Expression of a consensus dengue virus envelope protein domain III ...

Plant Cell Tiss Organ Cult (2012) 109:509–515 DOI 10.1007/s11240-012-0116-y

ORIGINAL PAPER

Expression of a consensus dengue virus envelope protein domain III in transgenic callus of rice Mi-Young Kim • Moon-Sik Yang • Tae-Geum Kim

Received: 11 October 2011 / Accepted: 15 January 2012 / Published online: 28 January 2012 Ó Springer Science+Business Media B.V. 2012

Abstract An effective dengue vaccine should elicit immune responses against all four different dengue virus serotypes. This study optimized the codon usage of a gene encoding consensus dengue virus envelope protein domain III (cEDIII) with cross-neutralizing activity against four dengue virus serotypes for plant expression. Then, a plant expression vector was constructed with this gene under the control of the rice amylase 3D promoter (RAmy3D), which is a strong inducible promoter under sugar starvation conditions. The synthetic cEDIII gene was fused with the RAmy3D signal peptide and ER retention signal, SEKDEL, and was introduced into rice callus by particle bombardment-mediated transformation. The integration and expression of cEDIII gene in transgenic rice callus was confirmed by genomic DNA PCR amplification, Northern blot analysis, and western blot analysis, respectively. Densitometric analysis determined that the highest expression level of the cEDIII protein in lyophilized rice callus was approximately 0.45 mg g-1. These results suggest that it is feasible to use transgenic rice callus to produce the consensus dengue virus envelop protein domain III for edible vaccine purposes. Keywords Dengue virus Envelope protein Consensus domain III Tetravalent Plant-based edible vaccine

M.-Y. Kim M.-S. Yang T.-G. Kim (&) Department of Molecular Biology, Chonbuk National University, Jeonju, Republic of Korea e-mail: [email protected] M.-S. Yang Jeonju Center, Korea Basic Science Institute, Jeonju 561-756, Republic of Korea

Introduction One of the most important emerging mosquito-borne viral diseases is dengue; dengue virus infection continues to be a threat, particularly to children, in over 100 tropical and sub-tropical countries that contain over two-fifths of the world’s population. A safe and effective dengue vaccine is critical to prevent the rapid spread of dengue caused by changing demographics, urbanization, environment, and global travel. Although attempts have been made to develop an effective vaccine for dengue for the last 60 years, no effective vaccine is currently available, and treatment is supportive only (Murrell et al. 2011). Dengue is caused by the dengue virus, which consists of four antigenically different serotypes (DEN 1–4). A person infected with one of these serotypes with non-neutralizing antibodies is at greatest risk for developing dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during a second, heterotypic dengue virus infection. This is referred to as antibody-dependent enhancement (ADE) (Huang et al. 2006). Hence, a vaccine must not only be cost-effective, but also must be protective against all four serotypes of dengue virus. Tetravalent dengue vaccines including a live attenuated vaccine (Brandler et al. 2010); chimeric vaccines based on attenuated dengue virus (Osorio et al. 2011; Etemad et al. 2008) or Yellow Fever 17D (Guy et al. 2010); and recombinant DNA vaccines (Raviprakash et al. 2006; Ramanathan et al. 2009; Lima et al. 2011) have been developed as candidate vaccines to protect against all serotypes of dengue virus infection (Swaminathan et al. 2010). Among these, a consensus dengue virus envelope protein domain III (cEDIII) was obtained by aligning amino acid sequences from different isolates of the four serotypes of dengue virus. It was shown that mice immunized with the recombinant cEDIII expressed in

