होम In Vitro Cellular & Developmental Biology - Plant Development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia...

Development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli

, , , , , , , , , ,
यह पुस्तक आपको कितनी अच्छी लगी?
फ़ाइल की गुणवत्ता क्या है?
पुस्तक की गुणवत्ता का मूल्यांकन करने के लिए यह पुस्तक डाउनलोड करें
डाउनलोड की गई फ़ाइलों की गुणवत्ता क्या है?
खंड:
38
भाषा:
english
पृष्ठ:
7
DOI:
10.1079/ivp2001247
Date:
January, 2002
फ़ाइल:
PDF, 124 KB
0 comments
 

To post a review, please sign in or sign up
आप पुस्तक समीक्षा लिख सकते हैं और अपना अनुभव साझा कर सकते हैं. पढ़ूी हुई पुस्तकों के बारे में आपकी राय जानने में अन्य पाठकों को दिलचस्पी होगी. भले ही आपको किताब पसंद हो या न हो, अगर आप इसके बारे में ईमानदारी से और विस्तार से बताएँगे, तो लोग अपने लिए नई रुचिकर पुस्तकें खोज पाएँगे.
In Vitro Cell. Dev. Biol.—Plant 38:11–17, January –February 2002
q 2002 Society for In Vitro Biology
1054-5476/02 $10.00+0.00

DOI: 10.1079/IVP2001247

2000 CONGRESS SYMPOSIUM ON MOLECULAR FARMING:
DEVELOPMENT OF AN EDIBLE SUBUNIT VACCINE IN CORN AGAINST ENTEROTOXIGENIC STRAINS OF
ESCHERICHIA COLI
STEPHEN J. STREATFIELD*, JOCELYNE M. MAYOR, DONNA K. BARKER, CHRISTOPHER BROOKS, BARRY J. LAMPHEAR, SUSAN L. WOODARD,
KATHERINE K. BEIFUSS, DEBRA V. VICUNA, LEIGH ANNE MASSEY, MICHAEL E. HORN, DONNA E. DELANEY, ZIVKO L. NIKOLOV,
ELIZABETH E. HOOD, JOSEPH M. JILKA, A ND JOHN A. HOWARD

ProdiGene, 101 Gateway Boulevard, College Station, TX 77845
(Received 10 May 2001; accepted 7 August 2001; editor J. M. Widholm)

Summary
Advances in the development of subunit vaccines and in the production of foreign proteins in plants together offer the
prospect of stable and inexpensive vaccine delivery systems. Various bacterial and viral proteins stably produced in plants
have been shown to elicit immune responses in feeding trials. We have extended this approach by using Zea mays as the
plant production system. Corn has several advantages as a vaccine delivery vehicle, most notably established technologies
to generate transgenic plants, to optimize traits through breeding and to process the seed into a palatable form. Here we
report on the production in corn seed of the GM1 receptor binding (B) subunit of the heat-labile toxin (Lt) from
enterotoxigenic strains of Escherichia coli. Versions of the Lt-B gene were synthesized to give optimum codon usage for corn
and to target the protein to either the cell surface or the cytoplasm. These synthetic genes were fused to a strong promoter
and transformed into corn. Lt-B was highly expressed in corn seed at up to 1.8% of the total soluble protein and this was
further increased approximately five-fold through plant breeding. As in E. coli, Lt-B produced in corn forms a functional
pentamer that can bind to the GM1 receptor. Furthermore, Lt-B pentamer stored in corn seed is much more res; istant to heat
than is the pure protein, allowing the transgenic corn to be readily processed into an edible form. This work demonstrates
the potential of using products derived from transgenic corn seed as delivery vehicles for subunit vaccines.
Key words: enterotoxigenic Escherichia coli; heat-labile toxin; protein stabilization; subcellular targeting; transgene
expression; transgenic corn.
Daniell et al., 2001). In many cases the plant-delivered antigens
induce immune responses and recent trials show promise of
protection when the antigens are administered orally.
Several plant species have been used to express foreign proteins
including vaccine candidates. The choice of species depends on
experimental considerations, such as whether the plant can be easily
transformed, and the economics of large-scale production using that
crop. Early work focused on tobacco and potato since they are
relatively easy to transform. However, from a commercial standpoint,
seed crops including cereals are advantageous. Several recent
reviews address the choices available for expressing heterologous
proteins at a high level in plants (Cramer et al., 1999; Giddings et al.,
2000; Daniell et al., 2001).
In the case of vaccine components, particular antigens have
received particular attention by virtue of their inherent antigenicity
and the route of colonization of the microorganism that produces
them. In particular, the receptor binding B subunits of cholera toxin
of Vibrio cholerae and of the heat-labile toxin (Lt) of enterotoxigenic
Escherichia coli (ETEC) have been expressed in plants and are
efficacious in mice (Arakawa et al., 1998; Mason et al., 1998) and, in
the case of Lt, in humans (Tacket et al., 1998). ETEC are responsible
for over 650 million cases of diarrhea and about 800 thousand deaths
in children under five annually in developing countries (Black,
1986). In addition, approximately 20% of visitors to these countries
contract travelers’ diarrhea from ETEC (Black, 1990). It is the toxins

