ARTICLE IN PRESS

Immunobiology 214 (2009) 113–120

0171-2985/$ - se

doi:10.1016/j.im

Abbreviations

interferon; IL-4

cell-mediated im

reading frame.�CorrespondE-mail addr

www.elsevier.de/imbio

Cloning, expression and immunogenicity of the translation initiation factor

3 homologue of Brucella abortus

Marcela Gonzaleza, Edilia Andrewsa, Hugo Folchb, Darwin Saeza, Alex Cabreraa,Paulina Salgadoa, Angel Onatea,�

aMolecular Immunology Laboratory, Department of Microbiology, Faculty of Biological Sciences, Universidad de Concepcion,

P.O. Box 152-C, Concepcion, ChilebFaculty of Medicine, Department of Immunology, Universidad Austral de Chile, Valdivia, Chile

Received 2 April 2008; received in revised form 5 June 2008; accepted 3 July 2008

Abstract

The infC gene of Brucella abortus encoding the translation initiation factor 3 (IF3) was cloned, sequenced andexpressed in Escherichia coli. The amino acid sequence analysis predicted a product with 74–80% identity with the IF3proteins from Mesorhizobium loti, Sinorhizobium meliloti, Aurantimona sp. and Mesorhizobium sp. This protein alsoshow 54% amino acid sequence identity with the E. coli IF3, sharing most of the residues which were described asresponsible for the biological activity of this protein. Since we have previously reported the immunoprotective capacityof this Brucella protein, we stimulated lymphoid cells from animals immunized with purified recombinant Brucella IF3protein ‘‘in vitro’’ with this antigen. The lymphocytes were able to mount a strong proliferative response withconcomitant production of gamma interferon, but without the secretion of either IL-4 or antibodies. Thus,immunization with the Brucella recombinant IF3 protein promotes a TH-1 polarized response, allowing us to proposeit as a promising candidate antigen for the development of subunit vaccines against Brucella.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Brucella abortus; Brucellosis; Translation initiation factor 3; IF3; Immunogenicity

Introduction

Brucella abortus, a facultative intracellular pathogen,is the etiological agent of brucellosis, a disease thataffects man and several other animal species (Corbel,1997). B. abortus is the most important Brucella species

e front matter r 2008 Elsevier GmbH. All rights reserved.

bio.2008.07.004

: IF3, translation initiation factor 3; IFN-g, gamma

, interleukin-4; CBPs, crude Brucella proteins; CMI,

munity; CTL, cytotoxic-T-lymphocytes; ORF, open

ing author. Tel.: +5641 2204118; fax: +5641 2245975.

ess: aonate@udec.cl (A. Onate).

involved in bovine brucellosis, which is characterized byabortion and infertility of infected animals. Vaccinationis the only method of control suitable for countries witha high incidence of this disease (Moriyon et al., 2004).Vaccination with live attenuated B. abortus strains 19and RB51 (Stevens et al., 1994, 1996) has been used inthe field to control brucellosis in cattle. However, forhumans, the vaccine strains used in animals areconsidered virulent or unsafe (Ashford et al., 2004;Corner and Alton, 1981; Palmer et al., 1997; Young,1983). Because Brucella is a facultative intracellularbacterium, resistance to B. abortus depends on acquiredcell-mediated immunity (CMI) (Zhan et al., 1993), with

ARTICLE IN PRESSM. Gonzalez et al. / Immunobiology 214 (2009) 113–120114

production of gamma interferon (IFN-g) by CD4+ Tcells, followed by macrophage activation leading toenhanced bacterial killing (Jiang and Baldwin, 1993;Jones and Winter, 1992; Stevens et al., 1992; Zhan andCheers, 1993), plus potentiation of cytotoxic-T-lympho-cytes (CTL) that also appeared to play an importantrole in protective immunity against brucellosis (He et al.,2001).

