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EvaluationofparticulateacellularvaccinesagainstBrucellaovisinfectioninrams

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valuation of particulate acellular vaccines against Brucella ovis infection in rams

aquel Da Costa Martinsa, Juan M. Irachea, José M. Blascob, M. Pilar Munozb, Clara M. Marínb,. Jesús Grillób, M. Jesús De Miguelb, Montserrat Barberánb, Carlos Gamazoa,∗

Immunoadjuvant unit, Department of Pharmacy and Pharmaceutical Technology and Department of Microbiology and Parasitology, University of Navarra, Pamplona, SpainAnimal Health Unit, SIA-DGA, Zaragoza, Spain

r t i c l e i n f o

rticle history:eceived 3 July 2009eceived in revised form 8 October 2009ccepted 14 October 2009vailable online 1 November 2009

eywords:

a b s t r a c t

The attenuated Brucella melitensis Rev 1 vaccine, used against brucellosis infection, interferes with sero-logical diagnosis tests, may induce abortions in pregnant animals, and may infect humans. In order toovercome these drawbacks, we developed acellular vaccines based on a Brucella ovis antigenic com-plex (HS) containing outer membrane proteins and R-LPS entrapped in poly(anhydride) conventionaland mannosylated nanoparticles (NP-HS and MAN-NP-HS) or in poly(�-caprolactone) microparticles(HS-PEC) as antigen delivery systems and immunoadjuvants. Brucellosis free rams were vaccinated sub-

rucellosisaccineicroparticleanoparticledjuvantam

cutaneously with a single dose of particles containing 3 mg of HS, and challenged 6 months thereafter.Protection was evaluated by clinical, bacteriological and serological examinations, in comparison withnon-vaccinated control rams. HS-PEC vaccine induced protection (7 out of 13 animals were infected)equivalent to that induced by the reference Rev 1 vaccine (8/14). In contrast, animals immunized withNP-HS were not protected, showing similar results to that obtained in the control unvaccinated rams. Fur-thermore HS-PEC vaccine did not interfere against B. melitensis serodiagnostic tests. In summary, HS-PEC

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. Introduction

Brucellosis is considered as one of the major extended bacte-iological diseases worldwide [1], and constitutes an importantocioeconomic and sanitary problem. Its incidence is beingncreased in certain parts of the World, and remains of particu-ar concern in developing countries throughout the Mediterraneanountries, Middle East, Eurasia and some regions of Africa and Latinmerica. Ovine brucellosis is produced by Brucella melitensis andrucella ovis; the former concerns to both ovine and humans and,onversely, the infection by B. ovis affects exclusively the ovinepecies, being characterized by genital lesions and reduced fertilitys the main clinical consequences.

The best effective alternative to control animal brucellosis withxtensive production systems is the use of vaccination programs.mmunoprophylaxis against ovine brucellosis based on the con-unctival inoculation of the live attenuated strain B. melitensisev 1 is relatively effective [2], nevertheless: (i) Rev 1 vaccine

nduce antibodies against the O chain of the smooth lipopolysac-haride (S-LPS), which is the main antigen in serological diagnosis,nd therefore, disables the differentiation between the vaccinatednimals and the infected by B. melitensis; (ii) Rev 1, although

∗ Corresponding author. Tel.: +34 948 425600x9251; fax: +34 948 425619.E-mail address: cgamazo@unav.es (C. Gamazo).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.10.073

s a safe and effective vaccine against brucellosis in rams.© 2009 Elsevier Ltd. All rights reserved.

attenuated, maintains residual virulence for the vaccinated ani-mals; (iii) there are been reported cases of human infection withRev 1 [3] which is streptomycin resistant, and (iv) its use is notallowed in those countries where the infection by B. melitensis hasbeen eradicated. In consequence, there is a need to find alternativesto the Rev 1 vaccine. With the aim of avoiding the Rev 1 derivedproblems we propose, as an alternative, the use of acellular vaccinescontaining O chain free antigens from B. ovis, thus being innocuousand avoiding serological interferences in the S-LPS based tests. Toinduce an effective protective immune response after a single shootinoculation, particle antigen delivery systems were used as vaccineadjuvants.

Previous studies revealed the effectiveness against B. ovis ofa formulation based on biodegradable poly(�-caprolactone) (PEC)microparticles in the mouse animal model against an experimentalB. ovis infection by either subcutaneous or oral routes [4]. Further-more, this formulation was also able to significantly protect againstB. ovis in rams, but the protection conferred was somewhat lowerthan that induced by Rev 1, probably due to the low amount of anti-genic extract used [5]. Taking this in consideration, the objective ofthis study was to determine the efficacy of that formulation by the

use of higher doses of the antigenic complex, which was obtainedfrom a single bacterial batch incubated in novel culture conditions.Moreover, an additional objective of this work was to determinethe protective efficacy of the same antigenic complex incorporatedin new immunoadjuvants based on nanoparticles of the copolymer

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ethylvinylether and maleic anhydride (Gantrez®AN, ISP Corp.),hich have been proven to exhibit high in vivo performance when

dministered with antigenic proteins [6–8].

