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.

B-cell epitopes recognized by Chinese water buffaloes (Bos buffelus) on the 22 kDa tegumental membrane-associated antigen (Sj-22) of the Asiatic bloodfluke, Schistosoma japonicum.

The 22.6 kDa tegumental membrane-associated antigen of schistosomes is of recognized importance in immunity to schistosomiasis. In China, bovines are known to play an important role in the transmission of Schistosoma japonicum. Ten buffaloes (Bos buffelus) were vaccinated with a recombinant form (reSj-22) of the S. japonicum 22.6 kDa tegumental antigen (Sj-22) and the sera were used to identify and map possible linear B-cell epitopes on this molecule using a series of 18 overlapping synthetic peptides (P1-P18). Sera from all of the ten vaccinated buffaloes reacted strongly with Sj-22 in western blots and in ELISA, while sera from a further ten adjuvant (Quil A) control buffaloes did not. Four peptides (P3, P8, P9 and P10) were predominantly recognized by at least 90% of the buffalo sera. This pattern of recognition is similar to that obtained in a previous study we undertook in mice immunized with the same antigen whereby peptides 3, 8, 9 and 10 were recognized by over 80% of CBA strain mice. The peptide most frequently recognized by mice (peptide 6), and mapping to an EF-hand calcium binding domain, was recognized by six of the ten vaccinated buffaloes. The major difference between buffaloes and mice occurred with peptide 1 which was recognized very frequently by all three strains of mice tested but was only weakly recognized by three of the ten buffaloes. This study provides a valuable reference for further study on the immunity stimulated by the 22.6 kDa tegumental antigen in the murine model and a natural bovine host of Schistosomiasis japonica.

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.

DNA immunization by intramuscular injection or gene gun induces specific IgG antibodies against a Schistosoma japonicum 22 kDa antigen, Sj22, when fused to the murine Ig K-chain secretory leader sequence.

The 22 kDa tegumental surface membrane-associated antigen of Schistosoma japonica, Sj22, is of recognized interest in schistosomiasis vaccine research. However, previous attempts to induce antibody responses against Sj22 by DNA immunization have been unsuccessful. In this report we demonstrate that fusing the Sj22 cDNA to the murine immunoglobulin Ig kappa-chain secretory leader sequence results in the generation of antigen-specific IgG antibodies following DNA immunization. Mice were immunized into the skin with DNA-coated gold microspheres using a gene-gun, or into the quadriceps muscle by intramuscular injection. Both methods of delivery generated antigen-specific IgG antibodies against the 22 kDa schistosome antigen. The use of a secretory leader sequence, such as the murine Ig-kappa chain used in this study, may facilitate the induction of host antibody responses following DNA immunization with other parasite cDNAs.

Production of interleukin-10 by peripheral blood mononuclear cells from residents of a marshland area in China endemic for Schistosoma japonicum.

Interleukin-10 (IL-10) cytokine production was assessed using peripheral blood mononuclear cells (PBMC) from 67 individuals living in an area endemic for schistosomiasis japonica in China (Dongting Lake, Hunan Province), and 11 control subjects from a non-endemic part of the same Province. Production of IL-10 was measured following in vitro stimulation of PBMC using whole parasite extract (SWAP) or a panel of recombinant Schistosoma japonicum antigens (22-kDa tegumental membrane-associated antigen, glyceraldehyde-3-phosphate dehydrogenase, paramyosin, 14-kDa fatty acid-binding protein and 28-kDa glutathione S-transferase) which are of recognized interest in the development of protective immunity to schistosomiasis. Significantly, PBMC isolated from the exposed population compared with the non-exposed population produced higher levels of IL-10. There was a trend towards higher mean levels of IL-10 release in putatively resistant (insusceptible) (consistently egg negative but highly exposed) individuals compared with susceptible (egg-positive) subjects from the exposed population. Analysis of individual exposure (the duration of water contact and the percent body surface area in contact with water, expressed as m2 h/day) vs. IL-10 production indicated a weak but consistent and statistically significant inverse correlation, with lower levels of exposure being associated with higher levels of IL-10. These results suggest an association between IL-10 production and resistance to S. japonicum in subjects from this Chinese population exposed to infection.

