Vaccine 26 (2008) 4639–4646

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Outer membrane vesicles as acellular vaccine against pertussis

Roy Roberts a, Griselda Morenob, Daniela Botteroa, Maria Emilia Gaillarda,Matías Fingermanna, Augusto Graieba, Martin Rumbob, Daniela Hozbora,∗

a Instituto de Biotecnología y Biología Molecular (IBBM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata,Centro Científico Tecnológico CONICET La Plata, Calles 47 y 115, 1900 La Plata, Argentinab Laboratorio de Investigaciones del Sistema Inmune (LISIN), Facultad de Ciencias Exactas, UNLP, 47 y 115, 1900 La Plata, Argentina

a r t i c l e i n f o a b s t r a c t

mentes agsociam B.ownussismetrith cyis intrtes. Kunizale-ceationith sud gro

zed e

Article history:Received 8 April 2008Received in revised form 27 June 2008Accepted 1 July 2008Available online 18 July 2008

Keywords:Bordetella pertussisVaccineOuter membrane vesiclesSurface proteomeIntranasal

In this study the developdetella pertussis as vaccinand gel electrophoresis asof the OMVs obtained froence of the main well-kncyclase—haemolysin, pertidentified by mass spectrolocation and the others wused in murine B. pertussdelivered by different rouintraperitoneal (i.p.) immOMVs as well as killed who6 and CCL20, i.n. immunizintranasally challenged wanimals and the PBS treateobserved in mice immuni

with both types of immunizatio

In view to their capacity to inobtained from B pertussis are ca

1. Introduction

Pertussis or whooping cough is an acute respiratory tract infec-tion mainly caused by Bordetella pertussis, fastidious gram-negativebacteria. This disease, though preventable by vaccination, remainsone of the top ten causes of death worldwide in childhood. Nowa-days morbidity of pertussis is significant not only in childrenyounger than 1-year-old [1] but also in adolescent and adults [2–4].It was believed that immunization conferred lifelong immunitydue to the paucity of pertussis cases in school aged children andadults in pre-vaccine and early vaccine period. Since from the 1980sthe number of reported cases has increased steadily among younginfants, 11- to 18-year-old people (adolescents) and adults, the tenetof lifelong protection after pertussis or childhood immunization

∗ Corresponding author. Tel.: +54 221 425 0497x31.E-mail address: hozbor@biol.unlp.edu.ar (D. Hozbor).

0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2008.07.004

and evaluation of outer membrane vesicles (OMVs) obtained from Bor-ainst pertussis disease is described. SDS-PAGE, immunoblot techniquested to tandem mass spectrometry were used to describe the compositionpertussis Tohama CIP 8132 strain. These techniques revealed the pres-

pertussis surface immunogens in the OMVs such as pertactin, adenylatetoxin, as well as the lipo-oligosaccharide (LOS). A total of 43 proteins werey. Some of them were predicted to have outer membrane or periplasmictoplasmic or unknown location. The characterized pertussis OMVs wereanasal (i.n.) challenge model to examine their protective capacity when

illed detoxified whole-cell B. pertussis bacteria were used as reference. Fortion, aluminum hydroxide was used as adjuvant. Since i.n. treatment withll bacteria enhanced markers of innate immune response such as TNF�, IL-s were performed with no adjuvant added. Immunized BALB/c mice wereblethal doses of B. pertussis. Significant differences between immunizedup were observed (p < 0.001). Adequate elimination rates (p < 0.005) were

ither with OMV or whole-cell bacteria. Comparable results were obtainedn route.duce airways innate and protective immunity in the mouse model, OMVsndidates to be used to protect against pertussis.

© 2008 Elsevier Ltd. All rights reserved.

has been dismissed [5]. Furthermore, adolescents and adults areincreasingly recognized as vectors for B. pertussis transmission toyoung infants, who have the higher risk of pertussis-related com-plications, hospitalization and death [6].

In response to the ongoing problem of pertussis, research wasfocused to design better strategies of disease control. In this sense,acellular vaccines have gained space in pertussis control, in par-ticular in industrialized countries, because of the reactogenicityassociated to whole-cell vaccines (wP) and also due to the epi-demiological pertussis scenario that include adolescents and adults[7,8]. The most modern acellular vaccines composed by two (per-tussis toxin and filamentous hemagglutinin), three (pertussis toxin,filamentous hemagglutinin and pertactin) or five immunogens(pertussis toxin, filamentous hemagglutinin, pertactin and fimbriae2 and 3) seem to be suitably not only for primary immunization [9]but also for boosting adolescents and adults [10,11]. It is acceptedthat the best acellular vaccine equals the efficacy of the traditionalwP but bearing a much lower reactogenicity, especially concerningserious side effects. However either wP or present acellular vaccines

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need to be improved since their efficacy rounds 70–90%, depend-ing on the formulation considered and the technical definition of aclinical case [12–14].

