ARTICLE IN PRESS

Immunobiology 214 (2009) 467–474

0171-2985/$ - se

doi:10.1016/j.im

Abbreviations

virus; CBPs, cru�CorrespondE-mail addr

www.elsevier.de/imbio

Vaccination with recombinant Semliki Forest virus particles expressing

translation initiation factor 3 of Brucella abortus induces protective

immunity in BALB/c mice

Alex Cabrera, Darwin Saez, Sandra Cespedes, Edilia Andrews, Angel Onate�

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

Received 14 August 2008; received in revised form 19 November 2008; accepted 25 November 2008

Abstract

Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression ofheterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinantRNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challengewith B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies atday 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) orcrude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, butnot interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challengewith the virulent B. abortus strain 2308 (Po0.01). These findings indicate that an SFV-based vector carrying RNAencoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.r 2008 Elsevier GmbH. All rights reserved.

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

Introduction

Brucella abortus, a gram-negative facultative intracel-lular bacterium, is the causative agent of brucellosis, achronic disease of cattle and other animals, includinghumans (Corbel, 1997). Resistance to B. abortus

depends on acquired cell-mediated immunity (CMI),especially on antigen (Ag)-specific T cell activation(Zhan and Cheers, 1993). The CD4+ T helper (Th) 1subset and CD8+ T cells are known to be involved in

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

bio.2008.11.016

: CMI, cell mediated immunity; SFV, Semliki Forest

de Brucella proteins; IF3, translation initiation factor 3.

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

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

mediating protection against B. abortus challenge inmice (Oliveira et al., 1998; He et al., 2001). Animportant T cell-mediated mechanism responsible forprotection is through secretion of interferon gamma(IFN-g), which up-regulates anti-Brucella activity ofmacrophages, the main host cellular reservoir for thebacterium (Oliveira et al., 1998). Live attenuatedvaccines, such as the B. abortus strains S19 and RB51,which are able to stimulate a strong CMI, are usuallyvery effective against brucellosis. Therefore, they havebeen exploited to control brucellosis in domestic animals(Cheville et al., 1996; Confer et al., 1985). However,these vaccine preparations are far from ideal, since theyexhibit several disadvantages, e.g., they are consideredtoo virulent or unsafe for human use, and they can

ARTICLE IN PRESSA. Cabrera et al. / Immunobiology 214 (2009) 467–474468

induce abortion in pregnant cattle (Corner and Alton1981; Smith and Ficht, 1990; Palmer et al., 1996;Ashford et al., 2004). Hence, a vaccine that is notinfectious for humans but remains able to stimulatestrong protective responses is necessary to controlbrucellosis. To develop the next generation of Brucella

vaccines, several research groups are pursing differentstrategies including the development of subunit vaccines(Oliveira and Splitter, 1996), utilization of bacterialvectors (Onate et al., 1999), DNA vaccines (Velikovskyet al., 2002; Al-Mariri et al., 2001; Onate et al., 2003),and RNA vaccine (Onate et al., 2005).

Animal cell expression vector systems based on RNAreplicons of alphaviruses have recently emerged as apotent tool for delivering genetic vaccines, mainlybecause of their ability to infect different types of cells,thereby achieving transfection efficiencies that are muchhigher than those obtained by conventional DNAvaccines (Liljestrom and Garoff, 1991; Zhou et al.,1994; Polo et al., 1999). RNA vaccines based on SemlikiForest virus (SFV) possess a self-replicating positivestrand RNA called replicon encoding the viral replicaseand the cloned foreign gene, which is packaged intoinfectious but replication-defective particles. Because ofthe absence of genes coding for structural proteins, theSFV particles cannot produce infectious progeny, whichis in turn an ideal feature for increased biosafety ofvaccines based this expression system (Zhou et al., 1994;Polo et al., 1999; Schlesinger, 2001). After cell infectionby viral particles, RNA replication allows expression ofthe foreign gene in infected cells, thereby resulting in theinduction of strong humoral and CMI (Berglund et al.,1999; Fleeton et al., 1999; Fleeton et al., 2000; Nilssonet al., 2001). Our previous research showed that therecombinant SFV particles containing the gene forCu–Zn superoxide dismutase of Brucella can inducesignificant levels of protection in mouse and bovinemodels of brucellosis (Onate et al., 2005; Saezet al., 2008). We have previously demonstrated thata 22.9-kDa protein of B. abortus can induce CMIand protection in mice (Cespedes et al., 2000). Thisprotein was later identified as the translation initiationfactor 3 (IF3) (Gonzalez et al., 2008). The objective ofthe present study was to evaluate the immunogenicityand protective efficacy of a vaccine based on SFVparticles carrying the Brucella IF3 gene (SFV-IF3particles).

