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Vaccine 24 (2006) 6534–6541
Nano- and microparticles as adjuvants in vaccine design:Success and failure is related to host natural antibodies
Michael S. Sinyakov a,∗, Moti Dror a, Tammy Lublin-Tennenbaum b,Samuel Salzberg a,�, Shlomo Margel b, Ramy R. Avtalion a
a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israelb Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
Received 9 February 2006; received in revised form 30 May 2006; accepted 14 June 2006Available online 28 June 2006
bstract
Bovine serum albumin (BSA) and the surface A-layer protein (AP) of an atypical strain of fish bacterial pathogen Aeromonas salmonicidaere covalently linked with polymeric nano- and microparticles, and antigenicity of the resulted conjugates was compared in mice andoldfish. Distinct albeit different levels of natural BSA and AP antibodies were present in both animal species. Significant stimulation of thenti-AP antibody response in mice strikingly contrasted to unresponsiveness or even suppression in fish. The results negatively correlate with
he levels of respective natural antibodies in the host and are discussed in context of problems related to fish vaccination. The work reinforceshe instructive role of natural antibodies in adaptive immune response.2006 Elsevier Ltd. All rights reserved.
eywords: Natural antibodies; Acquired antibodies; Aeromonas salmonicida
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. Introduction
Inefficiency of currently used conventional vaccines isrequently due to lack of appropriate adjuvants. The new-eneration vaccines consisting of purified proteins and pep-ides (isolated from microorganisms, produced by recombi-ant DNA technology or by direct chemical peptide syn-hesis, or expressed by relevant DNA constructs) are ofteneakly immunogenic. To be effective, these vaccines require
mmunostimulating compounds, adjuvants, which act non-pecifically to increase the immune response to a definedntigen. Search for harmless and effective adjuvants remainso be an urgent need in modern vaccinology.
Three kinds of the most frequently used adjuvants cane distinguished: (i) particulate (exemplified by aluminumalts, oil emulsions and liposomes), (ii) non-particulate (such
∗ Corresponding author. Tel.: +972 3 5318205; fax: +972 3 5351824.E-mail address: [email protected] (M.S. Sinyakov).
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264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2006.06.021
s saponins, lipid A and muramyl dipeptide derivatives),nd (iii) combined adjuvant compositions (Syntex adjuvantormulation, Ribi adjuvant system, and immune stimulatingomplexes), each one exerting its own type of immune mod-lation [1]. The adjuvant effect of a particular system cane mediated via three different mechanisms: (i) slow releasef antigens at the injection site (a depot effect), (ii) target-ng of antigens to the relevant antigen-presenting cells of themmune system, i.e. macrophages, and (iii) direct activationf cells in the immune system, e.g. bacterial adjuvants andytokines [2–4]. The former two mechanisms underlie thedjuvanticity of microparticles [4,5]. Microparticles (mostrequently, synthetic polymer microspheres) offer a promis-ng option to oil emulsions and mineral salt adsorbents, andheir beneficial use as carriers for vaccine delivery has beeniscussed [6–10]. An association of antigen(s) with micropar-
icles can be achieved by covalent linkage or physical entrap-ent. Compared to the latter technique, where the antigens non-covalently, physically incorporated in the micropar-icle’s interior, covalent coupling offers distinct advantages:
accine
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ewer antigens is required, processing and presentation byntigen-presenting cells is more efficient, stability duringtorage, and any excess of material can easily be regained11–14]. With the use of microparticles, a very low dose ofntigen can give rise to an optimal humoral response.
The structure and the properties of microparticles mayhange markedly with slight alterations in production condi-ions, which can lead to significant differences in the immuneesponse elicited. For this reason, adjuvants on the basis ofubmicron polymeric particles, so-called nanoparticles, wereeveloped and suggested for use as potent adjuvants [15–19].anoparticles can be prepared in a physico-chemically repro-ucible manner within narrow size limits [20]. The informa-ion accumulated in the last years has emphasized the impor-ance of the size and revealed the advantages of nanoparticlesver microspheres [21,22].
