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FMDV 아단위 백신의 면역반응 증진을 위한
고분자 전달체의 개발
August
,2016
By
Soyeon Yoon
Depar
t
mentofAgr
i
cul
t
ur
alBi
ot
echnol
ogy
Gr
aduat
eSchool
Abst
r
act
SoyeonYoon AnimalScienceandBiotechnology DepartmentofAgriculturalBiotechnology TheGraduateSchool SeoulNationalUniversity
Foot-and-mouth disease(FMD)isahighly contagiousdisease susceptible to cloven-hoofed animals such as cattle,pigs,goats,
etc.affecting livestock industry.In this aspect,FMDV subunit vaccineshavebeen developed topreventthespread ofthisfatal animalepidemic,because they provide severaladvantages such as no need for attenuation and serological tests that can differentiateinfected animalsfrom vaccinated onesand they can be produced with epitopes and have less side effects than live attenuated/inactivated vaccine although FMDV is continuously evolving and mutating,making it difficult to develop FMDV vaccine(mainlyliveattenuatedorinactivatedvaccines)toprotect animalsfrom disease.
However,there are severallimitations such as for practical application of subunit vaccines the low stability of subunit vaccine, easy degradation by enzyme and physiological environment.In addition,theirlow immunogenicity compared to liveattenuatedvaccineslimitstheefficacyofsubunitvaccine.Th erefore,enhancementofstability and immunogenicity ofsubunit vaccinesismainbottleneckinvaccinedevelopment.
introduced to enhance the immunogenicity of subunit vaccines becausethey introduceimmunomodulatory propertiesand provide the flexibility in the route ofvaccine delivery depending on the vaccination strategies. Furthermore, immune response can be greatly regulated by single factor or combination of multiple factors by modification ofthe polymeric adjuvants such as size of polymeric particle, surface charge, hydrophilicity, molecular weightandchemicalproperties.
In chapter I,pH-sensitive and mucoadhesive thiolated CAP (T-CAP) as a polymeric carrier was developed for efficient delivery ofmucosalsubunitvaccineM5BT through oralroute.In this study,cellulose acetate phthalate (CAP),the pH-sensitive polymerthatdissolveat> pH 6.2wasmodified by thiolation to introduce mucoadhesive property and to dissolve at ileum pH. FMDV recombinant antigen M5BT was encapsulated into thiolated CAP microparticles (T-CAP MPs) using double emulsion solventevaporation method.As a result,T-CAP MPs showedsustainedreleaseofencapsulatedM5BT from theMPsat intestinalpH (pH 7.4),whilereleasing lessM5BT atgastricpH (pH 2)due to its pH-sensitive property.Also,porcine mucosa assay showed 1.4-fold enhanced mucoadhesiveness of T-CAP MPsthan non-modified CAP MPsin vitro dueto theformation of disulfide bond between thiol group in T-CAP and mucin glycoproteins in mucus layer by thiol/disulfide exchange reactions.Finally,M5BT deliveredbyT-CAP MPselicitedhigher IgA production than only M5BT in in vivo mouse experiment. Therefore,this study represents an effective mucosal subunit
vaccinedeliverythroughoralroute.
In chapterII,mannan-decorated inulin acetate(M-INAC)MPs as an immunostimulatory polymeric carrier were developed for efficientdelivery ofsubunitvaccineM5BT.In this study,inulin was modified by acetylation to introduce hydrophobic moiety. AndvaccineFMDV recombinantantigenM5BT wasencapsulated into INAC MPs and decorated with mannan using double emulsion solventevaporation method.Asaresult,M-INAC MPs showed released more than 90% ofloaded antigen for6 days, whilelessthan50% ofM5BT wasreleasedfrom INAC MPs.
As a resultofin vivo immunization in murine model,after4 weeks ofimmunization,M5BT/M-INAC MPs and M5BT/INAC MPsshowedsimilarlevelofFMDV serotypeO specificantibody with the M5BT group coinjected with conventional adjuvant CFA.And M5BT encapsulated in M-INAC MPs elicited higher IgG titerthan M5BT/INAC MPs groups exhibiting similarlevel ofIgG titerwith M5BT group coinjected with CFA.Itindicates that antigen-loaded INAC MPs can enhance antigen specific immuneresponsecomparableto theconventionalgroup implying the potential polymeric adjuvant system for subunit vaccine. Therefore,this study representsan effectivesubunitvaccine for thebetterenhancementofadaptiveimmuneresponse.
Keywords :FMD subunitvaccine,polymericadjuvant,thiolated CAP,mannan-decoration,inulinacetate,oraldelivery
Cont
ent
s
Abstract I
Contents IV
ListofTablesandFigures VIII
ListofAbbreviations XI
GeneralIntroduction 1
Review ofLiterature 4
1.Foot-and-Mouth diseasevirussubunitvaccine 4
1)FMD 4
2)FMDV vaccine 4
2.Subunitvaccinedelivery strategy 7
1)Conventionaladjuvant 7
2)Polymericadjuvant 8
3.PolymericadjuvantcarrierforFMDV subunitvaccine11
1-1)MucosalimmunityandM cells 11
1-2)Passiveimmunization 12
1-3)Mucoadhesivepolymericcarrierfororalvaccination 13 2)Immunostimulatorypolymericcarrier 16 Chapter I.Development of pH-sensitive and mucoadhesive T-CAP MPsforefficientdelivery ofsubunitvaccineM5BT
through oralvaccination 19
1.Introduction 19
2.MaterialsandMethods 22
1)SynthesisofthiolatedCAP 22
2)QuantificationofthiolgroupcontentinT-CAP 22
3)PreparationofM5BT protein 23
5)MorphologybyFE-SEM 27 6) Determination of loading content and encapsulation
efficiency 27
7)Invitroreleasebehaviortest 28 8)StructuralintegrityoftheM5BT protein 28
9)Exvivoporcinemucosaassay 28
10)Invivooralimmunizationinmurinemodel 28
11)Bloodandfecalsampling 29
12)Anti-M5BT antibodydetectionbyELISA 30 13)Flow cytometric detection ofMHC class II-expressing
cellsinPeyer’spatches 31
14)Statisticalanalysis 32
3.Resultsanddiscussion 33
1)PreparationandcharacterizationofT-CAP 33 2)PreparationandcharacterizationofM5BT/T-CAP MPs
34 2-1)IsolationandpurificationofM5BT protein 34 2-2)PreparationandcharacterizationofM5BT/T-CAP MPs 35 3)MorphologyofM5BT/CAP andM5BT/T-CAP MPs 36 4)Loadingcontentandencapsulationefficiency 37 5) In vitro release behavior of M5BT from CAP and
T-CAP MPs 39
6)MucoadhesivepropertyofT-CAP MPs 41 7)Flow cytometric detection ofMHC class II-expressing cellsinPeyer’spatchesinileum 43 8) M5BT-specific antibody production after oral
4.Summary 49
Chapter II. Development of mannan-decorated inulin microparticles forenhancing the immunogenicity ofsubunit
vaccine 50
1.Introduction 50
2.MaterialsandMethods 52
1)SynthesisofINAC 52
2) Preparation of M5BT-loaded INAC MPs and
M5BT-loadedM-INAC MPs 52
3)MorphologyandsizedistributionofMPs 53 4)Identificationofmannan-decorationintoMPs 53 5) Determination of loading content and encapsulation
efficiency 54
6)Invitroreleasebehaviortest 55 7)Invivoimmunizationinmurinemodel 55
8)Bloodandfecalsampling 56
9)FMDV serotypeO specificantibodyproduction 57 10)Anti-M5BT antibodydetectionbyELISA 57
11)Statisticalanalysis 58
3.Resultsanddiscussion 59
1)PreparationandcharacterizationINAC 59 2)Preparation and characterization ofM5BT-loaded INAC
MPsandM5BT-loadedM-INAC MPs 61
3)MorphologyofMPs 62
4)Confirmationofmannan-decorationinINAC MPs 62 5)Loadingcontentandencapsulationefficiency 63 6)InvitroreleasebehaviorofM-INAC MPs 64 7)FMDV serotypeO specificantibodyproduction 65
8)M5BT-specificimmuneresponseafterimmunizationwith
MPs 67
4.Summary 69
Conclusion andFurtherProspects 70
LiteratureCited 73
Li
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<Figure>
Figure1.Experimentalflow chartofthisstudy 3 Figure 2. Polymeric adjuvant strategies for substituting
conventionaladjuvants 9
Figure3.GraphicalabstractofchapterI 21 Figure4. CompositionofM5BT protein 24 Figure 5. Equation for encapsulation efficiency and loading
content 27
Figure6.Invivooralimmunizationschemeinmurinemodel 29 Figure7.SynthesisschemeofthiolatedCAP 33 Figure8.1H 600MHzNMR spectra of(a)CAP and (b)T-CAP
MorphologybyFE-SEM 34
Figure 9. M5BT protein purification by his-tag affinity
chromatography 35
Figure 10.Procedure ofM5BT-loaded T-CAP MPs by double
emulsionmethod 36
Figure11.AnalysisofmorphologyofMPsbyFE-SEM 37 Figure12.EvaluationofproteinstructureofM5BT releasedfrom
M5BT/CAP MPsandM5BT/T-CAP MPsbySDS-PAGE 39