An interesting material to be analyzed as a new acellular vaccineis the outer membrane vesicles (OMVs) since they contain mainimmunogens used in currently acellular vaccines but expressedin the context of the pertussis membrane, allowing the immuneresponse to preferentially target surface-exposed epitopes in theirnative conformation.

In this report we described the development and characteri-zation of OMVs derived from B. pertussis cells as acellular vaccinecandidate either for intraperitoneal and nasal administration. Thislast route has been considered as an alternative that may possiblyimprove pertussis vaccination [15–18]. This route closely mimicsthe natural B. pertussis infection, which has been considered toinduce strong and long-lasting immunity. Isaka et al. [15] examinedthe effectiveness of recombinant B subunit of cholera toxin (rCTB) asa mucosal adjuvant for intranasal co-administration of an acellularpertussis vaccine, which consists of formalin-treated purified per-tussis toxin (PTXd) and filamentous hemaglutinin. Though furtherstudies are needed, the results obtained by these authors showedthat the dose of PTXd included in an acellular pertussis intranasalvaccine should be as low as possible and the addition of rCTB maynot be always necessary for the case of this nasal formulation. Haleet al. [16] showed that neonatal mice immunized intranasally withpertactin together with Escherichia coli heat-labile enterotoxin (LT)were protected against virulent B. pertussis challenge. Mielcarek etal. [17] showed that a single nasal administration of a geneticallyattenuated strain BPZE1 protects against challenge with virulentB. pertussis better than two administrations of acellular vaccine ininfant mice. These promising results encourage the development ofmucosal pertussis vaccines that could be administrated in differentpopulation groups.

The OMVs characterized here were found to induce protectionin mice after either intraperitoneal or intranasal administration.OMVs protective capacity was comparable to the protection pro-vided by wP by both routes. These results show that OMVs aregood candidates to be used as acellular vaccine to protect againstpertussis.

2. Materials and methods

2.1. Bacterial strains and growth conditions

B. pertussis Tohama strain (CIP 8132) and B. pertussis WHO refer-ence strain Bp18323 were used throughout this study. B. pertussisstrains were grown in Stainer–Scholte liquid medium (SS) as indi-cated previously [19].

2.2. Isolation of outer membrane vesicles (OMVs)

OMVs were isolated from bacterial cells. Briefly, as previouslydescribed [20], culture samples from the decelerating growth phasewere centrifuged at 10,000 × g for 20 min at 4 ◦C and the bacterialpellet obtained was resuspended in 20 mM Tris–HCl, 2 mM EDTApH 8.5 (TE buffer). Five milliliters of TE buffer were used to resus-pend approximately 1 g (wet weight) of bacteria. The suspensionwas sonicated in cool water for 20 min. After two centrifugationsat 10,000 × g for 20 min at 4 ◦C, the supernatant was pelleted at100,000 × g for 2 h at 4 ◦C. This pellet was resuspended in 1.5% (w/v)deoxycholate (DOC) in TE buffer. Six milliliters of this suspensionwere added on 2 ml of sucrose 60% (w/v).

After centrifugation at 100,000 × g for 2 h at 4 ◦C, the OMVband was observed at TE/sucrose interphase. This procedure was

6 (2008) 4639–4646

repeated once. The OMVs were stored with glycerol 1% and sodiumazide 0.001% at 4 ◦C. The sample obtained was negatively stainedand then examined with an electron microscope.

2.3. Electron microscope negative stains

Electron microscopy was performed by suspending OMVs in0.1 M ammonium acetate (pH 7.0). A droplet of this suspensionwas placed on a grid coated with a carbon-reinforced fomvar film.After 30 s, the excess fluid was removed by absorbing with filterpaper and the grids stained with 2% (w/v) phosphotungstic acidpH 5.2 (with KOH). Examination was done with a JEM 1200 EX Jeolmicroscope.

2.4. Protein assay

Protein content was estimated by the Bradford method [21]using bovine serum albumin as standard.

2.5. Immunoblots

Samples of OMV obtained from B. pertussis cells were treatedwith Laemmli sample buffer [22] and run on 10% SDS gels. Theelectrophoresis was performed at room temperature and constantvoltage, with molecular weights being estimated by means of thePharmacia Calibration Kit. After electrophoresis, the proteins weretransferred from the polyacrylamide to a polyvinylidenephosphatemembrane (Immobilon P, Millipore) and incubated with mousepolyclonal immune sera directed against the adenylate cyclase (AC-Hly) or pertussis toxin (PTx) of B. pertussis. (The immunochemicaldetection was performed using alkaline phosphatase-labeled sheepanti-mouse immunoglobulins.)