Materials and methods

Animals

Seven- to eight-week-old female BALB/c mice(obtained from Instituto de Salud Publica, Santiago,

Chile) were acclimated and randomly distributed intoexperimental groups. The mice were kept in conven-tional animal facilities and received water and food adlibitum.

Bacterial strains

The virulent B. abortus strain 2308 and the attenuatedstrain RB51 were obtained from our culture collection.The strain RB51 was originally obtained from theVirginia–Maryland Regional College of VeterinaryMedicine (Virginia Polytechnic Institute and StateUniversity, Blacksburg). Bacteria were grown underaerobic conditions in tryptose soy broth (Difco Labora-tories, Detroit, Mich.) for 72 h at 37 1C. All experimentswith live Brucellae were performed at a biosafetylevel 3 facility. The Escherichia coli strain DH5a (LifeTechnologies, Gaithersburg, Md.) was used for produ-cing the necessary plasmid constructs. E. coli wereroutinely grown at 37 1C in Luria–Bertani broth or agarsupplemented, when required, with 100 mg of ampicillinper ml.

Plasmid preparation

The vectors pSFV4.2, pSFV helper-S (pSFVhS),and pSFV helper-CS219A (pSFVhC) were generouslyprovided by Peter Liljestrom (Karolinska Institute,Stockholm, Sweden). The recombinant plasmidpBGt11, containing the gene encoding B. abortus IF3(infC), was initially obtained from a pUC19 genomiclibrary of B. abortus RB51 (Gonzalez et al., 2008). A536-bp fragment containing the infC gene was PCRamplified from the plasmid pBGt11, and subcloned inSFV4.2 downstream to the viral replicase gene andunder the control of the SP6 RNA polymerase promoter(Liljestron an Garoff, 1991). The recombinant plasmid,named pSFV-IF3, was electroporated into E. coli

DH5a, and a single recombinant colony was selected.The in vitro transcription and packaging of SFVparticles was performed under the same protocoldescribed by Onate et al., 2005.

Immunization

Mice were immunized by intraperitoneal route with106 infectious units of recombinant SFV-IF3 particles in200 ml of PBS at day 1 and 21. Mice in the negativecontrol group received PBS only. A third group of micewas vaccinated intraperitoneally with 2� 108 CFU of B.

abortus strain RB51 in 0.2ml of PBS at time zero, as apositive control.

ARTICLE IN PRESSA. Cabrera et al. / Immunobiology 214 (2009) 467–474 469

ELISA for antibodies against rIF3

The presence of serum immunoglobulin G (IgG) andM(IgM) with specificity to IF3 was determined by indirectELISA following a standard protocol (Onate et al., 2003)Purified recombinant IF3 protein (rIF3) obtained asdescribed earlier (Gonzalez et al., 2008), and diluted to3mg/ml in carbonate–bicarbonate buffer (pH 9.6) to coatthe antigen at a polystyrene plate. Isotype-specific rabbitanti-mouse horseradish peroxidase conjugates (ICNBiomedicals, Inc., Aurora, Ohio) were added at a 1:1000dilution. The results of total IgM and IgG are expressedas the mean OD490 at a serum dilution of 1:100.

Splenocyte cultures and lymphocyte proliferation

Three weeks after the last immunization, five mice fromeach group were euthanized, and their spleens wereremoved under aseptic conditions. Single-cell suspensionswere prepared from the spleens according to a standardprocedure (Onate et al., 2003). Splenocytes from all fivemice of each group were first pooled and then cultured at37 1C with 5% CO2 in a 96-well flat-bottom plate at aconcentration of 4� 105 viable cells/well in the presence ofno additives (unstimulated control) or one of thefollowing different stimulants: 0.4mg of crude B. abortus

RB51 proteins (CBPs), an extract obtained from bacteriasubjected to a treatment with a hypertonic salt solutionand sonication (Onate et al., 2000); 0.04mg of purifiedrIF3; or 0.25mg of Concanavalin A (ConA). Splenocyteswere cultured in c-RPMI for 3 days and pulsed for 8hwith 0.35mCi of [3H]thymidine (50Ci/mmol; Amersham,London, UK) per well. The radioactivity incorporatedinto the cellular DNA was measured using a liquidscintillation counter. Cell proliferation was expressed asmean counts per minute obtained from triplicate culturesprepared from a cell pool of each experimental or controlgroup composed of five mice.

