The Use of Transient Expression Systems for the ...

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The Use of Transient Expression Systems for the Rapid Production of Virus-like Particles in Plants Eva C. Thuenemann1*, Paolo Lenzi1, Andrew J. Love2, Michael Taliansky2, Martina Bécares3, Sonia Zuñiga3, Luis Enjuanes3, Gergana G. Zahmanova4, Ivan N. Minkov5, SlavicaMati6, Emanuela Noris6, Ann Meyers7, Alta Hattingh7, Edward P. Rybicki7, Oleg I. Kiselev8 Nikolai V. Ravin9, Michael A. Eldarov9, Konstantin G. Skryabin9, and George P. Lomonossoff1 1

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK; 2Cell and Molecular Sciences, The James Hutton Institute (Dundee), Invergowrie, Dundee, DD2 5DA, UK; 3 Department of Molecular and CellBiology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain; 4 Department of Plant Physiology and Molecular Biology, Plovdiv University, 24 Tsar Assen Str. 4000 Plovdiv, Bulgaria; 5Genomics Research Center Ltd., 105 Ruski Boulevard, 4000 Plovdiv, Bulgaria; 6Istituto di Virologia Vegetale, Consiglio Nazionale delle Ricerche, Strada delle Cacce 73, 10135 Torino, Italy; 7Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7700, South Africa; 8Research Institute of Influenza, Ministry of Health and Social Development of the Russian Federation, Prof. Popova str. 15/17, St. Petersburg 197376, Russia; 9Centre "Bioengineering", Russian Academy of Sciences, Prosp. 60-let Oktiabria 7-1 Moscow 117312, Russia Abstract: Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.

Keywords: Transient expression, virus-like particle, hepatitis B core antigen, human papillomavirus, bovine papillomavirus, bluetongue virus, foot-and-mouth disease virus, influenza virus, porcine respiratory and reproductive syndrome virus, protective immunity. 1. INTRODUCTION The use of plants as bioreactors for the production of pharmaceutical proteins has undergone dramatic advances since it was first proposed more than 20 years ago. Initially all the work focussed on stable transformation to produce lines of plants transgenic for proteins of pharmaceutical interest. Given the time and effort associated with the production of homozygous lines of stably transformed plants, it was entirely reasonable that early work concentrated on those proteins with well-characterised pharmacological properties. The downside of this approach has been that the resulting plantexpressed proteins were in direct competition with existing products produced by more “conventional” technologies, such as mammalian cell culture. From the above, it became clear that for plants to fulfil their potential as a means of producing pharmaceutical proteins, it would be necessary to develop methods for the rapid production and characterisation of plant-expressed proteins, if only as a prelude to stable genetic transformation. As a result, methods for transient expression of proteins were developed during the 1990s based on either replicating virus vectors or on Agrobacterium-mediated gene transfer. These methods have been steadily refined to the point where they can be used not only for the rapid screening of candidate pharmaceuticals but also for their large-scale production. This article describes some of the work undertaken by the EC FP7 Plant *Address correspondence to this author at the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK; Tel:/Fax: ???????????????????????????????; E-mail: [email protected] 1381-6128/13 $58.00+.00

Production of Vaccines (PLAPROVA) consortium which investigated the use of transient expression technologies to produce viruslike particles (VLPs) of both human and veterinary pathogens. VLPs were chosen as the target because it is generally recognised that multivalent and especially particulate structures, such as VLPs, make the most efficient immunogens as they stimulate a repertoire of B- and T-cell responses [1, 2]. Furthermore, such entities are generally easier to purify than individual proteins and can serve as platforms for the presentation of immunogenic sequences from other pathogens. 2. TRANSIENT EXPRESSION SYSTEMS 2.1. Replicative Replicative transient expression systems involve the insertion of the sequence of interest into the genome of a plant virus, most commonly an RNA virus, in such a way that it does not interfere with viral replication. The resulting construct is used to infect plants (nowadays usually by agroinfiltration; see section 2.3) and the inserted sequence is expressed when the virus replicates and moves throughout the plant. Details of the types of plant viral vectors available are given in [3] and a particular application of a vector based on tobacco mosaic virus (TMV) is described in [4]. The principle advantages of the replicative systems are that the process of replication greatly amplifies the inserted sequence and the constructs are capable of spreading within the plant. These features mean that replicative vectors are capable of achieving very high levels of expression in a short time. The disadvantages of such systems are that the need to preserve efficient replication limits the size and nature of the sequences which can be inserted. Moreover, the © 2013 Bentham Science Publishers

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act of replication often leads to the accumulation of mutations within the inserted sequence and competition between constructs (virus exclusion) makes it difficult to co-express multiple proteins within the same cell. 2.2. Non-replicative Non-replicative expression systems rely on the process of agroinfiltration. In these systems the sequence to be expressed is flanked by a plant promoter and terminator in the T-DNA region of a binary vector which is capable of replication in both Escherichia coli and Agrobacterium tumefaciens. This enables all the initial manipulations to be carried out in E.coli before the expression plasmids are finally transformed into A. tumefaciens. Infiltration of leaves with suspensions of A. tumefaciens containing the desired plasmid results in transfer of the T-DNA to nuclei of the plant cells. Transcription then occurs from the promoter resulting in expression of the protein of interest. The agroinfiltration approach is very quick and straightforward and it is possible to deliver multiple constructs to the same cell. A potential weakness is that expression is limited to the infiltrated regions as the constructs cannot spread from cell to cell. Thus, it is essential that expression levels within the infiltrated tissue are maximised. Expression levels can be boosted by the inclusion of a virus-derived suppressor of gene silencing during the inoculation procedure and by the deployment of sequences which enhance the translation of the gene of interest. Both these features were used in the development of the CPMV-HT expression system. In this system expression is enhanced by positioning the gene of interest between a modified 5’untranslated region (UTR) and the 3’UTR from cowpea mosaic virus (CPMV) RNA-2 and the mRNA is stabilised by the presence of the P19 suppressor of silencing from tomato bushy stunt virus (TBSV) [5]. The features of CPMV-HT were subsequently incorporated in the pEAQ series of transient expression vectors [6] which allow the rapid insertion of target sequences using either restriction enzyme or GATEWAY-based cloning methods (Fig. 1). The pEAQvectors have a modular design and can be used for the efficient expression of multiple proteins from the same T-DNA [7, 8] ensuring that all proteins are expressed in each cell. The pEAQ vectors were widely used by the PLAPROVA consortium to express a number of target proteins. 2.3. Delivery of Expression Vectors into Plants Agroinfiltration is essential for the delivery of non-replicative plasmids and is nowadays generally the method of choice for the delivery of replicating vectors as well; however, with replicating constructs, inoculation of plants with plasmid DNA or in vitro transcripts can also be used. A number of different A. tumefaciens strains have been used for this purpose, although certain strain/ vector combinations work better than others. For example, A. tumefaciens strains LBA4404 and AGL1 have proved highly effective at delivering pEAQ-based plasmids while GV3101 has not; the rea-