2.6. Lipo-oligosaccharide extraction and sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

LOS from OMVs were solubilized in the sample buffer describedabove and heated at 100 ◦C for 10 min. Twenty-five micrograms ofproteinase K in 10 �l of buffer were added per 50 �l of LOS sus-pension. The mixtures were incubated in a water bath at 60 ◦C for1 h with occasional vortexing. Proteinase K-treated samples wereapplied to gels. Electrophoresis was performed at room tempera-ture and constant voltage. The LOS was visualized by the Bio-Rad

silver-staining technique.

2.7. 2-DE electrophoresis

Isoelectric focusing (IEF) was performed by using IPG strips(InmobilineTM DryStrip Amersham Biosciences-AB). Two hundredmicrogram of proteins were applied to 7 cm strips of pH range4.0–7.0. The IPG strips were rehydrated overnight at room tem-perature with 125 �l of rehydration Buffer (7 M urea, 2 M tiourea,10% isopropanol and 2% Triton X 100) plus 1.25 �l 28% DTT, 0.62 �l0.5% ampholyte (pH 4.0–7.0 [Amersham]) and 0,01% Bromophenolblue containing 200 �g of proteins. Three preset programs wereexecuted with slight modifications such that focusing conditionscomprised the conditioning step, voltage ramping, and final focus-ing.

After IEF, the strips were equilibrated in 50 mM Tris buffer(pH 8.8) containing 6 M urea, 2% SDS, 30% glycerol, 1% dithio-threitol (DTT), followed by another 1 h equilibration step with thesame buffer supplemented with 4.5% iodoacetamide. SDS-PAGEwas performed according to the Laemmli method [22] with a 12.5%resolving polyacrylamide gel without a stacking gel. Separation in

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the second dimension was carried out at 40 V at 4 ◦C until the run-ning dye reached the bottom.

2.8. Coomassie staining and gel drying

Proteins resolved on gels were visualized by using a colloidalCoomassie staining method (see also http://prospector.ucsf.edu)with slight modifications. Briefly, the gel was fixed in a solutioncontaining 30% ethanol, 2% phosphoric acid with double-distilledwater (dDW) three times 30 min each. All incubations were per-formed with gentle shaking. After a fixing step, the gel waswashed with a solution of 2% phosphoric acid in dDW three timesfor 20 min each. Washed solution was discharged and gels werefixed again with a solution containing 2% phosphoric acid (v/v),ethanol 18% (v/v) and ammonium sulphate 15% (w/v) in dDW for30 min. To this solution a 1.5% volume of suspension of colloidalCoomassie (G-250) 2% in dDW was added. Duration of this stepwas 24–72 h.

2.9. Image analysis

A gel image was captured in a UVP Bioimaging systems EpiChemi3 Darkroom with a Hamamatsu Photonic systems camera,model 1394 C8484-51-03G, controlled by Labworks Image acquisi-tion and analysis software Version 4.6.00.0. The eight-bit grey scale.tiff files obtained were later processed with the Image Master 2DPlatinum, Version 6.0 software.

2.10. In-gel tryptic digestion of proteins

Coomassie-stained spots were excised from 2-DE gels and trans-ferred into microcentrifuge tubes. Plugs were washed with 100 �lammonium bicarbonate 25 mM for 10 min and incubated for 30 minat 60 ◦C in 20 �l of DTT 5 mM in ammonium bicarbonate 25 mMsolution. This step was followed by another incubation of 15 minat room temperature in darkness with 20 �l iodoacetamide 55 mMin ammonium bicarbonate 25 mM. Solution was discharged anda new washing with 25 mM ammonium bicarbonate was per-formed followed by other wash with 100 �l acetonitrile. Then,the spots were subjected to dehydration step three times with

25 �l acetonitrile for 10 min at room temperature. When gel spotsbecame completely white, acetonitrile was eliminated by evapo-ration. Spots rehydration was performed with small volumes (upto 20 �l) of 50 mM ammonium bicarbonate containing trypsin(20 �g/ml, Promega) and incubated for 45 min at 4 ◦C. The tryp-tic digestion was carried out overnight at 37 ◦C. After digestionthe spots were washed twice with 20 �l acetonitrile: 0.1% tri-flouroacetic acid (TFA) 33:66 for 10 min and supernatants werecollected. Sample concentration was performed in a Speedvac cen-trifuge.

2.11. Peptide mass fingerprinting

A matrix solution composed of �-cyano-4-hydroxy cinnamicacid (0.2 g/l) in 50% acetonitrile and 0.1% TFA was prepared forpeptide mass fingerprinting. A 0.4 �l mixture of matrix and sam-ple solutions (1:1 in volume) was applied to the target well. Thesolution mixture was dried for 10 min at room temperature andsubjected to a matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) operation by using theUltraflex (Bruker) with the following parameters: 20-kV accelerat-ing voltage, 75% grid voltage, 0.02% guide wire voltage, 70-ns delay,and a mass gate of 800–3000.