123

510

Escherichia coli produced neutralizing antibodies that recognized all serotypes of dengue virus (Leng et al. 2009). Transgenic plant systems that express antigens (‘‘edible vaccines’’) have several advantages over conventional vaccines such as safety, low production costs, ease of production scale-up, and minimal risk of contamination by animal or human pathogens and toxins. A plant expression system is the most promising edible vaccine system and has the ability to deliver antigens to the mucosal immune systems because the edible part or purified proteins in a transgenic plant expressing antigen proteins can be administrated orally (Lau and Korban 2009; Kim et al. 2006, 2011; Loc et al. 2011; Youm et al. 2010; MartinezGonzalez et al. 2011). However, a large biomass of transgenic plants containing antigen proteins is required to avoid oral tolerance due to low levels of expression of the antigen and degradation prior to immune priming. Thus, most research on plant-based vaccines has focused on achieving high expression levels of antigen proteins in transgenic plants. Several molecular and genetic approaches including gene modification with plant-optimized codon usage, strong promoters, strong 50 and 30 untranslated sequences, sub-cellular targeting signals, fusion to stable carriers, plastid transformation, and plant viral expression systems have been evaluated (Streatfield 2006). This study optimized the codon usage of the gene encoding consensus dengue virus envelope protein domain III for plants. Endoplasmic reticulum (ER) retention sequences were added to the gene in a plant expression vector with the gene under the control of the rice amylase 3D (Ramy3D) promoter, which is a strong, sugar-deprivation inducible promoter. This construct was introduced into rice callus to develop a production system for an edible plant-based vaccine that can potentially protect against all four serotypes of dengue virus.

Materials and methods Construction of the plant expression vector In a previous study, the amino acid sequences (103 amino acids) of domain III of the dengue virus envelope glycoprotein (EDIII) from different isolates of four serotypes of dengue virus were aligned to obtain a consensus sequence (cEDIII) (Leng et al. 2009). The nucleotide sequence of the consensus domain III was deduced from the amino acid sequence of the consensus cEDIII protein (Fig. 1). In this study, the cEDIII gene was synthesized based on plantoptimized codon usage. This gene was fused with the RAmy3D signal sequence at the N-terminus while the endoplasmic reticulum (ER) retention signal, SEKDEL, was fused at the C-terminus. BamHI and SacI restriction

123

Plant Cell Tiss Organ Cult (2012) 109:509–515

enzyme sites were added 50 and 30 to the gene for subsequent subcloning. The synthetic cEDIII gene (scEDIII) was digested with BamHI and SacI, and cloned into a BamHI/SacI-digested plant expression vector under the control of the promoter and 30 untranslated region (30 UTR) of the rice amylase 3D gene, pMYV657. This plant expression vector harbors the hygromycin phosphotransferase gene (HPT) as a selection marker for plant transformation (Fig. 1). Rice callus transformation Rice callus (Oryza sativa L. cv. Dongin) were prepared and transformed with pMYV657 via particle bombardmentmediated transformation (Chen et al. 2002). After 3–5 days, the rice callus were transferred to N6 selection medium supplemented with 2,4 dichlorophenoxyacetic acid (2 mg l-1), sucrose (30 g l-1), proline (0.5 g l-1), glutamine (0.5 g l-1), casein enzymatic hydrolysate (0.3 g l-1), gelite (2 g l-1), and hygromycin B (35 mg l-1) as the selection antibiotic, for 2–3 weeks. Genomic DNA PCR analysis Genomic DNA was prepared from transgenic rice callus using a ZymoBeadTM Genomic DNA Kit (Zymo Research, Orange, USA) and integration of the target gene into the plant genome was confirmed by genomic DNA PCR analysis. The presence of the scEDIII gene, including the signal sequence, in transgenic rice callus was determined using a Corbett Research Palm-Cycler (Corbett Research, Sydney, Australia) with scEDIII-specific primers; forward primer 50 -GCT CTA GAA TCA GTA GTG AGC-30 and reverse primer 50 -AAG TTC ATC CTT TTC GGA-30 . PCR reactions contained 100 ng genomic DNA, 10 pmol primers, 200 lM dNTPs, 2 ll 109 Taq polymerase buffer (10 mM Tris–HCl, pH 8.8, 50 mM KCl, and 0.1% Triton X-100), 1.5 mM MgCl2, 2 U i-Taq polymerase (iNtRON Biotechnology, Seoul, Korea) in a total reaction volume of 20 ll. The PCR amplification profile was as follows: denaturation at 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. This was followed by a final extension step of 72°C for 5 min. Fifty nanograms of plasmid DNA pMYV657 were used as a positive control. The amplified DNA was analyzed on a 1% agarose gel by electrophoresis and bands were visualized by staining the gel in 1 lg/l ethidium bromide (Sigma– Aldrich, St. Louis, MO, USA) solution for 5 min. Northern blot analysis Total RNA was extracted from non-transgenic and transgenic rice callus 5 days after induction with sugar starvation


(A)

