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Plants as biofactories: Production of

pharmaceutical recombinant proteins
CHIARA LICO, ANGIOLA DESIDERIO, SELENE BANCHIERI AND EUGENIO BENVENUTO*
ENEA, UTS BIOTEC, Section of Genetics and Plant Genomics
C.R. Casaccia, Via Anguillarese, 301, 00060 S. Maria di Galeria, Rome, Italy
*Corresponding author: benvenutoe@casaccia.enea.it

Abstract
Plants can be used for the large-scale production of a variety of recombinant proteins, destined for agroindustrial, biomedical and pharmaceutical applications. In
fact, blood components, hormones, enzymes, cytokines, antibodies and vaccines
have been successfully produced in plants. Plant systems offer unique advantages in
comparison with conventional techniques (bacteria, mammal or yeast cells): i) lower
production costs; ii) synthesis of functional proteins, similar to those produced in
animal cells, absolutely free of animal pathogens; iii) easy scale-up and purification
technology. Furthermore, the engineering of edible plants may allow for the delivery
of the recombinant protein (e.g. vaccines) through fruits, tubers, leaves or seeds. In
this way the cold chain, necessary for the storage and the transport of purified
recombinant products could be avoided, as well as the administration procedures by
injection. This review describes gene transfer methods (including stable and transient transformation), plant species used and strategies to obtain high yields of protein, with attention focused towards plant-derived antibodies and vaccines, known
as plantibodies or plantigens. Up to now, several groups are working in this
promising field of research demonstrating that plants are able to produce proteins
derived from different kingdoms, with highly complex structures (e.g. immunoglobulins A and G) and the different, post-translational, modification patterns of plants
do not dramatically affect the properties or the biological activity of the recombinant
protein. Using plants as biofactories to produce green therapeutic proteins is not
only a proof of concept but a reality as an approach for agroindustry.

Introduction: Plants as biofactories

Genomic and proteomic approaches to the study of fundamental cell mechanisms
are rapidly contributing to broaden our knowledge of metabolic pathways for the
Tuberosa R., Phillips R.L., Gale M. (eds.), Proceedings of the International Congress
In the Wake of the Double Helix: From the Green Revolution to the Gene Revolution,
27-31 May 2003, Bologna, Italy, 577-593, 2005 Avenue media, Bologna, Italy.

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exploitation of the cell factory. In the last few years, this knowledge has led to
important advances in the large-scale production of diagnostic and therapeutic proteins in heterologous hosts (bacteria, yeasts, mammalian or insect cells, and transgenic animals and plants), allowing the comparison of the most efficient method in
terms of costs, product quality and safety.
The new branch of plant biotechnology defined as molecular farming is mainly focused on the exploitation of plants of agronomic relevance as factories for large
scale production of biomolecules. In particular, the possibility of producing biopharmaceuticals using plants, paves new ways and offers solutions to some of the
problems associated to traditional foreign-expression systems. For example, the production of complex multimeric proteins, such as antibodies, in bacteria is limited
by the absence of the complex enzymatic machinery involved in post-translational
modification of neo-synthesized proteins. Among eukaryotic expression systems,
yeast is not always appropriate as hyperglycosylation of the final product is often
encountered (Harashima 1994; Malissard et al. 1996). Insect and mammalian cell
cultures represent complex systems requiring cumbersome, expensive procedures
and may be easily contaminated with toxins, viruses or prions raising concerns
about the safety of the final product. Finally, transgenic animals pose analogous
risks, in addition to slow scaling-up (in terms of time required for reproduction)
and ethical concerns. Plant systems may represent the solution to most of these
problems. First of all, plant cells are able to produce proteins structurally and functionally equivalent to those synthesized in mammalian cells, guaranteeing
high-quality products free of human pathogens. A distinct advantage is represented by the possibility of using plants as natural-storing systems when the synthesis
of the biomolecule is properly targeted to seeds or storage organs, allowing its conservation at room temperature. Furthermore, it might be possible to directly deliver the product by oral administration of tissues (fruits, leaves and roots) of an edible plant. In this way, extraction and purification of the recombinant product is
avoided and the biomolecule is also protected by a sort of capsule (the cell wall) for
prolonged action on mucosal surfaces.
Finally, the established agronomic procedures of crops offer big economic
advantages, with the only drawbacks being represented by the control of production steps and containment of genetically modified plants. The first recombinant
protein to be synthesized in planta was the human growth hormone (Barta et al.
1986). Since then, many other proteins have been produced by plant systems
(Table 1) and some of them have been commercialized (Hood et al. 1997; Witcher et al. 1998) proving their efficacy and competitiveness compared to other systems (Evangelista et al. 1998; Kusnadi et al. 1998).
It is important to highlight that plants can also be used as biofactories of endogenous compounds (e.g. acetil-salicilic acid and opiates), by modifying the metabolic pathways to improve the natural expression levels. Seventy percent of the cur578

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Table 1. Recombinant proteins expressed in plants for pharmaceutical applications.

