Julie C. Scott

Purification of a recombinant Schistosoma japonicum antigen homologous to the 22-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate against schistosomiasis

We describe the cDNA cloning, overproduction and purification of a 22.6-kDa antigen from the human blood fluke Schistosoma japonicum. A 777-bp cDNA (C32) was isolated from a S. japonicum lambda ZAPII cDNA expression library immuno-screened with hyperimmune rabbit serum (HRS) raised against soluble adult S. japonicum proteins. The open reading frame of C32 encodes a protein of 191 amino acids (aa) which exhibits 71% identity to a 22.6-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate for schistosomiasis. We have identified a sequence motif known as an EF-hand calcium-binding domain in both the S. japonicum and S. mansoni aa sequences, suggesting that the 22.6-kDa antigens are able to bind Ca2+. Further, we have, for the first time, obtained the 22.6-kDa antigen in purified, non-denatured, recombinant form, and in sufficient quantity to assess the protective value of the molecule in vaccination/challenge experiments. This was achieved by synthesizing the schistosome antigen with a short polyhistidine tag fused to the N-terminus which was then used for subsequent affinity purification. The recombinant protein was purified under non-denaturing conditions using nickel-chelate affinity chromatography.

DNA-based vaccination using Schistosoma japonicum (Asian blood-fluke) genes

We have examined the efficacy of nucleic acid vaccination in inducing immunity to the multicellular parasite, Schistosoma japonicum, a trematode worm responsible for causing schistosomiasis in humans and other mammalian species. A panel of Schistosoma japonicum cDNAs were cloned into eukaryotic expression vectors, injected into animals, and tested for immunogenicity. The cDNAs tested encoded 26- and 28-kDa glutathione-S-transferases, calreticulin, glyceraldehyde-3-phosphate dehydrogenase, a 22.6 kDa membrane-associated antigen, a 14 kDa fatty-acid binding protein, fragments of paramyosin, full-length paramyosin, and a novel gene comprising the 26 kDa glutathione-S-transferase fused to a fragment of paramyosin cDNA. The paramyosin gene constructs, including the fusion, were all able to induce anti-paramyosin antibodies; with the fragments of paramyosin these were of the IgG1, IgG2a and IgG2b isotypes. In contrast, none of the other schistosome cDNAs tested were able to induce detectable antibody responses. The anti-paramyosin antibodies did not protect mice challenged with cercariae of S. japonicum.

Sj-FABPc fatty-acid-binding protein of the human blood fluke Schistosoma japonicum: structural and functional characterization and unusual solvent exposure of a portal-proximal tryptophan residue

Sj-FABPc of the blood fluke of humans, Schistosoma japonicum, is a member of the FABP/P2/CRBP/CRABP family of beta-barrel cytosolic fatty-acid-binding and retinoid-binding proteins. Sj-FABPc has at least eight different variants encoded by a single-copy polymorphic gene. In fluorescence-based assays, recombinant Sj-FABPc was found to bind 11-(dansylamino)undecanoic acid (DAUDA), inducing a shift in peak fluorescence emission from 543 to 493 nm. A similar spectral change was observed in dansyl-amino-octanoic acid (in which the dansyl fluorophore is attached at the alpha-carbon rather than the omega-carbon of DAUDA), indicating that the ligand enters entirely into the binding site. Sj-FABPc also bound the naturally fluorescent cis-parinaric acid, as well as oleic acid and arachidonic acid, by competition, but not all-trans-retinol. Dissociation constants were, for cis-parinaric acid, K(d)=2.5+/-0.1 microM (mean+/-S.E.M.) and an apparent stoichiometry consistent with one binding site per molecule of Sj-FABPc and, for oleic acid, K(i) approximately 80 nM. A deletion mutant from which alpha-II was absent failed to bind ligand. Sj-FABPc modelled well to known structures of the protein family; an unusually solvent-exposed Trp side chain was evident adjacent to the presumptive portal through which ligand is thought to enter and leave. Intrinsic fluorescence analyses of Sj-FABPc and of the deletion mutant (from which Trp-27 is absent) confirmed the unusual disposition of this side chain. Virtually all members of the FABP/P2/CRBP/CRABP protein family have prominent hydrophobic side chains in this position, with the exception of liver FABP and ileal FABP, which instead have charged side chains. Liver FABP is known to be distinct from other members of the protein family in that it does not seem to contact membranes to collect and deposit its ligand. It is therefore postulated that the unusually positioned apolar side chains in Sj-FABPc and others in the family are important in interactions with membranes or other cellular components.