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2.12. Identification of proteins

Search and identification of peptides were performed witha licensed Version of MASCOT software (Matrix Science athttp://matrixscience.com), in a database containing the 3436accession number entries derived from the complete B. per-tussis genome sequence (downloaded from http://www.ncbi.nlm.nih.gov/). The MASCOT search parameters: (i) species, Bacte-ria (Eubacteria); (ii) allowed number of missed cleavages (only fortrypsin digestion) 1; (iii) variable post-translational modifications,methionine oxidation; (iv) fixed modifications, carbamidomethy-lation; (v) peptide tolerance, ±50 ppm; (vi) peptide charge, +;and (vii) monoisotopic peptide masses, were used to search thedatabase, allowing a molecular mass range for 2-DE analyses of±15%. Only significant hits as defined by MASCOT probability anal-ysis were considered.

Prediction of protein localization was carried out using PSORTalgorithm available at http://psort.nibb.ac.jp.

2.13. Innate response characterization

2.13.1. Tissue processingFifty microliters of OMV preparation containing 5 �g of total

protein was administered intranasally to BALB/c mice. At differenttimes upon stimulation mice were sacrificed by cervical disloca-tion. Inferior lobe of right lung was immediately processed for totalRNA obtaining using Nuclespin RNAII system (Macherey-Nagel,Germany) following manufacturer instructions.

2.13.2. RNA isolation, reverse transcription and quantitativereal-time PCR

RT-qPCR was performed as described [23]. Briefly, total RNAwas isolated using Nuclespin RNAII system (Macherey-Nagel, Ger-many) following manufacturer instructions. Reverse transcription(RT) was performed on 100 ng RNA using MMLV-RT (Promega).Resulting cDNA was amplified in triplicates using SYBR® Green PCRassay (Bio-Rad Laboratories, Hercules, CA, USA) and products weredetected on an ICycler (Bio-Rad). PCR samples were incubated for2 min at 50 ◦C and for 10 min at 95 ◦C, followed by 40 amplificationcycles with 1 min annealing/extension at 60 ◦C and 15 s denatura-tion at 95 ◦C. The �-actin expression was used as normalizer. PCRspecificity was checked by melting curves. Relative mRNA levelswere determined by comparing the normalized PCR cycle thresh-

old (Ct) between cDNA samples of the gene of interest as previouslydescribed [23]. Specific primers for the different cytokines werealready described [23,24]. Animal experiments were performed atleast in duplicates.

2.14. Mouse weight gain test (MWG)

The MWG-test was carried out using groups of 8 Balb/c miceout bred mice (15–20 g) which were i.p. vaccinated with a volumecontaining 1/10 human dose of whole-cell vaccine and detoxifiedOMV, respectively. Toxicity was also analyzed when immunizationwas performed using intranasal route. In all cases control groupreceived an equal volume of sterile PBS. Animals were observedfor 7 days and body weight was recorded after 16 h, 3 and 7 days.Vaccines were considered non-toxic when passing the followingrequirements (WHO and EP requirements): (a) the total weight ofthe mice from the vaccine group 3 days after treatment was thesame or higher than the initial weight, (b) at the end of 7 days theaverage weight gain of the vaccine group was not less than 60%of the control group and (c) not more than 5% of the animals diedduring the test period.

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2.14.1. Active immunization and intranasal challengeFemale BALB/c mice were obtained at 4 weeks of age from Biol

SAIC, Argentina. Animal protocol was performed as we previouslydescribed [25]. Either OMVs and killed whole-cell bacteria used asvaccines were detoxified with formalin (0.37% at 37 ◦C overnight).

Groups of 15 BALB/c mice were immunized i.p. withdetoxified killed whole-cell bacteria (Tohama vaccine strain,2 × 108 CFU ml−1) or detoxified OMVs (derived from Tohama vac-cine strain, 5 �g of protein). In both cases immunogens wereemulsified with aluminium hydroxide (alum) as an adjuvant(0.2 mg/ml).

Intranasal immunization was performed with either 50 �lof detoxified killed whole-cell bacteria (Tohama vaccine strain,2 × 108 CFU ml−1) or OMVs (derived from Tohama vaccine strain,5 �g of protein) without adjuvant. Either i.p. or i.n. protocols com-prised a two-dose schedule over a period of 2 weeks. Mice werechallenged 2 weeks after the second immunization by nasal chal-lenge with sublethal dose (107–108 CFU/50 �l−1) of WHO referencestrain B. pertussis 18323 or Tohama strain. Lungs of challengedmice were collected for bacterial counts 2 h, 5 and 8 days after thechallenge. The lungs aseptically removed, homogenized in the ster-ile PBS, were serially diluted, and then plated on Bordet–Gengouplates supplemented with defibrinated sheep blood to determine

bacterial recoveries at different time points during the course ofinfection. At least three independent experiments were performed.

