Schistosoma mansoni: Immunoblot analysis of adult worm proteins

EXPERIMENTAL

PARASITOLOGY

Schistosoma

60,

195-206 (1985)

mansoni:

lmmunoblot

Analysis

of Adult Worm Proteins

ANDREASRUPPEL,*,~URSULA ROTHER,* HEIKE VONGERICHTEN,*RICHARD LUCIUS,~ AND HANS JOCHEN Dmmmt *Institut

fir

Immunologic, Im Neuenheimer Feld 305, and flnstitut fiir Tropenhygiene, Feld 324, D-6900 Heidelberg, Federal Republic of Germany

Im Neuenheimer

(Accepted for publication 21 March 1985) RUPPEL,

A.,

ROTHER,

U.,

VONGERICHTEN,

H.,

LUCIUS,

R., AND DIESFELD,

H. J.

1985.

Schistosoma mansoni: Immunoblot analysis of adult worm proteins. Experimental Parasitology 60, 195-206. Proteins of adult Schistosoma mansoni were separated by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 3 I-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-radirradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes. 6 1985 Academic press. I~C. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Trematode, parasitic; Proteins, adult worm; Polyacrylamide gel electrophoresis; Immunoblotting; Immunofluorescence; Mouse serum; Infection; Irradiation of cercariae; Vaccination; Resistance to challenge infections; Dulbecco’s modified Eagle’s medium (DMEM); Newborn calf serum (NCS); Bovine serum albumin (BSA).

periments (reviews by McLaren 1980; Smithers and Doenhoff 1982) suggest that a The development of partial resistance to number of immune mechanisms may conrepeated infections with Schistosoma man- tribute to such resistance. However, their soni is well documented for a number of role in vivo is not yet clearly understood host species (Smithers and Doenhoff 1982). (McLaren and Smithers 1982). In addition Following infection with normal cercariae, to the immune system, factors independent “concomitant immunity” (Smithers and of the immune response seem to participate Terry 1969) may develop, and in vitro ex- in the elimination of challenge parasites (Dean et al. 1981a; Harrison et al. 1982; Wilson et al. 1983) following a primary int To whom correspondence should be addressed. INTRODUCTION

195

0014-4894185$3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

196

RUPPEL ET AL.

fection with normal cercariae. Vaccination with radiation-attenuated cercariae has also been known to induce resistance in mice against a challenge infection (Villella et al. 1961). Yet, little is known about the factors contributing to this situation to the development of resistance (Dean 1983). Resistance induced by infection with normal cercariae or vaccination with irradiated cercariae appears to be based on different mechanisms (Smithers and Miller 1980; Dean et al. 1981a, b). In the absence of pathology due to schistosomiasis, the immune system of the host may be expected to play an important role in the development of resistance. The antigens possibly involved in a protective immune response following either mode of infection are as yet unknown as are the corresponding antibodies. In this communication, the antibody response against single schistosome proteins was studied in mice, the species probably most intensively studied as a model of human schistosomiasis (Dean 1983). Mice were infected according to a number of different schemes. Their antibody responses against schistosome proteins were determined in immunoblots (Towbin et al. 1979; Burnette 1981) against total schistosome proteins separated previously by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ruppel and Cioli 1977). Although this technique selects for detergentstable antigens, it has nevertheless proved to be suitable for efficiently detecting a number of antigens from very different sources including helminth homogenates (Lucius et al. 1983; Lingelbach and Hinz 1984; Harrison et al. 1984). We report here the application of this method to the S. mansoni-mouse model. Some of the data have been reported elsewhere (Ruppel et al. 1984). MATERIALS

AND METHODS

The strains and maintenance of the life cycle of Schistosoma mansoni have been described (Ruppel et al. 1982). Mice were locally bred (NMRI) or they

