Foods as Production and Delivery Vehicles for Human Vaccines

Review
Foods as Production and Delivery Vehicles for
Human Vaccines
Schuyler S. Korban, PhD, Sergei F. Krasnyanski, PhD, and Dennis E. Buetow, PhD
Department of Natural Resources & Environmental Sciences, 310 ERML (S.S.K., S.F.K.), Department of Molecular and
Integrative Physiology (D.E.B.), University of Illinois, Urbana, Illinois
Key words: food crops, transgenic plants, plant-based vaccines, oral edible vaccines
Vaccination is a great asset for eradication of infectious diseases in humans and animals. With the prevalence
of antibiotic resistant bacterial strains and an alarming increase in new and re-emerging pathogens, the need for
vaccination continues to be a high priority for mammalian diseases. In the last several years, a novel approach
for developing improved mucosal subunit vaccines has emerged by exploiting the use of genetically modified
plants. It has been demonstrated that plant-derived antigens are functionally similar to conventional vaccines and
can induce neutralizing antibodies in mammalian hosts. Using genetically engineered plants for the production
of immunogenic peptides also provides a new approach for the delivery of a plant-based subunit vaccine, i.e.,
oral delivery, provided these immunogenic peptides are expressed in an edible part of the plant, such as grain
or fruit. Thus, food crops can play a significant new role in promoting human health by serving as vehicles for
both production and delivery of vaccines.
Key teaching points:
Via genetic engineering and molecular biology, genes encoding immunogenic proteins of an infectious agent can be transferred into
the nuclear genome of a plant system such that the plant is capable of producing the desired immunogenic protein subunit vaccines.
The production of antigens in genetically engineered plants provides an inexpensive source of edible vaccines and, in turn,
increases the value of plants as novel sources of medicinal drugs.
Edible vaccines against cholera toxin B subunit, hepatitis B surface antigen, E. coli heat labile enterotoxin, and Norwalk virus
capsid protein have been developed and tested for efficacy in animal and human trials. Immunologic responses were provoked in
both mice and humans.
A plant-based oral subunit vaccine for the respiratory syncytial virus has been developed. This virus is a serious pathogen that
causes bronchiolitis and pneumonia-type diseases in all human age groups and is a leading cause of viral lower respiratory tract
illness in infants and children worldwide.
This new area of agriculture, referred to as “biopharming,” will provide new value to crops grown for the sole purpose of producing
and/or delivering biopharmaceutical or medicinal products.
INTRODUCTION
Typically, vaccines against human diseases have been composed
of killed or attenuated live organisms or those whose
host differs from the vaccinated species. A subunit vaccine
composed of one or more subunits of an antigenic protein from
a disease-causing organism also can be immunogenically protective.
Because of their relative ease of genetic manipulation
and rapid growth, genetically engineered mammalian and yeast
cells are the most widely used large-scale production systems
for recombinant proteins or subunit vaccines. These are usually
done in bioreactors or large fermentation equipment. In addition,
recombinant proteins over-expressed in these organisms
require extensive purification prior to use to remove host protein
and other compounds. Both the fermentation facility as
well as the purification necessary for recovering these
recombinant proteins add to the high cost of production of
these subunit vaccines. In addition, some vaccines (especially
Address reprint requests to: Schuyler S. Korban, PhD, Department of Natural Resources & Environmental Sciences, 310 ERML University of Illinois, Urbana, IL 61801.
E-mail: s-korban@uiuc.edu.
Journal of the American College of Nutrition, Vol. 21, No. 3, 212S–217S (2002)
Published by the American College of Nutrition
212S

parenteral vaccines) contain preservatives such as formaldehyde,
thiomersal (a mercury-based compound) and aluminum
phosphate [1].
Plants provide a very attractive alternative that can be scaled
up to a high production system for recombinant proteins
[2,3,4]. Developing transgenic plants that can express these
antigenic proteins will eliminate the need for fermentation
facilities and overcome the possible risks of contamination of
subunit vaccines by unknown mammalian pathogens that may
remain undetected in these cultures. Pathogens that infect
plants do not infect humans, whereas mammalian pathogens
can infect human and other animal populations. Moreover, to
increase production of a subunit vaccine in plants will only
require planting more acreage of these value-added plants. If an
“edible” plant is used to produce a vaccine, for example,
purification to remove host toxins should not be necessary.
Also, any required processing of an “edible vaccine,” in the
form of juice, powder or sauce, would be less complicated and
easier than purification.
