The 16MDvjbR as an ideal live attenuated vaccine candidate fordifferentiation between Brucella vaccination and infection

Yufei Wang a,1, Yaoxia Bai a,b,1, Qing Qu a,c,1, Jie Xu a,c, Yanfen Chen a,c, Zhijun Zhong a,Yefeng Qiu d, Tongkun Wang a, Xinying Du a, Zhoujia Wang a, Shuang Yu a,b, Simei Fu a,c,Jing Yuan a, Qing Zhen c, Yaqing Yu c, Zeliang Chen a,*, Liuyu Huang a,**a Department of Infectious Disease, Institute of Disease Control and Prevention, Academy of Military Medical Science, Beijing 100071, PR Chinab College of Pharmacy, Southwest University, Chongqing 400715, PR Chinac School of Public Health, Key Laboratory of Zoonosis, Ministry of Education, Jilin University, Changchun 130021, PR Chinad Laboratory Animal Center, Academy of Military Medical Science, Beijing 100071, PR China

Veterinary Microbiology 151 (2011) 354–362

A R T I C L E I N F O

Article history:

Received 27 December 2010

Received in revised form 22 March 2011

Accepted 28 March 2011

Keywords:

Brucella

Live attenuated vaccines

VjbR

A B S T R A C T

Brucellosis brings great economic burdens for developing countries. Live attenuated

vaccines are the most efficient means for prevention and control of animal Brucellosis.

However, the difficulties of differentiating of infection from vaccine immunization,

which is essential for eradication programs, limit their applications. Therefore, the

development of a vaccine that could differentiate infection from immunization will

overcome the limitations and get extensive application. VjbR is a quorum sensing

regulator involving in Brucella’s intracellular survival. The vjbR<Tn5 mutants have been

proven effective against wild type strain challenge, implying its possibility of use in

vaccine candidate development. To further evaluate this candidate gene, in the present

study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to

Brucella melitensis VjbR could be detected in sera from patients and animals with

brucellosis but not in control ones, implying the potential use of this protein as a

diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing

the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and

mice. Vaccination of BALB/c mice with 16MDvjbR conferred significant protective

immunity against B. melitensis strain 16M challenges, being equivalent to which induced

by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific

immunoglobulin G response and induced the secretion of gamma interferon and

interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in

association with diagnostic tests based on the VjbR antigen would allow the serological

differentiation between infected and vaccinated animals. These results suggest that

16MDvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves

further evaluation for vaccine development.

� 2011 Elsevier B.V. All rights reserved.

* Corresponding author at: Department of Infectious Disease Control, Beijing Institute of Disease Control and Prevention, No. 20, Dongdajie, Fengtai

District, Beijing 100071, PR China. Tel.: +86 10 66948434; fax: +86 10 66948434.

** Corresponding author at: Department of Infectious Disease Control, Beijing Institute of Disease Control and Prevention, No. 20, Dongdajie, Fengtai

District, Beijing 100071, PR China. Tel.: +86 10 66948301; fax: +86 10 66948301.

E-mail addresses: zeliangchen@yahoo.com (Z. Chen), huangly@nic.bmi.ac.cn (L. Huang).1 The authors contributed equally to this work.

Contents lists available at ScienceDirect

Veterinary Microbiology

jou r nal h o mep ag e: w ww .e ls evier . co m/lo c ate /vetm i c

0378-1135/$ – see front matter � 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2011.03.031

1. Introduction

Bacteria of the genus Brucella are Gram-negativefacultative intracellular pathogens of various domesticand wild mammals (Boschiroli et al., 2001; Young, 1983).Among the six species, Brucella melitenesis, Brucella suis andBrucella abortus are pathogenic and virulent for humans(Young, 1995). Due to serious economic loss and publichealth risk, extensive efforts have been conducted toprevent the disease in animals through vaccinationprograms. In the absence of protective antigen, liveattenuated vaccines have been proven to be the bestvaccines and are used worldwide for prevention againstanimal brucellosis (Nicoletti, 1990; Schurig et al., 2002).However, its use is known to induce antibody responsesindistinguishable by the current conventional serologicaltests from those observed in infected animals (Blasco,1997; Jimenez de Bagues et al., 1992; Moriyon et al., 2004).This fact limits the extended use of these vaccines incountries applying eradication programs based on ser-ological testing and slaughtering of sero-positive animals.Therefore, numerous efforts have been made to developnew vaccines by targeted deletion of virulence genes orantigenic ones (Alcantara et al., 2004; Briones et al., 2001;Cloeckaert et al., 2004; Grillo et al., 2006; Yang et al., 2010).

VjbR (BMEI1116), a member of the luxR-like quorumsensing-related transcriptional regulator, is required for virB

expression, virulence in mice, and survival in macrophages(Delrue et al., 2005; Uzureau et al., 2010). Previous study ofour lab indicates that virB also positively affect transcriptionof vjbR (Wang et al., 2009). The Tn5 insertion mutant of vjbR isproven to be attenuated in mice and macrophage. Encapsu-lated vjbR mutant induced a robust and sustained cellularand humoral response that correlated with high levels ofprotection against wild type strain challenge (Arenas-Gamboa et al., 2008). Research from the same lab alsoshowed that vjbR deletion mutant of S19 induces diminishedinflammation and is safer than S19 (Arenas-Gamboa et al.,2009). These data implied that vjbR maybe an ideal virulencegene for development of live attenuated vaccines.

To further test the possibility of vjbR mutant as a newvaccine candidate, in the present study, we used thepurified recombinant VjbR of B. melitensis to assess theantibody response to this protein in sera from patients andanimals with brucellosis. Then, we constructed a targetedvjbR deletion mutant 16MDvjbR and demonstrate themutant as a vaccine candidate against subsequent B.

melitensis exposure.

2. Materials and methods

2.1. Ethics statement

All animals were handled in strict accordance withExperimental Animal Regulation Ordinances defined byChina National Science and Technology Commission, andthe animal work was approved by Beijing Institute ofDisease Control and Prevention animal ethics committee(Ethical Approval BIDCP002-2009). Animals are providedwith humane care and healthful conditions during theirstay in the facility. All individuals who use animals

received instruction in experimental methods and in thecare, maintenance and handling of mice, and are under thecommittee’s supervision.

