Original Paper
J Biomed Sci 1999;6:357–363
Received: February 5, 1999
Accepted: March 30, 1999
A Nontoxic Pseudomonas Exotoxin A Induces
Active Immunity and Passive Protective Antibody
against Pseudomonas Exotoxin A Intoxication
Tzong-Yueh Chen a, b Chia Po Lin c, d Chien-Chang Loa d Tso-Ling Chen c
Huey-Fang Shang e Jaulang Hwang b Cho-Fat Hui a, f
a Institute of Genetics, School of Life Science, National Yang-Ming University, b Institute of Molecular Biology,
Academia Sinica, c Division of Drug Biology, National Laboratories of Foods and Drugs, Department of Health,
Executive Yuan, d Graduate School of Microbiology, Soochow University, e Department of Medicine,
Taipei Medical College, and f Institute of Zoology, Academia Sinica, Taipei, Taiwan, ROC
Key Words
Pseudomonas exotoxin A W Vaccination
Abstract
Pseudomonas exotoxin A (PE) is one of the most potent
cytotoxic agents produced by Pseudomonas aeruginosa.
In this study, we examined the possibility of using PE
with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(¢576–613), for active immunization
against PE-mediated disease. We first examined the
toxic effects of PE and PE(¢576–613) on 5- and 9-weekold ICR mice. The results show that the subcutaneous
administration of PE(¢576–613) at a dose of 250 Ìg was
still nontoxic to 5- and 9-week-old ICR mice, while native
PE was lethal at a dose of 0.5 and 1 Ìg, respectively.
PE(¢576–613) was then used to immunize ICR mice. The
minimum dose of PE(¢576–613) that could effectively
induce anti-PE antibodies in 5- and 9-week-old ICR mice
was found to be 250 ng. However, immunization with
250 ng PE(¢576–613) failed to protect the immunized
mice from a lethal dose of PE. The effective immunization dose of PE(¢576–613) that could protect mice
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against a 2 Ìg PE challenge was found to be 15 Ìg. In
addition, sera obtained from PE(¢576–613)-immunized
ICR mice were able to neutralize PE intoxication and
effectively protect mice from PE. Thus, PE(¢576–613)
may be used as an alternative route to new PE vaccine
development.
Introduction
Pseudomonas aeruginosa are gram-negative bacilli and
are among the most problematic opportunistic pathogens
[31]. They are widely distributed in nature and seldom
infect healthy people. Yet, these bacteria always infect
immunodeficient individuals, such as patients suffering
from burns, cystic fibrosis, or patients who take immunosuppressive drugs [3, 7, 26, 27]. In addition, treatment of
these infections is greatly impaired by the acquired resistance of P. aeruginosa to antibiotics [8, 22, 37]. Therefore,
immunization against Pseudomonas infection may offer a
promising approach to circumvent this difficulty. In the
past decade, approaches to prevent P. aeruginosa infec-
Dr. Jaulang Hwang
Institute of Molecular Biology
Academia Sinica, Nankang, Taipei 115, Taiwan (ROC)
Tel. +886 2 2789 9217, Fax +886 2 2782 6085
E-Mail [email protected]
tion have used the outer membrane components or detoxified secretory toxins such as lipopolysaccharides [9, 24,
32], common endotoxin proteins [39], toxoids [10, 25],
cross-reacting mutant toxins or nontoxic materials [20],
flagella [2, 19, 29], pili [40], high-molecular-weight polysaccharides [7, 33–35], and outer membrane proteins [16,
30] as antigens for vaccination. However, among all identified pathogenic factors released from P. aeruginosa,
Pseudomonas exotoxin A (PE) is the most toxic component and is considered to be the most important virulent
factor [6]. Thus, development of a vaccine against PE may
be an alternative route to P. aeruginosa vaccination.
The intoxication process is thought to proceed first by
the binding of PE to its specific receptor on the surface of
toxin-sensitive cells. The PE-receptor complex then enters
the cell through receptor-mediated endocytosis. Finally,
PE is translocated to the cytosol where it catalyzes the
transfer of the ADP-ribose moiety of NAD+ to elongation
factor 2 (EF-2). This renders EF-2 inactive in protein synthesis and leads to the death of PE-affected cells [15, 28].
