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Issue 1, June 2001
Biological & Biomedical Sciences
A Review of the Progression of Transgenic Plants Used to Produce Plantibodies
For Human Usage
Elisa Ferrante and David Simpson
University of Virginia
Advisor: Timothy C. Scott, Ph.D.
University of Virginia
Abstract
Antibody production
in plants has acquired significance as an emerging system for the
production of many recombinant proteins, particularly including
those intended for therapeutic purposes. The use of transgenic plants
would not have been possible without important advances in genetics
and medical engineering fields. Such advances include the sequencing
of DNA, the discovery of restriction enzymes and various evolving
techniques of molecular biology. These developments, combined with
the recognition that plants can be used as a human antibody production
system, have stimulated much research and discoveries. This article
presents a general review on the subject, discussing successful
accomplishments in research during the past years, the several applications
of plant-produced substances and the advantages and disadvantages
of these methods of production. Comments on methods of suppression
and past and current research are also included, with emphasis on
the use of plantibodies in oral vaccines for medical purposes.
Introduction
There was a time
not too long ago when most medicinal compounds came from plants:
the potent heart stimulant digitalis from foxglove, for example,
and opium from poppy plant. But beginning about 50 years ago, chemistry
took over from botany, with most new drugs being artificially produced
in pharmaceutical labs (Moffat, 1995). Nowadays, one of the most
promising methods of producing proteins and other medicinal substances,
such as antibodies and vaccines, is the use of transgenic plants.
A transgenic plant contains a gene or genes that has been artificially
inserted. The inserted gene sequence, known as the transgene,
may come from an unrelated plant, or from a completely different
species. One of the purposes of inserting a combination of genes
in a plant is to make it as useful and productive as possible. This
process provides advantages such as higher yield, improved quality,
pest or disease resistance, and tolerance to heat, cold and drought.
However, transgenic plants can also be produced in such a way that
they express foreign proteins with industrial or pharmaceutical
value (see Tables 1 and 2). Transgenic plants represent an economical
alternative to fermentation-based production systems. Plant-made
vaccines or antibodies (plantibodies) are especially striking,
as plants are free of human diseases, thus reducing screening costs
for viruses and bacterial toxins (Herbers et al. 1999).
The first transgenic plants were reported in 1983. Since then, many
recombinant proteins have been expressed in several important agronomic
species of plants including tobacco, corn, tomato, potato, banana,
alfalfa and canola (Hammond et al. 1999). The choice of plant
system was initially driven by convenience and the need to evaluate
genetic constructs quickly. For this reason, tobacco plants were
generally used, however, potatoes and bananas were considered for
the purpose of acquiring a delivery system of vaccines for humans.
Foreign gene expression in plants can be accomplished either by
stable integration of foreign DNA, which results in transformation
of the nuclear genome, or by transient expression using modified
plant viruses (Figure 1). Stable genomic integration is accomplished
by introducing foreign DNA in the plant either by Agro bacterium
T-DNA vectors or by direct means. The integration method has the
advantage of permitting large scale cloning, maintenance of selected
high-expressing genes and the ability to sexually cross transgenes
to obtain multiple proteins expressed in the same plant (Tacket
et al. 1998). On the other hand, transient expression using
viral vectors is harder to initiate, because the viral vector must
be inoculated into individual host plants, but gives a greater yield
of protein.
Advantages and Disadvantages
Recombinant protein
production using transgenic plants as bioreactors is likely to be
more economical than alternative systems, especially for large-scale
needs. Factors in favor of plant systems as sources of animal derived
proteins, compared with other conventional methods, include:
- The
potential for large-scale, low-cost biomass production using agriculture.
- Low
risk of product contamination by mammalian viruses, blood-borne
pathogens, oncogenes and bacterial toxins.
- The
capacity of plant cells to correctly fold and assemble, not only
antibody fragments and single chain peptides, but also full-length
multimeric proteins.
-
Low downstream processing requirements for proteins administered
orally.
-
Elimination of the purification requirement when the plant containing
the recombinant proteins is edible, such as potatoes.
-
The ability to introduce new or multiple transgenes by sexual
crossing of plants.
-
The avoidance of ethical problems associated with transgenic animals.
-
Formulated in seeds, plant-made enzymes have been found to be
an extremely convenient method for reducing storage and shipping
costs, for an indefinite amount of time, under ambient conditions.
-
Production size is flexible and easily adjustable to the needs
of changing markets.
Plants
are also capable of synthesizing and assembling virtually any kind
of antibody molecule, ranging from the smallest antigen-binding domains
and fragments, to full length, and even multimeric antibodies.
There are, however, potential issues of concern for plant protein
production:
- Allergic
reactions to plant protein glycans and other plant antigens.
- Plant
and product contamination by mycotoxins, pesticides, herbicides
and endogenous metabolites.
