Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni.

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.     

Inter-species variation of schistosome 28-kDa glutathione S-transferases.

The 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.     

Expression of a Schistosoma mansoni 28-kilodalton glutathione S-transferase in the livers of transgenic mice and its effect on parasite infection.

Schistosomiasis is a debilitating tropical disease for which an effective vaccine is needed. A 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) has been shown to induce protective immunity. Sm28GST possesses significant sequence identity to mammalian GST isoforms. In order to study self-reactivity in mice immunized with Sm28GST and the concomitant phenomena of immune tolerance and epitope suppression, as well as their consequences for the protective immunity induced by this vaccination, we developed transgenic (Tg) mice that express Sm28GST under the control of a part of the mouse transferrin gene promoter. A study of (P28)Tg mice showed that the expression of Sm28GST was strictly localized in pericentrolobular hepatocytes. No histological change, inflammatory infiltrates, or modification of seric L-aspartate: 2-oxoglutarate aminotransferase concentration was observed over an 18-month period, despite a cross-reactivity between Sm28GST and a mouse molecule of 30 kDa. The immunoglobulin G anti-Sm28GST response of (P28)Tg mice immunized with recombinant Sm28GST was lower (P < 0.001) than that observed in non-(P28)Tg littermates and inversely proportional of Sm28GST liver expression. The response of non-(P28)Tg mouse spleen cells to Sm28GST stimulation was greater (P < 0.01) than that observed with (P28)Tg mouse spleen cells. (P28)Tg mice infected with 40 S. mansoni furcocercariae harbored more worms (P < 0.05) than did non-(P28)Tg control mice. The increase in the level of infection in (P28)Tg mice was reflected in concomitant increases in the numbers of adult worms and schistosome eggs found in livers and intestines after whole-body perfusion at 56 days postinfection, but no relative increase in the fertility of individual female worms was observed. The results obtained argue for the involvement of Sm28GST in reducing levels of infection and support the view that this enzyme has a central role in the maintenance of parasite viability, at least during its migration through host tissues.     

Expression of a 28-kilodalton glutathione S-transferase antigen of Schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.     

A monoclonal antibody blocking the Schistosoma mansoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability

The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm 28 GST) were investigated. Two mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GST enzymatic activity (S13) or the lack of blockade (H12). When passively transferred into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALB/c mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transferred; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non-living eggs. No effect on egg production and egg hatching was observed in H12-treated mice. In addition, worm pairs recovered from S13-but not H12-treated mice laid significantly fewer eggs in vitro, and normal worm pairs incubated in vitro with the S13 mAb produced significantly fewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mansoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.     

GM-CSF and TNF-alpha synergize to increase in vitro granuloma size of PBMC from humans induced by Schistosoma mansoni recombinant 28-kDa GST

The 28-kDa Glutathione S-transferase of Schistosoma mansoni (Sm28 GST) was described as a protective antigen capable of reducing female fecundity and the number of eggs in mice hepatic tissues. The role of GM-CSF and TNF-alpha in the in vitro granuloma reaction of peripheral blood mononuclear cells (PBMC) from chronic intestinal schistosomiasis patients before and after chemotherapy treatment to S. mansoni recombinant Sm28 GST was evaluated. Treatment of PBMC with recombinant Sm28 GST caused a significant increase in granuloma formation when compared to SEA or SWAP. Contrary to SEA or SWAP, Sm28 GST was not capable of inducing significant cellular proliferation. Moreover, recombinant Sm28 GST promoted a significant elevation in GM-CSF and TNF-alpha levels. However, we did not detect any significant IL-10 production. When Sm28 GST was applied in the presence of anti-GM-CSF or anti-TNF-alpha antibodies in cultures, we observed a significant decrease in granuloma size. Indeed, our results demonstrated that Sm28 GST was capable of promoting high in vitro granuloma index, and this event was associated with the balance of GM-CSF and TNF-alpha. These evidences suggest a role for GM-CSF as a major mediator in increasing granuloma reaction in human schistosomiasis. This event may contribute to exacerbate the pathology resulting from egg deposition in host tissues.     

