Research Article A Plant-Produced Antigen Elicits Potent ...downloads.hindawi.com/journals/bmri/2014/952865.pdf · Research Article A Plant-Produced Antigen Elicits Potent Immune

Research ArticleA Plant-Produced Antigen Elicits Potent Immune Responsesagainst West Nile Virus in Mice

Junyun He1 Li Peng1 Huafang Lai1 Jonathan Hurtado12

Jake Stahnke12 and Qiang Chen12

1 The Biodesign Institute Arizona State University 1001 S McAllister Avenue Tempe AZ 85287 USA2 School of Life Sciences Arizona State University Tempe AZ 85287 USA

Correspondence should be addressed to Qiang Chen shawnchenasuedu

Received 10 February 2014 Accepted 8 March 2014 Published 3 April 2014

Academic Editor Luca Santi

Copyright copy 2014 Junyun He et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate forWest NileVirus (WNV) Using various combinations of vector modules of a deconstructed viral vector expression system DIII was producedin three subcellular compartments in leaves ofNicotiana benthamiana by transient expression DIII expressed at much higher levelswhen targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol with accumulation level upto 73 120583g DIII per gram of leaf fresh weight within 4 days after infiltration Plant ER-derived DIII was soluble and readily purifiedto gt 95 homogeneity without the time-consuming process of denaturing and refolding Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizesa conformational epitope Furthermore subcutaneous immunization of mice with 5 and 25 120583g of purified DIII elicited a potentsystemic responseThis study provided the proof of principle for rapidly producing immunogenic vaccine candidates againstWNVin plants with low cost and scalability

1 Introduction

West Nile Virus (WNV) belongs to the Flavivirus genus ofthe Flaviviridae family It is a positive-stranded envelopedRNA virus that infects the central nervous system (CNS) ofhumans and animals Once a disease that was restricted toOldWorld countries it entered into the Western hemispherethrough New York City in 1999 and has now spread acrossthe United States (US) Canada the Caribbean region andLatin America [1] The outbreaks of WNV have becomemore frequent and severe in recent years with 2012 as thedeadliest yet with 286 fatalities in the US [1] WNV infectioncauses fever that can progress to life-threatening neurolog-ical diseases The most vulnerable human population fordeveloping encephalitis meningitis long-term morbidityand death includes the elderly and immunocompromisedindividuals [2] Recent studies also identified genetic factorsassociated with susceptibility to the disease [3 4] Currently

no vaccine or therapeutic agent has been approved for humanapplicationThe threat of globalWNVepidemics and the lackof effective treatment warrant the development of vaccinesand production platforms that can quickly bring them tomarket at low cost

TheWNVEnvelope (E) glycoproteinmediates viral bind-ing to cellular receptors and is essential for the subsequentmembrane fusion [5] It is also amajor target of host antibodyresponses [5] Studies have shown that WNV E shares athree-domain architecture with E proteins of dengue andtick-borne encephalitis viruses [6] The domain III (DIII) ofWNVE protein contains the cellular receptor-bindingmotifsand importantly the majority of the neutralizing epitopesthat induce strong host antibody responses andor protectiveimmunity are mapped to this domain [7] As a result DIIIhas been targeted as a WNV vaccine candidate [8] Insectcell and bacterial cultures have been explored to express theWNV DIII protein [9 10] However these culture systems

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 952865 10 pageshttpdxdoiorg1011552014952865

2 BioMed Research International

are challenged by their limited scalability for large-scaleprotein production Moreover DIII expression in bacterialcultures often leads to the formation of inclusion bodieswhich requires a cumbersome solubilization and refoldingprocess to yield a recombinant DIII protein that resembles itsnative structure [10]

Expression systems based on plants may provide solu-tions to overcome these challenges because they providehighly scalable production of recombinant proteins at lowcost and have a low risk of introducing adventitious human oranimal viruses or prions [11 12] Stable transgenic plants werefirst explored to produce subunit vaccine proteins Whilefeasible the low protein yield and the long time period arerequired for generating and selecting transgenic lines hindera broad application of this strategy [13] Recently transientexpression systems based on plant virus have been developedto address these challenges While the infectivity of plantviruses has been eliminated through viral ldquodeconstructionrdquothese vectors still retain the robustness of the original plantvirus in replication transcription or translation [14] Thusdeconstructed plant viral vectors promote high-level produc-tion of recombinant protein within 1 to 2 weeks of vectordelivery [14ndash16]TheMagnICONsystem is a popular exampleof these vectors based on in planta assembly of replication-competent tobacco mosaic virus (TMV) and potato virusX (PVX) genomes from separate provector cDNA modules[17 18] The 51015840 module carries the viral RNA dependentRNA polymerase and themovement protein (MP) and the 31015840module contains the transgene and the 31015840 untranslated region(UTR) A tumefaciens strains harboring the twomodules aremixed together and coinfiltrated into plant cells along witha third construct that produces a recombination integraseOnce expressed the integrase assembles the 51015840 and 31015840modulesinto a replication-competent TMV or PVX genome underthe control of a plant promoter [18 19] This assembledDNA construct is then transcribed and spliced to generate afunctional infective replicon Geminiviral expression systemis another example a DNA replicon system derived fromthe bean yellow dwarf virus (BeYDV) [20 21] Anotherinteresting example is an expression vector system thatis based on the 51015840 and 31015840-untranslated region of Cowpeamosaic virus (CPMV) RNA-2 This vector system does notrequire viral replication yet allows high-level accumulationof recombinant proteins in plants [22] Thus these planttransient expression systems combine the advantages ofspeed and flexibility of bacterial expression systems andthe post-translational protein modification capability andhigh-yield of mammalian cell cultures As a result of thisdevelopment a variety of protein vaccine candidates havebeen produced in plants [11 12 23ndash26] The immunogenicityof a plant-produced vaccine candidate against WNV has notbeen described

Here we described the rapid production of the WNVDIII in Nicotiana benthamiana plants using the TMV-basedvectors of theMagnICON systemWedemonstrated thatDIIIcan be expressed in three subcellular compartments of theplant cell including endoplasmic reticulum (ER) chloroplastand cytosol with the highest accumulation level in ER within4 days after infiltration Plant ER-derived DIII was soluble

and was readily purified to gt95 homogeneity Further anal-ysis revealed that plant-produced DIII was folded properly asit exhibited specific binding to a monoclonal antibody thatrecognizes a large conformational epitope on WNV DIIIThe immunogenicity of plant-derivedDIII was demonstratedin mice as subcutaneous immunization elicited a potentsystemic response

