Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants

Protein Expression and Purification 74 (2010) 236–241

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Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants Tae-Geum Kim a, Mi-Young Kim a, Moon-Sik Yang a,b,* a b

Department of Molecular Biology, Chonbuk National University, Jeonju 561-756, Republic of Korea Jeonju Center, Korea Basic Science Institute, Jeonju 561-756, Republic of Korea

a r t i c l e

i n f o

Article history: Received 6 May 2010 and in revised form 21 July 2010 Available online 4 August 2010 Keywords: Dengue virus Envelope glycoprotein domain III Cholera toxin B subunit Plant-based vaccine Mucosal immunization

a b s t r a c t Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297–394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB–EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB–EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB–EIII fusion protein in transgenic plant extracts. Based on the GM1-ELISA results, the CTB–EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, GM1-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB–EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Dengue virus is transmitted by the Aedes mosquito and causes the most important arthropod-borne viral disease in humans. The disease worldwide, although mainly occurring in the sub-tropical regions [1], infects approximately 50–100 million individuals annually. The flavivirus consists of three structural proteins – the capsid (C),1 envelope (E), and membrane (M) proteins – and a single strand of infectious RNA. The structure of whole virus and its envelope glycoprotein E have been determined by fitting X-ray crystallography and cryo-electron microscopy (cryo-EM) [2,3]. The envelope glycoprotein E of dengue virus plays a crucial role in host cell attachment and entry, as well as elicits antibodies showing protective immunity [4]. This glycoprotein E contains three ectodomains (domains I–III) and one transmembrane domain. Domain III is the primary site of interaction between the virus and receptors at the target’s cellular surface and contains

* Corresponding author at: Department of Molecular Biology, Chonbuk National University, Jeonju 561-756, Republic of Korea. Fax: +82 63 270 4334. E-mail address: [email protected] (M.-S. Yang). 1 Abbreviations used: C, capsid; E, envelope; M, membrane; cryo-EM, cryo-electron microscopy; CT, cholera toxin; LTB, labile enterotoxin B subunit; CTB, cholera toxin B subunit; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.07.013

neutralizing epitopes which elicit monoclonal antibodies, thereby strongly blocking viral adsorption by cells [3,5,6]. Transgenic plants and transgenic plant cell suspension cultures have been increasingly used as vehicles to produce commercial and pharmaceutical proteins. One such vehicle is a plant-based edible vaccine to protect against a wide variety of human infectious and autoimmune diseases [7–13]. Plant-based vaccines have several advantages: low cost to produce, ease of storage and transport, safety from animal pathogen contamination, and lack of needle-associated injury and disease spread. Mice intramuscularly immunized with plant-produced dengue virus EIII protein, using a TMV-based vector system, induced neutralizing antibody activity against dengue virus, indicating that plant-produced EIII protein possesses appropriate antigenicity and immunogenicity [14]. Plant-based edible vaccines may induce immune tolerance and low immune responses because of the low expression level of target antigen in transgenic plants. The alternative to overcome this obstacle is to increase antigen uptake into mucosal immune systems by using the enterotoxin B subunit as a carrier for the fused antigen. Bacterial toxins consist of A and B domains, and B domains have been used to increase antigen uptake in gut epidermal cells, thereby increasing mucosal immune responses [15,16]. Cholera toxin (CT) is a typical representative of the heteromultimeric AB toxins produced by Vibrio cholerae and one of the most effective

