Research ArticleExpression and Immunogenicity of Two Recombinant FusionProteins Comprising Foot-and-Mouth Disease Virus StructuralProtein VP1 and DC-SIGN-Binding Glycoproteins

Xinsheng Liu12 Jianliang Lv12 Yuzhen Fang12 Peng Zhou12 Yanzhen Lu123 Li Pan12

Zhongwang Zhang12 JunwuMa1 Yongguang Zhang12 and YongluWang12

1State Key Laboratory of Veterinary Etiological Biology OIENational Foot and Mouth Disease Reference LaboratoryKey Laboratory of Animal Virology of Ministry of Agriculture Lanzhou Veterinary Research InstituteChinese Academy of Agricultural Sciences Lanzhou 730046 China2Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and ZoonosesYangzhou 225009 China3College of Veterinary Medicine Gansu Agricultural University Lanzhou Gansu 730070 China

Correspondence should be addressed to Yongguang Zhang zhangyongguangcaascnand Yonglu Wang wangyonglumdhotmailcom

Received 29 April 2017 Revised 22 August 2017 Accepted 28 August 2017 Published 8 October 2017

Academic Editor Marcelo A Soares

Copyright copy 2017 Xinsheng Liu 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

Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccinedevelopment In this study the VP1 gene of foot-and-mouth disease virus (FMDV) serotype A was fused with the gene encodinghuman immunodeficiency virus (HIV) membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV) envelopeglycoprotein E2 both of which are DC-SIGN-binding glycoproteins After codon optimization the VP1 protein and the tworecombinant VP1-gp120 and VP1-E2 fusion proteins were expressed in Sf9 insect cells using the insect cell-baculovirus expressionsystem Western blotting showed that the VP1 protein and two recombinant VP1-gp120 and VP1-E2 fusion proteins were correctlyexpressed in the Sf9 insect cells and had good reactogenicity Guinea pigs were then immunized with the purified proteins and theresulting humoral and cellular immune responses were analyzed The VP1-gp120 and VP1-E2 fusion proteins induced significantlyhigher specific anti-FMDV antibody levels than the VP1 protein and stronger cell-mediated immune responsesThis study providesa new perspective for the development of novel FMDV subunit vaccines

1 Introduction

Foot-and-mouth disease (FMD) is an acute severe andhighly contagious disease that is caused by foot-and-mouthdisease virus (FMDV) which infects cloven-hoofed animalssuch as cattle pigs and sheep FMDV is characterized byrapid transmission high morbidity and low mortality andcan cause serious economic losses and social impacts [12] Vaccination is the most reliable and effective meansof preventing and controlling FMD Although traditionalFMD vaccines play an important role in the prevention andcontrol of FMD they present a number of shortcomings such

as incomplete inactivation of the virus and escape of liveviruses from vaccine production facilities [3 4] Thereforethe development of safe and effective new genetically engi-neered vaccines is required for the prevention control andeventual elimination of FMD in the future Many geneticallyengineered FMDVvaccines have recently emerged includingsubunit vaccines edible vaccines synthetic peptide vac-cines gene-deleted vaccines live vector vaccines and nucleicacid vaccines However the immune effects of these newgenetically engineered vaccines are not superior to those oftraditional inactivated vaccines Therefore vaccine researchhas focused on the adoption of new design strategies to

HindawiBioMed Research InternationalVolume 2017 Article ID 7658970 9 pageshttpsdoiorg10115520177658970

2 BioMed Research International

further improve the immunogenicity of these new geneticallyengineered vaccines

Dendritic cells (DCs) are the most potent specializedantigen-presenting cells in the body DCs capture processand present antigens through their surface antigen recep-tors DCs participate in the activation of naıve T-cells andinduce their proliferation and differentiation to elicit a strongimmune response [5 6] AlthoughDCs have a potent antigencapture function their nonspecific mechanisms of antigencapture and presentation could affect vaccine presentationand further influence the immune effects of vaccines [7]Therefore improvement of the immunogenicity of vaccinesby targeting antigens to DCs has become an emerging newvaccine design strategy DC-SIGN (dendritic cell-specificintercellular adhesion molecule-3-grabbing nonintegrin)also known as CD209 is a C-type lectin receptor on the sur-face of DC membranes that can specifically bind to a varietyof ligands including highly glycosylated proteins Lewis-typeblood antigens (Lex Ley Lea and Leb) intercellular adhesionmolecules and some virus envelope glycoproteins [8 9] Theuse of chemical crosslinking or genetic engineering methodsto combine vaccine antigens with the DC-SIGN ligand canspecifically target vaccine antigens to DCs and thus signifi-cantly improve the immune effects of a vaccine [8 10ndash13]

The FMDV capsid protein consists of four structural pro-teins (VP1 VP2 VP3 and VP4) The VP1 protein is exposedon the surface of the viral capsid and is the main protein thatdetermines the serotype and genotype of FMDV The VP1protein contains several antigen epitopes that can stimulatethe production of effective humoral and cellular immuneresponses and induce the production of specific neutral-izing antibodies [1] Related studies have shown that the HIVmembrane glycoprotein gp120 and the HCV envelope glyco-protein E2 are both high-affinity ligands for DC-SIGN thatare capable of specifically binding toDC-SIGN tomediate theinternalization and presentation of exogenous antigens [14ndash18] In this study the DC-SIGN-specific ligands HIV mem-brane glycoprotein gp120 andHCV envelope glycoprotein E2were separately fused with the FMDV structural protein VP1and expressed using the insect cell-baculovirus expressionsystem The immunogenicity of these two DC-targetingrecombinant FMDV VP1 fusion proteins was evaluated inguinea pigs and compared with that of the VP1 protein

2 Materials and Methods

21 Ethics Statement All guinea pig experiments were per-formed in a biosafety level 3 laboratory at Lanzhou Vet-erinary Research Institute (LVRI) Chinese Academy ofAgricultural Sciences (CAAS) This study was performedwith the approval of the Institutional Animal Use and CareCommittee of CAAS and all experiments were performedaccording to local (Regulations for the Administration ofAffairs concerning Experimental Animals) and international(Dolan K 2007 Second Edition of Laboratory Animal LawBlackwell UK) guidelines on the ethical use of animals Allguinea pigs used in the present study were humanely bredduring the experiment and euthanized at the end of theexperiment

22 Virus Plasmids Cells and Reagents FMDV serotype Astrain AF72 was isolated and maintained in our laboratory(the strain was maintained and provided by the LanzhouVeterinary Research Institute (LVRI) Chinese Academy ofAgricultural Sciences (CAAS)) E coli TOP10 competentcells the baculovirus transfer vector pFastBac 1 and Ecoli DH10Bac competent cells were purchased from Invit-rogen (California USA) Spodoptera frugiperda (Sf9) insectcells (Invitrogen USA) were cultured in Sf-900 II SFMmedium (Invitrogen USA) containing 5 heat-inactivatedfetal bovine serum (FBS Gibco USA) at 27∘C in an incubatorwith 5 CO

2 Restriction enzymes were purchased from

New England Biolabs (NEB) the transfection Cellfectin IIReagent and Gracersquos Insect Medium were purchased fromInvitrogen the mouse anti-His tag monoclonal antibody andHRP-conjugated goat anti-mouse IgG were purchased fromAbbkine (California USA)

23 Sequence Design and Synthesis The RNeasy Mini Kit(Qiagen USA) was used to extract genomic RNA from theFMDV serotype A strain AF72 stored in our laboratoryThe sequence of the gene encoding the structural proteinVP1 was amplified using the primer pair VP1-FR (VP1-F 51015840-ACTACCACCACTGGTGAG-31015840 and VP1-R 51015840-TTACAG-CAGCTGCTTGGCAGG-31015840) The PCR product was clonedusing the TOPO TA Cloning Kit (Invitrogen USA) andthe resulting plasmidswere sent to SangonBiotech (ShanghaiChina) for sequencing The VP1 gene sequence of the AF72strain obtained from sequencing was optimized according tothe codon preference of the insect cells by Sangon Biotech(Shanghai China) along with introduction of a His tag atthe end of the VP1 sequence and the restriction enzymesites BamHI and HindIII The sequence of the C2-V3 regionof HIV glycoprotein gp120 (accession number CAA74763)or the HCV envelope glycoprotein E2 (accession numberJN870282) published in GenBank was fused with the VP1sequence of FMDV serotype A strain AF72 through a(Gly3Ser)5linker to obtain two recombinant fusion genes

with codon preferences consistent with that of insect cellsSimilarly the restriction enzyme sites BamHI and HindIIIand a His tag were introduced in the fusion sequence andsynthesized by Sangon Biotech (Shanghai China)

24 Construction of Recombinant Baculovirus Plasmids Thesynthesized gene products encodingVP1VP1-gp120VP1-E2and the pFastBac 1 vector (Invitrogen USA) were digestedwith BamHI and HindIII Then the extracted VP1 VP1-gp120 and VP1-E2 fragments were ligated separately withthe pFastBac 1 fragment using T4 DNA ligase at 16∘C for10 hours The ligation products were transformed into Ecoli DH5120572 competent cells and the positive clones obtainedby blue-white screening were inoculated into LB mediumcontaining ampicillin and cultured at 37∘C for 12 hours inan incubator shaker at 220 rpm The plasmids pFastBac-VP1pFastBac-VP1-gp120 and pFastBac-VP1-E2 were extractedfrom the positive clones and confirmed by restriction enzymedigestion The constructed recombinant transfer plasmidspFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E-2were used to transform E coli DH10Bac competent cells

BioMed Research International 3

Positive colonies were selected by blue-white screening andthe recombinant bacmids were extracted and characterizedusing PCR with the universal M13 primers (M13-F 51015840-GTT-TTCCCAGTCACGAC-31015840 and M13-R 51015840-CAGGAAACA-GCTATGAC-31015840) The correct recombinant bacmids werenamed rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E-2 respectively

25 Preparation of Recombinant Baculoviruses A 1120583g quan-tity of the recombinant bacmids and 6 120583L of Cellfectin IIReagent were separately diluted using 100 120583L of incompleteGracersquos medium (without antibiotics and FBS) After mixingwell the twomixtureswere combined gentlymixed and thenincubated at room temperature for 45 minutes to preparethe rBacmid liposomes Sf9 monolayer cells that were in agood growth stage in 6-well plates (9 times 105 cellswell) werewashed twice with incomplete Gracersquos medium and then cov-ered with the rBacmid liposomes The cells were incubatedat 27∘C for 5 hours The culture medium was discardedcomplete Gracersquos medium (containing antibiotics and FBS)was added and the cells were incubated at 27∘C After theappearance of cytopathic effects the cells were removed fromthe plate placed in a centrifuge tube shaken vigorouslyand centrifuged at 1000timesg for 15 minutes The supernatantscontaining the P1 recombinant baculoviral stocks (rBac-VP1 rBac-VP1-gp120 and rBac-VP1-E2) were collected Therecombinant baculovirus was subcultured in Sf9 cells to thesecond passage (titer of approximately 107 pfumL) and thenstored at 4∘C for later use

