U.S. patent application number 11/876671 was filed with the patent office on 2009-03-12 for enterohemorrhagic escherichia coli vaccine.
Invention is credited to B. Brett Finlay, Andrew A. Potter.
Application Number | 20090068230 11/876671 |
Document ID | / |
Family ID | 22986519 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068230 |
Kind Code |
A1 |
Finlay; B. Brett ; et
al. |
March 12, 2009 |
ENTEROHEMORRHAGIC ESCHERICHIA COLI VACCINE
Abstract
Compositions and methods for stimulating an immune response
against a secreted enterohemorragic Escherichia coli (EHEC) antigen
are disclosed. The compositions comprise EHEC cell culture
supernatants.
Inventors: |
Finlay; B. Brett; (Richmond,
CA) ; Potter; Andrew A.; (Saskatchewan, CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR, 2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Family ID: |
22986519 |
Appl. No.: |
11/876671 |
Filed: |
October 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10039760 |
Jan 3, 2002 |
7300659 |
|
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11876671 |
|
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60259818 |
Jan 4, 2001 |
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Current U.S.
Class: |
424/257.1 |
Current CPC
Class: |
A61P 37/04 20180101;
Y02A 50/30 20180101; A61P 31/04 20180101; A61P 31/06 20180101; A61P
31/00 20180101; A61K 2039/55566 20130101; A61K 39/0258 20130101;
C07K 14/245 20130101; Y02A 50/474 20180101 |
Class at
Publication: |
424/257.1 |
International
Class: |
A61K 39/108 20060101
A61K039/108; A61P 31/06 20060101 A61P031/06; A61P 37/04 20060101
A61P037/04 |
Claims
1. A method for eliciting an immunological response in a mammal
against a secreted enterohemorragic Escherichia coli (EHEC)
antigen, said method comprising administering to said mammal a
therapeutically effective amount of a composition comprising an
EHEC cell culture supernatant.
2. The method of claim 1, wherein the EHEC is EHEC O157:H7.
3. The method of claim 1, wherein the mammal is a ruminant.
4. The method of claim 3, wherein the ruminant is a bovine
subject.
5. The method of claim 1, wherein the composition further comprises
an immunological adjuvant.
6. The method of claim 2, wherein the composition further comprises
an immunological adjuvant.
7. The method of claim 5, wherein the immunological adjuvant
comprises an oil-in-water emulsion.
8. The method of claim 6, wherein the immunological adjuvant
comprises an oil-in-water emulsion.
9. The method of claim 7, wherein the immunological adjuvant
comprises a mineral oil and dimethyldioctadecylarnrnonium
bromide.
10. The method of claim 8, wherein the immunological adjuvant
comprises a mineral oil and dimethyldioctadecylarnrnonium
bromide.
11. The method of claim 9, wherein the immunological adjuvant is
VSA3.
12. The method of claim 10, wherein the immunological adjuvant is
VSA3.
13. The method of claim 1, wherein the composition further
comprises one or more recombinant or purified EHEC antigens
selected from the group consisting of EspA, EspB, EspD, Tir and
Intimin.
14. The method of claim 13, wherein EspA+Tir comprise at least 20%
of the cell protein present in the composition.
15. The method of claim 1, wherein said composition is not
supplemented with recombinant antigens.
16. The method of claim 15, wherein Type III antigens comprise at
least 5% of the total protein of said cell culture supernatant.
17. The method of claim 15, wherein Type III antigens comprise at
least 10% of the total protein of said cell culture
supernatant.
18. The method of claim 15, wherein Type III antigens EspA+Tir
comprise at least 10% of the total protein of said cell culture
supernatant.
19. A method for eliciting an immunological response in a ruminant
against a secreted enterohemorragic Escherichia coli O157:H7 (EHEC
O157:H7) antigen, said method comprising administering to said
ruminant a therapeutically effective amount of a composition
comprising an EHEC O157:H7 cell culture supernatant and VSA3.
20. The method of claim 19, wherein VSA3 is present in the
composition at a concentration of about 20% to about 40% (v/v).
21. The method of claim 20, wherein VSA3 is present in the
composition at a concentration of about 30% (v/v).
22. The method of claim 19, wherein said composition is not
supplemented with recombinant antigens.
23. The method of claim 22, wherein Type III antigens comprise at
least 5% of the total protein of said cell culture supernatant.
24. The method of claim 22, wherein Type III antigens comprise at
least 10% of the total protein of said cell culture
supernatant.
25. The method of claim 22, wherein Type III antigens EspA+Tir
comprise at least 10% of the total protein of said cell culture
supernatant.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/039,760 filed Jan. 3, 2002 which claims benefit U.S.
provisional application No. 60/259,818 filed Jan. 4, 2001, the
contents of which are incorporated herein by reference in their
entirety.
FIELD
[0002] The present invention relates to compositions and methods
for eliciting an immune response in mammals against
enterohemorragic Escherichia coli. In particular, the invention
relates to the use of cell culture supernatants for treating and
preventing enterohemorragic E. coli colonization of mammals.
BACKGROUND
[0003] Enterohemorragic Escherichia coli (EHEC), also called Shiga
toxin E. coli (STEC) and vertotoxigenic E. coli (VTEC) are
pathogenic bacteria that cause diarrhea, hemorrhagic colitis,
hemolytic uremic syndrome, kidney failure and death in humans.
While many Shiga-like toxinproducing EHEC strains are capable of
causing disease in humans, those of serotype O157:H7 cause the
majority of human illness. This organism is able to colonize the
large intestine of humans by a unique mechanism in which a number
of virulence determinants are delivered to host cells via a type
III secretion system, including the translocated Intimin receptor,
Tir (DeVinney et al., Infect. Immun. (1999) 67:2389). In
particular, these pathogens secrete virulence determinants EspA,
EspB and EspD that enable delivery of Tir into intestinal cell
membranes. Tir is integrated into the host cell membrane where it
serves as the receptor for a bacterial outer membrane protein,
Intimin. Tir-Intimin binding attaches EHEC to the intestinal cell
surface and triggers actin cytoskeletal rearrangements beneath
adherent EHEC that results in pedestal formation. EspA, EspB, Tir
and Intimin are each essential for the successful colonization of
the intestine by EHEC.
[0004] Although EHEC colonize the intestine of ruminants and other
mammals, they generally do not cause overt disease in these
animals. However, contamination of meat and water by the EHEC
serotype O157:H7 (hereinafter, "EHEC O157:H7") is responsible for
about 50,000 cases of EHEC O157:H7 infection in humans annually in
the United States and Canada that result in approximately 500
deaths. In 1994, the economic cost associated with EHEC O157:H7
infection in humans was estimated to be over 5 billion dollars
annually.
[0005] The first documented EHEC O157:H7 outbreak traced to
contaminated meat occurred in 1982. Subsequently, it was
demonstrated that healthy ruminants including, but not limited to,
cattle, dairy cows and sheep, could be infected with EHEC O157:H7.
In fact, USDA reports indicate that up to 50% of cattle are
carriers of EHEC O157:H7 at some time during their lifetime and,
therefore, shed EHEC O157:H7 in their feces.
[0006] Because of the bulk processing of slaughtered cattle and the
low number of EHEC O157:H7 (10-100) necessary to infect a human,
EHEC O157:H7 colonization of healthy cattle remains a serious
health problem. To address this problem, research has focused on
improved methods for detecting and subsequently killing EHEC
O157:H7 at slaughter, altering the diet of cattle to reduce the
number of intestinal EHEC O157:H7 and immunizing animals to prevent
EHEC O157:H7 colonization (Zacek D. Animal Health and Veterinary
Vaccines, Alberta Research Counsel, Edmonton, Canada, 1997).
Recently, the recombinant production and use of EHEC O157:H7
proteins including recombinant EspA (International Publication No.
WO 97/40063), recombinant Tlr (International Publication No. WO
99/24576), recombinant EspB and recombinant Initimin (Li et al.,
Infec. Immun. (2000) 68:5090-5095) have been described. However,
production and purification of recombinant proteins in amounts
sufficient for use as antigens is both difficult and expensive. At
the present time, there is no effective method for blocking EHEC
O157:H7 colonization of cattle and other mammals and, thereby, for
reducing shedding of EHEC into the environment.
[0007] Therefore, there is a need for new compositions and methods
for treating and preventing EHEC disease, as well as for reducing
EHEC colonization of mammals in order to reduce the incidence of
health problems associated with EHEC-contaminated meat and
water.
SUMMARY
[0008] The present invention satisfies the above need by providing
such compositions and methods. In particular, the methods of the
present invention make use of a composition comprising a cell
culture supernatant (hereinafter "CCS") derived from an EHEC
culture to elicit an immune response against one or more EHEC
secreted antigens, thereby treating and/or preventing EHEC
infection and/or reducing EHEC colonization of the mammal. The
compositions can be delivered with or without a coadministered
adjuvant. In certain embodiments, EspA and Tir comprise at least
20% of the cell culture supernatant protein. The EHEC culture
supernatant may be derived from any EHEC serotype, but is
preferably obtained from a culture of EHEC O157:H7 and/or EHEC
0157:NM (non-motile). The cell culture supernatant of the present
invention is easy and relatively inexpensive to prepare and is
effective at dose regimens that have minimal toxicity.
[0009] EspA, EspB, Tir and Intimin are necessary for activation (A)
of host epithelial cell signal transduction pathways and for the
intimate attachment (E) of EHEC to host epithelial cells.
Therefore, without being bound by the following hypothesis, it is
thought that administration of the CCS of the present invention to
a mammal stimulates an immune response against one or more secreted
antigens, such as EspA and Tir, that blocks attachment of the EHEC
to intestinal epithelial cells.
[0010] Accordingly, it is an object of the present invention to
provide a vaccine effective to stimulate an immune response against
EHEC secreted antigens, thereby treating and/or preventing EHEC
disease in a mammal.
[0011] Another object is to provide a vaccine effective to reduce,
prevent and/or eliminate EHEC colonization of a ruminant or other
mammal.