Fish vaccinology faces the problems similar to thosencountered in vaccine design for humans and mammals.
wide range of pathogens is associated with fish dis-ases. Among the bacteria, Aeromonas salmonicida, theausal agent of furunculosis, is one of the oldest knownnd one of the most important fish pathogens due to itsconomically devastating impact on both marine and cul-ivated fish [23]. Extracellular A-layer protein (AP) of A.almonicida has been suggested to be a major virulent factor24]. Lack of efficient vaccines against A. salmonicida justxemplifies the problems related to the fish vaccines design25].
We aimed to develop a candidate vaccine formulationased on a particulate antigen preparation with built-in adju-anticity and consisted of the isolated AP covalently linkedo nano- or microparticles as adjuvants. At the same time weanted to compare the antigenicity of this conjugated AP insh with that in a known mammalian model, the mouse. To
his end, a preliminary search for efficient adjuvant was per-ormed in mice with the use of bovine serum albumin (BSA)s a model antigen. The AP was isolated from an atypical. salmonicida strain and conjugated thereafter to the suc-essful adjuvant in expectation that the resulting conjugateould also be successful in immunization of goldfish, aneromonas-susceptible fish species. The results were found
o negatively correlate with the level of respective naturalntibodies in the host.
. Materials and methods
.1. Reagents and chemicals
Sepharose 4B (Pharmacia Biotech Inc.), polylysineSigma, MW of 50 kDa), BSA (Sigma), vinyl sulfoneAldrich, 97%), aluminum potassium sulfate dodecahydrate
Sigma), cyanogen bromide (Fluka), 1-ethyl-3-(3-dimethyl-minopropyl)-carbodiimide (EDC, Pierce), complex cultureedia BHI broth (Difco), bovine hemin (Sigma), molecular-orous cellulose ether dialysis membrane of 300 kDa
mbsl
24 (2006) 6534–6541 6535
olecular weight cut-off (Spectra/Por), incomplete Freund’sdjuvant (IFA, Sigma), ELISA microplates (Greiner),lkaline phosphatase labeled goat anti-mouse IgG (Sigma),-nitrophenyl phosphate disodium hexahydrate (Sigma04 phosphatase substrate tablets), magnetic columns MidiACS (Miltenyi Biotec GmbH), glycine (Sigma), salts for
uffers (Bio-Lab Ltd., Israel), sodium dodecyl sulfate (SDS),crylamide, N,N′-methylene-bis-acrylamide, N,N,N′,N′-etramethylene diamine (TEMED, Sigma), �-mercapto-thanol (Merck), Bromo-Thymol Blue (pH range 6.0–7.6,DH), Coomassie Brilliant Blue R-250 and the standard lowolecular weight markers (Biorad) were used throughout.
.2. Synthesis of polymeric nanoparticles
Magnetic nanoparticles (mNP, ca. 100–150 nm) were syn-hesized essentially according to procedure described else-here [26]. The mNP beads were coated with polylysine
PL) and activated with divinyl sulfone (DVS) in the aque-us suspension (at pH 10.5 and the ratio PL:DVS of 1:50) tontroduce active double bonds onto the particles surface. Acti-ation was carried out with constant shaking for 6 h at roomemperature. To remove the excess of DVS, the activated par-icles were washed thereafter (5×) with 3 ml bidistilled watern the magnetic column Midi MACS attached to a magnet.fter taking the column off the magnet, the modified parti-
les were extracted from the column 10 min later with 0.1 Mhosphate buffer (PB, pH 7.6).
Alternatively, monodispersed polyacrolein nanoparticlespaNP) with average diameter of 200 nm were prepared by-irradiation of acrolein in aqueous solution in the presence ofDS as a surfactant as described elsewhere [27]. Upon com-letion of polymerization, the excess of the free monomernd surfactant was removed by extensive dialysis against dis-illed water. The concentration of the paNP beads in the finaluspension was estimated to be 1.9% (w/v) when preparedor conjugation with BSA, and 3.2% (w/v) when preparedor conjugation with AP. This type of beads contains a largeumber of reactive functional aldehyde groups and does noteed any additional activation prior to use for coupling ofmino ligands.