Introduction
Using plant systems to synthesize foreign proteins at levels
compatible with commercialization is the subject of intense research
activity. The emphasis is on high levels of expression and
demonstrating that the activity of a protein synthesized in a plant
system is comparable to that of the protein purified from its native
source or over-expressed in a microorganism. In the area of human
health, considerable effort has been put into expressing antibodies
and vaccine candidates in plants. For vaccines, selected proteins of
disease agents are expressed in plant tissue and delivered as subunit
vaccines. The plant material containing the protein can be used
directly as an edible, oral vaccine. Alternatively, the protein can be
purified and delivered orally or by injection. Major advantages of
oral vaccines are their relative ease and low cost of administration.
These advantages are maximized when the vaccine comprises edible
plant tissue or a food-type product derived from plants. Set against
the advantages of oral vaccines, the proportion of the dose actually
delivered to the mucosal surface may vary among individuals. There
are many reported examples of expressing subunit vaccine
candidates in various plant species and these are addressed in
several reviews (Mason and Arntzen, 1995; Giddings et al., 2000;
*Author to whom correspondence should be addressed: Email
sstreatfield@prodigene.com

11

12

STREATFIELD ET AL.

harbored by these bacterial strains that are the causal agents of
diarrhea. Lt is the only toxin present in about a third of ETEC strains
and is present alongside a heat-stable toxin in a further third
(Svennerholm and Holmgren, 1995). Lt has a multi-subunit
structure, consisting of a pentamer of ganglioside receptor binding
(B) subunits and a single enzymatic (A) subunit, making Lt
structurally similar to cholera toxin. The B subunit pentamer
delivers the A subunit to the surface of the gut, where it is
internalized and exerts its ADP-ribosylation activity on the a
subunits of specific heterotrimeric G proteins, resulting in activation
of adenylate cyclase, changes in ion flux and diarrhea. Since Lt
invades the body through cells lining the gut, a mucosal vaccine
approach has been taken to combat ETEC. Such oral vaccines under
development consist of an inactivated whole cell E. coli component
together with a cholera toxin B subunit or Lt-B subunit component
(Wiedermann et al., 2000). Here we report on the expression of Lt-B
in Zea mays (corn) as a model for delivering an edible vaccine for
humans in a cereal crop.
Materials and Methods
Construction of Lt-B expression vectors for corn. The Lt-B gene of an E.
coli strain of human origin (Leong et al., 1985) was synthesized to optimize
codon usage for maize. Two versions of the gene were made, one encoding the
barley a-amylase secretion signal at the N-terminus of Lt-B, and one
encoding only Lt-B. Oligonucleotides spanning the gene were annealed and
ligated and the products amplified using the polymerase chain reaction
(PCR). The Lt-B sequences were placed 30 to a maize constitutive promoter
and untranslated leader sequence from the ubiquitin regulatory system,
designated here PGNpr1, and 50 to the potato proteinase inhibitor II
transcription terminator. The Lt-B expression cassettes were introduced into
a plant transformation vector with right and left border sequences of
Agrobacterium tumefaciens Ti plasmid origin, and the pat gene of
Streptomyces viridichromogenes, conferring resistance to glufosinate
ammonium. The Lt-B transformation vectors were designated PGN7101
(barley a-amylase secretion signal fused to Lt-B) and PGN8957 (Lt-B).
Agrobacterium-mediated corn transformation and plant breeding. Stable
transformation of corn followed a modified version of Ishida et al. (1996) as
described in Streatfield et al. (2001). Immature zygotic embryos of Hi-II
kernels were transformed with A. tumefaciens strain EHA 101 containing
PGN7101 or PGN8957. T0 plants were regenerated from embryogenic tissues
and transferred to a greenhouse. Pollen from inbred lines was crossed onto
transgenics to give T1 seed. Subsequent generations were grown in the field,
where single leaves of seedlings were treated with 1% glufosinate ammonium
to identify transgenics. Pollen from elite inbred Lancaster and Stiff Stalk lines
was crossed onto the transgenics to generate seed. For large-scale grain
production, pollen of transgenic T2 plants was crossed onto a hybrid line.
Analysis of the insertion of vector sequences into the maize genome. DNA
from wild-type and transgenic maize leaves was prepared using a DNeasy
Plant Maxi Kit (Qiagen, Inc., Valencia, CA). DNA samples (1 mg) were used
as templates for PCR analysis using primers located within Lt-B sequence.
Products were size-separated on a 1% agarose gel containing ethidium
bromide and viewed under ultraviolet light. Alternatively, DNA samples
(20 mg) were digested with NcoI restriction endonuclease and DNA fragments
were size-separated on a 0.7% agarose gel. DNA was transferred onto a
charged nylon filter (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and
cross-linked to the filter by exposure to ultraviolet light. Radionucleotide
(32P) was incorporated into an Lt-B gene sequence by random prime labeling
using the High Prime reagent mix of Roche Diagnostics GmbH (Mannheim,
Germany) and the filter was incubated with this Lt-B probe. The filter was
washed under conditions of high stringency (0.015 M NaCl, 0.0015 M
Na3C6H5O7.2H2O, 0.1% sodium dodecyl sulfate (SDS), 658C) and exposed to
imaging film (Eastman Kodak Co., Rochester, NY).
Detection of Lt-B mRNA in corn leaf tissue. Total RNA was isolated from
leaf tissue as described by Chatterjee et al. (1996). RNA samples (20 mg)
were size-separated on an agarose/formaldehyde gel, transferred onto a
charged nylon filter (Amersham Pharmacia Biotech, Inc.) and cross-linked by