Year

Protein

Transgenic species

References

1986
1990
1990
1993

Human growth hormone

Human albumin
Human albumin
Epidermal growth factor

N. tabacum
N. tabacum
S. tuberosum
N. tabacum

Barta et al. 1986

Sijmons et al. 1990
Sijmons et al. 1990
Higo et al. 1993

1994

Human interferon-

O. sativa

Zhu et al. 1994

1995

Eritropoietin

N. tabacum

Matsumoto et al. 1995

1997

-casein

S. tuberosum

Chong et al. 1997

1997

and hemoglobin

N. tabacum

Dieryck et al. 1997

1997

Human mucarinic cholinergic receptors

N. tabacum

Mu et al. 1997

1998

Interleukine 2 and 4

N. tabacum

Magnuson et al. 1998

1999

Human 1-antitrypsin

O. sativa

Terashima et al. 1999

2000

Somatotropine

N. tabacum

Leite et al. 2000

2000

Collagen

N. tabacum

Ruggiero et al. 2000

2000

Lactoferrin

S. tuberosum

Chong et al. 2000

2000

Somatotropin

N. tabacum

Staub et al. 2000

2001

Human acetylcholinesterase

L. esculentum

Mor et al. 2001

2002

Bovin aprotinin

Z. mays

Azzoni et al. 2002

2002

Human collagen

N. tabacum

Merle et al. 2002

2003

Lactoferrin

N. tabacum

Choi et al. 2003

2003

Interleukine 18

N. tabacum

Zhang et al. 2003

2003

Human granulocyte-macrophage colony

stimulating factor

O. sativa

Shin et al. 2003

rently available pharmaceutical products are natural or naturally-derived compounds of plant origin. Molecular farming could potentially extend the range of
plant applications in the pharmaceutical industry, offering a valuable alternative to
other biological systems used to produce complex macromolecules.

Transformation methods
The expression of heterologous proteins in plants can be performed using two different methodologies. One is based on the stable expression of the recombinant
protein in transgenic plants and the other on the transient expression obtained
through epichromosomal transformation.
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Stable expression in nuclear or chloroplast genome

The most employed method is based on the natural capability of Agrobacterium

tumefaciens to stably transfer DNA into plant cells (Tinland 1996). The transformation of plant tissues by this technique can be performed either in vitro, on small
leaf explants, or directly in planta (Feldman and Marks 1987; Bechtold et al. 1993;
Clough and Bent 1998).
For plant species that are recalcitrant to Agrobacterium infection (i.e. monocots),
nuclear transformation can be achieved using the so-called biolistic method. This
technique consists of shooting tungsten or gold microbeads coated in DNA vector
through a particle gun on plant tissues. The biolistic method is also used for plastid transformation by inserting foreign DNA into the genome of chloroplasts. The
fundamental difference between plastid and nuclear transformation is that due to
homologous recombination in the chloroplast, the insertion of the foreign gene is in
a defined position in the organelles genome, often allowing us to obtain high protein
yields. The prokaryotic origin of the chloroplast poses some limitations, as complex
proteins require post-translational modifications that are not produced. Nonetheless,
this strategy is currently under rapid diffusion for biosafety reasons, as this organelle
is maternally inherited which reduces the risk of transgenes spreading through pollen.
Since the first attempts at gene transfer more than 20 years ago, the choice of
plant species was based on its genetic and physiological features such as in vitro
plasticity and its ease of transformation. Nowadays, several plant species can be
successfully transformed and research interests in molecular farming are directing the choice of several crops, food or non-food, depending on the final goal of
the project.

