Note: Descriptions are shown in the official language in which they were submitted.
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Vaccines Against Escherichia coil 0157 Infection
FIELD OF THE INVENTION
This invention relates to conjugates of the O-specific polysaccharide of
Shiga toxin-producing bacteria, such as E. coli 0157, with a carrier, and
compositions
thereof, and to methods of using of these conjugates and/or compositions
thereof for
eliciting an immunogenic response in mammals, including responses which
provide
protection against, or reduce the severity of, bacterial infections. More
particularly it
relates to the use of polysaccharides containing the tetrasaccharide repeat
unit:
(-*3)-a-D-GalpNAc-(l-->2)-a-D-PerpNAc-(l >3)-a-L-Fucp-(1-->4)-(3-D-Glcp-(1-+),
and conjugates thereof, to induce serum antibodies having bactericidal
(killing)
activity against E. coli, in particular E. coli 0157. The conjugates, and
compositions
thereof, are useful as vaccines to induce serum antibodies which have
bactericidal or
bacteriostatic activity against against E. coli, in particular E. coli 0157,
and are useful
to prevent and/or treat illnesses caused by E. coli 0157.
The invention further relates to the antibodies which immunoreact with
the O-specific polysaccharide of E. coli 0157 and/or the carrier, that are
induced by
these conjugates and/or compositions thereof. The invention also relates to
methods
and kits for detection, identification, and/or diagnosis of E. coli 0157,
using one or
more of the polysaccharides, conjugates or antibodies described above.
BACKGROUND
The most successful of all carbohydrate pharmaceuticals so far have
been the carbohydrate-based, antibacterial vaccines [1]. The basis of using
carbohydrates as vaccine components is that the capsular polysaccharides and
the 0-
specific polysaccharides on the surface of pathogenic bacteria are both
protective
antigens and essential virulence factors. The first saccharide-based vaccines
contained capsular polysaccharides of Pneumococci: in the United States a 14-
valent
vaccine was licensed in 1978 followed by a 23-valent vaccine in 1983. Other
capsular polysaccharides licensed for human use include a tetravalent
meningococcal
vaccine and the Vi polysaccharide of Salmonella typhi for typhoid fever. The
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inability of most polysaccharides to elicit protective levels of anti-
carbohydrate
antibodies in infants and adults with weakened immune systems could be
overcome
by their covalent attachment to proteins that conferred T-cell dependent
properties [2].
This principle led to the construction of vaccines against Haemophilus
influenzae b
(Hib) [3] and in countries where these vaccines are routinely used, meningitis
and
other diseases caused by Hib have been virtually eliminated [4]. Extension of
the
conjugate technology to the O-specific polysaccharides of Gram-negative
bacteria has
provided a new generation of glycoconjugate vaccines that are undergoing
various
phases of clinical trials [5].
Escherichia coli 0157:H7, an emerging infectious agent, was first
recognized as a human pathogen in 1983 [6]. Diseases caused by this pathogen
have
subsequently been recognized worldwide [7]. Infection with E. coli 0157 causes
a
spectrum of illnesses with high morbidity and mortality, ranging from watery
diarrhea
to hemorrhagic colitis and the extraintestinal complication of hemolytic-
uremic
syndrome (HUS). HUS can lead to acute renal failure requiring dialysis, and in
children and infants this complication has a considerable mortality. In some
studies,
E. coli 0157 was the most common cause of dysentery in patients seen in
hospital
clinics [8].
E. coli strains associated with HUS produce at least one toxin identical
to the exotoxin of Shigella dysenteriae serotype 1, referred to herein as
Shiga toxin I
(Stxl). This toxin has been variously referred to in the literature as Vero
cytotoxin 1
(VT 1), Shiga-like toxin 1 (SLT-I), and Shiga toxin l(Stx-I or Stx l ). In
some cases a
second toxin (variously referred to as VT2, SLT-II, Stx-II, or Stx2),
structurally and
functionally related to Stxl and having a cross-reactive A subunit, is also
produced.
Infection with Stx-producing organisms has been correlated with HUS, and E.
coli
0157:H7 is a common serotype that produces these toxins. However, strains of
E.
coli 0157 without Stx have been isolated from patients with hemorrhagic
colitis.
The pathogenicity of E. coli 0157 has been compared to that of
Shigella dysenteriae type 1 [9, 10]. Both E. coli 0157 and S. dysenteriae type
1
secrete almost identical exotoxins (Stx1 or Stx2) and cause bloody diarrhea,
with its
complications, only in humans. Antibiotic treatment does not ameliorate the
course of
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enteritis caused by E. coli 0157, and it may in fact increase the incidence of
HUS
caused by E. coli and S. dysenteriae type 1 [11,12]. Unlike S. dysenteriae
type 1,
which is confined to humans, E. coli 0157:H7 lives in cattle and in other
domesticated animals without causing symptoms. The feces of infected animals
serve
as a source of E. coli 0 157 infection in humans, through contamination of
drinking
water and meat.
Most adults have low or nondetectable levels of serum antibodies to E.
coli 0157 O-SP and to Shiga toxins. High levels of O-SP antibodies and low or
nondetectable levels of antitoxin are regularly found following infection with
E. coli
0157 and the subsequent complication HUS. It is not known whether immunity
follows infection with this pathogen.
Although there is no consensus on the host factors that might confer
immunity to E. coli 0157, the O-specific polysaccharide portion of the
lipopolysaccharides of the similar genus Shigella have emerged as possible
protective
antigens [13,14]. These polysaccharides were shown to be essential for the
virulence
of Shigella, and it is now well-established that the protection is serotype
specific.
Since each serotype is characterized by a distinct O-specific polysaccharide,
it is fair
to say that protection against E. coli 0157 is also 0-specific polysaccharide
specific.
The safety and immunogenicity of a protein conjugate of the O-specific
polysaccharides of S. sonnei, S.flexneri 2a, and S. dysenteriae type 1 has
been
demonstrated in human volunteers, and preliminary clinical trials have
established the
efficacy of these vaccines [9, 15, 16, 17].
The immunogenicity of saccharides, alone or as protein conjugates, is
related to several variables: 1) species and the age of the recipient; 2)
molecular
weight of the saccharide; 3) density of the saccharide on the protein; 4)
configuration
of the conjugate (single vs. multiple point attachment); and 5) the
immunologic
properties of the protein.
Because high molecular weight polysaccharides can induce the
synthesis of antibodies from B-cells alone, they are described as T-
independent
antigens. Three properties of polysaccharides are associated with T-
independence; 1)
their repetitive polymeric nature, which results in one molecule having
multiple
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identical epitopes; 2) a minimum molecular weight that is related to their
ability to
adhere to and cross-link membrane-bound IgM receptors, resulting in signal
transduction and antibody synthesis; and 3) resistance to degradation by
mammalian
enzymes. Most capsular polysaccharides are of comparatively high molecular
weight
(>_150 kD), and elicit antibodies in older children and in adults but not in
infants and
young children. O-SPs are of lower molecular weight (<100 kD), and may be
considered to be haptens because they combine with antibody (are antigenic)
but do
not elicit antibody synthesis (are not immunogenic). The immunogenicity of O-
SPs
as conjugates may be explained by two factors: 1) the increase in molecular
weight
that allows the O-SP to adhere to a greater number of membrane-bound IgM and
induce signal transduction to the B-cell; and 2) their protein component,
which is
catabolized by the O-SP stimulated B cell resulting in a peptide-
histocompatibility II
antigen signal to T cells.