(DBAIZ, BalbicAn) were obtained from Zentralinstitut fur Versuchstiere (Hannover, FRG) and (C57B116) from Charles-River-Wiga (Sulzfeld, FRG). They were kept under conventional (as opposed to specific pathogen free) conditions. For each individual experiment, normal and irradiated cercariae were derived from a common pool. Cercariae were irradiated with a 13’Cs gamma radiation source (gamma cell 1000, Atomic Energy of Canada) at a rate of 1740 radimin for a total of 20,000 rad. This dose kills 90% of the larvae within 4 days and 99.9% after 3 weeks (Miller and Smithers 1980). Infections with normal and irradiated cercariae were applied in parallel within 5 hr of emergence from snails. Primary and challenge infections were applied to different sites (Miller and Smithers 1980) of anesthesized (Nembutal, Ceva, Paris, France) mice under the conditions specified in Table I. Infections were simultaneously applied to Groups A through to H, I through to S, and T through to W, respectively. Schistosomes were perfused (Smithers and Terry 1965) with warm medium (see below, supplemented with 10 units hepariniml) from the mesenteric veins onto a 25pm mesh screen and counted in a Petri dish at 8-30 x magnification. Livers were squashed between two glass plates and very carefully inspected for residual worms, especially those derived from the challenge infection. Worms derived from the primary and challenge infections were readily differentiated according to their length (Doenhoff et a/. 1978). Details concerning the infections and groups of mice are listed in Table I. The resistance of mice to a challenge infection is expressed as the percentage of reduction of challenge derived worms obtained from mice infected or vaccinated previously as compared to untreated animals. The significance of the reduction was calculated by Student’s t test. It was consistently noted that perfusion of challenge worms was incomplete from the livers of infected, but not of normal, mice. Challenge worms which had not been perfused were detectable by their dark intestine in the squashes of pigmented livers. In the calculations, the number of these worms was added to that of the perfused parasites, although very recent evidence suggests that juvenile schistosomes that are not removable from the livers of infected mice by perfusion may, in fact, not be viable (Dean and Mangold 1984). Schistosomula were obtained as described by Clegg and Smithers (1972) using modifications described previously (Ruppel et al. 1983). Lung schistosomula were prepared according to Sher et a/. (1974). DMEM (Gibco, Karlsruhe, FRG; no. 074-1600) supplemented with 5% heat-inactivated NCS (Gibco), streptomycin (0.1 pg/ml), and penicillin (100 units/ml), was used for all parasite preparations. Both control mice and those infected with S. mansoni were bled at the indicated times from the tail tip,

~ChiStOSOmU

munsoni:

and at the end of the experiments by cardiac puncture. In this way, the possible appearance of cross-reacting antibodies was excluded in all these studies, although infections with Syphacia obvetura (Oxyura) were occasionally detected, especially in older animals. Sera were stored in 30- to 50-ul portions at -40 C and were thawed up to three times without obvious effect on the results. For electrophoresis, skin schistosomula and at least 6-week-old adult schistosomes of both sexes were used. Parasites were washed twice in DMEM without serum, suspended in sample buffer (0.05 M Tris-HCI, pH 6.8, 10% glycerol, 5% f-mercaptoethanol, 3% sodium dodecyl sulfate, and traces of bromphenol blue), sonicated, and boiled. They were applied to polyacrylamide slab gels prepared according to modifications (Ruppel and Cioli 1977) of the procedures described by Laemmli (1970) and Studier (1973). Eight pairs or 20,000 schistosomula in about 1 ml of sample buffer were used per slab (2-mm thick, IO-cm long), if the spacer gel had no wells. Alternatively, 1 pair or 2000 schistosomula were applied per well (4 mm2). Molecular weight standard proteins (Pharmacia, Uppsala, Sweden) were run in parallel. Following electrophoresis, proteins were electrophoretically transferred (Towbin er al. 1979) from the gel to a nitrocellulose sheet (BA85, pore size 0.45 pm; from Schleicher and Schtill, Dassel, FRG) at 6 V/cm and 250 mA overnight at 4 C in 0.025 M Tris, 0.186 M glycine, and 25% methanol. The nitrocellulose sheet was cut into 30-40 lanes that were treated subsequently with individual sera. Immunorecognition was performed according to Burnette (1981) using serum dilutions of 1:150. Incubation and washing conditions were described previously (Lucius et al. 1983), except that BSA of >96% purity (Serva, Heidelberg, FRG) was used to saturate additional protein binding sites of the nitrocellulose. Mouse immunoglobulins bound to schistosome proteins on the nitrocellulose were detected with a 1:500 dilution of horseradish peroxidaseconjugated goat antibodies to mouse IgG (gamma and light chain specific) or mouse IgM (mu chain specific, both purified by atlinity chromatography; Tago, BurIingame, CA, USA). Visualization of immune complexes was with 6% of a stock solution of 4-chloro-lnaphthol (0.3% 4-chloro-I-naphthol in methanol; Merck, Darmstadt, FRG) and 0.02% H,O, in water (Hawkes et al. 1982). The color reaction was usually left to proceed until nonspecific staining of the background became visible. The reaction was stopped by several washes with water. For each gel, a few lanes were neither incubated with BSA nor with serum, but were stained briefly with 0.2% amide/black 10B (Merck) in 40% methanol and 7% acetic acid, and destained in this solution without dye in order to reveal the pattern of total proteins transferred to the nitrocellulose.