Thus PE should contain at least three functional domains
in order to be toxic, namely binding to the receptor on the
cell membrane, translocation across the membrane, and
ADP ribosylation activities. Recent studies have correlated the structural domains of PE with specific biological
functions [1, 4, 14, 18, 21]. Based on information obtained from the three-dimensional structure [1] and the
study of PE using recombinant DNA methods [14, 18,
21], binding, translocation and ADP ribosylation domains have been identified to reside in domain Ia (residues 1–252), domain II (residues 253–364), and domain
III (residues 405–613), respectively.
Recently, our studies indicated that PE with a deletion
of the carboxyl 38 amino acid residues, designed
PE(¢576–613), completely loses its ADP ribosylation activity and, correspondingly, loses its cytotoxicity. Furthermore, PE(¢576–613) is able to block PE cytotoxic activity
when it is assayed in NIH 3T3 cells [5]. Therefore,
PE(¢576–613) might be a safer and more useful antigen
for PE vaccination. The purpose of the present study was
to assess the immunogenic properties of PE(¢576–613),
and the possibility of using PE(¢576–613) for vaccine
development.
Materials and Methods
Animals
The ICR strain mice used in this study were obtained from the
Animal Center, College of Medicine, National Taiwan University
and were bred in the animal facilities of the National Laboratories of
358
J Biomed Sci 1999;6:357–363
Foods and Drugs. The animals were housed in standard cages and
supplied with rodent laboratory chow (Purina Mills, Inc.) and water
ad libitum. These animals did not show any detectable antibody
responses to PE before experiments.
Materials and Bacterial Strains
Pseudomonas exotoxin A was purchased from ICN Biomedicals.
Plasmid pJJ3, which encoded Pseudomonas exotoxin A with a deletion of 38 carboxyl-terminal amino acid residues, was obtained as
described previously [5]. BL21(DE3), which had been lysogenized
with a phage (DE3) carrying the T7 RNA polymerase gene under the
control of a lac UV5 promoter [38], was a gift from Dr. F. W. Studies,
Biology Department, Brookhaven National Laboratory, and used as
a host to express PE(¢576–613). Nutrients for bacterial broth were
purchased from Difco Laboratories. Reagents for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA), and standard molecular
weight markers were purchased from Bio-Rad Laboratories. All other
reagents were of analytical grade.
Expression and Purification of PE(¢576–613)
PE(¢576–613) was prepared from BL21(DE3) transfected with
plasmid pJJ3 [5]. BL21(DE3) containing pJJ3 was cultured in LB
broth with 50 Ìg/ml ampicillin at 37 ° C. When absorbance at 650 nm
reached 0.3, 1-isopropyl-ß-D-1-thiogalacto-pyranoside (IPTG) was
added to a final concentration of 0.5 ÌM. Cells were harvested
90 min later and were resuspended into 20-cell-pellet volumes of lysis
buffer (1.5 mg/ml lysozyme, 1 mM EDTA, 50 mM Tris, pH 8.0).
DNase I, MgCl2, and MnCl2 were then added to a final concentration
of 50 Ìg, 10 mM, and 1 mM per ml, respectively. The incubation was
performed at room temperature for 15 min. In order to lyse the cells
completely, 50 mM EDTA and 1% Triton X-100 were added and the
incubation was prolonged for 2 additional minutes. The lysates were
then centrifuged at 13,000 g for 10 min to pellet the inclusion bodies.
The pellets were washed several times with a large volume of wash
buffer (1% Triton X-100, 5 mM EDTA, 10 mM Tris, pH 8.0) in
order to remove residual cell debris. Finally, the pellets were
extracted with a 10-pellet volume of 8 M urea. The purity of
PE(¢576–613) in the 8 M urea extracts reached approximately 70%
purity.
The purification of PE(¢576–613) was performed as follows.
First, the 8 M urea extracts were dialyzed at 4 ° C against 500 vol of
buffer containing 1 mM EDTA, and 10 mM Tris, pH 8.0. After 5
changes of dialysis buffer, samples were loaded onto a DEAE-Sephacel column, which was pre-equilibrated with 1 mM EDTA, 10 mM
Tris, pH 8.1, and eluted with a NaCl gradient up to 1 M NaCl.
PE(¢576–613) was eluted at 0.4 M NaCl. The fractions containing
PE(¢576–613) were then collected and examined by SDS-PAGE.
