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Infection and Immunity, January 2000, p. 221-226, Vol. 68, No. 1
Division of Infectious Diseases, Massachusetts General
Hospital, Boston, Massachusetts 021141;
AVANT Immunotherapeutics Inc., Needham, Massachusetts
024942; Department of Microbiology,
University of Texas Health Science Center, San Antonio, Texas
782843; and Department of Microbiology
and Molecular Genetics, Harvard Medical School, Boston, Massachusetts
021154
Received 27 July 1999/Returned for modification 22 September
1999/Accepted 20 October 1999
We have previously shown that more prominent immune responses are
induced to antigens expressed from multicopy plasmids in live
attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a
Vibrio cholerae has a
number of attributes that make it an attractive candidate for use as a
vaccine vector for inducing mucosal immunity against heterologous
antigens. V. cholerae is a well-studied noninvasive organism
that induces long-lasting mucosal and systemic immune responses
(11, 18). Attenuated strains of V. cholerae have
already been developed that have been shown to be both safe and
immunogenic in humans (2, 12, 13, 16, 26, 28); moreover,
vaccine strains of V. cholerae have been developed that are
able to secrete large heterologous antigens through the use of
the Escherichia coli hemolysin A protein export system
(21). Attenuated vaccine strains of V. cholerae have also recently been developed that are able to
express immunoadjuvants in vivo, such as LT(R192G), a
nonenterotoxic mutant of E. coli heat-labile enterotoxin that retains immunoadjuvant activity (23). Previously, we
have shown that the magnitude of immune responses induced against
antigens expressed by attenuated vaccine strains of V. cholerae is directly related to the quantity of antigen produced,
with more prominent immune responses induced to antigens expressed from
multicopy plasmids than to antigens expressed from single-copy genes on the chromosome (22).
In enteric bacteria, glutamine and glutamate serve as the primary
nitrogen donors for cellular metabolism (8, 19). Glutamine synthetase, encoded by glnA, is an enzyme required for
synthesis of glutamine and is responsible for assimilation of ammonia
when extracellular nitrogen concentrations are low (8, 25).
The activity and synthesis of glutamine synthetase are regulated by availability of nitrogen (8, 17). Strains of V. cholerae have already been developed that are deficient in
glutamine synthetase; these strains are unable to grow on minimal
medium lacking glutamine (8-10).
Here we report whether complementation of a glnA chromosomal
deletion with a plasmid expressing GlnA could be used as a balanced lethal system for in vivo expression of an antigen from a multicopy plasmid in vaccine and vector strains of V. cholerae.
Bacterial strains and media.
The bacterial strains and
plasmids used in this study are described in Table
1. All strains were maintained at
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of a
glnA Balanced Lethal Plasmid
System for Expression of Heterologous Antigens by Attenuated
Vaccine Vector Strains of Vibrio cholerae
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
glnA derivative of V. cholerae vaccine
strain Peru2. This mutant strain, Peru2glnA, is unable
to grow on medium that does not contain glutamine; this growth
deficiency is complemented by pKEK71-NotI, a plasmid
containing a complete copy of the Salmonella typhimurium
glnA gene, or by pTIC5, a derivative of pKEK71-NotI
containing a 1.8-kbp fragment that directs expression of CtxB with a
12-amino-acid epitope of the serine-rich Entamoeba
histolytica protein fused to the amino terminus. Strain
Peru2glnA(pTIC5) produced 10-fold more SREHP-12-CtxB in
supernatants than did ETR3, a Peru2-derivative strain containing the
same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo,
we inoculated germfree mice on days 0, 14, 28, and 42 with
Peru2glnA,
Peru2glnA(pKEK71-NotI), Peru2(pTIC5),
Peru2glnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days
after primary inoculation, including Peru2glnA; strains containing plasmids continued to harbor pKEK71-NotI
or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of
animals. However, specific anti-CtxB immune responses in serum (P 
0.05) and bile (P 0.001)
were significantly higher in animals that received
Peru2glnA(pTIC5) than in those that received ETR3, confirming the advantage of higher-level antigen expression in vivo.
