Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice

Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from cDNA lambda gt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni beta-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blots with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within beta-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain lambda gt11-fusion protein clones.     

Modification of immune responsiveness in murine Schistosomiasis mansoni. I. Time course after cercarial exposure.

The immune responsiveness of mice infected with 90 cercariae of Schistosoma mansoni has been investigated. Post-infection kinetics of antibody responses, delayed hypersensitivity and mitogen responsiveness show that there are profound disturbances of these immunological parameters starting 2 weeks after the infection. Although the final outcome is immunodepression the S. mansoni infection can produce immunostimulation. We have observed differences in the kinetics of the depression of humoral antibody responses in two strains of mice. It seems that the worms and not the eggs are of major importance in determining the observed alterations of immune responsiveness.     

Immunity in Schistosoma mansoni using antigens of Fasciola hepatica isolated by concanavalin A affinity chromatography.

Antigens of Fasciola hepatica adult worms were chromatographed using concanavalin A-Sepharose 4B. Two unbound peaks appeared in the inclusion volume (DT-1 and DT-2), and one peak was eluted with alpha-methylglucoside (E1-1). At least seven peaks were obtained by isoelectric focusing of E1-1. The largest of these peaks, with an average pI of 4.0, contained the antigens reactive with antibodies to Schistosoma mansoni. Mice immunized with DT-2 or E1-1 and challenged with S. mansoni cercariae developed 39 to 82% fewer worms than controls. DT-1 had no protective effect. Combining DT-1 and DT-2 abolished this protection. These experiments demonstrate that F. hepatica glycoprotein antigens induce in mice significant protection to infection with S. mansoni and offer an interesting approach to the study of vaccines in experimental schistosomiasis.     

Isolation and characterization of Schistosoma mansoni constitutive androstane receptor

Full length cDNA clones encoding a Schistosoma mansoni homologue of vertebrate CAR/PXR/VDR group nuclear receptor, termed SmCAR were isolated from screening a S. mansoni adult worm cDNA library. SmCAR is a 702 amino acid protein which retains a typical domain organization of nuclear receptor superfamily members. A homology search demonstrated that SmCAR exhibits the highest homology with mouse constitutive androstane receptor (CAR). Like its orthologues from invertebrates, SmCAR contains a P box sequence of ESCKA in the DNA binding domain. The P box is important in determining the DNA binding specificity for nuclear receptors. SmCAR mRNA is expressed in every stage of S. mansoni life cycle with an elevated expression level in egg and cercaria stages. Two forms (78 and 81 kDa) of SmCAR protein were detected in schistosome worm extract by Western blot analysis. SmCAR protein was demonstrated to be widely distributed in adult worms by immunolocalization studies, being found in the subtegument in both male and female worms and in the ovaries, vitellaria and eggs in female worms. In vitro DNA binding assays demonstrated that SmCAR binds to the hsp27 ecdysone response element (EcRE) as well as schistosome p14 gene upstream region. The AGTGCA half site is essential for binding of SmCAR to the p14 gene upstream region. Therefore, AGTGCA probably serves as a high affinity binding half site for ESCKA containing nuclear receptors.     

Acidic thiol proteinase activity of Schistosoma mansoni cultured in vitro

Schistosoma mansoni cultured in vitro for periods from 12 days to 33 weeks were analyzed for proteinase activity using a fluorescent substrate and compared with adult S. mansoni worms from infected mice. The enzyme activity measured was that of an acidic thiol-dependent proteinase previously described. Proteinase activity appears early in development, and rises rapidly to an approximately constant proportion of the total extractable worm protein. Cultured worms have 5 to 20 times higher specific activity than in vivo-developed worms. However, cultured worms and in vivo worms have approximately equivalent activities per worm.     

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.     

Immune responses during human schistosomiasis mansoni. I. In vitro lymphocyte blastogenic responses to heterogeneous antigenic preparations from schistosome eggs, worms and cercariae.

