Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-HELICOBACTER VACCINE COMPOSITION FOR USE BY
THE SUBDIAPHRAGMATIC SYSTEMIC RO TE AND COMBINED
MUCOSAL/PARENTERAL IMM RATION METHOD
The subject of the present invention is the specific use of a vaccine
preparation
intended to induce, in a mammal, a protective immune response against a
pathogenic
organism infecting the mucous membranes, in particular against Helicobacter
bacteria.
Helicobacter is a bacterial genus characterized by Gram-negative helical
bacteria. Several species colonize the gastrointestinal tract of mammals.
There may be
mentioned in particular H. pylori, H. heilmanii, H. fells, and H. mustelae.
Although
H. pylori is the species most commonly associated with human infections, in
some
Is rare cases, it has been possible to isolate in man H. heilmanii and H.
fells. A
bacterium of the Helicobacter type, Gastrospirillum horninis, has also been
described
in man.
Helicobacter infects more than 50% of the adult population in developed
countries and nearly 100% of that of developing countries, thereby making it
one of
2o the predominant infectious agents worldwide.
H. pylori is so far exclusively found at the surface of the mucous membrane of
the stomach in man and more particularly around the crater lesions of gastric
and
duodenal ulcers. This bacterium is currently recognized as the aetiological
agent of
antral gastritis and appears as one of the cofactors required for the
development of
2s ulcers. Moreover, it seems that the development of gastric carcinomas may
be
associated with the presence of H. pylori.
It therefore appears to be highly desirable to develop a vaccine intended to
prevent or treat Helicobacter infections.
To date, several Helicobacter proteins have already been proposed as vaccinal
3o antigens and the method of vaccination that is commonly recommended
consists of
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delivering the antigen at the level of the gastric mucous membrane, that is to
say at the
very site where the immune response is desired. To do this, oral
administration was
therefore selected.
Still with the same aim, induction of an immune response at the level of the
stomach, it has been more recently proposed to deliver the antigen at a
mucosal site
other than the gastric mucous membrane, such as the nasal or rectal mucous
membrane, for example (WO 96/31235). Lymphocytes stimulated by the antigen in
a
so-called inducer mucosal territory can migrate and circulate selectively so
as to go
and induce an immune response in other so-called effector mucosal territories.
to A variant of these methods involves carrying out a first immunization by
the
systemic route before administering the antigen by the nasal route.
For administration by the mucosal route, the antigen, most often a bacterial
lysate or a purified protein, is combined with an appropriate adjuvant such as
cholera
toxin (CT) or the heat-labile toxin (LT) from E. coli.
1s When administration by the mucosal route is used, the humoral response that
is
observed is predominantly of the IgA type. This indeed indicates that there
has been a
local immune response.
Some authors thought very early on that there was a good correlation between a
strong response of the IgA type and a protective effect (Czinn et al., Vaccine
( 1993)
20 11: 637). Others gave a more reserved opinion (Bogstedt et al., Clin. Exp.
Immunol.
(1996) 105: 202). Although there is up until now no real certainty on this
subject, the
induction of antibodies that are in particular of the IgA type appears
nonetheless
desirable for most authors.
In general, the appearance of IgAs is indicative of the coming into play of a
25 response on the part of the type 2 T helper lymphocytes (Th2 response).
Indeed, the stimulation of the T helper lymphocytes by a particular antigen
makes it possible to obtain various subpopulations of T helper cells,
characterized by
different cytokine synthesis profiles.
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The Thl cells in particular produce selectively interleukin-2 (IL-2) and
interferon-y (IFN-y), whereas the Th2 cells secrete preferably IL-4, IL-S, and
IL-10.
Because of their differentiated production of cytokines, these two types of T
helper
cells have distinct roles: the Thl cells promote cell-mediated immunity, i.a.,
an
s inflammatory-type response, whereas the Th2 cells stimulate humoral response
of the
IgA, IgE, and certain IgG subclass types. It is also known that the cytokines
produced
by mouse Thl cells can stimulate antibody response and in particular that IFN-
y
induces an IgG2a response. Thus, from the various studies in the prior art,
the
view emerges according to which the induction of a Th2 response characterized
by the
to appearance of IgA is essential, if not enough, to obtain a protective
effect.
Surprisingly, it has now been discovered that even if a Th2 response is not
damaging, it is also necessary to induce a high Thl response. Indeed,
experimental
results now demonstrate that a protective effect may be more easily correlated
with a
Th 1 response than with a Th2 response.
1s Contrary to what was initially sought (D'Elios et al., J. Immunol. (1997)
158:
962), the present application therefore reveals the importance of inducing an
inflammatory-type Thl response at the time of immunization, without which a
protective effect cannot be observed.
Consequently, the subject of the present invention is:
20 (i) The use of an immunogenic agent derived from a microorganism capable of
infecting the gastroduodenal mucous membrane of a mammal, e.g., derived from
Helicobacter, in the manufacture of a pharmaceutical composition intended for
the
induction of a Thl-type immune response against the said microorganism, e.g.,
Helicobacter, for treating or preventing an infection, e.g., a Helicobacter
infection in a
25 mammal; and
(ii) a method for preventing or treating an infection promoted by a
microorganism capable of infecting the gastroduodenal mucous membrane of a
mammal, e.g., a Helicobacter infection, according to which there is
administered to
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the mammal, in one or more applications, at least one immunogenic agent
derived
from the said microorganism, e.g., from Helicobacter, and by which a Thl-type
immune response is induced against, e.g., Helicobacter.
The induction of a useful Th 1 response can be demonstrated for the purposes
of
the present invention by estimating the relative level of the Thl response
relative to
the Th2 response by comparing, for example, the IgG2a and IgGI levels induced
in
mice against Helicobacter, which are respectively indicative of the coming
into play
of the Thl and Th2 responses. Indeed, the Th 1 response which is sought is
generally
accompanied by a Th2 response. However, it is considered that the Th2 response
to should not be significantly predominant relative to the Thl response. The
IgG2a and
IgGl levels induced in mice can be assessed conventionally using an ELISA
test,
provided that the tests used for each of the two subisotypes are of the same
sensitivity
and, in particular, that the anti-IgG2a and anti-IgG 1 antibodies are of the
same
affinity.
~5 The quantities of IgG2a and IgGI can be measured in particular using an
ELISA test that is identical or similar to that described below. The wells of
a
polycarbonate ELISA plate are coated with 100 ,ul of a bacterial extract from
Helicobacter, e.g., H. pylori, at about 10 ~g/ml in carbonate buffer. The
ELISA plate
is incubated for 2 hours at 37°C and then overnight at 4°C. The
plate is washed with
2o PBS buffer (phosphate buffered saline) containing 0.05% Tween 20 (PBS/Tween
buffer). The wells are saturated with 250 ,ul of PBS containing 1 % bovine
serum
albumin to prevent nonspecific binding of the antibodies. After incubating for
one
hour at 37°C, the plate is washed with PBS/Tween buffer. The antiserum
collected
from mice, a number of days after the latter have received the composition
intended to
25 induce a Thl-type immune response against Helicobacter, is serially diluted
in
PBS/Tween buffer. 100 ~.1 of the dilutions are added to the wells. The plate
is
incubated for 90 minutes at 37°C, washed, and evaluated according to
standard
procedures. For example, a goat antibody to mouse IgG2a or IgGl, coupled to an
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enzyme such as peroxidase, is used. The incubation in the presence of this
antibody is
continued for 90 minutes at 37°C. The plate is washed and then the
reaction is
developed with the appropriate substrate, for example, O-phenyldiamine
dihydrochloride when the enzyme used is peroxidase. The reaction is evaluated
by
s colorimetry by measuring the absorbance by spectrophotometry. The IgG2a or
IgG 1
titre of the antiserum corresponds to the reciprocal of the dilution giving an
absorbance of 1.5 at 490 nm.
The induction of a useful Thl response for the purposes of the present
invention is marked by a ratio of the ELISA IgG2a:IgG 1 titers in mice which
should
i o be greater than 1 / 100, 1 /50, or 1 /20, advantageously greater than 1 /
10, preferably
greater than 1/3, most preferably greater than'/Z, S, or 10. When this ratio
is around 1,
the Thl/Th2 response is said to be mixed or balanced. When the ratio is
greater than
or equal to 5, the Thl response is then said to be preponderant.
The production of a Thl (or Th2) response in mice is predictive of a Thl (or
15 Th2) response in man. Although it is easier to evaluate the type of
response in mice, it
can also be done in man by measuring the levels of cytokines specific for the
Thl
response on the one hand and, on the other hand, for the Th2 response, which
are
subsequently induced. The ThI and Th2 responses can be evaluated directly in
man
relative to each other on the basis of the levels of cytokines specific for
the two types
20 of response (see above), e.g., on the basis of the IFN-y/IL-4 ratio.
