In vitro synthesis of a 28 kilodalton antigen present on the surface of the schistosomulum of Schistosoma mansoni

Molecular and Biochemical Parasitology, 17 (1985) 105-114

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Elsevier MBP 00588

IN VITRO S Y N T H E S I S O F A 28 K I L O D A L T O N A N T I G E N P R E S E N T ON T H E SURFACE OF THE SCHISTOSOMULUM OF SCHISTOSOMA

MANSONI

JEAN-MARC BALLOUL, RAYMOND J. PIERCE, JEAN-MARIE GRZYCH and ANDRE CAPRON Centre d'Immunologie et de Biologie Parasitaire, Unit~ Mixte I N S E R M U 167 - C N R S 624, Institut Pasteur, 15, rue Camille Gu~rin, 59019 - Lille Cedex, France

(Received 20 February 1985; accepted 5 June 1985)

Adult Schistosoma mansoni proteins were fractionated on polyacrylamideslab gels, recovered by electro° phoretic elution and used for immunization of Fischer rats. Three antisera recognizing, respectively,28, 78 and 85 kDa antigens were obtained. The 28 kDa antigen was found among the in vitro translation products from adult worm RNA, and among the ~ZSl-labelledsurface antigens of S. mansoni schistosomula. The isoelectric point of the 28 kDa antigen was 6.3-6.5. The 28 kDa antiserum mediated a cytotoxic activity against schistosomula when used in an in vitro assay in the presenceof a purified eosinophil cellpopulation. Key words: Protein fractionation; In vitro translation; Schistosoma mansoni; Schistosomulum surface

INTRODUCTION The first step towards the p r o d u c t i o n of a vaccine against S c h i s t o s o m a m a n s o n i is the identification of target antigens present on the s c h i s t o s o m u l u m surface a m o n g the in vitro t r a n s l a t i o n products of the parasite RNA. The general p a t t e r n of the antigens recognized by infected rat or h u m a n sera on the surface of the s c h i s t o s o m u l u m of S. m a n s o n i is characterized by a group of p r o m i n e n t molecules at 30-40 k D a [ 1]. A m o n g m o n o c l o n a l antibodies p r o d u c e d a n d that were specific for s c h i s t o s o m u l u m surface molecules, few were able to i m m u n o p r e c i p i t a t e in vitro t r a n s l a t i o n products. A n I g G 2 a m o n o c l o n a l a n t i b o d y has been p r o d u c e d recognizing a 38 k D a s c h i s t o s o m u l u m surface antigen [2]. This m o n o c l o n a l a n t i b o d y was able to kill S. m a n s o n i schistosomula in the presence o f e o s i n o p h i l s a n d also showed protective activity by passive transfer in the rat [3]. U n f o r t u n a t e l y this m o n o c l o n a l a n t i b o d y did not recognize the in vitro

Abbreviations: PBS, phosphate-buffered saline; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel

electrophoresis; EDTA, ethylene diamine tetraacetic acid disodium salt; MEM, Minimum Eagle's Medium; MNT, MEM/normal rat serum/tripeptide; NRS, normal rat serum; IRS, infected rat serum; NRtS, normal rabbit serum; HRtS, hyperimmune rabbit serum. 0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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t r a n s l a t i o n p r o d u c t s either in the ,eticulocytc lysate or Xenopus/a~,vts oocytc systcm~ [4,51. A n o t h e r m o n o c l o n a l a n t i b o d y which recognized a 20 k D a antigen on the surlace o f the s c h i s t o s o m u l u n l was also unable to recognize in vitro t r a n s l a t i o n p r o d u c t s [6]. H o w e v e r , two protective mouse m o n o c l o n a l a n t i b o d i e s recognizing m ~itro translation p r o d u c t s o1"28 and 37 k D a have been described ( L. Stein et al. (1984) ~-ed. Proc. 43 abstr. 1467), but the a u t h o r s did not correlate these antigens with activcJ~, s \ n t h e s i z e d adult w o r m antigens, nor with surface structures of the s c h i s t o s o m u l t m ~ The identification o f putative surlacc antigens a m o n g t r a n s l a t i o n p r o d u c t s has been p e r f o r m e d using indirect m e t h o d s . T w o main m e t h o d s have been e m p l o y e d : one using live s c h i s t o s o m u l a to a b s o r b a n t i b o d i e s recognizing surface antigens from sera [7], a n o t h e r using fixed s c h i s t o s o m u l a [8]. These indircct m e t h o d s ha,,e identified some p u t a t i v e p o l y p e p t i d e precursors ol the surface antigens o f the s c h i s t o s o m u h i m at 15, 22, 24, 27, 28, 29, 43. S6 and 100 k D a a c c o r d i n g to various authol> 15.v.81. No direct r e l a t i o n s h i p has until now been established between the in vitro ~ranslailon p r o d u c t s and the surface antigens o f the s c h i s t o s o m u l u m . In our l a b o r a t o r \ t,ao o t h e r antigens; of 22-26 k D a have been identified [91. These antigens were able tc) induce an IgE-dcp e n d e n t cytotoxic activity when injected into rats. Again, however, nu relation has been established Will3 !I1 vitro t r a n s l a t i o n product> In this work, we Imve p r o d u c e d p o l y c l o n a l antisera with restricted specificities against S. mansoni antigens. One such a n t i s e r u m recognized a 28 k I ) a antigen a m o n g the in vitro t r a n s l a i , m p r o d u c t s from adult worln R N A and a n l o n g the proteins actively synthesized bx adult w o r n > , l'his a n t i s e r u m also recognized ~l 28 k l ) a antigen from l a c t o p e r o x i d a s e ~:Sl-labelled s c h i s t o s o m u h i m surface antigen:, MATt{RIALS AND MFItt()I)S

Parasites.

We have used a Puerto Rican strain of ,S'. mansoni t h r o u g h o u t this work. A d u l t w o r m s were collected by portal perfusion from golden h a m s t e r s (Mesocricems auratus), and then w a s h e d in p h o s p h a t e - b u f f e r e d saline (PBS) or in M i n i m u m Eagle's M e d i u m ( M E M ) when used for m e t a b o l i c labelling. Skin s c h i s t o s o m u l a were collected 3 h after a p p l i c a t i o n of cercariae to isolated pieces o f Swiss mouse a b d o m i n a l skin [ 10],

Fractionation o/S. mansoni antigens.

A d u l t w o r m s were h o m o g e n i z e d in PBS buffer using a P o t t e r - E l v e h j e m h o m o g e n i z e r a n d the h o m o g e n a t e centrifuged tit 5 000 rpm for 20 rain. A b o u t 2 mg o f antigens were f r a c t i o n a t e d on 13% p o l y a c r y l a m i d e slab gels using the d i s c o n t i n u o u s buffer system of L a e m m l i [11]. A f t e r electrophoresis, gets were stained by C o o m a s s i e brillant blue. The stained b a n d s were c.ut ()tit with a scalpel a n d t r a n s f e r r e d to the elution gel consisting o f a s u p p o r t i n g gel cast as shown in Fig. i Elution was p e r f o r m e d at 40-60 V for 6-12 h. F h e rate o f elution d e p e n d s on thu percentage o f the s e p a r a t i n g gel a n d on the m o l e c u l a r size o f the proteins being eluted. The m i g r a t i o n of the samples was s t o p p e d at the 2 M N a C l - g l y c e r o l laver interface ay, previously described 1121. W h e n all the peptides were eluted into the glycerol l a v c r

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Fig. 1. Diagram showing the elution slab gel system. (a) 2 M NaCI in water; (b) glycerol layer, 40% glycerol in

62.5 mM Tris-HCl, pH 6.8 and 0.1% SDS; (c) samples in gel slice from the first separation; (d) supporting gel, 13% polyacrylamidein 380 mM Tris-HCl, pH 8.8 and 0.1% SDS.

electrophoresis was stopped and the samples were removed with a Pasteur pipette. Samples were then dialysed against water to remove sodium dodecyl sulphate (SDS) and salts before being concentrated by lyophilisation. The fractionated proteins were then reanalysed on a 13% polyacrylamide slab gel.

