Schistosoma mansoni, S. haematobium, and S. japonicum: Identification of genus- and species-specific antigenic egg glycoproteins

EXPERIMENTAL

PARASITOLOGY

58, 333-344 (1984)

Schistosoma mansoni, S. haematobium, and S. japonicum: Identification of Genus- and Species-Specific Antigenic Egg Glycoproteins AMY P. NORDEN Department

AND METTE

of Pharmacology and Experimental Therapeutics, of Medicine, 725 North Wolfe Street, Baltimore,

STRANDS The Johns Hopkins University Maryland 21205, U.S.A.

School

(Accepted for publication 27 July 1984) NORDEN, A. P., AND STRAND, M. 1984. Schistosoma mansoni, S. haematobium, and S. japonicum: Identification of genus- and species-specific antigenic egg glycoproteins. Ex58, 333-344. Immunoreactive egg glycoproteins of Schistosoma perimental Parasitology mansoni, S. haematobium, and S. japonicum which are genus- and species-specific, or

react with sera of patients infected with other parasites, have been identified. Egg proteins were labeled with Iodine-125, and the concanavalin A-binding glycoproteins were immunoprecipitated with sera of patients infected with one of four species of Schistosoma or Trichinella spiralis, Taenia solium, Echinococcus granulosus, Entamoeba histolytica, or Wuchereria bancrofti. These immunoprecipitates were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite the strikingly different patterns of glycoproteins of the African species, the antibody immune responses of patients infected with S. mansoni and S. haematobium were found to be so similar that differentiation could not be established. In contrast, sera of patients infected with S. japonicum, S. mekongi, or parasites not of the genus Schistosoma, immunoprecipitated fewer of the major S. mansoni or S. haematobium glycoproteins. Likewise, antibody immune responses of patients infected with the Oriental schistosomes (S. japonicum and S. mekongi) could not be differentiated. Only a few quantitative differences were noted between our S. mansoni egg glycoprotein extract and a standardized soluble egg antigen extract. This study provides an explanation for the extensive cross-reactivity observed in diagnostic assays which utilize various fractions of schistosomal egg extracts as the antigen. 0 1984 Academic press. h. INDEX DESCRIPTORS: Schistosoma mansoni; Schistosoma haematobium; Schistosoma japonicum; Schistosoma mekongi; Trematodes, digenetic; Glycoproteins; Eggs; Trichinella spiralis; Wuchereria bancrofti; Nematodes, parasitic; Taenia solium; Echinococcus granProtozoa, parasitic; Antigens; Human; Crossulosus; Cestodes; Entamoeba histolytica;

reactive; Genus-specific; Species-specific; Immunoreactive; Radioimmunoprecipitation; Electrophoresis, gel; Serodiagnosis; Sodium dodecyl sulfate (SDS); Polyacrylamide gel electrophoresis (PAGE).

In the past, attempts have been made to isolate and identify genus-, species-, and stage-specific schistosome egg antigens for diagnostic and immunoprophylactic purposes, and to further our understanding of the induction of disease. These studies utilized antigens extracted and partially puritied from schistosome eggs, including sol’ To whom correspondence should be addressed.

uble egg antigen (Boros and Warren 1970; Boros et al. 1977), major serological antigens (Pelley et al. 1976, 1977; Hamburger et al. 1976; Pelley 1977), soluble egg antigens (Agl and Ag2) (Brown et al. 1977), Schistosoma japonicum soluble egg antigens (JAG I, II, III, and IV) (Long et al. 1981a), polysaccharide egg antigen (Boctor et al. 1979, 1982), major egg glycoprotein (Hamburger et al. 1982a), q and ‘Ye(Dunne et al. 1981), and urea-soluble antigens 333

