Nano- and microparticles as adjuvants in vaccine design: Success and failure is related to host natural antibodies

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  • Vaccine 24 (2006) 65346541

    Nano- and microparticles as adjuvanhos

    myl b, R, Ramat, Rama

    May 22006


    Bovine se pical stwere covale icity ogoldfish. Di presenanti-AP anti even sthe levels of respective natural antibodies in the host and are discussed in context of problems related to fish vaccination. The work reinforcesthe instructive role of natural antibodies in adaptive immune response. 2006 Elsevier Ltd. All rights reserved.

    Keywords: Natural antibodies; Acquired antibodies; Aeromonas salmonicida

    1. Introdu

    Inefficiefrequentlygenerationtides (isolanant DNAthesis, or eweakly immimmunostispecificallyantigen. Seto be an urg

    Three kbe distingusalts, oil em

    CorresponE-mail ad Decease


    ncy of currently used conventional vaccines isdue to lack of appropriate adjuvants. The new-vaccines consisting of purified proteins and pep-ted from microorganisms, produced by recombi-technology or by direct chemical peptide syn-xpressed by relevant DNA constructs) are oftenunogenic. To be effective, these vaccines require

    mulating compounds, adjuvants, which act non-to increase the immune response to a defined

    arch for harmless and effective adjuvants remainsent need in modern vaccinology.

    inds of the most frequently used adjuvants canished: (i) particulate (exemplified by aluminumulsions and liposomes), (ii) non-particulate (such

    ding author. Tel.: +972 3 5318205; fax: +972 3 5351824.dress: (M.S. Sinyakov).d.

    as saponins, lipid A and muramyl dipeptide derivatives),and (iii) combined adjuvant compositions (Syntex adjuvantformulation, Ribi adjuvant system, and immune stimulatingcomplexes), each one exerting its own type of immune mod-ulation [1]. The adjuvant effect of a particular system canbe mediated via three different mechanisms: (i) slow releaseof antigens at the injection site (a depot effect), (ii) target-ing of antigens to the relevant antigen-presenting cells of theimmune system, i.e. macrophages, and (iii) direct activationof cells in the immune system, e.g. bacterial adjuvants andcytokines [24]. The former two mechanisms underlie theadjuvanticity of microparticles [4,5]. Microparticles (mostfrequently, synthetic polymer microspheres) offer a promis-ing option to oil emulsions and mineral salt adsorbents, andtheir beneficial use as carriers for vaccine delivery has beendiscussed [610]. An association of antigen(s) with micropar-ticles can be achieved by covalent linkage or physical entrap-ment. Compared to the latter technique, where the antigenis non-covalently, physically incorporated in the micropar-ticles interior, covalent coupling offers distinct advantages:

    see front matter 2006 Elsevier Ltd. All rights reserved..vaccine.2006.06.021Success and failure is related toMichael S. Sinyakov a,, Moti Dror a, Tam

    Samuel Salzberg a,, Shlomo Margea Faculty of Life Sciences, Bar-Ilan University

    b Department of Chemistry, Bar-Ilan UniversityReceived 9 February 2006; received in revised form 30

    Available online 28 June

    rum albumin (BSA) and the surface A-layer protein (AP) of an atyntly linked with polymeric nano- and microparticles, and antigenstinct albeit different levels of natural BSA and AP antibodies werebody response in mice strikingly contrasted to unresponsiveness orts in vaccine design:t natural antibodiesLublin-Tennenbaum b,amy R. Avtalion a

    -Gan 52900, Israelt-Gan 52900, Israel

    006; accepted 14 June 2006

    rain of fish bacterial pathogen Aeromonas salmonicidaf the resulted conjugates was compared in mice andt in both animal species. Significant stimulation of the

    uppression in fish. The results negatively correlate with

  • M.S. Sinyakov et al. / Vaccine 24 (2006) 65346541 6535

    fewer antigens is required, processing and presentation byantigen-presenting cells is more efficient, stability duringstorage, and any excess of material can easily be regained[1114]. Wantigen can

    The struchange mations, whicresponse elsubmicrondevelopedNanoparticducible mation accumtance of theover micro

    Fish vaencountereA wide raeases. Amcausal ageand one oeconomicativated fishsalmonicid[24]. Lackexemplifies[25].