IMMUNOGENIC

PROTEINS

197

Immunofluorescence assays were performed with living skin or lung schistosomula incubated with a l/IO dilution of serum in DMEM-NCS for 30 min at room temperature. After two washes in 100 vol of DMEM-NCS, they were incubated as above with a 1140dilution of fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG antibodies (Miles, Munich, FRG). Following two additional washes, fluorescence was scored for individual schistosomula on an arbitrary scale (Dean 1977)from negative (- ) to strongly positive (+ + + +). Adult worms were embedded in Tissue-teck II OCT (Miles). Cryostat sections (8 urn) were incubated with l/10 dilutions of serum, washed, and incubated with a l/80 dilution of fluorescein isothiocyanate-anti-mouse IgG. For each serum, the reaction of four to six sectioned schistosomes was determined separately for the tegument, parenchyma, and gut, and scored from negative to strongly positive on an arbitrary scale. RESULTS

Mice infected with a few hundred untreated Schistosoma mansoni cercariae or vaccinated with lethally irradiated cercariae developed partial, but significant, resistance against a challenge infection given 4 weeks later (Table I, Groups D, F, G, N, and P). A primary infection with about 50 cercariae induced significant resistance only if irradiated (Group 0), but not if normal cercariae (Group M) were used. Sera obtained from mice at the time of challenge, i.e., 4 weeks after a primary infection, were screened for IgG antibodies binding in immunoblots to adult schistosome proteins. One protein reacted strongly (Fig. 1) with sera from all DBA/2 mice infected with 286 (Group A) or 198 normal cercariae (Group C), but did not react with sera obtained from uninfected (Group H) or irradiated cercariae vaccinated (Group E) mice. The protein corresponded to none of the major schistosome bands detected by amido black staining of the nitrocellulose (Fig. 1) or by Coomassie blue staining of the acrylamide gel (not shown). The molecular weight of this protein was 31 kDa. The antibody response had the same characteristics in Balb/c mice (Fig. 2) with 4-week-old infections: the 31-

DBAl2 DBAIZ DBA!2 DBAI2 DBAl2 DBAIZ DBAIZ DBAIZ

Balbk Balbic Balbic Balbic Balbic Balbk Balbic Balbk Balb/c Balbk

NMRI NMRI DBA2 DBA2

A B C D E F G H

I K L M N 0

T U V W

6 I 5 5

6 6 11 IO 10 10 10 6 6 5

10 5 5 10 10 5 5 20

No.

228 228 228 228

50 + 11 314 t 39 None 50 f II 314 k 39 58 k 29 291 k 7 58 t 29 291 + 7 None

286 k 6 None None 286 f 6 223 AZ 21 223 k 21 223 k 21 None

No. k SD

Belly Belly Belly Belly

None 20,000 rad 20,000 rad 20,000 rad

infection.

Belly Belly Belly Belly

Ear Ear Ear Ear Ear Ear

None None 20,000 20,000 20,000 20,000 None None None None

Ear Ear

None None

rad rad rad rad

Belly

Site

None

Treatment

Cercariae of primary infection

” Untreated cercariae applied 28 days after primary b With respect to B. ’ With respect to C. d With respect to L. ’ One pair perfused from one mouse.

ii S

Strain

Group

Mice

Infection Schemes of Mouse

28 28

28 28 28

None None None None

None None 184 2 22 184 ?k 22 184 t 22 184 k 22 184 k 22 None None None

198 k 198 k 198 k None 198 k 198 k None

None

No. + SD

Belly Belly Belly Belly Belly

Ear Ear Ear Ear Ear Ear

Site

44 51 44 51

68 53 68 53 53 53 53 112 112

28 42 63 42 28 42 63

Perfusion (days after primary infection) 15

41 36 66 73

t -iit

22 19 13 19

21 t 6 146 + 18 0 0’ 0 0

16 k 4 127 c 10

65 t 17 0 0 0

64 i

Worms of primary infection (No. c SD)

TABLE I Exposed to Cercariae of Schisrosoma

Cercariae of challenge infection”

Groups

SD

62 54 10 44 37

k 2 k k f

13 21 5 20 14

55 * 20 78 f 8 19 f 11 None 16 t 9 36 + 8

No. i

mansoni

136 846 29d 40d

71” 54’