0014-4894/84$3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Tsang et al. 1981). Unfortunately, later studies have shown that most of these antigens are neither stage-, species-, nor genus-specific (Huldt et al. 1975; McLaren et al. 1978, 1981; Hillyer and Gomez de Rios 1979; Hillyer et al. 1980; Hillyer and Pelley 1980; Ambroise-Thomas et al. 1981; Long et al. 1981b; Tsang et al. 1981; Ogunba et al. 1982; Barrel-Netto et al. 1983; Stek et al. 1983). This study extends our previous investigation in which we identified concanavalin A-binding antigens from adult male and female worms of each of three species of Schistosoma which are species-specific, genus-specific, or cross-reactive (Norden and Strand 1984). This study was designed to identify antigens of eggs which are species-specific and cross-reactive. For this purpose, schistosome egg extracts were radiolabeled with Iodine-125, and glycoproteins were isolated. Radioimmunoprecipitates with sera of patients infected with various parasites were analyzed by twodimensional sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and were characterized with respect to molecular weight and isoelectric point. Both the concanavalin A-binding fraction and the unbound fraction were used in radioimmunoprecipitation reactions. Essentially all of the egg glycoproteins were immunoreactive, in agreement with previous studies of egg antigens (Boros et al. 1977; Carter and Colley 1979, 1981; Harrison et al. 1979; Deelder et al. 1980; Long et al. 1981a, 1982; Tracy and Mahmoud 1982). Despite the strikingly different two-dimensional SDS-PAGE pattern of egg glycoproteins of S. mansoni and S. haematobium, the antibody immune responses of patients infected with African schistosomes were found to be so similar that differentiation of S. mansoni and S. haematobium infections could not be established. Likewise, antibody immune responses of patients infected with the Oriental schistosomes (S. japonicum and S. mekongi) could not be

differentiated. Only sera from patients infected with Trichinella spiralis showed extensive cross-reactivity, whereas sera from patients infected with Echinococcus granulosus, Taenia solium, Wuchereria bancrofti, or Entamoeba histolytica precipitated relatively few of the schistosome egg glycoproteins. These results using egg glycoproteins are analogous to our previous findings when worm glycoproteins metabolically labeled with [35S]methionine were examined (Norden and Strand 1984). The genus- and species-specific immunoreactive schistosome antigens are discussed with respect to their relevance for serodiagnosis. MATERIALS AND METHODS Eggsof Schistosoma mansoni (Puerto Rican strain) were obtained from female mice (strain CD-l, Charles River, Wilmington, MA, USA) infected 8 weeks previously with 100 cercariae by tail immersion (Oliver and Stirewalt 1952).S. haemafobium (Egyptian strain) eggs were obtained from male golden hamsters infected 16 weeks previously with 500 cercariae by the pipet method. 5. juponicum (Chinese strain) eggs were harvested from female CD-l mice infected 7 weeks previously with 75 cercariae by hair-loop technique. All infected animals were obtained through a U.S. National Institutes of Health Supply Contract (AI 02656). The eggs were isolated by the method of Dresden and Payne (1981) with the following modifications. The livers and cleaned small intestines of infected animals were incubated in 1.5% NaCl at room temperature for 24 hr. They were then homogenized in 10 ml saline per liver in a Waring Blendor for 2 min. The homogenate was filtered through nylon mesh (149 pm), and the filtrate was washed through sieve no. 170 (90 pm). The eggs were collected on sieve no. 325 (45 pm), and then extensively washed with saline, sedimented by centrifugation, and stored as a pellet at -70 C. The egg proteins were solubilized by minor modifications of the method described previously (Strand ef al. 1982). A 1.O-ml pellet of eggs was suspended in 3 ml of lysis buffer (5 m&f Tiis-HCl, pH 9.2,400 m&f KCl, 1% Triton X-100, 1 mM EDTA, 2 m&f phenylmethylsulfonyl fluoride, 1 mM o-phenanthroline, 1 m&f p-chloromercuribenzoic acid, and 1 n& iodoacetamine). Following two cycles of sonic treatment, freezing, and thawing, o2 macroglobulin was added to the suspension for a final concentration of 10 pg/ml. The suspension was heated for 15 min at 37 C, followed by another cycle of sonication, freezing, and thawing. The suspension was centrifuged at 100,OOOg

