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Polypeptide surface antigens of Schistosoma mansoni recognized by schistosomiasis patients have been identified and their strain and species specificity investigated. Antibodies from individuals infected with S. mansoni were used in immunoprecipitation assays of 125I-labelled schistosomulum surface antigens. All individuals surveyed from St. Lucia strongly precipitated antigens of approximately Mr 38,000 to 32,000 and 20,000. These antigens were shown by two-dimensional gel electrophoresis to be the same as those recognized by experimentally immunized mice. Although individuals showed a highly heterogeneous response against total polypeptide antigens synthesized in vitro by cell-free translation of adult S. mansoni mRNA, all individuals recognized the same surface antigens. Immunoprecipitation with sera from patients infected with S. mansoni in many different parts of Africa resulted in generally the same antigens being precipitated, although a very high molecular weight antigen(s), not strongly recognized by the St. Lucian sera was also precipitated by most of the African patient sera. One serum from Ghana precipitated the high molecular weight antigen but not the low molecular weight antigens, raising the possibility of the existence of S. mansoni strain(s) exhibiting some diversity in surface antigens. The surface of S. mansoni schistosomula was found to bind strongly antibodies from individuals infected with S. haematobium, demonstrating that most surface antigens are cross-reactive. Immunoprecipitation demonstrated, however, that of the polypeptide surface antigens only the very high molecular weight antigen was recognized by anti-S. haematobium antibodies and that the 38,000 to 32,000 and 20,000 Mr antigens were species-specific. Immunoprecipitation of the polypeptide antigens derived from purified adult surface membranes demonstrated recognition of the same 32,000, 25,000 and 20,000 Mr antigens recognized by chronically infected mice. Again these antigens were found to be species-specific.

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.

The relationship between reinfection with Schistosoma haematobium and immunological parameters was studied in a group of Gambian children aged from 8 to 13 years. Each individual's exposure to infection was assessed from observations of water contact, cercarial densities and infected snail densities at water contact sites. Eosinophil counts were made and responses to egg antigen (SEA) and adult worm antigen (WWH) measured by ELISA. Low levels of reinfection were associated with a high eosinophil count, high levels of antibodies against WWH and SEA, increased age and low exposure. In a multiple regression analysis of the association of reinfection with eosinophil count, antibody levels, exposure, age and sex, the effects of eosinophil count and exposure were still very significant after allowing for all the other variables. The effects of the antibody levels were close to significance after allowance for exposure and eosinophil count (for WWH: P = 0.09; for SEA: P = 0.07), although the evidence was less clear after additional allowance was made for age and sex. The ability of sera from the children to recognize different parasite antigens was also examined by immunoprecipitation of labelled schistosomulum surface, WWH, SEA and S. haematobium adult worm mRNA in vitro translation products. Schistosomulum surface antigens were recognized by all the sera and there was little variation in this response. There was more variation in their responses to SEA and WWH and a marked heterogeneity in the response to in vitro translation products. However, the pattern of antigen recognition appeared unrelated to susceptibility to reinfection.

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.

Antibodies from mice vaccinated with highly irradiated Schistosoma mansoni or S. haematobium cercariae were used to characterize schistosomulum surface epitopes which were found to be diverse in their species and stage specificities. The epitopes recognized on the Mr greater than 200,000 and 15,000 schistosomulum surface antigens of S. mansoni and the Mr greater than 200,000 schistosomulum surface antigen of S. haematobium were found to be cross-specific whereas those on the Mr 38,000, 32,000 and 20,000 schistosomulum surface antigens of S. mansoni and the Mr 35,000, 30,000 and 24,000 schistosomulum surface antigens of S. haematobium were only immunoprecipitated by homologous antibody and are thus possible targets of the protective species-specific immunity stimulated by highly irradiated cercariae. The epitopes recognized on the Mr greater than 200,000 and 38,000 antigens of S. mansoni were shown to cross-react with both the egg and the adult worm whereas those on the Mr 32,000 and 20,000 antigens only cross-reacted with the adult worm, and those on the Mr 15,000 antigen cross-reacted with neither the adult worm nor the egg. In addition the epitopes on the Mr 38,000 and 32,000 antigens were demonstrated to be polypeptide in nature. Those on the Mr greater than 200,000, 20,000 and 15,000 antigens, on the other hand, could not be conclusively defined.

Observations were made on the development of acquired resistance to Schistosoma haematobium in the baboon following immunization with cercariae by the percutaneous route and by the transplantation of adult worms into the mesenteric veins. In the first experiment six baboons were immunized with 1000 S. haematobium cercariae given percutaneously. They were challenged with 10 000 cercariae given 73 weeks later and the results were compared with a similar infection in non-immunized animals. The results showed that the baboon can develop a strong resistance to reinfection with S. haematobium. The manifestations of the immunity were (i) the absence of any increase in egg output after challenge (ii) the substantially lower level of adult worms and eggs in the tissues of the immunized baboons compared with the challenge control animals (iii) a reduction in the egg laying capacity of the residual worms and (iv) the virtual absence of gross pathology and the mild lesions seen in the tissue sections of all the immunized animals. The depression in egg laying of the worms was confirmed by transplanting them into non-immune baboons. This experiment indicated that the non-egg-laying worms in the immune baboons were not irreversibly damaged since they survived, some even migrating to the vesical and ureteric vessels, and egg-laying was rapidly resumed after transplantation. A further experiment was designed to see if a similar degree of immunity could be produced by an adult worm infection without previous exposure to cercariae or schistosomula. The immunization dose consisted of 50-100 S. haematobium worm pairs which were transplanted into the mesenteric veins of each of six baboons and the animals were challenged percutaneously with 7000 cercariae 35-55 weeks later. There was little difference in the worm burdens of the immunized and control animals but the worms in the immunized baboons produced fewer eggs and the pathology seen in these animals was much milder than in the challenge control animals suggesting that some degree of resistance to reinfection was produced by the transplanted worms.