AAG GGC ATG TCC TAC G CT ATG TGC ACC GGC AAG TTC AAG TTG G AG AAG GAG GTG K G M S Y A M C T G K F K L E K E V GCT GAG ACC CAG CAC GGC ACC ATC TTG ATC AAG GTG AAG TAC GAG GGC GAT GGC A E T Q H G T I L I K V K Y E G D G GCT CCT TGC AAG ATC CCT TTC GAG ATC CAG GAT GTG GAG AAG AAG CAC GTG AAC A P C K I P F E I Q D V E K K H V N GGA AGG TTG ATC ACC GCT AAC CCT ATC GTG ACC GAT AAG GAG TCC CCT GTG AAC G R L I T A N P I V T D K E S P V N ATC GAG GCT GAG CCT CCT TTC GGC GAT TCC TAC ATC GTG ATC GGC GTG GGC GAT I E A E P P F G D S Y I V I G V G D AAG GCT TTG AAG TTG AAC TGG TTC AAG AAG GGC TCC TCC K A L K L N W F K K G S S

SacI

(B) XbaI

Fig. 1 Construction of a plant expression vector for rice transformation. The synthetic consensus dengue virus envelope glycoprotein domain III (scEDIII) gene was synthesized based on riceoptimized codon usage (a). The scEDIII gene fused to the rice amylase 3D signal sequence and ER retention sequence (SEKDEL) was under the control of the promoter (RAmy3D) and the 30 untranslated region (30 UTR) of the rice amylase 3D gene. The hygromycin phosphotransferase (HPT) gene was used as a selection marker for transgenic rice callus (b)

511

RB

RAmy3D

scEDIII

Signal peptide

using Trizol Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the supplier’s instructions. Thirty micrograms total RNA were fractionated on a 1.2% formaldehyde-containing agarose gel and then transferred to a Hybond-N? membrane (Amersham–Pharmacia Biotech, Piscataway, NJ, USA). The blot was hybridized with a 32P-labeled cEDIII probe using the Prime-a-Gene labeling system (Promega U1100, Madison, USA) at 65°C in modified church buffer (1 mM EDTA, 250 mM Na2HPO4 7H2O, 1% hydrolyzed casein, 7% SDS, and pH 7.4) in a hybridization incubator (FINEPCR Combi-H, Seoul, Korea). The blot was washed twice with 29 SSC plus 0.1% SDS and again washed twice with 29 SSC plus 1% SDS for 15 min at 65°C. The membrane was then exposed to X-ray film (Agfa, Mortsel, Belgium). The autoradiogram was developed and fixed using an X-ray film development kit (Agfa). Western blot analysis Non-transgenic and transgenic rice callus were analyzed to detect cEDIII protein by western blot analysis. Proteins from the transgenic rice callus were extracted using 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, and 0.05% Tween-20) 3 days after induction of transgenic protein expression by sugar starvation. The ratio of extraction buffer volume to the sample weight was 2:1. Thirty micrograms of total soluble protein (TSP), as determined by the Bradford protein assay (Bio-Rad, Inc., Hercules, CA, USA), were separated by 15% sodium dodecylsulfate polyacrylamide gel electrophoresis

3’UTR

35S-p

HPT

polyA

LB

SEKDEL

(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) using a Trans Blot SD Semi-Dry Transfer Cell (Bio-Rad) for 50 min at 15 V. Nonspecific antibody binding was blocked by incubation of the membrane in 10 ml of 5% non-fat dry milk in TBS buffer (20 mM Tris–Cl, pH 7.5, and 500 mM NaCl) overnight at 4°C, followed by washing in TBS buffer for 5 min. The membrane was incubated for 2 h in a 1:2,500 dilution of mouse anti-dengue virus monoclonal 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 a TBST buffer. The membrane was then incubated for 1 h in a 1:5,000 dilution of goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma–Aldrich) and then washed twice in TBST buffer 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–Aldrich). Quantification of cEDIII protein Transgenic rice callus expressing the cEDIII protein were grown in suspension culture to achieve sufficient expression of the target protein and produce large amounts of plant material. The transgenic suspension culture callus were harvested 3 days after induction by sugar starvation using a vacuum pump system and were then lyophilized. The amount of cEDIII protein produced in the transgenic plant callus was determined by western blot analysis. TSP

123

512


corresponding to the scEDIII gene was detected in nontransgenic rice callus (Fig. 2).