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enterocyte targeting molecules. The CTB subunit is composed of five identical polypeptides (11.5 kDa) and assembles into a highly stable pentameric ring structure in the bacteria. The CTB pentamer binds selectively to sugar–lipid GM1-ganglioside molecules embedded in the intestinal epithelial membranes (enterocytes) and microfold (M) cells [17]. The CTB has been shown to function as an effective carrier molecule for fused foreign proteins, including mucosal vaccine antigens and autoantigens [18]. The expression of dengue virus E glycoprotein domain III was reported in plant using TMV-based vector system and transgenic nicotine-free tobacco [14,19]. Plant-produced EIII protein did not elicited immune responses in mice without adjuvant [14]. To overcome this problem in transgenic plants, antigens need to be fused with ligands such as cholera toxin B subunit (CTB), and enterotoxigenic E. coli heat-labile enterotoxin B subunit (LTB), to increase antigen uptake efficiency into mucosal immune systems. In this work, we investigated the capacity of a transgenic tobacco plant, which contains low or no nicotine contents, to produce a fusion protein consisting of cholera toxin B subunit (CTB) and domain III of dengue virus envelope glycoprotein (EIII) under the control of duplicated 35S Cauliflower Mosaic Virus promoter, which can induce efficiently immune responses without adjuvant. The integration and expression of target fusion gene was confirmed by genomic DNA PCR and Western blot analysis, respectively. The feasibility of transgenic plant expressing CTB–EIII fusion protein as plant-based edible vaccine for improving immune responses and protecting host from dengue virus infection will be investigated in further experiments.

Materials and methods Construction of plant expression vector Domain III (amino acids 297–394) of dengue virus type 2 E glycoprotein (EIII) was amplified from plasmid pMYV497 [19] by PCR with EIIIFB and EIIIRK primers (Fig. 1). The cholera toxin B subunit (CTB) was amplified from plasmid pPCV701CTB-NSP4 [20] by PCR with CTBF and CTBRB primers (Fig. 1). The amplified CTB and EIII PCR products were digested with BamHI (included in the primers) and ligated with each other. After ligation, the ligated mixtures were used as template for PCR amplification with the CTBF and EIIIRK to amplify the CTB–EIII fusion gene. The amplified CTB–EIII PCR products were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). The correct DNA sequence of CTB–EIII fusion gene was confirmed by DNA sequence analysis. The Gly-Pro-Gly-Pro

sequence was located between CTB and EIII protein for hinge flexibility. The CTB–EIII fusion gene fused with the ER retention signal (SEKDEL) was introduced into a plant expression binary vector, pMY27 [21]. This plant expression vector was designated pMYV498 (Fig. 1). The plant expression vector pMYV498 was transformed into Agrobacterium tumefaciens strain LBA4404 by tri-parental mating method [22] and the plasmid DNA in the transformed Agrobacterium cells was confirmed by restriction endonuclease digestion and agarose gel electrophoresis prior to plant transformation.

Plant transformation The leaves of tobacco (Nicotiana tabacum L. cv. MD609) cultured under sterile conditions in Magenta GA-7 culture boxes (Sigma Chemical, Co., St. Louis, MO) on Murashige and Skoog (MS) basal medium [23] containing 3.0% sucrose and 0.8% plant agar at 25 °C were sliced 0.5 cm  0.5 cm and placed for 15 min in a culture dish containing a suspension of the A. tumefaciens LBA4404 strain harboring the CTB–EIII fusion gene. After blotting the explants on sterile filter paper, they were transferred on a MS medium, supplemented with 0.1 mg/L 2-naphthaleneacetic acid (NAA) and 0.5 mg/L 6-benzylamino purine (BA), pH 5.8, and incubated in the dark for 2d at 25 °C. To select transgenic plant cells and to counter-select against continued Agrobacterium growth, the explants were transferred to MS selection medium supplemented with the same growth regulators, 100 mg/L kanamycin and 500 mg/L cefotaxime, for 5 weeks. The developed shoots were transferred into a hormone-free MS medium that contained 100 mg/L kanamycin and 500 mg/L cefotaxime to stimulate root formation. The plantlets were transferred to the greenhouse to mature.

Detection of the CTB–EIII fusion gene in transgenic plants Genomic DNA was isolated from leaf tissues of non-transgenic and transgenic tobacco using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The concentration of genomic DNA was measured at 260 nm in a UV spectrophotometer. The presence of the CTB–EIII fusion gene in transgenic tobacco genomic DNA was determined by PCR analysis using the CTBF and EIIIRK primers, the same primer set as in amplifying the CTB–EIII fusion gene. The PCR products were separated by electrophoresis in a 1.0% agarose gel.

