Plants, edible vaccine producing bioreactors
Through the centuries plants have proved to be extremely flexible
organisms that adjust to man’s requirements both for agricultural
and ornamental purposes. About twenty years ago early studies
on the recombinant DNA method also found the possibility of introducing
genes resulting from other vegetable species and of bacterial
or animal origin into vegetable tissue to ensure their expression
in this context.
This discovery has opened new horizons concerning the extensive
number of applications, which could be proposed. Early studies
naturally focused on improving plants’ defence against pathogens
and infesting organisms. To this end resistant genes of bacterial
or fungal origin or isolated from other plants naturally endowed
with the desired defence have been introduced. It takes little
to move from this stage to imagining the possibility of making
plants produce other protein types, which are not necessarily
to its advantage. In particular, the possibility of producing
molecules for health purposes (i.e. antigens for the production
of vaccines, antibodies or proteins with a pharmacological function)
have given rise to considerable interest
How to Produce Innovative Vaccines
The three main categories of vaccines available today are:
Attenuated vaccines, formed by pathogens with reduced virulence;
Inactive vaccines, formed by microrganisms, whose virulence has
been neutralized;
Subunit vaccines, formed by purified elements of microrganisms.
In this case the vaccine will only hold part (formed by one or
more proteins) of the original organism, which will be adequate
to trigger an immune reaction;
Recombinant vaccines. This last category is based on recognizing
antigen molecules, which can induce an immune response, on isolating
the corresponding gene and on producing the vaccine in a heterologous
expression system, which has so far been a microganism or animal
tissue.
The best method of expression will naturally be the one that ensures
a safe final product, optimal biological activity and low production
costs. Antigen expression in mammal cells has the advantage of
giving suitable products, but the procedure is expensive and not
free of danger (possible presence of pathogens derived from the
animal used). The use of microrganisms as a method of expression
enables a more extensive production but it also has limits when
secondary changes typical of eukaryote cells (i.e. glycosylation)
or a special fold in the protein produced are required.
A new method of expression based on the integration of adequate
genes in superior plants has been recently proposed. This would
offer considerable advantages for the product’s safety thanks
to the absence of contamination with animal pathogens or toxins
that could be present in vaccines expressed in animal or bacteria
cells. Given the ease with which plants produce plentiful biomass
on an agricultural and industrial scale, the approach would considerably
cut down costs. Lastly vaccines made from plants would not depend
on the “cold chain”, which is required today for conservation
and distribution and which could enable oral administration as
an alternative to intravenous injections (Daniell et al., 2001;
Sala et al., 2003).
The possibility of administering edible vaccines expressed in
plants has given rise to great interest even in the veterinary
field, especially for animals bred for food purposes, considering
the social and economic implications related to their health.
The possibility of directly providing these animals with substances
that have pharmacological activity in the form of food would greatly
simplify drug administration. When vaccines too must be precisely
dosed, transgenic plants that express the gene can be easily dehydrated,
the active principle can be dosed and hence administered after
mixing it with the daily ration. This would considerably cut down
costs and would avoid the stress induced in animals by the injection
method.
Studies on vaccines produced in plants have developed in many
directions in the past 10 years. Described below are some examples
of the first results obtained with this technology: the expression
of the hepatitis B specific antigen in tobacco and lettuce (Mason
et al., 1992; Eheani et al., 1997), of the rabies specific antigen
in tomatoes (McGarvey et al., 1995), of a cholera specific antigen
in tobacco and potatoes (Arakawa et al., 1997) and of a cytomegalovirus
specific antigen in tobacco (Tackaberry et al., 1999). Experiments
conducted on laboratory animals have revealed these vaccines’
capacity to stimulate the immune system by inducing the synthesis
of antibodies against hepatitis B (Thanavakam et al., 1995), the
Norwalk virus (Mason et al., 1996) and bacterial enteritis (Haq
et al., 1995).
In early experimentation on human volunteers, the vaccine formed
by E. coli’s enterotoxin’s subunit B expressed in
tobacco produced an immune response both on mucous tissue and
systemically in individuals orally treated with an adequate dosage
of transgenic potato (Tacket et al., 1998). The achievement of
mucous tissue immunity is one of the points in favour of edible
vaccines. Many pathogens penetrate the body through mucous tissue.
The first defences are hence the ones present on the very mucous
tissue that cover airways, the digestive system and the urogenital
one. The possibility of building chimerical genes, which express
detoxificated forms of the cholera toxin (CT) and of E. coli’s
thermolabile enterotoxin (LT), bound as adjuvants to antigen proteins
(Di Tommaso et al., 1996; Kong et al., 2001) has been designed
to further improve vaccine potential expressed in plants to evoke
a mucous response
Besides vegetable cells are surrounded by a cellulose wall that
protects them from the action of gastric juices. In non-ruminant
animals vegetable tissues are carried directly to the intestinal
lumen where they undergo slow lysis and can interact with a slow
release natural system.
