Lec. Molecular pharming: Plant production of vaccines, antibodies, therapeutic proteins- transient expression of recombinant proteins in plant

using viral vectors.

Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants. Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical proteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspension cells. Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressed proteins are functional and almost indistinguishable from their mammalian counterparts.

Biopharmaceuticals While early biopharmaceuticals were mainly proteins for replacement therapy such as erythropoietin (EPO) or growth hormones, current blockbusters include monoclonal antibodies (mAbs) or other proteins for the treatment of cancer and in flammatory diseases. Sales of many top-selling biopharmaceuticals are increasing,with US sales of themonoclonal antibody Herceptin used for the treatment of breast cancer growing by 82% from 2005 to 2006 alone (Lawrence, 2007). In 2007, the three top-selling pharmaceutical products sold by the Swiss drug-maker Roche were therapeutic monoclonal antibodies (Rituxan/Mabthera, Herceptin, and Avastin), contributing to sales of 9311 million € (Roche, Annual Finance Report for 2007).

The microbial system like E.coli is commonly used for production of recombinant protein production. The main drawback of the E.coli system is that it cannot be used for the production of glycoprotein. Simple proteins that are not glycosylated (such as insulin and human growth hormone) or that, while glycosylated in their native form, do not require glycosylation for pharmacological activity (such as α-, β-, and γ-interferon (IF), interleukin-2 (IL-2)) are mainly produced in E. coli.

Apart from E.coli, some of the yeast like Pichia pastoris and Saccharomyces is also used for the production of recombinant protein. The main advantage is the it can produce glycoprotein and the yeast system is free from toxin that is associated with E.coli system. But some of the glycoprotein produced is immunogenic.

Most of the human protein are glycoprotein and needs glycosylation for their proper function. Mammalian cell culture system is commonly used for the production of glycoprotein. For the production of more complex proteins requiring posttranslational modifications (like glycosylation –addition of carbohydrate moiety to the protein) the Chinese hamster ovary (CHO) cell is the main workhorse used. Further mammalian cell types used for the commercial production of approved protein drugs are baby hamster kidney (BHK) cells, mouse myeloma cell lines. The main advantage of this is the glycoprotein produced is not immunogenic in human or animal. However, this is a very expensive system for production of recombinant protein. Both the microbial and mammalian cell culture system is expensive process that require huge capital investment, trained main power to operate the system and operation cost is high thereby increasing the cost of the recombinant protein produced. Plant is a cheap alternative for the production of the recombinant protein. It requires less capital investment and scale up can be easily done in case of requirement. The main drawback of the plant system is that the glycoprotein produced are immunogenic and purification of the recombinant protein form

the plant tissue following good laboratory practices is difficult. However in large number of recombinant proteins have been successfully expressed in the plant and some are in clinical trial before commercialization. It is estimated that the plant derived recombinant protein can be 10-20 fold less expensive than the fermenter based recombinant protein production. Also the initial investment for establishment is less and scale up is possible.

Molecular Pharming — plants as production hosts for high-value compounds Plant cells combine the advantage of a full posttranslational modi fication potential with simple growth requirements and basically unlimited scalability ofwhole plants in the field. Plants and plant cells are versatile production systems, also allowing targeting of the recombinant proteins produced to different organs or subcellular compartments, thus allowing improved protection against proteolysis. Plants are not known to harborhuman or zoonotic pathogens,making thema safe host for the production of biopharmaceuticals. Human growth hormone was the first recombinant protein with therapeutic potential that was successfully expressed in plants ( Barta et al., 1986). More complex proteins soon followed, and Hiatt et al. (1989) produced a correctly assembled complete IgG1 antibody with full binding functionality in tobacco plants. Over the following years, a vast variety of pharmaceutical proteins were expressed in different plant species (with tobacco remaining the main production host) and targeted to different organelles or organs. Some examples include the expression of human serum albumin in tobacco and potato (Sijmons et al., 1990), human α-IF in rice (Zhu et al., 1994), and, more recently, IL-12 in tomato (Gutiérrez- Ortega et al., 2005). A milestone was the successful production of recombinant secretory IgA (sIgA) in tobacco plants (Maet al., 1995). It is believed that such sIgAs have great potential for use in topical passive immunotherapy (Fischer and Emans, 2000).

Examples of whole-plant-derived biopharmaceuticals in preclinical and clinical development The first clinical trial of a plant-produced biopharmaceutical was conducted by Planet Biotechnology Inc. for CaroRx™ (also known as the secretory variant of Guy's 13), a secretory IgA against Streptococcus mutans, which is the main causative agent of tooth decay. CaroRx is produced in tobacco grown in the field. The antibody was shown to effectively eliminate recolonization of S. mutans in humans for at least 4 months after oral topical application (Ma et al., 1998). CaroRx has received approval for human use in the E.U. (Kaiser, 2008). Large amounts of CaroRx are required for topical applications and it is dosed at 22.5mgper course of treatment (Ma et al., 1998). If itwere administered to the child population in Europe alone, more than 1000 kg of the antibody would be required annually (Ma et al., 2005). Another company which is expecting to enter a plant-produced protein product into the market soon is Ventria Bioscience, which uses its ExpressTec technology for high-level production of recombinant proteins in field-grown rice. Two of Ventria's primary products are human lactoferrin and human lysozyme. Expression levels reported are extremely high, with the recombinant protein contributing to 60% of total soluble protein or 1% of rice flour weight (10 g/kg) in the case of lysozyme and to 25% of total soluble protein or 0.5% of rice flour weight (5 g/kg) in the case of lactoferrin (Huang, 2005). The company is developing several products containing human lactoferrin and human lysozyme. An oral rehydration solution containing both enzymes as a dietary supplement for acute pediatric diarrhea is expected to be released soon.

