Note: Descriptions are shown in the official language in which they were submitted.
WO 95133482 ~ ~ ~ ~ ~ ~ PCT/AU95100335
TREATMENT ANC1 PREVENTION OF HELICOBACTER INFECTION
FIELD OF THE INVENTION
This invention relates to protective Helicobacter antigens, especially H.
pylori antigens, and to the use of these antigens for the treatment and
prevention
of gastroduodenal disease associated with H. pylori infection in humans.
BACKGROUND OF THE INVENTION
Helicobacter pylori is a bacterium that infects the stomach lining (or gastric
mucosa) of perhaps half the world's population. Spiral organisms were first
microscopically observed in human gastric mucosa in 1906. However, H. pylori
was not successfully cultured until 1982. Infection with the organism is
usually
chronic, and results in continuing inflammation of the gastric mucosa. The
infection is often asymptomatic. However, in association with other cofactors,
a
proportion of infected people 40 on to develop sequelae including peptic
ulceration of the stomach or duodenum, gastric adenocarcinomas and gastric
lymphomas. Peptic ulcer treatment studies have shown that cure of H. pylori
infection is associated with a dramatic reduction in the relapse rate of this
usually
chronic disease. Long term infection with H. pylori leads to the development
of
chronic atrophic gastritis, which has long been recognised as a precursor
lesion
in the development of gastric cancer. Thus a number of studies have now linked
preceding H. pylori infection with an increased risk of developing gastric
cancer.
Therefore eradication of current infection and prevention of new infection
with this
organism has the potential to significantly reduce the incidence of diseases
that
result in considerable morbidity and mortality'~2.
Infection with H. pylori is difFcult to treat. Current experimental therapies
for treating the infection have problems with efficacy and significant levels
of
adverse effects. There are no prophylactic measures available. A solution to
WO 95133482 , ~ ~ PCTIAU95100335
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both the prevention and treatment of H. pylori infection would be the
development of an immunogenic preparation that, as an immunotherapeutic,
treated established infections, and as a vaccine, prevented the establishment
of
new or recurrent infections. Such a preparation would need to induce effective
immune responses to protective antigens, while avoiding inducing responses to
self antigens or other potentially harmful immune responses. This may be
achieved by identifying the specific protective component or components and
formulating immunotherapeutic or vaccine preparations including these
component(s).
The identification of such protective components of an organism, is often
accomplished through the use of an animal model of the infection. H. pylori
does not naturally infect laboratory animals. However, an animal model of
human
H. pylori infection has been developed using a closely related organism, H.
fells,
and specific pathogen free (SPF) mice3. These organisms are able to colonise
the gastric mucosa of SPF mice, ,where they establish a chronic infection with
many of the features of H. pylori infection in humans. H. fells infection in
the
mice induces a chronic gastritis and a raised immune response. As in the human
case, this response is not effective in curing the infection.
This model has been used to demonstrate that oral treatment of H. fells
infected mice with a preparation containing disrupted H. pylori cells and
cholera
toxin as a mucosal adjuvant, can cure a signficant portion of infected
mice°. This
effect is likely to be mediated through an immune response to a cross-reactive
antigen possessed by each of the closely related species.
In working by the inventors leading to the present invention, these cross-
reactive antigens were recognised by performing a Western blot using H. pylori
disrupted cells as the antigen, and probing the blot with serum from mice
immunised with H. fells and cholera toxin adjuvant. Sections of membrane
containing proteins recognised as cross-reactive were removed from the
WO 95133482 ~ ~ ~. 4 ~ PCTIAU95/00335
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membrane, the proteins bound to them were eluted, and their N-terminal amino
acid sequence determined by microsequencing.
The N-terminal amino acid sequence of one of the two proteins that
successfully yielded sequence data closely matched the previously published
sequence of the microbial enzyme, ureases. This enzyme has already been
shown to be a protective antigen when used in a vaccine to prevent infection.
The N-terminal amino acid sequence of the other protein closely matched
the previously published N-terminal sequence of the microbial enzyme,
catalases.
This enzyme has not previously been shown to be a protective antigen of H.