2.14.2. Statistical analysisMeans and standard deviations were calculated from Log10-

transformed CFU numbers. Differences among means wereassessed by two-tailed Student’s t-tests with significance acceptedat the p < 0.05 level.

3. Results and discussion

3.1. OMVs isolation and characterization

As described in our previous work [20] vesicle formation fromcell pellets could be induced by sonication. Following this method-ology, OMVs from Tohama strain with a mean size of 70 nm andranging from 40 to 100 nm were obtained (Fig. 1A). These OMVscould not be observed when cell lysis procedure was carried outsuggesting that OMVs do not come from lysed bacterial cells. Thepresence of the lipo-oligosaccharide (LOS), an ubiquitous compo-nent of the outer membrane of gram negative bacteria, (Fig. 1B) and

Fig. 1. Bordetella pertussis outer membrane vesicles characterization: panel A, negatively s(12,5%) of LOS from B. pertussis OMVs. Panel C, Western blot of OMVs using anti PTX andpurposes. Molecular weights are indicated at the left.

Fig. 2. Separation and identification of proteins in B. pertussis OMVs samples using2D-PAGE followed by MALDI-TOF-MS. Samples were resolved by IEF (pH 4–7) and12.5% SDS-PAGE. Protein spots were visualized by colloidal Coomassie staining. Pro-tein spots are labeled as indicated in the legend for Table 1.

the well known surface immunogens such as Adenylate Cyclase-Haemolysin (AC-Hly) and Pertussis Toxin (PTX) (Fig. 1C) allowedus to verify that those vesicles were derived from the outer mem-brane as described [20]. At least 10 independent replicates of OMVsisolation and characterization procedure were carried out. In allcases similar morphology, size distribution and presence of surfaceimmunogens were observed.

To go further in the characterization of OMV composition,2D electrophoresis associated to matrix-assisted laser desorptionionization-time-of-flight mass spectrometry analysis were done inorder to identify some of the proteins present in OMVs (Fig. 2).Forty three proteins were identified and correlated on the basis oftheir predicted locations within the bacterial cell using the PSORTbalgorithm, which predicts the subcellular locations of proteins ingram-negative bacteria according to the presence or absence ofleader peptides, homologies to known proteins, transmembranedomains, and outer membrane anchoring motifs. From the 43 pro-teins successfully identified, 10 were predicted to be associated toexternal membrane or with cytoplasmatic/membrane (harbouring

tained B. pertussis OMVs examined with an electron microscope. Panel B, SDS-PAGEanti AC-Hly sera. Total protein stain of SDS-PAGE analysis is shown for comparative

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Table 1Proteome of Bordetella pertussis outer membrane vesicles

Order Protein name/function Gene locusa MW (kDa) pI Protein localizationb Top score Matchedpeptides

Sequencecoverage (%)

1 Putative outer membrane protein bp 3077 77 6.11 Outer membrane 231 16 342 Conserved hypothetical protein bp 3441 19.8 5.1 Cytoplasmatic/membrane* 119 6 513 Hypothetical protein bp 3128 68.7 5.9 Unknown 83 5 144 Putative lipoprotein bp 1296 30.6 7.4 Unknown 159 10 425 Succinate deshydrogenase catalytic

subunitbp 2360 27.2 6.2 Cytoplasmic 92 5 20

6 Outer membrane protein OMPQ bp 3405 39.1 5.7 Outer membrane* 87 5 197 Outer membrane porin protein

precursorbp 0840 41 5.4 Outer membrane 216 13 58

8 Putative ABC transport ATP bindingprotein

bp 3757 29.6 5.1 Unknown 102 9 54

9 N utilization protein A bp 1246 55 4.55 Cytoplasmic 120 10 2710 Argininsuccinate synthase bp 3537 49.3 5.2 Unknown 98 7 2511 Putative membrane protein bp 1440 33.4 5.3 Unknown* 247 14 5212 Glutathione synthetase bp 1499 34.8 5.4 Cytoplasmic 91 8 3713 Serin protease bp 2434 52.1 8.8 Periplasmic 140 9 3014 Chaperonin 60 kDa bp 3495 57.4 4.9 Cytoplasmic** 139 5 2815 Serum resistance protein bp 3494 103.3 7.1 Outer membrane 137 16 3016 Elongation factor Tu bp 0007 42.9 5.1 Cytoplasmatic** 82 6 2617 Fructose-biphosphate aldolase bp 1519 38.5 5.6 Cytoplasmic 71 5 2218 DNA direct RNA � subunit polymerase bp 3642 36.1 5.7 Cytoplasmatic 146 9 4619 Pertactin bp 1054 93.4 10 Outer membrane 144 18 4720 Putative quino protein bp 2196 40 8.7 Unknown 167 14 4721 ATP dependent protease, ATPase

