INFECTION AND IMMUNITY, Oct. 2005, p. 6523–6529
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 10
Recombinant Shiga Toxin B-Subunit–Keyhole Limpet Hemocyanin
Conjugate Vaccine Protects Mice from Shigatoxemia
Paola Marcato,1Thomas P. Griener,2George L. Mulvey,2and Glen D. Armstrong2*
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2H7,
Canada,1and Department of Microbiology and Infectious Diseases, University of Calgary
Health Sciences Center, Calgary, AB T2N 4N1, Canada2
Received 8 April 2005/Returned for modification 12 May 2005/Accepted 2 June 2005
Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of
infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise
because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite
this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS
make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2
B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization
could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin
would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways
to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin
unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole
limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2
B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and
cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation
were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of
a Stx2 B-subunit-based human EHEC vaccine.
The enterohemorrhagic group of Escherichia coli (EHEC)
causes hemorrhagic colitis and, in anywhere from 5 to 15% of
infected individuals, primarily very young and elderly subjects,
a serious clinical complication called hemolytic-uremic syn-
drome (HUS) (8, 23, 45). HUS is characterized by a triad of
clinical features, including hemolytic anemia, thrombocytope-
nia, and ultimately, acute renal failure. As well, in the most
severe cases, various degrees of central nervous system involve-
ment can become apparent. EHEC is also referred to as Shiga
toxigenic E. coli because this organism expresses exotoxins that
are biochemically related to the Shiga toxin (Stx) produced by
Shigella dysenteriae type 1 (43). Once EHEC has colonized the
intestines, it is possible for Shiga toxins to be translocated into
the submucosal compartment of the gut (3, 19). From there,
the toxins can be transported, possibly on the surface of poly-
morphonuclear leukocytes (23, 46, 47), to extraintestinal or-
gans and tissues, primarily the kidneys, where Shiga toxin-
mediated damage to endothelial cells in the glomerular
capillaries induces a cascade of microangiopathic events lead-
ing ultimately to HUS (45).
The Shiga toxins produced by EHEC are classified into two
families, Stx1 and Stx2, also commonly referred to as verotoxin
or verocytotoxin 1 and 2, according to their genetic and anti-
genic relatedness to the prototypic Stx produced by S. dysen-
teriae. In this classification scheme, Stx1 is virtually identical to
the prototypic S. dysenteriae Stx (38). In contrast, Stx2 is more
distantly related to Stx, and at least 10 variant species of Stx2
(reviewed in references 8, 45, and 49) have now been described
in various EHEC strains and serotypes isolated from humans
Regardless of their relationship to one another, the Shiga
toxins all display a classic AB5structure in which one enzymat-
ically active A subunit is combined with five identical B sub-
units which form a homopentamer displaying fivefold radial
symmetry around a central pore (12, 13). In the Stx family, the
A and B subunits of prototypic Stx1 and Stx2 are 52% and 60%
identical at the primary amino acid sequence level, respec-
tively. With the exception of one of the Stx2 variants (Stx2e),
the B pentamers of the Shiga toxins recognize the glycan se-
quence of globotriaosylceramide (Gb3) receptors found on
many eukaryotic cell surfaces (22, 29, 42), including renal en-
dothelial cells (28). Upon receptor ligation, the toxin is inter-
nalized by the host cell, and the A subunit’s RNA N-glycosi-
dase activity becomes activated, resulting in the catalytic
removal of a specific adenine from the eukaryotic 28S rRNA
component of the 60S ribosomal subunit (11, 40). This Stx
A-subunit-mediated rRNA depurination activity causes eu-
karyotic cell death by, depending on the cell type, a number of
possible mechanisms, including apoptosis.
The universal expression of the Shiga toxins by antigenically
diverse EHEC strains and serotypes as well as their central role
in severe pathogenesis makes these exotoxins compelling tar-
gets for vaccine development. Moreover, there is evidently a
much greater correlation between EHEC isolates expressing
prototypic Stx2 and a more severe course of illness (16, 24, 39,
41). Considering these epidemiological findings, we previously
(32) proposed that an acellular vaccine consisting of the non-
toxic B subunit from prototypic Stx2 might provide safe and
effective protection against the most severe complications of
* Corresponding author. Mailing address: Department of Microbi-
ology and Infectious Diseases, University of Calgary Health Sciences
Centre, Calgary, AB T2N 4N1, Canada. Phone: (403) 220-6885. Fax:
(403) 270-2772. E-mail: email@example.com.