Schistosoma

SPECIES: EGG GLYCOPROTEIN

for 60 mitt, and the supematant fraction was extensively dialyzed against 0.1 M borate buffer, pH 8.2, containing 1 M NaCl. In a typical experiment, a l.Oml pellet of eggs yielded 6 mg protein. To remove mouse immunoglobulin, the extract was passed over a 1.O-ml column of CNBr-activated Sepharose 4B beads (Pharmacia Fine Chemicals, Piscataway, NJ, USA) to which goat anti-mouse immunoglobulin had been crosslinked (at a ratio of 10 mg Ig: 1 ml beads). The nonadsorbed protein was dialyzed against concanavalin A buffer (20 r&f Tris-HCl, pH 7.6, 100 n&f NaCl, 0.2% Triton X-100, 1 mM CaCl,, 1 r&f MgCl,, and 1 mM MnCl,). Protein was determined by the method of Schaffner and Weissman (1973). S. mansoni (Puerto Rican strain) soluble egg antigen was supplied by the UNDPWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases. Proteins were iodinated by the chloramine-T method (Hunter 1967), and the reaction was terminated by addition of excess cold tyrosine (Jensenius and Williams 1974). The ‘2sI-labeled glycoproteins were isolated by concanavalin A-Sepharose 4B (Pharmacia) affinity chromatography as previously described (Strand et al. 1982). To decrease nonspecific binding, the radiolabeled glycoproteins were adsorbed twice with Staphylococcus uurcus (strain Cowan; Calbiochem-Behring, La Jolla, CA, USA) as described previously (Norden and Strand 1984). The supernatant fraction was used for immunoprecipitations as described previously (Strand et al. 1982), with the following modifications: (1) 8.5 x 10’ cpm of the labeled proteins was incubated with 2 pl serum, and (2) the buffer solution for the immunoprecipitation reaction contained 1% fetal calf serum. Sera of patients infected with parasitic diseases were obtained from several sources (Norden and Strand 1984), and are identified in the figures by the corresponding letters: (E) Dr. J. Ellner, University Hospital, Cleveland, OH, USA; (B) Dr. M. Stek, Naval Research Institute, Bethesda, MD, USA; (S) Dr. M. M. A. Salama, Zagazig University, Zagazig, Egypt; (N) Dr. J. Stanley, Kenyatta National Hospital, Nairobi, Kenya; (P) Dr. X. Q. Pan, Institute of Parasitology, Shanghai, The People’s Republic of China; and (C) The Center for Disease Control, Atlanta, GA, USA. Additional sera, identified in the figures by the letter (T), were provided by Dr. T. Nash, National Institutes of Health, Bethesda, MD, USA-sera from seven patients infected with S. mekongi, three patients infected with S. juponicum, two patients infected with S. huemutobium, one patient infected with Entumoebu histolyticu, one patient infected with Echinoccus grunulosus, and one patient infected with Tueniu solium (cysticercosis). Control sera were obtained from

ANTIGENS

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normal young adults at The Johns Hopkins Medical School, Baltimore, MD, USA. Sera from sheep infected 14 weeks previously with 200 metacercariae of Fusciolu heputica were obtained from Dr. R. Fetterer, Animal Parasitology Institute, U.S. Department of Agriculture, Beltsville, MD, USA. Sera from mice infected for up to 20 weeks with irradiated Trichinellu spit&is larvae were obtained from Professor N. Weatherly, University of North Carolina, Chapel Hill, NC, USA. Gel electrophoresis was carried out as described previously (Strand et al. 1982). Eggs were also metabolically labeled with [35S]methionine to exclude the possibility that contaminating host proteins were labeled with Iodine-125, or that a significant number of proteins lacked tyrosine residues. Analogous patterns of polypeptides were observed irrespective of the radiolabel used (Weiss and Strand, unpublished observations). The two-dimensional SDS-PAGE studies were limited to proteins that were included in the isoelectric focusing pH gradient of 7.6 to 3.8. In addition, we have defined glycoproteins as those polypeptides that specifically bind to concanavalin A. RESULTS