SDS-Polyacrylamide Gel Electrophoresis
Samples containing PE(¢576–613) were dissolved in Laemmli
buffer and boiled for 5 min prior to application to a 0.1% SDS,
12.5% acrylamide slab gel. Gels were stained by Coomassie Blue
after electrophoresis as described by Laemmli [33]. A total of 20 Ìg
protein sample was loaded per lane for visualization. All reagents
used for gel staining and destaining were freshly prepared. Marker
proteins used were: phosphorylase b, MW 97,400; bovine serum
albumin, MW 66,200; ovalbumin, MW 42,700; carbonic anhydrase,
MW 31,000; soybean trypsin inhibitor, MW 21,500; and lysozyme,
MW 14,400.
Chen/Lin/Loa/Chen/Shang/Hwang/Hui
Animal Toxicity
To test the toxicity of native PE and PE(¢576–613) in animals,
various doses of PE and PE(¢576–613) were subcutaneously injected
into ICR strain mice. Groups of twelve mice aged 5 weeks (18–20 g
body weight) or 9 weeks (28–30 g body weight) were used for this
study. The days of spontaneous death were recorded. Mortality rates
were then compiled for 7 days after injection for estimating the 50%
lethal dose (LD50) as described by Reed and Muench [36].
Immune Response Studies
Five-week-old mice weighing from 18 to 20 g were immunized
with PE(¢576–613) which was well suspended in 0.18% AlPO4 solution prior to immunization. Four groups of ICR mice were then inoculated subcutaneously with various doses of PE(¢576–613) on days
0, 7, and 14 to measure the minimum effective dose of PE(¢576–
613) for inducing anti-PE(¢576–613) antibodies. After the minimum
effective immunizing dose was obtained, three groups of ICR mice of
the same age were used to examine different immunization protocols. The PE(¢576–613) was administered subcutaneously on day 0
(group a), days 0 and 14 (group b), or days 0, 7, and 14 (group c).
Venous blood samples from these immunized mice were then taken
on days 0, 7, 14, 21, 28, and 42 for quantitation of the response of
anti-PE antibody. Usually, sera were kept frozen at –20 ° C until
assayed.
Enzyme-Linked Immunosorbent Assay
To quantitate the magnitude of anti-PE antisera induced by
PE(¢576–613), sera from immunized animals were examined by
ELISA [11]. Native PE was used to coat the polyvinylchoride flatbottom, 96-well microtiter plates (Falcon) at a concentration of 3 Ìg/
ml. 100 Ìl serial diluted mouse serum was added to a well precoated
with PE and incubated at 37 ° C for 1 h. Peroxidase-conjugated goat
antimouse immunoglobulin (Zymed) was used as the secondary antibody. The substrate solution contained 0.54 mg/ml of 2,2)-azainobis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.03% H2O2 in 0.1 M
citric acid.
Results
Expression and Purification of PE(¢576–613)
As shown in figure 1, PE(¢576–613) was expressed in
BL21(DE3)/pJJ3 and could be directly visualized as a
major band at the expected molecular weight after Coomassie blue staining (fig. 1, lane 1). Since PE(¢576–613)
existed as a form of inclusion body, it was extracted with
8 M urea from the inclusion body fraction (fig. 1, lane 2),
followed by dialysis to renature it. By this approach,
PE(¢576–613) was purified to about 70% purity (fig. 1,
lane 3). The renatured PE(¢576–613) was then further
purified through a DEAE-Sephacel column to obtain over
95% purity (fig. 1, lane 4).
Fig. 1. SDS-PAGE of isolated PE(¢576–613). SDS-PAGE was per-
formed on a 12.5% polyacrylamide gel 12 ! 14 cm and electrophoresed with 8 mA of constant current for 16 h at room temperature.
After electrophoresis, gels were stained with Coomassie blue: lane 1,
PE(¢576–613) in whole cell lysates; lane 2, PE(¢576–613) in 8 M
urea extract; lane 3, PE(¢576–613) after dialysis; and lane 4,
PE(¢576–613) after DEAE-Sephacel column chromatography.