Cytokine ELISAs

For detection of cytokines, culture supernatants ofspleen cells were collected after 48 h of antigen stimula-tion and tested for the presence of the cytokines byantigen-capture ELISA using OptEIA Set Mouse IFN-gand IL-4 (BD Biosciences, San Diego, Calif.). All assayswere performed in triplicate. The concentration ofINF-g and IL-4 in the culture supernatants wascalculated by using a linear-regression equation ob-tained from the absorbance values of the standards.

Protection experiments

The protection experiments were performed as pre-viously described (Onate et al., 1999). Briefly, 6 weeks

after vaccination, six mice from each group werechallenged by intraperitoneal injection of 104 CFU ofB. abortus 2308 per animal. Two weeks later, infectedmice were sacrificed, their spleens were homogenized,and dilutions were plated to determine the number ofBrucella CFU per spleen. Log10 units of protection werecalculated by subtracting the mean log10 CFU forthe experimental group from the mean log10 CFU forthe corresponding control group.

Statistical analysis

The data corresponding to the evaluation of the levelof antibodies, lymphocyte proliferation, detection ofcytokines, and protection experiments were analysed bythe student’s paired t test. A P value of 0.05 or less wasconsidered statistically significant.

Results

Immune response in mice immunized with SFV-IF3

particles

Indirect ELISA was used to detect the level of anti-IF3 IgG and IgM antibodies in serum of miceinoculated with SFV-IF3, B. abortus RB51 or PBS. Asshown in Fig. 1A, 14 days after immunization the serafrom mice immunized with SFV-IF3 contained asignificant level of IF3-specific IgM [OD490 ¼ 0.40670.066] (Po0.01 in comparison with the PBS controlgroup). At day 28 [0.19270.01] and 42 [0.13670.004]only basal levels of IgM were observed in SFV-IF3,which were not significantly different from the valuesobserved in the PBS inoculated group. In miceimmunized with RB51, the level of specific IgM[OD490 ¼ 0.828570.043] was significantly differentfrom the PBS inoculated group at day 14 post-immunization (Po0.01), differences maintained at day28 [OD490 ¼ 0.7047 0.032] and 42 [OD490 ¼ 0.61770.035]. The level of specific IgG present in sera fromanimals of the RB51 immunized group was significantlydifferent (Po0.01) from the PBS inoculated controlgroup at all time points, showing a continuous risebetween days 28 and 42 (Fig. 1B). The results for theSFV-IF3 group were not significantly different fromthe values observed in the PBS inoculated group at anyof the assayed days.

To examine the generated CMI specific to CBP andrIF3, the proliferative responses and cytokine secretionprofiles of spleen cells from mice inoculated with SFV-IF3, B. abortus RB51 and PBS were determined. Asshown in Fig. 2, spleen cells from mice immunized withSFV-IF3 exhibit significant T cell proliferative responsesupon stimulation with CBP and rIF3 (Po0.05 in

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14 28 420.0

0.2

0.4

0.6

0.8

1.0

***

Days

O.D

.

14 28 420.0

0.2

0.4

0.6

0.8

1.0

*

*

**

Days

O.D

.

Fig. 1. IF3 specific antibodies. Mice in each group were inoculated with SFV-IF3 (’: 106 viral particles in 200 ml PBS), B. abortus

strain RB51 (m: 2� 108 CFU) or PBS (�: 200 ml). Sera obtained at days 14, 28 and 42 post-immunization were diluted 1:100 and

used in the assay. The IgM and IgG levels are shown in panels A and B, respectively. Each time point represents the mean

OD7standard deviation (error bars) of antibodies collected from seven animals. The statistical significances are represented by

*(Po0.01), as compared with the control PBS group.

0

5000

10000

15000

20000

25000 PBSSFV-IF3RB51

CBP's rIF3

∗

∗

∗

3H th

ymid

ine

Fig. 2. Lymphocyte proliferation assay. BALB/c mice were

inoculated with recombinant SFV-IF3 particles, B. abortus

RB51 or PBS. The T-cell proliferative response was measured

3 weeks after the last inoculation. Splenocytes (4� 105 cells per

well) from five mice of each group were pooled and stimulated

in vitro with CBPs (10 mg/ml) and purified rIF3 (2 mg/ml). Each

bar indicates the average number of counts per minute for

triplicate cultures of cells; the error bars indicate standard

deviations. Groups with asterisk are significantly different

from the corresponding PBS inoculated groups (Po0.05).