2 Results

21 Expression of WNV E DIII in ER Chloroplast and Cytosolof N benthamiana Leaves To demonstrate the feasibility ofusing plants to produce a candidate vaccine forWNV we firstdetermined what subcellular compartment was optimal forDIII accumulation Agrobacterium tumefaciens strain con-taining the 31015840 DIII construct module was codelivered into Nbenthamiana leaves alongwith the 51015840module and an integraseconstruct through agroinfiltration [27 28] Three different51015840 modules were specifically chosen to target DIII into ERchloroplast or the cytosol [24] Leaf necrosis was observed inthe infiltrated area 4 or 5 days post infiltration (dpi) in plantsfor all constructs with cytosol-targeted construct causing themost severe symptoms (data not shown) By 6 dpi necrosiswas too extensive to recover significant amounts of live tissuefrom the infiltrated leaf area As a result DIII expression wasexamined between 2 and 5 dpi by Western blotting For theconstruct targeted to accumulate DIII in ER a positive bandwith the predicted molecular weight for DIII (135 kDa) wasdetected on Western blot starting 3 dpi (Figure 1 Lanes 3ndash5)In contrast no positive band was detected for chloroplast orcytosol-targeted DIII construct even on 5 dpi (Figure 1 Lanes6 and 7) An E coli-produced DIII was used as a positivecontrol and as expected it was detected as a positive bandon the Western blot (Figure 1 Lane 8) The E coli-producedDIII appeared to be larger than that from plants (169 kDa)because it contained multiple polypeptide tags from thebacterial expression vector pET28a (EMD Milipore) Thelack of positive band in the negative control leaf samples(Figure 1 Lane 1) confirmed the specificity of the DIII bandThe expression of DIII was quantified by a sandwich ELISAusing two WNV specific antibodies (Figure 2) In leaves thatDIII was targeted to the cytosol or chloroplast the maximallevels of accumulation are below 116 120583g of DIII per gramof leaf fresh weight (LFW) or 001 of total soluble protein(TSP) confirming the result of Western blotting The ER-targeted DIII reached the highest level of production at 4 dpiwith an average accumulation of 73120583gg LFW or 063TSP approximately sim63 times more than that in cytosol orchloroplast (Figure 2)

22 Purification of DIII from N benthamiana Plants Theavailability of an efficient purification scheme is anotheressential component for plant-derived DIII to become aviable WNV vaccine candidate Since DIII was tagged witha His

6tag we developed a two-step purification proce-

dure based on acid precipitation and immobilized metalion affinity chromatography (IMAC) Samples from variouspurification steps were analyzed by Coomassie blue staining

BioMed Research International 3

1 2 3 4 5 6 7 8

-

-

-

-

-

-

-

-

Plant DIIIE coli DIII

(kD

a)

100

75

50

37

25

15

10

20

Figure 1 Western blot analysis of DIII expression in N benthami-ana DIII was extracted from N benthamiana leaves and separatedon 15 SDS-PAGE gels and blotted onto PVDF membranes MAbhE16 and a goat anti-human kappa chain antibody were incubatedwith the membranes sequentially to detect DIII Lane 1 proteinsample extracted from uninfiltrated leaves as a negative controlLanes 2ndash5 sample collected 2 3 4 and 5 dpi from leaves infiltratedwith ER-targetedDIII construct Lane 6 sample collected 5 dpi fromleaves infiltrated with chloroplast-targeted DIII construct Lane 7sample collected 5 dpi from cytosol-targeted DIII leaves Lane 8 Ecoli-produced DIII as a positive control

Chloroplast Cytosol ER0

5

10

15

20

25

3060

70

80

Day 2Day 3

Day 4Day 5

(120583g

DII

Ig L

FW)

Figure 2 Temporal expression patterns of DIII in chloroplastcytosol and ER Total protein from plant leaves infiltrated withchloroplast cytosol or ER-targeted DIII construct was extractedon 2ndash5 dpi and analyzed by an ELISA with mAb hE16 whichrecognizes a conformational epitope on DIII and a polyclonal anti-DIII antibody Mean plusmn SD of samples from several independentexperiments are presented

analysis of SDS-PAGE (Figure 3(a)) andWestern blot analysis(Figure 3(b)) Interestingly DIII was efficiently extracted inthe soluble protein fraction of plant leaves (Figures 3(a) and3(b) Lane 2) in contrast to the insoluble inclusion body inE coli [29] Precipitation with low pH (50) removed a largeproportion of endogenous plant proteins including the mostabundant host protein the photosynthetic enzyme RuBisCo(Figure 3(a) Lane 1) while leaving DIII in the supernatant(Figure 3(b) Lanes 1 and 3) The pH adjustment from pH

50 to pH 80 which was required for the binding of DIIIto the nickel (Ni) IMAC resin did not cause any significantchange in protein profile (Figures 3(a) and 3(b) Lane 4) NiIMAC efficiently removed the remaining plant host proteins(Figure 3(a) Lanes 5 and 6) and enriched DIII to greater than95 purity (Figures 3(a) and 3(b) Lane 7) A faint reactiveband was detectable in fractions of total soluble protein pH50 precipitation and IMAC flow through (Figure 3(b) Lanes2ndash5) suggesting a minor DIII degradation product Onlythe intact DIII band with the predicted molecular mass wasdetected in the purified DIII fraction Approximately 32mgof purified DIII was obtained from 100 g LFW These resultsdemonstrated that not only can DIII be rapidly produced inplants but also isolated and purified to high homogeneityusing a scalable purification method

23 Plant-Derived DIII Is Specifically Recognized by a Neu-tralizing Monoclonal Antibody against WNV DIII To estab-lish a similarity of structural and immunological proper-ties between plant-produced and the native viral DIII weexamined the binding of plant-derived DIII to a monoclonalantibody (mAb) hE16 generated against WNV E Our pre-vious studies have shown that hE16 not only had potentneutralizing activity but it also effectively protected micefrom a lethal infection of WNV in both prophylactic andpostexposure models [30 31] Since hE16 binds a confor-mational epitope that consists of 4 discontinuous secondarystructural elements of the nativeWNVDIII [32] recognitionof a recombinant DIII by hE16 will be informative of itsproper folding ELISA results showed that plant-producedDIII demonstrated specific binding to hE16 produced inmammalian cell culture (Figure 4) DIII also specificallybound to a plant-derived hE16 that showed potent thera-peutic efficacy in mice (Figure 4) [30] Similar results wereobtainedwith the sandwich ELISAused for the quantificationof DIII in plant extracts (data not shown) These resultsindicate that plant-produced DIII was folded into a tertiarystructure that resembled the native viral DIII on the surfaceof WNV

24 Plant-Produced DIII Elicits Potent Systemic ImmuneResponse in Mice To evaluate the immunogenicity of plant-derived DIII BALBc mice were injected subcutaneouslywith four doses of DIII over an 8-week time period (ondays 0 21 42 and 63) Two dosages of 5120583g and 25 120583g ofDIII were tested with alum as adjuvant Mice were dividedinto 5 groups (119899 = 6 per group) with group 1 as thenegative control group injected with alum + saline (PBS)groups 2 and 3 with plant-derived DIII and groups 4 and5 with E coli-produced DIII as a control Individual serumDIII-specific antibody responses were measured by ELISAand Geometric mean titer (GMT) was calculated for eachgroup at various time points (Figure 5) Samples collectedfrom the control PBS group throughout the entire experimentcourse and preimmune sera for all groups taken prior to thefirst immunization (day 0) were negative for the presenceof anti-DIII IgG (titer lt 10) (Figure 5) All mice in groupsimmunized with 25 120583g of DIII responded after the first