For brucellosis and other infectious diseases, it isimportant to identify the antigens of the etiologicalagent that are able to stimulate the host defensiveresponse. This knowledge is essential for designing newvaccination strategies, such as the use of recombinantmicroorganisms, peptides or naked DNA and RNA. Inthis regard, a number of immunogenic proteins ofB. abortus have been previously identified and char-acterized, the most studied molecules being bacteriofer-ritin (Denoel et al., 1995), the P39 protein (Denoel et al.,1997), HtrA (Roop et al., 1994), GroEL, GroES, UvrA(Oliveira et al., 1996), YajC (Vemulapalli et al., 1998)L7/L12 (Oliveira et al., 1994; Oliveira and Splitter,1996), outer membrane proteins (Omps) (Cloeckaertet al., 2002) and the Cu–Zn superoxide dismutase (SOD)(Onate et al., 1999; Vemulapalli et al., 2000). Inaddition, several years ago, we described a 22.9 kDaBrucella protein that was able to induce CMI with aconcomitant protection in mice (Cespedes et al., 2000).In this study, we report the cloning and sequencing ofthe gene encoding this 22.9 kDa protein. Sequenceanalysis demonstrated the high level of homology ofthis protein with the translation initiation factor 3 (IF3)of other bacteria. We also expressed the putative IF3 asrecombinant protein in E. coli, and evaluated theimmunological properties of the purified recombinantprotein in mice.

Material and methods

Cloning the gene coding for the 22.9 kDa Brucellaprotein

Genomic DNA was isolated from B. abortus RB51, astable rough mutant derived from virulent strain 2308,as described by Sambrook et al. (1989). The genomicDNA was digested for 3 h at 37 1C with 0.12U of EcoRI

(Promega, Madison, Wisconsin) per mg of DNA. Inparallel, based on the previously determined sequence ofthe amino-terminal peptide (FRRPFRATPVQKDGP)of the 22.9 kDa protein (Cespedes et al., 2000), a mixtureof degenerate oligonucleotide primers were synthesizedand labelled with [g-32P] ATP to be used as a probe(Bruc1) for Southern blot analysis. The design of thedegenerate oligonucleotides was based on reversetranslation of amino-acid sequence ATPVQKDGP.

Inosine was used at the third position of some codonsto encompass all possible codons of the specific aminoacid (Bruc1: 50GCI, ACI CCI, GTI, CAA(G), AAA(G),GAC(T), GGI, CC 30). In the Southern blot, hybridiza-tion was carried out for 12 h at 50 1C, and the singleDNA band of 1.5–2.0 kb detected was cloned into theEcoRI site of the pUC19 vector, which was thentransformed into the E. coli strain DH5a. One clonebearing the chimeric plasmid with only one EcoRI insertof about 1.5 kb was selected and named pBGt11.Sequence analysis of the cloned fragment showed100% similarity with the target amino-terminal peptideof the 22.9 kDa protein (data not shown).

DNA sequence analysis of the B. abortus RB51

insert

The recombinant plasmid pBGt11 bearing theB. abortus RB51 insert was sequenced using an ABIPRISM 310 automatic genetic analyzer (Applied Bio-systems, Inc., Foster City, California). The computeranalysis of the sequence data was performed by BLASTanalysis of sequence databases available at the NationalCenter for Biotechnology Information.

Expression of the recombinant IF3 fusion protein

In order to produce and purify recombinant Brucella

IF3, the protein was expressed in E. coli BL-21(Novagen, Madison, WI) using the pET-15b vector(Novagen). In this vector, the product of the clonedforeign gene was expressed as a fusion protein with sixhistidine residues at the amino-terminal portion; thismodification allows the purification of the recombinantfusion protein by metal affinity chromatography inQIAexpress Ni-nitrilotriacetic acid (NTA) agarosematrix (Qiagen). As the first step, the infC gene wasPCR amplified from the plasmid pBGt11 using a primerpair designed based on the nucleotide sequence. Aprimer complementary to the 50 end of the IF3 gene 50-CCC CAT ATG CGC CGA CCG TTC AGA GCG-30

