Induced immune response of DNA vaccine encoding an association MSP1a, MSP1b, and MSP5 antigens of Anaplasma marginale

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Vaccine 26 (2008) 3522–3527

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Induced immune response of DNA vaccine encoding an association MSP1a,MSP1b, and MSP5 antigens of Anaplasma marginale

Flora S. Kanoa, Katia Tamekunia, Adriana L. Coelhoa, Joao Luis Garciaa, Odilon Vidottoa,

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e signe stra

Eiko Nakagawa Itanob, Marilda C. Vidottoa,∗

a Protozoology Laboratory, Departamento de Medicina Veterinaria Preventiva, CCA, Unib Immunology Laboratory, Departamento de Patologia, CCB, Universidade Estadual de L

a r t i c l e i n f o

Article history:Received 22 September 2007Received in revised form 1 April 2008Accepted 9 April 2008Available online 8 May 2008

Keywords:Anaplasma marginaleDNA vaccineImmunogenicity

a b s t r a c t

The outer membrane proimproved vaccine againstpcDNA-msp1˛, pcDNA-mdetermine the immunogeation. Expression of proteishowed high antibodies rand induced significant prseroconversion and reacteisotype and did not inducplasmid can be an effectiv

1. Introduction

The outer membrane proteins of Anaplasma marginale have beenthe focus of direct research to obtain an improved vaccine againstbovine anaplasmosis [1]. Immunization with purified outer mem-branes induces protection against acute A. marginale infection anddisease [2]. Various major outer membranes have been describedbased on the proteomic and genomic approach, and 21 proteinswere identified within the outer membrane immunogen [3]. Somewell-characterized outer membranes proteins, designated majorsurface proteins (MSPs): MSP1a, MSP1b, MSP2, MSP3, MSP4, andMSP5 were evaluated as potential candidates for antigens of vaccineproduction and diagnostic evaluations [4–9].

The MSP1a and MSP1b are complexed by disulfide-bonded andnoncovalently associated with MSP5 [10]; and this intermolecularrelationship seems to be important in the induction of protec-tive immunity. The MSPs recombinant proteins rMSP1a, rMSP1b,rMSP4 and rMSP5 showed efficient humoral response in immu-nized mice with ISCOMATRIX/rMSPs [11]. The MSP5 was detected

∗ Corresponding author. Present address: Protozoology Laboratory, Departamentode Medicina Veterinaria Preventiva, Caixa Postal 6001, CEP: 86051-970, CampusUniversitario, Universidade Estadual de Londrina-UEL, Londrina, Parana, Brazil.Tel.: +55 43 3371 4485; fax: +55 43 3371 4714.

E-mail address: [email protected] (M.C. Vidotto).

0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2008.04.047

ade de Londrina-UEL, Londrina, Parana State, Brazila, Londrina, Parana State, Brazil

of Anaplasma marginale have been the focus of research to obtain ane anaplasmosis. We evaluated the capacity of the recombinant plasmidsand pcDNA-mp5 to express MSP1a, MSP1b, and MSP5 proteins, and toof BALB/c mice immunized with these plasmids individually or in associ-s confirmed in Vero cells by IFA. The combination of recombinant plasmidsse, produced better induction of Th1 response than individual plasmids,ation of splenocytes. The mice sera immunized with A. marginale showedth all native MSPs, but demonstrated predominance of the humoral IgG1ificant proliferation of splenocytes. The use of association of recombinanttegy for the immunoprophylaxis of anaplasmosis.

© 2008 Elsevier Ltd. All rights reserved.

in all Brazilian A. marginale isolates studied [8] and has been con-served in American, African, Israeli and Brazilian strains [5,12,13].The r-MSP5 and monoclonal antibody ANA F16C1 has been usedfor detection of anti-Anaplasma-specific antibodies by the CI-ELISA[12,13].

Immunization with DNA plasmids encoding for a determineantigen represents a novel and promising method in vaccineresearch and development. Studies have demonstrated that afternaked DNA immunization, the antigen is naturally processed andpresented on major histocompatibility complex class I and class IImolecules, inducing a broad range of immune responses includingantibody production, CD4+ T helper cell (Th1), and CD8+ cytotoxicT cells (CTLs) [14–16]. Incorporation of multiepitope in DNA immu-nization consists of a new method to increase immunogenicity andprotection of the vaccinated animal as compared to single epitopevaccines [17].