    We aimbased on avanticity anto nano- orwanted to cfish with ththis end, aformed in mas a modelA. salmonicessful adjwould alsoAeromonasto negativeantibodies

    2. Materia

    2.1. Reage

    Sepharo(Sigma, M(Aldrich, 9(Sigma), cyaminopropmedia BHIporous ce

    molecular weight cut-off (Spectra/Por), incomplete Freundsadjuvant (IFA, Sigma), ELISA microplates (Greiner),alkaline phosphatase labeled goat anti-mouse IgG (Sigma),

    rophenhosphS (Mrs (Biamide

    ethyol (M), Coocular w


    agnetzed ese [26]and auspenduce an waseraturwere

    e magtakinere e

    phatelternatP) witdiatioas a su

    n ofurfactwaternsion

    onjugaonjugaer ofany ao ligan

    BSA a

    . BSASA-p


    BSA sd to 2ed ovl of 2re theing re

    e witheightith the use of microparticles, a very low dose ofgive rise to an optimal humoral response.cture and the properties of microparticles may

    rkedly with slight alterations in production condi-h can lead to significant differences in the immuneicited. For this reason, adjuvants on the basis ofpolymeric particles, so-called nanoparticles, wereand suggested for use as potent adjuvants [1519].les can be prepared in a physico-chemically repro-nner within narrow size limits [20]. The informa-ulated in the last years has emphasized the impor-size and revealed the advantages of nanoparticles

    spheres [21,22].ccinology faces the problems similar to thosed in vaccine design for humans and mammals.nge of pathogens is associated with fish dis-ong the bacteria, Aeromonas salmonicida, thent of furunculosis, is one of the oldest knownf the most important fish pathogens due to itslly devastating impact on both marine and cul-

    [23]. Extracellular A-layer protein (AP) of A.a has been suggested to be a major virulent factorof efficient vaccines against A. salmonicida justthe problems related to the fish vaccines design

    ed to develop a candidate vaccine formulationparticulate antigen preparation with built-in adju-d consisted of the isolated AP covalently linkedmicroparticles as adjuvants. At the same time weompare the antigenicity of this conjugated AP inat in a known mammalian model, the mouse. Topreliminary search for efficient adjuvant was per-

    ice with the use of bovine serum albumin (BSA)antigen. The AP was isolated from an atypical

    cida strain and conjugated thereafter to the suc-uvant in expectation that the resulting conjugate

    be successful in immunization of goldfish, an-susceptible fish species. The results were foundly correlate with the level of respective naturalin the host.

    ls and methods

    nts and chemicals

    se 4B (Pharmacia Biotech Inc.), polylysineW of 50 kDa), BSA (Sigma), vinyl sulfone7%), aluminum potassium sulfate dodecahydrateanogen bromide (Fluka), 1-ethyl-3-(3-dimethyl-

    yl)-carbodiimide (EDC, Pierce), complex culturebroth (Difco), bovine hemin (Sigma), molecular-

    llulose ether dialysis membrane of 300 kDa

    p-nit104 pMACbuffeacryltetramethanBDHmole


    Mthesiwher(PL)ous s

    introvatiotempticlesin thAftercles wphos

    A(paN-irraSDSpletioand stilledsuspefor cfor cnumbneedamin


    2.3.1and B

    Cwas c

    Theaddeproce100mixtublocksalinlar wyl phosphate disodium hexahydrate (Sigmaatase substrate tablets), magnetic columns Midi

    iltenyi Biotec GmbH), glycine (Sigma), salts foro-Lab Ltd., Israel), sodium dodecyl sulfate (SDS),, N,N-methylene-bis-acrylamide, N,N,N,N-lene diamine (TEMED, Sigma), -mercapto-erck), Bromo-Thymol Blue (pH range 6.07.6,massie Brilliant Blue R-250 and the standard loweight markers (Biorad) were used throughout.

