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Research in Veterinary Science 83 (2007) 347–354

Immune response of BALB/c mouse immunized with recombinantMSPs proteins of Anaplasma marginale binding to

immunostimulant complex (ISCOM)

Paula M. Kawasaki, Flora S. Kano, Katia Tamekuni, Joao Luis Garcia,Elizabete R.M. Marana, Odilon Vidotto, Marilda C. Vidotto *

Universidade Estadual de Londrina, Departamento. Medicina Veterinaria Preventiva, CCA, Campus Universitario, Caixa Postal 6001,

86051-970 Londrina, PR, Brazil

Accepted 1 February 2007

Abstract

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regionsworldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5.The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporatedinto immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM andISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccinesproduced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that receivedantigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60–105 kDa), rMSP1b (100 kDa), rMSP4(47 kDa), and rMSP5 (29 kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanismsof this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Anaplasma marginale; MSPs; ISCOM; ISCOMATRIX; Adjuvant

1. Introduction

Anaplasma marginale is an obligate intraerythrocyticbacteria from the Anaplasmataceae family (Dumler et al.,2001) that causes bovine anaplasmosis and is transmittedby infected ticks or by contaminated fomites. This diseaseis endemic in many tropical and subtropical areas, andthe acute phase causes anemia, abortion, weight loss, andeventually death, resulting in significant losses to meatand milk production (Richey, 1981).

The commercially existing vaccines are constituted bylive or dead organisms, and present limitations due to the

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doi:10.1016/j.rvsc.2007.02.002

* Corresponding author. Fax: +55 021 43 33714485.E-mail address: macarlos@uel.br (M.C. Vidotto).

need for cryopreservation, efficacies, and the possibilityof induction of iso-antibodies against erythrocytes thatare transferred by colostrum, resulting in isoeritrolysis atbirth (Kuttler, 1984).

Cattle immunized with the outer membrane fraction ofA. marginale can induce protective immunity againstchallenge with homologous isolate and provide partialprotection against heterologous challenge (Tebele et al.,1991). The protective outer membrane of A. marginaleincludes six major surface proteins (MSPs) well character-ized and designated as MSP1, MSP2, MSP3, MSP4, andMSP5 (Palmer and McGuire, 1984; Tebele et al., 1991;Visser et al., 1992; Oberle et al., 1993; Alleman et al.,1997). These findings suggest the potential use of MSPsas components of recombinant protein vaccine forAnaplasmosis.

348 P.M. Kawasaki et al. / Research in Veterinary Science 83 (2007) 347–354

MSP1 is a dimer of two structurally unrelated poly-peptides, MSP1a (msp1a) and MSP1b (msp1b1 andmsp1b2) (Barbet et al., 1987; Barbet and Allred, 1991;Camacho-Nuez et al., 2000; Vidotto et al., 1994). MSP1avaries in sequence and molecular size due to the numberof tandem 28- to 29-amino-acid repeats (Allred et al.,1990). The epitope of MSP1a is conserved and may havean important function as adhesion molecule of A. mar-

ginale in tick cells and bovine erythrocytes (McGareyet al., 1994; McGarey and Allred, 1994). Brown et al.(2001) demonstrated the predominance of T CD4+lym-phocytic response in bovines immunized with the com-plex MSP1.

The MSP1a, MSP4, and MSP5 are present and con-served in all Brazilian isolates of A. marginale analyzed(Patarroyo et al., 1994; Kano et al., 2002; Oliveira et al.,2003).

The extraction and purification of native out membraneproteins (MSPs) of A. marginale in large scale is practicallyimpossible. Therefore, the incorporation of recombinantMSPs into immune stimulating complex (ISCOM) canorganize the natural arrangement of the recombinantmajor surface proteins, inducing a protective immunityagainst A. marginale.

ISCOMs are stable colloidal complexes with an opencage-like structure in the size range of 40–100 nm. Theyare composed of the saponin adjuvant Quil A, cholesterol,phospholipids, and antigen, which are effective carriers ofsubunit vaccine (Morein et al., 1984; Kersten et al., 1991;Copland et al., 2000; Andersson et al., 2000). The ISCOM-ATRIX adjuvant consists of the same components, exceptthe antigen and has essentially the same structure ofISCOM (Pearse and Drane, 2005). A simple process to pre-pare ISCOMATRIX was developed with the introductionof hydration that promotes intimate moisture between QuilA, cholesterol, and phospholipids (Copland et al., 2000).The combination of antigen with ISCOMATRIX adjuvantwas then denominated as ISCOMATRIX vaccine (Cop-land et al., 2000).

