U.S. patent number 8,168,195 [Application Number 12/471,049] was granted by the patent office on 2012-05-01 for vaccines against escherichia coli o157 infection.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Edward Konadu, Yvonne Ageyman Konadu, legal representative, John B. Robbins, Shousun Chen Szu.
United States Patent |
8,168,195 |
Szu , et al. |
May 1, 2012 |
Vaccines against Escherichia coli O157 infection
Abstract
This invention relates to conjugates of the O-specific
polysaccharide of E. coli O157 with a carrier, and compositions
thereof, and to methods of using 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:
(.fwdarw.3)-.alpha.-DGalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)--
.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.), and
conjugates thereof, to induce serum antibodies having bactericidal
(killing) activity against hemolytic-uremic syndrome (HUS) causing
E. coli, in particular E. coli O157. The conjugates, and
compositions thereof, are useful as vaccines to induce serum
antibodies which have bactericidal or bacteriostatic activity
against E. coli, in particular E. coli O157, and are useful to
prevent and/or treat illnesses caused by E. coli O157. The
invention further relates to the antibodies which immunoreact with
the O-specific polysaccharide of E. coli O157 and/or the carrier,
that are induced by these conjugates and/or compositions thereof.
The invention also relates to methods and kits using one or more of
the polysaccharides, conjugates or antibodies described above.
Inventors: |
Szu; Shousun Chen (Bethesda,
MD), Robbins; John B. (Chevy Chase, MD), Konadu;
Edward (Ashanti Region, GH), Konadu, legal
representative; Yvonne Ageyman (Bronx, NY) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
|
Family
ID: |
34136897 |
Appl.
No.: |
12/471,049 |
Filed: |
May 22, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090232840 A1 |
Sep 17, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10987428 |
Nov 12, 2004 |
7553490 |
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09744289 |
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6858211 |
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PCT/US98/14976 |
Jul 20, 1998 |
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Current U.S.
Class: |
424/194.1;
424/197.11; 530/389.5 |
Current CPC
Class: |
A61K
39/0258 (20130101); A61K 2039/6037 (20130101); Y02A
50/474 (20180101); Y02A 50/30 (20180101) |
Current International
Class: |
A61K
39/385 (20060101) |
References Cited
[Referenced By]
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|
Primary Examiner: Navarro; Albert
Assistant Examiner: Portner; Ginny
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of prior U.S. patent application
Ser. No. 10/987,428, filed on Nov. 12, 2004 now U.S. Pat. No.
7,553,490, which is a continuation of prior U.S. patent application
Ser. No. 09/744,289, filed on Aug. 1, 2001 now U.S. Pat. No.
6,858,211, which is the national stage under .sctn.371 of PCT
Application No. PCT/US98/14976, filed on Jul. 20, 1998, published
in English under PCT Article 21(2). The prior applications are
incorporated herein by reference in their entirety.
Claims
The invention claimed is:
1. A composition, comprising: (1) about 5 .mu.g to about 50 .mu.g
of E. coli O157 O-specific polysaccharide covalently bound to a B
subunit of Shiga toxin 1, a B subunit of Shiga toxin 2, a non-toxic
Shiga toxin 1 holotoxin, or a non-toxic Shiga toxin 2 holotoxin;
and (2) a physiologically acceptable agent.
2. The composition of claim 1, wherein the E. coli O157 O-specific
polysaccharide is covalently bound to the B subunit of Shiga toxin
1, the B subunit of Shiga toxin 2, the non-toxic Shiga toxin 1
holotoxin, or the non-toxic Shiga toxin 2 holotoxin with a
linker.
3. The composition of claim 1, comprising the B subunit of Shiga
toxin 1.
4. The composition of claim 1, comprising the B subunit of Shiga
toxin 2.
5. The composition of claim 1, comprising the non-toxic Shiga toxin
1 holotoxin.
6. The composition of claim 1, comprising the non-toxic Shiga toxin
2 holotoxin.
7. The composition of claim 1, further comprising an adjuvant.
8. The pharmaceutical composition of claim 1, comprising (1) about
5 .mu.g to about 50 .mu.g of E. coli O157 O-specific polysaccharide
covalently bound to a B subunit of Shiga toxin 1 or a non-toxic
Shiga toxin toxin 1 holotoxin and (2) a physiologically acceptable
agent.
9. The composition of claim 1, wherein the E. coli O157 O-specific
polysaccharide is covalently bound to the B subunit of Shiga toxin
1 by means of a dicarboxylic acid dihydrazide linker.
