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Productionofdengue2envelopedomainIIIinplantusingTMV-basedvectorsystem.Vaccine

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Vaccine 25 (2007) 6646–6654

Production of dengue 2 envelope domain III in plantusing TMV-based vector system

Wanida Saejung a, Kazuhito Fujiyama a,∗, Tomohiko Takasaki b, Mikako Ito b,Koichi Hori c, Prida Malasit d, Yuichiro Watanabe c, Ichiro Kurane b, Tatsuji Seki a

a The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japanb Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku,

Tokyo 162-8640, Japanc Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo,

Komaba 2-8-1, Meguro, Tokyo 153-8902, Japand Medical Molecular Biology Unit, Office for Research and Development, Adulyadejvikrom Building,Faculty of Medicine, Siriraj Hospital, Mahidol University, Prannok Road, Bangkok 10700, Thailand

Received 26 October 2006; received in revised form 7 April 2007; accepted 10 June 2007Available online 30 July 2007

bstract

The envelope protein of dengue virus is the major protein involved in host cell receptor binding for viral entry and induction of immunity. Aene fragment encoding domain III of the dengue 2 envelope protein (D2EIII, amino acids 298–400) was successfully expressed in Nicotinanaenthamiana plant using a tobacco mosaic virus (TMV)-based transient expression system. The N-terminal 5′ untranslated region-omegaequence located upstream of D2EIII increased protein production in infected plant tissues. The recombinant protein was reactive with

nti-D2EIII polyclonal and anti-His tag antibodies. The intramuscular immunization of mice with D2EIII induced the production of thenti-dengue virus antibody. The induced antibody demonstrated neutralizing activity against dengue type 2 virus. The result indicates that theMV expression system produces the dengue virus antigen in plant, which possesses appropriate antigenicity and immunogenicity.2007 Elsevier Ltd. All rights reserved.

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eywords: Dengue virus serotype 2 envelope domain III protein; Neutraliz

. Introduction

Recently, increasing knowledge in plant molecular biol-gy has served as an essential tool for developing plantxpression systems that are suitable for biomass produc-ion of therapeutic proteins in plants. Plants offer severaldvantages such as the production of foreign antigens free ofontamination by animal pathogens, relative ease of geneticanipulation, and economical production. Furthermore, they

osses a eukaryotic protein modification machinery, allow-

ng subcellular targeting, proper folding and post-translation

odification [1]. Among various plant viral vectors tested toate, a tobacco mosaic virus (TMV)-based expression vec-

∗ Corresponding author. Tel.: +81 6 6879 7238; fax: +81 6 6879 7454.E-mail address: fujiyama@icb.osaka-u.ac.jp (K. Fujiyama).

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body; Tobacco mosaic virus

or provides a promising approach to expressing biologicallyctive recombinant proteins in a short time, and it potentiallyircumvents the time constraints in the case of using stableransgenic plant expression systems [2]. Various human andnimal therapeutic proteins were successfully expressed inecombinant TMV via read-through fusions to a coat proteinCP) [3,4], direct fusion to a CP [5,6] and insertion underhe control of CP subgenomic promoters [7–12]. The firstwo approaches could express only short peptides of about–25 amino acids (aa) [13], whereas the newly developedxpression system in the third approach could produce largerecombinant proteins of up to 130 kDa such as the human

apillomavirus type 16 L1 protein [10].

Dengue virus is a mosquito-borne human pathogen andurrently the most important flavivirus causing human dis-ase in the tropical and subtropical regions of the world,

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ccounting for up to 100 million patients per year [14]. Fourntigenically related serotypes of dengue virus (dengue 1, 2,and 4 viruses) induce a spectrum of diseases ranging fromsimple febrile illness, dengue fever, to a fatal dengue hem-rrhagic fever/dengue hemorrhagic shock syndrome [15,16].urrently, no vaccines against dengue are available. Various

trategies have been attempted to develop dengue vaccineandidates: attenuated, recombinant, subunit, chimeric andNA vaccines [15].Most of the recombinant DNA-based strategies for vac-

ine development have focused on the envelope (E) proteinf dengue virus. The E glycoprotein (495 aa) is the majortructural protein exposed on the surface of a mature dengueirion. It has a major role in host cell attachment and entry.n addition, the E glycoprotein is the primary antigen thatnduces protective immunity; hence, the major antigen forirus neutralization [17]. The E glycoprotein consists of threetructurally distinct domains: I, II and III [18]. This E glyco-rotein has been produced in several heterologous expressionystems such as Escherichia coli [19], Pichia pastoris [20,21]nd baculovirus [22], with appreciable yields of the anti-enic and immunogenic [22] dengue 2 E protein. However,he expression level of the full length or ectodomain of theengue 2 E protein is low or the expressed protein is easilyegraded [19]. Thus, the use of a smaller fragment containinghe biologically critical domain of the E protein, domain III,as been explored. It was shown that chimeric proteins con-aining dengue 2 E domain III (D2EIII) are immunogenic,apable of inducing neutralizing antibodies in experimentalnimals [23].