A LB

B

CTBF

pNOS

NPT II

t-NOS

pdu35S

CTB-EIII

t-NOS

RB

: 5’-GCTCTAGAGCCACCATGATTAAATTAAAATTTG-3’

CTBRB : 5’-GCGGATCCCGGGCCTGGGCCATT-3’ EIIIFB : 5’-GCGGATCCATGTCATACTCTATGTGT-3’ EIIIRK : 5’-GCGGTACCTTTCTTGAACCAGTTGAG-3’ Fig. 1. Plant expression vector pMYV498. Genes located within the T-DNA sequence flanked by the right and left borders (RB and LB) include a DNA fragment consisting of the cholera toxin B subunit and domain III of dengue virus E glycoprotein gene (CTB–EIII) fused with an ER retention sequence (SEKDEL) under the control of the duplicated Cauliflower Mosaic Virus 35S promoter (pdu35S) and an NPT II (neomycin phosphotransferase II) expression cassette for kanamycin selection of transgenic plants. The pNOS promoter and tNOS terminator are from the A. tumefaciens nopaline synthase gene (A). The primers for amplification of CTB, FimA1 and FimA2 were listed in (B). Underlines represent restriction enzyme sites used for gene construction.

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Detection of CTB–EIII fusion gene transcripts in transgenic plants The total RNA was isolated from leaf tissues of non-transgenic and transgenic plants using the RNeasy Plant Mini Kit (Qiagen) and was separated by electrophoresis through agarose gel containing formaldehyde [24]. The separated RNA was then transferred to an Hybond N+ membrane (Amersham Pharmacia Biotech RPN82B, Piscataway, NJ, USA). The membrane was hybridized with a 32P-labeled CTB–EIII probe using Prime-a-Gene labeling system (Promega U1100, Madison, WI, USA) at 65 °C in hybridization incubator (FINEPCR Combi-H, Seoul, Korea). The membrane was washed twice with 2 SSC and 0.1% SDS and then washed twice again with 2 SSC and 1% SDS for 15 min at 65 °C. Hybridized bands were detected by autoradiography using X-ray film (Fuji Photo Film Co. HR-G30, Tokyo, Japan). Detection of CTB–EIII fusion protein in transgenic plants Transgenic tobacco leaf tissues were analyzed to detect the CTB–EIII fusion protein using immunoblot detection methods with anti-CTB and anti-EIII polyclonal antibodies. Transgenic leaf tissues were homogenized by grinding in a mortar and pestle adding liquid nitrogen and extracted with extraction buffer (1:1 w/v) (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). Tissue homogenates were centrifuged at 17,000g in a Beckman GS-15R centrifuge for 15 min at 4 °C to remove insoluble cell debris. An aliquot of the supernatant containing 100 lg total soluble protein, as determined by the Bradford protein assay (Bio-Rad, Inc., Hercules, CA, USA), was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) at 100 V for 1.5–2 h in Tris–glycine buffer (25 mM Tris–Cl, 250 mM glycine, pH 8.3, 0.1% SDS). The separated protein bands were transferred from the gel to Hybond C membrane using a Mini Trans Blot electrophoretic transfer cell (Bio-Rad 170–3930) for 2 h at 130 mA in the transfer buffer (50 mM Tris, 40 mM glycine, 0.04% SDS, 20% methanol, pH 8.3). Nonspecific antibody binding was blocked by incubation of the membrane in 20 mL of 5% non-fat dry milk in a TBS buffer (20 mM Tris–Cl, pH 7.5 and 500 mM NaCl) for 1 h, with gentle agitation on a rotary shaker (40 rpm), followed by washing in a TBS buffer for 5 min. The membrane was incubated overnight at room temperature with gentle agitation in a 1:2000 dilution of mouse anti-dengue virus monoclonal antibody (AbD Serotec MCA2277, UK) or a 1:5000 dilution of rabbit anti-CTB antiserum (Sigma) in TBST antibody dilution buffer (TBS with 0.05% Tween-20 and 1% non-fat dry milk), followed by three washes in a TBST buffer (TBS with 0.05% Tween-20). The membrane was incubated in a 1:7000 dilution of anti-mouse IgG or a 1:7000 dilution of anti-rabbit IgG conjugated with alkaline phosphatase (Promega) in an antibody dilution buffer for 1 h at room temperature with gentle agitation. The membrane was washed twice in a TBST buffer and once in a TMN (100 mM Tris, pH 9.5, 5 mM MgCl2 and 100 mM NaCl) buffer, as before. After washing, the color was developed with BCIP/NBT (USB 102185-33-1 and USB 298-83-9, Cleveland, OH) in TMN buffer. Quantification of CTB–EIII fusion protein The expression levels of plant-expressed CTB–EIII fusion protein and their affinity for GM1-ganglioside receptor were determined by GM1-ELISA [25]. The microtiter plate (Becton Dickinson Labware, USA) was coated with 100 ll per well of monosialoganglioside GM1 (3.0 lg/mL) (Sigma) dissolved in bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), covered with Saran wrap and incubated at 4 °C overnight. The wells were washed three times with PBST and blocked by adding 300 lL/well of 1% BSA in PBS

