Embed Size (px)
Citation preview
859
Review
www.expert-reviews.com ISSN 1476-0584© 2010 Expert Reviews Ltd10.1586/ERV.10.85
Genes encoding vaccine proteins can be expressed in plant tissues exploiting different strategies for stable transgene expression, such as nuclear genomic integration [1] or expression from the plastid genome [2,3]. Transient gene expression provides a rapid alternative to the material- and time-consuming generation of stably transformed plants. When DNA is delivered into a plant cell, only a tiny proportion will become integrated into the host chromosomes and episomal DNA mol-ecules can remain transcriptionally competent for several days. This transient expression does not depend on chromo somal integration and is not affected by position effects. Expression from extra-chromosomal transgenes can be detected even 3 h after DNA delivery, reaches the maximum between 18 and 48 h, and persists for 10 days. The obvious advantages of these plant cell factory systems are ease of manipulation, speed, low cost, high protein yield, scalability and tight control of both upstream and downstream processing during manufacturing of these plant-derived biologicals.
Recently, plant-based systems for the expres-sion of recombinant proteins for vaccines and therapeutics have led to the generation of products successfully assessed in clinical trials. IFN-a
2b), produced in transgenic duckweed
[301], used to combat hepatitis C virus (HCV) was assessed in a Phase I clinical trial, while human glucocerebrosidase produced in trans-genic carrot cell cultures to combat Gaucher’s disease has progressed into a Phase III clinical trial [4]. Up till now, there are only few exam-ples of recombinant proteins synthesized using transient expression systems entering clinical trials. A case study example is the personal-ized therapeutic vaccine for non-Hodgkin’s lymphoma based on recombinant single-chain variable fragment (scFv) antibodies transiently produced in Nicotiana benthamiana plants by using a tobacco mosaic virus (TMV)-derived vector, assessed in a Phase I clinical trial [5]. Very recently, a H5N1 pandemic influenza vaccine based on the production of virus-like particles
Tatiana V Komarova1, Selene Baschieri2, Marcello Donini2,Carla Marusic2, Eugenio Benvenuto†2 and Yuri L Dorokhov1
1N.I. Vavilov Institute of General Genetics, Russian Academy of Science and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia2ENEA-UTBIORAD Laboratorio di Biotecnologie, Casaccia Research Center, 00123 Roma, Italy †Author for correspondence:Tel.: +39 063 048 6347 Fax: +39 063 048 6545 [email protected]
In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems.
Keywords: Agrobacterium • monoclonal antibodies • plant virus • transient expression • vaccine • virus-like particles • virus peptide display
Transient expression systems for plant-derived biopharmaceuticalsExpert Rev. Vaccines 9(8), 859–876 (2010)
sPeCIAL FoCUs y Plant-Derived Vaccines
www.expert-reviews.com/toc/erv/9/8
For reprint orders, please contact [email protected]
Expert Rev. Vaccines 9(8), (2010)860
Review
(VLPs) in N. benthamiana by using the hypertranslatable cowpea mosaic virus protein expression system (CPMV-HT) has also entered a Phase I [6].
It is a common notion that this way to produce biopharmaceu-ticals could soon replace the time-consuming procedures involved in bioproducts derived from stable transgenics, showing promise of high competitiveness towards ‘classical’ established methods.
While a number of excellent reviews have partly dealt with aspects of relevance to the field of transient expression in plants [1,7–16], in this article we tried to bridge the gaps, describ-ing in thorough detail the two most promising transient expres-sion approaches based on plant pathogen vectors, namely Agrobacterium tumefaciens strains and plant viruses with a wide range of expression and replication strategies.
AgroinfectionAgrobacterium-mediated transfer of genes from bacteria into plant cells in transient expression systems occurs via a specially constructed so-called binary vector family. These vectors con-sist of two parts: the first component is T-DNA (the segment delimited by the border sequences, the right [RB] and left [LB] border) and may contain multiple cloning sites, a selectable marker gene for transformed plant cells, a reporter gene and other genes of interest; and the second component is the vec-tor backbone, which carries plasmid replication functions for
Escherichia coli and A. tumefaciens, selectable marker genes for bacteria and optionally genes encoding plasmid mobilization functions [17,18]. The binary vectors have been used for many years not only for analyzing gene function but also for the pro-duction of biopharmaceuticals (Table 1). This approach uses the plant host Lactuca sativa, Arabidopsis thaliana or Nicotiana taba-cum. Recently, N. benthamiana became the most widely used experimental host in plant virology, monoclonal antibodies (mAbs) and vaccine production [19].
An obvious advantage of transient Agrobacterium-based vaccine gene expression is speed. The full expression of a gene of interest in agroinjected leaves may be reached in 3–4 days after being infil-trated with Agrobacteria [20,21]. Another attractive feature of this system is simplicity. All experimental procedures do not require expensive supplies and equipment. Leaves of greenhouse-grown plants may be infiltrated by using a syringe without a needle, vacuum infiltration [22–25] or the ‘wound-and-agrospray’ inocula-tion method [26]. Supplementation of the infiltration media with either surfactants such as Triton X-100, Tween-20 or Silwet L-77 improved the levels of expression [20,25]. This method provides synchronous gene expression because Agrobacterium is known to simultaneously infect at least 96% of cells of injected leaves [27].
There are many factors influencing the yield and quality of transgenic proteins: promoter activity, gene silencing, codon usage, protein stability and subcellular targeting. Post-transcriptional
Table 1. Yield of recombinant molecules of immunological interest obtained by Agrobacterium-mediated nonviral transient expression systems†.
Type of inoculation Silencingsuppressor
Pharmaceuticalprotein
Highest yield Ref.
Nicotiana benthamiana leaf agroinfiltration No HIV-1 p24 (144 aa), p17/24 and Gag (500 aa)
From 44 µg/kg FW (Gag) up to 16,148 µg/kg FW (p24), partially purified
[118]
N. benthamiana leaf agroinfiltration Yes HIV-1 Nef (219 aa) 250 ng/g FW, affinity purified [28]
N. benthamiana leaf agroinfiltration No HPV-16 L1 (531 aa) with VLP formation
400 µg/g FW [119]
N. benthamiana leaf coagroinfiltration Yes SARS-CoV nucleocapsid protein (420 aa)
79 µg/g FW, partially purified [29]
N. benthamiana leaf coagroinfiltration with TBSV p19
Yes Mycobacterium tuberculosis ESAT6:Ag85B (342 aa)
100 µg/g FW, partially purified [72]
Vacuum infiltration of lettuce (Lactuca sativa L.) with two populations of Agrobacterium encoding the mAb light and heavy chains
No Full-size mAb (~160 kDa tetramer)
20–80 µg/g FW, affinity purified [22]
Vacuum infiltration of N. benthamiana leaves with three populations of Agrobacterium encoding the mAb light, heavy chains and AMCV p19
Yes Full-size mAb (~160 kDa tetramer)
50–100 µg/g FW, affinity purified [106]
N. benthamiana leaf agroinfiltration with three populations of recombinant Agrobacterium encoding the mAb light, heavy chains and TBSV p19
Yes Full-size mAb (~160 kDa tetramer)
100–300 µg/g FW, affinity purified [202]
†The vector used in each of these cases was a 35S-based binary vector.aa: Amino acid; AMCV: Artichoke mottled crinkle virus; FW: Fresh weight; HPV: Human papillomavirus; mAb: Monoclonal antibody; SARS-CoV: Severe acute respiratory syndrome coronavirus; TBSV: Tomato bushy stunt virus; VLP: Virus-like particle.
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 861
Review
gene silencing (PTGS) is one of the reasons why leaves infiltrated by Agrobacteria usually express low amounts of protein, but coin-jection with genes encoding silencing suppressors increases protein accumulation [28–30]. Moreover, correct protein folding and pro-tein stability in the target cell compartment have great influence on vaccine protein yield. Recently, different strategies have been adopted to improve protein accumulation:
• Green fluorescent protein (GFP) as a carrier and fusion partner helped antigen folding and increased hepatitis B virus (HBV) surface antigen (HBsAg) [31] and chicken anemia virus (CAV) VP1, VP2 and VP3 [32] production;
• Hydrophobin sequence from Trichoderma reesei increased the expression levels of plant recombinant proteins transiently expressed in N. benthamiana plants by Agrobacterium infiltration [33];
• The construct harboring the complete Newcastle disease virus (NDV) hemagglutinin–neuraminidase (HN) glycoprotein gene with its own signal peptide, fused to a KDEL retention peptide, increased accumulation of HN [34];
• b-glucuronidase (GUS) used as a stable fusion partner enhanced the accumulation of a peptide derived from canine parvovirus within the cytoplasmic environment [35];
• Antibody Fc fragment was used to increase protein stability and yield of HIV-1 p24 [36].
Leaf agroinfiltration is a widely exploited approach, but also the plant root is considered an alternative system for foreign pro-tein production. The gene encoding a vaccine protein may be transformed into root cells via direct virus inoculation [37,38] or Agrobacterium-mediated delivery of TMV vectors in N. bentha-miana clonal root cultures, using either A. tumefaciens [30] or Agrobacterium rhizogenes [39]. This system has several advantages in that it represents a continuous organ culture system for con-tained manufacturing, easy to scale-up with higher expression levels if compared with leaf infiltration.
Plant virus-based vectorsSince 1984, when Ahlquist et al. first achieved in vitro synthesis of infectious brome mosaic virus (BMV), RNAs from full-length cDNA copies of postive-strand RNA viruses of both plants and animals have been produced successfully in vitro and in vivo [40]. The synthesis of infectious transcripts in vitro was a hallmark for plant virus-based vectors for devising and expressing recombinant proteins in field-grown plants [10]. The gene of interest is delivered to plant cells using infectious nucleic acid copies of the vector or, preferentially, as mature viral particles. The plant virus-based vec-tor technology has become a rapidly growing research area with significant applications in biopharmaceutical production (Table 2).
TobamovirusesComplete sequencing of the TMV genome and the synthesis of the T7 RNA-polymerase-directed full-length infectious transcripts in vitro opened the way for construction of ‘added-gene’ plant
virus-based vectors. TMV U1 strain RNA encodes four major pro-teins. The 126- and 183-kDa replicase proteins are translated from the first open reading frame (ORF) within the genomic RNA, the latter by occasional read-through of the amber stop codon for the 126-kDa protein. The 30-kDa movement protein (MP) and 17.4-kDa coat protein (CP) are expressed via individual 3 -́cot-erminal subgenomic (sg) RNAs from the 3 -́proximal ORFs [41]. Whereas the MP and the CP are dispensable, the 126- and the 183-kDa replicase proteins are required for viral RNA replication. The sg bicistronic intermediate-length RNA-2, called I
2 sgRNA,
is translated to produce the MP, whereas the 3 -́proximal CP gene of I
2 RNA is translationally silent [42]. This gene is expressed from
the small monocistronic sgRNA called low-molecular compo-nent (LMC). The vectors based on TMV [43], crucifer-infecting tobamovirus (crTMV) turnip vein clearing virus (TVCV) [44] and tomato mosaic virus (ToMV) [45] genomes exploit the sg promoter of the CP gene, providing the synthesis of the protein of interest directly from its own or an additional CP sg promoter (Figure 1). In RNA plant viruses, the CP sgRNA promoter extends downstream of the transcription initiation site and its activity is the highest in vectors where the CP is fused with a small immunogenic epitope [14]. The internal ribosome entry site (IRES) sequence presents an alternative way of vector-directed foreign gene expression. Translation of TMV U1 strain CP mRNA occurs by traditional cap-dependent ribosome scanning. However, vectors based on the genome of crTMV present another expression strategy where the IRES can be exploited. IRES-mediated translation is intrinsi-cally less efficient than cap-dependent translation but can provide expression of polycistronic templates [46].
