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A cDNA clone from an adult Schistosoma mansoni lambda gt11 expression library (A12) encoding an antigenic polypeptide of 22 kDa is described. A12 is 797 bp long and has one open reading frame encoding a protein of 190 amino acids which does not contain a signal sequence or membrane anchor motif and has no homologies with any sequences on the currently available data bases. Its product (sm22.6) is recognised by antibodies from mice protectively vaccinated with purified adult S. mansoni tegumental membranes and by serum from S. mansoni-infected Brazilians. It is present in all post-snail life cycle stages except the egg, is not sex-specific, and is found in 9 species of Schistosoma, but not in a range of other helminths. Data are presented which suggest that sm22.6 is a soluble, peripheral membrane protein.

Sm25 is the principal antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental membranes of adult Schistosoma mansoni. The full-length amino acid sequence of this protein has been deduced from the sequence of two cDNAs, one isolated by screening a cDNA library and the other, including the 5' end of the gene, amplified directly from adult worm RNA using the polymerase chain reaction. The predicted sequence represents a nascent polypeptide of Mr 21,500. Following cleavage of a predicted signal sequence, the Mr of the resulting polypeptide is 17,600. The polypeptide contains 2 potential sites for N-linked glycosylation and a hydrophobic domain at the C-terminus that could facilitate membrane association. Analysis of the mature gene product confirmed that Sm25 is an N-glycosylated integral membrane protein and that the Mr of the deglycosylated polypeptide is between 15,000 and 20,000.

The structures of the 38, 32 and 20 kDa surface antigens isolated from schistosomula and adult worms of Schistosoma mansoni were compared by two-dimensional peptide mapping, by immunological analysis and by one- and two-dimensional electrophoresis. Peptide mapping showed a high degree of similarity between the isolated antigens from both parasite stages. The NIMP/M47 monoclonal antibody showed cross-reactivity between the 32 and the 20 kDa antigens under denaturating and non-denaturating conditions, as demonstrated by immunoprecipitation and Western blotting. It is concluded that these antigens constitute a homologous family of surface antigens.

Protective immunity has been demonstrated in experimental schistosomiasis and is also believed to occur in man. It can be mediated by antibodies from infected animals or animals immunized with attenuated organisms. Recombinant Escherichia coli synthesizing antigenic polypeptides from the three principal species of schistosome that infect man, Schistosoma mansoni, S. japonicum and S. haematobium, have been constructed. Libraries of adult worm cDNA were prepared from each species in the expression vector lambda gt 11 and directly screened with antibodies from animals experimentally immunized with S. mansoni and S. japonicum and from humans infected with S. haematobium. The S. mansoni clones have been analysed in greatest detail. At least four different types of clones were identified. All the detected recombinant polypeptide antigens were recognised by antibodies from chronically infected mice and most were also recognised by antibodies from mice immunized with attenuated cercariae and anti-surface membrane antibodies. Clones synthesizing species-specific antigens for both S. mansoni and S. japonicum were identified by simultaneous screening of both libraries. At least three types of S. haematobium clones were identified by screening with human infection serum, most of which were species-specific. All the antigens were in the form of fusion peptides with E. coli beta-galactosidase and their expression was induced by isopropylthiogalactopyranoside. Since known protective monoclonal antibodies recognise highly glycosylated membrane proteins which cannot be identified in the form of nascent polypeptides, the direct identification of polypeptide antigens defined by their reactivity, as reported here, is an essential step in producing reagents by recombinant DNA technology, suitable for vaccination and diagnosis.

When freshly transformed schistosomula of Schistosoma mansoni are incubated with human serum, a protein doublet (molecular weight approximately 45 kDa) is expressed on the surfaces of the parasites; parasites incubated in defined media fail to express the doublet proteins. A 30 min exposure to serum is sufficient to induce the expression of the doublet, and even when the parasites are separated from the serum by a dialysis membrane, the doublet is still expressed. The doublet proteins are shown to be synthesized by cercariae, but not by schistosomula; they can be extracted using detergents and immunoprecipitated by specific antisera. Following expression of the doublet, purified low density lipoproteins from human serum interact with the schistosomula, become adsorbed onto their surface membranes and are possibly internalized and degraded.