Plants are genetically modified to produce pharmaceuticals, also called plant-made pharmaceuticals (PMPs). The first recombinant plant-derived pharmaceutical protein (PDP) was human serum albumin, initially produced in 1990 in transgenic tobacco and potato plants. Fifteen years on, the first technical proteins produced in transgenic plants are on the market, and proof of

concept has been established for the production of many therapeutic proteins, including antibodies, blood proteins, cytokines, growth factors, hormones, recombinant enzymes and human and veterinary vaccines.

Plant production of vaccines Vaccinology is a rapidly expanding research field and new vaccination strategies have been developed thanks to modern technologies based on the rational design of attenuated pathogens, live recombinant vaccines and protein (antigen)- or peptide (epitope)-based subunit vaccines (Plotkin 2005). The aim of these approaches is to obtain efficient and safe vaccine formulations being able at the same time to induce effective, long lasting immunity against complex viral pathogens such as HIV-1 (Nabel 2001), influenza A/H5N1 virus (Stephenson et al. 2004), SARS-coronavirus (Stadler and Rappuoli 2005). To reach this goal, targets must be found able not only to generate long-term memory B cell producing neutralizing antibodies to block free pathogens, but also T cell mediated immunity for the control of pathogen spreading by the elimination of infected cells (Lambert et al. 2005). The possibility to produce subunit vaccines through plants paves new ways and offers solutions to some of the problems associated to traditional production systems.

Protein antigens from various pathogens have been expressed in plants and used to produce immune responses resulting in protection against diseases in humans. Plant-derived vaccines have been produced against Vibrio cholerae, enterotoxigenic E. coli, hepatitis B virus, Norwalk virus, rabies virus, human cytomegalovirus, rotavirus, and respiratory syncytial virus F. Edible vaccines may be particularly valuable as a low-cost delivery mechanism for immunization against various diseases in developing countries. In 1992, Mason et al. (1992) described the expression of Hepatitis B virus (HBV) surface antigen in tobacco, paving the way not only for the production of antigens for parenteral application in plants, but also for the production of “edible vaccines”. The HBV surface antigen was also expressed in transgenic lupin and lettuce plants. When mice and human volunteers ate the antigen containing plants they developed a specific antibody response (Kapusta et al., 1999). Other plants considered for oral vaccine delivery are tomato, carrot, banana, and corn. Medicinal and commercial utility of vaccines for human use produced in plants will depend on the performance in human clinical trials. According to Marshall (2007), several vaccines, including oral vaccines, were in clinical trials (or had completed clinical trials) in October 2007, conducted by the group of C. Arntzen at Arizona State University and by companies such as Large Scale Biology Corporation and Prodigene. Human safety testing has successfully been completed for orally delivered spinach expressing a rabies antigen (Yusibov et al., 2002), E. coli heat-labile toxin as a vaccine for diarrhea produced in transgenic maize (ProdiGene Inc.) and transgenic potato tubers (Tacket et al., 1998), Hepatitis B virus surface antigen in transgenic potato (Thanavala et al., 2005) and transgenic lettuce (Kapusta et al., 1999) and Norwalk virus capsid protein in transgenic potato (Tacket et al., 2000). There is a concern that edible vaccines might induce tolerance instead of immunity, thereby actually weakening an immune response to the antigen, although signs of such induced tolerance have not been reported yet (Vermij, 2004).

Several antigens have been successfully expressed in plants by stable nuclear transformation with different ends. In some cases, plants were considered only as “biofactories” for massive production, while in others edible plant varieties have been chosen for the direct oral delivery of the expressed antigens.

Plant production of antibodiesAntibodies are multi-subunit glycoproteins, produced by the vertebrate immune system. They recognize and bind to their target antigens with great affinity and specificity, which allows them to be used for many applications, including the diagnosis, prevention and treatment of human and animal disease. It is estimated that approximately 1000 therapeutic recombinant antibodies are under development, up to one quarter of which may already be undergoing clinical trials. A large proportion of these antibodies recognize cancer antigens but others have been developed for the diagnosis and treatment of infectious diseases, acquired disorders and even transplant rejection (Gavilondo and Larrick 2000). Most of the recombinant full-length immunoglobulins being developed as pharmaceuticals are produced in mammalian cell culture, a few in hybridoma lines but most in immortalized lines which have been cleared by the FDA and equivalent authorities in other countries.

Over the last 15 years, plants have emerged as a convenient, economical and scalable alternative to the mainstream antibody production systems which are based on the large-scale culture of microbes or animal cells (Chu and Robinson 2001; Wurm 2004). In this chapter, we discuss the advantages and disadvantages of plants for antibody production, the diverse plant-based systems which are now available, and factors governing the success of antibody production in plants.

Recombinant antibody technology Structure of natural antibodies The typical antibody format is the mammalian serum antibody, which comprises two identical heavy chains and two identical light chains joined by disulfide bonds (Figure 1).