26 Expression and Purification of Recombinant Proteins Sf9cells were seeded into 1000mL cell culture flasks at a densityof 2 times 106 cellsmL and infected with the P2 recombinantbaculovirus rBac-VP1 rBac-VP1-gp120 or rBac-VP1-E2 whenthe cells were in the logarithmic phase After culturing for48ndash72 hours the cells and culture medium were transferredto centrifuge tubes and centrifuged at 10000119892 for 20 minutesat 4∘C The cells were collected and lysed after the additionof protease inhibitor (1 100) by pulse sonication of 6 secondsat 250W at 3-second intervals for a total of 4 minutes Thecell lysate was centrifuged at 10000119892 for 10 minutes at 4∘CThe supernatant was collected and passed through a Ni-chelating affinity column at a flow rate of 05mLminuteTheNi column was equilibrated with 20mMPB buffer at a flowrate of 05mLminute until the OD280 of the effluent reachedbaseline The column was washed with Ni-IDA WashingBuffer (20mMPB 30mM imidazole and 015M NaCl pH80) at a flow rate of 1mLmin until the OD280 of the effluentreached baseline Then the target protein was eluted withNi-IDA Elution Buffer (20mMPB 300mM imidazole and015M NaCl pH 80) at a flow rate of 1mLmin and theeffluent was collected The collected recombinant proteinsolution was added to a dialysis bag dialyzed against 1x PBSovernight and then subjected to 10 sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)

27Western Blotting Thepurified proteins were separated bySDS-PAGE and the proteins in the gel were then transferred

to amembrane under a constant voltage of 100V for 15 hoursFollowing the completion of the transfer the membranewas washed with PBS 4 times for 5 minutes per wash Themembrane was blocked with 5 skimmed milk at 37∘C for 1hour followed by incubation with the primary mouse anti-His-tag monoclonal antibody (1 1000 dilution) and type AFMDVVP1monoclonal antibody (prepared and stored in ourlaboratory 1 1000 dilution) for 1 hour at 37∘C respectivelyThe membrane was washed with TBS-Tween (50mM Tris150mM NaCl and 005 Tween 20 pH 76) 4 times for 5minutes per wash followed by incubation with the secondaryhorseradish peroxidase- (HRP-) labeled goat anti-rabbit IgGantibody (SigmaUSA) at a 1 5000 dilution for 1 hour at 37∘CThe membrane was washed and visualized using an ECLchemiluminescent substrate reagent kit (Thermo ScientificUSA)

28 Guinea Pig Immunization All animal procedures wereapproved by the Ethics Committee of LZVR Twenty-fivespecific pathogen-free- (SPF-) grade healthy guinea pigsweighing 250ndash300 g were randomly divided into 5 groupswith 5 animals per group The experimental groups (threegroups each group containing 5 guinea pigs) were immunizedby intramuscular injection of 1mL of purified VP1 proteinor the VP1-gp120 or VP1-E2 fusion proteins containingapproximately 02mg purified proteins with ISA-206 adju-vant (China Agricultural Vet Bio Science and TechnologyCo Lanzhou China) at an adjuvant antigen ratio of 1 1on days 0 and 21 respectively A PBSISA 206 mixturewas used as the negative immunization control (119899 = 5)and the commercially available inactivated vaccine (ChinaAgricultural Vet Bio Science and Technology Co LanzhouChina) was used as the positive immunization control (119899 =5) Blood samples were collected on days 7 14 21 and 28 afterthe first immunization and serum was isolated for antibodyand cytokine detection

29 Detection of Anti-FMDV-Specific Antibodies by ELISAThe FMDV-specific IgG antibody titers were measured inthe collected guinea pig serum samples via an indirectenzyme-linked immunosorbent assay (ELISA) In detailthe inactivated whole-virus antigen of FMDV serotype A(Diagnostic Products Center LVRI Lanzhou China) wasdiluted with 01M bicarbonate buffer (pH 96) at a 1 5ratio Then a 96-well flat-bottomed plate was coated withthe diluted antigen at 100 120583L per well The plate was sealedand incubated at 4∘C overnight The plate was washed withPBST 3 times and blocked with 5 skimmed milk in PBSTfor 1 hour at 37∘C The plate was then washed with PBST3 times followed by the addition of 100 120583L of the serumsamples (1 10 dilution) and incubation at 37∘C for 1 hourControl wells were set aside for the negative positive andblank controls The plate was washed with PBST 3 timesfollowed by incubation with 100 120583L of HRP-labeled rabbitanti-guinea pig IgG diluted at 1 1000 (Diagnostic ProductsCenter LVRI Lanzhou China) for 1 hour The plate waswashed with PBST 5 times followed by the addition of 50120583Lof the o-phenylenediamine dihydrochloride (OPD) substrate

4 BioMed Research International

and incubation at 37∘C for 15 minutes Then 50 120583L of stopsolution was added and absorbance was determined at492 nm

210 Virus Neutralizing Antibody Test (VNT) The FMDV-specific neutralizing antibody titers from serum samples at 7days after the booster immunization were determined usingthe VNT with BHK-21 cells according to the OIE protocol[19] Briefly 50 120583L of twofold serial serum dilutions startingat 1 2 was coincubated with equal volumes of viral stockcontaining 100 TCID

50(50 tissue culture infective doses)

of FMDV AF72 in 96-well plates (Corning USA) at 37∘Cfor 1 h Then cells were added to the mixture as indicators ofresidual infectivityThe plates were incubated at 37∘C for 72 hand the cells were fixed and stained with 10 methanol and005 methylene blue solution (prepared with formaldehydesolution) The neutralizing antibody titers were evaluated asthe reciprocal log

10of the highest dilution that neutralized

100 TCID50of FMDV in 50 of the wells

211 Lymphocyte Proliferation Assay Blood samples werecollected from the guinea pigs in each group at 7 days afterthe booster immunization Peripheral blood lymphocyteswere isolated using the Guinea Pig Lymphocyte SeparationSolution Kit (Solarbio China) The isolated lymphocyteswere resuspended in RPMI 1640 medium containing 1antibiotics seeded into a 96-well plate at a density of 1 times105 cellswell and cultured in a 5 CO

2incubator at 37∘C

for 24 hours to allow attachment of the cells to the plateThen the FMDV antigen was added to each sample at afinal concentration of 10120583gmL For each sample RPMI 1640mediumwas added as a negative control and concanavalin A(ConA) at a final concentration of 5 120583gmL was added as thepositive control After incubation at 37∘C for 72 hours cellproliferation was examined using the MTT Cell ProliferationAssay Kit (Solarbio China) In detail the supernatant wascarefully removed and 90 120583L of fresh medium was addedfollowed by the addition of 10 120583L of MMT and incubationfor 4 hours After incubation the supernatant was discardedand 110 120583Lof formazan solubilization solutionwas addedTheplate was placed on a shaker at low speed for 10 minutes tofully dissolve the crystals The OD value of each well wasread at 490 nmThe results were expressed as the stimulationindex (SI ratio of stimulated sample unstimulated sample atOD490 nm)

212 Cytokine Analysis Serum samples were collected fromthe guinea pigs in each group at 7 days after boosterimmunization The levels of the cytokine IFN-120574 were exam-ined in the serum samples from each group using the RatCytokine Antibody Array CYT-1 (RayBiotech Norcross GAUSA) and differences in the IFN-120574 levels were analyzedThe fluorescence signals were read using an InnoScan 300Microarray Scanner and analyzed using the data analysissoftware QAM-CYT-1 The assay and data analysis wereperformed by the RayBiotech Technical Service Department(Guangzhou China)

213 Statistical Analysis Statistical significance among thedifferent experimental groups was determined using the one-way ANOVA Differences were considered significant whenthe 119875 value was less than 005

3 Results

31 Confirmation of the Recombinant Transfer Vectors andBacmids The synthesized gene products for the VP1 proteinsand the VP1-gp120 and VP1-E2 recombinant fusion proteinswere digested at the BamHI and HindIII recognition sitesand cloned into the pFastBac 1 vector to construct therecombinant transfer vectors pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 respectively (Figure 1(a)) Therecombinant transfer vectors were digested with BamHIand HindIII to obtain fragments of 675 bp 1073 bp and1944 bp respectively by agarose gel electrophoresis the sizeof each fragment was consistent with the expected size(Figure 1(b)) The sequencing results indicated that theVP1 VP1-gp120 and VP1-E2 sequences in the recombinanttransfer vectors were correct Additionally the recombinantbacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 were confirmed by PCR with the M13 universalprimers to yield specific amplification fragments of approx-imately 29 kb 33 kb and 42 kb respectively (Figure 1(c))indicating that the recombinant bacmids were constructedcorrectly

32 Expression and Characterization of the RecombinantProteins Apparent cytopathic effects were observed in theSf9 insect cells 72 hours after transfection with the recombi-nant baculoviral bacmids rBacmid-VP1 rBacmid-VP1-gp120and rBacmid-VP1-E2 These cytopathic effects manifestedas enlarged round cells with enlarged nuclei that filled theentire cytoplasm and poorly refractive particles in the nucleiThe cells and supernatants were harvested and lysed andthe expressed proteins were detected by western blottingThe results showed that the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins were expressedcorrectly in the Sf9 cells the molecular weights of theexpressed proteins were approximately 30 kDa 35 kDa and69 kDa respectively (Figure 2) Additionally the expressedproteins reacted not only with the mouse anti-His tagmonoclonal antibody (Figure 2(a)) but also with the anti-FMDV VP1 monoclonal antibody (Figure 2(b)) indicatingthat the expressedVP1 protein and theVP1-gp120 andVP1-E2recombinant fusion proteins had good reactogenicity

33 Antibody Responses during Immunization To assess theimmunogenicity of the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins in guinea pigs bloodsamples were collected from all guinea pigs at 7 days afterbooster immunization and anti-FMDV-specific IgG levelsin the serum were determined using indirect ELISA Asshown in Figure 3 the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine all effectively induced specific anti-FMDV serotypeA IgG antibodies and significant increases in serum IgG

BioMed Research International 5

VP1VP1-gp120VP1-E2

P pHSV40 pA

Tn7L

f1 ori

AmpPUC ori

Tn7R

Gentamicin

pFastBac 148 kb

(a)

100

250

500750

10001500200030005000

100

250

500

75010001500200030005000

100

250

500

7501000

1500200030005000

M 1 2 M 3 4 M 5 6

(b)

M 1 M 2 M 3

1000

2000

3000

4000500060008000

10000

1000

2000

30004000500060008000

10000

2000

3000

4000500060008000

10000

(c)

Figure 1 Design of the recombinant transfer vector for theVP1 protein and theVP1-gp120 andVP1-E2 fusion proteins and confirmation of therecombinant transfer vector and baculovirus bacmids119872 indicates the DNA size marker (a) Sites of insertion of the coding sequences for theVP1 protein and the VP1-gp120 andVP1-E2 fusion proteins in the baculovirus transfer vector pFastBac 1 (b)The recombinant transfer vectorspFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2were digestedwith the restriction endonucleasesBamHI andHindIII and subjectedto agarose gel electrophoresis Lanes 1 3 and 5 undigested plasmids Lanes 2 4 and 6 pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 digestedwithBamHI andHindIII respectively (c)The recombinant bacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2were amplified by PCR with the M13 universal primers and the PCR products were subjected to agarose gel electrophoresis Lanes 1 2 and3 PCR products from rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 respectively

6 BioMed Research International

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(a)

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(b)

