Cloning of a 21.7-kDa vaccine-dominant antigen gene of Schistosoma mansoni reveals an EF hand-like motif

Several cDNA clones encoding a 21.7-kDa antigen (Sm21.7) were detected from a Schistosoma mansoni sporocyst cDNA expression library using irradiated cercaria-vaccinated rabbit serum. The antigen was designated ‘vaccine dominant’ because parasite-derived Sm21.7 was recognised preferentially by mouse vaccine sera compared with mouse infection sera. The cDNA and corresponding gDNA sequences showed 64% identity at the nucleotide level and 47% identity at the amino acid level with the sequence of a previously described S. mansoni tegumental antigen, sma22.6 [23]. Whereas sma22.6 mRNA occurs almost exclusively in the adult worm, Sm21.7 mRNA was equally abundant in the sporocyst, schistosomular and adult stages. Both Sm21.7 and sma22.6 sequences reveal a motif strongly homologous to the EF hand calcium binding domain but lacking the invariant glycine in the calcium binding loop. The disruptive nature of the glutamine which in Sm21.7 replaces the glycine explains why the motif is non-functional, as shown by the inability of Sm21.7 to bind calcium.     

Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine

Schistosomiasis is a major public health problem that affects mainly developing countries. There are 200 million people worldwide infected with schistosomes resulting in more than 250,000 deaths per year. Although schistosomicidal drugs exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study we isolated a cDNA clone encoding the Schistosoma mansoni lung-stage Sm22.6 protein, which is 100% and 79% identical with the 22.6 kDa adult worm tegument antigen of S. mansoni and S. japonicum, respectively. Further, we produced recombinant (r) Sm22.6 and constructed an Sm22.6 DNA vaccine. Western blot analysis confirmed the identity of purified MBP-Sm22.6 fusion protein using anti-MBP (maltose binding protein) and anti-rSm22.6 antibodies. Additionally, C57BL/6 mice were immunized and specific anti-Sm22.6 IgG responses were produced when both vaccination strategies were used. Importantly, only rSm22.6 vaccine provided levels of protection against challenge infection (34.5%). Mice immunized with rSm22.6 induced production of IgG1 and IgG2a and synthesis of IFN-gamma and IL-4 in cultured mouse splenocytes. Finally, rSm22.6 vaccination induced a Th0 type of immune response and protective immunity that suggests Sm22.6 as a potential candidate to compose an anti-schistosome vaccine.     

Protective effect and granuloma down-modulation promoted by RP44 antigen a fructose 1,6 bisphosphate aldolase of Schistosoma mansoni

A Schistosoma mansoni adult worm cDNA expression library was screened using rabbit IgG against PIII, an adult worm protein fraction, already known to possess protective and immunomodulating effects to a challenge infection in mice. A positive cDNA clone was selected and characterized. The cDNA screened encodes a protein (P44) with an ORF of 1089 bp and an amino acid sequence of 363 residues with a predictable molecular weight of 44 kDa. The P44 amino acid sequence exhibits 100% identity to the fructose 1,6 bisphosphate aldolase of S. mansoni, 66% to Homo sapiens and 66% to Mus musculus. The cDNA was cloned into a pGEX-4T-3 vector and expressed in Escherichia coli as a fusion protein (GST/P44). Mice vaccinated with recombinant P44 were able to develop high levels of IgG or IgG1 and displayed low levels of IgG2a isotype. Moreover, immunization of mice with this antigen induced a significant protection of 57% against a challenge infection and significant decrease in hepatic granuloma formation. Our results demonstrate that granuloma modulation can be targeted for pathology elimination through vaccination. This represents an advance in schistosome vaccinology and allows for the development of a therapeutic as well as a prophylactic vaccine.     

Factors affecting human IgE and IgG responses to allergen-like Schistosoma mansoni antigens: Molecular structure and patterns of in vivo exposure

