key: cord-014901-d9szap94
authors: Permyakova, N. V.; Uvarova, E. A.; Deineko, E. V.
title: State of research in the field of the creation of plant vaccines for veterinary use
date: 2015-01-04
journal: Russ J Plant Physiol
DOI: 10.1134/s1021443715010100
sha: 
doc_id: 14901
cord_uid: d9szap94

Transgenic plants as an alternative of costly systems of recombinant immunogenic protein expression are the source for the production of cheap and highly efficient biotherapeuticals of new generation, including plant vaccines. In the present review, possibilities of plant system application for the production of recombinant proteins for veterinary use are considered, the history of the “edible vaccine” concept is briefly summarized, advantages and disadvantages of various plant systems for the expression of recombinant immunogenic proteins are discussed. The list of recombinant plant vaccines for veterinary use, which are at different stages of clinical trials, is presented.

For many thousands of years plants have served humanity as a source of medicinal substances. How ever, only at the turn of the century XXI with the appearance of DNA technologies, it became possible to modify plant genomes and to create new types of plants (transgenic plants), which are capable of syn thesizing and accumulating in their tissues recombi nant proteins from various heterologous systems. To date transgenic plants in which nuclear and chloro plast genomes have been transformed with genes encoding heterologous proteins that are important in the treatment of various diseases -antigens of infec tious agents, antibodies, immunomodulators, etc. have been created [1, 2] . Of principal importance of this work development was the creation of the "edible vaccine" concept, the essence of which is the use of genetically modified plants containing protein anti gens of infectious agents for oral delivery of relevant antigens to the mucosa of the gastrointestinal tract of warm blooded animals.

Vaccination based on the programming of the spe cific mechanisms of warm blooded animal protection against pathogens is the most efficient method for the struggle against infectious diseases, which often result in a mass mortality. In agriculture, there is no alterna tive to livestock vaccination, because there are no anti viral drugs that are suitable for a wide use in animal husbandry. The importance of animal vaccination indirectly affects human health, because the use of vaccines significantly reduces the amount of pharma ceuticals in the food chain.

As a rule, animal immune mechanisms are acti vated by the direct introduction of infectious agents or their components. At present, most of used vaccines are preparations on the basis of inactivated agents. Although these vaccines manifest the high immunoge nicity, they are not without serious shortcomings. Among such disadvantages are the increased sensitiv ity of the organism to them, the large load on the immune system, the reactogenicity of vaccines (side effects), their toxicity. etc.

The application of molecular biology and genetic engineering methods opened wide prospects for the manufacturing of vaccines of new generation, which immunogenic components can be biological mole cules or their fragments. DNA fragments or proteins of the infectious agent cell envelopes can serve as immu nogenic components. When the gene encoding the envelope protein of the infectious agent is transferred into the genome of another organism, for example, plant, then the cells of such plant will synthesize the protein antigen capable of formation of resistance to this agent. Thus, the introduction into the organism not the whole pathogen but only its part, which is not capable of inducing infection development, will pro vide for the effect of vaccination. one fourth of the total pharmaceutical market of drugs for veterinary use, and it is constantly expanding. [3] . Preparation of medicinal substances for the pro duction of veterinary products is based on various approaches, including biotechnology using genetically modified (transgenic) organisms for these purposes; such expression systems as bacteria, yeast, cells of insects and mammals are used. The application of genetically modified plants with genes encoding phar maceutic proteins inserted in the genome opens new prospects for obtaining recombinant proteins, includ ing plant vaccines [4] .

This review is devoted to the analysis of possibilities of producing recombinant immunogenic proteins for veterinary use on the basis of plant expression systems, and the history of the concept of "edible vaccines" for animal immunization.

OF PLANT VACCINES The idea of the usage of plant cells for the synthesis and accumulation of recombinant protein antigens was for the first time successfully realized in 1992 by C. Arntzen and his colleagues [5] . Just this team of researchers not only demonstrated a possibility of the accumulation of the surface HBsAg antigen of hepati tis B virus but also its capability for self assembling in the virus like particles in transgenic tobacco plants. The virus like particles isolated from plant tissues were identical to the particles of HBsAg antigen of indus trial recombinant vaccine obtained in the yeast expres sion system and also to virus like particles from the blood plasma of patients infected with hepatitis B virus. Thus, it became obvious that genetically modi fied plants producing and accumulating protein anti gens of various infectious agents can be used for oral delivery of corresponding agents to the mucosa of the gastrointestinal tract of warm blooded animals, i.e., as "edible vaccines."

The next important step in the development of the "edible vaccine" concept on the basis of genetically modified plants was the creation of transgenic plants producing the heat labile enterotoxin of Escherichia coli [6, 7] and B subunit of the cholera toxin [8] . Heat labile toxin of E. coli consists of two parts: LT A (enzyme) and LT B (pentamer of receptor binding polypeptides). LT B binds to the receptors on the sur face of membranes of epithelial cells of the mammal small intestine and transports LT A in the intestinal cells, where it induces changes in the cell metabolism and cell dehydration. When the two parts of the heat labile enterotoxin are separated, the appearance of the LT B protein complex on the surface of epitheliocytes will stimulate a strong immune response of intestine mucosa without the appearance of any disease signs. Just this feature was the basis for the research of C. Arntzen [6] team on the creation of plant vaccine providing for the resistance to enterotoxigenic E. coli toxins. The authors established that LT B synthesized in transgenic tobacco and potato plants and also LT B isolated from E. coli delivered orally to mice induced similar immune responses.