Figure 2 Western blotting analysis of the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (a)Western blotting analysis using themouse anti-His tag monoclonal antibody as the primary antibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 andVP1-E2 fusion proteins respectively (b) Western blotting analysis using the anti-type A FMDV VP1 monoclonal antibody as the primaryantibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins respectively

antibody levels were observed over time after the initialimmunization in all groups except the PBS negative controlgroup The serum IgG antibody levels induced by the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were significantly lower than the levels produced inthe traditional inactivated vaccine group (119875 lt 005 Figure 3)but significantly higher than the levels in the PBS group (119875 lt005 Figure 3) within 28 days after the first immunizationImportantly the serum IgG antibody levels induced by theVP1-gp120 and VP1-E2 recombinant fusion proteins werehigher than the levels induced by the VP1 protein alone(119875 lt 005 Figure 3) after booster immunization suggestingthat fusion of the VP1 protein to the gp120 and E2 proteinsenhanced the ability of theVP1 protein to induce specific anti-FMDV IgG antibodies in guinea pigs

Furthermore the specific neutralizing antibodies againstFMDV serotype A of the immunized guinea pigs in eachgroup at 7 days after the booster immunization were assessedby VNT (Figure 4) The neutralizing antibody titers inthe VP1 protein the VP1-gp120 and VP1-E2 recombinantfusion proteins and the inactivated vaccine groups showedsignificantly higher FMDV-neutralizing activity than theantibody responses of the PBS group (119875 lt 005 Figure 4)Meanwhile the neutralizing antibody titers of VP1-gp120 andVP1-E2 recombinant fusion proteins were higher than thetiters induced by the VP1 protein (119875 lt 005 Figure 4)indicating that recombinant fusion strategy could enhancethe ability of the VP1 protein to induce specific neutralizingantibody in guinea pigs

34 Cell-Mediated Immune Responses Peripheral blood lym-phocytes were isolated from the guinea pigs 7 days after

booster immunization The specific proliferative responsesof the peripheral lymphocytes from the guinea pigs wereexamined using theMTT colorimetric assayTheproliferativeresponses in splenic lymphocytes from all guinea pigs werestimulated by concanavalin A (Figure 5) with no significantdifferences between groups (119875 gt 005 Figure 5) Dif-ferences in lymphocyte proliferation were observed amongall groups incubated with the inactivated FDMV antigenIn detail lymphocytes from guinea pigs immunized withthe VP1 protein VP1-gp120 and VP1-E2 recombinant fusionproteins or inactivated vaccine exhibited significantly higherproliferation levels than the PBS control group (119875 lt 005Figure 5) The lymphocytes of the group immunized withthe inactivated vaccine showed the highest proliferation levelfollowed by the groups immunized with the recombinantproteins in the order VP1-gp120 VP1-E2 and VP1 proteinAlthough lymphocyte proliferation was higher in the VP1-gp120 and VP1-E2 immunization groups compared to theVP1 protein immunization group the differences betweenthe groups were not significant (119875 gt 005 Figure 5)Furthermore to assess the cytokine levels in guinea pig seraafter immunization the serum IFN-120574 content was measuredusing a commercially available cytokine antibody array Theresults showed that the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine induced higher IFN-120574 levels in guinea pigs than inthe PBS negative control (119875 gt 005 Figure 6) The abilityof the inactivated vaccine to induce IFN-120574 production wassignificantly greater than the ability of the three recombinantproteins (119875 gt 005 Figure 6) whereas no significantdifferences (119875 lt 005 Figure 6) were observed between theVP1 VP1-gp120 and VP1-E-2 recombinant proteins

BioMed Research International 7

0

1

2

3

4

OD492

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

14 21 287

(d)

Figure 3 The VP1 protein and the VP1-gp120 and VP1-E2 fusionproteins induced the production of specific anti-FMDV IgG anti-bodies in guinea pig serum (each group contained 5 guinea pigs 119899 =5) Serum samples were collected from all guinea pigs at 7 days afterbooster immunization and the specific anti-FMDV IgG antibodylevels were determined by indirect ELISA PBSISA 206 mixturewas used as the negative control (119899 = 5) and the commerciallyavailable inactivated vaccinewas used as the positive control (119899 = 5)Significant values (lowast119875 lt 005) are indicated by an asterisk

0

1

2

3

Neu

tral

izin

g an

tibod

y tit

ers

28 d

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowast

lowast

Figure 4 Serum neutralizing antibody titers of immunized guineapigs (each group contained 5 guinea pigs 119899 = 5) at 7 days after boosterimmunization Significant values (lowast119875 lt 005) are indicated by anasterisk

4 Discussion

The baculovirus insect cell expression system is one of themost commonly used eukaryotic expression systems and isfast and efficient Hundreds of genes from animals plantsviruses bacteria and fungi have been highly expressed ininsect cells [20ndash22] The expressed exogenous proteins canbe posttranslationally modified in the cell (ie glycosylation

ConAFMDV antigen

VP1 VP1-gp120 VP1-E2 Inactivatedvaccine

PBS

lowast

0

1

2

3

4

Stim

ulat

ion

inde

x (S

I)

Figure 5 Specific proliferative responses of peripheral blood lym-phocytes from guinea pigs detected by MTT colorimetry Stimula-tion index (SI) means the ratio of stimulated sample unstimulatedsample at OD490 nm Significant values (lowast119875 lt 005) are indicatedby an asterisk

VP1-gp120VP1

VP1-E2

Inactivated vaccinePBS

400

300

200

100

0

(pg

ml)

lowast

lowast

IFN-

Figure 6 IFN-120574 levels induced by the VP1 protein (119899 = 5) and theVP1-gp120 (119899 = 5) and VP1-E2 (119899 = 5) fusion proteins in guinea pigserum Significant values (lowast119875 lt 005) are indicated by an asterisk

phosphorylation and acylation) and therefore this expres-sion system can be used to obtain a large number ofsoluble recombinant proteins that are functionally similarto the natural proteins [20 23] The HIV gp120 and HCVE2 proteins are both viral envelope glycoproteins that playimportant roles in mediating binding of the virus to itsreceptor The glycosylation of these proteins has a greatimpact on their antigenicity Eukaryotic expression systemscan glycosylate the expressed proteins and maintain theirnatural structure to the maximum extent Therefore thisstudy used the baculovirus insect cell eukaryotic expressionsystem to express the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

BioMed Research International 3

Positive colonies were selected by blue-white screening andthe recombinant bacmids were extracted and characterizedusing PCR with the universal M13 primers (M13-F 51015840-GTT-TTCCCAGTCACGAC-31015840 and M13-R 51015840-CAGGAAACA-GCTATGAC-31015840) The correct recombinant bacmids werenamed rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E-2 respectively

25 Preparation of Recombinant Baculoviruses A 1120583g quan-tity of the recombinant bacmids and 6 120583L of Cellfectin IIReagent were separately diluted using 100 120583L of incompleteGracersquos medium (without antibiotics and FBS) After mixingwell the twomixtureswere combined gentlymixed and thenincubated at room temperature for 45 minutes to preparethe rBacmid liposomes Sf9 monolayer cells that were in agood growth stage in 6-well plates (9 times 105 cellswell) werewashed twice with incomplete Gracersquos medium and then cov-ered with the rBacmid liposomes The cells were incubatedat 27∘C for 5 hours The culture medium was discardedcomplete Gracersquos medium (containing antibiotics and FBS)was added and the cells were incubated at 27∘C After theappearance of cytopathic effects the cells were removed fromthe plate placed in a centrifuge tube shaken vigorouslyand centrifuged at 1000timesg for 15 minutes The supernatantscontaining the P1 recombinant baculoviral stocks (rBac-VP1 rBac-VP1-gp120 and rBac-VP1-E2) were collected Therecombinant baculovirus was subcultured in Sf9 cells to thesecond passage (titer of approximately 107 pfumL) and thenstored at 4∘C for later use

26 Expression and Purification of Recombinant Proteins Sf9cells were seeded into 1000mL cell culture flasks at a densityof 2 times 106 cellsmL and infected with the P2 recombinantbaculovirus rBac-VP1 rBac-VP1-gp120 or rBac-VP1-E2 whenthe cells were in the logarithmic phase After culturing for48ndash72 hours the cells and culture medium were transferredto centrifuge tubes and centrifuged at 10000119892 for 20 minutesat 4∘C The cells were collected and lysed after the additionof protease inhibitor (1 100) by pulse sonication of 6 secondsat 250W at 3-second intervals for a total of 4 minutes Thecell lysate was centrifuged at 10000119892 for 10 minutes at 4∘CThe supernatant was collected and passed through a Ni-chelating affinity column at a flow rate of 05mLminuteTheNi column was equilibrated with 20mMPB buffer at a flowrate of 05mLminute until the OD280 of the effluent reachedbaseline The column was washed with Ni-IDA WashingBuffer (20mMPB 30mM imidazole and 015M NaCl pH80) at a flow rate of 1mLmin until the OD280 of the effluentreached baseline Then the target protein was eluted withNi-IDA Elution Buffer (20mMPB 300mM imidazole and015M NaCl pH 80) at a flow rate of 1mLmin and theeffluent was collected The collected recombinant proteinsolution was added to a dialysis bag dialyzed against 1x PBSovernight and then subjected to 10 sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)

27Western Blotting Thepurified proteins were separated bySDS-PAGE and the proteins in the gel were then transferred

to amembrane under a constant voltage of 100V for 15 hoursFollowing the completion of the transfer the membranewas washed with PBS 4 times for 5 minutes per wash Themembrane was blocked with 5 skimmed milk at 37∘C for 1hour followed by incubation with the primary mouse anti-His-tag monoclonal antibody (1 1000 dilution) and type AFMDVVP1monoclonal antibody (prepared and stored in ourlaboratory 1 1000 dilution) for 1 hour at 37∘C respectivelyThe membrane was washed with TBS-Tween (50mM Tris150mM NaCl and 005 Tween 20 pH 76) 4 times for 5minutes per wash followed by incubation with the secondaryhorseradish peroxidase- (HRP-) labeled goat anti-rabbit IgGantibody (SigmaUSA) at a 1 5000 dilution for 1 hour at 37∘CThe membrane was washed and visualized using an ECLchemiluminescent substrate reagent kit (Thermo ScientificUSA)

28 Guinea Pig Immunization All animal procedures wereapproved by the Ethics Committee of LZVR Twenty-fivespecific pathogen-free- (SPF-) grade healthy guinea pigsweighing 250ndash300 g were randomly divided into 5 groupswith 5 animals per group The experimental groups (threegroups each group containing 5 guinea pigs) were immunizedby intramuscular injection of 1mL of purified VP1 proteinor the VP1-gp120 or VP1-E2 fusion proteins containingapproximately 02mg purified proteins with ISA-206 adju-vant (China Agricultural Vet Bio Science and TechnologyCo Lanzhou China) at an adjuvant antigen ratio of 1 1on days 0 and 21 respectively A PBSISA 206 mixturewas used as the negative immunization control (119899 = 5)and the commercially available inactivated vaccine (ChinaAgricultural Vet Bio Science and Technology Co LanzhouChina) was used as the positive immunization control (119899 =5) Blood samples were collected on days 7 14 21 and 28 afterthe first immunization and serum was isolated for antibodyand cytokine detection