BACKGROUND: The human IgE response is associated with allergy and with host defence against parasitic worms. A response to Sm22.6, the dominant IgE antigen in adult Schistosoma mansoni worms, correlates with resistance to re-infection after treatment. Sm22.6 is one of a family of EF-hand containing parasite proteins with sequence similarity to dynein light chain (DLC) and with major non-parasite allergens. Here we compare human IgE and IgG responses to other family members, Sm20.8 and Sm21.7, as well as to SmDLC1, relating these to antigen structure and expression in parasite life stages. METHODS: Recombinant antigens were used in ELISA to measure antibody isotype responses in 177 cases from an endemic area, before and 7 weeks after treatment. Parasite antigen expression was assessed by RT-PCR and Western blotting. RESULTS: Levels of antibodies to Sm22.6 and Sm20.8 (but not to Sm21.7 or SmDLC1) showed posttreatment increases in all but young children. Many produced IgE to Sm22.6 and Sm20.8 (2 EF-hands), few to Sm21.7 (1 EF-hand) or SmDLC1 (no EF-hands). Sm21.7 was expressed in cercariae, adults and eggs, Sm22.6 and Sm20.8 were concentrated in the adult. CONCLUSIONS: These studies suggest that IgE antigens Sm22.6 and Sm20.8 are only released to boost antibodies when adult worms die, whilst Sm21.7 and SmDLC1 are released constantly from eggs dying in host tissue. IgE responses to these allergen-like molecules may be influenced by patterns of exposure and the number of EF-hand motifs.     

Immunoprecipitation of surface antigen precursors from Schistosoma mansoni messenger RNA in vitro translation products

Messenger RNA has been isolated from adults and eggs of Schistosoma mansoni and translated in vitro in mRNA dependent rabbit reticulocyte lysate. The in vitro translation products have been immunoprecipitated using a wide variety of hyperimmune and infection sera from rabbits, mice and humans. A large number of in vitro translation products are precipitated by these sera, and we have identified a subset of the immunoprecipitated polypeptides which are expressed on the surface of the young schistosomulum. Messenger RNA from both adults and eggs directs the synthesis of these polypeptides suggesting that at least some of the surface proteins of the young schistosomulum are being synthesised throughout the life cycle. cDNA clone banks prepared from adult mRNA will therefore contain the genes for these schistosomulum surface antigens greatly facilitating their isolation.     

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.     

Immunization with rP22 induces protective immunity against Schistosoma mansoni: effects on granuloma down-modulation and cytokine production

Schistosomiasis remains a significant public health problem in tropical countries and it is recognized as the most important human helminth infection in terms of morbidity and mortality. Although the existing antischistosomal drugs are highly effective, they do not prevent against reinfection or granuloma formation. Therefore, vaccine strategies are essential for the control of schistosomiasis. Our group recently identified the recombinant (r) P22 protein, a component of the adult worm protein fraction PIII that has been shown to engender protective and immunomodulatory effects on murine schistosomiasis. A cDNA clone encoding rP22 was isolated from a Schistosoma mansoni adult worm cDNA library using anti-PIII rabbit serum; it exhibited complete identity with S. mansoni Sm21.7 EF-hand antigen. Confocal microscopy revealed that rP22 is a tegument protein localized on the surface of S. mansoni miracidia and adult worms. Mice immunized with rP22 exhibited a 51% and 22.5% decrease in adult worm burden and in hepatic eggs, respectively. Additionally, rP22 vaccine produced a reduction in 60% of liver granuloma size and 71% of fibrosis in mice, suggesting that rP22 might contribute to down-modulate granulomatous hypersensitivity to S. mansoni eggs. Protective immunity in mice was associated with high titers of rP22-specific IgG antibodies; elevated production of IFN-γ, TNF-α and IL-10; and a reduced level of IL-4. In conclusion, these findings indicate that rP22-based vaccines could be useful to elicit protection and reduce pathology associated to schistosomiasis.     

The murine Sm-D autoantigen: multiple genes, genetic polymorphism, evolutionary conservation and lack of intervening sequences in the coding region.

Antibodies to the Sm nuclear antigen are diagnostic of systemic lupus erythematosus (SLE). MRL/Mp-lpr/lpr mice develop a similar illness, and a proportion also develop anti-Sm. To understand better anti-Sm reactivity in this murine model, we have cloned the murine Sm-D autoantigen. One cDNA clone was 517 bp long with an open reading frame of 357 nucleotides, encoding a 13.3 kDa protein of 119 amino acids. At the nucleotide level, the murine Sm-D cDNA was 89.8% homologous with human Sm-D (94% in the coding region), yet there was identity at the protein level, including a Gly-Arg nine-fold repeated C-terminus motif. Southern blot analysis of PstI-digested genomic DNA from seven mouse strains demonstrated a 7.8 kb band in every strain; in addition, a 2.8 kb band was seen in AKR/J, LG/J and MRL/Mp-lpr/lpr. PCR amplification of genomic DNA showed a single Sm-D gene product of 360 bp, which indicated a lack of intervening sequences. The Sm-D protein is thus highly conserved in evolution, probably owing to its essential role in the physiology of the cell.     