Immunization procedure. The antigens, fractionated as described above, were injected intraperitoneally with Freund's complete adjuvant into 3-month-old female Fischer rats. After 2 weeks, the rats were bled and reinjected in the same way. The rats were maintained immunized by injection of antigen every 2 weeks and were bled monthly. Sources of other immune sera. Fischer rats were infected by percutaneous exposure to 1 000 cercariae of S. mansoni and sera (IRS) were recovered 78 days after infection. Hyperimmune rabbit serum (HRtS) was obtained by immunizing rabbits with homogenates of S. mansoni adult worms by the Vaitukatis method [13]. Extraction of parasite RNA. Total RNA was extracted by the method previously described by Chirgwin et al. [ 14] and modified by Grausz et al. [4]. Live parasites were homogenized in a solution of 4 M guanidium thiocyanate, 5% 2-mercaptoethanol, 0.02 M sodium acetate pH 5.0 and 0.1% antifoam A (Sigma, St. Louis, MO, U.S.A.). This homogenate, previously spun for 15 min at 10000 rpm in a Sorvall HB-4 rotor to remove insoluble material, was layered over 1.5 ml of 3.4 M CsC1. Centrifugation was for 18 h at 30000 rpm and 20°C in a Beckman SW-39 rotor. The RNA pelleted during the centrifugation was solubilized in RNase-free water and precipitated with ethanol at -20°C.

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Translation ofextractedRNA in a cellc/kee translation system.

We have used the rabbit reticulocyte system as previously described by Pelham and J a c k s o n [15] using a lysatc provided by A mersham (U.K.). The translation mix contained 250 ng tal-E of total aduh S. mansoni RNA, l ta('i p.d ' ol'[~SS]methionine(l 100 Ci mmol ~, Amersham), 162mM potassium acetate, 0.70 mM magnesium acetate and 50 tam anaino acid mix without unlabelled methionine. The final concentration of the lysate was aboul 65(~.

Metabolic labelling ~ladult worm polypeptides.

20 adult worms <~1 both sexes were incubated in 1.5 ml of M E M containing neither methionine nor cvsteine for 1 h ~tt 37°C. The culture medium was then changed and 50 taCt of [~:'S]methionine were" added. Labelling was performed for 6 h at 37°C. The worms were then washed in cold M E M and homogenized in 200 tal of immunoprecipitation buffer Isee below).

lmmunoprectpitation of metabolic labelled products and in vitro translation product.s I m m u n o p r e c i p i t a t i o n assays were performed as previously described [I]. Labelled polypeptides (5 X 1()~ cpm) were diluted in 500 tal of the immunoprecipitation buffer lit m M Tris-HCl, pH 7.4, 0.5% Triton X-100, 0,1% SDS,0.15 M NaCI, 2 mM E D T A and incubated with 10 tal of serum for 2 h at r o o m temperature. Protein A - S e p h a r o s e ( 10 mg) in precipitation buffer was then added and the samples incubated f o r 2 h at r o o n l temperature. The Sepharose beads were then washed 10 times with precipitation buffer and a final wash was performed with a solution of 10 mM "Fris-ttCl, ptt 7.4, 2 mM E D T A . The beads were then suspended in 40 tal of sample bulTer c~mtaining 1~i SI)S, 150 mM Tris-HCl, pH 6.8, 10% sucrose. After boiling for 3 rain i~> ~olubihze immune. complexes, the samples were electrophoresed through 13% polyacrylamide gels and fluorographed using Amplify Scintillator (Amersham).

Two dimensional gelanalysis.

T w o dimensional gel separation w~ts performed exactl? as described by O'Farrell [ 16].

Labelling orS. mansoni schistosomula surface antigen.

Radioiodination olschistosomula surface was performed according to the method of Marchalonis e~t al. [17] and modified by Dissous et al. [1].

Eosinophil-dependent c.vtotoxicity. The cytotoxicity assay was performed on S. mansent schistosomula as previously described [18,19]. Effector cells were obtained from L O U / C rats stimulated 48-78 h previously by intraperitoneal injection e l l 0 ml 0.9~~ sterile physiological saline; peritoneal cavities were washed with 20 ml MEM containing penicillin (100 tag ml ~), streptomycin (50 tag ml-~), 1~ normal rat serum (NRS), 20 ng ml -~ synthetic tripeptide (glycyl-histidyl-lysine, Calbiochem, San Diego, CA) and 25 IU ml -l calcium heparinate (MNT). Eosinophil-rich populations were prepared by allowing these peritoneal cells (5-7 X 106 ml -~ in MNT) to adhere to plastic Petri dishes lor 2 h at 37°C. The non-adherent cells of each Petri dish were recovered, pooled, and