.3. BSA antigenic preparations
.3.1. BSA conjugated with nanoparticles (BSA-mNPnd BSA-paNP)
Covalent immobilization of BSA on the activated mNPas carried out at room temperature with constant shaking.he BSA solution containing 5 mg BSA in 0.3 ml PB wasdded to 2 ml mNP suspension and reaction was allowed toroceed overnight. To block the residual functional groups,00 �l of 2% glycine solution were added to the reaction
ixture thereafter. The unbound BSA and the excess of thelocking reagent were removed by extensive dialysis againstaline with the use of dialysis bags with 300 kDa molecu-ar weight cutoff. Completion of removal of the unbound
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SA was verified by SDS-polyacrylamide gel electrophore-is (SDS-PAGE) of the final suspension using 10% separat-ng gel run under reducing conditions. Concentration of themmobilized BSA was estimated to be 210 �g BSA per mlf the final suspension as measured by the modified Lowryssay [28].
Alternatively, covalent binding of BSA with paNP waserformed at 4 ◦C with constant shaking. The BSA solu-ion containing 20 mg BSA in 0.2 ml PB was added to 3 mlaNP suspension. Following overnight incubation, residualldehyde groups were blocked with 0.1% ethanolamine solu-ion in PB (1 h incubation). Removal of excessive reagents,DS-PAGE and estimation of the bound BSA were carriedut similarly to the same steps in BSA-mNP binding proto-ol. Concentration of the immobilized BSA was found to be.348 mg BSA per ml paNP suspension, which correspondso binding efficacy of 220 mg BSA/g paNP.
.3.2. BSA emulsified in IFA (BSA-IFA)BSA solution (2% in saline) was dropwise added to and
horoughly emulsified with equal volume of incomplete Fre-nd’s adjuvant (IFA) at 4 ◦C until homogeneity.
.3.3. BSA adsorbed to aluminum salt (BSA-Alum)Two milliliters of the BSA solution (1%, w/v) were pre-
ipitated by dropwise addition of 2 ml aluminum potassiumulfate solution (2%, w/v) accompanied by gentle stirring andimultaneous neutralization of the formed suspension to pH.2 with 0.1N NaOH. The final precipitate was collected byentrifugation (1500 × g, 15 min) and resuspended in 4 mlaline.
.4. Bacterial strain and growth conditions
An atypical strain (F12.1) of A. salmonicida isolated in ouraboratory from cutaneous ulcers of diseased goldfish [29,30]as used throughout.
.5. A-protein (AP) antigenic preparations
.5.1. Isolated APSoluble monomeric form of the A. salmonicida extracel-
ular AP was isolated from whole bacterial cells with the usef the low pH extraction method as described elsewhere [29].he resulting AP acidic extract (pH 2.3) was collected, neu-
ralized and stored frozen until used. This form of AP wasolely used for conjugation with nano- and microparticlesnd booster immunizations.
.5.2. AP conjugated with polyacrolein nanoparticlesAP-paNP)
Covalent immobilization of the isolated AP on the surfacef paNP was initiated by simple mixing of the protein solu-ion with the beads suspension in the ratio of 1:5 (w/w) andllowed to proceed for 18 h at 4 ◦C. The rest of the steps were
Udts
24 (2006) 6534–6541
he same as indicated above for coupling of BSA. Concen-ration of the immobilized AP was estimated to be 1.6 mgrotein per ml of the final suspension.
.5.3. AP conjugated with Sepharose (AP-Seph)Activation of Sepharose 4B with cyanogen bromide and
he following covalent linking of the isolated AP were per-ormed as specified in the manufacturer’s guidelines.
.5.4. AP emulsified in IFA (AP-IFA)Equal volumes of incomplete Freund’s adjuvant (IFA) and
P solution (0.2% in saline) were mixed together and thor-ughly emulsified until homogeneity at 4 ◦C.
.5.5. AP adsorbed to aluminum salt (AP-Alum)Two milliliters of AP solution (2 mg/ml) were precipitated
y dropwise addition of 2 ml aluminum potassium sulfate4 mg/ml) accompanied by gentle stirring and simultaneouseutralization of the resulted suspension to pH 7.2 with 0.1NaOH. The final precipitate was collected by centrifugation
1500 × g, 15 min) and resuspended in 4 ml saline.
.6. Combined BSA and AP formulation (AP-BSA-Seph)
Sepharose 4B was activated with cyanogen bromide, andSA was covalently linked to BrCN-activated Sepharoseccording to the manufacturer’s guidelines. The coupled BSAas modified with EDC, and the isolated AP was covalentlyound with the modified BSA essentially as specified in theanufacturer’s coupling instructions.
