Attempts to manipulate specific responses to induce resistance to Schistosoma mansoni in Kenyan baboons (Papio anubis)

Attempts were made to manipulate specific responses of baboons to protect them from infection with Schistosoma mansoni. In Experiment 1, eosinophilia was induced in naive baboons with Trichinella spiralis larvae given intravenously before intraperitoneal injection of globulin fractions from S. mansoni-infected baboon sera and subsequent percutaneous exposure to S. mansoni cercariae. In Experiment 2, baboons with 8- or 32-week-old primary S. mansoni infections received T. spiralis i.v. before an S. mansoni challenge. In experiments 3 to 5 respectively, naive baboons received intramuscularly before challenge: formalin-fixed S. mansoni schistosomula, with Bordetella pertussis as an adjuvant; a preparation of S. mansoni adult worm teguments; and a preparation of IgE-immune complexes obtained from S. mansoni-infected rat sera, with Freunds Complete Adjuvant. Minor, but statistically insignificant, protection was obtained in Experiments 2 (32-week infections) and 3, but was far less than that given by intact, irradiated living vaccines. There are signs on the horizon of non-living vaccines protecting rodents against S. mansoni infection and it would be prudent, as with drugs, to test these in primates before proceeding to man. The results of our experiments, though essentially negative, should help the design of any future vaccine trials in primates.     

Schistosoma mansoni: control of hepatotoxicity and egg excretion by immune serum in infected immunosuppressed mice is schistosome species-specific, but not S. mansoni strain-specific.

Immunosuppressed mice infected with Schistosoma mansoni suffer from an acute hepatotoxicity reaction, and they fail to excrete as many parasite eggs as comparably infected immunologically intact control animals. The hepatotoxicity was shown here to be preventable, and egg excretion rates were enhanced, by transfer of serum from donors with chronic S. mansoni infections, but not by serum from donors with heterologous infections of Schistosoma haematobium, Schistosoma bovis, or Schistosoma japonicum. The effects of the transferred sera are considered to be due to specific antibody, but the possibility of cytokine involvement is discussed. A high degree of serological cross-reactivity was found between sera from mice infected with the different schistosome species and unfractionated egg homogenate (SEA) in ELISA. Cross-reactivity of the heterologous sera was, however, reduced against CEF6, a partially purified fraction of S. mansoni eggs that contains the putative hepatotoxin and has serodiagnostic potential. S. mansoni isolates from Puerto Rico, Brazil, Egypt, and Kenya shared similar characteristics with respect to the immune dependence of egg excretion and hepatotoxicity in immunosuppressed mice. The S. mansoni geographic isolates were also indistinguishable serologically, in terms of both the capacity of respective infection sera to neutralize hepatotoxicity and in their capacity to promote egg excretion of the other isolates in vivo. Complete immunological cross-reactivity of the geographically distinct isolates was also observed in ELISA with both CEF6 and SEA. Utilization of CEF6 for serodiagnosis of schistosomiasis mansoni is therefore unlikely to be restricted by geographical considerations.     

Characterization of proteins and immunogens released by adult Schistosoma mansoni

The proteins released in vitro by metabolically radiolabeled adult Schistosoma mansoni were identified by 2-dimensional gel electrophoresis. To determine the origin of these proteins, adult worms were fractionated into surface membrane, tegument, and remaining body components, and the electrophoretic patterns of the proteins in the 3 fractions were compared to those of the released proteins. The immunogens present in these fractions then were identified by immunoprecipitation with sera from humans infected with S. mansoni. This analysis indicated that essentially all of the proteins released from the worm were immunogenic, whereas most of the major membrane and tegumental proteins were not reactive with the immune sera. Thus, it appears that the adult worm is defended against immune attack by detection of the host's antibody response against released proteins rather than against proteins-exposed on the worm's surface.     

Schistosome antigen gp50 is responsible for serological cross-reactivity with Trichinella spiralis.

