The Protein Moiety of Brucella abortus Outer Membrane Protein … · 2017-06-09 · The Journal of...

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and Oral Acquired BrucellosisSelf-Adjuvanting Vaccine against Systemic Th1 Immune Response, and Is a PromisingActivates Dendritic Cells In Vivo, Induces a Pathogen-Associated Molecular Pattern ThatMembrane Protein 16 Is a New Bacterial

OuterBrucella abortusThe Protein Moiety of

Juliana CassataroHeribert Warzecha, Guillermo H. Giambartolomei and Barbosa Carvalho, Julia Borkowski, Sergio Costa Oliveira,Paula Barrionuevo, Fernanda Souza de Oliveira, Natalia

Ibañez,Coria, Silvia M. Estein, Astrid Zwerdling, Andrés E. Karina A. Pasquevich, Clara García Samartino, Lorena M.

http://www.jimmunol.org/content/184/9/5200doi: 10.4049/jimmunol.0902209March 2010;

2010; 184:5200-5212; Prepublished online 29J Immunol

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The Journal of Immunology

The Protein Moiety of Brucella abortus Outer MembraneProtein 16 Is a New Bacterial Pathogen-Associated MolecularPattern That Activates Dendritic Cells In Vivo, Induces a Th1Immune Response, and Is a Promising Self-AdjuvantingVaccine against Systemic and Oral Acquired Brucellosis

Karina A. Pasquevich,*,† Clara Garcıa Samartino,*,† Lorena M. Coria,*,† Silvia M. Estein,‡

Astrid Zwerdling,*,† Andres E. Ibanez,*,† Paula Barrionuevo,*,† Fernanda Souza de Oliveira,x

Natalia Barbosa Carvalho,x Julia Borkowski,{ Sergio Costa Oliveira,x Heribert Warzecha,{

Guillermo H. Giambartolomei,*,† and Juliana Cassataro*,†

Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus

outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external

adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus

infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the firstBrucella protein

thatwithout the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19.Moreover,

the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route.

Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is

endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to

activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells andmacrophages in vitro. The latter property and its

ability to induce a protective Th1 immune response againstB. abortus infection have been found to beTLR4 dependent. The facts that

U-Omp16 is anoral protectiveAgandpossesses amucosal self-adjuvantingproperty ledus todevelopaplant-madevaccine expressing

U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally,

indicating that a plant-based oral vaccine expressingU-Omp16 could be avaluable approach to controlling this disease. The Journal

of Immunology, 2010, 184: 5200–5212.

Brucella abortus is a zoonotic Gram-negative pathogen thatcauses abortion and infertility in ruminants. In humans, itcauses undulant fever, characterized by malaise, aches,

and fevers. Human brucellosis can be contracted by accidentalcontamination from infected animals, handling infected tissues, orconsuming undercooked meat or unpasteurized dairy productsfrom infected animals (1). Brucella is a facultative intracellularbacterial parasite; the pathogenesis of brucellosis and the nature of

the protective immune response are closely related to this property(2). Although both Ab- and cell-mediated immune responses caninfluence the course of infection with Brucella, the latter are es-sential for clearance of intracellular bacteria. In this respect, IFN-gplays a central role in acquired resistance against Brucella, upreg-ulating macrophage microbial killing (3, 4).Because brucellosis has serious medical and economic con-

sequences, prevention of animal infection by vaccination is key.

*Laboratory of Immunogenetics, Clinical Hospital Jose de San Martın, School ofMedicine and †Institute of Studies on Humoral Immunity (National Council of Re-search), School of Pharmacy and Biochemistry, University of Buenos Aires, BuenosAires; ‡Laboratory of Immunology, Department of Animal Healthcare and PreventiveMedicine, School of Veterinary Sciences, National University of the Center of theBuenos Aires Province, Tandil, Argentina; xDepartment of Biochemistry and Immu-nology, Institute of Biological Sciences, Federal University of Minas Gerais, BeloHorizonte-Minas Gerais, Brazil; and {Technische Universitat Darmstadt, Institute ofBotany, Darmstadt, Germany

Received for publication July 13, 2009. Accepted for publication February 25, 2010.

This work was supported by grants from the Agencia Nacional de Promocion Cientıfica yTecnologica:PICT2006No.1670andNo.1335 (toJ.C. andG.H.G.); fromConsejoNacionalde Investigaciones Cientificas y Tecnicas: PIP 5239 and 5213 (to J.C. and G.H.G.); fromUniversidaddeBuenosAires:M408andB819(to J.C. andG.H.G.); fromConselhoNacionalde Desenvolvimento Cientıfico e Tecnologico/Prosul No. 490485/2007-3, Conselho Nacio-naldeDesenvolvimentoCientıficoeTecnologico/AgenciaNacionaldePromocionCientıficayTecnologicaNo.490528/2008-2,andInstitutoNacionaldeCienciaeTecnologiadeVacinas(to S.C.O); and from Agencia Nacional de Promocion Cientıfica y Tecnologica/ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico PICT 2008 No. 18 (to J.C.). K.A.P.,

C.G.S., and L.M.C. are recipients of a fellowship fromConsejo Nacional de InvestigacionesCientificas y Tecnicas (Argentina). A.E.I. is a recipient of a fellowship from Agencia Na-cional de PromocionCientıfica yTecnologica (Argentina). S.M.E., P.B.,G.H.G., and J.C. aremembers of the Research Career of Consejo Nacional de Investigaciones Cientificasy Tecnicas.

Address correspondence and reprint requests to Dr. Juliana Cassataro, Laboratorio deInmunogenetica, Hospital de Clınicas Jose de San Martın, Facultad de Medicina,University of Buenos Aires, Cordoba 2351 3er Piso Sala 4 1120, Buenos Aires,Argentina. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this paper: BM, bone marrow; DC, dendritic cell; DTH, de-layed-type hypersensitivity; HS, B. ovis REO 198 hot saline extract; i.g., intragastri-cally; L-, lipidated; M, marker lane; Omp, outer membrane protein; Pam3Cys, N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine; PAMP, pathogen-associ-ated molecular pattern; PB, polymyxin B; U-, unlipidated; wt, wild-type.

Copyright� 2010 by TheAmerican Association of Immunologists, Inc. 0022-1767/10/$16.00

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Thus, efforts have beenmade to prevent the infection through the useof vaccines (3, 5). All commercially available brucellosis vaccinesare based on live, attenuated strains of Brucella. Although effective,these vaccines have disadvantages: they can be infectious for hu-mans; they can interfere with diagnosis; they may result in abor-tions when administered to pregnant animals; and the vaccine straincan spread in the region (3, 6). Currently, no vaccine against humanbrucellosis is available (7). Therefore, improved vaccines thatcombine safety and efficacy to all species at risk need to be de-signed. Within the past few years, we and others have made effortsto develop a vaccine without these drawbacks, a vaccine that wouldbe more effective and safer than those used at present (8–11).Subunit vaccines, like recombinant proteins, are promising

vaccine candidates, because they can be produced at high yield andpurity and can be manipulated to maximize desirable activities andminimize undesirable ones. Moreover, they are safer for manip-ulators, well defined, not infectious, and cannot revert to a virulentstrain. However, despite these advantages, they tend to be poorlyimmunogenic in vivo, and require the coadministration of adjuvantsthat indirectly enhance the immune response against recombinantproteins. Therefore, recombinant vaccine success is usually de-pendent on the use of substances endowed with immunomodulatoryproperties that instruct and control the selective induction of theappropriate type of Ag-specific immune response (12–14).A promising immune stimulator is the lipid moiety N-palmitoyl-