Fig. 1. Structure of a typical mammalian serum antibody, comprising two identical heavy chains (dark gray) and two identical light chains (light gray). Solid black lines indicate continuation of the polypeptide backbone (simple lines indicate the constant parts of the antibody, curly lines indicate the variable regions). Antibody domains are indicated by circles. Disulfide bonds are represented by thick black bars.

Each heavy chain is folded into four domains, two either side of a flexible hinge region which allows the multimeric protein to adopt its characteristic Y-shape. Each light chain is folded into two domains. The N-terminal domain of each of the four chains is variable, i.e. it differs among individual B cells due to unique rearrangements of the germ-line immunoglobulin genes. This part of the molecule is responsible for antigen recognition and binding. The remainder of the antibody comprises a series of constant domains, which are involved in effector functions such as immune cell recognition and complement fixation. Below the hinge, in what is known as the

Fc portion of the antibody, the constant domains are class-specific. Mammals produce five classes of immunoglobulins (IgG, IgM, IgA, IgD and IgE) with different effector functions. The Fc region also contains a conserved asparagine residue at position 297 to which N-glycan chains are added. The glycan chains play an important role both in the folding of the protein and the performance of effector functions. Antibodies are also found in mucosal secretions, and these secretory antibodies have a more complex structure than serum antibodies. They are dimers of the serum-type antibody, the two monomers being attached by an additional component called the joining chain. There is also a further polypeptide called the secretory component, which protects the antibodies from proteases (Figure 2).

Fig. 2. Structure of a mammalian secretory antibody, comprising a dimer of the typical serum antibody, and including two additional components, the joining chain (black disc) and secretory component (white disc). Heavy chains are shown in dark gray and light chains in light gray. Solid black lines indicate continuation of the polypeptide backbone (simple lines indicate the constant parts of the antibody, curly lines indicate the variable regions). Antibody domains are indicated by circles. Disulfide bonds are represented by thick black bars.

Production of secretory IgA in transgenic plants.

The assembly and expression of the multimeric, complex molecule secretory immunoglobulin (sIgA) was first described successfully in transgenic plants and plants remain the best system to express this molecule. Indeed, the large scale production of recombinant sIgA is a very challenging task for tow main reasons: (i) the components of this molecule are naturally produced by two distinct cell types (plasma and epithelial cells) and (ii) the final product is a large complex molecule of almost 400 kDa display numerous post-transcriptional modifications (intra and inter chain disulfide bonds and glycosylation sites). SigA has also been produced in CHO cells but the cost of production might bee too high to envisage commercialization.

AntibodiesThe secretory IgA consists of two IgA molecules linked by a joining (J) chain and associated with a secretory component (SC). To exert its protective activity on the mucosa, polymeric IgAs (pIgAs, mostly IgA dimmers) are transported across the epithelium after binding to the polymeric immunoglobulin receptor (pIgR), which is expressed basolaterally on the epithelial cells. During transport, the pIgR (also known as the transmembrane secretory component) is cleaved and the secretory component (SC) is

released in association with pIgA to form sIgA. Quantitatively, sIgA is the most important antibody class with 40- 60 mg/ kg produced every day, whereas the daily production of IgG is only of 30 mg/kg. Moreover, the covalent binding of SC enhances resistance to proteolytic degradation making their the most stable form of Ab. In humans, there are two IgA subclasses, IgA1 and IgA2, that differ only in the hinge region: IgA1 contains a 13 amino acid, proline rich sequence which is not present in IgA2. The amino acid composition of the IgA hinge region renders it more resistant to proteases then other immunoglobulins. However, IgA1 is particularly sensitive to proteases produced by Gram-negative bacteria, whereas IgA2 is relatively more resistant (sue to the absence of the proline rich region)

The J chain is 137 amino acid, 15.6 kDa glycoprotein that is added just before the secretion of pIgA by the plasma cells. The J chain is a key protein in the synthesis of sIgA because it promotes polymerization of IgA and because its presence in the polymer is required for their affinity of pIgR/secretory component.

The secretory component (SC), an 80 kDa glycoprotein is the extracellular domain of the pIgR synthesized by the mucosal epithelium. During basal-to-apical transport across the epithelial cells, the pIgR ectoplasmic domain is cleaved, releasing SC in association with pIgA, thus forming sIGA. The human pIgR contains 5 domain. Domain I of the pIgR is involved in the initial non-covalent attached to pIgA. Subsequently, during transcytosis, the pIgR becomes covalently attached to pIgA via cysteine residues in domain V.

Passive immunization with sIgA

Active immunization has been successful in protecting against several infectious diseases. However, vaccines are still not available for numerous pathogens (eg. Human immuno deficiency virus, respiratory syncytial virus, hepatitis virus). Furthermore, active immunization is generally less effective in immuno-compromised individuals. Passive immunization with sIgA should provide a better protection level against pathogens than monomeric antibodies (i.e. IgGs or IgAs). It has been shown that SC is essential for the stability of the whole sIgA molecule when targeting the gastrointestinal (GI) tract.

Furthermore, sIgAs have a higher binding avidity; of r their antigens than monomeric antibodies, because of their four antigen combining sites.

Production of recombinant sIgA.Thus, for only two-expression system have been successful in producing rec. sIgA.

1. Full length sIgA was first expressed in transgenic tobacco2. In the past 5-6 years, several groups have succeeded in expressing recombinant

sIgA in mammalian cells. The use of CHO (Chinese hamster ovary) cells to produce sIgA has been the most successful strategy, and has relied on the successive stable transfecion and selection of CHO clones capable of expressing monomeric, dimeric and finally sIgA recombinant molecules. However, the levels of antibody production is stable transfectomas have generally been lower than in murine hybridomas.