PotexvirusesPotexviruses have monopartite, positive-strand RNA genomes encoding five ORFs. The 5́ end has a cap and the 3´ end has a polyA tail. The first ORF encodes the viral replicase. The central region of the genome encodes three overlapping ORFs, known as the triple gene block (TGB). These proteins are required for virus cell-to-cell movement [47,48]. The final ORF is the viral CP, which is required for virion assembly and virus cell-to-cell movement. The first potato virus X (PVX)-based vector was designed accord-ing to the ‘added gene’ strategy, where reporter gene (GFP) was cloned under control of duplicated CP sg promoter resulting in pPVX201 where viral RNA synthesis was 35S cauliflower mosaic virus (CaMV) promoter dependent (Figure 1) [49]. The majority of PVX-based vectors for different protein of interest production utilize pPVX-201 as a template [26,50]. But the stability of the added gene PVX-based vector is discussed and is likely dependent on the insert length [51]. Therefore, several ‘replaced gene’ variants were also designed. Giritch et al. made a so-called ‘deconstructed’ viral vector (see section ‘The MagnICON expression system’) in which the CP gene was substituted with GFP or antibody light-/heavy-chain genes (Figure 1) [52]. Removal of CP led to a higher level of tar-get protein expression. Another group constructed a PVXdt–GFP viral vector by deleting the TGB and CP gene. GFP expression was directed by the 25-K sg promoter (Figure 1) [53]. This variant was completely movement dysfunctional but gave considerable increase
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)862
Review
Table 2. Yield of recombinant molecules of immunological interest obtained by plant virus-mediated transient expression systems.
Virus vector
Type of inoculation Pharmaceutical protein VLP Highest yield Ref.
TMV TMV-30B-based vector-directed RNA transcript inoculation of spinach leaves
HIV-1 Tat protein (86 aa) No 0.3–0.5 mg/g FW [120]
TMV TMV-30B-based vector-directed RNA transcript inoculation of Nicotiana benthamiana leaves
HPV-16 L1 protein (531 aa) Yes ~0.03 x 10-3 mg/g FW [121]
TMV ToMV-TocJ-based vector directed RNA transcript inoculation of N. benthamiana leaves
The dengue virus envelope protein (102 aa fragment)
No ~0.1 mg/g FW [122]
TMV N. benthamiana leaf agroinfiltration Mycobacterium tuberculosis Ag85B and ESAT6 proteins (342 aa)
No 0.8–1.0 mg/g FW [72]
TMV TMV TTO1A vector-directed RNA transcript inoculation of N. benthamiana leaves
Human scFv proteins (~30 kDa) derived from human tumor immunoglobulin genes of non-Hodgkin’s lymphoma patients
No 100–800 µg/ml of leaf interstitial fluid
[5]
TMV TMV launch vector pBID4 agroinfiltration of N. benthamiana leaves
hGH (~30 kDa) No 0.7 mg/g FW [123]
TMV and PVX
N. benthamiana leaf agroinfiltration with two populations of recombinant Agrobacterium encoding the mAb light and heavy chains
Anticancer full-size mAb (~160 kDa)
No 100–300 µg/g FW, affinity purified
[202]
TMV and PVX
MagnICON: N. benthamiana leaf agroinfiltration with two populations of recombinant Agrobacterium encoding the mAb light and heavy chains
Anticancer full-size mAb (~160 kDa)
No 0.3–0.5 mg/g FW, affinity purified
[52]
TMV and PVX
MagnICON: N. benthamiana leaf agroinfiltration with two populations of recombinant Agrobacterium encoding the mAb light and heavy chains
Anti-West Nile virus mAb (~160 kDa)
No 0.8 mg/g FW leaves, affinity purified
[108]
TMV MagnICON: N. benthamiana leaf agroinfiltration
Yersinia pestis F1 (362 aa) and V (150 aa) antigens
No 0.3–0.5 mg/g FW; 1.2 mg /g FW (purified product)
[76]
TMV MagnICON: N. benthamiana leaf agroinfiltration
VV B5 (275 aa) antigenic domain
No 0.1 mg/g FW [124]
TMV MagnICON: N. benthamiana leaf agroinjection Plasmodium antigen PyMSP4/5 (33 kDa)
No 1–2 mg/g FW [125]
TMV MagnICON: N. benthamiana leaf agroinfiltration
NV CP (~58 kDa) Yes 0.8 mg/g FW [82]
PVX Inoculation of N. benthamiana leaves with pPVX201 vector-based cDNA construct
HBV nucleocapsid protein (21 kDa)
Yes 0.005–0.01 mg/g FW [50]
PVX Agroinfiltration of N. benthamiana leaves with pPVX201-based vector cDNA construct
M. tuberculosis ESAT6 antigen fusion with PVX CP (31 kDa)
No 0.5–1% of the TSP [126]
PVX and TMV
N. benthamiana leaf agroinfiltration GFP fused with CAV VP1, VP2 and VP3 (40–51 kDa)
No 1.2–5.4% of the TSP [32]
BeYDV N. benthamiana leaf agroinfiltration HBc (21 kDa) and NV CP (58 kDa)
Yes HBc: 0.80 mg/g FW; NV CP: 0.34 mg/g FW
[71]
aa: Amino acid; BeYDV: Bean yellow dwarf virus; BHV: Bovine herpesvirus; CAV: Chicken anemia virus; CP: Coat protein; CPMV: Cowpea mosaic virus; FMDV: Foot-and-mouth disease virus; FW: Fresh weight; GFP: Green fluorescent protien; HBc: Hepatitis B virus core antigen; hGH: Human growth hormone; mAb: Monoclonal anitbody; NV: Norwalk virus; PPV: Plum pox virus; PVX: Potato virus X; RHDV: Rabbit hemorrhagic disease virus; scFv: Single-chain variable fragment; TMV: Tobacco mosaic virus; ToMV: Tomato mosaic virus; TSP: Total soluble protein; VLP: Virus-like particle; VV: Vaccinia virus.
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 863
Review
in target protein production achieved when delivered into plant cells by agroinjection (together with the silencing suppressor P19).
The other strategy in PVX-based vector design is the fusion
of target protein to CP. There are two variants: target protein fused to CP N-terminus [54] or linked to the CP via the foot-and-mouth disease virus (FMDV) 2A catalytic peptide [55]. The 2A
POL POLMP
CP
CP
CP
CPL
CPL
CPS
CPS
CP
CP
MP
MP
MP
MP
MP
MP
POL
POL
POLTGB1
TGB2TGB3
POL
POL
POL
P1-Pro HcPro P3 Cl CP
Tobamoviruses Comoviruses
Bromoviruses
Potyviruses
Potexviruses
Viral RNA Viral RNA
Viral RNA
1a
2a
2b
Vector RNA-2
Vector RNA-3
RNA-1
RNA-1
RNA-2
RNA-2
RNA-3
Viral RNA
Viral RNA
Vector RNAs
Vector RNAs
MP sgP CP sgP
CP sgP
25K sgP
25K sgP
12K sgP
CP sgP
Pro-C HEL Vpg
Vpg NIa-Pro NIb-POL
Pro
CP sgP
CP sgPCP sgP
CP sgP
CP sgP
TGB1TGB2
TGB3
TGB1TGB2
TGB3
Figure 1. Schematic representation of viral genomes and vectors used to express heterologous peptides and proteins in plants. Target protein genes are shown hatched and their positions in the viral vectors are indicated. 2A catalytic peptide is marked with a black box. Dashed line represents a leaky termination codon. 1a, 1b and 2b: Components of replicase; Cl: Cylindrical inclusion protein; CP: Coat protein; CPL: Large coat protein; CPS: Small coat protein; FMDV: Foot-and-mouth disease virus; HC-Pro: Helper component proteinase; Hel: Helicase; MP: Movement protein; NIa-Pro: Viral proteinase; NIb-POL: RNA-dependent RNA polymerase; P3: Protein P3; POL: RNA polymerase; Pro: Proteinase; ProC: Proteinase cofactor; sgP: Subgenomic promoter (marked with arrows); TGB1–3: Components of the triple gene block; VPg: Virus protein genome linked.
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)864
Review
sequence promotes cotranslational cleavage of the fusion protein and maintains virus infectivity allowing the synthesis of both cleaved and recombinant CPs [55]. These approaches are usually used for peptide display.
PotyvirusesPotyviral-based vectors exploit different strategies for the expres-sion of foreign proteins. Their genome is a single-stranded, posi-tive-sense RNA translated into a single polyprotein that is further processed by virus-encoded proteases. The first potyvirus tagged with a foreign gene was tobacco etch virus (TEV) engineered to express the GUS reporter enzyme fused to the N-terminus of helper component proteinase (HC-pro) [56]. A plum pox potyvirus (PPV)-based vector has been constructed for the expression of full-length vaccine proteins. The foreign sequences were cloned between the NIb replicase and CP cistrons (Figure 1). The hetero logous protein is split from the rest of the potyviral polyprotein by cleavage at the site where the NIb and CP proteins are originally separated and at an additional NIa protease recognition site engineered at the N-terminus [57]. Several other members of the group are used in vector design and foreign protein production. The next genera-tion of potyvirus-based vectors allows simultaneous expression of two [58] or more [59] target proteins from one vector (Figure 1).
BromovirusesCucumber mosaic virus (CMV) and alfalfa mosaic virus (AMV) are the members of this group. They have segmented, tripartite linear ssRNA-positive genome composed of RNA1, RNA2 and RNA3. Each genomic segment has a 3 -́tRNA-like structure and a 5́ -cap. RNA1 and RNA2 encode proteins 1a and 2a, respec-tively, both involved in genome replication and internal transcrip-tion of sgRNA4 from the minus-strand copy of RNA3. RNA3 and sgRNA4 are translated into movement and capsid proteins, respectively. In the first CMV-based vector, CP gene was usually replaced by foreign gene (Figure 1) that prevented the CP-dependent cell-to-cell movement. In this case, CP was provided in trans to ensure proper cell-to-cell movement [60], otherwise the viral vec-tor is limited to the initially infected cells. In a more advanced version, CMV inducible viral amplicon (CMViva), one of the key components of the viral replicase (from RNA1), was under the control of a tightly regulated expression system inducible by estradiol. All three CMV components were engineered on a single Ti plasmid. As compared with analogous vectors under control of the 35S promoter, the overall yield of target protein (human a1-antitrypsin [AAT]) obtained with CMViva was several-fold higher [61]. The next step in development of a CMV-based system was to retrieve cell-to-cell movement. Previous studies have shown that deletion of the C-terminal 33 amino acids of CMV 3a MP enabled viral cell-to-cell movement independently of CP, although at lower efficiency [62]. A new CMV-based expression vector was designed that exploits the mutant 3a MP for CP-independent cell-to-cell movement [63]. Two (RNA1 and RNA2) or all three CMV components were engineered on a single Ti plasmid and CMV genome sequences were placed under the control of the 35S CaMV promoter. High yields of GFP (~450 mg/kg leaf tissue)
and human growth hormone (hGH; ~170 mg/kg leaf tissue) were achieved in N. benthamiana. CMV-based expression vectors are particularly attractive because the virus has a very broad host range, including more than 1000 monocot and dicot plant species.
Another broad host range virus is AMV. Several species of legumes were screened for transient protein expression using AMV-based expression system [64]. This system consists of three constructs expressed under the control of CaMV 35S promoter: one for RNA1 and RNA2 (encoding AMV viral replicase), a second encoding the viral CP required for genome activation and replication and a further construct for RNA3 in which the CP coding region is replaced by the foreign gene. Expression levels of 420 ± 26.24 mg GFP/kg fresh weight (FW) in the green pea variety speckled pea were achieved. High expression levels were also achieved for the anthrax protective antigen fused to lichenase (LicKM–PAD4) [64].