Figure 13. In vitro release profile of M5BT protein from M5BT/CAP andM5BT/T-CAP MPsatsimulatedgastrointestinal
pH 41
Figure 14.Analysis ofmucoadhesive property ofMPs in small
intestine 42
cellsinPeyer'spatchesfrom immunizedmice 44 Figure 16. M5BT-specific immune response after oral
immunizationwithMPs 47
Figure17.Anti-M5BT IgA levelin fecesafteroralimmunization
withMPs 48
Figure 18.Calculation equation for encapsulation efficiency and
loadingcontent 54
Figure 19.In vivo immunization experimentschedule in murine
model 56
Figure20.Schematicdiagram ofFMDV typeO antigen blocking
ELISA 57
Figure21.ChemicalreactionschemeforsynthesisofINAC 59 Figure22.600MHz1H-NMR spectraof(a)inulin and(b)inulin
acetate 60
Figure 23. Graphical illustration of microparticle formation protocolofM-INAC MPsbydoubleemulsionmethod 61 Figure24.Themorphology ofM5BT-loadedINAC and M-INAC
MPsanalyzedbyFE-SEM 62
Figure 25.Confirmation ofmannan-decoration ofINAC MPs by
CLSM 63
Figure 26. In vitro release profile of M5BT protein from
M5BT/INAC orM-INAC MPsatPBS (pH 7.4) 65
Figure 27.ELISA for in vitro detection of antibodies against FMDV serotypeinserum from immunizedmice 66 Figure 28.Antigen-specific immune response afterimmunization
withMPs 68
<Table>
Table 1. Parameters of polymeric particles affecting immune
response 10
Table2.Characteristicsofthiomers 18 Table3.His-tagaffinitychromatographybuffercomposition 25 Table4.Loading contentand encapsulation efficiency ofM5BT-loaded
MPs 38
Li
stofAbbr
evi
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Ab:antibody Ag:antigen
APCs:antigenpresentingcells BSA :bovineserum albumin
BCA assay:bicinchoninicacidassay CAP :celluloseacetatephthalate
CMF HBSS :calcium-andmagnesium-freeHank’sbalancedsalt solution
DCs:dendriticcells
DCC :N,N’-dicyclohexylcarbodiimide DCM :dichloromethane
DMF :dimethylformamide DMSO :dimethylsulfoxide
DTNB :5,5-dithio-bis-(2-nitrobenzoicacid)
E.coli:Escherichiacoli
EDTA :ethylenediaminetetraaceticacid
ELISA :enzyme-linkedimmunosorbentassay FACS :fluorescence-activatedcellsorting FBS :fetalbovineserum
FDA :fluoresceindiacetate
FE-SEM :fieldemissionscanningelectronmicroscope FMDV :foot-and-mouthdiseasevirus
HRP :horseradishperoxidase IN :inulin
IPTG :isopropylβ-D-1-thiogalactopyranoside LB :Luria-Bertani
M-cells:microfold-cells MPs:microparticles NaOAc:sodium acetate NHS :N-hydroxysuccinimide NMR :nuclearmagneticresonance PBS :phosphatebufferedsaline
PMSF :phenylmethanesulfonylfluoride PPs:Peyer’spatches
PVA :polyvinylalcohol
RPMI1640:RoswellParkMemorialInstitute1640 SDS :sodium dodecylsulfate
SDS-PAGE :SDS-polyacrylamidegelelectrophoresis T-CAP :thiolatedCAP
TMB :tetramethylbenzidine TLR :toll-likereceptor
Gener
alI
nt
r
oduct
i
on
Foot-and-mouth disease(FMD)isahighly contagiousdisease susceptible to cloven-hoofed animals affecting livestock industry. FMDV iscontinuously evolving and mutating,making itdifficult to develop FMDV vaccine to protectanimals from disease.In this aspect,FMDV subunit vaccines have been developed to preventthespread ofthisfatalanimalepidemicasthey provide severaladvantagessuchasserologicalteststhatcandifferentiate infectedanimalsfrom vaccinatedones.
However, there are limitations for practical application of subunitvaccinesbecausethey canbeeasily degradedbyenzyme and physiological environment, and its low immunogenicity compared to live attenuated vaccines. So, enhancement of stability and immunogenicity of subunit vaccines is main bottleneckinvaccinedevelopment.
To improve the efficacy of subunit vaccines, two vaccine delivery strategiesweredesignedbasedon theadjuvanteffectof polymeric carriers.Experimentalflow chart of this study was showninFigure1.
In thisstudy,FMDV recombinantantigen M5BT wasusedfor model subunit vaccine. M5BT is consisted of multi-epitopes which contain five B-cellepitopes and one T-cellepitope from viralprotein 1 (VP1)region ofFMDV serotype O as wellas M-cell targeting peptide (CKSTHPLSC) in N-terminal region. VP1 region is the main targetofsubunitvaccine development because FMDV infect the host cells through binding of RGD
(arginine-glycine-aspartate) motif in VP1 region to integrin receptor (
Ca
o,Y.eta
l
.
,2
0
1
2
)
.Providing T-cellepitope with B-cellepitopecaninducedifferentiationofB-cellstoplasmacells because T-cellepitope can presentthe processed antigen from dendritic cells to B-cells. This subunit vaccine can protect various serotype O FMDV because offive B-cellepitopes that areoriginatedfrom fivedifferentserotypeO FMDV strains.In ChapterⅠ,FMDV subunitvaccineM5BT wasencapsulated in thiolated CAP microparticles (T-CAP MPs)forefficientoral delivery of mucosal subunit vaccine. To overcome low bioavailability andantigenuptakeoforalsubunitvaccine,thepH sensitive and mucoadhesive T-CAP polymer was used to encapsulate the subunit vaccine. Release of FMDV subunit vaccine to ileum (M-cell rich region), and mucoadhesion to mucus layer may maximize the antigen uptake by M-cells in Payer‘spatches.
In ChapterⅡ,FMDV subunitvaccineM5BT wasencapsulated inmannan-decoratedinulinacetatemicroparticles(M-INAC MPs) forenhancing immune response againstloaded antigen.Because itisknown thatINAC can function asavaccineadjuvantwhen antigen was encapsulated into INAC particles (DIEGO G SILVA
et al., 2014), formation of antigen-loaded INAC particles can enhanceimmuneresponseaboutsubunitvaccineto overcomeits low immunogenicity.In addition,mannan which is also agonist for TLR and MR (mannose receptor) was decorated on the surface of INAC MPs to enhance antigen uptake by immune
cells.
Both particulatevaccinemay function asvaccineadjuvantsby enhancing the uptake oftheantigen by antigen presenting cells including macrophages and dendritic cells and delivering the encapsulated antigen to immune cells resulting in improved immuneresponse(Adams,J.R.etal.,2014).
Revi
ew ofLi
t
er
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1.Foot-and-mouth diseasevirussubunitvaccine
1)FMD
Foot-and-mouth disease(FMD)isahighly contagiousdisease susceptibletocloven-hoofed animalsincluding pigs,cattle,goats, andsheepandcontinuouslyoutbreaksaffectinglivestockindustry. Foot-and-mouth disease is characterized by high fever,blisters in mouth,tongue and hoofs,excessive salivation,and anorexia. Foot-and-mouth disease is caused by an aphthovirus of the family Picornaviridae,which is a non-enveloped capsid virus with icosahedral symmetry and single strand positive RNA (Alexandersenetal.,2003).Becauseofthedifferentserotypes(A, O,C,SAT1,SAT2,SAT3 and Asia1)and its subtypes within each serotypes(E.Domingo etal,2002),FMDV iscontinuously evolving and mutating,making it difficult to develop FMDV vaccinetoprotectanimalsfrom disease.
2)FMD vaccine
The purpose ofvaccination is to generate immune response againstinfectious agentsuch as pathogens and viruses and to generate memory response by stimulating innate and adaptive immuneresponse.Vaccinescan becategorizedasliveattenuated vaccines,inactivated vaccines,recombinantvaccine,and subunit vaccine.
preventthespread ofthisfatalanimalepidemic.Especially,live attenuated vaccines have been developed and manufactured worldwide.
But, this traditional vaccines have several disadvantages althoughitshowedgoodprotectionabilityofFMDV inlivetstock. The disadvantages include the possibilities of insufficient inactivation ofFMDV ofvaccinestoavirulentform ortherisk ofthespread oflivevirusduring vaccineproduction (T.R.Doel
et al.,2003),the different virus population resulted from the adaptation step in serialpassages to make the virus infectthe new host cells such as chicken embryo kidney cell. So, attenuatedvirusisasimilarversion oforiginalvirusin thewild (so called ‘field strain’) making it hard to protect field strain virus.Inaddition,thiskindofvaccinecannotquickly responseto therapidly spreading ofnew virusstrainandimmunizedanimals aredifficulttobedistinguishedfrom infectedanimalsinnatureb ecause ofthe presence ofnon-structuralproteins ofFMDV in thevaccine(D.K.Mackay,etal.,1988).