subunitbp 1198 96.3 5.3 Cytoplasmic 74 6 11

22 Polysaccharide biosynthesis protein bp 3150 46.7 5.6 Cytoplasmic 80 7 1823 Outer membrane protein A precursor bp 0943 20.9 9.2 Outer membrane* 122 6 5124 Superoxide dismutase bp 2761 21.2 6.5 Unknown 83 4 3025 Asparate aminotransferase bp 1795 43.2 6.1 Cytoplasmic 181 11 3926 Putative binding protein dependent

transporter proteinbp 3322 40.9 6.9 Periplasmic 132 8 36

27 Dihydrolipoamide acetyltransferase bp 1125 41.7 5.3 Cytoplasmic 72 5 2428 Putative exported solute binding bp 2963 40.4 8.2 Cytoplasmatic/membrane* 99 6 23

4.57.16.5

4.97.76.5

3 5.83 4.7

4.966.2

protein29 Enolase bp 2386 45.930 Serum resistance protein bp 3494 103.331 Succinate deshydrogenase flavo

subunitbp 2361 64.8

32 Alkyl hydroperoxide reductase bp 3552 20.133 Lipoprotein bp 2750 23.134 Putative ABC transport solute binding

proteinbp 2747 40.6

5- Enoyl-acyl carrier protein bp 3215 27.66- Hypothetical protein bp 3559 37.937 Trigger factor bp 1774 47.538 Hypothetical protein bp 1203 42.739 Putative outer membrane ligand

binding proteinbp 1112 137

40 Aconitate hydratase bp 2014 98 6.941 Hypothetical protein BP2818 bp 2818 28 7.842 Transcription antitermination protein

NusGbp 0009 20 5.8

43 Succinyl-CoA synthetase subunit beta bp 2541 41 5.2

Ref. * signal peptide present, ** membrane associated in other bacteria.a Gene loci are named according to NCBI (http://www.ncbi.nlm.nih.gov/).b Protein localization is as predicted by PSORT (http://psort.nibb.ac.jp).

signal peptide) localization, three with periplasmic localization,one with cytoplasmic/periplasmic localization, 11 had unknownorigin (two having signal peptide) and 18 with cytoplasmatic local-ization (Table 1). From this last group, some of the proteins such asEF-Tu, enolase, 60-kDa chaperonin and some components of themultienzyme pyruvate deshydrogenase complex may be associ-ated to membranes as was observed before in Neisseria meningitidis[26–31].

As we described previously for B. pertussis enriched membraneprotein samples [25] some of the proteins separated by 2D elec-trophoresis were present as multiple spots exhibiting variabilityin pI values (horizontal spot patterns) (Fig. 2). Charge variantsincluded EF-Tu, 60-kDa chaperonin, outer membrane porin pro-

Cytoplasmatic** 128 11 35Outer membrane 178 5 18Cytoplasmic/periplasmic 159 5 36

Cytoplasmic 85 4 40Unknown 87 4 26Periplasmic 82 7 24

Unknown 89 6 25Cytoplasmic 87 9 36Cytoplasmic 82 8 25Unknown 87 5 17

6 Outer membrane 75 13 8

4 Cytoplasmic 158 4 22 Unknown 107 21 67 Cytoplasmic 108 33 5

3 Cytoplasmic 78 13 5

tein precursor, serum resistance protein, and serine protease. Thesemay represent either natural isoforms or an artifact caused bysample preparation or two-dimensional electrophoresis. Similarly,serum resistance protein was resolved in multiple spots of differingmasses and pIs, suggesting possible protein processing, degrada-tion and/or modifications (Fig. 2).

The predominant outer membrane proteins detected were thosethat have been well established as the major components of theouter membrane, namely, outer membrane porin precursor (spot7), OMPQ (spot 6) and pertactin (spot 19). This last protein is awell-known protective immunogen used as a constituent of manydifferent acellular vaccines [32,33]. Other identified proteins wouldbe involved in carbohydrate metabolism and some of them were

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recently described in other gram-negative bacteria as having rolesin pathogenicity [34] and immunogenicity [35–37].

The use of mass spectrometry and western blot allowed us toconfirm the presence of known pertussis immunogens in OMVstogether with a variety of putatively important antigens. The struc-ture of OMVs allows membrane proteins to keep their native foldingand consequently the elicited immune response will preferentiallytarget surface-exposed epitopes in their native conformation. Fromthe vaccinal point of view, another interesting feature of OMVsis their inherent multivalency: protection could be obtained by acooperative effect of the responses to multiple antigens, each ofwhich may be insufficient to confer immunity when administeredalone.