S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine (Pam3Cys). Thismoiety is found at the N terminus of bacterial lipoproteins and isubiquitous in and unique to bacteria. Synthetic peptides that are notimmunogenic themselves induce a strong Ab response when co-valently coupled to Pam3Cys (15, 16). In vitro and in vivo evidenceindicates that bacterial lipoproteins and synthetic lipoprotein ana-logs (lipopeptides) are potent activators of innate immune cells andthat these pathogen-associated molecular patterns (PAMPs) triggercellular activation by binding to pattern recognition receptors onthe surfaces of monocytes/macrophages and dendritic cells (DCs)(17, 18). Numerous studies also report different proteins with im-munoadjuvant properties, such as outer membrane protein (Omp)A from Klebsiella pneumoniae, Neisseria meningitidis porins, andthe Tat (transcriptional transactivator) protein of HIV-1. Theseproteins are able to induce strong immune responses in the absenceof external adjuvants (19–22).If we take into account that in brucellosis the infection route

usually involves the entry of the pathogen through the mucosalsurfaces (23), another key point is the induction of immune re-sponses on the mucosal surfaces. Therefore, an oral vaccine couldbe a promising candidate to control the disease or to enhance theimmune protection provided by currently available vaccines. Theprotective capacity of several vaccine preparations has alreadybeen evaluated by the oral route, including live attenuated strains(24, 25), live recombinant vectors (26, 27), and a few recombinantproteins with mucosal adjuvants (8, 28).Omp16 of Brucella spp. is a lipoprotein and is expressed in all

biovars of B. abortus, B. melitensis, B. suis, B. canis, B. ovis, andB. neotomae (29–31). We have previously reported that B. abortusOmp16 confers protection against a challenge with virulent B.abortus when administered i.p. with systemic adjuvants (IFA oraluminum hydroxide) or orally with a mucosal adjuvant (choleratoxin) (8). Omp16 is also able to activate monocytes, inducing theproduction of proinflammatory cytokines, a phenomenon medi-ated by its lipid moiety (32).We speculate that the N-terminal lipid moiety of Omp16 could

confer enough adjuvanticity to evoke a protective immune responseagainst Brucella, abolishing the need of external adjuvants. In thepresent work, we evaluate this hypothesis.

Materials and MethodsMice

Female 6- to 8-wk-old specific-pathogen-free BALB/c micewere purchasedfrom the Universidad Nacional de La Plata (La Plata, Argentina). The wild-type (wt) strain C57BL/6 mice were provided by Federal University ofMinas Gerais (Belo Horizonte, Brazil), and genetically deficient TLR22/2,TLR42/2, and TLR62/2 C57BL/6 mice were provided by S. Akira (OsakaUniversity, Osaka, Japan). Mice were housed in appropriate conventionalanimal care facilities and handled according to international guidelinesrequired for animal experiments. Animals were used in the experiments 8–10 wk postbirth.

Bacteria

The virulent B. abortus 544 and B. abortus 2308 (used in the protectionexperiments), B. ovis REO 198 (used for production of the antigenicpreparation B. ovis REO 198 hot saline extract [HS]), and the vaccinestrain B. abortus S19 and B. abortus RB51 were obtained from our ownlaboratory collection (8, 33). Bacterial growth and inocula preparationwere performed as previously described (33, 34). All live Brucella ma-nipulations were conducted in biosafety level 3 facilities.

Ags

The recombinant lipidated (L-) and unlipidated (U-) Omp16 proteins weremanufactured as previously described (8, 32). Briefly, recombinantL-Omp16 was isolated from bacterial membranes, and U-Omp16 wasisolated from bacterial cytoplasm and then purified by affinity chroma-tography with an Ni-NTA resin (Qiagen, Dorking, U.K.). Expression andpurification of the recombinant proteins were checked by SDS-PAGE,followed by silver staining. To confirm the identity of the Omp16, Westernblot was performed and developed with anti–Omp16-specific mAb (datanot shown). Protein concentration was determined by the bicinchoninicacid assay with BSA as a standard (Pierce, Rockford, IL). LPS contami-nation was adsorbed with Sepharose-polymyxin B (PB) (Sigma-Aldrich,St. Louis, MO). Endotoxin determination was performed with Limulusamoebocyte assay (Associates of Cape Cod, Woods Hole, MA). All proteinpreparations contained ,0.25 endotoxin U/mg protein.

HS was obtained as previously described (34). In some experiments, ascontrol, a U-Omp16 enzymatically digested preparation was used. U-Omp16 was treated with proteinase K-agarose from Tricirachium album(Sigma-Aldrich) for 2 h at 37˚C, following manufacturer indications. Theenzyme immobilized in agarose was then centrifuged out (2000 3 g, 5min), and the supernatants were incubated for 1 h at 60˚C to inactivate anyfraction of soluble enzyme. The complete digestion of the proteins waschecked by SDS-PAGE, followed by Coomassie blue staining.

Immunizations and experimental design

Groups of 5–8 mice were anesthetized with methoxyflurane (Mallinckrodt,Phillipsburg, NJ) and then immunized.

Some groups of mice were immunized on days 0 and 15 by the i.p. routewith 30 mg of 1) L-Omp16, 2) U-Omp16 in PBS, 3) U-Omp16 emulsifiedin IFA, or 4) PBS-immunized mice as control. As positive control in theprotection experiments, a group of mice received a single dose i.p. of 1 3104 CFU live B. abortus strain 19.

Other groups of mice were intragastrically (i.g.) immunized with threeconsecutive weekly doses of U-Omp16 (100 mg) or PBS in 200 ml 0.1 Mbicarbonate buffer (pH 8), as previously described (8, 28). As positivecontrol, a group of mice received a single dose of live B. abortus RB51(0.5 3 109 UFC) by the i.g. route.

At 30 d after the last immunization, mice were challenged with virulentB. abortus for protection experiments or were sacrificed to perform cellularin vitro experiments; other groups of immunized mice were injected intheir footpads to perform the delayed-type hypersensitivity (DTH) test.

Sera were obtained from blood samples on days 0, 15, 30, and 45 after thefirst immunization.

Brucella challenge and protective response evaluation

At 1 mo after the last immunization, mice were challenged i.p. with 43 104

CFU live B. abortus 544 or 2308 or i.g. with 3 3 108 CFU B. abortus2308. At 1 mo postchallenge, their spleens where aseptically removed,homogenized in sterile PBS, diluted, plated, and incubated as described (8,33), and the number of B. abortus 544 or 2308 CFU was counted. Resultswere represented as the mean log10 CFU 6 SD per group. Units of pro-tection were calculated as the difference between the mean log10 CFUfrom the PBS group and the mean log10 CFU from the experimental group.

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In vitro cellular responses

Single spleen cell suspensions from immunized and control mice werecultured in duplicate at 4 3 106 cells/ml in RPMI 1640 (Life TechnologiesBRL, Grand Island, NY) supplemented with 10% FCS (Life Technolo-gies), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U penicillin/ml,and 100 mg streptomycin/ml (complete medium) with stimuli. The dif-ferent stimuli were HS (20 mg/ml), U-Omp16 (50 mg/ml), or completemedium alone. When HS was used as the stimulus, the complete mediumalso contained PB (15 mg/ml; Sigma-Aldrich). After 72 h incubation at37˚C in a humidified atmosphere (5% CO2 and 95% air), cell culturesupernatants were collected. IFN-g, IL-2, IL-4, IL-5, and IL-10 productionwas analyzed by sandwich ELISA, using paired cytokine-specific mAbsaccording to the manufacturer’s instructions (Pharmingen, San Diego,CA). In some experiments, splenocytes were depleted of CD4+ or CD8+

T cells, using mouse CD4 (L3T4) or mouse CD8 (Lyt2) Dynabeads ac-cording to the manufacturer’s instructions (Dynal Biotech, Oslo, Norway).Depletion was performed before antigenic stimulation. Each T cell pop-ulation was depleted with an efficacy greater than 99%, as determined byflow cytometry analysis (not shown). After being depleted, cells weresuspended in the original volume of complete medium and used in thein vitro stimulation assay.

DTH test

At 4 wk after the last i.g. immunization, mice were injected intradermally inone footpad with 30 mg U-Omp16 and in the contralateral footpad with anequal volume of saline, as negative control. The footpad thickness wasmeasured 24, 48, and 72 h later by using a digital caliper with a precision of0.01 mm, and the mean increase in footpad thickness (expressed in mm)wascalculated according to the following formula: (footpad thickness)U-Omp162(footpad thickness)saline.