A repeated proline-rich sequence in Sm B/B' and N is a dominant epitope recognized by human and murine autoantibodies.

Reactivity of a murine monoclonal anti-Sm B antibody was localized to a cDNA clone T3/2 encompassing codons 177-221 of Sm N and to a series of 10-residue synthetic peptides containing a proline-rich motif. Human sera containing anti-B antibodies also bound to this proline-rich motif in the form of a heptamer, PPGMRPP, and could distinguish the N/B sequence from similar sequences present in the RNP A and C polypeptides in a number of cases. Monoclonal antibodies binding to either this peptide or to a determinant present on both B and D polypeptides inhibited the majority of anti-B binding in 50% of anti-B sera. The region of N/B containing PPGMRPP also bears a second epitope, sterically blocked by monoclonal antibody KSm 5. This hotspot binds 40-60% of anti-B activity in 30% of anti-B sera. These data suggest that the anti-Sm B epitope recognition repertoire is restricted in SLE patients.     

Identification and characterization of myophilin, a muscle-specific antigen of Echinococcus granulosus

A muscle-specific gene of Echinococcus granulosus has been identified and characterized. A λgt11 clone (10P1), containing an incomplete copy of the gene, was originally isolated from a larval E. granulosus cDNA library by serum antibodies from dogs infected with the parasite. The full-length cDNA sequence was obtained by PCR amplification of cDNA from an adult E. granulosus λgt22A library. Southern blot analysis indicated the presence of the gene as a single copy in the genome of E. granulosus and also detected homologous genes in genomic DNA of E. multilocularis and Taenia saginata. The 21.2-kDa protein deduced from the complete cDNA sequence contains two regions of 12 amino acids with similarity to the EF-hand motif of calcium binding proteins. Antibodies raised against the purified 10P1-GST fusion protein detected a 22-kDa antigen in the E. granulosus developmental stages examined. Immunoelectron microscopy localized the native protein in the muscle of the parasite. The amino-acid sequence of the E. granulosus protein shows significant homology to the muscle proteins mp20 of Drosophila melanogaster, chicken SM22 and mammalian calponin, and also to the neuronal protein NP25 of rats. A conserved carboxy-terminal motif of 17 amino acids is present in all the homologous proteins and is proposed to be the characteristics feature of a novel protein family. The term myophilin is proposed for the E. granulosus protein due to its localization and homology to other muscle proteins.     

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.     

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.     

An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes

A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.     

Specific serodiagnosis of visceral leishmaniasis using a Leishmania donovani antigen identified by expression cloning

A λgt11 expression library was constructed using mRNA from the promastigote form of Leishmania donovani (African isolate MHOM/ET/67/HU3). The library was screened with serum obtained from a patient who contracted visceral leishmaniasis in the Sudan. Several cDNA clones which expressed β-galactosidase/L. donovani antigen fusion proteins were isolated. One of these clones corresponded to a 60 kDa membrane-associated antigen. By a Western blot assay antibodies against the fusion protein were detected universally in the sera of visceral leishmaniasis patients. They were not detected in sera from patients with cutaneous leishmaniasis or other parasitic protozoan infections. The gene coding for this antigen was specific to the genus Leishmania as judged by DNA hybridisation, and its chromosomal location was conserved. A 20 kb mRNA was detected on Northern blots of promastigote RNA.     

The reaction of human SLE antibodies with native, single stranded RNA: analysis of nucleotide sequence specificities and correlation with Sm and nRNP activities

The antigenic specificities of RNA reactive IgG autoantibodies from three individual human SLE sera and pooled NZB/NZW mouse serum were studied by competition reactions with synthetic co-polymers and heteropolymers of varying nucleotide compositions. Although minor differences in the individual binding patterns were noted, the RNA reactive immunoglobulins showed a clear preference for G, C and G,C,U containing RNA sequences. All three SLE sera had Sm or RNP activities with rabbit thymus extractable nuclear antigen (ENA). Conversely, all SLE sera that were selected on the basis of their positive ENA reaction could also bind free, single stranded RNA. These results suggest that the autoimmune response to RNA in human and murine SLE is restricted to one or a very few antigenic determinants. They also raise the possibility that autoantibodies to native RNA in SLE sera are partly responsible for ENA reactivity.     

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.