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Vaccine 27 (2009) 4127–4135

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

m21.6 a novel EF-hand family protein member located on the surface ofchistosoma mansoni adult worm that failed to induce protection againsthallenge infection but reduced liver pathology

ebora O. Lopesa , Leonardo F. Paivab , Mauricio A. Martinsa , Fernanda C. Cardosoa , Matheus A. Rajãoa ,ean M. Pinhoa, Marcelo V. Caliari c, Rodrigo Correa-Oliveirad, Samantha M. Mellob, Luciana. C.C. Leiteb,ergio C. Oliveiraa,∗

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Avenida Antonio Carlos 6627, Pampulha, Belo Horizonte 31270-901, MG, BrazilCentro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, BrazilDepartmento de Patologia, Universidade Federal de Minas Gerais, Avenida Antonio Carlos 6627, Pampulha, Belo Horizonte 31270-901, MG, BrazilCentro de Pesquisas Rene Rachou, Av. Augusto de Lima 1715, Belo Horizonte, MG 30190-002, Brazil

r t i c l e i n f o

rticle history:eceived 2 December 2008eceived in revised form 26 March 2009ccepted 21 April 2009vailable online 14 May 2009

a b s t r a c t

Schistosomiasis continues to be a significant public health problem that affects 200 million people world-wide. This is one of the most important parasitic diseases, and one whose effective control is unlikely inthe absence of a vaccine. In this study, we have isolated a cDNA clone encoding the Schistosoma mansoniSm21.6 protein that has 45% and 44% identity with Sm22.6 and Sj21.7 EF-hand containing antigens, respec-tively. Confocal microscopy analysis revealed that Sm21.6 is a membrane-associated protein localized on

eywords:chistosoma mansoniaccinem21.6ytokinesecombinant protein

the S. mansoni adult worm. Mouse immunization with rSm21.6 induced a mixed Th1/Th2 cytokine profileand no protection against infection. However, vaccination with rSm21.6 reduced by 28% of liver granu-loma numbers, 21% of granuloma area and 34% of fibrosis. Finally, rSm21.6 was recognized by sera fromindividuals resistant to reinfection compared with patients susceptible to reinfection and this moleculeshould be further studied as potential biomarker for disease resistance. In conclusion, Sm21.6 is a newtegument protein from S. mansoni that plays an important role in reducing pathology induced by parasite

infection.

. Introduction

Human schistosomiasis is a chronic debilitating parasitic dis-ase that represents a major health problem in endemic areas,n various parts of South America, Africa, and Southeast Asia [1].chistosomiasis mainly occurs in developing countries and is theost important human helminth infection in terms of global mor-

ality. This parasitic disease affects more than 200 million peopleorldwide causing more than 250,000 deaths per year [2]. Fur-

hermore, schistosomiasis causes up to 4.5 million DALY (disabilitydjusted life year) losses annually [3]. Current schistosomiasis con-rol strategies are mainly based on chemotherapy but in spite ofecades of mass treatment, the number of infected people remains

onstant [4]. Extensive endemic areas and constant reinfection ofndividuals together with poor sanitary conditions in developingountries make drug treatment alone inefficient [5]. Many con-ider that the best long-term strategy to control schistosomiasis is

∗ Corresponding author. Fax: +55 31 34092666.E-mail address: scozeus@icb.ufmg.br (S.C. Oliveira).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.04.068

© 2009 Elsevier Ltd. All rights reserved.

through immunization with an anti-schistosomiasis vaccine com-bined with drug treatment [6]. A vaccine that induces even a partialreduction in worm burdens could considerably reduce pathologyand limit parasite transmission [1].

In this study, we have identified the membrane protein Sm21.6that is one member of the family of EF-hand containing para-site proteins with sequence similarity to dynein light chain (DLC),such as Sm22.6 and Sm21.7 [7]. Sm22.6 is a protein consideredto be a target for the human IgE response, and it correlates withresistance to reinfection after treatment [8]. Regarding Sm21.7,it was isolated from a cDNA expression library using irradiatedcercariae-vaccinated rabbit serum. This antigen was designated“vaccine dominant” because parasite-derived Sm21.7 was recog-nized preferentially by sera from vaccinated mice compared withsera from infected mice [9]. Regarding localization, both antigenswere detected at the tegument surface by proteomic analysis [10].

Schistosomes have a unique outer syncytial surface, the tegumentthat constitutes the host–parasite interface and therefore repre-sents an obvious tissue to target for development of new controlstrategies [11]. Indeed, so far the two surface exposed membraneproteins identified on biotinylated adult worms, Sm29 and TSP-2,

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128 D.O. Lopes et al. / Vac

ave been tested as vaccines and provided protection in murineodels and are selectively recognized by naturally resistant indi-

iduals [12,13].In this present study, we investigate the vaccine potential of

m21.6 protein, a new EF-hand family protein member identified byranscriptome approach. Herein, we detected Sm21.6 transcripts inhe cDNA libraries of worm, cercariae, egg and lung-stage of Schis-osoma mansoni. Furthermore, immunolocalization revealed thatm21.6 is present on the tegument of male adult worm. Next, immu-ization of mice with rSm21.6 induced a mixed Th1/Th2 cytokinerofile but fails to engender protective immunity. However, rSm21.6accination reduced liver pathology in vaccinated mice. Finally,Sm21.6 was recognized by sera from individuals resistant to rein-ection compared with patients susceptible to reinfection and this

olecule should be further studied as potential biomarker for dis-ase resistance.