2.2. Bacterial strains, plasmids and mice

B. melitensis strain 16M and the vaccine strain Rev.1 wasobtained from the Center of Chinese Disease Prevention andControl. Brucella was cultured in tryptic soy broth (TSB) ortryptic soy agar (TSA). E. coli strain DH5a was grown onLuria–Bertani (LB) medium. Plasmid pBBR1MCS-2, a broadhost range plasmid capable of replicating in Brucella, waskindly provided by Professor Kenneth M. Peterson (Kovachet al., 1995). Mice were obtained from Experimental AnimalCenter of Academy Military Medical Science. All experi-mental procedures and animal care were performed incompliance with institutional animal care regulations.

2.3. Construction of the 16MDvjbR deletion mutant

To obtain deletion mutants, a new plasmid pUC19K wasfirstly constructed as follows. A pair of primers pUC19K-F(ACG TGG ATC CCT CGA GGG GCC CGC CAC CTG GGA TGAATG TC) and pUC19K-R (ACG TGT CGA CTC TAG AGA TATCAC GCG TCG GTC ATT TCG AAC CCC AGA) were synthesized,with restriction enzyme sites of BamHI, XhoI and ApaI addedto the 50 end of pUC19K-F and EcoRV, XbaI, MluI and SalI tothe 50 end of pUC19K-R. The kanamycin gene was amplifiedfrom pBBR1MCS-2 with primer pUC19K-F and pUC19K-R.PCR products were purified and digested with BamHI andSalI and cloned into the similarly digested plasmid pUC19,generating pUC19K, with a multi-cloning site on each side ofkanamycin gene. The recombinant plasmid pUC19K wasverified by restriction digestion and DNA sequencing.

The deletion mutant 16MDvjbR was constructed withpUC19K as follows. Two pairs of primers with restrictionsites at 50 ends were designed for amplification of theupstream (449 bp) and downstream (426 bp) arms of the B.

melitensis 16M vjbR ORF. Primer sequences are thefollowing: up-F, ACG TGG TAC CGA AGC ACA GGC CGAAAC GC; up-R, ACG TCT CGA GTA GCG GCA TCC TAT TTC A;and dn-F, ACG TGT CGA CAA TCG CCG AAA TCC TCA G; dn-R, ACG TAA GCT TAC CGC CTT CCA TCA CCC TC. The twohomologous arms were sequentially cloned into the twomulti-cloning sites (MCS) of pUC19K (with vjbR-U clonedat KpnI–XhoI sites and vjbR-D at SalI–HindIII sites) togenerate suicide plasmid pUC19K–vjbR. Competent 16Mwas electroporated with pUC19K–vjbR and potential vjbR

deletion mutant 16MDvjbR was isolated by its ampS kanR

phenotype. The deletion mutant was further confirmed byPCR amplification with primer pUC19K-F and vjbR-I-R(TGATCTCCGGCTGGTTGA), which located in kanamycingene and downstream of homologous arm of vjbR,respectively. PCR products were sequenced to confirmthe sequence. The deletion mutant was further confirmedby RT-PCR as described previously (Wang et al., 2009).

2.4. Complementation of the 16MDvjbR mutation

Genomic DNA of 16M was amplified with up-F and dn-R, and the products were ligated with pMD19-T vector

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362 355

(Takara) to generate vjbR-T, which was then transformedinto E. coli DH5a. Plasmid vjbR-T was isolated andelectroporated into competent 16MDvjbR. Then thecomplementary strain was selected on TSA containing100 mg/ml ampicilin and 50 mg/ml kanamycin. The tran-scription restoration of vjbR in the complementary strain16M–vjbR was further confirmed by RT-PCR.

2.5. Evaluation of 16MDvjbR attenuation in J774A.1

macrophages

Murine macrophage-like J774A.1 were used to assesssurvival capability of 16MDvjbR mutant, 16M–vjbR andtheir wild type strain 16M. Macrophage survival assayswere performed as previously described (Zhong et al.,2009). Briefly, mono-layers of macrophages of 1 � 105

cells/well were cultured in 24-well plate for 16 h at 37 8C,infected with Brucella at a MOI of 50. At 45 minpostinfection, the cells were washed twice with PBSand then incubated with 50 mg/ml of gentamicin for60 min to kill extra-cellular bacteria. Then, the culturewas replaced with DMEM with 25 mg/ml of gentamicin.At 0, 4, 24, 48 and 72 h post the infection, the supernatantwas discarded and cells were lysed, and the live bacteriawere enumerated by plating on TSA plates. All assayswere performed in triplicate and repeated at least threetimes.

2.6. Evaluation of 16MDvjbR attenuation in mice

Female 6-week-old BALB/c mice were inoculated intra-peritoneally (i.p.) with a total of 1 � 106 CFU of 16MDvjbR,16M, or 16M–vjbR. Infected mice were held in micro-isolator cages in biosafety level 3 facilities. Survival orpersistence of the bacteria in mice was evaluated byenumerate the bacteria in the spleens at different timepoints post the infection. At 1, 3, 7, 14, and 28 days post theinoculation, mice were euthanized and spleens wereremoved aseptically. The spleens were homogenized in1 ml PBS and serially diluted, followed by being plate onTSA plates. Recovered bacteria were enumerated toevaluate the survival capability in mice.

2.7. Immunization and antibody surveillance

Female 6-week-old BALB/c mice were randomlydivided into three groups. Group I and II were inoculatedintraperitoneally (i.p.) with 200 ml PBS containing1 � 106 CFU of 16MDvjbR or Rev.1 respectively, and groupIII was inoculated i.p. with 200 ml PBS as negative control.Serum samples were obtained from immunized mice at 2,4, 6, and 7 weeks post the immunization and IgG wasdetermined by ELISA. Briefly, heat-killed and sonicated B.

melitensis whole-cell antigen was used to coat 96-wellplates at a concentration of 25 mg protein/well. Afterovernight incubation at 4 8C, plates were washed once with100 ml PBST buffer (PBS containing 0.05% Tween-20) andblocked with 200 ml blocking buffer (10% heat-inactivatedfetal bovine serum in PBS, pH 7.4) for 2 h at 37 8C. Miceserum samples diluted 1:400 in the dilution buffer wereadded to wells in triplicate and incubated for 2 h at 37 8C.

