Schistosoma mansoni: antigen preparations which induce antibodies to schistosomula surface antigens

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.     

Schistosoma mansoni: expression and role of cysteine proteinases in developing schistosomula

The adult stage of Schistosoma mansoni utilizes host hemoglobin as a nutrient source. A proteolytic enzyme (SMw32) that has "hemoglobinase" activity is secreted into the parasite gut where it appears to be rapidly activated by glutathione released from host red blood cells. In the present study the expression of this proteinase, in developing schistosomula, has been correlated with digestive tract development and a dramatic rise in enzyme activity as early as Days 8-10 of culture. No evidence of the SMw32 proteinase was found in eggs, cercariae, or in newly transformed larvae. Further, the proteinase expressed at Days 8-10 is indistinguishable from the adult worm enzyme. In the larvae, indirect immunofluorescence with an anti-SMw32 monoclonal antibody showed that the proteinase is found throughout the developing cecum. The importance of cysteine proteinases to parasite development was also studied using a specific enzyme inhibitor, Ep-459. In cultures containing Ep-459 most (75%) of the schistosomula failed to survive the 18-day study period. Moreover, those that did survive showed a decrease in their growth (body length). These data suggest that the SMw32 proteinase is a developmentally regulated enzyme and that cysteine proteinase activity is essential in providing nutrients for the growth and survival of this parasite in its mammalian host. Thus, this proteinase may be an important target for chemotherapeutic intervention.     

Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Depletion of Schistosoma mansoni lung-stage schistosomula cholesterol by methyl-beta-cyclodextrin dramatically increases specific antibody binding to surface membrane antigens

Schistosoma mansoni lung-stage larvae appear to not bind antibodies from radiation vaccine or infection sera in the membrane immunofluorescence test. However, treatment of ex vivo lung-stage schistosomula with methyl-beta-cyclodextrin, a hydrophobic oligosaccharide that specifically extracts cholesterol from plasma membranes, induced readily detectable binding of specific antibodies in a concentration- and time-dependent manner. Surface membrane antigen binding of specific antibodies was also conclusively demonstrated by quantitative absorption of anti-schistosome sera with intact ex vivo larvae. These data together suggest that confinement of lung-stage schistosomula surface membrane antigens in cholesterol-rich sites allows only monovalent antibody binding, which can be detected by absorption and not by direct serology.     

Expression of lectin-binding surface glycoproteins during the development of Schistosoma mansoni schistosomula

Tegumental glycoproteins of Schistosoma mansoni cercariae, mechanically produced 24-hr and 48-hr schistosomula, and adult worms were radioiodinated with the Bolton-Hunter reagent, then isolated by lectin affinity chromatography. SDS-PAGE revealed Con A binding glycoproteins with apparent molecular weights of 180,000, 150,000, 43,000, and 30,000 in detergent extracts of the tegument of cercariae. These glycoproteins are retained by 24-hr mechanically produced, cultured schistosomula and are accompanied by the appearance of 2 additional labeled glycoproteins, mol. wt. 66,000 and 57,000. In 48-hr schistosomula, there is a marked increase in the relative size of the 66,000 mol. wt. peak. In contrast, the 57,000 mol. wt. glycoprotein is the major radiolabeled Con A binding component of the adult tegument; the other peaks are either reduced or absent in adults. Similar findings were obtained following affinity chromatography using immobilized Lens culinaris lectin or Ricinus communis agglutinin, and following metabolic labeling of glycoproteins with tritiated galactose.     

Cell-free synthesis of Schistosoma mansoni surface antigens: stage specificity of their expression

Messenger RNA has been extracted from all stages of the life cycle of the parasitic multicellular helminth Schistosoma mansoni. In vitro translation of these mRNA preparations in rabbit reticulocyte lysates yielded in each case a large number of polypeptides. Immunoprecipitation of translation products either by serum from immune mice or from human patients demonstrated that relatively few, approximately 10, polypeptides are recognised as antigens. Two of the in vitro synthesised antigens, of mol. wts. 22 000 and 14 000, were demonstrated to correspond to schistosomula surface antigens. The expression of these antigens may show stage specificity. Both are readily detected from adult and sporocyst translation products, neither from schistosomula and only the 22 000 antigen from miracidia. This is an unexpected finding since similar polypeptide antigens occur on the surface of schistosomula. These results indicate that not only are schistosomula surface antigens preformed at the preceding sporocyst stage, i.e., within the snail host, but they also remain invariant throughout the life cycle in the vertebrate host. Two other prominent schistosomula surface antigens of mol. wts. 38 000 and 32 000, were not recognised amongst cell-free translation products directed by RNA from any life cycle stage. The demonstration that at least two schistosomula surface antigens are detectable amongst adult mRNA cell-free translation products demonstrates the feasibility of identifying the genes encoding them in cDNA libraries from adult worm mRNA.     