Alternatively, if the assay method described above is used, it is possible to
predict that the ELISA titre that reflects the quantity of IgG2a should be
equal to or
greater than 10,000, preferably equal to or greater than 100,000, in a
particularly
preferred manner, equal to or greater than 1,000,000; this then means that the
Thl
- 2s response is significant.
The mammal for which the pharmaceutical composition or the method is
intended is advantageously a primate, preferably a human.
It is possible to induce a Thl response against Helicobacter by adjusting a
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number of factors, such as, for example, the route of administration. It has
indeed
been demonstrated that by using the systemic or parenteral route, a level of
protection
can be obtained that is similar to or greater than that observed when the
mucosal route
is used.
Accordingly, the subject of the invention is in particular:
(i) the use of an immunogenic agent derived from Helicobacter, in the
manufacture of a pharmaceutical composition intended to be administered by the
systemic or parenteral route in the part of a mammal, especially a primate,
situated
under its diaphragm, for treating or preventing a Helicobacter infection; and
to (ii) a method for preventing or treating a Helicobacter infection in a
mammal,
according to which there is administered to the said mammal, in one or more
applications, by the systemic or parenteral route, at least one immunogenic
agent
derived from Helicobacter.
As regards the method, it is indicated that, advantageously, the
administration
~ s of the immunogenic agent by the systemic or parenteral route is repeated
once or
several times, preferably at least twice, for the desired immune response to
be induced.
A preferred method by which a protective effect is obtained is in particular a
method
according to which the immunogenic agent is administered exclusively by the
systemic or parenteral route (strict systemic route). "A method in which the
2o administration of the immunogenic agent is carried out by the strict
systemic route" is
defined as a method not using a route of administration other than the
systemic route.
For example, a method in which the immunogenic agent is administered by the
systemic route and by the mucosal route does not correspond to the definition
given
above. In other words, "a method in which the administration of the
immunogenic
2s agent is carried out by the strict systemic route" should be understood to
mean a
method in which the immunogenic agent is administered by the systemic route
excluding any other route, in particular the mucosal route.
Still as regards the method, the administration by the systemic or parenteral
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route is advantageously carried out in the subdiaphragmatic part of the
mammal.
The immunogenic agent derived from Helicobacter is advantageously selected
from a preparation of inactivated Helicobacter bacteria, a Helicobacter cell
lysate, a
peptide and a polypeptide from Helicobacter in purified form. The immunogenic
s agent can also be a polynucleotide molecule, especially a DNA molecule
including a
sequence encoding a peptide or a polypeptide from Helicobacter placed under
the
control of elements necessary for its expression in a mammalian cell, or
alternatively a
viral vaccinal vector including a sequence encoding a peptide or a polypeptide
from
Helicobacter placed under the control of elements necessary for its expression
in a
mammalian cell.
For the purposes of the present invention, a preparation of inactivated
bacteria
can be obtained according to conventional methods well known to persons
skilled in
the art. Likewise for a bacterial lysate. A dose of inactivated bacteria or
cell lysate,
appropriate for prophylactic or therapeutic purposes, can be determined by
persons
is skilled in the art and depends on a number of factors, such as the
individual for whom
the vaccine is intended, e.g., the individual's age, the antigen itself, the
route and
mode of administration,. the presence/absence or the type of adjuvant, as can
be
determined by persons skilled in the art. In general, it is indicated that an
appropriate
dose is from about SO ,ug to 1 mg at about 1 mg of lysate.
2o A peptide or a polypeptide derived from Helicobacter can be purified from
Helicobacter or obtained by genetic engineering techniques or alternatively by
chemical synthesis. The latter process is advantageous in the case of
peptides.
"Peptide" is any amino acid chain of less than about 50 amino acids. When the
size is
greater, the term "polypeptide," which is also interchangeable with the term
"protein,"
2s is used. A useful peptide or polypeptide for the purposes of the present
invention can
be identical or similar to that which exists under natural conditions. It is
similar in
that it is capable of inducing an immune response of the same type but it can
include
certain structural variations such as, for example, a mutation, the addition
of a residue
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of a lipid nature, or, alternatively, it can be in fusion polypeptide or
peptide form.
An appropriate dose of peptide or polypeptide for prophylactic or therapeutic
purposes can be determined by persons skilled in the art and depends on a
number of
factors, such as the individual for whom the vaccine is intended, e.g., the
age of the
s individual, the antigen itself, the route and mode of administration, the
presence/absence or the type of adjuvant, as can be determined by persons
skilled in
the art. In general, it is indicated that an appropriate dose is from about 10
,ug to about
1 mg, preferably at about 100 ,ug.
The DNA molecule can advantageously be a plasmid that is incapable both of
replicating and of substantially integrating into the genome of a mammal. The
above-
mentioned coding sequence is placed under the control of a promoter allowing
expression in a mammalian cell. This promoter can be ubiquitous or specific
for a
tissue. Among the ubiquitous promoters, there may be mentioned the
Cytomegalovirus early promoter (described in U.S. Patent No. 4,168,062) and
the
~s Rous sarcoma virus promoter (described in Norton & Coffin, Molec. Cell.
Biol.
(1985) 5_: 281). The desmin promoter (Li et al., Gene (1989) 7$: 244443; Li &
Paulin,
J. Biol. Chem. (1993) X68: 10403), which is a selective promoter, allows
expression in
muscle cells and also in skin cells. A promoter specific for muscle cells is,
for
example, the promoter of the myosin or dystrophin gene. Plasmid vectors that
can be
2o used for the purposes of the present invention are described, i.a., in WO
94/21797 and
Hartikka et al., Human Gene Therapy ( 1996) 7: 1205.
In a useful pharmaceutical composition for the purposes of the present
invention, the nucleotide molecule, e.g., the DNA molecule, can be formulated
or
otherwise. The choice of formulation is highly varied. The DNA can be simply
2s diluted in a physiologically acceptable solution with or without carrier.
When the
latter is present, it can be isotonic or weakly hypertonic and can have a low
ionic
strength. For example, these conditions can be fulfilled by a sucrose
solution, e.g., at
20%.
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Alternatively, the polynucleotide can be combined with agents that promote
entry into the cell. This can be (i) a chemical agent that modifies cell
permeability,
such as bupivacaine (see, for example, WO 94/16737), or (ii) an agent that is
combined with the polynucleotide and that acts as a vehicle facilitating the
transport of
s the polynucleotide. The latter may be in particular cationic polymers, e.g.,
polylysine
or a polyamine, e.g., derivatives of spermine such as spermidine (see WO
93/18759).
This can also be fusogenic peptides, e.g., GALA or Gramicidin S (see WO
93/19768)
or, alternatively, peptides derived from viral fusion proteins.
This can also be anionic or cationic lipids. The anionic or neutral lipids
have
been known for a long time to be capable of serving as transporting agents,
for
example, in the form of liposomes, for a large number of compounds, including
polynucleotides. A detailed description of these liposomes, of their
constituents, and
of the processes for their manufacture is, for example, provided by Liposomes:
A
Practical Approach, RPC New Ed., IRL press (1990}.
t 5 The cationic lipids are also known and are commonly used as transporting
agents for polynucleotides. There may be mentioned for example LipofectinTM
also
- known by the name DOTIvIA (N-[1-(2,3-dioleyloxy) propyl]-N,N,N-
trimethylammonium chloride), DOTAP (1,2-bis(oleyloxy)-3-(trimethyl-
ammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS
2a (dioctadecylamidoglycyl spermine), and cholesterol derivatives, such as DC-
chol (3-
beta-(N-(N',N'-dimethylaminoethane) carbamoyl) cholesterol). A description of
these
lipids is provided by EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO
91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. The cationic lipids are
preferably used with a neutral lipid such as DOPE
(dioleylphosphatidylethanolamine)
2s as is, for example, described in WO 90/11092.
Gold or tungsten microparticles can also be used as transporting agents, as
described in WO 91/359, WO 93/17706, and Tang et al., Nature (1992) 56: 152.
In
this particular case, the polynucleotide is precipitated on the microparticles
in the
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presence of calcium chloride and spermidine, and then the whole is
administered by a
high-speed jet into the dermis or into the epidermis using an apparatus with
no needle,
such as those described in U.S. Patent Nos. 4,945,050 and 5,015,580, and WO
94/24243.
