Research Article Transgenic Carrot Expressing Fusion...

Research ArticleTransgenic Carrot Expressing Fusion Protein ComprisingM tuberculosis Antigens Induces Immune Response in Mice

Natalia V Permyakova1 Alla A Zagorskaya1 Pavel A Belavin1

Elena A Uvarova1 Olesya V Nosareva12 Andrey E Nesterov12 Anna A Novikovskaya1

Evgeniy L Zavrsquoyalov1 Mikhail P Moshkin1 and Elena V Deineko1

1 Institute of Cytology and Genetics Russian Academy of Sciences Prospect Lavrentieva 10 Novosibirsk 630090 Russia2State Research Center of Virology and Biotechnology Vector Koltsovo Novosibirsk 630559 Russia

Correspondence should be addressed to Natalia V Permyakova puhbionetnscru

Received 13 January 2015 Accepted 20 March 2015

Academic Editor Pengjun Shi

Copyright copy 2015 Natalia V Permyakova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Tuberculosis remains one of the major infectious diseases which continues to pose a major global health problem Transgenicplants may serve as bioreactors to produce heterologous proteins including antibodies antigens and hormones In the presentstudy a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10 esat6 and dIFN genewhich encode deltaferon a recombinant analog of the human 120574-interferon designed for expression in plant tissues This constructwas transferred to the carrot (Daucus carota L) genome by Agrobacterium-mediated transformationThis study demonstrates thatthe fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots The protein is able to induce bothhumoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection Itshould be emphasized that M tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral bloodmononuclear cells

1 Introduction

A highly efficient vaccine againstMycobacterium tuberculosisis in great demand because tuberculosis is currently amongthe diseases with the highest mortality rate Every minuteapproximately 20 people worldwide are infected with tuber-culosis and as a rule four of them die [1] Since the early1990s the mortality rate among tuberculosis patients hasbeen steadily increasing which is the result of increase inboth drug-resistantM tuberculosis strains and HIV infection[2] Animals are also susceptible to tuberculosis In particularbovine tuberculosis which is caused by the closely relatedbacterium M bovis is also hazardous to humans Approxi-mately 10 of tuberculosis patients were infected by animals[3]

Currently the BCG vaccine which has been in use forover 100 years is the major tool for immunoprophylaxis oftuberculosis Despite certain shortcomings of this vaccine[1] there is yet no alternative for immune prevention of this

diseaseThis fact emphasizes the need for the development ofnew tools for tuberculosis prevention Subunit vaccines thatinvolve recombinant antigens in combination with cytokinesare promising for this purpose [1 4] The most appropriatecandidate subunit vaccines are antigens that activate CD4+and CD8+ T cells and induce protective immunity Currentlythe M tuberculosis secreted and cell wall proteins (ESAT6Ag85B MTB72F and LipY) are regarded as the most promis-ing antigens [5ndash7]

Secreted proteins [8] are of special interest among theantigens with a protective activity ESAT6 (early secretedantigenic target 6 kDa) and CFP10 (culture filtrate protein10 kDa) [5 7] are the most valuable of these proteins CFP10is a chaperone for the ESAT6 protein and together theyform heterodimeric complexes CFP10 and ESAT6 are amongthe most important M tuberculosis proteins involved inpathogen-host interactions [9 10]High immunogenicity andspecificity of CFP10 and ESAT6 are confirmed by a high level

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 417565 11 pageshttpdxdoiorg1011552015417565

2 BioMed Research International

of 120574-interferon synthesis by peripheral blood mononuclearcells in response to contact with these antigens [11] Theseproteins are encoded in the M tuberculosis RD1 genomicregion Of note this region is deleted in theM bovis genomeCorrespondingly BCG administration fails to induce theimmune response to the antigens encoded in this regionincluding CFP10 and ESAT6

To increase the mucosal immunogenicity of proteins orpeptides otherwise imperceptible to the mucosal immunesystem recombinant antigensmay be fusedwith proteins thatdisplay adjuvant or immunomodulatory properties [12] Apromising protein possessing such properties is deltaferonThis is a recombinant analog of 120574-interferon with a deletionof ten C-terminal amino acids and mutations introduced atpositions 129 and 130 As a result deltaferon is synthesized ina native soluble form which displays an increased resistanceto blood proteases and a decreased antiviral activity [13 14]Therefore deltaferon is able to concurrently serve as anadjuvant and an immunomodulator efficiently boosting theimmune response to tuberculosis antigens [15]

Various systems are used for delivering antigens as sub-unit vaccines to human and animal organisms includingrecombinant viral vectors [16] recombinant bacterial systems[17] and lipoglycans conjugated with proteins [18] Oraladministration within plant cells [7 19 20] is among thepromising systems Proteins from plant cells expressingvaccine antigens are protected in the stomach from acids andenzymes but are released to the immune or blood circulatorysystem when plant cell walls are digested by microbes thatcolonize the gut Vaccine antigens bioencapsulated in plantcells provide both mucosal and systemic immunity andprotection against bacterial viral or protozoan pathogens ortoxin challenge upon oral deliveryThis new platform offers alow cost alternative to deliver different therapeutic proteinsto combat infection by eliminating inactivated pathogensexpensive purification cold storagetransportation and ster-ile injections We have used this particular approach whenconstructing transgenic carrot plants that produce M tuber-culosis antigens Because the tuberculosis pathogen is a res-piratory agent mucosal vaccination is well suited to initiateboth the mucosal and systemic immune responses

We have previously produced transgenic carrot plantsexpressing CFP10 and ESAT6 individual proteins It has beenshown that oral immunization of mice with such CFP10 orESAT6 induces both the cell-mediated and humoral immu-nities but it has also been demonstrated that they are toxicto peripheral blood mononuclear cells [21] In the workof other researchers it was shown that vaccination with afusion protein consisting of Ag85B and ESAT6 in animaland human models was more effective than either antigenalone [22] Thus to increase the immune response and todecrease cytotoxic effect we designed a new genetic constructcomprising the fusion protein consisting of the cfp10 andesat6 genes of M tuberculosis and the dIFN gene encod-ing human deltaferon as adjuvant Agrobacterium-mediatedtransformation was used to transfer this construct into thecarrot (Daucus carota L) genomeThe fusion protein CFP10-ESAT6-dIFN was synthesized in the storage roots of trans-genic carrot plants and displayed immunogenicity in the tests

with laboratory animals In this paper we describe resultsfor the studies focused on the synthesis of the fusion proteinCFP10-ESAT6-dIFN in transgenic carrot plants followed byoral administration to animals

2 Materials and Methods

21 Design and Assembly of Fusion Gene The esat6 andcfp10 genes were cloned using genomic DNA extractedfrom biomass of an M tuberculosis isolate recovered froma tuberculosis patient meeting the bioethical requirementsThe protocol of that study was approved by the Com-mittee on the Ethics of State Research Center Virologyand Biotechnology ldquoVectorrdquo Koltsovo Novosibirsk regionRussia IRB00001360 registered in OHRP USA (PermitNumber 2 28052001) DNA was isolated with a DNeasyBlood and Tissue Kit (Qiagen Germany) according to themanufacturerrsquos protocolThe 120574-interferon gene was amplifiedfrom the pIFN-120574-trp2-Δ plasmid [13]