65h

Reduction of worm burden VW

P < o.M)5 P < 0.001

P i 0.001

Significance of reduction

P < 0.005

P < 0.05

Not significant P < 0.001

Worms of challenge

e

m ci

Schistosoma mansoni: IMMUNOGENIC

a

t

b

a

FIG. 1. Immunoblots of adult Schisl‘osoma mansoni proteins with sera of DBA/Z mice obtained 4 weeks after infection. Proteins were separated on a 7-13% acrylamide gradient gel, and blots were performed with sera obtained from individual mice that had been infected for 4 weeks with 286 (a) normal or(b) lethally irradiated cercariae; (c) sera from uninfected mice. A nitrocellulose strip was stained with amide/black (arrow). The position of a protein of 31 kDa is indicated.

kDa band was recognized readily only by antibodies from mice infected with 314 normal cercariae (Group N). In contrast, sera of mice infected with 50 normal cercariae (Group M) showed at most a faint reaction, and sera of mice vaccinated with irradiated cercariae (Groups P and 0) or sera of naive mice did not react at all. Attempts to detect IgM antibodies reacting with adult schistosome proteins were unsuccessful with the same sera (not shown). Immunoblotting under the same conditions showed that schistosomula did not contain a protein in the 31-kDa range reacting with any of the sera obtained 4 weeks after a primary infection (not shown). Sera obtained from 4-week-infected mice were characterized with respect to the morphological localization of antigens against which antibodies had been formed. IgG antibodies binding to the surface of living skin or lung schistosomula and to the tegument or internal tissues of adult schistosomes in cryostat sections were detected by immu-

199

PROTEINS

nofluorescence tests (Table II). Antibodies directed against the surface of skin schistosomula and of adult worms had the highest levels in groups of mice (A, E, N, 0, and P) that were partially resistant to a challenge at the time the serum was taken. Antisurface antibodies were rare in infected but nonresistant mice (Group M) and were absent from naive mice. No antibodies to the surface of lung schistosomula were detected. Regardless of the dose, antibodies against gut-associated antigens of adult worms were always formed only following infections with normal (Groups A, M, and N; Fig. 3B), but never with irradiated (Groups B, 0, and P; Fig. 3A), cercariae. The observations with 4-week-infected mice are summarized in Table III. The appearance of antibodies reacting in immunoblots with adult schistosome proteins was followed in mice infected for pe-

-bell a

e

t

FIG. 2. Immunoblots of adult Schistasoma mansoni proteins with sera of Balb/c mice obtained 4 weeks after infection. Proteins were separated on a 7-13% acrylamide gradient gel, and blots were performed with sera obtained from individual mice that had been (a) uninfected, or infected for 4 weeks with (b) 314 normal, (c) 50 normal, (d) 291 lethally irradiated, and (e) 58 lethally irradiated cercariae. A nitrocellulose strip was stained with amidojblack (arrow). The position of a protein of Ill-kDa is indicated.

200

RUPPEL ET AL.

+ + +

+ +

+

I

+ +

+

I

+

FIG. 3. Immunofluorescence of Schisfosoma mmsoni sections with sera of Balbic mice obtained 4 weeks after infection with (A) 291 irradiated or (B) 314 normal cercariae. Arrows indicate the intestine.

riods between 4 weeks and 4 months. The IgG response to the 31-kDa protein continued to be pronounced, but additional proteins with high molecular weights were recognized by sera obtained at later times after infection (Groups T-W; Fig. 4). Antibodies against proteins of 31 and 67 kDa were pronounced in all strains of mice tested (DBA/2, Balb/c, NMRI and C57Bl/ 6) harboring 2- to 4-month-old infections (Fig. 5). Antibody responses against other schistosome proteins were either less prominent, were observed with only some of the mice, or could not be reproduced in all experiments. The antibody response following a challenge infection was studied in DBA/2 mice

I

+ + + +

+ + +

+ +

+

SChiStOSOma

munsoni:

IMMUNOGENIC

201

PROTEINS

TABLE III Summary of Reactions Induced in Mice by 4-Week-Old Infections of Schistosoma

mansoni

Infection performed with Irradiated cercariae

Normal cercariae Properties of mice

Few

Many

Few

Many

Resistance to challenge infection Antibodies to 30-kDa protein Antibodies to surface of Skin schistosomula Lung schistosomula Adult worms Antibodies to adult gut