Synthesis of conjugates for use as vaccines in humans has special
considerations. LPS is not suitable for parenteral administration to humans
because
of toxicity mediated by the lipid A domain. Usually, O-SP is prepared by
treatment
of LPS with either acid or hydrazine in order to remove fatty acids from lipid
A. The
resultant products retain the core region and the O-SP with its heterogeneous
range of
molecular weights (Mr). Conjugates are prepared by schemes that bind the
carrier to
the O-SP at multiple sites along the O-SP, or attempt to activate one residue
of the
core region.
In the case of E. coli 0157, vaccine development has been hindered
because there is little information about mechanisms of immunity [9], and
there are no
valid animal models for diseases caused by E. coli 0157[l0].
There have been some efforts to date to attempt to obtain effective
vaccine compositions against E. coli. See, e.g., Cryz et al. (U.S. Patent
5,370,872),
which describes the isolation of O-SP derived from LPS of 12 serotypes of E.
coli and
their covalent linkage to P. aeruginosa toxin A as a carrier protein [18]. The
twelve
monovalent conjugates were combined to form a polyvalent vaccine, which was
described as being safe and immunogenic in both rabbits and humans when
administered by injection. An antibody response to both the O-SP and toxin A
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moieties was reported, and protection of rabbits against E. coli sepsis was
demonstrated upon passive immunization with the resulting IgG antibodies.
However, neither bactericidal activity of the antibodies nor protection after
vaccination with the conjugates was shown, and antibodies against E. coli
strain 0157
and protection against E. coli 0157 infection are not mentioned.
Because anti-LPS or anti-O-SP antibody-mediated protection is likely
to be serotype-specific, it is unlikely that the polyvalent vaccine described
in US
Patent 5,370,872 would induce a significant level of antibodies against E.
coli 0157
O-SP or LPS. There remains a need, therefore, for compositions and methods of
inducing a significant level of antibodies against E. coli 0157. There also
remains a
need compositions and methods for inducing antibodies which have bactericidal
activity against E. coli 0157, and which also prevent or ameliorate HUS.
BRIEF DESCRIPTION OF THE INVENTION
It is an object of the invention to produce antigens based on the 0-
specific polysaccharide of Shiga toxin-producing bacteria, particularly E.
coli 0157,
conjugated with a carrier, and compositions thereof, and to methods of using
of these
conjugates and/or compositions thereof for eliciting an immunogenic response
in
mammals, including responses which provide protection against, or reduce the
severity of, bacterial infections. More particularly, it is an object of the
invention to
provide conjugates having polysaccharides containing the tetrasaccharide
repeat unit:
(- >3)-a D-GalpNAc-(1-='2)-aD-PerpNAc-(1-->3)-a-L-Fucp-(1-44)-f3 D-Glcp-(1--
>),
and compositions thereof, to induce serum antibodies having bactericidal
(killing)
activity against E. coli, in particular E. coli 0157. The conjugates, and
compositions
thereof, are useful as vaccines to induce serum antibodies which have
bactericidal or
bacteriostatic activity against against E. coil, in particular E. coli 0157,
and are useful
to prevent and/or treat illnesses caused by E. coli 0157.
It is yet another object of the present invention to provide conjugates of
E. coli 0157 O-SP bound to the non-toxic B-subunit of Shiga toxin 1 (StxB 1),
or
mutated non-toxic holotoxin of Shiga toxin 1 or Shiga toxin 2. These
conjugates have
the advantage of inducing both (1) serum IgG anti-0157-LPS with bactericidal
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activity, and (2) neutralizing antibodies to Shiga toxin 1
or Shiga toxin 2 (Stxl or Stx2)[19,20,21].
It is also an object of the invention to provide
antibodies which immunoreact with the O-specific
polysaccharide of E. coli 0157 and/or the carrier, that are
induced by these conjugates and/or compositions thereof.
Such antibodies may be isolated, or may be provided in the
form of serum containing these antibodies.
It is also an object of the invention to provide
a method for the treatment or prevention of E. coli 0157
infection in a mammal, by administration of compositions
containing the antibodies of the invention, or serum
containing the antibodies of the invention.
The invention also provides methods and kits for
identifying, detecting, and/or diagnosing E. coli 0157
infection or colonization using the antibodies which
immunoreact with the 0-specific polysaccharide of E. coli.
The invention also relates to methods and kits for
identifying, detecting and/or diagnosing the presence of
Shiga toxins 1 or 2.
According to one aspect of the present invention,
there is provided a conjugate molecule comprising the
E. coli 0157 0-specific polysaccharide, covalently bound to
a carrier which is: the B subunit of Shiga toxin 1, the
B subunit of Shiga toxin 2, a non-toxic mutant Shiga toxin 1
holotoxin, or a non-toxic mutant Shiga toxin 2 holotoxin,
wherein upon injection into a human of a therapeutically
effective amount of said conjugate molecule produces in serum
of said human bactericidal activity against E. coli 0157,
wherein: the bactericidal activity in the serum kills 50% or
more of E. coli 0157 at a serum dilution of 1,300:1 or more.
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DETAILED DESCRIPTION OF THE INVENTION
The invention provides conjugates of an E. coli 0157 0-specific
polysaccharide covalently bound, either directly or through a linker, to a
carrier, and
compositions thereof. The present invention also encompasses mixtures of such
conjugates and compositions thereof. In a preferred embodiment, the carrier is
the
non-toxic B subunit of Shiga toxin 1 or 2 (StxB 1, StxB2), or a non-toxic
mutant of
Stxl or Stx2 holotoxin. In yet another preferred embodiment, the particular E.
coli
01 57-Stx conjugate is part of a composition containing O-SP-carrier
conjugates from
other E. coli strains that commonly cause HUS, to form a multivalent vaccine
for
broad coverage against HUS. Hyperimmune plasma containing both anti-LPS and
neutralizing antibodies to Stxs are expected to provide protective and
therapeutic
effects in at-risk individuals and in patients during outbreaks.
The invention also provides methods of using these conjugates or
compositions thereof to induce in mammals, in particular, humans, the
production of
antibodies which immunoreact with the 0-specific polysaccharide of E. coli
0157. In
the preferred embodiment, antibodies which immunoreact with Shiga toxin I or
Shiga
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toxin 2 are also produced. The antibodies which immunoreact with the O-
specific
polysaccharide of E. coli 0157 are useful for the identification, detection,
and/or
diagnosis of E. coli 0157 colonization and/or infection. Antibodies which have
bactericidal or bacteriostatic activity against E. coli 0157 are useful to
prevent and/or
treat illnesses caused by E. coli 0157. Antibodies which immunoreact with
Shiga
toxins I and 2 are useful to neutralize Shiga toxins 1 and 2, and either
decrease the
incidence and/or severity of hemolytic-uremic syndrome, or prevent the
increase of its
incidence and/or severity, in established infections.
Pharmaceutical compositions of this invention are capable, upon
injection into a human of an amount containing 25 g of E. coli 0157 O-
specific
polysaccharide, of inducing in the serum bactericidal activity against E. coli
0157,
such that the serum kills, in the presence of complement, 50% or more of E.
soli
0157 at a serum dilution of 1300:1 or more. Preferred compositions can induce
serum bactericidal activity against E. coli 0157 such that the serum kills 50%
or more
of E. coli 0157 at a serum dilution of 32,000:1 or more, and the most
preferred
compositions can induce serum bactericidal activity against E. coli 0157 such
that the
serum kills 50% or more of E. coli 0157 at a serum dilution of 64,000:1 or
more. The
O-SP conjugate vaccines of this invention are designed to induce serum IgG
antibodies that will inactivate an inoculum of E. coli 0157 at the entrance of
the
jejunum before an infection is established.