exposure to ultraviolet light. The filter was incubated with Lt-B probe, washed
under conditions of high stringency (0.015 M NaCl, 0.0015 M Na3C6H5O7.2H2O, 0.1% SDS, 658C) and exposed to film as for the filter with crosslinked DNA.
Preparation of soluble protein extracts from corn seed and corn
puffs. Soluble proteins were extracted from seed as described in Streatfield
et al. (2001) and were similarly extracted from extruded corn puffs described
below. Protein concentrations were determined by a protein-dye binding
assay (Bradford, 1976).
Detection of Lt-B by immunoblotting. Proteins in soluble plant extracts
were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA)
by electroblotting. The membranes were blocked for 1 h with 5% non-fat
dried milk in TBST (0.01 M Tris base, 0.15 M NaCl, 0.05% Tween, pH 8.0),
incubated overnight with a 2000-fold dilution of Lt-B antibody (rabbit) in the
blocking solution and washed with TBST. The membranes were then
incubated for 1 h with a 5000-fold dilution of anti-rabbit antibody conjugated
to horseradish peroxidase in the blocking solution, washed with TBST,
incubated with the ECL Western blotting detection reagent (Amersham
Pharmacia Biotech) and exposed to imaging film (Eastman Kodak Co.).
Detection of Lt-B pentamer by GM1-based ELISA. The GM1 ELISA is
based on that of Svennerholm and Holmgren (1978). Between incubations
96-well plates were washed three times with phosphate-buffered saline (PBS)
(137 mM NaCl, 8.097 mM Na2HPO4.7H2O, 2.68 mM KCl, 1.47 mM KH2PO4,
pH 6.9). Wells were coated with 1.5 mg ml21 GM1 in PBS by incubating
overnight at 48C and were blocked with 3% bovine serum albumin (BSA) in
PBS by incubating at 378C for 30 min. Soluble plant protein extract was
added to the wells and incubated at 228C for 1 h. Lt-B antibody (rabbit) in
PBS/1% BSA was then added to the wells and incubated at 228C for 1 h and
anti-rabbit antibody conjugated to alkaline phosphatase in PBS/1% BSA was
added to the wells and incubated at 228C for 1 h. para-Nitrophenyl-phosphate
was then added to the wells and incubated at 378C for 30 min. Absorbance
values at 405 nm were recorded using a SPECTRAmax PLUS384 plate reader
(Molecular Devices, Sunnyvale, CA). A dilution series of recombinant Lt-B
standard was included in the assay.
Quantification of Lt-B in corn seed. A sandwich ELISA was used to
quantify Lt-B in corn (Streatfield et al., 2001). An Lt-B antibody at
133 ng ml21 was used to capture Lt-B in protein extracts and a biotinylated
version of the Lt-B antibody at 50 ng ml21 was used to recognize bound Lt-B.
For detection streptavidin –alkaline phosphatase and para-nitrophenylphosphate were deployed.
Production of corn puffs containing Lt-B. Large impurities were picked
out of corn grain and small impurities removed using a 6 mm screen. Grain
was ground in a style 148/size 8 mill (Bauer Brothers Co., Springfield, OH)
until most particles would pass through an 850 mm but not a 250 mm mesh.
The ground grain was passed through a X-20 single screw extruder (Wenger
Manufacturing, Inc., Sabetha, KS) at a shaft speed of 450 revolutions per
minute. Water was added in the extruder barrel to about 30% moisture. The
barrel had five sections, the first two held at ambient temperature, the third at
1108C, the fourth at 1508C and the fifth at 1708C. Pressure was greatest in the
fifth section and was approximately 7  105 kg m22 . A rotating blade cut the
extruded corn into roughly spherical pieces of approximately 12 mm
diameter. These corn puffs were dried at 608C to about 10% moisture.