[0012] Another object is to reduce the number of animals shedding
EHEC into the environment.
[0013] Another object is to reduce the number of EHEC shed into the
environment by an infected animal.
[0014] Another object is reduce the time during which EHEC are shed
into the environment by an infected animal.
[0015] Another object is reduce EHEC contamination of the
environment.
[0016] Another object is reduce EHEC contamination of meat and/or
water.
[0017] Another object is to treat, prevent and/or reduce EHEC
infections in humans.
[0018] Another object is to provide a vaccine effective as an
adjunct to other biological anti-EHEC agents.
[0019] Another object is to provide a vaccine effective as an
adjunct to chemical anti-EHEC agents.
[0020] Another object is to provide a vaccine effective as an
adjunct to biologically engineered anti-EHEC agents.
[0021] Another object is to provide a vaccine effective as an
adjunct to nucleic acid-based anti-EHEC agents.
[0022] Another object is to provide a vaccine effective as an
adjunct to recombinant protein anti-EHEC agents.
[0023] Another object is to provide a vaccination schedule
effective to reduce EHEC colonization of a ruminant.
[0024] Another object is to provide a vaccination schedule
effective to reduce EHEC shedding by a ruminant.
[0025] Another object is to provide a vaccine effective to reduce
EHEC 0157 colonization of cattle, such as colonization of EHEC
O157:H7 and/or EHEC 0157:NM.
[0026] Another object is to provide a vaccine effective to prevent
EHEC 0157 colonization of cattle, such as colonization of EHEC
O157:H7 and/or EHEC 0157:NM.
[0027] Another object is to provide a vaccine effective to
eliminate EHEC 0157 colonization of cattle, such as colonization of
EHEC O157:H7 and/or EHEC 0157:NM.
[0028] Another object is to reduce the number of cattle shedding
EHEC 0157 into the environment, such as shedding of EHEC O157:H7
and/or EHEC 0157:NM.
[0029] Another object is to reduce the number of EHEC 0157 shed
into the environment by infected cattle, such as shedding of EHEC
O157:H7 and/or EHEC 0157:NM.
[0030] Another object is reduce the time during which EHEC 0157 are
shed into the environment by infected cattle, such as shedding of
EHEC O157:H7 and/or EHEC 0157:NM.
[0031] Another object is to provide a vaccine effective as an
adjunct to other anti-EHEC 0157 agents.
[0032] Another object is to provide a vaccination schedule
effective to reduce EHEC 0157 colonization of cattle.
[0033] Another object is to provide a vaccination schedule
effective to reduce EHEC 0157 shedding by cattle.
[0034] Thus, in one embodiment, the invention is directed to a
vaccine composition comprising an enterohemorragic Escherichia coli
(EHEC) cell culture supernatant and an immunological adjuvant. In
certain embodiments, the EHEC is EHEC O157:H7 and/or EHEC 0157:NM.
In additional embodiments, the immunological adjuvant comprises an
oil-in-water emulsion, such as a mineral oil and
dimethyldioctadecylammonium bromide. In yet additional embodiments,
the immunological adjuvant is VSA3. The VSA3 may be present at a
concentration of about 20% to about 40% (v/v), such as at a
concentration of 30% (v/v).
[0035] In still further embodiments, the vaccine composition
further comprises one or more recombinant or purified EHEC secreted
antigens selected from the group consisting of EspA, EspB, EspD and
Tir. In other embodiments, EspA+Tir comprise at least 20% of the
cell protein present in the composition.
[0036] In further embodiments, the subject invention is directed to
methods for eliciting an immunological response in a mammal against
a secreted enterohemorragic Escherichia coli (EHEC) antigen. The
method comprises administering to the mammal a therapeutically
effective amount of a composition comprising an EHEC cell culture
supernatant. In certain embodiments, the EHEC is EHEC O157:H7
and/or EHEC 0157:NM. In additional embodiments, the mammal is a
human or a ruminant, such as a bovine subject. In yet further
embodiments, the composition further comprises an immunological
adjuvant, such as an oil-in-water emulsion which comprises e.g., a
mineral oil and dimethyldioctadecylammonium bromide. In additional
embodiments, the adjuvant is VSA3. The compositions may further
comprise one or more recombinant or purified EHEC secreted antigens
selected from the group consisting of EspA, EspB, EspD and Tir. In
other embodiments, EspA+Tir comprise at least 20% of the cell
protein present in the composition.
[0037] In another embodiment, the invention is directed to a method
for eliciting an immunological response in a ruminant against a
secreted enterohemorragic Escherichia coli O157:H7 (EHEC O157:H7)
antigen. The method comprises administering to the ruminant a
therapeutically effective amount of a composition comprising an
EHEC O157:H7 cell culture supernatant and VSA3. In additional
embodiments, VSA3 is present in the composition at a concentration
of about 20% to about 40% (v/v), such as at about 30% (v/v).
[0038] In still a further embodiment, the invention is directed to
a method for reducing colonization of enterohemorragic Escherichia
coli (EHEC) in a ruminant comprising administering to the ruminant
a therapeutically effective amount of a composition comprising an
EHEC cell culture supernatant and an immunological adjuvant.
[0039] In yet another embodiment, the invention is directed to a
method for reducing shedding of enterohemorragic Escherichia coli
(EHEC) from a ruminant comprising administering to the ruminant a
therapeutically effective amount of a composition comprising an
EHEC cell culture supernatant and an immunological adjuvant.
[0040] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, various references are set forth
herein which describe in more detail certain procedures or
compositions, and are therefore incorporated by reference in their
entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 shows the electrophoretic profile of CCS proteins
separated by polyacrylamide gel electrophoresis.
[0042] FIG. 2 shows the electrophoretic profile of recombinant
EspA, Tir, EspB and Intimin separated by polyacrylamide gel
electrophoresis.
[0043] FIG. 3 shows fecal shedding of EHEC O157:H7 by cattle
immunized with a CCS vaccine following EHEC O157:H7 challenge.
[0044] FIG. 4 depicts reactivation of fecal shedding of EHEC
O157:H7 in previously infected cattle.
[0045] FIG. 5 shows the serological response to immunization with
recombinant EspA+Tir vaccine and with recombinant EspB+Intimin
vaccine.
[0046] FIG. 6 depicts fecal shedding of EHEC O157:H7 following
immunization with recombinant EspA+Tir vaccine and with saline
vaccine.
[0047] FIG. 7 shows the number of animals shedding E. coli O157:H7
on each day of the vaccine trial described in Example 6. Bacteria
were detected by direct plating of fecal samples which had been
resuspended in saline on Sorbitol MaConkey agar supplemented with
cefixime and tellurite. Solid bars, placebo group; hatched bars,
EHEC vaccine group.
[0048] FIG. 8 shows an immunoblot analysis of sera from vaccinated
animals against EHEC secreted proteins. Each blot contains secreted
proteins from wild-type E. coli O157:H7 (EHEC), type III secretion
mutant (.DELTA.SepB), tir mutant (.DELTA.Tir) and a purified
glutathione-s-transferase:Tir fusion protein (GST-Tir). Proteins
were separated by SDS-10% PAGE and stained with Coomassie blue (A,
upper left panel) or transferred to nitrocellulose and probed with
representative sera from animals which received 3 immunizations
with each vaccine formulation (A, upper panels). The lower four
panels (B) were probed with sera from one representative animal
which received the EHEC vaccine, taken on days 0, 21, 25 and 49 of
the trial.
[0049] FIG. 9 shows the percentage of each group of animals
shedding E. coli O157:H7 (Panel A) and the total number of bacteria
recovered (Panel B) on each day of the trial described in Example
6. Bacteria were detected in feces by plating on Sorbitol MaConkey
agar supplemented with cefixime and tellurite following
immunomagnetic enrichment as described in J. Van Donkersgoed et
al., Can. Vet. J (2001) 42:714. (A) Solid bars, placebo; hatched
bars, EHEC vaccine; open bars, .DELTA.Tir vaccine. (B) .box-solid.,
placebo group; EHEC vaccine; .tangle-solidup., .DELTA.Tir
vaccine.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0050] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA technology, and immunology, which are
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Sambrook, Fritsch & Maniatis,
Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second
Edition (1989); Perbal, B., A Practical Guide to Molecular Cloning
(1984); the series, Methods In Enzymology (S. Colowick and N.
Kaplan eds., Academic Press, Inc.); and Handbook of Experimental
Immunology, Vols. I-IV D. M. Weir and C. C. Blackwell eds., 1986,
Blackwell Scientific Publications).
[0051] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
A. Definitions
[0052] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0053] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "an EHEC bacterium" includes a
mixture of two or more such bacteria, and the like.
[0054] As used herein, the term EHEC "cell culture supernatant" or
"CCS" refers to a supernatant derived from a cell culture of one or
more EHEC serotypes, which supernatant is substantially free of
EHEC bacterial cells or the lysate of such cells, and which
contains a mixture of EHEC antigens that have been secreted into
the growth media. Generally, an EHEC "CCS" will contain at least
the secreted antigens EspA, EspB, EspD and Tir, and fragments or
aggregates thereof. The CCS of the present invention may also
include other secreted proteins, such as EspF and MAP, one or both
of Shiga toxins 1 and 2, as well as EspP which is an approximately
100 kDa protein which is not secreted by the type III system. The
proteins can be present in a native form, or a denatured or
degraded form, so long as the CCS still functions to stimulate an
immune response in the host subject such that EHEC disease is
lessened or prevented, and/or colonization of EHEC is lessened or
suppressed. In some instances, a CCS may be supplemented with
additional recombinant or purified secreted antigens, such as with
additional EspA, EspB, EspD and/or Tir, as well as with any of the
other secreted proteins, and may also be supplemented with Intimin.
In certain embodiments, EspA+Tir will comprise at least 20% of the
cell culture supernatant protein.