1 2 3 4 5 6 7 8 9

10075

50

37

25

15

10

20 (kD

a)

(a)

1 2 3 4 5 6 7 8 9

1007550

37

25

15

10

20 (kD

a)

(b)

Figure 3 Purification of DIII from N benthamiana leaves DIII was purified from leaves infiltrated with ER-targeted DIII construct andanalyzed on 15 SDS-PAGE gels and either visualized with Coomassie blue stain (a) or transferred to a PVDF membranes followed byWestern analysis with hE16 (b) Lane 1 pH 50 precipitation pellet Lane 2 total extracted protein Lane 3 pH 50 supernatant Lane 4 NiIMAC loading Lane 5Ni IMACflow through Lane 6Ni IMACwash Lane 7Ni IMACelute Lane 8E coli-producedDIII Lane 9molecularweight marker

0

005

01

015

02

025

03

035

500 250 125 625 3125 15625

OD

450

mAb concentration (ngmL)

Control IgGhE16phE16m

Figure 4 Specific binding ELISA of hE16 to plant-derived DIIISerial dilutions of hE16 purified from mammalian or plant cellswere incubated in sample wells coated with plant-produced WNVDIII and detected with an HRP-conjugated anti-human gammaantibody A commercial generic human IgG was used as a negativecontrol Mean plusmn SD of samples from three independent experimentsis presented

administration while a response was only detectable afterthe third DIII delivery for mice immunized with the lowerdosage (5 120583g) This dose-dependent trend was also reflectedin the amplitude of the response throughout the various timepoints of the immunization For groups receiving DIII IgGtiters increased after each of the first three antigenrsquos deliveryand reached its peak at week 8 two weeks after the thirdimmunization Antibody titers at week 11 (two weeks after thefourth dose) were similar to those of week 8 for all groupsexcept the 5 120583g E coli-DIII group (Figure 5) This indicatedthat the last immunization did not significantly further boostthe DIII-specific antibody response especially in mice thatreceived the higher dosage of DIII Compared with E coli-produced DIII plant-derived DIII showed at least equivalent

100

1000

10000

100000

PBS

Serum collection time point

GM

T

25120583g E coli DIII25120583g plant DIII5120583g E coli DIII

5120583g plant DIII

0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811

(week)

Figure 5 Time course of DIII specific antibody responses in miceupon subcutaneous delivery of plant-derived DIII BALBC mice(119899 = 6 per group) were injected on weeks 0 3 6 and 9 with theindicated dosage of antigen Blood samples were collected on theindicated weeks and serum IgG was measured by ELISAThe 119910-axisshows the geometric means titers (GMT) and the error bars showthe 95 level of confidence of the mean

potency (119875 gt 05) in eliciting humoral response againstWNV(Figure 5)

In order to evaluate the Th type of response inducedby DIII antigen-specific IgG subtypes IgG1 and IgG2a wereevaluated by ELISA for samples collected at week 11 frommice that were immunized with 25 120583g of E coli- or plant-derived DIII As shown in Table 1 gt99 of DIII-specific IgGbelonged to the IgG1 subtype indicating an overwhelminglyTh2-type response stimulated by DIII antigen with alum asthe adjuvant

25 Characterization of Antiserum against Plant-Derived DIIIAntigen Antisera obtained at week 11 frommice of the 25 120583gplant-DIII groupwere examined in a binding assay with yeastthat displayed DIII in its native conformation on its surfaceFlow cytometric analysis demonstrated that antibodies in


Table 1 Anti-DIII IgG subtypes (IgG1 and IgG2a) of pooled serum samples

Group 3 Group 5Concentration (120583gmL) SEM Subtypetotal Concentration (120583gmL) SEM Subtypetotal

IgG1 50633 5800 995 48800 4808 998IgG2a 267 070 05 098 044 02Serum samples collected at week 11 were pooled for each indicated group and analyzed by ELISA for IgG1 and IgG2a antibody concentration Meanconcentration (120583gmL) of the IgG subtype and the standard error of the mean (SEM) from several independent measurements are presented Group 3 micereceived 25 120583g per dosage of plant-derived DIII Group 5 mice received 25 120583g per dosage of E coli-derived DIII

the anti-DIII sera displayed positive binding to DIII on thesurface of the yeast (Figure 6(a)) This indicated that anti-DIII sera contained antibodies that can recognize the nativeviral DIII protein Similar positive binding was observedfor positive control mAb hE16 (Figure 6(c)) but not forequivalent antisera frommice that were immunized with PBS(Figure 6(b)) To investigate if plant-DIII elicited antibodiesthat bind to the same epitope as the protective mAb hE16antisera were further analyzed with a competitive ELISAResults showed that preincubation of DIII with antisera fromimmunization of plant-derived DIII significantly inhibitedits binding to hE16 (Figure 7) No reduction in DIII bind-ing to hE16 was observed when it was preincubated withpreimmune serum This indicated that plant-produced DIIIinduced the production of anti-DIII IgGs that bind to thesameprotective epitope as hE16 or at least to epitopes adjacentto that oneThis suggested some of the antibodies in the anti-DIII sera were potentially neutralizing and protective

3 Discussion

WNV has caused continuous outbreaks in the US since itsintroduction in 1999 While the number of cases fluctuatedand even dropped from 2008 to 2011 the illusion that itstransmission would remain at a low rate quickly evaporatedas a largeWNV epidemic with high incidence of neurologicaldisease broke out in 2012 WNV was also reported to expandinto new geographic areas in Europe and other parts ofthe world Therefore the world may face larger and moresevereWNVoutbreaks associatedwith humanmorbidity andmortality In the absence of an effective treatment the needfor an effective WNV vaccine is more urgent than ever tohalt its expansion and to protect human populations that arevulnerable for developing neurological complications

Previous studies showed that immunization of DIIIproduced in E coli or insect cell cultures with CpGoligodeoxynucleotide adjuvant or in fusion with bacterialflagellin elicited WNV-neutralizing antibodies in mice andin certain instances protected mice from WNV infection[29 33 34] While encouraging these expression systemsmay not be able to provide the scale and robustness forWNVmanufacturing as the global threat ofWNV epidemicsdemands a scalable production platform that can quicklyproduce large quantities of vaccines at low cost MoreoverDIII is often recovered in the insoluble inclusion bodiesin bacterial cultures thus requiring a cumbersome solubi-lization and refolding process to yield DIII proteins thatresemble their native conformation [29] The high level of

endotoxins in E coli-based expression system also raisesbiosafety concerns and demands an expensive process ofpurification and validation for their removal to ensure thesafety of the final product [10]