-

+ +

+ -

+ -

+ +

+ + +

+ + -

+ + -

that had been vaccinated 4 weeks earlier with 223 irradiated cercariae (Group P). Two weeks after the challenge, antibody formation was still not apparent. Four weeks after the challenge, however, antibodies against the 31-kDa protein were formed in both vaccinated and challenge control (Group L) mice (Fig. 6). Thus, the challenge infection did not induce the formation of antibodies different from those resulting from a single infection with normal cercariae after the same period of infection (Figs. 1 and 2). Vaccination in the absence of a challenge did not lead to detectable antibody formation 8 weeks later (not shown). Similarly, in vaccinated Balb/ c mice antibodies against only the 31-kDa protein were detectable 3 weeks after the challenge (Group P). Moreover, mice with only a primary infection formed antibodies to the same proteins as mice receiving a challenge (Groups K and N) and bled 3 weeks later (results not illustrated). DISCUSSION

We investigated the antibody response of mice with a known ability or inability to partially eliminate a Schistosoma mansoni challenge infection, Whenever resistance to a challenge was observed in this study, it could not be ascribed to either schistosomiasis pathology (Wilson et al. 1983), since mice were challenged roughly 2 weeks before the development of liver granulomas,

or to localized skin reactions (Miller and Smithers 1982), since primary and challenge infections were applied to different sites. The induction of a high degree of resistance in mice only 4 weeks after a heavy infection with normal cercariae has not been reported previously. The observation was only possible since the mice were per-

a-j-ty-----

d

t - e 7’7

FIG. 4. Immunoblots of adult Schistosoma mansoni proteins with mouse sera obtained at least 6 weeks after infection. Proteins were separated on a 7-13% acrylamide gradient gel, and blots were performed with individual sera obtained from uninfected (a) NMRI or (b) DBA/2 mice, from NMRI mice infected with 228 cercariae for (c) 6 or (d) 7 weeks, and from DBA/2 mice infected with 228 cercariae for (e) 6 weeks, (f) 7 weeks, or (g) 3 months. Three nitrocellulose strips were stained with amide/black (arrows). The positions of proteins of 31 and 67 kDa are indicated.

RUPPEL ET AL.

202

1 a

I b

tc

FIG. 5. Comparison of mouse strains with respect to the reaction of sera in immunoblots with adult Schistosoma mansoni proteins. Reactions with (a) sera of 10 NMRI mice infected with varying numbers of cercariae (50-300) for 2-3 months; 6 uninfected NMRI mice; (b) sera of 3 DBA/2 mice infected with 228 cercariae for 7 weeks, 1 uninfected DBAR mouse; (c) sera of 6 Balb/c mice infected with 50 cercariae for 9 weeks, 6 uninfected Balb/c mice; and (d) sera of 5 C57 B116mice infected for 2-4 months with 50-100 cercariae, 2 uninfected C57 B116 mice. Acrylamide gradients were 7.5-13.5% (a,b) and 7.0-13.0% (c,d). Two nitrocellulose strips were stained with amidoiblack (arrows). The positions of proteins of 31 and 67 kDa are indicated.

fused according to Doenhoff et al. (1978) and/or egg antigens, which lack stage specwithin 3 weeks after the challenge, i.e., 7 ificity, may induce antibodies which react weeks after the primary infection, and, with worm antigens. A 31-kDa protein is therefore, just before death of the mice due the immunodominant component recogto severe pathology would occur. Light in- nized 4 weeks after a heavy infection or fections with normal cercariae did not in- later after either heavy or light infections duce significant resistance within 4 weeks, with normal cercariae. We observed that antibodies of all indialthough this may occur at later times (Long et al. 1980). Vaccination of mice with vidual mice of any group recognized the both heavy and light doses of irradiated cer- same antigens. Moreover, quantitative difcariae induced resistance, but the dose de- ferences were observed in only a few mice pendency was less pronounced in our ex- of a particular group. Although this homoperiment than in a more extensive study by geneity with respect to the antibody reMiller and Smithers (1982). In agreement sponses is not surprising with inbred mice, with the findings of Doenhoff et al. (1978), we did not observe major qualitative differthe apparent degree of resistance was ences between several strains of mice, inhigher if mice were perfused after 3 weeks cluding outbred animals. This suggests that rather than at a later time following the the antigens detected here as being most challenge. readily recognized in early infections may The results of this study indicate that in have a more general importance. In fact, the first weeks following infection with 3 1-kDa protein also reacts predominantly normal cercariae, the spectrum of anti- with sera of schistosomiasis patients schistosome antibodies may be rather re- (Ruppel, unpublished observations). stricted compared to later times. Possibly, Sera of mice vaccinated with irradiated different schistosome antigens may induce cercariae and partially resistant to a chalantibodies at different times after infection lenge infection did not contain antibodies