EHEC infections in a majority of the at-risk population. In
support of this conjecture, we previously reported (32) that
rabbits immunized with a recombinant preparation of the pro-
totypic Stx2 B subunit were protected from a subsequent chal-
lenge with a lethal dose (LD) of Stx2 holotoxin. However,
effective vaccination in this study was found to be unpredict-
able unless lipopolysaccharide (LPS) was included with the
antigen. Others have also found that inducing an effective
immune response to the Stx2 B subunit is difficult to achieve (1,
7). Since the presence of a high concentration of LPS would be
unacceptable in a human vaccine preparation, the object of the
studies described herein was to investigate ways of effectively
augmenting, in mice, the immunogenicity of the recombinant
Stx2 B subunit containing ?1 endotoxin unit per ml.
MATERIALS AND METHODS
Toxin purification and cell lines. The recombinant Stx2 B subunit and Stx2
holotoxin were expressed and affinity purified as described previously (32, 34).
Endotoxin was removed from all the preparations by using a Detoxi-gel (Pierce,
Rockford, IL) LPS affinity column, as recommended by the manufacturer and by
employing the modifications described in our previous article (32). The colori-
metric Limulus amebocyte lysate assay (QCL-100; BioWhittaker, Walkersville,
MD) indicated that the purified Stx2 B-subunit preparations contained ?1 en-
dotoxin unit/ml. The lack of holotoxin contamination in the recombinant Stx2
B-subunit preparation was confirmed by assaying it for cytotoxic activity in Vero
and Ramos Burkitt’s lymphoma B cells (30–32). Whereas Stx2 displays 50%
cytolethal doses of 380 pg/105Vero cells and 20 pg/105Ramos B cells, the Stx2
B-subunit preparation, at a concentration of 1 mg/105cells, was found to be
completely nontoxic in these two cell lines.
Conjugation of Stx2 B subunit to KLH. Imject mariculture keyhole limpet
hemocyanin (KLH), high purity research grade (Pierce Biotechnology, Rock-
ford, IL), was conjugated to the Stx2 B subunit using 1-ethyl-3-(-3-dimethylamin-
opropyl) carbodiimide hydrochloride (EDC; Pierce Biotechnology) per the man-
ufacturer’s instructions. Briefly, 2 mg of Stx2 B subunit was admixed with 2 mg
of KLH in conjugation buffer [0.1 M 2-(N-morpholino)ethanesulfonic acid
(MES), 0.1 M NaCl, pH 4.7] with 250 ?g of EDC and incubated at room
temperature for 2 h. To remove excess EDC and any unconjugated Stx2 B
subunit, the conjugation reaction mixture was exhaustively dialyzed at 4°C over
a period of 4 days against 0.05 M NaPO4buffer (pH 7.2) containing 0.15 M NaCl
using a 300,000-molecular-weight exclusion membrane (Spectrum Laboratories
Inc., Rancho Dominguez, CA). Conjugation efficiency was analyzed qualitatively
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) us-
ing a Mini-PROTEAN II cell system (Bio-Rad, Mississauga, ON, Canada).
Mouse immunization and Stx2 holotoxin challenge protocols. The mouse
immunization and Stx2 holotoxin challenge experiments were conducted in a
randomized double-blind manner with adherence to the recommendations of the
Canadian Council on Animal Care. The adjuvants tested in the study were
Quil-A (saponin; Cedarlane Laboratories Ltd., Hornby, ON, Canada), Quil-A
plus LPS from E. coli O111:B4 (Sigma-Aldrich, Oakville, ON, Canada), the Ribi
adjuvant system containing synthetic trehalose dicorynomycolate (RAS-TDM;
Cedarlane), RAS-TDM plus monophosphoryl lipid A from Salmonella enterica
serovar Minnesota R595 (MPL; Corixa, Hamilton, MT), 2% Alhydrogel (Ce-
darlane), or 2% Alhydrogel plus MPL. LPS was included as one of the adjuvants
in the pilot study described herein because it had to be included to induce rabbit
immunity to the Stx2 B subunit, as reported in our previous article (32). It was
therefore used in the present study to provide a point of reference against which
we could relate the activity of the non-LPS-based adjuvants. Six-week-old, 20-g
female BALB/c mice were used in all the experiments. The mice were ear
notched for identification. Preimmunization blood samples were obtained from
all the mice via the jugular vein. The mice subsequently received two 0.1-ml
anterior dorsal subcutaneous injections containing a total of 30 ?g of Stx2 B
subunit administered with each of the adjuvant formulations. One group of mice
was sham immunized with pyrogen-free 0.9% NaCl irrigation solution (USP;
Baxter Corporation, Toronto, ON, Canada). Alternatively, the mice were immu-
nized with 30 ?g of Stx2 B-subunit–KLH conjugate or KLH alone administered
with RAS-TDM or 2% Alhydrogel. The mice were immunized at 3-week inter-
vals a maximum of three times. Seven days postimmunization, the mice were bled
from the jugular vein to obtain test sera. Fourteen days after receiving their last
injection, the mice received a single anterior dorsal subcutaneous LD (0.2 ng/g of
body weight) injection of a cocktail consisting of Stx2 holotoxin plus 7.5 ?g
Quil-A in 100 ?l phosphate-buffered saline (PBS). The lethal challenge was
administered with Quil-A because this provides a depot from which the toxins
are more slowly released into the circulation than if they were administered in
PBS. The rationale was to create a situation that would more accurately mimic
the release of the toxins from the intestines of an infected individual. Beginning
on the third day after initiation of the Stx2 challenge, the mice were monitored
every 2 to 4 h and immediately euthanized by CO2asphyxia when signs (obvious
lethargy or anterior paralysis) of Shigatoxemia became apparent (35).