Here we demonstrated that a transient plant expressionsystem provided a rapid production of WNV DIII in N ben-thamiana plants In contrast to forming insoluble aggregatesin E coli cultures DIII was produced as a soluble proteinin plant cells As a result it can be directly extracted andpurified to gt95 homogeneity by a simple and a scalablepurification scheme without the time-consuming process ofdenaturing and refolding This enhanced the likelihood ofproducing DIII protein that displays its native conformationIndeed plant-derived DIII appeared to fold properly as it wasspecifically recognized by hE16 a protective anti-WNVmAbthat binds a large conformational epitope spanning 4 distinctregions of DIII

Within the three subcellular compartments we testedDIII accumulated at much higher levels in ER than inchloroplast and cytosol The highest expression level wasachieved rapidly at 4 dpi with an average accumulation ofapproximately 73120583gg LFW This level is lower than that ofother pharmaceutical proteins we have produced with theMagnICON system [24 30 35]The induction of leaf necrosisby DIII may contribute to the lower expression level as itmay shorten the window for accumulation It is not clear ifthe observed leaf necrosis is caused by an inherent toxicityof DIII or by the employed overexpression system To ourbest knowledgeWNVDIII has not been produced in anotherplant species orwith another plant expression systemWe alsospeculate that the 73120583gg LFW was a conservative estimatefrom the early small-scale expression experiments as weroutinely obtained 30ndash70120583g of purified DIII from 1 g of Nbenthamiana leaves with 30ndash50 recovery rate in pilot scaleexperiments (Chen unpublished data) The underestimationcould be partially attributed to the fact that hE16 was usedas a capture antibody in the ELISA as it only detectedfully folded DIII that displayed the specific conformationalepitope Regardless this expression level of WNVDIII is stillthe highest compared with other plant-produced Flavivirusvaccine proteins including DIII of dengue virus expressedwith a TMV-based vector in tobacco [36] Since the pro-duction of DIII was performed under standard conditionsits accumulation level in plants can be further increased bygenetic and environmental optimizations

Production of DIII by using plant-expression systemsmay also overcome the challenge of limited scalability andcost issues associated with bacterial and insect cell culture


M1 M2Cou

nts

FL1-H

Anti-plant DIII serum

0

200

100 101 102 103 104

(a)

Anti-PBS serum

M1 M2Cou

nts

FL1-H

0

200

100 101 102 103 104

(b)

M1 M2

+control mAb hE16

Cou

nts

FL1-H

0

200

100 101 102 103 104

(c)

Figure 6 Binding of antibodies in anti-DlII serum to DIII displayed on yeast cell surface DIII displaying yeast cells were incubated withpooled sera collected on week 11 from mice injected with either 25 120583g of plant-produced DIII (a) or PBS (b) hE16 was used as a positivecontrol mAb (c) After incubation yeast cells were stained with an Alexa Fluor 488-conjugated goat anti-mouse (a and b) or goat anti-human(c) secondary antibody and processed by flow cytometry

systems The scalability of both upstream and downstreamoperations for transient plant expression systems has beenrecently demonstrated For example we used nontransgenicN benthamiana plants for DIII production in this studyAs a result the wild-type plant biomass can be cultivatedand produced in large scale with routine agriculture practicewithout the need to build extraordinarily expensive cellculture facilities [23 37ndash39] We previously demonstratedthat commercially produced lettuce could be used as aninexpensive and virtually unlimited source for pharmaceuti-cal protein production [40] Accordingly the agroinfiltrationprocess to deliver DIII DNA construct into plant cells hasbeen automated and can be operated in very large scales Forexample several metric tons of N benthamiana plants areregularly agroinfiltrated per hour by using a vacuum infil-tration procedure [27 28] For downstream processing ourextraction and purification procedure eliminated the hard-to-scale up steps of denaturing and refolding and allowedthe recovery of highly purified DIII with a simple two-stepprocedure of low pH precipitation and IMACThe scalabilityof the downstream process consisting of precipitation andaffinity chromatography has been extensively demonstratedby the pharmaceutical industry and by our studies with otherplant-produced biologics [30 41] This simple and scalabledownstream process from plants will also reduce the costsassociated with denaturing and refolding procedures and theoverall cost for DIII production The cost-saving benefit of

plant-expression systemswas also extensively documented byseveral case studies

Our results also indicated that plant-produced DIIIshowed at least equivalent potency in eliciting humoralresponse against WNV in mice as E coli-produced DIII Thedemonstration of antibodies in anti-plant DIII serum thatcompeted with hE16 for the same DIII epitope indicates theinduction of potentially protective antibodies against WNVIt is interesting that both plant- and E coli-produced DIIIevoked a Th2-type response with alum as the adjuvant Thisis in contrast to a previous report that E coli DIII with CpGadjuvant stimulated a Th1-biased response [33] This is nottotally unexpected as comparative studies with Flavivirusantigens showed that alum tends to induceTh2 type responsewhile CpG is likely to skew the response toward the Th1type [42] Since E coli-produced DIII was shown to beprotective in the mouse challenge model [29 33 34] theequivalent potency of plant-DIII in generating high IgG titersand the induction of hE16-like antibodies suggest that it ishighly likely that plant-DIII will induce protective immunitywhen a proper adjuvant is used Overall the rapidity of DIIIexpression the availability of a simple purification schemeand the low risk of contamination by human pathogen andendotoxin indicate that plants provide a robust and low-cost system for commercial production of subunit vaccinesagainst WNV and other flaviviruses


Buffer Anti-DIII Preimmune

0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)

Figure 7 Competitive ELISA of DIII binding by hE16 and antibod-ies in anti-DIII serum Plant-derivedDIII immobilized inmicrotiterplate wells was preincubated with 1 100 dilution of indicated serahE16 was then applied to sample wells to determine its binding toDIIIThe inhibition of serum preincubation on the subsequent hE16binding to DIII is presented as the of OD

450reduction by the

preincubation Mean plusmn SD of samples from three measurements ispresented

4 Experimental Procedures

41 Construction of DIII Expression Vectors The codingsequence of WNV E DIII (amino acid 296ndash415 GenbankAcc number AF196835) was synthesized with optimized Nbenthamiana codons [43] An 18 bp sequence coding for thehexa-histidine tag (His

6) was added to the 31015840 terminus of the

DIII gene and then cloned into the TMV-based expressionvector pIC11599 of the MagnICON system [30 43] TheMagnICON vectors were chosen because they have beendemonstrated to drive high-level accumulation of recombi-nant proteins in N benthamiana plants [30 31 38 41 43]

42 Expression of WNV E DIII in N benthamiana LeavesPlant expression vectors were transformed into A tume-faciens GV3101 by electroporation as previously described[24] N benthamiana plants were grown and agroinfiltratedor coagroinfiltrated with the GV3101 strain containing theDIII-His