Production of recombinant sIgA in plants.Production of full length antibodies in plants.

Plants have the ability to assemble immunoglobulin heavy chains and light chains to form full-length antibodies very efficiently. In plants, the assembly of immunoglobulin takes place in ER. Signal peptide sequences directs translocation into the lumen of the endoplasmic reticulum (ER). Both plant and non-plant signal sequences form a variety of sources are sufficient for ER-targeting. Plant chaperones homologous to mammalian BiP, GRP94 and PDI (Protein disulfide isomerase) have been described within the ER, and the expression of immunoglobulin chains in plants is indeed associated with increased BiP and PDI expression. Co-expression of heavy and light chains resulted in IgG assembly and displacement of BiP from the heavy chain as the amount of light chain increased.

Murine IgG1 (Guy’s 13) that binds to the adhesion protein of Streptococcus mutants, the primary cause of dental caries. The strategy used to produce this antibody in plants was to express each immunoglobulin chain separately in different plant lines and then to stack the two genes in the same plant line by crossing parental plants individually expressing the heavy and light chains. This involved two generation of plants, and using this

technique, the yield of recombinant antibody was consistently high (approximately 1% TSP). Guy’s 13 IgG is relatively easy to purify in large quantities from tobacco. Guy’s 13 can also be expressed in transgenic plants with a transmembrane sequence so that it is retained in the plasma membrane with variable regions protruding into the apoplasm. Transgenic plants expressing antibodies immobilized in such a fashion may have important applications in phytoremediation and phytomining.

Other groups have expressed IgG antibodies using double transformation technique or have cloned the light and heavy chain genes together in a single Agrobacterium T-DNA vector. Both strategies can save time and effort.

Production of multimeric antibodies: sIgA.

The ability to stack gene in transgenic plants by successive crosses between individually transformed parental plants is a considerable advantage in attempting to construct multimeric protein complexes, such as secretory antibodies.

In order to generate a secretory antibody version of Guy’s 13 in plants the carboxyl terminal domains of the Guy’s 13 IgG antibody heavy chain were modified by replacing the Cγ3 domain with the Cα2 and Cα3 domain of an IgA antibody, these being required for binding to the J chain and SC. Four transgenic plants were generated to express independently the Guy’s 13 kappa chain, the hybrid IgA-G antibody heavy chain, the mouse J chain and the rabbit SC. A series of sexual crosses was performed between these plants and filial recombinants in order to generate plants in which all four protein chains were expressed simultaneously. In the final, quadruple transgenic plant, three froms of the antibody was detectable by western blot anlysis of samples prepared under non-reducing conditions. These bands had approximate molecular mass of 210 kDa (monomeric IgA-G), 400 kDa (IgA-G dimerized with the J chain) and 470 kDa (dimeric IgA-G associated with the SC). The assembly was very efficient, with greater than 50% of the SC being associated with dimeric IgA-G.

Frigerio et al. demonstrated that secretion of sIgA-G proceeded at a very slow rate in tobacco leaf cells. After 24 hour only about 10% of newly assembled molecules had been secreted with the bulk probably remaining in the ER.

Finally, in a human trial, the plant-derived secretory Guy’s 13 antibody prevented oral colonization by Streptococcus mutants, thereby demonstrating for the first time the therapeutic application in humans of a recombinant product derived from plant.

The Guy’s 13 sIgA-G plantibody technology is licensed to Planet Biotechnology Inc. (USA) and is currently in clinical trials under the product name CaroRxTM.

Transient transformation To get round the difficulties encountered in obtaining high rate of expression of the heterologous gene in nuclear transformed plants, transient transformation strategies have been devised. In this case, the gene encoding the antigen of interest is inserted in the genome of a pathogen that is used as a vector for expression during plant infection. The

two major techniques used to get transient transformation are based on plant viruses- or Agrobacterium-mediated infection of fully developed plants.

Plant viral expression vectors for recombinant protein expression

The transgenic approach has some shortcomings, including the length of time required to obtain the transgenic producer lines, low levels of expression, and inherent difficulties in the modification of an existing product. In some expression hosts, scaling up production also takes a ling time.

The foreign gene is inserted into the viral genome so that, upon infection of the host plant cell, the transgene is replicated and expressed along with native viral genes. This method of transient expression adds several further advantages to plant-based expression, including improved time efficiency, higher levels of target protein expression, flexibility and convenience in the modification of existing products (or the development of new ones), ease of scale-up, flexibility in the selection of a production host and the potential for protein manufacture in contained facilities. Furthermore, target proteins (in particular antigenic peptides) can be genetically fused to viral structural proteins, such as coat proteins, so that the plant virus is used not only for expression but also for the delivery of the vaccine antigens. To date, the coat proteins from a number of plant RNA viruses have been successfully used as carrier for antigenic peptides derived from various pathogens.

Plant viral vectors are being successfully developed and exploited for the industrial-scale expression of heterologous proteins and as a research tool for studies of gene expression. The initial engineering strategy (the ‘full virus’ vector strategy) aimed to design a vector that was essentially a wildtype virus, which was modified to carry and express a heterologous sequence that encoded a gene of interest. The new emerging trend (the ‘deconstructed virus’ vector strategy) reflects an ideology that recognises the inherent limitations of the viral process. It attempts to ‘deconstruct’ the virus, by eliminating functions that are limiting or undesired, and to rebuild it, either by delegating the missing necessary functions to the host (which is genetically modified to provide those functions) or by replacing them with analogous functions that are not derived from a virus.