and incubated at 37 °C for 2 h, followed by washing three times with PBST. The wells were loaded with centrifuged transgenic plant leaf extracts (5 lg of protein) with serial dilutions (100 lL per well) and purified bacterial CTB (5 ng of protein) as a standard curve and incubated overnight at 4 °C. The wells were washed three times with PBST and loaded with 100 lL per well of a 1:8000 dilution of rabbit anti-CTB primary antibodies and incubated for 2 h at 37 °C, followed by washing the wells three times with PBST. The plate was then incubated with 100 lL per well of secondary antibody, a 1:20,000 dilution of alkaline phosphataseconjugated anti-rabbit IgG (Sigma), for 2 h at 37 °C and washed three times with 300 lL PBST per well. The plate was finally incubated with 100 lL per well of TMB substrates L (PharMingen, USA) for 30 min at room temperature. The plate was measured at a 405 nm wavelength in an ELISA reader (Packard Instrument, USA). The affinity of plant-expressed CTB–EIII fusion protein for GM1-ganglioside receptor was detected, and the amount of CTB– EIII fusion protein synthesized in the transgenic plant was estimated based on the known amount of purified bacterial CTB. Results Detection of the CTB–EIII fusion gene in transgenic plants Dengue virus envelope glycoprotein E domain III (EIII) gene and cholera toxin B subunit (CTB) were amplified by PCR amplification and were ligated to each other after restriction enzyme treatment. Domain III fused with CTB (CTB–EIII) fusion gene was amplified by PCR and introduced into a plant expression vector (Fig. 1). The Kozak sequence (GCCACC) in the front of the start codon [26] and an ER retention signal (SEKDEL) before the stop codon were applied to CTB–EIII fusion gene to improve the expression level of recombinant protein in transgenic plants (Fig. 1). The plant expression vector was named pMYV498 and was introduced into tobacco N. tabacum cv. MD609, which contains almost no nicotine, by Agrobacterium-mediated transformation methods. Nine independent, putative transgenic kanamycin-resistant tobacco plants formed roots 4–6 weeks after transgenic shoots were transferred to MS basal medium containing antibiotic kanamycin (100 lg/mL). A DNA fragment corresponding in size to the CTB–EIII fusion gene (720 bp) was amplified by genomic DNA PCR amplification in all putatively selected transgenic tobacco plants. No DNA band corresponding to the CTB–EIII fusion gene was detected in non-transgenic tobacco (Fig. 2). Detection of CTB–EIII fusion gene transcripts in transgenic plants The transcription of the CTB–EIII fusion gene was confirmed using a 32P-labeled CTB–EIII probe for Northern blot analysis of