The development of this balanced lethal system thus permits construction and maintenance of vaccine and vector strains of V. cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
70°C in Luria-Bertani (LB) broth medium (24) containing
15% glycerol. Streptomycin (100 µg/ml), ampicillin (100 µg/ml),
and chloramphenicol (15 µg/ml) were added as appropriate. Cultures
were grown at 37°C with aeration in either LB medium, LB medium
supplemented with 2 mM glutamine if growing noncomplemented glutamine
auxotrophic strains, M9 minimal medium supplemented with 0.05 mM
thiamine (Sigma Chemical Co., St. Louis, Mo.) and 0.3 mM cysteine
(Sigma), or M9 minimal medium supplemented with 10 mM glutamine
(Sigma), 10 mM NH4NO3 (Sigma), 0.05 mM
thiamine, and 0.3 mM cysteine if growing noncomplemented glutamine
auxotrophic strains. Culturing of stool was done on LB agar plates
containing appropriate antibiotics and supplemented with 2 mM
glutamine; isolates were confirmed as V. cholerae on thiosulfate-citrate-bile salts-sucrose plates. LB agar plates, made
without NaCl and supplemented with 10% sucrose, were used to select
for double homologous recombinants lacking the sacB gene
during construction of V. cholerae vaccine strains
containing the deletion in the chromosomal glnA gene
(5, 9, 10, 14).
TABLE 1.
Bacterial strains and plasmids used in this study
Genetic methods. Isolation of plasmid DNA, restriction enzyme digestion, and agarose gel electrophoresis were performed by standard molecular biological techniques (24).
Genetic constructs.
A mutant of V. cholerae Peru2
deficient in glutamine synthesis, Peru2glnA, was
constructed by using a derivative of the suicide vector pCVD442
(5, 15). This derivative, pKEK70, contains a copy of the
V. cholerae O395 glnA gene with an internal
354-bp deletion (corresponding to amino acids phenylalanine-134 to
glycine-251) (10). Plasmid pKEK70 was mobilized from
E. coli SM10 
pir into V. cholerae Peru2 by conjugation; recombinants were selected for by
ampicillin resistance. Recombinants were grown to turbidity in the
absence of selection pressure and plated on LB agar lacking NaCl
but containing 10% sucrose (1, 5, 9, 10, 14, 20).
Colonies of Peru2glnA that had undergone allelic
exchange to introduce the expected internal 354-bp deletion within
glnA were confirmed by PCR amplification; isolates were
confirmed as being nutritionally auxotrophic on M9 minimal medium
lacking glutamine.
ompA2::SREHP-12::ctxB)
was blunt end ligated into the BamHI site of
pKEK71-NotI (replacing the NotI adapter),
creating plasmid pTIC5. The lppP
lacPOompA2::SREHP-12::ctxB
fragment is identical to that inserted as a single copy into the
lacZ gene of Peru2 to create vaccine strain ETR3
(22).
Quantitation of in vitro expression of SREHP-12-CtxB.
Overnight cultures of Peru2glnA(pTIC5) and ETR3 grown in
LB and M9 minimal media were divided into supernatant, periplasmic, enriched outer-membrane, and whole-cell fractions as previously described (3, 6, 7). In vitro expression of SREHP-12-CtxB was assayed in a quantitative enzyme-linked immunosorbent assay for
CtxB (22, 23). Briefly, serial dilutions of cellular
fractions in phosphate-buffered saline-0.05% Tween 20 (PBS-T; Sigma)
were applied to 96-well microtiter plates previously coated with type III ganglioside (Sigma). Detection of CtxB was performed by using a
1:2,000 dilution of goat anti-CtxB antibody (List Biological Laboratories, Inc., Campbell, Calif.) in PBS-T, followed by a 1:2,000
dilution of rabbit anti-goat immunoglobulin G (IgG)-horseradish peroxidase conjugate (Southern Biotechnology Associates, Inc., Birmingham, Ala.). Plates were developed with a 1-mg/ml solution of
2,2-azinobis(3-ethylbenzthiazolenesulfonic acid) (ABTS; Sigma) with
0.1% H2O2, and the optical density at 405 nm
(OD405) was read in a Vmax microplate reader (Molecular
Devices Corp., Sunnyvale, Calif.). Measured optical densities were
compared to a standard curve provided by dilutions of purified CtxB (List).
Inoculation and colonization of germfree mice.
Immediately
upon removal of mice from the shipping container, five groups of 6 to
12 germfree female Swiss mice, 3 to 4 weeks old (Taconic Farms, Inc.,
Germantown, N.Y.), were orally inoculated via gastric intubation with
250-µl inocula containing approximately 108 organisms of
V. cholerae strains resuspended in 0.5 M NaHCO3 (pH 8.0) (4). Prior to inoculation, Peru2glnA
was grown in M9 minimal medium supplemented with glutamine,
NH4NO3, thiamine, and cysteine.