3.2. Protection against intranasal B. pertussis challenge aftervaccination with OMVs

3.2.1. Intraperitoneal administration of OMVsIn order to evaluate the protection capacity induced by OMVs,

animal assays using intranasal B. pertussis challenge were per-formed. In particular, the effect on subsequent colonization by theWHO reference strain Bp 18323 (2 × 108 CFU/50 �l) after two i.p.administrations of OMVs derived from Tohama was analyzed. Theresults obtained were compared with those obtained in mice i.p.immunized with whole-cell B. pertussis Tohama pertussis vaccine(wP) used as a positive control. Both wP and OMV vaccines weredetoxified by the formalin treatment described in Section 2. In allcases alum was added as adjuvant. Animals vaccinated twice withPBS were used as negative control.

Two hours later and at days 5 and 8 after challenge, the lungswere collected for bacterial counting. As expected, significant dif-ferences between immunized animals and control group wereobserved (p < 0.001) (Fig. 3). Adequate elimination rates (p < 0.005)were observed in mice immunized with both types of vaccines.While in mice immunized with PBS the number of recoveredcolonies from lungs until day 8 after challenge was similar to thosefound 2 h after challenge, in mice immunized with either wP vac-cine or OMVs, the number of lung recovered colonies at day 5post-challenge dropped at least four orders of magnitude in rela-tion to the counting on day 0. At day 8, even less colonies wererecovered in both cases (Fig. 3). The mouse weight gain test is usu-ally employed to measure the toxicity of pertussis formulations.Results of mouse weight gain test obtained with OMV formula-tion were compared with those of whole-cell vaccine used for theNational schedule as well as with wP. Both pertussis formulationspassed toxicity criteria. Though all vaccinated mice either with wPvaccine or OMV gained weight at day 3, higher weight gain was seenin OMV-vaccinated mice (weight gain was 11 ± 2% for OMV group,3 ± 2% for the commercial vaccine, 8 ± 3% for wP lab-made formula-tion and 13 ± 3% for control mice). Altogether, data presented here

Fig. 4. Levels of inflammatory chemokines and cytokines mRNA in lungs of mice intranasof five mice per group at each time. Panel A, mRNA levels of CCL20. Panel B, mRNA levels

6 (2008) 4639–4646

Fig. 3. Effect of intraperitoneal immunization with B. pertussis OMVs and killedwhole-cell bacteria vaccine in the mouse intranasal challenge model. The intranasalchallenge was performed with the WHO reference strain B. pertussis 18323. Threeindependent experiments were performed. Results from one a representative exper-iment are shown. Results depicted are mean of four mice per group at each time.

supports the use of OMVs as intraperitoneal acellular vaccine forprevention of pertussis infection.

3.2.2. Intranasal administration of OMVsIn order to analyze the possibility to use OMVs as intranasal

vaccine, we evaluated first the capacity of such material to triggerairways innate response by measuring the induction of expressionof several typical markers of innate response at the RNA level. Thisroute mimics closely the natural B. pertussis infection which hasbeen considered to induce strong and long lasting immunity. Inthese experiments killed B. pertussis whole cell was used as positivecontrol. In particular TNF�, IL-6 and CCL20, levels were evaluated at

2 and 8 h after intranasal stimulation with either B. pertussis wholecells or OMVs. In all analyzed cases, upregulation of TNF�, IL-6 andCCL20 was detected with highest levels of expression after 2 h stim-ulation, ranging from 10- to 20-fold increase followed by a quickshutdown of the response with significantly lower levels at 8 h post-treatment (Fig. 4), and almost similar to basal expression 24 h afterstimulation (not shown). At the early time points (2 h after activa-tion) we observed that OMV intranasal delivery was able to inducesimilar or higher levels of such different innate response markersthan whole-cell stimulation. In summary, a clear induction of allinnate response markers studied was observed for intranasal OMVadministration.

The detoxification process that is usually used in anti-pertussisvaccine production may affect the innate response since it consistin a formaldehyde treatment that reduces the reactogenicity of thevaccine preparation mainly by crosslinking agonists such as LOS, apotent inducer of innate response. However, no significant differ-ences were observed in the capacity of triggering innate responsebetween crude and detoxified OMVs (data not shown), indicatingthat in spite of formaldehyde treatment, there is a residual lung-activating biological activity.

ally treated either with killed whole-cell bacteria, OMVs or PBS. Results are meansof IL-6. Panel C, mRNA levels of TNF�. *p ≤ 0,05, **p ≤ 0,01, ***p ≤ 0,001.

cine 26

sis To

cteria

y 0

05 ± 023 ± 055 ± 0

R. Roberts et al. / Vac

Table 2Lung clearance efficiency of OMVs in the intranasal challenge model using B. pertus

Immunization Challenge strain Ba

Da

PBSbp Tohama

6.OMVs (i.n.) 6.Killed whole-cell bacteria (i.n.) 6.

aCFU were not recovered.