Gene cloning and complete nucleotide sequence of philippine Schistosoma japonicum paramyosin

The development of an effective vaccine is recognised as a necessary adjunct to the control of schistosomiasis japonica, a disease affecting several million people in China and the Philippines. Currently, recombinant Schistosoma japonicum molecules are considered most suitable for large scale vaccine production and a number of genes encoding vaccine candidate polypeptides have been cloned and expressed (see Waine et al., 1993a). One of the molecules providing most promise as a vaccine target is paramyosin (Butterworth, 1992), a major structural protein of thick filaments in the muscle of most invertebrates; paramyosin genes have now been cloned from a range of parasitic helminths, including schistosomes (Limberger and McReynolds, 1990; Laclette et al., 1991; Dahmen et al., 1993; Landa et al., 1993; Mühlschlegel et al., 1993, Nara et al., 1994). The cloning and nucleotide sequence of S. Japonicum paramyosin is described.

Molecular cloning and enzymatic expression of the 28-kDa glutathione S-transferase of Schistosoma japonicum: evidence for sequence variation but lack of consistent vaccine efficacy in the murine host.

Glutathione S-transferases (GSTs) have long been regarded as attractive vaccine (and drug) targets in schistosomes due to their suspected role in detoxification processes. Indeed, the 28-kDa GST of Schistosoma mansoni (SmGST28) has proven efficacy as an antigen for protective immunity reducing worm burden, female fecundity and egg viability. In contrast, the vaccinating effects of the bacterial expressed homologue of Philippine S. japonicum (SjpGST28) have proved disappointing, possibly because this recombinant form was an incomplete sequence, lacking five N-terminal amino acids which may have affected its vaccination efficacy. Here we describe the cloning and functional enzymatic expression of a complete cDNA encoding SjpGST28. We report also on the immunogenicity and vaccine efficacy of this molecule as a purified recombinant protein and as a DNA plasmid vaccine in the murine model. We further describe the cloning of several complete cDNAs encoding the Chinese homologue of SjpGST28 and the identification of 3 SjcGST28 sequence variants which are probably encoded by distinct alleles.

Molecular and immunological characterisation of a polymorphic cytosolic fatty acid binding protein from the human blood fluke of humans, Schistosoma japonicum.

Most organisms obtain their fatty acids through their diet or by de novo synthesis, but human blood flukes belonging to the genus Schistosoma lack the oxygen-dependent pathways required for the synthesis of sterols and fatty acids so they are entirely dependent on their hosts for these and other complex lipids. Fatty acid binding proteins (FABPs) of the FABP/P2/CRABP/CRBP family of beta-barrel cytosolic lipid binding proteins (cLBP) appear to be particularly important to schistosomes in the uptake, transport and compartmentalisation of host-derived fatty acids and may provide important targets for immuno- and chemotherapy. Here we describe the isolation of a set of cDNAs prepared from the Asiatic schistosome, Schistosoma japonicum, which encode two groups of cLBPs based on sequence homology and unique cDNA restriction sites. Representative clones from the two groups, one encoding a complete Sj-FABP (F10), and the other encoding a deletion mutant (F25) were characterised at the nucleic acid level by Southern and Northern hybridisation analysis, and at the protein level by immunoblotting. The presence and size of introns in the genes encoding F10 and F25 were determined and, because of the interest in the Schistosoma mansoni FABP homologue (Sm14) as a putative vaccine candidate, the immunogenicity and protective efficacy of the two proteins were also evaluated. A particularly interesting finding was the degree of Sj-FABP amino acid sequence polymorphism found to occur within the S. japonicum worm population, which appears to be greater than that described from cLBPs from vertebrates or, indeed, any other group of organisms investigated to date.