.7. Immunization protocols
BALB/c female mice (6 weeks’ age, 5–8 mice per group)ere alternatively immunized intraperitoneally (i.p.) withSA-IFA, BSA-Alum, BSA-mNP, and BSA-paNP. Threether groups were similarly injected with saline, solubleSA, and BSA-free nanoparticles (NP) and served as con-
rols. All mice, except for saline and NP controls, receivedsingle-shot injection of 100 �g BSA per mouse and were
ested for the level of anti-BSA antibodies after 10–30 daysost-immunization. The mice were anesthetized with combi-ation of ketamine and diazepam, the blood was taken fromhe orbital venous sinus, sera were collected and kept frozenntil use.
Goldfish (Carassius auratus L., 6-month-old) werebtained from a goldfish farm (Gan-Shmuel, Israel). Fishere maintained at 22 ± 1 ◦C and were acclimatized to lab-ratory conditions for 2 weeks before treatment. The fishere divided into 4 groups, 10–15 fish in each one, and
lternatively immunized with soluble AP, AP-IFA, AP-Alum,nd AP-paNP, a primary i.p. injection dose was 100 �g AP.
pon 6 weeks, on day 42 the fish were given a boosterose of 50 �g soluble AP alone. The fish were bled prioro immunization and at 3 weeks intervals after primary andecondary injections (i.e. on days 0, 21, and 63); the sera
accine 24 (2006) 6534–6541 6537
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M.S. Sinyakov et al. / V
ere collected and tested for the level of anti-AP antibod-es. When immunized with AP-Seph and AP-BSA-Seph, thesh were i.p. primed with 15 �g particulate AP from theelevant conjugate and boosted with 50 �g soluble AP 3eeks thereafter, sera being collected at 3 weeks intervals
i.e. on days 21 and 42). In a parallel trial, mice and fish were.p. injected with soluble AP or AP-Seph with alternativeoses of 10, 20, and 40 �g AP; sera were collected 2 weekshereafter.
.8. Detection of antigens and antibodies
This was accomplished by employing the enzyme-linkedmmunosorbent assay (ELISA). To detect BSA and AP cova-ently linked to nano- or microparticles (in order to makeure they retained their antigenicity upon coupling), ELISAicroplates were coated with serially diluted relevant anti-
ens in soluble and particulate forms and allowed to absorbvernight at 4 ◦C. Mouse anti-BSA or mouse anti-AP serumthe first antibody), alkaline phosphatase labeled goat anti-ouse IgG (the second antibody), and phosphatase sub-
trate were consecutively applied thereafter, each step per-ormed for 1 h at 37 ◦C. The color reaction was measured asbsorbance at 405 nm.
To test the availability of the BSA immobilized on theurface of magnetic biodegradable and polyacrolein-basedanoparticles for the native anti-BSA antibodies, both prepa-ations were appropriately diluted to the starting concen-ration of 10 �g BSA/ml and further treated as indicatedbove.
Detection of the relevant antibodies was essentially carriedut as previously described [29]. The plates were sensitizedy coating with soluble BSA or AP respectively and blockedith 0.4% gelatin at the initial two steps (each performed
or 2 h at 37 ◦C). Serial dilutions of immune mouse or gold-sh sera to be tested (the first antibody), mouse anti-BSA orouse anti-goldfish IgM serum (the second antibody), and
lkaline phosphatase labeled goat anti-mouse IgG (the thirdntibody) were applied thereafter, and the color reaction waseveloped by addition of phosphatase substrate.
.9. Statistics
The unpaired two-tailed T-test was applied for evaluationf differences between the groups.
. Results
.1. BSA formulations: antigenicity in mice
Upon immobilization of BSA on the surface of magnetic
iodegradable and polyacrolein-based nanoparticles (conju-ates BSA-mNP and BSA-paNP, respectively), the availabil-ty of the bound BSA for the native anti-BSA antibodies wasssentially the same in both conjugates (Fig. 1).FNi
ig. 1. Detection of BSA in soluble and particulate forms upon conjugationith mNP and paNP nanoparticles.