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sons for this are unclear. The plant most commonly used for infiltration and subsequent expression is Nicotiana benthamiana and this host was exclusively used for the work described in this manuscript. The delivery of A. tumefaciens into leaf tissue involves replacing the air in the intercellular spaces with a suspension of bacteria harbouring the desired expression plasmid. On a small scale this can be achieved by syringe infiltration which uses the positive pressure from a needle-less syringe to force the bacteria into the leaf. This method is useful for inoculating individual leaves, or even just part of a leaf, and is suitable for the production of milligram amounts of proteins. However, it becomes very laborious if large amounts of material are needed and the alternative method of vacuum infiltration is used in these cases. In this approach, the aerial parts of whole plants are immersed in an Agrobacterium suspension and the pressure above reduced for several minutes, leading to the air in the intercellular spaces being removed. On release of the vacuum, the bacterial suspension replaces the lost air leading the bacteria to come into contact with most of the leaf tissue. This method can be automated and has been used for large-scale production of plant-produced proteins by companies such as Medicago Inc. (Quebec City, Canada). 3. EXPRESSION OF VLPS FROM NON-ENVELOPED VIRUSES 3.1. Hepatitis B Core Antigen (HBcAg) Although hepatitis B virus is an enveloped virus it contains a core structure that can self-assemble into non-enveloped particles. The core antigen of hepatitis B virus (HBcAg) has attracted considerable attention as a potential source of vaccines. HBcAg has been shown to be capable of self-assembly into core-like particles (CLPs) when expressed in a number of heterologous systems, including transient expression in plants using both replicating and non-replicating expression vectors [5, 9, 10].These CLPs have antigenic structures identical to those of native HBcAg particles [11]and have attracted attention as potential carriers of foreign antigenic sequences[12]. The core protein consists of an N-terminal assembly “core” domain encompassing the first 140 amino acids and an argininerich “protamine” domain (residues 150-183) connected by a linker peptide [13]. The core domain dimerises to form building blocks capable of self-assembly into VLPs. HBV CLPs are icosahedral nanoparticles formed by the association of 90 or 120 core protein dimers [14]. Insertion of foreign sequences into the immunodominant c/e1 B cell epitope, a surface-exposed loop on the HBV capsid protein, preserved the antigenic characteristics of HBcAg [15]. The potential capacity of the c/e1 immunodominant loop appears to be high, allowing the insertion of the green fluorescent protein (223 amino acids; [16]) although much smaller insertions can disrupt correct self-assembly. These observations demonstrate the impor-

Fig. (1). Structure of T-DNA region of pEAQ vector series. The gene of interest is inserted between the UTRs of CPMV RNA-2 using either restriction enzyme- or GATEWAY-based cloning. Transcription of the inserted gene is driven by the cauliflower mosaic virus 35S promoter and terminated by the nos terminator. The T-DNA region also contains the sequence encoding the TBSV P19 suppressor of gene silencing and the kanamycin resistance gene nptII. RB and LB indicate Right Border and Left Border of the T-DNA, respectively.

The Use of Transient Expression Systems

tance of characteristics such as the charge, hydrophobicity, -strand index, and proximity of the termini of the sequence to be inserted. To optimise a plant-based HBcAg presentation system, a library of plant codon-optimised HBcAg genes was constructed in which the arginine-rich C-terminal portion of the protein, which is responsible for the incorporation of nucleic acid into core particles, was modified or replaced. In the most extreme case the entire Cterminal region was deleted to give HBcAg149.Arginine clusters were replaced with lanine or lysine residues and a single cysteine residue was added to the C-terminus. The C-terminal HBcAg mutants were expressed in plants using the non-replicating CPMV-HT system. Western blot analysis confirmed that all the C-terminally modified versions of HBcAg, as well as the full-length version (HBcAg183), could be successfully expressed in inoculated leaves. When assessed for their ability to form VLPs, HBcAg183, as expected, was found to be the most efficient though the particles contained host nucleic acid (Fig. 2). Of the C-terminal mutants, sucrose gradient analysis indicated the formation of VLPs but these appeared to be less stable than those from HBcAg183. Only HBcAg149 gave sufficient levels of particles for further analysis which showed that this mutant could act as a source of nucleic acidfree VLPs (Fig. 2). Both full-length and C-terminally deleted versions of HBcAg were investigated as carriers of a foreign epitope from influenza virus as described in Section 4.1.

Fig. (2). Electron microscope images of plant-produced HBcAg particles. (A) particles produced by particles produced by expressing full-length HBcAg (HBcAg183). (B) particles particles produced by expressing the Cterminal deleted HBcAg (HBcAg149).Scale bar = 100nm.

3.2. Human Papillomavirus (HPV) Papillomaviruses (PVs) are non-enveloped tumour-inducing DNA viruses about 55 nm in diameter with a double-stranded circular genome of approximately 8 kb encapsidated by the major (L1) and minor (L2) capsid proteins; they form a large family with more than 100 species, the majority of which infect humans. Infection by human PVs (HPVs) can be associated with benign and malignant epithelial proliferations of skin and internal squamous mucosae. High-risk HPVs, of which HPV-16 and -18 are the most prevalent, cause cervical cancer (one of the two major cancers of women; [17]) and anogenital, head and neck tumours in both men and women [18]. Some HPVs have a cutaneous tropism (cHPVs) and have been etiologically associated with non-melanoma skin cancer in immuno-compromised individuals [19, 20]. L1 is the major capsid protein of HPV; it self-assembles into highly immunogenic VLPs or into capsomers made of five L1 monomers when expressed in different cell types [21, 22]. HPV VLPs can exist either as T=7 icosahedral particles consisting of 72 L1 capsomers which are morphologically identical to the native virions or as T=1 particles consisting of 12 L1 capsomers [23, 24]. The structure of the T=1 particle has been solved crystallographically and the L1 protein has been shown to have a jellyroll sandwich structure formed by conserved sequences with externally protruding hypervariable loops [24]. Three C-terminal helices (h2, h3, and h4) that project laterally on the VLP surface are important