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washed twice in MNT. After staining with Discombe's diluent or toluidine blue, this population contained 40-90% eosinophils and 4-10% mast cells. The cytotoxicity assay was carried out in sterile plastic microplates with flat bottomed wells (Nuclon, Denmark). Fifty schistosomula were added to each well and incubated overnight with 100 lal normal, infected, or immunized rat serum at a final dilution of 1/16. After 18 h incubation at 37°C, effector cells were added in 50 lal of M N T at a ratio of 6 000 effector cells to 1 schistosomulum. The plates containing effector cells and sensitized targets were incubated in a 5% CO2 humidified atmosphere at 37°C. The percentage of cytotoxicity was evaluated after 24-48 h by microscopic examination. RESULTS

Proteinfractionation.

In this study, we analysed 10 groups of proteins ofS. mansoni adult worms. The molecular weights of these groups ofpolypeptides were respectively 28, 37, 40, 43, 55, 66, 68, 73, 78 k D a and an 85-90 kDa doublet. To control protein integrity after the elution procedure, we analysed the fractionated polypeptides by SDS polyacrylamide gel electrophoresis (SDS-PAGE). No modification of the mole1

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cular weights was observed (Fig. 2), indicating that the eluted proteins were not degraded during the fractionation steps.

lmmunoprecipitation of the metabolic products.

After two injections, Fischer rats were bled and the sera werc used to immunoprecipitate the metabolic products of,3', mansoni adult worms. The results are shown in Fig. 3a. Only three groups of proteins induced an antibody response. Their molecular weights were respectively 28, 78 and 85 kDa. When analysed on S D S - P A G E in the absence of 2-mercaptoethanol the 28 kDa antigen reassociatcd as a dimer of 56 kDa. Infected Fischer rat serum weakly recognized the 28 kDa group ol molecules and recognized the 78 and 85 k D a antigens. ~\ hyperimmune rabbit antiserum recognized thesc three groups ol antigens and the 37. 40, 43, 55, 66, 68 and 90 kDa antigens, indicating the loss ol the mamunogenicity ~! these structures when isolated after S D S - P A G E fractionation

Immunoprecipitation of the in vitro translation products.

The 28 kDa and 85 kI)a antisera immunoprccipitated, respectively, a 28 and 85 kDa group of antigens among the in vitro translation products from adult worm RNA (Fig. 3bL The inability of the 78 kDa antiserum to recognize the in vitro translation products could indicate that the antibody response was mainly directed against epitopes produced during the posttranslational maturation of that protein (probably a step of glycosylation L The 28 and

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Fig. 3. (a) l m m u n o p r e c l p i t a t i o n of [~SS]methioninc m e t a b o l i c labelled p r o d u c t ~, t r o m a d u l t v, o r m s ~ i t h n o r m a l r a t s e r u m ( N R S / , infected rat s e r u m ([RS), n o r m a l r a b b i t s e r u m (NRtS), hypcrtmt+nune r a b b i t s e r u m ( H R t S ) , a n t i - 2 8 k D a s e r u m , anti-78 k D a s e r u m , a n t i - 8 5 k D a sol urn. (bt l m m u n o p l e c i p i t a t i o n ot I'SSlme t h i o n i n e labelled in v i t r o t r a n s l a t i o n p r o d u c t s f r o m a d u l t w o r m R N A with N R S , IRS, N|~,~S, t t R t S , a n t i - 2 g k D a s e r u m , a n t i - 8 5 k D a s e r u m . (el l m m u n o p r e c i p i t a t i o n o l ~:~l-labellcd s u r i a c c antigen~ Irolll N. mans'on~ s c h i s t o s o m u l a with I R S . N R S , H R ' S . NR~S a n d anti-28 k D a s e r u m

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85 kDa antigens had the same molecular weight as among the metabolic products, suggesting that they were not processed after the translational step. Only the 28 kDa antiserum immunoprecipitated the in vitro translation products from S. mansoni schistosomulum RNA and recognized a 28 kDa polypeptide (data not shown).

Immunoprecipitation of the 12SI-labelled surface antigens of the S. mansoni schistosomulum. Only the 28 kDa antiserum immunoprecipitated the 28 kDa group of antigens +

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(Fig. 3c). When compared to the pattern obtained with an infected rat serum or a hyperimmune rabbit serum, the 28 kDa group of antigens comigrated with the 30-40 kDa complex.