In vitro lymphocyte blastogenesis assays were performed using peripheral blood lymphocytes obtained from patients with schistosomiasis mansoni. Their infection was well characterized both clinically and in regard to the duration and intensity of Schistosoma mansoni infection. Subjects who were either not infected with any helminths or who were negative for S. mansoni but infected with other helminths provided two control groups. Cultures were exposed to various concentrations of heterogeneous soluble antigens prepared either from S. mansoni eggs, worms, or cercariae. In this series there was no statistical correlation between the intensity of infection (as determined by eggs/ml of feces) and the degree of cell-mediated reactivity observed by lymphocyte blastogenesis to any of the antigenic preparations. Individual patient lymphocyte responsiveness varied considerably. However, analysis of the data by groups, based upon the longevity of their S. mansoni infection, demonstrated different patterns of reactivity in regard to the antigen preparations. Responses to the egg antigens were only present early in infection. In contrast, reactivities stimulated by either the worm or cercarial preparations increased in a direct relationship to the duration of S. mansoni infection.     

The genome of Schistosoma mansoni: isolation of DNA, its size, bases and repetitive sequences

DNA has been prepared from adults and cercariae of Schistosoma mansoni utilizing a technique that involves centrifugation through cesium chloride. The DNA isolated from S. mansoni adults and that isolated from cercariae were found to be indistinguishable in all analyses. No modified bases were detected by chromatography or comparative endonuclease restriction. Cot analysis demonstrated that the haploid genome of S. mansoni is 0.26 pg (2.7 X 10(8) base pairs) and that the genome contains both moderately and highly repeated components. Some of the repetitive fraction of DNA consists of tandemly repeated ribosomal genes of which there are 500-1000 copies per genome (1.8-3.6% of the total DNA). Four other non-ribosomal repetitive sequences (comprising at least a further 2.0% of the total DNA) have been isolated from a DNA clone bank and their arrangement within the S. mansoni genome investigated by restriction and Southern blot analysis. These cloned segments of DNA appear in many different locations within the genome and thus are reminiscent of the interspersed DNA sequences described in higher eukaryotic organisms.     

Cloning of a major developmentally regulated gene expressed in mature females of Schistosoma mansoni

A cDNA library constructed from RNA isolated from adult Schistosoma mansoni has been screened by differential hybridization to identify clones corresponding to genes highly expressed by female worms. Several such cDNAs encoding the same highly abundant mRNA species were identified. Studies with one of these (pSF10) which contained a 500 base pair insert demonstrated that this gene was not expressed in immature females or eggs and encoded a polypeptide of approximately 35 000 daltons. Quantitation of the levels of RNA showed that 10% of the total RNA of female parasites was homologous to pSF10. A single gene corresponding to pSF10 was identified in Southern blotting experiments using adult worm DNA. The cloning of this gene will facilitate study of the molecular and genetic events controlling female schistosome maturation.     

Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni.

Our laboratories previously isolated a putative extracellular or membrane-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene, designated a signal peptide-containing (SP) Cu/Zn-SOD, from Schistosoma mansoni. SOD activity was thus investigated throughout the life cycle of S. mansoni and found in all stages: eggs, miracidia, cercariae, schistosomula, lung-stage worms, and adult worms. The adult worms had the highest SOD activity (53 +/- 9 nitrite units), which was five times higher than that of eggs or miracidia and twice as high as that of 3-h-old mechanically transformed schistosomula. Cu/Zn-SOD constituted over 95% of the total SOD activity found in S. mansoni, compared with that of Mn-SOD. Most of Cu/Zn-SOD specific activity was associated with a detergent-extractable fraction of the parasite. Isoelectric focusing gel electrophoresis analysis revealed that there were four major pI variants of Cu/Zn-SOD present in the adult worms. Only two of these Cu/Zn-SOD pI variants were present in the 3-h-old mechanically transformed schistosomula. Fast protein liquid chromatography gel filtration fractionation of adult parasite extract was carried out to correlate the SP Cu/Zn-SOD with the SOD activity by using anti-SP Cu/Zn-SOD monoclonal antibodies, which separated the immunoreactive gene product and the SOD activity into different fractions. Quantitative tissue fractionation also revealed a discordant distribution of the gene product compared with that of Cu/Zn-SOD activity. These results indicated the existence of another Cu/Zn-SOD(s) in the parasite. Purification of the Cu/Zn-SOD activity from the adult worms showed that it represented the two lower-pI variants found in both adult worms and 3-h-old schistosomula. Peptide sequence analysis of the purified Cu/Zn-SOD confirmed that there is a second form of Cu/Zn-SOD in the parasite.     

