Note: Descriptions are shown in the official language in which they were submitted.
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33,427-00 PCT
NOVEL ANTIGENS of HELICOBACTER PYLORI
Field of the Invention
The present invention relates to novel nucleic acids and polypeptides
relating to Helicobacter pylori. The nucleic acid sequences and polypeptides
are useful
for diagnostic and therapeutic purposes.
Background of the Invention
Helicobacter pylori (H, pylori) is a gram-negative, S-shaped,
microaerophilic bacterium that was discovered and cultured from a human
gastric
biopsy specimen infection. [Warren, J.R. et al., Lancet, 1: 1273-1275, (1983)
; and
Marshall et al., Microbios Lett, 25: 83-88, (1984)]. H. pylori has been
strongly linked to
chronic gastritis and duodenal ulcer disease. [Rathbone et al., Gut, 27: 635-
641,
(1986)]. Additional evidence has developed for an etiological role of H.
pylori in non-
ulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. [Blaser,
M.J.,
Trends Microbiol., 1: 255-260, (1993)]. H. pylori colonizes the human gastric
mucosa,
establishing an infection that usually persists for many years. About 30-50%
of the
human population appear to be chronically infected. [Rainer, H. et al,
Biologicals, 25:
175-177, (1997)]. The current recommended treatment for chronic H. pylori
infection is
multiple antibiotic treatment combined with a proton pump inhibitor, or with
bismuth
salts. However, this treatment may not fully resolve the infection and
resistance to
antibiotics can occur. H. pylori infection in humans induces a strong local
and systemic
immune response; however, the immune response if often unable to clear the
infection.
Accordingly, work has led to developing potential H. pylori vaccines. Various
antigens,
proteins and genes have been reported in this area. [See Bolin et al., PCT
Application
No. 96/38475, filed June 3, 1996; Doidge et al., PCT Application No. 96/33220,
filed
April 19, 1996; Clancy et al., PCT Application No. 96/25430, filed February
15, 1996;
Chan et al., PCT Application No. 96/12965, filed October 19, 1995; Byrne et
al., PCT
Application No. 96/01273, filed July 3, 1995; Alemohammad M.M., in U.S. Patent
5,262,156, filed August 12, 1991; Allan et al., PCT Application No. 97/03359,
filed
June 28, 1996; and Dettmar et al., German Patent Application 195235554 A1
based on
Great Britain Application No. 9413074.] As an alternative to potential
vaccines and
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diagnostic currently provided in the art referenced above, novel methods of
curing or
preventing H. pylori infection through the use of novel H. pylori proteins, as
well as
genes encoding such proteins are described herein. Proteins were selected for
use as an
antigen and/or vaccine candidate based on the following criteria: (i) the
antigen is
located on the bacterial surface, (ii) the antigen is conserved among H.
pylori clinical
isolates, (iii) the antigen elicits functional antibodies, (iv) the antigen is
able to confer
protection to vaccinated mice from challenge with a live organism.
Summary of the Invention
This invention relates to novel H. pylori bacterial surface proteins and
nucleic
acid sequences encoding therefor, in particular novel H. pylori bacterial
surface proteins
having molecular weights of approximately 75, 77, and 79 kilo daltons (kDa).
The
mature processed forms of these proteins share a common amino-terminal amino
acid
sequence. The proteins and nucleic acid sequences of the present invention
have
diagnostic and therapeutic utility for H. pylori and other Helicobacter
species. They can
be used to detect the presence of H. pylori and other Helicobacter species in
a sample,
and to screen compounds for the ability to interfere with the H. pylori life
cycle or to
inhibit H. pylori infection. More specifically, this invention includes
embodiments
relating to isolated nucleic acid sequences corresponding to the entire coding
sequences
of H. pylori surface proteins or portions thereof, nucleic acids capable of
binding mRNA
from H. pylori surface proteins and methods for producing H. pylori surface
proteins or
portions thereof using peptide synthesis and recombinant techniques.
Additional
embodiments are also directed to antigenic and vaccine compositions based on
agents
prepared from the proteins and nucleic acids of this invention and methods for
treatment
and prevention of H. pylori infections employing such compositions.
Brief Description of the Drawings
Figure 1: Figure 1(A) depicts an SDS-PAGE gel with bands showing the 75kDa and
77kDa proteins in lane 3 in comparison to ZwittergentTM 3-14 crude extract of
H. pylori
outer membrane proteins in lane 2 and molecular weight standards in lane 1.
Figure
I(B) depicts a Western blot of monoclonal antibody 64-27 with the co-purified
75kDa
and 77kDa proteins from ATCC 43579, as described in Example I. Lane I is the
2
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molecular weight markers, lane 2 is the crude extract and lane 3 is the co-
purified
proteins.
Figure 2 depicts an electron micrograph of the surface labeling of H. pylori
strain PBCC
1105 with mouse polyclonal antisera to a co-purified mixture of 75/77 kDa
proteins, as
described in Example 2.
Figure 3 depicts two graphs of Flow Cytometry analysis of strain PBCC 1105
with
labeled an anti-75/77 mouse polyclonal antibody, at day 0 (Figure 3A) and day
49
(Figure 3B) following injection of mice with a mixture of 75kDa and 77kDa
proteins, as
described in Example 2. Profilel is ATCC 43579 (homologous strain); profile 2
is strain
PBCC 1105; profile 3 is strain ATCC 43504; profile 4 is strain SS-1 and
profile 5 is a
urease-negative strain.
Figure 4 is a graph depicting the bactericidal activity of anti-75/77kDa
polyclonal
mouse sera, as described in Example 3, with either incomplete Freund's
adjuvant or
MPLTM
Figure 5: This Figure depicts the mouse protection data (in colony forming
units) from
the H. pylori SS1 experimental challenge following vaccination of mice with a
mixture
of the co-purified 75kDa/77kDa proteins, as described in Example 4.
Figure 6: This Figure depicts a DNA (SEQ ID NO 19)for the 75kDa gene from
strain
ATCC 43579.
Figure 7: This Figure depicts the predicted translated protein sequence for
the DNA
sequence (SEQ ID NO 21)for the 75kDa gene from strain ATCC 43579 in figure 6.
Figure 8: This Figure depicts a DNA sequence (SEQ ID NO 20)for the 79kDa gene
from strain ATCC 43579.
Figure 9: This Figure depicts the predicted translated protein sequence (SEQ
ID NO
22)for the DNA sequence for the 79kDa gene from strain ATCC 43579 in figure 8.
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Figure 10: This Figure depicts SDS-PAGE gel bands from comparison expression
experiments from Example 13 for recombinant 75kDa protein expressed from a low
copy number plasmid-pBAD24 and the high copy number T7 expression plasmid
pRSETb. The bands show the increased amounts of protein 75 kDa expressed.
Figure 11: This Figure depicts SDS-PAGE gel bands from comparison expression
experiments from Example 13 for recombinant 77kDa protein expressed from a low
copy number plasmid-pETl7 and the high copy number T7 expression plasmid
pRSETb.
The bands show the increased amounts of protein 77 kDa expressed.
Figure 12: This Figure depicts SDS-PAGE gel bands from comparison expression
experiments from Example 13 for recombinant 79kDa protein expressed from a low
copy number plasmid pBAD24 and the high copy number T7 expression plasmid
pRSETb. The bands show the increased amounts of protein 79 kDa expressed.
Figure 13: This Figure depicts mouse protection data (in colony forming units)
from the
H, pylori SS1 experimental challenge following vaccination of mice with a
mixture of
the co-purified 75kDa/77kDa proteins
Figure 13: This Figure depicts therapeutic effect (in colony forming units) of
a mixture
of the co-purified 75kDa/77kDa proteins when vaccinating mice after infection
with H.
pylori SS 1 Experiments included intragastric vaccination and a subcutaneous
vaccination.
Detailed Description of the Invention
One aspect of the present invention provides an isolated, substantially
purified
H, pylori polypeptide selected from the group consisting of (i) a polypeptide
having a
molecular weight of about 75 kDa; (ii) a polypeptide having a molecular weight
of about
77 kDa; and (iii) a polypeptide having a molecular weight of about 79 kDa;
wherein the
mature processed form of each polypeptide has a starting sequence consisting
essentially
of EDDGFYTSVGYQIGEAAQMV (SEQ. ID N0.7). The present invention also relates
to isolated polypeptides. Preferred embodiments of the invention relate to an
isolated
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polypeptide of H. pylori selected from the group consisting of (i) a
polypeptide having a
molecular weight of about 75 kDa and having the amino acid sequence of SEQ. ID
NO.1 or SEQ ID NO. 19; (ii) a polypeptide having a molecular weight of about
77 kDa
and having the amino acid sequence of SEQ. ID N0.2; and (iii) a polypeptide
having a
molecular weight of about 79 kDa and having the amino acid sequence of SEQ. ID
N0.3 or SEQ ID NO. 20. Preferably, the polypeptides of this invention have
antigenic
properties, such as being reactive with H. pylori antibodies. Antigens can be
based on
the isolated polypeptides sequences, or allelic or other variants thereof,
which are
biological equivalents. Suitable biological equivalents have about 70 to about
80%, and
most preferably at least about 90%, similarity to one of the amino acid
sequences
referred to above, or to a portion thereof, provided the equivalent is capable
of eliciting
substantially the same antigenic properties as the isolated poiypeptide
sequences
specified hereinabove.
The biological equivalents are obtained by generating variants and
modifications to the isolated polypeptides of this invention. These variants
and
modifications to the isolated polypeptides are obtained by altering the amino
acid
sequences by insertion, deletion or substitution of one or more amino acids.
The
polypeptides are then selected for use as an antigen and/or vaccine candidate
based on
the following criteria: (i) the antigen is located on the bacterial surface,
(ii) the antigen
is conserved among H. pylori clinical isolates, (iii) the antigen elicits
functional
antibodies, (iv) the antigen is able to confer protection to vaccinated mice
from
challenge with a live organism. Modifying the amino acid, for example by
substitution,
the amino acids of the protein to create a polypeptide having substantially
the same or
improved qualities. The amino acid changes are achieved by changing the codons
of the
nucleic acid sequence. It is known that such polypeptides can be obtained
based on
substituting certain amino acids for other amino acids in the polypeptide
structure in
order to modify or improve antigenic or immunogenic activity (see, e.g. Kyte
and
Doolittle, 1982, Hopp, US Patent 4,554,101, each incorporated herein by
reference).
For example ,through substitution of alternative amino acids, small
conformational
changes may be conferred upon a polypeptide which result in increased activity
or
enhanced immune response. Alternatively, amino acid substitutions in certain
polypeptides may be utilized to provide residues which may then be linked to
other
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molecules to provide peptide-molecule conjugates which retain sufficient
antigenic
properties of the starting polypeptide to be useful for other purposes. For
example, a
selected polypeptide of the present invention may be bound to a solid support
in order to
have particular advantages for diagnostic applications.
One can use the hydropathic index of amino acids in conferring interactive
biological function on a polypeptide, as discussed by Kyte and Doolittle
(1982), wherein
it is found that certain amino acids may be substituted for other amino acids
having
similar hydropathic indices and still retain a similar biological activity.
Alternatively,
substitution of like amino acids may be made on the basis of hydrophilicity,
particularly
where the biological function desired in the polypeptide to be generated is
intended for
use in immunological embodiments. US Patent 4,554,101, which states that the
greatest
local average hydrophilicity of a "protein," as governed by the hydrophilicity
of its
adjacent amino acids, correlates with its immunogenicity. Accordingly, it is
noted that
substitutions can be made based on the hydrophilicity assigned to each amino
acid.
In using either the hydrophilicity index or hydropathic index, which assigns
values to each amino acid, it is preferred to conduct substitutions of amino
acids where
these values are +2, with +1 being particularly preferred, and those within +
0.5 being
the most preferred substitutions.
Preferable characteristics of the polypeptides of this invention include one
or
more of the following: (a) being a membrane protein or being a protein
directly
associated with a membrane; (b) capable of being separated as a protein using
an SDS
acrylamide (10%) gel; (c) generating antibody which exhibits bactericidal
activity upon
injection in a mouse; and/or (d) reducing the colonization of H. pylori in
mice upon
delivery thereto.
The isolated polypeptides having molecular weights of about 75, 77, and 79 kDa
are particular useful in antigenic compositions for several reasons. First,
using
monoclonal antibodies derived by injecting mice with whole bacteria, these
polypeptides were identified as closely related outer membrane proteins of X.
pylori.
Second, our analyses by a number of in vitro techniques indicate that the
polypeptides
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are surface localized and that they are expressed in a variety of clinically
relevant
strains. Third, the polyclonal antibodies generated by injecting mice with a
mixture of
the purified polypeptides are bactericidal. Fourth, we observed that following
oral
vaccination with the polypeptides admixed with cholera toxin (CT), 100% of
mice
challenged with H. pylori strain SS 1 showed a statistically significant
reduction in
colonization as compared to non-vaccinated control mice.
Accordingly, further embodiments of this invention relate to an antigenic
composition comprising (i) at least one isolated polypeptide as disclosed
above and (ii)
a pharmaceutically acceptable buffer, diluent, adjuvant or carrier. The
antigenic
composition may comprise a carrier, which in turn may be conjugated to said
polypeptide. In additional embodiments, the antigenic composition may further
comprise an adjuvant. Preferably, these compositions have therapeutic and
prophylatic
applications as vaccines in preventing and/or ameliorating H. pylori
infection. In such
applications immunologically effective amount (as discussed herein) of at
least one
polypeptide of this invention is employed.
The formulation of such prophylactic or therapeutic antigenic compositions is
well known to persons skilled in this field. Antigenic compositions of the
invention
containing antigenic components (e.g., H. pylori polypeptide or fragment
thereof or
nucleic acid encoding an H. pylori polypeptide or fragment thereof) preferably
include a
pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable
carriers
and/or diluents 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 term "pharmaceutically
acceptable
carrier" refers to a carrier that does not cause an allergic reaction or other
untoward
effect in patients to whom it is administered. Suitable pharmaceutically
acceptable
carriers include, for example, one or more of water, saline, phosphate
buffered saline,
dextrose, glycerol, ethanol and the like, as well as combinations thereof.
Pharmaceutically acceptable carriers may further comprise minor amounts of
auxiliary
substances such as wetting or emulsifying agents, preservatives or buffers,
which
enhance the shelf life or effectiveness of the antibody. The use of such media
and
agents for pharmaceutically active substances is well known in the art. Except
insofar
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as any conventional media or agent is incompatible with the active ingredient,
use
thereof in the antigenic compositions of the present invention is
contemplated.
Such antigenic compositions are conventionally administered parenterally,
e.g.,
by injection, either subcutaneously or intramuscularly. Methods for
intramuscular
immunization are described by Wolff et al. and by Sedegah et al. Other modes
of
administration include oral and pulmonary formulations, suppositories, and
transdermal
applications. Oral immunization is preferred over parenteral methods for
inducing
protection against infection by H. pylori (See Czinn et al.). Oral
formulations include
such normally employed excipients as, for example, pharmaceutical grades of
mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like.
The antigenic compositions of the invention can include an adjuvant,
including,
but not limited to aluminum hydroxide; aluminum phosphate; StimulonTM QS-21
(Aquila Biopharmaceuticals, Inc., Worcester, MA); MPLTM (3-O-deacylated
monophosphoryl lipid A; RIBI ImmunoChem Research, Hamilton, MT), IL-12
(Genetics Institute, Cambridge, MA) N-acetyl-muramyl--L-theronyl-D-
isoglutamine
(thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as
nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-
dipalmitoyl-
sn-glycero-3-hydroxyphos-phoryloxy--ethylamine (CGP 19835A, referred to a MTP-
PE); and cholera toxin. Others which may be used are non-toxic derivatives of
cholera
toxin, including its B subunit; and/or conjugates or genetically engineered
fusions of the
H. pylori polypeptide with cholera toxin or its B subunit, procholeragenoid,
fungal
polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide
derivatives, phorbol esters, labile toxin of E. coli, non-H. pylori bacterial
lysates, block
polymers or saponins.
Preferably also, this antigenic composition or an isolated polypeptide of this
invention is used in a vaccine composition for oral administration which
includes a
mucosal adjuvant.
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In a particularly preferred aspect of this invention, an oral vaccine
composition
comprising an antigenic composition in association with a mucosal adjuvant, is
used for
the treatment or prevention of H. pylori infection in a human host.
The mucosal adjuvant can be cholera toxin; however, preferably, mucosal
adjuvants other than cholera toxin which may 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
Helicobacter antigenic composition. The same techniques can be applied to
other
molecules with mucosal adjuvant or delivery properties such as Escherichia
coli heat
labile toxin (LT). Other compounds with mucosal adjuvant or delivery activity
may be
used such as bile; polycations such as DEAF-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. QS-21, MPLTM, and
IL-12,
which described above, may also be used
The Helicobacter antigenic composition of this invention may be delivered in
the form of 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 isolated polypeptides of
this
invention may also be incorporated into oily emulsions.
The Helicobacter isolated polypeptides of the present invention may be
administered as the sole active immunogen in an antigenic composition.
Alternatively,
however, the antigenic, or vaccine, composition may include other active
immunogens,
including other Helicobacter antigens such as urease, lipopolysaccharide,
Hsp60, VacA,
CagA or catalase, as well as immunologically active antigens against other
pathogenic
species.
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One of the important aspects of this invention relates to a method of inducing
immune responses in a mammal comprising the step of providing to said mammal
an
antigenic composition of this invention. Preferred embodiments relate to a
method for
the treatment or prevention of Helicobacter infection in a human comprising
administering to a human an immunologically effective amount of an antigenic
composition. Immunologically effective amount, as used herein, means the
administration of that amount to a mammalian host, either in a single dose or
as part of a
series of doses, sufficient to at least cause the immune system of the
individual treated
to generate a response that reduces the clinical impact of the bacterial
infection. This
may range from a minimal decrease in bacterial burden to prevention of the
infection.
Ideally, the treated individual will not exhibit the more serious clinical
manifestations of
the Helicobacter infection. The dosage amount can vary depending upon specific
conditions of the individual. This amount can be determined in routine trials
by means
known to those skilled in the art.
Another specific aspect of the present invention relates to using a vaccine
vector
expressing an isolated Helicobacter polypeptide, or an immunogenic fragment
thereof.
