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Many parasitic helminths produce large quantities of glycosylated proteins, some if which are believed to be involved in the skewing towards the dominant Th2 response observed during helminth infection. Galectin-3 is a member of a family of lectin-binding proteins produced by many different types of immune cells, including macrophages. Galectin-3 recognizes the GalNAcbeta1-4GlcNAc (LDN) epitope present on many helminth antigens, including those of the schistosome eggs. Here we show that galectin-3 is not involved in the development of the Th2 response nor in schistosome granuloma formation. Galectin-3-deficient mice were able to expel the gastrointestinal nematode Trichuris muris at the same speed as wild-type mice. Expulsion of T. muris is known to be dependent on a Th2 immune response and galectin-3-deficient mice showed no defect in their ability to produce Th2 cytokines or in their antibody responses, compared to wild-type mice. Furthermore, galectin-3-deficient mice were also able to mount a Th2 response to Schistosoma mansoni infection and they exhibited normal hepatic granuloma formation. The data presented here demonstrate that galectin-3 is not a critical component in the development of Th2 responses during helminth infection in vivo, nor is it essential for schistosome egg granuloma formation.

gamma-irradiated cercarial vaccines induce high levels of protection in mice against Schistosoma mansoni infection, however, the same has not been well established for S. japonicum. Here we describe vaccination studies in mice with gamma-irradiated S. japonicum cercariae testing the effectiveness of different irradiation doses, number of vaccinations, and mouse strains. In CBA/Ca mice, a single percutaneous exposure to 500 S. japonicum cercariae previously attenuated by 10, 20, 30, 40 or 50 krad gamma-irradiation induced significant, but comparable levels of protection (34-46%) against challenge infection. In a repeat experiment in C57Bl/6 mice, only groups vaccinated with 10 or 20 krad gamma-irradiated cercariae showed statistically significant, but lower levels of resistance (20-24%). Multiple vaccination of CBA/Ca mice with 500 20 krad gamma-irradiated cercariae did not improve the resistance level (40%). Analysis of IgG responses showed no clear correlation between antibody levels and levels of protection. Western blot analysis suggested that recognition of a 200-kDa antigen might be correlated with protection, that antigens of 42 and 50 kDa may be involved in the protection induced by single vaccination, but that different antigens might be protective in single vs multiple vaccinations. Sera from mice vaccinated with gamma-irradiated cercariae recognized many fewer antigens than more protective sera from mice vaccinated with UV-attenuated cercariae. These results suggest that the mouse may not be a suitable host for studies involving gamma-irradiated S. japonicum vaccines.

The Schistosoma japonicum homologue of the 62 kDa fragment of S. mansoni myosin (SmIrV-5), which has proved highly protective against S. mansoni infection in mice and rats, has been cloned and expressed as the full length 62 kDa equivalent, Sj62, and a truncated 44 kDa version, Sj44. DNA sequencing showed the Sj62 sequence to be 88.4% identical at the nucleic acid level and 96% identical in deduced amino acid sequence to that of SmIrV-5. The recombinant proteins (rSj44 and rSj62) were strongly recognized in Western blotting by sera from mice multiply vaccinated with UV-irradiated S. japonicum cercariae and weakly recognized by S. japonicum chronic infection mouse sera. Unlike SmIrV-5, mouse antisera against the recombinant S. japonicum proteins did not give positive recognition in immunofluorescence assay with the surface of newly transformed schistosomula of the homologous species, S. japonicum, nor did they react with S. mansoni schistosomula. However, the anti-rSj62 sera clearly localized the native antigen to the subtegumental muscle layers in male adult worm sections by immunoelectron microscopy. Vaccination of several groups of mice and/or rats with rSj44 and rSj62 incorporated into different adjuvants induced high titres of specific IgG but in only one experimental group was there a significant reduction in worm burden (27%, P < 0.05). The possible reasons for the disparity between the vaccination results presented here and those demonstrated in experiments using rSm62 (IrV-5) are discussed.