M P N 1 2 3 4 5 6 7 8 9

CTB-EIII

Fig. 2. Detection of the CTB–EIII fusion gene in transgenic plants. Genomic DNA (400 ng) isolated from transgenic plant leaf tissues was used to demonstrate the presence of the CTB–EIII fusion gene using PCR amplification with CTBF and EIIIRK primers. Lane M is k/HindIII, used as a DNA size marker; lane P is pMYV498 template DNA, used as a positive control for PCR; lane N is non-transgenic plant genomic DNA, used as a negative control; lanes 1–9 are transgenic plant genomic DNA, used as template.

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N

2

3

6

7

8

and similar band patterns with the anti-dengue virus monoclonal antibody except two low bands under non-boiled conditions (Fig. 4D). No band corresponding to CTB–EIII fusion protein was detected in non-transgenic plant extracts.

9 CTB-EIII

Quantification of CTB–EIII fusion protein in transgenic plants

Fig. 3. Northern blot analysis to detect the CTB–EIII transcripts in transgenic plants. The total RNA extracts in the transgenic plant tissues were used to detect the CTB– EIII transcripts. Lane N is total RNA extracts from non-transgenic plants, used as a negative control; lanes 2, 3, and 6–9 are total RNA extracts from transgenic plants. The lower picture shows the Et-Br stained gel showing total RNA.

total RNA from transgenic plant leaf tissues. All plants tested showed a positive signal for CTB–EIII, but no signal was detected in non-transgenic plants (Fig. 3). The transgenic plants showing high expression of CTB–EIII fusion gene (#6, #7, #8 and #11) were selected for further analysis. Detection of CTB–EIII fusion proteins The transgenic plants with CTB–EIII fusion gene transcripts in transgenic leaf tissues were tested to detect CTB–EIII fusion protein by immunoblot analysis with anti-dengue virus monoclonal antibody and anti-CTB polyclonal antibody. The results of immunoblot with anti-dengue virus monoclonal antibody showed two bands of CTB–EIII fusion protein in the boiled condition (Fig. 4A), and a higher band, corresponding to pentameric size, and two same bands in the non-boiled condition (Fig. 4B). The results with anti-CTB polyclonal antibody showed similar band patterns with the anti-dengue virus monoclonal antibody under boiled condition (Fig. 4C)

A kDa

M

PE

N

6

7

8

9

95 72 55 43 26

11

kDa 72 55 43 26 17

Discussion CTB consists of five identical polypeptides, forming a pentameric ring, and the CTB pentamer has been shown to bind selectively to sugar-lipid GM1-ganglioside, CTB’s biologically active receptor, in the plasma membrane of intestinal epithelial cells (enterocytes) and microfold (M) cells [17]. The immunoblot results with antiCTB and -dengue virus monoclonal antibodies showed plant-produced CTB–EIII fusion proteins with pentameric size under unboiled conditions. The monomer and pentamer of CTB–EIII fusion protein was detected with anti-dengue virus antibody under the unboiled condition, the amount of CTB–EIII fusion protein assembly was measured 60% of CTB–EIII monomer by densitometry (data not shown). In addition, the biological activity of plant-produced CTB–EIII fusion proteins for GM1-ganglioside was confirmed by GM1-ELISA. Mice immunized with a CTB fusion antigen showed higher antigen-specific IgG antibody titers than mice immunized with antigen alone [16,27]. CTB has been used as a carrier and an adjuvant for fused antigen protein in plant-based vaccines

B

kDa 170 130 95 72 55 43 26

M

PE

N

6

7

8

9

PC N

6

7

8

9

17

17

C

The amounts of CTB–EIII fusion protein expressed in the transgenic leaf tissues were measured by comparison with known CTB protein amounts using GM1-ELISA and expressed as a percentage of total soluble protein (TSP) extracted from transgenic plants (% CTB–EIII). The amount of CTB–EIII fusion protein was found to be 0.019–0.0053% of TSP (Fig. 5B). The GM1-ELISA results also showed that the plant-expressed CTB–EIII fusion protein has biological activities for its receptors, GM1-gangliosides (Fig. 5A).