631015840 module (pICH11599-DIII) along with one of

its respective 51015840 modules (pICH15579 for cytosol targetingpICH20999 for ER targeting or pICH20030 for chloro-plast targeting) and an integrase construct (pICH14011) asdescribed previously [27 28 30 38 41]

43 Extraction and Purification of DIII from N benthami-ana Leaves Agroinfiltrated N benthamiana leaves wereharvested 2ndash5 dpi for evaluating DIII expression Leaveswere harvested 4 dpi for other protein analysis Leaves werehomogenized in extraction buffer (100mMTris-HCl pH 80150mMNaCL 1mMPMSF tablet protease inhibitor cocktail(Sigma Germany) at 1mLg LFW) The extract was clarifiedby centrifugation at 18000timesg for 30min at 4∘C The pHof the clarified extract was adjusted to 50 and subjected to

centrifugation at 18000timesg for 30min at 4∘CThe supernatantwas recovered pH adjusted back to 80 and subjected toanother centrifugation The supernatant was then subjectedto Ni IMAC on a 4mL His Bind column in accordance withthemanufacturerrsquos instruction (Millipore USA)The purifiedWNV DIII was eluted with imidazole and the eluate wasdialyzed against PBS The purity of DIII was estimated byquantitating Coomassie blue-stained protein bands on SDS-PAGE using a densitometer as described previously [30]

44 SDS-PAGE Western Blot and ELISAs Samples contain-ing DIII were subjected to 15 SDS-PAGE under reducing(5 vv 120573-mercaptoethanol) conditions Gels were eitherstained with Coomassie blue or used to transfer proteinsonto PVDF membranes (Millipore USA) Membranes werefirst incubated with MAb hE16 [30] and then subsequentlywith a goat anti-human kappa antibody conjugated withhorseradish peroxidase (HRP) (Southern Biotech) Specificbindings were detected using an ldquoECL plusrdquo Western blotdetection system (Amersham Biosciences)

The expression of WNV DIII protein in leaves wasdetermined by a sandwich ELISA Ninety-six well ELISAmicrotiter plates (Corning Incorporated USA) were coatedat 1 120583gmL hE16 mAb in coating buffer (100mM Na

2CO3

pH 96) overnight at 4∘C After washing three times withPBST (PBS containing 01 Tween-20) plates were blockedwith blocking buffer (PBS containing 5milk) and incubatedwith plant extracts Purified bacterial WNV DIII was usedas a positive control to generate the standard curve Extractsfrom uninfiltrated plants were used as a negative controlAfter washing the plate was incubated with a rabbit anti-WNV DIII polyclonal antibody [43] followed by an HRP-conjugated goat anti-rabbit IgG (Southern Biotech) Theplates were then developed with TMB substrate (KPL Inc)Values from negative control leaves were used as ldquoback-groundrdquo of the assay and were subtracted from the cor-responding values obtained from DIII construct-infiltratedleaves

The hE16 recognition ELISA was performed as describedpreviously [30] Briefly purified plant-DIII was immobilizedon microtiter plates After incubation with hE16 purifiedfrom mammalian cells or from plants an HRP-conjugatedgoat anti-human-gamma HC antibody (Southern Biotech)was used to detect bound antibodies A generic human IgG(Southern Biotech) was used as a negative control

The titer of DIII-specific IgG in mouse serum was alsodetermined by an ELISA Microtiter plates were coated withplant- or E coli-derived DIII blocked with PBS with 1bovine serum albumin (BSA) and incubated with a serialdilution of serum After washing with PBST the plates wereincubated with an HRP-conjugated goat anti-mouse IgG(H+L) (Southern Biotech) After further washingwith PBSTthe plates were developed with TMB substrate (KPL Inc)Geometric mean titer (GMT) was calculated for each groupat various time points and was used to express the titer of theDIII specific IgG

The ELISA for determining the IgG1 and IgG2a subtypeswere performed also on plates coated with plant- or E coli-derived DIII as described above Serial dilutions of serum


were applied to sample wells and incubated for 2 hr at 37∘CAfter washing with PBST the plates were incubated with anHRP-conjugated goat anti-mouse IgG1 (Santa Cruz Biotech)or anti-mouse IgG2a (Southern Biotech) In parallel variousdilutions of mouse IgG1 and IgG2a (Southern Biotech) werecoated on the same set of plates for generating standardcurves The plates were developed with TMB substrate (KPLInc)

A competitive ELISAwas also performed on plates coatedwith DIII purified from plants After blocking plates werepreincubated with serial dilutions of serum from pooledpreimmune serum (Group 3) or pooled serum collectedat week 11 (Groups 1 and 3) After thorough washingwith PBST plates were incubated with hE16 subsequentlyan HRP-conjugated goat anti-human-gamma HC antibody(Southern Biotech) and developed with TMB substrate (KPLInc) The inhibition of hE16 binding to DIII by preincu-bation of sera was calculated by (Binding

(no pre-incubation) minus

Binding(pre-incubation with serum)) Binding(no pre-incubation)

All ELISA measurements were repeated at least threetimes with each sample in triplicate

45 DIII Expression in E coli and Yeast Surface DisplayThe synthesized DIII coding sequence was cloned into thepET28a bacterial expression plasmid (EMD Milipore) withEcoRI and HindIII sites DIII was expressed in E coli andpurified using an oxidative refolding protocol as describedpreviously [44] Refolded DIII protein was further purifiedwith aNiHis Bind IMACas described for plant-derivedDIII

Yeast expressing WNV DIII was generated and stainedwith mAbs as described previously [30] Briefly yeast cellswere first grown to log phase and subsequently induced forDIII expression by an additional 24 h culture in tryptophan-free media containing 2 galactose The yeast cells werethen incubated with pooled mice serum collected in week11 from the DIII immunization experiments or hE16 mAbas a positive control [30] Serum from the saline mock-immunized mice was used as a negative control The yeastcells were stained with a goat anti-mouse or goat anti-human secondary antibody conjugated with Alexa Fluor 488(Invitrogen) Subsequently the yeast cells were analyzed on aBD FACSCalibur flow cytometer (Franklin Lakes)

46 Mouse Immunization All animal work was approved bythe institutional animal care and use committee Five-weekold female BALBC mice were divided into 5 groups (119899 = 6per group) Group 1 received saline buffer (PBS) with alum asmock immunized control Groups 2 and 3 received 5120583g and25 120583g of plant-derived DIII per dosage respectively Groups4 and 5 received 5 120583g and 25 120583g of E coli-produced DIIIper dosage as controls On day 0 each mouse was injectedsubcutaneously with 100 120583L of material containing saline(Group 1) 5 120583g (Groups 2 and 4) or 25 120583g (Groups 3 and 5)purified DIII protein in PBS with alum as adjuvant (SigmaDIII Protein solution alum volume ratio = 1 1) Mice wereboosted three times (on days 21 42 and 63) with the samedosage and immune protocol as in the 1st immunizationBlood samples were collected from the retroorbital vein on

day 0 before the immunization (pre-immune sample) and ondays 14 (2 week) 35 (5 week) 56 (8 week) and 77 (11 week)after the 1st immunization Serum was stored at minus80∘C untilusage