The quantity of DNA that can be used to vaccinate an individual depends on a
number of factors such as, for example, the strength of the promoter used to
express
the antigen, the immunogenicity of the product expressed, the condition of the
mammal for whom the administration is intended (e.g., the weight, age, and
general
state of health), the mode of administration, and the type of formulation. It
is
to indicated in particular that the administration by the intramuscular route
requires a
larger quantity of DNA than the administration by the intradermal route using
an
apparatus with no needle. In general, an appropriate dose for prophylactic or
therapeutic use in an adult of the human species is from about 1 ~cg to about
5 mg,
preferably from about 10 ~g to about 1 mg, most preferably from about 25 ~g to
about
~5 500 ,ug.
Vaccinal vectors are among the immunogenic agents mentioned above.
Adenoviruses and poxviruses in particular are among the vectors of viral
origin. An
example of a vector derived from an adenovirus, as well as a method for
constructing
a vector capable of expressing a DNA molecule encoding a useful peptide or
2o polypeptide for the purposes of the present invention, are described in
U.S. Patent No.
4,920,209. Poxviruses that can be used likewise are, for example, the vaccinia
and
canarypox viruses. They are described respectively in U.S. Patents Nos.
4,722,848
and 5,364,773 (see also, e.g., Tartaglia et al., Virology ( 1992) 188: 217 and
Taylor et
al., Vaccine (I995) 13: 539). Poxviruses capable of expressing a useful
peptide or
25 polypeptide for the purposes of the present invention can be obtained by
homologous
recombination, as described in Kieny et al., Nature ( 1984) 312: 163, such
that the
DNA fragment encoding the peptide or polypeptide is placed under conditions
appropriate for its expression in mammalian cells. A bacterial vector such as
the bile
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Calmette-Guerin bacillus can also be used. .
In general, the dose of a viral vector intended for prophylactic or
therapeutic
purposes can be from about 1 X 104 to about 1 X 10", advantageously from about
1 X
10' to about 1 X 10'°, and preferably from about 1 X 10' to about 1 X
109 plaque
s forming units per kilogram.
The immunogenic agent derived from Helicobacter can be any polypeptide
from Helicobacter, e.g., H. pylori. This can be in particular a polypeptide
present in
the cytoplasm, a polypeptide of the inner or outer membrane, or a polypeptide
secreted
in the external medium. Numerous polypeptides from Helicobacter have already
been
described in the literature, either with reference to their amino acid
sequence deduced
from the sequence of the cloned or identified corresponding gene, or with
reference to
a purification process that makes it possible to obtain them in a form
isolated from the
rest of their natural environment. As a guide, the following documents are
mentioned
in particular: WO 94/26901 and WO 96/34624 (HspA), WO 94/09023 (CagA), WO
is 96/38475 (HpaA), WO 93/181150 (cytotoxine), WO 95/27506 and Hazell et al.,
J.
Gen. Microbiol. ( 1991 ) ~7: 57 (catalase), FR 2 724 936 (membrane receptor
for
human lactofernn), WO 96/41880 (AIpA), EP 752 473 (FibA) and O'Toole et al.,
J.
Bact. (1991) ~3: 505 (TsaA). Other polypeptides are also described in WO
96/40893, WO 96/33274, WO 96/25430, and WO 96/33220. A useful polypeptide for
2o the purposes of the present invention can be identical or similar to one of
those cited
as a reference insofar as it is capable of promoting an immune response
against
Helicobacter. In order to meet this last condition, the immunogenic agent can
also be
a peptide derived from a polypeptide cited as a reference.
Advantageously, a polypeptide selected from the UreA and Urea subunits of
2s Helicobacter urease is used (see WO 90/4030). Preferably, both are used,
combined
in urease apoenzyme form or alternatively in multimeric form (see WO
96/33732).
Likewise, a useful vaccinal vector or DNA molecule for the purposes of the
present invention includes a sequence that can encode any polypeptide or
peptide
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described above.
A DNA molecule, or preferably a viral vaccinal vector, can also include a
sequence encoding a cytokine, for example, a lymphokine, such as interleukin-2
or
interleukin-12, under the control of elements appropriate for expression in a
mammalian cell. An alternative to this option also consists in adding to a
useful
pharmaceutical composition for the purposes of the present invention
comprising a
DNA molecule or a vector, another molecule, or viral vector encoding a
cytokine.
A useful pharmaceutical composition for the purposes of the present invention
can contain a single immunogenic agent or several. For example, an
advantageous
composition can comprise UreA and Urea, e.g., in apoenzyme form, as well as
one or
more other polypeptides selected in particular from those mentioned above.
Likewise,
when a DNA molecule or a vaccinal vector is involved, the composition can
contain
several of them, each encoding a particular polypeptide or a single DNA
molecule or
vaccinal vector encoding several peptides or polypeptides.
is A useful pharmaceutical composition for the purposes of the present
invention
can, in addition, contain compounds other than the immunogenic agent itself,
the
nature of these compounds depending, to a certain extent, on the nature of the
lmmunogenic agent, inactivated bacteria, cell lysate, peptide, or polypeptide,
DNA
molecule, or vaccinal vector. Thus, as has already been seen above, when a DNA
2o molecule is involved, the pharmaceutical composition can include various
formulation
agents. A composition can also include an appropriate adjuvant for
administration by
the systemic or parenteral route, e.g., an aluminum compound, such as aluminum
hydroxide, aluminum phosphate, or aluminum hydroxyphosphate. In general, it is
indicated that inactivated bacteria may not require the addition of an
adjuvant. The
2s same is true as regards the DNA molecules. On the other hand, the presence
of an
adjuvant is preferable when the immunogenic agent is a bacterial lysate or a
purified
peptide or polypeptide. Finally, when the immunogenic agent is a vaccinal
vector, the
use thereof is preferably avoided so that the immune response towards the
vector itself
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remains minimal.
In addition to the aluminum compounds, a large number of appropriate
adjuvants for administration by the systemic or parenteral route exist in the
state of the
art among which persons skilled in the art are capable of selecting the one
that best
s corresponds to their needs; in particular a compound capable of promoting
the
induction of a Thl-type immune response or a balanced response of the Thl +
Th2
type. As a guide, there can be mentioned in particular liposomes; ISCOMS;
microspheres; protein chochleates; vesicles consisting of nonionic
surfactants; cationic
amphiphilic dispersions in water; oil/water emulsions; muramidyldipeptide
(MDP)
~o and its derivatives such as glucosyl muramidyldipeptide (GMDP), threonyl-
MDP,
murametide and murapalmitin; and QuilA and its subfractions; as well as
various other
compounds such as monophosphoryl-lipid A (MPLA) major lipopolysaccharide from
the wall of a bacterium, for example of E. coli, Salmonella minnesota,
Salmonella
typhimurium, or Shigella flexneri; algan-glucan; gamma-inulin; calcitriol; and
1s loxoribine.
Useful liposomes for the purposes of the present invention can be selected in
particular from pH-sensitive liposomes, such as those formed by mixing
cholesterol
hemisuccinate (CHEMS) and dioleyl phosphatidyl ethanolamine (DOPE); liposomes
containing cationic lipids recognized for their fusiogenic properties, such as
3-beta-
20 (N-(N',N'-dimethylamino-ethane)carbamoyl)cholesterol (DC-chol) and its
equivalents, which are described in U.S. Patent No. 5,283,185 and WO 96/14831,
dimethyldioctadecylammonium bromide (DDAB) and the BAY compounds described
in EP 91645 and EP 206 037, for example Bay RI005 (N-(2-deoxy-2-L-leucylamino-
beta-D-glucopyranosyl)-N-octa-decyldodecanoylamide acetate; and liposomes
2s containing MTP-PE, a lipophilic derivative of MDP (muramidyldipeptide).
These
liposomes are useful for adding as adjuvant to all the immunogenic agents
cited.
Useful ISCOMs for the purposes of the present invention can be selected in
particular from those compounds of QuilA or of QS-21 combined with cholesterol
and
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optionally also with a phospholipid such as phosphatidylcholine. These are
particularly advantageous for the formulation of the lipid-containing
antigens.
Useful microspheres for the purposes of the present invention can be formed in
particular from compounds such as polylactide-co-glycolide (PLAGA), alginate,
chitosan, polyphosphazene, and numerous other polymers.
Useful protein chochleates for the purposes of the present invention can be
selected in particular from those formed from cholesterol and optionally an
additional
phospholipid, such as phosphatidylcholine. These are especially advantageous
for the
formulation of the lipid-containing antigens.
to Useful vesicles consisting of nonionic surfactants for the purposes of the
present invention can be in particular formed by a mixture of 1-mono-palmitoyl
glycerol, cholesterol, and dicetylphosphate. They are an alternative to the
conventional liposomes and can be used for the fol~nulation of all the
immunogenic
agents cited.