ComovirusesCowpea mosaic virus, the type member of the comoviruses group, has a narrow host range, normally infecting legumes other than N. benthamiana as experimental host. The genome of CPMV consists of two separately encapsidated positive-strand RNA mol-ecules: RNA1 and RNA2. Each genomic RNA has a viral protein genome (VPg) covalently linked to the 5́ end, a 3 -́polyA tail and encodes a single ORF whose expression is achieved through the subsequent processing of a precursor polyprotein. RNA1 encodes proteins involved in the replication of viral RNAs and polyprotein processing. RNA2 encodes the MP and the two CPs, which are essential for cell-to-cell movement and systemic spread. The proteinase responsible for processing both the RNA1 and RNA2 polyproteins is encoded by the RNA1 [65]. When design-ing CPMV-based vectors, usually RNA1 component remains unmodified as it contains genes essential for replication. As for the RNA2, two different approaches are used. The first implies foreign gene insertion in RNA2 resulting in an increase in size which is still functional for systemic spreading. The second, most efficient, involves insertion of the foreign gene as an in-frame C-terminal fusion to the RNA2-encoded polyprotein (Figure 1). The rescue of target protein is performed by an inducible proteo-lytic shunt through the 2A catalytic peptide from FMDV inserted upstream of the foreign protein sequence [66]. The first strategy is usually pursued when production of viral (or virus-like) particles is devised, while the second allows larger inserts to be incorpo-rated, but abolishes the ability of the virus to spread both within and between plants. The system relies on the observation that the sequences necessary for replication of RNA2 by the RNA1-encoded replicase lie exclusively at the 5´ and 3´ ends of the RNA. This allows most of the RNA2 ORF to be deleted without affecting the ability of RNA2 to be replicated. Unfortunately, such vector system lacks natural CPMV silencing suppressor [67] so another component (i.e., Hc-Pro from potato virus Y) should be supplied. This is usually done through agroinfiltration that allows simultaneous delivery of several genetic expression con-structs in plant cells. Utilization of ‘deleted’ variant of RNA2 gives higher yield of target protein and is clearly advantageous in
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 865
Review
case of antibody production [68]. Moreover, binary vectors simpli-fying cloning and expression steps with the CPMV system were designed. CPMV expression cassettes and the sequence encoding P19 have been incorporated into the T-DNA region of the result-ing vector allowing high-level expression of multiple polypeptides by infiltration of a single construct [65].
GeminivirusesViruses of this family possess single-stranded circular DNA genomes that replicate to very high copy number in the nuclei of infected plant cells. Owing to these unique features, the potential for using the DNA-containing geminiviruses as extrachromo-somal gene amplification vectors for the expression of useful pro-teins in plants has been recognized [69]. At present, the improved expression of recombinant GFP using a replicating vector based on beet curly top virus in leaf disks and agroinfiltrated N. bentha-miana leaves was observed [70]. In addition, a vector replicon based on the bean yellow dwarf virus (BeYDV) DNA genome was cre-ated recently [71]. A system composed of two vectors containing different portions of the BeYDV genome was constructed. The ‘Rep supplying vector’ encodes Rep, whose expression can be controlled by specific promoters and the LSL vector that con-tains the gene of interest and the cis-acting elements (the long and short intergenic regions, LIR and SIR, respectively) required for replication of the viral genome. It was shown that forma-tion of BeYDV-based replicons in bombarded tobacco NT-1 cells resulted in increased transient expression of the GUS protein. The delivery of added vector components increased protein pro-duction [71]. Codelivery of the BeYDV-derived vector and the Rep/RepA-supplying vector by agroinfiltration of N. benthamiana leaves resulted in efficient replicon amplification and protein pro-duction within 5 days. Coexpression of the tomato bushy stunt virus (TBSV) P19 protein enhanced the stability of the mRNA and increased protein yield. A single replicon vector containing a built-in Rep/RepA cassette drove protein expression to levels similar to the three-component system, even in the absence of P19 silencing suppressor.
The MagnICON expression systemThe Agrobacterium-delivered plant viral vectors in the transient expression systems exploits the RNA polymerase (Pol) II–mediated nuclear export route including 5́ -end capping, splicing and 3 -́end formation. In contrast to DNA geminiviruses, plant RNA viruses replicate in the cytoplasm and are not adapted to nuclear splicing machinery, which recognizes and removes cryptic introns from viral RNA leading to degradation. The Agrobacterium-delivered, so-called ‘first-generation’ TMV and PVX vectors, have low pro-duction capacity with protein yields quite low (0.04–0.3% of the total soluble protein [TSP]), requiring coinjection of plasmids encoding silencing suppressors such as tombusvirus p19, potyvirus P1/HC-Pro [26,32,72], inhibitors of pectin methylesterase [73] or Pol II-directed short noncoding RNAs [201].
To adjust TMV infectious copy transcription to Agrobacterium-mediated vector delivery, Gleba and coworkers developed a new generation expression platform, also known as ‘Magnifection’, a
new approach for expressing recombinant biopharmaceuticals in plants [8,74]. The MagnICON system includes advantages of Agrobacterium-mediated delivery and upgraded TMV-based vec-tors where putative cryptic splice sites were removed and multiple plant introns were inserted [27,44].
The idea of ‘deconstructed virus’ (pro-vector) is based on the efficient assembly of DNA modules obtained by recombination in plant cells. In this system, Agrobacterium-delivered plasmids encoding 5́ -part and 3 -́part of a viral vector and, as an additional component, the plasmid encoding an integrase, essential for full vector assembly in planta are used. To make assembly of the viral vector precise and prevent mutations that could occur during recombination, a fragment of plant intron was inserted into each part of provector: the donor site just upstream of recombination site in the 5́ -part and the acceptor site downstream of recombina-tion site in the 3 -́part. Thus, the recombination site with possible mutations is deleted during splicing [44].
The adopted strategy of deconstructing and reconstructing the viral RNA vector provides enhanced versatility and efficiency due to varying gene combinations, targeting signals and tags can be tested without the need to engineer each individual variant construct [44]. The cloning procedure is made even simpler with ‘golden-gate’ shuffling method allowing to assemble (in one step and one tube) at least nine separate DNA fragments into an accep-tor vector, obtaining different combinations and then choosing the best variant for each protein of interest [75]. This system proved to be very efficient for the production of different proteins of interest and is now widely used [52,76].
Antigens or antigen domain production There are two main methods for the production of foreign proteins in plant transient expression systems: antigens may be expressed in the plant cell as a single vaccine protein or in fusion with a protein carrier (Table 3); or antigenic peptides may be localized (exposed) on the surface of plant virions (Table 4).
Recombinant soluble antigens Transient expression strategies can be used to express in plants both soluble or self-assembling antigens. As for soluble whole antigens or antigen domains, polypeptides derived from several different pathogens have been produced mainly in Nicotiana species with varying recoveries depending on the expression strategy adopted but generally with expression levels signifi-cantly higher compared with those obtained through nuclear transformation. The first original paper in this area described the expression of the structural VP1 protein of FMDV using a TMV-based gene insertion vector with a yield of approximately 0.5–1 µg/g FW [77]. Since then transient expression underwent a fundamental evolution thanks to the development of modular deconstructed expression systems and also to the exploitation of silencing suppressors, codon optimization, expression targeting to specific subcellular compartments and even identification of optimal harvesting time [10]. With these devices, it is now possible to obtain up to 800 µg of the protein of interest per gram of fresh leaf tissues [72]. At present, the major bottleneck of the technology
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)866
ReviewTa
ble
3. I
mm
un
olo
gic
al e
ffica
cy o
f re
spre
sen
tati
ve a
nti
gen
s o
r an
tig
en d
om
ain
s tr
ansi
entl
y ex
pre
ssed
in p
lan
t ti
ssu
es.
An
tig
en
(pat
ho
gen
/dis
ease
)Pl
ant
Exp
ress
ion
leve
lFo
rm o
f d
eliv
ery
Ad
juva
nt†
Do
se (
anim
al)
Do
ses
(n)
and
d
eliv
ery
rou
teTy
pe
of
resp
on
seIn
vit
ro
neu
tral
izat
ion
as
say
or
chal
len
ge
Ref
.
VP1
(FM
DV
)N
icot
iana
b
enth
amia
na5
0–1
50
µg
/g F
LWFE
+
0.5
–1 µ
g/1
50
µl
(M)
Five
ip.
Hum
oral
+
[77]
R9
mim
oto
pe
(HC
V)–
cho
lera
to
xin
B N
. ben
tham
iana
6–8
0 µ
g/g
FLW
0.2%
TSP
FE+
0.5
–1 µ
g/3
0 µl
(M
)Fi
ve in
.H
umor
alN
D[127]
VP6
0 (R
HD
V)
Nic
otia
na
clev
elan
dii
ND
FE+
1 m
l (R
)Tw
o sc
.H
umor
al+
[128]
E7 (
HPV
-16
)N
. ben
tham
iana
3–4
µg
/g F
LWFE
-0.
5 µ
g/5
00
µl
(M)
Four
sc.
Hum
oral
C
ellu
lar
+
[129]
gD
c (B
HV
-1)
N. b
enth
amia
na15
–20
µg
/g F
LWFE
+
200
µg
(M)
5 g
(C)
On
e ip
. (M
)Se
ven
(hal
f d
ose
im.
and
half
do
se s
c.) (
C)
Hum
oral
C
ellu
lar
+ (
C)
[130]
GA
733
-2 (
colo
rect
al c
ance
r)N
. ben
tham
iana
ND
PA
+20
µg
(M)
On
e sc
. + t
wo
ip.
Hum
oral
C
ellu
lar
ND
[131]
SAG
1 (T
oxop
lasm
a go
ndii)
Nic
otia
na
tab
acum
6–1
0 µ
g/g
FLW
0.0
6–
0.1%
TSP
FE+
200
ng (
M)
Four
sc.
Hum
oral
+[132]
Tat
(HIV
-1)
Spin
acia
ol
erac
eae
30
0 µ
g/g
FLW
FL
-3
00
µg
(M)
Thre
e or
al
Hum
oral
ND
[120]
F1 a
nd V
(Yer
sini
a p
estis
)N
. ben
tham
iana
1–2
mg
/g F
LWPA
+25
µg
(GP)
Thre
e sc
.H
umor
al+
[76]
PA (
Baci
llus
anth
raci
s)–
liche
nase
N. b
enth
amia
naN
DPA
+10
0 µ
g (M
)Th
ree
ip.
Hum
oral
+[133]
B5 (
VV
)N
. ben
tham
iana
100
µg
/g L
LPA
+2
µg
(M)
Two
sc. +
on
e ip
.H
umor
al+
[124
]
E7 (
HPV
-16
)–lic
hena
seN
. ben
tham
iana
40
0 µ
g/g
FLW
PA+
40
µg
(M)
Five
sc.
Hum
oral
C
ellu
lar
+[134
]
F1 a
nd V
(Yer
sini
a p
estis
)–lic
hena
seN
. ben
tham
iana
120
–38
0 µ
g/g
FLW
PA+
25–2
50
µg
(MC
)Th
ree
sc.
Hum
oral
+[135]
Dom
ain
III E
nv29
8–
40
0 (D
ENV
2)N
. ben
tham
iana
5.6
µg
/g F
LW0.
28%
TSP
PA+
10–2
0 µ
g (M
)N
ine
(fou
r ×
10
µg
and
five
× 2
0 µ
g) i
m.
