Legumes Biofarming and Biopharmaceutical Sciences: A Review
The objective is to delineate and review prominent commercial and emerging biotechnopharmaceutical sciences that are state-of-art intersections of plant, human and animal ecologies. A special focus is placed on alfalfa as a bountiful legume from animal agriculture and medicinal perspectives. Crops are steadily and increasingly regarded as human health inducers. The new insights are changing and expanding plants prospective roles in plant and human ecologies form biotechnology and medicine perspectives. As such, more emphasis is placed on links between plant chemicals with post-modern human and animal health. Such plant-origin therapeutics include pharmaceuticals, multi-constituent botanical drugs, dietary supplements, functional foods and plant-derived recombinant proteins and vaccines. These compounds and products are expected to complement conventional pharmaceuticals for cure, prognosis and diagnosis of diseases, while improving the quality of agricultural crops. As a result, a more effective exploration of diverse phytochemicals may be developed. In addition, new biotechnologies enable manipulating plants capability in synthesizing natural products and substances. Advanced biotechnologies to generate preferential Biopharmaceuticals (BPC) are receiving increased research and commercial interests. However, the cost, safety and accessibility of such BPC are among the major challenges. Nonetheless, plant BPC could be inexpensive and potentially safe to produce and store. Vegetables and leguminous plants are the fitting examples. A main advantage of leguminous crops, namely alfalfa (Medicago sativa), is their immense biomass yield since they can be cropped several times a year. Leguminous plants contain phytoestrogens that encompass several compounds, such as flavonoids, isoflavonoids, coumestans and lignans. Legumes such as peas, soybeans and alfalfa can be used to produce plant-derived inexpensive monoclonal antibodies (plantibodies) for human and animal therapeutics. Due to the presence of a multitude of bioactive substances in legumes, further studies particularly involving nutrigenomics and metabolomics are required to specify such effects on human health indicators of particular BPC. Definitive Quantitative and qualitative dietary inclusion guidelines for foods with legume origins may become feasible for different age groups. These achievements will form new perspectives that will contribute to healthier and more viable plant-human-animal ecologies in the new era.
January 05, 2012; Accepted: March 03, 2012;
Published: June 21, 2012
Crops are being increasingly viewed as human health agents besides supplying
viable foods. The new insights emphasize such contributions to animal and human
biotechnology and medicine. New plant-origin therapeutics encompass pharmaceuticals,
multi-constituent botanical drugs, dietary supplements, functional foods and
plant-generated recombinant proteins (Ma et al., 2003;
Nikkhah, 2012a, b, c).
These would complement traditional pharmaceuticals for cure, diagnosis and treatment
of diseases, whilst improving crops quality. Also, with new biotechnologies,
plants capacities to synthesizing natural products may be manipulated. Advanced
biotechnologies to generate Biopharmaceuticals (BPC) are a host to modern research
and commercial interests (Fischer and Emans, 2000).
However, their cost and availability limit application (Nikkhah,
2012d). Nonetheless, plant BPC production and storage could be inexpensive
and potentially safe. Legumes are among prominent plants in furthering BPC technologies
that will be described in detail later.
Phytoestrogens are a group with pathetic estrogenic activity. In seedlings
of soybeans (Glycine max), the isoflavones of genistein and daidzein are predominant
(Graham, 1995). Phytoestrogens, especially isoflavones,
may cause infertility in livestock (Bouea et al.,
2003). Ever since these discoveries, more than 300 plants with estrogenic
animal responses have been found. Phytoestrogens include several compounds,
including flavonoids, isoflavonoids, coumestans (coumestrol) and lignans (Mazur
et al., 1998). Although causing infertility in animals, phytoestrogens
may benefit human health via prevention of certain diseases (Tham
et al., 1998). Specifically, phytoestrogens could prevent cancer
(Barnes, 1997), scavenge free radicals and lower blood
cholesterol (Carroll and Kurowska, 1995) and be antiestrogenic
and antiproliferative (Barnes, 1997). Phytoestrogens
are most abundant in leguminous plants that are present in international diets.
Legume seeds besides other parts of whole plants are edible. Soybean is a prominent
legume rich in isoflavones daidzein and genistein (Franke
et al., 1994). Daidzein and genistein are additionally responsible
for some the health benefits of soybeans (Messina and Barnes,
1991). Biochanin A and formononetin are other isoflavones in legumes. Besides
isoflavonoids, flavonoids also exert estrogenic activity but are much less active
than isoflavonoids (Collins-Burow et al., 2000).
Positive epidemiological correlations exist between fruits and vegetables intakes
and prevention of atheroscelerosis, cancer, diabetes and arthritis (Kaur
and Kapoor, 2001). As such, they are called as youth fountains,
promoting functional health and alleviating diseases. Phenolic flavonoids, lycopene,
carotenoids and glucosinolates are among major antioxidants (Kaur
and Kapoor, 2001).
A world in which natural and designed, synthetic proteins can be generated
with safe, secure and cost-effective approaches in desired quantities would
be a great accomplishment for the man (Nikkhah, 2012b,
c). That is particularly true when utilizing simple nutrients,
water and sunshine. Whilst beyond mans crude imagination, such advancements
do not seem too far from reality. Exploitation of plant powers (cereals and
legumes; Fig. 1, 2) in molecular farming
and generating recombinant proteins and vaccines on agricultural and medicinal
scales have been a concrete proof. The initiatives on plant-derived recombinant
BPC have tackled commercial approval, promising more upcoming new biotechnoecologies.
This review study will delineate prominent commercial emerging biotechnologies
that are state-of-art intersections of plant, human, and animal ecologies. A
special focus will be made on alfalfa as a bountiful multi-advantage legume
from both animal agriculture and medicine perspectives. Health implications
of alfalfa utilization in postmodern pharmaceutical industries will be delineated.
Other objectives include describing recombinant proteins, immunoglobulins and
vaccines production in alfalfa platforms using most recent biotechnologies.
Prospectus public interests and concerns will also be discussed.