-
Regulatory uncertainty, particularly for proteins requiring approval
for human drug use (Doran 1999).
Applications
The production of foreign proteins in plants has become an attractive
alternative to conventional production systems for pharmaceutical
polypeptides. Potential proteins produced by this method include
cytokines, hormones, enzymes, epidermal growth factors, interferons,
human protein C, and pharmaceutical foodstuff considered for oral
immunization.
The capacity of plants to produce different classes of proteins
with pharmaceutical value and the need for new technology for the
production and delivery of inexpensive vaccines has led to the use
of transgenic plants (see Table 1). In addition, large amounts of
antibodies can be produced at relatively low cost, using agriculture
instead of sophisticated and expensive cell culture-based expression
systems. Oral vaccines, as opposed to parenteral vaccines, offer
the hope of more convenient immunization strategies and more practical
means for implementing universal vaccination programs throughout
the world.
The pathogens responsible for the greatest burden of human disease
make initial contact with the human host at mucosal sites in the
respiratory tract, gastrointestinal tract or genital tract. Therefore,
stimulation of immune responses of suitable strength and quality
to protect against illness is particularly desirable at these sites
(Tacket et al. 1999).
The best way of achieving mucosal immunization is to apply the vaccine
directly to the mucosal surface, inducing systemic and cellular
immune responses as well as local immune responses. Mucosal vaccines
have been sought because of the potential for stronger immune responses
directed at the initial site of interaction between the pathogen
and host. Many infectious agents colonize or invade epithelial membranes.
These include bacteria and viruses that are transmitted in contaminated
food, water or by sexual contact. Transgenic plants that express
antigens in their edible tissue might be used as an inexpensive
oral-vaccine production and delivery system (Mason et al.
1995). Therefore, immunization might be possible through consumption
of an "edible vaccine" to provide passive immunization. Also, it
may be possible to use genetically engineered plants and plant viruses
to produce vaccines against several human diseases from tooth decay
to life-threatening infections such as diphtheria, cholera and AIDS
(Moffat, 1995).
Several issues will still have to be resolved before this intriguing
idea can become a reality. Some of the proteins used in experiments
may be extraordinarily potent inducers of immune responses. On the
other hand, other immunizing proteins may not work as well when
taken orally. In fact, they can have the opposite effect, because
many proteins in a diet induce tolerance, making the immune system
less able to mount a response against them. In addition, some other
compounds in plants may compromise the ability of the vaccine protein
to induce immunity. The food containing it must be palatable and
some foods need to be heated before ingested, which could possibly
cause the vaccine protein to denature, reducing or eliminating its
ability to elicit immunity.
Oral vaccines, whether living or nonliving, must be protected during
passage through the hostile environment of the stomach and intestine
to the sites where immune stimulation occurs (Tacket et al.
1999). In recent years, a variety of delivery systems have been
developed for presenting nonliving antigens to mucosal surfaces,
which will allow these antigens to persist and survive in the hostile
gastric and enteric environments. These include polylactide/polyglycolide,
microspheres, liposomes, proteosomes, cochleates, virus-like particles,
and immune-stimulating complexes.
One of the most promising methods, however, is the production of
antigens in the plants themselves, which assemble into ordered structures
such as virus-like particles. This gives the hope that they will
be more resistant to digestion and more likely to reach the gut-associated
lymphoid tissue.
Methods of Production
Conventional Method
Pharmaceutical and therapeutic antibodies synthesized in plants
can be produced in a variety of ways. Conventional methods use stable
transformation and transient expression to introduce new genes into
a host cell. Once DNA from the transformant host cell is isolated
and purified, it can be injected into the embryo of a maturing plant.
The plant can then propagate in an open field allowing for large-scale
production of antibodies. As mentioned before, however, purification
of these proteins is generally long and tedious. Upon isolation
of the antibody, several proteins, organic molecules, glycan and
herbicides must also be isolated, leading to a complex purification
process (Kusnadi et al. 1997).
In Vitro Cell Tissue Cultures
Plant tissue cultures offer an economically favorable method for
producing antibodies from plants. Using this approach, plant cells
in differentiated or dedifferentiated states are grown in a nutrient
medium in bioreactors under controlled conditions, with foreign
proteins harvested from either the biomass or culture liquid or
a combination of both (Doran 1999).
This method of production of human antibodies is not suitable for
the production of edible vaccines (simply because the antibody is
produced in a cell culture and not in a fruit or vegetable) but
offers many advantages to the conventional methods of extracting
and purifying a protein from a live plant. First, plant tissue cultures
offer larger amounts of proteins in shorter amounts of time (Doran
1999). This is because the bioreactors provide a much more controlled
and reproducible environment than an open field.
The advantages of this method allow desired antibodies to be produced,
purified, and transferred to the consumer in a minimal amount of
time. Also, purification of the proteins becomes easier (Doran 1999).