The invention also provides a saccharide-based vaccine, which is
intended for active immunization for prevention of E. coli 0157 infection, and
for
preparation of immune antibodies as a therapy, preferably for established
infections.
The vaccines of this invention are designed to confer specific preventative
immunity
against infection with E. coli 0157, and to induce antibodies specific to E.
coli 0157
O-SP and LPS. The E. coli 0157 vaccine is composed of non-toxic bacterial
components, suitable for infants, children of all ages, and adults.
The conjugates of this invention, and/or compositions thereof, as well
as the antibodies thereto, will be useful in increasing resistance to,
preventing,
ameliorating, and/or treating E. coli 0157 infection in humans, and in
reducing or
preventing E. coli 0157 colonization in humans.
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This invention also provides compositions, including but not limited
to, mammalian serum, plasma, and immunoglobulin fractions, which contain
antibodies which are immunoreactive with E. coli 0157 O-SP, and which
preferably
also contain antibodies which are immunoreactive with Shiga toxins 1 or 2, in
particular with the B subunit of Shiga toxins 1 or 2. These compositions, in
the
presence of complement, have bacteriostatic or bactericidal activity against
E. coli
0157. These antibodies and antibody compositions are useful to prevent, treat,
or
ameliorate infection and disease caused by the microorganism. The invention
also
provides such antibodies in isolated form.
High titer anti-0157 sera, or antibodies isolated therefrom, could be
used for therapeutic treatment for patients with E. coli 0157 infection or
hemolytic-
uremic syndrome (HUS). Antibodies elicited by the O-SP conjugates of this
invention may be used for the treatment of established E. coli 0157
infections, and
are also useful in providing passive protection to an individual exposed to E.
coli
0157.
The present invention also provides diagnostic tests and/or kits for E.
coli 0157 infection and/or colonization, using the conjugates and/or
antibodies of the
present invention, or compositions thereof.
The present invention also provides an improved method for
synthesizing an O-SP peptide conjugate, particularly the E. coli 0157 O-SP
conjugated to the B subunit of Shiga toxin 1 or 2 (Stxl or Stx2), or to a
mutant, non-
toxic Stxl or Stx2 holotoxin.
A number of primary uses for the conjugates of this invention are
envisioned. The E. coli ALPS-protein conjugates of this invention, and the
antibodies
they induce, are expected to be useful for several purposes, including but not
limited
to:
1) a vaccine for high-risk groups (children under 5 and senior citizens);
2) high-titered globulin for plasmapheresis, for prophylaxis and treatment of
E. coli 0157-infected patients; and
3) diagnostic reagents for detecting and/or identifying E. coli 0157.
The invention is intended to be included in the routine immunization
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schedule of infants and children, and in individuals at risk for E. coli 0157
infection.
It is also planned to be used for intervention in epidemics caused by E. coli
0157.
Additionally, it is may be used as a component of a multivalent vaccine for E.
coli
0157 and other enteric pathogens, useful for example for the routine
immunization of
infants. The invention is also intended to prepare antibodies with
bacteriostatic
bactericidal activity toward E. coli 0157, for therapy of established
infection. The
invention is also intended to provide a diagnostic test for E. coli 0157
infection
and/or colonization.
Definitions
Gaip = galactosaminopyranosyl; Perp = perosaminopyranosyl; Fucp =
fucopyranosyl; Glcp = glucopyranosyl.
As used herein, the term "O-SP" when used alone refers generically to
O-specific polysaccharide, whether produced by acidolysis or hydrazinolysis of
lipopolysaccharide. When used in designating conjugates, however (e.g. O-SP-
rEPA,
DeA-LPS-rEPA, etc.) these products are differentiated by use of the term "O-
SP" for
O-specific polysaccharide produced by acidolysis, and the term "DeA-LPS" for 0-
specific polysaccharide produced by hydrazinolysis.
As used herein, the terms "immunoreact" and "immunoreactivity" refer
to specific binding between an antigen or antigenic determinant-containing
molecule
and a molecule having an antibody combining site, such as a whole antibody
molecule
or a portion thereof.
As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules.
Exemplary antibody molecules are intact immunoglobulin molecules,
substantially
intact immunoglobulin molecules and portions of an immunoglobulin molecule,
including those portions known in the art as Fab, Fab', F(ab')2 and F(v), as
well as
chimeric antibody molecules.
Polymeric carriers
Carriers are chosen to increase the immunogenicity of the
polysaccharide and/or to raise antibodies against the carrier which are
medically
beneficial. Carriers that fulfill these criteria are described in the art [22,
23, 24, 25].
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A polymeric carrier can be a natural or a synthetic material containing one or
more
functional groups, for example primary and/or secondary amino groups, azido
groups;
or carboxyl groups. The carrier can be water soluble or insoluble.
Water soluble peptide carriers are preferred, and include but are not
limited to natural or synthetic polypeptides or proteins, such as bovine serum
albumin,
and bacterial or viral proteins or non-toxic mutants or polypeptide fragments
thereof,
e.g., tetanus toxin or toxoid, diphtheria toxin or toxoid, Pseudomonas
aeruginosa
exotoxin or toxoid, recombinant Pseudomonas aeruginosa exoprotein A, pertussis
toxin or toxoid, Clostridium perfringens and Clostridium welchii exotoxins or
toxoids,
mutant non-toxic Shiga toxin holotoxin, Shiga toxins 1 and 2, the B subunit of
Shiga
toxins 1 and 2, and hepatitis B surface antigen and core antigen.
Examples of water insoluble carriers include, but are not limited to,
aminoalkyl SEPHAROSE, e. g., aminopropyl or aminohexyl SEPHAROSE
(Pharmacia Inc., Piscataway, NJ), aminopropyl glass, and the like. Other
carriers may
be used when an amino or carboxyl group is added, for example through covalent
linkage with a linker molecule.
Methods for attaching polymeric carriers
Methods for binding a polysaccharide to a protein are well known in
the art. For example, a polysaccharide containing at least one carboxyl group,
through carbodiimide condensation, may be thiolated with cystamine, or
aminated
with adipic dihydrazide, diaminoesters, ethylenediamine and the like. Groups
which
can be introduced by such known methods include thiols, hydrazides, amines and
carboxylic acids. Thiolated and aminated intermediates are stable, and may be
freeze
dried and stored cold. Thiolated intermediates may be covalently linked to a
polymeric carrier containing a sulfhydryl group, such as a 2-pyridyldithio
group.
Aminated intermediates may be covalently linked to a polymeric carrier
containing a
carboxyl group through carbodiimide condensation. See for example reference
[26],
where 3 different methods for conjugating Shigella O-SP to tetanus toxoid are
exemplified. Because the methods of the present invention better preserve the
native
structure of the antigen, they are preferred over methods which oxidize the
polysaccharide with periodate [18].