. Materials and methods

.1. Extraction and characterization of the antigenic complex

The hot saline antigenic complex (HS) was obtained from thetrain B. ovis REO 198 by a modification of the method describedreviously [9]. To obtain a large and homogenous batch of cellsor the antigenic extraction, a thawed vial of stock suspension wastreaked onto BAB (Blood Agar Base no. 2, Difco, Detroit, USA)lates, and the 24 h culture was inoculated into a 10 L bioreactor (B.raun Biotech, Germany), and incubated at 37 ◦C for 48 h. Live cellsere suspended in saline solution (0.85% NaCl, 10 g packed cells per

00 mL), and heated in flowing steam for 15 min. Following cen-rifugation at 12,000 × g for 15 min, the supernatant was dialyzedor 5 days at 4 ◦C against several changes of deionized water (dH2O).he dialyzed material was ultracentrifuged for 3 h at 60,000 × g andhe pellet (HS) washed in dH2O, freeze-dried and stored at roomemperature. Total protein was determined by the BCATM Proteinssay method [10] with bovine serum albumin (PierceTM, Rock-

ord, USA) as standard. The analysis for 2-keto-3-deoxyoctonateKDO, exclusive marker of LPS) (Sigma–Aldrich, Madrid, Spain) cor-ected for 2 deoxyaldoses was performed by the method of Warrenodified by Osborn [11].To evaluate the effect of the manufacturing process on the pro-

ein profile and antigenicity SDS-PAGE and immunoblotting, werearried out as described previously [9]. After the SDS-PAGE per-ormed in 12% Bis–Tris acrylamide slabs (Acrylamide CriterionTM

T Precast gels, 10 Comb, 30 �L, 1 mm, Bio-Rad, CA, USA), proteinntigens were transferred to Immovilon membranes (Immovilon, Millipore Corp., Bedford, MA, USA) using a semi-dry trans-lotter (Bio-Rad, Richmond, CA, USA) and the immunoblottingas performed with a pool sera from rabbits experimentally

nfected with B. ovis, and the reaction developed as describedreviously [12] using peroxidase-conjugate goat anti-rabbit IgGNordic Immunological Lab, Tilburg, Netherlands) and 4-chloro-1-aphtol (Sigma–Aldrich, Steinheim, Germany) as chromogen. Thepparent molecular masses of the proteins present in the anti-enic extracts were determined by comparing their electrophoreticobility with that of molecular mass markers (rainbow coloured

rotein molecular weight marker, Amersham Pharmacia Biotech,reiburg, Germany). A gel diffusion test (GDT) was also performedo check the antigenic quality of the HS obtained from this currentnnovative extraction methodology, according to the procedurereviously described [13].

.2. Preparation and characterization of the adjuvant based onntigen-loaded nanoparticles

Conventional (NP-HS) and mannosylated (MAN-NP-HS)anoparticles were prepared by a modification of the solventisplacement method described previously [14], and then freeze-ried using sucrose (5%) as cryoprotector (Genesis 12EL, Virtis,SA).

The particle size and the zeta potential were accessed by photonorrelation spectroscopy (PCS) and electrophoretic laser Dopplernemometry, respectively, using a Zetamaster analyser system, at5 ◦C (Malvern Instruments, Malvern, UK). The diameter of the

anoparticles was determined after dispersion in ultrapure water1/10) and measured at 25 ◦C with a dynamic light scattering anglef 90 ◦C. The zeta potential was determined as follows: 200 �L ofhe samples were diluted in 2 mL of a 0.1 mM KCl solution adjustedo pH 7.4 [15]. The average particle size was expressed as the

ne 28 (2010) 3038–3046 3039

volume mean diameter (vmd) in nanometers (nm), and the averagesurface charge in millivolts (mV).

Nanoparticles morphology was accessed by scanning electronmicroscopy (Zeiss DSM 940 A, Oberkochen, Germany) with a dig-ital imaging capture system (DISS, Point Electronic GmBh, Halle,Germany). For this purpose freeze-dried formulations were resus-pended in ultrapure water and centrifuged at 27,000 × g for 20 minat 4 ◦C. Then, supernatants were rejected and the obtained pelletswere mounted on a glass plates adhered with a double-sided adhe-sive tape onto metal stubs, coated with gold to a thickness of 16 nm(Emitech K550 equipment, UK).

The yield of the nanoparticles preparation process was deter-mined by gravimetry from freeze-dried nanoparticles as describedpreviously [16]. The mannosamine amount associated to nanopar-ticles was estimated by quantification of free mannosamine in thesupernatants obtained during the purification step using the O-phthalaldehyde (OPA) fluorimetric assay of primary amines [17].The in vitro agglutination assay of mannosylated nanoparticleswas performed by measuring the turbidity changes in continu-ous kinetic measurements at 405 nm, using Concanavalin A (ConA, Sigma–Aldrich–Aldrich, Barcelona, Spain), to confirm the bio-logical activity of the mannosamine associated to the surface of thenanoparticles, as described previously [7].

The HS loading in the nanoparticles was quantified by the BCATM

Protein Assay (PierceTM, Rockford, USA) method from the differ-ence between its initial concentration added and the concentrationfound in the collected filtrates obtained during purification. Eachsample was assayed in triplicate, and a calibration curve of free HSin the filtrates obtained from control conventional nanoparticles(r2 > 0.999), or in the filtrates obtained from control mannosylatednanoparticles (r2 > 0.997) was used, respectively for NP-HS andMAN-NP-HS. HS loading was expressed as the amount of HS (in�g) per mg nanoparticles, while the entrapment efficiency (E.E.)was determined by relating the total weight of antigen entrappedin the batch of nanoparticles to the initial weight of antigen andexpressed in percentage.