    sis of polymeric nanoparticles

    ic nanoparticles (mNP, ca. 100150 nm) were syn-sentially according to procedure described else-. The mNP beads were coated with polylysinectivated with divinyl sulfone (DVS) in the aque-sion (at pH 10.5 and the ratio PL:DVS of 1:50) toctive double bonds onto the particles surface. Acti-carried out with constant shaking for 6 h at roome. To remove the excess of DVS, the activated par-washed thereafter (5) with 3 ml bidistilled waternetic column Midi MACS attached to a magnet.g the column off the magnet, the modified parti-xtracted from the column 10 min later with 0.1 Mbuffer (PB, pH 7.6).ively, monodispersed polyacrolein nanoparticlesh average diameter of 200 nm were prepared byn of acrolein in aqueous solution in the presence ofrfactant as described elsewhere [27]. Upon com-

    polymerization, the excess of the free monomerant was removed by extensive dialysis against dis-. The concentration of the paNP beads in the finalwas estimated to be 1.9% (w/v) when preparedtion with BSA, and 3.2% (w/v) when preparedtion with AP. This type of beads contains a largereactive functional aldehyde groups and does notdditional activation prior to use for coupling ofds.

    ntigenic preparations

    conjugated with nanoparticles (BSA-mNPaNP)t immobilization of BSA on the activated mNPout at room temperature with constant shaking.

    olution containing 5 mg BSA in 0.3 ml PB wasml mNP suspension and reaction was allowed toernight. To block the residual functional groups,% glycine solution were added to the reactionreafter. The unbound BSA and the excess of theagent were removed by extensive dialysis againstthe use of dialysis bags with 300 kDa molecu-cutoff. Completion of removal of the unbound

  • 6536 M.S. Sinyakov et al. / Vaccine 24 (2006) 65346541

    BSA was verified by SDS-polyacrylamide gel electrophore-sis (SDS-PAGE) of the final suspension using 10% separat-ing gel run under reducing conditions. Concentration of theimmobilizeof the finalassay [28].

    Alternatperformedtion contaipaNP suspaldehyde gtion in PBSDS-PAGEout similarcol. Conce3.348 mg Bto binding

    2.3.2. BSABSA so

    thoroughlyunds adjuv

    2.3.3. BSATwo mi

    cipitated bysulfate solusimultaneo7.2 with 0.centrifugatsaline.

    2.4. Bacte

    An atyplaboratorywas used th

    2.5. A-pro

    2.5.1. IsolSoluble

    lular AP wof the low pThe resultitralized ansolely usedand booste

    2.5.2. AP c(AP-paNP)

    Covalenof paNP wtion with thallowed to

    the same as indicated above for coupling of BSA. Concen-tration of the immobilized AP was estimated to be 1.6 mgprotein per ml of the final suspension.

    . AP cctivatiollowied as s

    . AP eual vo

    olutioly em

    . AP ao mil

    ropwis/ml) aalizatiH. The0 g,



    rding todifi

    d withfactur




    , andAll m

    gle-shd for timmun of krbitaluse.

    oldfishned frmaintry condividativelP-pa6 w

    of 50munizdaryd BSA was estimated to be 210g BSA per mlsuspension as measured by the modified Lowry

    ively, covalent binding of BSA with paNP wasat 4 C with constant shaking. The BSA solu-

    ning 20 mg BSA in 0.2 ml PB was added to 3 mlension. Following overnight incubation, residualroups were blocked with 0.1% ethanolamine solu-(1 h incubation). Removal of excessive reagents,

    and estimation of the bound BSA were carriedly to the same steps in BSA-mNP binding proto-ntration of the immobilized BSA was found to beSA per ml paNP suspension, which corresponds

    efficacy of 220 mg BSA/g paNP.

    emulsied in IFA (BSA-IFA)lution (2% in saline) was dropwise added to andemulsified with equal volume of incomplete Fre-ant (IFA) at 4 C until homogeneity.