CBP 250

500

750

1000

1250PBS

SFV-IF3

RB51

*

∗∗pg

/ml I

NF-

γ

rIF3

Fig. 3. IFN-g production upon stimulation with different

Brucella antigens. Splenocytes (4� 105 cells per well) from five

mice of each group were pooled and stimulated in vitro with

CBPs (50mg/ml), rIF3 (2 mg/ml), or RPMI 1640 (negative

control) for 24 h. Each bar indicates the geometric mean of the

response. Groups with asterisk are significantly different from

the corresponding PBS inoculated groups (Po0.05).

A. Cabrera et al. / Immunobiology 214 (2009) 467–474470

comparison to the PBS inoculated group). On theother hand, splenocytes from mice immunized withB. abortus RB51 exhibited a significant level ofproliferative response (Po0.05) when stimulated withCBPs (Fig. 2A), but not with rIF3 (Fig. 2B). Only lowlevels of spontaneous proliferation occurred in cultureswith no antigen stimulation (medium control). Spleno-cytes from mice of all groups had similarly high levels ofproliferative response to the mitogen Concanavalin A(data not shown). With respect to the cytokine profiles,at 6 weeks after immunization, the supernatants fromcultures of rIF3 stimulated spleen cells from SFV-IF3and RB51 vaccinated mice contained significantly higherconcentration of INF-g (Po0.05) (Fig. 3). No signifi-cant increment in INF-g secretion was observed insupernatant fluids of CBP stimulated spleen cells of

SFV-IF3-immunized mice (Fig. 3). In addition, no IL-4was detected in any of the culture supernatantsof splenocytes stimulated with the specific antigens(data not shown).

SFV-IF3 particles induce significant protection

against virulent B. abortus infection

Protection experiments were carried out by challen-ging vaccinated and control mice by intraperitonealinjection of the virulent B. abortus 2308 strain. Theresults indicated that immunization with SFV-IF3resulted in a significant degree of protection (1.09 logincrease in protection) compared to the PBS-inoculatedcontrol (Po0.01). In comparison, vaccination with live

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Table 1. Protection of mice against challenge with B. abortus

2308 after immunization with Semiliki Forest virus coding for

translation initiation factor 3 (IF3) homologue of B. abortusa.

Vaccine

(dose)

Log10 CFU of B. abortus

2308 in spleen (mean7SD)

Log10 units

of protection

PBS 6.9670.46 0

SFV-IF3 5.8770.38 1.09b

Live RB51

(2� 108CFU)

5.0470.51 1.91c

aMice were challenged intraperitoneally with 104 CFU of the 2308

strain 2 weeks prior to sacrifice.bPo0.01.cPo0.001 compared to the control PBS group.

A. Cabrera et al. / Immunobiology 214 (2009) 467–474 471

B. abortus strain RB51 induced 1.99 log of protection(Table 1).

Discussion

Vaccination continues to be the most successfulintervention for preventing infectious disease in animalsand humans. The development of a new generation ofvaccines able to prevent brucellosis is critical in order toovercome the disadvantages of currently used livevaccines. New vaccination approaches against brucello-sis, such as the use of DNA vaccines, have showedvariable results (Oliveira and Splitter, 1996; Al-Maririet al., 2001; Velikovsky et al., 2002; Onate et al., 2003).Low transfection rates and the possibility of integrationof plasmid DNA into the host genome are majorroadblocks for the successful application this approach.However, the emerging RNA based approach using theSFV was demonstrated to be a potent tool towards theimplementation of nucleic acid vaccines. The mainadvantage of this system is its greater transfectionefficiency, which results in the delivery of the foreignproteins into a wide range of eukaryotic cells. Packagingof alphavirus replicons that contain exogenous genesinto recombinant particles can be achieved veryefficiently in culture systems (Liljestrom and Garoff,1991). Entry and replication of recombinant SFVparticles in cells result in shutting off host proteinsynthesis and redirection of the cellular metabolismtowards the expression of virus-encoded proteins. Infact, previous studies have shown that an SFV vectorcan be efficiently exploited to induce cellular andhumoral immune responses in a variety of animalmodels (Frolov et al., 1996; Lundstrom, 2003; Saez etal., 2008), which were superior to those obtained with aconventional DNA vaccine (Fleeton et al., 2000).