~s Useful oil/water emulsions for the purposes of the present invention can be
selected in particular from MF59 (Biocine-Chiron), SAF1 (Syntex), and the
montanides ISA51 and ISA720 (Seppic).
A useful adjuvant for the purposes of the present invention can also be a
fraction derived from the bark of the South American tree Quillaja Saponaria
Molina;
2o for example, QS-21, a fraction purified by HPLC chromatography as is
described in
U.S. Patent No. 5,057,540. Since some toxicity may be associated with QS-21,
it may
be advantageous to use the latter in liposomes especially based on sterol, as
is
described in WO 96/33739.
Finally, an adjuvant effect can also be obtained by adding lipid to the useful
2s peptide or polypeptide for the purposes of the present invention. The
combination, by
covalent bonding, of such a peptide or polypeptide with a lipid or a lipid-
containing
compound capable of promoting the induction of a Th 1-type immune response, so
as
to form a lipid-containing lipopeptide or polypeptide conjugate, can be
achieved in
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various ways known to persons skilled in the art. For example, it is possible
to use
one of the compounds described in EP 431 327 such as N-palmitoyl-S-2,3-
(bispalmitoyloxy) propylcysteinylseryl serine (Pam3CSS), which is coupled by
known
processes to the N-terminal end of the peptide or polypeptide.
The therapeutic or prophylactic efficacy of a method or of a use according to
the invention can be evaluated according to standard methods, e.g., by
measuring the
induction of an immune response or the induction of a therapeutic or
protective
immunity using, e.g., the mouse/H. fells model and the procedures described in
Lee et
al., Eur. J. Gastroenterology & Hepatology (1995) 7: 303 or Lee et al., J.
Infect. Dis.
io (1995) ~: 161. Persons skilled in the art will realize that H. fells can be
replaced in
the mouse model by another Helicobacter species. For example, the efficacy of
an
immunogenic agent derived from H. pylori is preferably evaluated in a mouse
model
using an H. pylori strain adapted to mice. The efficacy can be determined by
comparing the level of infection in the gastric tissue (by measuring the
urease activity,
~s the bacterial load, or the condition of the gastritis) with that in a
control group. A
therapeutic effect or a protective effect exists when the infection is reduced
compared
with the control group.
A useful pharmaceutical composition for the purposes of the present invention
can be manufactured in a conventional manner. In particular, it can be
formulated
2o with a pharmaceutically acceptable carrier or diluent, e.g., water or a
saline solution.
In general, the diluent or carrier can be selected according to the mode and
route of
administration and according to standard pharmaceutical practices. Appropriate
carriers or diluents, as well as what is essential for the preparation of a
pharmaceutical
composition, are described in Remington 's Pharmaceutical Sciences, a standard
25 reference book in this field.
The methods according to the invention, as well as the compositions useful for
these purposes, can be used to treat or prevent, i.a., Helicobacter infections
and
consequently the gastroduodenal diseases associated with these infections,
including
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acute, chronic, or atrophic gastritis, and peptic ulcers, e.g., gastric or
duodenal ulcers.
The systemic route that is used can be the parenteral route, which can itself
be
chosen from the intravenous, intramuscular, intradermal, intraepidermal, and
subcutaneous routes; the latter four being however preferred to the
intravenous route.
The intramuscular and subcutaneous routes are particularly recommended. In all
cases, the use that will be made of the pharmaceutical composition can call
into play a
site of administration situated under the diaphragm of an individual. The
dorsolumbar
region constitutes, for example, an appropriate site of administration.
To obtain a protective or therapeutic effect, the operation that consists of
administering, for example, by the subdiaphragmatic systemic route, a useful
pharmaceutical composition for the purposes of the present invention can be
repeated
once or several times, leaving a certain time interval between each
administration;
which interval is of the order of a week or a month. Its precise determination
is within
the capability of persons skilled in the art and can vary according to various
factors;
~s such as the nature of the immunogenic agent, the age of the individual, and
the like.
In this particular case, the administration is said to be of the strict
systemic type. By
way of a nonlimiting illustration, there may be mentioned a vaccination scheme
that
consists of administering the urease apoenzyme three times by the subcutaneous
route,
in the dorsolumbar region, with an interval of two to four weeks between each
2o administration.
According to an alternative mode, it is possible to envisage operating in a
strict
systemic mode of administration, but using immunogenic agents that vary during
the
administrations constituting the steps of the vaccination procedure. By way of
a
nonlimiting illustration, there may be mentioned a vaccination scheme by the
strict
2s systemic route, in three steps: a first administration (priming) consists
of
administering a pox vector encoding, e.g., UreA and Urea, followed by two
consecutive administrations (boosters) of the urease apoenzyme.
In general, the subject of the invention is therefore also a pharmaceutical
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composition intended to treat or prevent a Helicobacter infection which
includes, for
consecutive administration, several products, each of the products being
formulated so
as to be administered by the subdiaphragmatic systemic route and containing an
immunogenic agent derived from Helicobacter selected independently from a
s preparation of inactivated Helicobacter bacteria, a Helicobacter cell
lysate, a peptide,
a polypeptide from Helicobacter in purified form, a DNA molecule comprising a
sequence encoding a peptide or a polypeptide from Helicobacter placed under
the
control of the elements necessary for its expression, and a vaccinal vector
including a
sequence encoding a peptide or a polypeptide from Helicobacter placed under
the
control of the elements necessary for its expression, preferably provided that
when a
first product contains a peptide or a polypeptide and a second product
contains a DNA
molecule or a vaccinal vector, the coding sequence of the DNA molecule or of
the
vaccinal vector encodes the peptide or polypeptide contained in the first
product.
Finally, an alternative vaccination procedure comprising several
I5 administrations staggered over time, e.g., within time intervals of the
order of a week
or a month, to be determined by persons skilled in the art, can include a
first
administration by the subdiaphragmatic systemic route and a second
administration by
the mucosal route other than the intranasal route, e.g., by the ocular, oral,
e.g., buccal
or gastric, pulmonary, intestinal, rectal, vaginal, or urinary route. By way
of a
2o nonlimiting illustration, there can be mentioned a vaccination procedure
that consists
of administering a DNA molecule or a vaccinal vector by the subdiaphragmatic
systemic route and then in administering a polypeptide by the gastric route,
the DNA
molecule or the vaccinal vector preferably encoding the polypeptide
administered by
the gastric route.
2s In general, the subject of the invention is therefore also a pharmaceutical
composition intended to treat or prevent a Helicobacter infection that
contains, for
consecutive administration, several products; one of the products being
formulated so
as to be administered by the subdiaphragmatic systemic route and another
product
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being formulated so as to be administered by a mucosal route other than the
intranasal
route; each of the products containing an immunogenic agent derived from
Helicobacter selected independently from a preparation of inactivated
Helicobacter
bacteria, a Helicobacter cell lysate, a peptide, a polypeptide from
Helicobacter in
s purified form, a DNA molecule including a sequence encoding a peptide, or a
polypeptide from Helicobacter placed under the control of the elements
necessary for
its expression and a vaccinal vector including a sequence encoding a peptide
or a
polypeptide from Helicobacter placed under the control of the elements
necessary for
its expression, preferably provided that when a first product contains a
peptide or a
to polypeptide and a second product contains a DNA molecule or a vaccinal
vector, the
coding sequence of the DNA molecule or of the vaccinal vector encodes the
peptide or
polypeptide contained in the first product.
A vaccinal vector contained in a product intended to be administered by the
mucosal route can be chosen from those described above. In addition, it can be
Is selected from bacterial vectors such as Shigella, Salmonella, Vibrio
cholerae,
Lactobacillus, and Streptococcus.
Nontoxic mutant strains of Vibrio cholerae that can be useful as live vaccine
vectors are described, for example, in Mekalanos et al., Nature (1983) 306:
551 and
U.S. Patent No. 4,882,278 (strain in which a substantial part of the region
encoding
2o each of the two alleles ctxA has been deleted so that no functional toxin
can be
produced); WO 92/11354 (strain in which the irgA locus is inactivated by
mutation;
this mutation may be combined in the same strain with ctxA mutations); and WO
94/1533 (mutant obtained by deletion lacking functional ctxA and attRSl
sequences).
These strains can be modified genetically to express heterologous antigens as
2s described in WO 94/19482.
Attenuated strains of Salmonella typhimurium, genetically modified or
otherwise for the recombinant expression of heterologous antigens, as well as
their use
as vaccines, are described in Nakayama et al., BioTechnology ( 1988) 6: 693
and WO
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92/11361.