Hum
oral
+[122]
HA
17–5
32 (
influ
enza
A v
irus,
H
5N1)
N. b
enth
amia
na6
0 µ
g/g
FLW
PA+
15–4
5 µ
g (M
)45
–90
µg
(F)
Thre
e3 s
c.H
umor
al+
(F)
[136
]
† Ant
igen
del
iver
ed w
ith
(+) o
r w
ith
ou
t (-
) ad
juva
nt.
BH
V: B
ovi
ne
her
pes
vir
us;
C: C
attl
e; C
AV
: Chi
cken
an
emia
vir
us;
DEN
V: D
eng
ue
viru
s; D
LP: D
ried
leaf
po
wd
er; F
1: F
ract
ion
1; F
: Fer
ret;
FE:
Fo
liar
extr
act;
FL:
Fre
sh le
aves
; FLW
: Fre
sh le
aves
wei
ght
; FM
DV
: Fo
ot-a
nd
-m
ou
th d
isea
se v
iru
s; G
P: G
uin
ea p
ig; H
A: H
emag
glu
tini
n; H
CV
: Hep
atit
is C
vir
us;
HPV
: Hu
man
pap
illo
ma
viru
s; im
.: In
tram
usc
ula
r; in
.: In
tran
asal
; ip.
: Int
rap
erit
on
eal;
LL: L
yop
hiliz
ed le
aves
; M: M
ou
se; M
C: M
acaq
ue;
N
D: N
o d
ata;
PA
: Pu
rifi
ed a
ntig
en; R
: Rab
bit
; RH
DV
: Rab
bit
hem
orr
hag
ic d
isea
se v
iru
s; S
AR
S-C
oV: S
ever
e ac
ute
res
pir
ato
ry s
ynd
rom
e-co
rona
viru
s; s
c.: S
ub
cuta
no
us;
TSP
: Tot
al s
olu
ble
pro
tein
; V: V
ant
igen
; V
V: V
acci
nia
viru
s.
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 867
Review
seems to be represented by the extraction and purification steps. Even if in vivo experiments often demonstrated the ability of plant-derived antigens to protect animals from challenge, the immunological properties of plant-derived recombinant antigens or antigen domains have been mostly tested by orally deliver-ing to mice (through gavage) powdered freeze-dried leaves or plant extracts. Alternatively, mice have been injected with plant extracts (Table 3). This, of course, prevents a correct evaluation of the immune response that can be positively or negatively affected by unidentified plant components. Several strategies have been evaluated to facilitate purification of the recombinant antigen other than classical affinity tags [36,78,79].
Plant-derived virus-like particlesAn ideal vaccine against viral pathogens should mimic the native virus to induce a protective immune response. These vaccines tra-ditionally based on live-attenuated or inactivated viruses are effec-tive in inducing both cellular and humoral immune responses. However, a major concern about the live-attenuated vaccines is their potential ability to revert to a pathogenic form [80]. In this respect, a novel class of VLP-based vaccines provides a promising way to overcome safety concerns. VLPs are macromolecular structures that morphologically resemble those of the virus from which they are derived and consist of one or several self-assembling viral proteins expressed through recombinant technologies. For both animal and plant viruses, there are several examples described in the literature in which the structural proteins retain their ability to self-assemble in the absence of the viral genome. These features make VLPs safe and effective vaccine tools for inducing strong B- and T-cell-mediated immune responses. Moreover, VLPs could be used not only as vaccine against the cognate virus but also as carriers for an efficient presentation of foreign epitopes to the immune system.
Pioneering work on hepatitis B core antigen (HBcAg)-derived VLPs was carried out using the MagnICON transient expres-sion system in N. benthamiana leaves. High levels of antigen were obtained (2.38 mg/g FW) and immunogenicity assayed in mice [81]. The results clearly showed that plant-derived HBcAg VLPs were able to induce specific serum IgG and intestinal IgA when administered mucosally in mice without adjuvants. The same system was successfully used for the production Norwalk virus (NV) VLPs [82]. Similarly to HBcAg, NV VLPs were pro-duced at high levels (0.86 mg/g FW) and elicited both systemic and mucosal immune responses.
Rapid and cost-effective vaccine production is essential to allow rapid response to newly emerging viruses. Influenza virus-derived VLPs in agroinfiltrated N. benthamiana leaves could therefore repre-sent a novel vaccine technology against possible influenza pandem-ics. In fact, the results obtained by D’Aoust and colleagues showed that two doses of plant-produced influenza H5-VLPs were able to protect mice not only from lethal homologous virus challenge but also against heterologous strains [6,83].
An attractive approach for the production of epitope-based plant-derived vaccine formulations is the construction of chimeric VLPs based on plant viruses displaying immunogenic peptides. Several plant viruses (e.g., CPMV, papaya mosaic virus and PVX) Ta
ble
3. I
mm
un
olo
gic
al e
ffica
cy o
f re
spre
sen
tati
ve a
nti
gen
s o
r an
tig
en d
om
ain
s tr
ansi
entl
y ex
pre
ssed
in p
lan
t ti
ssu
es (
con
t.).
An
tig
en
(pat
ho
gen
/dis
ease
)Pl
ant
Exp
ress
ion
leve
lFo
rm o
f d
eliv
ery
Ad
juva
nt†
Do
se (
anim
al)
Do
ses
(n)
and
d
eliv
ery
rou
teTy
pe
of
resp
on
seIn
vit
ro
neu
tral
izat
ion
as
say
or
chal
len
ge
Ref
.
HA
17–5
32 (
influ
enza
A v
irus,
H
5N1)
N. b
enth
amia
naN
DPA
+2.
5–5
µg
(M)
Thre
e sc
.H
umor
al+
[137]
HA
1–33
0 (in
fluen
za A
viru
s,
H5N
1)
N. b
enth
amia
na1
µg
/g F
LWPA
+10
µg
(M)
Two
im.
Hum
oral
-[138]
PyM
SP4
/5(P
lasm
odi
um y
oel
ii)N
. ben
tham
iana
1–2
mg
/g F
LW10
% T
SPD
LP+
100
µg
(M)
Five
ora
l (af
ter
one
DN
A v
acci
ne
do
se)
Hum
oral
ND
[125]
N (
SAR
S-C
oV)
N. b
enth
amia
na79
µg
/g F
LW0.
8–1
% T
SPFE
+2–
4 µ
g (M
)Fo
ur ip
.H
umor
alN
D[29]
† Ant
igen
del
iver
ed w
ith
(+) o
r w
ith
ou
t (-
) ad
juva
nt.
BH
V: B
ovi
ne
her
pes
vir
us;
C: C
attl
e; C
AV
: Chi
cken
an
emia
vir
us;
DEN
V: D
eng
ue
viru
s; D
LP: D
ried
leaf
po
wd
er; F
1: F
ract
ion
1; F
: Fer
ret;
FE:
Fo
liar
extr
act;
FL:
Fre
sh le
aves
; FLW
: Fre
sh le
aves
wei
ght
; FM
DV
: Fo
ot-a
nd
-m
ou
th d
isea
se v
iru
s; G
P: G
uin
ea p
ig; H
A: H
emag
glu
tini
n; H
CV
: Hep
atit
is C
vir
us;
HPV
: Hu
man
pap
illo
ma
viru
s; im
.: In
tram
usc
ula
r; in
.: In
tran
asal
; ip.
: Int
rap
erit
on
eal;
LL: L
yop
hiliz
ed le
aves
; M: M
ou
se; M
C: M
acaq
ue;
N
D: N
o d
ata;
PA
: Pu
rifi
ed a
ntig
en; R
: Rab
bit
; RH
DV
: Rab
bit
hem
orr
hag
ic d
isea
se v
iru
s; S
AR
S-C
oV: S
ever
e ac
ute
res
pir
ato
ry s
ynd
rom
e-co
rona
viru
s; s
c.: S
ub
cuta
no
us;
TSP
: Tot
al s
olu
ble
pro
tein
; V: V
ant
igen
; V
V: V
acci
nia
viru
s.
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)868
ReviewTa
ble
4. I
mm
un
olo
gic
al p
rop
erti
es o
f re
pre
sen
tati
ve c
him
eric
pla
nt
viru
s p
arti
cles
pro
du
ced
in p
lan
ts.
An
tig
en (
pat
ho
gen
/d
isea
se)
Plan
t an
dvi
ral c
arri
erR
eco
very
Form
of
del
iver
yA
dju
van
t†D
ose
(an
imal
)D
ose
s (n
) an
d
del
iver
y ro
ute
Typ
e o
f re
spo
nse
In v
itro
n
eutr
aliz
atio
n
assa
y o
r ch
alle
ng
e
Ref
.
VP1
141–
159
(FM
DV
)G
P41 73
1–75
2 (H
IV-1
)V
P18
5–9
8 (H
RV-1
4)
Vig
na
ungu
icul
ata
CPM
V
1.2–
1.5
mg
/g F
LWPu
rifi
ed C
VPs
+10
0–5
0 µ
g (b
oo
st) (
RB
)Th
ree
im. +
sc.
H
umor
alN
D[139]
VP2
3–1
9 (M
EV)
V. u
ngui
cula
taC
PMV
1–2
mg
/g F
LWPu
rifi
ed C
VPs
+10
0 µ
g–1
mg
(20
µg
pep
tid
e)
(MK
)
On
e sc
. H
umor
al+
[140
]
gp12
0 V
3 lo
op
(HIV
-1)
Nic
otia
na
ben
tham
iana
TBSV
0.9
mg
/g F
LWN
DN
DN
DN
DN
DN
D[141]
GP 25
3–2
75, N
P 40
4–
418
(RV
) gp
120
V3
loo
p (H
IV-1
)N
. ben
tham
iana
AlM
VN
DPu
rifi
ed C
VPs
+/-
10 µ
g (M
)Se
ven
ip.
Hum
oral
+[142]
VP2
6–2
0 (C
PV)
Nic
otia
na
clev
elan
dii
PPV
ND
Puri
fied
CV
Ps+
0.05
–5 µ
g (M
)5
µg
-1 m
g (R
B)
Two
ip. (
M)
Two
im. (
RB
)H
umor
al+
[143]
FnB
P-D
2 1–3
0
(Sta
phyl
oco
ccus
aur
eus)
FnB
P-D
2 1–3
8
(Sta
phyl
oco
ccus
aur
eus)
V. u
ngui
cula
taC
PMV
N. b
enth
amia
naPV
X
1.2
mg
/g F
LW
0.2
mg
/g F
LW
Puri
fied
CV
Ps+
1–10
µg
(M)
1 m
g (R
)Tw
o sc
. (M
)Th
ree
sc. (
R)
Hum
oral
+[144
]
FP26
1–27
4
(Pse
udom
onas
aer
ugin
osa)
Nic
otia
na
taba
cum
TMV
ND
Puri
fied
CV
Ps+
10–5
0 µ
g/1
00
µl
(M)
Four
im.
Hum
oral
+
[145]
VP2
3–1
9 (C
PV)
V. u
ngui
cula
taC
PMV
ND
Puri
fied
CV
Ps+
7.5
mg
(15
0 µ
g p
epti
de)
(D
)Tw
o sc
.H
umor
al+
[146
]
gp41
654
–65
9 (H
IV-1
)N
. ben
tham
iana
PVX
0.4
–0.
6 m
g/g
FLW
Puri
fied
CV
Ps-
50
µg
(1.3
µg
pep
tid
e) (
M)
Six
ip.