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for inter-capsomeric interactions and VLP formation. Deletion of the full h4 helix has no impact on capsomer assembly, but prevents the formation of either T=1 or T=7 VLPs [25, 26]. Two types of HPV vaccines are currently marketed, targeting the mucosal HPV-16, -18, and in one formulation also HPV-6 and 11 that cause genital warts. By contrast, no vaccines against cutaneous HPVs are available. The above prophylactic vaccines consist of L1-based VLPs made in insect or yeast cells. Despite their efficacy, they suffer from high production costs, relatively low protection levels, the necessity of being administered via intramuscular injection and the requirement of a cold chain. Plant expression systems may overcome some of these problems, in particular regarding production costs. Successful expression of HPV-16 L1 protein has been reported in transgenic and transplastomic plants, and plantproduced VLPs have been shown to be immunogenic after administration to animals (reviewed in [27]). Due to the strong immunogenicity of HPV capsomers and VLPs [28, 29], they are also suitable as immune-enhancer carriers for the presentation of heterologous antigens. As part of the work of the PLAPROVA consortium, transient expression has been used for the rapid expression of the L1 proteins of a mucosal (HPV-16) and a cutaneous HPV (HPV-8) in plants [30, 31]. For the cutaneous HPV-8, successful expression was achieved using either a replicating TMV-based vector [32] or the non-replicating pEAQ-HT vector system [6], the latter approach giving 15-fold higher yield [30]. A truncated version of HPV8-L1 (L1C22), lacking the C-terminal 22 amino acids, accumulated at higher yields compared to the native full-length protein. Overall, yields ranged from 17 to 240 mg/kg of fresh weight of leaf material, according to the vectors and the protein length [30]. The plantexpressed HPV8-L1 formed capsomers or VLPs of T=1 or T=7 symmetry, as detected by electron microscopy; L1C22also accumulated in the cytoplasm in the form of VLPs or paracrystalline arrays (Fig. 3A). A purification protocol optimised for use with geminiviruses [33] efficiently isolated and purified HPV VLPs from leaf tissue (Fig. 3B, C). Immunogenicity trials of the HPV-8 VLPs are currently underway. In the case of HPV-16, a synthetic L1 gene with a codon usage adjusted to be similar to that used by human cells was expressed using the pEAQ-HT system and a yield of about 80 mg/kg was obtained. To obtain HPV-16 VLPs suitable for presenting foreign epitopes, the synthetic gene was designed to contain unique restriction sites flanking sequences that encode amino acid regions predicted to be exposed on the VLP surface, according to published structural models. To illustrate the potential of this approach, two putatively exposed regions (Helix 4 and the coil region connecting Helix 4 and the -J sheet) were substituted by two versions of a conserved epitope of the ectodomain of the M2 protein of the influenza A virus (see Section 4.1) , namely M2e2-24 or M2e2-9 [31]. The chimeric proteins were all expressed at yields ranging from 30 to 120 mg/kg of fresh weight of leaf material and were all recognized by linear- and conformation-specific anti-HPV-16 L1 monoclonal antibodies (MAbs); chimeras carrying the M2e2-24 epitope also reacted with an anti-M2 MAb. Electron microscopy showed that the foreign epitopes did not disrupt the formation of T=1 or T=7 VLPs or capsomers. This was the first report of the successful expression of chimeric HPV-16 L1 carrying foreign epitopes in plants. 3.3. Bovine Papillomavirus (BPV) Bovine papillomavirus (BPV) is endemic throughout the world and causes a variety of economically important tumorigenic pathologies in horses and cattle. To date, eight different species of BPV have been identified [34], all of which are 55-60 nm diameter non-enveloped icosahedral viruses with an ~8 kbp circular doublestranded DNA genome. Structurally and genetically they resemble HPV (see section 3.2). BPV induces the formation of benign facial,


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Fig. (3). Production of HPV particles and capsomers expressed in plants. (A) Subcellular location of the HPV VLPs in the plant cell (cw = cell wall; cy = cytoplasm; vac = vacuole). (B) and (C) are electron micrographs of HPV VLPs purified from plant tissue at low and high magnification, respectively. Most of the particles are T=1 VLPs. Scale bar = 100nm.

penile, teat, urinary bladder and alimentary tumours of epithelia and derma, which can impair reproduction and milking and invokes slaughter [35, 36]. Moreover, BPV infections in general promote carcinogenesis and immunosuppression, leading to a subtle decline in animal health, which may be wrongly attributed to other causes [37]. Initial vaccine trials in the early 1990s involved vaccinating calves with BPV-2 and BPV-4 virus particles isolated from tumours [38]. The prophylactic protection conferred by these vaccines was virus type-specific and indicated that the L1 and L2 capsid proteins were likely key in conferring immunity. Later studies which used purified E. coli-expressed L1 and L2 fused to -galactosidase, found that the L1 vaccine prevented BPV-2-induced tumour formation in calves only if it was given before the challenge, whereas L2 promoted tumour rejection irrespective of whether it was administered before or after the challenge [39]. BPV cannot be grown in culture for the preparation of traditional killed or attenuated live vaccines [40], and consequently yeast and insect cell expression systems have been used to produce L1- and L2-based vaccines. However, these methods are expensive and time-consuming - critical disadvantages for a vaccine intended for veterinary use. BPV L1 expressed in insect cells typically self-assembles into pentameric capsomers, which may in turn unite to form higher order structures such as T=1 or T=7 VLPs composed of either 12 or 72 capsomers, respectively[21, 40]. L1 capsomers and higher order structures have been found to be highly immunogenic and have been shown to confer protection against future infection by BPV types from which they are derived [21, 40]. Consequently, L1 VLPs and their precursors are regarded as suitable prophylactic vaccines. Recent work by Love et al. [41] has demonstrated that BPV-1 L1 can be expressed transiently to a high level in N.benthamiana using the pEAQ vector system [6]. In these studies it was found that appropriate codon optimization of the L1 gene for expression in Nicotiana species enhanced transient expression by an order of magnitude over the non-optimised gene [41]. Electron microscopy revealed that the plant-expressed L1 self-assembled into T=1 VLPs that were ~30 nm in diameter (Fig. 4). Following the same extraction procedure as previously used to purify plant-expressed HPV VLPs (section 3.2), yields of 183mg/kg fresh weight of leaf material of highly pure, structurally stable VLPs were obtained. The VLP yield of >40 mg of purified protein per kg fresh leaf weight [41] was approximately four-fold higher than the level of 10mg per kg fresh leaf weight estimated to be required for economical

Fig. (4). Electron micrographs at two magnifications of structures formed by BPV L1 protein purified from N. benthamiana. The larger particles are T=1 VLPs while the smaller particles are capsomers.

production of a vaccine in plants [42]. The immunogenicity of the VLPs was investigated by injecting rabbits with purified L1 VLPs in PBS and incomplete Freund’s adjuvant (150μg of per animal) The antisera generated was strongly cross-reactive with both the plant-produced VLPs and BPV VLPs produced in insect cells [41]. . The high yields of plant-produced BPV L1 VLPs, their capacity to invoke strong immune responses, and the rapidity and ease with which they can be produced [41] indicate that transient expression in plants has considerable potential for the large-scale production of potential BPV vaccines. 3.4. Bluetongue Virus (BTV) Bluetongue is a non-contagious, insect-transmitted, viral disease of domestic and wild ruminants [43]. At present 26 serotypes of the virus are recognized [44]. The virulence and mortality rate of the different virus strains vary considerably, depending also on the infected species. Bluetongue virus (BTV) is an orbivirus (genus Orbivirus, family Reoviridae), with a multicomponent doublestranded RNA genome encapsidated in a complex, multi-layered virion. The viral proteins (VP) 2 and 5 provide an outer shell, which