was extracted from the fine lyophilized powder (100 mg dried cell weight) by gradually adding 20 ml 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, and 0.05% Tween-20). Known amounts (10, 100 ng, 1, and 2 lg) of bacterial EDIII protein (serotype II) purified in E. coli as a standard protein and plant protein extracts (5 and 10 ll of TSP) were separated by SDS-PAGE, and then the bacterial EDIII and plant-produced cEDIII proteins were detected using anti-EDIII monoclonal antibodies. The amount of cEDIII protein in the plant protein extracts was determined by comparing the band intensities of the plant-produced cEDIII and the known amounts of bacterial EDIII protein. The intensity of the bands detected on the western blots was quantified using densitometric analysis.

Expression of scEDIII in transgenic rice callus Northern blot analysis was conducted with a [32P]-labeled scEDIII probe to detect scEDIII transcripts in transgenic rice callus. Thirteen transgenic rice callus showed a positive signal for scEDIII transcripts, but no signal was found in non-transgenic rice callus (Fig. 3). Expression of the scEDIII protein was evaluated in three transgenic rice callus lines that had high mRNA expression levels of scEDIII by western blot analysis. The cEDIII protein was detected using anti-dengue virus antibody from transgenic rice callus protein extracts and appeared as two slightly different bands, both approximately 12 kDa (Fig. 4). The size of the purified bacterial EDIII protein expressed in the pRSET vector expression system was approximately 16 kDa. However, no band corresponding to the cEDIII protein was detected in boiled non-transgenic rice callus protein extracts (Fig. 4).

Results Construction of the plant expression vector and PCR analysis of transgenic rice callus

Quantification of cEDIII protein expression in lyophilized rice callus

The gene encoding the consensus domain III protein of the dengue virus envelope protein (scEDIII) was shown in a previous study to induce cross-neutralizing antibodies against four serotypes of dengue virus (Leng et al. 2009). To express this target gene in rice callus, the codon usage of scEDIII was optimized for plant expression, the gene was fused to the RAmy3D signal sequence and the ER retention signal, and then the gene was constructed into a plant expression vector under the control of the sucrosestarvation inducible RAmy3D promoter expression system. The rice expression vector, pMYV657, which contained the scEDIII gene, was transformed into rice callus via particle bombardment transformation. Thirteen independent putative transgenic rice callus appeared under hygromycin selective pressure 2–4 weeks after transformation. A DNA fragment corresponding in size to the scEDIII gene including the signal sequence (490 bp) was amplified in all transgenic rice callus by genomic DNA PCR analysis with primer sets specific to the scEDIII gene. No DNA band

1.0kb

PC

M

NC

1

2

3

4

The transgenic callus line #13, which showed the highest expression of the cEDIII protein among the transgenic callus lines, was selected for suspension culture for scaleup and to maximize transgenic protein expression. Transgenic rice callus induced by sugar-starvation were harvested and lyophilized after 3 days. Total soluble protein was extracted from lyophilized transgenic callus to measure the amount of cEDIII protein by western blot analysis and densitometry analysis. Known amounts (10, 100 ng, 1, 2 lg) of purified bacterial EDIII proteins were used to construct a standard curve. The amount of cEDIII present in transgenic callus protein extracts was calculated by comparing the intensity of the plant-produced cEDIII protein to known amounts of bacterial protein. The greatest amount of cEDIII expressed in transgenic rice callus (#13) suspension culture was 0.45 mg g-1 in lyophilized material (Fig. 5).

5

6

7

8

9

10

11

12

13

0.5kb

Fig. 2 Genomic DNA PCR analysis of scEDIII in transgenic rice callus. The presence of the scEDIII gene in the genome of transgenic rice callus was evaluated by PCR analysis of transgenic rice genomic DNA (100 ng) with scEDIII-specific primers. Lane PC pMYV657 template DNA used as a positive control for PCR; lane M 100 bp

123

DNA ladder (ELPIS BIOTECH, Seoul, Korea); lane NC genomic DNA from non-transgenic rice callus used as a negative control; lanes 1–13 PCR products from amplification of genomic DNA isolated from transgenic rice callus

Plant Cell Tiss Organ Cult (2012) 109:509–515 NC

1

2

513 3

4

5

6

7

8

9

10

11

12

13

6kb 4kb 1.0kb 0.5kb

Fig. 3 Northern blot analysis of the scEDIII gene in transgenic rice callus. Total RNA (30 lg) extracted from transgenic rice callus 5 days after induction with sugar starvation was probed with a kDa

