Schistosoma mansoni: larval damage and role of effector cell(s) in the synergy between vaccine immunity and praziquantel treatment.

A number of authors have demonstrated that the schistosomicidal compound, Praziquantel (Pzq), depends for its action upon the immune status of the host (Sabah et al. 1985; Brindley & Sher, 1987; Doenhoff et al. 1987). We have attempted to define the synergistic interaction between immuno- and chemotherapy further, using the murine irradiated vaccine model of schistosomiasis mansoni. In vaccinated mice, resistance operates in the skin but not the lungs; drug targeted towards lung-stage worms exacerbates lung-phase immunity, however, as depicted by the increased number and size of inflammatory reactions in the pulmonary tissues. Parasites are often found trapped within such foci. In the present investigation, light and ultrastructural studies have been utilized to examine the nature and extent of damage inflicted upon lung-stage larvae recovered from day 6 Pzq-treated vaccinated mice. Such studies have revealed that damage involves muscle disorganization, internal disruption and occasionally, loss of the tegument; in the latter case, cells are often seen attached to the denuded lung worms. To identify the crucial cellular effector cell(s) involved in the synergy between immuno- and chemotherapy, cell depletion studies have been performed in vivo. It would appear from these experiments that eosinophils or lymphocytes rather than neutrophils or macrophages are important effector cells in this synergy. Histological studies argue in favour of eosinophils being the key effector cells.

Trichuris muris: antigen recognition and transfer of immunity in mice by IgA monoclonal antibodies.

Mesenteric node lymphocytes from mice that had been infected with the nematode Trichuris muris, and then boosted with adult worm excretory-secretory antigens were fused with myeloma cells to produce a panel of 9 monoclonal antibodies (MoAbs). Five of the MoAbs were of the IgA isotype. The antigen recognition profiles of these MoAbs were studied using SDS-PAGE and immunoblotting; three major profile patterns were identified. Five MoAbs recognized a major band in the MW range 43-48 kD; all recognized a range of antigens. Three MoAbs were used to localize antigens in the bodies of adult worms. Granules within the anterior stichocytes were recognized strongly, as was material within the eggs and pseudocoelom. Two MoAbs stained the cuticle. Although the phosphorylcholine (PC) determinant was widely distributed within worm tissues none of the MoAbs tested recognized PC. Passive transfer of immunity was achieved using two of the IgA monoclonals; no immunity was transferred by the IgM and IgG MoAbs used. The limited recognition profiles of these IgA MoAbs, and the ability to stain stichocyte granules, suggest that their protective activity results from an interaction with ES antigens.

Schistosoma mansoni: histological analysis of the synergistic interaction between vaccine immunity and praziquantel therapy in the lungs of mice.

Naive CBA mice and mice vaccinated 4 weeks previously with gamma-irradiated cercariae of S. mansoni were challenged percutaneously with normal cercariae and then treated with 500 mg/kg body weight of Praziquantel (Pzq). The drug was administered intradermally on day 1 or intramuscularly on day 6, thus targeting against skin stage or lung stage challenge larvae respectively. The skin site of challenge and/or the lungs were removed at various time points to provide samples for histological examination. As reported elsewhere (Flisser, Delgado & McLaren 1989) the efficacy of Pzq was significantly enhanced in vaccinated mice and was influenced by the treatment regime. Histological analysis revealed that when Pzq was administered I/D on day 1 to vaccinated mice, the inflammatory response to challenge differed in extent but not nature from that seen in vaccinated but untreated cohorts. This correlates with worm recovery data showing no (this study), or only marginal synergy between drug treatment and immunity using this regimen of drug treatment (Flisser et al. 1989). Following the day 6 protocol of drug delivery, however, lungs from treated vaccinated mice exhibited many large inflammatory reactions containing trapped challenge larvae. In contrast, lungs from untreated vaccinated mice had only few foci which were small and rarely contained trapped larvae. These data again correlate well with worm recovery data showing that there is a highly significant synergy between vaccination and drug treatment administered at this time (Flisser et al. 1989; this study). It would seem, therefore, that Pzq exacerbates lung phase immunity in the NIMR vaccine mouse model where skin phase immunity predominates and pulmonary attrition is normally minimal. The results are discussed in the light of published data concerning the effector mechanisms thought to characterize skin and lung phase vaccine resistance in the murine model.

Immune dependence of schistosomicidal chemotherapy: an ultrastructural study of Schistosoma mansoni adult worms exposed to praziquantel and immune serum in vivo.