is laid on to the foundation provided by assembly of VP3 and VP7, which constitute the essentials of the inner shell. VP2 and VP5 do not form a structure without VP3 and VP7. Vaccination is regarded as one of the most effective ways of controlling and eventually eradicating bluetongue disease in affected areas. Most neutralising antibody activity is against VP2, but VP5 elicits neutralising antibodies as well [45]. Current commercially available vaccines include attenuated and inactivated virus strains [46]; however, they are not always safe in the case of the former and do not provide long-lasting immunity in the case of the latter. The search for better vaccine candidates has shown that VLPs are much better in this regard [47]. French and Roy [48] showed that BTV VP3 and VP7 can form CLPs when expressed together in insect cells using a baculovirus expression system and that expression of all 4 VPs in insect cells results in the formation of VLPs resembling the wild-type virus [49]. Vaccination trials in sheep using BTV VLPs showed they were highly immunogenic and protected the sheep from challenge with live virus [50]. The multi-layered structure of BTV provides a challenging target for expression using transient plant expression systems as it requires the simultaneous expression of four proteins and their correct assembly at a complex stoichiometric ratio. Although VP2 and VP5 can be used alone or in combination as a vaccine if produced in insect cells via a baculovirus expression system, assembled particles are more immunogenic [51]. Initial attempts at transient expression of BTV VLPs in plants during PLAPROVA focussed on expressing native sequences from BTV serotype 10 (kindly provided by Prof. P. Roy, London School of Hygiene and Tropical Medicine) using the pEAQ vector system [6]. Although coexpression of VP3 and VP7 from separate pEAQ constructs gave rise to CLPs (Fig. 5), the yield was low. Furthermore, attempts to produce VLPs by co-expression with VP2 and VP5 were inconclusive as, once again, expression levels of VP2 and VP5 were low.

Fig. (5). Electron microscope images ofCLPs from BTV-10 by coexpression of VP3 and VP7 in N. benthamiana. Scale bar =100nm.

It was rationalised that the low levels of expression observed may have been due to the fact that native (i.e. not codon-optimised for plants) sequences were being used in the experiments. To address this possibility, Nicotiana-sp. codon-optimised versions of the 4 genes encoding the structural proteins (VP2, VP3, VP5 and VP7) of BTV serotype 8, the strain most relevant to European outbreaks, were synthesized. It was also decided to concurrently test different


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transient plant expression systems in order to screen for the system that gave the highest levels of intact BTV-8 VLPs in N. benthamiana. The different expression vectors included the following: pEAQ-HT[6]; a pTRA vector suite [52] which targets recombinant proteins in plant cells to the cytoplasm (pTRAc-HT), the apoplast (pTRAkc-AH), the endoplasmic reticulum (ER) (pTRAkc-ERH) or the chloroplast (pTRAkc-rbcs1-cTP); pRIC3.0-HT which is a replicating vector [53] derived from bean yellow dwarf virus (BYDV); and the Zera™ peptide [54] fused to pEAQ-HT which facilitates the production of protein bodies in the plant cell cytoplasm containing the appropriate recombinant proteins. The targeting of recombinant proteins to different subcellular compartments or into protein bodies was investigated as it may facilitate easier purification of recombinant proteins when it comes to scaling up production. Western blots of extracts from plant tissue co-infiltrated with recombinant constructs containing either VP3 and VP7, or all 4 VPs (2, 3, 5 and 7) showed that all proteins were produced after 7 days post infiltration (dpi) using the chloroplast-targeting plant expression vector (pTRAkc-rbcs1-cTP) as well as pRIC3.0-HT. However, no CLPs or VLPs were detected in extracts from leaves coinfiltrated with pTRAks-rbcs1-cTP expression vector constructs. In extracts from leaves co-infiltrated with pRIC3.0-HT, there was an abundance of 70 to 80m-sized protein aggregates detected by transmission electron microscopy. It is possible that the polyhistidine (6His) tag fused to the 5’ termini of each of the recombinant proteins may have interfered with the proper folding of VPs to form CLPs or VLPs. The above studies revealed that the pEAQ-HT system was the most suitable for producing quantities of assembled BTV-8 VLPs. Comparisons of protein expression levels using pEAQ-HT were also carried out using 2 different A.tumefaciens strains (GV3101 and LBA4404), and resulted in higher expression levels in LBA4404-infiltrated plant tissue. A detailed description of the production and properties of these VLPs is currently in preparation (Thuenemann, E., Meyers, A., Rybicki, E., Heath, J. and Lomonossoff, G.P., in preparation). 3.5 Foot-and-Mouth Disease Virus (FMDV) Foot-and-Mouth Disease Virus (FMDV) is a highly contagious and devastating disease that affects all species of cloven-hoofed animals, including economically important species such as cattle, pigs and sheep. The current FMDV vaccine is an inactivated wholevirus preparation that is formulated with adjuvant prior to use in the field. However, there are concerns about safety, cost-efficiency, stability and potency of existing vaccines. Thus the development of subunit anti-FMDV vaccines based on FMDV capsid proteins, peptides and epitopes have been the subject of intense research over the past years. These have included attempts to develop plant-expressed vaccines. Many of the initial attempts concentrated on the expression of capsid protein VP1 either in transgenic plants or transiently since this contains the immunodominant epitope from the virus [5557]. More recently, attempts have been made to express the entire capsid protein precursor P1 in transgenic plants either alone [58] or in combination with the 3C protease necessary for its processing [59]. Though various subunit vaccine candidates have shown good protection in lab animals, none has reached the efficiency of current killed vaccines in protecting the livestock. To develop more efficient FMDV subunit vaccines, two approaches were taken during PLAPROVA. In the first approach, codon-optimised genes encoding hybrid proteins, combining within a single polypeptide chain B-cell epitopes of proteins VP1 and VP4 and T-cell epitopes of proteins 2C and 3Dfrom the most widespread O serotype of FMDV isolate were constructed[60]. The recombinant protein, H-PE, was expressed in N. benthamiana plants using a replicating potato virus X (PVX)-based vector. The protein was expressed at the level of 0.5-1% of the total plant protein, as estimated by SDS-PAGE, but appeared to be insoluble. Thus, the pro-