To evaluate the effect of the manufacturing process on theHS protein integrity profile and antigenicity, proteins from thefreeze-dried nanoparticles were extracted with acetone/DMF (3:1,v/v) (Pancreac, Barcelona, Spain) and assayed by SDS-PAGE andimmunoblotting. Briefly, 10 mg of freeze-dried nanoparticles wereresuspended in 1 mL of ultrapure water and centrifuged (28,000 × g,20 min, 4 ◦C). Then, the precipitate was dissolved in 2 mL of ace-tone/DMF (3:1, v/v) and kept for 1 h at −80 ◦C. Samples werecentrifuged at 28,000 × g for 20 min at 4 ◦C and the pellet waswashed with 1 mL acetone and kept 30 min at −80 ◦C. After cen-trifugation under the same conditions, pellets were resuspendedin the electrophoretic sample buffer (Tris–HCl 62.5 mM, pH 6.8;10% glycerol; 2% SDS; 5% �-mercaptoethanol and 0.05% bromophe-nol blue) for SDS-PAGE analysis, where samples were analysed byusing a 12% acrylamide slabs (Acrylamide CriterionTM XT Precastgels, Bio-Rad, CA, USA) and stained with Coomassie brilliant bluefor proteins. The apparent molecular weight of the proteins wasdetermined by comparing their electrophoretic mobility with thatof the molecular mass marker (rainbow coloured protein molecularweight marker, Amersham Pharmacia Biotech, Freiburg, Germany).Immunoblotting was performed as described in the characteriza-tion of the antigenic complex.

2.3. Preparation and characterization of the adjuvant based onantigen-loaded microparticles

HS containing poly(�-caprolactone) (HS-PEC) microparticleswere prepared by the solvent extraction/evaporation method,using the Total Recirculation One-Machine System (TROMS) toprepare the W1/O/W2 multiple emulsion [18]. Then, the obtained

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icroparticles were resuspended in mannitol (Roquette, Lestrem,rance) at 5% as cryoprotector and Tween 80 (Sigma–Aldrich, St.ouis, USA) at 0.1%, frozen at −80 ◦C and lyophilised (Genesis 12EL,irtis, USA).

HS-PEC were sized by laser diffractometry using a Mastersizer-® (Malvern Instruments, Malvern, UK), and the average particleize was expressed as the volume mean diameter (vmd) in microm-ters (�m). Samples of microparticles were diluted in water andssayed using a DTS 1060 Disposable Zeta Cell to determine theurface charge of microparticles by electrophoretic laser Dopplernemometry, using a Zetamaster analyser system (Malvern Instru-ents, UK). Microparticles shape and morphology were examined

y scanning electron microscopy applying the same methodologys for the nanoparticles.

The HS loading, as well as the structural integrity and antigenic-ty of antigenic proteins associated to HS-PEC, were accessed byDS-PAGE and immunoblotting, after previous disruption of theystems. For that, 20 mg of HS-PEC microparticles were washedith deionized water and centrifuged in order to avoid the cry-

protector interference. Then, microparticles were dissolved inmL dichloromethane (Pancreac, Barcelona, Spain), stirred for5 min, and the organic solvent was eliminated under reduced pres-ure (Vortex Evaporator, Labconco Corporation, Lenexa, KS, USA).inally, samples were resuspended in the electrophoretic sampleuffer, heated at 100 ◦C for 10 min and centrifuged (18,000 × g,4 min, 4 ◦C). For SDS-PAGE, the supernatants were analyzed bysing a 12% acrylamide slabs (Acrylamide CriterionTM XT Precastels, Bio-Rad, CA, USA) and gels stained with the Silver Stain kitBio-Rad, CA, USA). The protein integrity was analysed and the con-entration was estimated by calculating the average band densityn SDS-PAGE using Micro Image® software (Version 4.0; Olym-us Optical Co., USA), running a HS standard calibration curve20.00–0.62 �g/well). Each sample was assayed in triplicate. Thentrapment efficiency was determined by relating the total weightf HS entrapped in the batch of microparticles to the starting weightf HS. The antigenicity of the HS loaded in microparticles wasssessed by immunoblotting as described above for the antigen-oaded nanoparticles.

.4. Ovine immunization, challenge and bacteriological studies

The experiments in rams were performed according the meth-ds described previously [5], and in compliance with the Europeanegislation on animal experiments (86/609/EU). A total of 75ragonesa rams from 3 to 4 months-old, belonging to the bru-ellosis free flock from the CITA (Zaragoza, Spain), were randomlyllotted in 5 groups (Table 2), and subcutaneously vaccinated withmL of the corresponding vaccines as following: (1) HS loadedoly(�-caprolactone) microparticles (HS-PEC), 13 animals; (2) HS

oaded conventional poly(anhydride) nanoparticles (NP-HS), 12nimals; (3) HS loaded mannosylated poly(anhydride) nanoparti-les (MAN-NP-HS), 12 animals; and (4) live attenuated B. melitensisev 1 vaccine Rev 1 (individual dose: 1.6 × 109 CFU), 14 animals.similar group of 14 animals was kept unvaccinated as control.

he corresponding microparticle and nanoparticle vaccine formu-ations were diluted in dH2O, and each individual dose (1 mL) wasontaining a total of 3 mg of the HS antigenic complex. All animalsere vaccinated the same day and in the same anatomic region

left elbow), then placed in isolated pens and fed ad libitum. Duringhe days following vaccination all rams were inspected for rectalemperature and local reactions at the inoculation site.

Six months after vaccination, all rams were experimentallynfected with 1.16 × 109 CFU of B. ovis PA contained in 50 �L, anddministered conjunctivally (25 �L) and preputially (25 �L). The B.vis PA freshly bacterial suspensions were prepared as describedreviously [19]. Briefly, the freeze-dried PA reference strain was

ine 28 (2010) 3038–3046

rehydrated in sterile Buffered Saline Solution (BSS; 0.015 M NaCl,7 mM KH2PO4, 10 mM K2HPO4; pH 6.85) and grown on Blood AgarBase # 2 (BAB, Biolife, Italy) containing 10% sterile bovine serum(Seromed, Biochrom, Spain) for 72 h at 37 ◦C in 10% CO2. Cellswere harvested in BSS, and then spectrophotometrically adjustedin BSS to the required concentration. Exact doses inoculated wereassessed retrospectively by dilution and plating.