In vivo analysis of DC activation

In vivo induction of DC activation was evaluated by measuring the ex-pression of various surface markers by flow cytometry. BALB/c mice wereinjected i.v. with 100 mg U-Omp16, either untreated or digested withproteinase K; with 50 mg Escherichia coli LPS (Sigma-Aldrich); or withPBS alone. At 20 h postinjection, mice were sacrificed, and spleens frommice were removed and treated for 45 min at 37˚C with 400 U/ml colla-genase type IV and 50 mg/ml DNase I (Boehringer Mannheim, Indian-apolis, IN) in RPMI 1640. After inhibition of collagenase with 6 mMEDTA and 0.5% FCS (Invitrogen Life Technologies, Carlsbad, CA),a single spleen cell suspension was prepared. Spleen cells were incubatedfor 20–45 min at 4˚C in 5% mouse serum in the presence of primary Abs:FITC-, PE-, CyChrome- or PE-Cy5–conjugated Abs specific for CD8a,CD11c, CD40, CD80, CD86, or isotype control. mAbs were purchasedfrom eBioscience (San Diego, CA)and BD Biosciences (Franklin Lakes,NJ). After staining, cells were fixed and analyzed by flow cytometry, usinga FACSAria II (BD Biosciences). Data were analyzed using FlowJo soft-ware (Tree Star, Ashland, OR).

In vitro activation of bone marrow-derived DCs andmacrophages

DCs and macrophages were generated from bone marrow (BM) mono-nuclear cells from wt, TLR22/2, TLR42/2, or TLR62/2 C57BL/6 mice, inmedium containing rGM-CSF, as previously described (35, 36). Briefly,femora and tibiae were collected from 4- to 12-wk-old mice. After removalof adjacent muscles, bones were flushed throughout their interior with 5 mlHBSS to extract marrow cells. The marrow suspension was filtered through70-mm cell strainers (Falcon; BD Biosciences) to remove unwantedfragments. To obtain BM-derived DCs, BM cells were centrifuged andplated on 100-mm petri dishes (13 107cells/10 ml) in DC culture medium(complete medium, 50 mM 2-ME, 200 U GM-CSF). On days 2 and 4,fresh DC culture medium was added. On day 5, cells were plated on 6-wellplates (3 3 106 cells/well) in 3 ml fresh DC culture medium. Afterovernight culture, on day 6, cells demonstrated differentiated morphologyand expressed DC markers (CD11c+, MHC class II (MHCII)low, andCD40low; data not shown).

BM-derived macrophages were obtained as previously described (37).Briefly, BM cell suspension was centrifuged, and cells were resuspended inDMEM (Life Technologies) containing 10% FCS and 10% L929 cell-conditioned medium as a source of M-CSF. Cells were plated in 24-wellplates and incubated at 37˚C in a 5% CO2 atmosphere. Three days afterseeding, another 0.1 ml L929 cell-conditioned medium was added. On day7, the medium was renewed. On day 10 of culture, cells were completelydifferentiated into macrophages.

Differentiated BM-derived DCs or macrophages were stimulated for 24 hwith complete medium alone or complete mediumwith: U-Omp16 (1, 5, 10,or 50 mg/ml), E. coli LPS (1 mg/ml), or Pam3Cys (1 mg/ml) (InvivoGen,San Diego, CA). A plant-made U-Omp16 was used as the stimulant proteinin some assays. For control groups, in some experiments, cells werestimulated either with proteinase K-digested U-Omp16 (50 mg/ml) or withU-Omp16 (50 mg/ml) plus PB (15 mg/ml). After stimulation, cell-freesupernatants were collected and assayed by sandwich ELISA for TNF-aand IL-12 (p40) production, using paired cytokine-specific mAbs accord-ing to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Transient expression of U-Omp16 inNicotiana benthamina plants

The sequence of omp16 lacking the segment coding for the N-terminalsignal peptide was amplified by PCR, using primers PO16-102 (59-TTTG-GTCTCAAGGTATGAACCTTCCGAATAATGCCGGTGATCTGGGAC-TCG-39) and PO16-201 (59-GCTCTAGATCAGTGGTGGTGGTGGTGG-TGCTC-39). Primer PO16-201 also included the nucleotides encoding fora 63histidine extension thatwas alreadypresent in the templatevector [pET-U-Omp16 (32)]. The PCR product was cloned into the vector pCRblunt(Invitrogen, Karlsruhe, Germany), and its identity was confirmed by se-quencing. For subcloning, a BsaI–XbaI fragment from this vector was in-serted into the vector pICH10990 (38); the resulting vector was termedPO16-6121 and mobilized into agrobacteria by chemical transformation(39). Infiltration of N. benthamina plants was carried out as described byMarillonnet et al. (38).

Plant-expressed protein extraction, purification, andimmunoblot analysis

For rapidproteindetection,100–200mg frozenplantmaterialwasgroundand200ml sample buffer (0.125MTris-Cl, pH 6.8; 20% [v/v] glycerin; 4% [v/v]SDS; and 0.02% [w/v] bromophenol blue) was added. Samples were heatedat 95˚C for 10 min and subjected to SDS-PAGE (15%; see below).

For extraction of total plant protein, 100–200 mg frozen plant materialwas thoroughly ground, mixed with 200 ml extraction buffer (23 mMNa2HPO4; 17 mM NaH2PO4; and 100 mM NaCl, pH 7.4), and incubatedon ice for 20 min. After centrifugation at 4˚C and maximum speed for20 min, the supernatant was collected and centrifuged for a second timeunder the same conditions.

Recombinant protein was purified from plant material, using the ProtinoProtein Purification SystemTM (Ni-TED 2000 packed columns; Macherey-Nagel, Duren, Germany) according to the instructions. Purified protein wasdesalted and concentrated, using Amicon Ultra centrifugal filter devices(Millipore, Carrigtwohill, Ireland) according to the supplier’s protocol.Total soluble protein and purified protein concentration were determinedwith the BCA Protein Assay Kit (Pierce).

Defined amounts of total plant protein (0.1 mg, 0.5 mg, 1 mg, 5 mg, and15 mg) and of purified Omp16 (0.05 mg, 0.1 mg, and 0.25mg) in a volume of20ml weremixedwith 5ml 53 SDS sample buffer (250mMTris-Cl, pH 6.8;10% [v/v] SDS; 0.05% [w/v] bromophenol blue; and 50% [v/v] glycerin).Samples were heated at 95˚C for 10min and subjected to SDS-PAGE (15%).Electrophoresis was performed in 13 SDS running buffer (25 mM Tris, pH8.3; 192mMglycine; and0.1% [v/v] SDS)with 40V for 1 h and 100V for theremaining run time. After electrophoresis, the gels were equilibrated intransfer buffer (25 mM Tris; 192 mM glycine; 20% [v/v] methanol) for10 min, and proteins were transferred to a polyvinylidenedifluoride mem-brane (Hybond-P; Amersham Biosciences, Freiburg, Germany), using Pe-qulab tank-blot equipment (Pequlab, Erlangen, Germany) at 100 V for 1 h.The membrane was blocked in 5% (w/v) dried milk powder in 1 3 PBS-T(5.6 mMNa2HPO43 12 H2O; 2.9 mMK2HPO4; 2.9 mMNaCl; 0.05% [v/v]Tween 20; and pH7.2–7.4). Subsequently, themembranewas incubatedwiththe primaryAb (murine anti-Omp16mAb) at a dilution 1:2000 in 13 PBS-Tfor 1 h at room temperature. After washing three times in 1 3 PBS-T for10 min, the membrane was incubated with a HRP-conjugated secondary Ab(goat anti-mouse IgG-HRP; Santa Cruz Biotechnology, Heidelberg, Ger-many; dilution 1:10000 in 13 PBS-T) for 1 h at room temperature. Washingwas repeated as described, and immunoblots were developed using theChemiluminescence Detection Kit (AppliChem, Darmstadt, Germany) ac-cording to the supplier’s instructions.

Sequence analysis

The amino acid sequence of U-Omp16 was used to perform searches ofnonredundant protein databases. The BLAST program available at www.ncbi.nlm.nih.gov/BLAST/ and also the Phylogeny.fr BLAST platform atwww.phylogeny.fr/version2_cgi/index.cgi were run (using MUSCLE formultiple alignment and PhyML for phylogeny) (40, 41).

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Statistical analysis

Statistical analysis and plotting were performed using GraphPad Prism 4software (GraphPad, San Diego, CA). Protection results were evaluatedusing one-way ANOVA and the Dunnett multiple-comparison posttest. Thedata from cytokine production, DTH response, and flow cytometry wereanalyzed using one-way ANOVA with Bonferroni’s posttest. When datawere not normally distributed, a logarithmic transformation was appliedprior to the analysis; then parameters showed normal distribution. A pvalue ,0.05 was taken as the level of significance.