This study examines the immune-dependence of praziquantel (PZQ) for the treatment of Schistosomiasis mansoni in mice. We have shown elsewhere from worm recovery data that the efficacy of PZQ is significantly enhanced when mice are treated concomitantly with antisera raised against antigens released from adult schistosomes, even though such antisera show no intrinsic helminthotoxic activity (Doenhoff et al. 1987, Doenhoff, Modha & Lambertucci 1988). Moreover, indirect immunofluorescence assays have shown that male worms exposed to the dual treatment regime in vivo bind antiserum to their dorsal surfaces in a pattern that seems to follow the outline of the dorsal tubercles. Scanning and transmission electron microscopy have now been used to further define the features of damage inflicted upon worms through exposure to antiserum alone, drug alone, or the two treatments in combination. Such investigations revealed that the antiserum induces a classical membrane repair process in worms of both sexes, but little other damage. PZQ causes the formation of spherical protuberances on the dorsal tubercles of male worms, while the dual treatment regime induces both kinds of damage in male schistosomes, but with much enhanced severity. The protuberances show evidence of explosion and some regions of the tegument become completely destroyed. Regions other than the dorsal surfaces of the male worms do not exhibit comparable trauma, and neither do the females. These data are discussed in relation to the known schistosomicidal activity of PZQ, the notion that male and female worms exhibit regional and sexual differences in susceptibility, documented evasive strategies of the parasite and the interdependence of immuno- and chemotherapy.

Evidence that radio-sensitive cells are central to skin-phase protective immunity in CBA/Ca mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni as well as in naive mice protected with vaccine serum.

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were subjected to 550 rad of whole body (gamma) irradiation and then challenged 3 days later with normal cercariae. The perfusion recovery data showed that this procedure reduced the primary worm burden in naive mice by 22% and the challenge worm burden in vaccinated mice by 82%. Irradiation also ablated the peripheral blood leucocytes of both mouse groups by 90-100% at the time of challenge. Histological data revealed that such treatment caused a dramatic change in number, size and leucocyte composition of cutaneous inflammatory skin reactions that characterize challenged vaccinated mice and are known to entrap invading larvae; cutaneous eosinophils were preferentially abolished by this treatment. Polyvaccine mouse serum that conferred protection passively upon naive recipient mice, failed to protect naive/irradiated mice when administered by the same protocol. Distraction of macrophages by treatment of mice with silica did not affect the establishment of a primary worm burden and reduced the protection exhibited by vaccinated mice by only 16%. These data indicate that radio-sensitive cells are important to both innate and specific acquired resistance in this mouse model and that macrophages contribute only marginally to the expression of vaccine immunity.

Schistosoma mansoni: analysis of the humoral and cellular basis of resistance in guinea-pigs vaccinated with radiation-attenuated cercariae.

This study addresses the humoral and cellular basis of specific acquired immunity in the guinea-pig irradiated vaccine model of schistosomiasis mansoni. Rodents vaccinated with 500 gamma-irradiated cercariae and then splenectomized 4.5 weeks later showed a 33% reduction in resistance to challenge as compared to vaccinated animals or vaccinated/sham splenectomized controls. Serum harvested from once vaccinated individuals conferred modest levels of resistance upon naive recipients in some experiments, but transfer was not achieved consistently. Serum from vaccinated and thrice boosted rodents (Vbbb) routinely transferred around 45% immunity, however, provided it was given in 4 ml aliquots on day 9 post-challenge; Vbbb serum thus transferred 50% of donor immunity. Interestingly, multiple doses of this protective serum given on and either side of day 9 did not enhance the protection achieved with a single 4 ml aliquot. Neither peripheral lymph node cells nor splenocytes from the polyvaccinated serum donors were able to transfer resistance to recipient guinea-pigs and they failed to augment the protection achieved with Vbbb serum. Foot-pad testing revealed no correlation between delayed hypersensitivity responses and immunity to challenge in vaccinated guinea-pigs. Although polyvaccine guinea-pig serum successfully protected homologous recipients, it failed to protect mice when administered either at the time of challenge (the optimal schedule for transfer of polyvaccine mouse serum), or around day 9 (the optimal schedule for guinea-pigs). Similarly, guinea-pigs could not be protected with polyvaccine rat serum that conferred 75% resistance upon naive recipient rats.

Evidence for enhancement of IgG1 subclass expression in mice polyvaccinated with radiation-attenuated cercariae of Schistosoma mansoni and the role of this isotype in serum-transferred immunity.