Identification of novel 70-kDa heat shock protein-encoding cDNAs from schistosoma japonicum

A diverse range of organisms respond to a variety of chemical, physiological and temperature-associated stresses by a rapid and transient increase in the synthesis of heat shock proteins. We immunoscreened a Uni-ZAP XR cDNA library, prepared from mRNA isolated from the Philippine strain of the Asian bloodfluke, Schistosoma japonicum, using hyperimmune rabbit sera raised against soluble adult S. japonicum proteins. Six 70-kDa heat shock protein-encoding cDNA clones were identified which, upon further analysis, were separated into two distinct protein groups within the 70-kDa heat shock protein family, the 70-kDa heat shock proteins and the immunoglobulin heavy chain-binding proteins/glucose-related proteins (Grp78). A representative from both groups was fully sequenced and compared with homologous sequences available in the GenBank/EMBL database as the first stage in determining the role of their expression products in the regulation of S. japonicum development, in the induction of immunity, and whether they act as molecular chaperones capable of modulating the correct folding or repair of proteins within this species of schistosome.

Molecular cloning and functional expression of a cDNA encoding the major endoplasmic reticulum-associated calcium-binding protein, calreticulin, from Philippine strain Schistosoma japonicum.

We describe the cloning of a full length calreticulin (CR)-encoding cDNA clone isolated by immunoscreening of a cDNA library prepared with mRNA from adult worms of the Philippine strain of Schistosoma japonicum, the cause of Asian schistosomiasis. The sequence of the cDNA is presented, and its molecular characterisation and functional expression as a Ca2+-binding protein described. The potential role of CR in inducing protective immunity in the schistosomes is discussed.

A molecular comparison of glyceraldehyde-3^phosphate dehydrogenase and a 22.6-kDa tegument membrane-associated antigen from Chinese and Philippine Schistosoma japonicum

Abstract We compared cDNAs encoding the 37-kDa glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 22.6-kDa tegument membrane-associated antigen (Sj22.6), both considered as promising anti-schistosomiasis vaccine targets, from Chinese and Philippine Schistosoma japonicum. As with a number of other genes we have compared for these two strains, they are remarkably conserved. Full length cDNA clones, P52a (SjpGAPDH) and P11 (Sjp22.6), were isolated from cDNA expression libraries constructed from Philippine strain S. japonicum. Nucleotide (nt) sequencing of P52a revealed 98.7% identity overall (two amino acid (aa) substitutions in the ORF) with the Chinese S. japonicum GAPDH cDNA but lower sequence homology (within the ORF: 82% nt homology, 90% aa identity) with Schistosoma mansoni GAPDH. The cDNA of clone P11 has an additional 24 bp and 252 bp, respectively, of nt sequence at the 5′ and 3′ ends of the non-coding region but the ORF (3 nt differences; 1 aa change) contains the same number of predicted residues as the cDNA encoding the Chinese S. japonicum 22.6-kDa antigen. A sequence comparison of P11 and the S. mansoni homologue indicated they share 79.1% nt homology and 73.7% aa identity within the ORF. The fact that GAPDH and Sj22.6 from Philippine and Chinese S. japonicum exhibit remarkable sequence conservation, and that Sjp22.6 and Sjc22.6 are antigenically cross-reactive, supports the concept of developing a single vaccine effective against both Phillipine and Chinese S. japonicum.

Characterisation and expression of a cDNA encoding the 80-kDa large subunit of Schistosoma japonicum calpain ☆

We describe the cloning of a full length calpain-encoding cDNA constructed from two truncated cDNAs isolated from a cDNA library prepared with mRNA isolated from adult worms of the Philippine strain of Schistosoma japonicum. The cDNA sequence is 2.456 kb in length and predicts a protein of 758 residues with a molecular mass of 86.61 kDa and an isoelectric point of 5.34. Probes spanning the entire calpain cDNA hybridised to multiple bands in genomic DNAs of Philippine (SjP) and Chinese (SjC) S. japonicum, with some restriction fragment length polymorphisms evident between the two strains. Northern hybridisation analysis indicated that the cDNA codes for a single RNA transcript between 2.6 and 3.6 kb in size in the SjP and SjC genomes. After subcloning in the QIA express vectors pQE-31 and pQE-40 and subsequent expression, the recombinant protein was purified and shown to bind calcium. The availability of recombinant S. japonicum calpain will allow its future evaluation as a vaccine candidate, especially in light of recent work with the S. mansoni homologue which has provided evidence that this protein may be a target of protective immunity.