The following primers (up1ndashlo3) were used for cloning thehybrid gene into various vectors

up1 CCCGGATCCATGGCAGAGATGAAGACC-GATup2 GCTTCGGCGCGGGGATGACAGAGCAGC-AGTGGAATTup3 TCGCAGGCGCGGGGATGCAGGACCCAT-ATGTAAAAGAAup cfp-XbaI CCCTCTAGAATGGCAGAGATG-AAGACCGATlo1 TGTCATCCCCGCGCCGAAGCCCATTTGC-GAGGAlo2 TGCATCCCCGCGCCTGCGAACATCCCAGT-GACGlo3 CCCAAGCTTACTGGGATGCTCTTCGACCTlo esat-BamHI CCCGGATCCACTGGGATGCTC-TTCGACCT

Figure 1(a) shows the scheme for assembly of the hybridgene cfp10-esat6-dIFN The primers specific for cfp10 esat6and dIFN genes were designed so that they would overlapand allow a single continuous ldquofusionrdquo gene to be synthe-sized from the individual fragments The primers that carrymutually complementary sequences at their 51015840 ends alloweda glundashalandashglu link joint to be introduced The presence ofhybrid gene inserts was confirmed by PCR and the absenceof mutations in the produced clones was confirmed bysequencing at SB RAS Genomics Core Facility (data notshown)

In the plasmid pBI121-cfp10-esat6-dIFN the fusion geneis under the control of the cauliflower mosaic virus 35S RNApromoter which provides for its expression in plant cellsTheT-DNA region of the plasmid pBI121 pBI121-cfp10-esat6-dIFN is shown in Figure 1(b)

22 Construction and Analysis of Transgenic Plants (PCR andWestern Blot) The callus tissues induced from zygotic carrot

BioMed Research International 3

BamHIXbaI

Link Link

dIFN (469bp)cfp10 (303bp)

esat6 (288 bp)997888rarrup1

997888997888rarrup2

997888997888rarrup3 larr997888lo3

larr997888lo2

larr997888lo1

cfp10-esat6-dIFN (1070bp)

(a)

CaMV35S tNOSRB LB

BamHIXbaI

tNOSpNOS dIFN uidAcfp10nptII esat6

cfp10-esat6-dIFN

(b)

Figure 1 Schemes for assembly of the fusion gene (a) and for the T region in the binary plasmid pBI121 carrying sequence of the fusion genecfp10-esat6-dIFN (b) cfp10 nucleotide sequence of the M tuberculosis cfp10 gene esat6 sequence of the M tuberculosis esat6 gene dIFNsequence of the human deltaferon gene cfp10-esat6-dIFN sequence of the fusion gene nptII E coli neomycin phosphotransferase II geneand uidA sequence of the E coli 120573-glucuronidase gene RB and LB are the repeats flanking the plasmid T region pNOS and tNOS nopalinesynthase gene of theA tumefaciens Ti plasmid promoter and terminator correspondingly CaMV35S cauliflower mosaic virus 35S RNA genepromoter Link glyndashalandashgly hinge joints and up1 up2 up3 lo1 lo2 and lo3 denote the positions of the corresponding primers Arrows showthe sites for BamHI and XbaI restriction endonucleases The lengths of the corresponding gene sequences in base pairs are in parentheses

(D carota L cultivar Nantskaya) embryos were transformedwith the use of agrobacteria [23] The transformants wereselected using the selective media supplemented with theantibiotic kanamycin (100mgL) The kanamycin resistantplants were grown in a hydroponic greenhouse at 2516∘Canda photoperiod of 168 (daynight) until development of thestorage roots The genomic DNA of recipient carrot plantswas isolated using an Axygen (United States) kit for DNAextraction according to the manufacturerrsquos protocol

Presence of the target gene in the genome of the obtainedtransgenic carrot plants was confirmed by PCR using up1 andlo2 primers The expected fragment length was 570 bp

For Western blot analysis storage roots (6 g) of at leastfive individual plants were used The fusion protein CFP10-ESAT6-dIFN was visualized according to the standardprotocols for Western blot assay [24] using a Pierce ECLWestern Blotting Substrate (United States) for chemilumi-nescent reaction The rabbit polyclonal serum for visualizinghas been raised against the recombinant antigen rESAT6Concentrations of proteins and TSP in the storage roots wereassayed according to Bradford [25] The optical density ofsolutions was measured in an Eppendorf BioPhotometer plus(Germany)

23 Production of Polyclonal Antibodies to rESAT6 Malerabbits (Gray Giant) at an age of 6 months were immunizedaccording to the following scheme day 1 100120583g of rESAT6with 500 120583L of CFA intracutaneously day 14 100120583g ofrESAT6 with 500120583L of FIA subcutaneously and days 28 and42 100 120583g of rESAT6 subcutaneously Animals were bled (5ndash10mL) from the auricular vein ondays 28 42 and 56The sera

were obtained by natural clotting with subsequent purifica-tion from the blood cells by centrifugation at 3000 rpm for10min The serum samples were frozen and stored at ndash20∘C

24 Production of the Recombinant Proteins rCFP10 rESAT6and rCFP10-ESAT6-dIFN in E coli and Their PurificationThe recombinant proteins rESAT6 and rCFP10 (fused withGST polypeptide) which were used in experiments as acontrol were earlier produced inE coli strain BL21(DE3)Therecombinant proteins were affinity purified on GlutathioneSepharose 4B (Pharmacia Sweden) [26] The fusion recom-binant protein rCFP10-ESAT6-dIFN (fused with His tag) wasproduced in E coli strain BL21(DE3) Fusion gene expressionwas inducedwith 1mMIPTGThe fusion proteinwas isolatedand purified using a Ni-NTA Spin Kit (Qiagen Germany)according to the manufacturerrsquos protocol The recombinantdeltaferon was kindly provided by L R Lebedev (SRC VBVector Koltsovo Novosibirsk oblast Russia)

25 Laboratory Animals The study was conducted at theCenter for Genetic Resources of Laboratory Animals atthe Institute of Cytology and Genetics Siberian BranchRussian Academy of Sciences (RFMEFI61914X0005 andRFMEFI61914X0010) This study was carried out in strictaccordance with the recommendations in the Guide for theCare and Use of Laboratory Animals of the Russian NationalCenter of Genetic Resources of Laboratory Animals basedon SPF Vivarium of Institute of Cytology and Genetics SBRAS The protocol was approved by the Committee on theEthics of Animal Experiments of the RussianNational Centerof Genetic Resources of Laboratory Animals based on SPF


Vivarium of Institute of Cytology and Genetics SB RASNovosibirsk Russia (Permit Number 10 July 3 2012) Allsurgery was performed under halothane anesthesia and allefforts were made to minimize suffering

SPF inbred BALBcmale mice at an age of 6-7 weeks usedin the work were obtained from the SPF Vivarium with theInstitute of Cytology and Genetics Siberian Branch RussianAcademy of Sciences (Novosibirsk Russia)The animals werekept in groups of two to three males per cage (OptimiceAnimal Care Systems Inc) The cages were placed in aroom with a 20-fold air exchange at a temperature of 24plusmn 2∘C humidity of 45ndash50 and artificial 1212 (daynight)photoperiod with a daybreak at 0300 amThe feed and litterwere autoclaved at 121∘C before use Animals were providedwith deionized water (produced in a Millipore device) witha Severyanka (Eko-proekt St Petersburg Russia) mineralsupplement and feed ad libitum

251 Experimental Model 1 Oral Antigen AdministrationThe mice were divided into four groups three experimen-tal groups of six individuals each and control group ofthree animals The mice from the experimental groups werefed transgenic carrot storage roots containing the antigensCFP10 ESAT6 or rCFP10-ESAT6-dIFN on days 1 and 14of experiment after a 12-h complete food deprivation Thecontrol mice were fed nontransgenic storage roots After12 h the unconsumed storage root remains were weighedIn the remaining days the animals received standard feedad libitum Before feeding the storage roots were processedas follows all storage roots were exposed to 120574-radiation(IGUR-1 Russia) with an intensity of 8400 Rh for 48 h (totalirradiation dose 400 kR) 5ndash7 storage roots wereminced and10 gmale was placed into feedboxes

