Proc. Natl. Acad. Sci. USAVol. 93, pp. 269-273, January 1996Medical Sciences

A Schistosoma mansoni fatty acid-binding protein, Sml4, is thepotential basis of a dual-purpose anti-helminth vaccineMIRIAM TENDLER*t, CRISTIANA A. BRITOt, MONICA MAGNO VILAR*, NICOLAU SERRA-FREIRE§, CATIA M. DIOGO*,MARILIA S. ALMEIDA*, ALEXANDRE C. B. DELBEM1, JOSEi FIGUEIREDO DA SILVAII, WILSON SAVINO*,RICHARD C. GARRATTF, NAFTALE KATZt, AND ANDREW J. G. SIMPSONt***Instituto Oswaldo Cruz-Fundacao Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; tCentro de Pesquisas Rene Rachou-Fundacao Oswaldo Cruz, Belo Horizonte,30190-002, MG, Brazil; §Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; lInstituto de Fisica de Sao Carlos, Universidade de Sao Paulo,Sao Carlos, SP, Brazil; and 'lUniversidade Federal de Pernambuco, Recife, PE, BrazilCommunicated by Warwick E. Kerr, Universidade Federal de Uberldndia, Uberlandia, Brazil, July 13, 1995

ABSTRACT Molecular cloning of components of protec-tive antigenic preparations has suggested that related parasitefatty acid-binding proteins could form the basis of the pro-tective immune crossreactivity between the parasitic trema-tode worms Fasciola hepatica and Schistosoma mansoni. Mo-lecular models of the two parasite proteins showed that bothmolecules adopt the same basic three-dimensional structure,consisting of a barrel-shaped molecule formed by 10 antipar-allel (8-pleated strands joined by short loops, and revealed thelikely presence of crossreactive, discontinuous epitopes prin-cipally derived from amino acids in the C-terminal portionsof the molecules. A recombinant form of the S. mansoniantigen, rSml4, protected outbred Swiss mice by up to 67%against challenge with S. mansoni cercariae in the absence ofadjuvant and without provoking any observable autoimmuneresponse. The same antigen also provided complete protectionagainst challenge with F. hepatica metacercariae in the sameanimal model. The results suggest that it may be possible toproduce a single vaccine that would be effective against at leasttwo parasites, F. hepatica and S. mansoni, of veterinary andhuman importance, respectively.

Schistosomiasis, caused principally by Schistosoma mansoni, S.haematobium, and S. japonicum, afflicts some 200 millionindividuals in tropical regions of the world. Fascioliasis causedby Fasciola hepatica is an economically important disease ofcattle and sheep in Europe, the Americas, Australia, and NewZealand. There are no vaccines against Schistosoma or Fasci-ola species; however, there is evidence for protective immunecrossreactivity between S. mansoni and F. hepatica. Hillyer andcoworkers (1-3) have isolated a low molecular weight F.hepatica fraction that protects against both S. mansoni and F.hepatica infections. A component of this fraction is an antigenwith homology to mammalian fatty acid-binding proteins thatis termed Fhl5 (4). A similar antigen, Sm14, was cloned fromS. mansoni following studies of a protective saline extract ofadult worms, SE (5). These results suggested that the pair ofsimilar parasite proteins could mediate immune crossreactionand represent the basis of a subunit vaccine effective againstboth species. We have investigated the molecular relationshipof Fhl5, Sml4, and mammalian fatty acid-binding proteins(FABPs) (6) in detail and found that Fhl5 and Sml4 containpotential discontinuous, crossreactive epitopes not present intheir mammalian counterparts. Furthermore, we have foundthat a recombinant fusion protein containing the completeSml4 polypeptide stimulates a protective response againstboth S. mansoni and F. hepatica infection. The data thussuggest that Sm14 could form the basis of a single vaccine thatis effective against both parasites. Such a dual-purpose vaccine,

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

aimed primarily for veterinary use against an economicallyimportant disease such as fascioliasis, may represent an at-tractive route to preclinical development of a vaccine againsthuman schistosomiasis.