Plant RNA viruses as expression vectors

The majority of viruses that infect plants have single-stranded, positive sense RNA genomes. It has therefore been necessary to use infectious cDNA clones for the in vitro manipulation of RNA viruses, allowing them to be developed as effective tools for the commercial production of target protein in plants. Siegel conceptualized the potential use of RNA viruses as expression vectors. Brome Mosaic Virus (BMV) and Tobacco Mosaic Virus (TMV) were the first two RNA viruses to be converted into expression vectors. The genomes of number of plant RNA viruses have been engineered to express target sequences. Based on genome structure and identification of virus gene functions, several approaches have been employed for the expression of foreign sequences using plant viruses as expression systems.

These include:

(i) replacing non-essential viral genes with target sequences. (ii) Inserting target sequences into the viral genome as an additional gene with

an additional promoter. (iii) Fusing target sequences with viral structural gene with a cleavage site or

read-through sequence. (iv) Functional complementation of defective viral components, and (v) Trans-complementation of viral genes through transgene expression in the

host plant.

Examples of plant viruses used in the development of expression vectorsVirus Strategies used Pathogen or protein

epitopeTobacco Mosaic Virus

CP replacement, second subgenomic promoter (sgp), CP fusion, Read through fusion with CP, fusion with cleavage site

Malaria, rabies virusFMDV, MAB, alpha-trichosanthin (Inhibitor of HIV), allergens, scFvs, HCV(Hepatitis C virus vaccine)

Potato Virus X Second sgp, CP fusion, CP fusion with FMDV 2A element, IRES elements.

GFP, rotavirus, scFv

Cowpea Mosaic Virus

Fusion with CP, fusion to CP with protease cleavage site, complementation

GFP, HIV

Alfalfa Mosaic Virus Fusion with CP, second sgp HIV, rabies, Measles, RSV

RNA Virus + and - strandIn RNA viruses, the viral RNA may be directly replicated or act as a template in the synthesis of DNA. The RNA of single-stranded RNA viruses may be the positive strand (the mRNA or the negative strand (complementary of the mRNA). RNA viruses with (-) strand and dsRNA genomes must carry the RNA polymerase in the viral particle because the incoming viral RNA into plant cell can be neither translated nor copied by the cellular machinery. Deprotinized RNA genome (only nucleic acid) are not non-infectious. In contrast (+) strand viral particles lack a virion polymerase and deprotinized RNA (only viral RNA) are infectious. All RNA viruses except retroviruses encode an RNA-dependent RNA polymerase (Replicase) to catalyze the synthesis of new genomes and mRNA. (The intact virus particle, which is referred to as a virion consists of a nucleic acid molecule encased by a protein capsid. In some of the more complex virions, the capsid is surrounded by a lipid bilayer and glycoprotein containing envelope, which is derived from a host cell membrane).

Tobacco Mosaic VirusTMV is (+) RNA virus. It causes leaf mottling and discoloration. TMV is a rod-

shaped particle. Its ~2130 identical copies of coat protein subunits (158 amino acid residue; 17.5 kDa) are arranged. TMVs single RNA strand (~6400 nt).

Tobacco Mosaic virus (TMV) genomic and subgenomic RNAs. A. 6400 nucleotide TMV genomic RNA acts as a messenger RNA for the expression

of the two replicase-associated proteins of 126kDa and 183 kDa. B. The other genes in TMV RNA are expressed form subgenomic RNAs (sgRNA)

during the replication cycle.

The rod shaped TMV virions are composed of the CP protein and the genomic RNA. 5’end of TMV RNA contain a typical 5’ terminal cap structure (m7 GpppG) was discovered in TMV RNA. Infectivity lost on removal of cap structure. The 3’ terminal five codons of the 180 kDa protein gene overlap with the third ORF, coding for the 30 kDa protein. This third ORF terminates two nucleotides before the initiation codon of the fourth ORFs which encodes CP. The genes located internally in the genome are not expressed from the genomic RNA, but from subgenomic RNAs, which are generated during the course of replication.

The viral genome codes for two replicase-associated proteins translated right from the genomic RNA, and a movement protein (MP), and a CP translated from 3’ coterminal subgenomic mRNAs. The genome of TMV employs two distinct strategies for protein expression: read-through of an amber stop codon and production of subgenomic RNAs. The 5’-ORF of TMV is translated from the genomic RNA resulting in the accumulation of a 126 kDa protein. A translational read-through of an amber stop codon results in synthesis of a 183 kDa protein instead of the 126 kDa protein. These two proteins are first translated from the genomic RNA and comprise the viral RNAdependent RNA polymerase (RdRp) synthesizing a fulllength minus-sense copy of the genome. These copies are then used as a template for the amplification of plus sense genomes and the subgenomic mRNAs for MP and CP synthesis. The MP, a 30 kDa protein, is producedearly in the infection cycle and is required for cell to- cell movement of progeny viral RNA genomes by modifying the size exclusion limit of plasmodesmatal junctions. Later in infection the production of genomic RNA, CP mRNA, and CP become the major events in the viral replication cycle. The CP, a 17.5 kDa structural protein produced at very high levels, is required for systemic movement of the virus through the vascular system in the plant and for encapsidation of the viral genome.