tein (containing 6His tag) was purified from plant tissue using metal-chelate affinity chromatography under denaturing conditions. When used to immunise guinea pigs, an emulsion vaccine containing the plant-produced H-PE protein induced the formation of virus-neutralizing antibodies to FMDV and no evidence of infection was seen on challenge with FMDV serotype O/Taiwan/99[60]. The second approach involved the development of an "empty capsid"-based FMDV vaccine. This approach requires coexpression of the polyprotein P1 (encoding VP0, VP3 and VP1 capsid proteins) from FMDV and a protease able to process the polyprotein into individual capsid components; the assembly of the "empty capsid" occurs simultaneously with processing. The cleavage sites within the P1 polyprotein were modified to be recognised by the CPMV 24K protease, rather than the native FMDV 3C protease, since this is less toxic than 3C when expressed in plants [61]. In an attempt to express "empty capsids" in plants, pEAQ-HT vectors encoding the P1 precursor and the CPMV 24K protease on separate constructs were co-infiltrated into N. benthamiana leaves. The presence and processing of recombinant capsid proteins was analysed by Western blotting using antibodies against FMDV. The data showed that the modified P1 polyprotein carrying the recognition sites for 24K protease was expressed in N. benthamiana plants and that this polyprotein was processed into shorter proteins if the CPMV 24K protease was co-expressed. However, no VLP-like particles were observed by electron microscopy of protein samples isolated from agroinfiltrated plant leaves. This suggests that processing of the P1 polyprotein in plant cells and/or some steps of assembly of capsids differs from that occurring in animal cells; alternatively, any empty capsids which did form might be unstable in plants, a plausible scenario given the acid-instability of FMDV capsids. 4. VIRUS-LIKE PARTICLES FROM ENVELOPED ANIMAL VIRUSES 4.1. Influenza Virus Recombinant vaccines against influenza are currently based on the highly immunogenic virion surface proteins - hemagglutinin (HA) and neuraminidase (NA). In the case of plants, HA has been successfully transiently expressed using a number of systems including CPMV-HT,and the resulting VLPs were shown to be capable of conferring protective immunity in experimental animals [62, 63].However, because of the high variability of HA and NA, it has not been possible to develop a broad range vaccine against influenza. Unlike HA and NA, one of the minor virion proteins, M2, present at 16-68 copies per particle, is highly conserved. M2 is a membrane protein and its extracellular domain, M2e, is a promising vaccine candidate since the M2e sequences are conserved in virtually all influenza strains of human origin while the M2e peptides of potentially pandemic strains of animal origin differ from the human consensus in several amino acids. Significantly, the highly pathogenic strains of avian influenza that caused hundreds of cases of illness and lethal outcomes in countries of Southeast Asia and the Near East, differ from human strains in their M2e sequences, and these differences are important for the specificity of vaccine. The M2 protein itself is, however, poorly immunogenic and needs to be attached to a carrier molecule such as a VLP. Within the PLAPROVA project we explored several different approaches to produce M2e-linked VLPs in plants that could become candidate recombinant influenza vaccines. The VLP selected to act as a carrier for the M2e epitope was HBcAg since this had already been shown to be able to enhance the immunogenicity of M2e [64] and to be capable of efficient self-assembly within plants (see section 2.1). Two approaches for transient expression were used, a replicating vector based on PVX and the non-replicating pEAQ-HT system. For expression from PVX, the M2e peptide of avian influenza strain A/Duck/Potsdam/1402-6/1986 was used.In initial experiments, a recombinant protein, M2eHBc, consisting of the M2e pep-

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tide fused to the N-terminus of HBc antigen, was expressed in E. coli. Electron microscopy of purified particles confirmed assembly of the bacterially expressed M2eHBc protein into VLPs. The preparation was evaluated in animal experiments that showed that mice immunized twice with 50 μg of M2eHBc particles developed a protective immune response against the lethal avian influenza challenge. Thus, this recombinant protein was chosen for expression in plants.To express the fusion protein in plants, the hybrid M2eHBc gene was codon optimised and the two cysteine residues in positions 17 and 19 of the M2e peptide responsible for formation of disulfide bonds were replaced with serine residuesto improve the solubility of the protein[65]; these substitutions do not affect the immunological properties of M2e [66]. The hybrid gene was cloned into the PVX-based vector, pA7248AMV [67], and introduced into N. benthamiana by agroinfiltrationin the presence the P19 suppressor of gene silencing [68]. M2eHBc was well expressed in plants (about 30-40 mg/kg fresh weight of leaf material) and, crucially, was present in the soluble fraction [65]. The levels of expression obtained with the PVX-based viral vector were found to be twice as high as those obtained with the pEAQ-HT expression system using the same hybrid gene. The recombinant VLPs were purified from agroinfiltrated plants using a two-step procedure, including ammonium sulfate precipitation of the soluble fraction and ultracentrifugation in sucrose and CsCl gradients [65]. The M2eHBc was assembled into spherical 30-35 nm diameter VLPs which were similar to VLPs formed by the native HBc antigen (Fig. 6). The antigenic properties of the plant-expressed M2eHBc particles were analyzed by an enzyme-linked immunosorbentassay (ELISA) with a set of MAbs raised against a synthetic M2e peptide. The data showed that all antibodies recognize the plant-produced M2eHBc, confirming that M2e is exposed on the surface of the particles and is accessible to antibodies. To characterize their immunogenicity and protective action, BALB/c mice were immunized intraperitoneally with the plant-produced preparation of M2eHBc particles at two-week intervals using Sigma system Adjuvant (Sigma) for the first vaccination and incomplete Freund adjuvant (Sigma) for the two subsequent vaccinations. [65]. The dose of the antigen was 20 g/mouse at the first immunization and 50 g/mouse at the subsequent ones [65]. Evaluation of the level of antibodies raised against M2e by ELISA indicated that three-fold immunization induced the production of high titres of IgG serum antibodies. The titre of the IgG2a subtype antibodies was significantly higher than that of the IgG1 subtype, suggesting a predominant induction of the Th-1 type immune response mainly responsible for the cellular response [65]. To assess the protective effect of the candidate vaccine, experimental and control non-vaccinated mice were challenged with 1 LD50 of the mouse-adapted avian influenza strain A/Chicken/Kurgan/05/ 2005(H5N1). Upon infection the body weight of the immunized animals decreased significantly less than in the control mice (to 90% and to 70% of the initial weight, respectively). The clinical symptoms of infection were less severe in theimmunized mice, indicating that immunization with the candidate vaccine significantly relieved the course of the illness. The candidate vaccine

Fig. (6). The structure of plant-produced M2eHBc particles analyzed by electron microscopy.


protected 90% of immunised mice, while in the control group only half of the animals survived under the conditions of the infection [65]. Overall, our results show that production of M2eHBc particles in plants using a viral expression system is a feasible and promising approach for production of recombinant vaccines against influenza. In an alternative approach, the ability of HBcAg to tolerate the insertion of the 23 amino acid M2e epitope from avian influenza virus at various positions in the immunodominant loop was modelled using the SWISS-MODEL Workspace program. The results indicated that insertion between amino acids 82 and 83 of HBcAg should be well tolerated. Based on the results obtained with the Cterminally truncated versions of HBcAg described in Section 3.1, the M2e epitope was inserted at this position in HBcAg149 to give the construct HBcAg149M2e,which was expressed in plants using the pEAQ-HT system. Chimeric HBcAg149M2e produced in plants reacted with specific antibodies produced to the M2e peptide from Avian influenza virus H5N1 strain. The results indicated that the yield of HBcAg149M2e was 48 g/g of leaf tissue. The immunogenicity of the chimaeric HBcAg149M2e extracted from inoculated N. benthamiana plants was assessed in BALB/c mice using subcutaneous immunization. Anti-M2e and anti-HBc antibodies were measured in murine sera by indirect solid-phase ELISA using purified E.coli-derived HBcAg VLPs (1g/ml) or M2e synthetic peptide (1g/ml) in PBS for capture and sheep anti-mouse IgG conjugated to horseradish peroxidase (total IgG) was used as the secondary antibody. The results showed the presence of antibodies specific to the both the HBcAg carrier and the inserted M2e epitope in the immune but not the preimmune sera, indicating that the particles were immunogenic. 4.2. Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Porcine reproductive and respiratory syndrome virus (PRRSV) is the agent causing the most important infectious disease affecting swine, resulting in increasing economic losses worldwide. Current vaccines against PRRSV have limited efficacy. Modified live vaccines protect against challenge with homologous isolates, but generally have a limited effect against challenge with heterologous viruses [69]. Furthermore, live vaccines provide partial protection against clinical disease but do not prevent infection and, more importantly, they can revert to virulence [70, 71]. In addition, although the attenuated vaccines induce an immune response resembling that elicited by PRRSV natural infection, they do not induce high levels of neutralizing antibodies. Therefore, an improvement of vaccination strategies is required to control the disease and improve the performance of the herds. PRRSV is a member of the Arteriviridae family, included in the order Nidovirales. PRRSV has a single-stranded positive-sense RNA genome of around 15 Kb. The 5’ two-thirds of the genome encode replicase polyproteins (pp1a and pp1ab), and the rest of the genome encodes the structural glycoproteins (GPs) 2, 3, 4 and 5, as well as the membrane (M) and nucleocapsid (N) proteins. Envelope (E) and 5a proteins are encoded from the same subgenomic mRNAs as open reading frames (ORFs) 2 and 5, respectively [72]. GP5 and M are the major components of the viral envelope and form a disulfide-linked heterodimer [72]. A GP5-M heterodimer interacts with sialoadhesin, present in the cell membrane, which acts as the PRRSV receptor [73]. In addition, inside the cells, minor envelope proteins GP2 and GP4 interact with a cell receptor CD163, and this interaction is required for efficient PRRSV infection [74]. Deletion of minor envelope proteins GP2, GP3 and GP4 does not affect the production of non-infectious VLPs [75], indicating that GP5 and M proteins, together with N protein and RNA could be enough for the formation of VLPs. Nevertheless, minor envelope proteins are required for the generation of infectious viral particles [75]. Synthetic vaccines based on VLPs could have a distinct advantage in terms of stimulating both humoral and cellular immune