11

M

PC N

6

7

8

9

DkDa M 170 130 95 72 55 43

11

26 17

Fig. 4. Immunoblot detection of the CTB–EIII fusion protein in transgenic plants. The leaf tissue protein extracts from transgenic plants were analyzed for CTB–EIII fusion protein expression with either anti-dengue virus monoclonal antibody (A and B) or anti-CTB polyclonal antibody (C and D) as the primary antibody under the boiled (A and C) or unboiled conditions (B and D). Lane M is Prestained Protein Ladder (Fermentas, Glen Burnie, MD); lane PE is EIII protein purified from E. coli as control for anti-dengue virus monoclonal antibody; lane PC is bacterial CTB protein as a control for anti-CTB antibody; lane N is protein extract of non-transgenic leaf tissues (100 lg protein per lane) as a negative control; lanes 6–9 are protein extracts of transgenic plant leaf tissues (100 lg protein per lane). Arrows indicate the bands for the monomer and assembly of CTB–EIII fusion proteins.

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OD(405nm)

A 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

WT #6 #7 #8 #9

2

B

0

2

-1

2

-2

-3

2 Serial dilution

2

-4

2

-5

0.02

% of TSP

0.016 0.012 0.008 0.004 0 #6

#7

#8

#9

Transgenic plant Fig. 5. GM1-ELISA quantification of the CTB–EIII fusion protein in transgenic plants. The protein extracts from transgenic plants were used to measure the expression level of CTB–EIII fusion protein bound to GM1-ganglioside coated on the wells of a microtiter plate, and primary antibody against CTB was added to detect the CTB–EIII fusion proteins bound to GM1-ganglioside. Samples #6–#9 are protein extracts of transgenic plants expressing CTB–EIII fusion proteins.

[28,29]. It is expected that the plant-produced CTB–EIII fusion proteins could be efficiently taken up by a mucosal immune system, resulting in increased immune responses against the dengue virus. The boiled protein immunoblot results showed two bands that were detected around 26 kDa. The lower band is expected to be CTB–EIII fusion protein monomer, and the upper band might contain signal peptides or might be glycosylated because CTB has glycosylation sites [30] but EIII has none. The two low bands (26 kDa) observed on the immunoblot with anti-dengue virus monoclonal antibody were either slightly detected or not detected on the immunoblot with anti-CTB antibody. An explanation for these results is that the anti-CTB antibody was developed with purified bacterial CTB pentamer and may not efficiently detect CTB monomers. Although genomic DNA PCR analysis showed the bands of CTB– EIII fusion genes in all of the transgenic plants, two transgenic plants (#6, #7) showed high mRNA bands of CTB–EIII, whereas the rest showed low expression in Northern blot analysis. These different expression levels of CTB–EIII fusion gene among the transgenic plants are due to the different incorporation sites of the target gene in the chromosomes of individual plants, known as the ‘position effect’ [31,32]. More recent experiments reported that the transgene expression in transgenic plants was knocked down by RNA silencing. A viral-encoded suppressor of gene silencing prevented gene silencing and enhanced transient expression system in plants [33]. In our previous study, transgenic rice callus has been constructed to express LTB. Mice immunized with 20 mg lyophilized transgenic rice callus powder containing 1.7 lg of LTB elicited humoral immune responses, and immunized serum inhibited the