11 in
.H
umor
al+
[147]
GP 25
3–2
75, N
P 40
4–
418
(RV
)
N. t
abac
umN
. ben
tham
iana
Spin
acia
ole
race
aA
lMV
0.4
mg
/g F
LW5
0 µ
g/g
FLW
Puri
fied
CV
PsIn
fect
ed
spin
ach
leav
es
+ (
M)
- (H
)25
0 µg
(1.3
µg
pept
ide)
(M
)15
0 g
(5 m
g C
VPs
, 70
0 µg
pep
tide)
(H
)
Thre
e ip
. (M
)Th
ree
oral
(H
)H
umor
al+
[111]
R9
mim
oto
pe
(HC
V)
N. t
abac
umC
MV
50
µg
–1 m
g/g
FL
WPu
rifi
ed C
VPs
ND
ND
ND
Hum
oral
ND
[148]
† Ant
igen
del
iver
ed w
ith
(+) o
r w
ith
ou
t (-
) ad
juva
nt.
AlM
V: A
lfal
fa m
osa
ic v
iru
s; C
MV
: Cu
cum
ber
mo
saic
vir
us;
CO
PV: C
anin
e o
ral p
apill
om
avir
us;
CPM
V: C
ow
pea
mo
saic
vir
us;
CPV
: Can
ine
par
vovi
rus;
CSF
V: C
lass
ical
sw
ine
feve
r vi
rus;
CV
P: C
him
eric
vir
us
par
ticl
e;
D: D
og
; FLW
: Fre
sh le
aves
wei
ght
; FM
DV
: Fo
ot-a
nd
-mo
uth
dis
ease
vir
us;
H: H
um
an; H
CV
: Hep
atit
is C
vir
us;
HR
V: H
um
an r
hin
ovi
rus;
im.:
Intr
amu
scu
lar;
in.:
Intr
anas
al; i
p.: I
ntra
per
ito
nea
l; M
: Mo
use
; MC
: Mac
aqu
e;
MEV
: Min
k en
teri
tis
viru
s; M
K: M
ink;
ND
: No
dat
a; P
PV: P
lum
pox
vir
us;
PV
X: P
otat
o vi
rus
X; R
: Rat
; RB
: Rab
bit
; RSV
: Res
pir
ato
ry s
yncy
tial
vir
us;
RV
: Rab
ies
viru
s; s
c.: S
ub
cuta
neo
us;
TBS
V: T
om
ato
bu
shy
stu
nt v
iru
s;
TMV
: To
bac
co m
osa
ic v
iru
s.
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 869
Review
are currently being used for the expression of het-erologous peptides but only few examples of plant virus-derived chimeric VLPs in plant tissue have been reported up to now. The first report published by Natilla and colleagues described the engineering of CMV to generate VLPs exposing neutralizing epitopes of NDV [84]. Different NDV peptides were inserted in the CMV CP gene and expressed in N. benthamiana plants using a PVX-based tran-sient expression system. The immunoreactivity of purified chimeric VLPs to NDV specific antibod-ies, demonstrated the potential of CMV CP as a carrier of immunogenic epitopes.
As extensively reported in the literature, CPMV is one of the most versatile epitope presentation sys-tems for a number of viral and bacterial antigens [85]. Recently, the generation of CPMV VLPs in plants was published [86]. In this work, the CPMV-HT was employed to produce CPMV-derived empty parti-cles free from any RNA. Therefore, CPMV-derived VLPs represent a very promising and safe tool for vaccine production.
Peptide display on plant virusesMolecular insights on the mechanisms of activation of adaptive immune responses indicate that peptides, rather than whole proteins, are responsible for the activation of T and B lymphocytes (even if at a lower extent) [87]. It is therefore possible to stimulate spe-cific clones and bias the immune response towards a humoral or cell-mediated response by appropriate peptides thus eliminating major safety concerns of using live-attenuated vaccine strategies. The selec-tion of peptides to be included in vaccine formula-tions require an epitope mapping of the antigen of interest to identify immunodominant sequences. The identification of B-cell-activating peptides is complicated by the fact that immunoglobulins often recognize conformational epitopes. In the attempt to mimic conformational structures (particularly those of viral envelopes), cyclic peptides, branched pep-tides, peptomers (cross-linked peptide polymers) and other complex multimeric structures, as well as pep-tides conjugated to other molecules have been devel-oped [88]. Mixtures of peptides or ‘mimotopes’ iden-tified in combinatorial peptide libraries that could mimic relevant structures and potentially inducing broad responses, have been successfully employed in some preclinical vaccination trials (e.g., respiratory syncytial virus, measles and hepatitis C) [89]. When the aim is to identify peptides activating cytotoxic T lymphocytes (CTLs), the task is complicated by the fact that immunodominant peptides vary as a func-tion of the individual immuno genetic background (MHC haplotype). To overcome the problem that Ta
ble
4. I
mm
un
olo
gic
al p
rop
erti
es o
f re
pre
sen
tati
ve c
him
eric
pla
nt
viru
s p
arti
cles
pro
du
ced
in p
lan
ts (
con
t.).
An
tig
en (
pat
ho
gen
/d
isea
se)
Plan
t an
dvi
ral c
arri
erR
eco
very
Form
of
del
iver
yA
dju
van
t†D
ose
(an
imal
)D
ose
s (n
) an
d
del
iver
y ro
ute
Typ
e o
f re
spo
nse
In v
itro
n
eutr
aliz
atio
n
assa
y o
r ch
alle
ng
e
Ref
.
GP 17
0–1
90
(RSV
)N
. tab
acum
AlM
VN
DPu
rifi
ed C
VPs
-5
00
µg
(MC
)Th
ree
im.
Hum
oral
Cel
lula
rN
D[149]
E279
0–
86
0, E
2 85
4–
89
4 (C
SFV
)N
. ben
tham
iana
PVX
120
µg
/g F
LWPu
rifi
ed C
VPs
+5
0 µ
g (R
B)
Thre
e sc
. H
umor
alN
D[150]
L261
–171
(C
OPV
)N
. ben
tham
iana
TMV
ND
Puri
fied
CV
Ps-
2–20
µg
(M)
Two
sc.
Hum
oral
ND
[151]
NP 3
66
–374
(In
fluen
za A
viru
s)N
. ben
tham
iana
PVX
1.1
mg
/g F
LWPu
rifi
ed C
VPs
-5
0 µ
g (2
µg
pep
tid
e) (
M)
Two
sc.
Cel
lula
rN
D[97]
M2 2–
24 (
Influ
enza
A v
irus)
V. u
ngui
cula
taC
PMV
15–3
3 µ
g/g
FLW
Puri
fied
CV
Ps+
(M)
Thre
e sc
.H
umor
al+
[152]
R9
mim
oto
pe
(HC
V)
Lact
uca
sativ
aC
MV
0.2
mg
/g F
LWIn
fect
ed
lett
uce
leav
es-
9 g
(1.8
mg
CV
Ps,
144
µg
pep
tid
e)
(RB
)
22 o
ral
Hum
oral
ND
[153]
R9
mim
oto
pe
(HC
V)
N. b
enth
amia
naPV
X75
–125
µg
/g F
LWPu
rifi
ed C
VPs
+25
–50
µg
(0.2
–0.
4 µ
g p
epti
de)
(M
)
Nin
e sc
. H
umor
alN
D[154
]
† Ant
igen
del
iver
ed w
ith
(+) o
r w
ith
ou
t (-
) ad
juva
nt.
AlM
V: A
lfal
fa m
osa
ic v
iru
s; C
MV
: Cu
cum
ber
mo
saic
vir
us;
CO
PV: C
anin
e o
ral p
apill
om
avir
us;
CPM
V: C
ow
pea
mo
saic
vir
us;
CPV
: Can
ine
par
vovi
rus;
CSF
V: C
lass
ical
sw
ine
feve
r vi
rus;
CV
P: C
him
eric
vir
us
par
ticl
e;
D: D
og
; FLW
: Fre
sh le
aves
wei
ght
; FM
DV
: Fo
ot-a
nd
-mo
uth
dis
ease
vir
us;
H: H
um
an; H
CV
: Hep
atit
is C
vir
us;
HR
V: H
um
an r
hin
ovi
rus;
im.:
Intr
amu
scu
lar;
in.:
Intr
anas
al; i
p.: I
ntra
per
ito
nea
l; M
: Mo
use
; MC
: Mac
aqu
e;
MEV
: Min
k en
teri
tis
viru
s; M
K: M
ink;
ND
: No
dat
a; P
PV: P
lum
pox
vir
us;
PV
X: P
otat
o vi
rus
X; R
: Rat
; RB
: Rab
bit
; RSV
: Res
pir
ato
ry s
yncy
tial
vir
us;
RV
: Rab
ies
viru
s; s
c.: S
ub
cuta
neo
us;
TBS
V: T
om
ato
bu
shy
stu
nt v
iru
s;
TMV
: To
bac
co m
osa
ic v
iru
s.
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)870
Review
different individuals respond to different peptides of a same antigen the possibility exists that a ‘cocktail’ of peptides containing the immunodominant epitope for each haplotype may be used to prime antigen-specific helper T cells and CTLs [90]. There exists, how-ever, still another major obstacle for peptide vaccines due to their intrinsic low immunogenicity and the requirement of appropriate carriers and/or adjuvants to activate the immune response. The effi-ciency of different plant-produced chimeric virus particles (CVPs) in inducing the activation of peptide-specific immune responses is illustrated in Table 4.
A candidate vaccine prepared by antigen epitope presentation on the surface of virus particles has two major advantages. First, the purified viral particles induce a proper antigenic response by vir-tue of an intrinsic adjuvanticity. Second, the use of standard virus purification protocols make the vaccine preparation fast and cheap.
The vectors are based on different viruses including rod-shaped (TMV), filamentous (PVX and PPV) and icosahedral (CPMV, CMV and TBSV) viruses and can be adapted for peptide display.
To construct CVPs, the peptide-encoding sequence is generally fused to the CP gene in a position known to be exposed on the viral surface [91]. To guarantee the production of large quantities of CVPs, the structural and functional characterization of the CP is essential to avoid possible interference of the heterologous peptide with viral fitness. To this aim, fundamental are the studies that define mechanisms of viral movement and proteins involved in infection spreading, CP/virus particle structures and factors influencing virus genetic stability.
Tobacco mosaic virus was the first virus to be used for vaccine development [14,92,93]. TMV replicates to high titers in Nicotiana hosts (N. tabacum and N. benthamiana) and is easily mechanically transmissible at the same time. During assembly, RNA origin of assembly (OA) interacts with partially assembled CP discs. The model suggests that, similar to the threading of beads onto a string, OA allows sequential stacking of coat disks until the nucleic acid is fully encapsidated. With the exception of the OA, assembly is RNA sequence independent. Early ana lysis of the secondary structure of TMV CP revealed that the N-terminus and C-terminus residues were surface-exposed and might accommodate peptide insertions. It has also been shown that tolerance to insertion of foreign peptides depends on the biophysical properties of the new amino acids. To enhance the probability of recovering good yields of recombinant virions, it is important to maintain an overall neutral or negative charge of the virus particle avoiding hydrophobic peptides [14], and further data indicated that the presence of a cysteine residue in the foreign peptides implied changes in yield, morphology and stabil-ity of TMV particles, regardless of the position and the peptide sequence [94]. Display of a large functional domain (133-amino acid fragment) of protein A has also been obtained on the surface of TMV by the use of a flexible linker (GGGGS)
3 [95].
The CPMV, PVX and CMV were also used as carriers for epitope display. The results demonstrated the ability to generate significant quantities of chimeric virions in plants. As an exam-ple, a significant increase of PVX CVP yield can be obtained by optimizing the isoelectric point and amino acid composition (in particular serine and threonine) of the displayed peptide [96].
Another important aspect that should not be underestimated when designing CVPs are also rules governing antigen processing. In particular, when peptides are expected to activate cytotoxic CD8+ T cells, they must be properly grafted on the viral car-rier as the way they are displayed can severely affect vaccine effi-cacy [97]. In Table 4, the immunological properties of representative chimeric plant virus particles produced in plants are summarized. The vast majority of peptide insertions is represented by short linear epitopes located at or near the C-terminus and rarely at the N-terminus of the CP.