The plates were washed three times with PBST to removethe unbound antibody, and then 100 ml of rabbit anti-mouse IgG–horseradish peroxidase conjugate at a dilutionof 1:1000 was added to each well and incubated at 37 8C for30 min. After two washes with PBS, 100 ml per well of TMBsubstrate solution was added and incubated at 37 8C indarkness for 15 min. The reaction was stopped by adding50 ml of H2SO4 and the absorbance was measured at450 nm (OD450).

2.8. Cytokine production assay

At selected time post vaccination, five mice from eachgroup were sacrificed and their spleens were asepticallyrecovered. Single cell suspensions from the spleens wereobtained by homogenization. Cells were pelleted at1000 rpm for 10 min, and then 10 ml red blood cell lysisbuffer (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA[pH7.3]) was added to the pellet for 5 min. After washingthree times with PBS, the cell suspension was re-suspended in complete RPMI 1640 medium (GIBCO BRL)supplemented with 10% (v/v) heat-inactivated fetal bovineserum and 2 mM L-glutamine. The cell were cultured in 96-well plates for 72 h at a concentration of 5 � 105 cells/wellin the presence of 25 mg of B. melitensis 16M lysate/well,0.5 mg of concanavalin A (ConA, positive control) ormedium alone (negative control), respectively. Then theplates were centrifuged at 1000 rpm for 10 min, the clearculture supernatants were collected and stored at �20 8C.IFN-g and IL-10 were determined using an ELISAQuantikine Mouse kit (R&D Systems). All assays wereperformed in triplicate and the concentration for eachcytokine in the culture supernatants was calculated using alinear regression equation obtained from the absorbancevalues of the standards according to the manufacturer’sprocedures.

2.9. Protection efficiency evaluation

At 6 weeks post vaccination, mice were challenged i.p.with 1 � 105 CFU per mouse (200 ml) of virulent strain16M. At 1 and 2 weeks post the challenge, mice were killedand the spleens were removed aseptically. Bacterial cellnumber in each spleen was determined as described above.To differentiate between the vaccine candidate and thechallenge strain, each dilution was also plate on TSA withkanamycin to identify any residual kanamycin-resistantvaccine strain present.

2.10. Cloning, expression, and purification of recombinant

proteins

The VjbR and L7/L12 open reading frame (ORF) wereamplified by PCR from DNA of B. melitensis 16M. Then theamplified DNA fragments were cloned into the pDEST17vector by using Gateway cloning technology, andexpressed in Escherichia coli BL21(DE3) as N-terminallyHis-tagged fusion proteins. The expression of the recom-binant protein was analyzed by SDS-PAGE. The recombi-nant fusion protein VjbR was then purified by affinitychromatography with Ni2-conjugated Sepharose.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362356

2.11. VjbR indirect ELISA

Serum samples from Brucella-infected goats wereprovided by China Institute of Veterinary Drug Control.Serum samples from patients with brucellosis wereprovided by Songyuan CDC, Jilin Province, China. Antibodyresponses to the purified recombinant VjbR protein wereassessed by VjbR based indirect ELISA as describedpreviously (Zygmunt et al., 1994).

2.12. Western blotting

The recombinant protein VjbR and L7/L12 cell lysateswere analyzed by SDS-PAGE (12.5%). Proteins were thentransferred to nitrocellulose for 40 min at 0.8 mA/cm2 bysemi-dry Western blotting with the transfer buffer(25 mM Tris, 192 mM glycine, and 20% methanol).Membrane was incubated in blocking solution (5% non-fat milk in TBST) for 2 h at room temperature. Then themembrane was incubated with Brucella-vaccinated seradiluted 1:300 in 2.5% milk/TBST at room temperature for1 h. After three washes, the bands were revealed withrabbit anti-mouse immunoglobulin G (peroxidase con-jugated) for 1.5 h at room temperature in 5% milk/TBST.After three washes, membrane was exposed to X-ray filmfor 1 h.

2.13. Statistical analysis

Bacteria survival in macrophage and in mice wasexpressed as the mean log10 CFU � the standard deviation(SD). Antibody response was expressed as the meanOD450� SD. Cytokine production in vitro was expressed asmean cytokine concentration � SD. The protective efficiencyat different time points was expressed as the meanlog10 CFU � SD. The differences between groups were ana-lyzed by ANOVA followed by Tukey’s honestly significantdifference post test comparing all groups to one another. ForANOVA, P values of <0.05 were considered statisticallysignificant.

3. Results

3.1. VjbR induced specific antibody response in Brucella-

infected animal and human sera

To test whether the VjbR protein induce antibodiesduring Brucella infection, a panel of human and animal sera,which were confirmed as positive or negative for Brucella

infection, were collected and analyzed both by slideagglutination test (SAT) and VjbR indirect ELISA. Antibodiesagainst VjbR were detected in 65 of 75 (86.7%) humanpositive sera, and 100 of 120 (83.3%) goat positive sera,respectively. On the other hand, antibodies were onlydetected in 3 of 25 (12.0%) negative human sera, and 5 of 40(12.5%) negative goat sera. This implied that VjbR inducedspecific antibody response in Brucella-infected human andgoat sera. To evaluate the specificity of VjbR as a diagnoseantigen, we compared the performances of VjbR based ELISAand SAT. As shown in Fig. 1, the number of SAT+ sera is higherthan that of VjbR-ELISA+ for both human and goat positivesera. However, on the other hand, the number of SAT+ sera isalso higher than that of VjbR-ELISA+ for both human andgoat negative sera. The higher number of SAT+ for negativesera indicated the higher cross reaction of SAT, which mightbe resulted from the lower specificity of Brucella LPS. Allthese data indicated that VjbR induce antibody responseboth in patients and animals with brucellosis, implying itspotential use as diagnostic antigen for Brucellosis.