[0055] As used herein, a "recombinant" EHEC secreted protein, such
as rEspA, rEspB, rEspD and rTir, as well as the "recombinant
Intimin", refers to the full-length polypeptide sequence, fragments
of the reference sequence or substitutions, deletions and/or
additions to the reference sequence, so long as the proteins retain
at least one specific epitope or activity. Generally, analogs of
the reference sequence will display at least about 50% sequence
identity, preferably at least about 75% to 85% sequence identity,
and even more preferably about 90% to 95% or more sequence
identity, to the full-length reference sequence. See, e.g., GenBank
Accession Nos. AE005594, AE005595, AP002566, AE005174,
NC.sub.--002695, NC.sub.--002655 for the complete sequence of the
E. coli O157:H7 genome, which includes the sequences of the various
O157:H7 secreted proteins. See, e.g., International Publication No.
WO 97140063, as well as GenBank Accession Nos. Y 13068, U80908,
U5681,254352, AJ225021, AJ225020, AJ225019, AJ225018, AJ225017,
AJ225016, AJ225015, AF022236 and AF200363 for the nucleotide and
amino acid sequences of EspA from a number of E. coli serotypes.
See, e.g., International Publication No. WO 99124576, as well as
GenBank Accession Nos. AF125993, AF132728, AF045568, AF022236,
AF70067, AF070068, AF013122, AF200363, AF113597, AF70069, AB036053,
AB026719, U5904 and U59502, for the nucleotide and amino acid
sequences of Tir from a number of E. coli. Sterotype. See, e.g.,
GenBank Accession Nos. U32312, U38618, U59503, U66102, AF081183,
AF081182, AF130315, AF339751, AJ308551, AF301015, AF329681,
AF319597, AJ275089-AJ275113 for the nucleotide and amino acid
sequences of Intimin from a number of E. coli serotypes. See, e.g.,
GenBank Accession Nos. U80796, U65681, Y13068, Y13859, X96953,
X99670, X96953,221555, AF254454, AF254455, AF254456, AF254457,
AF054421, AF059713, AF144008, AF144009 for the nucleotide and amino
acid sequences of EspB from a number of E. coli serotypes. See,
e.g., GenBank Accession Nos. Y 13068, Y13859, Y17875, Y17874,
Y09228, 25 U65681, AF054421 and AF064683, for the nucleotide and
amino acid sequences of EspD from a number of E. coli
serotypes.
[0056] "Homology" refers to the percent similarity between two
polynucleotide or two polypeptide moieties. Two DNA, or two
polypeptide sequences are "substantially homologous" to each other
when the sequences exhibit at least about 80%-85%, preferably at
least about 90%, and most preferably at least about 95%-98%
sequence similarity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified DNA or polypeptide sequence.
[0057] Percent sequence identity can be determined by a direct
comparison of the sequence information between two molecules by
aligning the sequences, counting the exact number of matches
between the two aligned sequences, dividing by the length of the
shorter sequence, and multiplying the result by 100. Readily
available computer programs can be used to aid in the analysis,
such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and
Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National
biomedical Research Foundation, Washington, D.C., which adapts the
local homology algorithm of Smith and Waterman (1981) Advances in
Appl. Math. 2:482-489 for peptide analysis. Programs for
determining nucleotide sequence identity are available in the
Wisconsin Sequence Analysis Package, Version 8 (available from
Genetics Computer Group, Madison, Wis.) for example, the BESTFIT,
FASTA and GAP programs, which also rely on the Smith and Waterman
algorithm. These programs are readily utilized with the default
parameters recommended by the manufacturer and described in the
Wisconsin Sequence Analysis Package referred to above. For example,
percent identity of a particular nucleotide sequence to a reference
sequence can be determined using the homology algorithm of Smith
and Waterman with a default scoring table and a gap penalty of six
nucleotide positions.
[0058] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0059] As used herein, "vaccine" refers to a CCS composition that
serves to stimulate an immune response to an EHEC antigen, such as
a type III secreted EHEC antigen, therein. The immune response need
not provide complete protection and/or treatment against EHEC
infection or against colonization and shedding of EHEC. Even
partial protection against colonization and shedding of EC bacteria
will find use herein as shedding and contaminated meat production
will still be educed. In some cases, a vaccine will include an
immunological adjuvant in order to enhance the immune response. The
term "adjuvant" refers to an agent which acts in a nonspecific
manner to increase an immune response to a particular antigen or
combination of antigens, thus reducing the quantity of antigen
necessary in any given vaccine, and/or the frequency of injection
necessary in order to generate an adequate immune response to the
antigen of interest. See, e.g., A. C. Allison J. Reticuloendothel.
Soc. (1979) 26:619-630. Such adjuvants are described further
below.
[0060] As used herein, "colonization" refers to the presence of
EHEC in the intestinal tract of a mammal, such as a ruminant.
[0061] As used herein, "shedding" refers to the presence of EHEC in
feces.
[0062] As used herein, "therapeutic amount", "effective amount" and
"amount effective to" refer to an amount of vaccine effective to
elicit an immune response against a secreted antigen present in the
CCS, thereby reducing or preventing EHEC disease, and/or EHEC
colonization of a mammal such as a ruminant; and/or reducing the
number of animals shedding EHEC; and/or reducing the number of EHEC
shed by an animal; and/or, reducing the time period of EHEC
shedding by an animal.
[0063] As used herein, "immunization" or "immunize" refers to
administration of CCS, with or without additional recombinant or
purified EHEC antigens such as EspA, Tir, EspB, EspD, and/or
Intimin, in an amount effective to stimulate the immune system of
the animal to which the CCS is administered, to elicit an
immunological response against one or more of the secreted antigens
present in the CCS.
[0064] The term "epitope" refers to the site on an antigen or
hapten to which specific B cells and/or T cells respond. The term
is also used interchangeably with "antigenic determinant" or
"antigenic determinant site."
[0065] An "immunological response" to a composition or vaccine is
the development in the host of a cellular and/or antibody-mediated
immune response to the composition or vaccine of interest. Usually,
an "immunological response" includes but is not limited to one or
more of the following effects: the production of antibodies, B
cells, helper T cells, suppressor T cells, and/or cytotoxic T cells
and/or .gamma..delta. cells, directed specifically to an antigen or
antigens included in the composition or vaccine of interest.
Preferably, the host will display either a therapeutic or
protective immunological response such that EHEC disease is
lessened and/or prevented; resistance of the intestine to
colonization with EHEC is imparted; the number of animals shedding
EHEC is reduced; the number of EHEC shed by an animal is reduced;
and/or the time period of EHEC shedding by an animal is
reduced.
[0066] The terms "immunogenic" protein or polypeptide refer to an
amino acid sequence which elicits an immunological response as
described above. An "immunogenic" protein or polypeptide, as used
herein, includes the full-length sequence of the particular EHEC
protein in question, analogs thereof, aggregates, or immunogenic
fragments thereof. By "immunogenic fragment" is meant a fragment of
a secreted EHEC protein which includes one or more epitopes and
thus elicits the immunological response described above. Such
fragments can be identified using any number of epitope mapping
techniques, well known in the art. See, e.g., Epitope Mapping
Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.
Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear
epitopes may be determined by e.g., concurrently synthesizing large
numbers of peptides on solid supports, the peptides corresponding
to portions of the protein molecule, and reacting the peptides with
antibodies while the peptides are still attached to the supports.
Such techniques are known in the art and described in, e.g., U.S.
Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA
81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all
incorporated herein by reference in their entireties. Similarly,
conformational epitopes are readily identified by determining
spatial conformation of amino acids such as by, e.g., x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols, supra. Antigenic regions of
proteins can also be identified using standard antigenicity and
hydropathy plots, such as those calculated using, e.g., the Omiga
version 1.0 software program available from the Oxford Molecular
Group. This computer program employs the Hopp/Woods method, Hopp et
al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining
antigenicity profiles, and the Kyte-Doolittle technique, Kyte et
al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.
[0067] Immunogenic fragments, for purposes of the present
invention, will usually include at least about 3 amino acids,
preferably at least about 5 amino acids, more preferably at least
about 10-15 amino acids, and most preferably 25 or more amino
acids, of the parent EHEC secreted protein molecule. There is no
critical upper limit to the length of the fragment, which may
comprise nearly the full-length of the protein sequence, or even a
fusion protein comprising two or more epitopes of the particular
EHEC secreted protein.
[0068] "Native" proteins or polypeptides refer to proteins or
polypeptides isolated from the source in which the proteins
naturally occur. "Recombinant" polypeptides refer to polypeptides
produced by recombinant DNA techniques; i.e., produced from cells
transformed by an exogenous DNA construct encoding the desired
polypeptide. "Synthetic" polypeptides are those prepared by
chemical synthesis.
[0069] The term "treatment" as used herein refers to either (i) the
prevention of infection or reinfection (prophylaxis), or (ii) the
reduction or elimination of symptoms of the disease of interest
(therapy).
[0070] By "mammalian subject" is meant any member of the class
Mammalia, including humans and all other mammary gland possessing
animals (both male and female), such as ruminants, including, but
not limited to, bovine, porcine and Ovis (sheep and goats) species.
The term does not denote a particular age. Thus, adults, newborns,
and fetuses are intended to be covered.
B. General Methods
[0071] Central to the present invention is the discovery that cell
culture supernatants derived from EHEC cultures which contain EHEC
secreted antigens, produce an immune response in animals to which
they are administered and thereby provide protection against EHEC
infection, such as protection against colonization. In certain
embodiments, the compositions comprise a mixture of EHEC secreted
antigens, including but not limited to EspA, EspB, EspD and/or Tir.
The CCS of the present invention may also include other secreted
proteins, such as EspF and MAP, one or both of Shiga toxins 1 and
2, as well as EspP which is an approximately 100 kDa protein which
is not secreted by the type I11 system. In other embodiments, the
CCS is supplemented with additional recombinant or purified EHEC
antigens, such as with additional EspA, EspB, EspD, Tir, Intimin,
and the like. In certain embodiments, EspA+Tir comprise at least
20% of the cell culture supernatant protein. The compositions can
comprise cell culture supernatants and additional adjuvants from
more than one EHEC serotype to provide protection against multiple
EHEC organisms. Moreover, a pharmaceutically acceptable adjuvant
may be administered with the cell culture supernatant. The
compositions are administered in an amount effective to elicit an
immune response to one or more of the secreted antigens, thereby
reducing or eliminating EHEC infection. In some instances, EHEC
colonization of the animal is reduced or eliminated. In preferred
embodiments, the animal is a cow or a sheep or other ruminant. In
particularly preferred embodiments, the cell culture supernatant is
derived from a cell culture of EHEC O157:H7 or EHEC 0157:NM.