Production of monoclonal antibodies Plants represent an ideal system for the production of functional full-size immunoglobulins. mAbs have been produced either through the generation of transgenic plants or by transient expres-sion systems [98]. Long and laborious procedures are required for the establishment of stable transgenic lines and usually the reported IgG expression yields in literature are quite low [99–101]. Conversely, transient expression systems based on A. tumefaciens-mediated delivery or virus-based vectors allow the rapid accumulation of recombinant protein (high yields in few days). Transient expression based on leaf infiltration of Agrobacterium was successfully used to produce fully functional mAbs in different plant species (tobacco, N. benthamiana, lettuce and tomato) [22,102–104]. It is worth noting that in the case of the antihuman IgG C5–1 murine antibody, transiently expressed in N. benthamiana leaves, very high expres-sion yields were reported (up to 750 µg/g FW) [105]. In addition, the tumor-targeting human IgG H10 was purified from agroinfiltrated leaves at levels of 10 µg/g FW [106,107]. In both cases, a substan-tial increase in IgG accumulation (up to tenfold) was obtained by the coagroinfiltration of plant virus gene-silencing suppressor constructs (Hc-Pro from potato virus Y and p19 from artichoke mottled crinckle virus, respectively). In fact, there are plenty of data supporting the link between the decrease in expression yield of heterologous proteins in agroinfiltrated tissues and the activation of PTGS in the plant host accumulation [28–30]. Further advantage of this transient expression technology is also the rapid scalability in that it is possible to process large numbers of plants by automated vacuum-agroinfiltration systems that has a profound impact on industrial-scale applications.
Another innovative and promising opportunity is identified in the transient expression mediated by recombinant viral vectors. Antibody assembly requires the coexpression of both heavy (HC) and light chain (LC) in the same plant cell. For this reason, a method was devised based on TMV and PVX noncompeting viral vectors [52]. These recombinant nonreplicating viral vectors har-boring the HC and LC coding sequences are delivered into plant cells by Agrobacterium infiltration (MagnICON technology). This system was successfully used for the production of a neu-tralizing humanized mAb against the West Nile virus to be used in postexposure prophylaxis. Antibody purification yields from infiltrated N. benthamiana leaves were in the range of 300 µg/g FW [108]. In parallel, a viral vector based on CPMV was devised in which coagroinfiltration of HC and LC and p19 gene-silencing suppressor of TBSV in N. benthamiana yielded 325 µg/g FW
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 871
Review
of the human anti-HIV neutralizing mAb 2G12 [109]. Recently, high-level production of an IgG against Ebola virus was achieved by using geminiviral recombinant vectors. A single-vector DNA replicon system based on BeYDV was devised, eliminating the need of coinfection with multiple expression modules [110].
Biosafety concerns over animal & human administration of transiently produced vaccine formulationsThe present technological scenario is faced with unprecedented problems owing to the capacity to redesign the living organisms and the potential to create new sources of animal and plant dis-eases. Hence, the deployment of plant viral recombinant vectors in ‘molecular farming’ in human and animal therapy is associated with adequate biosafety and testing regulations established for all bio-pharmaceutical products. Plant virus vectors employed in molecular farming do not pose a major threat to human and animal health as pathogenicity for mammals has been never demonstrated. This envisages the possibility that, under some conditions, edible plant tissues (transiently expressing recombinant antigens) may be used as an alternative method to deliver vaccine at mucosal level (i.e., by oral delivery) with the major advantage of eliminating the use of needles, thus ruling out costs and transmission of blood-borne dis-eases. It is worth mentioning that, by this approach, the antigen may be protected from degradation during the passage through the gut by a natural shield of plant material. Proof of positive application of this technology for human benefit has been previously reported [111]. In this work, a group of volunteers who had previously been immunized against rabies virus with a conventional vaccine showed detectable levels of rabies virus-neutralizing antibodies when fed with raw spinach leaves expressing a chimeric virus displaying as fusion to CP antigenic determinants from rabies virus [111]. There are many variables influencing immunization and the study of oral plant vaccines is still in its infancy. Ideally, the dose should be adjusted to prevent or, under some circumstances, to induce oral tolerance and to avoid unexpected allergies. Some of these issues have been addressed in oral vaccination studies using plant tissues stably expressing antigens or allergens [112–114].
As for Agrobacterium used in transient systems, it should be mentioned that it is a soil-borne microorganism nonpathogenic for humans but it can transfer the T-DNA not only into plant genomes but also in human cultured cells [115]. Wide exploita-tion of Agrobacterium for biotechnological purposes and numer-ous medical cases of Agrobacterium species isolated from human bloodstream infections required the examination of biosafety-related implications of invasions of Agrobacterium of mammalian organisms. Direct experiments showed that intravenously injected Agrobacterium remained viable in the bloodstream during at least 6 days after injection. However, despite its persistence in the mouse bloodstream, Agrobacteria failed to manifest detectable expression of the T-DNA in the host tissues [116]. However, the biopharma-ceuticals manufacturing process requires purity levels that ensure the absence of bacterial contamination. Another frequently raised biosafety issue is the presence of endotoxins, polyphenols and alka-loids that can be copurified in the final product, especially when the extraction is performed from agroinfiltrated tobacco leaves.
Recent works demonstrated that all these unwanted compounds can be easily removed during the purification process, therefore do not represent a major concern [107,108]. A further limitation to the applicability in therapy of plant-derived biopharmaceuticals could be represented by the difference in N- and O-glycan composition from their mammalian counterpart which could lead to immuno-genicity problems. Different strategies have been recently devised to address this limitation, which include: modulation of plant gly-cosylation using RNA interference; coexpression of specific human glycosyltransferases; exploitation of specific knockout transgenic lines that synthesize complex N-glycans lacking immunogenic xylose and fucose epitopes; and retention of the recombinant protein in the ER [117]. It must be noted that in some cases, plant-specific N-glycosylation could also represent an advantage in that in mucosal vaccines plant glycosylated proteins may be endowed with intrinsic adjuvanticity [117].
Expert commentaryIn approximately 10 years, transient expression technologies underwent a fundamental evolution especially with the advent of modular deconstructed systems. This evolution contributed to a significant increase of expression levels of heterologous protein in plants. While molecules such as immunoglobulins have competi-tive yields comparing favorably with classical cell culture systems, plant biotechnologists should now focus efforts primarily on issues related to extraction and purification of recombinant antigens from plant tissues with the final goal of optimizing recovery of pharma-ceutical grade molecules. In parallel, endeavors should be devoted, in syntony with immunologists, to identify optimal vaccine targets and adequate delivery strategies. This is particularly true for intrin-sically poorly immunogenic recombinant antigens. In this context, further development of plant-based delivery strategies constitutes an important research area to be explored and the identification of innovative approaches should not be underestimated.
Five-year viewCurrent literature in the field of molecular farming clearly indicates that transient expression systems will give a significant impulse to biopharmaceuticals production in plants. New means of Agrobacterium-mediated delivery will be found and/or surfactants will become widely used or even replace vacuum-infiltration systems for large-scale production. New viral vectors will be designed and/or new successful combinations will be implemented in the case of multicomponent systems. Recent promising data on mAb produc-tion using viral vectors indicate that these molecules will be the first clinical-grade biopharmaceuticals derived from plants.
Financial & competing interests disclosureSome of the experimental work illustrated in the present article was partially supported by a grant from the Italian Foreign Affairs Ministry in the frame-work of the Bilateral Projects of Great Relevance. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)872
Review
ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest
1 Hammond RW, Nemchinov LG. Plant production of veterinary vaccines and therapeutics. Curr. Top. Microbiol. Immunol. 332, 79–102 (2009).
2 Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci. 14, 669–679 (2009).
3 Chebolu S, Daniell H. Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality. Curr. Top. Microbiol. Immunol. 332, 33–54 (2009).
4 Aviezer D, Brill-Almon E, Shaaltiel Y et al. A plant-derived recombinant human glucocerebrosidase enzyme – a preclinical and Phase I investigation. PLoS One 4, e4792 (2009).
5 McCormick AA, Reddy S, Reinl SJ et al. Plant-produced idiotype vaccines for the treatment of non-Hodgkin’s lymphoma: safety and immunogenicity in a Phase I clinical study. Proc. Natl Acad. Sci. USA 105, 10131–10136 (2008).
6 D’Aoust MA, Couture MM, Charland N et al. The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. Plant Biotechnol. J. 8, 607–619 (2010).
• Presentsacompleteoverviewoftheproductionofinfluenzavirus-likeparticlesinplant.
7 Chichester JA, Haaheim LR, Yusibov V. Using plant cells as influenza vaccine substrates. Expert Rev. Vaccines 8, 493–498 (2009).
8 Gleba Y, Klimyuk V, Marillonnet S. Magnifection – a new platform for expressing recombinant vaccines in plants. Vaccine 23, 2042–2048 (2005).
9 Jones HD, Doherty A, Sparks CA. Transient transformation of plants. Methods Mol. Biol. 513, 131–152 (2009).
10 Lico C, Chen Q, Santi L. Viral vectors for production of recombinant proteins in plants. J. Cell Physiol. 216, 366–377 (2008).
11 Marusic C, Vitale A, Pedrazzini E et al. Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study. Transgenic Res. 18, 499–512 (2009).
12 McCormick AA, Palmer KE. Genetically engineered tobacco mosaic virus as nanoparticle vaccines. Expert Rev. Vaccines 7, 33–41 (2008).
13 Rybicki EP. Plant-produced vaccines: promise and reality. Drug Discov. Today 14, 16–24 (2009).
14 Smith ML, Fitzmaurice WP, Turpen TH, Palmer KE. Display of peptides on the surface of tobacco mosaic virus particles. Curr. Top. Microbiol. Immunol. 332, 13–31 (2009).
15 Yusibov, V, Rabindran S. Recent progress in the development of plant derived vaccines. Expert Rev. Vaccines 7, 1173–1183 (2008).
16 Vancanneyt G, Dubald M, Schröder W, Peters J, Botterman J. A case study for plant-made pharmaceuticals comparing different plant expression and production systems. Methods Mol. Biol. 483, 209–221 (2009).
17 Komori T, Imayama T, Kato N et al. Current status of binary vectors and superbinary vectors. Plant Physiol. 145, 1155–1160 (2007).
18 Lee LY, Gelvin SB. T-DNA binary vectors and systems. Plant Physiol. 146, 325–332 (2008).
19 Goodin MM, Zaitlin D, Naidu RA, Lommel SA. Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol. Plant Microbe Interact. 21, 1015–1026 (2008).
20 Kim MJ, Baek K, Park CM. Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell Rep. 28, 1159–1167 (2009).
21 Lee MW, Yang Y. Transient expression assay by agroinfiltration of leaves. Methods Mol. Biol. 323, 225–229 (2006).
22 Negrouk V, Eisner G, Lee H et al. Highly efficient transient expression of functional recombinant antibodies in lettuce. Plant Sci. 169, 433–438 (2005).
23 Medrano G, Reidy MJ, Liu J et al. Rapid system for evaluating bioproduction capacity complex pharmaceutical proteins in plants. Methods Mol. Biol. 483, 51–67 (2009).
24 Simmons CW, VanderGheynst JS, Upadhyaya SK et al. A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnol. Bioeng. 102, 965–970 (2009).
25 Tague BW, Mantis J. In planta Agrobacterium-mediated transformation by vacuum infiltration. Methods Mol. Biol. 323, 215–223 (2006).
26 Azhakanandam K, Weissinger SM, Nicholson JS, Qu R, Weissinger AK. Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants. Plant Mol. Biol. 63, 393–404 (2007).