29 Detection of Anti-FMDV-Specific Antibodies by ELISAThe FMDV-specific IgG antibody titers were measured inthe collected guinea pig serum samples via an indirectenzyme-linked immunosorbent assay (ELISA) In detailthe inactivated whole-virus antigen of FMDV serotype A(Diagnostic Products Center LVRI Lanzhou China) wasdiluted with 01M bicarbonate buffer (pH 96) at a 1 5ratio Then a 96-well flat-bottomed plate was coated withthe diluted antigen at 100 120583L per well The plate was sealedand incubated at 4∘C overnight The plate was washed withPBST 3 times and blocked with 5 skimmed milk in PBSTfor 1 hour at 37∘C The plate was then washed with PBST3 times followed by the addition of 100 120583L of the serumsamples (1 10 dilution) and incubation at 37∘C for 1 hourControl wells were set aside for the negative positive andblank controls The plate was washed with PBST 3 timesfollowed by incubation with 100 120583L of HRP-labeled rabbitanti-guinea pig IgG diluted at 1 1000 (Diagnostic ProductsCenter LVRI Lanzhou China) for 1 hour The plate waswashed with PBST 5 times followed by the addition of 50120583Lof the o-phenylenediamine dihydrochloride (OPD) substrate

4 BioMed Research International

and incubation at 37∘C for 15 minutes Then 50 120583L of stopsolution was added and absorbance was determined at492 nm

210 Virus Neutralizing Antibody Test (VNT) The FMDV-specific neutralizing antibody titers from serum samples at 7days after the booster immunization were determined usingthe VNT with BHK-21 cells according to the OIE protocol[19] Briefly 50 120583L of twofold serial serum dilutions startingat 1 2 was coincubated with equal volumes of viral stockcontaining 100 TCID

50(50 tissue culture infective doses)

of FMDV AF72 in 96-well plates (Corning USA) at 37∘Cfor 1 h Then cells were added to the mixture as indicators ofresidual infectivityThe plates were incubated at 37∘C for 72 hand the cells were fixed and stained with 10 methanol and005 methylene blue solution (prepared with formaldehydesolution) The neutralizing antibody titers were evaluated asthe reciprocal log

10of the highest dilution that neutralized

100 TCID50of FMDV in 50 of the wells

211 Lymphocyte Proliferation Assay Blood samples werecollected from the guinea pigs in each group at 7 days afterthe booster immunization Peripheral blood lymphocyteswere isolated using the Guinea Pig Lymphocyte SeparationSolution Kit (Solarbio China) The isolated lymphocyteswere resuspended in RPMI 1640 medium containing 1antibiotics seeded into a 96-well plate at a density of 1 times105 cellswell and cultured in a 5 CO

2incubator at 37∘C

for 24 hours to allow attachment of the cells to the plateThen the FMDV antigen was added to each sample at afinal concentration of 10120583gmL For each sample RPMI 1640mediumwas added as a negative control and concanavalin A(ConA) at a final concentration of 5 120583gmL was added as thepositive control After incubation at 37∘C for 72 hours cellproliferation was examined using the MTT Cell ProliferationAssay Kit (Solarbio China) In detail the supernatant wascarefully removed and 90 120583L of fresh medium was addedfollowed by the addition of 10 120583L of MMT and incubationfor 4 hours After incubation the supernatant was discardedand 110 120583Lof formazan solubilization solutionwas addedTheplate was placed on a shaker at low speed for 10 minutes tofully dissolve the crystals The OD value of each well wasread at 490 nmThe results were expressed as the stimulationindex (SI ratio of stimulated sample unstimulated sample atOD490 nm)

212 Cytokine Analysis Serum samples were collected fromthe guinea pigs in each group at 7 days after boosterimmunization The levels of the cytokine IFN-120574 were exam-ined in the serum samples from each group using the RatCytokine Antibody Array CYT-1 (RayBiotech Norcross GAUSA) and differences in the IFN-120574 levels were analyzedThe fluorescence signals were read using an InnoScan 300Microarray Scanner and analyzed using the data analysissoftware QAM-CYT-1 The assay and data analysis wereperformed by the RayBiotech Technical Service Department(Guangzhou China)

213 Statistical Analysis Statistical significance among thedifferent experimental groups was determined using the one-way ANOVA Differences were considered significant whenthe 119875 value was less than 005

3 Results

31 Confirmation of the Recombinant Transfer Vectors andBacmids The synthesized gene products for the VP1 proteinsand the VP1-gp120 and VP1-E2 recombinant fusion proteinswere digested at the BamHI and HindIII recognition sitesand cloned into the pFastBac 1 vector to construct therecombinant transfer vectors pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 respectively (Figure 1(a)) Therecombinant transfer vectors were digested with BamHIand HindIII to obtain fragments of 675 bp 1073 bp and1944 bp respectively by agarose gel electrophoresis the sizeof each fragment was consistent with the expected size(Figure 1(b)) The sequencing results indicated that theVP1 VP1-gp120 and VP1-E2 sequences in the recombinanttransfer vectors were correct Additionally the recombinantbacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 were confirmed by PCR with the M13 universalprimers to yield specific amplification fragments of approx-imately 29 kb 33 kb and 42 kb respectively (Figure 1(c))indicating that the recombinant bacmids were constructedcorrectly

32 Expression and Characterization of the RecombinantProteins Apparent cytopathic effects were observed in theSf9 insect cells 72 hours after transfection with the recombi-nant baculoviral bacmids rBacmid-VP1 rBacmid-VP1-gp120and rBacmid-VP1-E2 These cytopathic effects manifestedas enlarged round cells with enlarged nuclei that filled theentire cytoplasm and poorly refractive particles in the nucleiThe cells and supernatants were harvested and lysed andthe expressed proteins were detected by western blottingThe results showed that the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins were expressedcorrectly in the Sf9 cells the molecular weights of theexpressed proteins were approximately 30 kDa 35 kDa and69 kDa respectively (Figure 2) Additionally the expressedproteins reacted not only with the mouse anti-His tagmonoclonal antibody (Figure 2(a)) but also with the anti-FMDV VP1 monoclonal antibody (Figure 2(b)) indicatingthat the expressedVP1 protein and theVP1-gp120 andVP1-E2recombinant fusion proteins had good reactogenicity

33 Antibody Responses during Immunization To assess theimmunogenicity of the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins in guinea pigs bloodsamples were collected from all guinea pigs at 7 days afterbooster immunization and anti-FMDV-specific IgG levelsin the serum were determined using indirect ELISA Asshown in Figure 3 the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine all effectively induced specific anti-FMDV serotypeA IgG antibodies and significant increases in serum IgG

BioMed Research International 5

VP1VP1-gp120VP1-E2

P pHSV40 pA

Tn7L

f1 ori

AmpPUC ori

Tn7R

Gentamicin

pFastBac 148 kb

(a)

100

250

500750

10001500200030005000

100

250

500

75010001500200030005000

100

250

500

7501000

1500200030005000

M 1 2 M 3 4 M 5 6

(b)

M 1 M 2 M 3

1000

2000

3000

4000500060008000

10000

1000

2000

30004000500060008000

10000

2000

3000

4000500060008000

10000

(c)

Figure 1 Design of the recombinant transfer vector for theVP1 protein and theVP1-gp120 andVP1-E2 fusion proteins and confirmation of therecombinant transfer vector and baculovirus bacmids119872 indicates the DNA size marker (a) Sites of insertion of the coding sequences for theVP1 protein and the VP1-gp120 andVP1-E2 fusion proteins in the baculovirus transfer vector pFastBac 1 (b)The recombinant transfer vectorspFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2were digestedwith the restriction endonucleasesBamHI andHindIII and subjectedto agarose gel electrophoresis Lanes 1 3 and 5 undigested plasmids Lanes 2 4 and 6 pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 digestedwithBamHI andHindIII respectively (c)The recombinant bacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2were amplified by PCR with the M13 universal primers and the PCR products were subjected to agarose gel electrophoresis Lanes 1 2 and3 PCR products from rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 respectively

6 BioMed Research International

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(a)

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(b)

Figure 2 Western blotting analysis of the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (a)Western blotting analysis using themouse anti-His tag monoclonal antibody as the primary antibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 andVP1-E2 fusion proteins respectively (b) Western blotting analysis using the anti-type A FMDV VP1 monoclonal antibody as the primaryantibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins respectively

antibody levels were observed over time after the initialimmunization in all groups except the PBS negative controlgroup The serum IgG antibody levels induced by the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were significantly lower than the levels produced inthe traditional inactivated vaccine group (119875 lt 005 Figure 3)but significantly higher than the levels in the PBS group (119875 lt005 Figure 3) within 28 days after the first immunizationImportantly the serum IgG antibody levels induced by theVP1-gp120 and VP1-E2 recombinant fusion proteins werehigher than the levels induced by the VP1 protein alone(119875 lt 005 Figure 3) after booster immunization suggestingthat fusion of the VP1 protein to the gp120 and E2 proteinsenhanced the ability of theVP1 protein to induce specific anti-FMDV IgG antibodies in guinea pigs

Furthermore the specific neutralizing antibodies againstFMDV serotype A of the immunized guinea pigs in eachgroup at 7 days after the booster immunization were assessedby VNT (Figure 4) The neutralizing antibody titers inthe VP1 protein the VP1-gp120 and VP1-E2 recombinantfusion proteins and the inactivated vaccine groups showedsignificantly higher FMDV-neutralizing activity than theantibody responses of the PBS group (119875 lt 005 Figure 4)Meanwhile the neutralizing antibody titers of VP1-gp120 andVP1-E2 recombinant fusion proteins were higher than thetiters induced by the VP1 protein (119875 lt 005 Figure 4)indicating that recombinant fusion strategy could enhancethe ability of the VP1 protein to induce specific neutralizingantibody in guinea pigs

34 Cell-Mediated Immune Responses Peripheral blood lym-phocytes were isolated from the guinea pigs 7 days after

booster immunization The specific proliferative responsesof the peripheral lymphocytes from the guinea pigs wereexamined using theMTT colorimetric assayTheproliferativeresponses in splenic lymphocytes from all guinea pigs werestimulated by concanavalin A (Figure 5) with no significantdifferences between groups (119875 gt 005 Figure 5) Dif-ferences in lymphocyte proliferation were observed amongall groups incubated with the inactivated FDMV antigenIn detail lymphocytes from guinea pigs immunized withthe VP1 protein VP1-gp120 and VP1-E2 recombinant fusionproteins or inactivated vaccine exhibited significantly higherproliferation levels than the PBS control group (119875 lt 005Figure 5) The lymphocytes of the group immunized withthe inactivated vaccine showed the highest proliferation levelfollowed by the groups immunized with the recombinantproteins in the order VP1-gp120 VP1-E2 and VP1 proteinAlthough lymphocyte proliferation was higher in the VP1-gp120 and VP1-E2 immunization groups compared to theVP1 protein immunization group the differences betweenthe groups were not significant (119875 gt 005 Figure 5)Furthermore to assess the cytokine levels in guinea pig seraafter immunization the serum IFN-120574 content was measuredusing a commercially available cytokine antibody array Theresults showed that the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine induced higher IFN-120574 levels in guinea pigs than inthe PBS negative control (119875 gt 005 Figure 6) The abilityof the inactivated vaccine to induce IFN-120574 production wassignificantly greater than the ability of the three recombinantproteins (119875 gt 005 Figure 6) whereas no significantdifferences (119875 lt 005 Figure 6) were observed between theVP1 VP1-gp120 and VP1-E-2 recombinant proteins