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The polysaccharide can be covalently bound to a carrier with or
without a linking molecule. To conjugate without a linker, for example, a
carboxyl-
group-containing polysaccharide and an amino-group-containing carrier are
mixed in
the presence of a carboxyl activating agent, such as a carbodiimide, in a
choice of
solvent appropriate for both the polysaccharide and the carrier, as is known
in the art
[25]. The polysaccharide is often conjugated to a carrier using a linking
molecule. A
linker or crosslinking agent, as used in the present invention, is preferably
a small
linear molecule having a molecular weight of about 500 or less, and is non-
pyrogenic
and non-toxic in the final product form, for example as disclosed in
references [22 -
25].
To conjugate with a linker or crosslinking agent, either or both of the
polysaccharide and the carrier may be covalently bound to a linker first. The
linkers
or crosslinking agents are homobifunctional or heterobifunctional molecules,
e.g.,
adipic dihydrazide, ethylenediamine, cystamine, N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-N-(2-iodoacetyl)-
(3-alaninate-propionate (SIAP), succinimidyl 4-(N-maleimido-methyl)cyclohexane-
1-carboxylate (SMCC), 3,3'-dithiodipropionic acid, and the like. Also among
the
class of heterobifunctional linkers area omega-hydroxy and omega-amino
alkanoic
acids.
More specifically, attachment of the E. coli 0157 O-specific
polysaccharide to a protein carrier can be accomplished by methods known to
the art.
In a preferred embodiment, the attachment is accomplished by first cyanating
the 0-
specific polysaccharide with a cyanylation reagent, such as cyanogen bromide,
N-
cyano-N,N,N-triethylammonium tetrafluoroborate, 1-cyano-4-(N,N-
dimethylamino)pyridine tetrafluoroborate, or the like. Several such
cyanylation
reagents are known to those skilled in the art [27]. The resulting cyanated E.
coli
0157 O-specific polysaccharide may then be reacted with a linker, such as a
dicarboxylic acid dihydrazide, preferably adipic acid dihydrazide, so as to
form a
hydrazide-functionalized polysaccharide. This hydrazide-functionalized
polysaccharide is then coupled to the carrier protein by treatment with a
peptide
coupling agent, preferably a water-soluble carbodiimide such as 1-ethyl-3-(3-
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dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide
methiodide, or the like.
More preferably, the cyanated E. coli 0157 O-specific polysaccharide
is directly reacted with the carrier protein, without introduction of a
linker. It has
been found, surprisingly, that, in the exemplified conjugates, elimination of
the
customary linker provides a more effective immunogen in the case of the E.
coli 0157
O-specific polysaccharide.
Regardless of the precise method used to prepare the conjugate, after
the coupling reactions have been carried out the unbound materials are removed
by
routine physicochemicali methods, such as for example gel filtration or ion
exchange
column chromatography, depending on the materials to be separated. The final
conjugate consists of the polysaccharide and the carrier bound directly or
through a
linker.
Dosage for Vaccination
The present inoculum contains an effective, immunogenic amount of a
polysaccharide-carrier conjugate of this invention. The effective amount of
polysaccharide-carrier conjugate per unit dose sufficient to induce an immune
response to E. coli 0157 depends, among other things, on the species of mammal
inoculated, the body weight of the mammal, and the chosen inoculation regimen,
as is
well known in the art. Inocula typically contain polysaccharide-carrier
conjugates
with concentrations of polysaccharide from about 1 micrograms to about 10
milligrams per inoculation (dose), preferably about 3 micrograms to about 100
micrograms per dose, and most preferably about 5 micrograms to 50 micrograms
per
dose.
The term "unit dose" as it pertains to the inocula refers to physically
discrete units suitable as unitary dosages for mammals, each unit containing a
predetermined quantity of active material (polysaccharide) calculated to
produce the
desired immunogenic effect in association with the required diluent.
Inocula are typically prepared as solutions in physiologically tolerable
(acceptable) diluents such as water, saline, phosphate-buffered saline, or the
like, to
form an aqueous pharmaceutical composition. Adjuvants, such as aluminum
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hydroxide, may also be included in the compositions.
The route of inoculation may be intramuscular, subcutaneous or the
like, which results in eliciting antibodies protective against E. coli 0157.
In order to
increase the antibody level, a second or booster dose may be administered
approximately 4 to 6 weeks after the initial injection. Subsequent doses may
be
administered as indicated herein, or as desired by the practitioner.
Antibodies
An antibody of the present invention in one embodiment is
characterized as comprising antibody molecules that immunoreact with E. coli
0157
O-SP or LPS.
An antibody of the present invention is typically produced by
immunizing a mammal with an immunogen or vaccine containing an E. coli 0157
polysaccharide-protein carrier conjugate to induce, in the mammal, antibody
molecules having immunospecificity for the immunizing polysaccharide. Antibody
molecules having immunospecificity for the protein carrier, such as the B
subunit of
Shiga toxins 1 or 2, will also be produced. The antibody molecules may be
collected
from the mammal and, optionally, isolated and purified by methods known in the
art.
Human or humanized monoclonal antibodies are preferred, including
those made by phage display technology, by hybridomas, or by mice with human
immune systems. The antibody molecules of the present invention may be
polyclonal
or monoclonal. Monoclonal antibodies may be produced by methods known in the
art. Portions of immunoglobulin molecules, such as Fabs, may also be produced
by
methods known in the art.
The antibody of the present invention may be contained in blood
plasma, serum, hybridoma supernatants and the like. Antibody-containing serum
of
this invention will be capable of killing, in the presence of complement, 50%
of E.
coli 01 57at a serum dilution of 1300:1 or more, preferably will do so at a
dilution of
32,000:1 or more, and most preferably will be capable of killing 50% of E.
coli 0157
at a dilution of 64,000:1 or more.
Alternatively, the antibodies of the present invention are isolated to the
extent desired by well known techniques such as, for example, ion
chromatography or
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affinity chromatography. The antibodies may be purified so as to obtain
specific
classes or subclasses of antibody such as IgM, IgG, IgA, IgG1, IgG2, IgG3,
IgG4 and
the like. Antibodies of the IgG class are preferred for purposes of passive
protection.
The antibodies of the present invention have a number of diagnostic and
therapeutic
uses. The antibodies can be used as an in vitro diagnostic agents to test for
the
presence of E. coli 0157 in biological samples or in meat and meat products,
in
standard immunoassay protocols. Such assays include, but are not limited to,
agglutination assays, radioimmunoassays, enzyme-linked immunosorbent assays,
fluorescence assays, Western blots and the like. In one such assay, for
example, the
biological sample is contacted with first antibodies of the present invention,
and a
labeled second antibody is used to detect the presence of E. coli 0157 to
which the
first antibodies have bound.
Such assays may be, for example, of direct format (where the labeled
first antibody is reactive with the antigen), an indirect format (where a
labeled second
antibody is reactive with the first antibody), a competitive format (such as
the addition
of a labeled antigen), or a sandwich format (where both labeled and unlabelled
antibody are utilized), as well as other formats described in the art.
The antibodies of the present invention are also useful in prevention
and treatment of infections and diseases caused by E. coli 0157.
In providing the antibodies of the present invention to a recipient
mammal, preferably a human, the dosage of administered antibodies will vary
depending upon such factors as the mammal's age, weight, height, sex, general
medical condition, previous medical history and the like.