Results
Agrobacterium-mediated corn transformation with Lt-B yields
intact low-copy inserts. The Lt-B vectors PGN7101 and PGN8957
were constructed for plant transformation. PGN7101 includes the
sequence encoding the secretion signal of barley a-amylase fused to
the N-terminus of Lt-B and the entire coding sequence for both
vectors is codon-optimized for expression in maize. Considering
PGN7101, the Signal P prediction server of the Center for Biological
Sequence Analysis (Department of Biotechnology, The Technical
University of Denmark) forecasts that the signal sequence should be
cleaved from Lt-B. Also, when this signal sequence was fused to
avidin for expression in maize, the protein accumulated in
intercellular spaces (Hood et al., 1997). PGN7101 was introduced

AN EDIBLE VACCINE PRODUCED IN CORN

FI G . 1. PCR and Southern hybridization analysis of genomic DNA from
corn lines transformed with PGN7101. A, PCR analysis of nine independent
transgenic lines derived from LTA transformation events (03, 05, 06, 07, 08,
09, 10, 17, and 20), the untransformed wild-type control line (wt) and the
Lt-B plant transformation vector PGN7101 (v) using primers comprising
sequences within Lt-B. B, NcoI restriction enzyme digests of genomic DNA of
the nine independent transgenic lines and the untransformed wild-type
control line size separated by agarose gel electrophoresis and probed with
Lt-B sequence. NcoI sites span the Lt-B gene in the vector PGN7101.

into corn by A. tumefaciens to generate 21 independent
transformation events designated LTA01 through LTA21. T0 plants
were regenerated from 16 of these events and T1 seed was recovered
from plants representing 12 events following out-crossing. T1 seed
representing nine of these events was germinated and leaf tissue was
harvested from a representative glufosinate ammonium-resistant T1
plant for each event. Genomic DNA was prepared from these tissue
samples and in each case was shown by PCR and hybridization
analysis to harbor a fragment spanning Lt-B of approximately the
size predicted from vector sequence (Fig. 1). Using appropriate
restriction enzymes, Lt-B and pat gene sequence probes identify a
single insertion of one copy of vector sequence for eight events,
while one event has a more complex pattern of insertion with two
copies of vector sequence (data not shown). Thus, A. tumefaciensmediated transformation of immature zygotic corn embryos appears
to result in the insertion of predominantly single copies of complete
genes. A spectinomycin sequence probe did not identify any insert,
implying that this antibiotic resistance gene sequence, which is
present on the vector outside the T-DNA border sequences, was not
incorporated into any transformation event.
PGN8957 was also introduced into corn by A. tumefaciens to
generate 18 independent transformation events designated LTB01
through LTB18. T0 plants were regenerated from 16 of these events
and, following out-crossing, T1 seed was recovered from plants
representing all 16 events. Genomic DNA from plants representing
these events was not analyzed.
Full-size message and functional Lt-B accumulate in transgenic
corn seed. The synthesis of Lt-B message was assessed in leaf
tissue of a representative plant derived from a transformation event
(LTA17) generated with the vector PGN7101 encoding the
extracellular targeted version of Lt-B. The transgenic corn clearly
synthesizes a single Lt-B message species of approximately the
predicted size (Fig. 2A). Plant tissues carrying PGN7101 were
analyzed on immunoblots and Lt-B was detected in transformed
callus, in leaves of regenerated seedlings and in T1 seeds. Soluble
protein extract from the Lt-B corn seed contains Lt-B pentamer and,

13

FI G . 2. Northern hybridization analysis of RNA and Western (immunoblot) analysis of protein prepared from corn transformed with PGN7101. A,
RNA prepared from the untransformed wild-type control line (wt) and a
representative plant derived from transformation event LTA17 generated with
PGN7101 (Lt-B) size-separated by agarose gel electrophoresis and probed
with Lt-B sequence. B, A mixture of boiled and unboiled pure Lt-B (c), wildtype seed extract (wt) and seed extract from a transgenic line generated with
PGN7101 (Lt-B) size-separated by SDS-PAGE and incubated with Lt-B
antibody. nb, not boiled; b, boiled; p, pentamer; m, monomer.