Factors influencing exogenous gene expression

One of the major prerequisites for the construction of plant biofactories with
good exogenous gene expression levels is the preparation of transformation vectors.
Among the factors to be considered are the codon usage and the flanking regulatory regions of the heterologous sequence. Usually, to reach high level of expression
constitutive promoters such as the CaMV (Cauliflower Mosaic Virus) 35S promoter (ODell et al. 1985) for most plants or the maize ubiquitin-1 promoter
(Christensen and Quail 1996) for monocots are used. The expression in specific
plant tissues/organs such as in seeds and in tubers or the expression in response to
specific physical or chemical inputs is possible by using tissue-specific or inducible
promoters (Zuo and Chua 2000). Also, viral translational enhancers can be useful
tools to increase the yield of the heterologous protein (Gallie and Walbot 1992). In
cereals, to increase the synthesis of the heterologous protein, an intron derived from
the maize polyubiquitin gene has been inserted in the coding region of the transgene (Vain et al. 1996). An important role is also attributed to polyadenilation signals generally derived from the 35S transcript of CaMV, the nopaline synthase gene
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(nos) of A. tumefaciens or the small subunit of the ribulose-1,5-biphosphate carboxilase (ssu) of Pisum.
Since stable nuclear transformation of plants implies the random insertion of the
transgene in the genome, the level of expression may be influenced by the transcriptional activity of the surrounding chromosomic region. Furthermore, the insertion
can hinder the transcription of genes involved in fundamental morpho-physiological
pathways producing atypical or lethal phenotypes. For these reasons, molecular characterization of transformants (e.g. evaluation of the copy number of the transgene and
definition of its position within the plant nuclear genome) is fundamental. The insertion of a high number of copies is not always an advantage, as high levels of expression can induce the activation of mechanisms of gene silencing, resulting in the total
suppression of the transgene (Metzlaff 2002). Plastid transformation could represent
a solution in that it eliminates positional effects and any possible interference on genes
involved in vital cell functions. Furthermore, activation of gene silencing has not been
described in transplastomic plants, despite the high yield of exogenous protein due to
the high number of transformed chloroplasts per single cell. Unfortunately, not all
plant species can be transformed with this technique (up to now only tobacco and
tomato) and once the transformation have been done it is very difficult to reach the
homoplasmic condition.
The quality of the product can be improved by targeting the protein to the secretory pathway of the cell where it can fold correctly and post-translational modifications can be introduced. Retention to the cell compartments can be obtained by
adding defined signal peptides, such as the H/KDEL peptide (Conrad and Fiedler
1998). This sequence is used to retain the proteins in the endoplasmic reticulum, such
as in the case where it is detrimental to proceed further in the pathway (i.e. the Golgi
apparatus), to avoid unwanted plant-specific glycosilation (Gomord and Faye 2004).
In fact, sugar residues added to proteins in the secretory pathway of plant cells are different from those of mammalian cells (presence of xylose and fucose and absence of
galactose and syalic acid). This aspect could have functional and immunologic implications (e.g. induction of allergic response; Bardor et al. 2003), even if recent data
demonstrate the absence of side effects on humans, induced by new glycosilation pattern of the foreign protein (Chargelegue et al. 2000).
Targeting of proteins to the secretory pathway can be also used to direct their
secretion through roots (rhyzosecretion). This strategy, requiring the growth of
plants in hydroponic cultures, can appreciably simplify the purification steps
(Drake et al. 2003).

Epichromosomal expression

The epichromosomal expression does not imply the integration of heterologous

sequences in the plant genome (transient transformation) and, as a consequence,
does not allow transgene inheritance, thus restricting the synthesis of the foreign
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molecule to a single plant. The most popular and widely-used technique for transient transformation is based on the use of genetically-modified plant viruses as vectors of the heterologous sequence. Transient transformation is an easy and rapid
strategy of expression, ensuring very high yields of the product of interest. This is
due to the fast multiplication of the modified virus in plant cells resulting in accumulation of exogenous sequence in the complex of viral expression machinery.
Usually, for single strand positive RNA viruses, such as Potato Virus X, the heterologous sequence is inserted into the viral genome as an additional open reading
frame (ORF) whose translation is controlled by a duplication of viral subgenomic
promoter (Figure 1b). Since the duplication of subgenomic promoters can sometimes result in recombination events leading to the loss of the exogenous sequence
(Chapman et al. 1992), alternative cloning strategies have been developed. For
instance, attaching the exogenous gene to an IRES sequence (Internal Ribosome
Entry Sequence; Toth et al. 2001) allowing the independent synthesis of two proteins coded by a bicistronic mRNA. This new fragment can then be inserted

Figure 1. Strategies used to obtain the transient expression of a sequence of interest (black box)
through viral vectors.