Later, LT B sequence was optimized for expression in plant cells and transferred into the potato genome [7] . In potato tubers, the protein assembled correctly in oligomers and was accumulated in amounts suffi cient for the induction of the immune response at oral delivery to the organism. On the basis of clinical trials of the "candidate" plant LT B vaccine, it was estab lished that the consumption of raw potato tubers con taining 0.3-10 mg LT B by volunteers resulted in the formation of serum and mucosal immune responses with high titers of antibodies [9] .

The initial concept of "edible vaccine" was heavily criticized by researchers who believed that in the aggressive medium of the gastrointestinal tract the recombinant protein should be destroyed. However, later it was experimentally established that the recom binant B subunit of the cholera toxin fused with green fluorescent protein (GFP) protected by the plant cellu lose cell wall at oral delivery is capable of passing through the gastrointestinal tract and reaching the antigen containing cells of the mouse intestine [10] . The results obtained confirmed the possibility of using plants synthesizing protein antigens of various infec tious agents for oral delivery of antigens to the mucosa of the gastrointestinal tract and experimentally con firmed a consistency of the "edible vaccine" concept. It became evident that genetically modified plants can be used for the creation of plant vaccines as a raw mate rial, and separate plant parts (fruits, roots, berries, leaves, etc.) can be used directly in food without pre liminary heat treatment.

RESPONSE FORMATION In the process of evolution, mammals developed secondary lymphoid mucosal tissue capable of antigen absorption, processing them, and using for the induc tion of the mucosal response. It was established that in this case both cellular and humoral immunity were formed. It is of importance that adaptive mucosal immunity can distinguish between usual food and symbiotic antigens and infectious agents [11] . The scheme of the mechanism of mucosal immune response formation is presented in Fig. 1 .

In the digestive tract, which is one of the pathways for the penetration of a variety of pathogens into organism, the main associated lymphoid tissue is the Peyer's patches. Peyer's patches, inductive sites of the intestine, which contain the dome, underlying the fol licle (B zones with germinal center) and interfollicu lar region containing T cells. The surface of the dome is covered by a specialized follicle associated epithe lium containing the folded cells (M cells), which are able to absorb and transport antigens from the intesti nal lumen. After successful capture, the antigen is par tially cleaved and enters into dendritic cells (see Fig. 1 , step 1). Dendritic cells are especially important in the initiation of adaptive immune responses, since they migrate to the lymph nodes (see Fig. 1 , step 2), and the mediators act in the development of various subpopu lations of T helper cells from naive T lymphocytes and also can interact with B lymphocytes (see Fig. 1 , step 5). The activated B and T cells leave the Peyer's patches and penetrate into the circulatory system. Mature T helper cells then return to the mucosa sur Pathogens (antigen) Epithelium T helper cells face to function as effectors (see Fig. 1 , step 3). T helper 17 expressing interleukin 17 (IL 17) increases the expression of the receptors of polymeric immunoglobulin (pIg) and secretion of antigen spe cific IgA (Fig. 1, step 4) . Subsequent generation of mature plasma cells producing IgA leads to the induction of antigen specific protection of local and distal mucosal surfaces.

Since the mucosal immune response has a general ized nature, oral (i.e., mucosal) vaccination is not only the immune response of the mucous membranes, but also the overall immune response of the organism [11] [12] [13] [14] . It is proved that vaccination via the surface of mucosa membrane can specifically activate the immune response to infection without the develop ment of such processes related to the disease as inflammation or toxicity [14, 15] .

OF PLANT VACCINES In comparison with traditional expression systems, plant systems are attractive to researchers in many ways, primarily due to the absence of the risk of plant cell infection with animal pathogens, viruses, prions, etc. Plants are capable of the synthesis of most recom binant antigens with the same posttranslational modi fications as in animal cells [16] . Plant vaccines can play especially important role in the protection of ani mals against diarrheal diseases and diseases, which infectious agents penetrate into the organism through the mucosal tissues.

Modern techniques of genetic engineering allow to selectively direct the recombinant proteins expressed in plant cells to various plant organs (seeds, tubers, fruits, etc.) [17] . This possibility greatly simplifies the large scale production of plant vaccines and reduces their cost. According to the experts, the final price of the product (recombinant protein) produced in a plant expression system will be much less than the price of a similar protein produced, for example, in mammalian cell culture [18] .

Since recombinant proteins can be accumulated in the storage organs or seeds, they are able to be main tained without any changes and the loss of biological activity for a long time (months and years). It was established that the recombinant protein of cholera toxin B subunit remained stable in transgenic rice grains for at least 18 months, when grains were stored under room temperature conditions [19] . Recombi nant protein antigens remained stable in rice grains during three years and provided for the formation of the protective immunity in mice against cholera agent or against enterotoxigenic E. coli [20] ; in soybean seeds and soy milk, the preservation of antigen stabil ity was observed for four years [21] .Thus, grains of transgenic plants can be transported to the site of final destination without additional freezing and treatment, and this ensures the retention of activity of the recom binant protein activity, its stability, and the constancy of dosage.