PYlori.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides an antigenic preparation for
use in the treatment or prevention of Helicobacter infection, which comprises
an
at least partially purified preparation of the catalase of Helicobacter
bacteria.
The term "at least partially purified" as used herein denotes a preparation
in which the catalase content is greater, preferably at least 30% and more
preferably at least 50% greater, than the catalase content of a whole cell
sonicate
of Helicobacter bacteria. Preferably, the preparation is one in which the
catalase
is "substantially pure", that is one in which the catalase content is at least
80%,
more preferably at least 90%, of the total Helicobacter antigens in the
preparation.
It is to be understood that the present invention extends not only to an
antigenic preparation comprising the catalase of Helicobacter bacteria, but
also
to antigenic preparations comprising immunogenic fragments of this catalase,
that
is catalase fragments which are capable of eliciting a specfic protective
immune
response in a mammalian host. Such immunogenic fragments may also be
X191440
W0 95/33482 PCTIAU95100335
-4-
recognised by Helicobacter-specific antibodies, particularly monoclonal
antibodies
which have a protective or therapeutic effect in relation to Helicobacter
infection
or polyclonal antibodies contained in immune sera from mammalian hosts which
have been vaccinated against Helicobacter infection.
In another aspect, the present invention provides a vaccine composition
for use in the treatment or prevention of Helicobacter infection in a
mammalian
host, which comprises an immunologically effective amount of an antigenic
preparation as broadly described above, optionally in association with an
adjuvant, together with one or more pharmaceutically acceptable carriers
and/or
diluents.
in yet another aspect, the present invention provides a method for the
treatment or prevention of Helicobacter infection in a mammalian host, which
comprises administration to said host of an immunologically effective amount
of
an antigenic preparation as broadly described above, optionally in association
with an adjuvant.
In a related aspect, this invention provides the use of a vaccine
composition comprising an immunologically effective amount of an antigenic
preparation as broadly described above, optionally in association with an
adjuvant, for the treatment or prevention of Helicobacterinfection in a
mammalian
host.
In yet another aspect, the inventionprovides use of an antigenic
the
preparation as broadly described optionallyassociation with
above, in an
adjuvant, in the manufacture of a compositionfor the treatment
vaccine or
prevention of Helicobacter infection in a mammalian host.
Preferably, but not essentially, the antigenic preparation of this invention
is orally administered to the host, and is administered in association with a
WO 95133482 ~ ~ PCTlAU95100335
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mucosal adjuvant. However, the invention also extends to parenteral
administration of this antigenic preparation.
The present invention also extends to delivery of the antigenic preparation
of this invention to the host using a vector expressing the catalase of
Helicobacter
bacteria, or an immunogenic fragment thereof. Accordingly, in a further aspect
this invention provides a preparation for use in the treatment or prevention
of
Helicobacter infection in a mammalian host, which comprises a vector
expressing
the catalase of Helicobacter bacteria or an immunogenic fragment thereof.
In this aspect, the invention extends to a method for the treatment or
prevention of Helicobacter infection in a mammalian host, which comprises
administration to said host of a vector expressing the catalase of
Helicobacter
bacteria or an immunogenic fragment thereof.
Further, the invention extends to the use of a vector expressing the
catalase of Helicobacter bacteria or an immunogenic fragment thereof, for the
treatment or prevention of Helicobacter infection in a mammalian host.
The present invention also extends to an antibody, which may be either a
monoclonal or polyclonal antibody, specific for an antigenic preparation as
broadly
described above.
In this aspect, the invention further provides a method for the treatment or
prevention of Helicobacter infection in a mammalian host, which comprises
passive immunisation of said host by administration of an immunologically
effective amount of an antibody, particularly a monoclonal antibody, speck for
an antigenic preparation as broadly described above.
By use of the term "immunologically effective amount" herein in the context
of treatment of Helicobacter infection, it is meant that the administration of
that
amount to an individual infected host, either in a single dose or as part of a
CA 02191440 2005-03-23
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series, that is effective for treatment of Helicobacter infection. By the use
of
the term "immunologically effective amount" herein in the context of
prevention of Helicobacter infection, it is meant that the administration of
that
amount to an individual host, either in a single dose or as part of a series,
that
is effective to delay, inhibit or prevent Helicobacter infection. The
effective
amount varies depending upon the health and physical condition of the
individual to be treated, the taxonomic group of individual to be treated, the
capacity of the individual's immune system to synthesise antibodies, the
degree of protection desired, the formulation of the vaccine, the assessment
~o of the medical situation, and other relevant factors. It is expected that
the
amount will fall in a relatively broad range that can be determined through
routine trials.