Two general expression vector designs for TMV based vectors were developed, CP fusion vectors and dual subgenomic expression vectors.

1. CP Fusion vector In CP fusion vectors, a ‘‘read-through sequence’’ is introduced immediately after the CP stop codon and before the ORF of interest. Peptides of interest, e.g., the epitope of the murine hepatitis virus, are fused to the CP and are therefore incorporated into soluble virions presenting the peptide on the surface of the virion. TMV particles have also been used as an epitope-presentation system. The helical arrangement of 2130 copies of coat protein around the viral RNA allows the presentation of multiple copies of the foreign epitope on the virus surface. At the same time, this can be detrimental to virion stability because the extra peptide can destabilize the virion structure. To circumvent this problem, a more stable system was developed exploiting the read through sequence from the replicase gene, so that both wild type and fusion peptide containing coat proteins were produced.

2. Subgenomic expression vectorsA schematic diagram of the TMV-based dual subgenomic vector described herein

is shown in Fig. 1. This 30B-based hybrid vector is designed on the characteristics identified by a series of TMV expression vectors

Invitro production of infective recombinant TMV-RNA and inoculation of N. benthamiana plants.

Capped infectios viral RNA is generated in vitro from the linearised plasmid with the ribomax Large Scale RNA production system-T7 kit (Promega) and the Cap-Analog (Ambion) according to manufacture’s instructions.

T7 RibomaxTM Express Large scale RNA production system is an in vitro transcription system designed for the consistent production of milligram amounts of RNA in a short amount of time. DNA templates are usually linearized prior to in vitro transcription to produce RNA transcripts of defined length. Blunt end obtained by treating with DNA polymerase I Large (Klenow fragment)

T7 reaction components: Sample reactionRibomax Express T7 2X Buffer 10 µlLinear DNA template (1 µg total) 1-8 µlNuclease Free water 0-7 µlEnzyme Mix, T7 Express 2 µl

---------Final volume 20 µl

---------Mix gently and incubate at 37 C for 30 min.

rNTPs. Enzyme Mix: T7 RNA polymerase, recombinant RNasin- Ribonuclease inhibitor and Rec. inorganic pyrophosphatase.

Cap Analog (Ambion)- m7 G(5’) ppp (5’) G (Cap Analog) is used for the synthesis of 5’ capped RNA

molecules in vitro transcription reactions. Capped mRNAs are genrally translated more efficiently in reticulocyte lysate and wheat germ in vitro translation systems. Uncapped mRNAs are rapidly degraded after microinjection into cells.

Infection of tobacco plant with TMV RNAThe integrity of the transcribed RNA is assessed by electrophoresis on a 1% agarose gel. After dusting N. benthamiana leaflets (2 per plant) lightly with carborundum powder, 2 μg in vitro transcripts are gently rubbed on their surface to initiate infection.

Recombinant protein isolationBetween one and three weeks after inoculation, leaflets of infected plants are frozen in liquid nitrogen and pulverized to isolate the recombinant protein in PBS buffer.

TMV expression vector was used for the expression of subunit vaccine against hepatitis C virus (HCV). A consenses sequence matching hypervariable region 1 (HVR1) of HCV, encoding a potential neutralizing epitope of 27 amino acids was fused to the C-terminus of CTB. Mice immunized intranasally with plant extract containing ~0.5 – 1.0 µg CTB/HVR1 developed anti-HVR1 antibodies. The same epitope has been engineered as fusion with other plant viruses such as AIMV. Human volunteers (in FDA approved trials) fed with spinach containing recombinant particles generated both IgG and IgA response specific to the pathogen.

Due to size limitations, there is unstable long distance movement of the chimeric virus in tobacco.

Transfection of N. benthamiana plantsAbout four-week-old N. benthamiana plants approximately at a 10-leaf stage are suitable hosts for transfection with agrobacteria to subsequently produce viral RNA. Veins of two leaves per plant are wounded carefully by pricking with a needle. Stem and leaves of the plant are then submerged for twenty minutes into the overnight culture of Agrobacterium. The N. benthamiana plants treated in that way are then placed in the dark in a humid atmosphere for 24 h to recover from the treatment. Thereafter, plants are kept in a growth chamber under 16 h of daylight at 22 C.

TMV vectors have been used to produce of many different kinds of proteins in plants including allergens [5,6], antibodies [7] or antibody fragments [8], and vaccine candidates [9,10]. TMV is an RNA virus that expresses large amounts of coat protein from a viral subgenomic promoter. To convert TMV to an expression vector an additional, heterologous coat protein subgenomic promoter and restriction enzyme sites for cloning of foreign DNA sequences were inserted into a T7 promoter driven cDNA clone of TMV[11]. In vitro transcription of this plasmid with T7 RNA polymerase is needed to generate biologically active transcripts. Transcripts are typically rub-