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between the subgenomic promoter for the coat protein gene (CP) and the CP gene
itself (Figure 1c) to be successfully expressed.
A possible limit to transient expression strategy could be the restricted host range
of the virus. Nonetheless, many plant viruses have been used for epichromosomal
transformation: Bromovirus, Hordivirus (BSMV: Barley Stripe Mosaic Virus),
Tobamovirus (TMV: Tobacco Mosaic Virus), Potyvirus, Potexvirus (PVX: Potato
Virus X), Comovirus (CPMV: Cowpea Mosaic Virus), Tombusvirus (TBSV: Tomato Bushy Stunt Virus) and Alfamovirus (AMV: Alfalfa Mosaic Virus). These viruses infect a wide range of plant species (Pogue et al. 2002).
An alternative strategy to virus-induced transient expression is the so-called
Agroinfiltration. This technique is typically used to evaluate the quality of an
expression construct in terms of yield, conformation and functionality of the
desired product, before the production of stably transformed plants. Moreover, it
offers the unique opportunity to simultaneously express the genes encoding for the
subunits of multimeric proteins, providing the possibility to evaluate their assembly and functionality (Vaquero et al. 1999). Agroinfiltration is based on the use of
A. tumefaciens, like stable transformation, but does not necessarily imply the integration of the exogenous DNA into the plant genome.

Plants expressing antibodies

Antibodies are key molecules of the vertebrate immune system. The typical structure
of these glycoproteins consists of four polypeptides, two heavy chains and two light
chains, linked together by disulfide bridges and non-covalent bonds. These complex
proteins are involved in the specific recognition of antigens (i.e. substances extraneous to the organism able to induce the activation of the immune response) by recognising a molecular structure with high-binding affinity and specificity. For these features, antibodies can be considered ideal reagents useful for biomedical and industrial applications, both in therapy and in molecular diagnostics. In fact, antibodies are
successfully used in clinical trials to direct drugs, radioisotopes and interfering
molecules to unhealthy cells and tissues (e.g. towards a tumour) and to obtain targeted therapeutic effects, reducing alteration of other organs. Moreover, the possibility
to deliver antibody inside cells to immunomodulate pathology-related functions
has been demonstrated. Finally, in diagnostics approaches, properly targeted antibodies allow for the identification, characterization and localization of physiological or
pathological alterations (Peeters et al. 2001; Ross et al. 2003).
The possibility of obtaining antibody expression in plants (plantibodies) was
first demonstrated by Hiatt et al. in 1989. In that case, the assembling and the correct folding of a full-length immunoglobulin type G in a non-animal cellular system required critical experimental approaches. Since then, different forms of antibodies have been successfully expressed in planta.
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Although the production of complete antibody molecules is essential for therapeutic applications requiring the activation of the immune system, many other
uses require only the preservation of the antigen binding site. The domain structure of immunoglobulins allows for the design and construction by molecular
engineering of different forms of recombinant antibodies resulting in the production of smaller and versatile molecules (Nolke et al. 2003; Kipriyanov and Le
Gall 2004). Several of these molecules, such as single domain antibodies (dAb;
Benvenuto et al. 1991), Fab fragments (De Neve et al. 1993) and single chain
variable fragments (scFv; De Wilde et al. 1999; Fiedler et al. 1999) have been successfully expressed in plants. The scFv fragments consist of immunoglobulin variable domains, connected by a linker peptide. These recombinant molecules
showed a great applicability due both to their easy expression in heterologous systems and versatility as reagents. Plants also represent the ideal production system
for fully multivalent antibodies, such as the secretory IgA (SigA; Ma et al. 1995).
SIgA molecules result from the expression and the assembly, through transgenic
plant crossing, of four polypeptide chains to form a dimeric IgA antibody, which
is particularly resistant to proteolysis. The first example of SIgA expressed in
plants has been the GUYS 13 antibody specific for the adhesion protein of Streptococcus mutans (the causative agent of dental caries). GUYS 13 has been successfully used in clinical trials, demonstrating the validity of plant-derived antibody for passive immunotherapy (Ma et al. 1998).
The antibody expression levels in transgenic plant system are variable (up to 5%
of total protein), depending on the plant species used and on the tissue where it is
expressed (Fiedler et al. 1997). Expression levels can be further improved using
transient plant transformation mediated by viral vectors (Fisher et al. 1999).
The expression of full-length immunoglobulins and antibody fragments can be
targeted to different plant cell compartments such as the apoplast, endoplasmic
reticulum and cytoplasm (Conrad and Fiedler 1998). This possibility may represent
an advantage for purification. Also, the ability of antibodies to specifically bind to
antigens can be useful for purification through chromatography procedures. The
purity level of plantibodies depends on their intended use. When antibodies are
designed for intravascular immunotherapy or for fine diagnostics, an accurate
purification is required. In contrast, in the case of topical passive immunization, the
use of edible plant-expressed antibodies is conceivable, reducing or completely
eliminating the purification procedure (De Wilde et al. 1999).
Many full-length or recombinant plantibodies produced either by academic
groups or companies have been used as therapeutic molecules. Some examples are
listed in Table 2. In the future, it is plausible that the use of plants to produce a high
amount of safe and highly-specific reagents, such as antibodies, will be further
improved.