MATERIALS AND METHODSSequence Analysis and Molecular Modeling. The amino

acid sequences of Sm14, Fhl5, and nine human homologueswere obtained from the OWL nonredundant protein sequencedatabase (7). The crystallographic structures of FABP fromhuman muscle (8), mouse adipocyte (9), and rat intestine (10)were obtained directly from the Brookhaven Protein DataBank (accession codes 2HMB, 1ALB, and 2IFB, respectively).Sequences of known crystal structure were aligned by least-squares superposition of the molecules using C' coordinatesalone, and the remaining sequences were subsequently incor-porated into the alignment by the method of Barton andSternberg (11) as implemented in the AMPS package.For the construction of the Sm14 model, the backbone of the

10 (3-strands and three a-helices was based on that of 1ALB.(3-Turn types were determined on the basis of the position ofglycine and/or asparagine and aspartic residues within theturn, which normally dictates their conformation. Side chainswere based on the conformation of identical residues inhomologous positions, where available, or otherwise on pre-ferred rotamers for the given secondary structure. The modelwas energy minimized by means of GROMOS within the graph-ics program WHATIF (12). The final model shows good stereo-chemistry as evaluated by the program PROCHECK (13), with95% of the residues falling within the most favored regions ofthe Ramachandran plot. The model presents an Eisenbergenvironment quality of 47.65, which is acceptable for a proteinof 133 residues in length (14), and WHATIF quality of -0.52,which is above the threshold expected for correct structures(15). An analogous procedure was adopted for the modeling ofFhl5, yielding Eisenberg and WHATIF qualities of 59.92 and-0.33, respectively.Expression of Recombinant Sm14 (rSml4). The entire open

reading frame of Sm14 was excised from the constructpDSSml4 (5) with BamHI and Hindlll restriction endonucle-ases, cloned into pGEMEX-1 (Promega), and used to trans-form Escherichia coli strain BL21(DE3). Expression of a40-kDa fusion protein consisting of the first 260 aa of the majorbacteriophage T7 capsid protein together with the complete

Abbreviations: FABP, fatty acid-binding protein; rSml4, recombinantSml4; SE, saline extract of adult Schistosoma mansoni.tTo whom reprint requests should be addressed at: Departamento deHelmintologia, Insituto Oswaldo Cruz-Fundagao Oswaldo Cruz Av.Brasil, 4365-Manguinhos, Cx. Postal 926, CEP 21045-900 Rio deJaneiro, RJ, Brazil.**Present address: Ludwig Institute for Cancer Research, Sao Paulo,

SP, Brazil.

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sequence of Sm14 (rSml4) was induced during logarithmic-phase growth of transformed bacteria in the presence ofampicillin (100 ,ag/ml) by addition of 1 mM isopropyl /-D-galactopyranoside followed by a further 3 hr of culture. Torecover the expressed fusion protein, bacterial cells werecollected by centrifugation at 15,000 x g, suspended in 50 mMTris HCl/2 mM EDTA/1 mM dithiothreitol/0.2% lysozyme atpH 7.5, and incubated on ice for 15 min. The lysate was thensonicated for two 30-sec cycles and recentrifuged, and thepellet was suspended in 50mM Tris HCl/10mM EDTA/1 mMdithiothreitol/0.5% Triton X-100 at pH 7.5, and centrifugedagain. After a further round of resuspension and centrifuga-tion, the final pellet was suspended in water and subjected toSDS/PAGE. The major band representing the expressedfusion protein was electroeluted and shown to be homoge-neous by reelectrophoresis.Immunization Experiments. New Zealand White rabbits

were immunized with two intradermal/subcutaneous doses of600 ,ug of SE [a saline extract of adult S. mansoni (16)] or 80,ug of rSml4 in the presence of Freund's complete adjuvant atan interval of 7 days, followed 21 days later by an intraperi-toneal booster injection with the same amount of antigen in theabsence of adjuvant. For outbred Swiss mice the doses were300 ,tg and 10 ,ug respectively, in the presence or absence ofadjuvant and were administered by the same route and with thesame immunization regimen. For assays of protection againstS. mansoni, the rabbits were challenged percutaneously with1000 cercariae and the mice with 100 cercariae (except whereindicated otherwise) 60 days after the last immunization andperfused 45 days later. Overall protection was calculated by theformula [(C - V)/C] x 100, where C is the average numberof worms in control animals and V is the average number ofworms in vaccinated animals. Statistical analysis was done withStudent's t test. For histopathological analysis of the mice usedfor protection experiments, fragments of the liver, intestine,spleen, lungs, and kidneys were taken at the time of perfusionand fixed with 10% formaldehyde, embedded in paraffin,sectioned, and stained with hematoxylin/eosin. For assays ofprotection against F. hepatica, Swiss mice, permissive hosts forF. hepatica, were challenged orally with 3 metacercariae 45days after the last immunization and sacrificed 30 days later.Worms were recovered from the bile duct and the livers of allanimals analyzed by using sequential histological sectionsstained with hematoxylin/eosin. Protection was determined onthe basis of histopathology where intraparenchymal liver le-sions, characteristics of normal F. hepatica infection, weretaken as indicating nonprotected animals.