Peru2glnA(pKEK71-NotI) and
Peru2glnA(pTIC5) were grown in M9 minimal medium
supplemented with thiamine and cysteine but not containing glutamine.
Peru2(pTIC5) was grown in M9 medium containing chloramphenicol and
supplemented with thiamine and cysteine. ETR3 was grown in M9 medium
containing streptomycin and supplemented with thiamine and cysteine.
Mice were subsequently housed under non-germfree conditions. No
antibiotic selection pressure or specific nutritional supplementation
was continued in vivo. All mice received repeat inocula on days 14, 28, and 42. To assess colonization, fresh stool samples were collected immediately upon passage from all mice every 24 h for the first 96 h after the day 0, 14, and 28 inoculations; pellets were also collected every 48 h from day 4 to day 14 after primary
inoculation. Collected pellets were immediately resuspended in 500 µl
of M9 medium, vortexed, and allowed to settle. One-hundred-microliter aliquots were plated on LB medium containing streptomycin and supplemented with 2 mM glutamine. Colonies were then replica plated onto thiosulfate-citrate-bile salts-sucrose medium and LB medium containing chloramphenicol and supplemented with 2 mM glutamine to
assess the intestinal passage of V. cholerae strains of
interest (22).
Immunological sampling.
Mice were sacrificed on day 56, at
which time blood, stool, and bile were collected and processed as
previously described (23). Processed samples were divided
into aliquots and stored in 70°C for subsequent analysis.
Detection of vibriocidal and anti-CtxB antibodies. Serum vibriocidal antibody titers were measured by a microassay as previously described (22, 23). To detect specific anti-CtxB IgG and IgA antibodies in sera, 100-µl duplicate samples of 1:200 dilutions of sera in PBS-T were placed in wells of microtiter plates previously coated with ganglioside and CtxB (22, 23). Plates were incubated at room temperature overnight and washed, and a 1:2,000 dilution in PBS-T of goat anti-mouse IgG or IgA conjugated to biotin (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was applied to each well. After 1 h of incubation at 37°C, the plates were again washed and a 1:4,000 dilution of streptavidin-horseradish peroxidase conjugate (Zymed Laboratories, Inc., South San Francisco, Calif.) was applied to each well. The plates were then incubated at 37°C for 1 h, washed, and developed with ABTS and 0.1% H2O2; the OD405 was detected kinetically with a Vmax microplate reader as previously described (21, 23). Plates were read for 5 min at 19-s intervals, and the maximum slope for an OD change of 0.2 U was reported as milliunits of OD per minute (21, 23).
To detect specific IgA antibody responses in stool and bile, measurements of total stool and bile IgA were first taken. Duplicate serial twofold dilutions of stool (1:100 to 1:800) and bile (1:800 to 1:102,400) samples in PBS-T were added to wells previously coated with rat monoclonal anti-mouse IgA antibody R5-140 (PharMingen, San Diego, Calif.) (23). After incubation of plates, a 1:2,000 goat anti-mouse IgA-horseradish peroxidase conjugate (Southern Biotechnology Associates) in PBS-T was added to each well and the plates were subsequently developed for horseradish peroxidase activity as described above (23). Comparisons were made to a mouse IgA standard (Kappa TEPC 15; Sigma) (23). To detect specific anti-CtxB IgA antibody in stool and bile, duplicate 200-µl samples of bile or stool containing 200 ng of total IgA in PBS-T were added to wells previously coated with ganglioside-CtxB. After incubation of plates, a 1:2,000 dilution of goat anti-mouse IgA-biotin conjugate (Kirkegaard & Perry) in PBS-T was added. The plates were assayed for horseradish peroxidase activity, and the OD405 was determined kinetically as described above.Statistics and graphics. Statistical analysis for the comparison of geometric means was performed for normally distributed data with the independent-sample Student t test or with the Mann-Whitney U test for nonparametric data by use of SPSS for Windows 8.0 (23). Data were plotted with Microsoft Excel 7.0a and GraphPad Prism 3.0.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Construction of glutamine-deficient vaccine strains.