10. The composition of claim 9, wherein the dicarboxylic acid
dihydrazide is an adipic acid dihydrazide.
11. A method of inducing an immune response, comprising
administering to a subject a therapeutically effective amount of
the composition of claim 1, thereby inducing the immune
response.
12. The composition of claim 8, further comprising an adjuvant.
13. The composition of claim 7, wherein the adjuvant is aluminum
hydroxide.
14. A method of inducing an immune response, comprising
administering to a subject a therapeutically effective amount of
the composition of claim 8, thereby inducing the immune response.
Description
FIELD OF THE INVENTION
This invention relates to conjugates of the O-specific
polysaccharide of Shiga toxin-producing bacteria, such as E. coli
O157, with 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:
(.fwdarw.3)-.alpha.-D-GalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)-
-.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.), and
conjugates thereof, to induce serum antibodies having bactericidal
(killing) activity against E. coli, in particular E. coli O157. The
conjugates, and compositions thereof, are useful as vaccines to
induce serum antibodies which have bactericidal or bacteriostatic
activity against E. coli, in particular E. coli O157, and are
useful to prevent and/or treat illnesses caused by E. coli
O157.
The invention further relates to the antibodies which immunoreact
with the O-specific polysaccharide of E. coli O157 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 O157, 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 O-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 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 O157: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 O157 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
O157 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 1 (Stx1). This toxin has been
variously referred to in the literature as Vero cytotoxin 1 (VT1),
Shiga-like toxin 1 (SLT-I), and Shiga toxin 1 (Stx-I or Stx1). In
some cases a second toxin (variously referred to as VT2, SLT-II,
Stx-II, or Stx2), structurally and functionally related to Stx1 and
having a cross-reactive A subunit, is also produced. Infection with
Stx-producing organisms has been correlated with HUS, and E. coli
O157:H7 is a common serotype that produces these toxins. However,
strains of E. coli O157 without Stx have been isolated from
patients with hemorrhagic colitis.
The pathogenicity of E. coli O157 has been compared to that of
Shigella dysenteriae type 1 [9, 10]. Both E. coli O157 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
enteritis caused by E. coli O157, 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 O157:H7 lives in cattle and in other domesticated animals
without causing symptoms. The feces of infected animals serve as a
source of E. coli O157 infection in humans, through contamination
of drinking water and meat.
Most adults have low or nondetectable levels of serum antibodies to
E. coli O157 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 O157 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 O157, 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 O157 is also O-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 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
(.gtoreq.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 (.ltoreq.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 (M.sub.r). 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 O157, 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 O157 [10].
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.
Pat. No. 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 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 O157 and protection against E. coli O157 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 U.S. Pat. No. 5,370,872 would induce a
significant level of antibodies against E. coli O157 O-SP or LPS.
There remains a need, therefore, for compositions and methods of
inducing a significant level of antibodies against E. coli O157.
There also remains a need compositions and methods for inducing
antibodies which have bactericidal activity against E. coli O157,
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
O-specific polysaccharide of Shiga toxin-producing bacteria,
particularly E. coli O157, 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:
(.fwdarw.3)-.alpha.-D-GalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)-
-.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.), and
compositions thereof, to induce serum antibodies having
bactericidal (killing) activity against E. coli, in particular E.
coli O157. The conjugates, and compositions thereof, are useful as
vaccines to induce serum antibodies which have bactericidal or
bacteriostatic activity against E. coli, in particular E. coli
O157, and are useful to prevent and/or treat illnesses caused by E.
coli O157.
It is yet another object of the present invention to provide
conjugates of E. coli O157 O-SP bound to the non-toxic B-subunit of
Shiga toxin 1 (StxB1), 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-O157-LPS with bactericidal
activity, and (2) neutralizing antibodies to Shiga toxin 1 or Shiga
toxin 2 (Stx1 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 O157
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 O157 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 O157 infection or colonization
using the antibodies which immunoreact with the O-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.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides conjugates of an E. coli O157 O-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 (StxB1, StxB2), or a
non-toxic mutant of Stx1 or Stx2 holotoxin. In yet another
preferred embodiment, the particular E. coli O157-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 O-specific
polysaccharide of E. coli O157. In the preferred embodiment,
antibodies which immunoreact with Shiga toxin 1 or Shiga 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 O157
colonization and/or infection. Antibodies which have bactericidal
or bacteriostatic activity against E. coli O157 are useful to
prevent and/or treat illnesses caused by E. coli O157. Antibodies
which immunoreact with Shiga toxins 1 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 .mu.g of E. coli
O157 O-specific polysaccharide, of inducing in the serum
bactericidal activity against E. coli O157, such that the serum
kills, in the presence of complement, 50% or more of E. coli O157
at a serum dilution of 1300:1 or more. Preferred compositions can
induce serum bactericidal activity against E. coli O157 such that
the serum kills 50% or more of E. coli O157 at a serum dilution of
32,000:1 or more, and the most preferred compositions can induce
serum bactericidal activity against E. coli O157 such that the
serum kills 50% or more of E. coli O157 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 O157 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 O157
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 O157, and to induce antibodies
specific to E. coli O157 O-SP and LPS. The E. coli O157 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
O157 infection in humans, and in reducing or preventing E. coli
O157 colonization in humans.