Immunization against anaplasmosis with single DNA vaccine(pVCL/msp1˛) produced antibodies against MSP1a protein in miceand calves [18]. In another experiment, immunization with pcDNA-msp1ˇ elicited antibodies and cattle immunized showed partialprotection against a virulent homologous challenge.

The aim of the present study was to evaluate the capacity ofthe recombinant plasmids to express MSP1a, MSP1b, and MSP5in eukaryotic cells in vitro, and determine the immunogenicity ofBALB/c mice immunized with these recombinant plasmids individ-ually or in combination.

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2. Materials and methods

2.1. A. marginale isolate and DNA extraction

A. marginale isolate was obtained from infected cattle of ParanaState (PR), Brazil, and designed PR1 [19,8]. DNA was extracted fromstabilate PR1 A. marginale using Purigene DNA Extract Kit (GentraSystem, Minneapolis, MN, USA) according to manufacture’s recom-mendation.

2.2. Production of DNA vaccines

The recombinant plasmids (pcDNA-msp1˛, pcDNA-msp1ˇ,pcDNA-msp5) were constructed for a previous study (unpublishedresults). Large scales culture of TOP10 Escherichia coli containingrecombinant plasmids were grown in LB broth at 37 ◦C; and theQiagen Plasmid Mega Kit (Qiagen, Valencia, CA, USA) was used toprepare DNA. The recombinant plasmids were quantified by spec-trophotometry measuring the optical density at 280 nm.

2.3. Production of recombinant MSPs proteins

The recombinant plasmids, pET102-msp1˛, pET101-msp1ˇ [20],and pRSET-msp5 [21] were used to produce recombinant MSP1a,MSP1b, and MSP5 as a fusion product with 6xHis. The rMSPswere purified using Ni-NTA resin according to the manufacturer’sinstructions (Qiagen, Valencia, CA, USA).

2.4. In vitro transfection and expression

The recombinant plasmids were examined for antigen expres-sion by transfection into Vero cells using lipofectamine 2000(InvitrogenTM Life Technologies, Carlsbad, CA, USA).

After 72 h of transient transfection, the cells were fixed in coverslips with cold methanol for 10 min, acetone for 1 min, follow-ing indirect immunofluorescence assay (IFA). Non-transfected Verocells and pcDNA/LacZ-His transfected Vero cells were used as neg-ative and positive controls, respectively. The IFA was performedwith anti-histidine diluted 1:5000 (for MSP1b), anti-MSP1a mono-clonal antibody ANA22B1 diluted 2 �g/ml (MSP1a), and anti-MSP5monoclonal antibody ANAF16C1 2 �g/ml (MSP5) diluted in PBScontaining 2% BSA, and incubated for 45 min at 37 ◦C. After threewashes in PBS–Tween 20, the cover slips were incubated for 45 minwith FITC-anti-mouse IgG (Sigma–Aldrich Co, USA) diluted 1:900 in

PBS containing 2% BSA. The IFA was examined using a fluorescencemicroscope with appropriate filters.

2.5. Animal and experimental design

The mice were maintained according to the National Instituteof Health standards the use of laboratory animals, in accordancewith protocols approved by the Institutional Research Commit-tee from State University of Londrina (process number 26927/05).Seven groups of eight female BALB/c mice (7–8 weeks) were uti-lized. Two negative controls were used: one group was immunizedwith 100 �l PBS (G1) and the other with 100 �g of pcDNA3.1empty vector (G2). The positive control group (G3) was immu-nized with 20 �g of protein from initial corpuscle of A. marginaleemulsified with complete Freund’s adjuvant in the first immu-nization and with incomplete Freund’s adjuvant in subsequentimmunizations. Group animals four were immunized with 100 �gpcDNA-msp1˛, G5 with 100 �g pcDNA-msp1ˇ, G6 with 100 �gpcDNA-msp5 and G7 with pool of recombinant plasmids (33 �g foreach pcDNA-msp1˛, pcDNA-msp1ˇ, and pcDNA-msp5). The micewere anaesthetized before immunization with the recombinant

(2008) 3522–3527 3523

plasmids in sucrose–phosphate–glutamate buffer (SPG) adminis-tered in each leg muscle [22].

For the G3, three immunizations were administered by subcu-taneous injections after 14 days post-inoculation, and the mice ofthis group received boosters at 8 and 12 weeks. The mice were vac-cinated at 0, 2, 5, 8 and 12 weeks with DNA vaccine. Blood sampleswere collected at −1, 1, 4, 7, 11, and 15 weeks, and the sera samplesstored at −20 ◦C until analysis.