Two dimensional gel analysis of the 28 kDa groupof antigens,

Whether immunopreclpitated from the metabolic products, in vitro translation products, or ~:sI-labelled surface proteins of the S. mansoni schistosomulum, the 28 kDa antigen appeared in all cases homogeneous for its molecular weight and isoelectric point: pl 6.3-6, 5 as shown in Fig. 4. Gels were exposed for 1 month. The 28 kDa antiserum recognized two polypeptides very close together in charge, probably indicating a structural identit~ between these proteins. The two dimensional gel analysis of the fractionated 28 kDa group of proteins revealed no more than three major polypeptides in a range ot isoelectric points of 6.3-7,0 (not shown).

Biologicalactivity of the 28 kDa antiserum.

A lethal activity of the 28 kDa antiserum was revealed by an in vitro assay in the presence of a purified eosinophil cell population (Fig. 5). This cytotoxicity was surprisingly high, approaching that of an infected rat serum. Results are shown of a typical experiment. All experiments were performed in duplicate. After six experiments, percentages of cytotoxicity of an infected Fischer rat serum ranged between 41.0 ± 10.0 and 85.5 ± 5.5%. The lethal activity of the 28 kDa antiserum ranged between 38.0 ± 3.0 and 65.0 :k 8.1%. In all cases the cytotoxicity ot the infected Fischer rat serum and of the 28 kDa antiserum compared to a normal Fischer rat serum was statistically significant (0.002 < P < 0.003). The percentage of cytotoxicity depended essentially on the activity of the different eosinophil-rich population used in each experiment.

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Grausz, D., Dissous, C., Capron, A. and Roskam, W. (1983) Messenger RNA extracted from Schistosoma mansoni larval stages codes for parasite antigens when translated in vitro. Mol. Biochem. Parasitol. 7, 293-301. Pierce, R.J., Aimar, C., Balloul, J.M., Delarue, M., Grausz, D., Verwaerde, C. and Capron, A. (1985) Translation of Schistosoma mansoni antigens in Xenopus oocytes microinjected with RNA from adult worms. Mol. Biochem. Parasitol. 15, 171-188. Tavares, C.A.P., de Rossi, R., Payares, G., Simpson, A.J.G., McLaren, D.J. and Smithers, S.R. (1984) A monoclonal antibody raised against adult Schistosoma mansoni which recognizes a surface antigen on schistosomula. Z. Parasitenkd. 70, 189-197. Knight, M., Simpson, A.J.G., Payares, G., Chaudri, M. and Smithers, S.R. (1984) Cell free synthesis of Schistosoma mansoni surface antigens: stage specificity of their expression. EMBO J. 3, 213-219. Taylor, D.W., Cordingley, J.S. and Butterworth, A.E. (1984) Immunoprecipitation of surface antigen precursors from Schistosoma mansoni messenger RNA in vitro translation products. Mol. Biochem. Parasitol. 10, 305-318. Auriault, C., Damonneville, M., Verwaerde, C., Pierce, R.J., Joseph, M., Capron, M. and Capron, A. (1984) Rat IgE directed against schistosomula-releasedproducts is cytotoxic for Schistosoma mansoni schistosomula in vitro. Eur. J. lmmunol. 14, 132-138. Clegg, J. A. and Smit hers, S.R. (1972) The effects of immune Rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasitol. 2, 79-98. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, MendeI-Hartvig, 1. (1982) A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. Anal. Biochem. 121,215-217. Vaitukaitis, J., Robbins. J.B., Nieshlag, E. and Ross, G.T. (1971) A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. 33,988-991. Chirgwin, J.M., Przybla, A.E., Mac Donald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Pelham, R.B. and Jackson, J. (1976) An efficient mRNA dependent translation system from rabbit reticulocyte lysates. Eur. J. Biochem. 67,247-256. O'Farrell, P.H. (1975) High resolution two dimensional gel electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021. Marchalonis, J.J., Cone, R.E. and Santer, V. (1971) Enzymatic iodination. A probe for accessible surface proteins on normal and neoplastic lymphocytes. Biochem. J. 124, 921-927. Capron, M., Capron, A., Torpier, G., Bazin, H., Bout, D. and Joseph, M. (1978) Eosinophil dependent cytotoxicity in rat schistosomiasis. Involvement of IgG2a antibodies and role of mast cells. Eur. J. lmmunol. 8, 127-130. Grzych, J.M., Capron, M., Dissous, C. and Capron, A. (1984) Blocking activity of rat monoclonal antibodies in experimental schistosomiasis. J. lmmunol. 133,998-1004.

