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SUMMARYThe localization of the 28 kDaSchistosoma mansoniglutathione S-transferase (Sm28 GST) has been investigated using immunohistochemistry and electron microscopy and the results compared with previously published data. This study confirms the wide distribution of this antigen in the parasite. In male and female worms, Sm28 GST is localized in the tegument, the parenchyma, the oesophageal epithelium and in genital organs. Sm28 GST was clearly detected in germinal and sustentacular cells. The decrease of staining intensity during the differentiation of germinal cells suggests a down-regulated expression of the molecule. At the ultrastructural level, this antigen was abundant in nuclei and less present in the cytoplasm. The marked heterogeneity observed in the staining of individual worms indicates that Sm28 GST seems to be closely associated with the parasite’s metabolism. The results are discussed in relation to the biological and protective functions of the protein.

To characterize Th cell populations induced by helminth infection, spleen cells from mice infected with Schistosoma mansoni were stimulated with parasite (worm or egg Ag) or mitogen (Con A) and the supernatants assayed for the Th1-specific cytokines IFN-gamma and IL-2 and the Th2-specific cytokines IL-4 and IL-5. Th2 cytokine production was not detected in substantial quantity until the 6 to 8th wk of infection and after reaching peak levels at 8 to 12 wk declined slowly thereafter. The time courses of IL-4 and IL-5 production, whereas differing from each other, closely resembled corresponding published data on IgE and peripheral blood eosinophil levels during murine schistosome infection. In contrast, Th1 cytokine responses occurred only during the first 6 wk of infection and were virtually absent during the peak period of Th2 production. To assess the role of egg deposition in the observed pattern of Th response, cytokine production was assayed in mice carrying unisexual schistosome infections in which parasite eggs are absent. Splenocytes from these animals displayed only marginal Th2 cytokine synthesis but greater Th1 cytokine responses than the corresponding cells from mice with bisexual infections. Moreover, cultures of liver tissue or isolated granulomas from infected mice constitutively produced high levels of IL-4 and IL-5 but failed to synthesize significant amounts of IL-2 and IFN-gamma even when stimulated with egg Ag or mitogen. Taken together the data indicate that egg deposition is the major stimulus of Th2 cytokine response in S. mansoni-infected mice and suggest that T cells belonging to this subset must play a major role in egg granuloma formation.

In the mouse, infection with Schistosoma mansoni results in an egg-producing infection and associated disease, whereas vaccination with attenuated larval stages produces a substantial and specific immunity in the absence of egg-induced pathology. Preliminary data showing enhanced interleukin-5 (IL-5) production by T cells from infected mice and interferon gamma (IFN-gamma) synthesis by cells from vaccinated animals (7), suggested differential CD4+ subset stimulation by the different parasite stimuli. To confirm this hypothesis, lymphocytes from vaccinated or infected animals were compared for their ability to produce IFN-gamma and IL-2 (secreted by Th1 cells) as compared with IL-4 and IL-5 (characteristic Th2 cytokines). After stimulation with specific antigen or mitogen, T cells from vaccinated mice or prepatently infected animals responded primarily with Th1 lymphokines, whereas lymphocytes from patently infected mice instead produced Th2 cytokines. The Th2 response in infected animals was shown to be induced by schistosome eggs and directed largely against egg antigens, whereas the Th1 reactivity in vaccinated mice was triggered primarily by larval antigens. Interestingly, Th1 responses in mice carrying egg-producing infections were found to be profoundly downregulated. Moreover, the injection of eggs into vaccinated mice resulted in a reduction of antigen and mitogen-stimulated Th1 function accompanied by a coincident expression of Th2 responses. Together, the data suggest that coincident with the induction of Th2 responses, murine schistosome infection results in an inhibition of potentially protective Th1 function. This previously unrecognized downregulation of Th1 cytokine production may be an important immunological consequence of helminth infection related to host adaptation.

