants by obtaining
monoclonal antibodies after oral immunization of mice with either
Helicobacter pylori or H. felis sonicates and showing that some of
these antibodies, directed against Helicobacter pylori, would
crossreact with H. felis and vice versa, (Michetti, P. et al.
"Specificity of Mucosal IgA Response in Balb/C Mice Following H.
felis or Helicobacter pylori Challenges" Proceedings of the DDW,
American Gastroenterological Association, 1001, A-251 (May 10-13,
1992); Davin, C. et al. "Helicobacter pylori Urease Elicits
Protection Against H. felis Infection in Mice" Proceedings of the
DDW, American Gastroenterological Association, 1213, A-304 (May
16-19, 1993)), but the basis for these cross-reactivities were
unknown.
[0009] Based on the homology existing between the different known
urease amino acid sequences, it has been proposed that jack bean
urease could be used as a vaccine against Helicobacter pylori.
(Pallen, M. J. and Clayton, C. L. "Vaccination Against Helicobacter
pylori Urease" Lancet, vol. 336, 186-7 (1990)). Nevertheless,
despite the homology among the different urease sequences,
cross-reactivity is not the rule. Guo and Liu have shown years ago
that ureases of Proteus mirabilis, Proteus vulgaris and Providencia
rettgeri show cross-reactivity to each other, while ureases of jack
bean and Morganella morganii are immunologically distinct from the
three former ureases. (Guo, M. and Liu, P. V. "Serological
Specificities of Ureases of Proteus Species" J. Gen. Microbiol,
vol. 136, 1995-2000 (1965)). So, even if an antigenic
cross-reactivity of Helicobacter pylori urease with other
Helicobacter ureases was a reasonable postulate, no data existed
demonstrating that this was really the case until we showed that
some H. felis monoclonal antibodies cross-reacted with Helicobacter
pylori urease. (Davin, C. et al. "Helicobacter pylori Urease
Elicits Protection Against H. felis Infection in Mice" Proceedings
of the DDW, American Gastroenterological Association 1213, A-304
(May 16-19, 1993)). J. Pappo has further demonstrated that mice
which have been infected by H. felis produce antibodies which
crossreact with Helicobacter pylori urease but not jack bean urease
(J. Pappo, unpublished data, 1993). The fact that jack bean urease
does not fall in the same immunological category than Helicobacter
urease suggests that jack bean urease may not be useful for
immunization against Helicobacter infections, the way it was done
for enteric bacteria. (Pimentel, J. L. and Cook, M. E. "Improved
Growth in the Progeny of Hens Immunized with Jackbean Urease"
Poultry Sci., vol. 64, 434-439 (1988)). Furthermore, attempts to
immunize mice against H. felis infection by oral or intraperitoneal
delivery of jack bean urease resulted in the production of
antibodies against jack bean urease, but failed to protect the mice
from infection. (Chen, M. et al. "Failure of Immunization Against
Helicobacter Using Jack Bean Urease," Acta Gastroenterol. Belg.,
vol. 56, 94 (1993)).
[0010] The use of an antigen that is the reaction product of urease
and glutaraldehyde is described in U.S. Pat. No. 4,837,017, "Urease
Antigen Product and Process," issued Jun. 6, 1989, to LeVeen et al.
The patent describes the use of the antigen to reduce ammonia
toxicity caused by urea splitting organisms. LeVeen et al. disclose
the injection of glutaral-dehyde treated jack bean urease into the
bloodstream. The LeVeen patent does not disclose the administration
of the urease antigen to the mucosal surface of a mammal in order
to stimulate antibody production by the local immune system.
Furthermore, there is no evidence in the specification that the
injection of a jack bean urease antigen could prevent Helicobacter
infection or be used to treat gastroduodenal infection by
Helicobacter.
[0011] Eaton et al. have shown that mutant H. pylori cultures with
weak urease activity are unable to infect gnotobiotic piglets.
(Eaton et al., "Essential Role of Urease in the Pathogenesis of
Gastritis Induced by Helicobacter pylori in Gnotobiotic Piglets,"
Gastroenterology, vol. 98, A654 (1990)). Eaton does not describe
the use of a urease antigen as a vaccine to prevent Helicobacter
infection or as a method of treating Helicobacter infection.
[0012] The use of Helicobacter pylori urease, or of related
ureases, as a vaccine against Helicobacter pylori infection has
previously been proposed by A. Labigne, and incorporated among the
claims of a patent filed on Oct. 6, 1988 by Pasteur Institute,
Paris, France. (Labigne, A. "Sequences of Nucleotides Coding for a
Protein Having an Urease Activity". EPO patent application # EPO
367 644 A1, 1989. International Publication # WO 90/04030, 1990).
The specification of this document contains, however, no evidence
of vaccination of any mammal against any Helicobacter infection
with urease. This part of the Pasteur Institute patent, therefore,
has not been reduced to practice, and the related claims (claims 27
and 28, page 16) should not be considered as valid. Furthermore,
the claims of this document relate to a protein presenting a urease
activity, and it will be understood from the experiments described
below that enzymatic activity of the urease-based vaccine is not
required to induce protection after oral immunization.
[0013] Moreover, while sequence homology with other bacterial
ureases might support the use of urease as a vaccine candidate
against Helicobacter pylori infection, the current knowledge of
human Helicobacter pylori infection would certainly not. First,
despite the fact that infected individuals often mount a strong
antibody response to urease, the anti-urease immune response does
not result in clearance or control of the infection. Second,
Helicobacter pylori is able to transport urease out of the cell and
to shed it from its surface, (Evans, D. J. et al.
"Urease-Associated Heat Shock Protein of Helicobacter pylori"
Infect. Immun., vol. 60, 2125-2127 (1992), Ferrero, R. L. and Lee,
A. "The Importance of Urease in Acid Protection for the
Gastric-Colonizing Bacteria Helicobacter pylori and Helicobacter
felis sp. nov." Microb. Ecol. Health Dis., vol. 4, 121-134 (1991)),
thus urease may not represent an appropriate target for the
development of a protective mucosal immune response. Indeed,
mucosal immune protection is thought to be mainly mediated by
secretory IgA, the agglutinating activity of which would be
impaired when the recognized antigen can be shed by the target
pathogen. Third, urease appears to be toxic for epithelial cells in
culture, and has been suspected to play a role in mucus degradation
and in peptic ulceration in vivo (Mgraud, F. et al., "Further
Evidence of the Toxic Effect of Ammonia Produced by Helicobacter
pylori Urease on Human Epithelial Cells," Infect. & Immun.,
vol. 60, 1858-63 (1992); Murakami, M. et al., "Gastric Ammonia has
a Potent Ulcerogenic Action on the Rat Stomach," Gastroenterology
1993, vol. 105, 1710-15), thus its use as antigen may be toxic.
[0014] Nevertheless, we reasoned that this antigen could be a
potentially efficient vaccine if:
[0015] first, we would deliver it orally at a sufficiently high
dose to elicit a stronger immune response than the naturally
occurring one
[0016] second, the amount of antibodies produced would be high
enough to bind all the urease, shed or not shed
[0017] third, we would use subunits of urease or a molecular
species that was non toxic.
[0018] Another aspect of the invention describes the use of
antibodies directed against urease to prevent and to treat
Helicobacter infection. European Patent Application No. 91310049.1,
filed by Kunio Ando on Oct. 31, 1991, claiming priority on Japanese
Patent Application No. 296609/90 filed Nov. 1, 1990, titled "A
Method for Producing a new Medicine for Both Treating and
Preventing Peptic Ulcer Diseases and Gastritis and Thus Formulated
Medicines," describes the oral administration of polyclonal
antibodies derived from bovine colostrum and bovine serum to
patients with active chronic gastritis type B and to patients with
duodenal ulcer. The Ando application describes the use of an
antibody preparation directed against many antigens, including
Helicobacter pylori, and does not disclose the use of an antibody
directed against urease to treat or prevent Helicobacter pylori
infection. The use of antibodies to treat gastric disease in
gnotobiotic piglets was described in U.S. Pat. Nos. 5,258178 and
5,260,057, issued to Cordle and Schaller and titled "Method and
Product for the Treatment of Gastric Disease." The Cordle and
Schaller patents describe the use of an antibody preparation that
does not solely contain antibodies directed against Helicobacter
pylori, and does not disclose the use of an antibody directed
against urease to treat or prevent Helicobacter pylori infection.
Nagata et al. describe the preparation of a monoclonal antibody
directed against Helicobacter pylori that inhibits urease activity.
(Nagata, K., et al., "Monoclonal Antibodies Against the Native
Urease of Helicobacter pylori: Synergistic Inhibition of Urease
Activity by Monoclonal Antibody Combinations," Infect. and Immun.,
Vol. 60,4826 (1992)). Nagata et al. do not describe the use of
monoclonal antibodies directed against urease to prevent or to
treat Helicobacter pylori infection.
[0019] Very few examples of therapeutic vaccines are available in
the literature. Most of them are related to parenteral
immunizations aimed to stimulate the host's immune system against
malignant tumors, to modulate the immune system in autoimmune
diseases such as rheumatoid arthritis or as desensitization in
allergy states. Therapeutic vaccination procedures against
different infections were also performed, most of them via a
parenteral route of immunization. They included immunizations
against leprosy in humans (Zaheer S A et al. "Combined Multidrug
and Mycobacterium w Vaccine Therapy in Patients with Multibacillary
Leprosy" J. Infect Dis., vol. 167, 401-410 (1993), Mukherjee A. et
al., "Histopathological Monitoring of an Immunotherapeutic Trial
with Mycobacterium w." Int. J Lepr. Other Mycobact. Dis., vol. 60,
28-35 (1992)), in complementation of antibiotic therapy,
vaccination against Pythiosis insidiori, a mycological infection,
in horses (Mendoza L, et al., "Evaluation of Two Vaccines for the
Treatment of Pythiosis insidiosi in Horses" Mycopathologia, vol.
119, 89-95 (1992)), an uncontrolled study on the use of an
autovaccine in chronic osteomyelitis (Sologub VV. "Experience in
Using an Autovaccine in Treating Patients with Chronic
Osteomyelitis" Vrach, Delo, 122-125 (1992)) and systemic
immunization against Campylobacter fetus infection of female cattle
(Schurig, G. G. D., et al., "Bovine Venereal Vibriosis: Cure of
Genital Infection in Females by Systemic Immunization," Infect.
& Immun., Vol. 11, 245-51 (1975)). To date, only one oral
immunotherapy study aimed at stimulating the mucosal immune system
in order to treat (and to prevent recurrence of) a mucosal
infection has been performed, for urinary tract infection (Schulman
C C, et al. "Oral Immunotherapy of Recurrent Urinary Tract
Infections: A Double-Blind Placebo-Controlled Multicenter Study" J
Urol., vol. 150, 917-921 (1993)). In that study, Schulman et al.
used a lysate of selected E. coli strains, together with an
concomitant treatment of antibiotics, chemotherapeutics or urinary
tract disinfectants to treat the acute infection at entry in the
study. Therefore, no study has demonstrated so far the
effectiveness of a therapeutic vaccine, used as a monotherapy,
administered to the mucosal immune system, against a bacterial
disease.
[0020] The novelty of a therapeutic vaccine against Helicobacter
infection also comes from the observation that H. pylori persists
as a chronic infection in the gastric cavity for years, despite
inducing a vigorous local and systemic immune response. This
observation was conceptually already an obstacle to the development
of a prophylactic vaccine against Helicobacter infection, but was
even more an obstacle to the development of a therapeutic
immunization.
[0021] In summary, there remains a need for effective treatment and
prevention of Helicobacter pylori-induced gastric infection in
humans. Recent data suggested the possibility to generate a vaccine
against this infection, but have not provided a clear
identification of defined antigen(s), common to all strains of
Helicobacter pylori, that could be incorporated into a safe and
effective vaccine.
[0022] In this invention, we have identified the urease antigen of
Helicobacter pylori as a candidate vaccine and demonstrated its
efficacy in an animal model. We have also demonstrated the use of
the Helicobacter pylori urease antigen for the treatment and
eradication of Helicobacter infection. We have further demonstrated
that the B subunit of urease alone (ure B) is effective as a
vaccine useful for the prevention of and treatment of Helicobacter
infection. These results were unexpected in the light of the
natural history of Helicobacter infections.
SUMMARY OF THE INVENTION
[0023] We have determined that it is useful to immunize animals
with Helicobacter urease peptides for both prophylactic and
therapeutic treatment. Immunization of animals with Helicobacter
urease peptides prevents infection by Helicobacter and eradicates
infection in previously infected animals. This method, and the
vaccine compositions, are useful for the prevention and treatment
of gastroduodenal disease associated with Helicobacter
infection.