BioMed Research International 7

0

1

2

3

4

OD492

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

14 21 287

(d)

Figure 3 The VP1 protein and the VP1-gp120 and VP1-E2 fusionproteins induced the production of specific anti-FMDV IgG anti-bodies in guinea pig serum (each group contained 5 guinea pigs 119899 =5) Serum samples were collected from all guinea pigs at 7 days afterbooster immunization and the specific anti-FMDV IgG antibodylevels were determined by indirect ELISA PBSISA 206 mixturewas used as the negative control (119899 = 5) and the commerciallyavailable inactivated vaccinewas used as the positive control (119899 = 5)Significant values (lowast119875 lt 005) are indicated by an asterisk

0

1

2

3

Neu

tral

izin

g an

tibod

y tit

ers

28 d

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowast

lowast

Figure 4 Serum neutralizing antibody titers of immunized guineapigs (each group contained 5 guinea pigs 119899 = 5) at 7 days after boosterimmunization Significant values (lowast119875 lt 005) are indicated by anasterisk

4 Discussion

The baculovirus insect cell expression system is one of themost commonly used eukaryotic expression systems and isfast and efficient Hundreds of genes from animals plantsviruses bacteria and fungi have been highly expressed ininsect cells [20ndash22] The expressed exogenous proteins canbe posttranslationally modified in the cell (ie glycosylation

ConAFMDV antigen

VP1 VP1-gp120 VP1-E2 Inactivatedvaccine

PBS

lowast

0

1

2

3

4

Stim

ulat

ion

inde

x (S

I)

Figure 5 Specific proliferative responses of peripheral blood lym-phocytes from guinea pigs detected by MTT colorimetry Stimula-tion index (SI) means the ratio of stimulated sample unstimulatedsample at OD490 nm Significant values (lowast119875 lt 005) are indicatedby an asterisk

VP1-gp120VP1

VP1-E2

Inactivated vaccinePBS

400

300

200

100

0

(pg

ml)

lowast

lowast

IFN-

Figure 6 IFN-120574 levels induced by the VP1 protein (119899 = 5) and theVP1-gp120 (119899 = 5) and VP1-E2 (119899 = 5) fusion proteins in guinea pigserum Significant values (lowast119875 lt 005) are indicated by an asterisk

phosphorylation and acylation) and therefore this expres-sion system can be used to obtain a large number ofsoluble recombinant proteins that are functionally similarto the natural proteins [20 23] The HIV gp120 and HCVE2 proteins are both viral envelope glycoproteins that playimportant roles in mediating binding of the virus to itsreceptor The glycosylation of these proteins has a greatimpact on their antigenicity Eukaryotic expression systemscan glycosylate the expressed proteins and maintain theirnatural structure to the maximum extent Therefore thisstudy used the baculovirus insect cell eukaryotic expressionsystem to express the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

4 BioMed Research International

and incubation at 37∘C for 15 minutes Then 50 120583L of stopsolution was added and absorbance was determined at492 nm

210 Virus Neutralizing Antibody Test (VNT) The FMDV-specific neutralizing antibody titers from serum samples at 7days after the booster immunization were determined usingthe VNT with BHK-21 cells according to the OIE protocol[19] Briefly 50 120583L of twofold serial serum dilutions startingat 1 2 was coincubated with equal volumes of viral stockcontaining 100 TCID

50(50 tissue culture infective doses)

of FMDV AF72 in 96-well plates (Corning USA) at 37∘Cfor 1 h Then cells were added to the mixture as indicators ofresidual infectivityThe plates were incubated at 37∘C for 72 hand the cells were fixed and stained with 10 methanol and005 methylene blue solution (prepared with formaldehydesolution) The neutralizing antibody titers were evaluated asthe reciprocal log

10of the highest dilution that neutralized

100 TCID50of FMDV in 50 of the wells

211 Lymphocyte Proliferation Assay Blood samples werecollected from the guinea pigs in each group at 7 days afterthe booster immunization Peripheral blood lymphocyteswere isolated using the Guinea Pig Lymphocyte SeparationSolution Kit (Solarbio China) The isolated lymphocyteswere resuspended in RPMI 1640 medium containing 1antibiotics seeded into a 96-well plate at a density of 1 times105 cellswell and cultured in a 5 CO

2incubator at 37∘C

for 24 hours to allow attachment of the cells to the plateThen the FMDV antigen was added to each sample at afinal concentration of 10120583gmL For each sample RPMI 1640mediumwas added as a negative control and concanavalin A(ConA) at a final concentration of 5 120583gmL was added as thepositive control After incubation at 37∘C for 72 hours cellproliferation was examined using the MTT Cell ProliferationAssay Kit (Solarbio China) In detail the supernatant wascarefully removed and 90 120583L of fresh medium was addedfollowed by the addition of 10 120583L of MMT and incubationfor 4 hours After incubation the supernatant was discardedand 110 120583Lof formazan solubilization solutionwas addedTheplate was placed on a shaker at low speed for 10 minutes tofully dissolve the crystals The OD value of each well wasread at 490 nmThe results were expressed as the stimulationindex (SI ratio of stimulated sample unstimulated sample atOD490 nm)

212 Cytokine Analysis Serum samples were collected fromthe guinea pigs in each group at 7 days after boosterimmunization The levels of the cytokine IFN-120574 were exam-ined in the serum samples from each group using the RatCytokine Antibody Array CYT-1 (RayBiotech Norcross GAUSA) and differences in the IFN-120574 levels were analyzedThe fluorescence signals were read using an InnoScan 300Microarray Scanner and analyzed using the data analysissoftware QAM-CYT-1 The assay and data analysis wereperformed by the RayBiotech Technical Service Department(Guangzhou China)

213 Statistical Analysis Statistical significance among thedifferent experimental groups was determined using the one-way ANOVA Differences were considered significant whenthe 119875 value was less than 005

3 Results

31 Confirmation of the Recombinant Transfer Vectors andBacmids The synthesized gene products for the VP1 proteinsand the VP1-gp120 and VP1-E2 recombinant fusion proteinswere digested at the BamHI and HindIII recognition sitesand cloned into the pFastBac 1 vector to construct therecombinant transfer vectors pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 respectively (Figure 1(a)) Therecombinant transfer vectors were digested with BamHIand HindIII to obtain fragments of 675 bp 1073 bp and1944 bp respectively by agarose gel electrophoresis the sizeof each fragment was consistent with the expected size(Figure 1(b)) The sequencing results indicated that theVP1 VP1-gp120 and VP1-E2 sequences in the recombinanttransfer vectors were correct Additionally the recombinantbacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 were confirmed by PCR with the M13 universalprimers to yield specific amplification fragments of approx-imately 29 kb 33 kb and 42 kb respectively (Figure 1(c))indicating that the recombinant bacmids were constructedcorrectly

32 Expression and Characterization of the RecombinantProteins Apparent cytopathic effects were observed in theSf9 insect cells 72 hours after transfection with the recombi-nant baculoviral bacmids rBacmid-VP1 rBacmid-VP1-gp120and rBacmid-VP1-E2 These cytopathic effects manifestedas enlarged round cells with enlarged nuclei that filled theentire cytoplasm and poorly refractive particles in the nucleiThe cells and supernatants were harvested and lysed andthe expressed proteins were detected by western blottingThe results showed that the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins were expressedcorrectly in the Sf9 cells the molecular weights of theexpressed proteins were approximately 30 kDa 35 kDa and69 kDa respectively (Figure 2) Additionally the expressedproteins reacted not only with the mouse anti-His tagmonoclonal antibody (Figure 2(a)) but also with the anti-FMDV VP1 monoclonal antibody (Figure 2(b)) indicatingthat the expressedVP1 protein and theVP1-gp120 andVP1-E2recombinant fusion proteins had good reactogenicity

33 Antibody Responses during Immunization To assess theimmunogenicity of the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins in guinea pigs bloodsamples were collected from all guinea pigs at 7 days afterbooster immunization and anti-FMDV-specific IgG levelsin the serum were determined using indirect ELISA Asshown in Figure 3 the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine all effectively induced specific anti-FMDV serotypeA IgG antibodies and significant increases in serum IgG

BioMed Research International 5

VP1VP1-gp120VP1-E2

P pHSV40 pA

Tn7L

f1 ori

AmpPUC ori

Tn7R

Gentamicin

pFastBac 148 kb

(a)

100

250

500750

10001500200030005000

100

250

500

75010001500200030005000

100

250

500

7501000

1500200030005000

M 1 2 M 3 4 M 5 6

(b)

M 1 M 2 M 3

1000

2000

3000

4000500060008000

10000

1000

2000

30004000500060008000

10000

2000

3000

4000500060008000

10000

(c)

Figure 1 Design of the recombinant transfer vector for theVP1 protein and theVP1-gp120 andVP1-E2 fusion proteins and confirmation of therecombinant transfer vector and baculovirus bacmids119872 indicates the DNA size marker (a) Sites of insertion of the coding sequences for theVP1 protein and the VP1-gp120 andVP1-E2 fusion proteins in the baculovirus transfer vector pFastBac 1 (b)The recombinant transfer vectorspFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2were digestedwith the restriction endonucleasesBamHI andHindIII and subjectedto agarose gel electrophoresis Lanes 1 3 and 5 undigested plasmids Lanes 2 4 and 6 pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 digestedwithBamHI andHindIII respectively (c)The recombinant bacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2were amplified by PCR with the M13 universal primers and the PCR products were subjected to agarose gel electrophoresis Lanes 1 2 and3 PCR products from rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 respectively

6 BioMed Research International

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(a)

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(b)

Figure 2 Western blotting analysis of the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (a)Western blotting analysis using themouse anti-His tag monoclonal antibody as the primary antibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 andVP1-E2 fusion proteins respectively (b) Western blotting analysis using the anti-type A FMDV VP1 monoclonal antibody as the primaryantibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins respectively

antibody levels were observed over time after the initialimmunization in all groups except the PBS negative controlgroup The serum IgG antibody levels induced by the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were significantly lower than the levels produced inthe traditional inactivated vaccine group (119875 lt 005 Figure 3)but significantly higher than the levels in the PBS group (119875 lt005 Figure 3) within 28 days after the first immunizationImportantly the serum IgG antibody levels induced by theVP1-gp120 and VP1-E2 recombinant fusion proteins werehigher than the levels induced by the VP1 protein alone(119875 lt 005 Figure 3) after booster immunization suggestingthat fusion of the VP1 protein to the gp120 and E2 proteinsenhanced the ability of theVP1 protein to induce specific anti-FMDV IgG antibodies in guinea pigs

Furthermore the specific neutralizing antibodies againstFMDV serotype A of the immunized guinea pigs in eachgroup at 7 days after the booster immunization were assessedby VNT (Figure 4) The neutralizing antibody titers inthe VP1 protein the VP1-gp120 and VP1-E2 recombinantfusion proteins and the inactivated vaccine groups showedsignificantly higher FMDV-neutralizing activity than theantibody responses of the PBS group (119875 lt 005 Figure 4)Meanwhile the neutralizing antibody titers of VP1-gp120 andVP1-E2 recombinant fusion proteins were higher than thetiters induced by the VP1 protein (119875 lt 005 Figure 4)indicating that recombinant fusion strategy could enhancethe ability of the VP1 protein to induce specific neutralizingantibody in guinea pigs