Serum or immunoglobulin fractions of serum from CBA/Ca mice vaccinated three or four times with radiation-attenuated cercariae of Schistosoma mansoni have been investigated for their capacity to confer protection upon naive mice. The data confirm that around 35% protection can be transferred with polyvaccine mouse serum administered in 0.5-ml aliquots 1 h before challenge (intravenously) and 24 h post-challenge (intraperitoneally). We show in addition, however, that polyvaccine serum is also protective when injected into the skin site of challenge as a single 0.05-ml aliquot. In contrast, lymphocytes obtained from the donors of protective serum conferred only 13% protection upon recipient mice. The passive cutaneous anaphylaxis assay showed that IgG1 is incremented by polyvaccination, while passive transfer experiments revealed that of the different isotypes fractionated from whole protective serum, only IgG1 has the capacity to protect naive recipients against challenge. The resistance transferred by IgG1 represents more than 60% of that obtained with whole serum and can be achieved using either the intravenous/intraperitoneal or the subcutaneous administration regimen. Recipients of serum given via the subcutaneous route exhibit cutaneous inflammatory focal reactions which comprise 20% eosinophils and 80% mononuclear cells; these foci entrap challenge larvae. The importance of IgG1 subclass expression to the success of serum-transferred resistance is discussed.

Experimental animal models in vaccination against schistosomiasis.

Contrary to previous expectations, innate resistance to a primary schistosome infection is mediated predominantly in the lungs of many laboratory rodents. In addition, the phenomenon of non-permissiveness seen in a sub population of 129 strain mice, is associated with worm relocation from the liver to the lungs and is facilitated by dramatic alteration to the lung and liver vasculature; lung located adult worms exhibit gut damage and are ultimately destroyed within eosinophil-rich inflammatory focal reactions. It is now clear that the immunity induced by exposure to radiation-attenuated cercariae can be affected in the skin (mice), the lungs (mice and rats) or the liver (guinea pigs) of laboratory rodents. Moreover, the fact that skin phase resistances involves radio-sensitive cells, while lung and liver phase immunity centres on radio-resistant leucocytes, resolves current discord in the literature. Immobilisation and trapping of challenge larvae within focal inflammatory infiltrates is nevertheless common to both skin and lung phase attrition. Hyperimmunisation of rodents with irradiated cercariae is associated with a switch in immunoglobulin isotype and serum harvested from such donors is able to protect naive recipients passively; transferred serum recruits effector cells. Challenge parasites exhibit a broader window of sensitivity to vaccine immunity than was originally envisaged; stages ranging from the 3 to 4 day old skin/lung stage larva to the 3 week old juvenile liver worm constitute targets of protective resistance in vivo. This is at variance with the fact that newly transformed schistosomula constituting the primary targets of in vitro effector mechanisms, a feature perhaps related to our inability to mimic the process of intravascular parasite immobilisation and trapping in the test tube. Finally, schistosomicidal drugs such as Praziquantel can, by re-exposing disguised parasite antigens, facilitate the expression of vaccine immunity in sites additional to those at which resistance is normally mediated.

Effect of praziquantel on the migration and survival of developmental stages of Schistosoma mansoni in mice.

Compressed organ autoradiography has been used to determine whether the anthelmintic drug praziquantel (Pzq) modifies the migration of isotopically labelled Schistosoma mansoni during the first 16 days of infection in CBA/Ca mice. The mice were treated with 500 mg kg-1 body weight of the drug on day 1 or day 6. Treatment caused a marked delay in parasite migration from the skin when the drug was administered intradermally at the site of infection on day 1; migration from the lungs was also delayed after such treatment. Pzq injected either intradermally on day 1 or intramuscularly on day 6 effectively reduced the number of parasites that finally arrived in the lungs and the livers by 41 and 47%, respectively. Intramuscular administration of the drug on day 1 had a negligible effect. Worm recoveries assessed on day 38 by perfusion of the hepatic portal system were greatly reduced when Pzq was administered on day 14. The worms proved less susceptible when the drug was administered on day 21 and were completely resistant following drug delivery on day 28. The influence of drug preparation and route of delivery on parasite migration and survival are discussed.

Schistosoma mansoni: enhanced efficacy of praziquantel treatment in immune mice.