252 Experimental Model 2 Antigen Administration by Injec-tion Similar to experimental model 1 the mice were dividedinto four groups three experimental (six individuals each)and one control (three individuals) groups All animals weresubcutaneously injected twice in the interscapular regionwith 50120583L of antigen (rCFP10 rESAT6 or rCFP10-ESAT6-dIFN) with subsequent reimmunization after 14 days in theexperimental groups or with 50120583L of PBS in the controlgroup

26 Sampling and Sample Preparation Animals were intrav-itally bled (200120583L) from the retroorbital sinus on days 13and 21 after the first immunization On day 28 animalswere euthanized by decapitation and blood was sampledTheblood specimens were divided into two portions one portionwas placed into tubes with anticoagulant (5 sodium EDTAsolution) and the other portion into clean tubes withoutanticoagulant The blood specimens with anticoagulant wereused to isolate peripheral blood leukocytes for LTT (lympho-cyte transformation test) The blood without anticoagulantwas used to obtain blood serum through natural clottingTheserum specimens were frozen in Eppendorf tubes and storedat ndash80∘C

The humoral immune response was assayed by solid-phase immune assay of the antibody content in blood serumand the cell-mediated immune response was assayed by LTT

The spleen was aseptically excised from euthanized miceand placed into RPMI 1640 nutrient medium (2mM L-glutamine 80mgL gentamicin and 5 fetal bovine serum)The resulting splenocyte suspension was used to assess theproliferative activity of deltaferon and recombinant antigens

27 Enzyme Immunoanalysis (EIA) Estimation of HumoralImmunity Antibodies were detected in the blood specimensby solid-phase enzyme immunoassay [27] To assess thehumoral immunity Corning plates were sensitized withsolutions of the recombinant antigens (rESAT6 rCFP10or rCFP10-ESAT6-dIFN) Nonspecific binding sites weresaturated with 3 fetal bovine serum solution [27] Wells ofthe plates prepared with recombinant antigens were supple-mentedwith 50120583L of the assayed blood samplesThe reactionwas stopped by adding 100 120583Lwell of 09M sulfuric acid andoptical density was measured in an EnSpire multimode platereader (United States) at a wavelength of 450 nm

28 Estimation of Cell-Mediated Immunity by LTT To isolatethe peripheral blood mononuclear cells the blood withanticoagulant was layered onto a Histopaque-1119 (SigmaUnited States) gradient and centrifuged for 15min at 500 rpmThe suspension of mononuclear cells was collected in a cleantube washed with RPMI 1640 and suspended in the samemedium

Proliferative activity was assessed using the dye MTT(20120583L) which was added 3 h before the end of cultivationaccording to the recommendations for TASC MTT Assays(RampD Systems United States) [28] We used the nonspecificproliferation inducers phytohemagglutinin (PHA SigmaUnited States) or concanavalin A (ConA Sigma UnitedStates) (positive control) as well as specific inducers namelythe recombinant proteins dIFN rESAT6 rCFP10 or rCFP10-ESAT6-dIFN The cell proliferative activity was estimatedaccording to the stimulation index (SI the ratio of meanabsolute OD values for stimulated cells to the mean absoluteOD values for unstimulated cells)

29 Statistical Processing Because blood was sampled forestimation of the immunogenic effects of the tested vac-cines at three subsequent time points from each animalthe repeated-measures ANOVA test was used for normallydistributed or log-transformed traits The antigen adminis-tration route (feeding or injection) and the protein used asan antigen were the factors Additionally the experimentalgroups were compared with the controls using repeated-measures ANOVA In this process gradation of the antigentype factor was reduced to two levels control and antigenWhen analyzing the LTT data Studentrsquos 119905-test was used toassess whether the SI exceeded unity as (SI minus 1)SE The SEvalue was taken from the table of unweighted averages andtheir errors (SE) obtained by repeated-measures ANOVA Inthe experiment with splenocytes the SI difference from unitywas calculated using the nonparametric sign test


15001000

500

1 2 3 4 5 76

(a)

1 2 3 4 5

60kDa 2(CFP10-ESAT6-dIFN)

32kDa CFP10-ESAT6-dIFN

16kDa CFP10-ESAT66kDa ESAT6

75kDa

25kDa

150 kDa100 kDa

50kDa37kDa

20kDa16kDa

(b)

Figure 2 Analysis of transgenic carrot plants (a) PCR reaction Electrophoretic patterns of the PCR amplified genomicDNAof three selectedtransformants assayed for the presence of the fusion gene cfp10-esat6-dIFN (15 agarose gel) (1ndash3) DNA of transgenic carrot plants (4) DNAof a nontransgenic carrot plant (5) positive control (plasmid pBi121-CFP10-ESAT6-dIFN) (6) negative control (no template DNA) and (7)DNA length marker (bp) (b) Western blot assay of the extracts (total soluble protein) of transgenic carrot storage roots In (b) lane (1)molecular weight marker Precision Plus Protein Kaleidoscope Standards (BioRad USA) lane (2) control 1 purified rCFP10-ESAT6-dIFNprotein produced in E coli lane (3) control 2 rCFP10-ESAT6-dIFN protein from inclusion bodies of E coli lane (4) negative control extractof nontransgenic carrot storage roots lane (5) extract of transgenic carrot storage roots Molecular weights of the visualized fragments andtheir assumed composition are shown to the right marker proteins molecular weights are shown to the left

3 Results

31 Genetic Construct with Fusion Gene The design ofthe construct makes it possible to synthesize the full-sizedgene cfp10-esat6-dIFN It is composed of two individual Mtuberculosis genes esat6 and cfp10 and the gene encodinghuman deltaferon (dIFN) within one open reading frameThere are no internal stop codons between them but thereis a ldquohinge jointrdquo of three amino acid residues GlyndashAlandashGlythat creates a flexible link between the domains of the fusionprotein

32 Analysis of Transgenic Carrot Plants In the experimentsabout 100 transgenic carrot plants carrying the gene cfp10-esat6-dIFN were obtained the transgenic carrot plants withthe cfp10 and esat6 genes were obtained earlier [21]

To confirm the transgenic status of the resulting carrotplants the storage root of each one was tested for presenceof the cfp10-esat6-dIFN nucleotide sequence in the genomicDNA by PCR with the corresponding primers The elec-trophoretic patterns of PCR products for three carrot storageroots are shown in Figure 2(a) The length of the amplifiedfragments is consistent with the expected PCR fragmentobtained from the plasmid (570 Bp) in lanes 1ndash3 as comparedwith the amplified positive control fragment in lane 5 Thissuggests that the hybrid gene cfp10-esat6-dIFN was integratedinto the nuclear genome of the assayed carrot plants

The Western blot assay was used to confirm the presenceof hybrid protein in the storage roots of transgenic carrotplants The polyclonal serum for visualizing the studiedprotein has been raised against the recombinant antigenrESAT6The corresponding results are shown in Figure 2(b)