ResultsOmp16 confers protection against B. abortus challengewithout the need of external adjuvants or its lipid moiety

As stated before, it was already described that the lipid moiety oflipoproteins has adjuvant activity per se (42). To address whetherthis was the case for the B. abortus Omp16 lipoprotein, a group ofmice was immunized i.p. with the lipoprotein alone, whereas othergroups of animals were immunized with PBS (negative control) orthe vaccine strain B. abortus S19 (positive control). Immunizedmice were challenged with virulent B. abortus 544, and the po-tential of the L-Omp16 per se to impart protection without ad-juvants was evaluated on the basis of its ability to eliminate thebacterial burden (B. abortus 544) in spleens of immunized mice atday 30 postchallenge. Immunization with L-Omp16 without ad-juvants induced a significant level of protection (1.49 U of pro-tection, p , 0.01) when compared with the control PBS-immunized mice (Table I). This result indicates that L-Omp16 isendowed with an intrinsic adjuvant activity that allows it to conferprotection against a challenge with virulent B. abortus, without theneed for external adjuvants.To corroborate the finding that the intrinsic adjuvanticity of

L-Omp16 was due to its lipid moiety, another experiment has beenconducted in which mice were immunized with L-Omp16 or withU-Omp16, in both cases without adjuvants. As before, L-Omp16conferred protection (1.59 U of protection, p, 0.01versus PBS), butunexpectedly U-Omp16 also induced significant protection levelsagainst an experimental B. abortus infection (1.68 U of protection, p, 0.01 versus PBS). Notably, the protection levels elicited by U-Omp16 were similar to those elicited by its respective lipidatedversion (p . 0.05), indicating that the molecular region responsiblefor the observed self-adjuvanticity is the protein portion of this li-poprotein. It is worth mentioning that the protection levels elicitedby U-Omp16 were similar to those elicited by the attenuated vac-cine strain B. abortus S19 (p . 0.05) (Table II).It is important to bear in mind that the purification yield of

L-Omp16 is much lower than that obtained when producingU-Omp16. Moreover, it is necessary to make extra LPS depletionrounds to get the recombinant lipoprotein LPS-free (8). Those as-pects and its self-adjuvanticity led us to focus on the protein region

of Omp16. Besides, the addition of an external adjuvant, like IFA,did not improve the U-Omp16–elicited protective responses (U-Omp16 + IFA: 1.46 versus U-Omp16: 1.33 U of protection; p .0.05) (Table III).Altogether, these results suggest that the protein portion of the B.

abortus Omp16 has an inherent adjuvant activity that allows it toinduce a protective immune response against an in vivo Brucellachallenge.

Immunization with U-Omp16 induces a specific Th1 cellularimmune response

Given the intracellular nature of Brucella spp., it is well known thatcellular immune responses (in particular, Th1) are crucial forconferring protection against this pathogen (43). Thus, we decidedto test whether immunization with U-Omp16 without adjuvants isable to induce a specific cellular immune response. Splenocytesfrom U-Omp16–immunized mice were stimulated in vitro witha known Brucella membrane extract that contains native Omp16(HS). After stimulation, splenocytes from U-Omp16–immunizedmice produced large amounts of IFN-g in comparisonwith the samesplenocytes incubated with complete medium alone (p , 0.01).This effect was specific, because spleen cells from U-Omp16–immunized mice produced higher levels of IFN-g than did cellsfrom PBS-immunized mice in response to the same stimulus (p ,0.01) (Fig. 1A). The splenocytes from both groups of mice did notproduce IL-2, IL-4, IL-5, or IL-10 in response to HS (data notshown). To evaluate which cells were responsible for the specificIFN-g production, we selectively depleted CD4+ T cells or CD8+

T from whole splenocytes. When splenocytes from U-Omp16–immunized mice were depleted of the CD4+ T cell population, thespecific IFN-g production in response to HS was abrogated (p ,0.01 versus not depleted) (Fig. 1B). On the contrary, CD8+ T cellpopulation depletion did not affect the specific IFN-g production(Fig. 1B). This result indicates that U-Omp16 immunization

Table I. Protection against B. abortus 544 in BALB/c mice immunizedwith L-Omp16 without adjuvant

Vaccine (n = 6) AdjuvantLog10a CFU of B. abortus544 at Spleen (mean 6 SD)

Units ofProtection

L-Omp16 None 4.25 6 0.44b,c 1.49B. abortus strain 19 None 3.71 6 0.17b 2.03PBS None 5.74 6 0.15c 0

aThe content of bacteria in spleens is represented as the mean log CFU 6 SD pergroup.

bSignificantly different from PBS-immunized mice; p , 0.01 estimated by Dun-nett´s test.

cSignificantly different from B. abortus strain 19 immunized mice; p , 0.01estimated by Dunnett´s test.

Table II. Protection against B. abortus 544 in BALB/c mice immunizedwith L-Omp16 or U-Omp16 without adjuvant

Vaccine (n = 5) AdjuvantLog10

a CFU of B. abortus544 at Spleen (mean 6 SD)

Units ofProtection

L-Omp16 None 4.68 6 0.55b,d 1.59U-Omp16 None 4.59 6 0.42b 1.68B. abortus strain 19 None 3.98 6 0.26b 2.29PBS None 6.27 6 0.14c 0

aThe content of bacteria in spleens is represented as the mean log10 CFU 6 SDper group.


cSignificantly different from B. abortus strain 19 immunized mice; p , 0.01estimated by Dunnett´s test.

dSignificantly different from B. abortus strain 19 immunized mice; p , 0.05estimated by Dunnett´s test.

Table III. Protection against B. abortus 544 in BALB/c mice immunizedwith U-Omp16 without adjuvant or in IFA

Vaccine (n = 5) AdjuvantLog10

a CFU of B. abortus544 at Spleen (mean 6 SD)

Units ofProtection

U-Omp16 None 4.37 6 0.14b 1.33U-Omp16 IFA 4.24 6 0.18b 1.46PBS None 5.70 6 0.20 0





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without adjuvants induces specific CD4+ T cells that are the majorIFN-g producers in response to HS.Incontrast, immunizationwithU-Omp16without adjuvantsdidnot

elicit a specific humoral immune response, inducing nearly un-detectableanti-Omp16immunoglobulintitersinsera(datanotshown).Taken together, these results indicate that the protein region of

the Omp16 lipoprotein has the ability to induce a Th1 immuneresponse when delivered i.p. without adjuvants.

Oral delivery of U-Omp16 confers protection againstB. abortus challenge and elicits a Th1 immune responsewithout the need of external adjuvants

Oral infection is one of the principal ways in which the disease isacquired, in both humans and animals (23, 28). For that reason, wesought to evaluate the protection afforded when mice were im-munized i.g. with U-Omp16 without adjuvants. As negative con-trol, a group was immunized similarly with PBS, and as positivecontrol another group received the already reported oral protectivevaccine strain B. abortus RB51 (24, 25). Oral immunization withU-Omp16 without adjuvants induced a significant protective re-sponse against an oral challenge with B. abortus 2308 (Table IV).This immunization elicited a specific DTH response at 48 and 72 hpost–U-Omp16 intradermal injection (Fig. 2A). Furthermore,splenocytes from mice orally immunized with U-Omp16 secretedIFN-g after an in vitro stimulation with HS (p , 0.05 versus PBS-immunized mice) (Fig. 2B). In contrast, splenocytes from all im-

munizedmice did not produce IL-2, IL-4, IL-5, or IL-10 in responseto HS (data not shown).All in all, these results indicate that the protein region of the

Omp16 lipoprotein, when delivered by the oral route withoutadjuvants, has the ability to induce a Th1 immune response in vitroas well as in vivo while mediating immunoprotection against anoral Brucella challenge.

FIGURE 1. U-Omp16 induces a specific cellular immune response when

administered i.p. without adjuvants. A, Spleen cells of PBS- or U-Omp16–

immunized mice (4 3 106/ml) were stimulated in vitro with an extract of

Omps of Brucella (HS) or complete medium alone (–). Cell-free culture

supernatants were collected at 72 h poststimulation for the measurement of

IFN-g (pg/ml) by ELISA. Results are shown as mean 6 SEM for each

group and are representative of three independent experiments.ppSignificantly different from the same stimulus in PBS-immunized mice

(p , 0.01). B, CD4+ or CD8+ T cell IFN-g responses in U-Omp16–

immunized mice. Splenocytes from PBS- or U-Omp16–immunized mice

were depleted of CD4+ cells (2CD4+) or CD8+ T cells (2CD8+), using

mouse CD4 (L3T4) Dynabeads or mouse CD8 (Lyt2) Dynabeads or were

not depleted (T). These cells were stimulated, and IFN-g was measured in

culture supernatants as described in A. ppSignificantly different from

analogous treated cells from PBS-immunized mice (p , 0.01). ##Signif-

icantly different from nondepleted cells (T) from the same group stimu-

lated with the same stimulus (p , 0.01).