2.6. Antibody assays and isotypes

Optimal dilutions were established using check board titrationswith dilutions of sera and conjugates. ELISA plates (Nunc-ImmunoTM Maxisorp, Roskilde, Denmark) were coated with 100 �lof the rMSP1a (5 �g/ml) for G4; rMSP1b (5 �g/ml) for G5; rMSP5(5 �g/ml) for G6; and pool of rMSP1a, rMSP1b, rMSP5 (5 �g/ml) forG7 and A. marginale initial bodies (10 �g/ml) for G3, G1 and G2.After overnight incubation at 4 ◦C, samples of serum from groupsof immunized mice were diluted (1:50) in PBS–Tween 20 pH 7.4plus 5% non-fat dry milk. After which the diluted samples wereadded in duplicate to each well, and the plates incubated at 37 ◦Cfor 45 min. The positive and negative control sera were includedin each plate. Three secondary antibodies were used, horseradishperoxidase-labeled anti mouse whole anti-IgG (Sigma) (1:10,000),anti-mouse IgG1 (1:50,000), and anti-mouse IgG2a peroxidase con-jugate (1:50,000); each incubated for 45 min at 37 ◦C. The substratefor the enzyme was OPD (ortho-phenylenediamine) 0.4 mg/ml, inH2O2 0.04% in substrate buffer (0.1 M acid citric, 0.2 M Na2HPO4).The reaction was blocked by 50 �l of 1N HCl. The absorbance valueswere estimated and the optic density (OD) values were calculatedas previously described [23]. A serum was considered to be positivewhen OD sample >OD mean from negative control sera (n = 9) +3xstandard deviation from negative control.

2.7. Western blot analysis

rMSP1a (G4), rMSP1b (G5), rMSP5 (G6), a pool of rMSPs (G7), andA. marginale initial bodies (G3) were electrophoresed on 7.5–17.5%gradient SDS-PAGE [24]. The proteins were transferred onto a0.45 �m nitrocellulose membrane (Hybond-ECLTM, Amershan Bio-sciences, Sunnyvale, CA, USA), as previously described [25]. Afterblocking, the strips of nitrocellulose membranes were incubatedwith pre-immunized and post-immunized pools of serum frommice of each group (G1 to G7) diluted (1:50) in blocking solution.

Peroxidase-labelled protein G (1:5000) was used as the secondaryantibody, and was incubated for 1 h at room temperature. Theperoxidase activity was demonstrated using enhanced chemilu-minescence (ECL) (Amershan Biosciences, Sunnyvale, CA, USA).Protein molecular weight markers (BenchMark TM Invitrogen LifeTechnologies, Carlsbad, CA, USA) were used as standard.

2.8. Proliferation assay

The spleens from mice immunized with recombinant plasmids(G4, G5, G6 and G7), from mice immunized with protein from ini-tial corpuscle of A. marginale (G3), from the control group (G2) andfrom normal mice (G1), were removed aseptically and teased. Theerythrocytes were lysed with tris–ammonium chloride, and thecell suspension washed trice at RPMI 1640 medium. For prolifer-ative assays, 100 �l of the cells were cultured in triplicate wells at aconcentration of 1 × 106 cells/ml at RPMI 1640 containing 10% fetalcalf serum in 96-well flat-bottom culture plates. Homogenate A.marginale (5 �g/ml) was then added to each well, after which thecells were cultured for 5 days at 37 ◦C with 5% CO2, and were pulsedwith 1 �Ci of [3H] thymidine 18 h before harvesting on glass filter

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Anapanti-h(e) pc

Fig. 1. Immunofluorescence of Vero cells transfected with DNA vaccine encoding(400×). (a) Non-transfected Vero cells; (b) pcDNA-lacZ Vero cells transfected usingmonoclonal antibody; (d) pcDNA-msp1ˇ using anti-histidine monoclonal antibody;

strips. The radioactivity was determined by cintilator (Beckman LS6.800). The stimulation index was calculated as the triplicate ofstimulated cells (cells + Ag) divided by the cell control (cell + RPMI1640). A stimulation index (SI) of ≥2 was considered significant.

2.9. Statistical analysis

The Student’s t-test was performed for statistical evaluation ofdata from IgG2a and IgG1 production.