A somewhat different picture is observed when lyo philization is used as a method of the conservation of protein antigens synthesized by plant cells. It was established that the recombinant protein of the norovirus envelope retained its immunogenicity in tis sues of both lyophilized and air dried tomato fruits [22] , but the tested samples differed in immunogenic ity. The immunogenicity of this recombinant protein in air dried tomato fruits was somewhat higher in comparison with lyophilized fruits. Similar results were obtained in experiments on the lyophilization of potato tubers synthesizing the protein of norovirus envelope, e.g., the immunogenicity of lyophilized tubers was lower than that of fresh tubers [22] . How ever, additional studies are required for the final solu tion of this question.

Despite these advantages, plant vaccines are not without disadvantages. One of them is differences in protein posttranslational modifications in plants and animals, e.g., in glycosylation of the recombinant pro tein [23] . It is known that more than a half of proteins synthesized by eukaryotic cells are glycosylated and more than a third of currently applied biopharmaceu tics are glycoproteins [24] . Although the activity of most proteins does not depend on glycosylation, in some cases it may be critical. Specific features of pro tein glycoforms can affect their folding, stability, trans portation, and changes in their functional activity and immunogenicity. Examples of biopharmaceuticals, which functional activity depends on the specific gly coform, are erythropoietin, antibodies, blood anti gens, some interferons and hormones [24] .

Scheme of the N glycan complex formation in plants and animals (humans, for example) is shown in Fig. 2 . The most significant difference in glycosylation is that the plant β 1,2 xylose is attached to the core mannose residue and α 1,3 fucose -to N acetylglu cosamine residue of the core glycan. In human cells xylose is not used at all in glycosylation and a proximal fucose residue is attached to glycans through the α 1,6 bond. It was established that sugar residues attached at posttranslational modifications of recom binant proteins in plant cells themselves were capable of exhibiting the immunogenicity. Approximately in one quarter of patients with allergy symptoms, IgE antibodies specific for complex glycans, which include xylose or fucose, were revealed [25] . Differ ences in glycosylation during posttranslational modi fications of recombinant proteins in plant and mam mal cells can be eliminated by genetic engineering techniques, which was successfully demonstrated on the moss Physcomitrella patens, in which the genes encoding the enzymes β 1,2 xylosyltransferase and α 1,3 fucosyltransferase responsible, respectively, for xylosylation and fucosylation of proteins were switched off by the "knock down" method [25] .

It is known that when the foreign gene is inserted in the genome of the transgenic plant, the level of its expression depends on the site of its insertion, which determines the level of protein (antigen in particular) accumulation in plant tissues. In this connection, one of disadvantages of plant vaccines is the difficulty in the standardization of their dosage. It is at this stage the development of the "edible vaccine" concept has undergone substantial revision, since the possibility of oral delivery of the recombinant antigen with the raw plant material was untenable because of the variability of the recombinant protein content. According to the researchers involved in the development of "candi date" plant vaccines, the antigen dosage problem in plant tissues can be successfully solved by the intro duction of additional treatment of the plant material: its refinement (to equalize the concentration of the antigen), drying or lyophilization. It is also necessary to introduce an additional stage associated with the development of rapid methods for determining and monitoring the dosage of the recombinant antigen. After appropriate preparation, plant vaccines can be encapsulated, tableted, and used in practice under corresponding medical supervision [14, 26] .

Thus, performed studies allow a suggestion that the plant vaccine based on the genetically modified plants is capable of inducing protective immunity and open new opportunities for the creation of low cost and easy handling vaccines against infectious diseases of animals. It is of importance that developed to date, highly effective methods of cultivation of agro eco nomically important plant species, as well as the seed production system for a particular culture make the plants attractive to be used as "biofactories" for man ufacturing cheap recombinant proteins for medical purposes. roplasts). Among plant expression systems with stable integration of the transgene in the nuclear genome, a separate group includes the cultivated duckweed, microalgae, and cell culture systems in vitro: cell sus pensions, cultures of "hairy roots", moss protonemas, which cultivation conditions are a completely closed environment (bioreactors). Promising is the transient expression system, in which the target gene is intro duced into plant cells and is expressed for a short period of time (several days), but is not integrated into the genome. Each of these expression systems has its advantages and disadvantages; the main details of these systems are considered below.

The most widely used system of heterologous gene expression, in particular for plant vaccine production, are transgenic plants with the stable integration of the transgene into the nuclear genome. The creation of these plants involves the transfer of foreign genes into the genome of plant cells using Agrobacterium tumefa ciens or bioballistics and subsequent regeneration of transformed plants from these cells. Being inserted into the nuclear genome, transgene becomes its resi dent part, is stably expressed, and is maintained in subsequent generations. Using this expression system for producing recom binant proteins, including plant vaccines, is still ham pered by relatively low levels of transgene expression, which is, as a rule, less than 1% of total soluble protein (TSP) and also by its variability in different plant organs and tissues within a single plant and in different plants.