In general, it is desirable to provide the recipient with a dosage of
antibodies which is in the range of from about 1 mg/kg to about 10 mg/kg body
weight of the mammal, although a lower or higher dose may be administered. The
antibodies of the present invention are intended to be provided to the
recipient subject
in an amount sufficient to prevent, or lessen or attenuate the severity,
extent or
duration of the infection by E. coli 0157. Antibodies which immunoreact with
Shiga
toxin 1 or 2 are intended to be provided to the recipient subject in an amount
sufficient to prevent, or lessen or attenuate the severity, extent or duration
of the
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infection by Shigatoxin producing organisms, such as E. coli strains 0157,
0111,
026, and 017.
The administration of the agents of the invention may be for either
"prophylactic" or "therapeutic" purpose. When provided prophylactically, the
agents
are provided in advance of any symptom. The prophylactic administration of the
agent serves to prevent or ameliorate any subsequent infection. When provided
therapeutically, the agent is provided at (or shortly after) the onset of a
symptom of
infection. The agent of the present invention may, thus, be provided prior to
the
anticipated exposure to E. coli 0157 (or other Shiga toxin producing
bacteria), so as
to attenuate the anticipated severity, duration or extent of an infection and
disease
symptoms, after exposure or suspected exposure to these bacteria, or after the
actual
initiation of an infection.
For all therapeutic, prophylactic and diagnostic uses, the
polysaccharide-carrier conjugates of this invention, as well as antibodies and
other
necessary reagents and appropriate devices and accessories may be provided in
kit
form so as to be readily available and easily used.
The following examples are exemplary of the present processes and
incorporate suitable process parameters for use herein. These parameters may
be
varied, however, and the following should not be deemed limiting.
EXAMPLES
Example 1
Conjugation of E. coli 0157 O-SP with Various Polytpeytides
0157 LPS were detoxified by hydrolysis with acetic acid (designated
O-SP) or with hydrazine (designated DeA-LPS) and then covalently bound to
Clostridium welchii exotoxin C (Pig Bel toxoid [CW]), Pseudomonas aeruginosa
recombinant exoprotein A (rEPA), or bovine serum albumin (BSA) [8]. These
E.coli
0157:H7 polysaccharide-protein conjugates were given the following
designations:
O-SP-BSAI
O-SP-BSA2
DeA-LPS-BSA
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O-SP-CW
DeA-LPS-CW
O-SP-rEPA
DeA-LPS-rEPAI
DeA-LPS-rEPA2
Mice were immunized with these conjugate compositions containing
2.5ug of polysaccharide, with booster injections, and the determination of
antibody
levels and bactericidal antibody titers in mice were determined. Geometric
mean
antibody level (ELISA units) and immunoglobulin class composition of LPS
antibodies elicited by E. coli 0157-rEPA conjugates in mice are shown in Table
1.
TABLE 1.
Immunoglobulin class composition of LPS antibodies elicited by
E. coli 0157-rEPA conjugates in mice
Immunogen Geometic mean antibody level (ELISA units) (25 h-75th centiles)
After 15` injection After 2"d injection After 3d injection
IgG
O-SP-rEPA 0.08 (0.05-0.10) 2.50* (1.06-4.79) 6.26** (3.37-9.6)
DeA-LPS-rEPAI 0.07 (0.04-0.13) 1.37* (0.50-2.63) 4.49*** (1.49-16,4)
DeA-LPS-rEPA2 0.07 (0.06-0.07) 0.66* (0.07-3.73) 5.10** (2.23-10.0)
IgM
O-SP-rEPA 0.53 (0.36-0.72) 0.51 (0.31-1.12) 0.38 (0.22-0.59)
DeA-LPS-rEPAI 0.11 (0.04-0.34) 0.32 (0.08-0.89) 0.94 (0.28-2.94)
DeA-LPS-rEPA2 0.09 (0.06-0.11) 0.32 (0.06-1.53) 0.28 (0.21-0.45)
a. IgG and IgM components of the hyperimmune 0157 sera (see Materials and
Methods) were
used as standards and assigned a value of 100 ELISA U each. Injection of O-SP,
DeA-LPS, or
saline did not elicit detectable antibodies.
P<0.01 when compared with the value for O-SP-rEPA after the first injection;
**, P>0.02 when compared with the value for the same immunogen after the
second injection;
***, P<0.07 when compared with the value for the same immunogen after the
second injection.
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Bactericidal activity of serum LPS antibodies elicited in mice by
immunization with heat-killed E. coli 0157:H7 or O-specific polysaccharide-
protein
conjugates are shown in Table 2 below:
TABLE 2.
Bactericidal activity of serum LPS antibodies elicited in mice by immunization
with heat-killed E.coli 0157:H7 or O-specific polysaccharide-protein
conjugates
Vaccinea Antibody titer (ELISA units) Reciprocal
bacterial
Total IgG IgM titerb
Expt 1
O-SP-C W 79.25 100
DeA-LPS-CW 15.1 >100
DeA-LPS-CW 19.4 80
E.coli 0157:H7 100.0 35
Expt 2
DeA-LPS-rEPA 18.8 0.07 320
DeA-LPS-rEPA 56.8 0.33 640
DeA-LPS-rEPA. 32.8 0.45 640
O-SP-rEPA 18.6 0.44 640
O-SP-rEPA 15.8 0.59 640
E. coli 0157:H7 is pooled hyperimmune sera from mice injected with heat-killed
E. coli
0157. All other sera were from individual mice taken after the third conjugate
injection.
Serum dilutions were mixed with an equal volume of -103 E. coli 0157:H7
organisms per ml
and complement.
b The reciprocal bactericidal titer is expressed as the highest serum dilution
yielding 50%
killing. Absorption with LPS or DeA-LPS removed all of the bactericidal
activity from sera
from conjugate-injected mice and 90% from the hyperimmune sera prepared by
injection of
heat-killed E. coli 0157.
Example 2
Conjugation of E. coli 0157 O-SP with rEPA: Preparation of Vaccine
Compositions
As discussed above, O-SP of E. coli 0157, prepared by acetic acid
hydrolysis, and DeA-LPS 0157, prepared by hydrazinolysis, have been previously
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described. Conjugates of these polysaccharides to rEPA (O-SP 0157-rEPA, DeA-
LPS
0157-1, and DeA-LPS 0157-rEPA2) were prepared by the published procedure [8].
These conjugates were approved for investigation by the NIH (OH94-CH-N040),
the
FDA (BB-IND-5528) and the Institutional Review Board, Carolinas Medical
Center,
Charlotte, NC (08-94-08B). Pyrogen, sterility and safety testing of the final
containers
were performed by the Center for Biologics Evaluation and Research, FDA. All
three
conjugates elicited serum. IgG anti-LPS with bactericidal activity when
injected by a
clinically relevant scheme and dosage in mice[8].
Clinical protocol
Volunteers of either gender and any ethnic group between ages 18 and 44
years were recruited from the staff of Carolinas Health Care System and the
city of
Charlotte, NC. Exclusion criteria were: pregnancy or planned pregnancy in the
next six
months, positive stool culture for E. coli 0157 or a history of hemorrhagic
colitis,
chronic disease including HIV 1, hepatitis or inflammatory bowel disease,
acute illness
including diarrhea, taking controlled substances, hospitalization within the
year, taking
regular medications, participation in another research protocol during the
next two
months, abnormal liver function test or having received cholera vaccine [32,
28]. After
giving Informed Consent, a medical history and physical examination were
performed
and blood was obtained for assay of HIV 1, hepatitis b surface antigen,
pregnancy test,
liver function tests (LFT), antibodies to E. coli 0157 LPS and P. aeruginosa
exotoxin A
(ETA) and a culture of the stool for E. coli 0157. Eighty-seven volunteers
were
determined healthy and randomized into 3 groups of 29 to receive a injection
of 0.5 ml
of one of the experimental vaccines containing 25 p.g of O-SP. Injections were
delivered
intramuscularly into the deltoid muscle. The volunteers were observed for 30
minutes
after vaccination. Temperature and local or systemic reactions were recorded
at 6, 24,
48 and 72 hours following vaccination.