7

responses against PRRSV. However, one of the main problems in the development of such structures is the limited knowledge of the correlates of protection. In order to address this issue, a vector system based on transmissible gastroenteritis virus (TGEV) was used to express the PRRSV GP5 and M proteins in mammalian cells. The proteins have been described as the main inducers of neutralising antibodies and T-cell mediated immune responses, respectively. It was found that the immune response elicited by vectors expressing both GP5 and M proteins was more protective than that induced by vaccines expressing GP5 alone [76, 77]. Altogether, the data are in agreement with the presence of conformational epitopes, relevant for protection, in the GP5-M heterodimer. Therefore, the formation of GP5-M heterodimer when the proteins were expressed by rTGEV was evaluated. Confocal microscopy indicated that GP5 and M proteins co-localize after PRRSV infection and when expressed by rTGEV. Western blot analysis of protein extracts from infected cells in non-reducing conditions detected a band recognized both by GP5- and M-specific antibodies, suggesting that it corresponds to a GP5-M heterodimer (Fig. 7). These data strongly suggest that GP5 and M proteins expressed by rTGEV vectors form a heterodimer.

Fig. (7). Western blot analysis of interaction between PRRSV GP5 and M proteins.Protein extracts were obtained from PRRSV infected MARC145 cells (PRRSV), ST mock infected cells, and ST cells infected with rTGEV or rTGEV expressing GP5 and M proteins (rTGEV-5+6). PAGE was performed with these protein extracts in reducing (upper panels) or nonreducing conditions (lower panels). Proteins were transferred to nitrocellulose membranes, and Western blot analysis was performed using antibodies specific for GP5 (-GP5) or M (-M) proteins.

Protection experiments showed that animals vaccinated with PRRSV GP5 and M proteins, expressed using the TGEV vectors, developed a faster and stronger humoral immune response than the non-vaccinated ones. Furthermore, partial protection against homologous challenge was observed, as vaccinated pigs showed decreased lung damage compared with the non-vaccinated ones [76]. Nevertheless, full protection was not observed, most likely due to the limited stability of PRRSV antigens expressed by the rTGEV system. Currently, alternative antigenic structures were designed to improve rTGEV vectors stability, and the protection they conferred is being evaluated. Using TGEV-based vectors, GP5 and M proteins were identified as conferring partial protection against PRRSV (see above). Therefore, the full-length sequences of both proteins were inserted


into pEAQ vectors and tested for expression in N. benthamiana. Different versions of both genes were constructed, containing chloroplast targeting signals or the endoplasmic reticulum retention signal to analyse the effect of targeting expression to different subcellular compartments. It was not possible to detect the expression of either of the proteins by Western blot analysis, regardless of the site of its targeting. Furthermore, infiltrated plant tissues, particularly in the case of GP5, rapidly became severely damaged and necrotic (Fig. 8). This phenotype was not due to the action of Agrobacterium, as plants infiltrated with empty vectors did not show the same effect. Interestingly, similar necrosis occurred when plants were infiltrated with constructs expressing a variety of other membrane-associated proteins from other organisms, suggesting that this phenomenon is specific to the expression of membrane proteins.

Fig. (8). Necrosis of GP5-infiltrated plant tissue. A N. benthamiana leaf infiltrated with a construct carrying the GP5 gene at 3 days after infiltration (left) compared with a non-infiltrated leaf (right).

The failure to achieve expression in plants of full-length versions of the GP5 and M proteins, indicated that some engineering of the protein would be desirable to eliminate those sequences which were toxic to plants. To eliminate the sequences potentially causing necrosis in inoculated tissue, partially deleted versions of GP5 and M proteins were designed for expression in plants. In principle, improved GP5-M antigenic structures expressed using the plant system could lead to the production of vesicles exposing PRRSV antigens in a VLP-like structure. The deleted constructs were based on a modified GP5 proteincodon-optimised for plant expression and were expressed in plants using the pEAQ-HT system. Removal of transmembrane domains was found to alleviate the problems of necrosis and studies to confirm the immunogenicity and protection immunity conferred by the modified protein are currently under way. 5. CONCLUSIONS The work of the PLAPROVA consortium clearly demonstrates that transient expression, using either replicative or non-replicative systems, is a highly efficient system for the production of VLPs from non-enveloped animal viruses. The method can be used in cases where the VLPs are made up of a single type of capsid, as in the case of HPV and BPV, or multiple types of proteins (BTV). The method was also highly successful for the expression of the nonenveloped HBcAg protein. It was generally found that codon optimisation was required or at least highly desirable to achieve high levels of expression in plants. Electron microscopy confirmed that the plant-expressed capsid proteins could self-assemble into VLPs and immunological analyses confirmed that these had the antigenic properties of the virus from which they were derived. An important next step will be to evaluate the ability of the plant-expressed VLPs to provide protective immunity in both model and target animals. By contrast with the success with non-enveloped VLPs, expression of proteins from enveloped viruses, such as PRSSV, has proved more problematic, almost certainly due to the presence of transmembrane domains. One solution to this problem is to identify

Thuenemann et al.