as with E. coli-synthesized Lt-B, the pentamer dissociates following
a 1008C heat treatment for 5 min (Fig. 2B). The functionality of Lt-B
in transgenic corn was confirmed by a GM1 receptor binding ELISA.
The level of Lt-B detected in transgenic corn seed using this
functional assay was within 40% of the level detected using a
sandwich ELISA (data not shown). No Lt-B was detected in wildtype seed using either assay.
The level of Lt-B in corn is increased through targeting for secretion
and through plant breeding. Lt-B was quantified in transgenic T1
seed from multiple plants derived from 11 PGN7101 transformation
events and 16 PGN8957 events using a sandwich ELISA.
Considering the highest level of expression observed in a single
seed from multiple plants derived from each event, there was a wide
range in the expression level between PGN7101 events of from 0.013
to 1.8% of total soluble protein (TSP). However, only one of the
PGN8957 events gave a detectable level of expression, and even
then only at 0.0005% TSP. Thus, Lt-B accumulated at an over
3000-fold higher level in corn seed when it was targeted for secretion
(Fig. 3).
The highest expressing lines of Lt-B corn derived from PGN7101
and PGN8957 were backcrossed through two and one further
generation(s), respectively, into elite inbred lines. Breeding
improved the agronomic quality of the transgenic plants and also
further boosted accumulation of Lt-B in the seed by from 1.9- to
2.6-fold per generation (Fig. 3). In the highest expressing example of
T3 seed carrying the extracellular targeted version of Lt-B,
accumulation is elevated to 9.2% TSP in a single seed, an
approximately five-fold improvement over expression in T1 seed.
Lt-B expressed in corn is stabilized to heat, allowing extrusion
processing of seed. Purified Lt-B in solution dissociates from
pentamer to monomer if heated at 1008C for 5 min (Hirst et al.,
1983). This is also the case for Lt-B present in a soluble protein

14

STREATFIELD ET AL.

transgenic corn (Fig. 4A). Similarly, less severe temperatures of 658C
or 858C for longer periods of up to 4 or 2 h, respectively, do not affect
the stability of the pentamer, although an 858C treatment for 4 h does
result in a large proportion of the pentamer dissociating to monomer
(Fig. 4B). By contrast, an 858C treatment for 2 h completely
dissociates pure Lt-B pentamer spiked onto wild-type corn material
(Fig. 4C). It should be noted that there is some variation in the
monomer to pentamer ratio in different batches of Lt-B corn seed
(compare Fig. 2B and the unheated seed extract in Fig. 4B).
However, the pentamer form is always observed in untreated
extracts.
Given the improved heat stability of Lt-B in intact corn seed over
pure Lt-B, whole kernels of a bulk grain sample of T3 seed
expressing the extracellular targeted version of Lt-B were processed
by extrusion to produce a corn puff-type product. This process
included brief heat and pressure treatments of up to 1708C and
7  105 kg m22 , respectively, but the Lt-B pentamer did not
dissociate to monomer (Fig. 4D).
Discussion

FI G . 3. Expression levels of Lt-B achieved in corn lines transformed with
PGN7101 and PGN8957. A, The highest level of Lt-B as a percentage of total
soluble protein (TSP) in a single seed transformed with PGN7101
(extracellular targeted Lt-B) determined for T1 seed from plants initiated in
tissue culture and for T2 and T3 seed from plants grown in the field. B, The
highest level of Lt-B in a single seed transformed with PGN8957 (cytoplasmic
Lt-B) determined for T1 seed from plants initiated in tissue culture and for T2
seed from plants grown in the field. The 95% confidence levels are shown for
the mean values. A sandwich ELISA was used to determine expression levels.

extract of transgenic Lt-B corn generated with PGN7101 (Fig. 2B).
However, while in the intact matrix of the corn seed, Lt-B pentamer
is much more resistant to heat. Treatments of up to 1208C for up to
4 min do not greatly affect the ratio of pentamer to monomer in