[0024] We have discovered that immunity can be induced in mammals
susceptible to gastrointestinal Helicobacter infection by
exploiting urease epitopes displayed on or about the surface of
Helicobacter organisms and using them as a vaccine target. The
immunity can be induced by immunization with native urease, but can
also be induced with recombinant urease subunit, produced as an
enzymatically inactive, therefore non-toxic form. The invention
provides a method of inducing immunity to Helicobacter infection by
administering to a mucosal surface of a mammal a polyaminoacid
preparation, i.e. a mixture of peptides and/or proteins, together
with an appropriate adjuvant. This polyaminoacid preparation
presents a plurality of epitopes characteristic of and exhibited by
a urease enzyme endogenous to the infecting Helicobacter organism.
The administration of the polyaminoacid preparation may be
performed by the oral route.
[0025] The active ingredient of the preparation may comprise
natural or biosynthetic epitopes and may take various forms. A non
exhaustive list of possible preparations includes purified,
naturally occurring or recombinantly produced urease preparations
of bacterial or other origin, digests of urease, fusion proteins
comprising urease epitopes, truncated forms of urease enzyme, or
peptides homologous with the amino acid sequence of urease. Since
development of immunity depends on induction of humoral and/or
cellular immune responses which bind to the infecting Helicobacter
organism, preferred preparations are those which most closely
duplicate the epitopes of the urease endogenous to the infecting
organism. For example, preparations displaying the epitopes of
urease of Helicobacter pylori are preferred for administration in
humans susceptible to Helicobacter pylori, and preparations
displaying the epitopes of urease of H. felis are preferred for the
administration in humans susceptible to H. felis. However, in
accordance with an important aspect of the invention, it has been
discovered that urease from a heterologous species may be used. For
example, we have shown that H. felis infection in mice can be
prevented by administration of urease from Helicobacter pylori.
Thus, H. pylori urease can be used to protect against H. pylori as
well as H. felis. H. felis is an occasional cause of human
infection and disease. (Wegman, W. et al., Schweig. Med.
Wochenschr. vol. 121, 245-54 (1991)).
[0026] According to a first embodiment of the present invention, a
method is provided of eliciting in a mammalian host a protective
immune response to Helicobacter infection. According to a second
embodiment of the present invention, a method is provided of
treating a mammalian host that is infected with Helicobacter.
[0027] Thus, in a first aspect, the present invention provides a
method of eliciting in a mammalian host a protective immune
response to Helicobacter infection. The method comprises the step
of administering to a mucosal surface of the mammal, including
humans, an immunologically effective amount of a urease antigen,
preferably Helicobacter pylori urease, capable of eliciting such a
protective immune response. The term "comprising" is used herein as
it is recognized in the art.
[0028] According to one aspect of the invention, there is provided
a method of eliciting in a mammalian host a protective immune
response to Helicobacter infection wherein an immunologically
effective amount of a urease antigen capable of eliciting such a
protective immune response, preferably Helicobacter pylori urease
or Helicobacter pylori urease B subunit, is administered to a
mucosal surface of the host.
[0029] In a second aspect, the present invention provides a method
of eliciting in a mammalian host a protective immune response to
Helicobacter infection. The method comprises the step of
administering to a mucosal surface of the mammal, including humans,
an immunologically effective amount of recombinant, enzymatically
inactive, urease B subunit as antigen, preferably recombinant
Helicobacter pylori urease B subunit, capable of eliciting such a
protective immune response.
[0030] The invention also includes within its scope the treatment
or prophylaxis of mammals, including humans, for Helicobacter
infection, wherein an immunologically effective amount of a urease,
or its subunits, capable of eliciting a protective immune response
to Helicobacter infection, is administered to a mucosal surface of
a patient. Preferably, the urease is Helicobacter pylori urease or
Helicobacter pylori urease B subunit, and the urease is preferably
administered in particulate form in association with a hydroxylated
calcium phosphate, for example hydroxyapatite. Moreover, it is
preferred to administer the Helicobacter pylori urease in
association with a mucosal adjuvant, the B subunit of cholera
toxin, muramyl dipeptide or other such adjuvants.
[0031] According to another aspect of the present invention, there
is provided a vaccine composition suitable for prevention of
Helicobacter infection, comprising an effective amount of a urease
antigen, preferably Helicobacter pylori urease or Helicobacter
pylori urease B subunit, or recombinant Helicobacter pylori urease
subunits, capable of eliciting in a host a protective immune
response to Helicobacter infection, in association with a
pharmaceutically acceptable carrier or diluent. Suitable carriers
and diluents will be recognized by those skilled in the art and can
be found in, for example Remington's Pharmaceutical Sciences (18th
ed., 1990).
[0032] The vaccines of the invention are administered in amounts
readily determined by persons of ordinary skill in this art. Thus,
for adults a suitable dosage will be in the range of 10 .mu.g to
100 milligrams, for example 50 .mu.g to 50 mg. A suitable dosage
for adults will also be in the range of 5 .mu.g to 500 mg. Similar
dosage ranges will be applicable for children. Carrier systems in
humans may include enteric release capsules protecting the antigen
from the acidic environment of the stomach, and including urease
antigen in a insoluble form as fusion proteins. The vaccine can be
administered as a primary prophylactic agent in adults or in
children, as a secondary prevention, after successful eradication
of Helicobacter pylori in an infected host, or as a therapeutic
agent in the aim to induce an immune response in the host
susceptible to contribute to the eradication of Helicobacter
pylori.
[0033] As noted above, a suitable mucosal adjuvant is cholera
toxin. Others which may be used are non-toxic derivatives of
cholera toxin, including its B subunit, and/or conjugates or
genetically engineered fusions of the urease antigen plus cholera
toxin or its B subunit. Other suitable delivery methods include
biodegradable microcapsules or immuno-stimulating complexes
(ISCOMs) or liposomes, genetically engineered attenuated live
vectors such as viruses or bacteria, and recombinant (chimeric)
virus-like particles, e.g. bluetongue. The amount of mucosal
adjuvant employed depends on the type of mucosal adjuvant used. For
example, when the mucosal adjuvant is cholera toxin, it is suitably
used in an amount of 5 .mu.g to 50 .mu.g, for example 10 .mu.g to
35 .mu.g. When used in the form of microcapsules, the amount used
will depend on the amount employed in the matrix of the
microcapsules to achieve the desired dosage. The determination of
this amount is within the skill of a person of ordinary skill in
this art.
[0034] Suitable carriers for the vaccines of the invention are
enteric coated capsules and polylactide-glycolide microspheres.
Suitable diluents are 0.2N NaHCO.sub.3 and/or saline.
[0035] Particulate hydroxylated calcium phosphate (HCP) is
especially useful as a carrier for the Helicobacter pylori urease
to be applied to mucosal surfaces. It is believed that the
Helicobacter pylori urease-hydroxylated calcium phosphate conjugate
is transported across epithelium where it raises a poly Ig immune
response. Preferably, the hydroxylated calcium phosphate is in the
form of microparticles suitable for transport across the
epithelium, particularly by cells specialized for this purpose (M
cells). A preferred form of hydroxylated calcium phosphate is
hydroxyapatite, a commercially available crystalline hydroxylated
calcium phosphate Ca.sub.10(PO.sub.4).sub.6 (OH).sub.2.
[0036] Commercially available hydroxyapatite generally consists of
slab-like crystals that are chemically and physically analogous to
inorganic hydroxyapatite in normal bone tissue. Ingestion of
hydroxyapatite should therefore be safe, as evidenced by the
existence of nutritional calcium/phosphorus supplements derived
from ground bone, which are designed to be ingested.
Commercially-high resolution hydroxyapatite (from CalBiochem)
consists of crystals varying widely in size. Crystals over 1 .mu.m
in length are unlikely to be taken up by M cells. Therefore, for
use in the invention, commercial hydroxyapatite crystals are broken
into small, relatively uniform crystalline fragments such as by
sonication. Preferably, a substantial proportion of the
hydroxyapatite is present as fragments of about 0.01-1.0 .mu.m.
Fragmentation may be measured either by electron microscopy or
light scattering, using standard techniques.
[0037] Preferred modes of administration of the Helicobacter pylori
urease antigen are orally, nasally, rectally or ocularly. Oral
administration can provide delivery to other G.I.
(gastrointestinal) mucosa including the intestinal mucosa.
[0038] The vaccines of the present invention may be administered to
a mucosal surface in the form of an aerosol, suspension, capsule
and/or suppository. The method of administration will be readily
apparent to a person of ordinary skill in this art and can be found
in, for example, Remington's Pharmaceutical Sciences (18 ed.,
1990).
[0039] According to a further aspect of the present invention,
there is provided a method of imparting to a mammalian host passive
protection to Helicobacter infection, comprising administering to a
mucosal surface of the host an immunologically effective amount of
a urease specific antibody produced in a host immunized with a
urease, preferably Helicobacter pylori urease or Helicobacter
pylori urease B subunit, capable of eliciting a protective immune
response to Helicobacter infection.
[0040] The present invention further includes the passive
immunization of mammals, including humans, against Helicobacter
infection. This is achieved by administering to a mucosal surface
of the patient an effective amount of a urease specific antibody,
preferably, an effective amount of a Helicobacter pylori urease
specific IgA monoclonal antibody.
[0041] Since the urease of Helicobacter pylori is shown to
represent the antigen involved in inducing protective immunity, a
further aspect of the invention is the use of Helicobacter pylori
urease as a diagnostic reagent to measure the immune response of
persons who have received a vaccine based on urease or to determine
whether an individual is immune or susceptible (and thus in need of
vaccination). The present invention also includes the use of urease
and urease-specific antibodies to construct assays and kits for
diagnosis of Helicobacter immunity, assessment of Helicobacter
susceptibility, and definition of immune responses to vaccines.
[0042] In a third aspect, the invention provides a method of
treating gastroduodenal disease in a mammal. This method comprises
the step of administering a therapeutically effective amount of a
composition comprising Helicobacter urease peptides. The
gastroduodenal diseases included within the scope of the invention
include, but are not limited to, gastritis, peptic ulcer disease,
including both gastric and duodenal ulcers, gastric cancer, chronic
dyspepsia with severe erosive gastroduodenitis, refractory ulcer
dyspepsia, intestinal metaplasia, low grade MALT lymphoma,
Helicobacter infection, Helicobacter pylori infection and
Helicobacter felis infection. The term "urease peptides" refers to,
but is not limited to, any urease or subunit or urease, either
naturally occurring or obtained by recombinant DNA techniques, as
well as a digested fragment or peptide thereof, fusion proteins
comprising the whole urease, subunits, or fragments thereof, or
truncated urease constructs. Also included within the term "urease
peptides," are proteins or peptides that display epitopes
sufficiently homologous to epitopes displayed by Helicobacter
urease such that antibodies that recognize epitopes displayed by
Helicobacter urease will recognize epitopes displayed by said
peptides or proteins.
[0043] In a more particular related aspect, the invention provides
a method of treating gastroduodenal disease caused by Helicobacter
infection. The Helicobacter infection may be, but is not limited
to, Helicobacter pylori or H. felis infection.
[0044] More particularly, the invention provides a method of
treating gastroduodenal disease caused by Helicobacter infection
whereby the composition comprising Helicobacter urease peptides is
administered to a mucosal surface. Without limiting the type of
mucosal surface used for administration, the mucosal surface may be
oral, nasal, rectal, or ocular.
[0045] The invention can also feature the administration of a
composition comprising Helicobacter urease peptides in association
with a mucosal adjuvant. The mucosal adjuvant may be selected from,
but is not limited to, cholera toxin, procholer-agenoid, cholera
toxin B subunit, fungal polysaccharides including, but not limited
to, schizophyllan, muramyl dipeptide, muramyl dipeptide
derivatives, phorbol esters, microspheres, non-Helicobacter pylori
bacterial lysates, labile toxin of Escherichia coli, block
polymers, saponins, and ISCOMs. Other mucosal adjuvants will be
recognized to those in the art and can be found in, for example,
Azuma, I., "Synthetic Immunoadjuvants: Application to Non-Specific
Host Stimulation and Potentiation of Vaccine Immunogenicity"
Vaccine, vol. 10, 1000 (1992); Pockley, A. G. & Montgomery, P.
C., "In vivo Adjuvant Effect of Interleukins 5 and 6 on Rat Tear
IgA Antibody Responses" Immunology, vol. 73, 19-23 (1991); Adam, A.