3.2. Construction and complementation of the B. melitensis

16MDvjbR mutant

To obviate the possibility of Tn5 on phenotypes of vjbR

mutant, we attempted to construct a deletion mutant ofvjbR by resistance gene replacement. Firstly, a new suicideplasmid pUC19K was constructed by cloning the kanamy-cin gene into the multi-cloning site (MCS) of pUC19, withone MCS on either side of the resistance gene. Then, twohomologous arms of vjbR was cloned into the two MCSrespectively to generate recombinant suicide plasmidpUC19K-vjbR, followed by transformation into competent

Fig. 1. Induction of specific antibody against VjbR in human and goat sera. Human and goat sera were collected and defined as positive or negative according

to their epidemiologic background and laboratory confirmation by commercial kit. Sera samples were then analyzed by both SAT and VjbR–ELISA.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362 357

16M to generate 16MDvjbR. PCR verification and DNAsequencing indicated that the vjbR was correctly replacedby kanamycin gene (data not shown). The complementarystrain was constructed by integration of vjbR gene at thedisrupted locus of 16MDvjbR. RT-PCR showed that vjbR

was not transcribed in 16MDvjbR but were restored in16M-vjbR (data not shown). All these data indicated thatboth the vjbR mutant 16MDvjbR and complementarystrain 16M–vjbR were correctly constructed.

3.3. 16MDvjbR is attenuated for survival in macrophages

The vjbR is involved in Brucella’s intracellular survival inmacrophage. To further assess attenuation of 16MDvjbR,J774A.1 macrophages were infected with 16MDvjbR, 16M–vjbR, and 16M, then their survival and replication capabilityin macrophages were determined. Macrophages wereinfected with the three strains at a MOI of 50, and thesurviving bacteria were enumerated. At 0 h, there were nodifferences in the number of surviving bacteria in J774A.1cells, indicating the vjbR does not affect invasion of Brucella

into macrophage. By 4 h post-infection, equivalent bacterialoads were observed in the infected macrophages. However,at 24 h post-infection, there was a 1.3-log and 1.0-logdecrease (P < 0.001) in the bacteria number of 16MDvjbRcompared to that of 16M and 16M–vjbR respectively. And in72 h postinfection, this decrease was increased to 4.8-log(Fig. 2). These results showed that 16MDvjbR mutant has adecreased replication capability in J774A.1 macrophages,indicating that vjbR is involved in Brucella chronic infection.

3.4. 16MDvjbR is attenuated for survival in mice

To evaluate survival capability in vivo, BALB/c micewere inoculated intraperitoneally with 1 � 106 CFU of16MDvjbR, 16M, or 16M–vjbR. Compared to the wild-type strain 16M and the complementary strain 16M–vjbR,splenic CFU in 16MDvjbR infected mice were significantlyreduced at days 3, 7, 14, and 28 (data not shown). At 28days post-infection, only 0.5-log of 16MDvjbR was isolatedfrom the spleens of the infected mice, while 6 logs of 16M

and 16M–vjbR were isolated (P < 0.001). Moreover,spleens of 16MDvjbR infected mice were significant lighterthan that of 16M and 16M–vjbR infected ones (Fig. 3A andB), indicating 16MDvjbR-dosed mice have a significantlyreduced inflammation. All these results indicate that16MDvjbR is greatly attenuated in mice.

3.5. Protection of 16MDvjbR against 16M challenge

In order to determine the possibility of 16MDvjbR as avaccine candidate, the protection efficiency of 16MDvjbR

Fig. 2. Survival capability of 16MDvjbR in macrophage cells. J774.A1 cells

were infected with strains 16M, 16MDvjbR, 16M–vjbR at an m.o.i. of 50:1.

At the indicated time points p.i., cells were lysed and bacteria were

enumerated by plating serial dilutions on TSA plates. Significant

differences between the mutant and parent strain are indicated as

follows: *P < 0.001.

Fig. 3. Spleen weights in BALB/c mice during 16MDvjbR infection. BALB/c

mice were injected intraperitoneally with 1 � 106 CFU of 16M, 16MDvjbR

or 16M–vjbR. Infected mice were euthanized at 1, 3, 7, 14, and 28 days

post the inoculation. Spleens were aseptically removed and weighted. (A)

Splenomegaly of the infected mice at 14 days post the infection; (B)

Spleen weight changes during infection.

Fig. 4. Protective efficiencies of 16MDvjbR against 16M challenge. BALB/c

mice were immunized intraperitoneally with 1 � 106 CFU of either

16MDvjbR mutant or Rev.1. Control groups received PBS. At 6 weeks post

the infection, the mice were challenged intraperitoneally with

1 � 105 CFU of virulent strain 16M. At 1 and 2 weeks post the

challenge, the mice (n = 5/time point) were euthanized and spleens

were removed aseptically. The spleens were homogenated and bacteria

numbers were determined. Bacteria recovery numbers are expressed as

the means � SD of individual log10(CFU/spleen). Significant differences

between 16MDvjbR and other groups are indicated as follows: *P < 0.05,

with a and b indicating PBS and Rev.1 groups, respectively.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362358

was evaluated and compared with that of the Rev.1, anextensively applied vaccine strain. At 6 weeks post theimmunization, the mice were challenged with 1 � 105 CFUof B. melitensis virulence strain 16M, and 1 and 2 weekspost the challenge, Brucella in spleen were isolated andenumerated. Protection efficiency was defined as thedifference between the numbers of viable bacteriarecovered from spleens of inoculated mice and thoserecovered from spleens of mice receiving PBS, and wereexpressed as log10 units of protection. As shown in Fig. 4, at1 and 2 weeks post-challenge, mice vaccinated with16MDvjbR mutant exhibited a significant degree ofprotection against B. melitensis 16M (P < 0.001) whencompared with controls receiving PBS. In addition,compared with that of Rev.1 immunized mice, there wasa statistically decrease in the splenic bacterial loads in the16MDvjbR immunized mice, with a 0.6-log (P < 0.05)reduction at 1 week post-challenge. But at 2 weeks post-challenge, there were no significant difference (P > 0.05)between them. The results indicated that 16MDvjbRmutant could provide similar protection efficiency againstchallenge to that of the Rev.1 vaccine strain.