To overcome these limitations of traditional vaccines,it is important to design antigen (Ag) that can elicit neutralizing antibodies with less or no safety concerns. In this context, subunitvaccineisreceiving thespotlight.Thesubunitvaccineis made with a recombinantvirus protein by inserting an antigen gene into virus- or bacterial vector to produce recombinant subunit vaccine.Nowadays,subunit vaccine is being explored becausethey canbeconsistedofepitopesthatcan berecognized by antigen presenting cells (APCs) and are immunogenic.
Vaccination with the recombinant virus antigens can protect these problems described above,and these antigens can provide advantagessuch asno need forlargescalecultureofvirusand attenuations steps by producing antigens synthetically (Roitt’s essentialimmunology12th,353p).
Despite of these advantages of subunit vaccine, there are limitations of subunit vaccine in practical uses. Enzymatic degradation in physiologicalconditions and low immunogenicity severely limitthe efficacy ofsubunitvaccine.To improve the lowerimmunogenicity ofsyntheticsubunitvaccinescompared to traditionalvaccines as wellas bioavailability,the importance of introducingsubunitvaccinecarriersisbeingaddressed.
2.Subunitvaccinedelivery strategy
Conventionalvaccinesusually donotrequireadjuvantsbecause they have enough structures thatcan be recognized by immune cellsasinvader.However,theimmunogenicity ofsubunitvaccine which is composed of proteins/polypeptides is poor.So it is necessarytouseadjuvanttoinitiateproperimmuneresponse.
1)Conventionaladjuvant
Conventional adjuvant includes mineral salts, emulsion, immune-stimulatory complexes (ISCOMs),microorganism-derived adjuvants, virosomes and virus-like particles (VLPs), and cytokines (Adams and Mallapragada, 2014). The action of adjuvants can be categorized by antigen delivery and immunostimulation.
First,adjuvantthatacts as an antigen delivery vehicle can trap or disperse the antigen inside so thatitcan deliver and present it to the immune cells.This kind of action is called “depoteffect”.Due to the depoteffect,particulate vaccine can elicitimmuneresponsebetterthan solublevaccineby presenting antigenasamultimericform (Roitt’sessentialimmunology12th).
Second, adjuvant that acts as an immunostimulants can stimulateimmunesystem resulting inenhancedimmuneresponse. Examples are microorganism-derived components (such as TLR agonist,mannan,CpG,LPS,MPLA,CFA),ISCOMs,cytokines.It isimportantto design immunostimulantsin vaccinedevelopment because it can activate the dendritic cells (DCs),one of the professionalAPCs,leading toincreased antigen uptake,migration
to lymph nodes followed by induction of adaptive immune response.Many adjuvantsincluding mineralsalts(such asalum), emulsions (such as CFA, IFA), liposomes, ISCOMS and polymeric carriers can function as antigen delivery vehicle and immunostimulantsatthesametime(Roitt’sessentialimmunology 12th).
The most widely used adjuvants are complete freund’s adjuvant(CFA).Antigens are emulsified in the CFA,which is composed ofinactivated mycobacterium.So,the adjuvanteffect ofCFA resulted from the depoteffects and immunostimulatory effects. CFA is used in several FMD vaccines although its increased immune-boosting effect is related to the toxicity of CFA itself. For the safety concerns, its use in human is forbidden by regulatory authorities and its use in animals is regulatedbytheguidelines.
2)Polymericadjuvant
Biomaterial-based polymeric adjuvantcan function as delivery vehicle in forms of nanoparticles (NPs),microparticles (MPs), micelles, matrices and etc. offering more advantages than conventionaladjuvants through a number ofmechanisms.Itis possible to reduce immunization dose due to its depot effects releasingantigensinasustainedmanner.
Polymericadjuvantscanalsobedesignedinanumberofways to introduce immunomodulatory properties and to provide the flexibility in the route of vaccine delivery depending on the vaccination strategies.The summary of the multiple ways of
modifying and functionalizing polymers for antigen delivery is shown in Figure2.Forpolymericadjuvantdevelopment,immune responsecan begreatly affected by singlefactororcombination ofmultiplefactorsbymodificationofthepolymericadjuvantsuch a size of polymeric particle, surface charge, hydrophilicity, molecular weightand chemicalproperties as listed in Table 1 (AdamsandMallapragada,2014).
Figure 2. Polymeric adjuvant strategies for substituting conventionaladjuvants (This figure was modified from Adams andMallapragada,2014).
Parameters Relatedimmuneresponse
Modification Size Tissueuptake,cellularuptake
Surfacecharge Interaction with cell membrane and mucosal surface
Hydrophilicity Hydrophilicity↓ =identifiedbythebodyas foreign,enhancecelladhesionandphagocytosis ofmacrophages.anincreaseofadjuvanteffect Molecular
weight
Hydrolysisofpolymer Releasepattern Chemical
properties
Various
Functionalization Modification withother polymers
Stealtheffect(ex:PEGylation) Opsonization
Bloodcirculationtime(Poloxamerand poloxamineblockcopolymer)
Penetrationofmucosalsurface(ex:Chitosan) Conjugationof
antibodies/carb ohydrates
Targetingandstimulatingantigenpresenting cells(ex:DCs,Macrophages)
Table1.Parametersofpolymericparticlesaffecting immuneresponse (Reference:AdamsandMallapragada,2014)
3.PolymericadjuvantcarrierforFMDV subunitvaccine
1-1)Mucosalimmunity andM cells
A number ofpathogens gain entry to the body via mucosal surfacesandtheinductionofimmuneresponsesatthesesurfaces can be crucialin providing the bestprotection againstdisease. Thesemay occurby thecollectionsoflymphocytes,plasmacells and phagocytes throughout the lung and the lamina propria (connective tissue) of the intestinal wall, or as organized mucosa-associated lymphoid tissue (MALT) with well-formed follicles.
Gut-associated lymphoid tissue (GALT)is separated from the lumen by epithelium with tightjunctions and a mucous layer. This epithelium is scattered with microfold (M)-cells; specialized antigen-uptaking cells with short,irregularmicrovilli on theirapicalsurface thatendocytose antigens.The endocytic vesiclescarry theantigen to beexocytosed atthebasalsurface fortheattention ofintraepitheliallymphocytes,dendriticcellsand macrophages.Thecellsandtissuesinvolvedinmucosalimmunity form an interconnected secretory system which IgA-producing B-cells may circulate. Foreign material, including bacteria, is taken up by M-cells and passed on to the underlying Peyer’s patch antigen-presenting cells, which then activate the appropriatelymphocytes.Thus,thePeyer’spatchesconstitutethe inductive site for immune responses in the gut. After their activationisinduced,thelymphocytestravelviathelymphtothe mesenteric lymph nodes where additional activation and
proliferation may occur.A specialfeature ofantigen-presenting cells from Peyer’s patches, mesenteric lymph nodes and the lamina propria is that they contain a population of CD103 + dendriticcells.
The “imprinted”T cells then move via the thoracic ductinto the bloodstream and finally in to the lamina propria.In this responsivesite,they assisttheIgA-forming B-cellsthatprotect a wide area of the bowel with protective antibody.T- and B-cells also appearin the lymphoid tissue ofthe lung and in othermucosalsitesguidedby theinteractionsofspecifichoming receptorswith appropriateHEV addressins.Itisinteresting that mucosal immunization at inductive site can be effective at anothermucosaltractgeneratingantibodyproduction
Many of the adjuvants can be used as mucosaladjuvants although there are also a number of molecules that are particularly effectiveas mucosaladjuvants,mostnotably cholera toxin (CT) and E.coliheat-stable enterotoxin (LT).Modified forms ofthe toxins and theirsubunits can powerfully stimulate mucosal responses although their toxicity limits practical application of mucosal adjuvant (Roitt’s essential immunology 12th).
1-2)Passiveimmunization
AlthoughthemorbidityofFMD is100%,themortalityisabout 50% in pigletswhereaslessthan 5% in adultanimals(Brownlie, 1985).Passive immunization is the key pointthatgives piglets protection from FMD.Thepassiveimmunization can beachieved in the fetus by maternally derived antibodies acquired by
placentaltransferand in thenewborn by intestinalabsorption of colostral immunoglobulin (Roitt’s essential immunology 12th).
Secretory IgA (sIgA)accounts forthe majorimmunoglobulin in colostral milk and it remains in the intestine instead of the absorption,sothatitcan protectbacteriaorvirusatthemucus layers.
Moreover, oral vaccination of mucosal subunit vaccine has severaladvantagesasbelows:
1) immune responses at the site of primary colonization of pathogens,2)mucosalimmune response to othersites such as upperrespiratory tract,mammary gland,3)safety,efficacy and high patient compliance rates because the vaccines are administered by the natural route of infection, 4) vaccine formulation ofhigherstability such as solid tablets is available fororaladministration(Brownlie,1985).
1-3)Mucoadhesivepolymericcarrierfororalvaccination Mucoadhesivepolymericcarriersystemsaredesignedtoextend theresidenttimeofpolymerintargetedregion/tissuemaximizing the amount of absorbed drug in desired absorption site (oral, nasal,respiratory,gastrointestinal,vaginal,etc.) and protecting vaccinefrom enzymaticdegradation.