Schistosoma mansoni antigens differentially recognized by resistant WEHI 129/J mice.

Mice of the strain WEHI 129/J are genetically resistant to chronic Schistosoma mansoni infection. Resistance is expressed in at least 50% of mice, with the remaining mice showing normal susceptibility to infection. The serum antibody specificities in the resistant proportion of WEHI 129/J were analyzed at various times after exposure to cercariae by using both Western blotting and immunoprecipitation. Comparisons with the susceptible proportion of WEHI 129/J and other permissive mouse strains revealed four antigens that were differentially recognized by resistant mice at various times of infection: Sm25, an Mr 25,000 integral membrane protein of adult worms that was better recognized by resistant mice 40 to 50 days after exposure; Sm67, an Mr 67,000 water-soluble antigen of adult worms that was better recognized by resistant mice at days 30 to 40; Sm120, an Mr 120,000 antigen expressed by cercariae and adult worms that was differentially recognized, although inconsistently, at days 20 to 40 postexposure; and Sm26, an Mr 26,000 glutathione S-transferase that was uniquely recognized by resistant mice at day 20 in two of three experiments. Analysis of antibody specificities in (BALB/c x WEHI 129/J)F1 x WEHI 129/J backcross mice indicated that high responsiveness to Sm25 at days 40 to 50 correlated with resistance. The candidacy of these four molecules as vaccines for schistosomiasis mansoni is discussed.     

Characterisation of Sm20, a 20-kilodalton calcium-binding protein of Schistosoma mansoni

A cDNA clone encoding part of a 20-kDa antigen of Schistosoma mansoni (Sm20) has been isolated. The amino acid sequence of this antigen, as predicted from the sequence of the cDNA, has significant homology to the family of calcium binding proteins which include calmodulin, troponin C and the light chain of myosin. Although we have been unable to show any immunological cross-reactivity between Sm20 and calmodulins from a range of other species, we have verified that Sm20 is a functional calcium binding protein. Sm20 is encoded by a small multigene family and is expressed in schistosomula and adult worms but not in eggs. The 20-kDa nascent polypeptide appears to be post-translationally modified to give a 38-kDa species. Sm20 is present in preparations of tegumental membranes and is easily removed from intact schistosomula by detergent treatment, suggesting that it is associated with the tegument. However, the cloned portion does not appear to be exposed on the surface.     

A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate

Understanding the mode of Schistosoma mansoni larval invasion and the mechanism of immune evasion utilized by larvae and adult worms is essential for a rational development of vaccines or drugs to prevent or cure the disease. This parasite has a very complex molecular organization in all parasite stages, and identifying the major parasite proteins would give clues to schistosome metabolism and to the interaction of the parasite with the host immune system. Our goal was the evaluation of the protein parasite repertoire using a proteomic approach, and the characterization of protein extracts from two different parasite stages of a Venezuelan isolate, such as cercariae and adult worms, previously performed by other authors in some other strains. A comparison among authors was made. Besides, we aimed to identify different isoforms of one of the vaccine candidates, the gluthation-S-transferase protein (Sm28GST), by 2D SDS-PAGE and mass spectrometry, and to achieve its immunologic detection using sera from rabbits immunized with synthetic peptides derived from the Sm28GST protein. These techniques allowed the identification of some of the target molecules of the protective immune response that are being evaluated as potential members of a multi-component and multi-stage anti-S. mansoni vaccine and to clarify if the selected peptides induce antibodies that are able to recognize different isoforms of the Sm28GST.     