[0072] Immunization with CCS stimulates the immune system of the
immunized animal to produce antibodies against one or more secreted
EHEC antigens, such as EspA, EspB, EspD and Tir, that block EHEC
attachment to intestinal epithelial cells, interfere with EHEC
colonization and, thereby, reduce EHEC shedding by the animal. This
reduction in EHEC shedding results in a reduction in EHEC
contamination of food and water and a reduction in EHEC-caused
disease in humans. Moreover, the unexpected and surprising ability
of CCS immunization to prevent, reduce and eliminate EHEC
colonization and shedding by cattle addresses a long-felt
unfulfilled need in the medical arts, and provides an important
benefit for humans.
[0073] Additionally, the CCS of the present invention can be used
to treat or prevent EHEC infections in other mammals such as
humans. If used in humans, the CCS can be produced from a mutated
EHEC which has been engineered to hock out one or both of the Shiga
toxins 1 and 2 in order to reduce toxicity.
[0074] As explained above, the therapeutic effectiveness of CCS can
be increased by adding thereto one or more of the secreted antigens
in recombinant or purified form, such as by adding recombinant or
purified EspA, EspB, EspD, Tir, and the like, fragments thereof
and/or analogs thereof. Intimin may also be added. Other methods to
increase the therapeutic effectiveness of CCS include, but are not
limited to, complexing the CCS to natural or synthetic carriers and
administering the CCS, before, at the same time as, or after
another anti-EHEC agent. Such agents include, but are not limited
to, biological, biologically engineered, chemical, nucleic acid
based and recombinant protein anti-EHEC agents.
[0075] CCS from pathogenic bacteria, other than serotypes of EHEC,
that require proteins such as EspA and Tir to colonize a host, can
also be used to stimulate the immune system of an animal to produce
antibodies against secreted EHEC antigens that reduce bacterial
binding to intestinal epithelial cells of the animal. These
bacterial species include, but are not limited to Citrotobactev
rodentium.
[0076] The CCS for use herein may be obtained from cultures of any
EHEC serotype, including, without limitation, EHEC serotypes from
serogroups O157, O158, O5, O8, O18, O26, O45, O48, O52, O55, O75,
O76, O78, O84, O91, O103, O104, O111, O113, O114, O116, O118, O119,
O121, O125, O28, O145, O146, O163, O165. Such EHEC serotypes are
readily obtained from sera of infected animals. Methods for
isolated EHEC are well known in the art. See, e.g., Elder et al.,
Proc. Natl. Acad. Sci. USA (2000) 97:2999; Van Donkersgoed et al.,
Can. Vet. J. (1999) 40:332; Van Donkersgoed et al., Can. Vet. J.
(2001) 42:714. Generally, such methods entail direct plating on
sorbitol MacConkey agar supplemented with cefixime and tellurite or
immunomagnetic enrichment followed by plating on the same media.
Moreover, CCS may be obtained from EHEC serotypes that have been
genetically engineered to knock-out expression of Shiga toxins 1
and/or 2, in order to reduce toxicity.
[0077] Generally, CCS is produced by culturing EHEC bacteria in a
suitable medium, under conditions that favor type III antigen
secretion. Suitable media and conditions for culturing EHEC
bacteria are known in the art and described in e.g., U.S. Pat. Nos.
6,136,554 and 6,165,743 (incorporated herein by reference in their
entireties), as well as in Li et al., Infec. Immun. (2000) 68:
5090-5095; Fey et al., Emerg. Infect. Dis. (2000) Volume 6. A
particularly preferable method of obtaining CCS is by first growing
organisms in Luria-Bertani (LB) medium for a period of about 8 to
48 hours, preferably about 12 to 24 hours, and diluting this
culture about 1:5 to 1:50, preferably 1:5 to 1:25, more preferably
about 1:10, into M-9 minimal medium supplemented with 20-100 mM
NaHCO.sub.3, preferably 30-50 mM, most preferably about 44 mM
NaHCO.sub.3, 4-20 mM MgSO.sub.4, preferably 5-10 mM and most
preferably about 8 mM MgSO.sub.4, 0.1 to 1.5% glucose, preferable
0.2 to 1%, most preferably 0.4% glucose and 0.05 to 0.5% Casamino
Acids, preferably 0.07 to 0.2%, most preferably about 0.1% Casamino
Acids. Cultures are generally maintained at about 37 degrees C. in
2-10% CO.sub.2, preferably about 5% CO.sub.2, to an optical density
of about 600 nm of 0.7 to 0.8. Whole cells are then removed by
centrifugation and the supernatant can be concentrated, e.g.,
10-1000 fold or more, such as 100-fold, using dialysis,
ultrafiltration and the like. Total protein is easily determined
using methods well known in the art.
[0078] As explained above, the CCS can be supplemented with
additional EHEC secreted proteins, such as EspA, EspB, EspD and/or
Tir. Intimin may also be added. These proteins can be produced
recombinantly using techniques well known in the art. See, e.g.,
International Publication Nos. WO 97140063 and WO 99124576 for a
description of the production of representative recombinant EHEC
secreted proteins. In particular, the sequences for EspA, EspB,
EspD, Tir and Intimin from various serotypes are known and
described. See, e.g., GenBank Accession Nos. AE005594, AE005595,
AP002566, AE005174, NC.sub.--002695, NC.sub.--002655 for the
complete sequence of the E. coli O157:H7 genome, which includes the
sequences of the various O157:H7 secreted proteins. See, e.g.,
International Publication No. WO 97/40063, as well as GenBank
Accession Nos. Y13068, U80908, U56817Z54352, AJ225021, AJ225020,
AJ225019, AJ225018, AJ225017, AJ225016, AJ225015, AF022236 and
AF200363 for the nucleotide and amino acid sequences of EspA from a
number of E. coli serotypes. See, e.g., International Publication
No. WO 99124576, as well as GenBank Accession Nos. AF125993,
AF132728, AF045568, AF022236, AF70067, AF070068, AF013122,
AF200363, AF113597, AF070069, AB036053, AB026719, U5904 and U59502,
for the nucleotide and amino acid sequences of Tir from a number of
E. coli serotypes. See, e.g., GenBank Accession Nos. U32312,
U38618, U59503, U66102, AF081183, AF081182, AF130315, AF339751,
AJ308551, AF301015, AF329681, AF319597, AJ275089-AJ275113 for the
nucleotide and amino acid sequences of Intimin from a number of E.
coli serotypes. See, e.g., GenBank Accession Nos. U80796, U65681,
Y13068, Y13859, X96953, X99670, X96953, Z21555, AF254454, AF254455,
AF254456, AF254457, AF054421, AF059713, AF144008, AF144009 for the
nucleotide and amino acid sequences of EspB from a number of E.
coli serotypes. See, e.g., GenBank Accession Nos. Y13068, Y13859, Y
17875, Y17874, Y09228, U65681, AF054421 and AF064683, for the
nucleotide and amino acid sequences of EspD from a number of E.
coli serotypes.
[0079] These sequences can be used to design oligonucleotide probes
and used to screen genomic or cDNA libraries for genes from other
E. coli serotypes. The basic strategies for preparing
oligonucleotide probes and DNA libraries, as well as their
screening by nucleic acid hybridization, are well known to those of
ordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra;
Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,
supra; Sambrook et al., supra. Once a clone from the screened
library has been identified by positive hybridization, it can be
confirmed by restriction enzyme analysis and DNA sequencing that
the particular library insert contains a type III gene or a homolog
thereof. The genes can then be further isolated using standard
techniques and, if desired, PCR approaches or restriction enzymes
employed to delete portions of the full-length sequence.
[0080] Similarly, genes can be isolated directly from bacteria
using known techniques, such as phenol extraction and the sequence
further manipulated to produce any desired alterations. See, e.-g.,
Sambrook et al., supra, for a description of techniques used to
obtain and isolate DNA. Alternatively, DNA sequences encoding the
proteins of interest can be prepared synthetically rather than
cloned. The DNA sequences can be designed with the appropriate
codons for the particular amino acid sequence. In general, one will
select preferred codons for the intended host if the sequence will
be used for expression. The complete sequence is assembled from
overlapping oligonucleotides prepared by standard methods and
assembled into a complete coding sequence. See, e.g., Edge (1981)
Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al.
(1984) J. Biol. Chem. 259:6311.
[0081] Once coding sequences for the desired proteins have been
prepared or isolated, they can be cloned into any suitable vector
or replicon. Numerous cloning vectors are known to those of skill
in the art, and the selection of an appropriate cloning vector is a
matter of choice. Examples of recombinant DNA vectors for cloning
and host cells which they can transform include the bacteriophage
.lamda. (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230
(gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1
(gram-negative bacteria), pME290 (non-E. coli gram-negative
bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus),
pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces),
YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells).
See, Sambrook et al., supra; DNA Cloning, supra; B. Perbal,
supra.
[0082] The gene can be placed under the control of a promoter,
ribosome binding site (for bacterial expression) and, optionally,
an operator (collectively referred to herein as "control"
elements), so that the DNA sequence encoding the desired protein is
transcribed into RNA in the host cell transformed by a vector
containing this expression construction. The coding sequence may or
may not contain a signal peptide or leader sequence. Leader
sequences can be removed by the host in post-translational
processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437;
4,338,397.
[0083] Other regulatory sequences may also be desirable which allow
for regulation of expression of the protein sequences relative to
the growth of the host cell. Regulatory sequences are known to
those of skill in the art, and examples include those which cause
the expression of a gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Other types of regulatory elements may also be
present in the vector, for example, enhancer sequences.