These studies were designed to identify genus-specific, species-specific, and crossreactive antigenic egg glycoproteins of three schistosome species. Sera from more than 80 patients infected with various parasites precipitated major glycoproteins with patterns characteristic for each type of infection, and showed only minor individual variations in reactivity. The minor variations may be attributed to several factors which have been previously discussed (Norden and Strand 1984), namely intensity of infection, the patient’s age, genetically determined factors, disease status, general health, or previous exposure to schistosomes or cross-reacting antigens (parasitic or otherwise). To control for the last two variables, we obtained pathogen-free mice which were then chronically infected with Schistosoma mansoni, S. japonicum, or Trichinella spiralis, or hamsters with S. haematobium. The major glycoproteins immunoprecipitated by sera from these infected animals were the same as those precipitated by sera from the infected humans, except that fewer individual variations in

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the immune responses of the infected animals were observed. Immunoprecipitates of radiolabeled egg extracts of each of the three schistosome species are considered below, and the figures illustrate representative examples of each pattern. The polypeptides in brackets are numbered sequentially as they are referred to in the text. Due to the heterogeneity of glycoproteins with respect to molecular weight and isoelectric point, some brackets may encompass more than one polypeptide. For each species of schistosome, extracts of eggs were labeled with Iodine-125 and chromatographed with concanavalin A. The fractionation was monitored by twodimensional SDS-PAGE to obtain (1) total egg proteins before concanavalin A chromatography, (2) concanavalin A nonadsorbed proteins, and (3) specifically bound and eluted proteins. The specifically bound and eluted glycoprotein fraction contained numerous iodinated proteins, and although the fraction contained only 3% of the labeled extract, it was highly immunoreactive. In a typical experiment, about 30% of radiolabeled antigen was precipitated by the homologously infected sera, while only about 2% was precipitated by normal human sera. The specifically bound and eluted glycoproteins were used for all of the studies reported here. By far the major fraction (97%) of the total labeled extract did not bind to concanavalin A, and was composed of two iodinated components of 69 and less than 9 kDa at pZ 6.2. The 69kDa polypeptide may correspond to MSA,, a component of soluble egg antigen which does not bind to concanavalin A (Pelley and Pelley 1976). The lower-molecular-weight component which migrated before the dye front is possibly a lipid. Gross overexposure of the autoradiographs revealed other minor proteins. The nonadsorbed fraction was not very immunogenic, since only 1% of the radiolabeled counts was precipitated by either normal or infected human sera.

STRAND

The glycoproteins of S. mansoni eggs labeled with Iodine-125, as analyzed by twodimensional SDS-PAGE, comprised approximately 20 glycoproteins, ranging in apparent mass from 400 to 9 kDa and pZ from 7.4 to 4.0 (Fig. 1A). The majority of the glycoproteins were acidic, and were of molecular weight greater than 40,000. This glycoprotein extract was then compared to 1251-labeledS. mansoni soluble egg antigens and found to be similar, despite marked differences in extraction techniques. Quantitative differences between the two extracts were that our extract was enriched in the polypeptides of 140 kDa at pZ 6.6 to 6.4 (Number I), 180 to 140 kDa at pZ 4.4 (Number 2), and 68 kDa at pZ 6.2 (Number 3), while soluble egg antigens were enriched in the polypeptides of 20 to 15 kDa at pZ 5.4 to 5.0 (Number 4), and 35 to 25 kDa at pZ 3.8 to 3.5. After fractionation of the iodinated soluble egg antigens on concanavalin A, the pattern of soluble egg antigen glycoproteins was indistinguishable from ours. When sera from 15 patients infected with S. mansoni and 11 infected with S. huematobium were incubated with our radiolabeled preparation of S. mansoni egg glycoproteins, most of the glycoproteins were precipitated (Figs. 1B and C, respectively), and the patterns of precipitated polypeptides were virtually indistinguishable. The majority of the glycoproteins were recognized with the same apparent reactivity by all the patients infected with either S. mansoni or S. haematobium, indicating extensive cross-reactivity between the two African species. Interestingly, polypeptides of 350 to 200 kDa at pZ 5.8 to 5.4 (Number 5), 170 to 150 kDa at pZ 5.1 (Number 6), and Number 1, which were not major constituents of the egg glycoprotein extract (Fig. 1A), were highly immunoreactive, as shown by immunoprecipitation (Figs. lB, C). Variation in the quantity of several precipitated glycoproteins provided evidence