3. Results

3.1. Eukaryotic expression of MSPs antigens of A. marginale

Cells transfected with recombinant plasmids (pcDNA-msp1˛,pcDNA-msp1ˇ, and pcDNA-msp5) expressed specific proteins byIFA (Fig. 1). Non-transfected Vero cells did not express anyimmunoreactive protein with anti-histidine, ANA22B1, ANAF16Cmonoclonal antibodies by IFA (Fig. 1a). In the positive control, theVero cells transfected with pcDNA-lacZ-His presented fluorescencewith anti-histidine monoclonal antibody (Fig. 1b). The fluores-cence was verified in Vero cells transfected with pcDNA-msp1˛using ANA22B1 monoclonal antibody against MSP1a (Fig. 1c), withpcDNA-msp1ˇ using anti-histidine monoclonal antibody (Fig. 1d),and with pcDNA-msp5 using ANAF16C1 monoclonal antibodyagainst MSP5 (Fig. 1e).

3.2. Humoral immune response of mice elicited by DNAvaccination

Serological responses were evaluated throughout the immu-nizations by ELISA (Figs. 2 and 3) and Western blot analysis

lasma marginale MSP1a, MSP1b and MSP5 antigens, using monoclonal antibodiesistidine monoclonal antibody (positive control); (c) pcDNA-msp1˛ using ANA22B1DNA-msp5 using ANAF16C1 monoclonal antibody.

(Fig. 4). The whole IgG production in mice is shown at Fig. 2from pooled sera from DNA-vaccinated BALB/c mice at −1, 4, 11,and 15 weeks after initial immunization. All pre-immunized andcontrol group sera (G1 and G2) reacted below the cut-off level(G1 = 0.083, and G2 = 0.085) and were considered as negative. Themice sera from the group immunized with A. marginale (G3, cut-off = 0.88) showed seroconversion after the second immunizationand were stable throughout the entire experiment. In G4 (pcDNA-msp1˛, cut-off = 0.183), G6 (pcDNA-msp5, cut-off = 0.113), and G7(pool of plasmids, cut-off = 0.149), the seroconversion begun afterthe second immunization, but only after the third immunization

all animals demonstrated positivity for specific proteins. In con-trast, mice sera from the G5 (pcDNA-msp1ˇ, cut-off = 0.120) showedlower seroconversion and few animals had OD above cut-off afterthe fifth immunization (Fig. 2).

The reactions of IgG1 and IgG2a to MSP1a, MSP1b, and MSP5were analysed from pooled sera from DNA-vaccinated BALB/c miceat 0, 4, 11, and 15 weeks after initial immunization (Fig. 3). The ODvalues for IgG1 and IgG2a demonstrated IgG2a isotype polariza-tion in all groups immunized with the DNA vaccines (Fig. 3a–e).Significant IgG2a over IgG1 production were detected after thefourth immunization with the DNA vaccine. The G4, G6, and G7demonstrated strong polarization for IgG2a at week 4 (p < 0.01).Mice immunized with A. marginale (G3) demonstrated predomi-nance of the humoral IgG1 isotype and the significant IgG1 levelswere verified at week 4 (p < 0.01) (Fig. 3).

Western blot results using pool of sera are shown in Fig. 4A andB. All pre-immunized and post-immunized sera of mice from G1(PBS) and G2 (vector alone) (Fig. 4A, lanes 1 and 2) did not demon-strate any reactivity. Sera from mice immunized with A. marginaleinitial bodies (G3) reacted with all native MSPs (approximately 100,76, 38, 31 and 19 kDa) (Fig. 4A, lane 4). Sera from mice immunized

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Fig. 2. Whole IgG antibody response measured by the indirect enzyme-linked immunosoinitial bodies of A. marginale); (c) G4 (100 �g pcDNA-msp1˛); (d) G5 (100 �g pcDNA-msp1ˇand pcDNA-msp5). Mice were immunized at weeks 0, 2, 5, 8, and 11 and the blood colleccut-off.