. Materials and methods

.1. Mice and parasites

Female C57BL/6 mice aged 6–8 weeks were purchased from theederal University of Minas Gerais (UFMG) animal facility. The miceere treated with anti-parasitic drugs 14 days before the exper-

ments start as previously described [14]. All protocols involvingnimals were approved by the local Ethics Committee on Ani-al Care (CETEA–UFMG no. 023/05). Cercariae of S. mansoni wasaintained routinely on Biomphalaria glabrata snails at CPqRR

Centro de Pesquisa Rene Rachou-Fiocuz) and prepared by exposingnfected snails to light for 1 h to induce shedding. Cercarial num-ers and viability were determined using a light microscope prioro infection.

.2. Study population

Peripheral blood was obtained from individuals with differ-nt genetic background living in two different endemic areas forchistosomiasis (“Melquiades” and “Côrrego do Onca”). These indi-iduals were classified in four groups, regarding their infectiontatus and the selection of subjects was performed based onlyn the criteria for inclusion and exclusion of each group inde-endent of previous knowledge of immune responses for each

ndividual. Non-infected (NI) individuals are healthy people with-ut any parasite infection or contaminated water contact. Anotherroup showed stool-negative examination after treatment and waslassified as resistant to S. mansoni reinfection (RR). Individualslassified as susceptible to S. mansoni reinfection (SR) showed stool-ositive examination following treatment (praziquantel, 40 mg/kg).he sera from RR and SR groups were obtained 6 months afterraziquantel treatment and these individuals were examined for S.ansoni infection using the Kato–Katz technique before treatment

nd 1, 6 and 12 months after treatment to check for reinfection rates.he water contact exposure was objectively evaluated by observersnd SR and RR groups had at least one contact daily. Individualsrouped as infected (INF) showed stool-positive examination ando treatment history. These infected patients had infection levelshat varied from 48 to 224 epg (egg counts per gram of faeces). Addi-ionally, RR, SR and INF patients were negative for other helminthic

nfections. These patients or their legal guardians gave informedonsent after explanation of the protocol that had been previ-usly approved by the Ethical Committee of the Federal Universityf Minas Gerais. Details regarding sex and age of the individualsncluded in this study are described in Table 1.

7 (2009) 4127–4135

2.3. Isolation of Sm21.6 cDNA

The Sm21.6 DNA sequence was obtained from São Paulo S.mansoni transcriptome project by searching homologue proteinsto Sm21.6 [15]. The sequence obtained was compared with thosealready deposited in the GenBank database using the Basic LocalAlignment Search Tool (BLAST) algorithm available at the NCBIwebsite (http://www.ncbi.nlm.nih.gov/BLAST/). The amino acidsequence of Sm21.6 was obtained and the search for conserveddomains was performed using Pfam program. After that, Syfpeithialgorithm was used in the prediction of human MHC II binding epi-topes (http://www.syfpeithi.de/Scripts/MHCServer.dll). Primarystructure analysis and post-translational modification predictionof Sm21.6 were performed using Expasy (Expert Protein AnalysisSystem, http://ca.expasy.org/tools) computer program. Molecularweight (MW) and isoelectric point (pI) were calculated with theCompute pI/Mw tool (http://www.expasy.ch/tools/pi tool.html).The presence of signal peptide (SignalP 3.0, http://www.cbs.dtu.dk/services/SignalP), glycosylation sites (YinOYang, http://www.cbs.dtu.dk/services/YinOYang) and transmembrane proteintopology prediction (TMHMM, http://www.cbs.dtu.dk/services/TMHMM-2.0) was also performed by bioinformatics.

2.4. Sm21.6 cloning and sequencing

Specific oligonucleotides were designed using the S. man-soni EST assembly contig (SmAE 605436.1, http://bioinfo.iq.usp.br/schisto6/cgi-bin/ceditor/ceditor.pl). The 5′ and 3′ oligonu-cleotides, CAC CAT GTT GGA AGA ATT TAT CAA AG and TTA ACCTTT TGG TGT ACG C, were used to amplify the complete openreading frame of Sm21.6 from S. mansoni cDNA clone MA3-9999U-M275-F06 (GenBank accession CD079525). The PCR reaction wasperformed using Platinum Pfx enzyme (Invitrogen, São Paulo,Brazil), and initiated with one cycle of 5 min at 94 ◦C, followed by30 cycles of 30 s at 94 ◦C, 1 min at 55 ◦C, and 3 min at 68 ◦C. PCRproducts were purified from agarose gel electrophoresis and clonedinto pENTR/D TOPO cloning vector (Invitrogen) and sequenced toconfirm its identity.

