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.     

Schistosoma mansoni: surface membrane isolation by polycationic beads

The Schistosoma mansoni surface membrane complex was isolated by binding polycationic beads to the worm surface in a sucrose- or sorbitol-acetate buffer, pH 5.0, at 4 C. The ratio of incorporation [3H]cholesterol/[14C]arachidonic acid was measured as well as the specific activities of the alkaline phosphatase (EC 3.1.3.1), Type I phosphodiesterase (EC 3.1.4.1), and Ca2+-adenosine triphosphatase (EC 3.6.1.3). The results indicated that membranes isolated on beads were of comparable or greater purity than membranes isolated by sucrose gradient centrifugation. The isolation procedure was rapid (30 min) and produced membrane fractions whose cytoplasmic surfaces were probably exposed.     

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.     

The release of membrane antigens into culture by adult Schistosoma mansoni.

Antigens sharing determinants with surface membranes and soluble proteins of adult Schistosoma mansoni have been detected in culture media after incubation of radioactively labelled worms. The relative quantities of these antigens were measured with specific antisera raised in rabbits and with serum from an immune rhesus monkey. It was found that 12-16% of TCA-precipitable radioactivity in the culture medium consisted of membrane antigens and 6-8% consisted of antigens sharing determinants with proteins found in the soluble fraction of adult worms. Over half the membrane antigens were present in particulate form, while other antigens were present in solution. Surface labelling the adult worms with [125I]confirmed that some of the particles in the culture medium were derived from the surface membrane of the adult worm and electron microscope examination of such particles showed that large membrane fragments were present. These results support the hypothesis that antibodies against schistosome membrane antigens are induced by particulate membrane antigens released by the parasite.     

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: 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 erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aK _m value of 2.56 · 10^–4 m and an apparentV _max of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface. Abbreviations: PLC, phospholipase C; PIPLC, phospholipase C acting selectively on phosphatidylinositol; NeuAc,N-acetylneuraminic acid; MU, 4-methylumbelliferone; PBS, Dulbecco's phosphate buffer saline solution. Gangliosides were coded according to Svennerholm [42] and the IUPAC-IUB recommendations [43].     

Schistosoma mansoni: surface membrane stability in vitro and in vivo

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.     

The lateral diffusion of lipid probes in the surface membrane of Schistosoma mansoni

The technique of fluorescence recovery after photobleaching was used to measure the lateral diffusion of fluorescent lipid analogues in the surface membrane of Schistosoma mansoni. Our data reveal that although some lipids could diffuse freely others exhibited restricted lateral diffusion. Quenching of lipid fluorescence by a non-permeant quencher, trypan blue, showed that there was an asymmetric distribution of lipids across the double bilayer of mature parasites. Those lipids that diffused freely were found to reside mainly in the external monolayer of the outer membrane whereas lipids with restricted lateral diffusion were located mainly in one or more of the monolayers beneath the external monolayer. Formation of surface membrane blebs allowed us to measure the lateral diffusion of lipids in the membrane without the influence of underlying cytoskeletal structures. The restricted diffusion found on the normal surface membrane of mature parasites was found to be released in membrane blebs. Quenching of fluorescent lipids on blebs indicated that all probes were present almost entirely in the external monolayer. Juvenile worms exhibited lower lateral diffusion coefficients than mature parasites: in addition, the lipids partitioned into the external monolayer. The results are discussed in terms of membrane organization, cytoskeletal contacts, and biological significance.     

Pulmonary leukocytic responses are linked to the acquired immunity of mice vaccinated with irradiated cercariae of Schistosoma mansoni

Pulmonary cellular responses in C57BL/6 mice exposed to Schistosoma mansoni have been investigated by sampling cells from the respiratory airways with bronchoalveolar lavage. Mice exposed to cercariae attenuated with 20 krad gamma-radiation developed stronger and more persistent pulmonary leukocytic responses than animals exposed to equal numbers of normal parasites. Although vaccination with irradiated cercariae also stimulated T cell responses of greater magnitude and duration than normal infection, the lymphocytic infiltrate elicited by each regimen did not differ substantially in its composition, 5 wk after exposure. Studies with cercariae attenuated by different treatments established that a link exists between the recruitment of leukocytes to the lungs of vaccinated mice and resistance to reinfection. There was a strong association between pulmonary leukocytic responses and the elimination of challenge infections by vaccinated mice. Animals exposed to irradiated cercariae of S. mansoni were resistant to homologous challenge infection but were not protected against Schistosoma margrebowiei. Homologous challenge of vaccinated mice stimulated anamnestic leukocytic and T lymphocytic responses in the lungs, 2 wk postinfection, but exposure of immunized animals to the heterologous species failed to trigger an expansion in these populations of cells. Our studies indicate that pulmonary leukocytes and T lymphocytes are intimately involved in the mechanism of vaccine-induced resistance to S. mansoni. It remains unclear whether these populations of cells initiate protective inflammatory reactions against challenge parasites in the lungs, or accumulate in response to the activation of the protective mechanism by other means.     