Accordingly, in a further aspect this invention provides a method of inducing
an
immune response in a mammal, which comprises providing to a mammal a vaccine
vector expressing at least one, or a mixture of isolated Helicobacter
polypeptides of this
invention, or an immunogenic fragment thereof. The isolated polypeptides of
the
present invention can be delivered to the mammal 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~an antigenic polypeptide
or
immunogenic fragment as a foreign polypeptide. Particularly, bacteria that
colonize the
gastrointestinal tract, such as Salmonella, Shigella, Yersinia, Vibrio,
Escherichia and
BCG have been developed as vaccine vectors, and these and other examples are
discussed by Holmgren et al. (1992) and McGhee et al. (1992).
An additional embodiment of the present invention relates to a method of
inducing an immune response in a human comprising administering to said human
an
amount of a DNA molecule encoding an isolated polypeptide of this invention,
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optionally with a transfection-facilitating agent, where said polypeptide
retains
immunogenicity and, when incorporated into an antigenic composition or vaccine
and
administered to a human, provides protection without inducing enhanced disease
upon
subsequent infection of the human with Helicobacter pathogen, such as H.
pylori.
Transfection-facilitating agents are known in the art.
The present invention also relates to an antibody, which may either be a
monoclonal or polyclonal antibody, specific for antigenic polypeptides as
described
above. Such antibodies may be produced by methods which are well known to
those
skilled in the art. The antibodies of this invention can be employed in a
method for the
treatment or prevention ofHelicobacter infection in mammalian hosts, which
comprises
administration of an immunologically effective amount of antibody, specific
for
antigenic polypeptide as described above.
It is proposed that the monoclonal antibodies of the present invention will
find
useful application in standard immunochemical procedures, such as ELISA and
western
blot methods, as well as other procedures which may utilize antibodies
specific to H.
pylori proteins. While ELISAs are preferred, it will be readily appreciated
that such
assays include RIAs and other non-enzyme linked antibody binding assays or
procedures. Additionally, it is proposed that monoclonal antibodies specific
to the
particular H. pylori protein or polypeptides may be utilized in other useful
applications.
For example, their use in immunoadsorbent protocols may be useful in purifying
native
or recombinant H. pylori proteins or variants thereof.
It also is proposed that the disclosed H. pylori polypeptides of the invention
will
find use as antigens for raising antibodies and in immunoassays for the
detection of anti-
75177 kDa antigen-reactive antibodies. In a variation on this embodiment,
samples
suspected of containing H. pylori may be screened, in immunoassay format, for
reactivity against antibodies specific for 75, 77 and 79 kDa polypeptides of
this
invention. Results from such analyses may then be used to determine the
presence of H.
pylori and potential infection.
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Diagnostic immunoassays include direct culturing of bodily fluids or tissue,
either in liquid culture or on a solid support such as nutrient agar. A
typical assay
involves collecting a sample of bodily fluid from a patient and placing the
sample under
conditions optimum for growth of the pathogen. The determination can then be
made as
to whether the microbe exists in the sample. Further analysis can be carried
out to
determine the hemolyzing properties of the microbe.
Immunoassays encompassed by the present invention include, but are not
limited to those described in U.S. Patent No. 4,367,110 (double monoclonal
antibody
sandwich assay) and U.S. Patent No. 4,452,901 (western blot), which U.S.
Patents are
incorporated herein by reference. Other assays include immunoprecipitation of
labeled
ligands and immunocytochemistry, both in vitro and in vivo.
Immunoassays, in their most simple and direct sense, are binding assays.
Certain preferred immunoassays are the various types of enzyme linked
immunosorbent
assays (ELISAs) and radioimmunoassays (RIAs) known in the art.
Immunohistochemical detection using tissue sections is also particularly
useful.
However, it will be readily appreciated that detection is not limited to such
techniques,
and western blotting, dot blotting, FACS analyses, and the like may also be
used.
In one exemplary ELISA, the anti-75/77 kDa antibodies of the invention are
immobilized onto a selected surface exhibiting protein affinity, such as a
well in a
polystyrene microtiter plate. Then, a test composition suspected of containing
the
desired antigen, such as a clinical sample, is added to the wells. After
binding and
washing to remove non-specifically bound immune complexes, the bound antigen
may
be detected. Detection is generally achieved by the addition of another
antibody,
specific for the desired antigen, that is linked to a detectable label. This
type of ELISA
is a simple "sandwich ELISA." Detection may also be achieved by the addition
of a
second antibody specific for the desired antigen, followed by the addition of
a third
antibody that has binding affinity for the second antibody, with the third
antibody being
linked to a detectable label.
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In another exemplary ELISA, the samples suspected of containing an H. pylori
polypeptide are immobilized onto the well surface and then contacted with the
anti-
75/77 kDa antibodies. After binding and appropriate washing, the bound immune
complexes are detected. Where the initial antigen specific antibodies are
linked to a
detectable label, the immune complexes may be detected directly. Again, the
immune
complexes may be detected using a second antibody that has binding affinity
for the first
antigen specific antibody, with the second antibody being linked to a
detectable label.
Further methods include the detection of primary immune complexes by a two
step approach. A second binding ligand, such as an antibody, that has binding
affinity
for the primary antibody is used to form secondary immune complexes, as
described
above. After washing, the secondary immune complexes are contacted with a
third
binding ligand or antibody that has binding affinity for the second antibody,
again under
conditions effective and for a period of time sufficient to allow the
formation of immune
complexes (tertiary immune complexes). The third ligand or antibody is linked
to a
detectable label, allowing detection of the tertiary immune complexes thus
formed. This
system may provide for signal amplification if desired.
Competition ELISAs are also possible in which test samples compete for
binding with known amounts of labeled antigens or antibodies. The amount of
reactive
species in the unknown sample is determined by mixing the sample with the
known
labeled species before or during incubation with coated wells. (Antigen or
antibodies
may also be linked to a solid support, such as in the form of beads, dipstick,
membrane
or column matrix, and the sample to be analyzed applied to the immobilized
antigen or
antibody). The presence of reactive species in the sample acts to reduce the
amount of
labeled species available for binding to the well and thus reduces the
ultimate signal.
Irrespective of the format employed, ELISAs have certain features in common,
such as coating, incubating or binding, washing to remove non-specifically
bound
species, and detecting the bound immune complexes. These are described below.
In coating a plate with either antigen or antibody, one will generally
incubate
the wells of the plate with a solution of the antigen or antibody, either
overnight or for a
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specified period. The wells of the plate will then be washed to remove
incompletely
absorbed material. Any remaining available surfaces of the wells are then
"coated" with
a nonspecific protein that is antigenically neutral with regard to the test
antisera. These
include bovine serum albumin (BSA), casein and solutions of milk powder. The
coating
allows for blocking of nonspecific adsorption sites on the immobilizing
surface and thus
reduces the background caused by nonspecific binding of antisera onto the
surface.
After binding of antigenic material to the well, coating with a non-reactive
material to reduce background, and washing to remove unbound material, the
immobilizing surface is contacted with the antisera or clinical or biological
extract to be
tested in a manner conducive to immune complex (antigen/antibody) formation.
Such
conditions preferably include diluting the antisera with diluents such as BSA,
bovine
gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added
agents also tend to assist in the reduction of nonspecific background. The
layered
antisera is then allowed to incubate for from 2 to 4 hours, at temperatures
preferably on
the order of 25° to 27°C. Following incubation, the antisera-
contacted surface is
washed so as to remove non-immunocomplexed material. A preferred washing
procedure includes washing with a solution such as PBS/Tween, or borate
buffer.
Following formation of specific immunocomplexes between the test sample and
the bound antigen, and subsequent washing, the occurrence and even amount of
immunocomplex formation may be determined by subjecting same to a second
antibody
having specificity for the first. Of course, in that the test sample will
typically be of
human origin, the second antibody will preferably be an antibody having
specificity in
general for human IgG. To provide a detecting means, the second antibody will
preferably have an associated enzyme that will generate a color development
upon
incubating with an appropriate chromogenic substrate. Thus, for example, one
will
desire to contact and incubate the antisera-bound surface with a urease or
peroxidase-
conjugated anti-human IgG for a period of time and under conditions which
favor the
development of immunocomplex formation (e.g., incubation for 2 hours at room
temperature in a PBS-containing solution such as PBS-Tween).
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After incubation with the second enzyme-tagged antibody, and subsequent to
washing to remove unbound material, the amount of label is quantified by
incubation
with a chromogenic substrate such as urea and bromocresol purple or 2.2'-azino-
di-(e-
ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H202, in the case of
peroxidase as the
enzyme label. Quantification is then achieved by measuring the degree of color
generation, e.g., using a visible spectra spectrophotometer. Alternatively,
the label may
be a chemilluminescent one. The use of such labels is described in U.S. Patent
Nos.
5,310,687, 5,238,808 and 5,221,605.
Further embodiments relate to isolated nucleic acid sequences encoding the
polypeptides of this invention, in particular the nucleic acid sequences as
set forth in
SEQ. ID N0.4, 5, and 6, or being substantially similar to all or a portion
thereof. The
term substantially similar means having a least 50-70%, more preferably 70-
80%, and
most preferably 80 or 90% identity to one of said sequences. Such
substantially similar
nucleic acid sequences hybridize under high stringency southern hybridization
conditions with at least one of the nucleic acid sequences set forth in SEQ ID
Nos. 4, 5
or 6.
The nucleic acid molecule may be RNA or DNA, single stranded or double
stranded, in linear or covalently closed circular form. For the purposes of
defining high
stringency southern hybridization conditions , reference can conveniently be
made to
Sambrook et a1..1989, Book 2, pp 9.31-9.58.
It will be appreciated that the sequence of nucleotides of this aspect of the
invention may be obtained from natural, synthetic or semi-synthetic sources;
furthermore, this nucleotide sequence may be a naturally occurring sequence,
or it may
be related by mutation, including single or multiple base substitutions,
deletions,
insertions and inversions, to such a naturally occurring sequence, provided
always that
the nucleic acid molecule comprising such a sequence is capable of being
expressed as a
Helicobacter antigen as broadly described above.
The nucleotide sequence may have expression control sequences positioned
adjacent to it, such control sequences usually being derived from a
heterologous source.
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This invention also provides a recombinant DNA molecule comprising an
expression control sequence having promoter sequences and initiator sequences
and a
nucleotide sequence which codes for a Helicobacter antigen, the nucleotide
sequence
being located 3' to the promoter and initiator sequences. In yet another
aspect, the
invention provides a recombinant DNA cloning vehicle capable of expressing a
Helicobacter antigen comprising an expression control sequence having promoter
sequences and initiator sequences, and a nucleotide sequence which codes for a
Helicobacter antigen, the nucleotide sequence being located 3' to the promoter
and
initiator sequences. Cloning vehicles can be any plasmid (or vector) known in
the art,
including viral vectors, such as alphavirus pox viruses. In a further aspect,
there is
provided a host cell containing a recombinant DNA cloning vehicle andlor a
recombinant DNA molecule as described above.
Suitable expression control sequences and host cell/cloning vehicle
combinations are well known in the art, and are described by way of example,
in
Sambrook et al. (1989). One embodiment of this invention relates to expression
systems
that employ the use of plasmids. Preferred embodiments of the invention employ
plasmids which exhibit high copy number upon replication in a transformed host
cell.
Copy number refers to number of copies of the plasmid, or the genes contained
therein,
which replicate in a host cell upon induction of the plasmid. The copy number
within a
cell determines the copies of the desired nucleotide sequence (or gene). This
copy
number equates to the gene dosage. Certain plasmids are characterized as high
copy
number. Generally, high copy number refers to a plasmid which generates at
least about
100 copies per cell and preferably generating from about 100 to about 700 or
1,000
copies per cell. The selection of the appropriate copy number for the
expression of the
nucleotides sequences of this invention when combined with the selection of a
strong
promoter improves the amount of desired polypeptide that is generated in a
host cell. In
each high copy number plasmid, the nucleic acid sequence encoding a selected
polypeptide is operably linked to a strong promoter (Hannig et al.). Plasmid
copy
number is determined by the nature the origin of replication of the plasmid
and
corresponding cis acting control elements, together these genetic elements are
defined as
a replicon. In plasmids which normally reside in E. coli, there are several
different
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replicons. Most common is pMBI and its close relative, ColEl. Plasmids which
carry
these replicons maintain about 15-20 copies per cell under normal growth
conditions. A
small protein encoded by the rop gene adjacent to the origin of replication
negatively
regulates pMBI/ColEl plasmid replication. Plasmids with deletions in the rop
gene or
mutations in the site of rop action have increased replication and higher copy
number.
One example of this is in the pUC plasmids whose copy number is increased to
500-700
copies per cell. Other lower copy number replicons are plSA (10-12
copies/cell), and
pSC101 (5-10 copies per cell). Also, the moderate copy number of pMBI and
ColEl
plasmids can be modified to high copy numbers by inhibiting the E. coli
protein
synthesis.
It is further noted that the copy number exhibited in a specific host is the
proper
measurement of plasmid copy number. Plasmids which normally do not reside in
gram
negative hosts or E. coli hosts may not be regulated for replication when
introduced into
E. coli hosts. When such promoters are not regulated, they can exhibit high
copy
number in the selected host. An example of this is pNG2, a plasmid from the
gram
positive bacteria Corynebacterium diphtheriae. Whereas, the normal copy number
for
this plasmid in C. dipththeriae is 1-2 copies per cell, transformants of the
plasmid in E.
coli are estimated to have >100 copies of the plasmid (Serwold-Davis, T.et
aI.,PNAS,
84: 4964-8, 1987).
Strong promoters are selected such they are easily regulated in order that
they
may be repressed during culture growth "towards maximal cell numbers. In many
embodiments, the strong promoters can also be induced so that the host cell
overproduces the recombinant polypeptide at a desired level, usually in excess
of about
10 to about 30% total cellular protein. The promoter is selected to maintain
the desired
level of polypeptide expression. Exemplary promoters are T7 promoter from T7
bacteriophage, arabinose promoter for the araBAD operon, lambda phage
promoters
(such as PL and PR) the trc and tac promoters. The T7 promoter is controlled
by the T7
RNA polymerase gene which is a very active enzyme; it elongates RNA chains
five
times faster than the E. coli RNA polymerase. In addition the polymerase is
very
selective for specific promoter sequences and termination signals so that the
action of
the enzyme is targeted specifically to the gene of interest. In one system
developed by
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Studier et al. (referenced, infra), the T7 polymerise gene has been integrated
into the
host chromosome under the control of the lac promoter. Upon induction with
IPTG, T7
RNA polymerise is produced and acts to transcribe a T7 promoter gene housed on
an
expression plasmid in the cell. In addition, a second plasmid expressing T7
lysozyme is
included to control the background expression of the T7 polymerise. For T7, an
alternative induction system for the expression of genes by the T7 promoter is
to grow
E. coli cells containing the T7 recombinant plasmid to the desired density and
then
introduce the T7 polymerise gene by infecting with a bacteriophage which
carries that
gene.
Expression can also be enhanced based on the choice of cellular
compartmentalization. Outer membrane proteins are often difficult to
overexpress
recombinantly because of the requirement for transport to the outer membrane
and
correct insertion into that membrane. Overexpression of full length outer
membrane
proteins which contain the leader sequences will many times overcome the host
cell
export machinery leading to cessation of growth and low recombinant protein
yield.
Cloning the mature sequences of an outer membrane protein behind translation
start
signals provided by the expression vector can eliminate the need for the host
cell to
transport the protein to the membrane and allow the cell to overexpress the
recombinant
protein as inclusion bodies in the cytosol which are relatively stable and
resistant to
proteolysis.
One or more of the above considerations are included when selecting a desired
host. In general, one can select or optimize the host based on factors which
can
influence the yield of recombinantly expressed proteins. These factors include
growth
and induction conditions, mRNA stability, codon usage, translational
efficiency and the
presence of transcriptional terminators to minimize promoter read through. For
example, one can modify the ability of the host to produce proteases. Stress
induced
proteases in the host cell are induced during induction of recombinant protein
expression, therefore, the use of protease deficient hosts and/or coexpression
of
chaperones can be employed to minimize the proteolysis of the recombinant upon
induction of the protein in culture. In the case at hand, the polypeptides of
the invention
contain cysteine bonds. Accordingly, one can aid cysteine bond formation, for
example
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through the use of trx hosts, which can help with the proper folding in the
cytosol by
altering the redox potential in the cytosol.
Suitable host cell or host strains for the practice of this invention are omp
T lon
host strains such as BL21, BLR, B834; trxB host strains such as AD494, lon
clpA
mutant strains such as KY 42263 and lon clpA hsl i~U mutant strains such as KY
2266
(for the latter two strains the Kanemori, M. et al. J. Bact. 1997. 79:7219-
7225).
When host cells, which are modified or selected for enhanced expression, are
combined with the high copy number plasmids (also referred to as over-
expressing
plasmids) of this invention, the polypeptide is expressed as inclusion bodies.
Preferably,
the nucleotide sequence selected for forming inclusion bodies is the
nucleotide sequence
corresponding to the mature portion of the polypeptide. The signal sequence of
the
desired polypeptide is not included in this nucleotide sequence for the mature
polypeptide. A desired H. pylori protein can be obtained by a method
comprising: (a)
transforming a selected host cell with at least one high copy number plasmid,
which
comprises the nucleotide sequence of interest operably linked to a strong
promoter, and
(b) growing the transformed host cell in culture media. Often, it is
advantageous to use
an inducible promoter to control the timing of the expression of the
nucleotide sequence.
Basically, when the promoter is inducible, the rate of transcription increases
in response
to the inducing agent or inducing conditions for the promoter. A selectable
marker can
used in the plasmid in order to grow the transformed host in the presence of a
selecting
agent that works in combination with the chosen marker. The H. pylori
polypeptide
produced by the above method is expressed as inclusion bodies and is also
soluble in
detergent extractions, without the requiring denaturants, such as urea or
guanidine for
solubilizing the inclusion bodies. These two characteristics are important
since they
allow for the application of straightforward and practical means in isolating
and
purifying the polypeptide. In the absence of expressing the polypeptide as an
inclusion
body and as a soluble material, the difficulties in the purification steps are
generally
overwhelming.
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In yet further aspects, there are provided fused polypeptides comprising a
Helicobacter polypeptide of this invention and an additional polypeptide, for
example a
polypeptide coded for by the DNA of a cloning vehicle, fused thereto. Such a
fused
polypeptide can be produced by a host cell transformed or infected with a
recombinant
DNA cloning vehicle as described above and it can be subsequently isolated
from the
host cell to provide the fused polypeptide substantially free of other host
cell proteins.