Generally,mucoadhesivepolymers havecharacteristicssuch as hydrophilicity,high molecular weight,optimum surface tension, hydrogen bonding capacity, sticky to mucus glycoproteins, non-toxicity and non-allergenicity, chemical inertness and cost-effectiveness.
mucoadhesivepolymersand naturalmucoadhesivepolymers.The syntheticmucoadhesivepolymersincludeCarbopolⓇ,polycarbophil,
poly(acrylic acid), polyacrylate, poly (methylvinylether-c o-methacrylic acid), polymethacrylate, polyalkylcyanoacrylate, poly(hydroxyethylmethylacrylate),poly(ethyleneoxide),poly(vinyl pyrrolidone), poly(vinyl alcohol) and thiol-containing synthetic polymers.The naturalmucoadhesive polymers include cellulose derivatives (carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose,sodium carboxymethylcellulose,methyl cellulose, methylhydroxyethyl cellulose etc.), alginate, dextran, karaya gum,guar gum,xanthan gum,soluble starch,gelatin, pectin,chitosan,tragacanth,hyaluronic acid,and thiol-containing natural polymers etc. Among them, thiol-containing polymers havepaid attention so called a new generation ofmucoadhesive polymers.
Thiolated polymers, also known as ‘thiomers’, are modified from existing polymers by introducing free thiolgroup (-SH) given by thiol group donor such as cysteine, homocysteine, cysteamine, N-acetylcysteine, glutathione, thioglycolic acid, mercaptobenzoic acid, 4-aminothiophenol, and mercaptophenylacetic acid (A.Bernkop-Schnurch etal.,1999;S. Bonengel, A. Bernkop-Schnürch, 2014). Unlike other mucoadhesive polymers that adhere to mucus layer by non-covalentbonds like hydrogen bonds,van derWaal’s forces and ionic interactions,thiolated polymers are considered as a promising new generation ofmucoadhesivepolymerin theaspect offormation ofstrong covalentbond between thiolated polymer
andmucusglycoproteinsviathiol/disulfideexchangereaction and an oxidation process(Leitneretal.,2003).Duetotheubiquitous cysteine-rich domain in mucuslayer,thiomersmimicthenatural mechanism of secreted mucus glycoproteins, which are also covalently anchored in the mucus layer by the formation of disulfide bonds, the most commonly encountered bridging structure in biological systems (A. Bernkop-Schnurch et al.,
2000).Thiolated polymers have severaladvantages:(1)stability enhancement due to the formation of inter- and/or intra-molecular disulfide bonds,(2) acquisition ofmucoadhesion property, (3) controlled drug release, and (4) a permeation enhancing effect (Bernkop-Schnurch et al., 2000). Numerous thiolated polymers were developed including thiolated Eudragit (Bijay Singh et al., 2015), thiolated polycarbophil, thiolated carboxymethylcellulose(A.Bernkop-Schnurchetal.,2000b), thiolated poly(acrylic acid) (Leitner et al., 2003), thiolated hydroxypropylmethylcellulose(Singhetal.,2015;Lietal.,2016). CharacteristicsofthiomersarelistedinTable2.
Thiolated polymers as peptide drug carriers have been developed fororaldelivery systems in pharmaceutics,biomedical sciences, and biomaterial sciences and so on. Oral peptide delivery system with thiolated polymers can enhancethe uptake of peptide due to the high mucoadhesive and permeation enhancing property resulted in the significantly improved oral bioavailability (A.Bernkop-Schnurch et al,2005;Singh et al., 2015;Lietal.,2016).
2)Immunostimulatory polymericcarrier
An idealvaccineadjuvantmustbridgethegapbetween innate and adaptive immunity,to notonly initially engage the immune system,but also elicit a successfulmemory response against future infections. Immune cells such as macrophages and dendritic cells recognize invading pathogens through pattern recognitionreceptors(PRRs)whichrecognizepathogen-associated molecular patterns (PAMPs) on viruses, bacteria, and other pathogens.Recentdataalsosuggeststhatthecentralmechanism ofadjuvanticitythatwasonceattributedtoantigenadsorption,a process known as the depot effect, may be through the engagement of PRRs.Given the increased knowledge of how pathogens and adjuvantsinteractwith theimmunesystem,itis now possible to design novel vaccines to train the immune system torespondtospecificpathogens.
Polymers have been studied formany years as adjuvants as they offer a unique setofadvantages over more conventional adjuvants.Oneofadvantages is thatthe size,molecularweight andchemistry ofpolymerscan betailoredtotargetvariouscells of the immune system.Polymers that have been extensively evaluatedasvaccineadjuvantsincludepolysaccharides,polyesters and non-ionic block copolymers.These polymers can be further modified with other components including carbohydrates to activate PRRs that may provide an optimalimmune response againsta given pathogen.So,itis importantto design vaccine with polymeric adjuvants so that they can improve the effectivenessvaccines.
Typically,vaccinesinteractwith pathogen recognition receptors (PRRs) to initiate an innate immune response mimicking the infection ofpathogen (Roitt’s essentialimmunology 12th).Some polymerslikeinulincanalsoactivatePRRs.
Inulinacetate(INAC)isreferstoacetylatedinulin.Typicallyat leastabout90% ofavailable hydroxylgroups ofthe inulin are acetylated. INAC is insoluble in water even at elevated temperatures,butissoluble in various organicsolvents such as acetone, chloroform, dichloromethane, ethyl acetate, etc. It is reported that INAC can only exert the function of vaccine adjuvantwhen antigen wasco-injected with INAC,notamixed form.So antigen should beencapsulated into INAC particlesfor enhancing immune response against injected antigen. INAC microparticlesandnanoparticlesmayfunctionasvaccineadjuvant by enhancing the uptake of the antigen and delivering the encapsulatedantigentoimmunecellsforactivation.INAC isalso anovelTLR agonist.So,INAC can work asavaccineadjuvant byencapsulatingantigensasparticleforms.
Polymer Thiolgroupcontent Method References PAA-Cys 402.5±58.2-776.0± 47.1 umolthiol groups/gPAA-Cys Ellman’s method Leitneretal.,2003
Chitosan-TBA 1022umol/g polymer Ellman's
method Dünnhauptetal.,2011 PAA-Cys 955umol/g polymer
Chitosan-TBA
conjugate60 59.groups8±3./g1umol thiol meEllman'thods Bernkop-Schnürchetal.,2003 Chitosan-TBA
conjugate100 95.groups1±9./g0umol thiol Chitosan-Thiobutylam
idineconjugate 264umoltgroup/g hiol meEllman'thods Roldoetal.,2004 PCP-Cys 180-344umol/g
polymer tIodomeitrationtric Clausenetal.,2000 PCP-Cys 12.3umolthiol
groups/g tIodomeitrationtric Bernkop-Schnürchetal.,2000 CMC-Cys 22.3umolthiol
group/g Chitosan-TBA 203.7±40.9
μmol/g Elmelman'thods Bernkop-Schnürchetal.,2004 Chitosan-TBA 100
μmol/g Elmelman'thods ElhassanImam etal.,2005 Eudragit L100-Cys 390.3±13.4
μmol/g Elmelman'thods Zhangetal.,2012 HMW Chitosan-TBA 213
μmol/g meEllman'thods Bravo-Osunaetal.,2006 LMW Chitosan-TBA 473
μmol/g
Thiolated Chitosan 320 ± 50umol/g Ellman's
method Muelleretal.,2012 Thiolated
hydroxyethylcellulose umol131.58 ±11./g 17 meEllman'thods Sartietal.,2010 HA-Cys 201.3 ± 18.7umol/g Ellman's
method Kafedjiiskietal.,2007 PCP-Cys 100 ± 8umol/g Iodometric
titration Bernkop-Schnürchetal.,2000 CMC-Cys 1280 ±84umol/g Iodometric
titration
Chapt
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Oralvaccinationisconsideredasamostconvenientandeasiest waytovaccinatelivestockanimalsalthoughlow bioavailabilityof orally delivered antigen (Ag) in harsh gastrointestinal environmentseverely limits the wide application oforalvaccine in livestock industry.Vaccine delivery with polymeric carriers can provide advantages such as physicalprotection ofvaccine from physiologicalpH and enzymes,improved antigen stability, improved immunogenicity,controlled release and functionalization withfunctionalorimmune-enhancingmaterialstoovercomethese limitations.