At weekly intervals after challenge all rams were clinicallyexamined for eventual lesions in testicles and epididymides. Nineweeks after challenge all rams were slaughtered and submit-ted to individual necropsy for bacteriological and pathologicalexaminations. Cultures were performed on portions of spleen andepididymides, and the whole seminal vesicles, ampullae ductus def-erens, bulbourethral glands, and cranial (submaxillary, parotid andretropharyngeal), iliac, scrotal, prefemoral and prescapular lymphnodes of each animal. Each sample was suspended in the mini-mal amount of BBS required for homogenisation using a blender(Seward Medical, London, UK), and then cultured on at least toculture plates of the modified Thayer-Martin’s medium contain-ing 100.000 IU nystatin/L [19] and incubated for 7–10 days at 37 ◦Cin a 10% CO2. Brucella isolates were identified by colonial morphol-ogy, Gram staining, oxidase and urease tests, CO2 requirement andphage typing [20]. A ram was classified as infected if at least one B.ovis CFU was isolated from any of the organs and lymph nodes sam-pled at necropsy. The B. melitensis Rev 1 vaccine strain was neverisolated from any sample, and all Brucella isolates correspondedalways to B. ovis PA challenging strain.

2.5. Immunological studies

Each animal was bled before vaccination and then blood samplestaken weekly or fortnightly after vaccination for serological studies.All serum samples were submitted to the modified Rose Bengal test(RBT) and the standard Complement Fixation test (CFT) to deter-mine the serological interference induced against smooth Brucellaspp. The modified RBT using the standard B. abortus antigen wasperformed as described previously [21]. The CFT using the B. abortusantigen was performed using the standard warm microtechnique[20]. Moreover, all samples were tested in an indirect ELISA withHS antigen as described previously [13,22], to evaluate the serolog-ical response against B. ovis. The gel diffusion test (GDT) was alsoperformed with the same purpose, and as described previously [13].

A commercial ovine IFN-� microplate ELISA (Mabtech AB, Swe-den) was used to measure T-cell responses in heparinized bloodcultures after a 24-h in vitro antigen re-stimulation (final concen-tration of HS, 10 �g/mL). Measurements from the blood samplesof each individual ram taken 1 and 6 months after immunizationwere performed by triplicate and mean data expressed in pg/mL of�-IFN.

The immunoblotting assays were performed as described above,with serum samples taken from each animal during the course ofthe experiment, and using peroxidase-conjugate rabbit anti-sheepIgG (Nordic Immunological Lab, Tilburg, Netherlands) as conjugate.

2.6. Pathological studies

Skin surrounding the inoculation sites and samples of grossinjured epididymides, accessory genital glands and lymph nodes,were taken during necropsy, fixed in 10% buffered forma-lin, embedded in paraffin and 4–5 �m sections stained withhaematoxylin–eosin by standard procedures for microscopicexamination.

2.7. Statistical analyses

The t-test was used for microparticles and nanoparticles char-acterization studies. Data in these studies were expressed as the

R. Da Costa Martins et al. / Vacci

Fig. 1. SDS-PAGE and Coomassie blue stain profiles of the HS antigenic complex(rflb

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A) and Western blot against a pool of sera from B. ovis experimentally infectedabbits (B). Lanes: (1) HS antigenic extract obtained by the traditional method (1 L-ask incubation); (2) HS antigenic extract obtained by the scale up method (10 Lioreactor).

ean ± S.D. of at least three experiments. Bacteriological resultsfter challenge and statistical comparisons were analysed by thehi-square test (with Yates correction). When required, the non-arametric Kruskal–Wallis test, followed by Mann–Whitney U-testas also applied. Statistically significant differences were consid-

red when P < 0.05. All data processing was performed using thetatView SE + Graphics software for Windows (5.0, SAS Institutenc. Copyright©).

. Results

.1. Characterization of the HS antigenic complex

In order to obtain a large batch of cells, and thus, reducing het-rogeneity in the antigenic extract, the B. ovis REO 198 strain wasnoculated in a 10 L bioreactor. After several analyses, the condi-ions selected were the following: TSBY salt medium (1% yeast

xtract) during 4 days at 36 ◦C with O2 saturation at 18%. The batchf antigenic extract obtained was mainly composed of 66.4 ± 10.6%rotein and 39.5 ± 3.8% rough lipopolysaccharide (R-LPS). Whenompared to the extract obtained with the old 1 L-flask incubationystem, the new extract was enriched in total protein (66.4 ± 10.6%

able 1hysicochemical characteristics and antigen loading ability of the vaccine formulations.

Vaccine formulation Sizea Zeta potential (mV) PDIb Yield

MP-HS 1.16 ± 0.15 �m −5.51 ± 0.43 <0.20 88 ±NP-HS 211 ± 5 nm −37.7 ± 0.7 0.15 ± 0.08 77 ±MAN-NP-HS 235 ± 4 nm −34.6 ± 1.3 0.14 ± 0.02 83 ±P-HS: HS loaded poly(�-caprolactone) microparticles; NP-HS: HS loaded conventional na

s the mean ± S.D. of at least at least 3 different determinations.a Determination of the nanoparticles (nm) and microparticles (�m) volume mean dia

aser diffractometry.b Polydispersion index.c The percentage yield of the polymer transformed into microparticles or nanoparticled Amount of mannosamine coating the nanoparticles.e Determination of the protein content by SDS-PAGE densitometry (microparticles) orf Determination of the entrapment efficiency, expressed in percentage, by relating the

he initial weight of antigen.

ne 28 (2010) 3038–3046 3041

vs. 46.7 ± 4.7%) [5] and the yield of the extraction process wassuperior (1.69 ± 0.51% vs. 1.32 ± 0.62%) [4,18]. The protein profileof both antigenic extracts, revealed by SDS-PAGE, confirmed themaintenance of the immunodominant outer membrane proteins(Omp 19, Omp 22 and Omp 25–31), and adequate antigenicityassessed by immunoblotting (Fig. 1). Moreover, in the gel diffu-sion test (GDT), the extract obtained by the novel methodologydemonstrated added antigenic yield and richness, than the HSobtained using the old flask system (optimal reactive concentra-tions = 0.15 mg/mL vs. 0.62 mg/mL, respectively).