Analysis of mouse sera in the Ramos B-cell cytotoxicity neutralization assay.
Undiluted mouse serum (5 ?l) was preincubated at room temperature with 0.05
ng of Stx2 holotoxin in 5 ?l of saline for 20 min and then transferred to 0.5 ml
of RPMI 1640 containing 2.5 ? 105cultured Ramos B cells. After 2 h of
incubation at 37°C in an atmosphere of 5% CO2-95% air, the Ramos B cells were
washed two times by low-speed centrifugation and finally resuspended in fresh
RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. After
an additional 16-h incubation period, the Ramos B cells were labeled with
annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen, Mississauga,
ON, Canada) and propidium iodide (Sigma-Aldrich), as described by the sup-
pliers. The percentage of apoptotic cells was recorded by flow cytometry using a
FACscan flow cytometer (Becton Dickinson, Mountain View, CA).
Determination of mouse IgG1 and IgG2a titers by the enzyme-linked immu-
nosorbent assay (ELISA). One hundred microliters/well of a 2.5-?g/ml Stx2 PBS
solution was incubated overnight at 4°C in 96-well enzyme immunoassay-radio-
immunoassay plates. The plates were then washed five times with PBS Tween
(PBST). The remaining protein binding sites were saturated using 2.5% skim
milk in PBST for 2 h at 37°C. One hundred microliters of serial dilutions of sera
in PBST was added to the wells, and the plates were incubated overnight at 4°C.
Following this step, the plates were washed five times with PBST, and 100 ?l/well
of a 1/2,000 dilution of peroxidase-conjugated goat anti-mouse immunoglobulin
G1(IgG1)- or IgG2a-specific antibodies (Southern Biotechnology, Birmingham,
AL) was added for 2 h at 37°C. The plates were again washed five times with
PBST and subsequently developed for 20 min with 100 ?l/well of 10 mM citrate
buffer (pH 4.2), 0.06% H2O2, and 0.055% 2,2?-azino-bis (3-ethylbenzothiazoline-
6-sulfonic acid) diammonium salt (ABTS; Boehringer-Mannheim, Indianapolis,
IN). The resulting absorbance data were recorded using a Spectramax 340
microtiter plate reader set at a wavelength of 410 nm.
In a pilot study, groups of mice were immunized a total of
three times over a 2-month period with the recombinant Stx2
B-subunit preparation (?1 endotoxin unit/ml) in admixture
with one of the six adjuvants listed in the legend to Fig. 1. With
the exception of mice immunized with the Stx2 B subunit
mixed with RAS-TDM, none of the animals immunized with
the Stx2 B subunit in admixture with any of the other adjuvants
survived a subsequent challenge with an LD of Stx2 holotoxin.
However, because of the small sample size, this result was not
significant (P ? 1.00, Fisher’s exact test). Accordingly, to fur-
ther investigate if RAS-TDM could enhance the immunoge-
nicity of the Stx2 B subunit, the experiment was repeated with
a larger group of animals. The results presented in Fig. 1B
demonstrate that RAS-TDM did have a significant (P ? 0.006,
Fisher’s exact test) promotional effect on the immunogenicity
of the Stx2 B subunit but, once again, protection was less than
optimal, with the survival rate being only 42%. As a result of
these observations, we decided to investigate whether the mu-
rine immune response to the recombinant Stx2 B subunit could
be further improved by chemically coupling the B subunit to a
carrier protein such as KLH (15).
