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.     

The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni

Five low m.w. polypeptide antigens are expressed on the surface of freshly transformed schistosomula of Schistosoma mansoni, and were reproducibly identified by surface labeling with 125I by using IODOGEN and immunoprecipitating with immune mouse sera. These molecules have approximate m.w. of 38,000, 32,000, 20,000, 17,000, and 15,000. They correspond to antigens recognized previously by lactoperoxidase-catalyzed iodination. Analysis of the surface of developing schistosomulum demonstrated that the 38,000 and 17,000 dalton antigens were lost from the parasite surface during 48 hr of in vitro culture. This process was not dependent on the presence of host serum. The two antigens were not lost due to shedding into the culture medium but were apparently sequestered to a site where they were no longer available for surface labeling. The 32,000, 20,000, and 15,000 dalton antigens, however, remained exposed on the schistosomulum surface for up to 2 days of in vitro culture. The expression of two new antigens was also induced by culture in vitro: a doublet of approximately 45,000 daltons and an antigen of approximately 11,000 daltons. The expression of the former was dependent on the presence of serum. These results demonstrate that the development of the schistosomula surface is a complex process, with events both dependent and independent of the presence of serum. In addition, the expression of polypeptide antigens is not coordinated, and antigens are lost, retained, or appear on the schistosomulum surface during the early stages of maturation.     

Solubilization of antigens of Fasciola hepatica which react with antibodies to Schistosoma mansoni.

Extracts of Fasciola hepatica adult worms contain antigens reactive with antisera prepared against Schistosoma mansoni adult worms. These antigens are poorly solubilized when homogenized in a phosphate buffered saline (PBS) solution and pellet readily when subjected to high speed centrifugation. Solubilization is improved greatly by the addition of sodium dodecyl sulfate (0.03%) to the PBS. When this is done, one obtains approximately the same total amount of crude Lowry reactive material as with PBS extraction followed by high speed centrifugation but antigenic reactivity to an anti-S. mansoni antiserum increases enormously. The antigens liberated from F. hepatica SDS extraction are largely materials under 200,000 MW and over 60,000 MW.     

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.     

Failure of anti-Fasciola antisera to induce antibody-dependent, eosinophil-mediated killing of Schistosoma mansoni schistosomula

Sera from rabbits or humans infected with Fasciola hepatica were tested for their ability to kill Schistosoma mansoni schistosomula in an antibody-dependent, eosinophil-mediated in vitro assay. In addition, anti-F. hepatica antisera raised in rabbits or calves, including one to a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen, were also tested for their killing ability. None of these antisera induced damage to S. mansoni schistosomula in vitro, even when enhanced with mononuclear cell supernatants containing eosinophil-activating factor. Serum from humans with S. mansoni did induce schistosomulum killing in vitro when tested under these same conditions. These results suggest that the mechanism of immunity to schistosomes induced by Fasciola antigens at the level of the schistosomula is mediated by factors other than eosinophils.     

Schistosoma mansoni: in vitro conversion of cercariae to schistosomula.

Schistosoma mansoni cercariae were incubated for 3-5h at 37 degrees C in various test solutions, and the rate and extent of conversion of the cercariae to schistosomula were determined. Criteria used to identify schistosomula included: (1) loss of cercarial tail, (2) viability of organisms in saline but not in water, (3) pre-acetabular gland evacuation and (4) ability to survive in culture. Incubation of cercariae in rat chamber fluid resulted in organisms which were water sensitive, but retained their tails and pre-acetabular gland contents. Conversion to water sensitivity was not blocked by 0-01 M EDTA.     

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.     