[0084] The control sequences and other regulatory sequences may be
ligated to the coding sequence prior to insertion into a vector,
such as the cloning vectors described above. Alternatively, the
coding sequence can be cloned directly into an expression vector
which already contains the control sequences and an appropriate
restriction site.
[0085] In some cases it may be necessary to modify the coding
sequence so that it may be attached to the control sequences with
the appropriate orientation; i.e., to maintain the proper reading
Frame. It may also be desirable to produce mutants or analogs of
the protein. Mutants or analogs may be prepared by the deletion of
a portion of the sequence encoding the protein, by insertion of a
sequence, and/or by substitution of one or more nucleotides within
the sequence. Techniques for modifying nucleotide sequences, such
as site-directed mutagenesis, are described in, e-g., Sambrook et
al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,
supra.
[0086] The expression vector is then used to transform an
appropriate host cell. A number of mammalian cell lines are known
in the art and include immortalized cell lines available from the
American Type Culture Collection (ATCC), such as, but not limited
to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK")
cells, as well as others. Similarly, bacterial hosts such as E.
coli, Bacillus subtilis, and Streptococcus spp., will find use with
the present expression constructs. Yeast hosts useful in the
present invention include inter alia, Saccharomyces cerevisiae,
Candida albicans, Candida maltosa, Hansenula polymorpha,
Kluyveromyces fragilis, Kluyveromyces lactis, Pichia
guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and
Yarrowia lipolytica. Insect cells for use with baculovirus
expression vectors include, inter alia, Aedes aegypti, Autographa
californica, Bombyx mori, Drosophila melanogaster, Spodoptera
fmgiperda, and Trichoplusia ni.
[0087] Depending on the expression system and host selected, the
proteins of the present invention are produced by culturing host
cells transformed by an expression vector described above under
conditions whereby the protein of interest is expressed. The
protein is then isolated from the host cells and purified. The
selection of the appropriate growth conditions and recovery methods
are within the skill of the art.
[0088] The proteins of the present invention may also be produced
by chemical synthesis such as solid phase peptide synthesis, using
known amino acid sequences or amino acid sequences derived from the
DNA sequence of the genes of interest. Such methods are known to
those skilled in the art. See, e.g., J. M. Stewart and J. D. Young,
Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,
Rockford, Ill. (1984) and G. Barany and R. B. Merrifield, The
Peptides: Analysis, Synthesis, Biology, editors E. Gross and J.
Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254,
for solid phase peptide synthesis techniques; and M. Bodansky,
Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and
E. Gross and J. Meienhofer, Eds., The Peptides: Anabsis, Synthesis,
Biology, supra, Vol. 1, for classical solution synthesis. Chemical
synthesis of peptides may be preferable if a small fragment of the
antigen in question is capable of raising an immunological response
in the subject of interest.
[0089] Once the above cell culture supernatants and, if desired,
additional recombinant and/or purified proteins are produced, they
are formulated into compositions for delivery to a mammalian
subject. The CCS is administered alone, or mixed with a
pharmaceutically acceptable vehicle or excipient. Suitable vehicles
are, for example, water, saline, dextrose, glycerol, ethanol, or
the like, and combinations thereof. In addition, the vehicle may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants in the case
of vaccine compositions, which enhance the effectiveness of the
vaccine. Suitable adjuvants are described further below. The
compositions of the present invention can also include ancillary
substances, such as pharmacological agents, cytokines, or other
biological response modifiers.
[0090] As explained above, vaccine compositions of the present
invention may include adjuvants to further increase the
immunogenicity of one or more of the EHEC antigens. Such adjuvants
include any compound or compounds that act to increase an immune
response to an EHEC antigen or combination of antigens, thus
reducing the quantity of antigen necessary in the vaccine, and/or
the frequency of injection necessary in order to generate an
adequate immune response. Adjuvants may include for example,
emulsifiers, muramyl dipeptides, pyridine, aqueous adjuvants such
as aluminum hydroxide, chitosan-based adjuvants, and any of the
various saponins, oils, and other substances known in the art, such
as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA,
synthetic oligonucleotides and combinations thereof (Schijns et
al., Curr. Opi. Immunol. (2000) 12: 456), Mycobacterial phlei (M.
phlei) cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei
DNA (M-DNA), M-DNA-M. phlei cell wall complex (MCC). For example,
compounds which may serve as emulsifiers herein include natural and
synthetic emulsifying agents, as well as anionic, cationic and
nonionic compounds. Among the synthetic compounds, anionic
emulsifying agents include, for example, the potassium, sodium and
ammonium salts of lauric and oleic acid, the calcium, magnesium and
aluminum salts of fatty acids (i.e., metallic soaps), and organic
sulfonates such as sodium lauryl sulfate. Synthetic cationic agents
include, for example, cetyltimethylammonium bromide, while
synthetic nonionic agents are exemplified by glyceryl esters (e.g.,
glyceryl monostearate), polyoxyethylene glycol esters and ethers,
and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate)
and their polyoxyethylene derivatives (e.g., polyoxyethylene
sorbitan monopalmitate). Natural emulsifying agents include acacia,
gelatin, lecithin and cholesterol.
[0091] Other suitable adjuvants can be formed with an oil
component, such as a single oil, a mixture of oils, a water-in-oil
emulsion, or an oil-in-water emulsion. The oil may be a mineral
oil, a vegetable oil, or an animal oil. Mineral oil, or
oil-in-water emulsions in which the oil component is mineral oil
are preferred. In this regard, a "mineral oil" is defined herein as
a mixture of liquid hydrocarbons obtained from petrolatum via a
distillation technique; the term is synonymous with "liquid
paraffin," "liquid petrolatum" and "white mineral oil." The term is
also intended to include "light mineral oil," i.e., an oil which is
similarly obtained by distillation of petrolatum, but which has a
slightly lower specific gravity than white mineral oil. See, e.g.,
Remington's Pharmaceutical Sciences, supra. A particularly
preferred oil component is the oil-in-water emulsion sold under the
trade name of EMULSIGEN PLUS.TM. (comprising a light mineral oil as
well as 0.05% formalin, and 30 mcg/mL gentamicin as preservatives),
available from MVP Laboratories, Ralston, Nebr. Suitable animal
oils include, for example, cod liver oil, halibut oil, menhaden
oil, orange roughy oil and shark liver oil, all of which are
available commercially. Suitable vegetable oils, include, without
limitation, canola oil, almond oil, cottonseed oil, corn oil, olive
oil, peanut oil, safflower oil, sesame oil, soybean oil, and the
like.
[0092] Alternatively, a number of aliphatic nitrogenous bases can
be used as adjuvants with the vaccine formulations. For example,
known immunologic adjuvants include mines, quaternary ammonium
compounds, guanidines, benzamidines and thiouroniums (Gall, D.
(1966) Immunology 11:369-386). Specific compounds include
dimethyldioctadecylammonium bromide (DDA) (available from Kodak)
and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl) propanediamine
("pyridine"). The use of DDA as an immunologic adjuvant has been
described; see, e.g., the Kodak Laboratory Chemicals Bulletin
56(1):1-5 (1986); Adv. Drug Deliv. Rev. 5(3):163-187 (1990); J.
Controlled Release 7:123-132 (1988); Clin. Exp Immunol.
78(2):256-262 (1989); J. Immunol. Methods 97(2):159-164 (1987);
Immunology 58(2):245-250 (1986); and Int. Arch. Allergy Appl.
Immunol. 68(3):201-208 (1982). Avridine is also a well-known
adjuvant. See, e.g., U.S. Pat. No. 4,310,550 to Wolff, III et al.,
which describes the use of N,N-higher
alkyl-N',N'-bis(2-hydroxyethyl)propane diamines in general, and
pyridine in particular, as vaccine adjuvants. U.S. Pat. No.
5,151,267 to Babiuk, and Babiuk et al. (1986) Virology 159:57-66,
also relate to the use of pyridine as a vaccine adjuvant.
[0093] Particularly preferred for use herein is an adjuvant known
as "VSA3" which is a modified form of the EMULSIGEN PLUS.TM.
adjuvant which includes DDA (see, U.S. Pat. No. 5,951,988,
incorporated herein by reference in its entirety).
[0094] CCS vaccine compositions can be prepared by uniformly and
intimately bringing into association the CCS preparations and the
adjuvant using techniques well known to those skilled in the art
including, but not limited to, mixing, sonication and
microfluidation. The adjuvant will preferably comprise about 10 to
50% (v/v) of the vaccine, more preferably about 20 to 40% (v/v) and
most preferably about 20 to 30% or 35% (v/v), or any integer within
these ranges.
[0095] The compositions of the present invention are normally
prepared as injectables, either as liquid solutions or suspensions,
or as solid forms which are suitable for solution or suspension in
liquid vehicles prior to injection. The preparation may also be
prepared in solid form, emulsified or the active ingredient
encapsulated in liposome vehicles or other particulate carriers
used for sustained delivery. For example, the vaccine may be in the
form of an oil emulsion, water in oil emulsion,
water-in-oil-in-water emulsion, site-specific emulsion,
long-residence emulsion, stickyemulsion, microemulsion,
nanoemulsion, liposome, microparticle, microsphere, nanosphere,
nanoparticle and various natural or synthetic polymers, such as
nonresorbable impermeable polymers such as ethylenevinyl acetate
copolymers and Hytrel.RTM. copolymers, swellable polymers such as
hydrogels, or resorbable polymers such as collagen and certain
polyacids or polyesters such as those used to make resorbable
sutures, that allow for sustained release of the vaccine.
[0096] Furthermore, the polypeptides may be formulated into
compositions in either neutral or salt forms. Pharmaceutically
acceptable salts include the acid addition salts (formed with the
free amino groups of the active polypeptides) and which are formed
with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or organic acids such as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed from free carboxyl
groups may also be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0097] Actual methods of preparing such dosage forms are known, or
will be apparent, to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 18th edition, 1990.