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank J Kilbourne for her excellent technicalassistance in animal studiesThe authors also thank J Casper-meyer for the critical reading of the paper This work wassupported by a NIAID Grants nos U01 AI075549 and R21AI101329 to Q Chen

References

[1] L R Petersen A C Brault and R S Nasci ldquoWest Nilevirus review of the literaturerdquo Journal of the American MedicalAssociation vol 310 no 3 pp 308ndash315 2013

[2] A V Bode J J Sejvar W J Pape G L Campbell and A AMarfin ldquoWest Nile Virus disease a descriptive study of 228patients hospitalized in a 4-county region of Colorado in 2003rdquoClinical Infectious Diseases vol 42 no 9 pp 1234ndash1240 2006

[3] M S Diamond and R S Klein ldquoA genetic basis for humansusceptibility to West Nile virusrdquo Trends in Microbiology vol14 no 7 pp 287ndash289 2006

[4] J K Lim C Y Louie C Glaser et al ldquoGenetic deficiency ofchemokine receptor CCR5 is a strong risk factor for symp-tomatic West Nile virus infection a meta-analysis of 4 cohortsin the US epidemicrdquoThe Journal of Infectious Diseases vol 197no 2 pp 262ndash265 2008

[5] W D Crill and G-J J Chang ldquoLocalization and characteriza-tion of flavivirus envelope glycoprotein cross-reactive epitopesrdquoJournal of Virology vol 78 no 24 pp 13975ndash13986 2004

[6] G E Nybakken C A Nelson B R Chen M S Diamondand D H Fremont ldquoCrystal structure of the West Nile virusenvelope glycoproteinrdquo Journal of Virology vol 80 no 23 pp11467ndash11474 2006

[7] T Oliphant M Engle G E Nybakken et al ldquoDevelopment ofa humanized monoclonal antibody with therapeutic potentialagainstWest Nile virusrdquoNature Medicine vol 11 no 5 pp 522ndash530 2005

[8] H E Prince andW R Hogrefe ldquoAssays for detecting West NileVirus antibodies in human serum plasma and cerebrospinalfluidrdquo Clinical and Applied Immunology Reviews vol 5 no 1pp 45ndash63 2005

[9] J Alonso-Padilla J Jimenez de Oya A-B Blazquez EEscribano-Romero J M Escribano and J-C Saiz ldquoRecom-binant West Nile virus envelope protein E and domain IIIexpressed in insect larvae protects mice against West Nilediseaserdquo Vaccine vol 29 no 9 pp 1830ndash1835 2011

[10] J J H Chu R Rajamanonmani J Li R BhuvananakanthamJ Lescar and M-L Ng ldquoInhibition of West Nile virus entry byusing a recombinant domain III from the envelope glycopro-teinrdquo Journal of General Virology vol 86 no 2 pp 405ndash4122005


[11] Q Chen ldquoExpression and manufacture of pharmaceuticalproteins in genetically engineered horticultural plantsrdquo inTransgenic Horticultural Crops Challenges and OpportunitiesmdashEssays by Experts BMou andR Scorza Eds pp 83ndash124 Tayloramp Francis Boca Raton Fla USA 2011

[12] Q Chen ldquoExpression and purification of pharmaceutical pro-teins in plantsrdquo Biological Engineering vol 1 no 4 pp 291ndash3212008

[13] H M Davies ldquoCommercialization of whole-plant systemsfor biomanufacturing of protein products evolution andprospectsrdquo Plant Biotechnology Journal vol 8 no 8 pp 845ndash861 2010

[14] C Lico Q Chen and L Santi ldquoViral vectors for production ofrecombinant proteins in plantsrdquo Journal of Cellular Physiologyvol 216 no 2 pp 366ndash377 2008

[15] T V Komarova S Baschieri M Donini C Marusic E Ben-venuto and Y L Dorokhov ldquoTransient expression systems forplant-derived biopharmaceuticalsrdquo Expert Review of Vaccinesvol 9 no 8 pp 859ndash876 2010

[16] M C Canizares L Nicholson and G P Lomonossoff ldquoUse ofviral vectors for vaccine production in plantsrdquo Immunology andCell Biology vol 83 no 3 pp 263ndash270 2005

[17] A Giritch S Marillonnet C Engler et al ldquoRapid high-yieldexpression of full-size IgG antibodies in plants coinfectedwith noncompeting viral vectrosrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no40 pp 14701ndash14706 2006

[18] S Marillonnet A Giritch M Gils R Kandzia V Klimyukand Y Gleba ldquoIn planta engineering of viral RNA repliconsefficient assembly by recombination of DNAmodules deliveredby Agrobacteriumrdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 18 pp 6852ndash6857 2004

[19] Y Gleba V Klimyuk and S Marillonnet ldquoMagnifectionmdashanew platform for expressing recombinant vaccines in plantsrdquoVaccine vol 23 no 17-18 pp 2042ndash2048 2005

[20] QChen JHeW Phoolcharoen andH SMason ldquoGeminiviralvectors based on bean yellow dwarf virus for production ofvaccine antigens and monoclonal antibodies in plantsrdquoHumanVaccines vol 7 no 3 pp 331ndash338 2011

[21] Z HuangW Phoolcharoen H Lai et al ldquoHigh-level rapid pro-duction of full-size monoclonal antibodies in plants by a single-vectorDNAreplicon systemrdquoBiotechnology andBioengineeringvol 106 no 1 pp 9ndash17 2010

[22] F Sainsbury E C Thuenemann and G P LomonossoffldquoPEAQ versatile expression vectors for easy and quick transientexpression of heterologous proteins in plantsrdquo Plant Biotechnol-ogy Journal vol 7 no 7 pp 682ndash693 2009

[23] Q Chen H Mason T Mor et al ldquoSubunit vaccines producedusing plant biotechnologyrdquo in New Generation Vaccines M MLevine Ed pp 306ndash315 Informa Healthcare USA Inc NewYork NY USA 4th edition 2009

[24] L Santi L Batchelor Z Huang et al ldquoAn efficient plant viralexpression system generating orally immunogenic Norwalkvirus-like particlesrdquoVaccine vol 26 no 15 pp 1846ndash1854 2008

[25] Z Huang Q Chen B Hjelm C Arntzen and H Mason ldquoADNA replicon system for rapid high-level production of virus-like particles in plantsrdquo Biotechnology and Bioengineering vol103 no 4 pp 706ndash714 2009

[26] M Bendandi S Marillonnet R Kandzia et al ldquoRapid high-yield production in plants of individualized idiotype vaccines

for non-Hodgkinrsquos lymphomardquo Annals of Oncology vol 21 no12 pp 2420ndash2427 2010