Here,wedeveloped apH-sensitiveand mucoadhesivethiolated CAP (T-CAP) as a polymeric carrier for efficient delivery of mucosalsubunitvaccineM5BT through oralroute.In thisstudy, celluloseacetatephthalate(CAP),the pH-sensitivepolymerthat dissolve at > pH 6.2 was modified by thiolation to introduce mucoadhesive property and to dissolve at ileum pH. FMDV recombinantantigen M5BT wasencapsulated into thiolated CAP microparticles (T-CAP MPs) using double emulsion solvent evaporation method. As a result,T-CAP MPs showed more
release ofM5BT from M5BT-loaded MPs atintestinalpH (pH 7.4)than atgastricpH (pH 2)dueto itspH-sensitiveproperty. Also, porcine mucosa assay showed 1.4-fold enhanced mucoadhesiveness ofT-CAP MPs than non-modified CAP MPs
in vitro due to the formation of disulfide bond between thiol group in T-CAP and mucin glycoproteins in mucus layer by thiol/disulfide exchange reactions. Finally, M5BT delivered by T-CAP MPselicited higherIgA production than M5BT itselfin
in vivo mouse experiment.Therefore,this study represents an effectivemucosalsubunitvaccinedeliverythroughoralroute.
2.Mat
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1)SynthesisofthiolatedCAP
The synthesis of thiolated CAP (T-CAP) was carried out according to the method described previously (Singh,B etal., 2015).Briefly,4 g ofCAP wasdissolved in 100mlofdimethyl sulfoxide (DMSO) and the carboxylic acid moieties of the polymerwereactivatedby N,N’-dicyclohexylcarbodiimide(DCC) (4.189 g) and N-hydroxyl succinimide (NHS) (2.337 g) with constantstirring atroom temperaturefor24h undernitrogenous condition to avoid the oxidation of sulfhydryl groups by atmosphericoxygen.By-productswereremovedbyfiltrationwith buchnerfunnel(90mm)andthefiltratewasfurtherreactedwith L-cysteine hydrochloride monohydrate (0.355 g)for18 h under similar condition.The reaction mixture was filtered to remove by-productsand thefiltratewasdialyzed initially against3L of DMSO toremovetheunboundL-cysteinehydrochlorideandthen againstdistilled waterseveraltimes to remove DMSO.Finally, the polymer solution was lyophilized after dialysis and the product was stored at –20℃ until use. The conjugation of L-cysteine was confirmed by 600MHz1H NMR spectroscopy
(AVANCE 600,Bruker,Germany).
2)Quantification ofthiolgroupcontentin T-CAP
The degree of thiolgroup substitution in the T-CAP was determined by Ellman’s method according to the manufacturer’s instructions.Briefly,10 gm/mlofT-CAP solution was prepared
and diluted with 0.1 M sodium phosphate buffer (pH 8) containing 1 mM EDTA to prepare different dilutions.50 ul aliquotsofeachdilutionwereaddedto500ulof0.5M phosphate buffer (pH 8.0) and 10 ulofEllman’s reagent(0.4 mg/mlof DTNB in 0.5 mol/lphosphate buffer,pH 8.0).Controlreactions were carried out with non-modified CAP.The samples were shielded from lightand incubated atroom temperatures for 15 min.And then,100 ulofthe supernatantwas transferred to a microtitrationplateandtheabsorbancewasmeasuredat412nm using microplatereader(TECAN Infinite200 PRO).Theamount ofthiolgroupswascalculated from thestandard curveprepared by measuring the absorbance of L-cysteine hydrochloride monohydratesolutionasdescribedabove.
3)Preparation ofM5BT protein
E.coliBL21(DE3)harboring ageneencoding forM5BT protein was seed in 4 mlofLB medium supplemented with ampicillin and incubated overnightat37℃ with shaking at200 rpm.4 ml of seed culture was inoculated in 800 ml of LB medium supplementedwith ampicillin and incubated at37℃ with shaking at200 rpm.When the culture reached an opticaldensity (O.D 600)of0.5-0.6,theculturewasinduced with 0.5mM IPTG and incubatedat37℃ withshaking at200rpm for4h.AfterIPTG induction,thecellswereharvestedby centrifugationat6,000rpm for 10 min,washed twice with ice-cold PBS and pellets were resuspended in 20 mlofhis-binding bufferper200 mlculture volume.Then,thecellsuspensionsweresonicated(10spulseon;
5spulseoff)for8 min with ablunt-end tip.Thecrudeprotein wascollected aftercentrifugation at17,000 rpm for15 min at4 ℃.
Figure4.CompositionofM5BT protein
The crude protein was purified using histidine-tag (his-tag) affinity chromatography. Before purification, the crude protein was filtrated by 0.45 um syringe filter to remove celldebris. Crude protein solution was loaded onto his-bind resin (6 ml), equilibrated with 3volumeofbinding bufferand charged with 5 volume of charging buffer.After washing with 3 volume of binding buffer to remove un-charged nickelions,column was washed with washing buffers containing different imidazole concentration (5,40,and70mM)toremovenon-specificprotein. 6 histidine-tag bearing M5BT was eluted with elution buffer. Buffercompositions are listed in Table 3.Each fractions were analyzed by SDS-PAGE to check the purification quality and purity ofprotein.The purified protein was dialyzed against5 L ofdistilled waterat4℃ for24h with waterchangesforthree
Buffer Imidazole Tris-Cl NaCl NiSO4 EDTA pH Volume Flow rate (ml/min) Chargingbuffer - - - 50mM -- 5 5 Bindingbuffer 5mM 20mM 0.5M - 7.9 3 5 Washing buffer 1 5mM 10 1-2 2 40mM 15 1-2 3 70mM 1 1-2 Elutionbuffer 1M 6 1-2 Stripbuffer - 100mM 3 5
*1volume=resinvolume
Table3.His-tagaffinitychromatographybuffercomposition
times to remove the salts in elution buffer followed by lyophilization.
Endotoxin was removed by ToxinEraser™ Endotoxin Removal
Kit (GenScript) according to the manufacturer’s instructions. Briefly,1.5 mlofpre-packed column consisted ofthe matrix of modifiedpolymyxinB (PMB)isactivatedbyadding 5mlofcold regeneration bufferand letthe bufferdrain completely atspeed of0.25 ml/min and repeated twice more.And the column was equilibrated by adding 6 mlofequilibration buffer and letthe bufferdraincompletelyatspeedof0.5ml/minandrepeatedtwice more. After applying the sample to the column, endotoxin-removed protein was collected and endotoxin levelin proteinsamplewasdetectedbyToxinSensor.
4)Preparation ofT-CAP MPs
4-1)Preparation ofM5BT-loadedT-CAP MPs
water-in-oil-in-water (W1/O/W2) double emulsions solvent
evaporation methoddescribedpreviously with alittlemodification (Singh,B etal.,2015).200 ulofaqueous solution ofM5BT (5 mg)wasstabilizedwith100ulof10% PluronicF-127solutionto form an internalaqueous phase (W1).100 mg ofeach T-CAP
and CAP was dissolved in 5 mlofdichloromethane and ethyl acetate:ethanol(1:1)respectively.Organic phase was emulsified with the aqueous phase using an ultrasonic processor (Sonics, Vibra cells™)(4 outputwatts)on ice for1 min 30 secto form
1stW1/O emulsion.The mixture emulsion was added drop by
drop into 50 mlof1% (w/v)poly(vinylalcohol)(PVA)solution and then homogenized with Ultra Turrax (T25,IKA,Germany) at13,000rpm for1min 30sectoform W1/O/W2emulsion.The
resulting double emulsion was stirred for 4 h at room temperature to evaporate the organic solvent. After solvent evaporation,thehardenedMPswerecollectedbycentrifugationat 6,000rpm for10min,washedwithdistilledwater,andlyophilized undervacuum.M5BT-loadedT-CAP and CAP MPswerestored at–20℃ untiluse.
4-2)Preparation ofFDA-loaded T-CAP MPs (FDA/T-CAP MPs)
Fluorescein diacetate (FDA)-loaded T-CAP and CAP MPs were similarly prepared as above-mentioned M5BT-loaded T-CAP MPs. 5 mg of FDA was dissolved into 200 ul of dichloromethane, then added to T-CAP (100 mg) solution dissolved in 5 mlofdichloromethane,and homogenized with 50
mlof1 % (w/v)PVA solution using Ultra Turrax (T25,IKA, Germany)at13,000rpm for1.5mintoform O/W emulsion.
5)Morphology by FE-SEM
The surface topography was analyzed by field-emission scanning electron microscope (FE-SEM) using SUPRA 55VP-SEM (Carl Zeiss, Oberkochen, Germany). MPs were mounted on metalstubs with thin adhesive copper tape and coated with platinum undervacuum using coating chamber(CT 1500HF,OxfordInstrumentsOxfordshire,UK).
6)Determination ofloading contentandloading efficiency Loading contentwasdetermined asfollows.The MPs (5 mg) were dispersed into 0.5 mlof0.1 M NaOH containing 0.5 % (w/v)SDS.Thesuspensionwasincubatedin awaterbathat60 ℃ for2h.Following centrifugation at14,000rpm for5min,0.5 ml of the supernatant was withdrawn for BCA assay. The encapsulation efficiency ofthe M5BT into MPs was determined by measuring the unloaded protein concentration in the supernatant during the double emulsion method steps. The loading contentand encapsulation efficiency wascalculated using thefollowingequations(Figure5):
7)Invitroreleasebehaviortest
The in vitro release of M5BT from M5BT/CAP or M5BT/T-CAP MPs was determined as follows.The MPs were placed into1.5mltubeswith 0.5mlof0.2M sodium phosphate buffer(pH 7.4)or0.2M HCl-KClbuffer(pH 2)for24h at37 ℃ with 100 rpm shaking.A 0.5 mlaliquotwas withdrawn and replaced with an equalvolumeofeach bufferatapredetermined time,and theamountofM5BT released wasmeasured atusing spectrophotometer(NanoPhotomter™).