(Promega, Madison, WI), was designed to contain aNdeI site (underline). The reverse primer, 50-CCC GGATCC TCA GCG AGG TGC CAG CAC CAT-30(Promega, Madison, WI), was designed to containthe 30 end of the coding sequence and a BamHI site. Theamplified gene fragment was digested with an appro-priated restriction enzyme and cloned into pET-15b.The recombinant plasmid, named pET-15b-IF3, waselectroporated into the recipient E. coli BL21 strain, anda single recombinant colony was selected. The expres-sion of B. abortus IF3 was achieved in E. coli as His-Tagfusion protein.

ARTICLE IN PRESSM. Gonzalez et al. / Immunobiology 214 (2009) 113–120 115

Antigenicity of recombinant Brucella IF3 protein

In order to examine if the recombinant IF3 (rIF3)protein is able to induce a good cell-mediated response,

EcoRI

| . . . . .

GAATTCTGGCGCCGCAACGCTCATGACAAGTCTCCTCGTTTTGCGCGCCTGTTTTG

. . . . .

TTGAAATGACGGGAAATGTGACCGATATGCCGCTTGCTCCCCGCGAAATGTCCGCA

. . . . .

GCTATTGACTTCGCCGCTCGCTTGACGACATTTCCGCGATCAGCCGCGGTTTGATG

. . . . .

TAAACAATCAGGAGATAACGACCATGCGCCGACCGTTCAGAGCGACGCCGGTCCAG

M R R P F R A T P V Q

TCAGCTTATTGACGCCGAAGGCCAGAACCATGGGGACGTTTCCATTCAGGAAGCCA

Q L I D A E G Q N H G D V S I Q E A

CCGAACGCCGAACCGCCAGTTTGCAAGATCGTGGACCTTGGCAAGCTGAAATATCA

P N A E P P V C K I V D L G K L K Y Q

TCGAGATCAAGGAAATCAAGATGCGGCCGAACATCGACACCCATGACTATGAGGTG

V E I K E I K M R P N I D T H D Y E V

CAAGGTCACCTTGCGTTTCCGTGGCCGTGAAATGGCGCACCAGGAACTGGGTATGA

K V T L R F R G R E M A H Q E L G M

GTGGAATCCGAGCCGAAGCTCGAAGGCCGCCAGATGATGATGGTGCTGGCACCTCG

V E S E P K L E G R Q M M M V L A P R

CATGCCGTGAAAGAAACCATGTCTTGGAAAAGCCGCCTTTCGGGCGGTTTTTCTTT

. . . . .

GGTGAACTGGGGCTGTATCAGCGCCGCCGCCGCTTTACGATCGGTGGGGTGATTGT

. . . . .

GCTCGCTGCACGAATATATCGAAGCCTTCGGCATCAGTCTCATTGTTGCGGCCATT

. . . . .

CGCGGAAATCGTGCAGAGCGGCCCTATTCGGTGACGCGCAACCCGCTTTATGTCTT

. . . . .

TCATCGTCGCGGCAGCTTTCGGCGTGTTGTGTTATCTCGCCTTTTCGATTGTCATC

. . . . .

CGAAGCCTATTGTGCCAAGGTGCCGCGTTTTTTTCCGAAGTTTTCGCTGTTTCACG

. . . . .

TTTACGGACGGTCTGGTTTTCTTCGTGGCCTATCCGTTCTTTGAATT 1548

Fig. 1. Nucleotide sequence of a 1548 bp fragment containing the

initiation codon is indicated in bold type (position 324). A putative

Stop codon.

we followed a procedure similar to that used before toinvestigate the properties of the native protein purifiedfrom total Brucella extracts by salt precipitation andchromatography (Cespedes et al., 2000). Female BALB/c

. . . . .

GCATGTTCGCAAAAGGGAGAAAACCCGCCTTATGGATGGAACCC 100

. . . . .