Monoclonal antibodies to the 28kDa glutathione S-transferase of Schistosoma bovis have been constructed in mice and used to characterize the epitope(s) potentially implied in the induction of anti-fecundity and anti-egg viability immune responses. Among the MoAbs produced three were particularly studied: Sb4-50 (IgG2a) and Sb4-56 (IgG1) which inhibited Sb28GST activity and Sb4-10 (IgG1) which did not. The use of overlapping peptides covering the entire amino acid sequence of Sb28GST, allowed us to define the linear epitopes recognized by these anti-Sb28GST MoAbs. Amino acid residues 202-211 were recognized by both MoAbs Sb4-50 and Sb4-56 and MoAb Sb4-10 recognized amino acid residues 58-67. Their capacity to inhibit GST activity suggested binding to the active site or to neighbouring regions, which include the C-terminal domain (a.a. 190-211) of the protein. When passively transferred into BALB/c mice MoAbs induced a significant reduction in egg hatching and an increase in immature eggs. Effects on worm burdens were, however, variable and no clear-cut association between the inhibition of enzyme activity and anti-fecundity or anti-viability activities was recorded. Our data indicate that beside the anti-fecundity and anti-viability immunity related to the impairment of GST activity, immune response to epitopes located in other regions of the molecule also contribute to the reduction of egg viability.

The T-helper dependency of the IgA antibody response has been investigated in rats injected intravenously with Schistosoma mansoni eggs. This method, allowing the trapping of parasite eggs in the lung tissue, led to a strong anti-egg IgA antibody response in the bronchoalveolar lavage but not in the serum. To characterize the cytokine pattern associated with the IgA response, kinetic analysis of the cytokine mRNA expression in the lungs, periaortic nodes (PN) and spleen was undertaken. Under such conditions, significant levels of mRNA encoding IL-5 and IL-10 were recorded in spleen during the early period following egg injection, as well as a more prolonged expression of TGF-beta and IL-6 mRNAs. However, neither IFN-gamma nor IL-4 mRNA could be detected in these samples. Finally, in lungs and in PN, RT-PCR analysis revealed delayed production of cytokine mRNA. Taken together our data suggest that the rat mucosal IgA antibody response is predominantly linked to a Th2 response.

The potential of a recombinant Schistosoma bovis-derived glutathione S-transferase (rSb28GST) to protect cattle against S. mattheei infection was tested in Zambia. All animals were challenged 2 weeks after the second inoculation with either 0·250 mg rSb28GST in adjuvants (vaccinated calves, n=14) or adjuvants alone (controls, n=14). In a first experiment, 7 vaccinated and 7 control animals were exposed to 10000 S. mattheei cercariae percutaneously. All animals developed clinical schistosomiasis 7–8 weeks after challenge. At perfusion, 12 weeks after challenge, vaccinated and control groups had averages of 887 and 541 eggs per gramme (epg) faeces, 6515 and 5990 worms, and 4·2 and 3·4 million tissue eggs, respectively. These results indicate that the immunization protocol used did not protect these calves against the massive single experimental challenge. In a second experiment, another 2 groups (n=7) of vaccinated and control animals were challenged naturally over a period of 9 months on a farm known to be endemic for S. mattheei. The natural infections were much lighter in intensity, as indicated by the mean faecal egg count (13 epg), worm count (139) and tissue egg count (294000) in non-vaccinated controls. In vaccinated calves, significant reductions in female worm burdens (50%), faecal egg counts (89%) and miracidial counts (93%) were recorded. Total tissue egg counts were also reduced by 42% in vaccinated animals. It therefore appears that the rSb28GST can provide significant protection in cattle against S. mattheei under conditions of low to moderate natural infection.

The specific IgA antibody responses to the protective recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST) Ag and to derived synthetic peptides have been evaluated before and 6 mo after chemotherapy in S. mansoni-infected patients from Kenya. These studies revealed a parallelism between the age-dependent evolution of IgA antibody levels to Sm28GST and to one synthetic peptide (115-131) and the acquisition of resistance to reinfection. Functional analysis revealed that IgA antibodies to Sm28GST displayed a potent neutralizing effect on the enzymatic properties of the molecule, and also markedly impaired schistosome fecundity, by limiting both the egg laying of mature worms and the hatching capacity of schistosome eggs into viable miracidia. These results suggest that, in addition to IgE, IgA antibodies might participate in the protective immune response against schistosomiasis.

The incidence of the X-linked immunodeficiency (Xid) on the outcome of Schistosoma mansoni infection has been evaluated through a comparative analysis of parasitological and immune parameters in two different mouse strains: control BALB/c and BALB. Xid mice which carry the Xid mutation and lack B1 (CD5+ B) cells. This study clearly demonstrates that infected B1 cell-deficient animals display a higher susceptibility to S. mansoni infection as revealed by an increase in the tissue egg loads and a significantly elevated mortality, as well as an increase in the granuloma densities. The analysis of the humoral and the cellular responses, conducted in the same experimental conditions, indicates differences in terms of cytokine production after specific antigenic stimulation of splenocytes. Larger amounts of IFN-gamma and IL-4 are observed in BALB. Xid mice while IL-10 production is reduced. In parallel, the study of the specific antibody isotype profiles shows higher amounts of specific IgE and IgG1 antibodies and lower amounts of IgM and IgA in BALB. Xid mice. Taken together, these observations support the idea that B cells are playing a role in the ability of mice to tolerate infection with Schistosoma mansoni.