Most often, the variability in expression of the transgene is due to the random nature of its insertion into the nuclear genome (effect of position) and may be associ ated with partial or complete transgene inactivation [27, 28] . Experts believe that the use of plant expression sys tems for obtaining plant vaccines is economically bene ficial at the level of expression of a target gene, which allows the accumulation of recombinant protein in an amount not less than 1% of TSP [29] .

A lot of ways to increase the level of foreign gene expression in transgenic plants is developed; they are very fully discussed in the reviews [17, 30, 31] . Among them are the optimization of the codon composition of the target sequence, the usage of strong promoters, the addition of introns or regions of binding with the nuclear matrix (SAR), and others. The search for tis sue and organ specific promoters, i.e., promoters providing for target gene expression in definite plant tissues or organs, is of a special interest. For example, the usage of promoters directing transcription of the target gene predominantly in the seeds can increase the yield of the target protein by an order or several fold: up to 13-14% of TSP in rice grains and up to 25% of TSP in tobacco seeds [17] . It is of importance that as compared with leaves, seeds contain less pro teases and much less water; therefore, recombinant protein is saved better.

Despite the fact that transgenic plants with stable transgene expression in the nuclear genome are used most widely, it is just this expression system that induces a cautious attitude of human society. One of the fears is associated with the possibility of transgene transfer from the cross pollinated plants into the genomes of wild relatives at growing biotechnological crops in open field. To solve this problem, researchers developed various agricultural technologies as well as fixing male sterility in transgenic plants to prevent unwanted cross pollination of cultivated and wild spe cies. Examples of production of various immunogenic proteins using for this purpose transgenic plants with stable transgene integration into the nuclear genome are presented in Table 1 .

Chloroplasts are most attractive among plant expression systems. Genetically modified plants with the stable transgene integration into the chloroplast genome were called transplastome plants. The specific organization of chloroplasts allows achieving a high dose of foreign gene in transplastome plants, which provides for the efficient production of the target pro tein. The record of the recombinant protein yield was achieved by transplastomic tobacco plants with the bacteriophage lysin gene PlyGBS, encoding a hydro lase of the bacterial cell wall, the level of which accu mulation in the leaves amounted to 70% of TSP [32] . Although in some cases, negative physiological changes were observed in plants with such high level of foreign gene expression, usually there were no devia tions in the development of such plants. Problems of transplastome plant adaptation to the high level of for eign gene expression are discussed in the review of Bally et al. [33] .

Transgene delivery to chloroplasts is performed using bioballistics (gene gun); its integration into the chloroplast genome occurs via homological recombi nation [34] . The advantage of chloroplast expression system is in the absence of the effect of position observed in the case of random pattern of transgene distribution in the nuclear genome. In plastids, there is no transgene splicing (inactivation); therefore, its expression is stably preserved in subsequent genera tions. Due to the prokaryotic organization of chloro plast genome, there is a possibility of co expression of several genes within a single operon [35] . An impor tant specificity of plastids is that they are inherited through the maternal line and usually are absent from pollen. Therefore, as distinct from usual transgenic plants, transplastome plants are safe for environment because the uncontrolled spread of the transgene into other plants is prevented [36, 37] .

It should be noted that protein posttranslational modifications, e.g., assembling multimer proteins, the TMV-tobacco mosaic virus; CaMV-cauliflower mosaic virus; TSP-total soluble protein; (+) immune response is revealed at oral immunization; (++) immune response is revealed at oral immunization, animals did not die after virus infection. Species of immunized animals are indicated, the way of antigen delivery is indicated in the cases when it was not oral; the amount of survived infected animals is indicated in the cases when the survival was less than 100%.

No. 1 2015

formation of disulfide bridges, lipid modifications, etc., occur successfully in chloroplasts. It is estab lished that recombinant proteins synthesized in chlo roplasts do not differ from native ones in their func tional activities [38, 39] . Chloroplast attractiveness as the system of recombinant protein expression is in the fact that they are closed structures and it preserves the metabolites, which when released into the cytosol are toxic to plant cells, such as B subunit of cholera toxin [38] and trihalose in tobacco cells [40] . On the basis of the above works, it becomes appar ent that transplastome plants can be regarded as the most promising system for efficient production of pro tein antigens. However, the main disadvantage of such expression system at plant vaccine manufacturing is that chloroplasts cannot glycosylate proteins. At present, the creation of transplastome plants is also associated with some technological problems related to the absence of the efficient system of regeneration for most plant species and also with the absence of the efficient system of transgen delivery to the chloroplast genome providing for the high percent of transformed plant yield.

The suspension cell cultures on the basis of geneti cally modified plants attract the attention of research ers as promising potential systems for biopharmaceu tics production. Such cultures can be obtained from loose callus tissues induced from genetically modified explants or on the basis of co culturing of the cell sus pension and A. tumefasciens. After the assessment of growth characteristics and cell line screening to obtain promising lines capable of the accumulation of great amounts of recombinant proteins, such lines can be cultivated in bioreactors for target protein obtaining.