Interaction of cholera toxin with three life-cycle stages of Schistosoma mansoni: adult worm, egg and cercaria

We have previously reported that there is an immunological cross-reactivity between Schistosoma mansoni and cholera toxin (CT). In this study, using an immunofluorescence technique with anti-CT antibody, we provide further evidence for this cross-reactivity by demonstrating an antigen, localized in the tegument of S. mansoni adult worms which is cross-reactive with a CT antigen. Anti-CT antibodies also reacted with structures in S. mansoni cercariae and eggs. Additionally, CT itself was found to bind strongly to the gut of the adult worm, gut cells of cercaria and the egg shell. The binding of CT to the parasite was blocked when parasite sections were incubated with CT which had been incubated with the ganglioside GM1. Lipid extraction and isolation of gangliosides demonstrated the presence of GM1 in adult worms. For further analysis of CT-binding structures, the possible interaction of CT with two major schistosome gut antigens, circulating cathodic antigen (CCA) and circulating anodic antigen (CAA), was studied. We found that CT blocked the binding of anti-CCA antibody to the gut of adult worms and that anti-CCA blocked the binding of CT to the worm gut. These findings indicate that CT binds to CCA present in the gut of the parasite and thus has, in addition to GM1, a second binding specificity.     

Immunization against Schistosoma mansoni in rhesus monkeys and the requirement of activation of both cell-mediated and humoral mechanisms.

When groups of rhesus monkeys were pretreated with BCG plus hyperimmune serum from monkeys with chronic schistosomiasis or with dialyzable transfer factor from uninfected monkeys plus hyperimmune serum and were challenged with 1,500 cercariae of Schistosoma mansoni, the mean worm burdens were significantly lower than that of untreated controls. Pretreatment with neither BCG alone nor Corynebacterium parvum plus a membrane antigen of adult worms of S. mansoni affected susceptibility. Neither lymphocyte proliferation in the presence of mitogens or schistosome antigen nor serological responsiveness (as measured by gel diffusion, Cercarienhüllenreaktion, circumoval precipitation, or enzyme-linked immunoabsorbent assay) correlated with the degree of resistance of the animals to S. mansoni. The pretreatment procedures used did not cause any abnormal histopathological responses and did not alter the characteristic host response to schistosome eggs in the lungs, liver, mesenteric lymph nodes, and colon.     

Studies on experimental mixed infections ofSchistosoma mansoni andS. haematobium in hamsters

Golden hamsters were superinfected simultaneously with 100Schistosoma haematobium cercariae, 1 and 3 weeks after initial infection with 100S. mansoni cercariae. Results indicate that there was a higher degree of resistance to superinfection withS. haematobium at 1 week following initial infection withS. mansoni than that produced in the other two superinfections. This resistance was evidenced by a reduction in the number and size of worms of both species, decrease inS. haematobium egg extrusion per female and by a striking deviation in the egg distribution pattern of both species. Such an early host resistance was not recorded in previous works. Cross-mating was observed but no hybridization took place and the eggs produced were hatchable and typical of their species.     

Schistosomal cholecystitis.

A 51-year-old man with schistosomiasis had symptoms of cholecystitis due to Schistosoma mansoni eggs found in all layers of the gallbladder associated with all stages of inflammatory granulomatous reaction. Adult worms were found in a subserosal venule also. Why parasitism of the gallbladder by S mansoni eggs is apparently rare is not known.     

Comparative antigen analysis of different life stages of Schistosoma mansoni by crossed immunoelectrophoresis

Two-dimensional (crossed) immunoelectrophoresis was used for analysis of soluble antigen extracts obtained from the three developmental stages, cercariae, adult worms and eggs, of Schistosoma mansoni by using homologous hyperimmune sera produced in sheep. The antigenic relationships between the three stages as well as the possible relationship to the intermediate snail host were studied. Seven antigen components were shown to be shared between all three life stages of S. mansoni. Furthermore, one antigen was common to adult worm and snail, and one other antigen was shared between cercaria and snail. By using an intermediate gel containing lectin in the antigen-antibody system or by enzyme staining of the immune precipitates it was possible to identify schistosome antigens possessing lectin reactivity or enzyme activity. Characterization of enzyme activities revealed three individual precipitating antigens in adult worm of S. mansoni possessing esterase, leucyl-glycyl-glycine peptidase and phenylalanyl-leucine peptidase activities, respectively. One further precipitinogen with malate dehydrogenase activity was identified for all three life stages.     