Plant proteins and vaccines production-biofarming: Plant-origin Biopharmaceuticals
(BPC) are in the developing phase as the upcoming commercial states-of-art in
plant-human-animal biotechnologies (Nikkhah, 2012d). These
may have highly effective production scale and economics, global safety, storage
capacity and reasonably efficient distribution.
||Endosperm cells from tobacco, The tobacco cell is full of
oil drops occupying most of the internal space encompassing limited protein
|| Different forms of corn-based edible vaccines produced and
approved to date (Prodigene; College Station, TX, USA), Transgenic corn
kernels corresponding to a 1 mg dose of the B subunit of (a) E. coli
heat-labile toxin, (b) Being processed for a palatable whole corn snack
and (c) Fractionated to produce concentrated embryo or germs with antigens
being 6x more
These proteins offer promising opportunities to synthesize and supply cost-effective
medicines and vaccines, especially in the emerging and developing world (Table
1). However, disadvantages include doubtful regulatory grounds in platform-raised
crops and vacillating manufacturing practice regulations in field-grown plants.
The novel products must undergo vigilant cooperation and compromises of academic
and commercial property landscape to be widely useable. Authorized operation
licenses must be ensured for successful application and utilization in specific
countries and regions (Ma et al., 2005a).
|| Production systems properties for recombinant proteins production
These products will enable saving lives of human and animal populations where
environmental challenges concur, as beginning to be commercially releasable
(Ma et al., 2005a; Twyman
et al., 2005).
Plant-origin platforms have rather low production and storage costs, low contamination
risks, and reasonably high product quality and scale-up capacity, compared to
mammalian cells, bacteria, yeast and transgenic-animal based systems (Table
1). Numerous platforms have been utilized to produce Recombinant Proteins
(RP) from plants, such as leafy crops, cereal and legume seeds, oilseeds, fruits,
vegetables, cell cultures, algae and aquatic plants (Twyman
et al., 2003; Fischer et al., 2004).
Producing RP in seeds is particularly important, as they are the evolutionary
packages of storage proteins in a stable environment. In addition, antibodies
accumulate at high levels in seeds and remain functional for several years (Stoger
et al., 2002). Such effective storage is due to the desiccated nature
of mature seeds. The rich combinations of molecular chaperones and disulfide
isomerases ensure appropriate protein folding in the expanding seeds, at the
absence of proteases (Muntz, 1998). As a result of the
limited size of seeds, their RPs are found in high concentrations, despite the
lower yield than expected of biomassive leafy crops, such as tobacco and alfalfa.
This property is, however, beneficial for extraction and downstream processing.
The relatively simple seed proteome (less competing proteins) along with low
levels of the substances known to interfere with downstream processing steps
(e.g., phenolics and alkaloids in tobacco leaves and oxalic acids in alfalfa)
further facilitate processing. Oil drops in safflower and rapeseed are additional
advantages and can be exploited during the initial purification stages (Moloney
et al., 2003). Moreover, seed proteins do not usually interfere with
plant growth which limits adventitious contact with non-target organisms, such
as biosphere microbes and leaf-consuming herbivores (Commandeur
et al., 2003).
Most recently, steps have been taken in development of plant-made vaccines
for veterinary purposes. Enterotoxigenic E. coli can cause a variety
of diseases in livestock, including Newcastle disease, foot-and-mouth disease
and diarrheal diseases. Appropriate plant production platforms are being developed
and investigated as for plant species and processing and transformation technologies
in producing widely effective and well-preserved vaccines (Ling
et al., 2010). However, commercial and environmental challenges are
to be overcome before such vaccines can gain legislative approvals for regional,
continental and worldwide uses.
Medicinal perspectives of legumes biofarming: The plant-based production
of commercial biopharmaceutical recombinant proteins is usually named molecular
farming. Plants can be engineered using a multitude of methods including
nuclear transformation, plastid engineering, viral based transient expression,
agroinfiltration and magnifection techniques to act as more effective, minimally-perishable
and economical vehicles and platforms for protein and vaccine production (Kumar
et al., 2007). A variety of grain legumes have been used to express
RPs. However, two species of pea and soybean have been the generally explored
production hosts. Despite a relatively low annual grain yield than cereals of
mainly maize and rice, soybeans main advantage is its high seed protein
content. However, soybeans transformation procedures are time-consuming.
Limited molecular soybean farmings are reported (Philip
et al., 2001; Zeitlin et al., 1998).
Zeitlin et al. (1998) produced a humanized antibody
against herpes simplex virus which was conducted in the plant rather than seeds
alone. In the study of Philip et al. (2001),
casein was expressed in soybean seeds using a lectin expression cassette. Another
practical possible issue with soybean is its abundant oil content that might
interfere with downstream processing steps.
Pea is similar to soybean in annual grain yield and seed protein. As a result,
pea is similarly regarded as a suitable platform for reasonably high RP yields
per hectare. However, pea costs could be around 50% more than soybeans, thus,
necessitating greater seed yield to make pea a viable economical techno-plant.
The major pharmaceutical proteins expressed in peas include single-chain antibodies
(Perrin et al., 2000). An example is an antibody
expressed at low levels to act against a cancer antigen as controlled by a seed-specific
legumin A promoter (Perrin et al., 2000). Another
was an antibody expressed under Unknown Seed Proteins (USP) promoter,
reaching 2% of total seed proteins (Saalbach et al.,
Antibody generation biotechnologies: Many crops can now be used to produce
antibodies. These include tobacco (Nicotiana tabaccum and N. benthamiana),
cereals grains (rice, wheat, maize), legume seeds (pea, soybean, alfalfa) and
fruit and root crops (tomato, potato) (Schillberg et
al., 2002). Several critical factors must be taken into account in making
most appropriate choices (Kusnadi et al., 1997).
Leafy crops such as tobacco and alfalfa have very high biomass yields per hectare,
because they are harvested multiple times a year. Tomatoes also have a high
biomass yield but with increased production costs of greenhouses. Greenhouses,
on the other hand, enable better containment compared with field management
(Stoger et al., 2002).
For biovaluable proteins such as antibodies, more expensive production systems
are justified to secure the many advantages. For example, antibodies expressed
in potato tubers and cereal grains are stable at room temperature for months
or years without stability losses. However, tobacco leaves must be dried or
frozen before transport or storage to maintain RP activities. Protein extraction
from seeds is more costly than from watery tissue, such as tomatoes. A majority
of cost in the commercial production of RP including antibodies in plants belongs
to downstream processing. Toxic metabolites such as tobacco alkaloids are a
limitation. Thus, more edible plants without such toxins would preferably serve
as expression hosts.
