Production and testing of Schistosoma japonicum candidate vaccine antigens in the natural ovine host.

The objectives of this work were to clone and express Chinese strain Schistosoma japonicum antigens and evaluate their immunogenicity and protective efficacy in the natural ovine host in China. Recombinant antigens selected for testing were: isoforms of glutathione S-transferase Sj28GST and Sj26GST; the large hydrophilic domain of Sj23, the homologue of the protective S. mansoni membrane antigen Sm23; and a 3' fragment of S. japonicum paramyosin. In addition, Chinese strain S. japonicum native paramyosin and GST were purified and used for vaccination. Antigens were co-administered with Freund's adjuvants or BCG. We also examined the effects of co-administration of native unfractionated GSTs with keyhole limpet haemocyanin (KLH), which shares a cross-reactive protective epitope with schistosomes. These are the first side-by-side comparisons of candidate defined-antigen schistosomiasis vaccines in a natural host. Significant partial protection was obtained with each of the antigens tested. Less protection was obtained with a recombinant fragment of S. japonicum paramyosin compared with native paramyosin. Co-administration of native GST and KLH was no more effective than vaccination with either antigen alone. Although encouraging levels of protection against S. japonicum were demonstrated using each of these antigens, further work is needed to optimise vaccine delivery and vaccination schedules.

Cloning of a Schistosoma japonicum gene encoding an antigen with homology to calreticulin.

A recombinant lambda gt11 clone, IVGS3, encoding part of a 55-kDa antigen was isolated from an adult Schistosoma japonicum cDNA library. The protein expressed by this clone was recognised strongly by serum from rats that had been vaccinated with irradiated cercariae (VrS) rendering them highly immune to a challenge infection. Antibodies in VrS which were specific for IVGS3 did not recognise adult worm antigens of S. mansoni, suggesting that the recombinant antigen contains species-specific epitopes, although IVGS3 was weakly recognised by rat serum raised against irradiated S. mansoni cercariae, indicating the presence of a related antigen in this species. A further clone, AM1(p), was obtained which, together with IVGS3 encompasses the entire coding region of the gene which has been called Sj55. Sequence analysis revealed similarities with murine calreticulin, a protein resident in the endoplasmic reticulum. As with murine calreticulin, Sj55 was shown to be a calcium-binding protein. Antigens with homologies to calreticulin have also been described in two other helminths, S. mansoni and Onchocerca volvulus.

Schistosoma mansoni: adult males and females differentially express antigens encoded by repetitive genomic DNA.

Two repetitive DNA sequences have been characterized from Schistosoma mansoni which were transcribed into mRNAs and translated to give two families of cross-reactive proteins. One DNA element, which was present as a 230 bp Pst I fragment was arranged in tandem arrays of at least 17 copies in the genome. The second element, which could be localized to a 1800 bp Pst I fragment, was dispersed in the genome. The 1800 bp repeat was found on the mRNA encoding the 45 kDa polypeptide precursor of a potential surface antigen. This precursor was post-translationally modified to give a 50 kDa antigen (Sm50) which was expressed from the cercaria to the adult worm and in the egg. However, a proportion of this antigen was differentially modified in females and eggs to give a 60 kDa form. Two copies of the 230 bp repeat and one copy of the 1800 bp repeat were found on a second cDNA clone. The antiserum raised against the fusion protein of this clone recognized a family of cross-reactive proteins ranging from 14 to 70 kDa in size. The members of this family were also differentially expressed between the sexes. Consequently, two families of antigens have been identified which were both encoded by repetitive DNA elements and whose members were both differentially expressed in adult male and female worms.

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.

Cloning of the gene encoding a 50 kilodalton potential surface antigen of Schistosoma mansoni.

A cDNA library was constructed from the mRNA of adult worms of Schistosoma mansoni, in the expression vector lambda gt11. This library was screened with a pool of sera raised against either soluble egg antigens or purified schistosomulum tegumental membranes. An antiserum raised against the fusion protein of one clone immunoprecipitated a 45 kDa polypeptide from the in vitro translation products of adult worm mRNA and recognised a 50 kDa antigen in homogenates of adult worms. This serum gave positive fluorescence of the surface of schistosomula in indirect immunofluorescence assays and was able to mediate killing of schistosomula by human eosinophils in vitro, suggesting that this clone contained part of a gene encoding a surface antigen.