Induction of Cytotoxic T-Cell Activity by the Protective Antigen of Schistosoma mansoni Sm28GST or its Derived C-Terminal Lipopeptide

In a previous work the authors demonstrated that immunization with Schistosoma mansoni 28-kDa glutathion-S-transferase (Sm28GST) was able to reduce hepatic damage in infected mice and that the adoptive transfer of Sm28GST-specific T cells reproduced the protective effect obtained with the recombinant molecule. In the present paper, the authors show that Sm28GST is also able to stimulate an antigen-specific, cytotoxic T-cell response against Sm28GST-pulsed P815 target cells in normal mice and that effector cells induced in vivo were classical Class I MHC-restricted CD8⁺ lymphocytes. The authors found no spontaneous CTL activity against Sm28GST-pulsed target cells during the course of the infection by S. mansoni although Sm28GST is expressed at different developmental stages of the parasite. It was observed, however, that immunization with Sm28GST is sufficient to elicit a significant level of CTL response for 6 weeks in infected mice. The role of these Class I MHC-restricted CD8⁺ lymphocytes in the protection observed precisely at the same period in immunized mice remains to be elucidated. The authors also observe that immunization with the lipopeptidic form of the C-terminal peptide of the molecule (190–211 peptide) led to a CTL activation comparable to that observed after immunization with the whole molecule demonstrating the feasibility of using a synthetic lipopeptide as immunogen for a CTL response against Sm28GST epitopes. Moreover, like Sm28GST-specific CTLs, 190–211 lipopeptide-specific cells were also Class I MHC-restricted lymphocytes.     

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody.

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.     

Single-dose mucosal immunization with biodegradable microparticles containing a Schistosoma mansoni antigen

The purpose of this work was to assess the immunogenicity of a single nasal or oral administration of recombinant 28-kDa glutathione S-transferase of Schistosoma mansoni (rSm28GST) entrapped by poly(lactide-co-glycolide) (PLG)- or polycaprolactone (PCL)-biodegradable microparticles. Whatever the polymer and the route of administration used, the equivalent of 100 microg of entrapped rSm28GST induced a long-lasting and stable antigen-specific serum antibody response, with a peak at 9 to 10 weeks following immunization. Isotype profiles were comparable, with immunoglobulin G1 being the predominant isotype produced. The abilities of specific antisera to neutralize the rSm28GST enzymatic activity have been used as criteria of immune response quality. Pooled 10-week sera from mice receiving PLG microparticles by the nasal or oral route neutralized the rSm28GST enzymatic activity, whereas sera of mice receiving either PCL microparticles, free rSm28GST, or empty microparticles inefficiently neutralized this enzymatic activity. Finally, this study shows that a single administration of these microparticles could provide distinct and timely release pulses of microencapsulated antigen, which might greatly facilitate future vaccine development.     

Expression and characterization of glutathione peroxidase activity in the human blood fluke Schistosoma mansoni.

Antioxidants may play an important role in immune evasion by schistosome parasites. Previous studies have focused on the roles of superoxide dismutase and glutathione S-transferase. In the present study, glutathione peroxidase (GPX) activity was measured in different fractions of worm extracts from several developmental stages of Schistosoma mansoni. The enzyme activity was shown to be developmentally regulated, with higher specific activities being found in the tegument-enriched Nonidet P-40 extract of adult worms (the stage least susceptible to immune killing) than in the larval stages (which are most susceptible to immune elimination). In all extracts tested, the activity against cumene hydroperoxide, even when glutathione S-transferase activity was removed, was higher than that for hydrogen peroxide. The expression of GPX cDNA in pGEX-2T by bacteria produced a 50-kDa fusion protein and a 32-kDa truncated protein. The latter was due to termination at the internal UGA codon that codes for selenocysteine. GPX activity was detected in the recombinantly produced GPX but not with Sj26-glutathione S-transferase from the vector. Mutating the TGA codon to TGT produced a full-length product, GPXm (19 kDa), that was used to produce 19 monoclonal antibodies. Anti-GPXm monoclonal antibodies recognized a 19-kDa molecule in adult-worm extract which, upon removal by immunoprecipitation, resulted in the loss of over 90% of the GPX activity, suggesting that a single form of GPX exists in the schistosome.     

Protective effect of rSm28GST-specific T cells in schistosomiasis: role of gamma interferon.