& Lederer, E. "Muramyl peptides as Immunomodulators" ISI ATLAS
OF SCIENCE 205 (1988); Clements, J. D., et al. "Adjuvant Activity
of Escherichia coli Heat-labile Enterotoxin and Effect on the
Induction of Oral Tolerance in Mice to Unrelated Protein Antigens"
Vaccine, vol. 6, 269 (1988); Ben Ahmeida, E. T. S., et al.
"Immunopotentiation bf Local and Systemic Humoral Immune Responses
by ISCOMs, Liposomes and FCA: Role in Protection Against Influenza
A in Mice" Vaccine, vol. 11, 1302 (1993); and Gupta, R. K. et al.
"Adjuvants--A Balance Between Toxicity and Adjuvanticity" Vaccine,
vol. 11, 290-308 (1993).
[0046] The mucosal adjuvant may also be genetically or chemically
linked to the urease peptides. Examples of this type of fusion
peptide are known to those skilled in the art and can also be found
in Czerkinsky et al., "Oral Administration of a Streptococcal
Antigen Coupled to Cholera Toxin B Subunit Evokes Strong Antibody
Responses in Salivary Glands and Extramucosal Tissues" Infect.
Immun., vol. 57, 1072-77 (1989); Nashar et al., "Current Progress
in the Development of the B Subunits of Cholera Toxin and
Escherichia Coli Heat-Labile Enterotoxin as Carriers for the Oral
Delivery of Heterologous Antigens and Epitopes" Vaccine, vol. 11,
235-40 (1993); and Dertzbaugh and Elson, "Comparative Effectivess
of the Cholera Toxin B Subunit and Alkaline Phosphatase as Carriers
for Oral Vaccines," Infect. Immun., vol. 61, 48-55 (1993). For
example, the urease B subunit could be expressed as a chimeric
protein that is genetically linked to the cholera toxin B subunit
through the use of a DNA expression vector containing the ure B
nucleotide sequence linked to the cholera toxin B subunit
nucleotide sequence.
[0047] In another related aspect, the method involves the
administration of a composition comprising Helicobacter urease
peptides where such composition is delivered in particulate form.
The composition may be delivered in particulate form through
association with a carrier. The carrier may be a hydroxylated
calcium phosphate, for example, hydroxyapatite. The term
"hydroxyapatite" refers to, but is not limited to, its meaning as
recognized by those skilled in the art to mean a tribasic calcium
phosphate, also known as hydroxylated calcium phosphate or calcium
hydroxide phosphate. This is only an example, and is not meant to
be limiting as to the type of carrier that may be used.
[0048] In another related aspect, the administered dosage of the
composition comprising Helicobacter urease peptides may range from
100 .mu.g to 1 g, for example, 0.14 mg to 14.4 mg per kg of body
weight. Those of skill in the art will recognize that the optimal
dose may be more or less depending upon the patient's body weight,
disease, the route of administration, and other factors. The dosage
level is readily determinable by standard methods. The number of
doses will depend upon the disease, the formulation, and efficacy
data from clinical trials. For example, the dosage may be
administered over 3 to 8 doses for a primary immunization schedule
over 1 month. This course of treatment is an example and is not
meant to be limiting.
[0049] In one related aspect, the method involves the
administration of a composition comprising Helicobacter urease Ad
peptides wherein the composition is administered in association
with a microsphere carrier. Such microsphere carrier may be, for
example, but is not limited to, a polylactide-coglycode
biodegradable microsphere carrier.
[0050] In another related aspect, the method involves the
administration of a composition comprising the Helicobacter urease
peptides wherein such composition comprises a recombinant live
vector which expresses a Helicobacter urease peptide. Those skilled
in the art will recognize that such live vector may be, for
example, a bacterial or a viral vector. For example, without any
limitation, the live vector may be selected from the group
consisting of Salmonella typhimurium, Salmonella typhi, Shigella,
Bacillus, Lactobacillus, BCG, Escherichia coli, Vibrio cholerae,
Campylobacter, yeast, Herpes virus, Adenovirus, Poliovirus,
Vaccinia, and Avipox. In addition, a carrier system which expresses
a Helicobacter urease peptide, such as Bluetongue virus-like
particles, Rotavirus-like particles, and Ty particles, may be used
to deliver the urease peptide. In a preferred aspect, the live
vector or the carrier system may be administered to a mucosal
surface.
[0051] A preferred embodiment of the present invention comprises a
method of treating a human infected with Helicobacter pylori,
comprising orally administering a therapeutically effective amount
of a composition comprising the ure B subunit of Helicobacter
pylori urease, in association with a mucosal adjuvant selected from
the group consisting of cholera toxin, procholeragenoid, cholera
toxin B subunit, fungal polysaccharides, including schizophyllan,
muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters,
liposomes, microspheres, non-Helicobacter pylori bacterial lysates,
labile toxin of Escherichia coli, block polymers, saponins, and
ISCOMs, the composition being administered in a particulate form in
association with hydroxyapatite. The composition may also be
administered with concurrent oral administration of a chewable 1.0
g NaHCO.sub.3 tablet. For the purposes of this invention, the term
"in association with" includes any type of association, including
but not limited to, a chemical or genetic association, such as that
present in a fusion protein.
[0052] In a fourth aspect, the invention provides a method of
treating gastroduodenal disease in a mammal, comprising
administering a therapeutically effective amount of a composition
comprising an antibody that recognizes Helicobacter urease. The
gastroduodenal diseases included within the scope of the invention
are referenced above. These methods use the above-referenced
composition comprising Helicobacter urease peptides to elicit an
antibody response in a mammal. The antibodies produced by the
immunized mammal are isolated and administered to the subject
mammal. The preparation of antibodies that recognize a given
antigen, such as the above-referenced composition, is known to
those skilled in the art. For example, polyclonal and monoclonal
antibodies can be prepared following the disclosure in Harlow, E.
& Lane, D., Antibodies: A Laboratory Manual (1988).
[0053] In a more particular related aspect, the administered
antibody is a monoclonal antibody. The preparation of monoclonal
antibodies is known to those skilled in the art.
[0054] More particularly, the administered antibody is an IgA
antibody, either a polyclonal or monoclonal IgA antibody. The
preparation of monoclonal IgA antibodies is known to those skilled
in the art and may be found in, for example, Winner, L., et al.,
"New Model for Analysis of Mucosal Immunity: Intestinal Secretion
of Specific Monoclonal Immunoglobulin A from Hybridoma Tumors
Protects Against Vibrio Cholerae Infection" Infect. and Immun.,
vol. 59, 977-982 (1991); and Weltzin, R., et al., "Binding and
Transepithelial Transport of Immunoglobulins by Intestinal M Cells:
Demonstration Using Monoclonal IgA Antibodies Against Enteric Viral
Proteins" J. Cell Biol., vol. 108, 1673-1685 (1989).
[0055] The term "antibody" as used for the purposes of this
invention includes, but is not limited to, polyclonal antibodies,
monoclonal antibodies, and anti-idiotypic antibodies. The
antibodies may be naturally derived from any animal, synthesized in
bacteria or another non-animal source, chemically synthesized, or
genetically synthesized.
[0056] In a preferred aspect of the invention, a method is provided
of treating a human infected with Helicobacter pylori, comprising
administering a therapeutically effective amount of a composition
comprising an IgA monoclonal antibody that recognizes the ure B
subunit of Helicobacter pylori urease.
[0057] In a fifth aspect, the invention provides a composition
useful in the therapeutic treatment of gastroduodenal disease,
comprising Helicobacter urease peptides. The gastroduodenal
diseases included within the scope of the invention, as well as the
Helicobacter urease peptides and mucosal adjuvants included within
the scope of the invention, are referenced above.
[0058] In a related aspect, the composition comprises Helicobacter
urease peptides and a mucosal adjuvant.
[0059] In another related aspect, the composition is in particulate
form. The composition may exist in particulate form through
association with a carrier. The carrier may be, for example,
hydroxyapatite, as referenced above. This is only an example, and
is not meant to be limiting as to the type of carrier that may be
used.
[0060] More particularly, the composition is present in particulate
form, in a liquid suspension.
[0061] In a related aspect, the composition comprises Helicobacter
urease peptides in association with a microsphere carrier. Such
microsphere carrier may be, for example, but is not limited to, a
polylactide-coglycolide biodegradable microsphere. The urease
peptides may encapsulated in the biodegradable microspheres.
Polylactide-coglycolide microspheres slowly hydrolyze in the
presence of water and becomes water-soluble, thus delivering the
peptides that are incorporated in the microsphere.
[0062] In another related aspect, the composition comprises
Helicobacter urease peptides wherein the composition comprises a
recombinant live vector which expresses a Helicobacter urease
peptide. Those skilled in the art will recognize that such live
vector may be, for example, a bacterial or a viral vector, for
example, without any limitation, the live vector may be selected
from the group consisting of Salmonella typhimurium, Salmonella
typhi, Shigella, Bacillus, Lactobacillus, BCG, Escherichia coli,
Vibrio cholerae, Campylobacter, yeast, Herpes virus, Adenovirus,
Poliovirus, Vaccinia, and Avipox. In addition, a carrier system
which expresses a Helicobacter urease peptide, such as Bluetongue
virus-like particles, Rotavirus-like particles, and Ty particles,
may be used to deliver the urease peptide.
[0063] In a preferred embodiment, a composition is provided that is
useful in the therapeutic treatment of Helicobacter pylori
infection of a human, comprising the ure B subunit of Helicobacter
pylori urease, a mucosal adjuvant selected from a group consisting
of cholera toxin, procholeragenoid, cholera toxin B subunit, fungal
polysaccharides, including schizophyllan, muramyl dipeptide,
muramyl dipeptide derivatives, phorbol esters, liposomes,
microspheres, non-Helicobacter pylori bacterial lysates, labile
toxin of Escherichia coli, block polymers, saponins, and ISCOMs,
and further comprising hydroxyapatite, wherein the composition is
in a particulate form in a liquid suspension. The term "in
association with" is defined as referenced above.
[0064] In a sixth aspect, a composition is provided that is useful
in the therapeutic treatment of gastroduodenal disease, comprising
an antibody that recognizes Helicobacter urease. The gastroduodenal
disease included within the scope of the invention, as well as the
antibodies included within the scope of the invention, are
referenced above.
[0065] In a preferred embodiment of the invention, a composition is
provided that is useful in the therapeutic treatment of
gastroduodenal disease, comprising an IgA monoclonal antibody that
recognizes the ure B subunit of Helicobacter pylori urease.
[0066] While not being bound by any theory, the present inventors
believe that administration of the urease antigen, or B subunit
thereof, to a mucosal surface stimulates the common mucosal immune
system and perhaps local sites in the gastric mucosa inducing an
immune response, including the appearance of Helicobacter pylori
specific IgA antibodies in the gastric secretions, which prevent
Helicobacter infection. Because of this belief, the terms
"immunization" and "vaccine" are used herein by their common
meanings as recognized by those skilled in the art and also to
indicate methods and compositions used for the treatment of
Helicobacter infection, without limitation to these meanings, and
without being bound to any theory regarding the mechanism of
treatment.
[0067] Since it is a routine matter to conduct pre-clinical trials
of candidate vaccines for human use in animal models, it is
believed that the methodology of the present invention is effective
in humans, especially in the prevention and treatment of peptic
ulcers, gastritis, gastric malignancies and other conditions
arising as a result of the presence of Helicobacter pylori and/or
H. felis.
[0068] Based on the dosage and course of treatment that
successfully eradicated infection in the mouse model, a preferred
range of dosage would be 100 .mu.g to 10 g of Helicobacter urease
peptides. Those skilled in the art will recognize the appropriate
dosage level to test from research reported for other oral vaccines
such as, for example, the research performed with Escherichia coli
lysate (6 mg dose daily up to a total of 540 mg) and with an
enterotoxigenic E. coli purified antigen (4 doses of 1 mg).
(Schulman et al, "Oral Immunotherapy-of Recurrent Urinary Tract
Infections: A Double-Blind Placebo-Controlled Multicenter Study,"
J. Urol., vol. 150, 917-921 (1993); Boedeker et al., "Safety,
Immunogenicity and Efficacy in Human Volunteers of Biodegradable,
Biocompatible Microspheres Containing Colonization Factor
Antigen/II (CFA/II) as an Enteral Vaccine Against Enterotoxigenic
E. coli (ETEC)" American Gastroenterological Assoc., vol. 999,
A-222 (1993)). Without intending any limitation as to the course of
treatment, the treatment could be administered over 3 to 8 doses
for a primary immunization schedule over 1 month. (Boedeker,
American Gastroenterological Assoc., vol. 888, A-222 (1993)).