SChiStOsOma

SPECIES: EGG GLYCOPROTEIN

ANTIGENS

337

B .

FIG. 1. Identification of Schisrosoma mansoni egg glycoproteins reactive with sera from humans infected with four different species of Schistosoma and Trichinella spiralis. Autoradiographs after two-dimensional gel electrophoresis of i2*I-labeled S. mansoni egg glycoproteins. Glycoproteins were fractionated by isoelectric focusing in the first direction and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Radiolabeled egg glycoproteins before (A) and after immunoprecipitation with (B) serum of S. mansoni-infected patient No. 53E; (C) serum of S. haematobium-infected patient No. 1s; (D) serum of T. spiralis-infected patient No. 8C; (E) serum of S. japonicum-infected patient No. 8P; and (F) serum of S. mekongi-infected patient No. 1T. Autoradiographs (D), (E), and (F) were exposed twice as long as (B) and (C). In this and subsequent figures, the glycoproteins in brackets are referred to in the text. Molecular weight standards (not shown) were cytochrome c, M, 11,700; chymotrypsinogen, M, 25,000; ovalbumin, M, 43,000; bovine serum albumin, M, 68,000; phosphorylase A, M, 94,000; and RNA polymerase, M, 160,000.

of differences among the humoral responses of these patients against the glycoproteins. For example, the glycoprotein of 180 to 100 kDa at pZ 4.9 to 4.5 (Number 7, Fig. lB, C) showed marked individual variation. However, it was not possible to identify a polypeptide uniquely precipitated by S. mansoni-infected human sera. Sera from 20 patients infected with the

Oriental schistosomes (S. juponicum and S. mekongi) and 10 patients infected with T. spiralis precipitated most of the S. mansoni egg glycoproteins that were precipitated by sera from patients infected with the African schistosomes (Figs. IE, F, D). However, sera from patients infected with the Oriental schistosomes or T. spiralis were less reactive toward the S. mansoni egg glyco-

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proteins, since only half as many counts of radiolabeled antigen were precipitated. Notably different was a reversal in reactivity toward the polypeptide of 70 to 40 kDa at pZ 4.7 (Number 8) and the polypeptide of 75 to 30 kDa at pZ 4.5 to 4.2 (Number 9). In all cases studied, sera of patients infected with the African schistosomes recognized polypeptide Number 8 to a much greater extent than did those infected with the Oriental schistosomes or T. spiralis. Thus, polypeptide Number 8 could be a candidate for detecting infections with the African schistosomes. Sera from normal humans, patients infected with Taenia solium, Echinococcus or granulosus, Entamoeba histolytica, Wuchereria bancrofti, normal sheep, or sheep infected for 14 weeks with Fasciola hepatica did not specifically recognize any of the S. mansoni egg glycoproteins. The 1251-labeledS. haematobium egg glycoproteins were analyzed analogously by two-dimensional SDS-PAGE and were found to be remarkably different from those of S. mansoni (Fig. 2A). Although there were approximately 20 major polypeptides ranging in molecular weight from 400 to 9 kDa and in pZ from 7.4 to 4.0, the pattern of polypeptides was quite dissimilar. Except for polypeptide Number 2, the acidic polypeptides of pZ less than 5.0 (Numbers 7, 8, 9) were notably missing. Additional polypeptides were present, including those of 50 to 42 kDa at pZ 6.8 to 6.4 (Number lo), 45 to 28 kDa at pZ 5.5 to 5.0 (Number ll), and 65 to 45 kDa at pZ 5.6 to 5.2 (Number 12). Species-specificity and cross-reactivity of the radiolabeled S. haematobium egg glycoproteins were studied by immunoprecipitation with sera of patients infected with various parasites. Sera of humans infected with S. haematobium or S. mansoni precipitated all of the 20 major glycoproteins (Figs. 2B, C), again indicating the extensive cross-reactivity between the two African species. Of particular interest were the highly immunoreactive polypeptides,