After the demonstration of blocking antibodies during rat experimental schistosomiasis, the existence of such factors was investigated in human schistosomiasis. The depletion, in sera from S. mansoni-infected patients, of a given isotype (IgM) either by protein A-Sepharose (PAS) absorption or by fast protein liquid chromatography (FPLC) induced a significant increase in IgG-mediated killing of S. mansoni schistosomula by human eosinophils. Inhibition experiments showed that IgM-enriched fractions (PAS effluents) were able to inhibit eosinophil-dependent cytotoxicity mediated by IgG fractions (total sera or PAS eluates). Both IgG and IgM antibodies from infected human sera immunoprecipitated antigens of 30,000-40,000 Mr in the labeled detergent extracts of schistosomulum surface. The specificity of IgG and IgM for the 38,000 Mr antigen was suggested by competition experiments using two radiolabeled mAbs (IPLSm1, IPLSm3) directed against this antigen. Moreover, crossinhibition between IgG and IgM antibodies for the Mr 38,000 antigen could be directly demonstrated. The in vivo relevance of such IgM blocking antibodies in the context of human immunity to schistosomiasis was evaluated in two groups of children classified as resistant or susceptible to posttreatment reinfection. IgM antibodies specifically directed against the 38,000 Mr antigen were measured by a capture assay. The mean levels of IgM antibodies were significantly higher in the susceptible than in the resistant group both before and after treatment. These results are consistent with the idea that immunity to schistosomiasis could be attributable not only to the existence of antibodies with defined effector function, but also to the absence of blocking antibodies. The description of the existence in human schistosomiasis of antibody isotypes blocking the effector response against defined surface targets might lead to a new understanding of the mechanisms regulating immunity to reinfection against schistosomes and possibly other parasites.

Rat IgG2a monoclonal antibodies have been produced after fusion of spleen cells from LOU/C rats infected with S. mansoni for 5 wk and IR983F nonsecreting rat myeloma cells. The cell supernatants of one particular IgG2a-producing clone (IPL Sm1) as well as ascitic fluids induced by this clone revealed anti-S. mansoni activity detected by immunofluorescence on schistosoma sections. In vitro studies of the effector function of such antibodies revealed that the rat IgG2a monoclonal antibodies mediated high levels of rat eosinophil-dependent cytotoxic effect against S. mansoni schistosomula, similar to that obtained with 5-week infected rat serum. Passive transfer experiments carried out with IPL Sm1 ascitic fluid showed a significant level of passive protection against a challenge infection. These results indicate a possible use of the monoclonal antibodies in analyzing in vivo functions of IgG2a antibodies, as well as in isolating potentially protective target antigens.

Rat IgG2c monoclonal antibodies have been produced after fusion of spleen cells from LOU/C rats infected with Schistosoma mansoni for 5 wk and IRF983F nonsecreting rat myeloma. The cell supernatant of an IgG2c-producing clone (IPLSm3), as well as ascitic fluids induced by this clone, revealed anti-S. mansoni activity detected by immunofluorescence on schistosomula sections. Antigenic analysis performed with IPLSm3 IgG2c antibody allowed to isolate onto the S. mansoni schistosomula surface a 38,000 dalton antigen previously characterized with the protective IPLSm1 IgG2a monoclonal antibody. Although IPLSm3 IgG2c did not exhibit any killing activity in vitro against schistosomula in the presence of complement, macrophages, or eosinophils, it was shown to strongly inhibit the eosinophil-dependent cytotoxicity mediated by IPLSm1 IgG2a antibodies. The blocking activity of IgG2c antibody was further demonstrated in vitro by the use of F(ab')2 fragments and in vivo by the inhibition of passively transferred immunity conferred by the IgG2a protective monoclonal antibody. These results indicate that blocking antibodies could play an important role in the expression of protective immunity during schistosome infection.