[0069] A recommended method of immunization is administration of
the Helicobacter urease peptide composition in the form of a liquid
alone or suspension containing Na.sub.2HCO.sub.3 or similar
material to temporarily neutralize gastric acid. The neutralizing
material may also be delivered separately at the time the peptide
composition is administered, such as in the form of a chewable
Na.sub.2HCO.sub.3 tablet. Alternatively, the composition can be
administered in the form of enteric-coated capsules. These methods
will likely avoid the problem of degradation of the urease
composition during its passage in the upper gastrointestinal tract.
These methods are reviewed in, for example, Levine & Norlega,
"Vaccines to Prevent Enteric Infections," Ballieres Clin. Gastro.,
vol. 7, 501-517 (1993).
[0070] Although a high dose of cholera toxin is not preferred as a
mucosal adjuvant for human use, other mucosal adjuvants recognized
by those skilled in the art, and as referenced above, will be
useful for treatment of humans.
[0071] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] The invention will now be further described with reference
to the accompanying drawings, in which FIGS. 1 through 8 are
graphical representations of the results set forth in Tables 1
through 9.
[0073] FIG. 1 is a graphic representation of the results from Table
1 of the tests for antibodies in serum (IgG) and intestinal
secretion (IgA) in mice that were not protected after immunization
with urease.
[0074] FIG. 2 is a graphic representation of the results from Table
1 of tests for antibodies in serum (IgG) and intestinal secretion
(IgA) in mice that were protected after immunization with
urease.
[0075] FIG. 3 is a graphic representation of the results from Table
1 of tests for antibodies in serum (IgG) and intestinal secretion
(IgA) in mice that were not protected after immunization with
Helicobacter pylori sonicate.
[0076] FIG. 4 is a graphic representation of the results from Table
1 of tests for antibodies in serum (IgG) and intestinal secretion
(IgA) in mice that were protected after immunization with is
Helicobacter pylori sonicate.
[0077] FIG. 5 is a graphic representation of the results set out in
Table 2 comparing the level of protection obtained with
Helicobacter pylori urease as compared to that obtained with
Helicobacter pylori sonicate and with cholera toxin.
[0078] FIG. 6 is a graphic representation of the results set out in
Tables 5 and 6 which measures urease activity in mice that were
challenged after oral immunization with recombinant urease A and B
subunits.
[0079] FIG. 7 is a graphic representation of the results set out in
Table 8 which measures gastric tissue urease activity as a
reflection of Helicobacter infection in mice that were subsequently
treated with the Helicobacter pylori ure B subunit, cholera toxin
and hydroxyapatite only (sham immunized), or untreated.
[0080] FIG. 8 is a graphic representation of the results set out in
Table 9 which measures gastric tissue urease activity as a
reflection of Helicobacter infection in mice that were subsequently
treated with the Helicobacter pylori ure B subunit, or cholera
toxin and hydroxyapatite only (sham immunized). For FIG. 8A, the
mice were sacrificed and urease assays performed 2.5 weeks after
the last immunization. For FIG. 8B, the mice were sacrificed and
urease assays performed 8 weeks after the last immunization.
DETAILED DESCRIPTION OF THE INVENTION
[0081] The present inventors have discovered that oral
administration to mice of polyaminoacid preparations exhibiting the
epitopes of Helicobacter pylori urease gives rise to a protective
immunological response against H. felis in mice, an animal model of
generally-accepted value for the study of the immune response to
Helicobacter infection (Lee, A. et al. "A Small Animal Model of
Human Helicobacter pylori Active Chronic Gastritis"
Gastroenterology, vol. 99, 1315-1323 (1990)), and a recognized
pathogen causing gastritis in humans (Wegman, W. et al., Schweig.
Med. Wochenschr., vol. 121, 245-54 (1991)). The effect of the
protective immune response is that immunized animals, when
challenged with pathogen, have a greatly reduced incidence of
infection, in comparison to non-immunized animals. Furthermore, the
inventors have discovered that oral immunization in mice using
Helicobacter pylori urease B subunit, produced as an
enzymatically-inactive recombinant protein, gives rise to a
protective immunological response in mice against H. felis. The
effect of the protective immune response is that immunized animals,
when challenged with pathogen, have also a greatly reduced
incidence of infection, in comparison to non-immunized animals
which do become infected.
[0082] The present inventors have discovered that oral
administration of Helicobacter urease peptides to mice infected
with H. felis results in the clearance of the infection. This
result indicates that the oral administration of Helicobacter
urease peptides is an effective therapy for the treatment of
Helicobacter infection in mammals. The oral administration of
Helicobacter pylori urease B subunit, produced as an
enzymatically-inactive recombinant protein significantly decreases
the level of H. felis infection in infected mice, in comparison to
control infected mice.
[0083] The following examples are offered by way of illustration
and are not intended to limit the invention in any manner.
[0084] A. Bacterial Cultures and Urease Purification
[0085] The strain of Helicobacter pylori used in the study
originates from a patient with a duodenal ulcer, and has been
subcultured on BHI agarose plates to homogeneity. Helicobacter
pylori is cultured in a suitable medium, typically, BHI
(Brain-Heart Infusion) medium, containing 0.25% yeast extract and
10% fetal calf serum and supplemented with 0.4% Campylobacter
selective complement (Skirrow supplement; Oxoid 69). The bacteria
are incubated under microaerophilic conditions at 37.degree. C. in
bottles that are sealed and shaken at 37.degree. C. for 2 to 3 days
to produce a liquid culture. A culture may also be prepared in
agarose plates consisting of BHI with 0.25% of yeast extract and 5%
of sheep blood under microaerophilic conditions at 37.degree. C.
for 3 days. The quantity of bacteria is determined by optical
density of the BHI solution at 660 nm, with one optical density
unit corresponding to 10.sup.8 bacteria. Cultures on agarose plates
are first resuspended in 154 mM NaCl.
[0086] One currently preferred source of polyaminoacid displaying
urease epitopes is purified urease, e.g., Helicobacter pylori
urease obtained by following the general method of Dunn et al. J.
Biol. Chem. 265, 9464-9469, modified as described below. Following
culturing, the Helicobacter pylori is harvested in water, spun
vortexed and spun again to produce a supernatant. The solution
containing the urease activity of Helicobacter pylori (assessed by
rapid urease test, see below) is then chromatographed on a CL-6B
sizing column and the fractions which present a strong urease
activity are pooled and dialysed over-night and again
chromatographed on an anion exchanger gel. The fractions are eluted
in increasing NaCl buffer and the collected fractions with a strong
urease activity are individually submitted to a SDS gel followed by
Coomassie staining. Two distinct bands corresponding to a molecular
weight of about 63 and about 28 kDa are identified as urease. The
fractions containing urease are pooled to give purified
Helicobacter pylori urease having a purity in the region of 95% to
99%.
[0087] B. Oral Immunization with Urease Purified from Helicobacter
pylori
[0088] While it is preferred to employ purified Helicobacter pylori
urease obtained as described above as the antigenic material, it
will be understood that it is also possible to use, as the
antigenic material, any urease or subunit of urease, either
naturally occurring or obtained by recombinant DNA techniques, as
well as digested fragment thereof, fusion proteins comprising the
fragments or the whole urease, truncated urease constructs, or
other peptide or protein preparations exhibiting urease epitopes
which are capable of eliciting a protective immune response to
Helicobacter infection (See below). Thus, it is possible to employ
a urease having a substantial homology with respect to Helicobacter
pylori urease and which is effective in raising a cross-protective
immune response to Helicobacter. An example of such a urease is
jack bean urease, which possesses about 70% homology with
Helicobacter pylori urease. Although it was once thought that jack
bean urease would be an effective antigen to prevent infection by
Helicobacter, this is no longer believed to be the case. (See Chen,
M. et al. "Failure of Immunization Against Helicobacter Using Jack
Bean Urease," Acta Gastroenterol. Belg., Vol. 56, 94 (1993)). The
invention is therefore not limited to the use of intact urease, and
covers the use of any polyaminoacid preparation which displays
urease epitopes sufficiently homologous to Helicobacter pylori
urease to generate a protective immunological response in a host to
Helicobacter infection. A suitable urease must have sufficient
homology to H. pylori urease to elicit a protective immune response
against Helicobacter infection. Typically, a urease having a
homology of greater than 70%, for example, 80-90% homology, with
respect to Helicobacter pylori urease, may be employed as the
urease antigen in the invention.
[0089] A non-limiting list of sources of potentially useful urease
preparations includes endogenous urease enzymes of the different
Helicobacter species, urease from other bacteria such as Klebsiella
pneumoniae or Proteus mirabilis, and, by analogy, any other urease
which the condition that these ureases share cross-reactive
epitopes with Helicobacter pylori urease. The urease genes of all
the organisms mentioned above represent a potential tool for
expressing recombinant urease products as a whole protein or as a
part thereof.
[0090] A non-limiting list of potentially useful urease
preparations includes peptides generated from purified urease (the
sources are mentioned above), using physical and/or chemical
cleavage procedures (i.e. CnBr) and/or proteolytic cleavage (using
proteases e.g. V8-protease, trypsin or others); or peptides
synthesized chemically and retaining crossreactive epitopes with
urease.
[0091] Other sources of potentially useful epitopes include
epitopes identified by their crossreactivity with urease, as the
result of screening with anti-urease antibodies. These peptides can
be naturally occurring peptides or peptides resulting from chemical
synthesis. Furthermore such peptides can result from the expression
of recombinant random oligonucleotides.
[0092] Another source of potentially useful epitopes includes
epitopes similar to urease as a result of the generation of
anti-idiotypic antibodies to urease. Such anti-idiotypic
antibodies, generated in any immunocompetent host, are obtained by
immunization of this host with anti-urease antibodies, with the
goal of generating antibodies directed against anti-urease
antibodies, which share structural homologies with urease.
[0093] The discussion herein focuses on the use of urease naturally
produced by Helicobacter pylori (section B). However, it will be
appreciated that the urease or subunits or constructs thereof
mentioned above, capable of eliciting the desired protective immune
response, may be produced by recombinant DNA techniques well known
in the art. The efficacy of particular preparations may be
determined by routine administration using animal models, oral
administration of the candidate vaccine, and challenge with
pathogen using a protocol substantially similar or identical to the
procedure described below.
[0094] It will be recognized by those skilled in the art that other
methods may be used to administer the Helicobacter urease peptides.
For example, the urease peptides may be administered as part of a
microsphere carrier formulation. Without limiting the type of
microsphere carriers used, one example would be the administration
of the urease peptides as part of a polylactide-coglycolide
biodegradable microsphere carrier formulation.
[0095] Another method of administering the Helicobacter urease
peptides would be to express the urease peptides in a recombinant
form in a live vector, for example, a bacterial or a viral vector.
To construct such a live vector, nucleotide sequences coding for
urease peptides would be incorporated into the genetic material of
a live vector. Such live vector would be administered to an
individual for the purpose of preventing Helicobacter infection and
also for the purpose of treating an individual already infected
with Helicobacter. Those skilled in the art will recognize that
examples of appropriate live vectors include Salmonella
typhimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus,
BCG, Escherichia coli, Vibrio cholerae, Campylobacter, yeast,
Herpes virus, Adenovirus, Poliovirus, Vaccinia, and Avipox. In
addition, a recombinant carrier system which expresses a
Helicobacter urease peptide, such as Bluetongue virus-like
particles, Rotavirus virus-like particles, or Ty particles, may be
used to deliver the urease peptide. The aforementioned list is not
meant to be limiting. Preferably, the live vector or the carrier
system would be administered mucosally such that the recombinant
urease peptides expressed by the live vector would be administered
to a mucosal surface.
[0096] Tables 1 and 2 below and FIGS. 1-5 describe the results
obtained when mice were orally immunized with purified Helicobacter
pylori urease. In this first experiment, administration of the
Helicobacter pylori antigen was carried out by orally administering
to the mice Helicobacter pylori urease purified as described in
section A, and coupled to hydroxyapatite crystals, used as a
carrier to enhance M cell binding and uptake. Cholera toxin (Sigma)
was given as a mucosal adjuvant. In this experiment, groups of
female SPF BALB/c six-week old mice were each orally immunized with
30 .mu.g of purified Helicobacter pylori urease coupled to 1 mg of
hydroxyapatite plus 10 Ag of cholera toxin adjuvant at day 0, 7, 14
and 21. The mice were then challenged twice with 10.sup.8 H. felis,
at day 28 and 30. For comparison purposes, similar female SPF
BALB/c six-week old mice were orally immunized with whole
Helicobacter pylori lysate (sonicate) and 10 .mu.g cholera toxin at
day 0, 7, 14 and 21. The mice were challenged at day 28 and 30 with
H. felis. The Helicobacter pylori sonicate was prepared by
collecting Helicobacter pylori from cell cultures, pelleting by
centrifugation and resuspending the pellet in 0.9% sodium chloride
followed by sonication.