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 O157
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 O157. 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-O157 sera, or antibodies isolated therefrom, could
be used for therapeutic treatment for patients with E. coli O157
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 O157 infections, and are also
useful in providing passive protection to an individual exposed to
E. coli O157.
The present invention also provides diagnostic tests and/or kits
for E. coli O157 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
O157 O-SP conjugated to the B subunit of Shiga toxin 1 or 2 (Stx 1
or Stx2), or to a mutant, non-toxic Stx1 or Stx2 holotoxin.
A number of primary uses for the conjugates of this invention are
envisioned. The E. coli LPS-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 O157-infected patients; and 3) diagnostic
reagents for detecting and/or identifying E. coli O157.
The invention is intended to be included in the routine
immunization schedule of infants and children, and in individuals
at risk for E. coli O157 infection. It is also planned to be used
for intervention in epidemics caused by E. coli O157. Additionally,
it is may be used as a component of a multivalent vaccine for E.
coli O157 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 O157, for therapy of established infection. The invention
is also intended to provide a diagnostic test for E. coli O157
infection and/or colonization.
Definitions
Galp=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 O-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').sub.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]. 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, N.J.), 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].
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)-.beta.-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 O157 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 O-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 O157 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-dimethylaminopropyl)carbodiimide,
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, or the
like.
More preferably, the cyanated E. coli O157 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 O157 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 physicochemical 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 O157 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 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 O157. 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 O157 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 O157 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 O157 at 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 O157 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 affinity chromatography. The
antibodies may be purified so as to obtain specific classes or
subclasses of antibody such as IgM, IgG, IgA, IgG.sub.1, IgG.sub.2,
IgG.sub.3, IgG.sub.4 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 O157 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 O157 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 O157.
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 O157. 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 infection by
Shigatoxin producing organisms, such as E. coli strains O157, O111,
O26, and O17.
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
O157 (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 O157 O-SP with Various Polypeptides
O157 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 O157:H7
polysaccharide-protein conjugates were given the following
designations: O-SP-BSA.sub.1 O-SP-BSA.sub.2 DeA-LPS-BSA O-SP-CW
DeA-LPS-CW O-SP-rEPA DeA-LPS-rEPA.sub.1 DeA-LPS-rEPA.sub.2
Mice were immunized with these conjugate compositions containing
2.5 ug 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 O157-rEPA conjugates in mice are shown in Table
1.
TABLE-US-00001 TABLE 1 Immunoglobulin class composition of LPS
antibodies elicited by E. coli O157-rEPA conjugates in mice
Geometic mean antibody level (ELISA units) (25.sup.th-75.sup.th
centiles) Immunogen After 1.sup.st injection After 2.sup.nd
injection After 3.sup.rd 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-rEPA.sub.1 0.07
(0.04-0.13) 1.37* (0.50-2.63) 4.49*** (1.49-16.4)
DeA-LPS-rEPA.sub.2 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-rEPA.sub.1 0.11 (0.04-0.34) 0.32 (0.08-0.89)
0.94 (0.28-2.94) DeA-LPS-rEPA.sub.2 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 O157 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.