Key issues
• Molecular farming is a plant-based biotechnology enabling safe global future production of biopharmaceuticals from genetically engineered plants.
• Plant cell gene-transfer benefits from ease of delivery of heterologous sequences by virus and bacterial pathogens, exploiting their natural capability to redirect the cell machinery towards the synthesis of foreign proteins.
• Gene engineering is progressing to maximize expression of recombinant molecules in plant cells. Some successful strategies have already been devised and future research on the plant cell as a biofactory for pharmaceuticals will guarantee yields in the range of the most competitive eukaryotic systems of overexpression currently used.
• While antibodies produced transiently in plants entered the precompetitive development, most of the vaccine antigens are still on their way towards clinical management.
• The ‘green’ vaccine formulation as such requires a careful elucidation of the mechanisms of action of the intrinsic adjuvanticity and delivery systems.
• ‘Green’ biopharming research raises no major biosafety issues while no definite regulatory testing of plant-derived biologics is available.
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 873
Review
27 Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat. Biotechnol. 23, 718–723 (2005).
•• Presentingthe‘magnifection’technology.
28 Lombardi R, Circelli P, Villani ME et al. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke mottled crinckle virus. BMC Biotechnol. 96, 1–11 (2009).
29 Zheng N, Xia R, Yang C et al. Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice. Vaccine 27, 5001–5007 (2009).
30 Yang L, Wang H, Liu J et al. A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension. J. Biotechnol. 134, 320–324 (2008).
31 Huang Z, Mason HS. Conformational analysis of hepatitis B surface antigen fusions in an Agrobacterium-mediated transient expression system. Plant Biotechnol. J. 2, 241–249 (2004).
32 Lacorte C, Lohuis H, Goldbach R, Prins M. Assessing the expression of chicken anemia virus proteins in plants. Virus Res. 129, 80–86 (2007).
33 Joensuu JJ, Conley AJ, Lienemann M et al. Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana. Plant Physiol. 152, 622–633 (2009).
34 Gómez E, Zoth SC, Asurmendi S et al. Expression of hemagglutinin–neuraminidase glycoprotein of newcastle disease virus in agroinfiltrated Nicotiana benthamiana plants. J. Biotechnol. 337–340 (2009).
35 Escribano JM, Perez-Filgueira DM. Strategies for improving vaccine antigens expression in transgenic plants: fusion to carrier sequences. Methods Mol. Biol. 483, 275–287 (2009).
36 Obregon P, Chargelegue D, Drake et al. HIV-1 p24–immunoglobulin fusion molecule: a new strategy for plant-based protein production. Plant Biotechnol. J. 4, 195–207 (2006).
37 Shadwick FS, Doran PM. Infection, propagation, distribution and stability of plant virus in hairy root cultures. J. Biotechnol. 131, 318–329 (2007).
38 Shadwick FS, Doran PM. Propagation of plant viruses in hairy root cultures: a potential method for in vitro production of epitope vaccines and foreign proteins. Biotechnol. Bioeng. 96, 570–583 (2007).
39 Skarjinskaia M, Karl J, Araujo A et al. Production of recombinant proteins in clonal root cultures using episomal expression vectors. Biotechnol. Bioeng. 100, 814–819 (2008).
40 Ahlquist P, French R, Janda M, Loesch-Fries S. Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc. Natl Acad. Sci. USA 81, 7066–7070 (1984).
41 Goelet P, Lomonossoff GP, Butler PJ, Akam ME, Gait MJ, Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc. Natl Acad. Sci. USA 79, 5818–5822 (1982).
42 Palukaitis P, Zaitlin M. The rod-shaped plant viruses. In: The Plant Viruses (Volume 2). Van Regenmortel M, Fraenkel-Conrat H (Eds). Plenum Press, NY, USA, 105–131 (1986).
43 Chapman SN. Construction of infectious clones for RNA viruses: TMV. Methods Mol. Biol. 451, 477–490 (2008).
44 Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y. In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl Acad. Sci. USA 101, 6852–6857 (2004).
45 Dohi K, Tamai A, Mori M. Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector. Arch. Virol. 153, 1667–1675 (2008).
46 Dorokhov YL, Ivanov PA, Komarova TV et al. An internal ribosome entry site located upstream of the crTMV coat protein (CP) gene can be used for CP synthesis in vivo. J. Gen. Virol. 87, 2693–2697 (2006).
47 Verchot-Lubicz J. A new cell-to-cell transport model for Potexviruses. Mol. Plant Microbe Interact. 18, 283–290 (2005).
48 Verchot-Lubicz J, Ye CM, Bamunusinghe D. Molecular biology of potexviruses: recent advances. J. Gen. Virol. 88, 1643–1655 (2007).
49 Baulcombe DC, Chapman S, Santa Cruz S. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7, 1045–1053 (1995).
50 Mechtcheriakova IA, Eldarov MA, Nicholson L et al. The use of viral vectors to produce hepatitis B virus core particles in plants. J. Virol. Methods 131, 10–15 (2006).
51 Avesani L, Marconi G, Morandini F et al. Stability of potato virus X expression vectors is related to insert size: implications for replication models and risk assessment. Transgenic Res. 16, 587–597 (2007).
52 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 103, 14701–14706 (2006).
53 Komarova TV, Skulachev MV, Zvereva AS, Schwartz AM, Dorokhov YL, Atabekov JG. New viral vector for efficient production of target proteins in plants. Biochemistry (Mosc.) 71, 846–850 (2006).
54 Uhde K, Fischer R, Commandeur U. Expression of multiple foreign epitopes presented as synthetic antigens on the surface of potato virus X particles. Arch. Virol. 150, 327–340 (2005).
55 Santa Cruz S, Chapman S, Roberts AG, Roberts IM, Prior DA, Oparka KJ. Assembly and movement of a plant virus carrying a green fluorescent protein overcoat. Proc. Natl Acad. Sci. USA 93, 6286–6290 (1996).
56 Dolja VV, McBride HJ, Carrington JC. Tagging of plant potyvirus replication and movement by insertion of b-glucuronidase into the viralpolyprotein. Proc. Natl Acad. Sci. USA 89, 10208–10212 (1992).
57 Varrelmann M, Maiss E. Mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana. J. Gen. Virol. 81, 567–576 (2000).
58 Beauchemin C, Bougie V, Laliberté JF. Simultaneous production of two foreign proteins from a potyvirus-based vector. Virus Res. 112, 1–8 (2005).
59 Kelloniemi J, Mäkinen K, Valkonen JP. Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths. Virus Res. 135, 282–291 (2008).
60 Zhao Y, Hammond J, Tousignant ME, Hammond RW. Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus. Arch. Virol. 145, 2285–2295 (2000).
61 Sudarshana MR, Plesha MA, Uratsu SL et al. A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues. Plant Biotechnol. J. 4, 551–559 (2006).
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)874
Review
62 Kim SH, Kalinina NO, Andreev I et al. The C-terminal 33 amino acids of the cucumber mosaic virus 3a protein affect virus movement, RNA binding and inhibition of infection and translation. J. Gen. Virol. 85, 221–230 (2004).
63 Fujiki M, Kaczmarczyk JF, Yusibov V, Rabindran S. Development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein. Virology 381, 136–142 (2008).
64 Green BJ, Fujiki M, Mett V et al. Transient protein expression in three Pisum sativum (green pea) varieties. Biotechnol. J. 4, 230–237 (2009).
65 Sainsbury F, Liu L, Lomonossoff GP. Cowpea mosaic virus-based systems for the expression of antigens and antibodies in plants. Methods Mol. Biol. 483, 25–39 (2009).
66 Gopinath K, Wellink J, Porta C, Taylor KM, Lomonossoff GP, van Kammen A. Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants. Virology 267, 159–173 (2000).
67 Liu L, Grainger J, Canizares MC, Angell SM, Lomonossoff GP. Cowpea mosaic virus RNA-1 acts as an amplicon whose effects can be counteracted by a RNA-2-encoded suppressor of silencing. Virology 323, 37–48 (2004).
68 Sainsbury F, Lavoie PO, D’Aoust MA, Vézina LP, Lomonossoff GP. Expression of multiple proteins using full-length and deleted versions of cowpea mosaic virus RNA-2. Plant Biotechnol. J. 6, 82–92 (2008).
69 Jeske H. Geminiviruses. Curr. Top. Microbiol. Immunol. 331, 185–226 (2009).
70 Kim K, Sunter G, Bisaro DM, Chung IS. Improved expression of recombinant GFP using a replicating vector based on beet curly top virus in leaf-disks and infiltrated Nicotiana benthamiana leaves. Plant Mol. Biol. 64, 103–112 (2007).
71 Huang Z, Chen Q, Hjelm B, Arntzen C, Mason H. A DNA replicon system for rapid high-level production of virus-like particles in plants. Biotechnol. Bioeng. 103, 706–714 (2009).
72 Dorokhov YL, Sheveleva AA, Frolova OY et al. Superexpression of tuberculosis antigens in plant leaves. Tuberculosis (Edinb.) 87, 218–224 (2007).
73 Dorokhov YL, Frolova OY, Skurat EV et al. A novel function for a ubiquitous plant enzyme pectin methylesterase: the enhancer of RNA silencing. FEBS Lett. 580, 3872–3878 (2006).
74 Gleba Y, Klimyuk V, Marillonnet S. Viral vectors for the expression of proteins in plants. Curr. Opin. Biotechnol. 18, 134–141 ( 2007).
75 Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).
76 Santi L, Giritch A, Roy CJ et al. Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system. Proc. Natl Acad. Sci. USA 103, 861–866 (2006).
77 Wigdorovitz A, Pérez Fuilgueira 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. Virology 264, 85–91 (1999).
78 Mishra S, Yadav DK, Tuli R. Ubiquitin fusion enhances cholera toxin B subunit expression in transgenic plants and the plant-expressed protein binds GM1 receptors more efficiently. J. Biotechnol. 127, 95–108 (2006).
79 Patel J, Zhu H, Menassa R, Gyenis L, Richman A, Brandle JE. Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves. Transgenic Res. 16, 239–249 (2007).
80 Goldman M, Lambert P-H. Immunological safety of vaccines: facts hypothesis and allegations. In: Novel Vaccination Strategies. Kaufmann SHE (Ed.). Wiley-VCH, Weinheim, Germany, 595–611 (2004).
81 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 24, 2506–2513 (2006).
82 Santi L, Batchelora L, Huanga Z et al. An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles. Vaccine 26, 1846–1854 (2008).
83 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. 6, 930–940 (2008).
84 Natilla A, Hammond RW, Nemchinov LG. Epitope presentation system based on cucumber mosaic virus coat protein expressed from a potato virus X-based vector. Arch. Virol. 151, 1373–1386 (2006).
85 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 393, 329–337 (2009).
86 Cañizares MC, Nicholson L, Lomonossoff GP. Use of viral vectors for vaccine production in plants. Immunol. Cell. Biol. 83, 263–270 (2005).
87 Molnár E, Dopfer EP, Deswal S, Schamel WW. Models of antigen receptor activation in the design of vaccines. Curr. Pharm. Des. 15, 3237–3248 (2009).
88 Azizi A, Diaz-Mitoma F. Viral peptide immunogens: current challenges and opportunities. J. Pept. Sci. 13, 776–786 (2007).
89 Knittelfelder R, Riemer AB, Jensen-Jarolim E. Mimotope vaccination-from allergy to cancer. Expert Opin. Biol. Ther. 9, 493–506 (2009).
90 Sette A, Sidney J. Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50, 201–212 (1999).
91 Johnson J, Lin T, Lomonossoff G. Presentation of heterologous peptides on plant viruses: genetics, structure and function. Annu. Rev. Phytopathol. 35, 67–86 (1997).