3.2. Physicochemical characterization of microparticles andnanoparticles

Table 1 summarizes the main physicochemical properties ofboth microparticle and nanoparticle vaccine formulations. Themicroparticles preparation method, by the use of TROMS, dis-played the higher yield of the polymer transformation into particles(88 ± 3%). All freeze-dried formulations displayed homogeneousvolume mean diameter with polydispersity index under 0.2.

All freeze-dried formulations displayed homogeneous sizes,around 200 nm for HS containing nanoparticles, and of about1 �m for antigen loaded microparticles, with low polydispersion(PDI < 0.2). Interestingly, in what refers to nanoparticles, the coatingwith mannosamine increased the mean diameter of the result-ing carriers, as described previously [7]. Concerning zeta potential,conventional and mannosylated nanoparticles displayed negativesurface charges, in contrast with microparticles which showed amore neutral surface (−5.51 ± 0.43 mV surface charged).

Similarly, size and morphology of the vaccine formulations wereconfirmed by comparing SEM analysis (Fig. 2), and all systems werefound to be homogeneous and spherically shaped.

The HS antigenic complex was efficiently entrapped in bothsystems. By relating the total weight of antigen entrapped inthe batch of microparticles to the initial weight of antigen, theentrapment efficiency was about 70%. Similar encapsulation effi-ciency values were obtained for nanoparticles, in comparison withHS loaded poly(�-caprolactone) microparticles. Nevertheless, theentrapment into nanoparticles significantly increased the HS load-ing per milligram of particle. For conventional poly(anhydride) andmannosylated nanoparticles, HS loading was calculated to be 35and 28 �g HS per mg of nanoparticles, respectively.

The mannosamine content for mannosylated nanoparticles wasfound to be about 30 �g/mg of nanoparticles. The agglutinationtest in the presence of the mannose-specific Concanavalin A lectin

confirmed the mannose integrity and biological activity after man-nosylation of poly(anhydride) nanoparticles (data not shown).

Fig. 3 shows the SDS-PAGE and Western blotting profiles of theHS after encapsulation and further re-extraction from microparti-cles and nanoparticles. In both cases, the protein profile was similar

c (%) Mannosamined (�g/mg np) HS loadinge (�g/mg particle) E.E.f (%)

3 – 6.55 ± 1.91 72 ± 226 – 34.93 ± 2.38 70 ± 24 32.1 ± 4.7 28.06 ± 1.63 64 ± 5

noparticles; MAN-NP-HS: HS loaded mannosylated nanoparticles. Data is expressed

meter, before freeze-drying, respectively by photon correlation spectroscopy and

s.

BCATM protein assay (nanoparticles).total weight of antigen entrapped in the batch of microparticles or nanoparticles to

3042 R. Da Costa Martins et al. / Vaccine 28 (2010) 3038–3046

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o free HS. Moreover, the immunoblotting demonstrated the sameeactivity against a pool of sera from B. ovis experimentally infectedabbits, showing that the antigenicity of the main proteins involvedn infection (Omp 19, Omp 22 and Omp 25–31 family) was con-erved.

.3. Immunological results

One of the aims of this work was to develop protective vaccineelivery systems with no interference in the serodiagnosis [modi-ed Rose Bengal (RB) and standard Complement Fixation (CF) tests]f infected animals with smooth Brucella spp. According the resultsbtained, all the sera taken from the animals immunized with Revwere seropositive in both RB and CF tests since the first week afteraccination, and remaining seropositive all along the experimentnot shown). In contrast, no positive reactions in these tests wereecorded, at any post-vaccination time, when testing the sera fromhe rams vaccinated with the acellular HS formulations developednot shown).

The evolution of the IgG-specific antibody response against theS antigen elicited after vaccination in the indirect ELISA is shown

n Fig. 4A. All immunized groups including those vaccinated withev 1 developed a strong positive serologic response since the sec-nd week after vaccination, also demonstrated by immunoblotting

ig. 3. SDS-PAGE and alkaline-silver stain for proteins from free HS (HS) or extracted fromtain for proteins of free HS or HS extracted from conventional (NP) or mannosilated (MAool of sera from B. ovis experimentally infected rabbits (B1 and B2). The HS loading was 2

oaded poly(�-caprolactone) microparticles; NP: HS loaded conventional poly(anhydride)

s. (A) HS-PEC: HS loaded poly(�-caprolactone) microparticles; (B) NP-HS: HS loadedanoparticles.

(Fig. 5; sera taken from HS-PEC vaccinated rams were chosen asa representative example). At the 8th week post-vaccination, thepercentage of reactor animals decreased, respectively to 45% and85%, for the Rev 1 and HS-PEC vaccinated rams. At the time ofchallenge (week 24 post-immunization), only few of these ani-mals remained positive in the indirect ELISA. However, at the timeof the experimental infection, animals vaccinated with HS loadednanoparticles remained seropositive. The challenge with B. ovis PAinduced a quick anamnestic antibody response to HS antigens inall vaccinated animals that was maintained until the slaughter-ing. The intensity and high affinity of the elicited specific antibodyresponse after HS-PEC, NP-HS or MAN-NP-HS immunization wasalso demonstrated by GDT (Fig. 4B), with similar kinetic but lessintense response than that observed in the indirect ELISA.