2.5. Sm21.6 transcript analysis in parasite life-stage cDNAlibraries

In order to identify Sm21.6 mRNA transcripts in the cDNAlibraries from specific human developmental stages of S. mansoni,PCR was performed using Sm21.6 specific primers. Adult worm,lung-stage, egg and cercariae cDNA libraries were gifted by theMinas Gerais Genome Network. PCR parameters using 1 �L (100 ng)of each library as template were as follows: denaturation for 30 sat 94 ◦C, annealing for 30 s at 57 ◦C and extension for 1 min at 72 ◦C(35 cycles). Agarose gel (1%) eletrophoresis and ethidium bromidestaining were performed to visualize the Sm21.6 sequence amplifiedfrom each library.

2.6. Expression and purification of recombinant Sm21.6

To produce a recombinant (r) Sm21.6, the full-length DNAsequence was directionally cloned by recombination into pDEST17(to produce a protein that contains an N-terminal hexahistidinetag). For protein expression, transformed BL21(DE3) cells (Invit-rogen) were grown in 600 mL LB plus ampicillin (1 mg/mL) untilOD600 = 0.6. Isopropyl-�-d-thiogalactopyranoside (IPTG, Invitro-gen) was added to the culture to a final concentration of 1 mM,

and cells were incubated for 3–4 h at 37 ◦C. Cells were harvestedby centrifugation and resuspended in 50 mL of lysis buffer (10 mMNaHPO4 pH 7.5, 150 mM NaCl, 1× Protease Inhibitor Cocktail (PIC)(Sigma, St. Louis, MO)). The cell suspension was passed three times(1500 psi) through a French press and the crude homogenate was

D.O. Lopes et al. / Vaccine 27 (2009) 4127–4135 4129

Table 1Study population.

Group Infection status Description Age (mean ± S.D.) Sex (M/F)

Infected (I) n = 7 Infected with S.mansoni

Individuals living in an endemic area for schistomiasiswith stool-positive examinations

17.1 ± 14.9 5/2

Susceptible toreinfection (SR) n = 9

Infected with S.mansoni

Individuals living in a endemic area for schistosomiasisthat present stool-positive examination afterpraziquantel treatment

22.8 ± 12.1 3/6

Resistant to reinfection(RR) n = 9

Not infected Individuals living in a endemic area for schistosomiasisthat although water contact, present stool-negative

fter pr

18.8 ± 9.3 5/4

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examination aon-infected (NI) n = 8 Not infected Individuals living

schistosomiastheir lives

entrifuged at 20,000 × g for 30 min at 4 ◦C. For protein purification,he inclusion bodies of rSm21.6 were rinsed twice with washinguffer (10 mM NaHPO4 pH 7.5, 500 mM NaCl, 1 M Urea, 1× PIC), andnally resuspended in solubilization buffer (wash buffer plus 8 Mrea, 10 mM imidazole). The recombinant protein was then purifiedy metal affinity chromatography after on-column refolding usinghe Akta Prime system (GE Healthcare, São Paulo, Brazil). Briefly, therotein concentration was adjusted to 0.5 �g/�L with solubilizationuffer and the sample loaded onto a Ni2+-NTA column (HisTrap HPGE Healthcare, 5 mL bed volume) pre-equilibrated with the sameuffer. The column was washed with 12 bed volumes of the equili-ration buffer until the UV absorbance baseline was reached. Then,he refolding of the bound protein was performed on-column byhe use of a linear urea gradient from 8 to 0 M, starting with thequilibration buffer and finishing with a buffer containing 10 mMaHPO4, pH 7.5, 500 mM NaCl, 10 mM imidazole. The total gradientolume was 25 mL and the flow rate was 1.0 mL/min. The refoldedSm21.6 was eluted with 10–500 mM imidazole linear gradient.ractions encompassing the main peak were pooled and the purityf the preparation assessed by SDS-PAGE. The buffer was exchangedo PBS (pH 7.4) using a desalting column (GE Healthcare) before these of this antigen.

.7. Immunolocalization of Sm21.6 in male adult worm of S.ansoni

Adult worms used in confocal microscopy studies were recov-red from perfused mice, as previously described [12]. For sectionsssays, 7 �m slices were deparaffinized with xylol series. Parasitesere blocked with 1% BSA in PBST (Tween 20, 0.05%) for 1 h and

ncubated with anti-rSm21.6 serum diluted 1:20 in blocking buffer.erum from non-immunized mice was used as a negative control.amples were washed three times with PBST and incubated withnti-mouse IgG antibody conjugated to Alexa Fluor 594 (Molec-lar Probes, CA, USA) diluted 1:100 in blocking buffer containinghalloidin Alexa Fluor 488 (Molecular Probes) to stain actin micro-laments. The samples were washed four times and mounted inntifade reagent (Prolong gold–Molecular Probes). The parasitesere visualized in a Zeiss 510 Meta confocal microscope using an

mmersion objective. All the parameters and microscope settingssed were maintained throughout the process.