92 Bendahmane M, Koo M, Karrer E, Beachy RN. Display of epitopes on the surface of Tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus–host interaction. J. Mol. Biol. 290, 9–20 (1999).
93 McCormick AA, Corbo TA, Wykoff-Clary S et al. TMV-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models. Vaccine 24, 6414–6423 (2006).
94 Li G, Jiang L, Li M et al. Morphology and stability changes of recombinant TMV particles caused by a cysteine residue in the foreign peptide fused to the coat protein. J. Virol. Methods 140, 212–217 (2007).
95 Werner S, Marillonnet S, Hause G, Klimyuk V, Gleba Y. Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A. Proc. Natl Acad. Sci. USA 103, 17678–17683 (2006).
96 Lico C, Capuano F, Renzone G et al. Peptide display on potato virus X: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles. J. Gen. Virol. 87, 3103–3112 (2006).
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
www.expert-reviews.com 875
Review
97 Lico C, Mancini C, Italiani P et al. Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice. Vaccine 27, 5069–5076 (2009).
98 De Muynck B, Navarre C, Boutry M. Production of antibodies in plants: status after twenty years. Plant Biotechnol. J. 8, 529–563 (2010).
• Givesacompleteoverviewofmonoclonalantibodiesproducedintransgenicplantsorusingtransientexpressionsystemswithaspecialfocusonantibodydegradationandstrategiesusedtoimproveexpressionyield.
99 Ma JK, Drake PM, Chargelegue D, Obregon P, Prada A. Antibody processing and engineering in plants, and new strategies for vaccine production. Vaccine 23, 1814–1818 (2005).
100 Ko K, Koprowski H. Plant biopharming of monoclonal antibodies. Virus Res. 111, 93–100 (2005).
101 Brodzik R, Glogowska M, Bandurska K et al. Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells. Proc. Natl Acad. Sci. USA 103, 8804–8809 (2006).
102 Hull AK, Criscuolo CJ, Mett V et al. Human-derived, plant-produced monoclonal antibody for the treatment of anthrax. Vaccine 23, 2082–2086 (2005).
103 Orzaez, D, Mirabel S, Wieland WH, Granell A. Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant Physiol. 140, 3–11 (2006).
104 Vaquero C, Sack M, Chandler J et al. Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves. Proc. Natl Acad. Sci USA 96, 11128–11133 (1999).
105 Vézina LP, Faye L, Lerouge P et al. Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants. Plant Biotechnol. J. 7, 442–455 (2009).
106 Villani MA, Morgun B, Brunetti P et al. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies. Plant Biotech. J. 7, 59–72 (2009).
107 Lombardi R, Villani ME, Di Carli M, Brunetti P, Benvenuto E, Donini M. Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-
agroinfiltration. Transgenic Res. DOI: 10.1007/s11248–010–9382–9389 (2010) (Epub ahead of print)
108 Lai H, Engle M, Fuchs A et al. Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice. Proc. Natl Acad. Sci. USA 107, 2419–2424 (2010).
109 Sainsbury F, Lomonossoff GP. Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol. 148, 1212–1218 (2008).
110 Huang Z, Phoolcharoen W, Lai H et al. High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol. Bioeng. 106, 9–17 (2010).
111 Yusibov V, Hooper DC, Spitsin SV et al. Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 20, 3155–3164 (2002).
112 Tacket CC. Plant-based oral vaccines: results of human trials. Curr. Top. Microbiol. Immunol. 332, 103–117 (2009).
113 Takagi H, Hiroi T, Yang L et al.A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses. Proc. Natl Acad. Sci. USA 102, 17525–17530 (2005).
114 Smart V, Foster PS, Rothenberg ME, Higgins TJ, Hogan SP. A plant-based allergy vaccine suppresses experimental asthma via an IFN-g and CD4+CD45RBlow
T cell-dependent mechanism. J. Immunol. 171, 2116–2126 (2003).
115 Tzfira T, Citovsky V. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr. Opin. Biotechnol. 17, 1–8 (2006).
116 Petrunia IV, Frolova OY, Komarova TV et al. Agrobacterium tumefaciens caused bacteraemia does not lead to GFP gene expression in mouse organs. PLoS One 3, e2352 (2008).
117 Gomord V, Fitchette AC, Menu-Bouaouiche L et al. Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol. J. 8, 564–587 (2010).
118 Meyers A, Chakauya E, Shephard E et al. Expression of HIV-1 antigens in plants as potential subunit vaccines. BMC Biotechnol. 8, 53 (2008).
119 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. 88, 1460–1469 (2007).
120 Karasev AV, Foulke S, Wellens C et al. Plant based HIV-1 vaccine candidate: Tat protein produced in spinach. Vaccine 23, 1875–1880 (2005).
121 Varsani A, Williamson AL, Stewart D, Rybicki EP. Transient expression of human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector. Virus Res. 120, 91–96 (2006).
122 Saejung W, Fujiyama K, Takasaki T et al. Production of dengue 2 envelope domain III in plant using TMV-based vector system. Vaccine 25, 6646–6654. (2007).
123 Rabindran S, Stevenson N, Roy G et al. Plant-produced human growth hormone shows biological activity in a rat model. Biotechnol. Prog. 25, 530–534 (2009).
124 Golovkin M, Spitsin S, Andrianov V et al. Smallpox subunit vaccine produced in planta confers protection in mice. Proc. Natl Acad. Sci. USA 104, 6864–6869 (2007).
• Presentscompleteimmunologicalcharacterizationofasmallpoxsubunitvaccineproducedinplantusingthe‘magnifection’transientexpressionsystem.
125 Webster DE, Wang L, Mulcair M et al. Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system. Plant Biotechnol. J. 7, 1–10 (2009).
126 Zelada AM, Calamante G, de la Paz Santangelo M et al. Expression of tuberculosis antigen ESAT-6 in Nicotiana tabacum using a potato virus X-based vector. Tuberculosis (Edinb.) 86, 263–267 (2006).
127 Nemchinov LG, Liang TJ, Rifaat MM, Mazyad HM, Hadidi A, Keith JM. Development of a plant-derived subunit vaccine candidate against hepatitis C virus. Arch. Virol. 145, 2557–2573 (2000).
128 Férnandez-Férnandez MR, Mourino M, Rivera J, Rodriguez F, Plana-Duran J, Garcia JA. Protection of rabbits against Rabbit hemorrhagic disease virus by immunization with the VP60 protein expressed in plants with a Potyvirus-based vector. Virology 280, 283–291, (2001).
129 Franconi R, Di Bonito P, Dibello F et al. Plant-derived Human papillomavirus 16 E7 oncoprotein induces immune response and specific tumor protection. Cancer Res. 62, 3654–3658 (2002).
Transient expression systems for plant-derived biopharmaceuticals
Expert Rev. Vaccines 9(8), (2010)876
Review
130 Perez Filgueira DM, Zamorano PI, Dominguez MG et al. Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity. Vaccine 21, 4201–4209 (2003).
131 Verch T, Hooper DC, Kiyatkin A, Steplewski Z, Koprowski H. Immunization with a plant-produced colorectal cancer antigen. Cancer Immunol. Immunother. 53, 92–99 (2004).
132 Clemente M, Curilovic R, Sassone A, Zelada A, Angel SO, Mentaberry AN. Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity. Mol. Biotechnol. 30, 41–50 (2005).
133 Chichester JA, Musiychuk K, de la Rosa P et al. Immunogenicity of a subunit vaccine against Bacillus anthracis. Vaccine 25, 3111–3114 (2007).
134 Massa S, Franconi R, Brandi R et al. Anti-cancer activity of plant-produced HPV16 E7 vaccine. Vaccine 25, 3018–3021 (2007).
135 Mett V, Lyons J, Musiychuk K et al. A plant-produced plague vaccine candidate confers protection to monkeys. Vaccine 25, 3014–3017 (2007).
136 Shoji Y, Bi H, Musiychuk K et al. Plant-derived hemagglutinin protects ferrets against challenge infection with the A/Indonesia/05/05 strain of avian influenza. Vaccine 27, 1087–1092 (2009).
137 Shoji Y, Farrance CE, Bi H et al. Immunogenicity of hemagglutinin from A/Bar-headed Goose/Qinghai/1A/05 and A/Anhui/1/05 strains of H5N1 influenza viruses produced in Nicotiana benthamiana plants. Vaccine 27, 3467–3470 (2009).
138 Spitsin S, Andrianov V, Pogrebnyak N et al. Immunological assessment of plant-derived avian flu H5/HA1 variants. Vaccine 27, 1289–1292 (2009).
139 Porta C, Spall VE, Loveleand J, Johnson JE, Barker PJ, Lomonossoff GP. Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Virology 202, 949–955 (1994).
140 Dalsgaard K, Uttenthal A, Jones GB et al. Plant-derived vaccine protects target animals against a viral disease. Nat. Biotechnol. 15, 248–252 (1997).
141 Joelson T, Akerblom L, Oxelfelt P, Strandberg B, Tomenius K, Morris TJ. Presentation of a foreign peptide on the surface of tomato bushy stunt virus. J. Gen. Virol. 78, 1213–1217 (1997).
142 Yusibov V, Modelska A, Steplewski K et al. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc. Natl Acad. Sci. USA 94, 5784–5788 (1997).
143 Férnandez-Férnandez MR, Martinez-Torrecuadrada JL, Casal JI, Garcia JA. Development of an antigen presentation system based on plum pox potyvirus. FEBS Lett. 427, 229–235 (1998).
144 Brennan FR, Jones TD, Longstaff M et al. Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses. Vaccine 17, 1846–1857 (1999).
145 Staczek J, Bendahmane M, Gilleland LB, Beachy RN, Gilleland HE Jr. Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa. Vaccine 18, 2266–2274 (2000).
146 Langeveld JPM, Brennan FR, Martinez-Torrecuadrada JL et al. Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus. Vaccine 19, 3661–3670 (2001).
147 Marusic C, Rizza P, Lattanzi L et al. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J. Virol. 75, 8434–8439 (2001).
148 Natilla A, Piazzolla G, Nuzzaci M et al. Cucumber mosaic virus as carrier of a hepatitis C virus-derived epitope. Arch. Virol. 149, 137–154 (2004).
149 Yusibov V, Mett V, Mett V et al. Peptide-based candidate vaccine against respiratory syncytial virus. Vaccine 23, 2261–2265 (2005).
150 Marconi G, Albertini E, Barone P et al. In planta production of two peptides of the Classical swine fever virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X. BMC Biotechnol. 6, 29 (2006).
151 Smith ML, Lindbo JA, Dillard-Telm S et al. Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications. Virology 348, 475–488 (2006).
152 Meshcheryakova YA, Eldarov MA, Migunov AI et al. Cowpea mosaic virus chimeric particles bearing the ectodomain of matrix protein 2 (M2E) of the influenza A virus: production and characterization. Mol. Biol. 43, 685–694 (2009).
153 Nuzzaci M, Vitti A, Condelli V et al. In vitro stability of cucumber mosaic virus nanoparticles carrying a hepatitis C virus-derived epitope under simulated gastrointestinal conditions and in vivo efficacy of an edible vaccine. J. Virol. Meth. 165, 211–215 (2010).
154 Uhde-Holzem K, Schlössera V, Viazov S, Fischer R, Commandeur U. Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9. J. Virol. Meth. 166, 12–20 (2010).
Patents
201 Dorokhov YL, Komarova TV, Atabekov JG. Method of hyperproduction of target protein in a plant. WO2008063093 (2008).
202 Dorokhov YL, Komarova TV. Method for overproducing anti-HER2/Neu oncogene antibodies in plant. WO2009048354 (2009).
Website
301 Biolex www.biolex.com
Komarova, Baschieri, Donini, Marusic, Benvenuto & Dorokhov