In this study, we evaluated the immune responseselicited in mice upon inoculation with recombinant SFVparticles containing a replicon coding for Brucella IF3,as well as their capacity to confer protection against

bacterial challenge. Two weeks after the first immuniza-tion with SFV-IF3 particles, only low levels of IF3-specific IgM were detected in the serum samples. Perusalof the published literature indicates that the induction ofantigen-specific antibodies upon SFV particle immuni-zation appears to be highly variable. In some casesstrong antibody response were obtained (Brand et al.,1998; Berglund et al., 1999; Fleeton et al., 2000; Yuet al., 2006), whereas in other cases very weak or noantibody responses were detected (Andersson et al.,2001; Penttila et al., 2004; Koopman et al., 2004; Onateet al., 2005; Li et al., 2007). Probably the nature of theantigen, the immunization interval and, in some cases,the boosting with recombinant proteins, may influencethe outcome of the antibody responses followingimmunization with recombinant SFV particles.

The induction of T cell immune responses by SFV-IF3 particle immunization was evaluated by measuringthe proliferative responses and cytokines produced bysplenocytes after in vitro stimulation with either rIF3protein or CBPs. Immunizations with SFV-IF3 resultedin a strong T-cell proliferative response, similar to thatobserved after vaccination with native or recombinantIF3 protein (Cespedes et al., 2000; Gonzalez et al.,2008). In contrast, in the RB51 group strong prolif-erative responses were observed after stimulation withCBP’s, whereas basal responses were detected inresponse to rIF3. This finding confirms the specificityof the response against rIF3, since a strong response wasonly present in the SFV-IF3 group. The weak responsesobserved in the RB51 group can be explained by lowlevel expression of IF3 in the B. abortus RB51 strain.The presence of increased levels of INF-g, but not IL-4,in supernatant fluids of splenocytes in mice immunizedwith SFV-IF3 suggests that SFV-IF3 immunizationinduced a Th1 biased immune response. It is welldocumented that cellular immunity plays a major role inthe establishment of a protective response againstBrucella (Jiang and Baldwin, 1993; Jones and Winter,1992; Ko and Splitter, 2003; Oliveira and Splitter, 1995;Golding et al., 2001). This response is based onproduction of INF-g, a key factor for Brucella clearancein the host (Murphy et al., 2001). For this reason, thedesign of a preventive vaccine against brucellosis mustbe based on its capacity to generate strong CMI withhigh levels of INF-g secretion from antigen-specific Tcells.

Interestingly, in the RB51 group a surprising patternof lymph proliferative responses and INF-g productionemerged after stimulation with rIF3. Namely, basallevels of proliferation associated with high levels ofINF-g production, even higher than those observed inthe SFV-IF3 group. This can be explained, at least inpart, by pleiotropic and paradoxical activities of cyto-kines depending on their concentrations, microenviron-ments and the stage at which they are produced during

ARTICLE IN PRESSA. Cabrera et al. / Immunobiology 214 (2009) 467–474472

this stimulation of an adaptive immune response.Nevertheless, we cannot, so far, endorse any of theprevious reasons as explanation for this high IFN-gproduction and low lymphoproliferation. Nowadays,other functions has been reported for INF-g, like theregulatory properties observed during Bacille Calmet-te–Guerin (BCG) infection of IFN knockout (KO) mice,with a failure to induce apoptosis of activated CD4 Tcells during BCG infection. The CD4 T cell populationwas markedly expanded, suggesting a regulatory func-tion of INF-g in the induction of apoptosis in CD4activated cells (Dalton et al., 2000). This phenotypecould be explained by the role of INF-g in thestimulation of macrophages, which in turn leads tonitric oxide (NO) production that, in turn, restricts theexpansion of splenocytes by inducing apoptosis (Wil-liams et al., 1998; Murphy, 1999). In our work, weobserved high levels of INF-g and low levels of lymphproliferation in the group of mice immunized with theB. abortus strain RB51. Perhaps this mechanism mightexplain a regulatory function of INF-g in a secondaryresponse against Brucellae antigens, in our case rIF3.

Our protection studies demonstrated that immuniza-tion with SFV-IF3 result in high level of protectiveimmune response, similarly to immunization with thelive RB51 vaccine strain. In this context, our resultssuggest that SFV-IF3 may be a suitable vaccinecandidate meeting the above-mentioned requirements.This is in agreement with previous studies using purifiedIF3 protein emulsified with incomplete Freund’s adju-vant for immunization (Cespedes et al., 2000). Furtherstudies are required to delineate the specific role playedby different T-cell subsets in the protective responseinduced by SFV-IF3 particle immunization, as well as toexamine its usefulness for applications in the fields ofhuman and veterinary medicine.

Acknowledgements

This work was supported by the Grant ‘‘Anillo delPrograma Bicentenario en Ciencia y Biotecnologıa’’ACT 08-2006 (CONICYT), Santiago, Chile.

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