Since we have evidence that OMV administration can inducelung innate response and we have also shown that OMV con-tain pertussis immunogens that can confer protection by systemicroute, we decided to test the protective capacity of OMVs as amucosal vaccine against pertussis with no added adjuvant. To thisaim two i.n. immunizations of mice (3–4-week-old BALB/c) witheither OMV vaccine or B. pertussis whole-cell vaccine derived bothfrom Tohama strain were performed. Immunized mice were thenintranasally challenged with sublethal (108 CFU in 50 �l) doses ofthe WHO reference strain B. pertussis 18323. Animals vaccinatedwith PBS were used as control. As expected, significant differencesbetween immunized animals and the control group were observed(p < 0.001, Fig. 5). Adequate elimination rates were observed in miceimmunized either with whole-cell bacteria or OMVs. While in miceimmunized with PBS, the number of recovered colonies from lungsuntil day 8 after challenge was similar to those found at 2 h afterchallenge, in mice immunized with either whole-cell vaccine or

OMVs, the number of lung recovered colonies at days 5 and 8 post-challenge dropped two and three orders of magnitude, respectivelywith respect to the counting at day 0.

To analyze the toxicity of OMV when they were used throughi.n. route, weight gain test was performed as described above. Micewere weighed 16 h and 3 days after immunization. Paired sampletesting revealed weight loss of OMVs treated mice at 16 h post-immunization. Weight gains at 3 days were 8, 14 and 19% forOMV, wP lab-made formulation and control groups, respectively.Although this indicates that the formulation used is acceptableaccording to the WHO requirements, improvement in the for-mulation could be done to reach at least reactogenicity levels ofwhole-cell vaccine. Different strategies such as producing OMVsfrom LOS-deficient strains and/or supplementing with non-toxicLOS analogues [38] can be envisaged to optimize the safety for i.n.administration.

As the OMVs were prepared from Tohama strain we also ana-lyzed the OMVs intranasal protective capacity against Tohamavaccine strain. In this case the results obtained were comparablewith those employing the WHO reference strain in terms of protec-tion since in both cases OMVs protected against infection. However,

Fig. 5. Effect of intranasal immunization with B. pertussis OMVs and killed whole-cell B. pertussis bacteria vaccine in the mouse intranasal challenge model. The WHOreference strain B. pertussis 18323 was used as challenge bacteria. Three independentexperiments were performed. Results from one a representative experiment areshown. Results depicted are means of four mice per group at each time.

(2008) 4639–4646 4645

hama strain

recovered after challenge (Log10 CFU/lung ± S.D.)

Day 5 Day 8

.28 5.94 ± 0.40 2.54 ± 2.44

.12 2.10 ± .81 a

.43 3.31 ± 0.90 1.14 ± 1.59

a higher elimination rate was observed when the challenge wasdone with B. pertussis Tohama strain compared to the eliminationrates of WHO reference strain (p < 0.001, Table 2). This result mightbe explained at least in part because the WHO reference strain(18323) [39] expresses different variants of polymorphic antigens(i.e. pertactin type A with a second modification in region II and adifferent S1 type from the other strains (S1E)) compared to Tohamastrain and as it was reported this difference might have implicationsin protection at least in the mice model employed [25].

Compared to intraperitoneal route a slightly difference ininduced protective immunity was observed for intranasal formu-lation either for the case of OMV or for whole-cell formulation(p < 0.05). One possible explanation might be that for intraperi-toneal immunization vaccines, were prepared adding aluminumhydroxide as adjuvant while for intranasal formulation no adjuvantwas added.

Altogether, data presented here supports the use of OMVs asacellular vaccine for prevention of pertussis infection. OMV-basedvaccines possess inherent benefits including: (i) main immunogensused in acellular vaccines are present in the context of the pertussismembrane, thus allowing the immune response to preferentiallytarget surface-exposed epitopes in their native conformation, (ii)the vaccine should afford protection by a cooperative effect of theresponses to multiple antigens, each of which may be insufficient toconfer immunity when administered alone, and (iii) the inclusion ofmultiple antigens instead of few may reduces the likelihood that thepropensity to undergo antigenic variation will result in simultane-ous loss of all epitopes targeted by the protective immune response.All these characteristics differentiate the OMVs from soluble com-ponents of the currently acellular pertussis vaccines.

The use of OMVs derived from other microorganism suchas Neisseria has already been proposed as vaccine to preventmeningococcal bacteraemia based on the properties describedabove [40,41]. The availability of new vaccine formulations offers

new opportunities to be considered in an attempt to improve thecontrol of B. pertussis infection in the very youngs but also in ado-lescents and adults.

Acknowledgements

This work was supported by ANCPyT and CICBA (Argentina)grants to DFH and INCO CT-2006-032296 grant to MR and DFH.DFH is a member of the Scientific Career of CICBA. MR is memberof the Scientific Career of CONICET. MF, DB, GM and EG have fel-lowships from CONICET. AG has a fellowship from ANCPyT and RRis supported by an INCO (EU) grant.

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