All volunteers returned at 1, 4 and 26 weeks following vaccination for a
health history and reaction, and blood was drawn. LFTs were performed, total
protein/albumin), total bilirubin/direct and indirect, alkaline phosphatase
(AP), SGOT
(AST), SGPT (ALT), and GGT at each visit. Volunteers who had abnormal LFT
levels
at one week had repeated LFT tests at subsequent visits. Serum was collected
for LPS
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and ETA antibody assays. Stool cultures for E. coli 0157 were obtained prior
to and 4
and 26 weeks following vaccination. E. coli 0157 LPS and P. aeruginosa
exotoxin A
(ETA) antibodies of the volunteers were determined by ELISA [8].
Statistical methods
Antibody levels are expressed as geometric means (GM). Levels below
the sensitivity of ELISA were assigned the value of one-half of that level.
Comparison
of GM was performed with either the two-sided t-test, paired t-test or the
Wilcoxon test
where appropriate.
Results - clinical responses
One volunteer reported 3-6 cm diameter of erythema at the injection site
within 24 hours following vaccination; one reported 1-3 cm and one reported >6
cm.
Four volunteers reported erythema and induration after 72 hours observation:
one (1-3
cm), two (3-6 cm) and one (>6 cm); all erythemas resolved by the 17th day.
Six volunteers (6.9%) had asymptomatic elevations (up to 35% above the
normal range) of one or more serum LFT following vaccination. Four of these 6
volunteers had mild elevation of LDH and/or AP that returned to normal at 4-5
weeks.
One volunteer had a serum bilirubin of 2.2 mg/dl (normal 1.5 mg/dl) with
indirect
bilirubin of 1.9 mg/dl at four weeks, and normal values at 14 weeks. Another
volunteer
had ALT (SGPT) and GGT evaluations of 33% and 26% respectively at four weeks,
and
elevations 13% and 47% respectively at 24 weeks following vaccination.
Ninety percent of volunteers reported oral temperatures less than 37.2 C
at different observation times post-vaccination. The remainder of the
volunteers
reported oral temperatures 37.2-38 C with symptoms of acute respiratory
infections.
There was no significant correlation between the reported post-
vaccination observations and the lots of vaccine administered and no volunteer
was
hospitalized during the study.
One recipient of DeA-LPS 0157-rEPAI had a stool culture positive for
E. coli 0157 at the 1 week post-vaccination visit. This volunteer had no
adverse
reactions following vaccination and no complaints throughout the study, and
subsequent
stool cultures were negative for E. coli 0157.
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Results - antibody levels (Tables 3a and 3b)
IgG: Pre-vaccination GM IgG anti-LPS levels in the three groups were
low and similar. One week after vaccination, 71/87 (82%) responded with a >_4-
fold
rise. Four weeks after vaccination, there were further rises in GM levels in
all three
groups (p<0.0001): all vaccinees responded with a >_4-fold rise over the 1
week level.
The GM levels in recipients of O-SP-rEPA were slightly higher than in those
injected
with either of the two DeA-LPS-rEPA conjugates (61.9 vs. 46.3 NS, 61.9 vs.
36.3,
p<0.05). At 26 weeks, the GM levels of the 3 groups were similar (32.8, 31.2,
33.1,
NS). Although the decline from the four week level was significant for each
group
(p<0.05), the levels at 26 weeks were higher than those at one week following
vaccination in all three groups (32.8, 31.2, 33.1 vs. 7.93, 5.73, 4.12,
p<0.01); and 97% of
volunteers had >_10-fold higher levels at 26 weeks than their pre-injection
levels. Within
the 25-75 percentile range, geometric mean titers were increased 68-fold to
132-fold
after 4 weeks, and the overall result for the three conjugates at 4 weeks was
a 93-fold
increase in geometric mean titer. At 26 weeks, the results were increases of
61 -fold to
70-fold, and 64-fold increase overall for all conjugates. The volunteer who
had a stool
culture positive for E. coli 0157 at 1 week had IgG anti-LPS levels at pre-
immunization,
1-, 4-, and 26-week post-immunization of 0.81, 1.15, 7.73 and 7.01
respectively, that are
lower than the GM of all 3 groups.
IgM: Each conjugate elicited a significant rise in IgM anti-LPS at the 4
and 26 weeks intervals (p<0.0001). O-SP-rEPA elicited the highest level at
each post
vaccination interval but the difference was significant only at 4 weeks (32.8
vs.
18.1,19.1, p<0.05). At the 4 week interval, there was a >_4-fold rise in 61/87
(70%) and
in 34/86 (39.5%) at 26 weeks compared to pre-vaccination levels. There was a
significant decrease in serum IgM anti-LPS at 26 weeks in all of the three
groups
(p<0.02) but there were no significant differences in the GM levels among the
three
groups. The volunteer who had a stool culture positive for E. coli 0157 at 1
week had a
pre-immunization anti-LPS IgM level which was relatively high (11.9). The IgM
levels
declined 1, 4 and 26 weeks post-immunization (7.04, 10.6 and 5.94 units,
respectively).
These levels are lower than the GM of the three groups.
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.: Pre-vaccination levels of IgA anti-LPS were low. Similar to IgG
and IgM anti-LPS, about 90% of the volunteers responded with >_4-fold rise in
IgA anti-'
LPS at one week, and 99% at four weeks (p<0.001). IgA anti-LPS GM levels
declined
to about 70% of the levels at the 4 week interval.
Table 3a.
Geometric mean. titers of serum IgG, IgM, and IgA lipopolysaccharide (LPS)
antibodies
elicited in volunteers by injection of E. coli 0157 O-SP-rEPA conjugates.
ELISA units (25th - 75th percentiles)
Conjugate Preimmune 1 Week 4 Weeks 26 Weeks
IgG
O-SP-rEPA 0.47 (0.3-0.7) 7.93 (2.8-24) 61.9 (40-91) 32.8 (23-50)
DeA-LPS-rEPA, 0.51 (0.3-0.9) 5.73 (1.8-22) 46.3 (22-84) 31.2 (12-61)
DeA-LPS-rEPA2 0.54 (0.3-0.9) 4.12 (2.2-6.0) 36.6 (20-76) 33.1 (15-57)
IgM
O-SP-rEPA 8.10 (4.0-14) 32.8 (23.51) 64.7 (47-109) 28.6 (17-44)
DeA-LPS-rEPA, 7.19 (3.1-12) 19.1 (9.2-29) 43.5 (13-56) 22.5 (11-34)
DeA-LPS-rEPA2 7.41 (4.6-13) 18.1 (10-27) 42.7 (26-73) 25.3 (17-35)
IgA
O-SP-rEPA 0.06 (0.0-0.1) 0.98 (0.5-2.4) 1.73 (1.0-2.5) 1.17 (0.9-2.1)
DeA-LPS-rEPA, 0.06 (0.0-0.1) 0.58 (0.3-0.8) 1.26 (0.6-3.7) 1.01 (0.5-1.9)
DeA-LPS-rEPA2 0.07 (0.0-0.1) 0.90 (0.4-1.8) 2.13 (1.2-4.9) 1.40 (1.0-2.5)
NOTE: High-titered postvaccination sera were used as standards. IgG, IgM, and
IgA were
assigned value of 100 ELISA units. Each group had 29 volunteers.