Based on the above-identified specific sequences, one may obtain numerous
additional isolated nucleic acid sequences encoding the polypeptides of this
invention
due to the degeneracy of the genetic code. Amino acids and their codons are
well-
known. Accordingly, using site-directed mutagenesis of one polypeptide of H.
pylori,
one can generate additional nucleic acid sequences, as desired. These methods
of
generating nucleic acid sequences and fragments thereof provide a convenient
manner in
which to generate portions of the polypeptides for fusion molecules.
Using the nucleic acid sequence described herein, one can generate synthetic
polypeptides displaying the antigenicity of a Helicobacter isolated
polypeptide of this
invention. As used herein, the term "synthetic" means that the polypeptides
have been
produced by chemical or biological means, such as by means of chemical
synthesis or
by recombinant DNA techniques leading to biological synthesis. Such
polypeptides
can, of course, be obtained by cleavage of a fused polypeptide as described
above and
separation of the desired polypeptide from the additional polypeptide coded
for by the
DNA of the cloning vehicle by methods well known in the art. Alternatively,
once the
amino acid sequence of the desired polypeptide has been established, for
example, by
determination of the nucleotide sequence coding for the desired polypeptide,
the
polypeptide may be produced synthetically, for example by the well known
Merrifield
solid-phase synthesis procedure.
Once recombinant DNA cloning vehicles and/or host cells expressing a desired
Helicobacter polypeptide of this invention have been constructed by
transforming or
transfecting such cloning vehicles or host cells with plasmids containing the
corresponding Helicobacter nucleic acid sequence, cloning vehicles or host
cells are
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cultured under conditions such that the polypeptides are expressed. The
polypeptide is
then isolated substantially free of contaminating host cell components by
techniques
well known to those skilled in the art. In a preferred embodiment for
purifying the
desired polypeptide one can first follow the standard techniques of lysing the
host cells
and then isolating the inclusion bodies by removing soluble proteins and other
contaminants potentially. In a second step, it is preferred to solubilize the
inclusion
bodies in a zwitterionic detergent, which are well-known in the art. The
detergent may
be used with a denaturant; however, surprisingly, no denaturant is required.
Finally, the
solubilized inclusion body material, is purified using cationic exchange gel
chromatography, followed by eluting with a salt solution to collect the
purified
polypeptide. This method generates a polypeptide which is at least about 75 or
80%
pure, preferably at least about 90%.
This invention also provides for a method of diagnosing an H. pylori infection
comprising the step of determining the presence, in a sample, of an amino acid
sequence
EDDGFYTSVGYQIGEAAQMV (SEQ ID No.: 7), or preferably any of the isolated H.
pylori polypeptides of this invention. Any conventional diagnostic method may
be
used. These diagnostic methods can easily be based on the presence of an amino
acid
sequence or polypeptide. Preferably, such a diagnostic method matches for a
polypeptide having at least 10, and preferably at least 20, amino acids which
are
common to the polypeptides of this invention.
As noted, the present invention also relates to nucleic acid sequences
encoding
H. pylori polypeptides. The nucleic acid sequences disclosed herein can also
be used for
a variety of diagnostic applications. These nucleic acids sequences can be
used to
prepare relatively short DNA and RNA sequences that have the ability to
specifically
hybridize to the nucleic acid sequences encoding the polypeptides of this
invention.
Nucleic acid probes are selected for the desired length in view of the
selected
parameters of specificity of the diagnostic assay. The probes can be used in
diagnostic
assays for detecting the presence of pathogenic organisms in a given sample.
With
current advanced technologies for recombinant expression, nucleic acid
sequences can
be inserted into an expression construct for the purpose of screening the
corresponding
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oligopeptides and polypeptides for reactivity with existing antibodies or for
the ability to
generate diagnostic or therapeutic reagents.
In preferred embodiments, the nucleic acid sequences employed for
hybridization studies or assays include sequences that are complementary to a
nucleotide stretch of at least about 10 to about 20 nucleotides, although at
least about 10
to 30, or about 30 to 60 nucleotides can be used. Nucleotide stretches of at
least 10
nucleotides are beneficial for providing stability and selectivity when
testing a clinical
sample for Helicobacter infection. A variety of known hybridization techniques
and
systems can be employed for practice of the hybridization aspects of this
invention,
including diagnostic assays such as those described in Falkow et al., US
Patent
4,358,535. Depending on the application, one will desire to employ varying
conditions
of hybridization to achieve varying degrees of selectivity of the probe toward
a target
sequence. For applications requiring a high degree of selectivity, one will
select
relatively low salt and/or high temperature conditions, such as provided by
0.02M-
O.15M NaCI at temperature of about 50°C to 70°C. These
conditions are particularly
selective, and tolerate little, if any, mismatch between the probe and the
template or
target strand.
For some applications, if one desires to prepare mutants employing a mutant
primer strand hybridized to an underlying template, less stringent
hybridization
conditions are called for in order to allow formation of the heteroduplex. The
conditions
may be altered by using O.I SM-0.9M salt, at temperatures ranging from about
20°C to
about 55°C. In general, it is appreciated that conditions can be
rendered more stringent
by the addition of increasing amounts of formamide, which serves to
destabilize the
hybrid duplex in the same manner as increased temperature. Thus, hybridization
conditions can be readily manipulated, and the method of choice will generally
depend
on the desired results.
In certain embodiments, one may desire to employ nucleic acid probes to
isolate
variants from clone banks containing mutated clones. In particular
embodiments,
mutant clone colonies growing on solid media which contain variants of an H.
pylori
polypeptide sequence could be identified on duplicate filters using
hybridization
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conditions and methods, such as those used in colony blot assays, to obtain
hybridization only between probes containing sequence variants and nucleic
acid
sequence variants contained in specific colonies. In this manner, small
hybridization
probes containing short variant sequences of the H. pylori genes of the
invention may be
utilized to identify those clones growing on solid media which contain
sequence variants
of the entire genes encoding polypeptides of 75, 77 and 79 kDa as discussed
herein.
These clones can then be grown to obtain desired quantities of the variant
nucleic acid
sequences or the corresponding antigen.
In clinical diagnostic embodiments, nucleic acid sequences of the present
invention are used in combination with an appropriate means, such as a label,
for
determining hybridization. A wide variety of appropriate indicator means are
known in
the art, including radioactive, enzymatic or other ligands, such as
avidin/biotin, which
are capable of giving a detectable signal. In preferred diagnostic
embodiments, one will
likely desire to employ an enzyme tag such as urease, alkaline phosphatase or
peroxidase, instead of radioactive or other environmental undesirable
reagents. In the
case of enzyme tags, colorimetric indicator substrates are known which can be
employed to provide a means visible to the human eye or
spectrophotometrically, to
identify specific hybridization with pathogen nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein
will be
useful both as reagents in solution hybridizations as well as in embodiments
employing
a solid phase. In embodiments involving a solid phase, the test DNA (or RNA)
from
suspected clinical samples, such as exudates, body fluids (e.g., amniotic
fluid, middle
ear effusion, bronchoalveolar lavage fluid) or even tissues, is absorbed or
otherwise
affxed to a selected matrix or surface. This fixed, single-stranded nucleic
acid is then
subjected to specific hybridization with selected probes under desired
conditions. The
selected conditions will depend on the particular circumstances based on the
particular
criteria required (depending, for example, on the G+C contents, type of target
nucleic
acid, source of nucleic acid, size of hybridization probe, et.). Following
washing of the
hybridized surface so as to remove nonspecifically bound probe molecules,
specific
hybridization is detected, or even quantified, by means of the label.
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The nucleic acid sequences which encode for the H. pylori polypeptides of the
invention, or their variants, may be useful in conjunction with PCRTM
technology as set
out, e.g., in U.S. Patent 4,603,102, one may utilize various portions of any
of H. pylori
sequences of this invention as oligonucleotide probes for the PCRTM
amplification of a
defined portion of an H. pylori (75, 77 kDa) sequence may then be detected by
hybridization with a hybridization probe containing a complementary sequence.
In this
manner, extremely small concentrations of H. pylori nucleic acid may be
detected in a
sample utilizing the nucleic acid sequences of this invention.
The following examples are included to demonstrate preferred embodiments of
the invention. It should be appreciated by those skilled in the art that the
techniques
disclosed in the examples which follow represent techniques discovered by the
inventors
to function well in the practice of the invention, and thus can be considered
to constitute
preferred modes for its practice. However, those of skill in the art should,
in the light of
the present disclosure, appreciate that many changes can be made in the
specific
embodiments which are disclosed and still obtain a like or similar result
without
departing from the spirit and scope of the invention.
EXAMPLES
Example 1
1.1 Bacterial strains. H. pylori strains PBCC 1101, PBCC 1102, PBCC 1103,
PBCC 1105, and PBCC 1107 were isolated from human gastric biopsies obtained
from
the University of Rochester School of Medicine and Dentistry (Rochester, NY).
H.
pylori strains LET 13 and RSD 14 were isolated from human gastric biopsies
obtained
from the Syracuse Veterans Administration Medical Center (Syracuse, NY). H.
pylori
strains MH 60 EG 52, RJ 17, LJ 63, and MJ 34 were obtained as frozen stocks
from the
Clement J. Zablocki VA Medical Center (Milwaukee, WI). H. pylori strain SS1
was
originally isolated from a human gastric biopsy, and subsequently adapted to
infect mice
(obtained from A. Lee, University of New South Wales, Sydney, Australia). H.
pylori
strains obtained from American Type Culture Collection were ATCC 43504 and
ATCC
43579. The H. fells strain was obtained from T. Blanchard, Case Western
Reserve
University, Cleveland, OH.
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1.2 Culturing of Helicobacter strains. Cultures of H. pylori and H. fells were
grown at 37°C on Columbia broth agar plates with 10% defibrinated horse
blood and 10
pg/ml vancomycin in a microaerophilic chamber. Liquid cultures of H. pylori
strains
were grown at 37°C in BI-II medium with 4% fetal calf serum and 10 p,g
/ml
vancomycin in flasks infused with a gas mixture of 10% C02/ 6% Oz/ 84% N2
(vol/vol/vol).
1.3 Preparation of H. pylori PBCC 1103. Strain PBCC 1103 was grown as above.
The cell pellet was washed twice in a phosphate buffered saline (PBS)
solution, and
resuspended in a 0.3% formaldehyde solution for 1 hr. The fixed cells were
then
washed in and resuspended in PBS to an O.D.6oo of 0.1 (approximately 10~ cfu).
1.4 Injection of Mice with H. pylori PBCC 1103. Ten 6 to 8 wk old female
BALB/c mice were primed by interperitoneal injection with ca. 10~ formalin
fixed
PBCC 1103 cells at weeks 0, and boosted at weeks 2, 4 and 8. After a 34 week
rest
period a pre-fusion boost of approximately 10~ formalin fixed PBCC 1103 was
given
interperioneally at week 42.
1.5 Production of Monoclonal Antibodies, Hybridoma Techniques and
Screening Procedures. During immunization and the rest period, mouse sera were
obtained (week 6, 10) and tested for antibody activity by ELISA by using air-
dried,
formalin fixed PBCC 1103 cells, 0.1 ml per well at an absorbance of 0.100
(A62o) as the
coating antigen.
Spleens were recovered from five immunized mice about 72 hours after the last
injection, and were combined with nonsecreting X63Ag8.b53 mouse (BALB/c)
myeloma cells in 5:1 ratio (splenocytes:myeloma). The cells were fused for
four
minutes in 50% (v/v) polyethylene glycol 1500 and 10% dimethylsulfoxide in
Dulbecco's Modified Eagle medium (D-MEM). The fused cells were diluted in
selection medium, D-MEM supplemented with hypoxanthine, aminopterin,
thymidine,
10% fetal bovine serum and 10% NCTC-109 media supplement (Gibco-BRL). The
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fusion efficiency (wells with colony growth vs. number of wells seeded) was
100%
(900/900).
Primary screening was completed by ELISAs using PBCC 1103 whole cell
antigen as described above, partially purified protein mixture with urease and
heat shock
proteins) and lipopolysaccharide (LPS) purified from PBCC 1103 cells.
Secondary
selection was completed by SDS-PAGE Western blot using heterologous ATCC 43579
whole cell lysate as antigen. Positive reactors were identified and designated
1 through
90, coded as hybridoma (Hpy) and saved for further characterization.
Selected hybridomas of interest were subcloned once by limiting dilution
procedure. Monoclonal antibodies were provided as tissue culture supernatant
(TCS),
concentrated by 50% saturated ammonium sulfate precipitation (SAS-TCS) or
ascites.
1.6 SDS-PAGE and Western Biot Analysis.
SDS-PAGE was carried out as described by Laemmli using 10-18% (w/v)
acrylamide gels (Zaxis, Hudson, OH). Proteins were visualized by staining the
gels with Coomassie ProBlue (Owl Separation Systems, Woburn, MA). Gels
were scanned using a Personal Densitometer SI (Molecular Dynamics Inc.,
Sunnyvale, CA) and molecular weights estimated using the Fragment Analysis
software (version 1.1) and prestained molecular weight markers obtained from
Owl Separation Systems. Transfer of proteins to polyvinylidene difluoride
(PVDF) membranes was accomplished using a semi-dry electroblotter and
electroblot buffers (Owl Separation Systems). Membranes were probed with the
indicated antisera followed by goat anti-mouse or anti-rabbit alkaline
phosphatase
conjugate as the secondary antibody (BioSource International, Camarillo, CA).
Western blots were developed using the BCIP/NBT Phosphatase Substrate
System from Kirkegaard and Perry Laboratories (Gaithersburg, MD).
1.7 Conservation of the 75 and 77 kDa Proteins in H. pylori Strains.
Tissue culture supernatants from hybridomas were analyzed by whole cell ELISA
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and antigen ELISA, followed by Western analysis using whole cell lysates of a
number of H. pylori strains. Parent hybridoma designated Hpy 64 was
determined by these methods to react to proteins in the range of approximately
75-79 kDa. Subsequent cloning produced the monoclonal antibody (MAb)
designated Hpy 64-27, which has reactive epitopes in 10 of 12 H. pylori
strains
tested, but not in H. fells (Table 1 ).
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Table 1
Reactivity of MAb Hpy64-27 with Heterologous H. pylori
Strains and an H. fells strain (Conservation of 75/77 kDa Proteins)
Strain Reactivi Strain Reactivi
ATCC 43579++ EG52 ++
PBCC 1101 ++ X17 _
PBCC 1102 +/- LJ63 +/-
PBCC 1103*++ MJ34 +
PBCC 1105 ++ PBCC 1107 ++
MH60 ++ H. fells*
SS1 -
* -homologous isolate ** non pylori
++ : strongly reactive + : reactive
+/- : slightly reactive : no detectable reactivity
1.8 Purification of the 75 and 77 kDa Proteins. Bacterial cells (ca. 10 g wet
wt of H. pylori ATCC 43579) were resuspended in 70 ml of 0.05 M HEPES / 10
mM EDTA / 1.0 mM PMSF (pH 7.0) by homogenization using a Tekmar Ultra-
Turrex tissue homogenizer. The cells were disrupted by sonication using a
Branson Sonifier Cell Disrupter. The disrupted cells, including the membrane
fraction, were pelleted by centrifugation at 42,000 rpm using a Beckman 70Ti
rotor for 40 min at 4°C. Following centrifugation, the pellet was
resuspended in
70 ml of 0.01 M HEPES / 1.0 mM MgCl2 / 1.0 mM PMSF / 1.0% TX-100 (pH
7.4) and stirred for 1 hr at room temp. The suspension was centrifuged at
42,000
rpm using a Beckman 70Ti rotor for 40 min at 4°C. Following
centrifugation, the
pellet was resuspended in 70 ml of 0.05 M Tris-HCl / 10.0 mM EDTA / 1.0%
ZWITTERGENTTM3-14 (pH 7.4) and stirred for 1 hr at room temp. The
suspension was then centrifuged at 42,000 rpm using a Beckman 70Ti rotor for
40 min at 4°C. Following centrifugation, the supernatant containing the
75 and
77 kDa proteins was collected and stored at -20°C for further
purification.
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The ZWITTERGENT~' 3-14 crude extract was buffer exchanged by passage
over a 250 ml Sephadex G-25 (coarse) column (Pharmacia) equilibrated in 0.02 M
Tris-
HCI / 5.0 mM EDTA / 1.0% ZWITTERGENTTM3-14 (pH 8.0). Approximately one half
(35 ml) of the buffer exchanged ZWITTERGENT"" 3-14 crude extract preparation
was
loaded onto a 10 ml Pharmacia SP Fast Flow Sepharose column equilibrated 0.02
M
Tris-HCl / 5.0 mM EDTA / 1.0% ZWITTERGENT~" 3-14 (pH 8.0). Unbound protein
was washed through the column with an additional 2 bed volumes of
equilibration
buffer. The 75 and 77 kDa proteins were co-eluted using a Linear NaCI gradient
(0-15 M
NaCI) in 0.02 M Tris-HCI l 5.0 mM EDTA / 1.0% ZWITTERGENTTM 3-14 (pH 8.0).
Fractions were screened for the 75 and 77 kDa proteins by SDS-PAGE / Western
and
pooled. Twelve ml of pooled co-eluting 75 and 77 kDa was subsequently applied
to a
500 ml Pharmacia Superose-12 column equilibrated in phosphate buffered saline
(PBS)
/ 1.0% ZWITTERGENTTM 3-14. Fractions were screened for the co-eluting 75 and
77
kDa proteins by SDS-PAGE / Western and pooled. Material from two independent
SP
Sepharose / Superose-12 runs was combined and the co-eluted protein was
precipitated
by the addition of 9 volumes of ethanol overnight at -20°C. The
resulting suspension
was then centrifuged at 9,000 rpm using a Beckman SS31 rotor for 30 min at
4°C and
the pellet was resuspended in 10 ml of PBS / 1.0% ZWITTERGENTTM 3-14. Purified
75 and 77 kDa proteins were stored at -20°C. The protein isolated using
this protocol
exhibited two bands on SDS-PAGE corresponding to subunit molecular masses of
75
kDa and 77 kDa (Figure 1 A).
Protein Estimation. Protein concentration was estimated by the BCA assay
from Pierce (Rockford, IL) using BSA as a standard.
1.9 Reactivity of MAb 64-27 clone with 75 kDa and 77 kDa. Western blot
analysis of co-purified 75 kDa and 77 kDa proteins revealed that only the
75kDa
protein reacts strongly with the MAb 64-27 (Figure 1B).