An attractive feature of the cell cultures as expres sion systems for recombinant protein production, as compared to the use of whole plants for this purpose, is that cell culture can be unified in growth character istics, cell dimensions and types. Moreover, cells are grown under strictly controlled conditions, when the product accumulation does not depend on the sea sonal weather changes and allows the permanent product obtaining in bioreactors. The additional insertion of signal peptide nucleotide sequences into the construct permits a protein secretion into the intercellular space, which allows target protein isola tion directly from the culture liquid. The addition of recombinant protein stabilizers to the suspension cul ture increases the yield of the target protein [41] .

By their capabilities, plant cell cultures are compa rable in production of therapeutic recombinant pro teins with the conventionally used mammalian cell cultures, such as Chinese hamster ovary cells. How ever, as distinct from the mammal suspension cultures, they are not infected by any animal pathogens. To data, there are many examples of plant cell cultures with the yield of recombinant protein in the amount more than 10 mg/L, which is a threshold value for starting the commercial manufacture of the product [42] .

An example of a commercially successful produc tion of veterinary vaccine products is developed by Dow Agro Sciences company (United States) system Concert™, patented as an effective and safe system for the production of vaccine proteins in cultured plant cells, cultured in a bioreactor. The first plant vaccine against Newcastle disease virus of birds obtained in the tobacco cell culture was approved for use by the Min istry of Agriculture of the United States in 2006.

The disadvantages of this expression system are still insufficiently high yield of the target recombinant pro tein and the instability of foreign genes in cultured plant cells due to the epigenetic silencing of the trans gene transcription [43] . Advantages, disadvantages, and specific features of recombinant protein produc tion in the plant cell cultures are discussed in reviews [42, 44] .

There are many examples of successful use of aqueous plants, such as duckweed, unicellular algae, and mosses, which are cultivated similarly as plant cell suspensions in bioreactors, for recombinant pro tein expression.

Duckweed attracts the attention of researchers as a potential highly efficient system for recombinant pro tein expression due to its capability of a rapid biomass accumulation: it can be doubled for 24-48 h. Geneti cally modified duckweed as a potential producer of biopharmaceutic proteins can be used by animals as a raw or dried food. Duckweed is a monocotyledonous angiosperm; for foreign gene transfer into its genome, the methods of agrobacterial transformation and bioballistics are used. Examples are known when genetically modified duckweed accumulated recombi nant protein in the amounts up to 25% of TSP, as assessed after the accumulation of GFP [45] . Sequenc ing the duckweed chloroplast genome is close to com pleting, which opens up some prospects for a signifi cant increase in the yield of recombinant proteins.

The systems of biopharmaceuticals production using genetically modified microalgae are actively developed. Algae combine advantages of both bacteria (rapid growth and simplicity of cultivation) and higher plants (a capability of posttranslational modifications and photosynthesis). Chlamydomonas reinhardtii is most promising among algae: it has a short time of bio mass doubling (about 10 h), it is easily subjected to nuclear and chloroplast transformation, it can be grown under photoautotrophic conditions or with the addition of acetate as the source of carbon. Nuclear transformants usually give rather low yield of protein product; therefore, recombinant protein production by this alga is based on the transformation of chloro No. 1 2015 plast, which occupies about 40% of the cell volume [46] . C. reinhardtii nuclear and chloroplast genomes are sequenced, and this simplifies substantially any genetic engineering manipulations. Known examples of protein antigen production in chloroplasts of C. reinhardtii are B subunit of cholera toxin fused with the coat protein of foot and mouth disease virus [47] or with D2 fibronectin binding domain of Staphylococ cus aureus [48] , as well as protein 28 virus cryptokary osis (shrimp disease) [49] and E2 protein of swine fever [50] .

The green moss Physcomitrella patens is the only representative of bryophytes, the genome of which is currently completely sequenced and approaches to its transformation are developed. The peculiarity of this moss is that at the stage of the haploid juvenile game tophyte (protonema) this moss is morphologically similar to filamentous algae and easily enough culti vated in a bioreactor [25, 42] . Under certain culture conditions the moss can be in the stage of protonema indefinitely long. The attractiveness of this moss spe cies as the system for recombinant protein expression is that, as distinct from plants, fragments of foreign DNA can be integrated in its genome through homo logical recombination, which reduces substantially a possibility of transferred gene inactivation. The P. pat ens cells are capable of postranslational modification of proteins of eukaryotic origin. Since at the step of gametophyte the moss has the haploid number of chromosomes, it becomes possible to modify the func tioning of individual genes, in particular the moss lines conducting glycosylation of recombinant proteins as in mammalian cell type were obtained [25] . The firm Grenovation (Germany) is developing the technology of biopharmaceutical protein production on the basis of P. patens in bioreactors. Expression system for pro ducing biopharmaceuticals based on the green moss is not the part of the food chain and is characterized by a high degree of biosafety.