ISCOM and ISCOMATRIX vaccines induce strong Thelper 1 lymphocytic response (Th1) and helper 2 (Th2),and act as presentation and induce excellent lymphocyteT cytotoxic (CLT) response (McGarey and Allred, 1994;McGarey et al., 1994; Malliaros et al., 2004; Moreinet al., 1984; Classen and Osterhaus, 1992; Bengtsson andSjolander, 1996; Morein et al., 1995; Sanders et al., 2005).

The objective of this study was to evaluate the immunehumoral response in BALB/c mice of rMSP1a, rMSP1b,rMSP4 and rMSP5 of A. marginale incorporated intoISCOM.

2. Materials and methods

2.1. A. marginale strain

A. marginale strain PR1 was obtained from cattle withinthe State of Parana (PR), Brazil. The PR1 strain was puri-

fied following experimental infection of splenectomizedcattle. Blood samples were collected, and after washes inPBS, the erythrocytes were suspended to a final concentra-tion of 109 erythrocytes per ml. The initial bodies of A.

marginale were purified as previously described (Palmerand McGuire, 1984).

2.2. Expression of the msp genes in the Escherichia coli

strain and purification of rMSPs

The msp genes of PR1 A. marginale strain were ampli-fied by PCR and cloned into the pET102/D-TOPO� vector(Invitrogen, Carlsbad, CA, USA), producing msp-6xHis-V5-HP thioredoxin fusion gene construct. The recombi-nant clones were over-expressed in E. coli BL21(DE-3),and the recombinant protein was purified by Ni-NTA col-umn (Qiagen�) and dialyzed.

2.3. ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccine

preparation

ISCOM/rMSPs were prepared by a previously describedtechnique (Demana et al., 2004) with modifications. Thismethod involves the hydration of dried phospholipid/cho-lesterol films using an aqueous solution of Quil A, at a ratioof 2:1:2 of Quil A/cholesterol/PC. The concentration ofeach peptide was initially based on molar fraction obtainedfrom the native membrane (Vidotto et al., 1994), and thenadjusted as necessary to obtain the molar fraction neededfor the preparation of ISCOM. Phosphatidylcholine(8 mg) and cholesterol (4 mg) were dissolved in 0.5 mLchloroform, and evaporated until dried at 45 �C for 1 h,after which the lipid film was hydrated in 3 ml of Tris bufferpH containing 8 mg of saponin Quil A at room tempera-ture for 2 h. Then, rMSP1a, rMSP1b (Tamekuni et al.,2006), rMSP4 (Kawasaki et al., 2007) and rMSP5 (Maranaet al., 2006) were added in concentrations of 400 lg,400 lg, 400 lg, and 200 lg, respectively; after dialysis,ISCOM/rMSPs was formed spontaneously as the detergentis removed and the antigens were incorporated into theparticle.

The ISCOMATRIX adjuvant was prepared as ISCOM,except for the lack of antigen and the ISCOMATRIX/rMSPs vaccine was produced by the addition of the rMSPs(1:1) in the adjuvant.

2.4. Characterization of ISCOM

ISCOM/rMSPs and ISCOMATRIX adjuvants wereexamined using negative staining transmission electronmicroscopy to confirm the formation of characteristicvesicle.

The antigenic characteristic of ISCOM/rMSPs was ana-lyzed by Western blotting using antibodies produced fromrabbit for each MSP. An aliquot of 500 lL of ISCOM/rMSPs was centrifuged at 10.000 rpm for 5 min. The pelletwas then resuspended in buffer (0.025 M Tris–HCl, 2%

Fig. 1. Western blotting of ISCOM/rMSPs and ISCOMATRIX adju-vants with hyperimmune serum produced in rabbit against the rMSPs.Lanes 1, 3, 5 and 7 were ISCOMATRIX adjuvant and lanes 2, 4, 6 and 8were ISCOM/rMSPs. Lanes 1 and 2: hyperimmune serum to rMSP1a;lanes 3 and 4: hyperimmune serum to rMSP1b; lanes 5 and 6:hyperimmune serum to rMSP4; lanes 7 and 8: hyperimmune serum torMSP5.