~ChisfosomU

WZUnS0ni.’ IMMUNOGENIC

PROTEINS

203

explain these inconsistencies. We are now studying antigens in detergent-free systems using immunoprecipitation or a combination of isoelectric focusing of schistosome proteins with immunoblots. The specificity of the antibody response in mice with a primary infection was apparently not changed by a challenge infection in the sense that proteins with molecular weights of 31 and 68 kDa remained immunodominant. The response of vaccinated mice to a challenge infection was not different from the usual primary infection with normal cercariae. Correa-Oliveira et ---btc-j--te a al. (1984) found a three-fold increase of FIG. 6. Immunoblots of adult Schistosoma mansoni total anti-schistosomulum antibodies in proteins with sera of vaccinated and challenged DBA/ mice vaccinated 10 weeks prior to a chal2 mice. Proteins were separated on a 7-13% acryllenge infection as compared to unchalamide gradient gel, and blots were performed with inlenged animals. The use of different antidividual mouse sera. Sera were obtained (a) 2 or (b) 4 weeks after a challenge infection with 198 cercariae gens, antibody detection techniques, and given to mice that had been vaccinated 4 weeks earlier vaccination protocols may explain the conwith 223 lethally irradiated cercariae; and (c) 2 or (d) tradiction between their findings and ours. 4 weeks after an identical infection of naive mice and Our experimental evidence is consistent (e) from uninfected control mice. Two nitrocelhdose with the view that the 31-kDa protein destrips were stained with amidoblack (arrows). tected in our immunoblots may originate from the schistosome gut. First, the presdetectable in our blotting system. Although ence of antibodies to the 31-kDa protein althe use of a strong detergent and heat treat- ways correlated with the presence of antiment of schistosome proteins can be as- bodies binding to the gut, while sera from sumed to denature certain antigenic deter- vaccinated mice that did not recognize the minants irreversibly, this observation was 3 1-kDa protein never contained anti-gut anrather unexpected. In fact, in sera of vac- tibodies detectable by immunofluorescinated mice, we demonstrated antibodies cence. Second, the 31-kDa protein was dedirected against the surface of skin schis- tectable only in immunoblots using prepatosomula and adult worms by immunoflu- rations of adult schistosomes but not skin orescent techniques. Similarly, such sera schistosomula that still lacked a gut. Third, have been shown to contain antibodies to antibodies to the 31-kDa protein are formed the surface of skin schistosomula in a cell in mice that have never had contact with adherence assay (Bickle and Ford 1982) and cercariae or schistosomula, but have been by immunoprecipitation of surface antigens intravenously injected with lung-stage par(Simpson ef al. 1983). Sera obtained from asites (not shown). Fourth, vaccination of vaccinated mice did not contain antibodies mice with cercariae irradiated at a dose that binding to the surface of lung schistosomula kills the vast majority of the larvae well bein our immunofluorescence assays, where- fore they can develop a gut did not cause as antibodies against the surface of this par- production of antibodies against the 3 I-kDa asite stage were observed by Bickle and protein. The data obviously do not exclude Ford (1982). At present, we are unable to the possibilities that the same or cross-re-

204

RUPPEL ET AL.

acting antigens may also be expressed on the surface of schistosomula or adult worms, or that different antigens are detected by the different techniques. The resistance to S. mansoni challenge infections observed in 4-week-infected mice was probably immunological in nature, at least in the sense that our experimental protocol excluded localized skin reactions (Miller and Smithers 1982) and egg-induced pathology, which has been implicated in the resistance observed in mice with longer infections (Dean et al. 1981a; Harrison et al. 1982). In any event, the data do not permit the attribution of a role in immune resistance to any of the antigens recognized in our immunoblots. In fact, a possible intestinal location of the 31-kDa antigen in adult schistosomes argues against a role for antibodies to this protein in the immune rejection of a challenge infection. Indeed, antibodies with the ability to participate in immune killing of parasites are currently believed to be directed preferentially against surface molecules of schistosomula. However, the in vitro binding of antibodies to the surface of schistosomula did not necessarily correlate with the in vivo resistance against a challenge in serum donors (Bickle et al. 1983). In conclusion, we provide evidence for considerable differences between antibody specificities that are induced in mice following either infection with normal cercariae or vaccination with irradiated ones. This finding would fit into the concept that the two regimes induce rejection of a challenge infection by different immune mechanisms (Smithers and Miller 1980; Dean et al. 1981a,b). We have also demonstrated that natural infections in mice led to the early and predominant formation of antibodies against a 31-kDa protein of adult Schistosoma mansoni, the origin of which appears to be the gut rather than the tegument. The consistent reaction observed here with sera of naturally infected mice