Role of IgE in primary murine Schistosomiasis mansoni

Schistosoma mansoni infection proceeds in normal mice in the absence of detectable levels of polyclonal or specific immunoglobulin (Ig)E until worms mature and deposit eggs. Hence, the course of a primary S. mansoni infection is not expected to vary appreciably in mice with defects in the IgE production. Experimental increase of IgE production early after infection may, however, influence worm development. In the first approach towards this goal, BALB/c mice were injected with interleukin(IL)4 to raise the level of endogenously synthesized IgE. A significant increase in serum polyclonal IgE and antischistosome IgG1 during the prepatent period was not associated with significant changes in worm and egg burden or liver pathology. During the second approach, mice were injected with IgE which was affinity purified from serum of BALB/c mice infected for 16 weeks with S. mansoni. The purified IgE bound to carbohydrate-independent epitopes of soluble antigens from 3 h larvae, adult worms and eggs and recognized the schistosomular surface membrane. No differences in worm and egg load or granuloma number and size were noted between untreated and exogenous IgE-injected mice. Together, the data demonstrate that by itself IgE does not influence the outcome of infection in primary murine S. mansoni.     

Possible eggshell protein gene from Schistosoma mansoni

We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products.     

Effect of aestivation of Biomphalaria pfeifferi on the survival and infectivity of Schistosoma mansoni cercariae

Schistosoma mansoni cercariae from post-aestivated Biomphalaria pfeifferi remain motile for 20 hours after release. Thereafter, their activity decreases with age. The difference in mortality rate of cercariae from aestivated and non-aestivated B. pfeifferi studied here proved to be statistically significant (P < 0.05) within the first 10 hours of the experimental period. Results of the percentage recovery of worms from different mouse organs infected with cercariae from aestivated and non-aestivated snails varied. The two main organs infected were the liver and intestine. In conclusion, the penetration, migration and maturation of cercariae into adult worms were not affected by the aestivation of B. pfeifferi.     

Stage and tissue specific differences in SjBMI1, a Polycomb protein in Schistosoma japonicum

Polycomb group protein BMI1, plays a central role in the stem cell pluripotency and development in metazoans. A gene encoding BMI1 homologue in the Schistosoma japonicum (SjBMI1) was cloned and identified. The deduced amino acid sequence shows high identity to the homologues from Schistosoma mansoni and Homo sapiens. Quantitative real time polymerase chain reaction (RT-PCR) and Western blot analysis revealed that the SjBMI1 is highly expressed in adult worms and eggs, not in cercariae. By immunofluorescent studies, SjBMI1 was localized to testes, ovaries of mixed sex infected adult worms, but not of single sex infected adult worms. The study reveals the SjBMI1 expression profile in developmental stages and localization characteristic and provides a clue that it may be associated with reproductive development of S. japonicum.     

Kinetics and isotype distribution of parasite-specific antibody responses in the spleen of mice during primary infection with Schistosoma mansoni. Studies at the single-cell level

The solid-phase enzyme-linked immunospot assay has been adapted for enumerating cells secreting antibodies to crude antigenic extracts from the main developmental stages of Schistosoma mansoni. The frequencies of splenocytes secreting antibodies reactive with soluble antigenic extracts from cercariae, adult worms, and eggs, were examined in C57BL/6 mice during the first 12 weeks of a primary infection with S. mansoni. Despite the existence of cross-reactive moieties present in these antigenic preparations, the characteristics of the responses monitored differed with regard to kinetics, magnitude, and/or isotype distribution. The responses peaked between 8 and 10 weeks of infection, were characterized by a predominance of cells secreting IgM antibodies against all three antigens, and followed the patterns IgM greater than IgA greater than IgG for cercariae, IgM greater than IgG greater than IgA for adult worms and IgM greater than IgG greater than IgA for egg extracts. On the other hand, these patterns were not always reflected in serum, especially for cercariae-reactive circulating IgG and IgA responses. When examined for numbers of total IgM-, IgG- and IgA-producing cells, the spleen of S. mansoni-infected mice was shown to display considerable numbers of immunoglobulin-producing cells in all three isotypes studied. The most spectacular increases were noted for IgG-secreting cells (more than 10-fold) and for IgM-secreting cells (up to 6-fold) 4 weeks after initial cercarial exposure. Taken together, these observations indicate that primary infection with S. mansoni results in early immunoregulatory alterations which may contribute to the maintenance of specific as well as nonspecific B cell hyperactivity.(ABSTRACT TRUNCATED AT 250 WORDS)