3.6. 16MDvjbR immunization induces both humoral and

cytokine responses

The production of Brucella-specific antibodies wasmeasured in the sera of the immunized mice. Sera frommice inoculated with 16MDvjbR or Rev.1 were collectedfrom immunized mice at selected intervals post-immuni-zation to monitor total IgG level by ELISA. As shown inFig. 5, antibodies were detected in sera of mice immunizedwith both the 16MDvjbR and the Rev.1 vaccine just at 2weeks post-vaccination, but not in that immunized withPBS, indicating 16MDvjbR could induce Brucella-specificantibodies. For mice vaccinated with the 16MDvjbR orRev.1 vaccine, the IgG levels peaked at 6 weeks post-vaccination and there were no significant differencebetween these two groups (P > 0.05). To determine the

magnitude of the memory response induced by thedifferent vaccine, mice were challenged at 6 weeks post-vaccination, and the IgG recall response was evaluated 1week post-challenge. Both 16MDvjbR and Rev.1 immu-nized mice underwent a rapid antibody recall, indicating16MDvjbR could generate a humoral immune memoryresponse.

To characterize the cellular immune response, thesplenocytes of immunized mice were isolated andstimulated with heat-inactivated B. melitensis 16M, com-plete medium 1640 (negative control) or ConA (positivecontrol), and cytokine secretion in the supernatant wasanalyzed. As expected, ConA stimulation induced produc-tion of IFN-g and IL-10 in spleen cells from all the threegroups, and no cytokine productions were induced by PBSstimulation in any of these groups. When stimulated withheat-inactivated B. melitensis 16M, significant productionsof IFN-g and IL-10 (P < 0.001) were observed in spleen cellsof 16MDvjbR or Rev.1 immunized mice, but not in PBSimmunized ones. Furthermore, a slightly higher cytokineproduction levels were observed in 16MDvjbR immunizedmice than Rev.1 in immunized ones (Fig. 6). This indicatedthat immunization of 16MDvjbR could induce cellularimmune responses.

3.7. Differentiation of 16MDvjbR immunization from

infection using VjbR as test antigen

The observation that VjbR elicited antibody response inpatients and animals with brucellosis prompted us toconsider whether it could be used as a diagnose antigen fordifferentiation between infected and vaccinated animals.

Fig. 5. Anti-Brucella antibodies in serum from mice immunized

with 16MDvjbR. BALB/c mice (n = 5/time point) were inoculated

intraperitoneally with 1 � 106 CFU of either 16MDvjbR or Rev.1.

Control groups received PBS. At 2, 4, 6, and 7 weeks post the

immunization, serum samples were collected and IgG antibodies were

determined by ELISA. The values are means � SD (n = 5) of the absorbance

at 450 nm (OD450). Significant differences between 16MDvjbR and other

groups are indicated as follows: *P < 0.001.

Fig. 6. Production of cytokines in stimulated spleen cells from 16MDvjbR

vaccinated BALB/c mice. BALB/c mice (n = 5/time point) were inoculated

intraperitoneally with 1 � 106 CFU of either 16MDvjbR or Rev.1. Control

groups received PBS. At 4 weeks post-vaccination, mice were euthanized,

and splenocytes were harvested and stimulated with either ConA, heat-

inactivated B. melitensis 16M, or RPMI 1640. The supernatants were

collected and IFN-g (A) and IL-10 (B) productions (pg/ml) were assayed by

ELISA. Cytokine production was expressed as the mean cytokine

concentration � SD (n = 5) for each group of mice. Significantly different

from the same stimulus in PBS-immunized mice are indicated as follows:

*P < 0.001.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362 359

To test this, sera from mice immunized with 16MDvjbR,16M, and PBS were collected. Western blotting wasperformed to determine whether the antibodies to VjbRand L7/L12 were induced in these sera. L7/L12 protein is aknown antigen of Brucella and used as a positive control inthis test (Oliveira and Splitter, 1996). For the positivecontrol L7/L12, reaction band was observed not only in seraof 16M infected mice but also in 16MDvjbR vaccinatedones. While for the protein VjbR, a single reaction band wasobserved in sera of 16M vaccinated mice but not in that of16MDvjbR, indicating that antibodies against VjbR wasinduced in 16M infected mice but not in 16MDvjbRvaccinated ones (Fig. 7A). Antibodies against the twoproteins were also detected by indirect ELISA. Beingconsistent with Western blot results, antibodies to L7/L12 were detected in sera of the two strains immunizedmice, and antibodies to VjbR were detected only in 16Mimmunized ones (Fig. 7B). These results implied that VjbRprotein could be used as a diagnostic antigen fordifferentiation between immunization and natural infec-tion.

4. Discussion

Most of the present license vaccines have severallimitations, such as residual virulence, splenomegaly, andinterference of serodiagnosis (Ashford et al., 2004; Berkel-man, 2003; Davis and Elzer, 2002; Schurig et al., 2002).Given these limitations, great efforts have been made todevelop new vaccine strains. Quorum sensing (QS) is aregulatory system for controlling gene expression inresponse to increasing cell density and involved invirulence for a wide variety of Gram negative bacteria(Fuqua and Winans, 1994). To date, two QS genes havebeen predicted in Brucella, one of which (vjbR) is involvedin Brucella virulence (Delrue et al., 2005; Taminiau et al.,2002; Uzureau et al., 2007). Given the importance of vjbR

to Brucella survival, several works focused on deleting thevjbR gene and determined whether the mutants arevaccine candidates. Published reports have shown thatencapsulated B. melitensis vjbR<Tn5 mutant could gen-erate higher level of protection against wild type challengein BALB/c mice than non-encapsulated one (Arenas-Gamboa et al., 2008). S19DvjbR is another vaccine

candidate constructed by the same laboratory. The mutantwas safer than S19, induced protection in mice, and shouldbe considered as a vaccine candidate when administered ina sustained-release manner (Arenas-Gamboa et al., 2009).These data indicate that vjbR is probably an ideal candidategene for creation of new vaccines strains. However, therewere no reports about the antigenicity to VjbR. In addition,the 16MDvjbR:Tn5 was constructed by random transpo-son-based mutagenesis strategies, which is not appro-priate for use as vaccine because of its instability andeffects of transposon on the mutant strain. To furtherevaluate the possibility of vjbR mutant as a new vaccinecandidate, in the present study, we have used purifiedrecombinant VjbR from B. melitensis to assess the antibodyresponse to this protein in sera from patients and animalswith brucellosis. Then, a resistance gene replacementmutant 16MDvjbR was constructed and its virulence andprotection efficacy were analyzed.