As with 20 krad-irradiated infections in mice, the present study shows that the immunity induced by Ro11-3128 termination of unattenuated infections at the skin stage is species specific, not operating against S. japonicum. Treatment with the drug Ro15-5458, also effective at the skin stage, however, resulted in significantly lower levels of resistance than Ro11-3128. Sera from mice immunized by infection plus Ro11-3128 treatment on days 1 or 2 (Ro11S) coprecipitated essentially the same pattern of 125I-labelled surface antigens as the 20 krad vaccine serum (VMS), viz. Mr 38,000, 32,000, 23,000 and 15,000. However, recognition by Ro11S was markedly stronger. Sera from the infected and Ro15-5458-treated mice (Ro15S) failed to recognize the Mr 23,000 antigen and produced a weaker response than Ro11S or VMS against the Mr 38,000 or 32,000 antigens but a comparable response to VMS against the Mr 15,000 antigen. Ro11S and VMS also recognized the Mr 16,000 surface antigen seen by Western blotting but its recognition by Ro15S was weaker. Compared with sera from animals treated at the skin stage, sera from animals treated at the lung stage (day + 6) showed weaker recognition of the Mr 32,000 and 15,000 antigens and no recognition of the Mr 23,000 antigen. In contrast, sera from mice treated at 15 days recognized both the Mr 32,000 and 23,000 antigens but not the Mr 15,000 antigen. Mice treated at these times show progressively less immunity than at the skin stage. Infected but untreated animals only showed significant recognition of the Mr 32,000 antigen. Thus compared with infections treated with Ro11-3128 on days 1 or 2, treatment at later times or with the drug Ro15-5458 resulted in selective and differential absence or diminution of response against either the Mr 38,000, 32,000, 23,000, 16,000 or 15,000 antigens. In vitro, Ro11-3128, in contrast to Ro15-5458, caused multiple vesicle formation at the surface of skin stage schistosomula but this was progressively less pronounced with lung and liver stage worms. The vesicles were shown to express surface membrane antigens but were apparently not derived from the existing outer leaflet of the surface membrane. It is suggested that this altered antigen expression might explain the optimum immunity induced.

Levels of antibody a binding to the schistosomulum surface and b mediating in vitro eosinophil-dependent cytotoxicity of schistosomula were studied and compared to the in vivo levels of resistance to cercarial challenge in mice infected with irradiated cercariae, unirradiated cercariae of single or mixed sex, or injected with eggs. Antibody-binding was assessed by counting the number of IgG-Fc-receptor bearing cells (P388D1 cells) adhering to mechanically-transformed schistosomula. Significant levels of adherence occurred with sera taken from 1--2 weeks following exposure to irradiated cercariae, the level increasing gradually thereafter and being enhanced by repeated exposure. Sera from the bisexual infection showed a dramatic increase in binding activity between weeks 5--8, and with the single sex infections there was a steady rise up to week 10 followed by a sharp rise between weeks 10--12. Weekly injections of eggs produced a steady rise in serum binding activity. Sera taken just before challenge were also tested for their ability to mediate killing of schistosomula by eosinophil-enriched preparations of heterologous rat peritoneal exudate cells in vitro. Significant levels of killing occurred with all sera, but the greatest lethal activity was found in sera from the egg-injected, chronically-infected and 200 male cercariae-infected groups. This ranking did not correlate with in vivo resistance against challenge as assessed by worm recovery, the egg-injected and single sex-infected groups failing to manifest significant resistance.

We have characterized various anti-schistosomular surface MoAbs previously shown to partially block in-vitro killing of schistosomula by human sera and eosinophils (Dunne et al. 1987). Immunodiffusion analysis showed that four IgM and one IgG3 MoAbs recognized periodate sensitive epitopes on the same molecular species present in schistosomular antigen but their patterns of reactivity with soluble egg antigen demonstrated that at least three distinct epitopes were involved. SDS-PAGE analysis showed the IgMs to react with 125I-labelled surface antigens of Mr 35,000-38,000 and Mr 20,000, and the IgG3 to react with an Mr 38,000 antigen. In spite of their effect in vitro, transfer of the IgM MoAbs at the time of challenge of mice vaccinated with irradiated cercariae or of mice injected with an unrelated (anti-Mr 16,000) protective MoAb failed to produce in-vivo blocking. Similarly, injection of Schistosoma mansoni eggs, prior infection with worms of one sex, or passive transfer of serum from single-sex infected mice failed to influence the resistance conferred by vaccination with irradiated cercariae.