In recent years, development of plant-based vaccines directed
at human and animal diseases [2–5] has opened up an
innovative and exciting opportunity for adding new high value
to food crops, thus increasing the uses and profitability of these
crops. With the tools of genetic engineering and molecular
biology, genes encoding immunogenic proteins of an infectious
agent are transferred into the nuclear genome of a plant system
via genetic transformation protocols, and these transgenic
plants are then capable of producing the desired immunogenic
protein subunit vaccines. The production of antigens in genetically
engineered plants provides an inexpensive source of
edible vaccines and, in turn, increases the value of plants as
novel sources of medicinal drugs. This new field of biological
science, referred to as molecular “biopharming” has received
much attention in the past decade and promises to become more
important in the next decade.
Background
Thus far, there are several reports on development of transgenic
plants that express antigenic proteins of pathogenic human
and animal organisms [2–4]. In early work, tobacco was
used as a model plant system for expression of antigenic
proteins, such as that for hepatitis B surface antigen (HBsAg)
[6]. This early study showed that the plant-derived protein had
similar buoyant density and antigenicity as do human- and
yeast-derived HBsAg, suggesting that the protein assumed typical
folding characteristics in the plant. This was followed by a
mice immunization study with tobacco-derived recombinant
HBsAg (rHBsAg) that demonstrated stimulation of T-cell proliferation.
In a later study, it was conclusively demonstrated
that B- and T-cell epitopes of HBsAg were preserved when the
antigen was expressed in transgenic tobacco [7]. In another
effort, constructs carrying the gene encoding the binding subunit
of Escherichia coli heat-labile enterotoxin (LT-B) were
Foods as Vehicles for Human Vaccines
introduced into tobacco and potato plants [8]. Heat labile enterotoxin
is produced by enterotoxigenic E. coli (ETEC), the
causal agent of an enteric disease, and also immunogenically
interacts with the cholera toxin of Vibrio cholerae. Thus, LT-B
is a candidate vaccine against both ETEC and cholera. Transgenic
plants expressing the LT-B protein or a modified LT-B
protein with a microsomal retention sequence have been developed.
The toxin protein was expressed in potato microtubers
and at much higher levels in plants containing the fusion
protein. Mice immunized by orally feeding with transgenic
potato tubers (expressing the recombinant antigen) developed
serum immunoglobulin G (IgG) and mucosal immunoglobulin
A (IgA) that were specific for LT-B [8].
The potato has since been used for production of antigenic
proteins against various pathogens and/or diseases. The cholera
toxin B subunit (CTB) against Vibrio cholerae was successfully
expressed in potato tubers and found to accumulate at
high enough levels that induced both mucosal and serum immune
responses in mice [9]. The potato was also used for the
production and delivery of the human insulin antigen [9].
Mason et al. [10] optimized the LT-B enterotoxin antigen and
expressed it in potato tubers as well. Later, Tacket et al. [11]
conducted the first human clinical study where they demonstrated
that humans given a plant-derived oral vaccine (fed raw
transgenic potato tubers carrying the recombinant LT-B antigen)
produced both serum IgG- and mucosal IgA-specific
antibodies in humans. The capsid protein of the Norwalk virus
was also expressed in potato tubers and found to be immunogenic
in test mice as well [12]. Again, a human clinical
trial was conducted by feeding 24 healthy adult volunteers
two or three doses of these potato tubers and found that 19
of 20 volunteers fed the transgenic potato (carrying the
capsid protein of the Norwalk virus) developed an immune
response, although the level of serum antibody increases was
reported to be modest [13].
In a recent study, Yu and Langridge [14] produced the first
multicomponent vaccine in potato against three enteric diseases
including cholera, rotavirus and ETEC. Although the amount of
the recombinant fusion proteins used in their immunological
studies was about 3.3 g/g potato tuber, mice fed these tubers
produced both serum and mucosal antibodies against the three
human diseases.
Among other food crops used for the development of plantbased
vaccines, both lupine and lettuce were used to express a
hepatitis B surface antigen (HbsAg) either in the pods or leaves,
respectively, and these tissues were found to be useful systems
for production and delivery of this antigen vaccine [15]. Soybean
was used for production of the glycoprotein B antibody of
the herpes simplex virus 2 (HSV-2) [16], while corn was used
for production of an LT-B subunit vaccine [17]. Recently,
Stöger et al. [18] expressed the single-chain Fv (ScFv) antibody
of the human carcinoembryonic antigen (CEA), a marker antigen
to diagnose tumor onset, in both rice and wheat grains.