Alfalfa and soybean are the main legumes for RP production which usually produce
less leaf biomass than tobacco but are efficient in utilizing atmospheric N
through N fixation (Zeitlin et al., 1998). Thus,
they require less chemical inputs (Kapusta et al.,
1999; Khoudi et al., 1999). As an advantage
for alfalfa, recombinant antibodies are produced as a single glycoform instead
of heterogeneous sets of different glycoforms as found in other plants (Dixon
and Sumner, 2003). In addition, grain legumes are beneficial production
crops due to their high seed protein contents. For instance, despite low yield,
peas are being developed as production platforms (Perrin
et al., 2000).
Estrogenic nature of legumes: Extracts of kudzu root (Pueraria lobata
L.) and red clover blossom (Trifolium pratense L.) may competitively
bind to estrogen receptor α (ERα). When estrogenic activity was determined
using an estrogen-dependent MCF-7 breast cancer cell proliferation assay, kudzu
root, red clover blossom and sprout, mung bean sprout (Vigna radiata
L.) and alfalfa sprout extracts (Medicago sativa L.) displayed increased
cell proliferation above levels observed with estradiol. All the above extracts
have been shown to exhibit agonist activity toward ERα. The active components
in kudzu root extract were isoflavones puerarin, daidzin, genistin, daidzein
and genistein. Thus, several legumes possess phytoestrogens with high estrogenic
Alkaloids and nonprotein amino acids (NAA): Within the approximately
650 genera and >18,000 legumes species, quinolizidine (characteristic of
Lupinus species; Fig. 3), dipiperidine, pyrrolizidine,
carboline, phenylethylamine and indole alkaloids are the present alkaloids.
The Tyr-derived Erythrina alkaloids may be found only in the large genus
Erythrina. The NAAs are common within the Leguminosae, with canavanine, pipecolic
acid and djencolic acid derivatives as the most important groups. The NAAs are
often highly toxic and cause serious human toxicoses, among which is lathyrism,
a nonprogressive motor neuron disease due to high intakes of grasspeas (Hanbury
et al., 2000). In the 5th century BC, an irreversible leg weakness
was reported in ancient cities inhabitants at times of war and starvation, when
people had to eat high pulses diets. Grasspeas are ideally planted in arid regions,
such as Ethiopia and the Indian subcontinent. Grasspeas seeds contain high levels
of 3-N-oxalyl-L-2,3-diaminopropanoic acid (ODPA) (Fig. 3)
which is responsible for several neurological symptoms and bone malformation,
particularly in children. Although low-ODPA grasspeas lines have been developed
via traditional breeding to better serve animals as healthier feeds, seed neurotoxins
removal by transgenic approaches has not yet been effectively developed (Hanbury
et al., 2000).
Health implications of leguminous isoflavonoids: Flavonoids are abundant
in plants, whilst isoflavonoids are rather restricted. Isoflavonoids are prevalent
in the Papilionoideae subfamily of the Leguminosae which act as antimicrobial,
allelopathic or anti-insect and induce nodulation genes of symbiotic Rhizobium
bacteria (Dixon, 1999). Pterocarpan-type phytoalexins
such as medicarpin and constitutive isoflavone malonyl glycosides (Fig.
3) are typical isoflavonoids. Temporal and spatial correlations exist between
phytoalexin accumulation and disease resistance in legumes. Isoflavonoids
roles in disease resistance have recently been verified using genetic approaches
Isoflavonoids originate from flavanones via a rare aryl relocation catalyzed
by the cytochrome P450 enzyme CYP93C1 (2-hydroxyisoflavanone synthase, or Isoflavone
Synthase (IFS) (Jung et al., 2000). Apparently,
the IFS gene has been evolutionary expressed in taxonomically distinct families.
In addition to their occurrence in papilionoid legumes, isoflavonoids exist
in some members of other families, including the Rosaceae, Chenopodiaceae, Apocynaceae
and Pinaceae. Isoflavones possess estrogenic, antiangiogenic, antioxidant and
anticancer activities (Dixon and Ferreira, 2000). They
are now becoming accepted as dietary supplements (Palevitz,
2000). Genistein (Fig. 3) has been a subject of >3,500
published reports over the last two decades. Major sources of isoflavones for
humans are soybean (daidzein and genistein) and chickpea (biochanin A, Fig.
3) seeds. Isoflavones seem most likely to be responsible for the health-promoting
properties of soy-enriched diets (Lamartiniere, 2000;
||Biochemical diversities of natural products in selected eight
legume species. The compounds include (1) Seed coat proanthocyanidin
in alfalfa (Medicago sativa), (2) Formononetin malonyl glucoside,
an isoflavone conjugate of alfalfa and barrel medic (Medicago truncatula)
roots, (3) Genistein, an isoflavone in seeds of soybean (Glycine max),
(4) Avicin D, a complex triterpene saponin in seed pods of Acacia victoriae,
(5) isoliquiritigenin, a chalcone in roots of licorice (Glycyrrhiza glabra),
(6) Glycyrrhizin, a triterpene saponin from roots of licorice, (7) medicagenic
acid glucoside, a triterpene saponin in roots of alfalfa and barrel medic,
(8) Medicarpin, a pterocarpan phytoalexin in fungally infected barrel medic
and alfalfa, (9) A prenylated isoflavone (lupinol A) from roots of Lupinus
species, (10) Lupanine, a quinolizidine alkaloid in roots of Lupinus
species, (11) 3-N-oxalyl-L-2,3-diaminopropanoic acid (ODPA) from seed of
grass pea (Lathyrus sativus) and (12) biochanin A, an isoflavone
in seeds of chickpea (Cicer arietinum)
Soy isoflavones intake has been associated with reduced risks of breast and
prostate cancers as well (Lamartiniere, 2000; Setchell
and Cassidy, 1999). Isoflavones may additionally have preventive effects
on osteoporosis and bone strength (Adlercreutz, 1998).