According to one aspect of the present invention, there is provided a
vaccine composition for use in the treatment or prevention of Helicobacter
~s infection in a mammalian host, which comprises an immunologically effective
amount of an antigenic preparation which comprises substantially pure, full
length Helicobacter catalase having an approximate molecular weight of
53kD, or an immunogenic fragment thereof, together with one or more
pharmaceutically acceptable carriers or diluents.
2o According to another aspect of the present invention, there is provided
a use of an antigenic preparation which comprises substantially pure, full
length Helicobacter catalase having an approximate molecular weight of
53kD, or an immunogenic fragment thereof, for the manufacture of a vaccine
composition for the treatment or prevention of Helicobacter infection in a
25 mammalian host.
According to a further aspect of the present invention, there is provided
a preparation for use in the treatment or prevention of Helicobacter infection
in
a mammalian host, which comprises a live vaccine vector expressing full
length Helicobacter catalase having an approximate molecular weight of 53
so kD, or an immunogenic fragment thereof.
According to another aspect of the present invention, there is provided
an antibody specific for full length Helicobacter catalase having an
approximate molecular weight of 53 kD, or an immunogenic fragment thereof.
CA 02191440 2004-08-24
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According to a further aspect of the present invention, there is provided
a vaccine composition for use in the treatment or prevention of Helicobacter
infection in a mammalian host, which comprises an antibody as described
above, together with one or more pharmaceutically acceptable carriers andlor
diluents.
Throughout this specification, unless the context requires otherwise,
the word "comprise", or variations such as "comprises" or "comprising", is to
be understood to imply the inclusion of a stated integer or group of integers
but not the exclusion of any other integer or group of integers.
DETAILED DESCRIPTION OF THE INVENTION
Preferably, the antigenic preparation of this invention comprises a
preparation of the catalase of H. pylori or H. fells, most preferably H.
pylori
catalase. Preferably also, this antigenic preparation is used in a vaccine
composition for oral administration which includes a mucosal adjuvant.
In a particularly preferred aspect of this invention, an oral vaccine
composition comprising an antigenic preparation of at least partially purified
H. pylori catalase in association with a mucosal adjuvant is used for the
2o treatment or prevention of H. pylori infection in a human host.
The mucosal adjuvant which is optionally, and preferably, administered
with the at least partially purified Helicobacter catalase preparation to the
infected host is preferably cholera toxin. Mucosal adjuvants other than
cholera toxin which may
WU 95133482 21914 4 0 pCT~AU95100335
-7_
be used in accordance with the present invention include non-toxic derivatives
of
cholera toxin, such as the B sub-unit (CTB), chemically modified cholera
toxin,
or related proteins produced by modification of the cholera toxin amino acid
sequence. These may be added to, or conjugated with, the Helicobactercatalase
preparation. The same techniques can be applied to other molecules with
mucosal adjuvant or delivery properties such as Escherichia coli heat labile
toxin.
Other compounds with mucosal adjuvant or delivery activity may be used such
as bile; polycations such as DEAE-dextran and polyornithine; detergents such
as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics
such
as streptomycin; vitamin A; and other compounds that alter the structural or
functional integrity of mucosal surfaces. Other mucosally active compounds
include derivatives of microbial structures such as MDP; acridine and
cimetidine.
The Helicobacter catalase preparation may be delivered in accordance
with this invention in ISCOMS (immune stimulating complexes), ISCOMS
containing CTB, liposomes or encapsulated in compounds such as acrylates or
poly(DL-lactide-co-glycoside) to form microspheres of a size suited to
adsorption
by M cells. Alternatively, micro or nanoparticles may be covalently attached
to
molecules such as vitamin B12 which have specific gut receptors. The
Helicobacter catalase preparation may also be incorporated into oily emulsions
and delivered orally. An extensive though not exhaustive list of adjuvants can
be
found in Cox and Coulter'.