8)Structuralintegrity oftheM5BT protein
Theintegrity ofM5BT beforeand afterencapsulation in MPs wasassessedbySDS-PAGE.
9)Exvivoporcinemucosaassay
Mucoadhesive property ofMPs was investigated using porcine intestinal mucosa. 4 mg of each of FDA-loaded MPs was dispersed on a freshly excised porcine intestinalmucosa,and incubated at37℃ for1 h with shaking at100 rpm.The MPs attached on the mucosa were collected, and remaining concentration of each MPs was calculated by measuring the absorbanceofFDA at490nm (n=3).
10)Invivooralimmunization in murinemodel
5femaleBALB/cmiceof7weeksofagewereusedpergroup in this study.Mice were purchased from Samtako,Co.Ltd.
(Osan,Korea)and housed in cages providing ad libitum access tofeed andwaterin accordancewith theguidelinesforthecare and useoflaboratory animals(SeoulNationalUniversity).After 1 week ofacclimatization,mice were orally immunized by oral gavageofMPsequivalentto200ug ofM5BT protein suspended in200ulofPBS viaa1mlsyringefittedwithanoralzondefor mouse(20G,5cm).Allimmunization groupsreceivedatotalof 6dosesofvaccinefor2priming(day0,1)and4boosting(day7, 8,14,15)andinvivooralimmunization schemeinmurinemodel wasshowninFigure6.
Figure6.Invivooralimmunizationschemeinmurinemodel
11)Bloodandfecalsampling
Bloodsamplesofimmunizedmicewerecollectedat0,2,and4 weeks. Each blood collection was conducted before each immunization.The blood samples were collected from tailvein
using BD microtainerfollowed by isolation ofserum from blood by centrifugation at14,000rpm for3min andstoredin –20℃, and used fordetection ofantigen-specific antibodies by ELISA. Fecalpellets were homogenized in 10 volumes ofresuspension buffer (PBS containing 1 mM PMSF and 1% BSA) at 4 ℃ overnight,centrifuged at 14,000 rpm for 10 min,supernatants were collected and analyzed forthe presence ofantigen-specific IgA byELISA.
12)Anti-M5BT antibody detection by ELISA
Levels ofserum M5BT-specific immunoglobulin G (totalIgG) and levelsofIgA in thefecalsampleswith specificity toM5BT weredeterminedbyELISA.M5BT proteinantigen(1ug/ml)was dilutedincarbonatebuffer(pH 9.6)anddilutedantigenwasused for coating wells (100 ul/well) of 96-well immunoplate (SPL 32096).Theplateswereincubated at37℃ for2h and washed with PBS (200 ul/well)for 3 times and blocked with blocking buffer (PBS containing 1% BSA) (200 ul/well) at room temperature for 1 h.Following blocking at room temperature, mouse sera with a 1:100 dilution in blocking bufferwere added to the wells (100 ul/well).Forfecalsamples,1:25 diluentwere used. Plates were incubated at 37 ℃ for 2 h followed by washing threetimeswith PBST (PBS containing 0.05% Tween 20,200 ul/well).For specific antibodies detection,plates were incubated for1h atroom temperaturewith appropriately diluted HRP-labeled goatanti-mouseimmunoglobulin conjugatesspecific for IgG (1:5000 dilutions)or IgA (1:5000 dilutions).The plates
werewashedthreetimeswithPBST andthentreatedwithTMB substratesolution(100ul/well)for5mininthedarkfollowedby theadditionofstopsolution(0.16M H2SO4;100ul/well)inorder
tostoptheenzymaticreaction.Theabsorbancewasmeasuredat 450nm usingmicroplatereader(TECAN Infinite200PRO).
13) Flow cytometric detection ofMHC class II-expressing cellsin Peyer’spatches
Afterfinalsampling from theimmunized mice,themice were dissectedtocollectPeyer’spatchesfrom theileum.Immunecells werefurtherisolated asdescribed earlier(Geem D.etal.,JVis Exp JoVE,2012).Briefly,a shortileum fragmentwith Peyer’s patch was cutlongitudinally and incubated at37 ℃ in 2 mM EDTA in CMF HBSS buffer for three sequential 15 min incubationstoremovetheepitheliallayer.Tissuesweredigested with 1.5 mg/mlType VIIIcollagenase in CMF HBSS/FBS,and the resulting suspension ofcells was passed through a 100 um cellstrainerbeforecentrifugation at1,500rpm for5minat4℃. Thecellswerewashed twicein ice-cold CMF PBS and blocked with 2.4G2anti-FcγRIII/IIin ice-cold staining buffer(CMF PBS + 5% FBS)for10min on ice.Following washing withice-cold staining buffer,cellswerestainedwith antibody staining cocktail (CD11candMHC classII)for20minoniceinthedark.Finally, cells were washed with ice-cold staining buffer twice and resuspended in 400 ul of ice-cold staining buffer for FACS analysis.
14)Statisticalanalysis
Allresults are expressed as mean ± standard deviation (SD). Statisticalsignificancewasassessed using t-testand aone-way analysis of variance (ANOVA) and post-hoc Tukey multiple comparison test. All statistical analysis was performed using GraphPadPRISM software(GraphPadSoftware,Inc.)Allstatistic alsignificanceisdenotedby*P < 0.05,**P < 0.01,and***P < 0.001.
3.Resul
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1)Preparation andcharacterization ofT-CAP
T-CAP was prepared by conjugation with L-cysteine hydrochloridebyDCC/NHS chemistryundernitrogenouscondition topreventtheoxidationofsulfhydrylgroup.Thereactionscheme forsynthesis ofT-CAP is shown in Figure 7.The coupling of cysteine and CAP was confirmed by proton nuclear magnetic resonance (1H NMR) as shown in Figure 8.The thiolgroup
contentin T-CAP was16.04umole/g polymerasdetermined by Ellman’smethod.
Figure8.1H 600MHzNMR spectraof(a)CAP and(b)T-CAP
2)Preparation andcharacterization ofM5BT/T-CAP MPs 2-1)Isolation andpurification ofM5BT protein
To prepare FMDV recombinant antigen M5BT for model subunitvaccine,M5BT protein was expressed with E.coliBL21
(DE3) harboring a gene encoding for M5BT protein. M5BT protein wasinducedby addition of0.5mM ofIPTG followedby sonication for isolation of crude protein and purification by his-tag affinity chromatography.Purification quality of purified proteinwascheckedbySDS-PAGE shownasinFigure9.
Figure 9. M5BT protein purification by his-tag affinity chromatography. Purified M5BT was identified by SDS-PAGE.(M:Protein marker;S:Crude protein sample; SFT: Sample flowthrough; W1-W3: Washing fraction; E1-E3:Elutionfraction)
2-2)Preparation andcharacterization ofM5BT/T-CAP MPs M5BT-loaded T-CAP MPs were prepared by doubleemulsion solventevaporation method (Figure 10).Briefly,M5BT solution (W1 phase) stabilized with Pluronic F-127 was sonicated with
T-CAP solution dissolved in dichloromethane(O phase)to form primary W1/O emulsion.The primary emulsion was added into
1% PVA solution(W2phase)followedbyhomogenizationtoform
W1/O/W2 double emulsion. After organic solvent evaporation,
Figure10.ProcedureofM5BT/T-CAP MPsby doubleemulsionmethod (Modifiedfrom thefigurefrom Sander,Softmatter,2014)
3)Morphology ofM5BT/CAP andM5BT/T-CAP MPs
The morphology ofM5BT/CAP and M5BT/T-CAP MPs was observed by FE-SEM. Both MPs had well-formed spherical particleswith smooth surfaces(Figure11).MPssmallerthan 10 um canbeefficientlytakenupbyM-cells(antigenuptakingcell) ofPeyer’spatchesinileum (J.H.Eldridgeetal.,1990).Moreover, MPs with this range in diameter can be internalized through phagocytosisbyantigen-presentingcells(APCs)playing acrucial roleininitiatinginnateimmuneresponse.
Figure11.Analysisofmorphology ofMPsby FE-SEM.(a)CAP MPs; (b) T-CAP MPs; (c) M5BT/CAP MPs; (d) M5BT/T-CAP MPs (Magnification:2,000X,scalebar:10μm).
4)Loading contentandencapsulation efficiency
Theloading contentand encapsulation efficienciesofM5BT in the T-CAP MPs is shown in Table 4.The loading contentof M5BT/CAP MPs and M5BT/T-CAP MPs were 4.62% (w/w) 4.97% (w/w),respectively,showing similar antigen amountper microparticles.And encapsulation efficiency ofM5BT/CAP MPs and M5BT/T-CAP MPs were 82.2%(w/w) and 72.1% (w/w), respectively. When it comes to the encapsulation efficiency, differentsolventused in microparticle formation may affectthe result.