suggests its possible use for immunodiagnosis. ACKNOWLEDGMENTS We are grateful to Mrs. C. Kirsten for preparing the cryostat sections; to Dr. P. Symmons (Institut fiir Genetik, Diisseldorf) for correcting the English text; to Mrs. G. Giith and U. Schubert for typing the manuscript; and to the Deutsche Forschungsgemeinschaft for financial support (Ro 133112-3). REFERENCES BICKLE, Q. D., DOBINSON,T., ANDJAMES,E. R. 1979. The effects of gamma-irradiation on migration and survival of Schistosoma mansoni schistosomula in mice. Parasirology 79, 223-230. BICKLE, Q. D., AND FORD,M. J. 1982. Studies on the surface antigenicity and susceptibility to antibody dependent killing of developing schistosomula using sera from chronically infected mice and mice vaccinated with irradiated cercariae. Journal of Zmmunology 128, 2101-2106. BICKLE, Q. D., FORD, M. J., AND ANDREWS, B. J. 1983. Studies on the development of anti-schistosomular surface antibodies by mice exposed to irradiated cercariae, adults and/or eggs of S. mansoni. Parasite Immunology 5, 499-511. BLJRNE~E, W. N. 1981. “Western Blotting”: Electrophoretical transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112, 195-203. CLEGG,J. A., AND SMITHERS,S. R. 1972. The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International

Journal

for Parasitology

2, 79-98.

CORREA-OLIVEIRA,R., SHER, A., AND JAMES, S. L. 1984. Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. V. Anamnestic cellular and humoral responses following challenge infection. American Journal of Tropical Medicine and Hygiene 33, 261-268. DEAN, D. A. 1977. Decreased binding of cytotoxic antibody by developing S. mansoni. Evidence for a surface change independent of host antigen adsorption and membrane turnover. Journal of Parasitology 63, 418-426. DEAN, D. A. 1983. A review. Schistosoma and related genera: Acquired resistance in mice. Experimental Parasitology 55, 1- 104. DEAN,

D. A.,

BUKOWSKI,

M. A.,

AND

CHEEVER,

A. W. 198la. Relationship between acquired resistance, portal hypertension, and lung granulomas in

~Chist0SWza

WZUizSOni:

ten strains of mice infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 30, 806-814. DEAN, D. A., BUKOWSKI, M. A., AND CLARK, S. S. 1981b. Attempts to transfer the resistance of Schistosoma mansoni-infected and irradiated cercariaeimmunized mice by means of paraabiosis. American Journal of Tropical Medicine and Hygiene 30, 113120. DEAN, D. A., AND MANGOLD, B. L. 1984. Autoradiographic analysis of resistance to reinfection with Schistosoma mansoni in mice. Evidence that the liver is a major site of worm elimination. American Journal of Tropical Medicine and Hygiene 33, 97103. DOENHOFF, M., BICKLE, Q., LONG, E., BAIN, J., AND MCGREGOR, A. 1978. Factors affecting the acquisition of resistance against Schistosoma mansoni in the mouse. I. Demonstration of resistance to reinfection using a model system that involves perfusion of mice within three weeks of challenge. Journal of Helminthology 52, 173-186. HARRISON, R. A., BICKLE, Q., AND DOENHOFF, M. J. 1982. Factors affecting the acquisition of resistance against Schistosoma mansoni in the mouse. Evidence that the mechanisms which mediate resistance during early patent infections may lack immunological specificity. Parasitology 84, 93- 110. HARRISON, L. J. S., PARKHOUSE, R. M. E., AND SEWELL, M. M. 1984. Variation in ‘target’ antigens between appropriate and inappropriate hosts of Taenia saginata metacestodes. Parasitology 88, 659-663. HAWKES, R., NIDAY, E., AND GORDON, J. 1982. Dot immunobinding assay for monoclonal and other antibodies. Analytical Biochemistry 119, 142-147. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LINGELBACH, K., AND HINZ, E. 1984. Echinococcus multilocularis: Identification of proteins inducing antibody formation in metacestode infection. Journal of Helminthology 58, l-6. LONG, E., HARRISON, R., BICKLE, Q., BAIN, J., NELSON, G., AND DOENHOFF, M. 1980. Factors affecting the acquisition of resistance against Schistosoma mansoni in the mouse. The effect of varying the route and the number of primary infections, and the correlation between the size of the primary infection and the degree of resistance that is acquired. Parasitology 81, 355-371. LUCIUS, R., RAUTERBERG, E. W., AND DIESFELD, H. J. 1983. Identification of immunogenic proteins of Dipetalonema viteae (Filarioidea) by the “western blotting” technique. Tropenmedizin und Parasitologie 34, 133-136.