[0097] As a control, female SPF BALB/c six-week old mice were
orally sham-immunized with 10 .mu.g of cholera toxin and 1 mg of
hydroxyapatite at day 0, 7, 14 and 21. The mice were then
challenged at day 28 and 30 with H. felis. All mice were housed and
immunized in parallel. All mice subject to the study were
sacrificed on day 35.
[0098] C. Oral Immunization with Recombinant Urease Subunits of
Helicobacter pylori
[0099] Genes encoding the structural A and B subunits of
Helicobacter pylori urease were obtained by polymerase chain
reaction (PCR) cloning according to standard procedures, based on
previously published sequences. (Clayton, C. L. et al. S.
"Nucleotide Sequence of Two Genes from Helicobacter pylori Encoding
for Urease Subunits" Nucleic Acid Res., vol. 18, 362 (1990)). These
genes were inserted in a vector (named pEV40) designed for high
expression and easy purification of foreign genes in E. coli.
Briefly, the foreign gene is inserted down-stream of a
thermo-repressible promoter, and in frame of a sequence encoding
for a repeat of six histidines. An ampR gene is present on this
vector for selection of transformants. Under the appropriate
temperature conditions, the recombinant protein obtained is
supplemented by six histidines at the N-terminal, which allow for a
one-step affinity purification on a nickel column. Both
Helicobacter pylori recombinant urease A and B subunits were
expressed separately in E. coli, and purified on a nickel column to
>95% purity.
[0100] While it is preferred to employ recombinant Helicobacter
pylori urease obtained as described above as the antigenic
material, it will be understood that it is also possible to use, as
the antigenic material, any urease or subunit of urease obtained by
recombinant techniques (e.g. fusion protein) expressing antigenic
sites of urease, which is capable of eliciting a protective immune
response to Helicobacter infection. Thus, it is possible to employ
in a construct a urease gene having a substantial homology with
respect to Helicobacter pylori urease and which is effective in
raising a cross-protective immune response to Helicobacter.
Examples of such a urease is jack bean urease, which possesses
about 70% homology with Helicobacter pylori urease, or H. felis
urease, which possesses about 88% homology with Helicobacter pylori
urease. The invention is therefore not limited to the use of
Helicobacter pylori urease genes and their gene products, and
covers the use of any recombinant urease, or the subunits thereof,
which is sufficiently close antigenically to generate a protective
immunological response in a host to Helicobacter infection. The
invention includes within its scope the use of any urease or
subunit of urease, either naturally occurring or obtained by
recombinant DNA techniques, as well as a digested fragment or
peptide thereof, fusion protein comprising the whole urease,
subunit or fragment thereof, or truncated urease construct which is
effective in reducing the level of Helicobacter infection in an
infected mammal. Typically, a recombinant urease having a homology
of 70-95% homology, for example, 80-90% homology, with respect to
Helicobacter pylori urease, may be employed as the recombinant
urease antigen in the invention.
[0101] The discussion herein focuses on the use of recombinant
Helicobacter pylori urease A and B subunits produced by E. coli
(section C). However, it will be appreciated that recombinant
urease or subunits or constructs thereof mentioned above, capable
of eliciting the desired protective immune response, may be
produced using other recombinant DNA techniques and other
eukaryotic or prokaryotic expression vectors well known in the
art.
[0102] Tables 3, 4 and 5 below and FIG. 6 describe the results
obtained when mice were orally immunized with recombinant
Helicobacter pylori urease subunits produced in E. coli. In this
experiment, administration of the Helicobacter pylori antigen was
carried out by orally administering to the mice recombinant
Helicobacter pylori urease A or B subunits produced in E. coli and
purified as described above, and coupled to hydroxyapatite
crystals, used as a carrier to enhance M cell binding and uptake.
Cholera toxin (Sigma) was given as a mucosal adjuvant. In this
experiment, groups of female SPF BALB/c six-week old mice were each
orally immunized with 30 .mu.g of recombinant Helicobacter pylori
urease A subunit, coupled to 1 mg of hydroxyapatite plus 10 .mu.g
of cholera toxin adjuvant at day 0, 8, 14 and 21. The mice were
then challenged with 10.sup.8 H. felis, at day 32, 34 and 36. For
comparison purposes, similar female SPF BALB/c six-week old mice
were orally immunized with 30 .mu.g of recombinant Helicobacter
pylori urease B subunit coupled to hydroxyapatite plus 10 .mu.g
cholera toxin at day 0, 8, 14 and 21. The mice were challenged
three times, at day 32, 34 and 36, with H. felis. As a control,
female SPF BALB/c six-week old mice were each orally sham-immunized
with 10 .mu.g of cholera toxin and 1 mg of hydroxyapatite at day 0,
8, 14 and 21. The mice were then challenged at day 32, 34 and 36
with H. felis. All mice subject to the study were immunized and
challenged in parallel. Animals were sacrificed on day 48 (12 days
after challenge) or 10 weeks after challenge.
[0103] D. Analysis of Gastric Biopsies, Blood, and Intestinal
Secretions
[0104] Biopsies were taken from the stomach and blood was obtained
from the heart. The intestines were removed and washed with 1 mM
PMSF (Boeringher) in PBS buffer to obtain intestinal secretions for
ELISA analysis.
[0105] To evaluate protection against H. felis colonization,
gastric biopsies from each animal were screened for the presence of
H. felis by assessing rapid urease activity by the Jatrox HP test
(Rohm Pharma), according to the supplier's directions. Briefly,
gastric biopsies are immersed in 0.5 ml supplier's mixture of urea
and phenol red, a pH indicator. A non-commercial version of the
urea and phenol red mixture will be recognized by those skilled in
the art and may contain, for example, Bacto yeast extract (0.1 g),
monopotassium phosphate (0.091 g), disodium phosphate (0.095 g),
urea (20 g), and Bacto phenol red (0.01 g) at a final pH of 6.9 at
25.degree. C. Urease activity generates ammonia and bicarbonate
from urea, and is followed by the calorimetric change of the
solution towards a higher absorbance at 550 nm. Urease activity was
quantified by spectrophotometric analysis. Other methods of
assaying urease activity will be recognized by those skilled in the
art and may be found in, for example, Mobley, H. L. T. &
Hausinger, R. P., "Microbial Ureases: Significance, Regulation, and
Molecular Characterization," Microb. Reviews, Vol. 53, 85-108
(1989).
[0106] Gastric biopsies of each animal included in the experiment
described in section B were also cultured on BHI agarose plates,
supplemented as above, for the detection of H. felis. After
incubation for 3 to 10 days in microaerophilic conditions, the
presence of H. felis was confirmed by Gram staining and
determination of urease activity. As a very significant correlation
was obtained for the detection of H. felis in gastric biopsies
between urease tests and H. felis cultures during the first set of
experiments (see Table 3), only gastric biopsy urease tests were
performed for the detection of H. felis in the experiment described
in section C. Detection of H. felis was confirmed by microscopy by
two independent investigators, using two different colorations
(acridine orange and cresyl violet).
[0107] Blood samples were allowed to clot for 3 hours at RT (room
temperature), and serum harvested and frozen at -20.degree. C.,
until further analysis. Intestinal secretions were spun for 5
minutes at 4.degree. C. to remove debris, and kept frozen at
-20.degree. C. Serum and intestinal samples of each animal were
analyzed by ELISA for evaluation of anti-Helicobacter activity,
according to standard procedures. Briefly, 96-well plates were
coated with a sonicate of Helicobacter pylori, followed by
saturation with 5% fat-free milk. Samples were serially diluted
from 1:1 to 1:1000 and incubated overnight at 4.degree. C. on ELISA
plates. Biotinylated anti-mouse IgG (serum) and anti-mouse IgA,
followed by streptavidin-Horseradish peroxidase was used for the
determination of the antibody levels.
[0108] The results of H. felis challenges following immunizations
with purified Helicobacter pylori urease are set out in Tables 1-3
and FIGS. 1-4 and the results of H. felis challenges following
immunizations with recombinant Helicobacter pylori urease A and B
subunits are set out in Tables 4-6 and FIGS. 5 and 6.
1 TABLE 1 Immunoglobulins urease Intestinal mouse test culture
Serum secretion number Immunization 12 h Gram Ig Ig G Ig IgA 1
Urease + HF + H felis 27 0 25 258 2 Urease + HF 0 0 264 273 221 246
3 Urease + HF 0 0 84 44 318 354 4 Urease + HF + H felis 81 42 12 5
5 Urease + HF 0 0 98 137 126 234 6 Urease + HF + 0 968 2093 31 22 7
Urease + HF 0 0 98 0 96 34 8 Urease + HF 0 0 247 1010 214 128 9
Urease + HF 0 0 N.D. N.D. 48 23 10 Urease + HF 0 0 50 0 124 99 11
Urease 0 0 319 205 44 53 12 Urease 0 0 14 0 86 87 13 Urease 0 0 0 0
0 0 14 Urease 0 0 0 0 43 61 15 Urease 0 0 58 0 110 127 16 Urease 0
0 140 63 21 37 17 Urease 0 0 84 240 114 280 18 Urease 0 0 N.D. N.D.
93 148 19 Urease 0 0 45 0 135 216 20 Urease 0 0 261 197 161 261 21
CT + HF 0 0 0 0 0 2 22 CT + HF + H felis 63 0 310 303 23 CT + HF +
H felis 90 0 N.D. N.D. 24 CT + HF + H felis 31 0 150 192 25 CT + HF
+ H fells 197 250 250 440 26 CT + HF + H felis 105 135 214 138 27
CT + HF + H felis 140 47 109 55 28 CT + HF + 0 0 0 16 15 29 CT + HF
+ H felis 0 0 0 0 30 CT + HF + H felis N.D. N.D. N.D. N.D. 31 HP
sonicate + HF + H felis 0 0 76 103 32 HP sonicate + HF + H felis 77
0 11 33 33 HP sonicate + HF + H felis 549 748 57 36 34 HP sonicate
+ HF 0 0 660 153 180 286 35 HP sonicate + HF + H felis 730 192 0 5
36 HP sonicate + HF + H felis 32 0 5 64 37 HP sonicate + HF 0 0 400
400 312 1149 38 HP sonicate + HF + H felis 1007 1360 149 26 39 HP
sonicate + HF 0 0 220 186 133 122 40 HP sonicate 0 0 873 1016 352
514 41 HP sonicate 0 0 727 899 126 191 42 HP sonicate 0 0 109 68 44
83 43 HP sonicate 0 0 147 949 167 97 44 HP sonicate 0 0 845 1094
246 64 45 HP sonicate 0 0 1217 1198 210 157 46 HP sonicate 0 0 81 0
256 218 47 HP sonicate 0 0 329 210 241 276 48 HP sonicate 0 0 1049
737 197 211
[0109] In Table 1, which refers to the experiment described in
section B, "h" means hour, "Ig" means immunoglobulin, "ND" means
"not determined", "urease+HF" means that the mice were immunized
with urease (coupled to hydroxyapatite, with cholera toxin) and
then challenged with H. felis, "urease" means that the mice were
immunized with urease (coupled to hydroxyapatite, with cholera
toxin) and not challenged, "CT+HF" means that the mice were
sham-immunized with cholera toxin and challenged with H. felis, "HP
sonicate +HF" means that the mice were immunized with Helicobacter
pylori sonicate with cholera toxin and challenged by H. felis, and
"HP sonicate" means that the mice were immunized with Helicobacter
pylori sonicate with cholera toxin and not challenged. In Table 1,
the numbers for the antibody results are given as a measure of
absorbance at 595 nm multiplied by 1000. The background measured in
absence of the antibodies, was subtracted.
[0110] The results of experiment described in section B obtained on
the basis of the gastric biopsies urease tests and on Gram staining
of H. felis cultures are set out in Table 2. Infection was defined
by mice with one or more markers of colonization by H. felis,
including urease test or Gram staining of cultures.