Differential antigen-stimulated proliferation of human mononuclear cells by recombinant Schistosoma japonicum antigens in a Chinese population.

Peripheral blood mononuclear cells (PBMC) from 117 individuals living on two islands in an area (Dongting Lake) endemic for schistosomiasis japonica in China, and 15 control individuals from a non-endemic area of China, were assessed for antigen-stimulated proliferation against five recombinant Schistosoma japonicum antigens of recognized interest in the development of immunity to schistosomiasis. Two recombinant antigens, paramyosin and 28-kD glutathione-S-transferase, stimulated cellular proliferation (stimulation index > or = 3.0) in 38.5% and 42.5% of subjects, respectively, a level similar to that induced by a soluble whole parasite extract (51.3%). In contrast, three other recombinant antigens tested--a fatty acid binding protein, 22-kD tegumental membrane-associated antigen, and glyceraldehyde-3-phosphate dehydrogenase--stimulated PBMC proliferation in only 3-8% of subjects. Moreover, we also identified a positive association between the degree of exposure, and cellular proliferation following stimulation with recombinant paramyosin or whole parasite extract. Highly significant differences in antigen-stimulated proliferation were also observed between the two islands, Niangashan and Qingshan. The whole parasite extract stimulated proliferation in 90% of subjects from Niangashan island compared with only 42.1% of subjects from Qingshan island (chi2 = 16.88, P = 0.00004), while glutathione-S-transferase stimulated proliferation in 77.3% of subjects from Niangashan island compared with only 34.7% of subjects from Qingshan island (chi2 = 13.09, P = 0.003). A similar, but not significant, trend was observed for paramyosin and the fatty-acid binding protein. The identification of differential cellular proliferative responses to specific schistosome antigens within an infected human population may have important practical implications for vaccine development against schistosomiasis japonica.

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.

Mapping of linear B-cell epitopes on the 14-kDa fatty-acid binding protein of Chinese Schistosoma japonicum.

The 14-kDa fatty-acid binding protein (FABP) of schistosomes is of recognised importance as a potential vaccine and/or drug target against schistosomiasis, but little is known of its antigenicity. In this study, we have identified and compared linear B-cell epitopes present on the FABP of Chinese strain Schistosoma japonicum, using sera obtained from experimentally infected mice, or from mice immunised with the functionally active recombinant antigen (rSjFABP). Sera from three strains of mice, CBA, C57BL/6 and BALB/c, representing different genetic backgrounds, were reacted with a series of overlapping peptides in epitope-scanning studies. Sera from experimentally infected mice reacted predominantly with peptides 9-12, encoding amino acids 91-132, in the C-terminal region of the molecule. This was in contrast to sera from mice immunised with rSjFABP, which reacted predominantly with peptides 4-9, encoding amino acids 41-101, in the central portion of the molecule. The results presented here describing the epitope mapping of this molecule may prove important in research aimed at further defining immune responses to schistosomal antigens. They indicate that epitopes recognised during vaccination with functionally active rSjFABP, at least in the murine model, differ from those recognised during natural infection.

Measuring exposure to S. japonicum in China. II. Activity diaries, pathways to infection and immunological correlates.