.8. Measurement of specific anti-Sm21.6 antibodies

ELISA was performed to confirm the titer of specific IgG, IgG1nd IgG2a anti-Sm21.6 in the serum of immunized animals as pre-iously described [16]. Briefly, 96 well flat-bottom microtiter plates

Nunc, Rochester, NY) were coated overnight at 4 ◦C with 100 �Lf rSm21.6 at a concentration of 5 �g/mL in a 0.1 M carbonateicarbonate buffer (pH 9.6) per well. The plate was then washedith 0.05% Tween 20 and blocked with bovine fetal serum 10% in

BS for 2 h at room temperature. Further, the plates were washed

aziquantel treatmentf endemic area fornever present this disease in

28.3 ± 4.5 3/6

three times with PBS plus 0.05% Tween 20 (PBST). One hundredmicroliters of each serum diluted 1:100 in PBST were added perwell and incubated for 1 h at room temperature. Plate-bound anti-body was detected by peroxidase-conjugated anti-mouse IgG, IgG1and IgG2a (Southern Biotechnology, Birmingham, AL) diluted inPBST 1:10000, 1:5000 and 1:2000, respectively. After 1 h at 37 ◦C,the plate was washed and orthophenylenediamine (Sigma) plus0.05% hydrogen peroxide in phosphate citrate buffer (pH 5) wasadded (100 �L/well). This mixture was then incubated for 30 minat room temperature, and the reaction was stopped by addictionof 2 N H2SO4 (50 �L/well). Absorbance was read at 492 nm usinga microplate reader (Bio-Rad, Hercules, CA, USA). Animals thatreceived PBS were used as negative control.

2.9. Cytokine analysis

Cytokine experiments were performed using splenocyte cul-tures from individual mice immunized with rSm21.6 plus CFA/IFA(n = 5 for each group). Splenocytes were isolated from maceratedspleens of individual mice 10 days after the third immunizationand washed twice with sterile PBS. After washing, the cells wereadjusted to 1 × 106 cells per well for IL-4, IL-10, IFN-� and TNF-�assays in RPMI 1640 medium (Gibco, CA, USA) supplemented with10% FBS, 100 U/mL penicillin G sodium, 100 �g/mL streptomycinsulfate, 250 ng/mL amphotericin B. Splenocytes were maintained inculture with medium alone or stimulated with rSm21.6 (25 �g/mL)or concanavalin A (ConA) (5 �g/mL) as previously described [16].The 96-well plates (Nunc) were maintained in an incubator at37 ◦C with 5% CO2. For cytokine assays polymyxin B (30 �g/mL)was added to the cultures and this treatment completely abrogatethe cytokine response to LPS as previously described [17]. Culturesupernatants were collected after 24 h of ConA stimulation, 48 hof rSm21.6 stimulation for IL-4 and TNF-� analysis and 72 h ofrSm21.6 stimulation for IL-10 and IFN-�. The assays for measure-ment of IL-4, IL-10, IFN-� and TNF-� were performed using theDuoset ELISA kit (R&D Diagnostic, Minneapolis, MN) according tothe manufacturer’s directions.

2.10. Challenge infection and worm burden recovery

Fifteen days after the last boost, mice were challenged throughpercutaneous exposure of abdomen skin for 1 h in water con-taining 100 cercariae (LE strain). Forty-five days after challenge,adult worms were perfused by portal veins [18]. Two independentexperiments were performed to determine protection levels. Theprotection was calculated comparing the number of worm recov-

ered from each group of immunization with its respective controls,using the formula:

PL = WRCG − WREGWRCG

× 100

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130 D.O. Lopes et al. / Vac

here PL = protection level, WRCG = worms recovered from controlroup, and WREG = worms recovered from experimental group.

.11. Hepatic granuloma analysis

Liver sections from mice (10/group) of control and experi-ental group immunized with rSm22.4 and infected with 100

ercariae were collected 45 days post-infection to evaluate theffect of immunization in granuloma formation. The livers sectionsemoved from the central part of the left lateral lobe were fixedith 10% buffered formaldehyde in PBS. Histological sections wereerformed using microtome (4 mm) and stained in a slide withomory’s trichromic. The granulomas were counted in Axiolab Carleiss microscopy using 10× objective lens. All slides were digital-zed by scanner HP scanjet 2400, in the resolution of 300 dpi. Theixels of each histological section were fully screened, with subse-uent creation of a binary image and calculating the total area of theut. The area of the lower cutoff was used as a minimum standardf tissue to be statistically analyzed. The results were expressedy the number of granuloma per area of liver (mm2). The area ofranulomas was obtained through the KS300 software containedn Carl Zeiss image analyzer. Fifteen granulomas from each mouse

ith a single well-defined egg were randomly chosen at a micro-cope with 10× objective lens and scanned through microcameraVC TK-1270/RGB. Using a digital pad, the total area of granulomasas measured and the results were expressed in square microme-

ers (�m2). To calculate the area of fibrosis in the granulomas, theixels of the collagen areas were selected through the real imageith subsequent creation of a binary image and obtain the area inm2.