the antigenic regions (epitopes) of the protein(s) of interest and present them on the surface of a heterologous carrier VLP. This approach is exemplified by the presentation of the M2e epitope from influenza virus on the surface of HPV VLPs and HBcAg particles and the demonstration that the chimeric HBcAg particles can protect mice against challenge with influenza. The alternative approach is to attempt to eliminate those portions (essentially the transmembrane domains) of the protein of interest responsible for causing necrosis in inoculated tissue while preserving the antigenic sites. In terms of expression, this strategy has been shown to be successful in the case of GP5 of PRRSV, but the ability of the modified protein to elicit an antibody response is yet to be determined. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The work described in this review was supported, in part, by the EU FP7 “PLAPROVA” project (Grant Agreement No. KBBE2008-227056) and by the Russian Federation Ministry of Education and Science (state contract 02.527.11.0002). At the John Innes Centre, the work was also supported by BB/J004561/1 from BBSRC, the John Innes Foundation and through COST action AF0804. The work of AJL and MT was also partially funded by the Scottish Government’s Rural and Environmental Science and Analytical Services (RESAS) Division. The work of NVR was partly funded by the Russian Academy of Sciences program “Fundamental Studies on Nanotechnologies and Nanomaterials”. At the University of Plovdiv, the work was also supported by EU FP7 "BioSupport" project and the Bulgarian science fund. At CNB-CSIC the work was also supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705). Additionally, the research leading to the PRRSV results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under the project PoRRSCon (EC grant agreement number 245141). ABBREVIATIONS BPV = BTV = BYDV = CLP = CPMV = FMDV = HA = HBcAg = HPV = NA = PRRSV = PV PVX TBSV TGEV TMV VLP VP

= = = = = = =

Bovine papillomavirus Bluetongue virus Bean yellow dwarf virus Core-like particle Cowpea mosaic virus Foot-and-Mouth Disease Virus Hemagglutinin Hepatitis B core antigen Human papillomavirus Neuraminidase Porcine respiratory and reproductive syndrome virus Papillomavirus Potato virus X Tomato bushy stunt virus Transmissible gastroenteritis virus Tobacco mosaic virus Virus-like particle Viral protein

REFERENCES [1]

Roy P, Noad R. Virus-like particles as a vaccine delivery system: myths and facts. Hum Vaccine 2008; 4: 5-12.

The Use of Transient Expression Systems [2] [3]

[4]

[5] [6]

[7] [8]

[9]

[10]

[11] [12] [13]

[14] [15] [16]

[17]

[18] [19]

[20]

[21]

[22] [23]

[24] [25]

Roy P, Noad R. Virus-like particles as a vaccine delivery system: myths and facts. Adv Exp Med Biol 2009; 655: 145-58. Yusibov V, Streatfield SJ, Kushnir N, Roy G, Padmanaban A. Hybrid Viral Vectors for Vaccine and Antibody Production in Plants. Curr Pharm Des 2012; in press. Petukhova NV, Gasanova TV, Stepanova LA, et al. Immunogenicity and protective efficacy of candidate universal influenza A nanovaccines produced in plants by tobacco mosaic virus-based vectors. Curr Pharm Des 2012; in press. Sainsbury F, Lomonossoff GP. Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 2008; 148: 1212-18 Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 2009; 7: 682-93. Sainsbury F, Cañizares MC, Lomonossoff GP. Cowpea mosaic virus: the plant virus-based biotechnology workhorse. Annu Rev Phytopathol 2010; 48: 437-55. Montague NP, Thuenemann EC, Saxena P, Saunders K, Lenzi P, Lomonossoff GP. Recent advances of cowpea mosaic virus-based particle technology. Hum Vaccine 2011; 7: 383-90. Meshcheriakova IA, El’darov MA, Nicholson L, Shanks M, Skryabin KG, Lomonossoff GP. The use of viral vectors to produce hepatitis B virus core particles in plants. J Virol Meth 2006; 131: 10-15. Meshcheriakova IA, El’darov MA, Beales L, Skriabin KG, Lomonossoff GP. Production of hepatitis B virus core particles protein in plants, by using cowpea mosaic virus-based vector. Vopr Virusol 2008; 53: 15-20. Huang Z, Santi L, LePore K, Kilbourne J, Arntzen CJ, Mason HS. Rapid, high-level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice.Vaccine. 2006; 24: 2506-13. Pumpens, P., Grens, E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology 2001; 44: 98-114. Birnbaum F, Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol 1990; 64: 3319-30. Crowther RA, Kiselev NA, Böttcher B, et al. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell 1994; 77: 943-50. Stahl SJ, Murray K. Immunogenicity of peptide fusions to hepatitis B virus core antigen.ProcNatlAcadSci USA 1989; 86: 6283-87. Kratz, PA, Böttcher B., Nassal M. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci USA 1999; 96: 1915-20. Bosch FX, Manos MM, Muñoz Net al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer 1995; 87: 796-802. Steenbergen RD, de Wilde J, Wilting SM, Brink AA, Snijders PJ, Meijer CJ. HPV-mediated transformation of the anogenital tract. J ClinVirol 2005; 32: S25-S33. BouwesBavinckJN, Plasmeijer EI, Feltkamp MC.Betapapillomavirus infection and skin cancer. J Invest Dermatol 2008; 128: 135558. Asgari MM, Kiviat NB, Critchlow CW, et al. Detection of human papillomavirus DNA in cutaneous squamous cell carcinoma among immunocompetent individuals. J Invest Dermatol 2008; 128: 140917. Kirnbauer R, Booy F, Cheng N, Lowy DR, Schiller JT. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA 1992; 89: 12180-84. Modis Y, Trus BL, Harrison SC. Atomic model of the papillomavirus capsid. EMBO J2002; 21: 4754-62. Baker TS, Newcomb WW, Olson NH, Cowsert LM, Olson C, Brown JC. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys J 1991; 60: 1445-56. Chen XS, Garcea R, Goldberg I, Casini G, Harrison SC. Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol Cell 2000; 5: 557-67. Chen XS, Casini G, Harrison SC, Garcea RL. Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1. J Mol Biol2001; 307: 173-82.

Current Pharmaceutical Design, 2013, Vol. 19, No. 00 [26]

[27] [28] [29]

[30]

[31]

[32]

[33] [34] [35] [36] [37]

[38]

[39]

[40] [41]

[42] [43] [44]

[45]

[46]

[47] [48]

[49] [50]