The 50-kDa component (gp50) present in Schistosoma mansoni eggs and secretions of the various life stages of the parasite was recognized by experimentally infected mice and by humans with S. mansoni, Schistosoma haematobium, and Schistosoma japonicum infection. All sera reacting with crude S. mansoni-soluble egg antigens (SEA) also reacted strongly with gp50 in enzyme-linked immunosorbent assay. No reactivity against gp50 was seen with sera from individuals without schistosomiasis, with the exception of sera from patients with Trichinella spiralis infection. All of 10 sera from patients with trichinellosis also reacted with schistosomes by immunofluorescence essentially recognizing testes, ovaries, ootype epithelium and ducts of the reproductive system. Cross-reacting antigens were seen in T. spiralis hypodermis, stichocytes and possibly germinal primordia using anti-gp50 monoclonal antibodies and anti-gp50-positive schistosomiasis patient sera. The results suggest that the anti-gp50 antibody response constitutes a significant part of the anti-SEA antibody response in infected individuals and is a major reason for the previously recognized serological cross-reactivity between T. spiralis and schistosome species.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Characterization of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni

To identify and characterize Schistosoma mansoni proteins that are recognized by infected hosts, we have used a pool of sera from infected humans to screen cDNA libraries constructed from poly(A)+ mRNA of adult S. mansoni. The deduced amino acid sequences of the three isolated clones showed a high degree of similarity to the large subunit of calcium-activated neutral proteinase (CANP) from humans and chicken. These overlapping clones, which include a nearly full-length clone with an open reading frame of 758 amino acid residues, together encode the entire large subunit of CANP. The deduced sequence of this S. mansoni protein can be divided into four domains (I-IV) that include the two domains characteristic of other large subunits of CANP: a thiol-protease domain (II) and a calcium-binding domain (IV) containing EF hand motifs. However, the schistosome protein is unique in having only three EF hand motifs in the calcium-binding domain and in having an additional EF hand motif that is shared between domains II and III. We have shown that these EF hand motifs are capable of binding 45Ca2+. Furthermore, the large subunit is S. mansoni contains an NH2-terminal sequence of 28 residues that is absent from the mammalian CANPs and has a high degree of similarity to the presumed receptor binding sequence of colicin Ia and Ib.     

Serological differentiation of acute and chronic schistosomiasis using Schistosoma mansoni recombinant protein RP26

We obtained a recombinant protein encoded by Schistosoma mansoni gene which was able to differentiate acute from chronic schistosomiasis when applied as antigen in enzyme-linked immunosorbent assay (ELISA). A cDNA clone encoding a 26 kDa recombinant protein (RP26) was selected by screening of an adult worm S. mansoni λZAP expression library with rabbit sera produced against PIII, an adult worm protein fraction already known to possess protective and immunomodulating effects. The clone cDNA presented 99% identity with S. mansoni Sm22.3 gene. We assayed IgG reactivity of sera from 18 patients with acute, 25 patients with chronic S. mansoni infection and 20 uninfected donors with RP26 in ELISA. Our results showed that 89% of sera were positive in acute schistosomiasis group, and only 26% in chronic group, without false-positive reactions in uninfected group. In mice the immune response to RP26 increased up to week 9 after infection and then diminished. We proposed that production of antibodies binding to RP26 stopped at the chronic stage of disease. The testing of sera from eight other parasitic infections with RP26 revealed no positive reactions in majority of sera. However, we observed low positive reaction in sera from 20% of leishmaniasis patients. Our results indicate that a recombinant protein RP26 can be used as immunodiagnostic reagent for detection of acute phase of schistosomiasis mansoni.     

Schistosoma mansoni adult microsomal antigens, a serologic reagent. II. Specificity of antibody responses to the S. mansoni microsomal antigen (MAMA)

The purified Schistosoma mansoni adult microsomal antigen, MAMA, was used in the quantitative single-tube kinetic dependent enzyme-linked immunosorbent assay (k-ELISA) to measure antibody levels of various human patient sera. The 511 serum specimens tested were from patients with both homologous and heterologous infections. Sera from U.S., Egyptian, Brazilian, and Puerto Rican patients infected with S. mansoni reacted strongly with MAMA. Chinese patients infected with S. japonicum, and Nigerians or Egyptians infected with S. haematobium produced much lower responses to this antigen than those infected with S. mansoni. Sera from patients with echinococcosis, filariasis, paragonimiasis, clonorchiasis, trichinosis, amebiasis, and hepatitis and from healthy uninfected control individuals generally contained no detectable antibodies against this antigen. The S. mansoni adult microsomal antigen, MAMA, therefore, appears to be a highly potent and specific reagent for the serodiagnosis of S. mansoni infections.     