Bactericidal activity of serum LPS antibodies elicited in mice by
immunization with heat-killed E. coli O157:H7 or O-specific
polysaccharide-protein conjugates are shown in Table 2 below:
TABLE-US-00002 TABLE 2 Bactericidal activity of serum LPS
antibodies elicited in mice by immunization with heat-killed E.
coli O157:H7 or O-specific polysaccharide-protein conjugates
Reciprocal Antibody titer (ELISA units) bacterial Vaccine.sup.a
Total IgG IgM titer.sup.b Expt 1 O-SP-CW 79.25 100 DeA-LPS-CW 15.1
>100 DeA-LPS-CW 19.4 80 E. coli O157: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
.sup.aE. coli O157:H7 is pooled hyperimmune sera from mice injected
with heat-killed E. coli O157. All other sera were from individual
mice taken after the third conjugate injection. Serum dilutions
were mixed with an equal volume of ~10.sup.3 E. coli O157:H7
organisms per ml and complement. .sup.bThe 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 O157.
Example 2
Conjugation of E. coli O157 O-SP with rEPA; Preparation of Vaccine
Compositions
As discussed above, O-SP of E. coli O157, prepared by acetic acid
hydrolysis, and DeA-LPS O157, prepared by hydrazinolysis, have been
previously described. Conjugates of these polysaccharides to rEPA
(O-SP O157-rEPA, DeA-LPS O157-.sub.1, and DeA-LPS O157-rEPA.sub.2)
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, N.C. (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, N.C. Exclusion criteria were:
pregnancy or planned pregnancy in the next six months, positive
stool culture for E. coli O157 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 O157 LPS
and P. aeruginosa exotoxin A (ETA) and a culture of the stool for
E. coli O157. 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 .mu.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 and ETA antibody assays. Stool cultures for E.
coli O157 were obtained prior to and 4 and 26 weeks following
vaccination. E. coli O157 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.degree. C. at different observation times post-vaccination.
The remainder of the volunteers reported oral temperatures
37.2-38.degree. 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 O157-rEPA.sub.1 had a stool culture
positive for E. coli O157 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 O157.
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 .gtoreq.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 .gtoreq.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 .gtoreq.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 O157 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 .gtoreq.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 O157 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.
IgA: Pre-vaccination levels of IgA anti-LPS were low. Similar to
IgG and IgM anti-LPS, about 90% of the volunteers responded with
.gtoreq.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-US-00003 TABLE 3a Geometric mean titers of serum IgG, IgM,
and IgA lipopolysaccharide (LPS) antibodies elicited in volunteers
by injection of E. coli O157 O-SP-rEPA conjugates. ELISA units
(25.sup.th-75.sup.th 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.sub.1 0.51 (0.3-0.9) 5.73
(1.8-22) 46.3 (22-84) 31.2 (12-61) DeA-LPS-rEPA.sub.2 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.sub.1 7.19 (3.1-12) 19.1 (9.2-29) 43.5 (13-56) 22.5
(11-34) DeA-LPS-rEPA.sub.2 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.sub.1 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-rEPA.sub.2 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.
TABLE-US-00004 TABLE 3b Fold increases in geometric mean titers of
serum IgG, IgM, and IgA lipopolysaccharide (LPS) antibodies
elicited in volunteers. -fold increase in 25.sup.th-75.sup.th
percentiles Ab class Conjugate 1 Week 4 Weeks 26 Weeks IgG
O-SP-rEPA 17 132 70 DeA-LPS-rEPA.sub.1 11 91 61 DeA-LPS-rEPA.sub.2
7.6 68 61 Geometric mean 11 93 64 IgM O-SP-rEPA 4.0 8.0 3.5
DeA-LPS-rEPA.sub.1 2.7 6.0 3.1 DeA-LPS-rEPA.sub.2 2.4 5.8 3.4
Geometric Mean 3.0 6.5 3.3 IgA O-SP-rEPA 16 29 20
DeA-LPS-rEPA.sub.1 9.7 21 17 DeA-LPS-rEPA.sub.2 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.
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 O157:H7. Pre-vaccination sera had no
detectable bactericidal activity against E. coli O157: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-US-00005 TABLE 4 Bactericidal activity (reciprocal titer) of
serum anti-lipopolysaccharide (LPS) antibodies elicited in
volunteers by injection of E. coli O157 O-SP-rEPA conjugates.
Antibody level (ELISA units) Bactericidal Conjugate IgG IgM titer*
Preimmune 0.21 2.92 0 Preimmune 0.84 9.1 0 O-SP-rEPA 120.1 354.2
>6.4 .times. 10.sup.4 O-SP-rEPA 251.9 112.9 1.3 .times. 10.sup.4
O-SP-rEPA 156.3 183.6 >1.3 .times. 10.sup.3 DeA-LPS-rEPA.sub.1
231.4 59.9 >6.4 .times. 10.sup.4 DeA-LPS-rEPA.sub.2 77.5 68.2
1.3 .times. 10.sup.4 *Expressed as reciprocal of highest serum
dilution yielding 50% killing.