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Table 2. Examples of pharmaceutical antibodies expressed in plants.

Antibody

Antigen specificities

Transgenic
species

Application

References

IgG and
SIgA

Streptococcus mutans

N. tabacum

Therapy (topical)

Ma et al. 1998

IgG

Colon cancer antigen

N. tabacum

Therapy/Diagnostics

Verch et al. 1998

IgG

Herpes Simplex
Virus (HSV-2)

G. max
O. sativa

Therapy (topical)

Zeitlin et al. 1998

Z. mays

Therapy (inhalation)

EPIcyte

Humanized Respiratory Syncytial

IgG
Virus
IgG

Clostridium difficile

Z. mays

Therapy (oral)

EPIcyte

IgG

Sperm

Z. mays

Contraceptive
(topical)

ReProtect LLC, MD

IgG

Hepatitis B antigen

N. tabacum

Therapy/Diagnostics

Valdes et al. 2003

Diabody

Carcinoembryonic
Antigen (CEA)

N. tabacum

Therapy/Diagnostics

Vaquero et al. 2002

scFv

Non-Hodgkins
(NHL) Lymphoma

N. tabacum

Vaccine

McCormick et al. 1999

scFv

Carcinoembryonic
Antigen (CEA)

N. tabacum, Therapy/Diagnostics
P. sativum,
L. esculentum,
O. sativa,
Triticum sp.

Stoger et al. 2000

Plants as systems for vaccine production and delivery

The formulation of subunit vaccines is based on pathogen-derived macromolecules
(proteins or peptides) known to induce protective immune responses without
inducing the disease. The production of these antigenic molecules in plants has all
the advantages of the expression of biopharmaceuticals in plant systems described
earlier, with the additional opportunity to get the expression in edible plant species
for oral administration of the vaccine without the need of purification treatments
(Walmsley and Arntzen 2003).
The production of vaccine components in plants can be achieved by both stable
and transient expression (Mason et al. 2002).
Stable expression has been exploited to express several antigens in different plant
species (e.g. potato, lettuce, lupin, tomato, carrot and maize; Table 3). Three of
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these antigen-expressing plants are at the moment under clinical trial to evaluate in
humans the efficacy of oral immunization by using transformed plant tissues. The
plants currently under study are potato, expressing the B subunit of E. coli
heat-labile toxin (Tacket et al. 1998) or the caspid protein of Norwalk virus (resulting in virus-like particles (VLPs) assembly in plant tissues; Tacket et al. 2000) and
lettuce expressing the major coat protein (HBsAg) of Hepatitis B virus (Kapusta et
al. 1999). Despite these promising results, until now it has not been possible to estimate the antigen delivered dose of immunization. This is mainly due to the variation of the expression levels of the heterologous gene among different transgenic
plants. Oral delivery of transgenic tissues is able to stimulate antibody titers similar
to those induced by traditional vaccines and present the additional advantage of
activating both systemic and mucosal immune responses.
Also, transient expression technology can be a useful strategy to synthesize vaccines
in plants. Aside from the production of antigenic proteins by inserting the complete
Table 3. Representative antigens expressed in transgenic plants.
Pathogen