34 Cell-Mediated Immune Responses Peripheral blood lym-phocytes were isolated from the guinea pigs 7 days after

booster immunization The specific proliferative responsesof the peripheral lymphocytes from the guinea pigs wereexamined using theMTT colorimetric assayTheproliferativeresponses in splenic lymphocytes from all guinea pigs werestimulated by concanavalin A (Figure 5) with no significantdifferences between groups (119875 gt 005 Figure 5) Dif-ferences in lymphocyte proliferation were observed amongall groups incubated with the inactivated FDMV antigenIn detail lymphocytes from guinea pigs immunized withthe VP1 protein VP1-gp120 and VP1-E2 recombinant fusionproteins or inactivated vaccine exhibited significantly higherproliferation levels than the PBS control group (119875 lt 005Figure 5) The lymphocytes of the group immunized withthe inactivated vaccine showed the highest proliferation levelfollowed by the groups immunized with the recombinantproteins in the order VP1-gp120 VP1-E2 and VP1 proteinAlthough lymphocyte proliferation was higher in the VP1-gp120 and VP1-E2 immunization groups compared to theVP1 protein immunization group the differences betweenthe groups were not significant (119875 gt 005 Figure 5)Furthermore to assess the cytokine levels in guinea pig seraafter immunization the serum IFN-120574 content was measuredusing a commercially available cytokine antibody array Theresults showed that the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine induced higher IFN-120574 levels in guinea pigs than inthe PBS negative control (119875 gt 005 Figure 6) The abilityof the inactivated vaccine to induce IFN-120574 production wassignificantly greater than the ability of the three recombinantproteins (119875 gt 005 Figure 6) whereas no significantdifferences (119875 lt 005 Figure 6) were observed between theVP1 VP1-gp120 and VP1-E-2 recombinant proteins

BioMed Research International 7

0

1

2

3

4

OD492

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

14 21 287

(d)

Figure 3 The VP1 protein and the VP1-gp120 and VP1-E2 fusionproteins induced the production of specific anti-FMDV IgG anti-bodies in guinea pig serum (each group contained 5 guinea pigs 119899 =5) Serum samples were collected from all guinea pigs at 7 days afterbooster immunization and the specific anti-FMDV IgG antibodylevels were determined by indirect ELISA PBSISA 206 mixturewas used as the negative control (119899 = 5) and the commerciallyavailable inactivated vaccinewas used as the positive control (119899 = 5)Significant values (lowast119875 lt 005) are indicated by an asterisk

0

1

2

3

Neu

tral

izin

g an

tibod

y tit

ers

28 d

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowast

lowast

Figure 4 Serum neutralizing antibody titers of immunized guineapigs (each group contained 5 guinea pigs 119899 = 5) at 7 days after boosterimmunization Significant values (lowast119875 lt 005) are indicated by anasterisk

4 Discussion

The baculovirus insect cell expression system is one of themost commonly used eukaryotic expression systems and isfast and efficient Hundreds of genes from animals plantsviruses bacteria and fungi have been highly expressed ininsect cells [20ndash22] The expressed exogenous proteins canbe posttranslationally modified in the cell (ie glycosylation

ConAFMDV antigen

VP1 VP1-gp120 VP1-E2 Inactivatedvaccine

PBS

lowast

0

1

2

3

4

Stim

ulat

ion

inde

x (S

I)

Figure 5 Specific proliferative responses of peripheral blood lym-phocytes from guinea pigs detected by MTT colorimetry Stimula-tion index (SI) means the ratio of stimulated sample unstimulatedsample at OD490 nm Significant values (lowast119875 lt 005) are indicatedby an asterisk

VP1-gp120VP1

VP1-E2

Inactivated vaccinePBS

400

300

200

100

0

(pg

ml)

lowast

lowast

IFN-

Figure 6 IFN-120574 levels induced by the VP1 protein (119899 = 5) and theVP1-gp120 (119899 = 5) and VP1-E2 (119899 = 5) fusion proteins in guinea pigserum Significant values (lowast119875 lt 005) are indicated by an asterisk

phosphorylation and acylation) and therefore this expres-sion system can be used to obtain a large number ofsoluble recombinant proteins that are functionally similarto the natural proteins [20 23] The HIV gp120 and HCVE2 proteins are both viral envelope glycoproteins that playimportant roles in mediating binding of the virus to itsreceptor The glycosylation of these proteins has a greatimpact on their antigenicity Eukaryotic expression systemscan glycosylate the expressed proteins and maintain theirnatural structure to the maximum extent Therefore thisstudy used the baculovirus insect cell eukaryotic expressionsystem to express the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

BioMed Research International 5

VP1VP1-gp120VP1-E2

P pHSV40 pA

Tn7L

f1 ori

AmpPUC ori

Tn7R

Gentamicin

pFastBac 148 kb

(a)

100

250

500750

10001500200030005000

100

250

500

75010001500200030005000

100

250

500

7501000

1500200030005000

M 1 2 M 3 4 M 5 6

(b)

M 1 M 2 M 3

1000

2000

3000

4000500060008000

10000

1000

2000

30004000500060008000

10000

2000

3000

4000500060008000

10000

(c)

Figure 1 Design of the recombinant transfer vector for theVP1 protein and theVP1-gp120 andVP1-E2 fusion proteins and confirmation of therecombinant transfer vector and baculovirus bacmids119872 indicates the DNA size marker (a) Sites of insertion of the coding sequences for theVP1 protein and the VP1-gp120 andVP1-E2 fusion proteins in the baculovirus transfer vector pFastBac 1 (b)The recombinant transfer vectorspFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2were digestedwith the restriction endonucleasesBamHI andHindIII and subjectedto agarose gel electrophoresis Lanes 1 3 and 5 undigested plasmids Lanes 2 4 and 6 pFastBac-VP1 pFastBac-VP1-gp120 and pFastBac-VP1-E2 digestedwithBamHI andHindIII respectively (c)The recombinant bacmids rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2were amplified by PCR with the M13 universal primers and the PCR products were subjected to agarose gel electrophoresis Lanes 1 2 and3 PCR products from rBacmid-VP1 rBacmid-VP1-gp120 and rBacmid-VP1-E2 respectively

6 BioMed Research International

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(a)

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(b)

Figure 2 Western blotting analysis of the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (a)Western blotting analysis using themouse anti-His tag monoclonal antibody as the primary antibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 andVP1-E2 fusion proteins respectively (b) Western blotting analysis using the anti-type A FMDV VP1 monoclonal antibody as the primaryantibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins respectively

antibody levels were observed over time after the initialimmunization in all groups except the PBS negative controlgroup The serum IgG antibody levels induced by the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were significantly lower than the levels produced inthe traditional inactivated vaccine group (119875 lt 005 Figure 3)but significantly higher than the levels in the PBS group (119875 lt005 Figure 3) within 28 days after the first immunizationImportantly the serum IgG antibody levels induced by theVP1-gp120 and VP1-E2 recombinant fusion proteins werehigher than the levels induced by the VP1 protein alone(119875 lt 005 Figure 3) after booster immunization suggestingthat fusion of the VP1 protein to the gp120 and E2 proteinsenhanced the ability of theVP1 protein to induce specific anti-FMDV IgG antibodies in guinea pigs

Furthermore the specific neutralizing antibodies againstFMDV serotype A of the immunized guinea pigs in eachgroup at 7 days after the booster immunization were assessedby VNT (Figure 4) The neutralizing antibody titers inthe VP1 protein the VP1-gp120 and VP1-E2 recombinantfusion proteins and the inactivated vaccine groups showedsignificantly higher FMDV-neutralizing activity than theantibody responses of the PBS group (119875 lt 005 Figure 4)Meanwhile the neutralizing antibody titers of VP1-gp120 andVP1-E2 recombinant fusion proteins were higher than thetiters induced by the VP1 protein (119875 lt 005 Figure 4)indicating that recombinant fusion strategy could enhancethe ability of the VP1 protein to induce specific neutralizingantibody in guinea pigs

34 Cell-Mediated Immune Responses Peripheral blood lym-phocytes were isolated from the guinea pigs 7 days after

booster immunization The specific proliferative responsesof the peripheral lymphocytes from the guinea pigs wereexamined using theMTT colorimetric assayTheproliferativeresponses in splenic lymphocytes from all guinea pigs werestimulated by concanavalin A (Figure 5) with no significantdifferences between groups (119875 gt 005 Figure 5) Dif-ferences in lymphocyte proliferation were observed amongall groups incubated with the inactivated FDMV antigenIn detail lymphocytes from guinea pigs immunized withthe VP1 protein VP1-gp120 and VP1-E2 recombinant fusionproteins or inactivated vaccine exhibited significantly higherproliferation levels than the PBS control group (119875 lt 005Figure 5) The lymphocytes of the group immunized withthe inactivated vaccine showed the highest proliferation levelfollowed by the groups immunized with the recombinantproteins in the order VP1-gp120 VP1-E2 and VP1 proteinAlthough lymphocyte proliferation was higher in the VP1-gp120 and VP1-E2 immunization groups compared to theVP1 protein immunization group the differences betweenthe groups were not significant (119875 gt 005 Figure 5)Furthermore to assess the cytokine levels in guinea pig seraafter immunization the serum IFN-120574 content was measuredusing a commercially available cytokine antibody array Theresults showed that the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine induced higher IFN-120574 levels in guinea pigs than inthe PBS negative control (119875 gt 005 Figure 6) The abilityof the inactivated vaccine to induce IFN-120574 production wassignificantly greater than the ability of the three recombinantproteins (119875 gt 005 Figure 6) whereas no significantdifferences (119875 lt 005 Figure 6) were observed between theVP1 VP1-gp120 and VP1-E-2 recombinant proteins

BioMed Research International 7

0

1

2

3

4

OD492

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

14 21 287

(d)

Figure 3 The VP1 protein and the VP1-gp120 and VP1-E2 fusionproteins induced the production of specific anti-FMDV IgG anti-bodies in guinea pig serum (each group contained 5 guinea pigs 119899 =5) Serum samples were collected from all guinea pigs at 7 days afterbooster immunization and the specific anti-FMDV IgG antibodylevels were determined by indirect ELISA PBSISA 206 mixturewas used as the negative control (119899 = 5) and the commerciallyavailable inactivated vaccinewas used as the positive control (119899 = 5)Significant values (lowast119875 lt 005) are indicated by an asterisk

0

1

2

3

Neu

tral

izin

g an

tibod

y tit

ers

28 d

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowast

lowast

Figure 4 Serum neutralizing antibody titers of immunized guineapigs (each group contained 5 guinea pigs 119899 = 5) at 7 days after boosterimmunization Significant values (lowast119875 lt 005) are indicated by anasterisk

4 Discussion

The baculovirus insect cell expression system is one of themost commonly used eukaryotic expression systems and isfast and efficient Hundreds of genes from animals plantsviruses bacteria and fungi have been highly expressed ininsect cells [20ndash22] The expressed exogenous proteins canbe posttranslationally modified in the cell (ie glycosylation

ConAFMDV antigen

VP1 VP1-gp120 VP1-E2 Inactivatedvaccine

PBS

lowast

0

1

2

3

4

Stim

ulat

ion

inde

x (S

I)