While, Mor et al. [19] used tomato to express the human
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Foods as Vehicles for Human Vaccines
Fig. 1. Southern blot analysis of tomato lines. Lanes 1–5 correspond to
transformed tomato lines carrying the RSV-F transgene and showing a
single copy of the transgene; lane 6 corresponds to an untransformed
tomato plant (control), and lane 7 corresponds to the plasmid DNA
showing the RSV-F antigen.
acetylcholinesterase (AChE) that provides protection against
organophosphate poisoning.
DESCRIPTION OF SUBJECT
Vaccine for RSV
In our laboratory, we wanted to develop a plant-based oral
subunit vaccine for the respiratory syncytial virus (RSV), a
serious pathogen that causes bronchiolitis and pneumonia-type
diseases in all human-age groups. RSV is a leading cause of
viral lower respiratory tract illness in infants and children
worldwide and can lead to infant mortality. The United Nations
1992 Children’s Vaccine Initiative calls for the development of
an oral vaccine against RSV as no vaccine is available so far.
An oral vaccine is desirable for its ease of use. RSV infects
virtually all children worldwide and can cause symptomatic
infections throughout life. Risk factors for severe RSV disease
include congenital heart disease, bronchopulmonary anomalies,
immunodeficiency, prematurity, and age of less than six weeks
[20]. Several surveys of children hospitalized with RSV show
mortality rates of 0.1% to 2.5% [21]. Based on data available at
the Centers of Disease Control and Prevention, hospitalization
in the U.S. due to RSV alone is approximately 90,000 per year
with annual costs of hospitalization running close to $300
million. Approximately, 25% to 40% of infected infants, elderly
people and adults with immuno-compromised systems
develop symptoms of bronchiolitis or pneumonia.
RSV disease occurs throughout the world and is more
severe in underdeveloped countries where it results in increased
mortality. In adults, it usually takes the form of a “common
cold,” but can be more severe. In recent years, RSV infections
have been increasingly noted in nursing homes and in other
group settings serving the institutionalized elderly [22]. The
virus enters the human body mainly through the nose and eyes,
but also through the mouth [20]. In the northern hemisphere,
the yearly peak season for RSV infections occurs from December
through March and, in urban centers, the virus is detected
from the Fall through the Spring [20].
There is no consistently effective treatment available for
RSV infections, and these infections can occur repeatedly in the
same individual. A major difficulty in developing an RSV
vaccine that works via the serum immune system has been the
fact that natural infection confers, at most, only temporary
protection against reinfections [23]. Another problem is that the
mechanism by which even partial immunity to RSV develops is
not well understood [23].
Although our ultimate goal is to develop this RSV edible
oral vaccine in the apple, we elected to use the tomato first for
the production and delivery of such a vaccine. The tomato is
one of the most important vegetable crops of commercial
importance in the U.S. USDA figures report that tomato continues
to show an increase in planting acreage (fresh market
acreage for 1999 was up 15% over 1998 and 13% greater than
1997) and a rise in production (1999 production was 23%
higher than 1998 and 16% above 1997) (USDA Agricultural
Statistic Board, NASS, USDA, Sept. 1999). Developing new
uses for tomato (by creating a specialty crop) will expand the
Fig. 2. Fertile transgenic tomato plants carrying the RSV-F antigen
growing in the greenhouse.
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market for this important food crop and provide a competitive
advantage to U.S.-produced tomato specialty crops.
In our first set of experiments, we developed various plant
expression constructs carrying the RSV-F antigenic protein
gene. These constructs consisted of the universal constitutive
cauliflower mosaic virus (CaMV) 35S promoter along with
enhancer elements or leader sequences added to the RSV-F
protein gene. The CaMV 35S-driven gene constructs carrying
the antigenic F-protein were transformed into apple leaf protoplasts
to determine whether the F-protein could be correctly
processed in a plant cell. Using SDS-PAGE and Western analyses,
the correct size protein (68 kd) was found in transformed
apple protoplasts [24]. Upon immunoblotting with a monoclonal
antibody against the F-protein, it was confirmed that the F
protein was expressed in apple. Furthermore, the level of expression
of the F-protein increased in constructs carrying the
leader sequence AMV RNA4 and the P268 enhancer element
[24]. This suggested that we could successfully enhance the
level of expression of the F-protein in transgenic plants by
using these newly designed gene constructs developed in our
laboratory.
Then, gene constructs (one containing the constitutive
CaMV 35S promoter and the other containing the fruit-specific
E-8 promoter) carrying the antigenic F protein also have been
transferred into tomato using Agrobacterium-mediated transformation,
and transgenic tomato plants have been recovered.