Other adjuvants, as well as conventional pharmaceutically acceptable
carriers, excipients, buffers or diluents, may also be included in the
prophylactic
or therapeutic vaccine composition of this invention. The vaccine composition
may, for example, be formulated in enteric coated gelatine capsules including
sodium bicarbonate buffers together with the Helicobacter catalase preparation
and cholera toxin mucosal adjuvant.
The formulation of such therapeutic compositions is well known to persons
skilled in this field. Suitable pharmaceutically acceptable carriers and/or
diluents
WO 95133482 21914 4 0 PCTlAU95100335
_8_
include any and all conventional solvents, dispersion media, fillers, solid
carriers,
aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art, and it is
described,
by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack
Publishing Company, Pennsylvania, USA. Except insofar as any conventional
media or agent is incompatible with the active ingredient, use thereof in the
pharmaceutical compositions of the present invention is contemplated.
Supplementary active ingredients can also be incorporated into the
compositions.
As an alternative to the delivery of the Helicobacter catalase preparation
in the form of a therapeutic or prophylactic oral vaccine composition, the
catalase
or an immunogenic fragment thereof may be delivered to the host using a live
vaccine vector, in particular using live recombinant bacteria, viruses or
other live
agents, containing the genetic material necessary for the expression of the
catalase or immunogenic fragment as a foreign antigen. Particularly, bacteria
that
colonise the gastrointestinal tract, such as Salmonella, Yersinia, Vibrio,
Escherichia and BCG have been developed as vaccine vectors, and these and
other examples are discussed by Holmgren et al.° and McGhee et aL9.
The Helicobacter catalase preparation of the present invention may be
administered as the sole active immunogen in a vaccine composition or
expressed by a live vector. Alternatively, however, the vaccine composition
may
include or the live vector may express other active immunogens, including
other
Helicobacter antigens such as urease or the lipopolysaccharide (LPS) of
Helicobacterbacteria (see International PatentApplication No. PCT/AU95/00077),
as well as immunologically active antigens against other pathogenic species.
It is especially advantageous to formulate compositions in dosage unit form
for ease of administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary dosages for the
human
subjects to be treated; each unit containing a predetermined quantity of
active
CA 02191440 2004-08-24
ingredient calculated to produce the desired therapeutic effect in association
with
the required pharmaceutical carrier andlor diluent. The specifications for the
novel dosage unit forms of the invention are dictated by and directly
dependent
on (a) the unique characteristics of the active ingredient and the particular
therapeutic effect to be achieved, and (b) the limitations inherent in the art
of
compounding such an active ingredient for the particular treatment.
Data obtained from Western blots mentioned above, show that H. pylori
catalase is recognised by the serum of mice vaccinated with an H. fells
antigen
preparation (plus cholera toxin adjuvant). These mice can be shown to be
protected against H. fells infection. This data indicates the use of H, pylori
catalase as a protective antigen in human H. pylori infection, and purified or
recombinant catalase may be used as an antigenic component of a therapeutic
or prophylactic vaccine, either on its own, or in combination with other
antigens,
carriers, adjuvants, delivery vehicles or excipients.
Further details of the present invention are set out, by way of illustration
only, in the following Examples. It is to be understood, however, that this
detailed
description is included solely for the purposes of exemplifying the present
invention, and should not be understood in any way as a restriction on the
broad
description of the invention as set out above.
EXAMPLE 1
A. METHODS
Sonicated H. pylori cells were separated in a 12% discontinuous (i.e.
homogeneous) SDS-PAGE gel under denaturing conditions using a Mini-Protean
II apparatus (Bio-Rad). Proteins were transferred from the gel to ProBlott*
(Applied Biosciences PVDF-polyvinylidene difluoride) membrane using CAPS
buffer (3-(cyclohexylamino)-1-proanesuiphonic acid buffer) in a Mini
transblot~
system (Bio-Rad).