In vitro cell cultures contain fewer biological proteins or molecules
(along with herbicides and pesticides) than open field plants or
bacterial/yeast cell cultures, which may contaminate the product.
Furthermore, plant cells and organs can propagate indefinitely in
tissue cultures (Doran 1999). Therefore, sexual reproduction is
not needed to ensure the lifespan of the species. Without sexual
reproduction, transgene stability is increased because of the absence
of crossing over, segregation and recombination involved in sexual
reproduction.
Inducible promoters may offer a solution to the problems associated
with open field production of plantibodies. They would allow for
better efficiency in the production of plantibodies. They would
also provide the plant with a more stable mode of translation, leading
to transgene stability (Doran 1999).
Breeding and Sexual Crossing
In 1989, Hiatt was the first to use sexual crossing as a way to
produce a functional protein in plants (Hiatt et al. 1989).
In this experiment, transformation was used to introduce kappa-chains
of either light or heavy regions into tobacco plants. The same was
done with gamma-chains of either light or heavy regions (Whitelam
et al. 1994). Upon crossing one plant with kappa-chains and
another plant with gamma-chains, an antibody was produced that expressed
both chains (Hiatt et al. 1989). This experiment provided
an ingenious way to produce antibodies in plants without the need
for a double transformation.
Transgenic seeds
As mentioned above, using green plant tissue as a bioreactor is
a great method of producing antibodies. Also discussed are certain
limitations to this process. Further restrictions are gathered when
plants are used as a storage system. Plants cannot store antibodies
for an extended period of time. This is because certain proteases
degrade the protein piece by piece.
Ulrike Fieldler and Udo Conrad showed that seeds of transgenic tobacco
could be used for high-level production and long-term storage of
antibodies. In their experiments, functionally active single chain
Fv (scFv) accumulated up to 0.67% of the total soluble seed protein.
In addition, storage of ripe transgenic tobacco seeds for one year
at room temperature showed no loss of scFv or its antigen-binding
activity (Fieldler et al. 1995). Seeds contain a low level
of proteases that allows proteins to be stored without degradation
(Kusnadi et al. 1997). This experiment suggests that seeds
can be used as bioreactors and as natural storage organs.
Targeting and Compartmentalizing
In some cases, antibodies can be tagged with a small peptide sequence.
This peptide sequence allows plant cells to direct an antibody or
protein to specific organelles and compartments after processing.
Compartmentalizing to easily isolated organelles provides a less
complicated purification procedure (Kusnadi et al. 1997).
Targeting also allows antibodies to be protected from proteases
that exist in the cytoplasm of cells. Targeting, however, has to
be specifically controlled. This involves proper cleavage of the
targeting sequence. If incomplete processing occurs, the quality
and amount of protein is lowered (Kusnadi et al. 1997).
Methods of Suppression
Gene Silencing
Recent studies with transgenic production of antibodies have shown
that the transgene involved can undergo inactivation; this process
is termed gene silencing. Gene silencing has been observed to occur
where there exist multiple copy integrations at one or more sites,
different base-composition between rDNA and the integration site,
detrimental effects of sequences adjacent to the rDNA integration
site and over-expression effects (Kusnadi et al. 1997).
There are certain ways to prevent the occurrence of inactivation:
Screening/selection for plants with single copy rDNA; developing
methods for single-copy integration; avoiding repetitive homologous
sequences; flanking rDNA with scaffold attachment regions; selection/screening
for stable rDNA expression; and developing site-specific recombination
systems (Kusnadi et al. 1997).
Oral Tolerance
When vaccines are taken orally, either with potato tubers or with
bananas, the intestinal immune response to these food-antigens is
called tolerance. Oral tolerance is an active immunologic response,
which prevents the development of responses to the many antigens
ingested by a host (Tacket et al. 1999). Upon intake, the
oral antigens are taken up by M cells. These M cells present antigens
to specific suppressor T cells, which secrete cytokines that allow
for the propagation of specific antibodies. Tolerance is dose- schedule-,
and antigen-specific (Tacket et al. 1999). Therefore, an
antigen that is orally administered and is tolerant will show no
response, because the expression of that antigen has been suppressed.
When synthesizing a protein from a transgenic plant, oral tolerance
must be taken into consideration.
Past and Current Research
Creating SIgA
Secretory immunoglobulin A (SIgA) is the most abundant form of immunoglobulin
in mucosal secretions (Julian et al. 1994). It is composed
of two monomeric IgA antibody units, a small J-chain and a secretory
component (Figure 2). In an experiment, which four plants expressing
each of the polypeptides were sexually crossed, it was found that
a functional SIgA antibody could be produced (Julian et al.