.12. Measurement of human IgG anti-Sm21.6

Sera of schistosomiasis patients living in endemic areas inrazil were tested by ELISA as previously described by our group19] to measure the levels of IgG to rSm21.6 antigen. For thisssay, 96-well flat-bottom microtiter plates (Nunc) were coatedvernight at 4 ◦C with 100 �l of rSm21.6–6×HIS at a concentra-ion of 5 �g/mL in 0.1 M carbonate bicarbonate buffer (pH 9.6)er well. The plates were then washed with 0.05% Tween 20 andlocked with 10% bovine fetal serum in PBS (pH 7.4) for 2 h atoom temperature. Subsequently, the plates were washed threeimes with PBS plus 0.05% Tween 20 (PBST). Serum samples diluted:50 (IgG) in PBST (100 �L/well) were added in duplicate and thelates incubated for 1 h at room temperature. Peroxidase-labellednti-human IgG (Sigma, St Louis, MO) was added at dilutions of:10,000 (100 �L/well). After 1 h at 37 ◦C, the plates were washednd orthophenylenediamine (Sigma) plus 0.05% hydrogen peroxiden phosphate citrate buffer (pH 5) was added (100 �L/well). This

ixture was than incubated for 30 min at room temperature, andhe reaction was stopped by addition of 2 N H2SO4 (50 �L/well).bsorbance was read at 492 nm using a microplate reader (Bio-Rad,ercules. CA, USA).

.13. Statistical analysis

Statistical analysis was performed with Student’s t-test andsing a computer software package GraphPad Prism(La Jolla, CA).

. Results

.1. Cloning and molecular characterization of S. mansoni Sm21.6

The full-length sequence of the S. mansoni cDNA encodingm21.6 was obtained by PCR amplification from a S. mansoni tran-

7 (2009) 4127–4135

scriptome cDNA clone with specific oligonucleotides that weredesigned using the corresponding EST contig (SmAE 605436.1).The resulting full-length cDNA displayed an ORF of 552 bp, encod-ing a protein of 183 amino acids with a predicted molecular massof approximately 21.6 kDa and an isoelectric point of 5.18. BlastPcomparisons of the deduced S. mansoni protein sequence to Gen-Bank showed that the best match (E-value = 1 × 10−80) was to anunknown S. japonicum protein (SJCHGC01853), with 77% identityand 86% similarity, probably the Sj21.6 ortholog. This was followedby S. mansoni tegument antigen Sm22.6 (AAA29856) and the S.japonicum 21.7 kDa antigen (AAD13338) with 45% and 44% identity,respectively.

3.2. Bioinformatic analysis of Sm21.6

Bioinformatic analysis of Sm21.6 primary sequence showed thatthis protein did not possess a signal peptide and transmembranedomain and it was predicted as a soluble protein. Three glycosy-lation sites are presented in the mature sequence on the Thr83,Thr85 and Thr126 (data not show). The analysis performed revealedthat all aligned proteins shown in Fig. 1, have an EF-hand motifpresent in several antigens associated with the tegument. Sm22.6and Sj21.7 have two EF-hand motifs while Sm21.6 and the S. japon-icum unknown protein has only one (Fig. 1). Moreover, we detectedthe presence of a dynein domain in Sm21.6 as observed in the otherproteins aligned. Additionally, prediction of human MHC class IIepitopes revealed that Sm21.6 possess four promiscuous peptidesthat have high binding score for HLA-DRB1 0101, HLA-DRB1 0301,HLA-DRB1 0401, HLA-DRB1 0701, HLA-DRB1 1101 and HLA-DRB11501 alleles (data not show). The profile of predicted HLA peptidesis more similar between Sm21.6 and the S. japonicum unknownprotein when compared Sm21.6 with Sm22.6 or Sj21.7 (Fig. 1). TheHLA epitopes are more concentrated at the center to the C-terminalregion of Sm21.6.

3.3. Sm21.6 transcripts are detected in cDNA libraries of allparasite stages

Sm21.6 transcripts with 552 bp were detected in all stages of S.mansoni life cycle tested: adult worm, cercariae, egg and lung-stagecDNA libraries (Fig. 2). Since the lungs seem to be the major site ofparasite elimination in the irradiated cercariae model of vaccina-tion; we hypothesized that the use of Sm21.6 as a vaccine candidatecould be an interesting approach in the control of schistosomiasis[20].

3.4. Production of recombinant Sm21.6

The gene was cloned into pDEST17 expression vector, and theprotein was expressed in E. coli BL21 (DE3) strain. Protein extract oftransformed bacteria showed a band at ∼22 kDa when induced withIPTG (Fig. 3A, lane 2). The bacteria were then lysed by a French pressand the lysate separated into soluble and insoluble fractions. Theinclusion bodies were shown to contain the majority of the recom-binant protein, which was mostly solubilized by extraction with8 M urea (Fig. 3B, lane 3). The protein was bound to a nickel-chargedcolumn under denaturing conditions, and purified after on-columnrefolding by affinity chromatography through an imidazole linear

gradient from 10 to 500 mM (data not shown). Eluted fractions con-taining rSm21.6 were pooled, and protein yield after purificationwas estimated to be around 12.0 mg of rSm21.6/L (Fig. 3B, lane 5).After buffer exchange the protein was used in vaccination experi-ments.