Example 2
2.1 Production in Mice of Polyclonal Antisera to Purified 75 lcDa and 77 kDa
Proteins. Two groups of 10 Swiss Webster mice were used for production of
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polyclonal antisera to the purified 75 kDa and 77 kDa proteins. One group was
injected
with 25 pg of the purified protein with incomplete Freunds adjuvant (IFA) on
weeks 0
and 4. The other group was injected with 25 ~tg of the purified proteins with
SO ~g of
MPLTM on weeks 0 and 4. Both groups were bled on week 6.
2.2 Surface Labeling by Immunoelectron Microscopy (IEM). Twenty microliter
droplets of live PBCC 1105 in broth were placed on parafilm. Three Hundred
mesh
gold carbon-coated formvar grids were placed film side down on the droplets.
Grids
with cells were then rinsed in droplets of PBSBSA buffer and transferred to
PBS
containing I% cold water fish gelatin. After blocking, grids were incubated in
mouse
polyclonal antisera diluted 1:50 in PBSBSA for I hr, then rinsed in buffer.
Grids were
then incubated on a fifty-fold dilution ofNanogold (Nanoprobes, Inc., Stony
Brook,
NY), washed in buffer, and fixed with a solution of 1 % gluteraldehyde in PBS.
The
fixative was removed from the grids with deionized water. The HQ silver
enhancement
kit (Nanoprobes, Inc.) was used to nucleate the Nanogold. The grids were then
stained
with Nanovan (Nanoprobes, Inc.) and viewed on a Zeiss I OC transmission
electron
microscope operating at 100 Kv. The polyclonal antiserum obtained from
injecting
mice with live strain PBCC 1103 was able to label the surface of heterologous
strain
PBCC 1105. As seen in Figure 2, the 75/77 kDa proteins are surface localized
and that
the reactive epitopes are present in both H. pylori strains.
2.3 Flow Cytometry. Approximately 106-10' cells of PBCC 1105 (O.D.6oo range
of 0.2 to 1.6) were washed once in PCM buffer ( 10 mM NaP04, pH 7.4, 150 mM
NaCI,
0.5 mM MgClz, 0.15 mM CaCl2). The cell pellet was gently resuspended in 100 ml
PCM buffer. One microliter of polyclonal antisera was added and the cells
incubated at
°
37 C for 30 min on a rocker. Cells were diluted with 900 ~l PCM buffer, mixed
gently
and pelleted at maximum speed in a microcentrifuge for 1 min. The pellet was
gently
resuspended in 100 p,l of PCM buffer. One microliter of Oregon Green 514
conjugated
goat anti-mouse antibody (Molecular Probes) was used to fluorescently label
the cells
tagged with the polyclonal antisera at 37°C for 30 min. The cells were
then washed as
described above and diluted in 1 ml PCM for analysis on the FACSort (Becton- ,
Dickenson). Sera obtained from mice prior to injection with the mixture of the
75 kDa
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and 77 kDa proteins on day 0 showed only background labeling in all five
strains tested,
with a mean fluorescent intensity of less than 10 (see Figure 3A). Polyclonal
antisera
obtained from the same mice on day 49 following two injections of the protein
mixture
displayed a positive fluorescent peak with all strains tested (strains ATCC
43579, PBCC
1105, ATCC 43504 and SS-I : See Figure 3B). The mean fluorescent intensity
ranged
from 36.44 for SSI to 322.68 from the homologous strain ATCC 43579. This
demonstrates that there are conserved epitopes among these strains. Without
being
bound by theory, the difference in the fluorescent intensities could be a
function of the
number of antigen molecules expressed on the cell surface or the antigenic
variability of
the epitopes among the heterologous strains.
Example 3
3.1 Assay of Bactericidal (BC) Activity of Polyclonal Antisera. Target cells
of
H. pylori strain PBCC 1105 for the BC assay were provided on the morning of
the
assay. The optical density of the liquid culture at 600 nm was approximately
0.1. Cells
pelleted from 10 ml of this liquid culture were used to adsorb any nonspecific
bactericidal activity in the complement source (human complement). Cells and
serum
were allowed to react for at least 1 hour on ice with occasional agitation.
After
incubation, the serum and cell mixture was centrifuged, and the serum was
removed and
placed on ice until needed. The BC assay was performed when the liquid culture
attained an O.D.6oo of approximately 0.3. These cells were diluted in PCM
buffer to a
concentration of 106 cfu per ml . The reaction consisted of 10 ~,1 strain PBCC
1105 at a
concentration of 106 cfu per ml, 10 ~,1 adsorbed serum as a complement source,
S ~,1 of
diluted polyclonal antisera (heat inactivated 60°C for 10 min), and 25
Pl of PCM. The
reaction was incubated at 36°C in a 10% C02 chamber for 30 min. Two
hundred P.1 of
PCM was then added to the reaction and duplicate SO ~,l aliquots were plated
on
Columbia agar with 10% defibrinated horse blood and 10 ~,g/ml vancomycin.
Plates
were incubated in a microaerophilic chamber for at least 72 hours, and counted
on an
automated plate reader. Polyclonal antisera from mice injected with the 75/77
kDa
protein mixed with Incomplete Freunds Adjuvant (IFA) had a BCso at a dilution
of
greater than 1/3200 and 75/77 kDa protein mixed with MPLT"' had a BCso at a
dilution
of greater than 1/3200 , as compared to 1/800 in serum from control mice (see
Figure 4,
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complete data for MPL is not shown). This is another indication that the
epitopes of the
75/77 kDa proteins are surface exposed.
Example 4 - Iramunization and Challenge Tests
4.1 Immunizations. C57BI/6 mice were vaccinated intragastrically on days 0, 2,
14, and 16 with 100 p.g recombinant H. pylori (Hp) urease (rUre) or native Hp
75/77
kDa protein formulated with 10 ~g cholera toxin (CT); or were vaccinated
subcutaneously on days 0 and 16 with 10 ~.g recombinant Hp rUre or native Hp
75/77
kDa protein formulated with 100 p,g Aluminum Phosphate (AIP04). Control mice
received no vaccine.
4.2 Reduction of Colonization in the Mouse Model. Infection was established by
the intragastric deposition of one 0.1 ml inoculum of H. pylori strain SS 1 on
day 37.
The challenge inocula contained a suspension of H. pylori at a concentration
of 2 x 10g
cfu per ml. The amount of viable H. pylori in the stomachs of mice was
determined 2, 4
and 8 weeks following challenge (days 50 and 64). At each time point, 5 mice
were
sacrificed by cervical dislocation, their stomachs harvested and split into 2
longitudinal
sections. One section from each mouse was homogenized and the homogenate
diluted
and plated on Columbia agar containing defibrinated horse serum and the
appropriate
antibiotics. Plates were incubated at 37 C in a microaerophilic incubator for
5 days.
After incubation the number of viable H pylori isolated were enumerated. Total
cfu per
gram of stomach tissue were obtained by multiplying colony numbers per plate
by the
dilution factor and dividing by the weight of the sectioned stomach; numbers
were
transformed and are expressed as logo cfu per gram homogenized stomach tissue
+ 1
standard error of the mean. Mice which were vaccinated intragastrically with
the 75/77
kDa protein mixture admixed with CT showed a significant reduction in
colonization
following challenge with Hp strain SS 1, as compared to non-vaccinated
controls. At
four weeks post-challenge, using either oral or parenteral routes of
immunization, the
number of colony forming units recovered per gram of stomach tissue was
reduced by
approximately 1-2 logs in vaccinated mice (see Figure 5). This result is
comparable to,
if not improved over, the reduction that is afforded by vaccination with
recombinant
urease admixed with CT.
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Example 5 - Protein Analysis
5.1 Enzymatic Cleavage of the 75 IcDa and 77 lr,Da Proteins.
(i) Trypsin cleavage. Approximately 0.5 mg of the 75 kDa and 77 kDa protein
mixture was precipitated with 90% (v/v) ethanol and the pellet resuspended in
a total
volume of 1 ml of phosphate-buffered saline (PBS) / 0.1% ZWITTERGENTTM 3-14.
Ten microliters of a 0.5 mg/ml solution of trypsin was added (Boehringer
Mannheim,
Indianapolis, IN), and the reaction mixture incubated for 4 hr at
37°C.
(ii) Elastase cleavage. Approximately 0.5 mg of the 75 kDa and 77 kDa protein
mixture was precipitated with 90% {v/v) ethanol and the pellet resuspended in
a total
volume of 1.0 ml of PBS / 0.1% ZWITTERGENT 3-14. Five microliters of a 1.0
mg/ml
solution of elastase was added (Worthington), and the reaction mixture
incubated for 4
hr at 37°C.
(iii) Endoproteinase Lys-C cleavage. Approximately 0.5 mg of the 75 kDa
and 77 kDa protein mixture was precipitated with 90% (v/v) ethanol and the
pellet
resuspended in a total volume of 1.0 ml of PBS / 0.1 % ZWITTERGENT 3-14. This
preparation was added directly to a vial containing 5 mg of endoproteinase Lys-
C
(Boehringer Mannheim). The reaction mixture was incubated for 4 hr at
37°C.
(iv) Separation of peptides. The cleavage reaction mixtures from (i) -(iii)
above were centrifuged in an Eppendorf centrifuge at 12,000 rpm for 5 min and
the
supernatant loaded directly onto a Vydac Protein C4 HPLC column (The
Separations
Group, Hesperia, CA). The solvent system used consisted of 0.1% (v/v) aqueous
trifluoroacetic acid (TFA), (Solvent A) and acetonitrile : H20 : TFA,
80:20:0.1 (v/v/v)
(Solvent B) at a flow rate of 1.0 ml/min. Following initial wash with Solvent
A, the
peptides were eluted with a linear gradient from 0-100% Solvent B in 30 min
and
detected by absorbance at 220 nm. Suitable fractions were collected, dried
down in a
Speed-Vac concentrator (Jouan Inc., Winchester, VA) and subsequently
resuspended in
distilled water. The fractions were subjected to SDS-PAGE using 10-18% (w/v,
acrylamide) gradient gels (Owl Separation Systems) in a Tris-Tricine buffer
system
(Schogger and von Jagow). The fractions exhibiting a single peptide band were
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submitted for N-terminal sequence analysis. Fractions displaying multiple
peptide
bands in SDS-PAGE were electrophoretically transferred onto a PVDF membrane as
described above. The membrane was stained with Coomassie Brilliant Blue R-250
and
individual bands excised and submitted for N-terminal sequence analysis
(Matsudaira),
as discussed in Example 6 below.
5.2 Estimation of Molecular Weight By MALDI-TOF Mass Spectrometry.
Determination of molecular weight by Matrix Assisted Laser Desorption /
Ionization -
Time of Flight (MALDI-TOF) mass spectrometry (Hillenkamp and Karas) was
carried
out using a Lasermat 2000 Mass Analyzer (Finnigan Mat, Hemel Hempstead, UK)
with
3,5-dimethoxy-4-hydroxy-cinnamic acid as the matrix. For samples containing
1.0%
ZWITTERGENTT"' 3-14, cold ethanol precipitation was carried out twice to
remove the
detergent using a 90% (v/v) final ethanol concentration followed by
solubilization of the
precipitated protein in water.. MALDI-TOF analysis using 3,5-dimethoxy-4-
hydroxy-
cinnamic acid matrix in presence of 70% (v/v) aqueous acetonitrile / 0.1% TFA
resulted
in the identification of predominantly two species with average molecular
weights of
75,572 kDa and 77,633 kDa.
Examples 6 - N-terminal Sequence Analysis
6.1 Amino Acid Sequence Analysis. N-terminal sequence analysis was carried out
using an Applied Biosystems Model 477A Protein/Peptide Sequencer equipped with
an
on-line Model 120A PTH Analyzer (Applied Biosystems, Foster City, CA). The
phenylthiohydantoin (PTH) derivatives were identified by reversed-phase HPLC
using a
Brownlee PTH C-18 column (particle size 5 mm, 2.1 mm i.d. x 22 cm 1.; Applied
Biosystems).
6.2 N-terminal Sequence Analysis of Both Intact 75 lcDa and 77 kDa Proteins
and Internal Peptides. Determination of the N-terminal sequence of both 75 kDa
and
77 kDa from intact proteins blotted onto PVDF exhibited identical sequence
through the
first 20 amino acids, EDDGFYTSVGYQIGEAAQMV (SEQ. ID No.: 7) of the mature
processed form of these proteins, i.e. after cleavage of their signal
peptides. This
sequence was shown to match the N-terminus of three translated gene sequences
identified from the H. pylori genome (Note: the third gene corresponds to a 79
kDa H.
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pylori genome (Note: the third gene corresponds to a 79 kDa H. pylori protein
as noted
in Example 7). In order to determine which of the three genes encode the 75
kDa and
77 kDa proteins, internal peptide fragments were generated from the mixture
and
subjected to N-terminal sequence analysis. Table 2 shows the N-terminal
sequences
obtained for 'both the intact proteins as well as fragments generated from the
digestion of
the mixture as described in Example 5. Sequence matches with the primary
sequence
deduced from the respective gene sequence are also indicated for each
fragment. The
data suggests that the mixture includes the 75 kDa and 77 kDa gene products
because
there is sequence identity specific to the respective genes. This result, in
turn, is .
consistent with the mass results obtained by MALDI-TOF.
Table 2
Fragment Sequence Summary
(positions are listed starting from the first amino acid of the
leader sequence)
Sequence 75 77 79 SEQ ID
IcDa lcDa kDa No.
EDDGFYTSVGYQIGEAAQMV 21-4021-40 21-4034
EDDGFYTSVGYQIGEAAQMVK21-41 35
STSSTTIFNNEPGYR 135-I49 36
TGGKPN-P----WS 215-228 37
TTTQTIDGK 226-234 38
NSIAHFGTQE-QI 422-434447-459459-47139
VPNAQSLQNVVSK 468-481493-506505-51740
SKKNNPYSPQGIET 479-492504-517516-52941
NYYLNQN 493-499518-524530-53642
Example 7 - Identification of the 75 tcDa, 77 l:Da and 79 kDa Genes in the
Chromosome of N. pylori.
7.1 Genetic Methods. Isolation of plasmid and chromosomal DNA, agarose gel
electrophoresis and restriction enzyme digestion were performed following
standard
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protocols (Sambrook et al.). DNA ligation was performed using a TOPO ligation
kit
(Invitrogen) following the recommended protocol.
7.2 Oligonucleotide Synthesis. Single stranded PCR primers were synthesized on
an Applied Biosystems model 380B DNA synthesizer using 13-cyanoethyl
phosphoramidite chemistry.
7.3 DNA Sequencing. DNA sequencing was obtained by asymmetric PCR
amplification using the fluorescent dye-labeled dideoxynucleotide terminator
method
(Gyllensten et al., 1988). The dye-labeled single stranded DNA fragments were
separated and identified with an Applied Biosystem model 373A automatic
sequencing
apparatus. Primary sequence information was analyzed using MacVector DNA
analysis
program (IBI, New Haven CT).
7.4 PCR Amplification. PCR amplifications were performed in 500 p,l tubes
containing 100 l.~l reaction volume overlaid with mineral oil. Each reaction
contained
10 mM Tris-HCL, pH 8.3, 50 mM KCl and 1.5 mM MgCl2; 2.5 units Taq polymerase
(Boehringer Mannheim), 200 mM dNTPs; 20 mM oligonucleotide primers and 1-2 p,g
chromosomal DNA. Templates were amplified for 30 cycles with a 1 second
extension
on the annealing time of each cycle in a Perkin-Elmer Cetus thermocycler.
7.5 Search Programs. DNA alignment searches were performed using the
MacVector program (IBI, New Haven CT). Protein homology searches were
performed
using either MacVector or DNASTAR (DNASTAR, Inc.) clustal alignment programs.
7.6 Identification of Genome Homology to a Degenerate Oligonucleotide. The
H. pylori genome (unannotated) was downloaded from TIGR (The Institute for
Genomic
Research) ftp file and stored as a MacVector file. The N-terminal amino acid
sequence
of the 77 kDa H. pylori protein was entered into MacVector program and reverse
translated to give the degenerate DNA oligonucleotide which was aligned to the
genome. The oligonucleotide used to align to the H. pylori genome had the
following
sequence:
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5'GARGAYGAYGGNTTYTAYACNWSNGTNGGNTAYCARATHGGNGAR
GCNGCNCARATGGTN 3' (SEQ. ID NO. 33].
Three matches were observed, corresponding to nucleotide positions 1404865-
1404807,
722946-722888 and 352616-352558, all on the negative strand of the genome.
MacVector was used to outline open reading frames which would overlap the
above
regions in the genomic DNA. Three open reading frames based on translational
start and
stop codons were found. An open reading frame between nucleotides 1402991-
1404925
(negative strand) predicted a protein of 81,157 kDa. There was a putative
signal
sequence of 20 amino acids which when cleaved would create a protein of 79 kDa
starting with the amino acids EDDGFYTSVGYQIGEAAQMV (SEQ ID No.7). A
second open reading frame between nucleotides 720808-723006 (negative strand)
predicted a protein of 79,123 kDa. There was a putative signal sequence of 20
amino
acids which when cleaved would create a protein of 77 kDa starting with the
amino
acids EDDGFYTSVGYQIGEAAQMV (SEQ ID No.7). A third open reading frame
between nucleotides 350550-352672 (negative strand) predicted a protein of
77,687
kDa. There was a putative signal sequence of 19 amino acids which when cleaved
would create a protein of 75 kDa starting with the amino acids
EDDGFYTSVGYQIGEAAQMV (SEQ ID No.7).
The three proteins have identical 20 amino acids at the N-terminus and share
internal peptide residues empirically determined from purified protein
preparations
(compare SEQ ID Nos. 1, 2, 3 and 7).
Example 8
8.1 PCR Amplification and Cloning of the 77 lcDa Gene. PCR primers were
synthesized which hybridized to the translational start and stop codons of the
ORF. The
PCR primers synthesized to amplify the 77 kDa gene from the chromosome of H.
pylori
had the following sequences: Forward Primer: 5' GGC CAT ATG AAA AAA CAC
ATC CTT TCA TTA GCT TTA GGC TCG 3' (SEQ ID No.: 8) and Reverse Primer: S'
GGC AAG CTT GGG AGT TTC ACA AAA AGC TTA GTA AGC GAA CAC 3' (SEQ
ID No.: 9). Chromosomal DNA from strains ATCC 43504. ATCC 43579, PBCC 1103,
PBCC 1105, PBCC 1107, LET 13, RSD 14, and SS1 were used as template DNA.
