Gary J. Waine

stevor and rif are Plasmodium falciparum multicopy gene families which potentially encode variant antigens

Several multicopy gene families have been described in Plasmodium falciparum, including the var genes that code for the variant surface antigen PfEMP1, the stevor family of subtelomeric open reading frames and the rif interspersed repetitive elements. This report documents the chromosomal location of stevor genes, their transcription and characteristics of the deduced protein. On 14 chromosomes, 34 stevor copies were identified from the Dd2 parasite line. Most are in subtelomeric regions within 50 kb of the telomere. stevor genes are located close to var genes and rij. All stevor genes sequenced had two exons: a short exon 1 encoding a start codon and a transmembrane domain; exon 2 encoding for the remainder of the approximately 30 kDa protein and including two more transmembrane segments. A similar structure was found for copies of rif and its predicted protein. In both STEVOR and RIF proteins, a highly polymorphic region is predicted to be a loop on the outer side of the membrane. We propose that stevor and rif are members of a larger superfamily. The number of copies of stevor and rif, their location close to the var genes, their extreme polymorphism and the predicted structure of the proteins suggest that stevor and rif code for variant surface antigens.

Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPc) from the human blood fluke Schistosoma japonicum

We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj). As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target. Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins. The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa. The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S. mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis. Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli. The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica. The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity. The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.

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.

Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: A potential vaccine candidate against schistosomiasis

Paramyosin from the blood fluke, Schistosoma mansoni, has shown promise as a vaccine candidate for schistosomiasis mansoni. Here we report the cloning and partial nucleotide sequence of a cDNA encoding paramyosin from the related human parasite, Schistosoma japonicum. Affinity purified antibodies to this clone recognized a S. japonicum antigen of molecular weight 97 kDa, equivalent to the reported size of S. mansoni paramyosin. Alignment of the cDNA sequence with that of S. mansoni paramyosin revealed 90% identity. Comparison of the predicted amino acid sequences revealed 95% identity. Although these two parasites differ in many characteristics, the substantial homology demonstrated here between S. mansoni and S. japonicum paramyosin could have important implications for the development of a S. japonicum vaccine.

HLA class II antigens positively and negatively associated with hepatosplenic schistosomiasis in a Chinese population.

To identify possible associations between host genetic factors and the onset of liver fibrosis following Schistosoma japonicum infection, the major histocompatibility class II alleles of 84 individuals living on an island (Jishan) endemic for schistosomiasis japonica in the Poyang Lake Region of Southern China were determined. Forty patients exhibiting advanced schistosomiasis, characterised by extensive liver fibrosis, and 44 age and sex-matched control subjects were assessed for the class II haplotypes HLA-DRB1 and HLA-DQB1. Two HLA-DRB1 alleles, HLA-DRB1*0901 (P=0.012) and *1302 (P=0.039), and two HLA-DQB1 alleles, HLA-DQB1*0303 (P=0.012) and *0609 (P=0.037), were found to be significantly associated with susceptibility to fibrosis. These associated DRB1 and DQB1 alleles are in very strong linkage disequilibrium, with DRB1*0901-DQB1*0303 and DRB1*1302-DQB1*0609 found as common haplotypes in this population. In contrast, the alleles HLA-DRB1*1501 (P=0.025) and HLA-DQB1*0601 (P=0.022) were found to be associated with resistance to hepatosplenic disease. Moreover, the alleles DQB1*0303 and DRB1*0901 did not increase susceptibility in the presence of DQB1*0601, indicating that DQB1*0601 is dominant over DQB1*0303 and DRB1*0901. The study has thus identified both positive and negative associations between HLA class II alleles and the risk of individuals developing moderate to severe liver fibrosis following schistosome infection.

Antibody isotype responses, infection and re-infection for Schistosoma japonicum in a marshland area of China.

Antibody isotype responses to adult worm antigen (AWA) of Schistosoma japonicum and two recombinant proteins (paramyosin (PMY) and a 22 kDa tegumental membrane-associated antigen (TEG)) were analyzed in 137 individuals from an area moderately endemic for schistosomiasis in the Dongting Lake region, Hunan Province, China. The prevalence and geometric mean (GM) intensity of infection before the implementation of curative chemotherapy were 28.5% and 234.4 epg, respectively, but 9 months after treatment the prevalence (6.6%) and intensity (38.3 epg) had decreased. There was no significant difference in either the prevalence or intensity of infection between males and females. Specific IgG (total), IgG4, IgG2, IgA and IgE responses to AWA, PMY and TEG were measured by ELISA. Males produced significantly (P < 0.05) more anti-AWA total IgG, IgE, IgA, IgG4 and IgG2 antibodies, and anti-TEG IgG2 antibody than their female counterparts. The OD450 levels of anti-AWA, PMY and TEG antibody isotypes did not present clear age-dependent trends except for peak levels of anti-AWA IgG4 antibodies evident among subjects 20-29 years of age. The total IgG and IgG4 antibody profiles against AWA correlated well with current S. japonicum infections while anti-AWA IgG2, IgA and IgE antibodies did not show such an association. Anti-AWA-specific IgE antibody levels were positively correlated (r = 0.55) with anti-AWA specific IgG4 antibody levels. In addition, the overall percentage of responders (using a cut-off value obtained from normal controls) to all isotypes to AWA were higher than those observed for both the recombinant antigens. Only 18.2%, 16.8% and 7.3% of the study population were IgE responders to AWA, PMY and TEG. A longer follow-up period is required before we can more fully understand the role of IgE, if any, in protective immunity against schistosomiasis japonica.

Genetic immunization of mice with DNA encoding the 23 kDa transmembrane surface protein of Schistosoma japonicum (Sj23) induces antigen-specific immunoglobulin G antibodies

The 23 kDa transmembrane surface protein of schistosomes is of recognized interest in studies of immune responsiveness in schistosomiasis. To examine the immunogenicity of the 23 kDa antigen of Schistosoma japonicum, Sj23, when delivered by genetic immunization, mice were immunized using a DNA construct containing the Sj23 cDNA under the control of a CMV promotor. Serological analysis of peripheral blood from immunized mice demonstrated that this construct was able to induce the production of antigen‐specific IgG antibodies that recognized a schistosome antigen of 23 kDa in Western blots. Despite inducing antigen‐specific antibodies, the Sj23 DNA vaccine was unable to confer protection in immunized mice subjected to challenge with S.japonicum cercariae. Appropriate engineering of the unique structure of the Sj23 kDa transmembrane protein of schistosomes may provide a novel vehicle for expressing foreign epitopes from other infectious agents or, possibly, cancer antigens, anchored to the surface of transfected cells.

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.

HLA Class II antigens are associated with resistance or susceptibility to hepatosplenic disease in a Chinese population infected with Schistosoma japonicum

The major histocompatibility Class II alleles of 108 individuals living in an area endemic for schistosomiasis japonica in China were determined to identify possible immunogenetic associations with advanced schistosomiasis. Two alleles, HLA-DRB1*1202 (P = 0.002) and HLA-DQA*0601 (P = 0.001) were strongly associated with resistance to advanced disease. In contrast, HLA-DQB1*05031 (P = 0.02) was associated with susceptibility to advanced schistosomiasis. The remaining alleles showed no association with advanced disease. Allele DRB1*1202 co-occurred with allele DQA1*0601; therefore, their independent protective effects could not be ascertained. In contrast, alleles DQA1*0601 and DQB1*05031 never co-occurred and had opposite and significant effects on the occurrence of disease.