In this study we examine the pathways to schistosomiasis exposure and infection among residents residing on two islands (large, Qingshan; small, Niangashan) in the Dongting Lake region (Hunan province) of China. An exposure model, based on activity diaries, was used to quantify an individual's square-metre-minute (sq.m.min) daily water contact. Subjects living on the small island had a significantly higher (P=0.0002) degree of exposure (mean+/-S.D., 13.2+/-11.0 sq.m.min) than individuals dwelling on the large island (mean+/-S.D., 5.5+/-7.1 sq.m.min). Participants identified as stool egg positive (mean+/-S.D., 8.3+/-10.4 sq.m.min) had higher exposures than for those never treated (mean+/-S.D., 2.2+/-3.4 sq.m.min) for schistosomiasis, and these high exposures rose steadily to peak at 35-49 years of age and decline after age 50. This exposure pattern differs markedly from those reported for African or South American schistosomiasis. The majority of human water contact occurs on the lake. Egg-positive subjects reported significantly higher (P < 0.05) episodes of water contact on the lake versus their egg-negative counterparts, who reported significantly higher (P < 0.01) exposure at the aquaculture ponds. The results of path analysis revealed that sex, age, island of residence and whether a fisherman or not were the most highly significant independent predictors of lake exposure. This accounted for approximately 40% (R2=0.39) of the total lake exposure. Exposure to lake water was a strong predictor (P=0.0006) of past infection and a modest predictor (P=0.05) of current infection.

Epidemiological identification of Chinese individuals putatively susceptible or insusceptible to Schistosoma japonicum: a prelude to immunogenetic study of human resistance to Asian schistosomiasis.

An epidemiological method, field-tested in Hunan, China, to identify residents potentially susceptible or insusceptible to endemic schistosomiasis japonica is described, as a prelude to selection of subjects for immunogenetic studies. After an initial cross-sectional survey on two islands (Qingshan and Niangashan--population 2990) in 1995-1996, an informative cohort (N = 249) was selected for treatment and 9-month follow-up to measure exposure and re-infection. Both the population prevalence (15.8%) and the geometric mean intensity of infection (26.2 eggs/g faces) indicated that the islands were moderately endemic for schistosomiasis. Exposure measurements revealed a strong, positive, linear association (r = 0.70) between daily activity diaries and direct water-contact observation. Individuals identified as stool-positive for schistosomiasis had significantly more water contact than those who were egg-negative (P = 0.03). Almost all (93%) of the cohort had ultrasonographic evidence of periportal fibrosis before treatment but in only 1.2% was this fibrosis scored > 1 in terms of the stages identified by the World Health Organization. At the follow-up it was possible to classify the 249 subjects into three, distinct, exposure-infection epidemiological groups. The first group (N = 20) was susceptible to re-infection and constituted 8% of the cohort. The second group (N = 61) was apparently insusceptible to re-infection despite the continuing high levels of exposure and included 24% of the cohort. The other 68% of the cohort (N = 168) remained uninfected but were at most only moderately exposed, or had a status indeterminate due to non-compliance. This epidemiological identification of susceptibles and insusceptibles for schistosomiasis japonica' links field and ongoing laboratory studies aimed at characterising the genetic and immunological factors associated with resistance to re-infection and/or disease.

A dominant B-cell epitope on the 22 kDa tegumental membrane-associated antigen of Schistosoma japonicum maps to an EF-hand calcium binding domain.

The 22 kDA tegumental surface membrane-associated antigen of schistosomes is of recognized importance in immunity to schistosomiasis. Here, we have defined linear B-cell epitopes on the recently cloned and expressed recombinant Schistosoma japonicum 22 kDa antigen (reSj22), using mice immunized with this molecule. Sera from three strains of mice, CBA, C57BL/6 and BALB/c, representing different genetic backgrounds, were reacted with a series of overlapping synthetic peptides in epitope-scanning studies. The predicted hydrophilic N-terminal domain was found to be highly immunogenic, containing several sequences that were strongly recognized by all strains of mice. The most dominant epitope identified, corresponding to peptide 6, was located in a region previously identified as an EF-hand calcium binding domain. In contrast, the more hydrophobic C-terminal region of the molecule was poorly recognized, with the exception of a single peptide encoding residues 121-140 that was recognized by CBA and C57BL/6 but not BALB/c mice, suggesting that the latter epitope was genetically restricted between the strains. The data presented here describing the epitope mapping of this molecule may prove important in research aimed at further defining immune responses to schistosomal antigens.

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.

Schistosomiasis vaccine development--the current picture.