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were challenged with normal cercariae and then treated with 500 mg/kg body weight of Praziquantel (Pzq). The drug was administered intramuscularly or intradermally on day 1, but intramuscularly only on day 6. The results show clearly that in naive mice skin-stage larvae were susceptible to Pzq provided the drug was given intradermally on day 1. Lung worms were susceptible to Pzq given intramuscularly on day 6. Drug efficacy in naive mice is thus dependent upon treatment being given at the correct time and via the optimal route. The efficiency of Pzq treatment was enhanced in vaccinated mice, but was again affected by the treatment regime. Analysis of the data revealed a highly significant synergistic effect between drug treatment and vaccination when Pzq was given intramuscularly on day 6. Synergy was detectable but only marginally significant when the drug was administered intradermally on day 1, and could not be demonstrated when Pzq was given via the intramuscular route on day 1. These findings are discussed in the light of known sites and mechanisms of vaccine resistance in mice, as well as in relation to the mode of action of Pzq against schistosome parasites.

Effect of praziquantel treatment on lung-stage larvae of Schistosoma mansoni in vivo.

Mice infected heavily with Schistosoma mansoni cercariae were treated 6 days later with Praziquantel and the parasites studied 1 h post-treatment. Immunofluorescence experiments showed that parasite surface antigens became available for labelling in larvae harvested from Praziquantel-treated mice, but not from untreated mice. Red blood cell antigens acquired from the host were localized on all lung worms studied. The distribution of each set of antigens, as revealed by fluorescence-labelling was, however, quite different. The lungs of heavily infected, drug-treated mice, exhibited severe haemorrhages which occurred in a dose-dependent manner. Fewer haemorrhages were found when heavily infected mice were treated simultaneously with Praziquantel and Aprotinin, a proteinase inhibitor. These results indicate that Praziquantel treatment in vivo induces the exposure of parasite antigens on lung-stage worms and may cause the release of parasite-derived enzymes which induce haemorrhages in the lung tissue of the host.

Mesocestoides corti: parameters of infection in CBA/Ca mice and the effect of introducing a concomitant trematode infection.

CBA/Ca mice infected with the tetrathyridia of Mesocestoides corti were exposed to Schistosoma mansoni cercariae either simultaneously, or at varying intervals after the initial M. corti infection. Cohorts were infected with either parasite alone. The dual infected mice and the mice harbouring M. corti alone were significantly heavier than either naive controls or mice carrying the S. mansoni infection only. The livers and spleens from dual infection mice were also found to be significantly heavier than those from other groups. Free M. corti tetrathyridia were reduced in number in the peritoneal cavity of dual infected mice, as compared with mice harbouring a single infection. Furthermore, the intensity of the initial M. corti infection, as measured by the number of capsules surrounding parasites in the liver, was reduced when the mice experienced an S. mansoni infection 21 days later. The mice which were exposed to M. corti only exhibited more mast cells and eosinophils around encapsulated tetrathyridia in the liver than did dual infection mice, while cells surrounding S. mansoni egg granulomas in the liver were significantly increased in dual infection mice. An increase in serum alkaline phosphatase levels was detected in both the mice receiving the dual infection and the mice with the S. mansoni only infection, but not in mice harbouring M. corti alone.

Serum from CBA/Ca mice vaccinated with irradiated cercariae of Schistosoma mansoni protects naive recipients through the recruitment of cutaneous effector cells.

Passive transfer experiments showed that 76% of the resistance induced in CBA/Ca mice by exposure to radiation-attenuated cercariae of Schistosoma mansoni could be transferred to naive recipients by administration of donor serum. The level of protection achieved depended on the volume of serum administered and immunity was demonstrated most consistently with serum harvested from thrice vaccinated donors. The serum was twice as effective when given to the recipients at the time of cercarial challenge as compared to administration 5 days after challenge, a result which indicates that serum-dependent challenge elimination is probably accomplished in the cutaneous tissues. This view was confirmed by the observation that passive protection could be ablated by administration of a monoclonal antibody which we have shown elsewhere to deplete the cutaneous inflammatory reaction to cercarial penetration. Histopathological studies revealed that vaccine serum induced subdermal inflammatory reactions in challenged recipient mice which were identical both in induration and kinetics to those seen in conventionally vaccinated individuals; on days 4 and 5 post-challenge, the reactions comprised 60% mononuclear cells and 40% eosinophils. Challenge larvae, which had transformed from skin-stage to lung-stage parasites, became trapped within such reactions and eventually showed generalized vacuolation consistent with the onset of damage. Some foci were seen to contain degenerated leucocytes, free eosinophil granules and debris which is thought to represent the remnants of dead parasites. Small focal reactions were identified on occasion in naive challenged mice and in recipients of normal mouse serum, but these reactions comprised predominantly mononuclear cells and were rarely seen to encompass challenge parasites. These data show that serum-transferred resistance in the vaccinated CBA/Ca mouse model involves the induction of a cutaneous inflammatory response in the recipients.