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Table 3b.
Fold increases in geometric mean titers of serum IgG, IgM, and IgA
lipopolysaccharide (LPS) antibodies elicited in volunteers.
Ab class Conjugate -fold increase in 25th - 75th percentiles
I Week 4 Weeks 26 Weeks
IgG O-SP-rEPA 17 132 70
DeA-LPS-rEPA, 11 91 61
DeA-LPS-rEPA2 7.6 68 61
Geometric mean 11 93 64
IgM O-SP-rEPA 4.0 8.0 3.5
DeA-LPS-rEPA, 2.7 6.0 3.1
DeA-LPS-rEPA2 2.4 5.8 3.4
Geometric Mean 3.0 6.5 3.3
IgA O-SP-rEPA 16 29 20
DeA-LPS-rEPA, 9.7 21 17
DeA-LPS-rEPA2 13 30 20
Geometric Mean 13 26 19
NOTE: High-titered postvaccination sera were used as standards. IgG, IgM, and
IgA were
assigned value of 100 ELISA units. Each group had 29 volunteers.
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Results - serum bactericidal activity (Table 4)
Serum from high-responding volunteers (above the 75th percentile) was
diluted serially and the diluted samples were analyzed for their ability to
kill E. coli
0157:H7. Pre-vaccination sera had no detectable bactericidal activity against
E. coli
01 57:H7. The three conjugates elicited serum bactericidal activity that
roughly
correlated with the serum IgG and IgM anti-LPS antibody levels.
The results in Table 4 are those for serum from high-responding
volunteers. Typically, the bactericidal titer (reciprocal dilution) for 50%
killing ranged
from about 2400 to about 32000.
Table 4.
Bactericidal activity (reciprocal titer) of serum anti-lipopolysaccharide
(LPS)
antibodies elicited in volunteers by injection of E. coli 0157 O-SP-rEPA
conjugates.
Antibody level Bactericidal
Conjugate (ELISA units) titer*
IgG I
Preimmune 0.21 2.92 0
Preimmune 0.84 9.1 0
O-SP-rEPA 120.1 354.2 >6.4 X 104
O-SP-rEPA 251.9 112.9 1.3 X 104
O-SP-rEPA 156.3 183.6 >1.3 X 103
DeA-LPS-rEPA3 231.4 59.9 >6.4 X 104
DeA-LPS-rEPA2 77.5 68.2 1.3 X 104
* Expressed as reciprocal of highest serum dilution yielding 50% killing.
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Results - serum-anti-P. aeruginosa exotoxin A (Table 5)
Most volunteers had low or non-detectable ETA antibodies in their pre-
vaccination sera. All three conjugates elicited significant increases in GM
IgG anti-ETA
at the 1-week (p<0.002) and 4-week (p<0.001) intervals. At 26 weeks, the GM
levels
declined to those observed one week after vaccination. There were no
statistically
significant differences in the GM IgG anti-ETA at each bleeding interval among
the
three groups.
Table 5
Serum antibodies to Pseudomonas aeruginosa exotoxin A (ETA) elicited by
Escherichia coli 0157 O-specific polysaccharide-rEPA conjugates in volunteers
GM antibody level (ELISA Units*)
Conjugate n Preimmune 1 week 4 weeks 26 weeks
O-SP-rEPA 29 0.29 0.93 1.90 0.88
DeA-LPS-rEPAI 29 0.39 0.91 1.48 0.87
DeA-LPS-rEPA2 29 0.29 0.65 0.93 0.67
*A high titered volunteer serum was used as a standard and assigned a value of
100 ELISA
Units.
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Example 3
Conjugation of E. coli 0157 O-SP with STXB1 and Preparation of Vaccine
Compositions
E. coli 01.57 O-SP was prepared by treatment of LPS with acetic acid
as previously described [8, 9]. The B-subunit of Shigella toxin I (StxBl) was
synthesized by Vibrio cholerae strain 0395-N1 (pSBC32 containing the StxB I
gene)
and purified by affinity chromatography[20, 21 ]. SDS 7% PAGE of StxB 1 showed
one major band at 9 kDa and a faint band with slightly higher molecular
weight.
For conjugation, 0157 O-SP was bound to StxBl directly by treatment
with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or by
carbodiimide mediated condensation with adipic acid hydrazide linker [29, 30].
For
direct conjugation, CDAP (100 mg/ml in acetonitrile) was added to O-SP in
saline (5
mg/ml) at 0.3/1 (wt/wt) at room temperature, pH 5.8 to 6. 60 L of 0.2 M
triethylamine (TEA) added to bring the pH to 7.0 for 2 minutes. An equal
weight of
StxB 1 was added to the CDAP treated O-SP and the pH maintained at 8.0 to 8.5
for 2
hours. The reaction mixture was passed through a 1.5x90 cm Sepharose 6B column
in 0.2M NaCl, the void volume fractions collected, and designated as OSP-
StxBl.
Conjugate using a linker, adipic acid dihydrazide (ADH) was prepared
as described previously [8, 30]. Briefly after addition of TEA in the above
procedure,
an equal volume of 0.8 M ADH in 0.5 M NaHCO3 was added and the pH maintained
at 8.0 to 8.5 for 2 hours. The reaction mixture was dialyzed against saline
overnight
at 4 C and passed through a 2.5x3l cm P 10 column in water. The void volume
fractions were pooled, freeze-dried, and designated as OSP-AH. OSP-AH (10 mg),
dissolved in 2 ml of saline, was added to an equal weight of StxBI and the pH
brought to 5.1. The reaction mixture was put on ice and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) was added to 0.05M and the pH
maintained at 5.1 to 5.5 for 2 hours. The reaction mixture was passed through
a
1.5x90 cm Sepharose 6B column in 0.2 M NaCl, the void volume fractions
collected
and designated as OSP-AH-StxB1. Double immunodiffusion and ELISA were
performed as described [8].
Female general purpose mice (n=10/group) were injected
subcutaneously with saline or one of the conjugates containing 2.5 p.g
saccharide on
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days 0, 14, and 28. The mice were exsanguinated 7 days after each injection.
Pooled
sera from hyperimmunized mice were used as reference and assigned 100 ELISA
units for IgG and IgM respectively. Neutralization of Stxl and Stx2 toward
HeLa
cells was measured using HeLa (CCL-2) cell monolayers in 96-well flat-bottom
microtiter plates [21 ]. Each well was seeded with 1-6x 104 cells in 0.1 ml.
Monolayers were established by overnight incubation in 5% CO2. Toxin
neutralization was determined by incubating dilutions of mouse serum with Stx-
I or
Stx-II at a final concentration of 100 pg/ml. The serum and toxin mixture was
incubated at room temperature for 30 minutes and 0.1 ml was added to each
well.
Following incubation overnight, the surviving cells were determined spectro-
photometrically using the! crystal violet staining method of Gentry and
Dalrymple[31].
Toxin neutralization was determined from a dose response curve of either Shiga
toxin
on each 96-well plate. Bactericidal activity was assayed as described [8, 10].