STRAND

Number 10 and 11, which did not correspond to any of the S. mansoni egg glycoproteins. Although the reactivity of these sera against the 20 glycoproteins varied, each of the 26 sera tested precipitated these major glycoproteins to some degree. As with S. mansoni antigens, fewer of the major S. haematobium glycoproteins were precipitated by sera of humans infected with S. japonicum or S. mekongi (Figs. 2D, E), demonstrating the limited antigenic similarities between the African and Oriental schistosomes. Furthermore, sera from patients infected with the Oriental species were less reactive toward the S. haematobium egg glycoproteins, to the extent that only one-fourth as many counts of radiolabeled antigen were precipitated. Notably decreased were polypeptides Number 10 and 11, while polypeptide Number 2 appeared to be the most immunoreactive glycoprotein. Polypeptide Number 12 showed the greatest individual variation, since the immunoprecipitation pattern obtained with several patients’ sera indicated that this polypeptide was a major antigen while other patients’ sera barely recognized it as immunoreactive. Polypeptides Number 10 and 11 would be prime candidates for detecting infections with the African species because of their decreased reactivity with sera of patients infected with the Oriental species. Sera from patients infected with T. spiralis precipitated considerably fewer of the major S. haematobium egg glycoproteins (Fig. 2F), and only one-fifth as many counts of radiolabeled antigen were precipitated compared to those precipitated by sera of patients infected with the African species. Polypeptide Number 2 appeared to be the most immunoreactive glycoprotein when sera from patients infected with T. spiralis were used. Sera from normal humans or patients infected with parasites not of the genus Schistosoma or Trichinella did not specifically precipitate any of the S. haematobium glycoproteins.

SChistOsoma

SPECIES: EGG GLYCOPROTEIN

$6 7;

Pi

339

ANTIGENS

4? 73

6p

5!

4P

FIG. 2. Identification of Schistosoma haematobium egg glycoproteins reactive with sera from humans infected with four different species of Schistosoma and Trichinella spiralis. Autoradiographs after two-dimensional gel electrophoresis of tz51-labeled S. haematobium egg glycoproteins (A), and immunoprecipitates of the radiolabeled glycoproteins with (B) serum of S. haematobium-infected patient No. 1s; (C) serum of S. mansoni-infected patient No. 1s; (D) serum of S. japonicum-infected patient No. 8P; (E) serum of S. mekongi-infected patient No. 1T; and (F) serum of T. spiralis-infected patient No. 5C. Autoradiographs (D), (E), and (F) were exposed twice as long as (B) and (C).

S. juponicum egg proteins were similarly labeled with Iodine-125, and the glycoproteins were resolved by two-dimensional SDS-PAGE into approximately 30 glycoproteins, ranging in molecular weight from 400 to 9 kDa and pZ from 7.5 to 4.2 (Fig. 3A). The S. japonicum egg glycoproteins appeared strikingly different from the S. mansoni or S. haematobium egg glycoproteins, both in number and pattern. Notably enhanced in the S. juponicum extract were polypeptide Number 5 and those of molecular weight less than 50,000. The S. &onicum egg glycoproteins at pZ 4.7 to 4.2 were dramatically different from those of the Af-

rican species. In this case, the polypeptides appeared as a smear from 400 to 45 kDa (Number 13, Fig. 3A), whereas the S. mansoni egg glycoproteins were visible as distinct spots (Numbers 2,7,8, and 9, Fig. lA), and only major S. haematobium egg glycoprotein was present in this region (Number 2, Fig. 2A). Sera of humans infected with S. japonicum or S. mekongi precipitated most of the major S. juponicum egg glycoproteins, and the precipitation patterns were practically indistinguishable (Fig. 3B). Interestingly, the only polypeptide which showed any marked individual variation was polypep-