Protein A-sepharose affinity chromatography was used to isolate IgG subclasses from the serum of CBA mice chronically infected with Schistosoma mansoni. The subclasses were tested for the presence of two antibodies which are responsible for the death of young schistosomula in vitro; 'lethal antibody' (LA), which kills schistosomula in co-operation with complement and 'eosinophil adherence antibody' (EAA) which causes the death of schistosomula by promoting the adherence of eosinophils to the parasite. LA and EAA were detected only in the IgG fraction of the serum. LA was concentrated in the IgG2a fraction and EAA in the IgG1 fraction. The development of IgG subclasses specific for schistosomula was followed in mice exposed to twenty cercariae by the fluorescent antibody technique. IgG1, IgG2a and IgG2b antibodies were detected 2 weeks after infection and their titres rose steadily to reach high levels by weeks 12 or 14. IgM antibody was not detected until week 6 and IgA until week 10; both were present at lower concentrations than the IgG1 antibodies.

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.

An indirect fluorescent antibody technique was used to detect mouse host antigens and parasite antigens on the surface of Schistosoma mansoni. A rabbit anti-mouse rbc antiserum to detect host antigens, and serum from mice immune to S. mansoni was used to detect parasite antigens. Schistosomula prepared after penetration of isolated mouse skin did not possess host antigens but bound antibody from immune serum (immune antibody); schistosomula recovered from the lungs of mice five days after infection possessed host antigens and failed to bind immune antibody. In contrast, schistosomula recovered from the skin of normal or immune mice three and 20 hours after cercarial penetration, adult worms, and cryostat sections of adult worms, were positive for host antigens but also bound immune antibody. Strong binding of immune antibody only occurred with the cryostat sections. Anti-schistosome antibody can therefore bind to schistosomes in the presence of host antigen. The lung forms of schistosomula however, may have different surface properties as there is no evidence that immune antibody binds to these forms.

Serum harvested from Sprague-Dawley rats twice vaccinated with gamma-irradiated cercariae of Schistosoma mansoni is able to protect naive recipients against a challenge infection, but the challenge parasites are susceptible to immune elimination over a very short period of time. Thus, vaccinated rat serum protects recipients against a percutaneous cercarial challenge when transferred on Day +5 but not Day 0 and protects recipients against a tail vein challenge with 5-day-old lung worms when transferred on Days 0 or +1, but not Days +4 or +5. Rats challenged with lung worms via the tail vein and given serum on Day +3 exhibit approximately half the protection expressed by counterparts that received serum on Day 0. However, vaccinated rat serum does not protect naive recipients against a lung worm challenge introduced directly into the liver. These data indicate that immune elimination of challenge parasites in the vaccinated rat model is site-dependent rather than stage-dependent, and most probably occurs during the lung phase of parasite migration.

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.

To determine the easiest method of raising antibodies to antigens exposed on the surface of schistosomula of Schistosoma mansoni, several crude preparations of the parasite were used to immunize mice. Schistosomula released products, whole worm homogenate, and parasite eggs all raised antibodies which bound to the surface of live schistosomula, although the anti-egg antiserum did so less strongly. Anti-schistosomula released products antiserum recognized three schistosomula surface antigens of Mr 15,000, 20,000, and 32,000, anti-whole worm homogenate recognized 20,000, 32,000, and 38,000 Mr surface antigens, and anti-egg recognized a less than 200,000 Mr surface antigen. None of these antigens was recognized when the labeled preparation was immunoprecipitated with its homologous antiserum. When these antisera were used to immunoprecipitate cell free translation products of adult worm RNA, the antischistosomula released products and anti-whole worm homogenate recognized an 11,000 Mr doublet while the anti-egg precipitated 14,000 and 44,000 Mr antigens. Other crude preparations were used to immunize rabbits; Formalin-fixed schistosomula, denuded adult worms, and purified worm tegument all induced antibodies which recognized the 20,000, 32,000, and 38,000 Mr schistosomula surface antigens.

The carrier effect, using TNP-labelled schistosomula was used to measure the helper T-cell activity against the schistosomula surface in CBA mice exposed to 30 cercariae of Schistosoma mansoni. After infection the helper T-cell activity reached a peak in 8--10 days, but by 6 weeks it had declined to background levels. Five x 10(7) spleen cells from chronically (12-week) infected mice when injected into 9-day infected mice caused a specific suppression of the helper T-cell response to schistosomula. Subsequent fractionation of the spleen cell population using a nylon wool column and specific depletion of T cells from the spleen cell population with anti-Thy 1.2 antisera and complement, showed that the suppressive activity was due to T cells. We conclude that during infection of mice with S. mansoni a population of suppressor T cells is generated which partially regulates antibody production against schistosome surface antigens.

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.