Conditions on the first round of PCR were 1 sec at 95°C, 1 sec at
60°C, 2 sec at 72°C ,
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with a S min hot start at 95°C (30 cycles). Ten microliters
electrophoresed on an
agarose gel revealed a 2 kb DNA fragment for strains ATCC 43504, PBCC 1103,
PBCC
1107, and LET 13. The PCR was repeated under lower annealing temperatures
(55°C)
to amplify strains ATCC 43579 and PBCC 1105. Under these conditions,
amplifications with these DNA templates were observed to have a diffuse band
at 2 kb.
A 2 kb family of bands was also observed after PCR amplification of SS 1 DNA
at 50°C
annealing temperature.
Two microliters of each PCR amplification was added to a TOPO ligation mix
(Invitrogen) and transformed into ToplOF' competent cells. Ampicillin
resistance
colonies showing a white color on X-gal IPTG plates were picked and grown for
mini
plasmid preparations (three for each strain). A single transformant for each
strain which
was confirmed to have an insert of 2 kb was picked for large scale DNA
preparation and
sequence analysis.
Sequence analysis confirmed that the genes for the 77 kDa protein from strains
ATCC 43504, ATCC 43579, PBCC 1103, PBCC 1105 and the 79 kDa protein from
strains PBCC 1107, LET 13, and SS 1 have been cloned successfully as described
above.
Examples on Cloning, recombinant expression,
and purification of the 75, 77, and 79 lcDa proteins
Strains and growth conditions. E. coli strain BLR(DE3) pLysS was obtained from
Novogen. E. coli strains TOP 10 and Top 1 OF' were obtained from Invitrogen.
E. coli
strains were grown in I-IYSOY buffer containing 1% glucose or glycerol.
Amplicilin
(100 pg/mL) and chloramphenicol (30 pg/mL) were added when appropriate.
DNA Manipulations. Ligations into expression vectors were performed as
described by Sambrook et al.
Example 9
9.1 Cloning and expression of the 75 lcDa gene from H. pylori strain
ATCC43579.
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PCR amplification, cloning and sequencing of 70 kDa family genes from 7
strains of H.
pylori revealed genes for the 77K kDa and the 79 kDa genes, but not the 75 kDa
gene.
Alignments of the three gene sequences published by TIGR revealed that the ATG
start
region of 75 kDa was different from that of the 77 kDa and 79 kDa genes and
would not
be expected to be amplified by the primer pair A and B (Table 3) used to
amplify the 77
and 79 kDa genes. A primer corresponding to the 75 kDa gene start region could
not be
designed because a stretch of 11 CT dinucleotide repeats exists 10 by
downstream from
the ATG start codon. CT dinucleotide repeats are also found in other H. pylori
genes,
and therefore a primer containing the repeats would not be specific for the 75
kDa gene.
DNA primers were designed based on the predicted sequences in and surrounding
the 75
kDa gene sequence as published by TIGR. A primer was designed to anneal
upstream
from the 75 kDa coding region (Table 3, primer C). Attempts to PCR amplify the
75
kDa gene from strain ATCC 43579 using primers B and C under a variety of
annealing
conditions did not yield a PCR product. In order to obtain the 75 kDa gene
from strain
ATCC 43579, two complimentary internal primers were made based on empirically
determined internal peptide sequence (Table 3, primers F and G). The front and
back
halves of the 75 kDa gene were amplifed in separate PCR reactions using primer
pairs D
and G or E and F (Table 3), cloned and sequenced to verify that they were
homologous
to the predicted 75 kDa sequence.
Two additional primers (Table 3, H and I) were designed with BsmBI ends for
seamless
cloning to the two 75 kDa gene fragments. Upon PCR amplification of the front
and
back halves with primer pairs D and I or E and H, the resulting DNA fragments
were
digested with BsmBI and ligated to each other. The ligated DNA was amplified
with
primers D and E, corresponding to the ends of the mature open reading frame. A
2 kb
DNA fragment was detected by ethidium bromide stained agarose gel
electrophoresis.
This DNA band was cloned in plasmid pCR2.1 (Invitrogen, San Diego, CA )
resulting in
pPX7768 and sequenced to confirm that it was the 75 kDa gene (Figure 6, SEQ ID
NO.
21). The predicted protein translation from this sequence is shown in Figure
7, SEQ ID
NO 19.
The mature gene fragment was PCR amplified using primer J and K (Table 3) with
pPX7768 as template DNA. The fragment was cloned into pRSETb at the
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NdelmECoRV sites to produce plasmid pPX7811. This plasmid was transformed into
the expression strain BLR(DE3)pLysS, selecting for ampicilin and
chloroamphenicol
resistance. The strain was grown to mig-logarithmic phase in HYSOY broth(
Difco)
containing ampicilin and chloroamphenicoi and expression was induced by the
addition
of 1 mM isopropanol beta-D-thiogalactopyranoside (IPTG). Expression proceeded
for 2
hours, after which the cells were harvested by centrifugation. Whole cell
lysates were
run on 10%SDS-PAGE. The 75kDa protein band was overexpressed as determined by
coomassie stained gels and reacted with mAB Hpy 64-27 on Western blots
9.2 Purification of the recombinant 75 kDa protein
Bacterial cells (ca. 1 S g wet wt of E. coli BLR(DE3)pLysS / pPX781 I ) were
resuspended in
75 ml of 10 mM NaP04 / 150 mM NaCI / 5.0 mM EDTA / 1.0 mM Pefabloc (pH 7.2) by
homogenization using a Ultra-Turrex tissue homogenizes (IICA Works,
Wilmington, NC).
The cells were disrupted by sonication using a Branson Sonifier Cell
Disrupter. Inclusion
bodies containing the recombinant 75 kDa protein were isolated by
centrifugation at 10,000
rpm using a Sorvall SLA-1500 rotor for 30 min at 10°C. Following
centrifugation, the
pellet was resuspended by homogenization in 75 ml of 10 mM NaP04 / 150 mM NaCI
/ 5.0
mM EDTA / 4.0% TX-100 (pH 7.2) and stirred for 1.5 hr at 4°C. The
suspension was
centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at
10°C. Following
centrifugation, the pellet was resuspended by homogenization in 75 ml of 10 mM
NaP04 /
150 mM NaCI / 5.0 mM EDTA / I .0% Zwittergent 3-14 (Z 3-14) (pH 7.2) and
stirred for I .5
hr at room temp. The suspension was centrifuged at 10,000 rpm using a Sorvall
SS-34 rotor
for 30 min at 10°C. Following centrifugation, the pellet was
resuspended by
homogenization in 75 ml of 10 mM NaP04 / 1 SO mM NaCI / 5.0 mM EDTA / 1.0%
Zwittergent 3-16 (Z 3-16) (pH 7.2) and stirred for 2 hr at room temp. The
suspension was
centrifuged at 10,000 rpm using a Sorvall SS-34 rotor for 30 min at
10°C. Following
centrifugation, the Z 3-14 and Z 3-16 supernatants containing the recombinant
75 kDa
protein were collected and stored at -20°C for further purification.
The Z 3-14 extract was
buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column
(Pharmacia
Biotech Inc., Piscataway, NJ) equilibrated in 0.02 M Tris-HCl / 5.0 mM EDTA /
1.0% Z 3-
14 (pH 8.0). The buffer exchanged Z 3-14 crude extract preparation was loaded
onto a 10
ml SP-Sepharose Fast Flow column (Pharmacia Biotech Inc.) equilibrated with
0.02 M Tris-
HCl / S.0 mM EDTA / I .0% Z 3-14 (pH 8.0). Unbound protein was washed through
the
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column with an additional 2 column volumes of equilibration buffer. The
recombinant 75
kDa protein was eluted using a linear NaCI gradient (0-0.5 M NaCI) in 0.02 M
Tris-HCl /
5.0 mM EDTA / 1.0% Z 3-14 (pH 7.0) over 8 column volumes. Fractions were
screened for
recombinant 75 kDa protein by SDS-PAGE and Western analysis and pooled. Pooled
fractions were dialyzed into PBS / 1.0% Z 3-14 overnight at 4°C. The Z
3-16 extract was
processed as described for the Z 3-14 extract.
Example 10
10.1 Cloning and expression of the 77 kDa gene from H. pylori strain ATCC
43579
Based on the published DNA sequence from TIGR, DNA oligonuceiotide primers
corresponding to the gene start (5') and end (3') were designed (Table 3,
primers A and
B). Chromosomal DNA from H. pylori strains ATCC 43579, ATCC 43504, PBCC 1103
and PBCC 1105 was isolated and used as templates for PCR amplification.
Following
PCR amplification, a 2 kb band was cloned into PCR2.1 TA cloning vector, and
the H.
pylori insert was sequenced. DNA sequences of the 77 kDa genes from strains
ATCC
43504, PBCC 1103 and PBCC 1105 were only partial but showed more homology to
the
77 kDa gene from TIGR than to the 75 kDa or 79 kDa genes. The 77 kDa gene from
strain ATCC 43579 was completely sequenced (plasmid pPX7760b). The DNA
sequence of the complete coding region is shown in SEQ ID NO. 5. The predicted
protein translation from this sequence is shown in SEQ ID NO. 2.
The 77 kDa gene was cloned into expression vector pRSETb which directs
expression
of the foreign protein by the T7 promoter. The mature gene fragment was PCR
amplified using primers J and K (Table 3) with pPX7760b as template DNA. The
band
was cloned into PCR2.1 (pPX7792). pPX7792 was cleaved with NdeI and EcoRV, and
the 2 kb gene fragment was ligated into pRSETb cut with the same enzymes.
After
verification of the correct clone by restriction endonuclease digestion, the
resulting
plasmid, designated pPX7796, was transformed into the expression strain
BLR(DE3)pLysS, selecting for ampicillin and chloramphenicol resistance. The
strain
was grown to mid-logarithmic phase in HYSOY broth (Difco, Detroit, Mich.)
containing ampicillin and chloramphenicol, and expression was induced by the
addition
of 1 mM IPTG. Expression proceeded for 2 hours, after which the cells were
harvested
by centrifugation. Whole cells lysates were run on 10-12% SDS-PAGE. The 77 kDa
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protein band was overexpressed as determined by coomassie stained gels, and
reacted
with polyclonal serum raised against the native protein.
10.2 Purification of the recombinant 77 lcDa protein.
Bacterial cells (ca. 32 g wet wt of E. coli BLR(DE3)pLysS (pPX7796) were
resuspended in
150 ml of 10 mM NaP04 / I50 mM NaCI / 5.0 mM EDTA / 1.0 mM Pefabloc (pH 7.2)
by
homogenization using a Ultra-Turrex tissue homogenizer. The cells were
disrupted using a
Microfluidizer Model 1 I OY. Inclusion bodies containing the recombinant 77
kDa protein
were isolated by centrifugation at 10,000 rpm using a Sorvall SLA-1500 rotor
for 30 min at
10°C. Following centrifugation, the pellet was resuspended by
homogenization in 150 ml of
IOmM NaP04 / 150mM NaCI / 5.0 mM EDTA / 1.0% TX-100 (pH 7.2) and stirred for 2
hr
at 4°C. The suspension was centrifuged at 10,000 rpm using a Sorvall SS-
34 rotor for 30
min at 10°C. Following centrifugation, the pellet was resuspended by
homogenization in 75
ml of l OmM NaP04 / 150mM NaCI / 5.0 mM EDTA / I.0% Z 3-16 (pH 7.2) and
stirred
overnight at room temp. The suspension was centrifuged at 10,000 rpm using a
Sorvall SS-
34 rotor for 30 min at 10°C. Following centrifugation, the supernatant
containing the
recombinant 77 kDa protein was collected and stored at -20°C for
further purification. The
Z 3-16 extract was buffer exchanged by passage over a 180 ml Sephadex G-25
(coarse)
column equilibrated in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0).
The
buffer exchanged Z 3-16 crude extract preparation was loaded onto a 10 ml SP-
Sepharose
Fast Flow column equilibrated 0.02 M Tris-HCl / S.0 mM EDTA / 1.0% Z 3-14 (pH
8.0).
Unbound protein was washed through the column with an additional 2 column
volumes of
equilibration buffer. The recombinant 77 kDa protein was eluted using a linear
NaCI
gradient (0-0.5 M NaCI) in 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH
7.0) over 8
column volumes. Fractions were screened for recombinant 77 kDa protein by SDS-
PAGE
and Western analysis and pooled. Pooled fractions were dialyzed into PBS / I
.0% Z 3-14
overnight at 4°C.
Example 11
11.1 Cloning and expression of the 79 kDa gene from H. pylori strain PBCC 1107
The 79 kDa gene was cloned form 3 strains of H. pylori , PBCC 1107, Let 13,
and SS 1.
Primers J and K (Table 3) were used to amplify the mature coding region for
cloning
into pRSETb following the same protocol as for the 75 and 77 kDa constructs.
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Following transformation into BLR(DE3) pLysS and induction by IPTG, the
recombinant plasmid pPX5048 directed the overexpression of the 79 kDa protein.
The
recombinant protein was easily detected in coomassie stained gels and reacted
with both
polyclonal sera raised against native 75, and 77 kDa genes (due to its
homology) and
with peptide-conjugate antisera directed against a sequence unique to the 79
kDa protein
(described below).
11.2 Purification of the recombinant 79 kDa protein.
Bacterial cells (ca. 15 g wet wt of E. coli BL21(DE3)pLysS / pPX5048) are
resuspended in
75 ml of 10 mM NaP04 / 150 mM NaCI / 5.0 mM EDTA / 1.0 mM Pefabloc (pH 7.2) by
homogenization using a Ultra-Turrex tissue homogenizer. The cells are
disrupted by
sonication using a Branson Sonifier Cell Disrupter. Inclusion bodies
containing the
recombinant 79 kDa protein are isolated by centrifugation at 10,000 rpm using
a Sorvall SS-
31 rotor for 30 min at 10°C. Following centrifugation, the pellet is
resuspended by
homogenization in 75 ml of l OmM NaP04 /.150mM NaCI / 5.0 mM EDTA / 4.0% TX-
100
(pH 7.2) and stirred for I .5 hr at 4°C. The suspension is centrifuged
at 10,000 rpm using a
Sorvall SS-31 rotor for 30 min at 10°C. Following centrifugation, the
pellet is resuspended
by homogenization in 75 ml of l OmM NaP04 / I SOmM NaCI / 5.0 mM EDTA / I .0%
Z 3-
14 (pH 7.2) and stirred for 1.5 hr at room temp. The suspension is centrifuged
at 10,000
rpm using a Sorvall SS-34 rotor for 30 min at 10°C. Following
centrifugation, the pellet is
resuspended by homogenization in 75 ml of 10 mM NaP04 / 150 mM NaCI / 5.0 mM
EDTA / 1.0% Z 3-16 (pH 7.2) and stirred for 2 hr at room temp. The suspension
is
centrifuged at 10,000 rpm using a Sorvall SS-31 rotor for 30 min at
10°C. Following
centrifugation, the Z 3-14 and Z 3-16 supernatants containing the recombinant
79 kDa
protein are collected and stored at -20°C for further purification. The
Z 3-14 extract is
buffer exchanged by passage over a 180 ml Sephadex G-25 (coarse) column
equilibrated in
0.02 M Tris-HCl / 5.0 mM EDTA / I.0% Z 3-14 (pH 8.0). The buffer exchanged Z 3-
14
crude extract preparation is loaded onto a 10 ml SP-Sepharose Fast Flow column
equilibrated 0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 8.0). Unbound
protein is
washed through the column with an additional 2 column volumes of equilibration
buffer.
The recombinant 79 kDa protein is eluted using a linear NaCI gradient (0-0.5 M
NaCI) in
0.02 M Tris-HCl / 5.0 mM EDTA / 1.0% Z 3-14 (pH 7.0) over 8 column volumes.
Fractions
are screened for recombinant 79 kDa protein by SDS-PAGE and Western analysis
and
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pooled. Pooled fractions are dialyzed into PBS / 1.0% Z 3-14 overnight at
4°C. The Z 3-16
extract is processed as described for the Z 3-14 extract.
Example 12
DNA Sequence and Predicted Translation of 75, 77 and 79 kDa genes
The DNA sequence of the 75 kDa gene from strain ATCC 43579 corresponding to
the
mature protein is shown in Figure 6, SEQ ID NO. 21. The open reading frame is
2070
by long, the same as the TIGR predicted protein. The translated protein is 689
aas long
containing 6 cysteine residues with an estimated pI of 8.88 (Figure 7, SEQ ID
NO. 19).
Alignment of the 75 kDa proteins from TIGR strain 26695 and ATCC 43579 reveals
that 26 amino acid residues are not conserved.
The DNA sequence of the 77 kDa gene from strain ATCC 43579 is shown in SEQ ID
NO. 5. The open reading frame corresponding to the mature protein is 2166 by
long, the
same as the TIGR predicted protein. The translated protein is 721 aas long
containing 6
cysteine residues with an estimated pI of 8.89 (SEQ ID NO. 2). Alignment of
the 77
kDa proteins from TIGR strain 26695 and ATCC 43579 reveals substitutions in 67
aas.
An additional 3 aas are unique to the TIGR protein, and an additional 11 aas
are unique
to the ATCC 43579 sequence.
The DNA sequence of the 79 kDa gene from strain PBCC 1107 is shown in Figure
8,
SEQ ID NO. 22. The open reading frame corresponding to the mature protein is
2157
by long, 21 by shorter than the TIGR predicted protein. The translated protein
from
strain PBCC 1107 is 718 aas long containing 8 cysteine residues with an
estimated pI of
6.99 which is considerably less than the pI of 8.68 predicted by the TIGR
sequence
(Figure 9, SEQ ID NO. 20). Alignment of the 79 kDa proteins from the TIGR
strain
26695 and PBCC 1107 revealed 99 amino acid residue changes. There are 6 aas
unique
to the TIGR 79 kDa sequence, and 1 as unique to the PBCC 1107 79 kDa sequence.