The Western blot assays performed (Fig. 5) provided infor-mation about the development and persistence of IgG antibodiesraised after immunization against the outer membrane proteins ofthe HS extract. After immunization with HS-PEC, NP-HS and MAN-NP-HS, the most immunogenic proteins were Omp 31, Omp 43 and

L-Omp 19, which induced a strong immunoresponse maintained inall vaccinated groups until the moment of challenge.

Having in consideration that IFN-� up-regulation is essential forprotection against brucellosis, we determined the production ofthis important cytokine by lymphoid cells in the immunized rams.

HS-PEC poly(�-caprolactone) microparticles (A1); SDS-PAGE and Coomassie blueN-NP) nanoparticles (A2), and their corresponding Western blot analyses against a.5 �g (A1) or 20 �g (A2, B1 and B2) per well. HS: Free hot saline extract; HS-PEC: HSnanoparticles; MAN-NP: HS loaded mannosylated poly(anhydride) nanoparticles.

R. Da Costa Martins et al. / Vaccine 28 (2010) 3038–3046 3043

Fig. 4. Percentage of seropositive animals in indirect ELISA (A) or gel diffusion(B) tests against B. ovis HS antigens, after vaccination with B. melitensis Rev 1(�), HS-PEC: HS loaded poly(e-caprolactone) microparticles (�), NP-HS: HS loadedcs(e

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Fig. 6. IFN-� (pg/mL) response of lymphoid cells obtained from vaccinated and non-vaccinated control individual rams previously stimulated with HS antigenic extract,

onventional poly(anhydride) nanoparticles (�), or MAN-NP-HS: HS loaded manno-ylated poly(anhydride) nanoparticles (�). Sera from control non-vaccinated rams*) were also included in the study. At week 24th after vaccination, all animals werexperimental challenged (arrow) with the virulent B. ovis PA strain.

he results obtained (Fig. 6) show that the cytokine profile was

haracteristic of a Th1 immunological response, revealed by theigh IFN-� amounts found in most vaccinated rams, independentlyf the type of vaccine used. Therefore, the cytokine pattern identi-ed reflects the activation of antigen-specific Th1 clones previously

ig. 5. Evolution of the serologic response against outer membrane proteins con-ained in the Brucella ovis HS antigenic extract (20 �g of HS per well) using serarom HS-PEC vaccinated rams, as an example. Lanes: (1) before vaccination (ramsree); (2) 2 weeks post-vaccination; (3) 2 months post-vaccination; (4) 4 monthsost-vaccination; (5) 6 months post-vaccination; (6) 2 months post-challenge; (7)months and 2 weeks post-challenge.

respectively 1 month (�) or 6 months (�) after immunization, and previous to thechallenge. *P < 0.05 (Mann–Whitney U test). Statistically significant differences werealso found between the control and all the vaccine systems, at the different timesof the experiment (not shown in the figure).

differentiated after vaccination. Moreover, the high levels of IFN-�were maintained at least by 6 months after immunization.

3.4. Bacteriological results

The bacteriological results obtained after the necropsy are sum-marized in Table 2. As it can be seen, the intensity of the challengewas adequate enough for statistical comparisons, since the percent-age of infection found in the unvaccinated controls was of about93%. While none of both HS loaded nanosystems induced significantprotection, the HS-PEC microparticles were protecting 54% of vac-cinated rams, being this protection similar to that induced by Rev 1(49%). In agreement with that, the percentage of infected sampleswas, in general, significantly higher in the animals vaccinated withnanoparticles than in the animals vaccinated with the effective vac-cines HS-PEC and Rev 1. In fact, the percentage of infected samplesin the animals vaccinated with NP-HS was even significantly higherthan that found in the unvaccinated controls.

3.5. Pathological results

The subcutaneous inoculation of all vaccines (including Rev 1)induced variable degrees of local reactivity in the tissues surround-

ing the injection area. In general, this local reactivity was moderateto low and was resolved in most cases in a few weeks after vaccina-tion. However, moderate inflammatory reactions at the inoculationsites were evidenced at the time of necropsy in some animals

Fig. 7. Section of the skin, at the inoculation site, obtained at the time of necropsyin a ram vaccinated with the MAN-NP-HS formulation.

3044 R. Da Costa Martins et al. / Vaccine 28 (2010) 3038–3046

Table 2Bacteriological results and protective efficacy of the experimental vaccine formulations after challenge with the virulent B. ovis PA strain.

Vaccine groupa No. of infected rams/total (%) No. of infected samples/total (%) Statistical differences vs.b

Unvaccinated Rev 1 MAN-NP-HS NP-HS

HS-PEC 6/13 (46) 25/104 (24) A, C B, C B, C A, CNP-HS 12/12 (100) 81/96 (84) B, C A, C B, C –MAN-NP-HS 10/12 (83) 51/96 (53) B, D B, D – –Rev 1 8/14 (51) 49/112 (44) A, C – – –Unvaccinated 13/14 (93) 65/112 (58) – – – –

Rams were vaccinated with HS loaded poly(�-caprolactone) microparticles (HS-PEC), HS loaded conventional nanoparticles (NP-HS), HS loaded mannosylated nanoparticles(MAN-NP-HS) or B. melitensis Rev 1 vaccine.

a Each ram was immunized with 1 mL of the different vaccines given in a single dose by subcutaneous route. The dose of B. ovis HS entrapped in microparticle andnanoparticle formulations was 3 mg per individual ram in all cases. The individual dose of Rev 1 vaccine was 1.6 × 109 CFU. Six months after vaccination all animals werechallenged conjunctivally and preputially with 1.16 × 109 CFU of virulent B. ovis PA strain. Eight weeks after challenge, all rams were necropsied and selected organs andl

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ymph nodes were submitted to bacteriological analyses.b Statistical differences between the different vaccine groups (Chi-square test re

espectively, regarding the number of infected rams. C and D: P < 0.05 and not signi

accinated with the nanoparticle and microparticle formulationsNP-HS: 2/13 animals; MAN-NP-HS: 6/12 animals; HS-PEC: 7/13nimals) (Fig. 7). These reactions were of mild nature and charac-erized by necrotic foci delimited by granulation tissue composedf variable amounts of polymorphonuclear leukocyte cells, plasmo-ytes, lymphocytes, macrophages, foam cells and giant cells, andccasionally surrounded by a fibrous connective capsule.