We have purified a 28,000 dalton (P28) protein from Schistosoma mansoni adult worms and used it to immunize Fischer rats. Immunofluorescence assays demonstrated that the P28 antigen was mainly located in the parenchyma of the schistosomulum and of the adult worm, including the dorsal spines of the parasite. Western blot analysis revealed that this antigen was present in three species of schistosomes: S. mansoni, S. japonicum, and S. bovis. The antibody response raised against this protein was able to kill S. mansoni schistosomula in in vitro cytotoxicity assays in the presence of rat eosinophils. The inhibition of this cytotoxic activity by an aggregated myeloma IgG2a indicated that one of the major isotypes involved in this in vitro model is IgG2a. The passive transfer of P28 antisera induced a significant level of protection against experimental infection. Moreover, we have immunized Fischer rats and BALB/c mice with the purified 28,000 dalton protein and observed a marked decrease (up to 70%) in the parasite burden in both experimental infection models.

A rat IgE mAb specific for larval Ag (26 kDa, 56 kDa) has been shown to protect rats against Schistosoma mansoni infection. Immunizations of Lou/M rats performed with this IgE (Ab1) induced the production of antiidiotypic antibodies (Ab2). Moreover, after this Ab2 production, anti-antiidiotypic antibodies (Ab3) were revealed. The screening of Ab3 isotypes showed the presence of IgG Ab3 and more interestingly of IgE Ab3, i.e., the same isotype as Ab1. These IgE and IgG antibodies recognized predominantly the 26-kDa Ag and were cytotoxic for schistosomula in the presence of platelets for IgE Ab3 and eosinophils for IgG Ab3. Both IgE and IgG Ab3 conferred by passive transfer protective immunity to infected rats (up to 50%). Thus the immunization with an IgE mAb led in part to the production of Ab3 of the same isotype as Ab1. In conclusion, these results suggest that the isotype selection of the antibodies of the third generation (Ab3) might be influenced by the Ab1. The respective role of the idiotope and isotype of Ab1 in isotype regulation is discussed.

Over the past 35 yr, diethylcarbamazine (DEC) has been the most widely used agent for the treatment of filarial diseases, particularly in onchocerciasis. The microfilaricidal action of DEC has been recently shown to be mediated by blood platelets with the additional triggering of a filarial excretory Ag (FEA). This FEA could be detected by using mAb in the serum of infected patients. By using one mAb (IA2(23] directed against Onchocerca volvulus and recognizing circulating Ag (Ab1), we purified by affinity chromatography the target molecule of IA2(23) (an O. volvulus glycoprotein recognized by IA2(23) mAb). This compound had a dose-dependent effect on the cytotoxic action of DEC-treated platelets. We subsequently produced an anti-idiotype mAb to Ab1 (Ab2), and considered the possibility of replacing the O. volvulus glycoprotein recognized by IA2(23) mAb by Ab2. Ab2 was selected according to its ability to inhibit the binding of radioiodinated Ab1 to the filarial target Ag. It induced the production of anti-O. volvulus antibodies (Ab3) in rats. At a constant concentration of DEC platelets, the addition of increasing amounts of Ab2 led to a dose-dependent cytotoxic effect against parasite larvae. Experiments performed with Ab2 on detergent solubilized surface proteins of platelets identified four bands of Mr 18, 26, 43.5, and 100 kDa, supporting the idea of the presence of binding sites on the platelets for a FEA required for the microfilaricidal cytotoxicity of DEC-treated platelets.

The expression of similar antigenic determinants by trematode parasites and their intermediate (invertebrate) or definitive (vertebrate) hosts has been previously reported. Studies of experimental and human infection by the parasite Schistosoma mansoni have revealed the strong immunogenicity of a surface antigen with a relative molecular mass (Mr) 38,000 (38K). Here we provide evidence that the important protective epitope of the 38K molecule is expressed by the uninfected intermediate host of S. mansoni, Biomphalaria glabrata and is synthesized both by the mollusc and by the parasite throughout its life cycle, thus confirming our original hypothesis. Deglycosylation experiments indicate that the protective epitope is an oligosaccharide and in B. glabrata, is associated with a 90K component. Analysis of soluble extracts from different freshwater mollusc species shows that the same protective epitope is found in schistosome as well as in non-schistosome hosts. Moreover, it was also found on the haemocyanin of the keyhole limpet (Megathura crenulata), a carrier protein widely used in immunological studies.