Example 13
Comparison of expression constructs for the overproduction of the 77, 75 and
79kd
proteins of H. pylori
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13.1 - 77K. The full length gene was cloned into pETl7b using the NdeI site to
fuse the
5' ends of the 77K gene to the vector ATG start codon, resulting in the full
unaltered
gene fused to the strong T7 promoter (pPX7762, Table 4). The full length gene
was also
cloned into an arabinose expression vector which also has strong promoter and
which is
tightly regulated. In addition, a third construct cloned the mature 77K gene
to an ompT
leader in pETl2b which could enhance the translation and signal processing of
the
mature 77K gene (pPX7782). None of the constructs containing the gene with a
signal
sequence were overexpressed in excess of 1 % total cellular protein (Table 4).
Primers was made corresponding to the start of the mature protein and the end
of the gene (Table 3, primers D and E). DNA sequence corresponding to the
mature
protein was PCR amplified using the pPX7760b as template DNA and cloned into
PCR2.1 (pPX7769). The gene was cleaved from pPX7769 by digestion with Asp718
and SpeI and inserted into the T7 promoted pETl7b (Novagen, Madison, Wisc.)at
the
Asp718 and SpeI sites. The resulting plasmid fused 18 amino acid residues from
the
vector to the mature 77 kDa gene. Moderate expression of the 77 kDa protein
was
observed, but the induced protein was not visible by coomassie stained gels,
indicating
that the recombinant protein constituted less than 1 % of the total cellular
protein. The
mature gene fragment was also cloned into the arabinose expression vector
pBAD24,
which provided the ATG methionine and two additional vector-encoded amino
acids to
the mature gene. The recombinant plasmids were housed in the appropriate E.
coli
strains, BLR(DE3) pLysS for T7 promoted vectors and ToplOF(ara-) for arabinose
vectors. Overnight cultures were seeded into HYSOY media containing 0.5-1.0%
glycerol and the appropriate antibiotic for selection. Cultures in mid-
logarithmic phase
were induced either with 1 mM IPTG (T7 vectors) or 0.0002-0.2% L-arabinose
(arabinose vectors) and induction was allowed to proceed for 2 hours. Cells
were
harvested and resuspended in IX SDS-PAGE loading buffer and run on a 12% SDS-
PAGE gel. The pBAD24 construct produced the recombinant 77 kDa protein which
was
detected by Western blot but not by coomassie stained gels.
Figure 11 shows that the 77K protein expressed from pE717b was not above 1%
of the total cellular protein. The pRSETb based clone, pPX7796, was able to
overexpress the protein greater than 10-20% of total cellular protein.
Analysis of the
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differences between pETl7b and pRSETb reveals that both vectors have identical
promoter sequences. A significant difference between the two vectors is in the
copy
number. pRSET b is based on the pUC replicon which maintains I00-700 copies
per
cell whereas pET vectors are based on a pMBI/ColEl replicon maintaining 25-30
copies per cell. A strong promoter coupled with a relatively high gene dosage
resulted
in overproduction of the recombinant mature 77K protein.
Example -75K. The mature gene (pPX7768) was amplified with primers D and E
(Table
3) to obtain a fragment with appropriate restriction site ends for cloning
into the
arabinose expression vector pBAD24 (Guzman et al. 1995. J. Bacteriol. 177:4121-
4130), fusing the vector ATG start codon and two additional vector encoded
amino
acids to the mature gene (pPX7794). The 75 kDa gene was expressed by this
vector
following induction with arabinose, but the level was approximately 1% of
cellular
protein and the recombinant protein was prone to proteolytic cleavage. The
mature
coding region was cloned into the T7 polymerase expression system pRSETb
(Invitrogen, San Diego Ca) by amplifying the 75 kDa gene from pPX7768 using
primers
J and K (Table 3) and ligating the fragment into the NdeI and EcoRV sites of
pRSETb.
The resulting recombinant plasmid (pPX7811 ) fuses the vector ATG to the
mature 75
kDa coding sequences. When the recombinant plasmid was induced by IPTG in the
BLR(DE3) pLysS expression strain (Novagen, Madison, Wisc.), the recombinant
protein was approximately 10% of the cell mass and was resistant to
proteolysis.
Comparison of recombinant protein yields after induction reveals that pPX7811
was
clearly superior in overexpressing the 75K mature protein (Figure 11).
Example -79K The mature 79K gene from strain PBCC1107 was cloned into the
arabinose vector pBAD24 fusing the vector ATG and 3 other amino acids to the
start of
the 75K gene (pPX5043, Table 4) and to the pRSET vector, fusing the vector ATG
to
the start of the mature 75K gene (pPX5048, Table). Comparison of recombinant
protein
yields after induction reveals that pPX5048 was superior in overexpressing the
79K
mature protein (Figure 12).
Notes on Recombinant protein induction and purification for the production of
75,
77, 79K proteins
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For the production of high yield of recombinant protein, the ampicilin marker
used in
plasmids pPX7796, pPX7811 and pPX5043 is partially deleted and a gene for
Kanamycin resistance is cloned in the site. This allows for fermentation of
the proteins
for use in humans. Strain E. coli BLR(DE3)pLysS housing either pPX7796,
pPX7811 or
pPX5043 is grown in I-IYSOY media containing glycerol until logarithmic phase
and
induced with 0.5-1 mM IPTG. Induction proceeds for 2-4 hours after which the
bacteria
is harvested by centrifugation.
Table 3A
PrimerDescription SEQ.
ID
NO.
A 5' start of 77 and 79 kDa genes 8
(sense)
B 3' end of 75, 77, and 79 kDa genes9
(antisense)
C Promoter region of 75 kDa gene 10
(sense)
D 5' start mature 75, 77, and 7kDa 1
genes (sense) I
E 3' end of 75, 77, and 7kDa genes 12
(antisense)
F Internal 75 kDa specific (sense) 13
G Internal 75 kDa specific (antisense)14
H Internal 75 kDa seamless cloning 15
(sense)
I Internal 7kDa seamless cloning 16
(antisense)
J 5' end start mature 75, 77, and 17
7kDa genes (sense)
K 3' end of 75, 77, and 7kDa genes 18
(antisense)
Table
3B
PrimerSequence (5' to 3') SEQ.
ID
NO.
A GGCCATATGAAAAAACACATCCTTTCATTAGCT g
B GGCAAGCTTGGGAGTTTCACAAAAAGCTTAGTAAGCGAACAC 9
C CGAAATCTTGTGATAAGATC 10
D CCGGGCTTGGTACCGGAAGACGACGGCTTTTAC 11
E CCGGGCACTAGTTTAGTAAGCGAACACATAATTCAAATACACGC 12
F CGGGGGGCAAACCAAATACAC 13
G GTGTATTTGGTTTGCCCCCCG 14
H GCCGAACGTCTCCCCTAAATGAGGCATGCCCAAACT 15
I CTACGGCGTCTCTTAGGGTAGTGATAATGATGCTCGCTTG 16
J CGCGGCGATATGGAAGACGACGGCTTTTAC 17
K CCGGCCGGTACCTTAGTAAGCGAACACATA lg
47
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PAGE INTENTIONALLY LEFT BLANK
48
CA 02329264 2000-11-22
WO 00/00614 PCT/US99/14375
.5
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49
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Example 14
Generation of peptide specific polyclonal antibodies to
the 77 and 79 lcDa proteins from X. pylori
14.1 Summary.
Ten peptides representing unique sequences of the 77 and 79 kDa proteins (five
to each)
from H. pylori were conjugated to CRMlg7. CRMlg7 is a well known carrier
protein
as described in Uchida, T. et al., 1971 Nature New Biology, Vol. 233, 8-11.
All of the
conjugates were characterized by SDS-PAGE and amino acid analysis. The soluble
conjugates were characterized by MALDI-TOF mass spectrometry analysis. An
animal
study was performed using the peptide conjugates as the antigen in order to
produce
antisera that would specifically recognize either the 77 or 79 kDa protein of
H. pylori by
western blot for purification protocols.
14.2 Peptides Used For Conjugation.
All peptides were synthesized using the Gilson Multiple Peptide Synthesizer
with a N-
terminal cysteine residue necessary for covalent attachment to the carrier
CRM197
protein in the specific conjugation chemistry used. The amino acid sequences
of the
peptides representing the 77 and 79 kDa peptides can be found in Table 5. All
peptides
were purified to near homogeneity by reversed phased HPLC and characterized by
MALDI-TOF mass spectrometry and amino acid composition analysis.
14.3 Preparation of Peptide-Cl2Mlg7 Conjugates.
The 77 and 79 kDa peptides were conjugated to CRMlg7 using the crosslinking
reagent
N-succinimidyl bromoacetate (Bernatowicz et a1.). On the day of the
conjugation, the
peptides were reacted with 5,5'-dithio bis(2-nitrobenzoic acid) [Ellman's
reagent] to
verify the content of free SH groups (resulted in greater than 95% free SH
groups). Free
amino groups of CRMlg7 were bromoacteyiated by reaction with an excess of
bromoacetic acid N-hydroxysuccinimide ester (Sigma Chemical Co.). To an ice
cold
solution of CRMlg7, 10% (v/v) 1.0 M NaHC03 (pH 8.4) was added. Bromoacetic
acid
N-hydroxysuccinimide ester, equal in weight to that of CRMlg7 used, was
dissolved in
200 u1 dimethylformamide, added slowly to the CRMlg7 and gently mixed at room
temperature in the dark for 1 hour. The resulting bromoacetylated (activated)
protein
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was purified by passage through a P6-DG column using PBS, 1 mM EDTA (pH 7.0)
as
the eluent. Following purification, the fractions corresponding to activated
CRM197
were pooled and the protein concentration was estimated by BCA protein assay
(Pierce
Chemical Co.). To determine the extent of bromoacetylation, the protein amino
groups,
both before and after treatment with bromoacetic acid N-hydroxysuccinimide
ester,
were reacted with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Approximately 70-
75%
of the amino groups were found to be bromoacetylated (activated). Following
TNBS
assay, approximately 2.4 - 4.6 mg of peptide was dissolved in sterile
distilled water to
an approximate concentration of 20 mg/ml. The peptide was slowly added to cold
activated C1Z.M197 in a 1:1 ratio (w/w) and the pH was adjusted to
approximately 7.0-
7.2. The resulting material was gently mixed overnight at 4oC in the dark
followed by
dialysis in the dark against two 1 L changes of PBS, pH 7.2. Each conjugate
was then
transferred into a sterile 15 ml polypropylene tube, wrapped in aluminum foil
and stored
at 4oC.
14.4 Characterization of Peptide-CRM197 Conjugates.
To verify conjugation, all peptide-CRMI g7 conjugates were analyzed by amino
acid
analysis (for the presence of the characteristic S-carboxymethylcysteine
residues) and
SDS-PAGE. The soluble conjugates and activated C1tM197 were subjected to MALDI-
TOF mass spectrometry analysis. The protein concentration was determined by
the
average of values determined by amino acid analysis and BCA (Table 6).
14.4(a) Amino Acid Analysis of Peptide-C1tM197 Conjugates.
A suitable aliquot of each conjugate was hydrolyzed using 6N HCl in the
presence of
5% phenol (v/v) and 1% 2-mercaptoethanol (v/v) under vacuum for 22 hours at 1
lOoC.
Following evaporation and resolubilization in Beckman sodium citrate sample
dilution
buffer (pH 2.2), the samples were analyzed on a Beckman 6300 Amino Acid
Analyzer
according to manufacturer's instructions. The degree of conjugation was
determined by
the estimated amount of S-carboxymethylcysteine residues formed per mole of
CRM197. The degree of conjugation for each conjugate is described in Table 7.
14.4(6) SDS-PAGE Analysis of Peptide-CltMly7 Conjugates
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A suitable aliquot of each conjugates was mixed with reducing sample buffer
and heated
at 100oC for 5 minutes. The conjugates and Markl2 molecular weight (MW)
standards
(Novex) were loaded on a 10 % (w/v, acrylamide) gel and SDS-PAGE (Laemmli) was
carried out. Following SDS-PAGE, the gel was stained with Coomassie brilliant
blue
and destained. The samples exhibited a smear staining pattern across a wide MW
range
that is characteristic of similar peptide conjugates.
14.4(c) MALDI-TOF Mass Spectrometry Analysis of Peptide-CRM197
Conjugates.
A suitable aliquot of activated CRM197 and each soluble conjugate were
analyzed by
MALDI-TOF mass spectrometry using 3,5-dimethoxy-4-hydroxy-cinnamic acid
(sinapinic acid) as the matrix. Due to the addition of bromoacetyl groups (120
mass
units each) to 29 lys residues of CluVI197 (mol. wt. 58,408.7), which was
based on
approximately 75% activation from the TNBS assay, activated CluVI197 was
expected
to have a mol. wt. of [58,408.7 + (29x120)) = 61,889. The experimental mol,
wt. of
activated CRM197 determined by MALDI-TOF mass spectrometry was 61,208 for
activated CRM197 used for the 77k peptide conjugates and 61,009 for activated
CRM197 used for the 79k peptide conjugates. The degree of conjugation for each
conjugate was calculated by subtracting the mass value of activated CRM197
from the
mass value of each conjugate and dividing by the mass of the peptide used to
prepaxe the
conjugate. The degree of conjugation for the soluble 77k and 79k peptide
conjugates is
described in the table 8. The mass values for heavily precipitated conjugates
could not
be determined.
14.5 Animal Study.
An animal study using the peptide conjugates was performed to produce antisera
that
will differentially recognize the 77kDa protein from the 79kDa protein in H.
pylori blot.
Table 9 describes the protocol for the animal study.
14.6 Analysis of Immune Sera by Peptide ELISA.
All peptide ELISAs were run using Nunc Maxisorp 96-well plates. Weeks 0 and 6
peptide conjugate antisera were all titered against homologous peptides.
Plates were
coated with 100 pl of peptides diluted to 1.0 p.g/ml with SO mM sodium
bicarbonate (pH
52
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9.0) and incubated at 37oC overnight. Plates were washed and blocked for 1
hour at
37oC with 250 p,l of 3% BSA in 137 mM tris buffered saline (TBS), pH 7.6.
Peptide
conjugate antisera were serially diluted into 0.3% BSA in TBS containing 0.05%
Tween-20, 100 pl of each dilution was added to the plates and incubated at
37oC for 1
hour. The plates were washed and subsequently incubated for 1 hour at 37oC
with 100
pl of alkaline phosphatase conjugated goat anti mouse secondary antibody
(Zymed)
diluted 1:1500 in 0.3% BSA in TBS containing 0.05% Tween-20. The plates were
washed and finally incubated for 1 hour at room temperature with 100 pl p-
nitrophenyl
phosphate substrate prepared in diethanolamine containing 0.5 mM MgCl2. Plates
were
read using an automated 96-well plate reader with a 405 nm test and 690 nm
reference
filter. All endpoint titers were calculated at 60 minutes at 0.1 AU. Peptide
ELISA
endpoint titers are shown in table 6.
14.7 Analysis of Immune Sera by Western blot
Week 6 sera from mice immunized with peptide groups: 97-7; #1, #6, #7 and 97-
14;
# 14, # 17 was used, (as stated in the Western Blot protocol section ) as the
primary
antibody in a Western blot against whole cell lysates of Helicobacter pylori
and the
recombinant 75, 77, and 79 kDa proteins. Conjugate 97-14 #14 generated a
strong
specific signal to the 79 kDa protein in both the H.pylori sample and the
recombinant 79
kDa protein, but not to native or recombinant 75 and 77 kDa proteins.
Table 5. Sequences of peptides used in the 77 and 79 kDa conjugates.
53
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Conjugate Peptide Sequence
97-6 #3/CRM,9, (77k) CASGNTSHVITNKLDGVPDS (SEQ 23)
97-6 #7/CRM,9, (77k) CSPSVNGTKTTTQTIDGK (SEQ 24
97-6 #9/CRM,9, (77k) CYFHATNSSEANAPKFS (SEQ 25)
97-6 #15/CRM,9~ (77K) CNPENLSENFKN (SEQ 26)
97-6 #20/CRM,9, (77k) CSGQGNNN (SEQ 27)
97-7 #1/CRM,9? (79k) CVMKNNNNVNEKLAGFGKEEVM (SEQ
28)
97-7 #6/CRM,9, (79k) CKAKNGSSSSSNGGNGSS (SEQ 29)
97-7 #1 I/CRM,9, (79k) CTTTYNNNKATVKFDIT (SEQ 30)
97-7 #14/CRM,9~ (79k) CLVRSTNNENTPGGGQ (SEQ 31)
97-7 #17/CRM,9, (79k) CRQTADINGGVYQF (SEQ 32)
54
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Table 6. Concentration of 77 and 79kDa peptide conjugates. Concentration was
determined by averaging the values determined by amino acid analysis and BCA.
Conjugate Concentration
(mg/ml)
97-6 #3/CRM,9, (?7kDa) 3.5
97-6 #7/CltM,9, (77kDa) 2.6
97-6 #9/CRM,9, (77kDa) 3.8
97-6 #15/CRM,9, (77kDa) 2.5
97-6 #20/Cll;Ivl,9, 3.2
(77kDa)
97-7 #1/C1ZN1,9, (79kDa) 1.8
97-7 #6/CltM,9, (79kDa) 0.9
97-7 #11/CltM,9, (79kDa) 3.3
97-14 #14/CltM,9, (79kDa) 3.3
97-14 #17/CRM,9, (79kDa) 3.7
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Table 7. Degree of conjugation for each peptide conjugate based on amino acid
analysis.
Conjugate
Degree of Conjugation
(moles peptide per mole
CRM,9,)
97-6 #3/CRM,9, (77kDa) 1 I
97-6 #7/CRM,9, (77kDa) 9
97-6 #9/CRM,~, (77kDa) 17
97-6 # 15/Cluvl,9, g
(77kDa)
97-6 #20/Cluvl,9, (77kDa) ~ g
97-7 #1/CIUVI,9, (79kDa)
97-7 #6/Cluvi,9, (79kDa) 12
97-7 #11/CRM,9, (79kDa) 14
97-14 #14/CRM,9, (79kDa) 13
97-14 #17/CltM,9, (79kDa) 17
56
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Table 8. Degree of conjugation of 77k and 79kDa peptide conjugates.
The mass of the conjugate is approximate due to the irregular shape of
the peak (broad and sometimes rough) possibly representing a
distribution of conjugates containing different amounts of peptides
attached. ND = not determined due to precipitate.