Oral vaccines, and especially edible vaccines, are an attractive
means of vaccination for reasons of cost and the likely induction of
both mucosal and systemic immune responses. Subunit vaccines,
consisting of specific antigens of pathogenic organisms, are
generally preferable from a safety perspective to live attenuated or
inactivated vaccines. Thus, edible subunit vaccines are particularly
promising. However, a concern with edible subunit vaccines is the
likely instability of the chosen antigen in the stomach and intestine.
Associated with this, the dose actually delivered to the gut may be
much smaller than the administered dose and for any given
administered dose the actual delivered dose may vary considerably
between subjects. Efforts to improve antigen stability in the gut have
focused on delivery vehicles and include the use of recombinant
attenuated strains of Salmonella (Van de Verg et al., 1990) and
Vibrio cholerae (Ryan et al., 1997), the micro-encapsulation of
antigens in biodegradable polymers (Eldridge et al., 1991),
liposomes (Jackson et al., 1990) or proteosomes (Mallett et al.,
1995) and the expression of antigens in transgenic plants (Arakawa
et al., 1998; Mason et al., 1998). In addition to being stable in the
gut, the chosen antigen must also be stable under conditions of
storage in the encapsulation vehicle prior to delivery.
Here we show that Lt-B produced in corn seed is structurally
intact, with pentamer and monomer being detected. The presence of
pentamer in the seed indicates that functional Lt-B is synthesized,
since it is the pentameric form of Lt-B that has a high affinity for the
GM1 ganglioside receptor (Tsuji et al., 1995) and receptor binding is
necessary for the immunogenicity, adjuvanticity and toxicity of Lt
holotoxin (Nashar et al., 1996; Guidry et al., 1997). Indeed, the Lt-B
produced in corn is functional at binding the GM1 receptor.
Furthermore, corn-expressed Lt-B pentamer is stabilized to heat
relative to the molecule in solution. This increased heat stability of
the seed-encapsulated protein makes possible various cornprocessing alternatives that use heat to gelatinize starch, so
rendering the corn material palatable. Indeed, we demonstrate that
Lt-B pentamer expressed in corn seed is sufficiently stabilized to
allow a corn-processing procedure involving temperatures of up to
1708C and pressures of up to 7  105 kg m22 . In addition, Lt-B
pentamer is stable in corn seed for at least 6 mo. when stored at room

AN EDIBLE VACCINE PRODUCED IN CORN

15

FIG . 4. Western (immunoblot) analysis of protein prepared from corn transformed with PGN7101 following heat treatments or extrusion
processing. A, PGN7101 Lt-B seeds were ground up, brought to 30% moisture and incubated for 1, 2, or 4 min at 1208C. Soluble protein
extracts were prepared, size-separated by SDS-PAGE, and incubated with Lt-B antibody. B, PGN7101 Lt-B seeds were ground up, brought
to 30% moisture and untreated or incubated for 15, 30, 60, 120, or 240 min at 65 or 858C. Soluble protein extracts were prepared, sizeseparated by SDS-PAGE and incubated with Lt-B antibody. C, Wild-type and PGN7101 Lt-B seed were separately ground up and brought
to 30% moisture. Pure Lt-B was added to the wild-type seed material. The resulting wild-type plus pure Lt-B control (c) and PGN7101 (LtB) samples were incubated for 2 h at 228C or 858C. Soluble protein extracts were prepared, size-separated by SDS-PAGE and incubated
with Lt-B antibody. D, PGN7101 seed extract (seed) and PGN7101 corn puff extract (puff) were size-separated by SDS-polyacrylamide gel
electrophoresis and incubated with Lt-B antibody. b, Boiled; nb, not boiled; p, pentamer; m, monomer.

temperature (data not shown). The stability of corn-expressed Lt-B in
the gut is implied from animal feeding studies. Mice fed Lt-B corn
produce Lt-B-specific Ig in serum and IgA at the mucosal surface of
the gut, and show protection against Lt holotoxin in a gut-swelling
assay (Streatfield et al., 2001).
For edible vaccines based on plants, a further critical aspect is to
limit the size of the dose that a subject has to consume. This

generally requires a high level of expression of the candidate antigen
in target plant tissue. Corn seed has been demonstrated to express
foreign proteins at over 2% TSP when the gene encoding the protein
is codon-optimized for expression in maize, is under the control of a
strong promoter and is targeted to the cell surface (Hood et al.,
1997). The most favorable subcellular location for the expression of
a particular protein is likely to be dependent on the properties of the

16

STREATFIELD ET AL.

protein. In the case of Lt-B expressed in tobacco and potato,
retention of the protein in the endoplasmic reticulum results in
three- to four-fold increased accumulation of the protein over cell
surface-targeted versions (Haq et al., 1995). In corn, we have now
demonstrated that targeting to the cell surface results in a level of
expression of up to almost 2% TSP in T1 seed, over 3000-fold higher
than the level obtained with cytoplasmic Lt-B. Furthermore,
multiple rounds of breeding have been shown to boost and also to
stabilize the level of expression of a heterologous protein (Zhong
et al., 1999). By repeatedly breeding from the highest expressing
lines, those lines with more variable and reduced expression, the
latter possibly silenced, are selected against. Here we found that the
expression of Lt-B was increased five-fold through two rounds of
selectively breeding from high expressing lines and concomitantly
introducing the transgene into agronomically superior germplasm.
This breeding program both improved the quality of the grain
harvested and increased the maximum level of expression achieved.
By combining the strategies of using a strong promoter, targeting the
foreign protein for secretion and plant breeding, we reduced the size
of a typical dose for a human subject, harboring 1 mg of Lt-B, to 38 g
of a whole corn kernel product. This quantity compares favorably
with Lt-B potatoes, in which a 1 mg dose of Lt-B corresponds to
about 100 g of tuber (Tacket et al., 1998). Furthermore, given that
the expression level of Lt-B is at 0.5 –1% TSP in the bulk grain
sample of T3 seed used for extrusion processing, it is very probable
that the dose size could be reduced at least 10-fold by simply
continuing the plant breeding program with the highest expressing
T3 seed (9.2% TSP) and then using a bulk grain sample derived from
this for extrusion processing.
The favorable economics of administering edible vaccines make
them attractive as preventive healthcare treatments, especially in
developing countries. In particular, expressing specific antigens in
easily manipulated crops holds great promise for developing
practical and commercially viable vaccines. Corn is an excellent
expression system candidate based on the level of expression that
can be achieved, the options for downstream processing and the
long-term stability of proteins in the dry seed.