2TABLE 2 Immunization Challenge % infected % protected Urease H.
felis 3/10 (30%) 7/10 (70%) * Sonicate H. felis 6/9 (66%) 3/9 (33%)
** CT H. felis 9/10 (90%) 1/10 (10%) * p = 0.0198 (two tailed
Fisher exact test) compared to CT control ** p = 0.303 (two tailed
Fisher exact test) compared to CT control
[0111] It will be seen from the results set out in Tables 1 and 2
that statistically significant protection against H. felis
challenge is obtained with oral immunization using Helicobacter
pylori urease as compared to that obtained using either
Helicobacter pylori sonicate or cholera toxin. Referring to Table
2, it will be seen that from a total of 10 immunized animals, only
3 became infected, as compared to 6 of the animals immunized with
Helicobacter pylori sonicate and 9 of the animals immunized with
cholera toxin. Table 2 shows that 70% of the animals were protected
from challenge by H. felis as compared to 33% of the animals
immunized with Helicobacter pylori sonicate and 10% of the animals
immunized with cholera toxin and then subjected to H. felis
challenge. In other words, 90% of the control mice exposed to H.
felis became infected by that pathogen whereas, in contrast, in
mice immunized with Helicobacter pylori urease 28 days before
exposure to H. felis, the infection rate was only 30%. This
represents a significant reduction in infection (p=0.0198 in the
Fisher exact test, as compared to the control mice). When the mice
were orally immunized with Helicobacter pylori sonicate, the
infection rate was 67% (not significant versus the control). The
protection obtained using Helicobacter pylori urease is unexpected
and could not have been predicted on the basis of the results
observed using Helicobacter pylori sonicate.
[0112] Referring to FIGS. 1-4, FIG. 1 represents graphically the
results of tests for antibodies in serum (IgG) and intestinal
secretion (IgA) in mice not protected after immunization with
urease. These are mice numbers 1, 4 and 6 appearing in Table 1, and
constitute Group A. FIG. 2 shows the antibody responses of mice
that were protected after immunization with urease (Group B), i.e.
mice 2, 3, 5 and 7-10.
[0113] FIGS. 3 and 4 relate to the results obtained with mice
numbers 31-39. FIG. 3 (Group C) depicts antibody responses of mice
not protected after immunization with Helicobacter pylori sonicate
(mice numbers 31, 32, 33, 35, 36 and 38) and FIG. 4 (Group D)
depicts the antibody responses of mice protected after immunization
with Helicobacter pylori sonicate (mice numbers 34, 37 and 39). It
is of interest to note with respect to FIGS. 3 and 4 that the IgA
antibody responses (but not IgG) are higher in the mice exhibiting
protection than in the mice that are not protected, suggesting a
correlation between protection and IgA response. Serum IgG
responses did not exhibit a correlation. Mucosal IgA but not serum
IgG are known to play a role in protection against bacterial
infections of the gut % (McGhee, J. R and Kyono, H. "New
Perspectives in Vaccine Development: Mucosal Immunity to
Infections" Infect Agents Dis., vol. 2, 55-73 (1993)).
[0114] The results of the correlation between the detection of H.
felis in gastric biopsies by urease tests and by cultures are set
out in Table 3.
3 TABLE 3 Urease Test + Urease Test - Total H. felis culture (+) 16
0 16 H. felis culture (-) 2 30 32 Total 18 30 48 Two-tailed
Fisher's Exact Test: p < 0.00001
[0115] Table 3 shows that a very significant correlation exists
between the results of urease tests performed on gastric biopsies
and the identification of H. felis by cultures. All animals shown
in Table 3 that were positive for urease activity, were H. felis
positive by histopathology. As cultures detected less often H.
felis infection than urease tests, urease tests were preferred for
the diagnosis of H. felis infection in mice in the next
experiments, due to its better sensitivity. This it approach
allowed the duplication of urease tests with larger fragments of
the stomach of each mouse, and a further increase in the
sensitivity of the urease test. Furthermore, the use of the method
with the highest sensitivity prevent an overestimation of the
protection obtained by the vaccine preparation to be tested. When
positive culture is used as the standard for infection, the
protection induced after urease immunization during the experiment
depicted in section B is as significant as with the combined use of
urease test and culture (p=0.021 versus p=0.019).
[0116] The results of the experiments described in section C
(recombinant urease subunits), obtained on the basis of the gastric
biopsies urease tests, are set out in Table 4, 5 and 6 and depicted
in FIG. 6.
4 TABLE 4 immunization mice n.degree. Urease test Infection CT 20
0.49 + Sacrificed 12 days 21 0.31 + post challenge 22 0.62 + 23
0.67 + 24 0.55 + 50 0.50 + 51 0.37 + 52 0.29 + 53 0.79 + 54 0.32 +
ure A + HAP + CT 40 0.67 + Sacrificed 12 days 41 0.48 + post
challenge 42 0.42 + 43 0.65 + 44 0.56 + 45 0.52 + 46 0.33 + 47 0.63
+ 48 0.22 + 49 0.37 + ure B + HAP + CT 25 0.15 - Sacrificed 12 days
26 0.07 - post challenge 27 0.03 - 28 0.64 + 29 0.13 - 30 0.02 - 31
0.66 + 32 0.00 - 33 0.79 + 34 0.15 - ure A + HAP + CT 68 0.00 -
Sacrificed 10 weeks 69 0.07 - post challenge 70 0.42 + 71 0.00 - 72
0.00 - ure B + HAP + CT 73 0.37 + Sacrificed 10 weeks 74 0.00 -
post challenge 75 0.37 + 76 0.00 - 77 0.00 - 78 0.00 - 79 0.39 + 80
0.00 - 81 0.37 + 82 0.00 -
[0117] In Table 4, "CT" means cholera toxin; "UreA" means
recombinant Helicobacter pylori urease A subunit; "Ure B" means
recombinant Helicobacter pylori urease B subunit; and "HAP" means
hydroxyapatite crystals. Mice 20 to 54 were sacrificed 12 days post
challenge and mice 68 to 82 10 weeks (106 days) post challenge. The
results of the urease test performed from biopsies of the stomach
of each animal are expressed as OD values at 550 nm. The O.D. value
for the assay solution alone (0.075 O.D.) was subtracted as
background from the O.D. value obtained for each mouse. The
positive and negative signs depicts the final status of infection
of each animal, according to the positivity or negativity of the
urease test for detection of H. felis. Positivity: OD.sub.550
values >0.2. The 0.2 value was chosen to define a positive
infection because at this value, the color change to the Jatrox
solution could be observed with the naked eye.
5TABLE 5 Protection as measured 12 days post challenge Immunization
Challenge % infected % protected Urease A subunit H. felis 10/10
(%) 0/10 (0%) Urease B subunit H. felis 3/10 (30%) 7/10 (70%) * CT
H. felis 10/10 (100%) 0/10 (0%) * p = 0.0031 (two tailed Fisher
exact test) compared to CT control
[0118]
6TABLE 6 Protection as measured 10 weeks post challenge
Immunization Challenge % infected % protected Urease A subunit H.
felis 1/5 (20%) 4/5 (80%) * Urease B subunit H. felis 4/10 (40%)
6/10 (60%) ** * p = 0.003 (two tailed Fisher exact test) compared
to CT control ** p = 0.01 (two tailed Fisher exact test) compared
to CT control
[0119] It will be seen from the results set out in Tables 4, 5 and
6 that statistically significant protection against H. felis
challenge is obtained with oral immunization using recombinant
Helicobacter pylori urease B subunit as compared to that obtained
using either recombinant Helicobacter pylori urease A subunit or
cholera toxin. Referring to Table 4, it will be seen that, 12 days
post challenge, from a total of 10 immunized animals, only 3 were
found infected in the urease B subunit group, as compared to all 10
animals immunized with Helicobacter pylori A subunit of urease and
10 out of 10 of the animals immunized with cholera toxin. Table 4
shows that 70% of the animals were protected from challenge by H.
felis as compared to 0% of the animals immunized with Helicobacter
pylori urease A subunit and 0% of the animals immunized with
cholera toxin and then subjected to H. felis challenge. In other
words, 100% of the control mice challenged with H. felis became
infected whereas, in contrast, in mice immunized with recombinant
Helicobacter pylori urease B subunit the infection rate was only
30%. This represents a significant reduction in infection
(p=0.0031, Fisher exact test) as compared to the control mice. The
fact that the protection observed with Helicobacter pylori urease
is entirely conferred by immunization with the B subunit of urease,
and that the A subunit has no such effect, was not expected on the
basis of our experiment with purified urease. This definition of
the roles of the 2 structural subunits of urease in the development
of a protective immune response is therefore novel. The protection
obtained using recombinant urease subunits, which are enzymatically
inactive, also teaches that non toxic forms of urease can be used
as oral vaccine against Helicobacter infection. Furthermore these
results strongly suggest that recognition of the active site is not
required for protection, as an inactive urease B subunit is very
unlikely to induce antibodies that will recognize and inhibit the
catalytic site of native urease.
[0120] Referring to Table 6, it will be seen that, when mice are
sacrificed 10 weeks post infection, 60% (6 mice out of 10) of the
animals immunized with urease B subunit and 80% (4 mice out 5) of
the animals immunized with Helicobacter pylori urease A subunit
were protected against H. felis infection. The fact that protection
obtained through immunization with urease B subunit lasts over time
and that immunization with urease A induces a protection which is
delayed compared to the one induced by urease B subunit could not
be expected from our experiment with purified urease or with other
experiment performed earlier. The fact that urease A subunit
immunization confers protection definitively proves that
recognition of the active site is not required for protection.
[0121] FIG. 6 summarizes results obtained after oral immunization
with recombinant urease A and B subunits (described in Table 5 and
6).
[0122] A second set of mice was immunized and assayed for infection
with Helicobacter felis at 10 weeks post challenge according to the
protocol described in Section C. In this example, twelve mice were
sham immunized with cholera toxin alone, twelve mice were immunized
with recombinant ure A subunit, and ten mice were immunized with
recombinant ure B subunit. The mean urease activity level found in
stomach samples mice that were immunized with ure B, but not
infected (uninfected mice controls), was 0.045, and this amount was
subtracted as background from each O.D. value obtained. Mice were
considered to be infected when the O.D. value was greater than
twice the standard deviation of values obtained with the uninfected
mice controls; the standard deviation was 0.022. The data obtained
from this experiment is presented in Table 7.
7 TABLE 7 immunization mice no. Urease test Infection CT 135 0.40 +
Sacrificed 10 weeks 136 0.28 + post challenge 137 0.25 + 138 0.10 +
139 0.10 + 140 0.34 + 141 0.41 + 142 0.36 + 143 0.46 + 144 0.40 +
145 0.40 + 146 0.51 + ure A + HAP + CT 161 0.12 + Sacrificed 10
weeks 162 0.47 + post challenge 163 0.00 - 164 0.00 - 165 0.02 -
166 0.01 - 167 0.01 - 168 0.37 + 169 0.00 - 170 0.39 + 171 0.47 +
172 0.00 - ure B + HAP + CT 151 0.00 - Sacrificed 10 weeks 152 0.00
- post challenge> 153 0.00 - 154 0.03 - 155 0.00 - 156 0.00 -
157 0.02 - 158 0.00 - 159 0.01 - 160 0.00 -
[0123] using this alternative method of analysis, the data
presented in Table 4 was reanalyzed along with the data in Table 7.
Instead of subtracting the background O.D. value obtained with the
urease assay solution alone (0.075 O.D.), the mean as the
background level.
[0124] The mean urease level obtained from unifected mice
sacrificed at twelve days was 0.089. Mice numbered 20-54 were
considered infected when the O.D. value was greater than twice the
standard deviation of values obtained with the uninfected mice
controls; the standard deviation was 0.008. the background level
sutracted from the O.D. values of mice sacrificed at ten weeks was
0.045, and mice were considered infected when the O.D. values was
greater than 0.044
[0125] Using this alternative method of analysis, at twelve days
post challenge, no effect was seen after sham immunization or after
immunization with the ure A subunit. However, only a low-grade
infection (O.D. value <0.22) was observed in 70% of the animals
immunized with the ure B subunit (p <0.02, Mann-Whitney U-test,
compared to sham immunized control). When mice were sacrificed ten
weeks post challenge, 59% (10/17) of the mice immunized with the
ure A subunit were protected against H. felis infection (p=0.0019,
two-tailed Fisher's exact test, when compared to sham-immunized
mice). Furthermore, 80% (16/20) of the mice immunized with the ure
B subunit were protected against H. felis infection (p=0.00002,
two-tailed Fisher's exact test, when compared to control,
sham-immunized mice). Under this alternative analysis, the ure A
subunit also elicits protective immunity. Under either method of
analysis, the results obtained demonstrate that immunization with
recombinant Helicobacter pylori urease subunits elicits protective
immunity against Helicobacter infection.
EXAMPLES
[0126] The invention will now be further described by reference to
the following non-limiting examples.
[0127] a) The Bacterial Strains
[0128] H. felis was provided by J. Fox (Division of Comparative
Medicine, Mass. Institute of Technology, Boston, USA). Helicobacter
pylori was isolated from patients with ulcer disease (CHUV,
Lausanne, Switzerland).