Fig. 3. IgG isotype humoral responses of serum from BALB/c mice immunized withDNA-vaccine encoding A. marginale MSP1a, MSP1b, and MSP5 proteins. (a) The resultof G2 (only vector); (b) G3 (100 �g initial bodies A. marginale); (c) G4 (100 �g pcDNA-msp1˛); (d) G5 (100 �g pcDNA-msp1ˇ); (e) G6 (100 �g pcDNA-msp5); (f) G7 pool ofplasmids (33 �g each pcDNA-msp1˛, pcDNA-msp1ˇ, and pcDNA-msp5). MeasuredIgG1 and IgG2a responses were taken of pool of serum at −1, 4, 11 and 15 weeksafter initial immunization by ELISA.

rbent assay (ELISA) in immunized BALB/c mice. (a) G2 (only vector); (b) G3 (100 �g); (e) G6 (100 �g pcDNA-msp5); (f) G7 (33 �g for each pcDNA-msp1˛, pcDNA-msp1ˇt were at weeks −1, 1, 4, 7, 11 and 15 (back arrows). Dashed line indicates positive

Fig. 4. Reactivity of sera from mice immunized with A. marginale, and DNA vaccineencoding MSP1a, MSP1b, and MSP5 of A. marginale by Western blot. (A) A. marginalePR1 strain initial bodies. (B) Lane 1 and 2, rMSP1a; lane 3 and 4, rMSP1b; lane 5and 6, rMSP5; lanes 7 and 8, pool of rMSPs. In A pre and post-immune sera of miceimmunized with empty vector (G2: lane 1 and 2); with A. marginale initial bodies(G3: lane 3 and 4); In B pool of mice sera (at week 15) diluted 1:50 immunizedwith pcDNA-msp1˛ (G4: lanes 1 and 2); with pcDNA-msp1ˇ (G5: lanes 3 and 4);with pcDNA-msp5 (G6: lanes 5 and 6); with pool of plasmid (pDNA-msp1˛, pcDNA-msp1ˇ and pcDNA-msp5) (G7: lanes 7 and 8). The positions and molecular masses(kDa) of protein standards are also shown.

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Fig. 5. Proliferation of splenocytes cells from mice immunized with single DNAvaccine encoding MSP1a, MSP1b and MSP5 and in association. The data are pre-sented for splenocytes proliferation cultured for 5 days with 5 �g of A. marginalehomogenate/ml. Results are presented as means count per minute of triplicate cul-tures in stimulation index values (SI). SI values ≥2.0 (dashed line) were consideredsignificant. Experimental groups were: G1 (PBS); G2 (empty vector); G3 (100 �g A.marginale initial bodies); G4 (100 �g pcDNA-msp1˛); G5 (100 �g pcDNA-msp1ˇ);G6 (100 �g pcDNA-msp5) and G7 (33 �g for each pcDNA-msp1˛, pcDNA-msp1ˇ andpcDNA-msp5).

with pcDNA-msp1˛ (G4) showed reactivity of approximately100–70 kDa (rMSP1a); while mice immunized with pcDNA-msp1ˇ(G5) reacted poorly resulting in only 100 kDa (rMSP1b). More-over, sera from mice immunized with pcDNA-msp5 (G6) reactedin approximately 31 kDa (rMSP5). Mice sera from G7 that receivedmixed recombinant plasmids reacted with all recombinant proteins(rMSP1a, rMSP1b, and rMSP5) of A. marginale. The reaction of post-immunized sera from mice immunized with mixed recombinantplasmids with rMSPs suggest that no suppression was observed.

3.3. T cell-mediated immune responses

The proliferation of splenocytes from experimental groups ispresented in Fig. 5. A strong lymphoproliferation (SI = 12.2) wasobserved in spleen cells from mice immunized with the associa-tion of recombinant plasmids (G7). Poor lymphoproliferation wasobserved in spleen cells from mice individually immunized withrecombinant plasmids; only in cells from G4 animals that receivedpcDNA-msp1a was observed significant proliferation (SI = 2.6).

4. Discussion

During this study, the MSP1a, MSP1b, and MSP5 of A.marginale were successfully detected in Vero cells transfectedwith pcDNA-msp1˛, pcDNA-msp1ˇ, and pcDNA-msp5, respec-

tively, as demonstrated by IFA using monoclonal antibodies;while the administration of the recombinant plasmids (pcDNA-msp1˛, pcDNA-msp1ˇ and pcDNA-msp5) elicited specific antibodyresponse against MSP1a, MSP1b and MSP5 in BALB/c mice. Previ-ous studies also related that A. marginale DNA vaccines inducedimmune responses in BALB/c mice immunized with a DNA vac-cine encoding MSP1a [18]. These authors immunized calves withpVCL/msp1˛ and detected only IgG1 antibodies in the sera ofanimals following DNA vaccination, although sera from bovineimmunized with initial bodies of A. marginale demonstrated bothIgG1 and IgG2 antibodies. The few studies relative to the controlof anaplasmosis using DNA vaccination are based either on themsp1˛ or msp1ˇ genes, and have demonstrated partial protectionfor virulent challenge with A. marginale strain [18,26].