In Figure 2(b) lane (1) is loaded with molecular weightmarker Precision Plus Protein Kaleidoscope Standards (Bio-Rad USA) with fluorescent band of 75 kDa Lane (2) isloaded with purified rCFP10-ESAT6-dIFN protein (10 ng)produced in E coli as control 1 Lane (3) is loaded with

rCFP10-ESAT6-dIFN protein from inclusion bodies of E colias control 2 Lane (4) is loaded with extract of nontransgeniccarrot storage roots (22320120583g of TSP) as negative controlLane (5) is loaded with extract of transgenic carrot storageroots carrying the gene cfp10-esat6-dIFN (28140 120583g of TSP)Several bands visualized in lanes (2) and (5) of Figure 2(b)presumably correspond to the following peptides accordingto their molecular weights ESAT6 (6 kDa) CFP10-ESAT6(16 kDa) CFP10-ESAT6-dIFN (32 kDa) and a putative dimeror trimer of one of these species 2(CFP10-ESAT6-dIFN)(60 kDa)

The concentration of the fusion protein in carrot storageroot tissues was compared with the known concentration ofthe protein isolated from E coli 20120583L of TSP solution (TSPconcentration of 1407120583g120583L) of transgenic carrot storageroots was applied on membrane so we applied 1407 lowast20 = 28140 120583g of TSP on membrane Control sample wasapplied in amount of 10 ng Therefore in comparison withrCFP10-ESAT6-dIFN on lane (2) we see not less than 10 ng ofESAT6 on lane (5) suggesting that the CFP10-ESAT6-dIFNconcentration in the extracts of transgenic carrot storageroots amounted to not less than 1 ng120583L which is not less than0035 TSP

33 Establishment of Humoral Immunity Figure 3(a) showsthe level of the antibodies to the rESAT6 antigen inblood sera of mice immunized with three types of anti-gens (rESAT6 rCFP10 and rCFP10-ESAT6-dIFN) that weredelivered either by injection or orally According to the re-sults from a repeated-measures ANOVA the efficiency ofimmunization with rESAT6 was independent of the admin-istration route (119865

134= 003 119875 = 086 for log-transformed

data) which suggested pooling the data for oral and injec-tion immunizations However the type of antigen wasshown to be a significant factor for variation in the contentof anti-rESAT6 antibodies in the blood of experimental


0040

0045

0050

0055

0060

0065

0070

0075

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-ESA

T6le

vel

lowastlowast

lowast

(a)

0040

0090

0140

0190

0240

0290

0340

0390

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-CFP

10

-ESA

T6-d

IFN

leve

l

lowastlowastlowast

lowast

(b)

Figure 3 (a) Level of the antibodies to rESAT6 antigen in the blood serum of the mice immunized with different antigens lowast119865114= 514

and 119875 = 0039 and lowastlowast119865114= 1205 and 119875 = 00037 (b) level of the antibodies to rCFP10-ESAT6-dIFN with different methods of antigen

presentation blue bars antigen delivered by injection brown bars antigen delivered orally lowast11986517= 655 and 119875 = 0038 and lowastlowastlowast119865

174= 27610

and 119875 lt 0001 both when compared with the control for the mouse groups immunized with the corresponding antigen (repeated-measuresANOVA two gradations of control as a factor and one of the antigens)

animals (119865334= 1239 119875 lt 0001 for log-transformed

data) Compared with controls immunization with rCFP10-ESAT6-dIFN elevated the level of antibodies to rESAT6 inmouse blood serum whereas immunization with rCFP10decreased this level (Figure 3(a))

Independent of the administration route (oral or byinjection) none of the tested antigens had a statisticallysignificant effect on the content of antibodies to rCFP10 (datanot shown)

A statistically significant effect of the antigen type (119865334=

1182 and 119875 lt 0001) and administration route (119865134= 761

and 119875 = 0009) as well as the interaction of these factors(119865334= 1888 and 119875 lt 0001) was demonstrated when ana-

lyzing the level of antibodies to rCFP10-ESAT6-dIFN(Figure 3(b)) Taking into account the significant dependenceof the antibody level in the animal blood on the administra-tion route the data for antibodies to rCFP10-ESAT6-dIFNwere analyzed separately for the animals immunized orally(brown bars) or by injection (blue bars)

The rCFP10-ESAT6-dIFN injections induced the maxi-mal increase in the antibodies specific for this antigen in themouse blood serum (Figure 3(b)) It should be emphasizedthat level of the antibodies to rCFP10-ESAT6-dIFN strikinglyexceeded the control level (119865

17= 27610 and 119875 lt 0001)

The levels of antibodies to rCFP10 and rESAT6 in miceimmunized with rCFP10-ESAT6-dIFN were at the controllevel (Figure 3(b))

In the variant with oral rCFP10-ESAT6-dIFN admin-istration a statistically significant increase in the level ofantibodies as compared with control was observed only whenfeeding animals with the carrot storage roots containing

the rESAT6 protein (11986517= 655 119875 = 0038) Of note

the induction of specific antibodies in the animal bloodwas undetectable in the case of oral rCFP10-ESAT6-dIFNadministration (Figure 3(b))

34 Cell-Mediated Immunity Cell-mediated immunity wasestimated using the stimulation index (SI) for proliferation ofperipheral blood mononuclear cells induced by recombinantproteins (rESAT6 rCFP10 or rCFP10-ESAT6-dIFN) andnonspecific proliferation inducers (PHA or ConA)

The proliferative response of mononuclear cells torESAT6 (Figure 4(a)) and to rCFP10 (Figure 4(b)) dependedon the antigen used for immunization of experimentalanimals (119865

334= 1512 and 119875 lt 0001 for rESAT6 and

119865334= 2097 and 119875 lt 0001 for rCFP10) Proliferation

was independent of the two administration routes either byinjection or orally (119865

134= 000 and 119875 = 099 for rESAT6 and

119865134= 199 and 119875 = 017 for rCFP10) Of note the animals

immunized with rESAT6 rCFP10 or rCFP10-ESAT6-dIFNeither by injection or orally displayed a statistically significantincrease in response as compared with the control

The SI induced by the fusion protein (Figure 4(c)) addedto the culture medium was independent of both administra-tion route (119865

134= 069 and 119875 = 041) and type of antigen

(119865334= 107 and 119875 = 037) Notably the SI for mononuclear

cells of both control and immunized animals exceeded unityin a statistically significant manner which could result fromthe presence of deltaferon in the fusion protein

Addition of ConA (Figure 5(a)) or PHA (Figure 5(b)) tothe culture medium induced a statistically significant prolif-erative response in all animals and was most pronounced in


080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120

130lowastlowastlowast

lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)

Figure 4 The SI for the proliferation of peripheral blood mononuclear cells induced by recombinant proteins after immunization withdifferent antigens (a) The SI for rESAT6 lowastlowastlowast119865

114= 1536 and 119875 = 00015 and lowastlowast119865

114= 1221 and 119875 = 00036 (b) the SI for rCFP10

lowastlowastlowast


114= 1163 and 119875 = 00046 (c) the SI for rCFP10-ESAT6-dIFN All 119865

114as compared to the control

for the mouse group immunized with this antigen (repeated-measures ANOVA administration route as a factor two gradations of controlas a factor and one of the antigens) 119875 lt 005 as compared with unity (Studentrsquos 119905-test)

the control individuals However the degree of this responsedepended on the type of antigen used for immunization(119865334= 1611 and 119875 lt 0001 for ConA and 119865

334= 358

and 119875 = 0024 for PHA) rather than on the administrationroute (119865

134= 123 and 119875 = 027 for ConA and 119865

134= 174

and 119875 = 020 for PHA) Immunization with rESAT6 orrCFP10 induced a statistically significant decrease in the SI

as compared with control whereas statistically significantdifferences were not observed in the case of the fusionprotein