Alkaline phosphatase, a marker for tegumental membranes of Schistosoma mansoni, was extracted using Triton X-100 from membranes purified by sucrose density gradient centrifugation. The enzyme activity was purified 6 800-fold over parasite homogenates and 118-fold over the tegumental membranes released when parasites were incubated in phosphate-buffered saline. Purification of the solubilised enzyme was achieved by binding to a Con A agarose affinity column, gel filtration of the eluted glycoproteins, and Blue affigel chromatography. The purified enzyme was shown to consist of a single glycosylated polypeptide Mr 65 000 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Enzyme activity was associated with a possible tetramer, Mr 260 000 on gel filtration. Activity was associated with a band Mr 130 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis run in the absence of reducing agents. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents, the size of the enzyme was shown to be similar in cercariae, schistosomula, adult schistosomes and their eggs, but it was smaller than the activity (Mr 145 000) extracted from host liver and intestinal microsomal membranes. The topography of the enzyme in the schistosome tegument was investigated by using surface radio-labelling reagents followed by its purification. The enzyme could be radio-iodinated only with difficulty in adult worms. Bolton and Hunter reagent, used at high concentration and for prolonged time periods, resulted in labelling of enzyme activity and the Mr 65 000 polypeptide subunit was also iodinated under these extreme conditions. It was concluded that the enzyme is not exposed at the schistome's surface, and is probably buried in the tegumental membrane network.

A previously described cDNA clone, pSF10, of Schistosoma mansoni encoding the very dominant female specific polypeptide (FSP) has been used to characterize the gene and its expression. The gene is detectable in different isolates of S. mansoni and is estimated to be present in 3 copies per haploid genome. The gene is not sex linked and exhibits neither amplification nor rearrangement concomitant with expression. Expression of the gene by parasites maturing in hamsters is first detected after 5 weeks when the RNA is present at 1/10 the level of that of 6 week worms. Although the FSP gene is specifically and highly expressed by egg laying female worms a corresponding polypeptide produced by the cell-free translation of RNA is not detectable. It was confirmed, however, that pSF10 does indeed encode a mRNA by DNA sequence analysis. The sequence demonstrated a mRNA containing a poly(A) tail and two open reading frames. One reading contains no methionine but is very high (47%) in glycine. This amino acid composition could account for the inability to detect the gene product by cell-free translation in the presence of [35S]methionine.

Lectins labeled with 125I or conjugated with fluorescein were employed to study the carbohydrates on the surface of different stages of schistosomula of Schistosoma mansoni. Newly transformed schistosomula were shown to bind concanavalin A; the 60 000 and 120 000 dalton agglutinins from Ricinus communis; the fucose-binding protein from Lotus tetragonolobus; wheat germ agglutinin and peanut agglutinin. Soybean agglutinin, Ulex europaeus agglutinin and Dolichos biflorus agglutinin, on the other hand, failed to bind to the schistosomulum surface. The binding of peanut and soybean agglutinin was unaffected by pretreatment of the parasites with neuraminidase. Binding of concanavalin A, the 120 000 dalton agglutinin from Ricinus communis, wheat germ agglutinin and peanut agglutinin to the surface of 5-day schistosomula, recovered from the lungs of mice, was also demonstrated. In each case, however, the level of binding was approximately 70% less than that observed with newly transformed schistosomula and the binding of the fucose-binding protein from L. tetragonolobus practically disappeared. In contrast with newly transformed schistosomula, lung stage schistosomula, pretreated with neuraminidase, displayed a significant increase in the binding of peanut and soybean agglutinin. The results indicate that a significant alteration in the surface carbohydrates of S. mansoni occurs during in vivo maturation of the parasite. This change may contribute to the organism's ability to survive in the vertebrate host.