Increased levels of specific anti-BSA antibodies werenduced in mice following single-shot i.p. injection witharious particulate BSA constructs compared to the con-rol groups (non-immunized, injected with soluble BSAlone or nanoparticles alone) that exhibited negligible back-round levels of antibodies. The antibody response wasime-independent within 10–30 days post-immunization.
ice stimulated with BSA-paNP developed significantlyigher antibody response than those injected with BSA-NP (p < 0.0014). The antibody level induced by BSA-paNP
howed no significant difference from that induced by BSA-
ig. 2. Anti-BSA antibody response in mice upon single-shot immunization.I ctrl: non-immunized controls; BSA: injection with soluble BSA; NP:
njection with paNP alone. Shown are means and standard errors.
6 accine 24 (2006) 6534–6541
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.2. AP formulations: antigenicity in fish
Based on the preliminary screening in mice, the paNParrier was chosen as a potent adjuvant for the ensuing experi-ents on immunization of fish with AP antigen of A. salmoni-
ida. The isolated AP was covalently linked to paNP, andhe resulting AP-paNP conjugate as well as other particulateP constructs were used for goldfish immunization. Results
howed that priming with all formulations to be tested wasneffective in eliciting antibody response different from theevel of pre-existing anti-AP natural antibodies, except for theriming with IFA-adjuvanted AP that resulted in high level ofntibody activity (p < 0.05). Following secondary stimulationith AP, the antibody activity was evident in the AP-Alum
mmunized group (p < 0.014), only (Fig. 3).
.3. AP-Sepharose conjugate: antigenicity in mice andsh
The antibody response of fish and mice to AP was com-ared using soluble AP and AP-Seph conjugate given in dif-erent doses ranging from 10 to 40 �g AP. Initially, the levelf natural AP antibodies in mice was significantly smallerhan that found in goldfish (p < 0.015). In goldfish, all dosesf soluble AP reduced the original level of natural antibod-es (p < 0.05). Independently of the dose level employed, both
ice and goldfish were unresponsive to the soluble AP stimuli
Fig. 4a). In goldfish, injection of the AP-Seph at a minimalose of 10 �g resulted in significant reduction of the nat-ral antibody level (p < 0.012), while the higher doses (20nd 40 �g AP) significantly increased the levels of acquireds‘wa
ig. 4. Anti-AP antibody response in goldfish and mice injected with various dosentibodies in non-immunized animals. Shown are means and standard errors.
ig. 3. Anti-AP antibody response in goldfish. NI ctrl: non-immunized con-rols; AP: soluble AP; PR: primary response; SR: secondary response. Shownre means and standard errors.
ntibodies (p < 0.028 and <0.013, respectively). In mice, theame conjugate elicited direct elevation of antibodies startingrom the 10-�g dose (Fig. 4b).
.4. AP-BSA-Sepharose conjugate: antigenicity in fish
In attempt to further stimulate immune responsiveness insh, we synthesized a combined two-antigen construct con-
isted of AP covalently linked to Sepharose via BSA as abridging’ molecule. The results of goldfish immunizationith this construct are shown in Fig. 5. The antibodies levelsrose to both AP and BSA were strikingly different. While a
s of soluble AP (a) or AP conjugated with Sepharose (b). Control: natural
M.S. Sinyakov et al. / Vaccine
Fig. 5. Anti-AP and anti-BSA antibody responses in goldfish injected withAP conjugated with Sepharose via BSA ‘bridge’. Control: natural antibodiesibg
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owerful rise of anti-BSA antibodies was observed in therimary response, the anti-AP antibody level was signifi-antly reduced (p < 0.005), compared to the natural level, butnderwent a significant increase (p < 0.0002) in the secondaryesponse.
. Discussion
In this work, we attempted to evaluate the potency ofynthetic nano- and microparticles as immunostimulatingarriers in immunization of fish with AP, which is supposed toe a major virulent factor of A. salmonicida, one of the mostelligerent fish pathogens. Two kinds of nanoparticles, mag-etic biodegradable (mNP) and polyacrolein-based (paNP)ere covalently bound to BSA that served as a model protein
ntigen. While the bound BSA availability in both conju-ates was similar (Fig. 1), the antigenicity of the BSA-paNPonjugate proved to be superior over that of the BSA-mNPonjugate and was comparable to that of the BSA-Alum pre-ipitate when tested in mice (Fig. 2).