PC1

M

NC

#9

#10

#13

PC2

32

P-labelled scEDIII probe. Lane NC total RNA from a nontransgenic rice callus as a negative control; lanes 1–13 total RNA extracted from transgenic rice callus kDa

55 43 34

55 43 34

26

26

17

17

M

NC

1

2

3

4

5

6

Discussion

Fig. 5 Quantification of cEDIII protein expression in transgenic rice callus. The amount of cEDIII protein in protein extracts of lyophilized callus was measured 3 days after induction with sugar starvation. The intensity of bands detected on western blots was measured by densitometry. The amount of cEDIII present in transgenic callus protein extracts was calculated by comparing the intensity of the plant-derived band to that of the bands containing a known amount of bacterial protein. Lane M molecular weight markers (Fermentas); lane NC non-transgenic rice callus protein extract used as a negative control; lanes 1 and 2, 5 and 10 ll of TSP extracted from transgenic rice callus; lanes 3–6, 10, 100 ng, 1, and 2 lg of purified bacterial EDIII protein used to construct a standard curve

Dengue is a major public health problem because almost half of the world’s population live in tropical and subtropical areas that are at risk of dengue infection, and as many as 100 million people are infected per year (Swaminathan et al. 2010). Dengue fever, with the severe consequences of DHF and DSS, results in substantial morbidity, mortality, and economic losses. However, there is still no licensed dengue vaccine to prevent the disease. The Pediatric Dengue Vaccine Initiative (PDVI) plan, which is a program of the International Vaccine Institute, Seoul, Korea, has been accelerated to develop a successful vaccine (Murrell et al. 2011). A dengue vaccine must be protective against all four dengue virus serotypes, be costeffective, and have a good safety profile. For this reason, tetravalent vaccines are being developed by several research groups, including the group that conducted the present study. In this study, a plant-based vaccine was

produced based on the consensus dengue virus envelope protein, which can potentially induce neutralizing antibodies against all serotypes of dengue virus. Plant-based vaccines are cost-effective and safe, as they are free from human pathogens. The cost-effective nature of this vaccine is particularly important because the regions that are most at risk for dengue infections are also impoverished. Although transgenic plants expressing antigen proteins seem to be promising, they elicit weak immune responses in animals due to low expression levels of antigen proteins. The expression of the EDIII protein in transgenic tobacco leaf tissues reportedly ranges from 0.13 to 0.25% of the total soluble protein (Kim et al. 2009a, b). The expression of a KDEL-tagged dengue virus protein was observed in 0.3% of total soluble proteins at 5 days of culture in cell suspension cultures of Nicotiana tabacum (Martinez et al. 2011). Domain III of the dengue virus envelope glycoprotein has

Fig. 4 Western blot analysis of the cEDIII protein in transgenic rice callus. The cEDIII protein in transgenic plant protein extracts after induction with sugar starvation was detected using mouse anti-dengue virus monoclonal antibody after boiling of the protein samples for 5 min to ensure protein denaturation. Lane PC1 yeast-derived recombinant cEDIII protein; lane M molecular weight marker (Fermentas, Glen Burnie, MD); lane NC non-transgenic rice callus protein extract used as a negative control; lanes 1–3 transgenic rice callus protein extracts; lane PC2 purified bacterial cEDIII protein