AAAGTGGCATTTATCACATGGGGGTGATTTTTCCTGTCAAAGAT 200

. . . . .

GGGGAAATGCCGTCTGGGCAACGAGCATCCGATCGCTGTAGACG 300

. . . . .

AAAGACGGACCGCGCTCCAACCGTGATATCCGGGTTCCCCGGGT 400

K D G P R S N R D I R V P R V

TGGCCATGGCGGAAGAGGCGGGTCTAGATCTTGTTGAGATCGTG 500

M A M A E E A G L D L V E I V

AAATCAGAAAAAAGCCGCCGAAGCGCGCAAGAAGCAGAAGACGG 600

N Q K K A A E A R K K Q K T

AAGATGAAAGCAGCGCAGCGTTTCTTTGAGGAAGGCGACAAGGT 700

K M K A A Q R F F E E G D K V

AGCTTCTGCAACGCGTCAAGGAAGACACGGTTGAAATCGCCAAG 800

K L L Q R V K E D T V E I A K

CTGATTTTGCATCCGTTTTCGGGAAACGCCGTTTTCCTTGAAAC 900

*

GCTCGAAACGAATATTCAAAGATGGGACAACCATGAAGACATTG 1000

. . . . .

TCTTTTGGTCATCGCGCTTATCTTCGTGCGCTCGCAATCGACCG 1100

. . . . .

ATCGGGCGCATGTGGTGCACGCTTTATATCGGCGGACGCAAGAG 1200

. . . . .

CAGCAGCATTGGTGCGGTGGGCATCGGCGCCCAGACCGGCAGCC 1300

. . . . .

CGGACGGAGGAGAAATTCCTCAAACAGAATTTCGGGCGACCTTA 1400

. . . . .

ACGACAAGGAACTGATCGTGCGGCCAGACCGGATTTACCGTACC 1500

. . . . .

infC gene of B. abortus (base position 324–860). The ATG

ribosome binding site is shown at position 310 (underlined). *,

ARTICLE IN PRESSM. Gonzalez et al. / Immunobiology 214 (2009) 113–120116

mice (12 weeks of age) were immunized in the footpadwith either 3 mg of rIF3, amount based on ourexperience with native IF3 (Cespedes et al., 2000) or25 mg of crude B. abortus RB51 soluble proteins crudeBrucella proteins (CBPs), obtained as previously de-scribed (Onate et al., 2000), which were emulsified inincomplete Freund‘s adjuvant (IFA; Sigma ChemicalCo., St. Louis, MO). Control animals were injected withPBS emulsified in IFA. Seven days later, mice weresacrificed, the popliteal lymph nodes (LN) wereremoved, teased and the resulting lymphocyte suspen-sion was adjusted to 4� 106 cells/ml in RPMI 1640medium containing 10% fetal calf serum and antibiotics.Then, 100 ml of the cellular suspensions were inoculatedinto each well of 96-well culture plates at 37 1C in anatmosphere of 5% CO2 and 96% humidity. The cellswere further cultured for 3 days and each well waspulsed for 8 h with 0.4 mCi of thymidine (40 Ci/mmol;Amersham, London, UK). At the end of this incubationperiod, cells were harvested and the radioactivityincorporated into the DNA of replicating cells wasmeasured using a liquid scintillation counter. Cellproliferation was expressed as mean counts per minuteobtained from triplicate cultures of pooled cells fromeach group. In addition, a stimulation index (SI) wascalculated for each experimental group by dividing thecounts per minute of cells stimulated with antigen by thecounts per minute of control cultures without antigen.