We constructed the deletion mutant of vjbR and itssingle-copy complementary strain, aiming to confirm thatreduced survival capability of the mutant is directly relatedwith the deleted gene vjbR. Transcription analysis indicatedthat vjbR was correctedly deleted in deletion mutant16MDvjbR and restored in complementary strain 16M–vjbR. The 16MDvjbR was evaluated for survival andattenuation in the macrophage and mouse models. Asshown in this study, the 16MDvjbR was much moresusceptible to macrophage killing than the wild-type16M, and this increased susceptibility was recovered inthe complementary strain. Moreover, 16MDvjbR was muchmore attenuated as evident from the in vivo colonizationstudy showing only 0.5-log 16MDvjbR was detected at 28days postinfection. These results indicated that 16MDvjbRmutant was defective for survival in macrophages andrapidly cleared from the spleen in BALB/c mice. This isconsistent with previous results and substantiated that vjbR

is involved in virulence of Brucella. Similarly, splenomegalywas observed in 16M and 16M–vjbR infected mice, but notin 16MDvjbR infected ones, implying reduced inflammatoryresponse exhibited by 16MDvjbR. The reduced virulenceand inflammatory response make it advantage for16MDvjbR to be a vaccine candidate.

Previous studies suggest that survival capability andpersistence in host are key aspects for an efficient Brucella

Fig. 7. Reaction of VjbR and L7/L12 to 16MDvjbR immunization sera. Sera from mice immunized with 16MDvjbR, 16M, and PBS were collected. The

antibodies to VjbR and L7/L12 were detected in these sera by Western blotting (A) and ELISA (B). Antibodies against the VjbR protein were not detected in

sera from 16MDvjbR immunized mice.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362360

live attenuated vaccine (Ko and Splitter, 2003; Stevenset al., 1995). For example, the loss of residual virulence ofRev.1 is associated with loss of protection efficiency both inmouse model and its preferential host (Blasco, 1997;Bosseray, 1991). Therefore the protective efficacy of the16MDvjbR mutant was investigated. Protection assaysshowed that vaccination of either 16MDvjbR or Rev.1could provide protection against virulent strain challenge,and furthermore, immunization with 16MDvjbR provideda slightly higher protection than Rev.1, indicating that the16MDvjbR mutant conferred a level of protection at leastequivalent to that conferred by the Rev.1.

The antibodies response and the cytokine profileselicited from splenocytes were also investigated toevaluate the degree of protection conferred by the16MDvjbR. Mice infected with 16MDvjbR produce anti-Brucella IgG and show a pattern similar to that of Rev.1.Vaccination efficacy is relative to the effective generationand maintenance of immunological memory. After chal-lenge, 16MDvjbR induced anti-Brucella IgG in 7 days,indicating the mutant can induce an immunologicalmemory response. Effective immunity against Brucella

requires cell-mediated mechanisms (Eze et al., 2000; Heet al., 2001). In particular, Th1 immune responsescharacterized by production of gamma interferon (IFN-g) are associated with protective immunity to Brucella. Andthese responses are best stimulated by live vaccines(Golding et al., 2001; Paranavitana et al., 2005). 16MDvjbRinduced higher level of IFN-g than that observed in naivemice. IL-10, a Th2 cytokine generally considered as an anti-inflammatory molecule, was also detectable in spleen cellsof 16MDvjbR infected mice. Although IL-10 can down-regulate protective immunity during primary B. abortus

infection (Fernandes et al., 1995), the results of our studyindicates that the induction of IL-10 does not diminish thedegree of protection against B. melitensis challenge, whichis in agreement with results reported by Pasquali et al.(2002). Taken together, these results indicated that16MDvjbR induced a mixed Th1 and Th2 cytokineresponse.

Serological diagnosis is the most convenient andextensively applied method for Brucellosis diagnosis.The lipopolysaccharide (LPS) of smooth Brucella speciesis by far the strongest antigen when compared to otherantigenic molecules. Consequently, Brucella LPS has beenconsidered the most important antigen during immuneresponse in brucellosis (de Bagues et al., 1992). Theserological procedures being used in the diagnosis ofanimal brucellosis are complement fixation test, roseBengal plate test, standard tube agglutination test, milkring test and enzyme-linked immunosorbent assay (Niel-sen, 2002). These tests are mainly based on the detection ofantibodies directed against the LPS portion of the cellmembrane. Therefore it is difficult to differentiate betweensera of vaccinated animals and which of infected onesusing LPS-based serological tests. In addition, tests basedon anti-LPS antibodies could give false positives because ofcross-reactivity with other Gram-negative bacteria likeYersinia enterocolitica O:9, Salmonella species, E. coli (Baldiet al., 1996). To evaluate the possibility of using VjbRprotein as diagnostic antigen, the VjbR was expressed in

vitro and used as coating antigen for detection its antibodyprofiles in different sera. As shown in Fig. 1, antibodies toVjbR could be detected in sera of patients and animals withbrucellosis by ELISA, but not in control ones, indicating thatVjbR could be used as a diagnose antigen for serologicaldiagnosis of brucellosis. To further confirm its use indifferentiation between infected and vaccinated animals,Western blot and indirect ELISA were used to differentiateimmunized sera from infected ones. The results showedthat antibodies to VjbR can be detected in infected sera, butnot in 16MDvjbR immunized ones, indicating thatimmunization sera could be differentiated from vaccina-tion ones using VjbR as diagnostic antigen. Therefore,advantages of the live attenuated vaccine candidate16MDvjbR over conventional ones include reducedvirulence, high protection efficiency, and most impor-tantly, it provides choice for differentiation of vaccinationfrom infection.