A schistosomula surface antigen of 38,000 daltons has been isolated by using a rat monoclonal IgG2a that has been shown to confer protection against S. mansoni infection by passive transfer in rats. This antigen is one of the previously characterized surface proteins reacting with sera from various infected hosts, including rat, mouse, monkey, and human. Studies on parasites of different developmental stages indicated the presence of this antigen on cercariae and skin-derived or mechanically transformed schistosomula.

The complementary DNA sequence encoding the Mr 28,000 antigen of Schistosoma mansoni has been isolated and expressed in Escherichia coli. Experimental vaccination of rats, hamsters and monkeys with a recombinant fusion protein induces a strongly cytotoxic antibody response. Immunization of rats and hamsters with this protein leads to significant protection against a natural challenge infection with live cercariae.

Monoclonal antibodies against Schistosoma mansoni have been produced by fusion of splenic lymphocytes from S. mansoni infected Rats and P3-X63-Ag8 BALB/c cells. In vitro and in vivo studies of the biological activities of these antibodies have led to the identification of IgE antibodies with a high reaginic activity and antibodies which in a complement dependent or eosinophil dependent system were shown to have a marked cytotoxicity for schistosomula in vitro. This methodology seems to open new perspectives for the study of antibody function in immunity against parasites as well as for the isolation of the corresponding target antigens.

A monoclonal antibody of the IgM class recognizing Onchocerca volvulus circulating antigen (COA) was obtained. This monoclonal antibody was used in a radioimmunoprecipitation-PEG assay (RIPEGA) to detect circulating antigen in onchocerciasis patients' sera. COA could be detected in 63 (80%) of the 79 African patient sera tested, and in 126 (76%) of the 164 Indian (Venezuela) sera studied. There was no direct correlation between the presence of COA detected in the patient serum and the level of microfilarodermia. The RIPEGA using this monoclonal antibody detected COA in 91% of children under 10 years old, whereas the microfilarodermia in this group was positive in only 52% of the cases. The specificity of this test is improved compared to the results obtained with polyclonal antibodies. Immunofluorescence studies suggest that the COA might be located in the microfilaria cuticle.

Schistosomiasis is a chronic, debilitating disease that affects over 200 million people worldwide, and of which around 800 000 die annually according to WHO estimates. Three species of schistosome infect man (Schistosoma mansoni, S. haematobium and S. japonicum) while a fourth species (S. bovis) infects cattle, causing extensive economic loss, particularly in East Africa. Despite the existence of effective chemotherapeutic agents, and numerous programs aimed at controlling the fresh water snail intermediate host, progress towards circumscribing the parasite has been slow. This may in part due to inadequate planning of chemotherapy campaigns (Prescott, 1987). However, the problem of drug resistance (Coles et al. 1987) and the overall economic cost mean that other approaches are necessary. Since, unlike protozoan parasites, schistosomes do not replicate in the definitive vertebrate hosts, a partial non-sterilizing immunity would greatly diminish both transmission levels in endemic areas, and the incidence of human pathology caused by the deposition of parasite eggs in host tissues (Phillips and Colley, 1978).

SUMMARYA total of 129 children were treated forSchistosoma mansoniinfections, and followed for intensity of reinfection at3-monthly intervals over a 21-month period. Blood samples were taken before treatment and at 5 weeks and 6, 12 and 18 months after treatment. This paper presents a statistical analysis of the relationship between various immune responses and subsequent reinfection. Responses analysed were: blood eosinophil levels; IgE antibodies against schistosomulum antigens; IgG antibodies mediating eosinophil-dependent killing of schistosomula; antibodies inhibiting the binding to schistosomulum antigens of two rat monoclonal antibodies that also recognize egg antigens; the levels of anti-adult worm and of anti-egg (total, IgM and IgG) antibodies; and IgM anti-schistosomulum antibodies. Results for each assay were well correlated for each of the five separate blood samples. None of the assays were predictive of resistance to reinfection, butsusceptibilityto reinfection was strongly correlated with results in the preceding blood samples for total anti-egg antibodies and the inhibition of binding of one of the two monoclonal antibodies. Further analysis also revealed a correlation between reinfection intensities and both IgM anti-schistosomulum antibodies and IgM and IgG anti-egg antibodies. These results are consistent with the hypothesis that early infections elicit the development, in response to egg antigens, of antibodies that block immune mechanisms directed against schistosomula. Blocking antibodies may be IgM, but might also be of an ineffective IgG isotype. The existence of such antibodies in young children would explain the slow development of immunity in the face of a range of detectable, potentially protective immune responses.