inoculated by hand onto plants to initiate an infection [4]. The in vitro transcription and rub inoculation steps in particular, add significantly to the cost and complexity of using TMV vectors. Agroinfection [12,13] is a less-expensive and more reproducible strategy for infecting plants with RNA viruses. In agroinfection a plant-functional promoter and RNA virus cDNA are transferred as T-DNA from Agrobacterium tumefaciens into plant cells. The T-DNA is transcribed in planta, to generate biologically active viral RNAs that can initiate self-replication. Although agroinfection has been used for several different plant RNA viruses, it has not been routinely used with TMV-based vectors. Recently, an agroinfection-compatible TMV replicon was constructed with extensive modifications to the TMV cDNA. These modifications included (1) the deletion of the TMV coat protein (CP) gene, (2) generating nearly 100 point mutations to destroy cryptic introns in the viral cDNA, and (3) inserting multiple (up to 24) plant-gene introns into the TMV cDNA sequences in a binary vector [14]. The point mutations and inserted introns dramatically improved the infectivity of the TMV replicon delivered to plants by agroinfection. However, because of the deletion of the CP gene, this replicon cannot move systemically in plants. Plants were therefore infected in a process called "magnifection" [9,15] in which whole plants were submerged and infiltrated with A. tumefaciens cultures carrying intron-modified TMV sequences in a binary vector.

The ‘full virus’ strategy Under the ‘full virus’ scenario, the delivery of amplicons is achieved by infecting the host with a mature viral particle or with a DNA/RNA molecule that contains a complete copy of the viral vector. Depending on the vector and its ability to move systemically, 2–3 weeks will be necessary for the vector to move to as many organs and cells of the host plant as possible. For example, the ability of viruses to infect the host is naturally low and typically requires either mechanical injury of the host tissues or delivery by an insect vector. Furthermore, systemic spread is usually a very species-specific process that is easily impaired by genetic manipulation of the virus. The full-virus strategy by designing vectors that were capable of systemic spread. The limitations of this approach are obvious: inserts larger than 1 kb are often not expressed properly and do not move well systemically; only short epitopes (of 20 amino acids or less) can be presented effectively as fusions to coat protein; systemic vectors never infect all of the harvestable parts of the plant (e.g. they rarely infect lower leaves); the process is asynchronous as it progresses at different speeds in different leaves; and the vector is usually unstable, thus much of the infected tissue does not express the protein of interest. These of vectors based on the ‘full virus’ strategy seems to be constrained, primarily by the limitations on the number and type of proteins that can be expressed using this technology. The most obvious application for these vectors today seems to be for the production of vaccines as coat protein fusions of short antigenic peptides or through the chemical coupling of antigens to engineered viral particles.

The ‘deconstructed virus’ approach may rely on the use of Agrobacterium to efficiently introduce a DNA copy of the viral vector into a plant cell (Table 1). All of the processes necessary for the generation of a functional DNA or RNA amplicon from T-DNA occur in plant cells. In fact, ‘agroinfection’ has been used for many years because it is often much more efficient than infection using assembled viruses, and definitely much more efficient than the use of DNA or RNA infectious molecules. In case of RNA viruses, agroinfection also represents an inexpensive alternative to the in vitro transcription methods that are used to convert a DNA vector into an infectious RNA. The agrodelivery of the two genomic components of cowpea mosaic virus from a mixed suspension of bacteria cultures, each harbouring different subgenomic complements, was demonstrated in an interesting recent publication. Two elements of first-generation viral vectors that are limiting are the ability of the vector to move systemically, which is provided by the coat protein, and the low level of expression of the protein of interest, probably because a significant fraction of the cell metabolic resources are devoted to synthesis of a large amount of coat protein.

A simple solution was to eliminate the coat protein and replace the systemic movement ability by artificial delivery of the viral vector to the entire plant using Agrobacterium.

On the basis of these findings, a simple fully scalable protocol for heterologous protein expression in plants was designed that is devoid of stable genetic transformation of a plant, but instead relies on the transient amplification of viral vectors delivered to the entire plant using Agrobacterium. The process is in essence an infiltration of whole mature plants or of detached mature leaves with a highly diluted suspension of agrobacteria carrying a proviral replicon on the T-DNA. In this case, infiltration of agrobacteria replaces the conventional viral functions of primary infection and systemic movement (Figure 1). Amplification within each cell and movement from cell-to-cell is performed by the replicon. Depending on the vector used, the host organism and the initial density of bacteria, the process takes from 4 to 10 days and, depending on the specific gene of interest, can result in the expression of up to 5 g recombinant protein per kg of fresh leaf biomass or over 50% of total soluble protein. Furthermore, as the viral vector does not contain a coat protein gene, it can express longer genes (up to 2.3 kb inserts or up to 80 kDa proteins). The infiltration of plants/detached leaves with bacteria has been achieved in many ways, one simple process being vacuum infiltration after immersing the aerial part of the plant in a bacterial suspension and applying a weak vacuum (approximately –0.8–0.9 bar) for 10–30 s [13__,14,15]. This new expression strategy, called magnifection (Figure 2), combines the advantages of three biological systems: the speed and expression level/yield of the virus, the transfection efficiency of Agrobacterium, and the posttranslational capabilities and low production cost of plants.

Schematic description of infection and spread of replicons based on (a) first-generation and (b) second-generation viral vectors.

General scheme for recombinant protein production in plants using magnifection. Depending on the protein of interest, magnifection can produce up to 0.5–5 g recombinant protein per kg of leaf biomass. With a green biomass yield of 100–300 ton/hectare per year, the yield of recombinant protein is expected to be 50–600 kg/hectare/year. The expression time is 6–10 days, making magnifection especially attractive for those applications where rapid industrial manufacturing is required. Components (a), (e) and (f) are standard existing industrial processes. Components (b), (c) and (d) are new elements, because large-scale infiltration (b) requires special equipment and the use of bacteria in steps (b), (c) and (d) requires biological containment to prevent the release of genetically engineered bacteria into the open environment.