In summary, in the present study, we firstly constructeda resistance gene replacement mutant of vjbR and itscomplementary strain. 16MDvjbR showed reduced survi-val in macrophage and mice compared with its wild typestrain and complementary strain, confirming that vjbR isessential for Brucella’s virulence. Immunization of16MDvjbR induced a mixed humoral and cell-mediatedimmune response, and provided protection against B.

melitensis virulence 16M challenge. In addition, the VjbRprotein is an immunodominant antigen, inducing specificantibodies both in human and animals. Antibodies to VjbRare not detected in 16MDvjbR immunized sera, providingan ideal diagnostic antigen for differentiation of immuni-zation from infection. Taken together, 16MDvjbR is anideal vaccine candidate with reduced residual virulenceand high protection efficiency. And of the most impor-tance, VjbR is a better diagnostic antigen that could be usedto differentiate vaccinated and infected animals byserological diagnosis.

Acknowledgements

This work was supported by National Key Program forInfectious Diseases of China (2008ZX10004-015), NationalBasic Research Program of China (Grant No.2009CB522602), and the National Natural Science Foun-dation of China (30901071, 810713203,1000041), National863 program (2007AA02Z412).

References

Alcantara, R.B., Read, R.D., Valderas, M.W., Brown, T.D., Roop 2nd, R.M.,2004. Intact purine biosynthesis pathways are required for wild-typevirulence of Brucella abortus 2308 in the BALB/c mouse model. Infect.Immun. 72, 4911–4917.

Arenas-Gamboa, A.M., Ficht, T.A., Kahl-McDonagh, M.M., Gomez, G., Rice-Ficht, A.C., 2009. The Brucella abortus S19 DeltavjbR live vaccinecandidate is safer than S19 and confers protection against wild-typechallenge in BALB/c mice when delivered in a sustained-releasevehicle. Infect. Immun. 77, 877–884.

Arenas-Gamboa, A.M., Ficht, T.A., Kahl-McDonagh, M.M., Rice-Ficht, A.C.,2008. Immunization with a single dose of a microencapsulatedBrucella melitensis mutant enhances protection against wild-typechallenge. Infect. Immun. 76, 2448–2455.

Ashford, D.A., di Pietra, J., Lingappa, J., Woods, C., Noll, H., Neville, B.,Weyant, R., Bragg, S.L., Spiegel, R.A., Tappero, J., Perkins, B.A., 2004.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362 361

Adverse events in humans associated with accidental exposure to thelivestock brucellosis vaccine RB51. Vaccine 22, 3435–3439.

Baldi, P., Giambartolomei, G., Goldbaum, F., Abdon, L., Velikovsky, C.,Kittelberger, R., Fossati, C., 1996. Humoral immune response againstlipopolysaccharide and cytoplasmic proteins of Brucella abortus incattle vaccinated with B. abortus S19 or experimentally infected withYersinia enterocolitica serotype 0:9. Clin. Vaccine Immunol. 3, 472.

Berkelman, R.L., 2003. Human illness associated with use of veterinaryvaccines. Clin. Infect. Dis. 37, 407–414.

Blasco, J.M., 1997. A review of the use of B. melitensis Rev 1 vaccine in adultsheep and goats. Prev. Vet. Med. 31, 275–283.

Boschiroli, M.L., Foulongne, V., O’Callaghan, D., 2001. Brucellosis: a world-wide zoonosis. Curr. Opin. Microbiol. 4, 58–64.

Bosseray, N., 1991. Brucella melitensis Rev. 1 living attenuated vaccine:stability of markers, residual virulence and immunogenicity in mice.Biologicals 19, 355–363.

Briones, G., Inon de Iannino, N., Roset, M., Vigliocco, A., Paulo, P.S., Ugalde,R.A., 2001. Brucella abortus cyclic beta-1,2-glucan mutants havereduced virulence in mice and are defective in intracellular replica-tion in HeLa cells. Infect. Immun. 69, 4528–4535.

Cloeckaert, A., Jacques, I., Grillo, M.J., Marin, C.M., Grayon, M., Blasco, J.M.,Verger, J.M., 2004. Development and evaluation as vaccines in mice ofBrucella melitensis Rev.1 single and double deletion mutants of thebp26 and omp31 genes coding for antigens of diagnostic significancein ovine brucellosis. Vaccine 22, 2827–2835.

Davis, D.S., Elzer, P.H., 2002. Brucella vaccines in wildlife. Vet. Microbiol.90, 533–544.

de Bagues, M., Marin, C., Blasco, J., Moriyon, I., Gamazo, C., 1992. An ELISAwith Brucella lipopolysaccharide antigen for the diagnosis of B. meli-tensis infection in sheep and for the evaluation of serologicalresponses following subcutaneous or conjunctival B. melitensis strainRev 1 vaccination. Vet. Microbiol. 30, 233–241.

Delrue, R.M., Deschamps, C., Leonard, S., Nijskens, C., Danese, I., Schaus,J.M., Bonnot, S., Ferooz, J., Tibor, A., De Bolle, X., Letesson, J.J., 2005. Aquorum-sensing regulator controls expression of both the type IVsecretion system and the flagellar apparatus of Brucella melitensis.Cell. Microbiol. 7, 1151–1161.

Eze, M.O., Yuan, L., Crawford, R.M., Paranavitana, C.M., Hadfield, T.L.,Bhattacharjee, A.K., Warren, R.L., Hoover, D.L., 2000. Effects of opso-nization and gamma interferon on growth of Brucella melitensis 16M inmouse peritoneal macrophages in vitro. Infect. Immun. 68, 257–263.

Fernandes, D., Benson, R., Baldwin, C., 1995. Lack of a role for natural killercells in early control of Brucella abortus 2308 infections in mice. Infect.Immun. 63, 4029.

Fuqua, W., Winans, S., 1994. A LuxR–LuxI type regulatory system activatesAgrobacterium Ti plasmid conjugal transfer in the presence of a planttumor metabolite. J. Bacteriol. 176, 2796.