As is evident in the SDS-PAGE results presented in Fig. 2,
the EDC conjugation process resulted in the production of
very-high-molecular-weight complexes of KLH and Stx2 B sub-
unit, which barely penetrated the separating gel. There is no
evidence in Fig. 2, lane D, of the gel of unconjugated KLH or
6524MARCATO ET AL.INFECT. IMMUN.
Stx2 B subunit, but they are readily detected in lanes B and C,
Groups of 10 mice were then immunized with unconjugated
KLH or the Stx2 B-subunit–KLH conjugate preparation ad-
ministered with either Alhydrogel or RAS-TDM. The ELISA
data presented in Fig. 3 demonstrate that after only two injec-
tions, the postimmunization sera from individual mice receiv-
ing the Stx2 B-subunit–KLH conjugate preparation adminis-
tered with RAS-TDM displayed the highest Stx2-specific IgG1
responses (Fig. 3B). In Fig. 3, the differences between the
responses of the control mice immunized with unconjugated
KLH and those immunized with the Stx2 B-subunit–KLH con-
jugate administered with RAS-TDM (Fig. 3B) were highly
significant (P ? 0.001, Student’s t test). For the IgG2a titers
(Fig.3D), these differences were also significant (P ? 0.009,
Student’s t test). For the IgG1 titers, the differences between
the responses of the control mice immunized with unconju-
gated KLH and those immunized with the Stx2 B-subunit–
KLH conjugate administered with Alhydrogel (Fig. 3A) were
also highly significant (P ? 0.001, Student’s t test). For the
IgG2a titers (Fig. 3C), however, these differences did not
achieve significance (P ? 0.198, Student’s t test). As well, the
differences between the response of mice immunized with the
Stx2 B-subunit–KLH conjugate vaccine administered with
RAS-TDM (Fig. 3B) and that of mice immunized with the
conjugate vaccine plus Alhydrogel (Fig. 3A) were also signifi-
cant (P ? 0.002, Student’s t test). The differences, however,
between the IgG2a titers in mice immunized with the Stx2
B-subunit–KLH conjugate vaccine administered with RAS-
TDM (Fig. 3D) and those of mice immunized with the conju-
gate vaccine plus Alhydrogel (Fig. 3C) were not (P ? 0.077,
Student’s t test). The ELISA data correlated with the Ramos
B-cell apoptosis neutralization results in that the greatest neu-
tralization was obtained using sera from mice immunized with
the Stx2 B-subunit–KLH conjugate preparation administered
with RAS-TDM (Fig. 4).
When the mice immunized with the Stx2 B-subunit–KLH
conjugates were challenged with an LD of Stx2 holotoxin, all
10 of the mice vaccinated with the preparation administered
with RAS-TDM survived whereas only 8 of the 10 mice im-
munized with the Stx2 B-subunit–KLH conjugate preparation
mixed with Alhydrogel survived the challenge (Fig. 5). How-
ever, the difference between these two groups was not signifi-
cant (P ? 0.474, Fisher’s exact test).
Over the preceding decades, large outbreaks and continuing
sporadic cases of EHEC infections in North and South Amer-
ica, Japan, Europe, and Australia have prompted a global
research effort directed at minimizing or eliminating the threat
that these organisms pose to human health. The more recent
inclusion of the diarrheagenic E. coli on the NIAID Biodefense
Research Priority Pathogens Category B list has also spurred
efforts in countermeasure research directed at these organ-
isms. Essentially, three approaches have been advocated, in-
FIG. 1. Survival plots for mice immunized with the Stx2 B subunit in admixture with various adjuvants or saline control containing no Stx2 B
subunit. Saline (– – –), Quil A ( ), Quil A plus LPS (?), Alhydrogel (...), Alhydrogel plus MPL (—), RAS-TDM (
MPL (). (A) Groups of five mice were used per immunization protocol; (B) groups of 15 mice were used per immunization protocol.
), and RAS-TDM plus
FIG. 2. Analysis of the recombinant Stx2 B-subunit–KLH conju-
gate by SDS-PAGE. Lane A, low-molecular-mass prestained standard
proteins used to calibrate the gel; lane B, KLH; lane C, Stx2 B subunit;
lane D, Stx2 B-subunit–KLH conjugate vaccine. The gel was stained
with Coomassie blue.