Table IV. Protection against an oral challenge of B. abortus 2308 inBALB/c mice orally immunized with U-Omp16 expressed in E. coli or inleaves of the tobacco species Nicotiana benthamiana

Vaccine(n = 5)

Expressedin Adjuvant

Log10a CFU of

B. abortus 2308at Spleen

(mean 6 SD)Units ofProtection

U-Omp16 E. coli None 4.61 6 0.17b,c 0.94U-Omp16 Tobacco None 4.29 6 0.08b,d 1.26B. abortusRB51

– None 3.74 6 0.44b 1.81

PBS – None 5.55 6 0.05c 0



cSignificantly different from B. abortus RB51 immunized mice; p , 0.01 esti-mated by Dunnett´s test.

dSignificantly different from B. abortus RB51 immunized mice; p , 0.05 esti-mated by Dunnett´s test.

FIGURE 2. U-Omp16 induces a specific cellular immune response when

administered orally without adjuvants. A, To study DTH reactions in mice

orally immunized with U-Omp16 or PBS without adjuvants, mice were

injected in one footpad with U-Omp16 and in the contralateral footpad

with an equal volume of saline. At 24, 48, and 72 h later, the footpad

thickness was measured and its increment was calculated by: (footpad

thickness)U-Omp16 2 (footpad thickness)saline. Significant differences from

the same stimulus in PBS-immunized mice are denoted on the graph. pp ,0.05; ppp , 0.01. B, Production of IFN-g by spleen cells of PBS-immu-

nized or U-Omp16 orally immunized mice stimulated in vitro with HS or

complete medium alone (–). Cell-free culture supernatants were collected

at 72 h poststimulation, and IFN-g (pg/ml) was measured by ELISA.

Results are shown as mean 6 SEM for each group and are representative

of three independent experiments. pSignificantly different from the same

stimulus in PBS-immunized mice (p , 0.05).



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U-Omp16 activates DCs in vitro and in vivo

DCs are initiators and modulators of immune responses with theability to activate naive T cells. Therefore, we evaluated the ability

of U-Omp16 to induce the activation of DCs in vitro. BM-derived

DCs of C57BL/6 mice were incubated in vitro with U-Omp16, and

the TNF-a production was measured. As controls, BM-derived

DCs were incubated with U-Omp16 plus PB or with U-Omp16

completely digested with proteinase K (Fig. 3A); at the same time,

complete medium and E. coli LPS were used as negative and

positive controls, respectively. After being stimulated with U-

Omp16, DCs produced significant amounts of TNF-a (p , 0.001

versus complete medium-stimulated cells). The ability of

U-Omp16 to activate DCs was not modified by the addition of

PB in the medium (Fig. 3B), indicating that the result is not an

outcome of LPS contamination. In the same way, when U-Omp16

digested with proteinase K was used to stimulate DCs, its acti-

vating ability was lost (p . 0.05 versus complete medium-stim-

ulated cells) (Fig. 3B), indicating that the measured activity

resides in the protein. This result was also specific for U-Omp16

because in the same stimulation conditions, another B. abortus

recombinant protein (U-Omp19) was unable to stimulate DCs to

produce cytokines in vitro (data not shown).The latter results indicate that U-Omp16 is able to activate DCs

in vitro, but did not ensure that the same would occur in vivo;

therefore, we decided to evaluate the U-Omp16 ability to activate

DCs in vivo. To achieve this, BALB/c mice were injected i.v. with

U-Omp16 (Fig. 3A). As controls, other groups of mice were in-

jected with either U-Omp16 completely digested with proteinase

K (Fig. 3A), E. coli LPS (positive control), or PBS alone (negative

control). At 20 h postinjection, splenic CD11c+ DCs were ana-

lyzed directly ex vivo by flow cytometry for the expression of the

surface markers CD40, CD80, and CD86. DCs from U-Omp16–

inoculated mice showed an increased expression of the cos-

timulatory molecules CD40, CD80, and CD86, compared with the

expression on DCs from control mice receiving PBS (Fig. 3C).

Similarly, E. coli LPS induced the upregulation of the three cos-

timulatory molecules on splenic DCs from injected mice (Fig.

3C). The ability of U-Omp16 to induce DC maturation in vivo was

totally abrogated by protein digestion with proteinase K, whereas

the same treatment did not influence the E. coli LPS effect on

these maturation DC markers (Fig. 3C; data not shown).

FIGURE 3. U-Omp16 induces in vitro and

in vivo activation of DCs. A, Coomassie blue

stained 12.5% SDS PAGE of U-Omp16 un-

treated or digested with proteinase K (U-

Omp16 + PK) that was used for the in vivo DC

activation assay. B, In vitro stimulation of BM-

derived DCs. C57BL/6 BM-derived DCs were

incubated in vitro with complete medium (–),

U-Omp16 (50 mg/ml) (NT U-Omp16), U-

Omp16 (50 mg/ml) with PB (15 mg/ml) (Pol B

U-Omp16), proteinase K digested U-Omp16

(50 mg/ml) (PK U-Omp16), or E. coli LPS

(1 mg/ml) (LPS). At 24 h later, the TNF-a (pg/

ml) concentration was measured by ELISA in

supernatants. Results are shown as mean 6SEM for each stimulation condition. pppSig-

nificantly different from the TNF-a amounts in

complete medium stimulated cells (p, 0.001).

C, In vivo stimulation of splenic DCs. BALB/c

mice were injected i.v. with 100 mg of

U-Omp16 either untreated or digested with

proteinaseKorwithE. coliLPSor PBSalone as

controls. At 24 h postinjection, splenic CD11c+

DCswere analyzed for their activation status by

assessing the surface expression of CD40,

CD80, and CD86molecules by flow cytometry.

This experiment was conducted three times

with similar results. Histograms display results

from one representative experiment. D, CD40

upregulation on the different DC sub-

populations. BALB/cmicewere injected i.v., as

in C, and the surface expression of CD40 was

analyzedon total splenicCD11c+DCs,myeloid

CD11c+CD8a+ DCs, or lymphoid CD11c+

CD8a2 DCs. Histograms display results from

one representative experiment out of three. E,

Mean fluorescence intensities for the CD40

expression on the indicated subpopulations

from three different mice per group are shown

in the bar graph. Significant differences be-

tween U-Omp16– or LPS-injected mice and

PBS-injected mice are denoted on the graph:

pp , 0.05; ppp , 0.01. Results are represen-

tative of two independent experiments.



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Splenic conventional CD11c+ DCs can be subdivided pheno-typically into three different subsets based on surface markerexpression: CD8a+CD42 DCs (myeloid DCs), CD8a2CD4+ DCs,and CD8a2CD42 DCs; the last two subsets are often referred toas CD8a2 DCs (lymphoid DCs) (44, 45). The effect of U-Omp16on CD8a+ DC and CD8a2 DC subsets in vivo is shown in Fig.3D. Total splenic DCs of mice treated with U-Omp16 upregulatedthe expression of the costimulatory molecule CD40 (Fig. 3C, 3D),this upregulation involved both splenic DC subpopulations: my-eloid DCs (CD11c+CD8a+, Fig. 3D) and lymphoid DCs (CD11c+

CD8a2, Fig. 3D), the effect being more evident in the myeloidsubpopulation (Fig. 3E).Thus, U-Omp16 self-adjuvanticity could be mediated, at least in

part, by the stimulation of DC maturation in vivo, in particular,activation of myeloid DCs.