Golding, B., Scott, D.E., Scharf, O., Huang, L.Y., Zaitseva, M., Lapham, C.,Eller, N., Golding, H., 2001. Immunity and protection against Brucellaabortus. Microbes Infect./Inst. Pasteur 3, 43–48.

Grillo, M.J., Manterola, L., de Miguel, M.J., Munoz, P.M., Blasco, J.M.,Moriyon, I., Lopez-Goni, I., 2006. Increases of efficacy as vaccineagainst Brucella abortus infection in mice by simultaneous inoculationwith avirulent smooth bvrS/bvrR and rough wbkA mutants. Vaccine24, 2910–2916.

He, Y., Vemulapalli, R., Zeytun, A., Schurig, G.G., 2001. Induction of specificcytotoxic lymphocytes in mice vaccinated with Brucella abortus RB51.Infect. Immun. 69, 5502–5508.

Jimenez de Bagues, M.P., Marin, C.M., Blasco, J.M., Moriyon, I., Gamazo, C.,1992. An ELISA with Brucella lipopolysaccharide antigen for thediagnosis of B. melitensis infection in sheep and for the evaluation

of serological responses following subcutaneous or conjunctival B.melitensis strain Rev 1 vaccination. Vet. Microbiol. 30, 233–241.

Ko, J., Splitter, G.A., 2003. Molecular host–pathogen interaction in bru-cellosis: current understanding and future approaches to vaccinedevelopment for mice and humans. Clin. Microbiol. Rev. 16, 65–78.

Kovach, M., Elzer, P., Steven Hill, D., Robertson, G., Farris, M., Roop, R.,Peterson, K., 1995. Four new derivatives of the broad-host-rangecloning vector pBBR1MCS, carrying different antibiotic-resistancecassettes. Gene 166, 175–176.

Moriyon, I., Grillo, M.J., Monreal, D., Gonzalez, D., Marin, C., Lopez-Goni, I.,Mainar-Jaime, R.C., Moreno, E., Blasco, J.M., 2004. Rough vaccines inanimal brucellosis: structural and genetic basis and present status.Vet. Res. 35, 1–38.

Nicoletti, P., 1990. Vaccination against Brucella. Adv. Biotechnol. Process.13, 147–168.

Nielsen, K., 2002. Diagnosis of brucellosis by serology. Vet. Microbiol. 90,447–459.

Oliveira, S.C., Splitter, G.A., 1996. Immunization of mice with recombinantL7/L12 ribosomal protein confers protection against Brucella abortusinfection. Vaccine 14, 959–962.

Paranavitana, C., Zelazowska, E., Izadjoo, M., Hoover, D., 2005. Interferon-gamma associated cytokines and chemokines produced by spleencells from Brucella-immune mice. Cytokine 30, 86–92.

Pasquali, P., Adone, R., Gasbarre, L., Pistoia, C., Ciuchini, F., 2002. Effect ofexogenous interleukin-18 (IL-18) and IL-12 in the course of Brucellaabortus 2308 infection in mice. Clin. Diagn. Lab. Immunol. 9, 491.

Schurig, G.G., Sriranganathan, N., Corbel, M.J., 2002. Brucellosis vaccines:past, present and future. Vet. Microbiol. 90, 479–496.

Stevens, M.G., Olsen, S.C., Pugh Jr., G.W., Brees, D., 1995. Comparison ofimmune responses and resistance to brucellosis in mice vaccinatedwith Brucella abortus 19 or RB51. Infect. Immun. 63, 264–270.

Taminiau, B., Daykin, M., Swift, S., Boschiroli, M.L., Tibor, A., Lestrate, P., DeBolle, X., O’Callaghan, D., Williams, P., Letesson, J.J., 2002. Identifica-tion of a quorum-sensing signal molecule in the facultative intracel-lular pathogen Brucella melitensis. Infect. Immun. 70, 3004–3011.

Uzureau, S., Godefroid, M., Deschamps, C., Lemaire, J., De Bolle, X.,Letesson, J.J., 2007. Mutations of the quorum sensing-dependentregulator VjbR lead to drastic surface modifications in Brucella meli-tensis. J. Bacteriol. 189, 6035–6047.

Uzureau, S., Lemaire, J., Delaive, E., Dieu, M., Gaigneaux, A., Raes, M., Bolle,X., Letesson, J., 2010. Global analysis of quorum sensing targets in theintracellular pathogen Brucella melitensis 16M. J. Proteome Res. 9,3200–3217.

Wang, Y., Chen, Z., Qiao, F., Ying, T., Yuan, J., Zhong, Z., Zhou, L., Du, X.,Wang, Z., Zhao, J., Dong, S., Jia, L., Yuan, X., Yang, R., Sun, Y., Huang, L.,2009. Comparative proteomics analyses reveal the virB of B. meli-tensis affects expression of intracellular survival related proteins.PLoS One 4, e5368.

Yang, X., Thornburg, T., Walters, N., Pascual, D.W., 2010. DeltaznuADel-tapurE Brucella abortus 2308 mutant as a live vaccine candidate.Vaccine 28, 1069–1074.

Young, E., 1983. Human brucellosis. Rev. Infect. Dis. 5, 821–842.Young, E.J., 1995. An overview of human brucellosis. Clin. Infect. Dis. 21,

283–289 quiz 290.Zhong, Z., Wang, Y., Qiao, F., Wang, Z., Du, X., Xu, J., Zhao, J., Qu, Q., Dong, S.,

Sun, Y., Huang, L., Huang, K., Chen, Z., 2009. Cytotoxicity of Brucellasmooth strains for macrophages is mediated by increased secretion ofthe type IV secretion system. Microbiology 155, 3392–3402.

Zygmunt, M.S., Debbarh, H.S., Cloeckaert, A., Dubray, G., 1994. Antibodyresponse to Brucella melitensis outer membrane antigens in naturallyinfected and Rev1 vaccinated sheep. Vet. Microbiol. 39, 33–46.

Y. Wang et al. / Veterinary Microbiology 151 (2011) 354–362362