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Parasitic helminths possess surface glycoconjugates that are recognized by the serum collectin molecule, mannose-binding lectin (MBL). Once bound, MBL triggers the lectin pathway of complement. Mice have two MBL, MBL-A and MBL-C. We previously showed that MBL-A deficient (MBL-A(-/-)) mice have enhanced survival of Brugia malayi microfilariae and abrogated microfilariae-specific IgM responses. In this study we show that MBL-A deficiency does not alter immunity to either Trichuris muris or Schistosoma mansoni. However, anti-nematode IgM levels were significantly lower in T. muris infected MBL-A(-/-) than wild-type mice. Interestingly nematode-specific IgG1 and IgG2a levels were higher in MBL-A(-/-) mice. Although, larval schistosomes are surrounded by a complement-sensitive membranous tegument, neither adult worm development, egg output, egg granuloma size nor cellular composition was affected in MBL-A(-/-) mice. In contrast to anti-nematode IgM responses, anti-schistosome IgM (and also IgG1 and IgG2b) responses were unaltered from wild-type mice. Anti-schistosome IgG2a was elevated, while IgG3 was significantly lowered, in MBL-A(-/-) mice. These results suggest that MBL-A is not a necessary component for immunity to either T. muris or S. mansoni helminths, however, MBL-A appears to be necessary for the development of specific IgM responses to nematode antigens.

We have previously shown that it is the lung stage schistosomulum which is both the inducer and the target of immunity in rats vaccinated with irradiated cercariae but that rats can also be partially protected by immunization with detergent extracts of mechanically-transformed schistosomula. In the present study we therefore compared the immunogenicity of these and intermediate schistosomular stages. In the first experiment low levels of protection (13-24%) were induced by extracts of mechanically-transformed schistosomula cultured for 3 hours or 2 days but not by extracts of lung schistosomula. In a repeat experiment insignificant levels of protection were induced by extracts of 3 h schistosomula and lung schistosomula were again non-protective. Nevertheless, high levels of anti-schistosomula antibodies were demonstrated and sera from actively immunized rats conferred significant passive protection in five out of six trials. The levels of protection conferred by passive immunization (26-51%) were in each case higher than the levels of protection demonstrated in the respective serum donor groups, showing that some form of immunological blockade suppression is operating to prevent expression of protective immunity in the actively immunized rats.

Keyhole limpet haemocyanin (KLH) was compared with Schistosoma haematobium soluble egg antigen (SEA) in the enzyme-linked immunosorbent assay (ELISA) for detection of S. haematobium infection, using 187 human sera collected from the S. haematobium endemic area of Pemba Island, Tanzania, and 30 normal sera from blood donors in Europe. There was a clear separation in terms of immunoglobulin (Ig) G and IgM titres between parasitologically positive patients and the blood donors, but titres of many parasitologically negative individuals in the endemic area were significantly high in comparison with the normal controls. Using as cut-off point the mean optical density +2 SD of sera from the blood donors to define ELISA positivity, and comparing the results with urine egg counts, the sensitivity of IgG-ELISA using KLH or SEA was high (91.11% and 95.56%, respectively) but the specificity was poor (43.30% and 31.90%, respectively. Similar results were obtained with IgM. When the 'gold standard' of haematuria and/or egg positivity as indicative of infection was used, the sensitivity of the ELISAs was similar but the specificity was increased to 59.25% and 44.44%, respectively. These results suggest that the patients with haematuria were probably infected with S. haematobium, which further supported the diagnostic value of haematuria detection for S. haematobium infection in endemic areas, and KLH was found to have a potential use in immunodiagnosis of S. haematobium infection in endemic areas. With both KLH and SEA antigens, the trend of reactivity in ELISA provided a correlate of the egg output (parasite burden) of infected patients.

A Schistosoma haematobium adult worm cDNA library was screened with sera from demonstrably immune baboons (mean worm reduction 94%) vaccinated with highly-irradiated cercariae (VBabS) and also with sera from adult S. haematobium-infected humans (HIS Sh). As species-specificity is a characteristic feature of the immunity induced by irradiated cercariae, S. haematobium species-specific clones were identified by rescreening positives with sera from S. mansoni-vaccinated animals or infected humans. Six species-specific clones, 3 initially detected with vaccinated baboon sera (D11, D14 and D26) and 3 with human infection sera (E1, E2 and E16) were further characterized by Western blotting. The three HIS-selected clones did not react significantly with VBabS or acutely-infected baboon sera (ABabS) while D11, D14 and D26 showed increasing reactivity with successive vaccinations. In addition D11 and D14 but not D26 responded to ABabS. When tested against HIS Sh from 24 patients of varying ages, D11 and D26 reacted most frequently with sera from individuals in the older age groups (> 17 yrs). A species cross reactive clone, D12, which was used as a positive control throughout, was found to react with all schistosome specific sera tested.