As distinct from above described systems based on the stable expression of foreign genes integrated into nuclear or chloroplast genomes, during transient expression target proteins are synthesized in the plant cell during relatively short time (several days) without insertion into the plant genome. At present, the fol lowing approaches are used for transient gene expres sion in plants: gene delivery with the help of agrobac terium, the use of plant virus vectors, and magnifec tion [51] [52] [53] . Tobacco mosaic virus (TMV), potato X virus, alfalfa mosaic virus, and cowpea mosaic virus are used as virus vectors [52] . The availability of infec tious cDNA clones, the small size of the viral genomes, the short time required for the expression of a target gene, and a high level of expression provides for a high attraction of this system. The rapid development of this expression system led to substantial modifications of the first gene inser tion vectors or full virus vectors, which is a recombi nant virus that behaves as wild type virus but is capable of expressing additional genes. The next step was the creation of "disarmed vectors" (deconstructed vec tors) lacking a number of original virus genes, and gene replacement vectors, in which a portion of the viral genes is replaced by alien genes [51] . Viral vectors have several substantial disadvantages: a tendency to the loss of foreign insertion in the process of virus spreading over the plant and a potential risk for envi ronment related to the presence of infectious recombi nant viral particles.

Launch vectors represent an alternative to recom binant plant viruses; cDNA of these viruses is deliv ered to plants within T DNA region of agrobacterial Ti plasmid. Firstly, primary transcription of T DNA occurs in the nucleus; then viral RNA is released into the cytoplasm, where its further amplification, trans lation, and protein synthesis occur [52] .

By 2005, the system of agroinfiltration based on the use of plant viruses and agrobacterial binary plasmids was upgraded and named as magnifection [54] . At magnifection, multiple agrobacterial lines carrying different parts of the TMV genome are used simulta neously. After agrobacterial transfer into the plant cell nucleus, separate parts of viral genome are assembled in plants in the completely functional viral replicon [51] . The substantial modification of the viral genome, including numerous point mutations for the removal of potential sites of splicing, intron insertion, and the removal of the gene encoding envelope proteins, pro vided for the highly efficient system capable of recom binant protein synthesis (up to 5 g/kg of fresh tissue), which is more than 50% of TSP [51] .

Among disadvantages of transient system is a necessity for recombinant protein isolation and purifi cation immediately after its accumulation in the plant, because, as distinct from seeds and fruits, plant leaves and stems cannot be stored for a long time.

The systems of transient expression of recombinant proteins are rather promising in the cases when a rapid production of a small amount of proteins is required. Experiments with transient expression in plants are held indoors, which reduces the risks associated with biosafety to almost zero. Examples of manufacturing immunogenic proteins for veterinary using transient expression systems are presented in Table 1 . The sys tems of transient expression for the production of recombinant proteins are described in more details in reviews [1, 41, 51, 52, 54] .

"CANDIDATE" PLANT VACCINES FOR VETERINARY USE Table 1 presents examples of using various expres sion systems for the production of "candidate" plant vaccines for veterinary use. Main specific immunogenic No. 1 2015 proteins synthesized at respective diseases (structural proteins, hemagglutinins, glycoproteins) are usually used as antigens. The most commonly used method of transgene construct delivering into plant cells is still the agrobacterial transformation. In some cases, the level of target protein expression was rather high [55] [56] [57] [58] , espe cially in the systems of transient expression [59] [60] [61] , and suitable for product commercialization. In all experiments using the "candidate" plant vac cines, the formation of a specific immune response was demonstrated in vivo, and in most experiments immunogenic proteins were delivered to animals just orally. "Candidate" plant vaccines were usually tested on mice, but in approximately a quarter works the ani mals subjected to the disease were tested. Protein S of the transmissible swine gastroenteritis virus synthe sized in the cells of transgenic maize [55, 62, 63] or tobacco [64] and delivered into the body of pigs as a food supplement, provided for 100% survival of ani mals after infection [62] . Rabbit protein VP60 virus synthesized in potato [65] and other plants (tobacco, pea, rape) [66] and delivered orally enhanced protective immunity: after infecting rabbits with this virus all ani mals survived [66] . The effect of plant recombinant antigen was comparable with commercially used vac cines. The use of plant vaccines for the vaccination of wild animals using edible baits (e.g., vaccine against rabies) will lead to an increase in the proportion of wild animal populations having immunity to the rabies virus.

A potential possibility to reduce the cost of produc tion of biopharmaceutics using genetically modified plants served at the end of the XX century as an impe tus for more than twenty biotechnological companies to initiate commercial programs. As seen from the Table 1 , many biological products of plant origin are developed, expressed in different types of plants and plant cell cultures. For a variety of reasons, including the still skeptical attitude of the human community to the biosafety of genetically modified plants, many of these works remained in the framework of laboratory tests. At the moment three companies function on the biotechnology market of veterinary preparations, two from the United States and one from Canada (Table 2 ). In the United States the Dow Agro Sciences company presented a recombinant plant viral HN protein of the Newcastle disease virus (approved by USDA) and a mixture of antiviral vaccines at the first stage of clinical trials. The second American company at Thomas Jef ferson University has developed a plant anti rabies vaccine (completion of phase 1). The Canadian Guardian Biosciences company presented plant vac cine against chicken coccidiosis at the second phase of clinical trials.