123

514

been expressed in plants using a TMV-based vector system, resulting in a yield of 0.28% of TSP (Saejung et al. 2007). In order to improve antigen protein expression, the cEDIII dengue virus envelope protein gene in this study was modified based on the optimized codon usage for plant expression, and fused with the Ramy3D signal peptide and ER retention signal. The cEDIII gene was under the control of the Ramy3D promoter, which has a strong expression system in transgenic rice cell suspension culture. The rice cell expression system with a Ramy3D promoter allows for expression of foreign genes in transgenic rice callus. The accumulation of hGM-CSF under the control of the Ramy3D promoter in transgenic rice suspension culture was 1,000-fold better than that of hGM-CSF in tobacco cell suspension culture under the control of the CaMV 35S promoter (Shin et al. 2003). The Ramy3D expression system can therefore potentially overcome immune tolerance caused by low expression of antigens in transgenic plants. ER retention is achieved through the C-terminal tetrapeptide H/KDEL, which mediates the retrieval of the tagged protein from the cisternal Golgi apparatus (Pelham 1990). Plant ER plays a role in protein storage, especially during seed maturation (Herman and Larkins 1999; Vitale and Ceriotti 2004), and storage of recombinant proteins in the endoplasmic reticulum (ER) has been shown to improve the expression levels of recombinant proteins in plant systems. Yasuda et al. suggested that the ER of rice endosperm cells is a prime storage compartment for foreign gene products based on their observation of a correlation between expression and localization of a foreign gene product in the rice endosperm (Yasuda et al. 2006). In this study, the highest expression level of the cEDIII protein in lyophilized transgenic rice callus was 0.45 mg g-1 (0.81% of TSP), which is higher than that of previous reports. The production of cEDIII protein in transgenic rice callus was detected as two bands on the western blot analysis. The band of lower molecular weight is similar in size to the yeast-produced cEDIII protein. The upper band might contain signal peptides; the fact that it has a slightly higher molecular weight than the yeast-produced cEDIII is likely unrelated to N-glycosylation because cEDIII does not have any possible N-glycosylation sites (Asn-X-Ser/ Thr). The upper band corresponded in size to the bacterial EDIII protein containing an additional N-terminal peptide (approximately 4 kDa) that was expressed and purified from E. coli using the pRSET expression system. The purified EDIII protein in plants using a TMV-based vector system induced anti-dengue virus antibodies with neutralizing activity against dengue virus (Saejung et al. 2007). However, plant-produced EDIII protein did not elicit immune responses in the absence of an adjuvant. To induce efficient immune responses, plant-produced EDIII proteins should be genetically fused with ligands such as CTB or

123


LTB. These ligands can function as carriers and adjuvants for genetically fused antigens by enhancing antigen uptake into the mucosal immune system and improving immune responses (Kim et al. 2004; Kim et al. 2009a, b). This study constructed a rice callus suspension system expressing the consensus domain III of the dengue virus envelope glycoprotein for development of plant-based vaccines to prevent infection of all four serotypes of dengue virus. The expression level was 0.45 mg g-1 dried weight of plant material. The ability of the rice-produced cEDIII protein to generate immunogenicity against the four dengue virus serotypes will be tested in future animal experiments. Transgenic rice plants will be regenerated from transgenic callus showing a high expression level of antigen protein and their feasibility as plant-based edible vaccines will be tested. Acknowledgments This study was supported by research funds of Chonbuk National University in 2010 and the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea.

References Brandler S, Ruffie C, Naiburg V, Frenkiel MP, Bedouelle H, Despres P, Tanqy F (2010) Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses. Vaccine 28:6730–6739 Chen PW, Lu CA, Yu TS, Tseng TH, Wang CS, Yu SM (2002) Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice. J Biol Chem 277: 13641–13649 Etemad B, Batra G, Raut R, Dahiya S, Khanam S, Swaminathan S, Khanna N (2008) An envelope domain III-based chimeric antigen produced in Pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes. Am J Trop Med Hyg 79:353–363 Guy B, Saville M, Lang J (2010) Development of Sanofi Pasteur tetravalent dengue vaccine. Hum Vaccine 6:696–705 Herman EM, Larkins BA (1999) Protein storage bodies and vacuoles. Plant Cell 11:601–614 Huang KJ, Yang YS, Lin YS, Huang JH, Liu HS, Yeh TM, Chen SH, Liu CC, Lei HY (2006) The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol 176:2825–2832 Kim TG, Befus N, Langridge WH (2004) Co-immunization with an HIV-1 tat transduction peptide-rotavirus enterotoxin fusion protein stimulates a Th1 mucosal response in mice. Vaccine 22: 431–438 Kim YS, Kim BG, Kim TG, Kang TJ, Yang MS (2006) Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system. Plant Cell Tiss Organ Cult 87:203–210 Kim MY, Yang MS, Kim TG (2009a) Expression of dengue virus E glycoprotein domain III in non-nicotine transgenic tobacco plants. Biotechnol Bioprocess Eng 14:725–730 Kim TG, Huy NX, Kim MY, Jeong DK, Jang YS, Yang MS, Langridge WH, Lee JY (2009b) Immunogenicity of a cholera toxin B subunit Porphyromonas gingivalis fimbrial antigen fusion protein expressed in E. coli. Mol Biotechnol 41:157–164