TLR-4 is involved in U-Omp16 self-adjuvanticity

Signaling through TLRs has been shown to be immunostimulatory,inducing host cells to proliferate, secrete cytokines and chemokines,and upregulate costimulatory molecules. The latter characteristicrenders TLR ligands a potent addition to the vaccine adjuvant rep-ertoire by possessing the capability to link the innate and adaptiveimmunesystems, thus inducingnotonlyan inflammatoryresponsebutalso activation of the adaptive armof the immune system(46).Amongthe TLRs, TLR2 and TLR4 mediate the response to the most diverseset of molecular structures, including some bacterial proteins (47),and it has been reported that someOmps interactwith heterodimers ofTLR2 and TLR6 (48). To determine if TLR2, TLR4, or TLR6 is in-volved in the immunostimulant activity of U-Omp16, BM-derivedDCs or macrophages from TLR22/2, TLR42/2, TLR62/2, or wtC57BL/6 mice were stimulated in vitro with different doses ofU-Omp16 (1, 5, 10, and 50 mg/ml), complete medium alone (un-stimulatedcontrol),E. coliLPS, orPam3Cys.After 24hof stimulation,the TNF-a and IL-12 p40 concentrations in supernatants were mea-sured. Both BM-derived DCs and macrophages from C57BL/6 wtmice produced significant levels of both cytokines after stimulationwith U-Omp16 in a dose-dependent fashion (Figs. 4, 5, respectively)when compared with the same cells treated with culture media alone(p , 0.01), indicating that both cell types were stimulated by

U-Omp16 in vitro. The same pattern of response to U-Omp16 stimuliin vitro was elicited in BM-derived DCs and macrophages fromTLR22/2 or TLR62/2 C57BL/6 mice, indicating that neither TLR2nor TLR6 is involved in the activation of both cell types by U-Omp16in vitro. In contrast, U-Omp16 was unable to stimulate BM-derivedDCs or macrophages from TLR42/2 C57BL/6 mice to secrete thesecytokines, suggesting that the stimulating activity ofU-Omp16 invitrowould be mediated by this receptor. E. coli LPS and Pam3Cys stimuliwere includedas controls, and, as expected, they induced the activationof all cell types excluding those derived from TLR42/2 mice stimu-lated with E. coli LPS or TLR22/2mice stimulated with Pam3Cys.Then we sought to determine if in vivo TLR4 plays a role in the

generation of the adaptive response elicited by U-Omp16. For thatpurpose, C57BL/6 wt or TLR42/2 mice were vaccinated with U-Omp16 without adjuvants, and the elicited cellular and protectiveresponses were evaluated. Spleen cells of immunized mice werecultured in vitro in the presence of U-Omp16 or culture mediumalone. The antigenic stimulus induced the production of significantlevels of IFN-g from the splenocytes of U-Omp16–vaccinatedmice, compared with the production in spleen cells of the controlmice (p , 0.05) (Fig. 6). In agreement with our previous in vitroresults, splenocytes frommice lacking TLR4 did not respond to thisstimulus in vitro, indicating that U-Omp16 requires TLR4 in vivofor eliciting a cellular Th1 immune response. Similarly, immuni-zation with U-Omp16 did not elicit significant protection against B.abortus infection in TLR42/2 mice (0.51 U of protection, p. 0.05versus PBS). As expected, U-Omp16 delivery in the absence ofexternal adjuvants elicited 1.15 U of protection (p , 0.01 versusPBS) in C57BL/6 wt mice (Table V).Overall, these results indicate that U-Omp16 requires the

presence of TLR4 for displaying its self-adjuvanting properties,activating macrophages and DCs while eliciting a protective Th1immune response against Brucella.

U-Omp16 is a new bacterial PAMP

The ability of U-Omp16 to interact with the innate immune systemthrough TLR4 suggested that it would be a PAMP. As PAMPs aregenerally constituted by conserved structures that are widespreadon different pathogens (49), we decided to investigate whether

FIGURE 4. The production of cytokines induced

by U-Omp16 stimulation of DCs in vitro is TLR4

dependent. BM-derived DCs from wt, TLR22/2,

TLR42/2, or TLR62/2 C57BL/6 mice were treated

in vitro for 24 h with complete medium alone (–) or

complete medium containing either U-Omp16 (1, 5,

10, 50 mg/ml), E. coli LPS (1 mg/ml), or Pam3Cys

(1 mg/ml). Cell-free supernatants were collected and

the concentration of TNF-a (A) or IL-12 p40 (B) was

determined by ELISA. Results are represented as

mean6 SEM of triplicate measurements. These data

are representative of at least three independent ex-

periments. pp , 0.01, significant differences be-

tween stimulated and complete medium alone

treated cells; #p , 0.01, significant differences be-

tween the indicated group and cells from wt mice

exposed to the same stimulus.



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homologous proteins are present among different organisms.BLAST analysis of the protein portion of Omp16 (U-Omp16) re-vealed high homology—near to identity (identities, 100–76%;scores, 297–215 bits)—with other Omps of the a Proteobacteriaclass, order Rhizobiales, particularly in the Brucellaceae, Rhizo-biaceae, Aurantimonadaceae, and Phyllobacteriaceae families(identical proteins are present in 27 different organisms amongthem) (Supplemental Fig. 1). Moreover, U-Omp16 is highly con-served (identities, 67–49%; scores, 189–142) among the a Proteo-bacteria class, homologous proteins being present in 99 organismsfrom Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospir-illales, and Sphingomonadales, as well as in the d ε subdivision,such as in Helicobacter bizzozeronii (Supplemental Fig. 2).It is noteworthy that BLASTand pfam sequence analyses stated

that Omp16 belongs to a protein family with a peptidoglycan

binding domain similar to the C-terminal domain of OmpA fromE. coli. This domain has a b/a/b/a-b(2) structure and is found in

the C-terminal region of many Gram-negative bacterial Omps—for example, porin-like integral membrane proteins (such asOmpA), small lipid-anchored proteins (such as pal), and MotBproton channels. In terms of the innate immune system, pub-lished studies have indicated that K. pneumoniae and E. coliOmpA induce DC activation (19, 50); therefore, we conducteda multiple sequence alignment comparing B. abortus U-Omp16with E. coli OmpA and K. pneumoniae OmpA. Although aminoacids are different, U-Omp16 from Brucella has significant ho-mology with K. pneumoniae OmpA (24% identity; score, 45.4bits) and from E. coli OmpA (25% identity; score, 48.5) (Sup-plemental Fig. 3).Taken together, these results indicate that Omp16 would con-

stitute a highly conserved protein among a Proteobacteria. Byrecognizing this protein, the innate immune system could alsosense the presence of these particular types of bacteria.

A plant-based U-Omp16 vaccine confers protection againstB. abortus

The notion that U-Omp16 evokes a protective and cellular Th1immune response when given orally without adjuvants prompted usto develop a plant-made vaccine based on U-Omp16. One majorprerequisite for vaccine production in plants is the development ofa reliable system that allows the expression of the desired Ag. Asa first approach, we studied the transient expression of U-Omp16 inN. benthamiana plants.The protein portion of Omp16 was generated by PCR and cloned

into the 39-provector module pICH10990 for transient nuclearexpression. The resulting vector termed pO16 6122 was mobilizedinto agrobacteria and infiltration performed with the 59-modulepICH15879 and recombinase module pICH10881, as described(38). Expression of the recombinant U-Omp16 was monitored bySDS-PAGE and immunoblot. As illustrated in Fig. 7A, U-Omp16accumulated to significant amounts in infiltrated N. benthamianaleaves. Determination of Omp16 content by immunoblot revealedthat a fraction of 2% of the total soluble protein consists of the

FIGURE 5. The production of cyto-

kines induced by U-Omp16 stimulation of

macrophages in vitro is TLR4 dependent.

BM-derived macrophages from wt,

TLR22/2, TLR42/2, and TLR62/2

C57BL/6 mice were treated in vitro for

24 h with complete medium alone (–) or

complete medium containing either U-

Omp16 (1, 5, 10, or 50 mg/ml), E. coli

LPS (1 mg/ml), or Pam3Cys (1 mg/ml).

Cell-free supernatants were collected, and

the concentration of TNF-a (A) or IL-12

p40 (B) was determined by ELISA. Re-

sults are represented as mean 6 SEM of

triplicate measurements. These data are

representative of at least three independent

experiments. pp , 0.01, significant dif-

ferences between stimulated and complete

medium alone treated cells; #p , 0.01,

significant differences between the in-

dicated group and cells from wt mice ex-

posed to the same stimulus.