Production and wide distribution of biopharmaceu ticals is hampered by a number of circumstances. The first of them is related to the problem of biosafetythe cultivation of genetically modified plants in the field can lead to the accidental introduction of foreign genes into crops grown for human consumption. Therefore, most companies producing biopharma ceutics focused on plant species, which are absent from the food chain of humans and animals and also on growing of genetically modified plants preventing their cross pollination with other crops. The second difficulty is related to the necessity of plant material treatment for the removal of various undesired com pounds, such as lignin, proteases, phenolic com pounds, and pigments, especially in the case of plant species, which are not consumed. All these facts result in the requirement of additional studies. The third cir cumstance is due to the fact that until now all aspects of maintaining and growing of plants producing biop harmaceuticals are not settled at the legislative level. Ambiguity and vagueness of the existing legislation in this area lead to the fact that large biopharmaceutical and biotechnology companies do not tend to invest in the development of technological lines and research programs in this area, which significantly inhibits the development of the industry. A significant problem for the development of vac cines for veterinary use, especially those used in agri culture, is the need to minimize the price of the final product. The vaccine should be inexpensive for entre preneurs engaged in commercial animal breeding and fully subsidized, if you intend to use the vaccine for mass immunization and the prevention of the disease spread in underdeveloped regions. As a result, the potential income of manufacturers of vaccines for ani mals is much less than that for vaccines intended for humans. For example, in 2007, the volume of the mar ket for the vaccine against human papilloma virus was estimated at more than 1 billion dollars, but the mar ket for the most popular animal vaccines (against foot and mouth disease of cattle and against Mycoplasma hyopneumoniae in pigs) together amounted to only 10-20% of this amount [3] . Animal vaccines are cheaper and the volume of market for them is less; therefore, the investments in their development are substantially less as compared with investments in the production of vaccines for humans, whereas the com plexity and diversity of both hosts and pathogens in the case of vaccines for animals is much higher.

Expression systems based on the use of plant cells still have a limited application or are used primarily in some laboratories. Nevertheless, biopharming (the biotechnological production of various substances for medicine) in plants has attracted the attention of researchers and manufacturers in developed countries. First biopharmaceuticals of plant origin, such as anti bodies (Anti HBsAg required to purify the hepatitis B vaccine), therapeutic and dietary proteins ("intrinsic factor" required at vitamin B12 deficiency, gastric lipase), have entered the market, that is an excellent illustration of this progress [67] .

Despite the fact that today the number of biophar maceutical proteins expressed in plant cells is enor mous, many questions still remain unresolved. The methods for target recombinant protein quantification and purification are still not developed for most of products. The problems of transgene silencing and increased expression of target protein genes are still at the stage of research. The important task that has yet to be solved is to achieve a stable level of expression in different batches of plant raw material. Not much work appeared for judging about maintaining the sta bility of recombinant proteins after the harvest, pro cessing, and storage. All of these problems require additional expenses for further research.

One of the main obstacles for the leading research groups working on the development and production of plant vaccines, given the financial constraints, is the fulfillment of the relevant official regulations govern ing the use of oral medications. To date, the purified vaccines and therapeutic proteins of plant origin must meet the same standards relating to the production, biosafety, purification, storage, dosage, etc. as any other recombinant proteins for medical purposes.

Nevertheless, despite these difficulties, there were first biopharmaceuticals of plant origin that passed all the necessary tests and were approved for use by the relevant authorities. Some new products having spe cific advantages over similar products obtained in mammalian cell cultures were developed. Such com panies as SemBioSys Genetics Inc. (Calgary, Can ada), Medicago Inc. (Quebec, Canada), Protalix Bio Therapeutics (Karmiel, Israel), and ORF Genetics (Iceland) proved the possibility of quick establishing of the production of purified plant proteins, which are quite competitive in today's market. Progress has been made in the formation of the legal framework related to the cultivation of transgenic plants, testing and use of plant biopharmaceuticals. Several production pro cesses based on transgenic plants have already received a brand GMP (Good Manufacturing Practice), the interest of manufacturers to this field of biotechnology began to increase again.

This work was performed within the framework of the project VI.62.1.5 (no. 01201280334) Development and improvement of genetic constructs to optimize the expression of target genes and the production of recombi nant proteins for medical purposes in transgenic plants and animals.

Plant produced vaccines: promise and reality

Evolution of plant made pharmaceu ticals

Current status of veterinary vac cines

Clinical trials fuel the promise of plant derived vaccines

Expression of hepatitis B surface antigen in transgenic plants

Oral immunization with a recombinant bacterial anti gen produced in transgenic plants

Edible vaccine protects mice against Escher ichia coli heat labile enterotoxin (LT): potatoes express ing a synthetic LT B gene

Effi cacy of food plant based oral cholera toxin B subunit vaccine

Immunogenicity in humans of a recombinant bacte rial antigen delivered in a transgenic potato

Receptor mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chlo roplasts into the mouse circulatory system

Delivery of plant made vaccines and therapeutics

Defending the mucosa: adjuvant and carrier formula tions for mucosal immunity

Induction of secretory immunity and memory at mucosal surfaces, Vaccine

Oral delivery of human biopharmaceuti cals, autoantigens and vaccine antigens bioencapsu lated in plant cells

The mucosal immune response to plant derived vaccines

Posttranslational modifica tion of therapeutic proteins in plants

Seed based expression systems for plant molecular farming

The economic potential of plant made pharmaceuticals in the manu facture of biologic pharmaceuticals