Sera from rabbits vaccinated with irradiated cercariae mediated cell (P388D1 or mouse peritoneal macrophage) adherence to lung-stage schistosomula (LS) but such antibody-mediated cell adherence was short-lived in contrast to cell adherence to mechanically transformed schistosomula (MS). Thus LS lost 50% of their adherent cells within 3-6 h in culture and up to 90% by 24 h, whereas adherence to MS was undiminished during this time. Rapid loss of adherent cells was unique to schistosomula that had developed to the lung stage because schistosomula recovered from the skin up to 3 days post-infection did not exhibit the rapid cell loss shown by 3-day LS. To determine whether cell loss was caused by loss of surface antigenicity during culture LS were cultured on their own for up to 24 h and at various intervals samples of schistosomula were tested for antigenicity by addition of immune serum and cells. Levels of adherence to both MS and LS were maintained throughout the incubation period. When antibody-opsonized schistosomula were washed and indicator cells added at progressive intervals, persistence of adherence was again demonstrated, showing that antibody binding to LS had not promoted surface antigen loss or degradation of bound antibody. It was then shown, by adding fresh macrophages to cultures up to 24 h old that LS which had lost their adherent cells nevertheless retained bound antibody, and comparison of adherence of 'used' and 'fresh' cells to MS and LS showed that the cytoadherence properties of macrophages were not significantly reduced during their culture with LS from which cells had been lost.(ABSTRACT TRUNCATED AT 250 WORDS)

Starch-elicited murine peritoneal macrophages were able to kill schistosomula in vitro in the presence of a variety of immune sera. Dose response experiments revealed the superior "quality" of serum from mice vaccinated four times with highly irradiated cercariae (4xVMS) in mediating killing at titers comparable to the other sera tested. B3A, a partially protective mAb (IgG3) that recognizes a Mr 16,000 schistosomular surface Ag, mediated higher levels of killing than any of the sera at comparable titers. In contrast, H12, a partially protective mAb (IgG2a; anti-Mr 32,000), and C1C9, a nonprotective McAb (IgG3; anti-Mr 38,000) failed to mediate killing. Two anti-Mac-1 alpha-chain mAb (5C6 and M1/70) mediated substantial dose-dependent blocking of 4xVMS and B3A-mediated macrophage killing. In contrast, a mAb to the Mac-1-associated beta-chain was less effective, whereas the mAb F4/80 did not significantly block killing despite being present on this macrophage population. Although whole 5C6 Ig was the most efficient at inhibiting B3A-mediated killing, 5C6 Fab fragments were still effective at concentrations as low as 0.5 microgram/ml (10 nM). On a molar basis 5C6 appeared to be more effective at blocking 4xVMS-mediated killing than M1/70, while only M1/70 was capable of inhibiting macrophage adherence to schistosomula. These findings, together with the observation that anti-alpha chain mAb were far more effective at blocking killing than the anti-beta-chain mAb, rules out the possibility that 5C6 is nonspecifically inhibiting B3A-FcR interaction. The data also imply a functional relationship between Mac-1 and FcRIII, the receptor for B3A, in macrophage killing.

Sera from rabbits and rats vaccinated with highly irradiated cercariae of Schistosoma mansoni (VRabS, VRatS) were found to be of substantially higher affinity than sera from CBA mice vaccinated four times (4 X CVMS), single sex sera (SSS) or chronic infection sera (CIS). In contrast, VRabS and SSS appeared to possess the highest titres of antibody, followed by CIS and VRatS, with 4 X CVMS displaying the lowest titre. Two mouse strains selectively bred for high-affinity (HA) or low-affinity (LA) antibody following vaccination were tested for their ability to resist a challenge infection. LA mice, which produce high titres of low-affinity antibody, manifested significantly more resistance than HA mice, which produce low titres of high-affinity antibody. Immunoprecipitation studies demonstrated that sera from vaccinated LA mice (LVMS) recognized 125I-labelled schistosomular surface antigens more intensely than sera from vaccinated HA mice (HVMS). However, peritoneal macrophages from HA and LA mice in the presence of HVMS, LVMS or 4 X CVMS, and naive macrophages activated in vitro with interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS) mediated comparable levels of schistosomula killing in vitro. The experiments described here provide evidence that the titre of antibody rather than its affinity may be a more critical factor in the development of optimal immunity to S. mansoni.

Absorption of serum from chronically infected mice with homogenized schistosome eggs reduced antibody binding to the schistosomulum surface by 94%, indicating that almost all schistosomulum surface recognition during chronic infection is due to epitopes shared with the egg. Absorption of the serum with egg homogenate from which protein antigens had been removed by boiling and digestion with proteinase K resulted in a similar reduction of antisurface antibody demonstrating that all the shared epitopes that are recognized are carbohydrate in nature. Analysis of the time course of anticarbohydrate antibody production and the levels of antibody in mice infected with a single sex of schistosome indicated that eggs directly stimulated this response. Mouse mAb were identified that bound at very high levels to the schistosomulum surface and that recognized carbohydrate epitopes shared with the egg. Three of these had previously been demonstrated to passively transfer resistance, indicating that these surface carbohydrates are potential targets of protective immunity in the mouse. All the anticarbohydrate mAb also bound to the surface of schistosomula of other schistosome species. Thus, the strong immune response against these epitopes in chronic infection could account for the cross-specific immunity observed. Mice vaccinated with irradiated cercariae lacked high levels of anticarbohydrate antibodies and their recognition of the surface was largely due to antibody to species-specific polypeptide epitopes. With respect to the Mr greater than 200,000 and 38,000 antigens, it was demonstrated that these epitopes were present on the same antigens that bear the carbohydrate moieties recognized by antibodies from chronically infected mice. This specific polypeptide recognition is also reflected in the immunity generated by exposure to irradiated cercariae.

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.