1994). In this experiment, the functionality of the protein was
studied by enzyme linked immunosorbent assay (ELISA). SA I/II was
used as the antigen to be recognized on the transgenically produced
SIgA antibodies (Julian et al. 1995). SA I/II is a cell surface
protein of Streptococcus mutans (Julian et al. 1995),
which can cause dental caries in humans. This experiment provided
insight that allowed researchers to conclude that the nature of
association in plants is similar to that in mammals and therefore
antibodies produced in plants could be used in mammals. CaroRxTM:
An anti- S. mutans SigA CaroRxTM is a clinically
advanced SIgA plantibody that protects humans from dental caries
(Larrick et al. 1998). In a preliminary study of CaroRxTM,
anti-S. mutans antibodies were orally administered to 84
human subjects. Upon the application of SA I/II, monoclonal antibodies
prevented the colonization of artificially and naturally implanted
S. mutans (Larrick et al. 1998). In addition, protection
from recolonization (with just 3 weeks of application) lasted for
two years.
Production of an Oral Cholera vaccine
From 1995 to 1998, extensive research was conducted in the usage
of potatoes as an antigen delivery system to initiate an immune
response (Arakawa et al. 1998, Haq et al. 1995). Of
particular interest was the CT or cholera toxin antigen. CT is composed
of an A subunit and B pentamer subunit. CTA is an enzymatically
active protein, which enters epithelial cells of the gut and causes
water loss from these cells. CTB is enzymatically inactive and binds
to Gm-gangliosides of epithelial cells, which allows for the intake
of CTA (Tacket et al. 1998).
In one experiment, Arakawa, Chong and Langridge used mice to study
the effects of a CT oral vaccine. The mice were fed transgenic potato
tissue containing thirty micrograms or CTB/gram (Arakawa et al.
1998). They were fed four times, on days zero, six, seventeen, and
twenty-four, and received a booster feeding on day sixty-five. Ten
mice were fed one gram of transgenic potato expressing CTB while
eight mice were fed three grams of transgenic potato expressing
CTB. In addition, five mice acted as a negative control and were
fed one gram of untransformed potato while eight mice were used
as a positive control and fed thirty micrograms of bacterial CTB
in sodium bicarbonate buffer (Arakawa et al. 1998). It was
observed that during the 70-day experiment, mice immunized with
thirty micrograms of bacterial CTB showed 55% protection (here protection
is defined as the percent of reduction in intestinal fluid accumulation).
In addition, there was 42% protection for mice immunized with one
gram of transgenic potato, and 62% protection for mice immunized
with three grams of transgenic potato (Arakawa et al. 1998).
From these experiments, it could be concluded that oral delivery
of antibodies was an effective way to treat certain diseases, at
least in animals. Similar experiments were done with LT-B or heat
labile enterotoxin (Aratzen, 1998), which showed similar results
(Haq et al. 1995).
Human studies with transgenic potato
The successful outcomes with the studies involving CTB and LT-B
in mice finally led to experimentation with human subjects. It was
hypothesized that a mucosal immune response would occur upon ingestion
of raw potato tubers expressing CTB or LT-B (Tacket et al.
1999). Fourteen human subjects were fed one hundred grams or transgenic
potato, fifty grams of transgenic potato, or fifty grams of wild
type potato. Each transgenic potato contained a variable amount
of LT-B that ranged from 3.7 to 15.7 micrograms per gram (Tacket
et al. 1999). This variability is a result of the different
promoters used in the experiment. During the experiment, 91% of
subjects who ingested transgenic potatoes developed IgG and anti-LT
antibodies. In addition, 55% of the subjects who ingested transgenic
potatoes developed a four-fold rise in IgA and anti-LT(Tacket et
al. 1999). The results of this experiment suggest that vaccines
from plants can be taken orally through some food substances. The
only problem is that humans normally do not eat raw potatoes. It
was shown however, that transgenic potatoes could be boiled for
3 minutes with only a 50% loss of LT-B (Tacket et al. 1999).
In addition, new studies are being carried out using fruits, such
as bananas, to deliver antibodies produced in plants.
Conclusion
The advancements made with transgenic plants have and will continue
to have a great impact on the lives of many. Transgenic plants offer
a new approach to producing and administering human antibodies. The
recent research with transgenic plants has played a captivating role
in providing edible vaccines, which are cheap and easy to administer.
The progression of transgenic plant technology now has allowed for
the progression of human life and other medicinal advancements. Hiatt
was the first to demonstrate that plants could produce human antibodies
and since that time, researchers have continued to build upon his
findings. Since 1983, progress in the field of antibody production
in plants has drastically increased, and it is projected that in the
near future, many of the necessary human antibodies will have an origin
as a plantibody.
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Journal of Young
Investigators. 2001. Volume Four.
Copyright © 2001 by Elisa Ferrante, David Simpson and JYI. All rights reserved.
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