[27] K Leuzinger M Dent J Hurtado et al ldquoEfficient agroinfiltra-tion of plants for high-level transient expression of recombinantproteinsrdquo Journal of Visualized Experiments no 77 2013

[28] Q Chen H Lai J Hurtado et al ldquoAgroinfiltration as aneffective and scalable strategy of gene delivery for productionof pharmaceutical proteinsrdquo Advanced Techniques in Biology ampMedicine vol 1 no 1 p 9 2013

[29] B E Martina P Koraka P van den Doel G van AmerongenG F Rimmelzwaan and A D M E Osterhaus ldquoImmunizationwith West Nile virus envelope domain III protects mice againstlethal infection with homologous and heterologous virusrdquoVaccine vol 26 no 2 pp 153ndash157 2008

[30] H Lai M Engle A Fuchs et al ldquoMonoclonal antibodyproduced in plants efficiently treats West Nile virus infectionin micerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 6 pp 2419ndash2424 2010

[31] J He H Lai M Engle et al ldquoGeneration and analysis of novelplant-derived antibody-based therapeutic molecules againstWestNile virusrdquoPLoSONE vol 9 no 3 Article ID e93541 2014

[32] G E Nybakken T Oliphant S Johnson S Burke M SDiamond and D H Fremont ldquoStructural basis of West Nilevirus neutralization by a therapeutic antibodyrdquoNature vol 437no 7059 pp 764ndash769 2005

[33] J-H J Chu C-C S Chiang and M-L Ng ldquoImmunization offlavivirus West Nile recombinant envelope domain III proteininduced specific immune response and protection against WestNile virus infectionrdquo Journal of Immunology vol 178 no 5 pp2699ndash2705 2007

[34] J W Huleatt H G Foellmer D Hewitt et al ldquoA West NileVirus recombinant protein vaccine that coactivates innate andadaptive immunityrdquoThe Journal of Infectious Diseases vol 195no 11 pp 1607ndash1617 2007

[35] W Phoolcharoen S H Bhoo H Lai et al ldquoExpression of animmunogenic Ebola immune complex in Nicotiana benthami-anardquo Plant Biotechnology Journal vol 9 no 7 pp 807ndash816 2011

[36] W Saejung K Fujiyama T Takasaki et al ldquoProduction ofdengue 2 envelope domain III in plant using TMV-based vectorsystemrdquo Vaccine vol 25 no 36 pp 6646ndash6654 2007

[37] Q Chen ldquoTurning a new leafrdquo European BiopharmaceuticalReview vol 2 no 56 pp 64ndash68 2011

[38] Q Chen ldquoVirus-like particle vaccines for norovirus gastroen-teritisrdquo in Molecular Vaccines M Giese Ed pp 153ndash181Springer Vienna Austria 2013

[39] Q Chen and H Lai ldquoPlant-derived virus-like particles asvaccinesrdquo Human Vaccines amp Immunotherapeutics vol 9 no 1pp 26ndash49 2013

[40] H Lai J He M Engle M S Diamond and Q Chen ldquoRobustproduction of virus-like particles and monoclonal antibodieswith geminiviral replicon vectors in lettucerdquo Plant Biotechnol-ogy Journal vol 10 no 1 pp 95ndash104 2012

[41] H Lai and Q Chen ldquoBioprocessing of plant-derived virus-likeparticles of Norwalk virus capsid protein under current GoodManufacture Practice regulationsrdquoPlantCell Reports vol 31 no3 pp 573ndash584 2012

[42] S L Demento N Bonafe W Cui et al ldquoTLR9-targetedbiodegradable nanoparticles as immunization vectors protectagainst West Nile encephalitisrdquoThe Journal of Immunology vol185 no 5 pp 2989ndash2997 2010


[43] J He H Lai C Brock et al ldquoA novel system for rapid andcost-effective production of detection and diagnostic reagentsof West Nile virus in plantsrdquo Journal of Biomedicine andBiotechnology vol 2012 Article ID 106783 10 pages 2012

[44] T Oliphant G E Nybakken S K Austin et al ldquoInductionof epitope-specific neutralizing antibodies against West Nilevirusrdquo Journal of Virology vol 81 no 21 pp 11828ndash11839 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


are challenged by their limited scalability for large-scaleprotein production Moreover DIII expression in bacterialcultures often leads to the formation of inclusion bodieswhich requires a cumbersome solubilization and refoldingprocess to yield a recombinant DIII protein that resembles itsnative structure [10]

Expression systems based on plants may provide solu-tions to overcome these challenges because they providehighly scalable production of recombinant proteins at lowcost and have a low risk of introducing adventitious human oranimal viruses or prions [11 12] Stable transgenic plants werefirst explored to produce subunit vaccine proteins Whilefeasible the low protein yield and the long time period arerequired for generating and selecting transgenic lines hindera broad application of this strategy [13] Recently transientexpression systems based on plant virus have been developedto address these challenges While the infectivity of plantviruses has been eliminated through viral ldquodeconstructionrdquothese vectors still retain the robustness of the original plantvirus in replication transcription or translation [14] Thusdeconstructed plant viral vectors promote high-level produc-tion of recombinant protein within 1 to 2 weeks of vectordelivery [14ndash16]TheMagnICONsystem is a popular exampleof these vectors based on in planta assembly of replication-competent tobacco mosaic virus (TMV) and potato virusX (PVX) genomes from separate provector cDNA modules[17 18] The 51015840 module carries the viral RNA dependentRNA polymerase and themovement protein (MP) and the 31015840module contains the transgene and the 31015840 untranslated region(UTR) A tumefaciens strains harboring the twomodules aremixed together and coinfiltrated into plant cells along witha third construct that produces a recombination integraseOnce expressed the integrase assembles the 51015840 and 31015840modulesinto a replication-competent TMV or PVX genome underthe control of a plant promoter [18 19] This assembledDNA construct is then transcribed and spliced to generate afunctional infective replicon Geminiviral expression systemis another example a DNA replicon system derived fromthe bean yellow dwarf virus (BeYDV) [20 21] Anotherinteresting example is an expression vector system thatis based on the 51015840 and 31015840-untranslated region of Cowpeamosaic virus (CPMV) RNA-2 This vector system does notrequire viral replication yet allows high-level accumulationof recombinant proteins in plants [22] Thus these planttransient expression systems combine the advantages ofspeed and flexibility of bacterial expression systems andthe post-translational protein modification capability andhigh-yield of mammalian cell cultures As a result of thisdevelopment a variety of protein vaccine candidates havebeen produced in plants [11 12 23ndash26] The immunogenicityof a plant-produced vaccine candidate against WNV has notbeen described