IMMUNOGENIC

PROTEINS

205

MCLAREN, D. J. 1980. Schistosoma mansoni: The parasite surface in relation to host immunity. In “Tropical Medicine Research Studies” (K. N. Brown, ed.), Vol. 1, pp. l-229. Research Studies Press/ Wiley, Chichester. MCLAREN, D. J., AND SMITHERS, S. R. 1982. Immunity to schistosomiasis: In vitro versus in vivo modeis. In “Animal Models in Parasitology” (D. G. Owen, ed.). McMillan Press, London. MILLER, K. L., AND SMITHERS, S. R. 1980. Schistosoma mansoni: The attrition of a challenge infection in mice immunized with highly irradiated live cercariae. Experimental Parasitology 50, 212-221. MILLER, K. L., AND SMITHERS, S. R. 1982. Localized skin changes at the site of immunization with highly irradiated cercariae of Schistosoma mansoni are associated with enhanced resistance to a challenge infection. Parasitology 85, 305-314. RUPPEL, A., AND CIOLI, D. 1977. A comparative analysis of various developmental stages of Schistosoma mansoni with respect to their protein composition. Parasitology 75, 339-343. RUPPEL, A., LUCIUS, R., VONGERICHTEN, H., KIRSTEN, C., DIESFELD, H. J., AND ROTHER, U. 1984. Schistosoma mansoni antigens detected in immunoblots by antibodies of mice infected with normal or irradiated cercariae. Hoppe-Seyler’s Zeitschrift fir Physiologische Chemie 365, 1051- 1052. RUPPEL, A., ROTHER, U., AND DIESFELD, H. J. 1982. Schistosoma mansoni: Development of primary infections in mice genetically deficient or intact in the fifth component of complement. Parasitology 85, 315-323. RUPPEL, A., ROTHER, U., VONGERICHTEN, H., AND DIESFELD, H. J. 1983. Schistosoma mansoni: Complement activation in human and rodent sera by living parasites of various developmental stages. Parasitology 87, 75-86. SHER, A., MACKENZIE, P., AND SMITHERS, S. R. 1974. Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the mouse. Journal of Infectious Diseases 130, 626-633. SIMPSON, A. J., JAMES, S. L., AND SHER, A. 1983. Identification of surface antigens of schistosomula of Schistosoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irradiated cercariae. Infection and Zmmunity 41, 591-597. SMITHERS, S. R., AND DOENHOFF, M. J. 1982. Schistosomiasis. In “Immunology of Parasitic Infections” (S. Cohen and K. S. Warren, eds.). Blackwell, Oxford. SMITHERS, S. R., AND MILLER, K. L. 1980. Protective immunity in murine Schistosomiasis mansoni: Evidence for two distinct mechanisms. American

RUPPEL ET AL.

206 Journal of Tropical Medicine

and Hygiene 29, 832-

841. S. R., AND TERRY, R. J. 1965.The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 55, 69.5-700. SMITHERS, S. R., AND TERRY, R. J. 1969. Immunity in schistosomiasis. Annals of the New York Academy SMITHERS,

of Sciences 160, 826.

Molecular H.,

Biology

79, 231-248.

STAEHLIN,

VILLELLA,

T.,

AND GORDON,

J. 1979.

J. B., GOMBERG,

H. J., AND GOULD,

S. E.

1961. Immunization to Schistosoma mansoni in mice inoculated with radiated cercariae. Science (Washington, D.C.) 134, 1073-1075. WILSON,

F. W. 1973. Analysis of bacteriophage T7, early RNAs and proteins on slab gels. Journal of

STUDIER,

TOWBIN,

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences 76, 4350-4354.

R. A., COULSON,

P. S., AND MCHUGH,

S. M.

1983. A significant part of the “concomitant immunity” of mice to Schistosoma mansoni is the consequence of a leaky hepatic system, not immune killing. Parasite Immunology 5, 595-601.