RESULTSIdentification of Potential Crossreactive Epitopes on Sml4

and Fhl5. Sm14 is as closely related to several human pro-teins-including P2 myelin protein (-42% sequence identity),and FABP from cardiac muscle (%Z42%)-as it is to Fhl5(-44%). The schistosome and F. hepatica molecules do notexhibit simple, coordinated alterations in size or continuoussequence when compared with their human homologues how-ever. Sm14 shows a marked falloff in conservation with humansequences toward the C terminus (from about residue 85onward) whereas the two parasite sequences show -47% meanidentity within the same region (Fig. 1). This region is the mostpoorly conserved across the family as a whole (17-19), and thislow conservation suggests a possible role for this region indetermining Sml4/Fhl5 epitopes. Molecular models con-structed for both Sm14 and Fhl5 show that the two moleculesadopt the same three-dimensional topology as other membersof the FABP family. This consists of a 10-stranded antiparallel,B-barrel with short interstrand connections which generallyform (3-turns and is illustrated for Sml4 in Fig. 2. TheC-terminal portion of the molecule, which essentially consti-

my-lIn P2......aFA}DP ..........cREPI ..........cRASPI ........cRAP2 .........pPABP-hom ......iFABP ..........IFASP..........hFABP ..........

aFABP (MOUSE)..iPABP (PRAT) ....

Smi4 ...........Phl5...........

my-lin P2 ......FAPBP ..........cRBPI..........cRJkBPI .........cRABP2 .........pFABP-hom ......IFABP ..........lFABP ..........hFAEP ..........

aFADP (MOUSE)..iFABP (RAT)....

Sm14 ...........Fhl5 ...........

myliln P2 ......FABP ..........cRBPI ..........cRABPI .........cRABP2 .........pFABP-hom ......iFABP..........lFABP .........hJABP .........

aYABP (HOUSE)..lPABP (RAT)....

5m14 ...........FhlS ...........

SNKFLOTWKLVSSENFDDYHEALOGVGATRKLGNLA KPTVIISKKCDAFVOTWKLVSSENFDDYHKEVOVOFATRKVAGMA KPNHIISVHPVDFTGYWKMLVNENPEEYLRALDVNVALRRIANLL KPDKEIVQDPNFAOTWKMRSSENFDELLKALOVNAMLRKVAVAAASKPHVEIRQDPNFSNWKIXIR9ENFEELLKVLGVNOVLRKIAVAAASKPAVEIKOE

HATVQQLEORWRLVDSKGFDEYMRELGVOIALRKHGAMA KPDCIITCDAPDSTKVDRSENYDKFHEKHGVNXV RAEAAHD NLKLTITQEMSFSOKYQLOSQENrEAFMIAIQLPEELIQKOKDI KGVSEIVQN

VDAYLGTWALVDSKNFDDYHKSLGVQFATROVASMT KPTTIIEKN1 hi h2 2

CDAFV§fVtMi KN)IUMMAFDQ5TwKVDRNENYEKFHEKHQINVVKRALQAHD NLKLTITQE

VMSATRQIGNTV T FT4D0FYLVSEDtYFM DKILNA " FLL

10 20 30 40

GDIITIRTESTFKNTEISFKLQQEFEETT ADkRKTKSIVTLQ RCSLuQDVITIKSESTFKNTEISFIL OEFDEVT ADDRKVKSTITLD OGVLVODHMIIRTLSTFRNYIMDFQVOKEFEEDLTOIDDRKCMTTVSWD GDKLQODQrYIKTSTTVRTTEINFKVGEOFEEET VDGRKCRSLATWENENKIHODTFYIKTSTTVRTTEINFKVOEEFEEQT VDGRPCKSLVRWESENKMVGKNLTIKTESTLKTTQFSCTLaEKFEETT ADGRKTOTVCNFT DGALVONKFTVKESSAFRNIVVFELOVTFNYNL ADGTELRGTWSLE ONKLIGKIHFKPTITAGSKVIQNEFTVOEECELET MTOEKV1TVVQLEOIDNKLVODILTLKTHSTFENTEISFRLaVEFDETT ADDRKVKSIVTLD GGKLV