Table 1. Toxicity of PE and PE(¢576–613) in 5-week- and 9-weekold ICR micea
5-week-old mice
9-week-old mice
PE, Ìg
2
1
0.5
0.25
0.125
0.0625
–
12/12
12/12
7/12
0/12
0/12
12/12b
12/12
9/12
1/12
0/12
–
PE(¢576–613), Ìg
250
50
15
5
0
0/12
0/12
0/12
0/12
0/12
0/12
0/12
0/12
0/12
0/12
a
The toxicity of PE and PE(¢576–613) was determined by subcutaneous injection of toxin into 5-week-old (weighing 18–20 g) or 9week-old (weighing 28–30 g) female ICR mice. The LD50 of PE was
estimated according to a method described previously [36].
b
All values given are number of mice which died/number of mice
injected.
Animal Toxicity of PE and PE(¢576–613)
The toxicity of native PE and PE(¢576–613) in ICR
mice of different ages was examined as shown in table 1.
Vaccination against Pseudomonas
Exotoxin A
J Biomed Sci 1999;6:357–363
359
Since immunization in the experiments took 4 weeks, the
toxicity of PE and PE(¢576–613) was examined in 5- and
9-week-old ICR mice. Various doses of PE or PE(¢576–
613) were administered subcutaneously. Five-week-old
ICR mice were found to be more sensitive to PE toxicity
than 9-week-old ICR mice. For 5-week-old ICR mice, 7
out of 12 receiving 0.25 Ìg of native PE, and all mice
receiving 0.5 Ìg PE or more died. Among 9-week-old ICR
mice, 1 of 12 mice receiving 0.25 Ìg PE, 9 of 12 mice
receiving 0.5 Ìg PE, and all mice receiving 1 Ìg PE or
more died. According to the method of Reed and Muench
[36], the LD50 of ICR mice aged 5 and 9 weeks for PE was
0.23 and 0.38 Ìg/mouse, respectively. On the other hand,
the subcutaneous administration of PE(¢576–613) to
ICR mice at doses as high as 250 Ìg, which is equivalent
to 650 times the LD50 dose of native PE, was found to be
nontoxic in 5- and 9-week old ICR mice. These results are
consistent with previous observations that PE(¢576–613)
lacks its ADP ribosylation activity and consequently loses
its cytotoxicity [5]. Therefore, PE(¢576–613) may be a
useful antigen for vaccination against PE-mediated diseases.
Fig. 2. Effects of various doses of PE(¢576–613) and different
immunization protocols on the induction of anti-PE(¢576–613) antibody in ICR mice. a Immune response induced by subcutaneous
injection. Groups of twelve 5-week-old female ICR mice were used in
the experiment. Mice were given several doses of PE(¢576–613) well
suspended in 0.18% AlPO4 (0.4 mg Al +3/ml) solution subcutaneously
on days 0, 7, and 14. Blood samples from 2 mice in each experimental group were taken on days 0, 7, 14, 21, 28, and 42, and the response
of anti-PE(¢576–613) antibody was evaluated at a 1:50 dilution as
described in Materials and Methods. Each data point of absorbance
at 410 nm by ELISA was the average of two replicate readings of 2
mice: [ = 0 Ìg; P = 0.05 Ìg; o = 0.25 Ìg; d = 1.5 Ìg. b Immune
responses induced by various immunization time schedules. Twelve
5-week-old female mice per group received 0.25 Ìg of PE(¢576–613)
subcutaneously in 0.5 ml of 0.18% AlPO4 on day 0 (d); days 0 and
14 (o), or days 0, 7, and 14 (P); [ = control AlPO4-immunized
group. The response of anti-PE(¢576–613) antibody was evaluated
at a 1:50 dilution of the collected sera as described in Materials and
Methods. Data are expressed as the means of absorbance at 410 nm
by ELISA from 12 mice.
360
J Biomed Sci 1999;6:357–363
Immunogenic Properties of PE(¢576–613)
To examine the immunogenic properties of PE(¢576–
613), we first measured the minimum dose and time
course of PE(¢576–613) administration which could result in an ideal induction of anti-PE(¢576–613) antibody
in ICR mice. PE(¢576–613) was used at a dose-range of
0.05 Ìg–1.5 Ìg and was well suspended in a 0.18% AlPO4
solution before injection. As shown in figure 2a, no antiPE(¢576–613) antibody could be detected during the
whole course of immunization with 0.05 Ìg PE(¢576–
613). However, detectable anti-PE(¢576–613) antibody
was observed on day 21 of immunization with 0.25 Ìg
PE(¢576–613) and a higher anti-PE(¢576–613) antibody
response was found in mice immunized with 1.5 Ìg
PE(¢576–613) after day 28. We then examined the efficiency of immunization by 0.25 Ìg PE(¢576–613) under
three different immunization protocols. The induction of
an anti-PE immune response was measured on days 0, 7,
14, and 21 for all three immunization protocols, and the
results are summarized in figure 2b. The immunization
protocols of groups a and b failed to give a detectable antiPE immune response, while the ICR mice immunized
three times on days 0, 7 and 14 had an increase in anti-PE
immune response (fig. 2b). We, therefore, chose protocol
c for the active immunization against PE intoxication.