[0129] b) Bacterial Cultures
[0130] Liquid Culture--Bacteria were cultured on BHI (Brain-Heart
Infusion, BioMerieux) liquid medium containing 0.25% of yeast
extract (Difco) and 10% of fetal calf serum (Inotech) supplemented
with 0.4% of Campylobacter selective complement (Oxoid). The
bacteria were incubated under microacrophilic conditions at
37.degree. C. and shaken at 37.degree. C. for 2 to 3 days.
[0131] Frozen Culture--The bacteria were cultured in liquid media,
then assayed for urease activity, and evaluated for morphology by
Gram staining and for motility by microscopy. The bacteria were
then centrifuged and resuspended at a concentration of 30 O.D. per
ml in BHI plus 20% glycerol and frozen at -80.degree. C. Just
before use, frozen stocks were thawed on ice, washed in 20 ml BHI,
centrifuged, and resuspended at a concentration of 1 O.D. to 1.5
O.D. per 200 .mu.l in 5 mM NaHCO.sub.3.
[0132] Culture on Agarose plates--The bacteria were cultured on
agar plate consisting on BHI with 0.25% of yeast extract and 5% of
sheep blood under microacrophilic conditions at 37.degree. C. for 3
days.
[0133] Quantification--The quantity of bacteria was determined by
the optical density of the BHI solution at 660 nm (1 optical
density unit (O.D.) corresponding to 10.sup.8 bacteria). The number
of viable bacteria is measured on the number of colony forming
units.
[0134] c) Preparation of Sonicates
[0135] Helicobacter pylori was collected from 31 blood agar plates
in 0.15 M NaCl and spun 5 minutes at 1400g at 4.degree. C. The
pellet was resuspended in 3 ml of NaCl and sonicated for 4 minutes.
The amount of proteins was evaluated by a Bradford assay (BioRad
Kit according to supplier).
[0136] d) Coupling of Immunogen to Hydroxyapatite
[0137] Immunogen (urease or subunit thereof) was incubated for 1
hour at 4.degree. C. with hydroxyapatite. 1.0 mg of hydroxyapatite
was used for 30 .mu.g of immunogen per mouse. At the end of the
incubation, 10 .mu.g of cholera toxin was added in a final volume
of 200 .mu.l PBS.
[0138] e) Challenge with Helicobacter felis
[0139] Mice were lightly anesthetized prior to intragastric
challenge with Helicobacter felis. H. felis in a total volume of
200 .mu.l were delivered to the stomachs of the respective mice
using silicon tubing attached to a hypodermic syringe.
Example 1
[0140] a) Extraction
[0141] Helicobacter pylori from 30 blood agar plates was harvested
in 0.15 M NaCl on ice. The solution was spun 5 minutes at 1400 g at
4.degree. C. The pellet was resuspended in 20 ml of H.sub.2O and
vortexed for 45 seconds (maximum speed). The extract was then spun
20 minutes at 6700 g at 4.degree. C. The supernatant was recovered
and the quantity of protein was evaluated (see "Quantification"
above) and precipitated with 70% of ammonium sulfate.
[0142] b) Purification of Urease
[0143] The solution was chromatographed on a Sepharose CL-6B column
(Pharmacia) with PBS (phosphate buffered saline) as mobile phase.
The 22 collected fractions which presented a strong urease activity
were pooled and dialyzed overnight at 4.degree. C. against 3 liters
of PEB (20 mM phosphate buffer, pH 7) and then chromato-graphed on
a Q Sepharose fast flow (Pharmacia) with PEB as mobile phase. The
fractions were eluted by 0 to 500 mM NaCl gradient. Ten of the
collected fractions with a strong urease activity were individually
subjected to an SDS gel followed by a Coomassie staining. The 6
fractions presenting 2 distinct bands corresponding to MW-63 and
-28 KDa were pooled and were considered as the purified urease.
Example 2
[0144] (see also section B)
[0145] Mice employed in the immunization studies were lightly
anesthetized with ether prior to intragastric immunization. And
then, sonicate preparation or purified urease, hydroxyapatite and
cholera toxin was suspended in PBS and 200 .mu.l were delivered to
the stomach of the respective mice using a polyethylene tubing
attached to a hypodermic syringe. This procedure will be referred
to as oral immunization.
[0146] Three oral immunization protocols were evaluated. These are
described below.
[0147] Protocol B1--Vaccination with Purified Urease
[0148] Female BALB/c six-week old mice (20) were orally immunized
with 30 .mu.g of purified of Helicobacter pylori urease and 1 mg of
hydroxyapatite and 10 .mu.g of cholera toxin at day 0, 7, 14 and
21. Ten mice were challenged at day 28 and 30 with 5.times.10.sup.7
and 10.sup.8 H. felis from liquid culture.
[0149] Protocol B2--Vaccination with Helicobacter Sonicates
[0150] Female BALB/c six-week old mice (20) were orally immunized
with 2 mg of Helicobacter pylori sonicate solution at day 0, 7, 14
and 21. Ten mice were challenged at day 28 and 30 with
5.times.10.sup.7 and 10.sup.8 H. felis.
[0151] Protocol B3--Control
[0152] Female BALB/c six-week old mice were orally immunized with 1
mg hydroxyapatite and 10 .mu.g of cholera toxin at day 0, 7, 14 and
21. The mice were challenged at day 28 and 30 with 5.times.10.sup.7
and 10.sup.8 H. felis.
[0153] At day 35 all mice were sacrificed and biopsies from the
stomach were taken as well as intestinal secretions and blood.
[0154] Protection and Evaluation
[0155] To evaluate protection, biopsies were screened for the
urease activity by the Jatrox HP test (Rohm Pharma) according to
the instructions of the supplier. The urease is quantified by a
spectrophotometric measurement at 550nm. The biopsies were also
cultured for the presence of H. felis and the presence of H. felis
was determined by Gram staining. Gastric antral biopsies were
homogenized and diluted (1:10 and 1:1000) in 0.15 M NaCl and plated
onto blood agar plates and incubated under microaerophilic
conditions at 37.degree. C. for 4 to 10 days.
[0156] ELISA
[0157] Intestinal secretions and blood were analyzed by ELISA for
the evaluation of antibody titer. The ELISA was carried out as
follows. Polystyrene plates (96 wells) were coated with lug/well of
purified urease at 37.degree. C. for 2 hrs. Non specific binding
sites were blocked with 5% powdered milk in PBS 0.1% Tween at
37.degree. C. for 30 minutes. The plates were washed once with PBS
0.1% Tween. Blood samples were tested at dilution 1:1000 and
intestinal secretions 1:1. 100 .mu.l of each sample were added to
the antigen coated plates. After 2 hrs of incubation, plates were
washed 3 times with PBS containing 0.1% Tween. Anti-mouse
biotinylated whole antibody from goat and anti-mouse IgA, IgG and
IgM biotinylated (Amersham) were added (100 .mu.l) at dilution
1:500 except for IgA (1:250) and incubated at 37.degree. C. for 1
hr. The plates were washed 3 times with PBS 0.1% Tween and 100
.mu.l of 1:1000 dilution of streptavidin Horseradish peroxidase in
PBS containing 0.1% Tween were added and incubated at 37.degree. C.
for 30 minutes. The plates ware washed 3 times and 50 .mu.l of 1:50
dilution of o-phenyl-diamine in citrate buffer pH 5.0 with 1
.mu.l/ml of 30% H.sub.2O.sub.2 were added and incubated at room
temperature for 20 minutes. The absorbance at 495 nm was measured
in each well.
Example 3
[0158] (see also section C)
[0159] Mice employed in the immunization studies were lightly
anesthetized with ether prior to intragastric immunization. Then,
30 .mu.g recombinant Helicobacter pylori urease A or B subunit
produced in E. coli, bound with hydroxyapatite, and supplemented
with cholera toxin was suspended in PBS and 200 .mu.l were
delivered to the stomach of the respective mice using a
polyethylene tubing attached to a hypodermic syringe. This
procedure will be referred to as oral immunization.
[0160] Three oral immunization protocols were evaluated. These are
described below.
[0161] Protocol C1--Vaccination with Recombinant Urease A
Subunit
[0162] Female BALB/c six-week old mice (10) were orally immunized
with 30 .mu.g of purified recombinant Helicobacter pylori urease A
subunit and 1 mg of hydroxyapatite and 10 .mu.g of cholera toxin at
day 0, 8, 14 and 21. Ten mice were challenged at day 32, 34 and 36
with 10.sup.8 H. felis from liquid culture.
[0163] Protocol C2--Vaccination with Recombinant Urease B
Subunit
[0164] Female BALB/c six-week old mice (10) were orally immunized
with 30 .mu.g of recombinant Helicobacter pylori urease B subunit
and 1 mg of hydroxyapatite and 10 .mu.g of cholera toxin at day 0,
8, 14 and 21. Ten mice were challenged at day 32, 34 and 36 with
10.sup.8 H. felis from liquid culture.
[0165] Protocol C3--Control
[0166] Female BALB/c six-week old mice were orally immunized with 1
mg of hydroxyapatite and 10 .mu.g of cholera toxin at day 0, 8, 14
and 21. The mice were challenged at day 32, 34 and 36 with 10.sup.8
H. felis.
[0167] At day 42, or at day 106, mice were sacrificed and multiple
biopsies from the stomach were taken.
[0168] Protection and Evaluation
[0169] To evaluate protection, biopsies of the corpus and antrum of
the stomach were screened for urease activity by the Jatrox HP test
(Rohm Pharma) according to the instructions of the supplier. The
urease is quantified by a spectrophotometric measurement at 550 nm.
The total of corpus and antrum OD values were added to obtain a
final OD value for each mouse.
Example 4
[0170] To determine whether immunization with urease peptides would
be an effective treatment of Helicobacter infection animals, mice
were first challenged with H. felis and then immunized with
Helicobacter pylori ure B subunit. The utility of immunization with
Helicobacter pylori ure B subunit to treat Helicobacter infection
is demonstrated in both Example 4 and Example 5.
[0171] a) Infection of Mice with H. felis
[0172] Female BALB/c six-to-eight week old mice were challenged
with H. felis at days 1 and 3 with 1 O.D. of frozen culture. The
mice were challenged at day 5 with 1.53 O.D. of liquid H. felis
culture.
[0173] b) vaccination with Recombinant Helicobacter pylori Urease B
Subunit
[0174] Eight H. felis infected mice were orally immunized with 30
.mu.g of recombinant Helicobacter pylori urease B subunit, 1 mg of
hydroxyapatite and 10 .mu.g of cholera toxin (holoenzyme obtained
from Calbiochem) at days 23, 30, 37, and 44.
[0175] c) Controls Ten H. felis infected mice were orally immunized
with 1 mg of hydroxyapatite and 10 .mu.g of cholera toxin at days
23, 30, 37, and 44. These mice were designated as "sham"
immunized.
[0176] Seven H. felis infected mice were not subject to
immunization.
[0177] d) Sacrifice and Evaluation
[0178] At day 70 all mice were sacrificed. The stomachs were
removed and cut in half longitudinally. To evaluate protection,
one-half of the stomach of each mouse (both corpus and antrum
sections) was screened for urease activity 3-4 hours
post-sacrifice, by the Jatrox HP test (Rohm Pharma) according to
the instructions of the supplier. The urease was quantitated by a
spectrophotometric measurement at 550 nm. Ten naive (uninfected)
BALB/c mice served as controls. These mice were sacrificed at day
70 and the stomach samples evaluated for urease activity to
determine the background level of urease activity. The mean
background value, 0.033 O.D., was subtracted from the O.D. value
for each mouse. The standard deviation in urease activity among the
naive mice was 0.025 O.D. The results of the urease assays are set
out in Table 8 and depicted in FIG. 7. In FIG. 7, the dashed line
indicates the O.D. value for twice the standard deviation in urease
activity among the naive mice.
8TABLE 8 Treatment Mouse No. Urease test Infection Untreated 1 0.42
+ 2 0.36 + 3 0.41 + 4 0.33 + 5 0.38 + 6 0.52 + 7 0.48 + Sham (HAP +
CT only) 8 0.51 + 9 0.48 + 10 0.61 + 11 0.44 + 12 0.51 + 13 0.48 +
14 0.44 + 15 0.58 + 16 0.48 + 17 0.53 + Ure B + HAP + CT 18 0.36 +
19 0.01 - 20 0.43 + 21 0.07 +/- 22 0.39 + 23 0.10 +/- 24 0.38 + 25
0.06 +/-
[0179] In Table 8, "ure B" means recombinant Helicobacter pylori
urease B subunit as referenced above; "untreated" means the mice
were challenged with H. felis, but received no subsequent
immunization. The results of the urease test performed from
biopsies of the stomach of each animal are expressed as O.D. values
at 550 nm. The background value subtracted from the O.D. value for
each mouse was 0.033. The infection is rated as positive (indicated
by a "+") where the O.D. value at 550 nm is over twice the standard
deviation found with the naive mouse controls (0.05 O.D.). A "+/-"
indicates that the O.D. value is greater than background but less
than 0.08, and that the value is reduced compared to the sham
immunized controls.