Proteinase phenotypes and fixation properties of rat mast cells in parasitic lesions caused by Mesocestoides corti: selective and site-specific recruitment of mast cell subsets.

The distribution, fixation properties, and protease phenotypes of mast cells populating lesions caused by the metacestode stage of the cestode Mesocestoides corti in the rat were characterized. Intraperitoneal infection with M. corti induced severe granulomatous types of reactions around the pancreas and further lesions in the liver. These sites were infiltrated with mast cells which contained either rat mast cell protease I or II derived respectively from connective tissue (CTMC) or mucosal mast cells (MMC). A proportion of cells in pancreatic granulomas had staining and fixation properties identical to those of intestinal mucosal mast cells; others were typical connective tissue mast cells. Subcutaneous inoculation of parasites was associated with nodular dermal reactions, and all of the infiltrating mast cells had the fixation and staining properties of CTMC and contained RMCPI uniquely. Increased numbers of RMCPII-containing mast cells were present in the intestines of rats infected intraperitoneally. Significant quantities of RMCPII were present in homogenates of pancreatic granulomas and in livers of rats harbouring intraperitoneal infections but none was detected in skin. These findings suggest that mast cells of different phenotypes are selectively recruited to some, but not all, lesions.

Schistosoma mansoni: evidence that eosinophils and/or macrophages contribute to skin-phase challenge attrition in vaccinated CBA/Ca mice.

Naive CBA mice and mice vaccinated 4 weeks previously with gamma-irradiated cercariae were challenged percutaneously with normal cercariae and the skin site of challenge removed at various times to provide samples for histological examination. Neutrophil abscesses surrounding schistosomula as well as cast cercarial tails were identified at the skin surface in both naive/challenged and vaccinated/challenged mice. The dermis in both groups of animals became infiltrated with mononuclear cells and granulocytes. Neutrophils were the predominant granulocyte in naive/challenged skin but eosinophils replaced neutrophils by 48 h post-challenge in skin from vaccinated mice. Schistosomula with adherent mononuclear cells and granulocytes were identified preferentially in the dermis of vaccinated mice; such larvae frequently exhibited subtegumental vacuolation. A further major characteristic of vaccinated/challenged skin was the formation, from 18 h, of extensive subdermal inflammatory foci, consisting of up to 50% mononuclear cells and 50% eosinophils. These foci invariably surrounded one or more larval parasites which were seen by both optical and scanning electron microscopy to have a morphology typical of lung stage rather than skin stage schistosomula. Dead, infiltrated parasites were also recognized within the subdermal foci. Our data support the view that vaccine immunity in this mouse model is mediated primarily in the skin and indicate that challenge attrition involves immobilization and killing of larval parasites by effector cells within subdermal inflammatory foci.

Immunity to Schistosoma mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae. T-cell activation of macrophages for larval killing.

This study addresses macrophage activation in guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosoma mansoni. Peritoneal exudate macrophages elicited in vaccinated animals by mineral oil injection were activated to kill larval schistosomes in vitro. Killing efficiency is dependent upon the cell: target ratio employed and is enhanced by, but is not strictly dependent on, the presence of specific antibodies. Macrophages co-cultured with parasites release superoxide radicals and hydrogen peroxide, but the use of inhibitors has shown that neither of these reactive oxygen intermediates are the causal agents of cellular cytotoxicity in this system. Oil-elicited macrophages from naive guinea-pigs do not show comparable activation; they can, however, be activated in vitro by incubation with culture supernatant fluids from schistosome antigen-stimulated spleen, or lymph node cells harvested from vaccinated guinea-pigs. Naive macrophages activated in this way kill schistosomula in vitro and release the activation markers IL-1 and superoxide anion. The macrophage-activating factor (MAF) present in spleen cell culture supernatant fluids has a MW of 35,000-55,000, but does not have the chemical characteristics of gamma-interferon. In this study MAF is shown to be released by a population of lymph node cells that does not adhere to nylon-wool columns, that responds well in proliferation assays to schistosome antigens and to the T-cell mitogen concanavalin A, but does not respond to the B-cell mitogen lipopolysaccharide. These cells have been identified as small lymphocytes.