Acknowledgments
We thank Mian Riaz and Mark Barron (Texas A&M University) for
extrusion processing of corn.

References
Arakawa, T.; Chong, D. K. X.; Langridge, H. R. Efficacy of a food plant-based
oral cholera toxin B subunit vaccine. Nat. Biotechnol. 16:292–297;
1998.
Black, R. E. The epidemiology of cholera and enterotoxigenic E. coli
diarrheal disease. In: Holmgren, J., Lindberg, A., Mollby, R., eds.
Development of vaccines and drugs against diarrhea. Stockholm:11th
Nobel Conference; 1986:23–32.
Black, R. E. Epidemiology of traveller’s diarrhea and relative importance of
various pathogens. Rev. Infect. Dis. 12:S73–S79; 1990.
Bradford, M. M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem. 72:248–254; 1976.
Chatterjee, M.; Sparvoli, S.; Edmunds, C.; Garosi, P.; Findlay, K.; Martin, C.
DAG, a gene required for chloroplast differentiation and palisade
development in Antirrhinum majus. EMBO J. 15:4194–4207; 1996.

Cramer, C. L.; Boothe, J. G.; Oishi, K. K. Transgenic plants for therapeutic
proteins: linking upstream and downstream strategies. Curr. Top.
Microbiol. Immunol. 240:95– 118; 1999.
Daniell, H.; Streatfield, S. J.; Wycoff, K. Medical molecular farming:
production of antibodies, biopharmaceuticals and edible vaccines in
plants. Trends Plant Sci. 6:219– 226; 2001.
Eldridge, J. H.; Staas, J. K.; Meulbroek, J. A.; Tice, T. R.; Gilley, R. M.
Biodegradable and biocompatible poly(D L -lactide-co-glycolide)
microspheres as an adjuvant for staphylococcal enterotoxin B toxoid
which enhance the level of toxin-neutralizing antibodies. Infect.
Immun. 59:2978–2985; 1991.
Giddings, G.; Allison, G.; Brooks, D.; Carter, A. Transgenic plants as factories
for biopharmaceuticals. Nat. Biotechnol. 18:1151–1155; 2000.
Guidry, J. J.; Cardenas, L.; Cheng, E.; Clements, J. D. Role of receptor
binding in toxicity, immunogenicity, and adjuvanticity of Escherichia
coli heat-labile enterotoxin. Infect. Immun. 65:4943–4950; 1997.
Haq, T. A.; Mason, H. S.; Clements, J. D.; Arntzen, C. J. Oral immunization
with a recombinant bacterial antigen produced in transgenic plants.
Science 268:714– 716; 1995.
Hirst, T. R.; Hardy, S. J. S.; Randall, L. L. Assembly in vivo of enterotoxin
from Escherichia coli: formation of the B subunit oligomer.
J. Bacteriol. 153:21–26; 1983.
Hood, E. E.; Witcher, D. R.; Maddock, S.; Meyer, T.; Baszczynski, C.; Bailey,
M.; Flynn, P.; Register, J.; Marshall, L.; Bond, D.; Kulisek, E.;
Kusnadi, A.; Evangelista, R.; Nikolov, Z.; Wooge, C.; Mehigh, R. J.;
Hernan, R.; Kappel, W. K.; Ritland, D.; Li, C.-P.; Howard, J. A.
Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and
purification. Mol. Breed. 3:291–306; 1997.
Ishida, Y.; Saito, H.; Ohta, S.; Hiei, Y.; Komari, T.; Kumashiro, T. High
efficiency transformation of maize (Zea mays L.) mediated by
Agrobacterium tumefaciens. Nat. Biotechnol. 14:745–750; 1996.
Jackson, S.; Mestecky, J.; Childers, N. K.; Michalek, S. M. Liposomes
containing anti-idiotypic antibodies: an oral vaccine to induce
protective secretory immune responses specific for pathogens of
mucosal surfaces. Infect. Immun. 58:1932–1936; 1990.
Leong, J.; Vinal, A. C.; Dallas, W. S. Nucleotide sequence comparison
between heat-labile toxin B-subunit cistrons from Escherichia coli of
human and porcine origin. Infect. Immun. 48:73–77; 1985.
