High expression of consensus dengue virus envelope glycoprotein domain III using a viral expression system in tobacco
Although plant-based vaccines have many advantages, their use is limited by low expression of antigen genes in transgenic plants, which results in low immune responses and immune tolerance. To overcome this problem, Nicotiana benthamiana was used to produce the consensus domain III of dengue virus envelope glycoprotein (E) via agroinfiltration with a plant virus-based expression system. The binding of E glycoprotein to a receptor is important for dengue virus entry into host cells and results in human disease. Consensus domain III of dengue virus E glycoprotein (cEDIII) is immunogenic and can elicit neutralizing antibodies against all four serotypes of dengue virus. A DNA fragment encoding cEDIII and M cell-targeting ligand fused to cEDIII (cEDIII-Co1) were constructed in a viral vector and introduced into tobacco plant cells by Agrobacterium-mediated infiltration. The cEDIII and cEDIII-Co1 fusion proteins were detected in protein extracts from agroinfiltrated leaves by Western blot analysis. The plant-produced cEDIII and cEDIII-Co1 fusion proteins composed 5.2 and 4.8 mg/g of dry weight of leaf tissues, respectively. These results suggest that the high expression of dengue virus cEDIII in plants with a plant virus-based expression system can overcome the low expression level to improve the feasibility of plant-based vaccines.
KeywordsDengue virus Plant-based vaccine Plant viral expression systems Consensus domain III
Dengue is a flaviviral disease transmitted to humans by infected Aedes mosquitoes in tropical and sub-tropical areas. Recently, dengue has become a significant public health problem; the World Health Organization (WHO) reports that two-fifths of the world’s population is at risk of infection (Murrell et al. 2011). Advanced research is needed to produce effective and cheap vaccines against dengue because of the potential considerable economic burden in developing countries.
Dengue virus belongs to the Flaviviridae family and consists of four antigenically different serotypes. The antigenically different strains make developing an effective dengue vaccine difficult because infection with just one serotype can lead to the full spectrum of dengue symptoms: fever, potentially life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) after heterologous consecutive infections. Tetravalent dengue vaccines are considered to protect against all serotypes without the antibody-dependent enhancement (ADE) of dengue virus infection (Huang et al. 2006). The envelope E glycoprotein of dengue virus plays a central role in receptor binding and virus entry into host cells by fusing with the cellular membrane. The envelope E glycoprotein is the major target for neutralizing antibodies (Wahala et al. 2009). The envelope E glycoprotein has three distinct ectodomains: domains I, II, and III (EDIII) (Modis et al. 2004). Domain III is the primary site of interaction between the virus and receptors at the cellular surface and contains neutralizing epitopes to strongly block viral adsorption by cells (Crill and Roehrig 2001; Hurrelbrink and McMinn 2003; Mukhopadhyay et al. 2005). A consensus sequence was deduced by aligning amino acid sequences of EDIII from isolates of the four dengue viral serotypes (Leng et al. 2009). Mice immunized with the recombinant consensus envelope domain III (cEDIII) developed neutralizing antibodies against all four serotypes of dengue virus (Chiang et al. 2011).
Plant-based production systems offer safe and inexpensive vaccines with the capacity to deliver antigens to mucosal immune targets by oral vaccination (Arakawa et al. 1998; Kim et al. 2012, 2013a). Antigen proteins can be fused with ligands to improve immune responses in mucosal immune systems, which can deliver the fused antigen protein to mucosal immune systems to enhance antigen uptake by mucosal immune cells. The cholera toxin B subunit (CTB) and enterotoxigenic E. coli enterotoxin B subunit (LTB) are representative ligands in transgenic plants. In addition, the M cell-targeting peptide ligand, Co1, in orally-treated mice enhanced the uptake of fused antigen into effective sites of mucosal immune systems and increased immune responses against fused antigen compared with antigen alone (Kim et al. 2013b).