Development of a vaccine for schistosomiasis, a parasitic disease currently affecting over 200 million people worldwide, has been targeted as a priority by the World Health Organisation. Research demonstrating the ability of humans to acquire natural immunity to schistosome infection, together with the successful use of attenuated vaccines in animals both under laboratory and field conditions, suggest that development of a human vaccine is feasible. Attenuated vaccines for schistosomiasis are considered neither safe nor practicable for human use, however, and therefore other approaches must be considered. This review examines progress currently being undertaken in a number of different areas towards achieving the goal of a safe and effective human vaccine for schistosomiasis.

Production of IgE antibodies against the 22 kDa tegumental membrane-associated antigen of schistosomes is directed by the antigen itself.

It has previously been reported that the predominant target of immunoglobulin E (IgE) recognition in sera from humans infected with Schistosoma japonicum in The Philippines or with S. mansoni in Kenya, is a 22 kDa tegumental membrane-associated schistosome antigen. In the present study, we demonstrate that the 22 kDa antigen can direct the production of antigen-specific IgE antibodies independently of schistosome infection and in the absence of any other parasite components or adjuvant. Three strains of mice were immunized using the purified, recombinant 22 kDa antigen of S. japonicum without the use of any adjuvant. Sera from all three strains of immunized mice, but not control animals, generated IgE antibodies specific for the native 22 kDa schistosome antigen in Western blots. Thus, the 22 kDa antigen itself must contain signals (presumably encoded by the primary amino acid sequence or by the secondary or tertiary structures of the molecule, or by a combination of these) which are sufficient to direct the isotype switch required for production of antigen-specific IgE.

Antibodies to Schistosoma japonicum (Asian bloodfluke) paramyosin induced by nucleic acid vaccination.

Nucleic acid vaccination by intramuscular or intradermal delivery of DNA plasmids encoding antigenic proteins has been shown to confer protection in experimental animals against viruses and unicellular protozoan parasites. However, this revolutionary approach has not been tested for induction of immunity to multicellular parasites, such as trematode worms. We report here, for the first time, that murine antibodies can be induced by intramuscular injection with plasmid DNA encoding fragments of Schistosoma japonicum paramyosin (Sj97), a 97 kDa molecule and a promising vaccine candidate in schistosomiasis. An additional construct containing the gene encoding full-length glutathione S-transferase (Sj26), another recognised anti-schistosome vaccine target, failed to raise detectable levels of specific antibody.

Schistosoma japonicum: heterogeneity in paramyosin genes.

Paramyosin is an integral muscle protein found in many invertebrates including schistosomes, and is considered an important candidate vaccine antigen in schistosomiasis. The characterisation of natural molecular variation in vaccine antigens including paramyosin is important because strain-specific vaccination may be necessary against schistosomiasis japonica. We have isolated partial cDNAs encoding paramyosin from an adult, Chinese strain Schistosoma japonicum gene library. Two of these cDNAs (B6 and Y6) encode the same region of paramyosin but their nucleotide sequences differ at eight positions and their deduced amino acid sequences differ by an arginine/cysteine substitution, demonstrating intrastrain variation in paramyosin. Southern blot analysis of genomic DNA from the Chinese and Philippine strains of S. japonicum demonstrated strain-related RFLPs at the paramyosin locus, and suggested that more than one copy of the paramyosin gene was present in the S. japonicum genome. PCR-based RFLP analysis which exploited restriction site differences between B6 and Y6 showed that paramyosin genotype B6 was much more common in schistosome populations and verified the existence of introns in the paramyosin gene(s) of S. japonicum.

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.

Nucleic acids: vaccines of the future.

The recent successful immunization of experimental animals using nucleic acids has provided a revolutionary new approach in vaccinology. In this article, Gary Waine and Don McManus examine the potential of nucleic acid vaccines for their effectiveness not only against infectious and parasitic organisms exhibiting an intracellular phase during their life cycle, but also against parasitic helminths, whose life cycle stages are either predominantly or completely extracellular.

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.