[0098] The composition is formulated to contain an effective amount
of secreted EHEC antigen, the exact amount being readily determined
by one skilled in the art, wherein the amount depends on the animal
to be treated and the capacity of the animal's immune system to
synthesize antibodies. The composition or formulation to be
administered will contain a quantity of one or more secreted EHEC
antigens adequate to achieve the desired state in the subject being
treated. For purposes of the present invention, a therapeutically
effective amount of a vaccine comprising CCS with or without added
recombinant and/or purified secreted EHEC antigens, contains about
0.05 to 1500 .mu.g secreted EHEC protein, preferably about 10 to
1000 .mu.g secreted EHEC protein, more preferably about 30 to 500
.mu.g and most preferably about 40 to 300 .mu.g, or any integer
between these values. EspA+Tir, as well as other EHEC antigens, may
comprise about 10% to 50% of total CCS protein, such as about 15%
to 40% and most preferably about 15% to 25%. If supplemented with
rEspA+rTir, the vaccine may contain about 5 to 500 .mu.g of
protein, more preferably about 10 to 250 .mu.g and most preferably
about 20 to 125 .mu.g.
[0099] Routes of administration include, but are not limited to,
oral, topical, subcutaneous, intramuscular, intravenous,
subcutaneous, intradermal, transdermal and subdermal. Depending on
the route of administration, the volume per dose is preferably
about 0.001 to 10 ml, more preferably about 0.01 to 5 ml, and most
preferably about 0.1 to 3 ml. Vaccine can be administered in a
single dose treatment or in multiple dose treatments (boosts) on a
schedule and over a time period appropriate to the age, weight and
condition of the subject, the particular vaccine formulation used,
and the route of administration.
[0100] Any suitable pharmaceutical delivery means may be employed
to deliver the compositions to the vertebrate subject. For example,
conventional needle syringes, spring or compressed gas (air)
injectors (U.S. Pat. Nos. 1,605,763 to Smoot; 3,788,315 to Laurens;
3,853,125 to Clark et al.; 4,596,556 to Morrow et al.; and
5,062,830 to Dunlap), liquid jet injectors (U.S. Pat. Nos.
2,754,818 to Scherer; 3,330,276 to Gordon; and 4,518,385 to
Lindmayer et al.), and particle injectors (U.S. Pat. Nos. 5,149,655
to McCabe et al. and 5,204,253 to Sanford et al.) are all
appropriate for delivery of the compositions.
[0101] If a jet injector is used, a single jet of the liquid
vaccine composition is ejected under high pressure and velocity,
e.g., 1200-1400 PSI, thereby creating an opening in the skin and
penetrating to depths suitable for immunization.
[0102] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the present invention
and/or the scope of the appended claims.
EXEMPLARY ASPECTS
C. Experimental
Example 1
Preparation of Cell Culture Supernatant (CCS)
[0103] Wild type EHEC O157:H7 were grown under conditions to
maximize the synthesis of CCS proteins (Li et al., Infect. Immun.
(2000) 68:5090). Briefly, an overnight standing culture of EHEC
O157:H7 was grown in Luria-Bertani (LB) medium overnight at
37.degree. C. (.+-.5% CO.sub.2). The culture was diluted 1:10 in
M-9 minimal medium supplemented with 0.1% Casamino Acids, 0.4%
glucose, 8 mM MgSO.sub.4 and 44 mM NaHCO.sub.3. Cultures were grown
standing at 37.degree. C. in 5% CO.sub.2 to an optical density at
600 nm of 0.7 to 0.8 (6-8 h). Bacteria were removed by
centrifugation at 8000 rpm for 20 min at 4.degree. C. The
supernatant was concentrated 100 fold by ultrafiltration and total
protein was determined by the bicinchoninic acid protein assay
method.
[0104] FIG. 1 shows molecular weight markers (lane 1) and a typical
CCS protein profile obtained by electrophoresis of CCS in a SDS-10%
polyacrylamide gel (SDS-PAGE) followed by Coomassie blue staining
(lane 2). The positions of EspA (25 kD), EspB/EspD (40 kD),
undegraded Tir (70 kD) and degraded Tir (55 kD) are indicated. As
determined by densitometric analysis using an HP Scanjet 5100C and
the ID software program from Advance American Biotechnology
(Fullerton, Calif., USA), EspA was about 5% undegraded Tir about
20% and degraded Tir about 6% of the total protein. However, the
percentages of proteins determined by densitometric analysis of
Coomassie blue stained SDS-polyacrylamide gels is not exact due to
variations in background staining, variations in the uptake of the
Coomassie blue stain, variations in the density of the bands, and
other factors known to those skilled in the art.
Example 2
Preparation of Recombinant Proteins
[0105] The genes coding for EspA, EspB, Intimin and Tir were
isolated (Li et al., Infect. Immun. (2000) 68:5090). A clinical
isolate of EHEC O157:H7 was used as the source of DNA. EspA, EspB,
Tir, and the region of eae encoding the 280 carboxyl-terminal amino
acids of Intimin were amplified from chromosomal DNA using PCR to
introduce unique restriction sites, followed by cloning into
appropriate plasmids. The resulting plasmids were cleaved and
ligated to create histidine-tagged fusions. Plasmids were
electrocuted into an expression strain of E. coli and the E. coli
were propagated (Ngeleka et al., Infect. Immun. (1996) 64:3118).
Gene expression was driven using the Tac promoter following IPTG
(isopropyl-.beta.-D-thiogalactopyranoside) induction. Bacteria were
pelleted, resuspended in Tris-buffered saline and lysed by
sonication. The lysate was centrifuged to remove insoluble material
and the histidine-tagged proteins were purified by passage through
a solid-phase nickel affinity chromatography column that
specifically binds proteins containing the histidine tag. All
recombinant protein preparations were stored at -20.degree. C.
until use.
[0106] The purity of the recombinant proteins was assessed by
SDS-PAGE on 10% gels followed by Coomassie blue staining. Typical
gel profiles of the chromatographically purified recombinant (r)
proteins are shown in FIG. 2. rEspA (lane 2) rEspB (lane 3) and
rIntimin (lane 4), were recovered in relatively pure form, but rTir
(lane 5) was subject to some degradation.
Example 3
Vaccine Formulation and Delivery
[0107] Vaccines were formulated by mixing CCS or rEspA+rTir in 2 ml
of a carrier containing from 30 to 40% of an adjuvant. Vaccines
were delivered subcutaneously. Animals were immunized on day 1 and
again at a 3-4 week intervals (boost). Serum samples were obtained
prior to the first immunization, at the time of each boost and at
the end of the experiment.
[0108] The serological response to immunization was determined
using an enzyme-linked immunosorbent assay (ELISA). One hundred p1
of rEspA (0.16 .mu.g/well), rTir (0.1 .mu.g/well), rEspB (0.24
.mu.g/well) and rIntimin (0.187 .mu.g/well) were used to coat the
wells in microtiter plates and the plates were incubated overnight
at 4.degree. C. The wells were washed 3.times., blocked with 0.5%
nonfat dried milk in phosphate-buffered saline. Serial dilutions of
sera were added to each well and incubated for 2 h at 37.degree. C.
The wells were washed and blocked and 100 .mu.l of
peroxidaseconjugated rabbit anti-bovine immunoglobulin G antibodies
(1:5000) were added to each well for 1 h at 37.degree. C. The wells
were washed and plates were read at a wavelength of 492 nm.
Example 4
Experimental Animals
[0109] Cattle, between the ages of 8 and 12 months, were purchased
from local ranchers. Fecal samples were obtained daily from each
animal for 14 days. The number of EHEC O157:H7 in the fecal samples
was determined by plating on Rainbow Agar. The plates were
incubated at 37.degree. C. for 2 days and black colonies were
enumerated. Growth was scored from 0-5. Animals having a score of 0
(no EHEC O157:H7) were used in all experiments.
Example 5
Animal Colonization Model
[0110] A model for EHEC 057:H7 colonization of cattle, wherein the
infection was sustained for >2 months, was developed using a
dose-titration protocol.
[0111] EHEC O157:H7 were grown as in Example 1. Twenty-four cattle
were divided into 3 groups of 8 animals each. Group 1 received
10.sup.6, Group 2 10.sup.8 and Group 3 10.sup.10 CFU of EHEC
O157:H7 by oral-gastric intubation in a volume of 50 ml on day
0.
[0112] To monitor shedding, fecal material was collected on days 1
through 14. The fecal material was weighed, suspended in sterile
saline and inoculated into culture media. Culture density was
determined as in Example 1.
[0113] As shown in FIG. 3, there was no significant difference
between numbers of EHEC O157:H7 shed by Group 2 (10.sup.8 CFU) and
Group 3 (10.sup.10 CFU) cattle. Group 2 cattle shed the most EHEC
O157:H7 on each of the 14 days. The number of EHEC O157:H7 shed by
Group 2 cattle reached a maximum on day 6 and declined to zero by
day 14.
[0114] Animals shedding EHEC O157:H7 (hereinafter, "positive") were
kept an additional 40 days during which time the number of EHEC
O157:H7 shed decreased to an undetectable level. The shedding of
EHEC O157:H7 by previously positive animals (hereinafter,
"carriers") was reactivated by withholding feed for 24 hours and
vaccinating with commercially-available clostridial or H. somnus
vaccines. As shown in FIG. 4, the number of carrier animals
shedding EHEC O157:H7 reached a maximum of approximately 50% on
days 6 and 7 post-reactivation and declined to zero by day 15.
[0115] As a dose of 10.sup.8 CFU produced a detectable number of
shed EHEC O157:H7 during the 14 days post-infection (FIG. 3) and
resulted in persistently infected animals (FIG. 4), this dose was
used as the challenge dose in subsequent experiments.
Example 6
Protective Capacity of CCS
[0116] To test the vaccine potential of secreted proteins, CCS was
mixed with the oil-based adjuvant, VSA3 (U.S. Pat. No. 5,951,988,
incorporated herein by reference in its entirety; S. van Drunen
Littel-van den Hurk et al., Vaccine (1993)11:25) such that each 2
ml dose contained 200 .mu.g of CCS protein and 30% (v/v) of
adjuvant (CCS vaccine). For the control group, sterile saline was
mixed with VSA3, such that each 2 ml dose contained 0 .mu.g of CCS
protein and 30% (v/v) of adjuvant (saline vaccine).