Results--Serum Anti-P. aerueginosa 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-US-00006 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-rEPA.sub.1 29 0.39 0.91
1.48 0.87 DeA-LPS-rEPA.sub.2 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.
Example 3
Conjugation of E. coli O157 O-SP with STXB1 and Preparation of
Vaccine Compositions
E. coli O157 O-SP was prepared by treatment of LPS with acetic acid
as previously described [8,9]. The B-subunit of Shigella toxin I
(StxB1) was synthesized by Vibrio cholerae strain 0395-N1 (pSBC32
containing the StxB1 gene) and purified by affinity chromatography
[20, 21]. SDS 7% PAGE of StxB1 showed one major band at 9 kDa and a
faint band with slightly higher molecular weight.
For conjugation, O157 O-SP was bound to StxB1 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 .mu.L of 0.2 M
triethylamine (TEA) added to bring the pH to 7.0 for 2 minutes. An
equal weight of StxB1 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 1.5.times.90 cm Sepharose 6B column in 0.2M NaCl,
the void volume fractions collected, and designated as
OSP-StxB1.
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
NaHCO.sub.3 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.degree. C. and passed through a 2.5.times.31 cm P10 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 StxB1 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 1.5.times.90 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
.mu.g saccharide on 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 Stx1 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
1-6.times.10.sup.4 cells in 0.1 ml. Monolayers were established by
overnight incubation in 5% CO.sub.2. 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 O157 O-SP--STXB1 Conjugates
Derivatization of O-SP with adipic acid dihydrazide was 3.1%
(wt/wt), similar to previous E. coli O157 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-StxB1 and 3.4% for OSP-AH-StxB1. A single line of
precipitation in double immunodiffusion was formed by rabbit
anti-Stx1 and mouse hyperimmune anti-O157 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-StxB1 was 0.63 for IgG and
0.14 for IgM and for O-SP-AH-StxB1 were 1.7 for IgG and 0.25 for
IgM: the differences between two conjugates were not statistically
significant.
TABLE-US-00007 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 O157 O-SP with
StxB1. Anti-LPS Neutralization Titer (%).dagger. (ELISA)* Serum
Dilution Immunogen IgG IgM 1:100 1:1000 1:10,000 Saline <0.05
<0.05 0.dagger-dbl. 0 0 OSP-AH-StxB1 1.7 0.25 >99 90 34
OSP-StxB1 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).
.dagger.Geometric mean (n = 10) neutralization titer determined
with Stx1 and HeLa cells, .dagger-dbl.No neutralization at 1/100
dilution.
Sera from mice injected with saline or human sera from volunteers
injected with E. coli O157 O-SP-rEPA conjugates showed no
neutralization to Stx1 or to Stx2. Sera from mice injected 3 times
with either of the O157 O-SP--StxB1 conjugates showed complete
neutralization of Stx1 at 1/100 dilution. At 1/1,000 dilution, the
GM of neutralization titer was 90% for OSP-AH-StxB1 and 98% for
OSP-StxB1. At 1/10,000 dilution, the sera from mice injected with
OSP-StxB1 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 O157 that were roughly proportional to
the content of IgG anti-LPS; this activity was removed by
absorption with E. coli O157 LPS.
Discussion
The O-SP of E. coli O157 LPS is a linear copolymer composed of the
tetrasaccharide repeat unit:
(.fwdarw.3)-.alpha.-D-GalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)-
-.alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.). It is
non-immunogenic, probably due to its 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 .gtoreq.4-fold rise in serum
IgG anti-E. coli O157 LPS that was sustained 26 weeks after
injection. (Re-injection of the E. coli O157 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 O157 LPS probably induced by cross-reacting
antigens [32, 33, 34, 35]. This is typical for other bacterial
pathogens as well. Higher levels of anti-O157 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 O157 and V.
cholerae O1, 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-O157 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 O111 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 the potency of E. coli O157
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 O157 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 O157
infection during an outbreak. Another use for the E. coli O157
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 O157 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 O157 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 O157 LPS-protein conjugates
disclosed herein will generate high antibody levels in humans
(i.e., approximately 5-10 times more IgG in humans than in mice)
and high neutralization antibody titers in humans (i.e., 10.sup.3
to 10.sup.4 in humans as opposed to 10.sup.2 in mice). The data
also show that the various E. coli O157 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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* * * * *