[0180] It will be seen from the results set out in Table 7 and in
FIG. 7 that administration of the Helicobacter pylori ure B subunit
to mice infected with H. felis resulted in the clearance of
infection in one out of eight mice. According to another, less
conservative, interpretation of these results, defining a positive
result as a value greater than 0.2, four out of eight mice (numbers
2, 4, 6, and 8) treated with ureB would be found to be protected
from infection. However, under either of the two definitions of a
positive result these mice exhibited a reduced urease activity as
compared to the untreated and the sham-immunized mice. Using the
0.05 value to define infection, the level of infection in ure B
immunized mice as compared to sham immunized mice exhibits a
statistically significant (p value of less than 0.004) reduction in
the level of infection. Thus the results of Example 4 indicate that
administration of ure B to mice infected with H. felis results in a
reduced level of infection.
Example 5
[0181] a) Infection of Mice with H. felis
[0182] Female BALB/c five-to-eight week old mice were challenged
with H. felis at day 1 with 1 O.D. of liquid culture. The mice were
challenged at day 3 with 0.8 O.D. H. felis cultured on agarose
plates. The mice were challenged at day 5 with 1 O.D. of H. felis
cultured on agarose plates.
[0183] b) Vaccination with Recombinant Helicobacter pylori Urease B
Subunit
[0184] H. felis infected mice were orally immunized with 30 .mu.g
of recombinant Helicobacter pylori urease B subunit, 1 mg of
hydroxyapatite and 10 .mu.g of cholera toxin (holoenzyme obtained
from Calbiochem) at days 23, 30, 37, and 44. These mice were
designated as "immunized."
[0185] c) Controls
[0186] H. felis infected mice were orally immunized with 1 mg of
hydroxyapatite and 10 .mu.g of cholera toxin at days 23, 30, 37,
and 44. These mice were designated as "sham" immunized.
[0187] d) Sacrifice and Evaluation
[0188] At day 62, ten immunized mice and six sham immunized mice
were sacrificed. At eight weeks after the last immunization, ten
immunized and eight sham immunized mice were sacrificed. Stomach
samples were screened for urease activity as described in Example
4. The mean background found in 29 naive BALB/c mice, sacrificed at
various times, was 0.04 O.D. This level of activity was used as a
background measurement and subtracted from the O.D. value for each
mouse. The standard deviation in urease activity among the naive
mice was 0.02 O.D. The results of the urease assays are set out in
Table 9 and depicted in FIG. 8. In FIG. 8, the dashed line
indicates the O.D. value for twice the standard deviation in urease
activity among naive mice.
[0189] e) Analysis of Blood and Fecal Samples
[0190] Blood Sample Collection
[0191] Blood samples were allowed to clot for 3 hours at room
temperature, and serum harvested and frozen at -20.degree. C.,
until further analysis.
[0192] Fecal Pellet Collection
[0193] Fresh fecal pellets (3-8) were collected in 1.5 ml eppendorf
tubes containing 500 .mu.l of PBS +5% non-fat dry milk+protease
inhibitors (AEBSF 0.2 mM, Aprotinin 1 .mu.g/ml, Leupeptin 10 .mu.M,
Bestatin 3.25 .mu.M). The samples were frozen at -20.degree. C.
until use. They were then thawed on ice, mashed, and centrifuged at
10,000 g for 10 minutes at 4.degree. C. to yield a supernatant free
of debris, and yellowish brown in appearance.
[0194] ELISA
[0195] Serum and fecal pellet samples of each animal were analyzed
by ELISA for evaluation of anti-urease activity, according to
standard procedures.
[0196] Fecal pellets and blood were analyzed by ELISA for the
evaluation of antibody titer. Polystyrene plates (96 wells) were
coated with 0.5 .mu.g/well of purified recombinant urease at
37.degree. C. for 2 hrs. Non specific binding sites were blocked
with 5% powdered milk in PBS containing 0.1% Tween at 37.degree. C.
for 30 minutes. The plates were washed once with PBS containing
0.1% Tween. Blood samples were tested at a dilution of 1:200 and
fecal pellets at a dilution of 1:1. 100 ul of each sample were
added to the antigen coated plates. After 2 hrs of incubation,
plates were washed 3 times with PBS containing 0.1% Tween.
Anti-mouse biotinylated whole antibody from goat (Amersham) and
anti-mouse IgA coupled to Horseradish peroxidase (Serotec) were
added (100 .mu.l) at a dilution of 1:500 and incubated at
37.degree. C. for 1 hr. The plates were washed 3 times with PBS
containing 0.1% Tween and, for blood samples only, 100 .mu.l of a
1:500 dilution of streptavidin and Horseradish peroxidase in PBS
containing 0.1% Tween were added and incubated at 37.degree. C. for
30 minutes. The plates were washed 3 times and 50 .mu.l of a 1:50
dilution of o-phenyl-diamine in citrate buffer, pH 5.0, with 1
.mu.l/ml of 30% H.sub.2O.sub.2 were added and incubated at room
temperature for 20 minutes. The absorbance at 495 nm was measured
in each well.
[0197] For the evaluation of total IgAs in fecal pellets, the ELISA
was carried out as described above except that the plates were
coated with 1 .mu.g/ml of goat anti-mouse IgA (SIGMA) and the fecal
supernatant was tested at a 1:200 dilution.
9TABLE 9 Sacrifice Treatment Mouse No. Urease test Infection 2.5
weeks Sham 1 0.58 + (HAP + CT only) 2 0.51 + 3 0.50 + 4 0.54 + 5
0.47 + 6 0.36 + Ure B + HAP + CT 7 0.02 - 8 0.06 + 9 0.01 - 10 0.00
- 11 0.02 - 12 0.02 - 13 0.02 - 14 0.44 + 15 0.00 - 16 0.03 - 8
weeks Sham 17 0.30 + (HAP + CT only) 18 0.27 + 19 0.29 + 20 0.20 +
21 0.27 + 22 0.27 + 23 0.37 + 24 0.31 + ure B + HAP + CT 25 0.26 +
26 0.30 + 27 0.00 - 28 0.17 + 29 N.D. 30 0.00 - 31 0.00 - 32 0.00 -
33 0.00 - 34 0.00 -
[0198]
10 TABLE 10 Sacrifice Treatment % cleared 2.5 weeks ure B 8/10
(80%) * sham 0/6 8 weeks ure B 6/9 (67%) ** sham 0/9 * p = 0.007
(two-tailed Fisher exact test) compared to sham control ** p =
0.014 (two-tailed Fisher exact test) compared to sham control
[0199] in Tables 9 and 10, "ure B` means recombinant Helicobacter
pylori urease B subunit as referenced above; "CT" means cholera
toxin; and "HAP " means hydroxyapatite crystals. "sacrifice" means
the date of sacrafice as measured from the last immunization at day
44. "N.D." indicates that no data were available. The results of
the urease test performed form biopsies of the stomach of each
animal are expressed as O.D. values at 550 nm. The infection is
rated as positive (indicated by a "+") where the O.D. value at 550
nm is over twice the standard deviation found in the mature mice
(0.04 O.D.). The background value subtracted from the O.D. value
for each mouse was 0.04 O.D. In Table 10, "% cleared" indicates
clearance of infection as measured by the urease assay.
[0200] It will be seen from the results set out in Tables 9 and 10,
and in FIG. 8, that administration of the Helicobacter pylori ure B
subunit to mice infected with H. felis resulted in the clearance of
infection in eight out of ten mice as assayed when the mice were
sacrificed 2.5 weeks after the last immunization. When the assay
was performed when the mice were sacrificed eight weeks after the
last immunization, the infection was cleared in six out of nine
mice. If the higher value of 0.2 is chosen to define infection as
referred to in the discussion of Table 8, under this method of
interpretation, treatment with the ure B subunit resulted in
clearance of infection in nine out of ten mice sacrificed at 2.5
weeks.
[0201] Table 10 indicates that statistically significant
therapeutic treatment of H. felis infection is obtained with oral
immunization using recombinant Helicobacter pylori urease B subunit
as compared to that obtained by sham immunization with cholera
toxin and hydroxyapatite alone. Table 10 shows that, when measured
at 2.5 weeks after the last immunization, 80% of the infected mice
were cleared of infection. When measured at eight weeks after the
last immunization, 67% of the infected mice were cleared of
infection. None of the sham immunized mice were cleared of
infection when measured at either 2.5 or eight weeks after the last
immunization. The significance of the reduction in the percentage
of mice cleared of infection when measured at eight weeks as
compared to 2.5 weeks after the last immunization is unclear at
present.
11TABLE 11 ure B IgA's in Feces/ Total anti- Total Mouse Urease
ureB Igs in Total IgAs ureB IgA's IgA's in No. Test Blood in Feces
in Feces Feces Ex. 4: 8 0.51 0.27 0.77 0.05 0.06 9 0.48 0.23 0.65
0.02 0.03 10 0.61 0.25 0.43 0.08 0.18 11 0.44 0.36 0.69 0.03 0.04
12 0.51 0.31 0.58 0.00 0.00 13 0.48 0.32 0.67 0.11 0.17 14 0.44
0.27 0.51 0.03 0.05 15 0.58 0.29 0.82 0.07 0.08 16 0.48 0.30 0.77
0.18 0.23 17 0.53 0.30 0.53 0.09 0.18 18 0.36 0.22 0.51 0.00 0.00
19 0.01 0.47 0.62 0.47 0.75 20 0.43 0.35 0.57 0.15 0.26 21 0.07
0.25 0.64 0.11 0.17 22 0.39 0.27 0.50 0.15 0.30 23 0.10 0.27 1.08
ND ND 24 0.38 0.28 0.66 ND ND 25 0.06 0.28 0.53 ND ND Ex. 5: 7 0.02
0.11 0.61 0.06 0.11 8 0.06 0.13 0.62 0.02 0.03 9 0.01 0.15 0.62
0.04 0.07 10 0.00 0.15 0.59 0.03 0.05 11 0.02 0.19 0.60 0.15 0.24
12 0.02 0.16 0.60 0.00 -0.01 13 0.02 0.19 0.66 0.07 0.11 14 0.44
0.18 0.62 0.00 0.00 15 0.00 0.10 0.64 0.08 0.12 16 0.03 0.14 0.62
0.29 0.46 25 0.26 0.35 0.98 0.07 0.07 26 0.30 0.38 1.12 0.04 0.03
27 0.00 0.39 1.15 0.01 0.01 28 0.17 0.33 0.97 0.02 0.02 29 N.D.*
0.38 1.00 0.08 0.08 30 0.00 0.34 0.69 0.04 0.06 31 0.00 0.38 0.76
0.08 0.10 32 0.00 0.37 0.58 0.02 0.04 33 0.00 0.38 1.07 0.22 0.21
34 0.00 0.35 0.64 0.06 0.09
[0202] In Table 11, the mice that were treated with ureB, used in
Examples 4 and 5, were evaluated for the antibody content of the
blood and the feces. The mouse numbers used correspond to the
numbers used in Tables 8 and 9. The urease test results correspond
to the results reported in Tables 8 and 9. "ND*" indicates that no
data was available because one of the stomachs, from mice numbered
18-21, was lost. The inventors are unable to determine which of the
sets of data from mice numbered 18-21 is incomplete. Therefore, the
"ND" is not meant to be specifically assigned to mouse number
21.
[0203] In the assays of the mice from Example 4, and the mice
numbered 1-10 from Example 5, the inventors had difficulty
obtaining adequate samples for analysis.
[0204] The clearance of H. felis infection by oral administration
of the Helicobacter pylori ure B subunit was not expected and is
therefore novel. The results described herein also teach that the
ure B subunit can be used to treat Helicobacter infection.
[0205] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The urease peptides, mucosal adjuvants, carriers, and
antibodies, along with the methods, procedures, treatments, and
assays, are exemplary and are not intended as limitations on the
scope of the invention. Changes therein and other uses will occur
to those skilled in the art which are encompassed within the spirit
of the invention as defined by the scope of the claims.
* * * * *