3 4 5 6 7UtdZRStTFK*'lT Lad fflf ADDV.±±flJ G)ONKFTVkSiSNrRNIDVVFELCVDFAYSL ADGTELTGTWTME GNKLV

*% * % * *** e

MLToTEF L iqg 8 SGt 4lmv GEEK3 TEO ESI tII It

so so 70 go 90

OVORW NGKETTIKRKLVDG KMVAECKMKOVVCTRIYEKVEVQIOW DGKSTTIKRKREDD KLVVECVMKOVTSTRVYERACVQKQ EKEQRGWTQWIEEGD ELHLEHRVEQVVCKQVFKKVQCTQTLLEGDGPKTYWTRELAND ELILTFGADDVVCTRIYVRECEQKLLtOEOPKTSWTRELTNWDELILTNTADDVVCTRVYVREOHOEW DOKESTITRKLEDO KLVVECVMNNVTCTRIYEKVEQKFKRTD NCNELNTVREXIID ELVQTYVYEOVEAKRIFKKD

TT FKNIKSVTZLNGD IITNTHTLODIVFKRISKRIBLQKW DQQETTKVRELIDQ KLILTLTHGTAVSTRTYFKEA

7 6 9 10sVQ})~ DKSTTIKRR aD:...........LV1E.3KQVTTRVYSROKFKRVD HOKELIAVREISON ELIQTYTYEGVERIFKKE

'S * 'S **

(QVD N iY K RNYKRLSPKG &C3luEq TTLL

100 110 120 130

FIG. 1. Sequence alignment of members of the FABP familyconsidered in this study. The first nine sequences are all of proteinsderived from human tissues: myelin P2 from peripheral nerve; adi-pocyte FABP, aFABP; cellular retinol-binding protein I, cRBP1;cellular retinoic acid-binding proteins I and II, cRABP1 and cRABP2;psoriasis-related FABP homologue, pFABP-hom; intestinal FABP,iFABP; liver FABP, lFABP; and heart FABP, hFABP. FABPs frommouse adipocyte and rat intestine are also shown, as they were usedtogether with hFABP for the construction of molecular modes. The13-sheet strands of aFABP are indicated by the shaded blocks andnumbered consecutively; the two a-helices (hl and h2) are marked bythe solid bars. Identical residues in the two parasite sequences (Sml4and Fhl5) are boxed. The subset of these residues which are conservedin no more than three of the human sequences are indicated either bystars (for exposed residues) or by the percent symbol (for solvent-inaccessible residues).

tutes strands 7-10 of the barrel, is shown in blue. The interloopconnections were ruled out as candidates for continuousepitopes because they are in general extremely small, unlikethe flexible projecting loops which often correspond to anti-genic determinants in other proteins (21, 22). Possible discon-tinuous crossreactive epitopes were thus sought by identifyingresidues that are identical in Sm14 and Fhl5 but that occur inno more than three of nine human sequences. Twenty-eightsuch residues were found, 22 of which were exposed on thesurface and thus potentially contribute to antigenicity (indi-cated by a ball-and-stick representation in Fig. 2). Of these 22residues, 13 were derived from the C-terminal portion of theprotein. The internal variant residues represent conservativesubstitutions or are at highly variant positions and are expectedto have a minimum effect on overall structure. The externalvariant residues, on the other hand, cluster at the upper andlower ends of the barrel, potentially constituting functional

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FIG. 2. Ribbon diagram of the molecular model for Sm14 con-structed on the basis of the three-dimensional structures of thehomologous molecules from mouse adipocyte, rat intestine, andhuman muscle. The C-terminal region of the molecule (from residue85 onward) is shown in blue. Residues shown in ball-and-stick repre-sentation are identical in both Sm14 and FhlS but present in no morethan three of the nine human sequences. Residues which fulfill thiscriterion but which are buried within the interior of the molecule andtherefore not expected to participate in epitopes have been excluded.The figure was produced with the program RIBBONS (20).