340

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FIG. 3. Identification of Schistosoma japonicum egg glycoproteins reactive with sera from humans infected with three different species of Schisrosoma and Trichinella spiralis. Autoradiographs after two-dimensional gel electrophoresis of ‘251-labeled S. juponicum egg glycoproteins (A), and immunoprecipitates of the radiolabeled glycoproteins with (B) serum of S. juponicum-infected patient No. 5P; (C) serum of a normal human; (D) serum of S. haematobium-infected patient No., 4s; (E) serum of S. mansoni-infected patient No. 24E; and (F) serum of T. spiralis-infected patient No. SC. Autoradiograph (D) was exposed three times longer than the others.

tide Number 5. The polypeptide of 75 to 50 kDa at pZ 5.0 (Number 14) was highly immunoreactive, whereas polypeptide Number 13 and the polypeptide of 55 kDa at pZ 6.3 to 6.0 (Number 15) were not (compare Figs. 3A and B.) Sera of patients infected with S. haematobium or S. mansoni precipitated fewer of the major S. japonicum glycoproteins (Figs. 3D, E); in particular, the glycopro-

teins in the molecular weight range of 95 to 30 kDa at pZ 7.5 to 5.4 and polypeptide Number 5. As was observed for the African schistosomes, sera from patients infected with parasites not of the genus Schistosoma had a low reactivity against the S. japonicum glycoproteins. Sera from patients infected with T. spiralis specifically precipitated polypeptides Numbers 5 and 14 (Fig. 3F);

Schistosoma

SPECIES:

EGG GLYCOPROTEIN

ANTIGENS

341

et al. 1982). Recently, however, an enzymelinked immunosorbent assay utilizing a S. haematobium soluble egg antigen preparation was reported to be successful in distinguishing between sera from hamsters infected with the two different African schistosomes (Pacheco and Hillyer 1983). The molecular identity of their S. haematobium egg antigen was not reported, making further comparisons with our study difficult. Several groups of major S. juponicum egg glycoproteins were predominantly recognized by sera of S. japonicum- or S. meDISCUSSION kongi-infected patients; glycoproteins in In this study, we have identified immu- the molecular weight range of 95 to 30 kDa noreactive genus- and species-specific at pZ 7.5 to 5.3 and polypeptide Number 5. schistosome egg glycoprotein antigens. The immunoreactive S. juponicum glycoOne major Schistosoma mansoni glycopro- proteins described in this study share the tein (Number 8) and two major S. haema- same apparent molecular weight and pZ tobium glycoproteins (Numbers 10, 11) were with some of those previously described precipitated to a much greater extent by (Carter and Colley 1981; Tracy and Mahsera of patients infected with the African moud 1982; Ishii and Owhashi 1982). Furschistosomes (S. mansoni and S. haema- thermore, the results of this study would tobium) than by sera of patients infected indicate that the preponderance of S. jawith the Oriental schistosomes (S. jupon- ponicum polypeptides of molecular weight icum and S. mekongi) or parasites not of greater than 110 kDa, previously referred the genus Schistosoma. S. mansoni poly- to as “female-specific” (Aronstein and peptide Number 8 may correspond to Strand 1983; Norden and Strand 1984), may MSA, (Pelley and Pelley 1976), a concanav- be egg polypeptides since they were idenalin A-binding protein of 50 kDa at pZ 4.5 tified only in female adult worm extracts. to 3.5. Our results support the potential Although the pZ values of the worm and egg’ diagnostic usefulness of this polypeptide in glycoproteins are not identical in these comparison to any of the other reactive studies because different radioisotopes polypeptides (Pelley et al. 1977). MEG,-H, were used, the high-molecularlweight glya 70-kDa S. haematobium egg glycoprotein coproteins were the most immunoreactive (Hamburger et al. 1982b), may correspond in both cases. This study has identified egg to our polypeptide of 70 kDa at pZ 7.0 to glycoproteins which may be responsible for 6.7 (Number 16, Fig. 2A); however, it did the extensive cross-reactivity previously not appear to be one of the major immu- observed between the Oriental schistonoreactive glycoproteins (Fig. 2B). somes (Hillyer and Bruce 1980; BarrelWe were not able to distinguish between Netto et al. 1983). the sera of S. mansoni-infected patients and Irrespective of the species of schistothose of patients infected with S. haema- some eggs used, our results confirm pretobium on the basis of immunoreactivity. vious work (Tsang et al. 1981; Ogunba et Others have previously reported this difft- al. 1982; Kamiya et al. 1982) in which sera culty in diagnostic assays (Huldt et al. of patients infected with Trichinella spiralis 1975; Pelley and Pelley 1976; McLaren et showed more cross-reactivity with schisal. 1978, 1981; Hillyer et al. 1980; Ogunba tosome egg extracts than did sera from pa-