In this study, a TocJ, TMV-based vector that is the hybridomato mosaic virus (ToMV) cDNA clone [12], was uti-ized to produce the D2EIII protein in tobacco plants. Welso modified the construct to increase the expression levely incorporating the TMV 5′ untranslated region and plantignal peptide to the D2EIII gene. The recombinant proteinas expressed, purified and used for immunizing mice. The

ecombinant D2EIII produced in tobacco plant induced theroduction of the anti-dengue virus antibody with neutral-zing activity. To the best of our knowledge, this is the firsteport of the production of the dengue envelope protein usinghe TMV-based expression system with an ability to induceeutralizing antibodies.

. Materials and methods

.1. Construction of expression vectors

The D2EIII protein was expressed in tobacco plant byenerating two expression vectors on the basis of TocJ12]. pSP/D2EIII/SEKDEL/His6 consisted of D2EIII with

he N-terminal plant signal peptide (SP), the C-terminalndoplasmic reticulum (ER) retention sequence for plants,er-Glu-Lys-Asp-Glu-Leu (SEKDEL) and the hexa-histidineHis6) tag. p�/SP/D2EIII/SEKDEL/His6 had the 5′ untrans-

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ated region (UTR)-omega (�) enhance element upstream ofP in addition to pSP/D2EIII/SEKDEL/His6.

To generate these constructs, the DNA fragment encoding2EIII (aa residues 298–400 of the dengue virus type 2

strain 16,881), GenBank accession no. M24447) and the′ flanking sequence was amplified by polymerase chaineaction (PCR) using pTrcHisA-D2EIII [24] as a templatend primer I: 5′-GGTACCTCATACTCTATGTGCACAG-3′ith KpnI (as underlined) at the 5′ end and primer II: 5′-AGCTCTTAATGATGATGATGATGATGAAGTTCATC-TTTTCAGATTGGCCGATAGAACTTC-3′ with SEKDEL

25], His6 tag and SacI (as underlined) at the 3′ end. Twondependent 5′ flanking sequences containing 19 aa ofisum vicilin SP [26] with and without 68 bp the 5′ UTR ofMV RNA, � enhance element [27] were amplified fromynthetic oligonucleotides containing SacI and KpnI at the′ and 3′ ends, respectively.

The PCR products of each fragment were cloned into theGEM-T Easy vector (Promega, USA) and the sequencesere confirmed. The fragment for D2EIII/SEKDEL/His6as digested with KpnI and SacI and ligated with

ither SP or �/SP digested with the same enzymes andubcloned into the TocJ vector, the TMV-based plantxpression vector [12,28] containing a T7 promoter athe SacI site, obtaining pSP/D2EIII/SEKDEL/His6 and�/SP/D2EIII/SEKDEL/His6 expression cassettes, respec-ively, as illustrated in Fig. 1B and C.

.2. In vitro transcription

The recombinant vector and TocJ (wild-type TMV) wereinearlized by digestion with MluI and then used as theNA template in in vitro transcription to generate full-lengthiral RNA using T7 RNA polymerase (Invitrogen, USA) asescribed previously [12]. The RNA transcripts of the recom-inant TMV are referred to as SP/D2EIII/SEKDEL/His6 and/SP/D2EIII/SEKDEL/His6.

.3. Plant inoculation and protein extraction

Six-week-old Nicotiana benthamiana plants wereechanically inoculated with infectious RNA transcripts on

he upper surface of mature leaves. Plants were maintainedn growth chambers (Sanyo, Japan) with 16 h of light peray at 25 ◦C until harvest. For secondary inoculation, theomogenate of leaves infected after the primary infectionas used as inoculum.Leaves with systemic infection after the inoculation

ere harvested at 8 days postinoculation (dpi) and sol-ble proteins were extracted by grinding the leaves inml of extraction buffer (phosphate-buffered saline (PBS),mM EDTA, and protease inhibitor cocktail tablets (Roche,

ermany)) per gram of leaves. After centrifugation of

ell debris at 12,000 × g, for 15 min at 4 ◦C, the super-atant was obtained to determine protein concentrationy Bradford protein assay (Nacalai Tesque, Japan) and