Peru2glnA was constructed by in vivo marker exchange;
this strain contains an internal in-frame 354-bp deletion of the
glnA gene. Peru2glnA is deficient in
glutamine synthetase and is unable to grow on M9 minimal medium lacking
glutamine but is able to grow on LB medium or on M9 medium supplemented
with glutamine. Introduction of plasmid pKEK71-NotI
containing a copy of the Salmonella typhimurium glnA gene
under the control of a high-level sigma 54-independent mutant
S. typhimurium glnA promoter (glnAp356) (10) complements this deficiency in glutamine
synthesis in Peru2glnA and allows
Peru2glnA(pKEK71-NotI) to grow
on M9 minimal medium lacking glutamine. Plasmid pKEK71-NotI
is maintained in glnA-deficient V. cholerae strains grown on M9 minimal medium without the need for
antibiotic selection pressure.
ompA2::SREHP-12::ctxB)
expressing CtxB with a 12-amino-acid epitope of the serine-rich
Entamoeba histolytica protein fused at the amino
terminus (SREHP-12-CtxB) was inserted into plasmid
pKEK71-NotI to make plasmid pTIC5. Plasmid pTIC5 complements
Peru2glnA and allows strain
Peru2glnA(pTIC5) to grow on M9 minimal medium
lacking glutamine. As is the case for pKEK71-NotI, pTIC5 is
maintained in glnA-deficient V. cholerae strains
grown on M9 minimal medium without the need for antibiotic selection pressure.
Expression and localization of SREHP-12-CtxB by vaccine
strains.
In vitro analysis of cellular fractions for the presence
of SREHP-12-CtxB in vaccine strains Peru2(pTIC5) (a vaccine strain containing a wild-type V. cholerae glnA gene on the
chromosome), Peru2glnA(pTIC5), and ETR3 showed
that essentially all of SREHP-12-CtxB localized to the supernatant
fractions. In LB medium containing chloramphenicol, Peru2(pTIC5)
expressed 2,339 ± 652 ng/ml/OD600 (geometric
mean ± standard error of the mean) of SREHP-12-CtxB in the
supernatant; in LB medium containing chloramphenicol,
Peru2glnA(pTIC5) expressed 954 ± 445 ng/ml/OD600, and in LB medium supplemented with
streptomycin, ETR3 expressed 35 ± 4 ng/ml/OD600. In
M9 minimal medium lacking both glutamine and antibiotics,
Peru2glnA(pTIC5) expressed SREHP-12-CtxB at
174 ± 52 ng/ml/OD600 and ETR3 expressed 12 ± 21 ng/ml/OD600. In summary,
Peru2glnA(pTIC5) expressed approximately 10-fold more SREHP-12-CtxB than did ETR3; this ratio was the
same when strains were grown in medium containing antibiotics and when strains were grown in minimal medium lacking glutamine.
Intestinal colonization in mice following oral inoculation of
vaccine constructs.
No antibiotic selection pressure or
nutritional supplementation was maintained after oral inoculation of
any group of mice. Surprisingly, despite complete auxotrophy of
Peru2glnA in M9 minimal medium lacking glutamine in
vitro, Peru2glnA was recoverable from the stool of
mice for 8 days after primary inoculation (Fig. 1). Strains recovered on day 8 after oral
inoculation were confirmed to contain the mutant glnA gene
by PCR analysis; these isolates were confirmed to be auxotrophic on M9
minimal medium lacking glutamine in vitro. These results suggest that
glutamine is present in sufficient quantities in the intestinal lumen
of mice to overcome the deficiency in glutamine synthetase in the
glnA V. cholerae vaccine strains described in this
paper.
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glnA after
primary inoculation, V. cholerae vector strains were
recoverable for 10 to 12 days after primary inoculation of mice with
Peru2glnA(pKEK71-NotI) or
Peru2glnA(pTIC5), suggesting a slight in vivo
survival advantage for glnA vaccine strains
complemented for the mutant glnA gene. Plasmids pTIC5
and pKEK71-NotI were themselves recoverable for 8 to 10 days after primary inoculation of mice, and in vivo stability of
pTIC5 was confirmed by restriction digestion of plasmid DNA isolated
from strains recovered 6 days after primary inoculation. Corroborating
previous results, nonauxotrophic strain ETR3 (containing wild-type
glnA on the chromosome) was recoverable from stool for 8 days after primary inoculation (22).