Other bacteria useful as vaccinal vectors are described in High et al., EMBO
{1992) ,~.~.: 1991 and Sizemore et al., Science (1995) ~: 299 (Shigella
flexneri);
Medaglini et al., Proc. Natl. Acad. Sci. USA ( 1995) 92: 6868 (Streptococcus
s gordonii); and Flynn J.L., CeII. Mol. Biol. (1994) 40 (suppl. I): 31, WO
88/6626, WO
90/0594, WO 91/13157, WO 92/1796, and WO 92/21376 (Calmette-Guerin bacillus).
In bacterial vectors, the DNA sequence encoding a peptide or polypeptide from
Helicobacter can be inserted into the bacterial genome or alternatively remain
in the
free state, carried by a plasmid. Obviously, this sequence is placed under the
control
of the elements necessary for its expression in the bacterial vector.
These bacterial vectors for administration by the mucosal route can be used in
combination with an appropriate adjuvant. Such adjuvants may be chosen from
bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin
(LT), the
Clostridium difficile toxin, and the Pertussis toxin (PT), or combinations,
subunits,
rs toxoids, or mutants that are derived therefrom. For example, it is possible
to use a
purified preparation of the native cholera toxin B subunit (CTB). Fragments,
homologues, derivatives, and fusions of these toxins are equally suitable
provided they
retain the adjuvant activity. Preferably, a mutant is used whose toxicity is
reduced.
Such mutants are described in, e.g., WO 95/17211 (mutant CT Arg-7-Lys),
2o WO 96/6627 (mutant LT Arg-192-Gly), and WO 95/34323 (mutant PT Arg-9-Lys
and
GIu-129-Gly). Other LT mutants that can also be used carry at least one of the
following mutations: Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp.
Other compounds, such as MPLA, PLGA, DC-chol, and QS-21 can also be
used as adjuvants for the mucosal route.
25 The invention also includes immunization methods for treating or preventing
Helicobacter (e.g., H. pylori) infection that involve mucosal (e.g., oral,
intranasal,
intragastric, pulmonary, intestinal, rectal, ocular, vaginal, or urinary
tract)
administration, followed by parenteral (e.g., intramuscular, subcutaneous,
intradermal,
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intramuscular, intravenous, or intraperitoneal). In one example of these
methods,
mucosal administration is carried out to prime an immune response to an
antigen, and
parenteral administration is carried out to boost the immune response to the
antigen.
Other examples of these methods involve alternating parenteral and mucosal
s administrations, for example, the following pattern can be used:
intramuscular
administration, combined intragastric + intranasal administration,
intramuscular
administration, and combined intragastric + intranasal administration.
Antigens,
formulations, adjuvants, administration regimens, specific mucosal and
parenteral
routes, and dosages to be used can readily be determined by one skilled in the
art.
Specific examples of these parameters that can be adapted for use in these
methods are
provided above.
In the description above, reference was made essentially to Helicobacter
infections and to the means for combating them by way of prevention and
prophylaxis.
However, it should be understood that the principles and methods stated above
can be
~5 applied mutatis mutandis to any other infection induced by any
microorganism whose
seat is the stomach, the duodenum or the intestine.
It is specified, in addition, that all-the documents published and cited in
the
present application are incorporated by reference.
The invention is illustrated below with reference to the following figures.
2o Figure 1 refers to Example 1 and presents a study of the local response in
the
salivary glands (Figure 1 A) and in the stomach (Figure I B) evaluated by
ELISPOT by
measuring the quantity of anti-urease IgA induced, expressed as spots/10~
cells
(Figure 1 A) or as number of responding mice, exhibiting more than 2 IgA
spots/mouse, (Figure 1B), after (a) administration of urease at DO by the
subcutaneous
2s route (SC) in the left posterior sublumbar part [(a) and (c)] or in the
neck [(b) and (d)],
followed by a booster by the nasal route (N) and intragastric route (IG), at
D28 [(a)
and (b)] or at D28 and D56 [(c) and (d)].
Figure 2 refers to Example 1 and presents the levels of urease activity after
a
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challenge, measured 4 hours after sacrificing mice which have received 3
times, on
D0, D28 and D56, an inactivated bacterial preparation by the intragastric
route ((a)
and (c)] or subcutaneous route in the left posterior sublumbar part (b). In
experiment
(c), 10 ~cg of cholera toxin were added to the bacterial preparation.
Experiments (d)
s and (e) correspond respectively to the positive and negative controls.
Figure 3 refers to Example 1 and presents the levels of urease activity after
a
challenge, measured 4 hours after sacrificing mice which have received 3
times, on
D0, D28, and D56: (a) a urease preparation encapsulated at about 80% in DC-
chol
liposomes, in the dorsolumbar muscles; or (b) a urease preparation with
cholera toxin
to adjuvant, by the intragastric route. Experiments (c) and (d) correspond
respectively to
the positive and negative controls.
Figure 4 refers to Example 1 and presents the levels of urease activity after
a
challenge measured 4 hours after sacrificing mice which have received 3 times,
on D0,
D28, and D56: (a) a urease preparation with cholera toxin adjuvant, by the
intragastric
is route or (b) a urease preparation with QS-21 adjuvant, by the subcutaneous
route in
the left posterior sublumbar part. Experiments (c) and (d) correspond
respectively to
- the positive and negative-controls.
Figure S presents the quantities of serum immunoglobulins induced in monkeys
subjected to the immunization procedures described in Example 2, and expressed
as
2o ELISA titre. A control group comprising 4 monkeys and three test groups are
formed,
each of the test groups comprising 8 monkeys; each test group is divided into
two
subgroups of 4 monkeys, one receiving only the inactivated H. pylori
preparation ( 1,
2, and 3) and the other receiving the inactivated H. pylori preparation and
recombinant
urease ( 1 u, 2u, and 3u). Group 1 and 1 a corresponds to the administration
procedure
2s [nasal + intragastric, 4 times]; group 2 and 2u corresponds to the
administration
procedure [intramuscular, 4 times]; group 1 and lu corresponds to the
administration
procedure [nasal + intragastric, intramuscular, nasal + intragastric,
intramuscular].
The ELISA titre is measured three times: a first time at DO (white band), a
second
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time at D42 (shaded band), a third time at D78 (black band).
Figures 6A and 6B show the urease activity (Figure 6A) measured after 4 hours
(ODsso nm) using the Jatrox test (Procter & Gamble) and the bacterial load in
mice
infected with H. pylori and then submitted to various treatments A - H [A: LT
+
s urease, orally; B: QS-21 + urease, parenterally in the neck; C: QS-21 +
urease,
parenterally in the lumbar region; D: QS-21 alone, sub-cutaneously in the
lumbar
region; E: Bay 81005 + urease, parenterally in the neck; F: Bay 81005 +
urease,
parenterally in the lumbar region; G: Bay 81005 alone, sub-cutaneously in the
lumbar
region (control); H: saline, sub-cutaneously in the lumbar region (positive
control)]. I
to represents the negative control.
Figure 7 presents the results of immunization of mice with a mucosal
prime/parenteral boost strategy with urease induced the most efficacious
protection
against challenge with H. pylori. Mice were immunized either orally with 25 ~g
urease + 5 ~g LT or parenterally with 10 ,ug urease with or without 100 ~cg
alum
is adjuvant. The mice were primed with orally administered urease + LT, 2
booster
doses were administered three weeks apart by either the parenteral or oral
route, as
shown in the figure. Mice were challenged with H. pylori two weeks after the
last
immunization and euthanized 2 weeks after challenge. At necropsy, one-third of
the
stomach, dissected longitudinally, was homogenized and cultured for H. pylori.
2o Figure 8 shows the effect of urease immunization on experimental challenge
of
rhesus monkeys with H. pylori. Monkeys were immunized with urease by
parenteral
routes ( 100 ,ug urease + 1 mg alum or 800 ~g Bay) or by a mucosal prime
(orally
administered 4 mg urease +100 ~cg LT)/parenteral boost (urease + alum)
strategy with
3 doses administered every 3 weeks followed by a fourth dose administered 20
weeks
2s after the first priming dose. Monkeys were challenged one week after the
last booster
dose. The monkeys were euthanized 10 weeks after challenge, 10 punch biopsies
per
animal were harvested from the stomach and cultured to determine H. pylori
colonization. Each symbol above represents the mean CFU of 10 sites cultured
per
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monkey. The line represents the median CFU for the treatment group.