The mice immunized with pcDNA-msp1ˇ elicited a smallresponse compared to the response obtained from mice immunizedwith pcDNA-msp1˛ and pcDNA-msp5, as evaluated by ELISA. Onlytwo of eight G5 animals produced titer of specific antibodies withOD above cut-off, after the fifth immunization. However, the serafrom mice immunized with pcDNA-msp1ˇ reacted specifically tor-MSP1b by Western blot. The MSP1a and MSP1b are associated

(2008) 3522–3527

in the native membrane through disulfide bonds [10], and the IgGtitters specific for MSP1a and MSP1b were comparable in com-plex MSP1-immunized cattle [27]. Moreover, CD4+ T-cell responseswere detected only against MSP1a [27], suggesting that MSP1a-specific T cells provide cognate help to both MSP1a and MSP1bspecific B cells.

The mice immunized with A. marginale DNA vaccines (pcDNA-msp1˛, pcDNA-msp1ˇ, and pcDNA-msp5) exhibited predominanceof IgG2a antibody relative to IgG1 (ratio of IgG2a/IgG1, ≥2.0) (Fig. 3).The presence of the IgG2 isotype is often considered as evidence ofa Th1 immune response [28]. Thus, these results suggest that intra-muscular injection with plasmids encoding MSPs generate a Th1response in mice. In contrast, the group that received initial bod-ies of A. marginale elicited a predominantly elevated productionof IgG1 (Fig. 3). This is because antigens associated with Freund’sadjuvant preferentially stimulate the Th2 immunity, and vigorouslysuppress the Th1 type [29]. It is well established that protec-tion against infection by an intracellular pathogen, including A.marginale, requires the generation of a Th1-type immune responseover time [16,28,30].

In this study, only the mice immunized with the association ofrecombinant plasmids (G7) demonstrated strong proliferation ofsplenocytes (SI = 12.2). In mice that received pcDNA-msp1a (G4)was observed significant proliferation (SI = 2.6), however othersanimals of groups that received individual recombinant plasmidspresented poor or none lymphoproliferation of spleen cells (Fig. 5).These results indicate that the association of plasmids better stim-ulates the cell immune response, and agree with previous data(17). MSP1a contain variants and conserved amino acids within thecarboxy terminal region that response specifically, and stimulatehigh levels of IFN-� production by CD4+ cells [27,30,31]. Peripheralmononuclear cells and CD4+ T-cell lines from MSP1-immunizedcalves proliferated vigorously in response to the immunizationwith Florida strain and heterologous strains of A. marginale forMSP1a, although poor or no recognition of MSP1b proteins werealso described [27].

Mice immunized with the recombinant plasmids (pcDNA-msp1˛, pcDNA-msp1ˇ and pcDNA-msp5) showed high antibodyresponse by ELISA, were able to react with all recombinant pro-teins (rMSP1a, rMSP1b, and rMSP5) as observed by Western blot,and induced an intense lymphoproliferation. These results indicatethat both immune response and T-cell lymphocyte were generatedwhen mixed recombinants plasmids was used.

Whereas the of nine different plasmids encoding antigens of

Plasmodium falciparum, pooled in cocktail, and administrated in asingle injection site induced significant suppression or completeabrogation of responses [32]. By the other hand, the association ofthree DNA vaccine constructions against schistosomiasis showedhigher protection for BALB/c mice than the immunization withsingle or double vaccines [33] and a combination of many genesprovided strong and complete protection against papillomavirus,simian immunodeficiency virus (SIV), Plasmodium chabaudi adami,and Plasmodium yoelii [34–37]. Thus, the immunization with mixedrecombinant plasmids encoding MSPs can be an effective strategyfor the immunoprophylaxis of anaplasmosis.

In conclusion, the administration of plasmids encoding A.marginale MSPs in BALB/c mice produced better induction of Th1immune response than individual plasmids. For the future, multi-epitope DNA vaccines should be evaluated in bovine protection forchallenge of anaplasmosis.

Acknowledgments

We thank Kerlei Cristina Medici, MSc. (Virology Laboratory) forpreparation of the Vero cells, Dra Mari Sumigawa for assistance

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with the proliferation of splenocytes, and Dr. Selwyn ArlingtonHeadley for revising the manuscript. This study was financiallysupported by CNPq, CAPES, and Fundacao Araucaria do Parana.

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