discontinuous epitopes with significant variation from humanproteins. Indeed, the four interstrand connections which areincluded within the C-terminal part of the structure showpronounced spikes in the main-chain accessibility (19), oftencorrelated with antigenic determinants (22). Thus, the anti-genicity of Sm14 appears to depend on dispersed C-terminalamino acids brought together in three-dimensional space due

to the overall structure of the molecule. It was thus reasonedthat vaccination with the intact protein was more likely to besuccessful than vaccination with discrete peptides.Immunization of Experimental Animals with rSml4. Sm14

was expressed as a fusion protein (rSml4) with the bacterio-phage T7 major capsid protein. Western blotting studiesshowed that rSml4 was specifically and strongly recognized byantibodies from mice and rabbits infected with S. mansoni orhumans from endemic areas. Furthermore, anti-Sm14 anti-bodies produced by immunizing New Zealand White rabbitswith rSml4 in the presence of Freund's complete adjuvantstrongly labeled sections of adult S. mansoni worms andreacted with a 14-kDa molecule in Western blots. However,these antibodies did not label any cell of normal rabbittissues-including brain, heart, skeletal muscle, small intes-tine, pancreas, kidney, liver, spleen, thymus, and testis-asassessed immunocytochemically (data not shown).

Preliminary assessment of the protective activity of rSml4,using four immunized rabbits challenged with S. mansonicercariae (Table 1, Exp. 1), showed that rSml4 stimulated anextremely high level of protective immunity. Then the samerSml4/adjuvant preparation was used to vaccinate Swiss micefor experiments varying the magnitude and number of cer-carial challenges (Table 1, Exps. 2-6). Again a high level ofprotection was achieved in all experiments. Four large, inde-pendent experiments (Table 2, Exps. 7-10) it showed thatrSml4 conferred protection at a high level, with or withoutFreund's complete adjuvant. Vaccination with the T7 capsidprotein alone did not stimulate any measurable protection.Analysis of the distribution of worm burdens among theanimals in the four experiments demonstrated that vaccinationwith rSml4 resulted in a significant skewing with up to 66% ofthe animals harboring slO worms, in contrast to controlgroups where the majority of animals had from 21 to 30 wormsand none had less than 11 worms (data admitted but notshown). In all unvaccinated mice typical immunopathologicalreactions were found, including periportal infiltrate, largeperiovular granuloma, and areas of focal necrosis. In vacci-nated animals the extent of periportal infiltrate was greatlyreduced and was limited to mononuclear cells. In addition, thenumber of periovular granulomas was reduced and areas offocal necrosis were absent.

Swiss mice immunized with rSml4 were also challengedorally with F. hepatica metacercariae in two independentexperiments (Table 3, Exps. 11 and 12). Detailed histopatho-

Table 1. Protection of rabbits and mice against experimental infection with S. mansoni cercariae byvaccination with Sm14 or SE in the presence of Freund's complete adjuvant

Challenge, WormNo. per no. of burden %

Exp. Animals group cercariae Group SEM protection

1 Rabbits 4 1000 PBS 109.5 ± 1.66rSml4 12.0 ± 1.81 89.0*SE 7.4 ± 0.98 93.2*

2 Mice 20 1000 PBS 170.0 ± 3.23rSml4 58.0 ± 2.86 65.9*

3 Mice 20 500 PBS 62.6 ± 0.86rSml4 31.5 ± 0.9 49.7*

4 Mice 20 100 PBS 27.2 ± 0.84rSm14 11.2 ± 0.6 58.8*

5 Mice 20 2 x lOOt PBS 52.6 ± 0.7rSml4 33.0 ± 0.72 37.3*

6 Mice 20 3 x lOOt PBS 69.6 ± 0.86rSml4 42.3 ± 0.83 39.2*

PBS, phosphate-buffered saline.*p < 0.05.tChallenges were at 1-week intervals and perfusion was 45 days after final challenge.

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Table 2. Protection of Swiss mice against experimental infectionwith 100 S. mansoni cercariae by vaccination with Sm14 or SE inthe presence or absence of Freund's complete adjuvant (FCA)

Exp. Group

7 PBSrSml4

rSml4+FCACP

CP+FCA

8 PBSrSml4

rSml4+FCASE

SE+FCA

9 PBSrSml4

rSml4+FCASE+FCAFCA

10 PBSrSml4

rSml4+FCAFCA

No. ofmice1219202222

814102021

812111110

79910

Worm burden± SEM

24.5 ± 0.719.9 ± 0.59

12.1 ± 0.5727.7 ± 0.5328.4 ± 0.53

28.0 ± 0.6613.6 ± 0.489.6 ± 0.48

13.1 ± 0.477.8 ± 0.46

26.8 ± 0.768.6 ± 0.6

10.1 ± 0.5311.6 ± 0.5825.9 ± 0.88

28.0 ± 0.7712.5 ± 0.6610.1 ± 0.6423.0 ± 0.57

% protection

59.6*50.6*

00

51.4*65.7*53.2*72.1*

67.9*62.3*56.7*3.4

55.3*64.0*17.8

CP, bacteriophage T7 capsid protein.*p < 0.05.