FIGURE 6. The elicited U-Omp16–specific cellular Th1 immune re-

sponse is TLR4 dependent. Spleen cells of PBS- or U-Omp16–immunized

C57BL/6 wt or TLR42/2 mice were stimulated in vitro with complete

medium alone (–) or complete medium with U-Omp16. After 72 h, cell-

free culture supernatants were collected for the measurement of IFN-g (pg/

ml) by ELISA. Results are shown as mean 6 SEM for each group and are

representative of three independent experiments. pp , 0.05, significantly

different from the same stimulus in cells from PBS-immunized mice.



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recombinant bacterial protein. Total expression level was rathermoderate for this protein; however, sufficient plant material couldbe provided for purification of U-Omp16 for subsequent immu-nization studies. Purification of the recombinant protein wasachieved owing to the C-terminal poly-histidine extension (63Histag) by immobilized metal affinity chromatography. Up to 20 mgof U-Omp16 could be purified from 1 g of fresh leaf material. TheU-Omp16 preparation from plants showed a high purity, as ex-emplified by SDS-PAGE and subsequent Coomassie staining (Fig.7B). All visible protein bands also gave a positive signal onWestern blot with the appropriate anti-Omp16 Ab (Fig. 7A);therefore, we conclude that they constitute different manifes-tations of Omp16.To evaluate whether the plant-made U-Omp16 contained the

immunostimulating properties from its E. coli-made pair, an in vitroBM-derived DC or macrophage stimulation assay was performed.When wt-derived cells were incubated in vitro with plant-made U-Omp16, both cell types produced large amounts of TNF-a and IL-12 p40 in response to the stimulus (p , 0.001 versus nonstimulatedcells), whereas TLR42/2 cells did not produce any from the mea-sured cytokines (Fig. 8). This result indicates that, as occurs with E.coli U-Omp16, plant-made U-Omp16 stimulates the innate immunesystem by a TLR4-mediated pathway.

To assess the protective capacity of plant-made U-Omp16,groups of mice were orally immunized in parallel either with to-bacco-expressed purified U-Omp16 or with E. coli-expressedpurified U-Omp16, and the elicited protective response wasevaluated. Plant-made U-Omp16 was able to elicit a significantlevel of protection (1.26 U of protection, p , 0.01 versus PBS)against oral B. abortus infection. The elicited protection wassimilar to that induced by U-Omp16 produced in E. coli (p . 0.05tobacco versus E. coli) (Table V).Altogether, these results indicate that plant-made U-Omp16 can

be correctly expressed in a plant system and conserve its intrinsicimmunogenicity: it activates the innate immune system through itsinteraction with TLR4, allowing it to induce a protective responseagainst an oral Brucella challenge. Therefore, a plant-made vac-cine could be a successful approach to control of brucellosis.

DiscussionEffective vaccines have three key components: 1) an Ag againstwhich adaptive immune responses are generated, 2) an immunestimulus or adjuvant to signal the innate immune system to po-tentiate the Ag-specific response, and 3) a delivery system to ensurethat the Ag and adjuvant are delivered together at the right time andlocation (51).Available brucellosis vaccines are live attenuated strains that

have all these key components but also have several disadvantages;thus, new improved vaccines need to be developed. Highly purifiedAgs offer potential advantages over traditional vaccines, includinga high degree of safety and the capacity of eliciting highly specificimmune responses, but in general they need to be coadministeredwith additional immunostimulant substances (adjuvants) becausethey are poorly immunogenic (12–14).Knowing the inherent stimulatory properties of the lipid moiety of

bacterial lipoproteins (32, 52), we hypothesized that B. abortusOmp16 lipoprotein would be able to elicit a protective immune re-sponse without the need of external adjuvants. Our results confirmthat the lipoprotein Omp16 of B. abortus as a recombinant Ag is ableto induce a protective immune response against a challenge withvirulent B. abortus when given to mice without external adjuvants,indicating that Omp16 lipoprotein has an intrinsic adjuvanticity. Toour surprise, however, U-Omp16 administered without adjuvantselicited similar levels of protection, suggesting that the protein por-tion of Omp16 has enough intrinsic adjuvant activity.This property is not unique to Omp16, because several bacterial

proteins have intrinsic adjuvant activity that allows them to inducespecific and effective immune responses without the help of ex-ternal adjuvants, such as the K. pneumoniae OmpA or the Ompcomplex of N. meningitidis serogroup B. The self-adjuvantingproperties of these proteins were attributed to their ability to ac-tivate different cellular types, mainly DCs (19–21, 53). Recently,other investigators have identified a Borrelia burgdorferi lipo-protein (BmpA) whose protein portion stimulates the secretion ofproinflammatory cytokines in human synovial cells (54).

Table V. Protection against B. abortus 2308 in C57BL/6 wt or TLR42/2 mice immunized with U-Omp16 without adjuvants

C57BL/6 wt C57BL/6 TLR42/2

Vaccine(n = 5) Adjuvant

Log10a CFU of B. abortus2308 at Spleen(mean 6 SD)

Units ofProtection

Log10a CFU of B. abortus2308 at Spleen(mean 6 SD)

Units ofProtection

U-Omp16 None 4.60 6 0.34b 1.15 5.64 6 0.31 0.51PBS None 5.83 6 0.06 0.00 6.15 6 0.54 0.00

aThe content of bacteria in spleens is represented as the mean log10 CFU 6 SD per group.bSignificantly different from PBS-immunized mice; p , 0.01 estimated by Dunnett´s test.

FIGURE 7. Characterization of recombinant U-Omp16 expressed in

plants. A, To estimate the level of recombinant U-Omp16 in N. ben-

thamiana leaves, crude protein extracts of various amounts of total protein

were subjected to SDS-PAGE and subsequent Western blot, and the in-

tensity of the signals was compared with a series of Omp16 preparations of

defined concentration. B, Recombinant U-Omp16 from leaves was purified

via immobilized metal affinity chromatography; 5 mg of the protein was

separated on SDS-PAGE and visualized with Coomassie staining (the ar-

row points to U-Omp16). Apparent sizes are given on the left side in kDa.

M, marker lane.



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The ability of U-Omp16 to induce a protective immune responsewithout the addition of adjuvants is not a frequently occurringcharacteristic in the brucellosis field. Indeed, otherBrucellaproteinshave been evaluated for their ability to induce protective responseswhen administered without adjuvants, giving unsatisfactory results.None of the Brucella p39, BLS, or L7/L12 proteins elicited a sig-nificant protective response when administered without adjuvants(33, 55, 56), whereas immunization with the DnaK protein ora fusion protein of Omp16 and L7/L12 without adjuvants inducedmoderate protection levels, but lower than those induced by thecontrol attenuated vaccine strain B. abortus S19 or the same pro-teins with adjuvants (10, 11). In the case of the fusion proteincontaining Omp16, the lower protection levels elicited by thispreparation compared with those elicited by U-Omp16 in this re-port could be due to the lower doses used in immunization or todifferences between the immunization routes used.The idea that protection elicited by Omp16 without adjuvants

was independent of protein acylation represents an advantage invaccine development, particularly in the scale-up for bulk manu-facture and in the final cost of the product, the production ofL-Omp16 being a more time-consuming and expensive processthan the production of U-Omp16 (8). Moreover, the protectionlevels induced by U-Omp16 in the absence of external adjuvantswere statistically similar to those elicited by the vaccine strain B.abortus S19 and were not improved by the addition of an adjuvant,such as IFA, indicating that U-Omp16 is a worthy vaccine can-didate for brucellosis control.To our knowledge, Omp16 is the first Brucella protein able to

induce protection levels similar to those induced by the control

live vaccine strain without the requirement of external adjuvants.

This unique quality represents an exceptional benefit because

external adjuvants might sometimes present risk, inducing adverse

reactions, such as local inflammation at the injection site with the

induction of granuloma or sterile abscess formation (57).Given that most pathogens gain entry into their hosts via mucosal

surfaces, the induction of a robust mucosal immune response by

vaccination may be an effective means of preventing infectious

diseases. Oral delivery of vaccines is particularly appealing, as it

would avoid the use of needles and allow self-administration.