Protective monoclonal antibody against Schistosoma mansoni: antigen isolation, characterization, and suitability for active immunization

Monoclonal antibodies that bind to the surface of developing schistosomula were generated from the spleens of chronically infected mice that were boosted with cercarial glycoproteins. The two most reactive monoclonal antibodies, denoted 152-66-9B and 152-66-1C, were used for identification of surface antigens. The antigen detected by these monoclonal antibodies persisted on the surface of the developing larva for 72 hr posttransformation. This monoclonal antibody effected complement-mediated killing of schistosomula in vitro as efficiently as infected mouse sera. It was also very efficient in inhibiting the infectivity of both cercariae and schistosomula. The antigen reactive with the 152-66-9B monoclonal antibody contains two major polypeptides (45 and 30 KD). These polypeptide chains might have originated from the same protein, because they have the same isoelectric point in two-dimensional gel electrophoresis. Moreover, the affinity-purified antigen migrated as only one protein band of approximately 200 KD in SDS-PAGE in nonreducing conditions. The 9B antigen was isolated, purified, and used for immunization, resulting in an antigen dose-dependent partial protection against S. mansoni infection.     

Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

The humoral immune responses of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one- and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test antigen extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with [35S] methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.     

Schistosoma mansoni, S. haematobium, and S. japonicum: identification of genus- and species-specific antigenic egg glycoproteins

Immunoreactive egg glycoproteins of Schistosoma mansoni, S. haematobium, and S. japonicum which are genus- and species-specific, or react with sera of patients infected with other parasites, have been identified. Egg proteins were labeled with Iodine-125, and the concanavalin A-binding glycoproteins were immunoprecipitated with sera of patients infected with one of four species of Schistosoma or Trichinella spiralis, Taenia solium, Echinococcus granulosus, Entamoeba histolytica, or Wuchereria bancrofti. These immunoprecipitates were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite the strikingly different patterns of glycoproteins of the African species, the antibody immune responses of patients infected with S. mansoni and S. haematobium were found to be so similar that differentiation could not be established. In contrast, sera of patients infected with S. japonicum, S. mekongi, or parasites not of the genus Schistosoma, immunoprecipitated fewer of the major S. mansoni or S. haematobium glycoproteins. Likewise, antibody immune responses of patients infected with the Oriental schistosomes (S. japonicum and S. mekongi) could not be differentiated. Only a few quantitative differences were noted between our S. mansoni egg glycoprotein extract and a standardized soluble egg antigen extract. This study provides an explanation for the extensive cross-reactivity observed in diagnostic assays which utilize various fractions of schistosomal egg extracts as the antigen.     

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.     

Evidence for host-induced selection in Schistosoma mansoni

A population of Schistosoma mansoni from Kenya was isolated in 1968 and subsequently passaged simultaneously through 2 different vertebrate hosts: baboons and mice. Recent electrophoretic studies demonstrated that genetic differences in the degree of polymorphism and in allele frequencies of polymorphic loci existed between S. mansoni populations from the 2 hosts. The present study was undertaken to assess the importance of vertebrate host-induced selection against particular alleles as mechanism to account for the observed differences. A population of S. mansoni which had originally been passaged through baboons and subsequently passaged through murine hosts for 4 generations was studied. At least 20 infected snails served as the source of parasite for each mouse passage. Allele frequencies of 4 polymorphic loci were assessed for each generation using horizontal starch gel electrophoresis. All 4 polymorphic loci (PGM-2, MDH-2, MDH-1, PGI) showed a selective trend towards allele frequencies identical with that of a strain (from the same isolate) maintained in mice for 12 yr. These data suggest that vertebrate host-induced selection results in a decrease in parasite variability due to loss of alleles as field isolates of S. mansoni are passaged in murine hosts. The use of non-human primate hosts, on the other hand, maintains a higher level of parasite variability.     