Here we described the rapid production of the WNVDIII in Nicotiana benthamiana plants using the TMV-basedvectors of theMagnICON systemWedemonstrated thatDIIIcan be expressed in three subcellular compartments of theplant cell including endoplasmic reticulum (ER) chloroplastand cytosol with the highest accumulation level in ER within4 days after infiltration Plant ER-derived DIII was soluble

and was readily purified to gt95 homogeneity Further anal-ysis revealed that plant-produced DIII was folded properly asit exhibited specific binding to a monoclonal antibody thatrecognizes a large conformational epitope on WNV DIIIThe immunogenicity of plant-derivedDIII was demonstratedin mice as subcutaneous immunization elicited a potentsystemic response

2 Results

21 Expression of WNV E DIII in ER Chloroplast and Cytosolof N benthamiana Leaves To demonstrate the feasibility ofusing plants to produce a candidate vaccine forWNV we firstdetermined what subcellular compartment was optimal forDIII accumulation Agrobacterium tumefaciens strain con-taining the 31015840 DIII construct module was codelivered into Nbenthamiana leaves alongwith the 51015840module and an integraseconstruct through agroinfiltration [27 28] Three different51015840 modules were specifically chosen to target DIII into ERchloroplast or the cytosol [24] Leaf necrosis was observed inthe infiltrated area 4 or 5 days post infiltration (dpi) in plantsfor all constructs with cytosol-targeted construct causing themost severe symptoms (data not shown) By 6 dpi necrosiswas too extensive to recover significant amounts of live tissuefrom the infiltrated leaf area As a result DIII expression wasexamined between 2 and 5 dpi by Western blotting For theconstruct targeted to accumulate DIII in ER a positive bandwith the predicted molecular weight for DIII (135 kDa) wasdetected on Western blot starting 3 dpi (Figure 1 Lanes 3ndash5)In contrast no positive band was detected for chloroplast orcytosol-targeted DIII construct even on 5 dpi (Figure 1 Lanes6 and 7) An E coli-produced DIII was used as a positivecontrol and as expected it was detected as a positive bandon the Western blot (Figure 1 Lane 8) The E coli-producedDIII appeared to be larger than that from plants (169 kDa)because it contained multiple polypeptide tags from thebacterial expression vector pET28a (EMD Milipore) Thelack of positive band in the negative control leaf samples(Figure 1 Lane 1) confirmed the specificity of the DIII bandThe expression of DIII was quantified by a sandwich ELISAusing two WNV specific antibodies (Figure 2) In leaves thatDIII was targeted to the cytosol or chloroplast the maximallevels of accumulation are below 116 120583g of DIII per gramof leaf fresh weight (LFW) or 001 of total soluble protein(TSP) confirming the result of Western blotting The ER-targeted DIII reached the highest level of production at 4 dpiwith an average accumulation of 73120583gg LFW or 063TSP approximately sim63 times more than that in cytosol orchloroplast (Figure 2)

22 Purification of DIII from N benthamiana Plants Theavailability of an efficient purification scheme is anotheressential component for plant-derived DIII to become aviable WNV vaccine candidate Since DIII was tagged witha His

6tag we developed a two-step purification proce-

dure based on acid precipitation and immobilized metalion affinity chromatography (IMAC) Samples from variouspurification steps were analyzed by Coomassie blue staining


1 2 3 4 5 6 7 8

-

-

-

-

-

-

-

-


(kD

a)

100

75

50

37

25

15

10

20



5

10

15

20

25

3060

70

80

Day 2Day 3

Day 4Day 5

(120583g

DII

Ig L

FW)







1 2 3 4 5 6 7 8 9

10075

50

37

25

15

10

20 (kD

a)

(a)

1 2 3 4 5 6 7 8 9

1007550

37

25

15

10

20 (kD

a)

(b)


0

005

01

015

02

025

03

035

500 250 125 625 3125 15625

OD

450





100

1000

10000

100000

PBS


GM

T


5120583g plant DIII

0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811

(week)










3 Discussion








M1 M2Cou

nts

FL1-H


0

200

100 101 102 103 104

(a)

Anti-PBS serum

M1 M2Cou

nts

FL1-H

0

200

100 101 102 103 104

(b)

M1 M2

+control mAb hE16

Cou

nts

FL1-H

0

200

100 101 102 103 104

(c)







0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)


450reduction by the













2CO3

















Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8

-

-

-

-

-

-

-

-


(kD

a)

100

75

50

37

25

15

10

20



5

10

15

20

25

3060

70

80

Day 2Day 3

Day 4Day 5

(120583g

DII

Ig L

FW)







1 2 3 4 5 6 7 8 9

10075

50

37

25

15

10

20 (kD

a)

(a)

1 2 3 4 5 6 7 8 9

1007550

37

25

15

10

20 (kD

a)

(b)


0

005

01

015

02

025

03

035

500 250 125 625 3125 15625

OD

450





100

1000

10000

100000

PBS


GM

T


5120583g plant DIII

0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811

(week)










3 Discussion








M1 M2Cou

nts

FL1-H


0

200

100 101 102 103 104

(a)

Anti-PBS serum

M1 M2Cou

nts

FL1-H

0

200

100 101 102 103 104

(b)

M1 M2

+control mAb hE16

Cou

nts

FL1-H

0

200

100 101 102 103 104

(c)







0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)


450reduction by the













2CO3

















Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9

10075

50

37

25

15

10

20 (kD

a)

(a)

1 2 3 4 5 6 7 8 9

1007550

37

25

15

10

20 (kD

a)

(b)


0

005

01

015

02

025

03

035

500 250 125 625 3125 15625

OD

450





100

1000

10000

100000

PBS


GM

T


5120583g plant DIII

0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811 0 2 5 811

(week)










3 Discussion








M1 M2Cou

nts

FL1-H


0

200

100 101 102 103 104

(a)

Anti-PBS serum

M1 M2Cou

nts

FL1-H

0

200

100 101 102 103 104

(b)

M1 M2

+control mAb hE16

Cou

nts

FL1-H

0

200

100 101 102 103 104

(c)







0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)


450reduction by the













2CO3

















Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Discussion








M1 M2Cou

nts

FL1-H


0

200

100 101 102 103 104

(a)

Anti-PBS serum

M1 M2Cou

nts

FL1-H

0

200

100 101 102 103 104

(b)

M1 M2

+control mAb hE16

Cou

nts

FL1-H

0

200

100 101 102 103 104

(c)







0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)


450reduction by the













2CO3

















Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M1 M2Cou

nts

FL1-H


0

200

100 101 102 103 104

(a)

Anti-PBS serum

M1 M2Cou

nts

FL1-H

0

200

100 101 102 103 104

(b)

M1 M2

+control mAb hE16

Cou

nts

FL1-H

0

200

100 101 102 103 104

(c)







0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)


450reduction by the













2CO3

















Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

10

20

30

40

Serum sample

minus10

Inhi

bitio

n (

)


450reduction by the













2CO3

















Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Acknowledgments


References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article A Plant-Produced Antigen Elicits Potent ...downloads.hindawi.com/journals/bmri/2014/952865.pdf · Research Article A Plant-Produced Antigen Elicits Potent Immune