During the loading of vaccine into the MPs, the vaccine’s stability is importantfor retaining its immune activity because B-cell receptor (BCR) recognize antigen as linear or conformationalepitopes whereasT-cellreceptor(TCR)can only recognize the processed antigen by APCs. Therefore, the structuralintegrity of the M5BT released from M5BT/T-CAP MPswasevaluatedbySDS-PAGE (Figure12)
Table 4.Loading contentand encapsulation efficiency ofM5BT-loadedMPs
Figure 12. Evaluation of protein structure of M5BT released from M5BT/CAP MPs and M5BT/T-CAP MPs by SDS-PAGE.Lane 1 : proteinmarker;Lane2-5:nativeM5BT (2.5-20ug);Lane6-7:M5BT releasedform CAP MPs;Lane8-9:M5BT releasedfrom T-CAP MPs; Lane10:protein-unloadedT-CAP MPs.
5)InvitroreleasebehaviorofM5BT from CAP andT-CAP MPs
The in vitro release profile ofM5BT from CAP and T-CAP MPs was investigated at simulated gastric acid (pH 2) and simulated intestinalfluid (pH 7.4)for24 h and was shown in Figure 13. The release profiles of M5BT from MPs were presented as the percentage ofamountofM5BT released from MPswithrespecttotheamountofM5BT loadedinMPs.
The results indicated that the release of M5BT from M5BT/T-CAP MPs was higher atpH 7.4 compared to pH 2. Thiolation ofCAP exhibited gastro-resistantproperty ofCAP at simulated gastric solution (pH 2)with releasing 18.6 ± 1.18 %
and 17.1 ± 1.42 % from MPs at2 h,respectively.The burst releaseeffectofM5BT/CAP MPsatpH 7.4wasobtainedwithin 1–2hduetotherapiddissolutionofCAP abovepH 6.2.M5BT releaseprofileofM5BT/T-CAP MPsatsimulated ileum pH (pH 7.4) showed 43.5 ± 1.63 %,56.8 ± 1.49 % at 12 and 24 h respectively whileitreleased28.8± 3.68%,33.6± 3.92% at12 and24hrespectivelyatpH 2.ThereleasebehaviorofbothMPs at pH 2 might be resulted from the diffusion of protein inside/outsidetheMPs.
J.H. Eldridge et al. (1990) reported that total number of microparticles within Peyer’s patches was increased untilday 4 and microparticleslessthan < 5um accounted for76-82% ofthe observed microparticles.This resultimplies thatM5BT/T-CAP MPs can continuously release antigen from MPs after M5BT/T-CAP MPswereinternalizedintoPeyer’spatches.
Figure 13.In vitro release profile ofM5BT protein from M5BT/CAP and M5BT/T-CAP MPs at simulated gastrointestinal pH. MPs (10 mg/ml)were suspended in differentpH buffer(pH 2,pH 7.4).Protein concentration wasmeasured by micro BCA assay.Allvaluesrepresent themeans± SD (n=3).
6)Mucoadhesiveproperty ofT-CAP MPs
Mucoadhesive property ofT-CAP MPs was evaluated by ex
vivo experimentusing freshly excised porcine intestinalmucosa with FDA-loaded MPs as fluorescence marker.The amountof FDA-loadedMPsattachedon freshly excisedporcineintestineat 37 ℃ is shown is Figure 14.The results revealed that the mucoadhesion ofT-CAP MPswas1.48-fold higherthan thatof CAP MPs after 1 h of incubation. Due to the enhanced mucoadhesion ofT-CAP MPs,itispossiblethatM5BT/T-CAP MPscontinuously releaseM5BT from T-CAP MPsremaining on themucuslayer.
Figure 14. Analysis ofmucoadhesive property ofMPs in smallintestine.4 mg of each of FDA-loaded MPs was dispersed on a freshly excised porcine intestinalmucosa, and incubated at37℃ for 1 h with shaking at100 rpm. The MPs attached on the mucosa were collected, and remaining concentration of each MPs was calculated by measuring theabsorbanceofFDA at490nm (n=3).(*p<0.05 byt-test)
7) Flow cytometric detection of MHC class II-expressing cellsin Peyer’spatchesin ileum
Immune cells located throughoutthe intestinallamina propria, especially in Peyer’spatches,play acrucialrolein sampling and processing luminalantigen for presentation to B,T cells.To determine the population of antigen-presenting cells (APCs) interacting with theantigen toinitiateadaptiveimmuneresponse
invivo,immunecellsfrom Peyer’spatchesinileum wereisolated and analyzed by flow cytometry (Figure 15). Here, APCs populations in Peyer’s patches were analyzed using the MHC class II surface marker. After gating, major immune cell populations expressing MHC class IIwere identified.The mice fed with M5BT via MPs (M5BT/CAP MPs; 26.1 %, M5BT/T-CAP MPs;26.87 %) showed increased population of MHC class II-positive cells when compared to thatofmice fed withM5BT only (16.23%)althoughthereisnotmuchdifference ofMHC classII-positivecellsbetweenCAP andT-CAP MPs.
In addition, CD11c-positive cells in Peyer’s patches from immunizedmicewithM5BT/T-CAP MPswereincreasedintotal populations (N.T, M5BT; 0.30%, M5BT/CAP MP; 0.47%, M5BT/T-CAP MPs;0.6%).
Increased population of MHC class II-expressing cells (i.e.
APCs)inPeyer’spatchesmay influencetheproduction ofIgA in intestine.
Figure15.Flow cytometricdetection ofMHC classII-expressing cells in Peyer's patches from immunized mice.Peyer's patches were collected from the mice immunized with M5BT/CAP or M5BT/T-CAP MPs.Isolated cellswerestained with MHC class IImarkerspriortodetection by FACS.ThepercentageofMHC class II-positive cells is indicated.(a)Gating area;(b-f)MHC classII-positivecells.(b)N.T;(c)M5BT;(d)M5BT/CAP MPs; (e)M5BT/T-CAP MPs.(f)Themean percentageofMHC class II-positivecellsfrom totalpopulations(%total)(n=3).
8) M5BT-specific antibody production after oral immunization with MPs
To evaluate the immune-enhancing effect of M5BT-loaded MPs by oralroute,mice were immunized with M5BT alone, M5BT/CAP MPsandM5BT/T-CAP MPsbyoralgavage.
Antigen-specific immunoglobulin in serum and fecalsamples from immunized with M5BT/T-CAP MPs were analyzed by M5BT-specificELISA.M5BT-specificELISA was conducted by coatingimmunoplatewithM5BT recombinantantigen.
To assess the systemic immune response after oral immunization with M5BT/T-CAP MPs,anti-M5BT IgG levelsin serum samples from immunized mice were analyzed by ELISA (Figure16).Among theimmunized groups,only miceimmunized with M5BT/T-CAP MPs showed significantly higher M5BT-specificIgG levelscomparedtoN.T group.
As a result,anti-M5BT IgA in fecalsample from immunized micewith M5BT-loaded MPswassignificantly higherthan that ofmiceimmunized with M5BT only withoutcarrier(Figure17). Thisresultindicatethatpolymericcarriercan deliverantigen to lymphoid tissue to induce antigen-specific immune response. Whenitcomestotheoralvaccinecarrier,T-CAP MPsexhibited more IgA production compared to CAP MPs at2 and 4 weeks resulted from differentpH-sensitive property by modifying CAP with thiolation (Figure 17 a,b). However, difference in fecal samplingmethodbetween2and4weeksresultedinthedifferent finalconcentration offecalpellets affecting lowerabsorbance of fecalsamplesat4weeks. AfternormalizationofeachIgA levels
by IgA levels ofcontrolgroups to compensate the finalfecal concentration,M5BT-specificIgA levelsweresignificantly higher at4 weeks only in mice immunized with M5BT/T-CAP MPs (Figure17c).
Because thiolation of CAP altered its pH-sensitive property thatcan dissolve atabove pH 6.2 to dissolve atileum pH (pH 7.4).Therefore,encapsulated antigen can be released from MPs andresultedinminimizingexposureofantigentoharshintestinal environmentthatcan denaturetheantigen.Thus,instead ofthe exposure of antigen at upper intestine part, proper antigen delivery todistalintestinepartwhereM-cellabundantregion in ileum may beimportantforprotecting anddelivering theantigen to elicit antigen specific immune response. IgA secreted by plasma cellscan protectthehostfrom infection by pathogen or virusatthemucosalsite.
Figure16.M5BT-specificimmuneresponseafter oralimmunization with MPs.Anti-M5BT serum IgG levels at4 weeks afterimmunization were measuredusingELISA.Allvaluesrepresentsthe means± SD (n=5).(*P<0.05,one-wayANOVA)
Figure 17.Anti-M5BT IgA levelin feces after oralimmunization with MPs.Fecalsamplesweretaken from miceat(a)2weeks,(b) 4 weeks.(c) Relative anti-M5BT IgA level compared with N.T group.Antibody levels were analyzed by ELISA.(n=5,error bars representstandarddeviations;*p<0.05,one-wayANOVA)
Summar
y
In this chapter,pH-sensitive and mucoadhesive T-CAP were prepared for delivering FMD subunit vaccine because oral vaccination ofmucosalsubunitvaccine has limitation due to its poor immunogenicity and low bioavailability despite of many advantagescomparedtoparenteraladministration.