however, only half as many counts of radiolabeled antigen were precipitated compared to those precipitated by sera of patients infected with S. juponicum. Notably missing were the polypeptides of pZ greater than 5.4 and molecular weight less than 50,000. Sera from normal humans (Fig. 3C) or patients infected with other parasites, including E. granulosus, E. histolytica, T. solium, or W. bancrofti, did not specifically recognize any of the S. juponicum glycoproteins.

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tients infected with any of the other helminths we have studied. In this study, we have shown that, although there are several genus- and species-specific egg antigens, the majority of the egg glycoproteins are cross-reactive. Thus, it will be necessary to characterize the schistosome egg glycoproteins further if they are to be used as antigens for diagnostic purposes. For example, is the observed cross-reactivity due to binding of antibody to the carbohydrate moieties or are the proteins extensively homologous? Results obtained by use of monoclonal antibodies have already demonstrated that it is possible to identify genus- and speciesspecific epitopes (Cruise et al. 1981, 1983; Mitchell et al. 1981, 1983a, 1983b; Dissous et al. 1982; Norden et al. 1982; Smith et al. 1982; Taylor and Butterworth 1982; AbdelHafez et al. 1983; Dresden et al. 1983; Zodda et al. 1983). Such monoclonal antibodies will therefore be important for diagnostic assays of antigenemia. For measurement of antibody responses, however, it will be necessary to identify the antigenic regions of individual glycoproteins and to establish the cross-reactivity of each of these regions. The glycoproteins that we have shown to be enhanced for Schistocoma species-specificity will clearly be most useful for further analysis. ACKNOWLEDGMENTS We are indebted to Dr. J. Ellner, Dr. R. Fetterer, Dr. S. E. Maddison, Dr. T. Nash, Dr. X. Q. Pan, Dr. M. M. A. Salama, Dr. J. Stanley, Dr. M. Stek, and Prof. N. Weatherly for the sera. We are grateful to the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases for the supply of the Schistosoma mansoni soluble egg antigen. We appreciate the excellent technical assistance of A. George and L. Baker and the secretarial assistance of L. Poole. We thank Dr. Pamela Talalay for her editorial review of the manuscript. This work was supported by Grant AI-19217 from the U.S. National Institutes of Health and Grant 283-0088 from the Edna McConnell Clark Foundation. A.P.N. was supported by the U.S. Public Health Service thru Grant 2 T32 CA 09243-06.

REFERENCES S. K., PHILLIPS, S. M., AND ZODDA, D. M. 1983. Schistosoma mansoni: Detection and

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