Schistosoma mansoni: increasing saline concentration signals cercariae to transform to schistosomula

Cercariae of S. mansoni shed the surface glycocalyx, form a double lipid bilayer on their surface, and transform to schistosomula when tails are removed and parasites are transferred from pond water to 300 mOsm phosphate-buffered saline. To determine whether the absolute concentration of saline or the relative change in saline concentration was the signal for surface transformation, cercariae were isolated from the snail hepatopancreas, sheared to remove the tails, and incubated in defined media for 3 hr at 37 degrees C. Surface transformation was assayed using the binding of the fluorescein-conjugated lectin concanavalin A to the schistosomular double unit membrane but not to the cercarial glycocalyx. An increase in salinity either from 18 mOsm (artificial pond water) to 120 mOsm (the snail osmolarity) or from 120 to 300 mOsm (the mammalian osmolarity) triggered transformation to schistosomula. Organisms constantly exposed to 120 mOsm or shifted from 120 mOsm to pond water did not transform their surfaces. The signal for transformation appeared to be increasing salinity rather than increasing osmolarity because cercarial bodies did not become schistosomula in 300 mOsm mannitol. Surface transformation was inhibited when cercariae were incubated with the acetylcholinesterase inhibitor eserine sulfate during a 10 min time when the osmolarity was raised. We conclude that increasing salinity rather than the absolute saline concentration is the signal for surface transformation and that eserine sulfate may inhibit the receipt of this signal.     

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.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Schistosoma mansoni: protein phosphorylation during transformation of cercariae to schistosomula.

Infectivity of the multicellular pathogen Schistosoma mansoni for the human host is dependent upon the ability of free-living cercariae to transform rapidly into parasitic schistosomula. The biochemical pathways that regulate this transitional period are unknown. The role of protein phosphorylation was investigated by examining the incorporation of [32Pi]phosphate into proteins of S. mansoni. A sevenfold increase in total phosphorylation was found in 3-hr-old schistosomula as compared to cercariae. Analysis of radiolabeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that a 14-kDa protein served as a marker for transformation, being phosphorylated in schistosomula but not cercariae. The protein was phosphorylated on a serine residue. Phosphorylation was stimulated by a shift of parasites from water to salt-containing medium at 23 degrees C. Incubation of organisms in water at 37 degrees C did not initiate phosphorylation of this protein. The 14-kDa phosphoprotein was extracted from parasite homogenates with 1 M NaCl but was insoluble in 1% Triton X-100. Protein phosphorylation during the cercarial-schistosomula transformation may represent an important biochemical event that regulates infectivity of the parasite for the human host.     

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.     

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.     

Mast cell-mediated toxicity to schistosomula of Schistosoma mansoni: potentiation by exogenous peroxidase

Mast cells, when incubated in vitro with hydrogen peroxide (H2O2) and iodide, are cytotoxic to schistosomula of Schistosoma mansoni, as determined morphologically by dye exclusion, motility, and refractility and by transmission and scanning electron microscopy. When intact mast cells were incubated with schistosomula, mast cell degranulation with extracellular release of mast cell granules (MCG) was only observed in the presence of added H2O2 (10(-4) M). The secreted MCG, which contain small amounts of endogenous peroxidase activity, adhered to the surface of schistosomula. By 15 to 30 min, the mast cell-H2O2 system in the presence of iodide (10(-4) M) produced marked disruption of the tegumental and internal structures of the schistosomula. No helminthic damage was noted if any component of the incubation mixture (mast cells, H2O2 or iodide) was omitted. MCG could substitute for intact mast cells in the H2O2 and iodide-dependent cytotoxic system; MCG-mediated killing of schistosomula was inhibited by the hemeprotein inhibitor azide, suggesting that the cytotoxic reaction required endogenous peroxidase. The cytotoxicity was increased by eosinophil peroxidase bound to the MCG surface. These findings suggest a mechanism by which mast cells may contribute to the host cytotoxic response to helminths. H2O2 formed by nearby inflammatory cells may induce mast cell secretion, and the released MCG, through their endogenous peroxidase content (or bound eosinophil or neutrophil peroxidase), may react with H2O2 and a halide to form a system toxic to the adjacent helminth.     

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.