D.O. Lopes et al. / Vaccine 27 (2009) 4127–4135 4131

Fig. 1. In silico analysis of S. mansoni EF-hand domain containing proteins. The full amino acid sequences of Sm21.6, Sm22.6 (AAY57921.1), Sj21.7 (AAD13338.1) and S.japonicum unknown protein (AAW25529.1) are aligned to show homologous domains (Cluacid position of each protein. EF-hand motifs are boxed in light grey while dynein domaiSyfpeithi computer program and they are underlined.

Fig. 2. PCR analysis of cDNA libraries using specific primers for the completes((

3t

f

trol group and presented the highest level at 45 days after the first

Facfli

equence of Sm21.6. Lanes: (1) ladder (bp); (2) negative control; (3) egg cDNA library;4) lung-stage cDNA library; (5) adult worm cDNA library; (6) cercariae cDNA library;7) positive control.

.5. Sm21.6 is a membrane-associated protein located on the

egument of S. mansoni adult worm

A mouse serum raised against rSm21.6 revealed through con-ocal microscopy that Sm21.6 is located at the surface of the adult

ig. 3. Expression and purification of Sm21.6 in Escherichia coli. (A) SDS-PAGE (12%) profileclone before and after induction with 1 mM IPTG, respectively. (B) Inclusion bodies wereolumn and purified after on-column refolding; lanes (1) soluble fraction after lysis; (2) wow through; (5) pooled fractions of rSm21.6–6×HIS-tag fusion protein eluted after Ni2+ c

ndicate the rSm21.6.

stal W). Numbers after each row indicate the residue position relative to the aminons are boxed in dark grey (Pfam). HLA-DRB binding peptides were predicted using

worm stage of S. mansoni (Fig. 4B and C). This membrane-associatedantigen appears to be highly expressed on the surface of adult wormand is not found in internal tissues of the parasite body. No anti-Sm21.6 antibody staining was observed in sections incubated withpre-immune mouse serum (Fig. 4A).

3.6. Analysis of IgG isotype responses to rSm21.6 followingimmunization

To investigate the presence of specific anti-Sm21.6 IgG antibod-ies, sera from vaccinated mice were tested by ELISA. Experimentalgroup vaccinated with rSm21.6 showed high levels of IgG thatappeared 15 days after the first immunization compared to the con-

immunization (Fig. 5). To determine the type of immune responseinduced after vaccination, the subclasses IgG1 and IgG2a werealso analyzed. The levels of specific anti-rSm21.6 IgG1 and IgG2aat 60, 75 and 90 days after the first immunization revealed that

of E. coli (BL21DE3) transformed with the pDEST17-Sm21.6. Lanes 1 and 2 representextracted with 8 M urea and the denatured protein (lane 3) was bind to Ni2+ charged

ashed fraction from the inclusion bodies before solubilization with 8 M urea; (4),hromatography. Positions of molecular mass standards (kDa) are indicated. Arrows

4132 D.O. Lopes et al. / Vaccine 27 (2009) 4127–4135

Fig. 4. Immunolocalization of Sm21.6 on S. mansoni adult worm. Confocal microscopy images of male adult worm of S. mansoni are shown. Polyclonal anti-Sm21.6 andsecondary antibody coupled to Alexa-633 (red) were used for fluorescence detection of Sm21.6 on male adult worm sections. Serum from naive mice was used as negativecontrol. Phalloidin (green) was used for the actin localization. The Sm21.6 localization is represented in (a) and merges in (b) and the negative control is shown in (c). (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).

Fig. 5. Kinetics of specific IgG anti-Sm21.6 induced in mice immunized with rSm21.6.As control group, mice were injected with PBS plus Freund adjuvant (FA). Sera ofimmunized mice per group were collected at days 15, 30, 45, 60, 75 and 90 after thefirst immunization and assayed by ELISA. Arrows indicate the timing of vaccination.RRda

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Table 3Protection level induces by C57BL/6 mice immunization with rSm21.6 plus Freund’sadjuvant and infected with 100 cercariae.

Treatment Number of Level of p-Valueworm recovered protection (%)

1st experiment

PBS + CFA/IFA 30.2 ± 2.7rSm21.6 + CFA/IFA 28.8 ± 1.5 4.6a 0.27

2nd experiment

TI

D

679

esults are presented as the mean absorbance measured at 492 nm for each group.esults represent the mean of two independent experiments. Statistically significantifferences of vaccinated mice compared to FA + PBS control group is denoted by onesterisk for (p < 0.05).

ice immunized with rSm21.6 had high IgG1/IgG2a ratio, suggest-ng that the vaccination to induce a typical Th2 immune responseTable 2).

.7. rSm21.6 plus CFA/IFA immunization induces a Th1/Th2

ytokine profile

Then we decided to determine the cytokine profile producedy splenocytes following rSm21.6 stimulation. We measured IFN-, TNF-�, IL-4 and IL-10 in splenocyte culture supernatants from

able 2gG1 and IgG2a immune profile induced by vaccination with rSm21.6 or PBS.

aysa Groups

IgG1 IgG

PBS Sm21.6 PB

0 0.01 ± 0.009 1.95 ± 0.028b 0.5 0.007 ± 0.004 1.97 ± 0.023b 0.0 0.02 ± 0.016 2.04 ± 0.016b 0.

a Days after the first immunization.b Statistically significant compared to group of animals immunized with PBS.