VOL. 73, 2005 SHIGA TOXIN 2 B-SUBUNIT VACCINE6525
cluding therapeutic interventions (4, 37), preventing the organ-
isms from entering the food or water supply (17, 20, 33), and
vaccination strategies aimed at protecting humans or eliminat-
ing the organisms from their major zoonotic reservoir, mainly
cattle (18, 21). In the present study, we have continued our
research into the feasibility of producing a safe, chemically
defined, and effective vaccine for protecting humans from the
serious complications of EHEC infections.
FIG. 3. ELISA analysis for antigen-specific IgG1 (A and B) and IgG2a (C and D) titers of sera from individual mice after two injections of KLH
only (Œ) or the Stx2 B-subunit–KLH conjugate (?) administered with Alhydrogel (A and C) or RAS-TDM (B and D). The data represent the
average of triplicate determinations for each point.
FIG. 4. Ramos B-cell apoptosis neutralizing activity of sera obtained from individual mice immunized with KLH only or the Stx2 B-subunit–
KLH conjugate admixed with RAS-TDM or Alhydrogel. The error bars represent the standard errors of the mean values from triplicate
determinations. The unshaded bars represent serum samples that significantly (P ? 0.05, Student’s t test) reduced Stx2 toxicity relative to the
activity of control sera. K106 is a positive control serum sample obtained from a rabbit immunized with the recombinant Stx2 B subunit in the
presence of endotoxin, as described in our previous article (30).
6526 MARCATO ET AL.INFECT. IMMUN.
Although O polysaccharide-based EHEC vaccines have
been described (9, 10, 25–27, 44), such vaccines suffer from the
limitation that the resulting immunity is serogroup specific,
and a large number of different EHEC serogroups have now
been implicated in human cases of hemorrhagic colitis and
HUS (20). Therefore, to provide broad-spectrum protection,
we and others (2, 5–7, 14, 32, 50) have proposed vaccine strat-
egies focused on the Shiga toxins, the only virulence factors
common to all EHEC strains and serogroups.
Given that the amino acid sequences of the Shiga toxins are
60% identical, it is feasible that polyclonal antibodies to the
Stx2 B subunit might confer cross-protection against Stx1. Sim-
ilarly, Stx2 B-subunit-specific polyclonal antibodies might also
confer protection against the more closely related variant
forms of Stx2. However, prototypic Stx2 is the most prevalent
of all the Shiga toxins expressed by EHEC isolates obtained
from subjects who develop HUS (16, 24, 39). Consequently, a
vaccine composed of the Stx2 B subunit should provide broad-
spectrum protection against the extraintestinal complications
of EHEC infections regardless of its ability to provide cross-
protective immunity against Stx1 or any of the Stx2 variants.
Although RAS-TDM promoted a protective immune re-
sponse to the hypoimmunogenic Stx2 B subunit, this required
a primary injection followed by two boosters, and we found
that the protection elicited was only partial. We therefore
elected to investigate whether coupling the Stx2 B subunit to a
carrier protein would improve the protection provided by a
Stx2 B-subunit-based vaccine. We chose KLH for the study
because it is approved for human use and has been used as a
hapten carrier for stimulating an immune response to small
immunogens such as drugs, hormones, peptides, polysaccha-
rides, lipids, and oligonucleotides (15, 36, 48, 51). Due to its
mitogenic activity, carbohydrate content, highly organized qua-
ternary structure, high molecular mass, and propensity to ag-
gregate, KLH elicits a vigorous immune response to itself (15).
This immune response involves activated antigen-specific B
and T lymphocytes and the expression of transactivating cyto-
kines and lymphokines, which can also invigorate the response
of B and T lymphocytes activated by poor immunogens which
are coupled to KLH.
Although ELISA clearly revealed that a Stx2-specific im-
mune response was induced in mice immunized with the Stx2
B-subunit–KLH preparation, the Ramos B-cell neutralization
data suggested the possibility that the protective activity of the
Stx2 B-subunit–KLH vaccine preparations may not have been
related to a specific immune response to the Stx2 B subunit.
However, it is conceivable that antibodies generated to the
Stx2 B subunit in the immunized mice may not have been of
sufficient affinity to neutralize the apoptogenic activity of Stx2
holotoxin in Ramos B-cells. In this regard, cytotoxicity assays
represent a two-step process involving first the binding and
then the subsequent internalization of the bound toxins into
the cytoplasmic compartment of the cell. In these assays, toxin
binding to specific receptors on the cell surface is reversible, as
is antigen binding to specific antibodies. Once the toxin-recep-
tor complexes have been internalized, however, the process is
irreversible, and the cell is destined to die. Antibodies, which
remain external to the cell, lose their neutralizing function
once toxin internalization has occurred.