Figure 9 presents gastritis scores in immunized and unimmunized rhesus
monkeys following challenge with H. pylori. Monkeys were orally immunized with
a
priming dose of 4 mg urease + 100 ~.cg LT followed 3 weeks later with 2
parenteral
administered 20 weeks after the first priming dose. Monkeys were challenged
one
week after the last booster dose. The monkeys were euthanized 10 weeks after
challenge, 2 cm2 sections were taken from the corpus, antrum and corporal-
antral
junction, fixed in 10% buffered formalin, embedded in paraffin and sections
stained
with H & E. Gastritis, typified by infiltration of lymphocytes, plasma cells,
and
io polymorphonuclear cells, was scored by microscopic examination of stained
sections.
Each symbol above represents the mean gastritis score of the 3 regions from
each
monkey.
Figure 10 presents epithelial changes in immunized and unimmunized rhesus
monkeys following challenge with H. pylori. Monkeys were orally immunized with
a
priming dose of 4 mg urease + 100 ,ug LT followed 3 weeks later with 2
parenteral
doses of 100 ~g urease + 1 mg alum every 3 weeks and 1 parenteral dose of
urease +
alum administered 20 weeks after the first priming dose. Monkeys were
challenged
one week after the last booster dose. The monkeys were euthanized 10 weeks
after
challenge, 2 cm2 sections were taken from the corpus, antrum and corporal-
antral
2o junction, fixed in 10% buffered formalin, embedded in paraffin and sections
stained
with H & E. Epithelial changes, defined as metaplasia, atrophy and/or
hyperplasia,
was scored by microscopic examination of stained sections. Each symbol above
represents the mean gastritis score of the 3 regions from each monkey.
2s Example 1: Immunization studies in mice
lA - Materials and methods
Mice
6/8-week old female Swiss mice were provided by Janvier (France). During
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the whole experiment, sterilized materials were used; the cages were protected
by
"isocaps;" the mice were fed with filtered water and irradiated food.
Administration procedure
During each experiment, the mice received 3 doses of the same product; each
dose at 28-day intervals (days 0, 28, and 56). The administration of the
product was
carried out by the nasal route (up to SO gel on waking mice), by the oral
route (300 ,ul
in 0.2 M NaHCO~ by gastric gavage), or by the subcutaneous route (300 ,ul
under the
skin of the neck or under the skin on the left side of the lumbar region). In
some
~o cases, an intramuscular inoculation was carried out (50 ,ul) in the
dorsolumbar muscles
of anaesthetized mice. Ten ,ug of urease were administered by the nasal,
subcutaneous
or intramuscular route, and 40 ~g by the oral route. As regards the
inactivated
bacterial preparation, 400 ~sg of cells were administered by the subcutaneous
route or
by the oral route.
is
Antigens and adjuvants
The H. pylori urease apoenzyme was expressed in E. coli and purified as has
been described in Example S of WO 96/31235. In the remainder of the text, the
simple term of urease is used to designate this apoenzyme.
2o A preparation of inactivated H. pylori bacteria (WC) was prepared as
follows: a
bottle of frozen bacteria ATCC 43579 is diluted in a two-phase medium in a 75
cm2
flask (Costar). This medium is composed of a solid constituent ( 10 ml
Columbia agar
(BioMerieux) + 6% sheep blood (BioMerieux)) and a liquid constituent (3 ml of
TSB,
BioMerieux). The flask is placed in a generbag containing a microaer
(BioMerieux)
2s and incubated with gentle shaking for 48 hours at 37°C. Culture is
then analyzed
(mobility, urease, catalase, and production of oxidase) and centrifuged
(optionally
after having grouped together several flasks) at 3,000 rpm for 20 minutes at
4°C. The
pellet is resuspended in PBS (BioMerieux) containing 1 % formalin (37%
formalin,
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-2s-
Sigma). The volume is adjusted so as to obtain a final concentration of 2
mg/ml (1 ml
having an OD of 1 at 600 nm before centrifugation corresponds to 377 ,ug of
protein).
The product is mixed gently at 4°C for 4 hours, washed 3 times in PBS,
and the final
solution is concentrated to 100 ~cg of protein/50 ~1. The aliquots are kept at
-70°C.
DC-chol liposomes containing urease are prepared as follows: first of all, to
obtain a dry lipid film containing 100 mg of DC-chol (R-Gene Therapeutics) and
100 mg of DOPC (dioleylphosphatidylcholine) (Avanti Polar Lipids), these
products
are mixed in powdered form in about 5 ml of chloroform. The solution is
allowed to
evaporate under vacuum using a rotary evaporator. The film thus obtained on
the
io walls of the container is dried under high vacuum for at least 4 hours. In
parallel,
20 mg of a urease lyophilisate and 100 mg of sucrose are diluted in 13.33 ml
of
20 mM Hepes buffer pH 7.2. Ten ml of this preparation (which contains 1.5 mg
of
urease and 0.75% sucrose) is filtered on the 0.220 ~cm Millex filter and then
used to
rehydrate the lipid film. The suspension is stirred for 4 hours and then
either extruded
~ s ( 10 passes on a 0.2 ~m polycarbonate membrane) or microfluidized ( 10
passes at a
pressure of 500 kPa in a Microfluidics Co Y10 microfluidizer). In the liposome
suspension thus obtained, the level of encapsulated urease is from 10 to 60%.
This
suspension is lyophilized after having adjusted the sucrose concentration to
5%
(425 mg of sucrose are added per 10 ml). Before use, the lyophilisate is taken
up in an
2o appropriate volume of water or buffer and the suspension is purified on a
discontinuous sucrose gradient (steps of 0, 30, and 60%) so as to obtain a
preparation
in which the quantity of encapsulated urease is greater than about 70%
compared with
the total quantity of urease.
Cholera toxin is used as mucosal adjuvant in an amount of 10 ~g/dose of urease
2s or of bacterial preparation.
The QS-21 (Cambridge Biosciences; Aquila} is used as adjuvant in an amount
of 1 S ,ug/dose of urease.
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Challenge
Two weeks after the second booster, the mice were subjected to a gastric
gavage with 300 ,ul of a suspension of a strain of H. pylori adapted to the
mice, the
strain ORV2002 (1 X 10' live bacteria in 200 ~1 of PBS; ODSSO of about 0.5).
One
group which received no dose of antigen and which serves as control is
challenged
likewise.
Analysis of the challenge
Four weeks after the challenge, the mice were sacrificed by breaking the
cervical vertebrae. The stomachs were removed in order to evaluate the urease
activity and to make histological analyses. The urease activity was evaluated
after 4
and 24 hours (OD at 550 nm) with the Jatrox test, Procter & Gamble) and after
24
hours the number of mice still negative was noted.
~s Measurement o_f the local antibody response by ELISPOT (salivary glands and
stomach)
The ELISPOTs were performed in accordance with Mega et al., J. Immunol.
(1992) 148: 2030. The plates were coated with an extract of H. pylori proteins
at a
concentration of 50 ,ug/ml.
2o To test the antibody response at the level of the stomach, we modified the
method as follows: half of the stomach was cut into 1-mm2 pieces with an
automatic
apparatus for cutting human tissues (McIllwain Laboratories, Gilford, UK) and
the
digestion carned out with Dispase (2 mg/ml, Boehringer Mannheim) in 2 ml of a
modified Joklil solution to which 10% horse serum (Gibco), glutamine and
antibiotics
2s were added. Four half hour digestions were performed at 37°C with
gentle mixing.
The cells thus digested were filtered after each step using 70 ~m filters
(Falcon), and
then washed 3 times in a solution of RPMI 1640 (Gibco) supplemented with 5%
fetal
calf serum (FCS), and incubated in the same solution for at least 4 hours in
plates
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covered with nitrocellulose (Millipore) ( 100 ,ul/well, 4 wells). Between 1
and 3 X 105
cells are obtained per half stomach (the cells of large size and the
macrophages were
not counted).
The biotinylated IgA and the streptavidin-biotinylated peroxidase complex
s were obtained from Amersham. The spots were revealed under the action of the
AEC
substrate (Sigma) and as soon as the plates are dry, they were counted under a
microscope (magnification X 16 or X40). The mean values corresponding to the
number of IgA spots in four wells were calculated and expressed as the number
of
spotsllOG cells.
to
Analysis of the response by ELISA
The analyses by ELISA were performed in accordance with the standard
procedure (the biotinylated conjugates and the streptavidin-peroxidase were
obtained
from Amersham and the OPD (O-phenyl-diamine dihydrochloride) substrate from
is Sigma). The plates were coated with H. pylori extracts (5 ~g/ml) in
carbonate buffer.
A control serum from mice directed against the H. pylori extract was
introduced in
- each experiment. The titre corresponds to the reciprocal of the dilution
giving an OD
of 1.5 at 490 nm.
20 1B - Results
The results are presented in Figures 1 to 4 described above and by the
following
comments:
Figure 1 shows that when the subcutaneous route is used, much better results
are obtained in terms of the local response both in the salivary glands and in
the
25 stomach if the administration took place in the posterior part of the mice,
that is to say
in the sublumbar region.