Figure 5 Specific proliferative responses of peripheral blood lym-phocytes from guinea pigs detected by MTT colorimetry Stimula-tion index (SI) means the ratio of stimulated sample unstimulatedsample at OD490 nm Significant values (lowast119875 lt 005) are indicatedby an asterisk

VP1-gp120VP1

VP1-E2

Inactivated vaccinePBS

400

300

200

100

0

(pg

ml)

lowast

lowast

IFN-

Figure 6 IFN-120574 levels induced by the VP1 protein (119899 = 5) and theVP1-gp120 (119899 = 5) and VP1-E2 (119899 = 5) fusion proteins in guinea pigserum Significant values (lowast119875 lt 005) are indicated by an asterisk

phosphorylation and acylation) and therefore this expres-sion system can be used to obtain a large number ofsoluble recombinant proteins that are functionally similarto the natural proteins [20 23] The HIV gp120 and HCVE2 proteins are both viral envelope glycoproteins that playimportant roles in mediating binding of the virus to itsreceptor The glycosylation of these proteins has a greatimpact on their antigenicity Eukaryotic expression systemscan glycosylate the expressed proteins and maintain theirnatural structure to the maximum extent Therefore thisstudy used the baculovirus insect cell eukaryotic expressionsystem to express the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

6 BioMed Research International

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(a)

25

30

40

50

70

100

(kDa) M 1 2 3

69 kDa

35 kDa

30 kDa

(b)

Figure 2 Western blotting analysis of the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (a)Western blotting analysis using themouse anti-His tag monoclonal antibody as the primary antibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 andVP1-E2 fusion proteins respectively (b) Western blotting analysis using the anti-type A FMDV VP1 monoclonal antibody as the primaryantibody Lanes 1 2 and 3 correspond to the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins respectively

antibody levels were observed over time after the initialimmunization in all groups except the PBS negative controlgroup The serum IgG antibody levels induced by the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were significantly lower than the levels produced inthe traditional inactivated vaccine group (119875 lt 005 Figure 3)but significantly higher than the levels in the PBS group (119875 lt005 Figure 3) within 28 days after the first immunizationImportantly the serum IgG antibody levels induced by theVP1-gp120 and VP1-E2 recombinant fusion proteins werehigher than the levels induced by the VP1 protein alone(119875 lt 005 Figure 3) after booster immunization suggestingthat fusion of the VP1 protein to the gp120 and E2 proteinsenhanced the ability of theVP1 protein to induce specific anti-FMDV IgG antibodies in guinea pigs

Furthermore the specific neutralizing antibodies againstFMDV serotype A of the immunized guinea pigs in eachgroup at 7 days after the booster immunization were assessedby VNT (Figure 4) The neutralizing antibody titers inthe VP1 protein the VP1-gp120 and VP1-E2 recombinantfusion proteins and the inactivated vaccine groups showedsignificantly higher FMDV-neutralizing activity than theantibody responses of the PBS group (119875 lt 005 Figure 4)Meanwhile the neutralizing antibody titers of VP1-gp120 andVP1-E2 recombinant fusion proteins were higher than thetiters induced by the VP1 protein (119875 lt 005 Figure 4)indicating that recombinant fusion strategy could enhancethe ability of the VP1 protein to induce specific neutralizingantibody in guinea pigs

34 Cell-Mediated Immune Responses Peripheral blood lym-phocytes were isolated from the guinea pigs 7 days after

booster immunization The specific proliferative responsesof the peripheral lymphocytes from the guinea pigs wereexamined using theMTT colorimetric assayTheproliferativeresponses in splenic lymphocytes from all guinea pigs werestimulated by concanavalin A (Figure 5) with no significantdifferences between groups (119875 gt 005 Figure 5) Dif-ferences in lymphocyte proliferation were observed amongall groups incubated with the inactivated FDMV antigenIn detail lymphocytes from guinea pigs immunized withthe VP1 protein VP1-gp120 and VP1-E2 recombinant fusionproteins or inactivated vaccine exhibited significantly higherproliferation levels than the PBS control group (119875 lt 005Figure 5) The lymphocytes of the group immunized withthe inactivated vaccine showed the highest proliferation levelfollowed by the groups immunized with the recombinantproteins in the order VP1-gp120 VP1-E2 and VP1 proteinAlthough lymphocyte proliferation was higher in the VP1-gp120 and VP1-E2 immunization groups compared to theVP1 protein immunization group the differences betweenthe groups were not significant (119875 gt 005 Figure 5)Furthermore to assess the cytokine levels in guinea pig seraafter immunization the serum IFN-120574 content was measuredusing a commercially available cytokine antibody array Theresults showed that the VP1 protein the VP1-gp120 andVP1-E2 recombinant fusion proteins and the inactivatedvaccine induced higher IFN-120574 levels in guinea pigs than inthe PBS negative control (119875 gt 005 Figure 6) The abilityof the inactivated vaccine to induce IFN-120574 production wassignificantly greater than the ability of the three recombinantproteins (119875 gt 005 Figure 6) whereas no significantdifferences (119875 lt 005 Figure 6) were observed between theVP1 VP1-gp120 and VP1-E-2 recombinant proteins

BioMed Research International 7

0

1

2

3

4

OD492

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

14 21 287

(d)

Figure 3 The VP1 protein and the VP1-gp120 and VP1-E2 fusionproteins induced the production of specific anti-FMDV IgG anti-bodies in guinea pig serum (each group contained 5 guinea pigs 119899 =5) Serum samples were collected from all guinea pigs at 7 days afterbooster immunization and the specific anti-FMDV IgG antibodylevels were determined by indirect ELISA PBSISA 206 mixturewas used as the negative control (119899 = 5) and the commerciallyavailable inactivated vaccinewas used as the positive control (119899 = 5)Significant values (lowast119875 lt 005) are indicated by an asterisk

0

1

2

3

Neu

tral

izin

g an

tibod

y tit

ers

28 d

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowast

lowast

Figure 4 Serum neutralizing antibody titers of immunized guineapigs (each group contained 5 guinea pigs 119899 = 5) at 7 days after boosterimmunization Significant values (lowast119875 lt 005) are indicated by anasterisk

4 Discussion

The baculovirus insect cell expression system is one of themost commonly used eukaryotic expression systems and isfast and efficient Hundreds of genes from animals plantsviruses bacteria and fungi have been highly expressed ininsect cells [20ndash22] The expressed exogenous proteins canbe posttranslationally modified in the cell (ie glycosylation

ConAFMDV antigen

VP1 VP1-gp120 VP1-E2 Inactivatedvaccine

PBS

lowast

0

1

2

3

4

Stim

ulat

ion

inde

x (S

I)

Figure 5 Specific proliferative responses of peripheral blood lym-phocytes from guinea pigs detected by MTT colorimetry Stimula-tion index (SI) means the ratio of stimulated sample unstimulatedsample at OD490 nm Significant values (lowast119875 lt 005) are indicatedby an asterisk

VP1-gp120VP1

VP1-E2

Inactivated vaccinePBS

400

300

200

100

0

(pg

ml)

lowast

lowast

IFN-

Figure 6 IFN-120574 levels induced by the VP1 protein (119899 = 5) and theVP1-gp120 (119899 = 5) and VP1-E2 (119899 = 5) fusion proteins in guinea pigserum Significant values (lowast119875 lt 005) are indicated by an asterisk

phosphorylation and acylation) and therefore this expres-sion system can be used to obtain a large number ofsoluble recombinant proteins that are functionally similarto the natural proteins [20 23] The HIV gp120 and HCVE2 proteins are both viral envelope glycoproteins that playimportant roles in mediating binding of the virus to itsreceptor The glycosylation of these proteins has a greatimpact on their antigenicity Eukaryotic expression systemscan glycosylate the expressed proteins and maintain theirnatural structure to the maximum extent Therefore thisstudy used the baculovirus insect cell eukaryotic expressionsystem to express the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

BioMed Research International 7

0

1

2

3

4

OD492

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

14 21 287

(d)

Figure 3 The VP1 protein and the VP1-gp120 and VP1-E2 fusionproteins induced the production of specific anti-FMDV IgG anti-bodies in guinea pig serum (each group contained 5 guinea pigs 119899 =5) Serum samples were collected from all guinea pigs at 7 days afterbooster immunization and the specific anti-FMDV IgG antibodylevels were determined by indirect ELISA PBSISA 206 mixturewas used as the negative control (119899 = 5) and the commerciallyavailable inactivated vaccinewas used as the positive control (119899 = 5)Significant values (lowast119875 lt 005) are indicated by an asterisk

0

1

2

3

Neu

tral

izin

g an

tibod

y tit

ers

28 d

VP1VP1-gp120VP1-E2

Inactivated vaccinePBS

lowast

lowast

lowast

Figure 4 Serum neutralizing antibody titers of immunized guineapigs (each group contained 5 guinea pigs 119899 = 5) at 7 days after boosterimmunization Significant values (lowast119875 lt 005) are indicated by anasterisk

4 Discussion

The baculovirus insect cell expression system is one of themost commonly used eukaryotic expression systems and isfast and efficient Hundreds of genes from animals plantsviruses bacteria and fungi have been highly expressed ininsect cells [20ndash22] The expressed exogenous proteins canbe posttranslationally modified in the cell (ie glycosylation

ConAFMDV antigen

VP1 VP1-gp120 VP1-E2 Inactivatedvaccine

PBS

lowast

0

1

2

3

4

Stim

ulat

ion

inde

x (S

I)

Figure 5 Specific proliferative responses of peripheral blood lym-phocytes from guinea pigs detected by MTT colorimetry Stimula-tion index (SI) means the ratio of stimulated sample unstimulatedsample at OD490 nm Significant values (lowast119875 lt 005) are indicatedby an asterisk

VP1-gp120VP1

VP1-E2

Inactivated vaccinePBS

400

300

200

100

0

(pg

ml)

lowast

lowast

IFN-

Figure 6 IFN-120574 levels induced by the VP1 protein (119899 = 5) and theVP1-gp120 (119899 = 5) and VP1-E2 (119899 = 5) fusion proteins in guinea pigserum Significant values (lowast119875 lt 005) are indicated by an asterisk

phosphorylation and acylation) and therefore this expres-sion system can be used to obtain a large number ofsoluble recombinant proteins that are functionally similarto the natural proteins [20 23] The HIV gp120 and HCVE2 proteins are both viral envelope glycoproteins that playimportant roles in mediating binding of the virus to itsreceptor The glycosylation of these proteins has a greatimpact on their antigenicity Eukaryotic expression systemscan glycosylate the expressed proteins and maintain theirnatural structure to the maximum extent Therefore thisstudy used the baculovirus insect cell eukaryotic expressionsystem to express the VP1 protein and the VP1-gp120 andVP1-E2 recombinant fusion proteins