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Vegetable Transformation Methods
There are essentially two plant transformation methods: one based
on the use of Agrobacterium and the biolistic particle delivery
system. The former is based on vegetable pathogens A. tumefaciens
and A. rhizogenes’ property to integrate their DNA (T-DNA)
with infected cells’ nuclear genome (de la Riva et al.,
1988). The introduction of exogenous genes into the adequately
modified T-DNA of Agrobacterium cells and the following infection
of a vegetable tissue lead to the study gene’s stable integration
in the plant’s genome and to the production of a transgenic
protein (Figure 1).
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Figura
1
Infezione di tessuto fogliare con Agrobacterium tumefaciens.
A) Durante l'infezione, il gene di interesse
viene trasferito dalla cellula batterica al nucleo della cellula
vegetale, tramite il T-DNA batterico che ha la capacità
di excidersi dal plasmide vettore e ricombinarsi con il DNA
vegetale inserendosi in un cromosoma.
B) Le cellule infettate da Agrobacterium
iniziano a proliferare formando il così detto “callo”.
C) Alcune cellule del callo iniziano a differenziarsi
e danno origine ad un germoglio.
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The application of Agrobacterium-mediated transformation, first
limited to tobacco and to few other species, which are the infection’s
natural targets, has now been extended to most vegetable species
marked by agronomic interest, including Graminae and legumen (Lee
et al., 2001; Chikwamba et al., 2002). This opens interesting
new prospects for the development of edible vaccines for both
human and veterinary use.
The second approach is based on the microprojectile bombardment
method (Taylor and Fauquet, 2002). Selected DNA sequences are
precipitated onto metal microparticles and bombarded against the
vegetable tissue with a special tool (particle gun) at an accelerated
speed. Microparticles penetrate the walls and release the exogenous
DNA inside the cell where it will be integrated in the nuclear
genome through mechanisms that have yet to be entirely cleared,
Vegetable cells have cytoplasmic organelles called chloroplasts,
which contain chlorophyll, generally known for their photosynthetic
function. These organelles, which, like mitochondria, are supposed
to derive from ancient bacterial predecessors and which have penetrated
a larger cell as symbionts, have an independent chromosome complement,
but their characteristics are typical of prokaryote cells. The
biolistic particle delivery system “shoots” adequately
processed DNA particles, which, penetrate into the chloroplast
and integrate with its genome. The chloroplast’s transformation
is an interesting alternative to nuclear transformation (Maliga,
2002; Daniell et al., 2002). In fact, according to some published
data on the transformation of tobacco for the expression of the
Bacillus thuringiensis’ (BT) insecticide toxin, the introduction
of exogenous genes into the chloroplast’s genome has led
to a collection of active recombinant proteins amounting to 47%
of total soluble proteins (Decosa et al., 2001). This type of
transformation offers the advantage of producing many transgene
copies per cell.
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Figure 2
Dischetto fogliare infettato con Agrobacterium tumefaciens,
allo stadio di formazione del callo. |
Besides, unlike what occurs in the nucleus, the chloroplast’s
transformation is based on the exogenous gene’s insertion
for homologous recombination. The transgene can thus be inserted
in a precise point of the plastid chromosome. This avoids positions
that can have negative effects on the plant’s growth, which
often occur in nuclear transformations, following the random insertion
of a gene in the nuclear genome.
However so far chloroplast genetic engineering has been carried
out only in tobacco and partly in the potato. We can hope that
in time this method will be extended to other species that are
more interesting for the production of edible vaccines (i.e. corn,
lettuce and clover).
Our laboratories at the University of Milan’s Department
of Biology are currently studying the possibility of making plants
express various proteins marked by antigenic activity both for
human and veterinary use. In the framework of cooperation with
the Pasteur Institute in Paris, a polyepitope (a molecule comprising
a series of peptide fragments endowed with immunostimulating activity)
with antigenic features against human melanoma was expressed in
tobacco leaves. It is currently at an early experimentation stage
to evaluate its level of immunogenicity.
An earlier stage of this process is research targeted at producing
veterinary vaccines. This study, conducted in collaboration with
many Institutes of the University of Milan’s Faculty of
Veterinary Medicine, first focuses on checking a new vaccine producing
system’s potential through a step by step comparison of
“traditional” vaccines and vaccines expressed in plants.