DISCUSSION T h e d e t e c t i o n of p u t a t i v e s c h i s t o s o m u h l m surface antigens a m o n g in vitro translation p r o d u c t s has been u n d e r t a k e n recently by indirect means [5,7,8]. Whilst the results o b t a i n e d can give a g o o d i n d i c a t i o n o l p r o t e i n s o f interest tot subsequent cloning experiments, they do not provide a means either tot d e t e r m i n a t i o n of the relevance o f the molecules c o n c e r n e d in the protective i m m u n e response, ~r for the identification of c D N A clones expressing them. The w o r k described here d e m o n s t r a t e s that by p r e p a r a t i v e S D S - P A G E , and s u b s e q u e n t use of the isolated protein p o p u l a tions for i m m u n i z a t i o n , p o l y c l o n a l antisera of restricted specificity can be o b t a i n e d . O n e serum, in p a r t i c u l a r , recognizing a 28 k D a g r o u p o f molecules a m o n g m e t a b o l i c labelling p r o d u c t s , in vitro t r a n s l a t i o n p r o d u c t s from larval and a d u l t w o r m R N A a n d t251 surface-labelled antigens of s c h i s t o s o m u l a was shown to recognize only tw~ p o l y p e p t i d e s o f very similar pl, 6.3 and 6.5, after two d i m e n s i o n a l analysis. T h e r e f o r e the i m m u n e response was restricted to the p l 6.3-6.5 antigen present a m o n g the p o p u l a t i o n of molecules isolated by S D S - P A G E , even after r e p e a t e d i m m u n i z a t i o n s . T h a t the two p o l y p e p t i d e s can be r e g a r d e d as potential targets of the i m m u n e system was shown by the high degree of cytotoxicity against s c h i s t o s o m u l a o b t a i n e d in the presence of e o s i n o p h i l s with the 28 k D a antiserum. It was p a r t i c u l a r l y n o t a b l e that the 28 k D a b a n d seemed not to induce the p r o d u c t i o n of b l o c k i n g a n t i b o d i e s , as has been shown for the 38 k I ) a molecule [1911. Now that a S. m a n s o n i s c h i s t o s o m u l u m surface c o m p o n e n t has been directly d e m o n s t r a t e d a m o n g t r a n s l a t i o n p r o d u c t s from larval and adult R N A , the technique will be e x t e n d e d to o b t a i n sera against o t h e r surface c o m p o n e n t s . Such sera, recognizing d e n a t u r e d p r o t e i n s and thus p r o b a b l y p r i m a r y s t r u c t u r a l e p i t o p e s can be used for the screening o f e D N A expression libraries~ ACKNOWLEDGEMENIS T h e a u t h o r s w o u l d like to t h a n k Th~r~se Lepresle, ,lacques Trotlet, S t e p h a n e Torres, T h i e r r y J o u a u l t and F r e d e r i c D e l b r e u v e for their technical assistance: and S u z a n n e van W i n g e n e for the parasites. This w o r k was s u p p o r t e d bv the I N S E R M (167), by the C N R S (624) and by the W H O special p r o g r a m for Research and T r a i n i n g in T r o p i c a l Diseases. J.M.B. is s u p p o r t e d by the D . G . R . S . T . REFERENCES I 2 3

Dissous, C., Dissous, C, and ('apron, A. (1981) Isolation and characterization ol surface antlgcn~ from Schistosoma rnansoni schistosomula. Mol. Biochem. Parasitol. 3, 215-225 Dissous, (' , (~r,"~ch. ,I.M. and ('apron, , \ (1982) X~hi~to,somo, man~onl surface antigen dclmcd b', :t ra~ protecuve monoclonal lg(i2a..1, lmmumH 129, 2232-2234 Grzych, ,I.M., Capron, M., Bazin, H. and Capron, A. (1982) In vitro and in vivo ellotto1 |tlllCtl,,,n,,1|ral lgG2a monoclonal anti-,R', manroni antibodies. J. Immunol. 129, 2739-2743.