Schistosoma mansoni: immunoblot analysis of adult worm proteins

Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.     

Immune responses during human Schistosomiasis mansoni. XI. Immunologic status of patients with acute infections and after treatment

Sixteen patients, 8 to 30 yr of age, with acute (toxemic) phase schistosomiasis mansoni were studied immunologically within 2 to 3 mo of their exposure to Schistosoma mansoni cercariae, and were monitored after chemotherapy. Total leukocyte levels and peripheral blood eosinophilias were higher in these patients than in similar individuals with chronic schistosomiasis mansoni. In contrast to chronic patients, the eosinophilias of the acute cases were decreased rather than elevated upon treatment. Total lymphocyte population (T and B cell) percentages were not altered during acute infection. Lymphoid subset (T3+, T4+, and T8+) analysis revealed elevated levels of both T4+ and T8+ cells. In vitro blastogenic responses of peripheral blood mononuclear cells (PBMN) to heterogeneous schistosome-derived antigens (eggs, SEA; adult worms, AW; and cercariae, CERC) were evaluated. SEA responsiveness was considerably higher than that of patients with chronic S. mansoni infections. The ratios of SEA to AW responses in acute cases gave a mean of 2.0, as opposed to 0.5 for a comparable group of chronically infected patients. The sera of most acute patients already contained suppressive factors that specifically decreased schistosomal antigen-induced PBMN blastogenesis. Chemotherapy of acute cases lead to a diminution of PBMN responsiveness to SEA and CERC. Treatment of patients with chronic infections lead to the elevation of such responses. PBMN from patients with acute infections produced lymphokine leukocyte inhibition factor upon exposure of the cells to SEA but not AW. A similar pattern was true for production of the lymphokine activity mitogenic factor. Levels of antibody in sera of acutely infected patients against SEA, CERC, and AW were considerably higher than levels in sera of chronically infected patients matched for age and intensity of their infections. These high antibody titers persisted for at least 6 mo after treatment, and were unrelated to the intensity of infection. The immunologic status of these patients with acute schistosomiasis mansoni differed considerably from patients with chronic infections. These findings re-emphasize the immunoregulatory events that apparently develop upon continued exposure to schistosomes and their products during chronic infection.     

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.     

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.     

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.     

Cloning and gene expression of Schistosoma mansoni protease

Schistosomes utilize proteases (termed hemoglobinases) for degradation of host globin. cDNA clones encoding Schistosoma mansoni protease were isolated by immunologically screening an expression cDNA library with antisera raised against purified hemoglobinase. Confirmation of the identities of the clones was obtained immunologically and biochemically. The bacterially produced fusion protein encoded by one clone, lambda Hb2, degraded hemoglobin in vitro. The sequence of this clone suggested that this S. mansoni protease is synthesized in a precursor form in vivo. Gene titrations indicated that S. mansoni contains multiple genes corresponding to this cDNA. The expression of these genes may be regulated during the organism's life cycle since adult, female worms contained the highest abundances of homologous mRNA and protein compared to other stages.     

Diagnostic Mr31/32,000 Schistosoma mansoni proteins (Sm31/32): reaction with sera from Sudanese patients infected with S. mansoni or S. haematobium

Sera of Sudanese patients with active infections of Schistosoma mansoni or S. haematobium were tested in immunoblots for their reactivity with Mr31/32,000 proteins of adult S. mansoni (Sm31/32). All sera from patients with intestinal (n = 123) and all but one from those with urinary schistosomiasis (n = 35) had antibodies against Sm31/32. These and additional data suggest that both specificity and sensitivity of Sm31/32 to detect schistosome infections are close to 100%. Antibodies against these proteins developed also in monkeys after experimental infection with S. haematobium. Sm31/32 antigens reacted in immunoblots as a doublet with most S. haematobium-patient sera and as a broad band with many S. mansoni-sera suggesting that at least two components are present in the molecular weight region of Mr31/32,000. The data demonstrate the potential use of Sm31/32 from adult worms to diagnose patients with intestinal or urinary schistosomiasis in endemic areas.