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Figure 3
Dischetto fogliare infettato, in fase di rigenerazione; si
nota la formazione di un germoglio. |
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Research Stages
This goal was achieved by marking out diseases which already had
marketed protein-based vaccines (the antigen) with tested immunogenicity.
Concerning proteins, it is important to know their structure,
amino acid sequence and especially the nucleotide sequence of
the gene they derive from. This data must be carefully evaluated
because, though every living being’s DNA only comprises
4 nucleotides and though the genetic code is more or less universal,
every organism has its “preferences” and some special
features in its protein translation and maturing systems. For
example, some amino acids are encoded by more than one DNA triplet,
but every organism preferentially uses certain triplets compared
to others. Hence the guest organism must present all the triplets
required for the protein’s translation. Other problems could
result from proteins that require glycosylation, since the chemical
structure of saccharide groups added by plants during a protein’s
maturing phase can slightly differ from animal structures and
this could cause the formation of an antibody that does not perfectly
meet requirements. One must be aware of such points ahead to adequately
modify the gene, which must be introduced into the host plant
(i.e. with site-specific mutation).
Current studies are developing along various lines and focus on
transforming plants into defence agents against diseases caused
by many organisms and with equally distant target organisms. A
few examples are filariasis in animals, and also in humans, caused
by a nematode worm, a special form of bovine enteropathy caused
by Escherichia coli bacteria and a degenerative disease in horses’
respiratory and reproductive systems caused by a herpes virus
strain. A protein, which, reproduced with traditional methods,
can be used as a vaccine has already been marked out and extensively
studied for all these diseases.
In the study’s early phase we chose to use tobacco as a
vegetable system, the best known and most studied system, to minimize
the variable factors involved. Naturally in this case experimentation
on animals must be conducted with protein extracts free of alkaloids
normally present in plant cell juice. The following phase envisages
the transformation of an edible plant (i.e. rice).
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Figure 4
Dischetto fogliare infettato, in fase di rigenerazione avanzata;
si notano le foglioline dei germogli in espansione. |
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If the genes are adequate for expression in the chosen host,
they must be placed in the plant under the control of an active
promoter, which can be recognized by vegetable polymerases. This
promoter can be either constitutive, in other words it can be
functional in all host tissue, or tissue-specific, in which case
the protein will only be expressed in a certain tissue of the
plant (i.e. the seeds’ reserve tissue). The currently used
promoter is CaMV 35S, which is a DNA sequence derived from the
cauliflower mosaic virus. It encourages the very virus’
reproduction in any part of the plant where the infection has
occurred. It is a good practice to place a transcription terminator
downstream of the gene to facilitate the polymerase’s detachment
from the DNA filament.
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Figure 5
Dalla superficie del callo spunta una vera e propria piantina. |
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The promoter-gene-terminator complex is then inserted in a T-DNA
in a plasmid vector that can replicate both in Escherichia coli
and in Agrobacterium tumefaciens. As all plasmids used in genetic
engineering these vectors, called binary vectors, carry genes
for resistance to a certain antibiotic or weedkiller to enable
transformed cell selection. All the early phases of genetic manipulation
are conducted on detoxificated strains of Escherichia coli, a
fast multiplying bacterium that is much more flexible. Agrobacterium
cells are transformed only at the end of the process.
Parts of the leaf can be infected once adequate checks have been
performed on selective soil to ascertain the correct insertion
of the plasmid, which carries the gene of interest into the final
bacterial host.
Infected “leaf disks” are placed in strictly sterile
conditions, once again in selective soil, while waiting for a
callus to form, which is cell proliferation caused by the Agrobacterium
(Figure 1). At this point the presence of adequate phytohormones
in the soil and the typical totipotence of non differentiated
vegetable cells enable the offshoot of a miniature plant to develop
from cells in whose genome the T-DNA has penetrated with its resistance
and study gene (Figure 2, 3 and 4). Roots will issue after a certain
lapse of time (Fig. 5) and the plant can be then moved from agarized
soil to normal soil (Figure 6).
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Figure 6
Piantine di tabacco GM in via di radicazione su terreno agarizzato. |
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| Piantina di tabacco GM trasferita
in vaso. |
Concerning the two trends mentioned as an example - filariasis
and equine herpes virus - studies have reached this stage and
analyses are currently underway to evaluate the two proteins’
level of expression in GM tobacco plants.
As whoever is experienced in laboratory research in the biological
field can easily deduce, the above described study is all but
free of difficulties. But we too, like other groups who are conducting
such research in the world, are firmly convinced that it is a
highly promising course, both as an alternative to already existing
solutions and to solve some problems that have yet to find an
adequate solution.
Prof.ssa Barbara Basso
CNR - Universitá degli Studi di Milano
Dipartimento di Biologia
“Luigi Gorini”- Milano