P.M. Kawasaki et al. / Research in Veterinary Science 83 (2007) 347–354 349

SDS, 15% glycerol, 2.5% 2-mercaptoethanol, pH 6.8),boiled for 5 min and submitted to electrophoresis SDS7.5–17.5% polyacrylamide gradient gel (PAGE). The pro-teins were transferred to nitrocellulose membranes (Phar-macia Biotech), as previously described (Towbin andGordon, 1984), and the membranes were blocked by buffer(PBS + 0.1% Tween 20 + 5% non-fat dried milk) for 1 h atroom temperature under agitation. The membrane wasthen washed in PBS-T (PBS + 0.1% Tween 20) and incu-bated for 1 h with monospecific polyclonal serum producedfrom rMSP1a, rMSP1b, rMSP4, and rMSP5, and dilutedat 1:5000, 1:5000, 1:5000, and 1:10.000, respectively. Themembranes were then washed; MSPs were incubated withHRP anti-bovine IgG 1:2000 (Sigma Immuno Chemicals),and detected by an enhanced chemiluminescence (ECL)Western blotting system (Amersham International, Amer-sham, United Kingdom). Protein molecular mass markers(RainbowTM coloured, Amersham Life Science) were usedas standards.

2.5. Immunization

Animals were maintained according to the NationalInstitute of Health standards the use of laboratory ani-mals. Six groups of female BALB/c mice (4–5 weeks) wereimmunized with r-MSP4 to produce immune serum. Thepositive control group (G1) was immunized with 20 lgof protein from initial corpuscle of A. marginale emulsi-fied with complete Freund’s adjuvant in the first immuni-zation and with incomplete Freund’s adjuvant insubsequent immunizations. Two negative controls wereutilized: one group was immunized with 0.1 mL of saline(G2) and the other with ISCOMATRIX adjuvant (G3).Group 4 animals was immunized with a pool of rMSPs(5 lg of each), while mice from Group 5 were immunizedwith 0.1 mL (20 lg) of ISCOM/rMSPs. Group 6 micewere immunized with vaccine ISCOMATRIX:rMSPs(1:1). Three immunizations were administered by subcuta-neous injections after 14 days post-inoculation. After eachimmunization the animals were bled, the serum was sepa-rated, and the titer and specificity of antibodies were ana-lysed by enzyme-immunoassay (ELISA) and Westernblotting. Serum control was obtained from non-immu-nized animals.

2.6. Estimation of the level of specific antibody activity byELISA to detect IgG, IgG1, and IgG2a

A pool of sera (n = 8), obtained at day 42 of the exper-iment, from G1 mice (immunized with initial corpuscle ofA. marginale) was used as positive control serum. A poolof sera of G3 animals (n = 8) was used as negative control.Optimal dilutions were established using checkerboardtitrations with dilutions of sera, antigen, and conjugates.The anti-A. marginale antibody responses from Groups 1and 2 animals were evaluated by the use of A. marginale

antigens to coat wells in a 96 well microtiter plate (Nunc)

at 1 lg/well, while pools of rMSPs were placed in 0.5 lg/well to evaluate anti-rMSP antibody responses in Groups3 (ISCOMATRIX adjuvant), 4 (pool of rMSPs), 5(ISCOM/rMSPs), and 6 (ISCOMATRIX/rMSPs). Afterovernight incubation at 4 �C, serum samples from eachgroup of mice immunized were diluted to 1:50 in PBST buf-fer, pH 7.4, and with 5% skim med milk powder. Then100 lL of the diluted sample was added in duplicate toeach well and incubated for 45 min at 37 �C. Three second-ary antibodies were used: anti-mouse IgG horseradish per-oxidase conjugate (1:10.000), and anti-mouse IgG1 andIgG2a horseradish peroxidase conjugate (dilution1:20.000); each incubated for 45 min at 37 �C. The sub-strate for the enzyme detection was OPD (o-phenylenedi-amine) 0.4 mg/mL, H2O2 0.04% in substrate buffer(0.1 M acid citric, 0.2 M Na2HPO4). The reaction was thenblocked with 50 lL of 1 M HCl and the plates were read at490 nm. The absorbance values were estimated and the ODvalue was calculated. To control plate-to-plate variation,positive and negative control sera were included in everyplate and a corrected OD value was determined for eachsample as follows:

ODcorr ¼ ODsample �ODnegative control of plate

� ��

=ODpositive control of plate �ODnegative control of plate�:

A serum was considered to be positive when ODcorr > [ODmean (from negative control sera, n = 8)+ 3 SD (standarddeviation from negative control sera)] (Garcia et al., 2006).