A monoclonal antibody raised against adult Schistosoma mansoni which recognizes a surface antigen on schistosomula

A monoclonal antibody has been raised by immunizing a mouse with an isolated tegumental preparation of adult Schistosoma mansoni. The hybridoma designated NIMP/M.47, secreted an IgG2a antibody which was positive by indirect immunofluorescence with live schistosomula of S. mansoni, but not with live schistosomula of S. bovis, or with other living life cycle stages of S. mansoni. In complement-dependent, or cell-mediated in vitro cytotoxicity assays, the monoclonal antibody mediated levels of schistosomular killing as high as those obtained with sera from infected mice. No significant protection, however, was obtained in passive transfer experiments. NIMP/M47 was specific for a 20,000 dalton polypeptide in the schistosomular surface, which was also recognized by serum from infected mice.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis

Schistosomes are blood-dwelling flukes that infect 200 million people worldwide and are responsible for hundreds of thousands of deaths annually. Using a signal sequence trap, we cloned from Schistosoma mansoni two cDNAs, Sm-tsp-1 and Sm-tsp-2, encoding the tetraspanin (TSP) integral membrane proteins TSP-1 and TSP-2. We raised antibodies to recombinant TSP fusion proteins and showed that both proteins are exposed on the surface of S. mansoni. Recombinant TSP-2, but not TSP-1, is strongly recognized by IgG1 and IgG3 (but not IgE) from naturally resistant individuals but is not recognized by IgG from chronically infected or unexposed individuals. Vaccination of mice with the recombinant proteins followed by challenge infection with S. mansoni resulted in reductions of 57% and 64% (TSP-2) and 34% and 52% (TSP-1) for mean adult worm burdens and liver egg burdens, respectively, over two independent trials. Fecal egg counts were reduced by 65-69% in both test groups. TSP-2 in particular provided protection in excess of the 40% benchmark set by the World Health Organization for progression of schistosome vaccine antigens into clinical trials. When coupled with its selective recognition by naturally resistant people, TSP-2 seems to be an effective vaccine antigen against S. mansoni.     

Incubation of Schistosoma mansoni lung-stage schistosomula in corn oil exposes their surface membrane antigenic specificities

Several potential cues for increased surface antigenic expression of lung-stage schistosomula, such as lack of glucose and amino acids and extremes of pH or HCO3- concentration, failed to alter the negligible larval reactivity with control, infection, or irradiated cercariae-vaccine serum in indirect membrane immunofluorescence. In contrast, incubation of larvae in 90% corn oil for 6 hr led to surface membrane changes, which allowed specific and strong binding of antibody from antischistosome sera. The data together indicated that the lung-stage worms' confinement of antigenic molecules in lipid-rich sites of the outer membrane could be reversed in vitro after exposure to corn oil, in a concentration- and time-dependent manner.     

Schistosoma mansoni: long-term culture of in vitro derived schistosomula

In vitro derived schistosomula were cultured under various conditions. Variables tested included concentration of organisms, type of culture vessel, frequency of media change, time of erythrocyte addition, antibiotic levels, heated vs unheated serum in the media, and fresh vs stored serum or erythrocytes. No differences were observed between cultures changed every 3 or every 7 days, but worm growth and development were retarded when the culture medium was changed every 2 weeks. Organisms cultured without changing the medium for 3 weeks did not survive. High levels of antibiotics also inhibited growth and resulted in increased mortality. None of the other factors tested resulted in remarkable differences between groups. Some cultures lived for as long as 69 days, and pairing of adult worms was observed as early as 55 days. Most of the cultures, however, were lost before that time from the outgrowth of contaminants which were probably present in the cultures from the outset.     

The effects of gamma-irradiation on migration and survival of Schistosoma mansoni schistosomula in mice

Infections with irradiated Schistosoma mansoni were established by intramuscular (i.m.) injection of mechanically transformed schistosomula. A dose of 2.3 krad. allowed persistence of a small proportion of worms to adulthood, and of these survivors the majority of the female worms were sexually sterile. However, a small proportion of 2.3 krad.-irradiated females and a larger proportion of similarly irradiated males were capable of pairing successfully with non-irradiated partners. Radiation in the range 2.3 to 10 krad. resulted in slightly reduced peak recoveries from the lungs while 20 krad. resulted in a much reduced and 40 krad. a virtual absence of survival to the lung stage. Increasing doses of radiation in the range 2.3 to 10 krad. resulted in successively fewer parasites reaching the liver. Thus, the major sites of the radiation-induced mortality appeared to be as follows: 2.3 krad., mainly in the liver; 4 krad., in the lungs and liver; 10 krad., mainly in the lungs; 20 krad., at the injection site and in the lungs and 40 krad., mainly at the injection site. The infections studied here showed reduced survival following exposure to high doses of radiation compared with the infections, established as percutaneously applied cercariae, which have been reported by other workers. Possible reasons for the disparity are discussed.     