Fig. 2. Alignment of the predicted B. abortus IF3 sequence (BRU

Mesorhizobium loti (RHILO), Sinorhizobium meliloti (RHIME), A

Sequences were aligned by CLUSTAL W and highlighted by Boxsha

RHILO (Q98CP5), RHIME (Q92ST3), 9RHIZ (Q1YFN0) and ME

Cytokine detection

For detection of the cytokines secreted by thestimulated immune cells, culture supernatants of lym-phocytes were collected after 48 h of antigen stimulationand tested for the presence of IFN-g and IL-4 byantigen-capture ELISA using OptEIA Set Mouse INF-gand IL-4 kits (BD Biosciences, San Diego, CA). Allassays were performed in triplicate. The concentrationof IFN-g and IL-4 in the culture supernatants wascalculated by using a linear-regression equation ob-tained from the absorbance values of the standards.

Statistical analysis

The significance of the data for lymphocyte prolifera-tion and cytokine production was analyzed by theStudent’s paired t-test. A P-value of 0.05 or less wasconsidered as statistically significant.

Results and discussion

Cloning and sequence of the B. abortus IF3

Nucleotide sequencing of the 1542 bp EcoRI fragmentrevealed the presence of one complete and one partial

AB) with the amino acid sequences of the IF3 proteins of

urantimonas sp. (9RHIZ), and Mesorhizobium sp. (MESSB).

de. Source or references for sequences: BRUAB (AF_196569);

SSB (Q11KK4).

ARTICLE IN PRESSM. Gonzalez et al. / Immunobiology 214 (2009) 113–120 117

open reading frame (ORF). The complete ORF en-compassed 178 codons, starting at nucleotide position323 with an AUG as the start codon. The ORF extendedup to position 857, where a UGA stop codon waslocated (Fig. 1). The ORF sequence is preceded by apotential Shine-Dalgarno sequence (AGGAG) at �14 to�9 (Sambrook et al., 1989) (Fig. 1). The partial ORFwas homologous to the BMEI2009 gene coding for anickel–cobalt–cadmium resistance protein NCCN of B.

melitensis (Del Vecchio et al., 2002). The first ORF wascoding for a 20.4 kDa protein consisting of 178 aminoacids. Comparison of the complete ORF with thesequences available at the GenBank database showedthat this segment encoded a protein that showshomology with the IF3 protein expressed by manybacteria. The ORF showed identity with the IF3 ofMesorhizobium loti (80%; GenBank accession no.Q98CP5), Sinorhizobium meliloti (79%; GenBank acces-sion no. Q92ST3), Aurantimonas sp. (74%; GenBankaccession no. Q1YFN0) and Mesorhizobium sp. strainBNC1 (74%; GenBank accession no. Q11KK4) (Fig. 2).Further inspection of the alignment with the well-knownE. coli IF3 protein (IF3_ECOLI) showed that thededuced amino acid sequence of the putative infC gene

IF3_BRUAB MRRPFRATPVQKDGPRSNRDIRVPRV

IF3_ECOLI MKGGKRVQTARPN--RINGEIRAQEV

*: *. ..: : * * :**. .*

IF3_BRUAB NAEPPVCKIVDLGKLKYQNQKKAAEA

IF3_ECOLI NAEPPVCRIMDYGKFLYEKSKSSKEQ

*******:*:* **: *::.*.: *

IF3_BRUAB EEGDKVKVTLRFRGREMAHQELGMKL

IF3_ECOLI EEGDKAKITLRFRGREMAHQQIGMEV

*****.*:************::**::

IF3_BRUAB --

IF3_ECOLI KQ 180

Fig. 3. Comparison of the predicted B. abortus IF3 sequence (IF3_B

coli (IF3_ECOLI). Asterisks indicate identical amino acids, gaps a

conserved and semiconserved substitutions, respectively. Residues in

(see text).