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.     

KCl-Extractable surface antigens of Schistosoma mansoni: immunological characterization and applicability in immunodiagnosis

A Schistosoma mansoni antigen preparation was obtained by extraction of adult worms with a 3 M KCl solution. An indirect immunofluorescence reaction on cryostat sections of adult worms showed that the extracted antigens mainly originated from the tegument. The complex antigenic composition of the tegument extract was shown by immunoelectrophoresis against serum from infected mice and immunized rabbits, which gave up to 9 and 17 precipitation lines, respectively. When we compared the use of adult worm antigens and the tegument antigen preparation in the DASS and ELISA tests for immunodiagnosis of human schistosomiasis, the average sensitivity of the tests with the two preparations was about equal, although considerable differences between individual sera occurred. Analysis of tegument antigens, fractionated by gel filtration, showed that the main serological activity of the tegument antigen preparation was due to high molecular weight antigens.     

Schistosoma mansoni: immediate hypersensitivity to adult antigens in the rat

Antigen fractions from adult S. mansoni, obtained from infected mice, were isolated by a variety of methods. A readily soluble fraction was obtained in good yield by freezing and thawing the schistosomes, while the less soluble residue was fractionated by the use of a number of the methods currently used for the extraction of tissue and cell surface antigens. The dialyzed, centrifuged products were characterized by acrylamide gel disc electrophoresis methods, agar gel precipitin reactions with antisera from rabbits immunized with whole schistosome homogenate, and by Prausnitz-Kustner (P-K) assay with sera from schistosome infected rats. The pattern of P-K reactivity suggested that there were a number of different antigen specificities involved in the reaginic antibody response to schistosome infection in rats. With repeated infection and increased duration of infection, more different antigens seemed to be involved in the reagin response. The schistosome antigen fraction obtained by freezing and thawing was especially reactive with both early infection rat sera and sera from multiply infected rats. Both the soluble fraction isolated by freezing and thawing and residue solubilized materials were found to be able to induce the formation of reagin antibodies on immunization with alum and B. pertussis vaccine.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

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.     

Severe hepatic fibrosis in Schistosoma mansoni infection is controlled by a major locus that is closely linked to the interferon-gamma receptor gene.

Lethal disease due to hepatic periportal fibrosis occurs in 2%-10% of subjects infected by Schistosoma mansoni in endemic regions such as Sudan. It is unknown why few infected individuals present with severe disease, and inherited factors may play a role in fibrosis development. Schistosoma mansoni infection levels have been shown to be controlled by a locus that maps to chromosome 5q31-q33. To investigate the genetic control of severe hepatic fibrosis (assessed by ultrasound examination) causing portal hypertension, a segregation analysis was performed in 65 Sudanese pedigrees from the same village. Results provide evidence for a codominant major gene, with.16 as the estimated allele A frequency predisposing to advanced periportal fibrosis. For AA males, AA females, and Aa males a 50% penetrance is reached after, respectively, 9, 14, and 19 years of residency in the area, whereas for other subjects the penetrance remains <.02 after 20 years of exposure. Linkage analysis performed in four candidate regions shows that this major locus maps to chromosome 6q22-q23 and that it is closely linked (multipoint LOD score 3.12) to the IFN-gammaR1 gene encoding the receptor of the strongly antifibrogenic cytokine interferon-gamma. These results show that infection levels and advanced hepatic fibrosis in human schistosomiasis are controlled by distinct loci; they suggest that polymorphisms within the IFN-gammaR1 gene could determine severe hepatic disease due to S. mansoni infection and that the IFN-gammaR1 gene is a strong candidate for the control of abnormal fibrosis observed in other diseases.     

Genetic differences in BCG-induced resistance to Schistosoma mansoni are not controlled by genes within the major histocompatibility complex of the mouse.

Induction of nonspecific resistance to Schistosoma mansoni infection after the i.v. injection of viable BCG was investigated in outbred mice and a panel of inbred and H-2 congenic strains. Significant protection was induced in CF1, A/J, C57BL/6, C57BL/10, DBA/2, C57BR, and SJL mice. BALB/c mice were not protected whereas CBA and C3H mice expressed intermediate degrees of protection. Expression of the protective phenomenon is not controlled by genes within the MHC as shown by the marked differences in response between BALB/c and DBA/2 (H-2d) as well as between C57BR and C3H (H-2k) mice. H-2 congenic strains with C57BL/10 background (B10.A and B10.D2) were high responders. BALB.B10 mice carrying the high responder (B10) MHC on the nonresponder (BALB/c) background were not protected. The degree of splenic hypertrophy did not correlate with the expression of nonspecific resistance. These results demonstrate that, in addition to controlling specific immune responses, genetic differences influence the nonspecific protective phenomena related to BCG administration as well.