The use of plant expression systems for production of recombinant proteins is superior to other eukaryotic expression systems in several respects. The cost of producing proteins in plants is lower than in other expression systems such as transgenic animals, fermentation, bioreactors, and microbial or animal cell culture-based systems. Plant systems can be scaled up quickly. Additionally, plants have no known human or mammalian pathogens (Horn et al. 2004). However, transforming and regenerating plants are tedious, time-consuming, and often costly measures. Agrobacterium-mediated transient gene expression systems are useful because they do not require transgenic plants (Fischer et al. 1999), and they are fast, flexible, unaffected by chromosomal positional effects, and can be employed in fully differentiated plant tissues (Wydro et al. 2006). Agrobacterium can be infiltrated into plant leaves as a liquid culture, and mediates the transfer of transgenes from the T-DNA region of the bacterial Ti plasmid into the plant cells. Most plant cells within the infiltrated region express the transgene within a few days. Transient expression systems based on plant viral vectors (magnifection) have been reported to express high levels of foreign proteins (Marillonnet et al. 2005; Chen et al. 2011).
In this study, the rice codon-optimized, synthetic cEDIII gene, which protects against all four dengue virus serotypes, was fused to the M cell-binding peptide, Co1, to develop a plant-based vaccine with the potential for increased mucosal immune responses. The cEDIII and cEDIII-Co1 fusion proteins were highly expressed by infiltration with Agrobacterium containing plant viral vector system, based on Tobacco Mosaic Virus (TMV). The cEDIII and cEDIII-Co1 fusion proteins in N. benthamiana will be tested in a mouse model by oral administration.
Materials and methods
Construction of plant expression vectors
A. tumefaciens strain GV3101 harboring provector parts (5′ and 3′) or pICH14011 (integrase) was grown at 28 °C in yeast extract peptone (YEP) broth supplemented with 10 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 5.5), 10 mM MgCl2, 20 µM acetosyringone, 50 µg mL−1 kanamycin, and 100 µg mL−1 rifampicin. Two hundred microliters of each cultured Agrobacterium was harvested by centrifugation at 5000×g for 15 min, and the pellet was resuspended in MS basal medium containing 10 mM MES (pH 5.6), 10 mM MgCl2, and 200 µM acetosyringone. Each tube contained Agrobacterium cultures with 3′ provector (pMYV817 or pMYV818), one 5′ provector (pICH20111 for expression in cytosol, pICH20155 for expression in apoplast by rice alpha-amylase 3A signal peptide, or pICH20030 for expression in chloroplast via ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit chloroplast targeting sequences), and pICH14011 (PhiC31, integrase). The Agrobacterium solution was adjusted to a final OD600 of 1.0 and pre-incubated at room temperature for 2–3 h before infiltration. Pre-incubated cultures were infiltrated into the underside of the leaf with a 1-mL disposable syringe without a needle. Infiltrated leaves were incubated for 3–7 days in a green house.
Western blot analysis of cEDIII proteins in N. benthamiana
The agroinfiltrated leaf tissues were ground with a mortar and pestle in liquid nitrogen. The resulting fine powder was added to an equal volume of extraction buffer (200 mM Tris–Cl, pH 8.0, 100 mM NaCl, 400 mM sucrose, 10 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 0.05 % Tween 20) and thawed. Samples were centrifuged twice at 13,000×g for 10 min at 4 °C. The total soluble protein (TSP) concentration was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). Thirty micrograms of TSP were separated by 15 % sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 2–2.5 h after boiling for 5 min in Tris–glycine buffer (25 mM Tris–Cl, 250 mM glycine, pH 8.3, and 0.1 % SDS). The separated protein bands were transferred from the gel to a Hybond C membrane (Amersham Pharmacia Biotech RPN303C, Piscataway, NJ) using a mini-transblot apparatus (Bio-Rad) at 150 mA for 2 h. Nonspecific antibody binding was blocked by 5 % non-fat dry milk in TBS (20 mM Tris–Cl, pH 7.5, and 500 mM NaCl), followed by washing in TBS for 5 min. The membrane was incubated for 2 h in a 1:2500 dilution of mouse anti-dengue virus antibody (AbD Serotech, Oxford, UK) in TBST antibody dilution buffer (TBS with 0.05 % Tween-20 and 2 % non-fat dry milk), followed by three washes in TBST buffer (TBS with 0.05 % Tween-20). The membrane was incubated for 2 h in a 1:5000 dilution of goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma, St. Louis, MO). The membrane was washed twice in TBST and once in TMN buffer (100 mM Tris–Cl, pH 9.5, 5 mM MgCl2 and 100 mM NaCl). After washing, color was developed with premixed BCIP/NBT solution (Sigma).