of B. abortus (IF3_BRUAB) exhibits 54% identity withthe IF3_ECOLI. All residues which were described ascritical for IF3 biological functions were also conserved(either identical or similar) in IF3_BRUAB (Fig. 3).B. abortus IF3 possess the conserved Tyr and Lys atpositions 110 and 115, which correspond to theconserved Tyr107 and Lys110 residues of E. coli IF3that have been implicated in the ribosome bindingability of this protein (Bruhns and Gualerzi, 1980;DeBellis et al., 1992; Spurio et al., 1991). In E. coli,

proofreading activity has previously been attributed toTyr70 and Tyr75 (DeBellis et al., 1992; Spurio et al.,1991); in addition, Tyr75 has also been found to be acritical component of atypical start codon discrimina-tion. In the B. abortus IF3 Tyr70 is not present, but theother Tyr (position 75 in E. coli), is conserved atposition 78. Furthermore, Val54 and Ile15 of IF3 inE. coli are thought to be the hydrophobic residuesinvolved in contacting the C-terminal helix of theN-terminal domain (Garcia et al., 1995); in B. abortus

IF3 Val is conserved at position 57 and Ile15 is replacedby an uncharged polar residue, Ser18 (Fig. 3). Finally, inE. coli the residues 78–88 are known to correspond to abasic linker which possesses seven Lys residues (out of

QLIDAEGQNHGDVSIQEAMAMAEEAGLDLVEIVP 60

RLTGLEGEQLGIVSLREALEKAEEAGVDLVEISP 58

:* . **:: * **::**: *****:***** *

RKKQKTVEIKEIKMRPNIDTHDYEVKMKAAQRFF 120

KKKQKVIQVKEIKFRPGTDEGDYQVKLRSLIRFL 118

:****.:::****:**. * **:**::: **:

LQRVKEDTVEIAKVESEP-KLEGRQMMMVLAPR- 178

LNRVKDDLQELAVVESFPTKIEGRQMIMVLAPKK 178

*:***:* *:* *** * *:*****:*****:

RUAB) with the amino acid sequence of the IF3 protein of E.

re indicated by dashes, and double and single dots indicate

bold type are those thought to be essential for full IF3 function

ARTICLE IN PRESSM. Gonzalez et al. / Immunobiology 214 (2009) 113–120118

14) that connects the two domains of E. coli IF3 (Biouet al., 1995). In common with E. coli, the correspondingregion in Brucella possessed six basic residues (5 Lys and1 Arg), which indicate a similar function. In view of thevery high level of homology with the infC from otherbacterial species and the overall position similaritieswith the E. coli IF3, we conclude that the infC genecodes for IF3 homologue of the B. abortus strain RB51(AF196569). This sequence has 100% homology withthe infC gene from the standard virulent B. abortus

strain 2308 (GenBank accession no. YF_415442), B. ovis

strain ATCC 25840 (GenBank accession no.YF_001259916) and B. abortus strain 9941 (GenBankaccession no. YP_222750).

Fig. 4. Western blot showing the expression of B. abortus IF3

protein by recombinant E. coli. Proteins separated by 13%

SDS-PAGE were either stained using Coomassie blue (A) or

transferred to a nitrocellulose membrane and revealed using

peroxidase conjugated anti-His antibodies (B). Lanes 1–3

contain the antigens of E. coli, recombinant E. coli and

purified recombinant IF3 protein, respectively (10 mg of

protein per lane). Numbers at the left indicate the position

of the molecular mass markers in kDa.

Table 1. Murine lymphocyte proliferation in response to His-IF3a

Footpad injection Proliferative response to rIF3 at the fo

0 0.04

Saline 2540 2890 (1.1)

CBPs 3594 4401 (1.2)

rIF3 9119 52,129 (5.7)*

*Po0.01 and **Po0.05 when compared with the values from cells from maLymphocytes obtained on 7-day post-immunization were cultured at 4� 1

triplicate wells of cells obtained from a pool of five mice in each group. Nu

Expression of recombinant IF3 fusion protein

The whole protein extracts of E. coli cells containingthe recombinant protein were analyzed by Westernblotting using a mouse anti-His (C-term)-HRP mono-clonal antibody (Fig. 4B). The observed molecular massof the recombinant protein was slightly higher than thepredicted molecular weight of the IF3 fusion protein.The expressed recombinant His-IF3 protein was thenpurified by affinity chromatography on a His-Bindcolumn, according to the manufacturer’s procedure(Qiagen). Analysis of Coomassie blue stained polyacry-lamide gels showed that the purified His-IF3 runaberrantly in SDS-PAGE at a position equivalent to amass of 29 kDa (Fig. 4A). This may be due to the alteredmobility observed in some His6-tagged proteins (Babicet al., 1997).