Con a ate Mass of ConjugateMass of PeptideDegree of
~ g (Approximate) Conjugation
(# peptides
per
CRM,9,)
97-6 #3/CltM,9, 79,555 2,013 9.1
(77kDa)
97-6 #7/CRM,9, 74,121 1,836 7.0
(77kDa)
97-6 #9/CRM,9, 86,910 1,872 13.7
(77kDa)
97-6 #15/CRM,9~ 68,870 1,407 5.4
{77kDa)
97-6 #20/CRM,9, 72,036 792 13.7
(77kDa)
97-7 #1/CRM,9, ND 2,466 ND
(79kDa)
97-7 #6/CRM,9~ ND 1,627 ND
(79kDa)
97-7 #11/CRM,9~ ND 1,932 ND
(79kDa)
97-14 #14/CItIvI,9,79,231 1,645 11.1
(79kDa)
97-14 #17/CRM,9~ ND 1,570 ND
(79kDa)
57
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Table 9. 77k and 79kDa peptide conjugate animal study protocol. Swiss Webster
mice were
used for entire study. Injection volume = 100 ~1; B = bleed; V = vaccinate.
Conjugate Dose Adjuvant Wk 0 Wk 3 Wk Wk
~g 6 8
97-6 #3/CRM,9, 10 20 p,g QS-21B, V V B, B
(77kDa) V
97-6 #7/CRM,9~ 10 20 p,g QS-21B, V V B, B
(77kDa) V
97-6 #9/CRM,9, 10 20 pg QS-21B, V V B, B
(77kDa) V
97-6 #15/CRM,9~ 10 20 ~tg QS-21B, V V B, B
V
(77kDa)
97-6 #20/CRM,9, 10 20 llg QS-21B, V V B, B
V
(77kDa)
97-7 #1/CRM,9, 10 20 pg QS-21B, V V B, B
(79kDa) V
97-7 #6/CRM,9, 10 20 pg QS-21B, V V B, B
(79kDa) V
97-7 #I 1/CRM,9.,10 20 ~g QS-21B, V V B, B
V
(79kDa)
97-14 #14/CRM,9~10 20 ltg QS-21B, V V B, B
V
(79kDa)
97-14 #17/CRM,9,10 20 pg QS-21B, V V B, B
V
(79kDa)
58
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Table 10. Weeks 0 and 6 peptide ELISA endpoint titers of 77 and 79kDa peptide
conjugate antisera against homologous peptides
Conjugate Week 0 Titer Week 6 Titer
97-6 #3/CRM,~, (77Da) 478 1,397,000
97-6 #7/CItIVI,9, (77kDa) 37S 513,400
97-6 #9/Cluvl,9, (77kDa) 324 967,900
97-6 #IS/CltM,9, (77kDa) S04 1,066,000
97-6 #20/CRM,9, (77kDa) 511 105,600
97-7 #1/CRM,9, (79kDa) 369 > 2,187,000
97-7 #6/CRM,9, (79kDa) 468 120,400
97-7 #I I/CItM,9, (79kDa) 492 1,438,000
97-14 #14/CRM,9, (79kDa) 261 523,000
97-14 #17/CRM,9, (79kDa) 2,242 1,644,000
Example 15
Prophylactic and Therapeutic Mouse Models
with 75/77 kDa Proteins
15.1 Prophylactic Mouse Model.
CS7BL/6 mice were vaccinated intragastrically with 100 pg native 75.77 kDa
proteins
admixed with 10 ug CT, 10 p.g CT-E29H (an attenuated cholera toxin mutant as
disclosed in U.S. Provisional Application No. 60/102,430 which is hereby
incorporated
herein), 100 p.g CpG20mer (a CpG oligonucleotide sequence, see Davis et al.,
1998. J.
Immunol. 160:870-876), or 10 p,g CT-E29H + 100 wg CpG20mer on days 0, 2,14,
and
16. Mice were challenged intragastrically with 2 x 108 cfu H. pylori strain
SS1 on day
31. Organisms recovered on days 58 and 59 are expressed as loglp cfu per gram
of
stomach tissue +/- 1 standard error of the mean. The number of colony forming
units
recovered was reduced by approximately 1-2 logs in mice vaccinated with 75/77
kDa
protein admixed with CT, CT-E29H, and with CT-E29H + CpG20mer (Table 13).
59
CA 02329264 2000-11-22
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15.1(a) Immunogenicity of native 75/77 kDa proteins.
Pre-challenge pooled sera (day 30) from mice vaccinated as above were analyzed
for the
presence of antibodies to the native 75/77 kDa proteins by standard ELISA
(Table 11).
Samples containing mucosal antibodies were also screened for specific
antibodies to the
75/77 kDa proteins (Table 12).
15.2 Therapeutic Mouse Model.
C57BL/6 mice were infected intragastrically with 2 x 10g cfu H. pylori strain
SS1 on
day 0. For intragastric vaccinations, mice received native 100 p,g 75/77 kDa
proteins,
recombinant urease, or ICLH admixed with CT on days 31, 33, 45, and 47. For
subcutaneous vaccinations, mice were injected with 10 mg native 75/77 proteins
admixed with AIP04 on days 32 and 60. A challenged but unvaccinated control
group
was included to assess the role of nonspecific clearance from CT in the KLH/CT
group.
Recovery of organisms 2 and 4 wks after the last vaccination is expressed in
log 10 cfu
per gram of stomach tissue +/- 1 standard deviation. The number of colony
forming
units recovered was significantly reduced at the 2 wk time point for mice
receiving
native 75/77 kDa proteins intragastrically admixed with CT, and at the 4 wk
time point
for mice receiving native 75/77 kDa proteins subcutaneously admixed with A1P04
(Figure 14).
Example 16
16.1 Vaccine Characterization of Recombinant Proteins 75, 77 and 79.
The above protocols for the animal studies are repeated for each of the
proteins to
establish the specific attributes by each protein as a vaccine.
CA 02329264 2000-11-22
WO 00/00614 PCTNS99/14375
w oo ~ o ~.,
pp ~ N ~n ~ N
~ N ~ N ~ ~ n
V ~ et~
vi ~ M ~ ~OO
.b M N M V1
N
O
O
R.
N C
O c1N O
O t;~ o0 O
~CG ett~-~
N M N
'' _ c,
''
'o U
v
~ W o
M N N O
a N
M ~ M +
b W o.
N
fa W
a>~ appN ~ oho..M-
~ r.rM M .-.('~ O
fdlDC." M rM-..01~ L.
_ ' O
a..
O
, ~ O OvOvOWO ~ N
C V N oNO~O oNO ~ G.
s ~"~'iy U ai
a ~o~ ~' o
it '
4,~ '~x b
O N H 4i O
r~' O N G
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>~~ a U U W G.+ ~ O
' N 'o"
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ou
,:, y cdt~~ c~ 1 z ~ o
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A A A A ~ ~ Ip
r n ~ ~ .~
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m v,v,~n a b
O
~ 000.o .-. ~ ~ ~ '='
-' 0
o o~".o~.o',o~. '~ ~ 'b~'o
N '~a a
~ d Q
61
CA 02329264 2000-11-22
WO 00/00614 PCTNS99/14375
O v1 N N O~
Ow1 ~1' h
V N ~D N o0
M
O o00ot1
OvM ~ h M
C.o-rV M N
.'
~ td
OD
'b
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9
('O Ovo0h .-. ~
~Dh ~ M
V N
H
N
U a O M ~
V~ ~ 0
O ~ V 0 p
~n
icl
N G
b C7 ,
ao
U , G,
4:
~ ~ ~
cp ' V O~00
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O
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b ~ N -E cd
N
i-n O O ~ N
N
N ~ ~ oo N a
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...~ W N
~ 3
0
o ~ C7~ ~ o o~ oo ~ ,.
y ~ V N n M
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~ ~ N
cdU ,~ C
O ~ ~ N ~ O
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_ ~ M ( ..
r V - M O r
7,
h 7 ,Y O
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O
~, L
~,cd O
O ~ ~
N M
~ c~
C ~ ~ a' '~OD ~
>
~. 'd
V U W
.y U W
o
a. 5 U ~~
o
: . on ~
~ ~
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, O
.o,D ~ A ~ A N ~ d0
O h h h (~
3
h h h h y v . '
""'
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. pa ~. '
c'h
' ~ V' ~ ~ ~ ~ V' ' o
~'
. o . o o b ai
~ o , o , ,
~
H ~ a ~ a a ~ .~~ -~
z
62
CA 02329264 2000-11-22
WO 00/00614 PCT/US99/14375
REFERENCES
References referred to herein above are noted below and are incorporated
herein:
Bernatowicz, M. S., and Matsueda, G. R. ( 1986) Analytical Biochemistry 155,
95-
102.
Czinn et al. 1993. Vaccine. 11, 637-642.
Hannig, G. and Makrides, S.C. 1998. Trends in Biotechnology. 161: 54-60.
Hillenkamp, F. and Karas, M. 1990. Mass spectrometry of peptides and proteins
by
Matrix-Assisted Ultraviolet Laser Desorption/Ionization. Methods Enzymol. 193,
280-295
Gyllensten, U.B. and Erlich, H.A. Generation of single stranded DNA by the
polymerase chain reaction and its application to direct sequencing of the HLA-
DQA
locus. Proc. Nat. Acad. Sci. USA. 1988, 85, 7652-7656.
Holmgren, J., Czerkinsky C., Lycke, N. and Svennerholm, A. 1992. Mucosal
Immunity: Implications for Vaccine Development. Immunobiol. 184, 157-179.
Kyte and Doolittle. 1992. J. Mol.Biol. 157, 105-137.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the
head
of bacteriophage T4. Nature 227, 680-685.
Matsudaira, P. 1987. Sequence from picomole quantities of proteins
electroblotted
onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10035-10038.
63
CA 02329264 2000-11-22
WO 00/00614 PCT/US99/14375
McGhee, J., Mestecky, J., Dertzbaugh M., Eldridge, J., Hirasawa, M., and
Kyono,
H. 1992. The Mucosal Immune System: From Fundamental Concepts to Vaccine
Development. Vaccine. 10 (2), 75-88.
Sambrook, Fritsch and Maniatis. Molecular Cloning - A Laboratory Manual. Cold
Spring Press (Cold Spring Harbor, New York). 1989.
Schiigger, H. and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-
polyacrylamide gel electrophoresis for the separation of proteins in the range
from 1 to
100 kDa. Anal. Biochem. 166, 368-379.
Sedegah et al. 1994. Immunology. 91, 9866-9870
Serwold-Davis, T. et al. 1987. PNAS 84:4964-8.
Studier, F.W. and Moffatt, B.A. 1986. J. Mol. Biol. 189:113-130.
Wolff et al. 1990. Science 247, 1465-1468
64
CA 02329264 2000-11-22
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Fulginiti, James P.
Fiske, Michael J.
. . . . . . . . . .... . Dilts, Deborah A.
(ii) TITLE OF INVENTION: Novel Antigens of Helicobacter pylori
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: American Cyanamid Company
(B) STREET: One Campus Drive
(C) CITY: Parsippany
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07054
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Webster, Darryl L.
(B) REGISTRATION NUMBER: 34276
(C) REFERENCE/DOCKET NUMBER: 33927-OOL
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 973-683-2159
(B) TELEFAX: 973-683-4117
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 708 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: I:
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Phe Leu
1 5 10 15
1
CA 02329264 2000-11-22
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Leu His Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile
20 25 30
Gly Glu Ala Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Glu Leu
35 90 45
Ser Asp Asn Tyr Glu Lys Leu Asn Asn Leu Leu Asn Asn Tyr Ser Thr
50 55 60
Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Asp
65 70 75 80
Ala Arg Asp Asn Leu Gly Ser Ser Ser Arg Asn Leu Leu Asp Val Lys
85 90 95
Thr Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala Ala
100 105 110
Val Gly Leu Trp Gln Val Thr Ser Tyr Ala Phe Thr Ala Cys Gly Pro
115 120 125
Gly Ser Asn Glu Asn Ala Asn Gly Gly Ile Gln Thr Phe Asn Asn Val
130 135 140
Pro Gly Gln Asp Thr Thr Thr Ile Thr Cys Asn Ser Tyr Tyr Glu Pro
145 150 155 160
Gly His Gly Gly Pro Ile Ser Thr Ala Asn Tyr Ala Lys Ile Asn Gln
165 170 175
Ala Tyr Gln Ile Ile Gln Lys Ala Leu Thr Ala Asn Gly Ala Asn Gly
180 185 190
Asp Gly Val Pro Val Leu Ser Asn Thr Thr Thr Lys Leu Asp Phe Thr
195 200 205
Ile Asn Gly Asp Lys Arg Thr Gly Gly Lys Pro Asn Thr Pro Glu Lys
210 215 220
Phe Pro Trp Ser Asp Gly Lys Tyr Ile His Thr Gln Trp Ile Asn Thr
225 230 235 290
Ile Val Thr Pro Thr Glu Thr Asn Ile Asn Thr Glu Asn Asn Ala Gln
245 250 255
Glu Leu Leu Lys Gln Ala Ser Ile Ile Ile Thr Thr Leu Asn Glu Ala
260 265 270
Cys Pro Asn Phe Gln Asn Gly Gly Arg Ser Tyr Trp Gln Gly Ile Ser
275 280 285
Gly Asn Gly Thr Met Cys Gly Met Phe Lys Asn Glu Ile Ser Ala Ile
290 295 300
Gln Gly Met Ile Ala Asn Ala Gln Glu Ala Val Ala Gln Ser Lys Ile
305 310 315 320
Val Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro
2
CA 02329264 2000-11-22
WO 00/00614 PCT/US99/14375
325 330 335
Phe Asn Pro Tyr Thr Asp Ala Ser Phe Ala Gln Ser Met Leu Lys Asn
340 345 350
Ala Gln Ala Gln Ala Glu Ile Leu Asn Gln Ala Glu Gln Val Val Lys
355 360 365
Asn Phe Glu Lys Ile Pro Thr Ala Phe Val Ser Asp Ser Leu Gly Val
370 375 380
Cys Tyr Glu Val Gln Gly Gly Glu Arg Arg Gly Thr Asn Pro Gly Gln
385 390 395 400
Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Lys Gln Thr
905 910 415
Ile Thr Asn Leu Asp Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln
420 925 430
Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys
435 440 495
Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala
950 455 960
Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser Lys
465 970 975 480
Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu
485 990 495
Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly
500 505 510
Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn
515 520 525
Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe
530 535 540
Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr
545 550 555 560
Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val
565 570 575
Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp
580 585 590
Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu
595 600 605
Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr
610 615 620
Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val
625 630 635 640
3
CA 02329264 2000-11-22
WO 00/00614 PCT/US99/14375
Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala
645 650 655
Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu
660 665 670
Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met
675 680 685
Gay Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr
690 695 700
Val Phe Ala Tyr
705
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser
1 5 10 15
Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val GIy Tyr Gln
20 25 30
Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Asp
35 40 45
Leu Ser Asp Arg Tyr Glu Ser Leu Asn Asn Leu Leu Thr Arg Tyr Ser
50 55 60
Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn
65 70 75 80
Ala Ala Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu Ile Gly Asp
85 90 95
Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Ile Asn Ala
100 105 110
Ala Val Gly Phe Trp Asn Val Leu Gly Tyr Ala Thr Gln Cys Gly Gly
115 120 125
Asn Ala Asn Gly Gln Lys Ser Thr Ser Ser Thr Thr Ile Phe Asn Asn
130 135 140
Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys Ser Leu Asn Gly Tyr
145 150 155 160
4
CA 02329264 2000-11-22
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Thr Pro Gly Tyr Tyr Gly Pro Met Ser Ile Glu Asn Phe Lys Lys Leu
165 170 175
Asn Glu Ala Tyr Gln Ile Leu Gln Thr Ala Leu Lys Gln Gly Leu Pro
180 185 190
Ala Leu Lys Glu Asn Asn Lys Lys Val Asn Val Thr Tyr Thr Tyr Thr
195 200 205
Cys Sex Gly Gly Gly Asn Asn Asn Cys Ser Ser Glu Ala Thr Gly Val
210 215 220
Ser Asn Gln Asn Gly Giy Thr Lys Thr Thr Thr Gln Thr Ile Asp Gly
225 230 235 240
Lys Ser Val Thr Thr Thr Ile Ser Ser Lys Val Val Asp Ser Thr Ala
245 250 255
Ser Gly Asn Thr Ser Arg Val Ser Tyr Thr Glu Ile Thr Asn Lys Leu
260 265 270
Glu Gly Val Pro Asp Ser Ala Gln Ala Leu Leu Ala Gln Ala Ser Thr
275 280 285
Leu Ile Ser Thr Ile Asn Thr Ala Cys Pro Phe Phe Ser Val Thr Asn