2.7. Western blotting of immunized mice BALB/c serum

The rMSPs were electrophoresed through a SDS-PAGE7.5–17.5% and transferred to a nitrocellulose membrane;

350 P.M. Kawasaki et al. / Research in Veterinary Science 83 (2007) 347–354

Western blotting was realized according to the techniquedescribed above, utilizing the serum of animals of allgroups two weeks after the last immunization, and serumpre-immune at a dilution of 1:100.

2.8. Statistical analyses

The OD results from IgG2a and IgG1 were comparedby ANOVA test, using GraphPad Prism statisticalpackage, a P-value of 60.05 was considered assignificant.

G1

0.00.10.20.30.40.50.60.70.80.91.01.11.2

Experimental days

Experimental days

Experimental days

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490n

m

G5

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0 14 28 42

0 14 28 42

0 14 28 42

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Fig. 2. Humoral response of mouse evaluated by whole IgG ELISA. G1 (init(pool of rMSPs), G5 (ISCOM/rMSPs), and G6 (ISCOMATRIX/rMSPs). The

3. Results

3.1. Analysis of ISCOM and ISCOMATRIX

Electronic microscopy demonstrated the presence oftypical structure of ISCOM and ISCOMATRIX, and alsothe presence of lipid vesicle.

The reactivity of proteins incorporated to ISCOM, withhyper immune mono-specific serum produced in rabbits foreach r-MSP by Western blotting is shown in Fig. 1. Theantibody produced against the r-MSP1a reacted with pro-teins of different molecular masses varying from 60 to

Experimental days

Experimental days

Experimental days

G2

0.00.10.20.30.40.50.60.70.80.91.01.11.2

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G4

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G6

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0 14 28 42

0 14 28 42

0 14 28 42

ial bodies of A. marginale), G2 (PBS), G3 (ISCOMATRIX adjuvant), G4arrows indicate the immunizations moment.

IgG1

IgG2a

0.00

0.25

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IgG1IgG2a

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G1 G5

G6 G6 G5

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G3 G2

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Fig. 3. Humoral response of mouse evaluated by indirect ELISA IgG1and IgG2a. G1 (initial bodies of A. marginale), G2 (PBS), G3 (ISCOM-ATRIX adjuvant), G4 (pool of rMSPs), G5 (ISCOM/rMSPs), and G6(ISCOMATRIX/rMSPs).

P.M. Kawasaki et al. / Research in Veterinary Science 83 (2007) 347–354 351

105 kDa. The antibodies produced against the rMSP1b,rMSP4, and rMSP5 reacted with proteins of 100 kDa,47 kDa, and 29 kDa, respectively. The control adjuvant

Fig. 4. Western blot analysis of rMSPs with the sera of immunized Balb/c mou(c) G5 (ISCOM/rMSPs), and (d) G6 (ISCOMATRIX/rMSPs). Lanes 1 and 2rMSP5. Lanes 1, 3, 5, and 7, pre-immune sera of each group. Lanes 2, 4, 6, a

(ISCOMATRIX) that did not contain the rMSPs in itsstructure did not react with the anti-rMSPs.

3.2. Humoral response in mice induced by ISCOM/rMSPs

and ISCOMATRIX/rMSPs vaccines

The data of ELISA for whole IgG, IgG1, and IgG2aare shown in Figs. 2 and 3. G2 and G3 animals were neg-ative in either whole IgG-ELISA or IgG1 and IgG2a-ELI-SAs. All groups immunized with rMSPs (Groups 1, 4, 5,and 6) induced IgG production 14 days after the firstimmunization, while the levels of IgG increased graduallyafter the third immunization (Fig. 2). No significant differ-ence in the production of IgG was observed between ani-mals from G5 and G6, animals that were immunized withISCOM/rMSPs and ISCOMATRIX + rMSPs,respectively.

No statistical differences were observed between IgG2aand IgG1 ODs from G1, G4, G5, and G6 (Fig. 3,P = 0.52). Groups 1, 4, 5, and 6 induced the productionof IgG2a 14 days after the first immunization; however,

se groups. (a) G1 (initial bodies of A. marginale), (b) G4 (pool of rMSPs),, rMSP1a; lanes 3 and 4 rMSP1b; lanes 5 and 6, rMSP4; lanes 7 and 8,nd 8, immune sera of group.