Because one of the main pathways of acquiring brucellosis is the

oral route, several efforts have been reported to develop an oral

vaccine. Most studies involved the use of attenuated Brucella

strains (24, 58, 59) or live vectors expressing Brucella Ags (26,

60) or carrying DNA plasmids for DNA vaccine delivery at the

mucosal gut (27). Subunit vaccines have several advantages over

live vaccines. They are not infectious, the spread of strains to the

environment is avoided, and they are easier to transport. Currently,

three recombinant proteins of Brucella have been evaluated as oral

vaccine candidates: choloylglycine hydrolase (28), Omp16, and

Omp19 (8). In all cases, proteins were administered with the

mucosal adjuvant cholera toxin. In this report, U-Omp16 was also

able to induce a protective response without the need of adjuvants

by the oral route, indicating that this protein also has mucosal self-

adjuvanting properties. This is an outstanding property because

the mucosal adjuvant repertoire is scant at present.In contrast, evaluation of the cellular immune responses in orally

or i.p. immunized mice indicates that U-Omp16 immunization

induces a specific Th1 response in vitro as well as in vivo when

administered without adjuvants. This cellular Th1-specific re-

sponse could be responsible for the elicited protection against

Brucella infection, because IFN-g plays a central role in pro-

tective responses against Brucella (61).Induction of adaptive specific immune responses involves the

activation of specific T cells by APCs. Among them, DCs are thekey mediators of adaptive immunity. The quality of signals re-ceived by DCs in response to PAMPs influences the nature of theevoked adaptive response. Following stimulation with PAMPs, DCmaturation occurs, a process in which DCs undergo phenotypicchanges resulting in an improved ability to promote T cell res-ponses. Many immunopotentiators mediate their effect by acti-vating DCs (62, 63). Our results show that U-Omp16 is able tostimulate DC activation in vitro and in vivo. This stimulationcould not be due to LPS contamination in the preparations, be-cause the protein preparations were exhaustively depleted of LPSwith Sepharose-PB, as assessed by Limulus amoebocyte assay.Moreover, U-Omp16 lost its DC-activating capacity when com-pletely digested with proteinase K, indicating that the in vivoelicited DC activation was due to the protein rather than to anothernonprotein contaminant. The same treatment did not affect thestimulating properties of E. coli LPS.The DC subpopulation (CD8a+) is able to produce large

amounts of IL-12 when activated with several immunostimulants,being involved in the induction of Th1 adaptive immune responses(44, 45, 63–65). In the current study, we have demonstrated thatthe DC stimulating activity of U-Omp16 was more prominent inthe CD8a+ DC subpopulation. Therefore, our results suggest thatthe intrinsic adjuvant activity of the protein portion of Omp16could be mediated, at least in part, by the in vivo activation ofDCs, leading to the priming of specific naive T cells and evokinga protective Th1 immune response against Brucella infection.

FIGURE 8. Plant-made U-Omp16 stimulates DCs

and macrophages in vitro in a TLR4-dependent fashion.

BM-derivedDCs (A,B) andmacrophages (C,D) fromwt

and TLR42/2 C57BL/6 mice were treated in vitro for 24

h with complete medium alone (–) or complete medium

containing U-Omp16 (50 mg/ml for DCs or 1, 5, and

10 mg/ml for macrophages) or E. coli LPS (1 mg/ml).

Cell-free supernatants were collected, and the concen-

tration of TNF-a (A, C) or IL-12p40 (B, D) was de-

termined by ELISA. Results are represented as mean 6SEM of triplicate measurements. pp, 0.001, significant

differences between stimulated and complete medium

alone treated cells.



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It is now known that signaling of one or more receptors onimmune cells (for example, TLRs) results in a rapid inflammatoryresponse, leading to enhanced presentation of Ags to the immunesystem (47, 66, 67). In the current study, we have demonstratedthat the ability of U-Omp16 to stimulate innate immune cells(DCs and macrophages) as well as to induce an adaptive specificimmune response in the absence of adjuvants required the pres-ence of TLR4. Although U-Omp16 immunization did not inducea statistically significant protection against Brucella in TLR42/2

mice, the protection was not totally abrogated, suggesting thatother mechanisms (additive or redundant) would account for theelicited protection against Brucella. The best characterized TLR4agonist is E. coli LPS. However, there are several agonists for thisreceptor that are not related to LPS, including different proteins.Among the protein TLR4 agonists are endogen molecules, such asthe extradomain A of fibronectin (68, 69) and gp96 (70), andpathogen-derived proteins, such as the peptidyl-propil cys-transisomerase from Helicobacter pylori (71), the DnaK protein fromFrancisella tularensis (72), Hsp60 from Chlamydia pneumoniae(73), and the Brucella lumazine synthase BLS from B. abortus(74). Altogether, our results indicate that U-Omp16 could con-stitute a new PAMP recognized by TLR4.A previous report by our laboratory indicates that B. abortus

L-Omp16 possesses proinflammatory activity on human mono-cytes in a TLR2-dependent fashion. This activity was dependenton protein lipidation (32). In that report, assays were designed toimitate the lipoprotein concentration present on the bacterialsurface at the site of infection (0.01–1 mg/ml). The current study isdifferent because the protein concentrations used (5–50 mg/ml)attempted to resemble those present at the site of injection aftervaccine administration (150 mg/ml). Therefore, the absence ofresponse by human monocytes following U-Omp16 stimulus re-ported in our previous paper could be due to the lower concen-trations used in such assays.PAMPs are generally well conserved structures among different

pathogens (49). The U-Omp16 sequence is highly conservedamong a Proteobacteria, and BLAST and pfam sequence analysesrevealed that U-Omp16 belongs to a family of proteins witha peptidoglycan binding domain similar to the C-terminal domainof OmpA. Well-studied members of this family include the E. coliOmpA, the E. coli lipoprotein PAL, and N. meningitidis RmpM,which interact with the outer membrane, as well as the E. colimotor protein MotB and the Vibrio flagellar motor proteins PomBand MotY, which interact with the inner membrane. From thestandpoint of the innate immune system, it has been stated that K.pneumoniae OmpA induces DC maturation by binding TLR2 (19).In addition, E. coli OmpA induces DC maturation independentlyof TLR4 (50). Although U-Omp16 from Brucella has significanthomology with K. pneumoniae OmpA and E. coli OmpA, thereare some differences in their amino acid sequences that may ex-plain the differences in TLR binding. A similar situation has al-ready been described in the case of flagellin from H. pylori, whichno longer recognizes TLR5 as the flagellin from E. coli because ofchanges in its amino acid sequence (75). At present, it is notknown which domain of K. pneumoniae OmpA binds TLR2, but itis important to have in mind that the homology with B. abortusU-Omp16 is predominantly at the C-terminal portion.Vaccine production in plants is attractive in terms of safety and

cost effectiveness. From a safety perspective, the plant expressionsystem would produce a vaccine free of animal pathogens andanimal proteins. The production of plant-derived vaccines is, inprinciple, almost limitless and may require little or no downstreamprocessing. An edible vaccine could be very practical to administerin cattle, but in this situation Ag and adjuvant must be expressed

together. The latter notion, together with our findings showing thatoral U-Omp16 delivery without adjuvants induced protectionagainst Brucella, led us to develop a plant-made vaccine ex-pressing U-Omp16. As a first approach, we studied the transientexpression of U-Omp16 in N. benthamiana plants. The fact thatU-Omp16 expressed in N. benthamiana leaves demonstratesa TLR4-mediated capacity to activate in vitro DCs and macro-phages indicates that it retains its immunostimulating activity. Thelatter result, in conjunction with the demonstration that E. coli-made U-Omp16 retains its stimulating activity when it is co-incubated with PB and loses its activity when completely digestedwith proteinase K, contributes to the interpretation that the mea-sured immunostimulating activity is not an outcome of contami-nating LPS but rather a specific effect of the U-Omp16 protein.Moreover, tobacco-made U-Omp16 induced the same protectiveefficacy as E. coli-expressed U-Omp16, indicating that the plant-made U-Omp16 retains its self-adjuvanticity and immunogenicity.These findings suggest that future attempts to obtain an ediblevaccine for cattle in another plant system (for example, alfalfa orbarley) hold much promise.Finally, this report presents U-Omp16 as a new bacterial PAMP

that signals through TLR4, is able to activate in vivo DCs, inducesa Th1 immune response, and is a very promising self-adjuvantingvaccine against systemic as well as orally acquired brucellosis. Thisstudy also set the precedent for development of a plant-madevaccine against brucellosis.

AcknowledgmentsWe thank Icon Genetics (Halle, Germany) for providing vectors for transient

plant expression.

DisclosuresThe authors have no financial conflicts of interest.

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