    adsorbed to aluminum salt (BSA-Alum)lliliters of the BSA solution (1%, w/v) were pre-

    dropwise addition of 2 ml aluminum potassiumtion (2%, w/v) accompanied by gentle stirring andus neutralization of the formed suspension to pH1N NaOH. The final precipitate was collected byion (1500 g, 15 min) and resuspended in 4 ml

    rial strain and growth conditions

    ical strain (F12.1) ofA. salmonicida isolated in ourfrom cutaneous ulcers of diseased goldfish [29,30]roughout.

    tein (AP) antigenic preparations

    ated APmonomeric form of the A. salmonicida extracel-

    as isolated from whole bacterial cells with the useH extraction method as described elsewhere [29].

    ng AP acidic extract (pH 2.3) was collected, neu-d stored frozen until used. This form of AP was

    for conjugation with nano- and microparticlesr immunizations.

    onjugated with polyacrolein nanoparticles

    t immobilization of the isolated AP on the surfaceas initiated by simple mixing of the protein solu-e beads suspension in the ratio of 1:5 (w/w) and

    proceed for 18 h at 4 C. The rest of the steps were


    the fform


    AP sough


    by d(4 mgneutrNaO(150



    was m




    BSAotherBSAtrols.a sintestepost-natiothe ountil



    alternand AUpondoseto imsecononjugated with Sepharose (AP-Seph)on of Sepharose 4B with cyanogen bromide andng covalent linking of the isolated AP were per-pecified in the manufacturers guidelines.

    mulsied in IFA (AP-IFA)lumes of incomplete Freunds adjuvant (IFA) and

    n (0.2% in saline) were mixed together and thor-ulsified until homogeneity at 4 C.

    dsorbed to aluminum salt (AP-Alum)liliters of AP solution (2 mg/ml) were precipitatede addition of 2 ml aluminum potassium sulfateccompanied by gentle stirring and simultaneouson of the resulted suspension to pH 7.2 with 0.1Nfinal precipitate was collected by centrifugation

    15 min) and resuspended in 4 ml saline.

    ined BSA and AP formulation (AP-BSA-Seph)

    se 4B was activated with cyanogen bromide, andcovalently linked to BrCN-activated Sepharoseo the manufacturers guidelines. The coupled BSAed with EDC, and the isolated AP was covalentlythe modified BSA essentially as specified in the

    ers coupling instructions.

    nization protocols

    female mice (6 weeks age, 58 mice per group)atively immunized intraperitoneally (i.p.) withBSA-Alum, BSA-mNP, and BSA-paNP. Threeps were similarly injected with saline, solubleBSA-free nanoparticles (NP) and served as con-ice, except for saline and NP controls, received

    ot injection of 100g BSA per mouse and werehe level of anti-BSA antibodies after 1030 daysnization. The mice were anesthetized with combi-etamine and diazepam, the blood was taken fromvenous sinus, sera were collected and kept frozen

    (Carassius auratus L., 6-month-old) wereom a goldfish farm (Gan-Shmuel, Israel). Fishained at 22 1 C and were acclimatized to lab-ditions for 2 weeks before treatment. The fish

    ed into 4 groups, 1015 fish in each one, andy immunized with soluble AP, AP-IFA, AP-Alum,NP, a primary i.p. injection dose was 100g AP.eeks, on day 42 the fish were given a boosterg soluble AP alone. The fish were bled prioration and at 3 weeks intervals after primary andinjections (i.e. on days 0, 21, and 63); the sera

  • M.S. Sinyakov et al. / Vaccine 24 (2006) 65346541 6537

    were collected and tested for the level of anti-AP antibod-ies. When immunized with AP-Seph and AP-BSA-Seph, thefish were i.p. primed with 15g particulate AP from therelevant coweeks ther(i.e. on dayi.p. injectedoses of 10thereafter.

    2.8. Detec

    This waimmunosorlently linkesure they rmicroplategens in solovernight a(the first anmouse IgGstrate wereformed forabsorbance

    To testsurface ofnanoparticlrations wetration ofabove.

    Detectioout as prevby coatingwith 0.4%for 2 h at 3fish sera tomouse antialkaline phantibody) wdeveloped

    2.9. Statist

    The unpof differenc

    3. Results

    3.1. BSA fo

    Upon imbiodeg...


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