9

Bishop B, Dasgupta J, Chen XS. Structure-based engineering of papillomavirus major capsid L1: controlling particle assembly. Virol J 2007; 4: 3. Giorgi C, Franconi R, Rybicki EP. Human papillomavirus vaccines in plants. Expert Rev Vaccines2010; 9: 913-24. Rose RC, White WI, Li M, Suzich JA, Lane C, Garcea RL. Human papillomavirus type 11 recombinant L1 capsomers induce virusneutralizing antibodies. J Virol 1998; 72: 6151-54. Evans TG, Bonnez W, Rose RC, et al. A Phase 1 study of a recombinant virus-like particle vaccine against human papillomavirus type 11 in healthy adult volunteers. J Infect Dis 2001; 183: 148593. Mati S, Masenga V, Poli A, et al. Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods. Plant Biotechnol J 2012; 10: 410-21. Mati S, Rinaldi R, Masenga V, and Noris E. Efficient production of chimeric Human papillomavirus 16 L1 protein bearing the M2e influenza epitope in Nicotiana benthamiana plants. BMC Biotechnology 2011; 11: 106. Giritch A, Marillonnet S, Engler C, et al. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl Acad Sci USA 2006; 103: 14701-06. Luisoni E, Milne RG, Vecchiati M. Purification of tomato yellow leaf curl geminivirus. New Microbiol 1995; 18: 253-60. Nasir L, Campo MS. Bovine papillomaviruses: their role in the aetiology of cutaneous tumours of bovids and equids. Vet Dermatol 2008; 19: 243-54. Campo MS. Infection by bovine papillomavirus and prospects for vaccination. Trends Microbiol 1995; 3: 92-97. Borzacchiello G, Roperto F. Bovine papillomaviruses, papillomas and cancer in cattle. Vet Res 2008; 39: 45. Bogaert L, Martens A, Van Poucke M, et al. High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoidaffected and healthy horses. Vet Microbiol 2008; 129: 58-68. Jarrett WFH, Oneil BW, Gaukroger JM, Laird HM, Smith KT, Campo MS. Studies on Vaccination Against Papillomaviruses - A Comparison of Purified Virus, Tumor Extract and TransformedCells in Prophylactic Vaccination. Vet Rec 1990; 126: 449-52 Jarrett WFH, Smith KT, Oneil BW, et al. Studies on Vaccination Against Papillomaviruses - Prophylactic and Therapeutic Vaccination with Recombinant Structural Proteins. Virology 1991; 184: 3342. Campo MS. Vaccination against papillomavirus in cattle. ClinDermatol 2003; 15: 275-83. Love AJ, Chapman SN, Matic S, Noris E, Lomonossoff GP, Taliansky M. In planta production of a candidate vaccine against bovine papillomavirus type 1. Planta 2012; DOI 10.1007/s00425-0121692-0. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM. Plantbased production of biopharmaceuticals.Curr Opin Plant Biol 2004; 7: 152-158. Schwartz-Cornil I, Mertens PPC, Contreras, et al. Bluetongue virus: virology, pathogenesis and immunity. Vet Res 2008; 39: 46. Maan, NS, Maan S, Belaganahalli MN, et al. Identification and differentiation of the twenty six Bluetongue virus serotypes by RTPCR amplification of the serotype-specific genome segment 2. PloS ONE 2012; 7: 32601. Lobato ZIP, Coupar BEH, GrayCP, Lunt R, Andrew ME. Antibody responses and protective immunity to recombinant vaccinia virusexpressed bluetongue virus antigens. Vet Immunol Immunopath 1997; 59: 293-30. Savini G, Maclachlan N J, Sanchez-Vizcaino JM, Zientara S. Vaccines against bluetongue in Europe. Comp Immunol Microbiol Infect Dis 2008; 31: 101-20. Liu F, Ge S, Li L, Wu X, Liu Z, Wang Z. Virus-like particles: potential veterinary vaccine immunogens. Res Vet Sci 2012; 93: 553-59. French TJ, Roy P. Synthesis of Bluetongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Virol 1990; 64: 1530-36. French TJ, Marshall JJA, Roy P. Assembly of double-shelled, virus-like particles of Bluetongue virus by the simultaneous expression of four structural proteins. J Virol 1990; 64: 5695-700. Roy P.Genetically engineered structure-based vaccine for bluetongue disease. Vet Ital 2004; 40: 594-600.

10 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 [51] [52]

[53]

[54] [55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

Noad R, Roy P. Virus-like particles as immunogens. Trends in Microbiol 2003; 11: 438-44. Maclean J, Koekemoer M, Olivier AJ, et al. Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 2007; 88: 1460-69. Regnard GL, Halley-Stott RP, Tanzer FL, Hitzeroth II, Rybicki EP. High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnol J 2010; 8: 38-46. Torrent M, Llompart B, Lasserre-Ramassamy L, et al. Eukaryotic protein production in designed storage organelles. BMC Biol 2009; 7: 5. Carrillo C, Wigdorovitz A, Oliveros JC, et al. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J Virol. 1998; 72: 1688-90. Wigdorovitz A, Carrillo C, Dus Santos MJ, et al. Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1.Virology 1999; 255: 347-53. Wigdorovitz A, Pérez Filgueira DM, Robertson N, et al. Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein VP1.Virology 1999; 264: 85-91. DusSantos MJ, Carrillo C, Ardila F, et al. Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen. Vaccine 2005; 23: 1838-43. Pan L, Zhang Y, Wang Y, et al. Foliar extracts from transgenic tomato plants expressing the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease virus elicit a protective response in guinea pigs.Vet Immunol Immunopathol 2008; 121: 83-90. Andrianova EP, Krementsugskaia SR, Lugovskaia NN, et al. Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs. Biochemistry (Mosc) 2011; 76: 339-46. Saunders K, Sainsbury F, Lomonossoff GP. Efficient generation of cowpea mosaic virus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants. Virology 2009; 393: 329-37. D'Aoust MA, Lavoie PO, Couture MM, et al. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice. Plant Biotechnol J. 2008; 6: 930-40. D'Aoust MA, Couture MM, Charland N, et al.The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient

Received: November 30, 2012

Accepted: January 31, 2013

Thuenemann et al.

[64]

[65]

[66] [67]

[68]

[69] [70]

[71]

[72] [73]

[74]

[75]

[76] [77]

and safe response to pandemic influenza. Plant Biotechnol J 2010; 8: 607-19. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Min Jou W, Fiers W. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med 1999; 5: 1157-63. Ravin NV, Kotlyarov RY, Mardanova ES, et al. Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein. Biochemistry (Mosc) 2012; 77: 33-40. De Filette M, Min Jou W, BirkettA, et al. Universal influenza A vaccine: optimization of M2-based constructs. Virology 2005; 337: 149-61. Mardanova ES, Kotliarov R Yu, Ravin NV. Increased efficiency of recombinant proteins production in plants due to optimised translation of RNA of viral vector. MolBiol (Mosk)2009; 43: 568-71. Voinnet O, Rivas S, Mestre P, Baulcombe D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J2003; 33: 949-56. Meng XJ. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol 2000; 74: 309-29. Bøtner A, Strandbygaard B, Sørensen KJ, et al. Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine. Vet Rec 1997; 141: 497-9. Nielsen HS, Oleksiewicz MB, Forsberg R, Stadejek T, Bøtner A, Storgaard T. Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations. J Gen Virol 2001; 82: 1263-72. Dokland T. The structural biology of PRRSV. Virus Res. 2010; 154: 86-97. van Breedam W, Van Gorp H, Zhang JQ, Crocker PR, Delputte PL, Nauwynck HJ. The M/GP(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner.PLoS Pathog 2010; 6: e1000730. van Breedam W, Delputte PL, Van Gorp H, et al. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 2010; 91: 1659-67. Wissink EH, Kroese MV, van Wijk HA, Rijsewijk FA, Meulenberg JJ, Rottier PJ. Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus. J Virol 2005; 79: 12495-506. Cruz JL, Zúñiga S, Bécares M, et al. Vectored vaccines to protect against PRRSV. Virus Res 2010; 154: 150-60. Jiang Y, Fang L, Xiao S, et al.Immunogenicity and protective efficacy of recombinant pseudorabies virus expressingthe two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus. Vaccine 2007; 25: 547-60.