PBS + CFA/IFA 51.1 ± 2.6rSm21.6 + CFA/IFA 48.8 ± 4.7 4.5 0.87

a Level of protection comparing rSm21.6 group versus PBS.

vaccinated mice. Cells from immunized animals stimulated withrSm21.6 produced statistically significant amounts of cytokinescompared to the respective controls cultured with medium alone(Fig. 6). Statistically significant level of IL-4, a signature of Th2-typeof immune response, was detected in the animals vaccinated withrSm21.6 plus CFA/IFA compared with the control group (Fig. 6B).Additionally IFN- � and TNF-�, Th1 cytokines, were produced inrSm21.6 CFA/IFA vaccinated group compared with control group(Fig. 6C and D). Furthermore, high levels of the modulatory cytokineIL-10 were also observed (Fig. 6A). These results indicate that vac-cination with rSm21.6 with adjuvant induces a mixed Th1/Th2cytokine profile.

3.8. rSm21.6 vaccination failed to confer protective immunity

To measure the protection levels induced by rSm21.6 plusCFA/IFA, mice were challenged and the levels of protection weredetermined by the difference in the number of adult worms recov-ered in the experimental groups compared to each control group45 days post-challenge (Table 3). The animals immunized with

2a IgG1/IgG2a ratio

S Sm21.6

06 ± 0.009 0.36 ± 0.014b 5.4205 ± 0.011 0.36 ± 0.013b 5.4705 ± 0.013 0.45 ± 0.027b 4.53

D.O. Lopes et al. / Vaccine 27 (2009) 4127–4135 4133

F t imm� resultb d sple

rpm

3

trtiPmm

3

obansgtIfi

4

cbi

ig. 6. Cytokine profile of mice immunized with rSm21.6. One week after the lasproduction in response to rSm21.6 (25 �g/ml) or medium alone as control. The

etween cytokines produced after rSm21.6 stimulation compared with unstimulate

Sm21.6 plus CFA/IFA and infected with 100 cercariae showed norotection against challenge infection in two independent experi-ents when compared to the control group (PBS plus CFA/IFA).

.9. Liver pathology

Histological analysis by digital morphometry of Gomoryrichromic-stained sections obtained from mice immunized withSm21.6 showed 28% of reduction of liver granulomas, 21% reduc-ion of granuloma area and 34% less fibrosis when animals werenfected with 100 cercariae when compared to mice that receivedBS (Fig. 7A–C). These findings suggest the potential use of thisolecule in an anti-disease vaccination program against schistoso-iasis.

.10. IgG responses of schistosomiasis patients to rSm21.6

To investigate the presence of specific anti-rSm21.6 IgG in seraf individuals according to their status of resistance and suscepti-ility to S. mansoni reinfection, ELISA was performed. Fig. 8 showsnti-rSm21.6 total IgG levels in sera of schistosomiasis patients andon-infected individuals. All schistosomiasis patients studied hadignificant IgG levels anti-rSm21.6 compared to the non-infectedroup. In order to determine if rSm21.6 could be recognized by resis-ant versus susceptible individuals to reinfection, we compared thegG profile of the RR group with SR patients. The resistant to rein-ection (RR) group had increased levels of IgG when compared tondividuals susceptible to reinfection (SR).

. Discussion

Control of schistosomiasis is based on drug treatment, snailontrol, improved sanitation and health education. The great num-er of reinfected people suggests that the vaccination, alone or

n combination with anthelmintic drugs, would provide a major

unization, splenocytes were isolated and assayed for IL-4, IL-10, IFN-� and TNF-s are presented as mean ± S.D. for each group. Statistically significant differencesnocytes are denoted by an asterisk (p < 0.05).

boost in the disease control program [1,21]. The surface proteinsof the lung-stage and adult worm of S. mansoni are considered tobe important antigens recognized by the host immune system andcritical molecules to compose an anti-schistosome vaccine [22,23].Herein, we isolated and identified by transcriptome approach theSm21.6 that is a Sm22.6 paralog protein. A group of important par-asite antigens belong to the family of EF-hand containing proteins,such as Sm22.6, Sm21.7 and Sm20.8 [7]. These proteins are sur-face membrane molecules that are recognized by antibodies fromprotected mice and by serum from S. mansoni patients resistant toreinfection. Sm21.6 showed 77%, 45%, and 44% of identity with theS. japonicum unknown protein (SJCHGC01853), Sm22.6 and Sj21.7,respectively. The Sm21.6 amino acid sequence contains one EF-hand motif and a dynein light chain domain at amino acid positions39–67 and 87–181, respectively. Furthermore, analysis of HLA epi-tope prediction of Sm21.6 has shown this antigen has high bindingscore for HLA-DRB1 0101, HLA-DRB1 0301, HLA-DRB1 0401, HLA-DRB1 0701, HLA-DRB1 1101 and HLA-DRB1 1501 alleles (data notshown). The four HLA-DRB epitopes found in Sm21.6 are more con-centrated at the center to the C-terminal region of the molecule.