Accordingly, paNP nanoparticles were chosen as a potentarrier for the following experiments with AP. The isolatedP was similarly bound to paNP and the resulted conju-ate was tested for antigenicity in goldfish along with otherP-containing preparations. All groups failed to respond toriming stimulus, and the relevant anti-AP antibody levelsid not differ from those of natural anti-AP antibodies withhe only exception of the fish injected with IFA-adjuvantedP (Fig. 3). Furthermore, none of the groups exhibited a sec-ndary response significantly different from the primary one,xcept the AP-Alum group. The same carrier that revealed an
xcellent adjuvanticity for BSA when tested in mice, provedo be inefficient for AP when tested in fish.This discrepancy may be related to the host (different ani-al species) and/or to the peculiarity of AP (which may be
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24 (2006) 6534–6541 6539
olerogenic molecule). To address these options, antigenicityn goldfish and mice was tested in parallel for the isolatedP and the AP-Seph conjugate. Sepharose microspheres
epresent biocompatible particles, which possess a porousatrix-type interior and thus can covalently bind antigenicolecules at high density without affecting their immuno-
ogical properties [31,32]; they have been recently used asarriers for AP [29] and reported as an alternative adjuvant inmmunotherapy [33]. Soluble AP doses of 10–40 �g caused
sharp decrease of the natural antibody level in the fishnd failed to stimulate the antibody response in both ani-al species (Fig. 4a). However, a dose effect on the level
f antibody response in both goldfish and mice was evidentith the AP-Seph conjugate demonstrating positive correla-
ion between the AP dosage and antibody response (Fig. 4b).Thus, the above discrepancy cannot be attributed to either
f the two options considered, and the covalent binding of APo a potent carrier may surmount the apparent AP-inducedolerance in both animal species involved. Another evidenteature, the presence of high level of natural anti-AP antibod-es in goldfish versus low level of natural anti-BSA antibodiesn mice, led us to suggest that success of immunization mighte negatively correlated with the level of the relevant naturalntibodies in the host.
Bearing in mind that development of T-cell memory insh may depend on the presence of immunogenic carrierolecules [34], the AP antigen was alternatively conjugatedith Sepharose via a BSA ‘bridge’ in attempt to artificially
ntensify T-helper cells stimulus. This new formulation alsonduced neutralization of the natural AP antibody activity inhe primary response. However, significant but still poor sec-ndary response, which exceeded the original level of naturalntibodies, was evident following booster immunization withP. At the same time, injection of AP-BSA-Seph induced aighly stimulated anti-BSA antibody response that largelyominated over antibody response to AP (Fig. 5). Thus,he same fish that were highly responsive to microspheres-djuvanted BSA (low level of natural BSA antibodies) wereound unresponsive to the same microspheres-coupled APhigh level of natural AP antibodies). This is in line withur suggestion that the level of antibody response negativelyorrelates with the level of respective natural antibodies.
The problems encountered in fish vaccination may beartly attributed to the specificity of the immune systemf fish as poikilothermic vertebrates that contributes to fre-uently observed immune tolerance and inadequate devel-pment of humoral immune response, the secondary anti-ody response in particular. Additional impediments maye related to considering fish as a homogeneous populationqually susceptible to pathogen invasion, the approach thatay not reflect a real situation [29], and attempts to apply
accines that ignore the fact that the antibodies to certain
ntigen(s) they are supposed to stimulate already exist in fishs natural antibodies. The postulate on negative correlationetween the level of acquired antibodies and that of respec-ive natural antibodies is the main outcome of this work that
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einforces the instructive role of natural antibodies in adap-ive immune response [35–37]. Interrelation of innate anddaptive immunities is a balance of the respective networks.nnate inhibition of adaptive immunity at the cellular levelas been recently reported [38]. The mechanism underly-ng this phenomenon at the antibody level is still obscure.
hen natural antibodies to a distinct antigen are present atigh levels, active immunization with the same antigen mayesult in the exhaustion of the pool of natural antibodies athe first stage, which appears as “false tolerance” or “falseuppression”. If an appropriate potent carrier (e.g. Sepharoseor AP) and sufficient doses of modified antigen are applied,his effect may be overcome, and a dose-dependant syn-hesis of acquired antibodies takes place at the secondtage.
cknowledgements
The authors dedicate this publication to the memory ofamuel Salzberg, late professor and dean at the Departmentf Life Sciences, Bar-Ilan University, who initiated this work.ind assistance of Dr. I. Burdygin in preparation of polymericanoparticles is highly appreciated.
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