Plant Cell Tiss Organ Cult (2012) 109:509–515 Kim MY, Kim TG, Yoo HS, Yang MS (2011) Expression and assembly of ApxIIA toxin of Actinobacillus pleuropneumoniae fused with the enterotoxigenic Escherichia coli heat-labile toxin B Subunit in transgenic tobacco. Plant Cell Tiss Organ Cult 105:375–382 Lau JM, Korban SS (2009) Analysis and stability of the respiratory syncytial virus antigen in T3 generation of transgenic tomato plants. Plant Cell Tiss Organ Cult 96:335–342 Leng CH, Liu SJ, Tsai JP, Li YS, Chen MY, Liu HH, Lien SP, Yueh A, Hsiao KN, Lai LW, Liu FC, Chong P, Chen HW (2009) A novel dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity. Microbes Infect 11:288–295 Lima DM, de Paula SO, Franca RF, Palma PV, Morais FR, GomesRuiz AC, de Aquino MT, da Fonseca BA (2011) A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection. Vaccine 29:831–838 Loc NH, Song NV, Tien NQD, Minh TT, Nga PTQ, Kim TG, Yang MS (2011) Expression of the Escherichia coli heat-labile enterotoxin B subunit in transgenic watercress (Nasturtium officinale L.). Plant Cell Tiss Organ Cult 105:39–45 Martinez CA, Giulietti AM, Talou JR (2011) Expression of a KDELtagged dengue virus protein in cell suspension cultures of Nicotiana tabacum and Morinda citrifolia. Plant Cell Tiss Organ Cult 107:91–100 Martinez-Gonzalez L, Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Lopez-Revilla R, Korban SS, Guevara-Arauza JC, Alpuche-Solis AG (2011) Oral immunization with a lettucederived Escherichia coli heat-labile toxin B subunit induces neutralizing antibodies in mice. Plant Cell Tiss Organ Cult 107:441–449 Murrell S, Wu SC, Butler M (2011) Review of dengue virus and the development of a vaccine. Biotechnol Adv 29:239–247 Osorio JE, Brewoo JN, Silenqo SJ, Arquello J, Moldovan IR, TaryLehmann M, Powell TD, Livenqood JA, Kinney RM, Huang CY, Stinchcomb DT (2011) Efficacy of a tetravalent chimeric

515 dengue vaccine (DENVax) in cynomolgus macaque. Am J Trop Med Hyg 84:978–987 Pelham HR (1990) The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 15:483–486 Ramanathan MP, Kuo YC, Selling BH, Li Q, Sardesai NY, Kim JJ, Weiner DB (2009) Development of a novel DNA SynCon tetravalent dengue vaccine that elicits immune responses against four serotypes. Vaccine 27:6444–6453 Raviprakash K, Apt D, Brinkman A, Skinner C, Yang S, Dawes G, Ewing D, Wu SJ, Bass S, Punnonen J, Porter K (2006) A chimeric tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques. Virology 353:166–173 Saejung W, Fujiyama K, Takasaki T, Ito M, Hori K, Malasit P, Watanabe Y, Kurane I, Seki T (2007) Production of dengue 2 envelope domain III in plant using TMV-based vector system. Vaccine 25:6646–6654 Shin YJ, Hong SY, Kwon TH, Jang YS, Yang MS (2003) High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 82:778–783 Streatfield SJ (2006) Mucosal immunization using recombinant plantbased oral vaccines. Methods 38:150–157 Swaminathan S, Batra G, Khanna N (2010) Dengue vaccines: state of the art. Expert Opin Ther Pat 20:819–835 Vitale A, Ceriotti A (2004) Protein quality control mechanisms and protein storage in the endoplasmic reticulum. A conflict of interests? Plant Physiol 136:3420–3426 Yasuda H, Hayashi Y, Jomori T, Takaiwa F (2006) The correlation between expression and localization of a foregin gene product in rice endosperm. Plant Cell Physiol 47:756–763 Youm JW, Jeon JH, Kim H, Min SR, Kim MS, Joung H, Jeong WJ, Kim HS (2010) High-level expression of a human beta-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice. Transgenic Res 19:1099–1108

123