35 Proliferative Response of Splenocytes Deltaferon a com-ponent of the fusion protein rCFP10-ESAT6-dIFN could bethe factor that stimulated proliferation of mononuclear cells


080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)

Figure 5The SI for the proliferation of peripheral bloodmononuclear cells induced by nonspecific proliferation inducers after immunizationwith different antigens (a) the SI for ConA lowastlowastlowast119865


114= 1585 and 119875 = 00013 (b) the SI for PHA

lowastlowastlowast

119865114= 1387 and 119875 = 0002 lowastlowast119865

114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865

114as compared to the control for

the mouse group immunized with this antigen (repeated-measure ANOVA administration route as a factor two gradations of control as afactor and one of the antigens) 119875 lt 005 as compared with unity (Studentrsquos 119905-test)

in the control animals that received no antigens (Figure 6)To test this assumption we estimated the proliferativeresponse of splenocytes isolated from intact male BALBcmice (Figure 6) When dIFN and the fusion protein wereadded to the culture the recorded SI exceeded unity in astatistically significant manner (119885 = 204 and 119875 = 004 signtest) The average SI values were equal for dIFN and rCFP10-ESAT6-dIFN but exceeded the values for splenocyte culturesthat received rESAT6 or rCFP10

4 Discussion

Currently plant cells are an attractive alternative expressionsystem for recombinant proteins with medical purposes andare used in many leading biotechnological laboratories andcompanies [29ndash31] Recent advances in this field have enableda significant increase in the expression level of recombinantproteins [32ndash34] improvement in posttranslational modi-fications that fit more closely with mammalian cells [3536] and approaches to directed modification of the plantgenome [37 38] It has become evident that plant systemspossess a high potential competitive ability compared withother expression systems and are of interest for investmentcompanies

There are examples of successful expression of M tuber-culosis antigens in plant cells [3 7 19 20]

Cell-mediated and humoral immune responses inducedby edible vaccines are formed via presentation of an antigen

0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x

Figure 6The SI for spontaneous proliferation ofmouse splenocytesinduced by different antigens SI exceeds unity (119875 lt 005 sign test)

to the intestinal mucosae Importantly in most cases lungmucosa is the particular site where tuberculosis infectionsstarts and mainly progresses Correspondingly the immune


response in lung tissues is amajor factor in the initial stages ofthe disease development and colonization byM tuberculosisandM bovis in warm-blooded organisms [39] The fact thatthe bodymucosae function as an integral system inwhich theactivated lymphocytes and the corresponding interleukinscirculate allows us to speak to a unified mechanism thatunderlies establishment of mucosal immunity during diseasedevelopment or administration of an edible vaccine

The ESAT6 and CFP10 proteins secreted at an early stageof tuberculosis stimulate T cells to produce 120574-interferonand exhibit CTL activity (cytotoxic T lymphocytes) bothin animal models and in humans According to recentdata these two proteins together have a high potential as acandidate subunit vaccine [5] As has been experimentallyshown the efficiency of recombinant ESAT6 in inducingprotective immunity against tuberculosis is comparable to theefficiency of the BCG vaccine [6]

A promising direction in the development of subunitvaccines is combining the antigens with adjuvants andimmunomodulators Plant-based vaccines targeting differentdiseases through the use of chimeric proteins as immunogenshave been of great interest to vaccine developers Progressover the past decade in the design and evaluation of newbroad-protective proteins has demonstrated the feasibilityof this technology Genetic fusions allowed the expressionof fusion proteins carrying two or more components withthe aim to elicit immune responses against different targetsincluding antigens from distinct pathogens or strains Toincrease immunogenicity the ESAT6 antigen was fused withother tuberculosis antigens (Ag85B and Mtb72F) [3 2040 41] or adjuvants (CTB cholera toxin B subunit LTBEscherichia coli heat-labile enterotoxin B subunit LipY acell wall protein and ELP elastin-like peptide) [3 7 42]and expressed in various plant species (Arabidopsis thalianatobacco and lettuce)

Earlier in another experiment we have assessed immuno-genicity of the recombinant M tuberculosis proteins ESAT6and CFP10 in experiments with laboratory animals [21] Inthis novel work we have obtained a fusion protein thatconsists of a combination of the M tuberculosis CFP10and ESAT6 antigens and human deltaferon Our resultssuggest that the components of fusion protein expressed incarrot cells retain their antigenic properties However theexpression level of the fusion protein in the storage roots oftransgenic carrot plants in these studies is insufficient for acommercial product [43]

The experiments with laboratory animals have demon-strated that the fusion protein CFP10-ESAT6-dIFN is able toinduce both humoral (Figure 3) and cell-mediated (Figure 4)immune responses when administered orally or by injec-tion to warm-blooded animals (mice) We have previouslydemonstrated that individual rESAT6 is toxic to peripheralblood mononuclear cells [21] as well as transgenic plantsat the stage of regeneration (causing various morphologi-cal abnormalities in regenerants) The immunization withrESAT6 or rCFP10 induced a statistically significant decreasein the SI by nonspecific inducers (ConA PHA Figure 5) ascompared with the control whereas statistically significantdifferences were not observed in the case of the fusion

protein Thus the M tuberculosis antigens contained in thefusion protein have no cytotoxic effect on the peripheralblood mononuclear cells Of note the addition of the fusionprotein or its component deltaferon stimulates proliferationof splenocytes in unimmunized mice (Figures 4 and 6)

A lower level of antibodies to orally administered rCFP10-ESAT6-dIFN compared with injection delivery is potentiallyexplainable by two factors First it is possible that the cellwall of carrot cells is poorly destroyed in the gastrointestinaltract [44 45] and the protein amount released by preliminarycarrot mincing is insufficient for induction of a high immuneresponse Alternatively the acids and digestive enzymes of thegastrointestinal tract may destroy the immunogenic proteinInterestingly the transgenic carrot storage roots containingthe fusion protein CFP10-ESAT6-dIFN do not differ fromthe efficiency of injected recombinant fusion protein in theirability to induce cell-mediated immunity This is especiallyimportant because the Th1 type of immune response plays aleading role in resistance toM tuberculosis infection [46 47]

A Western blot assay for the fusion CFP10-ESAT6-dIFN protein detected several bands on the lanes loadedwith the total protein of transgenic carrot storage rootsand with purified recombinant fusion protein from E coli(Figure 2(b)) The polyclonal serum for visualizing theseproteins has been raised against the recombinant antigenrESAT6 correspondingly all the molecules containing thispolypeptide are detected by Western blot assay During theextraction of proteins from plants denaturing and reducingagents disrupt the trimeric and other oligomeric formsof fusion protein into the monomeric form Under theseconditions fusion proteins are exposed to proteases andcould result in cleavage [7] And it is known that theproteins synthesized in a plant expression system can beposttranslationally modified [48] Our experimental data areinsufficient to determine the causes of the formation of lowmolecular weight bands However we assume that in plantcells fusion protein remains in themono- ormultimeric formsince we did not observe the toxic effect of individual ESAT6protein in animals immunized with fusion protein

Summing up the novel fusion protein rCFP10-ESAT6-dIFN is of clear interest for further studies The next stagesin our work are improvement of the genetic constructaimed at an increase in the expression level of the fusionrecombinant protein in transgenic plant tissues modificationof its oral delivery that would provide for a better humoralimmune response and kinetic and a more detailed analysisof the immune response developed as a result of vaccinationprotocol in humanized animal model

5 Main Conclusion

Fusion protein comprising the M tuberculosis genes cfp10and esat6 and dIFN gene expressed in transgenic carrotinduces humoral and cell-mediated immune responses whenadministered orally or by injection