Industrialization of the magnifection technology is currently an essential part of the activity of several business and academic groups, and the most serious constraint is the need for a specialized facility that allows handling of plants treated with agrobacteria and that can operate under conditions compliant with current good manufacturing practice. TMV is an RNA virus that expresses large amounts of coat protein from a viral subgenomic promoter. To convert TMV to an expression vector an additional, heterologous coat protein subgenomic promoter and restriction enzyme sites for cloning of foreign DNA sequences were inserted into a T7 promoter driven cDNA clone of TMV[11]. In vitro transcription of this plasmid with T7 RNA polymerase is needed to generate biologically active transcripts. Transcripts are typically rub-inoculated by hand onto plants to initiate an infection [4]. The in vitro transcription and rub inoculation steps in particular, add significantly to the cost and complexity of using TMV vectors. Agroinfection [12,13] is a less-expensive and more reproducible strategy for infecting plants with RNA viruses. In agroinfection a plant-functional promoter and RNA virus cDNA are transferred as T-DNA from Agrobacterium tumefaciens into plant cells. The T-DNA is transcribed in planta, to generate biologically active viral RNAs that can initiate self-replication. Although agroinfection has been used for several different plant RNA viruses, it has not been routinely used with TMV-based vectors. Recently, an agroinfection-compatible TMV replicon was constructed with extensive modifications to the TMV

cDNA. These modifications included (1) the deletion of the TMV coat protein (CP) gene, (2) generating nearly 100 point mutations to destroy cryptic introns in the viral cDNA, and (3) inserting multiple (up to 24) plant-gene introns into the TMV cDNA sequences in a binary vector [14]. The point mutations and inserted introns dramatically improved the infectivity of the TMV replicon delivered to plants by agroinfection. However, because of the deletion of the CP gene, this replicon cannot move systemically in plants. Plants were therefore infected in a process called "magnifection" [9,15] in which whole plants were submerged and infiltrated with A. tumefaciens cultures carrying intron-modified TMV sequences in a binary vector. While the magnifection process is very efficient, it is not easily adapted to a high throughput workflow. Also, the increased size of the intron-modified vectors can make cloning into these vectors more challenging. In addition, it is not clear if the intron-modified vectors are absolutely required for efficient agroinfection of local and systemic infection of plant tissue with a TMV vector. Maps

TMV is a rod-shaped virus that has a single-stranded plus-sense RNA genome. TMV expresses four proteins from three open reading frames (ORFs). Two viral genes (the viral movement protein and the capsid protein) are expressed from separate subgenomic promoters. TMV has typically been modified to express foreign genes by either replacing a viral gene (such as the coat protein [CP] gene, for example) with a gene of interest (for review, see Scholthof et al., 1996) or by inserting an additional subgenomic promoter (Dawson et al., 1989; Donson et al., 1991; Pogue et al., 1998) into the viral genome to drive the expression of an inserted foreign gene. Plants can be inoculated with TMV vectors through a process called agroinfection. In agroinfection, A. tumefaciens was used to deliver T-DNA composed of 35S promoter-driven TMV cDNA to plant cells. Transcription of T-DNA in the plant nucleus generated RNA that was capable of initiating self-replication in the cytoplasm. Multiple reports have documented the low agroinfection efficiency of the typical 35S-driven TMV vector (Turpen et al., 1993; Marillonnet et al., 2005; Man and Epel, 2006). Here, we report on the construction of an improved agroinfection-compatible TMV vector that lacks the TMV CP gene coding sequence. This modification resulted in a vector with several significant improvements, such as (1)much higher agroinfection efficiency; (2) higher recombinant protein expression levels; and (3) inability to form virus particles during its infection/ replication cycle. This new expression vector is called the TMV RNA-based overexpression (TRBO) vector. Here, we demonstrate that the TRBO vector can produce up to 100 times more recombinant protein than the P19-enhanced agroinfiltration transient expression system described above. It is proposed that, because of its efficacy and ease of use, the TRBO vector will be a useful transient expression vector for production of recombinant proteins in plants for either research or production purposes.

Figure 1. Maps of plasmids used in this project. The T-DNA regions of binary plasmids used in this project are represented. Block arrow, CaMV duplicated 35S promoter. Black box, CaMV polyA signal sequence/ terminator. Dark gray box, Tobacco etch virus 5#-nontranslated leader sequence. Light gray box, Ribozyme. Bent arrows, Subgenomic promoters. ORFs are represented by white boxes. Identities of ORFs are labeled in white boxes. Replicase, TMV 126K/183K ORF; MP, movement protein; P19, 19-kD RNA-silencing suppressor gene from Tomato bushy stunt virus.

Another advantage of TRBO is that it does not produce TMV CP. Because TMV CP is required for systemic movement, TRBO is not capable of systemic movement in plants. It also will not produce virions in plants. This has definitive biocontainment and protein purification advantages. First, this feature reduces the chances for inadvertent plant-to-plant movement of the vector. Second, when extracting proteins from TRBO-infected tissue, the recombinant protein of interest does not need to be purified away from virion particles. If one is using a viral vector that does generate virus particles (such as JL24), efforts must be taken to both separate virion particles from the recombinant protein of interest and also to inactivate virus particles in any extracts of infected plant materials. These issues are not a concern with TRBO because it does not generate virus particles.