Antigen

Transgenic specific

Human hepatitis B
virus

Coat protein epitope

N. tabacum
S. tuberosum
Lupinus luteus
Lactuca sativa

Mason et al. 1992

Thanavala et al. 1995
Richter et al. 2000
Ehsani et al. 1997
Kapusta et al. 1999

Norwalk virus

Coat Protein

N. tabacum
S. tuberosum

Mason et al. 1996

Tacket et al. 2000

Enterotoxic E. coli

Heat-labile enterotoxin B
subunit

N. tabacum
S. tuberosum

Haq et al. 1995

Mason et al. 1998
Tacket et al. 1998

Rabies virus

Glycoprotein

L. esculentum

McGarvey et al. 1995

Cytomegalovirus

Glycoprotein B

N. tabacum

Tackaberry et al. 1999

Vibrio cholerae

Cholera toxin B subunit

S. tuberosum

Arakawa et al. 1997, 1998

N. tabacum
S. tuberosum
D. carota

Ma et al. 1997
Avesani et al. 2003

Diabetes-associated
autoantigen

References

Foot-and-mouth
disease virus

Structural protein VP1

A. thaliana
M. sativa

Carrillo et al. 1998

Porcine transmissible
gastroenteritis
coronavirus

Glycoprotein S

A. thaliana
N. tabacum
Z. mays

Gomez et al. 1998

Tuboly et al. 2000
Streatfield et al. 2001

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coding sequences as additional ORFs in viral genomes (Figures 1b and 1c; Franconi
et al. 2002), purified chimeric viral particles can also be used as primary vaccine components (Table 4). By this approach, the sequence coding for an antigenic epitope (i.e.
the portion of an antigen, generally comprised of a few amino acids, responsible for
the activation of the immune response) is fused with the sequence encoding the coat
protein of a plant virus, in a position that is known to be displayed on the viral surface (Johnson et al. 1997; Figure 1d). In some instances, for example when using
Potato Virus X, long epitope sequences can also be expressed (more than 15-20 amino
acids) by inserting between the coat protein and the epitope sequences an additional
Table 4. Representative antigens expressed in plants through viral vector approach.
Pathogen

Antigen

Plant virus

References

Plasmodium
falciparum

Several epitopes

TMV

Turpen et al. 1995

Influenza virus

Hemagglutinin epitope

TMV

Sugiyama et al. 1995

Human
Immunodeficiency
Virus 1 (HIV-1)

Capsid epitopes

PVX
CPMV

Porta et al. 1996

Yusibov et al. 1997
Marusic et al. 2001

S. aureus

Fibronectin binding protein

(FNBP) epitope

PVX

Brennan et al. 1999

Hepatitis B virus

Mimotope

TMV

Nemchinov et al. 2000

Rabies virus

Chimeric peptide

AIMV

Yusibov et al. 2002

Human Papilloma
Virus (HPV)

E7 Oncoprotein

PVX

Franconi et al. 2002

Colerectal cancer

GA733-2 Antigen

TMV

Verch et al. 2004

Hepatitis C virus

Mimotope

CMV

Natilla et al. 2004

sequence coding for the 2A peptide of Foot and Mouth Disease Virus (FMDV; Ryan
et al. 1991; Ryan and Drew 1994). This peptide mediates an autocatalytic, stochastic
processing event that leads to the accumulation in the plant cell of a pool of both
recombinant and wildtype coat proteins (Santa Cruz et al. 1996; Figure 1e), and the
consequent virion displays both kinds of proteins.
Chimeric virus particles (CVPs) produced in plant tissues by using these strategies
have been able to induce in animal models strong antibody responses also without
adjuvants delivery (i.e. substances generally used in vaccine formulations to potentiate the immune response; Yusibov et al. 1997; Marusic et al. 2001). The high
immunogenicity of this kind of vaccines can be related to the high number of epitopes that are displayed on the surface of a complex molecular structure such as a virus
particle. Additional results from studies focused on the oral delivery in humans (phase
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I clinical trial) of spinach leaves infected with CVP displaying a rabies virus-derived
peptide (Yusibov et al. 2002) further strengthened the idea that transient expression
can also be considered as a useful tool for vaccine production in plants.

Conclusions
Plants are good expression systems, and in some aspects can be considered more
efficient biofactories than bacteria or yeast/mammalian cells for the production of
high value molecules. Plants have been used for the large-scale production of several recombinant proteins, especially for biopharmaceutical applications with particular emphasis on antibodies and vaccine components. Applications of these
plant-produced recombinant molecules imply that they must be considered similarly to the traditional pharmaceuticals before their introduction to the market. To
reach this goal it is fundamental to cultivate these plants in contained greenhouses
for a complete control of the productive row, before entering the standardized
Good Laboratory Practice (GLP) of the pharmaceutical industry.

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