In what concerns to the pathological analysis of the animalsound infected at necropsy (results not shown), the animals vacci-ated with the microparticle and nanoparticle formulations were

n general less affected than unvaccinated or Rev 1 vaccinated rams.n fact, two unvaccinated controls and three rams vaccinated withev 1 that were found infected at necropsy, presented importantacroscopic and microscopic lesions, mainly located in the epi-

idymides and vaginal layers. In contrast, only one of the ramsmmunized with HS-PEC presented severe lesions (epididymitisnd fibrinous vaginalitis).

. Discussion

The aim of this project was to develop and evaluate acellularaccines against B. ovis to face the problems arising from the use ofhe live attenuated commercial vaccine B. melitensis Rev 1. Theselternative vaccines are based on the use of the HS membranousntigenic complexes exploited as the diagnostic antigen of choiceor B. ovis infection. In fact, the majority of rams infected by B. ovisroduces high levels of circulating antibodies against the proteinsf the outer membrane [23]. Our main postulates were that thecellular vaccines proposed would be innocuous enough, shouldvoid the interference induced with the serodiagnostic tests forhe detection of animals infected with B. melitensis, and finally,asy to produce to be commercialised at reasonable cost. Moreover,lthough it can be relatively easy to produce these vaccines at smallcale, the formulations made should be efficiently and rapidly pro-uced at larger scales for massive production. Accordingly, we firsturposed to scale up the HS production in a bioreactor and inves-igate either the production yield or the quality of the HS extractbtained. The results revealed that the novel experimental condi-ions employed increased significantly the antigenic properties ofhe extract, leading to the benefit of the industrial scale up.

The optimal protective immunity to Brucella infection is depen-ent of a coordinate interaction between different T-cell subsetshich leads to an antigen-specific T-lymphocyte-mediated acti-

ation of macrophages, the main cellular reservoir of Brucella.ffective vaccines require the induction of appropriate protectivemmune responses and, in the case of acellular vaccines, adjuvantsre essential to achieve it. Nanoparticles are adjuvants capablef increasing both mucosal and systemic immune responses [24].

previous Fisher’s correction when required): A and B: P < 0.05 and not significant,, respectively, regarding the number of samples infected.

To exploit this potential, conventional nanoparticles were surfaceenriched with mannosamine, since it has been shown that bothantigen loading in nanoparticles and mannosylation of these sys-tems are effective approaches to potentiate immunogenicity, dueto the higher antigen uptake and presentation by APCs [25,26]. Theeffectiveness of mannosylated devices in vaccinology is ascribedto their ability to target mannose receptors and other lectinswith mannose-binding activity, highly expressed in cells of theimmune system [i.e., macrophages and dendritic cells (DCs) trig-gering the enhancement of the innate immune response [27–30].B. ovis HS loaded conventional and mannosilated nanoparticleswere successfully obtained by the solvent displacement method,with homogenous size distribution, high yield and effective anti-gen loading. The antigenic complex was efficiently entrappedinto poly(anhydride) nanoparticles with encapsulation efficienciesbetween 64% and 70%. As shown in Table 1, significant differ-ences were found between both types of nanoparticle formulationsfor size and zeta potential, due to the mannosamine presence onthe surface of these carriers. These results are in agreement withother works in which it was also described an increase on theaverage size for the mannosylated nanoparticles [26]. The sur-face decoration of the nanodevices slightly affected the HS loadingratio since conventional nanoparticles displayed a higher antigenloading than the mannosylated ones, in agreement with previ-ous data reported [26,31]. SDS-PAGE and immunoblotting of theHS extracted from both nanoparticles types demonstrated thatthere were no apparent modifications due to the manufacturingprocedure, including antigenicity. In agreement with the alreadycommented, both NP-HS and MAN-NP-HS elicited a strong andlong-lasting antibody response in the inoculated rams. At the timeof challenge (week 24 post-immunization) 75–90% of the animalsvaccinated were still seropositive in the indirect ELISA (Fig. 5).Moreover, nanoparticle delivery systems allowed also an intenseIFN-� response (Fig. 6). Despite this, the overall protective effi-cacy of these formulations was not adequate. Surprisingly, ramsimmunized with NP-HS resulted even more infected than con-trol unvaccinated animals. The high ability of these formulationsto activate and fix complement factors of its surface (unpublishedresults), could be explaining these apparently paradoxical results.Having these important complement activation abilities, phago-cytosis of nanoparticles once opsonized by complement could bethen enhanced via cell receptors (CRs) expressed on the surface ofphagocytes. It is known that signal transduction via CRs can sig-

nificant affects macrophage activity, including the up-regulation ofcytokine expression [32]. Thus, CRs can act as negative regulatorsfor IL-12 production, a Th1 activator citoquine, and hence, be ableto bias immunity to a Th2-type response through IL-10 production[33]. This can result in a CRs-mediated suppression of host immune

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ystem against intracellular pathogens like Brucella. Furthermore,L-10 is a negative factor for IFN-� production, and this is in agree-

ent with our results showing that IFN-� levels elicited in ramsaccinated with nanoparticles decreased earlier and faster thanhose elicited by immunization with other vaccines. In summary,omplement-mediated uptake enhancement of the complementpsonized microparticles may contribute to a bias in the cytokineatterns with the ensuing modulation of the innate immune mech-nisms favouring B. ovis infection. New experiments to study IL-10volution after immunization with nanoparticles should be per-ormed to confirm this hypothesis.