Before any comment on the subject of Figures 2 to 4, it should be noted that
these figures present the results obtained with the antigen used in the form
with
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cholera toxin adjuvant and administered by the intragastric route. This
experiment is
termed standard reference experiment since the prior art CT/IG combination is
that
which gives the best results up until now.
Figure 2 compares the results obtained with a preparation of inactivated
bacteria without adjuvant, by the intragastric route and subcutaneous route.
It is clear
that much better results are obtained when the subcutaneous route is used
while
targeting the sublumbar region. Furthermore, the results obtained after
administration
by the subcutaneous route are identical to, if not slightly better than, those
which are
obtained in the standard reference experiment with the same preparation, this
time
1o with the cholera toxin adjuvant and administered by the intragastric route.
Furthermore, reference can be made to experiments (a) to (e) the results of
which in terms of urease activity 4 hours after the mice have been sacrificed
are
reported in Figure 2 and it is indicated that the number of mice which are
still negative
for the urease activity 24 hours after having been sacrificed is respectively
(a) 0/8, (b)
~ 5 4/8, (c) 4/8, (d) 0/8, and (e) 10/ 10. This is in agreement with what was
previously
concluded in the paragraph; namely that experiment (b) leads to results
similar to
those obtained during the standard reference experiment.
Figure 3 shows that a urease preparation encapsulated into DC-chol liposomes
and administered by the subcutaneous route in the sublumbar region gives
results as
2o good as those obtained in the standard reference experiment.
Furthermore, reference can be made to experiments (a) to (d) whose results in
terms of urease activity 4 hours after the mice have been sacrificed are
reported in
Figure 3 and it is indicated that the number of mice which are still negative
for the
urease activity 24 hours after having been sacrificed is respectively (a)
S/10, (b) 4/10,
25 (c) 0/10, and (d) 10/10. This is in agreement with what was concluded in
the
preceding paragraph; namely that experiment (a) leads to results similar to
those
obtained during the standard reference experiment.
Figure 4 shows that a urease preparation with QS-21 adjuvant and administered
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by the subcutaneous route in the sublumbar region gives results as good as
those
obtained in the standard reference experiment.
Furthermore, reference can be made to experiments (a) to (d) whose results in
terms of urease activity 4 hours after the mice have been sacrificed are
reported in
Figure 4 and it is indicated that the number of mice which are still negative
for the
urease activity 24 hours after having been sacrificed is respectively (a) 1/8,
(b) 5/8, (c)
0/8, and (d) 10/10. This is in agreement with what was concluded in the
preceding
paragraph; namely that experiment (b) leads to results similar to those
obtained during
the standard reference experiment.
to The table below presents the quantities of IgA, IgGI, and IgG2a induced
during
experiments whose results in terms of urease activity are reported in Figures
2 to 4 as
well as the number of mice whose urease activity is characterized by an OD of
less
than 0.1 after 4 and 24 hours after sacrifice. The quantities of IgA, IgGl,
and IgG2a
are expressed as ELISA titre.
urease' WC WC WC urease urease
CT CT lipo DC-cholQS-21
IG IG IG SC SC SC
IgA 4s 91 107 63 0 1
IgGI 6s700 1920 349 1273146620000 2970399
IgG2a 20200 399 3440 42900 321000 113609s
OD < 0.1 s/10 0/8 s/8 6/8 s/10 6/8
4 hours
OD < 0.1 4/10 0/8 4/8 4/8 s/10 s/8
24 hours
2s Example 2: Immunization studies in monkeys
2A - Materials and methods
Monkeys
Twenty eight 2-year old monkeys (Macaca fascicularis) obtained from
Mauritius were used in this study. Before subjecting the monkeys to the
various
3o immunization procedures described below, a biopsy showed that most of them
were
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chronically infected with organisms similar to Gastrospirillum hominis (GHLO)
or H.
heilmanii.
Administration procedures
s Since nearly all the monkeys were infected with GHLOs, it was decided to
test
the efficacy of the various procedures in therapy. Three procedures were used,
as
summarized in the table below:
Group DO D21 D42 D63
l0 1 and lu IN + IG IN + IG IN + IG IN + IG
2 and 2u IM IM IM IM
3 and 3u IM IN + IG IM IN + IG
It is specified that the administration by the intramuscular route was carried
out
is in the dorsolumbar muscles.
Antigens and adjuvants
Since there is a cross-reactivity between the GPLOs and H. pylori, it was
chosen to use a preparation of inactivated H. pylori bacteria, as described in
Example
20 1 A, alone or in combination with recombinant urease prepared according to
the
method referenced in Example lA.
The E. coli heat-labile toxin (LT) (Sigma) or the B subunit of the cholera
toxin
(CTB) (Pasteur Merieux serums & vaccins) was used as mucosal adjuvant whereas
DC-chol was used as parenteral adjuvant. DC-chol powder is simply rehydrated
with
2s an antigen preparation.
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The doses used are as follows:
Route Microorganisms Urease DC-chol LT CTB
IG 400 ,ug 2.5 mg - 25 ~g -
s IN 400 ,ug 400 ,ug - 25 ng 25 ,ug
IM 400 ,ug 100 ,ug 400 ,ug - -
Biopsies, urease test, and bacteriologicallhistological study
A biopsy was performed on each of the monkeys before and after immunization
to (one month after the third booster). Using the biopsies, a urease test and
a histological
study were performed.
The urease activity is evaluated using the Jatrox kit (Procter & Gamble). The
level of this activity is estimated as follows, in a decreasing manner: level
3, pink
color appearing during the first 10 minutes; level 2, pink color appearing
between 10
~s and 30 minutes after the addition of the reagents; level l, pink color
appearing
between 30 minutes and 4 hours and level 0, weak or no color after 4 hours.
The histological studies were performed using biopsies fixed in formalin and
the bacterial load was quantified as follows: absence of bacteria (0); a few
bacteria of
the Helicobacter type (0.5); fairly numerous bacteria ( 1 ); numerous bacteria
(2);
2o highly numerous bacteria (3). A difference of one level (for example from 1
to 2)
corresponds to a number of bacteria 5 times greater.
Analysis of the response by the ELISA test
An ELISA test is carried out as described in Example lA.
2s
2B - Results
The table below relates to the bacterial load which, before and after
immunization, is assessed using two tests: (i) by evaluating the urease
activity and (ii)
by carrying out a histological study. The results relating thereto are
presented in
3o columns 3 to 6. The last three columns indicate for each group (control, 1,
2, or 3) the
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number of monkeys for which the bacterial load remains unchanged after
immunization (-->) according to the two tests; or appears lower (~~) or
increased (~') in
at least one of the two tests, the other test indicating a stationary
bacterial load. When
the results of the two tests show a similar variation, the upwards or
downwards arrow
is double.
UreasectivityHistology Variation
a
MonkeysGroup beforeafter before after~. r
immunization immunization
H 282 C 2-2 3-2 2 3-2
J 005 C 2-2 2-1 2 1-0 1/4 1/4 2/4
J 852 C 0-0 2-0 0 1-1 (2/4~'~')
J 476 C 0-0 2-0 0 1-1
H 799 1 2-2 2-2 2 2-2
1$ J 845 1 2-2 3-2 2 2-1
J 205 1 1-1 2-2 0 1
J 328 1 2-2 1-2 3 3-2 1/8 5/8 2/8
J 197 lu 2-2 3-2 2 3 (1/8~'~')
H 025 lu 2-2 2-2 1 1-1
G 460 lu 2-2 3-2 3 2-3
J 607 1u 2-2 2-2 2 2
H 549 2 3-3 2-2 3 2-3
H 622 2 3-3 1-1 2 2-3
H 504 2 3-3 1-1 2 2-1
2$ H 798 2 1-1 0-1 1 1-1
J 367 2u 2-2 2-1 3 2-3 6/8 1/8 1/8
G 486 2u 2-2 2-2 1 2-2
J 522 2u 2-2 0-0 2 2-2
G 722 2u 3-3 2-0 2 2-3
H 820 3 3-3 2-2 3 2-2
J 557 3 2-2 1-0 2 1-2*
H 588 3 2-2 2-0 3 1-2
J 153 3 3-3 3-3 2 3-3 5/8 0 3/8
3$ H 480 3u 2-2 2-2 2 3-3 (3/8~~)
J 344 3u 3-3 2-0 3 2-2
H 710 3u 2-2 2-2 2 3-3
J 262 3u 3-3 2-2 3 3-2
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. Thus, this table reveals that in the group having been subjected to an
immunization procedure by the strict mucosal route, the results are
substantially
identical to those obtained with the negative control group. On the other
hand, in the
groups having been subjected to an immunization procedure by the mixed mucosal
s and intramuscular route or by the strict intramuscular route, a marked
reduction in the
bacterial load is observed. This highlights the importance of the immunization
conditions and in particular of the immunization rate; consequently, the use
of an
immunization procedure which employs the parenteral route targeted in the
subdiaphragmatic region, is recommended in order to obtain a protective
effect.
to These results are to be placed in perspective with other results relating
to the
serum antibody levels which are presented in Figure 3. This figure shows that
the
immunization scheme by the strict mucosal route ( 1 and 1 u) leads to results
which are
very similar to those of the negative control group. On the other hand, the
immunization scheme by the mixed mucosal and intramuscular route (2 and 2u),
and
is better still the immunization scheme by the strict intramuscular route (3
and 3u),
makes it possible to induce antibody levels substantially greater than those
of the
control group.