Quantification of plant-produced cEDIII proteins
The expression level of cEDIII and cEDIII-Co1 fusion proteins in infiltrated leaf tissues was measured with immunoblotting and densitometry. For immunoblot analysis of cEDIII and cEDIII-Co1 fusion proteins, 1 and 4 µg of TSP of agroinfiltrated leaf tissue were loaded onto an SDS-PAGE gel after boiling for 5 min. Protein extracts of non-infiltrated leaf tissue as a negative control and 100, 250, and 500 ng of purified bacterial EDIII2 (serotype 2) were used to make a standard curve. The Western blot involved immunoblotting with anti-dengue virus antibody. The amount of cEDIII and cEDIII-Co1 fusion protein in agroinfiltrated leaf tissue was measured by comparing the band intensities of the plant-produced cEDIII protein and a known amount of bacterial EDIII2 with a densitometry analysis program (Alpha Ease FC™ software Version 3.3.3, AlphaInotech). cEDIII and cEDIII-Co1 fusion proteins were quantified with a standard curve of purified bacterial EDIII.
Construction of plant expression vector
The cEDIII gene sequence corresponding to an amino acid sequence with cross-neutralizing activity against all four dengue virus serotypes was deduced and synthesized to optimize codon usage for plants in a previous study (Kim et al. 2012). The cEDIII and cEDIII-Co1 fusion genes were amplified by PCR and the DNA sequences of PCR products were confirmed. The cEDIII and cEDIII-Co1 fusion genes were introduced into a plant viral vector, based on TMV, under the control of the MP subpromoter, and designated pMYV817 and pMYV818, respectively (Fig. 1).
Transient expression of cEDIII protein in N. benthamiana leaves
Quantification of ligand fusion proteins
Transgenic plants expressing antigen proteins as edible vaccine candidates have been used to protect against a wide variety of human infectious and autoimmune diseases. However, the low expression level of antigen proteins in transgenic plants is a big problem in developing plant-based oral vaccines because they result in low immune responses or immune tolerance (Tremblay et al. 2010; Neutra and Kozlowski 2006). This problem can be overcome by improving the efficacy of the mucosal immune response to target antigens through antigen sampling and presentation to the mucosal immune system. Low expression of the antigen protein requires feeding an animal a large amount of transgenic plant material, which can cause stress and affect the immune response. Many efforts have been made to improve the expression of target genes with strong or tissue-specific promoters, codon optimization of target genes, transcriptional or translational factors with 5′ UTR sequences, and protein targeting to subcellular locations (Lau and Sun 2009).
The use of ligands, which can target fused antigens to mucosal immune systems and improve antigen uptake and presentation by antigen-presenting cells, is an alternative strategy to overcome weak immune responses and immune tolerances. The B subunits of E. coli heat-labile enterotoxin (LT) or cholera toxin (CT) are representative ligands. In this study, the M cell-binding peptide, Co1, was fused to an antigen to target M cells. M cells are specialized, antigen-sampling epithelial cells in the mucosal immune system and are accessible to microorganisms or migration molecules for tissue-specific consequences of lymphocyte priming in Peyer’s Patches (Kim et al. 2010a; Takahashi et al. 2009). The ability of cEDIII-Co1 fusion proteins produced in transgenic rice callus to bind Peyer’s Patches was confirmed in an M-cell binding assay in a mouse model. These results indicate that the cEDIII-Co1 fusion antigen protein could be delivered to M cells in the mucosal epithelium, which are responsible for antigen sampling, and successfully presented in antigen presenting cells (Kim et al. 2013a).