TM
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Strips were removed from the ends of the PVDF and reacted with immune
sera from mice vaccinated with H. fells plus cholera toxin and traced with an
HRP
labelled anti-mouse sera and developed using 4-chloro-1-naphthol as per
standard Western blot methods. The remainder of the PVDF was stained with
Coomassie blue (Bio-Rad) to visualise the protein bands. Six proteins
recognised
by the immune sera were selected and the corresponding Coomassie stained
bands on the PVDF were carefully excised for sequencing.
The six excised bands of PVDF were cut into small pieces {approx. 0.5 cm
long) and placed into the reaction cartridge of an Applied Biosystems Model
476A
Protein Sequencer Systerri All chemistry, HP~C separations, data quantitation
and protein sequencing reporting is automatically carried out in this system.
B. RESULTS
Four samples gave no signal in the Protein Sequencer System. Two
samples gave clear amino acid sequence data: sample 5, an approximately 53
kD protein (t10%), and sample 3, an approximately 66 kD protein (t10%). This
data is shown below.
(i) Sample 3:
DDN
MKKIVFKEYV
AP
Note: the first three cycles gave equivocal results.
The sequence data of sample 3 corresponds closely, but not
exactly, with the previously published N-terminal sequence for the enzyme
urease5. This enzyme has been shown to be a protective antigen in
studies using the H. fells/ mouse model.
~TM
CA 02191440 2004-08-24
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(ii) Sample 5:
MVNKDVKQTTAFGTP
The sequence data of sample 5 corresponds closely, with one
difference, to the previously published N-terminal sequence of the enzyme
catalase°. This enzyme has not previously been shown to be a protective
antigen however the fact that the enzyme is recognised by the immune
serum of mice vaccinated with an H. fells antigen preparation to protect
against H. fells infection, combined with the fact that mice vaccinated with
an H. pylori antigen preparation are protected against H. fells infection,
indicates the H. pylori catalase as a protective antigen in H. pylori
infection in humans.
EXAMPLE 2
1. PURIFICATION OF H. PYLORI catalase'°
Approximately 60 plates (CSA) of H. pylori (clinical strain 921023) were
grown in 10% C02 at 37°C for 48 hours. All following steps until
loading on the
column were undertaken on ice. The H. pylori cells were harvested in 0.1 M
sodium phosphate buffer pH 7.2 and the suspension spun down gently and
resuspended in no more than 5 mL of 0.1 M sodium phosphate buffer. The
suspension was then sonicated at 6 kHz 40°~6 duty cycle for 5 minutes.
Following
this, the sonicate was spun for 5 minutes at 10,000 g, the supernatant
collected
and passed through a 0.22 Nm filter into a sterile container.
TM
The fittrate was loaded onto a K26/100 gel filtration column of Sephacryl
S-300 HR and eluted using sodium phosphate buffer at a flow rate of 1.0 mL
miri'. The eluate was collected into fractions (100 dropslfraction) and those
containing catalase identified by testing for c~talase activity (1 drop of the
fraction
placed in HZOz diluted 1:10 in distilled water and examined for bubbling).
Fractions containing the strongest catalase adivit)r were pooled then diluted
1:10
CA 02191440 2004-08-24
-12-
in 0.01 M sodium phosphate (frltered). The fractions were then run through a
MEMSEP 1000 cm ion exchange capsule. 100 mL of the 0.01 M sodium
phosphate buffer was then run through the ion exchange capsule to remove any
excess proteins. 1 M NaCI in 0.1 M sodium phosphate buffer was run through
the ion exchange capsule to elute out the catalase. Catalase positive
fractions
were identified by their strong yellow colour and confirmed b testing for a
bubbling
reaction in Hz02.
The catalase positive fractions were stored at 4°C and protected from
light.
TM
Each fraction was tested for protein concentration using the Bio-Rad DC
protein
assay, and selected for immunising mice if it contained over 1.5 mg/mL of
protein.
Prior to immunising mice the purifred catalase was checked for contaminants
using 12% SDS-PAGE. Proteins were visualised by staining with Coomassie
Blue, which indicated that the catalase preparation was at least 95% pure.
Image
analysis indicated that the catalase's molecular weight was 52-53 kDa. The
purified catalase was also strongly recognised by a catatase monoclonal
antibody.