Thus, a high serum response may be correlated with a protective effect,
whereas a contrario, a low response is linked to the absence of a protective
effect.
2o The immunization conditions which make it possible to obtain the desired
effect (high
serum response and protective effect) include the use of the parenteral route
targeted
in the subdiaphragmatic region or that of a Thl adjuvant.
Example 3: Treatment of an H. pylori infection in mice
2s We compared the efficacy of immunization via the subcutaneous (SC) route
with that of the mucosal route, in order to treat an H. pylori infection in a
mouse
model.
OF 1 mice were infected with 1 OG colony-forming units (cfu) of the H. pylori
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strain ORV2001. After one month, verification that the infection was well-
established
was made by randomly sacrificing 10/100 mice and testing the urease activity
on a
quarter of the entire stomach. Since all of the results were positive, the
mice were
then immunized ( 10 per group) 3 times at 3 weekly intervals, either
subcutaneously
using 10 ,ug of recombinant urease supplemented with 15 ~cg of QS-21 (Aquila)
or 400
~g of adjuvant Bay 81005 (Bayer), or orally using 40 ~g of urease mixed with 1
~g of
LT. For each of the two adjuvants administered parenterally, the immunization
was
carned out either in the neck, in order to reach the lymphatic ganglions of
the upper
region of the body, or in the lumbar region, in order to reach the abdominal
lymphatic
io ganglions. Ten mice were left uninfected and unimmunized (negative
control),
whereas the mice of the positive control received a saline solution, QS-21, or
Bay
adjuvant subcutaneously (lumbar region).
One month after the third immunization, all of the mice were sacrificed and
the
stomachs removed to evaluate the extent of the colonization by measuring the
urease
1s activity (10/10 mice were analyzed in each group), as well as by carrying
out
quantitative culturing (5/10 were analyzed). Figures 6A (test relating to
urease) and
6B (culturing) show that in the mice immunized with urease supplemented with
QS-
21, subcutaneously in the lumbar region, the infection had virtually
disappeared (4/5
mice were negative in quantitative culturing). The mice immunized with urease
2o subcutaneously in the neck, in the presence of QS-21, and the mice that
received
urease plus LT orally exhibited a 10- to 100-fold decrease in the infection
when
compared with the unimmunized mice. The Bay adjuvant induced an identical
decrease, which was more pronounced in the mice immunized in the lumbar
region.
Histopathology carned out on these same mice did not reveal any gastritis that
2s was more extensive than in the controls.
As we observed in our previous prophylactic study (Example 1 ), the protected
mice had a high level of the two isotypes IgG 1 and IgG2 in the serum, which
is
representative of a Th2/Thl equilibrated response. Furthelnore, the mice
immunized
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subcutaneously in the lumbar region had the highest levels of IgA in the
serum, which
demonstrates a mucosal response.
These results show that targeted, systemic immunization is capable of curing
an
acquired H. pylori infection in mice, and that the use of adjuvants that
induce an
equilibrated mucosal response of Thl/Th2 type is desirable in order to achieve
this
aim.
Example 4: Mucosal prime/parenteral boost strategy for urease immunization
that elicits protection in mice against infection with H. pylori
io Swiss Webster mice were immunized with a mucosal prime/parenteral boost
strategy. A single oral dose of 25 ,ug recombinant H. pylori urease (urease)
and S ~cg
Escherichia coli heat labile enterotoxin (LT) was administered as a prime.
Three
weeks later, mice were boosted by the parenteral route with 2 doses, 3 weeks
apart,
with 100 ~cg urease + 100 ~g alum. Mice immunized by this prime/boost strategy
is exhibited a 2,000-fold reduction in the median H. pylori colony forming
units (CFUs)
compared to unimmunized controls (Figure 7). Immunization by this strategy was
more efficacious than 3 doses of mucosal vaccine when a 1,000-fold reduction
in
colonization was achieved. A single priming dose of orally administered urease
+ LT
only produced a 10-fold reduction in median H. pylori CFUs, boosting with
urease
2o administered parenterally without an adjuvant resulted in only a 2-fold
reduction in
medium CFUs (Figure 7).
This immunization strategy was used to immunize rhesus monkeys against
challenge with H. pylori.
2s Example 5: Protection of rhesus monkeys from H. pylori infection by urease
immunization using a mucosal prime/parenteral boost strategy
Rhesus monkeys, seronegative for H. pylori, were treated with quadruple
therapy and confirmed to be free of H. pylori infection by culture and
histologic
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examination of gastric biopsies. Nineteen monkeys were randomized into 4
groups: 4
monkeys were sham vaccinated, 5 received recombinant H. pylori urease (urease)
vaccine given by the intramuscular route with alum (aluminum hydroxide,
Rehydragel) as an adjuvant, 5 received a mucosal priming dose of urease
vaccine
given by the oral route with Escherichia coli heat labile enterotoxin (LT) as
an
adjuvant, followed by two parenteral boosts of urease vaccine given by the
intramuscular route with alum, and 5 monkeys received urease vaccine given by
the
intramuscular route with Bay adjuvant. Monkeys were immunized every three
weeks
for the first three immunizations, and a fourth dose was administered at 20
weeks.
to One week after the last immunization, the monkeys were challenged with H.
pylori. The monkeys were sacrificed 10 weeks after challenge. At necropsy, 10
gastric sites ( 1 cardiac, 2 corporal, 3 corporal-antral junction, 3 antral,
and 1 pyloric)
were sampled for bacterial culture by taking S mm diameter punch biopsies.
Additional tissues from the S regions of the stomach were harvested for
1s histopathology.
Although all of the monkeys were infected with H. pylori asa result of the
experimental challenge, monkeys immunized with urease + LT as a mucosal prime
followed by 3 parenteral booster doses of urease + alum showed statistically
significant reduction (p=0.05, Wilcoxon rank sums test) in colonization when
2o compared to the control, sham immunized monkeys (Figure 8). A greater than
20-fold
reduction in median bacterial colony forming units per bunch biopsy (5.8 x 102
CFU,
ranging from 1 x 10'- to 6.7 x 102 CFU) was found in monkeys that received the
vaccine as a mucosal prime/parenteral boost regimen, compared to a median CFU
of
1.3 x 104 (range 1.5 x 103 to 1.8 x 105 CFU) for the group of sham immunized
2s monkeys.
The group of monkeys receiving urease vaccine in a mucosal prime/parenteral
boost regimen had similar gastritis (Figure 9) and epithelial changes (Figure
10) after
challenge with H. pylori. There was no evidence that vaccination enhanced
either
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gastritis or epithelial alterations.
in contrast to the monkeys receiving the mucosal prime/parenteral boost
regimen, monkeys immunized by the parenteral route with urease + Bay showed no
difference in H. pylori colonization compared with the sham-immunized controls
(p =
1.00), while monkeys treated with urease + alum showed a partial effect
(p=0.33)
(Figure 8). Culture data was unavailable for one of the monkeys in the group
receiving urease + Bay, due to heavy contamination of gastric samples with
other
bacteria.
These results show that an immunization scheme utilizing a mucosal prime of
io urease + LT followed by parenteral boosting with urease + alum is
efficacious in
preventing H. pylori infection in non-human primates. This scheme was chosen
with
the rationale that the immune system must be primed to respond to a mucosal
infection, i.e., by mucosal immunization with an appropriate adjuvant.
However, once
the immune response is 'set' properly, a more conventional adjuvant, such as
alum,
15 can be used as a parenteral immunization to boost the response. This kind
of scheme
is not only more effective than a mucosal only regimen, but it utilizes less
antigen
because the parenteral antigen dose ( 100 ,ug urease) can be much less than a
mucosal
dose (4 mg).
Other embodiments are within the following claims.