Analysis and comparison of immune reactivity in guinea-pigs immunized with equivalent numbers of normal or radiation-attenuated cercariae of Schistosoma mansoni.

Guinea-pigs immunized with equivalent numbers of normal or radiation-attenuated cercariae of Schistosoma mansoni are known to develop close to complete resistance to reinfection at weeks 12 and 4.5 respectively. We here analyse and compare the immune responses induced by the two populations of cercariae. Both radiation-attenuated and normal parasites of S. mansoni elicited an extensive germinal centre response in guinea-pigs by week 4.5 post-immunization. The anti-parasite antibody titre and cytotoxic activity of serum from 4.5-week-vaccinated, or 4.5-week-infected guinea-pigs were approximately equal, but sera from 12-week-infected individuals had high titres of anti-parasite antibody, which promoted significant larvicidal activity in vitro. In all cases, larvicidal activity was mediated by the IgG2 fraction of the immune serum. Lymphocyte transformation tests conducted on splenic lymphocytes from 4.5-week vaccinated guinea-pigs revealed maximal stimulation against cercarial, 2-week and 3-week worm antigens, whereas spleen cells from 4.5-week-infected guinea-pigs were maximally stimulated by cercarial and 6-week worm antigens. The splenic lymphocyte responses of 12-week infected animals were dramatic against antigens prepared from all life-stages of the parasite.

A comparison of two procedures for labelling the surface of the hydatid disease organism, Echinococcus granulosus, with 125I.

Living, intact protoscoleces of the British horse and sheep strains of Echinococcus granulosus were subjected to surface radioiodination procedures using 125I and Iodogen and 125I-Bolton Hunter reagent. Subsequent combined electron microscopy and autoradiography revealed specific surface membrane labelling with the Iodogen procedure, but significant tegumental labelling with the Bolton-Hunter reagent. The two parasite strains yielded different profiles of electrophoretically separated labelled proteins; the Iodogen method, not surprisingly, resulted in a less complex pattern of labelled polypeptides than the Bolton and Hunter reagent.

Trichinella spiralis: immunocytochemical localization of surface and intracellular antigens using monoclonal antibody probes.

A panel of immunologically and biochemically defined monoclonal antibody probes has been used in conjunction with immunocytochemical techniques to localize target antigens in sections of different life-cycle stages of Trichinella spiralis. Monoclonals that immunoprecipitate surface components from adult worms, show reactivity with the surface but not with internal tissues of sectioned parasites. Reagents that immunoprecipitate radio-isotope labelled stage-specific surface components of muscle-stage larvae, however, react with the stichosome and gut lining of sectioned larvae, as well as with the surface. Monoclonal antibody probes that do not stain the surfaces of live, intact muscle larvae in immunofluorescence assays, but which immunoprecipitate solubilized surface glycoproteins, also show reactivity with cuticular and stichosomal antigens of sectioned larvae. The more powerful resolution provided by electron microscopy has localized the surface antigens to the epicuticle and the intestinal antigens to the brush-border microvilli. Of particular interest was the finding that antigens of muscle-stage larvae, known to confer protection upon recipient mice, also exist in the stichosome of adult parasites. This observation may shed some light on the fact that mice immunized with antigens from muscle-stage larvae show, in addition to reduced muscle larva burden, accelerated expulsion of adult worms. The implications of these data for stage specificity of immune responses to trichinosis are discussed.

Immunity to schistosomiasis mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae: I. Humoral responses against skin-stage schistosomula.

The anti-schistosomular humoral responses of guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosoma mansoni have been investigated in vitro. The sera of vaccinated animals contain schistosomulicidal complement-fixing antibodies which peak in titre at week 5 after vaccination and predominantly consist of IgG2 and IgM antibodies. The ability of the serum to arm macrophages from normal animals to bind to schistosomula, also peaks in titre at week 5 and is associated with IgG2 antibodies. Basophils from normal animals can be sensitized in vitro by vaccine serum to degranulate in the presence of schistosomular antigens. This anaphylactic antibody activity is associated with IgG1 but not IgE antibodies, and peaks in titre at week 10. Three antigens (14 kD, 20 kD and 43 kD) are specifically and transiently detected by vaccine serum on Western blots of schistosomular proteins; these antigens are first discernible at week 4, but were virtually undetectable at week 12.