Immunogenicity of the recombinant B. abortus IF3

protein

Next, we evaluated the immunological properties ofthe recombinant His-IF3 protein. To this end, mice werevaccinated in the footpads with either 3 mg of rIF3 or25 mg of CBPs emulsified in incomplete Freund‘sadjuvant. When lymphocytes from His-IF3 immunizedmice were restimulated in vitro with the recombinantprotein, significantly stronger T cell proliferative re-sponses were observed than in animals vaccinated withCBPs (Table 1). This indicated that the recombinantHis-IF3 antigen maintained intact its antigenic proper-ties. In mice, immunized with CBPs proliferativeresponses were only detected at a concentration of1 mg/well (Po0.05). This can be easily explained by lowamount of native IF3 antigen present in the wholeBrucella protein extracts. In vitro stimulation oflymphocytes from the saline control group neverreached a SI above 2, indicating that the responsesobserved in His-IF3 and CBPs-immunized mice wereIF3 specific. With respect to the cytokine profile,supernatant fluids of lymphocytes from IF3-immunizedanimals contain high levels of IFN-g when compared tocontrols (Po0.05 for every antigen concentration used).

llowing concentration (mg/well)

0.2 1

3256 (1.3) 2587 (1)

4638 (1.3) 8066 (2.3)**

55,976 (6.1)* 85,505 (9.4)*

ice in the saline control group.

05 cells per well. Each value represents the average counts per minute of

mbers in parentheses are the SIs for each group.

ARTICLE IN PRESS

0

50

100

150

200

250

300

350

400

10.20.040

INF-

γ [p

g/m

l]

IF3Crude ProteinPBS

*

*

*

Fig. 5. IFN-g secreted by lymphocytes upon stimulation with

His-IF3. Lymphocytes obtained from popliteal lymph nodes

from mice vaccinated in the footpad with either His-IF3 (K),

CBPs (’) or PBS (m) were stimulated with different

concentration of His-IF3 for 48 h (mg/ml). The concentration

of INF-g was then detected in supernatant fluids by ELISA.

Each point represents the geometric mean7standard deviation

(error bars) of the response in lymphocyte suspensions from

five individual mice. *, Po0.05 when compared with the values

from cells from mice in the saline control group.

M. Gonzalez et al. / Immunobiology 214 (2009) 113–120 119

In contrast, only a low level of IFN-g was detected insupernatants from cells of CBPs-immunized animals(Fig. 5). IL-4 was not secreted by lymphocytes from anyof the immunization groups (data not shown). Thisfinding is in agreement with our previous study in whichimmunization of mice intra-footpad with CPBs orB. abortus Cu–Zn SOD did not induce the productionof IL-4 (Onate and Folch, 1995; Onate et al., 2000). Wefailed to detect IF3-specific antibodies in the serum frommice vaccinated with His-IF3 at seven day post-immunization (data not shown). The time intervalbetween immunization and sampling was probably tooshort to see strong antibody responses.

Taken together, these results demonstrate that im-munization with the Brucella recombinant His-IF3protein promotes a strong Th-1 polarized immuneresponse. This information, together with our previouslypublished results (Cespedes et al., 2000), suggest thatHis-IF3 is a promising candidate antigen for thedevelopment of subunit vaccines against B. abortus.

Acknowledgments

This work was supported by Grants 1000431 and1050054 from the Fondo Nacional de InvestigacionCientıficay Tecnologica (FONDECYT), Santiago,Chile. We are grateful to Hugo Sepulveda (Universidadde Concepcion) for computer assistance in the align-ments of the sequences.

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