binding of bacterial LTB to its receptor [34]. Although this transgenic plant showed a lower expression level (0.019% of TSP) than the transgenic plants expressing LTB (0.12% of TSP), if the dose amount (300 mg of lyophilized tobacco sample per feeding) and feeding frequency were increased, it is expected that the plant-produced CTB–EIII fusion protein would elicit a significant immune response in mice and other animals by oral vaccination. Dengue virus is member of Flaviviridae family and has four closely related, antigenically distinct serotypes: dengue-1, dengue-2, dengue-3 and dengue-4 [35]. A secondary infection with a different serotype from a primary infection or vaccination often results in a more severe disease state, due to antibody dependent enhancement [36]. The new vaccine for controlling of dengue infections must affect all dengue serotypes. The tetravalent dengue vaccine using domain III of the four dengue virus serotypes was expressed in yeast, and mice immunized with the tetravalent protein showed the neutralizing activity against all four serotypes [37]. It is expected that the mixtures of transgenic plants expressing each E glycoprotein of four serotypes could effectively elicit immune responses for all dengue serotypes. In this work, we have constructed an edible tobacco plant (low or no content of nicotine) expressing detectable amounts of domain III of dengue virus E glycoprotein fused with the cholera toxin B subunit. This edible transgenic tobacco expressing CTB–EIII fusion protein could be used as a vaccine to effectively improve immune responses by increasing antigen uptake into intestinal mucosal immune systems and prevent the host from acquiring a dengue virus infection. The feasibility of using a plant-produced CTB–EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments. Acknowledgments This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea. References [1] D.J. Gubler, Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century, Trends Microbiol. 10 (2002) 100–103. [2] Y. Modis, S. Ogata, D. Clements, S.C. Harrison, A ligand-binding pocket in the dengue virus envelope glycoprotein, Proc. Natl. Acad. Sci. USA 100 (2003) 6986–6991. [3] S. Mukhopadhyay, R.J. Kuhn, M.G. Rossmann, A structural perspective of the flavivirus life cycle, Nat. Rev. Microbiol. 3 (2005) 13–22. [4] G.J. Chang, Molecular biology of dengue viruses, in: D.J. Gubler, G. Kuno (Eds.), Dengue and Dengue Hemorrhagic Fever, The University press, Cambridge, United Kingdom, 1997, pp. 175–198. [5] R.J. Hurrelbrink, P.C. McMinn, Molecular determinants of virulence: the structural and functional basis for flavivirus attenuation, Adv. Virus Res. 60 (2003) 1–42. [6] W.D. Crill, J.T. Roehrig, Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells, J. Virol. 75 (2001) 7769–7773. [7] H.S. Mason, J.M. Ball, J.J. Shi, X. Jiang, M.K. Estes, C.J. Arntzen, Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice, Proc. Natl. Acad. Sci. USA 93 (1996) 5335–5340. [8] Y. Thanavala, Y.F. Yang, P. Lyons, H.S. Mason, C.J. Arntzen, Immunogenicity of transgenic plant-derived hepatitis B surface antigen, Proc. Natl. Acad. Sci. USA 92 (1995) 3358–3361. [9] T.A. Haq, H.S. Mason, J.D. Clements, C.J. Arntzen, Oral immunization with a recombinant bacterial antigen produced in transgenic plants, Science 268 (1995) 714–716. [10] P.B. McGarvey, J. Hammond, M.M. Dienelt, D.C. Hooper, Z.F. Fu, B. Dietzschold, H. Koprowski, F.H. Michaels, Expression of the rabies virus glycoprotein in transgenic tomatoes, Biotechnology (NY) 13 (1995) 1484–1487. [11] T.G. Kim, W.H. Langridge, Synthesis of an HIV-1 Tat transduction domainrotavirus enterotoxin fusion protein in transgenic potato, Plant Cell Rep. 22 (2004) 382–387. [12] T.G. Kim, A. Gruber, W.H. Langridge, HIV-1 gp120 V3 cholera toxin B subunit fusion gene expression in transgenic potato, Protein Expr. Purif. 37 (2004) 196–202.

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[26]

[27]

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[32]

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