Chen/Lin/Loa/Chen/Shang/Hwang/Hui
Active Immunization against PE Intoxication
Groups of six 5-week-old ICR mice weighing between
18 and 20 g were used for active immunization against PE
intoxication. Each mouse was injected subcutaneously
with different doses of PE(¢576–613) ranging from 0.15
to 15 Ìg on days 0, 7, and 14. On day 28, mice were challenged with 2 Ìg of PE, equivalent to about 6 times the
LD50 of PE in 9-week-old ICR mice. Mortality was
recorded for 7 days post-challenge. As shown in table 2,
only the group receiving higher immunizing doses, i.e.
15 Ìg of PE(¢576–613), survived after PE challenge.
However, 3 of 6 mice immunized with 2.5 Ìg PE(¢576–
613), 5 of 6 mice immunized with 1.5 Ìg PE(¢576–613),
and all mice immunized with less than 0.25 Ìg PE(¢576–
613) died. These results indicate that immunization with
15 Ìg of PE(¢576–613) could evoke a protective immune
response against PE. In order to distinguish whether the
protection induced by 15 Ìg of PE(¢576–613) came from
the production of anti-PE(¢576–613) antibodies instead
of the competition between PE(¢576–613) and native PE,
we also injected 6 5-week-old ICR mice with 2 Ìg of
native PE which was mixed with 15 Ìg of PE(¢576–613).
All mice died in this control experiment. In addition, we
also quantitated the level of PE(¢576–613) antibodies
obtained from the surviving mice of the 1.5 Ìg immunizing group by ELISA. All the survivors exhibited significantly higher levels of PE(¢576–613) antibodies (data not
shown).
Blocking of PE Intoxication with Anti-PE(¢576–613)
Antibody in ICR Mice
The neutralization experiments were carried out to
clarify that the protective effects of PE(¢576–613) in mice
immunized against PE were due to the neutralization of
PE by anti-PE(¢576–613) which was raised during the
period of PE(¢576–613) immunization. Animals were
immunized with 15 Ìg of PE(¢576–613) on days 0, 7, and
14 and then challenged with 2 Ìg of native PE on day 28.
On day 35, sera collected from surviving mice were
diluted 10-, 50-, or 250-fold with PBS buffer and then
mixed thoroughly with 2 Ìg native PE, which was equivalent to about 8-fold the LD50 of PE for 5-week-old ICR
mice. As shown in table 3, all mice survived after 7 days of
subcutaneous administration of the mixture of 2 Ìg PE
with 10-fold diluted sera from ICR mice immunized with
15 Ìg PE(¢576–613).
In addition, we also demonstrated that the antisera
induced by PE(¢576–613) could block both 35S-PE binding ability on NIH 3T3 cells and ADP ribosylation activity (data not shown).
Vaccination against Pseudomonas
Exotoxin A
Table 2. Active immunization against PE intoxication in ICR mice
preimmunized with different doses of PE(¢576–613)a
Immune dose
Death after PE challenge/mice injectedb
PE(¢576–613), Ìg
0.15
0.25
1.5
2.5
15
6/6
6/6
5/6
3/6
0/6
a
Five-week-old female ICR mice were given 10 ! graded
PE(¢576–613), which was well suspended in 0.18% AlPO4 (0.4 mg
Al +3/ml), subcutaneously on days 0, 7, and 14, and then challenged
with 2 Ìg PE on day 28.
b
Number of dead mice was recorded over a 7-day period.
Table 3. Neutralization of PE by sera from the survivors among
the PE(¢576–613) immunized mice followed by native PE toxin
challengea
Serum dilutionb
Deathc
1:10
1:50
1:250
0/3
1/3
3/3
a
Five-week-old female ICR mice were used in the experiment.