352 P.M. Kawasaki et al. / Research in Veterinary Science 83 (2007) 347–354

the production of IgG2a from animals immunized with ini-tial corpuscle of A. marginale (G1) and animals immunizedwith pools of rMSPs (G4) was not uniform. The bestIgG2a response was obtained in G4 animals 14 days afterthe third immunization; however, this was not significantlydifferent when compared to the animals immunized withISCOM/rMSPs and ISCOMATRIX + rMSPs (G5 andG6).

All animals that received antigen (Groups 1, 4, 5, and 6)responded specifically against the rMSPs by Western blot-ting (Fig. 4). The sera from these groups reacted with therespective rMSPs as follows: rMSP1a (60 and 105 kDa),rMSP1b (100 kDa), rMSP4 (47 kDa), and rMSP5(29 kDa).

4. Discussion

The live vaccines or erythrocytes of infected bovineswith A. marginale induce protective immunity that reducedor prevented clinical disease; however, these vaccines didnot prevent those bovines that were persistently infectedby A. marginale.

This study evaluated the humoral immune response inBALB/c mice of the subunit vaccine for bovine anaplasmo-sis that consisted of the MSP1a, MSP1b, MSP4, and MSP5recombinant proteins incorporated into ISCOM and Fre-und adjuvant. The ISCOM/rMSPs and ISCOMatrix/rMSPs were prepared by a more simple process due tothe introduction of lipid film hydration that promoted anintimate union between Quil A, cholesterol, and phospho-lipids (Copland et al., 2000). The proportion of 2:1:2 of thereagents Quil A: cholesterol: phospholipids utilized duringthis study demonstrated excellent results, with the forma-tion of typical structures of ISCOM, as visualized by elec-tronic microscopy. These reagents can also form othercolloidal structures, such as lipid vesicles, depending onthe proportion of each component system (Demanaet al., 2004).

The technique using lipid film hydration proved to bequick and efficient for the preparation of ISCOM andISCOMATRIX; similar results were described (Demanaet al., 2004). The preparation of ISCOMATRIX/rMSPsis easier when compared to the use of ISCOM/rMSPs,because rMSPs are added only after the formulation ofthe ISCOMATRIX adjuvant. Some reagents utilized dur-ing the purification of rMSP can inhibit the formation oftypical vesicle when added before lipid film hydration(Malliaros et al., 2004).

Both ISCOM/rMSPs and ISCOMATRIX/rMSPs pre-sented satisfactory immune humoral response in mice;these responses were comparatively elevated as those pro-duced with Freund adjuvant. Similar results were demon-strated in experiments with many others pathogens,where the animals presented high production of wholeIgG and were protected against challenge (Kersten et al.,1988; Hubschle et al., 2003; Crouch et al., 2004; McArthuret al., 2004).

Also, the recombinant vaccines tested during this studyinduced the production of specific antibodies to eachrMSPs as demonstrated by Western blot. The sera fromall groups that received antigen (i.e., positive controlgroup, rMSPs with Freund adjuvant, ISCOM/rMSPs,and ISCOMATRIX/rMSPs vaccine) reacted with therMSP1a (60–105 kDa), rMSP1b (100 kDa), rMSP4(47 kDa), and rMSP5 (29 kDa), suggesting that a subunitvaccine containing rMSPs can be efficient in an initial infec-tion of A. marginale, where the antibodies develop impor-tant functions.

The immunity against A. marginale needs humoraland cellular mechanisms. The antibodies against theMSPs act as opsonins, facilitate phagocytosis and elimi-nation of A. marginale, as demonstrated during in vitro

assay with antibodies from cattle immunized with thecomplex MSP1 (Cantor et al., 1993). Another possiblefunction of antibodies is the inhibition of invasion oferythrocytes by riquettsia (McGarey and Allred, 1994).The cellular response was also demonstrated in calvesprotected from infection upon challenge, A. marginale-specific CD4+ T cells as helper cells induced immuno-globulin isotype switching to IgG1 and IgG2. This isconsistent with the hypothesis that a strong Th cellresponse characterized by interferon-c and IgG2 produc-tion is important for protective immunity of Anaplasmo-sis (Brown et al., 1998) .

In conclusion, the ISCOMATRIX/rMSPs was easilyprepared and showed efficient humoral response. Addi-tional studies will have to be performed in cattle to evaluatethe humoral and cellular mechanisms of this subunit vac-cine and their possible use as protective vaccines againsthomologous and heterologous strains of A. marginale.

Acknowledgements

The authors thank Dr. Selwyn Arlington Headley forrevision of the manuscript. This study was supported byCNPq, Fundacao Araucaria do Parana, and CAPES.

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