Abbreviations

ANOVA Analysis of varianceSI Stimulation indexLTT Lymphocyte transformation testEIA Enzyme immunoanalysis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to L R Lebedev for kindly providingdeltaferon specimens and to the technical staff of the SPFVivarium with the Institute of Cytology and Genetics Thisresearch was supported by Program of Siberian Branch ofRussian Academy of Science ldquofundamental bases of biotech-nology creating therapies and diagnosis of diseasesrdquo BudgetProject VI6215 (no 01201280334)

References

[1] S H E Kaufmann ldquoFact and fiction in tuberculosis vaccineresearch 10 years laterrdquo The Lancet Infectious Diseases vol 11no 8 pp 633ndash640 2011

[2] L P Ormerod R J Shaw and D M Mitchell ldquoTuberculosis inthe UK 1994 current issues and future trendsrdquoThorax vol 49no 11 pp 1085ndash1089 1994

[3] L Dedieu D M Floss M Mockey et al ldquoExpression andimmunogenicity of the mycobacterial Ag85BESAT-6 antigensproduced in transgenic plants by elastin-like peptide fusionstrategyrdquo Journal of Biomedicine and Biotechnology vol 2010Article ID 274346 14 pages 2010

[4] M P Girard U Fruth and M-P Kieny ldquoA review of vaccineresearch and development tuberculosisrdquo Vaccine vol 23 no50 pp 5725ndash5731 2005

[5] P Klucar P F Barnes Y Kong et al ldquoVaccination strategies toenhance local immunity and protection againstMycobacteriumtuberculosisrdquo Vaccine vol 27 no 12 pp 1816ndash1824 2009

[6] J Dietrich KWeldingh and P Andersen ldquoProspects for a novelvaccine against tuberculosisrdquo Veterinary Microbiology vol 112no 2ndash4 pp 163ndash169 2006

[7] P S Lakshmi D Verma X Yang B Lloyd and H DaniellldquoLow cost tuberculosis vaccine antigens in capsules expressionin chloroplasts bio-encapsulation stability and functional eval-uation in vitrordquo PLoS ONE vol 8 no 1 Article ID e54708 2013

[8] A S Mustafa ldquoDevelopment of new vaccines and diagnosticreagents against tuberculosisrdquo Molecular Immunology vol 39no 1-2 pp 113ndash119 2002

[9] M I de Jonge G Pehau-Arnaudet M M Fretz et al ldquoESAT-6 from Mycobacterium tuberculosis dissociates from its puta-tive chaperone CFP-10 under acidic conditions and exhibitsmembrane-lysing activityrdquo Journal of Bacteriology vol 189 no16 pp 6028ndash6034 2007

[10] R Simeone D Bottai and R Brosch ldquoESXtype VII secretionsystems and their role in host-pathogen interactionrdquo CurrentOpinion in Microbiology vol 12 no 1 pp 4ndash10 2009

[11] R L V Skjoslasht T Oettinger I Rosenkrands et al ldquoComparativeevaluation of low-molecular-mass proteins from Mycobac-terium tuberculosis identifies members of the ESAT-6 family as

immunodominant T-cell antigensrdquo Infection and Immunity vol68 no 1 pp 214ndash220 2000

[12] R E Soria-Guerra L Moreno-Fierros and S Rosales-Mendoza ldquoTwo decades of plant-based candidate vaccines areview of the chimeric protein approachesrdquo Plant Cell Reportsvol 30 no 8 pp 1367ndash1382 2011

[13] P N Miroshnikov and P N Lebedev ldquoDevelopment of medici-nal product deltaferon based on analog of recombinant humaninterferon-gammardquo Bulletin of Biotechnology and Physicochem-ical Biology vol 2 no 2 pp 5ndash10 2006 (Russian)

[14] V I Masicheva N M Poustoshilova and E D DanilenkoldquoDevelopment of drugs based on genetically engineeredcytokinesrdquoMedical Immunology vol 3 no 3 pp 369ndash378 2001

[15] S I Tatrsquokov O Y Smirnova R Y Tsivkovskii et al ldquoMutanthuman gamma-interferon with a truncated C-terminus and itspropertiesrdquo Doklady Biochemistry vol 372 no 1ndash6 pp 112ndash1142000

[16] S Sereinig M Stukova N Zabolotnyh et al ldquoInfluenza virusNS vectors expressing the Mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protectanimals against tuberculosis challengerdquo Clinical and VaccineImmunology vol 13 no 8 pp 898ndash904 2006

[17] J A Triccas ldquoRecombinant BCG as a vaccine vehicle to protectagainst tuberculosisrdquo Bioengineered Bugs vol 1 no 2 pp 110ndash115 2010

[18] B Hamasur M Haile A Pawlowski et al ldquoMycobacteriumtuberculosis arabinomannan-protein conjugates protect againsttuberculosisrdquo Vaccine vol 21 no 25-26 pp 4081ndash4093 2003

[19] M M Rigano S Dreitz A-P Kipnis A A Izzo and A MWalmsley ldquoOral immunogenicity of a plant-made subunittuberculosis vaccinerdquo Vaccine vol 24 no 5 pp 691ndash695 2006

[20] Y L Dorokhov A A Sheveleva O Y Frolova et al ldquoSuperex-pression of tuberculosis antigens in plant leavesrdquo Tuberculosisvol 87 no 3 pp 218ndash224 2007

[21] E A Uvarova P A Belavin N V Permyakova et al ldquoOralimmunogenicity of plant-made Mycobacterium tuberculosisESAT6 and CFP10rdquo BioMed Research International vol 2013Article ID 316304 8 pages 2013

[22] J T van Dissel S M Arend C Prins et al ldquoAg85B-ESAT-6adjuvanted with IC31 promotes strong and long-livedMycobac-terium tuberculosis specific T cell responses in naıve humanvolunteersrdquo Vaccine vol 28 no 20 pp 3571ndash3581 2010

[23] E Yakushenko J Lopatnikova E Khrapov et al ldquoUse oftransgenic carrot plants producing human interleukin-18 formodulation of mouse immune responserdquo in New Research onBiotechnology in Biology and Medicine A M Egorov and GZaikov Eds chapter 10 pp 97ndash107 Nova Science PublishersNew York NY USA 2006

[24] S H Kaufmann C M Ewing and J H Shaper ldquoThe erasableWestern blotrdquo Analytical Biochemistry vol 161 no 1 pp 89ndash951987

[25] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[26] S Tatrsquokov Y Tumanov O Nosareva et al ldquoUse of recombinantspecies-specificM tuberculosisproteins for serological diagnos-ing of the infectionrdquo Epidemiology and Vaccine Prophylaxis vol29 no 4 pp 42ndash47 2006 (Russian)

[27] P Tijssen Practice and Theory of Enzyme Immunoassays Else-vier BV Amsterdam The Netherlands 1985


[28] D Gerlier and N Thomasset ldquoUse of MTT colorimetric assayto measure cell activationrdquo Journal of Immunological Methodsvol 94 no 1-2 pp 57ndash63 1986

[29] V Yusibov S J Streatfield and N Kushnir ldquoClinical devel-opment of plant-produced recombinant pharmaceuticals vac-cines antibodies and beyondrdquoHuman Vaccines vol 7 no 3 pp313ndash321 2011

[30] J Kaufman and N Kalaitzandonakes ldquoThe economic potentialof plant-made pharmaceuticals in the manufacture of biologicpharmaceuticalsrdquo Journal of Commercial Biotechnology vol 17no 2 pp 173ndash182 2011

[31] K-C Kwon D Verma N D Singh R Herzog and H DaniellldquoOral delivery of human biopharmaceuticals autoantigens andvaccine antigens bioencapsulated in plant cellsrdquoAdvanced DrugDelivery Reviews vol 65 no 6 pp 782ndash799 2013

[32] P N Desai N Shrivastava and H Padh ldquoProduction of het-erologous proteins in plants strategies for optimal expressionrdquoBiotechnology Advances vol 28 no 4 pp 427ndash435 2010

[33] P Maliga and R Bock ldquoPlastid biotechnology food fuel andmedicine for the 21st centuryrdquo Plant Physiology vol 155 no 4pp 1501ndash1510 2011

[34] A O Viacheslavova O N Mustafaev A A Tiurin et alldquoSet of module vectors for stable or transient expression ofheterologous genes in plantsrdquoGenetika vol 48 no 9 pp 1046ndash1056 2012

[35] B Nagels KWeterings N Callewaert and E JM vanDammeldquoProduction of plant made pharmaceuticals from plant host tofunctional proteinrdquoCritical Reviews in Plant Sciences vol 31 no2 pp 148ndash180 2012

[36] D E Webster and M CThomas ldquoPost-translational modifica-tion of plant-made foreign proteins glycosylation and beyondrdquoBiotechnology Advances vol 30 no 2 pp 410ndash418 2012

[37] F Fauser N Roth M Pacher et al ldquoIn planta gene targetingrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 19 pp 7535ndash7540 2012

[38] M S Antunes J J Smith D Jantz and J I Medford ldquoTargetedDNA excision in Arabidopsis by a re-engineered homingendonucleaserdquo BMC Biotechnology vol 12 article 86 2012

[39] R K Salyaev M M Rigano and N I Rekoslavskaya ldquoDevel-opment of plant-based mucosal vaccines against widespreadinfectious diseasesrdquo Expert Review of Vaccines vol 9 no 8 pp937ndash946 2010

[40] N A Marveeva M I Vasilenko A M Shakhovskiı and NV Kuchuk ldquoAgrobacterium-mediated transformation of lettuce(Lactuca sativa L) with vectors bearing genes of bacterial anti-genes fromMycobacterium tuberculosisrdquo Cytology and Geneticsvol 43 no 2 pp 27ndash32 2009

[41] N A Matvieieva O M Kishchenko A O Potrochov A MShakhovsky and M V Kuchuk ldquoRegeneration of transgenicplants from hairy roots of Cichorium intybus L var FoliosumHegirdquo Cytology and Genetics vol 45 no 5 pp 277ndash281 2011

[42] M M Rigano M L Alvarez J Pinkhasov et al ldquoProductionof a fusion protein consisting of the enterotoxigenic Escherichiacoli heat-labile toxin B subunit and a tuberculosis antigen inArabidopsis thalianardquo Plant Cell Reports vol 22 no 7 pp 502ndash508 2004

[43] A G Lossl and M T Waheed ldquoChloroplast-derived vaccinesagainst human diseases achievements challenges and scopesrdquoPlant Biotechnology Journal vol 9 no 5 pp 527ndash539 2011

[44] E A TydemanM L Parker RM Faulks et al ldquoEffect of carrot(Daucus carota) microstructure on carotene bioaccessibility in

the upper gastrointestinal tract 2 in vivo digestionsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 17 pp 9855ndash98602010

[45] L Lemmens S van Buggenhout A M van Loey and ME Hendrickx ldquoParticle size reduction leading to cell wallrupture is more important for the 120573-carotene bioaccessibilityof raw compared to thermally processed carrotsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 24 pp 12769ndash12776 2010

[46] F C Minion S A Menon G G Mahairas and M J Wan-nemuehler ldquoEnhanced murine antigen-specific gamma inter-feron and immunoglobulin G2a responses by usingmycobacte-rial ESAT-6 sequences in DNA vaccinesrdquo Infection and Immu-nity vol 71 no 4 pp 2239ndash2243 2003

[47] J-S Kim W S Kim H-G Choi et al ldquoMycobacteriumtuberculosis RpfB drives Th1-type T cell immunity via a TLR4-dependent activation of dendritic cellsrdquo Journal of LeukocyteBiology vol 94 no 4 pp 733ndash749 2013

[48] R Fischer S Schillberg S Hellwig R M Twyman and JDrossard ldquoGMP issues for recombinant plant-derived pharma-ceutical proteinsrdquo Biotechnology Advances vol 30 no 2 pp434ndash439 2012

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


of 120574-interferon synthesis by peripheral blood mononuclearcells in response to contact with these antigens [11] Theseproteins are encoded in the M tuberculosis RD1 genomicregion Of note this region is deleted in theM bovis genomeCorrespondingly BCG administration fails to induce theimmune response to the antigens encoded in this regionincluding CFP10 and ESAT6

To increase the mucosal immunogenicity of proteins orpeptides otherwise imperceptible to the mucosal immunesystem recombinant antigensmay be fusedwith proteins thatdisplay adjuvant or immunomodulatory properties [12] Apromising protein possessing such properties is deltaferonThis is a recombinant analog of 120574-interferon with a deletionof ten C-terminal amino acids and mutations introduced atpositions 129 and 130 As a result deltaferon is synthesized ina native soluble form which displays an increased resistanceto blood proteases and a decreased antiviral activity [13 14]Therefore deltaferon is able to concurrently serve as anadjuvant and an immunomodulator efficiently boosting theimmune response to tuberculosis antigens [15]

Various systems are used for delivering antigens as sub-unit vaccines to human and animal organisms includingrecombinant viral vectors [16] recombinant bacterial systems[17] and lipoglycans conjugated with proteins [18] Oraladministration within plant cells [7 19 20] is among thepromising systems Proteins from plant cells expressingvaccine antigens are protected in the stomach from acids andenzymes but are released to the immune or blood circulatorysystem when plant cell walls are digested by microbes thatcolonize the gut Vaccine antigens bioencapsulated in plantcells provide both mucosal and systemic immunity andprotection against bacterial viral or protozoan pathogens ortoxin challenge upon oral deliveryThis new platform offers alow cost alternative to deliver different therapeutic proteinsto combat infection by eliminating inactivated pathogensexpensive purification cold storagetransportation and ster-ile injections We have used this particular approach whenconstructing transgenic carrot plants that produce M tuber-culosis antigens Because the tuberculosis pathogen is a res-piratory agent mucosal vaccination is well suited to initiateboth the mucosal and systemic immune responses

We have previously produced transgenic carrot plantsexpressing CFP10 and ESAT6 individual proteins It has beenshown that oral immunization of mice with such CFP10 orESAT6 induces both the cell-mediated and humoral immu-nities but it has also been demonstrated that they are toxicto peripheral blood mononuclear cells [21] In the workof other researchers it was shown that vaccination with afusion protein consisting of Ag85B and ESAT6 in animaland human models was more effective than either antigenalone [22] Thus to increase the immune response and todecrease cytotoxic effect we designed a new genetic constructcomprising the fusion protein consisting of the cfp10 andesat6 genes of M tuberculosis and the dIFN gene encod-ing human deltaferon as adjuvant Agrobacterium-mediatedtransformation was used to transfer this construct into thecarrot (Daucus carota L) genomeThe fusion protein CFP10-ESAT6-dIFN was synthesized in the storage roots of trans-genic carrot plants and displayed immunogenicity in the tests

with laboratory animals In this paper we describe resultsfor the studies focused on the synthesis of the fusion proteinCFP10-ESAT6-dIFN in transgenic carrot plants followed byoral administration to animals

2 Materials and Methods

21 Design and Assembly of Fusion Gene The esat6 andcfp10 genes were cloned using genomic DNA extractedfrom biomass of an M tuberculosis isolate recovered froma tuberculosis patient meeting the bioethical requirementsThe protocol of that study was approved by the Com-mittee on the Ethics of State Research Center Virologyand Biotechnology ldquoVectorrdquo Koltsovo Novosibirsk regionRussia IRB00001360 registered in OHRP USA (PermitNumber 2 28052001) DNA was isolated with a DNeasyBlood and Tissue Kit (Qiagen Germany) according to themanufacturerrsquos protocolThe 120574-interferon gene was amplifiedfrom the pIFN-120574-trp2-Δ plasmid [13]

The following primers (up1ndashlo3) were used for cloning thehybrid gene into various vectors

up1 CCCGGATCCATGGCAGAGATGAAGACC-GATup2 GCTTCGGCGCGGGGATGACAGAGCAGC-AGTGGAATTup3 TCGCAGGCGCGGGGATGCAGGACCCAT-ATGTAAAAGAAup cfp-XbaI CCCTCTAGAATGGCAGAGATG-AAGACCGATlo1 TGTCATCCCCGCGCCGAAGCCCATTTGC-GAGGAlo2 TGCATCCCCGCGCCTGCGAACATCCCAGT-GACGlo3 CCCAAGCTTACTGGGATGCTCTTCGACCTlo esat-BamHI CCCGGATCCACTGGGATGCTC-TTCGACCT

Figure 1(a) shows the scheme for assembly of the hybridgene cfp10-esat6-dIFN The primers specific for cfp10 esat6and dIFN genes were designed so that they would overlapand allow a single continuous ldquofusionrdquo gene to be synthe-sized from the individual fragments The primers that carrymutually complementary sequences at their 51015840 ends alloweda glundashalandashglu link joint to be introduced The presence ofhybrid gene inserts was confirmed by PCR and the absenceof mutations in the produced clones was confirmed bysequencing at SB RAS Genomics Core Facility (data notshown)

In the plasmid pBI121-cfp10-esat6-dIFN the fusion geneis under the control of the cauliflower mosaic virus 35S RNApromoter which provides for its expression in plant cellsTheT-DNA region of the plasmid pBI121 pBI121-cfp10-esat6-dIFN is shown in Figure 1(b)

22 Construction and Analysis of Transgenic Plants (PCR andWestern Blot) The callus tissues induced from zygotic carrot


BamHIXbaI

Link Link



997888997888rarrup2

997888997888rarrup3 larr997888lo3

larr997888lo2

larr997888lo1


(a)

CaMV35S tNOSRB LB

BamHIXbaI


cfp10-esat6-dIFN

(b)






















15001000

500

1 2 3 4 5 76

(a)

1 2 3 4 5




75kDa

25kDa

150 kDa100 kDa

50kDa37kDa

20kDa16kDa

(b)


3 Results












0040

0045

0050

0055

0060

0065

0070

0075

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-ESA

T6le

vel

lowastlowast

lowast

(a)

0040

0090

0140

0190

0240

0290

0340

0390

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-CFP

10

-ESA

T6-d

IFN

leve

l

lowastlowastlowast

lowast

(b)




174= 27610










17= 27610 and 119875 lt 0001)










134= 000 and 119875 = 099 for rESAT6 and









080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120


lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)




lowastlowastlowast






334= 358


134= 123 and 119875 = 027 for ConA and 119865

134= 174





080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


BamHIXbaI

Link Link



997888997888rarrup2

997888997888rarrup3 larr997888lo3

larr997888lo2

larr997888lo1


(a)

CaMV35S tNOSRB LB

BamHIXbaI


cfp10-esat6-dIFN

(b)






















15001000

500

1 2 3 4 5 76

(a)

1 2 3 4 5




75kDa

25kDa

150 kDa100 kDa

50kDa37kDa

20kDa16kDa

(b)


3 Results












0040

0045

0050

0055

0060

0065

0070

0075

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-ESA

T6le

vel

lowastlowast

lowast

(a)

0040

0090

0140

0190

0240

0290

0340

0390

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-CFP

10

-ESA

T6-d

IFN

leve

l

lowastlowastlowast

lowast

(b)




174= 27610










17= 27610 and 119875 lt 0001)










134= 000 and 119875 = 099 for rESAT6 and









080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120


lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)




lowastlowastlowast






334= 358


134= 123 and 119875 = 027 for ConA and 119865

134= 174





080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














15001000

500

1 2 3 4 5 76

(a)

1 2 3 4 5




75kDa

25kDa

150 kDa100 kDa

50kDa37kDa

20kDa16kDa

(b)


3 Results












0040

0045

0050

0055

0060

0065

0070

0075

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-ESA

T6le

vel

lowastlowast

lowast

(a)

0040

0090

0140

0190

0240

0290

0340

0390

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-CFP

10

-ESA

T6-d

IFN

leve

l

lowastlowastlowast

lowast

(b)




174= 27610










17= 27610 and 119875 lt 0001)










134= 000 and 119875 = 099 for rESAT6 and









080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120


lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)




lowastlowastlowast






334= 358


134= 123 and 119875 = 027 for ConA and 119865

134= 174





080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


15001000

500

1 2 3 4 5 76

(a)

1 2 3 4 5




75kDa

25kDa

150 kDa100 kDa

50kDa37kDa

20kDa16kDa

(b)


3 Results












0040

0045

0050

0055

0060

0065

0070

0075

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-ESA

T6le

vel

lowastlowast

lowast

(a)

0040

0090

0140

0190

0240

0290

0340

0390

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-CFP

10

-ESA

T6-d

IFN

leve

l

lowastlowastlowast

lowast

(b)




174= 27610










17= 27610 and 119875 lt 0001)










134= 000 and 119875 = 099 for rESAT6 and









080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120


lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)




lowastlowastlowast






334= 358


134= 123 and 119875 = 027 for ConA and 119865

134= 174





080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0040

0045

0050

0055

0060

0065

0070

0075

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-ESA

T6le

vel

lowastlowast

lowast

(a)

0040

0090

0140

0190

0240

0290

0340

0390

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

Ant

i-CFP

10

-ESA

T6-d

IFN

leve

l

lowastlowastlowast

lowast

(b)




174= 27610










17= 27610 and 119875 lt 0001)










134= 000 and 119875 = 099 for rESAT6 and









080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120


lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)




lowastlowastlowast






334= 358


134= 123 and 119875 = 027 for ConA and 119865

134= 174





080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


080

085

090

095

100

105

110

115

120

125

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

ESAT

6-in

duce

d SI

lowastlowast

lowast

(a)CF

P10-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

100

090

080

110

120


lowastlowast

(b)

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol100

105

110

115

120

125

130

CFP1

0-E

SAT6

-dIF

N-in

duce

d SI

(c)




lowastlowastlowast






334= 358


134= 123 and 119875 = 027 for ConA and 119865

134= 174





080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


080

100

120

140

160

180

200C

onA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast

(a)

080

100

120

140

160

180

PHA-

indu

ced

SI

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

trol

lowastlowastlowast

lowastlowast lowast

(b)




lowastlowastlowast


114= 897 and 119875 = 0001 and lowast119865

114= 621 and 119875 = 0026 Both 119865




4 Discussion




0

05

1

15

2

25

dIFN

ESAT

6

CFP1

0

CFP1

0-E

SAT6

-dIF

N

Con

A

Stim

ulat

ion

inde

x













5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











5 Main Conclusion



Abbreviations




Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abbreviations




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

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