Characterization of human low density lipoprotein binding proteins on the surface of schistosomula of Schistosoma mansoni.

Low density lipoproteins (LDL) bound to the surface of Schistosoma mansoni may protect the parasite from assault by the immune system and provide essential lipids for the parasite in human schistosomiasis. Here we have characterized the LDL binding sites on the surface of schistosomula by comparing the binding of fluorescently labeled LDL to the parasite with LDL binding proteins as seen by ligand blotting before and after enzymatic treatment of viable parasites. Ligand blotting revealed two LDL binding bands, 17.8 +/- 0.8 and 15.7 +/- 0.6 kDa, in intact schistosomula. Trypsinization eliminated all of the specific and approximately two-thirds of the total LDL binding capacity of schistosomula in a time and concentration-dependent manner. LDL did not bind to any bands on blots of trypsinized, viable worms. Specific LDL binding was also eliminated by phosphatidylinositol-specific phospholipase C (PIPLC). PIPLC treatment removed both LDL binding bands from the worms and caused the appearance of an LDL binding band, 16.6 +/- 0.3 kDa, in the culture medium. LDL binding to the parasite recovered within 24 to 48 h after trypsinization but the recovery was inhibited by either monensin or puromycin. Both LDL binding bands reappeared in ligand blots of cultured worms within 24 h; the reappearance was blocked by puromycin but not by monensin. These studies suggest that the specific binding of human LDL to schistosomula is mediated by GPI-linked low molecular weight proteins that are continually synthesized and transported to the parasite surface.     

Schistosoma mansoni: exposure in vitro of adult male surface antigens

Incubation of adult male Schistosoma mansoni for 24 hr in medium containing newborn calf serum or normal human plasma resulted in an increase in the amount of parasite antigen exposed at the worm surface. No effect was observed on the amount of host antigen which was present. The increase in the exposure of parasite antigens takes place progressively over 24 hr and is partially dependent on the presence of lipoproteins in the culture medium. The possibility is discussed that the increase is due to environmentally induced changes in surface membrane lipid composition.     

Human neutrophils endocytose multivalent ligands from the surface of schistosomula of Schistosoma mansoni before membrane fusion

Human buffy coat cells adhering to schistosomula of Schistosoma mansoni that were preincubated in fluorochrome-conjugated concanavalin A (Con A), wheat germ agglutinin, lentil lectin, or purified IgG from a hyperimmunized rabbit, were examined by fluorescence and transmission electron microscopy and by freeze-fracture. All four fluorochrome- conjugated multivalent ligands were homogeneously distributed on the parasite surface after preincubation. Within 1-3 h after the addition of cells, large areas of nonfluorescence, 10-20 micrometer in diameter, were seen on the parasite surface. In addition, the fluorochromes were observed in granules within the cells. Electron microscope autoradiography of worms preincubated with 125I-Con A showed silver grains evenly distributed over the tegumental membrane. After the addition of cells, grains were seen over phagolysosomes in the cytoplasm of neutrophils adhering to the parasites. In addition, no grains were present over large areas of the tegumental membrane, which still retained its normal architecture, or over fusions between the neutrophil plasma membrane and the outer tegumental membrane. Rabbit IgG formed an electron-dense layer on the tegumental membrane which was endocytosed by neutrophils. Both neutrophils and eosinophils fused with the parasite in areas containing no electron-dense material on the surface. It is concluded that human neutrophils will endocytose a variety of multivalent ligands from the surface of schistosomula, which probably accounts for the failure of neutrophils to kill the parasite and acts to clear the parasite surface of both antigen and antibody. Presumably, the components of the parasite surface which have originally bound the ligands are also endocytosed since surface components labeled by galactose oxidase and NaB3H4 are taken into cells when examined by light microscope autoradiography. Finally, membrane fusion occurs in areas devoid of multivalent glands, which suggests that these ligands serve to bring the cells and parasites close together, but the actual fusigens probably reside in the lipids in the outer tegumental membrane.