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

8 BioMed Research International

Protein subunit vaccines contain only viral capsid pro-teins without nucleic acids and therefore are very safe Thesevaccine types have become one of the main research direc-tions in research on FMDV vaccines [24] The VP1 proteinis the main antigenic protein in FMDV and can induce theproduction of protective neutralizing antibodies to provide agreater protective effect Therefore many studies of FMDVsubunit vaccines have focused on the VP1 protein [25ndash29]However FMDV subunit vaccines have lower immunologicaleffects than traditional inactivated vaccines [24] Thereforetargeting vaccines to dendritic cells to improve their immuneeffects has become an important strategy to improve theimmune effects of vaccines DC-SIGN is the main ligandused for DC targeting [10] Therefore in this study naturalligands (the gp120 and E2 proteins) with a high affinity forDC-SIGNwere selected as the targetingmolecules to enhancethe efficiency of targeted presentation of the VP1 protein toDCs through the expression of a VP1 fusion protein withthese two DC-SIGN ligands this approach was intended toimprove the immune effect of the VP1 protein as a subunitvaccine in animals

In gene expression studies much attention has been paidto the selection of appropriate expression vectors and hostsystemswhereas the issue ofwhether the gene itself is the bestmatch with the vector and host system is often overlookedIn fact each organism used for protein expression includingE coli yeast mammalian cells plant cells and insect cellsexhibits some degree of codon usage bias or preference [3031] Thus the codons of the gene sequences to be expressedgreatly determine their expression efficiency in a given host[32] Generally rare codons can be eliminated in proteinexpression by redesigning and synthesizing the gene withoptimal codons to achieve optimal expression of the targetgene [32] Therefore the gene sequences encoding the VP1protein and the VP1-gp120 and VP1-E2 recombinant fusionproteins were optimized based on the insect cell codonpreference to enable efficient and accurate expression of thetarget proteins in Sf9 insect cells

Western blotting analysis showed that the recombinantproteins not only reacted with the mouse anti-His-tag mon-oclonal antibody but also bound to the anti-type A FMDVVP1 monoclonal antibody indicating good reactivity of theexpressed recombinant proteins Moreover the His tag in theexpression vector facilitated subsequent protein purification

The VP1-gp120 and VP1-E2 recombinant fusion proteinsinduced higher anti-FMDV IgG antibody titers compared toexpression of the VP1 protein alone (119875 lt 005 Figure 3)suggesting that fusion of the VP1 protein with the gp120and E2 proteins enhanced the ability of the VP1 proteinto induce specific anti-FMDV IgG antibody production inguinea pigs However no significant differences in cellularimmunity were found between the VP1 protein and the VP1-gp120 and VP1-E2 fusion proteins (119875 gt 005 Figure 5)Thus further study is necessary to clarify whether the higheranti-FMDV IgG antibody titers induced by the VP1-gp120and VP1-E2 recombinant fusion proteins were a result ofenhanced specific DC targeting of the VP1 protein throughthe expression of the VP1 fusion protein with the DC-SIGNligand gp120 and E2 proteins

5 Conclusion

In this study the Bac-to-Bac baculovirus expression systemwas employed to successfully express the type A FMDVstructural protein VP1 and its recombinant fusion proteinsVP1-gp120 and VP1-E2 in insect cells and the expressedproteins showed good reactivity The immunization studyresults in guinea pigs showed that the VP1 protein and theVP1-gp120 and VP1-E2 recombinant fusion proteins inducedthe production of specific anti-FMDV IgG antibodies inguinea pigs with the VP1-gp120 and VP1-E2 recombinantfusion proteins exhibiting a stronger ability to induce specificanti-FMDV IgG antibodies than expression of the VP1 pro-tein alone Additionally the VP1 protein and the VP1-gp120and VP1-E2 recombinant fusion proteins stimulated thespecific proliferation of guinea pig lymphocytes and inducedincreased IFN-120574 levels In summary the immunogenicity ofantigens can be improved to a certain extent through fusionof the antigens with DC-targeting ligands which provides anew perspective for future studies of subunit vaccines

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the NationalPig Industrial System (CARS-36-068) the National KeyResearch andDevelopment Program (2016YFD0501505) andthe Coordination Program of Lanzhou Veterinary ResearchInstitute (Y2016CG23)

References

[1] M J Grubman and B Baxt ldquoFoot-and-mouth diseaserdquo ClinicalMicrobiology Reviews vol 17 no 2 pp 465ndash493 2004

[2] S M Jamal and G J Belsham ldquoFoot-and-mouth disease pastpresent and futurerdquo Veterinary Research vol 44 no 1 article116 2013

[3] Y Cao Z Lu and Z Liu ldquoFoot-and-mouth disease vaccinesprogress and problemsrdquo Expert Review of Vaccines vol 15 no6 pp 783ndash789 2016

[4] L L Rodriguez and C G Gay ldquoDevelopment of vaccinestoward the global control and eradication of foot-and-mouthdiseaserdquo Expert Review of Vaccines vol 10 no 3 pp 377ndash3872011

[5] O P Joffre E Segura A Savina and S Amigorena ldquoCross-presentation by dendritic cellsrdquo Nature Reviews Immunologyvol 12 no 8 pp 557ndash569 2012

[6] C Dresch Y Leverrier J Marvel and K Shortman ldquoDevelop-ment of antigen cross-presentation capacity in dendritic cellsrdquoTrends in Immunology vol 33 no 8 pp 381ndash388 2012

[7] I Caminschi and K Shortman ldquoBoosting antibody responsesby targeting antigens to dendritic cellsrdquo Trends in Immunologyvol 33 no 2 pp 71ndash77 2012

[8] Y van Kooyk W W J Unger C M Fehres H Kalay and JJ Garcıa-Vallejo ldquoGlycan-based DC-SIGN targeting vaccinesto enhance antigen cross-presentationrdquoMolecular Immunologyvol 55 no 2 pp 143ndash145 2013

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

BioMed Research International 9

[9] A A Bashirova L Wu J Cheng et al ldquoNovel member ofthe CD209 (DC-SIGN) gene family in primatesrdquo Journal ofVirology vol 77 no 1 pp 217ndash227 2003

[10] J J Garcıa-Vallejo W W J Unger H Kalay and Y van KooykldquoGlycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccinesrdquo OncoImmunology vol 2no 2 Article ID e23040 2013

[11] L J Cruz P J Tacken J M Pots R Torensma S I Buschowand C G Figdor ldquoComparison of antibodies and carbohydratesto target vaccines to human dendritic cells via DC-SIGNrdquoBiomaterials vol 33 no 16 pp 4229ndash4239 2012

[12] R Liu J Wang Y Yang I Khan Y Dong and N ZhuldquoA novel rabies virus lipopeptide provides a better protectionby improving the magnitude of DCs activation and T cellresponsesrdquo Virus Research vol 221 pp 66ndash73 2016

[13] A Pecora D A Malacari M S Perez Aguirreburualde etal ldquoDevelopment of an APC-targeted multivalent E2-basedvaccine against Bovine Viral Diarrhea Virus types 1 and 2rdquoVaccine vol 33 no 39 pp 5163ndash5171 2015

[14] U Svajger M Anderluh M Jeras and N Obermajer ldquoC-typelectin DC-SIGN An adhesion signalling and antigen-uptakemolecule that guides dendritic cells in immunityrdquo CellularSignalling vol 22 no 10 pp 1397ndash1405 2010

[15] S K Khattar S Samal C C LaBranche D C MontefioriP L Collins and S K Samal ldquoComparative immunogenicityof HIV-1 gp160 gp140 and gp120 expressed by live attenuatednewcastle disease virus vectorrdquo PLoS ONE vol 8 no 10 ArticleID e78521 2013

[16] S V Su P Hong S Baik O A Negrete K B Gurney and BLee ldquoDC-SIGN binds to HIV-1 glycoprotein 120 in a distinctbut overlapping fashion compared with ICAM-2 and ICAM-3rdquoJournal of Biological Chemistry vol 279 no 18 pp 19122ndash191322004

[17] L-J Zhao W Wang H Ren and Z-T Qi ldquoERK signaling istriggered by hepatitis C virus E2 protein through DC-SIGNrdquoCell Stress and Chaperones vol 18 no 4 pp 495ndash501 2013

[18] S Pohlmann J Zhang F Baribaud et al ldquoHepatitis C virusglycoproteins interact with DC-SIGN and DC-SIGNRrdquo Journalof Virology vol 77 no 7 pp 4070ndash4080 2003

[19] OIE OIE Manual of Diagnostic Tests and Vaccines for Terres-trial Animals Foot-and-mouth disease 2009 httpwwwoieintengA FMD2012docs20105 FMDpdf

[20] SN Beljelarskaya ldquoBaculovirus expression systems for produc-tion of recombinant proteins in insect and mammalian cellsrdquoMolecular Biology vol 45 no 1 pp 123ndash138 2011

[21] NN Koroleva P V Spirin A V Timokhova et al ldquoBaculovirusvectors for efficient gene delivery and expression inmammaliancellsrdquoMolecular Biology vol 44 no 3 pp 479ndash487 2010

[22] H Zheng C L Liu J Zhuang and S S Yuan ldquoBaculovirusexpression of cloned porcine arterivirus generates infectiousparticles in both insect and mammalian cellsrdquo Journal ofBiotechnology vol 150 no 2 pp 251ndash258 2010

[23] F M Boyce and N L R Bucher ldquoBaculovirus-mediated genetransfer into mammalian cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no6 pp 2348ndash2352 1996

[24] F Diaz-San Segundo G N Medina C Stenfeldt J Arzt andT de los Santos ldquoFoot-and-mouth disease vaccinesrdquoVeterinaryMicrobiology vol 206 pp 102ndash112 2017

[25] Y J Du P Jiang Y F Li et al ldquoImmune responses of tworecombinant adenoviruses expressing VP1 antigens of FMDV

fusedwith porcine granulocytemacrophage colony-stimulatingfactorrdquo Vaccine vol 25 no 49 pp 8209ndash8219 2007

[26] L Pan Y G Zhang Y L Wang J L Lv P Zhou and Z WZhang ldquoExpression and detection of the FMDV VP1 transgeneand expressed structural protein in Arabidopsis thalianardquoTurkish Journal of Veterinary and Animal vol 35 no 4 pp 235ndash242 2011

[27] J P Rao P Agrawal R Mohammad et al ldquoExpression of VP1protein of serotype A and O of foot-and-mouth disease virus intransgenic sunnhemp plants and its immunogenicity for guineapigsrdquo Acta Virologica vol 56 no 2 pp 91ndash99 2012

[28] M Wang L Pan P Zhou et al ldquoProtection against foot-and-mouth disease virus in Guinea pigs via oral administration ofrecombinant Lactobacillus plantarum expressing VP1rdquo PLoSONE vol 10 no 12 Article ID e0143750 2015

[29] C S Yin W Y Chen Q Q Hu et al ldquoInduction of protec-tive immune response against both PPRV and FMDV by anovel recombinant PPRV expressing FMDV VP1rdquo VeterinaryResearch vol 45 no 1 article 62 2014

[30] Y L Wang Y H Mao X D Xu S H Tao and H Y ChenldquoCodon usage in signal sequences affects protein expression andsecretion using baculovirusinsect cell expression systemrdquo PLoSONE vol 10 no 12 Article ID e0145887 2015

[31] R Buchan and I Stansfield ldquoCodon pair bias in prokaryotic andeukaryotic genomesrdquo BMC Bioinformatics vol 6 2005

[32] Z P Zhoua Y K Danga M Zhou et al ldquoCodon usage is animportant determinant of gene expression levels largely throughits effects on transcriptionrdquoProceedings of theNational Academyof Sciences of the United States of America vol 113 no 41 ppE6117ndashE6125 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology