Microcrystalline Cellulose for Delivery of Recombinant

Research ArticleMicrocrystalline Cellulose for Delivery of RecombinantProtein-Based Antigen against Erysipelas in Mice

Wooyoung Jeon1 Yeu-Chun Kim2 Minhee Hong1 Sanoj Rejinold2 Kyoungmoon Park3

Injoong Yoon4 Sungsik Yoo4 Hongweon Lee15 and Jungoh Ahn 15

1Biotechnology Process Engineering Center KRIBB Cheongju 363-883 Republic of Korea2Department of Chemical and Biomolecular Engineering KAIST Daejeon 305-701 Republic of Korea3Biological and Chemical Engineering Hongik University Sejong 339-701 Republic of Korea4Choong-Ang Vaccine Laboratory Daejeon 305-348 Republic of Korea5University of Science and Technology (UST) 217 Gajeong-ro Yuseong-gu Daejeon 305-350 Republic of Korea

Correspondence should be addressed to Jungoh Ahn ahnjokribbrekr

Received 17 January 2018 Revised 6 April 2018 Accepted 3 May 2018 Published 11 June 2018

Academic Editor Gamal Enan

Copyright copy 2018 Wooyoung Jeon et alThis 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

The study describes the development of a vaccine using microcrystalline cellulose (Avicel PH-101) as a delivery carrier ofrecombinant protein-based antigen against erysipelas Recombinant SpaA surface protective protein from a gram-positivepathogen Erysipelothrix rhusiopathiaewas fused to a cellulose-binding domain (CBD) fromTrichoderma harzianum endoglucanaseII through a S3N10 peptide The fusion protein (CBD-SpaA) was expressed in Escherichia coli and was subsequently bound toAvicel PH-101 The antigenicity of CBD-SpaA bound to the Avicel was evaluated by enzyme-linked immunosorbent (ELISA) andconfocal laser scanning microscope (CLSM) assays For the examination of its immunogenicity groups of mice were immunizedwith different constructs (soluble CBD-SpaA Avicel coated with CBD-SpaA whole bacterin of E rhusiopathiae (positive control)and PBS (negative control)) In two weeks after immunization mice were challenged with 1x107 CFU of E rhusiopathiae andAvicel coated with CBD-SpaA induced protective immunity in mice In conclusion this study demonstrates the feasibility ofmicrocrystalline cellulose as the delivery system of recombinant protein subunit vaccine against E rhusiopathiae infection in mice

1 Introduction

Vaccines are the therapeutic formulations given to patientsto elicit immune responses entailing antibody production(humoral) or cell-mediated responses that will eventuallyfight variety of malignancies [1] There have been manyapproaches for vaccine delivery via vaccinations which areconsidered the most efficient prophylactic method againstvarious infectious diseases Vaccine delivery systems (iemicro- or nanoparticles liposomes and virosomes) havebeen investigated for improving vaccine efficacy [2ndash5] Inaddition various vaccine types have been developed to over-come the disadvantages of conventional vaccines Amongvaccines recombinant protein antigens or their fragmentshave been used and considered as novel vaccine candidatesDevelopment of such kinds of vaccines avoids the safetyissues with attenuating virus or cell cultures when making

conventional viral vaccines [6 7] However some recom-binant protein antigens have fatal handicaps (low stabilityand immunogenicity) caused by lack of key elements thatcan stimulate immune response [8] Use of adjuvants alongwith recombinant protein vaccines has been suggested forimproved immunogenicity [9] Different strategies have beenextensively explored in order to improve the immunogenicityby protecting antigens through immobilization on inorganicor organic matrices [10]

Cellulose is a primary component of plant cell walls anda linear polymer of glucose residues It is naturally resistantto biological degradation owing to its insolubility rigidityand tendency to pack together to form long crystals Itis chemically inert pharmaceutically safe and inexpensivemaking it an effective immunosorbent or carrier materialfor protein purification and immobilization [11] Cellulose-binding domain (CBD) refers to protein modules that bind

HindawiBioMed Research InternationalVolume 2018 Article ID 7670505 7 pageshttpsdoiorg10115520187670505

2 BioMed Research International

firmly to various types of cellulose The strong attractionbetween cellulose and CBD has applications in many areasespecially for immunology [12] In previous studies cellu-lose beads (Sigmacell 20 or Orbicell cellulose particles)attached to CBD-fused recombinant protein were used forparenteral vaccination for goldfish [13] and the immuno-genic properties of CBD in its free form were comparedwith the CBD-cellulose complex using cytokine productionas an immunological indicator Small Orbicell beads (1ndash10120583M) induced antibody levels that were equal to the titersproduced by the adjuvanted protein and bacterin formulaecompared to the larger Sigmacell particles (10ndash20 120583M)Similarly Lunin et al (2009) studied the immobilization ofCBDon the cellulose sorbent which enhances the synthesis ofspecific antibodies and found that cellulose immunosorbentis not immunotolerant and could induce cytokine productioninvolved in the regulation of humoral immune response [8]

Erysipelas is a very serious swine disease causingtremendous economic loss E rhusiopathiae causative agentof erysipelas is a gram-positive rod shaped non-spore-forming non-motile and bacterial pathogen [14] Althoughlive-attenuated or recombinant vaccines are currently admin-istered to control this disease their effectiveness is notconsistent Even the mechanisms of pathogenicity have notyet been elucidated [15ndash18] Therefore it is necessary to havea more advanced and effective system of vaccination againsterysipelas

In this study we developed Avicel (cellulose microcrys-talline) correlated with antigen and investigated its char-acteristics In addition antigen-immobilized Avicel vac-cine was subcutaneously injected into mice to analyze theactual immunogenicity of our newly developed antigen-immobilized Avicel recombinant protein vaccine Overallthe study highlights the development of an efficient recom-binant protein vaccine-immunosorbent system to protectswine against E rhusiopathiae infection

2 Materials and Methods

21 Bacterial Strains and Culture Conditions The E rhu-siopathiae serotype 15 strain was isolated from a diseasedpiglet in Korea For it E coli DH5120572 [Fminus 12060180dlacZΔM15endA1 hsdR17 (rKminus mK+) supE44 thi-1 120582minus recA1 gyrA96]was used as a host strain for cloning and plasmid mainte-nance The E coli BL21 host strain [Fminus hsdS gal ompT rBminusmBminus] (Novagen USA) harboring a lambda derivative DE3was used for gene expression

22 Cloning and Recombinant Plasmid Construction Thegene coding CBD of Trichoderma harzianum endog-lucanase II with artificial S

3N10

linker was ampli-fied from pEKPM-EcGAD-Lk-H6 (Ahn 2004 HyeminPark 2012) using specific primers (Forward 51015840-GAAGGAGATATACATATGCAGCAAACTGTTTGGGGG-31015840 Reverse 51015840-CTTCTTATCGGATCCGTTGTTGTTGTT-GTTGTTGTTGTTGTTGTTGCTGCTGCTAATGCATTG-AGCGTAGTA-31015840) in which the underlined characters wereintroduced for the In-fusion Advantage PCR cloningkit (Clontech USA) The italic letters indicate the site of

the restriction enzymes The polymerase chain reaction(PCR) product containing CBD with S

3N10

linker wasintroduced into the pET-28b(+) plasmid (Novagen USA)previously digested with NdeI and BamHI For the isolationof genomic DNA of E rhusiopathiae a DNeasy Bloodand Tissue Kit (Qiagen Germany) was used accordingto manufacturerrsquos instructions The surface protectiveantigen A (SpaA) gene was amplified from E rhusiopathiaegenomic DNA by PCR using the primers (Forward 51015840-ACTCCGCCAACTAGCTCTGGATCCGATTCGACAGAT-ATTTCTG-31015840 Reverse 51015840-GTGGTGGTGGTGGTGGTG-CTCGAGTTTTAAACTTCCATCGTTC) in which un-derlined base pairs were introduced for In-fusion ligationThe italic letters indicate the sites of BamHI and XhoI

23 Expression and Purification of CBD-SpaA Protein Theplasmid harboring pKPM-CBD-Lk-SpaA-H6 was trans-formed intoE coliBL21(DE3)The transformantswere grownin 2YT medium containing 50 120583gml of kanamycin at 37∘CWhen an A

600of 05 was reached 05 mM isopropyl-120573-thio-

D-galactopyranoside (IPTG) was added and cultures weregrown for an additional 3h at 37∘C Cells were harvested bycentrifugation and pellets were disrupted by sonication forfurther analysis Recombinant fusion protein was purifiedfrom the supernatant by Ni-NTA agarose (Qiagen Ger-many) using an Econo-Pac Chromatography Column (Bio-Rad USA) according to the manufacturer instructions Theprotein concentration was estimated using the BCA proteinassay kit (Pierce USA)

24 Binding of CBD-SpaA onto Avicel Binding assays werecarried out according to methods previously described withslight modification [19] Briefly Avicel (PH-101 Sigma lt50 120583m) (01 mg) was mixed with 500 120583g of the CBD-SpaAproteins at 4∘C for 2 h The supernatant was collected aftercentrifugation and used to determine uncombined proteinusing a BCA protein assay kit The amount of protein boundto Avicel was determined from the difference between finaland starting values in the supernatant Nonspecific proteinsbound to Avicel were removed by washing with 05 M NaCl

25 SDS-PAGE and Western Blot Analysis SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)was performed according to the method of Laemmle[20] For the Western blot analyses proteins separatedby 10 SDS-PAGE gel were transferred to nitrocellulose(NC) membranes The NC membranes were blocked byphosphate buffered saline (PBS) containing 5 skimmedmilk and washed three times with PBST (01 Tween 20in PBS) The membranes were incubated with His-probemonoclonal antibody (Santa Cruz Biotechnology USA)or mouse-derived antiserum prepared with whole bacteriaof E rhusiopathiae for 1 h at room temperature (RT) Thespecific antigen reacted with IgG alkaline phosphatase(AP) antibody (Sigma USA) was visualized using an APconjugated substrate kit (Bio-Rad USA)

26 Preparation of Polyclonal Mouse Antiserum To pre-pare mouse polyclonal antiserum against E rhusiopathiae

BioMed Research International 3

a five-week-old specific-pathogen-free (SPF) mouse wasinjected subcutaneously with formalinized whole cell vaccineof E rhusiopathiae twice within a 2-week interval Then ablood sample was collected twoweeks later and sera antibodytiter was measured using enzyme-linked immunosorbentassay (ELISA)

27 Antigenicity Evaluation Microtiter assembly strips(Thermo Scientific Finland) were coated overnight at4∘C with 100 120583l per well of immobilized CBD-SpaA onAvicel Several dilutions (1times104 5times104 1times105 and 5times105Avicel particles) of Avicel coated CBD-SpaA were testedin triplet The plates were washed three times with PBSTand blocked with 5 skimmed milk in PBST for 1h at RTAntiserum derived from mouse against E rhusiopathiae(1500) was added to the plates and then placed on a rockerplatform for 2h at RT For the detection of immunogeniccharacteristics the plates were incubated with a 12000dilution of horseradish peroxidase (HRP) anti-mouse IgGwhole antibody (GE Healthcare UK) for 1 h at RT Opticaldensity was read at 450 nm using a TECAN Infinity 2000PRO plate reader (TECAN Austria)

28 Confocal Laser Scanning Microscope (CLSM) Avicelparticles (1times105) coated with CBD-SpaA were incubated withantiserum against E rhusiopathiae (1500) for 2h at RT Afterwashing three times with PBST the immobilized CBD-SpaAwas incubated with Fluorescein- (FITC-) AffiniPure F(ab)2FragmentGoat Anti-Mouse IgG (H+L) (Jackson ImmunoRe-search Lab USA) for 30 min at 4∘C The plate was washedagain and Avicel particles coated with CBD-SpaA were fixedwith 37 paraformaldehyde for 10 min at RT followedby mounting with VECTASHIELD mount medium (VectorLab USA) Immunofluorescence was evaluated using anLSM 510 META Laser Scanning Microscope (Carl ZeissGermany)

29 Mouse Immunization and Challenge Forty SPF mice (5weeks old) were randomly assigned to 4 groups of eight eachand injected subcutaneously with 4 120583g of soluble CBD-SpaAAvicel coated with CBD-SpaA ERT2T-A containing wholebacterin of E rhusiopathiae serovar 15 (positive control) andPBS (negative control) emulsified with an oil-based adjuvantrespectively Two weeks after injection all groups were chal-lenged subcutaneously with 1times107 CFU of E rhusiopathiae(serovar 15) Mouse mortality was monitored daily for thefollowing ten days

3 Results

31 Expression of the CBD-SpaA Fusion Protein in E coliAs illustrated in Figure 1 pKPM-CBD-Lk-SpaA-H6 wasconstructed for the expression of SpaA fused to CBD Inthe construct SpaA was fused to CBD from T harzianumendoglucanase II via an artificial S

3N10

peptide known tocompletely resist E coli endopeptidase at its N-terminusand six histidines at its C-terminus The fusion protein(CBD-SpaA) was expressed in E coli BL21(DE3) harboringpKPM-CBD-Lk-SpaA-H6 The results of the SDS-PAGE and

Figure 1 A schematic representation of the expression vector forCBD-SpaA-H6 fusion protein SpaA surface protective antigen A(SpaA) from E rhusiopathiae CBD the cellulose-binding domainof T harzianum endoglucanase II Linker S

3N10

peptide (His)66x histidine tag sequence T7lac T7 promoter sequence T7t T7terminator sequence

Western blot (Figure 2) indicated that the CBD-SpaA wassuccessfully expressed at the expected molecular weight (776kDa) without being degraded by proteolysis and that theCBD-SpaA was overexpressed at a high level (35) withrespect to the percentage of total cell protein Moreover mostof the expressed CBD-SpaA were in soluble form comparedto our previous work in which extreme reduction of thesolubility ofE coli-derived glutamate decarboxylase occurredafter fusion with CBD [21]

32 Coating of Avicel with CBD-SpaA Protein The CBD-SpaA proteins from crude cell lysates were purified througha Ni-NTA column with the elution of an imidazole (250mM) The elution fraction contained CBD-SpaA with apurity of 851 Both the purified CBD-SpaA and crude celllysates were bound to microcrystalline cellulose Avicel PH-101 and the concentration of CBD-SpaA bound to Avicelwas determined by stripping the protein from the beadsby boiling Avicel displayed a binding capacity of 311plusmn0015mgCBD-SpaAgAvicel (purity 94) for the purified CBD-SpaAand 192plusmn0001 mgCBD-SpaAgAvicel (purity 81) for crude celllysates (Figure 3)

33 Antigenicity of CBD-SpaA Bound to Avicel The anti-genicity of CBD-SpaA bound to Avicel was evaluated byindirect ELISA assay As shown in Figure 4 the increasedabsorbance values were detected in proportion to the numberof Avicel particles coated with CBD-SpaA whereas the Avicelwithout CBD-SpaA as a negative control showed a value aslow as the PBS Confocal laser scanning microscope (CLSM)was used for visualization of antigenic properties of theAvicel coated with CBD-SpaA (Figure 5) Purified fusionprotein was bound to Avicel and incubated with anti-serumof whole E rhusiopathiae followed by goat-mouse IgG-FITC CLSM images demonstrated that green fluorescencedispersed almost equally on the surface of the Avicel coatedwith CBD-SpaA as shown in Figure 5

34 Protective Immunity in Immunized Mice To examinewhether protection could be induced without side effects byimmunization with the Avicel coated with CBD-SpaA weinjected each protein (free CBD-SpaA and Avicel coated withCBD-SpaA ERT2T-1 containing whole cell bacterin (positivecontrol) from CVAVC in the Republic of Korea and PBS(negative control)) into mice and challenged the mice with


M STkDa

100

75

50

37

25

(a)

M T SkDa

100

75

50

37

25

(b)

M STkDa

100

75

50

37

25

(c)

Figure 2 SDS-PAGE andWestern blot analyses of the CBD-SpaA-H6 expressed in E coli BL21(DE3)pKPM-CBD-SpaA-H6 (a) SDS-PAGE(b) Western blot with anti-histidine antibody (c) anti-serum derived from mouse inoculated with whole E rhusiopathiae Lane M standardmolecular weight marker T total cell lysates and S soluble fraction proteins The arrows indicate the expressed CBD-SpaA

CBD-SpaA

M 1 2 3 M 4 5kDa75

50

37

Figure 3 Coating of Avicel with CBD-SpaA protein Lanes 1-3 crude CBD-SpaA and Lanes 4 and 5 purified CBD-SpaA Lane M standmolecular weight marker Lanes 1 and 4 proteins before binding to Avicel Lane 2 proteins not bound to Avicel and Lanes 3 and 5 proteinsbound on Avicel

Number of avicel particles

Opt

ical

den

sity

A45

0

Avicel without CBD-SpaA Avicel with CBD-SpaA

1x105 1x104 5x104 1x105 5x1050

00

05

10

15

20

25

Figure 4 ELISA assay of Avicel coated with CBD-SpaA (n=3)


(a) (b) (c)

Figure 5 CLSM images for CBD-SpaA-H6 immobilized on Avicel Purified CBD-SpaA protein was bound to Avicel and incubated withanti-histidine antibody followed by goat anti-mouse IgG-FITCThe confocal microscope image indicates that CBD-SpaA protein had a highbinding affinity to Avicel (a) Differential interference contrast images (b) FITC fluorescence (c) merging of the two split images

Surv

ival

rate

()

0

20

40

60

80

100

Avicel coated with CBD-SpaA Free CBD-SpaA Whole cell bacterin(-) Control

2 4 6 8 100Day of post-challenge

Figure 6 Cumulative mortality of immunized mice after challengeMicewere vaccinatedwith free CBD-SpaAAvicel coatedwithCBD-SpaA and ERT2T-A containing whole cell bacterin as a positivecontrol and PBS as a negative control After 14 days all the micewere challenged with highly virulent E rhusiopathiae and survivalwas monitored for ten days

E rhusiopathiae All of the negative control mice died withinseven days after challenge Compared with negative controlgroup 5 of 8 (p lt 00001 by Fisherrsquos exact test) mice inthe ERT2T-1 immunized group and 6 of 8 (p lt 00001 byFisherrsquos exact test) mice in the free CBD-SpaA immunizedgroup survived In the Avicel coated with CBD-SpaA theimmunized group of mice showed 100 (p lt 00001 byFisherrsquos exact test) immune-protection against challenge withE rhusiopathiae (Figure 6) However that group did not showsignificant difference with the free CBD-SpaA immunizedgroup (p = 0467 by Fisherrsquos exact test) These resultsdemonstrated that the microcrystalline cellulose can be usedas a delivery carrier of recombinant protein

4 Discussion

Antigen subunits or synthetic peptides are considered as apromising alternative for viral vaccines They have been alsoconsidered safer vaccine systems than killedinactivated orlive-attenuated whole cellsviruses Recombinant DNA tech-nology has made the development of subunit vaccines moreefficient because the production and purification procedurecan be carefully designed to obtain high yields of a well-defined product [22] However some studies have shown thatsoluble immunogens rarely induce high titers of antibodieswithout the use of strong adjuvants [23 24] To overcome thetypical low immunogenicity of protein-based vaccines and toaddress the need for effective vaccines and efficient deliverysystems researchers have moved in the direction of molec-ular biotechnology [25] Recently advances in recombinantbiotechnology have led to the development of geneticallyengineered polymers with exact order and accuracy of aminoacid residues Recombinant protein-based polymers such aselastin-like polymers (ELPs) silk-like polymers (SLPs) andsilk-elastin-like protein polymers (SELPs) have been reportedto bring controlled release longer circulating therapeuticsand tissue-specific treatment options [26] Nanoscale struc-tures such as gold nanoparticles and virus-like particles havealso recently peaked interests for drug delivery as they offermanifest benefits [27 28]

In our study Avicel was selected as immunosorbent forpurification and delivery of recombinant protein subunitvaccine because it is highly inert inexpensive and safebiomaterial This should protect the protein subunit vaccinefrom degradation in harsh conditions (extreme pH or tem-perature) As shown in Figure 4 immune response increasedwith increasing Avicel particle concentration confirming itssuitability as a better immunosorbent for vaccine systemsThe higher immunity achieved with increased Avicel concen-tration could be due to selective coating of SpaA by Avicel

Our findings clearly demonstrate that Avicel is specificfor the CBD-SpaA with good binding capacity for vaccinedelivery The binding capacity was visualized using CLSMimaging as shown in Figure 5 It has been well known that


the Spa proteins are potent protective antigens against Erhusiopathiae infection [29] Recent reports show that SpaAis the major Spa-type of serotypes 1a 1b and 2 [30] whichare most commonly implicated in swine erysipelas Theprotective domain of SpaA lies between amino acids 29-414[30] This particular amino acid sequence itself can inducehighly protective antibodies againstE rhusiopathiae infection[15] Being a well delivery agent and immune sorbent Avicelfurther enhanced its potency as we found in our experiments

5 Conclusions

In this study we developed a vaccine using microcrystallinecellulose Avicel PH-101 to deliver recombinant protein-basedantigen against erysipelas The recombinant protein CBD-SpaA was expressed in E coli and bound to Avicel PH-101 Our data perceptibly showed that a 100 survival ratecould be achieved using the Avicel coated with antigen in Erhusiopathiae-challenged mice Thus the in vitro immuno-genicity test has been validated by the in vivo challengeexperiment

In our newly developed vaccine system protein purifi-cation is unnecessary This cuts down production costs andenables a cost-effective recombinant protein vaccine systemSpecifically our delivery system using Avicel coated withCBD-SpaA could provide an appealing vaccination strategyagainst erysipelas

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Wooyoung Jeon and Yeu-Chun Kim contributed equally tothis work

Acknowledgments

This research was supported by a grant from KRIBBResearch Initiative ProgramThis work was supported by theAdvanced Biomass RampDCenter (ABC) of theGlobal FrontierProject funded by the Ministry of Science and ICT (ABC-2015M3A6A2074238)

References

[1] C Saroja P Lakshmi and S Bhaskaran ldquoRecent trends invaccine delivery systems A reviewrdquo International Journal ofPharmaceutical Investigation vol 1 no 2 pp 64ndash74 2011

[2] C-J Chiou L-P Tseng M-C Deng et al ldquoMucoadhesiveliposomes for intranasal immunization with an avian influenza

virus vaccine in chickensrdquo Biomaterials vol 30 no 29 pp5862ndash5868 2009

[3] C Moser M Amacker A R Kammer S Rasi N Westerfeldand R Zurbriggen ldquoInfluenza virosomes as a combined vaccinecarrier and adjuvant system for prophylactic and therapeuticimmunizationsrdquo Expert Review of Vaccines vol 6 no 5 pp 711ndash721 2007

[4] S T Reddy A J van der Vlies E Simeoni et al ldquoExploitinglymphatic transport and complement activation in nanoparticlevaccinesrdquo Nature Biotechnology vol 25 no 10 pp 1159ndash11642007

[5] C Wang Q Ge D Ting et al ldquoMolecularly engineeredpoly(ortho ester) microspheres for enhanced delivery of DNAvaccinesrdquo Nature Materials vol 3 pp 190ndash196 2004

[6] MHansson P-ANygren and S Stahl ldquoDesign and productionof recombinant subunit vaccinesrdquo Biotechnology and AppliedBiochemistry vol 32 no 2 pp 95ndash107 2000

[7] S Liljeqvist and S Stahl ldquoProduction of recombinant sub-unit vaccines Protein immunogens live delivery systems andnucleic acid vaccinesrdquo Journal of Biotechnology vol 73 no 1pp 1ndash33 1999

[8] V G Lunin N E Sharapova T V Tikhonova et al ldquoResearchinto the immunogenic properties of the recombinant cellulose-binding domain of Anaerocellum thermophilum in vivordquoMolekuliarnaia Genetika Mikrobiologiia I Virusologiia pp 21ndash27 2009

[9] A S McKee MWMunks and P Marrack ldquoHow do adjuvantswork Important considerations for new generation adjuvantsrdquoImmunity vol 27 no 5 pp 687ndash690 2007

[10] G Georgiou C Stathopoulos P S Daugherty A R Nayak BL Iverson and R Curtiss ldquoDisplay of heterologous proteinson the surface of microorganisms From the screening ofcombinatorial libraries to live recombinant vaccinesrdquo NatureBiotechnology vol 15 no 1 pp 29ndash34 1997

[11] D Klemm BHeublein H P Fink andA Bohn ldquoCellulose fas-cinating biopolymer and sustainable rawmaterialrdquoAngewandteChemie vol 44 no 22 pp 3358ndash3393 2005

[12] M Linder and T T Teeri ldquoThe roles and function of cellulose-binding domainsrdquo Journal of Biotechnology vol 57 no 1ndash3 pp15ndash28 1997

[13] S Maurice M Dekel O Shoseyov and A Gertler ldquoCellulosebeads bound to cellulose binding domain-fused recombinantproteins an adjuvant system for parenteral vaccination of fishrdquoVaccine vol 21 no 23 pp 3200ndash3207 2003

[14] Y Shimoji ldquoPathogenicity of Erysipelothrix rhusiopathiae Vir-ulence factors and protective immunityrdquoMicrobes and Infectionvol 2 no 8 pp 965ndash972 2000

[15] Y Imada N Goji H Ishikawa M Kishima and T SekizakildquoTruncated surface protective antigen (SpaA) of Erysipelothrixrhusiopathiae serotype 1a elicits protection against challengewith serotypes 1a and 2b in pigsrdquo Infection and Immunity vol67 no 9 pp 4376ndash4382 1999

[16] H I Cheun K Kawamoto M Hiramatsu et al ldquoProtec-tive immunity of SpaA-antigen producing Lactococcus lac-tis against Erysipelothrix rhusiopathiae infectionrdquo Journal ofApplied Microbiology vol 96 no 6 pp 1347ndash1353 2004

[17] Y Shimoji Y Mori and V A Fischetti ldquoImmunologicalcharacterization of a protective antigen of Erysipelothrix rhu-siopathiae Identification of the region responsible for protec-tive immunityrdquo Infection and Immunity vol 67 no 4 pp 1646ndash1651 1999


[18] G J Eamens J C Chin B Turner and I Barchia ldquoEvaluationof Erysipelothrix rhusiopathiae vaccines in pigs by intradermalchallenge and immune responsesrdquoVeterinaryMicrobiology vol116 no 1-3 pp 138ndash148 2006

[19] J A Santiago-Hernandez J M Vasquez-Bahena M ACalixto-Romo et al ldquoDirect immobilization of a recombinantinvertase to Avicel by E coli overexpression of a fusion pro-tein containing the extracellular invertase from Zymomonasmobilis and the carbohydrate-binding domain CBDCex fromCellulomonas fimirdquo Enzyme and Microbial Technology vol 40no 1 pp 172ndash176 2006

[20] F Hong N Q Meinander and L J Jonsson ldquoFermentationstrategies for improved heterologous expression of laccase inPichia pastorisrdquo Biotechnology and Bioengineering vol 79 no4 pp 438ndash449 2002

[21] J O Ahn E S Choi H W Lee et al ldquoEnhanced secretion ofBacillus stearothermophilusL1 lipase in Saccharomyces cerevisiaeby translational fusion to cellulose-binding domainrdquo AppliedMicrobiology and Biotechnology vol 64 no 6 pp 833ndash8392004

[22] C Andersson Production and Delivery of Recombinant SubunitVaccines Department of Biotechnology Royal Institute ofTechnology (KTH) 2000

[23] O Perez A Batista-Duharte E Gonzalez et al ldquoHumanprophylactic vaccine adjuvants and their determinant role innew vaccine formulationsrdquo Brazilian Journal of Medical andBiological Research vol 45 no 8 pp 681ndash692 2012

[24] SWangH Liu X Zhang and FQian ldquoIntranasal and oral vac-cination with protein-based antigens Advantages challengesand formulation strategiesrdquo ProteinampCell vol 6 no 7 pp 480ndash503 2015

[25] I P Nascimento and L C C Leite ldquoRecombinant vaccines andthe development of new vaccine strategiesrdquo Brazilian Journal ofMedical and Biological Research vol 45 no 12 pp 1102ndash11112012

[26] J L Frandsen and H Ghandehari ldquoRecombinant protein-based polymers for advanced drug deliveryrdquo Chemical SocietyReviews vol 41 no 7 pp 2696ndash2706 2012

[27] F Y Kong J W Zhang R F Li Z X Wang W J Wang andW Wang ldquoUnique roles of gold nanoparticles in drug deliverytargeting and imaging applicationsrdquoMolecules vol 22 no 9 p1445 2017

[28] B D Hill A Zak E Khera and F Wen ldquoEngineering virus-like particles for antigen and drug deliveryrdquo Current Protein ampPeptide Science vol 19 no 1 pp 112ndash127 2018

[29] J E Galan and J F Timoney ldquoCloning and expression inEscherichia coli of a protective antigen of Erysipelothrix rhu-siopathiaerdquo Infection and Immunity vol 58 no 9 pp 3116ndash31211990

[30] H To and S Nagai ldquoGenetic and antigenic diversity ofthe surface protective antigen proteins of Erysipelothrix rhu-siopathiaerdquo Clinical and Vaccine Immunology vol 14 no 7 pp813ndash820 2007

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firmly to various types of cellulose The strong attractionbetween cellulose and CBD has applications in many areasespecially for immunology [12] In previous studies cellu-lose beads (Sigmacell 20 or Orbicell cellulose particles)attached to CBD-fused recombinant protein were used forparenteral vaccination for goldfish [13] and the immuno-genic properties of CBD in its free form were comparedwith the CBD-cellulose complex using cytokine productionas an immunological indicator Small Orbicell beads (1ndash10120583M) induced antibody levels that were equal to the titersproduced by the adjuvanted protein and bacterin formulaecompared to the larger Sigmacell particles (10ndash20 120583M)Similarly Lunin et al (2009) studied the immobilization ofCBDon the cellulose sorbent which enhances the synthesis ofspecific antibodies and found that cellulose immunosorbentis not immunotolerant and could induce cytokine productioninvolved in the regulation of humoral immune response [8]

Erysipelas is a very serious swine disease causingtremendous economic loss E rhusiopathiae causative agentof erysipelas is a gram-positive rod shaped non-spore-forming non-motile and bacterial pathogen [14] Althoughlive-attenuated or recombinant vaccines are currently admin-istered to control this disease their effectiveness is notconsistent Even the mechanisms of pathogenicity have notyet been elucidated [15ndash18] Therefore it is necessary to havea more advanced and effective system of vaccination againsterysipelas

In this study we developed Avicel (cellulose microcrys-talline) correlated with antigen and investigated its char-acteristics In addition antigen-immobilized Avicel vac-cine was subcutaneously injected into mice to analyze theactual immunogenicity of our newly developed antigen-immobilized Avicel recombinant protein vaccine Overallthe study highlights the development of an efficient recom-binant protein vaccine-immunosorbent system to protectswine against E rhusiopathiae infection

2 Materials and Methods

21 Bacterial Strains and Culture Conditions The E rhu-siopathiae serotype 15 strain was isolated from a diseasedpiglet in Korea For it E coli DH5120572 [Fminus 12060180dlacZΔM15endA1 hsdR17 (rKminus mK+) supE44 thi-1 120582minus recA1 gyrA96]was used as a host strain for cloning and plasmid mainte-nance The E coli BL21 host strain [Fminus hsdS gal ompT rBminusmBminus] (Novagen USA) harboring a lambda derivative DE3was used for gene expression

22 Cloning and Recombinant Plasmid Construction Thegene coding CBD of Trichoderma harzianum endog-lucanase II with artificial S

3N10

linker was ampli-fied from pEKPM-EcGAD-Lk-H6 (Ahn 2004 HyeminPark 2012) using specific primers (Forward 51015840-GAAGGAGATATACATATGCAGCAAACTGTTTGGGGG-31015840 Reverse 51015840-CTTCTTATCGGATCCGTTGTTGTTGTT-GTTGTTGTTGTTGTTGTTGCTGCTGCTAATGCATTG-AGCGTAGTA-31015840) in which the underlined characters wereintroduced for the In-fusion Advantage PCR cloningkit (Clontech USA) The italic letters indicate the site of

the restriction enzymes The polymerase chain reaction(PCR) product containing CBD with S

3N10

linker wasintroduced into the pET-28b(+) plasmid (Novagen USA)previously digested with NdeI and BamHI For the isolationof genomic DNA of E rhusiopathiae a DNeasy Bloodand Tissue Kit (Qiagen Germany) was used accordingto manufacturerrsquos instructions The surface protectiveantigen A (SpaA) gene was amplified from E rhusiopathiaegenomic DNA by PCR using the primers (Forward 51015840-ACTCCGCCAACTAGCTCTGGATCCGATTCGACAGAT-ATTTCTG-31015840 Reverse 51015840-GTGGTGGTGGTGGTGGTG-CTCGAGTTTTAAACTTCCATCGTTC) in which un-derlined base pairs were introduced for In-fusion ligationThe italic letters indicate the sites of BamHI and XhoI

23 Expression and Purification of CBD-SpaA Protein Theplasmid harboring pKPM-CBD-Lk-SpaA-H6 was trans-formed intoE coliBL21(DE3)The transformantswere grownin 2YT medium containing 50 120583gml of kanamycin at 37∘CWhen an A

600of 05 was reached 05 mM isopropyl-120573-thio-

D-galactopyranoside (IPTG) was added and cultures weregrown for an additional 3h at 37∘C Cells were harvested bycentrifugation and pellets were disrupted by sonication forfurther analysis Recombinant fusion protein was purifiedfrom the supernatant by Ni-NTA agarose (Qiagen Ger-many) using an Econo-Pac Chromatography Column (Bio-Rad USA) according to the manufacturer instructions Theprotein concentration was estimated using the BCA proteinassay kit (Pierce USA)

24 Binding of CBD-SpaA onto Avicel Binding assays werecarried out according to methods previously described withslight modification [19] Briefly Avicel (PH-101 Sigma lt50 120583m) (01 mg) was mixed with 500 120583g of the CBD-SpaAproteins at 4∘C for 2 h The supernatant was collected aftercentrifugation and used to determine uncombined proteinusing a BCA protein assay kit The amount of protein boundto Avicel was determined from the difference between finaland starting values in the supernatant Nonspecific proteinsbound to Avicel were removed by washing with 05 M NaCl

25 SDS-PAGE and Western Blot Analysis SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)was performed according to the method of Laemmle[20] For the Western blot analyses proteins separatedby 10 SDS-PAGE gel were transferred to nitrocellulose(NC) membranes The NC membranes were blocked byphosphate buffered saline (PBS) containing 5 skimmedmilk and washed three times with PBST (01 Tween 20in PBS) The membranes were incubated with His-probemonoclonal antibody (Santa Cruz Biotechnology USA)or mouse-derived antiserum prepared with whole bacteriaof E rhusiopathiae for 1 h at room temperature (RT) Thespecific antigen reacted with IgG alkaline phosphatase(AP) antibody (Sigma USA) was visualized using an APconjugated substrate kit (Bio-Rad USA)

26 Preparation of Polyclonal Mouse Antiserum To pre-pare mouse polyclonal antiserum against E rhusiopathiae






3 Results


3N10



3N10







M STkDa

100

75

50

37

25

(a)

M T SkDa

100

75

50

37

25

(b)

M STkDa

100

75

50

37

25

(c)


CBD-SpaA

M 1 2 3 M 4 5kDa75

50

37



Opt

ical

den

sity

A45

0


1x105 1x104 5x104 1x105 5x1050

00

05

10

15

20

25



(a) (b) (c)


Surv

ival

rate

()

0

20

40

60

80

100





4 Discussion






5 Conclusions



Data Availability






Acknowledgments


References



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







3 Results


3N10



3N10







M STkDa

100

75

50

37

25

(a)

M T SkDa

100

75

50

37

25

(b)

M STkDa

100

75

50

37

25

(c)


CBD-SpaA

M 1 2 3 M 4 5kDa75

50

37



Opt

ical

den

sity

A45

0


1x105 1x104 5x104 1x105 5x1050

00

05

10

15

20

25



(a) (b) (c)


Surv

ival

rate

()

0

20

40

60

80

100





4 Discussion






5 Conclusions



Data Availability






Acknowledgments


References



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



M STkDa

100

75

50

37

25

(a)

M T SkDa

100

75

50

37

25

(b)

M STkDa

100

75

50

37

25

(c)


CBD-SpaA

M 1 2 3 M 4 5kDa75

50

37



Opt

ical

den

sity

A45

0


1x105 1x104 5x104 1x105 5x1050

00

05

10

15

20

25



(a) (b) (c)


Surv

ival

rate

()

0

20

40

60

80

100





4 Discussion






5 Conclusions



Data Availability






Acknowledgments


References



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(a) (b) (c)


Surv

ival

rate

()

0

20

40

60

80

100





4 Discussion






5 Conclusions



Data Availability






Acknowledgments


References



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




5 Conclusions



Data Availability






Acknowledgments


References



































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

Microcrystalline Cellulose for Delivery of Recombinant