[0117] Sixteen cattle were divided in 2 groups of eight animals
each. Group 1 cattle received 2 ml of CCS vaccine subcutaneously
(experimental) and Group 2 cattle received 2 ml saline vaccine
subcutaneously (control) on days 1 and 22 (boost). Seroconversion
was assayed by ELISA (Example 3), on days 1 (pre-immunization), 22
and 36. As shown in Table 1, at day 22, Group 1 animals showed
specific antibody titers to EspA and Tir and, at day 36, these
titers showed a significant increase. Group 2 animals showed no
specific antibody titers at days 22 and 36. In particular, the
group which received the EHEC vaccine showed a 13-fold increase in
specific antibody titer to type III secreted proteins after a
single immunization and following the first booster, the eight
animals in the EHEC vaccine group demonstrated a 45-fold increase
in specific antibody titer while only one of the placebo vaccine
group seroconverted (X.sup.2, =0.0002).
TABLE-US-00001 TABLE 1 Serological response to immunization with
CCS Specific Antibody Titers* - Group Means Boost Challenge Group
Pre-immunization (Day 1) (Day 22) (Day 36) 1. Experimental 350
5,000 12,500 2. Control 450 500 650 *Values are group means
expressed as the reciprocal of the highest dilution yielding a
positive result.
[0118] At day 36, Group 1 and Group 2 animals were challenged with
10.sup.8 CFU of EHEC O157:H7 by oral-gastric intubation and fecal
shedding was monitored for 14 days (Example 5). As summarized in
Table 2, fewer experimental animals shed EHEC O157:H7 than control
animals and experimental animals that did shed, shed EHEC O157:H7
for a shorter period of time than control animals (FIG. 7). In
particular, The median number of days during which the organism was
shed in the vaccinated animals was 1.5 compared to 3.5 in the
placebo group (Wilcoxin Signed Rank Test, p=0.08). Seven out of
eight placebo-immunized animals shed the bacteria during the trial
and four of those animals shed the bacteria for four or more
consecutive days, indicating that they were persistently infected.
Five out of eight EHEC vaccine-immunized animals shed bacteria at
some point during the trial but only one animal shed the organism
for more than two consecutive days, indicating that colonization
was transient and significantly less than the placebo group. The
total number of bacteria isolated from fecal samples was
significantly lower among the EHEC vaccinated group as compared to
the placebo group (Wilcoxin Signed Rank Test, p=0.05), with the
former having a median of 6.25 colony forming units (CFU) per gram
of feces recovered compared to a median value of 81.25 CFU/g for
the latter. Thus, vaccination with the type III-secreted proteins
appeared to reduce the ability of the organism to colonize the
intestine as reflected by the decrease in the number of days
animals shed the organism as well as the numbers of shed bacteria
detected by fecal culture.
TABLE-US-00002 TABLE 2 Shedding by experimental and control animals
Experimental Control Animals shedding >1 day 1/8 6/8 Number of
days with scores of >1 1 8 Average days of shedding per animal
0.875 2.5 Total days shedding per group 7 20
[0119] These data show that CCS induced an antibody response in
cattle that reduced both number of animals shedding EHEC O157:H7
and the number of days during which EHEC O157:H7 were shed.
[0120] In order to enhance the effectiveness of the vaccine
formulation, groups of 6 calves were immunized as described above
with one of three doses of secreted proteins (50 .mu.g, 100 .mu.g,
200 .mu.g) or a placebo and the serological response was measured
in serum samples taken at days 0, 21 (boost) and 35. No significant
difference in anti-EHEC, anti-Tir or anti-EspA responses were
observed between any of the groups which received the EHEC vaccine
at any time point but all three were significantly higher than the
placebo group on days 21 and 35. Thus, a second vaccine trial was
designed in which three groups of yearling cattle were immunized
three times with 50 .mu.g of secreted proteins (n=13), 50 .mu.g of
secreted proteins from a tir mutant (.DELTA.Tir, n=10) or a placebo
(n=25). The adjuvant used was VSA3 and animals were immunized by
subcutaneous injection on days 0, 21, and 35, followed by oral
challenge with E. coli O157:H7 on day 49. The serological response
to immunization is shown in Table 3 (days 0 and 49 only) and was
comparable to that observed in the trial described above. The group
which received the .DELTA.Tir vaccine showed a response of similar
magnitude against total secreted proteins as the group which
received the 25 vaccine prepared from the wild-type strain, but, as
expected, a significantly reduced response to Tir (Wilcoxin Signed
Rank Test, p=0.006). However, the former group did show an increase
in anti-Tir antibody levels (Wilcoxin Signed Rank Test, p=0.009),
indicating either exposure to an organism producing an
immunologically related molecule or natural exposure to E. coli
O157:H7. This is further supported by the observation that there
was a significant increase in the anti-Tir antibody titer in the
placebo group on the day of challenge (Wilcoxin Signed Rank Test,
p=0.002) but no difference between the placebo or .DELTA.Tir groups
(p=0.37, Kruskal-Wallis ANOVA). The response to EspA was similar in
both the EHEC and .DELTA.Tir vaccine groups (p=0.45, Kruskal-Wallis
ANOVA) and was significantly higher than the placebo-immunized
animals (p<0.0001).
TABLE-US-00003 TABLE 3 Median serological response to immunization
with secreted proteins prepared from wild-type E. coli O157:H7
(EHEC), an isogenic tir mutant (.DELTA.Tir) or a placebo. Titers
are expressed as geometric mean values of the last positive
dilution of sera ( ). Numbers in parentheses represent the
25.sup.th-75.sup.th percentile. Anti-EHEC Anti-Tir Anti-EspA Group
n Day 0 Day 49 Day 0 Day 49 Day 0 Day 49 EHEC 13 10 6400 100 1600
100 400 (10-100) (3200-12800) (10-200) (800-3200) (10-200)
(200-1600) .DELTA.Tir 10 10 6400 10 200 100 300 (10-100)
(3200-25600) (10-200) (100-800) (10-200) (100-1600) Placebo 25 10
10 100 200 100 100 (10-200) (10-200) (10-200) (10-400) (10-200)
(10-200)
[0121] The immune response against each vaccine formulation was
also analyzed qualitatively by Western blotting using sera from two
representative animals per group. The results for representative
animals are shown in FIG. 8 and demonstrate that the proteins
secreted by the type I11 system were highly immunogenic in cattle.
The response in the EHEC and .DELTA.Tir vaccine groups was similar
with the exception of the response against Tir which was absent in
the latter group (FIG. 8, top panels). EspB, EspD and Tir were all
reactive, and following the second immunization on day 21 a
significant response against lipopolysaccharide was also observed.
The kinetics of the immune response in a vaccinated animal (FIG. 8,
bottom panels) show that anti-Tir antibodies were detectable
following a single immunization, as were antibodies against 43-kDa
and 100-kDa proteins. The latter proteins were produced by the
wild-type strain as well as the sepB and tir mutants and the 100
kDa protein is probably EspP, a non-type III EHEC secreted
protein.
[0122] Following oral challenge with E. coli O157:H7 on day 49,
each group was monitored daily for fecal shedding of the organism
for 14 days. In this experiment, bacteria were cultured following
immunomagnetic enrichment (J. Van Donkersgoed et al., Can. Vet. J.
(2001) 42:714; Chapman and Siddons, J. Med. Micvobiol. (1996)
44:267) rather than direct plating since yearling cattle shed less
than calves in this infection model. On the day of challenge, two
animals in the placebo group were culture-positive for E. coli
O157:H7 and were eliminated from the trial. The placebo-immunized
animals shed the organism after challenge much more than those in
the two EHEC vaccine groups (FIG. 9). Those which received the
placebo vaccine shed the organism for a median of 4 days,
significantly longer than the median of 0 days by the other two
vaccine groups (p=0.0002, Kruskal-Wallis ANOVA). Significantly
fewer bacteria were recovered from the EHEC and .DELTA.Tir vaccine
groups (p=0.04, Kruskal-Wallis ANOVA). From day 2 post-infection
onwards, 78% of the placebo animals shed the organism for at least
one day as compared to 15% of the EHEC and 30% of the .DELTA.Tir
vaccinates (Table 4).
[0123] The data presented above demonstrate that virulence factors
of EHEC, namely those secreted by the type III system, can be used
as effective vaccine components for the reduction of colonization
of cattle by EHEC bacteria, such as EHEC O157:H7. These proteins
are major targets of the immune response in humans following
infection (Li et al., Infect. Immun. (2000) 68:5090), although
cattle do not usually mount a significant serological response
against these proteins following natural exposure to the organism.
However, animals vaccinated with these proteins are primed and show
an increase in anti-EHEC and anti-Tir titers following oral
challenge with the organism.
[0124] Tir is likely required for colonization of the bovine
intestine, and this is supported by the observation that a vaccine
containing secreted proteins from a .DELTA.Tir E. coli O157:H7
strain was not as efficacious as an identical formulation from an
isogenic wild-type isolate. However, the former vaccine was
significantly more efficacious than a placebo suggesting that
immunity against colonization is multifactorial in nature. This is
supported by the Western blot analysis of the 1 response to
immunization in which several protein components as well as
lipopolysaccharide were recognized. The contribution to protection
by lipopolysaccharide is not known, but the presence of antibodies
against this molecule does not correlate with protection in a
murine EHEC model (Conlan et al., Can. J Micvobiol. (1999) 45:279;
Conlan et al., Can. J Micvobiol. (2000) 46:283). Also, immunization
with recombinant Tir and EspA can reduce numbers of bacteria shed,
but not the actual numbers of animals nor the duration of
shedding.
[0125] The prevalence of non-0157 serotypes in North America
appears to be increasing and represents a significant portion of
EHEC infections in other geographical locations. Since the type
III-secreted antigens appear to be relatively conserved among
non-0157 EHEC serotypes, this vaccine formulation is likely broadly
cross-protective, in contrast to formulations based upon the 0157
LPS antigen.