Additionally, the complement-mediated phagocytosis enhance-ent of nanoparticles may also contribute to the deliver of bacterial

ypopolysaccharides to functional APCs. The Brucella LPS is con-idered a slowly processable structure either by the incapacity ofhe molecular machinery to digest it, or to its ability to alteringhe constitutive intracellular trafficking within the cell, remain-ng in non-functional lysosomal compartments [34]. Once in theate endocytic non-degradative compartment, the LPS may recruit

HC-II products, being then exported to the cell surface in the formf stable macrodomains. LPS macrodomains may remain intactn the membrane for at least 3 months [35]. As a consequence,PS impairs further MHC-II antigen presentation for long peri-ds, inducing some immunosuppressory effect, related with thehronic immunosuppression-induced state observed upon somerucellosis infected patients [36]. This impairing effect has beenpecially observed in the liver sinusoidal endothelial antigen pre-enting cells. The fact that these cells are the main target forhe fate of nanoparticles [37] makes this hypothesis more plau-ible. The efficient capture of C3b/iC3b-coated nanoparticles inhe sinusoidal lumen is likely to occur through the novel comple-

ent receptor, CRIg (Complement receptor of the immunoglobulinuperfamily), which is uniquely expressed on tissue resident andinusoidal macrophages. CRIg participates in the internalization of3-opsonized particles from the circulation better than CR3 [38].

n addition, CRIg has been reported to down-regulate T-cell prolif-ration and, in turn, IL-2 and IFN production through a mechanismhat remains unexplained.

By contrast, the animals vaccinated with HS-loadedannosamine-coated nanoparticles (MAN-NP-HS) were not

ound more infected than controls, as standard NP-HS formulationid (Table 2). The rational could be the preferential uptake byupffer macrophages, in contrast to NP-HS that are uptaken prefer-ntially by DCs [39]. It is known that liver DCs induce preferentiallyTh2-type immunoresponse, suggesting that these cells, instead

f Kupfer cells, could be the responsible of the immunologicalolerance observed in animals vaccinated with NP-HS but not in

AN-NP-HS nanoparticles [40,41]. Thus, we may hypothesize thatnce inoculated, NP-HS are avidly covered by complement factorsunpublished results), readdressing their fate to liver, where, in theinusoidal endothelial APCs, naturally predisposed to tolerance,timulate the long-term unprocessable LPS, where may impairuture antigen presentations. After infection via mucosae, B. ovisells reach the liver via portal circulation, where the bacteriaill found a sensible niche (immunosuppressed DC) where they

eplicate. In contrast, MAN-NP-HS nanoparticles would reachreferentially Kupfer cells (via MBL), that does not present thatolerant tendency [25].

In contrast, a high protection was obtained after HS-PEC immu-ization, similar to that induced after Rev 1 vaccination. HS-PEClicited a significant strong and long-lasting antibody response,

hich, at difference of that happening with Rev 1, did not inter-

ere with the conventional serological tests available against B.elitensis. Moreover, the HS-PEC uptake by the APCs promoted alsostrong Th1 response as demonstrated by the high IFN-� levels

nduced.

[

ne 28 (2010) 3038–3046 3045

PEC microparticles containing HS (HS-PEC) were prepared byTROMS [42]. This method is semiautomatic and easily reproduciblesystem, designed for the microparticle preparation at a semi-industrial scale, then minimising human manipulation during theproduction and facilitating the implementation of GMP conditions.A previous study was done to select the most suitable cryoprotec-tor and after testing different surfactants and/or sugars at variousconcentrations, Tween 80 at 0.2% and mannitol at a concentra-tion of 5% were observed to be the most effective to avoid particleagglomeration. Together with the selection of adequate pharma-ceutical auxiliaries, such as �-cyclodextrin and Pluronic® F68 [18],and the use of mannitol as most adequate cryoprotector, boththe TROMS and freeze-drying techniques enabled us to obtainwell resuspendable, homogenously sized, smooth and sphericallyshaped microparticles observed by SEM (Fig. 2). Overall, when mea-sured by laser diffractometry, HS loaded microparticles (HS-PEC)displayed a size of around 1.2 �m, an optimal mean diameter to betaken up by APC’s.

In summary, the results indicate that one single dose of HS-PEC, containing 3 mg of HS antigenic complex, confers effectiveprotection against B. ovis in rams. Moreover, the lack of interfer-ence in the B. melitensis diagnostic tests, the intrinsic avirulenceand the innocuousness of HS-PEC, makes this formulation a suit-able anti-Brucella vaccine candidate. In spite of the results obtained,the potential use of the HS loaded nanoparticle systems for mucosaladministration is now under investigation.

Acknowledgements

The authors want to thank Prof. Dr. Rafael Jordana (Zoology andEcology Department, University of Navarra, Pamplona, Spain) forthe SEM analysis and to Maite Hidalgo (Pharmacy and Pharma-ceutical Technology Department, University of Navarra, Pamplona,Spain) for the technical assistance. This work was supported by“Fundacão para a Ciência e Tecnologia” (SFRH/BD/41703/2007) inPortugal, “Asociación de Amigos de la Universidad de Navarra”,“Fundación Caja Navarra” (“Nanotecnología y Medicamentos”, ref.10828) and grants from the “Ministerio de Ciencia e Innovación”(AGL2004-07088) in Spain.

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