6648 W. Saejung et al. / Vaccine 25 (2007) 6646–6654

Fig. 1. Schematic diagram of genome of TocJ and insertion of dengue virus 2 envelope protein domain III (D2EIII) cassettes into TocJ. (A) The diagram showsthe location of the viral genes in the vector that encodes T7 (the T7 RNA promoter); replicase, RNA-dependent RNA polymerase (the protein required for TMVreplication); MP, movement protein; and CP, coat protein. The D2EIII gene cassette was inserted at the SacI site positioned downstream of the subgenomicRNA promoter sequence of the original CP gene. The locations of ToMV CP SgP, the subgenomic RNA promoter of tomato mosaic virus CP (original SgP);and TMGMV-J CP SgP, the subgenomic RNA promoter of tobacco mild green mosaic virus variant Japan CP (second SgP) are indicated by arrows. Insertionof D2EIII cassettes into TocJ at SacI site shown in (B) for pSP/D2EIII/SEKDEL/His6 and (C) for p�/SP/D2EIII/SEKDEL/His6. �, 5′ untranslated regionof TMV-� enhance element; SP, signal peptide of Pisum vicilin; D2EIII, dengue virus 2 envelope protein domain III; SEKDEL, plant ER retention peptide;H n of Pis1 rrowhep row indp f the si

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is6, hexa histidine tag. Nucleotide and protein sequences of fusion regio3–19 indicate aa residues of SP (13–15) and linker sequence (16–19). The aeptide after entering the lumen of the rough ER [25], whereas the solid arosition related to the cleavage site that corresponded to the (−3, −1) rule o

as subjected to SDS-PAGE, Western blot analysis andLISA.

.4. Western blot analysis of inoculated plant samples

Protein homogenates derived from the plants inoculatedith the wild-type or recombinant TMV virus were separatedy 15% SDS-PAGE and separated proteins were stained withoomassie Brilliant Blue R-250 or electroblotted onto an

mmobilon-PVDF membrane (Millipore, USA). Membranesere incubated with either a mouse anti-His tag monoclonal

ntibody (Mab) (Qiagen, USA) or a rat anti-D2EIII poly-lonal antibody. The polyclonal anti-D2EIII antibody wasaised in rats by immunization with a purified D2EIII pro-ein produced in E. coli [24]. The membranes were furtherncubated with antibodies conjugated with horseradish perox-dase (HRP), sheep anti-mouse IgG (Amersham Biosciences,K) or goat anti-rat IgG (Jackson ImmunoResearch Labora-

ories, USA). Specific bindings were detected using an ECLlus Western blot detection system (Amersham Biosciences).

.5. Purification of D2EIII protein

Plant samples were harvested at 8 dpi and extracted withinding buffer (20 mM sodium phosphate, 500 mM NaCl;H 7.4) at 2 ml/g of leaf materials. His6-tagged D2EIIIamples were purified by immobilized metal ion affinity chro-

atography (IMAC) using HiTrap Chelating HP (Amershamioscience), in accordance with the manufacturer’s instruc-

ions. The plant extracts were applied to a column containingMAC resin equilibrated with the binding buffer, and the col-

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um vicilin signal peptide and D2EIII gene in gene cassette (D). Numbersad indicates the putative proteolytical cleavage site for removal of the signalicates the actual proteolytical cleavage site and (−3, −1) indicates the aa

gnal peptide cleavage site.

mn was then washed with washing buffer containing 10 mMmidazole. D2EIII was eluted in a stepwise manner withxtraction buffer containing 20, 60, 100, 200 and 500 mMmidazole. Purified D2EIII protein in elution fractions wasoncentrated using Centriprep YM-3 (Amicon, Millipore,SA) and stored at −20 ◦C until further analysis.The purified D2EIII protein was electroblotted onto

mmobilon-PVDF membranes (Millipore, USA), stainedith Coomassie Brilliant Blue R-250, excised, and sequencedsing a Procise 494 cLC protein sequencer (Applied Biosys-ems, USA) performed at the Protein Research Institute,saka University, Japan.

.6. Detection of D2EIII in inoculated tobacco by ELISA

The D2EIII protein production levels in tobacco plantsnoculated with recombinant TMV viruses were determinedy indirect ELISA. Total soluble samples were extractedsing coating buffer (10 mM PBS pH 7.2). Ninety-six wellIA/RIA plates (Corning Incorporated, USA) were coatedt 100 �l/well with purified bacterial D2EIII protein plusninoculated plant extracts (positive control and standardurve) or uninoculated plant extracts alone (negative control),nd then incubated overnight at 4 ◦C. After washing threeimes with PBST (PBS containing 0.1% Tween 20), 150 �lf blocking buffer (PBS containing 1% BSA) was added toach well. The plates were incubated at 4 ◦C for 2 h, washednd incubated with the rat polyclonal anti-D2EIII antibody

100 �l /well, diluted 1:1000 in blocking buffer) at 37 ◦C forh. After washing, goat anti-rat IgG conjugated with alkalinehosphatase (Kirkegaard & Perry Laboratories, Inc., USA,00 �l/well, diluted 1:1000 in 50 mM Tris–HCl (pH 8.0) con-

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aining 0.5% BSA) was added and the plates were incubatedt 37 ◦C for 2 h. After washing four times, color developmentas analyzed using the p-nitrophenyl phosphate tablet set

Sigma, USA) after incubating at 37 ◦C for 1 h and stoppinghe reaction with 0.75 M sodium carbonate. The plates wereead at 405 nm using a Microplate reader model 680 (Bio-ad, USA) and proteins were quantified by comparison withnown amounts of the bacterial D2EIII–antibody complex.ll measurements were performed in triplicate.

.7. Mouse immunization

Groups of 5-week-old female C3H mice (Charles Riverapan, Inc., Japan) were injected intramuscularly at one thighith 10 �g in the first four immunizations on days 0, 21, 50

nd 64 and with 20 �g of the purified D2EIII protein in PBSmulsified with TiterMax Gold adjuvant (TiterMax USA,nc., USA) in later immunizations on days 80, 87, 101, 115nd 128. Blood samples were successively collected from theetro-orbital vein on days 0, 8, 15, 28, 71, 98, 108 and 128.

ice were bled on day 138. Serum samples from individualice were subjected to immunological assays.

.8. Detection of D2EIII mouse serum IgG by ELISA

The anti-dengue virus antibody level in mouse sera wasvaluated by dengue IgG indirect ELISA (PanBio, Australia)ccording to the manufacturer’s instructions except that theRP-conjugated anti-mouse IgG antibody (CAPPEL, USA)as used as the secondary antibody. ELISA was performedsing the following dilutions of antibodies: 1:100 dilution ofouse antisera and 1:500 dilution of HRP-conjugated anti-ouse IgG antibody.

.9. Plaque reduction neutralization test (PRNT)

To estimate the level of neutralizing antibody to dengueirus, the plaque reduction neutralization test (PRNT) waserformed. PRNT was based on a procedure previouslyescribed for Japanese encephalitis and West Nile virus [29]sing the dengue 2 virus, S16803 strain, and monkey kidneyells (LLC-MK2) instead. The neutralizing antibody titer wasefined as the highest serum dilution reduced the number ofnput virus plaques by 90% (PRNT90).

. Results

.1. Gene engineering and plasmid construct

The 309 bp fragment encoding D2EIII, the important anti-enic domain of dengue 2 virus with 5′ flanking sequences

or enhancing expression level, was cloned into a TocJ, TMV-ased viral vector. TocJ is the hybrid tomato mosaic virusToMV) cDNA clone modified for facile insertion of for-ign genes that retained the ability to multiply in plants [12].

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he foreign gene could be expressed under the control ofhe subgenomic promoter of the CP gene from the originaloMV, whereas the expression of the viral CP gene was reg-lated by a second heterologous subgenomic promoter and aP open reading frame derived from a closely related virus

tomato mild green mosaic virus, Japanese strain) as shownn Fig. 1A.

To facilitate translocation of D2EIII to the lumen of theR, the D2EIII fragment was inserted downstream of 15 aaf relatively short putative Pisum vicilin SP plus 4-aa (fromaa) linker sequence generated after fusion of vicilin SPith the �-glucoronidase (GUS) gene [25]. This SP togetherith the 8 aa fusion region was previously shown to direct

he targeting of the hybrid GUS protein into the rough ERf protoplasts, callus tissues and whole tobacco plants. The-terminus of the D2EIII cassette was fused to the His6

ag-coding sequence for detection and purification purposes.xpecting the increase in expression level of D2EIII inlants, the D2EIII cassettes with and without � element wereonstructed by adding the � element preceding the SP, to con-truct two D2EIII cassettes, p�/SP/D2EIII/SEKDEL/His6nd pSP/D2EIII/SEKDEL/His6, respectively (Fig. 1Cnd B). These constructs were used as the templatesor in vitro transcription to synthesize recombinantNA viruses. The RNA transcripts from these tem-lates were designated as �/SP/D2EIII/SEKDEL/His6 andP/D2EIII/SEKDEL/His6, respectively.

.2. Production of recombinant D2EIII in tobacco plants

The RNA transcripts prepared by in vitro transcriptionere utilized for inoculation to the upper surface of expand-

ng leaves of N. benthamiana. On day 7 after inoculation,he mosaic symptom was observed as mild leaf deformationn newly formed leaves and some leaf mottling in infectedeaves. Moreover, the systemic viral spread in inoculatedlants was confirmed by Western blot analysis.

At 8 dpi, the inoculated leaves of N. benthamiana werearvested and extracted for total soluble protein (TSP)nd then analyzed for the D2EIII protein by ELISA andestern blot analysis utilizing the rat anti-D2EIII antibody

nd anti-His tag Mab, respectively. The plants inoculatedith �/SP/D2EIII/SEKDEL/His6 showed a D2EIII proteinroduction level approximately 7.4 times more than thatf SP/D2EIII/SEKDEL/His6 (113.7 and 15.4 �g/mg TSP,espectively) as determined by ELISA. The high D2EIIIroduction level determined by Western blot analysis of/SP/D2EIII/SEKDEL/His6 (data not shown) was consis-

ent with the high D2EIII production level determined byLISA.

.3. Production and molecular characterization of

urified D2EIII protein

To prepare a purified D2EIII protein in large quantityor mouse immunization, transcribed RNA products of the

6650 W. Saejung et al. / Vaccine 25 (2007) 6646–6654

Fig. 2. Time course for accumulation of recombinant D2EIII protein inN. benthamiana plants infected with �/SP/D2EIII/SEKDEL/His6. Leavesinfected with �/SP/D2EIII/SEKDEL/His6 RNA sample (primary infection)were harvested at 5–9 days postinfection (dpi). The uninoculated plant sam-ple was used as the control sample. Protein samples were subjected to indirectELISA for analysis of recombinant D2EIII protein level in crude protein sam-ples. The recombinant D2EIII protein concentration in plant samples wasdcu

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Fig. 4. SDS-PAGE and Western blot analysis of purified D2EIII protein.Extracts from inoculated leaves were purified using IMAC (HiTrap chelat-ing column, Amersham Biopharmacia, UK). The purified D2EIII protein(0.25 �g) obtained from the fraction eluted with 100 mM imidazole wasdetected by Coomassie Brilliant Blue staining (lane 1), Western blotting withaLB

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etermined by extrapolation of the standard curve prepared from a knownoncentration of the E. coli recombinant D2EIII protein mixed with theninoculated plant protein.

ecombinant TMV construct �/SP/DIII/KDEL/H that pro-ided high D2EIII production level, were inoculated onto N.enthamiana plants. To obtain the highest D2EIII produc-ion in plants, the accumulation of the recombinant proteinas initially monitored over time after inoculation. ELISAsing the rat anti-D2EIII antibody showed an increase in the2EIII protein accumulation from 5 to 9 dpi (Fig. 2). How-

ver, the severe mosaic symptom led to the shrivelling ofnfected leaves after 8 dpi, making harvesting difficult. There-ore, we determined that 8 dpi was optimal for harvestingnfected plant materials.

The replication of wild type and recombinant TMV innoculated plants was confirmed by the appearance of theand corresponding to the 17.5-kDa TMV coat protein innoculated tissues (Fig. 3A, lanes 2–6). For large-scale pro-

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ig. 3. Analysis of the accumulation of recombinant D2EIII in N. benthamiana plataining (A) or Western blot analysis using anti-His tag Mab (B) and rat anti-D2Eninoculated leaves (lane 1), TocJ, wild-type TMV, infected leaves (lane 2), primar), in inoculated leaves (lanes 3 and 5) or upper uninoculated leaves (lanes 4 andioRad, USA). An asterisk indicates the possible degraded plant-produced D2EIII

nti-His tag Mab (lane 2) and rat anti-D2EIII polyclonal antibody (lane 3).ane M, protein molecular weight marker (Precision Plus Protein Standards,ioRad).

uction, homogenates of leaves showing systemic infectionfter the initial inoculation with RNA transcripts (primarynfection) were used to inoculate new plants (secondarynfection). The primarily infected-plant homogenates werenfectious, leading to production of D2EIII in leaves show-ng systemic infection after secondary infection in which theroduction level of the D2EIII protein in inoculated leavesas higher than that in leaves showing systemic infection asetermined by Western blot analysis using anti-His tag Mabnd rat anti-D2EIII polyclonal antibodies (Fig. 3B and C). Asxpected, no signal was detected for the uninoculated leavesr TocJ-inoculated leaves. These data revealed that the virusas stable and retained infectivity upon passage to the second

ound of infection, moreover, the systemic spread of recombi-ant TMV occurred both in primary and secondary infection.

urthermore, TocJ-based vectors not only have the capacity toxpress a foreign protein systemically but also have the abilityo retain the foreign sequences during passage from plants tolants. Recombinant D2EIII from secondarily infected leaves

nts infected with �/SP/D2EIII/SEKDEL/His6 by Coomassie Brilliant BlueIII polyclonal antibody (C). The plant extracts (10 �g) were isolated fromily infected leaves (lanes 3 and 4), secondarily infected leaves (lanes 5 and6). Lane M, molecular weight marker (Precision Plus Protein Standards,

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Table 1Induction of neutralizing antibody against dengue 2 virus in mice immunizedwith D2EIII protein produced from N. benthamiana plants

Mouse no. Neutralizing antibody titera

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ere used to purify under native conditions with IMAC. Theurified 13.8-kDa D2EIII protein was obtained using an elu-ion buffer containing 100 mM imidazole (Fig. 4, lane 1) andeacted to the anti-His tag and anti-D2EIII antibodies (Fig. 4,anes 2 and 3, respectively). From 200 g of infected leaves,.12 mg of the D2EIII protein was purified, which correspondo 0.28% of TSP.

To investigate whether D2EIII was produced and pro-essed properly in N. benthamiana plants, the N-terminala residues of purified D2EIII protein were determinedy protein sequencing. The directly determined N-terminala sequence was Tyr-Gly-Thr-Ser-Tyr-Ser-Met-Cys. The2EIII sequence is underlined. The cleavage site of signaleptidase in a previous report is predicted to be between Ser15nd Ala16 of SP (Fig. 1D) [25]. However, the SP was cleavedetween Gly18 and Tyr19; thus, the N-terminal aa sequencetarts from Tyr (after solid arrow in Fig. 1D). The signaleptidase cleavage site in this study did not occur at the puta-ive proteolytic cleavage site as expected [25]. A Ala-X-Glyequence in this study is more preferable than a Val-X-Serequence [25], which is consistent with the (−3, −1) rule30,31]. This result revealed that the SP was processed andleaved off, resulting in the mature protein synthesis in N.enthamiana plants.

.4. Induction of anti-D2EIII antibody in micemmunized with purified D2EIII protein

To evaluate whether the purified D2EIII protein wasmmunogenic, mice were immunized intramuscularly withhe purified D2EIII protein in TiterMax Gold adjuvant. Aengue IgG indirect ELISA kit containing dengue virusserotypes 1–4) coated on the well plate as the antigen

as used for testing the antibody induction directed againstengue virus in sera collected at different times during theourse of immunizations. The results shown in Fig. 5 demon-trated that all animals immunized with the plant-produced

ig. 5. Immune response of mice immunized by intramuscular injectionf purified D2EIII protein produced in N. benthamiana plants. Mouse seraollected from each mouse at different times during immunization were sub-ected to ELISA. Serum IgG antibodies specific to the dengue virus types 1–4ere determined by ELISA using a dengue IgG indirect ELISA kit (PanBio,ustralia). Each line represents the response of an individual mouse, mouseos. 1–5.

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a Represented as the maximum serum dilution yielding a 90% reductionn plaque number (PRNT90).

2EIII protein produced the anti-dengue virus antibody; inarticular higher levels were found in mouse nos. 3 and 4.n mouse nos. 3 and 4, antibody levels were low duringhe first period of immunization with 10 �g/dose and thenncrease rapidly after day 80 when the antigen level wasncreased to 20 �g/dose. These data revealed that the plant-roduced D2EIII antigen can induce dengue virus-specificntibodies in mice. In addition, sera collected 10 days afterhe last immunization have the ability to neutralize dengueirus serotype 2 as determined by PRNT90 (Table 1). Theseera were also examined for anti-D2EIII antibody produc-ion by Western blot analysis. The result showed that mouseera reacted with the D2EIII protein both in the purified formnd in plant extracts with strong reactivity at a dilution of:1000 (Fig. 6, lanes 3 and 4) and showed no reactivity tohe uninoculated leaf and wild-type TMV, TocJ-infected leafamples (Fig. 6, lanes 1 and 2). Sera from unimmunized micehowed no reactivity with D2EIII and plant extract, indicat-

ig. 6. Western blot analysis of infected plant sample using mouse anti-2EIII antibody. The protein samples were subjected to 15% SDS-PAGE

nd Western blot analysis with serum prepared from mouse No. 3 at dilu-ion 1:1000 and goat anti-mouse IgG-HRP conjugated at dilution 1:1000.ane 1, uninocluated leaves as negative control (10 �g); lane 2, wild-typeMV, TocJ inoculated leaves as control vector (10 �g); lane 3, purifiedlant-produced D2EIII protein (0.25 �g); lane 4, plant extract inoculatedith �/SP/DIII/KDEL/H (10 �g). The protein molecular weight marker

Precision Plus Protein Standards, BioRad) is shown in the left panel.

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. Discussion

We report here our successful utilization of a N.enthamiana-recombinant TMV-based viral vector as aioreactor system to produce the D2EIII protein. The D2EIIIrotein was assessed to determine whether it could inducehe production of the anti-dengue virus antibody and neu-ralizing antibody against dengue virus 2 when administeredntramuscularly to mice.

The immunization experiment showed that the plant-2EIII protein successfully induced the anti-dengue virus

ntibody as well as anti-D2EIII antibody production in miceFigs. 5 and 6, respectively). Moreover, the plant-D2EIII-nduced antisera have an efficient dengue virus type 2-specificeutralizing activity as shown by PRNT90 (Table 1). Its noteworthy that the higher the anti-dengue virus anti-ody level, the higher the neutralizing antibody activity wasbtained as in sera of immunized mouse nos. 3 and 4, show-ng the correlation of anti-dengue virus antibody level andeutralizing antibody activity of these immunized-mousentisera.

The level of induction of immunized-mouse antisera to theengue virus antigen was low (Fig. 5). The low level of anti-engue virus antibody induction might be due to the smallntigenic fragment resulting in insufficient induction of highntibody production, even though an adjuvant was used inhis experiment. No antibody induction was detected when

ice were immunized with plant-produced D2EIII proteinithout adjuvant (data not shown). Therefore, to insure that

mmunized mice could produce neutralizing antibody, miceere immunized with the plant-produced D2EIII protein withiterMax Gold adjuvant many times and for a long period.n the other hand, the immunological property of the recom-inant protein should be improved. One possible approach ishe fusion of cholera toxin B subunit (CTB) to the N-terminusf the D2EIII sequence. This CTB was shown to function asn effective carrier of vaccine antigens and used widely as andjuvant for plant-based oral vaccines [32]. Thus, the CTB-2EIII fusion protein could enhance the immune response in

nimals.The detection of neutralizing activity was an important

bservation. Protective immune responses to dengue virusnfection are not completely elucidated. However, it is gen-rally understood that type-specific neutralizing antibodieslay an essential role in the protection and recovery fromengue virus infection. Based on this consideration, vaccineevelopment should focus on high-level induction of neu-ralizing antibodies. The titer of the neutralizing antibodynduced by the plant-produced D2EIII protein was not high,ut quite promising. The neutralizing antibody titer can bencreased by changing the antigen dose, route of immuniza-ion, or using other adjuvants.

The TMV-based vectors were first developed and haveeen modified to improve the stability and increase thexpression level of foreign proteins by constructing hybridMV-based vectors that contained an extra closely related

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ubgenomic promoter element [33]. Some approaches wereeveloped to improve the movement and to widen host rangesy DNA shuffling of the movement protein [34]. The advan-ages of using TMV as an expression system are a high yieldf a foreign protein up to 10% of TSP in infected leaves [33], ahort time required for protein expression, and a low toxicity.ossible developmental problems associated with constitu-

ive expression of foreign proteins can be avoided becauseecombinant TMVs can be applied to mature plants [9]. Fur-hermore, the production of foreign protein in tobacco plantsing TMV transient expression system is safe for humannd environment. N. benthamiana is a member of non-foodrops where the foreign proteins are subsequently purified orrocessed to yield the desirable products. Therefore, it willot contaminate human food supply. However, we have to beware of the cases indicated by Scubbert et al. [35]. Addi-ionally, N. benthamiana plants should be grown under fullysolated green house facilities which can avoid human con-act or access by insects and birds, only treated waste wateras released from the facility.In this study, D2EIII was engineered by inserting

nto the TocJ, hybrid TMV vector [12]. We initiallynvestigated the expression levels of the D2EIII pro-ein in two different constructs, with and without the

enhance element in p�/SP/D2EIII/SEKDEL/His6 andSP/D2EIII/SEKDEL/His6, respectively. Both recombinantonstructs produced the same typical mosaic symptomsn inoculated tobacco plants within 1 week after infec-ion and spread systemically to the upper uninoculatedeaves similarly to ordinary TMV infection, facilitatingroduction of large amounts of the protein. How-ver, the D2EIII production level in the case of using/SP/D2EIII/SEKDEL/His6 was about 7.4-fold higher than

n that using SP/D2EIII/SEKDEL/His6. Apparently, thensertion of the � element in the recombinant TMV constructnhanced significantly the expression of D2EIII in tobaccolants. The enhancement of D2EIII expression in the pres-nce of the � element is consistent with the previous reportsf foreign gene expression in vivo and in vitro [36–39]. Inddition, to facilitate purification and immunological detec-ion of D2EIII produced in plant, the His6 tag was placedownstream of the SEKDEL sequence. The extension at the-terminus with SEKDEL improved accumulation of the for-ign protein [7]. Considering the plant D2EIII protein per ses an edible vaccine, the His6 tag is not necessary. Therefore,he construct SP/D2EIII/SEKDEL could be used instead foreveloping edible vaccines.

The RNA transcript �/SP/D2EIII/SEKDEL/His6 wassed in the experiments for preparing large quantities ofhe purified D2EIII protein for the immunization experi-

ent. The �/SP/D2EIII/SEKDEL/His6 was stable duringwo successive passages in N. benthamiana plants (Fig. 3).

nfected plant materials retain infectivity and antigenicityhen stored at −80 ◦C contributing to the ease of storingirus as inocula and antigens. The recombinant D2EIII pro-ein produced from �/SP/D2EIII/SEKDEL/His6-inoculated

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. benthamiana leaves was easily purified with single-stepMAC facilitated by introducing the His6 tag at the C-erminus. Nevertheless, plant-produced D2EIII protein wasulnerable to protease degradation. The degraded D2EIII pro-ein observed as a band in Western blot analysis when reactedith the rat anti-D2EIII polyclonal antibody but not with

he anti-His tag antibody (Fig. 3C and B). The IMAC resinound only with the intact D2EIII protein but not with theegraded D2EIII protein (Fig. 4) indicating that the degra-ation occurs at the C-terminus. This partial degradation of2EIII in plant extracts led to low recovery of plant-produced2EIII protein when purified with IMAC. Despite this par-

ial degradation of plant-produced D2EIII, we succeeded inecovering a sufficient amount of the protein for intramus-ular administration. In Western blot analysis using the ratnti-D2EIII antibody, the smearing of bands of 15–18 kDaas observed for samples obtained before and after IMACurification (Figs. 3, 4, 6), but not for E. coli-derived pro-eins [28]. The D2EIII protein does not have any possible-glycosylation sites (Asn-X-Ser/Thr). At the moment, weo not know the reason for these observations.

The yield of purified D2EIII protein was 0.28% TSP. Thexpression level of D2EIII in tobacco leaves in this study wasigher than those of hypervariable region 1, a potential neu-ralizing epitope of hepatitis C virus, in tobacco leaves usinghe same expression system as previously reported (0.04%SP or 0.005% fresh weight) [8], as well as malarial epi-

opes expressed on the surface of recombinant TMV using theead-through strategy that yields about 0.003% fresh weight40]. Thus, it is conceivable that the high expression levelf D2EIII was achieved by combination of several factorsncluding targeting of protein to ER by the signal peptidend 5′ UTR. Concerning the D2EIII content of total solu-le protein prepared from infected plants, though the partialegradation of D2EIII protein occurred, the proteins weremmunoreactive with anti-D2EIII antibody (Figs. 3 and 6).uring purification by immobilized metal ion affinity chro-atography (IMAC), only intact D2EIII was purified with

ield of 0.28% of total soluble protein.In conclusion, we have demonstrated that the TocJ, TMV-

ased vector, is suitable for the production of the recombinant2EIII protein in N. benthamiana plants. This recombi-ant protein retains its antigenicity and immunogenicitys well as induces neutralizing antibodies in vaccinatednimals. Thus, this is the first study demonstrating thathe TMV-based expression system can be used to produceecombinant D2EIII proteins that can effectively inducemmune responses in animal models. However, the immuno-ogical property of recombinant proteins should be furthermproved.

cknowledgement

P. Malasit is a Senior Research scholar supported by thehailand Research Fund.

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