In a previous experiment, EDIII and CTB-EDIII fusion proteins were produced in transgenic tobacco plants and expressed as 0.13–0.25 % of TSP based on Western blot analysis and 0.019 % of TSP based on GM1-ELISA, respectively (Kim et al. 2009, 2010b). The consensus domain III of dengue virus glycoprotein fused with CTB (CTB-cEDIII) had increased expression in transgenic rice callus under control of the rice α-amylase 3D promoter: 0.68 mg/g of lyophilized rice callus (Kim et al. 2013c). cEDIII-Co1 fusion protein composed approximately 0.77 % of TSP (0.35 mg/g lyophilized transgenic rice callus). In this study, instead of nuclear transformation of tobacco and rice, agroinfiltration based on a plant RNA virus [tobacco mosaic virus (TMV)] was used to increase expression compared with previous reports. cEDIII and cEDIII-Co1 protein expression levels in agroinfiltrated leaves were 5.2 and 4.8 mg/g of dry weight of leaf tissues, respectively. The protein bands were detected by Coomassie blue staining. The domain III protein of the dengue virus expressed in plants with a TMV vector system composed 0.28 % of TSP, and the purified domain III protein induced anti-dengue virus antibodies with neutralizing activity. Mice immunized with plant-produced EDIII protein without adjuvant did not elicit an immune response, but response did occur following adjuvant exposure (Saejung et al. 2007). cEDIII-Co1 fusion proteins expressed at a high level are expected to be efficiently taken up by the mucosal immune system via the M-cell targeting ligand and to improve immune responses and to prevent infection with all dengue virus serotypes.
In Western blot analysis using anti-dengue virus antibodies under boiled conditions, the cEDIII or cEDIII-Co1 fusion proteins were detected as two main bands of monomeric proteins with slightly different and these results showed a similar band pattern as that observed in previous reports for the expression of foreign proteins in transgenic plants (Kim et al. 2009, 2013a). Two bands shown in Western blot analysis may account for the presence of processed or unprocessed signal sequences because the secreted cEDIII-Co1 fusion protein into suspension culture medium was detected only one band representing processed RAmy3D signal peptide (Kim et al. 2013a). Dengue virus E glycoprotein contains two N-linked glycosylation sites at Asn-67 and Asn-153 but these sites were not included in cEDIII domain of dengue virus glycoprotein. Other faint bands, which were not detected in protein extracts from non-infiltrated leaf tissues, were likely assembly of cEDIII or cEDIII-Co1 fusion proteins.
Targeting of antigen protein in plant cell is considered for improving expression level of foreign proteins. The high expression of human papillomaviruses L1 capsid protein (HPV-16 L1) was reported in chloroplast by means of transient expression with A. tumefaciens binary vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER), or chloroplasts (Maclean et al. 2007). However, in this study, when the expression of antigen proteins were compared for targeting to cytoplasm, endoplasmic reticulum, and chloroplast, the high expression of antigen proteins was observed in transgenic plant with targeting to ER. This result was not coincidence with result of expression of human papilloma viruses L1 capsid protein.
In this study, we used agrobacterium-mediated infiltration based on a plant RNA viral expression system to overcome low expression level of antigen protein in plant expression systems, which can result in low immune responses and immune tolerance. We constructed a plant expression vector based on TMV using a plant codon optimized consensus EDIII gene and a fusion to the M cell binding peptide, Co1. The expression of cEDIII and cEDIII-Co1 proteins was confirmed by Western blot analysis of agroinfiltrated N. benthamiana leaves. The plant-produced cEDIII and cEDIII-Co1 proteins composed 5.2 and 4.8 mg/g of dry weight of leaf tissues, respectively. These results suggest that cEDIII and an M cell binding ligand, Co1, fusion protein can be highly expressed to improve immune responses by antigen targeting of the mucosal immune system.
This paper was supported by a research fund from Chonbuk National University in 2014 and by the National Research Foundation (NRF-2014K1B1A1073861) funded by Korean Ministry of Science, ICT and Future Planning.
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