Additionally, by PCR analysis we determined the presence ofSm21.6 transcripts in all cDNA libraries from S. mansoni life cyclestudied (cercariae, lung-stage, adult worm and egg). Furthermore,we confirmed by confocal fluorescence microscopy that Sm21.6is located on the surface of the adult worm of S. mansoni. Thismembrane-associated antigen appears to be highly expressed onthe tegument of this parasite and absent in the internal tissuesof the parasite body. However, it is interesting to observe thatby bioinformatic analysis we did not find a peptide signal or atransmembrane domain in the Sm21.6 amino acid sequence. In par-

allel with our confocal findings, Braschi et al. [10] have found byproteomic analysis that Sm22.6, Sm21.7 and other dynein-relatedproteins are present in tegument extracts. They concluded that thelayer immediately beneath the plasma membrane is largely com-posed of dynein homologues forming a macromolecular complex.

4134 D.O. Lopes et al. / Vaccine 2

Fig. 7. Liver pathology measured in rSm21.6 vaccinated mice. (A) Hepatic granulomanumbers, (B) granuloma area and (C) fibrosis area were measured in rSm21.6 immu-nized mice and PBS control group. The quantification of the number of granulomasin histological sections was performed using a microscope with 10× objective lensand the results were expressed in number of granuloma per area of liver (mm2). Thegranuloma and fibrosis area were also measured and the results were expressed insquare micrometers (�m2). Statistically significant difference of rSm21.6 comparedto PBS group is denoted by one asterisk (p < 0.05). Results represent data obtainedfrom two independent experiments.

Fig. 8. IgG profile of schistosomiasis patients to rSm21.6. Analysis of IgG antibodyresponses in sera of infected patients (INF), susceptible to reinfection (SR), resis-tant to reinfection (RR) and non-infected individuals (NI). Results are expressed asmeans ± S.D. Statistically significant differences compared to the non-infected group(NI) are denoted by an # for p < 0.05. Statistically significant differences betweenresistant and susceptible to reinfection groups are denoted by ** for p < 0.05.

7 (2009) 4127–4135

Dynein-related proteins, such as Sm21.6, are probably providingstructural scaffolding for the parasite tegument and they may playa role shuttling vesicles to the surface plasma membrane.

In the mouse model, rSm21.6 immunization induced high lev-els of anti-Sm21.6 IgG after the second immunization and showedhigh IgG1/IgG2a ratio at 45, 60 and 90 days after the first immu-nization. This finding suggests a tendency of a Th2 type of immuneresponse induced by rSm21.6 vaccination. Furthermore, we con-firmed by cytokine analysis that rSm21.6 immunization elicited amixed Th1/Th2 type of immune response characterized by high lev-els of IFN-� and IL-4. To determine if rSm21.6 conferred protectionagainst S. mansoni infection, mice were challenged with 100 cer-cariae 2 weeks after the last boost and 45 days later worm burdenwas analyzed. Immunization with rSm21.6 induced no protec-tion against cercarial challenge in C57BL/6. There was no apparentcorrelation between the antibody levels generated and protectiveefficacy in this group, since all immunized animals had high levelsof antibody. However, the pattern of the immune response inducedby rSm21.6 vaccination may have influenced the lack of protec-tive immunity. Several S. mansoni antigens tested by our group thathad a tendency to induce a Th2 type of immune response failed toengender protection in the mouse model [24,25]. In contrast, Th1antigens induced partial protection against infection [12,25,26].Nevertheless, recent findings by Siles-Lucas et al. demonstratedthat protection induced by vaccination with Sb14-3-3zeta antigenwas achieved independently of a Th1 response [27]. Another pointcould be the fact that rSm21.6 immunization induced high levelsof IL-10. IL-10 is a regulatory cytokine and its production modu-lates the inflammatory response that is important to protect miceagainst murine schistosomiasis. The modulation of immunity byschistosomes has been the subject of numerous studies reportingthe participation of cellular and molecular mechanisms in the regu-lation of the granulomatous process [28,29]. Even though, rSm21.6was not able to reduce adult worm burden it was able to pro-mote 28% reduction in granuloma numbers, 21% in the granulomaarea and 34% less fibrosis in mice infected with 100 cercariae. Thegranuloma modulation observed here which reduced the pathol-ogy associated with this disease may have been influenced by IL-10induced by rSm21.6.

Recently, Fitzsimmons et al. [7] reported that Sm22.6 andSm20.8 could be cryptic worm antigens, hidden during activeinfection but released from dying parasite during treatment. Theyproposed that such a release would vaccinate the host and boostantibody levels as a result of primary or secondary responses tothese antigens. Herein, we observed that patients resistant to rein-fection have increased levels of IgG anti-rSm21.6 compared toindividuals susceptible to reinfection after treatment. Similar towhat was observed for Sm22.6 [7], human IgG response to rSm21.6may be a potential biomarker for resistance to reinfection, howeverthis aspect requires further investigation.

Acknowledgments

This work was supported by CNPq, FAPEMIG, FAPESP,FINEP/SEBRAE and INCT de Doencas Tropicais/CNPq.

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