Rice based mucosal vac cine as a global strategy for cold chain and needle free vaccination

Secretory IgA mediated protection against V. cholerae and heat labile enterotoxin producing enterotoxigenic Escherichia coli by rice based vaccine

Stability of a soybean seed derived vaccine antigen following long term storage, processing and transport in the absence of a cold chain

Tomato is a highly effective vehicle for expression and oral immunization with Norwalk virus capsid protein

Production of plant made pharma ceuticals: from plant host to functional protein

Post translational modifica tions in the context of therapeutic proteins

Current achievements in the production of complex biopharmaceuticals with moss bioreactors

Low dose oral immunization with lyophilized tissue of herbicide resistant lettuce expressing hepatitis B surface antigen for prototype plant derived vaccine tablet formulation

RNA mediated chromatin based silencing in plants

Transcriptional gene silencing in plants

Plant based production of biopharma ceuticals

Production of heterologous proteins in plants: strategies for optimal expression

Expression of heterologous genes in plant systems: new possibilities

Exhaustion of the chloroplast protein synthesis capac ity by massive expression of a highly stable protein anti biotic

Metabolic adaptation in transplastomic plants massively accumu lating recombinant proteins

Transplastomic plants

Overexpression of the Bt cry2Aa2 operon a ¸n´l in chloroplasts leads to formation of insecticidal crys tals

Chloroplast vector systems for biotechnology applications

Determining the transgene containment level provided by chloroplast transformation

Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

Chloroplast expression of His tagged GUS fusions: a general strategy to over produce and purify foreign proteins using transplas tomic plants as bioreactors

Accumulation of trehalose within transgenic chloro plasts confers drought tolerance

Plants as bioreactors for the production of vaccine anti gens

Towards high yield production of pharmaceutical proteins with plant cell suspension cultures

Position effects and epigenetic silencing of plant transgenes

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

High expression of transgene protein in Spirodela

Micro algae come of age as a platform for recombinant protein production

Foot and mouth disease virus VP1 pro tein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast

Heat stable oral alga based vaccine pro tects mice from Staphylococcus aureus infection

Factors effecting expression of vaccines in microalgae

Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas rein hardtii chroloplasts

Viral vec tors for the expression of proteins in plants

Agrobacterium mediated transient expression as an approach to production of recombi nant proteins in plants

A novel two component tobacco mosaic virus based vector system for high level expression of multiple ther apeutic proteins including a human monoclonal anti body in plants

Magnifec tion -a new platform for expressing recombinant vac cines in plants

Plant based vaccines: unique advan tages

Expression of the Newcastle disease virus fusion protein in transgenic maize and immunological studies

Multimerization of peptide antigens for pro duction of stable immunogens in transgenic plants

Generation and immunogenicity of Japanese encepha litis virus envelope protein expressed in transgenic rice

Induction of protective immunity in swine by recombinant bamboo mosaic virus express ing foot and mouth disease virus epitopes

In planta production of two peptides of the classical swine fever virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X

Expression in plants and immunogenicity of plant virus based exper imental rabies vaccine

Delivery of subunit vaccines in maize seed

A corn based deliv ery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immu nity in swine

Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants

Oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein

Pea derived vaccines demonstrate high immunoge nicity and protection in rabbits against rabbit haemor rhagic disease virus

Plant made pharmaceuti cals: leading products and production platforms

Pro duction of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants

Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike parti cles

Expression of rotavirus capsid protein VP6 in transgenic potato and its oral immuno genicity in mice

Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants

Bovine herpes virus gD protein pro duced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity

Induction of a protective antibody response to foot and mouth disease virus in mice fol lowing oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1

Induction of a protective antibody response to FMDV in mice following oral immunization with transgenic Stylosanthes spp. as a feedstuff additive

Expression of hemagglutinin protein of rinderpest virus in transgenic tobacco and immunogenicity of plant derived protein in a mouse model

Systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model

Expression of hemagglutinin protein of rinderpest virus in trans genic pigeon pea

Oral immunogenicity of the plant derived spike protein from swine transmissible gastroenteritis coronavirus

Cloning and sequence analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutraliz ing epitope in plants

Successful oral prime immunization with VP60 from rabbit haemorrhagic disease virus pro duced in transgenic plants using different fusion strate gies

Mucosal and sys temic immunization elicited by Newcastle disease virus (NDV) transgenic plants as antigens

Expres sion of the fusion glycoprotein of Newcastle disease virus in transgenic rice and its immunogenicity in mice

Expression of immunogenic S1 glycoprotein of infectious bronchitis virus in trans genic potatoes

Transient expression of the ectodomain of matrix protein 2 (M2e) of avian RUSSIAN

Immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus H5N1 challenge infection

Immunogenicity study of plant made oral subunit vaccine against porcine reproductive and res piratory syndrome virus (PRRSV)

Expression of the rabies virus glycoprotein in transgenic tomatoes

Immunization against rabies with plant derived antigen

Development of an edible rabies vaccine in maize using the Vnukovo strain

Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein

Expression of the rabies virus nucleoprotein in plants at high levels and evaluation of immune responses in mice

Expression of rabies virus G pro tein in carrots (Daucus carota)