TABLE-US-00004 TABLE 4 Number of animals shedding E. coli O157:H7
at any time between day 2 and day 14 postchallenge. Number Percent
Vaccine Shedding n Shedding p-value EHEC 2 13 15.4 0.003 .DELTA.Tir
3 10 30 0.008 Placebo 18 23 78.3 1
Example 7
Protective Capacity of rEspA+rTir and rEspB+rIntimin
[0126] rEspA, rTir, rEspB and rIntimin were mixed with the
oil-based adjuvant, VSA3, such that each 2 ml dose contained 50
.mu.g of rEspA+rTir or of rEspB+rIntimin and 30% (v/v) of adjuvant.
Sterile saline was mixed with VSA3, such that each 2 ml dose
contained 0 .mu.g of rEspA+rTir or of rEspB+rhtimin and 30% (v/v)
of adjuvant.
[0127] Thirty four cattle were divided in 4 groups. Ten cattle,
Group 1, were immunized with rEspA+rTir vaccine (experimental) and
10 cattle, Group 2, were immunized with rEspB+rIntimin vaccine
(experimental) on days 1, 22 (boost) and 36. Seven cattle, Group 3,
and 7 cattle, Group 4, were immunized with saline vaccine (control)
an days 1, 22 (boost) and 36. Seroconversion was assayed by ELISA
(Example 3) on days 1 (pre-immunization), 22 and 36. As shown in
FIG. 5, at day 22, Group 1 animals showed specific antibody titers
to rEspA and to rTir and Group 2 animals showed specific antibody
titers to rEspB and to rhtimin. Also, as shown in FIG. 5, at day
36, Group 1 animals showed an increase in specific antibody titer
to rTir and no change in specific antibody titer to rEspA and Group
2 animals showed an increase in specific antibody titer to rIntimin
and a decrease in specific antibody titer to rEspB. Groups 3 and 4
animals showed no specific antibody titers at days 22 and 36.
[0128] At day 36, Groups 1-4 animals were challenged with 10.sup.8
CFU of EHEC O157:H7 and shedding was monitored daily for 14 days
(Example 5). As shown in FIG. 6, differences in shedding between
Group 1 (rTir+rEspA) animals and Group 3 (saline) animals was
minimal during the first 5 days post-challenge. However, during the
second week post-challenge differences in Group 1 animals and Group
3 animals were evident. Fewer Group 1 animals shed EHEC O157:H7
than Group 3 animals. Group 1 animals shed less EHEC O157:H7 in
their feces for shorter time periods than Group 3 animals.
Differences in shedding between Group 2 (rEspB+rIntimin) and Group
4 (saline) animals were not evident with respect to the number of
animals shedding, the number of EHEC O157:H7 shed and the time
period of shedding.
[0129] These data show that the antibody response induced by
rEspA+rTir vaccine interfered with EHEC O157:H7 colonization of
cattle, whereas the antibody response induced by rEspB+rIntimin
vaccine did not interfere with EHEC O157:H7 colonization of
cattle.
Example 8
Protective Capacity of CCS+rEspA+rTir
[0130] CCS, CCS+rEspA, CCS+rTir, CCS+rEspA+rTir and saline are
mixed with an adjuvant.
[0131] Twenty-five cattle are divided into 5 groups of five 5
cattle and are immunized an days 1 and 22 (boost). Group 1 receives
CCS vaccine, Group 2 CCS+rEspA vaccine, Group 3 CCS+rTir vaccine,
Group 4 CCS+rEspA+rTir vaccine, and Group 5 saline vaccine.
Seroconversion is assayed by ELISA (Example 3) on days 1
(pre-immunization), 22 (boost) and 36. On days 22 and 36 each of
Groups 1-5 animals show specific antibody titers against EspA and
Tir, whereas Group 6 animals show no specific antibody titers.
[0132] At day 36, Groups 1-5 animals are challenged with 10.sup.8
CFU of EHEC O157:H7 and shedding is monitored daily for 14 days
(Example 5). Fewer animals in Groups 1-4 shed EHEC O157:H7 than
animals in Group 5. Group 5 animals shed the most EHEC O157:H7;
Group 1 animals shed less EHEC O157:H7 than Group 5 animals and
Groups 2-4 animals shed less EHEC O157:H7 than Group 1 animals.
Example 9
Protective Capacity of CCS with Various Antigens
[0133] CSS is mixed with and adjuvant, such that each 2 ml dose
contains 0, 50, 100 or 200 .mu.g of CCS and 30% (v/v) of adjuvant
(Table 5).
TABLE-US-00005 TABLE 5 Protective capacity of CCS with various
adjuvants Anitgen Group .mu.g Adjuvant CCS 1 50 Emulsigen-Plus CCS
2 100 Emulsigen-Plus CCS 3 200 Emulsigen-Plus CCS 4 200 Carbigen
CCS 5 100 MCC CCS 6 200 MCC CCS 7 200 MCC + Carbigen CCS 8 200 VSA
CCS 9 0 (control) Emulsigen-Plus
[0134] Seventy-two cattle are divided in 9 groups of 8 cattle.
Groups 1-8 animals are immunized with CCS+adjuvant (Table 5) and
Group 9 cattle are immunized with saline+adjuvants on days 1 and 22
(boost). Seroconversion is assayed by ELISA (Example 3) on days 1
(pre-immunization), 22 (boost) and 36. Groups 1-8 (CCS+adjuvant)
animals show specific antibody titers to EspA and Tir on days 22
and 36. Group 9 (saline+adjuvant) animals show no specific antibody
titers on days 22 and 36.
Example 10
Protective Capacity of CCS in Dairy Cows
[0135] Twenty adult dairy cows are divided in 2 groups of 10 cows.
Group 1 is immunized with CCS vaccine and Group 2 is immunized with
saline-vaccine on days 1 and day 22 (boost). Seroconversion is
assayed by ELISA (Example 3) on days 1 (pre-immunization), 22 and
36. On days 22 and 36 Group 1 cows show specific antibody titers
against EspA and Tir, whereas Group 2 cows show no specific
antibody titers.
[0136] At day 36, Groups 1 and 2 cows are challenged with 10.sup.8
CFU of EHEC O157:H7 and shedding is monitored daily for 14 days
(Example 5). Fewer Group 1 cows shed EHEC O157:H7 than Groups 2
cows. Group 1 cows shed less EHEC O157:H7 for a shorter period of
time than Groups 2 cows.
[0137] Six months after the initial immunization, Group 1 and 2
cows are again immunized (2nd boost) via the subcutaneous route. On
day 14 following the 2nd boost, antibody titers are assayed by
ELISA (Example 3). Group 1 cows have specific antibody titers to
EspA and Tir, whereas Group 2 cows have no specific antibody
titers.
[0138] On day 14 following the 2nd boost, Groups 1 and 2 cows are
again challenged with 10.sup.8 CFU of EHEC O157:H7 and shedding is
monitored daily for 14 days (Example 5). Fewer Group 1 (CCS) cows
shed EHEC O157:H7 than Group 2 (saline) cows. Group 1 cows shed
less EHEC O157:H7 for a shorter time periods than Group 2 cows.
Example 11
Protective Capacity of CCS in Calves
[0139] Ten weaned calves (3-6 month old) are divided into 2 groups
of 5 calves and are immunized prior to entry into a feed-lot (day
0) and on the day of entry into a feed lot (day 1, boost). Group 1
calves receive CCS vaccine and Group 2 calves receive saline
vaccine. Seroconversion is assayed by ELISA (Example 3) on days 0,
1 and 14. On days 1 and 14 Group 1 (CCS) calves show specific
antibody titers to EspA and Tir, whereas Group 2 (saline) calves
show no specific antibody titers.
[0140] At day 14, Groups 1 and 2 calves are challenged with
10.sup.8 CFU of EHEC O157:H7 and shedding is assayed daily for 14
days (Example 5). Fewer Group 1 calves shed EHEC O157:H7 than Group
2 calves. Group 1 calves shed less EHEC O157:H7 for a shorter time
period than Group 2 calves.
[0141] Ten weaned calves (3-6 mouth old) we divided into 2 groups
of 5 calves and are immunized on the day of entry into a feed-lot
(day 1) and on day 22 (boost) in the feed lot. Group 1 calves
receive CCS vaccine and Group 2 calves receive saline vaccine.
Seroconversion is assayed by ELISA (Example 3) on days 1
(pre-immunization), 22 and 36. On days 22 and 36 Group 1 (CCS)
calves show specific antibody titers to EspA and Tir, whereas Group
2 (saline) calves show no specific antibody titers.
[0142] At day 36, Groups 1 and 2 calves are challenged with
10.sup.8 CFU of EHEC O157:H7 and shedding is assayed daily for 14
days (Example 5). Fewer Group 1 calves shed EHEC O157:H7 than Group
2 calves. Group 1 calves shed less EHEC O157:H7 for a shorter time
period than Group 2 calves.
Example 12
Protective Capacity of CCS in Sheep
[0143] Twenty adult sheep are divided in 2 groups of 10 sheep.
Group 1 is immunized with CCS vaccine and Group 2 is immunized with
saline vaccine on day 1 and day 22 (boost). Seroconversion is
assayed by ELISA (Example 3) on days 1 (pre-immunization), 22 and
36. On days 22 and 36 Group 1 sheep show specific antibody titers
against EspA and Tir, whereas Group 2 sheep show no specific
antibody titers.
[0144] At day 36, Groups 1 and 2 sheep are challenged with 10.sup.8
CFU of EHEC O157:H7 and shedding is monitored daily for 14 days
(Example 5). Fewer Group 1 sheep shed EHEC O157:H7 than Group 2
sheep. Group 1 sheep shed less EHEC O157:H7 for a shorter period of
time than Group 2 sheep.
[0145] Thus, compositions and methods for treating and preventing
enterohemorragic E. coli colonization of mammals have been
disclosed. Although preferred embodiments of the subject invention
have been described in some detail, it is understood that obvious
variations can be made without departing from the spirit and the
scope of the invention as defined by the appended claims.
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