During the 2-h incubation with the antigen-antibody com-
plexes, it is possible that an exchange reaction may have oc-
curred in which loosely complexed Stx2 was able to access Gb3
receptors and become irreversibly internalized into the Ramos
B-cells. Such an exchange reaction would be favored in a sit-
uation involving antibodies with a relatively low affinity for
their antigen, antibodies which would have been produced if
the immune response was still in the maturing phase in mice
which had only received two doses of the vaccine. In the chal-
lenge phase of the experiment, however, any Stx2 holotoxin-
antibody complexes that formed, regardless of their affinity,
would have been exposed to a disposal mechanism involving
cellular internalization which would avoid cytotoxicity. Such a
disposal mechanism may have been sufficiently effective at
eliminating the Stx2 challenge dose in the mice before the
circulating holotoxin had time to exchange with Gb3receptors
on target cells. An alternative but more remote explanation is
that the two injection immunization protocols may have simply
primed the murine immune system for it to respond in a boost-
er-like fashion to the challenge dose of Stx2 holotoxin and
quickly enough to produce de novo antibodies with an affinity
that was high enough to be protective. These de novo antibod-
ies would not have been present in the postimmunization but
prechallenge serum samples which were evaluated in the
Ramos B-cell apoptosis neutralization assays.
Several reports have indicated that the Stx1 B subunit, which
lacks the enzymatic activity of the A subunit, activates the
apoptosis program in certain tissue culture cells. This B-sub-
unit-specific apoptogenic activity was seen only at relatively
high concentrations, much higher than that needed by the
holotoxin to achieve the same end. Nonetheless, such activity
would still raise safety concerns about any vaccine preparation
containing the Stx2 B subunit. Although we previously re-
ported (30) that the Stx2 B subunit caused apoptosis in Ramos
FIG. 5. Survival plots for mice immunized with KLH alone or the
Stx2 B-subunit–KLH conjugate admixed with RAS-TDM or Alhydro-
gel (n ? 10 mice per group). KLH mixed with Alhydrogel (
B-subunit–KLH mixed with Alhydrogel (—), KLH mixed with RAS-
TDM (– – –), and Stx2 B-subunit–KLH mixed with RAS-TDM (
The differences in the survival rates of mice immunized with the Stx2
B-subunit–KLH conjugate preparations and those of the mice immu-
nized with KLH alone were highly significant (P ? 0.025 and 0.011,
Stx2 B-subunit–KLH administered with Alhydrogel or RAS-TDM,
respectively, Fisher’s exact test).
VOL. 73, 2005 SHIGA TOXIN 2 B-SUBUNIT VACCINE6527
B-cells, we subsequently discovered (31) that this activity was
in fact due to minute amounts of Stx2 holotoxin in the B-
subunit preparations used in those studies. As a consequence,
the recombinant Stx2 B-subunit preparations used in the
present immunization studies were found to be completely free
of any cytotoxic activity at a concentration which was 2.6 ? 106
to 5 ? 107times greater than a 50% cytolethal dose for Stx2 in
Vero and Ramos B-cells, respectively.
In summary, by conjugating the Stx 2 B subunit to KLH, it
appears that the hyporesponsiveness of the endotoxin-poor
Stx2 B-subunit preparation can thereby be overcome following
only a primary injection and a single booster injection, induc-
ing a protective immune response to Stx 2 holotoxin in mice.
Further, the Stx2 B-subunit preparation used in these studies
was found to contain below-detectable levels of apoptogenic
activity in Ramos B cells. These results support additional
evaluation of an endotoxin-poor Stx2 B-subunit-based EHEC
vaccine using adjuvants and carrier proteins which have been
approved for clinical use in humans. The vaccine could be used
alone or in combination with other virulence factors to provide
protection from the devastating consequences of foodborne or
waterborne EHEC infections in human subjects. The availabil-
ity of such vaccine preparations would also serve as a deterrent
to using EHEC to compromise the safety of food or water
intended for human consumption.
This work was supported by operating grants MWS 56081 from the
Canadian Institutes for Health Research (CIHR) and VP 17 from the
Canadian Bacterial Diseases Network to G.D.A. P.M. was supported
by doctoral scholarships from the CIHR and Alberta Heritage Foun-
dation for Medical Research.
We thank Stefanie Wee for her technical assistance.
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