290 295 300
Gln Ser Gly Gly Pro Gln Met Glu Pro Thr Lys Gly Lys Leu Cys Gly
305 310 315 320
Phe Thr Glu Glu Ile Ser Ala Ile Gln Lys Met Ile Thr Asp Ala Gln
325 330 335
Glu Leu Val Asn Gln Thr Ser Val Ile Asn Ser His Glu Gln Ser Thr
340 345 350
Leu Val Gly Gly Asn Asn Gly Lys Pro Phe Asn Pro Phe Thr Asp Ala
355 360 365
Gln Phe Ala Gln Gly Met Leu Ala Asn Ala Ser Ala Gln Ala Lys Met
370 375 380
Leu Asn Leu Ala His Gln Val Gly Gln Thr Ile Asn Pro Asn Asn Leu
385 390 395 400
Thr Gly Asn Phe Lys Asn Phe Val Thr Gly Phe Leu Ala Thr Cys Asn
405 410 415
Asn Pro Ser Thr Ala Gly Thr Gly GIy Thr Gln Gly Ser Ala Pro Gly
420 425 430
Thr Val Thr Thr Gln Thr Phe Ala Ser Gly Cys Ala Tyr Val Glu Gln
935 940 445
Thr Ile Thr Asn Leu Glu Asn Ser Ile Ala His Phe Gly Thr Gln Glu
450 455 460
Gln Gln Ile Gln Arg Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe
CA 02329264 2000-11-22
WO 00/00614 PCT/US99/14375
465 970 475 480
Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr
485 990 495
Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser
500 505 510
Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr
515 520 525
Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu
530 535 540
Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Gly Ser Gln Thr Asn
545 550 555 560
Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Glu Gln Phe
565 570 575
Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp
580 585 590
Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp
595 600 605
Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn
610 615 620
Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Sex Val Gly
625 630 635 640
Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu
645 650 655
Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn
660 665 670
Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu
675 680 685
Ala Arg Pro Lys Lys Asn Asp Ser Asp His Ala Ala Gln His Gly Ile
690 695 700
Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Sex Phe
705 710 715 720
Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn
725 730 735
Tyr Val Phe Ala Tyr
790
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser
1 5 10 15
Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln
20 25 30
Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln
35 40 45
Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser
50 55 60
Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn
65 70 75 80
Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu Ile Gly Asp
85 90 95
Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Phe Leu Ala Ile Asn Ala
100 105 110
Ala Val Gly Leu Trp Asn Thr Ile Gly Tyr Ala Val Met Cys Gly Asn
115 120 125
Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val Ile Phe Asn Asp Gln
130 135 140
Pro Gly Gln Asp Ser Thr Gln Ile Thr Cys Asn Arg Phe Glu Ser Thr
145 150 155 160
Gly Pro Gly Lys Ser Met Ser Ile Asp Glu Phe Lys Lys Leu Asn Glu
165 170 175
Ala Tyr Gln Ile Ile Gln Gln Ala Leu Lys Asn Gln Ser Gly Phe Pro
180 185 190
Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr Glu
195 200 205
Cys Arg Gln Thr Ala Asp Ile Asn Gly Gly Val Tyr Gln Phe Cys Lys
210 215 220
Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser Ser
225 230 235 290
Thr Gln Thr Thr Ala Thr Thr Thr Gln Asp Gly Val Thr Ile Thr Thr
245 250 255
Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp Ile Thr Asn Asn
260 265 270
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Ala Glu Gln Leu Leu Asn Gln Ala Ala Asn Ile Met Gln Val Leu Asn
275 280 285
Thr Gln Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro Gly
290 295 300
Gly Gly Gln Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser Ile
305 310 315 320
Phe Gln Gln Glu Phe Ser Gln Val Thr Ser Met Ile Lys Asn Ala Gln
325 330 335
Glu Ile Ile Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln
340 345 350
Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser
355 360 365
Phe Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Met Phe
370 375 380
Asn Leu Ser Glu Gln Val Lys Lys Asn Leu Glu Val Met Lys Asn Asn
385 390 395 qp0
Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val Met
405 410 415
Thr Asn Phe Val Ser Rla Phe Leu Ala Ser Cys Lys Asp Gly Gly Thr
420 425 930
Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala
435 440 445
Tyr Val Gly Glu Thr Ile Ser Ala Leu Thr Asn Ser Ile Ala His Phe
450 455 460
Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr
465 470 475 480
Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn
4B5 990 995
Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln
500 505 510
Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu
515 520 525
Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile
530 535 590
Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn
545 550 555 560
Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly
565 570 575
Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr
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580 585 590
Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn
595 600 605
Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr
610 615 620
Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys
625 630 635 640
Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp
645 650 655
Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn
660 665 670
Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val
675 680 685
Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala
690 695 700
Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn
705 710 715 720
Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser
725 730 735
Val Tyr Leu Asn Tyr Val Phe Ala Tyr
790 745
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:4:
ATGAAAAAAACCCTTTTACTCTCTCTCTCT CTCTCTCTCTCGTTTTTGCTCCACGCTGAA 60
GACGACGGCTTTTACACAAGCGTGGGCTAT CAAATCGGTGAAGCCGCTCAAATGGTGAAA 120
AACACCAAAGGCATTCAAGAGCTTTCAGAC AATTATGAAAAGCTGAACAATCTTTTGAAT 180
AATTACAGCACCCTAAACACCCTTATCAAA TTGTCCGCTGATCCGAGCGCGATTAACGAC 240
GCAAGGGATAATCTAGGCTCAAGCTCTAGG AATTTGCTTGATGTCAAAACCAATTCCCCC 300
GCGTATCAAGCCGTGCTTTTAGCACTCAAT GCTGCAGTGGGGTTGTGGCAAGTTACAAGC 360
TACGCTTTTACTGCTTGTGGTCCTGGCAGT AACGAGAATGCGAATGGAGGGATCCAAACT 420
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TTTAATAATGTGCCAGGACAAGATACGACGACCATCACTTGCAATTCGTATTATGAGCCA480
GGACATGGTGGGCCTATATCCACTGCAAATTATGCGAAAATCAATCAAGCCTATCAAATC540
ATCCAAAAGGCTTTGACAGCCAATGGAGCTAATGGAGATGGGGTCCCCGTTTTAAGCAAC600
ACCACTACAAAACTTGATTTCACTATCAATGGAGACAAAAGAACGGGGGGCAAACCAAAT660
ACACCTGAAAAGTTCCCATGGAGTGATGGGAAATATATTCACACCCAATGGATTAACACA720
ATAGTAACACCAACAGAAACAAATATCAACACAGAAAATAACGCTCAAGAGCTTTTAAAA780
CAAGCGAGCATCATTATCACTACCCTAAATGAGGCATGCCCAAACTTCCAAAATGGTGGT840
AGAAGTTATTGGCAAGGGATAAGCGGCAATGGGACAATGTGCGGGATGTTTAAGAATGAA900
ATCAGCGCGATCCAAGGCATGATCGCTAACGCTCAAGAAGCTGTCGCGCAAAGCAAAATC960
GTTAGTGAAAACGCGCAAAATCAAAACAACTTGGATACTGGAAAACCATTCAACCCTTAC1020
ACGGACGCCAGCTTTGCGCAAAGCATGCTCAAAAACGCTCAAGCGCAAGCAGAGATTTTA1080
AACCAAGCCGAACAAGTAGTAAAAAACTTTGAAAAAATCCCTACAGCCTTTGTATCAGAC1190
TCTTTAGGGGTGTGTTATGAAGTGCAAGGGGGTGAGCGTAGGGGCACCAATCCAGGTCAG1200
GTAACTTCTAACACTTGGGGAGCCGGTTGCGCGTATGTGAAACAAACCATAACGAATTTA1260
GACAACAGCATCGCTCACTTTGGCACTCAAGAGCAGCAGATACAGCAAGCCGAAAACATC1320
GCTGACACTCTAGTGAATTTCAAATCTAGATACAGCGAATTAGGCAACACCTATAACAGC1380
ATCACCACCGCGCTCTCCAAAGTCCCTAACGCGCAAAGCTTGCAAAACGTGGTGAGCAAA1440
AAGAATAACCCCTATAGCCCTCAAGGCATAGAGACCAATTACTACCTCAATCAAAATTCT1500
TACAACCAAATCCAAACCATCAACCAAGAACTAGGGCGTAACCCCTTTAGGAAAGTGGGC1560
ATCGTCAATTCTCAAACCAACAATGGTGCCATGAATGGGATCGGTATTCAGGTGGGCTAT1620
AAGCAATTCTTTGGCCAAAAAAGAAAATGGGGCGCTAGGTATTACGGCTTTTTTGACTAC1680
AACCATGCGTTCATTAAATCCAGCTTCTTCAACTCGGCTTCTGATGTGTGGACTTATGGT1790
TTTGGAGCGGACGCTCTTTATAACTTCATCAACGATAAAGCCACCAATTTCTTAGGCAAA1800
AACAACAAGCTTTCCGTGGGGCTTTTTGGAGGGATTGCGTTAGCGGGCACTTCATGGCTT1860
AATTCTGAGTATGTGAATTTAGCCACCGTGAATAACGTCTATAACGCTAAAATGAATGTG1920
GCGAATTTCCAATTCTTATTCAATATGGGAGTGAGGATGAATTTAGCCAGATCCAAGAAA19$0
AAAGGCAGCGATCATGCGGCTCAGCATGGGATTGAACTAGGGCTTAAAATCCCCACCATC2040
AACACGAACTATTATTCTTTCATGGGGGCTGAACTCAAATACAGAAGGCTTTATAGCGTG2100
TATTTGAATTATGTGTTCGCTTACTAA 2127
lU
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(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:2226 basepairs
(B) TYPE:
nucleic
acid
(C) STRANDEDNESS: le
sing
(D) TOPOLOGY:
linear
(ii) MOLECULE omic)
TYPE:
DNA (gen
(xi) SEQUENCE EQ ID
DESCRIPTION: N0:5:
S
ATGAAAAAACACATCCTTTCATTAGCTTTAGGCTCGCTTTTAGTTTCCAC TTTGAGCGCT60
GAAGACGACGGCTTTTACACAAGCGTGGGCTATCAAATCGGTGAAGCCGC TCAAATGGTA120
ACAAACACCAAAGGCATTCAAGATCTTTCAGATCGTTATGAAAGTTTGAA CAACCTTTTG180
ACCCGATACAGCACCCTAAACACCCTGATCAAATTGTCCGCTGATCCGAG CGCGATTAAT240
GCGGCGCGTGAAAATCTGGGCGCGAGCGCGAAGAATTTGATCGGCGATAA AGCCAATTCC300
CCAGCCTATCAAGCGGTGCTTTTAGCGATCAACGCGGCGGTAGGGTTTTG GAATGTCTTA360
GGCTATGCTACGCAATGCGGGGGTAACGCCAATGGTCAAAAAAGCACCTC TTCAACGACC420
ATCTTCAACAACGAGCCAGGGTATCGATCCACTTCCATCACTTGCTCTTT GAACGGGTAT480
ACGCCTGGATACTATGGCCCTATGAGTATTGAGAATTTCAAAAAGCTTAA CGAAGCCTAT590
CAGATCCTCCAAACGGCGTTAAAACAAGGCTTACCCGCGCTCAAAGAAAA CAACAAGAAG600
GTCAATGTTACCTACACTTACACATGCTCAGGGGGAGGGAATAATAACTG CTCGTCAGAA660
GCCACAGGTGTAAGCAATCAAAATGGCGGAACTAAAACCACCACCCAAAC CATAGACGGC720
AAAAGCGTAACCACCACGATCAGTTCAAAAGTCGTTGATAGCACAGCGAG TGGTAACACA780
TCACGTGTCTCCTACACCGAAATCACCAACAAATTAGAAGGTGTGCCTGA TAGCGCTCAA840
GCGCTCTTAGCGCAAGCGAGCACGCTCATTAGCACCATCAACACGGCATG CCCGTTTTTT900
AGTGTAACTAATCAAAGTGGTGGTCCACAGATGGAACCGACTAAAGGGAA GTTGTGCGGT960
TTTACAGAAGAAATCAGCGCGATCCAAAAGATGATCACAGACGCGCAAGA GCTGGTCAAT1020
CAAACGAGCGTCATTAATAGCCATGAACAATCAACCCTAGTGGGCGGTAA TAATGGCAAG1080
CCTTTCAACCCTTTCACGGACGCTCAATTCGCTCAAGGCATGCTCGCTAA CGCTAGCGCG1140
CAAGCTAAAATGCTCAATTTAGCCCATCAAGTGGGGCAAACCATTAACCC TAACAATCTT1200
ACTGGGAATTTTAAAAATTTTGTTACAGGCTTTTTAGCCACATGCAACAA CCCCTCAACA1260
GCTGGCACTGGTGGCACACAAGGTTCAGCTCCAGGCACGGTTACCACTCA AACTTTCGCT1320
TCCGGTTGCGCGTATGTGGAACAAACCATAACGAATTTAGAAAACAGCAT CGCGCACTTT1380
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GGCACTCAAGAGCAGCAAATACAACGAGCCGAAAATATCGCTGACACTCT AGTGAATTTC1440
AAATCTAGATACAGCGAATTGGGGAATACTTACAACAGCATCACCACTGC GCTCTCCAAA1500
GTCCCTAACGCGCAAAGCTTGCAAAACGTGGTGAGCAAAAAGAATAACCC CTATAGCCCG1560
CAAGGCATAGAAACCAATTACTACTTGAATCAAAATTCTTACAACCAAAT CCAAACCATC1620
AACCAAGAATTAGGGCGTAACCCTTTTAGGAAAGTGGGCATCGTCGGCTC TCAAACCAAC1680
AACGGCGCCATGAATGGGATCGGTATTCAGGTGGGCTACGAGCAATTCTT TGGCCAAAAA1740
AGAAAATGGGGCGCTAGGTATTACGGCTTTTTTGATTACAACCATGCGTT TATTAAATCC1800
AGCTTCTTCAACTCGGCTTCTGATGTGTGGACTTATGGTTTTGGAGCGGA CGCTCTCTAT1860
AACTTCATCAACGATAAAGCCACTAACTTTTTAGGCAAAAACAACAAGCT TTCTGTGGGG1920
CTTTTTGGCGGGATTGCGTTAGCGGGCACTTCATGGCTTAATTCTGAGTA TGTGAATTTA1980
GCCACCGTGAATAATGTCTATAACGCTAAAATGAACGTGGCGAACTTCCA ATTCTTATTC2040
AACATGGGAGTGAGGATGAATTTGGCCAGGCCCAAGAAAAACGACAGCGA TCATGCGGCT2100
CAGCATGGGATTGAGTTAGGGCTTAAAATCCCCACCATCAACACGAACTA CTATTCCTTT2160
ATGGGGGCTGAACTCAAATACAGAAGGCTTTATAGCGTGTATTTGAATTA TGTGTTCGCT2220
TACTAG 2226
(2) INFORMATION
FOR SEQ
ID N0:6:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:2238 base
pairs
(B) TYPE:
nucleic
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:
linear
(ii) MOLECULE
TYPE:
DNA (genomic)
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:6:
ATGAAAAAAC ACATCCTTTCATTAGCTTTAGGCTCGCTTTTAGTTTCCACTTTGAGCGCT60
GAAGACGACG GCTTTTACACAAGCGTAGGCTATCAGATCGGTGAAGCCGCTCAAATGGTA120
ACAAACACCA AAGGCATCCAACAGCTTTCAGACAATTATGAAAATTTGAACAACCTTTTA180
ACGAGATACA GCACCCTAAACACCCTTATCAAATTGTCCGCTGATCCGAGCGCAATTAAT240
GCGGTGCGGG AAAATCTGGGCGCGAGCACGAAGAATTTGATCGGCGATAAAGCCAACTCC300
CCGGCGTATC AAGCCGTGTTTTTAGCGATCAACGCGGCGGTAGGGTTGTGGAATACCATC360
GGCTATGCGG TCATGTGCGGGAACGGGAACGGCACAGAGAGTGGGCCTGGCAGCGTGATC420
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TTTAATGACCAACCAGGACAGGATTCCACGCAAATTACTTGCAACCGCTTTGAATCAACT480
GGGCCTGGTAAAAGCATGTCTATTGATGAATTCAAAAAACTCAATGAAGCCTATCAAATC540
ATCCAGCAAGCTTTAAAAAATCAAAGTGGGTTTCCTGAATTAGGCGGGAACGGCACAAAA600
GTGAGTGTTAATTACAATTACGAATGCAGACAAACTGCTGATATCAACGGCGGTGTGTAT660
CAGTTCTGCAAGGCTAAAAATGGTAGTAGTAGCAGTAGTAATGGCGGTAATGGCAGTAGC720
ACGCAAACAACCGCGACAACCACGCAAGACGGCGTAACGATCACCACTACCTATAATAAT780
AACAAAGCCACCGTCAAATTTGACATCACCAATAACGCTGAACAGCTGTTAAATCAAGCG840
GCAAACATCATGCAAGTCCTTAATACGCAATGCCCTTTAGTGCGTTCCACGAATAACGAA900
AACACTCCAGGGGGTGGTCAACCATGGGGTTTAAGCACATCCGGGAATGCGTGCAGCATC960
TTCCAACAAGAATTTAGCCAGGTTACTAGCATGATCAAAAACGCCCAAGAAATAATCGCG1020
CAAAGCAAAATCGTTAGTGAAAACGCGCAAAATCAAAACAACTTGGATACTGGAAAACCA1080
TTCAACCCTTACACGGACGCCAGCTTTGCGCAAAGCATGCTCAAAAACGCTCAAGCGCAA1140
GCAGAGATGTTCAATTTGAGCGAACAAGTGAAAAAGAACTTGGAAGTCATGAAAAACAAC1200
AATAATGTTAACGAGAAATTAGCAGGATTTGGGAAAGAAGAAGTAATGACCAATTTTGTT1260
AGCGCCTTTTTGGCAAGCTGCAAAGATGGTGGCACATTGCCTAATGCAGGGGTTACTTCT1320
AACACTTGGGGGGCGGGTTGCGCGTATGTGGGAGAGACGATAAGCGCCCTAACCAACAGC1380
ATCGCTCACTTTGGCACTCAAGAGCAGCAGATACAGCAAGCCGAAAACATCGCTGACACT1440
CTAGTGAATTTCAAATCTAGATACAGCGAATTAGGCAACACCTATAACAGCATCACCACC1500
GCGCTCTCCAAAGTCCCTAACGCGCAAAGCTTGCAAAACGTGGTGAGCAAAAAGAATAAC1560
CCCTATAGCCCTCAAGGCATAGAGACCAATTACTACCTCAATCAAAATTCTTACAACCAA1620
ATCCAAACCATCAACCAAGAACTAGGGCGTAACCCCTTTAGGAAAGTGGGCATCGTCAAT1680
TCTCAAACCAACAATGGTGCCATGAATGGGATCGGCATTCAGGTGGGCTATAAGCAATTC1740
TTTGGCCAAAAAAGAAAATGGGGCGCTAGGTATTACGGCTTTTTTGATTACAACCATGCG1800
TTCATCAAATCCAGCTTTTTCAACTCGGCTTCTGACGTGTGGACTTATGGTTTTGGAGCG1860
GACGCGCTTTATAACTTCATCAACGATAAAGCCACCAATTTCTTAGGCAAAAACAACAAG1920
CTTTCTTTGGGGCTTTTTGGCGGGATTGCGTTAGCGGGCACTTCATGGCTCAATTCTGAG1980
TACGTGAATTTAGCCACCGTGAATAACGTCTATAACGCTAAAATGAATGTGGCGAATTTC2040
CAATTCTTATTCAATATGGGAGTGAGGATGAATTTAGCCAGATCCAAGAAAAAAGGCAGC2100
GATCATGCAGCTCAGCATGGGATTGAGTTAGGGCTTAAAATCCCCACCATCAACACGAAC2160
TATTATTCCTTTATGGGGGCTGAACTCAAATACAGAAGGCTCTATAGCGTGTATTTGAAC2220
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TATGTGTTCG CTTACTAA 2238
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala
1 5 10 15
Ala Gln Met Val
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
GARGAYGAYG GNTTYTAYAC NWSNGTNGGN TAYCARATHG GNGARGCNGC NCARATGGTN 60
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GGCCATATGA AAAAACACAT CCTTTCATTA GCTTTAGGCT CG 42
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GGCAAGCTTG GGAGTTTCAC AAAAAGCTTA GTAAGCGAAC AC 42