Immunization with a single dose of 50 micrograms of recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (rSm28GST) was able to induce a reduction in the worm burden, the number of eggs, and the degree of hepatic fibrosis as quantified by the measurement of collagen content in the liver of S. mansoni-infected mice. No relationship was found between anti-Sm28GST immunoglobulin G and immunoglobulin A titers and the levels of protection obtained. Adoptive transfers of Sm28GST-specific total, CD4+, or CD8+ T cells reproduced the protective effect obtained with the recombinant molecule. Moreover, experiments studying in vivo T-cell depletion demonstrated that anti-CD4- or anti-CD8-treated mice showed a significant decrease in the protective effect conferred, suggesting a role of the two T-cell subpopulations in the expression of Sm28GST-mediated protection against hepatic damage. Sm28GST-specific cells produced little interleukin-4 and high levels of gamma interferon. Treatment of immunized mice with anti-gamma interferon antibody totally suppressed the Sm28GST-induced protective effect and led to the rapid death of infected animals, suggesting a role for this cytokine in the expression of the protective immunity obtained after immunization with rSm28GST.     

Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases

We have constructed cDNA clones containing the complete nucleotide sequences coding for two highly antigenic Schistosoma mansoni adult worm proteins, Sm31 and Sm32. The predicted amino acid sequence of Sm31 shows significant homology to mouse, rat and human cathepsin B. The nucleotide sequence of Sm32 is identical to that reported by others for S. mansoni "haemoglobinase'. The different nucleotide sequences demonstrate the existence of two different proteolytic enzymes, both of which are synthesised in the form of precursor molecules. Structural homology of the schistosome cathepsin B to the mammalian ones indicates that the mature protein is processed from a propeptide. The calculated molecular weight of haemoglobinase of 47,000 suggests that post-translational processing is also involved in generating an active protease.     

Production and characterization of a monoclonal antibody against recombinant glutathione S-transferase (GST) of Fasciola gigantica

A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.     

Expression of a 28-Kilodalton Glutathione S-Transferase Antigen of Schistosoma mansoni on the Surface of Filamentous Phages and Evaluation of Its Vaccine Potential

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.     

日本血吸虫大陆株26kDa谷胱甘肽S┐转移酶编码区基因的克隆及序列测定

从日本血吸虫大陆株成虫分离总ＲＮＡ，逆转录合成ｃＤＮＡ第一链，根据菲律宾株２６ｋＤａ谷胱甘肽Ｓ－转移酶（ＧＳＴ）的ｃＤＮＡ序列，设计并合成引物，扩增出２６ｋＤａ的编码区基因，并克隆到ｐＢｌｕｅｓｃｒｉｐｔ质粒。初步酶切鉴定后，从两端对插入片段进行核苷酸序列测定。结果与菲律宾株比较，核苷酸同源性为９９．８％，仅第５８２位碱基不同，菲律宾株为Ａ，而大陆株为Ｇ。比较从ｃＤＮＡ推导出的氨基酸序列，两者１００％相同。测序结果也与曼氏血吸虫进行了比较。     

Obtention of a human primary humoral response against schistosome protective antigens in severe combined immunodeficiency mice after the transfer of human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from healthy donors were injected into C.B.-17 severe combined immunodeficiency (scid) mice which were subsequently immunized with crude Schistosoma mansoni adult worm antigenic preparation (SWAP) or with recombinant S. mansoni 28-kDa glutathione transferase (r-Sm-28-GST) antigen. PBMC from a S. mansoni-infected patient were also transferred. The specific human anti-SWAP and anti-Sm-28-GST antibody responses were monitored. The presence in both cases of human specific antibodies in scid mouse sera was determined by enzyme-linked immunosorbent assay and Western blotting techniques using anti-human immunoglobulin reagents. No antibodies were detected in these sera using anti-mouse immunoglobulin antisera. These antibodies were functional since a cytotoxic activity against schistosomula was observed when monocytes were incubated with scid mouse sera positive for anti-Sm-28-GST antibodies.