Results with 0157 O-SP - STXB1 conjugates
Derivatization of O-SP with adipic acid dihydrazide was 3.1% (wt/wt),
similar to previous E. cocci 0157 preparations [8]. The saccharide/protein
ratios
(wt/wt) were about 0.5 for both conjugates. The yields, based on saccharide in
the
conjugates, were 2.3% for OSP-StxBl and 3.4% for OSP-AH-StxB1. A single line
of
precipitation in double immunodiffusion was formed by rabbit anti-Stxl and
mouse
hyperimmune anti-0157 reacted against either conjugate.
After three injections, both conjugates elicited statistically significant
rises of IgG and IgM anti-LPS (Table 6). The geometric mean (GM) anti-LPS
level
elicited by OSP-StxB 1 was 0.63 for IgG and 0.14 for IgM and for O-SP-AH-StxB
1
were 1.7 for IgG and 0.25 for IgM: the differences between two conjugates were
not
statistically significant.
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Table 6.
Geometric mean IgG and IgM serum LPS antibody levels and neutralization titers
against Shiga toxin 1 elicited in mice by conjugates of Escherichia coli 0157
O-SP
with StxB I.
Neutralization Titer (%)t
Immunogen Anti-LPS (ELISA)* Serum Dilution
IgG IgM 1:100 1:1000 1:10,000
Saline <0.05 <0.05 0$ 0 0
OSP-AH-StxBl 1.7 0.25 >99 901 34
OSP-StxB 1 0.63 0.14 >99 98 70
* Geometric mean of sera from 10 mice. Expressed in ELISA units using pooled
hyperimmune mouse sera as reference (100 units for IgG and IgM respectively).
t Geometric mean (n=10) neutralization titer determined with Stxl and HeLa
cells,
No neutralization at 1/100 dilution.
Sera from mice injected with saline or human sera from volunteers
injected with E. coli 0157 O-SP-rEPA conjugates showed no neutralization to
Stx1 or
to Stx2. Sera from mice injected 3 times with either of the 0157 O-SP - StxB1
conjugates showed complete neutralization of Stxl at 1/100 dilution. At
1/1,000
dilution, the GM of neutralization titer was 90% for OSP-AH-StxB 1 and 98% for
OSP-StxBl. At 1/10,000 dilution, the sera from mice injected with OSP-StxBl
had a
significantly higher neutralization titer (70%) than the sera elicited by O-SP-
AH
StxB1 (34%). None of the sera from mice injected with either conjugate showed
neutralization against Stx2. Both conjugates elicited levels of bactericidal
antibodies
against E. coli 0157 that were roughly proportional to the content of IgG anti-
LPS;
this activity was removed by absorption with E. coli 0157 LPS.
DISCUSSION
The O-SP of E. coli 0157 LPS is a linear copolymer composed of the
tetrasaccharide repeat unit: (--+3)-a-D-GalpNAc-(1-32)-a-D-PerpNAc-(1-->3)-a-
L-Fucp-(1-->4)-Pi D-Glcp-(1-->). It is non-immunogenic, probably due to its
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comparatively low molecular weight. As with other polysaccharides, its
immunogenicity is increased by binding it to proteins to form a conjugate. Of
the three
conjugates of the present invention shown in Table 1, none elicited fever or
significant
local reactions in human volunteers, and all volunteers responded with a >_4-
fold rise in
serum IgG anti-E. coli 0157 LPS that was sustained 26 weeks after injection.
(Re-
injection of the E. coli 0157 O-SP conjugates was not attempted because of the
failure
of other polysaccharide conjugates to induce a booster response in adults.)
These volunteers, like most adults, had low levels of "natural" serum anti
E. coli 0157 LPS probably induced by cross-reacting antigens [32, 33, 34, 35].
This is
typical for other bacterial pathogens as well. Higher levels of anti-O 157 LPS
antibodies
are found in patients with HUS, and in individuals involved in raising cattle
in certain
areas, probably as a result of previous contact with these organisms. Although
the
unusual monosaccharide perosamine is found in the O-SP of both E. coli 0157
and V.
cholerae 01, we have not been able to detect a cross-reaction between human
antibodies
to these two antigens. The conjugate prepared from the O-SP obtained by acetic
acid
hydrolysis (O-SP-rEPA) elicited significantly higher levels of anti-0157 LPS
at four
weeks than did conjugates prepared with hydrazine-treated LPS. The LPS and ETA
antibody levels, however, at 26 weeks post-injection were similar in all three
groups
(Table 1). As reported for patients with shigellosis and for adults vaccinated
with
Shigella conjugates, serum IgG anti-LPS rose to the highest level and was the
most
sustained of the three serum immunoglobulins [13,15, 34, 36, 37]. Similar
results were
obtained in mice for the IgG anti-LPS responses elicited by E. coli 0111
conjugates
[38].
The protective action of existing vaccines may be due to the induction of
a critical level of specific IgG antibodies that, in many cases, inactivate
the inoculum of
the pathogen on epithelial surfaces including the intestine [39, 40]. It is
not commonly
appreciated that serum IgG is a major immunoglobulin component of secretory
fluids
including that of the small intestine. As has been observed in mice [8], all
three
conjugates induced IgG anti-LPS with bactericidal activity in the volunteers
(Table 2).
Serum IgG anti-polysaccharide is the major, if not the sole host component,
that confers
immunity induced by these conjugates. Accordingly, it should be possible to
standardize
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WO 00/04922 PCT/US98/14976
the potency of E. coli 0157 conjugates by chemical assay and by measurement of
serum
IgG anti-polysaccharide as has been done for Haemophilus influenzae type b
conjugate
vaccines.
The 1995 outbreak of E. coli 0157 infection in Japan lasted several
months, partly due to the failure to identify the bacterial sources [41 ].
Most of the
volunteers (81%) responded with nearly a 10-fold increase in IgG anti-LPS 1
week
after vaccination, indicating that the vaccine of this invention could serve
to control E.
coli 0157 infection during an outbreak. Another use for the E. coli 0157
conjugates
of this invention would be to prepare high-titered IgG anti-LPS globulin for
prophylaxis and treatment of case contacts during an outbreak. It has been
suggested
that antibiotic treatment of patients increases the incidence of HUS, possibly
by
causing lysis of the E. coli 0157 with release of additional Shiga toxins.
Clinical and
experimental data point to LPS as the pathogenic agent for HUS and the other
extraintestinal lesions following infection with enteric Gram-negative
pathogens [42,
43]. There is also a suggestion of a direct role of Shiga toxins on renal
tissue
involvement in HUS [44]. The present invention provides a solution to this
problem
in the form of a conjugate of E. coli 0157 O-SP with the B subunit of Shiga
toxin 1.
In mice, this conjugate induces both serum IgG anti-LPS and neutralizing
antibodies
to Shiga toxin 1.
The data show that the various E. coli 0157 LPS-protein conjugates
disclosed herein will generate high antibody levels in humans (i.e.,
approximately 5-
times more IgG in humans than in mice) and high neutralization antibody titers
in
humans (i.e., 103 to 104 in humans as opposed to 102 in mice). The data also
show
that the various E. coli 0157 LPS-protein conjugates disclosed herein will
generate a
greater than 4-fold rise in IgG antibody levels in about 80% of human subjects
one
week after a single injection and in all human subjects 4 weeks after a single
injection.
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