Mallett, C. P.; Hale, T. L.; Kaminski, R. W.; Larsen, T.; Orr, N.; Cohen, D.;
Lowell, G. H. Intranasal or intragastric immunization with
proteosome–Shigella lipopolysaccharide vaccines protects against
lethal pneumonia in a murine model of Shigella infection. Infect.
Immun. 63:2382–2386; 1995.
Mason, H. S.; Arntzen, C. J. Transgenic plants as vaccine production systems.
Trends Biotechnol. 13:388–392; 1995.
Mason, H. S.; Haq, T. A.; Clements, J. D.; Arntzen, C. J. Edible vaccine
protects mice against Escherichia coli heat-labile enterotoxin (LT):
potatoes expressing a synthetic LT-B gene. Vaccine 13:1336–1343;
1998.
Nashar, T. O.; Webb, H. M.; Eaglestone, S.; Williams, N. A.; Hirst, T. R.
Potent immunogenicity of the B subunits of Escherichia coli heatlabile enterotoxin: receptor binding is essential and induces
differential modulation of lymphocyte subsets. Proc. Natl Acad. Sci.
USA 93:226–230; 1996.
Ryan, E. T.; Butterton, J. R.; Smith, R. N.; Carroll, P. A.; Crean, T. I.;
Calderwood, S. B. Protective immunity against Clostridium difficile
toxin A induced by oral immunization with a live, attenuated Vibrio
cholerae vector strain. Infect. Immun. 65:2941–2949; 1997.
Streatfield, S. J.; Jilka, J. M.; Hood, E. E.; Turner, D. D.; Bailey, M. R.; Mayor,
J. M.; Woodard, S. L.; Beifuss, K.; Horn, M. E.; Delaney, D. E.;
Tizard, I. R.; Howard, J. A. Plant-based vaccines: unique advantages.
Vaccine 19:2742–2748; 2001.
Svennerhom, A.-M.; Holmgren, J. Identification of Escherichia coli heatlabile enterotoxin by means of a ganglioside immunosorbent assay
(GM1-ELISA) procedure. Curr. Microbiol. 1:19–23; 1978.
Svennerholm, A.-M.; Holmgren, J. Oral B-subunit whole-cell vaccines
against cholera and enterotoxigenic Escherichia coli diarrhea. In:
Ala’Aldeen, D. A. A., Hormaeche, C. E., eds. Molecular and clinical
aspects of bacterial vaccine development. Chichester: Wiley;
1995:205–232.
Tacket, C. O.; Mason, H. S.; Losonsky, G.; Clements, J. D.; Levine, M. M.;

AN EDIBLE VACCINE PRODUCED IN CORN
Arntzen, C. J. Immunogenicity in humans of a recombinant bacterial
antigen delivered in a transgenic potato. Nat. Med. 4:607–609; 1998.
Tsuji, T.; Watanabe, K.; Miyama, A. Monomer of the B subunit of heat-labile
enterotoxin from enterotoxigenic Escherichia coli has little ability to
bind to GM1 ganglioside compared to its coligenoid. Microbiol.
Immunol. 39:817–819; 1995.
Van de Verg, L.; Herrington, D. A.; Murphy, J. R.; Wasserman, S. S.; Formal,
S. B.; Levine, M. M. Specific immunoglobulin A-secreting cells in
peripheral blood of humans following oral immunization with a
bivalent Salmonella typhi–Shigella sonnei vaccine or infection by
pathogenic S. sonnei. Infect. Immun. 58:2002–2004; 1990.

17

Wiedermann, G.; Kollaritsch, H.; Kundi, M.; Svennerholm, A.-M.; Bjare, U.
Double-blind, randomized, placebo controlled pilot study evaluating
efficacy and reactogenicity of an oral ETEC B-subunit-inactivated
whole cell vaccine against travelers’ diarrhea (preliminary report).
J. Travel. Med. 7:27–29; 2000.
Zhong, G.-Y.; Peterson, D.; Delaney, D. E.; Bailey, M.; Witcher, D. R.;
Register, J. C. (III); Bond, D.; Li, C.-P.; Marshall, L.; Kulisek, E.;
Ritland, D.; Meyer, T.; Hood, E. E.; Howard, J. A. Commercial
production of aprotinin in transgenic maize seeds. Mol. Breed.
5:345–356; 1999.