AndT-CAP MPsasamucosaloralvaccineadjuvantproducing more IgA,the most important immunoglobulin for preventing FMD attheearly stageofinfection,wasprepared.Itwasfound thatT-CAP MPs resulted in the elevated population ofMHC class II+ cells in Peyer’s patches because antigen uptaken by
M-cellsfrom lumencan beuptaken andbetransportedtolymph nodebyimmunecells(DCs,Macrophages,B cells)toactiveboth innate immunity and adaptive immunity.This indicate thatoral immunization of mucosal subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer‘s patches eliciting mucosal IgA response. It will make a step forward into promisingoralsubunitvaccinedevelopmentinlivestockindustry.
Chapt
er I
I
.Devel
opment of mannan-decor
at
ed
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nul
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mi
cr
opar
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i
cl
es
f
or
enhanci
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he
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mmunogeni
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t
y ofsubuni
tvacci
ne
1.I
nt
r
oduct
i
on
The goal of vaccination is to provide long-term protection againstinfection by generating astrong immuneresponsetothe administered antigen. Conventional live attenuated vaccines typically do notrequireadjuvants.However,theimmunogenicity ofproteinsistypically poorandtheuseofadjuvantsisrequired. Vaccinesoftenrequiretheaddition ofimmunestimulatory agents called adjuvants to boost the specific immune response to antigens.Themostwidely used adjuvantsarecompletefreund’s adjuvant (CFA), which is composed of inactivated
mycobacterium.CFA isused in severalFMD vaccines,although itsincreased immune-boosting effectisrelated tothetoxicity of CFA itself.To substitute the conventionaladjuvant like CFA, development of new vaccine adjuvant is required for FMD vaccine which can enhance the antibody response. An ideal vaccine adjuvant should stimulate both humoral and cellular immuneresponsesagainstco-injectedantigens.
In thisstudy,tocoverthelimitation ofFMD subunitvaccines, inulin acetate microparticles decorated with mannan (M-INAC MPs)wereusedasapolymericadjuvantandcarriersystem.
Inulin is a storage carbohydrate of a number of plants including Jerusalem artichoke,chicory,dahlia,wheat,etc.Inulin
exists in several forms depending on its solubility and precipitation method.Recently,itwasreported thatparticleform ofinulin canactasan efficientadjuvantforvaccine(Kumar,S., & Tummala,H.,2013).So,particulation ofinulin is the main pointfordevelopinginulinasanadjuvantforvaccine.
To develop inulin adjuvant for subunit vaccine,INAC was prepared to make particle form. Inulin was acetylated by introducing acetylgroup from acety anhydride to inulin.INAC can be particulated in water due to self-assembled mechanism although it is soluble in various organic solvents including dichloromethane. Because it is known that INAC MPs can function as a vaccine adjuvantwhen antigen was encapsulated into INAC particles, antigen-loaded INAC MPs can enhance immune response ofthe subunitvaccine by overcoming its low immunogenicity.Particulated adjuvants may function as efficient adjuvants by enhancing the uptake ofthe antigen by immune cells such as macrophage, dendritic cells and delivering the encapsulatedantigentoimmunecellsforactivation.Also,subunit vaccine inside INAC particles can be released in a sustained manner known to induce long-term immune response of the antigen.Besides,itwas reported thatINAC is a novelTLR-4 agonist (Tummala, H., & Kumar, S., 2013). Through these benefits,INAC canworkasvaccineadjuvantandvaccinecarrier. Double emulsion solvent evaporation method was used to develop INAC microparticles to encapsulate subunit vaccine M5BT.In addition,INAC MPsweredecorated with mannan,the TLR-4agonist,asaspecificligandtorecognizeimmunecells.
2.Mat
er
i
al
sandMet
hods
1)SynthesisofINAC
The synthesis ofinulin acetatewas carried outaccording the method ofWu etal.(1999) with a little modification.Briefly, inulin (1 g)was added to 5 mlofdimethylformamide (DMF) and then 0.2 mlof 5% acetic anhydride was added.Sodium acetate (NaOAc,5% (w/v)) was used as a catalyst for the reaction.Theacetylation reaction wascarried at40 ℃ for24 h undernitrogen.After24h,INAC wasdialyzed againstDMF for 24 h to remove free acetic acid and againstdistilled waterto remove DMF and unreacted inulin.After dialysis,INAC was lyophilized and stored at–20 ℃ untiluse.The conjugation of acetylgroup was confirmed by 600 MHz1H NMR spectroscopy (AVANCE 600,Bruker,Germany) and acetylgroup contentin INAC wasquantified.
2) Preparation of M5BT-loaded INAC MPs and M5BT-loadedM-INAC MPs
M5BT-loaded INAC and M5BT-loaded M-INAC MPs were prepared by a double emulsion solvent evaporation method. Briefly,200ulof25mg/mlM5BT solution wasmixed with 100 ulof10% (w/v)Pluronic F-127 solution as an aqueous phase (W1). This aqueous phase was emulsified with 5 ml of
dichloromethane(DCM)asanoilphase(O)containing 100mg of INAC by sonication for 1.5 min,resulting in the formation of primary Wl/O emulsion.This primary emulsion was then added
dropwise into another aqueous (W2) phase (50 ml water)
containing 1% (w/v) poly(vinyl alcohol) (PVA) solution as a surfactant,with continuous stirring at13,000 rpm using Turrax resulting in theformation ofdoubleemulsion (W1/O/W2).In case
ofM5BT-loaded M-INAC MPs,0.75% PVA solution containing 0.25% mannan was used.The stirring was continued overnight forcomplete evaporation ofthe organic solvent.Resulting MPs werecollectedviacentrifugationat6,000rpm for10minat4℃. ThepelletedM5BT-loadedINAC orM5BT-loadedM-INAC MPs werewashedwithdistilledwaterandcentrifuged.ThefinalMPs wereresuspendedin10mlofdistilledwaterandfrozenbyliquid nitrogenfollowedbylyophilizationundervacuum.
3)Morphology ofMPs
The surface topography was analyzed by field-emission scanning electron microscope (FE-SEM) using SUPRA 55VP-SEM (Carl Zeiss, Oberkochen, Germany). MPs were mounted on stubs with adhesive copper tape and coated with platinum under vacuum using coating chamber (CT 1500 HF, OxfordInstrumentsOxfordshire,UK).
4)Confirmation ofmannan-decoration intoMPs
To confirm mannan-decoration in M-INAC MPs,FITC was conjugatedwith mannan.Briefly,100mg ofmannan dissolvedin 1 mlofdistilled waterwas slowly mixed with 5 mg ofFITC dissolved in 1 mlof DMSO.After stirring for 4 h at room temperaturein dark conditions,thereaction productwasdropped
into 8 ml of ethanol to remove the unreacted FITC. The precipitated FITC-mannan conjugate was washed with ethanol andcollectedby centrifugationat16,000rpm 3timesfor10min. FITC-mannan-decorated INAC MPs were visualized by confocal laserscanningmicroscope(CarlZeissLSM710).
5) Determination of loading content and encapsulation efficiency
Loading contentwasdetermined asfollows.TheMPs(5mg) were dispersed into 0.5 mlofdimethylsulfoxide (DMSO).The completely dissolved solution was used for measurement of protein concentration spectrophotometer (NanoPhotomter™).The
encapsulation efficiency ofthe M5BT into MPs was determined by measuring the unloaded protein concentration in the supernatant during the double emulsion method steps. The loading contentand encapsulation efficiency wascalculated using thefollowingequations(Figure18):
Figure18.Calculation equation forencapsulation efficiencyandloadingcontent
6)Invitroreleasebehaviortest
The in vitro release ofM5BT from M5BT-loaded INAC or M5BT-loaded M-INAC MPs was determined as follows.The M5BT-loaded MPs (5 mg)were placed into 1.5 mltubes with 0.5mlofPBS (pH 7.4)at37℃ with100rpm shaking.A 0.5ml aliquot was withdrawn and replaced with an equalvolume of PBS atapredetermined time,andtheamountofM5BT released wasmeasuredusingspectrophotometer(NanoPhotomter™).
7)Invivoimmunization in murinemodel
5femaleBALB/cmiceof7weeksofagewereusedpergroup in this study.Mice were purchased from Samtako,Co.Ltd. (Osan,Korea)and housed in cages providing ad libitum access tofeed andwaterin accordancewith theguidelinesforthecare and useoflaboratory animals(SeoulNationalUniversity).After 1 week ofacclimatization,mice were immunized intramuscularly with MPs in 50 ulofPBS orM5BT resuspended in 50 ulof PBS and CFA (1:1) via a 0.3 mlinsulin syringe.CFA was replacedwithIFA forboosting.Allimmunizationgroupsreceived a totalof 2 doses of vaccine for 1 priming (day 0) and 1 boosting (day 14).In vivo immunization experimentschedule in murinemodelwasshowninFigure19.