Genomics and metabolomics of legumes natural products: The development
and expansion of large-scale genome and Expressed Sequence Tag (EST) projects
have furthered natural products biosynthesis scales. Extensive DNA sequence
resources are now widely available for soybean. The sequences of the many genes
encoding enzymes of such products biosynthesis exist in the related databases.
A main question is how to identify relevant genes. A potential answer is to
apply functional genomics and metabolomics tools to globally assess transcriptomes
and the metabolomes. The extensive profiling of metabolites may be used to assess
gene function and to inquire biological systems responses to external stimuli
(Fiehn, 2002). This approach helps to qualitatively
and quantitatively define chemical phenotypes of genetically or environmentally
perturbed biological systems. No single technique may provide a thorough assessment
of such complex metabolomes, particularly in light of the vast chemical diversity
of natural products. Hence, multiple approaches must be utilized. These include
Thin-Layer Chromatography (TLC), infrared spectroscopy, NMR, Gas Chromatography-Mass
Spectrometry (GC-MS), liquid chromatography with UV or MS detection, liquid
chromatography-MS-capillary electrophoresis and capillary electrophoresis-MS
(Fiehn, 2002; Sumner et al.,
2002). The optimum techniques for chemical profiling of flavonoids and isoflavonoids
involve HPLC for separation, alongside UV absorption and mass selective detection
(Liu et al., 2002).
Specific protocols for a routine profiling of the flavonoid and isoflavonoid
from different legume species have been developed. These include red clover
(Trifolium pratense), soybean and lupin with high prenylated isoflavonoids
(Fig. 3) (Bednarek et al., 2001;
Lin et al., 2000). Saponins have poor chromophores.
Consequently, the optimum approach for profiling the triterpene glycoside of
legumes like alfalfa and barrel medic is reversed-phase HPLC coupled with electrospray-ionization
MS (Dixon and Sumner, 2003). The legume barrel medic
possesses more complex combinations of triterpenes compared to that studied
in alfalfa (Nikkhah, 2012a, b).
Therefore, the biological system can be a particular species, set of ecotypes,
or groups of species. In addition and instead, inducible systems such as elicited
roots or cell cultures may be utilized. As per triterpenes, cell suspension
cultures added with methyl jasmonate induce several compounds and related biosynthetic
enzymes. Depending on the metabolite profiles and present knowledge, a tentative
pathway is proposed, which includes P450- and GT-catalyzed reactions for triterpene
saponins (Suzuki et al., 2002). Next, cDNA libraries
are made from plant tissues with active chemistry, followed by high-throughput
EST sequencing (Suzuki et al., 2002). For instance,
in barrel medic, there are >250 expressed cytochrome P450s and almost 300
expressed GTs, based on EST counting of >30 cDNA libraries sequenced thus
Bioinformatics methodologies, namely self-organizing maps for in silico expression
analysis of EST libraries should be coupled with transcripts DNA array analysis
from chemical producers and nonproducers. These help to identify candidate genes
from a group of about 600 candidate P450s and GTs. The number of candidates
addressed (e.g., 20 of each class) (Dixon and Sumner, 2003)
would be adequately limited for direct expression studies in heterologous platforms,
such as Escherichia coli (E. coli) and yeasts. With more complicated
pathways at the absence of intermediates, multiple methods involving stable
or transient down-regulation of candidate genes combined with metabolite profiling
may be a necessity (Dixon and Sumner, 2003).
Plant pharmaceuticals: Modern and conventional approaches: In ancient
regions, traditional herbal medicines have long served human healthcare. Plant-structured
pharmaceutical platform can link agricultural sciences to medicinal sciences
and lead agriculture based economies to gradually improve human and animal healthcare
and welfare standards. As such, potential exists for a cost-effective generation
of molecules that aid in controlling infectious diseases, particularly HIV-AIDS.
The cost and logistics are among the key impediments in making and supplying
highly sensitive vaccines to vast populations in different remote world regions,
especially if lacking standard transportation and storage facilities. These
innovative proteins may be carried by fruit juice and sugarcane and cereal millings
(Ma et al., 2005b). Thus, appropriate facilities
for extraction, preparation, storage and transportation should more effectively
enable grater profits from plant-derived proteins in regions with suboptimal
infrastructures. Targeting recombinant proteins in seeds and using high-volume
economic food-processing technologies (e.g., freeze drying) can improve efficiency
from production to delivery (Ma et al., 2003,
Biosafety and bioethics are amongst the key concerns in adopting plant-derived
pharmaceutical proteins technologies in different world regions. Although crop
plants are the proven expression systems for novel proteins production (Streatfield
et al., 2003; Twyman et al., 2005),
specific designated regulations for various proteins will contribute to creating
food and environmental contaminations risks. Doubtful and in some instances
negative public impressions of world regions may unfavorably affect decision-making
in adopting such new biotechnologies. From a global market standpoint, agricultural
products are imported in considerable amounts from emerging markets to other
regions. As such, the consumers ongoing concerns of Genetically-Modified
(GM) contaminated foods have placed product labeling in great demands. Many
consumers even tend to accept premium payment for GM-free products. Overall,
early successful adoption of these emerging technologies depends on international
Nonetheless, some non-western countries are eager in using GM crops (James,
2004). These countries aim to become leaders in adopting plant biopharmaceutical
manufacturing technologies, having possessed the adequately advanced infrastructure
and lawmaking capacities for biotechnology development and adoption. China,
India, Brazil, Argentina, Iran, South Africa and Cuba are among the major countries.
South Africa has recently increased adoption of GM biotechnologies by enhancing
public awareness of the investment. This has encouraged similar initiatives
in African countries, such as Kenya. Production of foot-mouth disease vaccines
in Argentina (Dus-Santos et al., 2005), rabies
antibodies in India (Lamphear et al., 2002),
hepatitis B virus monoclonal antibodies in Cuba (Pujol et
al., 2005) and human papillomavirus antigens in South Africa (Rose
et al., 1999) have been reported. As such, emphases can be placed
on regionally important diseases in other countries. Several plant-origin clinical
biopharmaceutical proteins with their designated medical applications are presented
in Table 2.
Research and commercial perspectives for legumes: Plant-derived biopharmaceuticals
will be required to follow safety standards. However, since many herbal medicines
are classified as nutritional supplements, they are not closely scrutinized
for such standards. Should biopharmaceuticals be considered potentially detrimental,
persist in the environment and accumulate in non-target organisms, precautionary
measures are a must. Induction of biopharmaceutical production after harvesting
could be one approach to minimize environmental exposure, only ensuring that
viral vectors do not cause other regulatory concerns (Daniell
et al., 2001). Activation treatment of potentially harmful proteins
after expression is another approach to minimize risks of exposure.
For example, hirudin is produced as a fusion protein that is inactive. Hirudin
is activated only after purification from seeds (Cramer
et al., 1999). Another environmental concern is the possible outcrossing
of transgenic pollen to weeds or crops (Daniell, 1999a,
b). Expression of dangerous pharmaceutical proteins
in non-target plants due to such outcrosses could create public concerns and
negative perceptions. Several gene suppression methods are currently being studied
that include apomixis, incompatible genomes, transgenic mitigation, seed dormancy
or shattering control, suicide genes, infertility barriers, male sterility and
maternal inheritance (Daniell et al., 2001).
Foreign genes engineering via chloroplast genomes could suppress transgenes
effectively. An exception is the chloroplast genome that shows biparental inheritance
(e.g., pines, Daniell et al., 1998). As a different
strategy, RNAse genes can be expressed under the control of a tissue-specific
promoter to destroy the tapetum selectively during anther development, resulting
in male sterile plants (Mariani et al., 1990).
Another concern is the expression of destructive proteins in transgenic pollen.
For instance, the controversial toxic effects of Bacillus thuringiensis
(Bt) corn pollen on milkweeds (Asclepias spp.) fed to monarch butterfly
larvae impacted on public perception, despite questioning the validity of the
study. A solution could be engineering of biopharmaceuticals via chloroplast
genomes. Although the Cry protein of Bt was expressed in leaves (up to 47% of
total soluble protein), no toxicity occurred when milkweeds with transgenic
pollen were fed to monarch butterfly larvae (Cramer et
al., 1999). To date, chloroplast genetic engineering has been conducted
in tobacco and potato. Several recent laboratories have tried to extend this
technology to other crops. Reports are scarce on the production of glycoproteins
in transgenic chloroplasts. Additional public concerns are antibiotic resistance
genes and products in the edible parts of genetically modified crops. Now, several
approaches are available to generate transgenic plants in their nuclear or chloroplast
genomes without antibiotic selections (Daniell et al.,
2001; Richter et al., 2000).
Making most suitable choices of biopharmaceutical proteins and their production
crops is based on yield, storage conditions, control properties, initial set
up and running costs, purification methods, market size, environmental concerns,
public perception and competing technologies. Access to multiple alternative
approaches in optimizing plant protein synthesis in environmentally friendly
manners could promise a safe generation of biopharmaceuticals in transgenic
plants (Fig. 4).
|| Transgenic plant vaccines production process
Consequently, such plants and resulting proteins will become more available
to people and animals in demand of the products.
Alfalfa biofarming technologies: Alfalfa (Medicago sativa L.)
is a perennial herbaceous crop that is also known as lucerne. Alfalfa is a most
extensively planted crop worldwide and a major forage fed to livestock as hay,
silage and pasture. Alfalfa has a large dry biomass yield, high protein, minerals
and vitamins content and a vast capacity to fix N and enrich soils. These reduce
requirements for fertilization and improve growth conditions for other plants.
Improvement of alfalfa genetics, such as resistance to abiotic and biotic stress,
protein quality and digestibility, may increase alfalfa yield and nutritional
value (Vlahova et al., 2005). With high regeneration
ability, the fodder alfalfa is suitable for genetic modification via modern
approaches. Agrobacterium-mediated gene transfer has been used for transformation
of embryogenic alfalfa genotypes (Ninkovic et al.,
1995; Pezzotti et al., 1991) with efficiency
depending on genotype (Du et al., 1994). In addition,
a direct gene transfer has been applied (Vlahova et al.,
2005). Alfalfa is currently being used by the Canadian company Medicago
Inc. to develop multiple-cut, glasshouse production facilities (www.medicago.com).
Alfalfa use in the field may be discouraged by present regulatory guidelines
on open-pollinated species with compatible weedy relatives (Sparrow
et al., 2007).
In 1986 tobacco and sunflower calluses were first used to express recombinant
human growth hormone. Thus far, diverse plant systems have been utilized to
produce pharmaceuticals (Vlahova et al., 2005;
Fischer and Emans, 2000). Moreover, biotechnological
advances have furthered benefits of pharmaceuticals in alfalfa. Expression cassettes
are being optimized for protein expression in alfalfa leaves. New methods have
been renovated to express transient proteins. For instance, it has become feasible
to use agro-infiltration or protoplasts transformation for early demonstration
and validation. Alfalfa has a unique ability to produce recombinant glycoproteins
with uniform and homogenous glycosylation patterns. Alfalfa-derived C5-1 is
a diagnostic anti-human IgG that was developed in Canada for phenotyping and
cross-matching red blood cells for blood bank donors and recipients (Laurent
et al., 1993). The expression of biopharming represents
a cost-effective and reasonably safe alternate for antibody production (Yoshida
et al., 2004).
Alfalfa and recombinant molecules synthesis: Alfalfa may be harvested
up to 8-10 times annually in greenhouses with minimum equipment (Peterson
and Arntzen, 2004). A 10-ha greenhouse of well-matured alfalfa could produce
approximately 340 tons of fresh biomass plant per harvest. Thus, harvesting
8 times a year will yield about 2700 tons of alfalfa. To ease harvest and machinery
management throughout the year, grounds are divided into several smaller sections.
Soluble proteins extraction of alfalfa tissue starts with maceration in hammer
mills to break plant cells and facilitate effective and rapid extraction of
water and solubles. Subsequently, green juices are extracted after screw-pressing.
With a press capacity of 400 kg h-1 plant biomass input, about 800
L h-1 extract will be produced, thus, extracting about 6500 L juice/harvest.
Such a reasonable capacity is needed to avoid and minimize unnecessary delays
from harvest until juice extraction. The soluble protein concentrations in alfalfa
green juice are approximately 2%. Hence, a 10 ha greenhouse produces about 260
kg soluble proteins twice/week. With recombinant protein expression levels of
0.1 to 1% of soluble proteins, annual recombinant protein yield of a 10 ha greenhouse
will be estimably about 26-260 kg.
Recombinant proteins can be purified form alfalfa extracts using several strategies,
depending on protein application and the desired extent of purity. As for C5-1,
two methods have been studied. The first is the Québec method (Laurent
et al., 1993) purifying C5-1 from hybridoma cells, involving affinity
chromatography with a human IgG1 that is a matching antigen for C5-1 (Bardor
et al., 2003). The other method is a bed affinity chromatography
with staphylococcal protein A (Streamline rProteinA, Amersham Biosciences, Piscataway,
NJ). This method is more suited for large-scale C5-1 generation. These large-capacity
columns where liquids flow upward are loaded with unclarified green juices,
binding up to 20 mg of human IgG mL-1 of medium. Protein A, however,
is of minor affinity for mouse IgG. In pH of 9, about 5 mg C5-1 mL-1
of medium has been produced (DAoust et al.,
2004). To improve purification, the expanded bed column may be coupled with
streptococcal protein G, with greater affinity for mouse IgGs than for protein
A. Greenhouses, despite significant yields of recombinant proteins, will not
suffice should tons of proteins be required. Thus, field plants are a necessity.
In such production scales, molecular farming combines beneficially with animal-feed
industries. Wet fractionation (performed by Hall et al.
(2002) in Champagne, France) in large-scale production systems can process
about 750,000 tons of fresh alfalfa per year. During that process, temperature
and pH are monitored to separate alfalfa proteins and isolate protein-rich fractions
which are then dried into pellets for animal feed industries. Additionally,
the above large-scale fractionation has been refined in France (Viridis, Aulnay-aux-Planches)
to generate Rubisco that is an alfalfa-derived additive for human consumption.
Innovative alfalfa platforms for novel compounds production: Plant cells
are considered cost-effective and potentially safe manufactures of recombinant
proteins. Plant biopharming has greatly succeeded in producing many bioactive
pharmaceuticals, including diagnostic monoclonal antibodies (Khoudi
et al., 1999; Hiatt, 1990) and blood components
(Magnuson et al., 1998). Genetically modified
forages commonly fed to livestock are considered suitable for edible vaccines
production (Tuboly et al., 2000). Transgenic
alfalfa has been utilized to express the structural protein VP1 of
Foot-and-Mouth Disease Virus (FMDV) and immunize mice (Wigdorovitz
et al., 1999). As a bioreactor, transgenic alfalfa can produce commercial
enzymes, in a process that is much cheaper than fermentation facilities construction
(DAoust et al., 2004). Lactoferrin (Lf)
is an iron-binding glycoprotein present in human colostral whey proteins. It
is active against many pathogens. Thus, Lf is able to inhibit bacterial growth
by medium iron sequestration during microbial metabolism. A direct antibacterial
effect of Lf is related to its N-terminal region that can disrupt and penetrate
bacterial cell membranes (Yamauchi et al., 1993).
Also, Lf exhibits antiviral activities, being capable of suppressing human and
animal RNA- and DNA-viruses replication via blocking of cell-virus interactions.
Lactoferrin is a potent immunomodulator with anti-oxidant effects. It empowers
cell defence systems by stimulating macrophages and lymphocytes proliferation
and activity. Other functions include cytokine production, growth and likely
anti-tumor activity. Lf reduces free radicals which may in turn reduce disease
susceptibility Such antimicrobial and immune promoting effects of Lf have attracted
research to develop expression systems for massive production of recombinant
Lf. Lactoferrin has been expressed in Saccharomyces cerevisiae (Liang
and Richardson, 1993) and several mammalian systems, including baby hamster
cells (Stowel et al., 1991) and transgenic cows
and mice (Nuijens et al., 1997). Lactoferrin
was also produced in transgenic plants at 0.3% of total cellular protein (Salmon
et al., 1998).
Glycosylation studies suggest that alfalfa has the capability of producing
recombinant glycoproteins with homogenous glycosylation patterns. Efforts have
been made to produce human Lf in transgenic alfalfa plants. Barbulova
et al. (2002) used the highly embryogenic clone R4 from a commercial
Bulgarian cultivar Obnova 10. Agrobacterium tumefaciens-based gene system
has been utilized to transform alfalfa with a binary vector to carry human Lf
cDNA (Vlahova et al., 2005). Moreover, non-embryogenic
alfalfa variety of Furez has produced recombinant human Lf from cell cultures
with the Agrobacterium tumefaciens-based gene transfer system. Research
continues for further development of fast growing transgenic suspension cultures
(Vlahova et al., 2005).
Human clinical studies with plant-derived vaccines suggest steps forward (Yusibov
et al., 2002). Transgenic spinach expressing epitopes of rabies virus
glycoprotein (G protein) and nucleoprotein (N protein) were fused to Alfalfa
Mosaic Virus (AlMV) coat protein and were orally administered to 14 individuals.
Five of the people were previously immunized with a conventional rabies vaccine.
Three of those 5 and 5 of the 9 initially naive individuals exhibited specific
antibody responses to the rabies virus, whereas none in the control group showed
increases in rabies-specific antibodies. A week after completion of the feeding
regime, the 9 initially naive people were dosed with the conventional rabies
virus vaccine. Three of them showed rabies virus neutralizing antibodies but
none of the 5 controls did so. Despite detecting no neutralizing antibodies
before immunization with the commercial vaccine, potential for the plant-produced
rabies vaccine to complement oral vaccinations was logically evident (Walmsley
and Arntzen, 2003; Yusibov et al., 2002).
Recombinant immunoglobulins from alfalfa: Doses of recombinant IgG as
pharmaceutical are already developed or under clinical investigation (Bardor
et al., 2003; Raju, 2003). Due to increased
commercial interests in IgGs, conventional cell culture production systems do
not meet demands (Fig. 1). As a result, there have been demands
for alternative production methods (Fig. 1, 2).
Whole transgenic plants and animals are engineered to generate greater recombinant
IgG. In such a shift from a production system to another, key product criteria
must be carefully monitored to comply with stringent safety and efficiency requirements
through highly regulated clinical experiments. Another key issue is the high
batch-to-batch reproducibility which requires high products homogeneity. Heterogeneity
for IgG stems from variable maturation of N-glycans, affecting IgG products
quality. For instance, the US Food and Drug Association has made efforts in
improving the homogeneity of N-glycosylation in recombinant IgGs for pharmaceuticals
N-linked glycans in alfalfa may be processed into several mature oligosaccharides
with core-xylose and core α(1,3)-fucose and also terminal Lewis (a) epitopes.
The C5-1 monoclonal antibody expression in alfalfa has produced plant-derived
IgG1 that is N-glycosylated by a prime glycan with a α(1,3)-fucose and
a β(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since the core is
widespread in plant and mammal N-linked glycans, alfalfa plants appear to possess
capacities to produce recombinant IgG1 with a N-glycosylation. The latter fits
well for in vitro and in vivo glycan refinement into human suited plantibodies.
In vitro galactosylation of the alfalfa-derived C5-1 mAb has led to a
homogenous plantibody with terminal β(1,4)-galactoses similar to mammalian
IgGs (Bardor et al., 2003).
Legumes of especially alfalfa contain Saponins with possible anticarcinogenic
implications. Saponins possess an amphiphilic chemical structure that makes
them surface-active. Their anticarcinogenic effects are likely shown via direct
cytotoxicity, immune modulation, bile acid binding, and cell proliferation normalization.
Soybeans are amongst the most viable human dietary saponins because of being
key protein supplier. Non-dietary sources include alfalfa, sunflower, horse-chestnut
and many others (Price et al., 1987). The structure
of saponins differs considerably based on sugars amount, type and steroid sphere
composition. Fecal biliary excretion has been increased by alfalfa seeds ingestion
(Malinow et al., 1979), suggesting that saponins
may reduce bile acids conversion to secondary acids by intestinal microflora
and thus, may help in preventing colon cancers (Rao and
Sung, 1995). From a cardiovascular perspective, total circulating blood
cholesterol was lower in rats fed diets based on plant proteins of alfalfa,
fava, gluten, pea and soy compared to those fed animal proteins of casein, lactalbumin
and ovalbumin (Sautier et al., 1986). This cholesterol-lowering
effect is at least partially and possibly mediated through modulations of hepatic
LDL receptors (Friedman and Brandon, 2001).
Alfalfa possesses estrogenic activities. Estrogenic activity has been determined
using an estrogen-dependent MCF-7 breast cancer cell proliferation assay (Bouea
et al., 2003). Kudzu root, red clover blossom and sprout, mung bean
sprout, and alfalfa sprout extracts increase cell proliferation above levels
caused by estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell
proliferation induced by the extracts, suggesting that an Estrogen Receptor
(ER) related signaling pathway was involved. The ER subtype-selective activities
of legume extracts were examined using transiently transfected human embryonic
kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist
activity toward Erβ. Using HPLC to collect fractions and MCF-7 cell proliferation,
the active components in kudzu root extract were determined to be the isoflavones
puerarin, daidzin, genistin, daidzein and genistein. These results show that
several legumes are a source of phytoestrogens with high estrogenic activity
(Bouea et al., 2003).
Plant-derived edible vaccines aim to protect humans and animals against infectious
diseases, autoimmune diseases and various tumors (Jelaska
et al., 2005). Figure 4 shows edible vaccines production
process using transgenic plant platforms (Fig. 4). Transgenic
plants including alfalfa, potatoes, tomatoes, maize, rice and soybeans have
been used in vaccine development. Human studies with transgenic plants have
created promising data with no or minor safety concerns (Streatfield,
2006). As per livestock, animals have been fed transgenic plants (e.g.,
Arabidopsis thaliana, alfalfa and potato with antigens) to protect against
foot-and-mouth disease virus, BRV and bovine viral diarrhea virus (Streatfield,
2006). An Indian study has focused on Rinderpest disease in the Middle East,
Asia and Africa (Rice et al., 2005). Pigeon peas
have been fed to express protective viral hemagglutinin antigens in livestock.
Leukotoxin (Lkt) has been expressed recently in alfalfa and administered as
edible vaccines to attenuate stress and help to prevent diseases in animals
(Rice et al., 2005). Eating foods produced from
such plant-empowered animals will additionally provide similar health benefits
as do original plants. Storage and transportation costs of vaccines and related
facilities are saved, making the systems highly inexpensive. A commercial case
that has received much concern is mad cow disease. Prevention of
such health issues is central to the economic value of many livestock and food
Alfalfa is considered a natural laxative, a natural diuretic, an antifungal,
a liver detoxifier, a cure for kidney stones and urinary infections and a promoter
of pituitary gland function. Alfalfa is rich in vitamins and minerals and is
a major livestock forage feed worldwide. Alfalfa seeds contain amino acid L-canavanine
which is involved in pancytopenia occurrence in humans and systemic lupus erythematosus
induction in monkeys. It can cause allergies as well (Williamson
and Wyandt, 1997).
New promoters for high-level protein expression have been developed in alfalfa
leaves using alfalfa cell-culture and transient-expression technologies (DAoust
et al., 2004). Amongst alfalfas advantages are high biomass
yield, nitrogen fixing, soil nutrient enrichment and homogeneous glycan structures
of the glycoproteins synthesised in alfalfa leaves (Fischer
et al., 2004; Daniell and Edwards, 1995).
The latter is necessary for batch-to-batch consistency. On the other hand, alfalfa
leaves contain much oxalic acid that could interfere with optimal biofarming
processing (Fischer et al., 2004).
Properties of plant-made vaccines
||The plants that produce edible vaccines can be widely grown
in countries with developing and emerging markets
||Plants are routinely utilized in pharmaceuticals production with approved
purification and processing protocols
||Plant growth process is inexpensive and both body-and mind-friendly
||Plants may not have and generate human pathogens. As a result, the resulting
vaccines will not have major safety issues for humans
||Plant transportation is economically and commercially feasible worldwide
||Plants are the living dynamic organisms. Thus, any biological
changes that usually occur over time can severely alter vaccine production
quantity and quality
||Edible vaccines are very likely to be mistaken with normal fruits, and
thus, may be consumed more than formulated. Thus, education on public eating
behaviors and patterns are a necessity to ensure adequate safety
||Vaccines dosages may well vary, thus making intake guidelines development
more challenging. For example, bananas or legume tablets of different sizes
with different vaccines doses will not have similar public and medicinal
||For field and garden vaccines development, security is always an issue
||Glycosylation patterns between plants and humans differ which could affect
vaccine functionality and future status
Prospectus public and industrial trends of plant-animal-human biotechnologies:
Plant-produced vaccines will probably not face outcome 2 as illustrated in Fig.
5. A more realistic outcome would be that such vaccines possess gainful
outcomes, although the extent of success and public acceptance is still not
quite known. Certainly, given the safety, quality, security and consistency
challenges, the real world curve can hardly reach its fundamental and promising
status of 2002. It is obtaining pervasive community acceptance that some minimal
degree of high-quality manufacturing for genetically-modified products is required
in vaccine production. It is also becoming more favorable to believe in functioning
of injectable vaccines. Because the first licensed plant-derived pharmaceutical
recombinant proteins are antibodies including a poultry vaccine, a similar trend
is more likely to follow. Regulation-wise, quality control pathways should be
shortcut for the products that are intended for non-humans usage. The most feasibly
manufacturable products in the new time include high-merit proteins for diagnostics
purposes and/or kits for which no hurdles of clinical trials would be needed.
In addition, their required volumes are rather low. For instance, Farmacule
BioIndustries in Queensland, Australia, have recently produced vitronectin protein
in plants which is derived from animal serum and costs up to 5 million $US/g.
||The hype curve tracks public perception and attitudes
of plant-derived vaccines over time. Favorable publicity up until around
2002 fuelled mainly by edible vaccines ideas. However, fears of food chain
contaminations existed, followed by worries of regulatory problems and thus
no success for further development. Outcome 1 represents the perspectives
discusses. Outcome 2 represents a future when industries and governments
will not properly employ and fund the technology
Farmacule can produce up to 1 g/month vitronectin to meet world demands (ONeill,
2005). This suggests that an idealistic vision of plant-derived vaccines
for people with inadequate financial status may still be far away from reality.
Commercial exploitation of lower-hanging fruits for cheaper production and processing
may improve the future industrial and public standings of these new biotechnologies.
It is persuasive to realize the recent focus of a large tobacco company investing
in another company (i.e., Medicago Inc., of Canada), using alfalfa and tobacco
to produce a variety of vaccines. These include seasonal and H5N1 influenza
vaccines (Medicago, 2008). All in all, it is foreseen
that the technology at first can gain approval in manufacturing animal vaccines.
Later on, by demonstrating sufficient safety, the technology may create further
interest in human medicine. From a commercial investment viewpoint, at the beginning
the technology is being rather hosted by smaller companies and more independent
institutes. Involvement of larger philanthropic companies and investors could
be possible by taking global initiatives on more threatening diseases, such
as HIV. This can attract greater governmental interests also. Despite the fact
that vaccines such as Hepatitis B Virus (HBV) have been greatly targeted, it
is not likely for these to be the first products. The existing generic, licensed
vaccines are already very inexpensive. More probably, new types of vaccines
such as hepatitis and possibly rotavirus vaccines will be among those preferably
targeted since they are currently very costly. Later on inexpensive heat-stable,
suspension oral vaccines may receive more consideration (Rybicki,
2009). All of these are more critical where many preventable diseases challenge
animal and human life, where present vaccines are expensive, where no vaccines
exist for orphan diseases and where antibody therapy can effectively
treat prevalent diseases.
CONCLUSIONS AND IMPLICATIONS
The emerging biotechnologies of mainly plant-origin and legume-derived pharmaceutical
proteins production encounter real challenges. The biotechnologists and their
supporting sciences and industries should convince lawmaking and ethical organizations
and the major investing companies in the economical and viable nature of the
technologies compared to those established previously. Careful monitoring and
utilization of the initial products are a must for compelling validation of
the methods and regulatory frameworks. As such, the new proteins in forms of
vaccines and antibodies have the real opportunity to improve health and life
quality of large human and animal populations. Due to the presence of a multitude
of bioactive substances in legumes, further studies particularly involving nutrigenomics
and metabolomics are required to attribute and specify BPC effects on human
health indicators. Quantitative and qualitative dietary inclusion guidelines
for food and feed packages and preparations with legume origins may become feasible
for different animal and human age groups. These accomplishments will contribute
to forming healthy and viable plant-human-animal biotechnologies in the new
era. Genetic engineering helps to produce biopharmaceuticals most favorable
to human and animal health. Alfalfa is a multi-cut leguminous plant with high
annual biomass yield. Alfalfa leaves and seeds are main targets of biopharming
or biopharmaceuticals production of farming. With increasing demands
for Plant-Made Biopharmaceuticals (PMB), their production could be safer and
cheaper than animal-derived counterparts. Alfalfa is an inexpensive platform
for monoclonal antibodies, which are used as potential human therapeutics and
diagnostics. Plant expression systems to produce human and animal vaccines possess
many advantages. These include easily-established cultivation, inexpensive production,
no or little demand for cold-chain supply, rapid scale-up, simple seed distribution,
easy genetic manipulation, oral supply capacity and minor health concerns of
human pathogen and toxin contaminations. Plant-derived antibodies have passed
the early-stage clinical trials. It may well be feasible in the near future
to sweep away diseases by eating tasty vegetables and fruits. However, the existing
main concerns include biosafety, dosage uniformity, regulatory guidelines and
possible survival of herbicides and pesticides. The human vaccine technology
using plants may become feasible based on technical merits and approvals from
public and social sectors. Future research on enhancing the expression of special
genes is needed. It must be ensured that how and if transgenic plant vaccines
meet quality standards (purity, potency, safety and efficacy) of the World Health
The Ministry of Science, Research and Technology, and University of Zanjan,
Iran, are acknowledged for supporting the authors programs of optimizing
science education in the new millennium.
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