2. IMMUNISAT10N WITH H. PYLORI CATALASE.
Sufficient purified catalase for immunising 10 mice was obtained and
pooled. Mice were given 0.2 mg purified catalase +10 dug cholera toxin (GT) 4
times on days 0, 7, 14 and 21. Control groups were given cholera toxin alone
or
PBS buffer alone. The dose size was 150 NI for all groups. On the day of each
immunising dose, the catalase was checked for activity using 2 and for any
signs
of degradation using SDS-PAGE and Coomassie Blue staining. No signs of
declining activity or any degradation was observed throughout the immunisation
course. Three weeks after the last immunising dose all groups were challenged
twice with ~10° H. fells. Three weeks later mice were euthanased and
samples
(sera, saliva, bile and the stomach - half for histology and half the antrum
for the
direct urease test) were collected.
WO 95133482 21914 4 0 pCT1AU95100335
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Experiment Outline
TIME (days) CATALASE CT CT ALONE PBS ALONE
(10 Mice) (10 mice) (10 mice)
0 Cat + CT CT alone PBS alone
7 Cat + CT CT alone PBS alone
14 CAT + CT CT alone PBS alone
21 Cat + CT CT alone PBS alone
42 H. fells H. fells H. fells
Challenge Challenge Challenge
44 H. fells H. fells H. fells
Challenge Challenge Challenge
65 Collect 10 Collect 10 Collect 10
3. RESULTS
Urease
POSITIVE UREASE RESULT
(%)
Catalase + CT CT alone PBS alone
(10) (10) (10)
0/10 (0) 7/10 (70) 10/10 (100)
Western Blotting
Western blots of sera from mice showed strong recognition of H. pylori
catalase by the immunised mice, whereas mice from the other groups showed
weak or absent recognition.
Persons skilled in this art will appreciate that variations and modifications
may be made to the invention as broadly described herein, other than those
specifically described without departing from the spirit and scope of the
invention.
It is to be understood that this invention extends to include all such
variations and
modfications.
WO 95133482 ~ ~ 0 PCTIAU95100335
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REFERENCES
1. Helicobacter pylori Biology and Clinical Practice (1993). Edited by
C.Stewart Goodwin and Bryan W.Worsley. Published by CRC Press.
2. Halter, F., Hurlimann, S. and Inauen, W. (1992). Pathophysiology and
clinical relevance of Helicobacter pylori. The Yale Journal of Biology and
Medicine, 65:625-638.
3. Lee, A., Fox, J.G., Otto, G. and Murphy, J. (1990). A small animal model
of human Helicobacter pylori active chronic gastritis. Gastroenterology,
99:1316-1323.
4. Doidge, C.G., Gust, I., Lee, A., Buck, F., Hazel, S. and Mane, U. (1994).
Therapeutic immunisation against Helicobacter pylori - The first evidence.
Lancet 343(i):914-915.
5. Clayton, C.L., Pallen, M.J., Kleanthous, H., Wren, B.W. and Tabaqchali, S.
(1990). Nucleotide sequence of two genes from Helicobacter pylori
encoding for urease subunits. Nucleic Acid Res., 18(2):362
6. Westblom, T.U., Phadnis, S., Langenberg, W., Yoneda, K., Madan, E. and
Midkiff, B.R. (1992). Catalase negative mutants of Helicobacter pylori.
European Journal of Clinical Microbiology and Infectious Diseases,
11:522-526.
7. Cox, J. and Coulter, A. (1992). Advances in adjuvant technology and
application. In Animal Parasite Control Using Biotechnology. Edited by
W.K.Yong. Published by CRC Press.
8. Holmgren, J., Czerkinsky, C., Lycke, N. and Svennerholm, A-M. (1992).
Mucosal Immunity : Implications for Vaccine Development. Immunobiol.
184:157-179.
9. McGhee, J.R., Mestecky, J., Dertzbaugh, M.T., Eldridge, J.H., Hirasawa,
M. and Kiyono, H. (1992). The mucosal immune system : from
fundamental concepts to vaccine development. Vaccine 10(2):75-88.
10. Hazel, S.L., Evans Jr., D.J. and Graham, D.Y (1991). Helicobacter pylori
catalase. J. Gen. Microbiol. 137:57-61.