logical examination revealed 100% protection against parasitematuration and liver damage. This was also observed in agroup of mice vaccinated with SE. The livers of all the animalsshowed that in nonvaccinated animals complete parasite de-velopment and migration had occurred, as indicated by exten-sive damage to the hepatic parenchyma and destruction of thehepatic lobes. In addition, in four and three of the unvacci-nated animals in Exps. 11 and 12, respectively, adult wormswere found in the bile ducts. In none of the vaccinated animalswere parasites of any developmental stage observed, nor wereany intraparenchymal hepatic lesions indicative of successfulparasite maturation found. Nevertheless, capsular cicatriciallesions were seen in all vaccinated mice, indicating thatimmature parasites had successfully migrated to the liver buthad not developed further. Representative cross-sections ofthe livers of a vaccinated and unvaccinated mouse are shownin Fig. 3.

DISCUSSIONBefore embarking on vaccination experiments with rSml4, weconsidered the structure of Sml4 and Fhl5 in detail. Theanalysis revealed that the parasite molecules adopt three-dimensional structures which are identical in basic form tohomologous host proteins, preserving both the overwhelmingmajority of residues which contribute to the "hydrophobicbackbone" and one of the two previously identified alternativerecognition motifs for the fatty acid ligand (6). The few variant

Table 3. Protection of Swiss mice against experimental infectionwith three F. hepatica metacercariae by vaccination with Sm14

No. of No. withExp. Group mice hepatic lesions* % protection

11 PBS 15 15rSml4 15 0 100SE 15 0 100

12 PBS 14 14rSml4 15 0 100

*Intraparenchymal lesions were taken as indicating nonprotectedanimals.

FIG. 3. Vaccination of mice with rSml4 prevents intraparenchymalhepatic lesions following infection with F. hepatica. (a) Histopatho-logical profile of the liver from an unvaccinated mouse where there isgeneral disruption of the hepatocytes. (b) In contrast, the pattern froma rSml4-vaccinated mouse is normal. Material was fixed in 10%formaldehyde, embedded in paraffin, and stained with hematoxylin/eosin. CV, centrolobular vein (X770).

amino acids in the interior of the molecule are either in highlyvariable positions not essential for functions or representconservative substitutions. The putative epitope-formingamino acids are on the exterior of the molecules and areclustered in the tertiary, but not the primary, structure of theprotein.The vaccine trials provided evidence that Sm14 is potently

immunogenic and capable of stimulating protective immunityagainst both S. mansoni and F. hepatica infection, confirmingthat Sm14 and Fhl5 may represent the molecular basis for thepreviously observed cross-protection between these two par-asites. All the protection data were obtained with outbredanimals, thus showing that although the numbers of epitopeson rSml4 may be limited, due to its similarity to host proteins,this did not result in detectable genetic restriction of theprotective responses that it stimulates.The levels of protection that we have achieved with rSml4

vaccination against schistosome infection are as high as those

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achieved with other candidates for a subunit anti-schistosomevaccine (23-26). It will be of interest to determine whetherSml4 also stimulates protective immunity against other schis-tosome species, particularly since the homologous protein in S.japonicum contains almost all the amino acids associated withpredicted epitope formation (27). In our evaluation, thepossibility of a multipurpose vaccine for both veterinary andhuman use is of fundamental significance in terms of realprogress toward effective immunoprophylaxis against schisto-somiasis.

We acknowledge the Daresbury Laboratory (U.K.) for access to theSEQNET facility. We thank Erika Recone Borges for technical support.This investigation received financial support from the United NationsDevelopment Program/World Bank/World Health Organization Spe-cial Program for Research and Training in Tropical Diseases and fromthe Brazilian agencies Conselho Nacional de Pesquisas, Fundasao deAmparo a Pesquisa do Estado de Sao Paolo, Financidora de Estudose Projetos, and Fundficao Oswaldo Cruz.

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Medical Sciences: Tendler et al.

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