ICR mice were immunized subcutaneously on days 0, 7, and 14
by PE(¢576–613) and challenged with 2 Ìg native PE on day 28. Sera
from mice surviving 7 days after challenge were diluted 10-, 50-, or
250-fold with PBS and then mixed thoroughly with 2 Ìg native PE.
0.5 ml of each mixture was injected subcutaneously into ICR mice.
c
Mortality rates were compiled for 7 days after injection of native
PE. Values given are number of dead mice/number of mice injected.
b
Discussion
The potential role of PE in the pathogenesis of Pseudomonas infection in humans has been well documented
[41]. As reported by Cross et al. [6], there are distinct differences in the titers of anti-PE antibody in sera obtained
from dead and surviving patients after P. aeruginosa
infection [6]. These observations thus indicate that antiPE antibody might be one of the most effective factors in
protecting humans from life-threatening P. aeruginosa infection. Thus, to avoid the pathogenesis of Pseudomonas
infection, development of a vaccine against PE intoxication might be an appropriate approach. This study was
J Biomed Sci 1999;6:357–363
361
thus conducted in an effort to examine the possibility of
using a nontoxic PE fragment, PE(¢576–613), for active
immunization against PE intoxication. From a vaccination viewpoint, an ideal antigen would be highly antigenic
and nontoxic. Hwang et al. [21] demonstrated that the
ADP ribosylation domain is located in domain III of PE.
Therefore, deletion of the carboxyl-terminal portion of PE
would destroy ADP ribosylation activity, but retain the
majority of epitopes for PE antigenicity [21]. Based on
this concept, our recent study [5] showed that PE with a
deletion of 38 amino acid residues from the carboxyl terminus completely loses ADP ribosylation activity and,
consequently, loses cytotoxicity. Such experiments further demonstrate that PE(¢576–613) is a nontoxic component and can completely block PE intoxication in NIH
3T3 cells. In the present study, PE(¢576–613) as an active
immunogen for the prevention of PE intoxication was
evaluated in ICR mice. Our results indicate that using
PE(¢576–613) as an antigen for the prevention of native
PE intoxication is valid.
Previously, components of the outer membrane of
P. aeruginosa, such as lipopolysaccharides [7, 8, 24], protein F [17, 26] and conjugate vaccine [8, 11, 24], have
been used as antigens against P. aeruginosa infection.
Their abilities to induce protective immune responses in
certain experimental animals and even in human beings
have also been demonstrated [31]. In addition, vaccinations with lipopolysaccharides [7], high-molecular-weight
polysaccharide [14], and several other components [2, 16,
40] have also been shown to be effective against challenge
by PE [7, 11–13, 24, 35]. However, such preparations
require pooled surface antigens from viable cells of several different P. aeruginosa serotypes. Furthermore, because of the high possibility of endotoxin contamination
in LPS products, there is an inherent limitation in using
lipopolysaccharide products for vaccination. Thus, the
development of outer membrane components as vaccines
against Pseudomonas infection is impaired. In this study,
we have reported the simplicity of an engineered PE
derivative, PE(¢576–613), which can be used in active
immunization against PE intoxication. The advantages of
using PE(¢576–613) for vaccination against PE intoxication can be summarized as follows: (1) PE(¢576–613) is a
recombinant protein expressed in BL21(DE3)/pJJ3. The
purification procedure to obtain high-purity PE(¢576–
613) is simple. Thus, it is easy to obtain a large quantity of
PE(¢576–613) at low cost. (2) The application of
PE(¢576–613) in dosage form for vaccination is easier as
compared with the reported protective immunogens
found against P. aeruginosa infection so far. (3) PE(¢576–
613) after purification lacks any inherent endotoxicity.
Finally, like other available reported immunogenic
materials, PE(¢576–613) is effective in the treatment of
Pseudomonas infection. Our results showed that vaccination of ICR strain mice with PE(¢576–613) can protect
them against PE. Antibodies raised by PE(¢576–613) also
successfully protect mice against PE, and the protection
against PE intoxication in PE(¢576–613)-immunized
mice resulted from the neutralizing activity of antiPE(¢576–613) antibody. Thus, this study concludes that
the use of PE(¢576–613) as an antigen is a valuable alternative in PE vaccine development.
Acknowledgments
This work was supported by a grant from the National Science
Council (NSC87-2312-B-001-007) and Academia Sinica, Taiwan,
ROC.
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