In order to judge in vivo retention of plasmid pTIC5 by a vaccine
strain of V. cholerae not requiring complementation of a mutant glnA gene, a cohort of mice were inoculated with
Peru2(pTIC5) (containing wild-type glnA on the
chromosome). Compared with that of mice that received
Peru2glnA(pTIC5), qualitative intestinal colonization of mice that received Peru2(pTIC5) was not appreciably different. V. cholerae vector strains were recoverable for
10 days after oral inoculation, and pTIC5 was maintained by Peru2 in
vivo for 8 days after primary inoculation.
These results suggest that Peru2glnA is a glutamine
auxotroph in vitro but is not impaired for growth in vivo. Plasmids
that complement the introduced glnA mutation do, however,
appear to confer a slight in vivo survival advantage on glnA
V. cholerae vaccine strains. Colonization of the intestines
of mice by glnA strains containing plasmids that
complement the glnA mutation was equivalent to that of
strains containing wild-type glnA on the chromosome [ETR3
or Peru2(pTIC5)], and the retention of complementing plasmids by
strains deficient in glnA was similar to the in vivo retention of plasmids by the parental strain Peru2. Quantitative intestinal colonization studies to assess whether complemented strains
are more efficient at surviving in vivo were not performed.
As demonstrated in a previous study (22), V. cholerae vaccine strains were recoverable from stool for only 1 to
3 days following day 14 and later inoculations of mice, presumably due
to increased competition from normal intestinal flora of mice housed
under non-germfree conditions. Inoculations performed on days 14, 28, and 42 were, however, associated with boosting of the immune responses (data not shown).
Measurement of serum vibriocidal antibody responses. Vibriocidal antibodies were measured on day 56 samples (Fig. 2). Vibriocidal antibodies are a measure of immune responses against V. cholerae organisms themselves and reflect the ability of V. cholerae strains to colonize the intestine. In our experiment, all groups of mice developed vibriocidal antibody responses of comparable magnitudes, confirming the roughly equivalent abilities of the various V. cholerae vaccine strains to colonize the intestines of mice.
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Measurement of anti-CtxB antibodies.
Anti-CtxB antibody
responses were measured in day 56 samples of serum, stool, and bile
(Fig. 3). As seen previously, a poor but
statistically significant anti-CtxB antibody response was measurable in mice that received ETR3 [P 0.01;
serum anti-CtxB IgG compared to responses in mice that received
Peru2glnA or Peru2glnA(pKEK71-NotI)]
(22). The most prominent anti-CtxB responses, however, were
measurable in mice that received
Peru2glnA(pTIC5). Compared to the response in
mice that received ETR3 expressing SREHP-12-CtxB from a single
chromosomal copy, mice that were inoculated with
Peru2glnA(pTIC5) expressing SREHP-12-CtxB from
the multicopy plasmid had significantly more anti-CtxB IgG antibody in
serum (P 0.05) and mucosal anti-CtxB IgA in bile
(P 0.001). The anti-CtxB IgA response in stool was
also most prominent in mice that received
Peru2glnA(pTIC5) and approached but did not
achieve statistical significance compared to the response measured in mice inoculated with ETR3. It is worth noting that mice that received Peru2glnA(pTIC5) had a significantly higher
anti-CtxB response in bile than did mice that received Peru2(pTIC5)
(P 0.02), perhaps reflecting increased mucosal
immune responses related to improved retention of plasmid pTIC5 in a
glnA V. cholerae strain compared to a V. cholerae strain containing wild-type glnA.
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glnA V. cholerae strains are auxotrophic for glutamine, their ability to
survive in vivo appears to be equivalent to that of vaccine strains of
V. cholerae containing wild-type glnA on the chromosome. The development of this balanced lethal plasmid system thus
removes the need for antibiotic selection pressure in the construction
of V. cholerae vaccine strains expressing immunogenic antigens from plasmid-based systems and removes the need to grow oral
inocula of such strains in medium containing antibiotics. The
development of this balanced lethal plasmid system should, therefore,
assist in the development of improved vaccine and vector strains of
V. cholerae.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grants KO8 AI01332 (to E.T.R.) and AI40725 (to S.B.C.), both from the National Institute of Allergy and Infectious Diseases.
We are extremely grateful to Samuel L. Stanley, Jr.; Tonghai Zhang; and Lynne Foster for their assistance with CTB-SREHP-12 and to John J. Mekalanos for providing helpful input and V. cholerae Peru2.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114. Phone: (617) 726-3811. Fax: (617) 726-7416. E-mail: scalderwood{at}partners.org.
Editor: A. D. O'Brien
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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