The presence of the transgene was confirmed in 71 out of 74
tomato plants growing in the greenhouse using Southern blotting
(Fig. 1). The plants appeared normal and were not stunted
Foods as Vehicles for Human Vaccines
in growth. All plants were fertile (Fig. 2), and harvested fruit
was analyzed using ELISA. With the E-promoter, the F-protein
was expressed only in the fruit of all transgenic plants carrying
the F-antigenic protein gene. Variability in the expression level
of the F-protein among plants (even between and within each of
the two constructs) was observed, ranging from 9.0 to 32.5
g/g of fruit fresh weight (Fig. 3). Fruits from the highest
F-protein expressing plants (transgenic line #120 with 32.5
g/g of fruit fresh weight in plants transformed with the E-8
promoter construct) were fed to mice, and among 25 orally
immunized mice, 22 showed significant immune responses and
produced anti-RSV-F antibodies [25]. Pre-immunized and control
mice did not produce detectable anti-RSV-F antibodies.
This demonstrated successful oral immunization of mice and
showed the fruit-derived RSV-F was active as an oral immunogen.
T1 plants from the two highest F-protein expressing T0
lines were later grown in the greenhouse, and the expected 3:1
segregation ratio was observed for presence/absence of the
transgene (following Southern blot hybridization), suggesting
that the F-protein transgene is transmitted to its progeny and
that it is stably inherited in a Mendelian fashion in the progeny.
Upon analysis of the tomato fruit for the localization of the
antigenic RSV-F protein, it was found that the majority of the
antigenic protein is localized in the seed, while the pulp contained
only marginal levels of the antigen (Fig. 4). This suggested
that in order to deliver a high amount level of the RSV-F
antigen vaccine, the whole tomato fruit (seed and pulp) must be
homogenized and used for the delivery of the vaccine to insure
presence of high enough levels of the antigen.
Fig. 3. The level of recombinant RSV-F protein in fruit of different tomato plants. Tomato plants were transformed with a construct carrying the
RSV-F antigen gene driven by either the CaMV 35S promoter (plasmid pJSS-3) or the E-8 fruit-specific promoter (plasmid pJSS-4). Untransformed
tomato plant used as control.
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Foods as Vehicles for Human Vaccines
Fig. 4. Localization of the RSV-F antigen in a tomato fruit.
CONCLUSIONS
One of the advantages of oral vaccines is that they stimulate
production of mucosal antibodies more effectively than is the
usual case with injectable vaccines. The mucosal immune system
is known as the first line of defense against many disease
organisms, including RSV [2,4]. Eating fruit or drinking juice
of the transgenic tomato should induce the mucosal immune
system to produce antibodies against RSV. Communication
between the mucosal system and the serum system can be
expected to lead the latter also to produce immunoglobulins
against RSV. Since RSV can be repeatedly infectious throughout
life, the serum immune system, as noted earlier, does not
seem to be very protective against the virus. On the other hand,
repeated stimulation of the mucosal system before and/or during
the RSV infection season by an edible vaccine should
provide the appropriate protection. In addition, producing and
delivering this vaccine in tomato juice or apple juice will also
bypass the costs of hypodermic needles, and, in under-developed
and poor countries, this will also prevent hazards of
contamination of needles due to repeated use of needles from
one patient to another.
Plant-based vaccines will provide a new use for food crops
as these crops can then be grown for the sole purpose of
producing and/or delivering biopharmaceutical or medicinal
products. This leads to a new area of agriculture, now referred
to as “biopharming,” whereby agricultural crops with addedvalue
are grown in specialized areas and specifically used as
“factories” for production and delivery of edible vaccines
and/or other antimicrobial agents. Nevertheless, there are some
limitations to these recombinant antigens produced in plants
that should be overcome before this technology can make it to
the commercial sector. Among these limitations is the need to
achieve high enough yield of the recombinant antigen in the
plant tissue, and another limitation is dealing with the presence
of plant-specific glycans that might alter the properties of the
recombinant protein [26]. Research efforts to alleviate both of
these limitations are currently underway in various laboratories.
The use of foods as vehicles for production and delivery of
human vaccines is an exciting and novel field of biotechnology
and should pay dividends for both human health and the agricultural
sector in the near future.
ACKNOWLEDGMENTS
We wish to acknowledge the funding received from USDA-
NRI and Illinois Council for Food and Agriculture Research
(C-FAR) that allowed us to pursue the work described in this
review. We also like to acknowledge the research contributions
previously made by Dr. Leslie L. Domier (USDA-ARS, Department
of Crop Science), Dr. Jagdeep Sandhu (Department of
Crop Science), and Dr. Mark Osadjan.
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Received February 5, 2002
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